GENETICA POBLACIONAL Y MOLECULAR

- Electroforesis enzimática:
* Herramienta muy útil a contar de 1966
* Variabilidad genética poblacional
* Variación con significado adaptativo?
* Estructura genética poblacional
* Flujo génico
* Taxonomía
* Sistemática: evolutionary trees, etc.
 PROBLEMAS:
• Son marcadores neutrales?
• Tamaño de la muestra de tejido
• Mantención del tejido (evitar degradación)

Sin embargo, continúa siendo una técnica muy utilizada y por

consecuencia existe una enorme base de datos disponible.

Genética molecular y diversidad genética:

What is genetic diversity?
- it is possible to describe “genetic diversity” at various levels of biological organization.
1. an individual organism
- different individuals may differ in the number of different gene copies that they possess.
-in a diploid species, the maximum number of different forms of a gene that may be
present at any given genetic locus is two.
- these different forms of a gene are called alleles.
-suppose we analyzed the hexokinase (Hex) locus, an enzyme catalyzing the first reaction
in glycolysis.
HexA or “A” HexB or “B”
- if different alleles are present at the hexokinase gene it is said to be polymorphic.
- if an individual has two different alleles at a gene, it is said to be heterozygous - AB.
- if it has two copies of the same allele it is said to be homozygous – AA or BB.
- the number of heterozygous loci per individual may vary.

- an individual may be heterozygous at the hexokinase locus but homozygous at another

gene locus.
2. a population of organisms
- the amount of genetic variation present in an interbreeding population of organisms
usually greatly exceeds that present in individual organisms.
- this is a simple consequence of two factors.
- first, the total number of alleles present in a population at a given gene can be much
greater than two (the maximum number of different forms of the gene present in a
given organism.)
- second, if a gene is polymorphic in a population, not all individuals will be
heterozygous at that gene.
- some will be heterozygous, some will be homozygous.
- the proportions of individuals that are heterozygous and homozygous are determined
by the frequencies of alleles at that gene in the population.
- for example, consider a genetic locus with two alleles: A and B.
- if the species considered is diploid, then there can be three genotypes: AA, AB and
BB.
- let the frequency of the A allele be p and the frequency of the B allele be q.
- a fundamental principle of population genetics called the Hardy-Weinberg
equilibrium dictates that the frequencies of different genotypes is given by p2, 2pq
and q2.
Genotype: AA AB BB
Frequency: p2 2pq q2
- consider the following two examples.
Allele frequencies Genotype frequencies
A B AA AB BB
0.5 0.5 0.25 0.50 0.25
0.9 0.1 0.81 0.18 0.01
- the extent of genetic variability is clearly higher when the two alleles are present at
equal frequency.

- this principle holds true irrespective of allele numbers.

3. a species (a collection of species populations)
- different populations of a species may be genetically differentiated from each other to
varying degrees.
- if they are, the extent of genetic diversity in the entire species exceeds that present
within any one population.
- the term used to describe the genetic subdivision of a species is called population
structure.
- as shown later in class, to adequately conserve a species - marine or terrestrial - requires
knowledge of the degree of population structure of the species.
4. a group of related species.
- the final level that the term genetic diversity can be applied involves a group of related
species of organisms.
- here, the term “genetic diversity” is synonymous with “species diversity”.
- preserving genetic diversity in this sense means preserving a group of related species.
- by doing so, one preserves the legacy of their evolutionary history.
How do we measure genetic diversity?
- true measures of genetic diversity concern parameters that
are specific to populations, not individuals.
- to adequately assess levels of genetic diversity we need to
obtain a random sample of individuals from natural
populations and examine their genotypes at a number of
independent genetic loci.
- how do we go about estimating gene diversity?
The molecular genetics toolkit
1. Polymorphic proteins (allozymes)
- developed originally by Oliver Smithies in the late 1950’s.
- first applied to population-level variation by Harris (1966) on humans and Lewontin and
Hubby (1966) on Drosophila.
- one scores allozymes by obtaining tissue sample from a population - for example blood
samples.
- the next step is to grind up the tissue to liberate enzymes and other proteins from within
cells.
- the homogenized tissue is centrifuged and a small amount of the liquid supernatant is
applied to a gel - starch or acrylamide.
- the gel is exposed to an electric field and the proteins migrate in the gel because they
have a net negative charge.
- after the proteins have migrated a set distance, the positions of different enzymes are
localized by incubating a slice of the gel with a solution specific to a certain enzyme (i.e.,
has the substrate and cofactors necessary for catalysis).
- different bands may appear in different individuals caused by mutations in the protein
products that alter the net charge of the protein.
2. DNA markers
a. mitochondrial DNA (mtDNA)
- in most vertebrates, mtDNA is a circular molecule 16,000 to 18,000 bp in size.
- it is haploid and usually maternally inherited.
- it possesses 37 genes, 22 are tRNAs, 2 are rRNAs, and 13 are polypeptides.
- the mtDNA genome lacks introns, pseudogenes and intervening sequences that are
common in nuclear DNA (nDNA).
- various techniques have been developed to score polymorphisms in mtDNA.
- originally mtDNA are isolated from purified mitochondria, digested with a variety of
restriction endonucleases and the resulting products run on agarose gels.
- restriction endonucleases are enzymes that cut DNA when at highly specific
recognition sequences (usually 4 to 6 bp in size).
- for example, the restriction enzyme EcoRI cuts DNA at the sequence GATTC.
- suppose we isolated mtDNA from five individuals and cut the DNA with EcoRI.
- we may observe the following pattern:
Pcr.exe Cycseqpc.exe

DNA_DetectivePC.exe Gels PC.exe

- the frequencies of polymorphic sites for different enzymes
can be estimated and compared among different populations of
a species or among different species.
- because mtDNA is haploid, there is no heterozygosity.
- however, we can still estimate a measure of gene diversity
that reflects the number of polymorphic sites present in a
population and the frequencies of these sites.
- now we use the polymerase chain reaction (PCR) to amplify
smaller pieces of mtDNA for sequencing or scoring of
polymorphic restriction sites.
b. nuclear DNA (nDNA)

- restriction fragment length polymorphisms (RFLPs) can also

be scored in regions of nuclear DNA.
- the traditional way of scoring RFLPs is to isolate and label a
clone and then hybridize it to a membrane filter containing
DNA digested by a specific restriction enzyme.
- the location of the fragment is then located (ethidium
bromide)
- now we use PCR to amplify pieces of DNA of various sizes
for similar analyses.
c. microsatellites
- a more recently developed class of markers are called microsatellites.
- microsatellite arrays contain varying numbers of a small repeating unit that usually is 2-
4 bp in size (for example, CA or GC).
- within a specific chromosomal region, the size of microsatellite arrays differ because of
differences in the numbers of the repeating unit.
- for example, one microsatellite “allele” may contain 20 copies of CA.
- another allele may contain 19 copies.
- individuals may be homozygous for the 20 copy or 19 copy allele, or they may be
heterozygous and have both.
-----------CACACACACACACACA-------------- 8 “repeats”
-----------CACACACACACA---------------------- 6 “repeats”
- microsatellites are scored by using the PCR reaction to amplify the array using primers
constructed from unique flanking sequences of DNA.
- some microsatellites exhibit high levels of variability - and may have high mutation
rates (10-3 to 10-4 per gamete per generation).
Estimates of genetic diversity
- many thousands of species have now been surveyed for genetic diversity at
allozyme loci.
- the extent of variation present in most species is large.
- heterozygosity estimates are usually in the range of 8 to 20%.
- marine invertebrates - notably molluscs and echinoderms - have the highest levels
recorded.
- so an abundance of genetic diversity is there - why should we be concerned about
maintaining it?
Why is genetic diversity important?
- there are two ways in which genetic diversity is important for a species.
- one deals with present conditions, the other with the future.
1. Genetic diversity is selectively important
- genetic variation may be adaptive for the species.

- for example, if the geographic range inhabited by a species is large and variable some of the
genetic polymorphisms present may facilitate living in a varied set of conditions.
- an excellent example of a well-studied polymorphism that is experiencing natural selection is the
leucine aminopeptidase (Lap) locus in the blue mussel, Mytilus edulis.
- this enzyme catalyzes the cleavage of n-terminal amino acids from di-, tri- and tetrapeptides.
- the enzyme has two distinct functions.
- one is to serve as a digestive enzyme - it is abundantly expressed in the gut lumen.
- the other function is osmoregulatory.
- marine bivalves are osmoconformers - the osmolarity of their tissues is identical to that of
surrounding sea water. This osmoconformation is achieved by modifying intracellular levels of
free amino acids - notably proline, glycine and alanine.
- as salinity goes up, small peptides are cleaved and the a.a. pool increases.
- as salinity falls, these a.a.’s are removed from the pool to reform small peptides.
- some amino acids are exported to the haemolymph some are ultimately excreted. This results in a
net loss of nitrogen which may affect the animals energy budget.
- Lap functions in this capacity.
- a sharp cline exists in the frequency of the Lap94 allele at the entrance to Long Island Sound.
- the frequency of the 94 allele declines sharply from 0.55 in full oceanic salinity environments to
0.12 over a 50 km area.
- what is the cause of this cline?
- is there any environmental factor responsible for producing this cline?
- yes, at the entrance to Long Island Sound there is a drop in salinity from oceanic levels (33-35
ppt) to estuarine levels (25-30 ppt).
- the Lap-allele appears to be optimized for functioning in a high salinity environment.
- at the biochemical level it has been found to have a higher catalytic efficiency - about 20%
greater than the other alleles (96 or 98).
- find that genotypes possessing the 94 allele have higher catalytic activity.
- in the brackish water environment of Long Island Sound, the Lap-94 allele is at a disadvantage -
individuals possessing this allele suffer a higher loss of nitrogen and experience higher levels of
mortality than genotypes lacking the Lap-94 allele.
- the form of balancing selection acting to maintain the Lap-1 polymorphism is thus environmental
heterogeneity in salinity.
2. Genetic diversity may be adaptive for future conditions
- the presence of suitable levels of genetic variation may be crucial in
allowing a species to successfully adapt to changing environmental
conditions at some future date.
- if this variation is lost, the capacity of the species to adapt may be
compromised and it may face extinction.
- this is because the capacity of a species to adapt is dependent on its existing
reservoir of genetic variation.
- the process by which new genetic variation is introduced into a species -
mutation - is too slow to fuel this adaptive response.
Threats to genetic diversity
1. Genetic bottlenecks
- severe overexploitation of a marine species can cause the population to crash to low numbers.
- during the time that the population remains small, genetic diversity is easily lost.
- the population may or may not recover.
- if it does it has experienced what is called a “bottleneck”.
- an extreme example of a major genetic bottleneck involves the northern elephant seal (Mirounga angustirostis).
- the northern elephant seal was almost hunted to extinction in the late 19th century - it is estimated that only 10-30
individuals survived
- the species has now rebounded to a population size of approximately 130,000.
- in 1974, the Northern elephant seal was surveyed for genetic variation at 24 electrophoretic, or allozyme, loci by
Bonnel and Selander in 1974.
- surprisingly, it was found to possess no genetic variation!
- the Northern elephant seal is an interesting case in that the population has rebounded and appears to being do
fairly well without polymorphism that is commonly found in other large mammals.
- the Southern elephant seal was also extensively hunted in the last century but never experienced a decline below
an estimated size of several thousand animals. It possesses levels of protein polymorphism that are comparable to
other large mammals.
- the ultimate fate of the Northern elephant seal may ultimately be affected by this lost variation - it may be poised
on the brink of extinction if, for example, an epidemic were to strike.
2. Inbreeding
- inbreeding occurs in a population when matings occur among related individuals more commonly than
they would in a randomly mating population.
- there are two genetic consequences of inbreeding:
1. Increase homozygosity (i.e., prop. of homozygotes)
2. Decrease heterozygosity (i.e., prop. of heterozygotes)
- both consequences lead to inbreeding depression
- the loss of heterozygosity means that the population may lose adaptively significant variation.
- the increase in homozygosity is bad because it will result in the manifestation of homozygotes for deleterious
alleles normally “masked” in heterozygous state.
- both lead to what is called inbreeding depression.
- inbreeding depression causes a wide range of debilitating conditions:
1. reduced viability
2. sterility
3. reduced longevity
- inbreeding depression may contribute to population extinction as the fitness of the population erodes over time.
- inbreeding depression is a serious problem faced by the aquaculture industry.
- in maintaining small numbers of individuals in hatcheries as “broodstock”, over time there will be inbreeding.
- this makes it difficult for the broodstock to be successfully propagated over time.
- it is common to “outbreed” the stock by introducing new, unrelated individuals that introduce new genes into the
population.
3. Hybrization by introduced species
- a final threat to genetic diversity is caused by the inadvertent (or sometimes
purposeful) introduction of species to a new part of the world.
- these are commonly referred to as invasive species – you already had a lecture on
this problem earlier in the class.
- invasive species also can wreak serious genetic harm after their introduction.
- they can do this by intercrossing with closely related species that they have not been
exposed to in their evolutionary past.
- this is called hybridization.
- the two species form what is called a hybrid zone.
- the hybrid zone can have serious consequences to population “health” because
hybrids are usually inferior to either parental species and commonly are sterile or
have greatly reduced fecundities.
- this can cause dramatic declines in population size.
- an interesting (and scary) example of this problem is occurring right in our back door involving
mussels.
- a hybrid zone occurs in Monterey Bay between a native species of mussel, Mytilus trossulus
and an introduced species, M. galloprovincialis.
- galloprovincialis was introduced into southern California in the early part of the last century
from the Mediterranean.
- it has been rapidly moving up the coast and hybridizing with the native trossulus.
- it had reached Cape Mendocino about 5-6 years ago and stopped further expansion – we
thought it may be a “barrier” to further movement north.
- no such luck, it expanded past this cape during the last El Nino and is heading on up the coast.
- the consequence for trossulus is bad
- how many other examples are there like this?
- only a few others are known.
- many, many more are probably occurring right now but we don’t know it.
- we only discovered the mussel hybrid zone because mussels are extensively studied at the
genetic level.
- it is scary to consider that many other examples are likely out there waiting to be discovered.
 Conserving genetic diversity
- how do we go about preserving genetic diversity?
- recently, new approaches have been developed that utilize information provided by
phylogenetic trees.
- two approaches have been proposed:
1. Preserve the evolutionary history of the group.
- here, high value placed on phylogenetic “distinctiveness”.
- species (or populations) that are very different from others are given high priority for
conservation.
2. Preserve the evolutionary future of the group.
- here, priority is placed on trying to preserve young, rapidly diversifying groups
since these are viewed as having the highest “evolutionary potential”.
- which approach is correct?
- unfortunately, we don’t know…
Allozymes: have a simple and well understood genetic basis but their adaptative role, even
if minor or questionable, limits their utility as neutral markers for studies of population
structure.

RFLP (Restriction Fragment Length Polymorphism): Simple and not very expensive.
(Enzymatic digestion of the DNA at specific sites, which produces fragments of DNA that
can be separated by size using electrophoresis. Not to be used in nuclear DNA (produces
too many fragments). More suitable in more limited genomes such as mitocondrial DNA
(mtDNA)

Minisatellite DNA fingerprint: use non specific probes that reveals profiles unique to each
individual (expensive and difficult, but once developed they are very powerfull genetics
markers).

Microsatellites cDNA: use specific probes that recovers locus-pecific variation.(expensive

and difficult, but once developed they are very powerfull genetics markers).

RAPD (Random amplified Polymorphic DNA): A relative new technique, is simple to

develop, also identifies locus-specific variation, but does not require the use of radioactive
material. Compared to other DNA-based techniques, development and use of RAPD
markers is faster and less expensive. One disadvantage of RAPD polymorphisms compaed
to microsatellites is that the heterozygote cannot be distinguished from the positive
homozygote.
Test de paternidad:

El bird N° 2 puede excluirse de ser progenitor, ya que el descendiente

Posee A5A2 y el bird 2 no posee ninguno de estos alelos. El locus B
No excluye a ninguno de los potenciales progenitores.
RFLPs patterns using the ITS assay:
M.edulis (B), M. chilensis (C), M.
galloprovincialis (D), M. trossulus (E).
DNA ladder (A).
 bp A B B B B C C C C D D D D
1500
1000

500
450
300

200
180
100

Fotografía digital de un gel de agarosa al 3 % teñido con ethidium bromide sobre un transiluminador U.V. En
donde se observan los patrones de banda RFLP (restriction fragment length polymorphism) producidos por el
marcador nuclear ITS (internal transcriber spacer) en muestras de chorito chileno provenientes de Pto. Marín
Balmaceda (B), Bahía Laredo (Pta. Arenas) (C) y Yaldad (Chiloé) (D). Molecular weight marker (Promega’s 100
BP DNA) Ladder (A).
 A B B C C C D D
bp
A B B B B C C C C D D D D
1500

1000
300

600

500

300
200

100

A B B B B C C C C D D D D

Fotografía digital de un gel de agarosa al 3 % teñido con ethidium bromide sobre un transiluminador U.V.
En donde se observan los patrones de banda RFLP (restriction fragment length polymorphism) producidos
por el marcador nuclear GLU-5 en muestras de chorito chileno provenientes de Pto. Marín Balmaceda (B),
Bahía Laredo (Pta. Arenas) (C) y Yaldad (Chiloé) (D). Molecular weight marker (Promega’s 100 BP DNA)
Ladder (A).
 A B B C C C D D

bp
1500
126
1000

500

300

200

126
100

A B B C C C D D

Digital photo of an ethidium bromide stained 3% agarose gel transilluminated with ultraviolet light showing the
RFLP (random fragment length polymorphism) patterns produced by the Me nuclear marker for chilean blue
mussel samples from Pto. Marín Balmaceda (B), Bahía Laredo (Pta. Arenas) (C) and Yaldad (Chiloé Island)
(D). Molecular weight marker (Promega’s 100 BP DNA) Ladder (A).
 bp
1500

1000

500

300

200
180

126
100

A B B C C C D D

A B B C C C D D

Digital photo of an ethidium bromide stained 3% agarose gel transilluminated with ultraviolet
light showing the RFLP (random fragment length polymorphism) hybrid pattern produced by the
Me nuclear marker for chilean blue mussel samples from Pto. Marín Balmaceda (B), Bahía
Laredo (Pta. Arenas) (C) and Yaldad (Chiloé Island) (D). Molecular weight marker (Promega’s
100 BP DNA) Ladder (A).
 bp
1500

1000
800

500 500 500

300
200

100
A B B C C C D D

A B B C C C D D

Digital photo of an ethidium bromide stained 3% agarose gel transilluminated with

ultraviolet light showing the RFLP (random fragment length polymorphism) hybrid
patterns produced by the GLU-5 nuclear marker for chilean blue mussel samples from
Pto. Marín Balmaceda (B), Bahía Laredo (Pta. Arenas) (C) and Yaldad (Chiloé Island)
(D). Molecular weight marker (Promega’s 100 BP DNA) Ladder (A).
Biological species concept : “groups of actually or potentially interbreeding populations, which
are reproductively isolated from other such groups” (Mayr, 1970). Then: intrinsic (genetically
based) reproductive isolation (as opposed to extrinsic: e.g. geographic isolation) is central to
BSC. Hybridizing species, such as M. galloprovincialis and M. edulis or M. edulis and M.
trossulus cannot be viewed as species when the BSC is applied.

Evolutionary species concept: “an evolutionary species is a single lineage of ancestor-

descendent populations which maintains its identity from other such lineages and which has its
own evolutionary and historical fate” (Wiley, 1978) . In this case hybridization is not important,
what is the issue is whether the two species maintain their separate identities despite hybridization.
This is very subjective and allows for wide interpretations. When two species co-occur in a site
and it is almost impossible to distinguish between the two types, because there is such a wide
character overlap, it is difficult to consider that the individual identities are maintained.
Phylogenetic species concept: “the smallest diagnosable cluster of individual organisms within
which there is a parental pattern of ancestry and descent” (Cracraft, 1989). The same author notes
that “even if two sister-taxa hybridize, both can still be considered to be species if each is
diagnosable as a discrete taxon”. Thus this can be applied to Mytilus, however, the critical point is
that “both species have had a distinct phylogenetic and biogeographic history prior to
hybridization”, and we do not known much about the phylogenetic and biogeographic history
before hybridization. This concept do not consider reproductive isolation in the species definition,
however, put more emphasis on “diagnostic characters”, and there are now genetic markers that
allow us to distinguish between taxa within the genus Mytilus (Heath et al., 1995; 1996).
However, in general, the fact that the number of species recognized depends upon the resolving
power of the analytical tools available may impose a severe limitation to the PSC. For example, if
each individual organism is genetically unique at a high level of resolution, then the grouping of
individuals requires that we ignore differences that occurs below some arbitrary threshold.
Cohesion Species Concept, “species is the most inclusive population of individuals having the
potential for phenotypic cohesion through intrinsic cohesion mechanisms” Templeton (1989): The
focus of this concept, is on cohesion mechanisms such as gene flow, stabilizing selection, and
common ecological factors that keep species homogeneous. The problem becomes when we try
to make it operational, How similar is similar?, then it becomes a phenetic species concept. There
is little evidence that taxa within the genus Mytilus is cohesive, so under the CSC criteria these
are not good species.

As a conclusion: the different forms of Mytilus may not at this stage merit full species
status. According to present evidence, it is difficult to say if these taxa are in process of
differentiation (early sympatric speciation) or they are in a process of intergradation after a period
of allopatry. Then, we may treat each of these taxa as a semispecies (trinomial nomenclature) as
component members of the Mytilus edulis superspecies (note that we do not even agree on a
species definition) or may retain the binomial nomenclature, and eventually accept the
phylogenetic species concept.