COEVOLUTION

First some definitions: coevolution is a change in the genetic composition of one

species (or group) in response to a genetic change in another. More generally, the idea
of some reciprocal evolutionary change in interacting species is a strict definition of
coevolution.

At first glance (or thought), it might seem that everything is involved in coevolution.

This assumption might stem from the fact that virtually all organisms interact with
other organisms and presumably influence their evolution in some way. But this
assumption depends entirely on ones definition of the term Coevolution.

The term is usually attributed to Ehrlich and Raven's study of butterflies on plants
(1964) but the term was used by others prior to 1964 and the idea was very present in
the Origin of Species. Ehrlich and Raven documented the association between species
of butterflies and their host plants noting that plants' secondary
compounds (noxious compounds produced by the plant) determined the usage of
certain plants by butterflies. The implication was that the diversity of plants and their
"poisonous" secondary compounds contributed to the generation of diversity of
butterfly species.

Here we have a very general observation of one group of organisms having an

influence on another group of organisms. Is this coevolution? Some would argue that
it is not good evidence for coevolution because the reciprocal changes have not been
documented clearly. Like the issue of defining an adaptation, we should not invoke
coevolution without reasonable evidence that the traits in each species were a result
of or evolved from the interaction between the two species.

Lets consider plants and insects: there is little evidence to determine whether plants'
secondary compounds arose for the purpose of preventing herbivores from eating
plant tissue. Certain plants may have produced certain compounds as waste products
and herbivores attacked those plants that they could digest. Parasites and hosts:
when a parasite invades a host, it will successfully invade those hosts whose defense
traits it can circumvent because of the abilities it caries at that time. Thus presence of
a parasite on a host does not constitute evidence for coevolution. These criticisms are
quite distinct from the opportunity for coevolution once a parasite has established
itself on a host.
The main point is that any old interaction, symbiosis, mutualism, etc. is not
synonymous with coevolution. In one sense there has definitely been "evolution
together" but whether this fits our strict definition of coevolution needs to be
determined by careful 1) observation, 2) experimentation and 3) phylogenetic
analysis.

The classic analogy is the coevolutionary arms race: a plant has chemical defenses,
an insect evolves the biochemistry to detoxify these compounds, the plant in turn
evolves new defenses that the insect in turn "needs" to further detoxify. At present the
evidence for these types of reciprocal adaptations is limited, but the suggestive
evidence of plant animal interactions is widespread. An important point is
the relative timing of the evolution of the various traits that appear to be part of the
coevolution. If the presumed reciprocally induced, sequential traits actually evolved in
the plant (host) before the insect (parasite) became associated with it, we should not
call it coevolution. See different example figs. 22.6-22.7, pgs. 621-622 + text.

There are a variety of different modes of coevolution. In some cases coevolution is

quite specific such as those between two cellular functions. The endosymbiont theory
proposes that current day mitochondria and chloroplasts were once free-living
unicellular individuals. These cells entered the cytoplasm of other cells, an example of
the general phenomenon of endosymbiosis. Current-day mitochondrial and
chloroplast genomes are much smaller than the genome sizes of their presumed free-
living ancestors. Some of this reduction in genome size is due to the transfer of
genes from organelle genomes to the nuclear genome. Thus, being in the cellular
environment has influenced the evolution of organelle genomes. There is evidence
that the faster rate of evolution of animal mitochondrial DNA has accelerated the rate
of evolution of some of the nuclear genes that function in the mitochondria. Thus
there is some evidence for reciprocal phenomena

Other modes of coevolution involve competitive interaction between two specific

species. The Plethodon salamander study is a good example: two species are
competing: in the Great Smoky mountains the two species compete strongly as
evidenced by the fact that each species will increase population size if the other is
removed. Here there is a clear reciprocal interaction between the two populations
(species), each affecting the other.

[The role of competition between species, the coevolutionary responses to this

competition and the consequences for the evolution of communities is illustrated in
the Anolis lizard fauna of the Caribbean. There is coevolution because the competitive
interactions between resident and invading species of Anolis involve reciprocal
responses in the evolution of body size. These affect the structure of the lizard
community as evidenced by the general pattern of there being a single species of
lizard on each island.]

Character displacement also provides and example of a pattern we might interpret

as the result of coevolution. Mud snails show pattern of character displacement in
sympatry due presumably to competition for food items (don't confuse this with
reinforcement; the selective agent here is not reduced hybrid fitness). We might call
this co evolution because both species show a shift when compared to allopatric
samples of each species (mean of both ~ 3.2 in allopatry vs. ~ 4.0 and ~ 2.8 in
sympatry). If only one species exhibited character displacement and you were a really
picky evolutionist you might not be convinced of a reciprocal response.

Another strong case is the Ant - Acacia mutualism. Here specific traits in each

species appear to have evolved in response to the interaction. The ant
(Pseudomyrmex species) depends on the Acacia plant for food and housing; acacia
depends on ant for protection from potential herbivores (species that eat plant tissue).
Specific characters of the plant appear to have evolved for the maintenance of
this mutualism: 1) swollen, ~ hollow thorns (= ant home), 2) extra-floral nectaries
(source of nectar outside the flower [i.e., the usual location] providing ants with food),
3) leaflet tips = Beltian bodies (= 99% of solid food for larval/adult ants). Specific
characters in the ant that have evolved for the maintenance of this mutualism: 1)
defense against herbivores 2) removal of fungal spores from Beltian body break-point
(prevents fungal pathogens from invading plant tissues). The main point is that there
are traits in both the ant and the acacia that are traits not normally found in close
relatives of each that are not involved in similar mutualisms: mutualistic traits have
evolved for the interaction in reciprocal fashion. See another example : fig. 22.1 &
table 22.1, pg. 611.

Coevolution may be considered among broad groups of taxa, so called diffuse

coevolution (such as the general coevolution between plants and insects [assuming it
is real]). A nice idea, but in fact the real action must be going on between pairs of
species from each group. It is true that the Pierid butterflies (family Pieridae) are
associated with the plant family Cruciferae, so there may be something general about
each taxon that allows the coevolution to proceed. But the true reciprocal events must
be mediated at the host species-insect species level.

Mimicry presents a context were coevolutionary phenomena should be evident.

Generally, we would expect that Mullerian mimicry would be more likely to
exhibit reciprocal evolutionary patterns since both species involved are unpalatable
and therefore have an opportunity to affect the evolution of each other's color patters.
This does not mean that Batesian mimicry (one unpalatable model) will not involve
coevolutionary phenomena, but the evolution of warning coloration is certainly going
to be more asymmetrical since the palatable species will show a greater response to
the state of the model than will the model show to the evolving state of the mimic.

The Mullerian mimics Heliconius erato and H. melpomene. illustrate both the

frequency dependent nature of mimicry and the fact that each can influence the
evolution of the other. One would expect that the more abundant species would be
the model in a mullerian system, since it is what the selective agent (predation) is
cueing on. In general H. erato is the more abundant of the two species and H.
melpomene mimics the wing patterns of H. erato. In one area of overlap of the two
species, H. melpomene is the more abundant and H. erato assumes the hindwing band
pattern of H. melpomene (see figure below). Thus depending on local conditions, both
species are influencing the adaptive responses of the other and thus fits strict
definition of coevolution.

A crucial component of coevolution is phylogenetic analysis. If the cladograms of

the host and the cladograms of the parasite are congruent (e.g., figs. 22.2 - 22.3, pg.
612-613) this certainly suggests coevolutionary phenomena. But again, be careful
and think about it: cospeciation is just "association by descent". Have there been
reciprocal phenomena?; maybe just the speciation of the host induced the speciation
of the parasite and there was not parasite induced speciation of the host. One needs to
know the evolutionary history before we can make firm statements about
"co"evolution.

CONSERVATION GENETICS

Conservation biology is a rapidly growing discipline of cology and evolutionary

biology. In many ways the issues surrounding the conservation of endangered or
threatened species have rejuvenated aspects of population genetics and systematics
that were often viewed as "academic." Indeed, may aspects of conservation biology
can be view as "applied" ecology and evolutionary biology.

We will consider two different approaches to conservation genetics: 1) population

genetic issues relating to the maintenance of genetic variability, and 2) systematics
issues relating to the description of biodiversity and the recognition of evolutionary
"units" for preservation.

Due to the rapid destruction of habitat there are many species that are going extinct.
One estimate is in the neighborhood of 100 species per day! Habitat destruction is
generally attributable to human impact, but the causes of extinction are varied:
environmental variability, natural catastrophes, demographic variability
(stochasticity), genetic stochasticity, etc. Faced with this problem, biologist set out to
determine a Minimum Viable Population Size (MVP): a population size that ensure
the persistence of a species for specified period of time. One description is a 99%
chance of persistence of 1000 years.

Theoretical population genetics simulations have lead to some predictions. There

should be a positive relation between population carrying capacity (population size
the (local) environment can sustain) and the average time to population extinction.
Moreover, extinction times are exponentially distributed so a large proportion of
populations will go extinct in a period of time less than the mean time to extinction.

The implication from these simulation results is that Larger Populations are Better:
it will take longer for a larger population to go extinct, and larger populations will
lower the extinction curve. If environmental stochasticity is added to these models,
theory suggests that MVP should be 500 - 1000. If demographic stochasticity
(randomly fluctuating birth and death rates, i.e., random population growth rates),
MVP needs to be sustained at higher values (1000 - 5000). If this system is placed in
the context of an interwoven ecosystem, the MVP should probably be higher.

MVP and Genetic variation Population genetic theory indicates that inbreeding

depression will be likely with an effective population size of N e < 50. To avoid the
possibility of inbreeding, a lower limit of MVP = 500. These numbers may seem like
obscure conclusions from a series of complex Populus simulations, but they are the
working numbers for policy issues : N = 50 defines the critical list; N = 500 defines
the endangered list.

The values for MVP and "critical" versus "endangered" lists stem from some basic
issues relating to effective population size. N e refers to the effective number of
breeders in the population, and can be affected greatly by variance in reproductive
success A focus here is the ratio of effective size to census size Ne/N ratio.
Observations from field and laboratory experiments indicate that N e/N ratios are about
0.25 (range = 0.05 to 1.0). This means that either some individuals are breeding and
others are not, or that some individuals have different degrees reproductive success
that others. It is possible that a substantial proportion of the population does
reproduce, but that a small number of individuals produce most of offspring reducing
the genetic pool from which alleles are drawn. This reduces the N e/N ratio and hence
brings the population closer to the demographic danger zone.

With a Ne/N ratio of 0.25, the census size should be 4 times higher than the simple
numbers predicted by "critical" and "endangered" estimates. Now add population size
fluctuations: bottlenecks in census size affect N e more severely. Recall: = (1/Ne) =
(1/t)  (1/Ne)

The net effect of these factors is that MVP should be 5X to 10X Ne.

Given a finite amount of space for a nature preserve, do you establish a Single Large
or Several Small (SLOSS) system. The answer depends on the likely causes of
extinction in the particular system of concern. If the species is subject to demographic
fluctuations, it would be better to maintain one large system, since the plot above
suggests that extinction is more likely in small populations. In systems of species
where environmental stochasticity is a general problem , the Several Small approach
is probably better: many sub-reserves will reduce the chances of losing the entire
system.

The SLOSS debate is closely related to the dynamics of Metapopulations. The

maintenance of genetic diversity can be enhanced by structuring in a metapopulation
system. Alleles that might be lost in one deme can be fixed in another, and
the average of the metapopulation system may maintain more heterozygosity than a
simple population of similar total population size. The solution to this is not general
since metapopulation systems may have varying degrees of migration between demes
(at some level of migration, metapopulations become 'systems of subpopulations'
since in the strict sense the demes of a metapopulation experience little gene flow.)

Metapopulations can also contribute to the purging of deleterious recessive alleles.

With some level of inbreeding in demes, deleterious recessives will be selected
against. With limited amounts of gene flow, the system can effectively purge these
alleles that might not be expressed in a large random mating population. One
approach is to have semi- isolated subpopulations with corridors for dispersal.
There is no one solution to all these problems. The answers depend on 1) the species
and ecosystem in question, 2) the demographic issues (constant or variable) and 3) the
existing levels of genetic variation.

Many issues in conservation genetics have been centered around Zoo Biology. Most
zoos maintain rare or endangered species and are involved in captive breeding
programs with such species. Again, a central issue is the maintenance of genetic
variation. A number of recent studies have addressed the captive breeding
protocol to determine how mating systems affect the maintenance of genetic
variation.

Using Drosophila, several studies have shown that populations maintained with equal
founder size (EFS) retain more genetic variation that populations maintained by
random mating. EFS approaches equalize the number of founders that contribute to
the "captive" population each generation. Similarly, equal founder representation
(EFR) studies retain slightly more genetic variation than randomly mating
populations. EFR populations are maintained with a controlled pedigree where the
parentage of each contributing female and male is known. These types of studies
use allozyme electrophoresis to study directly the levels of heterozygosity in
experimental (EFS, EFR) and randomly mating control populations over time. In
addition, fitness studies can be performed by competing experimental flies
against tester stocks to determine if a higher fitness is maintained. The conclusion
from these studies is that controlled mating schemes can make a difference in retailing
genetic variation and attaining higher levels of fitness. The next step is how do you
translate these findings into captive breeding of Panda Bears??

Phylogenetic approaches to conservation biology have received a lot of attention.

This follows directly from the general concern about Biodiversity. To properly
appreciate and understand biodiversity, we must have a sense of phylogenetic
structure of the taxa involved. This applies to broad levels of organization (soil
bacteria, plants, animals) as well as to smaller taxonomic units (populations within
species). Molecular systematic approaches have been of great use since many new
techniques can be applied without harming wild individuals, and can even be applied
to museum skins for historical comparisons. In the context of the Endangered
Species Act several important issues come up: What is the phylogenetic relationship
of the endangered species? How much and what type of genetic variation (gene
trees)? What do we preserve? What IS a species? Any genetically distinct entity
has evolutionary potential.

Two case studies: The Dusky seaside sparrow: The species declined in the 1960's; by
1980 only 6 birds remained that were all male. A captive breeding program was
initiated and captive Scott's seaside sparrow was chosen as the females. When the
last male Dusky died Avise & Nelson analyzed its mitochondrial DNA (mtDNA) and
found that the Dusky and Scott's seaside sparrows were members of different clades
on the phylogeny of these sparrows. The implication is that more detailed
phylogenetic knowledge of the endangered species would have lead to different
management decisions in handling this captive breeding program (choosing a different
species to mate to the Dusky)

The red wolf was placed into a captive breeding program in 1974. By 1975 it was
extinct in the wild. Early data suggested that red wolves hybridized with coyotes.
Since coyote populations do well in human-disturbed habitats, hybridization may have
affected the survival of the red wolf. Wayne & Jenks studied the mitochondrial DNA
from captive red wolves and from 77 animals collected from the wild during the
capture program. They also used the polymerase chain reaction (PCR) to sequence
mitochondrial DNA from museum skins ("ancient DNA techniques") collected before
hybridization between red wolves and coyotes is thought to have begun. They found
that red wolves have either a gray wolf or a coyote mtDNA, indicating that the red
wolf "species" is entirely a hybrid. Other researchers disagree about the species
"status" of the red wolf. Nevertheless, this raises the question: What should we
protect? If the species isn't really a clear entity phylogenetically, does it deserve a
conservation/captive breeding effort? Wayne & Jenks argue that their data
should not be used to advocate the discontinuation of the conservation effort of the red
wolf.

These examples illustrate why the recognition of Evolutionarily Significant Units

(ESU) is an issue of great concern in conservation genetics. ESUs are defined various
ways, but they are recognized as populations with independent evolutionary histories.
Fixed allelic differences or strong phylogenetic support such as
multiple synapomorphies distinguishing one population from another are good
grounds for the recognition of distinct ESUs. Hence, a full understanding of how to do
molecular systematics is very important in molecular conservation genetics.

It should be emphasized that mitochondrial DNA markers are maternally inherited

and may not reflect the true evolutionary history of the entire populations. Hence it is
advisable to have additional nuclear markers for ESU recognition such as allozymes,
RAPDs or microsatellites. RAPDs are Randomly Amplified Polymorphic DNA.
This method uses the polymerase chain reaction to amplify random regions of the
genome with short random 10-base primers. Microsatellites are regions of the genome
that vary in the number of tandemly repeated sequences. The repeats are short (2-4
base pair repeat unit), but there may be many of them in a row. Individuals differ in
the number of repeat units they have and this difference can be determined by gel
electrophoresis.
Another relevant level of concern is a Management Unit (MU). These are defined as
populations that have different frequencies of alleles, but do not necessarily
show fixed differences between populations. Hence several MUs may exist within an
ESU. The figures below illustrate the difference. The general lesson is that molecular
approaches to conservation biology are potentially highly informative since many
overt phenotypic characteristics cannot reveal important differences that distinguish
populations. Since conserving endangered species is inherently a "genetic" endeavor,
to the extent that we recognize species as discrete reservoirs of historically unique
genetic material, the molecular approaches are very useful.

BIOGEOGRAPHY

We have referred to pattern and process throughout different sections of this course.
These concepts are central to the study of biogeography which, in turn, incorporates
many of the topics in evolutionary biology. Biogeography often leads us to infer
process from pattern.

Biogeography is the study of the distributions of organisms in space and time. It

can be studied with a focus on ecological factors that shape the distribution of
organisms, or with a focus on the historical factors that have shaped the current
distributions. Certain regions of the world have "Mediterranean climates" where ocean
current and wind patterns hit the west coast of N and S continents (Medit. region,
California coast, Chile coast, SW Africa coast). Similar climate has lead
to convergent , but unrelated (by definition) types of plants. To make sense of these
types of ecological patterns we require a phylogenetic (historical) perspective: we
need to focus on monophyletic groups.

The importance of a geographic scale was certainly appreciated by Darwin: the

Galapagos finches were morphologically distinct and geographically distinct and
there must be a connection. Moreover, the general view that speciation is a central
phenomenon in evolution, and that most speciation is allopatric speciation assumes
that geography plays a central role: some geographic feature divides a species range in
two or more parts and over time speciation is achieved (details in later lectures).

These sorts of observations were made by early biogeographers who recognized

certain types of distributions of organisms. Some species are restricted to a certain
region and are referred to as endemic species. Endemism needs to be defined with
relation to the taxonomic group: all life forms we know are endemic to the planet
earth; the genus Geospiza (Darwin's finches) are restricted to the Galapagos
islands; Geospiza fortis is endemic to specific islands; the spotted owl is endemic to
the old-growth forests of the pacific northwest. Cosmopolitan species have a world
wide distribution. They may be restricted to specific habitats, but occur on most
continents.

In addition to endemism, another important pattern that needed to be explained were

examples of disjunct distributions where clearly related species (or even the same
species) are found in different areas. Marsupials are found in Australia and South
America. Ratite birds (Ostrich, Emu&Cassowary, Rhea) are found in Africa,
Australia and South America, respectively.

Alfred Russell Wallace noticed that different regions of the world had congruent
patterns of endemic species and he drew up six biogeographic realms (see fig. 18.2,
pg. 510; nearctic, neotropical, holarctic, ethiopian, oriental and australian). Wallace
worked primarily in Malaysian region and had noticed a clear break between
Australian fauna and the fauna on the islands to the northwest. This break has come to
be known as Wallace's line (also a line between the Australian and the Asian
biogeographic zones). These patterns described long before continental drift was an
issue.

Different biogeographic areas can be quantified for levels of similarity in their biota
(biota=general term for flora+fauna, includes microbes). N 1 = number of species (or
other taxonomic unit) in one region, N2 = number in another region (N1 < N2) and C =
number of same species. Index of Similarity = C/N1. For Australia: New Guinea, I.S.
= 0.93 (93%), while Australia: Philippines, I.S. = 0.50 (50%). See table 18.1, pg 511.
This provides a simple quantification of Wallace's Line.

How do we account for these patterns? Early biogeographers tended to

invoke dispersal (prior to knowledge about continental drift). Potential problems: ad
hoc, could pull dispersal out of a hat whenever you needed to explain a peculiar
distribution. Leads to many wild scenarios of "gravid females" (pregnant, or
inseminated females carrying eggs) making there way to distant
regions. Muddyfooted duck carrying propagules in its feet; land bridges invoked
connecting disjunct regions. Criticized by many as unscientific: cannot falsify the
dispersal hypothesis because it is something we'll never know for sure, thus is of no
explanatory power.

Nevertheless, all these types of events probably have occurred at some point. The

Bering land bridge is well documented as an avenue of dispersal; the Opossum (a
marsupial) in North America clearly dispersed here from South America via the
Isthmus of Panama (see below); oceanic islands have life on them and it must have
gotten there by dispersal. Several modes of dispersal can be
described: Corridors between two regions on the same land mass, Filter bridges as
selective connections between two areas, Sweepstakes as rare chance events (e.g.
muddyfooted duck).

Dispersal hypotheses often associated with arguments about centers of origin: those

regions with the greatest species (or higher rank) diversity. Greater diversity should be
due to presence in that region longer (more time for speciation), hence should be the
region where the group originated and from which dispersal events took
place. Assumes that extant diversity has not moved from origin of diversity. Possible,
but not guaranteed for all taxa.

Alternative to Centers of Origin and subsequent Dispersal as a way to explain the

current distribution of species is vicariance where some barrier to genetic exchange
causes the separation of the related taxa. With the acceptance of continental
drift, vicariance biogeography became a discipline in which one could test
hypotheses (see below). See models in fig. 18.6, 18.8 and 18.9, pgs. 518-521.

As with most dichotomies in science: often need to invoke Both vicariance and

dispersal to account for distributions (not always in the same instance). Example:
Galapagos finches had to have dispersed to the archipelago from the mainland and in
so doing imposed a vicariance event on themselves. South American land
bridge when sea levels dropped in the Pliocene the isthmus of Panama rose and
served as an avenue of dispersal for terrestrial mammals (the "Great American
Interchange" where unique N.American mammals dispersed to S. America and unique
S. Amer. mammals moved north, 3 mil. years ago; see fig. 18.14, tables, 18.2, 18.3,
pgs. 528-529), but served as a vicariance event for marine life that was distributed in
the region. Lead to the formation of Geminate species (species pairs on either side of
the isthmus who are each other's closest relative and were probably one species before
the sea level dropped).

Pleistocene refugia nicely illustrate how vicariance and dispersal may need to be

invoked to explain current distributions. Glacial ice sheet forced species to new
distributions (vicariance event), after glacial retreat, the separated forms dispersed to
previous regions (or wider distribution). Relative roles of dispersal and vicariance in
determining species distributions can vary widely with a given species dispersal
abilities (see fig. 18.3, 18.4 pg. 514-515). Essential to realize that dispersal has two
components: the ability to move and the ability to become established. These two
properties may not be "optimized" in the same organism.

Continental Drift as source of vicariance events. Evidence for continental drift

provided by disjunct fossil specimens: Mesosaurus in South America and Africa.
Illustrates the space and time component of biogeography since the strata reflect the
same time (old) but are widely separated in space. Continents must have moved. (Fig.
18.5, pg. 517).

Major stages of the split-up of continents: Pangaea formed in Permian (> 250 MyBP)

and began to break up in the Triassic (200
MyBP). Laurasia and Gondwana separated at the Tethys seaway (135 MyBP).
Tropical corals, sea grasses and mangroves are related in Americas and old world
tropics reflecting earlier Tethyan distribution. Gondwana began to break up about 80
MyBP and the major continents were separated by late Cretaceous (65 MyBP). India
smashed into Asia crating the Himalayas. As the continents separated vicariance
events abounded and the fauna of various continents became
increasingly Provincialized. The South American mammals had many unique forms
with respect to the North American Fauna. Marsupials in Australian zone are distinct
form of mammal.

Testing biogeographic hypotheses with cladistic analysis. Brundin's midges (fig.

18.7, 18.8, pg. 519-520) a classic in vicariance biogeography. Sibley and
Ahlquist's Ratites and the Gondwana breakup. Testing hypotheses about the sequence
of vicariance events with cladograms from several species. Validity of biogeographic
hypothesis can be supported by congruence of independent cladograms from
unrelated species (see Cracraft, 1983, American Scientist vol. 71: pg273). By
considering the relationships of organisms and their geographic distributions, the
most parsimonious combination of the species cladograms can lead to an hypothesis
of vicariance events, a so-called area cladogram which presents the sequence of
splitting events.

Using cladistic methods, one can test biogeographic hypotheses by asking

whether area cladograms for other, unrelated taxa are congruent. If different taxa all
have similar area cladograms (i.e., are "congruent"), then the sequence of vicariance
events is supported. If one taxon is represented in a region where none of the other
taxa are found, then one might be forced to invoke dispersal to account for the
disjunct distribution. The strength of this approach is that hypotheses are testable and
one need not resort to ad hoc explanations that should be taken on faith.
Biogeography can be practiced in a scientific manner despite its historical nature.

EVOLUTION AND DEVELOPMENT I: SIZE AND SHAPE

First, some general background to the study of development and evolution. Evolution
of organisms involves a change in the developmental program, a change in a series
of developmental processes. We often refer to evolution as "descent with
modification" and the modification we often notice first is the overall appearance of
the organism. This appearance is a result of the development of the organism, thus
evolution is intricately involved with development.

Embryology played a major role in evolutionary theory in the 19th century, but was
largely ignored in the 20th. Development never really became part of the modern
synthesis. Some argue that this is due to the lack of communication
between geneticists and developmental biologists. The geneticists were concerned
with the rules of transmission of genetic material between generations and the
developmentalists were concerned with cellular changes that led to the transformation
of an egg into an adult organism. Mutations in adult phenotype were readily available
for the study of genetics, but there were precious few "developmental mutants" that
bridged the gap between development and genetics (such mutants were discovered in
growing numbers during the formulation of the "Modern Synthesis", and many more
discovered later).

The general approach is the same as we have taken with the evolution of other traits:
development has a genetic basis, if there is genetic variation for the developmental
program then development can evolve. We will first take a descriptive approach to
evolution and development and next lecture look at some of the more genetic and
cellular mechanisms of development.

Early embryologists noticed similarities between ontogeny (the development of an

organism) and phylogeny (ancestor descendant relationships in a group). The
common phrase "ontogeny recapitulates phylogeny" was put forward by Haeckel as
his biogenetic law (see fig. 21.3, pg. 588). Haeckel held that descendants, during their
ontogeny, passed through stages that resembled the adults of their ancestors. Before
this, Cuvier (1812) held that there were four major classes of organisms: vertebrates,
mollusks, articulates and radiates. Cuvier noticed that there was nothing in the
ontogeny of a vertebrate that resembled the adult stages of, say, a mollusk. This is
because evolution is a bush or a tree not a "ladder" of the great chain of beings. This
"branch-like" pattern to phylogeny was apparent to Haeckel, but he still claimed there
was "recapitulation".

von Baer made observations about ontogeny and phylogeny that seem obvious to us
today, but they are important in development and evolution as they run counter to
recapitulation: 1) more general characters appear early in development, 2) less
general forms develop from the more general forms, 3) embryos do not pass through
other forms they diverge from them, 4) embryos of higher forms only resemble
embryos of other forms (human, calf, chick and fish look similar at embryo stage but
diverge quickly). See section 17.8.2, and fig. 17.11, pgs. 478-479.

Putting these two views together, we see that there can be a sort of
recapitulation within a lineage (i.e., within an evolutionary sequence of ontogenies)
but there are many examples that refute the notion that phylogeny is reviewed during
ontogeny.

First efforts to place development and evolution in a quantitative, descriptive context

were provided by d'Arcy Thompson in On Growth and Form. Using simple rules of
geometric transformation he showed that one could obtain the varied forms of
organisms by "warping" or "bending" the relative positions of their body parts (see
fig. 21.10, pg. 599). These types of diagrams are helpful in identifying what changes
of form have taken place, but they do not identify how
developmental mechanisms have evolved (the same criticism might be leveled
towards Raup's computer snails (see figures 13.7 and 13.8, pgs. 356-357),
but mechanism was not the intention of these approaches).

One thing Thompson and Raup's diagrams did contribute was to focus attention on the
notion of size and shape. These two very simple words are deceptively complex in
the context of the evolution of development. A general paleontological pattern
is Cope's rule which states that the body sizes of species in a lineage of organisms
tend to get bigger through time. Horse evolution is a classic example. But what
happens when you get bigger? In most cases body parts do not grow at the same rate,
thus we have allometry.

Allometric growth is the differential rates of growth of two measurable traits of an

organism (often it is described as size-correlated changes in shape). It is quantified
as y = bxa where x is the measure of one trait, b is a constant, a is the allometric
coefficient and y is the other trait. In this form it describes a logarithmic relationship.
It can be made into a linear relationship by taking the logs of the values measured for
each trait (or by plotting on log x log graph paper):

log y = log b + a log x. This is the equation for a strait line with a being the slope of
the line. When a<1 we have negative allometry which means that as x gets bigger, y
gets bigger at a smaller rate. When a >1 we have positive allometry which means
that as x gets bigger, y gets bigger at a faster rate. When a=1 we have isometry (or
isometric growth) which means that there is no change in shape (i.e., the relative sizes
of body parts) during growth. See fig. 21.9, pg. 597.

We can describe different kinds of allometry: 1) interspecific allometry where traits

of individuals of the same age (usually adults) are compared between different
species, 2) intraspecific allometry where a) traits of individuals of all ages are
compared within a species (also called ontogenetic allometry), or b) traits of
individuals of the same age are compared within a species (also
called static allometry).

Some examples: interspecific=the Irish elk example (more below), intraspecific

(static)=measurements of body height and arm length in class, intraspecific
(ontogenetic)=measurements of body height and arm length with my daughter's day-
care measurements included. See figure demonstrating ontogenetic and interspecific
allometry of brain and body weight in the same graph.

Intraspecific allometry just describes growth, and alone is not an evolutionary

comparison. It is of interest that the allometric coefficient of Bio 48 males and
females is ~ 1.0, but if the toddler data are included the allometric coefficient goes up
to ~ 1.3. This means that as adults we have about the same proportions (a=1) but as
we grow from infant to adult, our arms get proportionally longer (a=1.3).

Allometry is useful in describing the evolution of size and shape. Different species
attain different morphologies by virtue of different timing of various developmental
processes. This change in timing is called heterochrony. Figures 21.5 - 21.8 and table
21.1, pgs. 590-594 review some of the typical examples of heterochrony. Using the
figure below, we can group these into two general classes: in figs B and C the
ancestor (dotted) and descendant (solid, but hard to see in C) have the same slope but
the descendant stops growing (=adult) at a different time; in figure D and E, the
descendant grows for the same amount of time (in these cases same amount of x but
different amount of y) but at a different slope. Both are heterochronic changes because
some aspect of timing (relative or absolute) has changed in evolution.

Notice that each axis of these graphs include both a measurement component and
a time component simply because growth by definition is both a temporal and a
dimensional phenomenon. Note that only one of these examples of different growth
plans (graph B) demonstrates "ontogeny recapitulates phylogeny": hypermorphosis.
Therefore, shape changes can be observed as 1) changes in the slope of an allometric
relationship, or 2) changes in the y intercept of an allometric relationship. Recalling
high school algebra, a change in the slope will change the intercept, but the intercept
can be changed without changing the slope (keep the line parallel and move it up or
down). All of these changes result in change in shape. Even with the same slope but
different intercepts the relative sizes of x and y will be different so there will be a
change in shape. The only case where there is a change in size with no change in
shape is when the allometric slope = 1.0 and growth continues (or retards) relative to
the ancestor.
Classic examples of allometry are neoteny in human evolution: as adults we look
like the juvenile stages of chimps; and neoteny in salamanders: the adult of
descendant retains gills (a juvenile morphology in the ancestor). Peramorphosis in the
evolution of deer: the "Irish elk" (actually a deer) has phenomenally large antlers and
are "disproportionately" large because there is an allometric relationship between
body size and antler size. In fact the Irish elk falls right on the line of allometry for
other species in the family (interspecific allometry). Thus, the antlers are larger than
usual, but they follow precisely the developmental program that seems to be a part of
its phylogenetic group. Previous adaptive (and maladaptive) stories had been told
about these huge antlers and how they probably drove the elk to extinction, thus a
challenge for "adaptive" evolution, but the allometry shows that they are not really
"abnormal" (probably went extinct due to climatic changes and hunting). See
discussion on pg. 356-358 of adaptive, vs. non-adaptive explanations of morphology.

Allometry is also important in the context of the criticisms to the "adaptationist

program". If you looked at a Titanothere with its bizarre horns pointing out of its
snout, you might say "what are those things for" as if they evolved for some function.
They may not be "for" anything but simply the result of a positive allometric
relationship between body size and horn size during evolution. Now the question
becomes: what causes Cope's rule? Since allometry is so common, changes in size
will produce changes in shape.

EVOLUTION AND DEVELOPMENT II: GENES AND MORPHOLOGY

Documenting allometry and patterns of size and shape changes in evolution are
helpful as descriptive approaches to the evolution of development. But these
phenomena are themselves the result of developmental mechanisms at the molecular
and cellular level. We can often say without reservation that there has been a change
in development during evolution, but how that change in development was achieved
is yet another question. We improve the description somewhat by saying that changes
in development result from changes in the: 1) spatial organization of cells, 2) timing
of gene action and tissue differentiation and 3) geometry of tissues and organs. But
how are these changes mediated?

Consider the comparison between humans and chimps: the adult morphology is

obviously distinct, but at the genetic level we are extremely close to chimps: >99%
similar at the genetic level. This is less genetic divergence that seen between sibling
species (can't tell them apart) of Drosophila and some mammals These observations
indicate that morphological evolution has proceeded faster than molecular evolution
suggesting that regulatory evolution has proceeded faster than DNA sequence
evolution. Where are the important mutations (short arrows)? in the coding sequences
of genes or in the regulatory sequences upstream from them? May depend on how the
product of a gene interacts with other genes (longer arrows).

Thus perhaps the key to understanding the evolution of development is the study the
evolution of the genetic regulatory mechanisms that control development. Now the
question becomes: what do we know about genetic regulation of development?

A fair amount is known in Drosophila. The exciting point here is that in recent years
there have been increasing numbers of papers describing the existence
of gradients across the egg or early embryo in the concentration of specific
proteins encoded by a handful of loci. These proteins can be thought of
as morphogens ("form creators"), molecules that, for years, were postulated to exist
by embryologists. With a gradient across the embryo of such a morphogen, there is
the possibility the other proteins that might interact with such a morphogen can
obtain position information from the gradient such that high concentration means
"anterior" (or "limb end" in vertebrate limb bud) and low concentration means
"posterior" (or "limb base").

The significant point in all this is that Drosophila geneticists have been able to
identify specific developmental mutations (mutations in the genes that code for
morphogens, or genes that code for molecules that interact with morphogens) that
disrupt specific events in development. One such example is the bicoid gene: when
this gene is mutated, its normal gradient is disrupted and the embryo has two tails (bi-
caudal). The point is that there are specific genes that determine the major body axes
and one can envision that evolution of major new developmental programs might
proceed by naturally occurring mutations in these genes that would move/alter the
gradient, or, equally as significant move/alter the cellular localization of the receptor
of a morphogen.

On a more theoretical level, morphogens have been hypothesized to operate in

a threshold-like manner in more localized examples of pattern formation such as the
generation of additional bristles in Drosophila or specific patterns of striping in
mammals (see figs. below). Specific molecules causing "prepattern" such as one sees
in zebras have yet to be identified, in contrast to the major advances made in
Drosophila, but mating zebras is a major undertaking.

There is solid support for such ideas in Drosophila development. The RNA encoded
by the bicoid gene is localized in the anterior portion of the embryo.
The protein translated from this localized RNA is distributed as a gradient from
anterior to posterior across the embryo. The bicoid protein affects the distribution of
the RNA of another gene, hunchback. This RNA (hunchback) is not distributed as a
gradient but in a discrete way: present in the anterior, absent in the posterior. Thus
there appears to be positional information in the concentration of bicoid which is
read by hunchback as a threshold. One could imagine that a mutation that affected
the localization of one morphogen could alter the localization of important thresholds
of different morphogens, which would in turn lead to the development of new
morphologies.

The genes controlling the early events in the development of Drosophila can be
classified into three broad categories: Gap genes are a set of genes that act to define
broad regions of the early embryo; these can regulate the expression and action
of Pair rule genes which further define the broad regions into more
numerous segments; the pair rule genes can affect the expression and action
of Segment polarity genes which will determine the fate of certain structure within
each segment. As with the gradients of morphogens described above, one can envision
mutations that alter the interactions between these broad classes of genes controlling
the developmental fate of parts of the organism which, if established in the
population, could lead to the evolution of new morphological "plans" (.g., a
new Bauplan).

There is good evidence for such a supposition in another very important set of genes:
the homeotic genes. Certain mutations in these genes result in homeotic
mutations where one body part is transformed into the structure of another body part.
The best examples are the Antennapedia complex and the Bithorax complex which
are large regions of the chromosome containing several genes each. The positions of
the genes on the chromosome have a remarkable correlation with the segment of the
body in which they are active! (see figures below). The genes contain a region of
DNA that codes for a highly conserved stretch of amino acids, known as
the homeobox that generally are involved in the determination of body
segments (but bicoid has a homeobox and it is more involved with anterior-posterior
determination). While mutations that move a leg to the position of an antenna (in the
Antennapedia complex) or transforms the balancing organs (halteres) into a pair of
wings (in the Bithorax complex) is of dubious fitness value to the organism, it does
show that modifications of the general body plan be achieved by mutations in one or a
few genes, i.e., there is genetic evidence that Hopeful (hopeless?) Monsters could be
produced.

These phenomena are compelling in light of the belief that arthropods (insects,
crustaceans, etc.) evolved from annelids (segmented worms; see figure below). One
can envision that sequential modification of body segments, through mutations such
as those described above, might allow for the evolution of insects from a worm-like
ancestor. Suggestive of this is the observation that when the Antennapedia
complex and the Bithorax complex are mutated the larval stage of the fruit fly is
transformed into a larva with many thoracic segments rather than the wild-type pattern
of differentiation into maxillary, labial and abdominal segments (see fig.below). This
"throw back" to the ancestral form (i.e., the middle segments of worms are relatively
undifferentiated) is called an atavism.

In thinking about all possible morphologies one might be able to get with bizarre
mutants in flies, and looking out at the incredible diversity of form in the natural
world we might best think of this problem in terms of the question: why this and not
that? Are there forbidden morphologies that development cannot produce?. There is
some nice evidence that the different forms seen between species may be the result of
the "playing out" of discretely different developmental programs. When the
developing limb bud of one salamander is treated with an inhibitor of mitosis,
the number and pattern of digits developing resembles that of another species
(section reading). This suggests that there are developmental constraints, i.e., that
development is constrained to proceed in a certain way. If different developmental
programs are carried by different lineages of organisms as they diverge from one
another, these developmental constraints become phylogenetic constraints: there is
no chance that horses will sprout wings because the lineage of horses (and ungulates
in general) are constrained to develop and use their forelimbs in very different ways
than bats, lets say.

A conceptual model for this notion of constrains is to think of there

being canalization of developmental programs. Waddington's model of development
as a ball rolling down a landscape suggests that the program is canalized to follow a
particular trough. Mutations and/or environmental fluctuations (next lecture) might
knock the ball around in the trough, and if these perturbations were strong enough
might throw the developmental program over into a new canalized ontogeny. In this
model the location of the troughs suggests that events that perturb development early
on are more likely to result in major changes in the developmental plan. Perhaps
developmental programs become more "canalized" as they proceed through
development. This is of particular significance in light of the network of genes
described above that establish body plan during early embryogenesis.
PHENOTYPIC PLASTICITY AND NORMS OF REACTION

Phenotypic plasticity is the ability of individuals to alter its physiology, morphology

and/or behavior in response to a change in the environmental conditions. This is
clearly demonstrated by the appearance of plants grown at different densities:
crowded plants look spindly and lanky, uncrowded plants look healthy and robust. In
the context of evolution, phenotypic plasticity demonstrates the two meanings of
adaptation: the plastic response is itself an example of a physiological adaptation and
it is widely held that the ability to be plastic is adaptive in the sense of increasing
fitness.

In thinking about phenotypic plasticity as a evolutionary adaptation it is important

to separate the trait in question from the plasticity for that trait. For example:
growing taller in response to plant crowding is adaptive in the sense that it increases
an individual's competitive ability for sunlight (lower fitness when shaded by other
plants). The "normal" height for a plant (lets assume there is such a thing) may have
evolved in response to pressures to allocate resources to growth versus reproduction in
a particular way. Thus there is a genetic basis for plasticity of plant height, and a
genetic basis for plant height itself. The point is that different genes probably control
these processes so the trait and its plasticity can (as opposed to must) evolve
independently.

Now consider the environment: certain physical properties of the environment can be
described by the mean (average) value or the range of values (highest - lowest).
Which aspect of an organism (the trait itself or the plasticity for that trait) will evolve
in response to which measure? It may be that the plasticity for a trait will evolve in
response to the range of values the environment throws at an organism (e.g., coldest -
hottest, driest-wettest days), whereas the trait itself (e.g., thickness of fur) will evolve
in response to the mean. This is not a rule! but would be an interesting thing to test
and/or think about.

The idea of plasticity is interwoven with the notion of canalization. In light of the ball
rolling down the trough of a developmental pathway (previous lecture), one can
consider the width of the trough as an indication of the amount of plasticity
"tolerated" in the organism in question. A highly canalized organism (or
developmental program) would have low plasticity.
Another variant form of the plasticity issue is that some organisms may
exhibit threshold effects where there is not a clear gradual transition between forms,
but a stepwise change of phenotype in response to a gradual environmental change.
See fig. 9.11, pg. 242, but note that these graphs do not have an environmental axis, so
a distinct from a norm of reaction. One example of this are plants that have distinctly
different growth forms in different environments. Question: is there an "environment"
that is half way in between air and water?, and if so would these plants exhibit a
graded response to such an environmental gradient?

A concept that places phenotypic plasticity in the context of a genotype-specific

response is the norm of reaction. A norm of reaction is an array of phenotypes that
will be developed by a genotype over an array of environments. The quantification of
a norm of reaction is conceptually quite simple: one obtains a number of different
genotypes (clonal pants are great for this) and grows each one in a variety of different
environments (e.g., different nutrient, light, water conditions). After a period of
growth one measures the desired trait(s) from each individual and plots the data out as
shown in figure below; this case for Drosophila bristles. Each line represents the data
for a different genotype. If all lines are perfectly horizontal and on top of one another
there is no effect of environment (E) or genotype (G) in case 1 below (each genotype
is x, y or z). If all lines are not horizontal but on top of each other there is an
environmental effect, but no genotype effect (case 2). If all lines are horizontal but at
different positions there is no effect of environment but there is an effect of genotype
(case 3 below). If lines not horizontal but are parallel there is an effect of environment
and genotype, but there is no genotype x environment interaction (figure and case 4
below). If the lines are anything other than horizontal, there is an effect of
environment. If the lines are neither horizontal nor parallel there is an effect of I)
environment (nonhorizontality), ii) genotype (lines not on top of each other) and iii)
genotype x environment interaction (not parallel; case 5 below).
The interesting case comes when the norms of reaction lines cross. Then there is a
range of environments where genotype 1 is "bigger" than genotype 2, where both
genotypes are about the same and where genotype 2 is "bigger" than the genotype 1
(see figure below). Thus determining what is the "best" genotype, or the "fittest"
genotype depends on the environment.

EARTH HISTORY (NOTE: this material covered in lecture on Origin of Life &
Fossil Record)

Much of evolutionary biology involves the history of organic diversity. Organic

diversity has been shaped and affected by the origin and history of planet earth. To
appreciate this history we need to acquire some knowledge of the geological processes
that have shaped the earth. One general theme to consider in this and the next lecture
is: if we were to start the history of earth over again from the "primeval soup" would
the results be the same? Almost certainly not (see Gould, 1989. Wonderful Life for a
detailed discussion). History is unique and events are contingent on what has
occurred previously. Much of the contingency of organic evolution is dependent on
the unique series of events that shaped the earth, this is why we need to understand
some basic geology.

How was the planet formed? What is its relationship to other matter in the universe?
A popular hypothesis for the formation of the earth is the nebular hypothesis. This
idea dates back to the philosopher Immanuel Kant (1755) and Laplace (1796) and has
been modified as empirical evidence and theory mount. Recent incarnations (chemical
-condensation-sequence model) start with the solar system forming from a rotating,
diffuse cloud of dust and gasses (a nebula). As the nebula cooled the matter
condensed into "planetesimals", near the sun where temperatures were highest
elements with the highest melting points (metals and heavy minerals) condensed first.
Lower melting temperature elements and compounds (water, methane, ammonia)
condensed more readily in the cooler areas further from the sun. This helps to explain
the density gradient in the solar system, the closest planets to the sun are terrestrial
while those further away are gaseous.

How did the earth form in the condensing nebula? The earth may have formed
through the accretion of many planetesimals and as the mass increased through
gravitational attraction and compression (overhead). The earth was probably initially a
homogeneous ball that heated from three sources: 1) energy of planetesimal impacts,
2) gravitational compression lowered potential energy releasing heat, and 3) heat from
radioactive disintegration (20 cals is released for 1 cm 3 of granite over 500 million
years). As the earth heated it began to differentiate into various zones of matter with
different properties (overhead). Differentiation was possible because molten material
could rise or sink depending on density, be moved by convective currents, and
localize due to chemical zonation (overhead). As the earth cooled outgassing of the
mantle released compounds (water vapor, carbon dioxide, hydrogen, nitrogen) into a
primitive atmosphere.

Early geologists tried to determine how old the earth was from observations about the
features of the earth. Age = Thickness of sedimentary rock/rate of sedimentation. Old
(<1.5 billion years) but not old enough. Age = salinity of sea/rate of salt deposition in
seas. Again old, but not old enough. Lord Kelvin (of absolute zero fame) calculated
the age of earth from its temperature, assuming it was molten at its formation. Gave
100 million years (and gave Darwin a bit of a problem: was this enough time??
Radioisotopes cleared things up (see below)

We can divide the processes that alter the earth's surface into two categories:
1) igneous processes (volcanism and mountain building) construct features by
increasing the average elevation of the land, 2) Sedimentary and erosive
processes (deposition and weathering) act as forces wearing down features created by
volcanoes and creating new horizontal features (e.g. river delta). The theory of Plate
tectonics provides a synthetic model for understanding how the dynamics of the earth
work. The plates move around, collide, move over or under one another. Divergent
boundaries are where plates move apart, convergent boundaries are where plates
move toward one another, transform boundaries (e.g. San Andreas fault) are where
plates move by each other. The continental plates (lithosphere) float on molten inner
layer (asthenosphere). Where plates meet there can be uplifting or
subduction. Uplifting results in mountain building through igneous activity and at the
boundaries between plates and actual scraping off of material from the subducted
plate. Subduction results in plates being forced downward and is seen is formations
such as ocean trenches.

The rock material of continental plates can be viewed as going through a rock
cycle that can be related to plate tectonics. Magma (molten rock) e.g., released from
volcanoes, crystallizes and forms igneous rocks ("fire formed rocks").
Through weathering and transport sediment is formed which by lithification become
sedimentary rock. Through exposure to high temperatures and pressure, sedimentary
rock (or any rock) can be changed into metamorphic rocks. If this rock is exposed to
extreme temperatures it can become molten again and form magma, and if released
through volcanic activity be reintroduced as igneous rock.

In what kind of rock would we expect to find fossils? Sedimentary rocks. Their
structure can tell us a lot about earth history. Laid down in strata of sedimentary
layers. Bedding planes generally mark the boundary between the end of one sediment
and the beginning of another.

Several logical rules can be used to determine the sequence of events: Relative

dating. generally one follows several principles: superposition the older rock is
below and the younger rock is above; original horizontality: the strata are laid down
originally in a horizontal position (gravity is what lays them down). Thus
nonhorizontality must have occurred after the deposition. The cross cutting
relationship states that the cut formation is older that the formation doing the cutting.

Another prominent feature is an unconformity which occurs when the rate of

deposition has been interrupted, the sediments eroded and deposition renewed. A clear
break in the sequence of events is apparent. One type of unconformity is an angular
unconformity where strata with originally horizontal bedding planes now have
bedding planes that intersect. Significant because it reflects a major episode of
geologic change.

All well and good for a given formation, but one would like to be able to make
general statements about larger regions. This can be done by correlation of strata
from different formations separated by some distance. Stratum "X" may lie near the
top of one formation and many miles away, X may be found near the bottom of a new
formation, at the top of which is a different layer "Y". Several miles further on, "Y"
may lie at the bottom of a third formation, and in this way one can link or correlate the
different strata.

This may work for a large region but one would like to do this for the entire earth. It
turns out that there are diagnostic fossils found in different formations around the
world. These Index fossils help correlate different formations on each of the major
land masses. This was recognized by William Smith (see lecture 2). The phenomenon
is more pronounced than an occasional fossil here and there: entire biotas go through
successive changes in sequential strata, illustrating the principle of faunal (biotic)
succession. We thus have the "age of trilobites" seen early in the fossil record. Later
the age of fishes, age of reptiles, age of mammals are clear in formations around the
world indicating the comparable ages of formations separated on different continents.

These fossil beds lead to the formation of the Geologic time scale, the names of each
period deriving from the locality where the characteristic formation was found. The
major divisions (eons, eras) are defined by the presence or absence of
fossils: proterozoic, phanerozoic (visible life or animals). Geological dating is often
problematic because geologists use fossils to date rocks and biologists use rocks to
date fossils. A measure independent of stratigraphy and fossil remains is necessary.
With the discovery of radioactive decay it became apparent that one could use the
ratio between the parent isotope and the daughter product (e.g., U238 decays through
several steps to Pb206). By measuring the amount of isotope and daughter product and
knowing the half life of the isotope one can estimate the absolute age of a rock
formation. Problems: when the daughter material escapes and hence produces an
inaccurate estimate. Additional tests with different isotopes can corroborate one
another.