Microturbellarians ( Platyhelminthes and Acoelomorpha ) in Brazil : invisible organisms ?

Microturbellarians typically belong to the benthos and may occur in a wide variety of environments. They are abundant in freshwater and marine ecosystems and may occur in moist terrestrial habitats. However, turbellarians are seldom taken into account in studies of biodiversity. Most studies on Brazilian microturbellarians had taxonomical purposes and were done in the years 1940-1950. Thus, information on their occurrence and ecological aspects are dispersed throughout several papers. We intend here to summarize the biogeographical distribution and ecological aspects of microturbellarians recorded for Brazil, indicating the main gaps in their knowledge and possible actions to enhance studies on this group. There are 239 species of microturbellarians registered for Brazil, with records distributed in 12 states. However, just three states located in southern Brazil have records of 94% of microturbellarian species. Thus, knowledge on the systematics and geographical distribution of Brazilian microturbellarians clearly reflect the scientific activity over many years or decades in two states of southeastern and southern Brazil. Considering the scant information on this group in Brazil, which is also the situation of the Neotropical microturbellarians in general, some actions should be proposed. First, it would be necessary to sample in the diverse biomes, as well as in the various river and sea basins, based on standardized sampling protocols. Second, it would be necessary to encourage diverse research groups to include microturbellarians and/or turbellarians in general into biodiversity inventories and studies on community structure of invertebrates. Third, it is necessary to increase the number of research groups on microturbellarians, in order to augment the studies on their morphology, systematics, and ecology. Considering their abundance, species richness and ecological importance in aquatic environments, despite some peculiarities regarding their sampling, sorting and identification procedures, the challenge to study microturbellarians and enhance knowledge about them in Brazilian ecosystems should be faced.


Introduction
Turbellarians are acoelomate, soft-bodied worms that have a sac-like gut and typically ciliated epidermal cells.Most turbellarians are hermaphrodites with cross fertilization following copulation, showing a complex reproductive system (Cannon, 1986;Rieger et al., 1991).Traditionally, flatworms used to belong to the phylum Platyhelminthes, which can be subdivided into three clades, viz.Acoelomorpha, Catenulida e Rhabditophora (Rieger et al., 1991), since Rhabditophora also includes the parasitic forms.The Acoelomorpha, however, has been removed from the Platyhelminthes into its own phylum (Baguñà and Riutort, 2004;Tyler et al., 2014;Littlewood, 2006;Larsson and Jondelius, 2008;Boll et al., 2013).The term turbellarians continues to be used and will be applied here without taxonomic connotation.
Turbellarians may occur in a wide variety of environments.They are abundant in freshwater and marine ecosystems and may occur in moist terrestrial habitats.They are, however, mainly marine animals, with only 1/5 of the known species occurring in freshwater (Hyman, 1951;Schockaert et al., 2008).Turbellarians typically belong to the benthos.Triclads and polyclads occur generally on hard bottom, under stones, or associated with macrophytes, algae or gravel (Hyman, 1951;Du-Bois Reymond Marcus and Marcus, 1968).Microturbellarians usually occur on sandy or muddy bottom.Most marine turbellarians are limited to the littoral zone of the ocean (Hyman, 1951).Freshwater forms may occur in temporary or permanent wetlands, such as lakes, natural or artificial ponds, pools, ditches, streams and rivers (Kolasa, 1991;Noreña-Janssen, 1995;Young, 2001), as well as in agroecosystems (Bambaradeniya et al., 2004;Vara and Leal-Zanchet, 2013).Some marine flatworms, such as acoels and polyclads, and freshwater rhabdocoels are planctonic (Hyman, 1951;Rocha et al., 1990;Dumont et al., 2014).Some microturbellarians may occur in humid terrestrial environments (Hyman, 1951;Tessens et al., 2014); land triclads may be a species-rich group in tropical and subtropical ecosystems (Carbayo et al., 2009).The temnocephalids, a group of the taxon Dalytyphloplanida, and some members of other taxonomic groups of microturbellarians live in association with other invertebrates or with turtles, usually as ectosymbionts (Hyman, 1951;Jennings, 1997).In addition, some acoels and rhabdocoels are parasites of molluscs, echinoderms and other invertebrates (Hyman, 1951;Bush, 1981).
Freshwater and land triclads were studied mainly in the Brazilian states of São Paulo and Rio Grande do Sul (Carbayo and Froehlich, 2008;Baptista et al. 2010;Leal-Zanchet et al., 2011;Amaral et al., 2014).Polyclads were mainly known from the coast of the states of São Paulo and Rio de Janeiro, as well as the northeastern coast of Brazil (Carbayo and Froehlich, 2008;Bahia and Padula, 2009;Bahia et al., 2012;Bulnes and Torres, 2014).
In contrast to triclads and polyclads, which are collected directly, microturbellarians should be sampled with some substratum using a fine-meshed sweep-net (Young, 2001).Material collected by the net should be transferred to plastic vials containing water from the sampling site.The vials should be transported to the laboratory and carefully examined under a stereomicroscope (Braccini and Leal-Zanchet, 2013;Vara and Leal-Zanchet, 2013).For sorting, samples may be treated by the method of reduction of oxygen (Schockaert, 1996) or by examining all sampling water under the stereomicroscope (Brusa et al., 2003).Live turbellarians should be transferred by a pipette to a petri dish and be examined alive for observation of general shape, size and colour pattern.After that, the microturbelários e/ou turbelários em geral em inventários da biodiversidade e estudos de estrutura de comunidades de invertebrados.Em terceiro lugar, é necessário ampliar o número de grupos de pesquisa em microturbelários, para aumentar os estudos sobre sua morfologia, sistemática e ecologia.Considerando sua abundância, riqueza de espécies e importância ecológica em ambientes aquáticos, apesar de suas peculiaridades de amostragem, triagem e identificação, o desafio de estudar e ampliar o conhecimento sobre microturbelários em ecossistemas brasileiros deve ser enfrentado.
Considering that the macroturbellarians are the best known group among turbellarians and that their study in Brazil was summarized by Carbayo and Froehlich (2008), we will focus on Brazilian microturbellarians herein.We intend to summarize the biogeographical distribution and ecological aspects of microturbellarians recorded for Brazil, by pointing out main gaps in the knowledge about them and possible actions to enhance studies on this group.

Methods
Original research articles were searched in the databases Thomson Reuters (ISI) and Scielo, between August 2014 and February 2015, and in the bibliographical assets of our laboratory in the Universidade do Vale do Rio dos Sinos (UNISINOS).The following key-words were used: Platyhelminthes, Acoela, Nemertodermatida, Catenulida, Prolecithophora, Rhabdocoela, Temnocephalida, Macrostomida, Lecithoepitheliata or Revertospermata and Brazil.
We organized the data thus obtained in order to summarize the following information: species, order or other representative taxonomic group, sampling place (city and state), and type of environment where the occurrence was recorded.Regarding the environment, the following data were considered: type of substratum (sand, mud, algae etc.), type of ecosystem (marine, freshwater or brackish water) and hydric regime (lentic or lotic).In the case of temnocephalids, the associated organism was informed.The classification of marine ecoregions follows Spalding et al. (2007).

Geographical Distribution and Ecological Aspects
A total of 44 articles with records of microturbellarians in Brazil was found, 26 of which were available on the online databases Thomson Reuters (ISI) and Scielo.Eighteen other articles were found in the bibliographical assets of our laboratory (UNISINOS).
There are 239 species of microturbellarians registered for Brazil, with records distributed in 12 states.The highest number of species was recorded for the state of São Paulo, followed by Rio Grande do Sul and Paraná (Table 1).The central, northern and northeastern regions of Brazil have no or just a few records (Figure 1).
Acoels, a group of marine flatworms, are represented by specimens sampled only in the state of São Paulo, mainly in sandy beaches, on algae or rock coasts (Tables 1 and 2).A few specimens were found on beach ponds.
Catenulids, which are mainly freshwater inhabitants, were registered in various continental environments, mainly in southeastern and southern Brazil (Table 1).They were frequently found in lentic environments, including rice fields and their canals, or in humid terrestrial environments.Six species were found in lotic environments and two species in water accumulated among leaves of Bromeliaceae (Table 3).Macrostomids inhabit marine and freshwater environments.They occur associated to the bottom or to the vegetation.In Brazil, 11 species were recorded in marine environments of the north coast of the state of São Paulo.Six other species were sampled in lentic continental ecosystems or in brackish water in various regions (Tables 1 and 4).
Rhabdocoels (Dalytyphloplanida and Kalyptorhynchia) inhabit mainly marine and freshwater environments, but some species are terrestrial.In Brazil, marine species were recorded in the north coast of the state of São Paulo, on algae, sand or mud.Freshwater species were found in lentic ecosystems, including rice fields and their canals, or lotic environments of southeastern and/or southern Brazil.Temnocephalids occur only in freshwater environments, with most species recorded in Brazil.They were found in the north, center, southeast and south of Brazil, as ectosymbiotes in turtles, decapods, insects and molluscs (Tables 1 and 5).
Lecithopitheliates may occur in freshwater, terrestrial and marine environments, but they are represented in Brazil by only two freshwater species, Prorhynchus stagnalis Schultze, 1851 and Geocentrophora applanata (Kennel, 1888).Prorhynchus stagnalis, considered worldwide distributed, was recorded in various localities in southeastern and southern Brazil, whereas G. applanata was sampled in two localities of central and southeastern Brazil (Tables 1 and 6).
Prolecithophorans inhabit freshwater and marine environments, but they have no records in Brazilian freshwater ecosystems (Vara and Leal-Zanchet, 2013;Braccini and Leal-Zanchet, 2013).The members of this taxon were sampled in marine environments on coastal areas of the state of São Paulo (Table 1).They were found on algae (Padina and Ulva) or on sandy and rocky coasts (Tables 1 and 6).
Revertospermats are exclusively marine flatworms.They are represented in Brazil by a single species, Urastoma cyprinae (Graff, 1882), found on the coast of the state of São Paulo (Tables 1 and 6).It is a parasite which inhabits the gills of bivalves.

Discussion and Conclusions
Just three Brazilian states, located in southeastern and southern Brazil, concentrate 94% of the microturbellarian species recorded for the country.Two of them, São Paulo and Rio Grande do Sul, house research groups on turbellarians, clearly reflecting the scientific activity over many years or decades.
Most marine microturbellarian diversity is known only in a small portion of the Warm Temperate Southwestern Atlantic, since the marine microturbellarian groups were almost exclusively studied in a small part of the northern littoral of the state of São Paulo (Marcus, 1948(Marcus, , 1949(Marcus, , 1950(Marcus, , 1952;;Hooge and Rocha, 2006).Just scattered records exist for the Tropical Southwestern Atlantic and Northern Brazil Shelf.
In addition to taxonomical studies, freshwater microturbellarian diversity was also investigated through recent inventories and studies on community structure done in the state of Rio Grande do Sul (Gamo and Leal-Zanchet, 2004;Braccini and Leal-Zanchet, 2013;Vara and Leal-Zanchet, 2013).In these studies, many unidentified species of catenulids, lecithopitheliates, macrostomids and rhabdocoels, among others, were recorded in agroecosystems and natural lentic environments of southern Brazil (Braccini and Leal-Zanchet, 2013;Vara and Leal-Zanchet, 2013).Thus, it is almost unnecessary to emphasize that the known microturbellarian diversity in Brazil represents only a very small part of the existing biodiversity.
Considering the scant information on the taxonomy and biogeography on Brazilian microturbellarians, which is also the situation of the Neotropical microturbellarians in general, some actions should be proposed.First, it would be necessary to sample in the diverse biomes, as well as in the various river and sea basins, following standardized sampling protocols, as for example, the guidelines of the Rapid Assessment Program, in order to quickly generate data about the local and regional diversity (Alonso et al., 2011).Second, it would be necessary to encourage diverse research groups to include microturbellarians and/or turbellarians in general in biodiversity inventories and studies on community structure of invertebrates.Third, it is necessary to increase the number of research groups on microturbellarians, in order to augment the studies on their morphology, systematic, and ecology.
There are, however, some problems for the inclusion of microturbellarians in ample biodiversity inventories.Microturbellarians should be sorted alive under the stereomicroscope and analyzed under the microscope to study their morphology with identification purposes (Young, 2001;Brusa et al., 2003).Thus, some adjustments in the sampling procedures used for other invertebrates are needed, such as the collection of water for sorting the live microturbellarians at the laboratory.Another problem is concerned with the identification of microturbellarians, because for the same reason mentioned above, it would be difficult to send live specimens for the identification at species level by specialists.
Besides the abundance and species richness of microturbellarians in freshwater and marine ecosystems, these organisms may feed on a wide range of benthic and planctonic invertebrates, such as oligochaetes, rotifers, cladocerans, copepods and nematodes, among other organisms including bacteria, algae and protozoans (Young, 2001).Thus, considering their abundance, species richness and ecological importance in aquatic environments, the challenge to study these organisms and enhance their knowledge in Brazilian ecosystems should be faced.

Figure 1 .
Figure 1.Records of microturbellarians in Brazil.Dots represent localities where microturbellarians were sampled.

Table 2 .
Species of Acoela recorded for Brazil, with localities of occurrence and type of environment and/or substratum where the microturbellarians were found.

Table 3 .
Species of Catenulida recorded for Brazil, with localities of occurrence and type of environment and/or substratum where the microturbellarians were found.

Table 4 .
Species of Macrostomida recorded for Brazil, with localities of occurrence and type of environment and/or substratum where the microturbellarians were found.

Table 5 .
Species of Rhabdocoela recorded for Brazil, with localities of occurrence and type of environment and/or substratum where the microturbellarians were found.
No information.* For temnocephalids, the host organism is indicated.
-No information.* For temnocephalids, the host organism is indicated.

Table 6 .
Species of Prolecithophora, Lecithoepitheliata and Revertospermata recorded for Brazil, with localities of occurrence and type of environment and/or substratum where the microturbellarians were found.

Table 7 .
Species of Proseriata recorded for Brazil, with localities of occurrence and type of environment and/or substratum where the microturbellarians were found.