A panel of tetranucleotide STR markers as an alternative approach to forensic DNA identification of wolf and dog.

Commercial panels of microsatellite (STR) loci are focused on the use of DNA of the domestic dog (Canis lupus familiaris) and are often inapplicable for genotyping the DNA of the gray wolf (Canis lupus lupus). We propose a CPlex test system, including one hexa- and 12 tetranucleotide autosomal STR loci, as well as two sex loci, that is equally efficient in DNA identification of biological samples of the wolf and the dog. Analysis of molecular variance between samples revealed significant differentiation values (FST = 0.0784, p < 0.001), which allows to use the panel to differentiate wolf and dog samples. Population subdivision coefficients (θ-values) were calculated for each of the 13 STR loci of the developed test system. It was shown that the values of the genotype frequency for dogs and wolves, without and with considering the θ-value, differ by three orders of magnitude (for dogs 8.9 × 10-16 and 2.1 × 10-14 and for wolves 1.9 × 10-15 and 4.5 × 10-14, respectively). The use of population subdivision coefficients will allow to identify the most reliable results of an expert identification study and the power of exclusion provided by the STR loci of the CPlex test system makes it possible to achieve a reliable level of evidence in forensic DNA analysis of both wolves and dogs. The test system has been validated for use in forensic identification of the dog and wolf based on biological traces found at crime scenes, as well as for individual identification and establishing biological relationship of animals of these species.

Antimicrobial resistance in wildlife: detection of antimicrobial resistance genes in Apennine wolves (Canis lupus italicus Altobello, 1921) from Central Italy.

The aim of this study was to molecularly investigate the presence of antimicrobial resistance genes (ARGs) in organ samples from 11 Apennine wolves (Canis lupus italicus) collected in Central Italy. Samples from lung, liver, spleen, kidney, tongue and intestine were investigated by PCRs targeting the following genes: tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tetA(P), tet(Q), tet(S), tet(X), sul1, sul2, sul3, blaCTX-M, blaSHV, blaTEM and mcr-1. A PCR positivity was highlighted for 13 out of the 21 tested genes; no positive results were obtained for tet(C), tet(D), tet(E), tet(G), sul3, blaCTX, blaSHV and mcr-1 genes. All 11 animals sampled showed positivity for one or more resistance genes. The results confirm the potential role of the wolf as an indicator and/or vector of antimicrobial-resistant bacteria or ARGs.

Relatedness-based mate choice and female philopatry: inbreeding trends of wolf packs in a human-dominated landscape.

Inbreeding can reduce offspring fitness and has substantial implications for the genetic diversity and long-term viability of populations. In social cooperative canids, inbreeding is conditioned by the geographic proximity between opposite-sex kin outside natal groups and the presence of related individuals in neighbouring groups. Consequently, challenges in moving into other regions where the species is present can also affect inbreeding rates. These can be particularly problematic in areas of high human density, where movement can be restricted, even for highly vagile species. In this study, we investigate the socio-ecological dynamics of Iberian wolf packs in the human-dominated landscape of Alto Minho, in northwest Portugal, where wolves exhibit a high prevalence of short-distance dispersal and limited gene flow with neighbouring regions. We hypothesise that mating occurs regardless of relatedness, resulting in recurrent inbreeding due to high kin encounter rates. Using data from a 10-year non-invasive genetic monitoring programme and a combination of relatedness estimates and genealogical reconstructions, we describe genetic diversity, mate choice, and dispersal strategies among Alto Minho packs. In contrast with expectations, our findings reveal relatedness-based mate choice, low kin encounter rates, and a reduced number of inbreeding events. We observed a high prevalence of philopatry, particularly among female breeders, with the most common breeding strategy involving the pairing of a philopatric female with an unrelated immigrant male. Overall, wolves were not inbred, and temporal changes in genetic diversity were not significant. Our findings are discussed, considering the demographic trend of wolves in Alto Minho and its human-dominated landscape.

Japanese wolves are most closely related to dogs and share DNA with East Eurasian dogs.

Although the domestic dog's origin is still unclear, this lineage is believed to have been domesticated from an extinct population of gray wolves, which is expected to be more closely related to dogs than to other populations of gray wolves. Here, we sequence the whole genomes of nine Japanese wolves (7.5-100x: Edo to Meiji periods) and 11 modern Japanese dogs and analyze them together with those from other populations of dogs and wolves. A phylogenomic tree shows that, among the gray wolves, Japanese wolves are closest to the dog, suggesting that the ancestor of dogs is closely related to the ancestor of the Japanese wolf. Based on phylogenetic and geographic relationships, the dog lineage has most likely originated in East Asia, where it diverged from a common ancestor with the Japanese wolf. Since East Eurasian dogs possess Japanese wolf ancestry, we estimate an introgression event from the ancestor of the Japanese wolf to the ancestor of the East Eurasian dog that occurred before the dog's arrival in the Japanese archipelago.

Comparative analysis of rare earth elements concentrations in domestic dogs and Apennine wolves of Central Italy: Influence of biological, nutritional, and lifestyle factors.

Rare Earth Elements (REEs) are strategical elements playing a crucial role in the industry, especially in producing high-tech materials. Therefore, REEs are new contaminants of emerging concerns. However, due to the lack of exposure data on REE occurrence in environmental matrices, especially in European countries, it is still tricky to establish environmental background levels to assess the ecotoxicological risk related to REEs exposure. The present study aimed to evaluate the liver concentrations of REEs in domestic dogs (Canis lupus familiaris) and Apennine wolves (Canis lupus italicus) living in the Abruzzo region, Italy. Moreover, for the scope of the present study, the dog's group was divided according to their sex, age, lifestyle, and diet. Wolves were categorized concerning their sex and genetic characteristics. Liver samples from dogs and wolves were collected during diagnostic necropsies from carcasses, sample mineralization was performed by a microwave digestion system with a single reaction chamber, and simultaneous determination of the presence of REEs was performed by Inductively Coupled Plasma Mass Spectrometer (Q-ICP-MS) using standard mode for all rare earth elements except scandium (Sc) which was acquired in kinetic energy discrimination (KED) mode. Hepatic concentrations of REEs were statistically significantly higher in wolves compared to dogs. Moreover, significant differences in REEs concentrations arose also from the genetic type of wolf, since "pure wolves" had higher liver concentrations of REEs compared to wolf-dog hybrids. Female and adult dogs also showed elevated REEs compared to male and juvenile dogs, while no significant differences were demonstrated for dogs' diet and lifestyle. The results of the present study confirm the exposure of domestic and wild carnivores to REEs, showing also the ability of REEs to accumulate in carnivore livers, suggesting the potential role of this species as an alternative bioindicator.

Demographic history shapes North American gray wolf genomic diversity and informs species' conservation.

Effective population size estimates are critical information needed for evolutionary predictions and conservation decisions. This is particularly true for species with social factors that restrict access to breeding or experience repeated fluctuations in population size across generations. We investigated the genomic estimates of effective population size along with diversity, subdivision, and inbreeding from 162,109 minimally filtered and 81,595 statistically neutral and unlinked SNPs genotyped in 437 grey wolf samples from North America collected between 1986 and 2021. We found genetic structure across North America, represented by three distinct demographic histories of western, central, and eastern regions of the continent. Further, grey wolves in the northern Rocky Mountains have lower genomic diversity than wolves of the western Great Lakes and have declined over time. Effective population size estimates revealed the historical signatures of continental efforts of predator extermination, despite a quarter century of recovery efforts. We are the first to provide molecular estimates of effective population size across distinct grey wolf populations in North America, which ranged between Ne ~ 275 and 3050 since early 1980s. We provide data that inform managers regarding the status and importance of effective population size estimates for grey wolf conservation, which are on average 5.2-9.3% of census estimates for this species. We show that while grey wolves fall above minimum effective population sizes needed to avoid extinction due to inbreeding depression in the short term, they are below sizes predicted to be necessary to avoid long-term risk of extinction.

Individual genotypes from environmental DNA: Fingerprinting snow tracks of three large carnivore species.

Continued advancements in environmental DNA (eDNA) research have made it possible to access intraspecific variation from eDNA samples, opening new opportunities to expand non-invasive genetic studies of wildlife populations. However, the use of eDNA samples for individual genotyping, as typically performed in non-invasive genetics, still remains elusive. We present successful individual genotyping of eDNA obtained from snow tracks of three large carnivores: brown bear (Ursus arctos), European lynx (Lynx lynx) and wolf (Canis lupus). DNA was extracted using a protocol for isolating water eDNA and genotyped using amplicon sequencing of short tandem repeats (STR), and for brown bear a sex marker, on a high-throughput sequencing platform. Individual genotypes were obtained for all species, but genotyping performance differed among samples and species. The proportion of samples genotyped to individuals was higher for brown bear (5/7) and wolf (7/10) than for lynx (4/9), and locus genotyping success was greater for brown bear (0.88). The sex marker was typed in six out of seven brown bear samples. Results for three species show that reliable individual genotyping, including sex identification, is now possible from eDNA in snow tracks, underlining its vast potential to complement the non-invasive genetic methods used for wildlife. To fully leverage the application of snow track eDNA, improved understanding of the ideal species- and site-specific sampling conditions, as well as laboratory methods promoting genotyping success, is needed. This will also inform efforts to retrieve and type nuclear DNA from other eDNA samples, thereby advancing eDNA-based individual and population-level studies.

Assessment of genetic diversity, population structure and wolf-dog hybridisation in the Eastern Romanian Carpathian wolf population.

The Carpathian Mountains have been constantly inhabited by grey wolves and present one of the largest distribution areas in Europe, comprising between 2300 and 2700 individuals in Romania. To date, however, relatively little is known about the Romanian wolf population. We aimed to provide a first assessment of genetic diversity, population structure and wolf-dog hybridisation based on 444 mostly non-invasively collected samples in the Eastern Romanian Carpathians. Pack reconstruction and analysis of population genetic parameters were performed with mitochondrial DNA control-region sequencing and microsatellite genotyping. We found relatively high levels of genetic diversity, which is similar to values found in previous studies on Carpathian wolves from Poland and Slovakia, as well as to the long-lasting Dinaric-Balkan wolf population. We found no significant population structure in our study region, suggesting effective dispersal and admixture. Analysis of wolf-dog hybridisation using a Single Nucleotide Polymorphism panel optimised for hybrid detection revealed low rates of admixture between wolves and domestic dogs. Our results provide evidence for the existence of a genetically viable wolf population in the Romanian Carpathians. The genetic data obtained in this study may serve as valuable baseline information for the elaboration of monitoring standards and management plans for wolves in Romania.

Dingoes, companions in life and death: The significance of archaeological canid burial practices in Australia.

The dingo, also known as the Australian native dog, was introduced in the late Holocene. Dingoes were primarily wild animals but a number resided in Aboriginal people's camps. Traditionally, these individuals were taken from wild litters before weaning and raised by Aboriginal people. It is generally believed that these dingoes were not directly provided for, and upon sexual maturity, returned to reproduce in the wild. However, some died while in the company of people and, were buried in occupation sites. This Australian practice parallels the burial of domestic dogs in many regions of the Asia-Pacific and beyond but has attracted very little research. We explore the historical and archaeological evidence for dingo burial, examining its different forms, chronological and geographic distribution, and cultural significance. Dingoes were usually buried in the same manner as Aboriginal community members and often in areas used for human burial, sometimes alongside people. This practice probably occurred from the time of their introduction until soon after European colonisation. We present a case study of dingo burials from Curracurrang Rockshelter (NSW) which provides insights into the lives of ancient tame dingoes, and suggests that domestication and genetic continuity between successive camp-dwelling generations may have occurred prior to European contact.

CanISO: a database of genomic and transcriptomic variations in domestic dog (Canis lupus familiaris).

BACKGROUND: The domestic dog, Canis lupus familiaris, is a companion animal for humans as well as an animal model in cancer research due to similar spontaneous occurrence of cancers as humans. Despite the social and biological importance of dogs, the catalogue of genomic variations and transcripts for dogs is relatively incomplete. RESULTS: We developed CanISO, a new database to hold a large collection of transcriptome profiles and genomic variations for domestic dogs. CanISO provides 87,692 novel transcript isoforms and 60,992 known isoforms from whole transcriptome sequencing of canine tumors (N = 157) and their matched normal tissues (N = 64). CanISO also provides genomic variation information for 210,444 unique germline single nucleotide polymorphisms (SNPs) from the whole exome sequencing of 183 dogs, with a query system that searches gene- and transcript-level information as well as covered SNPs. Transcriptome profiles can be compared with corresponding human transcript isoforms at a tissue level, or between sample groups to identify tumor-specific gene expression and alternative splicing patterns. CONCLUSIONS: CanISO is expected to increase understanding of the dog genome and transcriptome, as well as its functional associations with humans, such as shared/distinct mechanisms of cancer. CanISO is publicly available at https://www.kobic.re.kr/caniso/ .

ANTIMICROBIAL SUSCEPTIBILITY OF STAPHYLOCOCCUS SPP. ISOLATED FROM FELIDS AND CANIDS IN BELO HORIZONTE ZOO, BRAZIL.

The epidemiology of Staphylococcus spp. has become a major concern among humans and animals due to increasing antimicrobial resistance and frequent reports of infection. Despite the importance of animals as reservoirs for staphylococci, little is known about the epidemiology of Staphylococcus spp. in most nondomestic species, including canids and felids. This study evaluated the frequency, distribution, and patterns of antimicrobial resistance of staphylococcal species isolated from captive felids and canids from Belo Horizonte Zoo, Brazil. Rectal, oral, and nasal swabs from apparently healthy maned wolves (Chrysocyon brachyurus, n= 7), a lion (Panthera leo, n = 1), jaguars (Panthera onca, n = 3), and one swab of a cougar (Puma concolor) with an ear infection were streaked onto mannitol salt agar. Colonies identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, polymerase chain reaction for the Staphylococcus intermedius group (SIG), and 16S rRNA gene sequencing. Isolates were subjected to antimicrobial susceptibility tests and Staphylococcus pseudintermedius strains were subjected to multilocus sequence typing. Staphylococcus species were isolated from 24 of the 34 samples (70.6%). Among the isolated strains, S. pseudintermedius and Staphylococcus felis were the most frequent species (41.7 and 25%, respectively). Five novel sequence types were identified among the S. pseudintermedius isolates. Resistance to tetracycline (7/24, 29.2%) or penicillin (6/23, 26.1%) was significantly higher than the other antimicrobial agents tested (P < 0.05). One isolate, Staphylococcus nepalensis, was positive for mecA and resistant to five antimicrobials, and was thus classified as multidrug-resistant. The present work suggests that maned wolves are natural hosts of SIG and also reports the isolation of S. felis in sick and healthy, captive, nondomestic carnivores. The isolated staphylococci were susceptible to most classes of antimicrobials tested. However, the multidrug-resistance capability of an S. nepalensis strain reinforces the hypothesis that felids and canids act as reservoirs of pathogens with antimicrobial resistance.

The population bottleneck of the Iberian wolf impacted genetic diversity but not admixture with domestic dogs: A temporal genomic approach.

After decades of intense persecution, the Iberian wolf subspecies faced a severe bottleneck in the 1970s that considerably reduced its range and population size, nearly leading to its extinction in central and southern Iberian Peninsula. Such population decline could have impacted the genetic diversity of Iberian wolves through different processes, namely genetic drift and dynamics of hybridization with domestic dogs. By contrasting the genomes of 68 contemporary with 54 historical samples spanning the periods before and immediately after the 1970s bottleneck, we found evidence of its impact on genetic diversity and dynamics of wolf-dog hybridization. Our genome-wide assessment revealed that wolves and dogs form two well-differentiated genetic groups in Iberia and that hybridization rates did not increase during the bottleneck. However, an increased number of hybrid individuals was found over time during the population re-expansion, particularly at the edge of the wolf range. We estimated a low percentage of dog ancestry (~1.4%) in historical samples, suggesting that dog introgression was not a key driver for wolf extinction in central and southern Iberia. Our findings also unveil a significant decline in genetic diversity in contemporary samples, with the highest proportion of homozygous segments in the genome being recently inherited. Overall, our study provides unprecedented insight into the impact of a sharp decline on the Iberian wolf genome and refines our understanding of the ecological and evolutionary drivers of wolf-dog hybridization in the wild.

Special Issue: "Canine Genetics 2".

Wolves were the first animal species to become domesticated by humans, approximately 30,000-50,000 years ago. Human-directed dog breeding over thousands of generations has generated more than 350 recognized breeds displaying surprisingly different phenotypes with respect to morphology, behavior and disease predispositions. The domestication of wolves and the subsequent breeding of dogs can be viewed as one of humankind's oldest and largest genetic experiments and provides us with unique opportunities for research. Dogs have not only become human's best friend but were also described as geneticists' best friend in a past issue of Science. In recognition of the importance of canine genetics, this Special Issue, entitled "Canine Genetics 2", was compiled. It represents a sequel to the former Special Issue "Canine Genetics", which was published in 2019. During the last 15 years, the canine community has heavily relied on a reference genome derived from the female Boxer Tasha. "Canine Genetics 2" includes an article describing a greatly improved version of this important community resource. This Special Issue further contains several reports related to monogenic or complex inherited diseases in dogs. Finally, important aspects of wild canid research, genetic diversity in different populations and canine morphology were investigated.

Genome sequencing of 2000 canids by the Dog10K consortium advances the understanding of demography, genome function and architecture.

BACKGROUND: The international Dog10K project aims to sequence and analyze several thousand canine genomes. Incorporating 20 × data from 1987 individuals, including 1611 dogs (321 breeds), 309 village dogs, 63 wolves, and four coyotes, we identify genomic variation across the canid family, setting the stage for detailed studies of domestication, behavior, morphology, disease susceptibility, and genome architecture and function. RESULTS: We report the analysis of > 48 M single-nucleotide, indel, and structural variants spanning the autosomes, X chromosome, and mitochondria. We discover more than 75% of variation for 239 sampled breeds. Allele sharing analysis indicates that 94.9% of breeds form monophyletic clusters and 25 major clades. German Shepherd Dogs and related breeds show the highest allele sharing with independent breeds from multiple clades. On average, each breed dog differs from the UU_Cfam_GSD_1.0 reference at 26,960 deletions and 14,034 insertions greater than 50 bp, with wolves having 14% more variants. Discovered variants include retrogene insertions from 926 parent genes. To aid functional prioritization, single-nucleotide variants were annotated with SnpEff and Zoonomia phyloP constraint scores. Constrained positions were negatively correlated with allele frequency. Finally, the utility of the Dog10K data as an imputation reference panel is assessed, generating high-confidence calls across varied genotyping platform densities including for breeds not included in the Dog10K collection. CONCLUSIONS: We have developed a dense dataset of 1987 sequenced canids that reveals patterns of allele sharing, identifies likely functional variants, informs breed structure, and enables accurate imputation. Dog10K data are publicly available.

The far-reaching effects of genetic process in a keystone predator species, grey wolves.

Although detrimental genetic processes are known to adversely affect the viability of populations, little is known about how detrimental genetic processes in a keystone species can affect the functioning of ecosystems. Here, we assessed how changes in the genetic characteristics of a keystone predator, grey wolves, affected the ecosystem of Isle Royale National Park over two decades. Changes in the genetic characteristic of the wolf population associated with a genetic rescue event, followed by high levels of inbreeding, led to a rise and then fall in predation rates on moose, the primary prey of wolves and dominant mammalian herbivore in this system. Those changes in predation rate led to large fluctuations in moose abundance, which in turn affected browse rates on balsam fir, the dominant forage for moose during winter and an important boreal forest species. Thus, forest dynamics can be traced back to changes in the genetic characteristics of a predator population.

Wolf genetic diversity compared across Europe using the yardstick method.

Integrating data across studies with traditional microsatellite genetic markers requires careful calibration and represents an obstacle for investigation of wide-ranging species where populations require transboundary management. We used the "yardstick" method to compare results published across Europe since 2002 and new wolf (Canis lupus) genetic profiles from the Carpathian Mountains in Central Europe and the Dinaric Mountains in Southeastern Europe, with the latter as our reference population. We compared each population with Dinaric wolves, considering only shared markers (range 4-17). For each population, we calculated standard genetic diversity indices plus calibrated heterozygosity (Hec) and allelic richness (Ac). Hec and Ac in Dinaric (0.704 and 9.394) and Carpathian wolves (0.695 and 7.023) were comparable to those observed in other large and mid-sized European populations, but smaller than those of northeastern Europe. Major discrepancies in marker choices among some studies made comparisons more difficult. However, the yardstick method, including the new measures of Hec and Ac, provided a direct comparison of genetic diversity values among wolf populations and an intuitive interpretation of the results. The yardstick method thus permitted the integration of diverse sources of publicly available microsatellite data for spatiotemporal genetic monitoring of evolutionary potential.

Multiple Origins and Genomic Basis of Complex Traits in Sighthounds.

Sighthounds, a distinctive group of hounds comprising numerous breeds, have their origins rooted in ancient artificial selection of dogs. In this study, we performed genome sequencing for 123 sighthounds, including one breed from Africa, six breeds from Europe, two breeds from Russia, and four breeds and 12 village dogs from the Middle East. We gathered public genome data of five sighthounds and 98 other dogs as well as 31 gray wolves to pinpoint the origin and genes influencing the morphology of the sighthound genome. Population genomic analysis suggested that sighthounds originated from native dogs independently and were comprehensively admixed among breeds, supporting the multiple origins hypothesis of sighthounds. An additional 67 published ancient wolf genomes were added for gene flow detection. Results showed dramatic admixture of ancient wolves in African sighthounds, even more than with modern wolves. Whole-genome scan analysis identified 17 positively selected genes (PSGs) in the African population, 27 PSGs in the European population, and 54 PSGs in the Middle Eastern population. None of the PSGs overlapped in the three populations. Pooled PSGs of the three populations were significantly enriched in "regulation of release of sequestered calcium ion into cytosol" (gene ontology: 0051279), which is related to blood circulation and heart contraction. In addition, ESR1, JAK2, ADRB1, PRKCE, and CAMK2D were under positive selection in all three selected groups. This suggests that different PSGs in the same pathway contributed to the similar phenotype of sighthounds. We identified an ESR1 mutation (chr1: g.42,177,149 T > C) in the transcription factor (TF) binding site of Stat5a and a JAK2 mutation (chr1: g.93,277,007 T > A) in the TF binding site of Sox5. Functional experiments confirmed that the ESR1 and JAK2 mutation reduced their expression. Our results provide new insights into the domestication history and genomic basis of sighthounds.

Evolutionary history of the extinct wolf population from France in the context of global phylogeographic changes throughout the Holocene.

Phylogeographic patterns in large mammals result from natural environmental factors and anthropogenic effects, which in some cases include domestication. The grey wolf was once widely distributed across the Holarctic, but experienced phylogeographic shifts and demographic declines during the Holocene. In the 19th-20th centuries, the species became extirpated from large parts of Europe due to direct extermination and habitat loss. We reconstructed the evolutionary history of the extinct Western European wolves based on the mitogenomic composition of 78 samples from France (Neolithic-20th century) in the context of other populations of wolves and dogs worldwide. We found a close genetic similarity of French wolves from ancient, medieval and recent populations, which suggests the long-term continuity of maternal lineages. MtDNA haplotypes of the French wolves showed large diversity and fell into two main haplogroups of modern Holarctic wolves. Our worldwide phylogeographic analysis indicated that haplogroup W1, which includes wolves from Eurasia and North America, originated in Northern Siberia. Haplogroup W2, which includes only European wolves, originated in Europe ~35 kya and its frequency was reduced during the Holocene due to an expansion of haplogroup W1 from the east. Moreover, we found that dog haplogroup D, currently restricted to Europe and the Middle East, was nested within the wolf haplogroup W2. This suggests European origin of haplogroup D, probably as a result of an ancient introgression from European wolves. Our results highlight the dynamic evolutionary history of European wolves during the Holocene, with a partial lineage replacement and introgressive hybridization with local dog populations.

A multidisciplinary approach to estimating wolf population size for long-term conservation.

The wolf (Canis lupus) is among the most controversial of wildlife species. Abundance estimates are required to inform public debate and policy decisions, but obtaining them at biologically relevant scales is challenging. We developed a system for comprehensive population estimation across the Italian alpine region (100,000 km2 ), involving 1513 trained operators representing 160 institutions. This extensive network allowed for coordinated genetic sample collection and landscape-level spatial capture-recapture analyses that transcended administrative boundaries to produce the first estimates of key parameters for wolf population status assessment. Wolf abundance was estimated at 952 individuals (95% credible interval 816-1120) and 135 reproductive units (i.e., packs) (95% credible interval 112-165). We also estimated that mature individuals accounted for 33-45% of the entire population. The monitoring effort was spatially estimated thereby overcoming an important limitation of citizen science data. This is an important approach for promoting wolf-human coexistence based on wolf abundance monitoring and an endorsement of large-scale harmonized conservation practices.

Una estrategia multidisciplinaria para la estimación del tamaño poblacional de los lobos para la conservación a largo plazo Resumen El lobo (Canis lupus) está entre las especies de fauna más controversiales. Se requieren estimaciones de abundancia para informar al debate público y las decisiones políticas, pero es un reto obtenerlos en escalas con relevancia biológica. Desarrollamos un sistema para la estimación completa de la población en la región alpina de Italia (100,000 km2 ), con la participación de 1,513 operadores entrenados que representan a 160 instituciones. Esta red extensa permitió una colecta coordinada de muestras genéticas y análisis de captura-recaptura espacial que trascendieron las fronteras administrativas para así producir las primeras estimaciones de los parámetros clave para la evaluación del estado de la población de los lobos. Se estimó la abundancia en 952 individuos (95% intervalo de confianza 816-1120) y 135 unidades reproductivas (es decir, manadas) (95% intervalo de confianza 112-165). También estimamos que los individuos maduros representaban el 33-45% de toda la población. El esfuerzo de monitoreo se estimó espacialmente, por lo que sobrepasó una limitación importante de la ciencia ciudadana. Esta estrategia es importante para promover la coexistencia entre lobos y humanos con base en el monitoreo de la abundancia y el apoyo a las prácticas armonizadas de conservación a gran escala.

Diet-induced changes in fecal microbiota composition and diversity in dogs (Canis lupus familiaris): A comparative study of BARF-type and commercial diets.

BACKGROUND: Diet is known to strongly modulate the composition of the gut microbiota, thereby affecting health conditions and disease. Natural BARF-type and commercial diets have been used for feeding pets (e.g. dogs and cats) promoting changes in the canine microbiota in terms of abundance, richness, and diversity that may favor certain metabolic processes and resistance to certain infectious agents. Therefore, the present study sought to identify microbiota changes in dogs fed with a BARF-type diet versus dogs fed with a commercial diet by sequencing the V4 region of the 16S rRNA gene. METHODS: The microbiota of dogs fed with the BARF-diet (n = 20) and commercial-diet (n = 26) was studied using fecal samples. A metabarcoding strategy was employed by sequencing the V4 hypervariable region of the 16S rRNA gene using the Illumina HiSeq platform. DADA2 was used to assess the quality profile of the reads and to determine the core sample inference algorithm of the reads to infer amplicon sequence variants (ASVs). The taxonomic assignment was performed using sequences from the Silva v138 formatted reference database. The microbial diversity analysis was performed using the R package Phyloseq, which was used to calculate diversity and abundance indices and construct the respective graphs. Linear discriminant analysis (LDA) effect size analysis (LEfSe) was used to identify the differentially abundant taxa in the BARF group versus the commercial-diet group. RESULTS: The diet causes changes in fecal microbiota composition and diversity, with richness and diversity being higher in BARF-fed dogs. Beta diversity analyses confirmed that diet is directly related to microbiota composition regardless of breed or sex. Differentially enriched taxa were identified in each of the diets as Fusobacterium, Bacteroides, and Clostridium perfringens in BARF-fed dogs and Prevotella, Turicibacter, Faecalibacterium, and Peptacetobacter (Clostridium) hiranonis, mostly relevant in carbohydrate metabolism, in commercial-fed dogs. CONCLUSIONS: This study is the first one carried out in dogs from Colombia that seeks to identify changes in the intestinal microbiota concerning natural BARF type diet and commercial diet using a metabarcoding approach. Important differences were identified in terms of richness, diversity, and differentially enriched bacteria in each of the diets. The microbiota of dogs fed the BARF diet was characterized by higher richness and diversity compared to the commercial diet. However, it was identified that BARF-fed dogs can potentially acquire more opportunistic infections by pathogens of importance such as C. perfringens. Most of the taxa enriched in commercial diet-fed dogs are linked to carbohydrate metabolism, which may be directly related to diet composition.