65 (1): 135 – 150
4.5.2015© Senckenberg Gesellschaft für Naturforschung, 2015.

Phylogenetic relationships of endemic bunting species(Aves, Passeriformes, Emberizidae, Emberiza koslowi )from the eastern Qinghai-Tibet Plateau
Martin Päckert 1, Yue-Hua Sun 2, Patrick Strutzenberger 1, Olga Valchuk 3,
Dieter Thomas Tietze 4 & Jochen Martens 5, #Senckenberg Natural History Collections, Museum of Zoology, Königsbrücker Landstraße 159, 01109 Dresden, Germany — 2 Key Laboratory of Animal Ecology and Conservation, Institute of Zoology, Chinese Academy of Science, 100101 Beijing, China — 3 Institute of Biology
and Soil Sciences, Russian Academy of Sciences, Vladivostok, 690022 Russia — 4 Institute of Pharmacy and Molecular Biotechnology,
Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany — 5 Institut für Zoologie, Johannes Gutenberg-Universität,
55099 Mainz, Germany — # Results of the Himalaya-Expeditions of J. Martens, No. 281. – For No. 280 see: Genus (Wrocław) 25 (3): 351 –
355, 2014
1

Accepted 20.iii.2015.
Published online at www.senckenberg.de/vertebrate-zoology on 4.v.2015.

Abstract
In this study we reconstructed the phylogenetic relationships of a narrow-range Tibetan endemic, Emberiza koslowi, to its congeners andshed some light on intraspecific lineage separation of further bunting species from Far East Asia and along the eastern margin of the Tibetan
Plateau in China. The onset of the Old World bunting radiation was dated to the mid Miocene and gave rise to four major clades: i) one
group comprising mainly Western Palearctic species and all high-alpine endemics of the Tibetan Plateau; ii) a clade including E. lathami,
E. bruniceps and E. melanocephala; iii) one group comprising mainly Eastern Palearctic species and all insular endemics from Japan and
Sakhalin; iv) an exclusively Afrotropic clade that comprised all African species except E. affinis, whose phylogenetic relationships were
ambiguous and only poorly supported in all reconstructions. The Tibetan bunting, E. koslowi, turned out as an early offshoot of the Western
Palearctic-Tibetan clade 1 and thus represents an ancient relic lineage that dates back to a mid Miocene colonization event of its ancestors
to the alpine plateau habitats. This temporal scenario of an early Miocene origin of alpine Tibetan endemics coincides with recent results
for two further species, the Tibetan ground tit, Pseudopodoces humilis, and the Tibetan rosefinch, Carpodacus roborowskii. The origin
of extant intraspecific phylogeographic patterns and splits among sister species in Eastern Asia were dated back to the Pleistocene withearliest lineage splits occurring among taxa from the Japanese Archipelago including Sakhalin and their mainland counterparts. A similarly
ancient split separated a southern clade of E. godlewskii yunnanensis from S Sichuan and Yunnan from a northern clade including populations from central and northeastern China, Mongolia and S Siberia. Ecological segregation among breeding habitats of southern E. g.
yunnanensis at lower elevations and those of other conspecifics at high-alpine habitats might have played a key role in the spatial genetic
diversification of this species.

Key words
Emberiza, phylogeny, Tibetan bunting, endemics, Qinghai-Tibet Plateau, lineage splits, intraspecific differentiation.

Introduction
The Old World buntings of the genus Emberiza are widely distributed across the Palearctic, the Middle East, theHimalayas, East Asia and Africa. Throughout the genus’
entire range 42 currently accepted Emberiza species

ISSN 1864-5755(Rose, 2011; 38 species in Byers et al., 1995) occupy adiverse variety of breeding habitats, however most species tend to avoid very densely forested areas and prefersemi-open to open taiga forest habitats, Central Asian

135

Päckert, M. et al.: Phylogenetic relationships of Emberiza koslowi

Emberiza species count
1

12Fig. 1. Species richness patterns of Palearctic and Afrotropic Old World buntings, genus Emberiza based on year-round and breeding
ranges. Distribution data was obtained from the Birdlife International spatial data portal (Birdlife & NatureServe, 2012; distribution shape
files ‘native (breeding)’ and ‘native (resident’).

steppes and wetlands (Byers et al., 1995; Rose, 2011).
A geographically extensive hotspot of species richness
is located in the Eastern Palearctic (Fig. 1). There, many
bunting species occupy breeding habitats of open andsemi-open taiga forest and forest edges (all informationon habitat preferences according to Dementiev & Glad­kov, 1954; Panov, 1973; Rose, 2011). Among these E.
tristrami is typically bound to closed mixed and pine forests with dense bush undergrowth while E. elegans has apreference of mixed and deciduous forests with a presence of oak stands and E. leucocephalos even settles forest islands in steppe habitats. Other species have a clear
preference for wetland forests, marshes and riverbankswith sparse birch and willow stands (E. pusilla, E. aureola, E. rutila). Pallas’ bunting (E. pallasi) even breeds
at high latitudes of the subarctic tundra belt in shrub vegetation of dwarf willow and alder stands along river valleys and in subalpine tundra with Rhododendron bushes
and grasslands of the Siberian Altai. Twelve to sixteen
Emberiza species (wintering species included) have beenrecorded in local co-occurrence at different local field stations in Far East Russia (Mattes & Alfer, 2010; Mattes& Shokhrin, 2010; Heim et al., 2012). At Muraviovka
Park in the Middle Amur region the black-faced bunting(E. spodocephala) and Pallas’ bunting (E. pallasi) ranged
among the five locally most abundant species with respect
to the number of ringed individuals (Heim & Smirenski,2013). In southern Primorye, at Lazo Reserve highest
136

numbers in the period of migration were observed forE. rustica, E. elegans, E. spodocephala and E. tristrami(Mattes & Alfer, 2010), at Litovka river for the same
species and for E.rutila (Valchuk & Yuasa, 2002).
Besides Central and Far Eastern Siberia, further regions of high species richness in Asia are found at thenorthwestern margin of the Qinghai-Tibet Plateau and in
China at the southeastern plateau margin (Fig. 1). There,some species occupy bushy mountain slopes and montane steppe, such as Godlewski’s bunting (E. god­lews­kii;
Fig. 2) that has a patchy breeding distribution at the eastern, the northern and the western plateau margins. Someendemic species are confined to very small breeding
ranges at the northern plateau margins, such as the endangered rufous-backed bunting (E. jankowskii) from thegrass steppes of Inner Mongolia and W Jilin (Rose, 2011)
and at the eastern margins such as the Tibetan bunting (E.
koslowi; Fig. 2). The latter is restricted to a narrow breeding distribution around the border between the Chinese
provinces Xizang and Qinghai where it occupies alpine
shrubs of juniper, rhododendron and cotoneaster on steep
slopes and alpine grasslands between 3600 and 4600 m
a.s.l. (Schäfer, 1938; Olsson, 1995; Thewlis & Martins,
2000; Rose, 2011; Ju & Golok, 2013). The phylogenetic
relationships of this Tibetan endemic to other congeners
are so far unresolved.
A first near-complete molecular phylogeny of Old
World buntings, based on one mitochondrial and one

VERTEBRATE ZOOLOGY — 65  (1)  2014

Fig. 2. Endemics of the alpine high elevations of the Qinghai-Tibet Plateau or the plateau margins; left) Tibetan bunting, Emberizakoslowi; right) Godlewski’s bunting, Emberiza godlewskii; (pictures: MP, Qinghai 2013).

nuclear marker, was published by Alström et al., 2008.
Though some among- and within-clade relationships
were not resolved in the two-marker phylogeny the authors found evidence of four major Emberiza clades andconfirmed six sister species pairs previously identified bytraditional morphology-based systematics. Furthermore,they showed that three members of three traditionalmonotypic emberizid genera, Miliaria, Latoucheornis
and Melophus (cf. Byers et al., 1995) were firmly nested
within the Emberiza tree (Alström et al., 2008). These
results confirmed earlier taxonomic recommendations by
Sangster et al., (2004) for a transfer of the corn bunting(Miliaria calandra sensu Voous, 1977) into Emberizabased on early molecular studies with rather incompletetaxonomic samplings (Graputto et al., 2001; Lee et al.,2001). In contrast, contrary to molecular evidence fromthe near-complete bunting phylogeny (Alström et al.,2008) the other two monotypic Asian genera (Latoucheornis and Melophus) have been maintained and separated
from Emberiza by some authors still to date (Clements
et al., 2014). In a recent phylogenetic study on somepolytypic species of African brown buntings Olsson etal., (2013) found genetic support for the species statusof North African E. sahari as a sister of East Africanand Middle Eastern E. striolata (these two were alreadyseparated by Rose, 2011) and they recommended a further species-level split of E. goslingi from E. tahapisi.
Genetic distinctiveness of the latter two species furthermore coincides with differences of territorial songs (Osi­ejuk, 2011). Traditionally, the insular endemic of Socotra(E. socotrana) was thought to be affiliated with thisgroup (Rose, 2011). This relationship was recently confirmed by Schweizer & Kirwan (2014) who comparedsequence data obtained from museum specimens of the
Socotra bunting with the African brown bunting data set
by Olsson et al. (2013).
Apart from the latter study only little attention has
been drawn to intraspecific genetic variation of buntings.
Very few phylogeographic studies were dedicated tosub­specific variation in the reed bunting, E. schoeniclus(Kvist et al., 2011; Zink et al., 2008). Further evidence
of intraspecific lineage divergence among continental
and insular populations of E. spodocephala was inferredfrom RAPD analysis (Dolgova & Valchuk, 2008), from
DNA-barcoding (Saitoh et al., 2015) and from multi-locus phylogeographies and accompanying morphometricanalyses (Weissensteiner, 2013).
In our study we focused on the phylogenetic relationships of the Tibetan bunting (E. koslowi), a so far virtually
unstudied high alpine endemic of the Tibetan plateau, aswell as on intraspecific lineage separation in Asian bunting species at the southeastern Chinese plateau margins(such as E. godlewskii; Fig. 2) and in the Himalayas. We
added sequence data from taxa not included in previous
studies to the Emberiza tree and expanded the dataset toinclude three mitochondrial genes and two nuclear introns. In addition, we used a time-calibrated multi-locus
phylogeny in order to provide an evolutionary time scaleof Emberiza bunting evolutionary history.

Material and Methods
DNA analysis
We extracted DNA from 103 blood and tissue samplesof 34 Emberiza species plus a few focal subspecies from
China and the Himalayas (and two outgroup species,
Lapland longspur (Calcarius lapponicus) and snow bunting (Plectrophenax nivalis). For origin of samples andsequences see electronic supplement 1.
We amplified a 706-bp fragment of the mitochondrial
cytochrome-b gene for all samples available using theprimer combination L14841-Cytb (5’ – AAA AAG CTTCCA TCC AAC ATC TCA GCA TGA TGA AA – 3’,
Kocher et al., 1989) and H-15547-Cytb (5’ – AAT AGG
137

Päckert, M. et al.: Phylogenetic relationships of Emberiza koslowi

AAG TAT CAT TCG GGT TTG ATG – 3’, Edwards etal., 1991). The PCR protocol was 94ºC for 2 min followed by 35 cycles of 92ºC for 45 s, 56ºC for 1 min
and 72ºC for 1.5 min with a final extension in 72ºC for
5 min. A larger 1079-bp-long fragment was amplified
for at least one sample of each taxon investigated withthe primer combination O-L14851 (5’ – CCT ACC TAGGAT CAT TCG CCC T – 3’) and O-H16065 (5’ – AGT
CTT CAA TCT TTG GCT TAC AAG AC – 3’; Weir &Schluter, 2007). The PCR protocol was 94ºC for 10 min
followed by 35 cycles of 92ºC for 1 min, 53ºC for 1 min
and 72ºC for 2 min with a final extension in 72ºC for
10 min.
For a few toe pad samples obtained from whole skinsof target taxa we performed DNA extractions using thesbeadex® forensic kit (LGC Genomics). Extraction was
performed according to the manufacturer’s instructions
except for overnight incubation of tissue with proteinase K (instead of one hour) and only 60 µl elution volume (instead of 100 µl) in order to yield a sufficiently
high concentration of DNA extracts. All toe pad samples were analysed in a separate clean lab. There, eachstep of analysis (sampling, extraction and PCR) was
done on separate working benches. In order to avoid
cross-contamination working benches were cleaned with
DNA-away (Molecular Bio Products, Inc.), after eachstep both benches and lab rooms were decontaminatedwith UV-light for at least four hours. Using a set of sequences derived from DNA analysis of fresh samples
we designed several primer combinations for amplification of shorter gene fragments from toe pad DNA extracts (110 to 250 bp, depending on the quality and ageof template DNA). Primer design was carried out using
software OLIGOEXPLORER 1.2 (http://www.genelink.
com/tools/gl-oe.asp) and gradient PCRs were performed
in order to determine the optimum annealing temperature for each primer pair. PCR primer combinations for
amplification of short cytb fragments are provided inelectronic supplement 2. For comparison with our own
data set we obtained 99 further cytb sequences from
GenBank (see electronic supplement 1).
For reconstruction of a multi-locus phylogeny weamplified and sequenced four additional markers for a reduced taxon set with one representative sample per species (or mitochondrial lineage identified within species).
We added sequences of two further mitochondrial markers to the data set, 16S rRNA and the barcoding marker
cytochrome oxidase (COI) as well as two nuclear introns,
myoglobin intron 2 and fibrinogen intron 7. For amplification and sequencing of myoglobin-2 with the primer
combinations myo2, myo3 and myo3F we followed thenested PCR protocol in Ericson et al. (2003) and foranalysis of some problematic samples we used two further external primers previously applied by Tietze et al.
(2013) for rosefinches (CarpMyoF1 = 5’ – CAG CTG
TGT GAG AGT TGG – 5’ and CarpMyoR4 = 5’ – AGA
AAT GAA CTG TGA GGA AGG – 3’). We amplified
myoglobin introns in a hot-start touchdown PCR according to the protocols provided in Irestedt et al. (2006)
138

with annealing temperatures decreasing from 58°C
(5 cylces) and 56°C (5 cycles) to 54°C (30 cycles). For
primer combinations, PCR and sequencing protocols for
16S rRNA refer to Spicer & Dunipace (2004), for fib7
refer to Prychitko & Moore (1997) and for protocols for
the COI barcoding marker refer to Lijtmaer et al. (2012).
For hierarchical outgroup rooting we used sequences of representative species of New World Emberizini(Passerellidae sensu Dickinson & Christidis, 2014;
Ammodramus humeralis, Junco hyemalis, Passerculus sandwichensis, Zonotrichia albicollis and Z. leuco­
phrys), five species of longspurs and snow buntings(Ca­lcarius and Plectrophenax; Plectrophenacidae sensu
Dickinson & Christidis, 2014) plus a few distantly related taxa (Dendroica virens, Cardinalis cardinalis,
Stur­nella superciliaris, Fringilla coelebs) all of which
had also been used for outgroup rooting by Alström
et al. (2008). The final cytb alignment of 657 bp included 195 sequences. Sequences were aligned with
MEGA v5 (Tamura et al., 2011) and manually corrected after visual inspection. For comparison with ourown COI barcode sequences of target species we usedadditional sequence data from GenBank (mainly from
Kerr et al., 2009 and Saitoh et al., 2015) in order tocontrol for intraspecific mtDNA lineage divergencefound in our cytb data set. The multi-locus alignment for
all five markers was 3933 bp long (cytb: 1035 bp; 16S
rRNA: 838 bp; COI: 693 bp; fib7: 687 bp; myo: 680 bp;
Table 1).

Data analysis
We estimated the best-fitting substitution models for eachof the five molecular markers using MrModeltest v2 (Ny­lander, 2004). For both mitochondrial genes (COI andcytb) we estimated separate best-fit models for each codon position separately. Separate models were estimatedfor the full cytb data set (195 sequences) and for the reduced data set used for multi-locus reconstruction (66sequences). According to the Akaike Information Criterion (AIC), the best fitting model was GTR+I+Γ for all
mitochondrial genes, regardless of which cytb data set
was being used, and HKY+Γ was the best fitting modelfor the two nuclear introns (for model settings refer toTable 1).
We reconstructed a phylogeny based on our full cytb
data set using Maximum Likelihood (ML) with RAxML
v. 7.2.6 (Stamatakis, 2006; using the GUI python appli­
cation v. 0.93 by Silvestro & Michalak, 2010) and Bay­esian Inference with MrBayes v3.1.2 (Ronquist & Huel­senb­ eck, 2003). We performed two independent runs: In
the first run we treated the entire alignment as a single
partition, in the second run we analyzed each codon position as a separate partition. In the latter run we allowed
the overall rate to vary between partitions by setting thepriors < ratepr = variable > and model parameters suchas gamma shape, proportion of invariable sites and therate matrix were unlinked across partitions. BayesianVERTEBRATE ZOOLOGY — 65  (1)  2014

Table 1. Substitution models applied to the different partitions of single-locus and multi-locus alignments.
Cyt-b
bp

657

1035

single loc

multi loccodon1codon 2

codon 3COI

16S rRNA

fib7

myo

693

838

673

680
HKY + Γcodon1codon 2

codon 3

GTR + I + Γ

GTR + I + Γ

SYM + I + Γ

HKY + IGTR + I + Γ

GTR + I + Γ

GTR + ΓHKY

GTR + I + Γ

GTR + I + Γ

HKY + ΓπA

0.29880.31051.0

0.19230.35880.32160.23330.15760.42410.36440.3316

0.2775πC

0.41470.40981.0

0.24850.50870.35080.27310.27330.38600.26490.16880.2199πT

0.1096

0.09961.0

0.15630.03230.1256

0.31310.14470.05210.16900.17980.2424πG

0.17690.18011.0

0.40280.10010.20190.18050.42430.13770.20170.31990.26010.6851α0.91831.08690.43890.7411

1.7636

1.17350.1009—1.90380.55771.0725I

0.50740.54320.441100.03270.6115000.0316

0.651600

Ti/Tv ratio

—

—

—

1.589

—

—

—

4.0182——1.4204

1.502—

R(a)[A-C]

0.52210.5506

0.8964—0.5464

1.43170.1045—0.13375.3297—R(b)[A-G]

5.8351

6.3386

4.1498—52.9134

22.4627

2.5731—

10.489019.0417——R(c)[A-T]

0.5966

0.5876

0.5764—2.93391.97282.7748—0.04351

3.9780——R(d)[C-G]

0.19730.14330.3419—2.07790.15030.0001—0.27780.5238——R(e)[C-T]

4.92805.62302.6819—23.2161

16.7736

26.4879—

4.572659.6389——

analysis was performed using the Metropolis-coupled
Markov Chain Monte Carlo algorithm with two parallelruns, each with one cold and three heated chains. The
heating parameter l was set to 0.1. The chains ran for
10,000,000 generations, trees were sampled every 100th
generation. The first 3,000 samples were discarded asburnin and the model parameters and the posterior probabilities were estimated from the remaining samples.
The remaining trees were summarized in a 50% majority
rule consensus tree.
One sequence of E. lathami (from Alström et al.,2008) did not clade with three further sequences of thatsame species and the corresponding GenBank entry wascommented as ‘cytochrome-b-like’; due to this conflictamong clades we used a sequence set for E. lathami fromPrice et al. (2014) for reconstructions of the multi-locus
phylogeny.
For illustration of intraspecific mitochondrial lineage
separation we reconstructed haplotype networks for selected taxa (taxon pairs) with TCS v1.21 (Clement et al.,2000).
We reconstructed a multi-locus phylogeny based on
all five markers with BEAST v.1.8.1 (Drummond & Ram­baut, 2007). We ran BEAST for 30,000,000 generations(trees sampled every 1000 generations) under the uncorrelated lognormal clock model for all loci with the “auto-optimize” option activated and a birth-death process
prior (with incomplete sampling assumed) applied to thetree. As for the single-locus analysis, we performed two
different runs: i) 5 partitions: by gene only and 5 best-fit
substitution models applied to each partition; ii) 9 partitions: by gene and additionally by codon position for thetwo coding mtDNA markers with the best-fitting-model
settings applied to each codon position (for model settings compare Table 1).
In order to determine the best-fitting partitioning regime for our data we compared four partitioning schemes
using AIC (McGuire et al., 2007) and AICM (Baele etal., 2012). ML likelihood values were obtained with
RAxML v8.1.7 using 100 replicates of the new rapid hillclimbing algorithm under the GTR+ Γ model. AIC values
based on the likelihood of the best tree for each partitioning strategy were calculated with Microsoft Excel. AICM
values were obtained with Tracer v1.6 based on BEAST
v1.8.1 runs with 11 million generations and trees being
sampled at every 1000th state, the first 1000 trees werediscarded as burn-in. Substitution models applied to eachpartition were identical to the ones used in final analyses.
A GTR+I+ Γ model was applied to each partition of theone and two-partition schemes. The results of AIC and
AICM both give the same ranking of the four strategies
and show overwhelming support for the nine-partition
scheme over all other schemes tested (Table 2).
In the absence of reliable passerine fossils providing
appropriate node age constraints, molecular dating was
performed using an empirical substitution rate of 0.0105
substitutions per site per lineage per million years forcytb as evaluated by Weir & Schluter (2008). We applied this rate to the cytb partition and left the rates of allother loci to be estimated relative to the cytb rate. The log
files were examined with Tracer v1.4.8 (Drummond &Rambaut, 2007) in order to ensure effective sample sizes(ESS; which yielded reasonable values for all parameters
after 30,000,000 generations). Trees were summarizedwith TreeAnnotator v1.4.8 (posterior probability limit =0.5) using a burn-in value of 9,000 (trees) and the medianheight annotated to each node.
Node support in a ML framework was obtainedby 1,000 bootstrap replicates with RAxML (thoroughbootstrap option). In two separate runs, we partitioned
the concatenated matrix (5 partitions by gene; 9 partitions by gene and codon see above) and applied the
GTR + I + Γ model across partitions. We assigned multiple outgroups and treated only Old World buntings (Em­berizidae) and New World sparrows (Passerellidae) as
ingroups.
139

Päckert, M. et al.: Phylogenetic relationships of Emberiza koslowi

Table 2. AIC and AICM values for each partitioning strategy including partitioning of the two coding mitochondrial genes cytochrome-b(cyt-b) and ND2 by Codon position. K: number of parameters; w: Akaike weight.
DescriptionlnL(best tree)

K

AIC

DAIC

w

AICM

9 partitions; each locus, each codon position of cyt-b and ND2– 28334,36

1242

59152,72

0157535,90DAICM
02 partitions; nuclear loci and mitochondrial genes– 29706,08

276

59964,16

811,44059410,32

1874,42

1 partition; unpartitioned– 30132,77

13860541,53

1388,82059875,95

2340,05

5 partitions; one partition for each locus– 29616,12690

60612,25

1459,53059917,29

2381,38

Results
Topology of the Emberiza tree
The multi-locus phylogeny of Emberiza buntings isshown in Fig. 3. Tree topologies inferred from the two
independent runs with different partition schemes were
widely congruent and major clades were equally well
supported with one apparent exception (Fig. 3): The
5-partition scheme (by gene) reflected a monophyletic
African clade (with the exception of E. affinis) while the9-partition scheme (by gene and codon) yielded two nonmonophyletic African clades plus E. affinis as a separate
lineage with unresolved relationships of all three clades(for details see below).
A monophyletic clade of Old World buntings (Emberiza) received full Bayesian support in all reconstructions
and was sister to an equally well supported clade of New
World sparrows (with good support for this sister grouprelationship; Fig. 3). The Old World buntings were split
into four major clades that according to our moleculardating date back to a mid Miocene separation (12.3 – 16.6
Ma; Table 3, node Emberiza). However, the phylogenetic
relationships among these four clades (sister clades I+II
and III+IV) were poorly supported in all reconstructions.
A strongly supported clade I mostly comprised Western
Palearctic species and several endemics from the highalpine elevations of the Tibetan Plateau. The Tibetanbunting, E. koslowi, turned out as an early offshoot ofthat clade and the split age from the crown group wasdated to 9.0 – 11.9 Ma (Table 3). The basal split of clade I
separated corn bunting, E. calandra, as the oldest lineage
split (10.0 – 13.4 Ma; Table 3) from all other members ofthat clade. All nodes of clade I received strong to full
Bayesian support except for the relationships among two
clades of E. godlewskii and its closest relatives E. cia andE. cioides (see intraspecific patterns, below). Generally,
partitioning by gene and codon position yielded slightly
older age estimates for the deeper splits, however, estimates for younger nodes (e.g. sister species) were thesame for both partitioning schemes (Table 3).
The Western Palearctic-Tibetan Clade I was sister toa rather heterogeneous clade containing only four species(Fig. 3). Eastern Mediterranean E. melanocephala, Central Asian E. bruniceps and East Asian E. lathami formeda terminal monophyletic group with full support (Fig. 3,
clade II). Surprisingly, the only African species not to be
140

included in the African clade, E. affinis, was recovered assister to clade II, however without support from Bayesianposterior probabilities (Fig. 3). In the maximum likelihood analysis this species was sister to clade III, albeit
with equally weak bootstrap support (27%).
Clade III included several Eastern Palearctic species
with breeding ranges extending into northern and central
China as well as all bunting species endemic to Japan. A
basal split separated the sister species pair E. siemsseni
and E. elegans (clade IIIa) from a second clade (both subclades received strong support; Fig. 3). The crown clade(IIIb, Fig. 3) was comprised of two Japanese endemics(E. sulphurata and E. variabilis) and several Eastern
Palearctic species (four of them including populations
from Japan). Six of those species occur exclusively in the
Eastern and Central Palearctic with three of them reaching into northeastern Scandinavia (E. aureola, E. pusilla,
E. rustica; the western range limits of E. pallasi reach NE
European Russia) and only the reed bunting, E. schoeniclus occupies an extensive range in the Western Palearctic.
Finally, clade IV contained all African species except for E. affinis. A basal split separated E. cabanisi, E.
flaviventris and E. poliopleura (Fig. 3; clade IVa) froma terminal clade including some Middle East endemics
such as E. striolata and E. socotrana (Fig. 3; clade IVb).
Both subclades received full Bayesian support, however
their sister group relationship was only poorly supported
when the data set was partitioned only by gene but not bycodon position. When mtDNA markers were additionallypartitioned by codon positions the two subclades of theAfrican clade were not sister to each other and their relationships to the other three main Emberiza clades were
only poorly resolved (not shown).
Intra- and interspecific lineage separation

The tree based on the full cyt-b data set is shown in Fig. 4.
The same four major Emberiza clades as in the multi-locus analysis were recovered, however, with weaker support for main clades and relationships among the clades
were entirely unresolved. Strikingly, no remarkable EastWest Palearctic split could be detected in any bunting
species. No subclades coinciding with geographic distribution were recovered across trans-Palearctic breeding
ranges of focal species and the corresponding haplotype
networks did not show any phylogeographic structure ei-

VERTEBRATE ZOOLOGY — 65  (1)  2014

Table 3. Split ages estimates for selected nodes of the Old World bunting tree inferred from two independent runs with BEAST (means[95% highest posterior density intervals] in Ma); the African clade IV was not recovered as a monophyletic unit when the alignment waspartitioned by gene and codon (–); * = excluding E. affinis.
5 partitions by gene only

9 partitions by gene and codon positions

Emberiza

Node

Regional split

12.3 [10.5 – 14.1]

16.6 [14.8 – 18.6]

Clade I

10.0 [8.5 – 11.6]

13.4 [11.8 – 15.1]

Clade II*

6.4 [5.0 – 8.1]

8.0 [6.2 – 10.0]

Clade III

9.5 [8.0 – 10.9]

12.0 [10.5 – 13.5]

Clade IV

10.2 [8.5 – 11.9]—

E. koslowi

9.0 [7.7 – 10.5]

11.9 [10.5 – 13.5]

E. s. personata / E. s. spodocephala

Japan vs. cont. E Asia

1.6 [1.2 – 2.1]

1.7 [1.2 – 2.2]

E. variabilis / E. tristrami

Japan vs. cont. E Asia

2.5 [1.9 – 3.2]

2.8 [2.0 – 3.6]

E. godlewskii South / E. cia

S China vs. W Palearctic

2.2 [1.7 – 2.7]

2.5 [2.0 – 3.1]

E. godlewskii North / E. cioides

N China vs. W Palearctic

2.4 [1.9 – 2.9]

2.6 [2.0 – 3.2]

E. e. elegantula / E. e. elegans

C China vs. E Palearctic

1.0 [0.7 – 1.3]

1.0 [0.7 – 1.4]

E. c. flemingorum / E. c. cia

Himalaya vs. N Palearctic

0.5 [0.3 – 0.8]

0.5 [0.3 – 0.8]

ther (see E. rustica as an example; Fig. 4D; also E. schoeniclus, network not shown). Haplotypes of two Himalayan taxa turned out to be distinct from their northwesternor northeastern counterparts: E. fucata arcuata and E. cia
flemingorum (Fig. 4). Though the branching pattern in
E. cia received no support from the partitioned cytb dataset, E. c. cia and E. c. flemingorum were fully supported
sister taxa in the multi-locus analysis (and in the cytb tree
based on the unpartitioned data set) and were dated toa rather young, late Pleistocene origin (Fig. 3; Table 3).
Comparison of barcode sequences separated an Asianclade of E. cia (subspecies E. c. flemingorum and E. c.
par) from Western Palearctic E. c. cia (Fig. 5B).
A remarkable degree of intraspecific differentiation
was found in another three East Asian species. Far East
Russian E. elegans elegans were separated from Chinese
E. e. elegantula from Shaanxi and Sichuan by sevensubstitutions in the haplotype network (Fig. 4F). Likefor the Himalayan subspecies mentioned above the split
between Russian and Chinese E. elegans was dated to
the late Pleistocene (Fig. 3; Table 3). In contrast, Russian
E. spodocephala spodocephala were not notably distinct
from Chinese E. s. sordida, however one haplotype of theJapanese subspecies E. s. personata was separated fromall continental haplotypes by eleven substitutions (Fig.
4E). Comparison of COI barcode sequences confirmed
the strict separation of haplotypes from Japan and Sakhalin from continental haplotypes in Far Eastern Russia,
Mongolia and China (Fig. 5A). Unlike split ages in theprevious examples, the latter split within E. spodocephala was dated to the Pliocene-Pleistocene boundary androughly coincides with the slightly older split between
Far East Russian E. tristrami and Japanese E. variabilis(Fig. 3; Table 3).
The most striking and unexpected intraspecific diversification pattern was found in E. godlewskii. Southwestern Chinese haplotypes from Yunnan, Sichuan and
Qinghai were separated from northern and northeastern
haplotypes from Gansu, Ningxia and Hebei by a mini-

mum of 30 substitutions (Fig. 4C). The two clades of E.
godlewskii did not form a monophyletic clade neither insingle-locus nor in multi-locus reconstructions (Figs 3,4). In the latter, a sister group relationship of southern E.
godlewskii with E. cia received full support, while a sister group relationship of northern E. godlewskii with E.
cioides received moderate support (Fig. 3). The striking
differentiation between the two E. godlewskii clades was
also supported by COI barcodes: Along with our samples
from Chinese provinces Gansu and Ningxia the northerngodlewskii clade included further samples from adjacent
Mongolia and Russia (Fig. 5B).
In contrast to the findings outlined above, no mtDNA
lineage sorting could be neither found among E. hortulana and E. caesia nor among E. citrinella and E. leucocephalos (Fig. 4A, B).

Discussion
Genus-level systematics
Our five-gene tree topology generally confirmed the four
major clades of Old World Emberiza buntings alreadyfound by Alström et al. (2008). Likewise, our topology
is well in accordance with the recently revised genuslevel systematics of Old World buntings by Dickinson& Christidis (2014). They restricted the genus Emberiza
to those species contained in our clade I (including Tibetan E. koslowi), and our clade III fully reflects theirgenus Schoeniclus. The terminal sister species of ourclade II were separated as genus Granativora from themonotypic genus Melophus by Dickinson & Christidis(2014). This separation is compatible with our topology and seems well justified with respect to the strong
morphological distinctiveness of Melophus lathami as
the only Old World bunting having a prominent crest in141

Päckert, M. et al.: Phylogenetic relationships of Emberiza koslowi

0.64
29

1.0
68

*
1.0
82

0.91
49

0.80
43

*
0.99
67

*

1.0
98

*

Clade I 1.0
87 0.99
80

0.99
46

*

1.0
83
1.0
85

0.64
-

0.58
-

*

Clade II
Clade IIIa

1.0
65

*

*

1.0
88

1.0
97

1.0
95

0.96
-

0.99
65

0.88
Clade IV 39

*
*

*
1.0
99

0.89
52
0.88
37

*
1.0
87
Clade IIIb

0.94
63

1.0
99

1.0
93

0.54
-

*

*

1.0
98
0.83
50

Clade IVa

1.0
71

Clade IVb

1.0
97 0.88

*
0.76

*

0.52
45

10.0

7,5

* 45

1.0
98

0.25
-

12.5

1.0
99

*

0.99
77

70

15.0

*

1.0
78

*

0.81
-

Clade III

1.0
58

5.0

2.5

Dendroica virens
Cardinalis cardinalis
Calcarius lapponicus
Calcarius ornatus
Calcarius pictus
Plectrophenax mccownii
Plectrophenax nivalis
Ammodramus humeralis
Junco hyemalis
Zonotrichia albicollis
Zonotrichia leucophrys
Passerculus sandwichensis
Emberiza calandra
Emberiza fucata
Emberiza koslowi
Emberiza jankowskii
Emberiza cia cia
Emberiza cia flemingorum
Emberiza godlewskii South
Emberiza godlewskii North
Emberiza cioides
Emberiza stewarti
Emberiza citrinella
Emberiza leucocephalos
Emberiza cirlus
Emberiza buchanani
Emberiza cineracea
Emberiza caesia
Emberiza hortulana
Emberiza affinis
Emberiza lathami
Emberiza bruniceps
Emberiza melanocephala
Emberiza elegans elegantula
Emberiza elegans elegans
Emberiza siemsseni
Emberiza tristrami
Emberiza variabilis
Emberiza chrysophrys
Emberiza rutila
Emberiza sulphurata
Emberiza spodocephala personata
Emberiza spodocephala sordida
Emberiza spodocephala spodocephala
Emberiza rustica rustica
Emberiza rustica latifascia
Emberiza pusilla
Emberiza aureola
Emberiza pallasi
Emberiza schoeniclus
Emberiza yessoensis
Emberiza poliopleura
Emberiza flaviventris
Emberiza cabanisi
Emberiza capensis
Emberiza impetuani sloggetti
Emberiza impetuani impetuani
Emberiza socotrana
Emberiza tahapisi tahapisi
Emberiza tahapisi insularis
Emberiza tahapisi arabica
Emberiza goslingi
Emberiza sahari
Emberiza striolata

0.0 MaFig. 3. Dated multi-locus phylogeny of Old World buntings (Emberiza) including outgroup taxa from New World sparrows, longspurs andallies and others; BEAST 1.8.1 analysis, 5 partitions by gene only; 30,000,000 generations, burn-in = 9,000, birth-death prior (incomplete
sampling), mean heights; node support from posterior probabilities above nodes, node support from RAxML bootstrap (1000 replicates)
below nodes; * = full support from Bayesian posterior probabilities (1.0) and thorough likelihood bootstrap (100); comparison with tree
topology based on 9 partitions (by gene and codon): arrow upwards = increased node support from partition by gene and codon (> 0.95
Bayesian posterior probabilities), arrow downwards = strongly decreased node support from partition by gene and codon or node absent(different branching pattern); outgroups Fringilla coelebs and Sturnella superciliaris omitted.

both males and females. The only striking discordancewith the generic classification by Dickinson & Chris­tidis (2014) is the highly unexpected position of E. affinis that did not cluster with the other African buntings(Fringillaria sensu Dickinson & Christidis, 2014; ourclade IV) but was sister to the clade uniting Melophus
and Granativora sensu Dickinson & Christidis (2014) inthe Bayesian multi-locus tree. However, this placementof E. affinis is highly doubtful because it received poor
support in all analyses as it was based on evidence fromtwo mitochondrial markers only. In fact, close affinities
of E. affinis to other African species seem likely with respect to zoogeography but particularly with respect tostrong similarities in external morphology with E. flaviventris, E. cabanisi and E. poliopleura (our clade IVa).
142

These four African species were unified in a separate
subgenus by some authors (Cosmospina Wolters, 1972;
cf. Koblik, 2007) and likewise separated from other African species of subgenus Fringillaria. Within our clade
IVa we were able to confirm the supposed sister species
relationship between previously unstudied E. poliopleura and E. flaviventris (Rose, 2011). Notably, the twomain African clades (IVa and IVb) were not recovered assister clades in all multi-locus reconstructions, because
their position was highly dependent of sequence data
partition. Thus the phylogenetic relationships among
the subclades of African Fringillaria sensu Dickinson &Christidis (2014) remain subject of further multilocus or
genomic studies.

VERTEBRATE ZOOLOGY — 65  (1)  2014

0.92
-

*
*

1.0
94

arcuata

0.94
21

koslowi

1.0
99

1.0
97

clade Icalandra
fucata* buchanani
B 1.0

1.0
62

hortulana
caesia*cineracea*

cirluscia
flemingorum

*
1.0
98

1.0
90

*

0.94
53

C
*

affiniscioidesjankowskii
godlewskii South*melanocephala* lathami
lathami #*aureolarutilapusillarustica 1

D*rustica 2
spodocephalasordida

E*

0.94
59

* chyrsophrys*
tristrami
1.0 variabilis
99
sulphurata
0.96
elegansF * 83** yessoensis

0.95
61

clade IVaclade IVb
1.0
87

0.90
51

1.0
93

W

E
11

F
7

pallasischoeniclus 1* schoeniclus 2cabanisi
flaviventris
poliopleura*
capensis*
impetuanisahari* sahari # striolata
1.0tahapisi
0.99 0.92 82
1.0
96

86

66

socotrana

1.0
90

siemsseni

0.99
100

0.99
99

0.92
36

E

elegantula

*

0.94
58

0.93
27

D

personata1.0
92 0.99
84

0.93
57

clade III30

bruniceps*

Emberiza

Cgodlewskii North

*

1.0
93

B

* stewarti
0.95
80

clade IIA

citrinella
leucocephalos

99

0.99
84

A*

insularisarabica* goslingi

New World outgropus

0.2

Fig. 4. Single-locus phylogeny of Old World buntings (Emberiza) based on the mitochondrial cytochrome-b gene, Bayesian tree (no codon
partition applied); node support from posterior probabilities above nodes; node support from RAxML analysis below nodes; * = full support from Bayesian posterior probabilities (1.0) and thorough likelihood bootstrap (100), posterior probabilities < 0.9 not shown; haplotype
networks for selected species (pairs) are shown in boxes A – F at the right side of corresponding clades; D) two clades of E. rustica did notrefect a separation of East (grey) an d West (white) Palearctic samples; # = two sequences from Alström et al., (2008) that did not cladewith other sequences from the same species (for E. lathami, see material); non-Emberizidae outgroups omitted.

143

Päckert, M. et al.: Phylogenetic relationships of Emberiza koslowi

Inter- and intraspecific differentiationof buntings
The few studies dedicated to phylogeographic structure
within bunting species found remarkably low differentiation across the wide trans-Palearctic breeding rangeof the reed bunting, E. schoeniclus. Even though two
mtDNA lineages could be identified these reflected only
a shallow split and did not coincide with geographic
distribution (Zink et al., 2008) – similar to the lack ofwithin-clade differentiation of E. rustica (our study). In
all cases where significant genetic differentiation among
reed bunting populations could be confirmed, this wasreflected by distribution patterns of haplotype and allele
frequencies, e.g. on the Iberian Peninsula (Kvist et al.,2011). Evidently, the use of mitochondrial genes seems
to be problematic for the assessment of intraspecific phylogeographic structure and species identification in buntings. Moreover, in some cases discrimination between
sister species is not possible with mtDNA markers. The
best-known example is the case of the yellowhammer
and the pine bunting (E. citrinella, E. leucocephalos) thatshare a common mitochondrial gene pool but are strongly
distinguished by nuclear markers even in regions of local
sympatry (Irwin et al., 2009; for a lack of a barcode gap
compare Kerr et al., 2009). According to their close phylogenetic relationship, territorial songs of these two species were mutually understood in playback experiments(Tietze et al., 2012). A similar lack of genetic distinctiveness among species was documented for the subarctic
snow bunting (Plectophenax nivalis) and its parapatricsister species McKay’s bunting (P. hyperboreus; Maley& Winker, 2010) and could also be shown for E. hortulana and E. caesia in this study (although confirmationis required through sampling of further specimens andnuclear markers).
In contrast to these cases of low mtDNA divergenceamong species, there are examples of striking subspecificgenetic differentiation in buntings. Genetic distinctiveness of E. spodocephala populations from Japan and
Sakhalin (subspecies personata) as compared to mainland conspecifics was previously inferred from RADP
analysis (Dolgova & Valchuck, 2008) from and COI
barcoding analyses (Kerr et al., 2009; Saitoh et al.,2015). This differentiation pattern was also reflected bya relatively old split age in our multi-locus phylogeny. In
fact, E. s. personata also exhibits strong distinctivenessof external morphology against continental populations
where phenotypical variation follows a cline between
two extreme forms ‘oligoxantha Meise 1932’ in the West(southern central Siberia; Kuznetsk region) and ‘extremiorientis Sulphin 1928’ in the East (southern Ussuriland;
both taxa were synonymized with the nominate spodocephala; compare Vaurie, 1956, 1959). Despite slightdifferences in plumage coloration the Chinese subspecies
E. s. sordida does not seem to be genetically differentiated from nominate spodocephala at a considerable degree. Even though we have to rely on a very limited databasis, our findings on the intraspecific differentiation of144

E. spodocephala are in accordance with phylogeographic and morphometric analyses (Weissensteiner, 2013).
In contrast to E. spodocephala we found that the rather
subtle morphological differentiation among Russian E.
elegans elegans and Chinese E. elegans elegantula wasparalleled by a shallow genetic lineage split.
The unexpected deep split among the two polyphyletic clades of E. godlewskii must be discussed with respect to a controversy concerning the interpretation ofgeographical variation in external morphology and thus
on species boundaries between E. cia and E. godlewskii.
Based on his examination of whole skins Vaurie (1956)
assigned the latter to two different subspecies groups ofthe same species E. cia, thereby following the suggestion by Hartert (1928). Portenko (1960) and Voous(1962) later adopted that classification. In contrast, Mau­
ersberger (1972) separated E. godlewskii at the species
level from E. cia mainly based on variation in body size,feather proportion and plumage coloration. He stated thatvariation is clinal within each of the two taxa but at thesame time there is no evidence of clines or intermediate populations among vicariant populations of the two
species in Asia. Strikingly, clinal morphological variation of E. godlewskii does not reflect strong character
discontinuities, as one would expect from the deep split
in our phylogeny. The southernmost forms yunnanensis and khamensis differ from all other subspecies inhaving a less pronounced grayish neck-ring (Mauers­
berger, 1972), yunnanensis is rather short-winged withmean wing-lengths of 81 mm [75 – 86 mm] in males and77.5 mm [73 – 82 mm] in females (Vaurie, 1956). The
short wing length distinguishes yunnanensis from theneighboring subspecies omissa in the North, which has
slightly longer wings (Vaurie, 1956: measurements formales between 78.5 and 87 mm). Within the North-Southcline along the eastern Tibetan Plateau margin, the subspecies khamensis represents an intermediate form thatintergrades with the paler nominate godlewskii in the
North and with the more colourful reddish-brown yunnanensis in the South (Vaurie, 1956). Similar to the latter
form, its Himalayan counterpart E. cia flemingorum wasshown to be the most short-winged subspecies of E. cia(Martens, 1972). However, in that case similarity ofwing proportions may not necessarily indicate common
ancestry as suggested in our phylogeny but may rather
be due to the fact that migration of E. c. flemingorum isprobably limited to seasonal elevational movements likein many other Himalayan passerines. The morphological distinctiveness of the Nepalese subspecies flemingorum against its western counterparts stracheyi and par(Martens, 1972) is reflected by the clear split among one
haplotype of that form from all western E. cia included
in our molecular tree (but compare the separation of aWestern Palearctic cluster from a Central Asian/Himalayan cluster in the COI barcode analysis). However, allthese results have to be substantiated by more extensive
sampling and further integrative research including molecular, morphological and bioacoustic markers before
any taxonomic consequences should be drawn.

M A R 1383_s pod Russia, Amur Obl.
M A R 1383_s pod Russia, Amur Obl.

B
E. jankowskii

E. jankowskii

Fig. 5. Intraspecific genetic variation of Asian buntings based on a 613 bp alignment of COI barcodes; Bayesian inference of phylogeny with MrBayes; A) black-faced bunting, E. spodocephala; B) Godlewski’s
bunting, E. godlewskii; note that the E. cioides clade includes two GenBank sequences from samples of false species determination (EU847682 listed as E. cia, EU847683 as E. godlewskii).

G Q 481748_par Kazakhstan
G Q 481748_par Kazakhstan
G Q 481747_par Kazakhstan
G Q 481747_par Kazakhstan
E U 847686_s pod China
E U 847686_s pod China
F J465325_par IranF J465325_par IranE. cia 0.88
E. cia 0.88Nepal
G Q 481833_s pod Russia, K. Kray
G Q 481833_s pod Russia, K. Kray
F J465325_flem
F J465325_flem Nepal
G Q 481832_s pod Russia, Prim.GKray
Q 481832_s pod Russia, Prim. Kray
M A R 2586_c ia Germany
M A R 2586_c ia Germany
M0.9
A R 4003_c ia Morocco
M A R 4003_c ia Morocco
0.9 Kray
M A R 4853_s pod Russia, Prim. M
A R 4853_s pod Russia, Prim.
Kray
M A R 4952_yun China, Sichuan M A R 4952_yun China, Sichuan
M A R 4951_yun China, Sichuan M A R 4951_yun China, Sichuan
G Q 481830_s pod Mongolia, Dornod
G Q 481830_s pod Mongolia, Dornod
M A R 8156_ssp.
E. godlewskii SouthE. godlewskii
South China,
1.0
1.0Qinghai M A R 8156_ssp. China, Qinghai
G Q 481837_s pod Russia, Mag.GObl.
Q 481837_s pod Russia, Mag. Obl.
M A R 8528_yun China, Yunnan M A R 8528_yun China, Yunnan
G Q 481836_s pod Russia, Irk. Obl.
G Q 481836_s pod Russia, Irk. Obl.
M A R 4953_yun China, Sichuan M A R 4953_yun China, Sichuan
E U 847725_god China
E U 847725_god China
E U 847685_s pod China
E U 847685_s pod China
E U 847723_god Russia Irkutskaya
E UObl
847723_god Russia Irkutskaya Obl
G Q 481835_s pod Russia, Rep.GYakutia
Q 481835_s pod Russia, Rep. Yakutia
G Q 481765_god China
G Q 481765_god China
JN 698679_s pod China
JN 698679_s pod China 0.89
M A R 8580_omi
China, Hebei
M A R 8580_omi China, Hebei
0.89
JQ 174773_god Mongolia, SouthJQ
Gobi
174773_god Mongolia, South Gobi
E U 847684_s pod China
E U 847684_s pod China
M
A
R
6908_god
China,
Ningxia
M A R 6908_god
China, Ningxia
E. godlewskii
E. godlewskii
M A R 337_s pod Russia, Rep. Buryatia
M A R 337_s pod Russia, Rep. Buryatia
G Q 481766_god Mongolia, SouthG Gobi
Q 481766_god Mongolia, South Gobi
E. spodocephalaE. spodocephala
0.81
North
North
G Q 481838_s0.81
pod Russia, Irk. Obl.
G Q 481838_s pod Russia, Irk. Obl.
M A R 9314_god China, Gansu M A R 9314_god China, Gansu
A R 4962_s pod China, GansuM A R 4962_s pod China, Gansu
M A R 5770_god China, Gansu M A R 5770_god China, Gansu
continental E Asian clade
continental EMAsianclade
M A R 8577_omi China, Hebei
M A R 8577_omi China, Hebei
G Q 481831_s pod Russia, Khabarovsky
G Q 481831_s
Kray pod Russia, Khabarovsky Kray
JQ 174772_god Mongolia , SouthJQ
Gobi
174772_god Mongolia , South Gobi
JF 499132_s pod Japan, Chugoku
JF 499132_s pod Japan, Chugoku
M A R 5771_god China, Gansu M A R 5771_god China, Gansu
A B 843488_s pod Japan, Hokkaido
A B 843488_s pod Japan, Hokkaido
JF 499127_c io Japan, Chugoku JF 499127_c io Japan, Chugoku
A B 842742_s pod Japan, Hokkaido
A B 842742_s pod Japan, Hokkaido
JF 499126_c io Japan, Chugoku JF 499126_c io Japan, Chugoku
Japan, Hokkaido
A B 842736_s pod Japan, Hokkaido
A B 842736_s pod 0.4
M A R 655_c io captive
0.4 M A R 655_c io captive
G Q 481834_s pod Russia, Sakhalin
G Q 481834_s pod Russia, Sakhalin
JF 499125_c io Japan, Chugoku JF 499125_c io Japan, Chugoku
G Q 481752_c io Russia, Tyva G Q 481752_c io Russia, Tyva
A B 842737_s pod Japan, Hokkaido
A B 842737_s pod Japan, Hokkaido
G Q 481751_c io Mongolia, Dornod
G Q 481751_c io Mongolia, Dornod
A B 843490_s pod Japan, Honshu
A B 843490_s pod Japan, Honshu
E U 847693_c io China
E U 847693_c io China
A B 842741_s pod Japan, Hokkaido
A B 842741_s pod Japan, Hokkaido
JF 499124_c io Japan, Chugoku JF 499124_c io Japan, Chugoku
A B 843478_c io Japan, Shikoku A B 843478_c io Japan, Shikoku
G Q 481828_s pod Russia, Sakhalin
G Q 481828_s pod Russia, Sakhalin
G Q 481749_c io Russia Primorsky
GQ
Kray
481749_c io Russia Primorsky Kray
A B 842743_s pod Japan, TokyoA B 842743_s pod Japan, TokyoA B 843480_c io Japan, HokkaidoA B 843480_c io Japan, Hokkaido
G Q 481753_c io Kazakhstan
G Q 481753_c io Kazakhstan
A B 842739_s pod Japan, Hokkaido
A B 842739_s pod Japan, Hokkaido
E U 847683_god #E U 847683_god #G Q 481826_s pod Russia, Sakhalin
G Q 481826_s pod Russia, Sakhalin
A B 843479_c io Japan, Shikoku A B 843479_c io Japan, Shikoku
A B 843489_s pod Japan, Hokkaido
A B 843489_s pod Japan, Hokkaido
A B 842732_c io Japan, Chubu A B 842732_c io Japan, Chubu
1.0
E. cioides 1.0
E. cioides
A B 843477_c
io Japan, Kyushu A B 843477_c io Japan, Kyushu
A B 842740_s pod Japan, Hokkaido
A B 842740_s pod Japan, Hokkaido
E. spodocephala
0.77 E. spodocephala
0.77
A B 842731_c io Japan, Chubu A B 842731_c io Japan, Chubu
A B 842738_s pod JapanA B 842738_s pod JapanA B 842733_c io Japan, HokkaidoA B 842733_c io Japan, Hokkaido
G Q 481829_s pod Russia, Sakhalin
G Q 481829_s pod Russia, Sakhalinisland cladeisland clade
0.02
0.02 io Mongolia, Tov Aymag
G Q 481750_c
G Q 481750_c io Mongolia, Tov Aymag
E U 847682_c ia #E U 847682_c ia #G Q 481825_s pod Russia, Sakhalin
G Q 481825_s pod Russia, Sakhalin
E U 847692_c io China
E U 847692_c io China
0.02
0.02 G Q 481827_s pod Russia, Sakhalin
G Q 481827_s pod Russia, Sakhalin

A

VERTEBRATE ZOOLOGY — 65  (1)  2014145

Päckert, M. et al.: Phylogenetic relationships of Emberiza koslowi

Biogeographic history of Tibetan Plateau
species
The high elevations of the central Qinghai-Tibet Plateau
have been repeatedly considered a “cradle of evolution”
harbouring rather old relic genetic lineages of cold-adapted mammals (Deng et al., 2011; Tseng et al., 2013; Wanget al., 2014) and of passerine birds (Weigold, 2005; Leiet al., 2014; Päckert et al., 2015). According to our dated
multi-locus phylogeny the Tibetan bunting, E. koslowi
represents one of those rather ancient endemic speciesof the Tibetan Plateau. This species was already highlighted by Vaurie (1972) in his list of Tibetan endemics
and Weigold (2005) listed this species as one of his ‘first
degree endemics’ and a character species for the treelessgrasslands of the plateau region. Our split age estimatessuggest a mid Miocene separation of the Tibetan bunting
from its closest relatives and a likewise early colonization of the alpine plateau habitats by this species. This isin accordance with recent evidence that vast parts of theplateau region had already reached present-day altitudes
in the Miocene (Mulch & Chamberlain, 2006; Favre etal., 2014). The hypothesis of a rather ancient Miocene
colonization of alpine Tibetan Plateau habitats by ancestors of extant passerine species received recent support
from studies of other plateau endemics that have comparably isolated phylogenetic positions and were dated
back to similarly ancient split ages, such as the Tibetanrosefinch, Carpodacus roborowskii (Zuccon et al., 2012;
Tietze et al., 2013) and the Tibetan ground tit, Pseudopodoces humilis (Johansson et al., 2013; Qu et al., 2013).
There is recent evidence from a genomic study that during
long lasting separation the latter species strongly adapted
to ground-dwelling life at high-elevation environments
due to an effect of positive selection on genes related tohypoxia response and skeletal development and expansions in genes involved in energy metabolism (Qu et al.,2013). Whether similar genetic adaptations will be foundin other regional endemics, such as E. koslowi remains
the subject of forthcoming genomic studies.
In the Eastern Palearctic lineage splits among somesister-taxon pairs roughly coincide with the PliocenePleistocene boundary at 2.58 Ma (according to Cohen etal., 2013). However, our dated phylogeny hints to several
successive phases of faunal interchange among the Japanese Archipelago and the Far East Asian mainland withthe oldest colonization of southern Japan by ancestors of
E. sulphurata in the early Pliocene followed by three parallel events of faunal interchange at the Pliocene-Pleistocene boundary (for E. spodocephala compare Saitoh etal., 2015; Weissensteiner, 2013). A lack of considerable
differentiation among Japanese and continental mtDNA
haplotypes of E. cioides and E. yessoensis suggest two
further very recent events of interchange due to Holocene
range expansion from either an insular or a continental
glacial refuge (compare Saitoh et al., 2015). Generally,
these examples of Pleistocene lineage divergence between populations from Japan and Sakhalin against theirclosest relatives from the mainland are paralleled for ex146

ample in Phylloscopus leaf warblers (P. borealis group:
Alström et al., 2011; Saitoh et al., 2010; species pair P.
tenellipes vs. P. borealoides: Martens, 1988; Päckert etal., 2012).
Late Pleistocene lineage diversification among morphologically distinct subspecies along the eastern marginof the Qinghai-Tibet Plateau is evident in E. elegans but
not in E. spodocephala occurring in the Eastern Palearctic and China respectively. In contrast, the most striking phylogeographic pattern along the eastern plateau
margin is the deep north-south divide in E. godlewskii.
Even more unexpectedly this deep divergence does notseparate allopatric populations from central/ northern
China and southern Siberia (that are separated by a large
distribution gap) but bisects a continuous Chinese distribution range (see maps in Mauersberger & Portenko,
1971; Weigold, 2005; Rose, 2011). Signatures of Quarternary climate oscillations in the demographic history
and thus the extant phylogeographic structure of alpine
endemic species from the Tibetan Plateau are not uncommon and were previously documented for a number of
species (Qu et al., 2009, 2010; Yang et al., 2009; Leiet al., 2014). However, none of these previously investigated examples involved a deep north-south divide comparable to our finding in E. godlewskii. Strikingly, rangesof southern E. godlewskii (north of the Himalayan main
range) and E. cia (south of it) come close to each otheron the southern macroslope in the Central Himalayas.
Even though adjacent populations of their circum-Tibetan ranges are separated by distribution gaps in several
places, Mauersberger (1972) already drew a parallel tothe few classical examples of ‘circular overlap’. Previously, Mauersberger & Portenko (1971) already castinto doubt whether the assumed distributional gap insouthern Tibet was real or due to a lack of field records
from this region. In the Himalayas their easternmost record of E. godlewskii originated from Kharta in the eastern Mt Everest area (north of the main range). Martens(1972) commented on these easternmost Everest populations that the species had never been recorded West of
this location or anywhere throughout Nepal south of theHimalayan main range (not even as a vagrant or migrant)
and postulated an ecological component of the observed
distribution limits. Throughout its Tibetan range E. godlewskii seems to be restricted to the drier open habitats
at high elevations and strictly avoids the monsoon-humid southern flanks of the Himalayas (Martens, 1972).
This ecological segregation is paralleled on the easternplateau margins where Schäfer (1938) found three different forms of E. godlewskii co-occurring in elevational
parapatry in the surroundings of Batang. Generally, E. c.
yunnanensis rather occupies subtropical valleys at lower
elevations while E. c. khamensis occurs at the high alpine
and subalpine shrubs up to 4000 m (with omissa at intermediate elevations in areas of local parapatry where dense
forests separate the habitats of the lowland and the alpine
subspecies; Schäfer, 1938; Mauersberger & Portenko,1971). This kind of elevational segregation among alpine
and lowland breeding habitats of northern and southern

VERTEBRATE ZOOLOGY — 65  (1)  2014

E. godlewskii is in fact the only convincing hypothesisthat might explain the deep lineage separation among E.
g. yunnanensis and its northern conspecifics. Elevational
parapatry and ecological segregation of closely related
congeners is a common phenomenon in forest-dwelling
passerines of the southern and southeastern flanks of
the Tibetan Plateau (Johansson et al., 2007; Martens
et al., 2011; Päckert et al., 2012; Price, 2010; Price etal., 2014). In contrast, a similar phenomenon has rarely
been documented for alpine species of the high elevation
plateau habitats. Ecology and elevational distribution has
been assumed to play a role in lineage separation and speciation processes in the beautiful rosefinch and its allies(C. pulcherrimus, C. waltoni; Tietze et al., 2013; Päckert
et al., 2015), however, like in the case of E. godlewskii theknowledge on local habitat preferences and the breeding
biology of the populations involved is scarce and geographically more extensive sampling is required in orderto evaluate range-wide phylogeographic patterns.

Acknowledgements
These results were based on the rich material and field observations
from a multitude of nearly annual expeditions of JM to various partsof Asia from the year 1969 until today. This study received substantial funding from Deutsche Forschungsgemeinschaft, PA 1818/3-1
and from Staatsministerium für Wissenschaft und Kunst (SMWK)State of Saxony (AZ-4-7531.50-02-621-08/1). During the years JM
received several grants from Deutsche Ornithologen-Gesellschaft(DO-G), Gesellschaft für Tropenornithologie (GTO) and from
Feldbausch-Stiftung and Wagner-Stiftung both at Fachbereich Biologie, Johannes Gutenberg-Universität Mainz, Germany. Y-HS
received research grants from the National Natural Science Foundation of China, project No. 31272286. DTT was funded by theDeutsche Forschungsgemeinschaft (Ti 679/1-1, Ti 679/2-1). We
would like to thank the following colleagues who provided samples
for genetic analysis: M. Adams & R. Prŷs-Jones, Bird Collectionsof the Natural History Museum at Tring, UK; B. Haberl, Mainz;
J. Fjeldså, Natural History Museum of Copenhagen, Denmark; S.
Frahnert, Museum für Naturkunde Berlin, Germany; H. Grimm& M. Fischer, Naturkundemuseum Erfurt, Germany; P. Kessler,
Mainz; J.O. Kriegs, LWL Museum für Naturkunde, Münster, Germany; P. Lymberakis, Natural History Museum of Crete, Irakleio,
Greece; N. Rice, Bird Collection at the Academy of Natural Sciences at Drexel University, Philadelphia, USA; R. van den Elzen and
T. Töpfer, Forschungsmuseum Alexander Koenig, Bonn, Germany.
A part of the 657-bp cytochrome-b data set was generated and analysed by C. Waßmann within the scope of her diploma thesis at
Johannes Gutenberg University of Mainz, Germany. We cordiallythank A. Rauh for steady assistance and support during lab analysesof toe pad samples. We would furthermore like to thank D. Lu for
helping with the lab work in China and for providing sequences fortwo markers from one Tibetan bunting sample. We are particularly
grateful to Miss L. Li for establishing contact with a local Tibetan
community in south-eastern Qinghai, especially to Kenpo Tashi
Zangpo-Ju. We are very thankful for his hospitality and for giving

us guided tours in the habitats of the Tibetan bunting. We would
like to dedicate this work to to Kenpo Tashi Zangpo and his passionate scholarly efforts for the conservation of endemic wildlife in
the region of Nyanpo Yutse.

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