Ghosts of symbionts past: The hidden history of the dynamic association between filarial nematodes and their Wolbachia endosymbionts: Supplementary Data files

Supplementary data, Figures and Tables for manuscript "Ghosts of symbionts past: The hidden history of the dynamic association between filarial nematodes and their Wolbachia endosymbionts" Emmelien Vancaester, Guy R. Oldrieve, Alex Reid, Georgios Koutsovoulos, Dominik R. Laetsch, Benjamin L. Makepeace, Vincent Tanya, Sven Poppert, Jürgen Krücken, Adrian Wolstenholme, Mark Blaxter Many, but not all, parasitic filarial nematodes (Onchocercidae) carry intracellular, maternally-transmitted Wolbachia symbionts, and these alphaproteobacteria are targets for anti-filarial chemotherapeutic interventions for human disease. The symbionts of Onchocercidae derive from four of the major supergroups (C, D, F and J) defined within the genus Wolbachia. Using twenty-two whole genome sequences of filarial nematodes and genome sequences of their Wolbachia partners, we have explored the evolutionary history of the nematode-Wolbachia symbiosis. We screened the nuclear genome sequences of all the nematodes for nuclear Wolbachia transfers (NUWTs), fragments of the Wolbachia genome that have been integrated into the nuclear genome. Six species have no current Wolbachia infection. Setaria labiatopapillosa had no validated NUWTs and we interpret this to mean that this species was never infected with Wolbachia. In the other five species (Acanthocheilonema viteae, Cercopithifilaria (Ce.) johnstoni, Elaeophora elaphi, Loa (Lo.) loa and Onchocerca flexuosa) we found NUWTs, implying they have previously had and have now lost Wolbachia infections. For each NUWT locus, we identified the supergroup membership of the Wolbachia from which it originated, and found that the five Wolbachia-free species carried NUWTs derived from multiple supergroups, including a high shared rate of sequences derived from supergroup C. In Dirofilaria repens we identified a sample that carried two Wolbachia symbionts, one from supergroup C and one from supergroup F. In Dirofilaria immitis where live infection with a supergroup C Wolbachia is found, we identified NUWTs derived from an F Wolbachia, confirming that the F association predated the divergence of these Dirofilaria species. The supergroup D lineage of Wolbachia, as present in the human parasites Wuchereria bancrofti and Brugia malayi, derives from a replacement event. Madathamugadia (Md.) hiepei shows signs of multiple recent repeated endosymbiont replacement. From these data we infer that the history of Wolbachia in onchocercid nematodes includes not only cospeciation (as present in the Onchocerca-Dirofilaria group in association with supergroup C Wolbachia) and loss (in the five Wolbachia-free species), but also frequent symbiont replacement and dual infection. This dynamic pattern is challenging to models that assume host-symbiont mutualism. The zip archive contains: Scripts Python scripts written for data processing. FetchRegionSeq.py.gz Reduce_alignment.py.gz Reroot_rename_tree.py.gz SelectMatches.py.gz Data Directories containing intermediate data files generated for the analyses presented. 1_WolbachiaAnnotation - The prokka annotation of each Wolbachia genome analysed 2_WolbachiaOrthoFinder - The orthoFinder results for the clustering of the proteomes of the 165 selected Wolbachia proteomes 3_WolbachiaPhylogeny - The phylogeny of Wolbachia inferred using Astral 4_WolbachiaOrthoAlignments - Protein alignments of orthogroups from the orthoFinder analysis of 165 selected Wolbachia peoteomes 5_WolbachiaHMM - nucleotide hidden Markov models generated from the nucleotide alignments of orthogroups from the orthoFinder analysis of 165 selected Wolbachia proteomes 6_NUWTtrees - Phylogenies inferred using Astral of the Wolbachia orthogroups for which NUWT sequences were identified in the filarial nematode genomes. The NUWT sequences were profile aligned to the orthogroup nucleotide alignments. 7_NUWTmatches - File giving the nucleotide coordinates and scores of each orthogroup HMM in the filarial genomes. Tab delimited: columns are: Orthogroup number, Species abbreviation, Supergroup of origin of NUWT, Location of NUWT (Species, Contig/scaffold, base coordinates of match) 8_OnchocercidPhylogeny - Data supporting the onchocercid nematode phylogeny, including files of aligned orthogroups used in coalescent analyses in ASTRAL and the supermatrix of these alignments used for iQtree analysis. Supplementary Tables For completeness, the supplementary tables are replicated here. Table S1: Nematode genome data metrics Table S2: Wolbachia genome data Table S3: Dereplicated Wolbachia genomes Table S4: Contigs removed from filarial nematode genomes as likely contaminants. Table S5: Numbers and classification of NUWTs detected in each filarial nematode species genome Table S6: Span of NUWTs detected in each filarial nematode species’ genome. Table S7: Average length of NUWTs. Table S8: NUWTs from different Wolbachia supergroups. Supplementary Figures in a separate file Figure S1: BlobTools plot of initial Dirofilaria repens assemblyFigure S2: Genome phylogeny of WolbachiaFigure S3: Wolbachia proteome clusteringFigure S4: Phylogenetic tree of orthologous family OG0000236 and NUWTsFigure S5: Phylogenetic tree of orthologous family OG0000277 and NUWTsFigure S6: Phylogenetic tree of orthologous family OG0000301 and NUWTsFigure S7: Phylogenetic tree of orthologous family OG0000453 and NUWTsFigure S8: Phylogenetic tree of orthologous family OG0000206 and NUWTs论文"逝去共生体之魂：丝虫与其沃尔巴克氏体（Wolbachia）内共生体动态关联的隐秘历史"的补充数据、图表与表格 作者：Emmelien Vancaester、Guy R. Oldrieve、Alex Reid、Georgios Koutsovoulos、Dominik R. Laetsch、Benjamin L. Makepeace、Vincent Tanya、Sven Poppert、Jürgen Krücken、Adrian Wolstenholme、Mark Blaxter 多数而非全部寄生性丝虫（丝虫科Onchocercidae）均携带胞内、母系传播的沃尔巴克氏体（Wolbachia）内共生体；这类α-变形菌亦是人类丝虫病抗虫化疗干预的靶点。丝虫科的共生体源自沃尔巴克氏体属内定义的四大主要超群（C、D、F与J）。本研究依托22个丝虫的全基因组序列及其共生沃尔巴克氏体的基因组序列，探究了丝虫-沃尔巴克氏体共生关系的演化历史。我们针对所有丝虫的核基因组序列，筛查了核内沃尔巴克氏体转移序列（nuclear Wolbachia transfers, NUWTs）——即整合入宿主核基因组的沃尔巴克氏体基因组片段。 现有6个物种未检测到沃尔巴克氏体感染。其中，唇乳突丝线虫（Setaria labiatopapillosa）未检出经验证的NUWT，据此我们推测该物种从未被沃尔巴克氏体感染。其余5个物种（Acanthocheilonema viteae、约翰逊颈尾丝虫Cercopithifilaria (Ce.) johnstoni、鹿舌形丝虫Elaeophora elaphi、罗阿丝虫Loa (Lo.) loa以及屈曲盘尾丝虫Onchocerca flexuosa）中均检出NUWT，表明它们曾感染沃尔巴克氏体但现已丢失该共生体。针对每个NUWT位点，我们鉴定了其起源的沃尔巴克氏体所属超群，发现这5个无沃尔巴克氏体的物种携带的NUWT源自多个超群，其中源自超群C的序列共享比例极高。在犬皮下恶丝虫（Dirofilaria repens）的样本中，我们检出了两种沃尔巴克氏体共生体，分别源自超群C与超群F。在现已知感染超群C沃尔巴克氏体的人体寄生虫犬心丝虫（Dirofilaria immitis）中，我们检出了源自超群F的NUWT，证实了超群F的共生关联早于这些恶丝虫物种的分化。寄生于人体的寄生虫班氏吴策线虫（Wuchereria bancrofti）与马来布鲁线虫（Brugia malayi）所携带的沃尔巴克氏体超群D谱系，源自一次共生体替换事件。马达加斯加丝虫Madathamugadia (Md.) hiepei则表现出近期多次重复发生共生体替换的特征。 基于上述数据，我们推断丝虫科线虫体内沃尔巴克氏体的演化历史不仅包括协同成种（如盘尾丝虫属-恶丝虫属类群与超群C沃尔巴克氏体的关联模式）与共生体丢失（即上述5个无沃尔巴克氏体的物种），还频繁发生共生体替换与双重感染。这一动态模式对假设宿主-共生体互利共生的模型提出了挑战。 本压缩归档包含以下内容： ### 脚本 用于数据处理的Python脚本： FetchRegionSeq.py.gz、Reduce_alignment.py.gz、Reroot_rename_tree.py.gz、SelectMatches.py.gz ### 数据 包含本研究分析所用中间数据文件的目录： 1_WolbachiaAnnotation：所有分析的沃尔巴克氏体基因组的Prokka注释结果 2_WolbachiaOrthoFinder：针对165个筛选后的沃尔巴克氏体蛋白质组进行聚类的OrthoFinder分析结果 3_WolbachiaPhylogeny：利用Astral构建的沃尔巴克氏体系统发育树 4_WolbachiaOrthoAlignments：针对165个筛选后的沃尔巴克氏体蛋白质组的OrthoFinder分析所得同源组的蛋白质序列比对结果 5_WolbachiaHMM：基于165个筛选后的沃尔巴克氏体蛋白质组的OrthoFinder分析所得同源组的核苷酸序列比对结果构建的核苷酸隐马尔可夫模型（hidden Markov model, HMM） 6_NUWTtrees：针对在丝虫基因组中检出NUWT序列的沃尔巴克氏体同源组，利用Astral构建的系统发育树；其中NUWT序列已通过剖面比对整合至同源组核苷酸比对文件中 7_NUWTmatches：记录每个同源组HMM在丝虫基因组中的核苷酸坐标与匹配得分的制表符分隔文件，各列依次为：同源组编号、物种缩写、NUWT起源的超群、NUWT位置（物种、重叠群/支架、匹配的碱基坐标） 8_OnchocercidPhylogeny：支持丝虫科系统发育分析的数据，包括用于Astral溯祖分析的同源组比对文件，以及用于iQtree分析的上述比对的超矩阵文件 ### 补充表格 为确保完整，本文在此复刻全部补充表格： 表S1：丝虫基因组数据统计指标 表S2：沃尔巴克氏体基因组数据 表S3：去冗余沃尔巴克氏体基因组 表S4：从丝虫基因组中移除的疑似污染重叠群 表S5：各丝虫物种基因组中检出的NUWT数量与分类信息 表S6：各丝虫物种基因组中检出的NUWT序列跨度 表S7：NUWT序列平均长度 表S8：源自不同沃尔巴克氏体超群的NUWT ### 补充图表 单独归档的补充图表如下： 图S1：犬皮下恶丝虫（Dirofilaria repens）初始组装结果的BlobTools绘图 图S2：沃尔巴克氏体基因组系统发育树 图S3：沃尔巴克氏体蛋白质组聚类结果 图S4：同源家族OG0000236及其NUWT的系统发育树 图S5：同源家族OG0000277及其NUWT的系统发育树 图S6：同源家族OG0000301及其NUWT的系统发育树 图S7：同源家族OG0000453及其NUWT的系统发育树 图S8：同源家族OG0000206及其NUWT的系统发育树