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.

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.

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.

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.

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.

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.

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.

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.

Large scale pedigree analysis leads to evidence for founder effects of Hypertrophic Cardiomyopathy in Rhesus Macaques (Macaca mulatta).

Hypertrophic Cardiomyopathy (HCM) is the abnormal thickening of the ventricles and an increase in cardiac mass. Analyses of 108 rhesus macaque probands with pronounced HCM revealed a strong genetic predisposition to this disease. Macaques are ideal for investigating HCM because of their marked similarity to humans genetically, physiologically and anatomically.

Genetic diversity and population structure of long-tailed macaque (Macaca fascicularis) populations in Peninsular Malaysia.

BACKGROUND: The genetic diversity and structure of long-tailed macaques (Macaca fascicularis) in Peninsular Malaysia, a widely used non-human primate species in biomedical research, have not been thoroughly characterized. METHODS: Thirteen sites of wild populations of long-tailed macaques representing six states were sampled and analyzed with 18 STR markers. RESULTS: The Sunggala and Penang Island populations showed the highest genetic diversity estimates, while the Jerejak Island population was the most genetically discrete due to isolation from the mainland shelf. Concordant with pairwise F(st) estimates, STRUCTURE analyses of the seven PCA-correlated clusters revealed low to moderate differentiation among the sampling sites. No association between geographic and genetic distances exists, suggesting that the study sites, including island study sites, are genetically if not geographically contiguous. CONCLUSIONS: The status of the genetic structure and composition of long-tailed macaque populations require further scrutiny to develop this species as an important animal model in biomedical research.

Familial aggregation of chronic diarrhea disease (CDD) in rhesus macaques (Macaca mulatta).

Chronic diarrheal disease (CDD) is a critical problem for breeders of captive rhesus macaque (Macaca mulatta), as it results in significant levels of morbidity and death annually. As with other inflammatory disorders, CDD is thought to be caused by environmental and/or genetic factors. Although correspondence between the characters defined as Mendelian by pedigree or segregation analysis and functional genes is difficult to establish, such analyses provide essential entry points into understanding CDD in captive bred rhesus macaques. To investigate the familial aggregation of CDD in captive rhesus macaque, we performed pedigree, segregation and heritability analyses on genealogical data from 55 severely affected individuals (probands) through whom relatives with a history of CDD were ascertained from routine computerized colony records comprising vital and demographic statistics of 10,814 rhesus macaques. We identified 175 rhesus macaques with CDD and estimated its incidence as approximately 2% in the colony. The disease strongly clustered in eight multi-generation pedigrees. Inspection of the pedigrees, segregation analysis and heritability estimate of CDD suggest that susceptibility to the disease is under strong genetic control. Identification of the locations of susceptibility genes in the rhesus macaque genome could facilitate the reduction of their frequency in captive breeding facilities.

Diversity and molecular phylogeny of mitochondrial DNA of rhesus macaques (Macaca mulatta) in Bangladesh.

While studies of rhesus macaques (Macaca mulatta) in the eastern (e.g., China) and western (e.g., India) parts of their geographic range have revealed major genetic differences that warrant the recognition of two different subspecies, little is known about genetic characteristics of rhesus macaques in the transitional zone extending from eastern India and Bangladesh through the northern part of Indo-China, the probable original homeland of the species. We analyzed genetic variation of 762 base pairs of mitochondrial DNA from 86 fecal swab samples and 19 blood samples from 25 local populations of rhesus macaque in Bangladesh collected from January 2010 to August 2012. These sequences were compared with those of rhesus macaques from India, China, and Myanmar. Forty-six haplotypes defined by 200 (26%) polymorphic nucleotide sites were detected. Estimates of gene diversity, expected heterozygosity, and nucleotide diversity for the total population were 0.9599 ± 0.0097, 0.0193 ± 0.0582, and 0.0196 ± 0.0098, respectively. A mismatch distribution of paired nucleotide differences yielded a statistically significantly negative value of Tajima's D, reflecting a population that rapidly expanded after the terminal Pleistocene. Most haplotypes throughout regions of Bangladesh, including an isolated region in the southwestern area (Sundarbans), clustered with haplotypes assigned to the minor haplogroup Ind-2 from India reflecting an east to west dispersal of rhesus macaques to India. Haplotypes from the southeast region of Bangladesh formed a cluster with those from Myanmar, and represent the oldest rhesus macaque haplotypes of Bangladesh. These results are consistent with the hypothesis that rhesus macaques first entered Bangladesh from the southeast, probably from Indo-China, then dispersed westward throughout eastern and central India.

An empirical comparison of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) for relatedness estimation in Chinese rhesus macaques (Macaca mulatta).

We compare the effectiveness of short tandem repeat (STR) and single nucleotide polymorphism (SNP) genotypes for estimating pairwise relatedness, using molecular data and pedigree records from a captive Chinese rhesus macaque population at the California National Primate Research Center. We find that a panel of 81 SNPs is as effective at estimating first-order kin relationships as a panel of 14 highly polymorphic STRs. We note, however, that the selected STRs provide more precise predictions of relatedness than the selected SNPs, and may be preferred in contexts that require the discrimination of kin related more distantly than first-order relatives. Additionally, we compare the performance of three commonly used relatedness estimation algorithms, and find that the Wang [2002] algorithm outperforms other algorithms when analyzing STR data, while the Queller & Goodnight [1989] algorithm outperforms other algorithms when analyzing SNP data. Future research is needed to address the number of SNPs required to reach the discriminatory power of a standard STR panel in relatedness estimation for primate colony management.

Development and validation of a SNP-based assay for inferring the genetic ancestry of rhesus macaques (Macaca mulatta).

Rhesus macaques (Macaca mulatta) are an important primate model species in several areas of biomedical research. The wide geographic distribution of this species has led to significant genetic differentiation among local and regional populations. These regional differences can be important factors in the selection of the most appropriate subjects for particular research studies, as animals from different populations can respond differently to the same experimental treatment. Consequently, it is valuable to confirm the ancestry of individual rhesus monkeys from geographically distinct populations. Using DNA samples obtained from rhesus macaques from six National Primate Research Centers, we tested a set of 384 potential ancestry informative single nucleotide polymorphisms (SNPs) and identified a final panel of 91 SNPs that can reliably distinguish Indian-origin from Chinese-origin rhesus monkeys. This genetic test can be used to determine the ancestral origin of animals and to detect individuals that are hybrids between these two regional populations. To demonstrate use of the SNP panel, we investigated the ancestry of 480 animals from the Yerkes NPRC (YNPRC) for which the colony records were insufficient to clearly establish ancestry. Three of the YNPRC animals tested were determined to be hybrids. This SNP ancestry tool will be useful to researchers, colony managers, and others who wish to evaluate the ancestral origin of individual rhesus macaques, and therefore will facilitate more effective and efficient use of these animals in biomedical research.

Identifying human-rhesus macaque gene orthologs using heterospecific SNP probes.

We genotyped a Chinese and an Indian-origin rhesus macaque using the Affymetrix Genome-Wide Human SNP Array 6.0 and cataloged 85,473 uniquely mapping heterospecific SNPs. These SNPs were assigned to rhesus chromosomes according to their probe sequence alignments as displayed in the human and rhesus reference sequences. The conserved gene order (synteny) revealed by heterospecific SNP maps is in concordance with that of the published human and rhesus macaque genomes. Using these SNPs' original human rs numbers, we identified 12,328 genes annotated in humans that are associated with these SNPs, 3674 of which were found in at least one of the two rhesus macaques studied. Due to their density, the heterospecific SNPs allow fine-grained comparisons, including approximate boundaries of intra- and extra-chromosomal rearrangements involving gene orthologs, which can be used to distinguish rhesus macaque chromosomes from human chromosomes.

Single-nucleotide polymorphisms reveal patterns of allele sharing across the species boundary between rhesus (Macaca mulatta) and cynomolgus (M. fascicularis) macaques.

Both phenotypic and genetic evidence for asymmetric hybridization between rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques has been observed in the region of Indochina where both species are sympatric. The large-scale sharing of major histocompatibility complex (MHC) class II alleles between the two species in this region supports the hypothesis that genes, and especially genes involved in immune response, are being transferred across the species boundary. This differential introgression has important implications for the incorporation of cynomolgus macaques of unknown geographic origin in biomedical research protocols. Our study found that for 2,808 single-nucleotide polymorphism (SNP) markers, the minor allele frequencies (MAF) and observed heterozygosity calculated from a sample of Vietnamese cynomolgus macaques was significantly different from those calculated from samples of both Chinese rhesus and Indonesian cynomolgus macaques. SNP alleles from Chinese rhesus macaques were overrepresented in a sample of Vietnamese cynomolgus macaques relative to their Indonesian conspecifics and located in genes functionally related to the primary immune system. These results suggest that Indochinese cynomolgus macaques represent a genetically and immunologically distinct entity from Indonesian cynomolgus macaques.

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.