Copyright© 2024 by Department of Integrative Biology, University of California, Berkeley

Macroevolution of the Horse:

Investigating hypotheses about adaptive
radiation

Things to do before lab:

Please plan to have a printed or digital copy of this introduction with you in
lab. You will NOT have access to it on the lab computers.

1) Read this introduction before lab and bring a digital or printed copy to lab for
reference.

2) Become familiar with the following terms. You should be able to define these
terms before coming to lab:
● Macroevolution ● Adaptive radiation
● Microevolution ● Cladogram
● Speciation ● Phylogeny
3) Complete pre-lab videos and activities on bCourses

Goals and Objectives

1. Use morphological and stratigraphic data to construct a phylogeny
2. Use a phylogeny to test an evolutionary hypothesis
3. Interpret your phylogeny to draw conclusions about macroevolutionary processes and
patterns

Macroevolution
Most evolutionary biologists define macroevolution as evolution above the species level.
Macroevolution encompasses such events as the formation of higher taxa (genera, families,
orders, etc.), the origin of novel phenotypes, the appearance of new organisms in
unexploited habitats (adaptive radiations), morphological trends, and episodes of mass

Macroevolution of the Horse: Page 1 of 10

 Copyright© 2024 by Department of Integrative Biology, University of California, Berkeley

extinction. It includes neither evolutionary change within or among populations

(microevolution) nor the formation of new species (speciation).

Evidence for macroevolution is generally found in the fossil record. Therefore, the tasks of
describing and explaining macroevolution fall mostly to paleontologists. Paleontologists
describe macroevolutionary patterns by collecting data from fossils. Fossils are the
remains, impressions or traces of organisms that have been preserved in the rocks that
compose the earth’s crust. Fossils can be placed into an evolutionary framework with the
use of cladistics, and thereby can be used to understand the patterns of macroevolutionary
change. By combining fossil data with evidence for environmental change, it is possible to
understand the causes of macroevolution.

Adaptive Radiation
In this lab, it is important to differentiate the terms
evolutionary radiation and adaptive radiation. In an
evolutionary radiation, a clade undergoes a high rate of
speciation in a relatively short period of geological time.
To the right is an image of a phylogeny that shows an
evolutionary radiation (indicated in the dashed oval). The
forks in the lineage branches represent speciation events. A
non-adaptive evolutionary radiation occurs when a large
population is broken into many isolated locations, none of
which provides a wider range of habitat types than existed
previously. Even without new habitat types, a radiation can
happen as speciation proceeds independently in each isolated
location.
Adaptive evolutionary radiations usually occur in
response to the opening of new ecological niches. In
ecology, the term “niche” describes the role an organism plays in a community. A species’
niche includes the physical and environmental conditions it requires (such as temperature or
pH), the effects it has on resources (such as water or minerals), and the interactions it has
with other species (such as mutualism, predation, or competition).

Typically, new niches might arise from either of two possible scenarios. In one
scenario, an adaptive radiation occurs in response to the evolution of a new beneficial
phenotype. “Beneficial phenotype” refers to a new character (or combination of characters)
that results in higher fitness in a particular environment for those organisms that possess the
character(s). Organisms with this new character or characters might be able to out-compete
other species that were already in that environment, filling all of the ecological niches those
organisms previously occupied. Alternatively, the new character(s) might allow an
organism to find new ecological niches (also called “open niches”) that no existing
organisms had been able to utilize (as when plants and animals adapted to life on dry land).
If the radiation in the figure above were an adaptive radiation caused by a new beneficial
character (or characters), there would be important character changes at the base of the
radiation, where the dashed line crosses the lines of the phylogeny.

Another scenario that can produce an adaptive radiation occurs when the environment
changes and creates new niches that had not previously existed in a location. An adaptive
radiation driven by a major environmental change might be accompanied by other evidence
Macroevolution of the Horse: Page 2 of 10
 Copyright© 2024 by Department of Integrative Biology, University of California, Berkeley

produced by that environmental change (e.g., changes in atmospheric CO2 content, as

revealed in gas bubbles in ancient ice, or changes in rainfall that change sedimentation rates
revealed in the rock record). Such an adaptive radiation might also occur when organisms
are among the first to occupy a new and ecologically variable landscape. As, for example,
when members of a bird or plant species are among the first founders of populations on a
remote oceanic island archipelago such as the Galapagos Archipelago or the Hawaiian
Archipelago.

Because major environmental changes often impose strong selection on characters, which
can cause those characters to evolve rapidly, an environmental change can quickly make it
appear that the evolutionary radiation is being driven by the evolution of a new beneficial
character. Thus, it can be difficult to differentiate between these two root causes of any
particular evolutionary radiation. Nevertheless, an evolutionary radiation that coincides
with either the evolution of a beneficial phenotype or with a major environmental change
(or with both) is likely to fit the definition of an adaptive radiation. Therefore, evolutionary
biologists map information about both character transitions and environmental changes
onto phylogenetic trees to test hypotheses about possible causes of evolutionary radiations.

Biologists use different terminology to indicate the type of information contained in any
particular phylogenetic tree. In this lab we use the term cladogram to refer to a branching
diagram that only represents the evolutionary relationships among taxa.

Macroevolution of the Horse

Horse evolution has long been the subject of paleontological research, both because horses
have been important to people throughout history and because early paleontological
research recognized that horse fossils provide an interesting example of macroevolution.
Horses underwent a dramatic evolution in the relative crown height of their teeth (called
hypsodonty, which is defined and illustrated below), as well as in the shape of their
muzzles and feet, through the Tertiary period (see the geologic timescale on the last page).
While paleontologists have long suggested that evolutionary changes in horses are related
to a change in environment (i.e., the development of grasslands), recent data have begun to
highlight inconsistencies with this hypothesis. To test the hypothesis that horses
experienced an adaptive radiation in response to a major environmental change, we will
combine evidence related to the development of grasslands with a hypothesis of the
evolutionary history of these taxa.

Your Goals
In this lab, you will test the hypothesis that a major environmental change (the
development of grasslands) led to an adaptive radiation in horses. This study will
require an understanding of the evolutionary history of horses (as presented in a
cladogram), the rate of their taxonomic diversification, the timing of changes in their
morphology, and the timing of the development of the grasslands.
The steps in today’s labs are to:
1. Gather morphological data related to:
a. Hypsodonty
b. Skull morphology (muzzle shape and postorbital bar)
c. Foot morphology (number of functional toes)
2. Create a phylogeny and relate it to the hypsodonty scores of the taxa
Macroevolution of the Horse: Page 3 of 10
 Copyright© 2024 by Department of Integrative Biology, University of California, Berkeley

3. Interpret your phylogeny with respect to the adaptive radiation hypothesis

Gathering Morphological Data

Your group will develop criteria for determining the character state for each character
(hypsodonty, foot morphology, skull morphology). Record these criteria under
‘Methods’ on your worksheet before collecting your data. Feel free to visit the stations
to familiarize yourself with each character before you write your Methods.

Hypsodonty & Tooth Morphology

Mammalian teeth are composed of two main parts, the crown and the roots. The crown is
the part of the tooth used for chewing food, while the roots anchor the tooth in the jaw, just
like the roots of plants. The crown of the tooth is distinguished from the roots by the
presence of the enamel-dentine junction, the line below which there is no enamel
surrounding the tooth (the enamel-dentine junction is marked by a black line on the tooth
models you will measure). You will be focusing primarily on the shapes of the tooth
crowns.
For mammalian molars, the height of the crown relative to its length (see panels A and
B in the figure below) is an important indicator of how the teeth are used. Mammals
that eat soft foods, such as carnivores and many omnivores, generally have low-crowned
molar teeth, with a crown that is not much taller than its length. Mammals such as horses,
which eat more abrasive foods, generally have high-crowned teeth, with a crown much
taller than its length. In order to access the nutritious contents of plant cells, herbivorous
mammals use their molars to grind and crush plant material. Grasses have little stones in
their cells, which makes them more abrasive than the leaves of other plants, which lack
these mineral deposits. A diet of abrasive, fibrous foods such as grasses inevitably leads to
erosion of enamel from the surface of the crown. Having high-crowned teeth (high
hypsodonty index) slows the damage that results from a diet of grasses. Thus,
high-crowned teeth are considered one of the most important characters for recognizing
adaptation to eating grasses.

Macroevolution of the Horse: Page 4 of 10

 Copyright© 2024 by Department of Integrative Biology, University of California, Berkeley

One way to measure crown height of a species is by calculating a hypsodonty index,

which is the crown height divided by the crown length. See the illustration above for
how crown height and length are measured. By removing the absolute size of the tooth,
the hypsodonty index gives a relative measurement of tooth shape. Larger animals
generally have larger teeth, and the hypsodonty index prevents evolutionary changes in
overall body size from obscuring evolutionary changes in tooth shape.
For this week’s lab, you will calculate the hypsodonty index for each horse group from
measurements you obtain from the plaster casts of their teeth. The base of each tooth
you will be measuring is labeled with a number, which corresponds to its taxon, as
listed in the following table:
Taxon Identification number
Equus sp. 4
Nannippus sp. 7
Hipparion sp. 6
Merychippus sp. B 3
Megahippus sp. 5
Miohippus sp. A 2
Epihippus sp. 1

Use a ruler to take your measurements in millimeters and record them in Table 1
of your worksheet. Once you have completed your measurements, use a calculator to
determine the hypsodonty index for each species and enter all three values (height,
length, and hypsodonty index) in the data table.

Foot Morphology
Changes in foot morphology also reveal changes in the behavior of horses through time. In
general, animals that have more toes on the foot have greater maneuverability. Having more
toes results in more interaction with the ground and thus better control of movements.
However, greater maneuverability imposes trade-offs. In general, faster animals have fewer
toes (and thus lower overall foot weight). Having a lighter foot reduces the force necessary
to accelerate it at the end of a long limb and thus, the animal can accelerate faster. Long
limbs and lightweight feet also yield a very efficient limb motion, allowing animals to
cover great distances with relatively little expenditure of energy. As a result of this efficient
motion, animals with long limbs and lightweight feet are capable of rapidly escaping
predators and traveling long distances in search of food, both of which are important for
herbivores in open grasslands, where there are few places to hide and where long distance
travel is necessary to attain sufficient sustenance from low quality food (i.e., grass).

For this week’s lab you will be using the illustrations of horse feet provided in lab to
determine the number of functional toes on the foot of each taxon. You will count the
number of toes, including the main hoof, and then record this value in Table 2 on your
worksheet.

Skull Morphology
The shape of the skull often reflects the method of feeding in mammals. This week, you
will be analyzing two aspects of skull morphology to recognize adaptation to grazing on

Macroevolution of the Horse: Page 5 of 10

 Copyright© 2024 by Department of Integrative Biology, University of California, Berkeley

grass. First, you will examine the shape of the muzzle and second you will look for the
presence of a postorbital bar.
Muzzle shape
The shape of an animal’s muzzle, or its protruding snout, often reveals its feeding
habits. Animals that feed on grasses and other monocots are called grazers, whereas
animals that feed on trees and bushes (dicots) are called browsers. A flat or squared-off
muzzle (below right) enables grazers to efficiently crop grass at ground level, like a lawn
mower. Browsers tend to have a rounded muzzle with teeth that are oriented in different
directions, which enables animals to pull multiple leaves off trees or bushes at the same
time. A narrow, rounded muzzle (below left) is also more effective at selecting the best and
most succulent leaves from a branch.

rounded squared-off
Muzzle shapes of horses. These outlines represent the shape of the lower jaw,
looking down on the front lower teeth of the animal from above.

For this week’s lab, you will be looking at plaster casts of skulls to determine if each
taxon had a rounded muzzle or squared-off muzzle. Note your observations by
recording them in Table 2 of your worksheet.
Presence of a Postorbital Bar
The second character of skull morphology we will consider is the presence or absence
of a postorbital bar, the bone that closes the back of the eye socket (shown below). A
postorbital bar is the piece of bone that separates the eye from the temporalis muscle, the
main muscle used in chewing food. In order to break down tough, low-quality plant
material like grasses, grazers must chew each mouthful repeatedly, and as a result have
strong temporalis muscles. The development of a complete postorbital bar likely supported
the increased bulk of the temporalis muscle that was necessary for grazing, so that its
presence indicates an evolutionary shift towards grazing. Early horses, such as
Hyracotherium sp., lack a closed eye socket, but modern horses (Equus equus) have a
postorbital bar.

For this week’s lab you will need to determine which of the skull casts have complete
postorbital bars and which do not. In order for a skull to be considered as having a
postorbital bar, it must have a complete bar. Even if the skull has the beginnings of a
postorbital bar, if the bar does not completely enclose the eye socket, it should not be
considered as having a postorbital bar. In order to determine if a skull has a postorbital bar,

Macroevolution of the Horse: Page 6 of 10

 Copyright© 2024 by Department of Integrative Biology, University of California, Berkeley

closely examine each skull and look for a complete rod of bone behind the eye socket. Note
your observations by recording them in Table 2.

Taxon Stratigraphic range (MYA)

Equus sp. 3-0
Dinohippus sp. 9-5
Calippus sp. 15-7
Nannippus sp. 9-2
Hipparion sp. 14-9
Merychippus sp. A 22.5-17.5
Merychippus sp. B 19-15
Merychippus sp. C 17-13
Parahippus sp. 24-14
Megahippus sp. 15-12
Miohippus sp. B 32-26
Miohippus sp. A 37-29
Epihippus sp. 46-40
Hyracotherium sp. 55-49

Creating a Phylogeny Based on Hypsodonty

Step 1: Preparing your graph
To create your phylogeny, you will first need to create a graph with geologic time on
the vertical axis and hypsodonty on the horizontal axis. Using the graph paper provided,
plot the time axis with the most recent time (0 million years ago) towards the top of the
page. Use the table above, which includes the stratigraphic ranges for all taxa included in
this study, to determine the range of dates during which each taxon appears in the fossil
record.

To determine the range of hypsodonty index values necessary for your horizontal axis, refer
to the hypsodonty index values you recorded in your data table (Results Table 1).

Step 2: Plotting Hypsodonty

Plot the hyposodonty index values along the horizontal axis of the graph (at the bottom of
the page). To plot the hypsodonty index of each taxon on your phylogeny, draw a vertical
rectangle indicating the stratigraphic range of the taxon above its calculated hypsodonty
index. For example, if you found that Acritohippus sp. had a hypsodonty index of 1.4 and
was present from 24 to 20 million years ago, you would plot a vertical rectangle that
stretches from 24 to 20 million years ago (on the vertical axis) at 1.4 on the hypsodonty
index scale (horizontal axis) and label this rectangle as Acritohippus sp.

Step 3: Aligning your phylogeny with a cladogram

After you have plotted a graph in which the rectangles represent the taxa, you will use the
following cladogram to determine how the taxa are related. In other words, you will use
this cladogram to determine how to connect the rectangles with lines.

Macroevolution of the Horse: Page 7 of 10

 Copyright© 2024 by Department of Integrative Biology, University of California, Berkeley

Cladogram of horse species:

When drawing lines to connect species, connections should be made from the bottom of a
taxon’s stratigraphic range (i.e., flush with the bottom of the rectangle – see figure below).
By doing this, you are indicating that a speciation event occurred prior to the appearance of
daughter species in the fossil record. Although we do not know precisely when the common
ancestor (represented by a node) of two taxa occurred, we do know that the common
ancestor existed prior to the origin of the descendent taxa.

Connections also need to be drawn with the timescale in mind. Careful placement of the
nodes will ensure that when you are drawing connections, none of the lines are angled
downward, which would indicate a shift backwards in time (time always moves forward).
In addition, if a horizontal line must cross a vertical line or a bar representing the
stratigraphic range of another taxon, you should leave small gaps, such that you do not
cross the lines or bars with the connecting lines. Adding intersecting lines could
unintentionally indicate a common ancestor.

The figure below illustrates how to make the connections in your phylogeny, and how to
label the taxa. The vertical rectangles indicate the stratigraphic range (chronology, or
timing) of each taxon and are ordered left to right according to the hypsodonty index of
each taxon (not shown). The names of taxa are written vertically above the rectangles.
Connecting lines are drawn flush with the bottoms of the rectangles. Small gaps are left
where connecting lines would intersect with other lines or with the rectangles.

Macroevolution of the Horse: Page 8 of 10

 Copyright© 2024 by Department of Integrative Biology, University of California, Berkeley

Step 4: Plotting Other Morphological Character Transitions onto your

Phylogeny
After you have connected the taxa based on ancestral relationships, map the other three
morphological characters that you analyzed onto the phylogeny (as you did in the
Primate Phylogenetics lab). To describe the evolution of foot shape, you should mark on the
phylogeny where you think changes in the number of toes evolved. In order to track the
evolution of muzzle shape, you should mark on the phylogeny where you think the
character of the horse muzzle changed. Finally, in order to denote the development of a
complete postorbital bar, mark where this trait first appeared in completion on the
phylogeny. Indicating the evolution of these morphological traits on your phylogeny will
enable you to develop hypotheses about the pattern and possible causes of phenotypic
evolution in these taxa.

References:
Eldredge, N., and S. J. Gould. 1972. Punctuated Equilibria: an alternative to phyletic gradualism. Pp. 82-115. In T. J. M.
Schopf, ed. Models in Paleobiology. Freeman, Cooper, and Co., San Francisco, CA.
Froehlich, D. J. (2002). Quo vadis Eohippus? The systematics and taxonomy of the early
Eocene equids (Perissodactyla). Zool. J. Linn. Soc. 134: 141-256.
Hulbert, R. C., Jr. and B. J. MacFadden (1991). Morphological transformation and cladogenesis at the base of the adaptive
radiation of Miocene hypsodont horses. Am. Mus. Nov. 3000: 61.
Kelly, T. S. (1995). New Miocene horses from the Caliente Formation, Cuyama Valley
Badlands, California. Contributions in Science, Los Angeles County Museum 455:
1-33.
Kelly, T. S. (1998). New middle Miocene equid crania from California and their implications for the phylogeny of the
Equini. Contributions in Science, Los Angeles County Museum 473: 1-44.
MacFadden, B. J. (1998). Equidae. In: Evolution of Tertiary Mammals of North America.
Volume 1: Terrestrial Carnivores, Ungulates, and Ungulatelike Mammals, C. M. Janis, K. M. Scott and L. L. Jacobs, eds.
pp. 537-559, Cambridge University Press, Cambridge.
MacFadden, B. J. (2005). Fossil horses: evidence for evolution. Science 307: 1728-1730.

Macroevolution of the Horse: Page 9 of 10

 Copyright© 2024 by Department of Integrative Biology, University of California, Berkeley

Macroevolution of the Horse: Page 10 of 10