Bringing spiders to the multilocus era: novel anonymous nuclear markers for Harpactocrates ground-dwelling spiders (Araneae: Dysderidae) with application to related genera

2011·4.3 MB·English

by  Leticia Bidegaray-Batista

2011.TheJournalofAraclinology39:506-510
SHORT COMMUNICATION
Bringing spiders to the multilocus era: novel anonymous nuclear markers for Harpactocrates
ground-dwelling spiders (Araneae: Dysderidae) with application to related genera
Leticia Bidegaray-Batista', Rosemary G. Gillespie-and MiquelA. Arnedo': ’Institut de Recercade la Biodiversitat&
Departament de Biologia Animal, Universitat de Barcelona, Avenida Diagonal 643, 08020, Barcelona, Spain. E-mail:
[email protected]; -Department ofEnvironmental Science Policy and Management, University ofCalifornia, 137
Miilford Hall, Berkeley, California 94720-3114
Abstract. Multilocusapproachesareessentialforaccuratelyrecoveringtheevolutionaryprocessesunderlyingspeciesand
population history. However, historical inferences in non-model organisms are still almost exclusively based on
mitochondrial DNAdueto thedifficulty ofobtainingmultipleinformative loci. Here, weuseagenomiclibrarybased
approach, to generate 15 novel anonymous nuclear markers (ANMs) from the ground spider Harpactocratesglohifer
Ferrandez1986.TheANMscross-amplifyandsequencewellinthetargetspeciesanditstwoclosestrelatives,andsomeof
themalsointhemoredistantlyrelated//. ravasteUusSimon 1914. LevelsofnucleotidediversityoftheANMswithinH.
glohiferranged from0.05%to 1.4%andaveragesequencedivergencebetweenclosecongenericrelativesfrom0.02%to
13.9%,supportingtheutilityoftheselociinpopulationandspecies-levelanalyses.Moreover,across-speciesamplification
screenedinotherspidertaxashowedthatsomeofthelocicouldalsopotentiallybeusefulinmoredistantlyrelatedgenera.
Keywords: Genomiclibrary,nuclearloci,multilocusapproaches,non-modelorganisms
Multilocusapproachesbasedonunlinkedmarkers,includingboth Iberian Peninsula. From west to east, the species Harpactocrates
mitochondrial (mtDNA) and nuclear markers, are essential for an gredensisFerrandez 1986,H. glohiferFerrandez 1986andH. gurdus
accurate reconstruction of the evolutionary processes underlying Simon 1914arefoundalongtherange,showingnarrow,almostnon-
species and population history. Inferences based exclusively on overlapping distributions. As with other species (about 13) in the
mtDNA markers are hampered by processes such as sex-biased genus,theyaregenerallyfoundathighelevation(above 1000m)in
behaviors(e.g.,sex-biaseddispersal), lowdispersalabilityandsmall temperate and moist forests, suggesting a preference for cool and
populationsizes,incompletelineagesorting,introgressionorselective humidenvironments. Preliminaryphylogeneticandphylogeographic
sweeps(Avise2000;Hare2001;Irwin2002;Ballard&Whitlock2004; studieshaveshown that Pleistoceneglaciationshadamajorrolein
Wilkins2004;Kuo&Avise2005).Inrecentyearstherealizationthat structuringpopulations in Harpactocrates. These observationshave
gene trees and species trees can differ markedly in closely related led us to hypothesize that Harpactocrates species in the Sistema
specieshassparkedthedevelopmentofanewgenerationofmethods Central underwent population range expansion toward lower
for species tree inference, which rely strongly on the information elevations during cooler periods, facilitating gene flow, whereas at
provided by multiple markers (Maddison 1997; Edwards 2009). interglacial periods they retreated to high elevation refuges, which
Moreover, many studies have shown that the statistical power of then led to population fragmentation (e.g., Masta 2000; Knowles
parameterssuchasdivergencetimes,populationsizeandratesofgene 2001; DeChaine & Martin 2005; Carstens & Knowles 2007b;
flow,relyonthenumberoflociused(seeBrumfieldetal.2003;Brito Mardulyn et al. 2009; Schmitt 2009). With the aim oftesting this
& Edwards 2009 and references therein). Recent advances, both hypothesis usingamultilocusspecies/populationapproach,wehave
technological (see reviews of Hudson 2007; Thomson et al. 2010; developed 15 novel ANMs from a genomic library ofH. glohifer,
Ekblom&Galindo 2011)and methodological (e.g.. Hey& Nielsen whichcross-amplifyandsequencewellinthecloselyrelatedspeciesH.
2007; Heled& Drummond2010; Hey2010;Yang&Rannala2010), gurdusandH.gredeusisandsomeoftheminamoredistantspeciesH.
have greatly facilitated the implementation ofmultilocus analyses. ravasteUusSimon 1914.Additionally,wehavescreenedcross-species
Unfortunately, thescarcityofavailablegenomicinformationmakes amplificationinotherspidertaxainordertoevaluatetherangeand
findingmultipleinformativelociinnon-modelorganismsadaunting potentialapplicationofthenovelmarkers(seebelow).
task(Thomsonetal.2010).Thisisespeciallytrueforspiders,inwhich Weconstructed agenomic library from thepooled DNAoftwo
jautstthaehapnodpfuullatoifonmitaoncdhosnpderciiaelsalnedvelnuccalnearbemarrekleirabslythaatmpalriefiveadriaabnlde S/-/p.eegdloThoioflesr TsipsescuiemeDnsN.AToetxatlracgteinoonmkiict (DBiNotAoolwsa)sanedxtcroacntceedntruastiendg
sequenced(seee.g.,Hedin1997fornadi;Maddison&Hedin2003for viaethanolprecipitations.GenomicDNA(—10qg)wasdigestedwith
nadl. 16S and EF-1 alpha; Ayoub et al. 2007 for EF-1 gamma; EcoRI restriction enzyme (50 qL total volume). The enzyme was
Bidegaray-Batistaetal.2007forcoxl and 16S-nadl fragment;Bond selected following the recommendations of Carstens & Knowles
&al.S2t0o0c8kmfaonr2A0c0t8info5rC1;2SG-a1r6bSf&ragGimlelnetspained2I0T0S9-1foarndcoIxTlS,-2;16VSi-nnkadelt (m2i0t0o6c)h.onTdhreialgoaDlNwAasinttoo fernasgumreenttshatsmatlhleerentzhyanme1.d6idkilnoobtasceutpaitrhse
fragmentand EF-1 alpha; Musteretal.2009for ITS-1,5.8SrDNA (kb), therebyreducingthelikelihoodofcloningmtDNAfragments.
and ITS-2; DeBusschereelal.2010forcoxl and28S). EcoRlwasselectedbasedontheinformationprovidedbyacomplete
Here, we report on a method to circumvent this limitation by mitochondrial genome ofa new species ofParachtes Alicata 1964
isolating novel anonymous nuclear markers (ANMs) from a spider (pending formal description. Pons, Bidegaray-Batista & Arnedo
genomic library. Our target organisms are species belonging to the unpublished data), which is the sister genus to Harpactocrates.
ground-dwellinggenus Harpactocrates Simon 1914 (family Dysder- Digested DNA was visualized on a 1% agarose gel stained with
idae)whichinhabitthemountainrangesoftheSistemaCentralinthe ethidium bromide, and fragments between0.8 and 1.5 kbin length
506
BIDEGARAY-BATISTAETAL.—ANMSFOR HARPACTOCRATES 507
—
Table 1. PrimersequencesforfifteenanonymousnuclearlocidevelopedfromagenomiclibraryofHarpatocratesglohifer.Namesindicate
theloci,forwardandreverseprimers,PCRannealingtemperatureT(°C),sizeofampliconandGenBankAccessionNo.oftheoriginalcloned
fragment. Performancecodesindicatesuccessfulamplificationinthefollowingspecies; H. glohifer(1),H. gurdus(2), H. gredensis(3)and H.
ravastelliis(4),Parachtesromandiolae(a),Dysderuerythrina(b),Harpacteacorticcdis(c),Loxoscelesrufescens(d),Troglohyphcmtesliicifugaand
T.pedemontanus(e),NeniesiarandaandIberesiahrauni(0-PCRproductsresultingindoublebands(db)ormultiplebands(mb)areindicated
assuperscripts.
Locus Primersequences(5'-3') T(°C) Ampliconsize(bp) Accessionno. Performance
qgLl F:AGACAGCATTCAGAGTCCAAGCG 56 416 JN654497 1,2,3,4,a"’*^
R2:GCCGAAATAGTTTGAGCTCGTTTGCG
qgL5 F:TGCCCACGCCCCACTAAAATAGG 58 424 JN654498 1,2,3,4,a,b
R:GCCAGGTTGCCAGTTAAAATCACG
qgLlO F:AGCGACACATCCTTACCTGCGT 58 621 JN654506 1,2,3,4,a'’’^e
R:GCGCATCTGGAGAGCCTTTGA
qgLl2 F:TGGCACAGCAGTGGCCAGAA 55 617 JN654509 1,2,3
R:CATGTCAACCGAATAGAATC
qgL21 F:ACGCCCGAACCGACCTTTGC 58 580 JN654499 1,2,3,a‘^^f
R:ACGAGGGAGGTGCTAGAAGCG
qgL22 F;ACGATGCACCCTCGAAATGGTCG 58 508 JN6545!i 1,2,3,4,a°"’
R:TTGGCGCGGAACCTCTCAGC
qgL25 F:TGCCCACGCCACCCCTATCC 59 309 JN654510 1,2,3,4,a,b,d
R:AGGCCAACGCGAAAAGTCAGC
qgL26 F:TCCCCGGGTCACGTGGGAAG 57 586 JN654500 1,2,3,4,a"’”
R:TCCCCAACGTGAACGAACCGC
qgL28 F:GTCCCGTCGTCCGGGGTTTG 59 401 JN654508 L2,3,4,a
R:GCCACCCATGCTTTTTGTGCTCC
qgL29 F:TGGACTCCCGTTTCACAAGGCG 53 459 JN654502 1,2,3,a'"’’
R:CCACGCTATAATTGGCCCACAAGC
qgL32 F:AGCCCCAACATCCGTTTGACC 58 567 JN654503 1,2,3,4,a™'"
R:CGTCGGCAAAAGGGACCACCC
qgL33 F:ACGTGCCACACTCTCTCTCTTTCG 58 343 JN654504 1,2,3
R:TGCCTGCTCGAATCAAACCCAGC
qgL34 F:CGCGTCACCATAGGCTCTTCGC 58 408 JN654507 1,2,3,a'"'°,b''^
R:AGTTCGGTGTGTGGCCGAGC
qgL36 F:AGCGTAACGTAGGGCGTGCAG 58 627 JN654501 1,2,3,4,a"’^b"’^f
R:CGGCCGCTTGGAAGGTGGTC
qgL38 FR::TGCCACCGAAGGGCCGATTAAGCGGCACATAAGGCCGACTCAAC 58 454 JN654505 1,2.3,4,a
wereexcisedand purified from thegel using thekit QIAquick Gel ravaslellus from the Pyrenees). Each primer pair was tested in a
Extractionkit(QIAGEN). Excisedfragmentswereconcentratedvia standard25pLPCRmixwiththefollowingconditions:3minat94°
ethanolprecipitationsto8ng/pLandclonedusingtheCloneJET"^ Cfollowedby30cyclesofdenaturationat94°Cfor30s,annealingat
PCRCloningkit(Fermentas)andOneShotTOPIOcompetentcells threedifferenttemperaturesof52°C,55°Cand58°Cfor35s,and
(Invitrogen).Transformedcellswereplatedonagarplatescontaining extensionat72°Cfor1min,withafinalsingleextensionstepof72°C
ampicillin. Five hundred colonies were individually picked with a for 10min. Ofthe38 previouslyselected loci, 15producedasingle
pipette tip and added directly to a 25 pL PCR mixcontaining the band product forat least one oftheannealingtemperatures tested
primersincludedinthecloningkit.Amplifiedinsertswerevisualized and sequenced well in the three target species (Table 1). The
on a 1% agarose gel stained with ethidium bromide. Of the 500 annealing temperature of the PCR reaction was subsequently
amplifiedinserts, 206thatrangedinsizefrom 300to 1200bpwere optimized (see Table 1), and the extension time was adjusted
selectedforsequencing.Forty-onesequenceswerediscardedbecause accordingtothelengthofthefragment.
theywereeitheridenticaltoothersequencesordidnotsequencewell. Eight to 10 individualsofH. glohifer from different populations
The remaining 165 sequences were locally blasted against the were sequenced for each locus (Table2) to investigate how
Parachtes sp. mitochondrial genome to ensure that there were no informative the markers might be for population and phylogeo-
mtDNA sequencesamongtheselected inserts, and inallcasesthey graphicstudies.Inaddition,diversityindicesandpopulationstatistics
turned out to be part of the nuclear genome. Subsequently, we of the anonymous makers were compared with those of known
performednucleotideBLASTsearchesagainsttheGenBankdatabase mitochondrialgenesandnuclearintrons.Withthisaim,amitochon-
j tocharacterizesequences. Mostsequences(160outof165)reported drial fragment (IdS-nadl) spanning the 3' halfof the 16S rRNA
i!! tsnhouanbt-ssetiqhgueneyinftiwlceyarnestelneBocnLt-eAdcSofdToirnphgirtisr.meegriTohdniesr,stiygs-nueicbghahstaesdotfohnethfpeersaeetsuersneecsqeutehoanftcmeeusnlstwuierperldee oorffibtothsheoeNmasAligDsnuaHbludnreiehtcyo(dgrrnroniLgt)ie,onnatsheepacsrotuimbcuplnleiett5eI4t(-nRkaDNdiAA)alsenuudbu{tnihnieitLnl)u(,csrltpeh5ae4r)5i'nwtherarolenf
, stopcodonsineachofthesixreadingframes,andavoidingpoly-Aor amplifiedandsequencedfor10individualsof//,glohiferandasingle
j -Gruns,whicharedifficult tosequence. Theprimerpairsdesigned individualofH.gurdus,//.gredensisand//.ravastellus,respectivelly.
wereinitiallyscreenedinasetofindividualsconsistingofonefrom The16S-nadIfragmentwasamplifiedwithprimerpairsLR-N-13398
each of the three target species (//. glohifer, H. gurdus and H. (Simon et al. 1994) and Nl-J-12350 (5'-CCTARTTGRCTAR-
I gredensis)andonebelongingtoamoredistantlyrelated species(//. ARTTRGCRSATCARCCAATTG-3')andthesrp54withSRP54fl
508 THEJOURNALOFARACHNOLOGY
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Table 2. Overalldiversitymeasuresandstatisticsestimatedacross15anonymousloci,srp54intronandI6s-nadlmitochondrialfragmentfor
Harpactocratesglohifer. Samplesize(SS)indicatesthenumberofindividualssequencedforeachlocus,inbracketsthenumberofindividuals
usedintheestimationsconsideringthe0.9probabilitythresholdinPHASE.Thelength(L)inbpforeachlocusaftersequencesend-trimmingand
excludingsiteswithgaps.Thelengthsofindelsareindicatedastheyoccurredineachlocus(N/A:notapplicable,ifindelswerenotobserved).The
numberofsegregatingsites(S)andhaplotypes(H),nucleotidediversity(n),minimumnumberofrecombinationevents(Rm)ofHudson(1985),
linkagedisequilibriumstatistic(ZZ)ofRozasetal. (2001),Tajima’sDtest(D)ofTajima(1989),and theHKAtestofHudson(1987).Not
significant(ns)andsignificant(*)valuesatP<0.05ofstatisticsaftercoalescencesimulations.
H. glohifer HKATest(H.glohiferl
H.gredensis)srp54vs.
Locus SS L Indellength S H n Rm ZZ D otherlocus
qgLl 10(10) 313 N/A 8 3 0.0031 0 0.0000 -1.9072* p=0.4764ns
qgL5 10(10) 342 N/A 5 4 0.0039 2ns 0.0477ns -0.1917ns P=0.5292ns
qgLlO 9(6) 540 1,8 12 7 0.0057 0 0.1707ns -0.9653ns P=0.7549ns
qgL12 8(8) 542 1,2 16 7 0.0071 0 0.2002ns -0.8083ns P=0.2500ns
qqggLL2212 1100((190)) 442996 3,1,1,22 1156 105 00..00108480 1 n0s 00..10035670nnss -01..02047100nnss PP==00..47863489nnss
qqqgggLLL222856 101100(((1890))) 235211372 NN6,//AA1 821 623 000...000000600859 000 -000...010015077000nnss ---001...154529297909n*nss PPP===000...423385436194nnnsss
qqggLL3229 1100((99)) 439730 5, 1N1,/A3, 10 2112 65 00..00101790 1 n0s 00..01646655nnss --00..91476636nnss PP==00..59220515nnss
qqqgggLLL333364 110010(((11800))) 253651629 NN//1AA 111132 687 000...000011504782 1 n00s --000...000406477448nnnsss --000...414573782860nnnsss PPP===000...585862589040nnnsss
qgL38 10(9) 366 N/A 5 3 0.0067 0 0.2154ns 2.1431 ns P=0.3583ns
s1r6pS5-4nadl 1100((1100)) 815119 N/1A 767 98 00..00126357 71 nnss --00..00110534nnss --10..50416655nnss P= 0.-1021 ns
andSRP52rl (Jarmanetal.2002). PCRconditionswereasfollows: (qgL25) to 11.1% (qgLl) between H. glohifer and H. ravastellus,
94°Cfor2min;35X(94°Cfor35s;45°Cfor45sforI6S-uculIand 0.39%(qgL26)to8.5%(qgL28)betweenH.giirdusandH.gredensis,
from45°Cto50°Cfor35sforsrp54\72°Cfor45sforI6S-mulIand 1.3%(qgL25)to13.9%(qgL28)betweenH.gurdusandH.ravastellus,
35sforsrp54\72°Cfor5min). and 2.4% (qgL25) to 9.4% (qgLl) between H. gredensis and H.
Theallelicphaseofheterozygoticindividualswasresolvedusingthe ravastellus. Percent nucleotide diversity within H. glohifer ranged
algorithms provided by PHASE (Stephens et al. 2001; Stephens & from 0.05% (qgL26) to 1.4% (qgL33). Genetic diversity indices,
Donnelly 2003) and implemented in DnaSP v5 (Librado & Rozas recombination and linkage disequilibrium measures and neutrality
2009).Inthecasewheredirectsequencingdetectedmultiplecopiesof testswerecalculated foreach locus in H. glohiferusing DnaSPv5.
different lengths, either as a result of heterozygote individuals or Results are summarized in Table2. We applied the HKA test of
paralogy, the PCR productswereclonedand fourtoeightcolonies Hudson (1987) to assess whether levels of polymorphism and
sequenced. Cloning resultswerecompatiblewith allelesofdifferent divergencewerecorrelated, aspredicted by neutral theory. Thetest
length(i.e.,high sequencesimilarity)ratherthan paralogouscopies, was performed by comparing polymorphism in H. glohifer and
assuming that polymorphisms due to singleton mutations among divergencebetween H. glohiferand H. gredensisin theintronsrp54
sequencedcoloniesrepresentederrorsoftheTaqpolymeraseenzyme versuseachofthe 15anonymouslociandversusthemitochondrial
oCrumclmoinnigngsaerttiafla.ct2s01(0s;eeDVaiwllsaobnlanetcaale.t2a0l1.0)1.998;Calderonetal.2009; wferraegmefnotunId6.ast-naadnly.Nloeciutsh.erNorenceomobfintahteiHonKnAortlesitnskasgheodwiesdequdielviibatriiounm
The taxonomic range ofapplicability ofthe novel markers was fromneutralpredictions,althoughTajima’sDtestshowedsignificant
evaluatedbyscreeningasampleofspecimensbelongingtogroupsat negative values for the loci qgLl and qgL26. The mitochondrial
different hierarchical levels: ParachtesroniaiuliolaeCaporiacco 1949 fragment16S-nadlwas1.4timesmorevariablethantheintronsrp54,
andDysderaerythrinaWalckenaer 1802(Araneomorphae, Haplogy- and from 1.7 to 47 times more variable than the anonymous loci
nae, Dysderidae, Dysderinae), Harpactea corticalis Simon 1882 qgL33 and qgL26, which are the most and least variable ANM’s,
(Araneomorphae, Haplogynae, Dysderidae, Harpacteinae),Loxosce- respectively.
lesntfescensDufour1820(Araneomorphae,Haplogynae,Sicariidae), This study reports the first ANMs ever designed specifically for
Troglohyphantes hicifiiga Simon 1884 and T. pedenioiUanus Gozo spiders. These kinds ofmarkers, however, have been isolated and
1908 (Araneomorphae, Entelegynae, Linyphiidae), and Neniesia extensivelyappliedinotherorganisms;e.g.,grasshoppers(Carstens&
nindu Decae 2005 and Iherexia hrctiini L. Koch 1882 (Mygalomor- Knowles2006),halfbeaksand killifishes(DeBruynetal.2010a,b),
phae, Nemesiidae). Two independent PCR amplifications were oysters(Hareet al. 1996), birds(Lee& Edwards2008) and lizards
performed foreach locus using the following touchdown cycle: 94° (Rosenblum et al. 2007; Noonan & Yoder 2009). ANMs are
C2min;lOX[94°Cfor35s,63°Cfor35s(-0.5°C/cycle),72°Cfor promisingmarkersformultilocusapproachesatthepopulationand
CoIfmfitonhr]e;3a5mlpsO,lXi5f2[i°9c4aC°tiCfoonfros3ru5c3c5se.ss7,s25°a8r°CeCsfoufrmomr1a3mr5iinzs],e;7d722°i°nCCTafbfoorlre11m10.inm]i;n.15Rexsu[l9t4s° bsRpeieeccnhiaesrshdsolewv2ne0l0i7on;frKeccnleoonswtellsyetsurdeeilteasatle.(dC2a0ar0ns7dt;enrLesece&en&tKlnyEoddwwilaveresrdgs2e0d2007s0ap8;e)c.iCeaTsrh,setaesnnosvhea&ls
The average interspecific sequence divergence {p distance) ofthe ANMs designed here are potentially useful not only for the target
anonymous loci calculated with DnaSP v5 ranged from 0.02% species,butalsoformoredistantlyrelatedspeciesinthegenus(e.g.,
(qgL26)to 8.1%(qgL28)between H. glohiferand H. gurdiis,0.41% H.ravastellus),aswellasforspeciesinrelatedgenera(e.g.Parachtes),
(qgL26)to6.9%(qgL33)betweenH.glohiferandH.gredensis, 1.8% or even subfamilies (e.g., Dy.sdera, see Table 1). However, the
BIDEGARAY-BATISTAETAL.—ANMSFORHARPACTOCRATES 509
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nuclearmarkersfortheeasternfencelizard,Sceloporusimdiilatus.
MolecularEcologyNotes7:113-116. Manuscriptreceived25June2011, revised27September2011.
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