1. Biogeography and Phylogenetics

2. Phylogenetics
What did our phylogenetic trees from the past week show us?
What method was used to generate these trees? What do these methods assume? Were there clades that were always well-supported? Were there some species with uncertain relationships?

3. What is biogeography?
The study of the distribution of species and ecosystems in geographic space and through geological time.
How does this compare to what we did in last lab?
What types of trees did we get last week?
What do they show or tell us about the species?

4. Review What process does this part of a tree represent?
How can speciation occur?
How can we predict where allopatry might have occurred?
The node represents the common ancestor & divergence. Many ways for speciation to occur: reproductive isolation (such as chromosomal and genetic mutations or polyploidy), also geographic separation results in two distinct population evolving independently.

5. MEGA – Maximum Likelihood
Today we will be using MEGA again, but will be developing trees based on analysis of Maximum Likelihood.
When we used MEGA last week, what was the analysis based on?
How does a MEGA analysis using Maximum Likelihood differ from one using parsimony?
At the end of the analysis what additional information will we have?
What will still be missing?
This bootstrap consensus tree will utilize maximum likelihood rather than maximum parsimony (used in previous lab)
For non-coding regions of DNA, we assume point mutations tend to occur at relatively constant rates. This provides us with a “molecular clock”.
Greater number of differences in the sequences. between two species implies greater distance in time since they diverged from each other. So longer branches equate to longer divergence time.
GO TO NEXT SLIDE
Image source: 

6. Geography!! What program will we use to investigate this?
What does the acronym stand for and what does it do?
Who currently uses this program (as well as MEGA)?
What data do you enter into RASP?
What does RASP do with the data you enter?
Geography! Is still missing – an explanation of how these related organisms spread over the Earth to their current locations.
We will use another program to help us investigate this – RASP: Reconstruct Ancestral States in Phylogenies
These tools are the same tools used by research scientists studying phylogenetics and biogeography!
In association with MEGA maximum likelihood information RASP allows us to generate a prospective map for where these species may have been located (on the globe) at each of the branches in the phylogenetic history you developed using MEGA.
RASP takes our phylogenetic analyses in MEGA and combines these with currently known geographic locations of the species studied to generate a prospective map for where these species may have been located (on the globe) at each of the branches in their phylogenetic history.

7. Image source: https://en.wikipedia.org/wiki/File:Phylogenetic_tree.svg
RASP
Today, you will generate a phylogeny that utilizes the present-day location of the species along with the most-probable phylogenetic relationships to paint a prospective picture of the geographical ancestry for the species in the tree. 
Follow the instructions for RASP
Use MEGA Instruction Part2, RASP Instructions, Origins.csv
Image source: 

8. Everyone have RASP trees made?
Put your RASP tree on your screen and walk around to see other groups’ trees
Similarities? Differences?
What do the pie shapes and pieces mean?
Why are the graphs so similar?
What is missing from this tree?
How can we hypothesize more specific times in relation to our tree?
We can use phylogeny and current distribution to hypothesize: 1) a path on the planet explaining the current distribution of the species, and 2) a timescale over which this happened.
What do the pie shapes and pieces mean?
When the pie has multiple piece colors, it means that the program is weighting the potential geographic origin of the ancestor…and this will then need to be factored into the next level back (the ancestor previous to this.
Why are the graphs so similar? Remember, they are all working with the same .csv Origins file and they used this in conjunction with a consensus 200 pseudo-replicate bootstrap. So…any variability should only be the result of the bootstrap…and the bootstrap consensus trees should be VERY similar between groups.
What is missing from this tree? Age or time. We can see proportional time, but there’s no years associated.
How can we hypothesize more specific times?
The best first assumption, based off of lecture is that speciation often results from a physical (geographic) separation of members of a population into regions that are no longer in contact, say by a river that separates an interbreeding population…or…perhaps even a new sea…or possibly…an ocean. (See if they can make the connection from a small separation such as a river up to MUCH larger separations like a sea or ocean. How would we might obtain a new sea or ocean on geological timescales? – plate tectonics!)

9. Geography!! (and Continental Drift)
Today we’ll be using the Paleobiology Navigator and its ability to track continental drift to examine past relationships of land masses that are currently homes to the species under investigation.
1) Paleonavigator site:
2) Animation of Continental Drift and Time: 
Image source: 

10. How does this compare to the information you developed above?
Using the Paleobiology Navigator and other resources from the internet, consider how you can attach tentative dates to your RASP tree.
Do a search – when is the Crassulaceae family thought to have diverged from its closest ancestor? 
How does this compare to the information you developed above? 
Continue working through your worksheet.
Our tree makes some sense based on continental movements! Madagascar and mainland Africa used to be part of the same landmass, but then they separated. The students’ trees show them that these plants came from a common ancestor, but there was a divergence between the Madagascar and the mainland clades. Cool!
Based on this and comparing this information to their RASP results, they should be able to attach tentative dates to many of the branch nodes on their RASP outputs, based on the assumption that divergence in these plants corresponds to plate tectonics.
Important points students should research and think about:
The worksheet is designed to expose inconsistencies in the data analyses.
Using the Paleobiology Navigator and doing a web search for the age of the Crassulaceae will reveal that the continental split of Madagascar from mainland Africa occurred ~130mya, while the Crassulaceae family is only ~ million years old..So by their analysis of geographical separation, the genus of Kalanchoe diverged from other Crassulaceae ~130mya, but the entire family is at most 100my old!? Something is not right! They will need to address this, along with the fact that…
…certain Kalanchoe trace to mainland Africa after the supposed split of Madagascar (as deduced from the RASP output). Ask them to hypothesize how to explain this. This could have been errors in the phylogeny, or a migration event. What is a critical component is missing from these analyses that could strengthen their conclusions? Fossils…accurately dated and placed geographically.

11. Due at the end of lab:
the Maximum Likelihood bootstrap tree made in MEGA
the phylogenetic tree generated in RASP
screenshots of any pertinent geological continental appearances for key periods in the evolution and distribution of Crassulaceae over the planet
hypotheses and citations for how this group of organisms have evolved