Table of Contents
Abstract:
Deforestation is a rapidly growing threat to biodiversity and numerous animal species, particularly primate populations are affected by habitat fragmentation due to agricultural practices. Mantled howler monkeys in the La Suerte region of Costa Rica are especially vulnerable owing to habitat infringement by fruit plantations, which separate previously contiguous forest areas.
This habitat fragmentation may also affect individual primates’ dispersal patterns constraining gene flow within affected populations. Genetic diversity is crucial for the health and survival of primate species. In this study, we will analyze the effects of habitat fragmentation on genetic variation within howler monkey populations at the La Suerte Biological Research Station in Costa Rica.
Populations from an edge forest area and populations from an interior forest area will be specifically compared to assess changes in their dispersal patterns and gene flow. We will analyze isolated and purified DNA from fecal samples by utilizing Polymerase Chain Reaction (PCR) and gel electrophoresis. Genetic variation will then be determined through microsatellite loci analysis and statistical analysis. I hypothesize that howler monkey populations will have less genetic diversity in the edge forest area as compared to the interior forest area due to reduced population size and isolation.
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This study is crucial to better understand how habitat fragmentation influences howler monkeys’ population dynamics and genetic diversity. This project is well-aligned with Regis University’s mission and Jesuit values, especially due to its potential benefit for conservation efforts and global environmental awareness.
Project Description:
- Background and Significance
Rainforest deforestation and fragmentation are eminent issues that require advanced integrative research in order to implement effective management and long-term solutions. Clearing land for agricultural use is the primary cause of rainforest destruction (Estrada, 2015). Deforestation in turn results in habitat fragmentation as previously contiguous areas of rainforest are separated into disconnected regions by newly developed and cultivated areas. This process is a major threat to global biodiversity as well as to the health and survival of numerous plant and animal species, especially primate populations. Habitat fragmentation is largely detrimental to wildlife and has been determined as a major cause of declining primate populations globally (Arroyo- Rodriguez & Dias, 2010).
Primate populations are particularly vulnerable and more than 50% of all primate taxa are at risk of extinction due to habitat modification directly caused by humans (Mittermeier et al., 2012). The harmful effects of habitat fragmentation on primate populations are expressly evident in Costa Rica due to the country’s growing economy and agricultural practices. The rate of deforestation has continually increased over the past few decades, especially as a result of an increasing number of banana and pineapple plantations (Garber et. al., 2010). This increasing infringement on primate habitat requires more research and a better understanding of how habitat fragmentation affects primate species. An analysis of differing primate populations’ genetic diversity offers an effective and accurate illustration of how forest fragmentation may influence primate dispersal and gene flow.
This study specifically examines the impacts of habitat fragmentation on wild mantled howler monkeys (Alouatta palliata) living in fragmented areas at the La Suerte Biological Research Station (LSBRS) in Costa Rica. LSBRS is an ideal location of study as it’s considered one of the top 25 global biodiversity hotspots (Myers et al., 2000), and the La Suerte region has experienced growing forest fragmentation largely due to areas cleared for fruit plantations. Banana and pineapple plantations are particularly common and major companies operate in this area including Dole and Del Monte (Consumers International, 2010).
Despite the area’s ideal characteristics, relatively few research studies have been conducted at LSBRS, particularly in regard to the primates’ population structure (Pruetz & Leasor, 2002). In response to the fragmented forest areas, LSBRS members have implemented a reforestation program to form a corridor between the edge and the interior forest areas separated by fragmentation. This study will focus on the little-known effects of habitat fragmentation on the howler monkeys’ population dynamics, genetic diversity, and overall well-being. This study’s results will also be utilized to critically assess the efficacy of the LSBRS’ reforestation program.
Habitat fragmentation has been associated with numerous issues including decreases in habitat sizes, loss of ecological niches, population declines, loss of biodiversity, and decreases in resource availability (Arroyo-Rodriguez & Dias, 2010). Decreases in habitat sizes and increased habitat isolation can greatly influence a primate population’s genetic variation as both factors may change the group’s social organization through modified dispersal patterns and group size/composition (Arroyo-Rodriguez & Dias, 2010). In mantled howler monkey populations, individuals disperse from their natal group upon reaching sexual maturity (Glander, 1992).
This behavior benefits the various populations as it reduces inbreeding and increases genetic diversity. These dispersal patterns enable beneficial gene flow and subsequent genetic diversity that is necessary for a species’ health and survival (Oklander et al., 2010). As a consequence of habitat fragmentation, dispersal behaviors may be severely inhibited resulting in low genetic diversity within certain populations.
For example, a prior study found low levels of genetic variation within Argentinian black and gold howler monkeys (Alouatta caraya) living in fragmented forests compared to populations in larger forest areas (Oklander et al., 2010). With lack of necessary genetic diversity, health consequences and ultimately extinction are troubling possibilities for impacted primate species.
In this study, we will be analyzing various howler monkey populations’ genetic variability to compare how the genetic composition of populations differs in fragmented forest areas. Populations from an edge forest area and populations from an interior forest area will be specifically compared to determine the effects of habitat fragmentation on these populations’ dispersal patterns and gene flow.
Microsatellite loci analysis is an effective method for assessing genetic variation and levels of inbreeding (Bastos et. al., 2010; James, 1992; Pope, 1996; Winkler et. al., 2004). Microsatellites are repetitive segments of DNA that vary among individuals. Genetic variation can be determined by analyzing these microsatellite repeats and numbers (Morin & Woodruff, 1996; Pruetz & Leasor, 2002). From each microsatellite loci, individuals will be recorded as being homozygous (two of the same alleles present) or heterozygous (different alleles present) (Oklander et al., 2010).
In previous research, agarose gel electrophoresis was used. In this project, we will utilize polyacrylamide gel electrophoresis due to its greater resolving power than agarose gels (polyacrylamide gels can separate molecules of DNA whose lengths differ by as little as 0.1%), and its differentiation of the microsatellites at the necessary base pair range of less than 500 base pairs (Campbell et al., 1983). Agarose gel electrophoresis is also an ideal alternative due to its superior visualization and photographic results (one of the gel plates is UV-transparent), as well as its time and cost-effective methods. For example, conducting gel electrophoresis with MetaPhor agarose gels could also isolate the alleles of microsatellite markers, but the resolution is much lower and the cost is five times more than the same procedure conducted with polyacrylamide gels (Wang et al., 2003).
After the gel electrophoresis and statistical analysis are completed, populations can then be assessed as genetically healthy if high genetic diversity or high levels of heterozygosity are present, which indicates that beneficial gene flow is taking place (Woodruff, 1989).
Primarily, this project relates to the growing environmental issues we face today. Global environmental awareness is more important than ever before due to the increasingly greater threats of deforestation, loss of biodiversity, climate change, endangered ecosystems and species, pollution, land management, waste disposal, and overpopulation among others. It’s imperative that we better understand the effects of habitat fragmentation on various species, particularly vulnerable primate populations, in order to develop and implement realistic solutions to aid conservation efforts.
This challenging issue requires an integrative approach based off of the many stakeholders involved. It’s crucial that a future solution has taken into account the various groups that will be affected including the local community, the plant and animal species, conservationists, scientists, agricultural workers, and land owners.
This study’s purpose and implications truly exemplify the mission of Regis University as well as its Jesuit values. A Regis education perpetually challenges students to answer the profound question: “How ought we to live?” This complex environmental issue demands an intensive and thought-provoking discussion of how we as humans can best serve the environment, others, and future generations to come. This study evokes the particular Jesuit values of magis, men and women for and with others, and contemplatives in action.
With the magis principle we are required to strive for excellence in all things and to solve difficult problems to the best of our ability for the greater good. Men and women for and with others implores us to always provide service to those in need and to pursue justice on behalf of all persons and stakeholders involved with an issue. Contemplatives in action reminds us that social problems require more than just thought and contemplation, but rigorous action and reflection.
- Purpose and Specific Aims
The main goal of this research study is to assess and compare the genetic diversity present in mantled howler monkey populations from edge and interior forest areas constrained due to habitat fragmentation. This analysis of genetic diversity across various primate populations in the La Suerte forest could benefit this region’s conservation efforts, farming practices, and understanding of the effects of human-caused habitat fragmentation on population dispersal patterns.
Hypothesis
I hypothesize that howler monkey populations will have less genetic diversity in the edge forest area as compared to the interior forest area due to reduced population size and isolation. Habitat fragmentation can result in inbreeding depression and subsequently reduced gene flow and genetic variation. This study aims to draw attention to the potential issue of Costa Rican primates’ decreasing genetic diversity and population health due to forest fragmentation.
- Methods
Location of Study and Fecal Sample Collection
Fecal samples from mantled howler monkeys were previously collected in the summer of 2016 at the La Suerte Biological Research Station in Costa Rica (10 °26’N, 83°46’W). This station includes more than 300 hectares (ha) of primary forest, secondary forest, and regenerating pastures (Garber et al., 2010). This area includes an edge forest area and an interior forest area due to habitat fragmentation (Pruetz & Leasor, 2002). In 2005, a reforestation project was initiated with the goal of building a corridor between these fragmented forest sections (Garber et al., 2010).
Dr. Schreier and Regis University students followed mantled howler monkeys and collected their recent fecal samples from the forest floor. Samples were taken along with records of the specific individual’s sex and age-class. Approximately 5 grams of fresh feces were collected in 5-ml vials, which contained 2.5 ml of RNAlaterTM. Labeled vials were then shipped to the Regis University’s Biology Department and stored at -20°C.
DNA Extraction and Purification
DNA isolation from the frozen fecal samples was achieved with the QIAamp DNA Stool Mini Kit (Qiagen). This specific protocol enabled an increased ratio of primate DNA to non-primate DNA or microbiome DNA that may have been present due to contamination. It also allows high DNA yields with little chemical inhibition of Polymerase Chain Reaction (PCR) (Nechvatal et al., 2008), which is crucial with the limited DNA available from the gastro-intestinal epithelial cells in the primates’ fecal samples.
Determination of Nuclear Microsatellite Genotypes
Polymerase Chain Reaction
In order to determine microsatellite heterozygosity in two populations of monkeys at two loci each, microsatellite primers specific to the species will be used to perform PCR amplifications of the selected microsatellite loci. Specifically, the microsatellite primers APM1 and AB06 will be utilized (Goncalves et al., 2004; Ellsworth & Hoelzer, 1998). The necessary PCR procedure will be followed as previously detailed by Van Belle and utilized by Dr. Schreier and prior undergraduate research students (Van Belle et al., 2012).
Gel Electrophoresis
The PCR amplification products will be separated and visualized through gel electrophoresis. In previous research, agarose gel electrophoresis was used. In this project, we will utilize nondenaturing polyacrylamide gel electrophoresis due to its greater resolving power than agarose gels (polyacrylamide gels can separate molecules of DNA whose lengths differ by as little as 0.1%) and its ability to accommodate larger quantities of DNA (Campbell et al., 1983). Polyacrylamide gel electrophoresis is also ideal for photographing (one of the gel plates is UV-transparent) and it’s a time and cost-effective method (Wang et al., 2003).
Another crucial advantage includes the large plate sizes that enable DNA differentiation at a base pair length of about 200 base pairs. We have developed a successful protocol and acrylamide gel formula through five months of rigorous research and experimentation. We combine 24 mL of 30% acrylamide, 8 mL of 10x TBE, 4 uL of ethidium bromide, 48 mL of deionized water, 560 uL of APS, and 80 uL of TEMED.
The addition of TEMED as a final step is crucial as it rapidly polymerizes the gel and allows DNA differentiation at a specific resolution (Wang et al., 2003).We also add a dye to the electrophoresis gel wells that runs at approximately 150 base pairs in order to track the number and size of the microsatellite species. Every electrophoresis gel will contain a control in the first well that enables a direct size comparison between the microsatellite markers.
The specific DNA ladder used in the control is a low molecular weight DNA ladder that yields 11 bands for visualization of molecular weight standards. This digested plasmid includes fragments ranging from 25 to 766 base pairs with a distinct 200 base pair band (Biolabs, 2019). This permits the accurate resolving power to see less than 50 base pair differences. The electrophoresis gel is run at max voltage (about 250 V) for about 6 hours. Upon completion of the gel electrophoresis, the PCR products will be analyzed for genetic polymorphisms, a measure for genetic diversity.
The microsatellite sequences will then be evaluated for genetic diversity, as illustrated through various number of repeats, and overall heterozygosity for all individuals from the differing populations in the edge and interior forest fragments.
Statistical Analysis
We will analyze and compare the genetic diversity for mantled howler monkey populations within the edge and interior forest fragments separated by habitat fragmentation. Initially, the Hardy-Weinberg Equilibrium (HWE) can be utilized to test the null hypothesis (that there was no significant difference in genetic diversity between the different monkey populations). This evaluation will also determine the level of inbreeding in the differing populations.
This HWE data can then be analyzed using F-statistics to assess the ratio of heterozygous to homozygous individuals in the edge and interior forest fragments as well as within the population as a whole (Wright, 1951). The calculated fixation indices FIS and FST, which measure population genetic variability, are specifically used to determine the level of inbreeding (Oklander et al., 2010). These values will be crucial determinants in the overall comparison of genetic diversity between the monkey populations from the edge and interior forest fragments.
