biology phylogenies

Lab 12 Building Phylogenies

Objectives In this laboratory exercise, you will examine six species of agaricomycetes and predict the evolutionary

relationships among them. After completing this exercise you will be able to

• define ancestral characteristics, derived characteristics, branch point, and phylogeny. • predict ancestral and derived characteristics for agaricomycetes. • construct a phylogeny (phylogenetic tree). • support the phylogeny with data. • explain how evolutionary biologists discover evolutionary relationships.

Introduction One of the most compelling pieces of evidence for evolution is that organisms have amazing similarities. An

example that almost everyone has heard before is that the limbs of birds, bats, horses, moles, cats, frogs,

humans, turtles, and other vertebrates have virtually the same skeletal plan. Furthermore, even snakes and

whales show structural remnants of the limbs of their ancestors. The evolutionary interpretation of these

similarities is that the vertebrate limb has been modified by natural selection to perform different functions

(for example, running, digging, flying). Another commonly used example is that the embryos of turtles, mice, humans, chickens, and many other vertebrates are amazingly similar. Furthermore, the proteins and

DNA of organisms are remarkably similar. Why, do you suppose, can human diabetics use insulin extracted

from pigs to control their blood sugar levels? Well, the reason is that the chemical structure of human and

pig insulin is very similar.

In addition to these similarities, we discover that organisms that appear similar in one respect are often

similar in other respects (we can say the patterns are “concordant”). For example, organisms that are similar morphologically (in shape) have similar protein structures. Organisms that are less similar morphologically have less similar protein structures. This pattern holds for traits that are not easily

modified by evolution, but not so often by traits that are easily modified by selection. For example, flower

color might not be a good trait to use when looking for concordance because it is easily changed

genetically.

The concordance of traits is an important support of evolution. Imagine that we saw that organisms similar

in one set of characteristics were very different in a second set of characteristics and different again in a

third set of characteristics. This situation would be chaotic and we would be forced to question the reality

1

of evolution. The development of methods of DNA and protein analysis has shown dramatically that

organisms that are similar morphologically are also similar at the genetic level.

So, similarity among organisms provides evidence for evolution. We can then turn around and use the

similarities to try to reconstruct evolutionary relationships. That is the purpose of today’s lab: to construct a

hypothesized evolutionary relationship (a phylogeny or phylogenetic tree) of a group of organisms you probably know very little about: fungi.

Fungi are eukaryotic, heterotrophic, achlorophyllous organisms closely related to animals within Domain

Eukarya. A unifying character among fungi is that they are osmotrophic and possess cell walls containing

chitin. There are at least eleven major phylogenetic fungal lineages (Hibbett et al, 2007).

Fig. 1. Image modified from Hibbett et al. 2007. A higher level phylogenetic classification of the Fungi. Mycol. Res. 111:509-547.

Fungi are highly diverse in form and function and truly are cosmopolitan. Some fungi are aquatic and have

flagellate spores (Chytridiomycota) and are responsible for the demise of amphibians worldwide. Others are beneficial living inside digestive systems of herbivore mammals (Neocallimastigomycota) or hindguts of arthropods (Kickxellomycotina) while others kill nematodes (Zoopagomycotina). Most fungi are important as decomposers in all ecosystems such as the sugar fungi (Mucormycotina). Almost all vascular plants benefit from associations with fungi to their roots for increased water and nutrient uptake

(Glomeromycota) and humans have domesticated important strains of yeast and molds for industrial purposes (Ascomycota). And, people have always wondered about the magic emergence of mushrooms and their effects on the human body (Basidiomycota).

Evolutionary relationships in fungi have been reconstructed using morphology and DNA. Milestones in the evolution of fungal characters are shown In Fig. 2. Spores (reproductive and dispersal units of fungi) and hyphae (unit of filamentous fungi) are shown as earliest signs of fungal presence. Other traits emerged later giving rise to specialized structures and defining groups who exhibit them. This is the case of Basidiomycota

2

and Ascomycota, the most recently evolved fungal taxa, both of which share presence of dikaryotic hyphae

together with complex sexual and asexual reproductive structures.

Fig. 2. Evolution of fungal characters, based on molecular clock dating. a)Flagellate spore; b)Coenocytic filament; c)Septa; d)Clamp connections; e)Phragmobasidia; f)Asexual spores; g) Asci; h)Ascoma; i)Holobasidium; j)Basidioma. From Berbee & Taylor. 1993. Dating evolutionary relations of the true fungi. Can.J.Bot. 71:1114-1127

For this laboratory exercise you will study a handful of Basidiomycota, in the class Agaricomycetes. Fungi in this group include the mushrooms, puffballs, shelf fungi, jelly fungi, chanterelles, stinkhorns and others.

These fungi represent 98% of all described species in the Basidiomycota. Observe a working classification of this group in the image below. The specimens you have in front of you may be placed in several of the

orders within class Agaricomycetes (Fig. 3).

3

Fig. 3. Classification of Basidiomycota in its sub-lineages. Images taken from the website Tree of Life. URL: http://tolweb.org/Fungi/2377

How to reconstruct a phylogeny Think about the kind of diagram (phylogeny) you need to draw. Your goal is to develop and draw a phylogeny. A phylogeny is a reconstruction of relationships between taxa. The more characters organisms

share between them, the closer related they are. The basic assumption involved in making an evolutionary

tree (phylogeny) is that two organisms that have a more recent common ancestor will share more characters than two organisms with a less recent common ancestor. A character can be any descriptor:

from anatomical structures to DNA. One of the greatest strengths of constructing an evolutionary tree

using this method is that it is testable. The discovery or addition of characters should strengthen your

phylogenetic hypothesis. If, for example, new evidence is found you could add it to your evolutionary tree

to either support or reject your hypothesized evolutionary sequence.

How would you approach the reconstruction of a phylogeny for a lamprey, a shark, a frog, a dog, a cow, a monkey and a human? Probably you already know that the dog, cow, monkey and human are more related

between themselves than to the rest, but how so? How can you justify your assumption? First, you need to construct a table in which you write as many characters you can find and then score them (ideally with 0=absent, 1=present).

Lamprey Shark Frog Monkey Cow Human Vertebral column 1 1 1 1 1 1

Jaws 0 1 1 1 1 1 Hair 0 0 0 1 1 1

Loss of tail 0 0 0 0 0 1 Habitat marine marine amphibian arboreal terrestrial terrestrial

Based on your results you then decide which characters are ancestral (shared by more than one taxon) or derived (if they are unique to the taxon) to design your phylogeny (Fig 4a). The lamprey shares the one character with the rest and so it is at the base of the phylogenetic tree, serving as the outgroup, a group

4

that is thought to have traits similar to the ancestor of the group being studied. In other words, it is the

most ancestral taxon in this analysis. By default, the taxon at the tip of the phylogenetic tree will be the most evolved (the one with most derived characters). While building a phylogeny, it is important you understand that derived characters are the most useful to determine the position of taxa and that the more

characters you use the stronger your phylogenetic tree will be. Case in point: which mammal is more

related to the human: cow or monkey? How can you support a correct phylogenetic relationship among them based on the table above? Not possible. You need to elaborate on additional data. Better phylogenies have several traits showing the same result. This concordance of traits gives you more confidence in your conclusions.

Fig. 4. A phylogeny showing evolutionary relationships among vertebrates. The lines represent evolutionary

pathways that lead to groups. In this example, lamprey is the “outgroup” for the “jawed” vertebrates. Branching points show when two groups diverged. The “hatch” marks mark evolutionary innovations that unite descendant groups.

Finally, notice that not all characters you choose may end up being appropriate; this is the case of vertebral

column because all taxa (in our example) have it, or in the case of habitat a character harder to justify; in the end these may not be needed in your phylogeny. The phylogeny in Fig 4b is the same as in Fig 4a but it

appears to be more concordant in that it relies on more derived characters (resulting from a more detailed character table).

Phylogenetic relationships also vary depending on the number of taxa used. Observe Fig 5 and notice how

4a

4b

5

the use of characters changes its placement within the phylogeny.

Fig. 5. Two possible phylogenies showing evolutionary relationships after the addition of a horse to the earlier

phylogeny. In 4a, we hypothesize that the lengthened foot evolved once, in a lineage leading to both cow and

horse. In 4b, we hypothesize that the lengthened foot arose twice, once in the lineage leading to the cow and

once in the lineage leading to the horse. (Note that we wouldn’t put this character on the slanted line near the appearance of hair because it would then have to “disappear” before the divergence of monkeys.) So, because we have a complicated trait that is likely to evolve only once, we would use the first phylogeny—a.

It is likely you will end up building more than one phylogeny, all of which correctly address the problem.

But, which one is the best one? A better phylogeny preferably does not have reversals of character states, that is, you hypothesize fewer evolutionary changes to explain your phylogeny. This idea is called the

principle of parsimony. In Fig 5b, “walking on toes” requires an extra evolutionary step to separate cow from horse, whereas in Fig 5a it is assumed that the foot and toes only changed once.

Assignment: Build a Phylogeny of Fungi Your job is to construct a phylogeny for six agaricomycetes species. Your goal is to hypothesize evolutionary relationships among these species and show the characters that led you to your hypothesis. You will show your hypothesis and the table of characters that support your hypothesis in an evolutionary (or phylogenetic) tree, a diagram that shows where evolutionary innovations arose. 1. Examine the boxed set of agaricomycetes. Notice one of the specimens is labeled as “outgroup”. Write

down the names for all species and look for their file names in the “Phylogeny of Fungi” SAKAI webpage (see “instructions” below). Each file will include an image and a description. See the accompanying diagrams (Figs 5-7) to learn what the traits are.

2. Examine the characters of the agaricomycetes and of the outgroup (Cladina). Characters that are similar

5a

5b

6

between the outgroup and agaricomycetes (e.g., living on soil, dijkaryon) will not help build a phylogeny. You will use only evolutionary innovations in building your phylogeny; ancestral traits will not help you.

3. Next, choose characters that you think might be useful in building the phylogeny. Choosing which characters to use is one of the most important parts of building a phylogeny. The following tips will help

you choose characters, but different people will probably use different characters.

a. Avoid traits that you think are easily changed by evolutionary processes (e.g., color of basidiomata).

b. Choose traits either from the pictures or the list of traits and compare the “states” of those characters to those of the out group. The character state of the out group is considered to be

the ancestral state. For example, the presence of mycelium (see Fig. 2) is an ancestral trait because it also occurs in Cladina lichens. The presence of the basidium is an evolutionary innovation in Agaricomycetes because it does not occur in Cladina. Any change from that state is an evolutionary innovation. Non-varying traits won’t tell you anything about evolution within

the group.

c. Character states can be “presence” vs. “absence” or variations of a character that is “present” (e.g., hymenium enclosed or exposed in different shapes like gills or tubes).

4. When you find traits that vary among agaricomycetes species, list the various character states and then decide which of the character states is likely to be most advanced, least advanced, and intermediate.

For example, agaricomycetes in terms of basidiocarp shape: stipitate, sessile, gasteroid, etc. In this case,

you would need to decide which one is the most primitive. Reference to the character state of the out

group will help you make these decisions. One caution: loss of a character is relatively easy.

5. Think about the kind of diagram (phylogeny) you need to draw. 6. To start building a phylogeny, pick one trait (maybe basidiocarp or spore shape) and draw a phylogeny

using just the one trait. When building a phylogeny based on just one trait, you will probably not be

able to mark changes at all the points of evolutionary divergence.

7. Next, pick other traits and draw new phylogenies. You don’t need to draw phylogenies for all the traits listed but you should draw a good number. It is important to realize that some phylogenies are the

same even though the order of species is not the same.

8. Now you need to choose the better phylogeny using the principle of parsimony (the least amount of evolutionary changes to explain your hypothesis).

9. Strengthen your phylogeny by adding several traits to each taxon. Note that concordance of traits gives you more confidence in your conclusions.

10. Turn in your phylogeny (follow directions from your instructor). Also attach a list of the characters you used and show the ancestral and derived states of those characters.

Instructions on how to access Resources Login to USAOnline (Sakai). In “My Workspace” click on “Memberships” found on the left-hand column. Click on “Joinable Sites” and look for “Phylogeny of Fungi” (listed alphabetically). Upon joining the site you will see the link in your upper tab of Sakai. Under “Resources” you will find descriptions for all fungi; download and print (optional) files in your box. If you need extra help, there are useful weblinks and videos related to this assignment. Once you are finished with this class, you can unjoin this webpage.

7

Fig. 5. General line drawings for typical agaricomycetes. Taken from Hawksworth et al. 1983. Ainsworth & Bisby’s Dictionary of the Fungi.

8

Fig. 6. Terminology associated to mushrooms. Image taken from www.urbanmushrooms.com.

9

Fig. 7. Lichen forming fungi. Images taken and modified from: digilibraries.com

A, Thallus of foliose lichen (Parmelia) with apotthecia.

B, Cross section of one apothecium, × 5.

C, Section through the body of a gelatinous lichen (Collema), showing the photobiont (Nostoc) individuals surrounded by the fungus filaments, × 300.

D, a spermagonium of Collema, × 25.

E, a single Nostoc thread.

F, spore sacs and paraphyses of Usnea, × 300.

G, Protococcus cells and fungus filaments of Usnea.

A, Fruticose lichens on wood

B, Usnea, with apothecia

C, Sticta,

D, Peltigera, with apothecia

E, cross section of a single apothecium

F, Cladina, with apothecia

G, Crustose lichen (Graphis) on bark

H, Soredium of a lichen.

10

Your Name___________________________________________

11