Review: Wildlife forensic genetics-Biological evidence, DNA markers, analytical approaches, and challenges.

Wildlife-related crimes are the second most prevalent lawbreaking offense globally. This illicit trade encompasses hunting, breeding and trafficking. Besides diminishing many species and their habitats and ecosystems, hindering the economic development of local communities that depend on them, undermining the rule of law and financing terrorism, various cross-species transmissions (zoonoses) of pathogens, including COVID-19, can be attributed to wildlife crimes. Wildlife forensics applies interdisciplinary scientific analyses to support law enforcement in investigating wildlife crimes. Its main objectives are to identify the taxonomic species in question, determine if a crime has been committed, link a suspect to the crime and support the conviction and prosecution of the perpetrator. This article reviews wildlife crime and its implications, wildlife forensic science investigation, common forms of wildlife biological evidence, including DNA, wildlife DNA techniques and challenges in wildlife forensic genetics. The article also reviews the contributions of genetic markers such as short tandem repeat (STR) and mitochondrial DNA (mtDNA) markers, which provide the probative genetic data representing the bulk of DNA evidence for solving wildlife crime. This review provides an overview of wildlife DNA databases, which are critical for searching and matching forensic DNA profiles and sequences and establishing how frequent forensic DNA profiles and sequences are in a particular population or geographic region. As such, this review will contain an in-depth analysis of the current status of wildlife forensic genetics, and it will be of general interest to wildlife and conservation biologists, law enforcement officers, and academics interested in combating crimes against wildlife using animal forensic DNA methods.

Pedigree Data from Six Rhesus Macaque (Macaca mulatta) Matrilines at the California National Primate Research Center Indicate Inbreeding and Loss of Genetic Variation.

Relatedness and kinship structure in matrilines are a potential source of social stability. The current study aimed to analyze the extant pedigrees of 6 living matrilines in different field cages to assess rates of cross-generational inbreeding and loss of genetic variation over time. All 6 matrilines showed increasing levels of inbreeding over generation time, although the rates of increase were different. The female-to-male-adult sex ratio was correlated with average matriline inbreeding levels, while the number of adult males was positively correlated with average matriline genetic diversity. Over five times more paternal half-sibs than maternal half-sibs were present because paternity had been restricted to a few males yearly. Therefore, the relatedness through the paternal lines was over five times greater than that of the maternal lines. Overall, each matriline lost low to moderate levels of genetic variation with time. The current rates of gene flow between field cages by cross-fostered infants have not stopped inbreeding within these matrilines or loss of diversity due to genetic drift. This situation probably developed because translocated animals, especially males, may not breed successfully. Only 4 of the 22 translocated individuals, all females, eventually reproduced, resulting in 13 offspring and generating an overall breeding success of 0.59 across all 6 study matrilines. However, even this low rate of reproduction by the translocated animals reduced inbreeding and kinship among matrilines and increased genetic heterogeneity in the matrilines. Based on this study, we propose several colony management strategies, including equalizing adult sex ratios to increase the effective population size in the field cages, increasing the number of cross-fostered infants, and relying more on multigenerational pedigree data to aid the alignment of genetic and behavioral management techniques.

A global catalog of whole-genome diversity from 233 primate species.

The rich diversity of morphology and behavior displayed across primate species provides an informative context in which to study the impact of genomic diversity on fundamental biological processes. Analysis of that diversity provides insight into long-standing questions in evolutionary and conservation biology and is urgent given severe threats these species are facing. Here, we present high-coverage whole-genome data from 233 primate species representing 86% of genera and all 16 families. This dataset was used, together with fossil calibration, to create a nuclear DNA phylogeny and to reassess evolutionary divergence times among primate clades. We found within-species genetic diversity across families and geographic regions to be associated with climate and sociality, but not with extinction risk. Furthermore, mutation rates differ across species, potentially influenced by effective population sizes. Lastly, we identified extensive recurrence of missense mutations previously thought to be human specific. This study will open a wide range of research avenues for future primate genomic research.

Gene flow from rhesus (Macaca mulatta) to cynomolgus macaques (M. fascicularis) and effects of introgressive hybridization on reproduction in two biomedically relevant non-human primate species.

BACKGROUND: We compared the reproductive patterns of wild Indochinese and Sundaic cynomolgus macaques (Mf) exhibiting different levels of genetic admixture with rhesus macaques (Mm). METHODS: Ten adult females from each Indochinese (WHM) and Sundaic (KN/KTK) Mf populations, which exhibited 50% and 15% of Mm autosomal SNPs, were selected as focal animals. Animals were observed for 12 months, and the frequencies of sexual proceptivity, attractivity and receptivity, number of newborns, and changes in sex skin were recorded. RESULTS: Both populations showed all three sexual behaviors throughout the year, but they were classified as moderately seasonal breeders because their 3-month birth counts were as high as ~50%. The fecundity of WHM was lower than the KN/KTK. Changes in sex skin of WHM were more prone to Mm's pattern than the KN/KTK. CONCLUSION: The introgressive gene flow from Mm to Mf does not affect Mf's sexual behaviors; however, it can impact fecundity and physiological (sex skin) changes.

Streamlining the decision-making process for international DNA kinship matching using Worldwide allele frequencies and tailored cutoff log10LR thresholds.

The identification of human remains belonging to missing persons is one of the main challenges for forensic genetics. Although other means of identification can be applied to missing person investigations, DNA is often extremely valuable to further support or refute potential associations. When reference DNA samples cannot be collected from personal items belonging to a missing person, a direct DNA identification cannot be carried out. However, identifications can be made indirectly using DNA from the missing person's relatives. The ranking of likelihood ratio (LR) values, which measure the fit of a missing person for any given pedigree, is often the first step in selecting candidates in a DNA database. Although implementing DNA kinship matching in a national environment is feasible, many challenges need to be resolved before applying this method to an international configuration. In this study, we present an innovative and intuitive method to perform international DNA kinship matching and facilitate the comparison of DNA profiles when the ancestry is unknown or unsure and/or when different marker sets are used. This straightforward method, which is based on calculations performed with the DNA matching software BONAPARTE, Worldwide allele frequencies and tailored cutoff log10LR thresholds, allows for the classification of potential candidates according to the strength of the DNA evidence and the predicted proportion of adventitious matches. This is a powerful method for streamlining the decision-making process in missing person investigations and DVI processes, especially when there are low numbers of overlapping typed STRs. Intuitive interpretation tables and a decision tree will help strengthen international data comparison for the identification of reported missing individuals discovered outside their national borders.

Population Structure of Macaca fascicularis aurea, and their Genetic Relationships with M. f. fascicularis and M. mulatta Determined by 868 RADseq-Derived Autosomal SNPs-A consideration for biomedical research.

BACKGROUND: This study examined the population structure of Macaca fascicularis aurea and their genetic relationships with M. f. fascicularis and M. mulatta. METHODS: The study analyzed 868 RADseq-derived SNPs from samples representing the entire distribution range of M. f. aurea, including their inter- and intraspecific hybrid zones. RESULTS: The study supports a M. mulatta/Indochinese M. f. fascicularis, Sundaic M. f. fascicularis, and M. f. aurea trichotomy; M. f. aurea was genetically distinct from both forms of M. f. fascicularis and M. mulatta. Hybridization between M. f. aurea and M. f. fascicularis occurred in two directions: south-north (8°25' to 15°56') and west-east (98°28' to 99°02'). Low levels of M. mulatta introgression were also detected in M. f. aurea. CONCLUSION: This study showcases a complicated scenario of genetic relationships between the M. fascicularis subspecies and between M. fascicularis and M. mulatta and underscores the importance of these taxa's population structure and genetic relationships for biomedical research.

Effects of hybridization on pelvic morphology: A macaque model.

Ancient DNA analyses have shown that interbreeding between hominin taxa occurred multiple times. Although admixture is often reflected in skeletal phenotype, the relationship between the two remains poorly understood, hampering interpretation of the hominin fossil record. Direct study of this relationship is often impossible due to the paucity of hominin fossils and difficulties retrieving ancient genetic material. Here, we use a sample of known ancestry hybrids between two closely related nonhuman primate taxa (Indian and Chinese Macaca mulatta) to investigate the effect of admixture on skeletal morphology. We focus on pelvic shape, which has potential fitness implications in hybrids, as mismatches between maternal pelvic and fetal cranial morphology are often fatal to mother and offspring. As the pelvis is also one of the skeletal regions that differs most between Homo sapiens and Neanderthals, investigating the pelvic consequences of interbreeding could be informative regarding the viability of their hybrids. We find that the effect of admixture in M. mulatta is small and proportional to the relatively small morphological difference between the parent taxa. Sexual dimorphism appears to be the main determinant of pelvic shape in M. mulatta. The lack of difference in pelvic shape between Chinese and Indian M. mulatta is in contrast to that between Neanderthals and H. sapiens, despite a similar split time (in generations) between the hybridizing pairs. Greater phenotypic divergence between hominins may relate to adaptations to disparate environments but may also highlight how the unique degree of cultural buffering in hominins allowed for greater neutral divergence. In contrast to some previous work identifying extreme morphologies in first- and second-generation hybrids, here the relationship between pelvic shape and admixture is linear. This linearity may be because most sampled animals have a multigenerational admixture history or because of relatively high constraints on the pelvis compared with other skeletal regions.

DNA-based Determination of Ancestry in Cynomolgus Macaques (Macaca fascicularis).

Interest in the genetic composition of cynomolgus macaques (Macaca fascicularis) has increased due to the rising demand for NHP models in human biomedical research. Significant genetic differences among regional populations of cynomolgus macaques can confound interpretations of research results because they do not solely reflect differences in experimental treatment effects. Therefore, the common origin of cynomolgus macaques used as research subjects should be verified by using region-specific genetic markers to minimize the influence of underlying genetic variation among animals selected as research subjects on phenotypes under study. We compared the effectiveness of 18 short tandem repeat (STR) markers with that of 83 single-nucleotide polymorphism (SNP) markers to differentiate the ancestry of cynomolgus macaques from 6 different populations (Cambodia, Sumatra, Mauritius, Singapore, and the islands of Luzon and Zamboanga in the Philippines). Genetic diversity indices such as allele numbers and expected heterozygosity based on SNP were lower and exhibited lower standard errors than those provided by STR, probably because, unlike STR, most SNP are biallelic and consequently exhibit maximal expected heterozygosity values of 0.50. However, the standard error of estimates of observed heterozygosity based on SNP was higher than that for STR, perhaps reflecting sampling errors. Only 27 SNP were required to match the resolving power of 17 STR to detect population structure, that is, 1.6 SNP:1 STR. Whereas STR only differentiated the Mauritian population from all other populations, SNP detected 4 genetically distinct groups (Cambodia, Singapore-Sumatra, Mauritius, and Zamboanga). SNP are poised to become as valuable as STR for understanding and detecting genetic structure among cynomolgus macaques. Although STR will remain an important tool for cynomolgus macaque population studies, SNP have the potential to become the mainstream marker type.

Evaluating the genetic status of a closed colony of titi monkeys (Callicebus cupreus) using multigenerational pedigrees.

Pedigree metrics are essential for investigating colony genetic structure. The genetic structure of a closed Callicebus cupreus colony was examined using multigenerational pedigrees. Inbreeding was low, but genetic drift caused the loss of founder genome representation. Pedigrees can be used to detect founder representation and prevent bottlenecks and allele loss.

SNP-based genetic characterization of the Tulane National Primate Research Center's conventional and specific pathogen-free rhesus macaque (Macaca mulatta) populations.

BACKGROUND: The rhesus macaque is an important biomedical model organism, and the Tulane National Primate Research Center (TNPRC) has one of the largest rhesus macaque breeding colonies in the United States. METHODS: SNP profiles from 3266 rhesus macaques were used to examine the TNPRC colony genetic composition over time and across conventional or SPF animals of Chinese and Indian ancestry. RESULTS: Chinese origin animals were the least genetically diverse and the most inbred; however, since their derivation from their conventional forebearers, neither the Chinese nor the Indian SPF animals exhibit any significant loss of genetic diversity or differentiation. CONCLUSIONS: The TNPRC colony managers have successfully minimized loss in genetic variation across generations. Although founder effects and bottlenecks among the Indian animals have been successfully curtailed, the Chinese subpopulation still show some influences from these events.

Genetic analysis of samples from wild populations opens new perspectives on hybridization between long-tailed (Macaca fascicularis) and rhesus macaques (Macaca mulatta).

In the past decade, many researchers have published papers about hybridization between long-tailed and rhesus macaques. These previous works have proposed unidirectional gene flow with the Isthmus of Kra as the zoogeographical barrier of hybridization. However, these reports analyzed specimens of unknown origin and/or did not include specimens from Thailand, the center of the proposed area of hybridization. Collected specimens of long-tailed and rhesus macaques representing all suspected hybridization areas were examined. Blood samples from four populations each of long-tailed and rhesus macaques inhabiting Thailand, Myanmar, and Laos were collected and analyzed with conspecific references from China (for rhesus macaques) and multiple countries from Sundaic regions (for long-tailed macaques). Ninety-six single nucleotide polymorphism (SNP) markers specifically designed to interrogate admixture and ancestry were used in genotyping. We found genetic admixture maximized at the hybrid zone (15-20°N), as well as admixture signals of varying strength in both directions outside of the hybrid zone. These findings show that the Isthmus of Kra is not a barrier to gene flow from rhesus to long-tailed populations. However, to precisely identify a southernmost barrier, if in fact a boundary rather than simple isolation by distance exists, the samples from peninsular Malaysia must be included in the analysis. Additionally, a long-tailed to rhesus gene flow boundary was found between northern Thailand and Myanmar. Our results suggest that selection of long-tailed and rhesus macaques, the two most commonly used non-human primates for biomedical research, should take into account not only the species identification but also the origin of and genetic admixture within and between the species.

Genetic Characterization of a Captive Colony of Pigtailed Macaques (Macaca nemestrina).

Effective colony management is critical to guarantee the availability of captive NHP as subjects for biomedical research. Pigtailed macaques (Macaca nemestrina) are an important model for the study of human and nonhuman primate diseases and behavior. Johns Hopkins University hosts one of the largest captive colonies of pigtailed macaques in the United States. In this study, we used 56 single-nucleotide polymorphisms (SNP) to characterize this population of pigtailed macaques, understand their population structure, and assess the effectiveness of their colony management. The results demonstrate that the colony has maintained a high level of genetic diversity, with no loss of heterozygosity since its origin, and low levels of inbreeding and genetic subdivision.

The population genomics of rhesus macaques (Macaca mulatta) based on whole-genome sequences.

Rhesus macaques (Macaca mulatta) are the most widely used nonhuman primate in biomedical research, have the largest natural geographic distribution of any nonhuman primate, and have been the focus of much evolutionary and behavioral investigation. Consequently, rhesus macaques are one of the most thoroughly studied nonhuman primate species. However, little is known about genome-wide genetic variation in this species. A detailed understanding of extant genomic variation among rhesus macaques has implications for the use of this species as a model for studies of human health and disease, as well as for evolutionary population genomics. Whole-genome sequencing analysis of 133 rhesus macaques revealed more than 43.7 million single-nucleotide variants, including thousands predicted to alter protein sequences, transcript splicing, and transcription factor binding sites. Rhesus macaques exhibit 2.5-fold higher overall nucleotide diversity and slightly elevated putative functional variation compared with humans. This functional variation in macaques provides opportunities for analyses of coding and noncoding variation, and its cellular consequences. Despite modestly higher levels of nonsynonymous variation in the macaques, the estimated distribution of fitness effects and the ratio of nonsynonymous to synonymous variants suggest that purifying selection has had stronger effects in rhesus macaques than in humans. Demographic reconstructions indicate this species has experienced a consistently large but fluctuating population size. Overall, the results presented here provide new insights into the population genomics of nonhuman primates and expand genomic information directly relevant to primate models of human disease.

Mitigating Chinese-Indian rhesus macaque (Macaca mulatta) hybridity at the California National Primate Research Center (CNPRC).

The effectiveness of abating hybridity in a rhesus breeding colony was evaluated. STR data from the 2006 to 2015 newborns were analyzed. Hybridity decreased over successive years. Birth cohorts retained high genetic variability without signs of inbreeding and differentiation. Hybridity was minimized without compromising overall genetic variability.

Performing monkeys of Bangladesh: characterizing their source and genetic variation.

The acquisition and training of monkeys to perform is a centuries-old tradition in South Asia, resulting in a large number of rhesus macaques kept in captivity for this purpose. The performing monkeys are reportedly collected from free-ranging populations, and may escape from their owners or may be released into other populations. In order to determine whether this tradition involving the acquisition and movement of animals has influenced the population structure of free-ranging rhesus macaques in Bangladesh, we first characterized the source of these monkeys. Biological samples from 65 performing macaques collected between January 2010 and August 2013 were analyzed for genetic variation using 716 base pairs of mitochondrial DNA. Performing monkey sequences were compared with those of free-ranging rhesus macaque populations in Bangladesh, India and Myanmar. Forty-five haplotypes with 116 (16 %) polymorphic nucleotide sites were detected among the performing monkeys. As for the free-ranging rhesus population, most of the substitutions (89 %) were transitions, and no indels (insertion/deletion) were observed. The estimate of the mean number of pair-wise differences for the performing monkey population was 10.1264 ± 4.686, compared to 14.076 ± 6.363 for the free-ranging population. Fifteen free-ranging rhesus macaque populations were identified as the source of performing monkeys in Bangladesh; several of these populations were from areas where active provisioning has resulted in a large number of macaques. The collection of performing monkeys from India was also evident.

Identifying rhesus macaque gene orthologs using heterospecific human CNV probes.

We used the Affymetrix(®) Genome-Wide Human SNP Array 6.0 to identify heterospecific markers and compare copy number and structural genomic variation between humans and rhesus macaques. Over 200,000 human copy number variation (CNV) probes were mapped to a Chinese and an Indian rhesus macaque sample. Observed genomic rearrangements and synteny were in agreement with the results of a previously published genomic comparison between humans and rhesus macaques. Comparisons between each of the two rhesus macaques and humans yielded 206 regions with copy numbers that differed by at least two fold in the Indian rhesus macaque and human, 32 in the Chinese rhesus macaque and human, and 147 in both rhesus macaques. The detailed genomic map and preliminary CNV data are useful for better understanding genetic variation in rhesus macaques, identifying derived changes in human CNVs that may have evolved by selection, and determining the suitability of rhesus macaques as human models for particular biomedical studies.

Heterospecific SNP diversity in humans and rhesus macaque (Macaca mulatta).

BACKGROUND: Conservation of single nucleotide polymorphisms (SNPs) between human and other primates (i.e., heterospecific SNPs) in candidate genes can be used to assess the utility of those organisms as models for human biomedical research. METHODS: A total of 59,691 heterospecific SNPs in 22 rhesus macaques and 20 humans were analyzed for human trait associations and 4207 heterospecific SNPs biallelic in both taxa were compared for genetic variation. RESULTS: Variation comparisons at the 4207 SNPs showed that humans were more genetically diverse than rhesus macaques with observed and expected heterozygosities of 0.337 and 0.323 vs. 0.119 and 0.102, and minor allele frequencies of 0.239 and 0.063, respectively. In total, 431 of the 59,691 heterospecific SNPs are reportedly associated with human-specific traits. CONCLUSION: While comparisons between human and rhesus macaque genomes are plausible, functional studies of heterospecific SNPs are necessary to determine whether rhesus macaque alleles are associated with the same phenotypes as their corresponding human alleles.

Use of genome-wide heterospecific single-nucleotide polymorphisms to estimate linkage disequilibrium in rhesus and cynomolgus macaques.

Rhesus and cynomolgus macaques are frequently used in biomedical research, and the availability of their reference genomes now provides for their use in genome-wide association studies. However, little is known about linkage disequilibrium (LD) in their genomes, which can affect the design and success of such studies. Here we studied LD by using 1781 conserved single-nucleotide polymorphisms (SNPs) in 183 rhesus macaques (Macaca mulatta), including 97 purebred Chinese and 86 purebred Indian animals, and 96 cynomolgus macaques (M. fascicularis fascicularis). Correlation between loci pairs decayed to 0.02 at 1146.83, 2197.92, and 3955.83 kb for Chinese rhesus, Indian rhesus, and cynomolgus macaques, respectively. Differences between the observed heterozygosity and minor allele frequency (MAF) of pairs of these 3 taxa were highly statistically significant. These 3 nonhuman primate taxa have significantly different genetic diversities (heterozygosity and MAF) and rates of LD decay. Our study confirms a much lower rate of LD decay in Indian than in Chinese rhesus macaques relative to that previously reported. In contrast, the especially low rate of LD decay in cynomolgus macaques suggests the particular usefulness of this species in genome-wide association studies. Although conserved markers, such as those used here, are required for valid LD comparisons among taxa, LD can be assessed with less bias by using species-specific markers, because conserved SNPs may be ancestral and therefore not informative for LD.

Population genetics of the California National Primate Research Center's (CNPRC) captive Callicebus cupreus colony.

The California National Primate Research Center maintains a small colony of titi monkeys (Callicebus cupreus) for behavioral studies. While short tandem repeat (STR) markers are critical for the genetic management of the center's rhesus macaque (Macaca mulatta) breeding colony, STRs are not used for this purpose in the maintenance of the center's titi monkey colony. Consequently, the genetic structure of this titi monkey population has not been characterized. A lack of highly informative genetic markers in titi monkeys has also resulted in scant knowledge of the species' genetic variation in the wild. The purpose of this study was to develop a panel of highly polymorphic titi monkey STRs using a cross-species polymerase chain reaction (PCR) amplification protocol that could be used for the genetic management of the titi monkey colony. We screened 16 STR primer pairs and selected those that generated robust and reproducible polymorphic amplicons. Loci that were found to be highly polymorphic, very likely to be useful for parentage verification, pedigree assessment, and studying titi monkey population genetics, were validated using Hardy-Weinberg equilibrium and linkage disequilibrium analyses. The genetic data generated in this study were also used to assess directly the impact on the colony's genetic diversity of a recent adenovirus outbreak. While the adenovirus epizootic disease caused significant mortality (19 deaths among the 65 colony animals), our results suggest that the disease exhibited little or no influence on the overall genetic diversity of the colony.

Recovery of salivary DNA from the skin after showering.

PURPOSE: After sexual assault there is a limited amount of time before the DNA evidence on the surface of the victim's body is not recoverable. During an assault, the offender may leave saliva on the victim's skin. Traditional examination methods use a swabbing technique to collect saliva for DNA testing. Victim activity, especially hygiene activity such as showering, may negatively affect DNA recovery. METHODS: In this experiment, we compared two techniques for recovery of salivary DNA from the skin's surface after a victim showers. We compared the traditional swabbing method to a "wet-vacuum" method using the M-Vac© to collect saliva from four body regions (neck, arm, stomach, and leg). In our research, we tested whether either collection technique obtained enough salivary DNA for autosomal and Y-STR analysis. In addition, we tested whether the M-Vac© is more effective at collecting DNA from large surface areas than traditional methods, by determining the amount of DNA collected. RESULTS: With both collection techniques, we were able to obtain male salivary DNA from at least one body region of the female after she had showered. There was no statistical difference in the amount of DNA collected between the swabbing technique and the M-Vac©. Autosomal STR analysis failed to detect the male contributor's DNA; therefore, we used Y-STRs. With Y-STR analysis, 47 samples returned a full male profile, and 26 samples returned a partial male profile after sample concentration. CONCLUSIONS: This research shows that salivary DNA can be collected from skin after showering and successfully analyzed using Y-STRs.