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One of the most striking results found from the analyses of nuclear introns across ten Adélie penguin populations was the different patterns of variation observed for the marker AK1i5 compared to the other four sequenced. Due to the methodology used for phasing the haplotypes of the heterozygotic introns obtained, which relied on Bayesian probabilities implemented in PHASE, we cannot exclude the possibility that some haplotypes were incorrectly called. AK1i5 overall presented the most polymorphic sites (1 per 24.9 bp of sequence, compared to 1/26.9 (MPP4), 1/34.6 (UCHL3), 1/36.1 (ODC6), and 1/51.8 (HMG2)). PHASE utilizes the information from confirmed haplotypes to aid in assigning probabilities to putative haplotypes. For AK1i5, confirmed haplotypes showed high diversity. As a result, a number of potential phased haplotypes were almost equally probable (60%-40%). In order to address this uncertainty, cloning of PCR products from unphased heterozygotes and sequencing of ten clones or more per sample would be required, and/or increased sampling from further Adélie penguin individuals. At present this is beyond the scope of the current project, however, a smaller dataset for AK1i5 containing only unambiguously phased and homozygotic sequences showed similarly high diversities and a complicated network. The high reticulation and diversity at this marker may indicate a higher rate of evolution and potentially recombination. None of the analyses carried out could conclusively detect recombination. LAMARC estimates, despite not converging enough to provide a reliable estimate for all markers, did indicate varying amounts of recombination per marker. Estimates of recombination were highest for markers UCHL3, AK1i5 and ODC6, and lowest for HMG2 and MPP4. This is consistent with the statistical parsimony haplotype networks generated for these markers. Both ODC6 and UCHL3 presented several probable connections involving repeated substitutions (marked in red; Fig. 5.5). AK1i5 presented a much higher degree of reticulation still (Fig. 5.6). Recombination at these loci could provide one explanation for these patterns; however, a larger sample size for each intron would be necessary to fully address this possibility.

In the present study I sought to investigate whether variation in nuclear introns followed a similar pattern as that found in mitochondrial DNA, providing support for

the hypothetically refugial mitochondrial lineages, A and RS. No support was found; haplotype networks constructed and coded according to mitochondrial lineage showed a random distribution of haplotypes with no clustering based on lineage. Coalescence time for nuclear markers is twice that of the mitochondrial genome however, and previous research hypothesized that the time to the most recent common ancestor of the A and RS lineages was 75 kyr BP (37-122 kyr), during the last glacial maximum. Also, nuclear introns, presenting mutation rates much lower than mitochondrial DNA in general and the control region in particular, accumulate changes more slowly and therefore are more prone to incomplete lineage sorting. The influence of recombination also aids to remove any evidence of genetic structuring when barriers to gene flow have disappeared. The estimate of the tmrca of the mitochondrial A and RS lineages was based on a mitochondrial HVRI substitution rate calculated using radiocarbon-dated ancient Adélie subfossil bones (LAMBERT et al. 2002). Ritchie et

al (2004) justify their use of this faster rate (0.96 s/s/Myr) for their estimates of lineage divergence times, rather than the more frequently used phylogenetic rate (0.208 s/s/Myr) due to low likelihood that the A and RS lineages would have remained isolated through multiple glacial cycles. The lack of any A/RS split among the five introns sequenced for this study lends support to their use of this faster rate. If A and RS lineages had remained separated from 120 kya until the Last Glacial Maximum leading to the Holocene, nuclear regions would have presumably accumulated enough substitutions to affect haplotypic representations.

No other structuring was observed in these networks. Networks were not coded according to sample provenance, as a fine-scale population analysis was not the objective of the present study. To further investigate whether any nuclear intron structure or differentiation exists between Adélie penguin colonies, sampling sizes of introns for each colony need to be increased by at least ten individuals, and further loci should be added. However, considering evidence for sporadic gene flow between Adélie penguin colonies (DUGGER et al. 2010; SHEPHERD et al. 2005), it is unlikely

that any genetic structuring at the nuclear level would be evident. This finding supports prior research using microsatellite markers that also failed to find any evidence of structure among Ross Sea colonies (ROEDER et al. 2001).

Analysis carried out in this study indicated a probable historical demographic expansion. Haplotype networks were mostly star-shaped, which is thought to be characteristic of expanding populations (SLATKIN and HUDSON 1991). In support of this hypothesis, negative and significant Tajima’s D and Fu’s Fs were found for four of the introns, while Fu and Li’s F*

and D*

were non-significant. The mismatch distributions failed to reject the sudden expansion model, and growth estimates from

LAMARC were large and different from zero for two of the markers (for two others,

confidence intervals included zero, while marker AK1i5 did not support growth). Interestingly, the three markers showing evidence of recombination were those that least supported population expansion. Further sampling, identification of recombinants, and re-analysis with recombinant-free alignments could aid in determining whether the effect of recombination is masking the signature of demographic growth. While individually these tests may not be enough to distinguish between selection or demographic effects, and further LAMARC runs could be carried

out to increase confidence in the estimates obtained, taken together they indicate that Adélie penguins have the signature of an expanding population at nuclear loci as well as at the mitochondrial genome. Due to the difference in coalescence times for nuclear and mitochondrial regions, it is likely that they are identifying separate expansion events, since each glacial Antarctic cycle probably resulted in repeated population bottlenecks due to the loss of ice-free breeding areas (RITCHIE et al. 2004). Depending on the mutation rate used, the estimated times since expansion varied drastically. Averaged over the five loci, these values ranged from 1.95 Mya to 6.14 Mya. This range is wider still if one takes the confidence intervals into account (6.23 kya – 38.3 Mya). As a result, this reported time to expansion cannot be accurately determined at present. However, even the most recent estimate of time to expansion is well outside the confidence intervals for the divergence between the A and RS mitochondrial lineages (upper limit, 122 kya), which would have predated Adélie penguin expansion after the last glacial maximum. Almost certainly the signature of expansion reported here for nuclear introns originates from earlier population contractions and expansions. This illustrates the importance of using molecular markers that reflect different time periods within population history of a species.