Chromosome Analysis

Sometimes we don't have to look as far as the DNA sequence to understand the relatedness between organisms. When cells divide, strands of DNA become condensed in the nucleus into chromosomes. Each species has a characteristic number of chromosomes. By looking at the number of chromosomes, we can get an idea of how closely related two similar species are.

Take the mule, for example. A mule is what you get when you mate a donkey and a horse, two different species. While a mule can grow up and be very healthy, it is sterile (it can't produce offspring). This happens because even though the horse and the donkey are closely related (evidenced by the fact that they produce an offspring), they have different chromosome numbers.

During mitosis (cell division) each chromosome copies itself, so the mule can grow into a healthy adult. But during meiosis (the cell division that produces gametes) each pair of chromosomes (one from the mother and one from the father) has to pair up. Unfortunately for the mule, its chromosomes can't pair up, so meiosis can't be properly completed, making the mule sterile

Therefore, if we have a group of closely related species (or subspecies), taking a look at their chromosomes is a good first step to figure out how similar or different they really are.

How do you look at chromosomes?

Scientists observe chromosomes by making something called a karyotype, an organized profile of an where an individual's chromosomes are arranged and numbered by size from largest to smallest.

To make a karyotype, scientists take a picture of chromosomes during cell division, then cut them out and match them up according to size, banding pattern and centromere position.

But how do you take a picture of a chromosome?

Even though chromosomes are very small, they can be prepared so they can be seen under a microscope. Most of the time, chromosomes are an unorganized mass of material in the nucleus of the cell. But during mitosis, they condense and separate. At this stage, they can be taken from the dividing cell and put on a slide. Then, a special dye called Giemsa is applied to them. It stains the regions of DNA that are rich in adenine (A) and thymine (T), giving the chromosomes a striped appearance.

Tortoise Sequencing

You have seven tortoises:

1. [Gopherus polyphemus (North America)] 2. [Pyxis arachnoides (Madagascar)] 3. [P. planicouda (Madagascar)] 4. [Geochelone yniphora (Madagascar)] 5. [G. radiata (Madagascar)] 6. [G. pardalis (mainland Africa)] 7. [G. nigra (Galapagos)]

You know 4 of them are from Madagascar 4 are not. Presumably, these four should be more closely related to one another than the other tortoises.

In order to see which tortoises are most closely related, you sequenced 1355 base pairs (bp), including the 12S and 16S rRNA and cyt b genes from each tortoise.

Before you take a look at your gels, let's review some basics to help make sense of what you see.

Let's look at the gels and count the number of differences between the different tortoise species.

Click on any two gel plates to see how different each species is from the others. As you do, your data will be recorded in the charts below.

Parsimony

Now we'll use this information to show how closely related the different species are. Scientists use a type of analysis called maximum parsimony to come up with a tree based on this information.

First, start putting these species into groupings based on the genetic data you collected.

ß Pick the two species that are most closely related to each other. [Sp. 2 and 3 (.05) are a group.] ß Pick another two species that are closely related to each other.[Sp. 4 and 5 (.06) are a group.] ß How closely related are these two groups? Group them together. [These two groups 2-3 and 4-5 (.07) are closely related and can fall into one group.] ß Pick the species that you think is the next closest relative to the group. [Sp. 6 is closely related to Sp.5 (.07) so maybe it is the first one outside the grouping we just made.] ß Pick the next species that you think is the next closest relative to the group. [Sp. 7 has the same difference from all of the other Geochelone sp. (4,5,6) So it could be the next group outside of the group we just made.] ß You should have one species left. This is the outgroup. [Sp. 1 is the least related to all of the species, therefore it is the outgroup.]

So now we have put together a tree that looks like this. Now, we'll test its accuracy by putting it through the test of maximum parsimony analysis.(I feel like we should provide a better explanation of what this means if we're going to include it as a “check” for their data)

Well, what do you know? The analysis agrees with us. Species 2 and 3 are in a group. Species 4 and 5 are in a group. Both of those groups fall into a bigger group.

Which do you think are the four tortoises from Madagascar? Why?

We've just got some new evidence - it turns out there was a list of the names of the seven species of tortoise we found initially. Can you identify which species of tortoise is which?

Today, these are only four species of tortoises native to Madagascar: 2- Pyxis arachnoides 3- P. planicouda 4- Geochelone yniphora 5- G. radiata

This species of tortoise is native to Africa. 6- G. pardalis

This is the Galapagos tortoise. 7- G. nigra

This is the gopher tortoise from North America. 1- Gopherus polyphemus

Walk or Float

Good job! Now you know the relationships among these tortoises and have figured out which ones are from Madagascar. But we can find out something even more interesting. Using the data we have, we can determine when the ancestors of these tortoises came to Madagascar, telling us whether they arrived through dispersal or vicariance. How? We can find out, by using something called the molecular clock, that looks at how quickly DNA has changed based on the known rate of different kinds of mutations. This can tell us if the group from Madagascar became distinct before or after Madagascar split from the other continents 80 million years ago. The rate of divergence (mutation) in turtles/tortoises is .4-.6& per million years (This is about 3 times slower than in mammals.) Knowing this, you can take the data from this chart, and translate it to time. Let's start with sp. 2 (P. arachnoides) and 3 (P. planicauda). The divergence for these two species is 5&. The rate of divergence for tortoises is .4-.6& per million years. If you divide the divergence by the divergence over time, you get time. 5&/.4& or .05/.004= 12.5 .05/.006=8.33 Therefore, the sp. 2 and sp. 3 diverged between 8.33 and 12.5 million years ago. Now lets do it with all of them. Fill in the chart below in your student guide. In the columns marked “my”, fill in the number of million years it took for that divergence. Gene fragment combined data Species P. ara my- my+ P. pla my- my+ G. yni my- my+ G. rad my- my+ P. arachnoides (MAD) P. planicauda (MAD) 0.05 8.33 12.5 G. yniphora (MAD) 0.09 15 22.5 0.09 15 22.5 G. radiata (MAD) 0.07 11.7 17.5 0.07 11.7 17.5 0.06 10 15 G. pardalis (AFR) 0.10 16.7 25 0.09 15 22.5 0.08 13.3 20 0.07 11.7 17.5 *my- means the lower end of the estimate my+ means the upper end of the estimate Write the correct numbers into the list below: here have the students fill in or maybe we give them options and they have to pick, just something to make it interactive ß The two Pixis sp. diverged between [8-12] mya. ß The other Madagascar species, G. yniphora and G. radiata, diverged from one another [10-15] mya. ß The African tortoise sp., G. pardalis, diverged from all of the Madagascar tortoises between [13 and 25 mya]. ß The closest relative on Madagascar to the African tortoise G. pardalis is G. radiata. These two sp. diverged between [12 and 18] mya. According to your data, when did tortoises on Madagascar and tortoises in Africa share a common ancestor? (between 12 and 25 mya) Did this ancestor arrive in Madagascar by walking or floating? Madagascar separated from Africa in the late Jurassic period, around 160 million years ago, and separated from India around 88 mya. So they must have FLOATED!