MOLECULAR

Molecular Phylogenetics and Evolution 33 (2004) 428–439

PHYLOGENETICSANDEVOLUTION

www.elsevier.com/locate/ympev

Phylogenetic relationships of the African bush-shrikesand helmet-shrikes (Passeriformes: Malaconotidae)

Jerome Fuchsa,b,*, Rauri C.K. Bowiec,d, Jon Fjeldsae, Eric Pasqueta,b

a FRE 2695 Origine, Structure et Evolution de la Biodiversite, Departement Systematique et Evolution, Museum National d�Histoire

Naturelle, 55 Rue Buffon, 75005 Paris, Franceb Service Commun de Systematique Moleculaire, IFR CNRS 101, Museum National d�Histoire Naturelle, 43, rue Cuvier, 75005 Paris, France

c Department of Zoology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africad Department of Zoology, Division of Birds, Field Museum of Natural History, 1400 S. Lake Shore Dr, Chicago, IL 60605, USA

e Zoological Museum, University of Copenhagen, DK-2100 Copenhagen, Denmark

Received 28 January 2004; revised 10 June 2004

Abstract

The African bush-shrikes and helmet-shrikes (Malaconotidae sensu [A Complete Checklist of the Birds of the World, third ed.,

Helm Editions, London, 2003]) include 10 genera and 52 species of predatory passerine birds for which monophyly, sister-group,

and inter-generic relationships are disputed. To resolve their relationships, we analyzed 2313bp of sequence data obtained from

two nuclear introns (myoglobin intron-2, b-fibrinogen intron-5) and a mitochondrial protein-coding gene (ND2) using parsimony,

maximum likelihood, and Bayesian inference. A strongly supported clade that included representatives of the Malaconotidae, Platy-

steiridae, and Vangidae was found in all analyses. Three main groups emerged within this clade but relationships between these three

groups were always poorly supported. The first group included the helmet-shrikes (Prionops), flycatcher-shrikes (Bias and Megab-

yas), and vangas (Cyanolanius and Pseudobias), currently placed in the families Malaconotidae, Platysteiridae, and Vangidae,

respectively. The second group consisted of four Platysteiridae genera (Lanioturdus, Batis, Platysteira, and Dyaphorophyia), with

the remaining Malaconotidae genera (�core malaconotids�) forming the last group. Two main clades emerged within the �core mala-

conotids,� with the position of the genus Nilaus being variable. The first clade includedMalaconotus, Dryoscopus, Bocagia, and Tcha-

gra and the second Chlorophoneus, Laniarius, Rhodophoneus, and Telophorus. Monophyly of the genus Chlorophoneus was never

recovered, a result that is consistent with morphological data.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Bush-shrikes; Helmet-shrikes; ND2; Myoglobin intron-2; b-Fibrinogen intron-5

1. Introduction

The African bush-shrikes and helmet-shrikes (Mala-

conotidae sensu Howard and Moore, 2003) include 10

genera and 52 species that are variable in size and

plumage, some being drab and other brightly colored.

These predatory birds mainly feed on large insects

(Coleoptera, Hymenoptera, and Orthoptera) and even

1055-7903/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.ympev.2004.06.014

* Corresponding author. Fax: +33-1-40-79-30-63.

E-mail address: fuchs@mnhn.fr (J. Fuchs).

small mammals (rodents). Bush-shrikes and helmet-shrikes have traditionally been grouped with all Old

World shrikes in the family Laniidae (Morony et al.,

1975; Rand, 1960). The Laniidae is a poorly defined

assemblage that is only supported by the presence of

two adaptive characters, a hooked bill and the reduced

state of the Musculus flexor perforatus digiti III muscle

(Raikow et al., 1980). Results based on biochemical,

behavioral, and morphological characters (Harris andArnott, 1988; Harris and Franklin, 2000; Wolters,

1975–1982) or on DNA–DNA hybridization data

J. Fuchs et al. / Molecular Phylogenetics and Evolution 33 (2004) 428–439 429

(Sibley and Ahlquist, 1990) suggest that the Malaco-

notidae are only distantly related to ‘‘true’’ shrikes

(genera Corvinella, Eurocephalus, and Lanius) and the

Bornean Bristlehead (Pityriasis gymnocephala). Instead,

they suggest that the Malaconotidae are more closely

related to the families Platysteiridae or Vangidae.However, due to insufficient taxon sampling or analyt-

ical problems, these studies failed to resolve the rela-

tionships of bush-shrikes and helmet-shrikes. In

particular, monophyly, sister-group, and inter-generic

relationships remain controversial. The Malaconotidae,

Platysteiridae, and Vangidae are biogeographically

interesting as they represent African and Malagasy

clades of a larger group of corvids that apparentlyoriginated in Australia, subsequently radiating

throughout the Oriental region and finally colonizing

Africa (Barker et al., 2002; Ericson et al., 2002a).

The putative closely related family Platysteiridae

(batises, flycatcher-shrikes, and wattle-eyes) is endemic

to Africa and comprises six genera and 28 species

(Howard and Moore, 2003). All these species share

some distinctive morphological traits, e.g., a flat broadbill and bristles around the nostrils and gape, adapted

for flycatching behavior. Despite these differences in

foraging behavior and overall similarity, the Platysteir-

idae share many biochemical and morphological char-

acters with bush-shrikes and helmet-shrikes (Harris

and Arnott, 1988; Harris and Franklin, 2000; Pocock,

1966; Traylor, 1970; Wolters, 1975–1982). The family

Vangidae (15 genera and 22 species sensu Howardand Moore, 2003), endemic to Madagascar (with one

subspecies in the Comoros), is difficult to define due

to the considerable morphological divergence exhibited

within the family. A molecular study of Vangidae rela-

tionships suggests that these taxa are closely related to

a bush-shrike (Laniarius luehderi) (Schulenberg, 1993).

Further, similarity in vocalizations between the Vangi-

dae and some Malaconotidae (especially Malaconotus

alius and Telophorus kupeensis) lead Harris and Frank-

lin (2000) to propose these as potential sister-groups.

Monophyly and inter-generic relationships within the

Vangidae are still debated (Howard and Moore,

2003; Schulenberg, 1993; Yamagishi et al., 2001) and

resolving these relationships are beyond the scope of

our study.

There are also some phylogenetic problems withinthe Malaconotidae. First, the limits and monophyly

of the family are unclear. The position of the genus

Prionops (helmet-shrikes) in the Malaconotidae is par-

ticularly controversial due to the helmet-shrikes differ-

ing from other bush-shrikes by several morphological

and ethological characters, which has lead many

authors to treat them as relatively distant from other

bush-shrikes (Harris and Arnott, 1988; Raikow et al.,1980; Sibley and Monroe, 1990; Urban et al., 1997;

Wolters, 1975–1982). Some other genera, Nilaus—due

to its particular nest building and plumage—and Dry-

oscopus—due to its reproductive and communication

behavior—are thought to belong to the Platysteiridae

(Benson et al., 1971; Mayr, 1943; Traylor, 1970; Wol-

ters, 1975–1982). Conversely, the genera Bias, Megab-

yas, and Lanioturdus (Platysteiridae) are believed tobe closer to the Malaconotidae (Morony et al., 1975;

Rand, 1960; Sibley and Monroe, 1990; Traylor,

1970). Finally, limits of some genera (Bocagia, Chloro-

phoneus, Malaconotus, Rhodophoneus, Tchagra, and

Telophorus) are unclear. For example, the species La-

nius cruentus Ehrenberg, 1828 has been assigned to

the genera Tchagra, Telophorus or Rhodophoneus,

depending on the authors (Harris and Franklin, 2000;Howard and Moore, 2003; Morony et al., 1975; Rand,

1960; Sibley and Monroe, 1990; Urban et al., 2000;

Wolters, 1975–1982).

Here, we use 2313bp of aligned sequence data ob-

tained from two nuclear introns (myoglobin intron-2

and b-fibrinogen intron-5) and a mitochondrial pro-

tein-coding gene (ND2) to address the following ques-

tions: (1) Are the Malaconotidae sensu Howard andMoore (2003) monophyletic? (2) What is the sister-

group of the Malaconotidae? (3) What are the phyloge-

netic relationships among Malaconotidae genera?

2. Materials and methods

2.1. Taxon sampling

The DNA–DNA hybridization work by Sibley and

Ahlquist (1990) suggests a rapid mid-Tertiary radiation

of bush-shrike-like birds, with densely packed inter-

nodes. We selected a suite of species that encompass

the scope of this radiation, but avoids multiple represen-

tation from groups of closely related species. Resolving

the associations of the Malaconotidae, Platysteiridae,and Vangidae with various Asiatic taxa (Philentoma,

Tephrodornis, Aegithinidae, Artamidae, and Cractici-

dae, see Sibley and Ahlquist, 1990; Yamagishi et al.,

2001; and Barker et al., 2002) was beyond the scope of

our present study.

We obtained samples from all recognized Malaconot-

idae genera (19 species from 10 genera) as well as sam-

ples from putative closely related taxa in thePlatysteiridae (six species representing all six genera),

and Vangidae (two species). Trees were rooted with

the Tropical Kingbird (Tyrannus melancholichus, Tyran-

nidae, Suboscines) which is outside the Infraorder Os-

cines (see Ericson et al., 2003 for a review). Alternative

rooting with GenBank sequences from a stem Oscines,

i.e., Menura novaehollandiae (ND2: AY064754, Myogli-

bin: AY064744, Ericson et al., 2002b) did not affect thetopologies. Sample origins and GenBank accession

numbers are reported in Table 1.

Table 1

List of taxa studied (following Howard and Moore, 2003), voucher number, and GenBank accession numbers

Species Family Sample No. Origin Myoglobin b-Fibrinogen ND2

Ingroup taxa

Batis poensis Platysteiridae MNHN CG 1998-783 Cameroon AY529907 AY529974 AY529941

Bias musicus Platysteiridae MNHN 3-23 Cameroon AY529908 AY529975 AY529942

Bocagia minuta Malaconotidae ZMUC 128785 Congo AY529909 AY529976 AY529943

Campephaga flava Campephagidae AY165803a

Campephaga flava Campephagidae RCKB613 Kenya AY529977 AY529944

Chlorophoneus dohertyi Malaconotidae FMNH 358005 Burundi AY529910 AY529978 AY529945

Chlorophoneus nigrifrons Malaconotidae ZMUC 120151 Tanzania AY529911 AY529979 AY529946

Chlorophoneus sulfureopectus Malaconotidae MNHN CG 1998-823 Malawi AY529912 AY529980 AY529947

Coracina melaschistos Campephagidae MNHN 6-69 Laos AY529913 AY529981 AY529948

Corvus corone Corvidae MNHN 13-16 France AY529914 AY529982 AY529949

Cyanolanius madagascarinus Vangidae MNHN E117 Comoros AY529915 AY529983 AY529950

Dicrurus paradiseus Dicruridae MNHN 5-57 Laos AY529916 AY529984 AY529951

Dryoscopus cubla Malaconotidae ZMUC 116780 Kenya AY529917 AY529985 AY529952

Dryoscopus gambensis Malaconotidae ZMUC 124320 Kenya AY529918 AY529986 AY529953

Dyaphorophyia chalybea Platysteiridae MNHN 3-19 Cameroon AY529919 AY529987 AY529954

Laniarius aethiopicus Malaconotidae FMNH 356738 Tanzania AY529920 AY529988 AY529955

Laniarius barbarus Malaconotidae ZMUC 116792 Cage-bird AY529921 AY529989 AY529956

Laniarius funebris Malaconotidae ZMUC 123466 Tanzania AY529922 AY529990 AY529957

Laniarius luehderi Malaconotidae ZMUC 119044 Uganda AY529923 AY529991 AY529958

Lanioturdus torquatus Platysteiridae US-001 Namibia AY529924 AY529992 AY529959

Lanius collaris Laniidae MNHN 2-26 Cameroon AY529925 AY529993 AY529960

Malaconotus blanchoti Malaconotidae ZMUC 116824 Kenya AY529926 AY529994 AY529961

Megabyas flammulatus Platysteiridae MNHN CG 1968-1160 Kenya AY529927 AY529995 AY529962

Nilaus afer Malaconotidae FMHN uncatalogued Bostwana AY529928 AY529996 AY529963

Oriolus xanthornus Oriolidae MNHN 4-10D Thailand AY529929 AY529997 AY529964

Platysteira cyanea Platysteiridae MNHN 2-22 Cameroon AY529930 AY529998 AY529965

Prionops retzii Malaconotidae ZMUC 119500 Kenya AY529931 AY529999 AY529966

Prionops scopifrons Malaconotidae ZMUC 117528 Kenya AY529932 AY530000 AY529967

Pseudobias wardi Vangidae FMHN 356702 Madagascar AY529933 AY530001 AY529968

Rhipidura albicollis Rhipiduridae MNHN 5-48 Laos AY529934 AY530002 AY529969

Rhodophoneus cruentus Malaconotidae US-002 Kenya AY529935 AY530003 AY529970

Tchagra australis Malaconotidae ZMUC 116831 Kenya AY529936 AY530004 AY529971

Tchagra senegala Malaconotidae ZMUC 116834 Kenya AY529937 AY530005 AY529972

Telophorus zeylonus Malaconotidae FMNH 390107 South Africa AY529938 AY530006 AY529973

Terpsiphone viridis Monarchidae MNHN 2-20 Cameroon AY529939 AY530007

Terpsiphone viridis Monarchidae AF407058b

Outgroup taxa

Tyrannus melancholicus Tyrannidae MNHN 28-56 South America AY529940 AY530008

Empidonax flaviventris Tyrannidae AF447629c

Acronyms are: FMNH, Field Museum of Natural History, Chicago, USA; MNHN, Museum National d�Histoire Naturelle, Paris, France; RCKB,

Rauri C.K. Bowie; US, University of Stellenbosch, South Africa; and ZMCU, Zoological Museum, University of Copenhagen, Denmark.a Johanson and Ericson (2003).b Sorenson and Payne (2001).c Cicero and Johnson (2002).

430 J. Fuchs et al. / Molecular Phylogenetics and Evolution 33 (2004) 428–439

2.2. Laboratory procedures

Genomic DNA was extracted from either alcohol-

preserved tissues, frozen tissues (muscle, blood) or from

skin of museum specimens (0.5–1cm2) using a CTAB

buffer with Proteinase K (0.1mgml�1) or a Puregene

DNA isolation kit (Gentra Systems, Minneapolis, Min-

nesota). Myoglobin intron-2 was amplified with primers

Myo 2 and Myo 3F (Heslewood et al., 1998; Slade et al.,1993) and in a few cases with internal primers (Myo2int:

50 TRA GAC CCA TAA AAC TAA GTG 30; Myo3int:

50 TGA TCT GCT TCA TGA CCT T 30). The ND2

gene was amplified with primers L5219 Met andH6313 Trp (Sorenson et al., 1999). b-Fibrinogen in-

tron-5 was amplified using the primers Fib5 (50 CGC

CAT ACA GAG TAT ACT GTG ACA T 30) and

Fib6 (50 GCC ATC CTG GCG ATT CTG AA 30).

The thermocycling conditions included a hotstart at

94 �C, an initial denaturation at 94 �C for 3min, fol-

lowed by 35–40 cycles at 94 �C for 40s, 52–56 �C for

30s, and 72 �C for 50s, and was completed by a finalextension at 72 �C for 5min. Three microliters of the

amplification products was electrophoresed on 1.5%

agarose gel and visualized under UV light with ethidium

J. Fuchs et al. / Molecular Phylogenetics and Evolution 33 (2004) 428–439 431

bromide to check for the correct fragment size and to

control for the specificity of the amplifications. For b-fi-brinogen intron-5, amplicons of the appropriate length

were cut out of the gel and purified using GELase (Epi-

centre Technologies, Madison, Wisconsin). Purified

PCR products were cycle-sequenced using the ‘‘CEQDye Terminator Cycle Sequencing’’ kit (Beckman) or

the Big Dye terminator chemistry (Applied Biosystems

[ABI]) in both forward and reverse direction with the

same primers used for PCR amplifications and run on

an automated CEQ2000 DNA Analysis System sequen-

cer (Beckman) or on an ABI 3100 automated DNA se-

quencer. Sequences were always obtained from both

DNA strands. All b-fibrinogen intron-5 sequences werescreened for the presence of conserved exon sequences at

both the 30 and 50 ends, to ensure that the whole intron

was sequenced and that any length variation was due to

the presence of insertions or deletions (indels).

Allelic variation within individuals, i.e., heterozygos-

ity, was apparent in the two nuclear loci. Heterozygosity

could occur in two different ways: (1) length variation

between alleles and (2) single nucleotide polymorphism(SNP). Length variation between alleles was only appar-

ent for b-fibrinogen intron-5. PCR products from such

individuals were cloned using the TOPO cloning kit

(Inviatron) and multiple clones (6–10) were cycle-se-

quenced. A total of four species were cloned. Length

variation was present in two taxa (Tyrannus melancholi-

cus and Lanius collaris) with the alleles differing by 1 or

2bp. Both alleles were included in initial parsimonyanalyses. In all instance the two alleles from a single

individual clustered together with high bootstrap sup-

port and we therefore used the consensus sequences of

the two alleles obtained with Sequencher 4.1 (Genes

Codes, Ann Arbor, Michigan) for further analysis.

Occurrence of SNP in some b-fibrinogen intron-5 and

myoglobin intron-2 sequences was suggested by the

presence of double peaks. These double peaks were trea-ted using the appropriate IUPAC code.

2.3. Sequences alignment

Multiple alignment of intron sequences was accom-

plished by hand modification after an initial alignment

by Sequencher 4.1. Final alignment indicated the pres-

ence of several insertions–deletions events in the two nu-clear introns (18 in the myoglobin intron-2 and 39 in the

b-fibrinogen intron-5). Alignment of these indels was

fairly straightforward, yielding no region of significant

ambiguity, i.e., that led to topological differences be-

tween different alignments, in the myoglobin intron-2 se-

quences. Indels in the b-fibrinogen intron-5 sequences

could not be unambiguously aligned in one zone, mainly

due to the presence of multiple insertions–deletionsevents within an A–T-rich zone which made hypothesis

about homology questionable. We deleted this particu-

lar zone from our analyses (nucleotides 77–90 in our

b-fibrinogen intron-5 alignment). After removing align-

ment ambiguities, the number of potential informative

indels were 4 and 12 for myoglobin intron-2 and b-fi-brinogen intron-5, respectively. Gaps were treated as

missing data.

2.4. Phylogenetic analyses

2.4.1. Search parameters

Phylogenetic trees were estimated using parsimony,

maximum likelihood, and Bayesian inferences as imple-

mented in PAUP* 4.0b10 (Swofford, 2001) and MrBa-

yes v.3.0 (Huelsenbeck et al., 2001). Parsimony (P)analyses were performed under the heuristic search op-

tion with 10 random addition replicates with the

branch-swapping option tree bisection and reconnection

(TBR) implemented. Support for nodes was estimated

using the non-parametric bootstrap (Felsenstein, 1985)

with 1000 replicates. Likelihood models and parameters

were estimated using Modeltest v.3.04 (Posada and

Crandall, 1998) and the Hierarchical Likelihood RatioTest criterion. We also tested the applicability of the

molecular clock for all individual data set as well as

for the combined data set by using the Likelihood Ratio

Test (LRT) (�2 lnA = 2(lnk1 � lnk2), where k1 is the

likelihood of the restricted model) (Huelsenbeck and

Rannala, 1997), which follows a v2 distribution with

n � 2 degrees of freedom where n is the number of taxa.

Tree searches were estimated by heuristic TBR branch-swapping with 10 random addition replicates. Nodal

support was calculated using the non-parametric boot-

strap (100 replicates). For comparing different a priori

and a posteriori topologies, we use the non-parametric

Shimodaira–Hasegawa test (SH test) (Shimodaira and

Hasagawa, 1999) implemented in PAUP. Scores of the

constrained trees were obtained by instructing PAUP

to find the best tree under a specific constraint. TheSH test between the best tree and the constrained tree

was then performed using the RELL option with 1000

bootstrap replicates.

For the Bayesian analyses, the optimal model param-

eterization as determined by ModelTest was assumed

for each data set. Bayesian analyses for the combined

data set were performed freeing different parameters to

vary between the three partitions (ND2, myoglobin in-tron-2, and b-fibrinogen intron-5) (using the prset and

unlink options). Each analysis was initiated from a ran-

dom starting tree. Analyses were conducted using

Metropolis coupling chains with four incrementally

heated Markov chains. Chains were run for 4 · 106 gen-

erations, and sampled every 100. We determined the

minimum number of generations to discard as ‘‘burn-

in’’ by plotting likelihood scores against generationnumber. Posterior probabilities for clades were esti-

mated by a 50% majority-rule consensus tree based on

432 J. Fuchs et al. / Molecular Phylogenetics and Evolution 33 (2004) 428–439

the remaining saved trees. Four independent Bayesian

runs were performed for each data set. Log-likelihood

values and posterior probabilities were also checked to

confirm that using a different starting tree did not alter

our results significantly.

2.4.2. Evaluation of congruence

Significant incongruences between the individual gene

trees were detected by comparing the topologies and no-

dal support obtained under different analytical methods

(parsimony, maximum likelihood, and Bayesian infer-

ences). Criterions for incongruence were set at 70% for

the bootstrap values, according to Hillis and Bull

(1993), and at 0.95 for posterior probabilities; these lat-ter are argued to have a straightforward statistical inter-

pretation (Huelsenbeck et al., 2001). The support or

conflict of individual data sets with particular nodes

recovered in the shortest parsimony trees was also eval-

uated using the Partitioned Bremer Support (Baker and

DeSalle, 1997; Baker et al., 1998; Bremer, 1994), calcu-

lated with TreeRot v. 2 (Sorenson, 1999).

Table 2

Average nucleotide composition for myoglobin intron-2, b-fibrinogen intron

Gene A C

Myoglobin intron-2 0.28 0.22

b-Fibrinogen intron-5 0.30 0.17

ND2 0.31 0.32

ND2, first position 0.34 0.29

ND2, second position 0.17 0.34

ND2, third position 0.41 0.35

Combined data set 0.30 0.26

Base proportions are also shown for each codon positions of ND2. The v2

(Swofford, 2001), with df = 102 (*p < 0.01).

Table 3

Data characteristics and estimated substitution parameters for myoglobin in

Gene Myoglobin b-Fib

Number of bases 654 625

Number variable/informative 256/113 282/12

Model selected K80 + G HKY

Ti/Tv 2.59 1.79

RA–C

RA–G

RA–T

RC–G

RC–T

RG–T

a 0.84 1.21

I 0 0

�ln likelihood (ML) 3265.8 3542.6

Bayesian analyses 3296.88 ± 1.41 3572.1

Tree length (MP) 401 470

Number of trees (MP) 12,343 214

CI/RI 0.74/0.70 0.75/0

Likelihood scores were estimated under the TBR branch-swapping option. asites. Score of the Bayesian analyses represents the mean of the four independ

length, and homoplasy measures are for the most parsimonious tree(s) obta

3. Results

3.1. Sequence variation and characteristics

The three gene regions concatenated resulted in a fi-

nal alignment of 2313bp. Average base frequencies forthe individual genes and for the combined data set are

presented in Table 2. The number of variable and infor-

mative characters and maximum likelihood parameters

estimated by ModelTest for all data sets are indicated

in Table 3. From the myoglobin intron-2, we obtained

between 625bp (Nilaus afer) and 653bp (majority of

taxa). The uncorrected pairwise distances range between

0.3 (between L. luehderi and L. barbarus) and 7.8% foringroup species (between Tchagra australis and Rhipi-

dura albicollis) and up to 12.2% when the outgroup

was included (between T. melancholichus—outgroup—

and T. australis). For b-fibrinogen intron-5 we obtained,

after deleting the ambiguous zone, between 536 (Terpsi-

phone viridis) and 574bp (Lanioturdus torquatus). The

uncorrected pairwise distances range between 0.9

-5, ND2, and the combined data set

G T v2

0.25 0.25 8.66

0.22 0.31 18.64

0.12 02.5 105.91

0.18 0.19 23.57

0.10 0.39 8.32

0.09 0.15 234.13*

0.18 0.26 53.13

values refer to the two-way independence test implemented in PAUP

tron-2, b-fibrinogen intron-5, ND2, and the combined data set

rinogen ND2 Combined data set

1034 2313

6 627/547 1165/786

+ G GTR + G + I TVM + G + I

0.37 1.32

9.55 7.16

0.55 1.07

0.35 0.65

4.28 7.16

1 1

0.75 0.41

0.35 0.22

2 15838.92 23497.98

7 ± 1.32 15879.16 ± 1.78 22843.67 ± 0.96

3860 4770

2 1

.68 0.28/0.34 0.36/0.38

corresponds to shape parameter and I to the proportion of invariable

ent runs with the standard deviations associated. Number of trees, tree

ined under equally weighted parsimony.

J. Fuchs et al. / Molecular Phylogenetics and Evolution 33 (2004) 428–439 433

(between L. luehderi and L. aethiopicus) and 10.9% for

ingroup species (between Dyaphorophyia chalybea and

Rhipidura abicollis) and up to 15.4% with the inclusion

of the outgroup (between T. melancholichus and Lanius

collaris).

For ND2, we obtained a total of 1034bp, corre-sponding to positions 5246–6279 of the mtDNA chicken

sequence (Desjardins and Morais, 1990). We could not

obtain a clearly resolved chromatogram for T. melan-

cholichus. Thus, we used the ND2 sequence of Empido-

nax flaviventris (GenBank Accession No. AF447626,

Cicero and Johnson, 2002) that also belong to the family

Tyrannidae (Suboscines) as an outgroup. The v2 test

implemented in PAUP detected no base compositionbias (p = 0.38) among the whole gene. Homogeneity

across the taxa was not rejected for first and second co-

don positions (Table 2), however, the test detected sig-

nificant heterogeneity across all taxa for the third

codon position, with a deficiency in guanine. Deficiency

in guanine at third codon position, as well as the absence

of insertions, deletions, and stop-codon in the reading

frame and the absence of abnormal amino acid substitu-tion pattern in closely related taxa are typical of mito-

chondrial DNA of birds (Barker, 2004; Bates et al.,

1999; Hackett, 1996; Sorenson and Quinn, 1998), sug-

gesting that we had not sequenced nuclear pseudogenes.

The numbers of parsimony informative sites per codon

position were: 159 (29%) at first codon position, 64

(12%) at second position, and 324 (59%) at third posi-

tion. The uncorrected pairwise distances range between8.8 (between L. funebris and L. aethiopicus) and 25.4%

for ingroup species (between Bias musicus and Telopho-

rus zeylonus) and up to 30.75% when including the out-

group (between E. flaviventris and T. zeylonus).

3.2. Evaluation of congruence between the three genes

Only two significant topological incongruences, as in-ferred from bootstrap and Partitioned Bremer Support

values, were found between the individual genes trees

(Table 4). The first one involves the positions of the gen-

era Batis and Lanioturdus relative to Platysteira and

Dyaphorophyia (node E on Fig. 1). The three molecular

markers are all in siginificant conflict for this node. b-Fi-brinogen intron-5 indicated a sister-group relationships

between Batis and Lanioturdus while myoglobin in-tron-2 suggested that Lanioturdus is more closely related

to Platysteira and Dyaphorophyia than to Batis. Finally,

ND2 strongly supported the basal position of Laniotur-

dus in relation to Batis, Platysteira, and Dyaphorophyia.

The second incongruent node involves the position of

the genera Dryoscopus and Malaconotus relative to Boc-

agia and Tchagra (node I on Fig. 1). ML and Bayesian

analyses of ND2 and all analysis undertaken with thefibrinogen sequences supported a sister-group relation-

ships between Malaconotus and Dryoscopus, while the

unweighted MP analysis of the mitochondrial data set

suggested that Dryoscopus is closer to Bocagia and

Tchagra than to Malaconotus, but with bootstrap values

close to 70%, i.e., the threshold of significant

incongruence.

The Molecular Clock model was rejected for themitochondrial gene, suggesting rate variation among lin-

eages, but not for the two nuclear introns (Table 5).

3.3. Phylogenetic results—combined data set

Parsimony analysis resulted in a single most parsimo-

nious tree (Fig. 1; length = 4770 steps, CI = 0.36,

RI = 0.38) with the Malaconotidae, Platysteiridae, andVangidae forming a well-supported clade (Boot-

strap = 91%; node A on Fig. 1); a result supported in

both the ML (�ln = 23497.98, TVM + I + G model,

see Table 3 for parameters) and BI (GTR + G + I model

for ND2, a K80 + G model for myoglobin intron-2, and

a HKY85 + G model for b-fibrinogen intron-5) analyses

(Fig. 2, Table 4). A molecular clock was rejected

(�2 lnA = 87.44; df = 33; p = <0.001) when the threedata sets were combined. No significant variations in

the posterior probabilities among the four independent

runs and log-likelihood value were detected (Table 5).

In all three analyses the Malaconotidae (sensu How-

ard and Moore, 2003) are not monophyletic due to Pri-

onops being more closely related to the vangas

(Cyanolanius and Pseudobias, Vangidae) and flycatch-

er-shrikes (Bias and Megabyas, Platysteiridae) than totraditional Malaconotidae genera (Figs. 1 and 2; Table

4). Monophyly of the Malaconotidae was rejected by

the Shimodaira–Hasegawa test (dL = 39.14, p = 0.017).

The Platysteiridae genera (Lanioturdus, Batis, Platy-

steira, and Dyaphorophyia) are united in a single clade

in all three analyses (node D on Fig. 1, hereafter called

the �core platysteirids�) with considerable support (Figs.

1 and 2, Table 4). In the MP analyses the �core platy-steirids� are sister to the �core malaconotids,� whereasin the ML and BI analyses this clade is sister to the �corevangids.� However, neither hypothesis is strongly sup-

ported (Figs. 1 and 2, Table 4) and the SH test did

not reject the MP hypothesis (dL = 1.06 p = 0.42).

In all three analyses two main clades emerged within

the �core malaconotids� (node F on Fig. 1). The first in-

cluded Malaconotus, Dryoscopus, Bocagia, and Tchagra

(clade H) and the second Chlorophoneus, Laniarius,

Rhodophoneus, and Telophorus (clade J). Position of

the genus Nilaus was variable and not well supported

in any of the three analyses. Monophyly of the genus

Chlorophoneus was never recovered as C. dohertyi is

more closely related to Telophorus and Rhodophoneus

than to other Chlorophoneus species. Monophyly of

the genera Dryocopus, Laniarius, and Tchagra were allstrongly supported (Figs. 1 and 2, Table 4). Slight re-ar-

rangements among taxa occurred within clade H (see

Table 4

Summary of topological arrangements resulting from combined and individual analyses of the three genes (myoglobin intron-2, b-fibrinogen intron-

5, and ND2)

Node Gene All data (2313bp) Myoglobin (654bp) b-Fibrinogen (625bp) ND2 (1034bp)

Analyses: P PBS ML BI P ML BI SI P ML BI SI P ML BI

A 91 2/8/2 100 1.00 94 94 1.00 64 63 0.97 / 71 1.00

C <50 3/�2/0 / / <50 / / / / / / / /

D + B / / 58 0.60 / <50 / / / / / <50 0.54

B (Core vangids) 61 1/3/�1 98 1.00 / <50 / 66 76 1.00 / 62 0.99

Prionops 100 15/8/30 100 1.00 100 100 1.00 100 100 1.00 15 100 100 1.00

Bias + Megabyas 65 2/0/4 62 0.72 / 60 0.8 / / / / / /

Bias + Megabyas + Prionops / / <50 0.81 / <50 0.74 / / / / / /

Bias + Megabyas + Vangidae <50 3/�3/1 / / / / / / / / / / /

Vangidae 74 2/1/2 96 1.00 / / / 93 89 1.00 60 62 0.95

D (Core platysteirids) 97 4/4/5 100 1.00 89 88 1.00 63 81 1.00 1,4,24 79 94 1.00

E 99 3/�4/16 89 1.00 / / / / / / 1 99 96 1.00

Lanioturdus + Platysteira +

Dyaphorophyia

/ / / / 65 64 0.99 / / / / / /

Lanioturdus + Batis / / / / / / / 94 91 1.00 / / /

Platysteira + Dyaphorophyia 91 3/14/�5 100 1.00 69 63 0.84 100 100 1.00 / / /

F (Core malaconotids) 84 6/0/0 100 1.00 53 59 1.00 / 73 1.00 <50 85 1.00

G <50 3/�3/0 74 0.89 / / / / / / / 50 0.87

H 92 2/0/4 100 1.00 / / / 81 93 1.00 71 96 1.00

I 65 �1/�2/5 / / / / / / / / 79 / /

Malaconotus + Dryoscopus / / 54 0.99 / / / 74 85 0.92 / 50 0.62

Dryoscopus 100 7/10/23 100 1.00 98 98 1.00 100 100 1.00 1 100 100 1.00

Bocagia + Tchagra 99 1/1/7 100 1.00 73 82 1.00 87 87 1.00 95 99 1.00

Tchagra 99 4/1/14 100 1.00 83 93 1.00 1 86 86 1.00 2 96 91 1.00

J 93 1/3/3 100 1.00 / / / 9 / / 0.85 83 91 1.00

Laniarius 100 7/3/17 100 1.00 98 99 1.00 77 92 1.00 1 100 98 1.00

K 51 1/1/�1 53 0.74 <50 <50 0.9 / / / 1 / / /

C. nigrifrons + C. sulfureopectus 100 6/4/16 100 1.00 99 98 1.00 100 99 1.00 1 98 99 1.00

L 97 2/8/1 100 1.00 <50 <50 0.71 100 99 1.00 72 87 1.00

Telophorus + C. dohertyi 54 3/�2/0 83 1.00 82 86 0.99 54 53 / <50 68 0.98

Nodes refer to those labelled in Fig. 1. Numbers refer to Bootstrap values (MP, ML), to Posterior probabilities (BA) and to Partitioned Bremer

Support (myoglobin/b-fibrinogen/ND2) (PBS). Acronyms are: P, Parsimony; PBS, Partitioned Bremer Support; ML, Maximum Likelihood; BA,

Bayesian Analyses; SI, Synapomorphic indels (number represents the length in bp of the indel). / indicates either that an alternative node was found

or that the node was not resolved (polytomy). Node that were only found once and that were supported by bootstrap values/posterior probabilities

less than 70%/0.95 are not listed in the table.

434 J. Fuchs et al. / Molecular Phylogenetics and Evolution 33 (2004) 428–439

Fig. 1) depending on the optimality criterion used (P,

ML or BI). The parsimony topology was not signifi-

cantly rejected by the Shimodaira–Hasegawa test(dL = 2.82, p = 0.39).

All but two indels have unique gains or losses when

mapped onto the tree. These two indels are deletions

of one nucleotide where more than one alignment is pos-

sible (note that none of these alternative alignments af-

fect the phylogenetic results). In general, indels

supported nodes that also received high bootstrap val-

ues except in three cases. Two of these exceptions sup-ported relationships between some Corvoidea

outgroups while the latter supported the Chlorophon-

eus–Rhodophoneus–Telophorus clade (node K). Synapo-

morphic indels are indicated in Table 4.

Combination of the nuclear and mitochondrial data

sets increased support values of nodes that were moder-

ately to well supported in the individual analyses (boot-

strap values between 50 and 85%) whatever the

analytical methods employed (Table 4). Nodes that were

marginally found or always poorly supported, e.g., thesister-group relationships between �core malaconotids�and �core platysteirids� or between �core malaconotids�and �core vangids,� remained poorly supported.

4. Discussion

4.1. Monophyly of the Malaconotidae and sister-group

relationships

The systematic position of Prionops (helmet-shrikes)

has remained controversial as members of this genus dif-

fer from other Malaconotidae by their tarsal scutella-

tion, nest building, and social behavior (Dorst, 1960;

Harris and Arnott, 1988; Raikow et al., 1980; Urban

Fig. 1. Parsimony tree (4770 steps; CI = 0.36; RI = 0.38) obtained with

the unweighted combined data set. Numbers below branches represent

bootstrap values greater than 50% (1000 replicates). The Malaconot-

idae (sensu Howard and Moore, 2003) are in bold type.

Fig. 2. Maximum likelihood tree (�ln = 23497.98) and Bayesian tree

(mean log-likelihood value = �22843.67, SD = 0.96) obtained with the

combined data set under a TVM + I + G model for the ML search and

with the combined data set with the best-fit models estimated by

Modeltest 3 assigned to each partition for the Bayesian analysis (a

GTR + G + I model for ND2, a K80 + G model for myoglobin intron-

2, and a HKY85 + G model for b-fibrinogen intron-5). ML parameters

estimated from Modeltest 3.04: bases frequencies = (0.3155, 0.2711,

0.1602, 0.2532), Rmat = (1.32, 7.16, 1.07, 0.65, 7.16), shape = 0,41, and

Pinvar = 0.22. Four heated Markov chains were run for 4 · 106

generations for the Bayesian analysis. The first 2000 trees (200,000

generations) were discarded (�burn-in�). The three gamma shape

distributions were specified as unlinked. Symbols */* next to branches

represent bootstrap values/posterior probabilities greater than 95%/

0.95. The Malaconotidae (sensu Howard and Moore, 2003) are in bold

type.

J. Fuchs et al. / Molecular Phylogenetics and Evolution 33 (2004) 428–439 435

et al., 2000). Interestingly, DNA–DNA hybridization

data suggested a placement of Prionops closer to the

Vangidae and Platysteiridae than to other Malaconoti-

dae (Sibley and Ahlquist, 1990), but its exact relation-

ships remained uncertain due to short internodes, the

fact that only four species were used as �drivers� and that

tests of robustness were lacking. Our analyses confirmedthe tentative result obtained by the DNA–DNA hybrid-

izations by providing strong support for a clade com-

prising Prionops, Bias, and Megabyas (Platysteiridae),

and Cyanolanius and Pseudobias (as representatives of

the Vangidae). Several authors have proposed a close

link between Prionops and the Vangidae because they

share characters such as tarsal scutellation, jaw muscula-

ture, bill structure, and gregarious behavior (Beecher,1953; Benson, 1985; Dorst, 1960). Our analyses are the

first to provide strong support for this hypothesis.

Table 5

Likelihood Ratio Test on the applicability of the molecular clock to the dat

Gene �lnka �lnkb

Myoglobin intron-2 3277.80 3265.80

b-Fibrinogen intron-5 3561.96 3542.62

ND2 15877.02 15838.92

Combined data set 23541.70 23497.98

a The log likelihood of the restricted model (application of the molecularb The log likelihood of the non-restricted model (non-application of the mc H0 accepted.d H0 rejected.

Previous studies that tackle themonophyly of the Van-

gidae only included one representative of the supposed

closest genus (Laniarius), providing thus only a weak test

of Vangidae monophyly (Schulenberg, 1993; Yamagishiet al., 2001). In our present study, the two Vangidae

a

�2 lnA df p

24 33 0.87c

38.68 33 0.77c

76.20 33 <0.001d

87.44 33 <0.001d

clock).

olecular clock).

436 J. Fuchs et al. / Molecular Phylogenetics and Evolution 33 (2004) 428–439

genera (Cyanolanius andPseudobias) clustered together in

all combined analyses. As we only sampled two vangid

species, we were unable to test the monophyly of the Van-

gidae. This needs to be further explored with representa-

tives of all Vangidae genera. Here, we show that an

appropriate taxonomic sampling for this test should in-clude a substantial number of genera of the �core malaco-

notids,� �core platysteirids,� and �core vangids.�Bias and Megabyas have sometimes been merged in

Bias due to their similar skull, plumage (black upper-

parts with green sheen in males, rufous in females), size,

and proportions (Erard, 1987; Traylor, 1970). Our anal-

yses failed to provide strong support for a sister-group

relationship between these two genera, although theyform a sister-group in the phylogeny. Molecular diver-

gence as well as differences in ecology, morphology, so-

cial organization, and general behavior are sufficiently

high for retaining two genera (Harris and Franklin,

2000). The position of Bias and Megabyas within the

�core vangids� is quite surprising because they were sus-

pected to be related to the �core platysteirids,� despitesome morphological differences (Erard, 1987). Never-theless, their relationships to other birds have remained

obscure mainly due to the lack of comparative data

(Harris and Arnott, 1988; Harris and Franklin, 2000;

Traylor, 1970).

The systematic affinities of Lanioturdus have for

many years been uncertain, as its name suggests. It has

been considered an atypical member of the �core malaco-

notids� (Morony et al., 1975; Rand, 1960; Sibley andMonroe, 1990) or a close relative of the helmet-shrikes

(Wolters, 1975–1982). All our analyses included it in

the �core platysteirids� with strong support (high boot-

strap values and three synapomorphic indels). Its posi-

tion within the �core platysteirids� is also supported by

jaw musculature and plumage pattern (Beecher, 1953;

Harris and Arnott, 1988; Harris and Franklin, 2000; Ur-

ban et al., 1997). Thus, Lanioturdus represents an aber-rant platysteirid adapted to a mostly terrestrial life in the

Namib desert.

The position of Lanioturdus as the first offshoot in the

�core platysteirids� was a significant source of incongru-

ence between our data sets. Analyses undertaken with

the b-fibrinogen intron-5 sequences placed it as the

sister-group of the genus Batis, myoglobin intron-2 se-

quences placed it between Batis and the Dyaphoro-

phyia–Platysteira clade, and the ND2 sequences placed

it at the same position as the combined data set. The

SH test did not reject a sister-group relationship between

Batis and Lanioturdus (dL = 12.05, p = 0.12) nor did it

reject a sister-group relationship between Lanioturdus

and the Dyaphorophyia–Platysteira clade (dL = 13.49,

p = 0.09). A synapomorphic insertion of one nucleotide

within the large 24bp insertion that supports the mono-phyly of the �core platysteirids� seems to confirm the

monophyly of the Batis–Platysteira–Dyaphorophyia

clade but it could alternatively be interpreted as an auta-

pomorphic deletion in Lanioturdus. The placement of

Lanioturdus as the basal branch in the �core platysteirids�needs to be confirmed by further studies including addi-

tional representatives of the genera Batis, Dyaphoro-

phyia, and Platysteira.Species from these two latter genera were tradition-

ally placed together in Platysteira (Sibley and Monroe,

1990; Traylor, 1970) but Erard (1987) and Harris and

Franklin (2000) pointed out sufficient behavioral and

morphological differences for retaining two genera.

These clustered together with high bootstrap values in

most of our analyses but our taxonomic sampling does

not allow us to clarify the generic limits.Monophyly of the �core malaconotids� was well sup-

ported in all our analyses and is in agreement with the

insertion pattern of the Musculus flexor digitorum longus

muscle (Raikow et al., 1980). The other synapomorphy

proposed by the authors (presence of fluffy back feath-

ers) is also present in all members of the �core platysteir-ids� and even (more or less developed) in other corvid

groups (J. Fjeldsa, personal observation). This characterwas not considered by Raikow et al. (1980), as members

of the Platysteiridae were not included in their study. It

has not been fully documented how broadly this charac-

ter is shared, or whether its development, and adaptative

significance for courtship suggests homology.

Representatives of the core �malaconotids,� �platy-steirids,� and �vangids� formed a well-supported clade

(clade A on Fig. 1) in all our analyses. However, rela-tionships between these three main groups depended

on the analytical methods employed and were generally

poorly supported, so the exact sister-group of the �coremalaconotids� remains undetermined. The individual

analyses of the two nuclear introns showed that the

molecular divergence between the three main groups

was nearly identical to the divergence observed between

genera in the �core malaconotids.� These results agreewith those obtained by Sibley and Ahlquist (1990) in

their large DNA/DNA hybridization study where the

short internodes between the Clade A genera suggest a

rapid speciation burst leading to several lineages of very

divergent appearance. Evolution of foraging behavior,

and thus beak morphology, is particularly known for

its evolutionary plasticity (Cibois et al., 2001; Grant

et al., 2000; Irestedt et al., 2004; Pasquet et al., 1999).

4.2. Relationships within the �core malaconotids�

Nilaus is morphologically divergent from other repre-

sentatives of the �core malaconotids� because its tarsal

scutellation resembles that of Prionops and the nest

building is more Batis-like (Harris and Franklin, 2000;

Mayr, 1943). Our data as well as hind limb musculatureand DNA/DNA hybridization data nest it within the

�core malaconotids� (Raikow et al., 1980; Sibley and

J. Fuchs et al. / Molecular Phylogenetics and Evolution 33 (2004) 428–439 437

Ahlquist, 1990). Traylor (1970) argued that Nilaus is the

link between the �core malaconotids� and the �core platy-steirids,� but this also implies that the �core platysteirids�is the sister-group of the �core malaconotids,� a state-

ment that was not clearly supported by our data. Our

analyses placed it as the sister-group of clade H, but al-ways with low bootstrap values. The SH test did not re-

ject the placement of Nilaus as the first offshoot in the

�core malaconotids� nor did it reject its placement as

the sister-group of clade J (dL = 1.54, p = 0.31 and

dL = 2.38, p = 0.17, respectively). Its position relative

to clades H and J must thus be considered as unresolved.

The remaining malaconotid genera clustered together

in two primary clades that were always supported byhigh bootstrap/posterior probability values. The first

clade (Clade H) included Bocagia, Dryocopus, Malacon-

otus, and Tchagra and the second clade (Clade J) con-

sisted of Chlorophoneus, Laniarius, Rhodophoneus, and

Telophorus.

The genus Malaconotus comprises six little-known

large bush-shrikes, most of which are very locally distrib-

uted (relictual), with unique character combinations.Malaconotus was traditionally believed to be the closest

relative of Chlorophoneus due to their closely similar col-

ors (Hall et al., 1966; Urban et al., 1997). However,Mal-

aconotus differs from Chlorophoneus by the lack of

mouth spots (in nestlings) and by general communica-

tion behavior (Harris and Franklin, 2000). DNA–DNA

hybridization data suggested that Malaconotus is basal

relative to other members of the �core malaconotids� (Sib-ley and Ahlquist, 1990). A sister-group relationship be-

tween Chlorophoneus and Malaconotus was strongly

rejected by our data (SH test, dL = 54.19, p = 0.004).

Our analyses generate some new hypotheses about the

relationships of Malaconotus and suggest an association

with Dryoscopus, Bocagia, and Tchagra. However, the

relative position of Malaconotus within clade J depends

on the phylogenetic reconstruction method employed,and was always moderately supported.

The genus Dryoscopus is morphologically very uni-

form (six small species with black upperparts and white

or grayish underparts). Dryoscopus was monophyletic in

all our analyses, a result that was always highly sup-

ported, and lent further credence by a synapomorphic

deletion. Linear classifications placed it between Nilaus

and Tchagra (including Bocagia) (Rand, 1960; Sibleyand Monroe, 1990), suggesting a close link between Dry-

oscopus and Tchagra, whereas DNA/DNA hybridiza-

tion data could not resolve its relationships (Sibley

and Ahlquist, 1990). Our data also suggest that Tchagra

and Bocagia are very close to Dryoscopus. More taxa

and sequences from species within the genus may be nec-

essary to assess its exact sister-group (Malaconotus or

Bocagia–Tchagra).Due to similar plumage aspect, egg color, and dis-

play-flights, some authors merge Bocagia and Tchagra

in the genus Tchagra (Rand, 1960; Sibley and Monroe,

1990; Wolters, 1975–1982). However, Harris and Frank-

lin (2000) retained two genera and pointed out that Boc-

agia differs from Tchagra in several characters (sexual

dimorphism, eye color, tail color, communication

behavior, nest decoration, and vocalization). They sug-gested that Bocagia may be closer to Dryoscopus than

to Tchagra. This statement is clearly refuted by our data,

suggesting that the characters proposed by Harris and

Franklin (2000) for uniting Bocagia and Dryoscopus

are plesiomorphic within clade H. The diagnostic char-

acters proposed by Harris and Franklin (2000) for

retaining Bocagia are nearly all utilized in courtship dis-

plays and thus could have an accelerated rate of evolu-tion due to sexual selection. While the molecular

evidence seems to support merging Bocagia with Tcha-

gra, a denser taxon sampling is desirable to better quan-

tify sister-group relationships and generic limits.

The genusTchagra (sensu stricto) consists of four open

habitat species of very uniform plumage pattern. Its

monophyly was always well supported (high bootstrap

values and two synapomorphic deletions) by our data.The two species we lack in our taxon sampling (T. jamesi

and T. tchagra) are believed to form a superspecies with

T. australis (Sibley and Monroe, 1990). This assumption

is emphasized by the fact that a hybrid between T. aus-

tralis and T. jamesi has been described (Keith and

Twomey, 1968). However, inclusions of T. jamesi and

T. tchagra in a further study are necessary to determine

the taxonomic status of T. jamesi and eventually to quan-tify the amount of gene flow between the two taxa.

The second clade (clade J) consisted of the genera

Chlorophoneus, Laniarius, Rhodophoneus, and Telopho-

rus. This clade was supported by high bootstrap values

and a synapomorphic deletion (nine nucleotides). An

ethological synapomorphy (body bobbing while calling)

also supports this clade (Harris and Franklin, 2000).

The genus Laniarius comprises 16 species that areuniform in shape and proportions. Although colors

are variable, monophyly was never questioned and the

only contradictory hypotheses concern intra-generic

relationships (Harris and Franklin, 2000; Smith et al.,

1991). Our data also strongly support monophyly of

Laniarius (high bootstrap values and a synapomorphic

deletion of one nucleotide). Laniarius is traditionally

considered closer to Telophorus and Chlorophoneus thanto other representatives of the �core malaconotids� (Har-

ris and Franklin, 2000). Our data suggest that the closest

relatives of the genus Laniarius are the genera Chloro-

phoneus, Rhodophoneus, and Telophorus.

Representatives of the three latter genera formed a

clade in all our analyses but always with moderate to

low bootstrap support. However, we detected support

for monophyly because all its members share a synapo-morphic insertion of one nucleotide. The genus Chloro-

phoneus is not monophyletic (SH test, dL = 39.05,

438 J. Fuchs et al. / Molecular Phylogenetics and Evolution 33 (2004) 428–439

p = 0.009) in our analyses due to C. dohertyi being more

closely related to the genera Rhodophoneus and Telopho-

rus than to other Chlorophoneus species. This result is in

agreement with several morphological characters such

as toe fusion, presence of the Musculus iliofemoralis

externus muscle, presence of a black pectoral bar, andecological differences (Clark, 1981; Raikow et al.,

1980), also supported at the molecular level. We propose

to merge the undergrowth species of the genus Chloro-

phoneus (sensu Howard and Moore, 2003) (dohertyi

and viridis—including quadricolor) and Rhodophoneus

in Telophorus, and to keep the forest, woodland, and

bushland species in the genus Chlorophoneus (remaining

species). Monophyly of the latter genus (sensu stricto)needs to be tested because one of its members, the Crit-

ically Endangered Mount Kupe Bush-shrike (Chloro-

phoneus kupeensis), is quite different from other

Chlorophoneus species and some characters suggest that

it might be related to the �core vangids� (Harris and

Franklin, 2000). These latter authors argue that Chloro-

phoneus is more closely related to Rhodophoneus due to

the presence of mouth spots in nestlings, nest structure,and egg coloration, although some behavioral and mor-

phological data suggest a close relationship between

Rhodophoneus and Telophorus. The characters proposed

by Harris and Franklin (2000) are certainly a melting

pot of plesiomorphic and synapomorphic characters,

but in all cases they support the close affinities of the

genera Chlorophoneus, Rhodophoneus, and Telophorus.

Acknowledgments

We are very grateful to J.M. Bates and D. Willard

(Field Museum of Natural History, Chicago) and C. Co-

hen (FitzPatrick Institute, University of Cape Town) for

providing some of the samples we used. To Annie Tillier,

Celine Bonillo, and Josie Lambourdiere who kindly pro-vided technical help during laboratory works at MNHN,

and to Francois Baillon (IRD) who helped E. Pasquet

while in Cameroon doing field work. Laboratory work

at MNHN was supported by the �Service Commun de

Systematique Moleculaire,� IFR CNRS 101, MNHN.

R.C.K.B. thanks the Skye Foundation and Charitable

trust for providing funding for this project while at the

FMNH. The first version of the manuscript benefitedfrom the comments of two anonymous referees.

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