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

https://doi.org/10.1016/j.ympev.2015.05.022Get rights and content

Highlights

  • Sequences for 26 loci were obtained for the extinct Steller’s sea cow with hybridization capture and 2nd generation sequencing.

  • Steller’s sea cow is more closely related to dugongs than to manatees.

  • The edentulous Steller’s sea cow exhibits an inactivating splice site mutation in the enamelin (ENAM) gene.

Abstract

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 >30 kb 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.

Introduction

Sirenia is one of two fully aquatic mammalian clades. Like Cetacea, Sirenia has a fossil record extending back to the early middle Eocene (Savage et al., 1994, Domning, 2000, Benoit et al., 2013) and includes quadrupedal forms that document the early evolution of this group from terrestrial ancestors (Domning, 2000, Domning, 2001). Recent sirenians include three manatee species (Trichechus inunguis, T. manatus, T. senegalensis), the dugong (Dugong dugon), and Steller’s sea cow (Hydrodamalis gigas). The latter was discovered in the North Pacific by Georg Wilhelm Steller in 1741 and became extirpated by 1768 as a consequence of human predation on H. gigas and/or the indirect effects of human predation on sea otters (Domning, 1978, Forsten and Youngman, 1982, Anderson, 1995, Turvey and Risley, 2006, Domning et al., 2007). Turvey and Risley (2006) employed a modeling approach and concluded that overhunting was sufficient to exterminate Steller’s sea cow and that the effects of sea otter removal on sea cow decline were minimal. In addition to their much larger size – up to 10 m in length and up to 11,000 kg in mass (Domning, 1978) – H. gigas are distinguished from extant sirenians by their lack of teeth, and instead used a pair of broad cornified horny pads to masticate kelp (algal seaweeds) (Steller, 1751, Steller, 1899, Domning, 1976, Forsten and Youngman, 1982). D. dugon exhibits an intermediate condition and possesses both horny pads and teeth (Lanyon and Sanson, 2006). However, the teeth are simple, peglike structures that lose their thin coat of surface enamel from wear shortly after eruption (Lanyon and Sanson, 2006).

Hydrodamalis has traditionally been placed in the family Dugongidae, along with Dugong, whereas manatees belong to the family Trichechidae (McKenna and Bell, 1997). Cladistic analyses of anatomical characters from the cranium provide some support for an association of Hydrodamalis and Dugong to the exclusion of Trichechidae (Domning, 1994, Buffrénil et al., 2010, Vélez-Juarbe et al., 2012). However, Voss (unpublished doctoral dissertation, 2013) concluded that Hydrodamalis is more closely related to trichechids than to dugongids based on a cladistic analysis of a matrix that included both cranial and postcranial characters. Thus, cladistic analyses of anatomical characters provide only mixed support for an association of Hydrodamalis and Dugong to the exclusion of Trichechidae. Molecular studies addressing this problem include immunological comparisons (Rainey et al., 1984) and analyses of partial sequences for the mitochondrial cytochrome b (CYTB) gene that were obtained with PCR and Sanger sequencing (Ozawa et al., 1997). Both of these studies recovered an association of Hydrodamalis and Dugong to the exclusion of Trichechus, although bootstrap values in the mitochondrial study were below 70% (Ozawa et al., 1997). By contrast, Crerar (unpublished doctoral dissertation, 2012) used PCR and Sanger sequencing to obtain partial sequences (several hundred base pairs) for two mitochondrial genes, CYTB and D-loop, with more extensive taxon sampling among paenungulates (Sirenia + Proboscidea + Hyracoidea) than Ozawa et al. (1997). Crerar’s analyses provided only limited support for dugongid monophyly (i.e., Hydrodamalis + Dugong) and in some analyses grouped Dugong and Trichechus to the exclusion of Hydrodamalis or placed Hydrodamalis within Trichechus (Crerar, unpublished doctoral dissertation, 2012).

Here, we address the phylogenetic position of Hydrodamalis gigas with both molecular and morphological data. Molecular targets (26 nuclear loci; ∼34 kb) were enriched via hybridization capture using probes predominantly designed from dugong and manatee DNA sequences. Probes using elephant and hyrax sequence as bait were also employed to capture the complete coding sequence of the tooth-specific gene enamelin (ENAM) from both modern (dugong) and ancient (Hydrodamalis) DNA samples. The morphological data set builds on Vélez-Juarbe et al.’s (2012) matrix and includes both cranial and postcranial characters for Recent and fossil Sirenia. We provide phylogenetic and timetree estimates based on molecular and morphological data sets, and outline important steps in the macroevolutionary history of Sirenia. We also provide evidence for an acceptor splice site mutation in the ENAM gene of H. gigas, thereby providing another example of molecular decay of ENAM that parallels morphological degeneration of enamel in the fossil record.

Section snippets

Sampling

Six Hydrodamalis gigas specimens (ZI 6842, ZI 6844, ZI 6846, ZI 6852, ZI 6853, and ZI 17170(2)) collected in the mid-to late 1800s and housed in the Zoological Institute of the Russian Academy of Sciences (St. Petersburg, Russian Federation) were sampled. Extreme care was taken to minimize damage to the specimens, with sampling primarily conducted using a hand-held Dremel Moto-Tool. Cutting disks were replaced for each sample to prevent cross-contamination. Following collection, extracted

Hybridization capture results (2011)

A total of 24.7 and 40.0 million trimmed singleton reads were obtained from the two Dugong dugon and three Hydrodamalis gigas specimens, respectively. As expected, the percentage of these reads aligning to the manatee genome (TriManLat1.0) was much higher for modern (∼70%) versus ancient (∼8%) samples (Supplementary Table S8). Hybridization probes designed using dugong and manatee nuclear sequence were highly effective (96–100%) in capturing both modern (dugong) and ancient (Steller’s sea cow)

Gene capture with phylogenetically divergent probes

To our knowledge, this is the first study to demonstrate both interfamilial and interordinal gene capture from ancient DNA samples, though successful confamilial capture has been reported for both older primate (50,000 year old Neanderthal; Burbano et al., 2010) and more recent (47–170 year old museum specimens) dermopteran samples (Mason et al., 2011). Most of the bait sequences that were employed to capture gene segments from Hydrodamalis gigas were designed using homologous coding segments

Acknowledgments

This research was supported by NSF (United States) Grants EF0629860 (M.S.S., J.G.), EAR-PF1249920 (J.V.J.), and DEB1132229 (M.H.), NSERC (Canada) Discovery and Accelerator Supplement Grants (K.L.C.), and an NSERC Alexander Graham Bell Canada Graduate Scholarship (A.V.S.). We thank two anonymous referees for constructive comments on an earlier version of this manuscript.

Glossary

AfroSINE
a novel family of short interspersed nuclear elements (SINEs) whose distribution is restricted to the genomes of afrotherian mammals
Relaxed molecular clock
a molecular clock model that relaxes the equal rates assumption of a strict molecular clock and allows for rate variation across lineages
Sirenia
an order of placental mammals that includes the first ancestor of Dugong dugon that is not also an ancestor of Loxodonta africana (African elephant), Cornwallius sookensis (desmostylian), or

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