ISSN 0869-4362
Русский орнитологический журнал 2007, Том 16, Экспресс-выпуск 356: 567-575

First insights into a DNA sequence based
phylogeny of the Eurasian Jay
Garrulus glandarius
A.Akimova 1) , E.Haring 2) , S.Kryukov 1) , A.Kryukov 1)
1) Institute of Biology and Soil Science,

Far East Branch of the Russian Academy of Sciences, Vladivostok, Russia
2) Museum of Natural History, Vienna, Austria

Received 23 April 2007

The Eurasian jay Garrulus glandarius Linnaeus, 1758 is a widespread
Palearctic species which includes 33-35 more or less differentiated subspecies. These subspecies, combined into eight groups (Stresemann 1940;
Vaurie 1959) were later classified in five morphologically and geographically defined groups (Goodwin 1986). Besides G. glandarius, two monotypic species belong to the genus: Lidth’s jay Garrulus lidthi Bonaparte,1850 (restricted to some southern Japanese Islands) and Black-headed jay
Garrulus lanceolatus Vigors, 1831 (Himalayas).
Up to now, molecular phylogenetic studies including jays were published mainly for inter-species and inter-genus comparisons. Cibois andPasquet (1999) investigated the phylogenetic relationships of 11 genera ofCorvidae using sequence of the mitochondrial (mt) cytochrome b gene(cytb) as a molecular marker. In that analysis a close relationship of G.
glandarius and the Siberian jay Perisoreus infaustus was rejected, a result which was confirmed later by Ericson et al. (2005) in an analysis
based on one mt and two nuclear genes. These authors revealed a closer
relationship of jays with several genera of the Old World corvids than to
the monophyletic group of New World jays.
Besides those studies, no phylogenetic analyses were performed concerning interspecific relationships within the genus Garrulus or the intraspecific variation within G. glandarius. Therefore, the aim of the study
was to provide insights into the phylogeography of jays and to assess intraspecific genetic variation and phylogeographic patterns. We analyzed
sections of two mt sequences with different substitution rates: the cytb
gene and the control region (CR). The latter one is in general consideredas the faster evolving sequence and therefore was supposed to be especially useful for intraspecific studies (e.g. Kryukov et al. 2004). On the territory of Russia and adjacent countries, five subspecies are recognized(Stepanyan 2003): G. glandarius glandarius (Linnaeus, 1758), G. glandarius brandtii Eversmann, 1842; G. glandarius iphigenia Sushkin etРус. орнитол. журн. 2007. Том 16. Экспресс-выпуск № 356

567

Ptuschenko, 1914; G. glandarius krynicki Kaleniczenko, 1839; G. glandarius hyrcanus Blanford, 1873. Besides samples of these subspeices weincluded also G. glandarius japonicus Temminck et Schlegel, 1847 fromJapan as well as G. lidthi. We wanted to find out whether the individuals
cluster according to subspecies assignment (i.e., geographic origin) as wellas the level of their genetic differentiation.
Material
The sample set included 26 specimens (Table 1) belonging to five subspecies
covering a huge range from Western Europe to Japan: G. g. glandarius (distributed in Europe), G. g. brandtii (Asia), G. g. krynicki (Caucasus and Turkey),
G. g. iphigenia (Crimea), G. g. japonicus (Japan), and one hybrid between G. g.
glandarius and G. g. krynicki (taken from South Russia), and also G. lidthi.
Samples of livers and muscles fixed in 96% ethanol and kept in -4°C were used.
As an outgroup, we used the magpie Pica pica (sequence determined in a previous study: Kryukov et al. 2004) and the chough Pyrrhocorax pyrrhocorax (thisstudy). Accession numbers of all sequences determined in this study as well asof published sequences are given in Table 1.
Methods
DNA-extraction was performed with the phenol-chloroform deproteinization
method (Maniatis et al. 1982). The two marker sequences were analyzed in two
different laboratories (IBSS, Vladivostok and NHMW, Vienna). A partial regionof cytb (length of PCR product: 586 bp) was amplified at the IBSS employing
several published primers used previously for corvid birds (L14827, H16065,
Helm-Bychowski, Cracraft 1993; L14990, Kocher et al. 1989; H15916, Edwards
et al. 1991; and SNL4 (L15196), Kryukov, Suzuki 2000). All these primers initiated amplification, but the most successful combination (yielding one clear
fragment on the gel) proved to be L15916 (ATGAAGGGATGTTCTACTGGTTG)/ H16065 (GGAGTCTTCAGTCTCTGGTTTACAAGAC). Polymerase chain reaction was carried out in a «Biometra» Thermoсycler (USA) in 20 µl of reaction
containing 2 µl of 10x buffer, 0.125 mM MgCl2, 0.1 mM of each dNTP, 1 pmol ofeach primer, 60 ng of template DNA and 1 unit of Taq-DNA-polymerase. PCR
was performed under the following conditions: 5 min of pre-denaturation at
94°С, 35 cycles of denaturation for 1 min at 95°С; primer annealing for 2 min at
55°С; elongation for 2 min at 72°С, and finally an elongation step for 7 min at
72°С before cooling to 4°С. The amplification products were analyzed by electrophoresis in 1.5% agarose gels before sequencing.
Automated sequencing was perfomed with an ABI Prism 310 (Applied Biosystems). Cycle sequencing of purified PCR products was performed with the
BigDye Terminator kit (Applied Biosystems) and the primers SNL4 and Н15916
at a final concentration of 1 pmol/µl. Conditions of cycle sequencing: 25 cycles ofdenaturation for 30 sec at 96°С, annealing for 10 sec at 55°С, and elongation for
4 min at 60°С, and finally cooling to 4°C. A partial section of the CR was amplified at the NHMW with the primers CR-Cor+ (ACCCTTCAAGTGCGTAGCAG)
and Phe-Cor- (TTGACATCTTCAGTGTCATGC) as described previously (Kryukov

568

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et al. 2004). PCR products (length: ~ 680 bp) were extracted from 1% Agarosegels using the Quiaquick Gel Extraction Kit (Qiagen) and cloned using the
TOPO TA cloning Kit (Invitrogen). Sequening of both strands was performed by
MWG Biotech (Germany) using M13 universal primers.
Table 1. Specimens and sequences from GenBank used in the study.
LabcodeGeographic origin

Source /collection number

Markersequences

Accession
numbers

Gglagla1
Gglagla2
Gglagla3
Gglagla4
Gglagla5
Gglagla6
Gglagla7
Gglagla8
Gglagla9
Gglagla10
Gglagla11
Gglagla12
Gglagla13
Gglagla14
Gglagla15
Gglagla16

Garrulus glandarius glandarius
Russia, Kirov Region
V.Sotnikov / 174
cytb
Russia, Kirov Region
V.Sotnikov / 227
cytb
Russia, Kirov Region
V.Sotnikov / 228
cytb
Russia, Kirov Region
V.Sotnikov / 233
cytb
Russia, Kirov Region
V.Sotnikov / 235
cytb
Russia, Kirov Region
V.Sotnikov / 239
cytb, CR
Russia, Kirov Region
V.Sotnikov / 243
cytb
Russia, Kirov Region
V.Sotnikov / 226
CR
Russia, Smolensk Region
Ya.Red’kin / 126
cytb
Russia, Tula Region
Ya.Red’kin / 128
CR
Russia, Moscow Region
V.Korbut / 175
cytb, CR
Russia, Moscow Region
M.Konovalova / 518
cytbFranceE.Pasquet / 381
cytb, CR
Austria, Upper Austria
A.GamaufCR
Austria, Upper Austria
A.Gamauf
CR
GenBank
Cibois, Pasquet 1999 cytb

EF602118
EF602119
EF602120
EF602121
EF602122
EF602123, EF602136
EF602124
EF602137
EF602125
EF602138
EF602126, EF602139
EF602127
EF602128, EF602140
EF602141
EF602142
U86034

Gglabra1
Gglabra2
Gglabra3
Gglabra4
Gglabra5

Garrulus glandarius brandtii
Russia, Primorsky Region
A.Kryukov / 354
CR
Russia, Primorsky Region
A.Tsvetkov / 347
cytb
Russia, Primorsky Region
Ya.Red’kin / 346
cytb, CR
Russia, Amurskaya Region N.Kolobaev / 239
cytb, CR
Russia, Primorsky Region
V.Sotnikov / 351
cytb

EF602146
EF602131
EF602132, EF602147
EF602133, EF602148
EF602134

Gglaiph1
Gglaiph2

Garrulus glandarius iphigenia
Russia, Crimea pen.
Ya.Red’kin / 747
CR
Russia, Crimea pen.
V.Arhipov / 748
CR

EF602144
EF602145

Gglakry2

Garrulus glandarius krynicki
Russia, Kislovodsk
Ya.Red’kin / 125
cytb, CR

EF602130, EF602149

Garrulus glandarius krynicki × G. glandarius glandarius
Gggxk1
Russia, Rostov Region
G.Bahtadse / 375
cytb, CR
EF602129, EF602143
Gglajap1

Garrulus glandarius japonicus
Japan, Honshu
W.Neunercytb, CR

Glid1
Glid2

Japan, Riukiu Isl.
GenBankPpicjan5

Pica pica jankowskii
Russia, Primorsky Region
A.Kryukov / 714
cytb, CR

Gpyrbra1
Gpyrbra2

Pyrrhocorax pyrrhocorax brachypus
Russia, Tuva Rep.
Ya.Red’kin / 104
CR
EF602152
Russia, Tuva Rep.
A.Tsvetkov / 133
CR
EF602153

Garrulus lidthi
E.Pasquet / 383
Cibois, Pasquet 1999

Рус. орнитол. журн. 2007. Том 16. Экспресс-выпуск № 356

CR
cytb

EF602135, EF602150
EF602151
U86035
AY701183, AY701171

569

Phylogenetic analysis
Experimental sequences and those obtained from the GenBank werealigned with the software SeaView (Galtier et al. 1996). Phylogenetic trees (NJ,
MP and ML) were calculated with PAUP, version 4.0b10 (Swofford 2002). NJ
trees were calculated with p-distances. MP trees were calculated by a heuristic
search with a random taxon addition sequence (1000 replicates) and the TBR(tree bisection reconnection) branch swapping algorithm and delayed charactertransformation (DELTRAN). Gaps were treated as missing character. ML trees
were calculated by a heuristic search with a NJ starting tree and TBR branchswapping using the GTR+Γ model. For all tree calculating algorithms (NJ, MP
and ML) a bootstrap analysis was performed with 1000 (NJ, MP) or 100 (ML)repeats, respectively. Pairwise P-distances and Kimura 2-parameter distanceswere calculated with the program DNASA (Kryukov et al., in press). Average
distances were calculated by hand from the tables obtained from DNASA.

Results and Discussion
The partial cytb sequence was isolated from 18 individuals comprising
the four subspecies: G. g. glandarius, G. g. brandtii, G. g. krynicki, and G.
g. japonicus. The partial sequence of the CR was determined from 16 Garrulus individuals. Besides the four subspecies mentioned above, the CR
data set includes also two samples of G. g. iphigenia. Moreover, two Garrulus sequences from GenBank were included: Gglagla16 and Glid2 (cytb).
Pica pica and Pyrrhocorax pyrrhocorax were used as outgroup: Ppicjan5(ctyb, CR; sequences from Kryukov et al. 2004) and Ppyrbra1, 2 (CR; thisstudy). Lengths of the alignments were 586 bp for cytb (about half of thetotal gene, 1143 bp) and 677 bp for the CR.
Within the cytb sequences no insertions or deletions were found. Nucleotide frequencies for the cyt b gene were: А = 0.2942, T = 0.2272, G =0.1120, C = 0.3666. The G-criterion was calculated as 1.0922 which means
equal nucleotide dispersion within the section studied. As expected, synonymous transitions at the third codon positions were found as the mostcommon substitution type, which is in agreement with published data formany animals. Among birds, up to 78% of informative substitutions arelocated in the third codon position (Helm-Bychowski, Cracraft 1993).
The neighbor-joining tree based on the cytb sequences is shown in
Fig. 1. The MP and ML trees calculated from this data set have in general
the same topology with the four subspecies of G. glandarius separated infour clusters (bootstrap values of all analyses are shown in Fig. 1 as
NJ/MP/ML). Among them, the most basal split separates G. g. japonicus
from the mainland taxa. G. g. brandtii splits off from the next node. Finally, a clade comprising the 12 individuals of G. g. glandarius is the sister group of the G. g. krynicki clade which includes also a hybrid between
G. g. glandarius and G. g. krynicki. The hybrid origin of this phenotypi-570

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cally intermediate bird was approved by Ya.Red’kin (pers. comm.). Withinsubspecies, haplotype variation is rather low which is especially interesting for the nominate race the samples of which originated from quite distant European regions, i.e. Kirov (East European Russia), Austria andFrance.
Gglagla3
Gglagla13
73,64,59

Gglagla5
Gglagla1
Gglagla6
Gglagla16

100,96,85

Gglagla7
Gglagla2
Gglagla4
Gglagla12

70,64,-

Gglagla11
Gglagla9
Gggxk1

100,100,90

Gglakry2

100,79,66

100,100,83

100,100,99

Gglabra4
Gglabra3
Gglabra5
Gglabra2

Gglajap1
Glid2
Ppicjan5
0.5%
Fig. 1. NJ tree based on cyt b sequences of Garrulus glandarius, G. lidthi and P. pica (outgroup).
Bootstrap values >50% are depicted at the nodes (NJ, MP, ML). Labcodes of specimens correspond to those in Table 1.

The NJ tree calculated on the basis of the CR data set is very similar
to the cytb tree (Fig. 2). It includes also G. g. iphigenia which is part of theG. g. glandarius clade. The order of splits is the same as in the cytb tree.
The fact that the Japanese subspecies splits from the basal node inboth trees is in accordance with the hypothesis about the first appearanceof corvids in South East Asia from Australia (Sibley, Ahlquist 1985). In
this context the origin of the genus Garrulus itself may have been locatedin South East Asia.
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571

Gggxk1

98,91,84

Gglakry2
Gglagla10

96,62,82

Gglagla14
Gglagla15

88,60,80

Gglagla8
Gglaiph1

97,90,89

Gglaiph2
Gglagla13
Gglagla6

100,100,100

Gglabra1

70,65,62
100,100,93
94,86,98

Gglabra4
Gglabra3
Gglajap1

Glid1
100,100,100

Ppyrbra1
Ppyrbra2

Ppicjan5
1%
Fig. 2. NJ tree based on CR sequences of Garrulus glandarius, G. lidthi and the outgroup species
Pica pica and Pyrrhocorax pyrrhocorax. Bootstrap values >50% are depicted at the nodes(NJ, MP, ML). Labcodes of specimens correspond to those in Table 1.

For both data sets we calculated average K2P distances between thefour clades: G. g. glandarius including G. g. iphigenia (its distance to G. g.
glandarius of 0.3% in the CR is neglectable), G. g. krynicki, G. g. brandtii,
and G. g. japonicus (Tables 2 and 3). For comparison the p-distances arealso shown. The differentiation of G. g. japonicus and G. g. brandtii isquite pronounced in both data sets. Average K2P distances between G. g.
japonicus and the mainland subspecies range from 5.3-6.1 (cytb) and 4.44.9 (CR), for G. g. brandtii the respective values are 2-2.7 (cytb) and 2.62.8 (CR). This differentiation corresponds well with the distinct geographic distribution of these two subspecies in the Russian Far East andJapan respectively. In contrast, the differentiation between G. g. krynicki
and G. g. glandarius is lower (1% in cytb and 2% in CR).
The average K2P distance between G. lidthi and G. glandarius is12.3-14.9 in cytb, which is in the same range as the divergence of the outgroup genus Pica (14-16.6%). This underlines (1) that the mt lineages of
the two species have split long ago and (2) that the cytb gene clearly hasreached saturation for this level of divergence. This obviously is not thecase in the CR data set as becomes apparent when comparing the dis-572

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tances between G. glandarius, G. lidthi, and the outgroup taxa (Table 3).
For example, the distances between Pica pica vs. ingroup are twice ashigh as those found for G. glandarius vs. G. lidthi.
Table 2. Average genetic distances between the four clades of Garrulus glandarius,
G. lidthi and the outgroup Pica pica, calculated from cytochrome b sequences.
Kimura 2-parameter distances are above diagonal, p-distances below diagonal.
All codon positions considered
Gglagla
Gglagla
Gglakry
GglabraGglajap
Glid
Ppic

Gglakry

GglabraGglajap

Glid

Ppic

1.0

2.72
2.04

5.34
5.01
6.08

14.24
14.32
14.90
12.34

15.28
15.54
16.58
14.88
13.99

0.99
2.66
5.09
12.57
13.62

2.0
4.78
12.63
13.82

5.75
13.11
14.64

11.09
13.31

12.63

Table 3. Average genetic distances between the four clades of Garrulus glandarius,
G. lidthi and the outgroup Pica pica and Pyrrhocorax pyrrhocorax,
calculated from Control Region sequences.
Kimura 2-parameter distances are above diagonal, p-distances below diagonal
Gglagla
Gglagla
Gglakry
GglabraGglajap
Glid
Ppyrbra
Ppic

1.99
2.72
4.51
16.34
25.0
28.53

Gglakry

GglabraGglajap

Glid

Ppyrbra

Ppic

2.03

2.79
2.62

4.69
4.86
4.36

18.53
18.06
18.18
18.65

30.48
30.69
29.26
28.67
30.22

35.97
36.37
36.40
35.77
33.64
31.31

2.56
4.66
15.97
25.14
28.77

4.19
16.07
24.21
28.8

16.42
23.82
28.4

24.85
27.07

25.59

Which general conclusions can we draw from our data? There is a deep
interspecific divergence of the two Palearctic jays, G. glandarius and G.
lidthi, which is in the range of differentiation among many bird genera(Moore, DeFilippis 1997). Within G. glandarius there is a subspecific differentiation at the genetic (mt) level with the exception of G. g. iphigenia.
The differentiation of G. g. japonicus and G. g. brandtii could be easily explained with a longer lasting (or repeated) isolation of these subspecies
during the Pleistocene. Nevertheless, the differentiation is not so pronounced as found for the west-east divergence in other corvid taxa (e.g., P.
pica, Corvus corone, C. frugilegus – Haring et al. submitted). Concerning
the western subspecies one could interpret the trees at first sight in thefollowing way: G. g. krynicki and G. g. glandarius are differentiated andthe hybrid bird (Gggxk) has originated from a cross between a female G.
g. krynicki and a male G. g. glandarius. Nevertheless, the sample size is
too low to draw such conclusions, especially concerning the differentiation
between the G. g. glandarius and G. g. krynicki clades, which appear very
closely related. It also has to be taken into consideration that some memРус. орнитол. журн. 2007. Том 16. Экспресс-выпуск № 356

573

bers of both clades come from geographic localities that are rather closecompared to the rest of the European samples: Crimea (Gglaiph1, 2) and
Rostov Region (Gggxk) in South Eastern Europe and Kislovodsk (Gglakry)
in the North Caucasus region, respectively. Without analyzing much bigger samples of the subspecies one cannot rule out the possibility that thedifferentiation of G. g. glandarius and G. g. krynicki on the basis of mt sequences is just a sampling artifact.
The present data can be regarded as a first survey of the genetic diversity within G. glandarius. Our future aim is to analyze a broader sample
covering the whole Palearctic which probably will be possible only with
the inclusion of museum specimens into the study. This will enable us toelucidate the phylogeographic history of this species in more detail and tocompare it to genetic patterns found in other widespread corvid birds.
Acknowledgements
Authors are grateful for Ya.Red’kin, V.Sotnikov, G.Bahtadse, V.Arhipov, A.Tsvetkov, N.Kolobaev, M Konovalova, V.Korbut for providing us with the samples from Zoological Museum of the Moscow University. We thank also A.Gamauf (NHM Vienna,
Austria), E.Pasquet (MNHN Paris) and W.Neuner (TLF, Innsbruck, Austria) for providing samples. The study was supported by the Program of RAS «Dynamics of genpools».

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