Integrating micro and macroevolution of development:

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Integrating micro and macroevolution of
development a case study on horned beetles
ARMIN P. MOCZEK Department of Biology Indiana
University
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Alternative frameworks for understanding evolution
Developmental Constraints
History
Adaptation
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Mediators of evolutionary change
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HORNED BEETLES THE ORIGINS OF DIVERSITY....
OBJECTIVES
I Social environments and morphological
evolution II Endocrine mechanisms as mediators
(and guides of) phenotypic evolution III.
Developmental genetics as a window into the
ancestry of diversity
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The origins of diversity a case study on horned
beetles
Onthophagus taurus
ARMIN P. MOCZEK University of Arizona Center for
Insect Science
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Typical habitat of O. taurus
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Typical (micro) habitat of O. taurus
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Life cycle of a tunneler
(after Tyndale-Biscoe (1990)
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Male O. taurus are dimorphic for horn length

horn length
horn length (mm)
body size (mm)
(after Moczek AP Emlen DJ (2000). Animal
Behaviour 59 459- 466)
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Male horn dimorphism in Onthophagus beetles
- Background

• Male horn length is not heritable but determined
• in response to larval feeding conditions.
• BUT Males may differ genetically in other
  aspects of horn expression, such as the threshold
  body size.
• 3) ........

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3) Male morphs use alternative reproductive
tactics
b) hornless males sneak
Drawing by Barrett Klein
(after Moczek 2004)
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- Background cont.-
4) Horned and hornless morphologies help in
fighting and sneaking behavior
a) Longer horns improve fighting success (Moczek
Emlen 2000) b) Short horns improve agility
(Moczek 1996 Moczek Emlen 2000)
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- Background cont.-

• Exotic populations evolved
• highly divergent allometries
• lt 40 years / 80 generations

Mediterranean ancestor
h o r n l e n g t h ( m m )
Eastern United States (US)
Eastern Australia (EA)
Western Australia (WA)
(Moczek 2003)
b o d y s i z e (mm)
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- Background cont.-
6) Allometric divergence in O. taurus
parallels differences between species
(c) between other Onthophagus species
(b) between sister species
(a) between O. taurus populations
horn length (mm)
b o d y s i z e ( mm )
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HORNED BEETLES THE ORIGINS OF DIVERSITY....
OBJECTIVES
I Social environments and morphological
evolution II Endocrine mechanisms as mediators
(and guides of) phenotypic evolution III.
Developmental genetics as a window into the
ancestry of diversity
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Horned and hornless males use alternative
reproductive tactics
a) horned males fight

(Moczek AP 1996 Moczek AP Emlen DJ 2000)
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• Male fighting success
• depends on...
• body size
• horn length
• number of competitors

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Social environments and morphological evolution
- body size of competing males
a) small mean male body size
t
horn length
frequency
b) large mean male body size
told
tnew
horn length
frequency
body size
body size
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Social environments and morphological
evolution.....
Hypothesis
Prediction
(a) Differential body size hypothesis
mean male body size
mean threshold body size
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Social environments and morphological evolution
- intraspecific competition
high
low

consequences
- most females manage to breed - few males
compete for few females - low levels of
male-male competition
- not all females manage to breed - many males
compete for few females - intense male-male
competition
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Social environments and morphological
evolution.....
Hypothesis
Prediction
(b) Intraspecific competition hypothesis
O. taurus density
mean threshold body size
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Methodology sample sites - transects in
Eastern US, Eastern and Western
Australia (8-12 sites/range)
recorded - dung beetle composition on
per dung pad basis - sex ratios and
allometries - re-sampled subset of sites
in 4 week intervals
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Ecological and demographic causes of threshold
evolution
a) Differential body size hypothesis
Prediction
male body size (mm)
mean threshold body size (mm)
(Moczek 2003)
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Ecological and demographic causes of threshold
evolution
a) Differential body size hypothesis
Prediction
Results
male body size (mm)
p lt 0.001
mean threshold body size (mm)
US
WA
EA
(Moczek 2003)
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Ecological and demographic causes of threshold
evolution
b) Intraspecific competition hypothesis
Prediction
O.taurus density (individuals/kg dung site)
mean threshold body size (mm)
(Moczek 2003)
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Ecological and demographic causes of threshold
evolution
b) Intraspecific competition hypothesis
Prediction
Results
O.taurus density (individuals/kg dung site)
p lt 0.001
mean threshold body size (mm)
US
WA
EA
(Moczek 2003)
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HORNED BEETLES THE ORIGINS OF DIVERSITY....
CONCLUSIONS
I Social environments and morphological
evolution
Differences in social and ecological conditions
may drive allometric divergences in nature.
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Competition for dung is moderate in US populations
On average Colonized by lt10 beetles
Majority of resource dries out before beetles
can bury it
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Competition for dung is intense in WA populations
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Alternative means of dung beetle collecting an
Australian experience
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THE ORIGINS OF DIVERSITY....
OBJECTIVES
I Social environments and morphological
evolution II Endocrine mechanisms as mediators
(and guides) of phenotypic evolution III.
Developmental genetics as a window into the
ancestry of diversity
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Initial findings
Horned males (US WA) grow larger and longer
during larval development WA larvae require
higher weights to develop into the horned
morph WA larvae require more time (30) to
complete the final instar 
(Moczek Nijhout 2002)
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HORMONAL CONTROL OF A THRESHOLD TRAIT
sensitive period
threshold for horns
JH concentration
late 3rd instar prepupa pupa
(after Emlen Nijhout 1999. J. Ins. Phys. 45
45-53)
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Endocrine mechanisms of threshold evolution
(Moczek Nijhout 2002)
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EXPERIMENTAL APPROACH
(c) concentrations (1) low (2µg/larva)
(2) medium (10 µg/larva ) (3) high
(50 µg/larva)
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Juvenile Hormone (JH) mediates horn expression
E-US
Acetone control
low
r e s p o n s e
JH-analogue Methoprene
medium
high
I II III IV
developmental stage
(Moczek Nijhout 2002)
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Populations differ in JH sensitivity and timing
W-Australia
E-US
Acetone control
low
r e s p o n s e
JH-analogue Methoprene
medium
high
I
II
III
IV
I
II
III
IV
V
(WA only)
developmental stage
(Moczek Nijhout 2002)
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THE ORIGINS OF DIVERSITY....
CONCLUSIONS
Novel allometries can evolve rapidly in
geographically isolated population Differential
intraspecific competition may drive allometric
divergences in nature Allometric divergences may
be mediated by changes in (i) degree and (ii)
timing of sensitivity to JH
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HORNED BEETLES THE ORIGINS OF DIVERSITY....
OBJECTIVES
I Social environments and morphological
evolution II Endocrine mechanisms as mediators
(and guides of) phenotypic evolution III.
Developmental genetics as a window into
the ancestry of diversity
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Beetle horns an evolutionary novelty with
tremendous diversity

• have evolved in at least 6 beetle families
• lack obvious homologues outside the Coleoptera
• several thousand species develop horns
• remarkable diversity on a variety of levels

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Onthophagus rich phenotypic variation on a
variety of levels

1. Alternative (nutrition-dependent) male morphs

horn length
horn length (mm)
O. taurus
body size (mm)
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Onthophagus rich phenotypic variation on a
variety of levels
2) Inter-population divergence
Eastern US
W-Australia
horn length (mm)
b o d y s i z e (mm)
(Moczek 2003)
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Onthophagus rich phenotypic variation on a
variety of levels
3) Sexual dimorphism
?
?
?
?
O. taurus
O. nigriventris
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Onthophagus rich phenotypic variation on a
variety of levels - species
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Onthophagus rich phenotypic variation on a
variety of levels
3) Between sexes (Females are hornless or
develop rudimentary horns)

1. Within genomes (alternative male morphs)

2) Between populations (populations differ in
scaling between body size and horn length)

• Between species
• (species differ in scaling, number,
• sizes, and locations of horns)

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Overview

1) Where, when, and how do beetle horns grow? 2)
Which patterning genes regulate horn
development? 3) Which changes in the genetic and
developmental basis of beetle horns mediated
the evolution of horned beetle diversity?
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When, where and how do horns develop?
Phase I Horns form via explosive growth of
epidermal tissue.....
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.... but which epidermal regions proliferate
differs between species.
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Differential prepupal growth generates sexual
horn dimorphism in O. taurus
?
?
?
?
horn length (mm)
body size (mm)
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But differential pupal resorption generates
sexual horn dimorphism in O. binodus
?
?
?
horn length (mm)
?
body size (mm)
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Differential resorption also generates reversed
sexual horn dimorphism in O. sagittarius
?
?
?
?
horn length (mm)
body size (mm)
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Pupal resorption reverses horn growth and sexual
horn dimorphism in O. taurus
?
?
?
horn length (mm)
?
body size (mm)
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Conclusions

1. Where, when, and how do beetle horns grow?

• Horns are of epidermal origin, grow explosively
  during the prepupal stage, and are subject to
  size modulation during the pupal stage
• Intraspecific diversity in adult horn expression
  is the product of two discrete processes
• (a) differential growth during the prepupal
  stage
• (b) differential loss during the pupal stage.
• Species in turn differ in the degree to which
  they rely on one or the other mechnanism in the
  genesis of intraspecific diversity

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Conclusions - continued

1. Where, when, and how do beetle horns grow?

• Beetle horns develop at least in part similar to
  more typical insect appendages

Hypothesis Beetle horn development is patterned
by some of the same genes that instruct the
development of regular insect appendages
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Overview

1) Where, when, and how do beetle horns grow?
2) Which patterning genes regulate horn
development? 3) Which changes in the genetic
and developmental basis of beetle horns
have mediated the evolution of
horned beetle diversity?
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A conserved gene network patterns a wide
diversity of appendages
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A
A conserved gene network patterns a wide
diversity of appendages
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Distal-less (Dll)

• Structure and function
• expressed typically in distal portion
• of appendage fields
• required for correct set up of p/d axis
• loss of Dll truncates appendages
• ectopic expression of Dll induces
• outgrowth of ectopic limbs

Possible role in horn development Dll may play
a role in specifying the proximo-distal axis of
horn expression expected expression domain in
distal horn regions.
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distal
O. taurus horned prepupa transverse section DAPI
proximal
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distal
O. taurus horned prepupa transverse section DAPI
proximal
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distal
O. taurus horned prepupa transverse
section DAPI Distal-less
proximal
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distal
proximal
proximal
O. taurus early prepupa pupal thoracic
horn sagittal section DAPI extradenticle
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O. taurus early prepupa pupal thoracic
horn sagittal section DAPI dachshund in-situ
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Different patterning genes are expressed in
different horn types
e.g. aristaless (al) expression in thoracic (top)
but not in head horns (bottom)
al
al
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Domain size and location of DLL expression covary
with degree of pupal horn resorption
?
O. nigriventris (no resorption)
p
d
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Two ways to evolve horn dimorphism
evolutionary time
(after Moczek 2004)
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Dll expression in horn rudiments supports
ancestral male horn monomorphism (O. taurus)
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Dll expression in horn rudiments supports
ancestral male horn monomorphism (O.
nigriventris)
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The origin of sexual horn dimorphism different
answers for different types of horns
Thoracic horns
m
f
f
m

• SIMILAR developmental events in both sexes, incl
• i) prepupal horn growth
• ii) expression of patterning genes

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Conclusions
2) Developmental genetics of beetle horns

• Beetle horns evolved via the at least partial
  re-deployment of traditional appendage patterning
  genes.
• Different types of horns rely on at least in part
  different developmental mechanisms and thus may
  have had different and independent evolutionary
  histories.
• Little to no phylogenetic distance is needed for
  the evolution of diverse modifier mechanisms that
  contribute to the differential expression of
  horns within and between species

• Adult morphology is deceptive.....

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(after Emlen et al. 2005, Evolution 59 1060-1084)
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(after Emlen et al. 2005, Evolution 59 1060-1084)
?
?
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(after Emlen et al. 2005, Evolution 59 1060-1084)
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(after Emlen et al. 2005, Evolution 59 1060-1084)
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(after Emlen et al. 2005, Evolution 59 1060-1084)
IMPLICATION maybe one single origin was
followed by differential maintenance of horn
primordia into adulthood
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Why bother growing a thoracic horn if it is not
maintained into adulthood ?
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(Pre)pupal thoracic horns may facilitate
shedding of larval head capsule
DORSAL
1st thorax segment
head
head capsule
VENTRAL
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(Pre)pupal thoracic horns may facilitate
shedding of larval head capsule
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(Pre)pupal thoracic horns may facilitate
shedding of larval head capsule
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Experimental removal of prepupal thoracic horn
results in failure to shed larval head capsule
(the remaining curicle sheds just fine)
control
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Conclusions - general

• Beetle horns and horned beetles offer a rich and
  complex microcosmos for exploring the mechanics
  of innovation and diversification in the natural
  world.

• We need more people to work on these organisms!

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Future directions
Development of RNAi techniques to dissect target
gene function Development of genomic
approaches The interactions of endocrine and
genetic regulators in morphological evolution
PostDoc position available!!! Please contact
armin_at_indiana.edu

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Future directions
How do ecology and development interact to
direct evolutionary diversification?
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Future directions long term
Common themes in innovation and diversification
in the organismal world

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COLLABORATORS D. Rose (Gene expression)
B. Kesselring (Gene expression) A. Shelby
(JH Sex dimorphhism) Michelle McDaniel
(expert beetle care) Melanie ODay (expert
beetle care)
THANKS TO...
FUNDING National Science Foundation
National Institutes of Health Indiana METACyt
Initiative
INSPIRATION H. F. Nijhout L. Nagy D.
Wheeler R. Raff
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Conclusions
1) Where, when, and how do beetle horns grow?

• Horns are of epidermal origin,
• and grow explosively during
• the pre-pupal stage
• Horns are subject to a variable
• degree of size modulation
• during the pupal stage

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Conclusions
1) Where, when, and how do beetle horns grow?

• Intraspecific diversity in adult
• horn expression is the product
• of two discrete processes
• (a, b) differential growth
• during the prepupal stage

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Conclusions
1) Where, when, and how do beetle horns grow?

• Intraspecific diversity in adult
• horn expression is the product
• of two discrete processes
• (a, b) differential growth
• during the prepupal stage

(c, d) differential loss during the pupal stage.

• Species in turn differ in the
• degree to which they rely on
• one or the other mechanism
• in the genesis of intraspecific
• diversity