Title:'The'origin'of'the'Hox/ParaHox'genes,'the'Ghost'Locus'Hypothesis'and'the'
complexity'of'the'first'animal'
!
David.!E.K.!Ferrier!
The!Scottish!Oceans!Institute,!Gatty!Marine!Laboratory,!University!of!St!Andrews,!East!
Sands,!St!Andrews,!Fife,!KY16!8LB,!UK.!
!
[email protected]!
tel.!(0)1334!463480!
!
Author'biography'
!
David!E.K.!Ferrier!is!a!Senior!Lecturer!in!the!Gatty!Marine!Laboratory,!University!of!St!
Andrews,!whose!research!group!focuses!on!animal!evolutionary!developmental!
genomics.!
!
!
Keywords:!animal!evolution,!homeobox!genes,!animal!phylogeny,!Ediacaran.!
!
Summary'points'
!
I!Differential!gene!loss!has!had!an!important!role!in!animal!evolution.!
I!Origin!of!the!Hox/ParaHox!developmental!control!genes!occurred!before!the!origin!of!
the!poriferan!lineage.!
!
1!
I!Differential!gene!loss!of!Hox/ParaHox!genes!in!basal!lineages!makes!it!difficult!to!
determine!whether!these!genes!arose!from!the!ProtoHox!state!in!the!last!common!
ancestor!of!all!animals,!or!slightly!higher!in!the!animal!phylogeny.!
I!The!last!common!ancestor!of!animals!was!genetically,!and!possibly!morphologically,!
more!complex!than!previously!appreciated.!
I!The!nature!of!the!first!animal!can!only!be!deduced!by!integrating!comparative!
developmental!biology,!phylogenetics,!evolutionary!genomics!and!palaeontology.!
!
!
!
!
2!
Abstract'
!
A!key!aim!in!evolutionary!biology!is!to!deduce!ancestral!states!in!order!to!better!
understand!the!evolutionary!origins!of!clades!of!interest!and!the!diversification!
process(es)!that!have!elaborated!them.!These!ancestral!deductions!can!hit!difficulties!
when!undetected!loss!events!are!misinterpreted!as!ancestral!absences.!With!the!everI
increasing!amounts!of!animal!genomic!sequence!data!we!are!gaining!a!much!clearer!
view!of!the!preponderance!of!differential!gene!losses!across!animal!lineages.!This!has!
become!particularly!clear!with!recent!progress!in!our!understanding!of!the!origins!of!the!
Hox/ParaHox!developmental!control!genes!relative!to!the!earliest!branching!lineages!of!
the!animal!kingdom:!the!sponges!(Porifera),!comb!jellies!(Ctenophora)!and!placozoans!
(Placozoa).!These!reassessments!of!the!diversity!and!complexity!of!developmental!
control!genes!in!the!earliest!animal!ancestors!need!to!go!handIinIhand!with!
complementary!advances!in!comparative!morphology,!phylogenetics!and!palaeontology!
in!order!to!clarify!our!understanding!of!the!complexity!of!the!last!common!ancestor!of!
all!animals.!The!field!is!currently!undergoing!a!shift!from!the!traditional!consensus!of!a!
spongeIlike!animal!ancestor!from!which!morphological!and!molecular!elaboration!
subsequently!evolved,!to!a!scenario!of!a!more!complex!animal!ancestor,!with!subsequent!
losses!and!simplifications!in!various!lineages.!
!
Introduction'
!
Hox/ParaHox!genes!are!a!distinctive!type!of!developmental!control!gene!in!animals,!
encoding!transcription!factors,!which!form!a!subset!of!the!ANTP!class!of!homeobox!
genes![1,2].!They!are!renowned!for!their!clustered!organisation!and!for!exhibiting!
!
3!
Colinearity!–!gene!order!in!the!cluster!corresponding!to!gene!activation!(which!can!take!
various!forms!–!spatial,!temporal,!quantitative,!virtual[3I7]),!although!this!is!not!
universal!and!plenty!of!examples!exist!of!Hox!and!ParaHox!genes!that!are!no!longer!
clustered!and!hence!do!not!exhibit!Colinearity![8I10].!Despite!this!diversity!in!
organisation,!or!in!some!cases!because!of!it,!the!Hox!(and!to!a!currently!lesser!extent!the!
ParaHox)!genes!have!become!established!as!a!paradigm!for!studying!gene!regulation!in!
gene!clusters,!as!well!as!a!major!focus!for!animal!evolutionary!developmental!biology!
(evoIdevo)!because!of!the!integral!roles!that!these!genes!have!in!constructing!animal!
form!and!its!diversity.!The!evolutionary!origin!of!such!genes!is!thus!of!implicit!interest.!
!
In!the!early!days!of!molecular!evoIdevo!the!Hox!genes!were!proposed!to!have!been!a!
central!element!of!the!Zootype!–!a!defining!aspect!of!being!an!animal!(metazoan)![11].!
However,!a!closer!look!at!the!earliest!branching!lineages!of!the!animal!kingdom!revealed!
that!some!lineages!seem!to!lack!Hox/ParaHox!genes!completely!and!it!was!concluded!
that!the!origin!of!the!Hox/ParaHox!genes!occurred!some!time!after!the!origin!of!the!
animal!kingdom![12I14].!Until!recently!this!was!based!on!searches!involving!approaches!
such!as!PCR!and!genomic!or!cDNA!library!screening,!and!so!there!was!always!the!
possibility!that!genes!could!have!been!missed!due!to!technical!reasons!(rather!than!
biological!–!i.e.!absence!from!the!genome).!With!the!advent!of!whole!genome!sequencing!
and!the!rapid!decrease!in!sequencing!costs,!along!with!improved!bioinformatics!
capabilities!and!sequencing!technologies,!the!search!for!Hox/ParaHox!genes!has!shifted!
to!searching!through!whole!genome!sequences.!Genes!can!still!be!missed!by!such!
searches,!due!to!the!inevitable!gaps!in!draft!genome!sequences,!however,!these!searches!
based!on!whole!(or!nearly!whole)!genome!sequences!are!clearly!much!more!thorough!
than!the!older!PCR/library!screening!approaches.!
!
4!
!
The!earliest!examples!of!whole!genome!sequences!from!“basal”!(early!branching)!
animal!lineages!enforced!the!previous!views!that!Hox/ParaHox!genes!were!absent!from!
the!earliest!stages!of!animal!evolution!(including!poriferans!and!ctenophores).!In!this!
context,!the!name!ParaHoxozoa!was!proposed!to!distinguish!all!of!the!animal!phyla!that!
contained!Hox!and/or!ParaHox!genes!from!the!earliest!lineages!(Porifera,!Ctenophora)!
that!supposedly!lacked!these!genes![14].!Note!that!terms!like!‘basal!lineages’!or!‘earliestI
branching!lineages’!do!not!imply!that!the!extant!organisms!at!the!termini!of!these!
lineages!are!necessarily!representative!of!the!ancestral!forms!at!the!nodes!from!which!
these!lineages!evolved.!An!example!that!illustrates!this!point!is!that!of!the!Ctenophora!
discussed!below,!for!which!extant,!modernIday!forms!are!likely!to!be!very!different!from!
the!forms!on!the!lineage!stem!soon!after!it!split!from!the!lineage!giving!rise!to!the!rest!of!
the!animal!phyla.!
!
In!what!follows!I!will!summarize!how!this!has!dramatically!changed!very!recently:!a!
ParaHox!gene!has!been!discovered!in!poriferans!and!wider!genome!sequencing!is!
enhancing!our!realisation!that!differential!gene!loss!is!rife!between!animal!lineages,!
producing!Ghost!Hox!and!ParaHox!Loci!for!example![15,16].!This!has!important!
implications!for!our!reconstructions!of!ancestral!states!for!animals!and!consequently!
our!understanding!of!the!origin!of!the!animal!kingdom!as!a!whole!and!what!the!very!
first!animal!(or!at!least!the!last!common!ancestor!of!current!animals)!was!like!in!terms!
of!its!genome!content.!Furthermore,!if!we!can!deduce!the!developmental!control!genes!
that!were!present!and!understand!what!these!genes!were!doing!and!building!(e.g.!by!
comparison!of!the!roles!of!these!genes!in!extant!animals),!we!will!almost!certainly!have!
to!revise!our!view!of!the!appearance!of!this!ancient!ancestor.!
!
5!
!
The'origin'of'the'Hox/ParaHox'gene'clusters'from'a'ProtoHox'state'
!
In!1998!Brooke!et!al![17]!revolutionized!our!understanding!of!the!origin!of!the!Hox!
genes!following!their!discovery!of!the!ParaHox!cluster!in!amphioxus!(Branchiostoma,
floridae).!They!hypothesized!the!ancestral!existence!of!a!ProtoHox!cluster!that!gave!rise!
to!the!Hox!and!ParaHox!clusters!via!a!wholeIcluster!duplication.!Prior!to!the!discovery!
of!the!ParaHox!cluster!the!consensus!was!that!the!Hox!cluster!was!unique!and!had!
evolved!via!a!series!of!tandem!gene!duplications!in!an!ancestral!animal.!The!Hox!cluster!
has!then!been!conserved!in!many!presentIday!animals!(but!with!various!further!lineageI
specific!changes,!including!extra!gene!duplications,!losses!and!rearrangements!in!
distinct!species).!However,!the!ProtoHox!hypothesis!separated!the!stage!of!initial!
tandem!gene!duplications!from!the!origin!of!the!Hox!cluster!by!positing!that!a!ProtoHox!
gene!cluster!was!generated!in!the!first!instance!and!that!this!ProtoHox!cluster!then!
underwent!a!change!that!gave!rise!to!both!the!Hox!and!ParaHox!clusters!simultaneously!
[13,!17,!18].!In!the!original!formulation!of!the!ProtoHox!hypothesis!this!transition!from!
the!ProtoHox!state!involved!a!wholeIcluster!duplication![17].!Subsequent!variations!of!
the!ProtoHox!hypothesis!have!debated!whether!this!cluster!duplication!was!a!trans!or!
cis!duplication,!or!whether!it!was!not!a!duplication!at!all!but!instead!involved!splitting!a!
cluster!into!two![18I20].!
!
Regardless!of!which!of!these!specific!scenarios!reflects!the!truth,!all!involve!relatively!
largeIscale!rearrangement!events!involving!multiple!Hox/ParaHox!genes!and!associated!
neighbouring!genes.!Several!types!of!duplications!and!rearrangements!that!are!known!
to!be!prevalent!in!the!evolution!of!genome!architecture!are!consistent!with!scenarios!of!
!
6!
ProtoHox!to!Hox/ParaHox!evolution.!These!include!such!things!as!whole!genome!or!
whole!chromosome!duplication!followed!by!gene!losses!around!the!new!distinct!Hox!
and!ParaHox!loci,!so!that!distinctive!neighbourhoods!(in!terms!of!gene!content)!result!
around!the!Hox!versus!ParaHox!loci!(Fig.1A).!Alternatively,!the!ProtoHox!cluster!could!
have!been!within!a!segmental!duplication,!the!vast!majority!of!which!occur!
intrachromosomally!at!their!point!of!origin!(at!least!in!genomes!of!those!extant!animals!
that!have!been!studied!(reviewed!in![21]).!Or!a!ProtoHox!cluster!containing!distinct!
precursors!of!both!Hox!and!ParaHox!genes!could!have!been!split!by!an!inversion!event.!
If!these!clusters!were!formed!by!either!an!intrachromosomal!segmental!duplication!or!
an!inversion,!the!newly!separated!Hox!and!ParaHox!clusters!will!of!initially!existed!on!
the!same!chromosome!and!thus!be!linked!to!the!same!neighbouring!genes!(Fig.1B).!To!
arrive!at!the!situation!found!in!extant!animals,!with!the!Hox!and!ParaHox!clusters!
located!on!different!chromosomes!and!surrounded!by!distinctive!gene!neighbourhoods,!
a!chromosome!fission!or!an!interchromosomal!arm!exchange!could!have!occurred!
(Fig.1B).!Such!mutations!again!are!very!common!in!modern!day!animal!genomes!(e.g.!
[22]).!In!contrast!to!the!first!possible!scenario!(of!whole!genome!or!chromosome!
duplication!followed!by!differential!gene!losses!around!the!Hox!and!ParaHox!loci)!the!
second!scenario!of!separated!Hox!and!ParaHox!clusters!on!the!same!chromosome!
followed!by!an!interchromosomal!arm!exchange!or!chromosome!fission!will!
immediately!result!in!distinctive!gene!neighbourhoods!around!the!Hox!and!ParaHox!loci!
without!the!need!for!differential!gene!losses.!There!are,!of!course,!alternative!routes!to!
the!Hox!and!ParaHox!clusters!being!on!different!chromosomes!and!surrounded!by!
distinctive!gene!neighbourhoods,!but!these!alternative!routes!are!less!likely!to!have!
occurred!judging!from!the!relative!rates!of!the!required!mutational!events!when!
compared!to!those!described!above!(as!discussed!in![15],!and!see!below).!
!
7!
!
_____________________________________________________________________________________________________!
Figure'1.'
Likely'potential'routes'to'the'origin'of'the'Hox'and'ParaHox'clusters'on'distinct'
chromosomes'after'evolution'from'a'ProtoHox'cluster.!Black!rectangles!represent!
the!homeobox!genes!of!the!ProtoHox,!ParaHox!and!Hox!clusters.!Triangles!represent!
genes!that!neighboured!(were!linked!to)!the!ProtoHox!cluster!and!then!became!Hox!
cluster!neighbours,!whilst!ovals!represent!genes!that!neighboured!(were!linked!to)!the!
ProtoHox!cluster!and!then!became!ParaHox!cluster!neighbours.!Note,!the!‘triangles’!and!
‘ovals’!do!not!take!on!their!specific!Hox!or!ParaHox!loci!identities!until!the!Hox!and!
ParaHox!clusters!have!arisen!and!separated!onto!distinct!chromosomes!and!any!
necessary!gene!losses!have!occurred!to!make!the!Hox!and!ParaHox!neighbours!largely!
mutually!exclusive!(shown!as!black!triangles!and!ovals!in!the!figure).!Any!paralogous!
genes!that!are!affiliated!with!both!Hox!and!ParaHox!loci!are!not!considered!here!(e.g.!
collagens!and!tyrosine!kinase!receptor!genes![19]).!(A)!WGD!=!Whole!Genome!
Duplication,!WCD!=!Whole!Chromosome!Duplication,!X!=!gene!loss,!(B)!SD!=!Segmental!
Duplication,!and!“cluster!split*”!involves!the!number!of!homeobox!genes!in!the!
ProtoHox!cluster!increasing!to!form!the!precursors!to!both!the!Hox!and!ParaHox!gene!
families!prior!to!the!ProtoHox!cluster!being!split,!which!is!likely!to!initially!result!in!the!
Hox!and!ParaHox!first!being!linked!due!to!the!split!initially!being!intrachromosomal!
prior!to!some!form!of!translocation!to!distribute!the!clusters!onto!distinct!
chromosomes.!Note,!it!is!possible!that!a!prior!intrachromosomal!inversion!event!is!not!
required!if!the!interchromosomal!arm!exchange!happened!to!have!a!break!precisely!
within!the!ProtoHox!cluster.!Whilst!this!is!formally!possible,!the!likelihood!intuitively!
seems!to!be!low.!
!
8!
_____________________________________________________________________________________________________!
!
!
Alternatives'to'the'ProtoHox'cluster'producing'Hox'and'ParaHox'clusters,'and'
why'these'are'less'likely'
!
1)!Secondary!coming!together!of!homeobox!genes!
!
An!alternative!to!the!ProtoHox!to!Hox/ParaHox!hypothesis!is!that!the!homeobox!genes!
came!together!secondarily,!to!separately!form!distinct!Hox!and!ParaHox!clusters!that!
both!then!evolved!Colinearity,!rather!than!being!generated!by!a!whole!cluster!
duplication!or!split.!This!view!has!never!gained!much!traction,!perhaps!because!the!
sequence!relationships!between!the!Hox!and!ParaHox!genes!imply!that!they!did!
originate!by!duplications!from!each!other.!Since!by!far!the!most!prevalent!mode!of!gene!
duplication!is!tandem!duplication!(reviewed!in![21])!then!the!alternative!of!Hox!and!
ParaHox!genes!coming!together!secondarily!would!most!likely!have!required!that!the!
genes!initially!were!adjacent,!then!separated!and!then!came!together!again.!This!strikes!
most!people!as!less!parsimonious!than!the!ProtoHox!to!Hox/ParaHox!explanation,!
particularly!when!the!independent!evolution!of!Colinearity!also!needs!to!be!invoked.!
!
However,!this!alternative!must!be!kept!in!mind!in!case!it!eventually!turns!out!that!there!
is!a!mechanistic!basis!that!could!have!enabled!ordered!clusters!to!form!secondarily!and!
independently.!Such!mechanisms!are!not!yet!known.!Note!that!such!ordered!clusters!are!
distinct!from!the!looser!coming!together!of!functionally!related!or!coIregulated!genes!
into!distinct!regions!of!some!genomes![23]!or!the!unordered!association!of!genes!into!
!
9!
Topologically!Associating!Domains!(TADs)![24].!Nevertheless,!there!are!some!intriguing!
pieces!of!data!that!hint!at!the!possible!existence!of!mechanisms!underpinning!a!
secondary!coming!together!into!highly!ordered!clusters.!
!
Secondary!coming!together!of!homeobox!genes,!at!least!along!the!same!chromosome!
(i.e.!intrachromosomally),!appears!to!have!happened!in!the!case!of!the!NK!genes!in!
distinct!Drosophila!lineages![25].!Also,!this!time!interchromosomally,!the!Post1!Posterior!
Hox!gene!of!Platynereis,dumerilii!has!translocated!out!of!the!Hox!gene!cluster!and!off!the!
HoxIbearing!chromosome,!and!of!all!of!the!other!chromosomes!that!it!could!have!moved!
to!(P.,dumerilii!2n=28)!it!has!secondarily!become!linked!to!the!NK!cluster!genes![26].!
Finally,!Duboule![27]!has!argued!that!the!vertebrate!Hox!clusters!have!been!
consolidated!during!evolution!and!secondarily!became!more!highly!ordered!via!the!
evolution!of!regulatory!mechanisms!that!may!well!be!specific!to!this!group!of!animals.!
This!uncertainty!about!the!evolutionary!dynamics!effecting!the!distribution!of!
duplicated!genes!across!genomes!needs!to!be!addressed!with!wider!taxon!sampling!and!
reconstruction!of!homeobox!clusters!within!the!context!of!their!associated!
chromosomeIscale!neighbourhoods.!In!addition,!deeper!understanding!of!the!regulatory!
mechanisms!operating!across!these!homeobox!gene!clusters!is!also!required.!
!
2)!ProtoHox!to!Hox/ParaHox!involving!smallIscale!transposition!
!
The!types!of!chromosome!rearrangements!associated!with!hypotheses!about!the!origin!
of!the!Hox!and!ParaHox!clusters!were!outlined!above,!namely!whole!genome!or!
chromosome!duplications!(followed!by!differential!gene!loss)!or!intrachromosomal!
duplications!followed!by!chromosome!arm!exchange!or!fission.!The!plausibility!and!the!
!
10!
prevalence!of!these!types!of!mutations!underpin!the!logic!of!the!Ghost!Locus!hypothesis,!
whereby!the!genomic!neighbourhoods!of!these!homeobox!genes!can!be!used!to!trace!
their!evolution![15].!Other!types!of!gene!duplications!and!translocations!do,!however,!
exist.!But!it!is!the!much!lower!rates!of!these!other!mutations!or!their!less!appropriate!
natures!(when!considering!the!overall!biology!of!the!Hox!and!ParHox!genes)!that!make!
them!much!less!likely!to!have!been!involved!in!Hox/ParaHox!evolution.!
!
In!the!original!formulation!of!the!Ghost!Locus!hypothesis!MendivilIRamos!and!
colleagues![15]!(see!also![21])!outlined!the!data!that!showed!the!much!lower!levels!or!
inappropriateness!of!these!alternative!mutations,!including!retrotransposition!and!
smallIscale,!DNAImediated!interchromosomal!translocations!at!the!point!of!duplicate!
origin!(see!Supplemental!Information!in![15]).!The!data!is!inevitably!drawn!mainly!from!
species!for!which!highIquality!genome!sequences!are!available!that!are!reliably!
assembled!to!the!chromosome!scale,!in!order!to!distinguish!intrachromosomal!from!
interchromosomal!locations.!This!currently!tends!to!restrict!the!analyses!to!
conventional!model!systems!like!drosophilids,!nematodes!and!some!vertebrates!like!
humans.!In!the!intervening!time!since!the!original!formulation!of!the!Ghost!Locus!
hypothesis,!further!data!has!provided!additional!support.!A!recent!example!is!the!
analysis!of!duplications!in!the!human!genome![28].!
!
In!their!analysis!of!young!segmental!duplications!in!humans,!Bu!and!Katju![28]!found!
that!intrachromosomal!duplications!were!clearly!most!prevalent,!accounting!for!100%!
of!the!youngest!category,!which!presumably!contains!the!duplications!closest!to!their!
point!of!origin.!Also,!human!segmental!duplication!sizes!are!larger!than!previously!noted!
([28];!see!Supplementary!Information!of![15]!and!references!therein),!but!are!still!on!the!
!
11!
small!side!when!considering!that!duplication!of!a!multigene!array!is!statisticallyI
speaking!most!likely!in!the!case!of!ProtoHox!to!Hox/ParaHox![29].!It!is!not!clear,!
however,!that!duplication!processes!in!humans!are!typical!for!animals!as!a!whole![30,!
31]!due!to!an!apparent!elevation!of!the!activity!of!mobile!elements!at!some!point!in!
primate!evolution.!This!logic!can,!of!course,!be!extended!to!the!last!common!ancestor!of!
all!animals!whose!genome!rearrangements!may!have!occurred!in!a!very!different!way!
from!presentIday!animals.!It!is!to!be!hoped!that!improved!sequencing!technologies!and!
assembly!algorithms!will!increase!the!taxonomic!range!of!wellIassembled!genomes!so!
that!more!generally!applicable!rates!for!the!various!genome!rearrangement!mutations!
can!be!deduced,!in!order!to!better!assess!the!logic!underlying!ideas!like!the!Ghost!Locus!
hypothesis!for!Hox/ParaHox!origins.!
!
Whilst!we!must!obviously!adopt!the!standard!scientific!approach!of!following!the!most!
likely!or!probable!hypothesis,!which!in!this!case!clearly!involves!the!types!of!mutations!
invoked!by!the!Ghost!Locus!hypothesis,!it!formally!remains!possible!that!an!alternative!
route!to!the!separation!of!Hox!and!ParaHox!genes!onto!distinct!chromosomes!may!have!
occurred.!In!this!vein,!there!are!intriguing!instances!of!multigene!interchromosomal!
translocations!having!occurred,!including!the!MEDEA!transposable!element!in!beetles!
including!several!integral!genes![32]!or!circular!DNA!translocations!in!cattle!spanning!
several!hundred!kilobases!or!possibly!moving!single!genes!like!the!vasa!genes!of!Tilapia!
[33,!34].!The!prevalence!of!such!phenomena!and!their!relative!rates!compared!to,!for!
example,!intrachromosomal!duplications,!requires!further!investigation!before!they!can!
be!reasonably!invoked!as!a!plausible!route!to!the!origin!of!the!Hox!and!ParaHox!clusters.!
!
The'sponge'revolution'
!
12!
!
These!plausible!genome!rearrangement!scenarios,!based!on!commonly!occurring!types!
of!mutations,!form!the!background!to!the!discovery!of!the!Hox!and!ParaHox!Ghost!Loci,!
in!which!distinct!Hox!and!ParaHox!neighbourhoods!can!be!detected!in!an!animal!like!the!
sponge!Amphimedon,queenslandica,!even!though!these!loci!do!not!contain!the!
Hox/ParaHox!homeobox!gene!themselves![15].!The!idea!of!Ghost!Loci!first!arose!from!
consideration!of!the!placozoan,!Trichoplax,adhaerens,!which!contains!a!single!
Hox/ParaHoxIlike!gene!(Trox<2)!that!was!of!debated!affinity:!it!was!either!viewed!as!a!
ParaHox!gene![35]!or!as!a!direct!descendant!of!a!ProtoHox!state![36,!37],!this!second!
hypothesis!requiring!subsequent!asymmetric!evolution!of!the!Hox/ParaHox!genes!to!
account!for!the!clear!grouping!of!Trox<2!with!the!Gsx!family!of!ParaHox!genes.!Since!
asymmetric!evolution!of!duplicated!genes!is!known!to!be!common!(e.g.![38I41]),!then!
the!molecular!phylogenetic!trees!struggle!to!distinguish!between!these!two!alternative!
and!plausible!hypotheses!for!the!identity!of!Trox<2.!
!
An!alternative!approach!to!distinguishing!whether!Trox<2!was!a!ParaHox!or!ProtoHox!
gene!was!required.!MendivilIRamos!and!colleagues![15]!used!a!synteny!approach!based!
on!the!evolutionary!scenario!outlined!above!for!the!origin!of!the!Hox!and!ParaHox!
clusters!from!the!ProtoHox!state,!which!entails!common,!largeIscale!(multigenic)!
chromosomal!rearrangement!mutational!events!that!lead!to!distinctive!gene!
neighbourhoods!around!the!newly!formed!Hox!and!ParaHox!clusters.!Another!key!
finding!that!enables!this!synteny!approach!is!that!gene!neighbourhoods!(synteny)!are!
conserved!in!some!lineages!from!these!ancient!evolutionary!times,!including!the!last!
common!ancestor!of!cnidarians!and!bilaterians![42]!and!the!last!common!ancestor!of!
placozoans!and!bilaterians![43].!
!
13!
!
Based!on!the!detectable!(statistically!significant)!conserved!synteny!between!T.,
adhaerens!and!the!last!common!cnidarianIbilaterian!ancestor,!MendivilIRamos!and!
colleagues![15]!found!that!Trox<2!resided!in!a!ParaHox!neighbourhood,!and!hence!on!the!
basis!of!synteny!as!well!as!the!previous!molecular!phylogenetic!trees!Trox<2!is!a!
ParaHox!gene.!However,!under!the!ProtoHox!to!Hox/ParaHox!hypothesis!one!expects!
that!an!animal!with!a!ParaHox!locus!should!also!have!a!Hox!locus,!and!so!the!lack!of!a!
Hox!gene!from!T.,adhaerens!to!complement!the!Trox<2!ParaHox!gene!was!problematic.!
This!was!solved!by!using!the!Putative!Ancestral!Linkage!groups!(PALs)!of!the!last!
common!cnidarianIbilaterian!ancestor!generated!by!Putnam!and!colleagues![42],!which!
contained!a!list!of!genes!that!neighboured!the!Hox!cluster!in!this!ancestor.!MendivilI
Ramos!and!colleagues![15]!could!clearly!detect!orthologues!of!many!of!these!last!
common!cnidarianIbilaterian!ancestor!Hox!neighbours!in!T.,adhaerens.!Also,!more!
importantly,!they!are!clustered!in!a!distinct!region!of!the!placozoan!genome,!which!even!
though!it!does!not!contain!a!Hox!gene!clearly!is!homologous!to!the!Hox!neighbourhoods!
of!the!last!common!cnidarianIbilaterian!ancestor!and!extant!cnidarians!and!bilaterians.!
The!Hox!neighbourhood!of!T.,adhaerens!is!thus!a!Ghost!Hox!Locus.!
!
With!the!discovery!of!the!placozoan!Ghost!Hox!Locus!and!lists!of!genes!in!Hox!and!
ParaHox!neighbourhoods,!or!PALs,!of!the!last!common!ancestor!of!placozoans!and!
cnidarians/bilaterians,!the!issue!of!the!absence!of!Hox!and!ParaHox!genes!from!sponges!
could!be!addressed!afresh.!In!the!first!sponge!genome!to!be!fully!sequenced,!that!of!A.,
queenslandica![44],!the!orthologues!of!these!Hox!and!ParaHox!neighbours!clustered!into!
two!distinct!regions!of!the!genome!to!statistically!significant!levels![15].!A.,queenslandica!
thus!appears!to!have!distinct!Ghost!Hox!and!ParaHox!loci.!
!
14!
!
This!Ghost!Locus!hypothesis,!involving!loss!of!Hox!and!ParaHox!genes!from!a!basal!
lineage!like!the!sponge!A.,queenslandica,!received!a!major!boost!with!the!subsequent!
discovery!of!different!species!of!poriferans!that!had!retained!a!ParaHox!gene.!The!
calcisponges!Sycon,ciliatum!and!Leucosolenia,complicata!were!found!to!contain!Cdx!
genes![16].!The!principal!line!of!evidence!supporting!this!assignment!of!these!
calcisponge!genes!to!the!Cdx!family!came!from!molecular!phylogenetic!trees.!When!
dealing!with!such!trees,!which!are!built!from!the!60Iamino!acid!homeodomain,!node!
support!values!can!be!low!due!to!the!unavoidable!use!of!such!short!sequences,!
particularly!when!including!sequences!from!basal!animal!lineages.!Nevertheless,!the!
node!support!values!in!a!number!of!the!trees!in!Fortunato!et,al![16]!were!at!levels!that!
are!conventionally!considered!to!be!significant.!Confidence!in!the!assignment!can!be!
boosted,!despite!occasional!low!support!values,!when!the!affiliation!between!the!sponge!
gene!of!interest!and!a!known!homeobox!family!arises!repeatedly!when!trees!are!
constructed!with!different!phylogenetic!approaches!(with!their!inherent!different!
assumptions!and!pitfalls),!varied!taxon!sampling!and!when!focusing!on!particular!
families!that!possess!amino!acid!residues!that!seem!to!be!relatively!distinctive!for!
certain!families.!Finally,!building!trees!with!and!without!difficultItoIclassify,!longIbranch!
sequences!from!basal!lineages!such!as!sponges!and!checking!the!stability!of!the!topology!
with!respect!to!the!assignment!of!the!gene!of!interest!also!helps!to!establish!confidence!
in!the!classification.!Fortunato!and!colleagues![16]!performed!all!of!these!checks!and!
consistently!found!that!the!calcisponge!genes!of!interest!grouped!with!the!Cdx!family.!!
!
Consistent!with!this!Cdx!identity,!S.,ciliatum!also!possesses!distinct!Hox!and!ParaHox!
neighbourhoods!in!its!genome!(the!L.,complicata!genome!assembly!was!not!suited!to!the!
!
15!
analysis).!Also,!intriguingly,!the!SAR1!gene!is!a!close!neighbour!of!both!S.,ciliatum,Cdx!
and!the!ParaHox!gene!cluster!of!the!cnidarian!Nematostella,vectensis![16,!45],!and!
another!S.,ciliatum,Cdx!neighbour,!PPWD1,!is!also!a!ParaHox!neighbour!in!some!
bilaterians!(including!humans).!Although!there!are!not!enough!neighbours!on!the!S.,
ciliatum,Cdx,scaffold!to!assess!a!ParaHox!affinity!statistically,!they!clearly!do!not!
contradict!the!Cdx!classification,!but!instead!provide!some!intriguing!potential!ParaHox!
associations.!Clearly,!future!work!involving!improvements!to!the!S.,ciliatum!and!L.,
complicata!assemblies!could!help!to!increase!the!number!of!known!neighbours!to!the!
Cdx!gene!so!that!a!valid!statistical!test!can!be!performed,!and!further!taxon!sampling!
may!find!still!further!species!that!have!retained!ParaHox!(or!Hox)!genes.!
!
The!origin!of!the!Hox/ParaHox!genes!in!their!distinct!genomic!neighbourhoods!has!thus!
been!pushed!deeper!in!animal!evolution!than!the!divergence!of!the!poriferan!lineage!
from!the!rest!of!the!animals.!Adopting!the!consensus!opinion!at!the!time,!that!the!
Porifera!were!monophyletic!and!also!were!the!basalImost!lineage!of!animals![46],!led!to!
the!conclusion!that!the!last!common!ancestor!of!the!animals!thus!had!distinct!Hox!and!
ParaHox!genes![15,!16].!Determining!whether!this!sponge!data!really!does!indicate!the!
state!in!the!last!common!ancestor!of!all!animals,!or!instead!leads!us!to!the!state!of!an!
ancestor!one!or!two!nodes!above!the!origin!of!the!animal!kingdom,!is!intimately!
connected!to!the!phylogeny!of!the!basal!lineages!of!the!animal!kingdom.!
!
Phylogeny'of'the'basal'lineages'of'the'animal'kingdom'
!
A!recent!major!area!of!debate!in!the!field!of!animal!phylogeny!has!been!about!which!of!
the!Porifera!or!Ctenophora!constitute!the!basalImost,!earliest!branching!lineage!in!the!
!
16!
animal!kingdom![46I52].!The!latest!contribution!to!this!debate,!which!represents!the!
most!thorough!analysis!so!far!with!increased!taxon!sampling!and!careful!testing!for!
various!possible!sources!of!systematic!error!(such!as!the!misleading!signal!contributed!
to!many!studies!by!the!prevalent!ribosomal!proteins),!robustly!locates!the!Ctenophora!
as!the!basalImost!animal!lineage![51].!Whelan!and!colleagues![51]!also!resolved!
poriferans!as!monophyletic!rather!than!paraphyletic,!which!has!been!another!recent!
area!of!debate.!With!regards!to!the!origin!of!the!Hox/ParaHox!genes,!whether!they!arose!
before!or!after!the!origin!of!the!Ctenophora!remains!to!be!seen!(Fig.!2).!A!mention!of!a!
Cdx!gene!in!the!Supplementary!Information!of![50]!requires!closer!examination!to!test!
its!veracity,!and!hence!establish!whether!the!ProtoHox!to!Hox/ParaHox!transition!
occurred!after!the!divergence!of!the!ctenophores!(and!before!the!origin!of!sponges)!or!
instead!occurred!in!the!last!common!ancestor!of!all!animals.!
!
_____________________________________________________________________________________________________!
Figure'2.'
Animal'phylogeny'with'origin'of'Hox'and'ParaHox'specific'to'the'animals'
(Metazoa)'and'prior'to'the'origin'of'poriferans,'but'with'ambiguity'with'regards'to'
the'ctenophore'condition.!‘Ticks’!represent!confirmed!presence!of!Hox/ParaHox!genes!
in!at!least!some!members!of!the!phylum,!whilst!‘X’!represents!absence!from!
choanoflagellates![15],!the!sister!group!to!the!Metazoa.!Ghost!cartoons!represent!the!
presence!of!ghost!loci!and!‘?’!indicates!the!ambiguity!about!the!ctenophore!condition!
and!hence!the!uncertainty!about!whether!the!ProtoHox!to!Hox/ParaHox!transition!
happened!before!or!after!the!divergence!of!the!ctenophore!lineage!(see!text!for!details).!
All!animal!pictures!are!taken!from!the!Phylopic!database!(http://phylopic.org)!except!
for!the!Sycon!poriferan!picture,!which!was!constructed!by!the!author!and!is!derived!
!
17!
from!a!photo!from!Maja!Adamska!(http://biology.anu.edu.au/research/labs/adamskaI
labIgenomicIandIevolutionaryIbasisIanimalIdevelopment).!The!phylogeny!is!based!on!
that!of![51].!It!should!be!noted!however,!that!the!relative!branching!order!of!the!
ctenophore!and!poriferan!lineages!at!the!base!of!the!animal!phylogeny!is!still!not!
resolved!to!the!extent!that!there!is!a!general!consensus![87].!If!the!poriferan!lineage!is!in!
fact!the!basalImost!lineage!then!the!conclusions!of!MendivilIRamos!et!al![15]!and!
Fortunato!et!al![16],!that!the!last!common!ancestor!of!all!extant!animals!had!
Hox/ParaHox!genes!in!distinct!loci,!would!stand.!
_____________________________________________________________________________________________________!
!
!
Ctenophores!as!the!basal!animal!lineage!could!well!imply!a!simplification!of!morphology!
and!range!of!cell!types!during!poriferan!evolution,!including,!for!example,!possible!
secondary!loss!of!neurons!and!muscles![53I55],!notwithstanding!the!alternative!
interpretation!favoured!by!some!authors!that!the!ctenophores!and!eumetazoans!evolved!
at!least!some!of!these!cell!types!independently![50,!52].!This!potential!secondary!
simplification!or!loss!of!cell!types!perhaps!mirrors!the!extensive!differential!gene!loss!
that!is!now!being!discovered!across!these!lineages.!
!
Differential'gene'loss'is'rife'across'basal'animal'lineages''
!
Implicit!in!the!Ghost!Locus!hypothesis!is!differential!gene!loss.!In!recent!years!a!greater!
appreciation!for!the!importance!of!gene!loss!across!animal!lineages!has!developed,!as!a!
certain!counterIbalance!to!the!prevalent!view!that!deep!homology!and!conservation!of!
developmental!control!genes!underlies!much!of!animal!evolution!(e.g.![56I58]).!Whilst!
!
18!
the!deep!conservation!of!developmental!control!genes!certainly!still!holds!to!a!large!
extent,!it!is!clear!that!there!is!a!much!greater!degree!of!evolutionary!lability!than!was!
appreciated!in!the!early!days!of!molecular!evoIdevo![59].!
!
Excellent!reviews!of!the!patterns!of!differential!gene!loss!impacting!on!transcription!
factors!are!already!available!for!basal!animal!lineages!(including![60I63]).!Differential!
gene!loss!is!also!seen!in!genes!that!encode!proteins!other!than!transcription!factors,!
including!signalling!molecules,!genes!involved!in!nervous!system!activity,!epithelial!
integrity!and!“immune!response”![64].!NonIcoding!genes,!such!as!microRNAs,!have!also!
been!observed!to!be!secondarily!lost!from!some!basal!lineages,!such!as!the!Placozoa!
[43].!!In!the!Hox/ParaHox!context,!differential!gene!loss!is!also!consistent!with!the!
remaining!ParaHox!gene!in!the!placozoan!T.,adhaerens!being!from!one!family,!Gsx,!
whilst!the!remaining!ParaHox!gene!from!sponges!is!from!another!family,!Cdx.!
!
On!balance!there!has!clearly!been!extensive!gene!loss!during!the!evolution!of!the!basal!
animal!lineages!and!the!last!common!ancestor!of!all!animals!had!a!much!larger!
developmental!gene!repertoire!than!was!widely!appreciated!until!recently!(e.g.![42,!63,!
65,!66]).!A!more!extensive!understanding!of!the!gene!content!of!nonIbilaterian!animals,!
particularly!via!wider!taxon!sampling,!is!clearly!needed!to!properly!understand!the!
genome!content!of!the!last!common!animal!ancestor.!
!
A'large'diversity'of'developmental'control'genes'implies'a'morphologically'
complex'animal'ancestor?'
!
!
19!
Complexity!can!often!be!a!loaded!or!ambiguous!term!in!evolutionary!biology!and!needs!
precise!definition!in!order!to!try!to!avoid!confusion.!In!terms!of!complexity!simply!
meaning!a!large!number!of!different!entities,!clearly!the!ancestral!animal!was!much!
more!complex!than!appreciated!even!just!a!few!years!ago!if!we!consider!developmental!
control!genes!themselves.!What!is!less!clear!is!how!this!genetic!complexity!relates,!and!
related,!to!morphological!complexity.!Previous!attempts,!for!example,!have!involved!
‘measuring’!the!numbers!and!variety!of!different!cell!types!or!tissue!types![67].!!
!
It!is!becoming!clear!that!we!need!to!carefully!reassess!our!views!about!the!“simplicity”!
of!basal!animal!lineages.!With!improvements!in!microscopy,!immunohistochemistry!and!
histology,!as!well!as!closer!examination!of!the!expression!of!the!large!variety!of!genes!
being!isolated,!it!is!apparent!that!there!is!plenty!of!cryptic!‘hidden’!biology!in!basal!
animal!lineages!and!they!are!not!quite!as!morphologically!simple!as!previously!believed!
[68,!69].!Also,!previous!conclusions!about!the!similarity!and!likely!homology!of!cell!
types!can!be!questioned!when!specifically!examined.!A!particularly!pertinent!case!in!the!
present!context!of!basal!animal!lineages!is!the!longIassumed!affinity!between!sponge!
choanocytes!and!the!cells!of!the!nonIanimal!sister!group,!choanoflagellates.!This!
homology!is!now!being!questioned![70].!Furthermore,!despite!the!extensive!secondary!
gene!loss!described!above,!sponges!are!proving!to!still!be!rather!molecularly!complex,!
with!large!gene!numbers!(for!example!A.,queenslandica!estimates!now!exceed!40,000!
genes![71])!and!a!diversity!of!lncRNAs!comparable!to!that!of!bilaterians![72].!Also,!
expansions!of!TaxonIRestricted!Genes!(TRGs)!are!just!as!prevalent!in!basal!animal!
lineages!as!they!are!in!bilaterians![73,!74].!
!
!
20!
Additionally,!the!question!mark!in!the!subIheading!above!is!important.!It!is!intended!to!
highlight!the!uncertainty!with!which!a!diversity!of!developmental!control!genes!can!be!
equated!to!a!diversity!of!developmental!functions,!which!by!extension!could!build!
complex!morphologies!composed!of!a!diversity!of!cell!types!and!structures.!This!
uncertainty!arises!from!the!fact!that!we!currently!have!a!poor!understanding!of!how!
developmental!genes!control!the!development!of!morphology!in!basal!animal!lineages.!
In!large!part!this!is!due!to!a!traditional!focus!on!a!small!number!of!model!organisms!in!
developmental!biology,!which!did!not!include!any!examples!of!basal!animal!lineages.!
Also!the!extensive!pleiotropy!of!developmental!genes!in!these!model!organisms,!with!
each!gene!having!roles!in!a!wide!variety!of!developmental!contexts,!makes!deductions!
about!ancestral!gene!functions!particularly!difficult.!The!recent!development!of!
functional!genetic!techniques!like!TALEN!and!CRISPR/Cas9,!that!are!gradually!being!
brought!to!bear!on!basal!lineage!taxa,!should!help!to!address!this!problem!in!the!near!
future![75].!Nevertheless,!for!the!present!it!seems!reasonable!to!conclude!that!the!
previously!underIappreciated!number!and!diversity!of!developmental!control!genes!in!
the!last!common!ancestor!of!animals!probably!did!equate!to!a!complex,!diverse!set!of!
functions!that!presumably!did!build!complex!morphologies.!!
!
This!paucity!of!molecular!description!of!morphological!development!(e.g.!generation!of!
cell!type!diversity)!in!basal!animal!lineages!becomes!less!of!an!issue!when!we!consider!
another!valuable!form!of!evidence!in!deducing!the!appearance!of!ancient!ancestors,!
namely!palaeontology.!Here!great!strides!have!also!been!made!in!recent!years,!with!new!
preparatory!techniques!(e.g.!serial!grinding!to!build!3D!digital!reconstructions![76]!and!
XIray!computed!tomography![77])!and,!crucially,!the!discovery!and!description!of!new!
specimens.!Ctenophore!fossils!are!not!so!well!known!and!the!possible!earliest,!most!
!
21!
ancient!examples!are!controversial!in!their!affinities!(discussed!in![68]).!The!most!
unambiguous!ctenophore!fossils!are!from!the!Lower!Devonian![68],!but!potential!
Ediacaran!ctenophores!have!also!been!found![78].!An!important!caveat!to!keep!in!mind!
in!this!context,!however,!is!that!extant!ctenophore!lineages!seem!to!have!diverged!from!
each!other!relatively!recently.!The!last!common!ctenophore!ancestor!is!thus!separated!
by!a!long!evolutionary!distance!from!the!last!common!ancestor!with!other!extant!animal!
lineages!([68]!and!references!therein).!Much!evolutionary!change!could!thus!have!
happened!between!the!last!common!ancestor!of!all!animals!and!the!last!common!
ancestor!of!extant!ctenophores!(including,!for!example,!the!evolution!of!skeletonization!
in!some!extinct!ctenophores![79]),!and!the!morphological!differences!between!these!two!
ancestors!are!far!from!clear.!This!perhaps!warrants!a!reappraisal!of!the!Ediacaran!fossils!
(635I541!million!years!ago)!and!the!likelihood!of!the!last!common!ancestor!of!animals!
being!present!within!this!difficultItoIclassify!diverse!group!of!organisms.!
!
The!classification!of!these!Ediacaran!organisms!is!noted!for!the!controversy!and!range!of!
opinions!on!their!phylogenetic!affinities,!and!their!relationships!with!modern!
recognised!phyla!or!even!kingdoms.!But!a!consensus!seems!to!have!emerged!in!recent!
years!that!at!least!some!Ediacaran!species!are!animals![80,!81].!Some!authors!have!
hypothesized!that!some!of!the!earliest!Ediacaran!forms!(e.g.!rangeomorphs)!are!stem!
ctenophores!or!cnidarians,!but!this!is!still!debated!([80]!and!references!therein)!and!
some!now!prefer!to!treat!rangeomorphs!as!organisms!in!their!own!right!without!trying!
to!shoeIhorn!them!into!a!classification!based!on!extant!kingdoms!and!phyla![82I84].!
These!early!Ediacarans,!including!but!not!restricted!to!the!rangeomorphs,!could!well!be!
the!earliest!animals,!but!with!body!plans!that!are!rather!distinct!from!modernIday!
!
22!
metazoans.!The!complexity!of!these!early!Ediacarans!and!how!this!might!relate!to!
developmental!gene!diversity!is!wide!open!for!debate!and!further!research!(e.g.![85]).!
!
Summary'
!
In!conclusion,!it!is!clear!that!great!strides!are!being!made!in!understanding!the!nature!of!
the!last!common!ancestor!of!animals!on!a!number!of!fronts:!genome!sequencing,!
developmental!gene!characterisation,!functional!genetics,!phylogenetics!and!
palaeontology.!One!of!the!key!aspects!to!this!improvement!in!our!understanding!is!
wider!taxon!sampling.!The!revolution!in!our!understanding!of!the!origin!and!subsequent!
evolution!of!the!important!Hox/ParaHox!developmental!control!genes,!which!have!long!
been!a!major!focus!in!evoIdevo,!is!a!case!that!illustrates!this!importance!of!wide!taxon!
sampling!particularly!well.!The!origin!of!the!Hox/ParaHox!genes!occurred!prior!to!the!
origin!of!the!poriferans,!clearly!illustrating!that!the!last!common!ancestor!of!sponges!
and!the!remaining!Eumetazoa!were!more!complex,!molecularlyIspeaking,!than!
previously!appreciated.!Whether!this!ancestral!state!represents!that!of!the!last!common!
ancestor!of!all!animals!will!be!determined!by!the!rapidly!progressing!advances!in!
phylogenetics,!with!the!current!indications!being!that!we!need!a!more!extensive!
understanding!of!ctenophores.!Advances!in!palaeontology!will!also!be!key,!particularly!
with!regards!to!a!better!understanding!of!the!Ediacaran!fossils,!which!are!renowned!for!
the!controversy!over!their!affinities!(or!lack!of!affinities)!with!modernIday!lineages,!and!
whether!the!origins!of!the!animal!kingdom!can!be!distinguished!amongst!them.!In!the!
current!context!it!is!perhaps!suffice!to!say!that!the!morphological!appearance!and!
relative!complexity!of!the!first!animal!is!far!from!clear,!and!a!morphologically!complex!
ancestor!is!perfectly!plausible,!even!more!so!given!the!shift!of!the!ctenophore!lineage!to!
!
23!
a!basal!position!in!the!animal!tree!and!the!continued!discovery!of!cryptic!complexity!in!
extant!basal!lineages.!Continued!wide!taxon!sampling!and!progress!in!all!of!the!research!
fields!listed!above!are!significantly!improving!our!chances!of!determining!the!
complexity!(both!molecularly!and!morphologically)!of!the!last!common!ancestor!of!
animals,!which!was!the!starting!point!for!the!diversification!of!the!entire!animal!
kingdom.!Darwin’s!view!that!“from!so!simple!a!beginning!endless!forms!most!beautiful!
and!most!wonderful!have!been,!and!are!being,!evolved”![86]!may!need!to!be!slightly!
revised!for!the!animals,!since!they!started!from!a!notIsoIsimple!beginning!after!all.!
!
!
Acknowledgements'
!
The!author!would!like!to!thank!Olivia!Mendivil!Ramos,!Maja!Adamska!and!Sofia!
Fortunato!for!discussions!that!shaped!the!ideas!outlined!in!this!review,!and!Joanne!
Ferrier!for!comments!on!the!manuscript.!I!would!also!like!to!thank!the!various!
colleagues!who!continue!to!query!and!challenge!the!hypotheses!discussed!here.!
!
References'
!
1.!McGinnis!W.!A!century!of!homeosis,!a!decade!of!homeoboxes.!Genetics!1994;!137(3):!
607I11.!
2.!Holland!PW,!Booth!HA,!Bruford!EA.!Classification!and!nomenclature!of!all!human!
homeobox!genes.!BMC,Biol!2007;!5:!47.!
!
24!
3.!Duboule!D.!Temporal!colinearity!and!the!phylotypic!progression:!a!basis!for!the!
stability!of!a!vertebrate!Bauplan!and!the!evolution!of!morphologies!through!
heterochrony.!Development!Supplement!1994:!135I142.!
4.!Ferrier!DEK.!Minguillón!C.!Evolution!of!the!Hox/ParaHox!gene!clusters.!Int,J,Dev,Biol!
2003;!47:!605I611.!
5.!Kmita!M.!Duboule!D.!Organizing!axes!in!time!and!space;!25!years!of!collinear!
tinkering.!Science!2003;!301:!331I333.!
6.!Tarchini!B,!Duboule!D.!Control!of!Hoxd!genes’!collinearity!during!early!limb!
development.!Devel,Cell!2006;!10:!93I103.!
7.!Montavon!T,!Le!Garrec!JF,!Kerszberg!M,!et,al.!Modelling!Hox!gene!regulation!in!digits:!
reverse!collinearity!and!the!molecular!origin!of!thumbness.!Genes,Dev!2008;!22(3):!346I
359.!!
8.!Ferrier!DEK,!Holland!PWH.!Ciona,intestinalis!ParaHox!genes:!evolution!of!
Hox/ParaHox!cluster!integrity,!developmental!mode,!and!temporal!colinearity.!Mol,Phyl,
Evol!2002;!24:!412I417.!
9.!Monteiro!AS,!Ferrier!DEK.!Hox!genes!are!not!always!Colinear.!Int,J,Biol,Sci!2006:!2.!
10.!Lemons!D,!McGinnis!W.!Genomic!evolution!of!Hox!gene!clusters.!Science!2006;!313:!
1918I1922.!
11.!Slack!JM,!Holland!PWH,!Graham!CF.!The!zootype!and!the!phylotypic!stage.!Nature!
1993;!361:!490I492.!
12.!Finnerty!JR,!Martindale!MQ.!The!evolution!of!the!Hox!cluster:!insights!from!
outgroups.!Curr,Opin,Genet,Dev!1998;!8(6):!681I687.!
13.!Ferrier!DEK,!Holland!PWH.!Ancient!origin!of!the!Hox!gene!cluster.!Nat,Rev,Genetics!
2001;!2:!33I38.!
!
25!
14.!Ryan!JF,!Pang!K,!NISC!Comparative!Sequencing!Program,!et,al.!The!homeodomain!
complement!of!the!ctenophore!Mnemiopsis,leidyi!suggests!that!Ctenophora!and!Porifera!
diverged!prior!to!the!ParaHoxozoa.!Evodevo!2010;!1(1):!9.!
15.!Mendivil!Ramos!O,!Barker!D,!Ferrier!DEK.!Ghost!Loci!imply!Hox!and!ParaHox!
existence!in!the!last!common!ancestor!of!animals.!Curr,Biol!2012;!22:!1951I1956.!
16.!Fortunato!SAV,!Adamski!M,!Mendivil!Ramos!O,!et,al.!Calcisponges!have!a!ParaHox!
gene!and!dynamic!expression!of!dispersed!NK!homeobox!genes.!Nature!2014;!514:!620I
623.!
17.!Brooke!NM,!GarciaIFernàndez!J,!Holland!PWH.!The!ParaHox!gene!cluster!is!an!
evolutionary!sister!of!the!Hox!gene!cluster.!Nature!1998;!392:!920I922.!
18.!Ferrier!DEK.!Evolution!of!Hox!complexes.!In!Hox,genes:,studies,from,the,20th,to,the,
21st,Century!Landes!Bioscience!and!Springer!Science!and!Business!Media!2010:!91I100.!
19.!Minguillón!C,!GarciaIFernàndez!J.!Genesis!and!evolution!of!the!Evx!and!Mox!genes!
and!the!extended!Hox!and!ParaHox!gene!clusters.!Genome,Biol!2003;!4(2):!R12.!
20.!Ryan!JF,!Mazza!ME,!Pang!K,!et,al.!PreIbilaterian!origins!of!the!Hox!cluster!and!the!
Hox!code:!evidence!from!the!sea!anemone,!Nematostella,vectensis.!PLoS,ONE!2007;!2(1):!
e153.!
21.!Mendivil!Ramos!O,!Ferrier!DEK.!Mechanisms!of!gene!duplication!and!translocation!
and!progress!towards!understanding!their!relative!contributions!to!animal!genome!
evolution.!Int,J,Evol,Biol!2012:!id!846421.!
22.!Ou!Z,!Stankiewicz!P,!Xia!Z,!et,al.!Observation!and!prediction!of!recurrent!human!
translocations!mediated!by!NAHR!between!nonhomologous!chromosomes.!Genome,Res!
2011;!21:!33I46.!
23.!Hurst!LD,!Pál!C,!Lercher!MJ.!The!evolutionary!dynamics!of!eukaryotic!gene!order.!
Nature,Rev,Genetics!2004;!5:!299I310.!
!
26!
24.!Nora!EP,!Dekker!J,!Heard!E.!Segmental!folding!of!chromosomes:!a!basis!for!structural!
and!regulatory!chromosomal!neighborhoods.!Bioessays!2013;!35:!818I828.!
25.!Chan!C,!Jayasekera!S,!Kao!B,!et,al.!Remodelling!of!a!homeobox!gene!cluster!by!
multiple!independent!gene!reunions!in!Drosophila.,Nat,Commun!2015;!6:!6509.!
26.!Hui!JHL,!McDougall!C,!Monteiro!AS,!et,al.!Extensive!chordate!and!annelid!
macrosynteny!reveals!ancestral!homeobox!gene!organization.!Mol,Biol,Evol!2012;!29(1):!
157I165.!
27.!Duboule!D.!The!rise!and!fall!of!Hox!gene!clusters.!Development!2007;!134:!2549I
2560.!
28.!Bu!L,!Katju!V.!Early!evolutionary!history!and!genomic!features!of!gene!duplicates!in!
the!human!genome.!BMC,Genomics!2015;!16:!621.!
29.!Lanfear!R,!Bromham!L.!Statistical!tests!between!competing!hypotheses!of!Hox!
cluster!evolution.!Syst,Biol!2008;!57(5):!708I718.!
30.!Bailey!JA,!Eichler!EE.!Primate!segmental!duplications:!crucibles!of!evolution,!
diversity!and!disease.!Nat,Rev,Genetics!2006;!7:!552I564.!
31.!Gokcumen!O,!Tischler!V,!Tica!J,!et,al.!Primate!genome!architecture!influences!
structural!variation!mechanisms!and!functional!consequences.!Proc,Natl,Acad,Sci,USA!
110:!15764I15769.!
32.!Lorenzen!MD,!Gnirke!A,!Margolis!J,!et,al.!The!maternalIeffect,!selfish!genetic!element!
Medea!is!associated!with!a!composite!Tc1!transposon.!Proc,Natl,Acad,Sci,USA!2008;!105:!
10085I10089.!
33.!Durkin!K,!Coppieters!W,!Drögemüller!C,!et,al.!Serial!translocation!by!means!of!
circular!intermediates!underlies!colour!sidedness!in!cattle.!Nature!2012;!482:!81I!84.!
34.!Fujimura!K,!Conte!MA,!Kocher,!TD.!Circular!DNA!intermediates!in!the!duplication!of!
Nile!Tilapia!vasa!genes.!PLoS,ONE!2011;!6:!e29477.!
!
27!
35.!Monteiro!AS,!Schierwater!B,!Dellaporta!S,!et,al.!A!low!diversity!of!ANTPIclass!
homeobox!genes!in!Placozoa.!Evol,Dev!2006;!8(2):!174I182.!
36.!Jakob!W,!Sagasser!S,!Dellaporta!S,!et,al.!The!Trox<2!Hox/ParaHox!gene!of!Trichoplax!
(Placozoa)!marks!an!epithelial!boundary.!Dev,Genes,Evol!2004;!214(4):!170I175.!
37.!Schierwater!B,!Kamm!K,!Srivastava!M,!et,al.!The!early!ANTP!gene!repertoire:!insights!
from!the!placozoan!genome.!PLoS,ONE!2008;!3(8):!e2457.!
38.!Conant!GC,!Wagner!A.!Asymmetric!sequence!divergence!of!duplicate!genes.!Genome,
Res!2003;!13:!2052I2058.!
39.!Cusack!BP,!Wolfe!KH.!Not!born!equal:!increased!rate!asymmetry!in!relocated!and!
retrotransposed!rodent!gene!duplicates.!Mol,Biol,Evol!2007;!24(3):!679I686.!
40.!Pegueroles!C,!Laurie!S,!Albà!MM.!Accelerated!evolution!after!gene!duplication:!a!
timeIdependent!process!affecting!just!one!copy.!Mol,Biol,Evol!2013;!30(8):!1830I1842.!
41.!Katju!V.!To!the!beat!of!a!different!drum:!determinants!implicated!in!the!asymmetric!
sequence!divergence!of!Caenorhabditis,elegans!paralogs.!BMC,Evol,Biol!2013;!13:!73.!
42.!Putnam!NH,!Srivastava!M,!Hellsten!U,!et,al.!Sea!anemone!genome!reveals!ancestral!
eumetazoan!gene!repertoire!and!genomic!organization.!Science!2007;!317:!86I94.!
43.!Srivastava!M,!Begovic!E,!Chapman!J,!et,al.!The!Trichoplax!genome!and!the!nature!of!
placozoans.!Nature!2008;!454:!955I960.!
44.!Srivastava!M,!Simakov!O,!Chapman!J,!et,al.!The!Amphimedon,queenslandica!genome!
and!the!evolution!of!animal!complexity.!Nature!2010;!466:!720I726.!
45.!Hui!JHL,!Holland!PWH,!Ferrier!DEK.!Do!cnidarians!have!a!ParaHox!cluster?!Analysis!
of!synteny!around!a!Nematostella!homeobox!gene!cluster.!Evol,Dev!2008;!10(6):!725I
730.!
46.!Dohrmann!M,!Wörheide!G.!Novel!scenarios!of!early!animal!evolution!–!is!it!time!to!
rewrite!textbooks?!Integr,Comp,Biol!2013;!53:!503I511.!
!
28!
47.!Dunn!CW,!Hejnol!A,!Matus!DQ,,et,al.!Broad!phylogenomic!sampling!improves!
resolution!of!the!animal!tree.!Nature!2008;!452:!745I749.!
48.!Philippe!H,!Derelle!R,!Lopez!P,!et,al.!Phylogenomics!revives!traditional!views!on!deep!
animal!relationships.!Curr,Biol!2009;!19:!706I712.!
49.!Philippe!H,!Brinkmann!H,!Lavrov!D,!,et,al.!Resolving!difficult!phylogenetic!questions:!
why!more!sequences!are!not!enough.!PLoS,Biol!2011:!9:!e1000602.!
50.!Moroz!LL,!Kocot!KM,!Citarella!MR,!et,al.!The!ctenophore!genome!and!the!
evolutionary!origins!of!neural!systems.!Nature!2014;!510:!109I114.!
51.!Whelan!NV,!Kocot!KM,!Moroz!LL,!et,al.!Error,!signal,!and!the!placement!of!
Ctenophora!sister!to!all!other!animals.!Proc,Natl,Acad,Sci,USA!2015;!112:!5773I5778.!
52.!Ryan!JF,!Pang!K,!Schnitzler!CE,!et,al.!The!genome!of!the!ctenohore!Mnemiopsis,leidyi!
and!its!implications!for!cell!type!evolution.!Science!2013;!342:!1242592.!
53.!Ryan!JF.!Did!the!ctenophore!nervous!system!evolve!independently?!Zoology!2014;!
117:!225I226.!
54.!Marlow!H,!Arendt!D.!Evolution:!ctenophore!genomes!and!the!origin!of!neurons.!Curr,
Biol!2014;!24(16):!R757IR761.!
55.!Jékely!G,!Paps!J,!Nielsen!C.!The!phylogenetic!position!of!ctenophores!and!the!
origin(s)!of!nervous!systems.!EvoDevo!2015;!6:!1.!
56.!De!Robertis!EM.!EvoIDevo:!variations!on!ancestral!themes.!Cell!2008;!132:!185I195.!
57.!Takahashi!T,!McDougall!C,!Troscianko!J,!et,al.!An!EST!screen!from!the!annelid!
Pomatoceros,lamarckii!reveals!patterns!of!gene!loss!and!gain!in!animals.!BMC,Evol,Biol!
2009;!9:!240.!
58.!Foret!S,!Knack!B,!Houliston!E,!et,al.!New!tricks!with!old!genes:!the!genetic!bases!of!
novel!cnidarian!traits.!Trends,Genetics!2010;!26(4):!154I158.!
!
29!
59.!Carroll!SB,!Grenier!JK,!Weatherbee!SD.!From,DNA,to,Diversity:,molecular,genetics,and,
the,evolution,of,animal,design.!Blackwell!Publishing,!Second!edition!2005.!
60.!Larroux!C,!Luke!GN,!Koopman!P,!et,al.!Genesis!and!expansion!of!metazoan!
transcription!factor!gene!classes.!Mol,Biol,Evol!2008;!25(5):!980I996.!
61.!SebéIPedrós!A,!de!Mendoza!A,!Lang!BF,!et,al.!Unexpected!repertoire!of!metazoan!
transcription!factors!in!the!unicellular!holozoan!Capsaspora,owczarzaki.,Mol,Biol,Evol!
2011;!28(3):!1241I1254.!
62.!Gyoja!F.!A!genomeIwide!survey!of!bHLH!transcription!factors!in!the!placozoan!
Trichoplax,adhaerens!reveals!the!ancient!repertoire!of!this!gene!family!in!metazoans.!
Gene!2014;!542(1):!29I37.!
63.!Fortunato!SAV,!Adamski!M,!Adamska!M.!Comparative!analyses!of!developmental!
transcription!factor!repertoires!in!sponges!reveal!unexpected!complexity!of!the!earliest!
animals.!Marine,Genomics!2015;!in!press,!doi:10.1016/j.margen.2015.07.008.!
64.!Riesgo!A,!Farrar!N,!Windsor!J,!et,al.!The!analysis!of!eight!transcriptomes!from!all!
poriferan!classes!reveals!surprising!genetic!complexity!in!sponges.!Mol,Biol,Evol!2014;!
31(5):!1102I1120.!
65.!King!N,!Westbrook!MJ,!Young!SL,!et,al.!The!genome!of!the!choanoflagellate!Monosiga,
brevicollis!and!the!origin!of!metazoans.!Nature!2008;!451:!783I788.!
66.!Suga!H,!Chen!Z,!de!Mendoza!A,!et,al.,The!Capsaspora!genome!reveals!a!complex!
unicellular!prehistory!of!animals.!Nat,Commun!2013;!4:!2325.!
67.!Valentine!JW.!On,the,origin,of,Phyla.!University!of!Chicago!Press!2004:!pp75.!
68.!Dunn!CW,!Leys!SP,!Haddock!SHD.!The!hidden!biology!of!sponges!and!ctenophores.!
Trends,Ecol,Evol!2015;!30(5):!282I291.!
!
30!
69.!Marlow!HQ,!Srivastava!M,!Matus!DQ,!et,al.!Anatomy!and!development!of!the!nervous!
system!of!Nematostella,vectensis,!an!anthozoan!cnidarian.!Dev,Neurobiol!2009;!69(4):!
235I254.!
70.!Mah!JL,!ChristensenIDalsgaard!KK,!Leys!SP.!Choanoflagellate!and!choanocyte!collarI
flagellar!systems!and!the!assumption!of!homology.!Evol,Dev!2014;!16(1):!25I37.!
71.!FernandezIValverde!SL,!Calcino!AD,!Degnan!BM.!Deep!developmental!transcriptome!
sequencing!uncovers!numerous!new!genes!and!enhances!gene!annotation!in!the!sponge!
Amphimedon,queenslandica.,BMC,Genomics!2015;!16:!387.!
72.!Gaiti!F,!FernandezIValverde!SL,!Nakanishi!N,!et,al.!Dynamic!and!widespread!lncRNA!
expression!in!a!sponge!and!the!origin!of!animal!complexity.!Mol,Biol,Evol!2015;!32(9):!
2367I2382.!
73.!Milde!S,!Hemmrich!G,!AntonIErxleben!F,!et,al.!Characterization!of!taxonomicallyI
restricted!genes!in!a!phylumIrestricted!cell!type.!Genome,Biol!2009;!10:!R8.!
74.!Khalturin!K,!Hemmrich!G,!Fraune!S,!et,al.!More!than!just!orphans:!are!taxonomicallyI!
restricted!genes!important!in!evolution?!Trends,Genet!2009;!25:!404–413.!
75.!Ikmi!A,!McKinney!SA,!Delventhal!KM,,et,al.!TALEN!and!CRISPR/CAs9Imediated!
genome!editing!in!the!earlyIbranching!metazoan!Nematostella,vectensis.!Nat,Commun,
2014;!5:!5486.!
76.!Sutton!MD,!Briggs!DE,!Siveter!DJ,!et,al.!A!threeIdimensionally!preserved!fossil!
polychaete!worm!from!the!Silurian!of!Herefordshire,!England.!Proc,R,Soc,Lond,B,Biol,Sci!
2001;!268:!2355I2363.!
77.!Hagadorn!JW,!Xiao!S,!Donoghue!PCJ,!et,al.!Cellular!and!subcellular!structure!of!
Neoproterozoic!animal!embryos.!Science!2006;!314:!291I294.!
78.!Tang!F,!Bengtson!S,!Wang!Y,!et,al.!Eoandromeda!and!the!origin!of!Ctenophora.!Evol,
Dev!2011;!13(5):!408I414.!
!
31!
79.!Ou!Q,!Xiao!S,!Han!J,!,et,al.!A!vanished!history!of!skeletonization!in!Cambrian!comb!
jellies.!Sci,Adv!2015;!1:!e1500092.!
80.!Xiao!S,!Laflamme!M.!On!the!eve!of!animal!radiation:!phylogeny,!ecology!and!
evolution!of!the!Ediacara!biota.!Trends,Ecol,Evol!2008;!24(1):!31I40.!
81.!Droser!ML,!Gehling!JG.!The!advent!of!animals:!the!view!from!the!Ediacaran.!Proc,Natl,
Acad,Sci,USA!2015;!112(16):!4865I4870.!
82.!Cuthill!JFH,!Conway!Morris!S.!Fractal!branching!organizations!of!Ediacaran!
rangeomorph!fronds!reveal!a!lost!Proterozoic!body!plan.!Proc,Natl,Acad,Sci,USA!2014;!
111(36):!13122I13126.!
83.!Ghisalberti!M,!Gold!DA,!Laflamme!M,!et,al.!Canopy!flow!analysis!reveals!the!
advantage!of!size!in!the!oldest!communities!of!multicellular!eukaryotes.!Curr,Biol!2014;!
24:!305I309.!
84.!Mitchell!EG,!Kenchington!CG,!Liu!AG,!et,al.!Reconstructing!the!reproductive!mode!of!
an!Ediacaran!macroIorganism.!Nature!2015;!524:!343I346.!
85.!Erwin!DH.!Wonderful!Ediacarans,!wonderful!cnidarians?!,Evol,Dev!2008;!10(3):!263I
264.!
86.!Darwin!C.!The,origin,of,species,by,means,of,Natural,Selection,or,the,preservation,of,
favoured,races,in,the,struggle,for,life.!Penguin!Classics!1987!(reprinted!from!John!
Murray,!1859).!
87.!Telford!MJ,!Budd!GE,!Philippe!H.!Phylogenomic!insights!into!animal!evolution.!Curr,
Biol!2015;!25(19):!R876IR887.!
!
!
!
32!