Seed predation by monkeys and macaws in eastern Venezuela: Preliminary findings

Feeding data collected concurrently on bearded sakis (Chiropotes satanas) and red and green macaws (Ara chloroptera) on a large island in Guri, Venezuela provides preliminary evidence that these two seed predators have similar diets. Individuals of both species were equally capable of opening very hard, protected fruits. Of the seven fruit species used by macaws during the study period, four species were also ingested by sakis at the same stage of ripeness, two species were ingested at different stages of ripeness (macaws earlier than sakis), and one species was never observed to have been eaten by sakis. The second finding, that macaws ingest young seeds of the Anacardiaceae and Burseraceae families and the bearded sakis ingest only the ripe mesocarp of these species suggests that the most distinguishable difference in their diets might be a tolerance of toxins by the macaws that act as feeding deterrents for the monkeys. Although we did not document the location of local clay licks in eastern Venezuela, the use of clay licks by macaws in Peru (Munn, 1992) suggests that this activity (that is not practiced by the sakis) may be helpful in detoxifying or ameliorating the effects of ingesting chemically protected fruit. deceased.     

Molted feathers from clay licks in Peru provide DNA for three large macaws (Ara ararauna, A. chloropterus, and A. macao)

ABSTRACT Conservation genetic analyses of wildlife have increased greatly in the past 10 yr, yet genetic studies of parrots are rare because of difficulties associated with capturing them and obtaining samples. Recent studies have demonstrated that molted feathers can provide a useful source of DNA, but success rates have varied considerably among studies. Our objective was to determine if molted macaw feathers from Blue‐and‐yellow Macaws (Ara ararauna), Scarlet Macaws (A. macao), and Red‐and‐green Macaws (A. chloropterus) collected from rainforest geophagy sites called clay licks could provide a good source of DNA for population genetic studies. Specific objectives were to determine (1) how nuclear DNA microsatellite amplification success and genotyping error rates for plucked macaw feathers compared to those for molted feathers collected from clay licks in the Amazon rainforest, and (2) if feather size, feather condition, species, or extraction method affected microsatellite amplification success or genotyping error rates from molted feathers. Amplification success and error rates were calculated using duplicate analyses of four microsatellite loci. We found that plucked feathers were an excellent source of DNA, with significantly higher success rates (P < 0.0001) and lower error rates (P= 0.0002) than for molted feathers. However, relatively high success rates (75.6%) were obtained for molted feathers, with a genotyping error rate of 11.7%. For molted feathers, we had higher success rates and lower error rates for large feathers than small feathers and for feathers in good condition than feathers that were moldy and broken when collected. We also found that longer incubation times and lower elution volumes yielded the highest quality DNA when extracting with the Qiagen DNeasy tissue kit. Our study demonstrates that molted feathers can be a valuable source of genetic material even in the challenging conditions of tropical rainforests, and our results provide valuable information for maximizing DNA amplification success rates when working with shed feathers of parrots.     

Parrot behavior at a Rio Manu (Peru) clay lick: temporal patterns,associations, and antipredator responses

Although eating clay at licks (a form of geophagy) has been described, there are few behavioral data on temporal patterns, social interactions, species associations, or reactions to potential predators. We examined the behavior of nine species of macaws, parrots, and parakeets at the Machiguenga Ccolpa, a clay lick on the Rio Manu, Peru in the dry season. Three distinct mixed-species groups used the licks: in the early morning (parrots and small macaws), in mid-morning (large macaws), and in the early afternoon (parakeets), although the latter two groups used the licks at other times of day as well. The first parrots to begin eating at the lick in the early morning were yellow-crowned parrots (Amazona ochrocephala) and dusky-headed parakeets (Aratinga weddellii), followed by blue-headed parrots Pionus sordidus, and then by mealy (Amazona farinosa) and orange-cheeked (Pionopsitta barrabandi) parrots, and chestnut-fronted macaws (Ara severa). Although blue-headed parrots fed in dense groups of over 50, the others rarely exceeded 20 individuals. Scarlet macaws (A. macao) sometimes fed alone or joined the early morning groups, but most associated with a large group of red and green macaws (A. chloroptera) that arrived, often scaring off the smaller birds. On average, about 100 macaws and parrots fed in the early morning, macaw feeding groups averaging just over 40, and the parakeets averaged over 70. Average time at the lick ranged from 28 min for yellow-crowned parrots to 47 min for tui parakeets. Of the early morning group, blue-headed and mealy parrots were the most aggressive and orange-cheeked parrots were the least aggressive. Red and green macaws were more aggressive than scarlet macaws; the parakeets were equally aggressive. All species had more aggressive interactions with conspecifics than with other species. Responses to intruders and predators varied by species of parrot/macaw and type of intruder. In response to intruders or loud calls, responses could be partial (some individuals flew away, circled, and returned), temporary (all individuals flew away but returned within a few minutes), or total (all flew away and abandoned feeding for at least a half hour). The large macaws showed the lowest rate of total abandonment and the parakeets showed the highest. People passing up or down river in boats scared birds from the lick. The local residents (Machiguenga tribespeople in boats) elicited a much greater response than did the researchers. In the recent past, macaws and parrots were hunted for food, feathers, and the pet trade, and the birds response, as well as the presence of parrot and macaw feathers in local villages we visited, suggests some continued exploitation, or a long-term memory in the birds.Communicated by R.F. Oliveira     

Forensic species identification of large macaws using DNA barcodes and microsatellite profiles

Using mitochondrial and nuclear markers species identification was conducted in the case of seized feathers. Earlier, we had sequenced cytochrome c oxidase subunit I (COI) both from 10 seized specimens and 43 validation specimens from captive macaws belonging to 4 Ara species (A. macao, A. chloropterus, A. ararauna, and A. ambiguus) and identified 19 haplotypes based on COI sequences. Species-level identification using Barcode of Life Data Systems showed that seized feathers shared the highest similarity with scarlet macaws (A. macao), and this result was supported by the tree-base identification with high bootstrap values. Moreover, microsatellite profiles in AgGT17 locus showed that patterns of allelic distribution in the seized feathers were apparently distinct from those of red-and-green macaw (A. chloropterus), but were overlapped with those of A. macao, suggesting that all of seized feathers were derived from several individuals of A. macao. We also determined the parentage of hybrid macaws by the combination of COI barcodes and microsatellite profiles. The technique presented here will contribute to forensic identification and future conservation of large macaws that have been lost due to deforestation.     

A non-invasive genetic evaluation of population size,natal philopatry,and roosting behavior of non-breeding eastern imperial eagles (<Emphasis Type="Italic">Aquila heliaca</Emphasis>) in central Asia

Roughly one-third of all eagle species are considered to be threatened or endangered, but the ecology of most eagles remains poorly understood. While the pronounced territoriality of breeding adults facilitates behavioral studies, the demography of non-breeding individuals (pre-adults and non-territorial floaters) is almost completely unknown. Traditionally, limited data on pre-adult and floater movement come from wing-tagging and/or telemetry studies. As an alternative to these methods we used genetic analyses of non-invasively collected feathers to investigate the population biology of non-breeding eastern imperial eagles (Aquila heliaca) in Kazakhstan. Microsatellite profiles of shed feathers indicate that eastern imperial eagles roost communally with other raptors. Furthermore, roosts are large and dynamic: 287 non-breeding eastern imperial eagles were detected in our sample, and a mark-recapture analysis estimated the total number to be 308 ± 8. The natal origins of these individuals were investigated by comparing their microsatellite profiles to those available for >90% of the eastern imperial eagle chicks hatched at the study site over the six previous breeding seasons. Only 4% of the individuals genetically matched a chick, suggesting that the reserve may serve as a critical refugium for pre-adults and itinerant floaters. Feathers have long been recognized as a suitable source of DNA, but few studies have used wide-scale, non-invasive collections of feathers (>1,000 samples) to investigate fundamental aspects of avian biology. Our research demonstrates that non-invasive genetic analyses of feathers can be used to evaluate population size, natal philopatry, and local movements of birds that are difficult to study using traditional means.     

Influence of clay licks on the diversity and structure of an Amazonian forest

The spatial heterogeneity of resource availability is a major driver of biodiversity patterns. Some environmental conditions and resources are characterized by large‐scale patterns of variation within the landscape. Clumped local discontinuities or discrete elements also increase spatial heterogeneity, promoting local ‘biodiversity hot spots’ by modifying habitat characteristics and promoting plant–animal interactions. Clay licks are faunal attractors owing to their role in the nutritional ecology of the user species; nevertheless, the effect of their presence on the surrounding vegetation has been poorly quantified. Here, we use data from 100 × 10 m transects and evaluate the effects of the presence of clay licks on forest diversity and structure at local and landscape scales. In clay lick areas, there was a higher abundance of certain species, which helps to homogenize species composition between localities counteracting the natural distance‐decay of compositional similarity between transects without clay lick influence (controls). Compared to control sites, clay lick′s forests had higher palm densities, shorter but more variable individuals in the canopy and understory, a thinner canopy layer, and denser herbaceous and ground level covers. These differences were found along the whole length of transects in both sampled areas types. These results reveal that the presence of discrete elements (i.e., clay licks) may help to explain the compositional and structural heterogeneity of Amazonian forests influencing ecological processes such as seed dispersal and trampling. These considerations may be relevant for other biomes where clay licks are present and give weight to their inclusion in conservation initiatives in tropical forests.     

DNA sequence analysis to guide the release of blue-and-yellow macaws (Ara ararauna, Psittaciformes, Aves) from the illegal trade back into the wild

The illegal wildlife trade is one of the major threats to Brazil’s biodiversity. Approximately 80 % of illegally captured animals are birds, and 4 % of those are parrots. Although many seized birds do not survive, those that are recovered may be returned to the wild. The release of seized individuals into the wild should be conducted with caution, as local populations may suffer adverse effects if genetically different individuals are introduced. In this study, we evaluated the genetic relationships between 13 illegally captured blue-and-yellow macaws selected for release in northeastern Goiás, Brazil, and previously studied Brazilian macaw populations. We identified the seized macaws that were genetically similar to those from northwestern Goiás and that were therefore most suitable for release in that area. The genetic relationship was evaluated by sequence analysis of 403 bp of mitochondrial DNA control region. Relationships between mitochondrial haplotypes were computed via a median-joining network. Only six of the seized macaws were closely related to the macaws of northeastern Goiás, indicating that those macaws were potential candidates for release in that area. However, the release of these birds should follow all technical recommendations required by the Brazilian environmental authorities.     

Applications and techniques for non-invasive faecal genetics research in felid conservation

Non-invasive genetic techniques utilising DNA extracted from faeces hold great promise for felid conservation research. These methods can be used to establish species distributions, model habitat requirements, analyse diet, estimate abundance and population density, and form the basis for population, landscape and conservation genetic analyses. Due to the elusive nature of most felid species, non-invasive genetic methods have the potential to provide valuable data that cannot be obtained with traditional observational or capture techniques. Thus, these methods are particularly valuable for research and conservation of endangered felid species. Here, we review recent studies that use non-invasive faecal genetic techniques to survey or study wild felids; provide an overview of field, laboratory and analysis techniques; and offer suggestions on how future non-invasive genetic studies can be expanded or improved to more effectively support conservation.     

Conservation Genetics of Jacquemontia reclinata (Convolvulaceae), an Endangered Species from Southern Florida: Implications for Restoration Management

Guidelines designed to aid in the restoration of rare species have been previously proposed using two primary strategies to select individuals for augmentation and reintroduction: mixing progeny from different populations or separating individuals from different populations. Understanding the genetic structure and diversity of an endangered species can offer insights into conservation management strategies. We used random amplified polymorphic DNA markers to assess the genetic structure and diversity of Jacquemontia reclinata , a federally endangered species endemic to Southeastern Florida. We sampled 20 percent of total number of individuals from eight of the ten known wild populations. Across individuals high levels of polymorphic loci (94.7%) were found and larger populations had greater genetic diversity. Cluster and ordination analyses found that one population was genetically differentiated from all the others; this population grows in a unique habitat. Most genetic variation (77.5%) was found within populations, and genetic distances between populations were not explained by their geographic distances. We recommend the use of two management units in restoration programs for J. reclinata , one consisting of the genetically differentiated population and the second consisting of the other seven populations sampled.     

Genetic patterns and conservation of the Scarlet Macaw (<Emphasis Type="Italic">Ara macao</Emphasis>) in Costa Rica

Once widely distributed throughout the lowland forests of Costa Rica, scarlet macaws (Ara macao) have been reduced to two major, geographically separated, populations along the Pacific slope. Past demographic declines raise conservation concerns regarding the detrimental effects of population fragmentation. This investigation aimed to evaluate the current status of scarlet macaws along the Pacific slope by examining levels of genetic variation and patterns of genetic structure within and among remnant populations. Statistical analyses using multilocus genotypes revealed strong differentiation between Central and South Pacific populations, suggesting local geographic barriers have historically restricted gene flow between these localities. High genetic diversity suggests neither population suffers from genetic erosion, likely resulting from relatively large population sizes and high dispersal capacity and longevity. However, evidence of disequilibrium within the Central Pacific population infers anthropogenic threats have disrupted natural population dynamics. These results advocate on focusing available resources on habitat restoration and nest protection, as a means to assist in reestablishing demographic stability and maintain the genetic health of wild scarlet macaws in Costa Rica.     

Faeces as a source of DNA for molecular studies in a threatened population of great bustards

With recent advances in molecular biology, it is now possible to use the trace amounts of DNA in faeces to non-invasively sample endangered species for genetic studies. A highly vulnerable population of approximately 100 great bustards (Otis tarda) exists in Morocco necessitating the use of non-invasive protocols to study their genetic structure. Here we report a reliable silica-based method to extract DNA from great bustard faeces. We found that successful extraction and amplification correlated strongly with faeces freshness and composition. We could not extract amplifiable DNA from 30% of our samples as they were dry or contained insect material. However 100% of our fresh faecal samples containing no obvious insect material worked, allowing us to assess the levels of genetic variation among 25 individuals using a 542 bp control region sequence. We were able to extract DNA from four out of five other avian species, demonstrating that faeces represents a suitable source of DNA for population genetics studies in a broad range of species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic markers validate using the natural phenotypic characteristics of shed feathers to identify individual northern goshawks Accipiter gentilis

The recognition of individual animals is essential for many types of ecological research, as it enables estimates of demographic parameters such as population size, survival and reproductive rates. A popular method of visually identifying individuals uses natural variations in spot, stripe or scar markings. Although several studies have assessed the accuracy of these methods in mammals, crustaceans and fish, there have been few attempts to determine whether phenotypic characteristics are accurate when used for birds. Furthermore, even less is known about whether shed or moulted body parts can be reliably used to visually identify individuals. Here we assessed the accuracy of using phenotypic characteristics to identify avian individuals using a double‐marking experiment, whereby nine microsatellite genetic markers and natural markings on shed feathers were used to independently identify northern goshawks Accipiter gentilis. Phenotypic and genetic identification of individuals was consistent in 94.4% (51/54) comparisons. Our results suggest that the phenotypic characteristics of shed feathers can be reliably used as a non‐invasive and relatively inexpensive technique to monitor populations of an elusive species, the northern goshawk, without having to physically re‐capture or re‐sight individuals. We posit that using natural markings on shed feathers will also be a reliable method of identifying individuals in avian species with similar phenotypic characteristics, such as other Accipiter species.     

Genetic tagging: contemporary molecular ecology

Population genetic analyses have been highly successful in deciphering inter- and intra-specific evolutionary relationships, levels of gene flow, genetic divergence and effective population sizes. Parameters estimated by traditional population genetic analyses are evolutionary averages and thus not necessarily relevant for contemporary ecological or conservation issues. Molecular data can, however, also provide insight into contemporary patterns of divergence, population size and gene flow when a sufficient number of variable loci are analysed to focus subsequent data analyses on individuals rather than populations. Genetic tagging of individuals is an example of such individual-based approaches and recent studies have shown it to be a viable alternative to traditional tagging methods. Owing to the ubiquitous presence of hyper-variable DNA sequences in eukaryote genomes it is in principle possible to tag any eukaryote species and the required DNA can be obtained indirectly from substrates such as faeces, sloughed skin and hair. The purpose of this paper is to present the concept of genetic tagging and to further advocate the extension of individual-based genetic analyses beyond the identification of individuals to other kinds of relationships, such as parent-offspring relations, which more fully exploit the genetic nature of the data.     

Geographic variation in relict populations: genetics and phenotype of bush-cricket Pholidoptera frivaldskyi (Orthoptera) in Carpathians

A decreasing population size is often causing species extinction, however, relict species persisting in small-sized populations counter this. We analysed spatial genetic variation and past changes in population size at the maternally-inherited mitochondrial DNA level to clarify the origin of all recently known isolated populations of Pholidoptera frivaldskyi occurring in the range of Carpathian Mountains. Along with that we analysed also morphological variation as some phenotypic traits can retain useful information on population genetic structure. We found a relatively low genetic diversity within isolated populations as 778 bp COI gene sequences revealed only 13 unique haplotypes (n = 173 individuals from 10 populations). The spatial analysis of molecular variance identified three geographically homogenous genetic clusters (one in Slovakia and two in Romania) with a high level of differentiation among them, suggesting restricted gene flow, whilst Bayesian skyline simulation reconstructed a negative demographic change through evolutionary time. Inferred genetic pattern clearly coincides with differences in males’ colour phenotype as the extent of pigmentation on the lateral pronotum varied significantly among genetic lineages. We suggest that geographical variation in the species populations has relict-like character and their isolated occurrence is not a result of recent introduction events. Identification of ‘evolutionary units’ may help in the conservation and management of this rare insect species.     

A noninvasive method for extracting bivalve DNA from the water-filled mantle cavity

Genetic studies play a great role for determining the biology of bivalves, particularly those covering population genetics, phylogeny, breeding, stock management, and conservation. However, DNA sampling methods that require removal of bivalves from the water and/or opening of their shells often cause stress and damage to bivalves, which can be lethal. The invasiveness of DNA sampling has made it difficult to conduct genetic studies in threatened species, rare species, and/or breeding lineages. In the present study, we developed a non-invasive method for bivalve DNA sampling using the water-filled mantle cavity (WMC). Our method can extract DNA from a small WMC sample (about 100 µl), collected using a fine needle and syringe without opening the shell. We demonstrated that the WMC sample contains intact mitochondrial and nuclear DNA. DNA contamination from other organisms, such as adjacent bivalve individuals, did not affect the resulting PCR and DNA sequencing analyses. Finally, the individuals from whom WMC was collected remained alive for more than 2 months after the experiments. This non-invasive method will be of great assistance in investigating the genetics of bivalves.

     

Genetic stability of the D1Z2 region: implications for DNA genotyping and paternity testing.

The aim of the present study was to examine a single locus variable number tandem repeat for the purpose of DNA genotyping ("fingerprinting"). DNAs of 175 individuals from five ethnic groups (Black, Chinese, Japanese, Caucasian, and Melanesian) were analyzed. Restriction fragment length polymorphic analysis of random individuals revealed individual specific DNA patterns in all but one group. Among 20 Melanesian inhabitants of the Vanuatu islands in the southwest Pacific, three individuals were found to share a common pattern. This island population represents a "genetic isolate" and illustrates the importance of carrying out population studies on individual ethnic groups of interest. The complexity and the genetic stability of the D1Z2 region as revealed by the probe hMF1 make it an excellent candidate for DNA genotyping in paternity testing as 101 Caucasian individuals each had unique patterns for PstI and SinI digests.     

Species identification and genetic structure of threatened seahorses in Gran Canaria Island (Spain) using mitochondrial and microsatellite markers

A non-invasive DNA analysis of seahorse populations was carried out after extensive underwater surveys in Gran Canaria Island (Spain). In this geographical area, the presence of two species, Hippocampus hippocampus and H. guttulatus, has been previously reported. Sequencing of 16S ribosomal DNA (16S rDNA) was used for specific identification of live seahorses sampled in situ, as a previous step to evaluate genetic structure based on ten microsatellite markers. Phylogenetic analyses revealed the presence of a single species, H. hippocampus, in the seahorse communities found at Gran Canaria. No evidences of H. guttulatus or interspecific hybrids were found based on 16S rDNA and microsatellite data. The nuclear markers revealed low genetic diversity and lack of population structure across populations of Gran Canaria Island, with evidence of small population sizes. This study provides critical information to support conservation strategies of Gran Canaria seahorses.     

Phylogeographical Study on Eriophyton wallichii (Labiatae) from Alpine Scree of Qinghai Tibetan Plateau

In this study, we examined the nuclear ribosomal DNA internal transcribed spacer (ITS) sequence variation of Eriophyton wallichii, a perennial endemic to alpine scree of the Qinghai Tibetan Plateau. Nineteen haplotypes were detected by analyzing 187 individuals from 20 populations of E.wallichii. Most populations hold the unique haplotype, which showed different population with different haplotype. An analysis of molecular variance (AMOVA) for populations showed that the genetic variation mainly resided among populations (89.54%), the level of differentiation among populations was very high (GST=0.863; NST=0.957) but didn’t show a significant phylogeographical structure. It is different from foregoing other species reported in Qinghai Tibetan Plateau that the haplotypes of E.wallichii showed a distribution pattern that almost one population hold one unique haplotype, hardly share same halotype among populations.We hypothesized the unique genetic structure of E.wallichii might result from allopatric fragmentation in the “islands of alpine” due to the extremely isolated alpine scree and rough topography of the region, through the Quaternary climate oscillation dramatically caused population expanding or shrinking.     

Targeted multiplex next‐generation sequencing: advances in techniques of mitochondrial and nuclear DNA sequencing for population genomics

Next‐generation sequencing (NGS) is emerging as an efficient and cost‐effective tool in population genomic analyses of nonmodel organisms, allowing simultaneous resequencing of many regions of multi‐genomic DNA from multiplexed samples. Here, we detail our synthesis of protocols for targeted resequencing of mitochondrial and nuclear loci by generating indexed genomic libraries for multiplexing up to 100 individuals in a single sequencing pool, and then enriching the pooled library using custom DNA capture arrays. Our use of DNA sequence from one species to capture and enrich the sequencing libraries of another species (i.e. cross‐species DNA capture) indicates that efficient enrichment occurs when sequences are up to about 12% divergent, allowing us to take advantage of genomic information in one species to sequence orthologous regions in related species. In addition to a complete mitochondrial genome on each array, we have included between 43 and 118 nuclear loci for low‐coverage sequencing of between 18 kb and 87 kb of DNA sequence per individual for single nucleotide polymorphisms discovery from 50 to 100 individuals in a single sequencing lane. Using this method, we have generated a total of over 500 whole mitochondrial genomes from seven cetacean species and green sea turtles. The greater variation detected in mitogenomes relative to short mtDNA sequences is helping to resolve genetic structure ranging from geographic to species‐level differences. These NGS and analysis techniques have allowed for simultaneous population genomic studies of mtDNA and nDNA with greater genomic coverage and phylogeographic resolution than has previously been possible in marine mammals and turtles.     

Erodium maritimum (Geraniaceae), a species with an uneven and fragmented distribution along the Western Mediterranean and European Atlantic coasts,has a weak genetic structure

Erodium maritimum L. is an annual species presenting heterogeneous, sometimes very small, and distant populations, distributed along a discontinuous coastal strip of the European Atlantic and the central and western Mediterranean basin. The aim of this study is to investigate genetic variation and geographic structure changes across its large distribution. Fourteen populations of E. maritimum were studied using AFLP fingerprints, together with their population sizes, reproductive systems and flower visitors. AFLP markers revealed the genetic structure of the species to be weak. Many individuals from one population clustered together with those of other populations, showing a high degree of genetic admixture. Despite having a self‐compatible reproductive system, populations (especially the largest ones) showed high levels of genetic polymorphism, and the majority of genetic variation was contained within populations. The low genetic structure suggests high levels of gene flow, which might be explained through the dispersability of the species’ fruits. Finally, recommendations are provided for management strategies to facilitate the conservation of this endangered species.