Last updated: 2020-11-18

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knitr::opts_chunk$set(message = FALSE) 
library(tidyverse)
-- Attaching packages ------------------------------------------------------------------------------------------------------------------- tidyverse 1.3.0 --
v ggplot2 3.3.2     v purrr   0.3.4
v tibble  3.0.2     v dplyr   1.0.0
v tidyr   1.1.0     v stringr 1.4.0
v readr   1.3.1     v forcats 0.5.0
-- Conflicts ---------------------------------------------------------------------------------------------------------------------- tidyverse_conflicts() --
x dplyr::filter() masks stats::filter()
x dplyr::lag()    masks stats::lag()
library(patchwork)
library(ggsci)
library(dabestr)
Loading required package: magrittr

Attaching package: 'magrittr'
The following object is masked from 'package:purrr':

    set_names
The following object is masked from 'package:tidyr':

    extract
library(dabestr)
library(cowplot)

********************************************************
Note: As of version 1.0.0, cowplot does not change the
  default ggplot2 theme anymore. To recover the previous
  behavior, execute:
  theme_set(theme_cowplot())
********************************************************

Attaching package: 'cowplot'
The following object is masked from 'package:patchwork':

    align_plots
library(ggsignif)
library(ggforce)
library(lme4)
Loading required package: Matrix

Attaching package: 'Matrix'
The following objects are masked from 'package:tidyr':

    expand, pack, unpack
library(lmerTest)

Attaching package: 'lmerTest'
The following object is masked from 'package:lme4':

    lmer
The following object is masked from 'package:stats':

    step
library(sjPlot)
Learn more about sjPlot with 'browseVignettes("sjPlot")'.

Attaching package: 'sjPlot'
The following objects are masked from 'package:cowplot':

    plot_grid, save_plot
library(dotwhisker)
library(pals)
theme_set(theme_cowplot())

Data loading

npg_col = pal_npg("nrc")(9)
col_list <- c(`Wild`=npg_col[8],
   Landrace = npg_col[3],
  `Old cultivar`=npg_col[2],
  `Modern cultivar`=npg_col[4])

pav_table <- read_tsv('./data/soybean_pan_pav.matrix_gene.txt.gz')
nbs <- read_tsv('./data/Lee.NBS.candidates.lst', col_names = c('Name', 'Class'))
nbs
# A tibble: 486 x 2
   Name                   Class
   <chr>                  <chr>
 1 UWASoyPan00953.t1      CN   
 2 GlymaLee.13G222900.1.p CN   
 3 GlymaLee.18G227000.1.p CN   
 4 GlymaLee.18G080600.1.p CN   
 5 GlymaLee.20G036200.1.p CN   
 6 UWASoyPan01876.t1      CN   
 7 UWASoyPan04211.t1      CN   
 8 GlymaLee.19G105400.1.p CN   
 9 GlymaLee.18G085100.1.p CN   
10 GlymaLee.11G142600.1.p CN   
# ... with 476 more rows
# have to remove the .t1s 
nbs$Name <- gsub('.t1','', nbs$Name)
nbs_pav_table <- pav_table %>% filter(Individual %in% nbs$Name)
names <- c()
presences <- c()

for (i in seq_along(nbs_pav_table)){
  if ( i == 1) next
  thisind <- colnames(nbs_pav_table)[i]
  pavs <- nbs_pav_table[[i]]
  presents <- sum(pavs)
  names <- c(names, thisind)
  presences <- c(presences, presents)
}
nbs_res_tibb <- new_tibble(list(names = names, presences = presences))
Warning: The `nrow` argument of `new_tibble()` can't be missing as of tibble 2.0.0.
`x` must be a scalar integer.
This warning is displayed once every 8 hours.
Call `lifecycle::last_warnings()` to see where this warning was generated.
groups <- read_csv('./data/Table_of_cultivar_groups.csv')
groups <- rename(groups, Group = `Group in violin table`)
groups <- groups %>% 
  mutate(Group = str_replace_all(Group, 'landrace', 'Landrace')) %>%
  mutate(Group = str_replace_all(Group, 'Old_cultivar', 'Old cultivar')) %>%
  mutate(Group = str_replace_all(Group, 'Modern_cultivar', 'Modern cultivar')) %>%
  mutate(Group = str_replace_all(Group, 'Wild-type', 'Wild'))

groups$Group <-
  factor(
    groups$Group,
    levels = c('Wild',
               'Landrace',
               'Old cultivar',
               'Modern cultivar')
  )
groups
# A tibble: 1,069 x 3
   `Data-storage-ID` `PI-ID`   Group   
   <chr>             <chr>     <fct>   
 1 SRR1533284        PI416890  Landrace
 2 SRR1533282        PI323576  Landrace
 3 SRR1533292        PI157421  Landrace
 4 SRR1533216        PI594615  Landrace
 5 SRR1533239        PI603336  Landrace
 6 USB-108           PI165675  Landrace
 7 HNEX-13           PI253665D Landrace
 8 USB-382           PI603549  Landrace
 9 SRR1533236        PI587552  Landrace
10 SRR1533332        PI567293  Landrace
# ... with 1,059 more rows
nbs_joined_groups <-
  inner_join(nbs_res_tibb, groups, by = c('names' = 'Data-storage-ID'))

Linking with yield

Can we link the trajectory of NLR genes with the trajectory of yield across the history of soybean breeding? let’s make a simple regression for now

Yield

yield <- read_tsv('./data/yield.txt')
yield_join <- inner_join(nbs_res_tibb, yield, by=c('names'='Line'))
yield_join %>% ggplot(aes(x=presences, y=Yield)) + geom_hex() + geom_smooth() +
  xlab('NLR gene count')

Protein

protein <- read_tsv('./data/protein_phenotype.txt')
protein_join <- left_join(nbs_res_tibb, protein, by=c('names'='Line')) %>% filter(!is.na(Protein))
protein_join %>% ggplot(aes(x=presences, y=Protein)) + geom_hex() + geom_smooth() +
  xlab('NLR gene count')

summary(lm(Protein ~ presences, data = protein_join))

Call:
lm(formula = Protein ~ presences, data = protein_join)

Residuals:
     Min       1Q   Median       3Q      Max 
-11.8479  -2.1274  -0.3336   1.9959  10.0949 

Coefficients:
            Estimate Std. Error t value Pr(>|t|)    
(Intercept) -7.98158    7.24125  -1.102    0.271    
presences    0.11786    0.01624   7.258 8.07e-13 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 3.106 on 960 degrees of freedom
Multiple R-squared:  0.05203,   Adjusted R-squared:  0.05104 
F-statistic: 52.69 on 1 and 960 DF,  p-value: 8.075e-13

Seed weight

Let’s look at seed weight:

seed_weight <- read_tsv('./data/Seed_weight_Phenotype.txt', col_names = c('names', 'wt'))
seed_join <- left_join(nbs_res_tibb, seed_weight) %>% filter(!is.na(wt))
seed_join %>% filter(wt > 5) %>%  ggplot(aes(x=presences, y=wt)) + geom_hex() + geom_smooth() +
  ylab('Seed weight') +
  xlab('NLR gene count')

summary(lm(wt ~ presences, data = seed_join))

Call:
lm(formula = wt ~ presences, data = seed_join)

Residuals:
     Min       1Q   Median       3Q      Max 
-12.2910  -2.8692   0.1462   2.7771  19.6962 

Coefficients:
            Estimate Std. Error t value Pr(>|t|)    
(Intercept) 91.40656   14.67990   6.227 8.28e-10 ***
presences   -0.17636    0.03298  -5.348 1.21e-07 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 4.714 on 690 degrees of freedom
Multiple R-squared:  0.0398,    Adjusted R-squared:  0.0384 
F-statistic:  28.6 on 1 and 690 DF,  p-value: 1.213e-07

Oil content

And now let’s look at the oil phenotype:

oil <- read_tsv('./data/oil_phenotype.txt')
oil_join <- left_join(nbs_res_tibb, oil, by=c('names'='Line')) %>% filter(!is.na(Oil))
oil_join
# A tibble: 962 x 3
   names presences   Oil
   <chr>     <dbl> <dbl>
 1 AB-01       445  17.6
 2 AB-02       454  16.8
 3 BR-24       455  20.6
 4 ESS         454  20.9
 5 For         448  21  
 6 HN001       448  23.6
 7 HN002       444  18.5
 8 HN003       446  17.5
 9 HN004       442  18.9
10 HN005       440  15.5
# ... with 952 more rows
oil_join %>%  ggplot(aes(x=presences, y=Oil)) + geom_hex() + geom_smooth() +
  xlab('NLR gene count')

summary(lm(Oil ~ presences, data = oil_join))

Call:
lm(formula = Oil ~ presences, data = oil_join)

Residuals:
     Min       1Q   Median       3Q      Max 
-10.4376  -1.9081   0.4846   2.2401   9.0361 

Coefficients:
             Estimate Std. Error t value Pr(>|t|)    
(Intercept) 118.03941    7.31646   16.13   <2e-16 ***
presences    -0.22591    0.01641  -13.77   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 3.139 on 960 degrees of freedom
Multiple R-squared:  0.1649,    Adjusted R-squared:  0.1641 
F-statistic: 189.6 on 1 and 960 DF,  p-value: < 2.2e-16

OK there are many, many outliers here. Clearly I’ll have to do something fancier - for example, using the first two PCs as covariates might get rid of some of those outliers.

Boxplots per group

Yield

nbs_joined_groups %>% 
  filter(!is.na(Group)) %>% 
  inner_join(yield, by=c('names'='Line')) %>% 
  ggplot(aes(x=Group, y=Yield, fill = Group)) + 
  geom_boxplot() +
  scale_fill_manual(values = col_list) + 
  theme_minimal_hgrid() +
  theme(axis.text.x = element_text(size=12),
        axis.text.y = element_text(size=12)) +
  geom_signif(comparisons = list(c('Old cultivar', 'Modern cultivar')), 
              map_signif_level = T) +
  guides(fill=FALSE) +
  ylab('Protein') +
  xlab('Accession group')

And let’s check the dots:

nbs_joined_groups %>% 
  filter(!is.na(Group)) %>% 
  inner_join(yield_join, by = 'names') %>% 
  ggplot(aes(y=presences.x, x=Yield, color=Group)) +
  geom_point() + 
  scale_color_manual(values = col_list) + 
  theme_minimal_hgrid() +
  theme(axis.text.x = element_text(size=12),
        axis.text.y = element_text(size=12)) +  
  ylab('NLR gene count')

nbs_joined_groups %>% 
  filter(!is.na(Group)) %>% 
  inner_join(yield_join, by = 'names') %>% 
  filter(Group != 'Landrace') %>% 
  ggplot(aes(x=presences.x, y=Yield, color=Group)) +
  geom_point() + 
  scale_color_manual(values = col_list) + 
  theme_minimal_hgrid() +
  geom_smooth() +
  theme(axis.text.x = element_text(size=12),
        axis.text.y = element_text(size=12)) +  
  xlab('NLR gene count')

## Protein

protein vs. the four groups:

nbs_joined_groups %>% 
  filter(!is.na(Group)) %>% 
  inner_join(protein, by=c('names'='Line')) %>% 
  ggplot(aes(x=Group, y=Protein, fill = Group)) + 
  geom_boxplot() +
  scale_fill_manual(values = col_list) + 
  theme_minimal_hgrid() +
  theme(axis.text.x = element_text(size=12),
        axis.text.y = element_text(size=12)) +
  geom_signif(comparisons = list(c('Wild', 'Landrace'),
                                 c('Old cultivar', 'Modern cultivar')), 
              map_signif_level = T) +
  guides(fill=FALSE) +
  ylab('Protein') +
  xlab('Accession group')

Seed weight

And seed weight:

nbs_joined_groups %>% 
  filter(!is.na(Group)) %>% 
  inner_join(seed_join) %>% 
  ggplot(aes(x=Group, y=wt, fill = Group)) + 
  geom_boxplot() +
  scale_fill_manual(values = col_list) + 
  theme_minimal_hgrid() +
  theme(axis.text.x = element_text(size=12),
        axis.text.y = element_text(size=12)) +
  geom_signif(comparisons = list(c('Wild', 'Landrace'),
                                 c('Old cultivar', 'Modern cultivar')), 
              map_signif_level = T) +
  guides(fill=FALSE) +
  ylab('Seed weight') +
  xlab('Accession group')

Wow, that’s breeding!

Oil content

And finally, Oil content:

nbs_joined_groups %>% 
  filter(!is.na(Group)) %>% 
  inner_join(oil_join, by = 'names') %>% 
  ggplot(aes(x=Group, y=Oil, fill = Group)) + 
  geom_boxplot() +
  scale_fill_manual(values = col_list) + 
  theme_minimal_hgrid() +
  theme(axis.text.x = element_text(size=12),
        axis.text.y = element_text(size=12)) +
  geom_signif(comparisons = list(c('Wild', 'Landrace'),
                                 c('Old cultivar', 'Modern cultivar')), 
              map_signif_level = T) +
  guides(fill=FALSE) +
  ylab('Oil content') +
  xlab('Accession group')

Oha, a single star. That’s p < 0.05!

Let’s redo the above hexplot, but also color the dots by group.

nbs_joined_groups %>% 
  filter(!is.na(Group)) %>% 
  inner_join(oil_join, by = 'names') %>% 
  ggplot(aes(x=presences.x, y=Oil, color=Group)) +
  geom_point() + 
  scale_color_manual(values = col_list) + 
  theme_minimal_hgrid() +
  theme(axis.text.x = element_text(size=12),
        axis.text.y = element_text(size=12)) +  
  xlab('NLR gene count')

Oha, so it’s the Wilds that drag this out a lot.

Let’s remove them and see what it looks like:

nbs_joined_groups %>% 
  filter(!is.na(Group)) %>% 
  inner_join(oil_join, by = 'names') %>% 
  filter(Group %in% c('Old cultivar', 'Modern cultivar')) %>% 
  ggplot(aes(x=presences.x, y=Oil, color=Group)) +
  geom_point() + 
  scale_color_manual(values = col_list) + 
  theme_minimal_hgrid() +
  theme(axis.text.x = element_text(size=12),
        axis.text.y = element_text(size=12)) +  
  xlab('NLR gene count') +
  geom_smooth()

Let’s remove that one outlier:

nbs_joined_groups %>% 
  filter(!is.na(Group)) %>% 
  inner_join(oil_join, by = 'names') %>% 
  filter(Group %in% c('Old cultivar', 'Modern cultivar')) %>% 
  filter(Oil > 13) %>% 
  ggplot(aes(x=presences.x, y=Oil, color=Group)) +
  geom_point() + 
  scale_color_manual(values = col_list) + 
  theme_minimal_hgrid() +
  theme(axis.text.x = element_text(size=12),
        axis.text.y = element_text(size=12)) +  
  xlab('NLR gene count') +
  geom_smooth()

Does the above oil content boxplot become different if we exclude the one outlier? I’d bet so

nbs_joined_groups %>% 
  filter(!is.na(Group)) %>% 
  inner_join(oil_join, by = 'names') %>% 
  filter(names != 'USB-393') %>% 
  ggplot(aes(x=Group, y=Oil, fill = Group)) + 
  geom_boxplot() +
  scale_fill_manual(values = col_list) + 
  theme_minimal_hgrid() +
  theme(axis.text.x = element_text(size=12),
        axis.text.y = element_text(size=12)) +
  geom_signif(comparisons = list(c('Wild', 'Landrace'),
                                 c('Old cultivar', 'Modern cultivar')), 
              map_signif_level = T) +
  guides(fill=FALSE) +
  ylab('Oil content') +
  xlab('Accession group')

Nope, still significantly higher in modern cultivars!

Mixed modeling

Alright here’s my hypothesis: There’s a link between cultivar status (Old, Wild, Landrace, Modern), r-gene count, and yield, but it’s ‘hidden’ by country differences.

Great tutorial here: https://ourcodingclub.github.io/tutorials/mixed-models

So we’ll have to build some lme4 models!

Oil

nbs_joined_groups$presences2 <- scale(nbs_joined_groups$presences, center=T, scale=T)
hist(nbs_joined_groups$presences2)

oil_nbs_joined_groups <- nbs_joined_groups %>% inner_join(oil_join, by = 'names') 
oil_nbs_joined_groups$Oil2 <- scale(oil_nbs_joined_groups$Oil, center=T, scale=T)
basic.lm <- lm(Oil2 ~ presences2, data=oil_nbs_joined_groups)
ggplot(oil_nbs_joined_groups, aes(x = presences2, y = Oil2)) +
  geom_point() +
  geom_smooth(method = "lm")

Hm looks messy, you can see two groups

plot(basic.lm, which = 1)

which is confirmed by the messy line

plot(basic.lm, which = 2)

and this garbage qqplot.

So let’s build an lmer model!

mixed.lmer <- lmer(Oil2 ~ presences2 + (1|Group), data=oil_nbs_joined_groups)
summary(mixed.lmer)
Linear mixed model fit by REML. t-tests use Satterthwaite's method [
lmerModLmerTest]
Formula: Oil2 ~ presences2 + (1 | Group)
   Data: oil_nbs_joined_groups

REML criterion at convergence: 1872.4

Scaled residuals: 
    Min      1Q  Median      3Q     Max 
-4.5879 -0.5672  0.0869  0.6631  3.2111 

Random effects:
 Groups   Name        Variance Std.Dev.
 Group    (Intercept) 1.3349   1.1554  
 Residual             0.4075   0.6384  
Number of obs: 951, groups:  Group, 4

Fixed effects:
             Estimate Std. Error        df t value Pr(>|t|)  
(Intercept)  -0.04360    0.57867   2.99844  -0.075   0.9447  
presences2   -0.05350    0.02394 947.27006  -2.234   0.0257 *
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Correlation of Fixed Effects:
           (Intr)
presences2 -0.004

So the Variance for Group is 1.3349, that means it’s 1.3349/(1.3349+0.4075) *100 = 76% of the variance is explained by the four groups!

plot(mixed.lmer)

qqnorm(resid(mixed.lmer))
qqline(resid(mixed.lmer))

These still look fairly bad - better than before, but the QQ plot still isn’t on the line.

Let’s quickly check yield too

Yield

yield_nbs_joined_groups <- nbs_joined_groups %>% inner_join(yield_join, by = 'names') 
yield_nbs_joined_groups$Yield2 <-scale(yield_nbs_joined_groups$Yield, center=T, scale=T)
mixed.lmer <- lmer(Yield2 ~ presences2 + (1|Group), data=yield_nbs_joined_groups)
summary(mixed.lmer)
Linear mixed model fit by REML. t-tests use Satterthwaite's method [
lmerModLmerTest]
Formula: Yield2 ~ presences2 + (1 | Group)
   Data: yield_nbs_joined_groups

REML criterion at convergence: 2060.4

Scaled residuals: 
    Min      1Q  Median      3Q     Max 
-3.1643 -0.6819  0.0316  0.6948  2.8002 

Random effects:
 Groups   Name        Variance Std.Dev.
 Group    (Intercept) 0.6466   0.8041  
 Residual             0.8600   0.9274  
Number of obs: 761, groups:  Group, 3

Fixed effects:
             Estimate Std. Error        df t value Pr(>|t|)    
(Intercept)   0.23641    0.46910   1.98335   0.504 0.664692    
presences2   -0.15364    0.04172 757.46580  -3.683 0.000247 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Correlation of Fixed Effects:
           (Intr)
presences2 0.025 

Percentage explained by breeding group: 0.6466 / (0.6466+0.8600)*100 = 42%

plot(mixed.lmer)

qqnorm(resid(mixed.lmer))
qqline(resid(mixed.lmer))

:O

p-value of 0.000247 for the normalised presences while accounting for the breeding group, that’s beautiful.

ggplot(yield_nbs_joined_groups, aes(x = presences2, y = Yield2)) +
      facet_wrap(~Group, nrow=2) +   # a panel for each mountain range
      geom_point(alpha = 0.5) +
      theme_classic() +
      geom_line(data = cbind(yield_nbs_joined_groups, pred = predict(mixed.lmer)), aes(y = pred), size = 1) + 
      theme_minimal_hgrid() +
      theme(legend.position = "none") +
      xlab('Scaled and centered NLR gene count') +
      ylab('Scaled and centered yield') +
      scale_color_manual(values=as.vector(isol(40)))

Making the breeding group fixed

We have < 10 possible factors in the group, so making that fixed instead of random

# this doesn't work because you need at least one random effect
# mixed.lmer <- lmer(Yield2 ~ presences2 + Group, data=yield_nbs_joined_groups)

Adding country

We should also add the country the plant is from as a random effect, that definitely has an influence too (perhaps a stronger one???)

Yield

country <- read_csv('./data/Cultivar_vs_country.csv')

names(country) <- c('names', 'PI-ID', 'Country')

yield_country_nbs_joined_groups <- yield_nbs_joined_groups %>% inner_join(country)
mixed.lmer <- lmer(Yield2 ~ presences2 + (1|Group) + (1|Country), data=yield_country_nbs_joined_groups)
summary(mixed.lmer)
Linear mixed model fit by REML. t-tests use Satterthwaite's method [
lmerModLmerTest]
Formula: Yield2 ~ presences2 + (1 | Group) + (1 | Country)
   Data: yield_country_nbs_joined_groups

REML criterion at convergence: 1957

Scaled residuals: 
     Min       1Q   Median       3Q      Max 
-3.09429 -0.56737  0.03072  0.65680  2.89981 

Random effects:
 Groups   Name        Variance Std.Dev.
 Country  (Intercept) 0.3807   0.6170  
 Group    (Intercept) 0.4178   0.6464  
 Residual             0.7614   0.8726  
Number of obs: 741, groups:  Country, 40; Group, 3

Fixed effects:
             Estimate Std. Error        df t value Pr(>|t|)   
(Intercept)   0.07150    0.40194   2.28533   0.178  0.87336   
presences2   -0.11258    0.04116 726.98206  -2.735  0.00639 **
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Correlation of Fixed Effects:
           (Intr)
presences2 0.051 

Nice! Yield is negatively correlated with the number of NLR genes when accounting for breeding group AND country

ggplot(yield_country_nbs_joined_groups, aes(x = presences2, y = Yield2, colour = Country)) +
      facet_wrap(~Group, nrow=2) +   # a panel for each mountain range
      geom_point(alpha = 0.5) +
      theme_classic() +
      geom_line(data = cbind(yield_country_nbs_joined_groups, pred = predict(mixed.lmer)), aes(y = pred), size = 1) + 
      theme_minimal_hgrid() +
      theme(legend.position = "none") +
      xlab('Scaled and centered NLR gene count') +
      ylab('Scaled and centered yield') +
      scale_color_manual(values=as.vector(isol(40)))

Some diagnostics:

plot(mixed.lmer)

qqnorm(resid(mixed.lmer))
qqline(resid(mixed.lmer))

Hm, the qqplot looks slightly worse than when I use maturity group alone, interesting!

BIG DISCLAIMER: Currently, I treat country and group not as nested variables, they’re independent. I think that is the way it should be in this case but I’m thinking.

Making the breeding group fixed

Since we have too few factors in the breeding groups we have to make that fixed, not random

mixed.lmer <- lmer(Yield2 ~ presences2 + Group + (1|Country), data=yield_country_nbs_joined_groups)
summary(mixed.lmer)
Linear mixed model fit by REML. t-tests use Satterthwaite's method [
lmerModLmerTest]
Formula: Yield2 ~ presences2 + Group + (1 | Country)
   Data: yield_country_nbs_joined_groups

REML criterion at convergence: 1951.8

Scaled residuals: 
    Min      1Q  Median      3Q     Max 
-3.1864 -0.5700  0.0305  0.6525  2.8982 

Random effects:
 Groups   Name        Variance Std.Dev.
 Country  (Intercept) 0.3776   0.6145  
 Residual             0.7616   0.8727  
Number of obs: 741, groups:  Country, 40

Fixed effects:
                      Estimate Std. Error        df t value Pr(>|t|)    
(Intercept)           -0.15265    0.13488  33.06645  -1.132  0.26588    
presences2            -0.11149    0.04117 726.45969  -2.708  0.00693 ** 
GroupOld cultivar     -0.29578    0.16164 734.95182  -1.830  0.06768 .  
GroupModern cultivar   1.00458    0.22335 360.40725   4.498 9.28e-06 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Correlation of Fixed Effects:
            (Intr) prsnc2 GrpOlc
presences2   0.119              
GrpOldcltvr -0.112 -0.008       
GrpMdrncltv -0.194  0.065  0.156

Non-normalised yield

Let’s see whether the ‘raw’ values perform the same.

mixed.lmer <- lmer(Yield ~ presences.x + (1|Group) + (1|Country), data=yield_country_nbs_joined_groups)
summary(mixed.lmer)
Linear mixed model fit by REML. t-tests use Satterthwaite's method [
lmerModLmerTest]
Formula: Yield ~ presences.x + (1 | Group) + (1 | Country)
   Data: yield_country_nbs_joined_groups

REML criterion at convergence: 1679.6

Scaled residuals: 
     Min       1Q   Median       3Q      Max 
-3.09429 -0.56737  0.03072  0.65680  2.89981 

Random effects:
 Groups   Name        Variance Std.Dev.
 Country  (Intercept) 0.2602   0.5101  
 Group    (Intercept) 0.2856   0.5345  
 Residual             0.5205   0.7215  
Number of obs: 741, groups:  Country, 40; Group, 3

Fixed effects:
              Estimate Std. Error         df t value Pr(>|t|)    
(Intercept)   9.011013   2.481360 677.994843   3.631 0.000303 ***
presences.x  -0.015192   0.005555 726.982171  -2.735 0.006389 ** 
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Correlation of Fixed Effects:
            (Intr)
presences.x -0.991

Oh, lower p-values for the intercept

ggplot(yield_country_nbs_joined_groups, aes(x = presences.x, y = Yield, colour = Country)) +
      facet_wrap(~Group, nrow=2) +   # a panel for each mountain range
      geom_point(alpha = 0.5) +
      theme_classic() +
      geom_line(data = cbind(yield_country_nbs_joined_groups, pred = predict(mixed.lmer)), aes(y = pred), size = 1) + 
      theme_minimal_hgrid() +
      theme(legend.position = "none") +
      xlab('NLR gene count') +
      ylab('Yield') +
      scale_color_manual(values=as.vector(isol(40)))

plot(mixed.lmer)

qqnorm(resid(mixed.lmer))
qqline(resid(mixed.lmer))

Making the breeding group fixed

mixed.lmer <- lmer(Yield ~ presences.x + Group + (1|Country), data=yield_country_nbs_joined_groups)
summary(mixed.lmer)
Linear mixed model fit by REML. t-tests use Satterthwaite's method [
lmerModLmerTest]
Formula: Yield ~ presences.x + Group + (1 | Country)
   Data: yield_country_nbs_joined_groups

REML criterion at convergence: 1675.1

Scaled residuals: 
    Min      1Q  Median      3Q     Max 
-3.1864 -0.5700  0.0305  0.6525  2.8982 

Random effects:
 Groups   Name        Variance Std.Dev.
 Country  (Intercept) 0.2581   0.5081  
 Residual             0.5206   0.7216  
Number of obs: 741, groups:  Country, 40

Fixed effects:
                       Estimate Std. Error         df t value Pr(>|t|)    
(Intercept)            8.760056   2.465570 726.660933   3.553 0.000406 ***
presences.x           -0.015045   0.005556 726.459065  -2.708 0.006929 ** 
GroupOld cultivar     -0.244554   0.133648 734.951816  -1.830 0.067680 .  
GroupModern cultivar   0.830604   0.184672 360.407255   4.498 9.28e-06 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Correlation of Fixed Effects:
            (Intr) prsnc. GrpOlc
presences.x -0.999              
GrpOldcltvr  0.003 -0.008       
GrpMdrncltv -0.074  0.065  0.156

Oh, lower p-values for the intercept

ggplot(yield_country_nbs_joined_groups, aes(x = presences.x, y = Yield, colour = Country)) +
      facet_wrap(~Group, nrow=2) +   # a panel for each mountain range
      geom_point(alpha = 0.5) +
      theme_classic() +
      geom_line(data = cbind(yield_country_nbs_joined_groups, pred = predict(mixed.lmer)), aes(y = pred), size = 1) + 
      theme_minimal_hgrid() +
      theme(legend.position = "none") +
      xlab('NLR gene count') +
      ylab('Yield') +
      scale_color_manual(values=as.vector(isol(40)))

plot(mixed.lmer)

qqnorm(resid(mixed.lmer))
qqline(resid(mixed.lmer))

plot(resid(mixed.lmer))

These are the final numbers for the paper.

Plotting effect of each covariate

(re.effects <- plot_model(mixed.lmer, type = "re", show.values = TRUE))

#lmerTest breaks these other packages so I better unload it and reload only lme4
detach("package:lmerTest", unload=TRUE)

yield_country_nbs_joined_groups_renamed <- yield_country_nbs_joined_groups
names(yield_country_nbs_joined_groups_renamed) <- c('names', 'presences.x', 'PI-ID', 'Group', 'presences2', 'presences.y', 'Yield', 'Yield2', 'Country')
mixed.lmer <- lmer(Yield2 ~ presences2 + Group + (1|Country), data=yield_country_nbs_joined_groups_renamed)

dwplot(mixed.lmer,
       vline = geom_vline(xintercept = 0, colour = "grey60", linetype = 2))

library(stargazer)
stargazer(mixed.lmer, type = "text",
          digits = 3,
          star.cutoffs = c(0.05, 0.01, 0.001),
          digit.separator = "")

==================================================
                          Dependent variable:     
                     -----------------------------
                                Yield2            
--------------------------------------------------
presences2                     -0.111**           
                                (0.041)           
                                                  
GroupOld cultivar               -0.296            
                                (0.162)           
                                                  
GroupModern cultivar           1.005***           
                                (0.223)           
                                                  
Constant                        -0.153            
                                (0.135)           
                                                  
--------------------------------------------------
Observations                      741             
Log Likelihood                 -975.906           
Akaike Inf. Crit.              1963.812           
Bayesian Inf. Crit.            1991.460           
==================================================
Note:                *p<0.05; **p<0.01; ***p<0.001
library(ggeffects)
ggpredict(mixed.lmer, terms = c("presences2",  'Group'), type = "re") %>% 
   plot() +
   theme_minimal()

Let’s also plot that for non-normalised data

mixed.lmer <- lmer(Yield ~ presences.x + Group + (1|Country), data=yield_country_nbs_joined_groups_renamed)
ggpredict(mixed.lmer, terms = c("presences.x",  'Group'), type = "re") %>% 
   plot() +
   theme_minimal_hgrid() +
  xlab('NLR count') +
  ylab('Yield')

# alright back to regular programming
library(lmerTest)
mixed.lmer <- lmer(Yield2 ~ presences2 + Group + (1|Country), data=yield_country_nbs_joined_groups)

More complex models

If I add random slopes to either groups not much changes, I do get warnings indicating that there’s not much in the data:

mixed.lmer <- lmer(Yield2 ~ presences2 + (presences2|Group) + (1|Country), data=yield_country_nbs_joined_groups)
summary(mixed.lmer)
Linear mixed model fit by REML. t-tests use Satterthwaite's method [
lmerModLmerTest]
Formula: Yield2 ~ presences2 + (presences2 | Group) + (1 | Country)
   Data: yield_country_nbs_joined_groups

REML criterion at convergence: 1954.8

Scaled residuals: 
     Min       1Q   Median       3Q      Max 
-3.09789 -0.56422  0.04471  0.67067  2.88976 

Random effects:
 Groups   Name        Variance Std.Dev. Corr
 Country  (Intercept) 0.3920   0.6261       
 Group    (Intercept) 0.3858   0.6211       
          presences2  0.0310   0.1761   0.30
 Residual             0.7564   0.8697       
Number of obs: 741, groups:  Country, 40; Group, 3

Fixed effects:
            Estimate Std. Error       df t value Pr(>|t|)
(Intercept)  0.02964    0.38964  2.29148   0.076    0.945
presences2  -0.22515    0.12370  1.66243  -1.820    0.235

Correlation of Fixed Effects:
           (Intr)
presences2 0.269 
mixed.lmer <- lmer(Yield2 ~ presences2 + Group + (1 + presences2|Country), data=yield_country_nbs_joined_groups)
summary(mixed.lmer)
Linear mixed model fit by REML. t-tests use Satterthwaite's method [
lmerModLmerTest]
Formula: Yield2 ~ presences2 + Group + (1 + presences2 | Country)
   Data: yield_country_nbs_joined_groups

REML criterion at convergence: 1951.5

Scaled residuals: 
    Min      1Q  Median      3Q     Max 
-3.1904 -0.5715  0.0314  0.6551  2.9003 

Random effects:
 Groups   Name        Variance Std.Dev. Corr
 Country  (Intercept) 0.397511 0.63048      
          presences2  0.002137 0.04623  1.00
 Residual             0.761491 0.87263      
Number of obs: 741, groups:  Country, 40

Fixed effects:
                     Estimate Std. Error       df t value Pr(>|t|)    
(Intercept)           -0.1470     0.1387  28.1820  -1.060   0.2982    
presences2            -0.1162     0.0430  30.8335  -2.702   0.0111 *  
GroupOld cultivar     -0.3010     0.1615 733.1173  -1.863   0.0628 .  
GroupModern cultivar   1.0093     0.2192 166.8461   4.605 8.15e-06 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Correlation of Fixed Effects:
            (Intr) prsnc2 GrpOlc
presences2   0.334              
GrpOldcltvr -0.109 -0.017       
GrpMdrncltv -0.188  0.044  0.158
convergence code: 0
boundary (singular) fit: see ?isSingular

Oh, a significant p-value, let’s plot plot that and compare with he previous plot:

ggplot(yield_country_nbs_joined_groups, aes(x = presences2, y = Yield2, colour = Country)) +
      facet_wrap(~Group, nrow=2) +   # a panel for each mountain range
      geom_point(alpha = 0.5) +
      theme_classic() +
      geom_line(data = cbind(yield_country_nbs_joined_groups, pred = predict(mixed.lmer)), aes(y = pred), size = 1) + 
      theme_minimal_hgrid() +
      theme(legend.position = "none") +
      xlab('Scaled and centered NLR gene count') +
      ylab('Scaled and centered yield') +
      scale_color_manual(values=as.vector(isol(40)))

Quite similar, mostly downwards trajectories for each country.

Let’s do that non-normalised:

mixed.lmer <- lmer(Yield ~ presences.x + Group + (1 + presences.x|Country), data=yield_country_nbs_joined_groups)
Warning in checkConv(attr(opt, "derivs"), opt$par, ctrl = control$checkConv, :
unable to evaluate scaled gradient
Warning in checkConv(attr(opt, "derivs"), opt$par, ctrl = control$checkConv, :
Model failed to converge: degenerate Hessian with 2 negative eigenvalues
Warning: Model failed to converge with 1 negative eigenvalue: -9.3e+04
summary(mixed.lmer)
Linear mixed model fit by REML. t-tests use Satterthwaite's method [
lmerModLmerTest]
Formula: Yield ~ presences.x + Group + (1 + presences.x | Country)
   Data: yield_country_nbs_joined_groups

REML criterion at convergence: 1675.2

Scaled residuals: 
    Min      1Q  Median      3Q     Max 
-3.1863 -0.5679  0.0294  0.6532  2.8982 

Random effects:
 Groups   Name        Variance  Std.Dev.  Corr 
 Country  (Intercept) 5.191e-01 0.7204930      
          presences.x 2.402e-07 0.0004901 -0.98
 Residual             5.206e-01 0.7215284      
Number of obs: 741, groups:  Country, 40

Fixed effects:
                      Estimate Std. Error        df t value Pr(>|t|)    
(Intercept)            8.73673    2.46492 709.51652   3.544 0.000419 ***
presences.x           -0.01499    0.00555 412.58876  -2.702 0.007186 ** 
GroupOld cultivar     -0.24420    0.13365 733.16976  -1.827 0.068096 .  
GroupModern cultivar   0.83046    0.18493 218.59478   4.491 1.15e-05 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Correlation of Fixed Effects:
            (Intr) prsnc. GrpOlc
presences.x -0.999              
GrpOldcltvr  0.002 -0.007       
GrpMdrncltv -0.076  0.067  0.156
convergence code: 0
unable to evaluate scaled gradient
Model failed to converge: degenerate  Hessian with 2 negative eigenvalues
ggplot(yield_country_nbs_joined_groups, aes(x = presences.x, y = Yield, colour = Country)) +
      facet_wrap(~Group, nrow=2) +   # a panel for each mountain range
      geom_point(alpha = 0.5) +
      theme_classic() +
      geom_line(data = cbind(yield_country_nbs_joined_groups, pred = predict(mixed.lmer)), aes(y = pred), size = 1) + 
      theme_minimal_hgrid() +
      theme(legend.position = "none") +
      xlab('NLR gene count') +
      ylab('Yield') +
      scale_color_manual(values=as.vector(isol(40)))

Quite similar, mostly downwards trajectories for each country.

And now both random slopes:

mixed.lmer <- lmer(Yield2 ~ presences2 + (presences2|Group) + (1 + presences2|Country), data=yield_country_nbs_joined_groups)
summary(mixed.lmer)
Linear mixed model fit by REML. t-tests use Satterthwaite's method [
lmerModLmerTest]
Formula: Yield2 ~ presences2 + (presences2 | Group) + (1 + presences2 |  
    Country)
   Data: yield_country_nbs_joined_groups

REML criterion at convergence: 1953.7

Scaled residuals: 
     Min       1Q   Median       3Q      Max 
-3.11214 -0.56909  0.04459  0.66469  2.91084 

Random effects:
 Groups   Name        Variance Std.Dev. Corr
 Country  (Intercept) 0.42704  0.6535       
          presences2  0.01045  0.1022   0.81
 Group    (Intercept) 0.37523  0.6126       
          presences2  0.04201  0.2050   0.18
 Residual             0.75392  0.8683       
Number of obs: 741, groups:  Country, 40; Group, 3

Fixed effects:
            Estimate Std. Error       df t value Pr(>|t|)
(Intercept)  0.03595    0.38772  2.35869   0.093    0.933
presences2  -0.23848    0.14300  1.96304  -1.668    0.240

Correlation of Fixed Effects:
           (Intr)
presences2 0.231 
ggplot(yield_country_nbs_joined_groups, aes(x = presences2, y = Yield2, colour = Country)) +
      facet_wrap(~Group, nrow=2) +   # a panel for each mountain range
      geom_point(alpha = 0.5) +
      theme_classic() +
      geom_line(data = cbind(yield_country_nbs_joined_groups, pred = predict(mixed.lmer)), aes(y = pred), size = 1) + 
      theme_minimal_hgrid() +
      theme(legend.position = "none") +
      xlab('Scaled and centered NLR gene count') +
      ylab('Scaled and centered yield') +
      scale_color_manual(values=as.vector(isol(40)))

Yeah, nah

Oil

oil_country_nbs_joined_groups <- oil_nbs_joined_groups %>% inner_join(country)

mixed.lmer <- lmer(Oil2 ~ presences2 + Group + (1|Country), data=oil_country_nbs_joined_groups)
summary(mixed.lmer)
Linear mixed model fit by REML. t-tests use Satterthwaite's method [
lmerModLmerTest]
Formula: Oil2 ~ presences2 + Group + (1 | Country)
   Data: oil_country_nbs_joined_groups

REML criterion at convergence: 1808.3

Scaled residuals: 
    Min      1Q  Median      3Q     Max 
-4.5285 -0.5605  0.0983  0.6472  3.2209 

Random effects:
 Groups   Name        Variance Std.Dev.
 Country  (Intercept) 0.07703  0.2775  
 Residual             0.39128  0.6255  
Number of obs: 929, groups:  Country, 41

Fixed effects:
                      Estimate Std. Error        df t value Pr(>|t|)    
(Intercept)           -1.66466    0.09388  57.11719 -17.732   <2e-16 ***
presences2            -0.03579    0.02416 917.65081  -1.481    0.139    
GroupLandrace          1.93042    0.06953 911.95172  27.763   <2e-16 ***
GroupOld cultivar      2.25628    0.11897 922.58914  18.965   <2e-16 ***
GroupModern cultivar   2.46605    0.14998 209.92967  16.443   <2e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Correlation of Fixed Effects:
            (Intr) prsnc2 GrpLnd GrpOlc
presences2  -0.232                     
GroupLandrc -0.644  0.434              
GrpOldcltvr -0.405  0.249  0.509       
GrpMdrncltv -0.447  0.246  0.432  0.338

No significance here.

Protein

protein_nbs_joined_groups <- nbs_joined_groups %>% inner_join(protein_join, by = 'names') 
protein_nbs_joined_groups$Protein2 <- scale(protein_nbs_joined_groups$Protein, center=T, scale=T)
protein_country_nbs_joined_groups <- protein_nbs_joined_groups %>% inner_join(country)

mixed.lmer <- lmer(Protein2 ~ presences2 + Group + (1|Country), data=protein_country_nbs_joined_groups)
summary(mixed.lmer)
Linear mixed model fit by REML. t-tests use Satterthwaite's method [
lmerModLmerTest]
Formula: Protein2 ~ presences2 + Group + (1 | Country)
   Data: protein_country_nbs_joined_groups

REML criterion at convergence: 2472.7

Scaled residuals: 
    Min      1Q  Median      3Q     Max 
-3.6001 -0.6747 -0.0423  0.6274  3.5103 

Random effects:
 Groups   Name        Variance Std.Dev.
 Country  (Intercept) 0.07042  0.2654  
 Residual             0.81212  0.9012  
Number of obs: 929, groups:  Country, 41

Fixed effects:
                      Estimate Std. Error        df t value Pr(>|t|)    
(Intercept)            0.52736    0.11992  77.94050   4.397 3.43e-05 ***
presences2             0.04347    0.03466 923.71757   1.254     0.21    
GroupLandrace         -0.72486    0.09980 923.95960  -7.263 8.04e-13 ***
GroupOld cultivar     -1.20226    0.17045 923.25626  -7.053 3.42e-12 ***
GroupModern cultivar  -1.10372    0.20076 140.80066  -5.498 1.76e-07 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Correlation of Fixed Effects:
            (Intr) prsnc2 GrpLnd GrpOlc
presences2  -0.277                     
GroupLandrc -0.712  0.438              
GrpOldcltvr -0.445  0.253  0.511       
GrpMdrncltv -0.507  0.270  0.463  0.340

No significance here.

Seed weight

seed_nbs_joined_groups <- nbs_joined_groups %>% inner_join(seed_join, by = 'names') 
seed_nbs_joined_groups$wt2 <- scale(seed_nbs_joined_groups$wt, center=T, scale=T)
seed_country_nbs_joined_groups <- seed_nbs_joined_groups %>% inner_join(country)
mixed.lmer <- lmer(wt2 ~ presences2 + Group + (1|Country), data=seed_country_nbs_joined_groups)
summary(mixed.lmer)
Linear mixed model fit by REML. t-tests use Satterthwaite's method [
lmerModLmerTest]
Formula: wt2 ~ presences2 + Group + (1 | Country)
   Data: seed_country_nbs_joined_groups

REML criterion at convergence: 1676

Scaled residuals: 
    Min      1Q  Median      3Q     Max 
-2.9614 -0.6215  0.0110  0.5831  4.8138 

Random effects:
 Groups   Name        Variance Std.Dev.
 Country  (Intercept) 0.08566  0.2927  
 Residual             0.70082  0.8371  
Number of obs: 664, groups:  Country, 38

Fixed effects:
                       Estimate Std. Error         df t value Pr(>|t|)    
(Intercept)           -2.348857   0.183189  90.849260 -12.822  < 2e-16 ***
presences2            -0.007261   0.040531 656.652662  -0.179    0.858    
GroupLandrace          2.412123   0.166724 600.989922  14.468  < 2e-16 ***
GroupOld cultivar      2.727120   0.224422 656.159635  12.152  < 2e-16 ***
GroupModern cultivar   2.782167   0.259555  67.478942  10.719 3.26e-16 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Correlation of Fixed Effects:
            (Intr) prsnc2 GrpLnd GrpOlc
presences2  -0.267                     
GroupLandrc -0.864  0.364              
GrpOldcltvr -0.666  0.274  0.710       
GrpMdrncltv -0.663  0.298  0.648  0.539

Again, no significance here.

The final yield model

This is the final yield model for the paper

mixed.lmer <- lmer(Yield ~ presences.x + Group + (1|Country), data=yield_country_nbs_joined_groups)
summary(mixed.lmer)
Linear mixed model fit by REML. t-tests use Satterthwaite's method [
lmerModLmerTest]
Formula: Yield ~ presences.x + Group + (1 | Country)
   Data: yield_country_nbs_joined_groups

REML criterion at convergence: 1675.1

Scaled residuals: 
    Min      1Q  Median      3Q     Max 
-3.1864 -0.5700  0.0305  0.6525  2.8982 

Random effects:
 Groups   Name        Variance Std.Dev.
 Country  (Intercept) 0.2581   0.5081  
 Residual             0.5206   0.7216  
Number of obs: 741, groups:  Country, 40

Fixed effects:
                       Estimate Std. Error         df t value Pr(>|t|)    
(Intercept)            8.760056   2.465570 726.660933   3.553 0.000406 ***
presences.x           -0.015045   0.005556 726.459065  -2.708 0.006929 ** 
GroupOld cultivar     -0.244554   0.133648 734.951816  -1.830 0.067680 .  
GroupModern cultivar   0.830604   0.184672 360.407255   4.498 9.28e-06 ***
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Correlation of Fixed Effects:
            (Intr) prsnc. GrpOlc
presences.x -0.999              
GrpOldcltvr  0.003 -0.008       
GrpMdrncltv -0.074  0.065  0.156
ggplot(yield_country_nbs_joined_groups, aes(x = presences.x, y = Yield, colour = Country)) +
      facet_wrap(~Group, nrow=2) +   # a panel for each mountain range
      geom_point(alpha = 0.5) +
      theme_classic() +
      geom_line(data = cbind(yield_country_nbs_joined_groups, pred = predict(mixed.lmer)), aes(y = pred), size = 1) + 
      theme_minimal_hgrid() +
      theme(legend.position = "none") +
      xlab('NLR gene count') +
      ylab('Yield') +
      scale_color_manual(values=as.vector(isol(40)))

plot(mixed.lmer)

qqnorm(resid(mixed.lmer))
qqline(resid(mixed.lmer))

plot(resid(mixed.lmer))

detach("package:lmerTest", unload=TRUE)

yield_country_nbs_joined_groups_renamed <- yield_country_nbs_joined_groups
names(yield_country_nbs_joined_groups_renamed) <- c('names', 'presences.x', 'PI-ID', 'Group', 'presences2', 'presences.y', 'Yield', 'Yield2', 'Country')
mixed.lmer <- lmer(Yield ~ presences.x + Group + (1|Country), data=yield_country_nbs_joined_groups_renamed)

dwplot(mixed.lmer,
       vline = geom_vline(xintercept = 0, colour = "grey60", linetype = 2))

library(stargazer)
stargazer(mixed.lmer, type = "text",
          digits = 3,
          star.cutoffs = c(0.05, 0.01, 0.001),
          digit.separator = "")

==================================================
                          Dependent variable:     
                     -----------------------------
                                 Yield            
--------------------------------------------------
presences.x                    -0.015**           
                                (0.006)           
                                                  
GroupOld cultivar               -0.245            
                                (0.134)           
                                                  
GroupModern cultivar           0.831***           
                                (0.185)           
                                                  
Constant                       8.760***           
                                (2.466)           
                                                  
--------------------------------------------------
Observations                      741             
Log Likelihood                 -837.565           
Akaike Inf. Crit.              1687.131           
Bayesian Inf. Crit.            1714.779           
==================================================
Note:                *p<0.05; **p<0.01; ***p<0.001
library(ggeffects)
ggpredict(mixed.lmer, terms = c("presences.x",  'Group'), type = "re") %>% 
   plot() +
   theme_minimal_hgrid() +
  xlab('NLR count') +  theme(plot.title=element_blank())

mixed.lmer <- lmer(Yield ~ presences.x + (presences.x|Group) + (1|Country), data=yield_country_nbs_joined_groups_renamed)
summary(mixed.lmer)
Linear mixed model fit by REML ['lmerMod']
Formula: Yield ~ presences.x + (presences.x | Group) + (1 | Country)
   Data: yield_country_nbs_joined_groups_renamed

REML criterion at convergence: 1679.5

Scaled residuals: 
     Min       1Q   Median       3Q      Max 
-3.08887 -0.56698  0.03087  0.65972  2.89927 

Random effects:
 Groups   Name        Variance  Std.Dev.  Corr 
 Country  (Intercept) 2.590e-01 0.5089558      
 Group    (Intercept) 6.794e-01 0.8242671      
          presences.x 4.472e-07 0.0006687 -1.00
 Residual             5.206e-01 0.7215562      
Number of obs: 741, groups:  Country, 40; Group, 3

Fixed effects:
             Estimate Std. Error t value
(Intercept)  9.088288   2.507413   3.625
presences.x -0.015366   0.005566  -2.761

Correlation of Fixed Effects:
            (Intr)
presences.x -0.991
convergence code: 0
boundary (singular) fit: see ?isSingular
ggpredict(mixed.lmer, terms = c("presences.x",  'Group'), type = "re") %>% 
   plot() +
   theme_minimal_hgrid() +
  xlab('NLR count') +  theme(plot.title=element_blank())


sessionInfo()
R version 3.6.3 (2020-02-29)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 17134)

Matrix products: default

locale:
[1] LC_COLLATE=English_Australia.1252  LC_CTYPE=English_Australia.1252   
[3] LC_MONETARY=English_Australia.1252 LC_NUMERIC=C                      
[5] LC_TIME=English_Australia.1252    

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] ggeffects_0.16.0     stargazer_5.2.2      pals_1.6            
 [4] dotwhisker_0.5.0     sjPlot_2.8.6         lme4_1.1-21         
 [7] Matrix_1.2-18        ggforce_0.3.1        ggsignif_0.6.0      
[10] cowplot_1.0.0        dabestr_0.3.0        magrittr_1.5        
[13] ggsci_2.9            patchwork_1.0.0      forcats_0.5.0       
[16] stringr_1.4.0        dplyr_1.0.0          purrr_0.3.4         
[19] readr_1.3.1          tidyr_1.1.0          tibble_3.0.2        
[22] ggplot2_3.3.2        tidyverse_1.3.0      workflowr_1.6.2.9000

loaded via a namespace (and not attached):
  [1] TH.data_1.0-10      minqa_1.2.4         colorspace_1.4-1   
  [4] ellipsis_0.3.1      sjlabelled_1.1.7    rprojroot_1.3-2    
  [7] estimability_1.3    ggstance_0.3.4      parameters_0.9.0   
 [10] fs_1.5.0.9000       dichromat_2.0-0     rstudioapi_0.11    
 [13] glmmTMB_1.0.2.1     hexbin_1.28.1       farver_2.0.3       
 [16] fansi_0.4.1         mvtnorm_1.1-1       lubridate_1.7.9    
 [19] xml2_1.3.2          codetools_0.2-16    splines_3.6.3      
 [22] knitr_1.29          sjmisc_2.8.5        polyclip_1.10-0    
 [25] jsonlite_1.7.1      nloptr_1.2.1        broom_0.5.6        
 [28] dbplyr_1.4.4        effectsize_0.3.0    mapproj_1.2.7      
 [31] compiler_3.6.3      httr_1.4.2          sjstats_0.18.0     
 [34] emmeans_1.4.5       backports_1.1.10    assertthat_0.2.1   
 [37] cli_2.0.2           later_1.1.0.1       tweenr_1.0.1       
 [40] htmltools_0.5.0     tools_3.6.3         coda_0.19-3        
 [43] gtable_0.3.0        glue_1.4.2          maps_3.3.0         
 [46] Rcpp_1.0.5          cellranger_1.1.0    vctrs_0.3.1        
 [49] nlme_3.1-148        insight_0.10.0      xfun_0.17          
 [52] ps_1.3.4            rvest_0.3.5         lifecycle_0.2.0    
 [55] getPass_0.2-2       MASS_7.3-51.6       zoo_1.8-8          
 [58] scales_1.1.1        hms_0.5.3           promises_1.1.1     
 [61] sandwich_2.5-1      RColorBrewer_1.1-2  TMB_1.7.16         
 [64] yaml_2.2.1          stringi_1.5.3       bayestestR_0.7.5   
 [67] boot_1.3-25         rlang_0.4.7         pkgconfig_2.0.3    
 [70] evaluate_0.14       lattice_0.20-41     labeling_0.3       
 [73] processx_3.4.4      tidyselect_1.1.0    plyr_1.8.6         
 [76] R6_2.4.1            generics_0.0.2      multcomp_1.4-13    
 [79] DBI_1.1.0           mgcv_1.8-31         pillar_1.4.4       
 [82] haven_2.3.1         whisker_0.4         withr_2.2.0        
 [85] survival_3.2-3      performance_0.5.1   modelr_0.1.8       
 [88] crayon_1.3.4        utf8_1.1.4          rmarkdown_2.3      
 [91] grid_3.6.3          readxl_1.3.1        blob_1.2.1         
 [94] callr_3.4.4         git2r_0.27.1        reprex_0.3.0       
 [97] digest_0.6.25       xtable_1.8-4        httpuv_1.5.4       
[100] numDeriv_2016.8-1.1 munsell_0.5.0