Molecular Phylogenetics and Evolution 57 (2010) 1173–1183

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Molecular Phylogenetics and Evolution

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Molecular phylogeny and evolutionary habitat transition of the flower bugs(Heteroptera: Anthocoridae)

Sunghoon Jung a, Hyojoong Kim a,1, Kazutaka Yamada b, Seunghwan Lee a,⇑a Laboratory of Insect Biosystematics, Division of Entomology, Research Institute for Agricultural and Life Sciences, School of Agricultural Biotechnology, Seoul NationalUniversity, San 56-1 Shilim-dong, Gwanak-gu, Seoul 151-742, South Koreab Tokushima Prefectural Museum, Bunka-no-Mori Park, Hachiman-chô, Tokushima 770-8070, Japan

a r t i c l e i n f o

Article history:Received 16 April 2010Revised 27 August 2010Accepted 17 September 2010Available online 1 October 2010

Keywords:AnthocoridaeFlower bugsMinute pirate bugsPhylogenyAncestral character statesBayes traitsLyctocoridaeLasiochilidae

1055-7903/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.ympev.2010.09.013

⇑ Corresponding author. Fax: +82 2 873 2319.E-mail address: seung@snu.ac.kr (S. Lee).

1 Present address: Department of Life Sciences andUniversity, Seoul 120-750, South Korea.

a b s t r a c t

We performed a molecular phylogenetic study of the Anthocoridae, the flower bugs, based on maximumlikelihood, maximum parsimony, and Bayesian analyses of �3000 base pairs (bp) of DNA sequence fromthe mitochondrial 16S rRNA and nuclear 18S rRNA and 28S rRNA genes for 44 taxa. Our phylogeneticanalyses indicates that (i) the tribe Cardiastethini (Dufouriellini) could be a paraphyletic group, as thegenera Amphiareus and Dysepicritus are not included in the tribe; (ii) the main subgroups, Oriini andAnthocorini, are monophyletic within Anthocoridae; (iii) three tribes of Blaptostethini, Xylocorini, andScolopini are separated from the main anthocorid clade which is composed of Anthocorini, Cardiastethini,and Oriini, suggesting that Anthocoridae could not be monophyletic. We compared our molecularphylogeny to previous hypotheses of evolutionary relationships within Cimicoidea based on differentanthocorid classification systems using alternative hypothesis tests (Kishino–Hasegawa and Shimodaira–Hasegawa tests). BayesTraits were used to examine the ancestral character states inferring historicalhabitat patterns of the Anthocoridae. Reconstruction of the ancestral habitat patterns of the Anthocoridaesuggests that dead plants may have served as an important habitat for the common ancestor of anthocorids.The biological events such as diversification of angiosperms and anthocorid prey might have providedanthocorids with more habitat options, such as living plants; thereafter, Anthocorini and Amphiareusappeared to have evolved increasingly specialized habitat relationships.

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1. Introduction

Insects in the family Anthocoridae (Hemiptera: Heteroptera),sometimes referred to as flower bugs or minute pirate bugs, aresmall in size (1.4–4.5 mm). This family contains approximately400–600 species that are distributed worldwide (Péricart, 1972;Schuh and Štys, 1991; Péricart, 1996). Most species of Anthocoridaeare predaceous as nymphs and adults (Anderson, 1962c; Péricart,1972; Lattin and Stanton, 1992), thus some species, such as Oriuslaevigatus, O. strigicollis and O. insidiosus, are used as biological con-trol agents and are commercially produced and traded (Yasunaga,1997a; Kim et al., 2008). However, some species in this family con-sume both plants and other insects and their eggs (Carayon andSteffan, 1959; Chu, 1969; Péricart, 1972; Salas-Aguilar and Ehler,1977; Armer et al., 1998). A few species appear to be entirely phy-tophagous (Bacheler and Baranowski, 1975). Lattin (1999)published a comprehensive review of the general biology and

ll rights reserved.

Division of EcoScience, Ewha

bionomics of Anthocoridae, including a discussion of their use asbiological control agents.

Following the proposal of the infraorder Cimicomorpha byLeston et al. in (1954), Carayon (1972) proposed a classificationof Anthocoridae that included three subfamilies: Anthocorinae,Lyctocorinae, and Lasiochilinae (Fig. 1A; Table 1). Subsequently,Ford (1979) and Schuh (1986) pointed out that Carayon’s Anthoco-ridae may not be a monophyletic group. Schuh and Štys (1991)constructed a cladistic tree of relationships within Cimicomorpha,and based on this tree, they elevated Carayon (1972)’s threesubfamilies to family level (Lasiochilidae, Anthocoridae, and Lycto-coridae) (Fig. 1B), and moved all the tribes in the subfamily Lycto-corinae to the family Anthocoridae, except for the tribe Lyctocorini,which was treated as a separate family (Table 1). After this revi-sion, Cassis and Gross (1995) downgraded the rank of the threefamilies to the subfamily-level (Table 1). By elevating the threesubfamilies of Anthocorinae, Lyctocorinae, and Lasihchilinae tofamily level (sensu Schuh and Štys), the hypotheses of relation-ships within Cimicoidea also changed (e.g., the position of Plokio-philidae and Nabidae, Figs. 1A and B). Therefore, to perform aphylogenetic study of the Anthocoridae, the relationships of highertaxa within Cimicoidea should also be considered.

Fig. 1. Current hypotheses of family relationships within Cimicoidea. (A) Clado-gram after Kerzhner (1981), redrawn, and classification system by Štys andKerzhner (1975). (B) Cladogram after Schuh and Štys (1991) and their classificationsystem.

1174 S. Jung et al. / Molecular Phylogenetics and Evolution 57 (2010) 1173–1183

The main aim of this study was to apply molecular evidence toconstruct the phylogenetic relationships of the Anthocoridae forthe first time, and to test the two current hypotheses of higher taxarelationships within Cimicoidea (Kerzhner, 1981; Schuh and Štys,1991; Fig. 1) to clarify the various classifications of Anthocoridae(Table 1) based on molecular data. In addition, the ancestral habi-

Table 1Various classification of Anthocoridae (taxa used in this study; modified after Jungand Lee, 2007).

Carayon (1972) Schuh and Štys (1991) Cassis and Gross (1995)

ANTHOCORINAE ANTHOCORIDAE ANTHOCORINAEANTHOCORINI ANTHOCORINI ANTHOCORINIAnthocoris Anthocoris AnthocorisTetraphleps Tetraphleps TetraphlepsBLAPTOSTETHINI BLAPTOSTETHINI BLAPTOSTETHINIBlaptostethus Blaptostethus BlaptostethusORIINI CARDIASTETHINI

(Dufouriellini)CARDIASTETHINI(Dufouriellini)

Orius Amphiareus AmphiareusBilia Buchananiella BuchananiellaMontandoniola Cardiastethus Cardiastethus

Dysepicritus DysepicritusLASIOCHILINAE Physopleurella PhysopleurellaLasiochilus ORIINI ORIINI

Orius OriusLYCTOCORINAE Bilia BiliaCARDIASTETHINI

(Dufouriellini)Montandoniola Montandoniola

Amphiareus SCOLOPINI SCOLOPINIBuchananiella Scoloposcelis ScoloposcelisCardiastethus XYLOCORINI XYLOCORINIDysepicritus Xylocoris XylocorisPhysopleurellaLYCTOCORIINI LASIOCHILIDAE LASIOCHILINAELyctocoris Lasiochilus LasiochilusXYLOCORINIXylocoris LYCTOCORIDAE LYCTOCORINAESCOLOPINI Lyctocoris LyctocorisScoloposcelis

tat patterns of anthocorids were assessed using a Bayesian ap-proach with node-free rates of habitat exchange. Based on ourresults, we propose an evolutionary habitat history of theAnthocoridae.

2. Materials and methods

2.1. Taxon sampling

A total of 44 species including six outgroup species were se-lected for molecular analysis (Table 2). Family relationships withinthe infraorder Cimicomorpha based on morphological, molecular,or combined data (Kerzhner, 1981; Schuh and Štys, 1991; Tianet al., 2008; Schuh et al., 2009) are largely similar. However, recentmolecular-based phylogenetic studies of cimicomorphan familyrelationships are incomplete, because Lasiochilidae or/and Lycto-coridae were not included (Tian et al., 2008; Schuh et al., 2009).

We selected outgroup taxa according to hypotheses of cimicom-orphan relationships based on morphological or/and biologicaldata as proposed by Kerzhner (1981) and Schuh and Štys (1991)(Fig. 1A and B). We selected three outgroup species from Nabidaeand three from Cimicidae and Plokiophilidae as these families areclosely related to the Anthocoridae (Fig. 1A and B). The most recentstudy of family-level relationships within Cimicomorpha usingmolecular and morphological data was conducted by Schuh et al.(2009); nabids formed a sister group to the Cimicoidea (Anthocori-dae + Lyctocoridae + Lasiochilidae + Cimicidae + Plokiophilidae),which is why we used nabids as an outgroup in this study. Addi-tionally, according to Schuh and Štys’s (1991) cimicomorphan rela-tionships (Fig. 1B), Plokiophilidae is positioned between twoelevating families, Lyctocoridae and Lasiochilidae (sensu Schuhand Štys, 1991), and Cimicidae is the most derived group withinthe Cimicoidea, thus species in Plokiophilidae and Cimicidae werealso selected as sub-outgroups in this study.

Because the classification and taxon rank of Anthocoridae arecontroversial (Table 1), 38 species from three subfamilies (Antho-corinae, Lyctocorinae and Lashiochilinae) were selected as ingrouptaxa based on Carayon’s (1972) classification (Table 2), regardlessof the diverse subfamily or tribal designations.

2.2. DNA extraction, PCR amplification, and sequencing

Total genomic DNA was extracted from single individuals usingGENEALL� (Exgene� Tissue SV) according to the manufacturer’sprotocol. A hole was made in the cuticle of each individual, andthe sample was incubated at 56 �C in AE buffer and proteinase K.After incubation, cuticle samples with a genital segment were usedto make macerated slide specimens for voucher specimens. PCRswere performed using Advantage PCR II Taq polymerase (BDAdvantage™) and the reactions were performed in 20 ll volumescontaining 0.4 lm of each primer, 200 lm dNTPs, 2.5 lm MgCl2,and 0.05 lg genomic DNA template. The thermal cycling programconsisted of 40 cycles of 92 �C/30 s, 43–52 �C/30 s, and 72 �C/60 s,followed by a final extension at 68 �C/10 min. The PCR productswere cleaned using a QIAquick� PCR purification kit (QIAGEN,Inc.), and directly sequenced at NICEM (National InstrumentationCenter for Environment Management, Seoul National University,Republic of Korea). The nuclear 18S ribosomal gene, 28S ribosomalgene D3 region, and a portion of the mitochondrial 16S ribosomalgene were chosen as molecular markers. Those genes were used inmolecular systematics, especially within the cimicomorphans (In-secta: Heteroptera), and were targeted due to their relative easeof amplification and the possibility of integrating data into a broad-er framework within Heteroptera (Wheeler et al., 1993; Tian et al.,2008; Schuh et al., 2009; Weirauch and Munro, 2009).

Table 2Taxa used in this study with GenBank accession numbers, employing Carayon (1972)’s classification; Note: Taxa, whose sequence accession number started with GQ were sequenced from this research. Other sequence from NCBI(Tian et al., 2008; Schuh et al., 2009); DC: habitats are confirmed by direct collection. Type coding is explained in the text.

Family: Tribe Species Collecting country Type coding Habitat references Accession Number

Subfamily 18S rDNA 28S rDNA 16S rDNA

Anthocoridae: Oriini Orius atratus Japan AB Yasunaga (1997a); DC GQ258414 GQ258449 GQ258387Anthocorinae O. laevigatus Netherland A Péricart (1972) GQ258416 GQ258451 GQ258371

O. niger Nepal A Péricart (1972); DC GQ258418 GQ258453 GQ258392O. agilis A Kerzhner (1988) EF487296 EF487333 EF487274O. insidiosus U.S.A A Péricart (1972) GQ258415 GQ258450 GQ258390O. sauteri Korea AB Yasunaga (1997b); DC GQ258419 GQ258454 GQ258373O. minutus Korea AB Yasunaga (1997b); DC GQ258417 GQ258452 GQ258372O. strigicollis Netherland AB Yasunaga (1997b); DC GQ258420 GQ258455 GQ258374Bilia sp. Nepal A DC GQ258406 GQ258439 GQ258363Montandoniola moraguesi Korea AB Péricart (1972); DC GQ258413 GQ258448 GQ258370

Anthocorini Anthocoris confusus Korea B Kerzhner (1988); DC GQ258401 GQ258431 GQ258359A. hsiaoi A Bu and Zheng (2001) EF487306 EF487325 EF487285A. montanus Unknown EF487307 EF487326 EF487284A. chibi Korea B Hiura (1959); DC GQ258403 GQ258437 GQ258362A. flavipes B Péricart (1972) EF487309 EF487318 EF487289A. miyamotoi Korea B Kerzhner (1988); DC GQ258405 GQ258438 GQ258361A. thibetanus Unknown EF487314 EF487335 EF433419A. gracilis Nepal B DC GQ258402 GQ258432 GQ258391A. japonicus Korea B Hiura (1959); DC GQ258404 GQ258436 GQ258360A. pilosus AB Bu and Zheng (2001) EF487302 EF487334 EF487277A. zoui Unknown EF487303 EF487330 EF487275Tetraphleps aterrimus Japan B Kerzhner (1988); DC EF487295 EF487323 EF487273

Anthocoridae: Dufouirellini Amphiareus ruficollaris Japan C Yamada and Hirowatari (2003); DC GQ258394 GQ258430 GQ258383Lyctocorinae A. obscuriceps Korea C Yamada and Hirowatari (2003); DC GQ258393 GQ258429 GQ258358

A. constrictus Japan C Péricart (1972); DC GQ258397 GQ258427 GQ258359A. morimotoi Japan C Yamada and Hirowatari (2003); DC GQ258398 GQ258428 GQ258361Buchananiella crassicornis Malaysia C Yamada and Hirowatari (2007b); DC GQ258407 GQ258441 GQ258364B. leptocephala Malaysia C Yamada and Hirowatari (2007b); DC GQ258408 GQ258442 GQ258365Dysepicritus rufescens Japan C Yamada and Hirowatari (2002); DC GQ258399 GQ258444 GQ258386Physopleurella armata Korea BC Yamada and Hirowatari (2007a); DC GQ258421 GQ258456 GQ258375Cardiastethus exguus Korea BC DC GQ258409 GQ258443 GQ258366

Scolopini Scoloposcelis albodecussata Japan D Yamada and Hirowatari (2005a); DC GQ258422 GQ258457 GQ258376S. sp. Korea D DC GQ258423 GQ258458 GQ258377

Xylocrini Xylocoris cerealis Thailand C Yamada et al. (2006); DC GQ258395 GQ258459 GQ258384Blaptostethini Blaptostethus aurivillus Malaysia AC Yamada (2008); DC GQ258400 GQ258440 GQ258388Lyctocorini Lyctocoris beneficus Korea C Hiura (1966); DC GQ258412 GQ258447 GQ258369

Anthocoridae: – Lasiochilus japonicus Korea D Jung and Lee (2007); DC GQ258410 GQ258445 GQ258367Lasiochilinae L. luceonotatus Japan D Yamada and Hirowatari (2005c); DC GQ258408 GQ258446 GQ258368Nabidae Nabini Nabis stenoferus Korea GQ258426 GQ258434 GQ258379

Nabini N. flavomarginatus Korea GQ258424 GQ258433 GQ258380Nabini Himacerus apterus Korea GQ258425 GQ258435 GQ258381

Cimicidae – Cimex lectularius U.S.A GQ258396 GQ258460 GQ258382C. sp. AY252231 AY252474 AY252702

Plokiophilidae – Lipolophila eberhardi AY252148 AY252432 AY252661

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The complete 18S rRNA gene was amplified using primer pairs18S-1 (5’-CTGGTTGATCCTGCCAGTAGT-3’)/18S-4 (5’-GATCCTTCTGCAGGTTCACC-3’) (Campbell et al., 1995), and sequenced usingthe internal primers 18S-2 (5’-AGATACCGCCCTAGTTCTAACC-3’)and 18S-3(5’-GGTTAGAACTAGGGCGGTATCT-3’) with 18S-1 and18S-4, respectively. The annealing temperature was 48–50 �C.The D3 region of 28S rRNA gene and a partial fragment of the mito-chondrial 16S rRNA were amplified using primer pairs 28S-DD(5’-GGGACCCGTCTTGAAACAC-3’)/28S-FF (5’-TTACACACTCCTTAGCGGAT-3’) (Hillis and Dixon, 1991) and 16S-A (5’-CGCCTGTTTAACAAAAACAT-3’) (Simon et al., 1994)/16S-B (5’-CCGGTTGAACTCAGATCA-3’) (Kambhampati and Smith, 1995). The annealing tempera-tures were 45 �C and 50 �C, respectively.

2.3. Alignment and characterization of gene fragments

For the alignments, we retrieved GenBank reference sequencesfor the three gene regions (Tian et al., 2008) with which to compareour sequences. Raw sequences were examined and corrected usingSeqMan™II (version 5.01, 2001; DNA-star™). All DNA sequenceswere aligned using CLUSTAL X (version 1.83, 2003; Thompsonet al., 1997; default settings), MEGA 3.1 (Kumar et al., 2004), andMAFFT (Katoh et al., 2002; Katoh et al., 2005) of the online sever(v.6; <http://align.bmr.kyushu-u.ac.jp/mafft/online>). As the threegenes used in this study contain lots of gaps, to avoid bias in refin-ing alignments, we compared the result using the three aligningprograms. Some ambiguous sites in the three gene fragments wereremoved using GBLOCK 0.91b (Castresana, 2002; default settingswere used except for choosing the gap option ‘with half’).

2.4. Saturation tests

Given that nucleotide saturation can result in incorrect phylo-genetic inferences (Swofford et al., 1996), uncorrected pairwise se-quence distances were plotted against the numbers of transitionand transversion substitutions using MEGA 3.1 (Kumar et al.,2004) to estimate the extent to which the DNA sequences weresaturated.

2.5. Phylogenetic analyses

Maximum parsimony (MP) analysis was implemented inPAUP*4.0b10 (Swofford, 1998) using a heuristic search procedure,tree bisection reconnection (TBR) branch swapping, and 1000 ran-dom sequence additions with 10 trees held at each pseudoreplicate.All characters were treated as unordered and equally weighted forMP analysis. One thousand MP bootstrap replicates were performedusing a heuristic search procedure, with a maximum tree setting of200 trees. Decay index values (Bremer, 1988) were also calculatedto determine branch support using TreeRot V3 (Sorenson andFarnzosa, 2007). The partition-homogeneity test (Farris et al.,1994), as implemented in PAUP*, was performed to test for signifi-cant phylogenetic conflict between the three gene fragments.

For maximum likelihood (ML) analysis, MODELTEST version3.06 (Posada and Crandall, 1998) was used to select the best-fittingnucleotide substitution model, and then PAUP* settings were opti-mized using data of the selected model before searching. ML anal-yses were also performed with PAUP* using the heuristic searchprocedure with TBR branch swapping, 100 random additions of se-quences, and 10 trees held at each pseudoreplicate. One hundredML bootstraps were performed using a heuristic search procedureand a maxtree setting of 200 trees.

Bayesian phylogenetic (BP) analysis was implemented inMRBAYES (version 3.1.1; Ronquist and Huelsenbeck, 2003) forthe single and combined datasets. We analyzed the combineddataset using two different methods (Supplementary data 4): (1)

partitioned Bayesian analysis (PBA), in which the data were parti-tioned into 18S rRNA, 16S rRNA and 28S rRNA segments, with BPperformed using a partition scheme that maximized the likelihoodbased on the GTR + I + G model with specific model scores esti-mated by the MODELTEST for each gene region; (2) non-specificmodel scores Bayesian analysis (NBA), in which, as for ML analysis,we applied the GTR + I + G nucleotide substitution model with var-iable model scores to the partitioned dataset. For the BP analyses ofthe above two methods, four chains (three heated and one cold)were run, starting from a random tree and proceeding for 10 mil-lion Markov chain Monte Carlo (MCMC) generations, samplingthe chains every 100th cycle. To ensure that the distribution hadstabilized, Tracer version 1.4 (Rambaut and Drummond, 2008)was used to view the graphical representation of MCMC chain mix-ing. Burn-in was set at 15% of the sampled number of trees. Con-vergence was confirmed by monitoring likelihood valuesgraphically. A 50% majority-rule consensus tree was constructedfrom the remaining trees to estimate posterior probabilities (PP).

2.6. Evaluation of alternative hypotheses of relationships withinCimicoidea

To assess whether there was significant conflict between previ-ous phylogenetic hypotheses (Fig. 1) within Cimicoidea and ourmolecular phylogeny, we employed the Kishino–Hasegawa (KH)test (Kishino and Hasegawa, 1989) and Shimodaira–Hasegawa(SH) test (Shimodaira, 2001, 2002). First, we reconstructed alterna-tive, fully-resolved tree topologies consistent with the previoustaxonomic schemes or hypotheses of Kerzhner (1981) and Schuhand Štys (1991) (Fig. 1) using Mesquite version 2.6 (Maddisonand Maddison, 2007). To test Kerzhner’s hypothesis (Fig. 1A) basedon the classification of Carayon (1972), Table 1) for Anthocoridae,we removed Velocipedidae and Polyctenidae, and combinedMedocostinae, Nabidae, and Prostemmatinae into a single group,Nabidae, and constrained the anthocorid taxa based on Carayon’sclassification (1972) (Table 1). To test Schuh and Štys’ hypothesis(Fig. 1B), we also excluded Polyctenidae, and made Nabidae(Naboidea) the sister group of all the taxa within Cimicoidea basedon the hypothesized family relationships within Cimicomorpha,and constrained the anthocorid taxa based on the Anthocoridaeclassifications of Schuh and Štys (1991) (Table 1). Second, we per-formed ML heuristic searches under a GTR + I + G model for eachgene partition, and topological constraints were incorporated inRAxML v.7.0 (Stamatakis, 2006) to evaluate the highest-likelihoodtopology that satisfied the given hypothesis. In the third step, wecompared the ML tree obtained from this study with the two topol-ogies from the first and second steps using 2000 bootstraps andRELL optimization of the concatenated dataset in PAUP*4.0b10.

2.7. Ancestral character states of anthocorids

To explore the relationships between divergence and ancestralhabitat patterns of Anthocoridae, we used the habitat types ofanthocorids, and applied the Bayesian estimation of ancestralcharacter states referencing phylogenetic result. Outgroup taxa,Nabadae: Nabinae, damsel bugs including sub-outgroup taxa,Cimicidae and Plokiophilidae, ectoparasites and cobweb dwellers,respectively, were not included in the part of the study.

2.7.1. Habitat typesThe ancestral habitats of Anthocoridae were inferred based on

the habitats occupied by extant taxa. Table 2 indicates the habitatsfor each species used in this study. Although the host plants of spe-cific taxa are usually reported in taxonomic papers, we collectedmost anthocorid species in this study to confirm their habitats.Although we collected Blaptostethus aurivillus from live flowers of

S. Jung et al. / Molecular Phylogenetics and Evolution 57 (2010) 1173–1183 1177

Macaranga (Euphorbiaceae) from Malaysia, most taxa belonging toBlaptostethus inhabit dead plant materials such as clusters of veg-etable refuse and the nests of weaverbirds (Carayon, 1972). There-fore, we specified that this species inhabits both living and deadplants in our analysis.

Habitat types were coded according to the references (Table 2)as follows: (A) living forbs (including flowers of plants); (B) livingtrees (including shrubs); (C) dead leaf clusters; (D) under the barkof dead tree. Each species was assigned to one or two habitat typesbased on reference papers and information collected in this study,with the exceptions of Anthocoris montanus, A. thibetanus, andA. zoui because of lack of habitat information.

2.7.2. Bayesian estimation of ancestral character statesA Bayesian approach as implemented in the BayesTraits (PC ver-

sion 1.0) software package (Pagel et al., 2004; Pagel and Meade,2007) was used to reconstruct ancestral habitat character statesfor selected nodes in the partitioned Bayesian phylogenetic analy-sis (PBA). BayesTraits uses reversible-jump Markov chain MonteCarlo (MCMC) methods to derive posterior probabilities and thevalues of traits at ancestral nodes of phylogenies (Pagel et al.,2004). BayesMultiState was selected as model of evolution andMCMC as the method of analysis. Because some taxa occurred intwo habitats, we used the multiple character state option of Bayes-Multistate. For example, the code AB signified that the habitat typecould be state A or B (with equal probability) but not state C or D(Pagel and Meade, 2007). The rate deviation was set to 10. A hyper-prior approach was employed with an exponential prior seededfrom a uniform prior in the interval 0–10. Thus, acceptance ratesin the preferred range of 20–40% were achieved as recommended(Pagel et al., 2004; Pagel and Meade, 2007). A total of 50 millioniterations were run for each analysis with the first one millionsamples discarded as burn-in, with sampling every 1000th gener-ation. Because the posterior probabilities for ancestral habitatpatterns of the single runs differed slightly, we calculated thearithmetic mean of all samples for reconstruction of ancestralhabitat types.

3. Results

3.1. Characteristics of the three gene fragments

The alignment results of the each gene region used in this studyusing the three aligning programs are almost identical to eachother. The mitochondrial 16S rRNA dataset comprised 419 alignedbase pairs (bp), but some ambiguous sites were further excludedusing GBLOCK 0.91b with the half-option. Among the selected399 bp, 253 bp were variable and 212 bp were parsimony informa-tive. The average of the uncorrected sequence divergence amongtaxa for 16S rRNA was 16.3%, and the average proportions ofT:C:A:G were 44:10:31:15. The AT richness observed is character-istic of insect mitochondrial DNA sequences (Tauz et al., 1988;Crozier and Crozier, 1993). The nuclear 18S rRNA dataset com-prised 1949 bp of aligned sequences, but some ambiguous siteswere excluded using GBLOCK 0.91b with the half-option. Amongthe selected 1565 bp, 408 bp were variable and 245 bp were parsi-mony informative. The average of the uncorrected sequence diver-gence among taxa for 18S rRNA was 4.8%, and the averageproportions of T:C:A:G were 24:23:25:28. For the nuclear 28SrRNA dataset, 657 bp of sequences were aligned for all taxa, butsome ambiguous sites were excluded using GBLOCK 0.91b withthe half-option. Among the final 552 bp, 136 bp were variableand 85 bp were parsimony informative. The average uncorrectedsequence divergence of the 28S rRNA gene among taxa was16.3%, while the average proportions of T:C:A:G were 27:21:26:26.

3.2. Substitution patterns

Uncorrected P-distances were plotted against the number oftransitions (Ts) and transversions (Tv). Tests were performed forall three genes and the combined dataset. All three genes and com-bined dataset were free of saturation; Ts and Tv values increasedlinearly according to increasing uncorrected P-distances.

3.3. Phylogenetic analyses

3.3.1. Analyses of the single-gene datasetsSingle datasets were analyzed by Bayesian analyses using the

GTR + I + G model selected as the best-fit model for each of thethree genes by MODELTEST. For the individual-level phylogeneticanalyses of the three individual datasets (18S/16S/28S rRNA; Sup-plementary Figs. 1–3), the branch support values were insufficientto resolve relationships between lower taxa, but major nodes sub-tending generic or tribal clusters were highly supported. Themonophyly of the Oriini was supported robustly in all three anal-yses (PB = 100), and the monophyly of the Anthocorini and the Car-diastethini except for the two genera Amphiareus and Dysepicrituswere clearly supported in all three analyses. The monophyly ofthe genus Amphiareus was clearly supported by the 18S/16S rDNAdata (PB = 100).

3.3.2. Analyses of the combined datasetFor MP analyses, the partition–homogeneity test found signifi-

cant phylogenetic conflict among all possible data partitions (gen-erally P = 0.001). Nevertheless, there was no conflict betweenmajor nodes found in the analysis of individual datasets. Thus,the concatenated data set was analyzed. MP analysis yielded threeequally parsimonious trees with a tree length of 2722. The MP (PP)and decay index (DI) values for the strict consensus cladogram ofthe three most parsimonious trees are shown in Table 3. For MLanalyses, the GTR + I + G model selected by MODELTEST analysisas the most suitable for the combined dataset were used. ML anal-ysis produced a single tree which was identical to the post-burn-inconsensus tree from NBA based on the GTR + I + G model for thecombined dataset. Bootstrap P-values (PL) for nodes in the ML tree(ln L = �16765.98997) and PB of the NBA analysis are presented inTable 3. Nodal supports for several nodes differed between the PBAtopology and the ML and NBA topologies. The topology and nodalsupport values for major nodes based on the PBA analysis aremarked with capital letters in Fig. 3 and Table 3.

The monophyly of the superfamily Cimicoidea, all the remain-ing taxa excluding Nabidae: Nabinae, were well supported by highP-values (Fig. 2, node A). The family Lyctocoridae with tribes, Blap-tostethini and Xylocorini, was poorly resolved in all analyses, butXylocorini clustered with Cimicidae + Plokiophilidae (Fig. 2, nodeL), and Blaptostethini was positioned most basally except Lasiochi-lidae within Cimicoidea (Fig. 2 node B), both of which separatefrom the main Anthocoridae node (Fig. 2, node D) in the PBA(ML, NBA) tree. The main Anthocoridae (Fig. 2, node D), includingthe three tribes Oriini, Cardiastethini, and Anthocorini, was well-supported (Table 3). The monophyly of the tribe Anthocorini(Fig. 2, node E; Table 3) had strong support and included twogenera, Anthocoris and Tetraphleps. The genus Anthocoris was recov-ered as paraphyletic group, clustering together with Tetraphlepsaterrimus. The monophyly of the genus Amphiareus (A. ruficollaris,A. obscuriceps, A. constrictus and A. morimotoi) (Fig. 2, node F; Table 3)was well supported. In contrast, the two genera, Amphiareus andDyspicritus were separated from the main clade of tribe Cardiasteth-ini (Fig. 2, node I), and clustered with Anthocorini (Fig. 2, nodeN + R). The remaining members of Cardiastethini (Cardiastethusexguus, Physopleurella armata, Buchananiella crassicornis andB. leptocephala) (Fig. 2, node I; Table 3) formed a clade with strong

Fig. 2. Phylogenetic relationships inferred from Bayesian analysis based on a GTR + I + G model with specific model scores for each gene partition (PBA) with posteriorprobabilities (PP) under nodes. Thick branches indicate nodes also recovered in the other analyses. Capital letters on the nodes refer to nodes discussed in the text and supportvalues from other analyses are provided in Table 3. Higher-taxa names on the right side of the topology are based on Schuh and Štys (1991)’s classification with colored barregarding to the general habitat type for each higher taxon group. Gray boxes indicate taxa used as outgroup in this study. The habitat types of each species are color-codedaccording to the four patterns of habitats (Table 2). BayesMultistate analysis results of ancestral habitat type reconstructions are indicated as pie charts under the nodesshowing the relative likelihoods of each habitat type at respective nodes.

1178 S. Jung et al. / Molecular Phylogenetics and Evolution 57 (2010) 1173–1183

support. Therefore, Cardiastethini is not monophyletic due to theseparation of nodes F, node I, and Dysepicritus rufescens. Oriini wascomposed of 10 species from three genera (Bilia sp., Montandonilolamoraguesi, Orius atratus, O. laevigatus, O. niger, O. agilis, O. insidiosus,O. sauteri, O. minutus and O. strigicollis) and was monophyletic withstrong support (Fig. 2, node G; Table 3) while the subgenus Heteroriuswas strongly supported by all analyses (Fig. 2, node P; Table 3).

In terms of the relationships between tribes and genera, Oriiniand Cardiastethini without the two genera Amphiareus andDysepicritus clustered together with moderate support on the ML,BP (NBA, PBA), and MP trees (Fig. 2, node H; Table 3). Amphiare-us + Dysepicritus and Anthocorini were sister clades on ML/BP(NBA, PBA) and MP trees with relatively low support values(Fig. 2, nodes N, R; Table 3). In the ML/BP (NBA, PBA) analyses,Dysepicritus rufescens was basal to both node E (Anthocorini) + node

F (Amphiareus) with high support values (Table 3), and node F(Amphiareus) clustered with node E (Anthocorini) with low supportvalues (Table 3).

3.4. Comparisons of alternative hypotheses within Cimicoidea

One of the two previous phylogenetic hypotheses within Cimi-coidea was rejected on the basis of the Kishino–Hasegawa (KH)and Shimodaira–Hasegawa (SH) tests (Table 4, P-value <0.05).The hypothesis of Kerzhner (1981) differs from our molecular phy-logeny in all the placements of families within Cimicoidea, regard-less of the rank and the classification of the three subfamiliesLashiochilinae, Lyctocorinae, and Anthocorinae (Carayon, 1972).The hypothesis of Schuh and Štys (1991) conflicts with themolecular phylogeny in several areas, particularly the relationship

Fig. 3. Simplified view of higher taxa relationships for the hypothesized Anthocoridae (node D) found in this study showing the patterns of the change of habit type andrange. Capital alphabets and splits 1–3 for the nodes are corresponded with ones in Fig. 2 and discussed in the text, respectively. Species used as representatives of highergroups are as follows: Physopleurella armata for Cardiastethini, Orius sauteri for Oriini, Amphiareus obscuriceps for Amphiareus, and Anthocoris confusus for Anthocorini.

Table 3Clade recovered in the four analyses, maximum parsimony (MP), maximum likelihood (ML), and Non-specific model scores Partitioned Bayesian Analysis (NBA) and PartitionedBayesian Analysis with specific model scores for each partition (PBA) using PAUP and MrBayes. Values for MP with Decay Index (DI) and ML are boostrap values, those for theMrBayes posterior probabilities. ‘‘–” Indicates that a particular clade is not recovered in that analysis or values below 50.

Taxa clades Node on Fig. 2 MrBayes PAUP

NBA PBA MP DI ML

Cimicoidea A 1 1 98 29 95Cimicoidea excluding Lasiochilidae B 1 0.85 100 30 100Cimicoidea excluding Blaptostethini and Lasiochilidae C 0.78 1 65 1 57Anthocoridae D 1 1 67 2 90Anthocorini E 1 1 100 5 100Amphiareus F 1 1 100 26 100Oriini G 1 1 100 52 100Cardiastethini + Oriini H 1 1 85 2 75Cardiastethini I 1 1 100 21 100Anthocoridae + Lyctocoridae J – 0.57 – – –Cimicidae + Plokophilidae K 1 1 68 1 95Xylocorini + Cimicidae + Plokophilidae L 0.95 0.94 – – 57Lasiochilidae M 1 1 100 12 100Anthocorini + Amphiareus + Dysepicritus N 0.99 0.97 – – 90Scolopini O 1 1 100 48 100Subgenus Heterorius P 1 1 100 12 99Cimicoidea excluding Lasiochilidae, Blaptostethini and Scolopini Q 0.78 0.98 –Amphiareus + Anthocorini R 0.88 0.85 – – 59

Table 4Result from Kishino–Hasegawa (KH) and Shimodaira–Hasegawa (SH) tests amongtree topologies (within Cimicoidea). Previously proposed hypotheses (see Fig. 1) wereused to find a ML tree under those constraints, and those trees were compared withthe ML tree (best). Maximum likelihood values (L) are reported for each tree and thedifference in log likelihoods (dif Ln L) between the proposed tree and the best tree isused to assess statistical significance of the test. Tests were run in PAUP. *P < 0.05.

�ln L Dif ln L KH-test P SH-test P

Kerzhner (1981) 16861.76850 96.99891 0.007* 0.0045*Schuh and Štys (1991) 16779.24136 14.47177 0.3245 0.438Best 16764.76959

S. Jung et al. / Molecular Phylogenetics and Evolution 57 (2010) 1173–1183 1179

between Cimicidae and Plokiphilidae, and the placement of severaltribes of the Anthocoridae. However, as the P-values of the KH andSHtests were greater than 0.05, our molecular phylogeny partlysupports Schuh and Štys (1991)’s hypotheses of relationshipswithin Cimicoidea and the classification of Anthocoridae (withLyctocoridae and Lasiochilidae, Table 1), including the family levelrank of Lasiochilidae.

3.5. Ancestral character states analysis

Ancestral habitat types were estimated for the 10 nodes of high-er taxa relationships in the Bayesian trees (Fig. 2; pie charts).BayesMultiState analyses allowed free rates of habitat typesexchange between the four habitat types (Table 2). The analysissuggests that the habitat type coded as C + D (dead leaf

clusters + bark of the dead tree: dead plant; light blue and dark bluein Fig. 2, node A) at the root of Cimicoidea (Schuh and Štys, 1991)

1180 S. Jung et al. / Molecular Phylogenetics and Evolution 57 (2010) 1173–1183

had a reconstructed probability of greater than 90% compared toalternative habitat types. On the next-most distal bifurcations afterthe split of Lasiochilidae from the remaining taxa within Cimicoi-dea, the ancestral habitat types are reconstructed as types C thatcode for dead leaf clusters with the reconstructed probabilitiesfor the type was more than 90% (Fig 2, nodes B). The habitat rangesfor ancestral character states for the ancestor of the three tribes,Anthocorini, Oriini, and Cardiastethini (Fig. 2, nodes H, R + N) sug-gest that a mixture of live and dead plants were the ancestral hab-itat type for the main lineages of Anthocorini, Oriini andCardiastethini. However, the analyses indicated that dead plants,especially dead leaf clusters, were more likely to have been thehabitat type of the common ancestor of these main groups than liv-ing plants (Fig. 2, node D) although the ancestral habitats of thetwo tribes, Oriini and Anthocorini, were reconstructed as livingplants (Fig. 2, nodes E, G). The origin of habitat type for Anthocorini(Fig. 2, node E) and for Oriini (Fig. 2, node G) is clear; it was recon-structed as living trees with a probability of more than 99%. Theancestral habitat types for the genus Amphiareus (Fig. 2, node F)and for Cardiastethini except for the two genera, Amphiareus andDysepicritus(Fig. 2, node I) are also clear – the habitat type of deadleaves had a probability of greater than 99% for both these nodes,and is almost the identical habitat seen for the ancestor of theAnthocoridae (Fig. 2, node D; sensu Schuh and Štys, 1991), exclud-ing the three tribes, Blaptostethini, Scolopini and Xylocorini.

4. Discussion

4.1. Monophyly and paraphyly within Anthocoridae

The MP, ML, and BP (PBA, NBA) trees supported the monophylyof Anthocorini (Anthocoris + Tetraphleps) and Oriini (Ori-us + Bilia + Montandoniola) in the combined analyses with highsupport values (Fig. 2and Table 3). In the single gene analyses,the monophyly of Anthocorini and Oriini was also supported withrelatively strong PB nodal support values (18S rRNA: PB = 1, 1; 28SrRNA: PB = 1, 1; 16S rRNA: PB = 0.98, 1, respectively; Supplemen-tary Figs. 1–3). These results support Carayon’s (1972) morpholog-ical classification of tribes. Anthocorini, containing 12 genera,including the large genus Anthocoris with at least 50 described spe-cies worldwide, is strongly represented in the Holarctic Region(Schuh and Slater, 1995). Though most of the Palaearctic specieswere sampled in this study, all species in the genus Anthocorisand Tetraphleps aterrimus clustered together, thus the monophylyof the tribe Anthocorini is confirmed (Fig. 2, node E). The tribe Ori-ini contains 15 genera (Carayon, 1972). Carayon (1958) proposedthe new tribe Oriini based on reproductive morphology and otherstructures (e.g., left paramere of male in the form of a spiral, endo-soma of aedeagus short, copulatory tube single and short). Thoughmore sampling is required (e.g., Bilianella, Dokkiocoris, Kitocoris,Wollastoniella) to confirm the classification proposed by Carayon(1958), the clustering of Orius, Bilia, and Montandoniola in a cladewith high support values supports the monophyly of the tribe Ori-ini (Fig. 2, node G).

However, our results do not support the monophyly of the Car-diastethini in the single and combined analyses (Fig. 2, nodes F, I;Table 3; Supplementary Figs. 1–3). The genus Amphiareus (fourspecies: A. ruficollaris, A. obscuriceps, A. constrictus and A. morimo-toi) and Dysepicritus rufescens clustered together with Anthocorinioutside of the main clade of Cardiastethini (with three genera: Car-diastethus, Physopleurella, and Buchananiella). Additionally, thegenus Amphiareus (Fig 2, node F) clustered with Anthocorini sepa-rate from Cardiastethini (Fig. 2, node I) with high support values;this indicates that the current classification within the Anthocori-dae is labile. As Schuh and Slater (1995) indicated, the tribe Dufou-riellini (= Cardiastethini, restored by Carpintero and Dellapé, 2009)

may not be monophyletic, and our molecular data indicate thatAmphiareus and Dysepicritus should not be treated as members ofthe tribe Cardiastethini. Carayon (1972) provided some key charac-ters for the tribe: a short rostrum, fossula spongiosa in males re-duced or absent on foretibia, absent or vestigial on middle tibia,and ovipositor greatly reduced. Although these morphologicalcharacters are found in species in the genera Amphiareus andDysepicritus except for the short rostrum (Yamada and Hirowatari,2003), the biological trait of living only in dead leaf clusters andrelatively longer rostrum among taxa in the tribe Cardiastethinican be used as a reasonable character to exclude these genera fromthe tribe Cardiastethini.

4.2. The systematic status of Lasiochilidae and related taxa

As indicated previously for the various classifications of thefamily Anthocoridae (table 1), the rank of Lasiochilidae (Lasiochili-nae) and Lyctocoridae (Lyctocorinae) is controversial, and thetribes Cardiastethini, Xylocorini, and Scolopini have been classifiedas contained within Anthocoridae (sensu Schuh and Štys, 1991) orin Lyctocorinae (sensu Carayon, 1972) (Table 1). The family rank ofLasiochilidae proposed by Schuh and Štys (1991) is strongly sup-ported by molecular data positioning most basally within Cimicoi-dea (Fig. 2, node A). MP, ML, and BP (NBA, PBA) analyses supportthe monophyly of Lasiochilidae separate from Anthocoridae withinCimicoidea (Fig. 3, node M). Though Schuh and Slater (1995) indi-cated that lasiochilids have the fewest novel features of all of thecimicoid families, except for the presence of dorsal laterotergiteson abdominal segments 1 and 2 (Carayon, 1972), no traumaticinsemination is present in lasiochilids, in contrast to other cimic-oids (Schuh and Štys, 1991), which in light of our molecular data,is an important biological characteristic that differentiates thisgroup from the cimicoids.

Three families, namely Nabidae, Cimicidae, and Plokiophilidae,were used as outgroups in our study to determine the relationshipsbetween Anthocoridae and higher related taxa (Fig. 1, Table 1). Likethe previous studies on family relationships of Cimicomorpha(Schuh and Štys, 1991; Tian et al., 2008; Schuh et al., 2009), Nabi-dae was positioned more basally than Nabidae and Plokiophilidaefrom the main clade of Anthocoridae (Fig. 3, node A) in this study.Therefore, our results support the monophyly of the Cimicoidea(Anthocoridae, Cimicidae, Lasiochilidae, Lyctocoridae, and Plokio-philidae) which were also supported by morphological data (e.g.,male genital structure) in the cimicomorphan relationships (Schuhand Štys, 1991). Additionally, by using several outgroup taxa in thisstudy, we found that the three tribes Xylocorini, Scolopini andBlaptostethini were excluded from the main Anthocoridae clade(Fig. 2, node D), and instead clustered with Cimicidae + Plokiophil-idae and Lasiochilidae (Fig. 2, nodes C, Q, L). Either Cimicidae + Plo-kiophilidae or Nabidae: Nabinae was the most basal node in thisstudy, while the three tribes, Xylocorini, Scolopini, and Blaptos-tethini were always separated from the main Anthocoridae node(Fig. 2D) and clustered with Cimicidae + Plokiophilidae orNabidae + Lasiochilidae + Lyctocoridae.

The rank of Lyctocoridae (Schuh and Štys, 1991) and the phylo-genetic relationships of the three tribes, Blaptostethini, Scoloponi,and Xylocorini, could not be confirmed in this study due to rela-tively low support values which must be caused from relativelyless number of sampling than the remaining higher taxa. However,the three tribes were positioned between the groups Cimici-dae + Plokiophilidae and Lasiochilidae with relatively high supportvalues (Fig. 2, nodes B, C, L, Q) on the PBA tree. Even though Lycto-coridae proposed by Schuh and Štys (1991) has unique morpholog-ical features such as a modified left paramere (which does notserve as a copulatory organ, but rather the vesica) and the posses-sion of an apophysis on the female sternum VII, the molecular data

S. Jung et al. / Molecular Phylogenetics and Evolution 57 (2010) 1173–1183 1181

did not resolve the relationships between Lyctocoridae and thesetribes, and the position of Lyctocoridae itself was ambiguous dueto low nodal support (Fig. 2; Table 3, node J). Therefore, more sam-pling of these and related taxa and/or additional molecular mark-ers are required to infer their relationships.

4.3. The evolutionary implications of habitats and host plantspreferences within Anthocoridae

Based on our results, the family Anthocoridae is restricted tonode D (Fig. 2), which contains Oriini, Cardiastethini [nodeH + node F (Amphiareus) + Dysepicritus refescens], and Anthocorini(node E). Within the hypothesized Anthocoridae (Figs. 2 and 3,node D), the relationships of tribes or genera and their habitat pat-terns were mapped onto the simplified phylogram (Fig. 3); wefound that species in the hypothesized Anthocoridae evolved froma broad-range of habitats to specific habitats (Fig. 3, large arrow).We first ruled out the exceptional habitat cases, such as Orius agilis,which lives in the litter layer (Elov, 1976), and also excluded agro-ecosystems, ornamental plants, artificial habitats (e.g., green-houses), and stored products facilities as habitat types, asanthocorids have long been used as biological control agents. Wetherefore only used habitat (host plants) information from naturalecosystems.

Species within the tribe Cardiastethini used in this study, exceptfor the two genera Dysepicritus and Amphiareus, have been col-lected from both living and dead plants (Lattin, 1999; Yamadaand Hirowatari, 2005b, 2007a, 2007b). Oriini species have beencollected from living plants (Yasunaga, 1997b), especially from for-bs and flowers of living plants including shrubs and trees (Péricart,1972; Yasunaga, 1997b). Thus, habitat patterns suggest that spe-cies in the tribe Cardiastethini (except for the two genera, Dysepicr-itus and Amphiareus) have a relatively broader host range thanspecies in the tribe Oriini, as Oriini species inhabit only livingplants, especially various kinds of forbs (Fig. 3, nodes I, G). A fewtaxa of Oriini have been reported to feed chiefly on pollen (e.g.,O. insidiosus, O. pallidicornis, O. tristicolor, and Paratriphleps laevius-culus) (Barber, 1936; Carayon and Steffan, 1959; Bacheler andBaranowski, 1975; Salas-Aguilar and Ehler, 1977), and in oneexceptional case, O. insidiosus has been reported to feed on blood(Malloch, 1916). Furthermore, Elov (1976) reported O. agilis livingin the litter layer, an unusual habitat for species in this genus. Gen-erally, species of Oriini feed on aphids, thrips, and mites or theireggs, and usually inhabit living plants of forbs and flowers of trees(including agricultural crops) (Péricart, 1972; Yasunaga andMiyamoto, 1993; Buchholz et al., 1994; Luettge and Sell, 1994;Fiume, 1996; Frescata and Mexia, 1996). These species usuallyaggregate with the host prey of specific plants, which may explainwhy species of Oriini have been more effective as biological controlagents than species from other related anthocorid taxa.

Our results separate the two genera Dysepicritus and Amphiare-us from the main clade of Cardiastethini. Species in these two gen-era have been found in dead leaf clusters (Yamada and Hirowatari,2002, 2003). We have yet to collect these species from livingplants, but have collected them from clusters of dead leaves fromvarious kinds of plants. Sometimes one or two individuals of thisgroup are collected from living plants, which may be accidental,as they usually aggregate in the same habitat (Yamada andHirowatari, 2003). Péricart (1972) reported that Amphiareus inhab-ited broadleafed trees, but it is unclear whether the trees theywere referring to were dead or alive. Thus, the habitats of speciesin the genus are restricted to dead plants, which implies thattheir prey would also be restricted to small arthropods living ondead plants.

There are few biological studies of the Anthocorini. However,their habitats are known to be more restricted to deciduous shrubs

and trees (Anderson, 1962a, 1962b; Hill, 1957, 1961; Péricart,1972; Evans, 1976a–d) than that of other anthocorids. SomeAnthocorini species have been reported to be specialist predatorsagainst tree pests. For example, Anthocoris japonicus are usuallyfound on Zelkova serrata and Ulmus davidiana var. japonica (Ulma-ceae) (even during winter hibernation) where Colopha moriokaensis(Pemphigidae) occurs (field observations by the first author), andA. whitei was reported as a specialist gall-forming aphid on Manza-nita (Valenti et al., 1996). Anderson (1962a) reported A. bakeri inaphid galls on Manzanita in southwestern Oregon, and Lattin(1999) reported A. whitei on Cercocarpus ledifolius associated withpsyllids in Oregon. Most species in the tribe have been collectedfrom specific host plants at the genus or higher level, which mayindicate a preference for specific prey on the host plant (Sands,1957; Anderson, 1962a; Madsen et al., 1963; Lussier, 1965; Cobbenand Arnoud, 1969; Péricart, 1972; Mendel et al., 1991; Lattin andStanton, 1992, 1993) (e.g., Elatophilus stigmatellus from Pinus syl-vestris, Temnostethus ulmi from Ulmus pumila, and Anthocoris chibifrom Pinus densiflora, etc.; see Kerzhner, 1988; Lattin, 1999). There-fore, the strong preference of members of this tribe for specific hostplants implies that this group is a relatively more specialist groupthan other related taxa within the hypothesized family Anthocori-dae (Fig. 3).

The common Oriini ancestor appears to have evolved to live onlive plants (Fig. 3, node G, split 1; especially on forbs) from anancestral state of the mixed habitat (Fig. 3H, split 2). The commonancestor of Cardiastethini and Amphiareus and Dysepicritus (Fig. 3,nodes I, N + R, split 1) appears to have evolved to live on dead plant(leaf clusters) from the mixed habitat (Fig. 3, node H, split 2). Thecommon ancestor for Anthocorini appears to also have evolved tolive on live plants (Fig. 3, node E, split 1) from an ancestral charac-ter type of mixed habitat (Fig. 3, N + R, split 2). Those habitat tran-sitions might be related with the emergence of deciduous orherbaceous angiosperm plants providing various habitats whichcould have allowed the common ancestors of anthocorids tochoose between two different habitat types (dead plants or livingplant; Fig. 3, nodes, H, N + R ? I, G, F, E). Putting these biologicaldata together with molecular phylogenetic analyses indicates thatthe higher taxa within Anthocoridae evolved as a result of a nar-rowing range of habitats that resulted in them selecting a differenttype of habitat. The proliferation of angiosperms (Wikström et al.,2001) is thought to have driven the diversification of major herbiv-orous hemipteran groups (Grimaldi and Engel, 2005) such as anradiation of aphids known as the prey of anthocorids (von Dohlenand Moran, 2000), and it would appear that anthocorid diversifica-tion, too, closely tracks the rise of angiosperm-dominated forestsand their prey; anthocorids either chose new habitat, living plants(Fig. 3, nodes D ? H ? G and D ? N + R ? E), or shared theirancestors’ habitat of dead plant (Fig. 3, nodes D ? N + R ? F andD ? H ? I). Morphologically, most extinct anthocorids are flat-bodied (Yao et al., 2006), which appears to be an adaptation to livein dead plants whose habitat (under the bark and dead leafclusters; Fig. 2, node A) derived from the common ancestor. Addi-tionally, extant anthocorids that inhabit living plants also have aflat-body as a symplesiomorphic character, and this differentiatestheir feeding strategy from other predatory cimicomorphan spe-cies (e.g., hunting behavior of Anthocoris spp., Anderson 1962a;Valenti et al., 1996).

Acknowledgments

We would like to thank Dr. Tomohide Yasuanga, Dr. Ernst Heiss,Dr. Tadashi Ishikawa, Mr. Sangwook Park, Mr. Gary Pixsely, Mr.Jongok Lim, Ms. Ram Duwal, Dr. Eun Chan Yang, and the late Drs.M. Josifov and I. M. Kerzhner for providing samples and theirencouragements. We are grateful to Dr. Randall T. Schuh, Dr.

1182 S. Jung et al. / Molecular Phylogenetics and Evolution 57 (2010) 1173–1183

Pavel Štys, and Dr. Christiane Weirauch for the discussion aboutthis research during 4th IHS meeting. This research was supportedby a grant from the Agenda Project (Grant No. 102966290) and Bio-green 21 Program (Grant No. 034032), by the Rural DevelopmentAdministration in the Republic of Korea, and the Eco-technopia21 project ‘‘Construction of a DNA barcode system for the conser-vation and management of Korean insect fauna” and partly byGrant-in-Aid for Young Scientists (B) from the Japan Ministry ofEducation, Culture, Sports, Science and Technology to the thirdauthor (No. 20780043). This research was also supported by theResearch Institute for Agriculture and Life Sciences (RIALS).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ympev.2010.09.013.

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