Marine flatworms (Platyhelminthes: Polycladida) found in empty barnacle shells including a new species, from southern Mexican Pacific Author Ramos-Sánchez, Mariela Posgrado en Ecología Marina, Universidad del Mar, Campus Puerto Ángel, Cd. Universitaria S / N, 70902, San Pedro Pochutla, Oaxaca, México. & Laboratorio de Sistemática de Invertebrados Marinos (LABSIM), Universidad del Mar, campus Puerto Ángel, Ciudad Universitaria, Apdo. Postal 47, Puerto Ángel, Oaxaca, 70902, México. Author Carrasco-Rodríguez, Dahani S. 0000-0002-5367-4397 Laboratorio de Sistemática de Invertebrados Marinos (LABSIM), Universidad del Mar, campus Puerto Ángel, Ciudad Universitaria, Apdo. Postal 47, Puerto Ángel, Oaxaca, 70902, México. & carrascoshanti @ gmail. com; https: // orcid. org / 0000 - 0002 - 5367 - 4397 carrascoshanti@gmail.com Author García-Madrigal, María Del Socorro 0000-0003-4279-9116 Laboratorio de Sistemática de Invertebrados Marinos (LABSIM), Universidad del Mar, campus Puerto Ángel, Ciudad Universitaria, Apdo. Postal 47, Puerto Ángel, Oaxaca, 70902, México. & ms _ garcia _ m @ hotmail. com; https: // orcid. org / 0000 - 0003 - 4279 - 9116 ms_garcia_m@hotmail.com Author Bastida-Zavala, J. Rolando 0000-0003-4802-1348 Laboratorio de Sistemática de Invertebrados Marinos (LABSIM), Universidad del Mar, campus Puerto Ángel, Ciudad Universitaria, Apdo. Postal 47, Puerto Ángel, Oaxaca, 70902, México. & rolando _ bastida @ yahoo. com. mx; https: // orcid. org / 0000 - 0003 - 4802 - 1348 rolando_bastida@yahoo.com.mx text Zootaxa 2021 2021-04-28 4965 2 301 320 journal article 7010 10.11646/zootaxa.4965.2.5 0eea33ca-1f95-4da6-9cee-239291922331 1175-5326 4750207 F957C6AA-5592-47E2-91F5-1AA1F33513F8 Stylochus mistus sp. nov. ( Figures 2A–F , 3A–G , 4 A–G , 5A–C ) Type locality: Tangolunda, Bahías de Huatulco , Oaxaca , México ( 15°46’15.8” N , 96°5’43.2” W ) . Type material. Twenty-eight specimens. Holotype : as sagittal sections: UMAR-PLAT 40, reproductive structures in seven slides; Tangolunda Bay , Oaxaca , southern Mexican Pacific ; in the empty shell of Paraconcavus pacificus , 20–25 m in SEAPHOX ® , March 07, 2017 , Coll. CCB , MAAS , HMMO , PGRP , and SVM ). Paratypes : Five specimens as whole mounts: UMAR-PLAT 41A-D, 5 specs. ( Tangolunda Bay , same as holotype ). One specimen in histology sections, UMAR-PLAT 41E, 1 spec. in the frontal section of reproductive structures in two slides ( Tangolunda Bay , same as holotype ). Other specimens reviewed: Twenty-one specimens preserved in 70% ethanol: UMAR-PLAT 41F, 13 specs. fixed in 96% ethanol (damaged specimens, Tangolunda Bay , same as holotype ); UMAR-PLAT 41G, 8 specs. fixed in formalin 10% (damaged specimens; Tangolunda Bay , same as holotype ) . Description of external features Color. The background color is beige with a scant orange to light brown pigmentation; black transverse spots which, when fusing together, give the appearance of lines, mainly in the anterior marginal region of the body; nuchal tentacles orange; body margin orange or pink ( Figs 2A–C ). When fixed the color is beige, with maculae, spots and small dots black or olive green ( Fig. 2D ); and when stained with Mayer´s carmalum it has a purple to pink color ( Figs 2E–F ). Form. Oblong shape or slightly pyriform (after to fixed) ( Figs 2A–F , 3A , 5A ), measuring between 2–9 mm long (n=26, µ= 4.77 mm , SD= 1.79) and 1– 5.2 mm (n=26, µ= 2.77 mm , SD= 1.07) wide. Tentacles. Circular to pyriform nuchal tentacles ( Figs 3C , 5A ), positioned between 0.8–1.2 mm from the anterior margin of the body. They measure 0.25–0.3 mm in length (n=4, µ= 0.27 mm , SD= 0.02) and 0.2 mm in width (n=4, µ= 0.2 mm , SD= 0.02) and with 0.5 mm intertentacular distance. Eyes. About 90 to 295 (n=4, µ= 156, SD= 94) marginal eyes ( Figs 3B , 5A ) distributed in the anterior marginal region of the body; between 15–49 (n=4, µ= 28, SD= 16) tentacular eyes ( Figs 3B–C , 5A ) mostly located within the nuchal tentacles and one or two pairs of these located in the marginal-posterior region of the tentacles; between 26–59 (n=4, µ= 35, SD= 13) cerebral eyes arranged in two lines ( Figs 3B–C , 5A ), located anterior to the brain and oriented towards the anterior region of the body; few frontal eyes ( Fig. 3B , 5A ) distributed towards the anterior region of the body, arranged slightly in a fan-like manner. Brain. Oval, measuring 0.3 mm in length and 0.2 mm width, with scarcely visible globuli cell masses, positioned in the anterior region of the brain ( Fig. 3D ). Digestive system. Folded pharynx ( Figs 3E , 5A ), 2–3.6 mm long (n=4, µ= 2.9 mm , SD= 0.76) and 1.2–2.2 mm (n=4, µ= 1.5 mm , SD= 0.42) wide, located at 2.2 mm from the anterior margin of the body; with 6–7 (n=4, µ= 6.8, SD= 0.4) pharyngeal lobes. The mouth is located at the end of the first third of the pharynx ( Fig. 3E ). Gonopores. Separated gonopores, positioned at the end of the last third of the body; the male gonopore is at a distance of 5.7–6 mm from the anterior margin of the body, at 1.4 mm from the pharynx and 0.05 mm from the female gonopore ( Figs 3F–G , 4A , 5A–C ). Description of reproductive features Male reproductive system. Male gonopore almost terminal; the rest of male reproductive system oriented towards anteriorly from male gonopore; it presents spermiducal ducts which are oriented towards the anterior region of the body and extend to the second third of the pharynx, to descend later to the posterior region of the body. Prostatic vesicle prominent, oval and polyglandular ( Figs 4 A-B, E, 5B–C), located dorsal to the seminal vesicle. Seminal vesicle elongated and prominent ( Figs 3E–F , 4A–B , 5C ), connected through the spermiducal ducts to the spermiducal vesicles ( Figs 3G , 5C ). Penis papilla short ( Fig. 4C ), 1 mm long, unarmed; narrow male atrium ( Figs 4–F ) with a glandular epithelium lining ( Figs 4D–F ) in the peripheral region. Female reproductive system. Oviducts ( Figs 4C , 5C ) run from the anterior region of the body, around the pharynx, and connect directly with the proximal end of the vagina. Scarcely developed, narrow and short vagina, oriented towards the anterior region of the body ( Figs 4C–D , 5C ). Cement glands ( Fig. 5C ) are scarce and are found surrounding the vagina; lacks Lang’s vesicle. Habitat. The polyclads were found inside the shells of Paraconcavus pacificus ( Fig. 2G ), which were part of the fouling of a marine sensor (SeapHOx®), at a depth of approximately 20– 25 m . The sensor was installed on December 6, 2016 , and extracted on March 7, 2017 ; the dominant fouling fauna was represented by barnacles, decapods and polychaetes. Distribution. Tropical Eastern Pacific. Central coast of Oaxaca : Tangolunda Bay, Bahías de Huatulco. FIGURE 2. Stylochus mistus sp. nov. A–F (paratype UMAR-PLAT 41). A–C. specimens in vivo ; D. specimen preserved in ethanol; E–F. specimen stained in Mayer´s carmalum and cleared in methyl salicylate; G. Paraconcavus pacificus . Scaling bar of the figure A, B, D–F= 1 mm, G= 1 cm. FIGURE 3. Stylochus mistus sp. nov. A–G ( paratype UMAR-PLAT 41). A–G. specimen stained in Mayer´s carmalum and cleared in methyl salicylate; B–G. details of A, in whole mount; B–C. anterior region of the body, D. brain; E. pharynx; F–G. male and female reproductive system. Structures: b= brain, ce= cerebral eyes, fe= frontal eyes, fg= female gonopore, m= mouth, me= marginal eyes, mg= male gonopore, nt= nuchal tentacle, ovi= oviducts p= pharynx, sv= seminal vesicle, spv= spermiducal vesicle, te= tentacular eyes. Scaling bar of the figure A= 1 mm. FIGURE 4. Stylochus mistus sp. nov. A. ( paratype UMAR-PLAT 41), B–G. ( holotype UMAR-PLAT 40). A. male and female reproductive system, in frontal histological sections; B–G. male and female reproductive system, in sagittal sections; E. prostatic vesicle of type polyglandular. Structures: fg= female gonopore, gp= glandular epithelium, ma= male atrium, mg= male gonopore, ovi= oviducts, pp= penis papilla, pv= prostatic vesicle, sv= seminal vesicle, u= uterus, va= vagina. FIGURE 5. Stylochus mistus sp. nov. A–B. ( paratype UMAR-PLAT 41), C. ( holotype UMAR-PLAT 40). A. diagrammatic representation of complete specimen; B. diagrammatic representation of the male and female reproductive systems, in whole mount; C. diagrammatic representation of the male and female reproductive systems, in sagittal section. Structures: ce= cerebral eyes, cg=cement gland, ed=ejaculatory duct, fe= frontal eyes, fg= female gonopore, gp= glandular epithelium, me= marginal eyes, mg= male gonopore, nt= nuchal tentacle, ovi= oviducts p= pharynx, pv= prostatic vesicle, sv= seminal vesicle, spv= spermiducal vesicle, te= tentacular eyes, ovi= oviducts, u= uterus, va= vagina. Etymology. The specific epithet, mistus , derives from the Zapotec subdialect word mistu’ , which means “cat”, in reference to the color and dorsal color pattern of the specimens ( Figs 2A–C ), a pattern similar to the wild cat Leopardus wiedii Schinz , distributed along the coast of Oaxaca . Taxonomic remarks: Du Bois-Reymond Marcus & Marcus (1968) established two subgenera within Stylochus : S . ( Stylochus ) and S. ( Imogine ), based on the shape of the seminal vesicle. The first subgenus has a simple thinwalled seminal vesicle, while S. ( Imogine ) has on anchor-shaped (tripartite) seminal vesicle. Jennings & Newman (1996) raised subgenus Imogine to the genus level. Based on this separation, the new species was placed within the genus Stylochus . In this way, the genus Stylochus is represented by 34 species ( Tyler et al. 2006 -2020), of which four were described from the Eastern Pacific: Stylochus atentaculatus Hyman, 1953 , from San Mateo Beach, California; S. californicus Hyman, 1953 , from California; S. franciscanus Hyman, 1953 , from San Francisco Bay, California and S. insolitus Hyman, 1953 , from San Pedro Breakwater, California. Prior to this study, the genus Stylochus had not been recorded in the Tropical Eastern Pacific. Seven of the species described in this genus are associated with barnacles: Stylochus alexandrinus Steinböck, 1937 , from Alexandria , Egypt ( Galleni 1976 ); S. ellipticus ( Girard, 1850 ) , from south of Massachusetts ( Hyman 1940 ); S. neapolitanus ( Delle Chiaje, 1841 -1844), from Sicilia (Barnes 2002); S. pilidium ( Goette, 1881 ) , from Sevastopol Bay, Black Sea ( Rzhepishevskii 1979 ); S. pygmaeus Merory & Newman, 2005 , from Victoria , Australia ( Merory & Newman 2005 ); S. tauricus Jacubowa, 1909 , from the Black Sea ( Murina et al. 1995 ), and S . zanzibaricus Laidlaw, 1903 , from Zanzibar Island , Africa ( Skerman 1960 ). The comparison of Stylochus mistus sp. nov. was made taking into consideration the external and internal characters of the four species from California as well as the seven species associated with the barnacles. Stylochus mistus sp. nov. has frontal eyes and marginal eyes distributed in the first third of the body; it differs from S. neapolitanus , S. pygmaeus and S . zanzibaricus in that those lack frontal eyes ( Laidlaw 1903 ; Merory & Newman 2005 ; Marquina et al. 2014 ; Noreña et al. 2015 ), and from S. atentaculatus , S. californicus , S. insolitus , and S. tauricus because the marginal eyes are distributed over the entire body margin ( Hyman 1953a ; Murina et al. 1995 ). Stylochus mistus sp. nov. has separate gonopores and an unarmed penis papilla, and differs from S. franciscanus , S. alexandrinus , species which have a common gonopore ( Hyman 1953a ; Galleni 1976 ); and from S. ellipticus ( Hyman 1939a ; Hyman 1940 ) because this species has a penis papilla with stylet. The species of the genus Stylochus can be differentiated based on the color and pattern of coloration, presence/ absence of frontal eyes, the distribution of marginal eyes, the morphology of the hermaphroditic reproductive system, the presence/absence of armament of the penis papilla and the shape of the vagina ( Table I ) ( Hyman 1939a ; 1940 ; 1953a ). Other taxonomic characters for the specific definition, suggested by the present study, are: 1) the internal arrangement of the prostatic vesicle, which may be of the polyglandular or monoglandular type (reviewed in Bulnes et al . 2005 ); 2) presence of a glandular lining of the male atrium, described previously only in S. ellipticus ( Hyman 1939a , 1940 ), and also present in Stylochus mistus sp. nov.