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Detecting factors associated with transmission is important to understand disease epidemics, and to design effective public health measures. Clustering and terminal branch lengths (TBL) analyses are commonly applied to genomic data sets of Mycobacterium tuberculosis (MTB) to identify sub-populations with increased transmission. Here, I used a simulation-based approach to investigate what epidemiological processes influence the results of clustering and TBL analyses, and whether differences in transmission can be detected with these methods. I simulated MTB epidemics with different dynamics (latency, infectious period, transmission rate, basic reproductive number R0, sampling proportion, sampling period, and molecular clock), and found that all considered factors, except for the length of the infectious period, affect the results of clustering and TBL distributions. I show that standard interpretations of this type of analyses ignore two main caveats: (1) clustering results and TBL depend on many factors that have nothing to do with transmission, (2) clustering results and TBL do not tell anything about whether the epidemic is stable, growing, or shrinking, unless all the additional parameters that influence these metrics are known, or assumed identical between sub-populations. An important consequence is that the optimal SNP threshold for clustering depends on the epidemiological conditions, and that sub-populations with different epidemiological characteristics should not be analyzed with the same threshold. Finally, these results suggest that different clustering rates and TBL distributions, that are found consistently between different MTB lineages, are probably due to intrinsic bacterial factors, and do not indicate necessarily differences in transmission or evolutionary success.

Background: Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world's new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Methods: We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. Results: We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics. For L1 we identified signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Conclusions: Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans.

Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC). The MTBC comprises several human-adapted lineages known as M. tuberculosis sensu stricto, as well as two lineages (L5 and L6) traditionally referred to as Mycobacterium africanum . Strains of L5 and L6 are largely limited to West Africa for reasons unknown, and little is known of their genomic diversity, phylogeography and evolution. Here, we analysed the genomes of 350 L5 and 320 L6 strains, isolated from patients from 21 African countries, plus 5 related genomes that had not been classified into any of the known MTBC lineages. Our population genomic and phylogeographical analyses showed that the unclassified genomes belonged to a new group that we propose to name MTBC lineage 9 (L9). While the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely ancestral distribution for both L5 and L6 was the Eastern part of West Africa. Moreover, we found important differences between L5 and L6 strains with respect to their phylogeographical substructure and genetic diversity. Finally, we could not confirm the previous association of drug-resistance markers with lineage and sublineages. Instead, our results indicate that the association of drug resistance with lineage is most likely driven by sample bias or geography. In conclusion, our study sheds new light onto the genomic diversity and evolutionary history of M. africanum , and highlights the need to consider the particularities of each MTBC lineage for understanding the ecology and epidemiology of TB in Africa and globally.

The wheat Pm3 resistance gene against the powdery mildew pathogen occurs as an allelic series encoding functionally different immune receptors which induce resistance upon recognition of isolate-specific avirulence (AVR) effectors from the pathogen. Here, we describe the identification of five effector proteins from the mildew pathogens of wheat, rye, and the wild grass Dactylis glomerata, specifically recognized by the PM3B, PM3C and PM3D receptors. Together with the earlier identified AVRPM3A2/F2, the recognized AVRs of PM3B/C, (AVRPM3B2/C2), and PM3D (AVRPM3D3) belong to a large group of proteins with low sequence homology but predicted structural similarities. AvrPm3b2/c2 and AvrPm3d3 are conserved in all tested isolates of wheat and rye mildew, and non-host infection assays demonstrate that Pm3b, Pm3c, and Pm3d are also restricting the growth of rye mildew on wheat. Furthermore, divergent AVR homologues from non-adapted rye and Dactylis mildews are recognized by PM3B, PM3C, or PM3D, demonstrating their involvement in host specificity.

Blumeria graminis f. sp. tritici (B.g. tritici) is the causal agent of the wheat powdery mildew disease. The highly fragmented B.g. tritici genome available so far has prevented a systematic analysis of effector genes that are known to be involved in host adaptation. To study the diversity and evolution of effector genes we produced a chromosome‐scale assembly of the B.g. tritici genome. The genome assembly and annotation was achieved by combining long‐read sequencing with high‐density genetic mapping, bacterial artificial chromosome fingerprinting and transcriptomics. We found that the 166.6 Mb B.g. tritici genome encodes 844 candidate effector genes, over 40% more than previously reported. Candidate effector genes have characteristic local genomic organization such as gene clustering and enrichment for recombination‐active regions and certain transposable element families. A large group of 412 candidate effector genes shows high plasticity in terms of copy number variation in a global set of 36 isolates and of transcription levels. Our data suggest that copy number variation and transcriptional flexibility are the main drivers for adaptation in B.g. tritici. The high repeat content may play a role in providing a genomic environment that allows rapid evolution of effector genes with selection as the driving force.

Whole genome sequencing (WGS) of Mycobacterium tuberculosis has rapidly progressed from a research tool to a clinical application for the diagnosis and management of tuberculosis and in public health surveillance. This development has been facilitated by drastic drops in cost, advances in technology and concerted efforts to translate sequencing data into actionable information. There is, however, a risk that, in the absence of a consensus and international standards, the widespread use of WGS technology may result in data and processes that lack harmonization, comparability and validation. In this Review, we outline the current landscape of WGS pipelines and applications, and set out best practices for M. tuberculosis WGS, including standards for bioinformatics pipelines, curated repositories of resistance-causing variants, phylogenetic analyses, quality control and standardized reporting.

Recognition of the AVRPM3A2/F2 avirulence protein from powdery mildew by the wheat PM3A/F immune receptor induces a hypersensitive response after co‐expression in Nicotiana benthamiana. The molecular determinants of this interaction and how they shape natural AvrPm3a2/f2 allelic diversity are unknown. We sequenced the AvrPm3a2/f2 gene in a worldwide collection of 272 mildew isolates. Using the natural polymorphisms of AvrPm3a2/f2 as well as sequence information from related gene family members, we tested 85 single‐residue‐altered AVRPM3A2/F2 variants with PM3A, PM3F and PM3FL456P/Y458H (modified for improved signaling) in Nicotiana benthamiana for effects on recognition. An intact AvrPm3a2/f2 gene was found in all analyzed isolates and the protein variant recognized by PM3A/F occurred globally at high frequencies. Single‐residue alterations in AVRPM3A2/F2 mostly disrupted, but occasionally enhanced, the recognition response by PM3A, PM3F and PM3FL456P/Y458H. Residues enhancing hypersensitive responses constituted a protein domain separate from both naturally occurring polymorphisms and positively selected residues of the gene family. These results demonstrate the utility of using gene family sequence diversity to screen residues for their role in recognition. This approach identified a putative interaction surface in AVRPM3A2/F2 not polymorphic in natural alleles. We conclude that molecular mechanisms besides recognition drive AvrPm3a2/f2 diversification.

Powdery mildew is an important disease of cereals. It is caused by one species, Blumeria graminis, which is divided into formae speciales each of which is highly specialized to one host. Recently, a new form capable of growing on triticale (B.g. triticale) has emerged through hybridization between wheat and rye mildews (B.g. tritici and B.g. secalis, respectively). In this work, we used RNA sequencing to study the molecular basis of host adaptation in B.g. triticale. We analyzed gene expression in three B.g. tritici isolates, two B.g. secalis isolates and two B.g. triticale isolates and identified a core set of putative effector genes that are highly expressed in all formae speciales. We also found that the genes differentially expressed between isolates of the same form as well as between different formae speciales were enriched in putative effectors. Their coding genes belong to several families including some which contain known members of mildew avirulence (Avr) and suppressor (Svr) genes. Based on these findings we propose that effectors play an important role in host adaptation that is mechanistically based on Avr-Resistance gene-Svr interactions. We also found that gene expression in the B.g. triticale hybrid is mostly conserved with the parent-of-origin, but some genes inherited from B.g. tritici showed a B.g. secalis-like expression. Finally, we identified 11 unambiguous cases of putative effector genes with hybrid-specific, non-parent of origin gene expression, and we propose that they are possible determinants of host specialization in triticale mildew. These data suggest that altered expression of multiple effector genes, in particular Avr and Svr related factors, might play a role in mildew host adaptation based on hybridization.

Tuberculosis (TB) affects humans and other animals and is caused by bacteria from the Mycobacterium tuberculosis complex (MTBC). Previous studies have shown that there are at least nine members of the MTBC infecting animals other than humans; these have also been referred to as ecotypes. However, the ecology and the evolution of these animal-adapted MTBC ecotypes are poorly understood. Here we screened 12,886 publicly available MTBC genomes and newly sequenced 17 animal-adapted MTBC strains, gathering a total of 529 genomes of animal-adapted MTBC strains. Phylogenomic and comparative analyses confirm that the animal-adapted MTBC members are paraphyletic with some members more closely related to the human-adapted Mycobacterium africanum Lineage 6 than to other animal-adapted strains. Furthermore, we identified four main animal-adapted MTBC clades that might correspond to four main host shifts; two of these clades are hypothesized to reflect independent cattle domestication events. Contrary to what would be expected from an obligate pathogen, MTBC nucleotide diversity was not positively correlated with host phylogenetic distances, suggesting that host tropism in the animal-adapted MTBC seems to be driven by contact rates and demographic aspects of the host population rather by than host relatedness. By combining phylogenomics with ecological data, we propose an evolutionary scenario in which the ancestor of Lineage 6 and all animal-adapted MTBC ecotypes was a generalist pathogen that subsequently adapted to different host species. This study provides a new phylogenetic framework to better understand the evolution of the different ecotypes of the MTBC and guide future work aimed at elucidating the molecular mechanisms underlying host range.

Blumeria graminis (Ascomycota) includes fungal pathogens that infect numerous grasses and cereals. Despite its economic impact on agriculture and its scientific importance in plant-pathogen interaction studies, the evolution of different lineages with different host ranges is poorly understood. Moreover, the taxonomy of grass powdery mildew is rather exceptional: there is only one described species (B. graminis) subdivided in different formae speciales (ff.spp.), which are defined by their host range. In this study we applied phylogenomic and population genomic methods to whole genome sequence data of 31 isolates of B. graminis belonging to different ff.spp. and reconstructed the evolutionary relationships between different lineages. The results of the phylogenomic analysis support a pattern of co-evolution between some of the ff.spp. and their host plant. In addition, we identified exceptions to this pattern, namely host jump events and the recent radiation of a clade less than 280,000 years ago. Furthermore, we found a high level of gene tree incongruence localized in the youngest clade. To distinguish between incomplete lineage sorting and lateral gene flow, we applied a coalescent-based method of demographic inference and found evidence of horizontal gene flow between recently diverged lineages. Overall we found that different processes shaped the diversification of B. graminis, co-evolution with the host species, host jump and fast radiation. Our study is an example of how genomic data can resolve complex evolutionary histories of cryptic lineages at different time scales, dealing with incomplete lineage sorting and lateral gene flow.

BACKGROUND: Grass powdery mildew (Blumeria graminis, Ascomycota) is a major pathogen of cereal crops and has become a model organism for obligate biotrophic fungal pathogens of plants. The sequenced genomes of two formae speciales (ff.spp.), B.g. hordei and B.g. tritici (pathogens of barley and wheat), were found to be enriched in candidate effector genes (CEGs). Similar to other filamentous pathogens, CEGs in B. graminis are under positive selection. Additionally, effectors are more likely to have presence-absence polymorphisms than other genes among different strains. RESULTS: Here we identified effectors in the genomes of three additional host-specific lineages of B. graminis (B.g. poae, B.g. avenae and B.g. infecting Lolium) which diverged between 24 and 5 million years ago (Mya). We found that most CEGs in B. graminis are clustered in families and that most families are present in both reference genomes (B.g. hordei and B.g. tritici) and in the genomes of all three newly annotated lineages. We identified conserved protein domains including a novel lipid binding domain. The phylogenetic analysis showed that frequent gene duplications and losses shaped the diversity of the effector repertoires of the different lineages through their evolutionary history. We observed several lineage-specific expansions where large clades of CEGs originated in only one lineage from a single gene through repeated gene duplications. When we applied a birth-death model we found that the turnover rate (the rate at which genes are deleted and duplicated) of CEG families is much higher than for non-CEG families. The analysis of genomic context revealed that the immediate surroundings of CEGs are enriched in transposable elements (TE) which could play a role in the duplication and deletion of CEGs. CONCLUSIONS: The CEG repertoires of related pathogens diverged dramatically in short evolutionary times because of rapid turnover and of positive selection fixing non-synonymous mutations. Whil

There is a large diversity of genetically defined resistance genes in bread wheat against the powdery mildew pathogen Blumeria graminis (B. g.) f. sp. tritici. Many confer race-specific resistance to this pathogen, but until now only the mildew avirulence gene AvrPm3a2/f2 that is recognized by Pm3a/f was known molecularly. We performed map-based cloning and genome-wide association studies to isolate a candidate for the mildew avirulence gene AvrPm2. We then used transient expression assays in Nicotiana benthamiana to demonstrate specific and strong recognition of AvrPm2 by Pm2. The virulent AvrPm2 allele arose from a conserved 12 kb deletion, while there is no protein sequence diversity in the gene pool of avirulent B. g. tritici isolates. We found one polymorphic AvrPm2 allele in B. g. triticale and one orthologue in B. g. secalis and both are recognized by Pm2. AvrPm2 belongs to a small gene family encoding structurally conserved RNase-like effectors, including Avra13 from B. g. hordei, the cognate Avr of the barley resistance gene Mla13. These results demonstrate the conservation of functional avirulence genes in two cereal powdery mildews specialized on different hosts, thus providing a possible explanation for successful introgression of resistance genes from rye or other grass relatives to wheat.

Throughout the history of agriculture, many new crop species (polyploids or artificial hybrids) have been introduced to diversify products or to increase yield. However, little is known about how these new crops influence the evolution of new pathogens and diseases. Triticale is an artificial hybrid of wheat and rye, and it was resistant to the fungal pathogen powdery mildew (Blumeria graminis) until 2001 (refs. 1,2,3). We sequenced and compared the genomes of 46 powdery mildew isolates covering several formae speciales. We found that B. graminis f. sp. triticale, which grows on triticale and wheat, is a hybrid between wheat powdery mildew (B. graminis f. sp. tritici) and mildew specialized on rye (B. graminis f. sp. secalis). Our data show that the hybrid of the two mildews specialized on two different hosts can infect the hybrid plant species originating from those two hosts. We conclude that hybridization between mildews specialized on different species is a mechanism of adaptation to new crops introduced by agriculture.

BACKGROUND: Helitrons are Class II transposons which are highly abundant in almost all eukaryotes. However, most Helitrons lack protein coding sequence. These non-autonomous elements are thought to hijack recombinase/helicase (RepHel) and possibly further enzymes from related, autonomous elements. Interestingly, many plant Helitrons contain an additional gene encoding a single-strand binding protein homologous to Replication Factor A (RPA), a highly conserved, single-copy gene found in all eukaryotes. RESULTS: Here, we describe the analysis of DHH_Mothra, a high-copy non-autonomous Helitron in the genome of rice (Oryza sativa). Mothra has a low GC-content and consists of two distinct blocs of tandem repeats. Based on homology between their termini, we identified a putative mother element which encodes an RPA-like protein but has no RepHel gene. Additionally, we found a putative autonomous sister-family with strong homology to the Mothra mother element in the RPA protein and terminal sequences, which we propose provides the RepHel domain for the Mothra family. Furthermore, we phylogenetically analyzed the evolutionary history of RPA-like proteins. Interestingly, plant Helitron RPAs (PHRPAs) are only found in monocotyledonous and dicotyledonous plants and they form a monophyletic group which branched off before the eukaryotic "core" RPAs. CONCLUSIONS: Our data show how erosion of autonomous Helitrons can lead to different "levels" of autonomy within Helitron families and can create highly successful subfamilies of non-autonomous elements. Most importantly, our phylogenetic analysis showed that the PHRPA gene was most likely acquired via horizontal gene transfer from an unknown eukaryotic donor at least 145-300 million years ago in the common ancestor of monocotyledonous and dicotyledonous plants. This might have led to the evolution of a separate branch of the Helitron superfamily in plants.