PhD Proposal Demographic analysis and population genetic structure of large macaws in South-eastern Peru

Introduction The Amazon basin contains a highly diverse, incredibly complex, and globally important ecosystem that 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, are lacking or poorly understood (Munn 1992). Approximately 26% of the Neotropical parrots are classified as threatened (Birdlife International 2006) and the psittacines are considered the most endangered large bird family in the world (Bennett and Owens 1997). It is impossible to measure or predict the effects of deforestation, habitat fragmentation, human impact, hunting, etc. on species whose ecology is unknown. Recent studies, like the Tambopata Macaw Project in South-eastern Peru provide more insight into these species breeding ecology and natural history (Brightsmith 2005), but studies at the demographic and population genetic level in a wider range of species and study sites are still needed. Molted feathers are a promising and
underutilized source of data for genetic studies of birds in temperate and tropical ecosystems (Gebhardt et al. 2009). In this study we aim to reconstruct

demography and spatial ecology by using genetic tagging (the identification of individuals by genetic markers) of the following species in South-eastern Peru: Red-and-green Macaw (Ara chloropterus), Blue-and-yellow Macaw (Ara ararauna), Mealy Parrot (Amazona farinosa), and Blue-headed Parrot (Pionus menstruus). These species are quite different in ecology and also body size, which would provide a good reason for comparison. Knowledge of the movements of individuals across the landscape, and patterns of spatial genetic structure including variation between sexes, age-classes, and time promise to provide important clues about dispersal and other aspects of a species biology (Peakall et al. 2003). Underlining the conservation importance of our proposed project, a highway is about to be built running from the Atlantic to the Pacific Ocean (Tickell 1993, Conover 2003). This Interoceanic (or Transamazonian) Highway will run through Puerto Maldonado and our proposed study area in Southeastern Peru and will increase human use of the protected area in this region. Increasing the accessibility of tropical rainforest habitat by road development often has negative impacts on animal populations through habitat destruction (due to selective logging and deforestation), chemical pollution, edge effects, road-related mortality or capture, barrier effects, exotic species invasion and human invasions (hunters, miners, etc.) (Laurance et al. 2009). Parrot 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 due to

selective logging can reduce parrot reproduction and threaten population viability. Parrots also face an additional hazard as in many parts of the Amazon they are hunted for food, their feathers, or for the illegal pet trade, so increased human use of areas can negatively impact parrot numbers as a result of these processes. Another conservation concern in the study area is that oil companies are starting to explore areas with still largely intact ecosystems, like the Candamo Basin (Finer et al. 2008).

Study site The study would place in the Madre de Dios and Puno Region in Southeastern Peru. The core region of the research would be around Puerto Maldonado, the capital of the Madre de Dios Region. The protected areas included in the proposed research are as follows: Tambopata National Reserve (2,747 km2), 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). The rivers included in the investigation are: Madre de Dios, Tambopata, La Torre, Malinowsky, Chuncho, Tvara, Candamo, Piedras, Amigos, Colorado and Heath.

Previous genetic research in the region and results To date, only Gebhardt`s M.Sc. thesis (2007) used non-invasive genetic sampling of feathers to evaluate the genetic diversity, population genetic

structure, and the effective population size of Red-and-green Macaw (Ara chloropterus) and Scarlet Macaw (Ara macao) 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 the mitochondrial control region. They found that diversity was high across intact habitat for both macaws, but was higher for Scarlet Macaw than for Red-and-green Macaw. For each species, their analyses 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 Scarlet Macaws have more restricted gene flow than Red-and-green Macaws. Trends of female-biased gene flow for Scarlet Macaws were also observed and they suggest further investigation with expanded genetic sampling to prove this. Their effective population size estimates for Scarlet Macaws were three to five times larger than for Red-and-green Macaws. The researchers concluded that the processes that drive the genetic diversity and population structure of both Scarlet and Red-and-green Macaws operate at a large spatial scale and additional sampling outside of Peru is needed to evaluate genetic neighborhood size. They expect the genetic structure of macaws to be driven by isolation by distance because there are few suspected barriers to these highly vagile species within intact forest. The lack of differentiation among the studied locations indicates that the sampling scale was too small and they had too few sampling spots. They caution against using their estimates of effective population size as absolute values for conservation, mainly because of the potential for high gene flow with unsampled areas. No other population genetic studies of macaws and particularly parrots were carried out in this region.

Project Objectives In the pristine rainforests of South-eastern Peru populations of most native species remain large and undisturbed, suggesting that it is safe to assume that these populations reflect natural dynamics. South-Eastern Peru provides an ideal place to investigate the demography and population genetics of these poorly understood species. The main aim of this study is to reconstruct demographic traits of a variety of parrot species using population genetic methods in this intact habitat of Peru, and also to provide a baseline to measure how they may be affected by habitat loss in other areas. To achieve that, we would like to estimate the genetic diversity, genetic population structure and effective population size. Since 2003 a group of parrot researchers, conservation organizations, and manufacturers have been working together to design and test satellite collars for tracking large macaws. To date, this consortium has developed two prototypes which were tested e.g. on captive macaws in the Tambopata Research Center (TRC), South-eastern Peru (Brightsmith and Boyd 2006). The preliminary data indicate long distance movements of

Blue-and-yellow Macaws, during the periods when food supply was low in the areas around TRC (Brightsmith 2008). As part of our research we aim to do the next step in these analyses: to identify the habitat features in the distant areas where the birds are settling for these periods. Those sites may contain keystone food resources which allow the birds to survive the seasons of low food availability near the capture site. We also want to collect genetic samples from those sites to be able to describe their demography on their entire suspected home range. However, satellite telemetry is still very expensive and does not always provide the desired information. It also requires catching wild birds, which for macaws is a huge effort. Because of the high cost of each satellite-collar it can be applied for only a few birds, which can provide misleading generalization for the 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 different dispersal patterns. In our study we want to provide information about the demography of the population but instead of tracking individuals, like in mark-recapture studies, using genetic samples of the population. The upcoming data of the satellite telemetry study on macaws would also help to understand the demography of these species, as it would provide detailed information about their movement. These complementary data can be combined with our demographic results about their home-range. Parrots 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. A recent study (Lee et al. 2009) showed that the highest clay lick distribution in South America is found in South-eastern Peru, in our study area. We would use the genetic information of feathers collected from claylicks in combination with DNA obtained from nests of the same species. A good sample of nest trees will allow us to map out true 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 breeding system. Knowing the exact location of the nests where DNA was collected, and using forensic method of microsatellites, we could also map out for each family the patterns of claylicks they visit from their breeding location and estimate a home-range according to these data. Collected feather samples from the clay licks, in addition, would provide a true demographic snapshot of the number, sex, and age of individuals using the clay licks. Genetic tagging is a newly emerging technique in molecular ecology, which enables the unique identification of individuals by their hypervariable genetic markers, and has proven to be a highly effective method in other studies (Peakall et al. 2003, Peakall et al. 2006). This would mean representative sampling in the area, most cost efficiency, and would provide similar results like the capture-mark-recapture studies, which otherwise would be almost impossible for macaws.

Brightsmith et al. (2009) conducted research about the spatial distribution and physical characteristics of clay licks in Madre de Dios, Peru. We want to continue their work which was the first attempt of its kind to map out where the clay licks are located in that region. This will closely fit to our study, because (1) we need to visit many clay licks for collecting feathers for genetic material (see below), and (2) we also want to investigate the effects of clay licks on demography. Dispersal plays a crucial role in maintaining genetic diversity and fitness of populations (Reed and Frankham 2003). It shapes distribution patterns, affects population regulation and stability, influences extinction and recolonization dynamics in patchy populations and metapopulations, and controls patterns of gene flow, yet, despite the importance of dispersal in animal ecology, remarkably little is known about it (Peakall et al. 2003). Thus, we would evaluate the dispersal patterns of these species through the genetic diversity and genetic population structure. Dispersal is also a fundamental process that influences the response of species to landscape change and habitat fragmentation. The construction of the Interoceanic Highway just in the middle of our proposed study area (running through Puerto Maldonado) provides a strong conservational outcome for this study. Obtaining population genetic data during the construction should enable us to determine the impacts of the Interoceanic Highway on the studied populations. Rainforests and their biota are especially vulnerable to the environmental changes associated with roads and linear clearings (Laurance et al. 2009). Candamo Basin lies between the Amazon Lowland Rainforests of Southeastern Peru and the Andes. The scope of natural habitats contained within this area result in spectacular biodiversity. Around the Candamo Basin, the foothills of Andes form a high barrier, like a fortress. This place will be an important sampling site for our study, since due to its locality it can contain isolated populations of species. Investigating this, we would like to compare the genetics of macaws inside and outside of Candamo. In September 2007 a proposed bill was to be deliberated by the Peruvian Congress to reduce the size of the Bahuaja-Sonene National Park by 200 000 ha (19.22%.), 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 a major threat to Candamo as it has a massive hydrocarbon deposit. The beauty of the huge National Parks of

Peru is that they are areas designated to be free of human disturbance. Not even tourism is permitted, and scientific research only takes place with special permission. These areas are sanctuaries for the wildlife of the area, and a source of pride for the Peruvian people. Our research in Candamo could provide a strong scientific basis built on genetic data, which was not carried out before, urging the authorities to maintain the protected status of this region.

Materials and methods

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

Feather samples from clay licks and roosting sites Blood and feather from chicks in the nest (in TRC and other areas) Blood and feather from trapped parrots/macaws at the clay lick of TRC (Collpa Colorado) and nearby sites Captured parrots/macaws by the local trade

DNA will be extracted from the samples for: -

microsatellite genotyping mtDNA amplification species and individual identification molecular sexing

We will analyse our data for:

-

Genetic diversity by calculating heterozygosity, and allelic richness

observed

and

expected

Genetic population structure by Bayesian Clustering Analysis and differentiation (pairwise FST, GST, and allele frequency distributions) Effective Population Size Breeding dispersal distances for males and females Forensic style tracking of individuals across the landscape

The huge abundance of clay licks in South-eastern Peru offers us a perfect place to collect dropped feathers by macaws and parrots year-round. For finding these necessary samples for DNA we will search clay licks by boat intensively in the study area. Given the fact that Brightsmith et al. (2009) have already recorded many locations of clay licks, we could use these coordinates to find that places again to collect samples. We also expect to discover clay licks that haven`t been described before. The observations of Brightsmith et al. (2009) showed that clay licks are a dynamic resource and their availability and position in the landscape slowly shift over time.

In addition to the clay licks, we could also collect feathers from below the roosting sites of the species.

Our other method for finding a different source of DNA, and compare these to the DNA from the feathers collected at the clay licks, would be blood and plucked feather sampling from captured adults, and chicks. Plucked feathers were found to be an excellent source of DNA, with significantly higher success rates and lower error rates than for molted feathers (Gebhardt et al. 2009). As a continuation of the satellite telemetry research (Brighthsmith 2009), plan to catch more parrots and macaws at the Collpa Colorado and other sites nearby TRC in the upcoming years (Brightsmith pers. com.). This will be greatly complementary to our planned research, and will also lead to further blood and plucked feather samples from these wild birds. In TRC long term research (Tambopata Macaw Project) continues of the natural history and breeding biology of large macaws (Brightsmith 2005) and it will be possible to gain blood samples and plucked feathers from the chicks in the artificial and natural macaw nests around TRC and sometimes even from their parents captured inside the nest (George Olah pers. obs.) for our study. Since we need samples from a broader area, we strongly intend to search new natural nests during our boat journeys up to the smallest rivers and streams. This can be achieved by following the parrots` activity, searching nests in the rainforest, and even with the help of local people and the satellite telemetry data. Once a new nest is found, we can slingshot a line up the tree, climb it by using ropes and ascenders, and finally get our desired blood and plucked feather samples. Tambopata Research Center provides a good facility to be used as a base for the study. The Tambopata Macaw Project monitors nests in a 2 km radius around TRC. We also propose to go further and search through a much larger area for new nests by the above described methods. This would mean camping in the rainforest for weeks and returning to TRC regularly for supply. At the entrance of Candamo valley, Estacion Biologica la Nube is another research center previously owned by the local authority of the protected area but it is abandoned for now. Making some improvement on the building, we could use this station as a base for our investigations in Candamo.

Combining all genetic, demographic and ecological data gained during these research projects will lead to an excellent basis for Population Viability Analysis (PVA).

Further projects

1) Characteristics of endangered parrots

This will be a literature review on the endangered parrots worldwide, focusing on habitat modelling, treat risks, body size, foraging habit, food and distribution.
2) GIS analysis on spatial distribution of nests and clay licks

We would like to analyse how nest and clay lick distributions are related to demography and habitat use of the macaws. We will use GIS software with variables like coordinates of nests, clay licks, trees ideal for nesting, geography, water bodies, vegetation, etc. We will collect these data during our field trips. 3)Collpa mapping and distribution Since we need to visit the clay lick sites for collecting dropped feathers, we can also collect soil and plant samples from those areas and take the necessary measurements of the clay licks. 4)Habitat loss in Bolivia To try to quantify a genetic measure for how macaws are affected by habitat loss, we would like to get samples from Beni province, Northern Bolivia. This location is relatively near (500 km) to our intended core research area (TRC) in South-eastern Peru, but the habitat differs a lot. Earlier it contained grassland, but today it`s used for cattle ranching with a big human influence. No protected area exists on this part of Bolivia. Red-and-green Macaw and Blue-and-yellow Macaw also inhabit this region, offering a good study on habitat loss influencing natural dynamics in population genetic level, as we can also find their populations in the intact South-eastern Peru. We would include the Blue-throated Macaw (Ara glaucogularis) in the study as well, because this is the most endangered macaw today, only ~150 individuals left in the wild, and living only in Beni province, Bolivia. Collecting genetic samples from the remained populations of the Blue-throated Macaw would allow us to compare their population genetic structure to similar macaw species still maintaining healthy population in South-eastern Peru. Two NGO projects (WPT and ARMONIA) are running to conserve the Blue-throated Macaw in its original habitat. We would initiate cooperation with these organizations to collect the necessary genetic samples from the area.

5)Small parrot breeding biology in TRC In case of the smaller parrot species in and around TRC, the nesting records are poor, undocumented, and less studied. Conducting nest searches year-round and mainly in the peak of the small species breeding season (between May and August) would reveal significantly more small species breeding.

References

Bennett, P. M., Owens, I. P. F. 1997. Variation in extinction risk among birds: chance or evolutionary 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 Bulletin 116:134-145. 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 report to the Wildlife Protection Foundation, Schubot Exotic Avian Health Center, Texas A&M University, College Station, Texas. Brightsmith, D. J. 2009. Satellite telemetry of large macaws in Tambopata, Peru. Unpublished report to the Wildlife Protection Foundation, Schubot Exotic Avian Health Center, Texas A&M University, College Station, Texas. Brightsmith, D. J. and J. Boyd. 2006. Testing satellite telemetry tags for psittacines in Tambopata, Peru. Unpublished report to the Loro Park Foundation, North Star Technologies, and Amigos de las Aves Psittacine Conservation Fund, Schubot Exotic Avian Health Center, Texas A&M University, College Station, Texas. Brightsmith, D, G Vigo, and A Valds-Velsquez. 2009. Spatial distribution and physical characteristics of clay licks in Madre de Dios, Peru. Unpublished report. Texas A&M University, College Station, Texas. Burger, J. and M. Gochfeld. 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., Pimm, S.L., Keane, B. and Ross, C. 2008 Oil and Gas Projects in the Western Amazon: Threats to Wilderness, Biodiversity, and Indigenous Peoples. PLoS ONE 3(8): e2932. Gebhardt, K. J. 2007. Using Molted Feathers as a Source of DNA to Study Genetic Diversity and Population Structure of Macaws in the Amazon Rainforest of Per. M.Sc. Thesis, College of Graduate Studies, University of Idaho. Gebhardt, K. J., Brightsmith, D., Powell, G. and Waits, L. P. 2009. Molted feathers from clay licks in Peru provide DNA for three large macaws (Ara ararauna, A. chloropterus, and A. macao). Journal of Field Ornithology 80(2): 183-192. Gilardi, J. D., S. S. Duffey, and C. A. Munn. 1999. Biochemical functions of geophagy in parrots: detoxification of dietary toxins and cytoprotective effects. Journal of Chemical Ecology 25:897-922. Laurance, W.F., Albernaz, A.K.M., Schroth, G. 2002. Predictors of deforestation in the Brazilian Amazon. Journal of Biogeography 29:737748. Laurance, W.F., Goosem, M. and Laurance, S.G.W. 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., Brightsmith, D. J. and Marsden, S. J. 2009. Parrot claylick distribution in South America: do patterns of where help answer the question why? Ecography 32: 1-11. Munn, C. A. 1992. Macaw biology and ecotourism, or When a bird in the bush is worth two in the hand. Pages 47-72 in S.R. Beissinger and N.F.R. Snyder, eds. New World Parrots in Crisis: Solutions from Conservation Biology. Smithsonian Institution Press, Washington and London. Peakall, R., Ebert, D., Cunningham, R. and Lindenmayer, D. 2006. Mark recapture by genetic tagging reveals restricted movements by bush rats (Rattus fuscipes) in a fragmented landscape. Journal of Zoology 268: 207216. Peakall, R., Ruibal, M. and Lindenmayer, D.B. 2003. Spatial autocorrelation analysis offers new insights into gene flow in the Australian bush rat, Rattus fuscipes. Evolution 57(5): 11821195. Reed, D.H., and R. Frankham. 2003. Correlation between fitness and genetic diversity. Conservation Biology 17:230-237. Tickell, O. 1993. Highway threatens Tambopata. Geographical 65:7-9.