Robby Bonanno
Biology 303
Term Paper
Forensic DNA Evidence Used in Trial against Wolf Poacher in Italy:
Application of Genetics to Prosecute Wildlife Poachers
The population of native wolves in the Northern Italian Alps and Apennine has undergone a
recent boom due to conservation and governmental efforts after centuries of decline according to a
study conducted by Boitani (Boitani 2003). The Italian Ministry of Environment has extensively
monitored wolf repopulation and has concluded that local hunters and livestock breeders are having
increasingly common conflicts with the rejuvenated wolf population in this region due to the misguided
views that wolves compete for the same types of wild game that hunters do and more accurately due to
uncommon, yet significant, occurrences when wolves prey on domesticated livestock populations
(Caniglia 334). The local and national government has attempted to offset this economic loss for
livestock breeders by implementing compensation policies, however, these policies are often poorly
enforced, if at all, leading to frequent wolf killings and poaching despite the legal protection wolves in
Italy have been given since 1976 (Genovesi 2002). Approximately twenty percent of the Italian wolf
population, numbering approximately 800 native wolves, is illegally or accidently killed each year
(Boitani 2003). This statistic has driven authorities to step up enforcement of wolf conservation and
prosecution of those alleged to be responsible for the death of these protected animals.
Figure 1. Confiscated Tooth Necklace
In 2008, a necklace composed of ten canine (suspected to be wolf) teeth was confiscated by
authorities from a local hunter in the northern Italian Apennine region of Liguria in the Genova Province
(Fig. 1). Additionally, authorities also found a dead male wolf carcass nearby whose muzzle had been
removed, prompting the teeth and tissue samples to be sent to Laboratory of Genetics of ISPRA
(Institute for Environmental Protection and Research) to undergo DNA extraction, multilocus genotyping
of the individual samples through analysis of mtDNA (Randi and Lucchini 2000), autosomal
microsatellites (Randi et al. 2002), and Y-linked STRs (Sundqvist et al. 2001), and gender determination
using analysis of male-specific ZFX/ZFY restriction-site (Garcia-Muro et al. 1997). There already exists a
substantial database of dog and wolf genotypes from Italy as the result of the joint efforts of the Italian
government and the European community which have been attempting to monitor and study the
populations of large predators in Europe since the late 1990s and this database was used to crossreference the tissue and tooth samples collected by the Italian authorities in an effort to identify the
species of origin and the gender of the sample and to determine whether or not the sample was from a
previously sampled/marked member of the Italian wolf population or from the mutilated wolf found
dead near the hunter’s residence (Caniglia et al.). An analysis of the compiled data from four
independent conservation studies was prepared to make the above-mentioned identifications possible.
A great deal of emphasis was placed on a database of multilocus genotypes gathered from the DNA of
417 wolves found dead in the wild of the known Italian wolf range and another 130 wolves noninvasively sampled from the Western Alps over the last 15 years (Fabbri et al.). This compiled data
proved integral to this criminal investigation because without such resources and databases available,
identification of individual tissue samples as to their species of origin, population, and even exact
identity would have been impossible.
The inspiration for the utilization of these markers for analysis was the result of numerous
studies that had been conducted in the past in regards to identifying, monitoring, and genotyping
wolves. In 2000, a study investigated the effectiveness of isolating and analyzing mitochondrial DNA to
determine population segregation and establishing an additional means of identifying and discriminating
members of the Northern Italian wolf population from those of other Eastern European populations
(Randi and Lucchini, 2000). Although the intent of this study was not related to forensic analysis of
potential incriminating evidence in a criminal investigation, the scientists involved were interested in
studying the relationship of the small Italian wolf population size and instances of hybridization within
the population as a result of their small population and threatened status. The same researchers’ group
published another article that was the inspiration for the inclusion of the Bayesian admixture analyses of
the wolf teeth and tissue samples to identify variation in specific microsatellite regions of the DNA
(Randi et al. 2002). The results of this particular investigation lead to a novel approach to identifying the
presence of domesticated dog genes in the native Italian wolf genotype as a result of interbreeding
among these populations. The 12 autosomal microsatellite markers identified in this 2002 study ended
up being an integral component of the sample analysis performed to determine the species of origin of
the teeth from the necklace and ultimately linking these teeth to those of wolves previously identified
through the non-invasive studies. The final study these researchers drew inspiration from when
analyzing the collected samples involved the analysis of Scandinavian wolf DNA microsatellites with
particular interest in Y-chromosome haplotyping (Sundqvist et al. 2001). This study led to the decision
for the samples to be tested for the presence of three different Y-linked STR’s, which were previously
identified as being characteristic of the species of Canis lupis and thus provided a solid method for
distinguishing whether the samples were actually from wolves or simply from domesticated dogs.
DNA was successfully extracted from nine of the ten teeth from the necklace, as well as from
the tissue sample taken from the found-dead wolf and it was determined that every one of the samples
was a positive match for the Italian wolf mitochondrial DNA control-region haplotype, designated W14.
With these results, it was determined that the teeth from the necklace all belonged to individuals of the
Italian wolf population (Caniglia 336). Next, each of the individual tooth samples was genotyped and it
was concluded that the teeth originated from six different wolves (three males and three females) and
furthermore four of the teeth were traced back to a single male wolf, labeled WGE9M (Caniglia 336).
The Y-chromosome haplotype analysis confirmed that the three male animals were indeed from
members of the Italian wolf population (Caniglia 336). In addition, one of the teeth found on the
necklace was found to be a perfect genetic match with the DNA of the mutilated wolf found near the
hunter’s residence and two of the additional teeth were identified as belonging to wolves that were
non-invasively sampled by the joint efforts of the Italian government and conservation groups (Caniglia
336). The evidence for all of these conclusions can be found in Table 1 below, which is a summary of all
of the tests run on the tooth and tissue samples. Figure 3 below the table is a plot of the microsatellite
genotyping results and how they clearly show that all of the tooth samples (black triangles) were from
members of the Italian wolf population.
It is clear and indisputable from the results of this study, that the teeth samples from the
necklace confiscated from the local hunter are derived from members of the Italian wolf population and
that there is also a definite link between a tooth on the necklace and the mutilated wolf carcass found
near the hunter’s residence. It was also determined that at least two of the individual wolves whose
teeth were a part of the necklace had previously been non-invasively sampled and monitored as part of
a joint conservation project between the Italian government and international conservation agencies.
All of this data will be presented as incriminating evidence in the criminal trial against the alleged serial
wolf killer and will mark the first time in Italian history that wildlife poachers have been identified and
prosecuted for the killings of members of the still-recovering wolf population (Caniglia 334). The only
way for this type of effort to be effective is if national governments work together with international
and national conservation agencies to monitor and sample threatened and endangered species
populations within their countries and set up databases where information can be easily stored and
accessed, a scenario which was clearly and effectively demonstrated through the situation in Italy where
such databases existed due to the efforts of mergers between the government and wildlife conservation
organizations.
References
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