Integrative taxonomy of a new Redudasys species (Gastrotricha: Macrodasyida) sheds light on the invasion of fresh water habitats by macrodasyids

The order Macrodasyida (Gastrotricha) includes over 350 marine species, and only 3 freshwater species (Marinellina flagellata, Redudasys fornerise, R. neotemperatus). Herein we describe a new freshwater species of Macrodasyida, Redudasys brasiliensis sp. nov., from Brazil through an integrative taxonomic approach. The external morphology and internal anatomy were investigated using differential interference contrast microscopy, confocal microscopy, scanning and transmission electron microscopy. The systematization of the new taxon was inferred by nuclear (18S and 28S) and mitochondrial (COI) genes, and its intra-order relationships were assessed using data from most of available macrodasyids. Phylogenetic analyses yielded congruent trees, in which the new taxon is nested within the family Redudasyidae, but it was genetically distinct from the other species of the genus Redudasys. The new species shares the gross morphology and reproductive traits with other Redudasyidae and the presence of only 1 anterior adhesive tube per side with Redudasys neotemperatus, but it has a specific pattern of ventral ciliation and muscle organization. Results support the hypothesis that dispersion into fresh water habitats by Macrodasyida and Chaetonotida taxa occurred independently and that within Macrodasyida a single lineage invaded the freshwater environment only once. Furthermore, the Neotropical region seems to be peculiar for the evolution of the freshwater macrodasyid clade.

putative new species of Redudasys from the Edwards Aquifer, Texas (U.S.A.): that was the first record of the genus from the Nearctic biogeographic region. Three years later, Kånneby & Kirk 9 formally described the unnamed species reported by Kånneby & Wicksten 12 as Redudasys neotemperatus based on specimens collected in Oregon (U.S.A.). An additional finding of a macrodasyid gastrotrich specimen much likely belonging to Marinellina flagellata was made by J.M. Schmidt-Araya in a different Austrian stream (Todaro et al. 8 ).
Integrative taxonomy is a new approach for taxonomic and systematic studies that has spread in recent years [13][14][15] . The basic idea is to integrate information from different sources: ecological data, molecular data from nuclear and mitochondrial DNA, and morphological characters. In order to better understand the evolutionary history of the studied taxa, the data integration can be done by cumulation or congruence frameworks 13 . In the former case, the species identification is performed by a cumulative set of characters, without necessarily requiring concordance among all of them. Thus, a single set of characters considered good can be used to support the hypothesis about the new species. On the other hand, the framework of integration by congruence is based on a lineage divergence and the hypothesis under the assumption is based on the integration or coherent pattern of two or more independent sources of characters 13 . Considering that the advent of digital techniques in optical and electron microscopy have speed up the finding of new morphological characters and that new cybernetic infrastructures have been developed for analyzing and integrating data, the documentation and dissemination of data have greatly been improved 16 .
Herein, we formally describe a new species of freshwater Macrodasyida from Brazil through the approach of integrative taxonomy with the aim to investigate the relationships of the new taxon within the order. Morphological techniques (DIC, CLSM, SEM, TEM) and multigene molecular analyses (18S rRNA, 28S rRNA and COI mtDNA) were applied. We chose to integrate the data examined in this study by congruence: this framework has high confidence to promote taxonomic stability because the hypothesis regarding the description of the new species is supported by several character sets 13 . A study of additional specimens of Redudasys fornerise allowed a comparison within the genus.

Taxonomic account
Phylum  Kisielewski, 1987 emended diagnosis of the family Redudasyidae. Macrodasyids from 302 to 400 µm in total length, with rounded head bearing several sensory cilia but without tentacles or ocelli. Lateral trunk margins even, without indentations or protrusions. Posterior end two-lobed, without a peduncle. Cuticular covering smooth, without scales or spines. Adhesive apparatus consisting of anterior (TbA) and posterior tubes (TbP); ventrolateral tubes (TbVL) may also be present (Anandrodasys). TbA, 1-3 per side: 1 tube per side arising from a lateral pouch, or 2-3 tubes of unequal length per side, borne from a common base and emerging from a ventrolateral furrow (Redudasys) or inserted in parallel (Anandrodasys), protruding obliquely to the rear. TbP, 4-12 in total, distributed symmetrically at the end of the two caudal lobes. TbVL if present at all, 5-6 per side, along the anterior intestinal region. Dorsal tubes (TbD) and lateral tubes (TbL) absent. Longitudinal muscles visibly cross-striated. Ventral ciliation of different patterns in the 2 genera: Redudasys: paired fields posterior to the mouth and along the pharyngeal region (U04-U36), and unpaired patches along the median line of the trunk region (U39-U50) or arranged in two large longitudinal bands close to each other, extending from the mouth ring to the anterior trunk (U48) and then merging into a single unpaired band to the caudal end; Anandrodasys: an unified field posterior to the mouth splits into 2 paired longitudinal bands, with the medial along the pharyngeal region, and the lateral extending up to the anus posterior to which is an isolated unpaired patch. Redudasys brasiliensis sp. nov. (Figs 1-4) Diagnosis. Macrodasyid 302-376 µm in total body length. Body separated into head, trunk and caudal regions. Head rounded, bearing several sensory cilia, without tentacles or ocelli. Cylindrical trunk, posterior end two-lobed, without a peduncle. Cuticular covering smooth, with no ornamentation. Paired tactile bristles of equal length along the body sides. Adhesive tubes only at the anterior and posterior body ends. TbA, only one tube on each head side, arising from a ventrolateral pouch. TbP, two symmetrical groups, each composed of two tubes of unequal length arising from a common base. TbD and TbL absent. Ventral ciliation arranged in two large longitudinal bands close to each other, extending from the mouth ring to the anterior trunk then merging into a single band up to the caudal body end. Mouth terminal; buccal cavity inconspicuous, lined with thin cuticle. Cylindrical pharynx with pharyngeal pores at base. Circular muscles surround the entire pharynx and are in turn surrounded by helicoidal muscles. Ten to twelve longitudinal muscles are external to both circular and helicoidal muscles, and extend from the mouth ring to the caudal end. Two of these muscles, the ventrolateral bands, insert anteriorly on a transverse muscle at the region of the TbA, and caudally on a second transverse muscle. Muscles of the trunk include longitudinal bands along the intestine that are surrounded by somatic circular muscles;     Table S1).
Description. The description is based on both the holotype and 7 paratypes (Fig. 1a,b; Table 1). Worm-like body, flattened ventrally, vaulted dorsally, 302-376 μm in total length. Body well-delimited into head/trunk/ caudal regions. Head width (U17) 44-65 μm, anterior trunk width (U40) 30-64 μm, midtrunk width (U60) 33-68 μm, and caudal base width (U93) 18-30 μm. Head rounded anteriorly, without tentacles or ocelli. Caudal end bearing two symmetrical groups each of two tubes of unequal length arising from a common base.  Ciliation. Sensory cilia distributed irregularly, scattered around the mouth (ca.10 μm in length) and along the anterolateral head margin (ca. 30 μm in length). Six paired dorsolateral sensory bristles of equal length (ca. 20 μm in length) along the body sides and one paired bristle on each caudal appendage (Fig. 2c,d). Ventral locomotory ciliation arranged in two large longitudinal bands close to each other, extending from the mouth ring to the anterior trunk (U48) and then merging into a single band to the caudal body end (Fig. 1b).  Reproductive system. Male reproductive organs not seen, species probably parthenogenetic. Two ovaries lateral to the posterior intestine (ca. U82); usually one fully-grown egg (120 μm long) is visible dorsally (Fig. 2a).
Muscular system. Thin circular muscles, very numerous, surround the pharynx for its whole length (U01 to U43). Circular muscles of the intestine (U44 to U82) are less numerous and more regularly spaced; muscles noticeably closer each to other at the caudal end (U83 to U93) (Fig. 4a,c). Along the pharynx, 10 to 12 large, longitudinal muscles are inserted at the mouth ring and extend to the PhJIn (U43): they get thinner posteriorly (Figs 3c and 4a-c) and are external to circular muscles. One pair of dorsal longitudinal muscles extends from the mid pharynx to the caudal end (U23-U95) (Fig. 4a,c). They are external to circular muscles, except in the posterior pharyngeal region (U31-U37) and in the caudal body region (U83-U93), where they lie beneath the circular muscles (Fig. 4a,c). One pair of lateral longitudinal muscles extend from the PhJIn (U43) and branch into 3 parallel, thin bundles on each side from U61 to the body posterior end (U93). These longitudinal muscles are surrounded by circular muscles for their whole length (Fig. 4a,c). The largest muscles in the body are two longitudinal muscles that are inserted laterally to the pharynx in the region of the TbA (U14) and run ventrolaterally extending up to the caudal appendages (Figs 3a,b and 4a-c). These muscles are connected anteriorly (U16) by a thin ventral transverse muscle and posteriorly (U96) by another transverse semicircular muscle (Fig. 4b,c).
Ecology. Species relatively common in sediment of the Jequitinhonha drainage basin sites; rare in sediment of the São Francisco drainage basin sites. Kisielewski, 1987 (Figures 5, 6) Redudasys fornerise -Todaro et al. 8 . Description. The external and internal morphology are in accordance with the original description 7 and the subsequent redescription 8 (Figs 5a-f and 6a-c). It is important to highlight that this species has 2 TbA per side, the inner tube shorter than the outer, with a common base that arises from a ventrolateral furrow (Fig. 5c-e).
Helicoidal muscles were observed along the pharynx, ending at around the PhIJ (U51), but their insertion point could not be observed.
For the analysis of the COI mtDNA, the gene tree only showed the monophyly of the families Dactylopodolidae and Redudasyidae. Redudasys brasiliensis sp. nov. resulted as the sister-group of a clade composed by R. fornerise and R. neotemperatus, all supported by high bootstrap and Bayesian posterior probability values. All specimens of Redudasys brasiliensis sp. nov. were resolved as a monophyletic group, but with relative low bootstrap (61; not shown in Supplementary Fig. S1) and Bayesian posterior probability values (0.83; not shown in Fig. 8). The specimens of R. brasiliensis sp. nov. from São Gonçalo do Rio Preto and from Diamantina formed two distinct monophyletic clades highly supported by bootstrap (respectively, 98 and 97; Supplementary Fig. S1) and Bayesian posterior probability values (respectively, 1 and 1; Fig. 8). The genetic distance ( The parsimony network representing 10 sequences detected a total of seven haplotypes of Redudasys brasiliensis sp. nov. (Supplementary Fig. S2). As in the COI mtDNA gene tree, the haplotypes from the two populations were divided into two lineages. The latter species is separated from R. fornerise and R. neotemperatus by high number of mutations in the network (Supplementary Fig. S2).

Discussion
In this section, we discuss some morphological and biogeographical patterns that may be associated with the diversification of freshwater macrodasyids.

Comparison among members of Redudasyidae. Members of family Redudasyidae show few external
and internal morphological characters and hence the few characters concerning the adhesive system and locomotory cilia take on systematic significance 12 . Originally 7,8 , the main diagnostic characters of the genus Redudasys were the possession of 2 TbA per side and a ventral locomotory ciliation arranged into separate ciliary fields of unequal size (paired in the pharyngeal region and unpaired along the median trunk region). Anandrodasys, the only marine genus of the family, shows instead 3 TbA per side and ventral cilia arranged into an unified field posterior to the mouth and splitting into 2 paired longitudinal bands of different length. Kånneby and Kirk 9 described the first Redudasys species with the presence of a single pair of anterior adhesive tubes, as observed in R. brasiliensis sp. nov. Garraffoni et al. 11 . also reported an unnamed species of Redudasys with a single TbA per side from Brazil, but in this case that observation was a misinterpretation. During the present study we had the opportunity to collect fresh material and analyze Redudasys specimens from one of the sites  previously investigated 11 . This allowed us to verify that these individuals in fact had 2 TbA of unequal size per side arising from a common base just like in R. fornerise. Thus, we considered that the Redudasys specimens found by Garraffoni et al. 11 . can be treated as R. fornerise.
The new species can be distinguished from Redudasys neotemperatus by: a different pattern of locomotory ciliation (i.e., two large longitudinal bands close to each other, extending from the mouth ring to the anterior trunk, then merging into a single band up to the caudal body end instead of regularly spaced paired tufts in R. neotemperatus), a greater body size (302-376 µm instead of 220-284 µm), a lower number of dorsolateral sensorial cilia (6 pairs instead of [13][14]. Furthermore, Redudasys brasiliensis sp. nov. is also characterized by pharyngeal pores very poorly developed, unlike the other species of the family.
Redudasys brasiliensis sp. nov. and R. fornerise possess a quite similar muscular system, despite some specific muscles showing clearly distinct morphological patterns. The circular muscles of R. brasiliensis sp. nov. are external to the dorsal longitudinal muscles in the posterior pharyngeal region whereas in R. fornerise, they are internal to all longitudinal muscles. Moreover, the main longitudinal body muscles are ventrolateral in R. brasiliensis sp. nov. but clearly ventral for almost the whole length in R. fornerise. Finally, both species show two semicircular transverse muscles connecting the two ventrolateral muscles, but the anterior one is clearly thinner in R. brasiliensis sp. nov. than in R. fornerise.
The two populations of Redudasys brasiliensis sp. nov. were considered two separated species based on "4X rule" approach to delimiting species using DNA sequences 15,17 . However, this result can be biased due to limited number of individuals and candidate species (respectively, 12 and 3-4) and also because two of these 3-4 candidate species (R. fornerise and R. neotemperatus) had only 1 sequence each available in the GenBank (Supplementary Table S1). Thus, until more specimens of the three species can have their DNA sequenced, we still considered the individuals of both São Gonçalo do Rio Preto and Diamantina populations belonging to the same species, i.e., R. brasiliensis sp. nov.
Araújo et al. 10 . identified two freshwater macrodasyid specimens found in Brazil as belonging to genus Marinellina, basically because they had a single TbA per side. A subsequent study 18 on the spatial and temporal distribution patterns of freshwater meiofaunal taxa in a lotic system in Brazil found additional macrodasyid specimens with a single TbA per side and reported them as an unidentified species of Redudasyidae. Considering the diagnostic character of the number of TbA, the metric data which fall in the measurement range of Redudasys brasiliensis sp. nov., as well as the geographic origin of these specimens, we consider the specimens found in both studies as belonging to the new species. Furthermore, the putative assignment of these specimens to the genus Marinellina 10 cannot be supported with certainty due to several reasons: (a) insufficiency of the original description of the genus Marinellina 1,6,7 , (b) uncertainty about the exact position of TbA in Marinellina specimens 2 (c) absence of pharyngeal pores in Marinellina specimens, (d) geographic distance between the sampling sites of european individuals and of the specimens found by Araújo et al. 10 , respectively.
The new species joins the few other species of macrodasyids that lack a male system including the frontal and caudal accessory organs, useful to store foreign spermatozoa and to act as a copulatory organ -sensu Ruppert and Shaw 19 -, respectively. In these species, that are Anandrodasys agadasys, Redudasys fornerise, R. neotemperatus (Redudasyidae), Paradasys pacificus (Cephalodasyidae) and Urodasys viviparus, Thaidasys tongiorgii (Macrodasyidae) 20,21 , a probable parthenogenetic condition occurs. Only the latter species has a muscular organ of unknown function, which appears similar in position and structure to the caudal organ of other Macrodasyida 20 .
Colonization of freshwater habitats by Macrodasyida. Marine Macrodasyida are widely distributed along coastlines of the world and some level of endemism can be found in the North Hemisphere 22 . However, the levels of diversity and endemism of freshwater macrodasyids are considerably lower than in marine ones and, until now, no hypothesis or factor was presented to shed light on this great faunistic heterogeneity.  The Neotropical region seems to be a special area for the speciation of freshwater Macrodasyida. In Brazil, Kisielewski 7 described the first undoubted macrodasyid from inland waters (Redudasys fornerise); subsequently Garraffoni et al. 11 and Araújo et al. 10 reported other freshwater specimens of Macrodasyida and in the present study we describe Redudasys brasiliensis sp. nov. These findings highlight the presence and the relatively wide distribution of Macrodasyida in the inland waters of this geographical area.
As pointed out by Ribeiro 23 the hydrographic basins where populations of Redudasys fornerise and R. brasiliensis sp. nov. were found have an ancient biogeographic component. The populations of R. fornerise from State of São Paulo and the specimens of R. fornerise and R. brasiliensis sp. nov. from State of Minas Gerais are all very far from the Atlantic shore, respectively around 280 km and 420-450 km. However, these distances were only established during the last sea-level fluctuations in the Holocene transgression, about 5,600 years ago 24,25 . It is important to highlight that the South American continent has not always responded as a single rigid blocks during most of its geological history. The old history of South America shows that this large landmass has an intricate mosaic of amalgamation and break-up of several distinct continental fragments record in different periods 23,26 . Looking into the tectonic-sedimentary evolution of some provinces in the South American Platform, such as areas of São Paulo and Minas Gerais States, it is possible to observe specific orogenic cycles and depositional ages [27][28][29] . The sampling site in the State of São Paulo is located in the Bauru Basin represented by sandstones and reddish siltstones formed mainly in the Upper Cretaceous (99.6 to 65.5 Mya) 27 . On the other hand, the sampling sites from State of Minas Gerais lie in Lower Espinhaço Basin that is a mountain chain built up mainly of quartzitic rocks formed by a sequence of an intracontinental rift-sag basin system that developed around 1,500 Mya (Mesoproterozoic) to 600 Mya (Neoproterozoic) 28,29 .
These mosaics of smaller microblocks were surrounded by seaway and the current sampling sites could have been much closer to shallow marine environments than today. In this perspective, environment-specific diversification of freshwater macrodasyids within continental waters happened after the incursions of ancestral marine populations. The phylogenetic position of the monophyletic lineages composed by all Redudasys species is clearly nested into Macrodasyida clade, suggesting that the freshwater macrodasyids invaded inland environment only once. Furthermore, the phylogenetic hypothesis supports an independent colonization of freshwater habitats by Macrodasyida and Chaetonotida. This scenario is in agreement with the hypothesis advanced by Todaro et al. 8 , and in contrast with that proposed by Kieneke et al. 4 , implying a single colonization event of freshwater habitats in the stem lineage of Abursata ((Redudasys+Marinellina) + Paucitubulatina).
Some questions about the biogeographical and evolutionary history of the freshwater Macrodasyida remains unanswered: (a) how did Redudasys lineages achieve their current distributions in independent freshwater systems of Nearctic and Neotropics?; (b) why appear Redudasys populations found in freshwater habitats of the Austral hemisphere to be more widespread than those in Boreal hemisphere?; (c) how can the awareness of the existence of Marinellina, a taxon incertae sedis, affect our current biogeographic and evolutionary understanding of Redudasyidae? Faunistic observations. Until now, 76 freshwater species of Gastrotricha from Brazil have been repor ted 7,8,10,11,[30][31][32][33] . As highlighted by Garraffoni et al. 30 , due to the low number of sampled sites and wide variety of Brazilian freshwater habitats (with distinct geological and abiotic conditions) the number of new species will increase considerably in the next years. As an example for this statement, both the recently described new genus and species Cephalionotus kisielewskii 30 and Redudasys brasiliensis sp. nov. are psammic and from the same region, Diamantina Plateau in the Espinhaço Range. The geological structure of this area is composed of high altitude formations (more than 900 m), has a very specific vegetation known as rocky fields ("campos rupestres"), and shows special conditions of climate, soil, and water causing high rates of endemisms, especially of plant species 34 10 .
Living individuals were located by sorting the sediment poured into Petri dishes under a stereomicroscope Leica EZ4; they were mounted singly on glass slides, observed in vivo and anaesthetized with 2% MgCl 2 under a PrimoStar Zeiss light microscope, and photographed and filmed using a Leica DM2500 microscope equipped with differential interference contrast optics (DIC) and a camera Moticam 2300 of 3.0 megapixel. The videos are available upon request from the first author.
The positions of morphological characters along the body were measured using the percentage units (U) 35 Supplementary Table S2.
The amplification products were electrophoresed in 1% agarose gels containing SYBR Green (Life Technologies). The bands with expected sizes were excised and then purified using a QIAquick Gel Extraction Kit (Qiagen) or the PCR product was directly purified using Illustra ExoProStar 1-Step (GE Healthcare). The DNA fragments were sequenced using BigDye Terminator reactions in a 3730XL DNA Analyzer (Applied Biosystems) at the facility of the LaCTAD laboratory (Campinas, Brazil). The 18S rDNA, 28S rDNA and COI mtDNA partial DNA sequences of Redudasys brasiliensis sp. nov. were deposited in GenBank (Supplementary Table S2).
sequences, alignments and data analyses. 18S rDNA, 28S rDNA and COI mtDNA sequences were aligned separately with Mafft v.7.215 using the L-INS-I approach 37 . The best-fit substitution model was determined with jModelTest 2.1.4 38 . As the number of species analyzed with three genes and deposited in the GenBank is low, we run two distinct analyses (combined dataset and only COI) under maximum likelihood (ML) and Bayesian inference methods (BA) frameworks. ML analysis using RAxML 39 was run with a GTRCAT model with 1000 bootstrap replicates. BA analysis was done using MrBAYES v.3.2.3 40 using two different runs with four chains each for a maximum of 20 million generations (sampled every 500 generations). Best-fit evolutionary model was selected using Akaike Information Criterion. The analysis was stopped when the two runs reached convergence (average standard deviation of split frequencies lower than 0.01). Convergence and estimated sample size (ESS) were verified using TRACER v.1.5, and 10% of each run was discarded as burn-in. Both ML and BA analyses were performed using the CIPRES Science Gateway, San Diego Supercomputer Center 41 . For combined dataset, sequences of 37 species were retrieved from GenBank (2 Cephalodasyidae; 3 Dactylopodolidae; 2 Macrodasyidae; 1 Planodasyidae; 1 Lepidodasyidae; 18 Thaumastodermatidae; 6 Turbanellidae; 4 Redudasyidae) and for COI mtDNA, sequences of 27 species deposited in GenBank were included in the analysis (2 Cephalodasyidae; 3 Dactylopodolidae; 1 Macrodasyidae; 1 Lepidodasyidae; 13 Thaumastodermatidae; 4 Turbanellidae; 3 Redudasyidae) (Supplementary Table S2). Three species belonging to the order Chaetonotida, family Xenotrichulidae: Draculiciteria tesselata (Renaud Mornant, 1968), Xenotrichula intermedia Remane, 1934 and X. velox Remane, 1927 were used as outgroups (Supplementary Table S2). These species were selected as outgroups because members of Xenotrichulidae appeared as the sister group of all other Chaetonotida (e.g. Kånneby et al. 42 ).
Genetic distances. MEGA7 software 43 was used to calculate average uncorrected pairwise distances (p-distances) within and between species and haplogroups; alignment gaps were not considered. Moreover, the reliability of the new species diversification was tested applying the K/ϴ model (formerly known as 4X rule, Birky 17 , Fontaneto et al. 15 ). statistical parsimony networks. To visualize possible intraspecific relationships between the COI haplotypes for Redudasys fornerise, R. neotemperatus and Redudasys brasilensis sp. nov., statistical parsimony networks were calculated using the program PopART 44 . The raw networks produced by PopART were redrawn using software Adobe Photoshop CS6 (Supplementary Fig. S2).