Litter Breakdown and Microbial Succession on Two Submerged Leaf Species in a Small Forested Stream

<div><p>Microbial succession during leaf breakdown was investigated in a small forested stream in west-central Georgia, USA, using multiple culture-independent techniques. Red maple (<i>Acer rubrum</i>) and water oak (<i>Quercus nigra</i>) leaf litter were incubated <i>in situ</i> for 128 days, and litter breakdown was quantified by ash-free dry mass (AFDM) method and microbial assemblage composition using phospholipid fatty acid analysis (PLFA), ribosomal intergenic spacer analysis (RISA), denaturing gradient gel electrophoresis (DGGE), and bar-coded next-generation sequencing of 16S rRNA gene amplicons. Leaf breakdown was faster for red maple than water oak. PLFA revealed a significant time effect on microbial lipid profiles for both leaf species. Microbial assemblages on maple contained a higher relative abundance of bacterial lipids than oak, and oak microbial assemblages contained higher relative abundance of fungal lipids than maple. RISA showed that incubation time was more important in structuring bacterial assemblages than leaf physicochemistry. DGGE profiles revealed high variability in bacterial assemblages over time, and sequencing of DGGE-resolved amplicons indicated several taxa present on degrading litter. Next-generation sequencing revealed temporal shifts in dominant taxa within the phylum <i>Proteobacteria</i>, whereas γ-<i>Proteobacteria</i> dominated pre-immersion and α- and β-<i>Proteobacteria</i> dominated after 1 month of instream incubation; the latter groups contain taxa that are predicted to be capable of using organic material to fuel further breakdown. Our results suggest that incubation time is more important than leaf species physicochemistry in influencing leaf litter microbial assemblage composition, and indicate the need for investigation into seasonal and temporal dynamics of leaf litter microbial assemblage succession.</p></div

Mean (± 1SE) % relative abundance (%) of fungal lipid markers of red maple and water oak leaf packs over the 128-d incubation In Kings Mill Creek, GA, USA.

<p>(* = <i>p</i><0.001, ns = not significantly different).</p

Unifrac-based unweighted pair group method with arithmetic mean (UPGMA) clustering of maple and oak sequences following 0, 32, and 128 days of instream incubation.

<p>Values at nodes represent level of clustering support expressed as a decimal ranging from 0 to 1.0.</p

Comparison of alpha diversity metrics calculated for maple and oak leaf litter bacterial assemblages from paired-end sequencing results following 0, 32, and 128 days instream incubation at an even sampling depth of 4260 sequences.

<p>Superscript letters beside metric values represent Tukey’s multiple comparison groupings for each metric (α = 0.05).</p><p>Comparison of alpha diversity metrics calculated for maple and oak leaf litter bacterial assemblages from paired-end sequencing results following 0, 32, and 128 days instream incubation at an even sampling depth of 4260 sequences.</p

Dendrogram of ribosomal intergenic spacer analysis (RISA) electropherograms displaying bacterial assemblage similarities calculated using Ward’s method based on Jaccard’s similarity coefficient.

<p>Dendrogram of ribosomal intergenic spacer analysis (RISA) electropherograms displaying bacterial assemblage similarities calculated using Ward’s method based on Jaccard’s similarity coefficient.</p

Mean (± 1SE) ash free dry mass (AFDM) remaining over time during breakdown of red maple (closed circles), water oak (open circles), and mixed litter (inverted solid triangle) leaf packs incubated for 128 d in Kings Mill Creek, GA, USA.

<p>Mean (± 1SE) ash free dry mass (AFDM) remaining over time during breakdown of red maple (closed circles), water oak (open circles), and mixed litter (inverted solid triangle) leaf packs incubated for 128 d in Kings Mill Creek, GA, USA.</p

Intra and inter-species ANI values for the 44 <i>Aeromonas</i> genomes and identification on the basis of the MLPA and ANI (mislabeled genomes in bold).

<p>Intra and inter-species ANI values for the 44 <i>Aeromonas</i> genomes and identification on the basis of the MLPA and ANI (mislabeled genomes in bold).</p

Photos of extract and algal application.

<p>Photos illustrate three experimental plates (shade control, solvent control, and macroalgal extract) on: A. <i>M. faveolata</i> and B. <i>P. astreoides</i> corals. Experimental application of C. <i>Dictyota</i> sp. foliose brown macroalgae and algal mimic to a <i>P. astreoides</i> coral colony in Belize (2009), and D. <i>Lobophora variegata</i> decumbant brown macroalgae and algal mimic to a <i>M. faveolata</i> coral colony in Belize (2009).</p

SOD and CAT concentration results for experiments conducted in Belize (2009).

a<p>Algal treatments are organic (org.), aqueous (aqua), or live algal thalli (live) applications.</p>b<p>Mean concentration ± SE in coral tissue after 3 day exposure to treatments (n = 5). <i>P</i>-values generated with one-way ANOVA and Dunnett post-hoc tests.</p>c<p>Control values are collected from coral samples under the solvent plate control.</p

PERMANOVA and MRPP results for algal extract application and live algal thalli application.

a<p>Function of initial controls, experimental controls (solvent + shade + post controls combined), and treatment extracts,</p>b<p>Function of initial controls, post controls, algal mimic, live algae,</p>*<p>P<0.05,</p>**<p>P<0.001.</p