Utility of H3-genesequences for phylogenetic reconstruction - a case study of heterobranch Gastropoda

2004·5.8 MB·English

by  Angela Dinapoli

Bonnerzoologische Beiträge Band 55 (2006) Heft 3/4 Seiten 191-202 Bonn, November2007
Utility of H3-Genesequences for phylogenetic reconstruction
- a case study of heterobranch Gastropoda -*
Angela Dinapoli", Ceyhun Tamer", Susanne Franssen",
Lisha Naduvilezhath" & Annette Klussmann-Kolb"
)DepartiTient ofEcology, Evolution and Diversity - Phylogeny and Systcmatics, .1. W. Goethc-Univcrsity,
Frankfurt am Main, Gemiany
*Paperpresentedto the2nd Intemational Workshop on Opisthobranchia, ZFMIv, Bonn, Germany, September20th to 22nd, 2006
Abstract. In the present study we assessedthe utility ofH3-Genesequences forphylogenetic reconstruction ofthe He-
terobranchia(Mollusca,Gastropoda).ThereforehistoneH3 datawerecollected for49species includingmostofthema-
jorgroups.Thesequencealignmentprovidedatotal of246sitesofwhich 105 werevariableand96parsimony infonna-
tive. Twenty-four(of82) fust base positions were variable as were 78 ofthe third base positions but only 3 ofthe se-
condbase positions.
H3 analyses showed ahigh codon usage bias. The consistency index was low (0,210) and a substitLition saturation was
observedinthe3"^codonposition.ThealignmentwiththetranslationoftheH3 DNAsequencestoamino-acidsequences
hadno sitesthat were parsimony-informative within the Heterobranchia.
Phylogenetic trees were reconstructed using maximum parsimony, maximum likelihood and Bayesian methodologies.
Nodilittorina iiuifascicitci was used asoutgroup.
Theresolutionofthedeepernodes was limited in this molecularstudy. The data themselves were not sufficient toclar-
ifyphylogenetic relationships within Heterobranchia. Neitherthemonophyly ofthe Euthyneura nora step-by-step evo-
lutionbythe"basal"groupswassupported.AconclusionaboutthemonophylyofOpisthobranchiaandPulmonatacould
notbeextracted fromourdata becausewedid nothaveany resolution at this point.
Webelieve histone H3 alone provides nonew marker forstudyingdeep molecularevolution ofthe Heterobranchiadue
tothehigh grade ofconservation and the low phylogenetic signal.
Surprisinglytherewasagoodresolutiononthegeneralevel.Analysesconductedwithmaximumparsimonyand Bayesian
inference(usingalldata)recoveredall (ornearlyall)generamostlywith statistically significantlysupportednodes. Fur-
therstudiesfocusingon thepossibleutilityofhistone H3 forthe resolutionofrecent splits will be necessary.
Keywords. Heterobranchia, Opisthobranchia, histone H3, molecularphylogeny.
INTRODUCTION
1.
Many questions regardinggastropodphylogeny have not 2003; Knudsen et a!. 2006) in others not (Thollesson
yetbeen answered such as the molecularconfinnation of 1999). The Pulmonata and Opisthobranchia can be sepa-
theHeterobranchiaconceptbasedonmoiphological stud- ratedbycharactersrespectiveofthenervous system(pres-
ies from Haszprunar (1985, 1988). This taxon contains ence ofa procerebiaim and cerebral bodies in pulmonates
the Pentaganglionata Haszprunar, 1985 also known as and presence ofa rhinophoral nerve in Opisthobranchia
Euthyneura Spengel, 1881 (withtheOpisthobranchiaand and Pyramidclloidea). However the molecular confirma-
Pulmonata)andseveral mostly littleknown"basal"groups tion regarding the monophyly of the Opisthobranchia
(e.g. Valvatoidea, Omalogyroidea, Architectonicoidea, (VoNNEMANN et al. 2005; Grande et al. 2004a) and the
RissoelloideaandPyramidclloidea)whichpresentastep- Puhnonata(Tillieretal. 1996, Dayratetal. 2001) isstill
by-stepevolutiontowardstheeuthyneuran leveloforgan- a inatterofdebate. There is no coinprehensive investiga-
isation(Haszprunar 1988). Thehyperstrophyofthepro- tion concerning the "basal" groups. Only a few represen-
toconch is the most important autapomorphous character tative taxa (e.g. Valvatoidea- Coniirosti-apelliicida,Ar-
ofthe Heterobranchia. The Euthyneura are characterised chitectonicoidea - Pliilippea lutea, Pyramidclloidea -
bythepresenceoftwoadditional (so-calledparietal) gan- Pyramidelladolabrata) havebeen included incurrentmo-
glia. However, the monophyly ofthe Euthyneura has not lecularstudies(Colgan etal. 2000; Grande etal. 2004a,
been clarified by molecular studies, yet. In some studies 2004b).
they are recovered tnonophyletic (Colgan et al. 2000,
192 Angela Dinapoli et al.: Utility ofH3-Gene in Heterobranchia
In recent years molecular systematic analyses in gas- In the present study we wanted to test the utility ofH3
tropods have utiliseda varietyofgenetic markers,e.g. nu- genesequences forphylogeneticreconstnictionwithinthe
clear 28S ribosomal RNA and/or 18S ribosomal RNA or Heterobranchia (focusing primarily on the Opistho-
mitochondrial 16Sribosomal RNAand/orcytochromeox- branchia). We were especially interested in testing
idase subunit I (TiLLiER et al. 1994, 1996; Dayrat et al. whetherH3 is suitabletoresolvedeepernodeswithinhet-
2001; VoNNEMANN et al. 2005; Thollesson et al. 1999; erobranch phylogeny. Therefore, partial histone H3 data
Remigio & Hebert 2003). Nevertheless, new genetic were collected for 49 species including most ofthe ma-
markers are needed forthe resolution ofcertain phyloge- jor groups (Euthyneura with Opisthobranchia and Pul-
netic relationships (especially regarding deeper nodes). monataand"basal"groupswithValvatoidea,Architecton-
Pailial fragmentsofthegenecodingforthe extremelycon- icoidea, Omalogyroidea, Rissoelloidea and Pyramidel-
servative H3 protein(Maxson etal. 1983)were first used loidea).
to clarify arthropod molecular evolution (Colgan et al.
1998) and later polychaete (Brown et al. 1999), gastro- MATERIALSAND METHODS
pod(Colgan ctal. 2000,2003), polyplacophoran (Okusu 2.
etal. 2003),cephalopod(Lindgren etal. 2004) andhexa-
pod (Kii R et al. 2006) phylogcny.All studiesusedacom- 2.1. Specimens and DNA extraction
bined datasct in their approaches. In their study ofgas-
tropod phylogeny, Colgan et al. (2000) did not find a The studied taxa and the accession numbers are listed in
nionophyletic Heterobranchia while within the Euthyneu- Table Twenty ofthe49 sequences are taken from Gen-
I.
ra, the Opisthobranchia arc paraphyletic with respect to Bank. Opisthobranchiaare representedby26 species (in-
the pulmonates. Very similar phylogenetic relationships cluding 11 suborders). Nodilittoriua uuifasciata
were shown in Colcían et al. (2003). The Heterobranchia (Caenogastropoda Cox, 1960) was used as an outgroup.
as well as the Opisthobranchia and Pulmonata are rarely
recovered as monophylctic in these studies. DNAwasextractedfromethanol-preservedindividualsus-
ing the DNeasy Tissue Kit from Qiagen (Hilden, Ger-
many).
Table 1 . Taxonomicpositionsand collecting locationsofthe sampled taxa.Accessionnumbers ofsequences included in theana-
lyses (ZSM = Zoologische Staatssammiung); published sequences taken from GenBank arc marked with an asterisk.
MajorTaxon Species Locality GenBankAccessionNumber
Caenofjastropoda
Littorinoidea
Littorinidae Nodililtorinunnifusciala(Gray. 1S26) Genbank AF033705*
Conidae Conusmiles Linnaeus, 175X Genbank AF033684*
Campanilidae Cainpaiiilcsvmlialicum Iredale. 1917 Genbank AF033683*
Opisthobranchia
NudibiaiK'hia
Tethydidae Tclhystliiihria Linne. 1767 Blanes, Spain EF133468
Discodorididae Discodnrisalniiuaculala(Bergh, lXi<0) Genbank DQ280013*
Arminidae Armiiui ncaptililana (DelleChiaje. 1824) Banyuls-sur-Mer. Franee EF133469
Pleurobranchoidea
Pleurobranchidac Pli-iirohninclHiCiimcckeli Leue, 1X13 Blanes, Spain EF133470
Tylodinoidea
Unibraciilidae Uinhnuiiliiiii iimhraciiluiu (Lightfoot. 17S6) AtlantieOeean. MeteorBank EF133471
Ccphalaspidea
Scaphandi'idae Scaphanderlignariiis (Linne, 175X) Blanes, Spain EF133472
Philinidae Philineapena(Linnaeus. 1767) Genbank DQ0935O8*
Gastropteridae GastropleronmeckeliKosse, 1813 Blanes, Spain EF133473
Anaspidea
Akcridae Akera Inillata Müller, 1776 Kattegat, Denmark EF133474
Aplysiidae Aplysiaeaiiforniea Cooper, 1863 Miami, USA EF133475
Aplysiidae Aplysiacf.juliana Quoy& Gaimard, 1832 Genbank AF033675*
Aplysiidae Bursaielhi leaehiide Blainville. 1817 Dingo Beach,Australia EF133476
Thecosomata
Cavoliniidae Cliopvraniidala Linne. 1767 Canai7 Islands; Spain EF133477
Creseidae Creseissp. Genbank DQ280012*
Gymnosomata
Pneumodermatidae Pnewnodennaej. allanliea(Okcn, 1815) USA,Atlantic EF133478
)
Bonnerzoologische Beiträge 55 (2006) 193
IVliiini"TüYnn Sp6CÍ6S vJCIIUcl11tV AAlUII l^UIIIUCI
Sscoglosss
Plscobränchidtic Eh'siaíitíudo(Risso, 1818) Blanes, Spain EF133479
PlapnhriinpViiHap EhsiapnsiUa (Bergh. 1872) ("ipnHnnk [5QS14792*
Pläcobr3nchidac Elvsicicrispüfü Morch. 1863 Genbank DQ534790*
Piäcobrsnchidiic Eh'siü viridis{Montagu, 1804) Genbank r)O5'í4790*
i^vlinHrnhnlliHnf C^yliiidrohiillübcütiiiFischer 18^7 Florida USA FFI^(480
Acochlidis
Hedylopsidae Hedvlopsisspiculifera(Kovvalewsky, 1901) Rovinj,Croatia EF133481
Microhedylidae Unelagiandulifera(Kowalewsky. 1901) Rovinj, Croatia EF133482
Architcctibranciiia
Hydatinidae Micf'oiueloundatus(Brucuiere, 1792) Genbank DQ093513*
Actconoidca
ArtpnniHíip Piipcisoliditlü(Linné. 1758) DiH'^o Bcach Australia FFI^^483
Actconidac Rictüxispiiuctocaclütus(Cai'penter, 1864) Cayucos, Calitornica, USA. EFl33484
Bullinidac Bidliuüliucatü(Gray, 1825) Gcnbank AF033680*
Pulmonata
Systellommaiophora
Onchidiidac OucJiidiiiftisp. Genbank AF033706*
Onchidiidac Onchidellafloridana(Dali, 1885) Florida,USA EFl33485
Onchidiidae Onchidella sp. Genbank DQ093511*
Stylommatophora
Charopidae Hedleyoconchadelta(Pfeiffer, 1857) Genbank AF033693*
Siphonariidae Siphonariaserrata(Fischer, 1807) SouthAfrica EFl33486
Siphonariidae Siphonariaconcinna Sowerby,1824 SouthAfrica EFl33487
Siphonariidae Siphonariazelandica(Quoy& Gaimard, 1832) Genbank AF0337n*
Amphibolidae Salinatorsolida (Schacko, 1878) Genbank AF033712*
Eupulmonata
EUobiidae Ophicardelitsornatiis(Ferussac, 1821 Genbank AF033707*
"basal"Heterobranchia/Triganglionata
Pyramidelloidea
Pyramidellidae Titrbonillaláctea(Linné. 1758) Roscoff France EFl33488
Pyramidellidae Tiirbonillasp. WellnT^^ton NewZealand EFl33489
Architectonicoidea
Architectonicidae Heliacusvariegatus(Gmelin, 1791) Tropical aquarium (ZSM 200121^)3) EFl33490
Architectonicidae Philippealutea(Lamarck, 1822) Genbank AF033708*
Valvatoidea
Comirostridae Cornirostrapellucida(Laseron, 1954) VJV.11LiCL111\ AF033685*
Orbitestellidae Orbitestella vera Powell, 1940 Wellington,NewZealand EF56I623
Orbitestellidae Orbitestella sp. Leigh,NewZealand EF56I624
Omalogyroidea
Omalogyridae Omalogyrahurdwoodiana Strebel, 1908 Antarctic (ZSM Mol-20021228) EFl33491
Rissoelloidea
Rissoelidae Rissoelluelongalospira Ponder, 1966 Wellington,NewZealand EF56I622
Rissoelidae RissoellamieraFinlay, 1924 Wellington, NewZealand EF561620
Rissoelidae RissoellacystophoraFinlay, 1924 Wellington,NewZealand EF561621
2.2. DNA amplification and sequencing (Krefeld, Germany). First we sequenced only one frag-
ment per specimen. Moreover, to avoid mistakes accord-
The following degenerated primers were used: H3-F: 5'- ing to the conservative character ofthe H3 gene, we se-
ATG GCT CGTACC AAG CAG AC(ACG) GC-3' and quenced a random sample of 5 species a second time
H3-R: 5'-ATATCCTT(AG)GGCAT(AG)AT(AG)GTG whereby no varieties could be detected.
AC-3' (Colgan et al. 1998) and produced a 246 bp prod-
uct.ThePGRprofilewasasfollows: 95 °C for5 min, fol- 2.3. Sequence alignment
lowed by 35 cycles of30 s at 95 °C, 25 s at 52 °C, 45 s
at 72 °C and a final extension at 72 °C for 5 min and Taq Sequenceswerealignedmanually usingthesoftwarepack-
Polymerase, recombinant from Invitrogen (Karlsruhe, ageBioEditversion 7.0.5 (Hall 1999). The H3 DNAse-
Germany) was used.All products werepurified usingthe quences were translated into the amino acid sequences in
QIAquick Gel Extraction Kit from Qiagen (Hilden, Ger- GeneDoc version 2.6.002 (Nicholas & Nicholas 1997).
many)andsequencedinbothdirectionswithaCEQ2000 Thealignment isavailablefrom theauthors uponrequest.
Beckmann Coulterusingthe CEQ DTCS Quick Start Kit
194 Angela Dinapoli et al.: Utility ofH3-Gene in Heterobranchia
2.4. Statistical tests Bootstrapping (Felsenstein 1985) was performed for
maximum parsimony with 1000 replicates and formaxi-
Codon usage statistics were calculated using GCUAver- mum likelihood with 100 replicates.
sion 1.2 (McInerney 1997). The purpose ofthis function
istocalculatetheNumber(N) oftimesaparticularcodon The following analyses were conducted using MrBayes
is observed in an alignment and also to calculate the Rel- version 3.1.2 (Ronquist& Huelsenbeck2003): Bayesian
ative Synonymous Codon Usage (RSCU) values for the inference: a) all data (with one model forall three codon
dataset. RSCU values defme the number oftimes a par- positions), b) all data (with codon specific models) and
ticular codon is observed relative to the number oftimes c) only codon position one and two (third codonposition
that the codon would be observed in the absence ofany excluded;withonemodel forcodonpositiononeandtwo).
codon usage bias. Without any codon usage bias, the
RSCU value wouldbe 1.00.Acodon that is used less fre- For Bayesian inference a Metropolis Chain Monte Carlo
quently than expected will have a value ofless than 1.00 analysis with four chains and 1 000 000 generations was
and a codon that is used more frequently than expected performed with the first 1000 trees ignored as bum-in.
will haveavolumeofmorethan 1.00 (McInerney 1997).
RESULTS
Thedegreeofbias(^X"/''') which isthe sum oftheX" val- 3.
ues for the individual amino acids divided by the total
numberofinferredresidues (n) forthecombination ofda- 3.1. Statistical tests
ta from all species (Shields et al. 1988) was determined.
The sequences provided atotal of246 sites ofwhich 105
The substitution saturation wascalculated forall 3 codon werevariableand96parsimonyinformative.Twenty-four
positions usingthe methoddevelopedby XiAetal. (2003) (of82) firstbasepositionswerevariableaswere78 ofthe
DAMBE
implemented in the software package version third base positions but only 3 ofthe second base posi-
4.2.13 (XlA & XlE 2001). tions. Inseilion/deletionevents(indels)werenotobserved
in any ofthe groups. The amino acid alignment had no
2.5. Phylogciietic reconstruction sites thatwere parsimony-informative within the Hetero-
branchia.
Appropriate models for the analyses were selected after
running Modeltest version 3.4 (Posada & Crandall H3 analyses showedahighcodon usagebias(Tab. 3).The
1998) and using the Akaikc information criterion (AlC) biaswasprincipally againsttheuseofAandU in thethird
(see tab. 2). codonposition. X" testswereperfonnedforall aminoacids
and revealed thatthe null hypothesiswhich isthe expect-
The followinganalyseswereconducted using PAUP* ver- edequal usageofthe codonscanberejectedforallamino
sion4.0 blO(SwoFEoRD, 2002) (settings: heuristic search acids with a significance level of0,001 excepting histi-
strategy; tbr; gaps were treated as fifth bases): a) Maxi- dine (p<0,05). For aspartic acid the null hypothesis can
mum parsimony for all data and b) Maximum likelihood not be rejected (p=0.2l). The degree of bias (21X"M
for all data. showed a high value of0,617.
Table 2.Information on used models.
C'odoii-P(»siti(»ii Model Gamma distribution shape parameter Proportion ofinvariable sites
1st Position GTR+I a=equal Pinvar=0.5692
2"^' Position TVMcf+I a=eqiial Pinvai-=0.8647
3'"^' Position TVM+G a=0.8835 Pinvai"=equal
1"' and 2"^' Position GTR+I+G a=0.9167 Pinvai-=0.6909
ls',2"^' and 3'^' Position GTR+l+G a=1.0265 Pinvai-=0.5408
)
Bonnerzoologische Beiträge 55 (2006) 195
Table3. Codon Usage Bias. N = number oftimes a particular codon is observed in a datasct (alignment). RSCU values = num-
beroftimesaparticularcodon isobserved, relativetothe numberoftimesthatthecodon would beobserved in theabsenceofany
codonusagebias.Amino acids (AA) are indicated by the three letter abbreviations.
AA Codon N RSCU ' AA Codon N RSCU
Phe uuu 6 (0.08) Ser ucu 45 (1.38)
Phe uuc 142 (1.92) Ser ucc 34 (1.04)
Leu UUA 3 (0.05) Ser UCA 13 (0.40)
Leu UUG 43 (0.66) Ser UCG 8 (0.24)
Tyr UAU 2 (0.04) Cys UGU 0 (0.00)
Tyr UAC 96 (1.96) Cys UGC 0 (0.00)
UAA UGG
Ter 0 (0.00) Trp 0 (1.00)
Ter UAG 0 (0.00) Pro ecu 83 (1.36)
Ter UGA 0 (0.00) Pro ccc 107 (1.75)
Leu CUU 48 (0.74) Pro CCA 42 (0.69)
Leu cue 81 (1.24) Pro CCG 13 (0.21)
Leu CUA 2 (0.03) Arg CGU 233 (2.59)
Leu CUG 214 (3.28) Arg CGC 127 (1.41)
His CAU 31 (1.27) Arg CGA 18 (0.20)
His CAC 18 (0.73) Arg CGG 9 (0.10)
Gin CAA 41 (0.28) Thr ACU 53 (0.87)
Gin CAG 252 (1.72) Thr ACC 146 (2.38)
lie AUU 21 (0.43) Thr ACA 45 (0.73)
lie AUC 126 (2.57) Thr ACG 1 (0.02)
He AUA 0 (0.00) Ser AGU 8 (0.24)
Asn A AII U iñ r\r\\ öer OßOö \J..Oy
Asn AAC 0 (0.00) Arg AGA 74 (0.82)
Lys AAA 135 (0.61) Arg AGG 78 (0.87)
Lys AAG 306 (1.39) Ala GCU 201 (1.49)
Val GUU 9 (0.18) Ala GCC 279 (2.07)
Val GUC 89 (1.82) Ala GCA 47 (0.35)
Val GUA 4 (0.08) Ala GCG 12 (0.09)
Val GUG 94 (1.92) Gly GGU 32 (0.87)
Asp GAU 38 (0.78) Gly GGC 38 (1.03)
Asp GAC 60 (1.22) Gly GGA 72 (1.96)
Glu GAA 69 (0.70) Gly GGG 5 (0.14)
Glu GAG 128 (1.30) Met AUG 49 (1.00)
3.2. Phyiogenetic analyses
The consistency index in maximum parsimony analyses The maximum parsimony 50 % majority-nale consensus
waslow(0,210)aswell astheretention index (0.444). Us- tree(Fig. 1) showedall genera(Aplysia, Elysia. Ouchidcl-
ing the method developed by XiA et al. (2003) a substi- la, Siphonaria, Orbitestella, TurbonillaandRissoella)re-
tutionsaturationwasobservedinthethirdcodonposition coveredasmonophyletic. However,someofthebootstrap
(Ijs0,543 >¡ss.j.0,298).Tosupportthisobservationtheen- suppoils were lowandtherewasnobootstrap suppoitfor
tropyforeachpositioninthealigninentwascalculatedus- a monophyletic Rissoella. Beyond the genera level, only
ing BioEdit Version 7.0.5.2. Codon position one showed Architectonicoidea were detected as monophyletic. All
an average entropy value of0,09 whereas the value for other nodes lacked support.
thesecondcodonpositionwas0,009 and0,69forthethird
codonposition. Ifthenucleotidesoccurmoreorlessequal- The genera Orbitestella, Turbonilla, Ouchidella and
ly at a ceilain position within the alignment the entropy Aplysiaandagain, theArchitectonoicoideawerefoundto
is highest with the value of 1,36. bemonophyletic inthemaximum likelihoodanalyses.The
196 Angela Dinapoli et al.: Utility ofH3-Gene in Heterobranchia
Fig. 1. 50 % majoiity-rulc consensus tree ofmaximum parsimony of 14 most parsimonious trees based on histone H3 data set
(nucleotides), numberofparsimony infonnative characters = 96, consistency index (CI) = 0,210.
Nodilittorina unifasciata
Campanile symbolicum
Conusmiles
Cliopyramidata
Omalogyra burdwoodiana
Akera bullata
106 Bursatella leachii
Unela glandulifera
Pneumodermaof. atlántica
Cylindrobulla beauil
Aplysia californica
781 Aplysiacf.juliana
Philine aperta
Creseissp.
Cornirostrapellucida
Elysia timida
Elysiapusilla
Elysia crispata
Elysia viridis
Gastropteron meckeli
Orbitestella vera
Orbitestella sp.
Tethysfimbria
Heliacus variegatus
Philippealutea
Salinatorsolida
Pleurobranchaea meckeli
Scaphanderlignarius
Umbraculum umbraculum
Hedleyoconcha delta
Onchidium sp.
Hedylopsisspiculifera
Pupa solidula
Rictaxispunctocaelatus
Micromelo undatus
Bullinalineata
Turbonilla láctea
Turbonilla sp.
Ophlcardelusornatus
Armina neapolitana
Discodorisatromaculata
RIssoella elongatospira
Rissoella miera
RIssoella cystophora
Onchidella floridana
Onchidella sp.
Siphonaria concinna
Siphonaria serrata
Siphonaria zelandica
Bonnerzoologische Beiträge 55 (2006) 197
Fig. 2. 50 % majorityrule consensus Bayesian inference cladograni forthe histonc H3 dataset (based on nucleotides); Bayesian
posteriorprobabilities provided at the branches.
Nodilittorina unifasciata
Conusmiles
Omalogyraburdwoodiana
Campanilesymbolicum
Creseissp.
Unelaglandulifera
Philineaperta
0.771 Bursatellaleachii
Aplysiacalifornica
Aplysia of. Juliana
E Cliopyramidata
0.56 Heliacus variegatus
Philippealutea
Akerabúllate
0.73 Pneumodermaof. atlántica
0 Cylindrobullabeauii
Cornirostrapellucida
Pupa solidula
Rictaxispunctocaelatus
0.1)7
Micromeloundatus
Bullinalineata
Turbonillaláctea
Turbonillasp.
Gastropteronmeckeli
Orbitestella vera
Orbitestella sp.
Elysiapusilla
Elysiatímida
Elysiacrispata
Elysia viridis
Tethysfimbria
Salinatorsolida
Ophicardelusornatus
Hedleyoconcha delta
Onchidiumsp.
Hedylopsisspicullfera
Scaphanderlignarius
Umbraculum umbraculum
Pleurobranchaeameckeli
Discodorisatromaculata
Armina neapolitana
Onchidella floridana
Onchidellasp.
Rissoellaelongatospira
Rissoellamiera
Rissoellacystophora
Siphonaria concinna
Siphonaria serrata
Siphonariazelandica
198 Angela Dinapoli et al.: Utility ofH3-Gene in Heterobranchia
Fig. 3. 50 % majority rule consensus Baycsian inference phylogram forthe histone H3 dataset (based on nucleotides, 3^'^ codon
position excluded); Bayesian posteriorprobabilities provided at the branches.
—'Nodilittorina unifasciata
Campanile symbolicum
Omalogyraburdwoodiana
0.571 Conus miles
—
Discodorisatromaculata
—
Comirostrapellucida
—————
Micromelo undatus
•Sal—ina—torsolida
^—
Ophicardelus ornatus
Siphonana zelandica
Siphonaria concinna
Siphonaria serrata
• —
— ^—Hedleyoconcha delta
—^Onc—hidella sp.
———Onchidella floridana
— Onchidium sp.
Bullina lineata
•Rictaxispunctocaelatus
Pupa solídala
—
Unela glandulifera
Hedylopsisspiculifera
•Cylindrobulla beauii
•Elysia viridis
Elysiapusilla
Creseis sp.
Cliopyramidata
0.99 Aplysia cf.juliana
-Gastropteron meckeli
—
Scaphanderlignarius
—
Umbraculum umbraculum
Pleurobranchaea meckeli
Elysia tímida
I
0.62 •Elysia crispata
Tethys fimbria
•
0.701 •Armina neapolitana
Philine aperta
0.72 —Pneumoderma cf. atlántica
Heliacus variegatus
•
0.781 'Philippea lutea
Orbitestella vera
'
0.811 — Orbitestella sp.
Turbon^illa láctea
——
1.00 Turbonilla sp.
Akera bullata
— —
Bursatella leachii
0.53
Aplysia californica
Rissoella elongatospira
0.99 •Rissoella miera
•Rissoella cystophora
0.1
Bonnerzoologische Beiträge 55 (2ÜU6) 199
basis ofthe 50 % majority-rule bootstrap tree (tree not In this study we wanted to present a primary molecular
shown)resembledacomb. Therewasnoresolution ofthe insight into heterobranch phylogeny while simultaneous-
deep nodes. ly testingtheutilityofthegenecoding forthe highlycon-
served protein histone H3 for resolving the deepernodes
%
The 50 majority-rule consensus Bayesian inference within this taxon.
cladogram (with one model forall threecodon positions)
(Fig. 2) also recovered all genera as monophyletic. Only Unfortunately, the present study did not provide a robust
the Bayesianposteriorprobability ioxSiphonaria was low phylogenetic hypothesis for the relationships among dif-
(0.58)becauseonlyvaluesabove0.95 arestatisticallysig- ferent lineages ofHeterobranchia based on H3-Genese-
nificant. Beside thegenera level Architectonicoideawere quences. Neither the monophyly ofthe Euthyncura nora
monophyleticandtheCaenogastropodafomiedacladeto- step-by-stepevolution bythe"basal"groups wassupport-
gether with Omalogyra. All other nodes had no statisti- ed.Aconclusion aboutthemonophylyofOpisthobranchia
cally significant support. and Pulmonata could not be extracted from our data be-
cause we did not have any resolution at this point.
%
The 50 majority-rule consensus Bayesian inference
cladogram(withcodon specific models) (treenotshown) The firstto investigatethevalueofhistone H3 wereCol-
was quite similarto Figure. 2. All genera exceptRissoel- gan et al. (1998). They wanted to combine small nuclear
la andArchitectonicoidea were found to be monophyle- ribonucleic acid U2 data and histone H3 to investigate
tic.TheremainingnodesweresupportedbyBayesianpos- arthropod molecularevolution. However, partitioned da-
teriorprobabilities below 0.95. ta for H3 and U2 were incongment according to Incon-
gruence Length Difference tests. Using H3 data only,
%
The50 majoritymleconsensus Bayesianinferencephy- anomalous nodes appeared in their phylogenies while
logram (with 3'''' codon position excluded and with one some possessed decay indices of 1. Therefore, their data
model forcodonposifiononeand two) (Fig. 3)recovered were not sufficient to clarify relationships within major
only the genera Rissoella and Turhonilla as monophylet- arthropod groups.
ic while the Caenogastropoda together with Oiualogyra
weregroupedseparatelyfromtherestofthetaxa.All oth- Brown et al. (1999) investigated the DNA sequence da-
er nodes had no statistically significant support. taof34Polychaeta speciesforpartial histone H3, U2 snR-
NA and two segments of28S rDNA (Dl and D9-10 ex-
pansion regions). When using H3 only. Brown et al.
DISCUSSION
4. 1999) found a lack ofconcordance with morphological
(
results and argued that the inclusion ofall H3 data is in-
Molecularinvestigationsofdeep-level relationshipswith- appropriate for the phylogenetic levels under investiga-
in the Gastropoda have been made difficult due to a lack tions.
ofslowly evolving genes. Hence, a number ofdifferent
markers have been utilized to solve this problem. Analy- Colgan etal. (2000) and laterColgan et al. (2003) used
ses ofnuclear genes like the 28S ribosomal RNA and/or partialhistone H3 (327bp)fortheinvestigationofgastro-
the 18S ribosomal RNA have provided a number ofim- podphylogeny. InColganetal. (2000),wheretheauthors
portantinsights intogastropodrelationshipsatseveral lev- used 36 sequences ofhistone H3 only, using the chiton
els (TiLLiER et al. 1994, 1996; Dayrat et al. 2001; Von- Ischnochito)} ciiistralis as an outgroup, no cladeswere re-
NEMANN et al. 2005). The same applies to mitochondrial tained in the bootstrap analyses. H3 with the third codon
genes like the 16s ribosomal RNA (Thollesson et al. position excluded, retained only the higherVetigastropo-
1999) or the cytochrome oxidase subunit I (Remigio & da (bootstrap support = 68 %).
Hebert 2003). Colgan et al. (2000, 2003) used the his-
tone H3 protein in combination with other genes to clar- In Colgan et al. (2003) in which H3 alone was used to
ifygastropodmolecularevolution. However,manyaspects recoverphylogenetic relationshipswithinGastropoda, on-
ofgastropod phylogeny remain unclear such as the mo- lythe clade ofthe Patellogastropodawith a support of52
lecularconfirmationoftheHeterobranchiaconceptbased % was recovered. When thethird codonpositionwas ex-
on morphological studies from Haszprunar (1985, cluded, none ofthe expected groups were recovered.
1988). At the moment there is no comprehensive molec-
ular study ofheterobranch phylogeny especially one in- Okusoetal. (2003)werethe firsttoapply DNAsequence
cludingthe"basal"taxa(e. g. Pyramidelloidea,Architec- data to reconstruct the phylogeny ofthe molluscan class
tonicoidea, Valvatoidea, Omalogyroidea and Rissoel- Polyplacophora. Theiruse of59 sequences ofhistone H3
loidea). resolved deeper nodes than the mitochondrial genes did
while the strict consensus tree nested the two Gastropo-
200 Angela Dinapoli et al.: Utility ofH3-Gene in Heterobranchia
da Vivipariis georgianus and Sip/ioaariapectinaia with- (Colgan et al. 2000) and polychaetes (0.665) (Brown et
in Polyplacophora. al. 1999) and higher than the values of Drosophila
melcmogaster (FiTCH & Strausbaugh 1993) and arthro-
A combined approach to the phylogeny ofCephalopoda pods (0.37) (Colgan et al. 1998).
(Mollusca) using 18S rRNA, 28S rRNA, histone H3 and
CO! underscoredthe aim ofthe study presented by Lind- Another indication suggesting the problems ofH3 as a
GREN et al. (2004). The strict consensus tree forthe over- markerforstudyingdeepmolecularevolutionwasthehigh
all optimal parameter set for 66 sequences ofhistone H3 grade ofconservation indicated by the lack ofparsimo-
alonedidnotshowmonophylyforanyclasses investigat- ny-informative sites in the amino acid alignment.
ed.
Additional impoilant evidence was the observed substi-
KiER et al. (2006) investigated the molecular phylogeny tution saturation at the third codon position. Asaturation
of Hexapoda (supcrmatrix approach with 137 taxa; is caused by multiple-hits which render homoplasious
375bp). Theyonlyrecoveredoneordinal levelnodewhen changes. Homoplasy on the basis ofsaturation in substi-
using only H3. tution is one ofthe major problems in molecular phylo-
genetics(Tillieretal. 1996). Thisproblemgenerallybe-
According to our phylogenetic results and the results in comes more relevant at progressively higher taxonomic
thepapers listedabovehistone H3 aloneprovides nonew levels(Boore& Brown 1998). Ifnumeroussubstitutions
markerforstudyingdeep molecularevolution ofthe Het- occur at the same position, a hiding or completely eras-
erobranchiadue to the high gradeofconservation andthe ing ofthe ancient phylogenetic signal could be the result
low phylogenetic signal for deeper nodes. (Lopez et al. 1999). In order to avoid a decrease ofthe
phylogenetic information contained in the sequences, we
There were several indices defined by the results ofour excluded the third codon position in further analyses.
statistical tests supporting this assumption. We observed However, thiswasthepositionwiththemostvariablesites
ahighcodonusagebias(seetab. 2) inouralignmentwhich (78 of 82 positions) in our data set. With the exclusion
was also indicated by an increasing frequency ofC- and there was no phylogenetic information left for a resolu-
G-ending codons and fewer A- and U-ending codons tion ofthe deepernodes. Trees which resemble combs at
(Shields et al. 1988). High C+G contentat silent sitesre- the base resulted (see fig. 3). The entropy was calculated
flects the effect ofselection (Shields et al. 1988) while foreach position in the alignmentto furtherassessthe in-
selective constraints against certain codons might reduce fluenceofthe 3 codon positions.Ahighentropyvalueim-
theamount ofphylogenetic noise caused by synonymous plies that the nucleotides occuralmost equally atthis po-
substitution at either first or third codon positions sition within the alignment. An almost equal distribution
(Brown etal. 1999). However, ourdata suggest thatabias ofthe nucleotides at one position indicates a less selec-
in codon usage will not necessarily be indicative of the tive constraint allowing a higher frequency of substitu-
phylogenetic utility ofa sequence. Despite a high codon tions. The averagevalues foreach ofthe three codonpo-
usagebiasourcomputedphylogenetictrees showedapoor sitions indicate a less selective constraint for the third
resolution of the deeper nodes. An explanation for this codonpositionhence supportingthepreviousresultofthis
could be that the pressure to obtain the favomed codon codon position being saturated.
had partially obscured the phylogenetic signal. Colgan
etal. (1998,2000) made similarobservationsandconclud- It is questionable ifgiven a larger data set, the noise will
ed that apparent, high codon-usage bias as found for the eventuallysuccumbtothesignal. Therearefewexamples
H3 data does not necessarily result in high phylogenetic where the expansion ofan ambiguous datasethas result-
consistency for DNAsequences. In the studies presented ed in a convincingphylogeny (Boore & Brown 1998).
by Brown et al. (1999) a lack of agreement of the H3
analyseswith morphologyoccursdespitevery high codon Suiprisingly, there was a good resolution on genera lev-
usage bias. They concluded that whilst selective con- el. Analyses conducted with maximum parsimony and
straintsmayhavereducedtheabsoluterateofsynonymous Bayesian inference (with all data) recovered all (ornear-
substitutions, the pressure in favorof(homoplastic) resti- ly all)generamostlywith statistically significantsupport-
tution ofthe favoured codon has at least a paitially ob- ed nodes. However, our findings should be considerpre-
scuredphylogenetic signal. Hence, codon usagebiasdoes liminary and further studies including more genera are
not necessarily mean that a gene sequence will be phylo- necessarytotest the possible utility ofhistone H3 forthe
genetically useful. resolution ofrecent splits. In otherstudies (Colgan etal.
1998; Okusoetal. 2003; Lindgren etal. 2004)some(but
The degree ofbias (^^X"'") showed a high value of0,617. not all) genera were foundto be monophyletic butdue to
Itwas similarto the values observed in gastropods (0.60) the question the authors intended to answer, their studies