Wildlife Forensics is when a crime has been committed either by an animal or against an animal. There are many types of cases that occur such as, poaching, trafficking, abuse/neglect, and crimes caused by animals. In these cases, species identification techniques are often required to aid in investigations. In cases where an attack by an animal has occurred, for example, a dog biting a human, it is necessary to identify that it was a dog that attacked the human and also, to identify which dog it is, similar to that of excluding/including human suspects with DNA evidence.
In trafficking cases, it is necessary for species detection and identification to be used to identify what species has been trafficked and if they are any protection orders for that specific species.
The key methods of species identification are as follows:
Morphological:
Morphology is the study of the form and structure of organisms and their specific structural features.
This includes aspects of the outward appearance of a species (shape, structure, colour, pattern, size), as well as the form and structure of the internal parts such as bones and organs, i.e. internal morphology. The best known aspect of morphology, usually called anatomy, is the study of gross structure, or form, of organs and organisms.
Morphological taxonomy is looking for similarities in characteristics within a species. Morphological taxonomy uses morphology and phenotypic characteristics to distinguish between one species and another.
However, Identification is more a matter of opinion rather than fact as different scientists can interpret the morphology differently to that of other scientists.
Detailed comparisons of the morphological features of different animals, called comparative anatomy, provide strong arguments for the evolutionary relationships among different species. In the course of evolution, animals and plants tend to undergo adaptive morphological changes that enable them to survive under certain environmental conditions.
Molecular/Phylogenetic:
Molecular- DNA barcoding is a tool used to quickly identifying species using existing information of gene sequence in the gene bank. DNA barcoding uses molecular information to classify species, this allows for a more accurate identification of species which is necessary in Wildlife Forensic cases, especially when the case is brought to trial.
DNA barcoding is a novel technology to provide rapid, accurate, and automated species identifications using short orthologous DNA sequences. Orthologous means that it is inferred the species in question is to have descended from the same ancestral sequence.
DNA barcodes consist of short sequences of DNA between 400 and 800 base pairs that can be routinely amplified by PCR (polymerase chain reaction) and sequenced of the species studied. PCR is a technique used by molecular biologists to amplify a single copy or a few copies of a segment of DNA thus generating thousands to millions of copies of a particular DNA sequence. (Bartlett and Stirling, 2003)
Mitochondrial DNA (MtDNA) typing is more commonly used in forensic wildlife applications for three main reasons.
The first is that mitochondrial loci are regularly used in molecular taxonomy and phylogenetics for the purpose of species assignment and therefore there is already a wealth of data available in the scientific literature.
The second is that universal primers exist that can be applied to almost any unknown sample and generate a result.
Finally, many of the samples that are encountered within wildlife forensics are usually highly degraded and hence nuclear markers have a low amplification success rate compared to MtDNA which is present in up to 1000x higher quantities than the nuclear genome depending on the type of tissue.
(Johnson, Wilson-Wilde, and Linacre, 2013)
Phylogenetic- A phylogenetic tree or evolutionary tree is a branching diagram or “tree” showing the inferred evolutionary relationships among various biological species or other entities—their phylogeny—based upon similarities and differences in their physical or genetic characteristics.
Morphological or molecular techniques could be used depending on the state of the specimen being analysed. Usually, alleged wildlife crime cases requiring individualisation, so DNA is typically used. However, if the markers do not exist for the species in question, then they must be developed and peer group validated.
Morphology-based keys support accurate identification of many taxa. Nonetheless, for taxa that have not been well studied, or for which distinguishing morphological characters have not been identified, identification can be difficult. Accurate identification is also especially problematic for very small organisms, for members of cryptic species complexes, for eggs, and for immature stages.
DNA species barcoding:
Blast searches:
According the the BLAST website, “the Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families.â€
A BLAST search enables a researcher to compare a query sequence with a database of sequences, and identify database sequences that resemble the query sequence above a specific threshold.
With the use of BLAST, it can possibly identify a species or find homologous species. However this is only possible if the species has already been submitted into the database. This can be useful, for example, when working with a DNA sequence from an unknown species. (National Centre for Biotechnology Information, 2018)
Sanger Sequencing:
Sanger sequencing, also known as the chain termination method, is a technique for DNA sequencing based upon the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication. It was developed by Frederick Sanger in 1977. Sanger sequencing is the most common technique used by molecular laboratories to detect the exact nucleotide composition of PCR-amplified DNA fragments. It uses chain-terminating dideoxynucleoside triphosphates (ddNTPs), which terminate its elongation at a particular nucleotide upon incorporation into the growing DNA strand. This results in a mixture of DNA fragments of various lengths, with each fragment corresponding to one of four nucleotides positioned at a specific distance from the beginning of the sequence. However, next-generation sequencing (NGS) methods have replaced Sanger sequencing.
Next Generation Sequencing (NGS):
In NGS, vast numbers of short reads are sequenced in a single stroke. These are 3 types of sequencing:
Ion torrent: Proton / PGM sequencing,
Illumina (Solexa) sequencing,
Roche 454 sequencing.
(Hodkinson and Grice, 2014)
Ion torrent: Proton / PGM sequencing:
Ion torrent: Proton or PGM sequencing is a method of DNA sequencing based on the detection of hydrogen ions that are released during the polymerisation sequence of DNA. This is a method of synthesised sequencing, during which a complementary strand is built based on the sequence of a template strand. Unlike Illumina and 454, Ion torrent and Ion proton sequencing do not make use of optical signals. Instead, they exploit the fact that addition of a dNTP to a DNA polymer releases an H+Â ion. (EMBL-EBI, 2018)
Illumina (Solexa) sequencing:
Illumina sequencing technology, sequencing by synthesis (SBS) is a next-generation sequencing (NGS) technology. According to the Illumina website, the technology works as follows: “A fluorescently labeled reversible terminator is imaged as each dNTP is added, and then cleaved to allow incorporation of the next base. Since all 4 reversible terminator-bound dNTPs are present during each sequencing cycle, natural competition minimises incorporation bias.
The end result is true base-by-base sequencing that enables accurate data for a broad range of applications. The method virtually eliminates errors and missed calls associated with strings of repeated nucleotides (homopolymers).â€
Roche 454 sequencing:
Roche 454 sequencing can sequence much longer reads than Illumina. Like Illumina, it does this by sequencing multiple reads at once by reading optical signals as bases are added. Roche 454 sequencing has the longest but fewest reads per slide, a fast runtime, however, out of the 3 choices, it is the most expensive per run.
Morphological vs DNA species barcoding
Morphological systematics has been used for over 250 years and is the foundation of all current species hypothesis.
Hillis (1987) argues that that it is difficult to compare the two techniques as they originated for different purposes; morphological systematics for comparing anatomy and molecular systematics for population genetics. He suggests that both methods are inadequate on their own in defining, identifying and comparing one species from another. Where one method succeeds, the other has a downfall. To make this process as accurate as possible, both systems should be required and used in combination to identify and compare species, thus reducing the likelihood of errors occuring. Due to the complimentary nature of morphological identification and DNA species barcoding, the amalgamation of these two techniques has proven to have an increased rate of success on multiple occasions (Best et al., 1986), (Duellman and Venegas, 2005), (Gould et al., 1974), (Miyamoto, 1981), (Nixon and Taylor, 1977), (Shaklee and Tamaru, 1981).
Alone however, the usage of morphological methods for species identification has many problems, as does DNA species barcoding.
Morphology- Primarily, identification and comparison is a matter of the personal opinion of the researcher rather than a proven fact, as different scientists can interpret the morphology differently to that of other scientists. This is especially true between sub-sections of species, especially species that look similar but have a slight difference in their genetic make-up.
DNA species barcoding- Creating a database for this is both time consuming and expensive. Also, creating a database that is available to everyone means that anyone can add any sequence. But also, anyone can make mistakes when adding information and claim it is a sequence belonging to something other than what it actually is. This would decrease the accuracy of the database, and therefore provide false results to scientists who use the system. (Herbert and Gregory, 2005)
Another factor is that with the increase of technology available, there has been a large surge in the numbers of new species being discovered, and old species being reclassified. This in turn makes it much more difficult to define what a species is exactly. (Herbert and Gregory, 2005).
Also, due to this rapid discovery of new species and new information, there is a larger number of new species being genetically sequenced. However, there are not enough taxonomic experts available to name these new or reclassified species. Therefore, these new individuals go into the database as identification numbers. (Landry et al., 2003).
There are also problems if the specimen used has degraded to the point that they cannot be identified.
An improvement that could be made to these databases is that each submission of a sequence should also require several photographs of that individual organism and, descriptions of any major morphological characteristics.
Both techniques should be used in conjunction with one another therefore complimenting the other and produce the most accurate means of species identification and comparison. For scientific purposes, both methods of species identification should be universally accepted and used in unison to allow for the most accurate means of species identification. (Friedheim, 2016)
NGS vs Sanger sequencing
Technologies have made spectacular progresses in terms of accuracy, read depth and genome coverage, read length, capacity, and turnaround time. Costs of sequencing a whole genome have significantly dropped. Efforts to provide bench top, portable systems open avenues to the generalisation of genomic technologies in laboratories.
Sanger sequencing was the original for DNA sequencing. Sanger sequencing has a 99.99% accuracy and is the original “gold standard†for clinical research sequencing. However, newer NGS technologies are also becoming common in research labs due to their higher capabilities and lower costs per sample.
NGS ended up replacing first generation DNA sequencing method, Sanger sequencing. This was partly due to the fact that Sanger sequencing produced only one forward and reverse read of the DNA strand, whereas NGS allows for millions of fragments to be sequenced within a single run. This makes it much more cost effective for laboratories.
Next-Generation Sequencing has changed the way that molecular biology and genomic studies are carried out. It has made it possible to sequence and annotate genomes at a much faster rate. It has also made it possible to study variation, expression and DNA binding at a genome-wide level.
NGS is significantly cheaper, quicker, needs significantly less DNA and is more accurate and reliable than Sanger sequencing. Let us look at this more closely. For Sanger sequencing, a large amount of template DNA is needed for each read. Several strands of template DNA are needed for each base being sequenced (i.e. for a 100bp sequence you’d need many hundreds of copies, for a 1000bp sequence you’d need many thousands of copies), as a strand that terminates on each base is needed to construct a full sequence. In NGS, a sequence can be obtained from a single strand. In both kinds of sequencing multiple staggered copies are taken for contig construction and sequence validation.