![\"thumbnail\"](\"\/content\/figures\/s12915-016-0264-3-1.gif\")
Fig. 1.<\/strong><\/a> Comparative analysis of Magnaporthales genomes. a<\/strong> Evolutionary rate comparison between Sordariomycetes and vertebrates. All interior\n nodes have 100\u00a0% bootstrap support using a multi-protein concatenated dataset. Magnaporthales
\n and vertebrates are highlighted using thick branches in pink and black, respectively.
\n b<\/strong> Phylogenetic relationships among 19 lineages of Sordariomycetes, showing their genome
\n sizes (Mbp) and predicted gene numbers. The outgroup species are not shown in this
\n phylogeny. All interior nodes have 100\u00a0% bootstrap support using a multi-protein concatenated
\n dataset (shown in Additional file 1<\/a>). The numbers shown at the selected nodes are gene-support frequencies\/internode
\n certainty values. The black dots mark the five branches at which independent gene
\n losses are required to explain Magnaporthales-Colletotrichum<\/em> gene sharing under the assumption of vertical gene transmission\n <\/p>\n
Extended majority rule consensus and majority rule consensus (MRC) trees built using To search for HGT candidates, we employed a phylogenomic approach to build single-gene When allowing the NP-derived foreign genes to be shared with one other Pezizomycotina Given the overall paucity of NP-derived genes in M. incrustans<\/em> and two instances of exclusive sharing of such foreign gene markers with Colletotrichum<\/em>, we tested the magnitude of gene transfers between M. incrustans<\/em> and Colletotrichum<\/em> using the two-way phylogenomic approach. Out of 9154 single gene phylogenies generated Of the 93 putative instances of HGT, 45 likely resulted from gene transfers from Magnaporthales About one-quarter of the gene transfers occurred in the stem lineage of Magnaporthales We identified eight M. incrustans<\/em> genomic segments (containing 18 genes) that contain two or more physically linked We then asked, what is the functional significance of HGT between Magnaporthales and Given that DNA transfer and integration are largely independent of gene functions, We examined three potential issues that might weaken our case for the 93 HGTs between For the second issue, we applied a novel implementation of two-way phylogenomics and Overview of Magnaporthales genomes Magnaporthales comprises a group of fungal lineages with an evolutionary depth comparable to tetrapods (i.e., human-frog divergence; Fig.\u00a01a). The Magnaporthales lineages possess comparable genome sizes (39\u201342 Mbp) and total gene numbers (12\u201313\u00a0K), which are typical of Sordariomycetes (Fig.\u00a01b). To reconstruct a robust Sordariomycetes phylogeny, we identified 1453 highly conserved single-copy genes Read More<\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[],"tags":[],"class_list":["post-76755","post","type-post","status-publish","format-standard","hentry"],"_links":{"self":[{"href":"http:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/posts\/76755","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/comments?post=76755"}],"version-history":[{"count":0,"href":"http:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/posts\/76755\/revisions"}],"wp:attachment":[{"href":"http:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/media?parent=76755"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/categories?post=76755"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/tags?post=76755"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}
\n the corresponding 291 single-gene ML trees resulted in the same topology (Fig.\u00a01b<\/a>). Of the 11 internodes that define or link orders (Fig.\u00a01b<\/a>), 10 internodes have more than 50\u00a0% gene-support frequencies (GSF) or are supported
\n by more than 50\u00a0% (146) of the single-gene ML trees (Fig.\u00a01b<\/a>). All of these internodes have more than 0.3 internode certainties (IC, see 23<\/a>] for details), suggesting the defined bipartitions are more than four times more likely
\n to exist than the most likely alternative bipartitions. The same topology and ML bootstrap
\n support values were obtained when using the 583 (40\u00a0%) genes with the strongest phylogenetic
\n signal and when using the full set of 1453 genes, although with decreasing GSF and
\n IC values (Additional file 1<\/a>). These results show that Magnaporthales and Colletotrichum<\/em> are distinct lineages separated in the tree by multiple, well-defined Sordariomycetes
\n lineages.\n <\/p>\nHGT marker genes derived from non-Pezizomycotina sources<\/h4>\n
\n phylogenies for protein sequences from the specified query species. This approach
\n is conservative because many genes do not lead to highly supported phylogenies (or
\n no phylogenies at all) for different reasons such as lack of phylogenetic signal,
\n short sequence length, and few detectable homologs in the database (see Methods for
\n details). From the available Magnaporthales genomes, we used Magnaporthiopsis incrustans<\/em> (a grass pathogen in Magnaporthales) as a representative species. We used the M. incrustans<\/em> proteins as query against a local database that included NCBI RefSeq (version 55)
\n and genome and transcriptome data from 110 Pezizomycotina species (Additional file
\n 2<\/a>). We identified three instances in which M. incrustans<\/em> genes and their Magnaporthales orthologs were derived from non-Pezizomycotina (NP)
\n sources via HGT (Additional file 3<\/a>) with 85\u00a0% or more SH-like branch support 24<\/a>] and 85\u00a0% or more UFboot support 25<\/a>]. Limited numbers of foreign gene candidates were previously reported in its sister
\n lineage Pyricularia oryzae<\/em>10<\/a>], 12<\/a>], 15<\/a>], 26<\/a>].\n <\/p>\n
\n genus, we identified two NP-derived genes that are exclusively shared between M. incrustans<\/em> (and Magnaporthales orthologs) and Colletotrichum<\/em> (Fig.\u00a02<\/a>). An example is the monophyly of the Magnaporthales and Colletotrichum<\/em> major facilitator superfamily transporter proteins that are nested within bacterial
\n homologs (Fig.\u00a02a<\/a> and Additional file 4<\/a>). The other case represents the exclusive sharing of a putative alpha-1,2-mannosidase
\n that is derived from distantly related fungal lineages (Fig.\u00a02b<\/a> and Additional file 4<\/a>). These two instances of exclusive gene sharing were confirmed using a two-way phylogenomic
\n approach. The principle behind this method is analogous to the reciprocal-best hit
\n approach widely used with BLAST searches. More specifically, in this case, we subjected
\n the Colletotrichum<\/em> sequences in Fig.\u00a02a, b<\/a> to our phylogenomic pipeline to search its sister lineages and recovered exclusive
\n gene sharing with Magnaporthales (see Methods for details).<\/p>\n![\"thumbnail\"](\"\/content\/figures\/s12915-016-0264-3-2.gif\")
Fig. 2.<\/strong><\/a> Exclusive sharing of non-Pezizomycotina-derived horizontal gene transfer gene markers
\n in Magnaporthales and Colletotrichum<\/em>. a<\/strong> Maximum likelihood (ML) tree of a major facilitator superfamily transporter. b<\/strong> ML tree of a putative alpha-1,2-mannosidase that participates in carbohydrate transport
\n and metabolism\n <\/p>\nExtensive gene transfer between Magnaporthales and Colletotrichum<\/em><\/h4>\n
\n using M. incrustans<\/em> proteins as queries, we identified 93 (1.0\u00a0%) M. incrustans<\/em> genes with a Colletotrichum<\/em> provenance with 85\u00a0% or above SH-like branch support 24<\/a>] and 85\u00a0% or above UFboot support 25<\/a>] (Additional file 5<\/a>). These 93 candidates represent 89 distinct transfer events followed by independent
\n duplications of four different genes (Additional file 5<\/a>). These HGTs are located in relatively long M. incrustans<\/em> contigs (coding???5 genes) and have orthologs in other Magnaporthales species. In
\n 91\u00a0% (86\/93) of the cases, at least one of the associated Colletotrichum<\/em> genes is located in contigs or scaffolds encoding five or more genes. In 80\u00a0% (75\/93)
\n of the instances, shared genes are present in two or more Colletotrichum<\/em> species. Transfers of five genomic segments comprising 2\u20133 HGTs were identified between
\n the two lineages (Additional file 5<\/a>). In all but one case, only limited regions of the entire length of contigs were
\n impacted by HGT in both lineages. One example is the transfer of a two-gene Magnaporthales
\n segment to the common ancestor of Colletotrichum<\/em>. The phylogenies of the two genes with Magnaporthales-Colletotrichum<\/em> groupings are shown in Additional file 6<\/a>. These results, corroborated by the overall high quality of the fungal genome data,
\n suggest that most of the identified HGT instances between Magnaporthales and Colletotrichum<\/em> are not explained by sequence contamination.\n <\/p>\nThe nature and significance of HGT between Magnaporthales and Colletotrichum<\/em><\/h4>\n
\n to Colletotrichum<\/em> (Additional file 5<\/a>). One example is the phylogeny of a putative dimethylaniline monooxygenase in which
\n Colletotrichum<\/em> sequences are nested within homologs from Magnaporthales (Fig.\u00a03a<\/a> and Additional file 4<\/a>). Another 19 HGT instances were in the opposite direction (Additional file 5<\/a>) including a NACHT and TPR domain-containing protein, whose phylogeny shows Magnaporthales
\n to be nested within Colletotrichum<\/em> and its sister-group lineage Verticillium<\/em> (Fig.\u00a03b<\/a> and Additional file 4<\/a>). The directions of gene transfers for the remaining instances are unclear.<\/p>\n![\"thumbnail\"](\"\/content\/figures\/s12915-016-0264-3-3.gif\")
Fig. 3.<\/strong><\/a> The nature of horizontal gene transfer (HGT) between Magnaporthales and Colletotrichum<\/em>. a<\/strong> Maximum likelihood (ML) tree of a putative dimethylaniline monooxygenase. This phylogeny
\n provides an example of a gene transfer from Magnaporthales to Colletotrichum<\/em>. b<\/strong> ML tree of a NACHT and TPR domain-containing protein. This phylogeny provides an
\n example of a gene transfer from Colletotrichum<\/em> to Magnaporthales. c<\/strong> Random sampling analysis of HGT gene clustering in the M. incrustans<\/em> genome. We randomly sampled 93 genes from the M. incrustans<\/em> data 5000 times (see Methods) and the number of genomic segments derived from these
\n replicates (represented by the histogram) ranged from 0 to 7. In over 99.9\u00a0% (4955)
\n of the replicates, six or less genomic segments resulted. Therefore, the chance is
\n less than 0.1\u00a0% to generate the eight genomic segments that were observed in the empirical
\n data (the thick black arrow). Similarly, the range of the genes that were included
\n in the genomic segments was 0\u201314 with over 99.9\u00a0% of the gene numbers being 12 or
\n less. Therefore, the chance is less than 0.1\u00a0% to generate a total of 18 genes that
\n are contained in genomic segments. These results suggest that the enrichment of physical
\n linkage in our HGT data cannot be explained solely by chance. d<\/strong> The proportion of carbohydrate-activating enzymes, transporters, and peptidases among
\n the HGT set (gray color) in comparison to those in complete-genome data (white color).
\n The results of significance test are indicated for each comparison\n <\/p>\n
\n (e.g., Figs.\u00a02a<\/a> and 3b<\/a>, and Additional file 4<\/a>). Considering the relatively recent emergence of Colletotrichum<\/em>, these HGTs likely occurred between the Magnaporthales common ancestor and an ancient
\n lineage leading to extant Colletotrichum<\/em>. Other HGT instances occurred more recently and are restricted to particular Magnaporthales
\n lineages (e.g., Fig.\u00a03a<\/a> and Additional file 4<\/a>). Given the uncertainties that result from the varying sequencing depth and differential
\n gene loss among Magnaporthales clades, predictions about the timing of gene transfer
\n should be treated with caution. Nevertheless, these results strongly suggest that
\n Magnaporthales exchanged genes with the lineage leading to modern-day Colletotrichum<\/em>.\n <\/p>\n
\n genes of HGT origin (allowing one intervening non-HGT gene) (Additional file 5<\/a>). We manually examined the genomic locations of the relevant Colletotrichum<\/em> genes associated with the five genomic segments without non-HGT interruption (discussed
\n earlier). In almost all cases, the corresponding genomic segments were also found
\n in Colletotrichum<\/em> genomes. Random sampling 18 genes (5000 times) from the 9154?M. incrustans<\/em> genes with single-gene phylogenies showed that the physical linkage of HGT genes
\n is significantly more than expected by chance alone (Fig.\u00a03c<\/a>). A similar result was obtained when using the Ophioceras dolichostomum<\/em> (instead of M. incrustans<\/em>) proteome as the input for the two-way phylogenomic analysis (Additional file 7<\/a>). A total of 51 HGTs (51 distinct transfer events) were inferred between O. dolichostomum<\/em> and Colletotrichum<\/em> (Additional file 8<\/a>). These results suggest that HGT between Magnaporthales and Colletotrichum<\/em> often occurred as segmental transfers involving more than one gene.\n <\/p>\n
\n Colletotrichum<\/em>? From the perspective of taxonomy, out of the 1453 highly conserved single-copy orthologous
\n genes that were identified across 22 Pezizomycotina lineages (see Methods), none were
\n implicated in HGT. This suggests that Magnaporthales-Colletotrichum<\/em> HGTs have a limited impact on highly conserved genes and likely does not pose significant
\n challenges for the reconstruction of a fungal tree of life. From the perspective of
\n functional impacts, we examined several functional categories associated with the
\n plant pathogenic lifestyle, including carbohydrate-activating enzymes (CAZymes) 27<\/a>] involved in cell wall degradation, membrane transporters, and peptidases involved
\n in pathogenesis 28<\/a>]. We found a 2.6-fold enrichment of CAZymes in the M. incrustans<\/em> gene set derived from HGT (31.2\u00a0%; 29\/93; regardless of direction and timing of HGT,
\n Fig.\u00a03d<\/a>) when compared to the 9154-gene background data (11.7\u00a0%; 1075\/9154). This enrichment
\n was statistically significant (P<\/em>?=?1?\u00d7?10
\n \u20138<\/sup>
\n ; ?
\n 2<\/sup>
\n test) and was not explained by post-HGT duplication of CAZyme encoding genes in Magnaporthales.
\n The 29 transferred CAZymes represent 27 independent HGT events with only two genes
\n having resulted from post-HGT gene duplication. Enrichment of CAZymes among genes
\n that were transferred between Magnaporthales and Colletotrichum<\/em> (P<\/em>?=?0.052; 19.6\u00a0% (10\/51) in HGTs versus 11.0\u00a0% (999\/9047) in genome background; ?
\n 2<\/sup>
\n test) were also observed when analyzing the O. dolichostomum<\/em> genome data (Additional file 7<\/a>). Weak or non-significant differences were however found in the distribution of transporter
\n and peptidase genes (Fig.\u00a03d<\/a> and Additional file 7<\/a>).\n <\/p>\n
\n these results suggest that HGTs with cell wall degradation functions were selectively
\n retained (twice as likely than average) after insertion into host genomes. This function-driven
\n selection is likely linked to the plant pathogenic lifestyles found in both lineages.
\n The Magnaporthales-Colletotrichum<\/em> HGT connection may therefore have been facilitated by a shared ecological niche and
\n host. HGT occurs commonly between species that are in close proximity or have physical
\n contact (e.g., 29<\/a>]\u201331<\/a>]).\n <\/p>\nAlternative explanations for Magnaporthales-Colletotrichum<\/em> gene sharing
\n <\/h4>\n
\n M. incrustans<\/em> and Colletotrichum<\/em> (i.e., poor sampling and extensive gene loss among taxa, phylogenetic artifacts,
\n and random chance). Regarding the first issue, when the corresponding genes were absent
\n in all other Sordariomycetes lineages (e.g., Fig.\u00a02a<\/a>), the explanation for HGT due to poor sampling and extensive gene losses in closely
\n related lineages would require the complete absence or loss of the impacted genes
\n in all five Sordariomycetes lineages (Fig.\u00a01b<\/a> and Additional file 9<\/a>: Figure S1) that were well-sampled in this study (Additional files 2<\/a> and 10<\/a>). When assuming the existence of the node uniting Magnaporthales and Colletotrichum<\/em> to be the Sordariomycetes common ancestor, a total of five gene losses are required
\n to explain all Magnaporthales-Colletotrichum<\/em> HGTs (HGT type I, see Additional file 9<\/a>: Figure S1 for details). However, careful examination of the HGT gene trees derived
\n from the M. incrustans<\/em> genome data revealed a total of 33 independent HGT events [type II (4 genes), type
\n III (12 genes), and type IV (17 genes)] that require more than five gene losses when
\n vertical inheritance with gene loss is assumed (Additional file 9<\/a>: Figures S2, S3 and S4). For HGT types II and III, the corresponding genes are present
\n in additional Sordariomycetes lineages and form a sister group relationship (?85\u00a0%
\n UFboot support) to the Magnaporthales-Colletotrichum<\/em> monophyletic clade (e.g., Verticillium<\/em> in Fig.\u00a03b<\/a>). This leads to phylogenetic conflicts because Magnaporthales and Colletotrichum<\/em> are separated by additional Sordariomycetes lineages in the species tree shown in
\n Fig.\u00a01b<\/a> (see Additional file 9<\/a>: Figures S2 and S3 for details). To explain these phylogenetic conflicts, one ancient
\n gene duplication and 11 independent gene losses are required when assuming vertical
\n inheritance and gene loss, whereas only one gene transfer (type II) and an additional
\n gene loss (type III) are required when HGT is allowed (Additional file 9<\/a>: Figures S2 and S3). We also identified HGT cases (type IV), in which Colletotrichum<\/em> species are nested among Magnaporthales or vice versa (with???85\u00a0% UFboot support
\n at the relevant nodes, Fig.\u00a03a<\/a> and Additional file 9<\/a>: Figure S4). The phylogenetic conflicts raised in these HGTs require a total of one
\n ancient gene duplication and 11 independent gene losses when assuming vertical inheritance
\n and gene loss, whereas only one gene transfer (Type IV, scenario b) and an additional
\n gene duplication (Type IV, scenario a) are required when HGT is allowed (see Additional
\n file 9<\/a>: Figure S4 for details). Whereas we cannot definitively exclude the possibility of
\n vertical inheritance and gene loss as an explanation for each HGT candidate identified
\n in this study, a total of 33 HGT cases (corresponding to HGT types II\u2013IV, explained
\n in Additional file 9<\/a>) are highly unlikely to be explained by the vertical inheritance and gene loss scenario.
\n The topologies and supporting values of these high confidence HGTs (available in Additional
\n file 11<\/a>) were confirmed via examination of gene trees generated from two-way phylogenomics
\n and from the HGT validation procedure (see Methods). A total of 15 independent HGTs
\n (types II\u2013IV) were found in O. dolichostomum<\/em> genome data (Additional file 11<\/a>).\n <\/p>\n
\n an additional round of phylogenomic analysis to search for and validate HGTs. These
\n analyses involve different sequence sampling strategies (taxonomically dependent and
\n independent sampling, and BLASTp hits sorted by bit-score and by sequence identity)
\n and different tree building methods (FastTree and IQtree) (see Methods for details).
\n The Magnaporthales-Colletotrichum<\/em> HGTs are therefore unlikely to be primarily explained by phylogenetic artifacts.
\n Regarding the third issue, it is possible that analysis of large genomic datasets
\n might lead to observations of HGT that are explained solely by chance. However, random
\n sampling of the Magnaporthales gene set (see Methods) is unlikely to generate as many
\n physical linkages as we report in the empirical data (Fig.\u00a03c<\/a> and Additional file 7<\/a>). The enrichment of physical linkages among HGT candidates (0.1\u00a0% chance by random
\n sampling, Fig.\u00a03c<\/a> and Additional file 7<\/a>) is therefore unlikely to be accounted for solely by chance due to the large amount
\n of genome data being analyzed. Likewise, the observed enrichment of CAZyme genes (P<\/em>?=?1?\u00d7?10
\n \u20138<\/sup>
\n in M. incrustans<\/em> data, Fig.\u00a03d<\/a>; and P<\/em>?=?5?\u00d7?10
\n \u20132<\/sup>
\n in O. dolichostomum<\/em> data, Additional file 7<\/a>) in our HGT data is unlikely to be explained by random chance.\n <\/p>\n","protected":false},"excerpt":{"rendered":"