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1.1 The effects of habitat fragmentation on species. Habitat fragmentation is considered to be the main driver of extinction globally (Young & Clarke, 2000; Honnay & Jacquemyn, 2007; Hobbs, 2007; Klanderud et al., 2010). It is considered to have three major components (1) the pure loss of habitat, (2) reduction in patch size and (3) the increasing isolation of patches (Honnay et al., 2005). Habitat fragmentation affects both plants and animals and these effects are often integrated. Plant species that require animals to disperse the seeds can be just as affected by something as simple as a small cleared strip of land for telegraph poles as the more severe clearing for development and agriculture; if the matrix becomes inhospitable or not traversable by dispersal vectors (Soons & Ozinga, 2005; Aguilar et al., 2006). Fragmentation can also result in the increased severity of the effects of environmental events. Many authors have described the effects of fragmentation and increased edges on ecosystems (Laurence et al., 2006; Newmark, 2008; Olupot, 2009). Laurence et al. (2006) found that edge effects such as increased mortality and decreased recruitment success in the Amazon went as deep as 100m from the fragment boundaries. However when judging the significance of edge effects on a species, the individual responses of individual species must be considered as not all species will be affected in exactly the same way (Tilman & Downing, 1994; Laurence et al., 2006).
One of the biggest effects of habitat fragmentation is thought to be decreased population size and increased isolation of patches (Honnay et al., 2005). Demographic effects of small population size and isolation would be the most obvious due to the unpredictability of the environment. Studies claim that these small patches of habitat often contain only small populations which are highly susceptible to environmental stochasticity, and therefore extinction (Hobbs, 2007; Shaffer, 1981; Lande, 1988). Rare species are believed to be the worst affected, as they often do not have the numbers or the genetic diversity to withstand any disruption to their natural cycles (Ellstrand & Elam, 1993; Byer and Waller; 1999). However it has been argued that whilst rare species are under a high amount of stress from these disturbances, some species are able to persist if they are able to maintain dispersal and gene flow (Shapcott, 2000). For example, it was found by Shapcott (2000) that isolated populations of Syzygium nervosum had no significant correlations with population isolation, patch size of genetic diversity. It was concluded that this was due to gene flow between populations via seed dispersal by mobile frugivores. Conversely it has been found that species with naturally limited dispersal mechanisms such as Ceanothus venecosus were not affected by increasing isolation of species, only by a reduction in patch size (Lawson et al., 2010). This indicates that although many general assumptions can be made about species in isolated populations, it is necessary to understand the characteristics of the species before conclusions can be drawn.
Metapopulation theory explains the relationship of patches of habitat embedded in a matrix of non-habitat (Hanski, 1991). This essentially isolates the patches, however if the species within the patches are within the dispersal range of other populations the species can persist. This means that even though the smaller populations are under higher risk from environmental stochasticity, other nearby populations are still able to repopulate the area. This phenomenon is known as a 'rescue effect' (Hanski, 1991). This effect may still be nullified by increasing isolation, as if the patches of populations are further isolated from each other to the point where they are outside each others dispersal range, the genetic integrity of the metapopulation and of the species as a whole will be threatened.
Land use change is another cause of fragmentation, specifically by changed fire regimes (Lawson et al., 2010; McConnell & Sweeny, 2005). Managing fire regimes is an important part of conservation, as the ecosystems have adapted to the natural fire regimes in their habitat and may not react well to changes. However it has been discovered that if the interval between fires is too long, it may result in an increase in fire frequency and intensity which could be detrimental to the natural values (Whitehead et al., 2008). Mismanagement of fire regimes can be detrimental to species that are obligate seeders and require fire for reproduction. They are exposed to two risks: (1) senescence risk where fires occur at overly long intervals and the seed banks are depleted; and (2) an immaturity risk, where fires intervals are shortened to the point where seed banks are not developed enough to survive the frequent burnings (Lawson et al., 2010). Frequent burnings of landscapes for, for example, increased pick for cattle grazing, could expose any small immature populations newly growing in the landscape to irreparable damage.
1.2 The effects of habitat fragmentation on the genetics of a species
Rare species, compared to common and widespread species, are thought to have less genetic diversity, lower reproductive success and higher levels of inbreeding (Frankham, 1996), especially in small populations. For example Dowe, Benzie and Ballment (1997) found very little genetic diversity in the endangered palm species Carpoxylon macrospermum and Shapcott (1998) found extremely low levels of genetic diversity in the endangered palm species Ptychosperma bleeeseri. These observations are supported by many studies which in addition to this also state the importance of gene flow between populations to maintain an acceptable level of genetic variation and the effects of geographical isolation on the genetic variation of a species (Sinclair & Hobbs, 2009; Ægisdóttir et al., 2009; Honnay & Jacquemyn, 2007). For example, Honnay and Jacquemyn (2007) found that based on the results for 52 plant species, smaller populations consistently contained less genetic variation than larger populations. On the contrary, it was found by Shapcott et al., (2007) that the endangered palm species Beccriophoenix madagascariensis exhibited a considerable level of diversity.
Gonzales et al. (2010) explained the importance of understanding the effects of isolated population spatial genetic structure of species when assessing the evolutionary dynamics of a population, a concept which is agreed upon by other studies (Shapcott et al., 2007). However, there are arguments that the development of local genetic structure in populations is weak or does not occur at all when there is even a small amount of gene flow to counter the effect of population isolation (Peakall & Beattie, 1996). For example Peakall and Beattie (1996) found that seed dispersal was sufficient to minimize genetic differentiation.
Habitat fragmentation is one of the main reasons for the development of isolated populations. Many studies are consistent in the predictions that small populations are highly subject to genetic drift, resulting in the loss of potentially very important alleles and a decrease in the overall genetic diversity of the species (Ellstrand & Elam, 1993; Byers & Waller, 1999; Ægisdóttir et al., 2009; Barrett & Kohn, 1991; Young et al., 1996; Lowe et al., 2004; Trizio et al., 2005; Fulgione et al., 2009). However, in the case of long lived species the effect of genetic drift is reduced by extended time between generations, therefore reducing the number of alleles lost through drift (Young et al., 1996; Honnay & Bossuyt, 2005). For example, Ægisdóttir et al. (2009) found high within-population genetic diversity for the rare perennial Campanula thyrsoides which they believed could be explained by the longevity of the individuals.
Increasing the isolation of a species geographically can result in an increase in the levels of inbreeding. Depending on the species, increased inbreeding can lead to a reduced fitness for its environment and hence has implications for the survival of the species (Ellstrand & Elam, 1993). Byers and Waller's (1999) results support this theory, they found that although some species are able to cope with inbreeding depression by preferentially eliminating the detrimental alleles through purging, it can reach the point where the persistence of the species is threatened. Diversity and fitness for the environment depend on the species, for example severe inbreeding of a species is usually considered harmful in terms of the fitness of the species when considering its ability to adapt to environmental changes (Keller & Waller, 2002; Reed & Frankham, 2003). However there are palm species which contain high levels of apparent inbreeding within populations, for example studies of the Pinaga species of palm found high levels of genetic variation between populations, but high levels of inbreeding within populations (Shapcott, 1999). This was attributed to the species self-compatibility and that they probably self-fertilize.
Isolated populations are, as the name suggests, sparsely distributed throughout an area. This means that even in a terrestrial scenario, the island biogeography theory can be easily applied as the matrix between populations can be akin to the ocean which is a very effective dispersal barrier (Janzen, 1968). Plants that can exist for example, only on balds or limestone outcrops often find dispersal difficult, especially if their reproductive traits limit dispersal (Wyatt, 1997; Loehle, 2006). This has implications for the persistence of a species and its ability to adapt to changing from outcrossing to inbreeding.
1.3 The restoration of plant species within fragmented habitats
Restoring a species or ecosystem that has been fragmented is a complex issue which must take into consideration many different aspects and influences. Conserving just one species requires a very different approach than when attempting to conserve a whole ecosystem (Shapcott et al., 2007). Understanding the species' genetic variation and genetic partitioning across its geographic range is important when determining restoration or conservation plans for single species (Coates & Hopper, 2000). Unfortunately lack of funding can often results in conservation and restoration projects being tailored to have the most effect possible for the least amount of money. Consequently conservation and restoration efforts in fragmented habitats is often focused on a smaller number of populations that would give the best results or provide the best chance for the persistence of the targeted species or ecosystem (Pareliussen et al., 2006). Understanding the genetic diversity within these isolated populations provides essential information when restoration efforts are being assigned (Shapcott et al., 2007). Only very thorough and costly analysis of the species would provide a highly accurate restoration program, which would, due to the nature of the ever-changing environment, still have risks associated with it. The benefits of finding a rare, restricted species to study are that there is a greater chance to study the species as a whole, not just a representative. Subsequently any analysis is much more robust as every individual is accounted for and any unexpected results are found.
2.0 Justification of project
The species Tahina spectabilis is a rare palm species that was discovered in 2006 in Madagascar (Dransfield et al., 2008). Madagascar is one of the worlds 'biodiversity hotspots' (Myers et al., 2000) with over 80% of its flora being endemic (Gautier & Goodman, 2003). Dransfield and Beentje (1995) found 170 species, only 6 of which weren't endemic; however this number has since grown by approximately 27 new species in various genus's (Dransfield & Marcus, 2002; Dransfield, 2003; Hodel & Marcus, 2004; Britt & Dransfield, 2005; Hodel, Marcus & Dransfield, 2005; Rakotoarinivo, Ranarivelo & Dransfield, 2007). Tahina spectabilis is one of the newest additions, occupying a new genus, Tahina. T. spectabilis, more commonly known as the Blessed Palm, was found in the north-western coast of Madagascar, an area which is not know for its extensive diversity, and is one of the last places expected to have such a unique specimen of flora (Dransfield et al., 2008). This area is however well know for its extreme, seasonal climate that can support relatively few species compared to the species rich eastern coastline (Dransfield et al., 2008). T. spectabilis was an enigma in that its morphological characteristics, whilst unique, placed it in the tribe Chuniophoeniceae of subfamily Coryphoideae (Dransfield et al., 2008). However it did not fit within any current genus present and so a new genus was created and named after the species. The extreme climatic conditions, along with the apparent fragility of T. spectabilis, make it susceptible to the stochastic environment and to mismanagement of the area (i.e. changes to fire regimes, anthropogenic effects on the landscape)
It would seem that there have been few studies done regarding species who are seemingly restricted to one single population and that have no apparent means of genetic rescue. Tahina spectabilis is such a species, consisting of only one population found in the remote savannah of western Madagascar (Dransfield et al., 2008). It is known only to exist on naturally fragmented outcrops of 'tsingy', a karst limestone. This area of Madagascar, due to historical political events and agricultural requirements, is burnt frequently (Kull, 2002). Its isolation and small population size, as well as being a potential relic with the only known relatives existing in Asia, makes this species a very well suited research specimen for this topic. However its full suitability and potential as a research subject is still indefinite as there is so little information about the species. The uniqueness of T. spectabilis in itself with regard to its history, its extreme distance from its taxonomical relatives and its distinctive morphological characteristics makes it worth studying by itself. The afore-mentioned relation to species in Asia makes T. spectabilis the only representative of its tribe in Madagascar, making its conservation even more important (Dransfield et al., 2008). This project aims at developing a better understanding of the species in order to provide accurate knowledge about the species' current situation in order to develop a plan involving the restoration of the species as well as determining what it's listing in the IUCN Red List should be i.e. threatened, endangered.
This research aims to:
Determine the genetic variability of the sampled individuals in the species and therefore assessing population characteristics such as levels of inbreeding. The genetic composition of the species will be utilised to establish the existence of potential new populations.
The demographic data collected in the field will be used to reveal and investigate any relationships between size cohorts.
Using both the demographic and genetic data, changes in genetic diversity over generations can be investigated.
The frequency and success of recruitment events will be examined and the overall status of the population will be assessed.
All the collected information will be compared with the criteria for listings on the IUCN red list to determine the appropriate conservation status for T. spectabilis.
4.1 Identification of the boundaries of the methodology
Due to time and money constraints, the primers used will consist of those already available in the primer library at the University of the Sunshine Coast. Constraints on time mean that primers that have already been developed will have to be used rather than designing new primers.
4.2 Mode of data collection
Every individual found during the field work was mapped relative to other individuals. Data was collected from every individual such as height, number of fronds, and whether the plant was sampled. Samples consisted of mature leaf material and were collected from every plant over 30cm to avoid damage to smaller seedlings. Sample collection was undertaken by Dr. Alison Shapcott and was assisted by the VERAMA cashew estate of GROUPE UNIMA and RBGKEW and funded by the University of the Sunshine Coast. Samples were sent to the University of the Sunshine Coast for genetic analysis. Additional samples will be sourced from the Royal Botanic Gardens Kew including DNA from a plant found flowering in 2006 as well as seedlings propagated from this adult. These plants will be tested for outcrossing to determine if the plants are self-fertilising or require other flowering adults to reproduce.
Microsatellite markers will be used to undertake the genetic analysis. Microsatellites are locus-specific codominant markers which allow for the detailed study of population structure, parentage and relatedness, assessing genetic diversity and recent population history (Zhang & Hewitt, 2003). The genetic analysis of the samples will consist of extracting genomic DNA using the QIAGEN DNeasy Plant Kit. Extraction of DNA involves cooling and grinding each sample to lyse the cell walls. RNA is removed from the sample using RNase and the samples are then purified of any potentially damaging contaminants such as proteins or enzymes. Buffers are added to precipitate any proteins and polysaccharides and the DNA is bound to a filter, purifying the sample. Finally the sample is eluted through the filter and is ready for further use.
The microsatellite region at the particular loci in the DNA will then be amplified using polymerase chain reactions (PCR), using a range of primers believed to be suitable for the species Tahina spectabilis. The microsatellite loci have already been developed previously and are considered highly transferable between species. The purpose of these trials is to identify loci that are transferable to T. spectabilis and then optimise the conditions to gain the best possible result. A selection of successful primers from the trials will be used for the whole species.
Lastly the PCR product for each locus will be run through a fragment analyser to document the size of the different alleles and determine the genotype. This information will provide an account of each individual's genotype, and consequently outline the overall levels of diversity and inbreeding in the population. The data will then be used to undertake the genetic analysis.
4.3 Consideration of controls
A number of controls will be implemented throughout the lab procedures to eliminate the possibility of confusing contaminants with the desired DNA. During the trial PCR runs, a blank will be included with each run so that any contaminants amplified during the process will appear in the blank sample as well as the other samples containing the DNA. The samples can then be compared and the unwanted artefacts can be excluded from the analysis. A test species chosen for each locus depending on their suitability will also be included to check if the amplified product is the microsatellite and not an artefact and if the PCR protocol was successful.
4.4 Mode of statistical analysis
Each individual will be mapped using their (x, y) co-ordinates. This data will allow us to look at the population structure of the species for the analysis of spatial relationships. Due to the infrequent flowering events, it was possible to determine which seedlings came from which flowering event.
Once the genetic data has been obtained, various measures of diversity will be determined using the software package GenAlEx, including the number of alleles per locus in the population (A), the expected heterozygosity within the population (He) and the observed heterozygosity (Ho); also the frequency of particular alleles will be established throughout the population (p), as well as levels of inbreeding using the fixation index (F), namely the variation among individuals within the total species population (FIT). Levels of inbreeding will also be assessed by the level of significance and deviation from the Hardy-Weinberg equilibrium. Changes in diversity throughout generations will be measured using expected and observed levels of heterozygosity (He and Ho). To determine the level of genetic distance between size cohorts, a map will be constructed according to their multi-locus genotypes using a principal coordinate analysis. This will also help to determine where the outlying individuals belong in the main population in terms of the size cohorts and genotypes.
The geographic and genetic data will be tested for random or non-random distribution using Mantel test. This will test for spatial correlation between the genetic distances between individuals and their geographic distance. With regard to the overall status of the species, the demographic and genetic data will be used to investigate any spatial autocorrelation between the distribution of size classes and the distribution of genotypes which will provide information on the dispersal patterns of the species. The net reproductive rate (Ro) will establish the significance of the reproductive rate of Tahina spectabilis and whether the species is stable or is decline. Estimates of the species growth rates will also be done to achieve this.
4.5 Application of data
Once all this information has been collected and analysed, a comparison will be done against the criteria of the IUCN Red List to determine what level of conservation should be given to T. spectabilis. A contribution will be made to the application of Tahina spectabilis to be entered into one of the categories in the IUCN Red List, in an attempt to have it protected under its laws.
4.6 Consideration of ethics
Considerations of animal and human ethics were made during the collection of samples in the field; however they are not required for this project. Biosafety protocols will be observed during the laboratory work.
Table 1. Projected timeline for research.
Field Data Analysis
PCR Primer Trials
Genetic Data Analysis
Table 2. Projected Budget for March 2010-November 2010.
Projected Budget for March 2010-November 2010
Thesis Printing and Binding
Extra funds from grants awarded by International Palm Society (IPS) to Dr. Alison Shapcott will be used to cover any expenses incurred by the use of lab consumables. These consumables include PCR chemicals (Taq, dNTP's, PCR buffer, MgCl2) and disposable plastic wear eg. PCR plates, pipette tips. Lab chemicals consist of the purchase of 6 new custom-made fluorescently labelled microsatellite primers that will be used in the PCR procedures, as well as other consumables. These primers will expand the range of the current library of primers available for palms and will ensure that there are sufficient loci to generate significant results. Primers are approximately $215 a pair.
7.0 Projected Outcomes
We expect to determine the genetic variability in T. spectabilis and levels of inbreeding. Particularly we expect to confirm if the 2006 flowering plant produced seedlings by outcrossing with a plant external to the known population.
The genetic composition of the species will be utilised to establish the existence of potential new populations. The demographic size structure and number of cohorts will be determined and we hope that by using this data we will be able to estimate the frequency of reproductive pulses.
Maps will be created showing the relationships between all individuals according to their size and locations and genotype. The relationship between the outlier individuals and the main population will be resolved i.e. were they dispersed from the main known population or from another unknown population and do their genotypes match particular size cohorts that reflect their size and stage of development?
We also expect to determine if genetic diversity appears to have been lost between different generations. The frequency and success of recruitment events will be estimated. Overall the population viability of the species will be assessed. Finally the information will be compared with the criteria for listings on the IUCN red list to determine what its conservation status should be.