Philippa Kaur

Sample collection and incubationThree replicates of soil samples were collected from the top 10 cm in of plant-free patches in four ecosystems along the C. Hart Merriam elevation gradient in Northern Arizona25 beginning at high desert grassland (1760 m), and followed at higher elevations by piñon-pine juniper woodland (2020 m), ponderosa pine forest (2344 m), and mixed conifer forest (2620 m). Soils were air-dried for 24 h at room temperature, homogenized, and passed through a 2 mm sieve before being stored at 4 °C for another 24 h. Soil incubations were performed on soils with mass of 20 g of dry soil for measurements of CO2 and microbial biomass carbon (MBC), while 2 g of dry soil aliquots were incubated separately (but under equivalent conditions) for quantitative stable isotope probing (qSIP). We applied three treatments to these soils through the addition of water (up to 70% water-holding capacity): water alone (control), with glucose (C treatment; 1000 µg C g−1 dry soil), or with glucose and nitrogen (C + N treatment; [NH4]2SO4 at 100 µg N g−1 dry soil). All samples for qSIP were incubated with 18O-enriched water (97 atom%) and matching controls necessary to calculate the change in 18O enrichment across the microbial community. We applied water at natural abundance (i.e., no 18O-enriched water) to the larger soil samples prepared for measurement of carbon flux. All soils were incubated in the dark for one week. Following incubation, soils were frozen at −80 °C for 1 week prior to DNA extraction.Soil, CO2, and microbial biomass measurementsWe analyzed headspace gas of soils for CO2 concentration and δ13CO2 three times during the week-long incubation using a LI-Cor 6262 (LI-Cor Biosciences Inc. Lincoln, NE, USA) and a Picarro G2201 (Picarro Inc., Sunnyvale, CA, USA), respectively. Prior to incubation we analyzed soil MBC using the chloroform-fumigation extraction method on 10 g of soil. One sub-sample was immediately extracted with 25 ml of a 0.05 M K2SO4 solution, while a second sub-sample was first fumigated with chloroform (for 5 days), after which it was similarly extracted. Following K2SO4 addition, we agitated soils for 1 h, filtered the extract through a Whatman #3 filter paper, and dried the filtered solution (60 °C, 4 days). Salts with extracted C were ground and analyzed for total C using an elemental analyzer coupled to a mass spectrometer. MBC was calculated as the difference between the fumigated and immediately extracted samples’ soil C using an extraction efficiency of 0.45 (as per Liu et al.26).Quantitative stable isotope probingWe performed DNA extraction and 16S amplicon sequencing on 18O-incubated qSIP soils11,12,13. The procedures targeted the V4 region of the 16S gene as specified by the Earth Microbiome Project (EMP, http://www.earthmicrobiome.org) standard protocols27,28. We used PowerSoil DNA extraction kits following manufacture instructions to isolate DNA from soil (MoBio laboratories, Carlsbad, CA, USA). We quantified extracted DNA using the Qubit dsDNA High-Sensitivity assay kit and a Qubit 2.0 Fluorometer (Invitrogen, Eugene, OR, USA). To quantify the degree of 18O isotope incorporation into bacterial DNA, we performed density fractionation and sequenced 15–18 fractions separately following methods modified from the canonical publication7. We added 1 µg of DNA to 2.6 mL of saturated CsCl solution in combination with a gradient buffer (200 mM Tris, 200 mM KCL, 2 mM EDTA) in a 3.3 mL OptiSeal ultracentrifuge tube (Beckman Coulter, Fullerton, CA, USA). The solution was centrifuged to produce a gradient of increasingly labeled (heavier) DNA in an Optima Max bench top ultracentrifuge (Beckman Coulter, Brea, CA, USA) with a Beckman TLN-100 rotor (127,000 × g for 72 h) at 18 °C. We separated each sample from the continuous gradient into approximately 20 fractions (150 µL) using a modified fraction recovery system (Beckman Coulter). We then measured the density of each separate fraction with a Reichart AR200 digital refractometer (Reichert Analytical Instruments, Depew, NY, USA) and retained fractions with densities between 1.640 and 1.735 g cm−3. We cleaned and purified DNA in these fractions using isopropanol precipitation, quantified DNA using the Quant-IT PicoGreen dsDNA assay (Invitrogen) and a BioTek Synergy HT plate reader (BioTek Instruments Inc., Winooski, VT, USA), and quantified bacterial 16S gene copies using qPCR (primers: Supplementary Table 1) in triplicate. We used 8 µL reactions consisting of 0.2 mM of each primer, 0.01 U µL−1 Phusion HotStart II Polymerase (Thermo Fisher Scientific, Waltham, MA), 1× Phusion HF buffer (Thermo Fisher Scientific), 3.0 mM MgCl2, 6% glycerol, and 200 µL of dNTPs. We amplified DNA using a Bio-Rad CFX384 Touch real-time PCR detection system (Bio-Rad, Hercules, CA, USA) with the following cycling conditions: 95 °C at 1 min and 44 cycles of 95 °C (30 s), 64.5 °C (30 s), and 72 °C (1 min).We sequenced the 16S V4 region (primers: EMP standard 515F—806R; Supplementary Table 1) on an Illumina MiSeq (Illumina, Inc., San Diego, CA, USA). Sequences were amplified using the same reaction mix as qPCR amplification but cycling at 95 °C for 2 min followed by 15 cycles of 95 °C (30 s), 55 °C (30 s), and 60 °C (4 min). In addition to post-incubation soils, we extracted, amplified, and sequenced DNA of the bacterial community at the start of the incubation.Sequence processing and qSIP analysisThe raw sequence data of forward and reverse reads (FASTQ) were processed within the QIIME 2 environment (release 2018.6)29,30, denoising sequences with the available DADA2 pipeline31. We clustered the remaining sequences into amplicon sequence variants or ASVs (at 100% sequence identity) against the SILVA 132 database32 using an open-reference Naïve Bayes feature classifier33. We removed global singletons and doubleton ASVs, non-bacterial lineages, and samples with less than 4000 sequence reads. Removal of global singletons and doubletons resulted in the removal of 2241 unique ASVs from the feature table yielding 115,647 out of 117,888 (a retention of 98% of all ASVs) as well as the loss of 4018 sequences leaving 37,765,678 (a retention >99% of all sequences). We combined taxonomic information and ASV sequence counts with per-fraction qPCR and density measurements using the phyloseq package (version 1.24.2), in R (version 3.5.1)34. Because high-throughput sequencing produces relativized measures of abundance, we converted ASV sequencing abundances in each fraction to the number of 16S rRNA gene copies per g dry soil based on the known amount of dry soil added and the amount of DNA in each soil sample. All data and analytical code have been made publicly accessible35.To perform qSIP analysis and calculate per-capita growth rates of each ASV, we used our in-house qsip package (https://github.com/bramstone/qsip) based on previously published research7,10. Because rare and infrequent taxa are more likely to be lost in samples with poor sequencing depth with their absences affecting DNA density changes, we invoked a presence or absence-based filtering criteria on ASVs prior to calculation of per-capita growth rates. Within each ecosystem, we kept only ASVs that appeared in two of the three replicates of a treatment (18O, C, and C + N) and at that appeared in at least five of the fractions within each of those two replicates. ASVs filtered out of one treatment were allowed to appear in another if they met the frequency threshold.For all remaining ASVs (1081 representing less than 1% of all ASVs but 58% of all sequence reads), we calculated per-capita gross growth (i.e., cell division) rates observed in each replicate using an exponential growth model10. We applied these per-capita rates to the number of 16S rRNA gene copies to estimate the production of new 16S rRNA gene copies of each ASV per g dry soil per week using the following equation:$$frac{{rm{d}}{N}_{{rm{i}}}}{{{rm{d}}t}}={N}_{{rm{i,t}}}-{N}_{{rm{i,t}}}{e}^{-{g}_{{rm{i}}}t},$$
(1)
Where Ni,t is the number of 16S rRNA gene copies of taxon i at time t (here after 7 days) and gi represents the per-capita growth rate (calculated as a daily rate). See Supplementary Fig. 3 for results on the production of 16S gene copies.Calculation of 16S rRNA gene copy numbers and cell massIn parallel to taxonomic assignment, we compared quality-filtered 16S sequences against a database of 12,415 complete prokaryote genomes obtained from GenBank. From these genomes, we extracted data on 16S rRNA gene copy number, total genome size, and 16S gene sequence. We used BLAST to find matches against this database to the ASVs generated from QIIME 2 to make per-taxon assignments of 16S rRNA gene copy number and total genome size13. For ASVs that did not find an exact match, we assigned 16S rRNA gene copy number values and genome sizes based on the median values observed in the most specific possible taxonomic rank. We estimated the mass of individual cells for each population using published allometric scaling relationships between genome length and cellular mass from West and Brown:36$${{{log }}}_{10}({M}_{{rm{i}}})=frac{{{{log }}}_{10}left({G}_{{rm{i}}}right)-9.4}{0.24},$$
(2)
where Mi indicates cellular mass (g) and Gi indicates genome length (bp) for taxon i. We obtained this relationship by digitizing Fig. 436 using DataThief III and re-fitting the trend line in log–log space. We estimated that 20% of the cellular mass was carbon37. To validate this approach, cellular mass estimates and initial 16S copy number measurements were used to estimate population-level biomass C values which were summed and compared to initial community-level MBC. We found that these values overestimated initial MBC by an order of magnitude. As such, cellular carbon mass was divided by 10 in our final calculations. We applied cellular mass and 16S copy number estimates to the production of 16S copies to estimate the production of biomass carbon for each taxon during the incubation period (t):$${P}_{{rm{i}}}=frac{{rm{d}}{N}_{{rm{i}}}/{{rm{d}}t}}{C_{{rm{i}}}}cdot {M}_{{rm{i}}}cdot 0.2,$$
(3)
where Pi indicates production of biomass carbon (µg C g dry soil−1 week−1) and Ci indicates 16S copy number per cell for taxon i. The 0.2 coefficient represents an estimate that 20% of cellular mass is composed of carbon.Efficiency and respiration modelingWe estimated rates of respiration using qSIP-informed growth rates and community-level carbon use efficiency (CUE). CUE estimates were based on the incorporation of 18O-water into DNA as a measure of gross biomass production38,39 and measured CO2 in headspace gas from soil incubations. We calculated the production of 18O-labeled biomass carbon (18P) at the community-level for each sample by summing the products of per-taxon 18O enrichment (excess atom fraction, EAF) and relative abundance:$${, }^{18}{P}=mathop{sum }limits_{i=1}^{n}({,}^{18}{{{rm{EAF}}}}_{{rm{i}}}cdot {y}_{{rm{i}}})cdot {rm{DN}}{rm{A}}_{0}cdot fleft({{rm{MB}}}{rm{C}}_{0} sim {rm{DN}}{rm{A}}_{0}right),$$
(4)
where 18P indicates the gross production of 18O-labeled microbial biomass carbon per gram of dry soil per week, 18EAFi indicates the enrichment of DNA of taxon i and yi indicates its relative abundance, DNA0 indicates the concentration of DNA per gram of dry soil prior to incubation, and MBC0 indicates the microbial biomass carbon per gram of dry soil prior to incubation. Here, the MBC0 ~ DNA0 function indicates the linear relationship between MBC and DNA concentration. We used the output from Eq. 4 to calculate community CUE for each sample:$${{rm{CUE}}}=frac{{,}^{18}{{P}}}{(!{,}^{18}P+R)},$$
(5)
where R indicates the total CO2 respired per gram dry soil per week.We used the community CUE values from each sample (Eq. 5) to constrain/as upper and lower limits our estimates of per-taxon CUE. For a group of three replicates from a given ecosystem and treatment, we used the minimum and maximum observed community-level CUE values as the acceptable range of per-taxon CUE values. These constraints were used to control the shape of the function of per-taxon CUE and growth rate, though functions were modeled both with and without constraints (i.e., per-taxon CUE values were bounded only by 0 and 0.7). The range of community-level CUE values for each treatment were 0.18–0.53 for control soils, 0.04–0.13 for carbon amended soils and 0.03–0.08 for carbon and nitrogen amended soils and did not vary much between ecosystems. As a result of uncertainty in the literature about the relationship between growth rate and CUE14, several different relationships were postulated to model per-taxon CUE as a function of per-taxon growth rate: linear increase, linear decrease, exponential decrease, unimodal with peak CUE at growth rate of 0.5, and unimodal with peak CUE at a growth rate of 0.05 (the median of all per-taxon growth rates in the data). Comparisons between functions were made by calculating AIC values from per-taxon respiration, summed, and regressing against measured respiration values. Likewise, for each function, we tested how well per-taxon CUE estimates reconstructed community-level CUE by weighting the CUE value of each taxon by its relative abundance, summing, and regressing against community-level CUE. To select the best per-taxon CUE function, AIC values from both scaling efforts were combined. To make AIC values comparable, all respiration and CUE terms were z-transformed prior to regression scaling. To reflect our priority of estimating per-taxon respiration, AIC values from the respiration scaling regression models were multiplied by two and summed with AIC values from CUE scaling such that AICTotal = 2(AICResp) + AICCUE. Across these comparisons, the best estimate of per-taxon CUE was the unimodal function of growth rate, constrained by community-level CUE and peaking at growth rates of 0.5 (Table 1), such that:$${{rm{CUE}}}_{{rm{i}}}=-4({{rm{CUE}}}_{{rm{E}}{rm{:}}{rm{T}}{rm{:}}{{rm{range}}}})cdot {left({g}_{{rm{i}}}-0.5right)}^{2}+({{rm{CUE}}}_{{rm{E}}{rm{:}}{rm{T}}{rm{:}}{max }}),$$
(6)
where CUEi indicates per-taxon CUE, CUEE:T:max indicates the maximum CUE values observed for a group of replicates within a given ecosystem and treatment (E:T). With this function, higher per-capita growth rate values were parameterized to produce higher CUE values initially and then decrease reflecting a growth-CUE tradeoff14, here bound by the difference in maximum and minimum CUE values. We applied per-taxon CUE estimates from Eq. 6 to per-taxon growth rates to yield estimates of per-taxon respiration:$${r}_{{rm{i}}}={r}_{{rm{g,i}}}+{r}_{{rm{m,i}}}=left(frac{{g}_{{rm{i}}}}{{{rm{CUE}}}_{{rm{i}}}}-{g}_{{rm{i}}}right)+left(frac{{g}_{{rm{i}}}}{{{rm{CUE}}}_{{rm{i}}}}-{g}_{{rm{i}}}right)cdot beta,$$
(7)
where ri indicates per-capita respiration for taxon i, rg,i indicates growth-related respiration, rm,i indicates maintenance-related respiration, and β is a constant of 0.01 that represents the maintenance requirements as a proportion of total energy use40. We used these values of per-taxon, per-capita respiration rates to estimate per-taxon respiration per gram of dry soil per week:$${R}_{{rm{i}}}={P}_{{rm{i}}}cdot {r}_{{{rm{g,i}}}}+{P}_{{rm{i}}}cdot {r}_{{{rm{m,i}}}},$$
(8)
where Ri indicates respiration of CO2–C (µg C g dry soil−1 week−1) for taxon i.In addition to per-taxon respiration estimates based on 18O enrichment, we used another model for comparison. Here, respiration was calculated based on 16S abundance alone:$${R}_{{rm{i}}}={N}_{{rm{i}}}cdot f(R sim N+0),$$
(9)
where Ni indicates final 16S abundance for taxon i, R indicates microbial respiration of CO2-C (µg C g dry soil−1 week−1) and N indicates total 16S abundance at the end of the incubation. Here, the R ~ N function indicates the linear relationship, with an intercept of 0, between CO2 respiration and 16S gene concentration across all samples.Diversity, compositional, and statistical analysisFor patterns of evenness in bacterial carbon use and relative abundance, we used Pielou’s evenness which is the quotient of Shannon’s diversity and the observed richness. For each sample, we applied Pielou’s evenness to bacterial abundances as well as bacterial carbon use (relativized to sum to one, in both cases).We created a linear mixed model to test the relationship between the carbon use (the sum of biomass production and respiration) and relative abundance of bacterial genera from the dominant phyla, which accounted for >90% of all C flux. Here, we averaged carbon use and relative abundance for all replicates in a given ecosystem and treatment. We used the lme4 R package (version 1.1-20)41 and obtained p-values using the Satterthwaite method in the lmerTest R package (version 3.1-0)42. To limit pseudo-replication, we accounted for differences in carbon use across ecosystems and due to bacterial Genus by implementing random intercepts. We selected for the optimal random and fixed components by dropping individual terms and comparing models with likelihood ratio tests, disregarding models that failed to converge. Our final model fit was:$${{{log }}}_{10}({C}_{{rm{i}}}) sim {{{log }}}_{10}left({y}_{{rm{i}}}right)ast T+left(1|Eright)+(1|{{rm{Genus}}}),$$
(10)
where Ci indicates the relativized carbon use for taxon i (averaged across all three replicates in a given ecosystem and treatment), yi indicates the relative abundance of taxon i (averaged across all three replicates), T indicates soil treatment, and E indicates ecosystem.For differences in composition, we created species abundance tables using the traditional abundances, as well as measures of carbon use (growth and maintenance respiration) of each ASV in each sample. To account for differences in absolute abundances and flux rates between sites, we relativized all abundance tables. We summarized compositional differences using Bray–Curtis dissimilarities then identified multivariate centroids for all replicates in a site by treatment group. We tested the effect of site and nutrient amendment on the resulting group centroids using PERMANOVA tests implemented with the adonis function in the vegan package (version 2.5-3)43. We related compositional shifts in relative abundance to those in relativized growth and maintenance using Mantel tests with the mantel function in vegan.To test for changes in the type of soil C preferred by microbial genera (either 13C-labeled glucose or 12C soil carbon) in response to nitrogen addition, we used Levene’s test with the car package (version 3.0-10)44. Specifically, we analyzed the relationship between 13C use and 12C use (both relativized) on bacterial genera across all replicates and in C and C + N treatments using a linear model. We then extracted model residuals and tested whether variance was significantly different across treatments by focusing on the interaction between individual replicates and treatment. This produced a significance test describing treatment-level differences in 13C–12C use.Reporting summaryFurther information on research design is available in the Nature Research Reporting Summary linked to this article. More

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The fossils described in the following section have been collected from two different layers presenting slightly different assemblages but with several overlapping taxa. The descriptions are presented here in a taxonomic order. However, plates have been kept separate for the two localities. Plates 1–3 present the assemblage of the UPL while plates 4–9 present the plants that were collected from the LPL.Incertae sedis Bryophyta

Sporogonites
37

Sporogonites sp. A
Fig. 3a–d; Fig. 7a–b

Figure 3(a–d) Sporogonites sp. A, (e–g) Sporogonites sp. B., (a) Specimen AM 7944. Scale = 2 cm. Gross view of specimen. Several parallel slender non branching axes terminated by elongate sporangia. (b) Specimen AM 7944. Scale = 5 mm. Detail of the top part of the plant showing the shape of one sporangium. (c) Specimen AM 7944. Scale = 5 mm. Detail of the top part of the plant showing the shape of one sporangium. (d) Specimen AM 7944. Scale = 1 cm. Detail of an isolated specimen. Shape of one sporangium is visible. (e) Specimen AM 7953. Scales = 1 cm. Gross view of specimen showing the non-branching slender axis bearing terminally an elongate sporangium. (f) Specimen AM 7953. Scales = 1 cm. Detail of the sporangium. (g) Specimen AM 7953. Scales = 5 mm. Detail of the distal part of the sporangium characterized by a small notch (at arrow).Full size image
MaterialThis plant is very abundant in some layers where the elongated stems cover the whole bedding plane. Individual stems are in most cases difficult to identify.DescriptionThe plant consists of bunches of elongated non-divided axes, each ending in one sporangium when complete (Figs. 3a–d, 7a–b). In places, dichotomies seem to occur, but they result from the superimposition of axes. Axes are arranged in parallel. They are 10–11.5 cm long and 0.9–1.3 mm wide. Width is constant along their entire length.The distal part of the stalk is marked by a progressive but clear flaring which identifies the position of the proximal part of the sporangium (Figs. 3b–d, 7b). Sporangia are 7–7.5 mm long and 3–3.5 mm wide.Sporangia are elongated in shape and were probably ovoid to ellipsoid before compression. Their distal part is rounded in outline. The surface of the sporangia is unclear due to the coarse nature of the preservation. In some specimens, a small notch is observed on either side, approaching the tip of the sporangia, defining a small hemispheric structure (see arrows on Figs. 3d, 7b).Identity and comparisonsThe occurrence of elongated sporangia borne singly at the top of smooth unbranched axes unambiguously points to the genus Sporogonites Halle2737. This genus is comprised of 4 (or 5) species: S. exuberans Halle37, S. chapmanii Lang and Cookson33, S. excellens Frenguelli29 and S. yunnanense Hsü10. An additional species was described by Gonez31 but was not validly published. Gonez31 named it Sporogonites punctatus in his unpublished PhD manuscript. However, according to the International Code of Nomenclature for algae, fungi and plants art. 30.9, this publication is not effective and hence not valid (op. cit., art. 32.1).All species chiefly differ in the shape and size of their sporangia. Sporogonites yunnanense presents notably small sporangia ranging from 3.2–4.5 mm in length and 1.4–1.8 mm in width. By contrast, S. excellens is characterized by generally big sporangia up to 5 mm in width and 7 mm in length that are borne on up to 5 mm wide stalks. Our specimens do not compare favourably to either of these two species. The occurrence of a rounded apex to the sporangia in our specimens exclude them from S. chapmanii which is characterized by pointed sporangia. The above described South African specimens conform in shape and size range of the sporangia to S. exuberans. However, similar sporangia were previously reported as Sporogonites sp. A by Gerrienne et al.2 and as a Sporogonites punctatus in Gonez31 from the Paraná basin (Brazil). Sporogonites exuberans and Sporogonites “punctatus” differ mainly by the occurrence in the latter of a minute conical ornamentation on the upper half of the sporangia. The Brazilian material is comparable in size and shape to the Impofu Dam material, however the nature of the preservation of the latter precludes determination of the presence or absence of the diagnostic sporangial ornamentation. In order to avoid misleading paleogeographic interpretations we therefore prefer to leave the taxonomy of this plant open.Age and distributionThe genus Sporogonites is a common component of the earliest floras. It is most frequently reported from Emsian aged deposits in which it constitutes a common and widespread taxon (for full list see31). Its oldest reported occurrences are from assemblages from the Late Silurian of Vietnam31. In the Lochkovian, it has thus far only been found in the Brazilian Ponta Grossa Formation2,31. It is noteworthy to mention that sporangial ornamentation aside, our material is comparable to this sole Lochkovian occurrence.Sporogonites

sp. B
Fig. 3e–g

MaterialOnly one specimen of this plant has been collected as a relatively well-preserved isolated stem.DescriptionThis plant consists of a long unbranched stem distally bearing a large elongated sporangium (Fig. 3e). The stem is straight and measures 66.0 mm long and 1.9–2.1 mm wide. The distal end of the stem is marked by a progressive widening corresponding to the beginning of the sporangium (Fig. 3f). From the point of widening to the tip, the sporangium measures 14.3 mm long and 6.1 mm wide. The sporangium reaches its maximum width after 9 mm (2/3 of total length). The sporangium is terminated by a hemispherical structure marked by a clear depression of the lateral outlines and demarcated by a line of denser mineralisation (see arrow on Fig. 3g). This structure is 3.4 mm wide and 2.1 mm high.Identity and comparisonAs for Sporogonites sp. A, the occurrence of an elongated sporangium borne singly on a smooth non branched axis suggests the genus Sporogonites Halle37. Lack of bifurcation cannot be unambiguously established as a result of the lack of preservation of the base. Nonetheless the size of the plant mitigates against other explanations. Moreover, the general shape of the sporangium conforms to the genus Sporogonites, being very similar to both Sporogonites exuberans and Sporogonites “punctatus”. This specimen is, however, much larger than any formally described Sporogonites species. Further taxonomic discussions are nonetheless deferred on account of the mediocre preservation of the single specimen.The presence in both this specimen and Sporogonites sp. A of a small hemispherical structure terminating the sporangium is significant. A similar structure was observed by Halle37 on the Sporogonites (Sporogonites exuberans) type material from Röragen. In the most complete specimens, the tip of the sporangia is described as rounded but with a little break differentiating darker material around the extremity of the sporangium. Alternately this structure may be absent, and the sporangial tip characterised by a depression. Cyrille Prestianni (CP) has confirmed presence of this structure in specimens of Sporogonites exuberans from Belgium. From the perspective of the common identification of Sporogonites, as a bryophyte, this recurring structure likely represents the operculum of the capsule.Polysporangiophyta

23

Cooksonia
32

Cooksonia paranensis2 .
Fig. 4a–n; Fig. 7c and f

Figure 4(a–n) Cooksonia paranensis2,4. (a) Specimen AM 7908. Scale = 1 cm. specimen showing three branching orders and a terminal sporangium. (b) Specimen AM 7906. Scale = 1 cm. Truss of the plant showing a number of branching orders and terminal sporangia. (c) Specimen AM 7910a. Scale = 5 mm. Sporangium. (d) Specimen AM 7887. Scale = 5 mm. Sporangium. (e) Specimen AM 7913. Scale = 5 mm. Sporangium. (f) Specimen AM 7912a. Scale = 5 mm. Sporangium. (g) Specimen AM 7888. Scale = 5 mm. Sporangium. (h) Specimen AM 7950b. Scale = 5 mm. Sporangium. (i) Specimen AM 7964. Scale = 5 mm. Sporangium. (j) Specimen AM 7958a. Scale = 5 mm. Sporangium. (k) Specimen AM 7965. Scale = 5 mm. Sporangium. (l) Specimen AM 7955a. Scale = 5 mm. Sporangium. (m) Specimen AM 7959a. Scale = 5 mm. Sporangium. (n) Specimen AM 7954b. Scale = 5 mm. Sporangium.Full size image
MaterialThis plant is the most abundant in the UPL. It occurs either as isolated sporangia, sporangia connected to small stem fragments or as bunches of fertile axes. By contrast only two specimens have been identified in the LPL.DescriptionSeveral specimens of this plant have been discovered (Figs. 4, 7c,f). They consist of isotomously branched axes, 0.7–1.2 mm in width. In many cases, it is difficult to distinguish individual branching systems as, when not fragmentary, plants occur in bunches (Fig. 4b). This is particularly the case on one specimen that shows several isotomously branched plants arising from the same point (Fig. 4b). The specimens in Fig. 4a,b show the ultimate branching orders with sporangia attached. Branching systems always bear terminal sporangia (Fig. 4). Sporangia are trumpet- to cup-shape in outline and measure 2.5–5.0 mm in diameter and 2.0–3.0 mm in height. The axis/sporangium transition is progressive (Fig. 4c–m). It is thus difficult to identify with precision the base of the sporangium. The sporangial cavity gives the impression of being sunken into the subtending axis (Fig. 4f–k). The upper part of the sporangium is flat and marked by the presence of an apical plateau. The shape of the apical plateau seems very variable, but it results from differences in compression orientation (Fig. 5). In most cases, it is folded up, which results in a bulge (Figs. 5a, 4c–e) or in wrinkles (Figs. 5b, 4g). Several specimens seem to present a more spherical structure rather than an apical plateau (Fig. 4f,j–n). The apparent rounded shape of the sporangia is the result of the tilting of the apical plateau (Fig. 5c). This configuration was already noted by Gonez and Gerrienne7.Figure 5Schematic reconstruction of the sporangia of Cooksonia paranensis showing different position of the operculum (op) in regard to the sporangial chamber (sc): (a) in growth position with the operculum in place, (b) with the operculum compressed laterally showing several wrinkles and (c) with operculum completely tilted.Full size imageIdentity and comparisonsPlants with smooth isotomously branched axes, gradually widening distally into a single, terminal, cup- or trumpet shaped sporangium with an apical plateau can be attributed either to the genus Cooksonia Lang emend6 or to the genus Concavatheca52.The genus Cooksonia was originally described by Lang32 on the basis of compression fossils exhibiting “dichotomously branched, slender, leafless stems, with terminal sporangia that are short and wide [with an] epidermis composed of elongate, pointed, thick-walled cells [and a] central vascular cylinder consisting of annular tracheids”. Thanks to the works of Edwards and collaborators (see among others:35,36,7,9), the genus is now known in great detail, on the basis of both compression and coalified specimens. The type species C. pertoni is considered the earliest eutracheophyte23. The genus diagnosis was emended in 20106 at which time it included three well defined species: C. pertoni, C. paranensis and C. banksii6, however C. banksii has later been transferred to another genus (see below and Morris et al.52). C. pertoni and C. paranensis are morphologically similar, but, according to Gonez and Gerrienne6, C. paranensis can be distinguished from C. pertoni by its slender axes and the more gradual transition between axis and sporangium. As a result of this gradual transition, the sporangial cavity of C. paranensis is sunken in the subtending axis. The genus Cooksonia also includes three less well-preserved species, C. hemisphaerica32, C. cambrensis35 and C. bohemica38. All of these are considered doubtful by Gonez and Gerrienne6 because they are based on poorly preserved specimens. A restudy of the fossil material of C. bohemica has led Kraft et al.41 to place this plant within the genus Aberlemnia under the combination A. bohemica. Recently, a new species, C. barrandei64 has been described. The plant is morphologically close to C. pertoni and C. paranensis, but with more robust axes and bigger sporangia64.The specimens originally described under the binomial Cooksonia banksii by Habgood et al.7 have been transferred to the genus Concavatheca by Morris et al.52. The genus Concavatheca includes plants with smooth axes and single terminal sporangia. The subtending axis gradually widens distally into a cup-shaped, sunken sporangial cavity. The specimens of Concavatheca banksii differ from those of Cooksonia. pertoni because the spore mass of Concavatheca banksii is sunken in the subtending axis whereas Cooksonia pertoni has a discoidal spore mass subtended by an axis that gradually increases in diameter. Other differences exist, both in sporangial structure and spore ultrastructure. In having a sunken sporangial cavity, the species Concavatheca banksii is very similar to Cooksonia paranensis and the two species are difficult to distinguish. They are mainly differentiated by their preservation type: compression for C. paranensis and charcoalification for Concavatheca banksii. Accordingly, detailed comparisons are not possible, and the two species can be kept in separate genera, on the basis of art. 11.1 of the International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code)18, which states that “the use of separate names is allowed for fossil-taxa that represent different parts, life-history stages, or preservational states of what may have been a single organismal taxon or even a single individual”18, art. 11.1).The specimens here described are morphologically very close to Cooksonia pertoni, Cooksonia paranensis and Concavatheca banksii. The very gradual transition between the sporangia and the subtending axes as well as the “sunken” aspect of the sporangial cavity are reminiscent of both C. paranensis and Concavatheca banksii. As our material, like previously described material of Cooksonia paranensis, is preserved as compression fossils it cannot be accurately compared with Concavatheca banksia, and we accordingly choose to name it Cooksonia paranensis.Age and distributionCooksonia paranensis was first described from the Ponta Grossa Formation at Jackson de Figueiredo (southern Paraná Basin, Brazil) as well as in four other localities of the Paraná Basin2. A Lochkovian age has been proposed for these plant bearing beds based on palynology2. One single other putative occurrence has been recorded. In the Lochkovian Talacasto Formation, at Talacasto creek (Argentina) Edwards et al.43 report one poorly preserved specimen that could be assigned with doubts to either Cooksonia paranensis or Concavatheca banksii.
Cooksonia hemisphaerica32
Fig. 6a–b.

Figure 6(a,b) Cooksonia hemisphaerica, (c) Cooksonia cambrensis Edwards35, (d–f) Tortillicaulis sp., (g–h) cf. Cooksonia hemisphaerica, (a) Specimen AM 7914. Scale = 1 cm. Specimen showing the gross morphology of the plant. (b) Specimen AM 7914. Scale = 5 mm. Detail of a rounded sporangium that is clearly distinct from the widening subtending axis. (c) Specimen AM5828. Scale = 2 mm. Sporangium and subtending axis. (d) Specimen AM 7956a. Scale = 5 mm. Specimen showing the organisation of the sporangia. An oblique striation is visible at the surface of the sporangium. (e) Specimen AM 7943. Scale = 5 mm. Specimen showing naked axes bifurcating one time before terminating in elongate sporangia. Note the oblique striation at the surface of the sporangia. (f) Specimen AM 7921a. Scale = 5 mm. A dichotomizing naked axis terminally bearing two sporangia. The sporangia bear a distinct oblique ornamentation and are made of two halves that are twisting on each other. (g) Specimen AM 7915a. Scale bar = 1 cm. Specimen showing the gross morphology of the plant. (h) Specimen AM 7925. Scale bar = 5 mm. Detail of specimen Fig. 6g showing the organisation of the sporangia.Full size image
Figure 7(a,b) Sporogonites sp. A, (c) and (f) Cooksonia paranensis, (d–e) and (g–h) Steganotheca striata. (a) Specimen AM 7927. Scale = 1 cm. A truss of non-branching more or less parallelly oriented axes distally bearing one single sporangium. (b) Specimen AM 7927. Scale = 5 mm. Detail of the distal ends and of the sporangia. The arrow point towards the limits of the small hemispheric structure present at the tip of the sporangium. (c) Specimen AM 7891a. Scale = 1 cm. Gross morphology of the specimen showing several superimposed individuals. (d) Specimen AM 7893. Scale = 1 cm. Gross morphology of the specimen showing several intertwined axes. (e) Specimen AM 7997. Scale = 5 mm. Isolated termination showing the ultimate dichotomy and two sporangia. (f) Specimen AM 7886. Scale = 5 mm. Detail of an isolated sporangium. (g) Specimen AM 7987. Scale = 5 mm. Distal part of the plant showing two more or less parallel sided sporangia. (h) Specimen AM 7998. Scale = 5 mm. Distal part of the plant showing two more or less parallel sided sporangia. (i) Specimen AM 7973. Scale = 5 mm. Isolated specimen showing the organisation of the sporangium.Full size image
MaterialOne single moderately preserved specimen from the UPL.DescriptionThe specimen consists of a 58 mm long dichotomous branching system terminally bearing sporangia (Fig. 6a). Two isotomous dichotomies are observed (see arrows on Fig. 6a). The first order axis is broken at its base and measures 20 mm long and 1 mm wide. Second order axes are 10–12.5 mm long and 0.8–1 mm wide. Finally, third order axes are short and measure 5 mm long and 1 mm wide.Sporangia are globular, they measure between 1.3–2.0 mm long and 2.0–2.4 mm wide. Within the sporangium, a circular structure can be observed that we interpret as the sporangial cavity (Fig. 6b). This cavity measures between 1.5 and 1.8 mm in diameter. Sporangial wall is 0.3–0.4 mm thick. No dehiscence line could be observed.The subtending axis is 2.9–3.6 mm long. It gradually flares and distally reaches 2.0–2.4 mm in width. The sporangium-axis contact is relatively large as the axis is almost as wide as the sporangium (Fig. 6b).Identity and comparisonDichotomous branching systems distally bearing elongate structures are relatively common in the Lower Devonian. Identification is made difficult by the scarcity of morphological traits. In addition to Cooksonia hemispherica, four taxa are known to show such organisation. They are: Tortilicaulis Edwards35, Tarrantia Fanning et al.36, Salopella Edwards and Richardson44 and Uskiella Shute and Edwards12. They however all present an elongated sporangial cavity and a sharp transition at the sporangium-axis contact. The occurrence of isotomously branched axes distally bearing globular sporangia at the end of gently tapering subtending axes conforms to Cooksonia hemisphaerica Lang32,35,36. The here recorded size range conforms to the larger forms recorded from the British Isles.Age and distribution. Cooksonia hemisphaerica was originally described from the Targrove quarry32. The Targrove quarry deposits have been dated by means of fishes and spores and are Lochkovian in age36. Later, Edwards35 reported specimens of Cooksonia hemisphaerica from Freshwater East in the South Dyfed region (South Wales). This occurrence has been dated through palynology and is considered Pridoli in age. Another occurrence is the Lochkovian Brown Clee Hill locality (Shropshire-England)46. Cooksonia hemisphaerica has also been recorded in the Bryn Glas borehole (Anglo-Welsh Basin)42.
Cf. Cooksonia hemisphaerica32
Fig. 6g–h
MaterialThis plant is only known from a single more or less complete specimen collected from UPL.DescriptionIt is characterized by a dichotomous branching system distally bearing globular sporangia (Fig. 6g–h). It is 130 mm long and consists of a three-times isotomously dichotomizing axis. The branching angles are small measuring less than 10°. First order axis is proximally incomplete and measures 35 mm long and 1.9 mm in width. Second order axes are 49 mm in length and 1.0 mm in width. The third order axes are 25–27 mm in length and 0.8–0.9 mm in width. The fourth ultimate order axes are 16–19 mm in length and 0.6–0.8 mm in width. Each third order axis bears one sporangium. The subtending axes of the sporangia gradually flare at a length of 6.0–7.0 mm to approximately reach the width of the sporangia (Fig. 6h). The sporangia are globular and measure 1.0–1.5 mm long. When measured at their widest point, sporangia are 2.0–2.2 mm in width.Identity and comparisonsDespite notable differences, the shape of the Impofu Dam specimens is most reminiscent of Cooksonia hemisphaerica. In this plant, sporangia are globose and borne on axes strongly widening just below them32,36. The recorded size range for the sporangia in C. hemisphaerica is compatible with the Impofu Dam material. However, C. hemisphaerica differs in that its sporangia are produced shortly after the ultimate dichotomy. Moreover, the identity of the “rounded tip” as a sporangium is equivocal as no clear division from the axis is visible. Therefore, we cannot unequivocally assign this material to C. hemisphaerica and so name the fossil cf. Cooksonia hemisphaerica.
Cooksonia cambrensis35
Fig. 6c
MaterialOne single isolated sporangium from the UPL.DescriptionSporangium born singly at the end of an unbranched smooth axis (Fig. 6c). The axis is 18 mm long and 0.7 mm wide. It slightly tappers distally to reach 1 mm at the base of the sporangium. The sporangium-axis junction is clear and flat. The sporangium is elliptical in outline, but we here suspect some deformation to have occurred. It measures 2.8 mm wide and 1.4 mm high.Identity and comparisonAlthough occurring as a single isolated specimen, it remarkably conforms to Cooksonia cambrensis to which we assign it based on the shape of the sporangium and the very limited tapering of the subtending axis35,36.Age and distributionCooksonia cambrensis has been identified in the Pridoli of Wales35,47 and the Lochkovian of England36,47.
Steganotheca48
Steganotheca striata48
Fig. 7d–e; 7 g–h.
MaterialThis plant has exclusively been found in the LPL. Four specimens have been recovered occurring either as isolated stem fragments or as a relatively densely occurring plant mat.DescriptionOnly the ultimate one or two branching orders have been recovered, up to 35 mm in overall length. The axes are isotomously branched (Fig. 7d–e, g–h). Their width is relatively constant throughout specimens and range from 0.6–1.4 basally to 0.8–1.4 mm distally. The axial surface is smooth. The sporangia are borne singly. They measure 2.9–4.2 mm long and 1.9–3.0 mm wide. The subtending axis widens rapidly at the transition to the sporangium. The width of the sporangium then remains constant for most of its length. The tip of the sporangium is truncated and seems to be topped by a denser looking lens-shaped apical plateau. The whole structure has the shape of a mug.Identity and comparisonThe occurrence of smooth isotomous branching systems bearing isolated sporangia characterized by a flaring of the axes and topped by an apical plateau is indicative of either the genera Cooksonia32 or Steganotheca48. They chiefly differ in the shape of the sporangia. The occurrence of mug-shape terminal sporangia, that are longer than wide, parallel sided and truncated at the apex conforms to the genus Steganotheca. Despite the lack of minute details such as the striation observed on the sporangia of the original material from South Wales, we consider the similarities sufficient to attribute the Impofu dam material to the species Steganotheca striata.Age and distributionSteganotheca striata has been recorded in the Silurian (Ludfordian and Pridoli) of South Wales (Capel Horeb Quarry)48,49.
Aberlemnia5
Aberlemnia caledonica (Edwards)5
Fig. 8a–d

Figure 8(a–d) Aberlemnia caledonica, (e) Tortilicaulis sp., (f–h) Uskiella spargens. (a) Specimen AM 7970. Scale = 1 cm. Gross morphology of the plant showing its dense organisation. (b) Specimen AM 7970. Scale = 5 mm. Detail showing the organisation of the sporangia. (c) Specimen AM 7985. Scale = 5 mm. Detail of a subcircular sporangium. (d) Specimen AM 7980. Scale = 5 mm. Detail several subcircular sporangia. (e) Specimen AM 7984a. Scale = 5 mm. Ultimate dichotomy of the plant bearing two sporangia. Note the occurrence of an oblique striation at the surface of the sporangia. (f) Specimen AM 7966a. Scale = 1 cm. Isolated specimen showing the gross morphology of the plant. Note the horizontal axis and the marked curvature. (g) Specimen AM 7968a. Scale = 1 cm. Specimen showing the gross morphology of the plant. (h) Detail of specimen in fig. 7f showing two sporangia. Scale = 0.5 cm.Full size image
MaterialFour specimens including a more or less complete branching system have been recovered from the LPL.DescriptionThe largest specimen of this plant is figured in Fig. 8a. It is relatively densely packed and therefore it is difficult to describe individual branching systems. It consists of branched axes terminated by reniform to transversely elongated sporangia (Fig. 8a,b). The axes are smooth and mostly dichotomize isotomously. They are, however, some indications of anisotomous division, but preservational limitations precludes any definite statement thereon (Fig. 8a). Axial width is constant throughout specimens and measure 0.5–0.7 mm. Branch length decreases distally. The ultimate division gives rise to short axes. Sporangia are sub-circular in outline and measure 1.0–1.8 mm in width and 0.9–1.6 mm in length (Fig. 8b–d). Subtending axes widen sharply just beneath sporangia and reveal a curved axis-sporangium junction.Identity and comparisonThe occurrence of sub-circular terminal sporangia on mostly isotomously dichotomizing axes points towards the genera Aberlemnia and Sporathylacium. The prior was erected by Gonez and Gerrienne5 in order to accommodate specimens with reniform sporangia formerly included in the genus Cooksonia. The latter was established by Edwards et al.50 for bivalved reniform anatomically preserved sporangia. In the absence of any evidence for bivalved sporangia and considering the lack of anatomical details and of in situ spores in the Impofu dam material, we assign this material to Aberlemnia caledonica.Age and distributionAberlemnia has been recorded from the Lochkovian of Scotland48, the late Silurian to Lochkovian of Wales35,36,47,49, the Lochkovian of Brazil2, and possibly from the Ludlow of Bolivia51,74. Recently, Kraft et al.41 have suggested that the Late Silurian Cooksonia bohemica should be reassigned to the genus Aberlemnia. However, further studies are necessary to ascertain validity of this proposition.
Tortilicaulis35
Tortilicaulis sp.
Fig. 6d–f; Fig. 8e;
MaterialSpecimens of this plant consists of 3 specimens from the UPL and one specimen from the LPL.DescriptionIn all cases only one (Fig. 6d) or two (Figs. 6e–f, 8e) ultimate branching orders have been preserved. The preserved plant comprises large elongate sporangia terminating dichotomous axes. Sporangia are fusiform and taper distally to a blunt tip. The tip is obscured in specimen Fig. 6d by the occurrence of a transversely oriented axis. The sporangia show a clear twisted organization (see arrows on Fig. 6e,f). An obliquely oriented striation is observable on the surface of most sporangia. The measured angle of this striation is relatively variable and measures 24° (Fig. 6e), 28° (Fig. 6d) and 38° (Fig. 8e) from the vertical. A similarly oriented longitudinal splitting here interpreted as a dehiscence line is repeatedly observed (Figs. 6e–f, 8e). This is particularly visible on specimen Fig. 6f. This specimen is dehisced, and the putative two valves of the sporangia are slightly separated and twisted around each other. The junction with the subtending axis is unclear except on Fig. 6f where it is marked by the beginning of the putative dehiscence line. Maximum width of the sporangia occurs at mid-height. They measure 1.2–1.4 mm long and 0.3–0.4 mm wide in the UPL and 0.6 mm long and 0.2 mm wide in the LPL. The height to width ratio ranges between 3 and 3.5. The subtending axes are parallel sided but a faint widening upwards can be seen. Obliquely oriented cellular patterns could be observed on the subtending axes. Dichotomies are largely isotomous, branching at an acute angle between 10° and 15°.Identity and comparisonThe occurrence of relatively large fusiform sporangia terminating isodichotomously branched smooth axes with relatively low branching angles, points towards three genera, namely Salopella Edwards and Richardson44, Tortilicaulis35 and Teruelia53. Salopella is characterized by longitudinally aligned elongated cells on the sporangia whereas these are obliquely oriented in Tortilicaulis and Teruelia. The latter however differs in being characterized by a multi-slit dehiscence whereas Tortilicaulis exhibits only one dehiscence slit. The oblique striation and the single dehiscence slit observed on the Impofu dam sporangia therefore support assignment to the genus Tortilicaulis. The only two species included in this genus are T. transwalliensis and T. offaeus. The Impofu dam material all falls within the size range of T. transwalliensis as described from the Targrove quarry36. The height to width ratio is however slightly smaller in the South African material but falls within the range of T. transwalliensis in general35,36. Tortilicaulis transwalliensis includes a very large size range from very small to relatively large forms. Size and proportion of the sporangia therefore appear to provide a relatively weak taxonomic guideline. Besides the characteristics of the sporangia, the main difference between the Impofu dam material and previously described T. transwalliensis is the presence of a very long subtending axis. Although, morphologically very close to T. transwalliensis we cannot unequivocally assign our new material to this species and so assign the fossil to Tortilicaulis sp.Age and distributionThe genus Tortilicaulis was first described from the Pridoli of South Wales35. The species T. transwalliensis has further been reported from the Ludlow Targrove Quarry (Shropshire, UK)36 and from the Lochkovian Bryn Glas Borehole (South Wale, UK)42. Tortilicaulis offaeus has been described from the Lochkovian of North Brown Clee Hill locality (Shropshire, UK)46. Tortilicaulis cf. offaeus from the Lochkovian Tredomen Quarry (South Wales, UK). Another hypothetical record comes from the Lower Devonian (Lochkovian?) Argentinian Villavicencio Formation with the record of a cf. Tortilicaulis54.
Uskiella12
Uskiella spargens12
Fig. 8f–h
MaterialSeven specimens were recovered from the LPL.DescriptionThis plant consists of isodichotomous naked axes terminated by ovate structures. We assume these structures to be sporangia even though no in situ spores were observed.One exceptionally large specimen, AM7968, is 51 mm long and consists of an axis that branches six-times (Fig. 8g). The first order axis is broken and measures 1.3 mm in diameter. It is considered to have been horizontal. After the first dichotomy, it gives rise to two axes that curve upwards 6 and 7 mm following an angle of 91° and 94°. They both measure 14 mm long and 1.3 mm wide. Each of these further dichotomizes at most three more times. The branching pattern is obscured by the density of the branching. All divisions appear to occur isodichotomously even though a slight anisotomy is possible. Third order axes are 9–13 mm long and 0.7–0.8 mm wide; fourth order axes are unclear but measure between 3 and 7 mm long and 0.6–0.8 mm wide; fifth order axes are 5–8 mm long and 0.6–0.8 mm wide. The sixth order axes constitute the ultimate one and each terminate in a sporangium. These are very short and measure 1.2–3.5 mm long and 0.6–0.8 mm wide. The gradual axis/sporangium transition makes precise measurement of the sporangium and its subtending axis difficult. Thus, we arbitrarily considered the place where the subtending axis is no longer parallel sided to mark the base of the sporangium. The sporangia are ovate in shape. They are characterized by a widening of the subtending axis that gives to the whole structure the shape of a tennis racket. They measure 3.4–4.7 mm long and 1.3–2.3 mm wide. No specialized structure for dehiscence is observed. The outline of the sporangia is very variable. The axis/sporangium junction is difficult to observe with precision but is marked by a slightly convex to almost straight line.Another large specimen is AM7966 (Fig. 8f). It is 45 mm long and consists of an axis that branches three-times. The first order axis is 25 mm long and is presumed to have been horizontal for the first 14 mm, after which it is inflected by 108°. It measures 1.2 mm wide before the inflection and 1.4 thereafter. The plant subsequently dichotomizes three times. Second order axes are 9.5–10.8 mm long and 12–1.3 mm wide; third order axes are 5.4–6.8 mm long and 0.9–1.0 mm wide; fourth order axes are 4.1–5.9 mm long and 0.7–0.8 mm wide. The ultimate order axes measure 1.8–2.5 mm long and 1.0–1.2 mm wide. Only two sporangia are sufficiently well preserved to be accurately studied (Fig. 8h). They are rounded to ovate in shape and measure 6.2 and 3.4 mm long and 2.9 and 3.9 mm wide respectively. As with the previous specimen the axis/sporangium junction is marked by a slightly convex to almost straight line.In summary, this plant seems to be characterized by a horizontal axis that dichotomizes at least once before curving at an angle approaching 90° and giving rise to what we interpret as the erect part of the plant. It subsequently dichotomizes three more times. Dichotomies seem to occur more or less isotomously but branching is relatively difficult to interpret in both specimens. The sporangia are tennis-racket shaped in outline..Identity and comparisonsThe presence of longitudinally elongated sporangia showing a tapering base is initially suggestive of the genus Salopella Edwards and Richardson44. However, this genus differs significantly in shape as it is consistently described as having sporangia with acute tips and tapering apices44,46.Rounded to elongate relatively large sporangia have repeatedly been reported in the literature56. The shape of these sporangia resembles two specimens originally described by Croft and Lang56 as Cooksonia sp. but later identified as cf. Sporogonites by Cookson57. They were both rediscussed by Shute and Edwards who highlighted the occurrence of a longitudinal slit on the sporangia which defined two valves. The material was therefore reassigned to the species Uskiella spargens. We believe that the Impofu Dam material is more or less identical to adpression material thereof from both Wales (UK) and Victoria (Australia). Evidence for the occurrence of two valves in our material is tenuous, however a double valved sporangium would explain the variability of the shape of the sporangia observed in several specimens (Fig. 8h). In addition, the Impofu Dam specimens fall within the same size range as Uskiella spargens. Uskiella reticulata36 is characterized by much smaller sporangia. Considering the many similarities existing between the Impofu Dam material and both the Australian and Welsh material and despite the lack of definite evidence of a longitudinal dehiscence slit, we attribute the here described material to Uskiella spargens.RemarksOne of the important features of this plant is the description of an extensive branching system. The occurrence of a dichotomizing horizontal axis giving rise to an erect plant has repeatedly been observed in plants of more or less the same age such as Aglaophyton majus, Rhynia Gwynne-vaughannii or Nothia aphylla (see Hetherington and Dolan for references). The lack of anatomical preservation in the Impofu Dam material precludes a rigorous interpretation of these axes. It is, however, hypothesised that these dichotomizing horizontal axes performed the sporophyte rooting function. This suggest that Uskiella spargens lacked a true rooting system.Age and distributionUskiella spargens was originally described from the Pragian of Wales12 Specimens identified as Cooksonia sp. by Croft and Lang56 and later synonymized with U. spargens were collected from the Lochkovian of Allt Du (South Wales)59. Specimens originally described as cf. Sporogonites but synonymized with U. spargens by Shute and Edwards12 were collected from the Lochkovian to lower Pragian Humevale Siltstone Formation of Lilydale (Australia)57.GenusKrommia gen. nov.Type species: Krommia parvapilla sp. nov.Derivation of the name: Krommia from the Kromme River (from Afrikaans meaning curved).Diagnosis: Plant with smooth, three dimensional, isotomously branching axes; Sporangia small and rounded borne singly and terminally.SpeciesKrommia parvapila sp. nov.Derivation of the name: parvapila, from Latin a small ball referring to the sporangium.Diagnosis: Same as for genus. Dichotomizing up to three times. Branching angle variable (40°–110°). First and second order axes U shaped. Axes 1.5–3.0 mm long and 0.3–0.35 mm wide below sporangia. Small constriction at junction between subtending axis and sporangium. Sporangia rounded between 0.7 and 0.8 mm in diameter.Holotype: AM 7928a (part) and AM 7928b (counterpart), Fig. 9a.Figure 9(a–c) Krommia parvapilla gen. nov. sp. nov., (d–l) Elandia itshoba gen. nov. sp. nov. (a) Specimen AM 7929. Scale = 5 mm. Gross morphology of the plant showing the U-shaped first and second order axes. (b) Specimen AM 7928a. Scale = 5 mm. Slightly laterally compressed specimen showing the different branching orders and the terminal rounded sporangia. (c) Specimen AM 7969b. Scale = 5 mm. Specimen the U-shaped first order axes and the terminal sporangia. (d) Specimen AM 7894a. Scale = 5 mm. (e) Specimen AM 7897a. Scale = 5 mm. (f) Specimen AM 7975a. Scale = 5 mm. (g) Specimen AM 7988a. Scale = 5 mm. (h) Specimen AM 7975b. Scale = 3 mm (i) Specimen AM 7988a. Scale =  4 mm(j) Specimen AM 7932a. Scale = 5 mm. (k) Specimen AM 7931a. Scale = 3mm (l) Specimen AM 7975a. Scale = 3 mm.Full size imageParatypes: AM 7929 and AM 7969.Repository: Albany Museum, Devonian Lab, Beaufort Street, Makhanda, Eastern Cape, South Africa.Type locality: Impofu Dam, Kouga Municipality, Eastern Cape, South Africa (Fig. 1).Horizon: Kareedouw Member, Baviaanskloof Formation, Nardouw Subgroup, Table Mountain Group, Cape Supergroup.Age: Lower Devonian, Lochkovian?Synonymy: Minutia fragilis nomen nullum Gonez31, Fig. 1 p. 178, from Jackson de Figueiredo, Jaguariaiva county, Brazil.
Fig. 9a–c
MaterialFive specimens of this plant have been recovered from the LPL.DescriptionIn all specimens, only the ultimate parts of the plants have been preserved. They measure up to 20 mm in length and consist of smooth axes that branch isotomously at relatively variable angles (40–110°). This variability suggests that the branching system was three dimensional. We think that relatively wide angles (70–110°) were originally present and compressed during taphonomical processes. A maximum of three dichotomies has been observed however the occurrence on Fig. 9a of two superimposed similar branching systems in the same orientation suggests that more dichotomies can be expected. On this specimen only a small part of the first branching order has been preserved which measures 0.9 mm wide. It branches to produce two slightly different axes measuring 8–10 mm long and 0.6 and 0.7 mm wide respectively. The exact branching pattern is then obscured by the superimposition of at least one other branching system; however, it clearly branches two more times. The first and second branching orders are characterized by a slight curvature that give to the pair of axes a U rather than a V shape. The specimens are terminated by small rounded sporangia measuring 0.7–0.8 mm wide. The junction between the subtending axes and the sporangia is marked by a small constriction of the axis. Subtending axes measure 1.5–3.0 mm long and 0.3–0.35 mm wide.A similar branching pattern is observed in specimens illustrated in Fig. 9b and c. First axes orders are incomplete and measure 6.5–7.5 mm long and 0.5–0.8 mm wide respectively. Second branching orders measure 3.8–4.8 mm long and 0.3–0.4 mm wide in Fig. 9b and 5.0–8.0 mm long and 0.6–0.7 mm wide in Fig. 9c. Third order branches measure 2.0–3.5 mm long and 0.3 and 0.5 mm wide in Fig. 9b and 2.5–3.5 mm long and 0.4–0.6 mm wide in Fig. 9c. The fourth order axes also are the subtending axes of the sporangia. They measure 2.4–2.8 mm long and 0.3–0.5 mm wide in Fig. 9b and 3.4–3.5 mm long and 0.3–0.4 mm wide in Fig. 9c. They always end in a small constriction that marks the base of the sporangia. The sporangia are rounded and measure 0.7–0.8 mm in diameter. No dehiscence feature was observed.ComparisonSmall plant remains with minute (mesofossil sized) sporangia have repeatedly been observed in Silurian to Lochkovian deposits60,62,11,46,73. Occurring as isolated plant fragments most of them were kept in open taxonomy. Several plant fragments resembling the Impofu Dam material were illustrated by Morris et al. 73. Our specimens most closely resemble their morphotype C however the characteristic constriction at the base of the sporangia has not been reported. One of their illustrated specimens does however show a similar structure. Croft and Lang56 published several specimens as Cooksonia sp.. Mainly consisting of isolated sporangia, they could bear some superficial resemblance to the SA specimens however detailed comparison is made difficult by the absence of vegetative structures. Our specimens more closely resemble the more fragmentary Brazilian specimens illustrated in the unpublished thesis of Gonez31. They share the same overall organization including the curvature of the second and third order axes. The characteristic constriction at the base of the sporangia is also present and the sporangia are comparable in shape and size, suggesting that they represent the same species. The Brazilian material has however never been validly published. Considering the extensive branching system preserved and the occurrence of this plant both in Brazil and South Africa we chose to erect a new genus and species.GenusElandia gen. nov.Type species: Elandia itshoba sp. nov.Derivation of the name: after Eland (Taurotragus oryx), from Elandsjacht, original farm name of locality, meaning Eland hunt in Afrikaans.Diagnosis: Plant forming dense trusses of fine, smooth, isotomously branching axes. Axes bifurcating at a low angle and terminating in minute, elongate ovate sporangia; multiple axes united by a basal structure.SpeciesElandia itshoba sp. nov.Derivation of the name: from isiXhosa, itshoba, a ritual fly whisk made from a bulls tail, sometimes with the hair tips decorated with tiny beads.Diagnosis: As for genus, bifurcating up to four times, sporangia straight sided with broadly rounded apices. Sporangia 1.3–1.6 mm long and 0.4–0.7 mm wide.Holotype: AM 7932a (part) and AM 7932b (counterpart), Fig. 9j.Paratype: AM 7894, AM 7897, AM 7975, AM 7988.Repository: Albany Museum, Devonian Lab, Beaufort Street, Makhanda, Eastern Cape, South Africa.Type locality: Impofu Dam, Kouga Municipality, Eastern Cape, South Africa (Fig. 1).Horizon: Kareedouw Member, Baviaanskloof Formation, Nardouw Subgroup, Table Mountain Group, Cape Supergroup.Age: Lower Devonian, Lochkovian?
Fig. 9d–l
MaterialSeven specimens from the LPL.DescriptionThis plant most often occurs as densely packed trusses of axes. This renders description of individual axes’ organization difficult. In all specimens, it consists of up to 60 mm long thin smooth axes that branch up to four times and bear minute terminally elongate ovate sporangia.The organization of the plant is best seen in AM 7932a (Fig. 9j), which is proximally incomplete. It measures 33 mm long and branches 4 times. 7.2 mm of the first order axis is preserved which is 0.3 mm wide. Second order axes are 7.0 and 9.5 mm long and 0.2 and 0.3 mm wide respectively. Fourth order axes are 6.5–8 mm long and 0.2–0.3 mm wide. Only one fifth order axis is preserved and measure 4.5 mm long and 0.3 mm wide. The sixth order axes correspond to the subtending axes of the sporangia. They measure 2.4–3.1 mm long and 0.2–0.3 mm wide. The junction between the sporangium and the subtending axis is clear.The base of the plant is best seen on Fig. 9f,g. When preserved, first order axes are long, only dichotomize after 18 to 19 mm and are 0.3 mm wide. Several more or less parallelly disposed axes converge basally on a poorly preserved structure of indefinite shape (Fig. 9h,l). Up to 6 axes are observed to arise from this structure that is here interpreted as a remnant of the gametophyte. It is up to 4 mm wide. Figure 9d and e however suggest that in life a larger number of axes were probably attached to a more extensive gametophyte or cluster of gametophytes.The sporangia are vertically elongated and straight sided with broadly rounded apices. They measure 1.3–1.6 mm long and 0.4–0.7 mm wide.Identity and comparisonAs discussed for Krommia parvapilla, the occurrence of very small sporangia has been reported many times, mostly from Wales. In the majority of cases however, they are found isolated or connected to very fragmentary branching systems. The very limited available information (mostly the shape of the sporangium) makes comparison and identification not only difficult but also very likely misleading. The extensive branching system preserved in the Impofu Dam material allows for a better description of the plant to be made. Three features are noticeable, the shape of the sporangia (elongated, parallel sided and rounded tips), the delicate branching system with relatively small branching angles and the occurrence of possible gametophytic tissues at the base of the plant. As already discussed above, even if the shape of the sporangia is by far the most informative character in early land plants, it is also misleading as they are very simple. When comparing the sporangia alone, the Impofu Dam material most closely resembles Tarrantia salopensis Fanning et al.36 and Uskiella reticulata Fanning et al.36. The sporangia are however distinct, being much smaller and presenting a height/width ratio of 3.3 which is much higher than that encountered in these two species. The sporangia further lack the characteristic reticulation of U. reticulata. When comparing the whole plant, the Impofu Dam material very closely resembles Eogaspesia gracilis Daber75. This plant has been described as small slender dichotomizing axes being borne on a thicker dichotomous rhizome and bearing small ovate sporangia. Based on the illustrations, the connection between the axes and the so-called rhizome are dubious. Despite superficial resemblance, the south African material differs from Eogaspesia by being smaller (80–90 mm long for Eogaspesia as opposed to 60 mm for Elandia) and by presenting isotomous divisions only. The sporangia though very simple and thus difficult to compare differ in shape being more elongate (length/width ration of 3.8 in Elandia as opposed to 2.5 in Eogaspesia) are more parallel sided and have more rounded tips. We therefore exclude the Impofu Dam material from these two taxa. As far as we know, the combination of characters described above has never been encountered before. We thus chose to erect a new genus and a new species.GenusMtshaelo gen. nov.Type species: Mtshaelo kougaensis sp. nov.Derivation of the name: isiXhosa, a traditional broom.Diagnosis. Plant with multiple (at least 6) elongate sporangia, spindle-shaped in profile and evenly tapering to acute terminations, truncated proximally at point of attachment; arranged in a truss of sporangia that terminates elongate parallel sided isotomously bifurcating axes.SpeciesMtshaelo kougaensis sp. nov.Diagnosis. As for genus, robust axes bifurcate at least twice and widen slightly towards the terminal truss of sporangia; individual sporangia 0.74 to 0.8 mm wide and 4–6 mm long with a longitudinal dehiscence line. Vegetative axes 1.0 to 1.4 mm wide.Derivation of the name: Kouga is the name of the district in which the site is found, from Khoisan meaning ‘place of plenty’, -ensis from Greek meaning from.Holotype: AM 7999a (part), Fig. 10a.Figure 10(a–g) Mtshayelo kougaensis gen. et sp. nov., (h–i) Yarravia oblonga, (j–k) incertae sedis bilobed sporangia. (a) AM 7999a. Scale = 1 cm. Holotype. Bifurcating naked axis terminated by two synangiate structures. (b) Detail of AM 7999a. scale = 5 mm. Two synangiate structures. (c) Detail of AM 7999a. Scale = 5 mm. Detail showing the organization of a synangiate structure with the dehiscence line clearly visible. (d) Am 7902. Scale = 5 mm. Isolated synangiate structure. (e) AM 7904. Scale = 2.5 mm. Isolated branching axis showing two dichotomies and two terminal synangiate structures. (f) Detail of AM 7904. Scale = 5 mm. Two terminal synagiate structure. The arrow indicates where individual sporangia are starting. (g) AM 7990. Scale = 1 cm. Specimen showing several superimposed plants. (h) AM 7983. Scale = 5 mm. Gross morphology of the plant. (i) Detail of AM 7983. Scale = 5 mm. Detail of the synangiate structure showing four closely adpressed individual sporangia with a pointed tip. (j) AM 7933. Scale = 1 cm. Gross morphology of the plant. (k) Detail of AM 7933. Scale = 5 mm. Sporangia.Full size imageParatypes: AM 7902, AM 7904, AM 7933, AM 7983, AM 7990.Repository: Albany Museum, Devonian Lab, Beaufort Street, Makhanda, Eastern Cape, South Africa.Type locality: Impofu Dam, Kouga Municipality, Eastern Cape, South Africa (Fig. 1).Horizon: Kareedouw Member, Baviaanskloof Formation, Nardouw Subgroup, Table Mountain Group, Cape Supergroup.Age: Lower Devonian, Lochkovian?
Fig. 10a–g, Fig. 11.

Figure 11Proposed reconstruction of Mtshaelo kougaensis (a) and of its synangiate structure (b).Full size image
MaterialFive specimens of this plant have been collected from the LPL.DescriptionThis plant most often occurs as isolated branched axes (Fig. 10a–f) but can in some cases occur as densely packed trusses of axes (Fig. 10g). In the latter the organization of the branching system is obscured by the many superimpositions. The plant consists of robust smooth parallel-sided axes that dichotomize up to at least two times and terminate in trusses of elongate structures. The lack of preserved anatomy or spore contents prevents demonstration of the fertile nature of these structures. Considering their position and in analogy with other plants of similar organization we will consider them as sporangia. All sporangia seem to be attached at the same level giving to the whole structure the aspect of a synangium.The organization of the vegetative parts is best seen in specimen AM 7999 (Fig. 10a). This specimen is 80 mm long and has a generally flexuous appearance despite the robust aspect of the axes. It branches at least two times but only two synangium-like structures are preserved, only one of which exhibits clear attachment to the full branching system. The first order axis is 17 mm long and 1.4 mm wide. It branches at an angle of 32°. Second order axes are both quite flexuous and marked by a 90° curvature in the same direction. The left hand more completely preserved axis measures 20 mm long and 1.4 mm wide. It is difficult to say whether the evident curvature was originally present or the consequence of taphonomical processes. It could represent a horizontal part of the sporophyte as proposed in Fig. 4.Only one third order axis is fully exposed. It measures 39 mm long and is1.2 mm wide. Its width remains constant up to the distal end where it flares slightly up to 2.2. A second less complete termination flares to 2.6 mm from a subtending axis 1.0 mm wide. The axes then give the impression of being subdivided into several elongated structures that we interpret as synangiate sporangia.Several terminal structures are preserved (Fig. 10a–g). The distal end of the ultimate axis is marked by a slight and progressive widening (Fig. 10b–e). The axis then gives rise to several elongate sporangia that are all attached at the same level (Fig. 10d,e). The detailed organization of the termination is difficult to decipher. Although truncated, individual sporangia are particularly visible on the lower specimen in Fig. 10b. In this case three sporangia are visible and separated by a darker line of sediment. In other specimens, up to 4 sporangia can be identified (Fig. 10d,e). Distally, several additional tips can be seen suggesting that there are more sporangia hidden behind. Three to four additional tips can be identified in some cases (Fig. 10d,e). We consequently interpret the termination as being of 6 to 8 sporangia very likely organized in a circle. Individual sporangia are 0.4 to 0.8 mm wide and 4–6 mm long. They are spindle-shaped in profile, truncated proximally at the site of attachment and tapering distally to an acute tip. In some cases a slightly darker line can be observed within the sporangia. It could be interpreted as a longitudinal dehiscence line (Fig. 10c).
Yarravia64
Yarravia oblonga64
Fig. 10h and i
MaterialOne specimen from the LPL.DescriptionThis plant consists of robust smooth parallel-sided axes (Fig. 10h). Only one dichotomy is preserved. The axes are terminated by a truss of sporangia resembling a synangium. This specimen is 23 mm long and only shows the ultimate dichotomy and the axes subtending the terminal structures. The penultimate axis order is 9.8 mm long and 0.8 mm wide. It dichotomizes at an angle of 58° forming two axes measuring 4.5 and 7.0 mm long and 1.0 and 1.1 mm wide respectively. Only one synangium-like structure is preserved. Just before the insertion point of the sporangia, the subtending axis widens to 2.1 mm. Three elongate sporangia appear to all be inserted at the same level and are 3.5–4.2 mm long and 0.7–1.0 mm wide (Fig. 10i). They are parallel-sided. Their apices narrow abruptly and terminate in a slightly recurved beak-like structure. The tip of a possible fourth distal sporangium is also apparent.Identity and comparisonPlants presenting dichotomizing axes terminated by synangiate structures are rare in the Lower Devonian. The Impofu Dam material strongly recalls material attributed to the genus Yarravia that was reported from several Lower Devonian localities in Australia65,28,57 . Two species were identified Yarravia oblonga and Yarravia subsphaerica. The Impofu Dam material conforms more strongly to Yarravia oblonga. Of this plant, only the synangium-like structures and part of their subtending axes are known. The subtending axes present a massive aspect like that observed in the Impofu Dam material and the synangium like structures share the same organization. The size of the South African specimen is however smaller than the original material from Yarra Track but conforms almost exactly to material referred to as Yarravia cf. oblonga from Lilydale. Other occurrences of the genus Yarravia have been reported from France and Russia66,67, however these specimens need further study in order to be properly compared. Finally, specimens attributed to the genus Yarravia have been collected from the Devonian of Arizona but would as well need additional investigation68.
Incertae sedis heart-shape termination.
Fig. 10j and k.
MaterialA single specimen of this plant has been recovered from LPL.DescriptionThe plant measures 47 mm long. The branching system is apparently isotomous and branches only once (Fig. 10j). The first order is 33 mm long and 0.6 mm wide, slender and smooth. Second order are 9.2 and 9.8 mm long and 0.5 mm wide. The apex of each ultimate axis consists of a heart-shape structure measuring 4.0 mm long and 2.3 to 2.6 mm in width (Fig. 10k). This structure is complex and comprised of at least two more or less independent units. The two structures are easy to distinguish however they never seem to separate completely before their tips. These structures, here interpreted as sporangia, seem to occur after a dichotomy of the axis. Each one then progressively widens to reach 1.0 to 1.3 mm wide at two third of its length. Thereafter it forms a rounded tip, giving to the whole sporangium a club-shape.Identity and comparisonsThis plant bears a superficial resemblance to the Australian Yarravia Lang and Cookson28. Yarravia is characterised by several elongated sporangia apparently all attached at a single point. Our material rather seems to be composed of only two sporangia. A similar organisation was also described from the Lochkovian north Brown Clee Hill locality, Welsh Borderland (UK)60,61,62 . Although much smaller and preserved anatomically, Grisellatheca salopensis Edwards et al.62 presents a heart-shape fertile region made of two sporangia. Further comparison is however made difficult by the lack of anatomical details in our material. More

Mesocosm setupThe mesocosm experiment AQUACOSM VIMS-Ehux was carried out between 24th May (day 0) and 16th June (day 23) 2018 in Raunefjorden at the Marine Biological Station Espegrend, Norway (60°16′11 N; 5°13′07E) as previously described [7]. Four light-transparent enclosure bags were filled with surrounding fjord water (day −1; pumped from 5 m depth), and continuously mixed by aeration (from day 0 onwards). Each bag was supplemented with nutrients at a nitrogen to phosphorous ratio of 16:1 (1.6 µM NaNO3 and 0.1 µM KH2PO4 final concentration) on days 0–5 and 14–17, whereas on days 6, 7, and 13 only nitrogen was added. Nutrient concentrations were measured daily [18].Enumeration of phytoplankton cells by flow cytometryFor E. huxleyi enumeration by flow cytometry, water samples were collected in 50 mL tubes from ~1 m depth. Water samples were pre-filtered using 40 µm cell strainers and immediately analyzed with an Eclipse iCyt flow cytometer (Sony Biotechology, Champaign, IL, USA) as previously described [19]. A total volume of 300 µl with a flow rate of 150 µl min−1 was analyzed. A threshold was applied on the forward scatter to reduce background noise. Four groups of phytoplankton populations were identified in distinct gates by plotting the autofluorescence of chlorophyll (em: 663–737 nm) versus phycoerythrin (em: 570–620 nm) and side scatter: calcified E. huxleyi (high chlorophyll and high side scatter), Synechococcus (high phycoerythrin), nanophytoplankton including calcified and non-calcified E. huxleyi (high chlorophyll and phycoerythrin), and picophytoplankton (low chlorophyll and low phycoerythrin) [20]. See Fig. S1 for further details of gating strategy.Enumeration of EhV-like particles and bacteria by flow cytometryFor EhV and bacteria counts, 200 µl of sample were fixed a final concentration of 0.5% glutaraldehyde for one hour at 4 °C and flash frozen in liquid nitrogen. For analysis, they were thawed and stained with SYBR gold (Invitrogen, Carlsbad, CA, USA) that was diluted 1:10,000 in 0.2 μm filtered TE buffer (10:1 mM Tris:EDTA, pH 8), incubated for 20 min at 80 °C and cooled to room temperature [21]. Bacteria and EhV-like particles were counted and analyzed using an Eclipse iCyt flow cytometer (ex: 488 nm, em: 500–550 nm), and identified by comparing to reference samples containing fixed EhV201 and bacteria from lab cultures. EhV gating was very stringent in order to minimize the misidentification of other large viruses such as Micromonas pusilla virus (MpV) in the samples (see Fig. S2 for further details of gating strategy for EhV counts).Enumeration of extracellular EhV by qPCRWater samples (1–2 l) were sequentially filtered by vacuum through polycarbonate filters with a pore size of 20 µm (47 mm; Sterlitech, Kent, WA, US), then 2 µm (Isopore, 47 mm; Merck Millipore, Cork, Ireland), and finally 0.22 µm (Isopore, 47 mm; Merck Millipore). Filters were immediately flash-frozen in liquid nitrogen and stored at −80 °C until further processing. DNA was extracted from the 0.22 µm filters using the DNeasy PowerWater kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Each sample was diluted 100 times, and 1 µl was then used for qPCR analysis. EhV abundance was determined by qPCR for the major capsid protein (mcp) gene [22] using the following primers: 5ʹ-acgcaccctcaatgtatggaagg-3ʹ (mcp1F[23],) and 5ʹ-rtscrgccaactcagcagtcgt-3ʹ (mcp94Rv; Mayers, K. et al., unpublished). All reactions were carried out in technical triplicates. For all reactions, Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen) was used as described by the manufacturer. Reactions were performed on a QuantStudio 5 Real-Time PCR System equipped with the QuantStudio Design and Analysis Software version 1.5.1 (Applied Biosystems, Foster City, CA, USA) as follows: 50 °C for 2 min, 95 °C for 5 min, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. Results were calibrated against serial dilutions of EhV201 DNA at known concentrations, enabling exact enumeration of viruses. Samples showing multiple peaks in melting curve analysis or peaks that were not corresponding to the standard curves were omitted.Vesicle concentration and separationLab samplesE. huxleyi CCMP2090 was grown in 20 l filtered sea water (FSW) supplemented with K/2 nutrient mix at 18 °C, 16:8 h light:dark cycle, 100 μmol photons m−2 s−1. Uninfected cultures were grown to ~ 106 cells ml−1. Infected cultures were inoculated with EhV201 at a multiplicity of infection (MOI) of ~1:1 plaque forming unit (pfu) per cell and incubated under normal growth conditions for 120 h, at which time the culture had cleared. The entire 20 l volume was then filtered through a GF/C filter (Whatman, Maidstone, United Kingdom) followed by an 0.45 µm PVDF filter (Durapore, Merck Millipore) to eliminate cells and cellular debris.Mesocosm samplesOn days 2, 4, 5, 8, 12, 15, 18, and 23 we collected 25 l from bags 1–4 and combined them to produce one sample of 100 l for each sampling time. The samples were pre-filtered using a 200 µm nylon mesh, and then filtered through a GF/C filter (Whatman) followed by an 0.45 µm PVDF filter (Durapore, Merck Millipore) to eliminate cells and cellular debris.Particle concentrationParticles in the flow-through from the filtration stage were concentrated on a 100 kDa tangential flow filter (Spectrumlabs, Repligen, Waltham, Massachusetts, USA) to a final volume of ~500 ml. At this stage, mesocosm samples were stored in the dark at +4 oC and shipped back to the home lab. All samples were further concentrated to a final volume of 1–2 ml using 100 kDa Amicon-ultra filters (Merck Millipore).Vesicle separationVesicles were separated from other particles (including viruses) using an 18–35% OptiPrep gradient (MilliporeSigma, St. Louis, Missouri, USA). Gradients were centrifuged in an ultracentrifuge for 12 h at 200,000 × g. Fractions (0.5 ml) were collected from the top of the gradient and the fraction material was cleaned by washing three times and resuspended in 0.02 µm-filtered FSW using 100 kDa Amicon-ultra filters (Merck Millipore). Vesicles were detected in fractions with densities of 1.05–1.07 g ml–1 (fractions 3–5 from the top).Vesicle concentration in samples from lab cultures was measured by NTA using the NanoSight NS300 instrument (Malvern Instruments, Malvern, UK) equipped with a 488 nm laser module and NTA V3.2 software. Samples were diluted so that each field of view contained 20–100 particles. Three 60 s videos were recorded for each biological replicate, representing different fields of view. All the videos for a given experiment were processed using identical settings (screen gain of one and detection threshold of five).RNA extraction and sequencingIn order to eliminate RNA molecules that are not packed into vesicles, we subjected vesicle samples to RNase treatment prior to RNA extraction. Samples were incubated for 60 min at 37 oC with 10 pg µl−1 of RNase A (Bio Basic, Toronto, Canada). RNase activity was inactivated by adding 100 unites of Protector RNase Inhibitor (Roche, Basel, Switzerland). Total RNA (including RNA from ~18 nucleotides or more) was extracted using the miRNeasy kit according to the manufacturer’s instructions (Qiagen). Libraries were prepared using the TruSeq Small RNA Library kit (Illumina, San Diego, CA, USA), according to the manufacturer’s protocol. Each sample was indexed twice with the same index, one with polynucleotide kinase I treatment (according to manufacturer’s instructions, NEB, Ipswich, Massachusetts, USA) and one without. After 15 cycles of PCR amplification, libraries were cleaned with the QIAquick PCR Purification Kit according to the manufacturer’s instructions (Qiagen). Libraries were sequenced on the NextSeq platform (Illumina).sRNA bioinformatics analysisLow-quality read ends were trimmed and adaptors were removed using the cutadapt program [24], version 1.18. Reads shorter than 17 bp after the trimming were removed from further analyses. The remaining reads were mapped to an E. huxleyi integrated reference transcriptome shortly described in [6] using the RSEM software [25], version 1.3.1, with the default option of bowtie, version 1.1.2 [26]. Genes that had at least 5 reads in any of the samples were selected. For the heatmap (Fig. 1d), read counts were scaled to one million reads mapped to the E. huxleyi transcriptome and log2 transformed.Effect of vesicles on natural populations—experimental design and analysisOn days 14 and 20 of the mesocosm experiment (blue and red arrows in Fig. 1a, respectively), we combined equal volumes of water samples from bags 1–4 and filtered them through a 10 µm nylon mesh to eliminate zooplankton predators. We then supplemented the natural populations with f/50 nutrient mix and divided them into flasks, each containing 10 ml. In total, 30 flasks were treated with vesicles from uninfected lab cultures of E. huxleyi CCMP2090, at a ratio of ~500 vesicles cell−1 (calcified E. huxleyi determined by flow cytometry), and then all flasks were incubated in a growth chamber (15 °C, 16:8 h light:dark cycle, 100 μmol photons m−2 s−1). Once a day, samples were taken for flow cytometric quantification of live cells (see “Enumeration of phytoplankton cells by flow cytometry” above), or fixed for virus and bacteria counts (see “Enumeration of EhV-like particles and bacteria by flow cytometry” above). For statistical analysis, we used two-tailed t test with equal variance.Decay rate of EhV virions- experimental design and analysisTo determine the decay rate of infectivity of natural EhV virions, water was sampled from bag 4 on day 18, at a time point when viral infection was detected (green cross in Fig. 1a). This sample was filtered through a 0.45 PVDF filter (Durapore, Merck Millipore) to eliminate algal and most bacteria cells. EhV-like particles were counted by flow cytometry as described above and divided into nine tubes, each containing 1 ml. Triplicate samples were either treated with vesicles from EhV201-infected (VirusVesicles) or uninfected (controlVesicles) lab cultures (see above) at a ratio of ten vesicles per EhV-like particle, or not treated at all. All tubes were incubated in an on-land mesocosm facility that mimics the light and temperature conditions found at ~ 1 m depth within the fjord water. We used the most probable number (MPN) method [27] to determine the half-life of EhV within these samples. Briefly, a series of five-fold dilutions was prepared for each sample. Each dilution (10 μl) was then added, in eight technical replicates, to 100 μl of exponentially growing E. huxleyi CCMP374 cultures in multi-well plates and incubated under normal growth conditions for five days. This was repeated for four consecutive days for all samples. Clearance (infection) of the cells in the multi-wells was measured using an EnSpireTM 2300 Multilabel Reader (PerkinElmer, Turku, Finland) set to in vivo fluorescence (ex:460 nm, em:680 nm). MPN was calculated using the MPN calculation program, version 5 [28]. For the samples treated with controlvesicles, we could only obtain a positive MPN value for one time point, as the decay was faster than expected. Therefore, the minimum detectable infectivity values were used in order to calculate the maximum possible half-life. For statistical analysis, each treatment was compared to the untreated control, using ANOVA with Dunnett’s post-hoc test. More

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