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Experimental spidersExperimental spiders were maintained as previously reported37. Briefly, spiders were the F1 to F4 offspring of mated females collected from hallways of the Burnaby campus of Simon Fraser University (Burnaby, BC, CA). Upon hatching, juvenile spiders were housed individually in petri dishes (100 mm × 20 mm) and provisioned with the vinegar flies Drosophila melanogaster. Subadult spiders were fed with larvae of the mealworm beetle Tenebrio molitor. Each adult female spider was kept in a separate translucent 300-mL plastic cup (Western Family, CA) maintained at 22 °C under a reversed light cycle (12:12 h). Adult males and females were fed with black blow flies, Phormia regina. All spiders had access to water in cotton wicks. Water and food were provided once per week. Laboratory experiments were run during a reversed scotophase (0900 to 1700).Identification of contact pheromone components: Preparation of web extracts (summer 2017; spring and summer 2018)Each of the 100 spiders was allowed to build her web for three days on a wooden triangular prism scaffold (30 cm × 25 cm × 22 cm)44 of bamboo skewers (GoodCook, CA, USA) (Fig. 1b). After the spiders were removed from the scaffold, their webs were reeled up with a glass rod (10 cm × 0.5 cm) and deposited in a 1.5-mL glass vial. Per web, 50 µL of methanol (99.9% HPLC grade, Fisher Chemical, ON, Canada) were added and the silk was extracted for 24 h at room temperature. Prior to analysis, the silk was removed and the sample was concentrated under a steady nitrogen stream to the desired concentration.Identification of contact pheromone components: analyses of web extracts by gas chromatography–mass spectrometry (GC-MS)Aliquots (2 µL) of pooled and concentrated web extract (100 webs in 400 µL of solvent) were analysed by GC–MS, using a Varian Saturn Ion trap 2000 (Varian Inc., now Agilent Technologies Inc., Santa Clara, CA 95051, USA) and an Agilent 7890B GC coupled to a 5977 A MSD, both fitted with a DB-5 GC-MS column (30 m × 0.25 mm ID, film thickness 0.25 µm). The injector port was set to 250 °C, the MS source to 230 °C, and the MS quadrupole to 150 °C. Helium was used as a carrier gas at a flow rate of 35 cm s−1, with the following temperature programme: 50 °C held for 5 min, 10 °C min−1 to 280 °C (held for 10 min). Compounds were identified by comparing their mass spectra and retention indices (relative to aliphatic alkanes67) with those of authentic standards that were purchased or synthesised in our laboratory (Supplementary Table 1).Identification of contact pheromone components: high-performance liquid chromatography (HPLC) of web extractsWeb extract of virgin adult female S. grossa was fractionated by high-performance liquid chromatography (HPLC), using a Waters HPLC system (Waters Corporation, Milford, MA, USA; 600 Controller, 2487 Dual Absorbance Detector, Delta 600 pump) fitted with a Synergy Hydro Reverse Phase C18 column (250 mm × 4.6 mm, 4 µ; Phenomenex, Torrance, CA, USA). The column was eluted with a 1-mL/min flow of a solvent gradient, starting with 80% water (HPLC grade, EMD Millipore Corp., Burlington, MA, USA) and 20% acetonitrile (99.9% HPLC grade, Fisher Chemical, Ottawa, CA) and ending with acetonitrile after 10 min. A 60-web-equivalent extract was injected and 20 1-min fractions were collected. Each HPLC fraction (containing 20 web-equivalents) was tested in T-rod bioassays (Fig. 1c) for the presence of contact pheromone components. All eight fractions that elicited courtship responses by males (Supplementary Fig. 1) were analysed by HPLC-tandem MS/MS.Identification of contact pheromone components: HPLC-tandem MS/MS of bioactive HPLC fractionsThe bioactive HPLC fractions were analysed on a Bruker maXis Impact Quadrupole Time-of-Flight HPLC/MS System. The system consists of an Agilent 1200 HPLC fitted with a spursil C18 column (30 mm × 3.0 mm, 3 µ; Dikma Technologies, Foothill Ranch, CA, USA) and a Bruker maXis Impact Ultra-High Resolution tandem TOF (UHR-Qq-TOF) mass spectrometer. The LC-MS conditions were as follows: The mass spectrometer was set to positive electrospray ionisation (+ESI) with a gas temperature of 200 °C and a gas flow of 9 L/min. The nebuliser was set to 4 bar and the capillary voltage to 4200 V. The column was eluted with a 0.4-mL/min flow of a solvent gradient, starting with 80% water and 20% acetonitrile and ending with 100% acetonitrile after 4 min. The solvent contained 0.1% formic acid to improve peak shape.Identification of contact pheromone components: 1H NMR analyses of a bioactive fractionIn HPLC-MS analyses, a single bioactive fraction (9–10 min) appeared to contain only a single compound. This fraction was then further investigated using 1H NMR spectroscopy. The 1H NMR spectrum was recorded on a Bruker Advance 600 equipped with a QNP (600 MHz) using CDCl3. Signal positions (δ) are given in parts per million from tetramethylsilane (δ 0) and were measured relative to the signal of the solvent (1H NMR: CDCl3: δ 7.26).Identification of contact pheromone components: syntheses of candidate pheromone componentsThe syntheses of candidate pheromone components and synthetic intermediates are reported in the SI.Identification of contact pheromone components: T-rod bioassays (general procedures)The T-rod apparatus37 (Fig. 1c) consisted of a horizontal beam (25 cm × 0.4 cm) and a vertical beam (30 cm × 0.4 cm) held together by labelling tape (3 cm × 1.9 cm, Fisher Scientific, Ottawa, ON, CA). A piece of filter paper (2 cm2) was attached to each distal end of the horizontal beam. For each bioassay, an aliquot of web extract (in methanol), or a blend of synthetic candidate pheromone components, was applied to the randomly assigned treatment filter paper, whereas methanol was applied to the control filter paper. The solvent was allowed to evaporate for 1 min before the onset of a 15-min bioassay. A randomly selected naïve male spider was placed at the base of the vertical beam and the time he spent courting on each filter paper was recorded. In response to the presence of female-produced or synthetic pheromone on a filter paper, the male engaged in courtship, pulling silk with his hindlegs from his spinnerets and adding it to the paper. Sensing contact pheromone, the male essentially behaves as if he were courting on the web of a female. On a web, the male engages in web reduction prior to copulation, a behaviour that entails cutting sections of the female’s web with his chelicerae and wrapping the dismantled web bundle with his own silk pulled from his spinnerets41,56. Each T-rod apparatus was used only once. Replicates of experiments as part of specific research objectives were run in parallel to eliminate day effects on the responses of spiders. The sample size for each experiment was set to 20 unless otherwise stated.Identification of contact pheromone components: T-rod bioassays (specific experiments) (fall 2017; spring and summer 2018)Experiment 1 (fall 2017) tested a synthetic blend of volatile compounds 5–11 unique to mature S. grossa females (Fig. 1c and Supplementary Table 1) vs a solvent control. Parallel experiment 2 tested one web equivalent of virgin female web extract, followed by testing each of the 20 HPLC fractions in six replicates for the occurrence of courtship (spring 2018).Parallel experiments 3–6 (summer 2018) tested web extract at one female web equivalent (1 FWE) (Exp. 3), a ternary blend of the candidate contact pheromone components 12, 16 and 17 (Fig. 2d, Exp. 4), the same ternary blend (12, 16 and 17) in combination with the volatile compounds 5–11 (Exp. 5), and 5–11 on their own (Exp. 6).Parallel dose-response experiments 7–11 (summer 2018) tested the ternary blend of 12, 16 and 17 at five FWEs: 0.001 (Exp. 7); 0.01 (Exp. 8); 0.1 (Exp. 9); 1.0 (Exp. 10); and 10 (Exp. 11).Parallel experiments 12–15 tested the ternary blend, and all possible binary blends, of 12, 16 and 17. Parallel experiments 16–18 tested 12 and 16 in binary combination (Exp. 16) and singly (Exps. 17, 18).Origin of contact pheromone components (fall 2020)To trace the origin of contact pheromone component 12 (and coeluting 16), cold-euthanized female spiders were dissected in saline solution55 (25 mL of water and 25 mL of methanol, 160 mM NaCl, 7.5 mM KCl, 1 mM MgCl2, 4 mM NaHCO3, 4 mM CaCl2, 20 mM glucose, pH 7.4). Samples were homogenised (Kimble Pellet Pestle Motor, Kimble Kontes, USA) in methanol for 1 min, kept 24 h at room temperature for pheromone extraction, and then centrifuged (12,500 rpm, 3 °C for 20 min; Hermle Z 360 K refrigerated centrifuge; B. Hermle AG, Wehingen, DE) to obtain the supernatant for HPLC-MS analyses (see above) for the presence of 12 and 16. Three sequential sets of dissections aimed to determine (1) the pheromone-containing body tagma, (2) the pheromone-containing tissues or glands in that tagma and (3) the specific gland or tissue producing 12 & 16.To identify the pheromone-containing tagma, 11 spiders were severed at the pedicel, generating two tagmata: the cephalothorax with four pairs of legs and the abdomen. Each tagma was then extracted separately in 100 µL of methanol. Eight of 11 abdomen samples contained 12 and 16, whereas only one of 11 thorax samples contained 12 and 16 (Exp. 19), albeit at only trace amounts. With 12 and 16 being present in the abdomen, 20 additional abdomens were dissected68 to obtain separate samples of (i) haemolymph (25 µL), (ii) ventral cuticle (~0.5 cm2 near the pedicel, (iii) the ovaries, (iv) all silk glands combined, and (v) the gut (with anus, cloaca and Malpighian tubules). The remaining spider tissues (vi) were pooled as one sample, and 20 µL of the dissection buffer solution (vii) was obtained to detect potential pheromone bleeding. To each tissue sample, 50 µL of methanol were added. Only silk gland samples contained 12 and 16 (Exp. 20). Having established that only silk gland samples contained 12 and 16, the silk glands of 30 additional spiders were excised in the following order: (i) major ampullate gland, (ii) minor ampullate gland, (iii) anterior aggregate gland, (iv) posterior aggregate gland, (v) tubuliform, (vi) aciniform and flagelliform glands combined and (vii) pyriform gland. The glands from three spiders were combined in each sample and extracted in 30 µL methanol. Seven of ten posterior aggregate gland samples contained 12 and 16, with other silk gland samples not containing 12 and 16 or in only trace amounts (Exp. 21).Transition of contact pheromone components to mate attractant pheromone components: evidence for hydrolysis of contact pheromone components (12, 16 and 17) (spring 2021)To test for the hydrolysis of the contact pheromone components 12, 16 and 17, we compared their breakdown ratio (18/(12 + 16 + 17 + 18) on independent webs aged 0 days and 14 days (Exp. 24). Each of 140 spiders was allowed to spin a web on bamboo scaffolds for three days. Then, the spiders were removed and webs—by random assignment—were extracted immediately (0-day-old webs) or after 14 days of aging (14-day-old webs). On each web, the amount of contact pheromone components 12, 16 and 17, and of amide 18 as a breakdown product, was quantified using HPLC–MS, with 12 and 18 at 25 and 50 ng/µL as external standards.Transition of contact pheromone components to mate attractant pheromone components: Y-tube olfactometer bioassays (general procedures)The attraction of male spiders to web extracts and to candidate mate attractant pheromone components was tested in Y-tube olfactometers56 (Fig. 4a) lined with bamboo sticks to provide grip for the bioassay spider. Test stimuli were presented in translucent oven bags (30 cm × 31 cm; Toppits, Mengen, DE) secured to the orifice of side-arms. Test stimuli consisted of a triangular bamboo prism scaffold (each side 8.5 cm long) bearing a spider’s web, or bearing artificial webbing30 (40 ± 2 mg; Bling Star, CN) that was treated with web extract or synthetic chemicals in methanol (100 µL) as the treatment stimulus or with methanol (100 µL) as the control stimulus. For each experimental replicate, a male spider was introduced into a glass holding tube and allowed 2 min to acclimatise. Then, the holding tube was attached via a glass joint to the Y-tube olfactometer and an air pump was connected to the holding tube, drawing air at 100 mL/min through the olfactometer. Air entered the olfactometer through a glass tube secured to the oven bags’ second opening. A male that entered the olfactometer within the 5-min bioassay period was classed a responder and his first choice of oven bag (the oven bag he reached first) was recorded. Whenever a set of 30 replicates was completed by the same observer, using 30 separate Y-tubes, the Y-tubes were cleaned with hot water and soap (Sparkleen, Thermo Fisher Scientific, MA, United States) and dried in an oven at 100 °C for 3 h, whereas the bamboo sticks and the oven bags were discarded.Transition of contact pheromone components to mate attractant pheromone components: Y-tube olfactometer bioassays (specific experiments) (summer 2018)In experiments 22, 23 and 25–27, males were offered a choice between a solvent control stimulus and a treatment stimulus. The treatment stimulus consisted of (i) virgin female web-extract (1 web-equivalent) (Exp. 22, N = 24), (ii) the volatile compounds 5–11 unique to sexually mature females (Fig. 1d) (Exp. 23, N = 24), (iii) all breakdown products of the contact pheromone components 12, 16 and 17, consisting of the amide N-4-methylvaleroyl-l-serine (18) and the corresponding carboxylic acids 19, 20 and 21 (Exp. 25, N = 30), (iv) a blend of the acids 19, 20 and 21 (Exp. 26, N = 30) and (v) the amide 18 as a single compound (Exp. 27, N = 30). Compounds were tested at quantities as determined in virgin female web extract (50 webs in 150 μL of dichloromethane), following silyl-ester derivatization69 of acids in the extract, with valeric acid (200 ng; ≥99%, Sigma Aldrich, St. Louis, USA) added as an internal standard. Per web equivalent, there were 103 ng of 19, 3 ng of 20 and 54 ng of 21. The amide 18 was present at 200 ng per web equivalent, as determined using N-3-methylbutnaoyl-l-serine methyl ester as an external standard.Transition of contact pheromone components to mate attractant pheromone components: hallway of buildings experiment (fall 2018)As the ternary blend of the carboxylic acids 19, 20 and 21 attracted male spiders in Y-tube olfactometers (see Results), we aimed to confirm their functional role as mate attractant pheromone components also in ‘field’ settings (Exp. 28). To this end, we set up ten replicates of paired traps in building hallways on the Burnaby campus of Simon Fraser University. Adhesive-coated traps (Bell Laboratories Inc., Madison, WI, USA) were spaced 0.5 m within pairs and 20 m between pairs. By random assignment, one trap in each pair was baited with the carboxylic acids 19, 20 and 21 formulated in 200 µL of mineral oil (Anachemia, Montreal, CA; 2.8 mg of 19, 0.112 mg of 20 and 1.52 mg of 21), whereas the control trap received mineral oil only. Test stimuli were disseminated from a 400-μL microcentrifuge tube (Evergreen Scientific, Ranco Dominguez, CA, USA) with a hole in its lid punctured by a No. 3 insect pin (Hamilton Bell, Montvale, NJ, USA). Every week for 4 months (September to December 2018), traps were checked, lures were replaced, and the position of the treatment and the control trap within each trap pair was re-randomised.Communication function of amide breakdown product 18 (fall 2018)As the amide 18 did not attract males in Y-tube olfactometer experiments (see Results), we tested its alternate potential function as a contact pheromone component which, if active, would induce courtship by males. Using the T-rod apparatus (Fig. 1c), we treated one piece of filter paper with a solvent control and the other with a blend comprising both the contact pheromone components 12, 16 and 17 and the amide 18 (Exp. 29), a blend of 12, 16 and 17 (Exp. 30), and 18 alone (Exp. 31).Mechanisms underlying the transition of contact pheromone components to mate attractant pheromone components: relationship between web pH and breakdown rates of contact pheromone components (summer 2020)We allowed each of the 70 spiders to spin two webs, using one web to quantify the amide breakdown product (18) of the contact pheromone components (see above), and the other web to determine its pH according to the slurry method57 (Exp. 32). To this end, we first measured the pH of 50 µL water (HPLC grade, EMD Millipore Corp., Burlington, MA, USA) and then of a web with the water functioning as a conductor for the pH metre (LAQUAtwin pH 22 (Horiba, Kyoto, JP). Between web measurements, the pH metre was rinsed with water and regularly re-calibrated using a pH 7 and a pH 4 buffer (Horiba, Kyoto, JP).Mechanisms underlying the transition of contact pheromone components to mate attractant pheromone components: testing for pH-dependent saponification of contact pheromone components (12, 16 and 17) (summer 2021)To test whether pH alone catalyses saponification of the ester bond of contact pheromone components (12, 16 and 17), synthetic 12 was added to a 40% aqueous pH 7 buffer solution (Exp. 34), a pH 4 buffer solution (Exp. 34), and to acetonitrile (Exp. 35) as a polar aprotic solvent control (N = 12; 100 ng/µL each). pH-Dependent breakdown of 12 over time was assessed by analysing (HPLC-MS) diluted aqueous aliquots (2.5 ng/µl) of each sample at day 0 and after 14 days of storage at room temperature.Mechanisms underlying the transition of contact pheromone components to mate attractant pheromone components: testing for the presence of a carboxylesterhydrolase (CEH) (summer 2021)To test for the presence of a carboxylesterhydrolase (CEH), for each of three replicates we extracted (i) five webs of adult virgin female L. hesperus (positive control, known to have a CEH45), (ii) 20 webs of subadult S. grossa (deemed to have not yet produced a CEH) and (iii) ten webs of adult virgin female S. grossa, accounting for the different amounts of silk produced by these three groups of spiders. For each replicate, webs were extracted in 200 µL 0.05 M Sørensen buffer58 and analysed by Bioinformatics Solutions (Waterloo, ON, CA). After web samples were incubated for 20 min at 60 °C in 2× sample volumes of 10% SDS (lauryl sulfate; protein-denaturing anionic detergent), they were sonicated for 20 min. Then, the supernatant was withdrawn, reduced with dithiothreitol (DTT), and alkylated with iodoacetamide (IAA). Alkylated samples were treated further with a protein solvent (S-Trap kit; Protifi, Farmingdale, NY, USA). Briefly, samples were acidified by phosphoric acid to pH ≤1. Then 6× of sample volume S-trap buffer was mixed in. The mixture was loaded by centrifugation onto an S-Trap Micro Spin Column and washed 3× with S-trap buffer. Using the serine protease trypsin, protein digestions were carried out at 47 °C for 1 h in 50 mM triethylamonium bicarbonate (TEAB) buffer within the S-Trap Micro Spin column. Digestion products were eluted sequentially with 40 µL 50 mM TEAB and 0.2% formic acid. Eluates were dried and re-suspended in 0.1% formic acid.Eluates were analysed by HPLC-MS/MS in positive ion mode on a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher, San Jose, CA, USA), equipped with a nanospray ionisation source and a Thermo Fisher Ultimate 3000 RSLCnano HPLC System (Thermo Fisher). Peptide mixtures were loaded onto a PEPMAP100 C18 trap column (75 µm × 20 mm, 5 µm particle size; Thermo Fisher) at a constant flow of 30 μL/min and 60 °C isothermal. Peptides were eluted at a rate of 0.2 μL/min and separated using a Reprosil C18 analytical column (75 μm × 15 mm, 1.9 μm particle size; PepSep, DK) with a 60-min solvent gradient: 0–45 min: 4–35% acetonitrile + 0.1% formic acid; 45–55 min: 90% acetonitrile + 0.1% formic acid; 55–60 min: 4% acetonitrile + 0.1% formic acid.MS data were acquired in data-dependent mode with a cycle time of 3 s. MS1 scan data were acquired with the Orbitrap mass analyser, using a mass range of 400–1600 m/z, with the resolution set to 120,000. The automatic gain control (AGC) was set to 4e5, with a maximum ion injection time of 50 ms, and the radio frequency (RF) lens was set to 30%. Isolations for MS2 scans were run using a quadrupole mass analyser, with an isolation window of 0.7. MS2 scan data were acquired with the Orbitrap mass analyser at a resolution of 15,000 m/z, with a maximum ion injection time of 22 ms, and the AGC target set to 5e4. Higher energy collisional dissociation (HCD; fixed normalised collision energy: 30%) was used for generating MS2 spectra, with the number of microscans set to 1.Statistics and reproducibilityData (Supplementary Table 2) were analysed statistically using R70. Data of experiments 1–18 and 29–31 (testing courtship by male spiders in response to contact pheromone components) were analysed with a Wilcoxon test or Kruskal–Wallis two-tailed rank-sum test with Benjamini–Hochberg correction to adjust for multiple comparison. Data of experiments 19–21 (revealing the presence of contact pheromone components in the abdomen, silk glands, and posterior aggregate silk gland) were analysed with two-tailed, rather than one-tailed, Wilcoxon test or Kruskal–Wallis rank tests because we had no strong assumption as to whether or not pheromone would be present in any of these potential pheromone sources. The p values were adjusted for multiple comparison using the Benjamini–Hochberg method. Y-tube olfactometer data of experiments 22, 23 and 25–27, as well as the hallway experiment 28 (revealing attraction of male spiders to volatile pheromone components) were analysed using an one-tailed71 binomial test, anticipating attraction of spiders to volatile mate attractant pheromone components rather than to solvent control stimuli. Data of experiment 32 (revealing a correlation between web pH and breakdown of web-borne contact pheromone components) were analysed using generalised linear models. Data of experiments 33–35 (showing pH-dependent breakdown of synthetic contact pheromone) were compared using a two-tailed Kruskal–Wallis test with Benjamini–Hochberg correction.Reporting summaryFurther information on research design is available in the Nature Research Reporting Summary linked to this article. More

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Study speciesTo increase our ability to generalize the results, we conducted two multispecies experiments34. The experiments were designed independently, but, as they used similar treatments, we analyzed them jointly to further increase generalizability. For the experiment in China, we selected eight species that are either native or non-native in China (Supplementary Table 1). For the experiment in Germany, we selected 16 species that are either native or non-native in Germany (Supplementary Table 1). All 24 species, representing seven families, are herbaceous, mainly occur in grasslands, and are common in the respective regions. To control for phylogenetic non-independence of species, we selected at least one non-native and one native species in each of the seven families. All non-native species are fully established (i.e. naturalized sensu Richardson et al.35) in the country where the respective experiment was conducted, and, as they are common, most of them could be considered invasive36,37. We classified the species as naturalized non-native or native to China or Germany based on the following databases: (1) “The Checklist of the Alien Invasive Plants in China”38, (2) the Flora of China (www.efloras.org), and (3) BiolFlor (www.ufz.de/biolflor). Seeds or stem fragments of the study species were obtained from local botanical gardens, local commercial seed companies, or from wild populations (Supplementary Table 1).The experiment in ChinaFrom 21 May to 27 June 2020, we planted or sowed the eight study species into plastic trays filled with potting soil (Pindstrup Plus, Pindstrup Mosebrug A/S, Denmark). We sowed the species at different times (Supplementary Table 1) because they were known to require different times until germination. Three species were grown from stem fragments because they mainly rely on clonal propagation, and the others were propagated from seeds (Supplementary Table 1).On 13 July 2020, we transplanted the cuttings or seedlings into 2.5-L circular plastic pots filled with a mixture of sand and vermiculite (1:1 v/v). Three competition treatments were imposed: (1) competition-free, in which plants were grown alone; (2) intraspecific competition, in which two individuals of the same species were grown together; (3) interspecific competition, in which two individuals, each from a different species were grown together. We grew all eight species without competition, in intraspecific competition, and in all 28 possible pairs of interspecific competition. For the competition-free and intraspecific-competition treatments, we replicated each species seven times (i.e. we had seven technical replicates). For the interspecific-competition treatment, for which we had many pairs of species (i.e. biological replicates), we replicated each pair two times.The experiment took place in a greenhouse at the Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences (Changchun, China). The greenhouse had a transparent plastic film on the top, which reduced the ambient light intensity by 12%. It was open on the sides so that insects and other organisms could enter. To vary nutrient availability, we applied to each pot either 5 g (low-nutrient treatment) or 10 g (high-nutrient treatment) of a slow-release fertilizer (Osmocote® Exact Standard, Everris International B.V., Geldermalsen, The Netherlands; 15% N + 9% P2O5 + 12% K2O + 2% MgO + trace elements). To vary light availability, we used two cages (size: 9 m × 4.05 m × 1.8 m). One of them was covered with two layers of black netting material, which reduced the light intensity by 71% (low light-intensity treatment, where the light intensity was on average 233.5 μmol m−2 s−1, measured on a sunny day). The other was left uncovered (high light-intensity treatment, where the light intensity was on average 826.7 μmol m−2 s−1).The experiment included a total of 672 pots ([8 no-competition × 7 replicates + 8 intraspecific-competition × 7 replicates + 28 interspecific-competition × 2 replicates]×2 nutrient treatments × 2 light treatments). The pots were randomly assigned to positions and were randomized once on 15 August within each block (i.e. the low or high light-intensity treatment). The initial height of each plant was measured on 15 July 2020, two days after the transplanting. We watered the plants daily to avoid water limitations. On 1 September 2020, we harvested the aboveground biomass of all plants. The biomass was dried at 65°C for 72 h to constant weight and then weighed to the nearest mg.The experiment in GermanyOn 15 June 2020, we sowed seeds of the 16 species into plastic trays filled with potting soil (Topferde, Einheitserde Co). On 6 July 2020, we transplanted the seedlings into 1.5-L pots filled with a mixture of potting soil and sand (1:1 v/v). Like the experiment in China, we imposed three competition treatments: competition-free, intraspecific competition, and interspecific competition. However, in this experiment, which had two times more species than the experiment in China, we only included 24 randomly chosen species pairs for the interspecific-competition treatment, and all of these pairs consisted of one naturalized non-native and one native species. For the competition-free treatment, we replicated each species two times (i.e. we had two technical replicates). For the competition treatments, we did not use technical replicates for any of the species combinations for logistic reasons. However, as we had a large number of species pairs in the inter-specific competition treatment, we had many biological replicates.The experiment took place outdoors in the Botanical Garden of the University of Konstanz (Konstanz, Germany). To vary nutrient availability, we applied to each pot once a week either 100 ml of a low-concentration liquid fertilizer (low-nutrient treatment; 0.5‰ Universol ® Blue oxide fertilizer, 18% N + 11% P + 18% K + 2.5% MgO + trace elements) or 100 ml of a high-concentration of the same liquid fertilizer (high-nutrient treatment; 1‰). In total, pots in the low- and high-nutrient treatment received 0.4 and 0.8 g fertilizer, respectively. To vary light availability, we used eight metal wire cages (size: 2 m × 2 m × 2 m). Four of the cages were covered with one layer of white and one layer of green netting material, which reduced the ambient light intensity by 84% (low light-intensity treatment; where the light intensity was on average 219.0 μmol m−2 s−1, measured on a sunny day). The remaining four cages were covered only with one layer of the white netting material, which served as a positive control for the effect of netting and reduced light intensity by 53% (high light-intensity treatment; where the light intensity was on average 678.4 μmol m−2 s−1). In other words, the low light-intensity treatment received 34% (66% reduction) of the light intensity in the high light-intensity treatment.The experiment included a total of 320 pots ([16 no-competition × 2 replicates + 16 intraspecific-competition + 32 interspecific-competition]×2 nutrient treatments × 2 light treatments). The eight cages were randomly assigned to fixed positions in the botanical garden. The pots were randomly assigned to the eight cages (40 pots in each cage) and were re-randomized once within and across cages of the same light treatment on 3 August 2020. Besides the weekly fertilization, we watered the plants two or three times a week to avoid water limitations. On 7 and 8 September 2020, we harvested the aboveground biomass of all plants. The biomass was dried at 70 °C for 96 h to constant weight and then weighed to the nearest 0.1 mg.Statistical analysesAll analyses were performed using R version 3.6.139. To test whether resource availability affected competitive outcomes between native and non-native species, we applied linear mixed-effects models to analyze the biomass of the plants in the two experiments jointly and separately, using the nlme package40. For the model used to analyze the two experiments jointly, we excluded interspecific competition between two non-natives and interspecific competition between two natives from the experiment in China, because non-native-non-native and native-non-native combinations were not included in the experiment in Germany. When we analyzed each experiment separately, the results were overall similar to the results of the joint analysis. Therefore, we focus in the manuscript on the joint analysis and present the results of the separate analyses in Supplementary Note 2.Because plant mortality was low and mainly happened after transplanting, we excluded pots in which plants had died. The final dataset contained 1180 individuals from 871 pots. In the model, we included the aboveground biomass of individuals as the response variable. We included the origin of the species (non-native or native), competition treatment (see below for details), nutrient treatment, light treatment and their interactions as fixed effects; study site (China or Germany), and identity and family of the species as random effects. In addition, we allowed each species to respond differently to the nutrient and light treatments (i.e. we included random slopes). To account for pseudoreplication41, we also included pots as random effects and cages (ten cages, eight from Germany and two from China) as random block effects. In the competition treatment, we had three levels: (1) no competition, (2) intraspecific competition, and (3) interspecific competition between native and non-native species. To split them into two contrasts, we created two variables42 testings (1) the effect of the presence of competitors, and (2) the difference between intra- and interspecific competition (see Supplementary Note 3 for details). To improve the normality of the residuals, we natural-log-transformed aboveground biomass. To improve the homoscedasticity of the residuals, we allowed the species and competition treatment to have different variances by using the varComb and varIdent functions43. Significances of the fixed effects were assessed with likelihood-ratio tests (type II) with the car package44.To determine the ‘competitive outcome’, i.e. which species will exclude or dominate over the other species at the endpoint for the community45,46, one should ideally conduct a long-term study. Alternatively, one could vary the density of each species, which mimics the dynamics of species populations across time (see refs. 47,48 for examples). However, applying this space-for-time-substitution method would have largely increased the size of the experiment, especially when combined with the light and nutrient treatments. Still, by growing plants alone, in intraspecific competition and in interspecific competition, our experiments meet the minimal requirement for measuring competitive outcome, at least in terms of short-term biomass production46,49.In the linear mixed-effects model of individual biomass, a significant effect of origin would indicate that native and naturalized non-native species differed in their biomass production, across all competition and resource-availability (light and nutrients) treatments. This would tell us the competitive outcome between non-natives and natives across different resource availabilities. For example, an overall higher level of biomass production of non-native species would indicate that non-natives would dominate when competing with natives. A significant interaction between a resource-availability treatment and the origin of the species would indicate that resource availability affects the biomass production of native and non-native species differently, averaged across all competition treatments. In other words, it would indicate that resource availability affects the competitive outcome between natives and non-natives. A significant interaction between a resource-availability treatment and the competition treatment would indicate that resource availabilities modify the effect of competition (e.g. no competition vs. competition). Other studies frequently have inferred competitive outcomes from the effect of competition by calculating the relative interaction intensity50. However, while the competitive outcome and effect of competition are often related, they are not equivalent45. This is because the competitive outcome is both determined by the effect of competition and intrinsic growth rate48,49. For example, a plant species that strongly suppress other species but has a low intrinsic growth rate still cannot dominate the community.Reporting summaryFurther information on research design is available in the Nature Research Reporting Summary linked to this article. More

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Cone characteristics: the entire set and individual variantsCones used in all the test variants did not differ from each other in terms of height (coefficient of variance in the Student t-test–F = 1.33 at p = 0.23), diameter (F = 1.77 at p = 0.08), or initial weight (F = 0.86 at p = 0.55). Analysis of variance revealed a significant difference for cone humidity (F = 2.52 at p ˂ 0.05).Cone parameters such as height, diameter and initial weight are factors that can determine the course of the extraction process. Therefore, the relationship between diameter and height for all cones used in the study was described using a linear regression equation ((y=0.2794x+8.3195)), which means that cone diameter increased by 0.28 mm per 1 mm of cone height, ((R=0.778 >0.104-{R}_{kr})).The initial weight of cones may be associated with their harvest time or storage conditions. A linear regression equation was also used to describe the relationship between the height and initial weight of the examined cones (y = 0.238x–3.918), which means that initial weight increased on average by 0.238 g per 1 mm of height, (R = 0.795  > 0.104).Table 2 shows means with standard deviations, the minimum and maximum values of the measured parameters, the range of variance, the coefficient of variation and the standard error for the entire set of studied cones and seeds. The Shapiro–Wilk test showed that the examined characteristics had a normal distribution.Table 2 Cone and seed parameters for the entire study set.Full size tableThe cones used in the study had a height of 21.4–44.1 mm and a diameter of 12.5–24.3 mm. The mean height of a cone was 33.8 (± 3.4) mm and the mean diameter was 17.8 (± 1.6) mm. The initial weight of cones ranged from 2.137 to 9.111 g, with a mean of 4.144 (± 1.019) g. The initial moisture content of cones was from 27.6 to 57.1%, with a mean of 40.4 (± 4.5)%. Analysis was performed for individual extraction variants. The mean values of cone height h, diameter d, initial weight m01, and moisture content W were calculated (Table 3).Table 3 Mean parameter values and standard deviations for the nine process variants.Full size tableThe HSD Tukey test revealed one homogeneous group for cone height encompassing all variants and two homogeneous groups for diameter. The first group consisted of all variants except 7, and the second group included all variants except 2. One homogeneous group was obtained for initial weight. Two homogeneous groups were found for moisture content, one consisting of all variants except 7, and the other one containing variants 1, 4, 5, 6, 7, 8, and 9.Seed extraction results for the studied stepsSeed extraction conditions and timeThe change in cone weight in each step of the extraction process depended on its duration, temperature and humidity conditions in the extraction cabinet, as well as on the initial moisture content of the cones.Humidity inside the drying chamber decreased to an average of 30% after 2 h of the process in each step as a result of increasing temperature. Over the subsequent 4 h of the process, after increasing the temperature, the humidity inside the chamber declined significantly, and then (over 2 and 4 h) it decreased further only slightly, stabilizing at approx. 5% for the 10 h variants, 6% for the 8 h variants, and 8% for 6 h variants on average.Moisture content changes in cones during the seed extraction processThe initial moisture content (u01) of the studied cones was much greater than 0.20 ({mathrm{kg}}_{mathrm{water}}cdot {mathrm{kg}}_{mathrm{d}.mathrm{w}.}^{-1}), which means that special care must be taken during seed extraction, which should be conducted at a temperature of up to 50 °C8.The relatively high moisture content of the cones could be attributed to the absence of preliminary drying in airy storage places prior to seed extraction (which is typically the case in commercial practice) and the early date of cone harvest, at the beginning of the extraction season. The initial (u0x) and final (ukx) moisture content of cones used in each process variant is given with standard deviation in Table 4.Table 4 Initial and final moisture content of cones used in each process variant.Full size tableThe initial moisture content of cones (u0x) in most variants increased with each extraction step due to immersion. In most variants, the final moisture content (ukx) was the highest in the first extraction step and decreased or remained at the same level with each subsequent step.The mean initial moisture content for the three process variants with 10 h of drying was 0.411 ({mathrm{kg}}_{mathrm{water}}cdot {mathrm{kg}}_{mathrm{d}.mathrm{w}.}^{-1}). After 10 h of drying, the mean moisture content decreased to 0.130 ({mathrm{kg}}_{mathrm{water}}cdot {mathrm{kg}}_{mathrm{d}.mathrm{w}.}^{-1}). The mean initial moisture content in the fifth extraction step was 0.437 ({mathrm{kg}}_{mathrm{water}}cdot {mathrm{kg}}_{mathrm{d}.mathrm{w}.}^{-1}), and the final moisture content in that step was 0.071 ({mathrm{kg}}_{mathrm{water}}cdot {mathrm{kg}}_{mathrm{d}.mathrm{w}.}^{-1}) . Cones dried for 10 h reached on average 7% moisture content after extraction steps 4 and 5.The mean initial moisture content for the three process variants with 8 h of drying was 0.412 ({mathrm{kg}}_{mathrm{water}}cdot {mathrm{kg}}_{mathrm{d}.mathrm{w}.}^{-1}). After 8 h of drying, the mean moisture content decreased to 0.128 ({mathrm{kg}}_{mathrm{water}}cdot {mathrm{kg}}_{mathrm{d}.mathrm{w}.}^{-1}) . The mean initial moisture content in the fifth extraction step was 0.440 ({mathrm{kg}}_{mathrm{water}}cdot {mathrm{kg}}_{mathrm{d}.mathrm{w}.}^{-1}), and the final moisture content in that step was 0.064 ({mathrm{kg}}_{mathrm{water}}cdot {mathrm{kg}}_{mathrm{d}.mathrm{w}.}^{-1}) . Cones dried for 8 h reached on average 7.1% moisture content after extraction step IV and 6.4% after step V.The mean initial moisture content for the three process variants with 6 h of drying was 0.389 ({mathrm{kg}}_{mathrm{water}}cdot {mathrm{kg}}_{mathrm{d}.mathrm{w}.}^{-1}). After 6 h of drying, the mean moisture content decreased to 0.129 ({mathrm{kg}}_{mathrm{water}}cdot {mathrm{kg}}_{mathrm{d}.mathrm{w}.}^{-1}) . The mean initial moisture content in the fifth extraction step was 0.415 ({mathrm{kg}}_{mathrm{water}}cdot {mathrm{kg}}_{mathrm{d}.mathrm{w}.}^{-1}), and the final moisture content in that step was 0.084 ({mathrm{kg}}_{mathrm{water}}cdot {mathrm{kg}}_{mathrm{d}.mathrm{w}.}^{-1}) . Cones dried for 6 h reached on average 8.9% moisture content after extraction step IV and 8.4% moisture content after step V, which means that their final moisture content was higher than that of cones dried for 8 h and 10 h.The cones with the longest immersion time (15 min) were characterized by the highest initial moisture content in each extraction step as compared to the other two variants (immersion of 5 min and 10 min) with the same drying time. The final moisture content in a given extraction step differed between cones with different immersion times. Cones with an immersion time of 15 min were characterized by the highest final moisture content in individual extraction steps, and those with 5 min immersion revealed the lowest final moisture content.The Tukey HSD test revealed homogeneous groups in terms of initial moisture content (u01, u02, u03, u04, u05) and final moisture content (uk1, uk2, uk3, uk4, uk5) in each step, as shown in Table 4. For instance, four homogeneous groups were found for the final moisture content after extraction step V (uk5): the first one consisted of all variants except for 7, 8, and 9, the second one included variants 1, 2, 3, and 7, the third one comprised of variants 7 and 8, while the fourth one was constituted by variant 9 alone.Using Eq. (1), changes in moisture content were described for each of the tested cones over all five steps of each variant. The equation included the initial and final values of moisture content and the b coefficient for individual cones. The average values of the b coefficient and standard deviations for each extraction step are presented in Table 5 for individual extraction variants.Table 5 Mean values of the b coefficient and standard deviations for the five steps of the studied process variants.Full size tableThe lowest value of the b coefficient was recorded for the first step of the 10h_15min variant (b1 = 0.34), while the highest value was obtained for the fifth step of the 8 h_15 min variant (b5 = 0.60). In the process variants involving 10 and 8 h of drying , the b coefficient increased with each extraction step until the third one; in the fourth step it slightly decreased and in the fifth step it remained constant. In the variants with 6 h of drying the b coefficient almost peaked in the second extraction step and remained at a similar level until the fifth step. In the first steps of the variants with 6 h of drying, the mean value of the b coefficient was 0.54 and did not differ significantly from the coefficients obtained during the other steps. It was noted that in the 8 h_15 min variant, the b coefficients increased over successive steps.Figures 2–3 show examples of curves of actual and model changes in moisture content and the rate of extraction for sample cones, one each for variants 10 h_15 min and 8 h_15 min.Figure 2Diagrams: (a) actual and predicted changes in cone moisture content, (b) extraction rate in five extraction steps for larch cone no. 32 in the 10 h_15 min variant throughout effective extraction.Full size imageFigure 3Diagrams: (a) actual and predicted changes in cone moisture content, (b) extraction rate in five extraction steps for larch cone no. 17 in the 8 h_15 min variant throughout effective extraction.Full size imageEquations for changes in moisture content and extraction rate in consecutive extraction steps are given below for the graphically for the cone shown in Fig. 2 (no. 32 in the 10 h_15 min variant):Step I: ({u}_{1}=0.264cdot {mathrm{e }}^{left(-0.38 cdot {tau }_{i}right)}+0.107) ,(frac{d{u}_{1}}{d{tau }_{1}}=-0.100cdot {mathrm{e }}^{(-0.38 cdot {tau }_{i})})Step II: ({u}_{2}=0.372cdot {mathrm{e }}^{left(-0.44 cdot {tau }_{i}right)}+0.095) , (frac{d{u}_{1}}{d{tau }_{1}}=-0.164cdot {mathrm{e }}^{(-0.44 cdot {tau }_{i})})Step III: ({u}_{3}=0.397cdot {mathrm{e }}^{left(-0.49 cdot {tau }_{i}right)}+0.086) , (frac{d{u}_{1}}{d{tau }_{1}}=-0.195cdot {mathrm{e }}^{(-0.49 cdot {tau }_{i})})Step IV: ({u}_{4}=0.536cdot {mathrm{e }}^{left(-0.44 cdot {tau }_{i}right)}+0.080) , (frac{d{u}_{1}}{d{tau }_{1}}=-0.236cdot {mathrm{e }}^{(-0.44 cdot {tau }_{i})})Step V: ({u}_{5}=0.485cdot {mathrm{e }}^{left(-0.46 cdot {tau }_{i}right)}+0.076) , (frac{d{u}_{1}}{d{tau }_{1}}=-0.223cdot {mathrm{e }}^{(-0.46 cdot {tau }_{i})})Equations for changes (Fig. 3) in moisture content and extraction rate in consecutive extraction steps are also given for this cone (no. 17 in the 8 h_15 min variant):Step I: ({u}_{1}=0.304cdot {mathrm{e }}^{left(-0.53 cdot {tau }_{i}right)}+0.113) ,(frac{d{u}_{1}}{d{tau }_{1}}=-0.161cdot {mathrm{e }}^{(-0.53 cdot {tau }_{i})})Step II: ({u}_{2}=0.292cdot {mathrm{e }}^{left(-0.55 cdot {tau }_{i}right)}+0.085) , (frac{d{u}_{1}}{d{tau }_{1}}=-0.161cdot {mathrm{e }}^{(-0.55 cdot {tau }_{i})})Step III: ({u}_{3}=0.369cdot {mathrm{e }}^{left(-0.70 cdot {tau }_{i}right)}+0.077) , (frac{d{u}_{1}}{d{tau }_{1}}=-0.258cdot {mathrm{e }}^{(-0.70 cdot {tau }_{i})})Step IV: ({u}_{4}=0.379cdot {mathrm{e }}^{left(-0.71 cdot {tau }_{i}right)}+0.059) , (frac{d{u}_{1}}{d{tau }_{1}}=-0.269cdot {mathrm{e }}^{(-0.71 cdot {tau }_{i})})Step V: ({u}_{5}=0.428cdot {mathrm{e }}^{left(-0.77 cdot {tau }_{i}right)}+0.060) , (frac{d{u}_{1}}{d{tau }_{1}}=-0.330cdot {mathrm{e }}^{(-0.77 cdot {tau }_{i})})Finally, equations for changes in moisture content and extraction rate in consecutive extraction steps are given for cone no. 5 in the 6 h_15 min variant:Step I: ({u}_{1}=0.308cdot {mathrm{e }}^{left(-0.58 cdot {tau }_{i}right)}+0.0904) ,(frac{d{u}_{1}}{d{tau }_{1}}=-0.179cdot {mathrm{e }}^{(-0.58 cdot {tau }_{i})})Step II: ({u}_{2}=0.346cdot {mathrm{e }}^{left(-0.63 cdot {tau }_{i}right)}+0.1070) , (frac{d{u}_{1}}{d{tau }_{1}}=-0.218cdot {mathrm{e }}^{(-0.63 cdot {tau }_{i})})Step III: ({u}_{3}=0.368cdot {mathrm{e }}^{left(-0.63 cdot {tau }_{i}right)}+0.0837) , (frac{d{u}_{1}}{d{tau }_{1}}=-0.232cdot {mathrm{e }}^{(-0.63 cdot {tau }_{i})})Step IV: ({u}_{4}=0.387cdot {mathrm{e }}^{left(-0.68 cdot {tau }_{i}right)}+0.0838) , (frac{d{u}_{1}}{d{tau }_{1}}=-0.263cdot {mathrm{e }}^{(-0.68 cdot {tau }_{i})})Step V: ({u}_{5}=0.396cdot {mathrm{e }}^{left(-0.65 cdot {tau }_{i}right)}+0.0743) , (frac{d{u}_{1}}{d{tau }_{1}}=-0.257cdot {mathrm{e }}^{(-0.65 cdot {tau }_{i})})Figures 2a, 3a show the curves of actual changes in the moisture content of three sample cones subjected to different drying times (10 and 8 h) but the same immersion time (15 min); the curves were fitted to a model which is widely used in descriptions of drying at constant temperature (mostly for vegetables). The present study used variable temperature, which may have influenced the fit of the model, in addition to the input variables (drying and immersion times). The best fit was found for the cone subjected to the variant with 8 h of drying (Fig. 3), with a slight deviation in the first three extraction steps, and with a very good fit in the fourth and fifth steps. The lowest fit was found for the cone subjected to 6 h drying, which may be caused by insufficient drying time (the cone was exposed to 35 °C for 2 h, and to 50 °C for only 4 h).Figures 2b, 3b show diagrams for cone extraction rates at different drying times (10 h and 8 h) at the same immersion times (15 min). As can be seen, extraction rates decreased in the very beginning, which is characteristic of the so-called second period of solid drying (Pabis44).Seed extraction dynamicsTable 2 presents data on the number of scales and seeds for the studied cones. There were from 33 to 70 open scales per cone, with an average of 48 (± 6). From 1 to 76 seeds were extracted per cone, with an average of 36 (± 18). Finally, each cone contained from 5 to 97 seeds, with an average of 52 (± 19). The weight of the extracted seeds ranged from 0.001 g to 0.651 g, on average 0.193 (± 0.109) g.Cones obtained from different process variants did not differ in terms of the number of seeds extracted (F = 0.862 at p = 0.55) or their weight (F = 0.720 at p = 0.674). However, ANOVA did reveal significant differences in the number of scales (F = 3.561 at p ˂0.05) and the total number of seeds per cone (F = 2.93601 at p = 0.003645). Table 6 gives mean scale and seed numbers per larch cone (with standard deviations) for the various extraction variants and homogeneous groups.Table 6 Mean numbers of cone scales and seeds for each process variant.Full size tableOn average, 70% of the seeds were extracted from cones used in all nine study variants, with 30% of the seeds remaining in the cones. Table 7 shows the number of seeds extracted in individual variants and the number of seeds remaining in the cones, expressed as a percentage.Table 7 Number of seeds extracted from and remaining in the cones for each process variant.Full size tableThe greatest number of seeds was obtained in process variants 2–73%, closely followed by variants 3, 1, and 7 (72%), and 8 (70%). The lowest seed yield was obtained from variant 4 (65%).In all study variants, some of the seeds were obtained in the process of extraction in the chamber and some in the process of shaking in the drum (Table 7). The highest number of seeds in the chamber was obtained in variant 2 (69%), and the lowest in variant 9 (56%). On average, the largest quantity of seeds was obtained in the chamber in the 10 h variants, and the lowest quantity in the 6 h variants. Comparing different process variants of the same drying duration, the greatest number of seeds in the chamber were obtained in variants 2, 5, and 7 (and also in variant 8—only 1% fewer). The greatest quantity of seeds extracted by shaking in the drum was obtained in variant 9 (44%), and the lowest in variant 2 (31%). On average, 38% of seeds extracted in all variants were obtained by shaking in the drum.It can be seen that in each of the variants and their individual steps, the highest number of seeds was obtained after 6 h of the process. Figure 4a–c shows the percentage of seeds obtained during the effective extraction time, where the number of seeds extracted at a given step was added cumulatively to those from the previous steps.Figure 4Percentage seed yield dynamics for each step of a five-step extraction process: (a) 10 h of drying, (b) 8 h of drying, (c) 6 h of drying.Full size imageThe diagrams in Fig. 4 show the percentage of seeds obtained throughout the entire process. Each step consists of drying, shaking, immersion, and soaking, except for step V, which involved only drying and shaking without immersion or soaking. Analysis of seed yield over 10 h of drying (Fig. 4a) shows that on average 37% of all extracted seeds were obtained in the first step, 26% in the second step, approx. 20% in the third step, 11% in the fourth step, and about 6% in the fifth step.As regards the 8 h process (Fig. 4b), on average 30% of all extracted seeds were obtained in the in the first extraction step in the 8 h_5 min and 8 h_15 min variants, and as much as 53% in the 8 h_10 min variant. An average of 27% of all seeds were extracted in the second step, 15% in the third step, about 11% in the fourth step, and approx. 5% in the fifth step. The 8 h_10 min variant was characterized by the highest seed yield, beginning in the first step of the process (as compared to the 8 h_5 min and 8 h_15 min variants).As far as the variant with 6 h of drying is concerned (Fig. 4c), on average approx. 46% of all extracted seeds were obtained in the first step, 24% in the second step, 15% in the third step, approx. 11% in the fourth step, about 4% in the fifth step.When extracting seeds from larch cones, scale deflection and the number of obtained seeds are not assessed during the process, as is the case with pine and spruce cones due to the difficulties caused by the aforementioned morphology of larch cones (Tyszkiewicz, 1949). The presented diagrams show that a satisfactory seed yield (60%) was obtained in variants with 8 and 6 h of drying already after 10 h of effective extraction time.The seed yield coefficient, α (3), and the cone mass yield coefficient, β (4), for each extraction variant are presented in Table 8.Table 8 Seed yield coefficient and cone mass yield coefficient for each process variants.Full size tableThe seed yield coefficient was the highest for variants 2 (0.73) and 3 (0.72), and the lowest for variants 4 (0.65) and 6 and 9 (0.67). The cone mass yield coefficient was the highest for variant 5, and the lowest for variant 9.Seed viabilityTable 9 presents germination energy (E) and capacity (Z) for the control seeds as well as for seeds obtained from the various steps of the nine process variants, as well as their corresponding quality classes.Table 9 Germination energy and capacity for the control seeds as well as for seeds obtained from the various extraction process variants.Full size tableGermination energy and capacity for the control sample were 45% and 57%, respectively, meaning that naturally released seeds, not subjected to any thermal or mechanical treatments, were classified in quality class I18. Importantly, seeds obtained from all the studied process variants were also placed in the same class; their germination energy ranged from 30 to 59%, and their germination capacity from 35 to 61%. When analyzing each extraction step separately, no correlation was found between decreasing germination energy and successive steps. However, the average germination energy was 46% for seeds obtained in the first extraction step of all nine variants, 45% for those from the second and third steps, 41% for seeds from the fourth step and 40% for those from the fifth one. Thus, in each subsequent step the average germination energy of seeds was equal or lower than in the previous step, which is consistent with literature reports that prolonged drying may reduce the quality of seeds8. This is also corroborated by the fact that the highest germination energy and capacity was revealed by seeds from variants with 6 h of drying while the lowest germination indicators characterized seeds from the 10 h variants. Furthermore, seeds from variant 1 exhibited the lowest germination energy and capacity and seeds from variant 8–the highest.Another reason for the higher quality of seeds from variants with 6 h of drying may be the lower initial moisture content of the cones due to the longer time they were kept at room temperature immediately before the test (u01 = 0.391 ({mathrm{kg}}_{mathrm{water}}cdot {mathrm{kg}}_{d.mathrm{w}.}^{-1}) as compared to u01 = 0.411 ({mathrm{kg}}_{mathrm{water}}cdot {mathrm{kg}}_{d.mathrm{w}.}^{-1}) for seeds from variants with 8 and 10 h of drying). These results are in line with the study of Tyszkiewicz8, who noted that under the same temperature and humidity conditions, the quality of seeds from cones with a lower moisture content did not deteriorate, in contrast to the quality of seeds obtained from cones with a higher moisture content.The germination capacity of seeds calculated from the mean capacity of seeds obtained from the same extraction steps of all process variants was similar at 45% for each of the steps.In summary, in the study the authors investigated a five-step process of extracting seeds from larch cones involving immersion and heat treatment to maximize seed yield. It was found that the two-step process widely used in extractories is insufficient, while a four-step process does not lead to a significantly higher number of obtained seeds. Thus, a three-step process appears to be optimal. More