Ne) jointly determine intraspecific genetic diversity, but the role of mutation rate is often ignored. Here we investigate genetic diversity, spontaneous mutation rate and Ne in the giant duckweed (Spirodela polyrhiza). Despite its large census population size, whole-genome sequencing of 68 globally sampled individuals reveals extremely low intraspecific genetic diversity. Assessed under natural conditions, the genome-wide spontaneous mutation rate is at least seven times lower than estimates made for other multicellular eukaryotes, whereas Ne is large. These results demonstrate that low genetic diversity can be associated with large-Ne species, where selection can reduce mutation rates to very low levels. This study also highlights that accurate estimates of mutation rate can help to explain seemingly unexpected patterns of genome-wide variation.Mutation rate and effective population size ( Ne) in explaining variation in genetic diversity has received much attention, the role of spontaneous mutation rate is largely ignored. Here, Xu et al. show that giant duckweed has a high Ne yet low genetic diversity, likely due to its low mutation rate.While the role of effective population size ( While intraspecific genetic diversity is known to vary widely among species, the underlying causes remain controversial4. According to population genetic theory, the population mutation parameter (θ) is determined by the product of the spontaneous neutral mutation rate (μ) and effective population size (Ne), and in diploid species θ = 4 × Ne × μ5. In practice, the parameter θ is often estimated by the average pairwise nucleotide diversity (π) at putatively neutral sites6. While the role of Ne in explaining variation in genetic diversity among taxa has received much theoretical and empirical attention, the influence of variation in mutation rate remains largely ignored7.Explaining within-species genetic diversity—measured as the level of intraspecific DNA sequence variation—is one of the major goals in evolutionary and conservation biologys ≪ 1/Ne, where s is the selection coefficient against the increase of mutation rate) as genetic drift overrides the effect of natural selection. This ‘drift-barrier’ hypothesis can explain variation in mutation rates and the observed logarithmic-scaled negative relationship between Ne and μ among species8. An important prediction of this model is that a large effective population size could result in the evolution of a low mutation rate. One consequence of this is that populations with very large effective population sizes may have very low genetic diversity, when selection has driven mutation rate to an extremely low level. However, to our knowledge, whether this pattern is present in eukaryotes is unknown, largely due to the paucity of studies quantifying both genome-wide diversity and spontaneous mutation rates.As most spontaneous mutations are deleterious, selection should favor lower mutation rates, but in small populations the efficacy of selection to lower the mutation rate is limited  (“giant duckweed”). This species is one of the fastest growing angiosperms; under suitable growth conditions, it reproduces predominantly by asexual budding with a duplication rate of 2–3 days10. Consequently, S. polyrhiza often achieves extremely high census population sizes in nature as millions of individuals can be found in a single pond. However, previous studies using a limited number of genetic markers found low genetic diversity12, and whole genome resequencing of two genotypes revealed overall low heterozygosity13. Here, by sequencing 68 world-wide distributed genotypes and measuring spontaneous mutation rate under natural conditions, we show that low genetic variation in S. polyrhiza is associated with low mutation rate in the giant duckweed.To better understand the relationship between genetic diversity, mutation rate, and S. polyrhiza, we sequenced the genomes of 68 genotypes representing the global distribution of the species, using Illumina short-read sequencing with 29× average coverage  as well as 214,262 small indels. This represents on average one SNP per 145 bp in the S. polyrhiza genome, which is low compared to an average of one SNP per 23 bp in Arabidopsis thaliana when a comparable number of genotypes are sequenced16. Among all biallelic SNPs, 14,191 nonsynonymous and 8865 synonymous SNPs were found (Supplementary Table S. polyrhiza range-wide pairwise nucleotide diversity at synonymous sites (πS) was 0.00093, which is among the lowest values reported for any multicellular eukaryote for which genome-wide genetic diversity has been estimated  Asia  and 0.00013 (European versus American population). Within populations, π calculated from all sites ranged from 0.00018 (American population) to 0.00056 (SE Asian population)  of 0.33 varied from 8.6 kb in the SE Asian population to 86.8 kb in the European population. The relatively slow decay of LD in S. polyrhiza may be attributed to its predominantly clonal reproduction. Comparing across populations, we observed much faster LD decay in the SE Asian population, suggesting more frequent  sexual reproduction in this region and/or higher Ne. Together, these results establish that genome-wide nucleotide diversity in S. polyrhiza is extremely low and sexual reproduction might be frequent in the SE Asian population.Population structure analysis based on genome-wide polymorphisms revealed four population clusters in sia Fig. . A few son) Fig. . The aveS. polyrhiza can be explained by universally low mutation rate or, alternatively, low effective population size, we estimated the spontaneous mutation rate and used our estimates of mutation rate and genomic diversity to estimate effective population size. Because environmental factors, such as ultraviolet (UV) light, which prevails in the native habitats of S. polyrhiza, can affect mutation rates21, mutation rates measured in the lab may not necessarily reflect mutation rates in nature. In an attempt to get an estimate of mutation rate more similar to what would be observed in nature we estimated the genomic mutation rate in indoor and outdoor mutation accumulation (MA) experiments, and manipulated UV light in the outdoor experiments to further assess the effect of environmental factors . Among the 15 offspring, four de novo mutations were identified and confirmed by Sanger sequencing. These mutations all originated from the outdoor MA experiments, and located in non-coding regions. One mutation . As so few mutations were observed, we were unable to perform robust statistical analysis to test for treatment effects. However, the higher number of mutations found in outdoor samples and in the presence of UV light is consistent with the hypothesis that outdoor environmental factors increase the spontaneous mutation rate.To investigate if the observed low genomic diversity in S. polyrhiza is within the range of mutation rates reported for unicellular eukaryotes and Eubacteria, but is more than seven times lower than the reported rates for multicellular eukaryotes  were close to 0  or by substitutions of beneficial alleles at linked loci (genetic hitchhiking)Ne can be explained by extremely low mutation rate in S. polyrhiza. Linked selection may have further reduced the genome-wide genetic diversity. The role of mutation rate in driving variation in genetic diversity has been largely ignored, because obtaining accurate estimates of genome-wide genetic diversity and spontaneous mutation rate in a range of organisms has been difficult in the past. Our study emphasizes that accurate estimates of mutation rates are important for explaining patterns of genetic diversity within species.In this study, we show that the unexpected pattern of low genetic diversity in a species with large S. polyrhiza for 20 generations. Spirodela polyrhiza plants were propagated under three conditions: (i) indoors in the absence of UV light, (ii) outdoors in the absence of natural UV light, and (iii) outdoors in the presence of natural UV light. Spirodela polyrhiza genotype 7498 was pre-cultivated for three weeks in N-medium—which supports optimal growth 2 × 4 H2O, 8 mM KNO3, 5 µM H3BO3, 13 µM MnCl2 × 4 H2O, 0.4 µM Na2MoO4 × 2 H2O, 1 mM MgSO4 × 7 H2O, 25 µM FeNaEDTA)—in a climate chamber operating under the following conditions: 16 h light, 8 h dark; light supplied by vertically arranged neon tubes  on each side; light intensity at plant height: 186 ± 3 µmol s−1 m−2 outside polystyrene tubes and 142 ± 3 µmol s−1 m−2 inside polystyrene tube; temperature: 28 °C constant; humidity: 41%. The genotype 7498 originating from North Carolina (USA) was selected based on the existence of a clone-specific reference genome14. A single frond (S1) was transferred to a transparent 50 ml polystyrene tube  containing 30 ml N-medium, covered with foam cap and incubated in a climate chamber under the above specified conditions. To obtain 6 MA lineages per treatment, the S1 ancestor was propagated according to the propagation scheme  with 150 bp paired-end reads. For the 68 S. polyrhiza genotypes, all genotypes of S. polyrhiza  with 150 bp paired-end reads. On average, 48.2 million reads per genotype were generated.The plant tissue was ground by vigorously shaking the Eppendorf tubes with three metal beads for 1 min in a paint shaker  at 50 Hz. All DNA samples were isolated using the CTAB method33 with the parameters: –collapse –trimns –trimqualities –minlength 36. All of the trimmed reads were then mapped to the S. polyrhiza reference genome14 using BWA-MEM34 with default parameters. All reads with multiple mapping positions in the genome were removed and only the mapped reads were kept. PCR duplicates were removed using the “rmdup” function from SAMtools35. The aligned reads were then used for variant  calling using GATK v3.515 following the suggestions on best practices37. In brief, the aligned reads around indels were re-aligned using “IndelRealigner”, and variants were called using the UnifiedGenotyper function with the option -stand_call_conf 30 -stand_emit_conf 10. The variants were then filtered with the option MQ0 ≥ 4 && ((MQ0/(1.0 × DP)) > 0.1) & QUAL < 30.0 & QD < 5.0, which removes all variants that either have more than four samples with MappingQualityZero  and 10% of the mapped reads (DP) with low mapping quality, or have low Phred-scaled probability that a polymorphism exists at the site (QUAL), or low qual score normalized by allele depth (QD). The variant clusters were further annotated as more than three variants within 50 bp using the GATK VariantFiltration function. Only biallelic loci were kept for downstream analysis. The synonymous and non-synonymous variants were annotated using snpEFF (version 4.3 m)38. Due to low sequencing coverage, three individuals from the MA experiments were removed from downstream analysis . Variants from mitochondrial and chloroplast regions and clustered variants were removed using vcftools39. The population structure among the sequenced 68 genotypes was analyzed using fastSTRUCTURE v1.040. To this end, the loci that were not in Hardy-Weinberg equilibrium (P < 0.01) and tightly linked loci (r2 > 0.33) were removed using vcftools and bcftools41, respectively. Multiple K values (refers to number of populations) ranging from 1 to 10 were analyzed and the value K = 4 was selected using the chooseK.py function from the fastSTRUCTURE package. The genome-wide intraspecific diversity was analyzed using Popgenome v2.2.042 using a data set with no missing genotypes , and diversity at synonymous and non-synonymous sites was analyzed using SNPGenie43 using all variants. Overall, more than 88.0% of the genome and ~92.8% of the coding region had sufficient coverage for variant calling. The estimated population genomic summary statistics were then corrected based on the callable sites. Plink44 was used to calculate pairwise linkage disequilibrium (LD) from the dataset, for which related individuals were removed and only SNPs with MAF greater than 0.05 were kept. To model the decline of LD with physical distance, pairwise r2 between sites was used as the use of D′ is sensitive to small sample sizes46, and the decline of LD was modeled using Sved’s equation: E(r2) = (1−/(1 + 4 βd)) + 1/n, where β is the decline in LD with distance d47 and 1/n accounts for small sample size48. The extent of useful LD for mapping can be defined as r2 = 0.3349. In this study we use mean r2 for non-overlapping 100-bp bins to fit Sved’s equation.To analyze genetic diversity and population genomics of the 68 genotypes, additional filtering steps were performed using vcffilter 53. (1) To reduce false positives, we only considered the mapped and properly paired reads with insertion size greater than 100 bp and less than 600 bp using bamtools54. (2) We also excluded all genomic regions that were supported by fewer than nine or greater than 75 reads per sample from both variant counting and genome size calculation, as the variants from the regions that have low or high coverage are likely due to mapping errors (such as repetitive or duplicated regions). On average, 79.7% of the genomic region was kept. (3) Because spontaneous mutations should be only found in the offspring samples but not the ancestor, and the likelihood of a mutation occurring at the exact same position in multiple samples is extremely low , any variants that appeared in more than two samples were removed. (4) Only the heterozygous variants that were supported by at least three reads for both alleles were kept. After these filtering steps, 86 variants were found  and three were loss-of-heterozygosity (LOH) mutations . We further validated these SNPs using a Sanger-sequencing approach. Two LOH loci were very close to the gap of the genome assembly and the PCR primers could thus not be designed. We validated the remaining seven loci (six point-mutations and one LOH). In total, four out of the six point-mutations were confirmed, and the loss of heterozygosity mutation turned out to be a false positive. The confirmed point-mutations are listed in Table The relatively stringent parameters in the variant filtering process theoretically could result in a high rate of false negatives. To control this, we further estimated the false negative rate using the sequence data. We first identified all high-quality heterozygous SNP loci  from the ancestor using the same filtering parameters  and compared them with the heterozygous SNPs in the offspring using a custom script. In theory, all these variants should be found in the clonally produced offspring. Thus, the number of SNPs that could not be identified from the offspring was used to estimate the highest boundary of the false negative rate from our sequencing and variant calling/filtering pipeline, as some of these cases could be a true loss of heterozygosity.56. We introduced 1000 non-reference mutations to the callable regions using BAMsurgeon57 , with a frequency of 0.5 (standard deviation = 0.1). Using the same variant calling and filtering pipeline, we identified 94.5% (945 out of 1000) of the synthetic mutations that were successfully introduced to the BAM files, yielding an average false negative rate of 5.5% (95% CI 4.1–7.2%).In addition, we also estimated the false negative rate by simulating synthetic mutations to the sequence alignments, an approach that has been used previouslyBecause the total amount of DNA from a single individual was limited, the variant validation was performed using the descendants of the ancestor and offspring individuals. Specifically, at the end of the MA experiments, one individual of each line was propagated for four more generations under indoor conditions, after which the plants were frozen in liquid nitrogen for subsequent variant validation.To validate the candidate variants, DNA was isolated as described above. PCR primers were designed based on the 500 bp flanking sequences. The PCR reactions were performed with goTaq DNA polymerase (Promega) using 30 PCR cycles with an annealing temperature of 58 °C. The primer information is listed in Supplemental Data Further information on experimental design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Reporting Summary


More than half of the global ocean floor is draped by nutrient-starved sediments characterized by deep oxygen penetration and a prevalence of oxidized nitrogen. Despite low energy availability, this habitat hosts a vast microbial population, and geochemical characteristics suggest that nitrogen compounds are an energy source critical to sustaining this biomass. However, metabolic rates of nitrogen transformation and their link to microbial survival in this global-scale ecosystem remain virtually unknown. Here we provide quantitative constraints on microbial nitrogen cycling in open ocean oligotrophic sediments from seafloor to basement, spanning approximately 8 million years. We find active microbial nitrogen transformation throughout the sediment column but at very low rates. Local peaks in diversity and abundance of nitrifiers and denitrifiers occur at redox transition zones deep within the sediments, strongly indicating that these microbes are revived from their maintenance state and start growing again after millions of years of attrition. By mediating the flux of chemical compounds in and out of the sediment, this hidden biosphere influences the composition of the ocean and the atmosphere. Many of the processes constituting the global element cycles can occur without microbial catalyzation, albeit often at a considerably lower rate; the nitrogen cycle, however, is unique in that it is almost exclusively dependent on redox reactions regulated by microorganisms5. Hence, not only are the microbes essential for keeping the cycle turning6, but also for making nitrogen bioavailable and thereby a key controlling factor on primary production.Microbial communities in marine sediments impact all major global biogeochemical cycles, including those of oxygen, carbon, nitrogen, manganese, iron, and sulfur6, but despite recent progress in our understanding of nitrogen transformations in the marine environment11, our knowledge of key processes, fluxes, turnover rates, energy yields, spatial distributions, and population sizes in deep-sea sediments is strikingly limited12. One major reason for this limitation is the historical focus on organic-rich sediments on continental margins or in upwelling zones13. In such systems, the availability of energy, in the form of organic carbon, is accompanied by high microbial activity, restricting oxygen and nitrate penetration to a depth of centimeters or less15. Hence, the importance of nitrogen transformation in sediments with high organic load is dwarfed by other redox reactions such as sulfate reduction, for which the reaction zones can extend to depths of several hundred meters4. However, as researchers turn their attention to the vast oligotrophic regions on the ocean floor, it is becoming apparent that deep oxygen penetration and persistent nitrate throughout the sediment column represent a widespread geochemical scenario that differs markedly from the classic diagenetic redox sequence of organic-rich sediments21. Considering that more than half of the ocean floor is draped by oligotrophic sediments22, these new insights suggest that nitrogen transformation is a pervasive process in the deep sedimentary biosphere.It is clear that microorganisms in marine sediments play a substantial part in balancing the global nitrogen cycle23. North Pond sediments are characterized as oligotrophic, with a total organic carbon content of <0.3 weight percent24. With deep oxygen penetration and nitrate present throughout the sediment25, these cores offer an ideal opportunity to study nitrogen transformation processes in deep oligotrophic subseafloor sediments. Our approach is to 1) study the nature and long term fate of the nitrogen transforming population as a function of depth, by characterizing the diversity and structure within this part of the community. 2) Combine in situ reaction rates predicted by reaction-transport models with quantitative microbial abundance data to derive the mean cell-specific metabolic rates of functional groups involved in nitrogen cycling. We find a predominance of nitrifiers and denitrifiers surviving in a maintenance state, with strong indication of revival and local growth driven by increased energy supply in transition zones between oxic and anoxic regimes deep within these oligotrophic sediments.We present a comprehensive investigation of microbial nitrogen cycling in oligotrophic deep subseafloor sediments, using samples collected from North Pond, a small sediment filled basin, on the western flank of the Mid-Atlantic Ridge visited during the Integrated Ocean Drilling Program (IODP) Expedition 336. Two sediment cores from Holes U1383E and U1384A, hereafter referred to as site 3E and site 4A, respectively, provide access to the entire sediment column from seafloor to basement25  at site 3E and 26–30 mbsf at site 4A, while the AOTZs is located at 28–33 mbsf at 3E and 54–58 mbsf at 4A, respectively  accounts for 4% of total O2 influx from seawater at 4A (low data resolution prevented estimates for the OATZ at 3E), while the AOTZs account for 29% and 44% of O2 influx from the underlying basement at 4A and 3E, respectively  and the underlying oceanic crust (0.010–0.011 mmol m−2 yr−1), with the latter accounting for 19–24% of total sedimentary nitrate efflux  was highest near the surface, with ~10 4A Fig. . However 4A Fig. .Figure 2amoA gene, account for almost the entire archaeal population in the surface sediments, but decrease sharply with depth by four to five orders of magnitude. However, we found local archaeal amoA increases of up to 100 fold in the OATZ and AOTZ at both sites  and nitrite reductase (nirK and nirS), which are key enzymes that catalyze the first two steps of denitrification, respectively (NO3− → NO2− → NO). The abundance of the narG gene in 3E shows an initial decrease of two orders of magnitude, after which it stabilizes around 104 copies per gram sediment from five mbsf and until the AOTZ where a further decline is observed , DNRA bacteria (nrfA genes) and nitrogen-fixing bacteria (nifH genes), suggesting that these microbes play a negligible role in nitrogen cycling at the investigated depths.The abundance of functional groups involved in nitrogen transformation was quantified by targeting their diagnostic functional genes Fig. . Out of tes Fig. . Ammoniares Fig. . NitritemoA Fig. , includived Fig. . The narnes Fig. . The nitNO3− → NO− → NO. TNO3− → NO− → NO. T28], within the phylum Thaumarchaeota occur in most samples and account for 21% and 66% of total richness in near-surface sediments at 3E and 4A, respectively  and denitrifiers, based on the 16S rRNA gene amplicons, and their vertical distribution is largely consistent with the abundance variation of the corresponding functional genes Figs  and 3. Pely Fig. . Their rcus Figs  are consths Figs . AlthougTZs Fig.  and S6. When examining the OTU-level community structure of AOA as a function of sediment depth/age Fig. , we find−7–2.5 × 10−4 in 3E and 1.3 × 10−7–3.4 × 10−5 mol m−3 yr−1 in 4A) and are higher in the upper and lower oxic zones than in the anoxic zone, while the opposite trend was predicted for denitrification rates  over a wide range (0.01–50 µM) and found a best-fit h1 value of 10 µM ; Fig.  µM Fig. , suggest–5–10–2 fmol e− cell−1 d−1 similar to the cell-specific rates of nitrifiers and denitrifiers −1 hour−1 and characterize the nitrifiers as being in maintenance state with cell turnover times spanning approximately 3 orders of magnitude, from months to several hundred years   Fig. . This re39, both the reaction-transport model results . Furthermore, with our estimated oxygen consumption rates, which are always higher than the nitrification rates , and assuming it to be involved in aerobic heterotrophic metabolism, the cell-specific oxygen consumption rates, at North Pond fall within those estimated for communities at similar depths from oligotrophic and oxygenated sediments beneath the North and South Pacific gyres46 Fig. . Our estin situ rates estimated in surface environments, e.g. freshwater surface sediments [29–65 fmol cell−1 d−1 49], as well as rates obtained from cultured representatives of AOB  and AOA .Our data enable us to go beyond bulk respiration estimates, where all cells are treated as equal, and to resolve cell-specific rates of nitrogen transformation, specifically nitrification rates Fig. . In contOur analysis also provides reliable estimates of total denitrification rates and predicts active denitrification throughout both cores Figs –3. Howev52. The recent proposal of a basal power requirement (BPR) that limits subseafloor microbial persistence is based on estimated aerobic heterotrophic respiration and sulfate reduction rates48 and our results suggest that the lower BPR limit (~10−5 fmol e− cell−1 day−1) also holds true for autotrophic nitrifiers  in organic-rich sediments4. Taken together, these findings suggest that the increased energy available at redox transition zones where reduced and oxidized chemical species meet, such as the OATZ, AOTZ and SMTZ, allows starved cells to grow and divide in situ.At depth greater than 20 mbsf the OATZs and the AOTZs have been isolated from fresh organic carbon input and surface community recruitment for several million years. Nonetheless, local peaks in archaeal and bacterial cell abundance at both the OATZs and the AOTZs  and by a resurrected community structure resembling that of the surface layers  of abundant members, congruent with the recent finding that genomic evolution in subseafloor sediments is negligible58.Recent advances in our basic understanding of microbial evolution and community assembly support the notion that microbes currently populating the deep sedimentary biome are direct descendants of a persistent subset of those cells once deposited on the seafloorers Fig. . This paers Fig. . This fiAn increase in nitrifier abundance at the AOTZs  is in agreement with the general understanding of deep-sea sediments acting as a source of dissolved nitrogen for the overlying water masses61. We note that although our upward nitrate efflux estimates for the topmost active layer are probably biased due to the relatively low sampling effort in the surface sediments, they are comparable to those reported from the oligotrophic South Pacific Gyre18. Moreover, we estimate that 19–24% of total sedimentary nitrate efflux diffuses into the underlying oceanic crust  diffuses up from deeper anoxic layers and provides additional substrate, which otherwise is limited. This redox transition zone hosts a higher abundance of cells and a greater microbial diversity, particularly of nitrifiers, relative to adjacent horizons. We argue that these isolated peaks serve as indicators of local microbial growth, even though these cells have been sequestered from fresh organic matter input for millions of years. The microbial community composition in the OATZ suggests that the original surface nitrifying community has been resurrected at depth after a protracted period of dormancy. Despite a suggestive increase in AOA cell abundance at the AOTZ, the absence of a comparable diversity peak implies that the majority of the nitrifying community cannot rebound after passing through the anoxic zone.To summarize, a conceptual model of the nitrogen cycling processes and the associated community dynamics in the oligotrophic sediments of North Pond is provided in Fig. 25. The cores were cut into 1.5 meter-long sections on deck and immediately sampled for microbiological and sediment pore-water analyses, as described elsewhere23. For this study, sediment from the interior part of intact whole cores was sub-sampled using autoclaved cut-end 10 ml plastic syringes and stored in sterile Whirlpak® bags at −80 °C until further analysis. We note that a few horizons at site 3E showed signs of weak to moderate coring disturbance19.The two cores used in this study were obtained during the Integrated Ocean Drilling Program (IODP) Expedition 336 to North Pond, an isolated small sediment pond (8 km × 14 km) above basaltic ocean crust located on the western flank of the Mid-Atlantic Ridge at 22°49′N; 46°05′E. The water depths of the two coring sites were 4425 (U1383E) and 4475 (U1384A) meters below sea level. Temperature of the bottom seawater is assumed to be 1.5 °Cet al.25 and nitrate profiles reported in56, the diffusive fluxes of nitrate between sediments, the overlying seawater and the underlying basement, as well as fluxes of oxygen and nitrate in and out of the oxic-anoxic and anoxic-oxic transition zones were calculated using Fick’s first law of diffusion (details can be found in SI). Due to relatively low spatial resolution of data points in the surface sediments where the most radical curvature of geochemical profiles occur, the values reported should be regarded as minimum estimates.Based on the pore-water profiles of oxygen reported in Orcutt, 68, which discretizes the advection-diffusion-reaction equation. The model used in this study considers three primary reactions: aerobic respiration (R1), heterotrophic denitrification (R2), Manganese reduction (R3); and two secondary reactions: nitrification (R4) and Mn2+ oxidation (R5), between six chemical species . The model simulations assume that the geochemical profiles, including all implicit reactive intermediates, are near steady state. For details see SI.Geochemical interactions for the entire sedimentary sequence at both sites were simulated using a one-dimensional reaction-transport model® DNA Isolation Kit  following the manufacturer’s instruction with two modifications. First, the special bead coating G2 DNA/RNA enhancer  was used as described in Bælum et al.69. Second, 200 μg of sterile filtered polyadenylic acid70 (Sigma) was added to each lysis mixture prior to bead beating . Bead beating was performed using the MP-Biomedical FastPrep®-24 for 45 seconds (speed setting 6). To track potential contaminants introduced during the drilling and experimental processes, DNA from the drill mud, the plastic bag carrying the fluorescent microspheres, and the kit reagents were also extracted as describe elsewhere71. DNA extracts from each sample was finally eluted into 100 μl PCR-grade double-distilled water (ddH2O), and preserved at −20 °C until further analysis.A total of 43 sediment horizons were selected for DNA extraction  from sites 3E (19 horizons) and 4A (25 horizons). Genomic DNA was extracted using PowerLyseamoA and AOB amoA, respectively), nitrite oxidation (nxrB), nitrate reduction (narG and napA), nitrite reduction (nirS and nirK), nitrous oxide reduction (nosZ), anaerobic ammonium oxidation (hzsA and hzo), dissimilative nitrate reduction to ammonium (nrfA), and nitrogen fixation (nifH). In addition we screened for the presence of sulfate reducers, by targeting the dsrB marker gene. A complete list of primers and specific PCR conditions can be found in Table The functional genes encoding the key enzymes of the following nitrogen transformation processes were screened using conventional PCR in all 43 horizons: archaeal and bacterial ammonium oxidation  were subsequently enumerated by quantitative PCR (qPCR). In addition, archaeal and bacterial 16S rRNA gene abundances were quantified as described elsewhere43. All standards were quantified using BIO-analyzer  and 10-fold serial diluted to 10–106 copies µl−1. Details can be found in SI, including a complete list of primers and thermal conditions Table Based on initial PCR screening, the six functional genes successfully amplified in at least one of the 43 sediment horizons (i.e. AOA −) transferred per cell per day, under a number of assumptions, which can be found in the SI. For comparison with other relevant studies, cell specific reaction rates were recalculated into carbon metabolic rates as described in the SI.Mean cell-specific rates of nitrifiers and denitrifiers were calculated by dividing the total volumetric reaction rates predicted from the reaction-transport model by the total abundance of the respective functional groups measured by qPCR (details in SI). Due to the possible presence of multiple copies of a given functional gene in one genome, the calculated cell-specific rates should be considered minimum values. The cell-specific rates were normalized to femtomoles of electrons (e72. 16S rRNA genes were PCR amplified using primers Uni519F (5′-CAGCMGCCGCGGTAA-3′) and 1392 R (5′-ACGGGCGGTGWGTRC-3′) for samples from 4A, and Uni519F and 806 R (5′-GACTACHVGGGTATCTAATCC-3′) for 3E, to create the initial amplicon libraries. More details can be found in the SI section.DNA extracted from site 3E and 4A  was amplified in duplicate reactions using a previously described two-step amplification strategy, minimizing amplification bias73. Taxonomy of the representative sequences of each OTU were assigned using the software packages CREST against the SilvaMod reference database74 using a common ancestor algorithm. OTUs with taxonomic affiliation relevant to nitrogen transformation were manually assigned into functional groups as follows: the order Nitrosopumilales was assigned into ammonium oxidizing archaea (AOA), the genus Nitrosospira, Nitrosomonas and Nitrosococcus were considered ammonium oxidizing bacteria, AOB, the genus Nitrospira and Nitrospina were treated as nitrite oxidizing bacteria (NOB), and denitrifying bacteria i encompass the taxa of Pseudomonas, Arcobacter, Aeromonas, and Woeseiaceae (Tables 75. For diversity measures (richness) each sample was randomly subsampled to 1,000 reads in USEARCH v1076, prior to any comparison between samples.After quality control (details in SI) the filtered reads were clustered into Operational Taxonomic Units (OTUs) at 97% nucleotide similarity cutoff using UPARSEe Tables , S7. OTUsupplementary information, figures and tables


I read with interest the article by Vinod et al.  entitledRelated to this article, I would like to add that Özsoylu  reportedIn our department, eight patients with infantile hemangiomas were treated with interferon-a2a plus oral prednisolone without observing severe complications. In this study, the median age was 4 months (range: 3-12 months); six of them were female and two were male. Three cases were not followed and three patients achieved complete regression while a partial regression was observed in two others. The beneficial results were obtained with interferon-a2a plus oral prednisolone treatment in these cases .On the other hand, vascular endothelial growth factor (VEGF) is recognized as an essential regulator of blood vessel growth. The use of anti-VEGF abolishes this vascular endothelial growth-promoting activity in vitro . Three p We would like to thank Gözdaşoğlu  for showBest Regards,


The expanded rock-salt sublattice of these compounds shifts the valence band maximum to the middle of the Σ line, increasing the band degeneracy by a factor of three. Meanwhile, resonant bonding in the PbTe-like sublattice and soft Tl–Bi (In–Bi) bonding interaction is responsible for intrinsic low lattice thermal conductivities. Our results present an alternative strategy of designing high performance thermoelectric materials.High-efficiency thermoelectric materials require simultaneously high power factors and low thermal conductivities. Aligning band extrema to achieve high band degeneracy, as realized in PbTe, is one of the most efficient approaches to enhance power factor. However, this approach usually relies on band structure engineering, e.g., via chemical doping or strain. By employing first-principles methods with explicit computation of phonon and carrier lifetimes, here we show two full-Heusler compounds Li Finding new thermoelectric materials that can achieve high performance is vital to realizing the potential of future energy harvesting technology. Here, through computation methods, the authors identify two high-performance full-Heusler thermoelectric compounds with high intrinsic band degeneracy. In order to maximize zT, both electronic transport properties and lattice thermal conductivity have to be optimized carefully. Many strategies have been successfully used to suppress κL1. However, there are fewer approaches that can effectively improve the electronic properties, i.e., the power factor (PF = S2σ)4. One effective route is to increase the band degeneracy (Nv) and decrease the inertial effective mass zT of a material is proportional to 5. Although a high density of states (DOS) effective mass 5.Thermoelectric (TE) materials have important applications in energy harvesting, thermoelectric coolers, and thermal detectors as they can directly convert heat into electricity and vise versa. Highly efficient TE materials are required for practical applications and are characterized by the figure of merit Nv can be achieved either from a high valley multiplicity (the number of the carrier pockets of a band in the Brillouin zone) or a high orbital degeneracy (the number of bands with the same energy). Take the well studied TE material PbTe  is located at a low symmetry point of the first Brillouin zone, such as the Σ line of the rock-salt structure2. In addition to alloying, the band convergence could, in principle, be achieved through strain engineering. The lattice constant plays an important role on the alignment of Σ and L in PbTe9. However, a completely alignment requires an extremely large strain, which is not reachable in practice. Therefore, an alternative material design strategy is desired.A high value of XYZ; space group 12 and excellent dopability14. Since semiconducting full-Heusler (FH)   and one metastable (Li2InBi) FH compounds with PbTe-like electronic structure, are discovered by combining a TE material design strategy and high throughput ab initio thermodynamic screening17, see Supplementary Note 2TlBi (Li2InBi) is the interpenetration of Li2 cubic and TlBi (In–Bi) rock-salt sublattices. The electronic structure of [Li+]2[Tl+Bi3−] ([Li+]2[In+Bi3−]) is isoelectronic with PbTe (Pb2+Te2−) since the electrons donated by two Li atoms are delocalized in the whole system. However, the bond length of Tl–Bi (In–Bi) is considerably extended in the FH lattice. Consequently, both the VBM and CBM of these two compounds are located in the middle of the Σ line, with band degeneracy of Nv = 12 in the intrinsic compounds due to their large lattice constants (~7.15 Å). Our transport calculations, explicitly including phonon–phonon and electron–phonon interactions, show that these two compounds have low κL and high PF at room temperature. Benefiting from their low κL and high PF, Li2InBi and Li2TlBi are therefore identified as promising room-temperature TE materials with ideal zT values of 1.5 and 2.0 at 300 K, respectively.In this work, one stable . The convex hull distance (stability) is the formation energy difference between the target compound and its competing phases included in the Open Quantum Material Database (OQMD)18, which contains over 650,000 DFT calculations consisting of experimentally observed compounds from the international crystal structure database (ICSD)20 and hypothetical compounds with prototype structures. We also performed a ground state crystal structure search by using 21 distinct X2YZ prototype structures  up to 500 K, see Supplementary Fig. 2InBi as synthesizable compound since such small convex hull distance is well within the threshold of Heusler compounds24.Our density functional theory (DFT) calculations show that FH Li2YBi (Y = In and Tl) band structure are determined by [Y+Bi3−]2−, which is isoelectronic with Pb2+Te2− even though In/Tl (Bi) is cubic-coordinated with eight Li atoms as the nearest neighbors and octahedrally coordinated with six Bi (In/Tl) as the next nearest neighbor. This is because Li is the most electropositive element in these compounds and it donates its 2s electron to the crystal system. As shown in Fig. s electron is completely delocalized in these compounds and therefore it has very limited influence on the electronic structures of Li2YBi (Y = In and Tl) except for raising the Fermi level and opening the band gap, which is similar to the Li stabilized quaternary Heusler semiconductors16. We further verified this conclusion by performing band structure calculations for [TlBi]2− with the −2 charge balanced by a +2 Jellium background, see Supplementary Fig. 2InBi and Li2TlBi have very similar band structures with PbTe, as shown in Fig. 2InBi and Li2TlBi have much larger lattice constants than PbTe because of the inserted Li2 cubic sublattice, which plays an important role in raising the energy level of the VBM at the middle of Σ line. As a consequence, the Nv of Li2YBi reaches to 12, as observed in the PbTe under significant hydrostatic expansion. As depicted in Fig. 2TlBi (Li2InBi). At the same time, the large bonding distance (softer bonding interaction) between Bi and Y (Y = In and Tl) contributes to reducing κL as we will see later5.The main features of the Lid by Y+Bi−2−, whic2InBi has a very similar electronic structure to Li2TlBi, we only take Li2TlBi as an example here. The electronic structure of Li2TlBi is shown in Fig. 2InBi is shown in Supplementary Fig. 2TlBi is a small band gap semiconductor , which is consistent with a previous calculation25. These calculated gaps are well comparable with many high zT TE materials, such as PbTe: 0.19 eV26 and CoSb3: 0.05 ~ 0.22 eV28). In Li2TlBi, the band gap opens between the fully occupied Bi 6p and fully unoccupied Tl 6p states due to charge transfer from Tl to Bi. Tl atom loses its one 6p electron to the more electronegative Bi atom and becomes Tl+, and its 6s2 electrons are deeply (~−5 eV below the Fermi level) buried below the Bi 6p orbitals , forming stereochemically inactive lone-pair electrons. Two electropositive Li atoms lose their 2s electrons to Bi as well. Therefore, the 6p orbitals of Bi3− (from −4 to 0 eV below the Fermi level) are fully filled with six electrons. The splitting of three occupied Bi 6p orbitals into two groups, ~−2 eV (single degeneracy) and ~−0.5 eV (double degeneracy) at the Γ point is due to SOC. The conduction bands are mainly from the Tl+ 6p orbitals. The electron localization function (ELF) is shown in Fig. + 6s2 are clearly observed in Fig. 2+ in PbTe. Finally, the ELF of Li2TlBi is very similar to that of PbTe, which is identified as a typical resonant bonding system30.Since Li2TlBi band structure is that its VBM lies in the middle of Σ line of the first Brillouin zone of the FCC FH structure Nv = 12), see the Fermi surface in Fig. Nv = 12 of the VBM reaches a record high value, which only has been previously matched in the heavily doped PbTe and CoSb3 systems31. The second hole pocket, which is ~40 meV lower than VBM, is located at the middle of the Δ line (between Γ and X) and possesses a valley degeneracy of 6. Therefore, an extremely high Nv = 18 is reachable in Li2TlBi by means of hole doping. Although the CBM is located at L with the valley degeneracy of 4, the energy difference between CBM and the second highest electron pocket (in the middle of the Σ line) is only 7 meV. Therefore the Nv of the conduction band can potentially reach as high as 16 through light electron doping. The Fermi surfaces of the valence and conduction bands are displayed in Fig. Nv is sufficiently large, since As expected from the previous analysis, a remarkable feature of the Li2YBi (Y = In and Tl), we calculate S and σ based on the semiclassical Boltzmann transport equation under relaxation time approximation. We assume that the predominant carrier scattering mechanisms at 200 K and above are all based on phonons: (1) deformation potentials of acoustic and optical phonons and (2) Fröhlich coupling due to polar optical phonons33. Since the best thermoelectric efficiency is always achieved in the heavily doped region where the scattering on polar optical phonons is sufficiently screened and the dielectric constant is usually large in narrow band gap semiconductors34, we mainly take into account deformation potential scattering using first-principles calculated electron–phonon interaction (EPI) matrix elements. As shown in Fig. 2TlBi, the imaginary part of the electron self-energy Im(Σ) shows a strong energy dependence and is roughly proportional to the density of electronic states. States with a long lifetime appear near the VBM and CBM. This indicates that the lifetime is linked to the phase space availability for electronic transitions, i.e., electrons and holes near the band edges are less scattered due to limited phase space35.To quantitatively characterize the electron transport properties of Li−1 K−2 was recently measured at room temperature36. Figure −1 K−2 for FeNbSb at 300 K, representing the upper limit without considering other scattering sources such as defects and grain boundaries. Our calculated S, σ, and PF of FeNbSb at optimized carrier concentration and temperatures from 200 to 500 K also compare well with a recent theoretical study that employs the same methodology37. The good agreement between our calculations and experimental data confirms our assumption that electron–phonon coupling dominates carrier scattering in this system. It is noteworthy that the optimal PF of FeNbSb is significantly higher than that of PbTe at 300 K39.To validate our calculations, we also computed the thermoelectric properties for a well studied p-type HH compound FeNbSb, for which a PF as large as 10.6 mW m2TlBi and Li2InBi by comparing to FeNbSb. Despite the fact that S is generally much higher in FeNbSb  and 0.15 eV (Li2InBi), the S of Li2TlBi and Li2InBi is comparable with that of FeNbSb at optimal carrier concentration, particularly at 300 K, as shown in Fig. S of Li2TlBi and Li2InBi at higher temperatures. However, owing to the smaller band effective mass Nv), both Li2TlBi and Li2InBi have significantly higher σ than FeNbSb from 300 to 500 K with a carrier concentration about one order of magnitudue lower than FeNbSb  achieves exceptional PFs of 30.4/20.1 (26.3/19.0) mW m−1 K−2 at 200/300 K, nearly twice that of FeNbSb at 300 K. The outperformance of Li2TlBi and Li2InBi over FeNbSb is due to a comparable S and a higher σ at the optimized carrier concentrations, supporting our previous discussion.Next, we illustrate the ultrahigh PFs of Li2TlBi (Li2InBi) primitive cell contains four atoms and therefore 12 phonon branches. The mode decomposition in the zone center (Γ point) is 3Tu1 ⊕ 1Tg2. As shown in Fig. + cation instead of the heaviest atom Bi, which is consistent with the weaker bonding between Tl atom and its neighbors. As expected, the light lithium atom has much higher phonon frequencies 200 ~ 250 cm−1 and its phonon bands are completely separated from Tl and Bi. It is worth noting that these compounds possess two main differences from the previously reported alkali metal based rattling (R) Heusler15: (i) higher acoustic phonon frequencies, and (ii) higher frequency of crossing bands between acoustic and optical modes, meaning Tl (In) atom has a slightly stronger interaction with its neighbors than R-Heusler compounds.The LiκL is calculated by using first-principles compressive sensing lattice dynamics (CSLD) and solving the linear Boltzmann transport equation (see Methods for details) and the results are shown in Fig. κL of Li2TlBi and Li2InBi are isotropic (κL) and the calculated κL are 2.36 (1.55) Wm−1 K−1 at 300 K and 0.55 (0.52) Wm−1 K−1 at 900 K for Li2TlBi (Li2InBi), which are much lower than most FH and HH (≥7 Wm−1 K−140) compounds without doping or nanostructuring and also lower than PbTe .The lattice thermal conductivity 2InBi, and Li2TlBi have low-lying transverse optical modes (TO), as shown in Supplementary Figs. 7 and 8, further confirming the presence of the resonant bonding29, as expected from earlier electronic structures analysis. The long-range interaction caused by the resonant bonding leads to strong anharmonic scattering and large phase space for three-phonon scattering processes and, therefore, significantly suppresses lattice thermal transport29. Moreover, the weak Tl–Bi (In–Bi) bonding resulting from the large bonding distance between Tl and Bi (In and Bi) gives rise to low group velocities. Finally, the high-frequency optical modes associated with the Li atoms provide extra scattering channels for low-lying acoustic modes.Similar to PbTe, Li+: λ + λ′ → λ″) and emission (Γ−: λ″ → λ + λ′) processes in Fig. R-Heusler compounds15.The mechanism of the strong scattering of heat carrying acoustic phonon modes can be directly understood from phonon–phonon interactions. We show the phonon–phonon scattering rates in the absorption , respectively, which implies that Li2TlBi is the TE material with the highest zT at room temperature. Note that our calculated κL might be overestimated due to neglect of the phonon–phonon interaction beyond the third-order and phonon scattering by defects and grain boundaries. Furthermore, the zT of these FH materials could be further enhanced by suppressing heat transport through nanostructuring precipitates and optimizing grain size as commonly used in other Heusler compounds if the optimized carrier concentration can still be achieved. The optimized hole concentrations within the rigid band approximation for the maximum zT at room temperature are 1.3 × 1019 and 1.6 × 1019 cm−3, respectively, for Li2TlBi and Li2InBi  of Li (Tl) in Li2TlBi is 0.29 (0.31) eV per vacancy in Li (Tl) poor condition. These values are comparable with that of the Na doped PbTe (0.27 eV per defect)41, where the hole concentration can reach 1020 cm−3 at room temperature43. These values strongly suggest that the required hole concentrations for maximizing zT are achievable in Li2TlBi.For experiments, it is important to ascertain thermodynamic limit to the achievable dopant concentration. To estimate the defect solubility of forming p-type semiconductor, neutral defects of Li and Tl vacancy were considered. The calculated vacancy formation energy  Li2TlBi and Li2InBi have high zT at room temperature as well, due to the high conduction band degeneracy (at Σ line and L point) and low lattice thermal conductivity. A material with high zT for both hole and electron doping is important for fabricating TE devices. Therefore, Li2TlBi and Li2InBi are promising materials for room temperature thermoelectric applications.Owing to the small band gap, the maximum 2TlBi and Li2InBi FHs, by high throughput stability screening and TE material design strategy of creating the analogs with isovalent electronic structures to PbTe with much expanded lattices. We demonstrate that Li2TlBi and Li2InBi possess intrinsic high PFs and low κL by using the state-of-the-art computational methods which combines the electron Boltzmann transport theory with ab initio carrier relaxation-time from electron–phonon coupling and phonon transport theory with phonon lifetime from first-principles CSLD. The high TE performance of the p-type Li2TlBi and Li2InBi at room temperature are mainly due to the high Nv induced by the extended lattice and the low κL caused by the resonant bonding as observed in PbTe and weak bonding interactions of the extended lattice, respectively. Our TE material design strategy enhances the band degeneracy and suppresses lattice thermal conductivity of the PbTe-type materials simultaneously. It is also straightforward to be extended to design or discover other TE materials.In summary, we discover two promising room-temperature TE materials, Li45. The projector augmented wave (PAW47) pseudo potential, plane wave basis set, and Perdew–Burke–Ernzerhof (PBE48) exchange-correlation functional were used. The qmpy18 framework and the Open Quantum Material Database (OQMD)18 was used for convex hull construction. The band gap was computed by means of the screened hybrid functional HSE0649, including spin–orbit coupling (SOC).In this study, most DFT calculations are performed using the Vienna Ab initio Simulation Package (VASP)2YBi were confirmed by prototype structure screening15 and crystal structure searching using the particle swarm optimization method as implemented in the CALYPSO code22.The lowest energy structure of Li50. The disordered competing phases were simulated using the special quasirandom structures (SQS) as implemented in the alloy theoretic automated toolkit (ATAT)51. Vibrational entropies of the ordered phases and SQSs of the disordered phases are calculated by using phonopy package50. Configuration entropy is included if the competing phases have atom disorder. The calculated results are shown in the Supplementary Table The lattice dynamic stability and vibrational entropy were computed by performing frozen phonon calculation as implemented in phonopy package52 technique was employed to obtain the third-order force constants, which were used to iteratively solve the linearized phonon Boltzmann transport equation with the ShengBTE package53. The carrier lifetime due to electron–phonon coupling was computed by using Quantum Espresso and Electron–phonon Wannier (EPW) codes with SOC included56. Thermoelectric properties including Seebeck coefficient (S), electrical conductivity (σ), and electronic thermal conductivity (κe) were computed using BoltzTrap code57 with the adjusted band gap from HSE06 calculations and mode-resolved carrier lifetime from EPW.The CSLDSupplementary Information


AbstractInsecta: Phthiraptera) are permanent ectoparasites of birds and mammals; there are approximately 5,000 species described and many more undescribed, particularly in the Neotropics. We document the louse genera collected from birds sampled in Peru (2006–2007) and Colombia (2009–2016), from 22 localities across a variety of ecosystems, ranging from lowland tropical forest and Llanos to high elevation cloud forest. We identified 35 louse genera from a total of 210 bird species belonging to 37 avian families and 13 orders. These genera belong to two suborders and three families of lice: Amblycera, families Menoponidae (present on 131 bird species) and Ricinidae (39 bird species); and Ischnocera, family Philopteridae (119 bird species). We compared our bird-louse associations with data in Price et al. (2003) and recently published Neotropical studies. The majority of bird-louse associations (51.9%) were new, with most of these coming from Passeriformes, the most diverse avian order, with the most poorly known louse fauna. Finally, we found geographical variation in louse infestation and prevalence rates. With this study, we report the first comprehensive documentation of bird-louse associations for Colombia and substantially increase the known associations documented for Peru.The diversity of permanent ectoparasites is likely underestimated due to the difficulty of collecting samples. Lice ( Parasites are one of the most common forms of life on the planet . They haInsecta: Phthiraptera) are permanent parasites occurring on both birds and mammals. There are approximately 5,000 described species of lice, about 3,000 of which are known from birds  and Colombia 2009–2016)  post-mortem ruffling method for euthanised avian hosts. These hosts were collected and prepared as museum voucher specimens. To collect ectoparasites, JEA placed each euthanised host in a Ziploc bag with cotton soaked in ethyl acetate for 20 minutes. He then removed the bird from the bag and ruffled the plumage for 60 seconds over a white sheet of paper. Each specimen was returned to its Ziploc bag (with cotton soaked with ethyl acetate), ruffling the plumage two additional times, at intervals of 15 minutes. The ectoparasites were collected from the paper with a small brush and placed in a vial with 96% ethyl alcohol with a label including host specimen metadata. Bird voucher specimens were deposited in the bird collection of Instituto de Ciencias Naturales (ICN) of Universidad Nacional  and the Museum of Natural History (MHNU) at Universidad de los Llanos . Lice were separated from the other ectoparasites, placed into individual vials and identified to genus using taxonomic keys We compared our findings with the world checklist of chewing lice in 0) Not previously reported - avian species with no louse association data reported.1) Same as reported - avian species for which our study found the same louse genera as reported.2) Fewer than reported - avian species for which our study found fewer louse genera than reported3) More than reported - avian species for which our study found more louse genera than reportedAmblycera (Menoponidae and Ricinidae); and suborder Ischnocera (Philopteridae).In Colombia, we sampled 1,032 individual birds from 280 species. Just over half, 51.6% (532), of these birds were infected with ectoparasites  and we found lice on 30% (310) of individual hosts from 138 avian species, 36 avian families and 13 avian orders (Table AmblyceraSuborder Menoponidae - Six menoponid louse genera were distributed on 131 bird species: MyrsideaMenacanthusPsittacobrosusCarriker 1954 (3), MachaerilaemusEureumNitzsch 1818 (1) and OsborniellaRicinidae – Three ricinid louse genera were distributed on 39 bird species: RicinusTrochiliphagusTrochiloecetesIschnoceraSuborder Philopteridae – Twenty six philopterid genera were distributed on 119 bird species: PhilopterusNitzsch 1818 (42 bird species), BrueeliaKéler 1936 (30), RallicolaPenenirmusFormicaphagusPicicolaAlcedoffulaColumbicolaFurnariphilusMulcticolaClay and Meinertzhagen 1938b (2), PhysconelloidesAustrophilopterusEwing 1929 (1), CampanulotesCotingacolaCuculoecusEwing 1936 (1), DegeeriellaNeumann 1906 (1), GoniodesLipeurusNitzsch 1818 (1) OxylipeurusParagoniocotesPseudocophorusCarriker 1940 (1), RhynonirmusStrongylocotesSaemundssoniaSturnidoecusEichler 1944 (1) and VernoniellaIn total, including the two louse suborders, 131 bird species had one louse genus, 61 had two louse genera, 16 had three louse genera, 1 had four and 1 had five louse genera.We compared our findings with the world checklist of chewing lice in Amblycera (Menoponidae and Ricinidae); and suborder Ischnocera (Philopteridae) Suppl. material 1The dataset is the result of several trips to 22 localities to study Neotropical bird communities in Colombia and Peru Table In the present study, we report the genera of lice collected from 210 bird species at 22 sites in Colombia and Peru. We compared the louse-host association found in our study with the known genera of lice from these species of birds. We used We report 109 novel host-louse generic associations. This was not unexpected as we sampled several lowland and Andean habitats which have previously had few studies of bird-louse associations.Passeriformes. Knowledge of lice from many Passeriformes is relatively poor compared to non-passerines TyrannidaeThraupidaePrice and Johnson 2009FurnariidaeParulidaeCardinalidae, Emberizidae and FringillidaeMyrsidea harbour large numbers of undescribed species. Our data show that the majority of Passeriformes sampled (64.5%) have Myrsidea and many of them are likely to be new species.The majority (87.1%) of these new records were from Ricinidae are known to infect hummingbirds and small Passeriformes, whereas members of the Menoponidae are widely distributed across most avian families Ricinus on 36 species of Passeriformes from 11 host families. Myrsidea is a broadly distributed, mega-diverse genus Passeriformes and is considered to have a high degree of host-specificity Myrsidea, from the family Menoponidae, was distributed on 120 bird species, two of which were non-Passeriformes.The distribution of lice is related to the distribution of their hosts Ischnocera, the family Philopteridae is widely distributed on birds Philopteridae are often specialised morphologically and behaviourally for living on a single microhabitat in the plumage  where lice can avoid host preening Philopteridae found in our study were Philopterus, Brueelia and Rallicola. Of these, Philopterus was the most widely distributed genus in this family, occurring on a diverse array of passerine host families and a single non-passerine host species (42 bird species). Brueelia, the most speciose genus of lice in the family Philopteridae, infects avian hosts from many orders, including Coraciiformes, Passeriformes and PiciformesBrueelia on 30 bird species, including two species of Coraciiformes, two species of Piciformes and 26 species of Passeriformes. Finally, the third most frequently collected genus was Rallicola, found on 18 bird species, including one host species in the order Charadriiformes and 17 host species in the order Passeriformes. Rallicola is one of the most speciose of ischnoceran louse genera and has been reported from the avian host orders Apterygiformes, Charadriiformes, Gruiformes and PasseriformesIn Leptopogonamaurocephalus, in Brazil, L.amaurocephalus individuals were infected. However, other host species had similar prevalence rates as reported in previous studies. For example, in Brazil, Turdusleucomelas sampled (n=3) were infected with lice and we found that all individuals of Turdusleucomelas sampled at two localities by us (n=4) were infected. However, a second Brazilian study conducted by Thirty percent of the Colombian birds sampled (138 host species) were infected with lice. In Peru, This manuscript presents data on avian lice from 210 host species. We report and document significant new host-louse association records from poorly sampled yet diverse regions of the world. This information provides an important basis for future studies in the tropics and further enriches our knowledge of the parasite fauna associated with Neotropical birds.Supplementary material 1Lice from Colombian and Peruvian birdsData type: TaxonomicFile: oo_159704.xlsxJuliana Soto-Patiño, Gustavo A Londoño, Jorge Enrique Avendaño, Jill E Jankowski, Andrew T Cook and Julie Allen


A potential correlation between kinetics and in vivo profiles, especially cariprazine’s action on negative symptoms in schizophrenia, is investigated. The binding kinetics of four ligands were indirectly evaluated. After the receptor preparations were pre-incubated with the unlabelled ligands, the dissociation was initiated with an excess of [3H]spiperone. Slow dissociation kinetics characterizes aripiprazole and cariprazine at the D2 receptor. At the D3 receptor, aripiprazole exhibits a slow monophasic dissociation, while cariprazine displays a rapid biphasic behaviour. Functional ß-arrestin assays and molecular dynamics simulations at the D3 receptor confirm a biphasic binding behaviour of cariprazine. This may influence its in vivo action, as the partial agonist could react rapidly to variations in the dopamine levels of schizophrenic patients and the ligand will not quantitatively dissociate from the receptor in one single step. With these findings novel agents may be developed that display rapid, biphasic dissociation from the D3R to further investigate this effect on in vivo profiles.The dissociation behaviours of aripiprazole and cariprazine at the human D The origin of the symptoms is not clarified, but a neurotransmitter dysregulation in different parts of the brain is likely. Hyperactive dopamine transmission and increased D2 receptor (D2R) activation may cause positive symptoms. Hypoactive dopamine transmission and hence less D1 receptor activation in the prefrontal areas may be responsible for negative symptoms1. D3 receptors (D3Rs) seem to contribute to the origin of negative symptoms2. To date schizophrenia is treated only symptomatically. Typical first generation antipsychotic drugs reduce the positive symptoms but display severe and therapy limiting adverse drug effects (ADE) like extrapyramidal motoric symptoms (EPMS). The second generation, also known as atypical antipsychotic drugs reduce these adverse effects, while remain highly effective against positive symptoms. Aripiprazole, an atypical antipsychotic agent, exhibits good efficacy on the positive symptomatic of schizophrenia and a rather beneficial ADE profile. However it is not able to treat the negative symptoms of the disease sufficiently. Cariprazine, a novel antipsychotic agent4, entered the US market in 2016 and obtained European Medicines Agency (EMA) approval in May 2017. Unlike most other antipsychotics it is effective against negative symptoms in clinical trials5, although the mechanism of this action remains elusive.Schizophrenia is a group of neurological diseases characterized by specific symptom complexes. Positive symptoms, which are exaggerated in schizophrenic patients, include hallucinations or delusions. Negative symptoms  determines the duration of receptor occupancy it may influence signalling pathways or ADE8. Several studies address the RTs of antipsychotic drugs at the D2R and the correlation to efficacy or ADE9, however, to date such approaches have not been made for the D3R. A recent review highlighted the relevance of conformational receptor states and biased signalling at G-protein coupled receptors (GPCRs)10. With increasing insights into the crystal structure and signal transduction of GPCRs, it becomes more important to evaluate pharmacological characteristics of novel agents. We hypothesize that cariprazine’s dissociation behaviour elucidates possible mechanisms for its action on negative symptoms in schizophrenia. Its profile is compared to the partial agonist aripiprazole, the antagonist spiperone and the agonist rotigotine. In doing so, the understanding of ligand binding to the D3R could be improved and drug development might benefit from clinically optimized drugs, focused on specific symptoms.Distinct residence times (RTs) are discussed to influence drug profiles off) of [3H]spiperone are 0.013 min−1 (±0.003 min−1) at the D2R and 0.033 min−1 (±0.019 min−1) at the D3R. The koff of unlabelled spiperone in the indirect assay matches the koff of [3H]spiperone at both receptors (Tables Dissociation rate constants (k2R with 38 min for aripiprazole and slightly faster (24 min) for cariprazine  share a large, lipophilic aromatic motif, connected through a linker to a second motif with different hydrogen-bond acceptor and donor groups .The evaluation of kne Table . Caripra50 values of 23.7 nM for aripiprazole and 10.2 nM for cariprazine (monophasic fit). However, a biphasic fit of cariprazine is more appropriate (p = 0.0004) with EC50 values of 5.52 nM and 4.19 µM  contribute to the stabilization  also contribute to the stabilization  on the binding kinetics of bivalent ligands (ligands bearing two pharmacophores connected by a linker22). Aripiprazole also first interacts with the ECL2 of the receptor, then forms a π–π stacking interaction with Tyr7.35. The lock opens and the ligand enters the binding site. A stable intermediate state was not observed  and hydrophobic interactions  stabilize its binding mode. Stability assessments and unbinding simulations suggest that aripiprazole might also reorient in the binding pocket to a similar pose as cariprazine.The root-mean-square deviation of atomic positions (RMSD) against the timeframe in binding trajectories are consistent with the observed biphasic kinetics of cariprazine Fig.  and the ion Fig. . Tyr7.35ion Fig.  then reaion Fig. . These tion Fig. , howeverion Fig.  of the bved Fig. ; the ligved Fig.  varies cved Fig. . Both H-Even though the unbinding simulations offer less reliable structural insight, the trajectories display some characteristic dissociation features. Cariprazine relocates into the SBP after 17 ns Fig.  and full2R, but differ at the D3R. Aripiprazole exhibits similar RT at the D3R and the D2R, whereas cariprazine shows the shortest RT and the most pronounced biphasic behaviour of the tested ligands at the D3R. This could influence cariprazine’s in vivo action, as it can react rapidly to variations in the dopamine level. Recent research revealed that metastable receptor states may determine binding orientation of bivalent drugs in MD simulations22 and may influence their binding kinetics23. Our findings suggest that cariprazine’s biphasic properties are connected to a metastable binding pose. Different binding poses may influence a biased agonism at the D3R, by stimulating different signalling pathways depending on the natural ligand’s concentration. It has to be mentioned that there may be other explanations for the biphasic in vitro behaviour. Given the high affinity and the fast koff from the D3 receptor, the kobs should be fast, which could result in rebinding effects in the assay. To check this possibility, different concentrations of cariprazine  were evaluated, however, the biphasic nature of its dissociation remained. Although other models may also describe the obtained data, the combination of the experimental data and the MD simulations strengthen a biphasic in vitro dissociation profile of cariprazine and a pronounced interaction with different receptor sites. Various studies have shown that the ß-arrestin pathway at the D2R plays an important role in clinical efficacy of antipsychotics. Genetic ß-arrestin depletion led to a loss in antipsychotic activity of tested agents with an increase in motoric ADE24. A BRET assay revealed the antagonism of antipsychotic drugs, including aripiprazole, on the D2R mediated ß-arrestin recruitment25. Given the biphasic nature of cariprazines ß-arrestin recruitment and dissociation from the D3R, increases in local dopamine concentration may not lead to a complete displacement of the ligand, but a certain amount will be able to act at the target for a longer period.Cariprazine and aripiprazole display similar dissociation behaviours at the D26 criticizes the use of the RT as sole explanation of a drug’s profile and claims for individual evaluation of its impact. In the case of antipsychotic agents however, the correlation between RT and drug profiles seems to be a reasonable hypothesis for different in vivo profiles9. Cariprazine displays a different dissociation profile than other tested ligands and shows concurring properties in functional assays and MD simulations at the D3R. Its profile differs especially from aripiprazole, also a highly active antipsychotic, only lacking the effectivity on the negative symptoms. Therefore, the characteristic receptor interaction may be one possible cause behind the efficacy of cariprazine on the negative symptoms of schizophrenia.A recent review3R, there may be additional factors. The translation of an in vitro/in silico biphasic behaviour to in vivo effects remains to be elucidated and requires more research on the origin of negative symptoms. However, with these findings, it is possible to rationally develop compounds with biphasic kinetics and characterize them in vivo regarding their effects on the negative symptomatic. Within this work, we show that:2RCariprazine and aripiprazole share similar dissociation properties at the D3RCariprazine and aripiprazole display different dissociation profiles at the D3R than from the D2RCariprazine dissociates faster from the D3RCariprazine exhibits biphasic kinetics at the DThe biphasic behaviour is also observed in functional assays with cariprazineMD simulations support the experiments with structural interpretation.Although the clinical effects of cariprazine seem to correlate to its binding behaviour at the D13 emphasizes the significance of evaluating and comparing binding modes to understand and advance drug development. By elucidating the binding profile of cariprazine the insights on receptor interaction at the D3R were broadened and future evaluation of novel antipsychotic drugs with special focus on the negative symptoms has gained an interesting aspect. By this, a next step in the understanding and improvement of antipsychotic therapy was made and future approaches may benefit from specialized agents for this diverse symptom complex.Recent work27. CHO cells stably expressing the human dopamine D2shortR or D3R were cultured in DMEM . CHO-D2 cells were collected in PBS buffer, CHO-D3 cells in medium and centrifuged at 3,000 × g for 10 min at 4 °C. The pellet was resuspended in binding buffer , disrupted and centrifuged at 23,000 × g for 30 min (4 °C). The resulting pellet was stored in binding buffer at −80 °C.Cell culture and membrane preparations were performed as reported previously with modifications28 with modifications. Briefly, membrane preparations  were co-incubated with [3H]spiperone (0.2 nM) and the test ligand. Nonspecific binding (NSB) was measured with haloperidol (10 µM) and separation of bound radioligand was performed using VE-water. Assays ran in triplicates at least in three independent experiments. Data was analysed using non-linear regression and equation “one site competition”. The Ki values were calculated from the IC50 values using the Cheng-Prusoff equation29.Displacement assays were performed as reported previouslyoff of [3H]spiperone (RL) was measured, using an excess of haloperidol30. Cell preparations  were incubated  with 0.2 nM RL in 0.2 mL. The dissociation was initiated at different time points with an excess of haloperidol . Assays ran in quadruplicates with eleven time points with n = 4 at the D2R and n = 7 at the D3R. NSB was determined with haloperidol (10 μM), total binding with 0.2 nM RL for the total time of the experiments. Bound RL was separated as described above. Binding data was analysed using non-linear regression and fitting to “one phase exponential decay”.The koff of unlabelled ligands (UL) were measured indirectly by the dilution method, similar as described previously31. 50 µL of the membrane preparations  were incubated with the UL at four times its Ki value for 120 min. Afterwards the dissociation was initiated at different time points with an excess of RL . This dilution results in the majority of the receptors being occupied by RL at the end of the experiment. As the RL may only bind when the UL has dissociated, kobs of the RL reflects the koff of the UL . This model assumes that RL and UL bind to the same binding site. Assays ran in triplicates with eight time points with at least n = 3 independent experiments. Total binding was measured in the absence of UL; at the end of the experiments 80–100% of the total binding were achieved (spiperone and rotigotine 70% at the D3R). Rotigotine was measured at 5 times its Ki value at the D3R. NSB and separation are described above. The koff were obtained by applying non-linear regression and fitting to “one phase exponential association” or “two phase exponential association”. As this indirect method underlies model theories and assumptions the resulting koff are approximations that serve mainly as comparison criteria. The effects of Gpp(NH)p, the omitting of sodium-ions in the buffer and of using [3H]raclopride as RL were evaluated with the same method (n ≥ 2).The k3R (protocol of agonists for aripiprazole and cariprazine). Briefly, U2OS cells were incubated at 37 °C for 48 h. Agonist dilutions were added to the respective wells and incubated for 90 min at 37 °C. Afterwards the detection reagent was added and incubated for 60 min at room temperature in the dark. Luminescence was read with the Infinite 1000 Reader (Tecan). Assays ran in duplicates with n = 2 independent experiments. Data was analysed using non-linear regression and fitting to equations “log(agonist) vs. normalized response - variable slope” and “biphasic”. For biphasic fitting “bottom” was constrained to zero, “top” to 100 and nH1/nH2 to 1.The PathHunter® ß-arrestin eXpress GPCR assay kit was used to measure ß-arrestin recruitment at the D3R X-Ray structure (Protein database entry code: 3PBL)32, mutated residues were transformed to their natural form, eticlopride was removed. The protein was embedded into a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer, the complexes were solvated in TIP3P waters and neutralized with chloride ions. Parameterization was based on Amber ff14SB force field33 and restrained electrostatic potential (RESP)34 charges were calculated for the ligands. All preparatory steps were carried out with the default parameters in the BiKi Life Sciences suite35. The structures were minimized and equilibrated following the default protocol of the program package (see details in the Supplementary Information). Binding studies against the D3R were carried out applying BiKi Life Sciences MD Binding tool implemented in GROMACS-4.6.136 as published recently37. This approach is based on adaptive forces attracting the ligand into the predefined binding site, which residues were identified by NanoShaper38 and refined by visual inspection  ensemble at 300 K without the explicit membrane environment. The applied parameters are summarized in Supplementary Table MD simulations were performed on cariprazine and aripiprazole. The initial structures were constructed from the DIn vitro assays were analysed with Prism 6  and are given as means ± standard deviation (s.d.). In some instances the number of experiments was increased to characterize binding properties into detail (e.g. biphasic dissociation of cariprazine). This is marked in the tables. The signal of the indirect kinetic experiments and the ß-arrestin assays were normalized (zero was set as zero) to allow comparison of different curve shapes. Significance of biphasic fitting over one-phasic fitting or “biphasic” over “log(agonist) vs. normalized response” was tested with the globalized data sets using the “extra sum-of-squares F Test” provided by GraphPad. Comparisons were considered significant if p-value was <0.05 (α = 0.05). All in vitro assays were performed at room temperature.3H]Radioligands were purchased from Perkin Elmer . Cariprazine was synthesized at RCNS, Hungary. The authors confirm the identity and purity of the given compound. The ß-arrestin kit was purchased from DiscoverX .[Supplementary Information