Jennifer Gardner May 2, 2012

Fish 475 Preproposal Draft

Nuclear Gene Analysis to Determine Phylogeny of Orcinus orca Populations in
the Eastern North Pacific
A Preproposal By: Jennifer Gardner
Introduction
There are currently three distinct ecotypes of killer whale (Orcinus orca) in the Eastern North
Pacific (ENP) (Morin et al. 2006). These three ecotypes vary based on a number of different characters.
Arguably the most important of these characters is feeding ecology. The transient killer whale
population feeds on marine mammals (Ford et al. 1998, Saulitis et al. 2000). Their acoustic patterns
(Barrett-Lennard et al. 1996), morphology (Baird and Stacey 1988), and social structure (Sautilis et al.
2000) also vary from other sympatric populations. The offshore killer whale population is thought to
feed on fish, but their foraging ecology is not well known (Morin et al. 2006). Lastly, there are three
distinct populations of resident killer whales. All resident whales are known to feed on fish and
thought to be salmon specialists (Ford et al. 1998, Saulitis et al 2000). The distinct populations of
residents vary based on geographic region and are not known to interact with each other. There is the
Alaskan population, whose range is the Alaskan coast. There is the Northern resident population, which
inhabits the waters along the coast of British Colombia (Hoelzel et al. 1998). Finally, there is the
Southern resident population, which lives along the coast of Washington State and may possibly range
down the coast of the United States past Oregon to California. It is this population that inhabits the
Puget Sound (Hoelzel et al. 1998).
Currently the resident populations are recognized as distinct population segments (DSPs) for the
purposes of categorization under the US Endangered Species Act (ESA). This is significant because
currently only the southern resident population is listed and endangered under the ESA (Morin et al.
2006). However, the ESA treats DSPs and subspecies differently than it does total species (Haig et al.
2006). In order to properly manage the killer whale populations, it is imperative that their true
relationships be determined. The transient population has been shown to differ genetically from the two
other populations found in ESP (Morin et al 2010, Hoelzel 2002). The question this study will address is if
that variation is enough to consider the transient population a separate species.
Morin et al. (2010) claims the genetic variation is in fact enough to classify the transient
population a separate species. Based on comparisons of full mitochondrial sequences of all killer whale
populations, the transient population of the Eastern North Pacific has been diverging from the other
killer whale populations for the longest amount of time. The transient population has been genetically
isolated for roughly the past 700,000 years (Morin et al. 2010). I hypothesize that by analyzing long
segments of nuclear genes these findings will be supported and there will be enough data to resolve the
ecotypes into two separate species.
There has been much debate among taxonomists as to which type of analysis is preferable when
constructing phylogenies. There is the question if more genes create a better phylogeny or if simply
adding more samples and taxa will create a stronger phylogeny. Number of genes has been shown to be
associated with stronger more accurate phylogenies whereas number of taxa has no effect on the
strength and accuracy (Rokas and Carroll 2005).
Additionally there is debate over which type of genetic analysis will provide a more accurate
phylogenetic tree. It has been argued that mitochondrial genes are better for resolving species
phylogenies, especially in rapidly or recently evolved species (Moore 1995). However, a direct rebuttal
to this suggestion states that there are certain situations where mitochondrial genes will actually be less
informative than nuclear genes (Hoelzer 1997). One such scenario is when a species experiences
female philopatry combined with male dispersal (Hoelzer 1997). Long term studies indicate that killer
whales experience this type of mating structure to some extent (Hoelzel et al. 2007, Pilot et al. 2010).
While killer whale pods are tightly knit family based groups, they are matrilineal. However, males and
females remain with family groups throughout their lifetime, thus exhibiting a type of female philopatry.
Since the groups are family based, males are forced to venture out and breed with other pods to avoid
inbreeding. This is where male dispersal comes in (Houghton 2012). Based on this it would clearly be
beneficial to sequence major parts of the nuclear genome of killer whales for phylogenetic analysis.
Similarly, since the entire mitochondrial genome for the species has already been sequenced and
compared (Morin et al. 2010), the benefit of sequencing nuclear genes for phylogenetic comparison is
obvious.
Nuclear gene studies in the ENP killer whales could provide information for conservation of
these populations as well as a methodology that could then be expanded to the rest of the killer whale
populations worldwide. Due to the fact that previous studies found low rates of genetic variation among
the killer whale ecotypes (Hoelzel et al. 2002, Hoelzel et al. 1998), divergence found by Morin et al.
(2010) needs to be further supported before new species can be described with any credibility. Since
morphological and ecological differences have already been well studied the next logical step is to show
nuclear gene differentiation.
Developing a methodology for sequencing and comparing long segments of the killer whale
nuclear genome would be incredibly useful in a number of ways. First, if a few key regions of the killer
whale genome can be identified as variable, these regions could be used to help resolve questions of
killer whale taxonomy of the ecotypes present in the ENP but also worldwide. For example, some of the
ecotypes in the Antarctic region show the possibility of being one or more new species (Morin et al.
2010, LeDuc et al. 2008). If nuclear regions variable enough to be useful in phylogenetic analysis can be
identified, these same regions could be used to help resolve this issue as well.
It makes the most sense to start the search for useful nuclear regions in the ENP populations for
a number of reasons. One reason being that travel and research costs in this region and substantially
lower than in the Antarctic. In addition, the transient killer whale population has been shown to be very
genetically different from the resident population in terms of mitochondrial and microsatellite DNA
(Hoelzel et al. 1998, Morin et al. 2010). Thus it logically follows that if high amounts of variation are
going to be seen in a nuclear region, it will most likely be between two groups that are known to vary in
other region of the genome.
The most important reason for resolving the differences in killer whale populations is so that
they can be managed and protected in the most effective and efficient way possible. Currently only the
southern resident species of the Eastern North Pacific is listed as endangered as a DSP under the
Endangered Species Act (Haig 2006). However, as noted above, DSP management and conservations is
handled in a different fashion than that for entire species. It is also very difficult to get a population to
be listed as a DSP. The time and energy put in to getting a population even listed as a DSP, is time and
energy that could be put toward actual conservation work if it were listed as its own species. Being able
to resolve the ecotypes in separate species will make a management and conservation regarding the
species simpler and more effective.

Methods
Because this study is only focusing on whales in the Eastern North Pacific, it is imperative to get
as many DNA samples from as many different whales as possible. To this end the samples that have
already been collected would be assessed and if possible used for the study. The Vancouver Aquarium
claims to have over 500 samples from animals around Alaska and the coast of British Colombia
(Vancouver Aquarium 2011). The Northwest Fisheries Science Center and the National Marine Mammal
Laboratory have also been sampling these populations for many years and have biopsy samples in
reserve that could also be used (Houghton 2012). By using these samples only minimal field collecting
would be done to supplement underrepresented groups from this sample.
All field collecting will be focused on sampling the offshore ecotype as the majority of previous
sampling efforts have focused on resident and transient popluations (Vancouver Aquarium 2011,
Houghton 2012). This sampling will be done in areas of known high whale activity. Field collection will
involve the use of air guns or crossbows loaded with floating skin biopsy darts (Amos 1992). Skin
biopsies will be preserved at sea in a 20%DMSO/salt solution (Amos 1992). Ideally, around 200 samples
would be used in the final study. These 200 would include 50 samples each from the transient stock, the
offshore stock, the northern resident stock, and the southern resident stock. However, due to collection
difficulties the numbers may vary. In addition to field sampling, samples from stranded whales would
also be obtained through contact with the stranding response network that is active up and down the
coast. Ecotype of these animals will be determined by stomach contents and well as possible picture
identification. Field collecting permits will be obtained before any work is done.
Once samples are collected, DNA will be extracted using common extraction techniques,
including those found in Morin et al. (2010). Once DNA is extracted, initial PCR will be run using primers
for 20 different genes (McGowen 2011). These genes will be used as a base line to see divergence. If
little variation is seen, other primers will be designed for different segments of linked genes, the goal
being to sequence long fragments of the nuclear genome. The designing of primers and identifying of
genes to study will take place using only a few samples (n=20). However, once a region of high
representative variance is found, all samples will be amplified for this region. Amplified regions will then
be sent out for sequencing. The returned sequences will be run on software BEAST (Drummond and
Rambaut 2007, Morin et al. 2010) as well as SMOGD (Crawford 2010) to determine both Bayesian
phylogenies and degrees of genetic divergence respectively. The results of the phylogenies will
hopefully support the hypothesis that nuclear genes will show transient killer whales in the Eastern
North Pacific to be a separate species. With support of this hypothesis from both mitochondrial and
nuclear genes as well as morphology and ecology, work could then be done describing and naming the
new species.
Time Schedule
Work will begin as soon as possible. Samples will be obtained from previous collections and lab
work in identifying the nuclear regions to be analyzed will begin at once. This work should be done at
most in nine months (February 2013). Field collecting will be done April 2013-September 2013. Sampling
will take place for the whole summer until as many samples as possible are obtained or enough samples
are obtained to supplement collection samples. Lab work in amplifying, sequencing and analyzing the
DNA will take place concurrently with field collecting and will be finished six months after the last field
collections are done (March 2014). Time until the study can be published should be an additional three
to four months (June 2014-July 2014).


Literature Cited
Amos B, Hoelzel AR. 1992. Applications of molecular genetic techniques to the conservation of small
populations. Biological Conservation 61: 133-144.
Baird, RW. 1988. Variation in saddle patch pigmentation in populations of killer whales (Orcinus orca)
from British-Columbia, Alaska, and Washington state. Canadian Journal of Zoology 66 (11): 2582.
Barrett-Lennard LG, Ford JKB, Heise KA.1996. The mixed blessing of echolocation: differences in sonar
use by fish-eating and mammal-eating killer whales. Animal Behavior 51: 553-565
Crawford NG. 2010. SMOGD: software for the measurement of genetic diversity. Molecular Ecology
Resources 10: 556-557.
Drummond AJ, Rambaut A. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC
Evolutionary Biology 7: 214.

Ford JKB, Ellis GM, Barrett-Lennard LG, Morton AB, Palm RS, Balcomb III KC. 1998. Dietary specialization
in two sympatric populations of killer whales (Orcinus orca) in coastal British Columbia and
adjacent waters. Canadian Journal of Zoology 76 (8): 1456.

Haig SM, Beever EA, Chambers SM, Braheim HM, Bugger BD, Dunham S, Elliott-Smith E, Fontain JB,
Kesler DC, Knaus BJ, Lopes IF, Loschl P, Mullins TD, Sheffield LM. 2006. Taxonomic
Considerations in Listing Subspecies Under the U.S. Endangered Species Act. Conservation
Biology 20 (6): 1548-1594.

Hoelzel AR, Hey J, Dahlheim ME, Nicholson C, Burkanov V, Black N. 2007. Evolution of Population
Structure in a Highly Social Top Predator, the Killer Whale. Molecular Biology and Evolution 24
(6): 1407-1415

Hoelzel AR, Natoli A, Dahlheim ME, Olavarria C, Baird RW, Black NA. 2002. Low worldwide genetic
diversity in the killer whale (Orcinus orca): Implications for demographic history. Proceedings:
Biological Sciences 269: 14671473.

Hoelzel AR, Dahlheim M, Stern SJ. 1998. Low Genetic Variation Among Killer Whales (Orcinus orca) in the
Eastern North Pacific and Genetic Differentiation Between Foraging Specialists. Journal of
Heredity 89: 121-128.

Hoelzer GA. 1997. Inferring Phylogenies from mtDNA Variation: Mitochondrial-Gene Trees Versus
Nuclear Gene Trees Revisited. Evolution 51(2): 622-626.

Houghton J. 2012. Southern Resident Killer Whales. Lecture: May 4, 2012 for Fish 475 at Univerisity of
Washington, Seattle.

LeDuc RG, Robertson KM, Pitman RL. 2008. Mitochondrial sequence divergence among Antarctic killer
whale ecotypes is consistent with multiple species. Biological Letters 4: 426429.

McGowen MR. 2011. Toward the resolution of an explosive radiation- A multilocus phylogeny of oceanic
dolphins (Delphinidae). Molecular Phylogenetics and Evolution 60: 345-357.

Moore WS. 1995. Inferring Phylogenies from mtDNA Variation: Mitochondrial-Gene Trees Versus
Nuclear Gene Trees. Evolution 49 (4): 718-726.

Morin PA, LeDuc RG, Roberston KM, Hedrick NM, Perrin WF. 2006. Genetic analysis of killer whale
(Orcinus orca) historical bone and tooth samples to identify western U.S. ecotypes. Marine
Mammal Science 22(4): 897-909.

Morin PF, Archer FI, Foote AD, Vilstrup J, Allen EE, Wade P, Durban J, Parsons K, Pitman R, Li Lewyn,
Bouffard P, Able Nielsen SC, Rasmussen M, Willerselv E, Gilbert MTP, Harkins T. 2010 Complete
Mitochondrial genome phylogeographic analysis of killer whales (Orcinus orca) indicates
multiple species. Genome Research 20: 908-916.

Pilot M, Dahlheim ME, Hoelzel AR. 2010. Social cohesion among kin, gene flow without dispersal and the
evolution of population genetic structure in the killer whale (Orcinus orca). Journal for
Evolutionary Biology 23: 20-31.

Rokas A, Carroll SB. 2005. More Genes or More Taxa? The Relative Contribution of Gene Number and
Taxon Number to Phylogenetic Accuracy. Molecular Biology and Evolution 22(5): 1337-1344

Saulitis, E., C. Matkin, L. G. Barrett-Lennard, K. Heise And G. M. Ellis. 2000. Foraging strategies of
sympatric killer whale (Orcinus orca) populations in Prince William Sound, Alaska. Marine
Mammal Science 16:94109.

Vancouver Aquarium. 2011. Sleuthing the Secret Lives of Killer Whales with DNA.
http://killerwhale.vanaqua.org/page.aspx?pid=1357


Budget

Item Cost
PI Salary $48,000.00
Graduate Student Salary $19,968.00
Graduate Student Benefits $3,215.00
Student Hourly $14,400.00
Student Hourly Benefits $2,146.00
Travel $18,000.00
Boat Charter $60,000.00
Dart Equipment $15,000.00
Lab Bench and Material $1,000.00
Sequencing Fees $1,500.00
Total (Direct) $183,229.00
Indirect $128,644.00
Equipment
Graduate Student Operating Fees $15,291.00
Total $327,164.00

Budget Justification
PI salary is covering the cost of six months, because this project will span at least two summers. Thus the
salaries from 15 June 2012- 15 September 2012 as well as the salary from 15 June 2013-15 September
2013 are included. The costs of hiring a Graduate student (Tier I) are also included, as well as the costs
of hiring 2 hourly employs for 20 hours a week for six months (the two summer periods).
Boat charter fees are calculated for the maximum amount of time needed on the boat and dart
equipment, including darts, crossbow, and air gun, are included for the maximum amount needed.
Assuming that most samples are obtained from the Vancouver Aquarium collection and the NWFSC
collection, these costs will in reality be drastically lower.