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Life history evolution and aging in African annual fishes across an environmental gradient

From an evolutionary perspective aging reduces individual fitness, so the question arises as to
why natural selection hasn’t eliminated it. The classic evolutionary explanations (Medawar 1952;
Williams 1957) posit that the ultimate cause of aging is the declining force of natural selection with
age. If an organism has a very low probability of surviving to a given age, due to factors such as
predation, then natural selection will be inefficient at maintaining somatic tissue beyond this age, and
intrinsic deterioration, or aging, will result. The mathematical refinements of this theory predict how
aging and other life history features are expected to evolve in response to age specific mortality rates
(Charlesworth 1980). I propose a research program that will allow for the testing of life history theory
and the evolutionary theory of aging in natural populations of annual fish found across an
environmental gradient. My hypothesis is that populations native to a very ephemeral environment
(e.g. a short duration aquatic environment) reach maturity earlier, exhibit higher reproductive effort,
are shorter lived, and exhibit more rapid aging than populations from localities with a more permanent
aquatic environment.
My study organism is Nothobranchius, a genus of fish found in sub-Saharan Africa. Due to the
annual drying of their aquatic habitat, these fish have evolved an annual life cycle; as pools dry out
and the fish die, the developing eggs remain buried in the mud, in a dormant state, until the following
rainy season. In south east Africa three different species of these annual killifish are sympatric
(Reichard 2009) and found across a habitat characterized by variation in the duration of the rainy
season and total annual rainfall (Terzibasi et al 2008). My proposed study involves a laboratory and
field component. A common garden laboratory experiment, performed on second generation,
laboratory reared fish, will be used to quantify life histories. Under standard feeding and care
protocols (Genade 2005) fish from each population will be raised from birth to death. Life history
variables including: 1) growth rate 2) age and size at first reproduction 3) egg output 4) egg size 5) and
mortality rate will be quantified for the females. A simultaneous field effort will characterize the
environmental conditions of each population locality. These data, used in conjunction with historical
rainfall records, will be used to understand the selective forces shaping the life histories of each
I would use this fellowship to pay for travel to Gonarezhou National Park and Malilangwe
Private Wildlife Reserve in Zimbabwe in April/May of 2010. These two adjacent parks are found in
the southeast corner of Zimbabwe, have a semi-arid climate, and are reported to contain extremely
short lived populations of Nothobranchius that were described in the 1970s (Jubb 1971) and have not
been thoroughly studied since. One goal of this trip would be to collect live fish from multiple
localities throughout the parks and bring them back to UC Riverside for the common garden life
history studies. Furthermore, I would characterize habitat variables including water depth, pH, and
temperature and demographic variables including species composition, density, and sex ratio. Dario
Valenzano is my project associate; he is a post-doctoral fellow at Stanford University and has
previously organized an expedition to Mozambique in 2004 to collect Nothobranchius. We have
developed African collaborators to help with logistics (Hugo and Elsabe van der Westhuizen) and have
received enthusiasm and formal permission from the chief ecologist at Gonarezhou National Park to
conduct this work.

Charlesworth, B. 1980. Evolution in age-structured populations. Cambridge: Cambridge University
Genade, T. (2005) Laboratory Manual for culturing N. furzeri.
Jubb R.A. (1971) A new Nothobranchius (Pisces, Cyprinodontidae) from Southeastern Rhodesia.
Journal of the American Killifish Association 8: 12-19.
Medawar, P. B. 1952. An unsolved problem of biology. London: Lewis.
Reichard, M., Polacik, M. & Sedlacek, O. 2009. Distribution, colour polymorphism and habitat use of
the African killifish Nothobranchius furzeri, the vertebrate with the shortest life span. Journal

of Fish Biology 74: 198-212.
Terzibasi, E., Valenzano, D. R., Benedetti, M., Roncaglia, P., Cattaneo, A., Domenici, L. & Cellerino,
A. 2008. Large differences in aging phenotype between strains of the short-lived annual fish
Nothobranchius furzeri. PLoS ONE 3.
Williams, G. C. 1957. Pleiotropy, natural selection, and the evolution of senescence. Evolution: 398-

Proposed Budget

Explorer’s Club Exploration Fund (total requested)…………………………………$1500
Expense Breakdown:
Roundtrip airfare from Los Angeles to Johannesburg…………………………………....$1400
Supplies including dip nets, seine nets, minnow traps……………………. ……………..$100

*Note: Other expenses associated with the trip are vehicle rental in Johannesburg, fuel, and additional
research supplies such as a handheld GPS. My research associate, Dario Valenzano, has funding for
some these items. Lodging will not be a significant expense as we have received permission to camp
within Gonarezhou National Park.

Other funding applied for:
Society of Comparative and Integrative Biology, Fellowship for Graduate student travel
National Science Foundation, Graduate Research Fellowship Program

Statement of career objectives

My ultimate career aspiration is to become an evolutionary biologist and professor at a research
university. As I travel this path I would like to make scientific contributions that further our
understanding of biological process and have relevance to others outside the academic community. I
am in my second year as a graduate student and in the process of developing my dissertation project.
My organism of study is a group of annual fishes native to the plains of East Africa. Many types of
evolutionary questions can be addressed on such short lived vertebrates and an Explorer’s club
fellowship would permit the development of a dissertation project that has fieldwork in a remote part
of the world as an integral component. My proposal presents a unique system for studying the
evolution of aging in natural populations. More importantly, this aging comes in the context of
vertebrates of sufficiently short lifespan to make it practical to study the entire life history in a
laboratory setting. Because of Nothobranchius furzeri’s emergence as a model organism for studying
the genetics of aging, genomics resources are becoming available. My proposed comparative studies
will enhance our gain from this model system by integrating field studies of the organism’s natural
history and selective environment with laboratory studies of aging.

David Reznick
VOICE: (951) 827-5903
FAX: (951) 827-4286
12 December 2009

Dear Colleagues:
I submit this letter in support of Andrew Furness’ application for an Explorer’s Fund travel
grant. I am a professor in the Biology Department at the University of California, Riverside. I first
made Andrew’s acquaintance via e-mail when he inquired about coming to UCR for graduate studies
and have had much more extensive interactions with him in the 18 months since he moved to
Riverside. I am his major professor, so we interact at weekly lab meetings and in the lab. He was a
student on my graduate level course in Evolution. We have also worked together for two weeks in the
field in Trinidad. I will argue that Andrew is among the top one or two percent of the graduate
students that I have worked with in terms of his academic abilities, but more so in terms of his
potential to do original research.
At the time that Andrew first inquired about pursuing graduate studies with me, I felt that I
had a full lab and had been discouraging all graduate school inquiries for the prior two years.
Something about Andrew’s inquiry was different, so I encouraged him to apply. I was so impressed
with him after his interview that I then actively pursued him in a way that I have not done before; I
had significant competition to contend with because others were as impressed with him as I was. The
aspects of his application and personality that stood out were the remarkable breadth and
sophistication of his prior experience with science and his high enthusiasm for studying evolution. He
had already done research in three very different contexts – monitoring of invasive species, functional
anatomy of frogs and the molecular biology of membrane bound protein complexes. I found that he
could talk knowledgably about the basic research associated with all of these projects and that he had
been successful in pursing independent research on all of them. In the case of his frog work, his
accomplishments after a three-month internship at the Smithsonian Institution were sufficient for him
to generate a publication that is now in review. I also found that he was unusually capable in
navigating the primary literature and had well developed skills in synthesizing information from basic
research articles, generating research ideas, and writing proposals. He knew of me because he had
been interested in the evolution of senescence and had found articles that I had written on the subject
while doing recreational reading in the library. The article in question was in Nature, which means
that it was highly compressed and not intended for a general audience, yet he clearly understood it
very well.
I was also very interested in recruiting him as a graduate student because of his clarity of
expression in both his writing and in our personal conversations and in the ease with which he
interacted with the others in my lab when he came for a visit. He was outgoing, highly inquisitive,
well informed, well rounded, and genuinely interested in all that he encountered during his visit. There
were other indications that he was interactive and would fit in very well with an interactive lab and a
research team. For example, he had mentored first year students at Marquette who were first
generation college attendees. He had also traveled to Guatemala with his father to provide health care
to remote communities.
Andrew has lived up to my highest expectations. He was the standout student in my advanced
evolution class. The students are required to read 50 or more pages of original literature for every
class, then participate in class discussions. We emphasize the historical development of the major
questions in evolutionary biology and the methods used to pursue them, so we begin with the Origin of
Species and Mendel. We then review representative works from the late 19th and early 20th centuries,
review the development of science through that period by reading Provine’s “Origins of Theoretical
Population Genetics”, then move on to representative works from the modern synthesis era and current
science. Last year we emphasized adaptive radiation, so we read Simpson’s “The Major Features of

Evolution” (1953) and Schluter’s “The Ecology of Adaptive Radiation” (2000), among other works.
The discussions are a good probe of the students’ background and readiness for doing advanced study
in evolutionary biology. Andrew was the standout student because of the quality of his background,
but more so because of his ability to grasp difficult concepts and willingness to go the extra distance to
understand what each reading was about. He was also adventurous in his choice of a term paper
project, since he used it as an opportunity to learn about a new subject, rather than stay close to an area
in which he had prior experience. He wrote about sex-ratio evolution and demonstrated again that he
has a sophisticated ability to navigate the primary literature and synthesize material that ranges from
basic theory to empirical studies. He has been a standout in all of his other graduate courses and has
received either and A or A+ in all of them.
Andrew has been equally independent in developing a research proposal and in laying the
groundwork for executing it. He volunteered to work in my fish lab so that he could begin to develop
the skills necessary to pursue his project. He has now spent five months in the field in Trinidad so that
he can master our methods for doing mark-recapture and life history studies on small fish, plus he has
executed an independent research project on the native killifish in Trinidad to hone his skills for work
in Africa. He has also demonstrated that he is physically tough and not deterred by the uncomfortable
conditions that may be encountered in a field setting. In fact, he seems to thoroughly enjoy spending
late nights collecting fish in mosquito-invested jungle streams and long days over a microscope
collecting data.
Andrew has made contact with Alessandro Cellerino, Martin Reichard, and Dario Valenzano (a
former graduate student of Cellerino’s and now a post-doc at Stanford) to inquire about the fish that he
hoped to work with; they are the reigning experts in the study of the target species for Andrew’s
proposed research. Andrew has developed a collaboration with Dr. Valenzano to do field work in
Africa this coming spring. Dr. Valenzano has prior experience working on the annual killis in Africa
and can cover Andrew’s initial research efforts under his permits. Andrew has also visited Dr.
Valenzano’s lab to learn about the lab culture of the fish, has obtained eggs from him and from
hobbyists, and has successfully reared them in the laboratory. Dr. Valenzano has visited our
laboratory to advise us on fish culture and to gain some training in the use of otoliths for aging fish, so
we have developed a working relationship that has reciprocal benefits.
I think that Andrew’s choice of a research topic holds unusual promise. The study system,
annual killifish that inhabit ephemeral pools in east Africa, is outstanding for laboratory research for
multiple reasons. One of them, Nothobranchus fuerzi, has the distinction of being the vertebrate
equivalent of rapid-cycling mustard plant Arabidopsis thaliana; it has a lifespan of 12-20 weeks, so it
is a good workhorse for lab studies. The other two species are congeners that are likely to have similar
life histories, but they are not as well studied. The eggs of all three species can remain viable in a
dormant state for well over a year, so it is possible to generate eggs for studies, then store them on a
shelf until you are ready to begin work. There is a large community of hobbyists who keep these fish,
so all of the equipment and know-how for rearing them is readily available; Andrew has had the
foresight to join the local chapter of the American Killifish Society and has already met many of the
local experts. Their research potential has been realized by others, mostly scientists interested in the
study of senescence, so I think they will soon emerge as a model species for laboratory research.
These fish also hold great potential for field work. Enough preliminary field work has been done to
identify a large number of breeding sites that span a rainfall gradient, so there is the necessary baseline
for pursuing comparative work on natural populations.
Andrew has shown excellent academic skills in developing his proposal. He recognized that
these fish were the animal equivalent of annual plants, so he mastered the theoretical and empirical
literature that deals with life history evolution and adaptive phenotypic plasticity in these plants. He
made good use of the theory to make testable predictions for his study system and has designed
appropriate experiments to establish an association between habitat type and life history in the three
study species. If successful, then his study will be only the third that integrates of theory for the
evolution of senescence with organisms that experience natural variation in life span and have proven

genetic variation in lifespan. The prior comparative studies that show such genetic correlations were
done on guppies and Daphnia. Similar progress has been made in the study of garter snakes, but they
are less amenable to laboratory genetics.
The discovery of natural variation in lifespan will make it possible for one to later study the
genetic basis of lifespan evolution and the extent to which it is correlated with other features of the life
history. The field of aging genetics is large and well-funded, but is almost exclusively devoted to the
study of genetic variation discovered in model organisms (mostly yeast, Caenorhabditis, Drosophila
and mice) that have been subjected to many generations of lab culture. The lab culture of virtually all
organisms inadvertently selects for rapid development in the face of high resource availability while at
the same time carrying a risk of inbreeding. The genetic variation that has been discovered may well
represent adaptation to the lab environment or mutations that restore fitness lost due to inbreeding
depression. Also, selection in the lab environment can accommodate the evolution of correlated traits
that may well be deleterious and selected against in a natural setting. Andrew has instead designed a
project with a new organism that may enable him to discover similar genetic variation as it has
evolved under the constraints of a natural setting. If the aging genes that have prevailed in laboratory
studies also play an important role in nature, then we can truly say that they have a general impact on
lifespan. If not, then we would have the ability to reassess the value of the research done to date on
model organisms while at the same time have new candidate genes for study .
With regard to broader impacts, there is no question that Andrew will successfully complete a
thesis and move on to a productive career. He is a good contributor to lab discussions, even though he
is junior among some very advanced and loquacious fellow graduate students. He works well with
undergraduate volunteers and attends our weekly undergraduate meeting so that he can help to mentor
students and incorporate them into his research program. While in Trinidad, he also lead a half day
class for high school students in which he introduced them to our project and took them into the field
to show them how we do our work. Given his prior success as a tutor and what I have seen of him so
far, I am confident that our students will find working with him during the lab-phase of his dissertation
research to be a valuable experience. Since our university has one of the most diverse student bodies
in the nation, with a very high percentage of students who are the first of their family to go to college,
he will also be working with under-represented minorities along the way. The fundamental
importance of his chosen research questions and the promise of his contributing to the development of
a new model study organism add to the potential broader impacts of his research.
To summarize, I consider Andrew to be the best all-round student that I have seen because of
his academic abilities, his ability to synthesize ideas from the primary literature, his ability to generate
interesting research questions, then convert them into competitive proposal, his prior success in three
very different research programs and current success in his field work in Trinidad, his outgoing
personality and excellent speaking skills, and his prior success as a tutor. I am confident that your
investment in his future will be very well repaid.


David Reznick, Professor of Biology