A Hybrid Genetic Linkage Map of Two Ecologically and Morphologically Divergent Midas Cichlid Fishes (Amphilophus spp.) Obtained by Massively Parallel DNA Sequencing (ddRADSeq).

1.3 MB·English

by  Recknagel, Hans

INVESTIGATION
A Hybrid Genetic Linkage Map of Two Ecologically
and Morphologically Divergent Midas Cichlid Fishes
Amphilophus
( spp.) Obtained by Massively Parallel
DNA Sequencing (ddRADSeq)
HansRecknagel,1KathrynR.Elmer,1andAxelMeyer2
LehrstuhlfürZoologieundEvolutionsbiologie,DepartmentofBiology,UniversityofKonstanz,78457Konstanz,Germany
ABSTRACT Cichlid fishes are an excellent model system for studying speciation and the formation of KEYWORDS
adaptive radiations because of their tremendous species richness and astonishing phenotypic diversity. Midascichlid
Most research has focused on African rift lake fishes, although Neotropical cichlid species display much double-digest
variabilityaswell.AlmostonedozenspeciesoftheMidascichlidspeciescomplex(Amphilophusspp.)have RADSeq
been described so far and have formed repeated adaptive radiations in several Nicaraguan crater lakes. synteny
Hereweapplydouble-digest restriction-site associatedDNA sequencingtoobtainahigh-densitylinkage segregation
map of an interspecific cross between the benthic Amphilophus astorquii and the limnetic Amphilophus distortion
zaliosus,whicharesympatricspeciesendemictoCraterLakeApoyo,Nicaragua.Atotalof755RADmarkers RADmarkers
weregenotypedin343F hybrids.Themapresolved25linkagegroupsandspansatotaldistanceof1427cM mutationrate
2
with an average marker spacing distance of 1.95 cM, almost matching the total number of chromosomes
(n=24)inthesespecies.Regionsofsegregationdistortionwereidentifiedinfivelinkagegroups.Basedon
thepedigreeofparentstoF offspring,wecalculatedagenome-widemutationrateof6.6·1028mutations
2
pernucleotidepergeneration.Thisgeneticmapwillfacilitatethemappingofecomorphologicallyrelevant
adaptive traits in the repeated phenotypes that evolved within the Midas cichlid lineage and, as the first
linkage map of a Neotropical cichlid, facilitate comparative genomic analyses between African cichlids,
Neotropical cichlidsandotherteleostfishes.
Cichlidfishesareawell-knownevolutionarymodelsystemforstudy- 2007).Becauseofthismega-diversity,RiftLakeAfricancichlidshave
ingthemechanismsofspeciationandtheformationofadaptiveradi- been the primary focus of genomic research in this family to date
ations.AfricanRiftLakecichlidsinparticulardisplayanastonishing (reviewed in Fan et al. 2012), but the .60 million year divergent
variabilityinphenotypesandcompriseanenormousnumberofspe- (Matschiner et al. 2011) Neotropical lineage of cichlids is pertinent
cies, which makes Cichlidae one of the mostspecies-richfamiliesin if we are to understand cichlid diversity at the family level and in
vertebrates (Fryer and Iles 1972; Salzburger and Meyer 2004; Berra a phylogenetic context. For example, the Neotropical Midas cichlid
lineagealonefeaturessomeofthekeytraitsthatcharacterizeAfrican
Copyright©2013Recknageletal. cichlidfishdiversity:polymorphismsincolor,jaw,lip,tooth,andbody
doi:10.1534/g3.112.003897
shape(Meyer1990a,b;Elmeretal.2010b).
ManuscriptreceivedAugust8,2012;acceptedforpublicationNovember5,2012
This is an open-access article distributed under the terms of the Creative Because of their geographic distribution, which includes the two
CommonsAttributionUnportedLicense(http://creativecommons.org/licenses/ NicaraguanGreatLakesandtheindependentcolonizationsofatleast
by/3.0/),whichpermitsunrestricteduse,distribution,andreproductioninany eightcraterlakesofNicaragua,Midascichlidcommunitiescomprise
medium,providedtheoriginalworkisproperlycited.
repeatedlyevolvedpopulationsatdifferentevolutionarystagesoflocal
Supportinginformationisavailableonlineathttp://www.g3journal.org/lookup/
suppl/doi:10.1534/g3.112.003897/-/DC1. adaptation andspeciation (Wilson etal.2000; BarluengaandMeyer
1Present address: Institute of Biodiversity, Animal Health & Comparative 2010).Thetwooldestcraterlakeshousemostofthedescribedspecies
Medicine, College of Medical, Veterinary & Life Sciences, University of (9of 11).Interestingly, someofthe youngercrater lakes harbor no-
Glasgow,Glasgow,UK.
2Correspondingauthor:DepartmentofBiology,UniversityofKonstanz, tablydifferentecotypesinsympatrythataregeneticallynotdifferen-
Universitätsstrasse10,78457Konstanz,Germany.E-mail:[email protected] tiated from each other (e.g., Elmer et al. 2010c). What makes this
Volume3 | January 2013 | 65
systemsointerestingisthatsimilarmorphologies,suchaselongated general and perhaps cichlids in particular (Meyer and Schartl 1999;
body shapes or hypertrophied lips, have evolved independently and KurakuandMeyer2008).Theevolutionofacichlid-specificphysical
repeatedly in different crater lakes (Elmer et al. 2010b; Manousaki orderof particular genes along the chromosomes, and certain inter-
et al. 2013). These two key aspects, (1) the crater lakes as “natural actionsbetweenthesegenes,mighthaveresultedinagenomicarchi-
experiments” that contain replicates of cichlid evolution at various tecture in cichlid lineages that favored diversification (Salzburger
stagesofdiversification,and(2)theecomorphologicalvariabilitythat 2009). As an alternative mechanism hypermutability of the genome
evolvedindifferentenvironmentsinparallel,facilitatestheinvestiga- orspecificgenomicregionsmightalloworganismstoadaptatahigher
tion of mechanisms and discovery of principles of how and which pacetochanging selectionpressures.Throughagreaterneutral mu-
genetic materialunderliesadaptation and the formation of newspe- tation rate, the chance that beneficial mutations might emerge is
cies(ElmerandMeyer2011).Forexample,knowingthegeneticsthat enhanced, therefore allowing neofunctionalization to occur faster, as
leadtophenotypicchangesviaquantitativetraitloci(QTL)mapping has been proposed for the facilitation of morphological diversity in
and association studies in closely and more distantly related species teleosts(RaviandVenkatesh2008).
willultimatelyhelptoelucidatehowselectionhasledtotheastonish- The mechanisms responsible for the incomparable diversity of
ingbiodiversityofAfricanandNeotropicalcichlidsobservedtoday. cichlidsareunknown.Onlyanaccumulationandcomparisonofmore
In at least two instances, Midas cichlid radiations have formed andmoreanalyzedgenomicresources,suchastranscriptomes,whole
repeatedly in Nicaraguan crater lakes; specifically the independent genomes,andlinkagemapsofcichlidsandrelatedorganismscanshed
formationofseveralhigh-bodiedendemicspeciesthatoccupybenthic lightonthese unresolvedquestions.Someprogresshasalready been
nicheandoneelongatedlimneticspecieseachintwolakes(Vivasand madeinthisdirection(Albertsonetal.2003;Leeetal.2005;Sanetra
McKaye2001;Barluengaetal.2006;Elmeretal.2010b).Apartfrom et al. 2009; Elmer et al. 2010a; Fan et al. 2012; Cichlid Genome
bodyshape,limneticandbenthicspeciesalsodifferindiet,pharyngeal Consortium, 2006). The present study seeks to contribute to this
jawmorphology,behavior,andcoloration(BarlowandMunsey1976; much-needed resource and analysis with a Neotropical lineage and,
Baylis1976a,b;VivasandMcKaye2001;Barluengaetal.2006;Elmer basedon the pedigreeof parentstoF offspring,give anestimateof
2
etal.2010b;Lehtonenetal.2011).InLakeApoyo,whichistheoldest agenome-widemutationrate.
of all Nicaraguan crater lakes at 22,000 years of age, speciation into For decades, the analysis of genomic data were restricted to
these two distinct ecotypes hasoccurredinsituin the form ofsym- a few model organisms and organisms of economical importance.
patric species by ecological speciation (Barluenga et al. 2006). Evi- Next-generation sequencing platforms allow for the cost-effective
dence suggests that benthic-limnetic divergence in Xiloa was also in sequencing of whole or partial genomes in a relatively short time;
sympatry (Kautt et al. 2012). The linkage mapping analysis in this this is greatly accelerating genomic research in non-model
study is based on a cross of the sympatrically occurring limnetic organisms(Stapleyetal.2010;ElmerandMeyer2011).Apartfrom
AmphilophuszaliosusandbenthicAmphilophusastorquiifromcrater whole genome sequencing, with restriction site-associated DNA
LakeApoyo. sequencing(RADSeq)apowerfulnewtooltoexaminethegenomes
There is a paucity of evidence about the genetic basis of early of organisms has become available (Baird et al. 2008; reviewed in
divergence between two lineages as they emerge from a common Davey andBlaxter2010). The advantage of RADSeq is the reduc-
ancestor(reviewedinCoyneandOrr2004;NosilandSchluter2011). tion of the genomic information of an organism to a usable and
Forspeciesthatarosethroughallopatricspeciationwithoutexchang- comparable fraction of homologous single nucleotide polymor-
ing genetic material, the randomaccumulation of genetic incompat- phismsacrossasetofindividuals.Bychoosingenzymesthatdiffer
ibilitiesbytimemight explain theirreproductiveisolation.However, intheircuttingfrequency,RADSeqallows forthedevelopmentof
especiallyinstagesofspeciationwithgeneflow,itisofgreatinterestto variablenumbersofrepeatablegeneticmarkers(RADmarkers)to
ask(1)whetherfewormany genesandwhere theyarelocated, e.g., be sequenced by Illumina next-generation sequencing technology
clumped,inthegenome;and(2)whetherthesametypeofgenesare (Baird et al. 2008). RAD markers are polymorphic loci (i.e.,
involved in the processof speciation (Elmer and Meyer 2011; Feder sequenceswithatleastonesingle-nucleotidepolymorphism[SNP])
et al. 2012). In only a few wild vertebrate populations have these within or between individuals. Here, we apply a newly developed
questionsbeenaddressedviagenemappingandQTLmappingstud- double-digest RADSeq (ddRADSeq) protocol (based on Peterson
ies;thus,onlyveryfewcrucialgenesresponsibleforecologicaldiver- et al. 2012) to create a linkage map of various species of Midas
gence (e.g., Colosimo et al. 2005; Chan et al. 2010) and speciation cichlids.
(CoyneandOrr2004;NosilandSchluter2011)havebeenidentified In the present study, we construct a linkage map with the F
2
sofar.ThedevelopmentofaMidascichlidlinkagemapwillaidsub- progeny of an interspecific cross between the benthic A. astorquii
stantially in identifying the genes that underlie phenotypically and andthelimneticA.zaliosusMidascichlidsasasteptowarduncover-
ecologically important traits in fishes early during their adaptations ing the genetic basis of adaptive traits in this species complex. The
tolocalconditionsandtheprocessofincipientspeciation. linkagemapwillimprovegenomicresourcesforcichlidfishesin
Researchershave longquestionedwhy somecichlid fishesevolve general, and Neotropical cichlids in particular, and along with
in such a diverse and fast manner compared with other vertebrate whole-genome sequencing, contribute to insights into how cichlids
lineages (Meyer 1990a; Meyer 1993; Wittbrodt et al. 1998), or even achievedtheirastonishingphenotypic variability.
otherfishesin the sameenvironment(Elmeretal.in press).Behav-
ioral and morphological characters that have been proposed to be
MATERIALSAND METHODS
responsiblefortheelevatedspeciationrateofcichlidsincludeparental
care,sexualselection,functionaldesign,andphenotypicplasticityand Pedigree
polymorphisms (Meyer 1987; Stiassny and Meyer 1999; Salzburger A wild-caught male A. zaliosus (2007, Lake Apoyo, Nicaragua) and
2009). A particular genomic architecture, perhaps derived from awild-caughtfemaleA.astorquii(2007,LakeApoyo,Nicaragua)were
whole-genome duplications, has been proposed as a driver of rapid reared separately in the laboratory until sexual maturity and then
response to selection and higher diversification rates in fishes in crossedtoobtainF hybridprogeny.Theoffspringwereraisedtosexual
1
66 | H.Recknagel,K.R.Elmer,andA.Meyer
maturity in a community tank to allow full-sib pairing. When a pair sorted by individual. Processing of the 347 F -offspring individuals
2
formed,theyweremovedtoaseparatetankandafterspawningnatu- wasperformedusingthedenvo_map.plscript,whichcreatesstacksof
rallytheireggswereharvestedandrearedinanothertank.F offspring loci (i.e., RADSeq stacks) within individuals, followed by building
2
werekilledwhentheyreachedapproximately2cmtotallengthtoget a catalog of loci present in the two parents and then mapping all
enough tissue for DNA extraction. The F pair that had the greatest stacks from the offspringtothe parentalcatalogandfinally creating
1
numberofF offspringwaschosenforconstructionofthelinkagemap. theoffspringgenotypesforthoseloci.MultipleSNPsinoneRADSeq
2
stack were treated as a single polymorphic marker (i.e., one RAD
ddRADSeq librarypreparationandsequencing marker). The parameters were optimized and set as follows:
GenomicDNAwasextractedfromthetwoparents(“Pgeneration”), the minimum amount of identical reads to create a stack in each
theF hybridpairand347F individualsusingtheQIAGENDNeasy parent and progeny was set to three (2m 3, 2P 3) the number of
1 2
Blood&TissueKitfollowingthemanufacturer’sinstructionsinclud- mismatches allowed between loci from the parents was set to three
ing RNase A treatment. Then, 1 mg of DNA template from each (2n 3), and highly repetitive RAD tags were removed or disjointed
individual was double-digested using the restriction enzymes PstI- (2t),otherparametersweresettodefault.
HF(20units/reaction)andMspI(20units/reaction)inonecombined JoinMap4(VanOoijen2006)wasusedtoperformlinkage-map-
reaction for 3 hr at 37(cid:2). Subsequently, each fragmented sample was pinganalysis.GenotypeswereexportedfromStacksandwerefiltered
purifiedusingtheQIAGENMinEluteReactionCleanupKit,elutedin toonlyincludethosemarkersforwhichthereweregenotypecallsinat
20 mL of Elution Buffer (EB), and quantified. Fragments were then least90F2individualswithacoverageofatleast5·.Linkageanalyses
ligatedtoP1adaptersthatbindtoPstI-HFcreatedrestrictionsitesand wereperformedusingallmarkers(ofthetypeaa/bb,aa/ab,ab/aa,ab/
P2adaptersbindingtooverhangsgeneratedbyMspI(seeSupporting ab,ab/ac,ab/ccinthePgenerationasdefinedbyStacks)containing
Information,TableS1foradaptersequences).Ineachreaction,~0.4mg allelesfromtheF1pairthatcouldbematchedwithconfidencetoeach
ofDNA,1mLofP1adapter(10mM),1mLofP2adapter(10mM), parentalunitanddidnotdeviatefromMendeliansegregationratios.
1 mL of T4 ligase(1,000 U/mL), 4mL of 10· T4ligation buffer,and Markers under segregation distortion (P = 0.0520.001) were added
double-distilledwaterwerecombinedtoatotalvolumeof40mL.The stepbystepfromthelesssignificanttothemostandtheireffectonthe
ligationwasprocessedonapolymerasechainreaction(PCR)machine map was checked. Marker groupings were calculated using an inde-
usingthefollowingconditions:37(cid:2)for30min,65(cid:2)for10min,followed pendent logarithm of odds (LOD) score of 6.0, but the number of
byadecreaseintemperatureby1.3(cid:2)perminuteuntilreaching20(cid:2). calculatedlinkagegroupsdidnotchangebetweenLODscoresof6.0
After we ligated each individual’s DNA to the adapters, samples toa more stringent LOD scoreof 10.0. Eachlinkage group was cal-
were pooled and size-selected from an agarose gel. A preliminary culatedusingtheregressionmappingalgorithmandKosambi’smap-
IlluminasequencingruncontainingtheparentalandF DNArevealed ping function (Kosambi 1943) with the following thresholds:
1
that the pilot analysis gel size range of 2502500 bp resulted in se- recombination frequency of 0.400, LOD value of 1.0, and a good-
quencingtoomanyfragmentsandthereforearangeof~3302400bp ness-of-fitjumpof5.00.Markerorderwasoptimizedbyperforming
waschosenforsubsequentlibraries.Manualsizeselectionfromaga- aripplefunctionaftereachaddedlocus.
rose gelelectrophoresiswasperformedonthe library containingthe
parental and F samples and samples were then cleaned using the Comparativeanalysis
1
QIAGEN MinElute Gel Purification Kit and eluted in 10 mL of EB. All RAD markers used in the linkage map were indexed with the
Fifty F individuals were pooled per library and size selected using respectivelinkagegroupandpositionandcomparedwiththegenomes
2
Pippin Prep technology (Sage Science, Beverly, MA). Seven PCRs of the fishes tilapia (Oreochromis niloticus; version from 25.01.2012),
(severalarenecessarytominimizePCRbias)wereperformedinrep- stickleback(Gasterosteusaculeatus;versionBROADS1.61),andmedaka
licateonsize-selectedfragments[(Petersonetal.2012)Figure1].Each (Oryzias latipes; version MEDAKA1.48) using blastn searches and an
PCRcontained10220ngoflibraryDNAtemplate, 4mL of dNTPs e-value cut-off of 10215 for tilapia (a basal cichlid) and 10210 for
(100mM),4.0mLof5·PhusionHFbuffer(NEB),0.2mLofPhusion sticklebackandmedaka.Tilapiawasusedasarepresentativeofthe
Taq polymerase (NEB), 1.0 mL of each RAD primer (10 mM), and African cichlid lineage, whereas stickleback and medaka were used
filled up to 20 mL with double-distilled water. PCR conditions were asphylogeneticallycloseteleostoutgroupstothecichlids.Ifamarker
performed as follows 98(cid:2) (30 sec), [98(cid:2) (10 sec), 65(cid:2) (30 sec), 72(cid:2) mapped to more than one locus it was either excluded or, if the
(30 sec)] · 10, 72(cid:2) (300 sec). Gel electrophoresis was performed on differenceinthebitscorebetweenbesthitandsecond-besthitwas
pooled products to remove remaining oligo dimers and other con- more than 20, the best hit was retained. Markers that mapped to
taminants.Fragmentsrangingfrom330to400bpwereexcisedfrom unlinkedscaffoldswereexcludedfromthesyntenyanalyses.Synteny
a gelafter electrophoresis and cleaned usingthe QIAGENMinElute wascalculatedinpercentageofmarkersmappingtoasingleorthol-
GelExtractionKitandelutedin10mLofEB.Theeightlibraries(one ogouslinkagegroupincontrasttothosemappingtodifferentlink-
containing the parental and F DNA and the remainder seven each agegroups.Markersthatsolelymaptoasinglelinkagegroup,i.e.,ifno
1
containing 50 F individuals) were diluted to 1 nM and sequenced othermarkersmappedtothatsamelinkagegroup,wereexcluded.
2
singleend to150 bp lengthwiththe IlluminaTruSeq SBS Kit v5in
eightlanesonanIlluminaGenomeAnalyzerIIx(GeCKo:Genomics Mutationrateestimation
CenterUniversityofKonstanz)overtwoconsecutiveruns,eachcon- Denovomutationsareexpectedtoberecognizedashigh-confidence
tainingasinglePhiXcontrollane. SNPs present in one or a few offspring but not in the parents. Loci
from two F individuals and eight randomly chosen F individuals
1 2
Linkagemappinganalysis with high coverage were compared to the parental (P) loci and
Bioinformatic processing of the reads (i.e., RADSeq tags) was per- checkedforSNPsonlypresentintheprogeny.Thecoverageforalocus
formed using Stacks (Catchen et al. 2011). Reads were trimmed to tobeincludedintheanalysiswassettoatleast8·(minimally4·per
alengthof110bp,cleanedfromerroneousandlow-qualityreads,and alleletoreducethechanceofcandidatenewmutationsbeingsequencing
Volume3 January2013 | MidasCichlidLinkageMap | 67
Figure1 SchematicoftheddRADSeqprotocol.Arrows
indicate restriction sites for a rare-cutting enzyme in
green and a frequent-cutting enzyme in orange. P1
adapters specifically bind to the rare-cutting enzyme
(green) and P2 adapters bind to the frequent-cutter
(orange)restrictionsites.Eachindividualcontainsadif-
ferentbarcodeoffivenucleotidebasesspecifiedbythe
P1adapter.SequencedRADtagsstartwiththebarcode
(individual one: red barcode, individual two: blue
barcode). The bioinformatic software Stacks allows for
the detection of single nucleotide polymorphisms
between individuals (outlined by red box in sequence
alignment).
errors).Inaddition,anempiricaldistributionofallele-to-allelecoverage analyses.Oftheseloci,755wereinformative(allelesthatsegregatedin
ratio was estimatedfrom allheterozygous RAD markers in the F a1:2:1segregationratiointheF generation)andcouldserveforthe
2 2
generation. New mutation candidates in the progeny were checked constructionofthelinkagemap(Figure2).Becausedifferentlibraries
against this distribution, and only those mutations that had an allele didnotexactlycontainthesamelociandsomeofthelocididnotpass
ratio within the 95% confidence interval of the loci present in the thecoveragethreshold,weobtained~25%missingdata.Almost50%
empiricallyestimateddistributionwereretained.Thisstepyieldedloci ofthelociwerepresentinmorethan300individuals(FigureS1).
witharatiolowerthan0.5orgreaterthan2.0tobediscardedandwas
performedtoexcludecandidatelociwithoddallele-to-alleleratios(e.g., Linkagegroups
repetitivesequencesconfoundedwithalleles).Forexample,alocuswith Of the 347 F2 individualsgenotyped with ddRADSeq,four were ex-
amutationcandidateand10readsintotal,includingsevenreadsofthe cludedbecauseoflowcoverage(,6·perindividual;seeTableS3for
originalalleleandthreereadsoftheallelewiththenewmutation,would genotype data). From linkage mapping analysis, 25 linkage groups
beexcludedbecausetheallele-to-alleleratioislowerthan0.5. were identified with a total size of 1427 cM and an average marker
spacingdistanceof1.95cM(Figure2,TableS4).Linkagegroupswere
RESULTS between51.5and77.1cMinlengthexceptforthetwosmalllinkage
groups LG24 and LG25 with 2.5 and 2.0 cM, respectively. With the
SNPandmarkerdetection
exceptionoftwosmalllinkagegroupswithonlytwoandthreelinked
Atotalof254millionreadswereused,withanaverageof31.8million markers, all linkage groups consisted of at least 10 markers (mini-
reads per library (SD: 3.1 million reads). Onaverage,approximately mum: 10 markers, maximum: 54 markers). On average, a typical
588,000readswereobtainedperindividual(SD:201,454reads)with linkagegroupcomprised30markers(TableS4).Atotalof17markers
15· coverage per read (SD: 5.1·). Within the parental A. astorquii (2.3%ofallmarkers)werecompletelylinked(norecombinationwas
individual,aSNPfrequencyof0.80SNPs/kb(11,172SNPsin139,109 observed between them). The maximum distance between two
loci)andforA.zaliosus0.89SNPs/kb(12,764SNPsin142,740loci) markersonasinglelinkagegroupwas30.7cMonLG20.
were detected (Table S2). Of 118,213 loci that were scored in both
parents,12,429werepolymorphicwithonetothreeSNPs,withatotal Segregation distortion
of 15,035 SNPs and a frequency of 1.2 SNPs/kb. Of those loci that Almost 10% of all markers (74 of 755 markers) deviated from
were polymorphic, 18.5% were fixed between parents. A total of Mendeliansegregation ratios witha P-value between0.05and 0.001
38,203lociweredetectedintheparentsandwerepresentin atleast (TableS3).MarkersthatweredistortedsignificantlywithP,0.0001
90 of the 343 F individuals. The number of loci shared between werenotconsideredbecauseratiosthataretooskewedmightcreate
2
parents and F progeny was smaller than that between parents and inaccurate distances and false linkages (Cervera et al. 2001). Closely
2
F becausetheF progenygelsizerangewasnarrowerandtherefore linkedmarkerswithsimilarpatternsofsegregationdistortionproba-
1 2
included fewer fragments. A total of 3,184 loci that were shared be- bly can be attributed to biological causes, rather than genotyping
tweenparents,F andF swerepolymorphicandretainedforfurther error. Of these 74 markers, in fact 19 could be found on LG1 (39%
1 2
68 | H.Recknagel,K.R.Elmer,andA.Meyer
genome, excluding those markers that mapped to a single linkage
groupwithonlythisonesyntenicmarker),abasalAfricancichlidthat
last shared a common ancestor at least 60290 million years ago
(SalzburgerandMeyer2004).Fourlinkagegroups intilapia (LGs 1,
4,10,and21)andsixlinkagegroupsinMidas(LGs8,12,16,23,24,
and25)arenot representedby morethan two markers thatmap to
anyorthologouslinkagegroup.Midascichlidlinkagegroup23map-
pedto14lociofdifferentscaffoldsinthetilapiagenome.Fortheother
five Midas linkage groups that did not map to orthologous linkage
groupsintilapia,thisiscausedbyaninsufficientnumberofmarkers
usedtoanchorthetilapiagenome(TableS5)orsmalllinkagegroup
sizes(duetoinsufficientdataintilapiagenomicresources)intilapia.
In addition to the linkage groups that share homologous markers
(conservedsynteny),therespectivepositionofthesemarkers(linkage
order) is mostly the same,suggesting retained syntenic relationships
among those loci(Figure 3). However, divergent marker orders, pre-
sumablycausedbyintrachromosomalrearrangements,canbedetected
tosomeextentbetweentheMidascichlidandtilapia(e.g.,LGs13,15,
and21intheMidascichlid).Ingeneral,however,itisclearthatoverall
levels of synteny between these two cichlid lineages are quite highly
conserved, with 86% of all markers mapping to a single orthologous
linkage group when those linkage groups containing two or more
markersareconsidered.
Asexpected,fewerhomologousmarkerswerefoundforstickleback
(44markers)andmedaka(38markers)comparedwithtilapiaandthe
Midascichlid(Figure3).Thesticklebackshowsmanymarkersofcon-
servedsynteny(78%)withtheMidascichlid.Incontrast,fewermarkers
showconservedsynteny(38%)betweenmedakaandMidascichlids.
Mutationrate
TwonewmutationsintheF progenyandfivenewmutationsinthe
1
F progeny were found. The same mutation was observed in two
2
differentF individualsandwasthereforeonlycountedasonemuta-
2
tion.TheF mutationratewascalculatedto7.4·1028andtheF 5.8
1 2
·1028,leadingtoanaveragemutationrateof6.6·1028mutations
pernucleotidepergeneration.
DISCUSSION
Figure 2 Linkage groups constructed with 755 RAD markers and Here,wereportthefirstlinkagemapofaNeotropicalcichlidspecies.
ordered by size. Blocks of segregation distortion are indicated by
Itwillserveasimportantadditiontothegenomicresourcesforcichlid
brackets.Markersusedforsyntenyanalysesareshowninorange.For fishesandfacilitateinsearchingforgenesthatcontributetoecological
furtherinformationonmarkerIDs,position,segregationdistortionand
andphenotypicdiversityin Midascichlids inparticular andcichlids
markersusedinsyntenyanalysessee,TableS3.
moregenerally.Withatotalof25linkagegroups,thepresentlinkage
map closely matches the number of chromosomes (n = 24) in the
distortedmarkersonLG1)withmostofthemdirectlystrungtogether. Midascichlid(Feldbergetal.2003).ThesmallgroupsLG24andLG25
Inallthesemarkerstheratiowasskewedtowardahigherfrequencyof mightrepresentlargerlinkagegroupsorcollapsewithanotherlinkage
the paternal allele. Apartfrom the large segregationdistortion block groupwhenfurthermarkerscanbeincorporated.Alternatively,inthe
onLG1,fourothersmallerblocksofsegregationdistortionwereiden- caseofLG24,markersingreaterdistancemightnothavebeeniden-
tified:fiveadjacentmarkersonLG6,threemarkersincloseproximity tifiedbecauseofhighlydistortedsegregationratiosratherthanmiss-
atthedistalpartofLG15,sevenmarkersonLG23spanningadistance ing genotypes. Significant parts of a linkage group might be
of~15 cMandallthree markersonLG24.LG6 andLG24distorted completely missing if it contains distorted regions (Cervera et al.
markers exhibit a higher ratio of the maternally inherited allele, 2001;Doucleff et al.2004).Alternatively,the distortedmarkers con-
whereasdistortedmarkersbothonLG7andLG23displayanexcess stitute false linkages (e.g., Nelson et al. 2010), though at least in the
of heterozygotes. Thus, half of all distorted markers (37 of 74) are caseofLG24thisseemsimprobablegiventhatallthreemarkersarein
placedonthesefiveblocks(Figure2,TableS3). very close proximity and a small number of recombination events
haveoccurredbetweenthem.
Comparative genomics Severalgenesassociatedwithphenotypicdivergencebetweencichlid
The vast majority of markers present in any given particular Midas species have been identified to date (Kijimoto et al. 2005; Kobayashi
cichlidlinkagegroupalsomaptoasingleorthologouslinkagegroup etal.2006;AlbertsonandKocher2006;Salzburgeretal.2007;Roberts
in tilapia (92 of 107 markers that uniquely mapped to the tilapia et al. 2009; Fan et al. 2011; Roberts et al. 2011). In African cichlids,
Volume3 January2013 | MidasCichlidLinkageMap | 69
Figure 3 Oxford grid showing
relationship between Midas
cichlidandotherteleostfishes.
Eachlinkagegroup(inthecase
of medaka each chromosome)
issize-normalizedtoequalone
and marker positions were
placedrelativetotheirrespective
linkage group (or chromosome)
size.Oxfordgridsshowingmarker
relationships between (A) tilapia
andMidascichlid,(B)stickleback
and Midas cichlid, and (C) me-
daka and Midas cichlid. Color
refers to markers found on a
Midaslinkagegroup.
a specific linkage group (LG5) has been proposed to constitute a of loci, more missing data are accepted, which might result in in-
“speciation chromosome” containing genes responsible for sex deter- correct distances in linkage map construction and in QTL analyses
mination,toothshape,visualsensitivity,andcolorpattern(Streelman (HackettandBroadfoot2003).
and Albertson 2006). This linkage group is orthologous to the Midas To perform synteny analyses and achievea high marker density,
LG4.Itwillbeinterestingtotestwhetherthesegenesandthisparticular we allowed more missing data for the purpose of this study.
linkagegrouparealsoimportantfortraitsunderselectioninNeotrop- Compared with the five other linkage maps that have been
icalcichlids. constructed with RADSeq technology so far (Amores et al. 2011;
As the number of genetic markers that can be quickly generated Baxter etal. 2011; Chutimanitsakun etal. 2011; Pfender etal. 2011;
hasexplodedwithnewsequencingtechnology,thelimitingfactorfor Parnell et al. 2012), the map of the present study has the second
constructingahigh-resolutionlinkagemapisthebiologicalresources: highestnumberofprogenygenotypedformarkersandhenceahigh
the number of individuals and recombination events. Thousands of number of recombinations. The gar linkage map consisted of more
markerscanbeproducedinashorttime withddRADSeqbut high- markers(~5500),althoughitwasbasedonfewerindividuals(Amores
resolutionmappingcanonlybeachievedbymaximizingthenumber et al. 2011). The average spanning distance between two markers is
ofrecombinations, i.e., the individuals. Underlaboratory conditions, 1.95cMinourstudy,whichisthelowestvaluepublishedtodatefor
Midascichlidscanspawnnaturallyalmostoncepermonthwithmore a RADSeq study. In comparison with the published cichlid linkage
than a 300 offspring per clutch, which allows for harvesting a high maps from A. burtoni (Sanetraet al. 2009), tilapia(Lee etal. 2005),
number of individuals. Because of the Midas cichlids’ amenable life andLakeMalawicichlids(Albertsonetal.2003;Parnelletal.2012),
history,wewereabletocreateageneticmapbasedonmorethan340 thepresentMidascichlidlinkagemaphasthesecondhighestnumber
F individualsfromoneF family.Thenumberofmarkersthatcanbe of individuals, the highest number of markers, and lowest average
2 1
generatedandtheamountofmissingdatathatisacceptedconstitute distance between two markers. This result highlights the promise of
a trade-off. When minimizing missing data, markers that are not next-generation sequencing based genotyping, and especially the
present in all individuals (e.g., through library effects or coverage ddRADSeqprotocol,for linkage mapping experiments whenbiolog-
thresholds) will be lost. Accordingly, while maximizing the number icalmaterialissufficient.
70 | H.Recknagel,K.R.Elmer,andA.Meyer
AlthoughA. astorquiiandA. zaliosus haveclearly distinct ecolo- ofconservedsyntenywiththesespeciesislowerthancomparedwith
gies,their divergence timeisveryrecent[shouldbe lessthan 10,000 tilapia and (2) medaka shares considerably less conserved synteny
years (Barluenga et al. 2006) and maximally 20,000 years (Kutterolf with the Midas cichlid compared with stickleback. As chromosomal
etal.2007)].Forthisreason,only(cid:1)10.5%ofourmarkerswerepoly- rearrangements leading to linkage and synteny disruptions accumu-
morphic and of those 18.5% were fixed between species. Given this late with time, stickleback (divergence time ~140 mya, Matschiner
young divergence, it is of great interest how these species developed et al. 2011) and medaka (divergence time ~100 mya, Matschiner
and maintain reproductive isolation. Besides premating isolation, et al. 2011) display less conserved synteny to the Midas cichlid, as
chromosomal rearrangements or genetic incompatibilities might act expected,comparedwiththeyoungersplitofAfricanvs.Neotropical
as isolating barriers to gene flow in postzygotic reproduction cichlids. However, synteny disruption seems to be greater between
(reviewed in Coyne and Orr 2004). As the species are young and medaka and the Midas cichlid compared with stickleback, which
can be crossed easily, incompatible loci seems more probable than wasunexpectedbecausemostphylogeniesplacethesticklebackbasal
large genomic rearrangements (Moyle and Graham 2005). Genetic tocichlidsandmedaka(Chenetal.2003;Mabuchietal.2007;Azuma
incompatibilitiesbetweenspeciesdetectedonthebasisofsegregation etal.2008,Matschineretal.2011).Incorrecthomologydetermination
distortionhavebeensuggestedinmanycases,includinglakewhitefish or mapping errors might account for a lower degree of conserved
(RogersandBernatchez2007;Rogersetal.2007),Drosophila(Phadnis synteny between the Midas cichlid and medaka. Alternatively, the
andOrr2009),Nasonia(Gadauetal.1999;Niehuisetal.2008),and medaka lineage has undergone considerable more rearrangements
someplantspecies(Fooladetal.1995;Whitkus1998;Fishmanetal.
comparedwithcichlidsandstickleback,and/orphylogeneticrelation-
2001;Myburgetal.2004).
ships are incorrect, as relationships between these teleosts remain
Inourlinkagemappinganalysis,wefoundthat10%ofthemarkers
controversial(Chenetal.2003;Steinkeetal.2006).
deviated(P,0.05)fromexpectedMendeliansegregationratio.Single The mutation rate(u) estimation of 6.6 · 1028 persiteper gen-
markersdeviatingfromtheexpectedratiomightresultfromgenotyp-
erationinMidascichlidsindicatesasomewhatelevatedmutationrate
ingerrors,thoughblocksofdeviatinglinkedmarkersasonLGs1,6, compared to estimates from other vertebrates [mouse: 3.8 · 1028,
15,23,and24probablyhaveabiologicalbasisandmightbecausedby human: 3.0 · 1028 (Lynch 2010)]. A mutation rate based on the
segregation distortion genes (e.g., Lyttle 1993) or genetic incompati-
synonymous substitution rate and UTRs from pooled Midas cichlid
bilitiesbydifferentialfixationofallelesbetweenparents(Dobzhansky expressedsequencetagsyieldedanestimate(~1.25·1026persiteper
1937;Muller1942;FishmanandWillis2001).Ifgenomicincompat-
year)higherthanthatofthepresentstudy,althoughitwasthoughtto
ibilities between species exist, chromosomal regions should consis-
be elevated by population-level polymorphism (Elmer et al. 2010a).
tentlyexhibitsegregationdistortion.Asourdataonlyoriginatesfrom
FormitochondrialDNAcontrolregion,thesubstitutionratehasbeen
a single family, further testing in laboratory bred or wild A.astorquii estimatedas7.1·1028persiteperyear(BarluengaandMeyer2004),
andA.zaliosushybridswillbenecessary(DanzmannandGharbi2001)
which,despitethefactthatcontrolregionshouldhaveahighmuta-
torevealwhetherthefivelinkagegroupsexhibitingblocksofdistorted
tion rate, is fairly well in agreement with our current genome-wide
markersresultfrominterspecificgeneticincompatibilities.
mutationrateestimate.However,allcalculationsofmutationorsub-
Genomic architecture has been suspected of being a main factor
contributingtothecichlids’evolutionarysuccessintermsofbiodiver- stitutioninrecentlydivergedpopulationsdifferbecauseoftheroleof
lineage sortingindecreasingrates withtime(i.e., pedigreevs. popu-
sity(Salzburger2009).Thecomparativegenomicanalysisbetweenthe
lation vs. phylogenetic rates) [see e.g., (Ho et al. 2005; Subramanian
Midascichlidandthetilapiarevealsahighlyconservedsyntenywith
etal.2009)].Althoughvertebrateestimatesonlyincludemodelspecies
86% of the markers mapping to a single orthologous linkage group.
Thisfindingpointstowardahighlevelofconservedsyntenybetween such as human, mouse, and rat, this finding is congruent with in-
terspecific comparisons of mutation rates in teleost compared with
Neotropical and African cichlids, in spite of their relatively ancient
mammalianlineagesthatrevealedthatteleostsexhibitahigherneutral
split(60myaaccordingtoVencesetal.2001;Matschineretal.2011;
mutationrate(RaviandVenkatesh2008).Itmightbearguedthatan
but see Farias et al. 1999; Salzburger and Meyer 2004; Azuma et al.
elevatedmutationratemightexplaintheteleostmorphologicaldiver-
2008).Arelativelyhighlevelofconservatisminnumberofchromo-
sityandspeciesrichness,sincethechancethatabeneficialmutation
somesacrosscichlidgeneraandspeciesisobserved(seeFeldbergetal.
arises on which selection can act is increased. More estimates of
2003), indicating that interchromosomal rearrangements are rare.
Thisobservationandourresultssuggestthatatleastlarge-scalechro- vertebrate mutation rates are needed in order to ascertain whether
mosomalrearrangementsareprobablynotamainfactorcontributing thecichlidlineage,andspecies-richlineagesingeneral,exhibithigher
tothespeciesrichnessincichlidsandalsoagreewithpreviousstudies mutationrates.
demonstrating a high degree of conserved synteny between cichlid Cichlidfishesrepresentwonderfulexamplesforphenotypicdiversity
genomes(Sanetraetal.2009).Asacomparison,thehumanandlemur and convergence, rapid speciation, and adaptive radiation (Fan et al.
lineages diverged~74 mya (e.g., Huchon etal. 2007), althoughtheir 2012).Yet,wehavestilltounderstandhowtheunderlyinggeneticsof
numberofchromosomesdiffersconsiderably(Homosapiens:23chro- thisdiversityoperatetotranslateintotheubiquitousphenotypicrichness
mosomes; Microcebus murinus: 33 chromosomes), including several of this evolutionary lineage. The present linkage map adds valuable
interchromosomal rearrangements that occurred after their split genomic information to tackle these fundamental questions in evolu-
(Dutrillaux1979). tionary biology. Future genome-wide analyses on the level of popula-
Medaka and stickleback show less conserved synteny with the tions,species,andamongdistantlyrelatedlineagesincichlidfisheswill
Midascichlid(medaka:38%,stickleback:78%markerswithconserved profitfromthisresourceanditwillhelptoshedlightonthegenomicsof
synteny). Although the data for genomic comparisons between the adaptationinthisextraordinarilydiverseevolutionarylineage.
Midascichlidandthenoncichlidfishspeciessticklebackandmedaka
are limited because of the large phylogenetic distance of more than ACKNOWLEDGMENTS
100millionyearssincetheselineageslastsharedacommonancestor WethankF.Henningforhelpfulconversations.WethankS.Selent,
(Matschineretal.2011),itallowsfortwoconclusions:(1)thedegree C.Chang-Rudolf,andL.Beckforlaboratoryassistance.Wealsothank
Volume3 January2013 | MidasCichlidLinkageMap | 71
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