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COVID-19 vs. MoMoThe COVID-19 official deaths and MoMo ED time series overlaped for the period from 3 March 2020 to 1 January 2021 for Spain and its 19 regions (Fig. 2). In general, there was good agreement between both datasets, meaning that most of MoMo ED were related to COVID-19 deaths. During the first wave, the most important differences were observed in Spain, Madrid, Cataluña, Castilla-La Mancha, and Castilla y León. Before 22 June in Spain, MoMo ED showed 15,445 accumulated deaths more than the official COVID-19 deaths, which is beyond the error band. That difference comes basically from the four regions with the largest numbers of deaths (Madrid, Cataluña, Castilla-La Mancha, and Castilla y León). Table 1 shows the accumulated values before 22 June, which were used to estimate the CFR for Spain and its 19 regions according to the third phase of the National Seroprevalence Study4,5. For all regions, the CFR estimated from MoMo ED was larger than the CFR estimated from COVID-19 deaths. In particular, Asturias, Canarias, and Murcia were twice as large. Ceuta and Melilla dramatically increased their CFR from MoMo ED, although that may be biased due to their small populations and numbers of deaths.Similarly, the same variables for the period from 23 June to 29 November 2020 are reported in Table 2. In Spain, MoMo ED showed 6173 accumulated deaths more than the official COVID-19 deaths. This difference is a third of the difference observed prior to 22 June; because this is within the error band, there was a significant improvement in the detection of COVID-19 deaths in this period. Figure 2 also shows a general agreement between MoMo ED and official COVID-19 deaths time series after the first wave, with the exception of late July and early August. These differences were due to two heat waves that were responsible for at least 25% of the MoMo ED16.Infections estimated from COVID-19 deathsTo illustrate the delay between official daily infections data and REMEDID estimated daily infections, we applied REMEDID from COVID-19 deaths assuming CFR = 100%. Figure 3 shows the current IO21 and the infections associated with COVID-19 deaths for the first wave. The latter in Spain reached a maximum on 13 March 2020 (Table 3), the day before the national government decreed a state of emergency and national lockdown. Thus, the adopted measures had an immediate effect, which was observed in the official data IO21 7 days later (20 March). This delay is similar to the incubation period (mean 5.78 days2), which could be explained because official infections were reported when symptoms appeared. This delay reached 16 days when we compared with earlier version IO20 (not shown), which highlights the usefulness of the methodology to reinterpret official data from very early stages of the pandemic. On the other hand, the maximum number of deaths was reached on 1 April, which was 19 days after the inferred infection maximum, bringing this delay close to the 20 days expected between infection and death (Figs. 1, 3).Figure 3Official COVID-19 infections and deaths, and estimated infections with case fatality ratio (CFR) of 100% in Spain during the first wave. Left y-axis: COVID-19 daily infections IO21 (blue curve). Right y-axis: COVID-19 deaths (orange curve) and its REMEDID-estimated infections with CFR = 100% (red curve). All curves are for Spain. Thin blue and orange curves are daily data, and thick curves are smoothed by 14-day running mean. Arrows show delays between the maximum of inferred infections and maxima from COVID-19 deaths (orange arrows) and COVID-19 infections (blue arrows). Solid arrows are expected delays, dotted while arrows are observed delays.Full size imageTable 3 Date of first infection for REMEDID estimated daily infections from COVID-19 deaths (IRO) and from MoMo Excess Deaths (ED) (IRM), and for official COVID-19 daily infections released on June 2020 (IO20) and on February 2021 (IO21).Full size tableWe applied REMEDID to the official COVID-19 deaths with the corresponding estimated CFRs (see “Data” section) to obtain the time series of estimated daily infections, hereafter referred to as IRO. Figure 4 shows IRO and the accumulated infections for Spain and its 19 regions. Note that in Spain, IRO are amplified versions of inferred infections in Fig. 3. In Spain, the first infection, according to IRO,, is on 8 January 2020 (Table 3), 43 days before the first infection was officially reported on 20 February 2020 according to IO20. By contrast, IO21 places the first infection on 1 January 2020. Spain reached the maximum number of IRO on 13 March, a day before the state of emergency and lockdown were enforced (Table 4). On 14 March, IO20 = 1832, and IO21 = 7478; however, IRO = 63,727 (CI 95% 60,050–67,403), 35 and 9 times IO20 and IO21, respectively (Table 5). This implies that on that day, IO20 and IO21 only reported 2.9% (CI 95% 2.7–3.1%) and 11.7% (CI 95% 11.1–12.5%) of new infections, respectively. Although detection of infections clearly improved from IO20 to IO21, almost 90% of the infections are still not documented in the peak of the first wave. The situation is similar for the accumulated infections before 22 June 2020, as reported by the National Seroprevalence Study4,5.Figure 4Daily and accumulated infections for official COVID-19 daily infections (IO21), and daily infections estimated from COVID-19 deaths (IRO). Lines are daily infections and refer to the y-axis on the right; bars are accumulated infections and refer to the y-axis on the left. Red lines and cyan bars are official COVID-19 data; orange lines and blue bars are inferred infections with case fatality ratio (CFR) in Table 1. Thin orange lines correspond to the CFR confidence interval.Full size imageTable 4 Date of the most prominent relative maxima, for Spain and the 19 regions, of the REMEDID estimated daily infections from COVID-19 deaths (IRO) and from MoMo excess deaths (ED) (IRM), and official COVID-19 daily infections (IO21).Full size tableTable 5 REMEDID estimated daily infections from COVID-19 deaths (IRO) and from MoMo excess deaths (ED) (IRM) on 14 March, and for official COVID-19 daily infections released on June 2020 (IO20) and released on February 2021 (IO21).Full size tableIn almost all regions, IO20 showed a delay of 1 month or more between the first infection and IRO (Table 3). No delay in IO21 occurred in Islas Baleares, Castilla-León, and Galicia, while in three regions (Cataluña, Madrid, and La Rioja), the first case occurred earlier than the first case of IRO. However, 6 regions had delays of 15 days and other 6 regions had delays of 1 month. According to IRO, all regions except Ceuta and Melilla had some infections in January, but in IO21 only 6 regions had infections in that month. In all scenarios, the first infections were in Madrid and Cataluña.During the first wave, according to IRO most of the regions had maximum daily infections around 14 March. In Madrid, the maximum was reached on 11 March, coinciding with the educational centres closing and an official warning by the regional government (Table 4). Asturias was the last region to reach peak infections (25–26 March). The maximum percentage of documented cases (12.6%, CI 95% 9.2–18.4%) occurred in Asturias on 14 March, but in the other regions, only between 1.2 and 8% of the infections were documented (Table 5).Figure 4 shows how the IO21 and IRO curves of Spain and the 19 different regions fluctuated following the same pattern until the middle of June 2020, but thereafter, they showed different patterns. This reflects the fact that the Spanish government had decreed the control measures for the whole nation until June, but thereafter, each regional government implemented its own control measures. For example, some regions (e.g., Aragón, Islas Baleares, Cantabria, Comunidad Valenciana, Extremadura, Galicia, Murcia, País Vasco, and La Rioja) had two peaks, but others had only one. An apparent maximum on 22 June in Islas Baleares is an artifact produced by the interpolation for transition from the two CFRs. Although beyond the scope of this work, it would be very interesting to investigate the effects of the different control measures implemented on the corresponding IRO for the 19 regions.The Spanish COVID-19s wave reached a maximum of daily infections on 22 October from IRO and on 26 October from IO21. The delay of 4 days is similar to the mean incubation period (5.78 days2). The estimated number of new infections is still larger than the documented cases, but the shapes of the two curves are more similar in the second wave than in the first wave (Fig. S1). The same is true for the 19 regions, most of which had the largest peak around 22–26 October, with the exceptions of Canarias and Madrid, which reached maxima in late August and early September, respectively.Infections from MoMo excess deathsAssuming that MoMo ED accounts for both recorded and non-recorded COVID-19 deaths, negative deaths are meaningless, and they were set to zero. Then, the associated daily infections can be estimated, as in “Infections estimated from COVID-19 deaths” section, with a CFR of 100% from MoMo ED for Spain (Fig. 5). Note two main differences between this time series and that estimated from official COVID-19 deaths: (1) MoMo data present an error band that was inherited by the estimated infections; (2) MoMo ED estimated infections reached a maximum of 1443 (CI 99% 1329–1547), doubling the 776 inferred daily infections from official COVID-19 deaths in Fig. 3. This is because maximum MoMo ED was 1,584 (CI 99% 1468–1686) and maximum COVID-19 official deaths was 828, both estimated from the 14-day running mean time series. The maximum of inferred infections was reached on 13 March, just one day prior to the state of emergency and lockdown. The expected and observed delays with respect to official infections and MoMo ED were similar to those observed for estimated infections from official COVID-19 deaths. Error bounds of the estimated infections in Fig. 5 were computed from the MoMo ED error bounds. However, it should be highlighted that the combination of the error bounds from MoMo ED and the estimated CFRs might lead to unrealistic error estimates. To avoid this, the error estimates in Fig. 6 were estimated from the MoMo ED time series (no error bounds) and the error bounds of the estimated CFRs.Figure 5Official COVID-19 infections, MoMo Excess Deaths (ED), and estimated infections with case fatality ratio (CFR) of 100% in Spain during the first wave. Left y-axis: COVID-19 daily infections IO21 (blue curve). Right y-axis: MoMo ED (orange curve) and its REMEDID-estimated infections with CFR = 100% (red curve). All curves are for Spain. Thin blue and orange curves are daily data, and thick curves are smoothed by 14-days running mean. Dashed curves represent the error estimate of MoMo ED (orange) and inferred infections (red). Arrows show delays between the maximum of inferred infections and maxima from MoMo ED (orange arrows) and COVID-19 infections (blue arrows). Solid arrows are expected delays, dotted while arrows are observed delays.Full size imageFigure 6Daily and accumulated infections for official COVID-19 daily infections (IO21), and daily infections estimated from MoMo Excess Deaths (ED) (IRM). Lines are daily infections and refer to the y-axis on the right; and bars are accumulated infections and refer to the y-axis on the left. Red lines and cyan bars are for official COVID-19 data; and orange lines and blue bars are for inferred infections with case fatality ratio (CFR) in Table 1. Thin orange lines represent the error estimate of inferred infections.Full size imageThe REMEDID was applied to the MoMo ED with the corresponding CFRs (see “Data” section) to obtain the estimated daily infections, which will be referred hereafter as IRM. The IRM were calculated for Spain and its 19 regions and are depicted in Fig. 6, as well as the accumulated IRM. In Spain, the first infection shown by IRM happened on 9 January, with an error estimate from 9 to 10 January, 41 to 42 days before the first documented infection of IO20 on 20 February 2020 (Table 3). The maximum IRM was 77,855 (CI 95% 73,364–82,347) reached on 13 March. On 14 March, IRM showed 14,128 infections more than IRO (Table 5). Notice that the CFR used with MoMo ED data was larger than the one used with official COVID-19 deaths data, which makes this difference even more remarkable, because the larger the CFR the lower the estimated infections. Therefore, if the true CFRs, which are unknown, were used in both cases, IRM would double IRO on 14 March, as happened when a CFR of 100% was used (Figs. 3, 5). Notice that with the CFRs used, the IRM and IRO resulted in the same accumulated infections on 22 June and 29 November, matching the results of the seroprevalence study. Nevertheless, IRM showed 42 times more cases than IO20 and 10 times more than IO21 on 14 March, detection of official cases of only 2.4% (2.2–2.5%) and 9.6% (9.1–10.2%), respectively.Table 3 shows the estimated date of first infection for Spain and by region. Note that the first cases of IRM in Spain were on 9 January and in Aragón, Canarias, and Navarra on 8 January, which is possible because significant excess deaths in a region may not become significant for the whole country. In general, the maxima of daily infections were closer to those on 14 March in IRM than in IRO. During the first wave, all regions showed a single maximum, except for Ceuta, Melilla, and Murcia, which showed two maxima (Fig. 6). In general, the IRM time series in all regions were similar during that period. The official data clearly under-detected infections during the first wave. On 14 March, IRM were comparable to IRO, overlapping CI in all regions, but not in Spain as a whole (Table 5). During the second wave, there was improved detection of cases with differences among regions. More

Study site and speciesThe sub-tropical forest analysed in this study is located in China, Yunnan, Xishuangbanna, Mengla county, Village Quingyanzhai (#94) N 21.802068, E 101.380214, geodetic datum WGS84 (Fig. 6). The site is characterized by a rocky outcrop rising 30–50 m over surrounding rubber plantations, harbouring about 20 ha of relict dry tropical forest. The outcrop sides are steep and mainly covered with bamboo. The top area is colonized by shrubs and 10–15 m high trees (a few trees on the slopes are much higher). The most conspicuous species is Quercus yiwuensis Y.C. Hsu & H.W. Jen. In March 2017 we selected four individual trees of Q. yiwuensis, and an equal number of Pistacia weinmannifolia Franch, and Beilschmiedia percoriacea C.K. Allen that were also numerous on the site. Plants were identified by the authors in the field and labelled. Botanical specimens were deposited in the School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China.Figure 6Map of the study site with approximate position of analysed trees (aerial perspective from Google Maps 2018). GPS positions were obtained less than 1 m from the tree trunks. The distance from N3 to B1 is approximately 60 m.Full size imageQ. yiwuensis was designated P-tree (P1, P2, P3, P4) because we consistently found the orchid Panisea uniflora Lindl. growing on this phorophyte species. P. weinmannifolia was designated B-tree (B1, B2, B3, B4) because it harboured the orchid species Bulbophyllum odoratissimum (Sw) Lindl. (Supplementary Fig. S2 a-d). On P. weinmannifolia trees no P. uniflora was observed, while B. odoratissimum was never found on Q. yiwuensis trees. Both tree species were richly colonized by several other orchid species. Beilschmiedia percoriacea trees were designated neutral tree, N-tree (N1, N2, N3, N4), because neither of the two target orchid species grew on them. The latter tree species carried several lichens and a single fern species (Lepisorus sp.), but only in one instance was observed to carry an orchid epiphyte (Coelogyne sp.).GPS positions of investigated trees were obtained less than 1 m from the trunk (Fig. 6). Accuracy is about 3 m. Accuracy in altitude readings is about 100 m. Distance between degrees of latitude is 111 km. At N 21.78978 the distance between degrees of longitude is 103 km, which means that the last digit in the 5-digit decimal degrees corresponds to 1.11 m in latitude and 1.03 m in longitude.The trees were labelled with different colours as follows:

P-trees (carrying P. uniflora and other epiphytes, but not B. odoratissimum), identified as Q. yiwuensis, with red labels (P1 N 21.79880, E 101.37909, 1073; P2 N 21.79882, E 101.37923, 1072; P3 N 21.79878, E 101.37947, 1074; P4 N 21.79878, E 101.37904, 1073).

B-trees (carrying B. odoratissimum and other epiphytes, but not P. uniflora), identified as P. weinmannifolia, with blue labels (B1 N 21.79878, E 101.37950, 1074; B2 N 21.79880, E 101.37938, 1078; B3 N 21.79881, E 101.37931, 1083; B4 N 21.79884, E 101.37923, 1076).

N-trees (carrying epiphytes, but neither B. odoratissimum nor P. uniflora), identified as B. percoriacea, with yellow labels (N1 N 21.79873, E 101.37905, 1064; N2 N 21.79868, E 101.37908, 1072; N3 N 21.79883, E 101.37893, 1071; N4 N 21.79879, E 101.37895, 1071).

The point of access to the outcrop top area was located at the Western edge (N 21.79880, E 101.37827, 1058, Fig. 6).

SamplingFor each of the twelve selected trees, breast height circumference (BH = 130 cm above ground) was measured. Approximate total height was determined by Nikon Laser Forestry Pro or estimated if sighting lines were interfered by other vegetation.The lowermost individual of the target orchid species was recorded in relation to BH. Bark samples were collected, and bark features recorded at BH, by target orchid, and 50 cm above target orchid or BH, whichever was highest point. In N-trees, where there were no target orchids, sampling was thus at BH, BH + 50 cm, and BH + 100 cm.Sampling on each tree involved approximately 12 cm2 bark cut out with a sterile knife and rubber gloves to prevent cross-contamination, for pH-analysis, metabarcoding, fungal isolation and chemical analysis. Besides, 3 bark cores were taken by trephor sampler (16 mm, 2 mm diam., Costruzioni Meccaniche Carabin Carlo) for water holding measurement.Roots of target orchids were sampled, from three adult individual plants on each P- and B-tree. No permissions were necessary to collect plant samples, using a protocol that avoided plant damages. All plants were left in the exact location where they were found in the sampling site, after collecting the small portions of bark and root material for the study. All experiments including the collection of plant material in this study are in compliance with relevant institutional, national, and international guidelines and legislation.All fresh material collected from the sampling site was first kept in cool boxes, brought to the laboratory, and processed within three days.Fungal isolation from barkFor each sample, half of the bark material and orchid roots were kept at − 80 °C for subsequent metabarcoding analysis. The rest of bark (about 2 g for each sample) was immediately processed for fungal isolation. The large bark portions were ground into powder using a sterile mortar and pestle; 5 ml were reserved for pH measurement, while the rest was suspended in a final volume 50 ml sterile water solution in a sterile centrifuge tube. The tube was shaken with Vortex vibration meter thoroughly and solution aliquots were spread homogenously onto isolation medium plates. For each bark sample, aliquots of 500, 300, 200, and 100 μl, were spread per triplicate to one plate each of PDA (Potato Dextrose Agar) medium, containing ampicillin (50 μg/mL) and streptomycin (50 μg/ml) to inhibit bacterial growth49,50. A diluted solution was also made by mixing 1 ml of the original solution with 9 ml sterile water and plated. Petri dishes were incubated at room temperature (23–25 °C) in the dark for up to 2 months to allow the development of slow-growing mycelia. Fast growing fungal strains started to grow after about two days. Colonies showing different morphology and appearance were transferred to fresh plates to obtain pure cultures. In the following days, other slower growing mycelia were available in the Petri dishes and were also regularly picked up and isolated onto new PDA plates every 2 days. All isolated fungal strains were stored at 4 °C for further analysis. All strains were deposited in the LP Culture Collection (personal culture collection held in the laboratory of Prof. Lorenzo Pecoraro), at the School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China.Molecular and morphological analysis of bark culturable fungiThe identification of isolated fungal colonies was performed using DNA sequencing combined with microscopy. Total genomic DNA from isolated fungi was extracted following the cetyltrimethyl ammonium bromide (CTAB) method modified from Doyle and Doyle51. Fungal ITS regions were PCR-amplified using the primer pair ITS1F/ITS452 following the procedure described in Pecoraro et al.37 for PCR reaction, thermal cycling, and purification of PCR products. Controls with no DNA were included in every amplification experiment in order to test for the presence of laboratory contamination from reagents and reaction buffers. Purified DNA amplicons were sequenced with the same primer pair used for amplification. DNA sequencing was performed at the GENEWIZ Company, Tianjin, China.Sequences were edited to remove vector sequences and to ensure correct orientation and assembled using Sequencher 4.1 for MacOsX (Genes Codes, Ann Arbor, MI). Sequence analysis was conducted with BLAST searches against the National Center for Biotechnology Information (NCBI) sequence database (GenBank; http://www.ncbi.nlm.nih. gov/BLAST/index.html) to determine the closest sequence matches that enabled taxonomic identification. DNA sequences were deposited in GenBank (Accession Nos. MW603206 – MW603451). Fungal morphological characters (hyphae, pseudohyphae, conidiophores, conidia, poroconidia, arthroconidia, etc.) were examined using a Nikon ECLIPSE Ci microscope for the identification of isolates following the standard taxonomic keys53,54,55,56,57.Assessment of bark and orchid associated fungal community using Illumina sequencingBark and orchid root samples were pulverized in a sterile mortar, and genomic DNA was extracted using the FastDNA® Spin Kit as described by the manufacturer (MP Biomedicals, Solon, OH, USA)58,59. In total, this resulted in 60 DNA samples, including 36 from bark (3 sampling points for each tree × 12 trees) and 24 from orchid roots (3 orchid individuals sampled on each P- and B-tree × 8 trees; the 4 individual N-trees were not used for orchid sampling because they did not carry the study orchid species). Subsequently, amplicon libraries were created using two primer combinations targeting the internal transcribed spacer 2 (ITS-2): ITS7F and ITS4R60 was used as universal fungal primer pair to target nearly all fungal species, while ITS361 and ITS4OF62 was used to more specifically target orchid mycorrhizal fungi. Previous research has shown that most universal fungal primers have multiple mismatches to many species of the orchid-associating basidiomycetes, in particular in Tulasnellaceae family46,58,63. Since the goal of the present work was to analyse the total fungal community in the orchid-phorophyte environment (bark and orchid roots), as well as more specifically detect the orchid mycorrhizal fungi in the studied samples, it was necessary to combine two different primer pairs to characterise the whole investigated fungal diversity47,64,65,66. Polymerase chain reaction (PCR) amplification was performed in 50 μl reaction volume, containing 38 μl steril distilled water, 5 μl 10 × buffer (100 mM Tris–HCl pH 8.3, 500 mM KCl, 11 mM MgCl2, 0.1% gelatin), 1 μl of dNTP mixture of 10 mM concentration, 0.25 μM of each primer, 1.5 U of RED TaqTM DNA polymerase (Sigma) and approximately 10 μg of extracted genomic DNA. PCR conditions were as follows: 1 cycle of 95 °C for 5 min initial denaturation before thermocycling, 30 cycles of 94 °C for 40 s denaturation, 45 s annealing at various temperatures following Taylor and McCormick62, 72 °C for 40 s elongation, followed by 1 cycle of 72 °C for 7 min extension. To minimize PCR bias, three PCRs were pooled for each sample. The resulting PCR products were electrophoresed in 1% agarose gel with ethidium bromide and purified with the QIAEX II Gel Extraction Kit (QIAGEN). Amplicon libraries were generated using the NEB Next Ultra DNA Library Prep Kit for Illumina (New England Biolabs, USA) following the manufacturer’s instructions to add index codes. Samples were sequenced using the Illumina MiSeq PE 250 sequencing platform (Illumina Inc., San Diego, CA) at Shanghai Majorbio Bio‐Pharm Technology Co., Ltd. (Shanghai, China).Bioinformatics of fungal sequencesSequences originated from the total (ITS7F and ITS4R primers) and orchid-associated (ITS3 and ITS4OF primers) fungi datasets were processed separately. Raw reads were merged with a minimum overlap of 30 nucleotides, and the primer sequences were trimmed off. Subsequently, reads were filtered by discarding all sequences with expected error  > 1. The quality-filtered reads were denoised using the UNOISE3 algorithm67 to create zero-radius operational taxonomic units (zOTUs), with chimera removal. All the steps were performed using USEARCH v.1168. Raw sequences have been deposited in the Sequences Read Archive (SRA) of NCBI as BioProject ID PRJNA702612. The fungal zOTUs were assigned to taxonomic groups using the Blast algorithm by querying against the UNITE + INSD fungal ITS database (version 7.2, released on 10 October 2017)69 using the sintax algorithm with 0.8 cutoff70. The zOTUs originated with the orchid-associated fungal primers were manually screened for possible orchid-associated mycorrhizal families based on the information provided in Table 12.1 in Dearnaley et al.71, and only these were retained for further analysis in this dataset.To attempt removing spurious counts due to cross-talk (assignment of reads to a wrong sample) we removed all the zOTUs represented by less than 0.02% of reads in each sample, which is more conservative than previous error estimates72. The datasets were rarefied to the minimum sequencing depth (23,419 for total fungi and 13,074 for orchid-associated fungi), zOTUs present in less than three samples and low abundant zOTUs (with relative abundance  More

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