GBSN - Microbiology (Unit 3)Defense Mechanism of the body
Sigma Xi - Community Diversity of Foraminifera
1. Community diversity of
foraminifera over time,
space, and in response
to temperature
Giovanna Noe-Wilson
Undergraduate in Biology and Marine Science at Smith College
2. Hypothesis: the biodiversity of living benthic
foraminifera varies spatially (i.e. elevation) and
over time (e.g. seasonally)
5. GPS locations rarely reported with molecular data
Grattepanche & Katz (2018)
● When searching for geography in GenBank records of
Alveolates, Stramenopiles and Rhizaria (including
Foraminifera)
7. Foraminifera are:
● Protists: single celled eukaryotes
● That have shells or tests
○ made up of calcium carbonate
(CaCO3)
● Have pseudopods
○ Arm-like projection that allows
GNW
8. My study organisms: Foraminifera
● Protists: single celled eukaryotes
● That have shells or tests
● Have pseudopods
Two main types of foraminifera:
● Planktic - mainly living in the upper water
column
● Benthic - mainly living bottom/within
sediment
They are among the most abundant shelled
organisms in marine environments
GNW
9. Previous work on benthic forams
● Collected at various salinities &
heights above sea level
● Investigated community
structure during changing sea
levels
● Sampled various locations
within a salt marsh
● Investigating spatial variation
among living and nonliving
forams
10. Previous work on benthic forams
● Collected at various salinities &
heights above sea level
● Investigated community
structure during changing sea
levels
● Sampled various locations
within a salt marsh
● Investigating spatial variation
among living and nonliving
forams
Both papers base their studies solely
on morphological (physical) features
alone!
There is no molecular perspective!
11. Molecular Perspective - importance
● Taxonomy and biodiversity of benthic foraminifera is frequently studied
● Most taxonomy is still based on morphology (physical features)
This can lead to:
● “Lumping” or “splitting” - process that results in merging of species that have
similar physical features
Problem:
Can harm and under score species diversity when studying ecological research or
when creating environmental reconstructions (where biodiversity and identification is
critical
Morphological perspective can:
Help strengthen identification of species as well as identification of new or cryptic
species
12. Methods: microscopy (some examples)
(GNW)
These three images are all benthic foraminifera - two are the same species!
Identification through morphological (physical) features:
○ can lead to errors in species taxonomy and identification
○ Can miss cryptic species
13. subtidal
tidal mudflat
low marsh
mid-marsh
High marsh
upper border & upland
(above the tidal zone)
Methods: field work
● Soil samples taken at
different months (during
low tide) in the same
location using GPS
● Only the top 1 cm of soil
was taken from the
different locations and
filtered on site. They
were placed in lysis
buffer for preservation
● Locations were chosen
based on differences in
soil/mud composition
ranging from silt to sand
● Higher elevations were
mainly dry and also
contained vegetation
● Water and air
temperature were also
taken
15. Methods: molecular work (HTS analyses of
amplicons)
Filtration
Used 100 µm
filter to filter out
large
particulates
They were then
placed in -80 ℃
freezer to help
lysis open cells
DNA/RNA
Extraction
using zymo kit
PCR amplification
with 18F and 19R
Primers
Miseq
sequencing
Assembly &
data
analysis
16. Methods: Foram-specific primers
Polymerase chain reaction (PCR) amplification - allows for amplification of specific regions of either
DNA or RNA in a specific sample
Foram specific primers (18F/19R) are used because they amplify regions that are only specific to
foraminifera and no other species in the samples
17. Methods: Foram-specific primers
● Line represents the small subunit
Ribosomal DNA
● Dark green - common to all
Eukaryotes
● Light green - inserts only found in
foraminifera
18F/19R amplify regions (shown in light
green) that are unique only to
foraminifera
18. Methods: PCR using foram primers
DNAs RNAs
● Multiple bands shown here are not errors
○ Represent different foram species
○ Showing diversity in those light green regions (shown on previous slide)
19. Insights
● GPS and mapping allow for accurate site identification
○ Mapping and elevation allows identification of foram spatial patterns
○ Sheds light on importance of spatial awareness
20. Elevation
● Use of LiDAR (light
detection & ranging) data
gives accurate elevation of
each site
● Accuracy led to the
discovery that all sites are
not created equal
○ Farther up tidal mudflat ≠
increase in elevation
Site 1
Map by: GNW
Site 2
21. Insights
● GPS and mapping allow for accurate site identification
○ Mapping and elevation allows identification of foram spatial patterns
○ Sheds light on importance of spatial awareness
● Identifying foraminifera across sites and times
22. My focus = abundant species (>100 reads)
● Smaller read numbers were not considered
○ Fewer read counts could mean transcriptional noise making it difficult to know the difference
between actual presence or instances of gene expression or randomly present gene
expression due to sequencing errors
24. Species and elevation
● No clear pattern in
the number of
species
○ Less species in
lower
elevations?
● Roughly each site
had 6 - 18 species
● The ratio of “not
active” (DNA) to
“active” (RNA)
varied between
sites
Elevation at each site
Numberofspecies
Tidal mudflat Mid-marsh High Marsh Upper marsh DNA
RNA
25. Insights
● GPS and mapping allow for accurate site identification
○ Mapping and elevation allows identification of foram spatial patterns
○ Sheds light on importance of spatial awareness
● Considerable variation in foram diversity across sites and
times, and between “present” (DNA) and “active” (RNA)
26. Presence/absence of species
● Compared the top 15 OTUs (OTU - my proxy for species)
First present (DNA) then active (RNA)
Black = absent (no species found or species containing less than 100 reads)
Grey = 1000 - 100 reads
White = over 1000 reads
Note:
DNA: present - species can be present in a location or
sample however, they might be dormant
RNA: active - species are active (during the time of
collection)
27. Presence absence of species - DNA
Top species:
Arnoldiellina fluorescens
Highly abundant and
found in all the locations
Trochammina sp.
Highly abundant in only
one location
28. Presence absence of species - RNA
RNA has similar trends
as DNA
Arnoldiellina fluorescens
is active mostly
everywhere all the time
Trochammina sp.
even though highly
abundant in a specific
location is not also highly
active in the same
location
29. Insights
● GPS and mapping allow for accurate site identification
○ Mapping and elevation allows identification of foram spatial patterns
○ Sheds light on importance of spatial awareness
● Considerable variation in foram diversity across sites and
times, and between present” (DNA) and “active” (RNA)
● Individual species show complex patterns
30. Rank Abundance Curves
● Portray relative
abundance and
species diversity
within a
community Shows higher species
richness and higher
species evenness
Note: A lower slope
shows greater species
richness and species
evenness (shown in
red)
31. Rank Abundance of DNA
● Similar curves indicate
similar dominant trends
● Hyperdiverse sites!
● Results show
similarities in
community dynamics
by season
32. Rank abundance curve for RNA
● Rank abundance curve
shows similar curves,
indicating similar
dominance trends
● Much less variance than
DNA
33. Insights
● GPS and mapping allow for accurate site identification
○ Mapping and elevation allows identification of foram spatial patterns
○ Sheds light on importance of spatial awareness
● Considerable variation in foram diversity across sites and
times, and between present” (DNA) and “active” (RNA)
● Individual species show complex patterns
● Communities present differ in the summer
34. Conclusions
● Environmental conditions play a crucial role in foraminiferal
assemblages in salt marshes of Madison, CT
● Most sites were dominated by a monothalamous species,
Arnoldiellina fluorescens
● Seasonality is an important variable that control species
distribution
○ Also affects species diversity
● Having accurate GPS locations along with species
identification help aid in future research
35. Future Work
● Onsite elevational data should be collected to assess
elevational changes at each location and with better
accuracy
● Time, space, and other variants need to be compared to
better assess differences among foraminiferal assemblages
○ Examples: salinity, sediment characteristics, temperature,
etc.
36. Take away:
Identifying spatial variation of benthic forams using
a combination of morphological & molecular data
will provide a critical baseline for studies tracking
impacts of changing climates
37. Key Literature
Burki, F., Keeling, P. J. Rhizaria. Current Biol. 24(3): R103-R107.
Cunha, Camila., Kukimodo, Isabela., Semensattoo, Decio., Foraminiferal assemblages along the intertidal zone of Itapanhau
River, Bertigoa (Brazil). South American Earth Sci. (2017)
Katz, Laura., Grattepanche, Jean., Walker, Laura M., Ott, Brittany M., Pinto, Paim., Delwiche, Charles F., Lane Christopher E.,
Microbial diversity in Eukaryotes SAR clade: Illuminating the Darkness Between Morphology and Molecular Data
BioEssays.(2018) 40, 1700198
Milker, Yvonne., Horton, Benjamin P., Nelson, Alan R., Engelhart, Simon E., Witter, Robert C., Variability of intertidal foraminiferal
assemblages in a salt marsh. Marine Micropaleontology. (2015)
Pawlowski, Jan., Holzmann, Maria., Diversity & geographic distribution of benthic foraminifera: a molecular perspective. Biodivers
Conserv (2007) 17:317-328
Nettersheim, Benjamin J., Putative Sponge Biomarkers in unicellular Rhizaria question early rise of animals. Nature Ecology &
Evolution (2019)
Prazeres, Martina., R, T.,Variation in sensitivity of large benthic Forams to the combined effects of ocean warming and local
impacts. Sci Report (2017)
T. Cavalier‐Smith, Int. J. Syst. Evol. Microbiol. 2002, 52, 297.
Photo O. opaca source: https://www.flickr.com/photos/32133972@N05/
Photo Trochammina source - http://www.marinespecies.org/aphia.php?p=taxdetails&id=114348
Editor's Notes
They can be effectively used for monitoring coastal environments (5) tracking sea-level changes 6 and be important bio-indicators of pollution like that of heavy metal. 7
Benthic are paeffectively rticularly important as they comprise 90% of deep sea biomass (source 4)
They can be used for monitoring coastal environments (5) tracking sea-level changes 6 and be important bio-indicators of pollution like that of heavy metal. 7
and to exemplify this is what these people did look for GPS data in Genbank records of these groups including Rhizaria where my target taxa is found
What you see now is the number of records associated with each with each location and the biggest pie is no country reported
GPS allows us to make better decisions based on more up to date information
They live in lots of environments
Image: Giovanna Noe-Wilson - hand drawn
They have been broken down into these two types ones in the water column and and the benthics which is what I am studying
With the continuous stressors on marine and coastal habitats, taxonomy and biodiversity of benthic foraminifera is frequently studied; however, even with the increase in research done on these eukaryotes, they are still understudied (Grattepanche et al., 2018, Pawlowski & Holzmann, 2007). Most taxonomy has been, and is still, based largely on morphology classification (Grattepanche et al., 2018). The primary problem with this taxonomic approach is that it can lead to incorrect and inconsistent identification of species (Roberts et al., 2016, Saad & Wade, 2016, Pawlowski & Holzmann, 2007). Which then leads to “lumping” or “splitting,” a process that often results in a merging of species based on similar physical features; which then incorrectly groups species together. This causes erroneous foraminifera taxa or assemblages and inconsistent distribution patterns (Roberts et al., 2016, Saad & Wade, 2016 Pawlowski & Holzmann, 2007). This is extremely problematic when it comes to studying ecological research or creating environmental reconstructions where biodiversity and identification is critical (Roberts et al., 2016, Pawlowski & Holzmann, 2007)
Recently, taxonomic identification has moved away from relying solely on morphological features and focusing more heavily on a combination of morphological and molecular techniques (Grattepanche et al., 2018, Roberts et al., 2016, Pawlowski & Holzmann, 2007). Much of the molecular data has helped in the identification of new species as well as in identifying cryptic species (Roberts et al., 2016, Pawlowski & Holzmann, 2007). Molecular data enhances our understanding of the diversity of foraminifera and demonstrates the need for continued research. Recent studies which focus on bridging the gap between molecular and morphological data offer new perspectives on foraminiferal species and improved taxonomic stability (Roberts et al., 2016).
Photos taken by:
Figure 1: Rabindra
Figure 2: Elly Goetz
If you look in a microscope at these three you wouldn’t be able to differentiate or identify what species they are
They are all “snail-like” multichambered forams but this one is a genetic species
Collection location and pictures
Pictures: taken by Giovanna Noe-Wilson (using fulcrum)
Drawing hand drawn by Giovanna Noe-Wilson
Fulcrum - map!
Created by GNW
Really don’t understand the methods that much - why do we do these steps? - molecular
Kit helps amplify
The key thing is that I use foram specific primers and what means is that these primers - which means they only amplify the forams in my samples no other organisms
And the reason why I know why these are so specific is
They are amplifying the regions in light green that are unique to forams
On this green line represents the the small subunit Ribosomal DNA gene and the dark green is what all eukaryotes have and only forams have these inserts (light green)
My primers amplify two of these inserts that are unique to forams
This is a gel these are ladders for size
Here you can see bands - some or many contain multiple bands - these are not error they represent different foram species
Biologically this can come off as error but really the diversity in those light green regions
Photos provided by Laura Katz
Light Detection and Ranging—is a remote sensing method used to examine the surface of the Earth
My focus is abundant species which was species larger than 100 reads
Such few reads usually represent transcriptional noise. With so few reads it is difficult to ascertain whether these are truly actual instances of expression or randomly present due to sequencing errors. This can easily be ascertained by having multiple replicates of the same sample. If these reads are present in one, but not in other replicates then they most probably are transcriptional noise. We used this approach to ask the same question of small RNA expression library. To answer this question we used K-S statistics applied to frequency distribution plots of replicates. The rationale here was that the cut off should be the lowest number of reads where the distance between the two frequency distribution plots of replicates should be minimized. This was a valid assumption, since if the replicates were exactly the same the frequency distribution curves would overlap and the distance would be zero. Thus we defined noise as the minimum number of reads that prevents frequency distribution curves of replicates being close to each other. I am attaching the paper that describes this in more detail.
Pictures: taken by Giovanna Noe-Wilson (using fulcrum)
Drawing hand drawn by Giovanna Noe-Wilson
Remember that my eyes saw and plant people can tell us that there is a really big differences along these gradients the plants are really different here so I predicted there would be very obvious differences in the number of species of forams - and it's not what I found
These are the actual elevations because the elevations I calculated here in the SAL lab but when we sampled we were not exact so we are in the same ecology not the same altitude
Teach the axises - that is the estimated number of species which I estimated from my molecular data
And these are my four different locations I intended to to go back but as you can see the actual altitude really varied when we did go back
3 conclusions:
1) roughly each site had 6-18 species
2) the ratio of active to quesent species varied by different sites - here we can see a site with many more active species that quesent species and here its the other way
3) there isn't a clear pattern in the number of species except for that there seems to be not many species in the lowest altitude
There is not a clear trend along the altitude???
Closely related
These are 15 of the top species found at all the locations
Here when I think about presence absence of DNA here are the top 15 species of forams
Here is the top species and it is present everywhere and all the time
There is ovammina opaca which is super abundant sometimes but not other times
And then there is other guys that
Trochammina sp - which is highly abundant at one time and place.
Photo O. opaca source: https://www.flickr.com/photos/32133972@N05/
Photo Trochammina source - http://www.marinespecies.org/aphia.php?p=taxdetails&id=114348
When we look at the RNA we can see similar trends
Arnoldiellina is still everywhere all the time
Ovammina Opaca is more patchy so it present everywhere but not active all the time
And trohcammina even though highly abundant in that specific location it is not highly active in that location
Rank Abundance curve for all DNA collection sites shows similarities in community dynamics by season. Collection sites within the season have similar curves, indicating similar dominant trends.
Talk about what a rank abundance curve is
Which plots relative abundance of species against rank in abundance
A lower slope shows greater richness and species evenness
Communities are really different
Divide into two different colors - later
I found two different kinds of communities - these a had all roughly 30 species and similar distributions and these three had 30 -50 species - what this is telling us these guys have fewer abundances and many intermediate species (june)
These are march and May and these are June
Which have the species abundance
I don’t see the same distinct patterns and trends I do for DNA
Most sites were dominated by related to a monothalamous species, Arnoldiellina fluorescens (OTU1)
In general diversity (evenness) and species richness decrease both in elevation and distance. Higher elevation along with increasing from the intertidal zone tend to have the most effect on lowering species diversity. I conclude that elevation and climate (maybe salinity ? only tested the water not the salinity of samples) are important variables controlling species distribution (which can be seen and compared with in other papers)
Environmental conditions play a crucial role in foraminiferal assemblages in Madison CT
With my research I have found a large amount of heterogeneity and so we need to look at time and space
Figure 6. Benthic foraminiferal species distribution changes with increasing water depth and distance from the shore. It is not depth per se that is the controlling factor. Availability of food (flux of organic matter out of the surface ocean) and dissolved oxygen are probably the two most important influences on benthic foraminiferal abundance and species distributions. Other factors include sunlight, substrate, temperature, and pH. From Leckie and Olson, 2003; In, Olson and Leckie, eds., Micropaleontologic Proxies for Sea-Level Change and Stratigraphic Discontinuities, SEPM Special Publication 75: 5-19.
