Monroe Community College Molecular Systematics Palame Trees Lab Report please do the lab activities as it’s constructed in the lab sheet and all the report
Monroe Community College Molecular Systematics Palame Trees Lab Report please do the lab activities as it’s constructed in the lab sheet and all the report questions. follow the content exactly as it it’s. thank you. Palame Trees (4)
? Is Palame crassimana monophyletic in either the NJ1 or ML1 tree? Why or
why not?
? Is the genus Palame monophyletic in either the NJ1 or ML1 tree? Why or why
not?
? Is Palame crassimana monophyletic in either the NJ2 or ML2 tree? Why or
why not?
? Is the genus Palame monophyletic in either the NJ2 or ML2 tree? Why or why
not?
? What taxon is consistently sister to P. crassimana unicolor?
? Do you think that P. crassimana unicolor and its sister taxon are actually the
same species? Why or why not?
? What is the % similarity for P. crassimana unicolor and its sister taxon?
? Using the ML tree including all samples, which species is currently included in
the genus Palame (according to its name), but does not seem to be part of a
monophyletic genus (according to what you see in the tree)?
? If you were going to arrange the gastropods in pairs, without making a
detailed analysis of characters and character states, which ones seem the
most similar?
? ___________________________ and _____________________________
? ___________________________ and _____________________________
? ___________________________ and _____________________________
Gastropoda Trees (2)
? Are any of the pairs that you guessed about (based on overall morphology)
recovered as sister taxa in the NJ tree? If so, which ones?
? Can you find any similar morphological traits between the species that the NJ
tree indicates are sisters?
? Look at the gastropod NJ tree. Does your NJ tree show any of the
relationships shown in Colgan et al. 2007 (http://bit.do/Bio228GastropodTree, see last slide)?
? Which taxa are in the same clades in both your NJ tree and the Colgan tree?
? Does your ML tree show any of the relationships shown in Colgan et al. 2007
(http://bit.do/Bio228-GastropodTree, see last slide)?
? Which taxa are in the same clades in both your ML tree and the Colgan tree?
Fig. 3. A maximum
likelihood tree based on six
genes, including nuclear
and mitochondrial data,
from: Colgan et al. 2007.
Molecular phylogenetics of
Caenogastropoda
(Gastropoda: Mollusca).
MPE 42: 717-737.
The families analyzed in
your project are highlighted.
Lab 9: Molecular Systematics
Lab objectives:
1.
2.
3.
4.
Learn how scientists use DNA sequences to reconstruct evolutionary relationships
Practice phylogenetic reconstruction
Investigate the monophyly of three morphological forms of a species, and its
genus (Project A)
Investigate gastropod relationships (Project B)
Key concepts and calculations:
1. Molecular character: Usually a DNA sequence trait; often a base site or region in a
particular gene
2. Character state: alternative conditions of the character (A/T/C/G; can also be the
presence or absence of insertions or deletions, or chromosome duplications)
3. Homology/homologous characters: characters inherited, with or without modification,
from a common ancestor
4. mtDNA: mitochondrial DNA, which tends to evolve rapidly, relative to nuclear DNA
(nDNA); mtDNA is present in many copies per cell
5. COI: cytochrome oxidase subunit I, a protein involved in the electron transport chain of
oxidative phosphorylation; its gene, part of the mitochondrial genome, is often used for
animal species barcodes
6. Cladogram or phylogenetic tree: a branching tree, showing hypothesized evolutionary
relationships
7. Parsimony: one method of determining evolutionary relationships (other methods use
models of evolution); here, the simplest explanation, with the fewest number of
evolutionary changes, is considered the most parsimonious
8. Neighbor-joining: a similarity-based method for inferring relationships, in which
terminals (taxa) are grouped in pairs, and then pairs are grouped in pairs, etc.
9. Maximum likelihood: a method that aims to determine the tree topology that maximizes
the probability of observing the sequences at hand
10. Ingroup: the group studied by the investigator (the investigator starts with the hypothesis
that this group is monophyletic)
11. Outgroup: a taxon included for comparison; ideally, the outgroup will be a closelyrelated taxon (see sister group, below) and determine the polarity of character states to
reveal the direction of evolution (states that are ancestral vs. derived)
12. Terminology regarding phylogenetic groupings:
a. Monophyletic group (i.e. clade): a group composed of an ancestor and all its
descendants; contemporary organismal classification is based on monophyletic groups
b. Paraphyletic group: one or more of the descendants of the common ancestor has been
excluded (for instance, reptiles excluding birds)
c. Polyphyletic group: a group that consists of unrelated lineages, more closely related to
species not included in the group (for instance, winged animals including birds, bats, and
flying insects)
d. Sister group: the clade most closely-related to a particular clade or species of interest
e. Sister species: species most closely-related to a particular clade or species of interest
Activities during lab period:
1
a. Deeper discussion of key phylogenetic concepts, led by lab instructor
b. Use DNA Subway to select and align sequences
c. Use DNA Subway to generate phylogenetic trees using Neighbor-Joining and
Maximum Likelihood approaches
d. Use phylogenetic trees to make inferences about niches
Lab exercises:
Project A, Palame Beetles
Background:
Cryptic organisms (those that look very similar despite belonging to separate species) tend to
be poorly studied, and it can be challenging to find morphological traits that distinguish them. In
this project, you will use mitochondrial COI sequences to:
1. select and align genetic sequences from different taxa;
2. create Neighbor-Joining and Maximum Likelihood trees using one outgroup (a species
from a different genus but still in the same larger group of beetles);
3. create Neighbor-Joining and Maximum Likelihood trees using one outgroup (and
including two additional species from different genera);
4. determine if the three morphological forms of the Palame crassimana species form a
monophyletic group; and
5. determine if the genus Palame is consistently recovered as monophyletic
Palame is a genus of cryptic, crepuscular (active at dawn & dusk), wood-boring beetles that
colonize senescing (aging) wood of trees in the Brazil nut family. The trees are icons of old growth
Amazonian forests, and the beetles are important in forest nutrient cycling. The genus Palame was
first described by the great Amazonian explorer Henry Bates, who wrote the first scientific
description of mimicry (recall Batesian mimicry). He also sent back specimens of >14,000
Amazonian species, many of which were new to scienceincluding species of Palame. Although
the genus is small, currently including five species (four are shown below), there is considerable
intraspecific (within-species) variation in size, color, and the patterns of pubescence. This has led
to disagreement in how the species should be delimited (what separates one species from the
others). Palame crassimana was categorized in the past as existing within three different genera,
but these variants are currently considered morphological forms within a single species.
P. anceps
P. crassimana
bicolor
P. crassimana
unicolor
P. crassimana
olivaceous
2
P. mimetica
P. vitticolle
Fig 1. Four of the five recognized species of Palame, including three morphological forms of Palame
crassimana.
Rearing projects in French Guiana suggested that these forms had different host and stratum
associations (Berkov & Tavakilian 1999, Berkov 2002), lending support to the idea that they might
represent independent lineages. If the three morphological forms are part of a single species,
they should form a monophyletic group in a phylogenetic tree.
1.
Download beetle sequence data to your computer: http://bit.do/Bio228-PalameFasta
2.
Access the DNA Subway website https://dnasubway.cyverse.org/
a.
Log in as Guest (no need to register/create an account)
b.
Start a new project Determine Sequence Relationships (blue subway line)
c.
Project Type choose: mtDNA
d.
Select Sequence Source: Upload (first option, find downloaded FASTA)
e.
Give your project a descriptive Project Title
3.
Click on Sequence Viewer to see the 9 sequences included in the project
4.
Click on Select Data. Check P. crassimana bicolor, and P. crassimana unicolor and Save
Selections
5.
Click on MUSCLE to align the sequences. Click on MUSCLE a second time to view
Sequence Variation. Are there many base changes that distinguish the two morphological
forms?
6.
Close the window and return to select data. Choose all Palame sequences and Oedopeza
leucostigma (as a potential outgroup). Click on MUSCLE to align these sequences.
7.
First create a Neighbor-Joining tree, by clicking on Phylip NJ. Set O. leucostigma.
Download the resulting tree as an image and save it as ‘NJ1.png’.
8.
Now try a Maximum Likelihood tree, by clicking on Phylip ML. Make sure that O.
leucostigma is still selected as the outgroup. Download the resulting tree as an image and save
it as ‘ML1.png’, then close the window.
9.
Insert the two trees into the PowerPoint document, and resize them so that each occupies 1/4th
of a slide. Label them NJ1 and ML1.
Is Palame crassimana monophyletic in either the NJ1 or ML1 tree? Why or why not?
(NOTE: scientific names should be italicized, but since the rest of the question is italicized here, the
scientific name is underlined instead)
3
Is the genus Palame monophyletic in either the NJ1 or ML1 tree? Why or why not?
10.
Return to Select Data and add Xylergates lacteus and Neobaryssinus altissimus (two additional
species in the same tribe). Repeat the alignment with MUSCLE and rerun each of the trees
(make sure that O. leucostigma is still selected as the outgroup). Download the images, and
name them NJ2 tree and ML2 tree, respectively. Insert them into your PowerPoint and resize
them so that each occupies 1/4th of a slide. Label them NJ2 and ML2.
Is Palame crassimana monophyletic in either the NJ2 or ML2 tree? Why or why not?
Is the genus Palame monophyletic in either the NJ2 or ML2 tree? Why or why not?
What taxon is consistently sister to P. crassimana unicolor?
Do you think that P. crassimana unicolor and its sister taxon are actually the same species?
Why or why not?
11.
To see the percent similarity between P. crassimana unicolor and its sister species, click
MUSCLE to view the alignment, and click on Sequence Similarity %.
What is the % similarity for P. crassimana unicolor and its sister taxon?
Using the ML tree including all samples, which species is currently included in the genus Palame
(according to its name) but does not seem to be part of a monophyletic genus (according to what
you see in the tree)?
Project B: Gastropods
Gastopoda (snails and slugs) is an ancient lineage that has been documented in the Late
Cambrian (>485 million years ago). It is considered second in diversity only to the insects, with
an estimated 65,000 80,000 species in >600 families. In this project, you will observe
relationships among a select group of these taxa. In some cases, species were substituted for those
you used in the lab, according to the availability of sequence data. You will use mitochondrial
COI sequences to:
4
a. select and align taxa;
b. create Neighbor-Joining and Maximum Likelihood trees using a bivalve, the Atlantic
jackknife clam, as an outgroup; and
c. compare your trees to a previously published molecular phylogeny
http://bit.do/Bio228-GastropodFigure2
Fig 2. A bivalve outgroup taxon and six gastropod ingroup taxa.
If you were going to arrange the gastropods shown in Fig. 2 in pairs, without making a detailed
analysis of characters and character states, which ones seem the most similar?
___________________________
and
_____________________________
___________________________
and
_____________________________
___________________________
and
_____________________________
1. Grab the gastropod sequence data from here: http://bit.do/Bio228-GastropodFasta
2. Start a new project by clicking on My Projects and then on Determine Sequence
Relationships
a. Project Type: mtDNA
b. Select Sequence Source: Upload
c. Give your project a descriptive Project Title
3. Click on Sequence Viewer to see the 7 sequences included in the project.
4. Click on Select Data (Select All) and Save Selections.
5
5. Use MUSCLE to align the sequences and then click MUSCLE a second time to view
Sequence Variation. Trim the alignment, so that the sequences are the same length.
6. Create a Neighbor-Joining tree, by clicking on Phylip NJ. Set the Atlantic jacknife
clam as the outgroup. Download the image as NJ Gastropod tree.
7. Now try a Maximum Likelihood tree (Phylip ML). Make sure that the Atlantic
jacknife clam is still selected as the outgroup. Save this image as ML Gastropod tree.
8. Insert the two trees into your PowerPoint document and resize them so that each
occupies half of a slide. Label them NJ Gastropod and ML Gastropod, respectively.
Are any of the pairs that you guessed about (based on overall morphology) recovered as sisters
in the NJ tree? If so, which ones?
Can you find any similar morphological traits between the species that the NJ tree recovers as
sisters?
Look at the gastropod NJ/ML trees in your PowerPoint document. Does your NJ tree show any
of the relationships shown in Colgan et al. 2007 (http://bit.do/Bio228-GastropodTree)?
Which taxa are in the same clades in both trees?
Does your ML tree show any of the relationships shown in Colgan et al. 2007
(http://bit.do/Bio228-GastropodTree)?
Which taxa are in the same clades in both trees?
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