# Analysis of community ecology data in R

David Zelený

### Site Tools

en:forward_sel_examples

Section: Ordination analysis

## Variable selection (constrained ordination)

### Example 1: tb-RDA with forward selection: Which chemical variables measured in fen water are important for vegetation of Carpathian wetlands?

We will use data about species composition of vascular plants in Carpathian wetlands, and ask which chemical variables, measured in fen water running through these wetlands, are the most important to explain the compositional changes.

First, prepare the data:

# Carpathian wetlands - import data

# transform data using Hellinger transformation to prepare them for tb-RDA:
library (vegan)
vasc.hell <- decostand (vasc, 'hell')

# the last variable in the 'chem' dataset is 'slope', which is not a chemical variable - remove it:
chem1 <- chem[,-15]

Here, we will use transformation-based RDA ordination (tb-RDA). First, we need to test the global model (including all explanatory variables from which you plan to do selection) to see whether it is significant; if it is not, the forward selection should not be done.

tb_rda.all <- rda (vasc.hell ~ ., chem1)
anova (tb_rda.all)  # 0.001 *** - it is significant
adjR2.tbrda <- RsquareAdj (tb_rda.all)$adj.r.squared # 0.2011048 - adjusted R2 explained by all 14 variables is 20.1% #### Option a) Use of forward.sel function The function forward.sel from the package adespatial is elaborated forward selection approach based on linear constrained ordination (i.e. based on RDA - if you want to calculate CCA, you cannot use this function and need to resolve to use ordiR2step from vegan instead). Along to the testing significance of selected variable, this function includes also other stopping rules, published by Blanchet et al. (2008). The method based on significance is prone to be inflated, which may result into models with too many variables; Blanchet et al. (2008) came with another stopping rules along to the significance one. First, adjusted R2 of the model including all variables (from which the selection is made) is calculated. While selecting variables during the selection process, the selection stops if (1) the next selected variables is not significant (does not significantly improve the explained variation), and (2) if the R2adj of the model including this variable exceeds the R2adj of the global model. # install.packages ('adespatial') # run this code if you haven't install the adespatial package before library (adespatial) sel.fs <- forward.sel (Y = vasc.hell, X = chem1, adjR2thresh = adjR2.tbrda) # NOTE: forward.sel calculates only RDA (or tb-RDA in case of Hell. transformed data) Testing variable 1 Testing variable 2 Testing variable 3 Testing variable 4 Testing variable 5 Testing variable 6 Testing variable 7 Testing variable 8 Procedure stopped (alpha criteria): pvalue for variable 8 is 0.118000 (> 0.050000) See the table with selected variables saved in sel.fs object: sel.fs  variables order R2 R2Cum AdjR2Cum F pval 1 Ca 1 0.13898260 0.1389826 0.1263206 10.976337 0.001 2 conduct 14 0.03248342 0.1714660 0.1467337 2.626796 0.001 3 Si 6 0.02722659 0.1986926 0.1622696 2.242529 0.001 4 NH3 10 0.02398868 0.2226813 0.1748463 2.005953 0.002 5 NO3 9 0.02112004 0.2438013 0.1847233 1.787470 0.001 6 Mg 2 0.01914995 0.2629513 0.1927562 1.636862 0.003 7 pH 13 0.01744989 0.2804012 0.1991562 1.503467 0.024 Or, if you want to see only the names of selected variables, subset it from the sel.fs object: sel.fs$variables
[1] "Ca"      "conduct" "Si"      "NH3"     "NO3"     "Mg"      "pH"

Since there is (a potentially high) number of tests of significance during the forward selection procedure, it is better to apply a correction for multiple testing issue. The most common correction (but rather conservative) is Bonferroni, which multiplies each calculated P-value by the overall number of tests. In the forward selection, the overall number of tests (which can be potentially done) equals to the number of variables from which the selection is made (i.e. not the number of selected variables), and this should be considered while applying the adjustment.

The function available for adjusting the P-values is p.adjust. It can be applied on a single P-value or a vector of P-values, and optionally accepts also the argument n indicating the number of tests.

The P-values from the forward selection above are in the object sel.fs$pval, and the adjustement can be done in the following way (Holm's correction): n.tests <- ncol (chem1) # number of tests equals to number of all variables from which is being selected pval.adj <- p.adjust (sel.fs$pval, method = 'holm', n = n.tests)
sel.fs$pval.adj <- pval.adj sel.fs  variables order R2 R2Cum AdjR2Cum F pval pval.adj 1 Ca 1 0.13898260 0.1389826 0.1263206 10.976337 0.001 0.014 2 conduct 14 0.03248342 0.1714660 0.1467337 2.626796 0.001 0.014 3 Si 6 0.02722659 0.1986926 0.1622696 2.242529 0.001 0.014 4 NH3 10 0.02398868 0.2226813 0.1748463 2.005953 0.003 0.033 5 NO3 9 0.02112004 0.2438013 0.1847233 1.787470 0.005 0.050 6 Mg 2 0.01914995 0.2629513 0.1927562 1.636862 0.014 0.126 7 pH 13 0.01744989 0.2804012 0.1991562 1.503467 0.020 0.160 After the correction, the last two variables (Mg and pH) are already not significant. It is possible to help the intrepretation by adding the significance stars (e.g. three stars for P < 0.001). This can be done by many ways; perhaps the simplest is to use the function stars.pval from the package gtools (you need to install it if you haven't used it before): # install.packages ('gtools') sel.fs$pval.adj.stars <- gtools::stars.pval (pval.adj)
sel.fs
  variables order         R2     R2Cum  AdjR2Cum         F  pval pval.adj pval.adj.stars
1        Ca     1 0.13898260 0.1389826 0.1263206 10.976337 0.001    0.014              *
2   conduct    14 0.03248342 0.1714660 0.1467337  2.626796 0.001    0.014              *
3        Si     6 0.02722659 0.1986926 0.1622696  2.242529 0.001    0.014              *
4       NH3    10 0.02398868 0.2226813 0.1748463  2.005953 0.003    0.033              *
5       NO3     9 0.02112004 0.2438013 0.1847233  1.787470 0.005    0.050              *
6        Mg     2 0.01914995 0.2629513 0.1927562  1.636862 0.014    0.126
7        pH    13 0.01744989 0.2804012 0.1991562  1.503467 0.020    0.160    

Here, even the first selected variable has the significance P < 0.05 (one star). The reason is the number of permutations used in the forward.sel function, which by default equals to 999 (argument nperm). The lowest P-value which can be obtained with 999 permutations is 1/(999+1) = 0.001, and the correction will considerably increase this value (to 0.014 in case of 14 variables). The workaround is to increase the number of permutations to be able to get lower P-values, which even after correction could still fall below 0.001 threshold (here using 49,999 permutations to achieve P-value 1/50000 = 0.00002).

sel.fs <- forward.sel (vasc.hell, chem1, nperm = 49999)
pval.adj <- p.adjust (sel.fs$pval, method = 'holm', n = ncol (chem1)) sel.fs$pval.adj <- pval.adj
sel.fs$pval.adj.stars <- gtools::stars.pval (pval.adj) sel.fs  variables order R2 R2Cum AdjR2Cum F pval pval.adj pval.adj.stars 1 Ca 1 0.13898260 0.1389826 0.1263206 10.976337 0.00002 0.00028 *** 2 conduct 14 0.03248342 0.1714660 0.1467337 2.626796 0.00004 0.00052 *** 3 Si 6 0.02722659 0.1986926 0.1622696 2.242529 0.00016 0.00192 ** 4 NH3 10 0.02398868 0.2226813 0.1748463 2.005953 0.00080 0.00880 ** 5 NO3 9 0.02112004 0.2438013 0.1847233 1.787470 0.00304 0.03040 * 6 Mg 2 0.01914995 0.2629513 0.1927562 1.636862 0.00824 0.07416 . 7 pH 13 0.01744989 0.2804012 0.1991562 1.503467 0.02094 0.16752  Since the forward.sel function is not optimised, the calculation with 49,999 permutation take some time, but the reward is the P-values (after correction) below 0.001 in case of the first two variables. You can see that forward.sel function selected (after the correction) five environmental variables, which together explain 18.5% of the variance (adjusted R2, the column AdjR2Cum in the table above showing cumulative adjusted R2 of all variables). This is just slightly less than 20.1% which is explained by global model with 14 variables (see the result of RsquareAdj (rda.all)$adj.r.squared above).

In this case, the results of forward.sel function are the same as ordiR2step and ordistep functions below (option b and c), with seven included environmental variables (if calculated without correction for multiple testing).

#### Option b) Use of ordiR2step function

The vegan's function ordiR2step does similar job as forward.sel from adespatial, but it's use is a bit more complex. On the other side, the method is more general - it allows to use also CCA or db-RDA methods, while forward.sel is based purely on linear constrained ordination (RDA, and tb-RDA in the case that the species composition data are pre-transformed e.g. by Hellinger transformation). In the default setting the criteria for including the variable is based on both significance of the newly selected variables, and the comparison of adjusted variation (R2adj) explained by the selected variables to R2adj explained by the global model (with all variables); if the new variable is not significant or the R2adj of the model including this new variable would exceed the R2adj of the global model, the selection will be stopped.

To use the ordiR2step function, you need to specify two models - the “empty” model containing only intercept, and the “full” model including all variables (aka global model). Also, the argument direction allows for selecting the selection direction (forward, backward, both, with the default to both):

tb_rda.vasc.0 <- rda (vasc.hell ~ 1, data = chem1) # model containing only species matrix and intercept
tb_rda.vasc.all <- rda (vasc.hell ~ ., data = chem1) # model including all variables from matrix chem1 (the dot after tilda (~) means "include all from data")

The function ordiR2step then uses the empty model and the scope (variables) from the full model to proceed the selection. Set direction = 'forward'  to ensure that forward instead of stepwise selection is done, and increse the number of permutations to 999 (instead of default 499) to make results comparable to forward.sel (which by default is based on 999 permutations):

sel.osR2 <- ordiR2step (tb_rda.vasc.0, scope = formula (tb_rda.vasc.all), R2scope = adjR2.tbrda, direction = 'forward', permutations = 999)

Step: R2.adj= 0
Call: vasc.hell ~ 1

<All variables>  0.201104784
+ Ca             0.126320581
+ conduct        0.109418731
+ Mg             0.106926067
+ Corg           0.076260217
+ pH             0.074169805
+ Na             0.043030978
+ NH3            0.031875777
+ Si             0.022863900
+ SO4            0.018929801
+ K              0.015918981
+ NO3            0.008112568
+ Fe             0.004863012
+ PO4            0.002933349
<none>           0.000000000
+ Cl            -0.002347612

Df     AIC      F Pr(>F)
+ Ca  1 -47.149 10.976  0.001 ***
---
Signif. codes:  0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

Call: vasc.hell ~ Ca

<All variables>   0.2011048
+ conduct         0.1467337
+ NH3             0.1417066
+ Na              0.1403837
+ Si              0.1403827
+ pH              0.1403289
+ Mg              0.1386000
+ Corg            0.1375824
+ NO3             0.1340217
+ K               0.1302515
+ Fe              0.1299960
+ SO4             0.1266017
<none>            0.1263206
+ Cl              0.1254155
+ PO4             0.1235944

Df     AIC      F Pr(>F)
+ conduct  1 -47.841 2.6268  0.001 ***
---
Signif. codes:  0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

Call: vasc.hell ~ Ca + conduct

<All variables>   0.2011048
+ Si              0.1622696
+ NH3             0.1592776
+ NO3             0.1572584
+ Mg              0.1565375
+ Corg            0.1538422
+ Na              0.1533884
+ Fe              0.1517167
+ pH              0.1513672
+ K               0.1511453
+ SO4             0.1495485
+ Cl              0.1470011
<none>            0.1467337
+ PO4             0.1444723

Df    AIC      F Pr(>F)
+ Si  1 -48.18 2.2425  0.002 **
---
Signif. codes:  0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

Call: vasc.hell ~ Ca + conduct + Si

<All variables>   0.2011048
+ NH3             0.1748463
+ NO3             0.1739710
+ Mg              0.1708815
+ Corg            0.1689184
+ pH              0.1667252
+ SO4             0.1666206
+ Na              0.1661906
+ Fe              0.1655162
+ K               0.1653789
<none>            0.1622696
+ Cl              0.1620351
+ PO4             0.1599773

Df     AIC     F Pr(>F)
+ NH3  1 -48.308 2.006  0.001 ***
---
Signif. codes:  0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

Call: vasc.hell ~ Ca + conduct + Si + NH3

<All variables>   0.2011048
+ NO3             0.1847233
+ Mg              0.1835775
+ SO4             0.1799079
+ pH              0.1798906
+ Corg            0.1793391
+ Fe              0.1786200
+ K               0.1766585
+ Na              0.1756240
<none>            0.1748463
+ Cl              0.1737180
+ PO4             0.1725072

Df     AIC      F Pr(>F)
+ NO3  1 -48.236 1.7875  0.005 **
---
Signif. codes:  0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

Call: vasc.hell ~ Ca + conduct + Si + NH3 + NO3

<All variables>   0.2011048
+ Mg              0.1927562
+ pH              0.1907475
+ Corg            0.1890699
+ SO4             0.1876281
+ Fe              0.1869468
+ K               0.1868746
+ Na              0.1848251
<none>            0.1847233
+ Cl              0.1834910
+ PO4             0.1826904

Df     AIC      F Pr(>F)
+ Mg  1 -48.032 1.6369  0.011 *
---
Signif. codes:  0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

Call: vasc.hell ~ Ca + conduct + Si + NH3 + NO3 + Mg

<All variables>   0.2011048
+ pH              0.1991562
+ Corg            0.1963215
+ SO4             0.1958876
+ K               0.1956246
+ Fe              0.1953612
<none>            0.1927562
+ Na              0.1922729
+ Cl              0.1915194
+ PO4             0.1907648

Df     AIC      F Pr(>F)
+ pH  1 -47.709 1.5035  0.028 *
---
Signif. codes:  0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

Call: vasc.hell ~ Ca + conduct + Si + NH3 + NO3 + Mg + pH

+ Corg            0.2023822
+ Fe              0.2015249
+ K               0.2014550
+ SO4             0.2012822
<All variables>   0.2011048
<none>            0.1991562
+ Na              0.1988753
+ PO4             0.1974424
+ Cl              0.1974018
sel.osR2
Call: rda(formula = vasc.hell ~ Ca + conduct + Si + NH3 + NO3 + Mg + pH, data = chem1)

Inertia Proportion Rank
Total          0.5674     1.0000
Constrained    0.1591     0.2804    7
Unconstrained  0.4083     0.7196   62
Inertia is variance

Eigenvalues for constrained axes:
RDA1    RDA2    RDA3    RDA4    RDA5    RDA6    RDA7
0.09590 0.01904 0.01205 0.01027 0.00871 0.00718 0.00595

Eigenvalues for unconstrained axes:
PC1     PC2     PC3     PC4     PC5     PC6     PC7     PC8
0.03601 0.03279 0.02383 0.01908 0.01687 0.01593 0.01504 0.01358
(Showed only 8 of all 62 unconstrained eigenvalues)

The summary table is part of the ordiR2step function output, stored in the component $anova: sel.osR2$anova
                 R2.adj Df     AIC       F Pr(>F)
+ Ca            0.12632  1 -47.149 10.9763  0.001 ***
+ conduct       0.14673  1 -47.841  2.6268  0.001 ***
+ Si            0.16227  1 -48.180  2.2425  0.002 **
+ NH3           0.17485  1 -48.308  2.0060  0.001 ***
+ NO3           0.18472  1 -48.236  1.7875  0.005 **
+ Mg            0.19276  1 -48.032  1.6369  0.011 *
+ pH            0.19916  1 -47.709  1.5035  0.028 *
<All variables> 0.20111
---
Signif. codes:  0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

The result is exactly the same as results of forward.sel function and also as results of ordistep method based on P-values and AIC criteria (below). P-values can be adjusted for multiple testing (e.g. by Holm's correction) as follows:

sel.osR2_adj <- sel.osR2
sel.osR2_adj$anova$Pr(>F) <- p.adjust (sel.osR2$anova$Pr(>F), method = 'holm', n = ncol (chem1))
sel.osR2_adj$anova  R2.adj Df AIC F Pr(>F) + Ca 0.12632 1 -47.149 10.9763 0.014 * + conduct 0.14673 1 -47.841 2.6268 0.014 * + Si 0.16227 1 -48.180 2.2425 0.022 * + NH3 0.17485 1 -48.308 2.0060 0.014 * + NO3 0.18472 1 -48.236 1.7875 0.050 * + Mg 0.19276 1 -48.032 1.6369 0.099 . + pH 0.19916 1 -47.709 1.5035 0.224 <All variables> 0.20111 --- Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 You may consider to increase the number of permutations (argument permutations in ordiR2step to reach lower P-values; in the forward.sel example above we increased the permutations to 49,999 to make sure that even after adjustement of P-values the most significant one could be smaller than 0.001. #### Option c) Use of ordistep function Function ordistep performs step-wise selection of environmental variables based two criteria: if their inclusion into the model leads to significant increase of explained variance (the same as in forward.sel and ordiR2step), and if the AIC of the new model is lower than AIC of the more simple model. In contrary to previous functions, the function does not consider as criteria whether the adjusted R2 of the model exceeds the adjusted R2 of the global model. The use of ordistep function has the same logic as ordiR2step. rda.vasc.0 <- rda (vasc.hell ~ 1, data = chem1) # model containing only species matrix and intercept rda.vasc.all <- rda (vasc.hell ~ ., data = chem1) # model including all variables from matrix chem1 (the dot after tilda (~) means ALL!) sel.os <- ordistep (rda.vasc.0, scope = formula (rda.vasc.all), direction = 'forward') Start: vasc.hell ~ 1 Df AIC F Pr(>F) + Ca 1 -47.149 10.9763 0.005 ** + conduct 1 -45.808 9.4775 0.005 ** + Mg 1 -45.612 9.2612 0.005 ** + Corg 1 -43.249 6.6964 0.005 ** + pH 1 -43.091 6.5277 0.005 ** + Na 1 -40.775 4.1026 0.005 ** + NH3 1 -39.964 3.2718 0.005 ** + Si 1 -39.316 2.6145 0.005 ** + SO4 1 -39.034 2.3314 0.005 ** + K 1 -38.820 2.1162 0.010 ** + NO3 1 -38.267 1.5643 0.060 . + Fe 1 -38.038 1.3372 0.105 + PO4 1 -37.902 1.2030 0.150 + Cl 1 -37.532 0.8384 0.700 --- Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 Step: vasc.hell ~ Ca Df AIC F Pr(>F) + conduct 1 -47.841 2.6268 0.005 ** + NH3 1 -47.430 2.2190 0.005 ** + pH 1 -47.318 2.1081 0.005 ** + Na 1 -47.322 2.1125 0.010 ** + Si 1 -47.322 2.1124 0.010 ** + Mg 1 -47.177 1.9694 0.010 ** + Corg 1 -47.095 1.8880 0.010 ** + NO3 1 -46.806 1.6047 0.035 * + K 1 -46.502 1.3073 0.110 + Fe 1 -46.482 1.2873 0.115 + SO4 1 -46.209 1.0219 0.435 + Cl 1 -46.114 0.9296 0.590 + PO4 1 -45.968 0.7885 0.890 --- Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 Step: vasc.hell ~ Ca + conduct Df AIC F Pr(>F) + Si 1 -48.180 2.2425 0.005 ** + NH3 1 -47.931 1.9997 0.005 ** + NO3 1 -47.763 1.8367 0.005 ** + Mg 1 -47.703 1.7788 0.005 ** + Corg 1 -47.480 1.5629 0.020 * + Na 1 -47.442 1.5266 0.035 * + K 1 -47.257 1.3482 0.040 * + pH 1 -47.275 1.3658 0.050 * + SO4 1 -47.125 1.2218 0.085 . + Fe 1 -47.304 1.3936 0.095 . + Cl 1 -46.916 1.0210 0.420 + PO4 1 -46.709 0.8229 0.810 --- Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 Step: vasc.hell ~ Ca + conduct + Si Df AIC F Pr(>F) + NH3 1 -48.308 2.0060 0.005 ** + NO3 1 -48.234 1.9350 0.005 ** + Mg 1 -47.972 1.6855 0.020 * + Corg 1 -47.807 1.5280 0.025 * + SO4 1 -47.614 1.3446 0.055 . + pH 1 -47.622 1.3529 0.075 . + Na 1 -47.577 1.3104 0.095 . + Fe 1 -47.521 1.2568 0.105 + K 1 -47.509 1.2459 0.130 + Cl 1 -47.229 0.9815 0.470 + PO4 1 -47.058 0.8199 0.805 --- Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 Step: vasc.hell ~ Ca + conduct + Si + NH3 Df AIC F Pr(>F) + NO3 1 -48.236 1.7875 0.005 ** + Mg 1 -48.138 1.6951 0.005 ** + SO4 1 -47.824 1.4012 0.035 * + pH 1 -47.822 1.3998 0.045 * + Corg 1 -47.775 1.3558 0.045 * + Fe 1 -47.714 1.2986 0.125 + K 1 -47.547 1.1431 0.235 + Na 1 -47.459 1.0613 0.370 + Cl 1 -47.298 0.9112 0.665 + PO4 1 -47.195 0.8163 0.805 --- Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 Step: vasc.hell ~ Ca + conduct + Si + NH3 + NO3 Df AIC F Pr(>F) + Mg 1 -48.032 1.6369 0.010 ** + pH 1 -47.858 1.4764 0.040 * + Corg 1 -47.713 1.3430 0.040 * + SO4 1 -47.588 1.2288 0.135 + Fe 1 -47.530 1.1750 0.170 + K 1 -47.523 1.1693 0.210 + Na 1 -47.347 1.0080 0.480 + Cl 1 -47.233 0.9034 0.600 + PO4 1 -47.164 0.8408 0.785 --- Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 Step: vasc.hell ~ Ca + conduct + Si + NH3 + NO3 + Mg Df AIC F Pr(>F) + pH 1 -47.709 1.5035 0.040 * + SO4 1 -47.424 1.2453 0.095 . + Corg 1 -47.462 1.2795 0.100 . + Fe 1 -47.378 1.2040 0.115 + K 1 -47.401 1.2247 0.135 + Na 1 -47.110 0.9623 0.580 + Cl 1 -47.044 0.9036 0.650 + PO4 1 -46.979 0.8450 0.775 --- Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 Step: vasc.hell ~ Ca + conduct + Si + NH3 + NO3 + Mg + pH Df AIC F Pr(>F) + Corg 1 -47.130 1.2508 0.140 + Fe 1 -47.054 1.1839 0.150 + K 1 -47.048 1.1785 0.175 + SO4 1 -47.033 1.1650 0.185 + Na 1 -46.823 0.9783 0.595 + PO4 1 -46.697 0.8676 0.715 + Cl 1 -46.694 0.8645 0.735 Results are stored in sel.os object: sel.os Call: rda(formula = vasc.hell ~ Ca + conduct + Si + NH3 + NO3 + Mg + pH, data = chem1) Inertia Proportion Rank Total 0.5674 1.0000 Constrained 0.1591 0.2804 7 Unconstrained 0.4083 0.7196 62 Inertia is variance Eigenvalues for constrained axes: RDA1 RDA2 RDA3 RDA4 RDA5 RDA6 RDA7 0.09590 0.01904 0.01205 0.01027 0.00871 0.00718 0.00595 Eigenvalues for unconstrained axes: PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 0.03601 0.03279 0.02383 0.01908 0.01687 0.01593 0.01504 0.01358 (Showed only 8 of all 62 unconstrained eigenvalues) Selected variables are Ca, conduct, Si, NH3, NO3, Mg and pH. ### Example 2: Which soil chemical variables are important for vegetation of tropical forest? (Barro Colorado Island) Here we will use the dataset from the forest dynamics plot in tropical rainforest on Barro Colorado Island (Panama), namely data about woody species composition (dataset BCI in library vegan) and chemical variables from soil samples (BCI.soil). We will use forward selection to select the subset of soil variables that are important for explaining species composition and to plot the resulting ordination diagram. Since the plot is a regular grid of 10 columns and 5 rows, we need to also choose an appropriate randomization schema when testing the explained variation. Read the data about composition of woody species (dataset BCI in vegan) and upload also data about soil variables (from the dataset description). Since species composition data are represented by the number of individuals of given species in the subplot (100 m x 100 m), we log-transform them before the analysis to reduce the importance of dominant species. We also calculate DCA on the species composition data to see how heterogeneous they are. library (vegan) data (BCI) BCI.soil <- read.delim ('https://raw.githubusercontent.com/zdealveindy/anadat-r/master/data/bci.soil.txt') BCI.spe.log <- log1p (BCI) # log-transform the species data in BCI BCI.soil2 <- BCI.soil[,-1:-2] decorana (BCI.spe.log) # The length of DCA1 is 1.37 = homogeneous dataset! From DCA we see that the dataset is homogeneous, and we can use linear constrained ordination method (RDA). Define global model (including all explanatory variables we are gonna to select from), and test its significance (only if significant, we can do forward selection). When doing the permutation model in the function anova, we need to specify how to permute the samples. Since subplots are sampled in contiguous regular grid with equal distances between neighbours in the same row or column, they are spatially dependent (closer samples are more likely to be similar to each other than distant ones), and we need to preserve this dependence in the permutated data. We will choose the toroidal shift permutation for the grid (see theory), with the appropriate size of the grid (10 columns, 5 rows, see the schema below). rda_all <- rda (BCI.spe.log ~ ., BCI.soil2) anova (rda_all, permutations = how (within = Within (type = 'grid', ncol = 10, nrow = 5, mirror = TRUE), complete = TRUE)) # Permutation test for rda under reduced model # Permutation: grid mirrored 5 rows 10 columns # Number of permutations: 199 # # Model: rda(formula = BCI.spe.log ~ Al + B + Ca + Cu + Fe + K + Mg + Mn + P + Zn + N + N.min. + pH, data = BCI.soil2) # Df Variance F Pr(>F) # Model 13 24.838 2.3429 0.005 ** # Residual 36 29.358 # --- # Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 adjR2 <- RsquareAdj (rda_all)$adj.r.squared
adjR2  # 0.2626876

To continue with forward selection, we need to in addition to the full model rda_all define also the null model (with no predictor), rda_0:

rda_0 <- rda (BCI.spe.log ~ 1, data = BCI.soil2)  # includes only intercept

Function ordiR2step starts from the model with only intercept to full model; important is the argument scope, which indicates the list of variables from which the selection is done; the scope can be taken from the full model using the function formula; also, the argument R2scope should include the adjusted R2 of the general (whole) model as another stopping criteria of the selection.

rda_sel <- ordiR2step (rda_0, scope = formula (rda_all), direction = 'forward', R2scope = adjR2,
permutations = how (within = Within (type = 'grid', ncol = 10, nrow = 5,
mirror = TRUE), complete = TRUE))
rda_sel
# Call: rda(formula = BCI.spe.log ~ pH + P + Zn + Cu + Fe + Mn, data = BCI.soil2)
#
# Inertia Proportion Rank
# Total         54.1967     1.0000
# Constrained   18.1174     0.3343    6
# Unconstrained 36.0793     0.6657   43
# Inertia is variance
#
# Eigenvalues for constrained axes:
#   RDA1  RDA2  RDA3  RDA4  RDA5  RDA6
# 5.816 5.601 2.771 1.590 1.360 0.981
#
# Eigenvalues for unconstrained axes:
#   PC1   PC2   PC3   PC4   PC5   PC6   PC7   PC8
# 3.190 2.750 2.150 1.833 1.477 1.386 1.359 1.240
# (Showing 8 of 43 unconstrained eigenvalues)

rda_sel$anova # R2.adj Df AIC F Pr(>F) # + pH 0.073892 1 197.75 4.9096 0.005 ** # + P 0.132305 1 195.44 4.2314 0.005 ** # + Zn 0.176470 1 193.75 3.5206 0.005 ** # + Cu 0.210740 1 192.53 2.9973 0.005 ** # + Fe 0.229719 1 192.19 2.1087 0.005 ** # + Mn 0.241399 1 192.28 1.6775 0.010 ** # <All variables> 0.262688 # --- # Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 The “anova” table above contains the column R2.adj with adjusted R2 explained by that variable (for pH) or with adjusted R2explained by given variable in addition to all previous ones included in the model (e.g. for P it is variation explained by P in addition to the variation already explained by pH). The problem of the table is inflated Type I error rate for the P-values, caused by multiple testing issue. We need to adjust them (here by False Discovery Rate, FDR, correction): rda_sel$anova$Pr(>F) <- p.adjust (rda_sel$anova$Pr(>F), method = 'fdr', n = ncol (BCI.soil2)) rda_sel$anova
# R2.adj Df    AIC      F  Pr(>F)
# + pH            0.073892  1 197.75 4.9096 0.01300 *
# + P             0.132305  1 195.44 4.2314 0.01300 *
# + Zn            0.176470  1 193.75 3.5206 0.01300 *
# + Cu            0.210740  1 192.53 2.9973 0.01300 *
# + Fe            0.229719  1 192.19 2.1087 0.01300 *
# + Mn            0.241399  1 192.28 1.6775 0.02167 *
# <All variables> 0.262688
# ---
# Signif. codes:  0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

After the adjustement, we see that all results became less significant (P < 0.05). This is the cost for the fact that grid based randomization schema with 10 x 5 = 50 subplots returns (after allowing for mirroring) only 50 x 4 = 200 permutations (this including also the original, non-permuted data), and the miminal P-value is then 1/200 = 0.005. After FDR adjustment for 13 permutation tests (since there is 13 variables to select from), the P-values considerably increased (0.005 after adjustement becomes 0.013). But still all six selected variables remained significant.

Finally, we can draw the ordination diagram (using rda_sel ordination object created by forward selection process):

ordiplot (rda_sel, display = c('sites', 'bp'), type = 't')

Truly interesting is that there seem to be two rather strong and almost perpendicular environmental gradients - pH (correlated also with Zn, Fe, Mn and Cu) vs P. Also, if we check the eigenvalues of the first two ordination axes (RDA1 and RDA2 in RDA_sel), both of them explain a high proportion of variation (variation 5.8 and 5.6, respectively, out of total variation of 54.2). I am not familiar with the locality of BCI, but I know that there is a small depression in the western part of the plot, which sometimes gets flooded; I guess this is where the values of P are lower (samples 13 and 18 in the ordination diagram); for the distribution of pH in the plot, check dataset description).