Philippa Kaur

Screening of solvent and adjuvant
The results of solvent screening are shown in Table 1. The original pesticide could not be completely dissolved using a single solvent. However, 5% N-methyl-2-pyrrolidone + 10% cyclohexanone could completely dissolve the original pesticide. There was no solid precipitation at room temperature, so the formulation could be used for the subsequent experiment. According to Table 2, a mixture of sulfonate adjuvants (70b) and fatty alcohol polyoxyethylene ether adjuvants (AEO-4, -5, -7, -9, 992) could stabilize the system in a single, transparent, homogeneous phase. Therefore, sulfonate adjuvant (70b) was selected and mixed with five adjuvants of the AEO series to prepare thidiazuron·diuron ultra-low-volume sprays, numbered 1–5 (as shown in Table 3).
Table 1 Selection of solvent type and dosage (%: mass fraction).
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Table 2 Selection of adjuvants type and dosage (%: mass fraction).
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Table 3 Ultra-low-volume formulations used in this study.
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Surface tension measurement
The critical surface tension of cotton leaves is 63.30–71.81 mN/m. Figure 1 shows that the surface tension of each sample was 31.67–33.37 mN/m, which was much lower than the critical surface tension of the leaf, indicating the agent was able to completely wet the leaf and be fully distributed on the leaf surface. The maximum surface tension of the reference product was 38.90 mN/m. Under the same dosage of adjuvant, sample 5 with adjuvant 992 had the smallest surface tension of 31.67 mN/m.
Figure 1

Surface tensions of different samples. Different letters (a–d) indicate significant differences between means. Means followed by the same letter are not significant at the 5% significance level by the LSD test (LSD = 0.05). Vertical bars indicate a standard deviation of the mean. The detailed data of the histogram is shown in Supplementary Table S1.

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Contact angle measurement
According to Young’s equation, the smaller the surface tension, the smaller the contact angle40,41. Figure 2 shows the contact angle of different samples on cotton leaves and the change in contact angle over time. The contact angles of oil agents containing the adjuvant 992, AEO-7 and AEO-9 were smaller than that of the reference product, and the spreading effect was superior to that of the reference product. In the surface tension test, sample 5 had the smallest surface tension of 31.67 mN/m; this sample showed the minimum initial contact angle (39°) and a static contact angle (22°). The surface tension of the reference product was 38.90 mN/m., with the maximum initial contact angle (65.5°). Therefore, the relationship between surface tension and contact angle conformed to Young’s equation.
Figure 2

Contact angles of different samples on cotton leaves in 0–10 s. The detailed data of drawing the contact Angle curve is shown in Supplementary Table S2.

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Volatilization rate measurement
As shown in Fig. 3, the volatilization rate of the oil agent was much lower than that of the reference product. The volatilization rate of the five treatments was 5.80–8.74%, while the volatilization rate of the reference product was 22.97%. The volatilization rate of the oil agent met the quality requirements of an ultra-low-volume spray (≤ 30%). A low volatilization rate helps with spraying defoliants in hot and dry areas such as Xinjiang, effectively preventing evaporation of the droplets and increasing deposition.
Figure 3

Volatilization of different samples on filter paper. Different letters (a–e) indicate significant differences between means. Means followed by the same letter are not significant at the 5% significance level by the LSD test (LSD = 0.05). Vertical bars indicate a standard deviation of the mean. The detailed data of the histogram is shown in Supplementary Table S3.

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Viscosity measurement
Viscosity is an important factor affecting the atomization performance of a formulation42. Figure 4 shows that the viscosity of the five oil agents ranged from 12.9 to 18.3 mPa s, meeting the quality requirements of an ultra-low-volume spray ( 20 V), the droplet size distribution tended to be stable. This coincided with data shown in Fig. 6, where the inflection point appeared when rotation speed was 9600 rpm (voltage = 20 V).
Figure 6

Relationship between the rotation speed of the centrifugal spray atomizer and droplet size. D10: 10% cumulative volume diameter, D50: 50% cumulative volume diameter, D90: 90% cumulative volume diameter. The detailed data of drawing the curve is shown in Supplementary Table S6.

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Figure 7

Relationship between the rotation speed of the centrifugal spray atomizer and the fog droplet spectrum. The detailed data of drawing the curve is shown in Supplementary Table S6.

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Therefore, we determined that the optimal working conditions for the rotary atomizer were achieved by setting the DC voltage stabilized power supply current to 1.00 A and voltage to 20 V, which were used for subsequent experiments.
Atomization performance
The relationship between viscosity and droplet spectrum are shown in Table 4 and Fig. 8. The cumulative volume diameter for the five treatments was less than 150 μm meeting the requirements of the ULV spray32. The cumulative volume diameter for the five treatments was larger than that for the reference product, the width of the droplet spectrum was narrower, and the droplet distribution was more uniform. Droplet size affects the drift of droplets43. The D10 of the reference product was 25.62 μm under these working conditions. This droplet size was highly susceptible to drift and deposition on non-target organisms. Water suspension was not suitable for this application at low dosage.
Table 4 Droplet size and droplet size distribution of different sample sprays.
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Figure 8

Relationship between formulation viscosity and droplet spectrum. The detailed data of drawing the figure is shown in Supplementary Table S7.

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As presented in Table 4, droplet size increased with increasing viscosity, which influenced the droplet spectrum. The results in Fig. 8 show that the span of droplet size decreased with the increase of viscosity, indicating that droplets with more uniform distribution could be obtained by increasing the viscosity of the formulation41.
Droplet deposition effect
We tested the efficacy of the ULV spray formulation by spraying cotton plants using an UAV. The test results in Table 5 indicate that increasing the dosage of application would increase droplet size, coverage, and deposition density. At the same application dosage, the droplet size of the ultra-low-volume spray was slightly larger than that of the reference product, and the coverage and deposition density were greater than those of the reference product. The droplet spectral width (Rs) of the five treatments was less than 1, and the coefficient of variation was less than 7%, indicating that the droplet distribution was relatively uniform. Among treatments, T2 had the narrowest Rs and coefficient of variation (CV), where the droplet size distribution was the most uniform. For the ultra-low-volume spray, at the application dosage of 4.5–9.0 L/ha, the droplet coverage gradually increased from 0.85 to 4.15%; the droplet deposition densities were 15.63, 17.24, 28.45, and 42.57 pcs/cm2, which were larger than requirements suggested in the literature. The droplet coverage of the reference product (T5) was 0.73%, and the deposition density was only 11.32 pcs/cm2.
Table 5 Droplet size, coverage, deposition density, spectral width and variation coefficient for each treatment.
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Efficacy trials
The efficacy of cotton defoliant is reflected in the defoliation rate and boll opening rate of cotton after application. Therefore, we surveyed the defoliation rate and boll opening rate of cotton in the test area 3–15 days after application. The results are shown in Figs. 9 and 10.
Figure 9

Defoliation rate 3–15 days after treatment. The detailed data of drawing the curve is shown in Supplementary Table S8.

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Figure 10

Boll opening rate 3–15 days after treatment. The detailed data of drawing the curve is shown in Supplementary Table S9.

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Figure 9 indicates that the defoliation rates of the five treatments 15 days after the pesticide treatment were 59.82%, 63.96%, 71.40%, 77.84%, and 54.58%, respectively. The defoliation rates of T1, T2, and T5 were less than 70%.
Application of the ultra-low-volume spray at 4.50 L/ha or 6.00 L/ha and the reference product at 6.00 L/ha had a poor defoliation effect. T4 (9.00 L/ha) was superior to the others, and the defoliation rate reached 77.84% 15 days after application. As shown in Fig. 10, the boll opening rates of the five treatments were 58.54%, 67.74%, 95.35%, 100%, and 44.68% 15 days after application. Similarly, the boll opening rates of T1, T2, and T5 were poor, with the boll opening rate of the control T5 only 44.68%. We analyzed significant differences between the defoliation rates and boll opening rates of the five treatments. The results showed that the defoliation rate and boll opening rate associated with the thidiazuron·diuron ultra-low-volume spray on cotton plants were significantly different from those of the reference product.
Overall, the defoliation rate and boll opening rate produced by the ultra-low-volume spray were superior to those produced by the reference product. This result was consistent with data shown in Table 5. The higher the droplet coverage rate, the higher the droplet deposition density and the higher the defoliation rate and boll opening rate. T1, T2 and T5 had poor deposition effect on cotton plants, and the effective pesticide utilization rate was low, resulting in dissatisfactory defoliation rates and boll opening rates. Both the droplet coverage rate and the droplet deposition density of T3 and T4 were large. Therefore, droplets of pesticide solution could deposit more easily and uniformly on cotton leaves, allowing the plants to defoliate and open their bolls easily. More

Indian prime minister Indira Gandhi meets Maurice Strong, who chaired the 1972 Stockholm Conference on the Human Environment. Gandhi saw UNEP’s potential at a time when other countries doubted its value.Credit: Yutaka Nagata/UN Photo

The United Nations Environment Programme (UNEP) will be 50 next year. But the globe’s green watchdog, which helped to create the Intergovernmental Panel on Climate Change (IPCC), very nearly didn’t exist.
During talks hosted by Sweden in 1972, low- and middle-income countries were concerned that such a body would inhibit their industrial development. Some high-income countries also questioned its creation. UK representative Solly Zuckerman, a former chief scientific adviser to prime ministers including Winston Churchill, said the science did not justify warnings that human activities could have irreversible consequences for the planet. The view in London was that, on balance, environmental pollution was for individual nations to solve — not the UN.
But the idea of UNEP had powerful supporters, too. India’s prime minister, Indira Gandhi, foresaw its potential in enabling industry to become cleaner and more humane. And the host nation made a wise choice in picking Canadian industrialist Maurice Strong to steer the often fractious talks to success. He would become UNEP’s first executive director. Two decades later, Strong re-emerged to chair the 1992 Earth Summit in Rio de Janeiro, Brazil, which created three landmark international agreements: to protect biodiversity, safeguard the climate and combat desertification.
UNEP has chalked up some impressive achievements in science and legislation. In 1988, working with the World Meteorological Organization, it co-founded the IPCC, whose scientific assessments have been pivotal to global climate action. It also responded to scientists’ warnings about the hole in the ozone layer, leading to the creation of the 1987 Montreal Protocol, an international law to phase out ozone-depleting chemicals.
Strong’s successors would go on to identify emerging green-policy issues and nudge them into the mainstream. UNEP has pushed the world of finance to think about how to stop funding polluting industries. It has also advocated working with China to green its rapid industrial growth — including the Belt and Road Initiative to develop global infrastructure. It is essential that this work continues.
UNEP also accelerated the creation of environment ministries around the world. Their ministers sit on the programme’s governing council; at their annual meeting last week, they reflected on what UNEP must do to tackle the environmental crisis. Although the environment is a rising priority for governments, businesses and civil society, progress on the UN’s flagship Sustainable Development Goals — in biodiversity, climate, land degradation, pollution, finance and more — is next to non-existent. Moreover, the degradation of nature is putting hard-won gains at risk, argues a report that UNEP commissioned as part of its half-century commemorations.
The report, Making Peace with Nature, assesses much of the same literature as would a climate- or land-degradation assessment, but its key strength is in how it brings together researchers from across environmental science. In doing so, UNEP is helping to accelerate a mode of working that should be standard. If, for example, there is to be an assessment of how climate change affects biodiversity, it makes much more sense for this to be carried out by a joint team from the IPCC and the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services (IPBES) than by researchers from just one of these organizations.
The UNEP report’s authors stop short of recommending such changes to the architecture of the UN’s scientific advisory bodies. That is a missed opportunity. Also missing is a discussion and recommendations on how to make countries more accountable for their environmental pledges.
Both these actions are sorely needed if the world is to take more meaningful steps to battle climate change and biodiversity loss. Countries have become expert in capturing data and reporting them to UN organizations. But there is no mechanism that holds nations to account. For example, there is no system to ensure compliance with targets for the Sustainable Development Goals.
Last week, the UN produced a report in which countries published their progress towards commitments under the 2015 Paris climate agreement, known as nationally determined contributions. The agreement includes almost 200 countries, but just 75 reported their data. There are few incentives for success and no penalties for failure. Without such measures, it is hard to see how meaningful change could ever happen.
In the past, researchers have proposed that UNEP’s member states upgrade its powers so it becomes more of a compliance body — a World Environment Organization that, like the World Trade Organization, has the power to censure countries for failing to keep to agreements. But this has been resisted as too radical a step, which would upend the autonomy of the UN biodiversity and climate organizations that UNEP itself helped to bring into being.
Twenty years ago, there might have been some justification for such a view, but now, with the world on a path to extreme climate change, any action will need to be radical, including considering how to give UNEP more teeth.
UNEP helped to lay the foundations for a scientific consensus on environmental decline, and it should be proud of the body of law that has been enacted globally. Alas, such measures risk being too little, too late. As it embarks on a year of reflection ahead of its anniversary, member states must consider what more they need to do to empower UNEP to tackle the planetary emergency. More

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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