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Creating a pedigree for California ground squirrels (Otospermophilus

beecheyi) to study kin-structured social networks

Chloe Annand

Fall 2015 BIO 191

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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.

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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

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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.

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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

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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

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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

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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.  

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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.   

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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

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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

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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.

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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

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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.

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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).  

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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

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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

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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

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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.

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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

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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

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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

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mechanism underlying altruistic and cooperative behaviors in California ground squirrels,

we can add to the wealth of knowledge supporting kin selection theory.

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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.

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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.

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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.

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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.

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Figure 4. Mean ± S.E. band brightness for each of the eleven optimized primers in comparison with the ladder. Error bars denote standard deviation.

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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

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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

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