Why be original? Two new species of Choeradoplana resembling the type species of the genus in their external aspects (Platyhelminthes, Continenticola)

Abstract The genus Choeradoplana Graff, 1896 encompasses 16 species, most of them found in Brazil. Herein two new species of this genus are described from remnants of Araucaria moist forests, located in the states of Paraná and Santa Catarina, south Brazil. Both species resemble the type-species of the genus, C.iheringi, showing brownish dorsal surface covered by dark-brown flecks. However, regarding their anatomy, the new species differ from C.iheringi and other congeners by a long and horizontal disposed permanent papilla. In such aspects, both species resemble C.benyai, but differ from this species, as well as from each other, in details of the prostatic vesicle, ejaculatory duct, and penis papilla.


Introduction
The genus Choeradoplana, proposed by Graff (1896), has a Neotropical distribution, with most species recorded from Brazil. Its representatives show a cephalic region with a glandulo-muscular organ and longitudinal cutaneous musculature with a portion sunk into the mesenchyme (Graff 1899, Ogren andKawakatsu 1990). The genus was reviewed by Froehlich (1955). Carbayo and Leal-Zanchet (2003) and Carbayo and Froehlich (2012) suggested the inclusion of other features in its diagnosis. Currently, the genus encompasses 16 species, six of them occurring in areas of ombrophilous Araucaria forest (Graff 1899, Ogren and Kawakatsu 1990, Leal-Zanchet and Souza 2003, Carbayo and Froehlich 2012, Negrete and Brusa 2012, Lemos et al. 2014, Álvarez-Presas et al. 2017.
The Araucaria forest is a phytophysiognomy of the Atlantic Forest, which harbours a high species richness of land flatworms (Sluys 1998, Leal-Zanchet and Carbayo 2000, Castro and Leal-Zanchet 2005, Antunes et al. 2008, Leal-Zanchet and Baptista 2009Baptista et al. 2010, Leal-Zanchet et al. 2011, Amaral et al. 2014. A recent inventory of land flatworms in areas of Araucaria moist forest located in the states of Paraná and Santa Catarina, in south Brazil, indicated the occurrence of brownish specimens with dark-brown flecks over the dorsal surface, similar to the type-species of the genus, Choeradoplana iheringi Graff, 1899. Anatomical and histological analyses indicated that they belong to two different species that are herein described.
Just after sampling, colour pattern, body shape and dimensions of live specimens were recorded. They were then euthanised using boiling water and fixed in neutral formalin 10%. After fixation, specimens were maintained in 70% ethyl alcohol. Methods described by Rossi et al. (2015) were used for histological processing of material and analysis of external and internal characteristics. The material was sectioned at intervals of 6 µm and stained with Masson's trichrome method or Haematoxylin and Eosin (Romeis 1989 Description. External features. Body elongate with parallel margins (Figs 1, 2), elliptical in cross section; anterior end expanded, posterior end slight pointed. Cephalic region (ca. 3 mm long) with two glandular cushions separated by a median longitudinal slit in the ventral surface. Maximum length 67 mm when crawling; 50 mm after fixation (Table 1). Mouth at median third of body; gonopore at posterior third of body (Table 1).
Live specimens with dorsal surface covered by dark-brown pigmentation constituted by irregular, small flecks. Yellowish ground colour visible on cephalic region, on body margins, as well as on thin median stripe occurring along the body except for cephalic region (Figs 1, 2). Ventral surface whitish before and after fixation. After fixation, dorsal pigmentation fades.
Eyes absent on anterior tip (first 1.5 mm of body, corresponding to 3% of body length). Afterwards, eyes initially monolobate and uniserial. Eyes become trilobate and plurisserial after 3 mm and sparser towards posterior tip. No clear halos around eyes. Pigment cups between 20 µm and 30 µm in diameter.
Sensory organs, epidermis and body musculature. Sensory pits, as simple invaginations (15-20 µm deep), absent on anterior tip, occurring in a single row between 3% and 10% of body length. Three types of glands discharge through whole epidermis of pre-pharyngeal region: rhabditogen glands with xanthophil rhammites (ventrally with smaller rhabdites) and cyanophil glands with amorphous secretion, as well as few erythrophil glands with fine granular secretion (Figs 3, 4). Creeping sole occupies 82% of body width. Glandular margin absent. Glands discharging through anterior tip of body with similar arrangement as in other species of the genus. Table 1. Measurements, in mm, of specimens of Choeradoplana longivesicula sp. n. Abbreviations: * after fixation; DG distance of gonopore from anterior end; DM distance of mouth from anterior end; DMG distance between mouth and gonopore; DPVP distance between prostatic vesicle and pharyngeal pouch. The numbers given in parentheses represent the position relative to body length.  Cutaneous musculature with usual three layers (circular, oblique, and longitudinal layers), showing part of ventral longitudinal layer, as well as some muscle bundles of dorsal longitudinal layer, imbedded in mesenchyme (Fig. 3, Table 2). Longitudinal layer between four and eight times thicker than other two cutaneous layers in pre-pharyngeal region ( Table 2). Cutaneous musculature as thick paramedially as medially. Ventral musculature slightly thinner than dorsal in pre-pharyngeal region. Ratio between cutaneous musculature and body height (mc:h) ca. 19% (Table 2). In cephalic region, cutaneous musculature with similar arrangement as in other species of the genus (Fig. 5).
Digestive System. Pharynx bell-shaped, ca. 6% of body length, occupies ca. 90% of pharyngeal pouch. Mouth slightly posterior to dorsal insertion next to end of median third of pharyngeal pouch (Fig. 7). Oesophagus absent.
Reproductive organs. Testes in one or two irregular rows on either side of body, located beneath dorsal transverse mesenchymal muscles, between intestinal branches (Fig.  3), begin slightly anteriorly to ovaries, in anterior third of body, and extend to next to root of the pharynx (Table 1). Sperm ducts dorsal to ovovitelline ducts, under or among fibres of sub-intestinal transverse mesenchymal musculature, in pre-pharyngeal region ( Figure 3). They form spermiducal vesicles posteriorly to pharynx. Sperm ducts enter common muscle coat, ascend slightly and open laterally into proximal wall of prostatic vesicle. Intrabulbar prostatic vesicle, tubular and unpaired, traverses both penis bulb and papilla (Fig. 10), narrowing to open through tip of papilla as an ejaculatory duct (Fig.  11). Penis papilla, cylindrical and almost symmetrical, filling entire common atrium. Dorsal insertion of penis papilla slightly shifted posteriorly. Common atrium oval-elongate and unfolded, without differentiation between male and female atria (Figs 8,9).
Prostatic vesicle lined with high pseudostratified epithelium with few ciliated cells, receiving numerous openings of glands with ill-stained, coarse granular secretion, sometimes containing an erythrophil core. In addition, sparse openings of other two types of glands occur throughout the epithelium of prostatic vesicle: cells with xanthophil, coarse granular secretion, and cell with amorphous, cyanophil secretion. Vitelline follicles situated between intestinal branches, well developed. Ovaries ovoid, ca. twice longer than wide, measuring 0.2 mm in its antero-posterior axis. They are located dorsally to the ventral nerve plate, in anterior third of body. Ovovitelline ducts emerge laterally from median third of ovaries, then run posteriorly immediately dorsal to the nerve plate. Behind the gonopore, the ovovitelline ducts ascend posteriorly and medially inclined, and unite, dorsally to the female canal, forming the common glandular ovovitelline duct. Female canal relatively long and C shaped. This canal opens into bottom of posterior part of atrium, where a constriction occurs (Figs 8,9).
Female canal lined with erythrophil, pseudostratified epithelium. Three types of glands open through the epithelium of female canal: abundant cells with finely granular, erythrophil secretion, cells with coarse granular, xanthophil secretion, and scarce cells with amorphous, cyanophil secretion. Muscularis of female canal composed of longitudinal and circular interwoven fibres (20-30 µm) Gonopore canal almost vertical at the sagittal plane. Common muscle coat highly developed, especially at penis bulb (Figs 8,9), with interwoven oblique, circular and longitudinal fibres.
Etymology. The name is composed of the Latin adjective longus (long) and the Latin vesicula, alluding to the elongate prostatic vesicle.
Distribution. known only from the type-locality, General Carneiro, Paraná, Brazil. slides; pre-pharyngeal region: transverse sections on 7 slides; pharynx and copulatory apparatus: sagittal sections on 25 slides. Diagnosis. A species of Choeradoplana with dorsal surface covered by irregular small dark-brown flecks; pharynx bell-shaped; sperm ducts opening subterminally into prostatic vesicle; prostatic vesicle oval-elongate and folded, becoming funnelshaped proximally and forming an elongate duct inside penis papilla; penis papilla, conical, long and almost symmetrical, with dorsal insertion shifted posteriorly, filling the whole atrium.
Description. External features. Body elongate with parallel margins (Fig. 12), sub-cylindrical in cross section; anterior end expanded, posterior end slight pointed. Cephalic region (ca. 3 mm long) with two glandular cushions and a median slit in the ventral surface. Maximum length 20 mm when resting; 50 mm after fixation (Table 3). Mouth at median third of body; gonopore at posterior third of body (Table 3).
Live specimens with dorsal surface covered by irregular, small dark-brown flecks over all body length including cephalic region (Fig. 12). Yellowish ground colour visible on cephalic region, on body margins, as well as on thin, inconspicuous median stripe occurring along the anterior body half except for cephalic region. Ventral surface pale yellow. After fixation, dorsal pigmentation remains brownish; ventral surface becomes whitish with darker body margins.
Eyes absent on cephalic region (first 1.2 mm of body, corresponding to 2.4% of body length). After that, eyes initially monolobate and uniserial. Eyes become trilobate and plurisserial after 3 mm, becoming sparser towards posterior tip. No clear halos around eyes. Diameter of pigment cups between 24 µm and 32 µm in diameter.
Sensory organs, epidermis and body musculature. Sensory pits, as simple invaginations (15-18 µm deep), absent on anterior tip, occurring in a single row between 3% and 10% of body length.
Three types of glands discharge through whole epidermis of pre-pharyngeal region: rhabditogen glands with xanthophil rhammites (ventrally with smaller rhabdites) and cyanophil glands with amorphous secretion, as well as few xanthophil glands with coarse granular secretion (Fig. 13). Creeping sole occupies 89% of body width. Glandular margin absent. Glands discharging through anterior tip of body with similar arrangement as in other species of the genus.
Cutaneous musculature with usual three layers (circular, oblique, and longitudinal layers), with part of ventral longitudinal layer, as well as few muscle bundles of dorsal longitudinal layer, imbedded in mesenchyme (Fig. 13, Table 4). Longitudinal layer between five and eight times thicker than other two cutaneous layers in prepharyngeal region (Table 4). Cutaneous musculature as high paramedially as medially. Ventral musculature thinner than dorsal in pre-pharyngeal region. Mc:h 22% (Table 4). In cephalic region, cutaneous musculature with similar arrangement as in other species of the genus.
Mesenchymal musculature (Fig. 13) weakly developed, mainly composed of three layers: (1) dorsal subcutaneous, located mainly close to cutaneous musculature, with decussate fibres (2 fibres thick), (2) supra-intestinal transverse (2-4 fibres thick) and (3) sub-intestinal transverse (5-7 fibres thick). In cephalic region, mesenchymal musculature with similar arrangement as in other species of the genus. Table 3. Measurements, in mm, of the holotype of Choeradoplana cyanoatria sp. n. Abbreviations: * after fixation; DG distance of gonopore from anterior end; DM distance of mouth from anterior end; DMG distance between mouth and gonopore; DPVP distance between prostatic vesicle and pharyngeal pouch. The numbers given in parentheses represent the position relative to body length. Digestive system. Pharynx bell-shaped, as long as 7% of body length, occupies almost entire pharyngeal pouch. Mouth almost at the same transversal level as dorsal insertion in the beginning of median third of pharyngeal pouch (Fig. 14). Oesophagus absent.
Reproductive organs. Testes in two or three irregular rows on either side of body, located beneath dorsal transverse mesenchymal muscles, between intestinal branches (Fig. 13). They begin slightly anteriorly to ovaries, in anterior sixth of body, to just the root of the pharynx (Table 3). Sperm ducts dorsal to ovovitelline ducts, medially displaced, under or among fibres of sub-intestinal transverse mesenchymal musculature, in pre-pharyngeal region (Fig. 13). They form spermiducal vesicles posteriorly to pharynx. Sperm ducts enter common muscle coat, recurve, and open subterminally into prostatic vesicle. Intrabulbar prostatic vesicle, oval-elongate and folded, becoming funnel-shaped both proximally and distally (Fig. 17). Inside penis papilla, prostatic vesicle narrows and forms an elongate duct that opens through tip of the papilla. Penis papilla, conical, long and almost symmetrical, filling the whole atrium. The dorsal insertion of the penis papilla is posteriorly shifted (Figs 15, 16). Folded atrium without anatomical or histological differentiation between male and female regions. Close to papilla insertions, longitudinal folds represent part of papilla wall.
Prostatic vesicle and proximal third of ejaculatory duct receive abundant openings of cells with coarse granular, erythrophil secretion, besides sparse amorphous, cyanophil secretion, besides a third type of gland containing heavy cyanophil granules. Distal two thirds of ejaculatory duct receives openings from numerous glands with amorphous, cyanophil secretion. Muscularis of ejaculatory duct thin (5µm) composed of longitudinal fibres. Abundant glands with densely distributed, coarse granular, xanthophil secretion and numerous glands with amorphous, cyanophil secretion open through epithelial lining of penis papilla, besides sparse erythrophil glands through lining of penis papilla. Numerous glands with amorphous, cyanophil secretion and scattered glands with erythrophil, fine granular secretion open through epithelial lining of the atrium, which is cyanophil. Muscularis of penis papilla (40-80µm) composed of subepithelial layer with circular fibres followed by layer with longitudinal fibres, both layers well developed. Posteriorly to the gonopore, necks of cyanophil glands concentrate subepithelially; subepithelial muscle fibres of atrium scattered among these cell necks (Fig. 18). Vitelline follicles, situated between intestinal branches, well developed. Ovaries ovoid, 1.5 times longer than wide, measuring 0.3 mm in its antero-posterior axis. They are located dorsally to the ventral nerve plate, in anterior sixth of body. Ovovitelline ducts emerge laterally from median third of ovaries, and run posteriorly immediately above nerve plate. Behind gonopore, the ovovitelline ducts ascend posteriorly and medially inclined, uniting to form a common glandular ovovitelline duct. This duct is situated dorsally to the relatively long, C shaped female canal, which opens into the atrium (Figs 15, 16).
Shell glands of two types: with coarse granular, erythrophil secretion, as well as with coarse granular, xanthophil secretion, the cells bodies of which occur among cell Gonopore canal vertical at the sagittal plane. Common muscle coat highly developed, especially at penis bulb (Figs 15,16), with interwoven oblique, circular and longitudinal fibres.
Etymology. The name is a composite of the Latin adjective cyano (blue) and the Latin atria, referring to the abundant cyanophil secretion opening through the atrium.
Distribution. Known only from the type-locality, Três Barras, Santa Catarina, Brazil.

Notes on ecology and distribution
Choeradoplana longivesicula was recorded only in its type-locality, the Araucaria Natural Heritage Private Reserve, state of Parana, in a site showing an initial stage of regeneration with poorly developed understorey Leal-Zanchet 2017, Amaral et al. 2018). Choeradoplana cyanoatria occurred only in its type-locality, the Três Barras National Forest, state of Santa Catarina, located ca. 150 km east from the type-locality of C. longivesicula, in an area of Araucaria moist forest. Both species showed low abundance during night samplings.

Discussion
Both new species described herein match the diagnostic features of the genus Choeradoplana, namely a cephalic region that is curved backwards, a cephalic glandulo-muscular organ, and a cutaneous longitudinal musculature with a portion internal to the subcutaneous nerve plexus throughout the body, among others (Ogren and Kawakatsu 1990, Carbayo and Froehlich 2012, Carbayo et al. 2013.
Both species share with C. benyai a long penis papilla with horizontal orientation. However, by having a cylindrical and long prostatic vesicle that traverses the penis papilla. Choeradoplana longivesicula differs from C. benyai that shows a globose prostatic vesicle with folded wall restricted to the penis bulb (Lemos et al. 2014). In addition, a long ejaculatory duct traverses the penis papilla of C. benyai, whereas in C. longivesicula the prostatic vesicle opens into the tip of the penis papilla through a constriction, as if it were a short ejaculatory duct. Regarding shape of the prostatic vesicle and ejaculatory duct, C. cyanoatria is quite similar to C. benyai and can be easily differentiated from C. longivesicula. The elongate conical penis papilla of C. cyanoatria distinguishes it from C. benyai, which has a cylindrical and relatively longer penis papilla (Lemos et al. 2014). Choeradoplana longivesicula and C. cyanoatria show a long atrium with a continuous muscle coat and without anatomical or histological distinction between male and female regions, thus differentiating them both from C. benyai. The latter shows male and female atria with independent muscle coats and different gland types opening into these regions (Lemos et al. 2014).