Ibis (2016), doi: 10.1111/ibi.12377

The phylogenetic position of the world’s smallest
passerine, the Pygmy Bushtit Psaltria exilis
ULF S. JOHANSSON,1* PER G. P. ERICSON,1 JON FJELDS
A2 & MARTIN IRESTEDT3
Department of Zoology, Swedish Museum of Natural History, Box 50007, Stockholm 10405, Sweden
2
Center for Macroecology, Evolution and Climate, Natural History Museum of Denmark, University of Copenhagen,Copenhagen, Denmark3
Department of Bioinformatics and Genetics, Swedish Museum of Natural History, Box 50007, Stockholm 10405,
Sweden1

The Pygmy Bushtit is conﬁned to the montane forests of Java. It is the world’s smallest
passerine and morphologically resembles a small, drab long-tailed tit or bushtit (Aegithalidae). In its behaviour the Pygmy Bushtit show similarities with the members of the Aegithalidae, but owing to its small size and isolated geographical distribution relative to theother members of the Aegithalidae, it has always been placed in a monotypic genus within
the family. The afﬁnities of the Pygmy Bushtit have never been tested in a phylogenetic
context and the species has to date not been included in any molecular studies. In thisstudy we use sequence data from four different genetic markers to place it in the passerine
phylogenetic tree. Our results conﬁrm the inclusion of the Pygmy Bushtit in the Aegithalidae, but rather than being an isolated lineage, our results strongly suggest that it is nested
in the Aegithalos clade, and most closely related to the Black-throated Bushtit Aegithalos
concinnus. The range of the Black-throated Bushtit extends south into subtropical Indochina, with an isolated subspecies occurring in southern Vietnam. The Black-throatedBushtit contains several morphologically and genetically distinct lineages, which could represent distinct species, but the phylogenetic relationships within this complex are poorly
resolved and partly in conﬂict with current taxonomic treatment based on morphology.
Keywords: Aegithalidae, Aves, biogeography, Passeriformes, phylogeny.

The Pygmy Bushtit Psaltria exilis is a Javan endemic conﬁned to montane forests in the highlandsof western and central Java. It is a nondescript
drab grey bird with whitish underparts, whitish irides and yellow legs and feet (Harrap & Quinn
1996, Harrap 2008). In spite of its long tail, it is
only 8.5 cm in length, which makes the Pygmy
Bushtit the smallest passerine species (Harrap &Quinn 1996). Morphologically it resembles asmall, dull bushtit and although very little is
known about its biology, it also appears to behave
very much as other members of the Aegithalidae.
For instance, it lives in small family groups thatmove actively through the vegetation, builds a‘pouch-like’ nest and resembles other long-tailed
tits and bushtits in its contact calls and apparent*Corresponding author.
Email: ulf.johansson@nrm.se© 2016 British Ornithologists’ Union

lack of a territorial song (Harrap & Quinn 1996,
Harrap 2008). Based on this resemblance it haslong been placed in the Aegithalidae (e.g. Snow
1967, Sibley & Monroe 1990, Dickinson 2003,
Dickinson & Christidis 2014). As it is smaller andduller than other aegithalids and is the only tropical member of the clade, the Pygmy Bushtit has
been placed in the monotypic genus Psaltria.
The genera Aegithalos, Psaltriparus and Psaltria
have been grouped together in most classiﬁcations
since the mid-1800s. Until recently this group wasin turn often associated with the tits and chickadees (Paridae). Once designed a separate family,
Aegithalidae (Vaurie 1957), the bushtits nevertheless remained linked with the parids due to thelack of evidence suggesting an alternative placement. It was not until molecular data were available, ﬁrst DNA–DNA hybridization (Sibley &Ahlquist 1990) and later DNA sequencing

2

U. S. Johansson et al.

(Alstr€om et al. 2006, Johansson et al. 2008, Fregin
et al. 2012), that the relationship with parids was
rejected and it was shown that Aegithalidae areinstead most closely related to leaf warblers (Phylloscopidae) and bush warblers (Cettiidae) in the
Sylvoidea. These latter studies also revealed thatthe more warbler-like Leptopoecile species are partof the Aegithalidae and sister taxa to Aegithalos
and Psaltriparus. However, the systematic positionof the Pygmy Bushtit has been addressed in few
systematic studies and to date it has not been
included in any molecular phylogeny.
Aegithalos, with between six and nine species,
has the largest distribution of the four genera in thefamily. Members of the genus extend from the Iberian Peninsula and the British Isles across the
Palaearctic to Kamchatka and Japan, and south to
the Himalayas and Indochina. This large distributional range, however, comprises primarily a single
species, the Long-tailed Tit Aegithalos caudatus.
Another fairly widespread species is the Blackthroated Bushtit Aegithalos concinnus, which is distributed in the Himalayan foothills and over muchof eastern China and along mountain ridges in tropical Indochina. The remaining species in the clade all
have rather restricted distributions conﬁned to small
regions in the Himalayas or in the mountains ofsouthwestern China. The two Leptopoecile species
inhabit the mountains north and east of the Tibetan
Plateau, primarily above 3000 m, whereas the solemember of the genus Psaltriparus, the American
Bushtit Psaltriparus minimus, inhabits western
North America, extending from southwestern
Canada to southern Mexico and Guatemala.
Here we address the systematic position of the
Pygmy Bushtit of Java by including it in a molecular phylogenetic study for the ﬁrst time. We ﬁrst
verify its position among the bushtits by including
it in a large phylogeny of passerine bird species,
and then speciﬁcally address its relationships tothe other species in the Aegithalidae in an analysis
with a comprehensive coverage of the taxa currently recognized within this family.
METHODS
DNA extraction, PCR-amplification andsequencing
DNA for this study was obtained from toe-pads ofmuseum study skins. The DNA from P. exilis was
obtained from a specimen that was collected in© 2016 British Ornithologists’ Union

western Java on 12 April 1920 (NRM 571647).
We also extracted DNA from additional specimensof the Black-throated Bushtit complex including
two specimens of the subspecies A. concinnus
annamensis and representatives of the subspecies
A. c. talifuensis and A. c. concinnus (Table 1).
As the DNA obtained from old museum study
skins is generally heavily degraded, the laboratory
work was conducted under strict conditions to
avoid contamination, following the procedures
described in Irestedt et al. (2006, in press). We
speciﬁcally designed primers that PCR-ampliﬁed
the individual loci in short c. 200-bp fragments(Supporting Information Table S1). We sequenced
four loci, the mitochondrial cytochrome-b gene,and three nuclear intron regions: ornithine decarboxylase (ODC) introns 6 and 7, myoglobin intron
2, and transforming growth factor beta 2 (TGFB2)
intron 5.
Phylogenetic analyses
We ﬁrst aimed to evaluate the phylogenetic positionof the Pygmy Bushtit relative to other passerine
birds to ascertain its relationships with the Aegithalidae, as suggested by behavioural and morphological
data. To cover as broad a range of potential closely
related groups as possible, we made use of a previously published dataset of passerine birds (Johansson et al. 2008; Supporting Information Table S2).
This dataset consists of myoglobin and ODC
sequences from a broad representation (90 taxa) ofprimarily Passerida lineages, with the densest sampling within the Sylvioidea, including all Aegithalidae
genera(Aegithalos,
Psaltriparus
andLeptopoecile). We also included additional Aegithalos
sequences from GenBank: White-throated Bushtit
Aegithalos niveogularis (KJ454741, GU434033),
Black-browed
BushtitAegithalos
bonvaloti(KJ790385, GU434032) and Aegithalos concinnus
iredalei (DQ008570, GU434027).
While this ﬁrst analysis conﬁrmed the placement of the Pygmy Bushtit in the Aegithalidae, italso suggested a previously unrecognized afﬁnity
for this species within the genus Aegithalos near
A. concinnus. We therefore performed an extended
analysis of the relationships within this family
based on three markers (ODC, TGFB2 and cytochrome-b) and additional sequences primarily
obtained from P€ackert et al. (2010) (Table 1).
However, as recent studies of the A. concinnus
complex (P€ackert et al. 2010, Dai et al. 2011)

talifuensis

annamensis

annamensis

Aegithalos concinnus

Aegithalos concinnus

Aegithalos concinnus

talifuensis

iredaleimanipurensis

minimus

Aegithalos concinnus

Aegithalos concinnus

Aegithalos concinnus

Psaltriparus minimus

Aegithalos
glaucogularis

Aegithalos caudatus

Aegithalos
leucogenys
Aegithalos
niveogularis

Aegithalos sharpei

Aegithalos bonvaloti

taiti

talifuensis

Aegithalos concinnus

Aegithalos fuliginosus

concinnus
concinnus

Subspecies

Aegithalos concinnus
Aegithalos concinnus

Psaltria exilis

SpeciesIndonesia, western
Java
TaiwanChina, Anhui
province
o CaiVietnam, La
province, Sapa
o CaiVietnam, La
province, Sapa
Vietnam, Lam Dong
province, Da Lat
Vietnam, Dac Lac
province, B’sre
China, Shaanxi, Taibai
Shan, Houzhenzi
China, Yunnan, Jizu
Shan
Myanmar, Mt. Victoria,
Pakokku
Afghanistan,Dar-e-Nur
Nepal, Dolpo District,
Ringmo/Lake
Phoksumdo
neesFrance, Pyre
Orientales,
Serralongue
China, Henan,
Dongzhai Nature
Reserve
China, Sichuan, Emei
Shan, Hongchun
Nepal, Rasuwa District,
W Syabrubesi
Myanmar, Chin State,
Natmataung NP
USA, Washington,
Paciﬁc C., Grayland

Locality

GU434016

MAR3951

MAR79247

MAR4722

MAR4128

MAR2068

MAR7814GU434008

GU434012

GU434011

GU434013

GU434018

GU434017

GU434019

MAR3955

MAR540GU434020

GU434015

GU434014

KX014772

KX014771

KX014776

KX014775

MAR50885

MAR3232

MAR809NRM 571729

NRM 571725

NRM 571743

NRM 571740

KX014774
KX014773

KX014777

NRM 571647
NRM 571736
NRM 571732

Tgfb2

Specimen no.

Table 1. GenBank accession numbers for the samples used in the three gene analysis of this study.

2

2

2

2

2

2

2

2

2

2

2

1

1

1

1

1
1

1

Ref.

GU244415

GU244428

GU244426

AY755556

GU244480

GU244456

GU244437

GU244439

GU244435

GU244433

GU244444

KX014764

KX014763

KX014768

KX014767

KX014766
KX014765

KX014769

cytb

2

2

2

2

2

2

2

2

2

2

2

1

1

1

1

1
1

1

Ref.

GenBank accession no.

2

2

2

2

2

2

2

2

2

2

2

1

1

1

1

1

1

Ref.

(continued)

GU434022

GU434028

GU434027

GU434026

GU434029

GU434030

GU434033

GU434035

GU434034

GU434032

GU434031

KX014779

KX014778

KX014782

KX014781

Failed
KX014780

KX014783

ODC

Phylogenetic position of the Pygmy Bushtit
3© 2016 British Ornithologists’ Union

2
JX518521
2
JX518496
2
JX518503© 2016 British Ornithologists’ Union

Outgroup
Pnoepyga pusilla

Leptopoecile sophiae
Leptopoecile elegans€ckert et al. (2010).
References: 1 = This study; 2 = Pa

MAR49312
2
GU434025
GU434024
2
2
GU244482
GU244414
2
2
GU434010
GU434009
MAR2117
MAR5767

GU434023
2
GU244416
2
GU434007
MAR56377
plumbeus
Psaltriparus minimus

USA, Colorado,
Fremont C., Camon
City
AviaryChina, Sichuan, Bang
Ba village

ODC
Ref.
cytb
Subspecies
Species

Locality

Specimen no.

Tgfb2

Ref.

GenBank accession no.
Table 1. (continued)

2

U. S. Johansson et al.

Ref.

4

suggest that the current treatment of this taxon asa single polytypic species is questionable, we also
included several of the genetically and morphologically distinct subspecies of this species, including
two sequences of A. c. annamensis from the Da
Lat Plateau, southern Vietnam, sequences fromtwo specimens morphologically identiﬁed as
A. c. talifuensis from Sapa, Vietnam, and two
sequencesfrom
specimens
morphologically
identiﬁed as A. c. concinnus from Taiwan and theAnhui Province in China, respectively (Table 1).
The taxa A. c. manipurensis and A. c. iredalei
included in the study by P€ackert et al. (2010) werealso included.
The study by Dai et al. (2011) furthermore suggests that the delineation of species boundaries
within concinnus–talifuensis is more complex than
indicated by current taxonomy, with at least three
distinct lineages being present. Dai et al. (2011)refer to these clades as A, B and C, and concluded
that ‘[i]n terms of the current taxonomy, subspecies A. c. talifuensis includes lineages A and B,
whereas lineages C, . . . exhibits strong concordance between the geographical mtDNA lineage
and the currently described nominate subspecies,
A. c. concinnus’. Furthermore, the two ‘talifuenisis’clades A and B do not form a monophyletic group,
as clade B appears as the sister-group of ‘concinnus’(clade C) with high support, and there is little
genetic divergence between the two clades. In contrast, the two ‘talifuensis’ clades are separated by agenetic distance of 4.8% (cytochrome-b, uncorrected p-distance). As no morphological characters
are yet known to distinguish between the two ‘talifuensis’ clades and their respective geographicaldistributions have not been circumscribed, theselineages can at present only be identiﬁed using
molecular data. Therefore, we performed a third
analysis in which we included cytochrome-b
sequences from 112 Aegithalos individuals,
primarily sourced from GenBank (Supporting
Information Table S3), in order to fully investigate
the relationship of the Pygmy Bushtit to the A.
concinnus complex. This analysis includes all currently recognized species in Aegithalidae (including the Rufous-fronted Bushtit Aegithalos
iouschistos) and of the subspecies in the Blackthroated Bushtit complex we lacked sequence data
only from
A. c. pulchellus from eastern Myanmar.
We used the same basic approach for all threedatasets. The sequences were aligned by eye usingPhylogenetic position of the Pygmy Bushtit

BIOEDIT 7.2.5 (Hall 1999). For the analysis of thephylogenetic position of the Pygmy Bushtit within
Passeriformes we used the same alignment as
Johansson et al. (2008), excluding the 625-bp-longinsertion observed in ODC for Motacilla andAmaurocichla (Johansson et al. 2008). Phylogenetic
analyses were conducted with Bayesian inference(BI) using MRBAYES 3.2.1 (Huelsenbeck & Ronquist 2001, Ronquist & Huelsenbeck 2003) andmaximum likelihood (ML) using RAXML v8.1.21(Stamatakis 2014), as implemented in RAXMLGUI v1.5b1 (Silvestro & Michalak 2012). The
model of sequence evolution was selected with theBayesian information criterion (BIC) using PARTITIONFINDER (Lanfear et al. 2012). In the higherlevel study (‘Passeriformes’) the GTR+Γ4+I model
was selected for ODC and a GTR+Γ4 substitution
model was selected for myoglobin intron 2. For
the Aegithalidae ODC/TGFB2/cytochrome-b analysis the HKY model was selected for ODC andTGFB2, and GTR+Γ4 was selected for cytochrome-b. For this latter three-gene dataset we ran
four separate analyses, one on the concatenateddata, and one on each of the individual gene trees
using the same models suggested by PARTITIONFINDER. The GTR+Γ4 model was also selected for the
more inclusive cytochrome-b analysis. In all
MRBAYES analyses we used default priors. For eachof the different datasets, two independent runs,
each with four Metropolis-coupled MCMC chains,
were run for 20 million generations with trees
sampled every 500 generations. Trees sampled
before the chain reached apparent stationarity(burn-in) were discarded and the posterior probabilities for the topology were estimated from theremaining generations. To ascertain whether the
chains had reached stationarity we: (1) plotted the
log-likelihood values for each run and compared
the posterior probabilities among runs, (2)checked that the average standard deviation of
split frequencies had converged towards zero, (3)determined that the potential scale reduction factor approached 1.0 for all parameters and (4) used
TRACER v1.6 (Rambaut & Drummond 2007) todetermine whether the sampling of the posterior
distribution had reached an adequate effective
sample size. The ML analyses were performed
with rapid bootstrap (1000 replicates) and a thorough ML search for one run and 1000 replicates
under a GTR+G model. The dataset was partitioned by gene and branch-lengths were calculated
independently for each partition.

5

RESULTS
Phylogenetic position within the
Passeriformes
Our analysis of the higher-level relationships of
the Pygmy Bushtit based on myoglobin intron 2
and ODC sequences conﬁrms that it belongs in
the Aegithalidae. The monophyly of the Aegithalidae (Aegithalos, Psaltriparus, Leptopoecile and Psaltria) was recovered with strong support (posterior
probability (PP) = 1.00), and within this clade
Leptopoecile is placed basal relative to Aegithalos
and Psaltriparus (Fig. 1). However, the Pygmy
Bushtit is not placed as an isolated lineage within
the Aegithalidae as assumed by its current taxonomic treatment by recognition of a monotypic
genus, but is nested within Aegithalos and sister to
A. concinnus, represented here by the subspecies
A. c. iredalei (Supporting Information Fig. S1).
This group in turn is the sister-group to a clade
containing A. caudatus, A. niveogularis and A. bonvaloti.
Phylogenetic position within the
Aegithalidae
In the analysis addressing phylogenetic relationships within Aegithalidae based on ODC, TGFB2
and cytochrome-b sequences, the Pygmy Bushtit
also falls inside the Aegithalos clade (Fig. 1) andwithin this clade there is strong support(PP = 1.00, ML bootstrap = 98%) for placing it ina clade together with the taxa in the Blackthroated Bushtit complex. Indeed, it is nested
within this complex together with A. c. manipurensis, A. c annamensis and A. c. iredalei as the sistergroup of A. c. concinnus and A. c. talifuensis. However, the support for this placement is weak
(PP = 0.89, ML bootstrap = 57%) and in fact noneof the relationships within Black-throated Bushtit
complex is strongly supported.
The close afﬁnity of the Pygmy Bushtit with
the Black-throated Bushtit complex is supported
in all gene trees (Fig. S2a–c). However, the exact
placement differs among the different analyses.
The ODC gene tree is mostly unresolved, but oneof the few supported nodes in this tree places the
Pygmy Bushtit with some of the taxa in the Blackthroated Bushtit complex (PP = 0.60, ML bootstrap = 52%; Fig. S2a). The TGFB2 gene tree ismuch more resolved although many relationships© 2016 British Ornithologists’ Union

6

U. S. Johansson et al.

Pnoepyga pusilla
Leptopoecile sophiae
1100

Leptopoecile elegans
Psaltriparus m. minimus
1
100

Psaltriparus minimus plumbeus
Aegithalos caudatus taiti
1
100

Aegithalos glaucogularis
1
100

0.53
X

Aegithalos niveogularis
1
94

Aegithalos bonvaloti
1
95 Aegithalos fuliginosus

0.51
83

1
100

Aegithalos sharpei

Aegithalos leucogenys
Aegithalos concinnus iredalei

0.92
90

0.89
57

Psaltria exilis
Aegithalos concinnus manipurensis (MAR4722)
Aegithalos concinnus annamensis (NRM571725)

1
100

1
98

Aegithalos concinnus annamensis (NRM571729)
Aegithalos concinnus talifuensis (MAR2068)
1
95 Aegithalos concinnus concinnus (NRM571736)

0.69
54

Aegithalos concinnus concinnus (NRM571732)

0.55
42

Aegithalos concinnus talifuensis (NRM571740)

0.88
62

Aegithalos concinnus

0.85
X

Aegithalos concinnus talifuensis (NRM571743)

0.1

Figure 1. Molecular phylogeny of the Aegithalidae. The tree is a Bayesian consensus tree based on three genes, nuclear ODC andTGFB2 and mitochondrial cytochrome-b (total 1586 bp). Numbers above the branches indicate Bayesian posterior probabilities andnumbers below bootstrap support values from the maximum likelihood analysis.

have comparably low posterior probabilities orbootstrap support values (Fig. S2b). Of the three
gene trees, the cytochrome-b gene tree is the mostresolved and is also the tree in which most nodes
have comparably high support (Fig. S2c). Overall,
the TGFB2 and the cytochrome-b gene trees
recover a similar topology but differ at a few© 2016 British Ornithologists’ Union

poorly supported nodes. With respect to the placement of the Pygmy Bushtit, the TGFB2 gene treeplaces it basal relative to a monophyletic Blackthroated Bushtit complex, whereas in the cytochrome-b gene tree the species is placed within thisclade as the sister-group of A. e. iredalei. In bothgene trees the support for the Pygmy Bushtit in a

Phylogenetic position of the Pygmy Bushtit

clade with the taxa in the Black-throated Bushtit
complex has high support (TGFB2: PP = 1.00,
ML bootstrap = 89%; cytochrome-b: PP = 0.99,
ML bootstrap = 84%) whereas the support for its
relationship to the taxa within this complex islower, but at least in the TGFB2 gene tree its position as basal relative to a monophyletic Blackthroated Bushtit has a posterior probability 0.96
and a ML bootstrap support value of 77%.
Phylogenetic position with dense
sampling of Aegithalos
The tree based on the more inclusive cytochromeb analysis (Fig. 2) is congruent with that of themulti-locus concatenated analysis (Fig. 1), with the
exception that the previously identiﬁed concinnus/
talifuensis clade is not monophyletic. Similar to theconcatenated analysis a group with A. c. concinnus
and A. c. talifuensis forms the sister-group of allother Black-throated Bushtit taxa (including the
Pygmy Bushtit), but separated from this is a cladeof ‘talifuensis’ specimens from Yunnan that emerge
as the sister-group to A. c. manipurensis (Fig. 2).
This clade corresponds to lineage A of Dai et al.
(2011). All our samples from the concatenatedanalysis of concinnus and talifuensis fall into the former clade, and thus this ‘talifuensis’ lineage A is atpresent unsampled by the nuclear data.
DISCUSSION
The Pygmy Bushtit of Java is reminiscent of asmall long-tailed tit (Aegithalidae), both in morphology and in behaviour (Harrap 2008) and ourmolecular phylogenetic analyses conﬁrm this relationship. More surprisingly, our analyses suggestthat the Pygmy Bushtit is not an isolated lineage
within the Aegithalidae, but rather is nested in thegenus Aegithalos and is particularly closely related
to the Black-throated Bushtit.
The Black-throated Bushtit, although signiﬁcantly larger than the Pygmy Bushtit, is also
among the smaller members of the Aegithalidae,
being about 10.5 cm in length. It inhabits mixed
forest and lightly wooded and bushy habitats,including Rhododendron and bamboo at mid-elevations. It has a relatively large distribution that
extends further south than any other member ofthe genus and includes the Himalayan foothills
from northern Pakistan to eastern China and Taiwan, and reaches south to 20°N in the mountains

7of Myanmar and northern Laos. There is also an
isolated population at 11–15°N on the Da Lat
Plateau of southern Vietnam. Within this range it
is divided into six subspecies (concinnus, talifuensis, manipurensis, iredalei, pulchellus and annamenis(Harrap 2008)), which differ primarily in the colour of the crown and the amount and intensity of
rufous on the body underparts. Previous molecular studies have shown that these taxa areseparated by large genetic distances, but that thesystematics of this group is not entirely congruent
with the recognized subspecies boundaries. The
subspecies A. c. annamensis and A. c. iredalei,
which occupy the extreme southeast and west of
the species’ range, form at least two, possibly
three genetically distinct lineages well differentiated from the other populations in this complex(P€ackert et al. 2010).
The situation with the non-monophyletic talifuensis, ﬁrst identiﬁed by Dai et al. (2011), appears
to be more intricate than the phenotypic variationsuggests. At present, no distinguishing charactershave been identiﬁed that separate the two talifuensis groups and there is no obvious geographicalbarrier to prevent interaction between the two
clades (Dai et al. 2011). Dai et al. (2011) suggested that the two clades evolved in isolation in
the early Pleistocene (c. 2.4 million years ago, 95%
CI = 4.29–0.88 Ma), but later came into secondary contact. The two lineages may reﬂect thepresence of two cryptic species in this region, but
it seems more plausible to interpret the apparent
sympatry of the two clades in Yunnan as a case of
introgression of mtDNA following an expansion of
the widespread subspecies A. c. concinnus of southeastern China into the range of talifuensis in the
Hengduan Mountains in the southwest. Several
similar cases have been described in recent years,
where distinct mitochondrial haplotype groups
have been mixed over sometimes very large geographical areas (e.g. Zink et al. 2008, Webb et al.
2011, Pentzold et al. 2013). In some cases highly
divergent lineages are found to coexist in a panmictic population, such as in the Common Redstart Phoenicurus phoenicurus over most of Europe(Hogner et al. 2012). For a deﬁnitive interpretation it would be necessary to obtain a broader geographical sampling and to include sequence datafrom nuclear genes, including Z-linked markers. A
detailed analysis of the Black-throated Bushtit
complex is beyond the scope of this paper, but inview of these ﬁndings we agree with P€ackert et al.
© 2016 British Ornithologists’ Union

8

U. S. Johansson et al.

1
100

GU244414 Leptopoecile elegans
GU244482 Leptopoecile sophiae
DQ008518 Leptopoecile sophiae
1GU244415 Psaltriparus m. minimus
AF074597 Psaltriparus m. minimus
GU244416 Psaltriparus minimus plumbeus
0.99
GU244418
Psaltriparus minimus plumbeus
94
GU244417 Psaltriparus minimus plumbeus
1
GU244436 A. niveogularis
HIMALAYA97
GU244437 A.niveogularis
1
GU244430 A. fuliginosus
W. SICHUAN
87
GU244444 A. fuliginosus
1
GU244433 A.bonvaloti
95
GU244431 A. i. iouschistos
GU244432 A. i. iouschistos
E. HIMALAYA, SICHUAN
GU244434 A. sharpei
GU244435 A. sharpei
GU244476 A. glaucogularis
GU244480 A. glaucogularis
E. CHINA
GU244479 A. glaucogularis

1
100
1

1
100
0.99
65

GU244478 A. glaucogularis
GU244451 A. c. caudatus
AB159171 A. c. magnus
AB159169 A. c. trivirigatus
GU244466 A. c. europaeus
GU244459 A. c. europaeus
GU244452 A. c. caudatus
GU244455 A. c. taiti
GU244447 A. c. caudatus
PALEARCTIC
AB159170 A. c. trivirigatus
GU244454 A. c. europaeus
GU244470 A. c. irbii
GU244467 A. c. europaeus
GU244458 A. c. caudatus
GU244457 A. c. europaeus
GU244456 A. c. taiti
GU244450 A. c. taiti
GU244448 A. c. europaeus
GU244438 A. leucogenys
HINDU KUSH MTS.
GU244439 A. leucogenys
Psaltria exilis
AY755555 A. c. iredalei - Irukuwa Khola, Bhojpur District, W Bengal, India1
0.99
1 GU244426 A. c. iredalei - W Syabrubesi, Rasuwa District, Nepal
100
75
100 GU244427 A. c. iredalei - W Syabrubesi, Rasuwa District, Nepal
0.99
DQ008519 A. c. iredalei - W Bengal, India
55
1
A. c. annamensis - Da Lat, Vietnam (NRM571725)
100
A. c. annamensis - B'sré, Dac Lac, Vietnam (NRM571729)
0.87
1GU244428 A c. manipurensis - Myanmar
48
HQ605484 A. concinnus/talifuensis - Lijiang (LJ), Yunnan, China
58
HQ605489 A. concinnus/talifuensis - Lijiang (LJ), Yunnan, China
HQ605470 A. concinnus/talifuensis - Dahaoping (DHP), Yunnan, China
0.95
HQ605472 A. concinnus/talifuensis - Dahaoping (DHP), Yunnan, China
69
1
HQ605460 A. concinnus/talifuensis - Baoshan (BS), Yunnan, China
97
HQ605458 A. concinnus/talifuensis - Shuangjiang (LC), Yunnan, China
HQ605453 A. concinnus/talifuensis - Dahaoping (DHP), Yunnan, China
HQ605475 A. concinnus/talifuensis - Dahaoping (DHP), Yunnan, China
HQ605456 A. concinnus/talifuensis - Kungming (KM), Yunnan, China
HQ605488 A. concinnus/talifuensis - Lijiang (LJ),Yunnan, China
HQ605487 A. concinnus/talifuensis - Lijiang (LJ), Yunnan, China
HQ605466 A. concinnus/talifuensis - Lijiang (LJ), Yunnan, China
HQ605457 A. concinnus/talifuensis - Yuanxian (LC), Yunnan, China
HQ605455 A. concinnus/talifuensis - Dahaoping (DHP), Yunnan, China
HQ605459 A. concinnus/talifuensis - Gengma (LC), Yunnan, China
A. concinnus talifuensis - Sapa, Vietnam (NRM571740)
HQ605394 A. concinnus/talifuensis- Yanyuan (PZH), Sichuan, China
HQ605462 A. concinnus/talifuensis - Lijiang (LJ), Yunnan, China
Lineage B
HQ605486 A. concinnus/talifuensis - Lijiang (LJ), Yunnan, China
sensu
HQ605418 A. concinnus/talifuensis - Yanbian (PZH), Sichuan, China
Dai et al. 2011
HQ605419 A. concinnus/talifuensis - Panzhihua (PZH), Sichuan, China
HQ605421 A. concinnus/talifuensis - Miyi (PZH), Sichuan, China
GU244440 A. concinnus/talifuensis - Dongzhai, Henan, China
HQ605476 A. concinnus/talifuensis - Xianju (XJ), Zhejiang, China
HQ605408 A. concinnus/talifuensis - Miyi (PZH), Sichuan, China
HQ605398 A. concinnus/talifuensis - Guangyuan (GY), Sichuan, China
HQ605395 A. concinnus/talifuensis - Moxi (MX), Sichuan, China
HQ605380 A. concinnus/talifuensis - Wuxiu (WuX), Jiangxi, China
1
HQ605376 A. concinnus/talifuensis - Yuanjiang (YJ), Hunan, China
98
HQ605370 A. concinnus/talifuensis - Yangxin (YX), Hubei, China
HQ605348 A. concinnus/talifuensis - Chishui (CS), Sichuan, China
HQ605331 A. concinnus/talifuensis - Guilin (GL)Guangxi, China
HQ605315 A. concinnus/talifuensis - Jianyang (JY), Fujian, China
A. concinnus talifuensis - Sapa, Vietnam (NRM571743)
A. concinnus concinnus - Anhui, China (NRM571732)
JX398849 A. c. concinnus - Dawei Shan NP, Hunan, China
HQ605360 A. concinnus/talifuensis - Guiyang (GZ), Guizhou, China
Lineage C
HQ605423 A. concinnus/talifuensis - Shengnongija (SNJ), Hubei, China
sensu
HQ605383 A. concinnus/talifuensis - Nanjing (NJ), Jiangsu, China
Dai et al. 2011
HQ605299 A. concinnus/talifuensis - Jixi (JX), Anhui, China
HQ605441 A. concinnus/talifuensis - Haoping (HP), Shaanxi, China
HQ605356 A. concinnus/talifuensis - Guiyang (GZ), Guizhou, China
HQ605341 A. concinnus/talifuensis - Jinzhongshan (JZS), Guangxi, China
HQ605327 A. concinnus/talifuensis -Wenxian (WX), Gansu, China
HQ605322 A. concinnus/talifuensis - Jianou (JO), Fujian, China
HQ605295 A. concinnus/talifuensis - Jixi (JX), Anhui, China
HQ605293 A. concinnus/talifuensis - Jixi (JX), Anhui, China
GU244442 A. c. talifuensis - Longxi-Hongkou, Sichuan, China
GU244441 A. c. concinnus - Dongzhai, Henan, China
HQ605425 A. concinnus/talifuensis - Shengnongjia (SNJ), ; Hubei, China
HQ605340 A. concinnus/talifuensis - Jinxiu (JiX), Guangxi, China
HQ605373 A. concinnus/talifuensis - Pingjiang (PJ), Hunan, China
A. c. concinnus - Taiwan (NRM571736)
GU244422 A. concinnus - Dongzhai, Henan, China
HQ605367 A. concinnus/talifuensis - Guiyang (GZ), Guzhou, China
HQ605305 A. concinnus/talifuensis - Dongzai (DZ), Henan, China
AY755556 A. c. talifuensis - Emei Shan, Sichuan China (MAR3292)
HQ605420 A. concinnus/talifuensis - Panzhihua (PZH), Sichuan, China
HQ605407 A. concinnus/talifuensis - Yanbian (PZH), Sichuan, China
HQ605397 A. concinnus/talifuensis - Liziping (LZP), Sichuan, China
1
100

1
100

1
100

1
96

1
95

Lineage A
sensu
Dai et al. 2011

Aegithalos concinnus

0.1

Figure 2. Bayesian consensus tree of the analysis of 112 cytochrome-b sequences. Numbers above the branches indicate Bayesianposterior probabilities and numbers below bootstrap support values from the maximum likelihood analysis. Sequences used in thethree gene analysis are indicated in bold.

© 2016 British Ornithologists’ Union

Phylogenetic position of the Pygmy Bushtit(2010) that it is premature to make any taxonomic
recommendations for the group at this point.
The situation within the Black-throated Bushtit
complex aside, the close afﬁnity of the Pygmy
Bushtit with this group seems to be strongly supported. Both the higher-level phylogeny of the
Passeriformes based on myoglobin intron 2 andODC, as well as the more speciﬁc analysis of therelationships within the Aegithalidae based on
ODC, TGFB2 and cytochrome-b, place the Pygmy
Bushtit with the Black-throated Bushtit complex
with high posterior probabilities. Whether the two
species are sister-groups or the Pygmy Bushtit isnested within the latter species remains unresolved.
The Pygmy Bushtit is found in montane forests
in western and central Java. Of the subspecies in
the Black-throated Bushtit complex, A. c.
manipurensis occurs along the border between
western Myanmar and India, A. c. pulchelles ineastern Myanmar and northwestern Thailand, A. c.
annamensis in the highlands of southern Laos/Vietnam and A. c. iredalei in the Himalayas. To thenorth and to the east, A. c. concinnus is widely distributed in southeastern China, whereas the two
talifuensis lineages are distributed primarily insouthwestern China. The Pygmy Bushtit is thus
found 3000 km and 26 degrees of latitude southof the range of the nearest population of A. c. pulchellus in eastern Myanmar. Our data do not conﬁdently establish the relationship of the Pygmy
Bushtit with the taxa within the Black-throatedBushtit complex, but they do, with weak support,
indicate that it may have originated from an ancestral population of the iredalei–manipurensis–annamensis clade (Fig. 1).
Several other Javan taxa show a similar disjunctdistribution with its closest relative on the Asian
mainland. The most prominent example concerns
the mammalian megafauna, with the occurrenceof subfossil and extant oxen, rhinos and elephants
in Java (Morley 2012), but there are also several
examples of mainly dry-habitat birds on Java withsimilar distributions (Sheldon et al. 2015).
Over the last 2.5 Ma, Sundaland has repeatedly
been connected with and disconnected from mainland Asia due to cyclical sea level ﬂuctuations
when development of arctic glaciers tied up oceanic water (Hall 2011). Thus, for much of the
Pleistocene, the Gulf of Thailand and the area
between the Malay Peninsula, Sumatra, Borneo
and Java/Bali was dry land, which resulted in a9continental climate with drier conditions in theinterior of this land area. Pollen studies have givenrise to a theory that central Sundaland was the siteof a savanna corridor extending along the east
coast of the Malay Peninsula to Java during thepeaks of the glacial periods (Heaney 1991, Bird
et al. 2005), which explains how the Asian megafauna may have reached Java in the late Pliocene
through the mid-Pleistocene (Cranbrook 2000)
and how birds could adapt to drier environments.
However, the savanna corridor may not explain
the dispersal of birds associated with humid montane forests, such as the long-tailed tits. Such habitats in the Sinohimalayan Mountains have long
been isolated by deciduous forests from the evergreen montane forests of Java (Morley 2012). In
this context it may be more relevant to consider
vegetation formations associated with special soils.
Coarse-textured and often poorly drained soils ineastern Sumatra and western Borneo generally
underlie extremely nutrient-poor peat swamps andheath forests, and this sediment type extends
across the sea south of the Malay Peninsula,
between eastern Sumatra and western Borneo(Slik et al. 2011). Heath-forests, as found in many
nutrient-poor parts of Sundaland today, differ
from tropical rainforests in terms of structure andproductivity and are more similar to the forest of
nutrient-poor mountain ridges. We therefore suggest that the dispersal of montane forest birds may
have been possible along the ridge tops between
the Myanmar–Thailand border and Malay Peninsula, and then onward to the highlands of Javaduring periods of cold climate and low sea levels.
This research was supported by grants from the Swedish
Research Council (no. 621-2013-5161 to P.E. and 6212014-5113 to M.I.). J.F. thanks the Danish National
Research Foundation for support to the Center for
Macroecology, Evolution and Climate. We also wish to
thank three anonymous reviewers and Rauri Bowie forvaluable comments on the manuscript.

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Received 28 October 2015;
revision accepted 23 March 2016.
Associate Editor: Martin Collinson.

SUPPORTING INFORMATION
Additional Supporting Information may be foundin the online version of this article:

Phylogenetic position of the Pygmy Bushtit

Figure S1. Molecular phylogeny based on thedata set from Johansson et al. (2008) indicating
the position of the Pygmy Tit Psaltria exilis relative to the Aegithalidae and other passerine
lineages. The tree is a Bayesian consensus tree ofthe combined, mixed-model analysis of two
nuclear data partitions: myoglobin intron 2 andODC introns 6 and 7, along with intervening exon
7 (total 1597 bp). Numbers above the branches
indicate Bayesian posterior probabilities and numbers below bootstrap support values from the
maximum likelihood analysis.
Figure S2a. Bayesian consensus tree based on
introns 6 and 7, along with intervening exon 7, of
the ornithine decarboxylase (ODC) gene (ODC).
Numbers above the branches indicate Bayesianposterior probabilities and numbers below bootstrap support values from the maximum likelihood
analysis.

11

Figure S2b. Bayesian consensus tree based on
the ﬁfth intron of the transforming growth factor
b2 (TGF b2) gene. Numbers above the branches
indicate Bayesian posterior probabilities and numbers below bootstrap support values from the
maximum likelihood analysis.
Figure S2c. Bayesian consensus tree based on
mitochondrial cytochrome b gene. Numbers above
the branches indicate Bayesian posterior probabilities and numbers below bootstrap support values
from the maximum likelihood analysis.
Table S1. Primers used in this study.
Table S2. GenBank accession numbers for the
samples used in the ‘higher level’ phylogeny.
Table S3. GenBank numbers for the sequencesused in the extended cytochrome b analysis.

© 2016 British Ornithologists’ Union