Reeves' muntjac: a molecular genetic study of an invading species

The possibility of screening large numbers of individuals directly for genetic variability at the DNA level has only become feasible within the last decade. Prior to this, gel electrophoresis was employed to resolve small differences in the amino acid sequences of proteins. This thesis describes the application of three molecular genetic techniques to elucidate the relationships within sub-populations as well as the distribution and relationships between groups of sub-populations of an invading species of deer; Reeves muntjac (Muntiacus reevesi). Samples were taken from eighteen U.K. sub-populations of muntjac, representing increasing geographic distances centred on Woburn Abbey (the putative centre of origin) and one group from Taiwan were investigated. The polymerase chain reaction was used to amplify the control region of muntjac mitochondrial DNA prior to digestion with restriction enzymes. The restriction fragments produced were found to be sufficiently informative to identify eight maternal lineages and demonstrate that genetically closely related groups are in fact geographically widely separated, a finding counter to the hypothesis of a regular mode of dispersal. Genetic partitioning between sub-populations, a result which may be expected given the short time since the introduction of muntjac to the U.K. An appropriate DNA fingerprinting protocol was established, based on published protocols and two commercially available minisatellite probes. Detailed analysis of the banding patterns produced by these probes allowed an estimate of the level of inbreeding within sub-populations to be established. Tentative relationships were also able to be established between the sub-populations sampled. Although there was some overlap between probes, they generally detected independent loci. The mean band sharing coefficients were found to range from 0.19 to 0.33 (probe 33.15) and 0.15 to 0.34 (probe 33.6). The polymerase chain reaction was used to amplify the control region of muntjac mitochondrial DNA prior to digestion with restriction enzymes. The restriction fragments produced were found to be sufficiently informative to identify eight maternal lineages and demonstrate that genetically closely related groups are in fact geographically widely separated, a finding counter to the hypothesis of a regular mode of dispersal. Genetic partitioning between sub-populations was assessed by three agglomerative clustering methods which demonstrated that there is very little geographic partitioning between sub-populations, a result which may be expected given the short time since the introduction of muntjac to the U.K. Genetic variability between sub-populations was also assessed by an investigation of the geographic distribution of seven microsatellite loci. Most microsatellite loci were found to be highly polymorphic and there was substantial variation in the number of alleles detected per sub-population. Heterozygosity values were found to be high, ranging from 0.48 to 0.74. Fourteen 'rare' alleles were uncovered distributed between ten of the eighteen sub-populations. Wright's F$_{IS}$ was calculated as a metric of the level of inbreeding within sub-populations. The mean values of this estimator ranged from -0.181, indicating heterozygosity, to 0.222, indicating homozygosity. Nei's G$_{ST}$, an analogue of Wright's F$_{IS}$ was calculated to give an indication of the level of genetic sub-structuring between sub-populations. These calculations indicated that differentiation by distance is not significant over the geographic range of the sample area, again indicating that the muntjac have no dispersed in a 'natural' way. The use of three different molecular techniques has allowed comparisons to be made between their relative merits in terms of the level of genetic information they provide and in their ability to define population structure

Life history strategies in anadromous trout, 'Salmo trutta L.' : with special reference to osmoregulatory physiology

1. Juvenile trout, Salmo trutta L., from three parental groups - sympatric Sea trout and freshwater-Resident trout, and isolated trout from above a waterfall impassable by upstream migrating anadromous trout - were reared under three ration regimes to manipulate growth rates. The development of seawater tolerance was studied by measuring drinking rates after periodic salinity challenges during the first two years of juvenile growth. No trout were observed to undergo the parr-smolt transformation in any of the parental form/ration combinations after two years in freshwater. However, a considerable proportion did mature during this time period. The proportion of maturing trout was directly related to ration level but was also influenced by parental form, with isolated trout demonstrating a greater tendency to mature early. Seawater tolerance increased with age in all groups. However, mean drinking rates upon salinity challenge were generally lower, from Experiment 2 onwards, in Resident trout than in either of the other two groups. 2. Eight immature sea trout (finnock) were radiotracked in the River Eden, Fife, during September, October and November 1994. The individual finnock displayed considerable variation in patterns of movement; two remained in freshwater for at least 27 days whereas others moved downstream out of the river within days or even hours of release. In general, this highlighted the transient nature of the freshwater migrations of some finnock, indicating that they move in and out of rivers over brief periods of time and apparently do not necessarily remain in freshwater continuously throughout the winter. 3. The hypo-osmoregulatory ability of finnock during the winter was assessed in two experiments. The number of finnock was limited in Experiment 1. Therefore, this was designed as a preliminary assessment of the physiological response of finnock to acute freshwater-seawater transfer. Osmoregulatory abilities were assessed by measurement of drinking rates, plasma ion and plasma cortisol concentrations after acute freshwater-seawater challenge and compared with freshwater-adapted and seawater-adapted control groups. Finnock displayed physiological responses typical of euryhaline teleosts upon seawater challenge; a rapid increase in drinking rate, an increase in plasma ion concentrations (but only to levels similar to, or slightly greater than, those of seawater-adapted fish), and increased plasma cortisol concentrations. The second experiment, in which numbers of finnock were greater, made use of the same techniques to assess the longer term acclimation of finnock to both freshwater-seawater and seawater- vi freshwater challenge, to establish whether finnock might suffer from a more subtle reduction in seawater-tolerance which would not have been necessarily apparent in the acute challenge of Experiment 1. Finnock did not appear to be physiologically compromised by seawater challenge during the winter months, and therefore, a breakdown in hypo-osmoregulatory abilities alone cannot be considered a reason for finnock returning to estuaries and rivers during the winter. 4. The physiological effects of low to medium levels of infestation of the ectoparasitic copepod Lepeophtheirus salmonis (Kroyer) on wild sea trout post-smolts were assessed at intervals during the development of the parasite. A mean infestation level of 18 parasites caused significant disruption to the osmoregulatory ability of hosts, as demonstrated by significantly higher plasma osmolality and chloride ion concentrations when compared with naive post-smolts. In addition, since no skin lesions were apparent on the hosts, these physiological effects were considered to be the consequence of larval attachment to the gill filaments, thereby possibly puncturing the epithelia and also damaging vital branchial ion excretory cells. 5. The modern molecular genetic RAPD-PCR technique was used to screen DNA of Lepeophtheirus salmonis collected from wild and fanned salmonid hosts from around the Scottish coasts. This technique indicated markedly different patterns of genetic variation amongst L. salmonis of farmed and wild origin, and between different farms. A number of genetic markers were found to be exclusive to, or at considerably higher frequency amongst, sea lice collected from farmed salmonid hosts. This technique established the possibility of assigning provenance to L. salmonis collected from wild hosts