A confidence interval analysis of sampling effort, sequencing depth, and taxonomic resolution of fungal community ecology in the era of high-throughput sequencing

<div><p>High-throughput sequencing technology has helped microbial community ecologists explore ecological and evolutionary patterns at unprecedented scales. The benefits of a large sample size still typically outweigh that of greater sequencing depths per sample for accurate estimations of ecological inferences. However, excluding or not sequencing rare taxa may mislead the answers to the questions ‘how and why are communities different?’ This study evaluates the confidence intervals of ecological inferences from high-throughput sequencing data of foliar fungal endophytes as case studies through a range of sampling efforts, sequencing depths, and taxonomic resolutions to understand how technical and analytical practices may affect our interpretations. Increasing sampling size reliably decreased confidence intervals across multiple community comparisons. However, the effects of sequencing depths on confidence intervals depended on how rare taxa influenced the dissimilarity estimates among communities and did not significantly decrease confidence intervals for all community comparisons. A comparison of simulated communities under random drift suggests that sequencing depths are important in estimating dissimilarities between microbial communities under neutral selective processes. Confidence interval analyses reveal important biases as well as biological trends in microbial community studies that otherwise may be ignored when communities are only compared for statistically significant differences.</p></div

Effect of sequencing depths on estimates of geographic community dissimilarity and 95% confidence intervals for FFE communities that differ in geography.

<p>FFE communities between nine <i>P</i>. <i>taeda</i> plots across varying distances (1–107 km) were compared. ANOSIM R (grey) and PermANOVA R² (red) values were calculated with Bray-Curtis dissimilarity and 95% confidence interval based on 1000 subsamples. Sequencing depth tested at 100, 1000, 5000, 10000, 20000. X-axes are on a log-scale. Dotted lines indicate 95% confidence interval. Solid lines indicate the mean.</p

Effect of sequencing depths on estimates of ANOSIM R and 95% confidence intervals based on Bray-Curtis dissimilarity indices.

<p>Comparisons were made between FFE communities of a) bases and tips of <i>P</i>. <i>taeda</i> needles at 5, 10, 20 and 60 samples per group and b) between <i>P</i>. <i>torreyana</i> needles between Santa Rosa and San Diego, CA at 3, 6, 9, and 14 samples per group. Sequencing depths were tested at 100, 1000, 5000, 10000, 20000. X-axes are on a log-scale. Dotted lines indicate 95% confidence intervals for different sampling sizes. Solid lines indicate the mean. Trends for different sampling efforts were overlapped in a single plot.</p

β-diversity patterns are revealed with increasing sequencing depths between communities that differ only by random processes.

<p>Non-metric multidimensional scaling of two simulated communities drifted for 20 generations from one community with 5 million individuals and 974 OTUs (taxa) have beta-diversity patterns revealed only after 50,000 sequences per sample, which corresponds to ~1% of the community per sample. Ordinations are based on between-sample dissimilarity calculated with Bray-Curtis (left panel), Jaccard (middle panel). OTU accumulation curves (right panel) demonstrate community sampling with varying sequencing depths. From top to bottom, sequencing depths correspond to a) 100, b) 1000, c) 5000, d) 10000, and e) 50000 sequences. Insets represent the accumulation curves from the previous sequencing depth.</p

Effect of sampling effort on estimates of ANOSIM R and PERMANOVA R² effect sizes and 95% confidence intervals.

<p>Sampling effort represents random subsamples of each comparison group with replacement. ANOSIM R (grey) and PERMANOVA R² (red) values were calculated with Bray-Curtis dissimilarity and 95% confidence interval based on 1000 subsamples. a) Comparison of FFE communities between bases and tips of <i>P</i>. <i>taeda</i> needles at five-sample intervals from five to 60 samples each. b) Comparison of FFE communities between <i>P</i>. <i>torreyana</i> needles between different geographic locations (Santa Rosa and San Diego, CA) at one-sample intervals from three to 14 samples each.</p

Image_5_Restoring bigcone Douglas-fir post-fire in drought-stricken Southern California: Assessing the effects of site choice and outplanting strategies.JPEG

IntroductionForest restoration is a powerful tool to combat the dual threats of drought and fire, both of which have been increasing in frequency and severity in recent years in the Western United States. The hard-hit region of Southern California is home to the endemic bigcone Douglas-fir, Pseudotsuga macrocarpa (Vasey), whose abundance and range have been impacted by multiple large fires within the last two decades.MethodsTo better understand the ecology of these trees, and thereby improve the potential for restoration in light of predicted future conditions, we outplanted 1,728 seedlings into burned areas with varying levels of pre-planting fire severity and proximity to water (near canyon bottom and upslope). Within each planting area, seedlings were planted into one of four microhabitats; under conspecifics, under the codominant oak species [Quercus chrysolepis (Liebm)], in the open (no woody canopy), or in the open within a microsite feature such as a log, rock or a small shaded hole. At each site and within each microhabitat, half the seedlings were treated with oak-soil amendments (soil from under the oak) and half with spring watering (4 months after planting). To better understand the influence of outplanting conditions, we tracked the survival of the seedlings over the next two years.Results and discussionShort-term (March to June) survivorship following planting was quite high and the most significant die-off of seedlings occurred during the first dry season (June to November) despite strong droughts in the second and third dry seasons. Overall, only 7.3% (127 of 1,728) of seedlings survived. Seedling success depended strongly on the microhabitat and summer watering, though not in necessarily intuitive ways. Seedlings that received supplemental water during the first summer did worse than unwatered seedlings. The most successful microhabitats for planting were open sites with microsite features and sites underneath canyon live oak, while sites under mature bigcone Douglas-firs had the lowest rates of survival. Position on the slope had no effect on outcomes and soil amendment had a weak negative effect on seedling survival.</p

Image_3_Restoring bigcone Douglas-fir post-fire in drought-stricken Southern California: Assessing the effects of site choice and outplanting strategies.TIFF

IntroductionForest restoration is a powerful tool to combat the dual threats of drought and fire, both of which have been increasing in frequency and severity in recent years in the Western United States. The hard-hit region of Southern California is home to the endemic bigcone Douglas-fir, Pseudotsuga macrocarpa (Vasey), whose abundance and range have been impacted by multiple large fires within the last two decades.MethodsTo better understand the ecology of these trees, and thereby improve the potential for restoration in light of predicted future conditions, we outplanted 1,728 seedlings into burned areas with varying levels of pre-planting fire severity and proximity to water (near canyon bottom and upslope). Within each planting area, seedlings were planted into one of four microhabitats; under conspecifics, under the codominant oak species [Quercus chrysolepis (Liebm)], in the open (no woody canopy), or in the open within a microsite feature such as a log, rock or a small shaded hole. At each site and within each microhabitat, half the seedlings were treated with oak-soil amendments (soil from under the oak) and half with spring watering (4 months after planting). To better understand the influence of outplanting conditions, we tracked the survival of the seedlings over the next two years.Results and discussionShort-term (March to June) survivorship following planting was quite high and the most significant die-off of seedlings occurred during the first dry season (June to November) despite strong droughts in the second and third dry seasons. Overall, only 7.3% (127 of 1,728) of seedlings survived. Seedling success depended strongly on the microhabitat and summer watering, though not in necessarily intuitive ways. Seedlings that received supplemental water during the first summer did worse than unwatered seedlings. The most successful microhabitats for planting were open sites with microsite features and sites underneath canyon live oak, while sites under mature bigcone Douglas-firs had the lowest rates of survival. Position on the slope had no effect on outcomes and soil amendment had a weak negative effect on seedling survival.</p

Image_6_Restoring bigcone Douglas-fir post-fire in drought-stricken Southern California: Assessing the effects of site choice and outplanting strategies.JPEG

IntroductionForest restoration is a powerful tool to combat the dual threats of drought and fire, both of which have been increasing in frequency and severity in recent years in the Western United States. The hard-hit region of Southern California is home to the endemic bigcone Douglas-fir, Pseudotsuga macrocarpa (Vasey), whose abundance and range have been impacted by multiple large fires within the last two decades.MethodsTo better understand the ecology of these trees, and thereby improve the potential for restoration in light of predicted future conditions, we outplanted 1,728 seedlings into burned areas with varying levels of pre-planting fire severity and proximity to water (near canyon bottom and upslope). Within each planting area, seedlings were planted into one of four microhabitats; under conspecifics, under the codominant oak species [Quercus chrysolepis (Liebm)], in the open (no woody canopy), or in the open within a microsite feature such as a log, rock or a small shaded hole. At each site and within each microhabitat, half the seedlings were treated with oak-soil amendments (soil from under the oak) and half with spring watering (4 months after planting). To better understand the influence of outplanting conditions, we tracked the survival of the seedlings over the next two years.Results and discussionShort-term (March to June) survivorship following planting was quite high and the most significant die-off of seedlings occurred during the first dry season (June to November) despite strong droughts in the second and third dry seasons. Overall, only 7.3% (127 of 1,728) of seedlings survived. Seedling success depended strongly on the microhabitat and summer watering, though not in necessarily intuitive ways. Seedlings that received supplemental water during the first summer did worse than unwatered seedlings. The most successful microhabitats for planting were open sites with microsite features and sites underneath canyon live oak, while sites under mature bigcone Douglas-firs had the lowest rates of survival. Position on the slope had no effect on outcomes and soil amendment had a weak negative effect on seedling survival.</p

Image_2_Restoring bigcone Douglas-fir post-fire in drought-stricken Southern California: Assessing the effects of site choice and outplanting strategies.TIFF

IntroductionForest restoration is a powerful tool to combat the dual threats of drought and fire, both of which have been increasing in frequency and severity in recent years in the Western United States. The hard-hit region of Southern California is home to the endemic bigcone Douglas-fir, Pseudotsuga macrocarpa (Vasey), whose abundance and range have been impacted by multiple large fires within the last two decades.MethodsTo better understand the ecology of these trees, and thereby improve the potential for restoration in light of predicted future conditions, we outplanted 1,728 seedlings into burned areas with varying levels of pre-planting fire severity and proximity to water (near canyon bottom and upslope). Within each planting area, seedlings were planted into one of four microhabitats; under conspecifics, under the codominant oak species [Quercus chrysolepis (Liebm)], in the open (no woody canopy), or in the open within a microsite feature such as a log, rock or a small shaded hole. At each site and within each microhabitat, half the seedlings were treated with oak-soil amendments (soil from under the oak) and half with spring watering (4 months after planting). To better understand the influence of outplanting conditions, we tracked the survival of the seedlings over the next two years.Results and discussionShort-term (March to June) survivorship following planting was quite high and the most significant die-off of seedlings occurred during the first dry season (June to November) despite strong droughts in the second and third dry seasons. Overall, only 7.3% (127 of 1,728) of seedlings survived. Seedling success depended strongly on the microhabitat and summer watering, though not in necessarily intuitive ways. Seedlings that received supplemental water during the first summer did worse than unwatered seedlings. The most successful microhabitats for planting were open sites with microsite features and sites underneath canyon live oak, while sites under mature bigcone Douglas-firs had the lowest rates of survival. Position on the slope had no effect on outcomes and soil amendment had a weak negative effect on seedling survival.</p

Image_8_Restoring bigcone Douglas-fir post-fire in drought-stricken Southern California: Assessing the effects of site choice and outplanting strategies.JPEG

IntroductionForest restoration is a powerful tool to combat the dual threats of drought and fire, both of which have been increasing in frequency and severity in recent years in the Western United States. The hard-hit region of Southern California is home to the endemic bigcone Douglas-fir, Pseudotsuga macrocarpa (Vasey), whose abundance and range have been impacted by multiple large fires within the last two decades.MethodsTo better understand the ecology of these trees, and thereby improve the potential for restoration in light of predicted future conditions, we outplanted 1,728 seedlings into burned areas with varying levels of pre-planting fire severity and proximity to water (near canyon bottom and upslope). Within each planting area, seedlings were planted into one of four microhabitats; under conspecifics, under the codominant oak species [Quercus chrysolepis (Liebm)], in the open (no woody canopy), or in the open within a microsite feature such as a log, rock or a small shaded hole. At each site and within each microhabitat, half the seedlings were treated with oak-soil amendments (soil from under the oak) and half with spring watering (4 months after planting). To better understand the influence of outplanting conditions, we tracked the survival of the seedlings over the next two years.Results and discussionShort-term (March to June) survivorship following planting was quite high and the most significant die-off of seedlings occurred during the first dry season (June to November) despite strong droughts in the second and third dry seasons. Overall, only 7.3% (127 of 1,728) of seedlings survived. Seedling success depended strongly on the microhabitat and summer watering, though not in necessarily intuitive ways. Seedlings that received supplemental water during the first summer did worse than unwatered seedlings. The most successful microhabitats for planting were open sites with microsite features and sites underneath canyon live oak, while sites under mature bigcone Douglas-firs had the lowest rates of survival. Position on the slope had no effect on outcomes and soil amendment had a weak negative effect on seedling survival.</p