Whole genome sequencing analysis of alpaca suggests TRPV3 as a candidate gene for the suri phenotype.

BACKGROUND: Alpaca is a domestic South American camelid probably arising from the domestication of two wild camelids, the vicugna and the guanaco. Two phenotypes are described for alpaca, known as huacaya and suri. Huacaya fleece is characterized by compact, soft, and highly crimped fibers, while suri fleece is longer, straight, less crimped, and lustrous. The gene variants determining these phenotypes are still unknown, although previous studies suggested a dominant inheritance of the suri. Based on that, the aim of this study was the identification of the gene variants determining alpaca coat phenotypes through whole genome sequencing (WGS) analysis. RESULTS: The sample used includes two test-cross alpaca families, suri × huacaya, which produced two offspring, one with the suri phenotype and one with the huacaya phenotype. The analyzed sample was expanded through the addition of WGS data from six vicugnas and six guanacos; this because we assumed the absence of the gene variants linked to the suri phenotype in these wild species. The analysis of gene variant segregation with the suri phenotype, coupled with the filtering of gene variants present in the wild species, disclosed the presence in all the suri samples of a premature termination codon (PTC) in TRPV3 (transient receptor potential cation channel subfamily V member 3), a gene known to be involved in hair growth and cycling, thermal sensation, cold tolerance and adaptation in several species. Mutations in TRPV3 were previously associated with the alteration of hair structure leading to an impaired formation of the hair canal and the hair shaft in mouse. This PTC in TRPV3, due to a G > T substitution (p.Glu475*), results in a loss of 290 amino acids from the canonical translated protein, plausibly leading to a physiological dysfunction. CONCLUSION: The present results suggest that the suri phenotype may arise from a TRPV3 gene variant which may explain some of the suri features such as its longer hair fibre with lower number of cuticular scales compared to huacaya.

Hypothalamic-pituitary-gonadal axis response to photoperiod changes in female guanacos (Lama guanicoe).

The guanaco, a wild South American camelid, is renowned for its remarkable resilience to extreme conditions. Despite this, little is known about how reproductive hormones in female camelids are influenced during their seasonal breeding period, which occurs during long photoperiod. To explore this, the study investigated the response of the hypothalamic-pituitary-gonadal axis in female guanacos during short days (10L:14D; July) and long days (16L:8D; December) in the Mediterranean ecosystem (33°38'28″S, 70°34'27″W). Blood samples from 14 adult animals were collected, and measurements of melatonin, 17ß-estradiol, FSH, and LH concentrations were taken. The results showed that melatonin concentration was lower (P < 0.05) during long days than short days, whereas 17ß-estradiol, FSH, and LH concentrations were higher (P < 0.05) during long days compared to short days. Furthermore, the study detected the expression of the melatonin receptor 1A and kisspeptin in the hypothalamus and pituitary, suggesting that the pineal gland of female guanacos is sensitive to seasonal changes in day length. These findings also indicate a seasonal variation in the concentration of reproductive hormones, likely linked to the distinct modulation of the hypothalamic-pituitary-gonadal axis of female guanacos during short and long days.

Genome-wide scan for runs of homozygosity in South American Camelids.

BACKGROUND: Alpaca (Vicugna pacos), llama (Lama glama), vicugna (Vicugna vicugna) and guanaco (Lama guanicoe), are the camelid species distributed over the Andean high-altitude grasslands, the Altiplano, and the Patagonian arid steppes. Despite the wide interest on these animals, most of the loci under selection are still unknown. Using whole-genome sequencing (WGS) data we investigated the occurrence and the distribution of Runs Of Homozygosity (ROHs) across the South American Camelids (SACs) genome to identify the genetic relationship between the four species and the potential signatures of selection. RESULTS: A total of 37 WGS samples covering the four species was included in the final analysis. The multi-dimensional scaling approach showed a clear separation between the four species; however, admixture analysis suggested a strong genetic introgression from vicugna and llama to alpaca. Conversely, very low genetic admixture of the guanaco with the other SACs was found. The four species did not show significant differences in the number, length of ROHs (100-500 kb) and genomic inbreeding values. Longer ROHs (> 500 kb) were found almost exclusively in alpaca. Seven overlapping ROHs were shared by alpacas, encompassing nine loci (FGF5, LOC107034918, PRDM8, ANTXR2, LOC102534792, BSN, LOC116284892, DAG1 and RIC8B) while nine overlapping ROHs were found in llama with twenty-five loci annotated (ERC2, FZD9, BAZ1B, BCL7B, LOC116284208, TBL2, MLXIPL, PHF20, TRNAD-AUC, LOC116284365, RBM39, ARFGEF2, DCAF5, EXD2, HSPB11, LRRC42, LDLRAD1, TMEM59, LOC107033213, TCEANC2, LOC102545169, LOC116278408, SMIM15, NDUFAF2 and RCOR1). Four overlapping ROHs, with three annotated loci (DLG1, KAT6B and PDE4D) and three overlapping ROHs, with seven annotated genes (ATP6V1E1, BCL2L13, LOC116276952, BID, KAT6B, LOC116282667 and LOC107034552), were detected for vicugna and guanaco, respectively. CONCLUSIONS: The signatures of selection revealed genomic areas potentially selected for production traits as well as for natural adaptation to harsh environment. Alpaca and llama hint a selection driven by environment as well as by farming purpose while vicugna and guanaco showed selection signals for adaptation to harsh environment. Interesting, signatures of selection on KAT6B gene were identified for both vicugna and guanaco, suggesting a positive effect on wild populations fitness. Such information may be of interest to further ecological and animal production studies.

The effect of photoperiod and melatonin on plasma prolactin concentrations in female guanaco (Lama guanicoe) in captivity.

The present study examined the effects of different photoperiods and melatonin treatment on plasma prolactin concentrations in guanacos, a South American camelid, in captivity. Fourteen adult female guanacos, not gestating or lactating and isolated from males, were studied. The control group was exposed to natural daylight, during short days (N = 7, 10L:14D) and long days (N = 7, 16L:8D). The treatment group (N = 7, 10L:14D) received melatonin implants every 23 days for 6 weeks during long days. Blood samples were taken at intervals of 1 week for 3 weeks, starting the third week of treatment. Prolactin concentrations were measured using competitive ELISA. Plasma concentrations of prolactin in non-lactating female guanacos have seasonal changes, with a higher concentration (p < .001) in short days (3.50 ± 2.24 ng/ml) than long days (1.10 ± 0.91 ng/ml). Melatonin treatment significantly decreases (p < .05) plasma concentrations of prolactin on the 21st day after the treatment. These findings are the first report of an endogenous circannual rhythm of plasma prolactin concentration and the action of melatonin treatment on prolactin secretion in this wild camelid.

Conflict between Farmers and Guanacos (Lama guanicoe cacsilensis): Field Surveys, Remote Sensing, and Interviews Provide Information for Conservation of a Critically Endangered Species in Southern Peru.

The Peruvian guanaco (Lama guanicoe cacsilensis) is classified as being "in critical danger of extinction" by the government. In this study, we evaluate how the conflict between farmers and guanacos in the Susapaya and Estique Districts, Tacna Department (Southern Peru) may represent a threat to their survival. To evaluate the situation, we 1. Conducted field surveys to monitor guanaco presence, 2. Used available remote sensing data to map guanaco movement, and 3. interviewed the impacted farmers concerning their losses. Remote sensing data showed that sedentary guanaco family groups located in prime steppe vegetation habitats never entered agricultural areas, while field surveys showed that bachelor bands and solitary individuals did, perhaps seeking forage due to growing population pressure. Interview data found that 90% of community farmers felt that guanacos were a problem best resolved by better fencing (45%), hunting (19%), or increased security (16%), and 92% saw no value in the conservation of the species. Hunting is illegal, given the critically endangered status of guanacos in Peru, so additional efforts are needed to both educate those who feel guanacos are a menace and involve them in efforts to preserve the species.

Histomorphological changes between short and long days in ovary guanacos (Lama guanicoe).

This is the first morpho-histological comparison of guanaco ovaries between reproductive (long-days) and non-reproductive (short-days) seasons, and oestrogen receptor-alpha (ERα) and beta (ERß) detection. Different stages of follicle development were found in the cortical area, but no corpus luteum was detected. The size and frequency of antral follicles and large atretic follicles were higher in long-day ovaries than short-days, consistent with ovarian activity in this season. Differential expression of ERα and ERß was observed in follicles at different stages of development between short and long days. These data reveal histological and molecular differences between reproductive and non-reproductive seasons of guanaco ovaries.

Behavioural Indicators of Intra- and Inter-Specific Competition: Sheep Co-Grazing with Guanaco in the Patagonian Steppe.

In extensive livestock production, high densities may inhibit regulation processes, maintaining high levels of intraspecific competition over time. During competition, individuals typically modify their behaviours, particularly feeding and bite rates, which can therefore be used as indicators of competition. Over eight consecutive seasons, we investigated if variation in herd density, food availability, and the presence of a potential competitor, the guanaco (Lama guanicoe), was related with behavioural changes in domestic sheep in Chilean Patagonia. Focal sampling, instantaneous scan sampling, measures of bite and movement rates were used to quantify behavioural changes in domestic sheep. We found that food availability increased time spent feeding, while herd density was associated with an increase in vigilant behaviour and a decrease in bite rate, but only when food availability was low. Guanaco presence appeared to have no impact on sheep behaviour. Our results suggest that the observed behavioural changes in domestic sheep are more likely due to intraspecific competition rather than interspecific competition. Consideration of intraspecific competition where guanaco and sheep co-graze on pastures could allow management strategies to focus on herd density, according to rangeland carrying capacity.

Utility of genetic variation in coat color genes to distinguish wild, domestic and hybrid South American camelids for forensic and judicial applications.

A molecular genetic protocol for distinguishing pure and hybrid South American camelids was developed to provide strong, quantifiable, and unbiased species identification. We detail the application of the approach in the context of a criminal case in the Andes Mountains of central Chile where the defendants were alleged to have illegally hunted three wild guanacos (Lama guanicoe), as opposed to hybrid domestic llama (Lama glama)/wild guanaco crosses, which are unregulated. We describe a workflow that differentiates among wild, domestic and hybrid South American camelids (Lama versus Vicugna) based on mitochondrial cytochrome b genetic variation (to distinguish between Lama and Vicugna), and MC1R and exon 4 variation of the ASIP gene (to differentiate wild from domestic species). Additionally, we infer the population origin and sex of each of the three individuals from a panel of 15 autosomal microsatellite loci and the presence or absence of the SRY gene. Our analyses strongly supported the inference that the confiscated carcasses corresponded with 2 male and 1 female guanacos that were hunted illegally. Statistical power analyses suggested that there was an extremely low probability of misidentifying domestic camelids as wild camelids (an estimated 0 % Type I error rate), or using more conservative approached a 1.17 % chance of misidentification of wild species as domestic camelids (Type II error). Our case report and methodological and analytical protocols demonstrate the power of genetic variation in coat color genes to identify hybrids between wild and domestic camelid species and highlight the utility of the approach to help combat illegal wildlife hunting and trafficking.

Are there different vigilance strategies between types of social units in Lama guanicoe?

Group vigilance is a cooperative behaviour in social species that reduces individual risk of predation. Lama guanicoe is a social species of camelid performing cooperative vigilance, but little is known about the vigilance behaviour of different social units. We analysed the vigilance behaviour in different types of social units of L. guanicoe, to better understand the complexity of this behaviour. The best supported models for both the frequency of vigilance and the proportion of time vigilant included the type and size of social units as the most important predictors that affect the vigilance behaviour. Solitary males devoted proportionally more time in vigilance behaviour than family, mixed or bachelor groups, whereas females in female groups spent more time vigilant than guanacos in mixed groups. Frequency of vigilance was higher in family individuals and solitary males than in bachelor or mixed groups. It is likely that that in family and females groups, the presence of offspring would increase the vigilance behaviour to detect possible predators. Topography and habitat characteristics that determine the predation risk, affected vigilance behaviour of mixed groups and solitary males. Our results suggest that vigilance behaviour should not be generalized for this species, since there are differences between the types of social units, probably related to intrinsic characteristics of each one and the perception of risk.

Sarcocystosis in South American camelids: The state of play revisited.

Members of the genus Sarcocystis (Apicomplexa: Sarcocystidae) are intracellular protozoan parasites that infect a wide range of domestic and wild animals, resulting in economic losses in production animals worldwide. Sarcocystis spp. have indirect life-cycles where canids and felids serve as main definitive hosts while a range of domestic and wild animals serve as intermediate hosts, including South American camelids (SACs) such as alpacas, llamas and guanacos. These animals primarily occur in South American countries on Andean, elevated plains but in recent years, alpacas and llamas have become emerging animal industries in other parts of the world such as Australia, Europe and the USA due to their high-quality fiber, meat and hides. For instance, alpaca meat is becoming popular in many parts of the world due to its lower cholesterol content than other red meat, thereby it has the potential of a valuable product for both local and international markets. However, SAC meat can be degraded and/or even condemned due to the presence of macroscopic sarcocysts in skeletal muscles, leading to significant economic losses to farmers. The infection is generally asymptomatic, though highly pathogenic or even fatal Sarcocystis infections have also been reported in alpacas and llamas. Despite the economic importance of sarcocystosis in SACs, little is known about the life-cycle of parasites involved, disease transmission, epidemiology, pathogenesis, diagnosis, control and public health significance. This review article provides an in-depth analysis of the existing knowledge on the taxonomy, epidemiology, clinicopathology and diagnosis of Sarcocystis in SACs, highlights knowledge gaps and proposes future areas of research that could contribute to our better understanding of sarcocystosis in these animals.

Genetic Variation in Coat Colour Genes MC1R and ASIP Provides Insights Into Domestication and Management of South American Camelids.

The domestication of wild vicuña and guanaco by early pre-Inca cultures is an iconic example of wildlife management and domestication in the Americas. Although domestic llamas and alpacas were clearly selected for key, yet distinct, phenotypic traits, the relative patterns and direction of selection and domestication have not been confirmed using genetic approaches. However, the detailed archaeological records from the region suggest that domestication was a process carried out under significant control and planning, which would have facilitated coordinated and thus extremely effective selective pressure to achieve and maintain desired phenotypic traits. Here we link patterns of sequence variation in two well-characterised genes coding for colour variation in vertebrates and interpret the results in the context of domestication in guanacos and vicuñas. We hypothesise that colour variation in wild populations of guanacos and vicunas were strongly selected against. In contrast, variation in coat colour variation in alpaca was strongly selected for and became rapidly fixed in alpacas. In contrast, coat colour variants in llamas were of less economic value, and thus were under less selective pressure. We report for the first time the full sequence of MC1R and 3 exons of ASIP in 171 wild specimens from throughout their distribution and which represented a range of commonly observed colour patterns. We found a significant difference in the number of non-synonymous substitutions, but not synonymous substitutions among wild and domestics species. The genetic variation in MC1R and ASIP did not differentiate alpaca from llama due to the high degree of reciprocal introgression, but the combination of 11 substitutions are sufficient to distinguish domestic from wild animals. Although there is gene flow among domestic and wild species, most of the non-synonymous variation in MC1R and ASIP was not observed in wild species, presumably because these substitutions and the associated colour phenotypes are not effectively transmitted back into wild populations. Therefore, this set of substitutions unequivocally differentiates wild from domestic animals, which will have important practical application in forensic cases involving the poaching of wild vicuñas and guanacos. These markers will also assist in identifying and studying archaeological remains pre- and post-domestication.

Landscape connectivity among remnant populations of guanaco (Lama guanicoe Müller, 1776) in an arid region of Chile impacted by global change.

Connectivity between populations plays a key role in the long-term persistence of species in fragmented habitats. This is of particular concern for biodiversity preservation in drylands, since water limited landscapes are typically characterized by little suitable habitat cover, high habitat fragmentation, harsh matrices, and are being rapidly degraded at a global scale. In this study, we modelled landscape connectivity between 11 guanaco Lama guanicoe populations in Chile's arid Norte Chico, a region that supports the last remnant coastal populations of this emblematic herbivore indigenous to South America. We produced a habitat suitability model to derive a regional surface resistance map, and used circuit theory to map functional connectivity, investigate the relative isolation between populations, and identify those that contribute most to the patch connectivity network. Predicted suitable habitat for L. guanicoe represented about 25% of the study region (i.e., 29,173 km2) and was heterogeneously distributed along a continuous stretch along the Andes, and discontinuous patches along the coast. As a result, we found that high connectivity current flows in the mid and high Andes formed a wide, continuous connectivity corridor, enabling connectivity between all high Andean populations. Coastal populations, in contrast, were more isolated. These groups demonstrate no inter-population connectivity between themselves, only with higher altitude populations, and for two of them, animal movement was linked to the effectiveness of wildlife crossings along the Pan-American highway. Our results indicate that functional connectivity is an issue of concern for L. guanicoe in Chile's Norte Chico, implying that future conservation and management plans should emphasize strategies aimed at conserving functional connectivity between coastal and Andean populations, as well as the protection of habitat patches likely to act as stepping stones within the connectivity network.

The Mitochondrial Genome of the Guanaco Louse, Microthoracius praelongiceps: Insights into the Ancestral Mitochondrial Karyotype of Sucking Lice (Anoplura, Insecta).

Fragmented mitochondrial (mt) genomes have been reported in 11 species of sucking lice (suborder Anoplura) that infest humans, chimpanzees, pigs, horses, and rodents. There is substantial variation among these lice in mt karyotype: the number of minichromosomes of a species ranges from 9 to 20; the number of genes in a minichromosome ranges from 1 to 8; gene arrangement in a minichromosome differs between species, even in the same genus. We sequenced the mt genome of the guanaco louse, Microthoracius praelongiceps, to help establish the ancestral mt karyotype for sucking lice and understand how fragmented mt genomes evolved. The guanaco louse has 12 mt minichromosomes; each minichromosome has 2-5 genes and a non-coding region. The guanaco louse shares many features with rodent lice in mt karyotype, more than with other sucking lice. The guanaco louse, however, is more closely related phylogenetically to human lice, chimpanzee lice, pig lice, and horse lice than to rodent lice. By parsimony analysis of shared features in mt karyotype, we infer that the most recent common ancestor of sucking lice, which lived ∼75 Ma, had 11 minichromosomes; each minichromosome had 1-6 genes and a non-coding region. As sucking lice diverged, split of mt minichromosomes occurred many times in the lineages leading to the lice of humans, chimpanzees, and rodents whereas merger of minichromosomes occurred in the lineage leading to the lice of pigs and horses. Together, splits and mergers of minichromosomes created a very complex and dynamic mt genome organization in the sucking lice.

Guanaco abundance and monitoring in Southern Patagonia: distance sampling reveals substantially greater numbers than previously reported.

BACKGROUND: Guanacos (Lama guanicoe) are thought to have declined in Patagonia mainly as a result of hunting and sheep ranching. Currently accepted estimates of total population size are extrapolated from densities obtained through strip transects in local studies. We used road surveys (8,141 km) and distance sampling to estimate guanaco density and population size over major environmental gradients of Santa Cruz, a large region in southern Patagonia. We also calculated the survey effort required to detect population trends in Santa Cruz. RESULTS: We found considerable spatial variation in density (1.1 to 7.4 ind/km2), with a mean value of 4.8 ind/km2, which is more than twice the mean value guessed for central and northern Patagonia. Consequently, guanaco numbers in Santa Cruz were estimated at 1.1 million individuals (95% CI 0.7 to 1.6), which almost doubles current estimates of guanaco population size in South America. High guanaco abundance was found in arid lands, overgrazed and unable to support profitable sheep stocks. Detecting a 50% change in guanaco population size over a 10-year period requires substantial monitoring effort: the annual survey of between 40 and 80 30-km transects, which becomes up to 120 transects if trends are to be detected over 5 years. CONCLUSIONS: Regional patterns in guanaco density can only be detected through large-scale surveys. Coupling these surveys with distance sampling techniques produce robust estimates of density and its variation. Figures so obtained improve currently available estimates of guanaco population size across its geographic range, which seem to be extrapolated from strip counts over small areas. In arid lands degraded by sheep overgrazing, sustainable use of guanaco populations would help harmonize guanaco conservation, socio-economic progress of rural areas, and eventually the restoration of shrub-steppes.

Macroscopic and histological investigation of guanaco footpads (Lama guanicoe, Müller 1776).

The surface of guanaco footpads is characterized by hairless skin with up to 4-mm-thick stratum corneum that protects from abrasion. The horny layer is pliable and elastic, and ensures firm contact with irregular ground. It is padded with a particular structure of the subcutaneous layer, the digital cushion. The flat cushions of each of the two digits are of elongated ovate shape, each about 45-mm long, up to 20-mm wide, and 8-mm thick. The cushions are lined by a 1-2-mm-thick capsule that resembles a tunica albuginea. The capsule consists of coarse collagen fibers, with elastic fibers absent. The cushion capsule and dermis approach each other, and fuse along a line that runs parallel to the longitudinal axes of cushion and digit. Loose connective tissue rich in elastic fibers and acidic glycosaminoglycans separates dermis and cushion capsule lateral to the narrow interconnecting zone. The cushion capsule encloses cloudy yellowish, gelatinous material. Microscopy shows bundles of elastic fibers in abundant mucinous matrix. Tightly gathered elastic bundles adjoin the inner surface of the capsule. Rough cords of elastic fibers branch out from there and traverse to the opposite side. The cushion is pressed flat, and elastic fibers are stretched when bearing weight. With relief of load, elastic fibers contract and reset the cushion's shape. Contractile cells are absent. A resistant capsule and easily malleable mucinous contents establish the functioning as a gel pad. Mucinous connective tissue between elastic fiber bundles contains abundant basophilic matrix. Hyaluronan, chondroitin sulfate, and dermatan sulfate are main matrix constituents. Spindle-shaped or stellate fibroblasts contain vimentin, S100 protein, and neuron specific enolase. Moprhology, staining characteristics and synthesis activities of these cells meet the criteria to be classified as myxoid cells. The connective tissue in guanaco digital cushions represents myxoid tissue.

Assessing the effect of radiocollars on juvenile guanaco survival.

To evaluate the potential impact of our research activities on guanacos (Lama guanicoe), we tested for effects of radiocollaring on juvenile (≤1 year of age) survival in Torres del Paine National Park, Chile during 1992-1996. The survival of collared (40.1%) and uncollared (38.2%) juveniles was not significantly different (G=0.08; P=0.77). Our results suggest that radiocollaring does not adversely affect the survival of juvenile guanacos. Although we observed no effect of radiocollaring, we still underline the importance of testing for effects of radiocollars in other wildlife investigations when feasible.

Density but not climate affects the population growth rate of guanacos ( Lama guanicoe) (Artiodactyla, Camelidae).

We analyzed the effects of population density and climatic variables on the rate of population growth in the guanaco ( Lama guanicoe), a wild camelid species in South America. We used a time series of 36 years (1977-2012) of population sampling in Tierra del Fuego, Chile. Individuals were grouped in three age-classes: newborns, juveniles, and adults; for each year a female population transition matrix was constructed, and the population growth rate (λ) was estimated for each year as the matrix highest positive eigenvalue. We applied a regression analysis with finite population growth rate (λ) as dependent variable, and total guanaco population, sheep population, annual mean precipitation, and winter mean temperature as independent variables, with and without time lags. The effect of guanaco population size was statistically significant, but the effects of the sheep population and the climatic variables on guanaco population growth rate were not statistically significant.