Three new species of Urocleidoides (Monogenoidea: Dactylogyridae) parasitizing characiforms (Actinopterygii: Characiformes) in Tocantins River, states of Tocantins and Maranhão, and new record for U. triangulus in Guandu River, state of Rio de Janeiro, Brazil

Three new species of Urocleidoides Mizelle & Price, 1964 are described from the gills of characiform fishes in the Tocantins River and its tributaries. Urocleidoides boulengerellae sp. nov. is described from Boulengerella cuvieri (Spix & Agassiz, 1829) and differs from all its congeners by the dorsal bar with a long posteromedial projection; male copulatory organ with 2–3 counterclockwise rings and a base with a flange; an accessory piece comprising a robust Y-shaped unit and a sheath-like unit; and a highly sclerotized vaginal canal. Urocleidoides paratriangulus sp. nov., described from Psectrogaster amazonica Eigenmann & Eigenmann, 1889, Cyphocharax gouldingi Vari, 1992, Caenotropus labyrinthicus (Kner, 1858) and Mylesinus paucisquamatus Jégu & Santos, 1988, is most similar to Urocleidoides triangulus (Suriano, 1981) Rossin & Timi, 2016 based on the shape of the anchors and bars but differs from U. triangulus in the morphology of the projection of the dorsal bar, the number of rings of male copulatory organ, and by the smaller size of members of hook pairs 1 and 5 compared with those of the remaining pairs. Urocleidoides tocantinensis sp. nov. is easily distinguished from all other species of the genus by the morphology of the vagina, which present a vaginal vestibule with a membranous cap. Urocleidoides triangulus is reported from its type host in the Guandu River, state of Rio de Janeiro. The present study increases the number of Urocleidoides species to 37 recognized species that fit all the generic characters.


INTRODUCTION
Urocleidoides was proposed by Mizelle and Price (1964) for a new species, Urocleidoides reticulatus Mizelle & Price, 1964 from the gills of Poecilia reticulata Peters (Poeciliidae), which was collected from Capitol Aquarium, Sacramento, California, USA. A few years later, 12 species were described within this genus, from the gills of fish belonging to the orders Characiformes, Cyprinodontiformes, Gymnotiformes and Siluriformes (Mizelle et al. 1968, Mizelle and Kritsky 1969, Kritsky and Leiby 1972. Subsequently, Molnar et al. (1974) described eight new species from Trinidad and Tobago and transferred to this genus two species from Cleidodiscus Mueller, 1934, based on the emended diagnosis provided by Mizelle et al. (1968), which expanded the bounds of Urocleidoides. Kritsky et al. (1986) revised the generic diagnosis, described new species and reviewed the material belonging to known species. These authors provided redescriptions and restricted the genus to the species that presented a sinistral vaginal sclerite. Twenty-three species were thus considered incertae sedis, which reduced the number of species in the genus to five. Currently, the genus comprises 34 recognized species described in the Neotropical Region (Argentina, Brazil, Colombia, El Salvador, Panama, Peru, Trinidad and Tobago and Mexico), that are parasites of Characiformes, Gymnotiformes and Cyprinodontiformes (Kritsky and Thatcher 1974, Molnar et al. 1974, Suriano 1997, Kritsky et al. 1986, Mendoza-Franco et al. 2007, Mendoza-Franco and Reina 2008, Rosim et al. 2011, Moreira et al. 2015, Rosin and Timi 2016, Ferreira et al. 2017, Oliveira et al. 2020, Zago et al. 2020. Although species of Urocleidoides have a wide distribution in the tropics, there are still few studies documenting the diversity of Monogenoidea in the Neotropical Region, considering the great biodiversity of host species. Among the species that were considered incertae sedis, eight remain uncertain and 16 have been allocated to other genera (Kritsky et al. 1986, 1989, 2000, Jogunoori et al. 2004, Mendoza-Franco et al. 2009, Yamada et al. 2015, Acosta et al. 2019. During studies on the helminth fauna of fish in the Tocantins River, characiform fish, including Boulengerella cuvieri (Spix & Agassiz, 1829) & Santos, 1988 (Serrasalmidae) and Caenotropus labyrinthicus (Kner, 1858) (Chilodontidae) were examined. Characiformes is the one of the largest orders of freshwater fish, with at least 2300 valid species distributed in 520 genera (Nelson et al. 2016). These fish harbor a great diversity of helminths. However, no monogenoids have previously been described or reported from B. cuvieri, P. amazonica, C. labyrinthicus or C. gouldingi, while Notozothecium bethae Kritsky, Boeger & Jégu, 1996 was reported from M. paucisquamatus collected in the Tocantins River by Kristky et al. (1996). In the current paper, three new species of Urocleidoides are described and a new distributional record for Urocleidoides triangulus (Suriano, 1981) Rossin & Timi, 2016 is presented.

MATERIAL AND METHODS
During expedition carried out in 2010 from the middle part of the Tocantins River, in the states of Maranhão and Tocantins, were collected 32 samples of B. cuvieri (14.5-40.8 cm in standard length and 35.3-551.0 g in weight), six of C. gouldingi (16.1-17.9 cm in standard length and 50-76 g in weight), six of M. paucisquamatus (12.3-15.5 cm in standard length and 65.2-158.2 g in weight) and seven of C. labyrinthicus (11.1-14.1 cm in standard length and 36-71 g in weight). These were identified by the ichthyologists from the Museu Nacional, Universidade Federal do Rio de Janeiro (UFRJ). These fish were caught with the aid of local fishermen and were examined for parasites immediately afterwards.
Ninety-seven samples of P. amazonica (12.0-16.8 cm in standard length and 43.0-100.0 g in weight) were collected from the urban zone of the municipality of Imperatriz, state of Maranhão, and in the village of Embiral, which is part of its rural zone, between March 2018 and August 2019. These were identified by the specialists of the Laboratório de Sistemática e Ecologia de Organismos Aquáticos (LASEOA), Universidade Estadual do Maranhão (UEMA). These fish were caught in nets and immediately packed in Styrofoam boxes filled with ice and taken to the Laboratório de Anatomia, Universidade Estadual da Região Tocantina do Maranhão (UEMASUL), for material processing. The fish sample collection protocol and laboratory procedures were approved by the Research Ethics Committee of Universidade Estadual do Maranhão, under protocol number 21/2017, and the environmental collection license was obtained from the System for Authorization and Information on Biodiversity (SISBIO), under protocol number 61650-1. The gills were removed from the fish and placed in vials containing hot water (65 °C), which were then shaken. Formalin was added to reach a concentration of 5%.
Parasitological indexes were calculated as proposed by Bush et al. (1997), followed by standard deviation.
Etymology. the specific name refers to the scientific name of the host.
Etymology. The specific name refers to the similarity with Urocleidoides triangulus.
Remarks. Urocleidoides paratriangulus sp. nov. is similar to U. triangulus considering the shape of the anchors and bars. The new species differs from U. triangulus by the projection of the dorsal bar (prominent postero-median process in U. triangulus and slender or a more robust projection with lateral allae in the new species); the number of rings of MCO (2.5 to 3 in U. triangulus and about two in the new species); and by the reduced size of hooks (pairs 1, 5 and 7 reduced in size in U. triangulus and 1 and 5 only in U. paratriangulus sp. nov.). Only five species of Urocleidoides possess a medial projection on the posterior margin of the dorsal bar: Urocleidoides curimatae Molnar, Henek & Fernando, 1974;U. neotropicalis;U. piriatiu;Urocleidoide tenuis Zago, Yamada, Yamada, Franceschini, Bongiovani & Silva, 2020;and U. boulengerellae sp. nov. The medial projection is slenderer than the bar arms in the five species, whereas in U. triangulus and in some specimens of U. paratriangulus sp. nov., the projection is thicker than the arms. Urocleidoides paratriangulus is similar to U. curimatae and U. tenuis also in terms of the triangular shape of the ventral anchor, but it can be differentiated by the number of coils and the length of the MCO (3 coils in U. paratriangulus sp. nov. vs 1.5 coils in U. curimatae and 7.5 in U. tenuis), as well as through the morphology of the accessory piece (curved shaft in U. paratriangulus sp. nov., straight in U. curimatae and pincer-shaped in U. tenuis). During a study carried out in the Guandu River by the laboratory team, specimens of U. triangulus were found parasitizing Cyphocharax gilbert (Quoy & Gaimard, 1824). The morphometry of the specimens studied herein were used to make comparisons with those of U. paratriangulus sp. nov. This was the first record in this host in Brazil. Urocleidoides tocantinensis sp. nov. 35,36 http://zoobank.org/96A69E56-0836-4AC2-85A3-3C486B873C33 Type host. Psectrogaster amazonica Eigenmann & Eigenmann (Curimatidae).
Etymology. The specific name refers to the locality, Tocantins River.
Remarks. Urocleidoides tocantinensis sp. nov. resembles Urocleidoides falxus Zago, Yamada, Yamada, Franceschini, Bongiovani & Silva, 2020 by the absence of coils on the MCO and Urocleidoides surianoae Rosin & Timi, 2016 in the shape of accessory piece, but differs in the morphology of the MCO (MCO reverse J-shaped, with expanded proximal end in U. surianoae and a straight tube in the new species). The new species differs from all other species of the genus by the morphology of the vagina, which present a vaginal vestibule with a membranous cap.

DISCUSSION
To date, 34 species of Urocleidoides are known, described from 44 different host species. Urocleidoides is mainly characterized by the presence of a sclerotized structure located in the median region of the body, named vaginal sclerite. At present, species of Urocleidoides are found in fishes from Argentina, Brazil, Colombia, El Salvador, Guatemala, Mexico, Panama and Trinidad and Tobago. Although there is not enough information to reconstruct the historical associations of Urocleidoides spp., there is plenty of evidence that their lineages can parasitize a diverse range of host, from the fact that they are parasites of 10 different families belonging to three orders (Characiformes, Cyprinodontiformes and Gymnotiformes) (Mizelle and Price 1964, Kritsky et al. 1986, Moreira et al. 2015, Ferreira et al. 2017. A phylogenetic analysis using morphological and molecular data has suggested that Characiformes is a sister group of Gymnotiformes (Briggs 2005). However, the parasite data is still at too early a stage to be able to say whether divergence between these two latter fish groups in South America might also represent the earliest phylogenetic split of Urocleidoides in the tropics (Saitoh et al. 2003, Briggs 2005, Mendoza-Franco and Reina 2008.
Species of Urocleidoides have also been found in aquarium fishes, generally those collected from the native habitats in the Neotropical Region, such as U. reticulatus from Poecilia reticulata and Urocleidoides vaginoclaustrum Jogunoori, Kritsky & Venkatanarasaiah, 2004, from Xiphophorus hellerii Heckel, 1848. Hosts of these two species have been introduced into aquaria and ponds in Israel, Czech Republic, India, California (USA), and central Mexico (see Kritsky et al. 1986, Jogunoori et al. 2004, Mendoza-Palmero & Aguilar-Aguilar 2008. Urocleidoides boulengerellae sp. nov. was found only in one host species, B. cuvieri, with a high prevalence, mean intensity, and abundance. In Brazil, the distribution of B. cuvieri is restricted to the Amazon and Tocantins-Araguaia basins, in the states of Pará, Amapá, and north of Mato Grosso. The new species was not found in any other host examined in the present study, and thus can be considered to be a specialist, as the majority species of the genus. On the contrary, U. tocantinensis sp. nov. was found in host species belonging to different families: M. paucisquamatus (Serrasalmidae) and P. amazonica (Curimatidae), likewise, U. paratriangulus sp. nov. was found in hosts of different families, thus demonstrating that these species are more generalist. This scenario is similar to what has been observed for a few other species of this genus, such as Urocleidoides eremitus Kritsky, Thatcher & Boeger, 1986 which was found parasitizing hosts belonging to Anostomidae and Erythrinidae. All of the remaining species that occur in more than one host species are restricted to a single host family. Rosim et al. (2011) postulated that the presence of a ventral bar with enlarged extremities may be also a characteristic that can be considered to be diagnostic for Urocleidoides, in addition to those proposed by Kritsky et al. (1986) in the revision of the genus. Zago et al. (2020) proposed an amended diagnosis for the genus, considering the recent discovery of new Urocleidoides species. Among these features considered was the absence of coil in the MCO, which groups together U. falxus and Urocleidoides tocantinensis sp. nov.
Urocleidoides triangulus was originally described from C. gilbert, as Andropira triangula from the Chascomús lagoon, Argentina, by Suriano (1981). Suriano (1997) provided a redescription of the species and proposed a new combination, allocating the species in Palombitrema, as P. triangulum. Rosin and Timi (2016) studied the monogenoidean fauna of Cyphocharax voga (Hensel, 1870): they found this species and proposed a new combination, U. triangulus, based on having identical morphology of the copulatory complex, hooks and internal organs, specially by the presence of a vaginal sclerite. Urocleidoides triangulus has been reported parasitizing different species of Cyphocharax: C. voga (Rosin and Timi 2016), C. gilbert in Argentina (Suriano 1981(Suriano , 1997, C. modestus (Fernández-Yépez) (Abdallah et al. 2015) and C. nagellii in Brazil (Steindachner) (Vieira et al. 2013, Abdallah et al. 2015. The present report provides the first record in the type host in Brazil. The data obtained in the present study increases the number of Urocleidoides species to 37 and shows that further studies are necessary to clarify the morphological characteristics that limits the diagnosis of this genus.