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We have analyzed the inbreeding-purging process in four captive populations of different ungulate species with effective sizes ranging 4–40 and with available pedigrees as well as survival and productivity records. This allows us to explore the role of inbreeding and purging in determining the evolution of fitness traits in a range of scenarios relevant in the context of conservation.In A. lervia (Ne ≈ 4), purging is expected only for the most severely deleterious alleles (those giving dNe  > 1, which implies d  > 0.25 as, for example, in completely recessive alleles with deleterious homozygous disadvantage s  > 0.5). Thus, it could be that purging has not been detected for this species because such severely deleterious alleles had been purged during the demographic decline in the wild, before the foundation of the captive population. This would be consistent with the low and non-significant inbreeding load estimated in this species. It is also possible that these estimates are non-significant due to the relatively small number of individuals available.G. cuvieri and N. dama have significant initial inbreeding loads that, adding up the direct and maternal components, is about 1.25 in both cases, which is on the order of other estimates published for captive populations (Ralls et al. 1988). Since in both species Ne  > 10, purging should be efficient against less severely deleterious alleles than in A. lervia (d  > 0.1). Purging is detected for both species with very low P values. This result is in agreement with Moreno et al. (2015), who suggested that purging had occurred in G. cuvieri as they found an increased juvenile survival parallel to an increased inbreeding coefficient. The relative contribution of severe and mild deleterious effects to the inbreeding load of populations is under a scientific debate with direct implications in conservation biology (Ralls et al. 2020, Kyriazis et al. 2021, Pérez-Pereira et al. 2021). The large d estimates obtained in our analysis indicate that a substantial fraction of the initial inbreeding load is being purged under modest effective population sizes, implying that such substantial fraction is due to relatively severe deleterious mutations in these two populations. As far as we are aware, these are the first estimates of this purging parameter obtained in managed, non-experimental populations. Previous estimates of d were obtained in D. melanogaster bottlenecked populations, first for egg-to-pupae viability in lines with Ne = 6 or 12 under noncompetitive conditions (d = 0.09, Bersabé and García-Dorado 2013), and second in lines with higher Ne ≈ 40–50 under more competitive conditions, giving a larger estimate of d, of the order of that estimated in these two ungulate endangered species (d ≈ 0.3, López-Cortegano et al. 2016).Regarding G. dorcas, given its larger population size, purging is expected even against alleles with mild recessive component of the deleterious effect (d  > 0.025). However, although a significant (if modest) inbreeding load was estimated, no significant purging was detected. Nevertheless, the number of equivalent complete generations by the end of the pedigree (EqG = 7) was smaller than our proposed minimum number of generations required to detect purging (tm = 10). This suggests that, due to the large size of this population, more generations are needed to detect purging.The results above support the use of tm to get an approximate idea about when a pedigree is too shallow for purging to be detected. Should the number of generations available be larger than tm, IP predictions could additionally be computed to search the d values that can be expected to produce detectable purging. Supplementary Fig. S3 shows that the true number of generations required to detect purging becomes increasingly larger than tm for alleles with smaller d values, as they suffer weaker purging each time they are exposed in homozygosis. The tm approach helps to understand the failure of many studies to detect purging. Such is the case of the extensive meta-analyses on 119 zoo populations by Boakes et al. (2007), where the median Ne value was 22.6 while the median number of generations was t = 3 meaning that, for most species, at least 5 more generations were needed before purging could be detectable. On the contrary, and in agreement with this tm approach, purging was experimentally detected in lines of D. melanogaster with Ne = 43 (i.e., tm ≈ 10) where, after an initial period of inbreeding depression, fitness experienced a substantial recovery beginning between generations 10 and 20 (López-Cortegano et al. 2016).A reason why detecting purging in captive populations is challenging is that a fitness rebound can also be due to adaptation to captive conditions or to environmental effects, such as those derived from improved husbandry (Clifford et al. 2007). In fact, this might have been the case in Speke’s gazelle breeding program, where the observed rebound of fitness was first ascribed to purging (Templeton and Read 1984, 1998), while Kalinowski et al. (2000) suggested that husbandry improvements could also be responsible for these findings. Our estimates of d and δ, however, are based on the association between the fitness trait and purged inbreeding at the individual level (Wi, gi) which, in our data, is mainly expressed within cohorts while average survival showed little variation through time. In addition, the analyses included temporal factors (YOB or POM) that should have removed confounding effects from adaptation to captivity or improved husbandry. Therefore, adaptive processes or time-dependent environmental factors are not expected to have biased our IP estimates.For productivity, the estimates of inbreeding load were high (overall inbreeding load ~5, P value  More

Insects and bamboosFive internodes (length: mean ± SD = 44.8 ± 1.1 cm, n = 5; diameter in the middle part of internodes: 21.4 ± 0.8 mm, n = 5) of five living mature culms of P. simonii bamboo were sampled at Kawaminami, Miyazaki Prefecture, Japan [32°9′ N, 131°29′ E] on 6 June, 2019. Per internode, four semi-cylindrical strips (ca. 15 × 2 cm) were made and stored at − 25 °C until use.To obtain fungus-free larvae of D. bucculenta, we sampled five beetle eggs from P. simonii bamboo obtained at Toyota, Aichi Prefecture, Japan [35°9′ N, 137°13′ E] on 9 May, 2019 in the laboratory from ovipositing females collected at Kawaminami on 10 and 11 April, 2019. The eggs were immersed in 99.5% ethanol for 10 s followed by 70% ethanol for 10 s for surface sterilization and then individually placed on potato dextrose agar (PDA) (Difco, Detroit, MI, USA) plates. The plates were incubated at 25 °C in the dark until 30 days after larval hatching to confirm the absence of the formation of yeast or other microbial colonies. Consequently, all five larvae hatched successfully and aseptically.The bamboo used in this study was morphologically identified using the literature29. This is native to the study areas and no other host bamboo species are distributed there29. Therefore, no voucher specimen of this bamboo has been deposited in a publicly available herbarium. No specific permits were required for the described field studies. The location is not privately-owned or protected in any way. The field studies did not involve endangered or protected species. All applicable international, national, and/or institutional guidelines for the care and use of animals and plants were followed. This study is reported in accordance with ARRIVE guidelines.Component analyses of bamboo tissuesFor YP and LP, the yeast W. anomalus originating from D. bucculenta in Kawaminami (strain: DBL05Kawaminami) was cultured on a 9-cm PDA plate to obtain enough biomass for further experiments. Afterwards, yeast cells were suspended in ca. 10 mL of sterilized water, and were inoculated on the inner surface of the autoclaved internode strips using an autoclaved tissue paper immersed with the yeast suspension. For LP, additionally, the fungus-free 2nd instar larvae (weight: mean ± SD = 2.4 ± 0.4 mg, n = 5) were individually placed on the yeast-inoculated strips. Each of these yeast-inoculated and yeast-and-larva-inoculated strips was then put in a sterilized test tube (3.0 cm in diameter and 20 cm tall) with moistened cotton placed at the bottom. Each of the test tubes was covered with a sterilized polypropylene cap, sealed with Parafilm Sealing Film (Pechiney Plastic Packaging, Chicago, IL, USA) on which three small holes were made using a fire-sterilized insect pin to avoid oxygen shortage, and individually put in a plastic zipper bag. These yeasts and insects were incubated at 25 °C in the dark for 47 days for YP (n = 5), and 47 (n = 4) and 73 (n = 1) days until these larvae reached adulthood for LP (adult elytral length: mean ± SD = 9.2 ± 0.4 mm, n = 5). Microbial contamination was invisible to the naked eye.For FP, YP and LP, the inner surface (up to 0.3 mm in thickness, dry weight: 336 to 935 mg) of a strip was sampled using a small U-shaped gouge. In the case of FX, first, the pith of a strip was completely removed, and then xylem tissue (up to 0.5 mm in thickness, dry weight: 729 to 872 mg) was sampled using a small U-shaped gouge. These tissues were individually sampled from five strips derived from five different internodes for each tissue type.Samples were extracted by aqueous ethanol and hydrolyzed by sulfuric acid with reference to the literature30,31,32 as follows. Four types of samples were freeze-dried and pulverized using a rotor-speed mill (Fritsch, PULVERISETTE 14, 0.2 mm mesh). About 80 mg of powdered sample was extracted using 5-mL 80% ethanol aqueous solution (aq.) at 63 °C three times. The volume of the extracts was adjusted to 25 mL, filtered, and analyzed using ion exchange chromatography measurements (extractable sugar analysis). Their extracted residues were hydrolyzed using sulfuric acid as follows: 50-mg samples were immersed in 1.64-g 72% sulfuric acid aq. at 30 °C for 2 h, boiled in 39.4-g 3% sulfuric acid aq. for 4 h, and filtered to collect sulfuric acid residues as sulfuric acid lignin fractions. The volumes of the filtrates were fixed to 100 mL, passed through a sulfuric acid-removing filter (DIONEX OnGuard IIA), and submitted to ion exchange chromatography measurements (structural sugar analysis). For the uronic acid measurements, the sulfuric acid-removing filter was not used.Ion exchange chromatography measurements were conducted using a DIONEX ICS-3000 apparatus. The measurement conditions were as follows: column, CarboPac PA-1 (2.0 mm I.D. × 250 mm L, Dionex corp.); flow rate, 0.3 mL min−1; column temperature, 30 °C; injection volume, 25 µL; eluent, H2O (solvent A), 100 mM NaOHaq. (solvent B), aqueous solution containing 100 mM NaOH and 1.0 M CH3COONa (solvent C), and aqueous solution containing 100 mM NaOH and 150 mM CH3COONa (solvent D). The gradient conditions for monomers, dimers, and uronic acids were as follows: for monomers, with a gradient of B 0.5% C 0% 45 min, C 100% 10 min, B 100% 10 min, B 0.5% C 0% 20 min; for dimers, with a gradient of B 50% C 0% 50 min, C 100% 10 min, B 100% 10 min, B 50% C 0% 15 min; for uronic acids, with a gradient of D 100% 10 min. These extraction, hydrolysis, and measurement procedures were conducted using n = 5 samples. For the structural sugars, their yield was calculated as the dehydrated state. The values of other extractives % were calculated by the subtraction of total extractable sugars % from total extractives %.Elemental analysis (carbon, hydrogen, nitrogen) was conducted by 2400 CHNS Organic Elemental Analyzer (PerkinElmer Japan, Yokohama, Japan). About 1-mg dried samples were burned completely and the produced CO2, H2O, and N2 (after reduction of NOx species) gasses were quantified by a thermal conductivity detector.Means of components of bamboo tissues were compared among tissue types using the Steel–Dwass test after the Kruskal–Wallis test. Calculations were performed using R 3.5.133.Carbon assimilation testThe yeast W. anomalus (DBL05Kawaminami) was cultured aerobically in 20 mL of yeast nitrogen base (YNB) (Difco) containing 0.5% glucose at 25 °C in the dark for 2 days with shaking at 85 rpm. The culture media were centrifuged and cell pellets were suspended in sterile water, in which the OD600 was adjusted to 0.10. Fifty μL of the cell suspension was added into a tube (2 mL) with 1 mL of each of 14 different media containing YNB and one of the following carbon sources: d-glucose, d-galactose, d-mannose, d-xylose, l-arabinose, d-fructose, d-galacturonic acid, d-glucuronic acid, sucrose, cellobiose, starch from corn, xylan from corn, carboxymethyl cellulose, and no carbon source (n = 5 to 6). The concentration of each carbon source was 0.5 g L−1, except for xylan at 1.5 g L−1. The tubes were shaken at 85 rpm and incubated at 25 °C in the dark for 7 days. Afterwards, the presence of visible pellets of yeasts and OD600 were recorded to determine the growth of the strain. The degree of assimilation was scored according to the presence of the pellets and the difference in the turbidity increase (ΔOD600) between culture media containing no and a given carbon source as follows: no growth (without a pellet, ΔOD600  More

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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

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