Camelid Species Research Articles

Abstract 3853: TAS266, a novel tetrameric nanobody agonist targeting death receptor 5 (DR5), elicits superior antitumor efficacy than conventional DR5-targeted approaches

Abstract Preferential induction of apoptosis in cancer cells has been the objective of therapeutic strategies targeting apoptotic pathways. To this end, multiple therapeutic agonists of Death Receptors 4 and 5 (DR4, DR5), have been developed and are under clinical evaluation. Although these agonists, including antibodies and soluble ligand TRAIL, demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation for the discrepant pre-clinical and clinical results is that DR5 may play a more prominent role in in vitro model systems as opposed to cancers in humans. Alternatively, these results might indicate that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients. In particular, naturally dimeric antibody agonists require secondary cross-linking via Fcα receptors expressed on immune cells present in the tumor microenvironment to achieve optimal clustering of DR5 into a ternary active state. Because immune cell content in the tumor can be heterogeneous, reliance on this secondary mechanism for activity may limit the potency of these antibodies. To overcome this limitation, a novel nanobody approach was taken to eliminate the need for cross-linking and improve receptor activation with the goal of generating a significantly more potent DR5 agonist. Nanobodies are a class of therapeutic proteins based on single, high affinity heavy chain domain (VHH) antibodies that naturally occur in camelid species, and these VHH domains can be linked to form multivalent structures (di-, tri-, tetra-, etc). This approach led to the development of a tetrameric DR5 targeted agonist, TAS266, with significantly greater avidity for DR5 binding. TAS266 activates downstream caspases with more rapid kinetics and is up to 1000-fold more potent in cell death assays when compared to a cross-linked DR5 antibody or TRAIL. In vivo, TAS266 elicits single dose tumor regressions in multiple tumor xenograft models and sustained tumor regressions after treatment cessation. TAS266 showed superior anti-tumor activity compared to a DR5 agonist antibody and TRAIL, including the ability to induce tumor regression in a patient-derived primary pancreatic tumor model that is insensitive to the agonist antibody. Thus, TAS266 has the potential for superior clinical activity in settings insensitive to the conventional therapeutic approaches to DR5. First-in-man trials are expected to begin in 2012. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3853. doi:1538-7445.AM2012-3853

A long life among ruminants: giraffids and other special cases

In order to investigate differences in the relative maximum longevity and other life history parameter between ruminant species, we collated data on mean body mass, maximum longevity, gestation period and newborn mass in wild ruminant and camelid species. Among ruminants, giraffids (giraffe Giraffa camelopardalis and okapi Okapia johnstoni) have particularly high longevities, long gestation periods, and low intrauterine growth rates. A particularly high absolute and relative longevity is also achieved by the anoa (Bubalus depressicornis), a member of the bovinae (cattle-type ruminants) and an insular dwarf (inhabiting the Indonesian island of Sulawesi). The fact that some (but not all) other small ruminants also achieve surprisingly high longevities leads to the hypothesis that extreme relative longevities in this group are an indication for secondary body size reduction.

Genetic parameters for growth of fiber diameter in alpacas1

The alpaca is the most important fiber producer of the South American camelid species, and is an important source of income for the Andean communities. Nowadays, fiber diameter is considered the main selection objective in alpaca populations throughout the world. However, fiber diameter increases with the age of the animals, and it would be preferable to select those animals that maintain a thin fiber throughout their life span. The goal of this study was to describe the genetic relationship between fiber diameter at weaning age (6 mo) and the evolution of fiber diameter along the life span. The analysis of the evolution of fiber diameter was studied as a useful model for canalization and as a longitudinal trait by hierarchical Bayesian analysis. The results suggested that substantial genetic variation exists for fiber diameter and also for the variability and linear growth of the fiber diameter. Thus, a genetic selection program is plausible to modify the evolution of fiber diameter with time, together with a favorable correlated decrease in fiber diameter.

Serosurvey of zoo ungulates in central Europe

In total, 926 individuals representing 69 different species of bovids, cervids and camelids from one Czech and ten German zoos were tested for antibodies against five viral and three bacterial pathogens that can be transmitted across species borders. In addition, the archived veterinary data of all bovids, cervids and camelids, kept at eight of the 11 zoos between 1998 and 2004, were reviewed. The results demonstrate that both malignant catarrhal fever and Chlamydophila psittaci were endemic in zoo ruminants in Germany. Epidemiologic factors are discussed that might have a qualitative implication for cross-species transmission within zoos. The factors included are population size, population density and exhibit type (single species/mixed species).

113 EFFECT OF SOME FACTORS ON CONCEPTION RATE IN ALPACAS UNDER PERUVIAN HIGHLAND CONDITION

Alpacas are a domestic species of South American camelids with a great importance to highland people because of their high-quality fibre production. However, their reproductive performance has been reported to be poor, with a birth rate of 50% under the Andean community’s conditions. Two experiments were designed to evaluate the effect of some factors on the pregnancy rate of alpacas at first service in lactating, 3- to 8-years old, without parity problems. The objective of the first experiment (n = 85) was to evaluate the effect of age (G1: 3, G2: 4 to 5, G3: ≥ 6 years old); mating time (G1: 15 min, G2: 16–24 min, G3: ≥ 25 min), and interval from parturition to mating (G1: 20 days, G2: ≥ 20 days) on first service conception rate. A second experiment (n = 174) evaluated the effect of month of calving (January, February, or March) on conception rate in females with a postpartum interval ≥20 days. Animals were mated with male of good fertility after a receptivity test and confirmation of presence of a dominant follicle ≥7 mm by ultrasonography. Pregnancy was determined by ultrasonography 25 days after mating. Proportional data were compared by Fisher’s exact test. In the first experiment, conception rates were 57.9, 66.7, and 47.2% in females of G1, G2, and G3, respectively (P ≤ 0.05); 50.0, 54.5, and 59.0% in females with mating time of 15, 16–24, or ≥ 25 min of mating; 48.0 and 59.4% in those with a postpartum interval <20 days and ≥20 days (P ≤ 0.05). In the second experiment, conception rates were 58.3, 70.7, and 82.1% in alpacas calving in January, February, and March, respectively (P ≤ 0.05). The results suggest that age of female, postpartum interval, and month of calving are factors that affect conception rates in alpacas. Therefore, a reproductive management system that includes these factors would improve the pregnancy rate in alpacas under highland Peruvian conditions. Consejo Superior de Investigacion – UNMSM.

Ovarian function in South American camelids (alpacas, llamas, vicunas, guanacos)

Ultrasound technology and hormone assays have provided a better understanding of folliculogenesis and ovulation in South American camelids in the last two decades. Females exhibit waves of ovarian follicular growth and are induced ovulators and therefore do not exhibit oestrous cycles in the manner of spontaneously ovulating species such as sheep and cattle. There is much variation in inter-wave interval among camelid species (alpaca/llama 10–22 days, vicuna 4–11 days), within species and within individual animals as the range of each phase of follicular growth is wide. Ovulation occurs 24–30 h after mating and luteolysis occurs approximately 10 days later if conception fails to occur.

From Marvelous Antidote to the Poison of Idolatry: The Transatlantic Role of Andean Bezoar Stones During the Late Sixteenth and Early Seventeenth Centuries

Abstract Introducing intertwined themes of profit, the hunt for treasure, and the excavation of camelid bodies; and drawing from the analysis of Renaissance medical books, bilingual Aymara–Spanish and Quechua–Spanish dictionaries, inquisition records, and inventories of curiosity cabinets, this essay considers how Peruvian bezoar stones acquired transatlantic importance following their discovery in 1568 by a Spanish soldier. In spite of its less-than-glamorous physiological genesis as a calcinated concretion formed in the digestive tract of ruminants, including the four species of Andean camelids, the bezoar stone played a significant yet academically overlooked role in the social and economic history of modern Europe and Spanish America for its use as an antidote to poisons, and the stones constituted one of the most sought-after objects for the fashionable cabinets of curiosities belonging to Europe’s powerful elites. However, for indigenous pastoral peoples, bezoars were central to the reproduction of native cultural practices and directly linked to the foundational myths of Andean cosmology. The stones were believed to protect the herds and the shepherds, for whom the camelids represented the primary source of wealth. Consequently, the bezoar stone takes on unforeseen significance as a neglected site where the colonial drama of competing epistemologies was enacted.

Osteometría y genética de los camélidos mochica, costa norte del Perú

El objetivo de este trabajo es evaluar los resultados de la osteometría a dos muestras óseas de camélidos de sitios de la época Mochica (siglos I y VII d.C.) en la costa norte de Perú. Los huesos analizados en este estudio son primeras falanges que provienen de dos sitios arqueológicos Mochica (Cerro Chepén y Zona Urbana Moche). Se realizaron análisis discriminantes a una muestra de 44 primeras falanges, tomando en cuenta las cinco variables que son medidas en este hueso. Los resultados obtenidos mostraron la identificación de dos especies de camélidos domésticos en ambos sitios, Lama glama “llama” y Lama pacos “alpaca”. La interpretación de los resultados es discutida mediante la explicación de un modelo de especiación geográfica o vicariante de los camélidos que habitaron la costa peruana en la época Mochica. Este modelo toma en cuenta que actualmente en esta ecología no hay crianza de camélidos, así como factores genéticos y evolutivos de una nueva forma doméstica, incluyendo el efecto de la Regla de Bergmann y los nuevos pastos en la morfología de esta nueva forma de camélido doméstico que vivió en la época Mochica.

308 GUANACO SPERM CHROMATIN EVALUATION USING TOLUIDINE BLUE

Guanacos, a wild species of South American camelids, have a high-quality fiber with great economic potential. To evaluate reproductive aptitude in guanacos, our laboratory has developed a reliable semen collection technique using electroejaculation and has applied various methods for evaluating semen characteristics. Studies for evaluating the state of sperm chromatin have also been initiated. Toluidine blue (TB) is a cationic stain that unites with the phosphate groups in the DNA, thus permitting differentiation between sperm heads according to the degree of chromatin decondensation. The objectives of this study were to determine the TB staining patterns of guanaco sperm chromatin, establish a positive control for the stain, and evaluate the effect of collagenase on sperm chromatin condensation. Semen was collected from 4 guanacos, between 6 and 9 years old, using electroejaculation. In Experiment 1, to establish a positive control for the stain, equal quantities of 1% dithiothreitol (DTT) and raw semen were incubated at room temperature for 30 s, 1.5 min, and 3 min. After incubation, smears were made and then dried, to avoid continuing the reaction, and finally were stained with 0.02% TB. A split-plot design was used with time as the splitting factor and considering the males as a block. In Experiment 2, raw semen was divided into 2 aliquots, one diluted 4 : 1 in 0.1% collagenase in HEPES-TALP-BSA medium and the other left without enzyme. Both aliquots were incubated 4 min at 37°C and, after centrifugation to remove the enzyme, smears were made and stained with TB. Spermatozoa were classified according to the degree of chromatin decondensation. Analysis of variance was performed using the males as a blocking factor and the treatment as a fixed factor. According to the degree of chromatin decondensation, three patterns of staining with TB were observed: light blue (negative, without alteration of chromatin condensation), light violet (intermediate, some degree of decondensation), and dark violet (positive, high degree of decondensation). A significant increase (P < 0.05) of sperm with highly decondensed chromatin was observed in semen incubated for 3 min with DTT when compared to 30 s of incubation. Therefore, 3 min of incubation with DTT was chosen as the positive control for Experiment 2. No significant differences in any of the 3 patterns of TB staining were observed between semen incubated with or without 0.1% collagenase. In conclusion, it is possible to use TB to evaluate the degree of chromatin decondensation in guanaco spermatozoa and to use DTT as a positive control for the stain. Treatment of guanaco semen with 0.1% collagenase did not affect sperm chromatin condensation; therefore, this enzyme can be used to decrease semen viscosity and aid handling in the laboratory.

A one‐step exclusion‐binding procedure for the purification of functional heavy‐chain and mammalian‐type γ‐globulins from camelid sera

A new approach has recently been proposed for the purification of 'mammalian-type' IgG, consisting of exclusion binding. The technique uses a gel ('Melon gel'; Pierce) that binds to all plasma proteins, but not to IgGs, thus allowing IgGs to be recovered in the FT (flow-through) fraction. Here, the technique was applied to camelid IgGs, which are known to be composed of not only classic mammalian-type IgGs (IgG1) but also HC-IgGs (heavy chain IgGs). Both mammalian type and HC-IgGs can be purified in the FT fraction of dromedary (Camelus dromedarius) plasma samples with less than 8.5% contamination, by making minor improvements to the conditions recommended by the manufacturer. The contaminant proteins, as determined by LC-MS/MS (liquid chromatography-tandem MS), are mainly transferrin and albumin. The recovery rate is elevated for both types of IgGs (95+/-14% and 88+/-25% for IgG1 and HC-IgGs respectively). IgGs thus purified maintain their ability to bind to their antigen, as measured by surface plasmon resonance and Western ligand blotting. Furthermore, IgGs can be purified from plasma samples of all camelid species in a similar manner, although the ratio of HC-IgGs to total IgGs was lower for Lama (llama) and Vicugna (vicuña) than for Camelus species. The 'Melon gel' technique can thus be used to satisfactorily purify IgG1 and HC-IgGs from all camelid species.

Ovarian follicular dynamics and hormonal secretory profiles in guanacos ( Lama guanicoe)

The objective of the present study was to describe ovarian activity in 11 adult non-mated guanacos, evaluated every second day for 40 days by transrectal ultrasonography and by plasma estradiol-17β and progesterone concentrations. An inverse relationship ( r = −0.29, P < 0.001) was observed between the diameter of the largest ovarian follicle and the total number of follicles indicating that follicular growth in guanacos occurs in waves. The mean duration of follicular wave was 15.1 ± 4.2 days with a range from 9 to 26. The follicular growth phase was 7.0 ± 2.4 days, the static phase 3.0 ± 1.2 days, the regression phase 5.2 ± 2.1 days and the inter-wave interval was 12.6 ± 5.6 days. The maximum follicular diameter in each follicular wave was 10.2 ± 2.1 mm with a range from 7.2 to 16.1 mm. Inter-wave intervals of longer duration were associated with a larger maximum follicle diameter ( P < 0.001). Follicular activity alternated between ovaries in 93% of the waves with 48% of dominant follicles occurring in the left and 52% in the right ovary without differences ( P > 0.05). Plasma estradiol-17β concentrations showed a wave-like pattern, varying from 20.0 to 92.1 pmol/L. Plasma progesterone concentrations remained below 1 nmol/L without any ultrasonic evidence of ovulation during the study. These results in guanacos suggest a follicular wave pattern more similar to the llama ( Lama glama) than previously described in other South American and Old World camelid species.

Lamanema chavezi (Nematoda: Molineidae): Epidemiological data of the infection in South American camelids of Northwest Argentina

Faecal samples from llamas ( n = 708), vicuñas ( n = 171) and guanacos ( n = 4) were obtained between December 2004 and May 2009 in three Provinces of Northwest Argentina (Jujuy, Salta and Catamarca) to know the distribution, prevalence and intensity of Lamanema chavezi infection in these South American camelid species (SACs). Faeces were examined by a sedimentation-flotation technique using a Cl 2Zn + ClNa solution (specific gravity = 1.59). Eggs of L. chavezi occurred in 30.3% of 89 llama herds and in 18.5% of 708 llamas sampled with a mean intensity of 271.8 eggs/g (EPG) of faeces (range 20–2120). The highest values for all parameters of the infection were registered in llamas from Catamarca Province. Significant differences ( P < 0.001, Fisher's exact test) were detected only for the lower prevalence in llamas from Jujuy respect to those from the other two Provinces. The overall individual prevalence of L. chavezi in llamas was lower than in reports from adult domestic camelids of neighbour countries while mean intensity was higher. The individual prevalence of L. chavezi in guanacos was 75.0%, with a mean intensity of 66.0 EPG (range 40–120) while no vicuñas were detected as infected. Most of infected SACs were located at the phytogeographical region of Andean Patagonic Domain with a dispersion ranging between 22°10′ and 26°40′ South latitude.

Identification of camelid specific residues in mitochondrial ATP synthase subunits

ATP synthase is an enzyme involved in oxidative phosphorylation from prokaryotic to eukaryotic cells. In mammals it comprises at least 16 subunits from which the mitochondrial encoded ATP6 and ATP8 are essential. Mitochondrial genes variations have been suggested to allow rapid human and animal adaptation to new climates and dietary conditions (Mishmar et al. 2003). Camelidae taxa are uniquely adapted to extremely hot and dry climates of African-Asian territories and to cold and hypoxic environments of the South American Andean region. We sequenced and analyzed ATP6 and ATP8 genes in all camelid species. Based on the available structural data and evolutionary conservation of the deduced proteins we identified features proper of the group. In Old World camels the ATP8, important in the assembly of the F0 complex, showed a number of positively charged residues higher than in the other aligned species. In ATP6 we found the camelid specific substitutions Q47H and I106V that occur in sites highly conserved in other species. We speculate that these changes may have functional importance.

Verocytotoxigenic Escherichia coli O157 in animals on public amenity premises in England and Wales, 1997 to 2007

At the request of the public health authorities, 31 public amenity premises in England and Wales containing animals of various species were investigated for the presence of verocytotoxigenic Escherichia coli...

The giraffe (Giraffa camelopardalis) cervical vertebral column: a heuristic example in understanding evolutionary processes?

The current study considers the osteological morphology of the giraffe (Giraffa camelopardalis) vertebral column, with emphasis on evaluating both the adaptive and constraining features compared with other ungulates as a heuristic example in understanding evolutionary processes. Vertebral columns of giraffes varying in age from calf to adult were studied in order to understand the potential evolutionary scenarios that might have led to the modern phenotype. Data from the giraffe sample were then compared with the results from several other ungulate species, including the okapi and two species of camelids that also have visibly elongated necks. Our results show that the elongated neck of the modern giraffe appears to specifically result from evolutionary changes affecting the seven cervical vertebrae, independent of the remainder of the vertebral column. The cervical vertebrae comprise over half of the length of the total vertebral column in the giraffe. The increases in cervical vertebrae lengths also appear to be allometrically constrained, with alterations in the overall length of the neck resulting from the elongation of the entire cervical series, rather than from a single vertebra or subset of vertebrae. We place our results in the context of hypotheses concerning the origin and evolution of the giraffe neck, and the evolution of long necks in a broader sense. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155, 736–757.

Artificial insemination in South American camelids and wild equids

An overview of the present status of the use of artificial insemination (AI) in South American camelids and wild equids is offered. Technical aspects of semen collection, dilution and cryopreservation have limited the development and use of AI in camelid and equid species. To-date, efficiency is low but progress has been made and viable offspring have been produced through the use of AI in domestic South American camelids using both fresh and frozen semen. The origin, composition, and function of the viscous component of camelid seminal plasma remain a mystery and an obvious area for future research. A better understanding of the normal constituents of seminal plasma will enable the rational design of semen extenders suitable for camelids. Post-thaw sperm viability is very low, and studies are needed to address questions of optimal freezing and thawing procedures as well as the insemination dose. The basis for differences in reported pregnancy rates with sexed and frozen semen in domestic equids, and the ultimate success of AI in wild equids will require continued research into the “stallion effect”, extenders and cryoprotectants, optimal volume and number of spermatozoa, temperatures during handling, processing an transport, and insemination techniques. In both camelids and equids, research on domestic species under controlled conditions provides and excellent opportunity to develop effective semen handling techniques for application in wild and endangered species of the respective families.

Measuring the energy expenditure and water flux in free‐ranging alpacas (Lama pacos) in the peruvian andes using the doubly labelled water technique

Energy expenditure and water flux were measured in free-ranging alpacas Lama pacos, a South American camelid, on natural pastures of the Peruvian Andes (altitude: 4,400 m above sea level). Water influx rate (WIR) was estimated in 16 males (age 2 years, weight 48.5+/-8.6 kg) labelled with 2H. In addition, the field metabolic rate (FMR) was measured in four of these animals labelled with both an oxygen (18O) and a hydrogen (2H) isotope. The WIR averaged 3.62 L H2O/day and the mean total body water 33.1 kg, equal to 68.2% of body weight (BW). The FMR of the four doubly labelled animals was 14.05 MJ/day. New allometric equations were calculated describing the relationships between WIR or FMR and BW, respectively, including published data on ruminants and the present alpaca results. The regression equation indicates that daily WIR scales to a similar metabolic size (kilograms of BW(0.94)) in alpacas than in wild or domesticated ruminants and camelids originating from arid and semiarid habitats. The resulting regression equation for FMR explained over 99% of the variation and corresponded to the function FMR (kilojoules per day)=1079 (kilograms of BW0.668) (n=5, r2=0.995, P<0.001). The FMR measured in this study, the first reported for a South American camelid species, suggests that free-ranging alpacas have similar energy expenditures on a metabolic weight basis as other wild ruminants living under harsh climatic conditions.

Late Holocene camelid use tendencies in two different ecological zones of Northwestern Argentina

The human–camelid relation can be traced from the end of the Pleistocene to the present time. During that time span that relations changed transforming from merely extractive techniques to productive ones (e.g. herding). In order to recognize the camelid use tendencies within the late productive economies, archaeofaunal assemblages from southern Calchaquíes valleys are compared with other collections from the higher Argentinean Puna (Northwestern Argentina). Some possible signs of distinctive economical use of camelids between these two zones are discussed, as expressed in the archaeofaunal record of the Late Holocene. Three periods were observed. The earliest period shows for both zones the use of three camelid species ( Lama glama, Lama guanicoe and Vicugna vicugna) with the presence of more adult remains than subadults. The second period (Middle) shows more variability with less biodiversity in Puna and some regions of lower valleys while proportions of adults and subadults for Puna are the inverse of those during the earliest period. For the Late period the economy shows for Puna the presence of adult camelids while the subadults still are predominant in certain localities. Valley records do not register major changes during the three periods in age classes and species occurrence. The presence of two camelid use models in the region is established.

Cross-species chromosome painting among camel, cattle, pig and human: further insights into the putative Cetartiodactyla ancestral karyotype

The great karyotypic differences between camel, cattle and pig, three important domestic animals, have been a challenge for comparative cytogenetic studies based on conventional cytogenetic approaches. To construct a genome-wide comparative chromosome map among these artiodactyls, we made a set of chromosome painting probes from the dromedary camel (Camelus dromedarius) by flow sorting and degenerate oligonucleotide primed-PCR. The painting probes were first used to characterize the karyotypes of the dromedary camel (C. dromedarius), the Bactrian camel (C. bactrianus), the guanaco (Lama guanicoe), the alpaca (L. pacos) and dromedary x guanaco hybrid karyotypes (all with 2n = 74). These FISH experiments enabled the establishment of a high-resolution GTG-banded karyotype, together with chromosome nomenclature and idiogram for C. dromedarius, and revealed that these camelid species have almost identical karyotypes, with only slight variations in the amount and distribution patterns of heterochromatin. Further cross-species chromosome painting between camel, cattle, pig and human with painting probes from the camel and human led to the establishment of genome-wide comparative maps. Between human and camel, pig and camel, and cattle and camel 47, 53 and 53 autosomal conserved segments were detected, respectively. Integrated analysis with previously published comparative maps of human/pig/cattle enabled us to propose a Cetartiodactyla ancestral karyotype and to discuss the early karyotype evolution of Cetartiodactyla. Furthermore, these maps will facilitate the positional cloning of genes by aiding the cross-species transfer of mapping information.

Sistemática, taxonomía y domesticación de alpacas y llamas: nueva evidencia cromosómica y molecular

Four camelid species exist in South America: two wild, the guanaco (Lama guanicoe) and the vicuna (Vicugna vicugna), and two domestic, the alpaca (Lama pacos) and the llama (Lama glama). However, the origin of the domestic species has been a matter of debate. In the present study, variations in chromosome G banding patterns and in two mitochondrial gene sequences have been used to study the origin and classification of the llama and alpaca.-Similar patterns in chromosome G band structure were observed in all four Lamini species, and these in turn were similar to the bands described for camels, Camelus bactrianus. However, fine and consistent differences were found in the short arms of chromosome 1, separating camels, guanacos and llamas from vicunas and alpacas. This pattern was consistent even in a hybrid guanaco x alpaca. Equivalent relationship showed the complete cytochrome b gene sequences, and the minimum expansion tree of the partial control region sequence, grouping guanaco with llama and vicuna with alpaca. Phylogenetic analyses showed V. vicugna and L. guanicoe as monophyletic groups. Analysis of both gene sequences revealed two clades within vicuna, concordant with the two described subspecies, but the results for guanaco did not confirm existence of the four previously proposed subspecies. The combined analysis of chromosomal and molecular variation showed close genetic similarity between alpacas and vicunas, as well as between llamas and guanacos. Although directional hybridization was revealed, our results strongly support the hypothesis that the llama would have derived from L. guanicoe and the alpaca from V. vicugna, supporting reciassification as V. pacos.