Revision and description of six species of Choeradoplana (Platyhelminthes, Tricladida), with an emendation to the genus Author Lago-Barcia, Domingo Laboratorio de Ecologia e Evolucao, Escola de Artes, Ciencias e Humanidades (EACH), Universidade de Sao Paulo (USP), Av. Arlindo Bettio, 1000, Sao Paulo, SP, 03828 - 000, Brazil & Departamento de Zoologia, Instituto de Biociencias, Universidade de Sao Paulo (USP), Rua do Matao, Tv. 14, 321, Sao Paulo, SP, 05508 - 090, Brazil domingo.lagobarcia@gmail.com Author Silva, Marcos Santos Laboratorio de Ecologia e Evolucao, Escola de Artes, Ciencias e Humanidades (EACH), Universidade de Sao Paulo (USP), Av. Arlindo Bettio, 1000, Sao Paulo, SP, 03828 - 000, Brazil Author Carbayo, Fernando Laboratorio de Ecologia e Evolucao, Escola de Artes, Ciencias e Humanidades (EACH), Universidade de Sao Paulo (USP), Av. Arlindo Bettio, 1000, Sao Paulo, SP, 03828 - 000, Brazil & Departamento de Zoologia, Instituto de Biociencias, Universidade de Sao Paulo (USP), Rua do Matao, Tv. 14, 321, Sao Paulo, SP, 05508 - 090, Brazil & Programa de Pos-Graduacao em Sistematica, Taxonomia Animal e Biodiversidade, Museu de Zoologia, Universidade de Sao Paulo, Sao Paulo, SP, Brazil text ZooKeys 2021 2021-02-11 1016 1 48 http://dx.doi.org/10.3897/zookeys.1016.59617 journal article http://dx.doi.org/10.3897/zookeys.1016.59617 1313-2970-1016-1 133ADA73102F422D8D6CBD27A01F1551 44B553052CCF5F1A9DB2D1295302BFB2 Choeradoplana eudoxiae Silva & Carbayo sp. nov. Figures 10 , 11 , 12 , 13 Material examined. Holotype MZUSP 2272 (field code, F3417), sexually mature: Floresta Nacional de Sao Francisco de Paula, State of Rio Grande do Sul, Brazil, ( -29.43628 , -50.37369 ). coll. F. Carbayo and co-workers, 22 January 2009; transverse sections of the cephalic region on 7 slides; horizontal sections of ovaries on 4 slides; transverse sections of the pre-pharyngeal region on 4 slides; sagittal sections of the pharynx and copulatory apparatus on 8 slides; the posterior extremity on 3 slides. Distribution. Only known from the type locality, Floresta Nacional de Sao Francisco de Paula, State of Rio Grande do Sul, Brazil. Etymology. The specific epithet pays homage to the late Prof. Eudoxia Maria Froehlich, 21 October 1928 - 26 September 2015, for her insightful life lessons and lasting contribution to the knowledge of the neotropical planarian fauna for 60 years. Diagnosis. Choeradoplana species with pastel yellow back and brown fawn spots more concentrated in the paramedian region. Its anterior extremity has no lateral dilations or "neck" differentiating its head from its body. The ventral side of the cephalic region is concave, and without distinct glandular cushions. The extrabulbar portion of the prostatic vesicle has paired branches and an unpaired, roughly rounded section; the intrabulbar portion is a dilated vertical duct. Penis papilla is absent. Description. The live holotype measured 38 mm in length, and 1.5 mm in width. Preserved, it measured 27.5 mm in length and 2 mm in width. Its body is slender and subcylindrical, with the anterior 1/8 becoming progressively thinner towards the anterior tip. The anterior extremity is rounded and the posterior is pointed. The dorsal side is convex, while the ventral side is slightly convex. The anteriormost body portion is approximately five millimeters long and rolled up so that the ventral side is facing upwards (Fig. 10A-C ). This ventral surface is concave, without distinct glandular cushions. This ventral surface is flat in the preserved holotype. Its creeping sole is as wide as 75.5% of body width at the pre-pharyngeal region. Its mouth is 14.5 mm (52.7% of body length) from the anterior extremity, and the gonopore is 16.8 mm (61.1%). The dorsum background color of the body is pastel yellow (RAL 1034) with fawn brown (RAL 8007) spots (Fig. 10A-C ); these spots are more densely distributed in the paramedian regions, some merged with each other. The ventral side is cream (Fig. 10D ). The cephalic extremity is greyish dorsally and ventrally. The body color faded on the preserved holotype. The eyes are one pigmented-cup type of 25-30 µm in diameter. There are no clear halos around them (Fig. 10C, D ). Since the very anterior histological sections are lost, it could not be ascertained whether they occur in this body region. Posteriorly, the eyes are marginal along the body length. Sensory pits are 17.0-22.5 µm deep, distributed ventro-laterally in a uniserial row from the anterior sections of the body (approximately 0.2 mm of the anteriormost body were lost) to 4.5 mm behind it. Abundant rhabditogen cells open onto the dorsal surface of the body and its margins in the pre-pharyngeal region. The epithelium of the margins is also pierced by gland cells producing erythrophil granules (Fig. 11A, B ). The ventral epithelium is pierced by three types of gland cells, namely scarce gland cells producing granules of dark, cyanophil secretion, gland cells producing cyanophil granules, and gland cells producing an erythrophil secretion. There is no glandular margin. The cutaneous musculature consists of a subepithelial circular muscle, followed by a diagonal layer with decussate fibers, and a longitudinal muscle organized in bundles (Fig. 11A, B ). This longitudinal muscle is 57.5 μm thick dorsally and arranged in bundles of 50-90 fibers each, whereas ventrally it is divided into a 30 μm-thick muscle of bundles (with 8-15 fibers each), and an insunk muscle with 70 μm-thick bundles (with 16-32 fibers each) (Fig. 11A ). The thickness of the cutaneous muscle coat is 20% of the body height. In the pre-pharyngeal region, the same parenchymal muscles as in Ch. iheringi , namely the dorsal decussate muscle (52-55 μm thick), transverse supra-intestinal muscle (20-22 μm ); and transverse subintestinal muscle (12-15 μm ) (Fig. 11A, B ). The muscle retractor of the head is delta-shaped in a cross-section along ~ 0.5 mm (or 1.8% of body length) starting from 0.1 mm behind the anterior extremity of the body (Fig. 11C, D ), and its thickness equals 36% of the height of the cephalic region. The Muskelgeflecht is 32 μm thick (22% of body height). The subneural parenchymal muscle consists of a few transverse fibers. The glandular cushions are composed of a relatively small quantity of rhabditogen cells (Fig. 11C, D ). The central nervous system presents a ventral nerve plate (70-85 μm thick or 9-11% of body height) in the pre-pharyngeal region. The mouth is located in the middle of the pharyngeal pouch (Fig. 12A ). The pharynx is bell-shaped (Fig. 12B ). An esophagus is absent. The outer pharyngeal epithelium is underlain by an 8 μm-thick longitudinal muscle, followed by a 15 μm-thick circular muscle. The inner pharyngeal epithelium is underlain by a circular muscle layer with longitudinal fibers interspersed (20 µm thick). The testes are dorsal, 90-150 μm in diameter, located under the supra-intestinal transverse muscle layer, and partially placed between the intestinal diverticula. The anteriormost testes are located 0.9 mm anterior to the ovaries (or 21% of the body length); posteriormost near the root of the pharynx (49% of the body length). Sperm ducts run immediately above the subintestinal muscle layer, dorsally and slightly internal to the ovovitelline ducts. Distal portions of sperm ducts contain sperm and are surrounded by a 20 μm-thick circular muscle. These ducts communicate with the respective short branch of the prostatic vesicle (Fig. 12C, D ). The paired branches run posteriorly. The extrabulbar portion of the prostatic vesicle consists of these paired branches and an unpaired, roughly rounded section with pleated wall receiving the branches. The intrabulbar portion of the prostatic vesicle is elongate and runs dorsally and posteriorly. The passage of the prostatic vesicle to the proximal region of the male atrium is narrowed by an annular fold (Fig. 12C, D ). There is no ejaculatory duct, nor penis papilla. The penis bulb is very thick and consists of numerous muscle fibers which are continuous with those underlying the epithelium of the male atrium. The prostatic vesicle is lined with a columnar, ciliated epithelium underlain by a dense layer (20 μm thick) of interwoven circular and longitudinal muscle fibers. The epithelium of the diverticula and that of the anterior section of extrabulbar section of prostatic vesicle are pierced by numerous gland cells producing strong erythrophil (pinkish) granules. The epithelium of the posterior section of the extrabulbar portion is pierced by gland cells producing abundant erythrophil granules. Two types of gland cells pierce the epithelium of the intrabulbar portion of the vesicle; one type is very abundant, and produces cyanophil granules, while the second type produces erythrophil granules. Figure 10. Choeradoplana eudoxiae Silva & Carbayo, sp. nov. Holotype A living animal in dorsal view B, C detail of anterior extremity in a dorsal view D detail of anterior extremity in ventro-lateral view in the preserved specimen. Abbreviations: cm common muscle coat, co common glandular ovovitelline duct, dd decussate dorsal cutaneous muscles, dm diagonal decussate muscles, e eye, ej ejaculatory duct, ep esophagus, er erythrophil secretion, fa female atrium, fd female genital duct, g gonopore, i intestine, lc longitudinal cutaneous muscles, ma male genital atrium, mk Muskelgeflecht (Graff, 1899), mo mouth, o ovary, ov ovovitelline duct, ph pharyngeal pouch, pp penis papilla, pv prostatic vesicle, px pharynx, rg rhabditogen glands, r retractor muscle, sb subintestinal transverse muscles, sd sperm duct, sg shell glands, sk sunken longitudinal cutaneous muscles, sm spermatophore, sn subneural transverse muscles, sp supra-intestinal transverse muscles, t testis, vi vitellaria, vn ventral nerve plate. The male atrium is elongated. The proximal half is horizontal, slightly folded and narrow. The distal half is wider, and provided with two large transverse and oblique folds. The atrium is lined with a 7 μm high epithelium, and pierced by two types of gland cells producing erythrophil and cyanophil granules, respectively. The atrial epithelium is underlain by a 20 to 35 μm-thick circular muscle with longitudinal fibers intermingled (Figs 12D , 13A ). Figure 11. Choeradoplana eudoxiae Silva & Carbayo, sp. nov. Photomicrographs of transverse sections of the holotype A dorsal portion of the pre-pharyngeal region ofreion B ventral portion of the pre-pharyngeal region C anterior region at 0.1 mm from the anterior tip of the body. of the pre-pharyngeal region D anterior region at 0.5 mm from the anterior tip of the body. Abbreviations: cm common muscle coat, co common glandular ovovitelline duct, dd decussate dorsal cutaneous muscles, dm diagonal decussate muscles, e eye, ej ejaculatory duct, ep esophagus, er erythrophil secretion, fa female atrium, fd female genital duct, g gonopore, i intestine, lc longitudinal cutaneous muscles, ma male genital atrium, mk Muskelgeflecht (Graff, 1899), mo mouth, o ovary, ov ovovitelline duct, ph pharyngeal pouch, pp penis papilla, pv prostatic vesicle, px pharynx, rg rhabditogen glands, r retractor muscle, sb subintestinal transverse muscles, sd sperm duct, sg shell glands, sk sunken longitudinal cutaneous muscles, sm spermatophore, sn subneural transverse muscles, sp supra-intestinal transverse muscles, t testis, vi vitellaria, vn ventral nerve plate. The ovaries are mature, rounded, ~ 100 µm in diameter. They are located 8.9 mm from the anterior extremity of the body (24.7 % of body length), and above the ventral nerve plate. The ovovitelline ducts emerge from the dorso-lateral aspect of the ovaries and run above the nerve plate; their proximal section contains sperm. Laterally to the female atrium, they rise posteriorly to unite dorsally to the common glandular ovovitelline duct (Fig. 13A, B ). This common duct is located behind the female atrium, and runs downwards to open into a canalicular projection of the posterior section of the female atrium. Figure 12. Choeradoplana eudoxiae Silva & Carbayo, sp. nov. Photomicrographs of sagittal sections of holotype A, B pharynx C prostatic vesicle and male atrium D copulatory apparatus. Abbreviations: cm common muscle coat, co common glandular ovovitelline duct, dd decussate dorsal cutaneous muscles, dm diagonal decussate muscles, e eye, ej ejaculatory duct, ep esophagus, er erythrophil secretion, fa female atrium, fd female genital duct, g gonopore, i intestine, lc longitudinal cutaneous muscles, ma male genital atrium, mk Muskelgeflecht (Graff, 1899), mo mouth, o ovary, ov ovovitelline duct, ph pharyngeal pouch, pp penis papilla, pv prostatic vesicle, px pharynx, rg rhabditogen glands, r retractor muscle, sb subintestinal transverse muscles, sd sperm duct, sg shell glands, sk sunken longitudinal cutaneous muscles, sm spermatophore, sn subneural transverse muscles, sp supra-intestinal transverse muscles, t testis, vi vitellaria, vn ventral nerve plate. Figure 13. Choeradoplana eudoxiae Silva & Carbayo, sp. nov. Holotype A diagrammatic reconstruction of copulatory apparatus B photomicrograph of a sagittal section of the female atrium. Abbreviations: cm common muscle coat, co common glandular ovovitelline duct, dd decussate dorsal cutaneous muscles, dm diagonal decussate muscles, e eye, ej ejaculatory duct, ep esophagus, er erythrophil secretion, fa female atrium, fd female genital duct, g gonopore, i intestine, lc longitudinal cutaneous muscles, ma male genital atrium, mk Muskelgeflecht (Graff, 1899), mo mouth, o ovary, ov ovovitelline duct, ph pharyngeal pouch, pp penis papilla, pv prostatic vesicle, px pharynx, rg rhabditogen glands, r retractor muscle, sb subintestinal transverse muscles, sd sperm duct, sg shell glands, sk sunken longitudinal cutaneous muscles, sm spermatophore, sn subneural transverse muscles, sp supra-intestinal transverse muscles, t testis, vi vitellaria, vn ventral nerve plate. The female atrium is irregular, provided with two or three lateral and dorsal folds (Fig. 13 ). This atrium is as long as half the male atrium, and is lined with an 8-10 μm high epithelium, the cells of which are erythrophil in their subapical portion. This epithelium is pierced by gland cells producing cyanophil granules, and is underlain by a 10 µm-thick muscle of circular and longitudinal muscle fibers. The common muscular coat is well developed and continuous with the penis bulb. This coat wraps the intrabulbar portion of the prostatic vesicle, and the male and female atria. The length:height ratio of the copulatory apparatus enveloped by the common muscle coat is 2.2:1. Behavioral note. When touched with a finger on the posterior end, the animal reacted by rolling forward. Firstly, it lifted its posterior extremity forward to touch the ground at the level of the midbody so that the body forms a loop. Next, the loop moved forwards until the anterior third of the body detached from the ground, which subsequently lengthened and touched the substrate. By doing so, the animal moved forward a distance equivalent to half its body length in approximately one second. It then immediately repeated this whole movement repeatedly. Remarks. The species described herein matches all diagnostic characteristics of Choeradoplana , except for the glandular cushions of the cephalic region, which are not developed. This is so notable that the species was initially assigned to Cephaloflexa upon examination of the live and preserved specimen. Regarding the body shape, Cephaloflexa is characterized by having "the anterior third very gradually narrowing, without constriction or widening and without grooves on the ventral surface. The anterior tip rolled upwards and is ventrally concave" ( Carbayo and Leal-Zanchet 2003 ). Ch. albonigra is similar in this aspect to the genus Cephaloflexa and to Ch. eudoxiae Silva & Carbayo, sp. nov. Unfortunately, it was not possible to sequence DNA from the small tissue available of Ch. eudoxiae Silva & Carbayo, sp. nov., and the homology test of the particular body shape of these two species remains an open question. The remaining diagnostic attributes of Choeradoplana are present in Ch. eudoxiae Silva & Carbayo, sp. nov., such as the ventral cutaneous longitudinal muscle partially sunken into the parenchyma; a retractor muscle of the cephalic extremity with a delta shape in transverse section; and a dorsal decussate parenchymatic muscle modified in the cephalic region into the Muskelgeflecht. The body color of the Ch. eudoxiae Silva & Carbayo, sp. nov. resembles that of some congeners in the brownish background color with dark black or dark brown spots over it, namely Choeradoplana abaiba Carbayo et al., 2018 , Ch. agua Carbayo et al., 2018 , Ch. banga Carbayo & Froehlich, 2012, Ch. benyai Lemos & Leal-Zanchet, 2014, Ch. bocaina Carbayo & Froehlich, 2012, Ch. cyanoatria Iturralde & Leal-Zanchet, 2019, Ch. longivesicula Iturralde & Leal-Zanchet, 2019, Ch. pucupucu Carbayo et al., 2018 , and the herein-described Ch. onae Lago-Barcia & Carbayo, sp. nov., Ch. riutortae Lago-Barcia & Carbayo, sp. nov. and Ch. claudioi Lago-Barcia & Carbayo, sp. nov. However, the general aspect of Ch. eudoxiae is lighter. Moreover, only Ch. eudoxiae Silva & Carbayo, sp. nov. and Ch. albonigra lack the cephalic glandular cushions among all species of the genus, with the latter having a different, black-striped dorsum. With respect to the internal morphology, Ch. eudoxiae Silva & Carbayo, sp. nov. is similar to Ch. albonigra , Ch. tristriata , Ch. bocaina , Ch. riutortae Lago-Barcia & Carbayo, sp. nov., Ch. claudioi Lago-Barcia & Carbayo, sp. nov., and Ch. onae Lago-Barcia & Carbayo, sp. nov. in that the prostatic vesicle is extrabulbar and it is lacking a penis papilla. However, in the latter four species an extrabulbar portion of the prostatic vesicle is dish-shaped, whereas this organ is pear-shaped (in Ch. eudoxiae Silva & Carbayo, sp. nov.) or has two short tubular branches (branches (with a pear-shaped organ) Ch. tristriata ( paired tubes ) and Ch. albonigra ). Ch. eudoxiae sp. nov. is distinguished from Ch. tristriata in the relatively compact copulatory apparatus of the latter, and from Ch. albonigra in that the copulatory apparatus in this species is relatively longer, the paired portions of the prostatic vesicle are shorter, and the common glandular ovovitelline duct is relatively shorter.