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The age structure of populations is an important and often unresolved factor in ecology and wildlife management. Parameters like onset of reproduction and senescence, reproductive success and survival rate are tightly correlated with age. Unfortunately, age information of wild animals is not easy to obtain, especially for birds, where few anatomical markers of age exist. Longitudinal age data from birds banded as chicks are rare, particularly in long lived species. Age estimation in such species would be extremely useful as their long life span typically indicates slow population growth and potentially the need for protection and conservation. Telomere length change has been suggested as a universal marker for ageing vertebrates and potentially other animals. This method, termed molecular ageing, is based on a shortening of telomeres with each cell division. In birds, the telomere length of erythrocytes has been reported to decline with age, as the founder cells (haematopoietic stem cells) divide to renew circulating red blood cells. I measured telomere length in kakapo, the world largest parrot and four other bird species (Buller’s albatross, kea, New Zealand robin and saddleback) using telomere restriction fragment analysis (TRF) to assess the potential for molecular ageing in these species. After providing an overview of methods to measure telomere length, I describe how one of them (TRF) measures telomere length by quantifying the size distribution of terminal restriction fragments using southern blot of in-gel hybridization (Chapter 2). Although TRF is currently the ‘gold standard’ to measure telomere length, it suffers from various technical problems that can compromise precision and accuracy of telomere length estimation. In addition, there are many variations of the protocol, complicating comparisons between publications. I focused on TRF analysis using a non-radioactive probe, because it does not require special precautions associated with handling and disposing of radioactive material and therefore is more suitable for ecology laboratories that typically do not have a strong molecular biology infrastructure. However, most of my findings can be applied to both, radioactive and nonradioactive TRF variants. I tested how sample storage, choice of restriction enzyme, gel Abstract II electrophoresis and choice of hybridization buffer can influence the results. Finally, I show how image analysis (e.g. background correction, gel calibration, formula to calculate telomere length and the analysis window) can not only change the magnitude of estimated telomere length, but also their correlation to each other. Based on these findings, I present and discuss an extensive list of methodological difficulties associated with TRF and present a protocol to obtain reliable and reproducible results. Using this optimized protocol, I then measured telomere length of 68 kakapo (Chapter 3). Almost half of the current kakapo population consists of birds that were captured as adults, hence only their minimum age is known (i.e. time from when they were found +5 years to reach adulthood). Although molecular ageing might not be able to predict chronological age accurately, as calibrated with minimum age of some birds, it should be able to compare relative age between birds. Recently, the oldest kakapo (Richard Henry) was found to show signs of reproductive senescence. The age (or telomere length) difference to Richard Henry could have been used to approximate the remaining reproductive time span for other birds. Unfortunately, there was no change of telomere length with age in cross sectional and longitudinal samples. Analysis of fitness data available for kakapo yielded correlations between telomere length and fledging success, but they were weak and disappeared when the most influential bird was excluded from analysis. The heavy management and small numbers of kakapo make conclusions about fitness and telomere length difficult and highly speculative. However, telomere length of mothers and their chicks were significantly correlated, a phenomena not previously observed in any bird. To test if the lack of telomere loss with age is specific to kakapo, I measured telomere length of one of its closest relatives: the kea (Chapter 4). Like kakapo, telomere length did not show any correlation with age. I then further assessed the usefulness of molecular ageing in birds using only chicks and very old birds to estimate the maximum TL range in an additional long lived (Buller’s albatross) and two shorter lived species (NZ robin and saddleback). In these Abstract III species, telomere length was on average higher in chicks than in adults. However, age matched individuals showed high variations in telomere length, such that age dependent and independent telomere length could not be distinguished. These data and published results from other bird species, coupled with the limitations of methodology I have identified (Chapter 2), indicate that molecular ageing does not work in most (if not all) birds in its current suggested form. Another way to measure telomere length is telomere Q-PCR, a real-time PCR based method. Measurement of the same kakapo samples with TRF and Q-PCR did not result in comparable results (Chapter 4). Through experimentation I found that differences in amplification efficiency between samples lead to unreliable estimation of telomere length using telomere Q-PCR. These differences were caused by inhibitors present in the samples. The problem of differential amplification efficiency in Q-PCR, while known, is largely ignored by the scientific community. Although some methods have been suggested to correct for differing efficiency, most of these introduce more error than they eliminate. I developed and applied an assay based on internal standard oligonucleotides that was able to corrected EDTA induced quantification errors of up to 70% with high precision and accuracy (Chapter 5). The method, however, failed when tested with other inhibitors commonly found in DNA samples extracted from blood (i.e. SDS, heparin, urea and FeCl3). PCR inhibition was highly selective in the probe-polymerase system I used, inhibiting amplification of genomic DNA, but not amplification of internal oligonucleotide or plasmid standards in the same reaction. Internal standards are a key feature of most diagnostic PCR assays to identify false negatives arising from amplification inhibition. The differential response to inhibition I identified greatly compromises the accuracy of these assays. Consequently, I strongly recommend that researchers using PCR assays with internal standards should verify that the target DNA and internal standard actually respond similarly to common inhibitors.

The South Mountains are composed of two fundamentally different terranes. The western half of the range consists of Precambrian metamorphic and granitic rocks, whereas the eastern half is dominated by a composite middle Tertiary pluton. North-northwest-trending, middle Tertiary dikes have extensively intruded both terranes. A major episode of middle Proterozoic metamorphism and deformation produced a steep crystalloblastic foliation that generally strikes northeast. Middle Tertiary plutonism was accompanied by intense mylonitization that affected Precambrian and middle Tertiary rocks alike. Discrete phases of mylonitization were associated with each intrusive pulse between 28 and 25 m.y.B.P. Mylonitization generally produced a lowangle foliation and east-northeast-trending lineation. The attitude of mylonitic foliation defines a broad, east-northeast-trending anticline that controls the topographic axis of the range. Structurally low rocks in the core of the anticline are nonmylonitic, but intensity of mylonitic fabric increases progressively toward higher structural levels. Mylonitic Tertiary plutonic rocks are exposed as a gently dipping carapace overlying their less deformed equivalents. Mylonitic fabric cuts through the Precambrian terrane as a broad, west-dipping zone. Rocks above and below this mylonitic zone are lithologically identical and mostly retain their Precambrian structure. Fabrics in all rock types indicate that mylonitization resulted from extension parallel to east-northeast-trending lineation and flattening perpendicular to subhorizontal foliation. Mylonitization occurred under conditions of elevated temperature but relatively low confining pressure. Gold-bearing quartz veins occur in tension fractures that are late- to post-kinematic with respect to mylonitic deformation. Mylonitization was succeeded by more brittle deformation that produced chloritic breccia and microbreccia in the footwall of a major detachment fault that dips gently to the east. The detachment fault and underlying breccia were formed by normal faulting and brittle extension in an east-northeast direction. Rocks above and immediately below the detachment fault were antithetically rotated during faulting. Mylonitization, detachment faulting, and formation of the main east-northeast-trending anticline are all manifestations of eastnortheast-directed, middle Tertiary extension. Evidence for a possible continuum between mylonitization and detachment faulting has important implications regarding the evolution of Cordilleran metamorphic core complexes.