Conservation, innovation, and the origins of novelty: a case study on horned beetles

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Conservation, innovation, and the origins of
novelty a case study on horned beetles Armin
P. Moczek Indiana University
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In the frequent fits of anger to which the males
especially are subject, the efforts of their
inner feelings cause the fluids to flow more
strongly toward that part of their head in some
there is hence deposited a secretion of horny
matter, and in others of bony matter mixed with
horny matter, which gives rise to solid
protuberances thus we have the origin of horns
and antlers. J. B. Lamarck 1809, Zoological
Philosophy
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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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Beetle horns are major structures
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Beetle horns are ecologically important
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Beetle horns are highly diverse
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Beetle horns are without obvious homology
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Onthophagus rich phenotypic variation on a
variety of levels
3) Between sexes (typically, females are
hornless or develop rudimentary horns)

• 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) When, where, and how do horns grow?
• Regulation of horn growth and patterning
• 3) Regulation and significance of pupal
  remodeling
• The origin(s) of horns and horn diversity
• Current frontiers....

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

• Horns originate during
• prepupal development as
• epidermal outbuddings

(Moczek Nagy, Evolution Development 2005)
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1) Where, when, and how do beetle horns grow?

• Horns originate during
• prepupal development as
• epidermal outbuddings

(Moczek Nagy, Evolution Development 2005)
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1) Where, when, and how do beetle horns grow?

• Horns originate during
• prepupal development as
• epidermal outbuddings

(Moczek Nagy, Evolution Development 2005)
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1) Where, when, and how do beetle horns grow?

• Horns originate during
• prepupal development as
• epidermal outbuddings

• Horn precursors evaginate
• upon pupation and are
• visible externally

(Moczek Nagy, Evolution Development 2005)
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1) Where, when, and how do beetle horns grow?

• Horns originate during
• prepupal development as
• epidermal outbuddings

• Horn precursors evaginate
• upon pupation and are
• visible externally

• Pupa molts into final adult
• phenotype

(Moczek Nagy, Evolution Development 2005)
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1) Where, when, and how do beetle horns grow?

• Horns originate during
• prepupal development as
• epidermal outbuddings

• Horn precursors evaginate
• upon pupation and are
• visible externally

• Pupa molts into final adult
• phenotype

• Intra- and interspecific
• diversity were generally
• assumed to be the result of
• differential prepupal growth

(?)
(Moczek AP, American Naturalist, 2007)
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The significance of pupal remodeling......
Pupal remodeling can generate sexual dimorphism
Example O. binodis
?
?
?
?
(Moczek AP, American Naturalist, 2007)
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The significance of pupal remodeling......
Pupal remodeling can generate reversed sexual
dimorphism
Example O. sagittarius
?
?
?
?
(Moczek AP, American Naturalist, 2007)
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The significance of pupal remodeling......
Pupal remodeling can eliminate sexual dimorphism
Example O. taurus
?
?
?
?
(Moczek AP, American Naturalist, 2007)
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Conclusions

• 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 mechanism in the
  genesis of intraspecific diversity

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Overview

• 1) When, where, and how do horns grow?
• Regulation of horn growth
• and patterning
• 3) The significance of pupal remodeling
• The origin(s) of horns and horn diversity
• Current frontiers....

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Histological observations suggest similarities in
the formation of horns and traditional 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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Target pathways for the origin and
diversification of beetle horns
known functions
pathway
candidate genes
leg gap genes
p/d axis patterning
Dll, dac, exd, hth al
hedgehog / wingless / dpp
appendage initiation
hh, wg, en, dpp, fz, arm
Hox
regional identity
Dfd, Scr, Antp, Ubx, Abd B, lab, pb
EGFR
growth
egf, lim, dlg1, rho, sp, ker, argos
growth, joint formation, cell shape
Notch
N, fng, Dl
Phosphatidylinositol 4-kinase II IRS-1 Insulin
receptor substrate 1 Insulin-like peptide
receptor
Insulin signaling
growth
PCD-5,
Programmed cell death
growth
JH epoxide hydrolase JH acid methyl transferase
Juvenile Hormone signaling
growth
Ec-R, br, usp, E75B, par-1, Ecd-hydroxylase
Ecdysone signaling
growth
MAPK signaling
growth
Ras, MAPKK, MAPK-NK2, MESK2
Onthophagus ortholog cloned
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A conserved gene network patterns p/d axis
formation in a wide diversity of appendages
hh
en
wg
dpp
hth
Dll
dac
EGFR ligands
exd
al bar bb
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A conserved gene network patterns p/d axis
formation in a wide diversity of appendages
hh
en
wg
dpp
hth
Dll
dac
EGFR ligands
exd
al bar bb
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n-EXD, HTH, and DLL show conserved patterns of
expression during horn development.....
d
p
(Moczek, Rose, Sewell, Kesselring, Development
Genes and Evolution 2006)
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...but dachshund (dac) does not
(Moczek, Rose, Sewell, Kesselring, Development
Genes and Evolution 2006)
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AL expression domain matches predicted location
but not relative size
DAPI
d
p
DLL
(Moczek Nagy, Evolution Development 2005)
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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)
d
al
p
al
(Moczek Nagy 2005, Evolution Development)
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Evolution of male horn dimorphism

horn length
horn length (mm)
body size (mm)

-
larval food quantity quality
(Moczek Emlen 2000. Animal Behaviour)
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Two ways to evolve horn dimorphism
evolutionary time
(after Moczek 2006)
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Dll expression in horn rudiments supports
ancestral male horn monomorphism (O. taurus)
(Moczek Nagy, Evolution Development 2005)
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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) Regulation of horn growth and patterning
The evolution of horn development appears to have
been mediated, in part, by the redeployment of
p/d patterning genes DAC expression is
ubiquitous suggesting a lack or loss of function
in horn patterning. Different types of horns
may rely on different developmental
mechanisms and thus may have had different and
independent evolutionary histories. Little to no
phylogenetic distance appeared to have been
necessary for the evolution of diverse modifier
mechanisms that contribute to the differential
expression of horns within and between species

The next step comparative functional analysis of
horn patterning via larval RNAinterference
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RNAinterference down-regulates RNA transcript
abundance in a gene-specific manner
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The first attempt dacRNAi
A)
B)
(i) T2 leg
(i) T3 tibia/tarsi
(ii) antenna
(iii) eye
control
control
-t1
-t1-3
-t2
-t4
-t3
-t4
RNAi
-t5
-t5
RNAi
control
RNAi
- wing margin
RNAi
(Moczek et al. in prep)
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hthRNAi affects horn growth in thorax but not
head
A)
(Moczek et al. in prep)
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Overview

• 1) When, where, and how do horns grow?
• Regulation of horn growth and patterning
• 3) The regulation and significance of pupal
  remodeling
• The origin(s) of horns and horn diversity
• Current frontiers....

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Pupal remodeling of horns (including complete
resorption) is widespread and labile
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Pupal remodeling of insect appendages is
ubiquitous (but rarely involves complete
resorption)
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Pupal remodeling of insect appendages is
ubiquitous (but rarely involves complete
resorption)
But exceptions exist. Example Tussock moth
(Orygia recens) ?-specific degeneration of
pupal wings via massive programmed cell
death (Lobbia et al. 2003, Journal of Insect
Science 311)
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Pupal remodeling of horn involves sex-specific
up-regulation of programmed cell death
(Kijimoto, Andrews Moczek, in prep)
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Candidate genes for the regulation of pupal
remodeling Sex combs reduced (Scr)
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Candidate genes for the regulation of pupal
remodeling Sex combs reduced (Scr)
(Moczek et al. in prep)
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ScrRNAi affects horn remodeling but not growth in
male (but not female) O. nigriventris
horn length (mm)
horn length (mm)
(Moczek et al. in prep)
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ScrRNAis effect on horn remodeling is sex
reversed in O. sagittarius (and only in the
thorax but not head)
(Moczek et al. in prep)
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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
(Moczek, Cruickshank, Shelby, Evolution 2006)
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Experimental removal of prepupal thoracic horn
results in failure to shed larval head capsule
(the remaining curicle sheds just fine)
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Experimental removal of prepupal thoracic horn in
O. binodis results in failure to shed larval head
capsule
O.binodis
control
ablated
ablation treatment
none
sham control
TH horn

p lt 0.01
h e a d c a p s u l e f a i l u r e
0
(28)
(35)
(36)
(Phylogeny after Emlen et al. 2005 Evolution)
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O. gazella
Same effect in O. gazella .....
?
?
(Phylogeny after Emlen et al. 2005 Evolution)
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... but not in the sister genus Oniticellus!
ablated
control
ablation treatment
none
TH horn
h e a d c a p s u l e f a i l u r e
ns
0
(24)
(22)
(Phylogeny after Martin-Piera 2000)
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Conclusions II

• (Pre)pupal thoracic horns may facilitate shedding
  of larval head capsule

• If correct, this may explain maintenance of horn
  development in larvae despite absence of horns in
  adults

• Dual function of prepupal horn growth may be
  unique to the genus Onthophagus

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Are beetle horns an exaptation?
Could beetle horns have evolved initially as an
adaptation to larval/pupal development, and only
secondarily have been co-opted to become
precursors to adult traits?
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Pupae in Scarabaeinae are famous for (i) their
pupal support structures and (ii) resorbing
them prior to the adult molt
Implications
Pupal outgrowths and their resorption are likely
ancestral to adult horns
Adult horns may have originated through a simple
failure to remove pupal outgrowths prior to
pupal-adult molt (e.g. vial failure to activate
PCD)
Evidence
Anecdotal failure-to-remove in extant species
(Moczek, Cruickshank, Shelby, Evolution 2006
images courtesy of Alberto Ballerio, University
of Turin)
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Now you see me, now you dont Pterorthochaetes
insularis
(Moczek, Cruickshank, Shelby, Evolution 2006
images courtesy of Alberto Ballerio, University
of Turin)
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Now you see me, now you dont Onthophagus vacca
?
?
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Current frontiers
The major challenges I
From correlational gene expression to gene
function analysis
From genes to genomes
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Dungbeetleonomics
Collaborators Teiya Kijimoto, Justen Andrews,
Emilie Snell-Rood, Amy
Cash, IU
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Dungbeetleonomics
Collaborators Teiya Kijimoto, Justen Andrews,
Emilie Snell-Rood, Amy Cash, IU

• 2,781 unique Onthophagus sequences (on average
• 900 bp fragment size lt 1 redundancy gt 65
• with initiating Methionine
• 693 ESTs without significant sequence similarity
• to existing data bases (but 335 with ORF gt 100
  AA)
• most major developmental functions, signaling
• pathways, and endocrine regulatory pathways are
• represented

(Kijimoto et al. Genome Biology, in review)
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Dungbeetleonomics
Collaborators Teiya Kijimoto, Justen Andrews,
Emilie Snell-Rood, Amy Cash, IU
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Dungbeetleonomics
Collaborators Teiya Kijimoto, Justen Andrews,
Emilie Snell-Rood, Amy Cash, IU
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Dungbeetleonomics
Collaborator Yoram Yerushalmi, U. of Arizona
O. taurus 2D SDS page Proteins expressed in
hornless male female both
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The big picture
The major challenges II
Microevolution of horn development
Macroevolution of horn development
Behavioral ecology of horned beetles
Endocrine regulation of horns and horn diversity
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Behavioral ecology of horned beetles
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Behavioral ecology of horned beetles
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Behavioral ecology of horned beetles
b) hornless males sneak
Drawing by Barrett Klein
(after Moczek 2005, BioScience)
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How does behavioral ecology guide morphological
evolution?
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How do endocrine mechanisms mediate between
diverging ecologies and morphological evolution?
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The big picture
The major challenges II
Microevolution of horn development
Macroevolution of horn development
Behavioral ecology of horned beetles
Endocrine regulation of horns and horn diversity
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Conclusions - general

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

• We need more people to work on these organisms!

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THANKS TO...
COLLABORATORS
Evolutionary Developmental Genetics Debra
Rose, Teiya Kijimoto Bethany Kesselring
FUNDING
Genomics Justen Andrews, Teiya Kijimoto Amy Cash,
Emilie Snell-Rood
Phylogenetics Tami Cruickshank
Proteonomics Yoram Yerushalmi
INSPIRATION H. F. Nijhout L. Nagy D.
Wheeler R. Raff
Endocrinology Andrew Shelby, Mary Ann
Rankin, Tina Taub-Montemayor
Speciation and the evolution of size shape
Harald Parzer, Joseph Tomkins, Astrid Pizzo,
Antonio Rolando