Using next generation sequencing to generate whole mitochondrial genomes for population genetics and phylogeography of cetaceans
Dr. Phillip Morin, Protected Resources Division, Southwest Fisheries Science Center
Abstract and bio
Live blog notes:
Hoelzel et al 2002 found extremely low genetic diversity in control region (1000 base pairs): only 13 haplotypes from 100 samples from global killer whales. LeDuc et al 2008 increased to 35 haplotypes in ~>180 samples, but still very little global structure in phylogenetic tree.
But there are good reasons to use whole mitochondrial genome (16.4 kilobase genome) broken into 2-3 overlapping products (4.8-9.4 kb). Next generation sequencing uses highly parallel sequencing of small (30-350bp) fragments, but generate 100 million to 10 billion copies very economically and quickly.
Gathered north pacific samples (only 5 offshore), including ENA (Eastern North Atlantic who differ most in tooth wear) type 1 and 2, offshore, resident, transient, unknown. Also had samples from Antarctic whales and by Andy Foote from N Atlantic whales. We used Baysian techniques and publicly available mitochondrial priors from a wide range of marine mammals and managed to date divergence in killer whales to ~700,000 years ago.
Killer whale mitogenetics show that transients diverged ~700ky ago. In comparison, residents and offshores diverged much more recently, ~175ky ago (e.g. conventional wisdom: beginning of the pliocene). Antarctic B/C diverged from each other 150ky ago, and from A/GoM 335ky. Nuances are: proximity of ENA (1/2) and a Hawaii whale to North Pacific residents/offhores hints of exchange through the Northwest passage; some Antarctic A individuals have a haplotype close to transients, suggesting there may be even more types of killer whales in Antarctica (Bob plans to find out).
De Queiroz, 2007: helps in defining of species/subspecies — a hot topic for killer whales
- B/C Antarctic types have strong morphological, feeding behavior and prey, group size, and genetic differences. Foote et al. 2010.
- N Pac transients: should be distinct species, primarily due to genetic divergence, though they also differ in morphology, feeding behavior and prey, group size, acoustics, fatty acids, contaminants.
- Resident/Offshores we tend to believe are different sub-species, or species awaiting more evidence. We have especialluy little info about offshores (only 5 samples and minimal behavioral differences).
- North Atlantic situation is undetermined.
So, we had this low world-wide diversity (even in microsatellites — why?). With whole mitogenome, we have strong association of ecotypes and genotypes. For species with low mtDNA sequence diversity or poor phylogenetics, these new techniques can be very useful!
Other species that could benefit:
- Blue whales (taxonomy and population structure, using SNPs)
- Fin whales (150 mitogenomes sequenced but not analyzed; clear need for analysis of whether N Pac and Atlantic are really the same species (likely a historic taxonomic mistake)
- Sperm whales (even less diverse than KWs — globally about 30 haplotypes, but 90% of samples fall into 3 haplotypes)
- Turtles (effectively dinosaurs — been around for millions of years w/only 7 species and handful of haplotypes; SNPs may help describe population structure of leatherback and green turtles that move around the globe and are currently hard to genotype to source location when caught in longline fisheries)
Mike Ford Q: have you estimated historic population sizes from your results? We’ve only recently started those analyses and we’re overwhelmed with data. A current Masters student is looking at rates of patterns of evolution in mitochondrial genome. Hoping to fund a post-doc (or any other collaborators!) to look at historic population size.
Q: Did you differentiate between N Pacific residents: We had 1? southern resident and a couple from Russia, but no BC residents.
Q: What’s difference between ecotype and subspecies? It’s a really tough call (demographically distinct, DPS, evolutionarily distinct…). In my mind, a subspecies is one in which you have multiple lines of evidence (not necessarily including genetic) suggesting distinctive evolutionary trajectories. There is likely gene flow in delphinids (some evidence from microsatellite data, but some is suspect inference).
Q: Is there an issue with nodes evolving at different rates? Our MS student is working on that and has a manuscript in preparation, but we’re still confident in our times.
Q: What are the different potentials of mitochondrial, microsatellites, and SNPs as tools for understanding evolution? I hate microsatellites because we don’t understand them, especially their mutation rates (overestimate gene flow and underestimate divergence time)! They indicate divergence, but aren’t diverging linearly in time. SNPs are so simple in comparison!