University of Iowa
Iowa Research Online
Theses and Dissertations
Spring 2015
Ecological and biogeographical patterns associated
with genetic differentiation in a diverse genus of
Neotropical fruit flies
Kristina Jane Ottens
University of Iowa
Copyright 2015 Kristina Ottens
This thesis is available at Iowa Research Online: http://ir.uiowa.edu/etd/1716
Recommended Citation
Ottens, Kristina Jane. "Ecological and biogeographical patterns associated with genetic differentiation in a diverse genus of Neotropical
fruit flies." MS (Master of Science) thesis, University of Iowa, 2015.
http://ir.uiowa.edu/etd/1716.
Follow this and additional works at: http://ir.uiowa.edu/etd
Part of the Biology Commons
ECOLOGICAL AND BIOGEOGRAPHICAL PATTERNS ASSOCIATED WITH GENETIC DIFFERENTIATION IN A
DIVERSE GENUS OF NEOTROPICAL FRUIT FLIES
by
Kristina Jane Ottens
A thesis submitted in partial fulfillment
of the requirements for the Master of Science
degree in Biology in the Graduate College of
The University of Iowa
May 2015
Thesis Supervisor: Assistant Professor Andrew Forbes
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
____________________________
MASTER’S THESIS
_________________
This is to certify that the Master’s thesis of
Kristina Jane Ottens
has been approved by the Examining Committee for
the thesis requirement for the Master of Science degree
in Biology at the May 2015 graduation.
Thesis Committee:
____________________________________________
Andrew Forbes, Thesis Supervisor
____________________________________________
Josep Comeron
____________________________________________
Maurine Neiman
ACKNOWLEDGMENTS
I would like to thank my advisor, Dr. Andrew Forbes, for providing time and support throughout
my project as well as pushing me to think about the implications of my results. I would also like to thank
my committee members, Dr. Josep Comeron and Dr. Maurine Neiman, for providing insightful questions
and comments that led to a well-rounded thesis. I also thank my lab mates, Gaby Hamerlinck, Alaine
Hippee, Amanda Nelson, and Eric Tvedte, as well as the other graduate students within the Biology
department for stimulating conversations that prompted me to think about my project from many
different angles. Finally, I would like to thank Marty Condon and Matthew Lewis for providing me with
samples and the opportunity to assist with field collections along with thought-provoking discussions
about my work.
ii
ABSTRACT
Understanding the processes that generate biodiversity is a major goal of evolutionary biology.
The ultimate cause of biodiversity is the evolution of barriers to gene flow between populations of
organisms, but the proximate mechanisms are often more complex. I am interested in disentangling the
roles of geographic isolation and ecological selection in the diversification of a species-rich genus of
tropical tephritid fruit flies. Blepharoneura are highly specialized and host specific flies; most species
specialize on a single plant host and flower sex although multiple species may exploit the same resource.
At one location in Peru, two plant species (two sexes – four plant niches) are host to 14 Blepharoneura
species. Phylogenetic analyses of mitochondrial DNA sequences reveal that some species may be
diverging as a result of shifts to new host plants (suggesting possible ecological selection acting in
speciation), while other species show an apparent pattern of geographic divergence in addition to or
without host shifts. To further investigate these ecological and geographic signals underlying the history
of Blepharoneura speciation, more rapidly evolving molecular markers are required. Here, I use
microsatellites to address this question for seven Blepharoneura species (sp1, sp4, sp8, sp10, sp21, sp28,
and sp30) characterized by differing patterns of host-plant use and geographic distribution.
Microsatellite data indicates patterns of ecological divergence associated with host use in at least five
species (sp1, sp4, sp10, sp21, sp30) and patterns of geographic divergence in all seven species.
iii
PUBLIC ABSTRACT
Insects constitute >50% of the world’s species, many of which are either agriculturally or
pharmaceutically important. Understanding why this group is so diverse and what generates that
biodiversity could provide new insights into preserving economically important ecosystems. The
ultimate cause of biodiversity is the evolution of barriers that separate populations of organisms. I am
interested in discovering what barriers are important in the diversification of a species-rich group of
tropical fruit flies. Blepharoneura are highly specialized and host specific flies. Most species feed on a
single plant host although multiple species may exploit the same resource. For example, at one location
in Peru, two plant species are host to 14 Blepharoneura species. In addition, Blepharoneura occupies a
large range, occurring from Mexico to Bolivia. To examine how ecological and geographic barriers are
leading to diversification in Blepharoneura, I studied seven species characterized by differing patterns of
host-plant use and geographic distribution. I found patterns of ecological diversification associated with
host use in five species and patterns of geographic divergence in all seven species.
iv
TABLE OF CONTENTS
LIST OF TABLES ............................................................................................................................................. vi
LIST OF FIGURES ...........................................................................................................................................vii
CHAPTER
I.
INTRODUCTION ................................................................................................................... 1
II.
METHODS............................................................................................................................ 9
Focal species ....................................................................................................................... 9
DNA extraction and species delineation ............................................................................. 9
Library construction and microsatellite evaluation .......................................................... 10
Clustering analyses ........................................................................................................... 10
III.
RESULTS ............................................................................................................................ 19
Structure in mtDNA trees ................................................................................................. 19
Microsatellites: Host-associated structure in Blepharoneura .......................................... 21
Microsatellites: Geographic structure in Blepharoneura ................................................. 22
IV.
DISCUSSION ...................................................................................................................... 52
Host-associated structure in Blepharoneura .................................................................... 52
Geographic structure in Blepharoneura ........................................................................... 53
Synthesis: Host-associated and geographic structure in Blepharoneura ......................... 55
REFERENCES ................................................................................................................................................ 56
v
LIST OF TABLES
Table
1. Eighteen primer pairs use to amplify microsatellite loci for Blepharoneura flies. Primers were
optimized for different melting temperatures and did not amplify for all seven focal species. ......... 13
2. All seven species showed significant deviations from Hardy-Weinberg equilibrium (HWE) and
linkage disequilibrium. However, because deviations from HWE and linkage disequilibrium
were not consistent across multiple populations and are therefore not locus-specific the
deviating loci did not need to be excluded from the study. Loci that were either not
amplified for a specific population or were homozygous are denoted by “N/A”. Male and
female G. acuminata and G. spinulosa are referred to as m and f G. acum and G. spin
respectively. ......................................................................................................................................... 14
3. MtCOI data show evidence for host-associated divergence in two species (sp1 and sp21) and
geographic divergence in five species (sp1, sp4, sp8, sp21, and sp28). Species that did not
show either host or geographic divergence are denoted with an “X” while species that were
found in a single host are denoted with “N/A”. ................................................................................... 25
4. Microsatellite data indicate that sp1, sp4, sp10, sp21, and sp30 support host-associated
divergence within at least one biogeographic zone and all seven species (sp1, sp4, sp8, sp10,
sp21, sp28, and sp30) support geographic divergence (C=Caatinga; HG=Humid Guyana;
N=Napo; P=Puna; PT=Pantanal; VC=Venezuelan Coast; WE=Western Ecuador; Y=Yungas)
within at least one host type (m=male; f=female; G. acum=G. acuminata; G. spin=G.
spinulosa). Populations that did not show host or geographic divergence are denoted with
an “X” while populations reared from a single host or biogeographic zone are denoted with
an “N/A”. .............................................................................................................................................. 26
vi
LIST OF FIGURES
Figure
1. Ancient (>2mya) speciation events were associated with geographic shifts (blue) 36.0% of
the time and host shifts (orange) 52.5% of the time (Winkler and Condon unpublished).
Among recent (≤2mya) splits, 54.6% were related to geographic isolation and 50.0% were
host related. ............................................................................................................................................. 8
2. Locations of sample sites and identities of Blepharoneura species collected at each site. Sp1
samples used were found in male and female G. acuminata in Humid Guyana, Pantanal, and
Yungas. Sp4 was reared primarily in male G. spinulosa in Caatinga, Humid Guyana, Pantanal,
and Yungas. Sp8 was found primarily in male G. spinulosa flowers in Humid Guyana,
Pantanal, and Yungas. Sp10 was found mostly in female G. spinulosa in Humid Guyana and
Pantanal. Sp21 was reared from male G. spinulosa and male and female G. acuminata in
Humid Guyana, Venezuelan Coast, Western Ecuador, Pantanal, and Yungas. Sp28 was only
reared from male G. acuminata in Pantanal, Puna, and Yungas. Sp30 was reared from male
and female G. acuminata and male and female G. spinulosa in Caatinga, Humid Guyana,
Napo, Pantanal, and Yungas. .................................................................................................................... 12
3. The number of populations was determined using Evanno’s ΔK; however this method does
not allow for the differentiation between two populations and one population. a) Evanno’s
ΔK shows a defined peak at three populations in female G. acuminata and the DISTRUCT
plot supports geographic structure. b) Evanno’s ΔK supports two populations in sp30 from
Humid Guyana, but unlike sp21, the peak is not defined as ΔK could be higher for one
population. However, the DISTRUCT plot supports two host-associated populations. c)
Similar to sp30 in Humid Guyana, Evanno’s ΔK shows two populations in sp30 reared from
Napo but the peak is not defined. The DISTRUCT plot does not show host-associated
structure. .................................................................................................................................................. 18
4. Sp1 shows two major clades when analyzed with mtCOI. Sp1a (n=93) was reared from male
and female G. acuminata and male and female G. spinulosa found in Humid Guyana. Sp1b
(n=99) was reared from male and female G. acuminata found in Pantanal and Yungas. ....................... 27
5. Sp4 shows five major clades when analyzed with mtCOI. Sp4a (n=2) was reared from male G.
spinulosa. Sp4b (n=176) was reared from male G. acuminata and male and female G.
spinulosa found in Humid Guyana. Sp4c (n=71) attacked male G. spinulosa and G. acuminata
in Caatinga. Sp4d (n=4) was reared from male G. spinulosa in Caatinga and Napo. Sp4e
attacked male and female G. acuminata and G. spinulosa flowers in Humid Guyana, Napo,
Pantanal, and Yungas.. ............................................................................................................................. 28
6. Sp8 shows four major clades when analyzed with mtCOI. Sp8a (n=1) was reared from male
G. spinulosa in Napo. Sp8b (n=25) attacked male and female G. spinulosa found in Napo.
Sp8c (n=15) attacked male G. spinulosa in Napo and Pantanal. Sp8d (n=183) was reared
from male G. spinulosa in Caatinga and Napo. ........................................................................................ 31
7. Sp10 shows five major clades when analyzed with mtCOI. Sp10a (n=2) was reared from
female G. spinulosa in Western Ecuador. Sp10b (n=29) attacked female G. acuminata and
vii
male and female G. spinulosa. Sp10c (n=1) attacked male G. spinulosa in Humid Guyana.
Sp10d (n=60) was reared from male and female G. spinulosa in Humid Guyana, Pantanal,
and Yungas. Sp10e (n=79) attacked male G. acuminata and male and female G. spinulosa. ................. 33
8. Sp21 shows seven major clades when analyzed with mtCOI. Sp21a (n=20) were all reared
from male G. spinulosa in Pantanal. Sp21b (n=9) were reared from male and female G.
acuminata in Yungas. Sp21c (n=1) attacked male G. acuminata in Yungas. Sp21d (n=34)
attacked male and female G. spinulosa in Humid Guyana and Venezuelan Coast. Sp21e (n=2)
was reared from female G. acuminata in Western Ecuador. Sp21f (n=10) was reared in
Pantanal and Yungas from male G. acuminata. Sp21g (n=58) was found in male and female
G. acuminata and male G. spinulosa. ....................................................................................................... 34
9. Sp28 shows two major clades when analyzed with mtCOI. 28a (n=40) is found in Pantanal,
Puna, and Yungas. 28b (n=99) is found in Pantanal and one individual is found in Yungas.
Sp28 was only reared from male G. acuminata. ...................................................................................... 35
10. Sp30 shows four major clades when analyzed with mtCOI. Sp30a (n=3) were all reared from
male G. spinulosa in Caatinga. Sp30b (n=1) attacked male G. acuminata in Humid Guyana.
Sp30c (n=3) was reared from male and female G. acuminata in Yungas. Sp30d (n=439)
attacked male and female G. acuminata and G. spinulosa in Humid Guyana, Napo, Pantanal,
and Yungas. .............................................................................................................................................. 36
11. Sp1 is found in male and female G. acuminata in Yungas, male G. acuminata and G.
spinulosa in Humid Guyana, and male G. acuminata in Pantanal. No host-associated
structure was determined for Pantanal since only one host was available. a) Sp1 reared in
Yungas shows two genetically differentiated populations (K=2): one primarily in female G.
acuminata (n=13) and one in male G. acuminata (n=5). b) Sp1 reared from Humid Guyana
(n=85) supported two populations but because the clustering appears to be random this
may be an artifact of the inability of Evanno’s Δ K to assess the likelihood of a K=1. ............................. 40
12. Sp4 reared from Humid Guyana supported two populations (K=2): one primarily associated
with male G. acuminata (n=1) and the other associated with in female (n=4) and male
(n=25) G. spinulosa. Four individuals reared from male G. spinulosa and one individual from
female G. spinulsoa show some amount of similarity to the individual reared from male G.
acuminata................................................................................................................................................. 41
13. Sp8 was reared from male and female G. spinulosa in Yungas and male G. acuminata and G.
spinulosa in Humid Guyana. a) Evanno’s ΔK supported two populations which are both
found in male (n=30) and female (n=1) G. spinulosa from Yungas. However because the
structure does not match any apparent ecological pattern, we suggest that the real number
of populations is likely one (K=1). b) Similarly, male G. acuminata (n=2) and G. spinulosa
(n=19) from Humid Guyana show no obvious pattern, likely indicating that the real number
of populations is one (K=1)....................................................................................................................... 42
14. Sp10 was reared from male and female G. spinulosa in Humid Guyana and female G.
acuminata and G. spinulosa in Pantanal. a) Sp10 reared from Humid Guyana supported
three populations (K=3): two with no obvious structure in female G. spinulosa (n=38) and
one accounting for much of the variation in male G. spinulosa (n=3). b) Sp10 reared from
viii
Pantanal supported three populations (K=3). All three populations are present in female G.
spinulosa (n=21) and one population is present in female G. acuminata (n=1). ..................................... 43
15. a) Sp21 reared from Yungas supported two genetically distinct populations (K=2): one in
male (n=4) and female (n=6) G. acuminata and one in male G. spinulosa (n=2). One
individual reared from male G. acuminata is more genetically similar (>75%) to male G.
spinulosa flies than to other individuals found in male G. acuminata. b) Sp21 found in
Pantanal support two populations (K=2): one in male (n=14) and female (n=6) G. acuminata
and one in male G. spinulosa (n=20). Three individuals from male G. acuminata show
similarity to male G. spinulosa; two of which are more similar (>50%) to male G. spinulosa
than to the G. acuminata population. c) Sp21 reared from Humid Guyana shows two distinct
populations (K=2). One population is found in male (n=36) and female (n=10) G. acuminata
and female G. spinulosa (n=1) and the second population is in male G. spinulosa (n=29). One
individual in each group shows genetic similarity (>50%) to the opposite population. .......................... 44
16. a) Sp30 found in Humid Guyana shows two genetically distinct populations (K=2). One
population is found primarily in male G. acuminata (n=7) and the other population is found
in male (n=28) and female (n=31) G. spinulosa. Some individuals from both male G.
acuminata and male/female G. spinulosa show genetic similarity to the opposite population.
b) Sp30 found in Napo supported two populations but structure appears to be random,
indicating that this is really one population (K=1; male [n=6] and female [n=8] G. spinulosa).
c) Sp30 reared from male (n=19) and female (n=12) G. acuminata and male (n=37) and
female (n=32) G. spinulosa from Pantanal supported five populations (K=5). All five
populations are found randomly dispersed through all four groups which indicates the real
number of predicted populations is one. ................................................................................................. 45
17. Sp1 reared from male G. acuminata support two populations (K=2); one in Humid Guyana
(n=84) and another in Pantanal (n=3) and Yungas (n=5). Three individuals from Humid
Guyana show some genetic similarity to individuals in Pantanal and Yungas. ........................................ 46
18. Sp4 found in male G. spinulosa supports two genetically distinct populations (K=2). One
population in Caatinga (n=31) and Humid Guyana (n=25), and another population in
Pantanal (n=47) and Yungas (n=12). ........................................................................................................ 46
19. Sp8 reared from male G. spinulosa supports two weakly structured populations (K=2).
Humid Guyana (n=19) and Yungas (n=30) sites were most differentiated in their assignments
to the two populations, while individuals from Pantanal (n=24) were more intermediate. ................... 47
20. Sp10 reared from female G. spinulosa supports two populations (K=2): one primarily
represented in Pantanal (n=21) and one primarily in Humid Guyana (n=38). ......................................... 47
21. Sp21 was found in Pantanal, Humid Guyana and Yungas in female and male G. acuminata
and Western Ecuador, Panatanal, Yungas, Venezuelan Coast, and Humid Guyana in male G.
spinulosa. a) Sp21 from female G. acuminata shows three populations (K=3): one in
Pantanal (n=6), one in Humid Guyana (n=10), and one in Yungas (n=6). b) Sp21 reared from
male G. acuminata supported two populations (K=2). One population is in Yungas (n=4) and
Pantanal (n=9) and another population is in Humid Guyana (n=36). c) Sp21 found in male G.
spinulosa supports three populations (K=3). One population is found primarily in Western
ix
Ecuador (n=2), Pantanal (n=20), and Yungas (n=2), one population is found in Venezuelan
Coast (n=1), and all three populations with no obvious structure are found in Humid Guyana
(n=24). The one individual found in Venezuelan Coast shows genetic similarity to both
Western Ecuador/Pantanal/Yungas and Humid Guyana but is more similar (>50%) to the
population found in Humid Guyana. ........................................................................................................ 48
22. Sp28 reared from male G. acuminata supports two populations (K=2): one represented
primarily in Yungas (n=10) and Puna (n=2) and the other represented more in Pantanal
(n=46). ...................................................................................................................................................... 49
23. Sp30 is found in Humid Guyana, Pantanal, and Napo in female G. spinulosa, Yungas, Humid
Guyana, and Pantanal in male G. acuminata, and Yungas, Pantanal, Napo, and Humid
Guyana in male G. spinulosa. a) Sp30 found in female G. spinulosa supports two populations
(K=2) with strong geographic structure. One population is found in Humid Guyana (n=31)
and the other population is found in Pantanal (n=32) and Napo (n=8). b) Sp30 reared from
male G. acuminata show two somewhat geographically structured populations (K=2): one
primarily represented in Yungas (n=1) and Humid Guyana (n=7) and one with more affinity
to Pantanal (n=32). Because individuals from Humid Guyana and Pantanal show genetic
similarity to the opposite population there are likely ancient polymorphisms. c) Sp30 from
male G. spinulosa supports two weakly structured populations (K=2). One population in
Yungas (n=3), Pantanal (n=37) and one Humid Guyana (n=29) and Napo (n=6)). d) Individuals
reared from female G. acuminata did not show a clear signal of geographic structure
between Pantanal (n=12) and Yungas (n=4). ........................................................................................... 50
24. Isolation by distance analyses in sp21 reared from G. acuminata do not show a significant
relationship (p=0.072). ............................................................................................................................. 51
25. Isolation by distance analyses in sp21 reared from G. spinulosa do not show a significant
trend (p=0.210)......................................................................................................................................... 51
26. Isolation by distance analyses in sp30 reared from male G. acuminata and G. spinulosa do
not show a significant trend (p=0.404). ................................................................................................... 51
27. Isolation by distance analyses in sp30 reared from female G. acuminata and G. spinulosa
show a significant relationship (p=0.042). ............................................................................................... 51
x
CHAPTER I
INTRODUCTION
Determining how biodiversity is generated and maintained is an important part of evolutionary biology.
New biodiversity is ultimately a consequence of speciation, the result of the accumulation of
reproductive barriers between populations. Reproductive barriers arise in many ways, including as a
result of natural selection in different environments (ecological speciation), sexual selection, or
stochastic processes during periods of geographic isolation. Geographic isolation is generally considered
important in the formation of reproductive barriers (although see Kondrashov and Mina 1986; Berlocher
and Feder 2002; Bolnick and Fitzpatrick 2007). However, what is not yet clear is whether geographicallyisolated populations diverge more often due to stochastic processes (drift, mutation-order selection,
etc.), or if environmental differences between habitats are more important in driving the evolution of
adaptions that result in reproductive isolation (Nosil 2012). Understanding the circumstances that lead
to species diversification, whether through natural selection and/or stochastic processes, will provide
new insights into the generation of biodiversity.
Natural selection is a major evolutionary force, and many reproductive barriers are linked
directly to changes in environments that may alter selective regimes. For instance, if selection favors
adaptation to a new environment, habitat isolation can result in preferences for distinct habitats or
microhabitats, reducing the probability that individuals from different populations will mate. Shifts in
habitats can also impact how sexual signals are sent and received (Endler 1992) and lead to changes in
reproductive timing, resulting in allochrony (Feder et al. 1997). Natural selection can even produce
reproductive isolation in sympatry when a population shifts hosts or habitats (Agosta 2006). For many
phytophagous insects that mate on their host plants, host shifting can “automatically” isolate
populations feeding on different plants and interrupt gene flow, leading to diversification (Rundle and
Nosil 2005).
Habitat isolation, here referred to as a reduction in gene flow between populations resulting
from their fidelity to different habitats, is often caused by shifts in host preference. Host shifting is a
common occurrence among insects that allows species to escape conspecific competition (Feder et al.
1995), predation (Gratton and Welter 1999), or the threat of host extinction. Competition between
conspecifics can lead to limited resources and reduced fitness; shifting to a new host provides new
resource opportunities. In the same way, predation can lead to host shifts. For instance, when a
predator becomes specialized on a host and is therefore habituated to locating the host in a specific
1
habitat, the host can escape predation by changing habitats or hosts and moving into “enemy-free”
space. Host shifts can also be caused by host extinction. When a host plant becomes endangered or
extinct, organisms that rely on the plant will either go extinct themselves or shift to a new and
potentially more abundant host plant. Because not all individuals in a population are likely to switch
hosts, gene flow between the sub-populations may cease, leading to genetic isolation and eventually
speciation.
In general, animal speciation without ecological change requires geographic isolation.
Populations may be geographically separated by anthropogenic habitat fragmentation (e.g.
urbanization, roadways, or logging [see Gerlach and Musolf 2000; Keyghobadi 2007; Vanderast et al.
2007; Holderegger and Di Giulio 2010]), geologic/natural processes (e.g. river cuts, glacial cycles,
mountain building events [see Hayes and Sewlal 2004; Chapple et al. 2005; Ruiz-Sanchez and Specht
2013]), or random dispersal events (see Zink et al. 2000; Weir and Price 2011). Under conditions of
geographic isolation, speciation may be driven by non-selective processes such as genetic drift (Wright
1931). Genetic drift causes shifts in allele frequencies that eventually lead to fixation, and because it acts
randomly, geographically separated populations may fix incompatible alleles that result in reproductive
isolation (Coyne 1992). Drift is the driving force behind Hubbell’s (2001) unified neutral theory of
biodiversity and biogeography, in which neutral processes drive speciation through a series of stochastic
events that lead to the accumulation of unique mutations. Stochastic changes in the form of ecological
drift, random dispersal, and random mutations lead to diversification.
As populations become separated by greater geographic distances, isolation by distance (IBD)
predicts that genetic differentiation will also increase due to ecological divergence and stochastic
mutations coupled with reduced gene flow (Wright 1943). However, the distance needed to identify
separate populations can vary based on the organism (Peterson and Denno 1998). For example, in the
damselfly Coenagrion mercuriale, populations are considered different at distances of less than 10 km
(Watts et al. 2004) while Aedes aegypti populations must be more than 250 km apart (GorrochoteguiEscalante et al. 2000).
Of course, ecological speciation and allopatric divergence are not mutually exclusive. Ecological
speciation requires only that divergent natural selection results in the creation of reproductive barriers,
which can occur under conditions of allopatry, sympatry, or any intermediate context. Populations can
become geographically isolated due to stochastic events and then adapt to divergent habitats, with that
adaptation driving the evolution of reproductive isolation. On the other hand, habitat shifts can also
promote expansion into new geographic space, leading to geographic isolation and stochastic evolution
2
of isolating barriers (Coyne 1992; Turelli et al. 2001). Teasing apart the contributions of natural selection
and stochastic events in divergence is a major goal of my thesis work.
I used the Neotropical fruit fly Blepharoneura to understand how host shifts and geographic
isolation contribute to diversification. Blepharoneura is an ideal system for these questions because of
its large diversity (>100 species), its extreme host specificity, its use of many plant hosts (at the genus
level), and its large geographic range. Blepharoneura is composed of at least 130 morphologically cryptic
species (~50 described; Condon et al. 2008) that attack plants within the family Cucurbitaceae.
Blepharoneura species that feed on plants within the subtribe Guraniinae are highly specialized and host
specific (>85% attack a single host; Condon et al. 2008).
Geographic isolation seems likely to have contributed to Blepharoneura diversity. The
Guraniinae, especially Gurania acuminata and G. spinulosa, are abundant and widespread, ranging from
Panama to Bolivia. While this continuity might promote gene flow and prevent populations from
diverging, the continent-spanning distances between populations on either end of some fly species’
distributions suggest that geographic isolation may play an important role in speciation. Aside from
sheer distance, the geologic history of the continent may have contributed to Blepharoneura speciation.
In South America there are two major geographic barriers known to reduce or prevent gene flow
between animals and plants: the Andes mountain range (Brower 1994; Bernal et al. 2004) and the
Amazon River/Basin (Collins and Dubach 2000; Hayes and Sewlal 2004); both of which cut through the
ranges of many Blepharoneura species.
Geologic processes, such as the formation of the Andes and the Amazon River, may create new
climates that can lead to changes in biogeographic patterns. Biogeographic patterns are described using
the history of landmasses or habitats on biotic factors such as plant and animal distributions, abiotic
factors like soil and climate, or a combination. Biogeographic patterns are often good indicators of
species range limits (Wiens and Graham 2005) as transitions in biogeographic zones also indicate a
transition in climate or soil composition to which many plants are sensitive. Changes in biogeographic
patterns and the environment can lead to the formation of new niche space and allow for host shifts.
Biogeographic patterns, along with ecological characters, can be used to infer the relative importance of
habitat changes and stochasticity in diversification for any given group of organisms.
Reproductive isolation resulting from host shifting and adaptation to new plants is another
hypothesis that may explain some of the diversity in Blepharoneura. Gurania host plants are functionally
dioecious: male plants transition to a female stage after reaching a particular size. Both male and female
plants produce multiple branches with several inflorescences or flowers on each branch. Most
3
Blepharoneura species oviposit eggs into the fused sepals (calyx) of one flower sex of a specific plant
species, a preference that exists despite an absence of discriminating structures associated with flowers.
Contrary to classic ecological theory stating that extreme specialists should experience limited niche
overlap compared to generalist species (Darwin 1859, Schluter 2000), many Blepharoneura species
apparently overlap in each flower-sex niche, potentially leading to competition between species and
selection for host shifts (Condon et al. 2008). For example, at one collection site in Peru two plant
species (two sexes; four plant niches) were host to a total of 14 Blepharoneura species. Blepharoneura is
also parasitized by Bellopius, a parasitic braconid wasp. Bellopius, like Blepharoneura, displays extreme
host specificity- nearly every wasp species ecloses or hatches out of its “own” fly species (Condon et al.
2014). However, some Blepharoneura species apparently protect themselves from parasitism better
than others. For instance, sp1 is never parasitized by Bellopius while sp21 is extremely vulnerable,
especially when it is found on male G. spinulosa (Fig. 1). Species that experience high parasitism
pressures may commonly shift to hosts where they find “enemy-free space” and potential for reduced
mortality.
Condon et al. (2008) delineated Blepharoneura species across South America using
mitochondrial cytochrome c oxidase I (COI). Lineages that were separated by more than 4% pairwise
sequence differentiation, a conservative but arbitrary value, were considered unique from other
Blepharoneura lineages. Mitochondrial DNA (mtDNA), COI in particular, is frequently used in DNA
barcoding for quickly and easily identifying species (Hebert et al. 2003a). COI is optimal for barcoding
and determining evolutionary histories: like other animal mtDNA, it does not contain introns, making it
easily comparable between groups; it is easily amplified with conserved primers; and it does not
recombine because it is maternally inherited (Hebert et al. 2003a; Saccone et al. 1999). In some groups
of organisms, COI also provides enough resolution to allow for the delineation of closely-related species
(Johns and Avise 1998; Hebert et al. 2003b). Also, unlike other mitochondrial genes such as 16S and 12S,
COI does not have as many insertions or deletions to confound sequence analyses (Doyle and Gaut
2000).
Winkler and Condon (unpublished) analyzed Blepharoneura COI sequences using a 4% level of
pairwise differentiation to delineate species (equal to lineages >2mya). They then used the phylogenetic
relationships between “species” to determine if geographic isolation or host shifts (either between host
plant species or host plant sexes) were correlated with species differentiation. They did not distinguish
between splits that occurred with only one form of isolation from those that included both host shifts
and geographic isolation. Winkler and Condon found that 36.0% of speciation events involved
4
geographic shifts as opposed to no geographic shifts and 52.5% included host shifts. Next, they
identified splits below the 4% cutoff at a much more liberal value, 0.5% pairwise sequence
differentiation, to identify whether recent (≤2mya) Blepharoneura lineage splits were more often
correlated with geographic changes or host shifts. They found that geographic isolation was involved in
54.6% of divergence events and host shifts were involved in 50.0% of events. This indicates that more
recent divergence events (below the 4% cutoff used by Condon et al. (2008)) are slightly more often
associated with geographic isolation than host-shifting events. However determining how recent splits
can affect diversification and lead to speciation events is dependent on the accuracy of mitochondrial
species delineations.
This sole reliance on mtDNA may have drawbacks. DNA barcoding in particular has received
criticism for only using a single gene to identify species, and for its justification of using particular
sequence divergence-based cutoffs to define species (Lee 2004; Mortiz and Cicero 2004; Will and
Rubinoff 2004; Will et al. 2005; Meier et al. 2006). Studies involving mtDNA and DNA barcoding vary in
the percent sequence differences used to determine species cutoffs (Meyer and Paulay 2005; Cognato
2006). Species cutoffs are usually determined using a combination of morphological characteristics and
intraspecific variation while attempting to limit nonmonophly (Wiens and Penkrot 2002). While there
are many ways to determine species thresholds (Liu et al. 2005), typically two main methods of choosing
an appropriate cutoff are used: a fixed value or 10x the average intraspecific divergence (Hebert et al.
2004). Using the second method is especially problematic when attempting to delineate an undescribed
genus as it requires knowledge of the divergence rates between previously described species. In
addition, there are many occurrences where neither method accurately separates intra and interspecific
divergence and delineates species (see Meyer and Paulay 2005; Cognato 2006). For example, when
Cognato (2006) looked at species thresholds in insects, using 2% as a standard cutoff, he found that 28
of his 62 comparisons overlapped leading to incorrect classifications. The problems caused by incorrect
species cutoffs can obscure patterns of more recent diversification. If species cutoffs are set to a
conservative value, any variation below this level will be disregarded, potentially “lumping” distinct
species together. If set too liberally, one risks “splitting” species based on conclusions arising from high
rates of intraspecific variation common within some taxonomic groups. MtDNA can also have a different
evolutionary history compared with the nuclear genome. The mitochondrial genome is maternally
inherited and non-recombining, so patterns inferred from mtDNA sequence may represent incomplete
lineage sorting or rare introgression during periods of hybridization (Funk and Omland 2003; Ballard and
Whitlock 2004). While mtDNA can evolve quickly and define species to a very fine level, sometimes
5
determining where species should be separated is problematic. To combat these issues and provide
support for COI identified species as well as understand how these lineages diversify, other markers
need to be examined to identify intraspecific patterns of variation.
To complement information contained in mtDNA, a variety of molecular markers can be utilized,
including nuclear DNA (nDNA) sequences, next generation makers (such as reduced-representation
libraries (RRLs), complexity reduction of polymorphic sequences (CRoPS), and restriction-site-associated
DNA sequencing (RAD-seq)), Amplified Fragment Length Polymorphisms (AFLPs), Restriction Fragment
Length Polymorphisms (RFLPs), Random Amplified Polymorphic DNA (RAPDs), and microsatellites, all of
which have pros and cons depending on the question. While mtDNA is relatively fast evolving and can
resolve most fine-scale taxonomic discrepancies, nDNA provides broader resolution and is more useful
for looking at relationships between more distantly-related species (Zhang and Hewitt 2003). Next
generation sequencing can provide data at a relatively low cost but often requires a large amount of
computational power and a reference genome (Davey et al. 2011). Other markers can be broken into
several categories: single-locus or multilocus and dominant or co-dominant (Mueller and Wolfenbarger
1999). Single-locus markers such as RFLPs and microsatellites use a single site in the genome while
multilocus markers such as RAPDs and AFLPs use sites spread across the genome. Dominant markers
(RAPDs, AFLPs) are only scored as present or absent while co-dominant markers (microsatellites, RFLPs)
can identify heterozygosity and homologous alleles, giving them more statistical power than dominant
markers (Mueller and Wolfenbarger 1999; Gerber et al. 2000).
In order to resolve patterns of diversity at and below the species-level for Blepharoneura, faster
evolving markers are needed. I used microsatellites because they are relatively cheap to manufacture in
large quantities, easily amplified even with poor quality DNA from samples collected under non-ideal
conditions, genus specific (which eliminates the possibility of contamination from parasitic or symbiotic
DNA), and provide better statistical power than dominant markers (Selkoe and Toonen 2006).
Microsatellites, also known as simple sequence repeats (SSRs,) are 1-6 nucleotide tandem repeats with
alleles that vary in length due to mutations caused mainly by strand slippage during replication that
either increases or decreases the length by one unit (Jarne and Lagoda 1996).
By using microsatellites I will be able to 1) determine how robust mtDNA 4% sequence
divergence species cutoffs are for Blepharoneura and identify at what level, if at all, they lead to
incorrect conclusions (determined by evidence of reduced or halted gene flow between populations)
and 2) resolve patterns of divergence below the 4% species level in Blepharoneura that are associated
with ecological and/or geographic factors. The ultimate aim of my thesis is to identify population
6
structure within Blepharoneura lineages and determine how habitat isolation and stochastic processes
such as geographic isolation influence diversification in this clade of South American fruit flies. In
Chapter II, I outline the methods I employed in this study. In Chapter III, I discuss the results related to
ecological and geographic structure. In Chapter IV, I discuss the implications of my results and
summarize my findings, including how host-associated and geographic differentiation together can lead
to genetically isolated populations.
7
BLEPHARONEURA SPECIES SHIFTS
PROPORTION SPECIATION EVENTS
Geography
Host Shifts
60%
50%
40%
30%
20%
10%
0%
ANCIENT (>2MYA)
RECENT (≤2MYA)
Figure 1. Ancient (>2mya) speciation events were associated with geographic
shifts (blue) 36.0% of the time and host shifts (orange) 52.5% of the time
(Winkler and Condon unpublished). Among recent (≤2mya) splits, 54.6% were
related to geographic isolation and 50.0% were host related.
8
CHAPTER II
METHODS
Focal species
I selected seven Blepharoneura species (defined at the 4% COI divergence level; sp1, sp4, sp8, sp10,
sp21, sp28, and sp30; Condon et al. 2008) representing species with a variety of patterns of host use and
geographic distribution. Some selected species attack two or more host plant-sex niches and are found
in multiple geographic zones (as defined by Morrone 2006; sp1, sp21, sp30). Other species primarily
attack one host niche across multiple biogeographic zones (sp4, sp8, sp10). One species (sp28) is only
found in a single plant niche in multiple biogeographic zones (Fig. 2). Because Gurania acuminata and G.
spinulosa were the most abundant and provided the largest geographic distribution (next most
abundant species is G. bignoniaceae n=7), only individuals that were reared from these host plants were
used.
DNA extraction and species delineation
I collected samples of Blepharoneura flies from natural populations across South America over a 10 year
period with Marty Condon and others (see Condon et al. 2014 for collection and rearing methods). I
extracted DNA from 363 adult Blepharoneura samples that had been preserved in 95% ethanol and
stored at -80° C. Matthew Lewis (USDA-ARS) extracted Blepharoneura DNA from all other adult samples
and from EtOH-preserved Blepharoneura pupae and empty Blepharoneura puparia (total n = 2,561)
from which parasitic wasps had emerged. Matthew Lewis homogenized pupal samples with disposable
microfuge pestles while we nondestructively extracted DNA from post-emergence (empty) puparia or
two legs from adult samples. We extracted genomic DNA from all samples using the DNeasy Blood &
Tissue Kit (Qiagen, Valencia, CA).
For all flies, either Matthew Lewis (USDA-ARS) or I amplified a 504 bp segment of mtCOI (Table
1) on a Mastercycler Gradient thermocycler (Eppendorf Scientific, Inc., Westbury, NY) with the following
‘touch-down’ program: 2 min at 92°C, 12 ‘touch down’ cycles from 58 to 46°C (10 s at 92°C, 10 s at 5846°C, 1.5 min at 72°C), 27 cycles at 10 s at 92°C, 10 s at 45°C, 1.5 min at 7°C, and 10 min at 72°C. We
sequenced segments using Big Dye sequencing kits (Applied Biosystems, Foster City, CA) on an ABI3100
(ML) or an ABI3730 (KO) and analyzed and aligned sequences using either Sequencher (Gene Codes
Corp., Ann Arbor, MI) or Geneious (Biomatters v6.1.7, Auckland, NZ).
I generated neighbor-joining trees using mtCOI sequences in Geneious using a Tamura-Nei
model with 1000 bootstrap replicates to delineate my focal species. I separated my focal species into
major clades (any host associated or geographic monophyletic group that was separated by at least
9
0.5% sequence divergence) to analyze divergence patterns. The final sample of mtDNA-scored flies
included 106 individuals of sp1, 122 of sp4, 76 of sp8, 63 of sp10, 126 of sp21, 58 of sp28, and 190
individuals of sp30.
Library construction and microsatellite evaluation
I used the most abundant species in our collection, sp30, to construct microsatellite libraries. DNA from
15 adult sp30 flies was sent to the Savannah River Ecology Laboratory (SREL; Aiken, SC), where samples
were sequenced using Illumina 100 bp paired-end reads. SREL provided us with a summary of putative
microsatellite loci along with motif and minimum repeat length. I tested several primers and selected
eighteen loci that produced a single strong band on a 1% agarose gel to limit confounding effects
produced by overlapping alleles. Not all loci amplified for all seven species (Table 1). I used a
Mastercycler Gradient thermocycler (Eppendorf Scientific, Inc., Westbury, NY) to carry out PCR
amplifications with the following program: 3 min at 94°C, 35 cycles at 1 min at 94°C, 1 min at a locusspecific temperature (Table 1), 1 min at 68.0°C, and 10 min at 68.0°C. I used fluorescent labels (HEX,
FAM, or TAMRA) on forward primers in combinations that allowed for multiplexing of several different
loci at once. I genotyped individuals on an ABI 3730 and called alleles using GeneMarker 2.2.0 (2013).
I used Microchecker (van Oosterhout et al. 2004) to check for null alleles and other genotyping
errors such as stuttering and large-allele dropout. I used Arlequin (Excoffier et al. 2005) to sample
individual loci for deviations from Hardy-Weinberg equilibrium as well as linkage disequilibrium (Table
2). I sampled each species at least once using a single biogeographic zone and host species. For two
species that had the most data (sp30 and sp21), I calculated Fst values in Arlequin and then compared
these to geographic distances in PASSaGE (Rosenberg and Anderson 2011) using 300,000 permutations
to test for isolation by distance.
Clustering analyses
I used InStruct (Gao et al. 2007) to identify genetic structure within each focal sample. Unlike other
Bayesian clustering programs, InStruct allows for inbreeding. I ran InStruct from K=1 to K=10 in mode 5
(infer population structure and individual breeding coefficients) for five independent chains. Each chain
was run with 100,000 burn-in steps, 200,000 iterations, and a thinning interval of ten steps. I found the
number of predicted populations were determined using Evanno’s Δ K (Evanno et al. 2003; Dixon et al.
2013). I used DISTRUCT (Rosenberg 2002) to produce graphical representations of InStruct boxplots for
K values with highest support. One major downfall of using Evanno’s Δ K is the inability to distinguish
between populations with no structure (K=1) and populations supporting two groups (K=2). To
differentiate between these two types of groups (ones with no real structure vs. those that actually
10
supported two populations) I used the structure seen in the DISTRUCT plot. Populations that were
randomly or evenly distributed across all groups or instances where there was no host-associated
pattern were assumed to consist of a single population (Fig. 3).
To determine if differences in host use within my focal Blepharoneura species (sp1, sp4, sp8,
sp10, sp21, sp28, and sp30, as defined by Condon et al. 2008) are associated with diversification I
analyzed flies using different host species and flower sexes within each single biogeographic zone (this
limits the total data used in any single analysis but avoids confounding ecological isolation with
geographic structure). I determined biogeographic zones using definitions determined by Morrone
(2006), who reviewed patterns of insect ranges in South America and defined zone boundaries as those
where multiple insect ranges ended. For example, to examine the host-associated divergence in sp1
between male and female G. acuminata and male G. spinulosa I first looked only at individuals found in
Humid Guyana. Next, I analyzed individuals found in Pantanal and finally individuals in Yungas. By
looking at each biogeographic zone in turn I can isolate the amount of differentiation associated with
host use differences. I found that of my seven focal species, five (sp1, sp4, sp10, sp21, and sp30)
displayed host-associated diversification.
In order to determine if there is geographic structure in my focal Blepharoneura species (sp1,
sp4, sp8, sp10, sp21, sp28, and sp30 as defined by Condon et al. 2008), I separately analyzed each fly
species within a single host species and flower sex to isolate differentiation caused by changes in
geographic regions. For example, to determine if sp1 showed geographic differentiation between Humid
Guyana, Pantanal, and Yungas I first only looked at individuals in male G. acuminata. Then I only
examined individuals in female G. acuminata and finally I looked at individuals in male G. spinulosa. I
found that all seven of my focal species (sp1, sp4, sp8, sp10, sp21, sp28, and sp30) supported
geographic differentiation.
11
Figure 2. Locations of sample sites and identities of Blepharoneura species collected at each site. Sp1
samples used were found in male and female G. acuminata in Humid Guyana, Pantanal, and Yungas.
Sp4 was reared primarily in male G. spinulosa in Caatinga, Humid Guyana, Pantanal, and Yungas. Sp8
was found primarily in male G. spinulosa flowers in Humid Guyana, Pantanal, and Yungas. Sp10 was
found mostly in female G. spinulosa in Humid Guyana and Pantanal. Sp21 was reared from male G.
spinulosa and male and female G. acuminata in Humid Guyana, Venezuelan Coast, Western Ecuador,
Pantanal, and Yungas. Sp28 was only reared from male G. acuminata in Pantanal, Puna, and Yungas.
Sp30 was reared from male and female G. acuminata and male and female G. spinulosa in Caatinga,
Humid Guyana, Napo, Pantanal, and Yungas.
12
Table 1. Eighteen primer pairs use to amplify microsatellite loci for Blepharoneura flies. Primers
were optimized for different melting temperatures and did not amplify for all seven focal species.
01233
Motif
Length
2
Size Range
(bp)
355-387
Species
Amplified
28, 30
01329
2
253-279
06083
2
215-237
11186
2
286-322
1, 4, 10,
21, 30
1, 4, 8, 10,
21, 28, 30
21, 28, 30
16166
4
98-138
21181
3
142-166
15860
2
396-436
01780
4
254-322
22613
2
255-287
07147
4
423-459
04598
4
188-240
01483
4
156-184
01435
4
264-352
25539
3
193-238
03484
3
188-230
27457
3
209-239
10004
3
189-213
13171
4
217-235
Locus
8, 10, 21,
28,30
1, 4, 8, 10,
21, 28, 30
1, 4, 8, 10,
21, 30
30
1, 4, 8, 10,
21, 28, 30
10, 21, 28,
30
8, 10, 21,
28, 30
1, 4, 8, 10,
21, 28, 30
30
1, 4, 8, 10,
21, 28, 30
1, 4, 8, 10,
21, 28, 30
1, 4, 8, 10,
21, 28, 30
1, 4, 8, 21,
28, 30
1, 4, 8, 10,
21, 28, 30
Melting
Primer Sequence 5'-3'
Temp (°C)
61.2 F- GACACACATTCACAAACATAAATGC
R- TCAGTTGGAGTGTTGGACGG
61.2 F- CTTATCATATCTTGCTTGCATACG
R- GGCGAGAATTCAATTTGTACG
61.2 F- ACCATGAATTGAGAGCGTGC
R- TGGCCGATGACATTACTTGC
61.2 F- TTCATTTATGCATTGCTTGGC
R- TGGCTCACTTTAAAGAACTTACGG
67.0 F- CGTTTCTGTGTCACCGACG
R- AGGAGACACGTCGCAATAAGG
67.0 F- TGTTTGCTTGTTGCCACTGC
R- TGTCATTGTTAAGTGAGCAGCTACC
56.1 F- ATTTACTTGCACGCACACGC
R- AAGGACAACCAGATTTACTCATTACG
65.2 F- GCTGCGCCAGTAAATATAGAAAGC
R- AACGAACTTTGCCCATACGC
63.2 F- TGAGTCAAGCTCCACACATGC
R- TCACGAATATTTCGCGCC
61.2 F- TAATCGCGCAAGTGTGTTCC
R- CCATTCGTTGTAGTCGCAGG
61.2 F- AAATTGAGCAGCGTGGGC
R- CCAGTTGCCACTACAAGTCGG
61.2 F- CTATCCAGTTCAACCGACCG
R- GCGCCCTTTGTACGTATTCC
61.2 F- GCAACTACAAATCAAATCATTTCACC
R- TTCGATGGAATTTGCCAACC
61.2 F- GTCTGTGTGAAACTTAGGCCG
R- GCGCAGATTTGATTTGTTGC
57.5 F- GCTAAACAATCGCTTCACTTGG
R- AGTGTGAATGTGTCTGGGCG
57.5 F- TTGTTGAAGTAGTTGGCGTGG
R- GCTGTACGCTCGTTTGTTGC
59.2 F- AATAATCGTCACCGTCACCG
R- ATGACCATGCCAACAACACC
59.2 F- CAATGAGGAACTCGGTGTTGG
R- TCGATCGTTTGACTTGTGTGC
13
Table 2. All seven species showed significant deviations from Hardy-Weinberg equilibrium
(HWE) and linkage disequilibrium. However, because deviations from HWE and linkage
disequilibrium were not consistent across multiple populations and are therefore not locusspecific the deviating loci did not need to be excluded from the study. Loci that were either not
amplified for a specific population or were homozygous are denoted by “N/A”. Male and female
G. acuminata and G. spinulosa are referred to as m and f G. acum and G. spin respectively.
Species Locus
Population
sp1
f G. acum
Yungas
sp4
sp8
01329
06083
21181
15860
22613
01483
25539
03484
27457
10004
13171
01329
06083
21181
15860
22613
01483
25539
03484
27457
10004
13171
01329
06083
21181
15860
22613
01483
25539
03484
27457
10004
13171
06083
16166
21181
m G. spin
Caatinga
m G. spin
Pantanal
m G. spin
Yungas
HWE
# Linked
p-value Loci
0.00
0
1.00
1
1.00
1
0.00
0
0.00
0
1.00
1
0.01
0
N/A
0
0.14
2
0.35
1
0.01
0
0.00
6
0.01
3
N/A
0
0.00
3
0.00
1
0.01
5
0.00
1
1.00
2
0.90
4
0.00
0
1.00
1
0.25
3
0.24
5
0.90
0
0.00
5
0.00
7
0.00
6
0.40
4
0.02
3
0.13
6
0.00
6
0.02
3
0.00
1
0.22
1
0.64
1
14
Population
m G. acum
Humid Guyana
m G. spin
Humid Guyana
m G. spin
Humid Guyana
HWE
# Linked
p-value Loci
0.20
8
0.00
6
1.00
1
0.00
6
0.04
5
0.00
4
0.00
4
0.00
8
0.15
7
0.00
8
0.00
7
0.02
4
0.03
2
0.01
4
0.00
3
0.02
1
0.58
1
0.00
4
0.00
5
0.03
4
0.31
3
0.04
1
0.15
0.00
0.05
2
6
1
sp10
sp21
15860
22613
04598
01483
25539
03484
27457
10004
13171
06083
16166
21181
15860
22613
04598
01483
25539
03484
27457
10004
13171
01329
06083
16166
21181
15860
22613
07147
04598
01483
25539
03484
27457
13171
01329
06083
11186
16166
21181
15860
22613
07147
04598
01483
m G. spin
Pantanal
f G. spin
Humid Guyana
m G. spin
Pantanal
0.00
0.00
0.10
0.01
0.21
0.80
0.78
0.00
0.00
0.07
0.01
0.65
NA
0.01
0.04
0.00
0.00
0.11
0.21
0.00
0.02
0.02
0.00
0.05
0.69
0.00
0.00
0.16
0.11
0.14
0.15
0.98
0.19
0.64
0.90
0.00
0.08
0.34
0.75
0.00
0.03
0.02
0.21
0.36
3
5
1
6
2
1
1
3
1
1
7
0
NA
4
2
2
4
2
3
2
1
3
7
3
2
8
5
3
1
4
4
2
7
1
0
3
3
0
4
4
6
2
4
2
15
f G. spin
Pantanal
m G. spin
Humid Guyana
0.00
0.14
0.58
0.00
0.00
0.34
0.43
0.00
0.00
6
3
1
4
4
1
2
2
2
1.00
0.00
0.63
0.76
0.00
0.00
0.00
0.04
0.00
0.35
0.76
0.01
0.02
0.00
0.10
0.00
0.00
0.05
0.00
0.00
0.01
0.00
0.13
2
7
3
2
4
6
4
6
5
2
0
5
2
9
4
2
3
7
8
11
8
7
2
sp28
sp30
25539
03484
27457
10004
13171
01329
06083
11186
16166
21181
15860
22613
07147
04598
01483
25539
03484
27457
10004
13171
01233
06083
11186
16166
21181
22613
07147
04598
01483
25539
03484
27457
10004
13171
01233
01329
06083
11186
16166
21181
15860
01780
22613
07147
m G. acum
Humid Guyana
m G. acum
Pantanal
m G. acum
Pantanal
0.10
0.01
0.61
0.00
0.04
0.07
0.00
0.29
0.00
0.00
0.00
0.00
0.56
0.56
0.01
0.49
0.00
0.00
0.00
1.00
0.00
0.12
0.00
0.60
0.03
0.12
0.00
0.48
0.00
0.44
0.00
0.12
0.00
0.00
0.00
0.04
0.04
0.00
0.05
0.07
0.00
0.00
0.10
0.00
2
2
3
4
5
4
7
8
5
4
5
7
2
1
3
2
3
4
5
0
9
1
5
1
4
3
5
0
5
2
1
2
4
4
10 f G. spin
5 Pantanal
3
9
4
3
6
8
7
2
16
0.00
0.00
0.08
0.01
0.01
13
7
1
7
3
0.00
0.00
0.05
0.00
0.61
0.04
0.00
0.00
0.86
0.00
12
8
6
12
7
3
12
12
1
10
04598
01483
01435
25539
03484
27457
10004
13171
01233
01329
06083
11186
16166
21181
15860
01780
22613
07147
04598
01483
01435
25539
03484
27457
10004
13171
01233
01329
06083
11186
16166
21181
15860
01780
22613
07147
04598
01483
01435
25539
03484
27457
10004
13171
f G. spin
Humid Guyana
m G. spin
Humid Guyana
0.24
0.06
0.29
0.36
0.00
0.22
0.47
0.90
0.00
0.00
0.11
0.00
0.29
0.00
0.00
0.04
0.00
0.24
0.00
0.00
0.00
0.02
0.00
0.00
0.00
0.04
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
3
3
5
5
6
1
3
1
15 m G. spin
10 Pantanal
2
14
3
14
14
15
16
12
14
13
14
13
13
15
8
9
8
1
3
10
0
4
7
6
0
6
3
3
4
3
2
0
2
0
17
0.02
0.00
0.00
0.10
0.00
0.06
0.00
0.41
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
7
7
10
4
5
1
9
2
11
4
8
13
10
8
11
10
4
10
6
7
7
3
7
3
6
2
a
Evanno's ΔK
sp21 female G. acuminata
300
200
100
0
2 3 4 5 6 7 8 9 10
# of Populations
b
Evanno's ΔK
sp30 Humid Guyana
300
200
100
0
2 3 4 5 6 7 8 9 10
# of Populations
c
Evanno's ΔK
sp30 Napo
150
100
50
0
2 3 4 5 6 7 8 9 10
# of Populations
Figure 3. The number of populations was determined using Evanno’s ΔK; however this method does
not allow for the differentiation between two populations and one population. a) Evanno’s ΔK shows
a defined peak at three populations in female G. acuminata and the DISTRUCT plot supports
geographic structure. b) Evanno’s ΔK supports two populations in sp30 from Humid Guyana, but
unlike sp21, the peak is not defined as ΔK could be higher for one population. However, the
DISTRUCT plot supports two host-associated populations. c) Similar to sp30 in Humid Guyana,
Evanno’s ΔK shows two populations in sp30 reared from Napo but the peak is not defined. The
DISTRUCT plot does not show host-associated structure.
18
CHAPTER III
RESULTS
Structure in mtDNA trees
Sp1 (defined at the 4% level by Condon et al. 2008) had two major clades: sp1a and sp1b (Fig. 4). Sp1a
(n=93) contained Blepharoneura that attacked male (n=89) and female (n=1) G. acuminata and male
(n=2) and female (n=1) G. spinulosa. Individuals in this clade were all found in Humid Guyana. Sp1b
(n=50) had four subclades. One subclade contained male G. acuminata found in Pantanal (n=17). The
second subclade had female G. acuminata from Yungas (n=13). The third subclade contained a mixture
of male (n=7) and female (n=8) G. acuminata from Yungas and the fourth subclade had reared male G.
acuminata from Yungas (n=5).
Sp4 had five major clades: sp4a, sp4b, sp4c, sp4d, and sp4e that were reared primarily from
male G. spinulosa (Fig. 5). Sp4a only contained two individuals. Both were from male G. spinulosa but
one was found in the Venezuelan Coast and the other in Humid Guyana. Sp4b (n=176) contained flies
reared from male flowers (n=165) and female flowers (n=7) of G. spinulosa and male flowers of G.
acuminata (n=4) all of which were found in Humid Guyana. Sp4c (n=71) contained Blepharoneura that
attacked male G. spinulosa (n=65) and male G. acuminata (n=6) in Caatinga. Sp4d (n=4) was reared from
male G. spinulosa in Caatinga (n=1) and Napo (N=3). Sp4e (n=193) was reared in male (n=2) and female
(n=1) G. acuminata and male (n=189) and female (n=1) G. spinulosa. Individuals were found in Humid
Guyana (n=1), Napo (n=33), Pantanal (n=148), and Yungas (n=11).
Sp8 had four major clades: sp8a, sp8b, sp8c, and sp8d that were found mostly in male G.
spinulosa (Fig. 6). Sp8a contained a single individual reared from male G. spinulosa in Napo. Sp8b (n=25)
attacked male (n=24) and female (n=1) G. spinulosa in Napo. Sp8c (n=15) were reared from male G.
spinulosa in Napo (n=3) and Pantanal (n=12). Sp8d (n=183) contained three subclades. The first subclade
contained individuals reared from male (n=77) and female (n=1) G. spinulosa in Pantanal (n=44) and
Yungas (n=34). The next subclade contained individuals reared from male G. acuminata (n=1) and male
G. spinulosa (n=24) from Pantanal (n=20) and Yungas (n=5). The last subclade attacked male G.
acuminata (n=2) and male G. spinulosa (n=78) flowers. All individuals were found in Humid Guyana.
Sp10 had five major clades: sp10a, sp10b, sp10c, sp10d and sp10e that were reared primarily
from female G. spinulosa (Fig. 7). Sp10a contained two individuals reared from female flowers of G.
spinulosa in Western Ecuador. Sp10b (n=29) has two subclades. One contained individuals reared from
female G. acuminata (n=1) and male (n=1) and female (n=7) G. spinulosa in Humid Guyana and the other
contained female G. acuminata (n=20) from Pantanal (n=18) and the Venezuelan Coast (n=2). Sp10c
19
contained a single individual reared from male G. spinulosa from Humid Guyana. Sp10d (n=60) attacked
male (n=3) and female (n=57) G. spinulosa in Humid Guyana (n=52), Pantanal (n=7), and Yungas (n=1).
Sp10e contains two subclades. One subclade attacked male G. acuminata (n=1) and male (n=2) and
female (n=25) G. spinulosa in Humid Guyana (n=20), Pantanal (n=4), and Yungas (n=1). The other
subclade was reared from male (n=1) and female (n=50) G. spinulosa in Napo (n=23), Pantanal (n=27),
and Yungas (n=1).
Sp21 had seven major clades: sp21a, sp21b, sp21c, sp21d, sp21e, sp21f, and sp21g (Fig. 8).
Sp21a were all found in male G. spinulosa (n=20) in Pantanal. Sp21b had two subclades: one attacked
male G. acuminata (n=4) in Pantanal and the other attacked female G. acuminata (n=5) in Yungas. Sp21c
contained one individual that attacked male G. acuminata in Yungas. Sp21d was found in male (n=33)
and female (n=1) G. spinulosa in Humid Guyana (n=33) and Venezuelan Coast (n=1). Sp21e attacked
male G. spinulosa (n=2) in Western Ecuador. Sp21f was reared from male G. acuminata (n=10) in
Pantanal (n=6) and Yungas (n=4). Sp21g had four subclades. One subclade contained two individuals
reared from female G. acuminata in Yungas and another subclade contained two individuals from
female G. acuminata in Pantanal. Another contained individuals found in male (n=38) and female (n=9)
G. acuminata and male G. spinulosa (n=2) in Humid Guyana. The last subclade contained individuals
reared from female G. acuminata in Pantanal (n=4) and Humid Guyana (n=1).
Sp28 had two major clades: sp28a and sp28b which were only reared from male G. acuminata
(Fig. 9). Sp28a (n=40) attacked flowers in Pantanal (n=28), Puna (n=2), and Yungas (n=10) and sp28b
(n=99) attacked male flowers in Pantanal (n=98) and Yungas (n=1).
Sp30 had four major clades: sp30a, sp30b, sp30c, and sp30d (Fig. 10). Sp30a had three
individuals reared from male G. spinulosa in Caatinga. Sp30b had one individual that attacked male G.
acuminata in Humid Guyana. Sp30c (n=3) was found in male (n=1) and female (n=2) G. acuminata in
Yungas. Sp30d was reared from male (n=37) and female (n=3) G. acuminata and male (n=248) and
female (n=151) G. spinulosa. Individuals were found in Humid Guyana (n=250), Napo (n=28), Pantanal
(n=157), and Yungas (n=4)
In summary, six of seven focal species show host- or geographic associated divergence between
major clades in their mtCOI trees (Table 3). Sp1 showed both host and geographic divergence: sp1a
mainly attacked male G. acuminata in Humid Guyana while sp1b attacked a mix of male and female G.
acuminata in Pantanal and Yungas. In sp4 there were no host-associated differences but there were
geographic differences. Sp4a and sp4b were reared mainly from Humid Guyana, sp4c were reared from
Caatinga, and sp4d were reared in Napo, while sp4e were found occurring in multiple biogeographic
20
regions across the continent. Sp8 displayed geographic but not host-associated differentiation. Sp8a and
sp8b were reared from Napo while sp8c and sp8d were reared primarily from Humid Guyana, Pantanal,
and Yungas. Sp10 did not show host-associated differences but did show geographic divergence. Sp10a
was only found in Western Ecuador while sp10b, sp10c, sp10d, and sp10e were found in Humid Guyana,
Venezuelan Coast, Pantanal, Yungas and Napo. Sp21 showed both host and geographic divergence.
Sp21a and sp21e were only reared from male G. spinulosa, sp21d were reared from both male and
female G. spinulosa, and sp21b, sp21c, sp21f, and sp21g were reared from male and/or female G.
acuminata. Sp21d and sp21e were only reared from zones north of the Amazon River and the Andes
(Western Ecuador, Humid Guyana, and Venezuelan Coast) while sp21a, sp21b, sp21c, and sp21f were
only reared from Pantanal and Yungas. Sp21g were reared from both Humid Guyana and Pantanal. Sp28
did not show geographic divergence and because it was found in a single host, it did not show hostassociated divergence. Sp30 did not display host-associated divergence but did have geographic
divergence. Sp30a is only found in Caatinga, sp30b is only found in Humid Guyana, sp30c is only found in
Yungas, and sp30d is found throughout Humid Guyana, Pantanal, Yungas, and Napo.
Microsatellites: Host-associated structure in Blepharoneura
Sp1 is found in three biogeographic zones – Yungas (n=18), Pantanal (n=3), and Humid Guyana (n=85) but only showed differentiation in host use in Yungas and Humid Guyana (see Table 4 for a summary of
all results). Evanno’s ΔK (Evanno et al. 2003) supported two genetically differentiated populations in
Yungas: one in female G. acuminata (n=13) and one in male G. acuminata (n=5; Fig. 11). In Pantanal and
Humid Guyana Evanno’s Δ K supported two populations, however, I determined that no host-associated
structure (K=1) was resolved in Pantanal or Humid Guyana. For the remainder of the study instances
where Evanno’s Δ K supported two populations but did not show supported host-associations, the
predicted structure was assumed to be an artifact of the method and I predicted one real population
(K=1).
Sp4 is found in four biogeographic zones – Caatinga (n=31), Humid Guyana (n=32), Pantanal
(n=47), and Yungas (n=12). Weak structure was found in Humid Guyana; one population was found in
male G. acuminata (n=1) and another population was found in female (n=4) and male (n=25) G.
spinulosa (Fig. 12). No structure (K=1) was found in Caatinga, Pantanal, or Yungas as they all were found
in the same host.
Sp8 is found in Humid Guyana (n=21), Pantanal (n=24), and Yungas (n=31). No host-associated
structure (K=1) was found in any biogeographic zone (Fig. 13).
21
Sp10 is in Pantanal (n=22) and Humid Guyana (n=41). Evanno’s ΔK for the Humid Guyana sample
supports three populations in Humid Guyana; two populations with no obvious structure in female G.
spinulosa (n=38) and one in male G. spinulosa (n=3). Pantanal shows three weakly associated
populations; all three populations are found in female G. spinulosa (n=21) with no obvious structure and
one population is found in female G. acuminata (n=1; Fig. 14).
Sp21 is found in Humid Guyana (n=69), Venezuelan Coast (n=1), Western Ecuador (n=2),
Pantanal (n=40), and Yungas (n=12). Host–associated differentiation occurs in Humid Guyana, Pantanal,
and Yungas (Fig. 15). Humid Guyana has two genetically differentiated populations; one in male G.
acuminata (n=36), female G. acuminata (n=10), and female G. spinulosa (n=1), and another in male G.
spinulosa (n=29). Pantanal has two genetically associated populations; one in female (n=6) and male
(n=14) G. acuminata and one in male G. spinulosa (n=20). Yungas also has two populations; one in male
(n=4) and female (n=6) G. acuminata and one in male G. spinulosa (n=2). Western Ecuador and
Venezuelan Coast had no structure (K=1; Fig. 15).
Sp28 is found in Pantanal (n=46), Puna (n=2), and Yungas (n=10). No structure (K=1) was
resolved in any biogeographic zone as all individuals were reared from male G. acuminata.
Sp30 is found in Humid Guyana (n=67), Napo (n=14), Pantanal (n=101), and Yungas (n=8) but
host-associated differences were only identified in Humid Guyana and Pantanal. I found evidence for
two populations in Humid Guyana; one in male G. acuminata (n=7) and male G. spinulosa (n=28) and
one in female G. spinulosa (n=31; Fig. 16). Individuals reared from Pantanal supported five populations
(K=5), however all five populations appear to be randomly dispersed throughout male (n=19) and female
(n=12) G. acuminata and male (n=37) and female (n=37) G. spinulosa which usually indicates there is
only one real population (K=1). No structure (K=1) was resolved for Napo and because Yungas only
contained eight individuals InStruct was not run.
Microsatellites: Geographic structure in Blepharoneura
Sp1 is found primarily in male (n=92) and female (n=13) G. acuminata but only showed patterns of
geographically-associated differentiation in male G. acuminata (see Table 4 for a summary of all results).
Evanno’s Δ K (Evanno et al. 2003) supports two populations in male G. acuminata; one in Humid Guyana
(n=84) and another in Pantanal (n=3) and Yungas (n=5; Fig. 17). Female G. acuminata does not show any
structure (K=1; Fig…).
Sp4 attacks mainly male G. spinulosa (n=115) which supported two genetically distinct
populations; one in Pantanal (n=47) and Yungas (n=12) and another in Caatinga (n=31) and Humid
Guyana (n=25; Fig. 18).
22
Sp8 also attacks predominantly attacks male G. spinulosa (n=73) and Evanno’s Δ K supports two
weakly structured populations. One population is found in Humid Guyana (n=19) and the other is in
Pantanal (n=24) and Yungas (n=30; Fig. 19).
Sp10 is found primarily in female G. spinulosa (n=59) and shows evidence of two geographicallyassociated populations; one in Humid Guyana (n=38) and one population in Pantanal (n=21; Fig. 20).
Sp21 mostly attacks male (n=54) and female (n=23) G. acuminata and male G. spinulosa (n=49).
In female G. acuminata there are three geographically-associated populations; one in Humid Guyana
(n=10), one in Pantanal (n=6), and one in Yungas (n=6; Fig. 21). Male G. acuminata displays two
genetically distinct populations; one in Humid Guyana (n=36) and one in Pantanal (n=9) and Yungas
(n=4). Male G. spinulosa shows three geographically-associated populations; two with no obvious
structure in Humid Guyana (n=24) and the Venezuelan Coast (n=1) and one in Pantanal (n=20), Yungas
(n=2), and Western Ecuador (n=2).
Sp28 is only found in male G. acuminata (n=58) and displays two geographically-associated
populations. One population is found in Puna (n=2) and Yungas (n=10) and another population is found
Pantanal (n=46; Fig. 22).
Sp30 is found in all four host types (male (n=27) and female (n=16) G. acuminata and male
(n=75) and female (n=71) G. spinulosa), but only shows geographically-associated differences in male G.
acuminata and female and male G. spinulosa. In male G. acuminata there are two geographicallyassociated populations with weak structure; one in Humid Guyana (n=7) and Yungas (n=1) and another
population in Pantanal (n=19; Fig. 23). In female G. spinulosa Evanno’s Δ K supports two populations.
One population is in Humid Guyana (n=31) and one population is in Napo (n=8) and Pantanal (n=32).
Male G. spinulosa supports two geographically-associated populations; one in Humid Guyana (n=29) and
one population in Napo (n=6), Pantanal (n=37), and Yungas (n=3). No structure (K=1) was found in
female G. acuminata.
Isolation by distance analyses were insignificant for both sp21 male and female G. acuminata
(p=0.072; Fig. 24) and male G. spinulosa (p=0.210; Fig. 25) and sp30 male G. acuminata and G. spinulosa
(p=0.404; Fig. 26). Female G. acuminata and G. spinulosa showed a significant trend (p=0.042; Fig. 27).
Individuals in sp21 reared from male and female G. acuminata flowers could be combined into a single
analysis because InStruct determined there were not host-associated divergences and were therefore a
single population. Sp30 reared from male G. acuminata and G. spinulosa could also be combined into a
single population and female G. acuminata and G. spinulosa because InStruct did not find hostassociated differences in biogeographic zones.
23
Table 3. MtCOI data show evidence for hostassociated divergence in two species (sp1 and
sp21) and geographic divergence in five
species (sp1, sp4, sp8, sp21, and sp28).
Species that did not show either host or
geographic divergence are denoted with an
“X” while species that were found in a single
host are denoted with “N/A”.
Species
sp1
sp4
sp8
sp10
sp21
sp28
sp30
Host
Divergence
Flower Sex
X
X
X
Host Plant
N/A
X
Geographic
Divergence
HG vs. PT, Y
C vs. HG vs. N
HG vs. PT, Y
WE vs. HG, VC, PT, N
HG, VC, WE vs. PT, Y
X
C vs. HG, Y, PT, N
24
Table 4. Microsatellite data indicate that sp1, sp4, sp10, sp21, and sp30 support
host-associated divergence within at least one biogeographic zone and all seven
species (sp1, sp4, sp8, sp10, sp21, sp28, and sp30) support geographic divergence
(C=Caatinga; HG=Humid Guyana; N=Napo; P=Puna; PT=Pantanal; VC=Venezuelan
Coast; WE=Western Ecuador; Y=Yungas) within at least one host type (m=male;
f=female; G. acum=G. acuminata; G. spin=G. spinulosa). Populations that did not
show host or geographic divergence are denoted with an “X” while populations
reared from a single host or biogeographic zone are denoted with an “N/A”.
Species
sp1
sp4
sp8
sp10
sp21
sp28
sp30
Host Divergence
Biogeographic
Type of
Zone
Divergence
Humid Guyana
X
Pantanal
N/A
Yungas
Host Plant
Caatinga
N/A
Humid Guyana
Host Plant
Pantanal
N/A
Yungas
N/A
Humid Guyana
X
Pantanal
N/A
Yungas
X
Humid Guyana
Host Plant
Pantanal
Flower Sex
Humid Guyana
Host Plant
Pantanal
Host Plant
Venezuelan Coast
N/A
Western Ecuador
N/A
Yungas
Host Plant
Pantanal
N/A
Puna
N/A
Yungas
N/A
Humid Guyana
X
Napo
Host Plant
Pantanal
X
Yungas
N/A
Geographic Divergence
Host Type
m G. acum
f G. acum
Type of Divergence
HG vs. PT, Y
N/A
m G. spin
C vs. PT, Y
m G. spin
HG vs. PT, Y
f G. spin
HG vs. PT
m G. acum
f G. acum
m G. spin
HG vs. PT, Y
HG vs. PT vs. Y
HG, VC vs. PT, WE, Y
m G. acum
PT vs. P, Y
m G. acum
f G. acum
m G. spin
f G. spin
HG, Y vs. PT
X
HG vs. N, PT, Y
HG vs. N, PT
25
Figure 4. Sp1 shows two major clades
when analyzed with mtCOI. Sp1a
(n=93) was reared from male and
female G. acuminata and male and
female G. spinulosa found in Humid
Guyana. Sp1b (n=99) was reared from
male and female G. acuminata found
in Pantanal and Yungas.
26
27
28
Figure 5. Sp4 shows five major clades when analyzed with mtCOI. Sp4a (n=2) was reared from male
G. spinulosa. Sp4b (n=176) was reared from male G. acuminata and male and female G. spinulosa
found in Humid Guyana. Sp4c (n=71) attacked male G. spinulosa and G. acuminata in Caatinga. Sp4d
(n=4) was reared from male G. spinulosa in Caatinga and Napo. Sp4e attacked male and female G.
acuminata and G. spinulosa flowers in Humid Guyana, Napo, Pantanal, and Yungas.
29
30
Figure 6. Sp8 shows four major clades when analyzed
with mtCOI. Sp8a (n=1) was reared from male G.
spinulosa in Napo. Sp8b (n=25) attacked male and
female G. spinulosa found in Napo. Sp8c (n=15)
attacked male G. spinulosa in Napo and Pantanal. Sp8d
(n=183) was reared from male G. spinulosa in Caatinga
31
Figure 7. Sp10 shows five major clades when analyzed
with mtCOI. Sp10a (n=2) was reared from female G.
spinulosa in Western Ecuador. Sp10b (n=29) attacked
female G. acuminata and male and female G. spinulosa.
Sp10c (n=1) attacked male G. spinulosa in Humid
Guyana. Sp10d (n=60) was reared from male and
female G. spinulosa in Humid Guyana, Pantanal, and
Yungas. Sp10e (n=79) attacked male G. acuminata and
male and female G. spinulosa.
32
Figure 8. Sp21 shows seven major clades when analyzed
with mtCOI. Sp21a (n=20) were all reared from male G.
spinulosa in Pantanal. Sp21b (n=9) were reared from
male and female G. acuminata in Yungas. Sp21c (n=1)
attacked male G. acuminata in Yungas. Sp21d (n=34)
attacked male and female G. spinulosa in Humid
Guyana and Venezuelan Coast. Sp21e (n=2) was reared
from female G. acuminata in Western Ecuador. Sp21f
(n=10) was reared in Pantanal and Yungas from male G.
acuminata. Sp21g (n=58) was found in male and female
G. acuminata and male G. spinulosa.
33
Figure 9. Sp28 shows two major clades
when analyzed with mtCOI. 28a (n=40)
is found in Pantanal, Puna, and Yungas.
28b (n=99) is found in Pantanal and
one individual is found in Yungas. Sp28
was only reared from male G.
acuminata.
34
35
36
37
Figure 10. Sp30 shows four major clades when analyzed with mtCOI. Sp30a (n=3) were all reared
from male G. spinulosa in Caatinga. Sp30b (n=1) attacked male G. acuminata in Humid Guyana.
Sp30c (n=3) was reared from male and female G. acuminata in Yungas. Sp30d (n=439) attacked male
and female G. acuminata and G. spinulosa in Humid Guyana, Napo, Pantanal, and Yungas.
38
a
b
Figure 11. Sp1 is found in male and female G. acuminata in Yungas, male G. acuminata and G.
spinulosa in Humid Guyana, and male G. acuminata in Pantanal. No host-associated structure was
determined for Pantanal since only one host was available. a) Sp1 reared in Yungas shows two
genetically differentiated populations (K=2): one primarily in female G. acuminata (n=13) and one in
male G. acuminata (n=5). b) Sp1 reared from Humid Guyana (n=85) supported two populations but
because the clustering appears to be random this may be an artifact of the inability of Evanno’s Δ K
to assess the likelihood of a K=1.
39
Figure 12. Sp4 reared from Humid Guyana supported two populations (K=2): one primarily associated
with male G. acuminata (n=1) and the other associated with in female (n=4) and male (n=25) G.
spinulosa. Four individuals reared from male G. spinulosa and one individual from female G.
spinulsoa show some amount of similarity to the individual reared from male G. acuminata.
40
a
b
Figure 13. Sp8 was reared from male and female G. spinulosa in Yungas and male G. acuminata and
G. spinulosa in Humid Guyana. a) Evanno’s ΔK supported two populations which are both found in
male (n=30) and female (n=1) G. spinulosa from Yungas. However because the structure does not
match any apparent ecological pattern, we suggest that the real number of populations is likely one
(K=1). b) Similarly, male G. acuminata (n=2) and G. spinulosa (n=19) from Humid Guyana show no
obvious pattern, likely indicating that the real number of populations is one (K=1).
41
a
b
Figure 14. Sp10 was reared from male and female G. spinulosa in Humid Guyana and female G.
acuminata and G. spinulosa in Pantanal. a) Sp10 reared from Humid Guyana supported three
populations (K=3): two with no obvious structure in female G. spinulosa (n=38) and one accounting
for much of the variation in male G. spinulosa (n=3). b) Sp10 reared from Pantanal supported three
populations (K=3). All three populations are present in female G. spinulosa (n=21) and one
population is present in female G. acuminata (n=1).
42
a
b
c
Figure 15. a) Sp21 reared from Yungas supported two genetically distinct populations (K=2): one in
male (n=4) and female (n=6) G. acuminata and one in male G. spinulosa (n=2). One individual reared
from male G. acuminata is more genetically similar (>75%) to male G. spinulosa flies than to other
individuals found in male G. acuminata. b) Sp21 found in Pantanal support two populations (K=2):
one in male (n=14) and female (n=6) G. acuminata and one in male G. spinulosa (n=20). Three
individuals from male G. acuminata show similarity to male G. spinulosa; two of which are more
similar (>50%) to male G. spinulosa than to the G. acuminata population. c) Sp21 reared from Humid
Guyana shows two distinct populations (K=2). One population is found in male (n=36) and female
(n=10) G. acuminata and female G. spinulosa (n=1) and the second population is in male G. spinulosa
(n=29). One individual in each group shows genetic similarity (>50%) to the opposite population.
43
a
b
c
Figure 16. a) Sp30 found in Humid Guyana shows two genetically distinct populations (K=2). One
population is found primarily in male G. acuminata (n=7) and the other population is found in male
(n=28) and female (n=31) G. spinulosa. Some individuals from both male G. acuminata and
male/female G. spinulosa show genetic similarity to the opposite population. b) Sp30 found in Napo
supported two populations but structure appears to be random, indicating that this is really one
population (K=1; male [n=6] and female [n=8] G. spinulosa). c) Sp30 reared from male (n=19) and
female (n=12) G. acuminata and male (n=37) and female (n=32) G. spinulosa from Pantanal
supported five populations (K=5). All five populations are found randomly dispersed through all four
groups which indicates the real number of predicted populations is one.
44
HG
PT Y
Figure 17. Sp1 reared from male G. acuminata support two populations (K=2); one in Humid Guyana
(n=84) and another in Pantanal (n=3) and Yungas (n=5). Three individuals from Humid Guyana show
some genetic similarity to individuals in Pantanal and Yungas.
C
HG
PT
Y
Figure 18. Sp4 found in male G. spinulosa supports two genetically distinct populations
(K=2). One population in Caatinga (n=31) and Humid Guyana (n=25), and another
population in Pantanal (n=47) and Yungas (n=12).
45
PT
Y
HG
Figure 19. Sp8 reared from male G. spinulosa supports two weakly structured populations (K=2).
Humid Guyana (n=19) and Yungas (n=30) sites were most differentiated in their assignments to the
two populations, while individuals from Pantanal (n=24) were more intermediate.
PT
HG
Figure 20. Sp10 reared from female G. spinulosa supports two populations (K=2): one primarily
represented in Pantanal (n=21) and one primarily in Humid Guyana (n=38).
46
a
b
Y
PT
HG
PT
HG
Y
c
WE
PT
Y
VC
HG
Figure 21. Sp21 was found in Pantanal, Humid Guyana and Yungas in female and male G. acuminata
and Western Ecuador, Panatanal, Yungas, Venezuelan Coast, and Humid Guyana in male G.
spinulosa. a) Sp21 from female G. acuminata shows three populations (K=3): one in Pantanal (n=6),
one in Humid Guyana (n=10), and one in Yungas (n=6). b) Sp21 reared from male G. acuminata
supported two populations (K=2). One population is in Yungas (n=4) and Pantanal (n=9) and another
population is in Humid Guyana (n=36). c) Sp21 found in male G. spinulosa supports three
populations (K=3). One population is found primarily in Western Ecuador (n=2), Pantanal (n=20), and
Yungas (n=2), one population is found in Venezuelan Coast (n=1), and all three populations with no
obvious structure are found in Humid Guyana (n=24). The one individual found in Venezuelan Coast
shows genetic similarity to both Western Ecuador/Pantanal/Yungas and Humid Guyana but is more
similar (>50%) to the population found in Humid Guyana.
47
Y
P
PT
Figure 22. Sp28 reared from male G. acuminata supports two populations (K=2): one represented
primarily in Yungas (n=10) and Puna (n=2) and the other represented more in Pantanal (n=46).
48
b
a
HG
N
PT
Y HG
c
PT
d
Y
PT
N
HG
PT
Y
Figure 23. Sp30 is found in Humid Guyana, Pantanal, and Napo in female G. spinulosa, Yungas, Humid
Guyana, and Pantanal in male G. acuminata, and Yungas, Pantanal, Napo, and Humid Guyana in male
G. spinulosa. a) Sp30 found in female G. spinulosa supports two populations (K=2) with strong
geographic structure. One population is found in Humid Guyana (n=31) and the other population is
found in Pantanal (n=32) and Napo (n=8). b) Sp30 reared from male G. acuminata show two
somewhat geographically structured populations (K=2): one primarily represented in Yungas (n=1)
and Humid Guyana (n=7) and one with more affinity to Pantanal (n=32). Because individuals from
Humid Guyana and Pantanal show genetic similarity to the opposite population there are likely
ancient polymorphisms. c) Sp30 from male G. spinulosa supports two weakly structured populations
(K=2). One population in Yungas (n=3), Pantanal (n=37) and one Humid Guyana (n=29) and Napo
(n=6)). d) Individuals reared from female G. acuminata did not show a clear signal of geographic
structure between Pantanal (n=12) and Yungas (n=4).
49
Figure 25. Isolation by distance analyses in sp21
reared from G. spinulosa do not show a
significant trend (p=0.210).
Figure 24. Isolation by distance analyses in sp21
reared from G. acuminata do not show a
significant relationship (p=0.072).
Figure 27. Isolation by distance analyses in sp30
reared from female G. acuminata and G.
spinulosa show a significant relationship
(p=0.042).
Figure 26. Isolation by distance analyses in sp30
reared from male G. acuminata and G. spinulosa
do not show a significant trend (p=0.404).
50
CHAPTER IV
DISCUSSION
Host-associated structure in Blepharoneura
Of the six focal species that used more than one plant host (sp1, sp4, sp8, sp10, sp21, and sp30) just two
(sp1 and sp21) showed host-associated structure between major clades in mtCOI trees. Of the lineages
that showed variation in host type in mtCOI (sp1, sp4, sp8, sp10, sp21, and sp30) half were found
primarily in a single host species and flower sex (sp4, sp8, sp10), which limited my ability to detect
structure. Sp4 flies were reared mostly from male G. spinulosa: ten individuals (2.2%) were reared from
female G. spinulosa and six individuals (1.3%) were reared from male G. acuminata. Sp8 were reared
primarily from male G. spinulosa: two individuals (0.9%) from female G. spinulosa and three individuals
(1.3%) from male G. acuminata. Sp10 were reared primarily from female G. spinulosa: one individual
(0.6%) reared from female G. acuminata, one individual (0.6%) reared from male G. acuminata, and
eight individuals (4.5%) reared from male G. spinulosa. Because none of these secondary host types
make up more than 5% of the total reared individuals, small sample sizes may be limiting the ability to
detect differentiation. With the inclusion of more individuals from these “rare” host types, additional
host-associated groups may emerge.
Microsatellites revealed more host-associated structure than mtCOI. Host-associated structure
was resolved for five species (sp1, sp4, sp10, sp21, and sp30) and only sp8 showed no structure. Hostassociated structure within each species tended to split in one of two ways - either by host plant species
or by flower sex - and these alternatives were generally consistent within each species. For example, in
sp1, InStruct split populations based on flower sex associations (male vs. female flowers, independent of
plant species), while sp4, sp21, and sp30 consistently grouped according to host species (but not flower
sex). Sp10 technically showed splits along both flower sex and host species, however host-associated
structure between species (Pantanal) only included a single individual reared from female G. acuminata
flowers, and this individual was genetically similar to some flies reared from female G. spinulosa. More
individuals are needed to determine if this is a real pattern. Another exception was sp21 in Humid
Guyana, which showed an unusual pattern where one population was associated with male and female
G. acuminata as well as female G. spinulosa and another population was in male G. spinulosa (Fig. 15).
Again, only one female G. spinulosa individual was included in the sample, so more female G. spinulosa
individuals should be scored to ascertain if this pattern is real. In one or both instances it may also be
possible that the “rogue” individual represents a rare fly ovipositing in the “wrong” host.
51
The different patterns of host-associated structure resolved for Blepharoneura species suggest
that the mode of fly specialization differs from species to species. Fly species that use a single flower sex
but multiple plant species must be able to discriminate between flower sexes (perhaps using visual or
volatile cues) despite a lack of apparent differences between male and female calyx tissues. Conversely,
other species that use both male and female flowers within a single plant species must be able to
discriminate between species (perhaps using volatile cues associated with other plant parts). Host-shifts
must require changes in host-preference that are sensitive to flower species or sex.
Geographic structure in Blepharoneura
Six species (exception: sp28) showed geographic structure in mtCOI trees, while microsatellites
indicated geographic structure within all seven species. In both mtCOI and microsatellites individuals in
the southwestern zones were genetically similar and individuals in the northeastern zones were
genetically similar while populations between the two groups were differentiated. Of the six lineages
that showed geographic differentiation in mtCOI trees (sp1, sp4, sp8, sp10, sp21, and sp30), all six were
associated with splits between northern (Western Ecuador, Humid Guyana, Venezuelan Coast, Napo)
and southern (Caatinga, Pantanal, Yungas) regions. These patterns are consistent with the riverine
barrier hypothesis, which proposes that the formation of the Amazon and its tributaries fragmented
ancestral populations and prevents gene flow between current populations (Collins and Dubach 2000;
Hayes and Sewlal 2004).
In most cases, flies of the same species at the Pantanal and Yungas sites were genetically similar.
In many other biogeographic studies (Cracraft 1985; Hall and Harvey 2002), Pantanal and Yungas both
fall into a single, large zone called Inambari. Much of my results follow the results of those studies and
support grouping these two areas into a single biogeographic region. One exception is sp30 reared from
female G. acuminata, but this includes just a single individual from Yungas; more individuals from
Yungas are needed to determine if this is a real pattern or if the individual contains an ancient
polymorphism. Other exceptions are sp21 reared from female G. acuminata and sp28 reared from male
G. acuminata. Individuals that attack male and female G. acuminata could experience a smaller home
range or be more sensitive to environmental changes than individuals reared from other hosts.
As in mtDNA, much microsatellite-based geographic structure within Blepharoneura species
reflected collections made north and south of the Amazon River or east and west of the Amazon Basin.
For example, sp1 reared from male G. acuminata, sp8 reared from male G. spinulosa, and sp21 reared
from male G. acuminata split between Humid Guyana and Pantanal/Yungas, sp4 reared from male G.
spinulosa split between Humid Guyana/Caatinga and Pantanal/Yungas, sp10 reared from female G.
52
spinulosa split between Humid Guyana and Pantanal, sp21 reared from male G. spinulosa split between
Humid Guyana/Venezuelan Coast and Pantanal/Yungas, and sp30 reared from female G. spinulosa split
between Humid Guyana and Pantanal (Table 4).
Two species (sp21 and sp30) also included harder-to-explain clustering patterns. Sp21 in male G.
spinulosa showed little differentiation between Western Ecuador, Pantanal, and Yungas and sp30 in two
hosts was similar at Napo and Pantanal sites. While both regions are north of the Amazon River and
Amazon Basin, the Amazon River is much narrower which may not effectively prevent gene flow
between biogeographic regions. Western Ecuador is located on the opposite side of the Andes from
Pantanal and Yungas. Although it has been shown that gene flow can be continuous across both sides of
the Andes in some species of bats (Ditchfield 2000; Hoffman and Baker 2003) and euglossine bees (Dick
et al. 2004), populations are generally thought to cross the Andes using a small pass in Colombia (Dick et
al. 2004). While the shortest low-elevation route is through the eastern Andes in Colombia and
Venezuela, this area is surrounded by dry arid savannas or ocean (Eva et al. 2002). To determine if
Blepharoneura populations from Pantanal and Yungas are mixing with populations in Western Ecuador
via this pass, collections from Columbia would be instructive. If Columbian populations are more similar
to Pantanal and Yungas, then we can predict that populations are migrating via a northern route.
However, if populations are more similar to other northern biogeographic zones, such as Humid Guyana,
we can assume that populations from Pantanal and Yungas are not mixing with Western Ecuador via a
pass through Colombia. In addition, more individuals from Western Ecuador should be collected and
scored as only two were available for this study and the small sample size could bias results.
Two subgroups, sp8 and sp30, both reared from male G. spinulosa, show weak geographic
structure. Sp8 supports two populations and while one population is represented more frequently by
flies in Pantanal and Yungas and the other population is more common in Humid Guyana; both
populations are represented in all three zones. This indicates that gene flow is likely occurring between
Pantanal/Yungas and Humid Guyana. However, because some amount of differentiation is still present
(one population is more common in Pantanal/Yungas vs. Humid Guyana), this could potentially
demonstrate the beginning stages of divergence between Humid Guyana and Pantanal/Yungas in sp8
and Humid Guyana/Napo and Pantanal/Yungas in sp30. If early differentiation is associated with weak
genetic structure, then identifying populations with weak structure should lead us to toward insights
into what might be driving diversification in Blepharoneura.
53
Synthesis: Host-associated and geographic structure in Blepharoneura
Host-associated differentiation and geographic divergence may not be mutually exclusive.
Microsatellites showed both host-associated and geographic structure in sp1, sp4, sp10, sp21, and sp30
while mtCOI only displayed both forms of diversification in sp1 and sp21. Host use and association with
particular biogeographic regions are often linked, as demonstrated by the mtCOI trees (e.g., see sp1,
sp4, sp10, sp21, and sp30) and therefore cannot be parsed apart. For example, mtCOI sequences for
sp21 show that subclades group by both host species and biogeographic region (Fig. 8). Therefore,
where this species shows differentiation according to host use, it also shows differentiation between
biogeographic zones. This could indicate that reproductive isolation is driven by geographic splits
followed by host shifts, or that reproductive isolation is driven by ecological changes and species
subsequently move to new biogeographic regions. A third possibility is that geographic shifts and
ecological shifts occur simultaneously.
While both host shifts and biogeographic changes are likely important for reproductive isolation,
biogeographic regions tend to be associated with stronger genetic differentiation in Blepharoneura. For
example, microsatellites reveal much finer-scale host-associated structure; possibly associated with
recent shifts in host use while mtCOI does not resolve much of this host-associated structure. This could
indicate that host shifts are responsible for more recent divergence as patterns associated with
geographic shifts are more deeply resolved and therefore older than ecological changes. On the other
hand, host shifts could occur before biogeographical shifts. Data from Winkler and Condon
(unpublished) support this: more ancient (>2mya) diversification events involved host shifts than
geographic changes (Fig. 1) possibly indicating that ecological shifts ultimately drive reproductive
isolation.
While neither prediction is fully supported by the current evidence, future work could focus on
parsing apart these two hypotheses. By examining more species that have either geographic or
ecological diversification one might ultimately determine the relative importance or necessity of both
geographic and ecological shifts in speciation.
My evaluation of Blepharoneura using microsatellites does not dispute analyses of genetic
differentiation based on mtCOI by Condon et al. (2008) and it permits a finer-scale analysis of ecological
and geographic structure. However, one take home message is that by using a 4% mtCOI sequence
divergence cutoff to define species we are overlooking important genetic structure, both above and
below the species level. My results show that within species defined by a 4% pairwise divergence cutoff
there are finer-scale patterns of diversity that reveal real barriers to reproduction.
54
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