Date post: | 18-Feb-2017 |
Category: |
Documents |
Upload: | chloe-annand |
View: | 25 times |
Download: | 2 times |
Annand
Creating a pedigree for California ground squirrels (Otospermophilus
beecheyi) to study kin-structured social networks
Chloe Annand
Fall 2015 BIO 191
1
Annand
Abstract
For centuries, the evolution of cooperative and seemingly altruistic behaviors have
baffled biologists. By definition, altruistic behaviors are costly to the actor and benefit
the recipient. How a behavior that decreases the direct fitness of the actor has evolved is
not yet well understood. It is thought that indirect fitness, the net gain of fitness benefit
from reproduction of a relative, may be one possible mechanism. In order to determine
how relatedness and cooperative behaviors intersect, we plan to use polymerase chain
reaction (PCR) primers amplifying neutral polymorphic microsatellite repeat loci to
assign parentage within our study population. We have optimized eleven primers for
California ground squirrels, Otospermophilus beecheyi. Thirteen of these loci are
potentially polymorphic. We suspect that there will be a low level of heterozygosity in
our study population, indicative of inbreeding. In the future, we can use these primers to
perform fragment analysis. From this, we will generate data to construct a pedigree that
can in turn be used to elucidate the extent to which kinship influences cooperative and
seemingly altruistic behaviors in California ground squirrels.
2
Annand
IntroductionThe selective pressures driving cooperative and seemingly altruistic behaviors represent
an evolutionary puzzle. A cooperative behavior is defined as one that increases the
fitness of the recipient, and has evolved due to the benefit of the recipient (Hamilton
1964, West et al. 2006). Because of this, cooperative behaviors may be beneficial,
neutral, or damaging for the individual performing the behavior. Altruistic behaviors are
beneficial to the recipient, but costly to the individual performing the behavior (West et
al. 2006). At first glance, there seems to be little evolutionary benefit to helping a
conspecific when an individual could be selfish and have greater access to resources.
Though common along a wide array of taxa, altruistic behaviors lead to decreased direct
fitness of the donor. For example, western honeybees (Apis mellifera) forage
cooperatively, with individuals alerting other foragers to high quality food sources
(Seeley and Visscher 1998). Moreover, long-tailed manakins (Chiroxiphia linear) mate
cooperatively. Beta males assist unrelated alpha males in performing courtship displays,
however the alpha male is the only one who regularly mates (McDonald and Potts 1994).
Rhesus monkeys (Macaca mulatta) altruistically aid conspecifics in fights by interfering,
which is risky for the actor (Kaplan 1978). How could a behavior that decreases the
actor’s fitness evolve? One possible mechanism is kin selection.
Kin selection, proposed by W.D. Hamilton (1964), suggests that altruistic traits
are selected for via net inclusive benefits from both direct and indirect reproduction.
Indirect fitness is gained by the reproduction of a close relative (Hamilton 1964). This is
in contrast to direct fitness, which is gained directly from individual reproduction. The
major concept underlying indirect fitness – the idea that an individual can increase their
fitness through the reproduction of a sibling – is explained by Hamilton’s equation. A
3
Annand
simple inequality, Hamilton’s equation, r x B > C, shows altruistic traits will be favored
when relatedness (r) and benefit in terms of offspring equivalents (B) are greater than the
cost in terms of offspring equivalents (C). Thus, inclusive benefits can act as a selective
pressure, conferring the evolution of nepotism, the preferential treatment of relatives.
Kin selection favors seemingly altruistic behaviors amongst a wide taxa of
animals, including mammals (reviewed by Smith 2014). Many social mammals have
evolved antipredator behaviors, suites of behaviors favored because they reduce the risk
of predation. While these behaviors are often beneficial to surrounding conspecifics,
antipredator behaviors tend to draw attention, increasing risk of predation, to the actor.
Alarm calls by Belding’s ground squirrel (Urocitellus beldingi) are thought to be
nepotistic, done for the benefit of nearby relatives (Sherman 1977). By alarm calling, the
actor not only alerts conspecifics to the danger, but also informs predators to the actor’s
exact location. This behavior probably evolved because the surrounding conspecifics are
closely related to the caller. Female Belding’s ground squirrels tend to remain close to
their natal burrow, surrounded by sisters, mother, aunts, and female cousins (Sherman
1977). By alarm calling, therefore, the individual increases her relative’s fitness and
reaps indirect fitness benefits. Meerkats (Suricata suricatta) on the other hand, seem to
gain direct fitness from guarding, an anitpredator behavior in which an individual, known
as a sential, stands on high ground and alerts conspecifics if predators are nearby
(Clutton-Brock et al. 1999). Guarding is potentially costly, depending on whether the
caller has eaten recently. Specifically, there is a trade-off between standing guard and
foraging, and it has commonly been believed that guards are more at risk for predation.
4
Annand
Because of this trade off, individuals are more likely to stand guard if they have recently
eaten, and are full.
Meerkats stand guard regardless of whether relatives are nearby. Dominant
females, who have the most related individuals within a group, were found to stand guard
significantly less than other adults in the group. Surprisingly unrelated immigrant
individuals spent roughly the same amount of time as other adults standing guard
(Clutton-Brock et al. 1999). This suggests that standing guard is in an individual’s best
interests. Standing guard can lead to decreases predation risk in the guard, a direct and
immediate fitness benefit. Thus, alarm calling can potentially evolve via direct benefits,
indirect benefits, or a combination of both through inclusive fitness benefits. Discerning
whether a behavior is nepotistic or selfish can be a challenge; the selective pressures
behind many social behaviors are not yet well understood. For many social animals, it is
not yet know if cooperative behaviors are altruistic or selfish.
The indirect fitness benefit gained from an action is largely dependent on
relatedness of the participating individuals. Because siblings share about 50% of their
genetic information, by enabling a sibling to reproduce, an individual can pass on 25% of
their genetic information. An altruistic behavior may only evolve via inclusive fitness if
the recipient and the actor share genes. Kin recognition mechanisms aid in selective
behaviors (Mateo 2003). In many species, phenotype recognition is the main mode of kin
recognition. Familiarity matching is another common mechanism for kin recognition, in
which individuals base recognition on shared associations (Wahaj et al. 2001). It is
possible for a species to evolve multiple mechanisms of kin recognition for different
contexts. Spotted hyenas (Crocuta crocuta) recognize maternal kin via familiarity
5
Annand
matching, whereas they recognize paternal kin via phenotypic matching (Wahaj et al.
2001).
Many mechanisms for kin recognition are commonly used by a variety of
Holarctic ground squirrels belonging to the tribe Marmotini. Among these species,
Belding’s ground squirrels are perhaps the best studied. Belding’s ground squirrels
produce at least two distinct scents that mediate kin recognition (Mateo 2003). Known as
kin labels, these odors from the oral and dorsal glands help individuals identify kin, both
familiar and unfamiliar. There is also evidence that kin selection in Columbian ground
squirrels, Spermophilus columbianus, relies on littermate bias (Hare and Murie 1996).
This study suggested that rearing association, not necessarily phenotype matching,
promotes cooperation among Columbian ground squirrels. Though Hare and Murie
(1996) interpreted these results to mean that cooperative behaviors have evolved by some
mechanism other than kin selection in Columbian ground squirrels, this is an
oversimplification of their data. In the vast majority of cases, rearing associations are a
strong indicator of relatedness. Though less accurate than phenotype matching or kin
labels, it is reasonable that rearing association would evolve as a mechanism to
distinguish between maternal kin and unrelated individuals.
California ground squirrels, Otospermophilus beecheyi, are facultatively social
and have evolved several altruistic anti-predator behaviors that may be nepotistic
(Owings et al. 1977; Smith et al., in review). Much like Belding’s ground squirrels,
California ground squirrels alarm call to warn nearby conspecifics of predators.
Individuals also tail flag (Putman et al. 2015) and throw substrate at rattlesnakes
(Swaisgood et al. 1999). Both of these behaviors increase the risk of predation to the
6
Annand
altruistic individual, while decreasing the risk of predation to other group mates.
California ground squirrels have been observed to perform cheek touches, in which one
squirrel puts its nose to the mouth area of another squirrel (Smith et al., in review). This
has been observed as a common greeting, and potentially allows individuals to scent
match and determine relatedness to the second individual. Although California ground
squirrels have been the focus of extensive study, their familial relationships remain
poorly understood, and we lack a fundamental understanding of how their social groups
are structured.
Our long-term goal is to explain patterns of potentially nepotistic behaviors by
studying relatedness among group-mates. This is the first study of its kind to be
conducted on California ground squirrels. Ultimately, we aim to discern the direct and/or
indirect fitness benefits seemingly altruistic behaviors have for California ground
squirrels. We will accomplish this by using fragment analysis to determine the exact
length in base pairs of each amplified microsatellite repeat at each loci. We can then
create a pedigree using the genetic tools detailed below to study the behavioral
implications of relatedness in an effort to explain cooperative behaviors in California
ground squirrels observed at Briones Regional Park in Contra Costa County, California,
U.S.A. Long-term social interactions data will be used in conjunction with the pedigree
we construct to examine how relatedness affects interactions between individuals. In our
current study, we identified polymerase chain reaction (PCR) primers that successfully
amplified specific segments of California ground squirrel DNA for the first time. The
genome of California ground squirrels has yet to be sequenced, and no papers have been
published detailing PCR primer sequences for California ground squirrels. Before we
7
Annand
can assign parentage, we must therefore optimize PCR primers so that we can eventually
compare fragment lengths at specific genetic loci between individuals.
PCR is a technique used to amplify short, specific sequences of DNA. Primers
anneal specifically to a singular location within the genome, directing amplification. We
can choose primers that target variable sequences of DNA outside the coding and
regulatory sequences, specifically microsatellites. For assignment of parentage, the
microsatellite loci we identify must be polymorphic within our study population. By
comparing unique alleles across potential parents at a specific locus, we can determine
which individuals are related to the offspring. In order to do this successfully, we need to
have a variety of different alleles throughout our population for each microsatellite loci.
We used the phylogeny constructed by Helgen et al. (2009) to determine relatedness of
California ground squirrels to candidate species.
Primers from hoary marmots (Marmota caligata) and alpine marmots (M.
marmota) were hypothesized to ultimately be most successful. This is because the genus
Marmota is more closely related to Otospermophilus than the other genera represented in
our candidate primers (Fig. 1). Because of this, we can infer the specific sequences that
the primers bind to have been conserved. Mutations within the genome can act as a sort
of “molecular clock.” Species that are more distantly related have a higher number of
differences within their genome. Of all the genera represented, less evolutionary time has
passed between the divergence of Marmota and Otospermophilus, suggesting similar
genomic sequences. We therefore hypothesized that the primers from Marmota species
will likely be the most successful at amplifying the DNA of California ground squirrels.
8
Annand
Methods
Study subjects
California ground squirrels live in distinct groups, called colonies, consisting of one or
more adult males and likely several matrilines of adult females and their immature
offspring (Smith et al. in review). We studied two colonies (Crow and Paradise) of free-
living California ground squirrels in Briones Regional Park, Contra Costa County,
California, USA from May 2013 to July 2015.
California ground squirrels are likely promiscuous and polygynandrous, meaning
that both males and females of the species mate with multiple partners (Smith et al. in
review). Female California ground squirrels are iteroparous, giving birth to one liter of 4-
11 pups a year, in many cases fathered by multiple males (Boellstorff et al. 1994,
Grinnell and Dixon 1919).
Behavioral data & live trapping
Behavioral data were collected during the daily observations of squirrels within our study
population. Data were collected on social interactions as well as cooperative behaviors
occurring in the context of predation. The latter include alarm-calling, tail-flagging, and
the throwing of dirt at Northern pacific rattlesnakes (Crotalus oreganus). Individuals
were trapped using Tomahawk traps (Tomahawk Live-Trap Company, Hazelhurst,
Wisconsin, USA) baited with black oil sunflower seeds and Skippy Extra Crunchy
Peanut Butter Super Chunk (Hormel Foods). Upon their initial trapping, individuals were
marked with unique identifying symbols using Nyanzol cattle dye (Greenville Colorants:
New Jersey), monel metal ear tags (National Band and Tag Co., Newport, Kentucky,
USA), and pit tags (Biomark, Inc., Boise Idaho) for permanent identification.
9
Annand
Collection of hair and tissue samples
We collected hair and tissue samples for genetic analysis from squirrels safely held in
handling cones (Koprowski 2002). Hair samples were collected from all squirrels
trapped during the summer months (May-July) from 2013 to 2015. One dozen hairs,
including the root, were taken from each animal, placed in envelopes, and stored in
Ziploc bags (S.C. Johnson and Son) on ice until they were transferred to the -20ºC
freezer. Tissue samples were collected in the summer of 2015 by clipping a 1-2 mm
sliver of skin tissue from the outer rim of the ear with sterilized surgical scissors. Tissue
samples were immediately submerged in 1.5 ml of lysis buffer solution (8.65 ml sterile
H2O, 250 µl 20% SDS, 500 µl 0.5 molar EDTA, and 100 µl 1.0 molar Tris, pH 8) and
kept temporarily on ice until they could be transferred to 22°C refrigerator for storage.
Collection of tissue samples caused only momentary discomfort and rarely resulted in
bleeding. This procedure is less invasive than taking a blood sample.
For this initial primer screening, we used DNA isolated from the livers of road
kill O. beecheyi (N=6) found by our team at Briones Regional Park. This was also done
to conserve the small amounts of DNA we have collected from our study population
using minimally invasive sampling methods. DNA was isolated from the liver tissue
samples using Qiagen DNeasy Blood & Tissue Kits (QIAGEN). We will use DNeasy
blood & tissue kits to isolate DNA from the roots of hair and tissue samples from our
study population to ultimately establish pedigrees for both of our study populations.
PCR screening procedure
10
Annand
In order to amplify the isolated DNA, we used polymerase chain reaction (PCR). PCR is
a technique used to amplify small, specific, repeated segments of DNA known as
microsatellites. The number of microsatellite repeats is variable from individual to
individual. When electrophoresed on a gel, the PCR product allows for the visualization
of the approximate length of each microsatellite. These sequences are heritable, meaning
that by determining the number of microsatellite repeats, indicated by total band size on
the gel, and comparing the individual in question with its known mother and potential
fathers, we can determine parentage in our study population. Primers are highly specific
sequences, approximately 20 base pairs in length, of single stranded DNA. Primers bind
to DNA via complementary base pairing. Typically a primer matches to only one
location within the entire genome.
Our goal is to identify polymorphic neutral markers. We are looking for neutral
markers (noncoding sequences) so that number of microsatellite repeats an individual has
are independent of selective pressures. The presence of polymorphic loci will help us to
more accurately assign parentage. By comparing allele length, which corresponds to
microsatellite repeat number, in an individual and their potential parents, we can
determine which squirrels the individual is related to. Unique repeat numbers within the
population will be crucial for identifying parents. However, if the majority of individuals
within our population have identical microsatellite repeat alleles at the loci we are
comparing, we will not be able to rule out individuals as possible parents. Because of
this, polymorphisms are key to accurately assigning parentage.
The genome of the California ground squirrel has yet to be sequenced, and no
PCR primers for our study species have been previously published. Thus, we gathered
11
Annand
primers known to amplify microsatellite loci for other closely related Sciurid species.
PCR primers have been published for 6 species from tribe Marmotini (Table 1). From
these, we identified 24 unique primers for screening. Of these, one primer was identified
from a study of Idaho ground squirrels (Urocitellus brunneus brunneus; May et al. 1997),
eight were from a study of Belding’s ground squirrels (Urocitellus beldingi; Nunes et al.
2014), seven from a study of Columbian ground squirrels (Spermophilus columbians;
Stevens et al. 1997), one from a study of European ground squirrels (Spermophilus
citellus; Hanslik & Krukenhauser 2000), one from a study of hoary marmots (Marmota
caligata; Kyle et al. 2004), and twelve from a study of alpine marmots (Marmota
marmota; Da Silva et al. 2003, Goossens et al. 1998, Hanslik & Krukenhauser 2000).
We have screened each primer a minimum of five times. For our purposes, we defined
primers with a percent success greater than or equal to 80% as optimized
PCR reactions were set up using wither OneTaq polymerase or GoGreen taq
polymerase. In the first case, we set up 50 µl PCRs using reagents from New England
Bio Lab. We set up reaction mixtures in 0.2 ml PCR tubes (Axygen Scientific)
containing 10 µl OneTaq Standard Reaction 5X buffer, 10 µl 1.25 mM dNTP, 0.4 µl
5000 U/ml OneTaq DNA polymerase, 5 µl 2 pmol/µl each forward and reverse primer.
The volume of template DNA varied, corresponding to approximately 100 µg DNA,
depending on the concentration of the sample. The remainder of the volume is nuclease
free water. When using GoGreen Taq polymerase (Promega), we set up 50 µl PCRs
containing 25 µl GoGreen Taq polymerase, 10 µl 2 pmol/µl each forward primer and
reverse primer, a volume of template DNA corresponding to approximately 100 µg DNA,
and nuclease free water makes up the additional volume.
12
Annand
We used a MJ Mini (BioRad) thermocycler for all PCR reactions. The protocol is
as follows: four minute 95°C initial denaturation step, followed by 30 repeated cycles of
one minute 95°C denaturation step, two minute 52-57°C annealing step, and two minute
72°C extension step, concluding with a ten minute 72°C final extension step. The
samples were kept indefinitely at 4°C until they are moved to the 4ºC freezer. Published
annealing temperatures varied between primers, as detailed in Table 2, and were
optimized for each candidate primer to maximize amplification of DNA, and therefore
the yield of PCR product for California ground squirrels.
To elucidate the optimal temperature for each primer, we carried out three PCR
reactions for each primer: one with an annealing temperature of 52°C, one with an
annealing temperature of 55°C, and one with an annealing temperature of 57°C. Optimal
annealing temperatures for each primer were determined by comparing band brightness
between the three trials. Band brightness correlates to the quantity of DNA within the
band. Thus, we can assume that the brightest band on the electrophoresed gel indicates
the most successful and abundant amplification. Brightness ranking protocol is discussed
in greater detail below.
Gel electrophoresis protocol
We ran our PCR products on either 2% or 4% agarose gel at 125 V for 30-60 minutes.
Gels were used to quantify the PCR reaction’s success. We used 2% agarose gels during
the initial phases of our screening process to determine whether our target primers would
amplify DNA. The 4% agarose gels were used in the later stages of screening, after we
had determined which primers were optimal, to analyze the base pair length of the bands
generated. The higher concentration gels are denser, which makes the amplified DNA
13
Annand
travel slower through the agarose matrix. This makes the banding pattern on the gel very
crisp and easy to visualize. When running both gels we used 100 bp DNA #N3231L
ladder (New England BioLabs).
Criteria for success
We considered a trial to be successful if band(s) were present and visible on the
electrophoresed gel after running the sample (Fig. 2). This result indicated that
amplification of the DNA was successful. Unsuccessful amplifications were defined as
trials were no bands were visible on the gel, indicating that the primer was unable to
anneal and amplification of the desired sequence were unsuccessful. The total number of
trials was defined as any trial where a sample was run on a gel, whether successful or
unsuccessful. Inconclusive or ambiguous trials were not included in our screening
process, such as those where the PCR reaction was potentially compromised, trials where
the gel was cast incorrectly, trials where the product potentially ran off the gel during
electrophoresis, or trials where the ladder was not visualized by the researchers.
Quantification of band brightness
We photographed the gels to determine the brightness of bands (Fig. 4). We ranked each
band present on the gel on a scale of 1 (faint) to 3 (bright). Brightness was determined in
comparison with the ladder, with 3 being equal to the brightness of the ladder (as seen for
the gel on the right in Fig. 2). This was done in preparation for future fragment analysis.
Band brightness was dependent on the type of PCR mixture used, with GoGreen Taq
polymerase yielding the brightest bands out of the two used. Annealing temperature also
affected brightness, as brightness is directly correlated with quantity of DNA.
14
Annand
Ethical note
All procedures are approved by the Mills College Institutional Animal Care and Use
Committee. We possess the necessary permits from the State of California Department
of Fish & Wildlife, as well as East Bay Regional Parks.
Results
Behavioral data
From 2013 to 2015, we have 1,462 captures of individual squirrels (N= 163 females, N=
149 males). From these squirrels, we observed thousands of social interactions.
Primer Screening
The number of trials ranged from 5 (N= 1 primer) to 40 (N= 1 primer), with a total of 327
trials combined for the entire set of 24 primers.
Overall, at least one primer from every species screened was successfully
optimized to amplify California ground squirrel DNA. Thus far, we have optimized 11
primers (Fig. 3). These 11 primers were ranked from highest to lowest percent success:
2g2, GS22, ST10, B108, D4, IGS-6, A116, MA018, GS20, SS-Bibl18, and D106 (Fig. 3).
Of the 11 optimized primers, two are from Alpine marmots, one from Hoary marmots,
one from the European ground squirrel, two from Colombian ground squirrels, four from
Belding’s ground squirrel, and one is from the Idaho ground squirrel. Nine primers had a
percent success of between 70-50%: GS26, GS14, B12, GS12, GS17, MA091, SS-Bibl4,
GS25, and SS-Bibl31 (Fig. 3). Four primers were deemed unsuitable for use of
California ground squirrels, with percent success of less than 50%; only one of these
seven primers, MS47, had a 0% success rate in our trials (Fig. 3).
15
Annand
Potential Polymorphic Loci
We observed polymorphisms for 13 primers: GS22, B108, D4, IGS-6, A116, MA018,
GS20, SS-Bibl18, D106, GS26, B12, GS12, GS25, and SS-Bibl31. We were able to
collaborate with the 2015 Genetics lab to collect polymorphism data for the eleven
optimized primers. Data collection for polymorphisms is still in the early stages; more
screening will have to be done in order to determine whether these loci are truly
polymorphic in our two populations of California ground squirrels. We also have yet to
thoroughly screen primers with percent success of between 50-70% for polymorphisms.
Discussion
In total, we have optimized 11 primers: 2g2, GS22, ST10, B108, D4, IGS-6, A116,
MA018, GS20, SS-Bibl18, and D106. Of the 11 optimized primers three, 2g2, GS22,
and ST10, had 100% success. 2g2 was taken from a study on Hoary marmots (Kyle et al.
2004), whereas GS22 was originally identified in a study of Columbian ground squirrels,
and ST10 was taken from a study of European ground squirrels. Because of the
consistent success of amplification, we can assume that the 20-22 bp sequences that these
primers anneal to have been conserved, and are identical or near identical in California
ground squirrels (Table 2).
Amplification success
The Marmota primers (Da Silva et al. 2003, Goossens et al. 1998, Hanslik &
Krukenhauser 2000, Kyle et al. 2004) were less successful as we anticipated. Three
Marmota primers were optimized in our initial trials, three are currently in the process of
16
Annand
being optimized, and one was deemed to be unsuitable for our purposes (Fig. 2). Because
the Marmota genus is so closely related to California ground squirrels we expected to see
a much higher number of the Marmota primers represented in our optimized category
(Fig. 1).
Unlike the Marmota primers, Belding’s ground squirrel primers were surprisingly
successful. Of the species represented in our candidate primers, Belding’s ground
squirrel is the most distantly related to California ground squirrels. Four primers
originally from Belding’s ground squirrel were optimized, one is in the process of being
optimized, and three were unsuitable for use on California ground squirrels.
Unsuccessful attempts at amplification may be because the primer sequence is
non-homologous to any loci in the California ground squirrel genome, as seems to be the
case for MS47. For primers with more variable success rates, such as the six with percent
success between 50-70%, amplification could have been unsuccessful due to incorrect
annealing temperature. Unsuccessful attempts at amplification could have also been due
to incorrect reagent concentrations or other errors within the PCR reaction.
Polymorphic loci
During our initial screening, we have also noted polymorphisms within the individuals
used for 13 primers: GS22, B108, D4, IGS-6, A116, MA018, GS20, SS-Bibl18, D106,
GS26, B12, GS12, GS25, and SS-Bibl31. Nine of these, GS22, B108, D4, IGS-6, A116,
MA018, GS20, SS-Bibl18, and D106 are optimized. We are hoping to optimize GS26,
B12, and GS12 in the near future. The last two polymorphic primers, GS25 and SS-
Bibl31, have percent success of <50%, and thus were deemed unsuitable for use on
California ground squirrel genome. Despite this low percent success, we are attempting
17
Annand
to find optimal annealing temperatures for both primers due to their promise as
polymorphic loci.
Band brightness
Band brightness was found to be variable both across the 11 optimized primers, and
within a primer. Determining band brightness will be useful for assigning fluorescent
dye tags to our PCR primers in order to carry out fragment analysis. The dyes we will be
using are 6-FAM (blue), VIC (green), NED (yellow), PET (orange), and LIZ (red) from
the Applied BioSystems DS-33 dye kit. Some of the dyes will fluoresce brighter than
others. We will assign dyes that fluoresce brightly to primers that produce faint bands (1-
2 on our ranking system), and primers that produce bright bands will be assigned the
dimly fluorescent dyes. This will maximize fragment visibilities when performing
fragment analysis.
Future research directions
We are currently attempting to optimize nine of the originally screened primers: GS26,
GS14, B12, GS12, GS17, MA091, SS-Bibl4, GS25, and SS-Bibl31. These primers had
percent success rates of between 50-70%. In order to optimize these primers, we are
testing different annealing temperatures. Our goal is to optimize at least four of these
primers, so that we have at least 15 primers to use for pedigree construction. We also
intend to screen our optimized primers for polymorphic loci more thoroughly.
The next step is to send samples of fluorescently labeled PCR product from our
study population to the University of California, Berkeley sequencing lab for fragment
analysis. Fragment analysis will determine the exact base pair length of specific
microsatellite repeats in individual squirrels, which will give us a more precise length in
18
Annand
base pairs than gel electrophoresis. Using these data we can determine parentage in the
same way as if we were using gel electrophoresis. By comparing the number of
microsatellite repeats in each of its two alleles to the number of repeats at a specific locus
in the potential parents, we can assign parentage. From our pedigree we also hope to
elucidate the mating system in California ground squirrels. While previous studies have
suggested that California ground squirrels are likely promiscuous and polygynyandrous,
genetic data is needed to confirm this (Smith et al., in review).
Heterozygosity and polymorphic loci
Heterozygosity, having many alleles for a specific gene within the population, will give
rise to polymorphisms. Many factors can influence the heterozygosity of a population,
including inbreeding, which we have hypothesized is prevalent within our study animals,
as is the case for the closely related yellow-bellied marmots, M. flaviventris (Olson et al.
2012). Polymorphisms are important indicators of genetic diversity. The amount of
genetic diversity within a population has implications for fitness (reviewed by
Kempenaers 2007). It is possible that, due to inbreeding, we will see a low level of
genetic diversity within our actual study population, and thus few polymorphic loci.
Many factors can affect heterozygosity within a population. Selective pressures, mating
preferences, and stressful environmental conditions could all contribute to low levels of
heterozygosity (Da Silva et al. 2006, Goossens et al. 2001). The longstanding drought in
California is a probably environmental stressor on our population of California ground
squirrel, which may increase inbreeding, decreasing heterozygosity. The numbers of kin
groups within a population can also impact heterozygosity. (Pilot et al. 2010). A greater
number of kin groups within the population leads to a greater level of heterozygosity.
19
Annand
Though we will be unable to determine kin groups in our population until a pedigree has
been constructed, this is still a possible source of inbreeding depression within our
population.
Heterozygosity has a known impact on survival in other Marmotini species. In
Alpine marmots, heterozygosity correlates with juvenile survival (Cohas et al. 2009, Da
Silva et al. 2006). Individuals with higher levels of heterozygosity, thus greater genetic
diversity, have increased fitness. Two major hypotheses have been proposed to explain
the heterozygote advantage. The local effect hypothesis suggests that heterozygosity at
neutral markers is indicative of heterozygosity at linked fitness loci (reviewed by
Hansson and Westerberg 2003). The general effect hypothesis proposes that
heterozygosity at neutral markers are the result of genome-wide heterozygosity (reviewed
by Hansson and Westerberg 2003). This hypothesis is somewhat dependent on partial
inbreeding, and so might be applicable to our study population of California ground
squirrels. Evidence in populations of Alpine marmots supports the local effect
hypothesis (Da Silva et al. 2006). Because the California ground squirrel is closely
related to Alpine marmots, it is possible that we will find support for the local effect
hypothesis in California ground squirrels as well. However, without microsatellite data
from our study population, we cannot make a conclusion about heterozygosity within our
population.
Evolution of cooperation
We have yet to elucidate the mechanism underlying cooperative and seemingly altruistic
behaviors in California ground squirrels. It is possible that cooperation can evolve
independently of kin selection in California ground squirrels, and that seemingly altruistic
20
Annand
behaviors are in fact selfish. There are five generally accepted mechanisms for the
evolution of cooperative behaviors: kin selection, which has been the focus of this paper;
direct reciprocity; indirect reciprocity; network reciprocity; and group selection (Nowak
2006).
Research suggests that Columbian ground squirrels, a closely related Marmotini
species, do not favor kin when alarm calling (Fairbanks and Dobson 2010). Cooperation
between individuals, both kin and non-kin, has been observed in Columbian ground
squirrels. This may be also be true for California ground squirrels, and will be something
we explore after the creation of pedigrees for our two study colonies. Cooperation
without nepotism may only confer direct fitness benefits, although there is the possibility
that seemingly altruistic behaviors such as alarm calling and standing guard may have
direct fitness benefits in California ground squirrels, as seen in meerkats (Clutton-Brock
et al. 1999).
The population size of our study colonies may also have effects on the evolution
of cooperation and altruistic behaviors. There is a trade off between having a closely
related kin group, which would be more conducive for the evolution of cooperative
behaviors, and group size, as seen in the yellow bellied marmot (Armitage & Schwartz
2000). Large groups are vulnerable to freeloaders and are associated with lower
reproductive rates (Armitage & Schwartz 2000). At this time, we have not elucidated
how group size in our population may affect cooperation and seemingly altruistic
behaviors in California ground squirrels.
We have hypothesized that kin selection is the driving force behind the evolution
of cooperative behaviors in California ground squirrels due to the highly social nature of
21
Annand
the animals and the potentially high degree of relatedness within a colony. Though kin
selection is strongly supported as the mechanism behind the evolution of altruistic
behaviors in social insects (Bourke 2005, Foster 2004, Queller & Strassman 1998,
Ratnieks 2006), there is a lack of publications exploring the evolution of altruism and
cooperative behaviors in social mammals on the level of individuals (Foster et al. 2006,
Hamilton 1964). We hope that our future conclusions will expand the understanding of
kin selection as it pertains to the evolution of cooperative and seemingly altruistic
behaviors.
Conclusions
The mechanism behind the evolution of cooperative and seemingly altruistic traits in
California ground squirrels has yet to be determined. It is possible, though we believe
unlikely, that these seemingly altruistic behaviors, such as tail flagging and throwing
substrate, have direct fitness benefits for the actor, and therefore are not truly altruistic.
Future research will also address what factors influence heterozygosity in California
ground squirrels, and how heterozygosity at microsatellite loci affects fitness. After
microsatellite data has been collected for our study population, we will be better equipped
to examine which heterozygote advantage hypothesis best aligns with our study
population.
Our current and future research will help to elucidate the evolutionary forces
favoring cooperative and seemingly altruistic behaviors in social free-living mammals.
There is still much to discover about the selective pressures shaping the evolution of
cooperative and seemingly altruistic behaviors. We hope that by elucidating the
22
Annand
mechanism underlying altruistic and cooperative behaviors in California ground squirrels,
we can add to the wealth of knowledge supporting kin selection theory.
23
Annand
Acknowledgements
A gigantic thank you to Dr. Jenn Smith, my thesis advisor and primary investigator on
this project, for all of her edits, suggestions, guidance, and knowledge. Thank you to
Heather Pearl, for carrying out the temperature screenings for the primers, as well as her
invaluable help troubleshooting. I would like to thank Valeska Muñoz, Kate Haughton
and Jenny Cumbie for their work on the genetics aspect of this project, as well as the rest
of Team Squirrel for collecting data in the field. Thank you to Dr. Robin Ball, Dr. Elaine
Tan and the genetics classes from Fall 2014 and Fall 2015 for their assistance. An
additional thank you to Dr. Robin Ball for PCR and primer consultation. Thank you Dr.
Jared Young and the Nematode Ninjas, the Swope lab, and the Barrett Fellowship and
Mills College for funding the progress of this research. I would also like to thank the
rangers at Briones Regional Park, and the California Department of Fish and Wildlife for
allowing us to do our research.
Literature citedArmitage, K. B., & Schwartz, O. A. (2000). Social enhancement of fitness in yellow-
bellied marmots. Proceedings of the National Academy of Sciences, 97(22), 12149-12152.
Boellstorff, D.E., Owings, D.H., Penedo, M.C.T., & Hersek, M.J. Reproductive behavior and multiple paternity of California ground squirrels. Animal Behaviour, 47, 1057-1064.
Bourke, A.F.G. (2005) Genetics, relatedness and social behaviour in insect societies. In Insect Evolutionary Ecology (Fellowes, M. et al., eds), pp. 1–30, CABI Publishing.
Clutton-Brock, T. H., O'Riain, M. J., Brotherton, P. N. M., Gaynor, D., Kansky, R., Griffin, A. S., & Manser, M. (1999). Selfish sentinels in cooperative mammals. Science, 284(5420), 1640-1644.
Cohas, A., Bonenfant, C., Kempenaers, B., & Allaine, D. (2009). Age‐specific effect of heterozygosity on survival in alpine marmots, Marmota marmota. Molecular Ecology, 18(7), 1491-1503.
24
Annand
Da Silva, A., Luikart, G., Allaine, D., Gautier, P., Taberlet, P., & Pompanon, F. (2003). Isolation and characterization of microsatellites in European alpine marmots (Marmota marmota). Molecular Ecology Notes, 3(2), 189-190.
Da Silva, A., Luikart, G., Yoccoz, N. G., Cohas, A., & Allaine, D. (2006). Genetic diversity-fitness correlation revealed by microsatellite analyses in European alpine marmots (Marmota marmota). Conservation Genetics, 7(3), 371-382.
Fairbanks, B., & Dobson, F. (2010). Kinship does not affect vigilance in Columbian ground squirrels (Urocitellus columbianus). Canadian Journal of Zoology, 88(3), 266–270.
Foster, K.R. (2004) Diminishing returns in social evolution: the not-so-tragic commons. Journal of Evolutionary Biology, 17, 1058–1072.
Foster, K. R., Wenseleers, T., & Ratnieks, F. L. (2006). Kin selection is the key to altruism. Trends in Ecology & Evolution, 21(2), 57-60.
Goossens, B., Graziani, L., Waits, L. P., Farand, E., Magnolon, S., Coulon, J., Bel, M., Taberlet, P., & Allainé, D. (1998). Extra-pair paternity in the monogamous Alpine marmot revealed by nuclear DNA microsatellite analysis. Behavioral Ecology and Sociobiology, 43(4-5), 281-288.
Goossens, B., Chikhi, L., Taberlet, P., Waits, L. P., & Allaine, D. (2001). Microsatellite analysis of genetic variation among and within Alpine marmot populations in the French Alps. Molecular Ecology, 10(1), 41-52.
Grinnell, J., & Dixon J. 1919. Natural History of the Ground Squirrels of California. Sacramento, California: California State Printing Office.
Hamilton, W. D. 1964. The genetical evolution of social behaviour, I and II. Journal of Theoretical Biology, 7, 1-52.
Hanslik, S. & L. Kruckenhauser. 2000. Microsatellite loci for two European sciurid species (Marmota marmota, Spermophilus citellus). Molecular Ecology, 9, 2163-2165.
Hansson, B., & Westerberg, L. (2002). On the correlation between heterozygosity and fitness in natural populations. Molecular Ecology, 11(12), 2467-2474.
Hare, J. F., & Murie, J. O. (1996). Ground squirrel sociality and the quest for the ‘holy grail’: does kinship influence behavioral discrimination by juvenile Columbian ground squirrels?. Behavioral Ecology, 7(1), 76-81.
Helgen, K. M., Cole, F. R., Helgen, L. E., & Wilson, D. E. (2009). Generic revision in the Holarctic ground squirrel genus Spermophilus. Journal of Mammalogy, 90(2), 270-305.
Kaplan, J. R. (1978). Fight interference and altruism in rhesus monkeys. American Journal of Physical Anthropology, 49(2), 241-249.
Kempenaers, B. (2007). Mate choice and genetic quality: a review of the heterozygosity theory. Advances in the Study of Behavior, 37, 189–278.
Koprowski, J.L. (2002). Handling tree squirrels with a safe and efficient restraint. Wildlife Society Bulletin, 101-103.
Kyle, C. J., Karels, T. J., Clark, B., Strobeck, C., Hik, D. S., & Davis, C. S. (2004). Isolation and characterization of microsatellite markers in hoary marmots (Marmota caligata). Molecular Ecology Notes, 4(4), 749-751.
Mateo, JM. (2003). Kin recognition in ground squirrels and other rodents. Journal of Mammalogy, 84(4), 1163-1181.
25
Annand
May, B., Gavin T.A, Sherman, P.W, & Korves, T.M. (1997). Characterization of microsatellite loci in the Northern Idaho ground squirrel Spermophilus brunneus brunneus. Molecular Ecology, 6, 399-400.
McDonald, D. B., & Potts, W. K.. (1994). Cooperative display and relatedness among males in a lek-mating bird. Science, 266(5187), 1030–1032.
Nowak, M. A. (2006). Five rules for the evolution of cooperation. Science, 314(5805), 1560–1563.
Nunes, S., Weidenbach, J. N., Lafler, M. R., & Dever, J. A. (2014). Sibling relatedness and social play in juvenile ground squirrels. Behavioral Ecology and Sociobiology, 1-13.
Olson, L. E., Blumstein, D. T., Pollinger, J. R., & Wayne, R. K. (2012). No evidence of inbreeding avoidance despite demonstrated survival costs in a polygynous rodent. Molecular ecology, 21(3), 562-571.
Owings, D. H., Borchert, M., & Virginia, R. (1977). The behaviour of California ground squirrels. Animal Behaviour, 25, 221-230.
Pilot, M., Dabrowski, M.J., Jancewiz, E., Schtickzelle, N., & Gilwicz, J. (2010). Temporally stable genetic variability and dynamic kinship structure in a fluctuating population of the root vole Microtus oeconomus. Molecular Ecology 19(13), 2800-2812.
Putman, B. J., Coss, R. G., & Clark, R. W. (2015). The ontogeny of antipredator behavior: age differences in California ground squirrels (Otospermophilus beecheyi) at multiple stages of rattlesnake encounters. Behavioral Ecology and Sociobiology, 69(9), 1447-1457.
Queller, D.C. and Strassmann, J.E. (1998) Kin selection and social insects. Bioscience 48, 165–175.
Ratnieks, F.L.W. et al. (2006) Conflict resolution in insect societies. Annual Review Entomology, 51, 581–608.
Seeley, T. D., & Visscher, P. K.. (1988). Assessing the Benefits of Cooperation in Honeybee Foraging: Search Costs, Forage Quality, and Competitive Ability. Behavioral Ecology and Sociobiology, 22(4), 229–237.
Sherman, P. W. (1977). Nepotism and the evolution of alarm calls. Science, 197(4310), 1246-1253.
Smith, J.E., Long, D.J., Russell, I.D., Newcomb, K.L., & Munoz, V.D. In review. Otospermophilus beecheyi (Rodentini Sciuridae). Mammalian species.
Smith, J.E. (2014). Hamilton’s legacy: kinship, cooperation and social tolerance in mammalian groups. Animal Behaviour, 92, 291-304.
Stevens, S., Coffin, J., & Strobeck, C. (1997). Microsatellite loci in Columbian ground squirrels Spermophilus columbianus. Molecular Ecology Notes, 3(2), 189-190.
Swaisgood, R. R., Owings, D. H., & Rowe, M. P. (1999). Conflict and assessment in a predator–prey system: ground squirrels versus rattlesnakes.Animal Behaviour, 57(5), 1033-1044.
Wahaj, S. A., Van Horn, R. C., Van Horn, T. L., Dreyer, R., Hilgris, R., Schwarz, J., & Holekamp, K. E. (2004). Kin discrimination in the spotted hyena (Crocuta crocuta): nepotism among siblings. Behavioral Ecology and Sociobiology, 56(3), 237-247.
26
Annand
West, S., Griffin, A., & Gardiner, A. (2007). Social semantics: altruism, cooperation, mutualism, strong reciprocity and group selection. Journal of Evolutionary Biology, 20(2), 415–432.
27
Annand
Figures and tables
Figure 1. Marmotini phylogeny from Helgen et al. (2009), as determined by analyzing the sequence of the entire cytochrome-b gene. This phylogeny was used to determine relatedness amongst species with candidate primers and California ground squirrels.
28
Annand
Figure 2. Gel demonstrating criterion for success. The gel on the right is a successful trial for B12, as indicated by the presence of bands. The gel on the left is an unsuccessful trial of D107, as indicated by the lack of bands visible on the gel.
29
Annand
Figure 3. Percentage of successful amplifications per loci using Polymerase Chain Reaction (PCR) based on DNA isolated from California ground squirrels. Primers are arranged from consistently most successful on the left to consistently least successful on the right. Black bars indicate the optimized primers, with 80-100% success. Gray bars indicate primers with 50-70% success. White bars indicate primers with success rates of 0-50%. Numbers above each bar indicate the total number of trials.
30
Annand
Figure 4. Mean ± S.E. band brightness for each of the eleven optimized primers in comparison with the ladder. Error bars denote standard deviation.
31
Annand
Table 1. List of candidate primers selected from the literature published for other Marmotini species. Primers appearing in bold were optimized for California ground squirrels.
Locus Literature size (bp)
Annealing Temp (ºC) Original Species Reference
2g2 103–127 49-50 C Marmota caligata Kyle et al. 2004
GS22 182-192 54-58 C Spermophilus columbianus Stevens et al. 1997
ST10 127–135 52 C S. citellus Hanslik & Kruckenhauser 2000
B108 279-298 58.5 C Urocitellus beldingi Nunes et al. 2014D4 271-343 56 C U. beldingi Nunes et al. 2014
IGS-6 137 52 C U. brunneus brunneus May et al. 1997
A116 300-316 57 C U. beldingi Nunes et al. 2014MA018 296-298 55-65 C M. marmota Da Silva et al. 2003GS20 228-238 54-58 C S. columbianus Stevens et al. 1997
SS-Bibl18 132–136 55 C M. marmota Goossens et al. 1998D106 164-176 58.5 C U. beldingi Nunes et al. 2014GS26 113-117 54-58 C S. columbianus Stevens et al. 1997GS14 243-281 54-58 C S. columbianus Stevens et al. 1997B12 213-225 57 C U. beldingi Nunes et al. 2014
GS12 146-162 54-58 C S. columbianus Stevens et al. 1997GS17 157-169 54-58 C S. columbianus Stevens et al. 1997
MA091 163-182 50-60 C M. marmota Da Silva et al. 2003SS-Bibl4 182–186 55 C M. marmota Goossens et al. 1998
GS25 132-152 54-58 C S. columbianus Stevens et al. 1997SS-Bibl31 154–164 55 C M. marmota Goossens et al. 1998
D108 199-223 56 C U. beldingi Nunes et al. 2014B6 129-147 55 C U. beldingi Nunes et al. 2014
D107 169-207 56 C U. beldingi Nunes et al. 2014
MS47 177–193 50 C M. marmota Hanslik & Kruckenhauser 2000
32
Annand
Table 2. List of candidate primers screened for California ground squirrels. Primers were compiled from literature on species closely related to California ground squirrels (see Fig. 1). Optimized primers are bolded here. Our observed band size and optimized annealing temperatures are shown.
Locus Candidate Primer Sequence Observed size (bp)
Annealing Temp (ºC)
2g2 F 5': TGA ACT GGG TCT TGA GGT CTR 5': GTC TGC TCT GCT CTC CAT CA -- 55
GS22 F 5': TCC CAG AGA ACA ACA TCA ACAG R 5': TCC GCA CAG GTC TTG GAC TT -- 55
ST10 F 5': TTG TGA TCC TCC AGG GAG TT R 5': GTG ATT TCC AAA CCC CAT TC ~100 57
B108 F 5': GGA GCG TCA ATG GAG AGG R 5': GGC AGA AGG CAG AAC TGG 200-300 52-57
D4 F 5': AGC AAG ACC CTA AGC AAC R 5': AGC ACC CTG TTA CAA AGG
300, various other bands 55-57
IGS-6 F 5': GGG CAT TAA TTC CAG GAC TTR 5': GGG CTG GAA TTA AAG GTA TCA -- 57
A116 F 5': TCT GTC TCA CCT CCT GTG TCR 5': GCA AAC TCA CCT CTA AGA TGG 300-400 57
MA018 F 5': ATC CGT CCA ATA AAG AAA TTC R 5': GTT TCT TGT GGC TCA GTG GTC AGA TG <100, 300-400 57
GS20 F 5': TCC AGA GTT TTT CAG ACA CA R 5': GCC CAG CCA TCA CCC TCA CC -- 55
SS-Bibl18 F 5': ATG GTC ATG GAA GGG AAG R 5': GGC ATC TTC ACA GTT GAT CT -- 55
D106 F 5': GGA CCA GAG TGG TAC TTC TGTG R 5': AGC ACC CAG AGA CTG TGA CTTA none 55
GS26 F 5': CCC AGG GAC CAC ATA GGA GGT AR 5': AGG ACT GGG GTT GTA GGT GAG T -- 57
GS14 F 5': CAG GTG GGT CCA TAG TGT TAC R 5': TTG TGC CTC AGC ATC TCT TTC <100 55-57
B12 F 5': CCA GCC TAC TTT GTT GTT CC R 5': CAC CAG GAC AGC ACA CA TAC -- 57
GS12 F 5': CCA AGA GAG GCA GTC GTC CAG R 5': TCA GAG CAG AGC ACT TAC AGA -- 55
GS17 F 5': CAA TTC GTG GTG GTT ATA TC R 5': CTG TCA ACC TAT ATG AAC ACA ~100 52
MA091 F 5': CCT GTG TGA GTC CTG GAG TC R 5': AGC CAT TTA GGT TAC ATC TGC <100 57
SS-Bibl4 F 5': CCT AGG TTC AGT CTT CAA CACAR 5': TGG TGT TGC CAT TGT TCT -- 55
GS25 F 5': CCA GCA TGG GGG AGA GAG AG R 5': CTT GTC ATT TAT CCA TTC ATA G -- 52
SS-Bibl31 F 5': TTA CAC CTT CTC TGG CTCC R 5': TCT GAG CGG ATT GTC TTT AT 100-200, >1000 52
D108 F 5': CAA CTC TGA ATC CCT CAC AG R 5': TCC AAG CTG AAT CCT CTA CTAG -- N/A
B6 F 5': CA CCC TCC ACC TTT TAG AAG R 5': TCC AAT GAA CTT TTC CAT CTC 100-200 N/A
D107 F 5': CAA CTC TGA ATC CCT CAC AG R 5': TCC AAG CTG AAT CCT CTA CTAG -- N/A
MS47 F 5': CCT GAT GTA GTC AGT CAG R 5': TGT GGG AAA TGG CAC ATC N/A N/A
33
Annand 34