Performance of amplicon and shotgun sequencing for accurate biomass estimation in invertebrate community samples

New applications of DNA and RNA sequencing are expanding the field of biodiversity discovery and ecological monitoring, yet questions remain regarding precision and efficiency. Due to primer bias, the ability of metabarcoding to accurately depict biomass of different taxa from bulk communities remains unclear, while PCR‐free whole mitochondrial genome (mitogenome) sequencing may provide a more reliable alternative. Here, we used a set of documented mock communities comprising 13 species of freshwater macroinvertebrates of estimated individual biomass, to compare the detection efficiency of COI metabarcoding (three different amplicons) and shotgun mitogenome sequencing. Additionally, we used individual COI barcoding and de novo mitochondrial genome sequencing, to provide reference sequences for OTU assignment and metagenome mapping (mitogenome skimming), respectively. We found that, even though both methods occasionally failed to recover very low abundance species, metabarcoding was less consistent, by failing to recover some species with higher abundances, probably due to primer bias. Shotgun sequencing results provided highly significant correlations between read number and biomass in all but one species. Conversely, the read–biomass relationships obtained from metabarcoding varied across amplicons. Specifically, we found significant relationships for eight of 13 (amplicons B1FR‐450 bp, FF130R‐130 bp) or four of 13 (amplicon FFFR, 658 bp) species. Combining the results of all three COI amplicons (multiamplicon approach) improved the read–biomass correlations for some of the species. Overall, mitogenomic sequencing yielded more informative predictions of biomass content from bulk macroinvertebrate communities than metabarcoding. However, for large‐scale ecological studies, metabarcoding currently remains the most commonly used approach for diversity assessment.     

Shotgun metagenomics and microscopy indicate diverse cyanophytes,other bacteria,and microeukaryotes in the epimicrobiota of a northern Chilean wetland Nostoc (Cyanobacteria)

Prokaryotic Nostoc, one of the world's most conspicuous and widespread algal genera (similar to eukaryotic algae, plants, and animals) is known to support a microbiome that influences host ecological roles. Past taxonomic characterizations of surface microbiota (epimicrobiota) of free‐living Nostoc sampled from freshwater systems employed 16S rRNA genes, typically amplicons. We compared taxa identified from 16S, 18S, 23S, and 28S rRNA gene sequences filtered from shotgun metagenomic sequence and used microscopy to illuminate epimicrobiota diversity for Nostoc sampled from a wetland in the northern Chilean Altiplano. Phylogenetic analysis and rRNA gene sequence abundance estimates indicated that the host was related to Nostoc punctiforme PCC 73102. Epimicrobiota were inferred to include 18 epicyanobacterial genera or uncultured taxa, six epieukaryotic algal genera, and 66 anoxygenic bacterial genera, all having average genomic coverage ≥90X. The epicyanobacteria Geitlerinemia, Oscillatoria, Phormidium, and an uncultured taxon were detected only by 16S rRNA gene; Gloeobacter and Pseudanabaena were detected using 16S and 23S; and Phormididesmis, Neosynechococcus, Symphothece, Aphanizomenon, Nodularia, Spirulina, Nodosilinea, Synechococcus, Cyanobium, and Anabaena (the latter corroborated by microscopy), plus two uncultured cyanobacterial taxa (JSC12, O77) were detected only by 23S rRNA gene sequences. Three chlamydomonad and two heterotrophic stramenopiles genera were inferred from 18S; the streptophyte green alga Chaetosphaeridium globosum was detected by microscopy and 28S rRNA genes, but not 18S rRNA genes. Overall, >60% of epimicrobial taxa were detected by markers other than 16S rRNA genes. Some algal taxa observed microscopically were not detected from sequence data. Results indicate that multiple taxonomic markers derived from metagenomic sequence data and microscopy increase epimicrobiota detection.     

Fish genomes: Sequencing trends,taxonomy and influence of taxonomy on genome attributes

594 fish genomes have been sequenced in past two decades, this represents 1.85% of the total reported fish species (32,000). Despite this no study represents the trends and only some studies have delved into how the genome size (GS) of the genomes are shaped by species taxonomy. However, all these studies have used data obtained by traditional cytometric methods and also have largely disregarded other genome attributes namely GC, number of chromosomes (CR), number of genes (GE), and protein count (PC). The present study used the most current data on genome attributes of fishes as generated by the whole genome sequencing projects to understand the trends, effect of taxonomy on the genome attributes (GS, GC, CR, GE, and PC) and the interrelation of genome attributes. The trends states that maximum number of fish genomes were sequenced in year 2020, order Cichliformes represents the highest number of published genomes, Illumina is the most used technology for sequencing fish genomes, etc. Our analyses exhibit some concrete trends for fishes as a whole and indicated a strong selection for smaller genomes among all vertebrates and a strong effect of taxonomy on all genome attributes. It also provides clear insights that the fish GS is significantly different from birds, amphibians, reptiles, mammals and insects while the GC only varied from insects. An inverse relation was observed between the GS and GC, and a direct relation was observed between the GS and CR, GE and PC. The results also signify that the per MB value of all the genome attributes decline with increasing GS.     

Comparative metagenomic,phylogenetic and physiological analyses of soil microbial communities across nitrogen gradients

Terrestrial ecosystems are receiving elevated inputs of nitrogen (N) from anthropogenic sources and understanding how these increases in N availability affect soil microbial communities is critical for predicting the associated effects on belowground ecosystems. We used a suite of approaches to analyze the structure and functional characteristics of soil microbial communities from replicated plots in two long-term N fertilization experiments located in contrasting systems. Pyrosequencing-based analyses of 16S rRNA genes revealed no significant effects of N fertilization on bacterial diversity, but significant effects on community composition at both sites; copiotrophic taxa (including members of the Proteobacteria and Bacteroidetes phyla) typically increased in relative abundance in the high N plots, with oligotrophic taxa (mainly Acidobacteria) exhibiting the opposite pattern. Consistent with the phylogenetic shifts under N fertilization, shotgun metagenomic sequencing revealed increases in the relative abundances of genes associated with DNA/RNA replication, electron transport and protein metabolism, increases that could be resolved even with the shallow shotgun metagenomic sequencing conducted here (average of 75 000 reads per sample). We also observed shifts in the catabolic capabilities of the communities across the N gradients that were significantly correlated with the phylogenetic and metagenomic responses, indicating possible linkages between the structure and functioning of soil microbial communities. Overall, our results suggest that N fertilization may, directly or indirectly, induce a shift in the predominant microbial life-history strategies, favoring a more active, copiotrophic microbial community, a pattern that parallels the often observed replacement of K-selected with r-selected plant species with elevated N.     

乐清湾贝类群落组成及其粒径谱结构特征

为了解乐清湾贝类资源情况及群落稳定性特征,基于2016—2017年乐清湾定点调查数据,分析了不同季节乐清湾贝类组成、优势种、资源密度、以及物种多样性,并首次建立乐清湾海域贝类生物量粒径谱与标准化生物量粒径谱。结果显示：1)本次共采集到贝类共计45种,隶属于12目,25科,31属。焦河蓝蛤(Potamocorbula ustulata)为乐清湾绝对优势种;2)资源密度表现为夏季最高,其次为春季,秋季与冬季次之;4个季节的丰富度指数(D)、多样性指数(H′)和均匀度指数(J′)平均为3.26、1.61、0.45,3种多样性指数均处于较低水平;3)ABC曲线显示乐清湾贝类的生物群落处于中度干扰状态;4)乐清湾生物量粒径谱谱型复杂,优势种控制着生物量粒径谱的峰值,且与我国其他海域的结果也存在差异;5)标准化生物量粒径谱的斜率范围为-1.5539—-0.7373,在我国近海海域中属于偏低水平,表明营养循环水平较低。截距范围在16.673—21.597,高于我国其他海域,说明乐清湾贝类生产力水平较高。     

Biochemical assessment of evolution and taxonomy of the morphologically poorly diverged geckos, Gekko yakuensis and G. hokouensis (Reptilia: Squamata) in Japan, with special reference to their occasional hybridization

We assessed the validity of two gekkonid species, Gekko yakuensis and G. hokouensis, in southern Japan. We first assigned all 398 specimens into 18 samples merely on the basis of localities. By conducting significance test for deviations of genotype frequencies from Hardy‐Weinberg at 11 allozyme loci, we checked the reproductive unity of constituents in each of those local samples, and where necessary, rearranged them into subsamples on the basis of genetic markers so that we recognized minimum reproductively cohesive units. We then compared allele frequencies among all samples and subsamples examined. Results clearly indicated that all but two can be classified into two groups that can be discriminated from each other by remarkable allele frequency differences at four diagnostic loci, and by large genetic distances even between sympatric subsamples. Observations of morphological features of the samples and subsamples confirmed that the two groups correspond to G. yakuensis and G. hokouensis, supporting validities of these two species. Allele frequency comparisons, however, also revealed that the remaining two samples, both from southern Kyushu, possessed ‘marker alleles' of both G. yakuensis and G. hokouensis at all four diagnostic loci. These samples thus were considered to represent populations that have been derived through hybridization of the two species. Detailed analyses for genetic structures demonstrated that all hybrid genotypes in the two samples are post‐Fi generations with only one individual resulting from the back‐cross with a pure line population of G. yakuensis. This finding negates the possibility that the hybrid populations are maintained by a constant supply of newly produced Fj hybrids, but suggests that the hybrid genotypes constitute stable breeding populations. This implies that the genealogical independence of G. yakuensis and G. hokouensis in several other sympatric areas has been maintained by operations of some isolation mechanisms at a pre‐mating phase. Investigations of the morphological variation in each sample or subsample revealed that although the two species can be externally largely discriminated from each other by slight modifications of the currently used diagnoses, it is difficult to detect their hybrids based solely on the morphological features.     

A comparison of natural and human determinants of phytoplankton communities in the Kentucky River basin,USA

Physical, chemical, and biological characteristics of the Kentucky River and its tributaries were assessed for one year to compare effects of seasonal, spatial, and human environmental factors on phytoplankton. Phytoplankton cell densities were highest in the fall and summer and lowest in the winter. Cell densities averaged 1162 (± 289 SE) cells m1^–1. Cell densities were positively correlated to water temperature and negatively correlated to dissolved oxygen concentration and to factors associated with high-flow conditions (such as, suspended sediment concentrations). Chrysophytes, diatoms, and blue-green algae dominated winter, spring, and summer assemblages, respectively. Ordination analyses (DCCA) indicated that variation in taxonomic composition of assemblages was associated with stream size as well as season.Spatial variation in phytoplankton assemblages and effects of humans was investigated by sampling 55 sites in low flow conditions during August. Phytoplankton density increased with stream size. Assemblages shifted in composition from those dominated by benthic diatoms upstream to downstream communities dominated by blue-green algae and small flagellates. Human impacts were assumed to cause higher algal densities in stream basins with high proportions of agricultural or urban land use than in basins with forested/mined land use. While density and composition of phytoplankton were positively correlated to agricultural land use, they were poorly correlated to nutrient concentrations. Phytoplankton diversity changed with water quality: decreasing with nutrient enrichment and increasing with conditions that probably changed species composition or inhibited algal growth. Human impacts on phytoplankton in running water ecosystems were as great or greater than effects by natural seasonal and spatial factors. Our results indicated that phytoplankton could be useful indicators of water quality and ecosystem integrity in large river systems.     

Ecological effects of cell-level processes: genome size,functional traits and regional abundance of herbaceous plant species

Background and Aims

Genome size is known to be correlated with a number of phenotypic traits associated with cell sizes and cell-division rates. Genome size was therefore used as a proxy for them in order to assess how common plant traits such as height, specific leaf area and seed size/number predict species regional abundance. In this study it is hypothesized that if there is residual correlation between genome size and abundance after these traits are partialled out, there must be additional ecological effects of cell size and/or cell-division rate.

Methods

Variation in genome size, plant traits and regional abundance were examined in 436 herbaceous species of central European flora, and relationships were sought for among these variables by correlation and path analysis.

Key Results

Species regional abundance was weakly but significantly correlated with genome size; the relationship was stronger for annuals (R² = 0·145) than for perennials (R² = 0·027). In annuals, genome size was linked to abundance via its effect on seed size, which constrains seed number and hence population growth rate. In perennials, it weakly affected (via height and specific leaf area) competitive ability. These relationships did not change qualitatively after phylogenetic correction. In both annuals and perennials there was an unresolved effect of genome size on abundance.

Conclusions

The findings indicate that additional predictors of regional abundance should be sought among variables that are linked to cell size and cell-division rate. Signals of these cell-level processes remain identifiable even at the landscape scale, and show deep differences between perennials and annuals. Plant population biology could thus possibly benefit from more systematic use of indicators of cell-level processes.     

The influence of vegetation type,soil properties and precipitation on the composition of soil mite and microbial communities at the landscape scale

Aim We used a landscape‐scale study of birch invasion onto heather moorland to determine the consistency of changes in vegetation type and soil properties and in the community composition of five soil organism groups. Our aim was to determine whether the degree to which soil organisms respond to natural changes and/or induced changes (e.g. changes in land‐use type and climate) in habitat is consistent across trophic and taxonomic groups in the context of conservation policies for birch woodland and heather moorland. Location Mainland Scotland. Methods We sampled mesostigmatid mites, oribatid mites, fungi, bacteria and archaea in adjacent patches of birch woodland (dominated by Betula pubescens) and heather moorland (dominated by Calluna vulgaris) at 12 sites for which annual rainfall ranged between 713 and 2251 mm. Differences in community composition were visualized using non‐metric multidimensional scaling based on Bray–Curtis dissimilarities. The factors contributing to differences between habitats within sites were explored using general linear models and those among sites using redundancy analysis. Results The communities of all groups differed between habitats within sites, but only the oribatid mites and fungi differed consistently between habitats across sites. Within sites, dissimilarity in fungal communities was positively related to the difference in C. vulgaris cover between habitats, whereas dissimilarities in bacteria and archaea were positively related to differences in soil pH and C:N ratio between habitats, respectively. Main conclusions The influence of vegetation type and soil properties differed between groups of soil organisms, albeit in a predictable manner, across the 12 sites. Organisms directly associated with plants (fungi), and organisms with microhabitat and resource preferences (Oribatida) were strongly responsive to changes in habitat type. The response of organisms not directly associated with plants (bacteria, archaea) depended on differences in soil properties, while organisms with less clear microhabitat and resource preferences (Mesostigmata) were not strongly responsive to either vegetation type or soil properties. These results show that it is possible to predict the impact of habitat change on specific soil organisms depending on their ecology. Moreover, the community composition of all groups was related to variation in precipitation within the study area, which shows that external factors, such as those caused by climate change, can have a direct effect on belowground communities.     

Effects of pine wilt disease invasion on soil properties and Masson pine forest communities in the Three Gorges reservoir region,China

Pine wilt disease (PWD) has caused significant Masson pine mortality in the Three Gorges reservoir region in central China. In this study, five uniform Masson pine stand types infected by PWD were selected and surveyed on slopes and aspects with similar environmental conditions. In sites that had been infected, soil bulk density was reduced, and the difference among the groups was statistically significant (P < 0.05) at the 0–10 cm and 10–20 cm soil layers, but not at 20–40 cm. Other soil water‐related physical properties, excluding noncapillary porosity, significantly differed among the groups in all soil layers. Additionally, the values of available phosphorus, sodium, potassium, calcium, and magnesium were higher in the invaded stands, but the total nitrogen and organic matter contents were lower. Masson pine does not become reestablished following PWD‐induced mortality but is instead replaced by broad‐leaved tree species. Among the 19 examined environmental variables, five were found to be significantly related with the ordination of plant community structure: Masson pine stumps (MPS), K⁺, capillary water holding capacity (CWHC), capillary porosity (CP), and soil water content (SWC). Among these factors, the plant community structure was principally related to MPS and K⁺. The findings of this study show that the outbreak of PWD has impacted Masson pine forest soil properties and altered forest community composition. The disease is negatively related with the presence of Masson pine and positively associated with that of broad‐leaved tree species.