Molecular Phylogenetics and Evolution
Vol. 11, No. 2, March, pp. 246–260, 1999
Article ID mpev.1998.0571, available online at http://www.idealibrary.com on

Molecular Phylogeny of Palearctic–African Acrocephalus andHippolais Warblers (Aves: Sylviidae)
Andreas J. Helbig1 and Ingrid Seibold
University of Greifswald, Vogelwarte Hiddensee, D-18565 Kloster, Germany
Received November 11, 1997; revised April 23, 1998

Phylogenetic relationships of the reed warbler group(genera Acrocephalus, Hippolais, Chloropeta; Aves: Passeriformes) and their potential relatives were studied
using nucleotide sequences (1 kb) of the mitochondrial
cytochrome b gene. This species-rich but morphologically poorly differentiated group of insectivorous passerines is distributed in Eurasia, Africa, and Australasia. Intergeneric relationships were poorly resolved,
but monophyly of the reed warbler group (including
Chloropeta) versus other Sylviidae was strongly supported. A basal polytomy within the reed warbler
group consists of seven branches and may indicate a
rapid early radiation. In the genus Acrocephalus threemajor clades were identified, which corresponded tophenotypic groups characterized by body size andplumage patterns. However, current delimitation ofsome subgenera (Acrocephalus, Lusciniola, Bebrornis)
is at variance with our phylogeny estimate, and appropriate revisions are proposed. The genus Hippolais,
which may or may not be monophyletic, consisted of
two well-supported clades of four species each. Some
Acrocephalus taxa whose species status had been
doubted (griseldis, tangorum, orientalis, australis)
proved to be highly distinct genetically. Genetic distances between members of two pairs of allopatricHippolais taxa (caligata/rama; opaca/elaeica) were aslarge or larger than between other closely related
warbler species. Overall, cytochrome b sequences resolved phylogenetically young relationships quite well,whereas more ancient nodes remained poorly
resolved. r 1999 Academic Press

INTRODUCTION
Warblers of the genera Acrocephalus (reed warblers,
29–34 species, number depending on ranking of distinct allopatric taxa) and Hippolais (6–7 species) are agroup of drab-colored, insectivorous passerine birds of

1 To whom correspondence should be addressed. Fax: (49)-3830050441. E-mail: helbig@rz.uni-greifswald.de.

1055-7903/99 $30.00
Copyright r 1999 by Academic Press
All rights of reproduction in any form reserved.

the Old World family Sylviidae. In terms of plumage
coloration the reed warblers are among the most monomorphic groups of songbirds, although there is considerable differentiation between taxa in size and vocalizations. The most recent comprehensive systematic listsof this group were given by Watson et al. (1986) andSibley and Monroe (1990, 1993), whereas Haffer (1991)
discussed Palearctic taxa and Erard et al. (1997) treated
African taxa in some more detail. There is a consensus
among systematists that the genera Acrocephalus andHippolais are closely related, but their monophyly withrespect to each other and to the Afrotropical genus
Chloropeta (3 species) has been doubted (Fry et al.,
1974; Wolters, 1980, 1990; Erard et al., 1997). A fourth
genus, the grasshopper warblers Locustella (8 species),
is also thought to be closely related to the reed warbler
group (Haffer, 1991). A classification of the first three
genera, taking into account results presented in thispaper, is listed in Appendix 1.
Acrocephalus species live primarily in reed beds andswamp vegetation (e.g., Papyrus in Africa), but sometaxa, especially island forms, have secondarily colonized scrub or even woodland habitats (e.g., brevipennis, sechellensis, many Pacific island taxa). Hippolais
and Chloropeta species live primarily in scrub, woodland, or tall grass habitats. All Acrocephalus and fourout of six Hippolais species have brownish or greybrown plumage, some (e.g., melanopogon) with strong
rusty tones. Two Hippolais (icterina, polyglotta) and all
three Chloropeta have yellow underparts and brownish
to greenish upperparts. Species of all three genera havea pale supercilium of varying length (indistinct in A.
aedon) and all lack wingbars. The only other obvious
plumage markings, a pale median crown stripe (paludicola, young schoenobaenus) and some dark streaking on
crown, back, or lateral underparts, are restricted to thefour species of the subgenus Calamodus within Acrocephalus.
The systematics of Acrocephaline warblers aboundwith problems at all taxonomic levels: (1) Intergenericlevel: Assuming that Acrocephalus/Hippolais warblers(perhaps including Chloropeta) form a monophylum,246

MOLECULAR PHYLOGENY OF REED WARBLERS

their sister group is unknown. Haffer (1991) suggested
that the genus Locustella might be most closely related,
representing a distinct, more terrestrial adaptive type.
The genera Cettia and Cisticola were supposed to be
more distant relatives of this group (Haffer, 1991: p.
21). (2) Generic level: The generic separation between
Acrocephalus and Hippolais is based mainly on tailshape (rounded in Acrocephalus, square-ended orslightly indented in Hippolais), bill shape (broaderbased, flatter in Hippolais), relative length of undertail
coverts (long in Acrocephalus, shorter in Hippolais),and egg color (ground color whitish or olive in Acrocephalus, pinkish in Hippolais). In contrast to Hippolais, thesong of most Acrocephalus species contains many repetitions and fewer imitations. Chloropeta is separated
from the other two genera by its flycatcher-like, broadbased bill. All these characters are rather weak fordefining generic limits.
Four species now generally regarded as Acrocephalus
warblers have been placed in separate (sub)genera by
various authors: melanopogon (in Lusciniola); sechellensis and rodericanus (in Bebrornis); aedon (in Phragmaticola). Such treatment stresses the phenotypic uniqueness of each of these species but, for lack of a rigorous
phylogenetic analysis, runs the risk of leaving Acrocephalus (or some of its subgenera) paraphyletic. (3)Subgeneric level: Intrageneric relationships and thus
subgenus delimitations are equally unclear, particularly with regard to the subgenera Acrocephalus(Palearctic and Australo-Pacific taxa with unstreaked
plumage) and Calamocichla (African taxa). (4) Species
level: Many allopatric taxa are very similar morphologically and the degree of genetic differentiation andspecies limits between these taxa are unknown. This isparticularly true for the arundinaceus–stentoreus group(Salomonsen, 1929; Mayr, 1948; Stresemann and Arnold, 1949; Pearson and Backhurst, 1988; Eck, 1994;
Shirihai et al., 1996), the scirpaceus–baeticatus group(Clancey, 1975; Dowsett-Lemaire and Dowsett, 1987;
Komen, 1988; Ash et al., 1989) and the Hippolais
caligata–pallida group (Cramp, 1992).
Here we present the first attempt at reconstructing
the phylogeny of the reed warbler group based onmolecular data, namely slightly over 1 kb of sequence ofthe mitochondrial cytochrome b gene (a preliminary
analysis was published by Helbig, 1996). The present
study involves a total of 37 ingroup taxa of four genera,
which belong to 28 traditionally recognized species(Table 1). The resulting phylogeny estimate is compared to the traditional classifications presented by
Watson et al. (1986) and Haffer (1991). Some of thephenotypic characters on which the traditional classifications were based are discussed in the light of our
phylogeny. Our results suggest some revisions of thetaxonomy of this problematic group. The detailed mapping of phenotypic attributes onto this phylogeny and247

the analysis of evolutionary trends within this radiation will be the subject of a separate publication (Helbig
and Seibold, in prep.).
MATERIAL AND METHODS
Sampling
Material was specifically collected for this study by
the authors and many collaborators over a period of 7
years, from a wide range of geographic locations (Table 1). Blood samples were taken in the field from theulnar vein of breeding birds (adults or nestlings) andindividuals caught and banded on migration stop-over.
Samples were preserved in NaF-EDTA buffer(Arctander, 1988) and stored at ⫺18°C. All birds wereidentified either by the authors or by expert bird
banders who had extensive experience with these difficult-to-identify species in the field and in the hand.
Laboratory Procedures
Total cellular DNA was extracted from the blood
samples and the cytochrome b gene (except 100 bp at its
58-end) was PCR amplified. Amplification and sequencing techniques were as described by Helbig et al. (1995)
with the following modifications. Recognizing the possibility of (co)amplifying chromosomal copies of the cytochrome b gene (nuclear pseudogenes), each sequence
was verified by sequencing products from two separate
PCR amplifications with different primer combinations: (1) primers A–F amplified a portion of 1100 bp
including most of the cytochrome b gene plus parts ofthe tRNA-Thr gene; (2) primers D–DDL amplified aregion about 2900 bp in length, including part of thecytochrome b gene (400 bp overlap with A–F product),four tRNAs genes, the entire control region, and partsof the 12s RNA gene. Sequences of amplification primers were as follows (H ⫽ heavy strand; L ⫽ light strand;
numbers give position of 38-end in Gallus gallus mitochondrial genome, cf. Desjardin and Morais, 1990):
primer A (L-14993), CCA TCC AAC ATC TCA GCY TGATGA AAY TT; primer F (H-16065), GGA GTC TTC AGT
TTT TGG TTT ACA AGA C; primer D (L-15569), ATC
CCA TTC CAC CCC TAC TAC TC; and primer DDL(H-1816), GCA CCG CCA AGT CCT TAG AGT T. In
many cases it was also possible to amplify with primers
A and DDL a 3500-bp fraction which overlapped completely with the A–F fraction (1100 bp). Excess amplification primers were digested enzymatically (Sequenase
product purification kit, USB) and both strands of thecytochrome b gene were cycle-sequenced directly (forsequencing primers, see Helbig et al., 1995).
Two to seven individuals were sequenced per taxon(if available) to insure against misidentifications, sample mix-ups, accidental contamination, or other potential problems. The study of intrataxon genetic variation
was not a goal of this study, so for multiple individuals

248

HELBIG AND SEIBOLDTABLE 1
Warbler Taxa Investigated in this Study with Number of Individuals Sequenced and their Geographic Origin
No. of individuals
Taxon
Genus Acrocephalus
A. a. arundinaceus

N

Origin1041 bp

300–900 bp

3

Lake Constance, Germany (2b)Lake Neusiedl, Austria (1b)Kasachstan (b), Kuwait (m)Hebei Prov., China (1m)Hong Kong (1m)Luzon, Philippines (2m)Tsavo Nat. Park, Kenya (m)Dead Sea, Israel (b)Shuqaiq, SW Saudi Arabia (3b)Oman, north. coast (1b)Luzon, Philippines (b)Middlesex, West Australia (b)Hebei Prov., China (m)Lake Constance, Germany (3b)
Eilat, Israel (m)Kasachstan (1b); Ukrania (1b)Tsavo Nat. Park, Kenya (1m)
Kenneret (1b) & Eilat (1m), Israel
Riad, Saudi Arabia (2b)Djoudj Nat. Park, Senegal (b)Shuqaiq, SW Saudi Arabia (b)SE Finland (b); Yenessei, Siberia (b)Tsavo NP, Kenya (m); Oman (m)Eilat, Israel (m)China (b)Crimea, Russia (b)Hebei Prov., China (2m)Khao Sam Roi Yot, Thailand (m)Kenya (b)Seychelles (b)Madagascar (b)Austria (2b)Greece (1m); Oman (1m)Yenessei River, Siberia (1b)Hebei Prov., China (3b)
Wladivostok, E Siberia (2b)Biebrza Marshes, Poland (b)Lake Neusiedl, Austria (2b)Hortobágy, Hungary (1b)Kuwait (m)

1
—
—
1
—
1
1
1
1
—
1
1
2
1
—
—
1
1
—
1
—
1
2
—
1
1
1
—
2
1
1
—
1
—
—
2
2
1
—
—

1
1
2
—
1
1
2
1
2
1
1
1
2
2
1
2
—
1
2
2
4
1
—
1
—
1
1
1
2
1
1
2
1
1
3
—
2
1
1
2

2

Tsavo Nat. Park, Kenya (m)Djoudj Nat. Park, Senegal (3m)Guinea, West Africa (1m)western Mongolia
Krasnovodsk, Turkmenistan
Tsavo Nat. Park, Kenya (m)Tsavo Nat. Park, Kenya (m)Germany (b); Greece (m)Crimea, Russia (m)Djoudj Nat. Park, Senegal (m)

2
2
—
1
1
2
2
1
—
1

1
1
1
—
—
1
2
1
1
1

2

Kisumu, Lake Victoria, Kenya (b)

2

—

2
3

Bavaria, Germany (b)Tsavo Nat. Park, Kenya (m)

1
2

1
1

A. arundinaceus ‘‘zarudnyi’’
A. orientalis

2
4

A. griseldis
A. stentoreus levantina
A. stentoreus brunnescens

3
2
4

A. stentoreus harterti
A. (stentoreus) australis
A. aedonA. s. scirpaceus

2
2
4
5

A. s. fuscus

7

A. baeticatus guiersi
A. (baeticatus?) avicenniae
A. dumetorum
A. palustris

3
4
2
3

A. agricola agricola
A. a. septimus
A. tangorum

1
2
3

A. gracilirostris
A. sechellensis
A. newtoni
A. schoenobaenus

4
2
2
5

A. bistrigiceps

5

A. paludicola
A. m. melanopogon

4
3

A. melanopogon mimicus
Genus Hippolais
H. pallida elaeica
H. pallida opaca
H. (c.) caligata
H. (c.) rama
H. languida
H. olivetorum
H. icterina
H. polyglotta
Genus Chloropeta
Chloropeta gracilirostris
Genus Locustella
L. luscinioides
L. fluviatilis

2
3
4
1
1
3
4
3

Note. The two columns on the right show the number of individuals for which complete (1041 bp) or partial (300–900 bp) sequences wereobtained. N, total number of specimens sequenced per taxon. m, migrant; bird caught on migration or on wintering grounds. b, breeding; birdcaught in breeding period within breeding area.

249

MOLECULAR PHYLOGENY OF REED WARBLERS

only partial sequences (300–900 bp) were obtained aslisted in Table 1.

RESULTSIntergeneric Relationships

Sequence Analysis
The separate PCR amplifications yielded an overlapof either 400 or 1100 bp of cytochrome b sequence,
which was carefully checked for discrepancies. For the
taxa studied here, separate amplifications from thesame target DNA always yielded identical sequences.
H- and L-strand sequences matched in all cases. Sequences were also inspected for double bands (whichmight indicate heterogeneous template), stop codons,
unusual frequencies of nonsynonymous substitutions,and unexpected transition–transversion ratios. None ofthese anomalies was detected, so we are confident thatsequences reported here are those of the active mitochondrial genes.
The complete sequences have been deposited in the
EMBL nucleotide data bank (Accession Nos.: z73474,
z73475, z73483, z73487, z73494, AJ004762–AJ004798).
For the purpose of clarity, genetic distances within thetaxon-rich genus Acrocephalus are shown separately(Table 3) from those within Hippolais and between
genera (Table 2).
Phylogenetic analyses were performed using two
standard methods: (1) maximum parsimony (heuristic
search, random sequence addition) using PAUP 3.1.1.
(Swofford, 1993) and (2) neighbor-joining (Saitou andNei, 1987) with Kimura 2-parameter distances calculated with MEGA 1.0 (Kumar et al., 1993). Bootstrap
tests were performed with each kind of analysis, andthe bootstrap percentages were used as heuristic indicators of clade support. Phylogenetic results are presented as consensus trees in which poorly supported
branches are collapsed.
The characteristics of sequence variation across thecytochrome b gene and the phylogenetic information
content of cytochrome b sequences are well known andhave been documented extensively (Kornegay et al.,
1993; Krajewski and King, 1996; Nunn and Cracraft,
1996; Randi, 1996; Griffiths, 1997; Moore and DeFilippis, 1997). The main conclusion from these studies isthat transitions, particularly at 3rd-codon positions,saturate rapidly through multiple substitutions. Since
we were interested in resolving relationships both
between very recently diverged taxa and among phylogenetically more ancient lineages, we performed two
types of analyses with respect to weighting of transitions: (1) all transitions and transversions were included in calculating Kimura 2-parameter distances(for neighbor-joining analyses) and weighted equally inparsimony analyses and (2) Kimura distances were
calculated using transversion differences only, andmaximum parsimony was performed with transitions
down-weighted by a factor of five relative to transversions (TV:TS ⫽ 5:1).

Choice of outgroup. To study the intergeneric relationships of the reed warbler group (Acrocephalus,
Hippolais, Chloropeta) we used the Goldcrest Regulus
regulus and the Blackcap Sylvia atricapilla as outgroup representatives. A DNA–DNA hybridization study(Sheldon and Gill, 1996) showed Regulus to be only
distantly related to other ‘Sylviidae’ and Sylvia to be
more closely related to babblers (Timaliidae) and bulbuls (Pycnonotidae) than to the other Sylviine genera
studied here. Both can thus be assumed to be reliable
outgroup taxa relative to the reed warbler group. The
genera Locustella, Cisticola, Cettia, and Phylloscopus
have variously been suggested to be more closely
related to the reed warbler group (Haffer, 1991), but
only for Phylloscopus was this based on phylogenetic
evidence (Sibley and Ahlquist, 1990). We include one
representative of each of these genera in our analysis.
Phylogeny. Using Empidonax minimus (Tyrannidae; sequence from Helm-Bychowski and Cracraft,1993) as a non-oscine outgroup taxon, the basal position of Regulus (but not of Sylvia) relative to all other
Sylviid genera was confirmed (bootstrap support ⫽ 90%;tree not shown). In all further analyses, Regulus wastherefore designated as the outgroup. Parsimony andneighbor-joining analyses showed that the reed warbler group comprises three closely related genera:
Acrocephalus, Hippolais, and Chloropeta (Fig. 1). Disregarding for the moment the problem of reciprocalmonophyly of these three genera, it is clear that they
form a tightly associated group well separated from therepresentatives of all other genera included here (bootstrap support 100%). Neither Locustella nor Phylloscopus, which had been hypothesized to be close to the reed
warbler group on phenotypic grounds and based on
DNA hybridization, respectively, are supported as its
sister taxon; neither are Cisticola, Cettia, or Sylvia.
While transversion distances suggest that Phylloscopus, and Sylvia may be closer to the reed warbler groupthan are Locustella, Cisticola, or Cettia, bootstrap
support (63%) is poor and all basal branches areprobably best regarded as unresolved.
The gap between the reed warbler group and theother sylviid genera also becomes clear from pairwisetransversion distances (Table 2), which can be assumed
to accumulate linearly with time over the range of
distances considered in this study. Whereas divergences range up to 3.75% within the group, divergencevalues between members of the reed warbler group andany other Sylviid species are above 4.9% and most areover 6%. It thus appears that the reed warbler group isa genetically well-defined radiation within the Sylviid
passerines and that cytochrome b sequences do not

250

HELBIG AND SEIBOLDTABLE 2
Genetic Distances (Percentage Nucleotides Differing, Uncorrected) between Representatives of All Major
Clades of the Reed Warbler Group Plus Potentially Related Sylviidae

1 Regulus regulus
2 Sylvia atricapilla
3 Cisticola juncidis
4 Cettia cetti
5 Phylloscopus coll.
6 Locustella luscini.
7 Locustella fluviat.
8 H. pallida opaca
9 H. pallida elaeica
10 H. caligata rama
11 H. c. caligata
12 H. languida
13 H. olivetorum
14 H. icterina
15 H. polyglotta
16 Chloropeta gracil.
17 A. m. melanopogon
18 A. schoenobaen.
19 A. s. scirpaceus
20 A. dumetorum
21 A. aedon
22 A. gracilirostris
23 A. arundinaceus

1 Regulus regulus
2 Sylvia atricapilla
3 Cisticola juncidis
4 Cettia cetti
5 Phylloscopus coll.
6 Locustella luscini.
7 Locustella fluviat.
8 H. pallida opaca
9 H. pallida elaeica
10 H. caligata rama
11 H. c. caligata
12 H. languida
13 H. olivetorum
14 H. icterina
15 H. polyglotta
16 Chloropeta gracil.
17 A. m. melanopogon
18 A. schoenobaen.
19 A. s. scirpaceus
20 A. dumetorum
21 A. aedon
22 A. gracilirostris
23 A. arundinaceus

1

2

3

4

5

6

7

8

9

10

11

12

—
9.41
8.28
7.85
8.74
8.27
8.76
8.26
8.74
8.84
8.84
9.22
8.93
8.65
8.47
8.74
7.41
7.59
8.84
8.27
8.17
8.55
9.03

17.10
—
7.99
7.66
6.63
7.02
7.60
7.49
7.20
7.88
7.88
6.92
6.82
7.12
7.12
7.97
6.35
6.82
6.72
6.15
6.92
6.63
6.72

16.75
16.65
—
6.31
7.60
7.71
7.43
7.89
8.18
7.89
8.08
7.51
7.60
7.90
7.51
7.41
7.33
7.41
8.08
7.51
7.90
7.03
7.12

17.25
17.64
15.92
—
6.01
6.69
6.98
6.69
6.69
7.36
7.17
6.01
6.49
6.50
6.80
6.49
6.02
5.91
6.98
6.69
6.01
7.07
6.59

16.43
15.56
15.30
14.73
—
7.12
7.31
5.86
5.76
6.44
6.44
4.90
5.19
6.06
6.06
6.53
5.49
5.76
5.86
5.87
6.25
6.15
6.44

17.02
15.67
15.61
17.46
14.52
—
1.35
8.27
8.37
8.27
8.46
7.79
8.46
8.08
8.09
8.46
7.60
7.31
8.37
7.99
7.70
8.27
8.17

17.52
15.50
14.37
16.88
14.53
7.90
—
8.57
8.57
8.47
8.47
7.51
8.18
8.09
8.10
8.37
7.52
7.41
8.08
7.90
7.80
8.37
8.28

16.52
16.43
15.78
15.89
14.41
16.54
15.78
—
1.44
2.69
2.11
3.07
2.98
2.88
2.89
2.98
2.02
2.21
3.46
2.50
2.69
2.59
2.88

17.00
15.75
15.98
15.50
13.35
16.25
15.88
9.61
—
2.59
2.40
2.79
2.50
2.79
2.60
2.88
1.92
2.31
2.98
2.40
2.60
2.50
2.79

17.96
15.85
15.78
16.47
15.27
17.60
16.75
11.14
10.09
—
0.96
3.07
2.98
3.08
2.50
3.75
2.41
2.98
3.46
2.88
3.08
2.79
2.69

18.16
16.81
16.17
14.73
14.79
17.21
16.17
10.18
9.13
6.72
—
3.07
2.98
3.08
2.89
3.55
2.21
2.59
3.27
2.69
2.69
2.59
2.50

17.39
16.62
15.69
16.47
12.97
16.54
16.36
11.05
10.85
11.72
11.14
—
1.25
1.92
1.92
2.98
2.50
2.79
2.69
2.31
2.69
2.79
3.07

13

14

15

16

17

18

19

20

21

22

23

18.25
17.48
16.27
16.96
13.26
18.37
17.32
12.01
10.95
12.01
12.30
8.65
—
1.44
1.44
2.69
2.21
2.50
2.98
2.21
2.79
2.50
2.98

17.79
16.35
16.28
15.62
14.62
16.75
16.28
12.50
11.25
12.88
12.79
10.48
11.15
—
1.16
3.56
2.60
2.79
3.46
2.69
2.89
2.98
3.46

17.32
16.55
14.84
16.31
13.38
15.90
15.53
11.07
10.39
10.88
10.78
8.37
10.11
6.45
—
3.37
2.41
2.79
3.27
2.50
3.08
2.79
3.08

17.58
17.19
15.59
15.99
14.31
17.02
16.36
11.82
10.95
12.20
11.82
12.30
12.10
14.04
12.42
—
2.79
2.69
3.36
2.79
2.79
3.07
2.98

16.55
15.88
15.53
16.21
14.63
16.46
15.72
10.01
10.30
11.55
11.26
11.65
11.36
12.04
10.99
10.97
—
0.87
2.31
1.54
1.93
2.02
2.31

16.23
15.56
14.73
14.63
12.39
16.06
13.96
9.41
8.84
10.76
10.66
10.18
10.76
10.96
10.39
9.99
7.60
—
2.79
1.73
1.83
1.92
2.40

17.39
15.37
16.65
16.96
13.93
17.21
16.46
11.62
10.57
12.39
11.43
10.95
11.91
12.40
11.36
11.53
10.39
10.37
—
1.54
2.88
2.98
2.88

17.31
14.90
16.38
17.75
15.00
17.23
16.67
11.44
11.06
12.12
11.54
11.15
12.02
11.84
10.79
11.92
11.85
10.29
10.58
—
2.12
2.02
2.12

17.60
16.73
15.41
15.81
15.29
17.71
16.67
11.83
11.35
12.12
11.63
12.21
12.79
12.32
11.75
11.83
11.46
10.00
12.88
12.70
—
2.40
2.50

17.87
16.33
16.75
17.44
15.37
17.79
17.52
11.24
10.85
11.82
11.91
12.01
13.06
13.17
11.93
11.82
12.22
10.85
12.20
12.60
12.79
—
1.06

17.39
15.56
15.50
15.89
13.93
17.88
16.46
10.57
10.47
10.47
11.24
10.18
10.66
11.83
10.30
11.43
10.88
9.61
10.37
11.54
11.25
9.61
—

Note. Above diagonal, transitions and tranversions; below diagonal, transversion differences only.

identify any close relatives of the group among the taxa
studied here.
Are the Genera Acrocephalus and Hippolais
Monophyletic?
The cytochrome b data did not support the monophyly of either Acrocephalus or Hippolais (Fig. 2),irrespective of the combination of outgroup taxa used
(Regulus plus any one or all of the other sylviid taxa in
Fig. 1). The ingroup topology was characterized by abasal polytomy, from which five well-supported clades
plus two single species (Acrocephalus aedon, Chloropeta gracilirostris) branched off. This basal polytomy
remained unresolved in the optimal trees from both

251

MOLECULAR PHYLOGENY OF REED WARBLERSTABLE 3
Genetic Distances (Percentage Nucleotides Differing, Uncorrected) between Acrocephalus Taxa

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28

bistrigiceps
melanopogon
mimicuspaludicola
schoenobaenus
baeticatus
scirpaceus 1
scirpaceus 2
fuscus 1
fuscus 2
avicenniae
dumetorum
palustris
agricola
septimustangorumaedon
griseldis
newtonisechellensis
gracilirostris 1
gracilirostris 2
arundinaceus
orientalis
australislevantina
brunnescens
harterti

bistrigiceps
melanopogon
mimicuspaludicola
schoenobaenus
baeticatus
scirpaceus 1
scirpaceus 2
fuscus 1
fuscus 2
avicenniae
dumetorum
palustris
agricola
septimustangorumaedon
griseldis
newtonisechellensis
gracilirostris 1
gracilirostris 2
arundinaceus
orientalis
australislevantina
brunnescens
harterti

1

2

3

4

5

6

7

8

9

10

11

12

13

14

—
0.67
0.81
0.67
1.15
1.92
2.12
1.65
1.92
1.92
1.92
1.92
1.92
2.02
2.02
1.73
2.31
3.08
2.12
2.12
2.21
2.22
2.69
2.60
2.88
2.50
2.50
2.50

8.00
—
0.14
0.58
0.87
2.12
2.31
1.80
2.12
2.12
2.12
1.54
1.92
1.64
1.64
1.54
1.93
2.69
2.02
1.93
2.02
2.03
2.31
2.21
2.50
2.12
2.12
2.12

8.14
2.17
—
0.81
0.54
1.90
2.17
1.81
1.90
1.90
1.90
1.36
1.63
1.63
1.63
1.49
1.77
2.71
2.31
2.18
2.31
2.31
2.17
2.17
2.44
2.04
2.04
2.04

8.86
8.10
8.41
—
0.96
2.22
2.41
1.95
2.21
2.22
2.22
1.83
2.21
1.93
1.93
1.64
2.12
2.79
1.93
1.84
1.92
1.93
2.41
2.31
2.60
2.22
2.21
2.22

7.40
7.60
7.73
7.80
—
2.60
2.79
2.25
2.59
2.60
2.60
1.73
2.40
2.31
2.31
1.83
1.83
2.59
1.73
1.74
1.92
1.92
2.40
2.31
2.59
2.21
2.21
2.21

10.97
10.69
11.40
10.89
11.15
—
0.19
0.00
0.00
0.00
0.00
1.35
0.58
1.25
1.25
1.25
2.69
3.27
2.98
2.90
2.79
2.79
2.69
2.60
2.69
2.60
2.50
2.50

10.48
10.39
11.67
11.16
10.37
1.92
—
0.00
0.19
0.19
0.19
1.54
0.77
1.44
1.44
1.44
2.88
3.46
3.17
3.09
2.98
2.98
2.88
2.79
2.88
2.79
2.69
2.69

10.03
10.04
10.54
11.08
9.73
2.40
0.45
—
0.00
0.00
0.00
1.50
0.60
1.05
1.05
1.20
2.25
2.99
2.70
2.55
2.54
2.55
2.25
2.25
2.54
2.10
2.10
2.10

10.77
10.88
11.67
11.36
10.76
1.83
1.63
2.10
—
0.00
0.00
1.35
0.58
1.25
1.25
1.25
2.69
3.27
2.98
2.90
2.79
2.79
2.69
2.59
2.69
2.60
2.50
2.50

10.49
10.89
11.28
10.89
10.67
2.41
1.83
2.55
1.54
—
0.00
1.35
0.58
1.25
1.25
1.25
2.69
3.27
2.98
2.90
2.79
2.79
2.69
2.60
2.69
2.60
2.50
2.50

10.20
10.21
10.87
10.89
10.38
1.44
0.87
1.35
0.77
1.35
—
1.35
0.58
1.25
1.25
1.25
2.69
3.17
2.98
2.90
2.79
2.79
2.69
2.60
2.69
2.50
2.50
2.50

10.67
11.85
11.53
9.83
10.29
11.07
10.58
10.63
10.67
10.49
10.11
—
1.15
1.06
1.06
0.77
2.12
2.50
2.12
2.12
2.02
2.02
2.12
2.02
2.12
2.02
1.92
1.92

10.58
10.78
11.13
10.39
10.28
7.98
8.26
7.78
7.97
7.98
7.69
10.19
—
1.44
1.44
1.15
2.69
3.07
2.60
2.51
2.40
2.41
2.31
2.21
2.31
2.21
2.11
2.12

10.49
9.06
10.85
10.89
9.90
10.30
10.00
9.88
10.10
10.68
9.82
10.11
9.71
—
0.00
0.77
2.60
3.17
2.69
2.61
2.50
2.50
2.60
2.50
2.60
2.50
2.40
2.41

15

16

17

18

19

20

21

22

23

24

25

26

27

28

10.21
9.06
10.87
10.61
9.82
10.21
9.91
9.75
10.01
10.60
9.73
9.83
9.62
0.19
—
0.77
2.60
3.18
2.70
2.61
2.50
2.51
2.60
2.50
2.60
2.50
2.41
2.41

9.72
10.79
11.14
9.92
9.62
10.49
10.19
9.90
10.29
10.11
9.82
9.82
9.13
7.89
7.71
—
2.12
2.69
2.21
2.13
2.02
2.02
2.12
2.02
2.12
2.02
1.92
1.92

10.97
11.46
11.82
11.18
10.00
12.80
12.88
12.87
12.79
12.80
12.70
12.70
12.60
12.70
12.62
12.22
—
2.60
2.41
2.32
2.40
2.41
2.50
2.40
2.69
2.41
2.31
2.31

12.02
11.55
12.89
11.84
10.28
11.92
11.24
11.23
11.34
11.73
11.06
11.63
11.82
11.73
11.65
11.63
11.83
—
1.83
1.93
1.63
1.64
1.34
1.44
1.73
1.25
1.34
1.35

10.97
11.95
13.04
11.56
10.48
12.42
12.69
12.14
12.40
12.03
12.22
12.42
12.60
11.84
11.66
1.107
12.70
11.83
—
0.19
0.58
0.58
1.25
1.15
1.44
1.15
1.06
1.06

10.04
11.41
12.11
11.50
9.27
12.27
11.97
11.11
11.87
11.69
11.40
12.16
11.39
11.11
10.93
10.43
11.88
10.14
4.15
—
0.68
0.68
1.35
1.25
1.54
1.26
1.16
1.16

12.02
12.22
12.48
12.03
10.85
12.02
12.20
10.63
12.01
11.92
11.83
12.60
12.20
11.73
11.65
11.35
12.79
11.43
7.50
6.95
—
0.00
1.06
0.96
1.25
0.87
0.86
0.87

11.46
11.96
12.52
11.76
10.49
11.66
11.65
10.36
11.65
11.56
11.27
12.62
11.65
11.56
11.48
10.98
12.04
11.36
7.51
6.77
1.15
—
1.06
0.96
1.25
0.87
0.87
0.87

10.00
10.88
11.94
10.68
9.61
10.67
10.37
9.88
10.47
10.19
9.81
11.54
10.85
11.44
11.16
9.90
11.25
9.80
9.23
8.01
9.61
9.05
—
0.29
0.58
0.29
0.19
0.19

10.48
10.30
12.62
10.78
9.41
10.67
10.37
10.18
10.47
10.58
10.00
11.73
10.57
10.29
10.20
10.48
10.58
9.13
8.65
7.72
9.03
8.66
5.86
—
0.48
0.19
0.10
0.10

10.87
11.07
12.48
11.84
9.80
11.63
11.34
10.78
11.43
11.35
10.96
11.83
11.53
11.25
10.97
10.87
10.77
9.61
9.42
8.20
9.22
8.57
6.24
4.90
—
0.48
0.38
0.38

9.43
10.02
11.68
10.40
8.94
10.11
9.90
9.60
9.90
9.53
9.43
11.26
10.48
10.59
10.50
9.43
10.68
8.56
7.80
6.86
8.37
7.80
4.81
3.08
4.42
—
0.10
0.10

9.62
10.11
11.80
10.59
9.13
10.19
10.09
9.88
9.99
9.71
9.52
11.44
10.28
10.38
10.30
9.42
10.67
8.55
7.88
6.85
8.55
7.99
4.80
3.07
4.42
0.19
—
0.00

9.34
9.83
11.55
10.40
8.94
10.01
9.90
9.75
9.81
9.53
9.34
11.16
10.29
10.20
10.12
9.53
10.30
8.65
7.80
6.96
8.27
7.71
5.29
3.17
4.52
1.06
1.06
—

Note. All haplotypes differing by at least four substitutions are included. Above diagonal, transitions and transversions; below diagonal,transversion differences only.

252

HELBIG AND SEIBOLD

FIG. 1. Bootstrap consensus tree of the reed warbler group with
one or two representatives of each major clade (compare Figs. 2 and 3;
A. ⫽ Acrocephalus; H. ⫽ Hippolais) and representatives of five potentially related Sylviid genera. Regulus (Goldcrest) was designated as
the outgroup taxon. Branches with less than 60% bootstrap support
were collapsed. Numbers are bootstrap values from 200 replicates
each; above branch, neighbor-joining, Kimura distances (transversions only); below branch, maximum parsimony (TV:TS ⫽ 5:1).

neighbor-joining and maximum parsimony analyses,
irrespective of whether both transitions and transversions were included, transversions were down-weighted
5:1 versus transitions (parsimony), or only transversions were used. If only Phylloscopus collybita was usedas the outgroup, there was moderate support for a basal
bifurcation separating four Hippolais species from theremaining Hippolais and all Acrocephalus species (bootstrap 78%, transversion distances; Fig. 2), thus rendering Hippolais paraphyletic. However, with additional
outgroup taxa (Sylvia, Locustella, Regulus) this branch
received only 48–50% bootstrap support and thus cannot be regarded as resolved.
Intrageneric Phylogeny of Hippolais
Hippolais warblers formed two clades of four taxa
each (Fig. 2): clade I consisted of opaca, elaeica, caligata,rama; clade II included olivetorum, languida, icterina,
polyglotta. Within each of the two Hippolais clades twopairs of sister taxa were identified and these wereconcordant with phenotypic similarities: (a) icterina,
polyglotta: These are the only two Hippolais species
with yellow-green plumage; their cyt b divergence is
6.57%, i.e., fairly high for a pair of parapatrically
distributed sister species which are known to hybridize
occasionally (Faivre and Ferry, 1989). (b) olivetorum,
languida: These are the two largest Hippolais species.
Both have uniformly grey plumage and their breeding

distributions are restricted to the eastern Mediterranean and Middle Eastern region. Their sister relationship was supported with transitions and transversions
included (91% bootstrap support), but was unresolved
with transversions alone. Given an uncorrected sequence divergence of 8.7% between the two species,
transitions are not expected to be strongly saturated, so
their inclusion should yield the more reliable phylogenetic estimate. (c) opaca, elaeica: These two allopatrictaxa have so far been classified as subspecies of H.
pallida, although they are morphologically quite different (Cramp, 1992). Their sequence divergence of 9.64%
is surprisingly large. In phylogeny reconstructions
using both transitions and transversions the two did
not appear to be sister taxa (elaeica grouped withcaligata; bootstrap 69%); only if transitions were excluded was their sister relationship supported (bootstrap 88%). (d) caligata, rama: The sister relationshipof these two western Asian taxa, treated as subspeciesof H. caligata by most authors, was strongly supported(99–100% bootstrap). They differed by almost the sameamount of sequence divergence (6.76%) as the morewesterly distributed parapatric species pair icterina,
polyglotta.
Intrageneric Phylogeny of Acrocephalus
Within the genus Acrocephalus as traditionally delimited, three fairly well-supported clades plus a single
isolated species emerged. These three basic Acrocephalus clades in the molecular phylogeny correspond exactly to morphologically defined groups based on plumage pattern and body size (Fig. 3).
Acrocephalus clade 1 (small size, streaked plumage;
Palearctic breeding distribution) includes the species
paludicola, schoenobaenus, bistrigiceps (subgenus Calamodus), and melanopogon (Lusciniola). Representatives of the nominate (western) and mimicus (eastern)subspecies of A. melanopogon differed considerably(2.17% sequence divergence). A fifth species, A.
sorghophilus, not studied here, undoubtedly belongs tothis clade. All five species are characterized by plumage
with streaking on the under- and/or upperparts as wellas contrasting head patterns, characters not occurringin any other Acrocephalus or Hippolais warbler. Interspecific sequence divergence values are in a rather
narrow range of 7.53–9.10%, indicating that all four
species are about equally divergent from each other. No
relationships could be resolved confidently among them(all bootstrap values below 60%). In particular, A.
melanopogon was not more distant from the other three
species than these were among each other.
Acrocephalus clade 2 (small size, unstreaked plumage; primarily Palearctic breeding distribution) includes eight taxa of brownish, uniformly colored (except
for pale supercilium) warblers of relatively small bodysize: the A. scirpaceus superspecies (see below) as well

MOLECULAR PHYLOGENY OF REED WARBLERS

253

FIG. 2. Phylogeny of the reed warbler group including all taxa differing by more than 3% sequence divergence (compare Fig. 3 for detailed
phylogeny of Acrocephalus). The bootstrap consensus tree is based on neighbor-joining (Kimura distance, transversions only; values above
branch) and maximum parsimony analyses (TV:TS ⫽ 5:1; values below branch). Branches supported by less than 60% bootstrap support werecollapsed (except one branch, see text for discussion).

as palustris, dumetorum, tangorum, and agricola. All ofthese are very similar morphologically except for wing
shape, which varies in close concordance with migratory behavior (wings of long-distance migrants are
longer and more pointed than those of sedentary taxa).
These species have so far been placed in the subgenus
Acrocephalus along with a number of larger bodied
species not belonging to this clade in the molecularphylogeny.
Two subclades are well supported: A. palustris is thesister species of the A. scirpaceus complex (bootstrap
94–97%), and the two central and east Asian Paddyfield
Warblers A. agricola and A. tangorum, sometimes
treated as conspecific, are well differentiated sistertaxa at 7.74–7.92% sequence divergence. A. dumetorum proved to be highly distinct and appeared almost

equally divergent from either subclade. Its relationships remain unresolved. Representatives of the two A.
agricola subspecies (nominate and A. a. septimus)
differed by only 0.2%. Specimens of A. tangorum fromnear the breeding grounds in eastern China had aslightly different haplotype (0.7%) from one caught on
their highly localized wintering grounds in Thailand.
Acrocephalus clade 3 (large size, unstreaked plumage) consists of a Palearctic and an Afrotropical subclade. Results were contradictory with respect to theposition of A. griseldis, depending on how much weight
was given to transitions. When both TS and TV were
included, neighbor-joining (Kimura distances) and maximum parsimony (equal weighting) supported griseldis
as the sister species of both the Palearctic and theAfrican subclades. With transversion distances (neigh-254

HELBIG AND SEIBOLD

FIG. 3. Phylogeny of the genus Acrocephalus based on 1041 nucleotides of the cytochrome b gene. All Acrocephalus taxa studied and all
haplotypes differing by more than five substitutions were included. A consensus tree is shown in which branches with less than 60% bootstrap
support were collapsed (except most basal branches). Numbers are bootstrap values (200 neighbor-joining replicates) using Kimura distances
calculated from transversion differences only (above branch) or with transitions and transversions (below branch). ⫺ ⫽ branch was notsupported with transversions only. Maximum parsimony analyses (TV:TS ⫽ 5:1) yielded the same topology as resolved here. Subgenera of the
traditional classification are shown (compare with Appendix 1 for suggested revision).

bor-joining) or down-weighting of transitions (5:1, maximum parsimony), griseldis appeared as the most basal
member of the Palearctic clade (bootstrap 72%). Within
the Afrotropical subclade the two island endemics
newtoni (Madagascar) and sechellensis (Seychelles) areclosely related sister taxa (4.17% divergence). Note thatsequences of three other species probably belonging tothis subclade (rufescens, brevipennis, rodericanus) were
not available. In Greater Swamp Warblers A. gracilirostris from Kenya, two haplotypes differing by 1.26%
were found. Relationships within the Palearctic subclade are further discussed below (see ‘A. arundinaceus–A. stentoreus complex’).

Acrocephalus clade 4 is represented by a single
species, A. aedon, the Thick-billed Warbler. It is highly
distinctive, both morphologically and genetically, differing from all other species by 10% sequence divergenceor more. It has no close relatives among the taxa
included in this study.
Problems with species limits
Two complexes of closely related Acrocephalus warblers are particularly problematic with respect to levelsof differentiation and species limits. We have obtained
sequence data of several taxa within each of the twocomplexes. Note, however, that a detailed analysis of

255

MOLECULAR PHYLOGENY OF REED WARBLERS

these was not a primary goal of this study. A fuller
sampling of geographic populations, particularly inpotential contact zones of parapatric taxa, remains to
be done.
A. scirpaceus complex. Four taxa of this complex,
represented by two to seven individuals each (Table 1),
were sequenced. All four are closely related genetically,
but with clear differences between them. In addition,
considerable sequence divergence was also found within
taxa: in nominate A. scirpaceus three cytochrome b
haplotypes were identified which differed by two to
three substitutions from each other. Sequence divergence between nominate A. s. scirpaceus and eastern A.
s. fuscus was much larger at 1.63–2.55%. Interestingly,
two quite divergent haplotypes were found in birds
identified as A. s. fuscus: one caught in March 1994 in
Eilat (Israel) differed by 1.54% from an individual
caught on migration (November) in Ngulia, Kenya.
African Reed Warblers of the West African subspecies
A. baeticatus guiersi (Colston and Morel, 1984) differedfrom other forms of the A. scirpaceus complex by
1.44–2.41%, whereas the population breeding in mangroves on the Red Sea coast of Saudi Arabia, describedas A. baeticatus avicenniae (Ash et al., 1989), was closer
genetically to both nominate scirpaceus (0.87%) and theindividual of A. s. fuscus from Israel (0.77%) than toWest African baeticatus (1.44%). Phylogenetic relationships within the A. scirpaceus complex could not be
resolved (Fig. 3).
A. arundinaceus–A. stentoreus complex. The largebodied Palearctic Reed Warblers of this group are
generally regarded as comprising three or four species:
A. arundinaceus, A. (a.) orientalis, A. griseldis, and A.
stentoreus. One of these, A. griseldis, is geneticallyhighly divergent, as discussed above (8.6–9.8% sequence divergence from all others). Among the remaining forms, haplotypes of nominate arundinaceus fromCentral Europe and its eastern subspecies ‘‘zarudnyi’’(Kasachstan) were identical (300 nucleotides compared). These taxa occupy a contiguous range from
Europe to Central Asia and are separated by a gap ofunknown extent from eastern Asian A. orientalis. Nominate arundinaceus differed from orientalis by 5.86%.
The third large Reed Warbler species, A. stentoreus, hasa more southerly and highly fragmented breeding
range, extending from Israel (A. s. levantina), NE
Africa (A. s. stentoreus), and Arabia (A. s. brunnescens)through south-central Asia east to the Phillipines (A. s.
harterti) and south to northern Australia (A. s. australis). The two Middle Eastern subspecies were very close
to each other (0.19% divergence) and also close to A. s.
harterti from the Philippines (1.06%). All three of these,
however, were much more divergent from the Australian A. (s.) australis (4.42–4.52%). This divergence islarger than the distance between A. orientalis and thethree Palearctic subspecies of A. stentoreus (3.07–
3.17%). Accordingly, the phylogeny reconstruction (Fig.
3) shows A. orientalis more closely related to A. stentoreus than to A. arundinaceus. Furthermore, the phylogeny indicates that a taxon A. stentoreus which
includes australis as a subspecies might be paraphyletic relative to A. orientalis.
DISCUSSION
The results of our analysis shed new light on theevolutionary history and systematics of one of the mostdifficult and most species-rich assemblages of Palearctic–
African passerine birds. Not surprisingly, given therelatively limited phenotypic differentiation of Acrocephalus and Hippolais warblers, a number of results
were at variance with traditional taxonomy.
Relationships at the Generic Level
First, we were able to reject the hypothesis that theGrasshopper Warblers (genus Locustella) are closely
related to the Acrocephalus group. This idea had been
based on a superficial resemblance in plumage pattern,a similar preference for marshy vegetation, and arounded tail in both of these genera. On the other hand,
Locustella species differ from Acrocephalus in theirinsect-like songs (four of the eight species), partially
down-covered nestlings, and three (rather than two)
black tongue spots in nestlings (Haffer, 1991). Our
analysis provides no evidence that Locustella is phylogenetically closer to the reed warbler group than are
many other Sylviid genera, such as Phylloscopus, Sylvia, Cettia, or Cisticola. The phenotypic resemblance
between Locustella and Acrocephalus may therefore beeither plesiomorphic or convergently evolved (particularly the rounded tail, which is an adaptation to life indense vegetation).
Second, we could neither confirm nor reject themonophyly of either Acrocephalus or Hippolais. The
whole assemblage seems to have differentiated in a
rapid basal radiation, which cannot be resolved bycytochrome b sequences and leaves a polytomy at thebase of the gene tree. The Afrotropical genus Chloropeta, of which only one species (gracilirostris) was included here, is part of this radiation, as had long beensuspected (Keith and Vernon, 1966; Erard et al., 1997).
Relationships at the Subgeneric Level
Subgroups within the reed warbler radiation were
well defined in the phylogenetic analysis and in agreement with relationships that had been inferred fromphenotypic similarities. Thus, three basal Acrocephalus clades, each characterized by a combination ofplumage features and body size, were well supported in
the molecular phylogeny. These clades were partly
congruent with traditional subgenera, the most signifi-

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HELBIG AND SEIBOLD

cant departure from current taxonomic practice being
the paraphyly of the subgenus Acrocephalus.
Within the large-bodied reed warbler clade, a further
bipartition identified the Palearctic and the Afrotropical taxa each as a monophyletic group and A. griseldis,
with a somewhat intermediate breeding distribution in
SW Asia, as their probable sister species. The subgenus
Calamocichla, as far as it was represented here, ismonophyletic and most likely includes A. rufescens(West and Central Africa), A. brevipennis (Cape VerdeIslands), and A. rodericanus (Rodrigues Island, Indian
Ocean). The only Australasian representative of thereed warblers, Acrocephalus australis, is clearly derived from the Palearctic clade. Therefore, the basic
phylogenetic division among reed warblers with unstreaked plumage is not between Palearctic and African species, as had previously been assumed, but
between large- and small-bodied species. Only secondarily, within the large-bodied clade, did a Palearctic
and an Afrotropical group differentiate. Note that we
had no material available of any of the at least six
species in the subgenus Conopoderas (Wolters, 1980),
which consists entirely of central and south Pacificisland endemics. Both the monophyly of this group andits relationships (probably with the eastern Palearctic
large-bodied species) remain to be established. The twocentral and east Asian species A. agricola and A.
tangorum both possess faint dark lateral crown margins, a character otherwise found only in the streakedplumage clade (subgenus Calamodus). The cytochromeb tree, however, places agricola/tangorum in the ‘small
unstreaked group’ (bootstrap support 95%).
Our findings of intrageneric relationships in Acrocephalus suggest some changes in subgeneric taxonomy: the small-bodied species with unstreaked plumage should be placed in a separate subgenus, for which
the name Notiocichla Oberholser, 1905 (see Wolters,1980) has priority. Furthermore, the molecular phylogeny clearly shows that the Seychelles Warbler, anendangered species (Collar and Stuart, 1995), is amember of the Calamocichla group. This is also supported by song characters (Dowsett-Lemaire, 1994).
Along with its close relative A. rodericanus (not included here), it should, therefore, not be placed in a
separate (sub)genus ‘Bebrornis’ (contra Watson et al.,
1986; Sibley and Monroe, 1990).
Of the two monotypic reed warbler subgenera thatare in widespread taxonomic use, Phragmaticola (A.
aedon) and Lusciniola (A. melanopogon), the first was
supported by the molecular phylogeny, but the second
was not. The Thick-billed Warbler A. aedon, based on
plumage pattern (no streaking) and size (large), might
be suspected to belong to the clade of large-bodied
Acrocephalus. However, it has many unique characters(e.g., proportionally long tail with narrow feathers,
thick bill, lack of supercilium) indicating that it repre-

sents a relatively ancient lineage among reed warblers.
This is confirmed by the molecular phylogeny (Fig. 3)
and agrees with its placement in a monotypic subgenus
Phragmaticola. Within the streaked plumage clade, A.
melanopogon also has a number of autapomorphic
features which prompted some earlier authors to allocate it to its own subgenus or even genus Lusciniola(see Parker and Harrison, 1963). The molecular phylogeny, however, does not support this treatment, because
melanopogon is not more distant from the other speciesof this clade than these are from each other.
The two Hippolais clades of four taxa each also
correspond to phenotypically well characterized groups:
birds of the pallida/caligata clade possess more brownish plumage than those of the olivetorum/icterina clade
and have a more Acrocephalus-like song and lesspinkish, more whitish ground color of the eggs. The
monophyly of each of these clades is reasonably well
supported both phenotypically and genetically (Fig. 2),
so classification in two subgenera (see Appendix 1) isjustified.
Relationships at the Species Level
Within each of the three major Acrocephalus groups,
a number of questions regarding species-level relationships was resolved. Within the clade of small-bodied
unstreaked species, A. palustris had been assumed tobe the sister species of A. dumetorum (Koskimies, 1980;
Haffer, 1991), with which it hybridizes locally, butrarely, in Finland (Koskimies, 1980, 1991). According to
the cytochrome b gene phylogeny, however, palustris is
the sister species of the scirpaceus complex (bootstrap
94–96%), while dumetorum is much more divergent
from the palustris/scirpaceus clade. It was not evenpossible to establish whether dumetorum is closer tothe palustris/scirpaceus group or the agricola/tangorum species pair. If palustris ⫻ dumetorum hybrids areindeed fertile, as has been reported (Koskimies, 1991),
this is another interesting example illustrating thatinterbreeding between non-sister taxa, even if they are
not particularly close genetically, is possible amongsome bird species (cf. Grant and Grant, 1992).
The odd suggestion by Fry et al. (1974) to regard theAfrican Reed Warbler A. baeticatus as conspecific with
A. dumetorum is strongly contradicted by our molecular phylogeny (Fig. 3). It had previously been rejectedon phenotypic grounds, especially song characters andmigration pattern (Dowsett-Lemaire and Dowsett,1987). A. baeticatus is much more closely related to thescirpaceus group than to dumetorum.
The Manchurian Reed Warbler A. tangorum has had
a particularly unsettled systematic history. While Williamson (1968) and Watson et al. (1986) regarded it as asubspecies of A. bistrigiceps, most authors treated it asconspecific with A. agricola (for review see Alström etal., 1991). Field experiments with song playback pro-

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MOLECULAR PHYLOGENY OF REED WARBLERS

vided evidence that it is closely related to agricola(Alström et al., 1991), a finding that is supported by ourmolecular phylogeny. In view of the distinctive phenotypic characters of this form and its large genetic
distance from agricola (7.7–7.9%), we recommend thatit be treated as a separate allospecies.
Among the large-bodied, unstreaked Acrocephalus
warblers our most interesting finding was the relatively primitive position of A. griseldis. Formerly treatedas a subspecies of A. arundinaceus (Vaurie, 1959;
Williamson, 1968), our data make it clear that griseldis
is genetically a highly distinctive taxon, not evenclosely related to arundinaceus. Rather, griseldis appears to be the sister species either to all other largebodied Acrocephalus warblers (except A. aedon) or to
the non-African large-bodied species. The position ofgriseldis in the optimal trees depended on the weighting of transitions versus transversions.
Species and Subspecies Boundaries
Representatives of the westernmost breeding population of A. agricola, on the Crimea Peninsula (subspecies
septimus; Watson et al., 1986), were poorly differentiated (0.2% sequence divergence) from the nominate
subspecies (specimen from China). Together with thelack of clear morphological differences, the genetic data
indicate that the current range disjunction separating
the westernmost populations around the Black Sea
from those of the main breeding range might be very
recent.
Previous taxonomic discussion of the A. scirpaceus
complex has centered mostly on the validity of a species
distinction between African (baeticatus) and Palearctic(scirpaceus) Common Reed Warblers (see DowsettLemaire and Dowsett, 1987, for review). Less attention
has been paid to differentiation within these two groupsof populations. Our mitochondrial sequence data, although limited by small sample size and incomplete
geographic sampling, allow two tentative conclusions.
(1) The total range of genetic divergence within thecomplex is small (up to 2.6% sequence divergence;
Table 2) compared to other groups of taxa previously
regarded as single species, e.g., Hippolais pallida (cyt bdivergence up to 9.6%), the A. arundinaceus/stentoreus
group (6.2%, see below), Phylloscopus bonelli (7.9%;
Helbig et al., 1995), or the Phylloscopus collybita complex (5.4%; Helbig et al., 1996). (2) There seems to be as
much genetic divergence between populations attributed to a single subspecies, A. s. fuscus, as there is
between Eurasian (scirpacus) and African (baeticatus)
Reed Warblers. The breeding range of fuscus extends
from Turkey and parts of Arabia east to NW Mongolia
and is highly fragmented. This situation might provide
ample opportunity for population divergence, a topic
which requires further study. The same is true of thefragmented range of A. baeticatus in Africa, where we

sampled only a single population (in Senegal). Regarding the mangrove-dwelling population avicenniae on
the Arabian Red Sea Coast (Ash et al., 1989), thegenetic distance data (Table 2) suggest closer relationships to scirpaceus than to baeticatus.
The systematics of the A. arundinaceus/stentoreus
complex have been debated for a long time and arecontroversial to this day (Salomonsen, 1929; Mayr,
1948; Stresemann and Arnold, 1949; Eck, 1994). Controversies have centered on the species distinction (a)between stentoreus and arundinaceus, which are supposed to hybridize in Kasachstan (Panov, 1989), (b)between arundinaceus and orientalis, and (c) between
griseldis and arundinaceus. The latter two have now
been firmly established as well-differentiated species(Pearson and Backhurst, 1988; this study), which are
not sister taxa (Fig. 3).
Among the remaining taxa, the range of genetic
divergences measured by cytochrome b sequence comparisons is clearly larger (6.2%) than in the A. scirpaceus complex (2.6%). Three populations described as
subspecies of A. stentoreus, levantina from Israel, brunnescens from Arabia, and harterti from the Philippines,
are all very closely related (maximum divergence 1%),
notwithstanding the huge geographic separation between them. However, genetic distances between thesetaxa and australis, orientalis, and arundinaceus, aswell as among the latter three, are much larger (4.4–
6.2%), suggesting species level divergence. The available evidence for hybridization between arundinaceus
and stentoreus at Lake Sorbulak in southeastern Kasachstan is inconclusive, consisting only of field observations of eight individuals with intermediate song.
Their alleged hybrid identity seems not to have been
confirmed by any other means (A. Pukas, cited in
Panov, 1989).
According to the molecular phylogeny, A. orientalis is
more closely related to A. stentoreus than to A. arundinaceus (bootstrap support 92%; Fig. 3) and should not,therefore, be treated as a subspecies of the latter(contra Cramp, 1992; Sibley and Monroe, 1993). The cyt
b sequence data also indicate that A. stentoreus in its
current delimitation, which includes the Australasiantaxon australis as a subspecies, might be paraphyletic
relative to A. orientalis. Therefore, our results on thegenetic divergence and phylogenetic relationships would
be best reflected by recognizing arundinaceus, stentoreus, orientalis, and australis as four separate species.
APPENDIX 1
Suggested classification of the genera Acrocephalus,
Hippolais, and Chloropeta with summary of approximate breeding ranges. Traditional generic limits were
accepted, because molecular data presented here nei-

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HELBIG AND SEIBOLD

ther confirm nor contradict them. Taxa are grouped
into subgenera and superspecies (as defined by Sibley
and Monroe 1990); superspecies names are set insquare brackets. Deviations from Watson et al. (1986)
and Sibley and Monroe (1990, 1993) are based on
results presented in this paper. The subgenus
Conopoderas (Acrocephalus) with many Pacific island
taxa is not listed, because none of these was included inthis study. Subspecies (ssp.) are listed only as far as
they were subjects of this study.
* ⫽ taxon subject of this study
Genus Acrocephalus
Subgenus Phragmaticola*A. aedon (Thick-billed Warbler); monotypic; eastern
Palearctic.
Subgenus Calamodus*A. melanopogon (Moustached Warbler); 3 ssp.; western Palearctic from Iberia to Ukrania (*melanopogon, albiventris) and from Caucasus region to southcentral Asia (mimicus).
*A. paludicola (Aquatic Warbler); monotypic; Palearctic from Central Europe to western Siberia.
*A. schoenobaenus (Sedge Warbler); monotypic; western Palearctic from Britain to western Siberia.
*A. bistrigiceps (Black-browed Reed Warbler); monotypic; eastern Palearctic.
A. sorghophilus (Streaked Reed Warbler); monotypic;
eastern Palearctic (breeding range unknown).
Subgenus Notiocichla*A. [scirpaceus] scirpaceus (Common Reed Warbler); 3
ssp.; western Palearctic east to Ukrania (*scirpaceus), Middle East to west-central Asia (*fuscus), Red
Sea coasts (mangroves) from SW Saudi Arabia to
Eritrea (*avicenniae).
*A. [scirpaceus] baeticatus (African Reed Warbler);
subSaharan Africa; 6⫹ ssp. (*A. b. guieri in N
Senegal).
*A. palustris (Marsh Warbler); Western Palearctic.
*A. dumetorum (Blyth’s Reed Warbler); monotypic;
Palearctic from S Finland to Lake Baikal.
*A. [agricola] agricola (Paddyfield Warbler); central
Palearctic from Black Sea to south-central Siberia; 2ssp. (*A. a. septimus in western part of range).
*A. [agricola] tangorum (Manchurian Reed Warbler);
Manchuria (China); monotypic.
A. [agricola] concinnens (Blunt-winged Warbler); 3
ssp.; range highly disjunct: eastern China (concinnens), Assam (stevensi), N Pakistan, Kashmir (haringtoni).
Subgenus indeterminate*A. griseldis (Basra Reed Warbler); monotypic; S Iraq.
[Note: Phylogenetic relationships of this species were
not fully resolved; it may require its own subgenus.]

Subgenus Acrocephalus*A. [arundinaceus] arundinaceus (Great Reed Warbler); western to central Palearctic; monotypic (‘‘zarudnyi’’ in eastern part of range hardly distinct).
*A. [arundinaceus] orientalis (Oriental Reed Warbler);monotypic; Eastern Palearctic.
*A. [arundinaceus] stentoreus (Clamorous Reed Warbler); 11 ssp.; fragmented range from NE Africa
through southern Asia to Philippines (with *levantina in Israel, *brunnescens from Red Sea to
India, *harterti in Philippines).
*A. [arundinaceus] australis (Australasian Reed Warber); 6⫹ ssp.; Australia, New Guinea (probably including populations from Lesser Sundas west to Java).
Subgenus Calamocichla
A. [rufescens] rufescens (Greater Swamp Warbler, Cape
Reed Warbler); west-central Africa; 3⫹ ssp.
A. [rufescens] brevipennis (Cape Verde Swamp Warbler); Cape Verde Islands off West Africa; monotypic.
*A. gracilirostris (Cape Reed Warbler); 9 ssp.; eastern
to southern Africa.
*A. [newtoni] newtoni (Madagascar Reed Warbler);
Madagascar; monotypic.
*A. [newtoni] sechellensis (Seychelles Warbler); Seychelles.
A. [newtoni] rodericanus (Rodrigues Warbler); Rodrigues Isld., Indian Ocean.
Genus Hippolais
Subgenus Hippolais*H. [icterina] icterina (Icterine Warbler); monotypic;
western Palearctic from France to SW Siberia.
*H. [icterina] polyglotta (Melodious Warbler); monotypic; western Mediterranean region.
*H. languida (Upcher’s Warbler); monotypic; southcentral Asia from E Mediterranean coast to Turkestan.
*H. olivetorum (Olive-tree Warbler); monotypic; eastern Mediterranean region.
Subgenus Iduna
H. [pallida] pallida (African Olivaceous Warbler); 3
ssp.: Northern Africa: Egypt along Nile Valley (pallida), scattered populations in Central Sahara (reiseri) and Chad Basin (laeneni).
*H. [pallida] elaeica (Eastern Olivaceous Warbler);monotypic; west-central Palearctic from Balkan Peninsula east to Tien Shan.
*H. [pallida] opaca (Western Olivaceous Warbler); monotypic; Iberia, NW-Africa.
*H. [caligata] caligata (Booted Warbler); monotypic;
Central Palearctic from W Russia to Altai.
*H. [caligata] rama (Sykes’ Warbler); monotypic; westcentral Asia from Caspian Sea to Afghanistan.

MOLECULAR PHYLOGENY OF REED WARBLERS

Genus Chloropeta
Chloropeta natalensis (African Yellow Warbler); 4 ssp.;
subSaharan Africa.
*Chloropeta gracilirostris (Papyrus Yellow Warbler); 2
ssp.; Eastern Africa.
Chloropeta similis (Mountain Yellow Warbler); monotypic; Eastern Africa.
ACKNOWLEDGMENTS
We thank the following people for providing blood samples for thisstudy: R. Barth, D. Brown, J. Dierschke, J. Fjeldså, W. Forstmeier, D.
Franz, J. Garcia-Moreno, G. Halmos, F. Heinzenberg, H. Jännes, J.
Morgan, G. Nikolaus, S. Rumsey, V. Salewski, K. Schulze-Hagen, H.
Shirihai, P. Symens, F. Wong, and R. Yosef. We also thank B. Leisler
and K. Schulze-Hagen for stimulating discussions about reed warbler
phylogeny.

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