Tardigrades of Kristianstads Vattenrike Biosphere Reserve with description of four new species from Sweden

Kristianstads Vattenrike Biosphere Reserve [KVBR] is a UNESCO designated area of Sweden possessing high biological value. Although several studies on tardigrades inhabiting Sweden have been performed, the KVBR area has been neglected. The current study investigates the tardigrade fauna of five areas of the biosphere reserve and includes 34 samples of different substrates analysed quantitatively and qualitatively. In total, 33 species of tardigrades were found in the samples, including 22 new records for the Skåne region, 15 new records for Sweden, and four species new to science. Mesobiotus emiliae sp. nov., Xerobiotus gretae sp. nov., Itaquascon magnussoni sp. nov., and Thulinius gustavi sp. nov. were described with an integrative approach (when possible) using morphological characters (light, electron scanning, and confocal laser scanning microscopies) and molecular markers (ITS2, 18S, 28S, cox1). A new protocol to increase morphological data was developed recovering mounted specimens within old slides for SEM analysis. Emended diagnoses for the genus Itaquascon and the transfer of Platicrista itaquasconoide to the genus Meplitumen are proposed. This study enriches the knowledge of the tardigrade biodiversity both within the KVBR and in Sweden and contributes to the rapidly increasing number of tardigrade species reported worldwide. The 33 species identified in the KVBR area represents 28% of all water bear species found in Sweden so far. The restricted study areas and limited number of samples collected suggests that the KVBR is very rich of tardigrades.

In the last five years, more than 60 species of tardigrades new to science have been discovered and described 1 . Taking advantage of the integration of morphological and molecular techniques, taxonomists are now able to identify cryptic species, and to DNA barcode species, improving the accuracy of species description and identification. In spite of the ever-growing understanding of the diversity and phylogeny of the phylum Tardigrada, the faunistic data are still very scarce and highly under-studied. Only a small part of the publications reporting species new to science focuses on the composition of the community of tardigrades inhabiting a substrate or an area, e.g. [2][3][4][5][6][7] , even if the importance of combining taxonomic and faunistic studies have been suggested, e.g. [8][9][10] . Without extensive faunistic studies, e.g. [11][12][13] , information on the distribution (biogeography), auto-and synecology, and adaptation of the species remain very limited.
The main aim of the current study was to provide information about the tardigrade diversity of an unexplored area of Sweden, the Kristianstads Vattenrike Biosphere Reserve [KVBR] (Skåne County, Sweden). This biosphere reserve is a UNESCO designated area in the southernmost region of Sweden known for its wetlands, rivers, and lakes and for successful coadaptive management of its natural resources 25 . Within the Kristianstads Vattenrike, both residential areas and biotopes possessing high biological value of international and national importance such as wetlands, forest, and sandy arable land are included. In fact, this area of about 1050 km 2 encompasses many sites of the "Natura 2000" network and nature reserves: it is one of the most biodiverse areas in Sweden, hosting 20% of the species considered threatened in the country 26   Molecular characterization. The analyses of the molecular markers were not possible due to the lack of alive specimens: the genomic material extracted from dead specimens gave no amplicons.  the table  the number of specimens measured for each species (for the number of measurements for each character see  Supplementary Table S1), the measures of the selected structures (characters; range in µm), and the relative pt indexes are displayed.    Fig. 4c). Legs of the first pair smaller than those of the second and third pairs. The area of the leg cuticle surrounding the claws with a swelling (forming a garter-like structure; Fig. 4a, d-f). These swellings appearing covered with microdigitations and few minute scattered granules (with SEM; Fig. 4d).  www.nature.com/scientificreports/ Bucco-pharyngeal apparatus with antero-ventral mouth (Fig. 3c,d). Buccal ring with ten peribuccal lamellae (Fig. 4g). Buccal tube of Macrobiotus type, curved in the first half, and ending with a thick cuticular ring within the pharynx (Fig. 3c,d). Ventral lamina with an antero-ventral thickening (Fig. 3d). Buccal armature (Figs. 3c,e, 4g) composed of: an anterior band of small teeth at the base of the peribuccal lamellae (with SEM); a thin posterior band of small teeth not always visible with LM, but clearly visible with SEM; three dorsal and three ventral transversal crests, medio-ventral crest appearing split in two or three mucrones in some specimens (with LM). Lateral cribrose areas posterior to the transversal crests visible with SEM (Fig. 4g). Stylet support, inserted at 77.0-81.2% of buccal tube, in shape of an elongated sigma with a distal flat enlargement (Fig. 3c). Typicallyshaped stylet furcae, with oval condyles supported by short branches provided with rounded apophyses (Fig. 3f). In the pharynx: large and triangular pharyngeal apophyses overlapping the first macroplacoid; two rod-shaped www.nature.com/scientificreports/ macroplacoids (in lateral view; Fig. 3d), and evident drop-shaped microplacoid. In frontal view (Fig. 3g), the first macroplacoid in shape of a drop with a medial slight constriction longer than the second, the second rectangular with rounded corners and with a small terminal slight constriction. Double-claws I-III different from claws IV (Figs. 3h,i, 4d,e): claws I-III of Xerobiotus type (without lunules), claws IV with a longer common tract, small and short claw branches, and pale lunules (more sclerified proximally than distally) sometimes visible (Fig. 3i). Internal and external claws of the same leg similar in shape, external (or posterior, in claw IV) claw slightly larger than the internal (or anterior, in claw IV). Proximal portion of the basal part of all claws with a small enlargement, larger in claw IV (Figs. 3h,i, 4d). Claws increasing in length from the first to the third pair. Primary branch of all claws with short accessory points (larger in claws IV). Cuticular bars under the base of the claw IV thick and with ragged margin (Fig. 3i): cuticular bar under the posterior claw wider and stretched toward the anterior claw, cuticular bar under anterior claw developed toward the front of the body (Fig. 3i).
Spherical eggs laid free (Fig. 2d,h), ornamented with processes in shape of inverted goblets with straight or concave cross section (according to Kaczmarek et al. 35 ; Fig. 2e-g,i,j). Processes base surrounded by a crown of dots (Fig. 2g); terminal disc slightly concave and divided by a septum from the trunk (Fig. 2e,f). The edge of the terminal disks indented, the indentations appearing like tapered tip (with LM; Fig. 2g) and like elongated processes ornamented with granules (with SEM; Fig. 2i); in several processes indentation-like structures (provided with granular ornamentation) occurring also in the upper surfaces of the disk (Fig. 2j). Wrinkled egg surface between the processes (Fig. 2g, i) and scattered with dot-like pores (with SEM; Fig. 2i). Egg with an embryo found.  Xerobiotus pseudohufelandi (Iharos, 1966) 44 by: the presence of a posterior band of teeth, shorter common tract in the claws I-III (pt 11.07-11.99 in X. pseudohufelandi; pt 9.19-9.91 in X. gretae), and the egg surface lacking reticulation.   Table S2).
Description. Body whitish, 135.9-509.3 µm in length (Fig. 5a). Eye-spots absent in mounted specimens. Cuticle smooth. Bucco-pharyngeal apparatus of Itaquascon type (Fig. 5b). Rigid and straight buccal tube, clearly longer than the apophyses for the insertion of the stylet muscles [AISM]. AISM symmetrical and flat ridge-shaped. Buccal tube followed by a pharyngeal tube almost of the same length (pharyngeal tube pt 91.6-113.8). Flexible pharyngeal tube formed by a rope-shaped thickening organized in a geometrical repeated pattern resembling an alternating hexagonal "wire meshes" (Fig. 5b,e,f); the "wire meshes" pattern begins in a more anterior position dorsally and ventrally than laterally (Fig. 5e,f). Very thin stylet supports present but hardly detectable and inserted on the pharyngeal tube in its anterior portion (Fig. 5b,e,f). Small stylet furca with short branches ending in drop-shaped condyles. Stylet coat more sclerotized in its proximal and distal portions than in its middle part. Pharyngeal tube ending within the pharynx with three small triangular apophyses (Fig. 5b). In the pharynx, only a single long, straight, and weakly thickened bar present (Fig. 5b). Double-claws of Hypsibius type (Fig. 5c,d), internal (anterior, in the claw IV) and external (posterior, in the claw IV) claws of the same legs different both in shape and size. Claws increasing in length from the first to the fourth legs. Basal part of all claws long, with enlarged base. Main branch of external claw (posterior, in the claw IV) long, quite straight, and poorly sclerotized throughout its length, with evident accessory points; its proximal part placed on a cuticular digit and connected with the secondary branch with a pair of filaments spanning from the tip of the branch (Fig. 5c). Main branch of internal (anterior, in the claw IV) claw shorter and more curved than the external, with evident accessory points. Thin and hardly detectable pseudolunules present under all claws (Fig. 5c,d). Straight cuticular bars (Fig. 5c), similar in size, with ragged margins on the internal side of the legs I-III, extending from the internal claw base to the anterior side of the leg, weakly visible only on the first pair of legs.
Description. Body whitish, 231.0-346.0 µm in length (Fig. 6a,j). Eye-spots present. Dorsal cuticle sculptured with large tubercles, with polygonal base, that gradually increase in size from the head to the posterior side of the body (Fig. 6b,k). www.nature.com/scientificreports/ Bucco-pharyngeal apparatus with antero-ventral mouth opening (Fig. 6c,n). Buccal tube straight. Twelve peribuccal lamellae present. Buccal armature formed by a posterior line of small round teeth, followed by a line of large rounded teeth in the position of the transversal crests (Fig. 6d,e). Two bigger rounded teeth present ventrally within the second line in correspondence of the stylet sheaths (Fig. 6d). Ventral and dorsal AISM crest-shaped and symmetrical with respect to the frontal plane (Fig. 6n). Long and straight stylet supports with a distal flat enlargement. Typically-shaped stylet furca, with long branches provided with large apophyses (Fig. 6f). In the pharynx, large pharyngeal apophyses overlapping the first macroplacoid (Fig. 6n); three rod-shaped (in lateral view) macroplacoids arranged in a curved line; first and second almost fuse together, third spaced from the second (Fig. 6g). In frontal view, the first macroplacoid in shape of a triangle, second in shape of a rectangle with rounded corners, and the third almond-shaped and slightly constricted in the middle; length sequence 3 > 1 > 2.
Double-claws of Isohypsibius type (Fig. 6h,i,l,m) increasing in length from the first to the fourth pair of legs, external claw slightly longer than the internal. Basal portions of all claws short and slender, enlarged in their proximal portion. Primary branch of all claws with thin and short accessory points (never reaching the end of the branch and not always visible) and divided from the rest of the claw by a basal septum, with a dorsal knoblike thickening (Fig. 6h,i). Pseudolunules hardly detectable and small on external claws of legs I-III (Fig. 6h,l), larger on posterior claw of legs IV (Fig. 6i,m).
Smooth oval eggs laid in exuvium (an exuvium with four eggs was found).   (Table 3) of Eutardigrada (18 genera and 29 species of Parachela, and one genus and two species of Apochela), and Heterotardigrada (one genus, two species). The identification of the morphospecies was carried out with morphological and morphometric approaches.
The species belonging to the family Macrobiotidae were the most represented, found in 23 samples. The 11 macrobiotid species belonged to five genera (Macrobiotus, Mesobiotus, Minibiotus, Paramacrobiotus, and Xerobiotus) and were found in 69.7% of the terrestrial substrates, with variable diversity (1-4 species per substrate) and variable density (0.1-22.4 ind/g; Table 3) within each sample. The genus Macrobiotus was the most represented among the Macrobiotidae and among all the genera identified in all samples (7 species distributed among 17 samples).
The sample SVC11 (C4340 in Bertolani's Collection) was the richer in terms of overall density (66.4 ind/g;  53 were initially morphologically identified, but the evidence of intraspecific variability for some characters led us to suspect the presence of cryptic species. The analyses were performed by genotyping the markers ITS2 and cox1. The analyses of the cox1 were unsuccessful, but the ITS2 sequences amplified from nine specimens (C4340 C-D, J-P; Gen-Bank accession numbers: XXXX) were sufficient to reveal the presence of three species: Macrobiotus polonicus, Macrobiotus cf. polonicus, and Macrobiotus aff. wandae. Macrobiotus polonicus, already identified via morphology, was confirmed also by a very low p-distance of its sequences (0.00-0.01%; 587 bp) with respect to those already attributed to this species (Supplementary Table S3). In the population, eight males with spermatozoans within the gonad were found. One specimen previously identified as M. polonicus was revealed to belong to a cryptic taxon that we named M. cf. polonicus (p-distance 0.04-0.05% with respect to M. polonicus sequences; Supplementary     www.nature.com/scientificreports/ of fine granules on the external side of all legs, for this species the egg morphology is unknown. Macrobiotus aff. wandae is probably a species new to science both for the ITS2 differences (p-distance 0.17-0.18% from the three available sequences of Macrobiotus wandae; Supplementary Table S3) and for the different shape of the egg having a more expanded distal disk on the processes. Since only one egg and few animals of this species were collected and the cox1 sequencing gave no result, further collection and analyses will needed before a possible new species description. The most common morphospecies in the samples was Ramazzottius oberhaeuseri (Doyére, 1840) 54 . It was retrieved from 39.4% (13 terrestrial samples) of the samples and from all the sampled areas except Balsberget, with a highly variable density: e.g., 70.8 ind/g in a lichen, 0.3 ind/g in a soil with grass, and 0.1 ind/g in a moss or in a leaf litter. Milnesium asiaticum Tumanov, 2006 55 was found in 33.3% (11 samples) of the terrestrial samples from all the sampled areas, but with low density (0.1-1.4 ind/g). Hypsibius convergens (Urbanowicz, 1925) 56 was found in 32.4% (11 samples) of both terrestrial and freshwater samples, and in all the sampled areas except the HKR campus, with a low density (0.1-6.2 ind/g). Macrobiotus persimilis Binda & Pilato, 1972 57 had a wide distribution, found in 30.3% (10 samples) of terrestrial samples, with low density (0.3-2.3 ind/g). All the other morphospecies have a more restricted distribution within the samples (Table 3).
Considering all samples, the species diversity within individual samples and between sampling areas was variable (number of morphospecies: 0-7; 12-19, respectively), but most of the samples (67.6%) had three to six morphospecies, and only within two samples (SVC5, 6; 0.5%) there were no tardigrades (Table 3).

Discussion
The species abundance of tardigrades has been shown to be mostly related to the sampling effort, rather than to the surface of the sampling area 58 . The limited number of visited sites and limited sampling effort (34 samples collected in five sites of a 1050 km 2 area) of our study suggest that the true tardigrade biodiversity of KVRB is much higher than recorded, and that there are considerably more tardigrade species to be found in this area. Moreover, the identifications of species were carried out mainly morphologically and hidden molecular diversity, linked to cryptic species, may have been undervalued. Nonetheless, this study documented 33 tardigrade species from the KVBR (Table 3), of which 22 are new records for Skåne, 15 are new records for Sweden, and four are new to science.
KVRB has a higher biodiversity of tardigrades (33 species in 34 samples; n° species / n° samples ratio 0.97) compared to areas similar in types of analysed substrates and sampling effort. As comparison, thirty-nine species (found in 64 samples; ratio 0.61) that were collected in a much smaller area (Monte Rondinaio Valley, Apennines, Northern Italy) 59 which was considered an area of high diversity 60 . Other faunistic studies, comparable in sampling effort and substrate diversity, found lower diversity, e.g. 26 58 . Therefore, this study indicates a high biodiversity of tardigrades in the KVBR, as suggested by the presence of many genera, species, and new taxa from different environments and habitats (i.e. freshwater sediments, soil, and moss). Additional sampling efforts in the KVBR area will likely reveal many more species. For example, in Bartels & Nelson 13 an increase of about six times in the sampling effort in the same sampling area (Great Smoky Mountains National Park, USA) 60 led to the identification of 78% more species, to a final total amount of 73 species in 401 samples, with a decrease in ratio to 0.18.
The description of X. gretae sp. nov. and I. magnussoni sp. nov. increases the number of species in Xerobiotus and Itaquascon and therefore enriches the knowledge of the synapomorphic characters of these phylogenetic lines. Xerobiotus gretae sp. nov. presents an area of the legs that surrounds the claws, corresponding to what we called "garter-like structure", that is present also in X. xerophilus. This structure was described for X. xerophilus by Dastych and Alberti 42 as "small swelling" (see SEM Figs. 8,12,17 in Dastych and Alberti 42 ) and is visible in the LM Fig. 6e in Pilato et al. 43 . Furthermore, in the original description of X. pseudohufelandi by Iharos 44 the garter-like structures are not reported, but they are visible in Fig. 6b in Pilato et al. 43 . Similarly, in X. euxinus this structure is not reported in the original description, but a pale refractive structure is visible in the leg shown in Fig. 5b in Pilato et al. 43 . Additionally, this garter-like structure, the smaller legs of the first pair compared to those of the second and third pairs, and the absence of ventral cuticular pores were always observed in three Xerobiotus populations from Portugal, France, and Italy (Guidetti R. personal observation). Smaller legs of the first pairs are also present in X. xerophilus (see Figs. 1, 6-8 in Dastych and Alberti 42 ).
Recently, the genus Xerobiotus has been proposed to be suppressed based on molecular analyses that would nest this genus within the Macrobiotus species 45 . In our opinion, the Xerobiotus suppression is premature (or not sufficiently supported) based on the presence of several clear synapomorphies of Xerobiotus species (see below) that allow distinguishing the genus from any other tardigrade genera. Macrobiotus (according to Stec et al. 45 ), the clade including Xerobiotus, is still not morphologically well defined and inhomogeneous (e.g., in claw and egg morphologies), is without any autapomorphy, and contains several lineages (one of which is composed by Xerobiotus species) recognized by molecular data, but to date not yet supported by morphological apomorphies 45 (excluding Xerobiotus). Moreover, the suppression of Xerobiotus would also imply the suppression of the genus Pseudohexapodibius that shares with Xerobiotus the morphology of claws of the first three pairs of legs and of the bucco-pharyngeal apparatus, together with the reduction of the leg size. Such important changes in the systematics (e.g., the erection and suppression of genera) need well supported results from both morphological and molecular point of view. According to our new findings, the garter-like structures are an additional apomorphy of Xerobiotus. This genus hence differs from the other genera of Macrobiotoidea in several synapomorphic characters, such as the presence of: smaller legs of the first pairs compared to those of the second and third pairs, garter-like structures (i.e. swelling of the leg with microdigitations) in each leg, different shape of the claws www.nature.com/scientificreports/ between the hind legs and those of the first three pair of legs, typical shape of the claw (of Xerobiotus type and without lunules) in the first three pair of legs, claws of the hind legs with long common tact and short branches, and very small cuticular pores only in the dorsolateral portion of the cuticle. Stec et al. 45 45 , all the Macrobiotus species share the same claw type apart from the species of the former genus Xerobiotus, further underlining the uniqueness of the characters of these species. Several new genera have been erected from Macrobiotus, since it was the first genus created in the phylum, see 1 , and probably other genera will be identified with related apomorphies, explaining the presence of several phylogenetic lineages in the cluster identified by Stec et al. 45 as Macrobiotus. Itaquascon (Hypsibiidae) is a genus belonging to the subfamily Itaquasconinae, i.e. eutardigrades with Hypsibius type of claws, a bucco-pharyngeal apparatus subdivided into a rigid buccal tube and a flexible pharyngeal tube provided with or without spiral thickening, and AISM in shape of wide flat ridges 46,63,64 . The thickenings in the pharyngeal tube do not always form a true annulation in the Itaquasconinae, but it is more likely a patterned thickening (i.e. simple, complex, net-like) 64 . According to Gąsiorek et al. 64 , the Itaquascon pharyngeal thickening pattern is formed by annuli forking and merging irregularly and vanishing in the ventral side (net-like). In contrast with their observation, the net-like thickening pattern of I. magnussoni sp. nov. and I. placophorum (in type material and other populations; see "Methods") that we observed resembles an alternating hexagonal wire mesh (Fig. 6e,f). Thus, our observations suggest that the pattern of the pharyngeal tube thickenings has a major variability within the genus in contrast to that pointed out by Gąsiorek et al. 64 . Analyses of more species, observing also type material, are needed to better understand the ultrastructure of the flexible pharyngeal tube of each genus of this subfamily. In addition, in the amended diagnosis of Itaquascon by Gąsiorek et al. 64 , it is reported that this genus is "devoid of placoids", but in I. placophorum (and I. magnussoni sp. nov.) thickenings (a.k.a. bulbar linings, cuticular ridges) within the pharynx are present (Supplementary Figure S1). For this reason, we propose an amended diagnosis for the genus Itaquascon (see "Taxonomic account").
Moreover, the observation of the holotype of Platicrista itaquasconoide (Durante Pasa & Maucci, 1975) 21 (slide: CT3552) revealed the presence of a lateral patterned thickening anteriorly to the stylet support insertion point (Supplementary Figure S1). This newly observed character and all the other characteristics of the species fit with the recently described genus Meplitumen Lisi et al. 65 . Thus, we propose to move the species, previously moved from Diphascon to Platicrista 66 , to this genus with the new name Meplitumen itaquasconoide comb. nov.
The presence of 29 morphospecies and the four new species identified in this study increased the known tardigrade biodiversity in Sweden by 11%. Furthermore, four new type localities are added to the 21 previously reported in literature for Sweden 24 . Thus, four of the 25 Swedish type localities (16%) and 33 of the 116 recorded Swedish species (28%) are located within the KVBR area. Despite the restricted sampling effort of this study, our study suggests that the KVBR conceals a very high diversity of tardigrades. In particular, further studies of the sandy habitats and the diverse freshwater environments in the KVBR area will likely reveal additional species, some of which may also be new to science. The new species found in this study increased our knowledge of global tardigrade diversity (more than 1330 species are now described 1 ), allowed the identification of new synapomorphic characters for a better definition of the taxa, and illustrated that a broad faunistic survey also can have an impact on the systematics of a phylum. Barros, 1939 (amended diagnosis). Cuticular thickening between the buccal tube and the pharyngeal tube absent in the known species. Sinusoidal stylet supports. Pharynx elongated. Cuticular thickenings within the pharynx present or absent. Claws of Hypsibius type, with external claws with primary branches markedly longer than secondary ones.

Methods
Tardigrade sampling and observations. Thirty-four samples of mosses, leaf litter, soil with grass, and one freshwater detritus sample from five different sampling areas within the KVBR (i.e. Balsberget, Gropahålet, Kristianstad University Campus, Näsby Fält, Sånnarna) were collected in June 2014 (Supplementary Table S4). Extraction of tardigrade animals and eggs were carried out using sieves (mesh size: 250 μm and 38 μm) after keeping the samples in distilled water for about 30 min. Specimens were isolated from the extraction using a needle and a Pasteur pipette under a stereo microscope, and then mounted on slides in Hoyer's or Faure-Berlese fluid for observations with LM and Confocal Scanning Laser Microscopy [CLSM]. Additional specimens were observed with SEM following the protocol of Guidetti et al. 7 .
Dry weight of mosses, lichens, soil with grass, and leaf litters were estimated after the tardigrades extraction by drying the samples in an oven at 50 °C for four days, and the density of tardigrades per sample was calculated as the number of tardigrades per gram of dry substrate.
Remaining subsamples were stored dry at room temperature at the Department of Environmental Science and Bioscience at HKR or/and at the Department of Life Science of Unimore.
Five specimens of Xerobiotus gretae sp. nov. were recovered in 2019 for SEM observation from a permanent slide mounted with Faure-Berlese fluid in 2014 with the following new protocol: in order to recover the specimens within the mounting medium, the slide was rinsed in distilled water overnight to rehydrate and dilute the Faure-Berlese fluid. The coverslip was then gently removed using a needle and the specimens were recovered with a glass pipette. The recovered specimens were washed in distilled water three times. Subsequently, the protocol of Guidetti et al. 7 was applied for preparation of specimens for SEM.
Observations with SEM (Nova Nano SEM 450, FEI company) and CLSM (Ti2-e with A1R HD Resonant scanning module, Nikon) were performed at the "Centro Interdipartimentale Grandi Strumenti" at Unimore. For the observation and 3D reconstruction with CLSM, the protocol of Guidetti et al. 7  The body length of the animals was measured excluding the hind legs; the buccal tube length was measured from the anterior end (at the level of the stylet sheath, or where generally the transversal crests are present) to its posterior end within the pharynx; the claws were measured only if they were in perfect frontal view. Claws of Itaquascon specimens were measured following Pilato et al. 67 , those of Mesobiotus and Xerobiotus following Kaczmarek et al. 35 , those of Thulinius following Beasley et al. 68 . The common tract of the posterior claw of the legs IV of Xerobiotus was measured from the base of the claw, excluding lunule, to the branching point of the secondary branch. Raw morphometric data are given in the Supplementary Table S1 and are organized and analysed using the template Parachela ver.1.6 by Michalczyk et al. 69 modified to perform the correction of the body size effect with the Thorpe's normalization of the measures. Thorpe's normalization calculation was carried out following the protocol from Bartels et al. 70 .
Genotyping. The genotypization was performed on selected specimens from the samples SVC11, 15, 22, 31, Table S5) for both the molecular characterization of the species new to science and for the detection of cryptic species. Before molecular analysis, animals and eggs were observed individually with LM following the protocol described by Guidetti et al. 7 allowing us to obtain pictures of the specimens (photographic voucher specimens).

(Supplementary
In particular, the evaluation of the presence of cryptic species was performed on the specimens belonging to the sample SVC11. In order to ensure the connection between the morphology of specimens and the DNA sequences, eggs were isolated and the newborns were left to hatch. Then, eggshells were mounted on permanent slides with Faure-Berlese fluid (hologenophore) and pictures of the hatched newborns were taken before the DNA extraction 28 .
Genomic DNA was extracted with QuickExtract kit (Epicentre) following the manufacturer protocol on live and dead specimens. Afterwards, when possible, the carcasses of the animals were recovered and placed on permanent slides (voucher specimens; available at the Department of Life Science of Unimore). For DNA extraction, four additional specimens of Xerobiotus gretae sp. nov. were recovered from the permanent slides C4341s1 following the protocol of Guidetti et al. 7 .
Molecular investigations were carried out using fragments of the nuclear ITS2, 18S, and 28S genes and the mitochondrial cytochrome oxidase 1 (cox1) gene (Supplementary Table S5). Several couples of primers and amplification protocols 2,46,71,72 were tested (Supplementary Table S5). The amplified products were gel purified using the Wizard Gel and PCR cleaning kit (Promega, Madison, WI, USA). Sequencing reactions were performed using the ABI Prism Big Dye Terminator v. 1.1 sequencing kit (Applied Biosystems, Foster City, CA, USA) on purified amplicons. Each sequencing reaction contained 0.2 mM of a single PCR primer to initiate the sequencing reaction, 2 µl of BigDye, 70 ng of purified products, 4 µl of sequencing suffer (BigDye Terminator v. 1.1, Applied Biosystems, Foster City, CA, USA), and distilled water for a final volume of 20 µl. Cycling conditions for sequencing reactions consisted of 25 cycles of 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min. Both strands were sequenced using an ABI Prism 3100 (Applied Biosystems, Foster City, CA, USA). Nucleotide sequences of the newly analysed specimens were submitted to GenBank, and accession numbers are reported in the Supplementary Table S5. Chromatograms obtained and nucleotide sequences were checked by visual inspection and the sequences were aligned with the MAFFT algorithm. In order to perform proper molecular comparisons, we included sequences from GenBank pertaining to other specimens in our analysis. Pairwise nucleotide sequence divergences between scored haplotypes were calculated using p-distance by using MEGA7 73 .