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Study area and samples
Ectemnorhinus weevil specimens were collected over three consecutive years (April 2001 – April 2003) from 28 localities (Fig. 1) on Marion Island. Samples from Prince Edward Island which has restricted access were collected along an altitudinal gradient (0 – 675 m a.s.l.) at 200 m intervals and from an additional locality consisting largely of Ditrichum strictum, in April 2003. Coordinates for all the sampling localities are summarised in Table 1. All specimens were collected by hand and preserved in absolute ethanol.
For the morphometric component of the study, between five and 30 Ectemnorhinus specimens per locality were measured. Due to uncertainty about the taxonomic status of Ectemnorhinus species on both Marion Island and Prince Edward Island, individuals were only identified as belonging to the genus, based on the generic descriptions provided by Kuschel and Chown (1995), with a priori rather than a posteriori multivariate morphometric analyses being used to define phenetic groupings.
Molecular characterisation
Following rehydration of ethanol-stored weevils with water, one to two weevil legs per specimen were frozen in liquid nitrogen before being ground and mixed with phosphatebuffered saline (PBS). DNA was extracted using a modified guanidinium thiocyanate (GuSCN)/silica-based method (Boom et al. 1990). Published primers C1-J-1718 and TL2-N-3014 (Simon et al. 1994) were initially used to generate partial sequence data for representatives of all six weevil species occurring on Marion Island namely, Bothrometopus elongatus (Jeannel 1953), Bothrometopus parvulus (Waterhouse 1885), Bothrometopus randi (Jeannel 1953), E. marioni (Jeannel 1940), E. similis (Waterhouse 1885) and Palirhoeus eatoni (Waterhouse 1879). As these primers generally delivered poor quality sequences, two Marion Island weevil-specific COI primers were designed from the aligned partial sequences, following the guidelines of Rychlik (1993). These Marion Island weevil-specific COI primers termed GF and GR1 (Table 2) amplified a 1059 bp PCR product under the following conditions: 1×Buffer, 0.2 mM dNTP, 0.4 μM of each primer and 1 U Taq polymerase in a final volume of 50 l containing 200 ng of template DNA. A typical temperature profile consisted of an initial denaturation step at 94ºC for 90 s, followed by 40 cycles at 94ºC for 22 s, 46ºC for 30 s and 72ºC for 1 min. PCR products were purified and DNA sequences were determined by automated cycle sequencing on an ABI PRISMTM 3100 Analyser using the ABI PRISM Big DyeTM Terminator version 3.0 sequencing standard.
Phylogenetic analyses
Three sequence datasets were compiled, a Marion Island dataset, a Prince Edward Island dataset and a combined Prince Edward Islands archipelago dataset. Neighbor Joining (NJ; Saitou and Nei 1987) and Minimum Evolution (ME; Rzhetsky and Nei 1992) algorithms in MEGA version 2 (Kumar et al. 2001) were used to construct phylogenies with nodal support being assessed by 10 000 bootstrap replications. Model Test version 3.06 (Posada and Crandall 1998) was used to identify the model of evolution that best fits the data with parameters identified under the Akaike Information.
Criterion (Akaike 1974) being used for subsequent Maximum Likelihood analyses (Felsenstein 1981). In each case, the TrN + I model with equal rates for all sites that correspond to the General time–reversible model, GTR + I (Rodriguez et al. 1990) was selected. The proportion of invariable sites (I) and three different substitution types estimated for each dataset was as follows: 1) Combined data set: I = 0.7935, rate [A-G] = 48.19, rate [C-T] = 11.65, and other rates = 1.00; 2) Prince Edward Island dataset: I = 0.8018, rate [A-G] = 40.61, rate [C-T] = 9.03, and other rates = 1.00; and 3) Marion Island dataset: I = 0.7985, rate [A-G] = 50.85, rate [C-T] = 12.11, and other rates = 1.00.
Chapter 1: General Introduction
Chapter 2: Morphometric measurement selection: an invertebrate case study based on weevils from sub-Antarctic Marion Island
Chapter 3: Molecular and morphometric assessment of the taxonomic status of Ectemnorhinus weevil species (Coleoptera: Curculionidae) from the sub-Antarctic Prince Edward Islands .
Chapter 4: Morphometric changes over time: an analysis based on weevils (Coleoptera: Curculionidae) from the sub-Antarctic Marion and Prince Edward Islands
Chapter 5: Morphometric changes over time in weevils (Coleoptera: Curculionidae) from the sub-Antarctic Marion Island: an analysis based on mire sediment sub-fossil remains and recent samples
Chapter 6: Synopsis and conclusion
