Free-living Microturbellarians (Platyhelminthes) from Wetlands in Southern Brazil, with the Description of Three New Species

© 2021 Academia Sinica, Taiwan Open Access Free-living Microturbellarians (Platyhelminthes) from Wetlands in Southern Brazil, with the Description of Three New Species Jhoe Reyes1,2 , Daniela Binow2, Rogério T. Vianna1,2,*, Francisco Brusa3,4 and Samantha E. Martins1,5 1Programa de Pós-graduação em Biologia de Ambientes Aquáticos Continentais (PPGBAC/FURG), Instituto de Ciências Biológicas, Universidade Federal do Rio Grande, RS, Brazil. *Correspondence: E-mail: [email protected] (Vianna). E-mail: [email protected] (Reyes) 2Universidade Federal do Rio Grande, Instituto de Ciências Biológicas, Laboratório de Biologia de Parasitos de Organismos Aquáticos, Av. Itália km 8, Rio Grande, RS 96203-900, Brazil. E-mail: [email protected] (Binow) 3División Zoología Invertebrados, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Boulevard 120 & 61, La Plata, Argentina. E-mail: [email protected] (Brusa) 4Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina 5Norwegian Institute for Water Research (NIVA), Ecotoxicology and Risk Assessment Section, Gaustadalléen 21, 0349, Oslo, Norway. E-mail: [email protected] (Martins) Received 6 June 2020 / Accepted 10 February 2021 / Published 27 May 2021 Communicated by Benny K.K. Chan The Taim strict nature reserve (ESEC Taim) is a protected wetland (Ramsar site) in southern Brazil. Although the ESEC Taim harbours a broad conspicuous flora and fauna, this area lacks studies on freeliving Platyhelminthes. Here, we describe three new species to science, one belonging to Macrostomorpha (Macrostomum itai n. sp.) and two belonging to Dalytyphloplanoida (Gieysztoria duopunctata n. sp. and G. hermes n. sp.). Macrostomum itai n. sp. differs from their congeners for its distinct stylet in “J”- shaped. Gieysztoria duopunctata n. sp. is distinguishable based on the crown-like penis stylet, this species possesses 12–18 spines of similar sizes and two smaller spines (one a bit longer than the other). Gieysztoria hermes n. sp. clearly differs from its congeners owing to its fibrous girdle, which bears two groups of complexes of spines and three different-in-length ventral spines. Moreover, we identified twentythree species, eleven belonging to Catenulida, three to Macrostomorpha, seven to Dalytyphloplanoida, one to Kalyptorhynchia, and one to Prorhynchida. We recorded three species for the first time in Brazil (Myostenostomum vanderlandi, Macrostomum quiritium and Strongylostoma elongatum). This new information on these free-living Platyhelminthes makes this protected area a more valuable and reinforces the actions that are being done to protect it. Key words: Coastal wetlands, Ramsar site, Taxonomy, Turbellaria, Neotropical region. BACKGROUND Freshwater microturbellarians are free-living Platyhelminthes that live in aquatic habitats such as streams, ponds, lagoons and wetlands, where they associate with the benthos or between roots or leaves of aquatic plants, or associate with filamentous algae in permanent or temporary waters (Noreña et al. 2015; Reyes and Brusa 2017). Most freshwater microturbellarians are known from the Holarctic region owing to the intensive research carried out in this region (Schockaert et al. 2008; Van Steenkiste et al. 2011a; Willems et al. 2007). However, there is still a lack of knowledge from distribution data of other Citation: Reyes J, Binow D, Vianna RT, Brusa F, Martins SE. 2021. Free-living microturbellarians (Platyhelminthes) from wetlands in southern Brazil, with the description of three new species. Zool Stud 60:22. doi:10.6620/ZS.2021.60-22. Zoological Studies 60:22 (2021) doi:10.6620/ZS.2021.60-22 1 © 2021 Academia Sinica, Taiwan biogeographical regions. In the Neotropical region, most studies on microturbellarians have been done in Argentina and Brazil. In Argentina, the knowledge of microturbellarians was increased and systematized by Noreña-Janssen (1995), Brusa et al. (2003 2008), Noreña et al. (2003 2004 2005abc 2006ab), Damborenea et al. (2005 2007 2011), Brusa (2006), and Adami et al. (2012). In Brazil, Marcus (1943 1944 1945ab 1946 1948 1949) studied the microturbellarian fauna from a wide variety of environments, and wrote one of the most important works on microturbellians in the area. In the Coastal Plain of Southern Brazil, freshwater microturbellarians have been studied for their diversity and composition (Braccini and Leal-Zanchet 2013; Vara and Leal-Zanchet 2013; Gamo and Leal-Zanchet 2004). Besides, Braccini et al. (2016) summarized all the records of the Brazilian microturbellarians, taking into account biogeographical and ecological aspects and suggesting actions to enhance the study of the group. More recently, six species of microturbellarians were recorded for the first time in wetlands in the Northeast region of Rio Grande do Sul. These findings increased the known diversity of freshwater microturbellarians to 109 species in Brazil (Braccini et al. 2017). The southern region of Brazil presents a vast number of freshwater wetlands, which are hot spots of biodiversity (Guadagnin 1999). The Taim nature reserve (ESEC Taim) is a protected mosaic of wetlands, and was recently designated as a Ramsar site (Ramsar 2017). Despite the high biodiversity found in the ESEC Taim, there are no reports of freshwater microturbellarians in this area. Knowledge on microturbellarians in this reserve would allow us to add value to the state of conservation of the natural reserve and reinforce protection actions. Thus, this study details the diversity of species of free-living microturbellarians occurring in this area, including the description of three new species of microturbellarians (Macrostomida and Dalytyphloplanida). We also comment on the morphology and distribution of the species reported in this protected area, some of which are recorded in Brazil for the first time. MATERIALS AND METHODS Research area The area of study is located in the ESEC Taim, located in the Coastal Plain of Rio Grande do Sul, southern Brazil (32°50'S, 52°26'W), near Brazil’s border with Uruguay between lake Mirim and the Atlantic Ocean (Bager et al. 2010) (Fig. 1). The ESEC Taim is Fig. 1. Maps showing the collection sites in the Taim nature reserve (ESEC Taim), Rio Grande do Sul, Brazil. page 2 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan comprised of a mosaic of ponds, channels and flooded areas (regionally called “banhados”) of different lengths and depths. Details on the sampled sites are given below: Lake Nicola (Site 1) (32°33'18.07"S; 52°31'18.50"W) Located within the wetland, it is one of the main ponds in the ESEC Taim. It is a permanent pond with a notorious seasonal variation on the water regime, including the marsh vegetation on its boundaries. This lake is mainly flooded by water from Lake Mangueira, which flows diffusely to lake Nicola through the swamp area. In addition, it receives water from different external channels, including agricultural water systems located in the peripheric area to the ESEC Taim. Water channel (Site 2) (32°35'50.52"S; 52°34'5.40"W) Site 2 presents a remarkable seasonal water fluctuation throughout the year, and it is connected to Lake Mangueira and Lake Nicola. This channel presents about 16 km in length, but this measurement may vary according to seasonal rainfalls and eutrophication processes. Lake Mangueira (Site 3) (32°50'11.42"S; 52°38'37.45"W) This lake is approximately 820 km2 and only its northern area is within the ESEC Taim. Lake Mangueira is the main hydric reservoir of the protected area, water from this lake flows through a channel parallel to the western limit of the wetland, connecting to Lake Nicola (Artioli et al. 2009). Sample collection and preparation Sampling campaigns were carried out bimonthly from June 2018 to July 2019. Sampling was conducted randomly in two different microhabitats, i.e., benthal and phytal environments. We delimit a square of 0.5 × 0.5 m to collect littoral floating vegetation on flooded areas (up to 50 cm in depth) in each sampling site. Floating vegetation was rinsed within the square area with a sieve (335 µm mesh size), washed with local water and returned to the environment. Samplings of floating vegetation were repeated three times in each site. The benthic sampling was also performed in shallow flooded areas using a core (10 cm diameter, 75 cm length). The core was introduced 10 cm into the sandy/muddy sediment, only the first 2 cm of the surface was collected. Samplings of the benthic environment were repeated five times at each site. All material collected by the sieve and core was then deposited into plastic jars (1.25 L) with water from the sampled environment. Jars were carried to the laboratory and maintained with air pumps in a temperate room (~20°C). Afterward, samples underwent Schockaert’s (1996) oxygen depletion method. When needed, we also examined the samples under the stereomicroscope as suggested by Brusa et al. (2003). Extracted animals were counted and examined in vivo by the squash method. We recorded the morphological traits of individuals through optical microscopy by making videos, photographs and sketches so we could identify the turbellarians at the species level. Whenever turbellarians present hard parts (sclerotic copulatory stylet), one or more specimens were whole-mounted using Hoyer’s medium according to Humason’s (1979) preparation. Other specimens were fixed in AFA (Humason 1979) or alcohol 96% for molecular analyses. Photographs of live and whole-mounted individuals were taken using a camera Olympus DP73/DP72 attached to a differential interference microscope Olympus CX4/ BX51. In addition, drawings were made with a camera lucida based on whole-mounted specimens. Measurements (generally made in micrometers “μm”) of soft and sclerotic structures were obtained using the software ImageJ (Schneider et al. 2012). The number of specimens or structures measured in different species is mentioned in each case as “n”. In the case of supernumerary structures in some species, “n” refers to that structure and not to the specimens, unless otherwise noted. Sizes were expressed as follows: average ± standard deviation (minimum value–maximum value; “n”). Vouchers, holotypes and paratypes studied herein were deposited at the Helminthological Collection of the Museu de Zoologia da Universidade de São Paulo (MZUSP), Brazil. Some animals were lost in the examination process, and in these cases it was not possible to deposit material. Sampling permits were granted by the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio), permit numbers 602912/2017 and 64551-1/2018. RESULTS Order Catenulida Graff, 1905 Family Catenulidae Graff, 1905 Genus Catenula Dugès, 1832 page 3 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan Catenula lemnae Dugès, 1832 (Fig. 2A) Synonyms: Anortha gracilis Leidy, 1851. Catenula gracilis Leuckart, 1854. Derostoma catenula Leydig, 1854. Stenostomum monocelis Schmidt, 1878. Stenostomum gracile Graff, 1882. Stenostomum lemnae Graff, 1882. Studied material: Live individuals studied, three of them observed in squashed preparations. The rest, fixed in alcohol (MZUSP PL 2257). Localities: Site 1 (23/06/2018; 28/02/2019) and Site 3 (24/06/2018; 18/02/2019; 05/06/2019), associated with benthal and phytal microhabitats. Description: Whitish mature individuals 2120 ± 1158.2 μm (1301.1–2939 μm; n = 2) long and 105.4 ± 48.5 μm (52.2–147.2 μm; n = 3) wide, with three to eight zooids. When it is measured only the first zooid, they are 275.4 ± 86.7 μm (195.3–367.4 μm; n = 3) long. They present the typical habitus of the species, i.e., elongated body, rounded anterior region of the body forming the prostomium, pre-oral swelling with ciliated furrows, with a statocyst located just at the ciliated furrow level (Fig. 2A). Taxonomic remarks: Morphology of individuals corresponds to descriptions given by Marcus (1945a b), Noreña-Janssen (1995) and Reyes et al. (2017), for the Neotropical region. Also, C. lemnae found here are following the descriptions, illustrations and pictures of material studied in the Palearctic and Nearctic region (Luther 1960; Gamo 1987; Noreña et al. 2007; Larsson and Willems 2010). Distribution: The species is widespread (cosmopolitan), it is found in Sweden (Larsson and Willems 2010), USA (Ruebush 1914), Ireland (Young 2001), United Kingdom (Young 2001), Spain (Gamo 1987; Noreña et al. 2007), Perú (Reyes et al. 2017), Brazil (Marcus 1945b) and Argentina (Noreña-Janssen 1995). Also, Siberia and Japan (see Luther (1960) and references therein). Family Stenostomidae Vejdovsky, 1880 Genus Myostenostomum Luther, 1960 Myostenostomum vanderlandi Rogozin, 1992 (Fig. 2B–D) Studied material: Four individuals studied, wholemounted individuals photographed alive, afterward a single individual fixed in alcohol (MZUSP PL 2258). Localities: Site 1 (19/11/2018; 2/05/2019), Site 2 (15/03/2019) and Site 3 (15/03/2019), associated with benthal and phytal microhabitats. Description: Specimens studied alive are vermiform, the body coloration varies from light orange to whitish. The anterior region is rounded. The body, 762.7 ± 218.7 μm (550.1–9871 μm; n = 3) long and 114 ± 18.3 μm (92.9–125.2 μm; n = 3) wide, consist of two to four zooids (Fig. 2B). The anterior region of the body bears two lateral ciliated pits, 48.1 ± 2.29 μm (46.4–50.7 μm; n = 3) long and 20.4 ± 4.1 μm (17.5–20.3 μm; n = 2) deep. The ciliated pits are connected to the anterior brain lobe which, in its anterior region, has 5–7 paired metameric ganglia while, in its posterior region, has two lateral trapezoidal lobes (Fig. 2C). The posterior brain lobe is somewhat triangular in shape with the apex directed to the anterior region. Besides, elongated ganglia are present at the base of both lateral angles of the posterior brain (Fig. 2D). Ventral and behind the posterior brain lobe, it is located the “V”-shaped mouth. This mouth leads to the pharynx, which is 60 ± 29.3 μm (41.7–93.8 μm; n = 3) long. At the anterior half of the intestine, is located the muscular gut, 76.9 ± 8.4 μm (71.5–86.6 μm; n = 3) long and 102.6 ± 28.9 μm (72.2–129.7 μm; n = 3) wide. The intestine continues tapering to an intestine-lacking region reaching the base of the caudal appendage, which is 53.9 ± 35.9 μm (28.5–79.3 μm; n = 2) long (Fig. 2B). Male and female reproductive systems not observed. Taxonomic remarks: Myostenostomum was established by Luther (1960), this genus is differentiated by the anterior muscular gut, and consequently, it erected two species M. bulbocaudatum and M. tauricum. Later, Rogozin (1992) reviewed the genus and recognized that the anterior and posterior cerebral ganglia can be used as a diagnostic character for species discrimination so, four species were allocated in Myostenostomum, including Myostenostomum sp. (sensu Van der Land (1970)) defined as M. vanderlandi. Individuals in this study are M. vanderlandi due to the similar morphological configuration of the cerebral ganglia, i.e., the anterior lobe has 5–7 paired metameric ganglia in the anterior region, while in its posterior region, has two lateral trapezoidal lobes which are in accordance with the descriptions of Van der Land (1970), Rogozin (1992) and Tokinova and Berdnik (2017). Moreover, the posterior triangular lobe with laterally elongated ganglia is also corroborated. However, on average, our specimens are smaller than those reported for the Palearctic region (Rogozin 1992; Tokinova and Berdnik 2017), except for the length of the muscular gut, which is longer. Since the type locality of M. vanderlandi is Suriname, we expand the range of distribution approximately 4242 km southwards in Rio Grande do Sul, Brazil, being the first record for the country. Distribution: This species was originally found page 4 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan Fig. 2. Live specimens of Catenulida. (A) Catenula lemnae, chain two zooids. (B–D) Myostenostomum vanderlandi: (B) habitus of whole-mounted individual, (C) detail of the anterior brain lobe and (D) detail of the posterior brain lobe. (E–F) Stenostomum glandulosum: (E) live individual, (F) free-hand schematic representation. (G) Stenostomum grande, a single zooid. (H) Stenostomum paraguayense, intestine with some food items. (I) Stenostomum hemisphericum, free-hand schematic representation. page 5 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan in Paramaribo, Suriname (South America) (Van der Land 1970). Recently, it was registered in Volga-Kama Reserve, Russia (Tokinova and Berdnik 2017). Genus Stenostomum Schmidt, 1848 Stenostomum cryptops Nuttycombe and Waters, 1935 Studied material: A single individual, afterward whole-mounted and studied alive. Localities: Site 2 (9/10/2018), associated with phytal microhabitat. Description: The body configuration is typical of the species, small and with two pairs of plate-like light-refracting bodies, the first pair located on the anterior brain lobe, while the second pair is located at the posterior brain lobe. Club-shaped pharyngeal glands distributed along the anterior half of the pharynx. Muscular pharynx. Several excretophores laterally distributed along the intestine. Taxonomic remarks: Our observations are in agreement with descriptions made by Marcus (1945b), and Nuttycombe and Waters (1938). However, Marcus (1945b) stated that specimens from São Paulo bear only a pair of light-refracting bodies associated with the anterior brain lobes. Nonetheless, our specimen presents two pairs of these structures which made them more similar to individuals studied by Nuttycombe and Waters (1938) in North America. This is the second record of S. cryptops for the country since it was found in São Paulo (Marcus 1945b). Distribution: The species is found in Georgia, USA (Nuttycombe and Waters 1938) and São Paulo, Brazil (Marcus 1945b). Stenostomum glandulosum Kepner and Carter, 1931 (Fig. 2E, F) Studied material: Five whole-mounted individuals studied alive. Localities: Site 2 (30/11/2018), associated with phytal microhabitat. Description: Specimens of 586.7 ± 71.3 μm (521.9–681 μm; n = 5) long, bear two zooids. The first zooid is 381.8 ± 49.4 μm (348.3–468.1 μm; n = 5) long. With large superficial lateral ciliated pits of 39.3 ± 6.4 μm (32.2–47.8 μm; n = 4) long. Anterior brain lobe divided into metameric masses with three pairs of platelike light-refracting bodies (Fig. 2E, F). Rounded mouth with several club-shaped pharyngeal glands on the margin. Pharynx 66.4 ± 13.7 μm (54.4–86.8 μm; n = 5) in length, tube-shaped, with several pharyngeal glands. Intestine with excretophores and is extended up to the caudal end. Longitudinal excretory channels extending from the anterior to the posterior region of the body (Fig. 2F). Taxonomic remarks: The specimens found here agree well with the descriptions made by Kepner and Carter (1931), and Nuttycombe and Waters (1938). However, our individuals are smaller than those described by Marcus (1945b) (first zooid: 600–800 μm; 16 zooids: 1200 μm), Kolasa and Young (1974b) (first zooid: 400 μm; three zoids: 1100 μm), but longer than those described by Kolasa and Young (1974a) (first zooid: 370 μm; two zooids: 900 μm). The most remarkable feature of this species are the light-refracting bodies. Here, the specimens from ESEC Taim have three pairs, while Marcus (1945b), Kolasa and Young (1974b), and Kolasa and Young (1974a) mentioned 2–3, 1 or none, and only 2 pairs respectively, suggesting a notable variation of this feature. Distribution: The species was recorded in North Europe (Lanfranchi and Papi 1978), USA (Kepner and Carter 1931; Nuttycombe and Waters 1938), Poland (Kolasa and Young 1974a), Suriname (Van der Land 1970), Brazil (Marcus 1945b), and Kenya (Kolasa and Young 1974b). Stenostomum grande Child, 1902 (Fig. 2G) Synonym: Stenostomum oesophagium Kepner & Carter, 1931. Studied material: Twenty-nine individuals studied alive; four whole-mounted individuals observed. The remaining, fixed in alcohol (MZUSP PL 2259). Localities: Site 1 (23/06/2018; 19/09/2018; 28/02/2019; 1/07/2019), Site 2 (9/10/2018; 30/11/2018; 15/03/2019; 29/05/2019) and Site 3 (24/04/2019; 5/06/2019), associated with benthal and phytal microhabitats. Description: Live specimens are whitish, with fusiform shaped. They have 1–3 zooids. The body length is 732.3 ± 340.9 μm (466.6–1226.8 μm; n = 4). Individuals follow the descriptions given by Kepner and Carter (1931), Nuttycombe and Waters (1938), Marcus (1945ab) and Noreña et al. (2005b) for S. grande. Taxonomic remarks: Recently, molecular analyses suggest that S. grande and S. leucops could be a species complex (Rosa et al. 2015). However, Rosa et al. (2015) did not change the status of S. grande owing to they find no morphological features to propose the taxonomic change. This species is a common inhabitant of lentic freshwater habitats (ponds and lagoons) in southern Brazil (Braccini et al. 2016; Braccini and Leal-Zanchet 2013; Vara and Leal-Zanchet 2013). page 6 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan Distribution: This species is widely distributed. It occurs in USA (von Graff 1911; Kepner and Carter 1931; Nuttycombe and Waters 1938), Russia (Nasonov 1923 and references therein), Finland (Nassonov 1924), Poland (Kolasa 1977), Suriname (Van der Land 1970), Brazil (Marcus 1945b), Peru (Noreña et al. 2006b) and Argentina (Noreña et al. 2005b). Stenostomum paraguayense (Martin, 1908) Luther, 1908 (Fig. 2H) Synonym: Weldonia parayguensis Martin, 1908. Stenostomum parayguensis (Martin, 1908). Studied material: Three individuals observed, two whole-mounted individuals studied. Localities: Site 2 (9/10/2018; 30/11/2018), associated with phytal microhabitat. Description: Animals are elongated, live specimens are whitish. Body with 2–4 zooids. The body length is 931.5 ± 144.1 μm (829.6–1033.4 μm; n = 2) long and 115 ± 24.9 μm (97.4–132.6 μm; n = 2) wide. This species is easily recognizable due to the three pairs of light-refracting bodies, the transverse cells line with refringent granules located at the first third of the intestine, and the two caudal finger-like extensions (the dorsal extension longer than the ventral) (Fig. 2H). Taxonomic remarks: Our individuals are longer than those described by Marcus (1945b) (first zooid: 350–800 μm), Noreña et al. (2005b) (first zooid: 800 μm) and Damborenea et al. (2011) (first zooid: ~680 μm). Moreover, individuals from the ESEC Taim have the dorsal caudal extension longer than the ventral, while Noreña et al. (2005b) indicate otherwise. The dorsal caudal extension could be absent or reduced as Marcus (1945b) noted. Also, the transverse cells line with refringent granules could be a variable feature since Damborenea et al. (2011) were not able to observe this feature in individuals from Peru. Distribution: Suriname (Van der Land 1970), Brazil (Marcus 1945b), Peru (Noreña et al. 2006b; Damborenea et al. 2011), Paraguay (Martin 1908) and Argentina (Noreña et al. 2005b). Stenostomum hemisphericum Nassonov, 1924 (Fig. 2I) Studied material: Three live whole-mounted individuals studied. Hand-drawings were made. Localities: Site 3 (24/04/2019), associated with benthal microhabitat. Description: Single live specimens are whitish, elongated. The anterior region of the body is somewhat pointed; while, the posterior, rounded. Brain lobes at the anterior region of the body, laterally paired (Fig. 2I). Two light-refracting bodies associated to each side of the posterior brain lobes. Mouth surrounded by pharynx glands. The pharynx is long, with lateral and oblique muscular fibers and glands associated along its extension. Excretophores along the intestine with no particular order (Fig. 2I). Taxonomic remarks: The most remarkable feature of this species is the long pharynx which bears oblique and lateral muscle fibers associated with the pharynx. Despite few sampled individuals, morphology observed corresponds well to the descriptions made by Nassonov (1924), Marcus (1945a), Damborenea et al. (2011) and Braccini et al. (2017). This species is recorded for the third time in Brazil. Distribution: Ukraine (Nassonov 1924), Suriname (Van der Land 1970), Brazil (Marcus 1945b; Braccini et al. 2017) and Peru (Damborenea et al. 2011). Stenostomum cf. pegephilum Nuttycombe and Waters, 1938 (Fig. 3A–C) Synonym: Anokkostenostomum pegephilum (Nuttycombe and Waters, 1938). Studied material: A single live whole-mounted individual studied. Photographs and videos of the live animal. Localities: Site 2 (9/10/2018), associated with phytal microhabitat Description: Live specimens have chains with two zooids, they are whitish. The body length is 557.6 μm long and 121.5 μm wide. Blunt anterior end with two lateral small ciliated pits (29.7 μm long). Anterior and posterior brain lobes located at the anterior region: the anterior brain lobe (ahead metameric; behind, lobes laterally paired) and the posterior brain lobe (laterally paired lobed) (Fig. 3A–B). Longitudinal mouth with associated glands (Fig. 3A–C). The muscular pharynx is 99.4 μm long, it has several small pharyngeal glands at its posterior half (Fig. 3C). Rounded posterior end with short cilia (Fig. 3B). Taxonomic remarks: General features are corroborated with descriptions given by Nuttycombe and Waters (1938), Marcus (1945b) and Noreña et al. (2005b). However, for two zooids, our specimen is smaller than those found by Nuttycombe and Waters (1938) (900 μm) and Marcus (1945b) (800 μm). Previous reports stated a transversal mouth (Nuttycombe and Waters 1938; Marcus 1945b), but we observed a longitudinal mouth with several surrounded glands. Nuttycombe and Waters (1938) states that the pharynx page 7 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan seems to form a posterior separated camera, while our specimen has two differentiated pharyngeal regions. Distribution: The species is found in Poland, Germany (Lanfranchi and Papi 1978), USA (Nuttycombe and Waters 1938), Italy (Noreña et al. 2005b and references therein), Suriname (Van der Land 1970) and Brazil (Marcus 1945b). Stenostomum saliens Kepner and Carter, 1931 (Fig. 3D) Synonyms: Stenostomum saltatum Kepner and Carter, 1931. Anokkostenostomum saliens (Kepner and Carter, 1931). Studied material: Two live whole-mounted individuals studied and photographed. Localities: Site 2 (9/10/2018), associated with phytal microhabitat. Description: Elongated whitish animals with 1–2 zooids, the anterior region of the body is pointed, while the posterior one is rounded (Fig. 3D1–2). The body is covered by cilia and has larger rigid cilia on the whole surface. The anterior region with brain lobes, which are connected to the lateral small ciliated pits. A V-shaped mouth is located in the first third of the body (Fig. 3D1). Intestine with excretophores located, mainly, at the anterior area of the gut (Fig. 3D2). Distal region of the intestine with lacking region. The body is 598.8 ± 363.7 μm (341.6–855.9 μm; n = 2) long and 79.2 ± 54.4 μm (40.7–117.6 μm; n = 2) wide. Taxonomic remarks: The features observed in individuals from the ESEC Taim correspond to the diagnostic features given by Kepner and Carter (1931), Nuttycombe and Waters (1938), Marcus (1945b), Kolasa (1973a) and Noreña et al. (2005b). In addition, our specimens are in the range of the length of individuals described by Marcus (1945b) (400–600 μm) and Kolasa (1973a) (600–800 μm), but are larger than those from USA (Nuttycombe and Waters 1938) (620 μm) and Argentina (when two zooids are measured) (Noreña et al. 2005b) (290 μm). Distribution: USA (Kepner and Carter 1931; Nuttycombe and Waters 1938), Poland (Kolasa 1973a), Brazil (Marcus 1945b), Suriname (Van der Land 1970), and Argentina (Noreña et al. 2005b). Stenostomum simplex Kepner and Carter, 1931 (Fig. 3E) Studied material: Two whole-mounted individuals photographed alive. Localities: Site 2 (9/10/2018), associated with benthal microhabitat. Description: Small whitish animals have a fusiform shape. The anterior region has two small ciliated pits connected to the brain lobes. Two lateral light-refracting bodies associated with the posterior brain lobe. The mouth is located in the first third of the body. The intestine has excretophores, which are located laterally along the entire gut. Taxonomic remarks: Identification is confirmed based on the habitus of live specimens and general morphology, both corresponding to the description and illustration given by Kepner and Carter (1931), Nuttycombe and Waters (1938) and Marcus (1945b). Distribution: USA (Kepner and Carter 1931; Nuttycombe and Waters 1938) and Brazil (Marcus 1945b). Stenostomum tuberculosum Nuttycombe and Waters, 1938 (Fig. 3F–G) Synonyms: Anokkostenostomum tuberculosum (Nuttycombe and Waters, 1938). Studied material: Two whole-mounted individuals studied and photographed alive. Localities: Site 2 (30/11/2018; 15/03/2019), associated with phytal microhabitat. Description: Live specimens whitish with three zooids. The body length is ± 438 μm long. The anterior end of the body has a small projection (tubercle) (Fig. 3F–G). The anterior brain lobe is metameric, while the posterior brain lobes are round-like. The anterior brain lobes are connected to the lateral ciliated pits. Oval mouth, rounded pharynx with two long lateral pharyngeal glands (?) (Fig. 3G). The intestine wall is lobulated. Taxonomic remarks: This species is easily recognizable owing to the small anterior tubercle located in the frontal region of the cephalic end. Our individuals are shorter than those studied by Nuttycombe and Waters (1938) (four zooids: 800 μm), Marcus (1945b) (four zooids: 1000 μm) and Noreña et al. (2005b) (two zooids: 720 μm), but it is almost in the range of individuals found by Luther (1960) (two zooids: 480–500 μm). Distribution: Finland (Luther 1960), Poland (Kolasa 1973a), Germany (Lanfranchi and Papi 1978), USA (Nuttycombe and Waters 1938), Suriname (Van der Land 1970), Brazil (Marcus 1945b), and Argentina (Noreña et al. 2005b). Order Macrostomorpha Doe, 1986 Family Macrostomidae Beneden E, 1870 Genus Macrostomum Schmidt, 1848 page 8 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan Fig. 3. Live specimens of Stenostomidae and Macrostomidae. (A–C) Stenostomum cf. pegephilum: (A) chain with two zooids, (B) schematic representation, (C) detail of the muscular pharynx. (D1–2) Stenostomum saliens: (D1) detail of the anterior region of the body and (D2) dorsal view of the habitus of a specimen. (E) Stenostomum simplex (scale bar not available). (F–G) Stenostomum tuberculosum: (F) individual with three zooids, (G) schematic representation. (H–I) Macrostomum quiritium: (H) dorsal view of a mature specimen, (I) detail of the penis stylet. (J) Macrostomum tuba, detail of the penis stylet. page 9 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan Description: Body of live individuals are 5.4 ± 2.3 mm (1.6–8.7 mm; n = 14) long and 1.5 ± 0.7 mm (0.4–2.9 mm; n = 13) wide, flattened and leaf-shaped, yellow-whitish. Round eggs of ± 0.24 mm in diameter (Fig. 6A). Taxonomic remarks: The general appearance and anatomy of our specimens are in accordance with descriptions made for the Neotropical region (Beauchamp 1939; Marcus 1943 1946; Ponce de León 1985; Noreña-Janssen and Faubel 1992; Gamo and Leal-Zanchet 2004; Noreña et al. 2006b) and other biogeographical regions (Fuhrmann 1894; Stout 1953; Luther 1963; Young 2001). However, the body size of our specimens is smaller than those described for the Neotropical region by Beauchamp (1939) (9 mm), Marcus (1946) (15 mm), Noreña-Janssen (1995) (8–10 mm) and Gamo and Leal-Zanchet (2004) (3, 4 and 6 mm); but larger than those found in Peru by Noreña et al. (2006b) (1–1.5 mm). Distribution: Widely distributed in freshwater ponds around the world (see Tyler et al. 2016 for a complete overview). Genus Bothromesostoma Braun, 1885 Bothromesostoma evelinae Marcus, 1946 (Fig. 6B) Studied material: A single individual studied alive, fixed in alcohol (MZUSP PL 2261). Localities: Site 2 (29/05/2019), associated with phytal microhabitats. Description: Body is 3.5 mm long and 0.5 mm wide, cylindrical or fusiform. Pigmentless spots, one pair dorsolateral at the anterior end of the body, connected to the peripheral unpigmented edge, while the dorsal surface has a dark blackish pigmentation (Fig. 6B). The ventral surface has clearer pigmentation than the dorsal surface. Taxonomic remarks: Our findings correspond to descriptions given for specimens found in the Neotropical region (Marcus 1946; Noreña-Janssen 1995; Noreña et al. 2006b; Ponce de León 1986). Distribution: The species is distributed in Brazil (Marcus 1946), Uruguay (Ponce de León 1986), Argentina (Noreña-Janssen 1995) and Peru (Noreña et al. 2006b). Subfamily Typhloplaninae Luther, 1904 Genus Strongylostoma Ørsted, 1844 Strongylostoma elongatum Hofsten, 1907 (Fig. 6C–E) Studied material: Eight individuals studied alive in whole-mounted preparations, one of them fixed in AFA (MZUSP PL 2262). Localities: Site 1 (19/09/2018), Site 2 (15/03/2019) and Site 3 (18/02/2019), associated with benthal and phytal microhabitats Description: Body, 1.3 mm long and 0.2 mm wide, fusiform, orange brownish. One pair of eyespots at the anterior region of the body, with rod tracks between them (Fig. 6C, E). Rosulatus pharynx situated in the first half of the body, anterior to the common genital pore. The male reproductive system has the ejaculatory duct with distal spines (Fig. 6D–E). Taxonomic remarks: A notorious feature that differentiates S. elongatum from other species of the genus Strongylostoma is the ejaculatory duct with distal spines. Hence, species with spines along all the ejaculatory duct (S. cirratum Beklemischew, 1922, S. dicorymbum Marcus, 1946 and S. radiatum Müller, 1774) and lacking them (S. simplex Meixner, 1915 and S. devleeschouweri Van Steenkiste et al. 2011b) are easily discriminated. Also, our specimens, when compared with S. coecum (Sekera 1906) Sekera 1912 (do not present eyes) and S. gonocephalum (see Graff 1913) (with oval spots beside and behind of the eyes) are easily differentiated. In conclusion, owing to the notable features observed, our specimens are S. elongatum. This is the first record for Brazil and the Neotropical region. Distribution: Widespread throughout the Palearctic and Nearctic region (see Van Steenkiste et al. 2011b). Family Dalyelliidae Graff, 1905 Genus Gieysztoria Ruebush and Hayes, 1939 Gieysztoria evelinae (Marcus, 1946) Luther, 1955 (Fig. 7A–C) Synonym: Microdalyellia evelinae Marcus, 1946. Studied material: A single individual studied alive, afterwards whole mounted in Hoyer’s medium (MZUSP PL 2236). Localities: Sites 3 (18/02/2019), associated with phytal microhabitat. Description: The body is 0.9 mm long and 0.2 mm wide; anterior end bluntly rounded, caudal end pointed, tail-like shape; brownish-orange pigmentation concentrated especially behind the pharynx. Two black bean-like eyes located at the anterior region of the body. The pharynx is 137.2 μm long and 111.2 μm wide. Two dorsal feathery vitellaria (357.9 μm long) with fingerlike prolongations (Fig. 7A). The male reproductive system comprised by two rear lateral testes, seminal vesicle (60.7 μm long, 95.4 μm wide), prostatic vesicle page 16 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan (60.4 μm long, 88.3 μm wide) and a penis stylet (Fig. 7B). The penis stylet is 72.9 µm in total length, with a dorsal opened girdle and distal spines. The girdle is composed by a proximal ring (24.77 μm in height and 47.61 μm in width) united by bridges (5.4 μm in height) to a distal ring (Fig. 7C1). The distal ring bears spines that are composed of two different groups. The first group is composed of four hollow well-developed spines (41.7 μm, n = 4, length) with a broad triangular slightly outward-curved base (18.4 μm, n = 1, width) (Fig. 7C1, C4). The second group bears five to seven thin fine spines (39.7 μm, n = 7, length) with its distal tip slightly curved outward, located interspersed with the first group of spines and are connected by its base to the distal ring (Fig. 7C1–2). The female reproductive system is composed of a saccular ovary (113.7 µm long), a seminal receptacle, uterus and a copulatory bursa. Male and female reproductive systems discharge into the common genital pore which is located at the posterior 1/3 of the body. Taxonomic remarks: The above-mentioned features follow descriptions given by Marcus (1946) and Brusa et al. (2008). The body length (1700 µm) of the original description (Marcus 1946) is bigger than Fig. 7. Live specimens of Gieysztoria. (A–C) Gieysztoria evelinae: (A) lateral view of a free-swimming individual, (B) detail of the posterior body region with details of the copulatory organs and (C1–4) detail of the penis stylet in different focal planes. (D–F) Gieysztoria hymanae: (D) dorsal view of a mature individual, (E) posterior body region with details of the copulatory organs and (F1–4) detail of the penis stylet in different focal planes. page 17 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan specimens from the ESEC Taim. Our specimens are bigger than those found in Argentina (483–583 µm) (Brusa et al. 2008). However, the total length of the stylet of our findings (72.9 µm) is smaller than those from Argentina (90–100 µm) (Brusa et al. 2008) and São Paulo (90–100 µm) (Marcus 1946). The same trait is observed when comparing the length of distal spines, which are 50-60 μm and 60 μm for Argentinian and Brazilian specimens, respectively (Brusa et al. 2008; Marcus 1946). Although stylet dimensions are smaller than those previously described, the stylet configuration remains the same. Distribution: The species was previously recorded from Brazil (São Paulo, Corôa lagoon) (Marcus 1946) and Argentina (Buenos Aires province, littoral of the Río de la Plata: Atalaya and La Matilde stream) (Brusa et al. 2008). Gieysztoria falx Brusa, Damborenea and Noreña, 2003 Comments: descriptions and remarks were made in Reyes et al. (2019). Gieysztoria hymanae (Marcus, 1946) Luther, 1955 (Fig. 7D–F) Synonyms: Microdalyellia (Gieysztoria) hymanae Marcus, 1946. Studied material: Seven individuals observed, four whole-mounted individuals studied alive. Afterwards, all individuals were whole mounted in Hoyer’s medium (MZUSP PL 2237–2243). Locality: Sites 3 (18/02/2019), associated with phytal microhabitat. Description: Body 912.5 ± 92.9 μm (798.3– 1025.6 μm; n = 4) long (Fig. 7D–E). Penis stylet, 49.9 ± 8.1 μm (43.9–66.8 μm; n = 7) long, with a dorsal opened girdle and distal spines. The girdle is composed by a proximal ring, 40.7 ± 7.7 μm (30.1–50.4 μm; n = 5) width, united by bridges to a distal ring (Fig. 7F1–4). The distal ring bears spines that are composed of two different groups. The first group is composed of four hollow grouped spines, 42.7 ± 5 μm (36.8–48.7 μm; n = 5) long, with a broad triangular base. The second group bears four to six thin fine spines, 38.7 ± 3.2 μm (34.1–42.6 μm; n = 7) long, with its distal tip slightly curved inward, located interspersed with the first group of spines and are connected by its base to the distal ring (Fig. 7F1, F3). Taxonomic remarks: The habitus and anatomical configuration of specimens found in the ESEC Taim are in accordance with original descriptions given by Marcus (1946), as well as those given by Brusa et al. (2008). Nevertheless, our individuals are larger than those found previously in Brazil (600–800 μm) (Marcus 1946) and Argentina (250–290 μm) (Brusa et al. 2008). However, when comparing the length of the penis stylet, our specimens are smaller than specimens of the original description from Brazil and the specimens from Argentina, both with 60 μm in length (Marcus 1946; Brusa et al. 2008). The most resembling species of G. hymanae is G. evelinae, but G. hymanae differs from G. evelinae due to the latter has four robust hollow spines while G. hymanae has four hollow spines with no robustness. Another feature not observed in wholemounted individuals was the proximal girdle, this structure may disappear when mounted in Hoyer’s medium, but it was observed in live animals. Distribution: The species was previously recorded only in Brazil (São Paulo, river Pirajussará) (Marcus 1946) and Argentina (Buenos Aires province, littoral of the Río de la Plata: Atalaya stream) (Brusa et al. 2008). Gieysztoria duopunctata n. sp. Reyes and Brusa (Figs. 8–10) urn:lsid:zoobank.org:act:F2160DDB-6595-4E08-B8EB023021FF140F Studied material: Holotype: one mounted specimen (MZUSP PL 2210). Paratypes: fifteen mounted specimens (MZUSP PL 2197–2209, 2211, 2212). Type locality: Site 3 (32°50'11.42"S; 52°38'37.45"W) (18/02/2019; 5/06/2019), associated with phytal microhabitats at the ESEC Taim. Another locality: Site 2 (15/03/2019), associated with phytal microhabitats Etymology: The specific name is originating from Latin, duo (two) and punctata (tip, point) The name refers to the dorsal spines located at both sides of the edges of the girdle, which are small, and different in length, one smaller than the other. Diagnosis: Crown-shaped stylet ± 43.1 μm long, comprises a fibrous-like girdle with a distal reinforced ring from which 12–18 spines of different lengths and widths arises. Spines gradually get longer along the rim of the girdle until the opposite region of the girdle opening. In this area, spines are the longest and the widest. Description: Body 791.2 ± 101.8 μm (650.9–945.3 μm; n = 11) long and 157.4 ± 36.75 μm (120–257.6 μm; n = 11) wide, rounded form (flattened whole-mounted) to elliptical; anterior region with a rounded end and caudal region tapering caudally abruptly, composed by a tail-like with 5–6 finger-like adhesive papillae (Fig. 10A–B). Body orange browndarkish pigmentation (live adults), it can be seen page 18 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan spotted along the body or dispersed, but some spots are especially concentrated behind the pharynx (Fig. 8A–C). Also, brown narrow longitudinal stripes along the body are seen in the biggest animals (Fig. 8D). Ciliated epidermal cells are polygonal in shape; rigid cilia not observed. Rhabdites are arranged in groups of 3–6 over the body surface. The brain is comprised of two globular ganglia from which lateral nerves extend towards the posterior region of the body, situated ventrally to the eyes. Black bean-shaped eyes located dorsally at the anterior region of the body; they are separated from each other by 44.5 ± 9.7 μm (31.2– 58.4 μm; n = 11) (Figs. 8A–C, 10A). Mouth ventral, subterminal. Doliiformis pharynx (see Luther 1955), 140.7 ± 19.3 μm (108.2–170.6 μm; n = 11) long and 103.7 ± 9 μm (85.5–117.1 μm; n = 11) wide (Figs. 8A–C, 10A). Male reproductive system posterior in the half body region and it is composed of testes, deferent ducts, seminal vesicle, prostatic vesicle, and penis stylet. Paired testes, 183 ± 50.4 μm (127.5–280.5 μm; n = 9) long and 28.3 ± 7.6 μm (21.6–40.4 μm; n = 7) wide, located at the posterior 1/3 of the body, at both sides of the body (Figs. 8A–B, 10A). Deferent ducts pair, thin and short, from the lateral-rostral region of the testes until connecting one in each side of the seminal vesicle (Figs. 8E, 10A–B). The globular seminal vesicle is 40.3 ± 5.5 μm (34.7–47.8 μm; n = 4) long and 47.5 ± 5.2 μm (41.3–53.3 μm; n = 4) wide. Prostatic vesicle is 31.8 ± 1.9 μm (30–34.14 μm; n = 4) long and 49.6 ± 6.4 μm (43.2–58.2 μm; n = 4) wide, globular with thickwalls, (Figs. 8A, E, 10A–B); saccular glands present, associated to the proximal region. Also, the prostatic vesicle is more voluminous than the seminal vesicle in mature animals (Figs. 8E, 10B). Distally, the prostatic vesicle is continued by a sclerotic penis stylet. The crown-shaped stylet, 43.1 ± 3 μm (37.6–47.7 μm; n = 14) long, comprises a well-defined fibrous-like girdle that has a distal reinforced ring from which 12–18 spines of different lengths and widths arises (Fig. 9B). The dorsally-opened girdle is 14.3 ± 2.2 μm (11–18.71 μm; n = 14) in height and 27.7 ± 3 μm (20.8–331.8 μm; n = 16) in width. Also, the girdle is 56.8 ± 5.3 μm (54–64.67 μm; n = 4) in circumference. In addition, the girdle is higher where the longest spines arise and gradually reduces in height as the spines shorten (Fig. 10C–D). Spines are fang-shaped with a long and broad funnel-shaped base, from caudal-transverse view, spines take the form from rectangular to quadrangular shape (Fig. 9A). The two smaller spines measure: 13.9 ± 3 μm (8.6–19 μm; n = 15) long and 4.3 ± 1.3 μm (2.3–6.3 μm; n = 12) wide; 19 ± 3.8 μm (13.3–23.4 μm; n = 8) long and 5.5 ± 2.7 μm (3.2–9.8 μm; n = 8) wide. Spines gradually get longer along the rim of the girdle until the opposite region of the girdle opening. In this area, spines are the longest (27.3 ± 4 μm (15.3–31.9 μm; n = 15)) and the widest (5 ± 1.4 μm (3.7–9.1 μm; n = 13)) (Figs. 9A–B, 10C–D). The width of the spines was measured from its base. Ovary single, saccular, in the left posterior 1/3 region of the body, posterior to the intestine (Figs. 8A, E, 10A–B), followed by a narrow oviduct associated to the seminal receptacle. The seminal receptacle connects to the uterus by the female duct. The uterus may contain one egg. Egg 166 ± 5.2 μm (162.2–171.9 μm; n = 3) long and 112 ± 5.5 μm (108.3–118.3 μm; n = 3) wide, oval, brown-yellowish (Fig. 10B). Copulatory bursa has thick walls, united to the uterus by distal region, both discharge on the common genital atrium which leads to the gonopore (Figs. 8E, 10A–B). Gonopore approximately on the 1/3 of the body length. Vitellaria 331.4 ± 75.3 μm (214.5–408 μm; n = 6) long, paired (smooth), dark greenish; filling in dorsoventrally the region comprising from the posterior end of the pharynx toward the ovary, where it forms a “V”-shaped common duct (Figs. 8B, 10A–B). Taxonomic remarks: Within Gieysztoria, species are grouped as Aequales and Inaequales following Luther’s (1955) criteria. The first group has spines with similar shapes and sizes, while the latter, has spines with different sizes and shapes. Further, Inaequales is subdivided into Falcatae (with a large, robust falcate stylet spine, and a variable number of plate-like spines), Fenestratae (girdle with one or more openings in the girdle), Radiatae (stylet with symmetrical morphology) and Aberrantes (stylet with an aberrant configuration) (Luther 1955; Van Steenkiste et al. 2012). Gieysztoria duopunctata n. sp. belongs to the Inaequales, subgroup Radiatae. Seven species resemble G. duopunctata n. sp., namely: G. bergi (Beklemischev, 1927), G.coronae Noreña-Janssen, 1995, G. foreli (Hofsten, 1911), G. pseudodiadema Noreña-Janssen, 1995, G. pulchra Wang and Deng, 2006, G. reggae Therriault and Kolasa, 1999 and G. tigrensis Noreña-Janssen, 1995. The stylet lengths of G. coronae, G. pseudodiadema and G. tigrensis were taken from figures in the respective descriptions, while the rest of the stylet lengths were obtained from the measurement mentioned by the respective author of each species. It is possible to differentiate G. duopunctata n. sp. from other Radiatae species due to several features. Gieysztoria foreli and G. reggae present in the girdle a distal ring joined with several bridges and spines of similar shape or size, 15– 22 and 8–12, respectively (Luther 1955; Therriault and Kolasa 1999; Lu et al. 2013). Gieysztoria tigrensis has a bigger stylet length (363.6 μm); this species has spines with remarkable differences in length and configuration (Noreña-Janssen 1995). Contrary, G. duopunctata n. page 19 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan sp. has a small stylet (43.1 μm long) with 12–18 fanglike spines of variable lengths. The stylet length of G. pulchra (48 μm) is bigger than those of G. duopunctata n. sp. (43.1 μm). Also, G. pulchra has two long lateral spines on the edges of the girdle, while G. duopunctata n. sp. has two small (but different in size) spines on Fig. 8. Live specimens of Gieysztoria duopunctata Reyes and Brusa n. sp. (A) Internal organisation from a live animal based on a whole-mounted preparation, dorsal view. (B) Habitus from live animal, dorsal view. (C) Detail of the anterior region of the body. (D) Detail of the epidermis surface, ventral view (anterior region of the body oriented at the left side). (E) Posterior body region with details of the copulatory organs. page 20 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan the edges of the girdle. The total number of spines of G. duopunctata n. sp. are 12–18, notably differentiated from G. pulchra which has 13 spines (Wang and Deng 2006). The stylet length of G. duopunctata n. sp. (43.1 μm) is similar to G. pseudodiadema (43.8 μm) (Noreña-Janssen 1995). However, G. pseudodiadema Fig. 9. Gieysztoria duopunctata Reyes and Brusa n. sp. Penis stylet in different focal planes. (A1–6) caudal view and (B1–6) lateral view (same scale bar for all photographs in A and B, respectively). page 21 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan has seven empty spines, spines at the left side are welldeveloped, while those at the right side are reduced (Noreña-Janssen 1995). So, G. pseudodiadema differs from G. duopunctata n. sp. owing to the number of spines and stylet configuration. Gieysztoria coronae has a ~41.2 μm long stylet with 25–26 similar-in-length spines, these spines are hollow and triangular in shape when observed in cross section (Noreña-Janssen 1995). Contrary, G. duopunctata has a 43.1 μm long stylet with 12–18 fang-like different-in-length spines, spines are quadrangular in shape when observed in cross section. Also, two small spines (one a bit longer than the other) arise from the edges of the girdle of G. duopunctata n. sp. As for G. bergi, this species has a 107 μm long stylet, 24 spines similar in length (30 μm) and are quadrangular in shape (Beklemischev 1927; Luther 1955; Lu et al. 2013). These features are not seen in G. duopunctata which has a small stylet (43.1 μm long), Fig. 10. Gieysztoria duopunctata Reyes and Brusa n. sp. (A) Schematic representation of the habitus from a live animal based on a whole-mounted preparation, dorsal view. (B) Detail of the male and female copulatory systems, dorsal view. (C) Schematic representation of a squashed stylet. (D) Penis stylet (from the holotype). page 22 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan 12-18 fang-like different-in-length spines and two spines notoriously different in size at the dorsal edges of the stylet. Therefore, the above-mentioned differences, mainly on the penis stylet morphology, indicate that individuals found in the ESEC Taim represent a new species within Gieysztoria. Distribution: Southern Brazil, Rio Grande do Sul state, Taim strict nature reserve (ESEC Taim). Gieysztoria hermes n. sp. Reyes and Brusa (Figs. 11–12) urn:lsid:zoobank.org:act:F98ED003-EAE2-4DB7-A16AC73AE5C9B9B1 Studied material: Holotype: one mounted specimen (MZUSP PL 2213). Paratypes: five mounted specimens (MZUSP PL 2214–2218). Type locality: Site 1 (32°33'18.07"S; 52°31'18.50"W) (19/11/2018), associated with benthal microhabitats at the ESEC Taim. Another locality: Site 1 (19/11/2018), associated with benthal and phytal microhabitats at the ESEC Taim. Etymology: The name of this species is originated from the name of the Greek Olympian god Hermes. This deity is usually depicted using the petasos, a widebrimmed hat, but with a pair of small wings, which resembles the stylet morphology of this species when observed upside down. Diagnosis: Stylet with a dorsally-opened fibrous girdle. The girdle has five different groups of spines: two groups of rows of spines (a and b) arising from the dorsal edges of the girdle, and three ventral spines. Spines from group a are a dorsal protrusion of the girdle, which bears several rows (toothbrush-like) of spines. Proximal spines are longer and have broader hollow bases, these spines gradually get shorter and thinner as spines reach its distal end. Spines from group b are a dorsal protrusion, distally, has several rows with a great number of very thin and short spines. Within the three ventral spines, the central one is the largest and strongest. The remaining flanking spines are shorter, but different in length. Description: Body 798.6 ± 164.4 μm (509.5–923 μm; n = 5) long and 225.2 ± 55.2 μm (164.4–304.6 μm; n = 5) wide; habitus typical of the genus, rounded anterior end, widening towards the middle region of the body, elongated and narrowed at the end where it bears 5–6 finger-like adhesive papillae (Fig. 11C–D). Orangebrown pigment in vivo, randomly dispersed all over the body (Fig. 11A–C). Body surface with several rhabdites and ciliated epidermal cells, rigid cilia not distinguished. Eyes, one pair, black kidney-like, in the anterolateral region, separated by 55.8 ± 4.35 μm (50.5–61 μm; n = 4) (Fig. 11A, D). Brain under the eyes. Mouth subterminal, ventral at the anterior region of the body. Mouth followed by a dolliform pharynx, 162.8 ± 18.4 μm (137.7–182 μm; n = 4) long and 125.6 ± 12.6 μm (112.2–140.2 μm; n = 4) wide (Fig. 11A, C– D). Ellipsoidal testes are 215.7 ± 50.7 μm (144.7–317.2 μm; n = 10) long and 37.5 ± 12.6 μm (26.4–63.1 μm; n = 8) wide, paired laterally. Deferent duct thin, paired, and extend from the anterior region of the testes, connecting these to the seminal vesicle (Fig. 11B, E). Seminal vesicle, 33 ± 10.5 μm (22–42.6 μm; n = 4) long and 41.5 ± 17.7 μm (25.1–59.3 μm; n = 4) wide, globular-shaped; circular notch in the posterior central region (Fig. 11B, E), distally connected to a prostatic vesicle. Prostatic vesicle 30.4 ± 9.7 μm (21.9– 39 μm; n = 4) long and 36.3 ± 13.7 μm (22.8–50.5 μm; n = 4) wide, globular, with lateral external glands associated; distally is continued by a sclerotic penis stylet (Fig. 11B, E). Stylet is 34.2 ± 3.5 μm (29.1–37.6 μm; n = 6) in axial length; is composed, proximally, by a dorsally-opened fibrous girdle, 24.8 ± 7.3 μm (19.4–39 μm; n = 6) in width and 11.5 ± 2.1 μm (9.1–14.7 μm; n = 6) in height, may present an oval fenestra associated to the strongest distal spine. Distally, the girdle presents five different groups of spines: two groups of rows of spines (a and b) arising from the dorsal edges of the girdle and three spines (blue-, redand green-colored spines) (Fig. 12C) arising from the ventral side (Fig. 12A–C). Spines from the group a, comprise a dorsal protrusion of the girdle, 26.7 ± 2 μm (23.3–28.3 μm; n = 6) long, which bears several rows (toothbrush-like) of spines. Proximal spines are longer (10.8 ± 1.6 μm (8.6–13 μm; n = 6)) and have broader hollow bases, these spines gradually get shorter and thinner as spines reach its distal end (shortest spines are 3.7 ± 0.8 μm (2.4–4.4 μm; n = 6)) (Fig. 12C: a). Spines from group b, comprise a dorsal protrusion which, distally, has several rows with a great number of very thin and short spines. These spines gradually align and lengthen towards its proximal area where they form a comb-like structure (Fig. 12C: b). A fang-shaped spine (colored in red) is 20.7 ± 1.7 μm (18.5–23.3 μm; n = 6) long, is curved, with a rounded distal tip outward. This is the largest and strongest spine, at the ventral median side of the girdle. Proximally, this spine has a large base (6.8 ± 0.9 μm (5.2–7.9 μm; n = 6) wide) for muscular insertion (Fig. 12C: red). The other fang-shaped spine (colored in blue) is 16.8 ± 1.5 μm (15–19.1 μm; n = 6) long, is slightly curved outwards, and is located between red spine and spines from the group a. Proximally, it presents a broad base for muscular insertion. Distally, it has a blunt tip. Also, a blue spine is associated with spines from the group a (Fig. 12C: blue). Spine (colored page 23 of 33Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan in green) is 11.9 ± 1.2 μm (10.4–13.5 μm; n = 6) long, blade-like, between red spine and spines from group b. Besides, the green spine is very close to spines from group b (Fig. 12C: green). The ovary is ± 127.3 μm long, single sac-like, at the right side of the body, posteriorly to the intestine and continued by the oviduct (Fig. 11B, E). Copulatory bursa thick-walled, releasing the contents to the common genital atrium. Seminal receptacle with sperm. Gonopore at the posterior 1/3 of the body. Vitellaria are 303.7 ± 613 μm (210.7–371.8 μm; n = 8) long, paired, smooth dark-greenish, from the posterior side of the Fig. 11. Gieysztoria hermes Reyes and Brusa n. sp. (A) Detail of the anterior region of the body. (B) Posterior body region with details of the copulatory organs. (C) Habitus from live animal, dorsal view. (D) Schematic representation of the habitus from a live animal. (E) Schematic representation of the male and female copulatory systems. page 24 of 33 Zoological Studies 60:22 (2021) © 2021 Academia Sinica, Taiwan pharynx to the end of the intestine, where they join forming a common duct (Fig. 11C–D). 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