Advances in modern-day biology and the
implication of new approaches in conservation
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Introduction
Current importance of conservation genetics
Erosion of global genetical resources
Taxonomic problems, hybridization
New research approaches
Role of genomics in conservation biology
High-througput technologies
Mass research of biodiversity
Breeding of endangered species, reproductive technologies and cloning
Genetically modifies organisms
Fossil DNA and Jurassic parks
Genetic resources in modern collections, silent biodiversity crisis
Future challenges
Introduction
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Conservation biology has been described as a crisis discipline which aims to an
understanding of the patterns and processes that make it such a critical subject.
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Crisis disciplines often see periods of expansion of the toolbox used to address the
dilemmas posed by the forces causing the crises. In conservation biology, these tools
have advanced accordingly.
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Some examples of this toolbox expansion include the use of global positioning
systems, satellite-based imaging of areas, the inclusion of new mathematical
approaches in conservation theory, and perhaps the most visible: genetics.
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The inclusion of genetics in conservation biology over the past decade has been
influenced by the proliferation of technology in genomics, phylogenetics, and
population biology.
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One of the major challenges to modern-day conservation genetics is the more
efficient and better defined incorporation of genetics into conservation decision
making in the context of complexities of social, cultural, and political issues.
Current scope of conservation genetics
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In a landmark paper, Lande (1988) pointed out that demographic factors were much
more important in explaining extinction than any of the genetic factors. Caughley
(1994) suggested that too much focus on technical approaches to conservation
(including conservation genetics) had resulted in neglect of more important issues
such as habitat loss and disease.
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The interest in genetic variation and structure should not result in neglecting of
ecological or epidemiological issues. Population genetics should be combined with
demographic data in the analytical approach of the population viability analysis.
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All the efforts of conservation biology are aimed to single goal to conserve living
systems, and it has no sense to differentiate them into mutually competing fields.
Modern conservation genetics demands combination of authentic field research and
excellent laboratory skills.
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Conservation genetics builds bridges to conservation management and education. It
is not appropriate to reduce this work to more service-oriented, uncreative, routine
work which ultimately not result in high quality work or synthetic understanding.
Erosion of global genetic resources
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Global extinction rates are estimated as 1,000 to 10,000 higher than at any time in
the last 65 milion years (Lawton a May 1995). Much of the current concern over the
extinction crisis centers on loss of species, but the scale of population extinction is
truly alarming.
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Considering that there are some 1,1 to 6,6 billion genetically distinct populations
globally, the rates of tropical forest loss suggest rates of local population extinctions
on the rate of 1,800 populations per hour (Hughes et al. 1997). This rate is 432 times
greater than that of species loss in the same region (approximately 100 species per
day; Lawton a May 1995).
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Gibbs (2009) estimated that relative contribution of loss of populations to erosion of
global genetic diversity may be 8-21% in vertebrates, and 11-12% of aggregate loss.
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At least for vertebrates, most remnant populations tend to persist in the periphery of
the species’ historical geographic range, and thus species ranges tend to collapse
outward rather than inward. It is still not clear what is the potential significance that
incremental loss of locally differentiated populations represents for global genetic
resources.
Taxonomic drawbacks
Modern taxonomy against conservation agenda.
Protection under act may not be flexible on taxonomic matters.
What is the conservation unit?
Species, subspecies, evolutionary significant unit, management unit ?
Taxonomic inflation
In well-studied groups, the number of species is increasing very rapidly, and this is in
large part from the elevation of subspecies to the species level. Known subspecies are
raised to species as a result in a change in species concept, rather than to new
discoveries.
DNA barcoding
• DNA barcoding has targeted the sequencing of a DNA marker of all the named
species on the planet. It uses a short DNA sequence from a standardized position in
the genome as a molecular diagnostic for species-level identification.
• DNA barcodes are short and can be obtained rapidly and cheaply as a result of highthroughput approaches. Barcodes are unique identifiers of commercial products
• DNA barcoding has provoked both excitement and criticism
• New bioinformatic tools: Consortium for the Barcode of Life (CBOL)
http://www.barcoding.si.edu; http://www.dnabarcoding.ca
Problem of hybridization
Human-induced hybridization events
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Simien wolf x domestic dog, Ethiopia
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Cuban x American crocodile, Cuba
Natural hybridizations
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The red wolf (Canis rufus) was believed to be an endangered unique taxon in southeastern US. It has now been demonstrated that „red wolves“ are the descendants of
a natural wolf – coyote hybrid zone.
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Polar bears – do they actually constitute a species?
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Asexual reproduction
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Geographical parthenogenesis
New research techniques
The expansion of the scope of conservation genetics over the past two decades has
been brought by the infusion of high-throughput DNA sequencing and genotyping
technology.
Sparingly used or extinct techniques
• Allozymes
• Random amplification of polymorphic DNA (RAPD)
• Restriction fragment length polymorphism (RFLP)
• Minisatelites
Used techniques
• DNA sequencing
• Amplified fragment length polymorphism (AFLP)
• Paired interspersed nuclear elements (PINEs)
PINE fragments used to identify
hybrids between two trout species
• Microsatelites
• Single nucleotide polymorphism (SNP); Inter-simple sequence repeat (ISSR)
• DNA typing (microarrays; DNA chips; expressed sequence tags, ESTs)
Conservation biology by definition should be non-invasive, and the development of new
techniques of non-invasive approaches is of major importance.
Conservomics?
The role of genomics in conservation biology
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Genomics is the culmination of three decades of technological development that has
paved the way for the creation of high-throughput biology.
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Genomic techniques have promised to expand and supplement the field of
conservation genetics, and there are various specific ways in which this expansion
might be possible. Population genomics refers to the study of many DNA markers in
many individuals from different populations.
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High-troughput refers simply to the ability of modern techniques to generate large
amounts of data by increasing the throughput of specimens analyzed.
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Conservomics – the union of high-troughput technology with conservation genetics
(Amato and DeSalle 2009)
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DNA sequencing, transcriptomics, proteomics, metabolomics
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Within a space of less than a decade, the development of high-throughput
technologies has transformed the task of sequencing a mammalian genome from the
year-long and extremely expensive endeavour, to a project that can be performed by
an individual laboratory within a few months.
High-throughput sequencing and
genotyping technology
New DNA sequencing technology
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Solexa (Illumina): A method of parallelization of DNA sequencing called bridge
polymerase chain reaction.
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Emulsion PCR (Margulies et al. 2005): Individual DNA molecules and primer-coated
beads in aqueous bubbles encased in an oil phase.
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The Margulies emulsion approach has been commercialized by 454 Life Sciences,
the Shendure emulsion approach (originally called „polony sequencing“).
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The 454Life Sciences sequencing method uses pyrosequencing approaches. This
method adds one nucleotide at time and then detects on the solid surface through
light emitted by the release of attached pyrophosphates.
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About 2,000 sequences can be handled by a single automated DNA sequencer in a
day. The new techniques have increased the throughput of sequencing by two or
three orders of magnitude (up to 100 million pb per run).
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All these methods have limitation that the fragments immobilized on the solid surface
usually are very short. Shotgun methods which produce short sequence reads (but
high error rate). This may be advantage for studies of fossil DNA.
New high-throughput DNA polymorphism
detection approaches
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All microarray approaches are based on DNA hybridization, RNA is used as a
template for hybridization to the array. The use of target DNA as a template enables
the re-sequencing approach, which can be used e.g. for scanning the state of
thousands of SNPs.
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Multiplex PCR
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Denaturing high-performance liquid chromatography (DHPLC): examination of SNPs;
the differences in DNA are detected using ultraviolet absorption.
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Temperature gradient electrophoresis.
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High-resolution melting of entire amplicons.
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MALDI-TOF: matrix-assisted laser desorption/ionization time-of-flight; mass
spectrometry.
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Single-strand conformation characterization.
Mass biodiversity research
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Environmental DNA – assaying of microbial diversity in environmental samples
Metagenomic approach – analysis of composition of metapopulations of organisms
High-throughput DNA polymorphism detection and conservation biology
Direct inferences of detrimental and adaptive variation in „genome-enabled“ taxa
QTL mapping techniques, MHC genes
Bioinformatic databases
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DNA DataBank of Japan
European Molecular Laboratory
GenBank at the National Center for Biotechnology Information, USA
BLAST, BLAT – tools to compare and visualize sequence information
Ensembl Genome Browser
Santa Cruz Genome Browser
NCBI MapViewer
New language of genomics and molecular biology
System approaches in biology
Genetic management of captive populations
Captive populations can serve a number of different roles in conservation efforts. Most
importantly, they can be used to educate, enlighten, and enchant people about the
diversity of species and their adaptations. Captive populations have also served as
sources for supplementation or restoration of wild populations.
Importantly, good genetic management is a prerequisite for any of these conservation
goals of captive populations to be achieved. Some animal populations in zoos have
become so inbred or otherwise compromised genetically that they are unlikely to persist
longer than few more generations.
Captive populations need not only to retain the wild characteristics but also to retain high
levels of genetic variation. We need to stop three causes of genetic change:
• Random genetic drift
• Artificial selection for specific, favoured phenotypes
• Unintended natural selection for traits that confer high fitness in captivity
Drift can be minimized by breeding strategies that maximize the effective population size.
Reproductive technologies
Is biotechnology the solution for stopping genetic change in captive populations?
Powerful tools for manipulating genes may provide us with methods the cessation of
evolution that we desire in our wildlife populations being conserved in captivity.
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Molecular characterization of genetic diversity
Cryopreservation of gametes and embryos
Propagation of nearly extinct species by cloning
DNA manipulations
Utilization of these high-tech approaches in conservation is still highly problematic.
Low-tech approaches to conservation genetic may find much better use.
High-tech and low-tech approaches should be viewed as complementary rather than in
conflict.
Low-tech conservation genetics
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Classic methods of pedigree analysis and management
GENES – programme for pedigree analyses
VORTEX – simulation software for population viability analysis
Testing of the effectiveness of conservation breeding strategies
The role of assissted reproduction
in animal conservation
The transfer of embryos to the uterus. The first calf from transferred embryos was born in
1950, in humans the first in vitro fertilization and embryo transfer in 1978.
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Artificial insemination is widely used in cattle industry, and it is particularly successful
in bovids and felines and even in pandas. Fertilization in vitro can be achieved with
much lower quantity of sperm compared to artificial insemination.
Cryopreservation of sperm works well only for certain groups., e.g. freezing of mouse
sperm was regarded for many years as difficult or impossible.
Cryopreservation of eggs is difficult because of the large volume of cytoplasm. There
is a real risk of inducing chromosome abnormalities.
Importance of the knowledge of endocrinology of the reproductive cycle. Transfer
between different species is difficult, possibly for immunological reasons.
Embryo splitting has not been widely used in farm animal breeding because of the
young tend to be oversized at birth. This form of cloning is unlikely to have a wide
application in conservation.
Nuclear transfer cloning
Conservation and cloning
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Cloning is the technique of transferring nucleus from a body cell to another cell,
usually an unfertilized egg, previously stripped of its own genetic material.
Cloning has yet to become practical procedure. Its success is rather limited, methods
are not optimalised. For every cloned animal that is born alive, a far greater number
have died at some stage during development.
Cloning techniques are quite demanding and often species-specific. Obtaining of
oocytes and surrogate mothers in sufficient number is difficult in endangered species,
The transfer between species had only limited success. Cloning is obviously possible
only between closely related species.
The only live birth produced from an interspecies reconstructed embryo is Noah.
Noah was created from the skin cell nucleus of a gaur and an enucleated oocyte from
domestic cow.
The mixture of mitochondrial and nuclear genes from the different species may also
disrupt genetic programming and development of hybrid embryo.
Cloning is a very poor and inefficient method of reproducing yielding only small
number of genetically identical individuals. The present methods of cloning are
unlikely to have a significant role in conservation of species.
GMO and transgenes
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The increasing trend of producing transgenic animals and plants and public
discussion of concerns surrounding the products of crop biotechnology.
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Once transgenes move into free range, they have the opportunity to multiply and
spread via sexual reproduction.
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Hybridization between genetically modified crops and wild unmanaged species.
There are the world’s 13 most important food crops in terms of area planted, and all
but one apparently hybridize naturally with wild relatives. Assumed hybridization and
gene flow rates seems to be biologically significant, greatly exceeding mutation rates.
Some small portion of hybrids will cause problems after transmission of pest and
herbicide tolerance to weeds. Most of genetically modified organisms contain
bacterial genes derived from Bacillus thurigiensis that produce insecticide proteins. Bt
varieties have resistance against insect pests.
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Sunflower is one of the most important oilseed crops. The wild form of the same
species is a noxious weed and can act as a weed to the crop form. Experiments have
shown that pollinating insects may move some of the crop’s pollen into the weed
populations, resulting in hybridization. Crop alleles have established and spread
through wild populations.
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Genetically modified salmons have a new gene that maintains steady production of
growth hormone (the production is cyclic naturally). What will happen if this gene
escapes to wild salmons or other fish species after hybridization?
Fossil DNA and Jurassic parks
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The generation of complete genomes initiated era of comparative genomics. Genome
sequencing projects offer only a source for subsequent research. Very few genomes
have been completed even in draft form, and even fewer are considered finished.
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Ancient DNA can be obtained from fossils up to about 100,000 years old.
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The entire genome of a 38,000-year-old Neanderthal has been sequenced by a team
of scientist from Germany in 2009. DNA has recently been extracted from the bones
of five other Neanderthals.
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Ancient DNA is highly fragmented, is present in only trace amounts, and is usually
swamped by bacterial and fungal DNA. Obtaining of reliable sequences requires
multiple sequencing coverage (around 10-20 fold).
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The techniques of the study of ancient DNA contain inherent problems, particularly in
regards to the generation of authentic and useful data. Research in this field must
take a more cognitive and self-critical approach. Criteria for authentication should be
adopted to reduce contamination and artefactual results. Only a few studies were
published that appear to have adopted all proposed criteria.
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Idea of reviving extinct species: Michael Crichton, Jurassic Park, 1990
Let’s make a mammoth?
Can we put flesh on the bones of the draft sequences and go from a genome to living
beast? It would require to master the following steps:
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defining exactly genome sequences
synthesizing a full set of chromosomes from these sequences
engulfing them in a nuclear envelope
transferring that nucleus into an egg
getting that egg into a womb that would carry it to term
None of these steps is currently possible.
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Receiving of a sufficiently error-free genome would be extremely costly and timeconsuming.
Millions of the tiny fragments of long-dead DNA should be assembled into a coherent
sequence.
Synthesis of whole the genome and wrapping naked DNA up in proteins that
condense into chromatin.
Karyotype.
Mitochondria and sperm problems.
Problems in reproductive biology of elephants.
Question of genetic variation.
Biodiversity, conservation and genetic
resources in modern collections
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The roles and approaches of modern museums and herbaria have changed
dramatically as a result of the use of more genomic approaches to specimen
analysis. The need to collect samples under a modern paradigm of preservation that
maintains also the integrity of biomolecules within collected specimens.
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Biological materials change and deteriorate with time. It is necessary to elaborate
approaches to maintain materials containing native DNA available for future analyses.
Banking of genetic resources.
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The most effective means of preserving biological materials is by freezing and
subsequently storing at low temperature. The ideal technique of ultracold
preservation is the storage in liquid nitrogen. Frozen zoos.
The Ambrose Monell Cryo Collection at the American Museum of Natural History.
Center for Reproduction of Endangered Species (CRES), the Zoological Society of San Diego.
CCTCs = Cryopreserved Cell and Tissue Collections (provide access to chromosomal analysis)
CCTC = DNA + RNA + Protein Banks = Genome Banks
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There is no recognized worlwide directory of genetic resource collection and no
universal information system. Creation of a genome database on a global scale is
urgently needed. Modern bioinformatics ultimately link tissue specimen collection
record with bibliographic citations.
The silent biodiversity crisis
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For many centuries biological materials have been collected. Ex situ genetic resource
collections. Genetic resources include any representation of genetic diversity, from
natural occurrences to highly domesticated and managed collections. Typically,
genetic collections include species, populations, genotypes or individuals, tissues,
genes, DNA or RNA sequences.
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It is estimated that there are 6,500 natural science collections worldwide, with more
than 2,4 billion specimens.
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The silent biodiversity crisis concerns the loss of biorepositories and biological
research collections.
Predominant problems
• Insufficient, reduced, fluctuating funding
• Under-evaluation of collections, the true value of these collections is not calculated
• Accidents, natural disasters, wars
• Insufficient taxonomic expertize (taxonomic impediment). There are only about 5,000
experienced taxonomist worldwide and their abundance is decreasing
• Inappropriate use of genetic resource collections
International Species Information System (ISIS, 675 members from 73 countries)
Future challenges
Conservation genetics on the age of genomics
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Rapid biodiversity assessment will become more and more important in modern
conservation and its heuristic potential is rapidly growing with availability of highthroughput technologies in the age of genomics.
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Utilization of conservation genetics in real effective programmes of nature
management (including landscape dynamics a area-based conservation).
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Integration of genetic data with other biological and abiotic factors. Population viability
analyses.
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Landscape genetics inheres in fusion of molecular population genetics and landscape
ecology with the use of new statistical tools. Recognizing of landscape features
limiting gene flow, cryptic boundaries, secondary contact zones.
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There is a great need to enhance the ability of conservation genetics to noninvasively
collect, archive, and use genetic resources of as broad an array of biodiversity as
possible.