Olah Proposal and Progress Report 2010

8/2/2019 Olah Proposal and Progress Report 2010

1/16

George Olah, Research PhD Proposal and Progress Report - 2010, ANU

1

Development of novel genetic techniques for conservation studies

of large macaws

Project Objectives

The aim of my PhD project is to develop powerful population genetic tools that will have wideapplicability to the study of wild parrots. The immediate aims are to use DNA extracted from feathersdropped by wild Scarlet Macaws (Ara macao) at clay licks to estimate population size and geneticstructure, and individual movements. This study will provide essential base-line data for understandingthe impacts of habitat fragmentation on large wide-ranging macaws, and for comparison to other moreendangered species. We will:

(1) identify individual Scarlet Macaws from the feathers they drop in the study area using genetictagging;

(2) use individual identities from genetic samples to study individual movements and to estimatehome range size;

(3) reconstruct demographic and genetic traits of the study populations, especially effectivepopulation size, genetic diversity, genetic differentiation among populations, and sex-biases indispersal;

(4) use knowledge of home ranges and population structure and size to assess the impact of humandisturbances to the environment including the new Inter-oceanic Highway through south-easternPeru and expanded oil exploration in wilderness areas such as the Candamo Valley;

(5) assess the feasibility of our novel genetic tagging techniques as a widely applicable tool inconservation biology.

Project Summary

Genetic studies are important tools for understanding conservation issues such as the impact on faunaof habitat fragmentation, degradation and loss. This study was initiated in October, 2009, with themajor objectives of (1) understanding and documenting the current population genetic structure ofScarlet Macaw (Ara macao) populations in SE Peru; (2) assessing the feasibility of genetic tagging, anovel non-invasive technique for identification of individuals by genetic markers, for measuringhome-range, dispersal, and population structure; and (3) applying our techniques to specificconservation issues. These include documenting the effect of fragmentation due to the recently builtInter-oceanic Highway running through the study site; investigating the degree of isolation of the

Scarlet Macaw population in the isolated Candamo Valley with the view to making informedrecommendations for protecting this biodiversity hotspot; and introducing the genetic taggingtechnique to other species of endangered macaws, such as the Blue-throated Macaw (Araglaucogularis) in Bolivia.

This is the first project of its kind, and is intended to provide a broad-based genetic tool for theconservation of wild parrots. We will capitalize on two enormous advantages. First, we will havedirect access to the research outcomes of the recently launched Scarlet Macaw Genome Project. Theextensive knowledge of the scarlet macaw genome will enable effective development of highlyvariable genetic markers that will enable powerful insights into the population processess of wildmacaws. Second, we will have access to extraordinarily large number of samples from feathersdropped at clay licks on the river systems of SE Peru. Macaws in Peru visit clay licks to supplement

their diet with minerals and toxin-absorbing clays (Gilardi et al. 1999; Burger and Gochfeld 2003;Brightsmith 2004), and large numbers of naturally dropped feathers are left behind. Our study area


2/16


2

includes the highest clay lick distribution in South America. This offers us a perfect place to collectmolted macaw feathers year-round.

The process of dispersal is poorly understood for most species yet plays a crucial role inpopulations. Dispersal shapes distribution patterns, affects population regulation and stability,influences extinction and recolonization dynamics in patchy populations and metapopulations, andcontrols patterns of gene flow. We will indirectly evaluate the dispersal patterns of Scarlet Macawthrough the analysis of population genetic structure and more directly by employing genetic tagging toassess the movement of individuals. This will provide a much needed perspective for understandingthe response of species to landscape change and habitat fragmentation.

Endorsements/Recognition

The fieldwork phase of the project requires collecting DNA samples from macaws in Peru. Themajority of the field work takes place in Madre de Dios Region of Peru. For collecting samples outsidethe protected areas in this region we have a permit in progress from the Peruvian Ministry of

Agriculture (Ministeria de Agricultura).The core region of this research is located in protected areas Tambopata National Reserve andBahuaja-Sonene National Park. We have permit from the Peruvian National Service of ProtectedAreas (SERNANP - Servicio Nacional de reas Naturales Protegidas por el Estado) that allows us tocollect genetic samples inside the protected areas. The permit number is: N 001-2010-SERNANP-DGANP-PNBS.

Exporting DNA out of Peru and importation into the United States requires CITES permits from eachnational government. The importation permit for USA has been granted under the number10US07071A/9. The exportation permit from Peru is in progress.

This project works in strong collaboration with the Tambopata Macaw Project, for that the Texas

A&M University provides academic background, and also giving recognition to our project. Ananimal research ethics clearance for taking blood samples from live birds has been attained from theTexas A&M University (AUP 2009-215).

20th August, 2010Canberra, Australia


3/16


3

Project description

Background information

The Amazon basin contains a highly diverse, incredibly complex, and globally important ecosystemthat is, even today, still poorly understood. It holds 60% of the worlds remaining tropical rainforest(Laurance et al. 2002). The general biology and natural history of many species from this area,including large charismatic species like parrots (order Psittaciformes), are lacking or poorlyunderstood (Munn 1992). Approximately 26% of the Neotropical parrots are classified as threatened(Birdlife International 2006) and the Psittaciformes are considered the most endangered large birdorder in the world (Bennett and Owens 1997). It is impossible to measure or predict the effects ofdeforestation, habitat fragmentation, human impact, and hunting, on species whose ecology isunknown. Recent studies, like the Tambopata Macaw Project in South-eastern Peru provide insightinto these species breeding ecology and natural history (Brightsmith 2005), but studies at thedemographic and population genetic level in a wider range of species and study sites are still needed.Molted feathers are a promising and underutilized non-invasive source of data for genetic studies of

birds in temperate and tropical ecosystems (Gebhardt et al. 2009). In this study we aim to reconstructdemography and spatial ecology by using genetic tagging (a novel technique for identification ofindividuals by genetic markers) of Scarlet Macaw (Ara macao) in South-eastern Peru. Knowledge ofthe movements of individuals across the landscape, and patterns of spatial genetic structure includingvariation between sexes, age-classes, and time promise to provide important clues about dispersal andother important aspects of a species biology (Peakall et al. 2003). The status of Scarlet Macaws isLeast Concern according to the IUCN Red List, thus providing a model system for developing andtrialing methods that are not practical initially in more endangered species and for providing apowerful genetic tool kit for the conservation of a wide range of more endangered macaw species.

This PhD project was initiated with three main objectives (1) to understand and document thebreeding biology, and current demographic and population genetic structure of Scarlet Macaw

populations in SE Peru; (2) to develop and test the newly emerging technique of genetic tagging onwild macaw populations; and (3) recommend further specific applications of genetic tagging in theconservation biology of endangered macaw species.

(1) The first step is to understand the demography, breeding biology and population geneticsof Scarlet Macawsstill maintaining healthy populations in the pristine forests of South-eastern Peru(Brightsmith 2005; Gebhardt et al. 2009). Although their habitat is still relatively intact today, ahighway is being built running from the Atlantic to the Pacific Ocean (Tickell 1993; Conover 2003).This Inter-oceanic (or Trans-amazonian) Highway runs through Puerto Maldonado and our study areain South-eastern Peru and will increase human use of the protected area in this region, and probablylead to habitat fragmentation. Increasing the accessibility of tropical rainforest habitat by roaddevelopment often has negative impacts on animal populations through habitat destruction (due toselective logging and deforestation), chemical pollution, edge effects, road-related mortality orcapture, barrier effects, exotic species invasion and human invasions (e.g. hunters, miners) (Lauranceet al. 2009). Macaw reproduction is limited mainly by available nesting sites in large over-story trees(Munn 1992), and since one cavity will often be used for decades, the removal of these large trees dueto selective logging can reduce parrot reproduction and threaten population viability. As a non-geneticcomponent in this PhD, I am analyzing 10 years of breeding biology data recorded in the TambopataResearch Center, the core region of our study site in SE Peru, to better understand the nesting sitepreferences and to evaluate breeding success trends of this species. Scarlet Macaws also face anadditional hazard as in many parts of the Amazon they are hunted for food, their feathers, or for theillegal pet trade, so increased human use of areas can negatively impact macaw numbers as a result ofthese processes. It is also important to document the original population genetic structure of these

populations, otherwise later studies will not have the base genetic data necessary to compare to, andevaluate habitat fragmentation on the ecosystem.


4/16


4

(2) We will develop and test a novel genetic technique for studying the spatial scale ofdispersal and movements in wild macaws. Large, wide ranging parrots like macaws (especially thosein smaller populations) are the hardest to study in this respect, mainly because these birds travel longdistances and they are hard to mark/recapture, unless a huge amount of money and work are invested.Since 2003 a group of parrot researchers, conservation organizations, and manufacturers have beenworking together to design and test satellite collars for tracking large macaws. To date, thisconsortium has developed two prototypes which were tested e.g. on captured macaws in theTambopata Research Center (TRC), South-eastern Peru (Brightsmith and Boyd 2006). Thepreliminary data indicate long distance movements of Blue-and-yellow Macaws, during the periodswhen food supply was low in the areas around TRC (Brightsmith 2008). Those sites may containkeystone food resources which allow the birds to survive the seasons of low food availability near thecapture site.

However, satellite telemetry is very expensive and does not always provide the desired information. Italso requires catching wild birds, which for macaws is a huge effort. Because of the high cost of eachsatellite-collar it can be applied for only a few birds, which can provide misleading generalization forthe whole population or species. Because of the few individuals captured, the method is not

representative and could generate biased data, e.g. the individuals are easy to capture show differentdispersal patterns.

Our study will complement the satellite telemetry by using non-invasive genetic tagging and allowmany more individuals to be tracked for providing information about the dispersal of the populations.Genetic tagging is a newly emerging technique in molecular ecology, which enables the uniqueidentification of individuals by their hypervariable genetic markers, and has proven to be a highlyeffective method in other studies (Palsboll 1999; Taberlet and Luikart 1999; Waits et al. 2001; Peakallet al. 2006). Genetic markers are DNA sequences with a known location on a chromosome that can bemeasured and quantified and used to identify individuals or species (Peakall and Smouse 2009).Genetic markers are visualized using laboratory procedures that detect variation at the DNA level. Oneparticularly powerful genetic marker type is a Short Tandem Repeat (STR or Microsatellite), a pattern

of very short (1-6 base pairs) nucleotide motifs that are repeated and the repeated sequences aredirectly adjacent to each other. The pattern is typically in the non-coding intron region of the genome.These markers target DNA regions where there is variations in the number of repeat lengths withinand among individuals. By identifying repeats of a specific sequence at specific locations in thegenome, it is possible to create a genetic profile (genotype) of an individual. STR has become thestandard genetic marker for determining genetic profiles in human forensic cases. Indeed, theseregions are so variable, that just 13 STR (microsatellite) loci are used in forensic analysis of humans.The population genetic calculation of the probability of identity provides an estimate of the averageprobability that two independent samples will have the same identical genotype (Peakall and Smouse2009). In the USA, the FBI has selected 13 STR loci to serve as panel of core loci for forensicinvestigations. With this set of loci, the probability of identity (a match between profiles of twounrelated persons in a randomly mating population of Caucasian Americans) is one in 575 trillion.

While the application of STRs for human forensic analysis is most well known, the same kind ofgenetic markers are being employed in a wide range of animals for genetic tagging. Peakall (the co-supervisor of this project) and colleagues have used genetic tagging in small mammals anddemonstrated the importance of this technique in modern conservation genetic studies (Peakall et al.2003; Peakall et al. 2006; Ruibal et al. 2009; Ruibal et al. 2010). With appropriate sampling, andgiven the new impossibility of capturing large numbers of macaws, genetic tagging provides the bestprospective method of studying the movement and dispersal of macaws among clay-licks. Theupcoming data of the satellite telemetry study on macaws would help to understand the small scalemigration of these species, as it would provide detailed reference information about their movement.These complementary data can be combined with our genetic results about their dispersal and home-range.

For effective use of the genetic tagging method, a variable set of microsatellite loci are needed. So faronly nine microsatellite loci have been described by optimization from cross-species amplification


5/16


5

(Gebhardt and Waits 2008). The College of Veterinary Medicine & Biomedical Sciences at TexasA&M University launched the Scarlet Macaw Genome Project (SMGP) early this year, coordinated byProf. Ian Tizard, and the entire Scarlet Macaw genome will be sequenced for next year. It is anextraordinary opportunity for our project to collaborate with the SMGP in developing microsatellitemarkers on the sequenced genome of our study species. This genome project will provideunprecedented access to suitable genetic markers.

(3) The last objective of this study will be to suggest specific applications concerning theconservation biology of wild parrots.

(a) One of the long term uses of this technique is to predict the effect of the highway built in the studyarea. Identifying individuals through the landscape (see details in the objectives) we could generalizethe home range used by the population which is very important (e.g. if the birds are still crossing thehighway for visit clay licks, nesting sites). Also, this study will provide baseline population geneticdata of the recent Scarlet Macaw populations for future investigations. Assuming that thesepopulations have not been affected yet by the fragmentation (given that macaws are long living birdsand genetic differentiation can be observed after a few generations), only upcoming studies can show

detectable genetic effects caused by the road construction. This will provide essential information forthe conservation biology of Scarlet Macaws.

(b) Another conservation concern in the study area is that oil companies are starting to explore areaswith still largely intact ecosystems, like the Candamo Basin (Finer et al. 2008). It lies between theAmazon Lowland Rainforests of South-eastern Peru and the Andes. The scope of natural habitatscontained within this area result in spectacular biodiversity. Around the Candamo Basin, the foothillsof Andes form a high barrier, like a fortress. This place will be an important sampling site for ourstudy, since due to its locality it can contain isolated populations of species. To prove this, we wouldlike to compare the genetics of Scarlet Macaws inside and outside of Candamo, as well as trackingindividuals by genetic tagging to see if they cross the hill barriers. In September 2007 a proposed billwas to be deliberated by the Peruvian Congress to reduce the size of the Bahuaja Sonene NationalPark by 200 000 ha (19 %), including the area of Candamo to be the target of oil companies. Although

the bill was rejected because of a high international attention, oil exploration can still be considered amajor threat to Candamo and other areas in the region as they have massive hydrocarbon deposit. Thebeauty of the huge National Parks of Peru is that they are areas designated to be free of humandisturbance. Not even tourism is permitted, and scientific research only takes place with specialpermission. These areas are sanctuaries for the wildlife of the area, and a source of pride for thePeruvian people. Our samples from Candamo could provide a strong scientific basis built on geneticdata, which was not carried out before, urging the authorities to maintain the protected status of thisbiodiversity hotspot.

(c) Once tested we would advocate the application of genetic tagging for Blue-throated Macaws (Araglaucogularis), currently the most endangered macaw species (Hesse and Duffield 2000). Only about150 individuals remain in the wild in Beni province, Bolivia (Yamashita and Machado de Barros

1997), relatively near (500 km) to our research site of SE Peru. Collecting non invasive geneticsamples (like feathers, as the principal DNA source for our technique) from the remained populationsof this species would allow researchers to compare their population genetic structure to ScarletMacaws still maintaining healthy population in South-eastern Peru. Genetic tagging of Blue-throatedMacaws would also contribute to understand the dispersal and movement of the remaining birds in thewild without capturing them for radio/satellite-collaring. Identifying and tracking the individual birdswould also be useful in future reintroduction efforts.

To date, only Gebhardt`s M.Sc. thesis (Gebhardt 2007) has used non-invasive geneticsampling of feathers to evaluate the genetic diversity, population genetic structure, and the effectivepopulation size of wild macaws (Red-and-green Macaw, Ara chloropterus, and Scarlet Macaw, Aramacao) in intact rainforest in Peru. They sampled macaws from three to four locations throughout the

Peruvian Amazon and generated genetic data for 12 nuclear microsatellite loci and 381 bp of themitochondrial control region. They found that diversity was high across intact habitat for both macaw


6/16


6

species, but was higher for Scarlet Macaws than for Red-and-green Macaws. For each species, theiranalyses suggested one genetic group across the areas sampled (3 in Madre de Dios and 1 in Iquitos,1000 km northern). Their results also indicated high gene flow across the region; however ScarletMacaws had more restricted gene flow than Red-and-green Macaws. Trends of female-biased geneflow for Scarlet Macaws were also observed and they suggest further investigation with expandedgenetic sampling to prove this. Their effective population size estimates for Scarlet Macaws were threeto five times larger than for Red-and-green Macaws. The researchers concluded that the processes thatdrive the genetic diversity and population structure of both Scarlet and Red-and-green Macaws operateat a large spatial scale and additional sampling outside of Peru is needed to evaluate geneticneighborhood size. They expect the genetic structure of macaws to be driven by isolation by distancebecause there are few suspected barriers to these highly vagile species within intact forest. The lack ofdifferentiation among the studied locations indicates that the sampling scale was too small and theyhad too few sampling spots. They caution against using their estimates of effective population size asabsolute values for conservation, mainly because of the potential for high gene flow with unsampledareas.

In the first year of this project we started to collect feather and small quantity blood samples of

Scarlet Macaws from the study area between December, 2009 and April, 2010. We also tested themodified Qiagen DNeasy tissue kit extraction protocol described by Horvth et al.(2005) on captivemacaws feathers in the laboratory of Szent Istvn University, Faculty of Veterinary Science, inBudapest, Hungary. For PCR sex identification we successfully tested the primers P2 and P8,described for parrots, that amplify female specific W chromosome marker (Miyaki et al. 1998).

No other population genetic studies of macaws have been carried out in this region with stillhealthy populations, though genetic studies are important tools to help understanding conservationissues, like fragmentation, perturbation (Peakall et al. 2006) and also apply non-invasive methods foralready endangered species by habitat loss and fragmentation. By combining ecology and genetics wecan frequently discover new insights not evident from either ecology or genetic studies alone (Peakalland Beattie 1996). While ecological studies tend to have a higher priority in conservation biology and

ongoing researches of this field are more often in the region, there is a lack of well designed geneticstudies on macaws. In addition, genetic results can also help us test predictions from our ecologicalknowledge and vice versa (Peakall and Smouse 2009).

Objectives

Macaws in Peru leave large numbers of dropped feathers at clay licks. A recent study (Lee et al. 2010)showed that the highest clay lick distribution in South America is found in our study area in South-eastern Peru. We will use the genetic information of feathers collected from clay-licks in combinationwith DNA obtained from blood samples taken from nestlings.

Dispersal plays a crucial role in maintaining genetic diversity and fitness of populations (Reedand Frankham 2003). It shapes distribution patterns, affects population regulation and stability,influences extinction and recolonization dynamics in patchy populations and metapopulations, andcontrols patterns of gene flow, yet, despite the importance of dispersal in animal ecology, remarkablylittle is known about it (Peakall et al. 2003). Thus, we will evaluate the dispersal patterns of ScarletMacaw through the genetic diversity and genetic population structure. Dispersal is also a fundamentalprocess that influences the response of species to landscape change and habitat fragmentation.Obtaining conservation genetic data from both sides of the Inter-oceanic Highway (running throughthe middle of our study area) could enable us to determine the impacts of the new highway on thestudied populations. Rainforests and their biota are especially vulnerable to the environmental changesassociated with roads and linear clearings (Laurance et al. 2009).


7/16


7

Using genetic tagging with microsatellites, combined with the exact location of the nests andclay licks where DNA was collected, we can map out for each macaw family the patterns of clay-licksthey visit from their breeding location and estimate a home-range according to these data. Comparingthe genetic samples around the region (both from nests and clay licks) will show us the estimatedrange that the individuals use. We mainly focus on re-sampling the most frequently used clay-licks byScarlet Macaws across the landscape (roughly in equal distance from each other, in both sides of thehighway) to investigate where we can find genetic clues (feathers) from the same individuals. We willgroup our major clay licks in pairs (e.g. 3x2 design; each pair separated by the highway) to comparethe feathers found at each clay licks to its pair. This way we can investigate if for example individualscross the highway, or it acts as a barrier and separates populations. New restrictions or barriers to geneflow (such as the highway) are unlikely to be detected in genetic diversity until after at least a fewgenerations, but by the genetic tagging method we can observe the effect of the highway indirectly bytracking individuals genetically in different geographic locations.

Feather samples from the clay licks, in addition, provide a true demographic snapshot of thenumber and sex of individuals using the clay licks. Sample of nest trees will also allow us to map outtrue sex-specific dispersal distances i.e. the distance young birds go to breed. This will lead to further

understanding of population genetic structure and could result in more insights into their breedingsystem. Brightsmith (the co-supervisor of this project) and colleagues (Brightsmith et al. 2009) starteda study about the spatial distribution and physical characteristics of clay licks in Madre de Dios, Peru.This is the first attempt of its kind to map out where the clay licks are located in that region. Thisclosely connects to our study, because (1) we need to visit many clay licks for collecting feathers forgenetic material (see below), and (2) we also investigate the effects of clay licks on demography. Wewould also rely on the indications of the satellite collars provide to estimate where the birds aremoving, and we want to collect genetic samples from those sites to be able to describe theirdemography on their entire suspected home range.

Justification

Documentation of the original population genetic structure of Scarlet Macaw populations in our studyarea will serve as base-line genetic data for future genetic studies on the species. This is necessary forevaluating the effects of habitat fragmentation on macaw populations. In particular, the evaluation ofhome-ranges of individual macaws in relation to the new Inter-oceanic Highway will facilitateconservation management and sustainable development plans.

Our results from the Candamo Valley could provide compelling scientific evidence for maintainingand raising the protected status of this biodiversity hotspot.

Assessing the feasibility of our novel non-invasive method to measure home-range, dispersal, andpopulation structure of Scarlet Macaws will provide a powerful new technique for studying theconservation biology of a wide range of parrot species. If we can confirm the utility of this techniquefor Scarlet Macaw, we can then apply this approach of non-invasive genetic tagging for smallendangered populations of other macaw and parrot species, like Blue-throated Macaws, threatened byhabitat loss and fragmentation.

Our research may also have forensic applications. If we detect population genetic differences acrossthe range of Scarlet Macaws, it may be possible to pinpoint geographic origin of traded birds, tradingroutes and where the hunting pressure is highest. This would contribute to the control of illegal pettrade of this species in the region.


8/16


8

Materials and Methods

Macaws are extremely difficult to capture for traditional sources of DNA such as blood. Howevermolted feathers have been evaluated recently as a relatively good source of DNA (Gebhardt et al.2009). For our research we will use the following sources of DNA:

- Feather samples from clay licks and roosting sites- Small blood samples from nestlings- Blood and feather samples from trapped macaws at the clay lick of Tambopata Research

Center (Collpa Colorado) and nearby sites- Macaws captured by local people

The huge abundance of clay licks in South-eastern Peru offers us a perfect place to collect droppedfeathers, as non-invasive samples, by Scarlet Macaws year-round. Previous studies found thatmicrosatellite amplification rates were good for molted feathers, the success rates were highest andgenotyping rates were lowest for large feathers in good condition (Gebhardt et al. 2009). For finding

these necessary samples for DNA we search known clay licks by boat intensively in the study area.We have also discovered new clay licks in the first season of this project. The observations ofBrightsmith et al. (2009) showed that clay licks are a dynamic resource and their availability andposition in the landscape slowly shift over time. In addition to the clay licks, we also collect feathersfrom below the roosting sites of macaws.

We also obtain blood samples and plucked feathers from a limited number of captured adults andnestlings. As a continuation of the satellite telemetry research (Brightsmith et al. 2009), plan to catchmore macaws at the Collpa Colorado and other sites nearby TRC (Brightsmith pers. com.). This isgreatly complementary to our research, and will also lead to further blood and plucked feather samplesfrom these wild birds.

In TRC long term research (Tambopata Macaw Project) continues on the natural history and breedingbiology of large macaws (Brightsmith 2005) and it is possible to gain blood samples and pluckedfeathers from the chicks in the artificial and natural Scarlet Macaw nests around TRC and sometimeseven from their parents captured inside the nest (George Olah pers. obs.) for our study.

Since we need samples from a broader area, we continue to search new natural nests and clay-licksduring our boat journeys up to the smallest rivers and streams in the region. In the first field seasonthis was achieved by following the macaws activity, searching nests in the rainforest, and even withthe help of local people and the satellite telemetry data. Once a new nest is found, we slingshot a lineup the tree, climb it by using ropes and ascenders, and finally get blood and plucked feather samples.

Tambopata Research Center provides a good facility to be used as a base for the study. At the entranceof Candamo Valley, Estacion Biologica la Nube is another research center we could use as a base forour investigations in Candamo.

DNA will be extracted from the samples for:

- microsatellite genotyping- mtDNA amplification- species and individual identification- molecular sexing

DNA will be extracted from the samples using standard techniques (e.g. Murphy et al. 2007). We willbe able to multiply the minute amounts of DNA in feather tips using the Polymerase Chain Reaction


9/16


9

(PCR) method. High quality nuclear and mitochondrial DNA can now be extracted from feathers.However, Horvth et al. (2005) found that the blood clot in the superior umbilicus of feathers providesmore and better quality DNA than the basal tip of the calamus. Despite their lower DNA yield, basaltip samples appeared to be as good a source of DNA as clot samples for mitochondrial DNA fragmentamplifications.

Collaborating with the Scarlet Macaw Genome Project we will develop microsatellite markers in theBiosystematics Center at the Texas A&M University where the molecular laboratories are equippedwith thermocyclers, an ABI 3130 Capillary Electrophoresis, and the means to design polymerase chainreaction (PCR) primers for amplifying DNA loci. Several mtDNA regions (e.g. control region, ND2,COI) will be examined for levels of diversity, and appropriate species specific primers constructed.Primers made for over 50 parrot loci deposited in GenBank will be also screened to identify variablemicrosatellite loci. We envision the use of 10 highly variable loci (or more if variation is low).

After scoring our codominant STR (microsatellite) DNA profiles we will analyze our data usingGenAlExGenetic Analysis in Excel (Peakall and Smouse 2005) and other genetic software.

The scored DNA profile will be analyzed for:

- Forensic style tracking of individuals across the landscape- Home-range estimation based on genetic tracking- GIS analysis of the highways effect on the home-ranges of individuals- Genetic diversity by calculating observed and expected heterozygosity, and allelic richness- Genetic population structure by Bayesian Clustering Analysis and differentiation (pairwise

FST, GST, and allele frequency distributions)- Effective Population Size- Inbreeding- Population fragmentation- Breeding dispersal distances for males and females

Combining all genetic, demographic and ecological data gained, will lead to an excellent basis forPopulation Viability Analysis (PVA) as well.


10/16


10

Logistics

The study is taking place in the Madre de Dios and Puno Region in South-eastern Peru. The coreregion of the research is around Puerto Maldonado, the capital of the Madre de Dios Region. Theprotected areas included in the research are as follows: Tambopata National Reserve (2,747 km 2),Bahuaja-Sonene National Park (10,914 km2), Los Amigos Conservation Concession (1,376 km2),Amarakaeri Communal Reserve (4,023 km2), Madidi National Park, Bolivia (18,958 km2), ManuripiHeath National Amazon Reserve, Bolivia (7,656 km2). The rivers included in the investigation are:Madre de Dios, Tambopata, La Torre, Malinowsky, Chuncho, Tvara, Candamo, Piedras, Amigos,Colorado and Heath. The Map 1. shows the satellite image of the research area.

Map 1. Research area of the study.

Fieldwork lasts 5-6 months each year over the 3-year research period. George Olah, the principal

investigator of this project has been awarded by Endeavour Europe Scholarship from the AustralianGovernment for 2010 and International University Research Scholarship from the Australian NationalUniversity for 2011-2013. Using the support of those grants, the research has already managed toconduct the first fieldwork, and to buy a boat and an eco-friendly (4 strokes) Mercury 40hp boatengine for the research. The Map 2. indicates the area sampled during the first year research. Duringfuture field seasons we will re-sample the area and also collect samples from the other side of theInter-oceanic Highway as well as from the northern part of Bolivia and the Acre province of Brazil(see Map 1.).


11/16


11

Map 2. Sampled area during the first field season of the research in 2010. Red lines indicate the riversystem monitored for nests and clay licks.

Public awareness

In collaboration with the partners outlined below, this PhD project will make the results public inseveral important ways. This will include scientific papers in respected journals, reports to nationalgovernments involved in rainforest road constructions (Peru, Bolivia, Brazil), internationalconservation agencies (e.g. IUCN), and articles in local newspapers both where the birds occur andelsewhere. We also want to give presentations in the local schools of the native communities (e.g.Infierno) teaching the children and their parents about the consequences of logging trees and huntingmacaws. This approach has helped to reduce hunting of several other parrot species in several projectsin Central and South America, such as Brazil (Kyle 2005), Guyana (Johnson and Melville 2005),

Argentina (Masello et al. 2007), and Belize (Studer 2008).Ecotourism and volunteer tourism now have huge potential to fund conservation research programs(Brightsmith et al. 2008). Ecotourism can provide benefits to local communities through generatingincome from nature-based attractions and creating new jobs for local people. During our work wewould encourage the local communities and investors to benefit more from ecotourism. In alreadyexisting eco-lodges (e.g. Posada Amazonas, Refugio Amazonas, Tambopata Research Center,Explorers Inn, Wasai, Inkaterra, etc.) we will give presentations about our work for the tourists.

A documentary film is planned to make about the effects of the Inter-oceanic Highway incollaboration with the Filmjungle.eu Productions (see below), a wildlife film-making initiative.Footage will be taken of the fieldwork of this research and combined with other material on macawsand their habitat, interviews with native land owners, representatives of nature parks, NGOs, oil

companies, road constructors about the conservation issue of the new highway. Footage can be used indifferent versions for specific purposes (e.g. some 5-10 min-long versions for schools, for local


12/16


12

communities, for researchers, for fundraising aims, for tourists). We would also target Youtube, localtelevision, and make DVDs for distribution where appropriate.

Finally, we will design printed materials about the conservation biology of macaws. Our bulletins andposters will be distributed in the local schools and universities. In addition a homepage of this projectwill be launched with all available information about the macaws and the research.

Collaboration

Tambopata Macaw ProjectThe Tambopata Macaw Project is a long term research project on the ecology andconservation of macaws and parrots in the lowlands of South-eastern Peru under the directionof Dr. Donald Brightsmith of the Schubot Exotic Bird Health Center at Texas A&MUniversity. The project has been working with wildlife and local communities since 1989. Along history of dedicated research and monitoring has provided many insights into variousaspects of parrot and wildlife of SE Peru.www.macawproject.org

Texas A&M UniversityWe have been working together with researchers and veterinarians from the Schubot ExoticBird Health Center, Department of Vet Pathobiology, College of Veterinary Medicine &Biomedical Sciences at TAMU. We collaborate with the Scarlet Macaw Genome Project ofthe TAMU (directed by Prof. Ian Tizard) in developing microsatellite loci. Anothercooperation aim with TAMU is to standardize a chromosome based telomere aging techniquefor wild Scarlet Macaws.www.cvm.tamu.edu/schubot

Filmjungle.eu ProductionsThis brand new and quite small production team was founded by documentary filmmakersfrom all over Hungary with a sole aim: to produce scientific- and wildlife films for the globalmarket. They have worked in many interesting corners of the world. From remote places likeAntarctica, Papua New Guinea, Greenland or Africa, to their closest neighborhood in theCarpathian Basin in Europe they have collected thousands of hours of SD and HD materialand produced several award winning documentaries. They appeared on the internationalmarket with the HD bluechip wildlife documentary 'Budapest Wild' which was awarded withmore than 15 international prizes. By now their broadcast partners include NDR (Germany),YLE (Finland), and RAI (Italy).www.filmjungle.eu

Rainforest Expeditions S.A.C.Rainforest Expeditions (est. 1989) operates three award winning Amazon lodges: PosadaAmazonas, Refugio Amazonas, and Tambopata Research Center. At Rainforest Expeditionsthey build sanctuaries that host unique experiences, engage local communities, and help hostsand guests learn to value their relation with the nature. The company has supported the firstfield season of this research by hosting it in the Tambopata Research Center and helped inmany aspects of the projects logistics.www.perunature.com

Outputs

PhD Thesis by 2013

Approximately five high quality scientific papers in international conservation and genetics journals

Each year: Report to the Peruvian National Service of Protected Areas (SERNANP - ServicioNacional de reas Naturales Protegidas)

End of the study: Final Report to SERNANP, the Peruvian Government, and NGOs

Documentary film of the macaws conservation in relation with the Inter-oceanic Highway
http://www.macawproject.org/http://www.macawproject.org/http://www.macawproject.org/http://www.cvm.tamu.edu/schubothttp://www.cvm.tamu.edu/schubothttp://www.cvm.tamu.edu/schubothttp://www.filmjungle.eu/http://www.filmjungle.eu/http://www.filmjungle.eu/http://www.perunature.com/http://www.perunature.com/http://www.perunature.com/http://www.perunature.com/http://www.filmjungle.eu/http://www.cvm.tamu.edu/schubothttp://www.macawproject.org/


13/16


13

Bulletins, posters and a homepage of the project

Timetable of activities

December 2009April 2010: First field research season of this project was conducted in SE PeruMayOctober 2010: Sequencing Scarlet Macaw genome (Texas A&M University);

Analyzing previous breeding biology data of Scarlet Macaws, andwriting grant applications (Australian National University)

November 2010April 2011: Second field season of the research in SE Peru/Northern Bolivia

MayOctober 2011: Developing genetic markers and primers on the sequenced ScarletMacaw genome; Extracting DNA from samples and running thegenetic tests on them (at the Texas A&M University)

November 2011April 2012: Third field season of the research in SE Peru/Brazil

MaySeptember 2012: Extracting DNA from samples and running the genetic tests on them(at the Texas A&M University)

October 2012January 2013: Statistical analysis of the genetic data collection (Australian NationalUniversity)

FebruaryJune 2013: Writing up PhD thesis and reports (Australian National University)


14/16


14

References

Bennett, P. M. and Owens, I. P. F. (1997). Variation in extinction risk among birds: chance orevolutionary predisposition? Proceedings of the Royal Society of London B 264: 401-408.

Brightsmith, D. (2004). Effects of weather on parrot geophagy in Tambopata, Per. Wilson Bulletin116: 134-145.

Brightsmith, D., Stronza, A., et al. (2008). Ecotourism, conservation biology, and volunteer tourism: Amutually beneficial triumvirate.Biological Conservation 141(11): 2832-2842.

Brightsmith, D., Vigo, G., et al. (2009). Spatial distribution and physical characteristics of clay licks inMadre de Dios, Peru. Unpublished report- Texas A&M University, College Station, Texas.

Brightsmith, D. J. (2005). Parrot nesting in southeastern Peru: seasonal patterns and keystone trees.Wilson Bulletin 117: 296-305.

Brightsmith, D. J. (2008). Satellite telemetry of large macaws in Tambopata, Peru. Unpublished reportto the Wildlife Protection Foundation - Schubot Exotic Avian Health Center, Texas A&M University,College Station, Texas.

Brightsmith, D. J. and Boyd, J. (2006). Testing satellite telemetry tags for psittacines in Tambopata,Peru. Unpublished report to the Loro Park Foundation, North Star Technologies, and Amigos de lasAves Psittacine Conservation Fund - Schubot Exotic Avian Health Center, Texas A&M University,College Station, Texas.

Burger, J. and Gochfeld, M. (2003). Parrot behavior at a Ro Manu (Per) clay lick: temporal patterns,associations, and antipredator responses.Acta Ethologica 6: 23-34.

Conover, T. (2003). Pers long haul: highway to riches, or ruin?National Geographic 203: 80-100.

Finer, M., Jenkins, C. N., et al. (2008). Oil and Gas Projects in the Western Amazon: Threats toWilderness, Biodiversity, and Indigenous Peoples. PLoS ONE3(8): e2932.

Gebhardt, K. J. (2007). Using Molted Feathers as a Source of DNA to Study Genetic Diversity andPopulation Structure of Macaws in the Amazon Rainforest of Per. M.Sc. Thesis College of GraduateStudies, University of Idaho.

Gebhardt, K. J., Brightsmith, D., et al. (2009). Molted feathers from clay licks in Peru provide DNAfor three large macaws (Ara ararauna, A. chloropterus, and A. macao).Journal of Field Ornithology

80(2): 183-192.

Gebhardt, K. J. and Waits, L. P. (2008). Cross-species amplification and optimization of microsatellitemarkers for use in six Neotropical parrots.Molecular Ecology Resources 8(4): 835-839.

Gilardi, J. D., Duffey, S. S., et al. (1999). Biochemical functions of geophagy in parrots: detoxificationof dietary toxins and cytoprotective effects.Journal of Chemical Ecology 25: 897-922.

Hesse, A. J. and Duffield, G. E. (2000). The status and conservation of the Blue-Throated Macaw Araglaucogularis.Bird Conservation International 10: 255-275.

Horvth, M., Martnez-Cruz, B., et al. (2005). An overlooked DNA source for non-invasive geneticanalysis in birds.Journal of Avian Biology 36: 84-88.


15/16


15

Johnson, M. and Melville, S. (2005). Guayana - land forsaken or preserved? Country at a crossroad.PsittaScene 17(4): 6-7.

Kyle, T. (2005). Gold Rush: The search for Golden Conures in western Par, Brazil. Psitta Scene 17:2-5.

Laurance, W. F., Albernaz, A. K. M., et al. (2002). Predictors of deforestation in the BrazilianAmazon.Journal of Biogeography 29: 737-748.

Laurance, W. F., Goosem, M., et al. (2009). Impacts of roads and linear clearings on tropical forests.Trends in Ecology and Evolution 24(12): 659-669.

Lee, A. T. K., Kumar, S., et al. (2010). Parrot claylick distribution in South America: do patterns ofwhere help answer the question why?Ecography 33: 503-513.

Masello, J. F., Failla, M., et al. (2007). Reserve status: parrot colony aims for legal protection.PsittaScene 19(4): 13-15.

Miyaki, C. Y., Griffiths, R., et al. (1998). Sex Identification of Parrots, Toucans, and Curassows byPCR: Perspectives for Wild and Captive Population Studies.Zoo Biology 17: 415-423.

Munn, C. A. (1992). Macaw biology and ecotourism, or When a bird in the bush is worth two in thehand. Page: 47-72. In: New World Parrots in Crisis: Solutions from Conservation Biology. S. R.Beissinger and Snyder, N. F. R., eds. Smithsonian Institution Press, Washington and London.

Murphy, S., Double, M., et al. (2007). The phylogeography of palm cockatoos, Probosciger aterrimus,in the dynamic Australo-Papuan region.Journal of Biogeography 34(9): 1534-1545.

Palsboll, P. J. (1999). Genetic tagging: contemporary molecular ecology. Biological Journal of the

Linnean Society 68: 3-22.

Peakall, R. and Beattie, A. J. (1996). Ecological and genetic consequences of pollination by sexualdeception in the orchid Caladenia tentactulata.Evolution 50: 2207-2220.

Peakall, R., Ebert, D., et al. (2006). Markrecapture by genetic tagging reveals restricted movementsby bush rats (Rattus fuscipes) in a fragmented landscape.Journal of Zoology 268: 207216.

Peakall, R., Ruibal, M., et al. (2003). Spatial autocorrelation analysis offers new insights into geneflow in the Australian bush rat,Rattus fuscipes.Evolution 57(5): 11821195.

Peakall, R. and Smouse, P. E. (2005). GenAlEx 6: Genetic Analysis in Excel. Population genetic

software for teaching and research, The Australian National University, Canberra, Australia.http://www.anu.edu.au/BoZo/GenAlEx

Peakall, R. and Smouse, P. E. (2009). An Introduction to Genetic Analysis for Population Studies.Australian National University, Canberra, Australia.

Reed, D. H. and Frankham, R. (2003). Correlation between fitness and genetic diversity. ConservationBiology 17: 230-237.

Ruibal, M., Peakall, R., et al. (2009). Field-based evaluation of scat DNA methods to estimatepopulation abundance of the spotted-tailed quoll (Dasyurus maculatus), a rare Australian marsupial.Wildlife Research 36: 721-736.
http://www.anu.edu.au/BoZo/GenAlExhttp://www.anu.edu.au/BoZo/GenAlExhttp://www.anu.edu.au/BoZo/GenAlEx


16/16


16

Ruibal, M., Peakall, R., et al. (2010). Advancement to hair-sampling surveys of a medium-sizedmammal: DNA-based individual identification and population estimation of a rare Australianmarsupial, the spotted-tailed quoll (Dasyurus maculatus). Wildlife Research 37: 27-38.

Studer, K. (2008). Leaving Wild to Save a Species. PsittaScene 20(3): 16-17.

Taberlet, P. and Luikart, G. (1999). Non-invasive genetic sampling and individual identification.Biological Journal of the Linnean Society 68: 41-55.

Tickell, O. (1993). Highway threatens Tambopata. Geographical 65: 7-9.

Waits, L. P., Luikart, G., et al. (2001). Estimating the probability of identity among genotypes innatural populations: cautions and guidelines.Molecular Ecology 10: 249-256.

Yamashita, C. and Machado de Barros, Y. (1997). The Blue-throated Macaw Ara glaucogularis:characterization of its distinctive habitats in savannahs of the Beni, Bolivia.Ararajuba 5(2): 141-150.

Olah Proposal and Progress Report 2010

Documents

Transcript of Olah Proposal and Progress Report 2010