Molecular Phylogenetics and Evolution 53 (2009) 384–393

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/locate /ympev

A new Indo-Malayan member of the Stenostiridae (Aves: Passeriformes) revealedby multilocus sequence data: Biogeographical implications for a morphologicallydiverse clade of flycatchers

Jérôme Fuchs a,b,*, Eric Pasquet c,d, Arnaud Couloux e, Jon Fjeldså f, Rauri C.K. Bowie a

a Museum of Vertebrate Zoology and Department of Integrative Biology, 3101 Valley Life Science Building, University of California, Berkeley, CA 94720-3160, USAb DST-NRF Centre of Excellence at the Percy FitzPatrick Institute, University of Cape Town, Rondebosch 7701, Cape Town, South Africac UMR5202 «Origine, Structure et Evolution de la Biodiversité », Département Systématique et Evolution, Muséum National d’Histoire Naturelle, 55 Rue Buffon, 75005 Paris, Franced Service Commun de Systématique Moléculaire, IFR CNRS 101, Muséum National d’Histoire Naturelle, 43, rue Cuvier, 75005 Paris, Francee Genoscope, Centre National de Séquençage. 2, rue Gaston Crémieux, CP5706, 91057 Evry Cedex, Francef Zoological Museum, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 August 2008Revised 25 June 2009Accepted 29 June 2009Available online 2 July 2009

Keywords:Rhipidura hypoxanthaStenostiridaeMulti-locus approachBiogeography

1055-7903/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ympev.2009.06.015

* Corresponding author. Address: Museum of Verment of Integrative Biology, 3101 Valley Life ScieCalifornia, Berkeley, CA 94720-3160, USA. Fax: +1 51

E-mail address: jerome.fuchs@berkeley.edu (J. FucURL: http://www.genoscope.fr (A. Couloux).

Recent molecular studies on passerine birds have highlighted numerous discrepancies between tradi-tional classification and the phylogenetic relationships recovered from sequence data. Among the tradi-tional families that were shown to be highly polyphyletic are the Muscicapidae Old World flycatcher. Thisfamily formerly included all Old World passerines that forage on small insects by performing short salliesfrom a perch. Genera previously allocated to the Muscicapidae are now thought to belong to at leastseven unrelated lineages. While the peculiarity of most of these lineages has been previously recognizedby Linnean classification, usually at the rank of families, one, the so-called Stenostiridae, a clade compris-ing three Afrotropical and Indo-Malayan genera, has only recently been discovered. Here, we address ingreater detail the phylogenetic relationships and biogeographic history of the Stenostiridae using a com-bination of mitochondrial and nuclear data. Our analyses revealed that one species, Rhipidura hypoxantha,previously attributed to the Rhipiduridae (fantails), is in fact a member of the Stenostiridae radiation andsister to the South African endemic genus Stenostira (Fairy Flycatcher). Our dating analyses, performed ina relative-time framework, suggest that the splits between Stenostira/R. hypoxantha and Culicicapa/Elmin-ia occurred synchronously. Given that the Stenostiridae assemblage has been consistently recovered byindependent studies, we clarify its taxonomic validity under the rules of the International Code of Zoo-logical Nomenclature.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

Recent molecular studies have revealed several evolutionaryand biogeographic surprises as the affinities of some passerinesthat were considered enigmatic by earlier systematists have beenresolved. For example, the New World genus Sapayoa, formerlyconsidered to be a member of the New World Pipridae, is nowthought to be nested within the Old World Suboscines (Fjeldsået al., 2003; Chesser, 2004; Irestedt et al., 2006; Moyle et al.,2006), which, given the pantropical distribution of the clade, im-plies a more complex biogeographic interpretation than previouslythought. Another puzzling example concerns the genus Hyliota, an

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tebrate Zoology and Depart-nce Building, University of

0 643 8238.hs).

African genus of four songbird species (Dickinson, 2003), formerlyassigned to several families (e.g. Platysteiridae, Muscicapidae, Syl-viidae), is now, according to mitochondrial and nuclear sequencedata, thought to represent a very old branch within the Passeridawith no obvious extant relatives (Fuchs et al., 2006a).

Among the Passeriformes families which have the most dis-puted taxonomic history are the Muscicapidae and Monarchidaesensu Watson et al. (1986a,1986b); these families consist of OldWorld small to medium-sized flycatchers with a flat bill and bris-tles around the nostrils and gape. The Muscicapidae and Monar-chidae are now considered to include at least seven unrelatedlineages of Old World flycatchers that are nested within the twoprimary Oscine radiations, the crown Corvidea and the Passerida(Pasquet et al., 2002; Barker et al., 2004; Fuchs et al., 2006a,b;Johansson et al., 2008), suggesting convergent evolution of flycat-ching foraging behaviour. Most of the Old World flycatcherfamilies have been the focus of recent phylogenetic studies(e.g. Monarchidae: Pasquet et al., 2002; Filardi and Moyle, 2006;

J. Fuchs et al. / Molecular Phylogenetics and Evolution 53 (2009) 384–393 385

Irestedt et al., 2008; Platysteiridae: Fuchs et al., 2004; Njabo et al.,2008; Muscicapidae: Cibois and Cracraft, 2004; Voelker and Spell-man, 2004). These studies highlighted that some genera weremisclassified at the subfamily or tribe levels and that the currentdelimitations of many genera and species are in need of revision.Even more surprisingly, some Muscicapidae and Monarchidaegenera fall in totally unexpected clades: the African genusErythrocercus, usually considered a monarch is now thought tobe a member of the Old World warbler radiation (Sylvioidea),probably related to the bush-warblers (Cettidae) (Pasquet et al.,2002; Alström et al., 2006; Fuchs et al., 2006a).

Another unusual lineage identified from molecular data is theso-called Stenostiridae, a name that entered the literature inBeresford et al. (2005). At present, the Stenostiridae includesthree genera (Indo-Malayan Culicicapa: two species, AfricanElminia: five species and African Stenostira: monotypic) that wereall considered at some point in their taxonomic history to bemembers of the Muscicapidae. Yet, the exact relationships of thisclade are highly uncertain, although apparently close to the baseof the Passerida radiation (Barker et al., 2004; Beresford et al.,2005; Fuchs et al., 2006a; Johansson et al., 2008). Current phylo-genetic hypotheses suggest that Culicicapa and Elminia are sister-groups (Beresford et al., 2005; Fuchs et al., 2006a; Johanssonet al., 2008; Nguembock et al., 2008b), suggesting an Africanancestral distribution for the clade followed by dispersal ofCulicicapa into the Indo-Malayan biome. As part of our ongoingeffort to unravel the biogeographic history of Old World corvids(Fuchs et al., 2004, 2006b, 2007; Fuchs, 2006; Pasquet et al.,2007; Jønsson et al., 2008; Irestedt et al., 2008), it was discoveredthat the Yellow-bellied Fantail (Rhipidura hypoxantha) was notrelated to other fantails but was instead a putative member ofthe Stenostiridae as defined by Beresford et al. (2005). Rhipidurahypoxantha, sometimes placed in its own monotypic genusChelidorhynx, differs from other fantails (1 genus and 43 species)by its tiny size and the presence of yellow in the plumage. Thefact that R. hypoxantha is part of the Stenostiridae was alsorecently suggested by Nyári et al. (in press: which was submittedand subsequently published online during the peer reviewingprocess of the present manuscript), using a comprehensivesampling of the ‘true’ Rhipidura and by Lei et al. (2007), althoughno other ‘true’ Rhipidura were sampled by Lei et al. (2007). Theexact relationships of R. hypoxantha within the Stenostiridae werenot confidently resolved in Nyári et al. (in press), who wereaiming to resolve the relationships among the ‘true Rhipiduridae,partly due to limited sampling within the Stenostiridae. Lei et al.(2007) did not include any of the two African Stenostiridaegenera nor any of the ‘true’ Rhipidura. Consequently neither studywas able to determine the exact relationships of R. hypoxantha orindeed the phylogenetic relationships among the member of theStenostiridae. In this study we make use of an extensive multilo-cus dataset (eight loci) to investigate the biogeographic history ofthe Stenostiridae including the placement of R. hypoxantha, andelaborate on the initial finding of Beresford et al. (2005) byformerly naming this clade (the avian family Stenostiridae)following the rules of the International Code of ZoologicalNomenclature (ICZN, 1999).

2. Material and methods

2.1. Sampling designs

Our analyses are performed at two levels. First, we analysed thenuclear myoglobin intron-2 (MB) and the mitochondrial ND2 se-quence data for all the primary Oscines lineages highlighted in re-cent higher-level phylogenies of Passeriformes (e.g. Ericson et al.,

2002; Barker et al., 2004; Johansson et al., 2008) to demonstratethat Rhipidura hypoxantha is not closely related to other fantails,nor to any other corvid clade, but is instead a member of theStenostiridae. We rooted our trees with a Suboscine (Pitta sordida,Pittidae) and include a comprehensive dense taxon sampling of Os-cines to critically determine if R. hypoxantha is part of the Stenos-tiridae and to rule out the possibility of long-branch attractiondriving this relationship. Given that several analyses performedon this dataset failed to recover significant support for some keynodes in order to be able to infer the biogeographic history ofthe Stenostiridae, we conducted a second set of analyses with amore reduced outgroup sampling but greatly expanded our genesampling to now also include: the mitochondrial ATP6 gene, aswell as the nuclear mos (or c-mos), b-Fibrinogen intron-5 (FGB),TGFb2 intron-5, Ornithine Decarboxylase introns 6 and 7 withthe intervening exon 7 (ODC) and GAPDH intron-11, for a total oftwo mitochondrial and six nuclear loci. In this second set ofanalyses, we rooted our trees using sequences from a corvid(Chlorophoneus sulfureopectus) and sampled a representative ofeach major Passerida lineage (‘Sylvioidea’, ‘Passeroidea’ and‘Muscicapoidea’) as proximate outgroups. All ATP6, mos, FGB,TGFb2, ODC, GAPDH were specifically generated for the purposeof this study as were sequences of MB and ND2 for R. hypoxanthaand some Oscine outgroups (Table 1).

2.2. Laboratory procedures

Tissues (blood, liver, muscle) were extracted using either aCetylTrimethylAmmonium Bromide-based protocol (Win-nepenninckx et al., 1993) with an overnight Proteinse-K digestion(0.1 mg ml�1) or by using a Quiagen Dneasy Extraction kit (Qiagen,Valencia, CA), following the manufacturers protocol. We amplifiedtwo mitochondrial (ND2 and ATP6) and six nuclear loci (mos, myo-globin intron-2, GAPDH intron-11, Ornithine Decarboxylase (ODC)intron-6 to intron-7, b-Fibrinogen intron-5 and TGFb2 intron-5)using the Polymerase Chain Reaction. The nuclear introns we se-quenced are located on different chromosomes in the chicken gen-ome: MB and GAPDH are on chromosome 1, mos on chromosome2, TGFb2 and ODC on chromosome 3, and FGB on chromosome 4.Primer pairs used for amplification were L5219/H6313 (ND2,Sorenson et al., 1999), L9245/H9947 (Eberhard and Bermingham,2004), Myo2/MyoF (myoglobin intron-2, Slade et al., 1993; Hesle-wood et al., 1998), TGF5/TGF6 (TGFb2 intron-5, Primmer et al.,2002), Fib5/Fib6 (Fuchs et al., 2004), OD6/OD8 (ODC, Allen andOmland, 2003), G3P13/G3P14b (Fjeldså et al., 2003) and 944/1550 (mos, Cooper and Penny, 1997). The thermocycling condi-tions included a hotstart at 94 �C, an initial denaturation at 94 �Cfor 3 min, followed by 35–40 cycles at 94 �C for 40 s, 52–58 �Cfor 30 s, and 72 �C for 50 s, and was completed by a final extensionat 72 �C for 5 min. Purified PCR products were cycle-sequencedusing the Big Dye terminator chemistry (ABI, Applied Biosystems)in both directions with the same primers used for PCR amplifica-tion, excepted G3P13 was replaced by G3PintL1 (Fjeldså et al.,2003), and run on an automated ABI 3100 DNA sequencer. Hetero-zygous sites in nuclear loci (double peaks) were coded using theappropriate IUPAC code.

2.3. Alignment and phylogenetic analyses

Alignment was performed by eye for all loci using Se–Al v2.0a11(Rambaut, 2007). All coding genes (ND2, ATP6, mos) were checkedfor the presence of stop codons or insertion/deletion events thatwould have disrupted the reading frame. All alignments are avail-able from the first author upon request. We used maximum likeli-hood and Bayesian inference (e.g., Holder and Lewis, 2003;Huelsenbeck and Ronquist, 2001), as implemented in RAxML

Table 1List of sampled species, including tissue/voucher and GenBank access numbers. Taxonomy follows Dickinson (2003). Species without information on Tissue/voucher number arefor taxa for which the two loci have been sequenced from different individuals.

Species Tissue/Vouchernumber

Family MB ND2 mos GAPDH FGB TGFb2 ODC ATP6 References

Aegithalos caudatus Aegithalidae AY228281 AY136588 1, 2Aegithina tiphia AMNH 22963 Aegithinidae AY816225 AY816132 3Alauda arvensis MNHN CG 1995-19 Alaudidae AY228284 DQ125975 1, 4Alethe poliocephala MNHN 01-11 Muscicapidae GQ369652 GQ369696 5Andropadus virens MNHN 02-01 Pycnonotidae GQ369653 GQ369697 5Arachnothera

longirostraMNHN 05-23 Nectariniidae GQ369655 GQ369699 5

Batis poensis MNHN CG 1998-783

Platysteiridae AY529907 AY529941 6

Bombycilla garrulus Bombycillidae AY228286 AY329412 1, 7Campephaga flava RB 613 Campephagidae DQ125949 AY529944 3, 6Cettia fortipes MNHN 15-34 Sylviidae DQ125948 DQ125976 4Chlorophoneus

sulfureopectusMNHN CG 1998-823

Malaconotidae AY529912 AY529947 EF052701 DQ406648 AY529980 GQ369680 GQ369669 EU554452 4, 6, 8, 9

Corvus corone MNHN CG 1995-41 Corvidae AY529914 AY529949 6Criniger ndussumensis MNHN 03-15 Pycnonotidae GQ369654 GQ369698 5Criniger pallidus MNHN 04-4I Pycnonotidae GQ369638 GQ369685 GQ369705 GQ369626 GQ369617 GQ369672 GQ369716 5Criniger pallidus MNHN JF1904 Pycnonotidae GQ369660 5Culicicapa ceylonensis MNHN 31-91 incertae sedis DQ125951 DQ125979 GQ369627 GQ369618 GQ369673 GQ369661 GQ369719 4, 5Culicicapa ceylonensis MNHN 04-09G incertae sedis GQ369706 5

GQ369662-

Culicicapa helianthea ZMUC 0783 incertae sedis EU669960 EU652703 GQ369707 GQ369628 GQ369619 GQ369674 GQ369663 GQ369720 5, 10Dicrurus aeneus MNHN 04-03A Dicruridae EF449726 EF449663 11Dumetella carolinensis MNHN CG 1997-

1374Mimidae GQ369657 GQ369701 5

Elminia albiventris MNHN 40-61 incertae sedis GQ369641 EU652710 GQ369713 GQ369634 GQ369679 GQ369668 GQ369724 5, 10Elminia albonotata ZMUC 02939 incertae sedis DQ125952 DQ125980 GQ369711 GQ369632 GQ369622 GQ369677 GQ369666 GQ369723 4, 5

GQ369675-

Elminia longicauda MNHN 01-03 incertae sedis DQ125953 DQ125981 GQ369710 GQ369631 GQ369621 DQ369676 GQ369665 GQ369722 4, 5GQ369687

Elminia nigromitrata MNHN 40-59 incertae sedis GQ369640 GQ369712 GQ369633 GQ369623 GQ369678 GQ369667 GQ369718 5GQ369658-

Enicurus schistaceus MNHN 05-07 Muscicapidae GQ369637 GQ369684 GQ369704 GQ369625 GQ369616 GQ369671 GQ369659 GQ369715 5Erythrocercus maccallii MNHN 03-25 incertae sedis DQ125954 DQ125982 4Estrilda nitidula MNHN CG 1998-

773Estrildidae GQ369643 GQ369688 5

Ficedula hypoleuca NRM 976132 Muscicapidae AY228300 DQ146345 1, 4Garrulax leucolophus MNHN JF344 Timaliidae GQ369644 GQ369689 5Hemipus picatus MNHN 33-6A Campephagidae DQ406637 DQ411309 3Hylia prasina MNHN CG 1998-

769Sylviidae DQ125905 AY136606 4

Hyliota flavigaster MOM 2003.2.118 Sylviidae DQ125956 DQ125983 4Hypsipetes

leucocephalusMNHN 15-60 Pycnonotidae GQ369645 GQ369690 5

Iole propinquua MNHN 31-83 Pycnonotidae GQ369646 GQ369691 5Lanius collaris MNHN 02-26 Laniidae AY529925 AY529960 6Locustella naevia MNHN CG 1996-

123Sylviidae GQ369648 AY382386 5, 12

Loxia curvirostra Fringillidae AY228303 AF447290 1, 13Macrosphenus flavicans MNHN CG 1998-

774Sylviidae DQ125967 DQ125997 4

Malaconotus blanchoti ZMUC 116824 Malaconotidae AY529926 AY529961 6Megabyas flammulatus MNHN CG 1968-

1160Platysteiridae AY529927 AY529962 6

Melocichla mentalis MNHN 01-51 Sylviidae DQ125958 DQ125998 4Menura novaehollandiae Menuridae AY064744 NC007883 14, 15Modulatrix orostruthus incertae sedis DQ125962 DQ125986 4Motacilla aguimp MNHN 17-41 Motacillidae GQ369649 GQ369693 5Nicator chloris MNHN CG 1998-

711incertae sedis DQ125964 4

Nicator chloris FMNH 391705 incertae sedis DQ402188 16Parus major Paridae AY228310 AY136587 1, 2Passer domesticus MNHN 28-43 Passeridae GQ369642 EF449664 EF449705 GQ369635 GQ369681 GQ369725 5, 8Passer montanus EF626752 EF625341 17Pericrocotus ethologus AMNH JGG991 Campephagidae EF052845 EF052790 8Philentoma velata LSUMZ B-38542 incertae sedis AY816221 AY816228 3Phragmaticola aedon MNHN 04-08D Sylviidae DQ125965 DQ125987 4Phyllastrephus

albigularisMNHN 01-42 Pycnonotidae GQ369650 GQ369694 5

386 J. Fuchs et al. / Molecular Phylogenetics and Evolution 53 (2009) 384–393

Table 1 (continued)

Species Tissue/Vouchernumber

Family MB ND2 mos GAPDH FGB TGFb2 ODC ATP6 References

Phylloscopus collybita MNHN 28-37 Sylviidae DQ125966 DQ125988 4Pica pica MNHN 23-10J Corvidae EF052763 EF052690 8Picathartes

gymnocephalaAMNH AC350 Picathartidae AY228314 DQ125989 4, 14

Pitta sordida MNHN 31-06 Pittidae GQ369647 GQ369692 5Prinia bairdii MNHN 02-45 Cisticolidae DQ125959 DQ126000 4Prionops retzii ZMUC 119500 Malaconotidae AY529931 AY529966 6Promerops cafer Promeropidae DQ125968 DQ125990 4Pycnonotus barbatus MNHN 02-29 Pycnonotidae GQ369651 GQ369695 5Rhipidura albicollis MNHN 5-48 Rhipiduridae AY529934 AY529969 6Rhipidura hypoxantha MNHN 14-38 Rhipiduridae GQ369639 GQ369686 GQ369709 GQ369630 GQ369620 GQ369664 GQ369717 5Rhipidura rufifrons Rhipiduridae DQ084100 DQ084073 18Riparia riparia MNHN JF254 Hirundinidae GQ374445 AY826015 5, 19Sitta europeae MNHN H18 Sittidae AY064257 GQ369702 5, 13Stachyris nigriceps MNHN 04-06H Timaliidae AY228321 DQ125992 1, 4Stenositra scita RB661 Sylviidae DQ125970 DQ125993 GQ369708 GQ369629 EU680689 EU680768 GQ369721 4, 5Stenositra scita VCB26 Sylviidae GQ369682 5Sturnus vulgaris NRM 19966615 Sturnidae AY228322 DQ146346 1, 4, 17Tephrodornis virgatus MNHN CG 1989-76 incertae sedis AY816220 AY816226 3Terpsiphone paradisi MNHN 05-53 Monarchidae EF052761 EF052688 8Thamnornis

chloropetoidesFMNH 356699 incertae sedis DQ125971 DQ125995 4

Troglodytes troglodytes MNHN CG 1995-130

Troglodytidae GQ369636 GQ369683 GQ369703 GQ369624 GQ369615 GQ369670 EU680775 GQ369714 5, 17

Turdus philomelos MNHN CG 1995-127

Turdidae GQ369656 GQ369700 5

Urosphena squameiceps Sylviidae DQ008563 AY382399 12, 20Vanga curvirostris MNHN CG 364-A Vangidae AY701505 AY701508 3

References: 1 Ericson and Johansson (2003), 2 Sefc et al. (2003), 3 Fuchs et al. (2006a), 4 Fuchs et al. (2006b), 5 This study, 6 Fuchs et al. (2004), 7 Voelker and Spellmann(2004), 8 Fuchs et al. (2007), 9 Nguembock et al. (2008a), 10 Nguembock et al. (2008b), 11 Pasquet et al. (2007), 12 Drovetski et al. (2004), 13 Yuri and Mindell (2002), 14Ericson et al. (2002), 15 Slack et al. (2006), 16 Moyle and Marks (2006), 17 Johansson et al. (2008), 18 Filardi and Moyle (2006), 19 Sheldon et al. (2005), 20 Alström et al.(2006).

J. Fuchs et al. / Molecular Phylogenetics and Evolution 53 (2009) 384–393 387

v7.0.4 (Stamatakis, 2006; Stamatakis et al., 2008), and MrBayes3.1.2 (Huelsenbeck and Ronquist, 2003; Ronquist and Huelsenbeck,2003) to estimate phylogenetic relationships. The most appropri-ate substitution models were determined with MrModeltest 2.0(Nylander, 2004), using the Akaike Information Criterion (Akaike,1973; Posada and Buckley, 2004).

Maximum likelihood and Bayesian analyses for the concate-nated data set were performed allowing the different parameters(base frequencies, rate matrix or transition/transversion ratio,shape parameter, proportion of invariable sites) to vary betweenthe four or fourteen partitions, i.e. mixed-model analyses (Ronquistand Huelsenbeck, 2003; Nylander et al., 2004). The relevance ofpartitioning the data set by gene and codon position was assessedusing Bayes Factor (Kass and Raftery, 1995; Nylander et al., 2004).We used the harmonic mean approximations of the marginal like-lihood of the two compared models (M0 and M1) to calculate theBayes Factor (BF), which is the ratio between the two comparedmodels. A value greater than 10 for 2 ln BF was considered asstrong evidence against the simpler model (M0) (Kass and Raftery,1995; Nylander et al., 2004). In all MrBayes analyses, four Metrop-olis-coupled Markov Chains Monte Carlo, one cold and threeheated, were run for five to fifteen million iterations with treessampled every 100 iterations. The number of iterations discardedbefore the posterior probabilities were calculated (i.e. the lengthof the ‘burn-in’ period) were graphically estimated using the plotcommand. Two independent Bayesian runs initiated from randomstarting trees were performed for each data set, and the log-likeli-hood values and posterior probabilities were checked to ascertainthat the chains had reached stationarity. We also checked thatthe Potential Scale Reduction Factor (PSRF) approached 1.0 for allparameters and that the average standard deviation of split fre-quencies converged towards zero. We used the program Tracerv1.4 (Rambaut and Drummond, 2007) to check that we reached

convergence for the posterior distribution of the parameter esti-mates and that our effective sample size of the underlying poster-ior distribution was large enough for a meaningful estimation ofparameters.

We searched for conflicting phylogenetic hypotheses betweenindividual gene trees by comparing the topologies and nodal sup-port they recovered. We set the threshold for a signal to be con-flicting if the compared nodes received bootstrap values (BS) of70% or greater (Hillis and Bull, 1993) or posterior probability (PP)of 0.95 or greater (Ronquist and Huelsenbeck, 2003).

2.4. Topology tests

We used Bayes Factors (Kass and Raftery, 1995; Nylander et al.,2004) to assess if we could definitely reject the hypothesis thatRhipidura hypoxantha is a member of the fantail radiation. Wecompared the harmonic mean approximations of the marginallikelihood of the models with and without any topology constraint.

2.5. Dating analyses

We used Beast v1.4.6 (Drummond et al., 2002, 2006; Drum-mond and Rambaut, 2007), to estimate divergence dates withinthe Stenostiridae. We assigned the best fitting model, as estimatedby MrModeltest2, to each of the fourteen partitions. The currentfossil record of passerine birds is still poorly understood and thephylogenetic relationship of many passerine fossils need to bedetermined more precisely (Manegold et al., 2004; Mayr andManegold, 2004; Manegold, 2008). A new fossil, assigned to theCerthioidea (treecreepers, gnatcatchers and nuthatches) hasrecently been described, but its exact relationships within theCerthioidea are unclear (Manegold, 2008). Given the lack ofconsensus concerning the relationships of many passerine fossil

'Crown Corvida'

Stenostiridae

0.1

1.00.97

0.99

1.0

1.0

0.98

0.97

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1.0

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Menura novaehollandiaePicathartes gymnocephala

Culicicapa ceylonensisCulicicapa helianthea

Stenostira scitaRhipidura hypoxantha

Elminia longicauda

Elminia albonotataElminia albiventris

Elminia nigromitrata

Terpsiphone paradisi

Pica pica

Rhipidura albicollisRhipidura rufifrons

Aegithina tiphia

Prionops retziiVanga curvirostris

Megabyas flammulatus

Malaconotus blanchotiChlorophoneus sulfureopectus

Tephrodornis virgatusHemipus picatus

Pericrocotus ethologusCampephaga flava

Dicrurus aeneusLanius collaris

Batis poensis

Phylloscopus collybitaAegithalos caudatus

Riparia riparia

Hylia prasina

Erythocercus mccalliiCettia fortipesUrosphena squameiceps

Melocichla mentalisMacrosphenus flavicans

Stachyris nigricepsGarrulax leucolophus

Phragmaticola aedonLocustella naevia

Thamnornis chloropetoides

Phyllastrephus albigularisCriniger ndussumensis

Andropadus virens

Criniger pallidusPycnonotus barbatus

Hypsipetes leucocephalus

Prinia bairdii

Arachnothera longirostra

Bombycilla garrulus

Modulatrix orostruthus Promerops cafer

Troglodytes troglodytesSitta europeae

Motacilla aguimpEstrilda nitidula

Passer domesticus

Loxia curvirostra

Turdus philomelosAlethe poliocephala

Enicurus schistaceusFicedula hypoleucaSturnus vulgarisDumetella carolinensis

Hyliota flavigasterParus major

Nicator chloris

Pitta sordida

Alauda arvensis

Philentoma velata

Corvus corone

Iole propinquua

Passeroidea

Muscicapoidea

Certhioidea

Sylvioidea

Passerida

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8795

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Fig. 1. Fifty percent majority rule consensus tree resulting from the four partition Bayesian analyses of the concatenated data set (72 taxa, myoglobin intron-2, ND2).Numbers close to nodes represent maximum likelihood bootstrap values greater than 70% and posterior probabilities greater than 0.95. The Stenostiridae are highlighted bythe gray block.

388 J. Fuchs et al. / Molecular Phylogenetics and Evolution 53 (2009) 384–393

forms and the methodological pitfalls associated with the use ofsecondary calibration points (Graur and Martin, 2004), we onlyperformed our dating analyses in a relative time framework. Ourprimary objective was to determine if divergence times betweenAfrican and Indo-Malayan members of the Stenostiridae couldhave occurred synchronously, that is if a vicariance event was dri-ven by the same external factors. For this purpose, we set the basalnode within the Passerida to the arbitrary value of 5 time units andused a normal distribution with the mean and standard deviationset to 5 and 0.25 time units, respectively. We assumed a Yule Spe-ciation Process for the tree prior and an uncorrelated lognormaldistribution for the molecular clock model (Ho, 2007). We used de-fault prior distributions for all other parameters and ran MCMCchains for 50 million generations.

3. Results

3.1. Two gene data set

The ND2 fragment we analysed corresponds to the positions5241–6281 of the Gallus gallus mitochondrial genome (X52392,Desjardins and Morais, 1990). The use of Bayes Factors indicatedthat the best partitioning strategy was to allow the three codonpositions to have different models and parameter values (ND2 nopartition ML�ln = 33274.48; BI harmonic mean �ln = 32922.01,ND2 partitioned by codon position ML = 31941.39; BI harmonicmean �ln = 32219.27; BF = 1405.48). All primary oscine lineages(e.g. Corvidea, Passerida, Muscicapoidea, Sylvioidea) were recov-ered in the ND2 tree, although support values differed among

J. Fuchs et al. / Molecular Phylogenetics and Evolution 53 (2009) 384–393 389

nodes (Supplementary Fig. 1). The monophyly of the Stenostiridaewas recovered in both the ML (BS: 77%) and BI (PP: 1.0) analyses.Rhipidura hypoxantha clustered as the sister group of Stenostira scita,although maximum likelihood bootstrap values and posteriorprobabilities were low (BS: 61%, PP: 0.61). Bayesian analyses withthe genus Rhipidura constrained as monophyletic yielded a poster-ior distribution of trees with significantly worse likelihood valuesthan without the constraint (BI harmonic mean �ln = 32256.08,BF = 73.62).

Analyses performed on MB sequences yielded a 50% majorityrule consensus tree (ML�ln = 7443.28, BI harmonic mean �ln =7592.91, Supplementary Fig. 2) where most of the primary passer-ine clades (e.g. Passerida, Muscicapoidea, Certhioidea) receivedposterior probabilities of 0.95 or greater. Rhipidura hypoxanthaclustered as the sister group of Stenostira scita but without support(BS 36%, BI: 0.50). Bayesian analyses with the genus Rhipiduraconstrained to be monophyletic yielded a posterior distributionof trees that was significantly worse than without the constraint(BI harmonic mean �ln = 7635.54, BF = 85.26).

Mixed-model analyses (four partitions) performed on the con-catenated data set (ND2 and MB, ML �ln = 39347.92, BI harmonicmean �ln = 40009.33) strongly recovered the monophyly of theStenostiridae, if we take into account that Rhipidura hypoxanthais part of this clade (BS 83%, BI: 1.0). Bayes Factors allowed us toreject the hypothesis that Rhipidura is monophyletic (BI harmonicmean �ln = 40082.38, BF = 146.10). Relationships within theStenostiridae were partially resolved: Elminia is monophyletic(BS: 100%, BI: 1.0) and the first taxon to branch off, the genus Culic-icapa is monophyletic (BS 100%, BI: 1.0) and the sister-group (BS:61%, B1: 0.99) of the Stenostira/R. hypoxantha clade (BS: 82%, BI:0.73) by Fig. 1).

3.2. Eight gene data set

3.2.1. Mitochondrial lociThe analysed fragments of mitochondrial origin correspond to

the positions 15449–15929 (ND2) and 9240–9923 (ATP6) of theGallus gallus mitochondrial genome (X52392, Desjardins andMorais, 1990). Number of aligned base pairs, the model selectedfor each partition and likelihood scores are detailed in Table 2.

Table 2Number of aligned base pairs, model selected for each partition and likelihood scores for(given in �ln). Likelihood values always refer to the non-partitioned analyses.

Loci Alignment length

ND2 1041MB 713ND2 1st codon position 347ND2 2nd codon position 347ND2 3rd codon position 347MB (reduced data set) 667ND2 (reduced data set) 1041ND2 1st codon position (reduced data set) 347ND2 2nd codon position (reduced data set) 347ND2 3rd codon position (reduced data set) 347ATP6 (reduced data set) 684ATP6 1st codon position (reduced data set) 228ATP6 2nd codon position (reduced data set) 228ATP6 3rd codon position (reduced data set) 228GAPDH (reduced data set) 356mos (reduced data set) 603mos 1st codon position (reduced data set) 201mos 2nd codon position (reduced data set) 201mos 3rd codon position (reduced data set) 201ODC (reduced data set) 742FGB (reduced data set) 576TGFb2 intron-5 (reduced data set) 579

Uncorrected-p distances within the Stenostiridae range between6% (C. ceylonensis and C. helianthea) and 22.2% (R. hypoxanthaand E. longicauda) (ND2) and between 4.9% (E. albonotata andE. albiventris) and 18.6% (R. hypoxantha and E. albiventris) (ATP6).Bayesian analyses performed on the two individual mitochondrialloci yielded 50% majority rule consensus trees (ND2 partitioned bycodon position: harmonic mean �ln = 6387.01, ATP6 partitionedby codon position: harmonic mean �ln = 3590.44) with a very sim-ilar level of resolution, with: 1) both Culicicapa and Elminia recov-ered as monophyletic (PP: 1.0 in all cases), and 2) relationshipsamong genera receiving poor support. Monophyly of the Stenostir-idae was strongly recovered with ND2 (PP: 1.0) but not with ATP6(polytomy involving Culicicapa, Stenostira/R. hypoxantha/Elminiaand Alophoixus). Bayesian analyses performed on the combinedmitochondrial data set (partitioned by gene and codon position,6 partitions, 13 taxa; ML�ln = 9825.30 BI harmonic mean:�ln = 9962.55) revealed three primary lineages supported by pos-terior probabilities greater than 0.95 within the Stenostiridae (BP:98%, PP: 1.0): Culicicapa (BS: 100%, PP: 1.0), Elminia (BS: 100%, PP:1.0) and R. hypoxantha/Stenostira (BS: 65%, PP: 0.96); the relation-ships between Culicicapa and R. hypoxantha/Stenostira did receivevarying support (BS: 80%, PP: 0.64).

3.2.2. Individual nuclear lociNumber of aligned base pairs, the model selected for each par-

tition and likelihood score values are detailed in Table 2. We couldnot obtain fibrinogen intron-5 sequence data for Elminia albiventrisor the TGFb2 intron-5 sequence for R. hypoxantha. Length variantheterozygosity was detected in three individuals: Culicicapahelianthea and Enicurus schistaceus in ODC and Elminia longicaudain TGFb2. Uncorrected-p distances within the Stenostiridaeranged between 0.6% (E. albonotata and E. albiventris) and 7.0%(R. hypoxantha and C. helianthea) (MB), between 0.3% (C. ceylonensisand C. helianthea) and 11.6% (R. hypoxantha and Stenostira)(GAPDH), between 0.3% (E. albonotata and E. albiventris) and 7.0%(C. helianthea and R. hypoxantha) (mos), between 0.4% (E. nigromitrataand E. albiventris) and 8.4% (E. albonotata and C. ceylonensis)(TGFb2), between 0.15% (C. ceylonensis and C. helianthea) and 8.6%(R. hypoxantha and Stenostira) (ODC), between 1.0% (E. albonotataand E. nigromitrata) and 12.6% (R. hypoxantha and C. helianthea)(FGB) for the various loci, respectively.

each data set. The likelihood scores refer to the harmonic mean of the Bayesian runs

Model ML score BI Score

GTR + C + I 33274.48 32922.01GTR + C 7443.28 7592.91GTR + C + IGTR + C + IGTR + C + IK80 + C 2092.37 2132.25GTR + C + I 6804.82 6814.62GTR + CHKY + IGTR + C + IGTR + C + I 3924.80 3943.50GTR + CGTR + IHKY + C + IHKY + C 1208.72 1245.44HKY + C + I 1793.22 1831.13GTR + CHKY + IK80 + IGTR + C 2411.32 2447.91HKY + C 2164.64 2184.47K80 + C 1999.99 2043.19

0

1.0

1.0

1.0

1.0

1.0

4

54/0.63

Chlorophoneus sulfureopectus

Passer domesticus

Troglodytes troglodytes

Enicurus schistaceus

Criniger pallidus

Stenostira scita

Rhipidura hypoxantha

Elminia longicauda

Elminia nigromitrata

Elminia albonotata

Elminia albiventris

Culicicapa helianthea

Culicicapa ceylonensis

cmos0.1

53/1.0

1.0

65/0.96

80/0.64

1.0

1.0

1.0

1.0

Chlorophoneus sulfureopectus

Passer domesticus

Troglodytes troglodytes

Enicurus schistaceus

Criniger pallidus

Stenostira scita

Rhipidura hypoxantha

Elminia longicauda

Elminia nigromitrata

Elminia albonotata

Elminia albiventris

Culicicapa helianthea

Culicicapa ceylonensis

mitochondrial0.01

0.60

1.0

0.57

57/0.61

1.0

1.0

0.56

Chlorophoneus sulfureopectus

Passer domesticus

Troglodytes troglodytes

Enicurus schistaceus

Criniger pallidus

Stenostira scita

Rhipidura hypoxantha

Elminia longicauda

Elminia nigromitrata

Elminia albonotata

Elminia albiventris

Culicicapa helianthea

Culicicapa ceylonensis

1.0

Myoglobin0.01

1.0

0.98

0.55

1.0

Chlorophoneus sulfureopectus

Passer domesticus

Troglodytes troglodytes

Enicurus schistaceus

Criniger pallidus

Stenostira scita

Rhipidura hypoxantha

Elminia longicauda

Elminia nigromitrata

Elminia albonotata

Elminia albiventris

Culicicapa helianthea

Culicicapa ceylonensis

GAPDH

0.01

1.0

1.0

1.0

1.0

27/0.69

1.0

0.63

Chlorophoneus sulfureopectus

Passer montanus

Troglodytes troglodytes

Enicurus schistaceus

Criniger pallidus

Stenostira scita

Rhipidura hypoxantha

Elminia longicauda

Elminia nigromitrata

Elminia albonotata

Culicicapa helianthea

Culicicapa ceylonensis

0.01

1.0

1.0

12/0.52

24/0.61

1.0

0.81

0.97

71/0.90

0.62

Chlorophoneus sulfureopectus

Passer montanus

Troglodytes troglodytes

Enicurus schistaceus Allele 1

Criniger pallidus

Stenostira scita

Rhipidura hypoxantha

Elminia longicauda

Elminia nigromitrata

Elminia albonotata

Enicurus schistaceus Allele 2

Culicicapa helianthea Allele 2

Elminia albiventris

fib5 ODC

Culicicapa ceylonensis

GFb20.01

1.0

1.0

1.0

0.97

Chlorophoneus sulfureopectus

Passer domesticus

Troglodytes troglodytes

Enicurus schistaceus

Criniger pallidus

Stenostira scita

Elminia longicauda Allele 1

Elminia nigromitrata

Elminia albonotata

Elminia albiventris

Culicicapa helianthea

Culicicapa ceylonensis

Elminia longicauda Allele 2

98

100

96

100

100

100

96

100

43

100

89

62

72

100

96

74/0.67

100

100

97

81

95

97

98

77

100

91

100

Culicicapa helianthea Allele 1

100

100

82/0.96

91

100

98

100

Fig. 2. Fifty percent majority rule consensus tree resulting from the Bayesian analyses of the individual loci (13 taxa). Numbers close to nodes represent maximum lihood boostrap values greater than 70% and posteriorprobabilities greater than 0.95. The Stenostiridae are highlighted by the gray blocks.

390J.Fuchs

etal./M

olecularPhylogenetics

andEvolution

53(2009)

384–393

.01

0.9

T

1.0

1.0

83

100

98

like

0.01

1.0

1.0

1.0

1.0

1.0

0.84

1.0

Chlorophoneus sulfureopectus

Passer sp

Troglodytes troglodytes

Enicurus schistaceus

Criniger pallidus

Stenostira scita

Rhipidura hypoxantha

Elminia longicauda

Elminia nigromitrata

Elminia albonotata

Elminia albiventris

Culicicapa helianthea

Culicicapa ceylonensis

99

96

100

89

100

99

99

Fig. 3. Fifty percent majority rule consensus tree resulting from the eight partitionBayesian analyses of the nuclear data set (13 taxa, myoglobin intron-2, GAPDHintron-11, ODC, TGFb2 intron-5, b-fibrinogen intron-5, mos). Numbers close tonodes represent maximum likelihood bootstrap values greater than 70% andposterior probabilities greater than 0.95. The Stenostiridae are highlighted by thegray block.

0.1

0.99

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Chlorophoneus sulfureopectus

Passer sp

Troglodytes troglodytes

Enicurus schistaceus

Criniger pallidus

Stenostira scita

Rhipidura hypoxantha

Elminia longicauda

Elminia nigromitrata

Elminia albonotata

Elminia albiventris

Culicicapa helianthea

Culicicapa ceylonensis

100

100

100

100

64

100

92

91

Fig. 4. Fifty percent majority rule consensus tree resulting from the fourteenpartition Bayesian analyses of the complete data set (13 taxa, ND2, ATP6, myoglobinintron-2, GAPDH intron-11, ODC, TGFb2 intron-5, b-fibrinogen intron-5, mos).Numbers close to nodes represent maximum likelihood bootstrap values greaterthan 70% and posterior probabilities greater than 0.95. The Stenostiridae arehighlighted by the gray block.

J. Fuchs et al. / Molecular Phylogenetics and Evolution 53 (2009) 384–393 391

Trees resulting from the analysis of the individual nuclear lociwere unevenly resolved (Fig. 2). GAPDH resulted in an overall poly-tomy whereas FGB recovered a fully bifurcating and well sup-ported topology within the Stenostiridae. Monophyly of theStenostiridae was recovered in the mos and FGB gene trees; as inthe mitochondrial loci, monophyly of the two polytypic genera(Culicicapa and Elminia) was strongly supported by all loci (BS:100%, PP: 1.0). Relationships among genera either formed a polyto-my or were poorly supported. A sister-group relationship betweenStenostira and R. hypoxantha was retrieved in three gene trees (MB,mos, FGB), although the level of node support was variable.

3.2.3. Combined nuclear lociAnalyses of the combined nuclear data set (ML �ln = 11518.58,

BI harmonic mean, �ln = 11717.96) yielded the same topology(Fig. 3) where (1) the Stenostiridae were monophyletic (BS: 99%,PP: 1.0), (2) the two polytypic genera (Culicicapa and Elminia) weremonophyletic (BS: 100, PP: 1.0), (3) Stenostira and R. hypoxanthawere sister-taxa (BS: 89%, PP: 0.84) and were in turn sister to theCulicicapa/Elminia clade (BS: 96, PP: 1.0). Thus the topology impliestwo splits involving African and Indo-Malayan taxa (Stenostira/R.hypoxantha and Culicicapa/Elminia).

3.2.4. Combined mitochondrial and nuclear dataAll nodes within the Stenostiridae were recovered by boostrap

values greater than 90% or by posterior probabilities of 1.0 usingthe 14 partition analytical strategy (ML�ln = 21343.47, BI harmonicmean, �ln = 21696.31, Fig. 4). Stenostira was recovered as the

sister-group of R. hypoxantha (BS = 91%, PP = 1.0) and Culicicapawas the sister-group to Elminia (BS = 92%, PP = 1.0).

3.2.5. Dating analysesResults from the dating analyses revealed that the split between

R. hypoxantha/Stenostira (3.61, 95% HPD: 2.97–4.24) and Culicicapa/Elminia (3.49, 95% HPD: 2.92–4.05) occurred synchronously. Thesesplits occurred soon after the initial split within the Stenostiridae(3.97, 95% HPD: 3.40–4.53).

4. Discussion

4.1. Clarifying the taxonomic status of the Stenostiridae

The term Stenostiridae was introduced in the literature byBeresford et al. (2005) to define the clade formed by Culicicapa,Elminia and Stenostira but without any formal description. Herewe formally name the family Stenostiridae under the rules of theinternational Code of Zoological Nomenclature (ICZN, 1999) to bethe clade that contains all taxa that are closer to Stenostira thanto any members of the Muscicapoidea, Bombycillidae, Parinae/Remizinae, Regulidae, Hyliota, Certhioidea, Sylvioidea andPasseroidea (sensu Johansson et al., 2008). The Stenostiridae hasbeen consistently recovered with strong support using severalgene combinations and independent samples by several researchgroups over the last few years (Pasquet et al., 2002; Beresfordet al., 2005; Fuchs et al., 2006a, 2006b; Johansson et al., 2008;Nyári et al., in press, this study), implying that the recovery of this

392 J. Fuchs et al. / Molecular Phylogenetics and Evolution 53 (2009) 384–393

clade is not a bias due to differences in gene or taxon sampling. Toour knowledge, no morphological characters unambiguously de-fine the Stenostiridae but this clade is supported by a synapomor-phic one base-pair insertion (A in position 330 of our alignment) inMB. The type genus for the family would be Stenostira Cabanis &Bonaparte, 1850, and not Stenostira (Vieillot 1818) (sic) as incor-rectly stated in Beresford et al. (2005). The name Stenostiridae isformed as a traditional Linnaean family name, following the Inter-national Code of Zoological Nomenclature (ICZN, 1999). The factthat Rhipidura hypoxantha is not related to other members of thegenus Rhipidura (Nyári et al., in press, this study) suggests theuse of the genus name Chelidorhynx, 1843 for Rhipidura hypoxanthamay be more appropriate. The Stenostiridae presently includesnine species in four genera: Stenostira Cabanis & Bonaparte, 1850(one species, Stenostira scita), Chelidorhynx Blyth, 1843 (one spe-cies, Chelidorhynx hypoxantha), Culicicapa Swinhoe, 1871 (two spe-cies, Culicicapa ceylonensis and Culicicapa helianthea), and ElminiaBonaparte, 1854 (five species, Elminia albonotata, Elminia albicauda,Elminia albiventris, Elminia longicauda and Elminia nigromitrata).

4.2. Relationships and first insights into the biogeographic history ofthe Stenostiridae

Whereas the monophyly of the Stenostiridae has been retrievedby several research groups using independent samples and mark-ers (Beresford et al., 2005; Fuchs et al., 2006a; Johansson et al.,2008, this study), great uncertainty still exists concerning theplacement of the Stenostiridae within the Passerida radiation.Our analyses based on myoglobin intron-2 and ND2 sequencesplaced the Stenostiridae among members of the basal Passeridapolytomy, along with the eight other primary Passerida lineagesrecently highlighted by Johansson et al. (2008). Johansson et al.(2008) also analyzed a six-gene data set (mitochondrial ND2 andnuclear myoglobin intron-2, b-fibrinogen intron-5, ODC region,RAG-1 and RAG-2) where they found some support for a cladeincluding the Stenostiridae, Paridae/Remizidae and the Sylvioidearadiation. The posterior probability for this node was higher than0.95 (0.98), but bootstrap values for parsimony and maximum like-lihood were low (below 50%). Hence, the relationships of theStenostiridae within the Passerida remain uncertain and await fur-ther sequence data.

In contrast, our taxon and gene sampling does allow us for thefirst time to provide a well-supported phylogenetic hypothesis ofrelationships within the Stenostiridae. We also highlight here an-other example of a sister-group relationship between a South Afri-can endemic genus (Stenostira) and an Indo-Malayan genus(Chelidorhynx). Although being each others closest relatives, thesetwo taxa appear to have diverged a long time ago as inferred fromthe branch lengths. Interestingly, this pattern is very similar to theone described by Jønsson et al. (2007) involving the sister-grouprelationship between the South African endemic genus Chaetopsand the Indo-Malayan Eupetes, adding thus further evidence forthe existence of a highly peculiar and old South African endemicvertebrate fauna whose closest relatives are not on the African con-tinent (see also van der Meijden et al., 2005 and Fjeldså and Bowie,2008). Whether this pattern reflects survival of lineages that neverdiversified in other parts of their respective continent or are theonly extant lineages of previously more diverse clades that havesubsequently encountered multiple extinctions requires furtherstudy, and may prove impossible to resolve given the poor fossilsongbird record. Our dating performed under a relaxed-clockassumption and in a relative time framework indicated that thetwo independent splits between African and Indo-Malayan taxaoccurred synchronously (Chelidorhynx/Stenostira: 3.61, 95% HPD2.97–4.24; Culicicapa/Elminia: 3.49, 95% HPD 2.92–4.05), suggest-ing that a similar event may have driven the respective isolation

of these ancestral lineages. Placement of the few existing passerinefossils in a rigorous phylogenetic framework is needed before wecan tie with tie confidence our observed synchrony of splits inthe Stenostiridae with particular geological or climate relatedevents.

Acknowledgments

We are very grateful to A. Tillier, C. Bonillo and J. Lambourdièrefor providing invaluable help in the laboratory. This work was sup-ported by the Plan Pluriformation ‘Etat et structure phylogénétiquede la biodiversité actuelle et fossile’, the ‘Consortium National deRecherche en Génomique’, and the ‘Service de Systématique Molé-culaire’ of the Muséum National d’Histoire Naturelle (IFR101). It ispart of the Agreement No. 2005/67 between the Genoscope and theMuséum National d’Histoire Naturelle on the project ‘Macrophy-logeny of life’ directed by Guillaume Lecointre. The Phongsaly For-est Conservation and Rural Development Project, a Lao-Europeancooperation and its staff, P. Rousseau, C. Hatten, Y. Varelides, R.Humphrey and Y. Tipavanh provided invaluable help and assis-tance during fieldwork in Laos, which was conducted with A. Ci-bois. We also thank CapeNature for issuing permits to collectStenostira. The ‘Société des Amis du Muséum’ provided fundingto JF for his fieldwork and is sincerely acknowledged. This researchwas supported by a postdoctoral fellowship to JF from the DST/NRFCentre of Excellence at the Percy FitzPatrick Institute, University ofCape Town.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ympev.2009.06.015.

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