Thesis (Honors)--College of William and Mary, 2009. / Includes bibliographical references (leaves 51-54). Also available via the World Wide Web.

The studies described here introduce a model for residue preservation on stone tools. They simulate stone tool manufacture in order to define parameters important for the study of DNA residues. Microscopic examination of stone tools has identified microcracks that trap DNA and protein from animal blood cells. Thorough investigation of different methods to recover residues from stone tools shows that surface washing leaves DNA and protein, trapped in subsurface microcracks. However, other extraction techniques are able to release 60-80% of DNA and protein residues. Previous research documents the identification of protein from stone tools sonicated in 5% ammonium hydroxide, but it remains untested whether the same treatment yields useable DNA. Using this treatment, I identified 13-year-old DNA residues from experimentally manufactured stone tools. In addition, results clearly indicate that washing procedures typically used to curate stone tools removed only a small fraction of the DNA deposited during animal butchery. Twenty-four pieces of chipped stone recovered from the Bugas-Holding site were studied to explore the validity of ancient DNA residue identifications. Nine tools yielded DNA residues. Modern humans did not touch three of these tools, which suggests that the DNA recovered from them was present prior to excavation. One tool, which was handled by excavators without gloves, harbored DNA from three species, and these templates competed during PCR. On at least two tools, handling after excavation introduced animal DNA unrelated to tool use. Careful testing of Bugas-Holding chipped stone suggests that stone tools may harbor both ancient and modern DNA, and that investigators must take great care to exclude modern DNA from ancient specimens. Ultimately, I developed and streamlined a method to analyze DNA-containing residues preserved on stone tools. This led to several technical improvements in ancient DNA residue analysis. These include a more effective DNA recovery protocol, methods to measure sensitivity and inhibition of PCR in each sample, and strategies to surmount competition between templates during amplification, which can occur in samples that contain DNA from multiple species. These new developments will help future investigators achieve the full potential of ancient DNA residue analysis. / Graduation date: 2003

This research focuses on scale pattern and cross sectional morphology of hair to identify fossil hairs from Parahyaena brunnea (brown hyaena) coprolites, from Gladysvale cave in the Sterkfontein Valley, South Africa. The coprolites are part of a brown hyaena latrine preserved in calcified cave sediment dated to the Middle Pleistocene (257- 195 ka). Forty-eight fossil hairs were extracted from a 75 x 30 x 15 cm block using fine tweezers and a binocular microscope. They were ultrasonic cleaned in analar ethanol and examined using scanning electron microscopy. Hair identification was based on consultation of standard guides to hair identification and comparison with my own collection of samples of previously undocumented guard hairs, from 15 taxa of indigenous southern African mammals. Samples were taken from the back of pelts curated at the Johannesburg Zoo and Ditsong National Museum of Natural History in Tshwane (formerly Transvaal Museum, Pretoria). Based on the fossil hairs identified here, this research has established that brown hyaenas shared the Sterkfontein Valley with warthog, impala, zebra, kudu and black wildebeest. These animals are associated with savanna grasslands, much like the Highveld environment of today. Fossil human hair was also noted in the coprolites. These findings provide a new source of information, on the local Middle Pleistocene fossil mammal community, and insight into the environment in which archaic and modern humans in the interior of the African subcontinent lived. Amid a scarce fossil and archaeological record for this time period, these results make a significant contribution to the ongoing debate about the role of climate change in the evolution and success of modern humans. In accordance with modern brown hyaena feeding behaviour, the presence of medium to large-sized mammal hairs in the coprolites suggests the co-existence of large feline predators in the area, or a period of active hunting, based on the behaviour of modern brown hyaenas when rearing cubs.

In this thesis human skeletal remains believed to be the victims of the Batavia Mutiny of 1629 were subjected to DNA analysis. So far the remains of 10 individuals (of which 9 were available for this study) have been exhumed from Beacon Island, in the Houtman’s Abrolhos, off the coast of Western Australia. The remains are now stored in the Western Australia Maritime Museum (WAMM) in Fremantle. In this research an attempt is made to type ancient DNA (aDNA) from the remains of the Batavia Mutiny, which are almost 400 years old. Previous anthropological studies have been performed on these remains in order to assign sex, age and stature. The aim of the present project is to study the familial relationships of the remains and to determine their sex based on molecular genetic analysis. In order to protect the invaluable museum specimens and minimise the risk of contamination from exogenous contemporary DNA, a tooth sample from each available individual (designated A15507, A16316, A15831, M3901, SK5, SK6, SK7, SK8 and SK9) was subjected to DNA extraction. Comparison and optimisation of DNA extraction methods from more recent teeth samples was performed in order to determine the most suitable method for the DNA extraction of the ancient teeth samples. Three types of genetic markers were analysed in an attempt to study the familial relationships and determine the sex of each individual. Multiplex primers (Hummel, 2003) which simultaneously amplify the HV1 and HV2 regions of mitochondrial DNA (mtDNA) were used in this research to analyse familial relationships. These primers were selected because of their ability to amplify small fragments (131bp, 168bp and 217bp) of DNA template, which suit the nature of aDNA samples. Primers published by Sullivan et al.(1993), which amplify a 106bp region on chromosome X and 112bp on chromosome Y of the amelogenin gene, were used to determine sex. In addition, short tandem repeat (STR) marker were also analysed to determine familial and sex using the AmpFlSTR®Profiler PlusTMPCR kit from Applied Biosystems. The PCR conditions of all primers were optimised before usage on the Batavia remains. As aDNA analysis is prone to contamination, stringent precautions were undertaken throughout this research. Despite this, contamination is suspected in some of the mtDNA sequences obtained (particularly from SK5, SK7, A15507 and A15831), which most probably came from the positive control used in the optimisation analysis. For these samples the sequences for the HV2 region were poor and polymorphisms relative to a reference were similar to each other and to the positive control profile. However, some conclusions have been made on other individuals (SK8, SK9, M3901, A16316) based on the HV1 and HV2 sequences obtained. Based on two or more different polymorphisms observed in the individuals it was concluded that it is likely there is no maternal relationship between individuals A16316 and SK8, SK9 and M3901 and between individuals SK8, M3901 and SK9. However these results require repetition for confirmation. The attempt to type the amelogenin gene on chromosomes X and Y was unsuccessful most likely due to the poor preservation of the remains. It is apparent from this research that although it was possible to extract aDNA (especially multicopy mtDNA) from teeth material that were almost 400 years old, the main hurdle in this aDNA analysis was contamination and DNA degradation.

The western embayment of Lake Ontario, Hamilton Harbour, is one of the most polluted sites in the Laurentian Great Lakes and in recent years has seen a reoccurrence of cyanobacterial blooms. This study uses a multidisciplinary approach to examine the presences of toxic Cyanobacteria in the harbour in order to gain insight into these recurrent blooms. Microscopic analyses of phytoplankton samples collected during the 2009 summer-fall sampling season from two locations within the harbour showed the spatial and seasonal diversity of the contemporary cyanobacterial community. Microcystis colonies relative abundances in relation to total algal numbers were estimated. The lowest and highest relative abundances of Microcystis in the phytoplankton population were 0.6% and 9.7%, respectively, and showed seasonal variability between stations. Fourteen cyanobacterial genera comprising six families and three orders were identified and for which the most abundant filamentous genera during the summer-fall sampling season were Planktothrix, Aphanizomenon and Limnothrix. Potential microcystin producers Microcystis, Planktothrix, Aphanizomenon and Dolichospermum were also present and during the sampling period Microcystis was recorded at both stations on all dates, however, its relative abundance was below 10 % throughout the study period. The composition and abundance of filamentous cyanobacteria were observed to be positively statistically correlated to water quality environmental parameters dissolved nitrates (NO3/NO2), dissolved inorganic carbon (DIC), and conductivity. Redundancy analysis (RDA) found that 53.35% total variance of Aphanizomenon was correlated to low water column NO3/NO2 and conductivity, and higher water column DIC. 58.13% of the relative abundance of Planktothrix was correlated to high concentrations of dissolved nitrates, while 51.69% of total variance of Limnothrix was correlated to higher DIC and lower water column dissolved nitrate concentrations. Information about past cyanobacterial communities was obtained from the sediment core analysis, using paleolimnological and modern molecular methods. The age of the 100.5 cm long sediment core retrieved from the deepest part of Hamilton Harbour was established to be 140 years (1869-2009), using the Constant Rate of Supply (CRS) 210Pb age model. This age was not sufficient to provide information of harbour’s environmental conditions, presence of the blooms, and triggers for their occurrence before European settlement in the area. Results of the HPLC analysis of fossil pigments indicated that the dominant members of the algal community have not changed over the 140 years and that cyanobacteria were regular members of the phytoplankton community. The composition of the major chlorophyll pigments indicated high presence of Chlorophyta and Bacillariophyta in the harbour at all times. The main algal groups identified on the basis of marker pigments presence, besides the Chlorophyta and Bacillariophyta, were the Dinophyta and the Cryptophyta. The presence of a scytonemin derivative, compound B, indicated that cyanobacterial blooms were occurring in past, before the first officially recorded blooms in the 1960s. Cyanobacterial pigments presence indicated that Cyanobacteria have been a regular but not dominant feature of Hamilton Harbour phytoplankton in the past. To our knowledge, this study is the first one examining fossil pigments from Hamilton Harbour. Results of the PCR-DGGE molecular analysis of 16S rRNA-V3 gene fragments from sedimentary DNA revealed the presence of thirteen cyanobacterial genotypes. The temporal change in the cyanobacterial community composition was indicated by the increasing number of species over time, from the oldest to the most recent sediment layers. The deepest sediment strata showed the lowest number (two bands) and intensity of bands. The most recent sediment layer had the greatest numbers (11) and intensity of bands. This increased diversity indicated changing environmental conditions in the harbour, primarily nutrient pollution and worsening water quality. Results of the PCR-DGGE molecular analysis of mcyE-AMT gene fragments showed that Microcystis aeruginosa and Planktothrix rubescens were two microcystin producers present in Hamilton Harbour over the last 80 years. The persistent presence and resilience of these two genera indicated a more serious and longer-term issue of toxic blooms than previously recognized. Historical records show that noticeable anthropogenic impact on Lake Ontario environment has been measurable since the 1780s, the first dramatic impact on the Lake Ontario watershed was evident from the mid1880s, the earliest evidence of eutrophication in the lake occurred between 1820 and 1850, while human induced environmental changes in Hamilton Harbour date back ca. 350 years. In the 1960s, cyanobacterial blooms were first officially recognized in the harbour and the lower Great Lakes. The present research is the first report of the mcyE module and AMT domain of microcystin genes being amplified from sediment of North American lakes, and showed that toxic Cyanobacterial have been regular members of Hamilton Harbour phytoplankton community for almost a century. This research considerably deepened the knowledge of the past toxic cyanobacterial blooms in Hamilton Harbour and their possible causes. It also showed that in the absence of historical records, both the PCR-DGGE method and the mcyE-AMT gene may be used for reconstruction of the past toxic blooms not only in the Laurentian Great Lakes, but also in other aquatic regions of the world impacted by toxic cyanobacterial blooms. Also, it demonstrated the utility of the combined molecular and paleolimnological analyses, which might become a useful tool in the determination of the bloom causes factors and in the mitigation of the future production of toxic blooms.