Gastrotrichs and tardigrades in a remnant of Atlantic Forest (Serra do Japi, SP, Brazil)

Serra do Japi, located in the southeast of São Paulo State, is considered a priority area for conservation, as it houses original Atlantic Forest cover remains. Despite the significant number of studies about vertebrates and invertebrates that were carried out in this region, the meiofauna biodiversity is completely unknown. Thus, the present study aimed to investigate for the first time freshwater Gastrotricha and limnoterrestrial Tardigrada in Serra do Japi Biological Reserve. Samples of sediments, periphyton and floating vegetation in reservoirs and natural lagoons, and mosses growing on native and non-native tree trunks were collected in May 2019. At least five gastrotrichs morphotypes were identified and three of them were formally described: Chaetonotus acanthocephalus, C. dadayi (first record in Brazil), and Heterolepidoderma mariae (first record outside the type locality). In regards to tardigrades, twelve morphotypes were identified and four of them were formally described: Pseudechiniscus juanitae, Minibiotus cf. acontistus, Echiniscus dreyfusi and Itaquascon umbellinae (last two species reported for the first time outside the type locality). This study reinforces that meiofaunal diversity and distribution have been underestimated, even in one of the five largest hotspots in the world.


Introduction
Meiofauna can be defined as an assembly of freshwater and marine organisms that pass through a coarse sieve of 500 μm and are retained by a finer sieve of 44 μm and do not have close phylogenetic relationships among them (Giere 2009). These animals can be classified as permanent, having an entire life as small organisms, or temporary, having one or more stages of the life cycle with 'meiofaunal' body size (Higgins & Thiel 1988). Beside in the water bodies of rivers and lakes, freshwater meiofauna can be found in distinct environments, such as rooted vegetation, macroalgae fronds, mosses, and some animal structures like worm tubes (Higgins & Thiel 1988, Boeckner et al. 2009, Giere 2009).
Due to the meiofaunal body size and challenging identification process, the knowledge about gastrotrichs and tardigrades' diversity in the world is underestimated (Vicente 2010, Appeltans et al. 2012Balsamo et al. 2014, 2020, Fonseca et al. 2018, Garraffoni et al. 2019b). Notwithstanding, a few studies have aimed to reveal the richness and distributional patterns of these two freshwater and limnoterrestrial meiofauna taxa in Brazil (de Barros R 1939a, b, 1942a, b, 1943, Kisielewski 1991, Assunção 1999, Pilato et al. 2002b, Garraffoni et al. 2010, 2017a, b, 2019a, Garraffoni & Melchior 2015, Araújo et al. 2013, Rocha et al. 2016, Garraffoni 2017, de Barros RC 2020. This study aims to contribute to this knowledge by presenting the first records of gastrotrichs and tardigrades at the Biological Reserve of Fundação Serra do Japi, an extensive area of Atlantic Forest located in São Paulo State. In addition, this manuscript provides taxonomic notes of the morphotypes and formally described species found at the Reserve.

Study site
Serra do Japi is located in the southeast of São Paulo, bordering four cities (Jundiaí, Pirapora do Bom Jesus, Cajamar and Cabreúva), between the geographical coordinates 23°14′0″S and 46°58′0″W. The elevation has an area of approximately 35,000 hectares and a native vegetation cover of semi-deciduous mesophyllous forests, semi-deciduous forests of altitude, and sparse rocky enclaves (Morellato 1992) and fragments of reforestation with pine and Eucalyptus sp., pastures, and small portions of agricultural crops. The altitudinal ranges vary between 700 and 1,000 meters (Pinto et al. 1972). In the coldest month, July, the average temperatures range between 11.8°C and 15.3°C, and in the warmest month, January, between 18.4°C and 22.2°C depending on the altitude (Morellato 1992). The rainfall cycle is variable, increasing in the months of December-January, with values greater than 250 mm per month, and decreasing in the winter, with values lower than 41 mm and 71 mm per month, depending on the location (Morellato 1992).

Collecting procedures and Data analysis
Samples were collected from May 18 th to 19 th , 2019. Gastrotricha specimens were sampled by collecting specimens of the floating vegetation belonging to Salvinia sp. from a small artificial reservoir (23°14'42.8"S, 46°56'12.4"W) and natural lagoon (23°14'47.3"S, 46°56'12.4"W) and stored in plastic buckets. Tardigrada specimens were sampled by collecting mosses belonging to family Pottiaceae on native (23°14'21.9"S, 46°56'07.6"W) and from Sematophyllum galipense (Müll.Hal.) Mitt. on exotic Pinus sp. (23°13'59.8"S, 46°56'01.5"W) trees ( Figure 1) and stored in paper bags. Freshwater and limnoterrestrial samples were brought back to the University of Campinas for further analysis. Water with sediment was sieved (42 µm mesh) and specimens were encountered by sorting small amounts of sediment poured into Petri dishes under a Zeiss Stemi 2000 stereomicroscope. Mosses were placed in a beaker filled with ddH 2 O, and after 20 minutes the water was transferred to Petri dishes and the specimens were scanned under a Zeiss Stemi 2000 stereomicroscope.
Alive specimens were isolated with micropipettes into a glass embryo dish. The gastrotrichs and tardigrades were anesthetized with 2% MgCl 2 and warm water (70°C), respectively. Subsequently, gastrotrichs were isolated and mounted on glass slides and tardigrades were stored in 70% ethyl alcohol and permanent slides were made using Fluoromount Aqueous Mounting medium. All specimens were documented under a Zeiss Axio Imager M2 light microscope equipped with Differential Interference Contrast optics (DIC) connected to a camera. Photomicrographs were taken using the software ZEN -blue edition. The most representative structures of tardigrades identified up to the specific level were measured. Methods and terminology used to measure the specimens of Echiniscus dreyfusi followed Bartylak et al. (2019); Pseudechiniscus juanitae followed Roszkowska et al. (2020); Itaquascon umbellinae followed Pilato et al. (2002a), Minibiotus cf. acontistus followed Kaczmarek & Michalczyk 2017. Morphometric data was handled using "Echiniscoidea'' and "Parachela" templates available from the Tardigrada Register (Michalczyk & Kaczmarek 2013).
Photos of Gastrotricha (due to the fragility of their bodies, they were destroyed during the microscopic examination and are no longer available - Balsamo et al. 2014, 2020, Garraffoni et al. 2019b) and photos and permanent slides of Tardigrada specimens are available at the Zoological Museum "Adão José Cardoso" (ZUEC), at the University of Campinas, Brazil.

Results
At least five morphospecies (38 specimens) of Gastrotricha belonging to two genera of Chaetonotidae were present. Among these five morphospecies, three were attributed to described species (Chaetonotus dadayi Schwank, 1990, Chaetonotus acanthocephalus Valkanov, 1937 and Heterolepidoderma mariae Garraffoni & Melchior, 2015). A total of 12 morphospecies (32 specimens) belonging to two classes of Tardigrada were present. Among these 12 morphospecies, eight were identified at the genus level and four were attributed to described species (Itaquascon umbellinae de Barros R, 1939a, Pseudechiniscus juanitae de Barros R, 1939b, Minibiotus cf. acontistus de Barros R, 1942b and Echiniscus dreyfusi de Barros R, 1942a). The remaining morphospecies could not be described to species level due to problems in the fixation process (see Discussion).

Chaetonotus (Primochaetus) acanthocephalus Valkanov, 1937
Remarks: C. acanthocephalus is easily recognized by the presence of five dorsal cephalic scales with long spines, two plates at the side of the hypostomium, and the arrangement of dorsal and lateral spines (e.g. two pairs at the neck, transversal row at the mid-dorsal surface). This species was previously reported in Poland, Germany, Romania, Bulgari (Valkanov 1937, Kisielewski 1981, Schwank 1990) and Brazil (Kisielewski 1991, Garraffoni et al. 2010, Araújo et al. 2013. Schwank, 1990 Figure 2 (D-F) Examined material: Fourteen specimens (ZUEC PIC 377-389). Remarks: Von Daday (1905) identified specimens found on the border between Paraguay and Brazil, in Estia Postillon, as Chaetonotus similis Zelinka, 1889. Later, Schwank (1990 recognized that the specimens analyzed by Von Daday (1905) had a pair of spines that protruded beyond the adhesive tubes. This feature is not observed in Chaetonotus similis and Schwank (1990) described a new species, C. dadayi, to accommodate the Paraguayans organisms. Several years later, d'Hondt (2006) found C. dadayi in French Guiana and presented the first photographic record of a specimen of this species. The Brazilian specimens are morphologically close to the previous descriptions due to the presence of spined scales of the posterior dorsal region smaller than the trunk ones, lateral spined scales of the furca base longer than adhesive tubes; two pairs of long, thick, and simple spines on the dorsal region and two pairs of scales with long, thin and simple spines in the ventral region. This is the first report from Brazil. Examined material: Six specimens (ZUEC PIC 391-396). Remarks: Heterolepidoderma mariae can be easily recognized by specific cuticular ornamentation of the dorsal body surface, keeled scales with three different shapes. It is the first report outside the type locality, Paulínia -São Paulo State, Brazil (Garraffoni & Melchior 2015).
Remarks: The specimen found in Serra do Japi has four features not mentioned in the original description of Echiniscus dreyfusi: thick granulation on the dorsal side of the legs; spine present on the first pair of legs; spurs on claws of the third and fourth pairs of legs; the dorsal pair of spines has a striated texture. This species was previously reported in New Guinea (Iharos 1963), Argentina (Claps & Rossi 1988), Japan (Utsugi 1988), andBrazil (de Barros R 1942a). In Brazil, it is the first report outside the type locality, São Vicente City -São Paulo State.

Figure 4 (B)
Examined material: One specimen, unidentified sex. Remarks: Body orange and plump. Dorsal plates with large pores irregularly distributed; spinulosus type. Trunk appendages in the form of spines; spines D d and E more robust and rough. There is a small denticle on each D d spine. Small spine on the first leg pair. Papilla and dentate collar present on the fourth leg pair.
Remarks: Body orange. Round black eyes present in two specimens. Cirrus internus, externus and A present. Clava visible in one specimen. Pseudosegmental plate present. Very visible cuticular granulation on the dorsal plates and legs. Papilla on the fourth pair of legs visible in one specimen. Dentate collar absent. Spurs on claws IV present. Spurs on claws I visible in one specimen. This species was previously reported in Austria (Mihelcic 1962, Maucci 1974, Italy (Ramazzotti & Maucci 1983), Galápagos Islands (Schuster & Grigarick 1966), China (Wang 2009), and Brazil (de Barros R 1939b, 1942a, du Bois-Reymond Marcus 1944

Figure 4 (D)
Examined material: Three specimens, unidentified sex. Remarks: Eyes present in only one specimen. Cuticle smooth. Oral cavity armature (OCA) composed of three bands of teeth. Pharyngeal apophysis present and very visible. Three macroplacoids present and separated by the same distance from each other; the second macroplacoid is the smaller; the third macroplacoid has a small constriction; very small triangular microplacoids present. Claws of the hufelandi type.

Figure 4 (E)
Examined material: Four specimens, unidentified sex. Remarks: Eyes present in two specimens. Cuticle smooth except for very visible granulation present on all legs. Oral cavity armature composed of three bands of teeth. Pharyngeal apophysis present; three macroplacoids present; the second is the smaller and the third is positioned more distant from the others; the third macroplacoid has a small constriction; microplacoids absent. Claws of the hufelandi type. Dentate lunules under claws IV.

Paramacrobiotus sp. 3
Examined material: Two specimens, unidentified sex. Remarks: Eyes absent. Cuticle smooth except for very visible granulation present on all legs. Oral cavity armature (OCA) composed of three bands of teeth. Pharyngeal apophysis present. Three macroplacoids present; the second is the smaller and the third is positioned more distant from the others; the third macroplacoid has a small constriction; very  Genus Minibiotus Schuster, 1980 Minibiotus cf. acontistus de Barros R, 1942b Table 3 Examined material: One specimen, unidentified sex (ZUEC PIC 398). Remarks: Eyes absent. Cuticle smooth. Buccal tube long and narrow. Three macroplacoids present; microplacoids absent. Claws of the hufelandi type (claws Y-shaped; symmetrically arranged with respect to the median plane of the leg; without basal spurs).

Figure 5 (B)
Examined material: Eight specimens, unidentified sex. Remarks: Eyes present only in two specimens. Cuticle smooth. Buccal tube long and narrow. Large pharyngeal apophyses present. Three macroplacoids present; microplacoids present and very small. Claws of the hufelandi type.

Itaquascon umbellinae de Barros R, 1939a
Figure 5 (C), Table 4 Examined material: One specimen, unidentified sex (ZUEC PIC 397).  Barros R, 1942b. The pt index is the ratio of the length of a given structure to the length of the buccal tube expressed as a percentage (Pilato 1981 Remarks: Eyes absent. Cuticle smooth. Buccal tube long and narrow; pharyngeal bulb oval without placoids. Claws of the Hypsibius type (the secondary branch and the basal section form a continuous curve; claws of the same leg are different in size and shape). This species was described from Itaquaquecetuba, São Paulo State, Brazil (de Barros R 1939a), and reported in Argentina (Maucci 1988). In Brazil, this is the first report outside the type locality.
Remarks: Eyes absent. Cuticle smooth. Cuticular thickening between the buccal tube (rigid) and the pharyngeal tube (flexible) absent. Pharyngeal apophyses are small. Three macroplacoids present; microplacoids present. Claws of the Hypsibius type.

Discussion
Meiofauna organisms are represented in ⅔ of all known metazoan taxa (Giere 2009, Cerca et al. 2018 and it is impossible to apply a unique methodological protocol to perform fixation and preparation for all meiofaunal taxa (Fonseca et al. 2018). As a consequence, a large amount of these tiny animals, that have small, fragile bodies, remains poorly studied by researchers since the process of sorting and identification is intensive and time sensitive (Appeltans et al. 2012, Fonseca et al. 2018, Garraffoni et al. 2019b, Balsamo et al. 2020.
The examination of living microscopic organisms requires patience from the taxonomists, especially those that are beginners in meiofaunal identification. The small size and transparency of the body of meiofaunal individuals make them difficult to be discovered among sediments and for subsequent steps such as collecting them by pipette and isolating them for further studying (Giere 2009, Balsamo et al. 2014, 2020, Nelson et al. 2015. Furthermore, gastrotrichs must be studied alive and are frequently destroyed or even lost during the process of preparation for observations under light microscopy (Balsamo et al. 2014, 2020, Garraffoni et al. 2019b. If the specimens are not properly fixed, they can easily vanish or not stay in a suitable position for future observations of the morphological features (Nelson et al. 2015, Fonseca et al. 2018, Garraffoni et al. 2019b, Balsamo et al. 2014, 2020. In these cases, due to losses of important morphological information, the accuracy of the species identification decreases, and misidentifications are inevitable (Schill 2018). Even in tardigrades that have an exocuticle that remains preserved after the fixation process, morphological information of taxonomic importance can be lost (Morek et al. 2016).
This study is a direct result of undergraduate lectures that aimed to teach not only meiofaunal diversity but also how to collect, sort, and identify these tiny animals, using gastrotrichs and tardigrades as study models. Thus, all the problems described above were considered and largely overcome by the students during the lectures. As a result, we found at least five morphospecies of Gastrotricha and 12 of Tardigrada, but within these 17 morphospecies, only seven of them (41,2%) were identified at a specific level. It is important to highlight that we could not find more morphospecies of Gastrotricha because many specimens were poorly fixed or deteriorated, preventing proper identification beyond the genus level.
Regarding the gastrotrichs, two of the three identified species and all unidentified morphotypes belong to the most specious genus in the taxon, Chaetonotus Ehrenberg, 1830 with more than 250 species formally described (Balsamo et al. 2020). The species Chaetonotus dadayi was found in Brazil for the first time and these samples will be important to provide material for a near future redescription of this species providing detailed morphological and ecological data.
The morphotype Chaetonotus sp. 1 has a remarkable feature of nine long spines arranged in three subsequent groups of three spines in the middle of the trunk on the dorsal surface. These conspicuous spines in the middle of the trunk are found in other representatives of this taxon and can be arranged in a "belt", in a subsequent transverse row, or inserted in large nearby scales forming a grouping (Araújo et al. 2013). Kisielewski (1991) reported two species of Chaetonotus in Brazil with long spines on the dorsal region, C. bisacer and C. succinctus.
Representatives of these species have spines arranged in a "belt" shape, differing from C. sp.1, which presents three spines closely inserted in three subsequent groups of spines. Araújo et al. (2013) also reported an unidentified species of Chaetonotus with long spines on the dorsal region, but with five and not nine as described for the present one. Due to the shape and distribution of the dorsal and ventral scales and a pair of spines over the furcal base, the morphotype Chaetonotus sp. 2 is similar to two species reported for Brazil by Kisielewski (1991), C. intermedius and C. breviacanthus. However, C. sp. 2 differs from both species by the presence of a double-edge scale and by the pair of long spines at the furcal base. In comparison to C. intermedius, it was not possible to visualize the two pairs of ventral terminal scales and the cuticular pharyngeal rod. Additionally, in comparison to C. breviacanthus, conspicuous protuberance at the anterior edges of the hypostomium and the posterior "U" edge of the scales were not observed in any specimens of C. sp.2.
Despite the fact that tardigrades have a rigid and armored integument and morphological investigations of living individuals are not required, from the 12 morphotypes of Tardigrada found in the present study, eight were not identified to species level. This occurred because in many species the study of adult morphology alone is not enough for species identification (Nelson et al. 2015, Schill 2018. Very often it is necessary to analyze the eggs because they have essential taxonomic significance   Dastych 1998, Meyer & Hinton 2009, can only be properly identified if the eggs are available. The genus Minibiotus has 50 described species morphologically diverse (Degma et al. 2020). According to recent studies, the results obtained with the use of integrative approach using genetic and detailed morphological data, e.g. in the description of the species Minibiotus ioculator Stec, Kristensen and Michalczyk 2020a, can clarify the species relationships and contribute with information for status polyphyletic of the group (Stec et al. 2020a Kaczmarek et al. 2015). Two Minibiotus morphospecies were sampled in the present study and one of them was very similar to M. acontistus described by de Barros R (1942b). Among the genus Macrobiotus, one of the most specious groups within Tardigrada, we were only able to highlight that Macrobiotus sp. 1 shows a very distinct dorsal cuticle ornamentation.
Three morphospecies of the genus Paramacrobiotus were found: Paramacrobiotus sp. 1, Paramacrobiotus sp. 2 e Paramacrobiotus sp. 3. Despite that species belonging to this genus have a very restricted biogeographic distribution range , for an accurate description of any Paramacrobiotus species it is important to use an integrative taxonomy approach (Stec et al. 2020b). Furthermore, recentely the subgeneric classification was revisited and nomenclature acts and diagnoses were changed (Marley et al. 2018).
The genus Adropion Pilato, 1987 is currently composed of 15 species (Degma et al. 2020), and considered a polyphyletic group, represented by three independent evolutionary lineages (Gasiorek & Michalczyk 2020). The unique species reported in Brazil was Adropion scoticum Murray, 1905(de Barros R 1943, du Bois-Reymond Marcus 1944, Kaczmarek et al. 2015. However, we only found a single specimen and a detailed observation of the morphological traits was not possible and A. scoticum is considered a complex of species and a reanalysis of the taxon is necessary with an integrative taxonomy approach (Kaczmarek et al. 2015, Duenãs-Cedillo et al. 2020). Thus, we decided to keep as Adropion sp.
The last morphotype, Echiniscus sp. is very similar to Echiniscus succineus Gąsiorek & Vončina 2019, only reported for the type locality in Madagascar. However, the specimen found in Serra do Japi differs from E. succineus by the presence of a small denticle on spines D d (absent in E. succineus) and lacks epicuticular thickenings at the edges of the dorsal plates (present in E. succineus). Given these morphological differences and considerable geographic distance, they are probably different species.
It is known that Brazilian freshwater and limnoterrestrial meiofauna organisms are poorly studied and their biodiversity is still underestimated (Garraffoni et al. 2017a, Kaczmarek et al. 2015, de Barros RC 2020. This perspective will only change if the number of surveys increases in the coming years. As the number of taxonomists specialized in these groups is very low in Brazil, the opportunity to show these lesser-known taxa to undergraduate students can potentially inspire the next generation of meiofaunal researchers. Despite some problems in sorting and identification routines due to students' inexperience, this study achieved important goals of teaching and provided an immersive experience for the students interested in these tiny animals.