Microsatellite Analysis for Identification of Individuals Using Bone from the Extinct Steller's Sea Cow (Hydrodamalis gigas).

Microsatellite DNA can provide more detailed population genetic information than mitochondrial DNA which is normally used to research ancient bone. The methods detailed in this chapter can be utilized for any type of bone. However, for this example, four microsatellite loci were isolated from Steller's sea cow (Hydrodamalis gigas) using published primers for manatee and dugong microsatellites. The primers DduC05 (Broderick et al., Mol Ecol Notes 6:1275-1277, 2007), Tmakb60, TmaSC5 (Pause et al., Mol Ecol Notes 6: 1073-1076, 2007), and TmaE11 (Garcia-Rodriguez et al., Mol Ecol 12:2161-2163, 2000) all successfully amplified microsatellites from H. gigas. The DNA samples were from bone collected on Bering or St. Lawrence Islands. DNA was analyzed using primers with the fluorescent label FAM-6. Sequenced alleles were then used to indicate a difference in the number of repeats and thus a difference in individuals. This is the first time that H. gigas microsatellite loci have been isolated. These techniques for ancient bone microsatellite analysis allow an estimate of population size for a newly discovered St. Lawrence Island sea cow population.

Interordinal gene capture, the phylogenetic position of Steller's sea cow based on molecular and morphological data, and the macroevolutionary history of Sirenia.

The recently extinct (ca. 1768) Steller's sea cow (Hydrodamalis gigas) was a large, edentulous North Pacific sirenian. The phylogenetic affinities of this taxon to other members of this clade, living and extinct, are uncertain based on previous morphological and molecular studies. We employed hybridization capture methods and second generation sequencing technology to obtain >30kb of exon sequences from 26 nuclear genes for both H. gigas and Dugong dugon. We also obtained complete coding sequences for the tooth-related enamelin (ENAM) gene. Hybridization probes designed using dugong and manatee sequences were both highly effective in retrieving sequences from H. gigas (mean=98.8% coverage), as were more divergent probes for regions of ENAM (99.0% coverage) that were designed exclusively from a proboscidean (African elephant) and a hyracoid (Cape hyrax). New sequences were combined with available sequences for representatives of all other afrotherian orders. We also expanded a previously published morphological matrix for living and fossil Sirenia by adding both new taxa and nine new postcranial characters. Maximum likelihood and parsimony analyses of the molecular data provide robust support for an association of H. gigas and D. dugon to the exclusion of living trichechids (manatees). Parsimony analyses of the morphological data also support the inclusion of H. gigas in Dugongidae with D. dugon and fossil dugongids. Timetree analyses based on calibration density approaches with hard- and soft-bounded constraints suggest that H. gigas and D. dugon diverged in the Oligocene and that crown sirenians last shared a common ancestor in the Eocene. The coding sequence for the ENAM gene in H. gigas does not contain frameshift mutations or stop codons, but there is a transversion mutation (AG to CG) in the acceptor splice site of intron 2. This disruption in the edentulous Steller's sea cow is consistent with previous studies that have documented inactivating mutations in tooth-specific loci of a variety of edentulous and enamelless vertebrates including birds, turtles, aardvarks, pangolins, xenarthrans, and baleen whales. Further, branch-site dN/dS analyses provide evidence for positive selection in ENAM on the stem dugongid branch where extensive tooth reduction occurred, followed by neutral evolution on the Hydrodamalis branch. Finally, we present a synthetic evolutionary tree for living and fossil sirenians showing several key innovations in the history of this clade including character state changes that parallel those that occurred in the evolutionary history of cetaceans.

Rewriting the history of an extinction-was a population of Steller's sea cows (Hydrodamalis gigas) at St Lawrence Island also driven to extinction?

The Kommandorskiye Islands population of Steller's sea cow (Hydrodamalis gigas) was extirpated ca 1768 CE. Until now, Steller's sea cow was thought to be restricted in historic times to Bering and Copper Islands, Russia, with other records in the last millennium from the western Aleutian Islands. However, Steller's sea cow bone has been obtained by the authors from St Lawrence Island, Alaska, which is significantly further north. Bone identity was verified using analysis of mitochondrial DNA. The nitrogen-15 (δ(15)N)/carbon-13 (δ(13)C) values for bone samples from St Lawrence Island were significantly (p ≤ 0.05) different from Bering Island samples, indicating a second population. Bone samples were dated to between 1030 and 1150 BP (approx. 800-920 CE). The samples date from close to the beginning of the mediaeval warm period, which could indicate that the population at St Lawrence Island was driven to extinction by climate change. A warming of the climate in the area may have changed the availability of kelp; alternatively or in addition, the animals may have been driven to extinction by the expansion of the Inuit from the Bering Strait region, possibly due to opening waterways, maybe following bowhead whales (Balaena mysticetus), or searching for iron and copper. This study provides evidence for a previously unknown population of sea cows in the North Pacific within the past 1000 years and a second Steller's sea cow extirpation event in recent history.