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(if my question should be cut up into sub-questions please let me know, since all those questions are related I decided to ask them here together as one long question)

Main question
As part of my research I try to perform multivariate analysis in order to establish which environmental factors are important in explaining the vegetation species composition over time in a newly constructed wetland (with 27 different basins, each sampled yearly for 4 years).
Apart from that, I aim to analyse whether those environmental factors change over time, and which of them influence biomass production 4 years after establishment of the wetland. For those last two questions I know how to perform the analyses, but for the multivariate analyses I am not sure which test to choose, how to interpret the results and which statistics to report in my manuscript.
Since I perform all my statistical analyses in R, I would like to also perform the multivariate analyses in R. I have read up on the vegan-package and think it contains all I need, but I cannot figure out some of the details.

Overview of my data
My species-data are Tansley-classes (abundance classes 0-9), with 77 species and 108 observations (27 basins * 4 years).
My environmental-data consist of two groups: sediment samples and water samples. The sediment samples were only taken in the last year (27 observations, 19 vector-variables, 2 factor-variables), the water samples were taken each year (108 observations, 40 vector-variables, 2 factor-variables, 1 column with 'year' as vector-variable).

  1. If I want to include the sediment samples in my multivariate analyses, can I simply use the same numbers for all years? If not, does this mean I cannot use them, since I don't have numbers for all rows in my species-data?
  2. I had some NA's for the water samples and got some problems when trying out some MVA-tests. Therefore, I filled them up with the average number per variable for the same basin. Are there any strong reasons why I should not do this?
  3. I am not sure whether or not to use 'year' as a variable for the environment. I measured everything over time, so it feels right to take it into account, but should I do this by taking it as a vector-variable? And should I then only use it in CCA, or also in CA with ENVFIT? I think I should only use 'year' in CCA, so I will still see which other factors influence species composition.

Tests which I tried with example data
To show you what I tried, I will first supply some example data (with species-data in spec (containing classes 0-9), and environment-data in env (containing numbers, factor, and year)):

data(dune)
spec = rbind(dune,dune)
spec = spec[1:36,]
v = paste0(rep(LETTERS[1:9], each=4), rep(1:4,9))
row.names(spec) = v
env = data.frame(replicate(40,sample(0.0:20.0,36,rep=TRUE)))
env = env/10
row.names(env) = v
env$fac1 = as.factor(rep(c("gr1","gr2","gr3"), each=12))
env$year = as.numeric(rep(1:4,9))
env_excl_year = subset(env, select = -year)

I think I should do the following with my data:

  1. Perform an unconstrained correspondence analysis (or RDA, how to choose?)
  2. Fit environmental vectors to the ordination
  3. Use significant environmental factors in constrained model
  4. Make useful plots

This is what I did in R (with questions in between):

library(vegan)
spec.ca = cca(spec)
spec.ca

What should I report from spec.ca? Since the inertia is mean squared contingency coefficient (instead of variance or correlation), I think it doesn't make sense to report the Eigenvalues. Or should I use: spec.rda = rda(spec, scale=TRUE)? With inertia being correlation, where it does make sense to report Eigenvalues?

ef_ca = envfit(spec.ca, env_excl_year, permu=999) #without year
ef_ca
(scores_ef_ca = scores(ef_ca,"vectors"))

What should I report from ef_ca? Why are the scores different from the number I see when inspecting ef_ca? Can I indeed use R2 as "the proportion of variation in species composition explained by this variable"? And can I use the p-value to establish whether the variable can explain species composition (with p<0.05: H0 of no explanation by this variable rejected)?

In my case, X13 and year had p<0.05 (since the data is made randomly, you may have a different outcome). I would also like to know more about 'fac1' (with my real data, this one also has p<0.05). If I would like to say more about those variables, specifically about how they can explain the development of the species composition in the different basins (see research question), should I add those to a constrained model (cca), inclusing year (which was not included in ca, was this a good decision?)?

spec.cca = cca(spec ~ X13 + year + fac1, env)
spec.cca
anova(spec.cca)
RsquareAdj(spec.cca)
anova(spec.cca, by="term", step=200)   #Type-I: sequential test, dependent on order
anova(spec.cca, by="margin", step=500) #Type-III: marginal effects, independent of order
anova(spec.cca, by="axis", step=1000)  #test of individual axes

And should I then report on the F- and p-value of the whole model, with R2, and F- and p-value for separate terms? For the terms: should I use "term" or "margin", and why (results differ)? How do you choose the amount for step? Should I perform VIF or correlation analysis to exclude certain variables from my analyses? What is a good rule of thumb to use here for exclusion?

I know you can also perform cca with all variables or using forward or backward selection.

spec.cca.all = cca(spec ~ ., env)
spec.cca.all
spec.cca.start = cca(spec ~ 1, env)
spec.cca.forward = step(spec.cca.start, scope = formula(spec.cca.all), test = "perm")
spec.cca.forward
spec.cca.backward = step(spec.cca.all, scope = list(lower=formula(spec.cca.start), upper = formula(spec.cca.all)), trace = 0)
spec.cca.backward

When I did this with forward selection, I got exactly the same model as when choosing variables by envfit p<0.05. Is this a coincidence, or logical? Which is preferred: using envfit or forward selection? I guess envfit, because I think the exact same results was just random, but I am not sure.

I would like to plot my data in a clear way. However, my plots tend to get a bit crowded. Also, I am not sure when to use which scaling (I think scaling = 1 means sites are weighted averages of species, scaling = 2 means species are weighted averages of sites, but then which one do you choose and why?). If I plot cca, the plot is immediately very crowded, if I plot ca I have a bit more freedom.

plot(spec.cca) #very crowded, seems not to be adaptable
plot(spec.ca, scaling=2) #less crowded, and adaptable
p1 = plot(spec.ca, scaling=2, type="n")
p1_sel = orditorp(spec.ca, display="species",labels = names(spec), pcol="green", pch="+")
p1_sel = orditorp(spec.ca, display="sites",labels = row.names(spec), pcol="blue", pch="o")
plot(ef_ca, p.max=0.05, col = "red")
with(env, ordispider(spec.ca, fac1, col=c("orange","purple","magenta"), label=T))

Can I adapt the cca-plot to be less crowded? Can I just also use scaling = 1 in the code above for ca? I am not sure if I can do this, since ef_ca keeps plotting the arrows in the same direction, even though the arrangement of species and sites differs. Probably I should just make multiple plots, each containing a part of the information. Any hints are very welcome!

My species-data can be grouped in three groups, based on another data.frame, say veg_groups = as.data.frame(t(rbind(names(spec),rep(c("sp1","sp2","sp3"), each=10)))). Can I also use this in my plots, say coloring the species in three different colours depending on veg_groups? How do I do this?

Thank you all very much in advance for helping me out, even if it is only with a little part of my question.

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