Interpreting Dog Evolution – Morphological and Genetic Evidence 3/16

Integrated Science 4 Name Per

Ø  Part I. Morphological Characteristics

Lineage from Ancestor Wolf

It is widely accepted that the domestic dog originated from the wild gray wolf (Canis lupus). While some still debate where and when exactly this happened, the general idea is that perhaps certain populations of wolves recognized that humans could serve as a means to obtain food. In turn, the humans may have recognized that the wolves were good companions and could ward off other predators in the wild. It is conceivable that the humans began to care for and travel with tame wolves. These wolves would breed, and perhaps their docile and trusting character traits were passed on to future generations. At some point, humans began to see other desirable traits in the domesticated wolves that led to directed breeding in the hopes of gaining traits beneficial to the humans – the ability to herd, hunt and do work. Eventually the focus then changed to appearance of the domesticated dog and the formation of different breeds.

Think about the dogs in your life – your dog, your friend’s dog, or your neighbor’s dog. There are plenty of them to consider, as 46% of United States households own at least one dog (Humane Society, 2014). Surely you can envision the differences between a golden retriever and a poodle. It isn’t as easy, however, to list the differences between a rough collie and a Shetland sheepdog. Evolutionary biologists have used many different methods to study and describe the evolutionary relationships of organisms. One method relies on careful observation of the appearance of the organisms. This method is founded upon the idea that closely related organisms will have more features in common than those that are distantly related. If you consider the rough collie and the Shetland sheepdog in terms of their evolutionary relatedness, one might think they are closely related... but what about other breeds? In this activity, you will be studying the evolutionary history of 7 dog breeds and the gray wolf. These breeds include: basset hound, cairn terrier, collie, German shepherd, Labrador retriever, pug and Siberian husky.

Domestication of the dog

§  Based on archaeological evidence, the dog was the first animal to be domesticated by humans.

§  Conflicting archaeological evidence estimates that the dog was domesticated in Germany around 14,000 B.C. or even earlier, around 135,000 years ago. Still other scientific data suggest origins in either East Asia or Eastern Europe.

§  There are currently 200-400 breeds of domestic dog worldwide.

§  When comparing the heads of wolves and dogs of similar height and weight, the head of the dog is smaller than the head of the wolf.

Before we begin this analysis, a few reminders about how to build a phylogenetic tree (cladogram). There are several types of phylogenetic trees, just as there are several ways to gather the data used to construct them. We will be building a type of tree called a cladogram. A cladogram is a type of phylogenetic tree in which the lengths of the lines drawn do not represent time in evolutionary history. Cladograms may be depicted in several ways, but the type we will be using here looks like the one presented at right.

In this type of cladogram, the organisms listed at the top are currently living (extant). As you move down the tree, you are moving into the past. Anywhere two lines meet represents a common ancestor. The most recent common ancestor of all of the living organisms is represented at the very bottom of the tree. To construct your cladogram, you will evaluate all of the organisms to determine the similarities between them. Let’s use the above tree and organisms A, B, and C as an example. The first step is to compare all of the organisms in order to find the closest relatives - those that have the most similaries.

Suppose you gather the following data:

Pairs / Similarities
A and D / 4
A and H / 1
A and B / 7

Using this data, we determine that A and B had the most similarities, more than A and H and more than A and D. We see this represented on the example tree because amount of nodes is the largest between A and H showing that there are more physical trait differences between organisms A and H.

When you begin building your own cladograms, you will use the information you enter in Table 2 to determine which pairs of organisms have the most similarities. Keep the following guidelines in mind:

• Organisms to the right of the node have the traits represented by the node. But organisms to the left do not have the trait.

• Organisms to the right are more complex because they contain more of the derived traits.

• Once you determine that a pair of organisms is closely related, you may need to evaluate how closely they are related to other organisms in order to determine where to place the pair on the tree and where common ancestors should be located. When all groups are linked by one common ancestor, the tree is complete. Note that in the example tree there is 1 ancestor, the most recent common ancestor of all of the letters is A.

Procedures

1.  Look at the various animals on the sheet provided by your instructor.

2.  Fill in the Data Table 1 using the guideline of noting the presence (“+”) or absence (“-”) of the trait. Real phylogeneticists would actually take measurements and use numerical data, when or if possible.

3.  Use the data collected and recorded in Data Table 1 to determine the number of similarities between each pair of animals. Record this information in Data Table 2.

4.  Create a cladogram for the various dog breeds being analyzed based on their morphology (appearance.)

Data

Note the presence (“+”) or absence (“-”) of the trait. Record this data in Data Table 1.

It may be helpful to use the following definitions:

§  Short Legs – Are legs well proportioned with the body or are they shorter than they should be to be in proportion? Use a “+” if short legs are present.

§  Skin Wrinkling – Is there an excess of skin such that folds and wrinkles are present? Use a “+” if skin wrinkling is present.

§  Curly Hair – Use a “+” if the hairs of the coat are tightly curled.

§  Floppy Ears – Are ears erect or floppy (laying alongside the head)? Use a “+” if floppy ears are present.

§  Size – Is the overall size of the dog “large” – height greater than 20”, weight greater than 50 lbs? Use a “+” if the size ears are large.

§  Short snout – The veterinary term for this condition is brachycephaly. It is characterized by a snout that accounts for a small percentage of the length of the head (nose to back of the head). Use a “+” if a short snout is present.

§  Furnishings – Long mustache and eyebrow growth. Use a “+” if furnishings are present.

§  Coat color – Is the color is “blended” (more than one color mixed throughout)? Use a “+” if a blended coat is present.

§  Curly Tail - Use a “+” if the tail is not straight and curls around itself.


Data Table 1 - Presence (+) or Absence (-) of Traits in each type of Animal

Data Table 2 – The number of similarities between each pair of animals.


Create a phylogenetic tree (cladogram) for the various dog breeds being analyzed based on the analysis of morphological traits shown in Data Table 2:

Ø  Part 2. DNA Analysis

Introduction

In this exercise, you will be evaluating the relatedness of Canis lupus and the various forms of Canis familiaris by means of assaying for similarities in their DNA. Changes in the DNA at a specific nucleotide position are called Single Nucleotide Polymorphisms (SNPs) and they can be detected by performing gel electrophoresis and RFLP analysis. Because the number of accumulated DNA differences reflects evolutionary relatedness, this information can then be used to construct a cladogram.

Your class has received samples of DNA from the wolf and from dogs of the different dog breeds. The DNA is in the form of a PCR product for a specific SNP in the genome that has been restriction enzyme digested. You will load these samples into an agarose gel and subject the gel to electrophoresis. You will then visualize the bands to perform RFLP analysis and record your observations.

Background

A mutation is a change in an organism’s DNA. Mutations in the wolf DNA have led to the domesticated dogs we are familiar with today. Biologists can use a variety of methods to detect mutations. The chosen method is largely determined by the type of mutation the scientist is attempting to detect. For the purposes of this lab, we will focus on one type of mutation and one method of detection: SNPs and RFLPs. One of the simplest types of mutation is that involving one nucleotide. Such mutations are referred to as single nucleotide polymorphisms (SNPs, often pronounced “snips”). As the name implies, this type of mutation involves a change (“poly” = many, “morphisms” =forms) of one nucleotide. You may think that the detection of one small change out of the millions of nucleotides in the entire dog genome may be nearly impossible (like finding a needle in a haystack). It is actually fairly easy using the tools of biotechnology. To find a SNP, first you need a large amount of the DNA sample. This can be done using a technique called Polymerase Chain Reaction (PCR). Next, a Restriction Fragment Length Polymorphism (RFLP) analysis is performed. Finding one SNP in the “haystack” of the genome is aided by making the genome a more manageable size by focusing in on a region of interest. It is similar to finding an item at the grocery store that you do not usually buy – it helps to know what aisle it is in, and if it is at the beginning, middle or end of the aisle on the right or left side. Often the part of interest involves a gene or a region of one gene. To focus on a region of the genome and replicate it millions of times, scientists use a technique called polymerase chain reaction (PCR). In attempting to determine if a SNP is present in an area of interest, a scientist might start by doing a PCR reaction. This will produce enough DNA in a focused region of interest to work with, but has not revealed any information about the DNA sequence within this region. Now the scientist may choose to make use of restriction enzymes. Restriction enzymes are useful to molecular biologists because they will cleave or cut DNA only within a particular recognition sequence. If the sequence is precisely in tact, the enzyme will cut the DNA. If the sequence is altered even by 1 nucleotide, the enzyme will not cut and the DNA will remain in one piece. This feature allows for DNA sequences to be checked for the loss (or gain) of restriction sites (the site within the recognition sequence where the enzyme actually cuts) and thus the presence or absence of a SNP. The DNA fragment(s) that result are then separated by size using gel electrophoresis. This method can result in segments of DNA that vary in length and generate a particular pattern on a gel. These patterns, restriction fragment length polymorphisms (RFLPs), allow researchers to distinguish changes in the DNA based on the presence or absence of a particular restriction site.

Materials

§  Pre –cut DNA samples from one SNP at one of eight different locations in the genome - The DNA samples within one SNP set are from the wolf, basset hound, cairn terrier, collie, German shepherd, Labrador retriever, pug and Siberian husky.

§  Electrophoresis Chamber

§  1 X TBE Buffer

§  Gel Tray

§  Pre-cast gel using agarose (0.8%)

§  Gloves

Procedures

1.  A 0.8% agarose gel has been prepared for you. Place the gel tray onto the platform in the electrophoresis chamber so that the comb slots are closest to the black or negative electrode. * You may need to remove the comb before placing the gel on the gel tray.

2.  Pour 1 X TBE buffer into the electrophoresis chamber until it covers the gel completely.

3.  Carefully remove the comb from the gel. Use both hands to grasp the ends of the comb and pull straight upward with a steady motion. Do not tear the agarose or the wells will be useless. Make sure the wells are completely submerged in buffer.

4.  Load the DNA Samples. Set the volume of a micropipetter to 10 µl. Load 10 µl of each DNA sample into its corresponding well in the agarose gel. Steady the pipet tip so that it is under buffer and over the well. Be careful not to puncture the bottom or sides of the well. The loading dye makes the sample denser than the buffer so that it sinks to the bottom of the well. TO AVOID CONTAMINATION, CHANGE TIPS FOR EACH DNA SAMPLE.

Gel Electrophoresis

a.  Put the cover tightly on the electrophoresis chamber and, with the power supply off, connect the electrical leads to the power supply. The leads should be connected anode (+) to anode (red to red) and cathode (-) to cathode (black to black).