Ancestry and Chance shape the Selection, Specialization and Speciation of Chlamydomonas ...

116 Pages·2017·2.03 MB·English

by  Andrea L

Ancestry and Chance shape the Selection, Specialization and
Speciation of Chlamydomonas reinhardtii in a Long-term
Evolution Experiment
by
Andrea Lofano
Department of Biology

McGill University
Montréal, Québec, Canada

April, 2017

A thesis submitted to McGill University in partial fulfillment of the requirements
for the degree of Masters of Science.

© Copyright: Andrea Lofano, 2017

[i]
Dedication

To my family and my friends who I consider family and for Science

[ii]
Abstract
This thesis explores how populations evolve and adapt to large-scale environmental
shifts over thousands of generations. Populations of Chlamydomonas reinhardtii of
independent ancestry were exposed to novel environments that provided ecological and
reproductive challenges for these unicellular autotrophs. Reciprocal transplant experiments
were used intermittently throughout 19 years (roughly 7000 generations) of selection, to
investigate the progression of evolutionary adaptation, specialization, and radiation.
Chapter 1 compares the growth of C. reinhardtii strains selected in a heterotrophic
environment with the ancestral autotrophically propagated population. Multiple replicates
were set up in order to observe the interactions between selection, chance and ancestry and
how they influence the repeatability of evolution. Traits under direct selection, namely
heterotrophic growth capacity, are heavily influenced by selection environment and
demonstrate large-scale allele frequency shifts occurring in independent replicates. Ancestry
and chance, on the other hand, play a supporting role in terms of population dynamics. Traits
under indirect selection, such as autotrophic and mixotrophic proficiency, ancestry greatly
influences the evolutionary outcome and the likelihood of observing extreme phenotypes non-
viable in the ancestral environment. This experiment shows that not only have the ecological
niches of the replicate samples shifted in response to direct selection, but are also directed by
historical contingencies within the populations.
Chapter 2 further explores the concept of diversification and how prolonged selection
can lead to specialization that can ecologically and reproductively isolate a formerly
homogeneous population. The same founding populations of Chlamydomonas reinhardtii from
the previous section were grown in opposing environments: liquid (ZIF populations) and solid
(MOP populations) media, which provide vegetative and reproductive growth challenges. A
classic reciprocal transplant experiment was conducted on the replicate populations of differing
ancestry, where fitness and reproductive vitality was approximated through counting the
number of zygotes arising from crosses between and within populations in both environments.
Consistently higher vegetative growth and unique morphological adaptations were observed
[iii]
when populations were surveyed in their native environment. Offspring number is significantly
higher when mating occurs within a single population in their preferred environment, while
crosses between MOP and ZIF populations produce intermediate zygote numbers. The
environment in which the cross occurs directly influences the number of viable zygotes
resulting from the mating, especially when inoculated in solid media. These environmental
barriers to gene flow coupled with significant ecological divergence among MOP and ZIF
populations exacerbates this reduction in gene flow and reinforces selective mating upon
secondary contact.

[iv]
Résumé
Cette thèse examine comment les populations évoluent et s'adaptent aux changements
environnementaux à long terme. Des populations de Chlamydomonas reinhardtii d'ascendance
indépendante ont été exposées à de nouveaux environnements qui ont fourni des défis
écologiques et reproductifs pour ces autotrophes unicellulaires. Les expériences de
transplantation réciproque ont été utilisées de façon intermittente au cours des 19 années
(7000 générations) de sélection, afin d'étudier la progression de l'adaptation évolutive, la
spécialisation et la radiation adaptative.
Le premier chapitre compare la croissance de C. reinhardtii sélectionnées dans un
environnement hétérotrophe avec la population ancestrale propagée autotrophiquement. De
multiples répétitions ont été mises en place afin d'observer les interactions entre sélection,
hasard et ascendance ainsi que sur la manière dont ils influencent la répétabilité de l'évolution.
Les traits sous sélection directe, à savoir la capacité de croissance hétérotrophe, sont fortement
influencés par l'environnement de sélection et démontrent des changements de fréquence
allélique à grande échelle se produisant dans des échantillons indépendants. L'ascendance et le
hasard, cependant, jouent un rôle de soutien. En revanche, dans les cas de traits sous sélection
indirecte, tels que la compétence autotrophique et mixotrophique, le résultat évolutif est
grandement influencé par l'ascendance et la probabilité d'observer de nouveaux phénotypes
augmente. Cette expérience montre que non seulement les niches écologiques durant
l’expérience sont déplacées en réponse à la sélection directe, mais sont également dirigées par
des contingences historiques au sein des populations.
Le chapitre deux examine comment la diversification écologique et la façon dont la
sélection prolongée peut mener à une spécialisation pouvant isoler sur le plan écologique et
reproductif, une population autrefois homogène. L’expérience consiste en trois populations
homogènes fondatrices de Chlamydomonas reinhardtii qui ont été cultivées dans les conditions
liquides (populations ZIF) ou solides (populations MOP), qui fournissent des problèmes en
termes de croissance végétative et reproductive. Une expérience de transplantation réciproque
classique a été menée où l'aptitude et la vitalité reproductive ont été estimées en comptant le
[v]
nombre de zygotes présents après des croisements entre et au sein des populations dans les
deux environnements. Une croissance végétative constamment plus élevée et des adaptations
morphologiques uniques ont été observées lorsque les populations ont été examinées dans
leur environnement naturel. Le nombre de descendants est significativement plus élevé lorsque
l'accouplement se produit entre les populations dans leur environnement de prédilection,
tandis que les croisements des populations MOP et ZIF produisent des nombres de zygotes
intermédiaires. L'environnement influence directement le nombre de zygotes viables résultant
de l'accouplement, en particulier lorsqu'il est inoculé dans des milieux solides où la
reproduction sexuelle est difficile. Ces obstacles environnementaux à l'écoulement des gènes
couplés à une divergence écologique significative parmi les populations MOP et ZIF aggravent
l'isolement reproductif et renforcent l'accouplement sélectif au contact secondaire.

[vi]
Acknowledgements
I would first and foremost like to thank my supervisor, Graham Bell for his invaluable
guidance and fortitude and for sharing his wealth of scientific knowledge and experience with
me. Most importantly, he transmitted his love and dedication to learning that continues to
inspire me daily. Thank you for your enthusiasm and commitment to this endeavour. I would
also like to acknowledge the members of my supervisory committee; Melania Cristescu, Irene
Gregory-Eaves and Andrew Hendry for reviewing my work and presenting thoughtful topics of
exploration and further research.
Secondly, I would like to thank my collaborator, Gemma De Martino and my lab mates;
Luke Anderson-Trocmé and Pedram Samani for hours of fruitful discussion and advice on
experimental design, execution and interpretation of results. Also, to my R and statistical
analysis mentors, Étienne Low-décarie and Brain Leung, whose patience and teachings have
allowed me to become a better scientist. To my office mates, Monica Granados and Genelle
Harrison and fellow biology students, Colin McDonnell and Clarissa Fleck for their friendship
and humor. Finally, to my tireless editors, Lucie Huyghe and especially Tiffany Chin, who took
the time to review my thesis and calm my panicking self.
Lastly, Kathy Tallon has been a great source of physical and emotional strength
throughout this process. Without her, neither myself, nor our beloved Chlamydomonas lines
would surely not be here.
Thank you also to funding services; the Natural Science and Engineering Research
Council (NSERC) and McGill University, for providing financial support for research in science.

[vii]
Contribution of authors
I am the main author of all the chapters included in this thesis. All data formatting,
statistical analysis and the majority of the text have been completed by me. Because of the long
length of the evolution experiments, many individuals have contributed to its initiation and
maintenance, primarily Kathy Tallon, who has diligently transferred and collected data from all
samples for several years. Professor Graham Bell is a co-author on all chapters. He has
significantly contributed to all aspects of this project including the development of research
questions (Chapter 1, 2), experimental design (Chapter 1, 2) and initiation of the ecological
assays (Chapter 1). Professor Gemma De Martino is responsible for the mating assays and data
collection related to the sexual mating of MOP and ZIF lines (Chapter 2). All co-authors have
agreed to be included on the papers and chapters of this thesis.

[viii]
List of Tables
Chapter 1
1) Mean growth, standard error and significant differences of heterotrophically selected
lines grown in autotrophy, mixotrophic and their native heterotrophic environment,
separated by date of assay.
2) ANOVA of growth comparisons between the two ancestral populations (MOP and ZIF)
and the dark selected lines and superior generalists (16 day incubation).
Chapter 2
3) ANOVA comparing the vegetative growth of ZIF and MOP replicates grown in bolds and
bolds acetate media
4) ANOVA comparing the total zygote production of MOP and ZIF populations, in nitrogen-
rich or nitrogen-free environments
5) ANOVAs analysing the difference in zygote formation between MOP only mating, ZIF
only mating and MOP/ZIF crosses in a nitrogen-free environment
6) ANOVAs analysing the difference in zygote formation between MOP only mating, ZIF
only mating and MOP/ZIF crosses in a nitrogen-rich environment
7) ANOVA comparing the differences in zygote production between individual replicates in
a solid nitrogen-rich environment with an extended 10 day incubation.

[ix]
List of Figures
Chapter 1
1) Boxplot showing the variation in heterotrophic growth across all four ecological assays
conducted assaying phenotypes of these lines grown in the dark.
2) Boxplot of autotrophic growth of lines selected in darkness across all four assays.
3) Boxplot of the mixotrophic growth of dark selected lines across 4 ecological assays.
4) Regression of heterotrophic growth versus autotrophic growth for dark selection lines
over 4 separate assays
5) Growth profiles of dark adapted lines in autotrophic, mixotrophic and heterotrophic
conditions
6) Time course of vegetative growth in a homogenized liquid environment
7) Diagram of the changing phenotypes of the dark lines at different points in their
adaptation to the dark
Chapter 2
8) Vegetative growth of sexually selected population inoculated into liquid
9) Colonies grown vegetatively on solid agar plates
10) Regression of the number of colonies present on plates (x axis) and the size of these
colonies (y axis). A linear regression output table is also included.
11) The total zygote production seen from mating occurring within and between
populations grown in Nitrogen-free solid media (2 day incubation)
12) The total zygote production seen from mating occurring within and between
populations grown in Nitrogen-free liquid media (2 day incubation)
13) The total zygote production seen from mating occurring within and between
populations grown in Nitrogen-rich liquid media (2 day incubation)
14) The total zygote production seen from mating occurring within and between
populations grown in Nitrogen-rich solid media (4 day incubation)
15) The total zygote production seen from mating occurring within and between
populations grown in Nitrogen-rich solid media (12 day incubation)
[x]