Genetic identification of hybrid camelids

North American zoos in 1984 had the first opportunity in many years to obtain South American camelids after a long embargo on imports. The Rio Grande Zoo in Albuquerque, New Mexico, purchased two alpacas and one presumptive llama during this period. The llama, however, appeared to be phenotypically intermediate between a llama and an alpaca. In an attempt to ascertain the identity of this animal, it and purebred individuals of llama and alpaca were compared at 22 presumptive genic loci using starch-gel electrophoresis. Genic differences between llama and alpaca suggest this animal to be a hybrid. If further tests prove consistent with the findings of this study, this technique will provide a simple assay using easily obtained blood for identifying llama and alpaca. The use of genetic management techniques such as this in zoos holds great potential for helping to preserve pure breeding lines of closely related interfertile animals.     

DNA composition in South American camelids I. Characterization and in situ hybridization of satellite DNA fractions

The DNA composition and the in situ hybridization of satellite fractions were analysed in the New World camelids llama, alpaca, guanaco and vicuña. In the four camelid forms, it was possible to identify a similar main band DNA and five satellite fractions (I–V) with G+C base contents ranging from 32% to 66%. Satellites II–V from llama were in situ reannealed on chromosomes from the four camelid forms. The results obtained were: (a) the four satellites hybridized with regions of C-banding (centromeric regions of all chromosomes and short arms of some autosomes); (b) in general, homologous hybridizations (llama DNA versus llama chromosomes) were more efficient than heterologous reassociations; there were however three exceptions to this rule (vicuña and alpaca satellite fraction II, chromosome group B; vicuña fraction V, chromosome groups A and B); (c) X chromosomes from the four camelids had satellites III–V but lacked satellite II, (d) no satellite fraction was detected on chromosome Y. The analysis of the in situ hybridization patterns allowed to conclude that most or all C-banded chromosome regions comprise several satellite DNA fractions. It is, moreover, proposed that there is an ample interspecies variation in the number of chromosomes that cross-react with a given satellite fraction. Our data give further support to the close genomic kinship of New World camelids.     

Genetic analysis reveals the wild ancestors of the llama and the alpaca.

The origins of South America's domestic alpaca and llama remain controversial due to hybridization, near extirpation during the Spanish conquest and difficulties in archaeological interpretation. Traditionally, the ancestry of both forms is attributed to the guanaco, while the vicu?a is assumed never to have been domesticated. Recent research has, however, linked the alpaca to the vicu?a, dating domestication to 6000-7000 years before present in the Peruvian Andes. Here, we examine in detail the genetic relationships between the South American camelids in order to determine the origins of the domestic forms, using mitochondrial (mt) and microsatellite DNA. MtDNA analysis places 80% of llama and alpaca sequences in the guanaco lineage, with those possessing vicu?a mtDNA being nearly all alpaca or alpaca-vicu?a hybrids. We also examined four microsatellites in wild known-provenance vicu?a and guanaco, including two loci with non-overlapping allele size ranges in the wild species. In contrast to the mtDNA, these markers show high genetic similarity between alpaca and vicu?a, and between llama and guanaco, although bidirectional hybridization is also revealed. Finally, combined marker analysis on a subset of samples confirms the microsatellite interpretation and suggests that the alpaca is descended from the vicu?a, and should be reclassified as Vicugna pacos. This result has major implications for the future management of wild and domestic camelids in South America.     

Trinucleotide Microsatellite Loci and Increased Heterozygosity in Cross-Species Applications in the Social Wasp, Polistes

Though microsatellite loci are usually found to be most polymorphic in the species in which they are first identified, we have found significant increases in polymorphisms in some cross-species applications. We present eight new trinucleotide microsatellite loci derived from two species of social wasps, Polistes annularis and Polistes bellicosus. We assessed the primers designed from these species and the degree of polymorphism in two additional species, P. dorsalis, which is very closely related to P. bellicosus, and P. dominulus, which is an Old World congener, thought to have diverged from New World Polistes over 80 million years ago. Cross-species applications for these microsatellite loci indicate that the priming sites from P. bellicosus loci are conserved in P. dorsalis and amplified similarly sized products with higher heterozygosities than the original species in two of three cases. A locus that was monomorphic in P. annularis had a heterozygosity of 1.0 in the distantly related P. dominulus. Cross-species applications of these loci indicated that alleles were generally of similar lengths in the new and original species when they retained their heterozygosity.     

Simple sequence repeats for genetic studies of alpaca

Trace sequences from the 2X alpaca genome sequencing effort were examined to identify simple sequence repeats (microsatellites) for genetic studies. A total of 6,685 repeat-containing sequences were downloaded from GenBank, processed, and assembled into contigs representing an estimated 4,278 distinct sequences. This sequence set contained 2,290 sequences of length > 100 nucleotides that contained microsatellites of length > or = 14 dinucleotide or 10 trinucleotide repeats with purity equal to 100%. An additional 13 sequences contained a GC microsatellite of length > or = 12 repeats (purity = 100%) were also obtained. Primer pairs for amplification of 1,516 putative loci are presented. Amplification of genomic DNA from alpaca and llama by PCR was demonstrated for 14 primer sets including one from each of the microsatellite repeat types. Comparative chromosomal location for the alpaca markers was predicted in the bovine genome by BLAT searches against assembly 4.0 of the bovine whole genome sequence. A total of 634 markers (41.8%) returned BLAT hits with score > 100 and Identity > 85%, with the majority assignable to unique locations. We show that microsatellites are abundant and easily identified within the alpaca genome sequence. These markers will provide a valuable resource for further genetic studies of the alpaca and related species.     

Polymorphic dinucleotide microsatellite loci in the sandfly Lutzomyia longipalpis (Diptera: Phlebotominae)

The sandfly Lutzomyia longipalpis, an important vector of visceral leishmaniasis in the New World, is believed to be a species complex. In an effort to better understand population dynamics and speciation in this vector we developed a panel of dinucleotide — (CA)_n— microsatellite loci using an enrichment technique. Eleven polymorphic loci that produced consistent allelic banding patterns were characterized using a laboratory population of L. longipalpis. These dinucleotide microsatellite loci were more polymorphic than trinucleotide microsatellites characterized in wild‐caught samples of two other sandfly species; the variability of these loci was unexpected because the laboratory flies were believed to be inbred.     

Tetranucleotide microsatellite loci from the striped dolphin (Stenella coeruleoalba Meyen, 1833)

Five tetranucleotide microsatellite loci were isolated from the striped dolphin (Stenella coeruleoalba). Characterization of these loci revealed between three and 11 alleles at each locus and observed heterozygosity ranged from 0.192 to 0.775. All sets of primers amplified homologous loci when tested on four related species and showed polymorphisms in most locus–species combinations. A sixth microsatellite, which appeared to be monomorphic in the striped dolphin, was also shown to be polymorphic in three related species.     

Heterologous amplification of microsatellite markers from colubroid snakes in European natricines (serpentes: natricinae)

Eighteen microsatellite loci developed for a range of snake species (New World natricines, elapids, crotalids) were tested against European natricines (Natrix natrix, N. maura, and N. tessellata) in cross-species amplification experiments. Five loci were polymorphic (average expected heterozygosity 0.749 for a population of N. natrix in Amsterdam, mean sample size 47.8) and three loci were monomorphic. The remainder could not be consistently scored or failed to amplify. Further tests on single individuals of a diverse set of eight species of colubroid snakes showed that 15 of the 18 loci could be cross-amplified in at least one of these species. We conclude that our results show promise for the utilization of these markers for experimental assessments of genetic variation in the phylogenetically closely related group of European natricine snakes with emphasis on N. natrix. The full suite of microsatellite markers now available for snakes may show additional potential for subsequent investigation across a broader range of colubroid snakes.     

Characterization of polymorphic microsatellite markers for the Carnaby's cockatoo (Calyptorhynchus latirostris) and related black cockatoo species

Microsatellite loci were isolated from Carnaby's black cockatoo (Calyptorhynchus latirostris: Aves), a highly valued, endangered, and endemic species of bird from Western Australia. This study describes three dinucleotide and one tetranucleotide microsatellite loci for which the primers produced clear and polymorphic amplification patterns with between two and nine alleles and moderate levels of variability. Two additional dinucleotide markers which were monomorphic in the Carnaby's cockatoo were able to amplify and were polymorphic in two other species of black cockatoo, greatly increasing the utility of these markers.     

Development of new polymorphic microsatellite markers for the New World screw‐worm Cochliomyia hominivorax (Diptera: Calliphoridae)

In this report, we describe the development of seven new polymorphic microsatellite markers for Cochliomyia hominivorax, a parasitic insect pest of primary agricultural and veterinary importance throughout the Neotropics. The number of alleles found ranged from 3 to 13 per locus, with the expected heterozygosity ranging from 0.4220 to 0.9045. The across‐taxa amplification of some of these new microsatellite loci was successful in four additional Calliphoridae species. In combination with the 10 polymorphic microsatellite markers previously described, the markers developed here should provide a high resolution for assessing the fine‐scale genetic structure of New World screw‐worms.     

Isolation and characterisation of polymorphic microsatellite loci in the vulnerable spectacled flying fox, Pteropus conspicillatus

The spectacled flying fox, Pteropus conspicillatus, is listed as vulnerable in Australia and is under threat from numerous impacts. Primers to amplify eight co-dominant microsatellite loci were designed for Pteropus conspicillatus, based on an enriched genomic library. Four loci were monomorphic in this species while the remaining four loci were highly polymorphic with 16–23 alleles. Two of the four monomorphic loci were found to be polymorphic in Pteropus alecto, a closely related congener. All but one of the six polymorphic loci were in Hardy Weinberg equilibrium. Additionally, six microsatellite loci isolated for Pteropus rodricensis were tested against individuals of P. conspicillatus with all loci amplifying reliably. These loci will be used to investigate population genetic structure in the vulnerable spectacled flying fox.     

Characterization of polymorphic microsatellite loci from Round Island Petrels (Pterodroma arminjoniana) and their utility in other seabird species

Six microsatellite loci were isolated from the petrels of Round Island, near Mauritius in the Indian Ocean (Pterodroma arminjoniana). Three loci were monomorphic in P. arminjoniana but were found to be polymorphic in other Procellariiforms. Cross-utility of all six loci was tested in 17 Procellariiform and 1 penguin species. In addition, 53 microsatellite loci developed for other species of birds were tested for cross-species amplification in P. arminjoniana. Six of these loci were found to be polymorphic.     

Isolation and characterization of 10 polymorphic dinucleotide microsatellite markers for llama and guanaco

We isolated and characterized five polymorphic dinucleotide microsatellite markers from llama (Lama glama) and five from guanaco (Lama guanicoe). All loci were assayed on wild llamas and guanacos from Argentina, as all of the primers were able to amplify in both species.     

Development of microsatellite markers for muskellunge (Esox masquinongy) and cross‐species amplification in two other esocids

We report the development of seven polymorphic microsatellite loci in muskellunge (Esox masquinongy) using an unenriched subgenomic library. Polymorphic loci exhibited 2–11 alleles with observed heterozygosities ranging from 0.190 to 0.917 (n = 24). All seven loci amplified by their respective primer pairs resulted in monomorphic products in northern pike (E. lucius) whereas three loci amplified monomorphic products in grass pickerel (E. americanus americanus); these results imply conservation of flanking sequence but loss of polymorphism between these closely related species. Only one of six microsatellite primers developed in a previous study in northern pike amplified polymorphic products in muskellunge.     

Speciation is not necessarily easier in species with sexually monomorphic mating signals

Should we have different expectations regarding the likelihood and pace of speciation by sexual selection when considering species with sexually monomorphic mating signals? Two conditions that can facilitate rapid species divergence are Felsenstein's one‐allele mechanism and a genetic architecture that includes a genetic association between signal and preference loci. In sexually monomorphic species, the former can manifest in the form of mate choice based on phenotype matching. The latter can be promoted by selection acting upon genetic loci for divergent signals and preferences expressed simultaneously in each individual, rather than acting separately on signal loci in males and preference loci in females. Both sexes in the Chrysoperla carnea group of green lacewings (Insecta, Neuroptera, Chrysopidae) produce sexually monomorphic species‐specific mating signals. We hybridized the two species C. agilis and C. carnea to test for evidence of these speciation‐facilitating conditions. Hybrid signals were more complex than the parents and we observed a dominant influence of C. carnea. We found a dominant influence of C. agilis on preferences in the form of hybrid discrimination against C. carnea. Preferences in hybrids followed patterns predicting preference loci that determine mate choice rather than a one‐allele mechanism. The genetic association between signal and preference we detected in the segregating hybrid crosses indicates that speciation in these species with sexually monomorphic mating signals can have occurred rapidly. However, we need additional evidence to determine whether such genetic associations form more readily in sexually monomorphic species compared to dimorphic species and consequently facilitate speciation.     

Isolation and characterization of 27 polymorphic microsatellite loci for the common snook,Centropomus undecimalis

Here we describe 32 di‐, tri‐ and tetranucleotide microsatellite loci isolated by PIMA, a polymerase chain reaction (PCR)‐based procedure, for the common snook (Centropomus undecimalis). Five loci were monomorphic, and the remaining loci averaged 6.7 alleles per locus in a sample of 45 common snook. For polymorphic loci, expected heterozygosities ranged from 0.02 to 0.91 (mean = 0.538). Significant departures from Hardy–Weinberg equilibrium expectations occurred in two loci. Exact tests for genotype disequilibrium gave evidence for linkage at one pair of loci. Many cross‐species primer assays yielded PCR fragments of the expected size for 11 species of Centropomus and two species of the confamilial genus Lates.     

Isolation and characterization of 15 microsatellite loci from mango (Mangifera indica L.) and cross‐species amplification in closely related taxa

We report here on the development and characterization of 15 microsatellite loci isolated from Mangifera indica L. These markers were evaluated using 59 Florida cultivars and four related species from the USDA germplasm collection for mango. Two loci were monomorphic and 13 polymorphic, with two to seven alleles per locus. Four loci departed significantly from Hardy–Weinberg equilibrium and have significant heterozygote deficiency. Nine loci exhibited significant linkage disequilibrium. Cross‐species amplification was successful in four related species. These loci are being used to investigate patterns of genetic variation within M. indica and between closely related species.     

Oxygen binding properties,capillary densities and heart weights in high altitude camelids

Summary The oxygen binding properties of the blood of the camelid species vicuna, llama, alpaca and dromedary camel were measured and evaluated with respect to interspecific differences. The highest blood oxygen affinity, not only among camelids but of all mammals investigated so far, was found in the vicuna (P₅₀=17.6 Torr compared to 20.3–21.6 Torr in the other species). Low hematocrits (23–34%) and small red blood cells (21–30 m³) are common features of all camelids, but the lowest values are found in theLama species. Capillary densities were determined in heart and soleus muscle of vicuna and llama. Again, the vicuna shows exceptional values (3720 cap/mm² on average in the heart) for a mammal of this body size. Finally, heart weight as percent of body weight is higher in the vicuna (0.7–0.9%) than in the other camelids studied (0.5–0.7%). The possibility that these parameters, measured in New World tylopodes at sea level, are not likely to change considerably with transfer to high altitude, is discussed.In the vicuna, a unique combination of the following features seems to be responsible for an out-standing physical capability at high altitude: saturation of blood with oxygen in the lung is favored by a high blood oxygen affinity, oxygen supply being facilitated by low diffusion distances in the muscle tissue. Loading, as well as unloading, of oxygen is improved by a relatively high oxygen transfer conductance of the red blood cells, which is due to their small size and which compensates the negative effect of a low hematocrit on the oxygen conductance of blood. Blood oxygen transport is presumably favored by two factors: a relatively large heart mass and, as a result of low hematocrit, a low blood viscosity. Both are advantageous for achieving a high maximal cardiac output.     

Microsatellite markers for woolly monkeys (Lagothrix lagotricha) and their amplification in other New World primates (Primates: Platyrrhini)

Seven polymorphic microsatellite loci were identified for woolly monkeys (Lagothrix lagotricha) from an ‘enriched’ genomic library. For a wild population of 66 animals, these markers averaged over 10 alleles per locus and provided a combined probability for excluding a random individual from parentage of over 98%. These loci were screened in up to 13 other genera of New World monkeys, and many were variable in multiple taxa. Few other platyrrhine‐specific microsatellite markers have been identified; thus, these loci should prove valuable for studying the population genetic structure and mating system not just of Lagothrix but also of other neotropical primates.     

A complete molecular phylogeny of Claravis confirms its paraphyly within small New World ground‐doves (Aves: Peristerinae) and implies multiple plumage state transitions

The three species in the genus Claravis (Aves: Peristerinae) are unique among members of the small New World ground‐dove clade. All three species inhabit forested areas rather than open scrubby habitat, and exhibit obvious sexual dichromatism. However, the phylogenetic relationships within Claravis remain unknown. The only molecular phylogenetic study to include more than one species of Claravis indicated the genus is paraphyletic. Here we include molecular data from all three Claravis species, including sequences from a museum skin of the previously unsampled Claravis geoffroyi (purple‐winged ground‐dove). Using both mitochondrial and nuclear loci, we estimate phylogenies and divergence times for the small New World ground‐dove clade. We also use ancestral state reconstruction methods to infer the evolution of male blue plumage (and thus sexual dimorphism) in the clade. As in the previous study we recover Claravis as a paraphyletic group, but with Claravis geoffroyi as the sister species to Claravis mondetoura (maroon‐chested ground‐dove). This result has important implications for the evolutionary history of the small New World ground‐dove clade. In particular, we recover multiple independent transitions between the monomorphic and dimorphic plumage states, which perhaps indicates sexual dimorphism arose twice in the group.