Molecular Genetic Approaches in Wildlife Conservation

Abstract

Wildlife conservation is of major biological importance due to the survivability of organisms in an ecosystem and population stability. The primary concern of the management and genetics of populations is inbreeding. The small population size can play a diminishing role in genetic variability and increasing inbreeding in animal populations. Inbreeding depression can be due to major factors such as rare, deleterious, recessive alleles which can be repressed under heterozygous conditions. The decreasing effect in heterozygosity can be significant upon severe bottleneck effect. The genetic variations between populations could be assessed using molecular techniques. Mitochondrial DNA variations for determining the founder’s effect can be widely applicable in the management of wild populations. The maternal lineages in a captive population can signify the variations in the population as well as the number of males contributing to the gene pool of the various population. Molecular markers can be used to differentiate between populations and identify the individuals contributing to the gene pool of the species.

1. Introduction

Wildlife populations are faced with several anthropogenic pressures such as climate change, habitat fragmentation, destruction of habitat, pollution, the threat of invasive species, harvesting, and ill effects of novel pathogens. This contributes to high extinction and challenges in sustaining wild populations [1]. While habitat fragmentation has been fundamentally the prime concern for conservation research. The evolutionary consequences of fragmentation are underestimated due to a reduction in the levels of gene flow among the formerly connected fragments of the forest. An increase in genetic drift and selection by the evolutionary processes may lead to differentiation among populations would [2]. Increased tendency for genetic differentiation and local adaptation are not considered for resource-based management. Fragmentation of habitat combined with management efforts may create a source or sink dynamics which is vital for selection [3].

The loss of genetic diversity often leads to affect individual fitness and poor adaptability to their surroundings [4]. A small population size is at risk of genetic changes within the population. The captive breeding programs of vulnerable or endangered animals are necessary for their conservation to increase their chances of long-term survival; however, this methodology often increases the chances of inbreeding causing poor fitness in populations [5]. It is under stood that inbreeding causes a decreased genetic diversity and leads to a reduction in reproductive rates resulting in increased extinction or survival risks. To recover genetically impoverished endangered populations, they may breed with individuals from other populations [6]. If genetic diversity is too low any wildlife population can be at a greater risk of extinction [7]. There are many molecular techniques for genetics studies that can reduce the extinction risk by suggesting localized monitoring population and management mechanisms wherever necessary that can reduce the chances of inbreeding. Breeding initiative programs are started assuming that the founders of the captive population are distinct from each other.

2. Wildlife genetics

Small population size contributes to inbreeding and loss in genetic variation. Thus, the measure of genetic variation in a given population serves as a measure of the extent to which the population is inbred. Effective recombination occurs only in individuals heterozygous at many loci. Inbreeding reduces the frequency of heterozygotes, thereby reducing the effective rate of recombination. Inbreeding causes an increase in homozygosity resulting in increased expression of deleterious effects of recessive homozygous genotypes and decreased frequency of heterozygous combinations which may be over dominant.

Random fluctuation in gene frequencies of alleles resulting in random genetic drift reduces the genetic variation, increasing the homozygosity and the loss of evolutionary adaptability to environmental changes within small populations. The maintenance of genetic variability in a real population can be understood by Wright’s concept of effective population size [8]. Populations with different selection effective population sizes are predicted to develop profoundly different genome architectures [9].

3. Genetic variation

Species in general are a set of individuals that are capable of interbreeding and producing fertile offspring which can be genetically alike. According to Darwinism, the species instead of being constant changes according to its environment causing variation over time. Genetic variation in biodiversity means that variation at all levels of biological organization [10] including diversity within or among species.

Genetic variation in populations results in short-range fitness and long survival rates (population level). Genetic variation is caused due to evolutionary driving forces like natural selection and genetic drift etc. which results in variability among individuals causing differentiation at the population level, species, and higher-order taxonomic groups. The study of variation among individuals, populations, and species is population genetics. Population genetics relates to the analysis of evolutionary and demographic factors affecting the genetic composition of a population [11] increasing the chance of an organism to survive and adjust to the ever-changing environment. However, genetic-based molecular markers are a powerful tool in analyzing the genetic distinctiveness of individuals, populations, or species [12]. For genetic analysis, modern sequence-based marker systems are being used now like single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) [13]. However, for fish population genetic studies, microsatellites have become the marker of choice [14]. Molecular markers are commonly used for wild-population studies [15].

4. Factors of evolution

Various evolutionary factors such as allele frequency, genetic drift, and mutation are affecting the wildlife populations. The Hardy-Weinberg theory proves that the relative frequencies of several gene alleles in a Mendelian population tend to remain constant [16]. The genotypic variability present in a population is passed on from one generation to the next. Evolution is defined as a change in the genetic composition of populations. If the genes frequencies in populations remained constant that means evolution could not occur.

The genetic forces that modify the gene frequencies in populations

• Mutation

• Selection

• Genetic drift

• Migration.

The assumption in the Hardy-Weinberg law is that the population is infinitely large. If the populations are small, it consists of small numbers of breeding individuals which tend to lose genetic diversity. That is true in populations from one generation to other. We assumed that the population is isolated and there is no immigration or emigration that carriers of some genotypes in preference to others. Migration in individuals of genetically diverse populations can increase or decrease the gene frequencies in a population gene pool.

5. Mutants in populations

In mendelian populations, mutation provides the ultimate source of genetic variation in that no two individuals have the same genotype. Genetic variability owes to mutation, unfixed genes which are represented in a population by two or more alleles. The existence of diverse alleles is because of a genetic mutation which is the fundamental force of evolution. Mutant alleles can persist in the gene pool of a population depending on natural selection for many generations according to Hardy Weinberg’s theory [16].

Most mutants that arise naturally in the populations are deleterious mutations and are harmful to the organism; in some cases, they are completely lethal. The accumulation of harmful mutations of hereditary diseases is opposed by natural selection. According to the theory of natural selection which is based that the progeny of any species survives and interbreed and produces a variety of surviving offspring. The population of a species includes genetic variables which occur more or less by natural selection in a given environment [17]. The better-adapted variants will constitute from one generation to another. Artificial selection is also a process similar to natural selection except that the variants leave the larger progeny which is chosen by human beings rather than the environmental factors.

6. Equilibrium between mutation and opposing selection

Deleterious mutants occur by natural selection in the populations. Some deleterious mutants are incorporated into the gene pool of every generation. A particular number of deleterious mutants are also removed in every generation by genetic death. If the elimination is higher in a population than mutation, the frequency of mutants will be minimized. When the numbers are produced to balance the numbers eliminated there will be an equilibrium. If the population will have a greater load of recessive than dominant mutants this will give rise to various diseases and deformities [18]. In a population; the magnitude of deleterious mutants will be determined by the rate of equilibrium of mutants.

7. Dynamic changes in populations

7.1 Genetics of inbreeding

Inbreeding depression can be manifested upon a decrease in litter size or infant mortality. A metapopulation structure can increase inbreeding in the case where a population is genetically isolated if fewer than one viable migrant is entering the population per generation. In the case of the patch population, each population would have gone through a recent genetic bottleneck during population colonization. Such kinds of bottlenecks will lead to more inbreeding depression [19].

7.2 Total variance

It is hypothesized that populations can go on fixation of some alleles and loss of others. The selection pressures may be different for different ecological patches. The genetic variation between isolated patches can help monitor the species from extinction. Diverse patches can lead to genetic variants that can survive under strong selection pressures such as diseases, or changes in natural vegetation [19]. Loss of genetic variation shows increased vulnerability to epidemic diseases because the population does not have a heterogenous immunological structure. Viral diseases have recently spread through populations of each species [20].

7.3 Demographic stochasticity

Demographic correlations occur along the time axis. Over time demographic changes such as weather conditions, epidemics, predators can be factored out leaving the residuals to be demographic stochasticity.

7.4 Environmental changes and catastrophes

Environmental changes can impact the population whereas volcanism, regional pollution, pandemics, and forest fires can affect metapopulation as a unit [21].

7.5 Extinction and recolonization

Slatkin hypothesized that extinction and recolonization can be a factor in gene flow. Further, suggested for evaluating the effect of extinction and recolonization on the genetic differentiation of populations depends how the colonization groups of individuals are formed and how much relation between colonization and migration [22]. It also states that if the average time in generations is less than the extinction of local populations or equal to the effective number of locally breeding adults will prohibit the genetic differentiation of local populations due to drift even though in the absence of migration, extinction and recolonization. This statement on extinctions and recolonizations corresponds to Wright’s rule for the exchange between permanent local populations [23].

8. Conservation and management strategies

Natural habitats are lost and fragmented at disturbing rates thereby natural resources require human involvement and management to sustain such pressures. Curtailing the tide of extinction due to habitat loss, deforestation, the introduction of alien species and overexploitation should and must continue to be urgent goals for conservation management. Advocacy toward evolutionarily enlightened management can help in advancing urgent goals. It is also necessarily need to recognize and understand the evolutionary correlates of anthropogenic forces in conservation biology. Upon environmental modification, a species must either respond to the selective pressures imposed by the environment or ultimately be lost to extinction [24].

According to Darwin’s theory of natural selection, organisms that are better adapted can acquire more resources and leave better-fit offspring. Thus, nature selects individuals with favorable traits that allow them to flourish and reproduce. This theory is popularly known as survival of the fittest. Genotypic variations arise from mutations that occur at the gene level during the copying of DNA. Natural selection leads to adaptation; a population’s character makes it suitable for a particular environment where favorable traits are passed from one generation to another. When the population is small, inbreeding is more likely to take place as the number of mates is limited. For several species, the offspring survivorship declines as the populations are inbred. Captive breeding populations of mammals such as ungulates, primates, and small mammals exhibit higher mortality from inbred mating than from noninbred mating [25].

9. Molecular advances in wildlife management

Less complex and exact methods for sex and species identification in animals are essential for applications in wildlife monitoring, wildlife conservation, and forensics [26]. Genetic applications provide a platform for using applied tools for estimating the effective population size and also the study of the changes in population demography for organisms that are difficult to census using ecological techniques [27]. There are various types of molecular markers nowadays scientist is using to estimate the genetic variability of animals classified as the non-PCR based and PCR based molecular marker. RFLP is the non-PCR based marker while DNA markers such as RAPD, RFLP, AFLP, VNTRs, and mitochondrial DNA markers is PCR based. Such markers have been used in genetic studies to understand the genetic divergence within and among populations or species [28].

10. Molecular applications toward conservation

Molecular markers are an advanced technique for the characterization of genetic resources. It compares the genetic diversities which can resolve taxonomic disputes and establish management elements within species. The molecular marker analysis can be used as a suitable tool for determining the genetic variation, biodiversity, illegal hunting and poaching for the effective implementation of the laws for the protection of endangered species. There are various DNA techniques available for genetic diversity analysis for conservation of wildlife populations [29]. The main application of mtDNA sequences in conservation genetics consists of population structuring, resolving taxonomic ambiguities, identification of interspecific hybridization, and combating illegal hunting and poaching activities of rare and endangered animals. The selection of appropriate bioinformatics tools often plays a vital role in constructing phylogenic inference with the help of mitochondrial and nuclear markers [30].

Molecular markers estimate the relativeness between the individuals by comparing the genotypes at several polymorphic loci. The vast majority of the systematic phylogenetic studies have applied mtDNA sequencing. Previous reports suggest that mitochondrial DNA and its phylogeny represent only the genealogy of a specific gene that is maternally inherited. There are many other molecular markers such as RAPD, AFLP, mitochondrial DNA or microsatellites can be used for population studies, species identification, biodiversity, phylogeny, and forensics investigation [29].

11. Molecular markers in wildlife conservation

11.1 RFLP (restriction fragment length polymorphism)

Restriction fragment length polymorphism is the most widely used molecular marker based on the hybridization technique. Data is analyzed based on the presence or absence of recognition sites for a specific restriction endonuclease. An RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments of the digested DNA after which they can be separated using agarose gel electrophoresis, characteristic of a specific genotype at a specific locus. The change in recognition site by point mutation or SNP produces fragments of different lengths in the same region of the genome. Although two individuals of the same species have an almost identical genome that will differ at only a few nucleotides due to the point mutation. The polymorphism in the hybridization pattern is created due to changes in restriction sites, such variation is called RFLP.

The main application of the RFLP marker is in the DNA fingerprinting technology in which repetitive elements are widespread across the genome for making multiple copies that are used as hybridization probes [31]. A study has also been conducted in Colombia for the identification, population structure, and genetic variability among bug species (Rhodnius prolixus) by RFLP markers [32].

11.2 Random amplified polymorphic DNA (RAPD) markers

RAPD markers are amplified products of the less functional part of the genome that do not vary upon selection pressure on the phenotypic level. It is a dominant marker. These markers are highly polymorphic [33]. Thereby certain regions of the DNA can gather more nucleotide mutations, and RAPD markers can assess interpopulation genetic differentiation. Use only a single arbitrary primer (8–12 nucleotides) which binds randomly throughout the genome and amplifies the gene. This marker produces different product sizes with the same primer in different organisms of the same species. This is detected in non-coding DNA sequences in the genome, but rare in coding sequences. RAPD loci are inherited as Mendelian markers which are dominant and can be scored as present or absent. RAPDs and PCR-based markers have similar benefits in that there is no need for information on the targeted DNA sequence or gene organization [34]. The primers are commercially available. Advantages of RAPDs are the ease of analyzing a large number of loci and individuals. The disadvantages of this marker are that this will not amplify in Mendelian inheritance of the loci and cannot distinguish between homozygotes and heterozygotes. The analysis follows assumes that the population is under the Hardy-Weinberg equation. RAPD markers can be used in identifying the presence of paralogous PCR products in which different DNA regions have the same lengths and appear in a single locus, with low reproducibility due to a low annealing temperature during the Polymerase chain reaction [35].

11.3 Single nucleotide polymorphism (SNP)

Single nucleotide polymorphisms (SNP) are the most common molecular marker analysis as they show abundant polymorphism in any genome. It includes both coding and non-coding region, and reveal hidden polymorphism which cannot be easily detected by other markers. SNPs can detect polymorphisms induced by point mutations which give rise to diverse alleles comprising alternative bases at a given nucleotide position within a locus [36]. A single-nucleotide variant (SNV) is a common definition for a single nucleotide change in DNA sequence. An SNP within a locus can replace the four bases (A, T, C, and G) in a certain stretch of DNA at the SNP site. Although most of the SNPs are restricted to only one of two alleles (either C/T or the A/G) and have been observed as bi-allelic in nature. SNP markers are inheritedas co-dominant markers. SSCP analysis has been commonly used for SNP identification, heteroduplex analysis, and direct DNA sequencing. Many SNPs do not alter cellular function so they show no effect but some SNPs have been associated with diseases such as cancer and to influence physiological responses to drugs. Now a days DNA sequencing analysis has been the most accurate approach for SNP discovery [37].

11.4 Microsatellite markers

Microsatellites recently have become a popular marker for population investigations. It is the simple sequence repeats of di, tri, and tetra nucleotides PCR-based markers. It is wildly used in fingerprinting technology. Microsatellites are short fragments of DNA generally multiple copies of tandemly arranged 1 to 6 bp long e.g., ACA or GATA, and are repeated up to about 100 times. Microsatellites are abundant in all species studied so far. They have been found in coding regions, introns, and non-gene sequences. Microsatellite markers are highly polymorphic, abundant, and evenly distributed throughout regions of chromosomes [38]. Irrespective of changes in the repeat units, there are large numbers of alleles at each microsatellite locus of a population. Microsatellites are inherited in a Mendelian fashion as codominant markers. Microsatellites are highly variable markers, and very much useful in individual identifications. However, the use of microsatellite markers is disadvantageous due to the requirement of a large upfront investment and effort. Each microsatellite locus has to be identified and its flanking region of the fragment of the marker should be sequenced for the design of primers for PCR. PCR amplification is done by slippage during replication, small differences in the size between alleles of a particular microsatellite locus are possible [39].

11.5 Mitochondrial DNA markers

Mitochondrial DNA (mtDNA) analysis is largely used in recent populations and phylogenetic assessment in wild populations. Mitochondrial gene is largely conserved across various animals, due to maternal inheritance with fewer duplications, no intron, and short intergenic regions [40]. Mitochondrial DNA is highly variable in nature due to its high mutation rate when compared to nuclear DNA. The mtDNA genome of vertebrates has been extensively analyzed in comparison to nuclear DNA for resolving species identification, phylogenetic and forensic investigation due to its unique property, lack of recombination, and high concentration of mtDNA per cell compare to nuclear DNA. Since mitochondria have a faster mutation rate, lack ofrepair mechanisms during replication, and have a smaller population size due to the maternal inheritance of the haploid mitochondrial genome [41].

Mitochondrial DNA analysis is useful in establishing phylogenetic analysis among closely associated species due to its high rate of mutation. It is known that different regions of the mitochondrial genome evolve at different rates. The complete mtDNA molecule is transcribed except for the control region or the Dloop which is approximately 1 kb where the replication and transcription of the molecule are initiated. Due to non-coding sequences of this region high levels of polymorphism are shown when in comparison with the coding regions of the mitochondria such as the cytochrome b and COI gene [42], this is due to reduced functional constraints and less selection pressure.

The 16S rRNA gene in the mitochondrial genome is the least evolving region of the mitochondria in comparison to rapidly evolving regions such as the control region [43]. In the non-Mendelian mode of inheritance, the mtDNA molecule is considered a single locus double-strand molecule because mtDNA is maternally inherited, and the phylogenies and population structures derived may not reproduce those of the nuclear genome if gender-biased migration or selection [41].

11.6 Next generation sequencing (NGS)

Next-generation sequencing (NGS) technologies are approaches that sequence nucleotides faster at a cheaper rate when compared to Sanger sequencing to study genetic variation associated with diseases or other biological phenomena. These are parallel to the DNA sequencing methods that have opened an innovative era of genomics and molecular biology. It came commercial use in 2005, this method was initially called “massively-parallel sequencing” because it can process millions of sequencing reactions at the same time. NGS can detect bases if performed cyclically and in parallel [44]. Population genomics is comparison of large sets of DNA sequences of various wildlife populations. This is a new model shift in the field of population genetics by combining genomics concepts [45]. Population genomics uses genome sampling to identify the phenotypic variation such as gene flow and inbreeding and to improve understanding of microevolution [46]. Due to the recent advancements in sequencing technology and data analysis, scientists are able to study thousands of loci from populations and understand genomic wide effects.

To measure the genetic variation in a species with the available reference genome in online databese, researchers can perform DNA/RNA-sequencing and epigenome sequencing. In a DNA-sequencing, NGS is generating whole genome, whole exome for eukaryotes, and target sequencing. Researcher are comparing the results to reference genomes and verify genetic variations such as SNPs, structural variations and other variations using software’s [47].

The advancement of NGS technology have aided scientists to examine biological systems with population-scale genomics in particular wildlife genomic research. The low cost and high output data of NGS provides more scope for population genomic studies along with newly developed data algorithms. The third-generation sequencing technologies also have been introduced recently. This third-generation sequencing technologies are aiding and supplementing NGS approaches. However, we may expect huge data in genomics soon and has opened up new experimental approaches in basic and applied clinical research [44].

Among the applications that benefit from NGS is the study of genetic diversity in heterogeneous samples. ShoRAH is a computational tool for quantifying genetic diversity in mixed sample and for identifying the individual clones. With the availability of NGS techniques, studying genetic diversity becomes faster and cost-effective when compared other sequencing. Now a days NGS are used in number of applications such as clinical genetics, microbiology, oncology, populations studies [48].

12. Wildlife sample collection and documentation

12.1 Wildlife sample collection kit

The investigator must have a forensic sample collection kit to collect and store the biological materials. While in the field, the items required for sample collection are gloves, mask, datasheet, sterile vials, Ethanol or spirit, forceps, scissor, measuring tape, silica, envelopes, ziplock, cello tape, permanent marker, surgical blade, collection card (Figure 1).

Figure 1.

Wildlife sample collection kit.

12.2 Collection and storage of biological samples

Proper collection and storage of biological samples for the molecular analysis is very important known as biological resources. So, the proper preservation of biological sample is crucial because the preservation and laboratory analyzing at a future date. The processing protocol that will give quality result for the intended laboratory analyses must be selected from among various possible protocols. To collect blood samples, blood can be stored in vacutainers using sterile syringe and stored at 4°C for examination. Sometimes, blood spot or stains might be present during field investigation. The FTA classic card a commercially available product can be suitable for isolation, purification and storage of DNA samples. A drop of blood can be loaded on the specific FTA card. The fresh tissue samples can be stored in vials using molecular grade ethanol (100%). It can be labeled using marker and stored at −20°C. DNA in dried specimens ordinarily remains in good condition for at least a year. Frozen specimens AT -80°C will be remains stable indefinitely (Figure 2).

Figure 2.

Collection and storage of biological samples.

13. Laboratory protocols for DNA analysis for species identification and population analysis

13.1 Extraction of DNA from various biological samples

There is a range of biological samples such as tissue, blood, hair, bone, scat, etc. available to analyze DNA recovered during field surveys. The protocol to apply for DNA extraction varies on the type of sample collected. To extract the DNA from stool, blood, or tissue samples Qiagen fast DNA kit, Germany is widely used and commercially available. Other kits are also available for DNA extraction. Extracted DNA will be useful for studying populations study and wildlife forensics and as well as for genetic studies and the yielded DNA can be checked under 0.8% agarose gel electrophoresis.

13.2 Quality assessment of isolated DNA using agarose gel electrophoresis

Agarose gel electrophoresis is a technique used to resolve DNA fragments on the basis of their molecular weight. Smaller fragments migrate faster than larger ones. The size of fragments can be known by using known size standards DNA ladder, and comparing the distance of unknown fragment. 0.8% agarose is used for the preparation of agarose gel. The contents are boiled in the oven. And 3 μl ethidium bromide (EtBr) is added to the gel. After polymerization, the DNA sample should be loaded and mixed with 5X gel loading dye. Finally, the samples are visualized under the gel documentation system.

13.3 Polymerase chain reactions (PCR)

Polymerase chain reaction is a laboratory technique for amplifying millions to billions of copies of a fragment of DNA. PCR involves using short synthetic DNA fragments called primers to select a segment of the genome to be amplified. To identify the species from the DNA samples, the targeted sequences can be amplified using universal or species-specific primers following the forward and reverse primer; 1x Buffer; 25 mM MgCl₂; 100 mM dNTP; 25x BSA; 1 U/ul taq; template DNA. The PCR program is to be set in the thermocycler.

13.4 Analysis of raw sequences

ABI files obtained through a genetic analyzer can be viewed and edited in the sequencing analysis software. After thorough checking of the quality of sequencing, the project file must be saved and exported in FASTA format. The edited sequences can be compared with NCBI or Genbank using the BLAST tool. The database can give results of the search revealing records that are he closest match in terms of sequence similarity [49].

14. Conclusion

Recent advances in molecular biology allow us to gather sufficient genetic data on any species without causing any harm to the organism involved. This chapter describes the various molecular markers which are being utilized for the study of wildlife conservation. Studies on the variation of DNA sequences allow one to differentiate genetically differentiated populations, understand inbred populations and determine the actual number of males and females contributing to the successive generations. Basic data on the genetic and cytogenetics of any species is necessary for the management program. Overall, reliability of molecular markers as a powerful tool for the identification of species and phylogenetic analysis as compared to traditional approaches for taxonomic studies. Applying molecular markers approaches will help solve problems in the management of wild populations and help in identifying the subsequent gene pools.

Acknowledgments

We thank the Director, Zoological Survey of India for her encouragement and support. We are thankful to the officer-in-charge Zoological Survey of India, Southern Regional Centre, and the staff for their constant support.

Conflict of interest

The authors declare no conflict of interest.