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plots.r
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library(ape)
library(tidyverse)
library(ggtree)
library(ggpubr)
#Read in metadata
metadata <- read.csv("metadata.csv")
## GENOME SIZE FIGURES ##
#Add a column for cytometric completeness
metadata$cytometric.completeness <- metadata$assembly / metadata$flow.cytometry * 100
#Make dataframe of BUSCO and cytometric completeness
completeness.comp.df <- metadata %>%
select(strain, taxon, assembly, flow.cytometry, busco.completeness, cytometric.completeness) %>%
filter(!is.na(cytometric.completeness)) %>%
gather(type, value, -strain, -taxon, -assembly, -flow.cytometry) %>%
mutate(label=paste0('"("*italic("', taxon, '")*")"'))
#Format labels
completeness.comp.df$label <- sub(' sp\\.")\\*")', '")~"sp\\.)', completeness.comp.df$label)
#Barplot of completeness for each strain
gg.completeness <- ggplot(completeness.comp.df, aes(x=paste("IMI", strain), y=value, fill=type)) +
geom_bar(stat="identity",
colour="black",
size=0.2,
width=0.4,
position="dodge") +
geom_text(data=completeness.comp.df[completeness.comp.df$type == "busco.completeness",],
aes(label=label, y=0),
vjust=4,
size=2,
parse=T,
show.legend=FALSE) +
labs(y="Completeness (%)") +
coord_cartesian(clip="off") +
scale_y_continuous(limits=c(0, 100),
expand=c(0, 0)) +
scale_fill_manual(values=c("white", "dimgrey"),
labels=c("Gene set (BUSCO)", "Cytometric genome size estimate"),
guide=guide_legend(title="Method of assessing assembly completeness",
title.position="top",
title.hjust=0.5)) +
theme(legend.position="top",
legend.key.size=unit(0.5,"line"),
legend.title=element_text(size=9.5, face="bold"),
legend.text=element_text(margin=margin(r=5)),
legend.margin=margin(0,0,0,0),
legend.box="vertical",
axis.text.x=element_text(size=8, colour="black"),
axis.title.y=element_text(margin=margin(0,10,0,0)),
axis.title.x=element_blank(),
plot.margin=unit(c(1,1,5,1), "mm"),
panel.grid.major.x=element_blank())
#Make dataframe of assembly and cytometry genome size
size.comp.df <- completeness.comp.df %>%
select(strain, taxon, assembly, flow.cytometry, label) %>%
distinct() %>%
gather(type, value, -strain, -taxon, -label)
#Barplot of size for each strain
gg.size <- ggplot(size.comp.df, aes(x=paste("IMI", strain), y=value, fill=type)) +
geom_bar(stat="identity",
colour="black",
size=0.2,
width=0.4,
position="dodge") +
geom_text(data=size.comp.df[size.comp.df$type == "assembly",],
aes(label=label, y=0),
vjust=4,
size=2,
parse=T,
show.legend=FALSE) +
labs(y="Genome size (Mbp)") +
coord_cartesian(clip="off") +
scale_y_continuous(limits=c(0, 50),
expand=c(0, 0)) +
scale_fill_manual(values=c("white", "dimgrey"),
labels=c("Total assembly size", "Cytometric genome size estimate"),
guide=guide_legend(title="Method of assessing genome size",
title.position="top",
title.hjust=0.5)) +
theme(legend.position="top",
legend.key.size=unit(0.5,"line"),
legend.title=element_text(size=9.5, face="bold"),
legend.text=element_text(margin=margin(r=5)),
legend.margin=margin(0,0,0,0),
legend.box="vertical",
axis.text.x=element_text(size=8, colour="black"),
axis.title.y=element_text(margin=margin(0,10,0,0)),
axis.title.x=element_blank(),
plot.margin=unit(c(1,1,5,1), "mm"),
panel.grid.major.x=element_blank())
#Write to file
#tiff(file=paste0("completeness_fig-", Sys.Date(), ".tiff"), height=6, width=6, units="in", res=300)
ggarrange(gg.size, gg.completeness, ncol=1, labels=c("C", "D"), align="v")
#dev.off()
## GENETIC MARKERS FIGURE ##
#Make dataframe summarising markers for each lineage
markers.df <- metadata %>%
select(lineage, acl1, Bml, cmdA, GAPDH, HIS3, mak.2, nik.1, PKC, LSU, RPB1, RPB2, TEF1, TUB2) %>%
distinct() %>%
gather(gene, copynum, -lineage) %>%
mutate(gene=factor(gene)) %>%
mutate(lineage=factor(lineage, levels=rev(levels(factor(lineage)))))
#Grid plot of markers used for each lineage
gg.markers <- ggplot(markers.df, aes(x=gene, y=lineage, fill=copynum)) +
geom_tile(colour="grey97", size=1) +
scale_fill_manual(values=c("white", "dimgrey")) +
scale_y_discrete(labels=str_to_sentence(levels(markers.df$lineage))) +
scale_x_discrete(position="top",
labels=c("acl1", "Bml", "cmdA", "GAPDH", "HIS3", "LSU", "mak-2",
"nik-1", "PKC", "RPB1", "RPB2", expression(paste("EF1", alpha)), "TUB2")) +
theme_minimal() +
theme(legend.position="none",
legend.title=element_text(face="bold", hjust=1),
axis.title=element_blank(),
axis.text.x=element_text(size=6, colour="black"),
axis.text.y=element_text(face="italic", size=7, colour="black"))
#Write to file
#tiff(file=paste0("markers_fig-", Sys.Date(), ".tiff"), height=2, width=6, units="in", res=300)
gg.markers
#dev.off()
## T-BAS FIGURE ##
#Read in T-BAS tree
tbas <- read.tree("tbas_tree_220804.tre")
#Plot tree to get node numbers for clades containing own strains
#tiff(file=paste0("tbas-", Sys.Date(), ".tiff"), height=35, width=8, units="in", res=600)
ggtree(tbas, size=0.1) +
geom_tiplab(size=0.8) +
geom_text2(aes(subset=!isTip, label=node), size=0.8, hjust=-0.3)
#dev.off()
#Format labels
tbas$tip.label[which(tbas$tip.label == "355091")] <- "355091/355093"
#Make dataframe with nodes to plot lineages separately
tbas.df <- data.frame(node=c(3249, 3232, 3222, 3207, 2393, 2151, 2077, 2058, 2006),
lineage=c("Didymellaceae", "Didymella", "Didymellaceae", "Didymosphaeriaceae", "Nectriaceae", "Colletotrichum", "Collariella", "Neurospora", "Gnomoniaceae"))
#Make vectors of strains
strains <- c("355080", "355082", "355084", "355091/355093", "356814", "356815", "359910", "360193", "360204", "364377", "366226", "366227", "366586", "367209")
#For each lineage...
for (i in 1:length(tbas.df$node)) {
#Extract lineage clade
tbas.tmp <- extract.clade(tbas, tbas.df$node[i])
#Plot tree
gg.tbas <- ggtree(tbas.tmp, size=0.3)
#Create vector of fontface for new genomes in this study
tip.face <- gg.tbas$data %>%
filter(isTip == "TRUE") %>%
pull(label)
tip.face <- ifelse(tip.face %in% strains, "bold.italic", "italic")
#Add formatted tip labels
gg.tbas <- gg.tbas +
xlim(0, 0.7) +
geom_tiplab(size=2,
font=tip.face,
show.legend=FALSE) +
ggtitle(label=tbas.df$lineage[i]) +
scale_colour_manual(values=c("black", "red")) +
theme(plot.title=element_text(size=8, hjust=0.5, face="bold.italic"))
assign(paste0("gg.tbas.", i), gg.tbas)
}
#Write to file
#tiff(file=paste0("tbas_fig-", Sys.Date(), ".tiff"), height=5, width=6, units="in", res=600)
ggarrange(plotlist=mget(ls(pattern="gg.tbas.")))
#dev.off()
## TREE FIGURES ##
#Make list of outgroups for each tree
outgroups <- list(neocosmospora=c("Fusarium_staphyleae_strain_CBS_125482",
"Fusarium_celtis-occidentalis_strain_CBS_125502",
"Fusarium_cicatricum_strain_CBS_125550"),
colletotrichum=c("Monilochaetes_infuscans_culture-collection_CBS_869.96"),
collariella=c("Melanocarpus_albomyces_strain_CBS_638.94",
"Ovatospora_brasiliensis_strain_CBS_130174",
"Achaetomium_globosum_strain_CBS_332.67"),
neurospora=c("Sordaria_tomento-alba_CBS_260.78",
"Sordaria_fimicola_FGSC_2918",
"Sordaria_brevicollis_FGSC_1904",
"Pseudoneurospora_amorphoporcata_CBS_626.80",
"Sordaria_sclerogenia_FGSC_2741",
"Sordaria_macrospora_FGSC_4818"),
gnomoniopsis=c("Melanconis_alni_strain_AR_3500_voucher_BPI_748444",
"Melanconis_marginalis_strain_AR_3442_voucher_BPI_748446"),
neocucurbitaria=c("Pyrenochaeta_acicola_CBS_812.95"),
neodidymelliopsis=c("Neoascochyta_desmazieri_strain_CBS_346.86",
"Neoascochyta_sp._2_NV-2016",
"Neoascochyta_europaea_strain_CBS_504.71"),
ascochyta=c("Phoma_herbarum_strain_CBS_615.75"),
didymosphaeriaceae=c("Massarinaceae_sp._DQD-2015a_voucher_MFLU_15-0057"),
didymella=c("Paraboeremia_putaminum_strain_CBS_130.69",
"Paraboeremia_adianticola_strain_CBS_187.83",
"Macroventuria_anomochaeta_strain_CBS_525.71",
"Macroventuria_wentii_strain_CBS_526.71",
"Paraboeremia_selaginellae_strain_CBS_122.93"))
#Make list of markers used for each tree
markers <- list(neocosmospora=c("acl1+cmdA+RPB1+\nRPB2+TEF1"),
colletotrichum=c("GAPDH+HIS3+TUB2"),
collariella=c("RPB2+TUB2"),
neurospora=c("Bml+LSU+mak-2+\nnik-1+PKC+TEF1"),
gnomoniopsis=c("TEF1+TUB2"),
neocucurbitaria=c("RPB2+TEF1+TUB2"),
neodidymelliopsis=c("RPB2+TUB2"),
ascochyta=c("RPB2+TUB2"),
didymosphaeriaceae=c("LSU+RPB2+\nTEF1+TUB2"),
didymella=c("RPB2+TUB2"))
#Make list of axis limits for each tree
xlims <- list(neocosmospora=0.25,
colletotrichum=0.7,
collariella=0.4,
neurospora=0.12,
gnomoniopsis=0.5,
neocucurbitaria=0.2,
neodidymelliopsis=0.3,
ascochyta=0.2,
didymosphaeriaceae=0.6,
didymella=0.25)
#Make list of tip label sizes for each tree
tip.sizes <- list(neocosmospora=3,
colletotrichum=2.3,
collariella=3,
neurospora=3,
gnomoniopsis=3,
neocucurbitaria=3,
neodidymelliopsis=3,
ascochyta=3,
didymosphaeriaceae=2.3,
didymella=3)
counter <- 0
#For each lineage...
for (i in c("didymella", "ascochyta", "neodidymelliopsis", "didymosphaeriaceae", "neocucurbitaria", "gnomoniopsis", "colletotrichum", "collariella", "neurospora", "neocosmospora")) {
counter <- counter + 1
#Read in tree
tree <- read.tree(paste0("phylogenetics/raxmlng/", i, "/", i, "_concat.raxml.support"))
#Root tree
tree <- root(tree, outgroups[[i]], edgelabel=TRUE, resolve.root=TRUE)
#If the lineage is one that contains an excessively long branch, truncate the branch
if (i %in% c("neocosmospora", "neocucurbitaria", "neodidymelliopsis")) {
shortened.edge <- tree$edge[which.max(tree$edge.length), 2]
assign(paste0("shortened.edge.", i), shortened.edge)
tree$edge.length[which.max(tree$edge.length)] <- tree$edge.length[which.max(tree$edge.length)] / 10
}
#Read in tree metadata
metadata <- read.csv(paste0("phylogenetics/raxmlng/", i, "/metadata.csv"))
#Plot tree
gg.tree <- ggtree(tree, linetype=NA) %<+% metadata
#Create vector of fontface for new assemblies in this study
tip.face <- gg.tree$data %>%
filter(isTip == "TRUE") %>%
pull(own)
tip.face <- ifelse(tip.face == "Y", "bold.italic", "italic")
#Colour branches by support and scale x axis
gg.tree <- gg.tree +
geom_tree(aes(colour=ifelse(as.numeric(label) < 70, "insig", NA)),
show.legend=FALSE) +
xlim(0, xlims[[i]]) +
scale_colour_manual(values="darkgrey",
na.value="black")
#If the lineage is one with species complexes to collapse...
if (i %in% c("colletotrichum")) {
#Make dataframe of species complex nodes
clades.df <- data.frame(
sc=unique(metadata %>%
filter(species.complex != "") %>%
pull(species.complex)),
node=NA
)
#Find the most recent common ancestor for each clade
for (j in 1:length(clades.df$sc)) {
clades.df$node[j] <- MRCA(tree,
metadata$tip[metadata$species.complex == clades.df$sc[j]])
}
#Make dataframe of clades to collapse
collapse.clades.df <- clades.df %>%
filter(!sc %in% c("gloeosporioides", "acutatum")) %>%
mutate(label=paste0("Colle. ", sc, " species complex"))
for (j in 1:length(collapse.clades.df$sc)) {
if (length(which(gg.tree$data$species.complex == collapse.clades.df$sc[j])) > 1) {
#Collapse clades
gg.tree <- ggtree::collapse(gg.tree,
node=collapse.clades.df$node[j])
}
}
#Add clade labels
gg.tree <- gg.tree +
geom_cladelab(data=collapse.clades.df,
mapping=aes(node=node, label=label),
fontface="italic",
fontsize=tip.sizes[[i]],
offset.text=0.005) +
geom_point2(aes(subset=node %in% collapse.clades.df$node),
shape=18, size=2)
label.clades.df <- clades.df %>%
filter(sc %in% c("gloeosporioides", "acutatum")) %>%
mutate(label=paste0("Colle. ", sc, "\nspecies complex"))
gg.tree <- gg.tree +
geom_cladelab(data=label.clades.df,
mapping=aes(node=node, label=label),
fontface="italic",
fontsize=tip.sizes[[i]],
barsize=1,
offset=0.23,
offset.text=0.01)
}
#Add tip labels and a label indicating markers used
gg.tree <- gg.tree +
geom_tiplab(aes(label=name),
size=tip.sizes[[i]],
font=tip.face) +
annotate(geom="text",
label=LETTERS[counter],
x=-Inf, y=Inf, hjust=0, vjust=1,
size=5, fontface="bold") +
annotate(geom="label",
label=markers[[i]],
x=max(na.omit(gg.tree$data$x))*0.17, y=max(na.omit(gg.tree$data$y))*0.92,
size=3)
assign(paste0("gg.tree.", i), gg.tree)
}
#Add labels indicating truncated branches
gg.tree.neocosmospora <- gg.tree.neocosmospora +
geom_label2(aes(x=branch, subset=node == shortened.edge.neocosmospora),
label="//",
label.padding=unit(0, "pt"),
label.size=0)
gg.tree.neocucurbitaria <- gg.tree.neocucurbitaria +
geom_label2(aes(x=branch, subset=node == shortened.edge.neocucurbitaria),
label="//",
label.padding=unit(0, "pt"),
label.size=0)
gg.tree.neodidymelliopsis <- gg.tree.neodidymelliopsis +
geom_label2(aes(x=branch, subset=node == shortened.edge.neodidymelliopsis),
label="//",
label.padding=unit(0, "pt"),
label.size=0)
#Write to file
#png(file="chapter4figure3.A.png", height=9, width=6, units="in", res=600)
#tiff(file=paste0("didymella_fig-", Sys.Date(), ".tiff"), height=9, width=6, units="in", res=600)
gg.tree.didymella
#dev.off()
#png(file="chapter4figure3.B.png", height=4, width=6, units="in", res=600)
#tiff(file=paste0("ascochyta_fig-", Sys.Date(), ".tiff"), height=9, width=6, units="in", res=600)
gg.tree.ascochyta
#dev.off()
#png(file="chapter4figure3.C.png", height=4.5, width=6, units="in", res=600)
#tiff(file=paste0("neodidymelliopsis_fig-", Sys.Date(), ".tiff"), height=9, width=6, units="in", res=600)
gg.tree.neodidymelliopsis
#dev.off()
#png(file="chapter4figure3.D.png", height=9, width=6, units="in", res=600)
#tiff(file=paste0("didymosphaeriaceae_fig-", Sys.Date(), ".tiff"), height=9, width=6, units="in", res=600)
gg.tree.didymosphaeriaceae
#dev.off()
#png(file="chapter4figure3.E.png", height=4.5, width=6, units="in", res=600)
#tiff(file=paste0("neocucurbitaria_fig-", Sys.Date(), ".tiff"), height=9, width=6, units="in", res=600)
gg.tree.neocucurbitaria
#dev.off()
#png(file="chapter4figure3.F.png", height=4.7, width=6, units="in", res=600)
#tiff(file=paste0("gnomoniopsis_fig-", Sys.Date(), ".tiff"), height=9, width=6, units="in", res=600)
gg.tree.gnomoniopsis
#dev.off()
#png(file="chapter4figure3.G.png", height=9, width=6, units="in", res=600)
#tiff(file=paste0("colletotrichum_fig-", Sys.Date(), ".tiff"), height=9, width=6, units="in", res=600)
gg.tree.colletotrichum
#dev.off()
#png(file="chapter4figure3.H.png", height=4.5, width=6, units="in", res=600)
#tiff(file=paste0("collariella_fig-", Sys.Date(), ".tiff"), height=9, width=6, units="in", res=600)
gg.tree.collariella
#dev.off()
#png(file="chapter4figure3.I.png", height=6.5, width=6, units="in", res=600)
#tiff(file=paste0("neurospora_fig-", Sys.Date(), ".tiff"), height=9, width=6, units="in", res=600)
gg.tree.neurospora
#dev.off()
#png(file="chapter4figure3.J.png", height=9, width=6, units="in", res=600)
#tiff(file=paste0("neocosmospora_fig-", Sys.Date(), ".tiff"), height=9, width=6, units="in", res=600)
gg.tree.neocosmospora
#dev.off()
#Read in supplementary LSU tree
supp.tree <- read.tree("phylogenetics/raxmlng/didymosphaeriaceae/didymosphaeriaceae_LSU.raxml.support")
#Root
supp.tree <- root(supp.tree, outgroups$didymosphaeriaceae, edgelabel=TRUE, resolve.root=TRUE)
#Truncate branch
shortened.edge <- supp.tree$edge[which.max(supp.tree$edge.length), 2]
supp.tree$edge.length[which.max(supp.tree$edge.length)] <- supp.tree$edge.length[which.max(supp.tree$edge.length)] / 3
#Read in metadata
metadata <- read.csv("phylogenetics/raxmlng/didymosphaeriaceae/metadata.csv")
#Plot tree
gg.supp.tree <- ggtree(supp.tree, linetype=NA) %<+% metadata
#Create vector of fontface for new genomes in this study
tip.face <- gg.supp.tree$data %>%
filter(isTip == "TRUE") %>%
pull(own)
tip.face <- ifelse(tip.face == "Y", "bold.italic", "italic")
#Plot tree
gg.supp.tree <- gg.supp.tree +
geom_tree(aes(colour=ifelse(as.numeric(label) < 70, "insig", NA)),
show.legend=FALSE) +
xlim(0, xlims[[i]]) +
scale_colour_manual(values="darkgrey",
na.value="black") +
geom_label2(aes(x=branch, subset=node == shortened.edge),
label="//",
size=3,
label.padding=unit(0, "pt"),
label.size=0) +
xlim(0, 0.15) +
geom_tiplab(aes(label=name),
size=2.2,
font=tip.face)
#Write to file
#tiff(file=paste0("didymosphaeriaceae_suppfig-", Sys.Date(), ".tiff"), height=9, width=6, units="in", res=600, compression="lzw")
gg.supp.tree
#dev.off()