Ecology

Taxonomic and morphological results
Four species new to science (Mesobiotus emiliae sp. nov., Xerobiotus gretae sp. nov., Itaquascon magnussoni sp. nov., and Thulinius gustavi sp. nov.) were found in the Kristianstad Vattenrike Biosphere Reserve, and their formal descriptions follow.
Morphometric data for the animals and the egg of these species are reported in the Tables 1 and 2 respectively (Supplementary Table S1 for the raw and the Thorpe’s normalized data). The morphometric data used in differential diagnoses of the species were not Thorpe’s normalized. After Thorpe’s normalization, only the lengths of very few characters in the new species resulted not isometric with respect to the length of the buccal tube (Supplementary Table S1). For these characters, the range (min–max) of pt indexes calculated on the Thorpe’s normalized data and the range (min–max) of pt indexes calculated on the non-normalized data did not change significantly (Supplementary Table S1).
Table 1 Summary of the morphometric data of the animals of the species new to science.
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Table 2 Summary of the morphometric data of the eggs of two of the species new to science.
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Mesobiotus emiliae sp. nov.
ZooBank: lsid:zoobank.org:act:3DA2F1C0-BEC8-4D9A-B111-5D5F8F1FCCA0.
Type locality
Sånnarna, west of the nature reserve (Kristianstad, Skåne, Sweden). Sandy soil with grass (55.928931 N, 14.246299 E), collected on June 10th, 2014; sample SVC22 (C4342 in Bertolani’s Collection). The species was also found in two other localities (SVC34, 35) and it is probably present in further four localities in which only animals were found (SVC2, 8, 27, 30; Supplementary Table S2).
Type repositories
The holotype (SVC22 s11o), 34 paratypes, and an egg (SVC22 s1, s5, s7, s8, s11) are at Kristianstad University [HKR], 19 paratypes and an egg (SVC22 s4, s10) are in the collection of the Swedish Museum of Natural History [SMNH], and 3 paratypes and two eggs (C4342 s3, s9) are in the Bertolani’s Collection of University of Modena and Reggio Emilia [Unimore].
Description
Body whitish, 96.8–342.0 µm in length (Fig. 1a). Eye-spots absent in mounted specimens. Cuticle smooth, with sparse granules on the posterior side of the legs IV (visible with Light Microscopy [LM]; Fig. 1g,i), and with granules covered with 1–5 dots on the external side of the legs I-III (Fig. 1h; visible with Scanning Electron Microscopy [SEM]).
Figure 1

Mesobiotus emiliae sp. nov. (a) In toto (ventro-dorsal view) (b) Bucco-pharyngeal apparatus (dorso-ventral view from multiplanar images stack), (c) Buccal armature (ventral view), (d) Bucco-pharyngeal apparatus (lateral view) (e) Claw III (lateral view from multiplanar images stack), (f,g) Claw IV (lateral view from multiplanar images stack), (h) Claw III (fronto-lateral view), (i) Claw IV (frontal view). Empty indented arrows: crests of the buccal armature; indented white arrows: placoids constrictions; white arrowhead: cuticular ring of the buccal tube pharynx ending; empty arrows: accessory points of the main claw branch; empty indented arrowheads: lunules; asterisks: granulation on the legs IV. (a–c,e) Holotype. (a–g) LM, PhC; (h,i) SEM. Scale bars (a) 100 µm; (b–d) 10 µm; (e–i) 5 µm.

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Bucco-pharyngeal apparatus with antero-ventral mouth (Fig. 1b, d). Buccal ring with ten lamellae on its external margin. Buccal armature composed of: an anterior band of small teeth; a posterior line of conical teeth; three dorsal and three ventral transversal crests, the medio-ventral crest reduced to two or four mucrones in some smaller specimens (Fig. 1c), the latero-ventral crests shorter than the latero-dorsal ones. Short and straight stylet supports with distal flat enlargement, inserted at the 73.8–79.4% of the buccal tube. Typically-shaped stylet furca, with spherical condyles supported by short branches provided with small apophyses. Buccal tube ending with a thick cuticular ring within the pharynx (Fig. 1d). In the pharynx: pear-shaped pharyngeal apophyses; three grain-shaped (in lateral view) macroplacoids and an evident drop-shaped microplacoid. In frontal view, first macroplacoid triangular, second and third rectangular with rounded corners, and third with a deep distal constriction (Fig. 1b); length sequence 3  > 2  > 1.
Double-claws of Mesobiotus type (Fig. 1e–i) with evident accessory points on the main branch. All claws similar in shape, external claws slightly larger than internal. Claws increasing in size from the first to the fourth, claws of hind legs clearly the largest. Smooth lunules under all claws, larger under claws of the hind legs (the anterior lunules clearly larger with respect to the posterior).
Spherical eggs free laid, ornamented with processes in shape of large and short cones or mammillated with tips of different lengths (generally short), sometimes terminating in a tuft of filaments (Fig. 2a–c). Process wall formed by two sides (an internal and an external), interspersed with trabecular structures forming irregular meshes (Fig. 2b), in shape of bubble-like structures in the longer process tips (Fig. 2c). Base of the processes with a crown of irregular and small thickenings: smaller thickenings in shape of large dots, the larger ones triangular-shaped (Fig. 2b). Filaments of the process tips mostly short, elongated in abnormal processes. Processes in numbers of 11–14 on the circumference. Egg surface between the processes smooth or sparsely dotted. Egg with embryos found.
Figure 2

Eggs of Mesobiotus emiliae sp. nov. and Xerobiotus gretae sp. nov. (a–c) M. emiliae sp. nov. (a) iIn toto, (b) Processes surface detail, (c) Abnormal processes (lateral view). (d–j) X. gretae sp. nov. (d) In toto, (e,f) Processes (lateral view), (g) Processes (frontal view), (h) In toto, (i) Processes and surface detail, (j) Process (fronto-lateral view). Arrowhead: tuft of filaments on the tip of the egg process; arrow: septum dividing trunk and terminal disk of egg process; empty arrowhead: crown of dots at process base; indented arrowheads: pore on egg surface; empty indented arrowheads: indentation-like structures (provided with granular ornamentation) occurring in the upper surfaces of the disk. (a–g) LM, PhC; (h–j) SEM. Scale bars (a,d,h) 10 µm; (b,c,e–g,i) 5 µm; (j) 1 µm.

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Differential diagnosis
According to the taxonomic key of Mesobiotus species32,33, M. emiliae sp. nov. is different from any other described species of this genus. Mesobiotus emiliae sp. nov. belongs to the harmsworthi group of species, and within this group Mesobiotus insuetus (Pilato, Sabella & Lisi, 2014)34, Mesobiotus lusitanicus (Maucci & Durante Pasa, 1984)35, Mesobiotus occultatus Kaczmarek, Zawierucha, Buda, Stec, Gawlak, Michalczyk & Roszkowska, 201836, Mesobiotus patiens (Pilato, Binda, Napolitano & Moncada, 2000)37, Mesobiotus pseudoblocki Roszkowska, Stec, Ciobanu & Kaczmarek, 201638, and Mesobiotus snaresensis (Horning, Schuster & Grigarick, 1978)39 share with Mesobiotus emiliae sp. nov. the following characteristics: smooth cuticle; buccal armature with both anterior and posterior rows of teeth visible with LM, and without accessory teeth between posterior row and transversal crests; smooth lunules in the claws of the hind legs; eggs with processes in shape of cone or hemisphere with elongated tip or inverted funnel surrounded by a basal crown of dots or digitations, and with smooth or wrinkled surface between them.
Mesobiotus emiliae sp. nov. differs from:
M. insuetus by: the presence of granulation on legs IV, the shorter first macroplacoid (pt 15.4–16.8 in M. insuetus; pt 7.9–12.7 in M. emiliae), the shorter macroplacoids row (pt 46.2–48.9 in M. insuetus; pt 35.2–46.0 in M. emiliae), the different morphology of the hind claws (i.e. not with secondary branches diverging distally and forming a right angle with the primary branches as in M. insuetus), the shorter primary branch of all claws (e.g., pt of the primary branch of the claws II, 31.9–36.0 in M. insuetus; 20.4–25.4 in M. emiliae), and the processes of the egg with larger meshes on the surface (with LM, as small separated dots in M. insuetus and bubbles in contact to each other in M. emiliae);
M. lusitanicus by: the presence of granulation on legs IV, the length sequence of macroplacoids (3  > 1  > 2 in M. lusitanicus), the more evident microplacoid (pt 4.2 in M. lusitanicus; pt 7.3–12.3 in M. emiliae), and the shape of egg processes (i.e. not in shape of hemispheres terminating with a cap-like structure or with a fringed cones as in M. lusitanicus);
M. occultatus by: the absence of granulation on the legs I-III (with LM), the length sequence of macroplacoids (1 ≥ 3  > 2 in M. occultatus), the smaller eggs (full diameter 97.4–126.6 µm in M. occultatus and 62.4–76.5 µm in M. emiliae), the shape of the egg processes with a base/height ratio (74–106% in M. occultatus and 144–164% in M. emiliae), the distance between the egg processes (mean 2.6 µm, 0.6 SD in M. occultatus and mean 1.1 µm, 0.4 SD in M. emiliae);
M. patiens by: the absence of granulation on the legs I-III (with LM), the length sequence of macroplacoids (1  > 3  > 2 in M. patiens), the smaller eggs (full diameter 90.5–100.0 µm in M. patiens and 62.4–76.5 µm in M. emiliae), absence of slender tips in the egg processes;
M. pseudoblocki by: the presence of granulation on legs IV, the length sequence of macroplacoids (1  > 3  > 2 in M. pseudoblocki), the smaller anterior claw of the hind legs (pt 27.5–33.5 in the primary branch and 19.8–27.4 in the secondary branch in M. pseudoblocki and pt 27.5–33.5 in the primary branch and 19.8–27.4 in the secondary branch in M. emiliae), the closer processes on the egg surface (mean 2.8 µm, 0.6 SD in M. pseudoblocki and mean 1.1 µm, 0.4 SD in M. emiliae), the processes of the egg not in shape of sharpened narrow cones, the processes base/height ratio (47–70% in M. pseudoblocki and 144–164% in M. emiliae);
M. snaresensis by: the presence of granulation on hind legs, the more evident microplacoid (pt 4.2–7.3 in M. snaresensis; pt 7.3–12.3 in M. emiliae), the processes of the egg not terminating with a sharp or bifid tips, and the absence of pseudoareolation on the egg surface between the processes.
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.
Etymology
We dedicate this species to Emilia Lonis, the beloved hundred-years-old grandmother of the coauthor Massa E., one of the last living workers that with their hand-on work for the reclamation of the “Piana di Terralba” (Sardinia, Italy; now site of the “Natura 2000” network) have contributed to the eradication of malaria in the island saving thousands of life.
Xerobiotus gretae sp. nov.
ZooBank: lsid:zoobank.org:act:E5265E82-86E9-4069-9B3B-B0F11D43DE79.
Type locality
Sånnarna, (Kristianstad, Skane, Sweden). Moss on ground (55.928056 N, 14.252694 E), collected on June 10th, 2014. Sample SVC15 (C4341 in the Bertolani’s Collection). An animal of this species was also found within a Saxifraga sp. (SVC19; Supplementary Table S2).
Type repositories
The holotype (SVC15 s2m), 51 paratypes, and an egg (SVC15 s2, s3) are at HKR, 50 paratypes (SVC15 s5) are in the collection of the SMNH, and 29 paratypes and an egg (C4341 s1, s4) are in the Bertolani’s Collection of Unimore. Five paratypes recovered from the old slide C4341 s1 (for the extraction and mounting protocol, see: Methods section) together with four paratypes and an egg freshly extracted were mounted on stubs for SEM observation.
Description
Body whitish or pale green, 177.5–438.3 µm in length (Figs. 3a, 4a,f). Orange eye-spots present in mounted specimens. Very small scattered pores (about 0.5 µm in diameter) in the dorso-lateral cuticle (Figs. 3b, 4b,c). Very small single granules, distributed almost regularly, present on the entire cuticle (only visible with SEM; 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).
Figure 3

Xerobiotus gretae sp. nov. (a) In toto (ventro-dorsal view), (b) Cuticular ornamentation (dorsal view), (c) Bucco-pharyngeal apparatus (dorso-ventral view from multiplanar images stack), (d) Bucco-pharyngeal apparatus (lateral view from multiplanar images stack), (e) Buccal armature (ventral view), (f) Stylet furca (frontal view), (g) Macroplacoids (frontal view from multiplanar images stack), (h) Claw III (frontal view), (i) Claw VI (lateral view). Arrow: cuticular pores; empty indented arrows: crests on the buccal armature; empty arrowheads: cuticular ring of the buccal tube pharynx ending; indented white arrows: placoid constrictions; empty arrow: accessory points of the main claw branch; arrowheads: basal rounded cuticular thickening of the claws; black arrowhead: lunules; indented white arrowheads: cuticular bars. (a,c) Holotype; (a–i) LM, PhC. Scale bars (a) 100 µm; (b–h) 10 µm.

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Figure 4

Xerobiotus gretae sp. nov. (a–e) Specimens mounted on stub from alive tardigrades. (f–g) Specimens mounted on stub from old permanent slide. (a) In toto (lateral view), (b,c) Cuticle (dorsal view), (d) Claw I (fronto-lateral view), (e) Claw IV (lateral view), (f) In toto (ventral view), (g) Buccal opening. White arrows: garter-like structure covered with microdigitations; indented white arrows: pores; arrowheads: very small single dots on the cuticle; empty arrows: accessory points; black arrows: basal rounded cuticular thickening of the claws; black arrowhead: granules on the garter-like structure; empty indented arrowhead: anterior band of small teeth at the proximal end of the peribuccal lamellae; empty arrowhead: posterior line of small teeth; indented white arrowhead: dorsal transversal crests; empty indented arrow: cribrose area in buccal tube. SEM. Scale bars (a,f) 100 µm; (b,c) 1 µm; (d,e,g) 5 µm.

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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). Typically-shaped 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 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.
Differential diagnosis
Xerobiotus gretae sp. nov. differs from all other Xerobiotus species by having pores on the cuticle visible with LM, an enlargement in the basal part of the claws, and cuticular bars under the claws IV.
Moreover, Xerobiotus gretae sp. nov. differs from:
Xerobiotus xerophilus (Dastych, 1978)41,42 by: the presence of a posterior band of teeth and the dorsal transversal crests not fused in the buccal armature, the shape of the egg processes (flattened hemispherical processes in X. xerophilus), and the egg surface lacking reticulation;
Xerobiotus euxinus Pilato, Kiosya, Lisi, Inshina & Biserov, 201143 by: the dorsal transversal crests not fused in the buccal armature, and the presence of cuticular bars under the claws of the hind legs;
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.
Molecular characterization
It was not possible to extract genetic material from the specimens recollected from the permanent slides (C4341 A–E). The analyses of the molecular markers amplified from four specimens (C4341 G–L; GenBank accession number: MW581665-8, cox1; MW588431-3, ITS2; MW588438-41, 28S; MW588434-7, 18S; Supplementary Table S5) revealed single haplotypes for ITS2, 18S, and 28S genes, and three haplotypes for cox1 gene (highest p-distance = 0.4%; Supplementary Table S3).
Xerobiotus gretae sp. nov., in comparison to the more similar GenBank sequences which belong to Xerobiotus sp. collected in South Africa (Cape of Good Hope, Western Cape)45, differs for p-distances of 1.6–2.6% for cox1 (796 bp), 1.0% for ITS2 (452 bp), and 0.0% for 18S (870 bp). Xerobiotus pseudohufelandi, collected in Italy (Monte Calvario)46, differs from X. gretae sp. nov. for p-distance of 16.9–17.8% for cox1, and 0.0–0.1% for 18S, Xerobiotus sp., collected in Poland (Błedowska Desert)45, differs from X. gretae sp. nov. for p-distances of 17.8–18.1% for cox1, 5.1% for ITS2, and 0.1–0.2% for 18S (Supplementary Table S3).
Etymology
We dedicate this species to the climate activist Greta Thunberg, for her brave and insightful efforts to open the eyes of the world leaders about the need for action against climate change. The achievements of Greta Thunberg give us hope that the challenges of changing the unsustainable path of human societies may still be possible, just like the tiny tardigrades are able to overcome seemingly impossible environmental challenges. But we have to act now!
Itaquascon magnussoni sp. nov.
ZooBank: lsid:zoobank.org:act:254843BC-60F7-4B8E-A94D-8783214F3399.
Type locality
Näsby Fält (Kristianstad, Skåne, Sweden), along a trail to Araslövssjön Lake. Moss on bark of Alnus sp. (56.059328 N, 14.136678 E), 2 m up on the tree, collected on June 10th, 2014. Sample SVC32 (C4344 in the Bertolani’s Collection). The species was also found in two other localities (SVC3, 27; Supplementary Table S2).
Type repositories
The holotype (SVC32 s4c) and 13 paratypes are at HKR, four paratypes (SVC32 s8) are in the collection of the SMNH, and eight paratypes (C4344 s12) in the Bertolani’s Collection of Unimore.
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).
Figure 5

Itaquascon magnussoni sp. nov. (a) In toto (ventro-lateral view), (b) Bucco-pharyngeal apparatus (dorso-ventral view from multiplanar images stack), (c) Claw II (lateral view from multiplanar images stack), (d) Claw VI (frontal view), (e) First section of the pharyngeal tube (3D dorsal reconstruction), (f) First section of the pharyngeal tube (drawn of dorsal view). White arrows: stylet supports; indented arrow: thickened bar in the pharynx; empty arrows: accessory points of the main claw branch; empty indented arrows: pseudolunules; arrowhead: cuticular bar under the claw. (a–c) Holotype. (a–d) LM, PhC; (e) CLSM; (f) schematic drawing. Scale bars (a) 100 µm; (b–d) 10 µm, (e,f) 5 µm.

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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.
Eggs unknown.
Differential diagnosis
Itaquascon magnussoni sp. nov. differs from all other Itaquascon species by having the stylet support inserted on the flexible pharyngeal tube. Considering the presence of the thickening within the pharynx, the most similar species of I. magnussoni sp. nov. are Itaquascon placophorum Maucci, 197347 and Itaquascon simplex (Mihelčič, 1971)48 (considered nomen dubium by Ramazzotti et al.49, thus not considered in this diagnosis).
Itaquascon magnussoni sp. nov. differs from I. placophorum by: the longer buccal tube with respect to the bucco-pharyngeal tube (buccal tube 16–17% of the bucco-pharyngeal tube in the holotype of I. placophorum and 48.0–48.4% in I. magnussoni), the longer thickening in the pharynx (calculated pt 31.3 in the holotype of I. placophorum; pt 44.8–63.5 in I. magnussoni), the claws with pseudolunules and evident accessory points on the main branch.
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.
Etymology
The species name is to honor Sven-Erik Magnusson, a sustainability visionary and leading person behind the development of Kristianstads Vattenrike Biosphere Reserve, and the first Coordinator of the Biosphere Reserve.
Thulinius gustavi sp. nov.
ZooBank: lsid:zoobank.org:act:149B5B73-580F-4BBE-BCB0-BCFD53E54EB5.
Type locality
Araslövssjön Lake, Näsby Fält (Skåne, Sweden). Upper layer of freshwater sediments in the bottom of the shore of the lake (56.059050 N, 14.135425 E), sample SVC31 (C4343 in Bertolani’s Collection).
Type repositories
The holotype (SVC31 s3b) and nine paratypes (SVC31 s2, s4, s5; SVC31b s2, s6, s8) are at HKR, one paratype (SVC31 s4) is in the collection of the SMNH, and one paratype (C4343 s1) in the Bertolani’s Collection of the Unimore. Two paratypes were mounted on stubs for SEM observation.
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).
Figure 6

Thulinius gustavi sp. nov. (a) In toto (dorso-lateral view), (b) Cuticular ornamentation (dorso-lateral view), (c) Bucco-pharyngeal apparatus (dorso-ventral view from multiplanar images stack), (d) Buccal armature (dorsal view), (e) Buccal armature (ventral view), (f) Stylet furca (frontal view), (g) Macroplacoids (frontal view from multiplanar images stack), (h) Claw I (lateral view), (i) Claw IV (frontal view), (j) In toto (lateral view), (k) Cuticular ornamentation (dorso-lateral), (l) Claw II (frontal-lateral view), (m) Claw IV (frontal view), (n) Bucco-pharyngeal apparatus (3D lateral reconstruction). Indented arrowheads: cuticular ornamentation; white arrowhead: peribuccal lamellae; empty indented arrowhead: posterior line of small round teeth; white arrow: second macroplacoid constriction; white indented arrows: accessory points of the main claw branch; empty arrow: pseudolunules; empty arrowheads: apophyses for the insertion of the stylet muscles. (a–c) Holotype. (a–f), (h,i). LM, PhC; (g) LM, DIC; (j–m) SEM; (n,o) CLSM; Scale bars (a) 100 µm; (b–j,n). 10 µm; (k) 2 µm; (l,m) 5 µm.

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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 knob-like 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).
Differential diagnosis
Thulinius gustavi sp. nov. differs from all other Thulinius species by having a dorso-lateral ornamented cuticle with tubercles. Considering the presence of pseudolunules only under the external (posterior, in claws IV) claws, the most similar species are Thulinius romanoi bertolani, bartels, Guidetti, Cesari & Nelson, 201450 and Thulinius saltarsus (Schuster, Toftner & Grigarick, 1978)51.
T. gustavi sp. nov. differs from:
T. romanoi by: the absence of ornamented cuticle in ventral side, the presence of eye-spots, the more narrow buccal tube (pt 21.9 and 13.4 in the holotype and a paratype of T. romanoi; pt 9.1–10.7 in T. gustavi), the stylet support inserted more posteriorly (pt 62.9 in the holotype of T. romanoi; pt 70.2–73.1 in T. gustavi), the macroplacoid length sequence (1  > 3  > 2 in T. romanoi), the shorter accessory points that never reach the end of the branch, and the presence of evident pseudolunules in claw IV;
T. saltarsus by: the dorso-lateral ornamented cuticle, presence of eye-spots, the macroplacoid length sequence (1  > 3  > 2 in T. saltarsus).
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.
Etymology
The species name has been chosen in honor of Gustav Thulin (1889–1945), the first internationally recognized Swedish tardigradologist, who made important contributions to the knowledge of the Swedish tardigrade fauna and to modern taxonomy and phylogenetics of tardigrades.
Faunistic results
The analysis of the 34 samples collected (33 terrestrial and one freshwater; see “Methods”) in the five sampled areas within the KVBR revealed the presence of 33 morphospecies belonging to 20 genera (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.
Table 3 Morphospecies identified with a morphological approach in the samples collected from five areas within Kristianstads Vattenrike.
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The highest densities of tardigrades (ind/g) were found in a lichen and a moss (Table 3, Supplementary Table S4). However, tardigrade densities in both lichens (five samples) and mosses (13 samples) were highly variable, ranging from 2.7 to 75.4 ind/g (mean: 27.6 ind/g, SD: 29.5, N = 5) in lichens, and from 0.2 to 66.4 ind/g (mean: 11.1 ind/g, SD: 17.9, N = 13) in mosses. In contrast, leaf litter (two samples) and soil with grass (three samples) were less abundant in animals and had a more homogeneous density: 0.1–1.9 ind/g (mean: 0.9 ind/g, SD: 0.68, N = 5; Table 3, Supplementary Table S4).
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; Table 3). Within this sample Macrobiotus polonicus Pilato, Kaczmarek, Michalczyk & Lisi, 200352 and Macrobiotus wandae Kayastha, Berdi, Mioduchowska, Gawlak, Łukasiewicz, Gołdyn, & Kaczmarek, 202053 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; GenBank 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 Table S3). Macrobiotus cf. polonicus differs morphologically from M. polonicus by the presence 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, 200655 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, 197257 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). More

In natural ecosystems, the efficiency of energy transfer from resources to consumers determines the biomass structure of food webs. As a general rule, about 10% of the energy produced in one trophic level makes it up to the next1–3. Recent theory suggests this energy transfer could be further constrained if rising temperatures increase metabolic growth costs4, although experimental confirmation in whole ecosystems is lacking. We quantified nitrogen transfer efficiency (a proxy for overall energy transfer) in freshwater plankton in artificial ponds exposed to 7 years of experimental warming. We provide the first direct experimental evidence that, relative to ambient conditions, 4 °C of warming can decrease trophic transfer efficiency by up to 56%. In addition, both phytoplankton and zooplankton biomass were lower in the warmed ponds, indicating major shifts in energy uptake, transformation and transfer5,6. These new findings reconcile observed warming-driven changes in individual-level growth costs and carbon-use efficiency across diverse taxa4,7–10 with increases in the ratio of total respiration to gross primary production at the ecosystem level11–13. Our results imply that an increasing proportion of the carbon fixed by photosynthesis will be lost to the atmosphere as the planet warms, impairing energy flux through food chains, with negative implications for larger consumers and the functioning of entire ecosystems. More

Evolution of ACE2 in bats inhabiting urban or rural areas
We collected ACE2 orthologues from 46 bat species across the phylogeny (Fig. 1 and Supplementary Table 1). These species contained 28 species that roost or forage in urban areas near humans and 18 species more restricted to rural areas and hence likely to have minimal contact with humans (Supplementary Table 2). In total, we examined 46 species representing 11 bat families (Supplementary Table 3). After aligning the protein sequences of bat ACE2 orthologues, we examined 25 critical residues involved in the binding of the surface spike glycoprotein (S protein) of SARS-CoV-2 (ref. 9) (Extended Data Fig. 1). Genetic variations were observed in nearly all these 25 sites, which may have led to different abilities to support entry of SARS-CoV and SARS-CoV-2 (ref. 9). Furthermore, we detected at least 22 amino acid sites that are putatively under positive selection (Supplementary Table 4), which is indicative of heterogeneous selection pressure across sites. Notably, four of these positively selected sites are in the binding region of ACE2 to the SARS-CoV-2 S protein (Supplementary Table 4).
Fig. 1: Phylogenetic tree of 46 bat species in this study.

The labels of bat species in our experiments are indicated. Expression levels determined by western blot (Fig. 2a) are shown with asterisk symbols compared with human ACE2: the triple asterisk indicates high expression, the double asterisk indicates medium expression and the single asterisk indicates low but detectable expression. The ability of bat ACE2 to support SARS-CoV and SARS-CoV-2 pseudovirus entry is shown with different signs (Fig. 3a,b): infection data are presented as percentage mean values of bat ACE2 supporting infection compared with the infection supported by human ACE2. Infection efficiency 50% with a double plus sign. Bat phylogeny was taken from previous studies28,29,30.

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Interaction between bat ACE2 orthologues and SARS-CoV or SARS-CoV-2 receptor binding domain
Efficient binding between the S protein and the ACE2 receptor is essential for SARS-CoV and SARS-CoV-2 entry. This binding is mainly mediated by the interaction between the critical residues on the receptor-binding domain (RBD) and ACE2. To characterize the receptor function of ACE2 orthologues in a range of diverse bat species, we generated a stable cell library consisting of cell lines expressing the respective 46 bat ACE2 orthologues through lentiviral transduction of 293T cells lacking ACE2 expression10. All bat ACE2 orthologues were exogenously expressed at a comparable level after puromycin selection, as indicated by western blot and immunofluorescence assays detecting the C-terminal 3×FLAG-tag (Fig. 2a,b).
Fig. 2: Expression of bat ACE2 orthologues and their interaction with the SARS-CoV and SARS-CoV-2 RBD.

a, Western blot detected the expression levels of ACE2 orthologues on 293T stable cells by targeting the C-terminal 3×FLAG-tag. Glyceraldehyde 3-phosphate dehydrogenase was employed as a loading control. b, Visualization of the intracellular bat ACE2 expression level by immunofluorescence assay detecting the C-terminal 3×FLAG-tag. Scale bar, 100 μm. c,d, Assessment of the interaction between different ACE2 orthologues and SARS-CoV-RBD-hFc (c) or SARS-CoV-2-RBD-hFc (d) proteins. Species that do not support efficient binding are underlined. 293T cells stably expressing the different bat ACE2 orthologues were incubated with 5 μg ml−1 of the recombinant proteins at 37 °C for 1 h; binding efficiency was examined by Alexa Fluor 488 goat anti-human IgG via fluorescence assay. Scale bar, 200 μm.

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To analyse the interaction, we produced recombinant SARS-CoV or SARS-CoV-2 RBD human immunoglobulin G (IgG) Fc fusion proteins (RBD-hFc), previously reported to be sufficient to bind human ACE2 efficiently11,12. Protein binding efficiency was tested on the bat ACE2 cell library by means of immunofluorescence or flow cytometry targeting the human Fc. As expected, binding was almost undetectable on mock 293T cells but a strong binding signal was detected in the 293T cells expressing human ACE2 (Fig. 2c,d). Consistent with previous reports13,14, SARS-CoV-2 RBD showed higher binding to human ACE2 than SARS-CoV, which can also be observed on many bat ACE2 orthologues (Fig. 2c,d). Previous reports have shown that only a small fraction of ACE2 orthologues from tested mammalian species could not bind with SARS-CoV-2 S protein (n = 6 of 49 species7; n = 5 of 17 species15). However, our study revealed that many bat species (n = 32 and n = 28 of 46 species) do not support efficient binding with SARS-CoV-RBD and SARS-CoV-2-RBD, respectively (Fig. 2c,d). The overall profiles of bat ACE2 to bind to SARS-CoV and SARS-CoV-2 RBD are generally comparable; a few showed contrasting modes of binding preferences (Fig. 2c,d). For instance, Bat22 could bind to SARS-CoV but not SARS-CoV-2, whereas Bat14, 21 and 40 could bind to SARS-CoV-2 but not SARS-CoV (Fig. 2c,d). Flow cytometry analysis showed consistent results (Extended Data Fig. 2).
Overall, the RBD-hFc binding assays demonstrated that bat ACE2 orthologues showed different affinity and selectivity levels to SARS-CoV and SARS-CoV-2, indicating that the ACE2 receptors of many bat species may not support efficient SARS-CoV and SARS-CoV-2 infection.
Receptor function of bat ACE2 orthologues to support the entry of SARS-CoV and SARS-CoV-2 using pseudotyped and live viruses
To further evaluate the receptor function of different bat ACE2 orthologues, we employed a vesicular stomatitis virus (VSV)-based rhabdoviral pseudotyping system to mimic the coronavirus spike protein-mediated single-round entry15. SARS-CoV and SARS-CoV-2 pseudotypes were generated by assembling the coronavirus spike proteins and replication-deficient VSV with the VSV glycoprotein gene replaced with a fluorescence protein (VSV-dG-GFP) or a firefly luciferase (VSV-dG-Luc) reporter15. Both viruses showed minimal background infection on 293T cells, but efficient infection on 293T-human ACE2 cells (Extended Data Fig. 3). The susceptibility of the 293T cells expressing bat ACE2 orthologues was then examined with SARS-CoV and SARS-CoV-2 pseudotypes. The results showed that bat ACE2 orthologues have varying abilities to support coronavirus entry and different preferences for SARS-CoV and SARS-CoV-2. (Fig. 3a,b and Extended Data Fig. 4). Pseudotypes with green fluorescent protein (GFP) reporter showed similar results (Extended Data Fig. 5). Notably, we found that 24, 21 and 16 of the 46 bat species showed almost no entry for SARS-CoV, SARS-CoV-2 and both viruses, respectively (Figs. 1 and 3a,b and Supplementary Table 5), suggesting that these species are not likely to be potential hosts of either or both coronaviruses. The bat species showing no viral entry include those that occur in urban areas and those more restricted to rural areas (Fig. 1), suggesting that there is no correlation between proximity to humans and probability of being natural hosts of SARS-CoV or SARS-CoV-2. Although horseshoe bats were suggested as potential natural hosts of SARS-CoV and SARS-CoV-2 (refs. 1,2,3), only one of the three species examined (Rhinolophus sinicus) supported SARS-CoV entry; this species was suggested as the potential host of SARS-CoV3,16. None of these tested horseshoe bats showed entry for SARS-CoV-2 (Figs. 1 and 3). These results unambiguously indicate that ACE2 receptor usage is species-dependent.
Fig. 3: Characterization of bat ACE2 orthologues mediating entry of SARS-CoV and SARS-CoV-2 viruses.

a,b, Ability of bat ACE2 orthologues to support the entry of SARS-CoV and SARS-CoV-2 pseudovirus. 293T cells expressing bat ACE2 orthologues in a 96-well plate were infected with VSV-dG-Luc pseudotyped with SARS-CoV (a) and SARS-CoV-2 (b) spike proteins, respectively. Intracellular luciferase activity was determined at 20 h post-infection. RLU, relative light unit. c, 293T cells expressing bat ACE2 orthologues were inoculated with the SARS-CoV-2 live virus at an MOI = 0.01. N protein (red) in the infected cells was detected through immunofluorescence assay at 48 h post-infection. Scale bar, 200 μm. Samples expressing the indicated ACE2 orthologues that showed almost no entry for SARS-CoV-2 live virus are underlined. Data shown are representative results from 3 independent experiments and are presented as the mean ± s.d. (n = 3 for a and n = 2 for b).

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The SARS-CoV-2 S protein used in this study for pseudotyping contains a D614G mutation, which is currently a dominant variation17. The D614G mutation remarkably improved the in vitro infectivity of SARS-CoV-2 but may not significantly affect the receptor interaction since it is not in the RBD18. Indeed, we identified a very similar susceptibility profile using an original strain without D614G (Extended Data Fig. 4). We further demonstrated that the pseudotyped entry assay mimics the entry of live viruses through a SARS-CoV-2 infection assay (Fig. 3c). As expected, the profile of SARS-CoV-2 N protein expression is highly consistent with the results from the VSV-dG-based pseudotyped virus entry assay, except for some ACE2 that showed relatively higher infection efficiency (for example, Bat43–46) compared with the pseudovirus infection assay, which may be attributed to the different virus strains used (Fig. 3c). In addition, the live virus infection resulted in the phenotype of plaque formation, while the pseudotypes showed evenly distributed, single-round infection (Extended Data Fig. 5), which also partially explains why some bat ACE2 showed higher infection in the live virus infection assay.
When comparing the RBD-hFc binding and pseudotyped entry profiles, we found that binding and susceptibility are not always consistent, although the phenotypes were reproducible. For instance, some species (Bat12, 13, 14) were able to bind to SARS-CoV-2 RBD-hFc efficiently but could not support infection of the same virus, indicating that high binding affinity does not guarantee efficient viral entry (Figs. 2 and 3). In contrast, some species (Bat3–8) were defective or less efficient in SARS-CoV RBD-hFc binding but supported the entry of the same virus to some degree (Figs. 2 and 3). We hypothesize that such minimal binding may be sufficient for viral entry mediated by those ACE2 orthologues; alternatively, additional residues outside the traditional RBD region might be required for efficient interaction. These hypotheses should be tested in the future. Together, our results demonstrated dramatic variation of susceptibility to SARS-CoV and SARS-CoV-2 infection among bat species, suggesting that SARS-CoV and SARS-CoV-2 can selectively use some bat ACE2 as functional receptors for viral entry and many—if not most—bat ACE2 are not favoured by one or both viruses.
Evaluation of critical residues in bat ACE2 orthologues affecting viral binding and entry efficiency or specificity
We comprehensively analysed the relationship between critical RBD binding sites in bat ACE2 sequences and their ability to support SARS-CoV and SARS-CoV-2 RBD binding and viral entry. Several critical residues were identified that may play critical roles in the determination of species specificity (Extended Data Fig. 1). According to the sequence alignment, two species pairs (Bat33 and Bat34 and Bat38 and Bat40) were selected to demonstrate the role of critical residues in RBD binding and viral entry because they were phylogenetically close but showed contrasting phenotypes for supporting RBD binding and viral entry. Specifically, Bat34 and Bat38 do not support SARS-CoV and SARS-CoV-2 RBD binding and infection, while Bat33 supports efficient binding and infection of both viruses and Bat40 supports infection of both viruses and to a lesser degree SARS-RBD binding (Figs. 2 and 3). We compared their protein sequences and highlighted the residues that may affect RBD interaction. For example, substitutions I27K, N31G and K42E were observed when comparing Bat33 with Bat34, while Q24L, E30K, K35Q and G354N were present between Bat38 and Bat40 (Fig. 4a). We hypothesized that the discrepancy in binding and infection phenotype is determined by their differences in critical residues for RBD interaction. To test this hypothesis, we designed a residue swap mutagenesis assay to investigate the role of critical residues on RBD binding and virus entry (Fig. 4a). We generated four swap mutations and corresponding 293T stable cell lines to test whether these substitutions could achieve gain-of-function and loss-of-function. All bat ACE2 orthologues and related mutants were expressed at a comparable level after lentiviral transduction, as indicated by the immunofluorescence of the C-terminal 3×FLAG-tag (Fig. 4b). Recombinant SARS-CoV and SARS-CoV-2 RBD-hFc proteins were applied to the cells expressing different ACE2 and binding efficiency was evaluated by immunofluorescence (Fig. 4c) and flow cytometry assays (Fig. 4d). As expected, the swap of critical residues on the selected four bat ACE2 changed their receptor function to the opposite, except for Bat38 mutant, which remained unable to bind SARS-CoV RBD-hFc (Fig. 4c,d). GFP (Fig. 4e) and luciferase levels (Fig. 4f) from the pseudotyped virus entry assay and the N protein staining from the live SARS-CoV-2 infection assay (Fig. 4g) further confirmed our hypothesis at the viral entry level. Structure modelling of bat ACE2/SARS-CoV-2-RBD complexes showed that the substitutions of I27K and N31G between Bat33 and Bat34 lead to a reduced packing interaction and the substitution of K42E disrupts the hydrogen bond with Y449, which may be related to the difference of susceptibility between Bat33 and Bat34 (Fig. 4h,i and Extended Data Fig. 6). In comparison, the substitutions of Q24L and E30K between Bat38 and Bat40 destroyed the favourable hydrophilic interactions with N487 and K417, respectively (Extended Data Fig. 6).
Fig. 4: Evaluation of the critical binding sites determining the species-specific restriction of SARS-CoV and SARS-CoV-2 binding and entry.

a, Swap mutagenesis assay to investigate the role of critical residues on bat ACE2 orthologues for tropism determination. Residues involved in RBD (according to the structure between SARS2-RBD and human ACE2, Protein Data Bank 6M0J) interaction are shown in the table. Residues that changed in the mutagenesis assay are marked in red. b, The expression level of the bat ACE2 orthologues and related mutants in transduced 293T cells was determined by an immunofluorescence assay recognizing the 3×FLAG-tag. Scale bar, 200 μm. c,d, Binding efficiency of SARS2-RBD-hFc and SARS2-RBD-hFc on 293T cells expressing bat ACE2 and related mutants. Cells were incubated with 5 μg ml−1 of recombinant proteins at 37 °C for 1 h and then washed and incubated with a secondary antibody recognizing human Fc. Immunostaining (c) and flow cytometry (d) were conducted to show binding efficiency. Scale bar, 200 μm. e,f, Ability of the indicated ACE2 and related mutants to support the entry of coronavirus pseudotypes. The 293T cells expressing the indicated ACE2 and their mutants were infected with SARS-CoV and SARS-CoV-2 pseudotypes expressing GFP (e) and luciferase (f). Infection was analysed at 20 h post-infection. Scale bar, 200 μm. Data are presented as the mean with s.d. (n = 2). g, 293T cells infected by the SARS-CoV-2 live virus at an MOI = 0.01; the infection was examined at 48 h post-infection through N protein (red) immunostaining. Nuclei were stained with Hoechst 33342 (blue). Scale bar, 200 μm. h,i, Comparison of the interface between Bat33/SARS-CoV-2-RBD and Bat34/SARS-CoV-2-RBD. Bat33 and its complexed RBD are coloured cyan and gold, respectively (h); Bat34 and its complexed RBD are coloured wheat and green, respectively (i). The mutated residues in ACE2 and the corresponding residues in SARS-CoV-2-RBD are shown and labelled. The red dotted lines between residues indicate hydrogen or ionic bonds.

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In addition, two bat cell lines, the lung epithelial cell line Tb 1 Lu of Tadarida brasiliensis (Bat31) and the kidney epithelial cell line of Pteropus alecto (Bat2), were used to validate our findings derived from human HEK293T cells. Endogenous ACE2 expression was almost undetectable in these two cell lines, accounting for at least 1,000 folds lower than the susceptible Vero-E6 cells (Extended Data Fig. 7a). Therefore, these cells cannot support the entry of SARS-CoV and SARS-CoV-2. We successfully generated Tb 1 Lu stable cell lines expressing human ACE2 and bat ACE2 (Bat2, 3, 31, 32) since the transduction efficiency of Tb 1 Lu is much higher than that of PakiT03 cells (Extended Data Fig. 7b). As expected, Tu 1 Lu were susceptible to both SARS-CoV and SARS-CoV-2 when human ACE2 or some bat ACE2 orthologues (Bat2, 3 and 31) were expressed, yet remained non-susceptible when an ACE2 of a closely related species (Bat32) was expressed (Extended Data Fig. 7c–e). Furthermore, we conducted SARS-CoV and SARS-CoV-2 pseudovirus entry assays on the two bat cell lines transiently transfected with various bat ACE2 (Bat2, 3, 31, 32, 33, 34, 38, 40) and their mutants (mutant Bat33, 34, 38 and 40m). The results were consistent with those derived from human cells, further confirming that ACE2 is the main receptor for the species-specific entry of SARS-CoV and SARS-CoV-2 in these bat cells (Extended Data Fig. 7f,g). More

Natural soundscapes have always called to me. As an eco- and electro-acoustics researcher, with a background in sound engineering and electronic music composition, I have always tried to strike a balance between art and science in my work.
In 1998, when I first heard about the extinction crisis — more than 35,500 species of flora and fauna are endangered — the idea for the Fragments of Extinction project came to me very quickly. My vision was to build a collection of 24-hour-long ‘acoustic fragments’, recorded at the highest definition possible, capturing the sonic heritage of ancient, biodiverse, untouched tropical rainforests — before climate change damages them irreversibly.
In these forests, some species vocalize from the canopy, some from the ground and others from big tree trunks that act like sound diffusers. To capture a 3D acoustic portrait of the forest, we simultaneously record on 38 audio channels and microphones.
In this photograph, I am standing in the Sonosfera, a geodesic theatre in Pesaro, Italy, in which audiences can experience rainforest soundscapes captured in the Amazon, Africa and Borneo. Forty-five high-definition loudspeakers are positioned in an isolated, acoustically perfect space, realistically reproducing the ecosystems’ natural sounds.
For the first 15 minutes of the performance, the Sonosfera is completely dark. Sound helps listeners to ‘build’ the forest space around them — the position of every insect and amphibian; the birds and mammals moving through the canopy. My team then projects the spectrograms shown here to explain the sounds, and present data showing that these ecosystems are disappearing.
We have captured the deep infrasound calls of elephants and have recorded insects that sound exactly like violins or trumpets. Our ecosystem recordings are very different. But I don’t have a favourite — they’re a collection. More

Genome sequencing and assembly
We performed a de novo genome sequencing and assembly of CBB using a hybrid approach by combining 454-FLX and Illumina reads from female and male individuals. A total of 3.02 Gb of high-quality 454-FLX sequences and 26 Gb of Illumina sequences were obtained in this study (Table S1), which represent approximate 19 × and 160 × genome coverage respectively based on a previously estimated CBB genome size of 163Mb21. The genome hybrid assembly approach we used involved an initial pre-assembly of the 454FLX data with Newbler and the Illumina data with ABySS22, followed by merging of these two pre-assemblies into a single genome consensus with Metassembler23. Our final hybrid H. hampei CENICAFE_Hham1.1 (Hham1.1) genome assembly had a size of 162.57 Mb, comprising 8198 genome scaffolds (Table 1). This assembly represents an improvement in sequence contiguity, containing a 36.3-Kb contig-N50; 340.2-Kb scaffold-N50 and 4.9 Mb for the largest genome scaffold, compared with a previously published CBB genome assembly21, which resulted in contig and scaffold N50 of 10.5-Kb and 44.7-Kb respectively and largest genome scaffold of 440-Kb. The Hham1.1 genome assembly completeness was assessed using Benchmarking Universal Single-Copy Orthologs (BUSCO)24. BUSCO recovered 98.22% of the 1066 Arthropoda core gene set, from which 96.25% were complete genes and 2% were fragmented genes (Fig. S1). BUSCO results indicate that almost the entire genome of H. hampei was sequenced and de novo assembled in this study.
Table 1 Hypothenemus hampei genome assembly (CENICAFE_Hham1.1) statistics.
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Transcriptome assembly
Illumina RNA-seq data obtained from whole-body female and male adults were de novo assembled using rnaSPades25 and sequence redundancy reduced by CD-HIT26. The resulting transcript assembly was composed of 64,244 contigs (available at NCBI TSA accession: GIPB00000000.1). The average transcript length was 1103-bp, transcript N50 of 2145-bp and largest transcript of 26,019-bp. The transcript assembly completeness with BUSCO recovered 99.6% (98.97% completed and 0.65% fragmented genes) of the 1066 Arthropoda core gene set. (Fig. S1). Using TransDecoder27, we extracted 35,558 protein-encoding transcripts with full Open Reading Frames (ORFs), from which 33,378 (95%) were annotated against InterPro and NCBI NR proteins. As expected, top BLAST hits were against the Coleoptera species, including D. ponderosae (61%) Sitophilus orizae (22%), Anoplophora glabripennis (3%) and Tribolium castaneum (5.7%); whereas the remaining hits were against other insect species (14%).
Gene prediction and functional assignations
We identified 18,765 gene models encoding 20,801 proteins on the Hham1.1 genome assembly using BRAKER2 gene predictor and all available RNA-seq evidence for H. hampei at NCBI. The number of gene models found here for our Hham1.1 assembly is slightly smaller than the previous gene prediction (19,222) performed on the first published H. hampei genome draft21. Completeness of the Hham1.1 gene set using BUSCO recovered 97.2% (94.1% completed and 3.1% fragmented genes) of the Arthropoda core gene set (Fig. S1). BLASTP found 18,364 (88.3%) Hham1.1 predicted proteins similar (e-value  More

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Overview of the GutCP algorithm
Our approach uses the idea that we can leverage cross-feeding interactions—which comprise knowing the metabolites that each microbial species is capable of consuming and producing—to mechanistically connect the levels of microbes and metabolites in the human gut. Several different mechanistic models in past studies have shown that this is indeed possible18,20,29,36,37. While GutCP is generalizable and can be used with any of these models, in this paper, we use a previously published consumer-resource model20. We use this model because of its context and performance: it is built specifically for the human gut and is best able to explain the experimentally measured species composition of the gut microbiome with its resulting metabolic environment, or fecal metabolome (compared with other state-of-the-art methods, such as ref. 29). To predict the metabolome from the microbiome, it relies on a manually curated set of known cross-feeding interactions9. It then uses these known interactions to follow the stepwise flow of metabolites through the gut. At each step (ecologically, at each trophic level), the metabolites available to the gut are utilized by microbial species that are capable of consuming them, and a fraction of these metabolites are secreted as metabolic byproducts. These byproducts are then available for consumption by another set of species in the next trophic level. After several such steps, the metabolites that are left unconsumed constitute the fecal metabolome.
We hypothesized that adding new, yet-undiscovered cross-feeding interactions would improve our ability to predict the levels of metabolites with our mechanistic and causal model. Specifically, we predict that the set of undiscovered interactions resulting in the most accurate and optimal improvement in predictions would be the most likely candidates for true cross-feeding interactions. Inferring such an optimal set of new cross-feeding interactions or reactions is the main logic driving GutCP. In what follows, we sometimes refer to cross-feeding reactions (i.e., metabolite consumption or production by microbes) as “links” in an overall cross-feeding network of the gut microbiome, whose nodes are microbes and metabolites (Fig. 1a; metabolites in blue, microbes in orange); the links themselves are directed edges connecting the nodes. Links can be of two types: consumption or nutrient uptake reactions (from nutrients to microbes) and production or nutrient secretion reactions (from microbes to their metabolic byproducts).
Fig. 1: Overview of the GutCP algorithm.

a Schematic of the original set of known cross-feeding interactions (top) and bar plot of the prediction error for each metabolite and microbe (bottom). The cross-feeding interactions are represented as a network, whose nodes are either metabolites (cyan circles) or microbial species (orange ellipses), and directed links represent the abilities of different species to consume (red arrows) and produce (blue arrows) individual metabolites. b GutCP adds a new consumption link (red) and production link (blue) as added links reduce the prediction errors for metabolites and microbes.

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The salient aspects of our method are outlined in Fig. 1. We start with the known set of consumption and production links that were originally used by the model; these links are known from direct experiments and represent a ground-truth dataset or original cross-feeding network9. These are shown in Fig. 1a through the pink and blue arrows connecting nutrients 1 through 6 with microbes (a) through (c). For each sample, using only the species abundance from the microbiome, we use the model to quantitatively estimate the microbiome’s species and metabolomic composition. Briefly, we assume that a defined set of polysaccharides, common to human diets, are available as the nutrient intake to the gut (nutrients 1 and 4 in Fig. 1a). We calculate the microbiome and metabolome profiles separately for each individual, which contain a different set of microbial species in their guts. At the first trophic level, all microbial species that are capable of using the polysaccharides (indicated by the pink arrows in Fig. 1a) consume each of them in proportion to their abundances (microbes a, b, and c in Fig. 1a). They subsequently secrete a fixed fraction of the consumed nutrients as metabolic byproducts; every species at this trophic level secretes all the metabolic byproducts it is known to secrete (blue arrows in Fig. 1a) in equal proportion (nutrients 2–6 in Fig. 1a). At the next trophic level, all species detected in the individual’s gut which can consume the newly secreted byproducts consume them as nutrients, secreting a new set of byproducts, and this continues for four trophic levels (not shown in Fig. 1a for simplicity). At the end of this process, all metabolites which remain unconsumed by the community comprise the metabolome of the individual and the microbial species which consume nutrients and grow comprise the microbiome of the individual (for a complete description, see “Methods” and previous work20).
For each metabolite and microbial species, there can be two kinds of prediction errors, or biases: individual (the sample-specific difference between predicted and measured levels) and systematic (average difference across all samples). We focused on the “systematic bias” for each metabolite and microbial species: the average deviation of the predicted levels from the measured levels across all samples in our dataset (Fig. 1a, bottom). The systematic bias for each metabolite and microbe tells us whether our model generally tends to predict their level to be greater than observed (overpredicted), less than observed (underpredicted), or neither (well-predicted). We assume that metabolites and microbes with a large systematic bias are most likely to harbor missing consumption or production links that are relevant across many samples. We prioritize adding links to them in proportion to their systematic biases.
After measuring the systematic bias for each metabolite and microbe, GutCP proceeds in discrete steps (Fig. 1a, b). At each step, we attempt to add a new link to the current cross-feeding network. This new link is chosen randomly from the entire set of combinatorially possible links (see “Methods”; for S species, M metabolites, and two kinds of links (consumption and production), there are a total of 2SM combinatorially possible links). We accept this link—keeping it in the current network—if it leads to an overall improvement in the agreement between the predicted and measured levels of microbes and metabolites. We repeat the process of adding new links—accepting or rejecting them—until the improvements in the levels of metabolites and microbes became insignificant. Overall, GutCP can add several links to improve the agreement between the predicted and measured levels of microbes and metabolites (in Fig. 1a, b, bottom, adding the extra red and blue link at the top results in improved predictions for metabolite (1), metabolite (3), and microbe (b). Figure 2a shows how the cross-feeding network improves over a typical GutCP run via the red trajectory, starting from the original network (Fig. 2a, top left) to the final network state (Fig. 2a, bottom right). Trajectories from 100 other runs are shown in gray. GutCP repeatably reduces both the error of the metabolome predictions (y axis; measured as ({text{log}}_{10}(frac{,text{pred}-text{meas}}{text{measurement},}))) and improves the correlation between the predicted and measured metabolomes (x axis).
Fig. 2: Improvement in predictions using GutCP.

a Improvement in log error (({text{log}}_{10}(frac{,text{pred}-text{meas}}{text{measurement},}))) and the correlation between the prediction and measured fecal metabolome during 100 typical runs of the GutCP algorithm. The gray point at the top left indicates the performance of the original cross-feeding network of Ref. 9, and the black points at the bottom right, that of improved networks predicted using GutCP. A trajectory example, highlighting how performance improves over a GutCP run, is shown in red, and others are shown in gray. b Rarefaction curve showing the number of unique cross-feeding interactions discovered by GutCP over 100 runs of the algorithm. c Prevalence of links, i.e., the number of GutCP runs in which they repeatedly appeared (red dots; total 100 runs) and for comparison, a corresponding binomial distribution with the same mean (black dotted line). P values for different prevalences are estimated using the one-sided binomial test.

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Cross-validating the newly predicted interactions
To test if the cross-feeding interactions predicted by GutCP are generalizable to unknown datasets, we performed fourfold cross-validation. We used a sample -omics dataset of the gut microbiome and metabolome sampled from 41 human individuals, comprising 221 metabolites and 72 microbial species (data from ref. 38). We split our -omics dataset into two subsets: training (three-fourths of the individuals) and test (one-fourth of the individuals) subsets. We then ran GutCP on the training subset to discover new interactions and added them to the ground-truth interactions taken from ref. 9. Doing so resulted in a network of cross-feeding interactions learned only from the training subset of the data. Finally, we evaluated the improvement in accuracy of metabolome predictions resulting from the trained network on the unseen, test subset of the data. We repeated this process three times, each time splitting the full dataset into a training subset (with a randomly chosen three-fourths of the individuals) and test subset (with the remaining one-fourth of the individuals); finally, we calculated the average improvement in prediction accuracy over all four splits.
We found that both the training and test set performances after using the links predicted by GutCP were significantly better than the baseline given by the original cross-feeding network (Table 1). Specifically, both measures of model performance, namely the logarithmic error and the average correlation, improved by 64% and 20%, respectively, after adding GutCP’s discovered interactions. In addition, the test set performance was comparable to the training set performance (6% difference; Table 1). This suggests that the cross-feeding interactions inferred by GutCP are not likely to be a result of over-fitting.
Table 1 Cross-validating the newly predicted interactions.
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Building a consensus-based atlas of predicted cross-feeding interactions
Having confirmed that GutCP is unlikely to over-fit data, we pooled the entire sample dataset of 41 individuals and ran 100 independent instances of our prediction algorithm on it; we verified that incorporating more instances did not qualitatively affect our results (Fig. 2b shows a rarefaction curve, which highlights the number of new links discovered by GutCP as we perform more runs the algorithm). Each run of the algorithm resulted in an average of 140 newly predicted cross-feeding interactions. Then, based on consensus from many runs, we assigned a confidence level to each predicted interaction, namely what fraction of GutCP runs it was discovered in. By calculating a null distribution (Fig. 2c, black), which predicts the fraction of GutCP runs where a random link would be discovered by chance, we assigned a P value to each link and set a threshold at P = 10−3 (Fig. 3c, red; see “Methods” for details). Doing so finally resulted in a complete consensus-based atlas of 293 predicted cross-feeding interactions, which we have provided as a resource for experimental verification in Supplementary Table 1. Figure 3a shows a condensed version of these interactions obtained from the simulation with the best performance (the trajectory example in Fig. 2a with the lowest log error and highest correlation coefficient) in the form of a matrix; specifically, newly added interactions are in dark colors, and old interactions in faded colors. Supplementary Fig. 3 shows a complete version of this matrix. Note that some of the predicted interactions in Fig. 3a are unrealistic, e.g., the production of certain sugars like D-Fructose and D-Sorbitol. Such interactions are unlikely to be predicted in repeated simulations, and thus will not be part of the final consensus set. This illustrates the power of pooling results from several simulations to arrive at a set of highly probable predictions.
Fig. 3: New cross-feeding interactions predicted by GutCP.

a Concise matrix representation of the improved cross-feeding network of the gut microbiome predicted by GutCP (the trajectory example in Fig. 2a with the best performance). The rows are metabolites, and columns, microbial species. Faded cells represent the original, known set of cross-feeding interactions, both production (light blue), consumption (light red), and bidirectional links (gray). The new cross-feeding interactions predicted by GutCP are shown in dark colors: production links in dark blue, consumption links in dark red, and bidirectional links in black. b Network of 293 new links predicted by GutCP (with a P value  More