3. Results and discussion
Levels of genetic variation at both mitochondrial and microsatellite loci are quite low in the Cerro Fatal population in contrast to the moderate to high levels exhibited in the Cerro Montura and La Caseta porteri populations (table 1). Overall, genetic variation among the three Santa Cruz populations is highly structured with most genetic variation being among, rather than within, populations (table 2). Genetic differentiation between populations was also revealed by highly significant fixation indices for all possible pairwise comparisons, observed for both the mitochondrial CR and microsatellite datasets (table 2). In fact, all three populations are reciprocally monophyletic as recovered from the mtDNA haplotype tree (data not shown) and population aggregation analysis revealed distinct aggregates among the domed porteri, with 31 fixed nucleotide differences across four mtDNA gene regions diagnosing Cerro Fatal porteri from La Caseta porteri (data not shown). From both a genealogical (Avise & Ball 1990) and phylogenetic species (Davis & Nixon 1992) perspective, the two domed lineages of porteri in Cerro Fatal and La Caseta represent distinct biological units. The presence of 100 private alleles between these two populations across nine microsatellite loci provides further evidence for this split, as do the results of assignment tests that identified 88 and 96% of La Caseta and Cerro Fatal individuals, respectively, as belonging to the populations in which they were collected. The three saddlebacked Cerro Montura individuals are also clearly genetically distinct from the two domed porteri on Santa Cruz, sharing all mtDNA haplotypes and the majority of microsatellite alleles (greater than 80%) with the saddleback ephippium population from the nearby island of Pinzón (figure 1). These individuals from north western Santa Cruz could represent the only remaining survivors of the source population from which the ephippium populations originated, or a recent secondary colonization from Pinzón (Fritts 1984).
On a higher level, phylogenetic analysis reveals that the lineages on Santa Cruz are paraphyletic, all exhibiting closer affinities with taxa on other islands than with each other (figure 2). The Cerro Fatal porteri are most closely related to chatamensis on San Cristóbal, with pairwise genetic distances indicating a stronger association with the extinct domed population (figures 1 and 2). In contrast, La Caseta porteri is the basal lineage within a clade that includes the extinct Floreana taxon and several lineages from the younger, western island of Isabela (figures 1 and 2). Of all pairwise comparisons involving each taxon, La Caseta porteri and the extinct Floreana taxon reciprocally exhibit the shallowest sequence divergence. Lastly, the Cerro Montura individuals form a well-supported sister group with the ephippium taxon on Pinzón, consistent with both morphology and mtDNA haplotype data (figures 1 and 2). Overall, topological tests rejected hypotheses of monophyly (pporteri alone (Cerro Fatal and La Caseta).
In addition to the distinct phylogenetic affinities of the three porteri lineages with taxa from different islands, application of a species-specific mtDNA CR rate (Beheregaray et al. 2004) suggests a temporal divide in their relative divergence times. The split between the Cerro Fatal porteri and San Cristóbal chatamensis lineages exhibits differing divergence times relative to the extinct, domed and extant saddlebacked chatamensis lineages, estimated at approximately 445000 and 490000 years ago (ya), respectively. Slightly deeper divergence times were reconstructed for La Caseta porteri and the extinct taxon on the southern island of Floreana, with a split dated between 584000 and 926000 ya. The timing of the saddleback lineages' separation from Cerro Montura on Santa Cruz and ephippium on Pinzón was much more recent (0-261000 ya).
Taken together, these findings suggest a significant revision of Galápagos tortoise taxonomy is in order. Of more immediate importance, the results of this molecular study unambiguously designate the La Caseta and Cerro Fatal porteri as distinct conservation units (Moritz 1994; Vogler & DeSalle 1994). This designation is especially pertinent for Cerro Fatal, as this population has suffered a dramatic decline in recent times due to heavy poaching and the conversion of their habitat to agricultural fields and lacks genetic variation compared with the fairly abundant and diverse population from La Caseta. From a broader perspective, despite years of study beginning with Darwin in 1835, modern DNA-based research coupled with thorough character and taxonomic sampling (extant and extinct) is still yielding insights into the taxonomy of the renowned giant tortoises of Galápagos. Of particular relevance are implications for conservation of the genetic, morphological and behavioural diversity of these extraordinary organisms, whose historical contribution to human intellectual history warrant them special attention.
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