Molecular Phylogeny of the Subfamilies in Geometridae

Molecular Phylogenetics and EvolutionVol. 20, No. 1, July, pp. 65–77, 2001doi:10.1006/mpev.2001.0949, available online at http://www.idealibrary.com on

(Geometroidea: Lepidoptera)David Abraham,* ,1 Nils Ryrholm,† Håkan Wittzell,* Jeremy D. Holloway,‡

Malcolm J. Scoble,‡ and Christer Lofstedt*

*Department of Ecology, Lund University, Solvegatan 37, SE-22362 Lund, Sweden; †Department of Ecology, Evolutionary BiologyCentre, Uppsala University, Norbyvagen 18 D, SE-752 36 Uppsala, Sweden; and ‡Department of Entomology,

Natural History Museum, Cromwell Road, London SW7 5BD, Great Britain

Received June 5, 2000; revised January 17, 2001; published online May 2, 2001

(e.g., Kennel and Eggers, 1933; Cook and Scoble, 1992;

Molecular sequence data from three gene fragments

were used to examine critically a provisional phyloge-netic classification based on morphological charactersof the Geometridae, one of the most species-rich fam-ilies of moths. The sister group relationship betweenGeometridae and Drepanidae gained further supportfrom the molecular analysis, which was based on theND1 mitochondrial gene and the first and second ex-pansion segments of the 28S ribosomal RNA gene. Al-though the alignment of the second expansion seg-ment contained regions with many gaps, it providedthe most resolution of the gene fragments. Parsimonyanalysis of the combined data resulted in a cladogramin which species belonging to Drepanidae, Larenti-inae, and Sterrhinae formed monophyletic groups.The Ennominae did not form a monophyletic groupbut rather were contained within a broader monophy-letic group including Archiearinae, Geometrinae, andAlsophilinae (represented by only one species pergroup in the present study). The molecular resultswere used to explore further the relationship betweenSterrhinae and Larentiinae, the question as towhether Ennominae actually represent a monophy-letic group, and the relationships between Ennominaeand some of the other subfamilies. © 2001 Academic Press

INTRODUCTION

In common with most families of Lepidoptera, mor-phological studies have enabled many groupings ofgenera of Geometridae to be assigned, with some con-fidence, to tribes and subfamilies. However, there arealso several areas where such groupings are ill-definedor where there exists much ambiguity over relation-ships between the groups. The family Geometridae isclearly monophyletic, defined particularly on theunique structure of the abdominal tympanal organs

1 To whom correspondence should be addressed. Fax: 1146 46 2224716. E-mail: David.Abraham@ekol.lu.se.

65

Scoble, 1992; Minet and Scoble, 1999). A review ofhigher classification in the Geometridae is presentedby Holloway (1994, 1996, and 1997) and Minet andScoble (1999). Relationships of the Geometridae withother families are considered by Holloway (1998) andMinet and Scoble (1999).

We are investigating diversity in pheromone sys-tems across the geometrid subfamilies (Zhu et al.,1996; Lofstedt and Kozlov, 1997) and therefore requirethe best hypothesis of geometrid phylogenetic relation-ships to provide a context for our observations. Giventhe weak resolution in many areas of geometrid clas-sification, we conducted a pilot study on molecular datafrom a sample of Scandinavian geometrid taxa to gainadditional information to help resolve some of the prob-lems. The Geometridae have much higher taxonomicdiversity in tropical or warm temperate latitudes; somany key groups are not represented, such as thesubfamilies Oenochrominae sensu stricto, Desmobath-rinae (a segregate of the old concept of the Oenochro-minae; Holloway, 1996), Orthostixinae, and manytribes of the Ennominae and Geometrinae. In addition,the works by Holloway cited above were not all avail-able at the time that this work was in progress, sosampling of some key groups such as the Cosymbiinilineage of the Sterrhinae was omitted.

In any character set, be it morphological or molecu-lar, homoplasy and reversals may be frequent enoughto generate sufficient noise in the data to mask clearphylogenetic signals. There has also been extensivedebate in recent years on whether it is better to com-pare data sets from different sources (e.g., morpholog-ical vs molecular) for congruence in phylogenetic struc-ture or to use total data sets (see Bull et al., 1993;Huelsenbeck et al., 1996; Ballard, 1996; Baker et al.,1998). We have to adopt the former course as no mor-phological data set of the major Geometridae cladeshas been subjected to numerical cladistic analysis. Fig-ure 5b presents the most recent consensus, based on

1055-7903/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

method. Furthermore, any gross inconsistencies be-TABLE 1

LE

66 ABRAHAM ET AL.

the references cited earlier. Our approach, therefore,has been to use the molecular results to examine crit-ically parts of the provisional classification synthesizedfrom morphological observations. If the molecular re-sults show a significant correlation with the best avail-able morphological consensus, then we can havegreater confidence in the cladograms derived from each

Full Taxon Names of the Species Includedin This Study

NoctuidaeNoctuinae

Agrotis segetum Denis and SchiffermullerAmphipyrinae

Spodoptera littoralis BoisduvalDrepanidae

Drepana falcataria LinnaeusDrepana curvatula BorkhausenDrepana lacertinaria LinnaeusCilix glaucata Scopoli

GeometridaeLarentiinae

Ecliptopera silaceata Denis and SchiffermullerHydriomena impluviata Denis and SchiffermullerHydriomena furcata ThunbergOperophtera fagata ScharfenbergOperophtera brumata Linnaeus

EnnominaeAgriopis aurantiaria HubnerColotois pennaria LinnaeusBupalus piniaria LinnaeusLycia lapponaria HarrisonErannis defoliaria Clerck

SterrhinaeIdaea straminata BorkhausenIdaea aversata LinnaeusIdaea biselata HufnagelScopula immorata LinnaeusScopula ornata Scopoli

AlsophilinaeAlsophila aescularia Denis and Schiffermuller

GeometrinaeJodis putata Linnaeus

ArchiearinaeArchiearis parthenias Linnaeus

TAB

Primer Sequence

Gene Sequence

28SD1Forward GGG GAG GAA AAG AAAReverse CAA CTT TCC CTT ACG G

28SD2Forward AGA GAG AGT TCA AGAReverse TTG GTC CGT GTT TCA A

ND1Forward TTC AAA CCG GTG TAA GReverse TAG AAT TAG AAG ATC A

tween the morphological and the molecular results willhighlight weaknesses in the existing morphologicalclassification and can therefore be used to focus effortin future work on geometrid phylogenetics. The pilotsample for the molecular study enables us to assessboth the suggested monophyly of some of the main taxaand the relationships between certain major taxa.

Sequence data from three gene fragments are usedas molecular characters. The mitochondrial ND1 gene(ND1) was chosen because it is useful below the super-family level (Pashley and Ke, 1992; Weller et al., 1994;Weller and Pashley, 1995). Mitochondrial and nuclearDNA have a different evolutionary rate (Ballard andKreitman, 1995; Naylor et al., 1995); we therefore in-cluded nuclear gene fragments in this study. The firstexpansion segment (D1; Larsen, 1992) and the secondexpansion segment (D2; Belshaw and Quicke, 1997) ofthe 28S ribosomal RNA (rRNA) gene was used. In theirarticle on the classification of noctuid moths, Weller etal. (1994) successfully used a geometrid species as out-group. We used the two noctuid species Agrotis seg-etum and Spodoptera littoralis as outgroup in ourstudy. Four species of Drepanidae, which is the onlyEuropean family in the Drepanoidea (the Epicopeiidaeare restricted to east and southeast Asia), are includedin this study because Minet (1991) placed them assister group to Geometroidea. By including theDrepanidae in this study, we can confirm that theoutgroup is well chosen if the drepanid species form amonophyletic group outside the Geometridae.

MATERIALS AND METHODS

Taxa Examined

The species analyzed are given in Table 1. Allgeometrid subfamilies are represented except forthe subtropical Australasian/Oriental subfamilyOenochrominae and the Orthostixinae. Within theGeometridae, representatives of Cosymbiini (Sterrhi-nae) and Trichopterygini (Larentiinae) were not in-cluded. Within the family Drepanidae, no Thyatirinae

2

Written 5* to 3*)

Reference

A AC Larsen (1992)CT

A CGT G Belshaw and Quicke (1997)CGG G

AGG Weller et al. (1994)CAG C

s (

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CCAC

67PHYLOGENY OF GEOMETRIDAE

FIG. 1. Sequence data for 28S D1. (3), Excluded sites; (z), identical to first sequence; (2), gaps (IUB ambiguous codes used). For full taxonnames refer to Table 1.

68 ABRAHAM ET AL.

FIG. 2. (a) Sequence data for 28S D2. (3) Excluded sites; (z), identical to first sequence; (2), gaps (IUB ambiguous codes used). For fulltaxon names refer to Table 1. (b) Gap data matrix for 28S D2. (E), Gap absent; (1), gap present; (2), inapplicable.

69PHYLOGENY OF GEOMETRIDAE

FIG. 2—Continued

were included, nor were representatives of the tropical

c4

placement of the gaps) were rejected, and the align-

70 ABRAHAM ET AL.

family Uraniidae (Geometroidea). All specimens werecollected in Sweden under the supervision of Dr. NilsRyrholm, except for Ecliptopera silaceata, collected byDr. F. Schlyter. All the noctuid species were reared inthe laboratory.

Living individuals were frozen and stored at 286°C.Sequencing was done for one individual of each species.The following gene fragments were used: the ND1 genefragment (Pashley and Ke, 1992; Weller et al., 1994;Weller and Pashley, 1995), and the D1 (Larsen, 1992)and D2 (Belshaw and Quicke, 1997) segments of the28S rRNA gene. The ND1 sequence is a 500-bp frag-ment of the mitochondrial ND1 gene and is locatedbetween the 16S rRNA and the cytochrome b genes inthe insect mitochondrial genome. The nuclear DNAsequence D1 and D2 gene fragments of the 28S rRNAgene are 300 and 500 bp, respectively. The D1 genefragment is located approximately 160 bp downstreamof the start of the 28S rRNA gene (Hancock et al.,1988), and the D2 gene fragment starts 20 bp upstreamof the second expansion segment of the 28S rRNA gene(Belshaw and Quicke, 1997).

Laboratory Protocols

Single individuals were frozen in liquid nitrogen andcrushed with a modified pipette tip. DNA was ex-tracted with the QIAamp Blood and Tissue kit (Qiagen)according to the manual, with the exception that in thefinal step DNA was resuspended in 80 ml AE-buffer.DNA was stored at 220°C until further use. PCRs werearried out in 100-ml volumes containing 4 ng DNA,00 mM dNTPs, 60 mM MgCl2, PCR buffer, 20 pM

forward primer, 20 pM reverse primer, 0.4 ml Ampli-Taq (Perkin–Elmer), and H2O (Milli-Q, sterilized).Primer sequences are listed in Table 2.

PCR conditions were as follows: 95°C denaturation(2 min), 30 cycles 95°C denaturation (30 s), 40–53°Cannealing (30 s), 72°C extension (60 s), and a finalextension step at 72°C for 5 min. The annealing tem-perature varied depending on species and gene frag-ment. The PCR product was cleaned and concentratedin 30 ml TE-buffer with Millipore tubes (30,000NMWL). A 10-ml sample was examined on an agarosegel for quality and quantity before the sequencing re-action was carried out. Of the cleaned PCR product, 4ml was used in the sequencing reaction with dRhod-amine-labeled terminators (Perkin–Elmer). Both senseand antisense strands were sequenced with an ABI 310automated sequencer.

Sequence Analysis and Alignment

Sequences were checked and edited with SeqEd (Per-kin–Elmer). The consensus sequences were aligned inCLUSTAL W 1.74 (Thompson et al., 1994), the align-ment was manually checked, the dubious data (i.e.,regions with gaps where we are not certain about the

ment was converted to Nexus format.The D1 sequences were easily aligned except for

some gap regions that were excluded (Fig. 1). TheClustal W alignment calculated for the D2 sequencescontained many gaps. We therefore aligned the D2sequences with Malign (Wheeler and Gladstein, 1994)using the commands Quick, Score 3, Iter, Treeswap,and Alignswap, giving a rapid heuristic search proce-dure with limited branch swapping after tree construc-tion. Gap to substitution cost ratios of 2:1, 3:2, and 1:1were tested. We manually checked and adjusted thealignments and excluded the regions with gaps (Fig.2a). Informative gaps in the excluded regions weretransformed into a data matrix following the “simpleindel coding” method (Simmons and Ochoterena,2000). In the “simple indel coding” method all gaps thathave different 59 and/or 39 termini are coded as sepa-rate presence/absence characters. Whenever gaps fromdifferent sequences are a subset of other gaps, se-quences that have these longer, completely overlap-ping gaps are coded as inapplicable for the gap char-acter being coded. Parsimony analysis was performedwith and without the gap data matrix (Fig. 2b). For theND1 gene fragment, we aligned our sequences one byone to the sequence from Spodoptera frugiperda(Pashley and Ke, 1992). This enabled us to find thestart of the ND1 gene and the sequences were easilyaligned in Clustal W and afterward manually checked(Fig. 3). The ND1 gene fragment was converted toamino acid data with the Drosophila mitochondrialgenetic code.

Phylogenetic Analysis

In PAUP*d65 (Swofford, 1999) we used maximum-parsimony to build strict consensus trees (commands:heuristic, TBR, addseq 5 random, nreps 5 100). For allgene fragments, branch support was assessed by boot-strapping as implemented in PAUP*d65 (commands:addseq 5 random, TBR, MULPARS, 100 bootstraps;Felsenstein, 1985). Consensus trees and branch sup-port for the amino acid sequences from the convertedND1 gene fragments were assessed in the same way.After the trees for the separate gene fragments werecompared, data were combined in a total evidence tree.The combined data were used unweighted. Branchsupport in the combined evidence tree was assessed bybootstrapping and Bremer support (commands: hserch,addseq 5 random, nreps 5 30, rseed 5 1; Bremer,1988, 1994). Acctran branch lengths (as implementedin PAUP*d65) are shown for the total evidence treeonly. In all trees, only bootstrap values over 50% areshown.

Data Partitions

For testing the heterogeneity between data parti-tions, we used the partition-homogeneity test, as im-

71PHYLOGENY OF GEOMETRIDAE

FIG. 3. Sequence data for the mitochondrial ND1 region. (3), Excluded sites; (z), identical to first sequence; (2), gaps (IUB ambiguouscodes used). For full taxon names refer to Table 1.

F

e

72 ABRAHAM ET AL.

plemented in PAUP*d65 (commands: heuristic, TBR,addseq 5 random, nreps 5 100; Swofford, 1999).

RESULTS

irst Expansion Segment Analysis

From the 344 bp of the D1 fragment, 46 sites werexcluded and 24 variable sites were parsimony infor-

FIG. 4. (a) Strict consensus of 2081 equally parsimonious trees bTree length, 57; consistency index, 0.75; retention index, 0.82. OnlyLarentiinae; S, Sterrhinae; A, Archiearinae; G, Geometrinae; E, Ennparsimony analysis of the 28S D2 gene fragment with the gap data mNumbers in parentheses are bootstrap values if only the DNA matrix0.57; retention index, 0.67. Only bootstrap values over 50% are shArchiearinae; G, Geometrinae; E, Ennominae; Al, Alsophilinae. (c)combined 28S D1 and 28S D2 gene fragments with the gap data mNumbers in parentheses are bootstrap values if only the DNA matrix0.56; retention index, 0.63. Only bootstrap values over 50% are shArchiearinae; G, Geometrinae; E, Ennominae; Al, Alsophilinae. (d) Sparsimony analysis of the mitochondrial ND1 gene fragment. Tree lparentheses are bootstrap values for amino acid sequences. Tree leretention index 0.60. Thick line indicates additional resolution gainevalues over 50% are shown. N, Noctuidae; D, Drepanidae; L, LarenAl, Alsophilinae.

mative (Fig. 1). The Drepanidae and Larentiinae formmonophyletic groups with high bootstrap values (94and 85%, respectively: Fig. 4a). Since Geometrinae andArchiearinae are represented by only one species, wecannot say whether they form monophyletic groups.The Ennominae do not form a monophyletic group andof the Sterrhinae only the Idaea occur as a monophy-letic entity. Alsophila aescularia (Alsophilinae) groups

d on unweighted parsimony analysis of the 28S D1 gene fragment.tstrap values over 50% are shown. N, Noctuidae; D, Drepanidae; L,nae; Al, Alsophilinae. (b) Strict consensus tree based on unweightedrix. Tree length, 425; consistency index, 0.62; retention index, 0.74.sed. Tree length if only DNA matrix is used, 218; consistency index,

n. N, Noctuidae; D, Drepanidae; L, Larentiinae; S, Sterrhinae; A,ict consensus tree based on unweighted parsimony analysis of theix. Tree length 482; consistency index, 0.64; retention index, 0.75.sed. Tree length if only DNA matrix is used, 301; consistency index,

n. N, Noctuidae; D, Drepanidae; L, Larentiinae; S, Sterrhinae; A,t consensus of two equally parsimonious trees based on unweightedth, 505; consistency index, 0.47; retention index, 0.45. Numbers in

th of 498 equally parsimonious trees, 204; consistency index, 0.63;y translation of the data into amino acid sequences. Only bootstrapae; S, Sterrhinae; A, Archiearinae; G, Geometrinae; E, Ennominae;

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73PHYLOGENY OF GEOMETRIDAE

with Erannis defoliaria and Colotois pennaria (Enno-minae) with a high bootstrap value (81%). The D1fragment alone is not able to resolve all the subfami-lies.

Second Expansion Segment Analysis

The D2 fragment was examined with and withoutthe gap data matrix (Fig. 2b). In Malign, a gap tosubstitution cost ratio of 2:1 gave the best alignment ofthree tested ratios when we manually checked thealignments and compared the amount of gaps and con-served regions in each alignment. Without the gapdata matrix, the data matrix comprises 573 bp, ofwhich 358 sites were excluded and 52 variable siteswere parsimony informative (Fig. 2a). With the gapdata matrix, the data matrix comprised 713 sites, ofwhich 358 were excluded and 136 sites were parsimonyinformative. As in the tree for the D1 fragment theDrepanidae and Larentiinae are monophyletic (Fig.4b). In contrast with the D1 fragment tree, the Ster-rhinae too form a monophyletic group. Further, Al-

FIG. 4—

sophila aescularia (Alsophilinae), Archiearis parthe-ias (Archiearinae), and Jodis putata (Geometrinae)

orm a monophyletic group together with the Ennomi-ae, although the Ennominae themselves do not ap-ear as a monophyletic entity. The D2 fragment withhe gap data matrix yielded the most resolution of thehree gene fragments.

ombined Analysis of the First and SecondExpansion Segments

The D1 and D2 fragments are both part of the 28SRNA gene and were therefore also analyzed together,lthough the partition-homogeneity test showed signif-cant heterogeneity (P 5 0.03) between the D1 and the2 fragments. Together with the gap data matrix from

he D2 fragment, the two fragments comprised 1057ites, of which 404 were excluded and 160 were parsi-ony informative. Without the gap data matrix, the

ata matrix comprised 917 sites, of which 404 sitesere excluded and 76 variable sites were parsimony

nformative. The Drepanidae, Larentiinae, and Ster-

ontinued

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rhinae form monophyletic groups in the tree, with boot-

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the grouping of the single species representing Al-sEgnv

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(wDascE

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74 ABRAHAM ET AL.

strap values of 96, 100, and 100%, respectively (Fig.4c). As in the tree for the D2 fragment (Fig. 4b) Al-sophilinae, Archiearinae, Geometrinae, and Ennomi-nae form a monophyletic group. The analysis of thecombined fragments reveals that addition of the D1fragment to the D2 fragment results in slightly differ-ent bootstrap values but does not give any topologicaldifferences between the D2 and the D1D2 tree (seeFigs. 4b and 4c). The bootstrap support for the groupcontaining the Ennominae is higher, compared to theD2 fragment tree.

ND1 Analysis

The data matrix for the ND1 gene fragment com-prises 399 bp, of which 93 sites were excluded and 112variable sites were parsimony informative (Fig. 3).Agrotis segetum and Spodoptera littoralis do not form amonophyletic outgroup; Spodoptera littoralis appearsas a sister group to the Drepanidae. The Drepanidaeform a monophyletic group (Fig. 4d). In this tree Al-sophila aescularia groups with Jodis putata, instead ofwith Agriopis aurantiaria (cf. Figs. 4b and 4c) or withErannis defoliaria and Colotois pennaria (cf. Fig. 4a).

he subfamily Sterrhinae is less well supported in thisree than in the D1, D2, or combined D1D2 fragmentrees. Only Idaea aversata and Idaea straminata groupogether (bootstrap value 90%), whereas in the D1ragment tree all the Idaea grouped together. Convert-ng the ND1 gene fragment to amino acid data by usef the Drosophila mitochondrial genetic code and sub-equent data analysis resulted in a tree in which thearentiinae formed a monophyletic group (data nothown).

ombined Data Analysis

The partition-homogeneity test revealed significanteterogeneity between D2 and ND1 (P 5 0.01) but notetween D1 and ND1 (P 5 0.44). The combined dataatrix including the gap data matrix comprises 1456

haracters, of which 501 sites were excluded and 272ariable sites were parsimony informative. Withouthe gap data matrix, the combined data matrix com-rises 1316 characters, of which 501 sites were ex-luded and 188 variable sites were parsimony informa-ive. The Drepanidae, Larentiinae, and Sterrhinae allppear as monophyletic groups (Fig. 5a). Alsophila aes-ularia, Archiearis parthenias, and Jodis putata form aonophyletic group with the Ennominae. The place-ent of the Drepanidae outside the Geometridae in the

ladogram indicates that the outgroup (Agrotis seg-tum and Spodoptera littoralis) was well chosen. Ex-luding the noctuids from analysis resulted in a tree inhich the drepanid species grouped basal to theeometrid species. The numbers between squarerackets in Fig. 5a are bootstrap values for the com-ined data without the Noctuidae. This indicates that

ophilinae, Archiearinae, and Geometrinae with thennominae is not an artifact due to the chosen out-roup. The basal grouping for the Larentiinae, Sterrhi-ae, and Drepanidae is well supported by bootstrapalues (97 or 100%).Minet (1991) gave Drepanidae as the sister group toeometroidea. A similar grouping is found in the com-ined evidence tree based on our data and is wellupported (bootstrap values for data with/without gapatrix are 99/97%, Bremer support 7).In the ND1 gene fragment tree Alsophila aescularia

Alsophilinae) formed a monophyletic group togetherith Jodis putata (Geometrinae). In the trees for the1, D2, and combined D1D2 gene fragments, Alsophilaescularia was placed within the Ennominae. Theame topology is found for Alsophila aescularia in theombined evidence tree, where it resides within thennominae.The three gene fragments combined together containore phylogenetic information than the individual

ragments analyzed separately. The better resolutions most evident in the Ennominae. Addition of the gapata matrix to the data matrix is here responsible for aetter resolution. Since the subfamilies Alsophilinae,eometrinae, and Archiearinae are only representedy one species each, further sampling is necessary be-ore it is possible to judge whether these groups areest included in Ennominae. In the combined evidenceree, the Larentiinae and, next, the Sterrhinae areasal to the rest of the Geometridae.

DISCUSSION

There are three main differences between the molec-lar cladograms resulting from the experimental anal-sis in the present work (Fig. 5a) and the provisionalhylogenetic hypothesis proposed by Holloway (1997)Fig. 5b), which was based on a synthesis of morpho-ogical observations across a considerable diversity ofeometrid moths and information gleaned from scat-ered primary and secondary literature on the family.

arentiinae and Sterrhinae

Whereas in the morphologically based classificationhe Larentiinae and Sterrhinae are represented as sis-er taxa, in the molecular cladogram these subfamilieso not combine to form a monophyletic group (Fig. 5c).he relationship between the Sterrhinae and the Lar-ntiinae is, in fact, only weakly supported by morpho-ogical evidence (Holloway, 1997). The evidence in-ludes the sharing of a hammer-headed ansa (a scleriteurving over the tympanum in each of the two antero-ateral tympanal organs of the abdomen) and the oc-urrence of structures (rugosity, pouches, and evagina-ions) on the second abdominal sternum of the male inome groups. The hammer-headed ansa is most

75PHYLOGENY OF GEOMETRIDAE

FIG. 5. (a) Strict consensus tree based on unweighted parsimony analysis of the combined data matrix. Tree length, 1000; consistencyindex, 0.54; retention index, 0.61. Numbers in parentheses are bootstrap values for combined data without the gap data matrix. Numbersin square brackets below branches are bootstrap values for the combined data without the Noctuidae. Numbers below branches indicateacctran branch lengths/Bremer support. Thick line indicates different resolution gained by combined data matrix without gap data matrix.Only bootstrap values over 50% are shown. (b) Provisional phylogenetic classification for the Geometridae; modified from Holloway (1997).(c) Comparison of the molecular cladogram (left) and the provisional phylogenetic classification (right) matched as far as possible to show theareas of discordance.

strongly developed in the Cosymbiini (Sterrhinae), a

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within the Geometridae in having a full complement oflafItcei“aps

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76 ABRAHAM ET AL.

group not represented in the molecular analysis andthus not tested for monophyly. The structures on thesecond abdominal sternum of the male occur in theSterrhini/Scopulini lineage of the sterrhines and inonly the Trichopterygini lineage of the larentiines.(Trichopterygini were not included in the molecularanalysis.) The weakness of the morphological supportfor a sister group relationship between Sterrhinae andLarentiinae is underlined by the molecular analysis.

Ennominae

In the morphological hypothesis, the Ennominae areportrayed as a monophyletic group forming a sistertaxon with Desmobathrinae 1 Geometrinae, whereasin the molecular analysis they do not form a monophy-letic group but rather lie within a broader monophy-letic group including Archiearinae, Geometrinae, andAlsophilinae (Figs. 5a and 5c). Morphologically, theEnnominae are defined principally on what is per-ceived to be the loss of a tubular vein M2 in the hind-wing. A morphological character noted by Holloway(1997) and providing some support for an associationbetween Ennominae, Desmobathrinae, and Geometri-nae is the presence, in various taxa across this group,but by no means in all, of a transverse setal comb(Ennominae) or a pair of setal patches (Desmobathri-nae and Geometrinae) on the third abdominal sternumof the male. Although the Desmobathrinae were notrepresented in the analysis, the molecular cladogramgives some support for the monophyly of those taxawith setae arranged in this way.

All ennomine taxa in the molecular sample exceptColotois fall within the broad concept of the Boarmiiniproposed by Holloway (1994). Three of these, with thenotable exception of Erannis defoliaria, are united

ith the only representative of the Alsophilinae (Fig.a). Holloway (1994) suggested that a number of enno-ine tribes might be related based on the shared char-

cter of a single, terminal pair of cremasteral spines.nterestingly, this feature is also seen in the Alsophili-ae. This might suggest that presence/absence of theubular condition of vein M2 in the hindwing might beomoplastic. Holloway (1996) noted that taxa such asrthostixinae and the southeast Asian genus Hetera-

ex were included in the old concept of Oenochrominae,ut that they had, in fact, a number of ennomine fea-ures, although hindwing vein M2 is present.

he Basal Geometrid Clade

The Archiearinae are placed as the sister taxon to allther Geometridae in the morphological classification,hereas in the molecular cladogram this position isccupied by the Larentiinae, with the Archiearinaeeing placed with a clade composed of Ennominae,eometrinae, and Alsophilinae (Fig. 5c). Archiearinaeave typically been viewed as somewhat peculiar

arval prolegs (although some are reduced) and lackingn accessory tympanum on the metapostnotum, theenestra media being extremely narrow (Minet, 1983).n “typical” geometrids, larval prolegs are reduced tohe pairs on abdominal segments 6 and 10, causing theharacteristic “looper” locomotion. In many primitivennomines, reduction in number or size of the prolegss frequent, although to a lesser extent than occurs intypical” geometrids. Here again, the support for anssociation between Archiearinae and Ennominae ap-arent in the molecular results suggests that furthercrutiny of this association would be worthwhile.The additional information gained from the molecu-

ar data will be used for the further examination of theifferent pheromone systems within the geometridubfamilies.

ACKNOWLEDGMENTS

We thank Dag Ahren, Lund University, Lund for interesting anduseful discussions about the practical work; Ruey-Jane Fan, LundUniversity, Lund for her comments on various drafts; and DickVane-Wright and Ian Kitching, Natural History Museum, Londonfor their suggestions on a final draft of the text.

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