文献—Emergent simplicity in microbial community assembly--论文全过程详细阅读整理与翻译

本文对Emergent simplicity in microbial community assembly—— 论文全过程详细阅读整理

原始文献Goldford J E , Lu N , Bajic D , et al. Emergent Simplicity in Microbial Community Assembly[J]. Science, 2018, 361(6401):469-474.
原始代码链接https://github.com/jgoldford/mcrm.

文章说明和系列内容

本系列更新主要是关于2018年一篇群落结构组成的文献进行阅读的分析和总结，将全文的过程进行分解。主要分为下面几个篇章。本系列的全部内容都为原创，由于个人水平有限，整理过程可能对一些理解不到位甚至错误，请辩证的阅读。

本文字数共（28960）大于阅读3分钟

• 论文全过程详细阅读整理与翻译
• 全文框架分析
• 文献模型过程推导
• 代码整理过程注释

文献整理过程存在的不足

缺少多篇文献对比，对文献的研究意义的认识不足
对文献中的方法进行延拓的内容不足
对实验部分的内容的背景知识不足，在实验理解过程有待提高

文献内容与整理翻译

Introduction：

【研究背景1：微生物群落在自然界中、动物发展过程和宿主体内都起着至关重要的作用。】microbial communities play critical roles in a wide range of natural processes, from animal development and host health to biogeochemical cycles. 【研究背景2：随着基因检测技术的提升，人们可以更加清晰的解析群落中微生物的物种变化。因此近期我们能对群落的功能和机制进行深入研究。】Recent advances in DNA sequencing have allowed us to map the composition of these communities with high resolution. This has motivated a surge of interest in understanding the ecological mechanisms that govern microbial community assembly and function (4). 【研究背景3：对微生物群落的定量分析和准确预测是设计和控制群落发展方向的重要前提。因此需要建立对群落定量、准确的预测模型。】A quantitative, predictive understanding of microbiome ecology is required to design effective strategies to rationally manipulate microbial communities toward beneficial states.
【研究背景4：综述文献,在过去的研究中发现在群落结构具有需要经验性的结构特征，这种特征广泛存在不同环境中。共同的特征包括1.群落中高度的多样性，2起着同样功能作用的菌群共存，3.群落中功能稳定尽管在物种水平变化程度较大，4. 在不同的系统发育水平上，营养物质与群落的分类组成之间的关系具有不同程度的相关关系。】Surveys of microbiome composition across a wide range of ecological settings, from the ocean to the human body (2, 3), have revealed intriguing empirical patterns in microbiome organization. These widely observed properties include high microbial diversity, the coexistence of multiple closely related species within the same functional group, functional stability despite large species turnover, and different degrees of determinism in the association between nutrient availability and taxonomic composition at different phylogenetic levels (3, 5–10). These observations have led to the proposal that common organizational principles exist in microbial community assembly (6, 7). 【转折句1：然而目前缺乏能解释前期发现这些特征的基础理论，只有对这些现象的机制进行探究才能推进微生物菌落的进一步发展。只有寻找到解释的机制才能区分自然形成的群落中那些是局部特征，哪些是群落共有的特征。】However, the lack of a theory of microbiome assembly is hindering progress toward explaining and interpreting these empirical findings, and it remains unknown which of the functional and structural features exhibited by microbiomes reflect specific local adaptations at the host or microbiome level (10) and which are generic properties of complex, self-assembled microbial communities.
【背景句5：目前对群落特征的研究主要采用较少的物种自下而上的方式进行研究】Efforts to connect theory and experiments to understand microbiome assembly have typically relied on manipulative bottom-up experiments with a few species (11–13). 【转折句2：尽管简单的群落在一定程度上是有利于探究一些群落中显著存在的相互作用机制，但是不能清楚的研究这个机制在广泛的群落结构中是否仍然有效。】Although this approach is useful for providing insights into specific mechanisms of interactions, it is unclear to what extent findings from these studies scale up to predict the generic properties of large microbial communities or the interactions therein. 【提出目前的问题矛盾1：目前就对群落中的竞争和共生对群落发展的作用贡献不明确，同时缺乏对在更加复杂系统中内部相互作用对群落形成的影响。】Of note is the ongoing debate about the relative contributions of competition and facilitation (14, 15) and the poorly understood role that high-order interactions play in microbial community assembly (11, 16, 17). 【本文核心解决的科学问题： 通过对大量收集菌群在高度控制和更加方便理解的基础自发形成群落并对形成的群落进行特征统计分析，建立实验结果和理论管理起来的模型，并对比实验结果和理论预测。】To move beyond empirical observations and connect statistical patterns of microbiome assembly with ecological theory, we need to study the assembly of large numbers of large multispecies microbiomes under highly controlled and wellunderstood conditions that allow proper comparison between theory and experiment.

结果1 群落的大量组装可以在单一的碳源的限制下进行

Assembly of large microbial communities on a single limiting resource
【实验方法：1.将自然界中收集的群落在M9（利于控制、采用唯一碳源、最少培养基原料原则）培养基中，采用高通量测序来监控群落中的形成过程。】To meet this challenge, we followed a high-throughput ex situ cultivation protocol to monitor the spontaneous assembly of ecologically stable microbial communities derived from natural habitats in well-controlled environments; we used synthetic (M9) minimal media containing a single externally supplied source of carbon, as well as sources of all of the necessary salts and chemical elements required for microbial life (Fig. 1A). 【材料来源：研究的群落来源于1.土壤中，2.植物叶片的表面。这些样本中的微生物具有高度的多样性和和丰富度。通过测量精确的序列变体，观察到群落中分布了110到1290可分辨菌种。】Intact microbiota suspensions were extracted from diverse natural ecosystems, such as various soils and plant leaf surfaces (methods). Suspensions of microbiota from these environments were highly diverse and taxonomically rich (fig. S1), ranging between 110 and 1290 exact sequence variants (ESVs). 【实验过程1：将收集的12个样品制成悬浮液，把悬浮液加入到基础培养基中并以葡萄糖作为唯一的碳源，放置在30°C下静止培养。】We first inoculated 12 of these suspensions of microbiota into fresh minimal media with glucose as the only added carbon source and allowed the cultures to grow at 30°C in static broth. 【实验过程2：之后将混合培养的样品每培养48小时就转移到新的培养基中，固定的稀释因子8 × 10−3，重复进行12次（24天）传代数目约84代（平均取6小时一代））】We then passaged the mixed cultures in fresh media every 48 hours with a fixed dilution factor of D = 8 × 10−3 for a total of 12 transfers (~84 generations). 【实验过程3:在最后一次培养转移过程后使用16s rRNA进行片段扩增测量群落中的组成。高分辨的精确的序列变体能在单核苷酸水平进行解析群落。 】At the end of each growth cycle, we used 16S ribosomal RNA (rRNA) amplicon sequencing to assay the community composition (Fig. 1A and methods). High-resolution sequence denoising allowed us to identify ESVs, which revealed community structure at single-nucleotide resolution (18).
【实验结果1：大多数的群落在60代之后就趋于稳定的结构。】Most communities stabilized after ~60 generations, reaching stable population equilibria in nearly all cases (Fig. 1B and fig. S2). 【实验结果2：对12个生态群落中观察到在1万个核酸序列可以分类4到17个ESVs。群落中群属保守估计具有丰富的组成。】For all of the 12 initial ecosystems, we observed large multispecies communities after stabilization that ranged from 4 to 17 ESVs at a sequencing depth of 10,000 reads; further analysis indicated that this is a conservative estimate of the total richness in our communities (figs. S3 and S4 and methods). 【实验结果3：通过单一碳源的培养方法确定了扩增长测序产生的分类排布，包括代表性群落中所有优势菌属的分离和表型特征】We confirmed the taxonomic assignments generated from amplicon sequencing by culture-dependent methods, including the isolation and phenotypic characterization of all dominant genera within a representative community (fig. S5).

结果2 群落在科水平的组装上具有趋同性

Convergence of bacterial community structure at the family taxonomic level
【测序结果分析：高分辨率的测序方法允许我们能够将群落培养在一个能够很好控制的生态系统。在物种水平上的分辨结果展示出12个自然群落组成上有较高的差异性。】High-throughput isolation and stabilization of microbial consortia allowed us to explore the rules governing the assembly of bacterial communities in well-controlled synthetic environments. At the species (ESV) level of taxonomic resolution, the 12 natural communities assembled into highly variable compositions (Fig. 1C). 【不同水平上的聚类差异：对ESVs在更高水平（分类等级）进行分组聚类发现复杂多样的12个自然界生态系统在科水平上出现一致的结构主要分为两类：1.肠杆菌科和假单胞菌科。】However, when we grouped ESVs by higher taxonomic ranks, we found that all 12 stabilized communities— with very diverse environmental origins—converged into similar family-level community structures dominated by Enterobacteriaceae and Pseudomonadaceae (Fig. 1D). 【递进句：尽管不同群落的起始来源和种类差异巨大，但都在科水平上表现为相似结构。】In other words, a similar family-level composition arose in all communities despite their very different starting points. 【差异小结：科水平上出现的短暂差异性（β多样性）与独立复制过程出现的差异性相当。但这种结构特点不行适用于属水平上的变化。】This is further illustrated in fig. S6, where we show that the temporal variability (quantified by the b diversity) in family-level composition is comparable to the variability across independent replicates. The same is not true when we compare taxonomic structure at the subfamily (genus) level.
【单一碳源控制的进一步验证：为了更好的理解在课水平上出现相似性的原因，本文从12个以葡萄糖作为唯一碳源的静止培养的群落每个做8个重复。】To better understand the origin of the taxonomic variability observed below the family level, we started eight replicate communities from each of the 12 starting microbiome suspensions (inocula) and propagated them in minimal media with glucose, as in the previous experiment. 【验证实验说明：这些复制的群落中都是来源于同一个样品，拥有相同的菌种；任何在这些样品中观察到群落组成的差异都可以认为是培养过程中随机产生的变化，微生物-微生物相互作用足以产生替代物种水平的群落组装。】Given that the replicate communities were assembled in identical habitats and were inoculated from the same pool of species, any observed variability in community composition across replicates would suggest that random colonization from the regional pool and microbe-microbe interactions are sufficient to generate alternative species-level community assembly.
【碳源控制实验的结果：对于12个样本中有9个样品的重复组都表现出在科水平上的趋同结构。】For most of the inocula (9 out of 12), replicate communities assembled into alternative stable ESV-level compositions, while still converging to the same family-level attractor described in Fig. 1E (see also fig. S6). 【单一碳源结果讨论：在重复组中有一组非常具有代表性，在8个的重复组内部都存在非常强烈的相似性，最后都收敛到同一科水平的结构上。这个相似的数量与不同来源的样本发现一致。】One representative example is shown in Fig. 1, F and G; all eight replicates from the same starting inoculum assembled into strongly similar family-level structures, which were quantitatively consistent with those found before (Fig. 1D). 【转折句：然而在属水平结构有明显的差异，包含假单胞科和大肠杆科多个不同的属。】However, different replicates contained alternative Pseudomonadaceae ESVs, and the Enterobacteriaceaefraction was constituted by either an ESV from the Klebsiella genus or a guild consisting of variable subcompositions of Enterobacter, Raoultella, and/or Citrobacter as the dominant taxa.
【实验结果：在其他3个群落中也表现出相似的群落组成结构，在属水平和科水平上都表现出相似性。】For the remaining (3 out of 12) inocula, all replicates exhibited strongly similar population dynamics to each other and equilibrated to similar population structures at all levels of taxonomic resolution (fig. S7).【实验结果分析：重复的实验结果的相似性表明出群落表现出的组成的趋同性不是有实验操作失误导致这种现象的出现。】 The reproducibility in population dynamics between replicate communities indicates that experimental error is not the main source of variability in community composition. 【种群瓶颈对结果的影响：在新鲜培养基中进行连续稀释所导致的种群瓶颈对观测到的种群动态变化影响不大】The population bottlenecks introduced by the serial dilutions in fresh media have only a modest effect on the observed variability in population dynamics (fig. S8).【稀释因子对结果的有影响：稀释因子可以通过引入种群瓶颈之外的方式影响群落的组装，例如，通过设定稀释之间的传代数，和通过在之前的生长阶段在一定程度上进行稀释。】However, the dilution factor can influence community assembly through means other than introducing population bottlenecks—for instance, by setting the number of generations in between dilutions and by diluting, to a greater or lesser extent, the environment generated in a previous growth period.
【结论1：尽管实验中观察在属水平上有多样性的组成差异，但在科水平上表现出相似的结构特点。】Despite the observed species-level variation in community structure, the existence of familylevel attractors suggests that fundamental rules govern community assembly. 【文献引用：最近关于自然群落的研究一致发现，在类似栖息地中环境过滤的选择作用会使功能趋同，同时允许每个功能类群内部存在分类变异。】Recent work on natural communities has consistently found that environmental filtering selects for convergent function across similar habitats, while allowing for taxonomic variability within each functional class (5, 6). 【结论句：通过葡萄糖作为唯一碳源，众所周知，肠杆菌科的磷酸转移酶系统和假单胞菌科的腺苷三磷酸结合转运蛋白使这类微生物具有葡萄糖摄取能力，由于这种竞争优势，肠杆菌科和假单胞菌科在葡萄糖培养基构建的群落中可能出现固定的比例。】In our assembled communities in glucose media, fixed proportions of Enterobacteriaceae and Pseudomonadaceae may have emerged owing to a competitive advantage, given the well-known glucose uptake capabilities of the phosphotransferase system in Enterobacteriaceae and ABC (adenosine triphosphate–binding cassette) transporters in Pseudomonadaceae . 【提出假设：如果添加不同的碳源到培养基中可能会改变最后形成的群落结构。】This suggests that the observed family-level attract or may change if we add a different carbon source to our synthetic media. 【实验方案：为了验证不同碳源对群落结构的改变，通过将12个群落做8个重复（96样本）中分别加入3种不同的碳源（葡萄糖、柠檬酸、亮氨酸）进行培养】To determine the effect of the externally provided carbon source on environmental filtering, we repeated the community assembly experiments with eight replicates of all 12 natural communities, this time using one of two alternative single carbon sources—citrate or leucine—instead of glucose. 【实验结果：对不同碳源的培养结果进行测序分析，每个培养条件下都测量1万条核苷酸链。亮氨酸的培养基中可以分类6到22类ESVs,柠檬酸的培养基中可以分类4到22类ESVs。这两种碳源上也在ESVs水平表现出广泛的差异性，但在科水平都收敛到相同的结构。】Consistent with previous experiments using glucose minimal media, communities that assembled on citrate or leucine contained large numbers of species: At a sequencing depth of10,000 reads, communities stabilized on leucine contained 6 to 22 ESVs, and communities stabilized on citrate contained 4 to 22 ESVs. As was the case for glucose, replicate communities assembled on citrate and leucine also differed widely in their ESV-level compositions, while converging to carbon source–specific family-level attractors (Fig. 2A and figs. S9 and S10).
【数据分析结果：相同碳源培养的群落在科水平上的结构更加相似性，相似性大于来自同一样本的群落】Family-level community similarity (Renkonen similarity) was, on average, higher between communities passaged on the same carbon source (median, 0.88) than between communities passaged from the same environmental sample (median, 0.77; one-tailed Kolmogorov-Smirnov test, P < 10−5 ; fig. S11). 【实验结果：以柠檬酸为碳源相比葡萄糖为碳源的培养基中大肠杆科明显减少、假单胞科更加丰富。大肠杆科在亮氨酸为碳源没有生长，其中黄杆菌科反而大量生长了。】Communities stabilized on citrate media had a significantly lower fraction of Enterobacteriaceae (Mann-Whitney U test, P < 10−5 ) and were enriched in Flavobacteriaceae (Mann-Whitney U test, P < 10−5) relative to communities grown on glucose;communities stabilized on leucine media had no growth of Enterobacteriaceae and were enriched in Comamonadaceae relative to communities grown on glucose (Mann-Whitney U test, P < 10−5 ) or citrate (Mann-Whitney U test, P < 10−5 ).
【总结句：实验的结果显示出群落的组成受碳源种类的调控。】These results suggest that the supplied source of carbon governs community assembly.【实验目的：为了量化这种现象设计了支持向量机进行训练学习（输入：群落中不同物种的丰富度，输出：对应培养碳源的种类。交叉验证）】 To quantify this effect, we used a machine learning approach and trained a support vector machine to predict the identity of the supplied carbon source from the family-level community composition. We obtained a cross-validation accuracy of 97.3% (Fig. 2B and methods). 【结论句：发现把菌群中丰富度较小的群落也加入到svm的训练过程能提高机器学习的准确率。这说明碳源不只是影响优势菌群而是有更加广泛的群落种类影响。(对群落分类影响的因素，群落组成整体结构性差异)】Importantly, we found that considering the tails of the familylevel distribution (as opposed to just the two dominant taxa) increased the predictive accuracy (Fig. 2B), which indicates that carbon source– mediated determinism in community assembly extends to the entire family-level distribution, including the rarer members.
【总结句：本文没有选择单一生长最适的菌种进行研究，而是选择能够共存且比例固定的多菌科复杂群落，这些菌科能够通过碳源进行有效预测。】Rather than selecting for the most fit single species, our environments select complex communities that contain fixed fractions of multiple coexisting families whose identities are determined by the carbon source in a strong and predictable manner (fig. S11). 【推论句：本文假设分类学上的聚集可能反映在科水平上保守的功能选择性。】We hypothesized that taxonomic convergence might reflect selection by functions that are conserved at the family level. 【扩展句：前面的发现支持这一想法，本文发现每种类型碳源中构建的群落宏基因组表现出了大量的聚类 ，并且在代谢通路中富集。当在琼脂板上涂布稳定群落，通常会在每个平板中发现多个可识别的菌落形态，表明每个群落内的多个分类群能够外加单一碳源的条件下独立生长。】Consistent with this idea, we find that the inferred community metagenomes assembled in each type of carbon source exhibit substantial clustering by the supplied carbon source (Fig. 2C) and are enriched in pathways for its metabolism (fig. S11). When we spread the stabilized communities on agarose plates, we routinely found multiple identifiable colony morphologies per plate, showing that multiple taxa within each community are able to grow independently on (and thus compete for) the single supplied carbon source. 【结果句：这表明，使每个群落中能够代谢单一外加碳源的基因和途径分布在群落的多个分类群中，而不是仅存于最佳竞争物种中。】This suggests that the genes and pathways that confer each community with the ability to metabolize the single supplied resource are distributed among multiple taxa in the community, rather than being present only in the best-competitor species.

结果3 广泛的新陈代谢促进稳定竞争和促进共存

Widespread metabolic facilitation stabilizes competition and promotes coexistence
【背景句：传统的消费者与资源模型描述的是当多个物种竞争同一个资源且这个资源是生长限制性关键物质时竞争排异是唯一可能的结果，除非其他特定情况。】Classic consumer-resource models indicate that when multiple species compete for a single, externally supplied growth-limiting resource, the only possible outcome is competitive exclusion unless specific circumstances apply (20–25). 【转折句（提出问题）：尽管实验室和一些环境中中对消费-资源模型已经有许多的证实，但是这个模型只反映了微生物相互作用的一个方面。】However, this scenario does not adequately reflect the case of microbes, whose ability to engineer their own environments both in the laboratory (26–29) and in nature (30, 31) is well documented. 【猜想句：因此，本文对实验过程观察到几个资源竞争的种属共同存在，猜想不同种群在代谢过程中产生的副产物可以供群落中的其他微生物使用。】Thus, we hypothesized that the observed coexistence of competitor species in our experiments may be attributed to the generic tendency of microbes to secrete metabolic by-products into the environment, which could then be used by other community members.
【主题句：为了确定代谢副产物介导的生态位创造的合理性，本文更深入地分析了一个具有代表性的葡萄糖群落。】 To determine the plausibility of niche creation mediated by metabolic by-products, we analyzed one representative glucose community in more depth. 【实验方法：选择葡萄糖群落中丰富度最高的4个属(假单胞菌属、拉乌尔菌属、柠檬酸菌属以及肠杆菌属)，共占该群落微生物总数的97% 。】We isolated members of the four most abundant generain this community (Pseudomonas, Raoultella, Citrobacter, and Enterobacter), which together represented ~97% of the total population in that community (Fig. 3A). 【实验方法：实验选择的菌种都具有不同性状和菌落表征。】These isolates had different colony morphologies and were also phenotypically distinct (fig. S5). 【实验方法：所有的菌群都能在唯一碳源中独立生长，都共同竞争唯一的资源。当将这四种分离的菌株混合在一起时，群落进行自下而上的重组，这四个菌株都能够稳定地共存。】All isolates were able to form colonies in glucose agarose plates, and all grew independently in glucose as the only carbon source, which indicates that each isolate could compete for the single supplied resource. All four species were able to stably coexist with one another when the community was reconstituted from the bottom up by mixing the isolates together (fig. S5). 【实验方法：为了测试这个群落交叉互养的可能性，将这四个分离菌株在含葡萄糖为唯一碳源的培养基上单一培养48个小时】To test the potential for cross-feeding interactions in this community, we grew monoculture of the four isolates for 48 hours in synthetic M9 media containing glucose as the only carbon source (Fig. 3B).【实验方法：在生长后期葡萄糖消耗到很少的剩余，剩余量几乎不可测量而培养基中任何碳源都来自于前一批微生物细胞分泌的代谢副产品。】 At the end of the growth period, the glucose concentration was too low to be detected, indicating that all of the supplied carbon had been consumed and that any carbon present in the media originated from metabolic by-products previously secreted by the cells.【实验方法：为了检测这些分泌物是否足以支持该群落中其他物种的生长，将剩余的培养液进行过滤移除细胞，并添加到新鲜的培养基（m9）中作为唯一碳源 】 To test whether these secretions were enough to support growth of the other species in that community, we filtered the leftover media to remove cells and added it to fresh M9 media as the only source of carbon (Fig. 3B).【结果句：本文发现所有的分离菌重都能生长其他分离菌种的分泌物中，形成交叉互样的代谢网络。】 We found that all isolates were able to grow on every other isolate’s secretions (e.g., Fig. 3C), forming a fully connected facilitation network (Fig. 3D). 【结果句：分离菌株能够在其他群落成员的分泌物中快速生长，通常包括多次二元变换，而且在分泌物中的生长量与其在葡萄糖培养基的生长量相当，这表明在这个代表性群落中，分泌的副产物是丰富多样的。】Growth on the secretions of other community members was strong, often including multiple diauxic shifts (fig. S12), and the amount of growth on secretions was comparable to that on glucose (fig. S13), suggesting that the pool of secreted by-products is diverse and abundant in this representative community.
【实验方法：对于寻找在代谢副产物的交叉互养是否是普遍存在的特征性，本文选择95个样品葡萄糖为单一碳源培养基进行培养群落，进行另外48h的生长。（12个样品每个做7到8个重复）】To find out whether growth on metabolic by-products is common among our communities, we thawed 95 glucose-stabilized communities (seven or eight replicates from each of 12 initial environmental habitats) and grew them again on glucose as the only carbon source for an extra 48-hour cycle.【扩展句：在全部95个群落中，葡萄糖在24小时就被完全消耗结束，但大多数群体任然能进一步生长】 In all 95 communities, glucose was completely exhausted after 24 hours of growth (Fig. 3E), yet most communities continued growing after the glucose had been depleted (Fig. 3E). 【结果句：此外，在分泌的副产物中，群落能够大量生长：平均而言，群落依赖分泌物增长的生物量相当于其依赖葡萄糖产生的生物量的25%】Moreover, community growth on the secreted by-products was strong: On average, communities produced ~25% as much biomass on the secretions alone as they did over the first 24 hours when glucose was present (Fig. 3F). 【扩展句：通过碘化丙啶染色的群落中成像鉴定只有低数量的渗透或很少明显溶解的细胞。】Propidium iodide staining and phasecontrast imaging of communities at the singlecell level identified low numbers of permeabilized or obviously lysed cells (fig. S14). 【主题句：虽然不能完全排除细胞死亡对结果的影响，仍验证了一种假设，即代谢副产物 (而不是细胞裂解) 是观测到的交叉互养的主要来源。】This supports the hypothesis that metabolic by-product secretion (rather than cell lysis) is the dominant source of the observed cross-feeding. 【转折句（扩展句）】However, lytic events that leave no trace in the form of empty bacterial cell envelopes would not have been detected in our micrographs, so a contribution from cell death to our results cannot be entirely ruled out. 【扩展句：其他的一些机制也能对生物高度多样性产生影响。】Other mechanisms may also operate together with facilitation in specific communities to support high levels of biodiversity (16, 24, 32–34). 【例句：在实验中，通过剧烈摇动，培养物很好地混合，我们还发现含有多个分类群的群落，表明空间结构是不需要共存的】In experiments where the cultures were well mixed by vigorous shaking, we also found communities containing multiple taxa, indicating that spatial structure is not required for coexistence (fig. S15). 【扩展句：此外，本文没有观察到群落被时间竞争生态位影响。】In addition, we did not observe effects from temporal competitive niches in our experiments (fig. S16).
【背景句：近期的一些工作发现pH对微生物的代谢生长同样是重要的限制性原因，能够驱动群落的组装形成。】Recent work has suggested that alteration of the pH by bacterial metabolism may also have important growth-limiting effects (35, 36) and can be a driver of microbial community assembly.【主题句：本研究表明，虽然单个分离菌株在葡萄糖作为单一碳源生长时可以大幅酸化环境，但是在葡萄糖培养基中生长的稳定群落对pH变化不大，大多数群落降低不超过1个单位，并且在生长48h后所有群落pH均稳定在6.5。】 Our results suggest that although individual isolates can substantially acidify their environment when grown in glucose as mono-cultures (e.g., the pH drops to 4.85 in Citrobacter mono-cultures and to 5.55 in Enterobacter mono-cultures after 48 hours), our stabilized communities exhibit only modest changes in pH as they grow in glucose minimal media, dropping by less than 1 unit in most communities and stabilizing to pH 6.5 in all cases after 48 hours of growth (fig. S17). 【例句：在其他碳源中，如亮氨酸，pH甚至比葡萄糖更稳定。总而言之，本研究结果表明，相对于单一培养中观测到的效果，群落可以“缓冲”发酵酸化作用】In other carbon sources, such as leucine, the pH is even more stable than in glucose (fig. S17).【扩展句：虽然超过了本研究的范围，但是阐明其他可能发生稳定竞争的机制的作用，如噬菌体捕食或非传递竞争网络，将更充分地描述这些微生态系统中相互作用的格局。】 Altogether, our results suggest that acidification by fermentation may be “buffered” by the community relative to the effect observed in monocultures. Although beyond the scope of this work, efforts to elucidate the roles of other mechanisms that may stabilize competition, such as phage predation or nontransitive competition networks , will more fully characterize the landscape of interactions in these microcosms.

结果4在竞争模型中加入代谢副产物互养的因素之后可以预测群落构建的结果。

A generic consumer-resource model recapitulates experimental observations
【主题句：实验结果表明，单一限制营养素的竞争可以通过非特异性代谢促进其稳定共存。】Our experiments indicate that competition for a single limiting nutrient may be stabilized by nonspecific metabolic facilitation, leading to coexistence. 【模型方法：为了测试这个特征是否能够促进共存，本文通过经典的MacArthur消费者-资源模型模拟了单个外加碳源的群落构建过程，该模型还包括非特异性交叉互养。】To test whether this feature alone promotes coexistence, we simulated a community assembly process on a single supplied carbon source, using a version of the classic MacArthur consumer-resource model , which was modified to include nonspecific cross-feeding interactions.【模型方法：交叉喂养是通过一个化学计量矩阵来模拟的，这个矩阵对作为代谢副产品的消耗资源的比例进行编码。】Cross-feeding was modeled through astoichiometric matrix that encodes the proportion of a consumed resource that is secreted back into the environment as a metabolic by-product (supplementary materials). 【模型方法：将此矩阵设置为零将导致不分泌副产品，并在一种资源的最小环境中恢复消费者资源模型的经典结果：限制性营养物质消耗率最高的物种竞争性地排除所有其他物种。】Setting this matrix to zero results in no by-products being secreted and recovers the classic results for the consumerresource model in a minimal environment with one resource: The species with the highest consumption rate of the limiting nutrient competitively excludes all others (Fig. 4A, inset).【模型设计：然而，当我们从均匀分布中提取化学计量矩阵（同时确保能量守恒）并用数百个“物种”（每个物种由每种资源的随机产生的吸收率定义）初始化模拟时，通常会观察到共存。】 However, when we drew the stoichiometric matrix from a uniform distribution (while ensuring energy conservation) and initialized simulations with hundreds of “species” (each defined by randomly generated rates of uptake of each resource), coexistence was routinely observed (Fig. 4A). 【模型设计：在这个模拟中，所有共存的“物种”都能代谢多种物质，能够在单一供应的资源和其他物种的分泌物上独立生长。】All of the coexisting “species” in this simulation were generalists, capable of growing independently on the single supplied resource and on each other species’s secretions. 【模型结果：本文的实验表明，限制性营养素的性质对科水平的群落组成有很大的影响，这可能归因于与每个科水平内部的相关的代谢能力。】Our experiments showed that the family-level community composition is strongly influenced by the nature of the limiting nutrient, which may be attributed to the metabolic capabilities associated with each family. 【模型结果：本文通过开发一个从四个代谢“家族”中抽样消费者系数的程序来模拟这种情况，以确保来自同一个科水平的消费者在新陈代谢方面是相似的】We modeled this scenario by developing a procedure that sampled consumer coefficients from four metabolic “families,” ensuring that consumers from the same family were metabolically similar (supplementary materials and fig. S18).【模型参数：本文模型随机生成100个样本和4个代谢家族，然后模拟了20个随机子集的50个物种在三种资源中的一种的生长。就像我们的实验数据一样】 We randomly sampled a set of 100 consumer vectors (or “species”) from four families, then simulated growth for 20 random subsets of 50 species on one of three resources. 【模型结果对比：正如前文实验的结果一致，尽管没有展示出在种水平上的多样性，但相似的群落结构出现在科水平上。】As in our experimental data (Fig. 2A), simulated communities converged to similar family-level structures (Fig. 4C), despite displaying variation at the species level (Fig. 4B).【结果句：本文通过计算每个模拟的群落的范围内的代谢能力来确认科水平收敛和功能收敛之间的对应关系，从而预测出每种资源的社区范围资源吸收率。】 We confirmed the correspondence between family-level convergence and functional convergence by computing the community-wide metabolic capacity per simulation, resulting in a predicted community-wide resource uptake rate for each resource (supplementary materials). 【模型结果：生长在同一资源上的群落收敛到相似的吸收能力，并增强了消耗限制性养分的能力】Communities grown on the same resource converged to similar uptake capacities with an enhanced ability to consume the limiting nutrient (Fig. 4D). 【扩展句：重要的是，这种功能趋同甚至在消费者来自均匀分布的情况下表现出来，没有强制的科水平的消费者结构，这表明群落层面的功能结构的出现是消费者资源模型的一个普遍特征】Importantly, this functional convergence was exhibited even when consumers were drawn from uniform distributions, with no enforced family-level consumer structure, suggesting that the emergence of functional structure at the community level is a universal feature of consumer-resource models (fig. S19).
【模型结果：在模型中经常可以观察到属于同一代谢家族的几个物种可以在平衡状态下共存。这些来自同一家庭的共存消费者的“行会”能够支持稀有（<1%相对丰度）分类群的稳定增长这与实验数据吻合。】We frequently observed that several species belonging to the same metabolic family could coexist at equilibrium. These “guilds” of coexisting consumers from the same family were capable of supporting the stable growth of rare (<1% relative abundance) taxa (Fig. 4E), similar to what we observed in our experimental data (Fig. 1, C and E). 【模型结果：模型表明，物种是由一个密集的易化网络稳定的，与实验中广泛的代谢促进作用的观察结果一致。】Our model suggests that species are stabilized by a dense facilitation network (Fig. 4F), consistent with observations of widespread metabolic facilitation in experiments (Fig. 3D).【主题句：因此，本文发现随机产生的代谢和资源吸收能力的群落动力学模拟捕捉了我们实验中广泛的定性观察结果，并概括了以前在自然群落中的经验观察结果】 Thus, we find that simulations of community dynamics with randomly generated metabolisms and resource uptake capabilities capture a wide range of qualitative observations from our experiments and recapitulate previous empirical observations in natural communities .

Discussion 讨论

【背景句：在缺乏微生物群落组装理论的情况下，通常很难确定自然微生物群落的经验观察特征是否是系统特定决定因素的结果，例如进化史和宿主水平上过去的选择压力，或者它们是否只是大型自组装社区的泛型涌现属性。】 In the absence of a theory of microbiome assembly, it is often difficult to determine whether empirically observed features of natural microbiomes are the result of system-specific determinants, such as evolutionary history and past selective pressures at the host level (10), or whether they are simply generic emergent properties of large self-assembled communities.【主题句：本文的研究结果表明，大型消费资源生态系统的一般统计特性包括：即使在简单的环境中也有很大的分类学多样性；在物种更替的情况下，具有稳定的群落级功能；在营养物质如何决定群落组成方面，在不同的分类学深度上具有可预测性和可变性。】 Our results show that the generic statistical properties of large consumer-resource ecosystems include large taxonomic diversity even in simple environments, a stable community-level function in spite of species turnover, and a mixture of predictability and variability at different taxonomic depths in how nutrients determine community composition.【扩展句：本文观察总结的规律不仅存在实验样本中，同样也广泛存在于其他体系中。】 All of these features are not only observed in our experiments, but also have been reported in systems as diverse as the human gut (3, 10), plant foliages (6), and the oceans (2, 38).
【主题句：本文的理论结果解释了这些经验发现的普遍性，并表明它们可能反映了大型自组装微生物群落的普遍性和通用性。】Our theoretical results thus provide an explanation for the ubiquity of these empirical findings and suggest that they may reflect universal and generic properties of large selfassembled microbial communities. 【扩展句：尽管消费者资源模型简单，但它不仅可以捕捉到实验中观察到的许多一般性的定性特征，而且可以概括出更微妙的方面，包括物种的大量生长的时间以及群落的家族级相似性】In spite of their simplicity, consumer-resource models may not only capture many of the generic qualitative features observed in the experiments, but also recapitulate the more subtle aspects, including the existence of temporal blooms in species that eventually go extinct and family-level similarity of communities (fig. S20 and Fig. 4A). 【模型局限性：然而，这些模型缺乏生物化学细节，因此无法解释其他实验结果。例如：pH值的变化，二次型转变变化，或者葡萄糖和柠檬酸盐的群落之间的相似性比它们在亮氨酸中稳定的群落更相似。】However, the models lack biochemical detail and thus do not have the resolution to explain other experimental results such as pH changes, diauxic shifts, or the fact that glucose and citrate communities are more similar to each other than they are to those stabilized in leucine (Fig. 2A). 【结果句：上述理论和简单的实验装置也使我们能够确定导致大型稳定社区聚集的广泛机制。】The theory and simple experimental setup described above also allowed us to identify widespread mechanisms that lead to the assembly of large, stable communities. 【扩展句：我们发现，密集连接的交叉供给网络可以稳定高度相关物种的公会内的竞争，这些物种都是供应碳源的有力竞争对手。】We find evidence that densely connected cross-feeding networks may stabilize competition within guilds of highly related species that are all strong competitors for the supplied carbon source. 【例句：这种交叉喂养网络自然会导致集体而不是成对的相互作用，这支持了高阶相互作用在复杂微生物群落中起关键稳定作用的假设。】Such cross-feeding networks naturally lead to collective rather than pairwise interactions, supporting the hypothesis that higher-order interactions play a critical stabilizing role in complex microbiomes (16, 17). 【研究的局限性：这些发现是否在具有大量外部资源的更复杂的环境中是通用的仍有待阐明。】Whether these findings are generic in more complex environments with a larger number of externally supplied resources remains to be elucidated. 【例句：在这项工作中提出的实验和理论表明，稳定的微生物群落由代谢通才组成，而不是代谢专家（39），能够消耗供应的资源和代谢副产品。】For instance, the experiments and theory presented in this work indicate that the stabilized microbial communities consist of metabolic generalists, rather than metabolic specialists (39), capable of consuming both the supplied resource and metabolic by-products. 【研究待解决问题：目前尚不清楚这些发现是否适用于适应静态环境的微生物群落，在这些环境中，代谢专一性可能会赋予适应能力优势。】It is unclear whether these findings are generalizable to microbial communities adapted to static environments where metabolic specialization may confer fitness advantages (39). 【全文总结：本研究提出，采用高通量自上而下 (top-down) 的方法对群落构建进行直接的数学建模，这是一种未被充分利用但非常有前景的途径，可用于确定微生物组构建的通用机制和统计规则，同时也为开发微生物组的定量理论提供了一个敲门砖。】We propose that high-throughput top-down approaches to community assembly that are amenable to direct mathematical modeling are an underused but highly promising avenue to identify generic mechanisms and statistical rules of microbiome assembly, as well as a stepping stone toward developing a quantitative theory of the microbiome.
讨论部分框架：

一、 文章的优点
1.科学问题的提出：从群落量化的角度去探讨群落形成过程的选择机制，将过往的经验性、实验观察到的结果以模型的形式进行反映。多种方法的结合使用。
2.研究思路：本文写作呈现有大到小从群落的不同结构进行向下挖掘，以逐步深入的方式展开问题的研究过程。同时文章紧贴主题的关键词实验与模型，文章前部分主要以实验中发现规律为主线进行描述，之后以数学模型为基础进行理论分析结合前期的一些实验模型进行改进并分析模型的特点。对模型进行数值模拟观察模拟结果与实验的吻合性。有模型能反映实验中的一些现象表面了模型的可行性，接着对模拟过程中数值的变化产生的一些特征来推理在实验过程是否存在类似的机制。
3.结论证明：本文最后通过模型模拟结果与实验现象的结合，总结出在群落中不同分类下存在的特征行为，从理论模型和实验现象都给予了验证。

文章的缺点

• 待续

1.写作方面：
2.文章结果：

对自己的益处

1.研究角度：
数据处理：（1）热图（2）三元图 （3）PCA（4）SVM（5）Renkonen similarities

对他人的益处

1.写作思路：
2.结论证明: