The short time span of our experiment together with the small number of sampling time points causes our results to be rather a short sequence of snapshots than a time course of microbial succession on litter. Therefore, we avoid interpreting the results as “trends” and concentrate on differences between pairs of time points. Nevertheless, we think that the results provide an interesting insight into early stages of pine litter decomposition at the molecular level.
We observed no influence of enzymatic treatment neither on the amount of DNA isolated nor on microbial diversity. This might have been caused by the effectiveness of bead beating, making the action of enzymes pointless. In our previous study, we observed the influence of enzymes, as the method of isolation was based on less effective thermal/chemical lysis . However, an alternative explanation is possible, assuming that cell envelopes of microbes in our samples were not digested by the enzymes used. Albeit possible, it is unlikely at least in the case of lysozyme, taking into consideration high numbers of Proteobacterial reads generated from the samples. We found that the enzyme worked well on E. coli DH10B in the buffer used for digestion. However, no increase in fungal DNA yield could be an effect of digestion conditions that were suboptimal for the chitinase used (according to the producer the optimal pH is 4, while the buffer used for digestion had pH 8). It seems that there is no reason for enzymatic pre-treatment of litter samples prior to bead-beating-based DNA isolation. Other studies report significant influence of lysozyme digestion on soil DNA yield, even in combination with bead-beating ; however, the bead-beating method was not as effective as in contemporary kits using dedicated bead-beaters.
DNA content of litter samples significantly increased during the studied decomposition period. This increase was caused by greater numbers of bacterial and fungal cells, as we found that pine DNA was almost completely degraded in t1 samples. This was confirmed by results of qPCR analysis of bacterial 16S rRNA and fungal ITS copy numbers. The number of bacterial sequences increased first (in the t1 samples), supporting our hypothesis that fresh litter is first colonized by bacteria, albeit, alternatively, this increase might have been caused by growth of phyllospheric organisms. It seems plausible that bacteria are responsible for pine DNA degradation.
We have found no archaeal reads in our libraries, which might mean that there were no Archaea in our samples. This is supported by results of a metaproteomic study concerning beech litter decomposition . Nevertheless, it is possible that Archaea in pine litter might belong to a group whose 16S rRNA sequences differ from those amplifiable with the primer system we used, e.g., Nanoarchaeota, Altiarchaeota, or Diapherotrites or, as in all phyla the coverage was well below 90%, they could be members of generally amplifiable groups having non-amplifiable variants. There are reports of Archaea being cultured from Scots pine ectomycorrhizas (Methanolobus, Halobacterium, and unknown member of 11.c Crenarchaeota) , and ~ 2% of sequenced transcripts from spruce litter were coming from Archaea  and both facts support the latter possibility.
Shannon’s diversity, evenness, and species richness were consistently higher for bacterial communities than for fungal ones, which might be caused by the number of bacterial taxa being around one order of magnitude greater than the number of fungal ones in soils . Moreover, bacterial diversity grew over time, while fungal communities became less diverse. This effect might have been caused by three factors: litter humidity, temperature, and time (decomposition stage). Although precipitation was lower in the t0–t1 period than in t1–t2 one, water content was the highest in t1 samples, which was probably caused by low temperature. During the initial period of the experiment (t0–t1), temperature was ~ 0 °C with occasional snow, and diversity did not change significantly, neither in the case of fungi, nor bacteria, which suggests weak selection. Later, warm period was hallmarked by bacterial species richness increase, showing possible colonization of litter by bacteria from the surrounding environment. Evenness was lower in t2 samples causing diversity to stay at the t0 level. In the case of eukaryotic community diversity, species richness and evenness decreased significantly in the t2 samples, suggesting strong selection.
t0 samples harbored a bacterial community similar to pine phyllosphere community , with high shares of Proteobacterial sequences, particularly members of the Acetobacteraceae and Pseudomonadaceae families being the hallmarks of phyllosphere. Differences might have resulted from tree species being different (Scots pine vs. limber pine), geographical distance (Central Europe vs. California), and physiological state of needles (dry, fallen vs. fresh ones). The eukaryotic t0 community consisted of Fungi, out of which Ascomycotal class Pezizomycotina prevailed, which was similar to fresh pine needles community studied by Millberg and colleagues  and unlike in senescent oak leaves, where Dothideomycetes prevailed . Again, the differences might have resulted from geographical distance (Sweden vs. Poland) and physiological state of the needles. Eukaryotic sequences that could be identified down to the genus level belonged mainly to known phyllospheric fungi, such as Phoma, Trichoderma, or Lophodermium, and the latter might act as an early decomposer in litter [67, 68]. Low number of sequences was assigned to Naemacyclus (Helotiales, synonym Cyclaneusma), a genus comprising fungi causing needle cast ; thus, it is plausible that certain amount of collected needles were shed prematurely due to its action.
Differences between t1 and t0 samples were profound, in spite of temperature being below 0 °C most of the time. After 3 months of field incubation, the samples harbored communities that were drastically different from t0 ones; this fact demonstrates that colonization of fresh litter by soil and older litter-inhabiting organisms is rapid, regardless of harsh environmental conditions. This is in line with results of earlier studies on mass loss, e.g.,  and with reports concerning decomposition of broadleaf litter [17, 19, 20]. Reads coming from phyllospheric organisms were less abundant than in the t0 samples (e.g., members of Acetobacteraceae, as well as fungi Lophodermium and Phoma), showing that, in spite of the lack of diversity decrease, selection operates at this stage of litter decomposition, but organisms unable to survive are replaced by colonizers. Obvious fungal colonizers were members of Sistotrema, ectomycorrhizal/saprophytic fungi of the Cantarellales order (Basidiomycota, Agaricomycetes), and Ceuthospora (synonym Phacidium, of Helotiales (Ascomycota, Dothideomycetes), , a genus comprising plant pathogenic fungi causing various diseases from needles rot in conifers to fruit rot in cranberries, apples, or pears [72, 73]. Reads derived from these organisms were virtually absent from t0 samples and constituted over 50% of all eukaryotic reads in t1 ones. Small numbers of reads obtained from t0 samples might be explained by spores sedimenting from air on the collected needles. The eukaryotic community at this stage of decomposition was more similar to the initial one than the bacterial one, suggesting that bacteria were able to grow more actively under winter conditions. C:N ratio was highest in t0 samples, indicating nitrogen depletion, probably due to organic nitrogen being consumed by microbes.
To no surprise, at the end of the experiment, the samples harbored communities different from both t0 and t1 ones. As seasonal differences in microbial community composition in spruce litter were not found to be large , we suggest that the differences visible in our data should be attributed to decomposition stage. Phyllospheric organisms were even less abundant in t2 than in t1, typical soil organisms could be found, such as nematodes of the Chromadorea class or bacteria of the Rhizobium genus, and the litter was colonized by organisms belonging to Dothideomycetes (Fungi, Ascomycota) and Sphingomonadaceae as well Bradyrhizobiaceae (Bacteria, Alphaproteobacteria), probably capable of degradation of recalcitrant compounds (lignocellulose, lignin) prevailing at this stage of decomposition . The presence of nematodes might be one of the causes of slower increase of bacterial 16S rDNA counts, as the most abundant phylotype of this group (Plectus) is a bacterivore. This is supported by the results of co-correspondence analysis, as the influence of eukaryotic community on the bacterial one is significant and explains ~ 10% of variation. On the other hand, interactions among Eukaryota cannot be excluded, e.g., certain fungivorous nematodes most abundant in the t1 samples (e.g., members of the Aphelenchoides genus ) might be involved in much lower Sistotrema abundance in the t2 samples. The influence of bacteria on the eukaryotic community was even stronger (~ 22%). It might be explained, e.g., by providing nitrogen (by N2 fixation) that seems to be a limiting factor, particularly in t0 samples. This is confirmed by the results of CCA, wherein C/N was identified as a significant environmental variable. It is also possible that bacteria provided fungi with phosphorus, another limiting nutrient, by solubilizing its soil resources . As the measured environmental variables were responsible for explaining of over 37% of variance, it seems plausible that they were the key drivers of microbial community structure changes.
Bacterial community structure at the level of genus was surprisingly similar to this found at the corresponding stages of decomposition in oak leaf litter , where Pseudomonas, Sphingomonas, and Burkholderia were among the dominating genera. Differences were mainly quantitative and were more pronounced in the phyllospheric communities. The most remarkable difference was the lack of Duganella (Oxalobacteriaceae, Betaproteobacteria) and Frigoribacterium (Micrococcaceae, Actinobacteria) in pine needles; these genera were replaced by unclassified members of the Acetobacteraceae family. Subsequent stages of pine litter decomposition harbored less Pedobacter. This fact suggests that, while plants assemble unique phyllospheric communities, decomposers’ assemblages, at least bacterial, may be more generic and similar regardless of the quality of litter.
Fungal communities decomposing pine litter are less similar to those degrading oak leaf litter described by Voriskova and colleagues ; however, general picture looks similar: phyllospheric communities are rapidly replaced by distinct communities characteristic for particular stages of decomposition. Certain fungi are abundant both in oak and pine decomposing litter, e.g., Athelia, Rhodotorula, and Sistotrema. Interestingly, Sistotrema, most abundant in 12-month-old oak litter , displays abundance peak in t1 samples. Thus, it is possible that fungal succession on decomposing pine litter “overtakes” the one on oak leaves.
A model of litter decomposition assumes that soluble compounds are degraded first, then degradation of hemicelluloses follows and the last stages comprise degradation of cellulose-lignin complex leading to increasing lignin/cellulose ratio . We used PICRUSt to model possible metagenomes of decomposing litter to learn if the probable gene content supports this model.
However, one should bear in mind that the results obtained with PICRUSt are only an approximation of the “true” metagenome, and many factors, such as all biases influencing the underlying 16S rRNA gene fragment sequencing or differences in gene content of closely related organisms due to lateral gene transfer, may skew the results. Moreover, the set of functions derived from PICRUSt is only potential one, i.e., we do not know if the functions are actually expressed. Another limitation is that, due to the lack of a eukaryotic database, it was not possible to perform such an analysis for eukaryotic sequences. Therefore, the results should be treated with caution.
We expected a shift in community composition from organisms capable of various carbohydrates utilization to cellulose-degrading specialists, to lignin degraders, although the latter only to some extent, as our experiment concentrated on the first 8 months of litter decomposition. Our results support this view, as cellulolytic genes (β-glucosidases) were most abundant in t2 samples, and certain genes that might be involved in lignin degradation (oxygenases and oxidases) were also found. The genes engaged in active transport of cellobiose (cellulose degradation product) into the cell (PTS system) were most frequent in t0 samples and permeases allowing for passive import of this sugar were most numerous in t1 samples. This picture might mean that during the initial stage of litter decomposition (t0) after depletion of other easily accessible sugars, active transfer of cellobiose is required, most probably due to the low concentration of this compound; then, fungi start producing cellulases causing increase of the cellobiose concentration, which allows passive transfer; and finally, bacterial cellulolytic activity may supplement fungal activity (t2). The latter claim was partially supported by results of Zifcakova and colleagues, who found that Fungi were responsible for only half of carbohydrates metabolism-involved transcripts production in spruce litter . Given the importance of coniferous litter decomposition and taking into account all the limitations of the methodology as well as the general scarcity of molecular studies devoted to coniferous litter decomposition, more work is needed to understand which organisms are responsible for particular processes during decomposition.