Independent Analysis II
Alexa Hershberger Methods
4/26/2021
Benthic Macroinvertebrate Community
Assemblage in the Bear Run Watershed
Objective
The data selected for this study is
from my undergraduate pilot study in the
Bear Run Watershed. This project is funded
by the Susquehanna River Basin
Commission. Acid mine drainage (AMD) is
a result of abandoned coal mines and refuse
piles. Bear Run Watershed is looted with
abandoned coal mines that have not been
properly treated once the company left. The
untreated mining soil releases heavy trace
metals into the stream that is detrimental to
different wildlife. In 2005, the Susquehanna
River Basin Commission conducted a nine-
phase AMD restoration project in the
Watershed. The restoration improved the
water quality and increased fish species
richness, but the recovery of the
macroinvertebrate community has been
slow, perhaps due to the poor habitat
quality. This pilot study examined the
chlorophyl a abundance, environmental
variables, and macroinvertebrate abundance,
but I will only focus on the
macroinvertebrate abundance for this
analysis. Our goal was to determine how
substrate type and site impairment affects
the macroinvertebrate recovery in the Bear
Run Watershed. We predicted that the
recovery would be stronger on substrates
lacking an iron residue and other
precipitates, thus a higher abundance on the
artifical substrate and control sites.
1
Sample Design
This study was conducted in the Bear
Run Watershed in northern Indiana County.
It is one of the largest AMD impact zones to
the headwaters section of the Susquehanna
River. The original data had six sites. Of
these six sites, there were three AMD sites
and three control/remediated sites. Each site
also had three Hester-Dendy Samplers to
replicate the artifical substrate and three
Hess Sampling collections to replicate the
natural substrate. To avoid pseudo-
replication, I combined the three subsamples
for each substrate type into two measures
per site. So, there was one measurement for
the artificial substrate macroinvertebrate
community and one measurement for the
natural substrate macroinvertebrate
community for each site.
Figure 1. Macroinvertebrate sampling methods. (Hester-
Dendy sampler on left and Hess sampler on
right)
The sample locations were chosen by the
project manager at the Susquehanna River
Basin Commission and Dr. Janetski. The
sites were based off a previous thesis student
in Dr. Janetski’ s lab. The environmental
variables collected at each site was:
substrate type, impairment, pH, water
temperature in Celsius, and conductivity.
The collected individuals were stored in
80% ethyl alcohol and identified to the order
taxonomic level.
Analysis
I based my analysis off the AW09,
AW10, and AW11 worksheets. To first
understand my dataset, I wanted to conduct
a community analysis examining the
patterns within the entire species
assemblage. In the species assemblage data,
there were 6 locations in Bear Run and a
total of ten species. I wanted to also explore
the beta diversity because it would compare
changes in macroinvertebrate communities Figure 1. Histogram of overall abundance across sites.
between sampled locations. The result of
the beta diversity will be used to analyze the The histogram for overall abundance is
second part of my hypothesis in comparing significantly skewed to the right. There are
community structure between AMD and two outlier species that has a higher
control sites. The third analysis workshop frequency compared to the others (Figure 1).
will link my community results to my There is evenness in this community expect
environmental data. This will be used to for the two species. I would expect to see a
look at the effect of substrate type of dominant species in this community, which I
macroinvertebrate community structure. The would assume is the Diptera and Plecoptera,
R packages used in this analysis were: readr, because they were listed as the most
here, dplyr, tidyr, tibble, ggplot2, betapart, abundant genera.
and vegan. I then looked at species occurrence
in the population. The total occurrence of
Results the most abundant species and least
The first step in my analysis was to abundance species is show in Table 1.
complete a species assemblages diversity Table 1. Species Occurrence in the Bear Run Watershed.
analysis. The main intention of this part of
the analysis was to examine different Species Species Occurrence
patterns throughout the entire species
assemblage. Diversity, species richness and Plecoptera 12
species evenness were all analyzed in this Ephermeroptera 11
portion. The macroinvertebrate counts were
from six locations in the Bear Run Trichoptera 10
Watershed. There was a total of 10 species.
The most abundant species were Diptera Diptera 10
(123), Plecoptera (114), Coleoptera (26),
and Annelida (22). The least abundant Annelida 9
species were Trichoptera (19), Megaloptera Coleoptera 8
(30), Tipuloidea (2), Pterygota (1), and
Copepod (1). Megaloptera 3

Tipuloidea 2
2
 Pterygota
Copepod
I then used the computed Shannon Diversity
to determine the biological significance of
differences in diversity. I chose the
Shannon Diversity Index because it is the
default index. This index reflects the
balance between species richness and
species evenness.
Figure 3. Histogram of effective species unit from Shannon
Diversity Index.
The range of the effective number of species
across the sample locations is from
approximately 3 to 5 (Figure 3).
The third part of this initial analysis was to
examine the rarefaction curve to look how
the species diversity varies from each site.
This may have been skewed due to the
difference in water quality between the
AMD and control streams. I added the
species rarefaction analysis to address any
detection probability of low abundance
issues in my study.
1
1
Figure 4. Rarefaction curve of species richness.
The results from the rarefaction curve
suggest that the minimum sample size from
our data set should be approximately 20
individuals. The trend does not appear to
fully stabilize once all the species have been
encountered (Figure 4). This does raise
some concern, but I am not sure how to
address it. I would assume that if I increase
my sample size, I should have a more
representative sample. This should stabilize
the species rarefaction curve.
Next, I looked at the gamma
diversity and species accumulation curves.
The species accumulation curve will look at
site level variation, which should provide a
more adequate comparison between AMD
and control sites compared to the rarefaction
curve. The gamma diversity is the
relationship between the overall observed
species richness/diversity based on the six
sites sampled. Based on the species
accumulation plot, I should encounter more
species if I sampled an addition 6 locations
(Figure 5). The curve appears to flatten out
the most at approximately 12 sites. I will be
interesting to see how the species
accumulation curves compare when I have
16 sites.
3

Figure 5. Species accumulation plot.
I then computed the beta diversity
for the dataset. The reason I computed this
was because I wanted to examine the
macroinvertebrate species assemblages
between sites. The nested patterns for beta
diversity were tested to detect whether
species loss was random or if it occurred in
a predictable pattern. This is especially
important in AMD sites.
Figure 6. Nested temperature plot.
In the nested temperature plot, most
of the red appears on the left side of the
curve. This shows that there is a strong
nested pattern. There are also a few rare
species indicated on the right side of the
curve (Figure 6). I then compared the
observed community to a pattern from the
random simulation of species across the sites
where the number of species occurrences
were held constant and the number of
species per site were held constant.
4
Table 2. Observed Community Simulation.
Statistic SES Mean Prob.
22.6 21.2 21.2 0.27
The p-value of the observed
community simulation is not significant, so I
know that this is equivalent to a random
structure (Table 2). If the p-value were
statistically significant, it would be a nested
design because of the null model is random.
The second portion of the second
large analysis was reviewing the
unconstrained ordination. The principle
component analysis relies on a Euclidean
distance.
Principle component one accounts for
approximately 40% of the variation in the
original dataset. The proportion explained
drastically drops after PC4. PC1 through
PC4 account for approximately 90% of the
variation.
Table 3. Kaiser-Guttman Criterion
PC1 PC2 PC3
0.114 0.061 0.026
According to the Kaiser-Gutman criterion
PC1, PC2, and PC3 should be retained
(Table 3).
 variation in the dataset. The points are fairly
close to the line, so it is a decent fit (Figure
9).

Figure 7. Biplot of community assemblage based on PC1

and PC2 with a scaling of 2.

The plotted PCA was based on Figure 9. Stress plot of non-metric multidimensional
scaling.
method 2. The species that is most strongly
represented by PC1 appears to be Diptera (~ Next, I added categorical
0.5). The sites that appear the most different environmental descriptions to the
is site 7 and site 12. They are both control unconstrained ordination plot.
sites but differ as artificial and natural
substrate. The species that is most strongly
represented by PC2 was Annelida (0.4).
The two sites that appear the most different
is site 3 and site 5. This is interesting
because both sites represent AMD sites on
natural substrate. Based on the eigenvalues,
CA1, CA2, and CA3 should be included in
the correspondence analysis (Figure 8).

Figure 10. Unconstrained ordination plot with

environmental variables.

The two most distinct communities are

Pterygota and Copepod. This may be
Figure 8. Correspondence analysis of eigenvalues. skewed because there was only one
Copepod found in the samples. The two
To determine the rank rather than the actual
most similar communities are Diptera and
values, I conducted a non-metric
Annelida because they are overlapping. This
multidimensional scaling, which uses a non-
makes sense because both species are
parametric form of ordination. The stress
pollution-tolerant (Figure 10).
plot showed that the NMDS does an
adequate job of characterizing the original

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 Lastly, I conducted a post hoc in the variables of substrate type,
comparison of the environmental variables temperature, and pH. The r-squared value
to unconstrained ordination via non-metric was also the same for all three approaches.
multidimensional scaling. This indicated that it did not matter what
approach I chose.
The ordination of this dataset was
examined by the variance, eigenvalues, and
accumulated constrained eigenvalues. The
original variation in the data set that is
captured in the first two constrained
components is about 0.55 (RDA1 + RDA2).
The amount of additional information that is
provided by the third constrained component
is approximately 0.035, which is not that
much.
I then made a triplot that displays my
Figure 11. Post Hoc comparison of environmental sites, species, and environmental variables
variables to unconstrained ordination.
from my Redundancy Analysis Model with
The variables that are almost contours.
perfectly aligned with NMDS axis 2 are the
substrate type and temperature. The
variable that appears to be the strongest
when explaining differences in species
communities is substrate type. The variable
that appears to change most similarly across
the sites is conductivity and pH. I then used
a redundancy analysis, which is a form of
multivariate linear regression. The amount
of variation that is explained by the
constrained portion of the ordination was
approximately 50% (r-squared = 0.73,
adjusted r-squared = 0.5). The variables that
have high variance inflation factors are
water temperature and conductivity. Figure 12. RDA plot of environmental variables.

There was no difference in the r- Substrate type and pH appear to be most

squared values in the apriori models and the
redundancy analysis. To determine which
variables to include in the model, I used the
step-wise variable selection approach. I will
run the forward selection, backward
selection, and forward-backward selection.
The outcome for each of the selection
approaches was the same. They all resulted
6
strongly related to the first axis because they
are the most parallel to the axis. All the
variables have a non-linear relationship with
the contour curves (Figure 12).
Conclusion
The results of this analysis support
my prediction that substrate type influences
the macroinvertebrate community.
Temperature and pH both have an influence
on the community assemblage which is
expected. It is interesting that pH has an
influence while site type does not. I would
predict that there would be a different
community present at the AMD sites. A
literature search indicated that there should
be a difference. It is possible that my
sample size was not large enough to pick up
the difference in site type. I will increase
my site number for my thesis, so this should
solidify my results. I will also add a
sediment analysis to my thesis to better
understand how the substrate plays a role on
macroinvertebrate community.
Code Chunks
tot.occur <- macro_BRW.dat %>%
summarise_each(list(~sum(.>0))) %>%
t %>% data.frame %>% arrange(desc(.))
tot.occur %>%
head(5) # 5 most abundnant species
tot.occur %>%
tail(5) # 5 least abundant species
tot.occur[,1] %>%
hist(breaks=10) # Display histogram of
overall abundance across sites
##Initial Diversity Indices
macro.Shannon <-
specnumber(macro_BRW.dat)
hist(macro.Shannon, breaks=10)

easypackages::libraries("readr", macro.H <- diversity(macro_BRW.dat)

"here", "dplyr", "tidyr", "tibble", #Basic Shannon Diversity Index
"ggplot2", "vegan") hist(macro.H, breaks=10)

#Species Assemblage Data macro.simpson <-

diversity(macro_BRW.dat,
macro_BRW.dat <- index="simpson") #Basic Simpson's
read_csv(here("Original Data", Diversity Index
"macro_specR.csv")) hist(macro.simpson, breaks=10)
View(macro_BRW.dat) macro.J <-
macro.H/log(macro.Shannon) #Pielou's
##Summarizing Species Assemblage Data eveness
tot.abund <- macro_BRW.dat %>% hist(macro.J)
colSums() %>% sort(decreasing = TRUE) ##Effective Species Unit
%>% data.frame
macro.Hsp <- exp(macro.H) ##Effective
tot.abund %>% species number
head(5) # 5 most abundnant species hist(macro.Hsp)
tot.abund %>% #Species Rarefaction
tail(5) # 5 least abundant species
macro.min <-
tot.abund[,1] %>% min(rowSums(macro_BRW.dat))
hist(breaks=20) # Display histogram of macro.Srare <- rarefy(macro_BRW.dat,
overall abundance across sites sample= macro.min)
macro.Srare

7
plot(macro.Shannon, macro.Srare,
xlab = "Observed No. of Species", ylab =
"Rarefied No. of Species")
rarecurve(macro_BRW.dat, step = 20,
sample = macro.min, col = "blue", cex
= 0.6)
##Gamma Diversity
macro.sac <-
specaccum(macro_BRW.dat)
plot(macro.sac, ci.type="polygon",
ci.col="yellow")
macroBRW_specRich<-
specpool(macro_BRW.dat)
macroBRW_specRich
##Beta Diversity
macro_dat.dist <-
vegdist(macro_BRW.dat,
method="jaccard") # Includes relative
abundance in the calculation
macro_bin.dist <-
vegdist(macro_BRW.dat, binary=TRUE,
method="jaccard") # Calculation is
based on presence-absence
betadiver(help='true') # Displays
different methods used to calculate
betadiver
macro.beta <-
betadiver(macro_BRW.dat, method="r",
order=TRUE)
macro.beta
#Nested Patterns for Beta Diversity
nestedchecker(macro_BRW.dat)
macro.temp <-
nestedtemp(macro_BRW.dat)
plot(macro.temp) # temperature
8
plot(macro.temp, kind="incid")
#incidence
oecosimu(macro_BRW.dat,
nestedchecker, "quasiswap")
#Beta Diversity ##Species Assemblage
Data
macro_env.tdf <-
read.csv(here("Original Data",
"macro_envR.csv"))
macro_sp.tdf <-
read.csv(here("Original Data",
"macro_spR.csv"))
macro_sp.tdf
#macro_sp.tdf <- macro_sp.tdf %>%
# mutate(SITE = as.factor(ï..SITE),
#SUBSTRATE =
as.factor(SUBSTRATE))
##Nested Temperature
oecosimu(macro_BRW.dat,
nestfun=nestedtemp, "quasiswap")
##Similarity and Distance Measures
betadiver(help='true') # Displays
different methods used to calculate beta
diversity
macro.beta <-
betadiver(macro_BRW.dat,
method="sor", order=TRUE)
macro_dat.dist <-
vegdist(macro_BRW.dat,
method="jaccard") # Includes relative
abundance in the calculation
macro_bin.dist <-
vegdist(macro_BRW.dat, binary=TRUE,
method="jaccard") # Calculation is
based on presence-absence
##Unconstrained Ordination ##Principle
Component Analysis
view(macro_BRW.dat)
macro.hel<-decostand(macro_BRW.dat,
method="hellinger")
macro.hel.pca<-rda(macro.hel)
##Number of Relevant Axes
(macro.hel.eig<-
macro.hel.pca$CA$eig)
macro.hel.eig[macro.hel.eig>mean(ma
cro.hel.eig)] #Kaiser-Guttman
evplot <- function(ev)
{ macro.ca.eig[macro.ca$CA$eig>mean(m
# Broken stick model (MacArthur
1957)
n <- length(ev)
bsm <- data.frame(j=seq(1:n),
p=0)
bsm$p[1] <- 1/n
for (i in 2:n) bsm$p[i] <-
bsm$p[i-1] + (1/(n + 1 - i))
bsm$p <- 100*bsm$p/n
# Plot eigenvalues and % of variation
for each axis
op <- par(mfrow=c(2,1))
barplot(ev, main="Eigenvalues",
col="bisque", las=2)
abline(h=mean(ev), col="red")
legend("topright", "Average
eigenvalue", lwd=1, col=2, bty="n")
barplot(t(cbind(100*ev/sum(ev),
bsm$p[n:1])), beside=TRUE,
main="% variation",
col=c("bisque",2), las=2)
legend("topright", c("%
eigenvalue", "Broken stick model"),
pch=15, col=c("bisque",2),
bty="n")
par(op)
} plots
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##Plotting the PCA
plot(macro.hel.pca, scaling=1)
plot(macro.hel.pca, scaling=2)
biplot(macro.hel.pca, scaling=1)
biplot(macro.hel.pca, scaling=2)
##Correspondence Analysis
macro.ca<-cca(macro_BRW.dat)
summary(macro.ca)
macro.ca.eig<-macro.ca$CA$eig
macro.ca.eig
#Kaiser-Guttman
acro.ca$CA$eig)]
evplot(macro.ca.eig)
plot(macro.ca, scaling=1)
plot(macro.ca, scaling=2)
##Non-metric Multidimensional Scaling
macro.nmds <-
metaMDS(macro_BRW.dat, k=2,
distance="bray")
macro.nmds
stressplot(macro.nmds)
plot(macro.nmds, main="NMDS_Bray")
plot(macro.nmds, type = "n")
points(macro.nmds, display = "sites",
cex = 0.8, pch=21, col="red",
bg="yellow")
text(macro.nmds, display = "spec",
cex=0.7, col="blue")
##Add categorical environmental
descriptions to unconstrained ordination
View(macro_env.tdf) ##Reducing Explanatory Variables
##Apriori Models
##Ellipses
macro.rda.KNPFe<-
plot(with(macro_env.tdf,macro.nmds, rda(macro.hel~CONDUCT+SUBSTRATE+TEM
type = "n")) P+PH+ï..SITE,macro.e.scl) #include
text(macro.nmds, display = "spec", all variables; r-squared value drops
cex=0.7, col="blue") drastically
#summary(pine.rda)
##Hulls macro.rda.KNPFe
plot(with(macro_env.tdf,macro.nmds,
RsquareAdj(macro.rda.KNPFe)
type = "n"))
text(macro.nmds, display = "spec", vif.cca(macro.rda.KNPFe)
cex=0.7, col="blue")
##Step-wise Variable Selection
#Post Hoc Comparison of Environmental
Variables to Unconstrained Ordination macro.mod.all<-rda(macro.hel~ . ,
##Non-metric multidimensional scaling macro.e.scl) #Set maximum model size
macro.mod.0<-rda(macro.hel~ 1 ,
##Fit Selected Variables macro.e.scl) #Set minimum model size
macro.ef <-
envfit(macro.nmds~SUBSTRATE+PH+TEMP+ macro.step.fwd=ordistep(macro.mod.0,
CONDUCT , data=macro_env.tdf, scope=formula(macro.mod.all),
perm=1000) direction="forward",
macro.ef permutations=how(nperm=1000))

plot(macro.nmds, dis="site") vif.cca(macro.step.fwd)

plot(macro.ef)
RsquareAdj(macro.step.fwd)
##Constrained Ordination ##Scale Data
macro.step.bwd=ordistep(macro.mod.al
macro.hel<- macro_BRW.dat %>% l, direction="backward",
decostand(method="hellinger") permutations=how(nperm=1000))
macro.hel
vif.cca(macro.step.bwd)
macro.e.scl<-macro_env.tdf %>% scale
%>% data.frame RsquareAdj(macro.step.bwd)
macro.e.scl
macro.step.both=ordistep(macro.mod.0,
##Redundancy Analysis scope=formula(macro.mod.all),
direction="both",
macro.rda.all<- permutations=how(nperm=1000))
rda(macro.hel~.,macro.e.scl)
(macro.rda.all) vif.cca(macro.step.both)
RsquareAdj(macro.rda.all) RsquareAdj(macro.step.both)
vif.cca(macro.rda.all) ##Examine Ordination
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summary(macro.step.bwd) text(macro.step.bwd, display = "spec",
cex=0.7, col="black", scaling=2)
coef(macro.step.bwd) text(macro.step.both, display= "bp",
anova(macro.step.bwd, cex=0.7, col="purple4", scaling=2)
permutations=1000)
with(macro_env.tdf,
anova(macro.step.bwd, by="axis", ordisurf(macro.step.fwd, SUBSTRATE,
permutations=1000) ###Tests add=TRUE, col="green4"))
signifiance of each axis
plot(macro.step.bwd, scaling=2,
#coef(macro.rda) main= "Triplot RDA; Scaling 2",
macro.rda.all type="n")
points(macro.step.bwd, display =
##Evaluate variables of the constrained "sites", scaling=2,cex = 1.1, pch=21,
ordination col="red", bg="yellow")
anova(macro.step.bwd, by="mar", text(macro.step.bwd, display = "spec",
permutations=1000) ####Marginal cex=0.7, col="black", scaling=2)
effects (Type III ANOVA where order text(macro.step.both, display= "bp",
should not matter cex=0.7, col="purple4", scaling=2)

coef(macro.step.bwd) with(macro_env.tdf,
ordisurf(macro.step.fwd, TEMP,
#Triplots add=TRUE, col="green4"))
plot(macro.step.bwd, scaling=2,
main= "Triplot RDA; Scaling 2",
type="n")
points(macro.step.bwd, display =
"sites", scaling=2,cex = 1.1, pch=21,
col="red", bg="yellow")
text(macro.step.bwd, display = "spec",
cex=0.7, col="black", scaling=2)
text(macro.step.both, display= "bp",
cex=0.7, col="purple4", scaling=2)

with(macro_env.tdf,
ordisurf(macro.step.fwd, PH,
add=TRUE, col="green4"))

plot(macro.step.bwd, scaling=2,
main= "Triplot RDA; Scaling 2",
type="n")
points(macro.step.bwd, display =
"sites", scaling=2,cex = 1.1, pch=21,
col="red", bg="yellow")

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