Tardigrades Research in Brazil: an overview and updated checklist

. Tardigrades are microscopic animals, commonly referred to as “water bears”, and comprise the phylum Tardigrada. They are found in diverse habitats in terrestrial, freshwater and marine environments worldwide. In this paper, it is presented a brief history of the study of tardigrades in Brazil and an updated species checklist of Brazilian tardigrades. Since the first report in 1913, the number of tardigrades records has increased, reflecting advances in the understanding the diversity of tardigrades in Brazil. A total of 100 species known from Brazil are listed, being 30 in marine and 70 in terrestrial and freshwater environments. The records are concentrated in Southeast (47.1%) and Northeast (41.3%) regions. Despite the advances, further research and sampling of new areas is still needed, besides reanalysis and confirmation of old records. Brazil, with its vast territory, extensive continental shelf and great diversity of biomes, has great potential to expand our knowledge of tardigrades fauna. de tardígrados. São listadas 100 espécies conhecidas para o Brasil, sendo 30 em ambientes marinhos e 70 em ambientes terrestres e de água doce. Os registros estão concentrados nas regiões Sudeste (47,1%) e Nordeste (41,3%). Apesar dos avanços, ainda são necessárias mais pesquisas e amostragens de novas áreas, além da reanálise e confirmação dos registros antigos. O Brasil, com seu vasto território, extensa plataforma continental e grande diversidade de biomas, tem um grande potencial para expandir nosso conhecimento sobre a fauna de tardígrados.


INTRODUCTION
Tardigrades are microscopic Ecdysozoans (Aguinaldo et al., 1997), with 0.5 mm to 1.2 mm in length (excluding the last pair of legs), four pairs of locomotor appendages (lobopodous), generally ending with claws of varying numbers and shapes . These animals are commonly referred to as "water bears" due to their bear-like appearance (legs with claws), and slow lumbering gait (Nelson et al., 2015).
Since the first observation in 1773, 1,298 species of tardigrades (142 genera and 30 families) have been described, including two fossils species (Degma et al., 2019). Two classes and four orders were accepted in the Tardigrada phylum. Eutardigrada constituted by Apochela and Parachela, and Heterotardigrada constituted by Echiniscoidea and Arthrotardigrada (Jørgensen et al., 2018). A third class, Mesotardigrada, was established by Rahm in 1937 based on the description of Thermozodium esakii Rahm, 1937 from a Japanese hot spring (Nelson et al., 2015). However, the type material was lost, so Mesotardigrada is considered nomen dubium until new evidence is found (Grothman et al., 2017). Recently, based on molecular data, Guil et al. (2019) proposed the creation of a new class, Apotardigrada, constituted by order Apochela, and consequently suppression of order Parachela and its superfamilies erected as orders (Fig. 1).
The tardigrades occupy a variety of habitats; they can be found in marine, freshwater, and terrestrial environments worldwide (Nelson, 2002). Heterotardigrada, with few exceptions, encompasses marine tardigrades that inhabit the inter-tidal zone and shallow waters of the continental shelf as well as the deep-sea benthic sediments. On the other hand, Eutardigrada, and the new class Apotardigrada, mainly contain both terrestrial and freshwater species (Nelson et al., 2015;Guil et al., 2019).
In Brazil, however, tardigrades are still poorly known; there are about 80 taxa recorded in eight Brazilian states: Ceará, Pernambuco, Rio Grande do Norte, Minas Gerais, São Paulo, Rio de Janeiro, Rio Grande do Sul and Paraná . The little information about the tardigrades fauna and their distribution in Brazilian regions are mainly a result of a scarcity of specialists and of taxonomic studies about the group. It is necessary to promote further studies and stimulate younger biologists to dedicate themselves to the study of the tardigrades in Brazil. To start this, it seems useful to remember the efforts made in the past and to know the current state-ofthe-art, including the species already known. Thus, this paper presents a brief history of the study of tardigrades in Brazil and provides an updated species checklist of Brazilian tardigrades.

On tardigrades research In Brazil
The first work to report a tardigrade species in Brazil dates back to the early twentieth century, when the Scottish naturalist James Murray recorded the occurrence of Macrobiotus occidentalis Murray, 1910; however, without specifying the Brazilian location (Murray, 1913). It was almost two decades after that new records of tardigrades were made. In 1931, the German zoologist Gilbert (Franz) Rahm (Rahm, 1931). In the next year, Rahm added Pseudechiniscus suillus papillata Ehrenberg, 1853 (mosses from Rio de Janeiro) in the previous list and dated the record of Milnesium tardigradum in Pernambuco state from 1928 (Rahm, 1932).
The German naturalist couple Ernest Gustav Gotthelf Marcus and Eveline du Bois-Reymond Marcus were the first researchers to study tardigrades in a Brazilian institution. In 1936, Marcus and his wife left Berlin to live in São Paulo, as Ernest Marcus had accepted an invitation to head the Zoology Department at the University of São Paulo, from 1936 to 1963 (Corrêa, 1991;Edmunds, 1991;USP, 2019). Eveline and Ernest Marcus received Brazilian citizenships in 1940 and never again left the country (Corrêa, 1991). Together, Eveline and Ernest Marcus published over 280 papers on varied taxonomic groups (Sawaya, 1970;Corrêa, 1991), 27 of them on anatomy, histology, physiology, embryology, ecology, and systematics of tardigrades. Some articles published in São Paulo were written in Portuguese, as a way of honoring Brazil (Mendes, 1994); these included important works as "Sôbre a anabiose dos Tardigrados, com descrição duma nova especie" (Marcus, 1937), and "Sobre Tardígrados Brasileiros" (du . Eveline and Ernest Marcus described several new species of tardigrades, such as Macrobiotus sawayai Marcus, 1937(Marcus, 1937, Batillipes pennaki , and Orzeliscus belopus du ; they were the pioneers in the study of marine tardigrades on the Brazilian coast.
Using morphological data, the biologist Cláudia Maria Leite Assunção was the first Brazilian researcher to explore the tardigrades phylogeny. In her PhD thesis, Assunção investigated the relationship between the subgroups Stygarctidae and Digitopoda, resulting in the establishment of Stygarctidae as a monophyletic taxon, and Digitopoda was branched into two clades (Assunção, 1994(Assunção, , 2001. The biologist Clélia Márcia Cavalcanti da Rocha maintains a research group on the systematics and ecology of tardigrades, working mainly with marine tardigrades of the northeastern coast of Brazil (Rocha, 2018). Recently, her group described the new species Ligiarctus alatus Gomes Júnior, Santos, da Rocha, Santos & Fontoura, 2018  . Rocha also organized the first tardigrades scientific collection in Brazil (Rocha, 2018). Since 2017, the biologist André Rinaldo Senna Garraffoni has been developing a project that intends to redescribe all species with missing type material, whose type locality is in the state of São Paulo, thus increasing the number of nuclear (18S and 28S) and mitochondrial (COI) gene sequences of the Brazilian tardigrades species deposited in Genbank (Garraffoni, 2019).
Until the late twentieth century, the tardigrades were sporadically studied in Brazil. However, despite of historical gaps, the future of tardigrades research in Brazil seems promising. The presence of researchers interested on tardigrades in the different regions of Brazil (Northeast, Southeast and South), the training of new researchers (e.g., Edivaldo Lima Gomes Júnior, Érika Santos, Mônica Marinho Verçosa, and Paola Visnardi Fassina), the increasing of Brazilian tardigrades records and the recent description of new species from Brazilian specimens, are pointing to new horizons. On the other hand, to strengthening the Brazilian tardigradology, it would be interesting the establishment of a cooperation network among Brazilian tardigrades researchers, in order to ensure the exchange of information and expertise.

On tardigrades diversity in Brazil
There are four relevant species surveys of tardigrades in Brazil: Assunção (1999a, b) listed six marine and 58 limnoterrestrial species for the state of São Paulo;  listed 27 marine species for the Brazilian coast; Gomes Júnior (2015) listed 80 tardigrades species, both marine and limnoterrestrial, and Rocha et al. (2016) updated the Brazilian limnoterrestrial checklist to 62 taxa. In the present update, the number of tardigrades records in marine environment increased to 30 (Table 1) and to 70 in terrestrial and freshwater environments ( Table 2). There are fewer studies on freshwater and marine species compared to terrestrial species, and the major samples investigated were sediment (mud, sand and gravel) to marine, and mosses to limnoterrestrial tardigrades. Compared to Gomes Júnior's (2015) checklist, it was not found any record on tardigrade species in the state of Rio Grande do Sul. On the other hand, it was found one record in the state of Amazonas and new records in the state of Alagoas. The records are concentrated in Southeast (47.1%) and Northeast regions (41.3%) and reflect the historical construction of the Brazilian tardigradology and, consequently, the distribution of researchers (Fig. 2).
This sampling effort represents minimal due to the extensive continental dimension of Brazil. According to  over 65% of the Brazilian terrestrial area is undocumented. However, if we take into account the geographic extension of the country (8.511 million km²), the territorial sea (3.6 million km²), the richness of habitats and variety of Brazilian biomes, the percentage should be much higher, perhaps over 99%. In other words, the vast Brazilian territory virtually remains unexplored with respect to tardigrades.
While recognizing the advances in understanding biodiversity of Brazilian tardigrades in the last years, further research and sampling of new areas is still needed, particularly in those states not yet sampled. Old records,  especially those that appeared only once, need to be confirmed with new specimens. Brazil, with its vast territory, extensive continental shelf and great diversity of biomes, has great potential to expand our knowledge of both marine and limnoterrestrial tardigrades fauna. Twenty-six tardigrade species (8 marine, 18 limnoterrestrial) have so far been originally described in Brazil, exhibiting this potentiality.

MATERIAL AND METHODS
Tardigrade species found in Brazil, according to the most recent classification (Degma et al., 2019), are shown in the following checklist. References are reported within parentheses '( )' . The species written in bold characters have Brazil as their type-locality. One species was invalidated, Echiniscus fischeri Richters, 1911, and 24 species were reallocated (for details, see species checklist). According to Santos et al. (2018b), the specimens from Brazil were wrongly identified for the species Batillipes tubernatis, so the previous records from the Pernambuco coast    Marcus, 1937 has been omitted from modern taxonomic literature and requires redescription , it was kept on the checklist once its distribution is restricted to Brazil. Two new records from state of Paraná were included by the author: Paramacrobiotus (Amicrobiotus) cf. areolatus  sampled in mosses from Curitiba, and Viridiscus viridis (Murray, 1910) in lichens from Vila Velha state Park, Ponta Grossa (unpublished).