Six freshwater microturbellarian species (Platyhelminthes) in permanent wetlands of the Coastal Plain of southern Brazil: new records, abundance, and distribution

Microturbellarians occur in a wide range of ecosystems, but their diversity and distribution are largely unknown. In this work, we report on the morphology, abundance, and distribution of 6 species of microturbellarians, namely Baicalellia evelinae, Catenula evelinae, C. leuca, C. turgida, Gieysztoria chiqchi, and Stenostomum hemisphericum. We provide photographs and schematic drawings of the specimens in vivo. Three of them were known only for their type localities. Most species showed low to moderate abundance and were found in a single wetland in this study. Gieysztoria chiqchi is reported from Brazil for the first time. All records extend the ranges of the species at least 800 km to the southeast.


Introduction
Microturbellarians are small, mostly free-living animals, usually less than 1 mm long (Young 2001). They are abundant in a wide range of environments and belong typically to the benthos. They may live directly associated with the bottom or on the roots of macrophytes and occur in fresh-, brackish and marine waters, in lotic or lentic, temporary, or permanent ecosystems (Young 2001, Van Steenkiste et al. 2013. Despite their abundance in many ecosystems, turbellarians are seldom taken into account in studies of biodiversity, perhaps due to the fact that they need to be studied alive to be identified (Schockaert et al. 2008). In addition, histological sections may be necessary. The distribution of microturbellarians in the Neotropical region is mainly known from taxonomic studies, most of which were done in Argentina and Brazil (e.g. Marcus 1945a, b, 1946, 1948, 1950, 1952, Noreña et al. 2004, Brusa 2006, Damborenea et al. 2007, Brusa et al. 2008, Adami et al. 2012, Martínez-Aquino et al. 2014, Braccini et al. 2016. In Brazil, the distribution of microturbellarian species is mainly known from taxonomical works done by Marcus (e.g. 1945aMarcus (e.g. , b, 1946Marcus (e.g. , 1948Marcus (e.g. , 1950Marcus (e.g. , 1952 in the state of São Paulo, as well as from inventories and studies on community ecology done in the state of Rio Grande do Sul (Braccini et al. 2016). There 239 species of microturbellarians recorded from Brazil, 108 of them NOTES ON GEOGRAPHIC DISTRIBUTION recorded in freshwater ecosystems. In southern Brazil, 52 microturbellarian species were recorded in freshwater ecosystems (Braccini et al. 2016).
In the present work, we report the morphology, abundance, and distribution of 6 species of microturbellarians, namely Catenula evelinae, C. leuca, C. turgida, and Stenostomum hemisphericum (Catenulida), Gieysztoria chiqchi and Baicalellia evelinae (Dalytyphloplanida), which are recorded for the first time for southern Brazil. In addition, we provide photographs of the specimens in vivo after squash preparation and schematic drawings of the main diagnostic characters, which is recommended for studying microturbellarians (Noreña et al. 2016).

Methods
The study areas are located on the Coastal Plain of Rio Grande do Sul (Fig. 1), southern Brazil. The regional climate is subtropical and humid, without extreme dry periods (Nimer 1979). The mean annual rainfall is about 1300 mm, varying from 1200 to 1750 mm/year, well distributed throughout the year; the mean annual temperature is about 18-20 °C, varying between -3 °C and 18 °C in the coldest season and between 22 °C and 32 °C in the warmest season (Nimer 1979, IBGE 1986, RADAM-BRASIL 1986, Pedrozo and Rocha 2007, Alvarez et al. 2013). The region is mainly covered by areas of savanna and remnants of semideciduous forest (IBGE 1986).
Surveys were carried out on the shore areas of 5 lentic, permanent, freshwater wetlands (Table 1) in accordance to federal environmental laws (permanent collection license SISBIO 24357-1). Two surveys were done in each wetland in 2012: between March and May (summer/autumn survey) and between July and September (winter/spring survey). Sampling was conducted, on benthic substrate and vegetation, with a fine-mesh sweep-net (335 µm) in 3 random sites of the littoral of each lagoon. The sampling effort was standardized at 10 minutes at each site. The material collected by the net was transferred to 500 ml plastic vials containing filtered water from the sampled site.
The vials containing water from the sampled sites were transported to the laboratory and carefully examined within 3 days after collection. All sampled water was gradually poured into a Petri-dish and examined under the stereomicroscope (Brusa et al. 2003). Each turbellarian was counted and transferred to a histological slide and their external aspect was examined under the stereomicroscope. Subsequently, their anatomy was analysed through optical microscopy, before and after  the squeeze preparation technique (Gamo 1987a). For taxonomical purposes, they were measured, sketched and photographed under the microscope. Abbreviations used in the figures: ab, anterior brain lobe; b, brain; c, cilia; cf, ciliated furrow; e, eyes; i, intestine; m, mouth opening; mc, Minot cells; mg, mouth gland; np, nephridiopore; pb, posterior brain lobe; ph, pharynx; phg, pharyngeal glands; pht, pharyngeal tentacle; pr, protonephridium; r, rhabdoid; rb, light reflecting bodies; st, statocyst; v, vitellaria; ov, ovary.

Results
Species were identified using the taxonomic keys of Cannon (1986), Gamo (1987b), Young (2001), and Noreña et al. (2005). The taxonomic works of Marcus (1945aMarcus ( , 1945bMarcus ( , 1946Marcus ( , 1948Marcus ( , 1949Marcus ( , 1950Marcus ( , 1952Marcus ( , 1954, Brusa et al. (2003), Damborenea et al. (2005), Larsson and Willems (2010), and the Turbellarian Taxonomic Database (Tyler et al. 2006(Tyler et al. -2016 were used for comparisons. Whenever possible, voucher specimens were fixed in absolute ethanol or Bouin (Romeis 1989)  General morphology. The body, with length varying between 0.6 and 0.8 mm, is elongate with truncated anterior tip and rounded posterior tip. The anterior region has long-ciliated sensorial cells and a statocyst with a statolith in the prostomium base (Figs 2, 3). In addition, there is a pre-oral swelling with ciliated furrows and occurrence of conspicuous epidermal inclusions. The intestine is located in the posterior 2/3 of the body. A protonephridium duct is visible in the posterior 1/6 of the body and the nephridiopore is subterminal (Fig. 3). Remarks on ecology and distribution. Species with low abundance in each wetland in the summer/autumn survey and absent from the winter/spring survey (Table  2). Catenula evelinae was previously reported only for its type locality, in the city of São Paulo, in southeast Brazil (Marcus 1945a). Thus, the present record represents a southeastern range extension of about 800 km.  Marcus, 1945 Figures 4, 5 General morphology. Elongated and slender body (length to 1 mm) with rounded anterior and posterior tips; pre-oral region longer than wide. There is a statocyst located approximately in the middle of the cephalic lobe; a ciliated pit is absent. The intestine is large, 2/3 of the body length (Figs 4, 5). Remarks on ecology and distribution: Species with moderate abundance in the summer/autumn survey, but absent in the winter/spring survey (Table 2). Similarly to C. evelinae, C. leuca was previously recorded only in its type locality, in the city of São Paulo (Marcus 1945a). The present record, thereby, increases its known distribution about 800 km to the southeast. (Zacharias, 1902)  General morphology. Body length (0.4-0.6 mm) similar to those from the Holarctic region; mean length a little shorter than that of São Paulo specimens (0.6-0.8 mm). Their main morphological characteristics are: absence of statocysts, body length 5 or 6 times longer than wide, preoral ring with 4 longitudinal grooves on each side (Figs 6, 7) and 3-4 µm long rhabdoids.  (Table 2). It was described from the Holarctic region (Zacharias 1902, Larsson andWillems 2010) and previously reported for the Neotropical region in the city of São Paulo, in southeast Brazil (Marcus 1945a, b). The present record represents an extension of its known distribution about 800 km to the southeast.

Family Stenostomidae Vejdovsky, 1880
Genus Stenostomum Schmidt, 1848 Stenostomum hemisphericum Nassonov, 1924 Figures 8, 9 General morphology. Specimen with 2 zooids, reaching a length of 5 mm (Figs 8,9). Similar to the specimens analyzed by Marcus (1945a), the brain is divided into 2 portions, the anterior lobes are separated in small independent parts and the posterior lobes are trilobated with 2 light-refracting bodies, constituted by a pear-shaped vesicle (0.01 mm in diameter) and a spherical refractory element in its inner part associated with the posterior lobe. The mouth is rounded. The simplex pharynx is long, consisting of strong muscles, with longitudinal, circular, oblique and lateral muscle fibers. The nephridiopore is subterminal (Fig. 9). The pharyngeal glands showed a restricted distribution around the mouth and the posterior portion of the brain was relatively smaller than that of the specimens from the type locality, in the Holarctic region (Nassonov 1924 Remarks on ecology and distribution. This species was represented by a singleton in the summer/autumn survey and was absent in the winter/spring survey (Table  2). It was described from the Holarctic region (Nassonov 1924); in the Neotropical region, it was reported at Belém, northern Brazil (Marcus 1945a) and in the department of Ucayali, Peru (Damborenea et al. 2011  Family Provorticidae Beklemischev, 1927Genus Baicalellia Nassonov, 1930 Baicalellia evelinae Marcus, 1946 Figures 10, 11A, B General morphology. Body 3 times longer than wide (body length to 0.6 mm), with rounded anterior and posterior tips (Figs 10, 11A). The pharynx measures up to 0.15 mm and shows pharyngeal tentacles forming an inverted cone towards the mouth. The male reproductive system consists of a pair of testes, a spherical seminal vesicle, and a C-shaped penis. The latter is constituted by a sclerotized tube, about 50 µm long, with a broad basis, gradually narrowing towards the tip (Fig. 11B). The female reproductive system shows a "bursa seminalis" located between the ovaries, and the uterus with an intestinal connection. The uterus contained 2 egg capsules, each with 2 eggs (Fig. 11A).

Remarks on ecology and distribution.
This species had moderate abundance in the winter/spring survey but was absent in the summer/autumn survey (Table 2). Baicalellia evelinae was previously reported from 3 localities in the state of São Paulo (Marcus 1946). The current record represents a southeastern range extension of about 800 km.
The arrangement, number, and size of spines of the sclerotized stylet, as well as the morphology of its fibrous girdle are identical to these of the type specimens. Remarks on ecology and distribution. This species had a high abundance in the summer/autumn survey, but most of them were dead during the sample analysis, probably due to some disturbance shortly before the survey in Capivari do Sul. It showed low abundance in the summer/ autumn survey in Osório (Table 2). Gieysztoria chiqchi was previously recorded only for its type locality, in the department of Loreto, Peruvian Amazon . Thus, the present record is the first for Brazil and represents an increase of the known distribution of this species of about 3800 km to the southeast.

Discussion
The diversity and distribution of microturbellarians are poorly known in the Neotropical region, and most knowledge is restricted to taxonomic works (Noreña et al. 2004, Brusa 2006, Damborenea et al. 2007, Brusa et al. 2008, Adami et al. 2012, Martínez-Aquino et al. 2014, Braccini et al. 2016). In the present work, we report new records of 6 microturbellarian species, 3 of them previously reported only from their type localities. All new records extend the known ranges of these species. In addition, the new records of C. evelinae and C. leuca present the possibility to develop further studies on these species, which were originally described in the 1940s from the city of São Paulo where the type localities no longer exist due to urban growth. The species richness and abundance of turbellarians in this area was previously studied in 9 wetlands of the Coastal Plain of southern Brazil (Braccini and Leal-Zanchet 2013). However, Braccini and Leal-Zanchet (2013) did not find the species reported here. A possible explanation is a differing methodology used for sampling and sorting the material. The specimens of turbellarians were treated by the oxygen reduction method by Braccini and Leal-Zanchet (2013), and after visual detection, they were transferred to a Petri-dish for examination (Gamo 1987a). In the present study, all sampled water was examined under the stereomicroscope (Brusa et al. 2003), which favored the detection of small specimens (≤ 1 mm), even when their abundance was low, which is the case for many species of microturbellarians. However, this method requires more time, which reduces the number of samples for sorting after each field trip, because the identification of microturbellarians needs to be done in vivo.
The present results augment the number of reported freshwater microturbellarians to 109 species in Brazil. An extensive area of the Neotropical region, as well as other parts of the world, has no or only scattered taxonomic and/or ecological studies , Schockaert et al. 2008, Martínez-Aquino et al. 2014, Braccini et al. 2016, and thus, the known diversity of this group is seemingly highly underestimated.