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Synergy between S. cerevisiae and R. etli in biofilm formationWhen S. cerevisiae Mat α Σ1278h and R.etli CE3 were grown in minimal medium with low glucose concentrations (0.1%), these species adhered to abiotic surfaces to form biofilms (Fig. 1). Interestingly, R. etli and S. cerevisiae formed a mixed biofilm whose biomass was ~ 3 times greater than that of either single-species biofilm (Fig. 1a). In addition, at 24 h, the number of colony-forming units (CFU)/cm2 of R. etli CE3 in the mixed biofilm was higher than that in the pure biofilm (Supplementary Fig. 1). Confocal laser scanning microscopy of biofilms stained with the Live/Dead Kit (propidium iodide and SYTO9) showed that in the mixed biofilm, the yeast cells formed patches, and the bacterial cells covered most of the surface (Fig. 1b). In contrast, monospecies biofilms of R. etli and S. cerevisiae had lower structural complexity and contained a greater (80%) number of dead cells, and their individual densities were lower than their populations in the mixed biofilm (Fig. 1b). These results suggest that in mixed biofilms, S. cerevisiae promotes bacterial growth.Figure 1The interaction between S. cerevisiae and Rhizobium etli CE3 results in the formation of a structurally complex and more productive biofilm in terms of biomass. (a) Biofilm formation of R. etli CE3 and S. cerevisiae Σ1278h Mat α and biofilm growth over time in minimal dextrose medium. The data are representative of 3 independent experiments +/− the S.D. values. (b) Top view and cross section of confocal micrographs of the S. cerevisiae-R. etli mixed biofilm and the single-species biofilms. Magnification 40 × . The images are representative of 3 independent experiments. Biofilms were developed on glass microscope slides and stained with a LIVE/DEAD viability kit. Red fluorescence indicates dead cells, and live cells are colored green. Images were acquired 24 h after inoculation.Full size image
S. cerevisiae secretes dicarboxylic acids that promote R. etli growth and biofilm formationWe found that the R. etli colonies that grew close to S. cerevisiae on solid glucose minimal medium were larger than those growing far from yeast colony (Fig. 2).Figure 2Yeast cells produce dicarboxylic acids that promote the growth of R. etli. (a) R. etli growth in coculture with S. cerevisiae BY4741 mutants (aco1Δ, fum1Δ, sdh1Δ and mdh1Δ) that accumulate dicarboxylic acids and a BY4741 strain with blockade of the aerobic respiratory chain (rho-). (b) Test on solid medium showing that S. cerevisiae BY4741 (*) secretes compounds that promote bacterial growth (  >). In contrast, BY4741 rho- cells (ρ), which do not produce dicarboxylic acids, do not promote the growth of R. etli CE3. R. etli CE3 cells were spread over MMD agar, and yeast cells were spotted in the center. (c) Top view of light micrographs of dual-species biofilms; S. cerevisiae (arrowhead) and R. etli (arrow). Biofilms were developed on glass microscope slides and stained with crystal violet. Magnification 20 × . The images are representative of 3 independent experiments. (d) Growth of R. etli strains in coculture with S. cerevisiae BY4741. The growth of the rhizobium strains was estimated at 24 h. R. etli CE3 strains: wild-type (wt), dctA- containing an empty expression plasmid (dctA-) and dctA- containing a plasmid expressing dctA (dctA-/dctA). The data are representative of 3 independent experiments +/− the S.D. values.Full size imageWe used a visual growth promotion assay on solid medium to screen for S. cerevisiae knockout strains (YKO library) that influenced bacterial growth. 159 yeast mutants were unable to promote R. etli CE3 growth (Supplementary Table 3). In general, these mutants were defective in mitochondrial function. Interestingly, we found that 5 strains with mutations in genes coding for enzymes involved in the TCA cycle showed an enhanced ability to promote bacterial growth compared to that of the wild-type strain (Fig. 2a).To determine how the S. cerevisiae mutants may affect the fungal-bacterial interaction, we analyzed factors that may be altered in mutants with mitochondrial function defects and a compromised TCA cycle.We compared the production of TCA intermediates between the wild-type and mutant yeast strains. Mutants defective in mitochondrial function (mef1Δ, gep5Δ, sdh2Δ, ppa2Δ, imp1Δ, cox7Δ, cyc1Δ and cyc2Δ) produced low amounts of tricarboxylic acids (Supplementary Fig. 2a). In contrast, the aconitase mutant (aco1Δ) produced 60% more citrate and succinate; the fumarase mutant (fum1Δ) resulted in fumarate accumulation; the succinate dehydrogenase mutants (sdh1Δ and sdh4Δ) produced 80% more succinate; and the mitochondrial malate dehydrogenase mutant (mdh1Δ) produced 60% more malate and succinate (Supplementary Fig. 2b). These results suggested that the large quantities of tricarboxylic acids secreted by the mutant yeast played a role in promoting bacterial growth in the cocultures.We analyzed the biomass of mixed biofilms formed by yeast cells defective in mitochondrial function (Σ1278B petit mutant). The ability of the wild-type and the petit mutant strains to form a monospecies biofilm was similar (Supplementary Fig. 3). In contrast, the mixed biofilm formed by yeast cells defective in mitochondrial function was significantly lower in biomass than that formed by the wild-type yeast strain (Fig. 2c). Also, Σ1278B petit mutant produced low amounts of tricarboxylic acids (Supplementary Fig. 2a).We next measured the biomass of the mixed biofilm formed by S. cerevisiae and a Rhizobium mutant unable to take up C4-dicarboxylic acids (dctA-). This evaluation revealed that C4-dicarboxylate uptake by R. etli is necessary to form mixed biofilms with high biomass (Fig. 2d).A symbiotic plasmid is involved in the phenotypic plasticity of R. etli.
The genome of Rhizobium etli CE3 is composed of a chromosome and 6 plasmids (pA, pB, pC, pD, PE and pF)11. To determine whether elements encoded by these replicons can participate in the establishment of commensalism, we evaluated the formation of biofilms by yeast and R. etli strains lacking these replicons12. We found that lack of pA, pB, pC or pF did not affect the ability of bacteria to coexist with yeast (Fig. 3a). Interestingly, a strain cured of plasmids pA-/pD- could not coexist with S. cerevisiae to form a mixed biofilm and obtain the benefits provided by the fungus (Fig. 3a).Figure 3Plasmids pA and pD encode proteins performing functions that are necessary for the coexistence of bacterial cells with yeast. Growth of R. etli strains in biofilms with S. cerevisiae S1278B. (a) Growth in mixed biofilms of R. etli strains lacking the plasmids; pA, pB, pC, pF and in one case of two plasmids, pA-/pD-. The growth of the rhizobia strains was assessed at 24 h. (b) Scheme of the genes contained in a cosmid that partially complements the growth of the pA-/pD- strain in mixed biofilms. Here, 3, 2 and only one gene was amplified to generate the plasmids AD1, AD2 and AD3, respectively, as indicated in the figure. (c) Growth of R. etli strains in mixed biofilms. Strains AD1 and AD2 are R. etli pA-/pD- cells that carried plasmids AD1 and AD2, respectively. The growth of rhizobium strains in mixed biofilms was estimated at 24 h. The data are representative of 3 independent experiments +/− the S.D. values.Full size imageTo determine the genetic elements from the symbiotic plasmid involved in the interaction with yeast, we complemented the R. etli pA-/pD- strain with a cosmid library containing fragments of partial digestion (EcoRI) of the R. etli CE3 genome13. We found that a cosmid containing 9 ORFs from plasmid pD (GenBank: U80928.5) partially restored the ability of R. etli pA-/pD- to form a mixed biofilm (Fig. 3b). This cosmid contains 7 insertion sequences (IS) and a predicted operon encoding a probable peptide pheromone/bacteriocin exporter (RHE_PD00332) and a probable bacteriocin/lantibiotic ABC transporter (RHE_PD00333) (Fig. 3b).The complete operon or only the ABC transporter gene, including its endogenous promoter and terminator regions, was cloned into plasmid pBBR1MCS-3, and the resultant plasmids were named AD1, AD2 and AD32, respectively (Supplementary table 1 and 2). We found that complementation with the complete operon (plasmid AD2) partially restored the ability of R. etli pA-/pD- to form a mixed biofilm with yeast (Fig. 3c). In contrast, complementing with the RHE_PD00332 gene (plasmid AD3) does not restore the phenotype. It is necessary to complement only with the RHE_PD00333 gene to determine if its product is involved in the phenotypic plasticity of R. etli. These results suggest that the ABC transporter gene (RHE_PD00333) is involved in the fungal-bacteria interaction.
S. cerevisiae produces a small molecule that affects R. etli growthTo determine how S. cerevisiae affects the growth of R. etli pA-/pD- (Fig. 4a), we evaluated the inhibitory activity of methanol extracts of S. cerevisiae culture supernatants.Figure 4S. cerevisiae s1278B produces a small molecule that only affects the growth of R. etli strains that do not harbor the symbiotic plasmid and plasmid A. (a) S. cerevisiae and R. etli strains were inoculated in close proximity onto MMD soft agar. R. etli pA-/pD- grew, forming a swarm far from the yeast colony. (b) Inhibition of R. etli pA-/pD- growth by 5 µg/mL of a purified compound from the yeast supernatant, which we named Sc2A. (c) Proposed molecular structure of Sc2A.Full size imageInterestingly, we found that the methanol extract inhibited R. etli pA-/pD- growth but had no activity against wild-type R. etli (Fig. 4b). We investigated the chemical constituents of the S. cerevisiae culture supernatants. After succesive organic solvent extractions, the methanolic extract was fractionated by HPLC and 8 fractions were obtained. Each fraction was tested for its determine its effect on the growth of R. etli pA-/pD-. Only a fraction with the ability to inhibit the growth of R. etli pA-/pD- was identified. This resulted in ~ 90% pure sophoroside, judging by its appearance as a dominant peak in the mass spectra obtained by Fast Atom Bombardment Mass Spectroscopy (FAB). As a result, a new sophoroside with bacteriostatic activity, named Sc2A, was isolated (Fig. 4c). The structure of Sc2A was elucidated by a combination of extensive spectroscopic analyses, including 2D NMR and HR-MS.Sc2A was isolated as a crystalline powder with a positive optical rotation ([α]D25 + 13.7°, c0.58, H2O). The molecular formula of Sc2A was determined to be C30H50O24 from its positive-mode FAB data (m/z 794.26 [M + H]+), which was consistent with the 13C NMR data. RMN1H (CD3OD, 400 MHz) data for Sc2A: δ 5.1 d (J = 3.6 Hz), 4.4 d (J = 8 Hz), 4.23 dd (J = 9, 4.8 Hz), 3.79 t (J = 10.8, 14.4 Hz), 3.73 m, 3.67 m, 3.639 m, 3.63 dd (J = 8, 9.2 Hz), 3.53 dd (J = 5.6, 5.2 Hz), 3.36 dd (J = 3.6, 4 Hz), 3.31 dd (J = 8, 8 Hz), 3.10 dd (J = 8, 7.6 Hz), 2.77 dd (J = 4.4, 6.8 Hz), 2.61 m, 2.46 m, 2.33 m, 2.12 m. RMN13C-DEPT (CD3OD, 400 MHz) data for Sc2A: δ 181.2 (C), 175.9 (C), 98.1(CH), 93.8 (CH), 78.05 (CH), 78.02 (CH), 76.30 (CH), 74.92 (CH), 73.80 (CH), 73.11 (CH), 71.78 (CH), 71.72 (CH), 64.37 (CH2), 62.87 (CH2),62.72 (CH2), 57.24 (CH), 30.70 (CH2), 26.19 (CH2), 28.21 (CH2).The IR spectrum of Sc2A displayed characteristic absorptions of 3416.34 cm-1 (O–H), 1642.10 (C = O), 1405.44 (C–OH), 1242.93 (C–O–C), 1040.36 (C-H), and 598.48 (O-C-O).Sc2A possesses a sophorose linked by 2,5 hexanedione to another molecule of sophorose (Fig. 4c).Sc2A induces the expression of genes involved in symbiosisExpression from the nifH and fixA promoters was studied in R. etli monocultures and cocultures with yeast by monitoring GUS activity in living cells. Cells were grown on solid PY-D medium for 1 day, and monitoring of GUS expression showed that the nifH promoter was strongly induced when R. etli was grown with yeast in liquid medium and on solid medium (Fig. 5).Figure 5The expression of Rhizobium etli genes involved in symbiosis is induced in cocultures with yeast or by exposure to the small molecule Sc2A. (a) Activity of different R. etli promoters in monoculture (Re) or in coculture with yeast (+ Sc). Cells were cultured for 24 h in 1 ml of PY-D in 1.5-mL tubes. The tubes were kept closed to generate an environment with a low oxygen concentration. (b) Activity of the nifH promoter in R. etli cells grown alone (Re) or in coculture with yeast (+ Sc) on PY-D agar. (c) Effect of Sc2A on the expression of the nodA gene in R. etli cells grown in liquid culture. Cells stimulated with the flavonoid naringenin were included as a positive induction control. The data are representative of 3 independent experiments +/− the S.D. values.Full size imageAt the beginning of the symbiosis, the legume roots exude flavonoids, which induces in R. etli the expression of a group of genes (nod) involved in the synthesis of lipochitooligosaccharides, also called nodulation factors (NFs). Recognition of NFs by the host plant triggers both rhizobial infection and initiation of nodule organogenesis14. NodA protein is involved in N-acylation of the chitooligosaccharide backbone of NFs. Given the participation of nodA in the interaction of R. etli with a eukaryote, we decided to evaluate the expression of this gene in response to exposure to 5 µg/mL of Sc2A (this concentration is similar to that found in cocultures). We found that Sc2A induces the expression of nodA (Fig. 5c). However, the levels of induction of nodA were moderated compared to the values obtained upon naringenin induction (Fig. 5c). More

All experimental procedures were permitted by the Institutional Animal Care and Usage Committees of the Grassland Science College of Gansu Agricultural University (GSC-IACUC-2015-0011). Our experiments were conducted according to their guidelines, which are in accordance with the Guide for the Care and Use of Laboratory Animals (the Constitution of Experimental Animal Ethics Committee of Gansu Agricultural University). All experiments were performed in accordance with ARRIVE guidelines.Animals and laboratory conditionsAdults of both sexes (three males and three females) were captured in April 2015. Specifically, the animals were captured in Mayin Tan (37°12′N, 102°46′E; Tianzhu Tibetan Autonomous County, China) using live traps25 set at fresh surface mounds. The individuals were then transported to the laboratory and housed in an acrylic box with a pipeline covered with soil. The box and pipeline were covered by black cloth to simulate the dark environment of plateau zokors. The temperature in the room was maintained between 20 and 25 °C. Food was supplied daily and consisted of potatoes, lettuce and carrot. After three days of acclimatization to the laboratory the animals were used in the different experiments. Our laboratory is located 2 km away from the field site. At the end of the experiments all animals were returned to the capture site in good health.Laboratory testing arenaThe experimental setup for the laboratory experiments was as follows (Fig. 1): A transparent Perspex tube (8 cm × 8 cm × 80 cm) was joined to the side of the dark acrylic box (40 cm × 40 cm × 40 cm). A rubber stopper was inserted into one end of the tube to avoid effects from the external environment. Treatment apparatus was placed into the rubber stopper (see “Laboratory treatment apparatus” section, below), and, to avoid the apparatus being damaged by the animals, wire mesh (8 cm × 8 cm × 0.5 cm) was placed about 15 cm from one end of the tube. A mercury thermometer was inserted into the tube in the middle to monitor the tube’s temperature. Between experiments with different animals, the box and tube were wiped with 95% alcohol and then with distilled water.Figure 1Schematic drawing of the setup used to test burrow-sealing behavior in plateau zokors in the laboratory. (1) acrylic box covered with soil 30 cm in depth; (2) experimental animal; (3) mercury thermometer; (4) transparent Perspex tube; (5) the pipe’s support clip; (6) wire mesh (8 cm × 8 cm × 0.5 cm); (7) rubber stopper (8 cm × 8 cm × 5 cm).Full size imageLaboratory treatment apparatusA rubber stopper with seven holes was used for plugging one end of the tube (Fig. 2). The oxygen concentration, light, temperature, sound and gas flow were considered in this design.Figure 2Schematic diagram of the rubber stopper used to simulate the entrance plug of the burrow. (1) power supply; (2) light bulb switch; (3) electric wire switch; (4) oxygen cylinder; (5) in situ three-parameter soil gas analyzer; (6) voice recorder; (7) negative pressure drainage device; (8) rubber plug; (9) LED bulb; (10) the iron rod; (11) heating cord; (12) AVOXIVY speaker with 5 cm diameter.Full size imageOxygen treatmentTo avoid the oxygen that was delivered into the tube causing the gas to flow too strongly, become drier, and create a sound, a steel oxygen cylinder and thin hose (0.3 cm in diameter) were selected, and one end of the hose was connected directly to the oxygen cylinder with a humidifier bottle, while the opposite end was inserted into the rubber stopper (Fig. 2). Before beginning the experiment, we allowed the oxygen cylinder to sit for two hours at laboratory temperature to remove any temperature effects. A three-parameter soil gas analyzer (13.05.03Pro, Shanghai SAFE Biotech Co., Ltd, China) was used to monitor the oxygen concentration in the tube (Fig. 2).Light treatmentThe average light intensity—that is, 360 Lux from 8:00 am to 8:00 pm—was measured in the field. One end of a wire was connected to an LED light (1 Watt), and the other end to the power supply (Fig. 2).Temperature treatmentThe temperature in the burrow entrance in the field was about 3 °C warmer than that at a tunnel depth of 10 cm. As such, one end of a wire was connected to a heater strip and the other end to the electrical power supply (Fig. 2). A thermometer was inserted into the tube to monitor the temperature inside the tube (Fig.1). During the experiment period in the laboratory, we switched on or off to make sure the relatively constant temperature inside the tube. The temperature range inside the tube was 3.2 ± 0.27 °C .Sound treatmentWhen a burrow is opened, wind whistle can be produced around the burrow entrance. Accordingly, a voice recorder (PCM-D50, frequency response 50 Hz–40 kHz, Sony, Japan) was placed at the burrow entrance in the field to record the burrow-entrance sound, the duration of which was 30 min. In the laboratory, the two ends of a wire were connected to an AVOXIVY loudspeaker (diameter: 5 cm; impedance: 4 Ω; 50 Hz–20 kHz) and a voice recorder, respectively (Fig. 2). The recorded sound was played back with a 60 dB sound pressure level, as measured at the burrow entrance in the field (XL2 sound level meter, Nti Audion, Switzerland). The sound was repeatedly played within one hour.Gas flow treatmentTo avoid ambient atmosphere entering the tube, a negative pressure drainage ball with plastic tube (12 cm long, 2 cm in diameter) connected the tube through a rubber stopper (Fig. 2). The tunnel gas was inhaled by the ball, then we pinched the ball to blow the gas into the tunnel as gas flow treatment.Field treatment apparatusFor the field experiment, the apparatus consisted of a tube (40 cm long, 8 cm in diameter) and an alarm device. The alarm device was made up of a loudspeaker, two slide rails (15 cm long), two metal plates (approximately 7 cm in length and 3 cm in width), and three coiled metal springs (5 cm long, 2 cm in diameter). The three springs were joined to one of the metal plates, while the other metal plate was fixed on the slide rails. The two metal plates were touched by the plateau zokor when it was plugging, which triggered the alarm device, thus enabling us to know whether or not burrow-sealing behavior was occurring (Fig. 3). The aluminum tube with an oxygen device was embedded into the burrow. The soil covering the tube served as an excellent insulator, buffering the tube from the aboveground temperature (Fig. 4A). A steel oxygen cylinder and thin hose (0.3 cm in diameter) were applied by connecting one end of the hose directly to the oxygen cylinder with a humidifier bottle, and then the opposite end of the hose was inserted into the tube (Fig. 4A). A three-parameter soil gas analyzer (13.05.03Pro, Shanghai SAFE Biotech Co., Ltd, China) was used to monitor the oxygen concentration in the tube (Fig. 4A). Allowing sunlight to enter the burrow, a glass bottle, open at one end but closed at the other, was embedded into the burrow. We also used soil to cover the bottle, and there was a 5 cm gap at the surface (Fig. 4B). The aluminum tube with high thermal conductivity was embedded into the burrow. Again, we used soil to cover the bottle and retained a 20 cm gap (Fig. 4B).Figure 3Schematic drawing of the apparatus used to test the burrow-sealing behavior of plateau zokor in the field. (1) tube; (2) loudspeaker; (3) slide rail; (4) metal plate; (5) coiled metal springs.Full size imageFigure 4(A) Schematic drawing of the apparatus used in the oxygen treatment placed in the tunnel of the plateau zokor. (B) Schematic drawing of the apparatus used for the temperature and light treatments placed in the tunnel of the plateau zokor. (1) tunnel of the plateau zokor; (2) oxygen cylinder; (3) three-parameter soil gas analyzer; (4) plateau zokor.Full size imageProcedureIn the laboratory experiment, we tested three males and three females for their responses to each treatment. To avoid generating stress and habituation to treatments, zokors were tested for 12 h each day and there was one hour interval between treatments, and five days interval between round of testing for the same individual (Table 1). We performed a control experiment in which a rod was inserted into the burrow but no further treatment was applied, which allowed us to evaluate whether it was the treatment that was causing the burrow-sealing behavior. Before beginning treatment experiment, each zokor was tested 24 times (12 h × 2 days) under the control experiment. We determined the rod movement as occurrence of burrow-sealing behavior.Table 1 Times of the experiments for each treatment in the laboratory simulation.Full size tableIn the field experiment, we tested three zokors (one male, two females), and six zokors were caught in the cold season and warm season (three males and three females, respectively). We then fastened radio collars (Ag357, Biotrack, Ltd., UK) to each captured individual to allow us to track the position in foraging tunnels of each zokor. Each zokor was used three times in the experiments under each treatment, and, after finishing each experiment, we changed the position of the foraging tunnel to ensure the test tunnel was not an abandoned tunnel. According to radio-tracking data, the straight-line distance between the test tunnel and the nest for each treatment was about 5 m. We conducted a control experiment that whether plateau zokor move to the test tunnel or not during the time between treatments. In the cold season, from 4 October 2015 to 2 November 2015, the burrow-sealing behavior of each zokor was tested under different treatments during their active time (12:00–18:00) and inactive time (09:00–11:00) for a total of 27 days (Table 2). The same was done in the warm season but for a total of 18 days from 15 May 2016 to 5 June 2016, in which the active time was 14:00–20:00 and the inactive time was 08:00–13:00 (Table 2).Table 2 Times of the experiments for each treatment in the active and inactive periods of plateau zokors during the warm and cold season.Full size tableData analysisThe occurrence of burrow-sealing was recorded as “1”, and non-sealing was recorded as “0”. The frequency of burrow-sealing was the number of times the burrow was sealed divided by the total number of experiments for each treatment26, and we considered the frequency for each individual as a replicate. The latency to reseal the burrow was the period from the start of the treatment to the sealing of the burrow, and we considered each instance of latency to reseal the burrow as a repeat. The latency to reseal the burrow for non-sealing under each treatment was unavailable data and was therefore removed. The presence of a normal distribution in the initial data was determined using the Kolmogorov–Smirnov test. All data followed a normal distribution. A comparison of males and females in their frequency of sealing the burrow and in their latency to reseal the burrow under each treatment was performed with an independent-samples T-test. Multiple comparisons were made for the frequency of burrow-sealing and the latency to reseal the burrow under different treatments by using the least significant difference method at the significance level of P = 0.05. In the field experiment, the number of replicates was fewer than three for frequency and the latency to reseal the burrow, we did not conduct multiple comparisons.Preliminary statistical analysis of the data was performed using Excel 2013 and SPSS 19.0. All the figures and tables were produced in GraphPad Prism 8.0 and Excel 2013. More

Site, meadow, and fertilisationThe grassland utilized in this study was located in Sedico (BL: 420 m a.s.l., eastern Italian Pre-Alps), where the annual mean temperature is 10.6 °C and the annual rainfall is approximately 1366 mm (389, 326, 401, and 250 mm in spring, summer, autumn, and winter, respectively). The site was level and had an alluvial calcareous substratum. The soil was sandy-loam textured with 12.2% gravel content, 14.6% total carbonate content, and a pH of 7.5. Since 1977, a section of the meadow has been used for a fertilisation trial organised as three completely randomized blocks with 24 m2 plots and twenty-seven treatments obtained by combining three levels of yearly N, P, and K applications per ha: 0, 96, and 192 kg N as ammonium nitrate; 0, 54, and 108 kg P2O5 as triple superphosphate; and 0, 108, and 216 kg K2O as K sulphate. Since 2010, the grassland has been cut twice per year and surveyed for seed production in three treatments: no fertilisation (000), fertilization with no N and intermediate levels of P and K (011), and fertilisation with the highest nutrient rates (222).The vegetation of the three treatments (Annex 1) corresponded to the following meadow types: type 000, vegetation intermediate between a poor-soil form of the Arrhenatherum elatius meadow (Ar0) and a Bromus erectus meadow (Br), with high species richness and low legume abundance; type 011, an Ar0 meadow with high species richness and legume abundance; and type 222, a grass-rich form of the Arrhenatherum elatius meadow with low species richness and legume abundance.Plant sampling and laboratory analysisDuring the two growth periods within each of the years from 2012 to 2017, fertile shoots were sampled from the three fertilization treatments. 15–30 shoots (5–10 in each plot) from each flowering species were collected at the optimal seed maturation stage (most fruits/inflorescences still intact, i.e., no seed shedding). At the sub-plot level, all fertile shoots were collected at the time of meadow mowing on one 1-m2 sub-plot per plot. Collected shoots were put separately per species into porous paper bags, dried, and preserved in a refrigerator until laboratory analysis.During the autumn and winter after collection, the 15–30 shoots of each species were analysed for the number of compound and/or simple inflorescences and the flowers per simple inflorescence or shoot. For species with flowers or inflorescences too numerous to be rapidly counted, an inflorescence length or diameter which could be related to the number of flowers was also measured (e.g., the panicle length in grasses). In sample flowers, intact fruits, or simple inflorescences, the number of ovules per flower and the number of ovules transformed to seed were observed under a binocular microscope. Mature seeds were weighed and tested for germinability and viability according to ISTA19. Germination trials were performed with three seed samples per species, which were placed on filter paper in petri-dishes and moved to a germinator for 4 weeks (8 h light/25 °C and 16 h darkness/15 °C) with weekly observation and extraction of germinated seeds. At the end of the germination test, seeds that had not germinated were checked for viability with the tetrazolium test. Total viability was calculated as the sum of germinability and viability of non-germinated seeds.All shoots collected on the sub-plots were counted and measured for the number of inflorescences and flowers. When inflorescences and flowers were too numerous to be counted rapidly (e.g., in all grasses), only the same length/diameter measured on the 15–30 shoot samples was recorded.A more detailed description of the laboratory analyses is available in Scotton20.Data analysisThe value of each reproductive trait was calculated for each year and growth period at the plot level for each species. The values of the traits describing the size of the reproductive system were obtained from the shoots collected on the sub-plots. However, for species with too many flowers per shoot, a relation was calculated between the flowers per shoot and the length/diameter of the inflorescences measured on the 15–30 shoot samples. This relationship was then used to calculate the flower number for each shoot. The number of ovules per flower, the portion of ovules transformed to seed (ovule site utilisation, i.e. the filled seed/ovule ratio), the 1000-seed weight, germinability, and viability were calculated from the results of lab analyses of the 15–30 shoot samples.Because all the species collected were not always present in the six study years, only the thirty-two species (fifteen grasses and seventeen forbs: Table 1) found in at least three of the study years were considered in this paper to obtain enough reliable results. For all grasses, data were available only for the first growth period. For four forbs (see Annex 3) data were available for the first and second growth periods: in these cases, the average values of the two periods were used in the analyses.Table 1 Species studied for reproductive traits in a grassland fertilisation trial in the Italian eastern Alps.Full size tableThe statistical analyses were performed at the levels of individual species and the two grassland functional groups (grasses and forbs). Nine main reproductive traits describing the whole process of gamic reproduction were considered: number of simple inflorescences per shoot, flowers per simple inflorescence, ovules per flower, ovules and viable seeds per shoot, OSU (ovule site utilization), percent viability, germinability, and seed weight. Percent dormancy (the difference between percent viability and germinability) and the shoot density recorded in the subplots were also considered in some analyses.Only sixteen species were present in all of the fertilisation treatments, presenting a challenge in the tests that included all of the species together because a balanced among-treatments comparison was only possible by discarding the data from species not present in all of the treatments. To overcome this issue, we assumed that due to symbiotic N-fixation, the high presence of legumes in the 011 treatment (fertilization with P and K) was equivalent to a yearly N fertilisation of about 3.5 kg/ha per percent point of legume abundance in the species composition21,22. Therefore, treatment 011 (30% more legume abundance than in treatment 222: Annex 1) was regarded as an intermediate N addition of 105 kg per ha per year (from 3.5 kg N × 30% legume abundance). The values of the reproductive traits were then calculated for two fertilisation levels, low (LowFert) and high (HighFert). For species present in 000 and 011, LowFert was 000 and HighFert was 011. For species present in 011 and 222, LowFert was 011 and HighFert was 222. For species present in three fertilisation treatments, LowFert was 000 and HighFert was the average between 011 and 222. Statistical analysis considering only the species present in all fertilisation treatments yielded a similar pattern of fertilisation effects to those found in analysis of the two separate fertilisation levels. The analysis of the two fertilisation levels was therefore utilized because it was representative of a larger number of species.Statistical analyses (see summary in Annex 2) were conducted with three main aims: 1. studying the fertilisation effect on the reproductive behaviour of individual species and the two species groups of grasses and forbs; 2. finding species biological and ecological traits explaining their response to fertilisation; and 3. identifying multispecies correlations among reproductive traits and the possible effects of fertilisation on their patterns.For the first aim, the fertilisation effect was tested for the reproductive traits of each individual species through application of a mixed linear model under a repeated measure approach. In the model, fertilisation treatment, year, and block were input as class factors, and a plot identifier was used as a random factor. In case of significant fertilisation effects, the among-treatment differences were tested using the Tukey multiple comparison adjustment. Prior to performing the mixed model, data were checked for homoscedasticity and normality and, if necessary, log-transformed.From the individual species mixed models, a table was calculated containing the frequency of cases with fertilisation effects (three levels: no, positive, or negative) for each reproductive trait and species group. To check if grasses and forbs differed for the obtained frequencies, for each trait a chi-square test on the frequency table “fertilisation effect x species group” was performed.In a following set of analyses, the effect of the grassland functional group (grasses or forbs) on the multi-year means of each reproductive trait was tested with general linear models (GLM). Prior to the analysis, the data were sometimes log-transformed to mitigate homoscedasticity and normality problems. In these analyses, species were considered as replicates within the species group (therefore not included as a class factor) and the fertilisation level was input as a class factor. The effect of the fertilisation level on each reproductive trait was tested separately for the two species groups. In this case, the GLM included both fertilisation level and species as class factors.For the second aim, possible biological and ecological traits explaining the species response to fertilisation were investigated by relating the percent variation due to fertilisation in two important size traits (ovule and viable seed number per shoot: variables Y) to the following explanatory (X) variables: average values of the nine reproductive traits, the seven Ellenberg bioindicator values23, and the percent variation of shoot density. The relationships were fitted according to a linear regression approach for grasses and forbs together or separately and checked for the parametric assumptions of residual normality and homoscedasticity. For the percent variation due to fertilisation of individual species of OSU, seed germinability, viability and weight, one-way analyses of variance were performed where three traits of the species reproductive biology (type of reproduction, breeding system and pollen vector4: Annex 1) were used as categorical factors. A GLM approach was also used in this case.For the third aim, multispecies correlations were analysed by in-pairs relating the reproductive trait values of individual species averaged across fertilisation treatments and years. Fertile shoot density recorded in the subplots. was used as a supplementary characteristic. Nonlinear relationships were made linear with a log-transformation. Because the purpose of the analysis was not to predict one trait from the other but to efficiently summarise the relationships between traits, the standardised major axis (SMA) approach was used instead of the linear regression method24. The analyses were performed for grasses and forbs both together and separately. In order to verify if fertilisation could affect the characteristics of the evaluated relationships, a second set of SMA analyses were performed by separating the two fertilisation levels and the lines obtained were tested for common slope and elevation according to Warton et al.24.The year effect will be reported in a forthcoming paper and is therefore not discussed here, despite its inclusion in the statistical analyses.We used SAS/STAT software 12.325 with procedures MIXED, GLM, REG, and UNIVARIATE, and R 3.0.026 with package SMATR.Additional statementsThe experimental research and the collection of plant material was done according to relevant institutional, national, and international guidelines and legislation. No grassland species considered in the research is included in the list of endangered species according to the IUCN, European Union, Italian national and regional classifications. The collection of plant specimen was done with permission of the grassland owner during the hay-making operations for forage production which do not need a special permission from the concerned local authorities. The plant species were identified by the first author, Michele Scotton. A voucher specimen of each plant species considered in the research was stored in the laboratory of the authors’ Department (DAFNAE) and the authors have provided an ID number for each voucher specimen. More

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Summary of sequencing dataWe obtained 168 million and 180 million 250 bp reads from Asian Badger and Northern Hog Badger, respectively. After removing transcripts and unigenes below 200 bp, we obtained 335,772 transcripts and 285,159 unigenes belonging to Asian Badger and 413,917 transcripts and 362,075 unigenes belonging to Northern Hog Badger (Table 1). Next, we analysed the length distribution of the unigenes and transcripts in these two species (Fig. 1). Their N50 of transcript length is longer than 1000 bp, and their N50 of unigene length is longer than 600 bp. The average GC content of the transcriptome data of Asian Badge was 52.71%, a value slightly higher than that of the Northern Hog Badger, which was 52.12% (Table 1).Table 1 Summary of the transcriptome of Asian Badgers and Northern Hog Badgers.Full size tableFigure 1Length and quantity distribution of transcripts and unigenes.Full size imageFunctional annotation and classification of the assembled unigenesThe success rate of annotation of these research data in the seven databases is shown in Table 2. In total, 34,150 (ZH) and 31,632 (GH) unigenes had GO terms (Table 2). Among them, there were three GO items related to digestion: positive regulation of the digestive system process (GH and ZH both have one gene), digestive tract development (GH and ZH both have four genes), and digestion (GH has five genes, ZH has three). Next, we compared the GO terms of Asian Badger and Northern Hog Badger transcriptomes and found that the distributions pattern of gene functions from these two species were particularly similar (Fig. 2). This predictable result indicates that there is no bias in the construction of the libraries from the Asian Badger and Northern Hog Badger. For both species, in the three main partitions (cellular component, molecular function, and biological process) of the GO classification, ‘Cellular process’, ‘Binding’ and ‘Metabolic process’, terms were principal individually (Fig. 2). In total, 8915 (ZH) and 10,203 (GH) unigenes had KOG terms (Table 2). In addition, 15,667 (ZH) and 17,823 (GH) were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (Table 2) and grouped into 32 subclasses. Interestingly, the digestive system subcategory contains 695 and 611 unigenes in Asian Badger and Northern Hog Badger, respectively, involving 9 pathways, namely, bile, gastric acid, pancreatic, salivary secretion, carbohydrate, protein, vitamin, fat digestion and absorption, and mineral absorption.Table 2 Gene annotation success rate statistics.Full size tableFigure 2GO term Top20 for GH and ZH.Full size imageAnalysis of orthologous genesThe transcriptome evolution of different species can be understood by comparing transcriptome data. We analysed the possible orthologous genes between the transcriptome of Asian Badger and Northern Hog Badger obtained in this study. We selected a total of 5227 homologous gene pairs from these four species. After 5227 pairs of homologous genes were optimized and screened, 943 orthologous gene pairs were obtained (Supplementary Table S1).To explore whether the genes related to small intestinal digestion in Asian Badger and Northern Hog Badger have undergone adaptive evolution. We can predict the genes that affect the evolution of the two species through selection pressure on orthologous genes12. We selected 473 orthologous gene pairs with Ka/Ks  > 1 called divergent orthologous genes from the Ka/ks analysis results. We obtained 1263 orthologous gene pairs with Ka/Ks  More

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