Turkish Journal of Zoology
Turk J Zool
(2016) 40: 749-757
© TÜBİTAK
doi:10.3906/zoo-1510-22
http://journals.tubitak.gov.tr/zoology/
Short Communication
CHD genes: a reliable marker for bird populations and phylogenetic analysis?
Case study of the superfamily Sylvioidea (Aves: Passeriformes)
1,2
1
1
1
1,
Mitică CIORPAC , Radu Constantin DRUICĂ , Gogu GHIORGHIȚĂ , Dumitru COJOCARU , Dragoș Lucian GORGAN *
1
Department of Biology, Faculty of Biology, “Alexandru Ioan Cuza” University of Iasi, Iasi, Romania
2
Interdisciplinary Research Department - Field Science, “Alexandru Ioan Cuza” University of Iasi, Iasi, Romania
Received: 07.10.2015
Accepted/Published Online: 09.04.2016
Final Version: 24.10.2016
Abstract: The chromo-helicase-DNA binding protein (CHD) genes are widely used markers for sex determination in birds and provide
a rapid and low-cost method with applicability to a large number of taxa. A good phylogenetic marker displays highly conserved
domains and a slow evolution rate, proprieties that CHD genes seem to have. This gives rise to the following question: is the CHD gene a
reliable marker for phylogenetic and bird population analysis? The aim of this study is to investigate whether the CHD gene is a reliable
tool for molecular phylogeny and for population analysis within the superfamily Sylvioidea. Our results reveal that CHD genes are good
markers for these two analyses, even better than myoglobin.
Key words: CHD genes, cytochrome b, myoglobin, Sylvioidea
Sex identification in birds based on their external
morphology is difficult (Griffiths et al., 1998), as they are
mostly sexually monomorphic (Ong and Vellayan, 2008).
With the discovery of the chromo-helicase-DNA binding
protein (CHD) gene (Griffiths and Tlwarl, 1995) in the
avian sex chromosome (Ellegren, 1996), molecular DNA
noninvasive sexing methods, such as the analysis of feathers, became possible and generated applicability to wildlife
DNA forensics (An et al., 2007). The avian CHD1 genes
belong to a family composed of a chromatin organization
modifier (An et al., 2007) domain, an SNF2-related helicase/ATPase domain, and a DNA binding domain; thus,
the acronym CHD stands for these (Fridolfsson and Ellegren, 1999). CHD genes have an important role in the
avian genome, as in other organisms, due to their involvement in chromatin remodeling in the control of transcription elongation (Simic et al., 2003). This gene has two introns with different lengths for the Z and W chromosome,
allowing the discrimination between the amplicons of the
Z and W chromosomes by gel electrophoresis (Dubiec
and Zagalska-Neubauer, 2006). Being a functional part of
the DNA and with a very slow evolution rate, the CHD
gene is highly conserved, even among distant species. The
alignment between the CHD-W sequence in birds and the
CHD sequence in mice does not include any gaps, except
for two 130- and 175-bp deletions (Vucicevic et al., 2013).
Its high degree of conservation across species has led to
*Correspondence: [email protected]
the design of universal primers for birds’ sex determination (Griffiths et al., 1998; Kahn et al., 1998; Fridolfsson
and Ellegren, 1999).
Genetic distances are a frequently used tool to identify
and assess the species status of closely related taxa (Wesson
et al., 1993; Hung et al., 1999; Burbrink et al., 2000; Bradley
and Baker, 2001; Cagnon et al., 2004; Parkin et al., 2004;
Olsson et al., 2005; Newman et al., 2012). This way, genetic
distances are frequently compared between different studies, even if different genetic markers are involved (Helbig
et al., 1995; Baker et al., 2003; Johnson and Cicero, 2004;
Zhang et al., 2007). Fast-evolving parts, like mitochondrial
sequences (Heidrich et al., 1998) or nuclear introns, are
suitable for resolving young evolutionary relationships, for
example those between species, whereas older relationships are better analyzed with more conservative genes like
nuclear exons (Lin and Danforth, 2004). Mostly, a combination of the two is used to target different parts of a
phylogenetic tree.
For the superfamily Sylvioidea, in addition to mitochondrial genes such as cytochrome b, control region,
or ND2 (NADH-ubiquinone oxidoreductase chain 2),
a number of nuclear genes are used for molecular phylogeny, genes like fibrinogen beta chain intron 5 (FGB),
glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
lactate-dehydrogenase B intron 3 (LDHB), ornithine-decarboxylase exon 6–8, intron 7 (ODC1), recombination
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CIORPAC et al. / Turk J Zool
activation gene 1 (RAG1), and the widely used myoglobin
intron 2 (myo) (Fregin et al., 2009). Considering this, the
aim of this study is to investigate whether the CHD gene
is a reliable tool for molecular phylogeny and population
analysis of the superfamily Sylvioidea.
Blood samples from three species of the genus Acrocephalus, A. schoenobaenus, A. arundinaceus, and A.
scirpaceus, were collected from five different bird ringing
camps in Larga Jijia (Iasi County, Romania) during 2010
and stored in Queen’s Lysis Buffer (Seutin et al., 1991). The
genomic DNA was isolated and purified using the DNA
IQ System (Promega, Madison, WI, USA), and quantified through spectrometry and electrophoresis. The CHD
genes were amplified using PCR methods with one pair
of specific primers: P2 5’-TCT GCA TCG CTA AAT CCT
TT-3’ (Griffiths and Tlwarl, 1995) and P8 5’-CTC CCA
AGG ATG AGR AAY TG-3’ (Griffiths et al., 1998). The
PCR reaction was carried out in 25 µL of total volume
containing GoTaq Green Master Mix (Promega), primers (0.2 µM final concentration), DNA template (~12.5
µg), and nuclease-free water up to the final volume. The
amplification was conducted in a SensoQuest Labcycler
(SensoQuest GmbH, Göttingen, Germany) under the following cycling conditions: 1.5 min of initial denaturation
at 95 °C, followed by 30 cycles of denaturation at 95 °C for
30 s, primer alignment at 48 °C for 45 s, and elongation at
72 °C for 45 s, followed by a final elongation step at 72 °C
for 5 min, in accordance with Griffiths et al. (1998). A 3%
agarose gel was run for the PCR products and visualized
in UV light. The distinction between males and females
was made by the presence of one band for males and two
bands for females.
A total of 21 sequences belonging to 7 taxa, with 6 from
the superfamily Sylvioidea (Table 1), were used to assess
the reliability of the CHD-Z gene in phylogenetic analysis. The cytochrome b (cytb [1012 bp; 337 variable sites, of
which 176 were informative]), myoglobin intron 2 (myo
[656 bp; 207 variable sites without gaps, of which 176 were
informative]), and chromo-helicase-DNA-binding protein
gene (CHD-Z exon and intron [326 bp; 62 variable sites
without gaps, of which just 13 were informative]) sequences were aligned and concatenated using MEGA 6 (Tamura
et al., 2013), defining 3 data sets for the same taxa: 1) cytb
sequences; 2) cytb and CHD-Z sequences concatenated; 3)
cytb and myo sequences concatenated.
The corrected HKY (Hasegawa et al., 1985) distance
between taxa for each gene was obtained using PAUP
v4.0b10 (http://paup.sc.fsu.edu/). The optimal substitution model was selected according to the Akaike information criterion (Akaike, 1974) using three substitution
schemes (+F; +I; +G, 4 nCat) and 8 different topologies
in jModelTest v2.1 (Guindon and Gascuel, 2003; Darriba
et al., 2012). The base composition heterogeneity and chisquare (χ2) test of deviation from base composition homogeneity of variable sites were calculated in PAUP v4.0b10
(http://paup.sc.fsu.edu/). Furthermore, the relative composition variability (RCV) was computed as a summary
statistic for each marker, following the method of Phillips
and Penny (2003).
The maximum likelihood (Baker et al., 2003) tree was
designed in SeaView (Gouy et al., 2010) using PhyML
(Guindon et al., 2010) and was visualized in DensiTree
v2.1.11 (Bouckaert, 2010). Bayesian inference (BI) analysis was conducted in BEAST v1.8.0 [Bayesian Evolutionary
Table 1. Phylogenetic analysis data set.
Taxon
Family
Acrocephalus_sechellensis
GenBank Acc. No.
cytb
CHD-Z
myo
Acrocephalidae
AJ004284 1
AM180353 2
FJ883122 3
Phylloscopus_collybita
Phylloscopidae
HQ608821 4
AF355151 5
DQ125966 6
Phylloscopus_trochilus
Phylloscopidae
Z73492 7
AF355150 5
AY887719 16
Pycnonotus_sinensis
Pycnonotidae
FJ487714 17
EF582413 18
GQ242100 8
Pycnonotus_taivanus
Pycnonotidae
NC013483 9
EU204971 9
GQ242099 8
Parus_atricapillus
Paridae
AF347937 10
DQ068391 11
KF183723 12
Gallus_gallus
out-group
AP003319 13
AF006659 14
NM001167752 15
Heidrich et al. (1998); 2Dawson et al. (2005); 3Fregin et al. (2009); 4Lei et al. (2010); 5Bensch et al. (2006); 6Fuchs et al. (2006); 7Helbig
et al. (1995); 8Zuccon and Ericson (2010); 9Chang et al. (2010); 10Gill et al. (2005); 11Harvey et al. (2006); 12Johansson et al. (2013);
13
Nishibori et al. (2005); 14Griffiths et al. (1998); 15Boardman et al. (2002); 16Alstrom et al. (2006); 17Lohman et al. (2010); 18Chang et al.
(2008).
1
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Analysis Sampling Trees (Drummond et al., 2012)] using
the substitution model selected by the AIC for each gene
and a Relaxed Clock: Uncorrelated Log-normal (Drummond et al., 2006) molecular clock model with a tree prior
setup to the Speciation: Yule Process (Yule, 1925; Gernhard, 2008) model, applied for a UPGMA starting tree.
One independent Markov chain Monte Carlo simulation
of 10 million iterations for each data set was run through
CIPRES Science Gateway v.3.3 (Miller et al., 2010). Convergence for the posterior distributions of the parameter
estimates was checked using Tracer v1.5 (http://tree.bio.
ed.ac.uk/software/tracer/), and then the summary maximum clade credibility (MCC) tree was computed using TreeAnnotator. The MCC trees were visualized and
graphically edited in FigTree v1.4.0 (http://tree.bio.ed.ac.
uk/software/figtree/). Finally, the posterior marginal likelihoods, treeness index (Phillips and Penny, 2003), Colless
tree imbalance (Colless, 1982), and mean posterior probability for each tree were used for model comparisons and
identification of the best tree model fit.
The CHD gene is an easy and reliable tool in bird sex
determination, defining the differences even from the size
of the PCR products. This gene presents two alleles of different sizes; the allele from the W chromosome is bigger
than that of the Z chromosome, allowing for discrimination between genera. Sex determination using the CHD
gene was conducted for three species of Acrocephalus: A.
schoenobaenus, A. arundinaceus, and A. scirpaceus. For
A. schoenobaenus, a 166.66% sex ratio from a sample size
of 32 was identified, which suggests sex disequilibrium
within the population, as the male percentage is 62.50%.
This sex ratio trend was constant during the sampling
dates (Figure 1). A 42.85% sex ratio was observed for A.
arundinaceus in a sample size of 20 individuals, with 30%
males and a constant trend during the sampling campaign.
A similar situation was observed for A. scirpaceus, with a
higher sex disequilibrium and a sex ratio of 311.11% in a
sample size of 37 individuals with 75.68% males.
Taking into account the possibility of hybridization between A. arundinaceus and A. scirpaceus, identified across
Europe, the correlation degree between these two higher
sex disequilibria was tested based on a correlation test that
showed a strong positive level of correlation (0.81) for the
males and females of both species and a negative correlation between the males and females of different species.
In the world of birds, quite often both sexes have similar phenotypic traits, and even an experienced ornithologist may have difficulty in identifying an ambiguous individual (Dubiec and Zagalska-Neubauer, 2006). Other
characteristics, such as sexually monomorphic colors for
around 60% of all songbird species (Price and Birch, 1996)
and the morphological uniformity of nestlings, increase
the difficulty of taxonomical classification. Therefore, the
utility of the CHD-based technique in molecular sexing
of bird species is unquestionable, having been proven by
numerous studies. Furthermore, it is a fast and accurate
Figure 1. Sex distribution for A. arundinaceus, A. scirpaceus, and A. schoenobaenus, shown by sampling date.
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CIORPAC et al. / Turk J Zool
method, but at the same time easy to perform and relatively cheap. Furthermore, numerous bird species or populations are protected by different conservation programs
and the sampling process must be as noninvasive as possible. Previously, Ong and Vellayan (2008) showed the applicability of CHD-based molecular sexing as a completely
noninvasive method, using feathers as a source of DNA.
The analysis of the sequence divergences for 7 taxa
representing 4 families of the superfamily Sylvioidea, plus
an outgroup, Gallus gallus, indicates that the HKY distance for CHD-Z was between 0.000 and 0.290 (data not
shown). Divergences between cytochrome b (cytb) and
myoglobin, intron 2 (myo), were higher than those of the
CHD-Z gene. A graphical representation of the pairwise
corrected sequence divergences (HKY distance) for CHDZ, cytb, and myo is shown in Figure 2 and indicates a linear relation among these three genes. The cytb sequence
generally diverges much faster than CHD-Z (the ratio of
cytb/CHD-Z is 1.41); the myoglobin gene also diverges
faster than CHD-Z (the ratio of myo/CHD-Z is 1.15) for
the same set of taxa. Considering just the ingroup taxa
(the taxa from superfamily Sylvioidea), the myoglobin
and CHD-Z genes have the same percentage of divergence
(0.086%), much lower than cytb, which presents a value
twice as large (0.178).
Comparing the sequence divergence and the current
classification within the superfamily Sylvioidea, the values
are closely related for each taxonomic position. The species from the same genus revealed a divergence between
0.000 and 0.004 (genera Phylloscopus and Pycnonotus) for
the CHD-Z gene compared to 0.003–0.102 for the cytb
gene and 0.007–0.028 for the myo gene. CHD-Z interfam-
ily distances showed a minimum value for Acrocephalidae/Phylloscopidae (0.066) and a maximum value for Acrocephalidae/Paridae (0.086). Myoglobin showed closer
values: a maximum of 0.086 for the same families, but a
minimum between Pycnonotidae/Phylloscopidae (0.051).
This last value is rather unexpected given that these two
families are taxonomically located in different clades. Divergence between taxa based on cytb gene analysis shows
high values that are uniformly distributed across taxa and
comparable to those of the CHD-Z and myo genes.
The evolution time of the CHD-Z gene indicates a
higher substitution rate for transitions (2.536) and a lower
rate for transversions (0.511–1.000) (Table 2). The optimal
substitution model for CHD-Z gene evolution, selected by
the AIC algorithm in jModelTest (Guindon and Gascuel,
2003; Darriba et al., 2012), was TPM1uf + G (Kimura,
1981), with 1.304 shape for 4 gamma categories.
The chi-square test of homogeneity of base frequencies
across taxa was nearly significant (χ2 = 23.829, P = 0.068)
for CHD-Z marker across 39 variable sites within the ingroup. Meanwhile, the chi-square test was clearly statistically insignificant for the myo and cytb markers (P = 0.384
for myo and P = 0.929 for cytb), suggesting a more stable
base composition among taxa. The base composition heterogeneity (RCV) confirms the chi-square test of homogeneity, showing low levels of variability for the widely used
markers cytb (RCV = 0.018) and myo (RCV = 0.025) and a
higher value for the new proposed marker CHD-Z (RCV =
0.046). Phillips and Penny (2003) showed that nucleotide
homogeneity within the ingroup and countable outgroup
heterogeneity can equally affect the root position and the
tree topology. The high nucleotide homogeneity and the
Figure 2. HKY distance of cytochrome b vs. CHD-Z (left) and myoglobin vs. CHD-Z (right). The line represents the relationship of
overall sequence divergences between two segments and indicates that the cytb and myo sequences are diverging faster than the CHD-Z
gene sequence (cytb/CHD-Z = 1.41 and myo/CHD-Z = 1.15).
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CIORPAC et al. / Turk J Zool
Table 2. Optimal substitution model selected based on AIC algorithm in jModelTest for the analyzed genes.
Gene
AIC model selected
R(AC)
R(AG)
R(AT)
R(CG)
R(CT)
R(GT)
ti/tv
gamma
p-inv
CHD-Z
TPM1uf+G
1.000
2.536
0.511
0.511
2.536
1.000
-
1.304
-
cytb
TVM+G
3.887
10.131
1.761
0.459
10.131
1.000
-
0.244
-
myo
HKY
1.914
-
-
-
-
-
1.914
-
-
higher distance within individuals from four different
families, observed for the cytb and myo markers, could
explain the presence of uncertain relationships within the
families of Sylvioidea. In contrast, the CHD-Z marker
presents higher nucleotide heterogeneity, determined by
the intron region, and a much lower distance within individuals, mainly determined by the exon region.
Knowing the implications of molecular marker selection for phylogenetic analysis and the risk of obtaining different topologies using an improper marker, the topology
given by the CHD-Z gene was tested along with those of
the cytb and myo genes, the most common markers used
in bird phylogeny. The ML tree of a single nuclear gene reveals a wired topology for both analyses, CHD-Z and myo
(Figure 3), probably caused by an improper tree algorithm
(ML usually gives good topology when the analyzed sequence has a high number of nucleotides) or by a short
distance between all studied taxa. Therefore, the phylog-
eny of a single nuclear gene, in our case CHD-Z or myo, is
insufficient and unreliable.
The mitochondrial sequence is a reliable tool for phylogenetic relationships between supraspecific taxa, but usually it is not a good indicator of intra- or interspecific relationships. Over time, combined data sets and nuclear and
mitochondrial markers have proven their usefulness in phylogeny inference by increasing the resolution. For example,
Pons et al. (2011) drew the green woodpecker’s phylogeography using mitochondrial marker cytb and another four
nuclear markers (one of them being Z-linked), showing a
clear distinction between the Iberian and European subspecies of green woodpecker, soon confirmed by another study
by Perktas et al. (2011) that used a different mitochondrial
marker over an increased number of individuals. Therefore,
combined mitochondrial and nuclear data sets are mandatory in order to eliminate any taxonomic uncertainties.
Combining the cytochrome b sequence with the CHD-Z
Figure 3. The ML tree for myoglobin gene (in blue) and for CHD-Z gene (in red); both trees are scaled in substitutions
and the length of each tree and different topologies indicate that both of them are improper for phylogeny using a single
gene.
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gene and myoglobin, two data sets were obtained for a complete BI phylogenetic analysis to test the reliability of the
CHD-Z gene in phylogeny. A topology comparison of the
MCC trees with cytochrome b highlights that all the MCC
trees have a similar topology, with differences given by the
total length of the branches (Figure 4), determined by the
mutation rates specific for each gene. All these three MCC
trees are scaled in substitutions and vary between 0.322 for
cytb and 0.175 for myo and cytb.
The BI analysis showed a clock rate for CHD-Z-cytb
genes (1.457), intermediary between myoglobin - cytb
(0.974) and the cytb gene (1.772) independently analyzed,
meaning that the myo gene is changing faster through time
and can negatively affect the time-calibrated trees, giving
wrong divergence species time. A marginal likelihood estimation was performed for the tree models comparison
by the AIC through Markov chain Monte Carlo (AICM)
algorithm, inferred using the method-of-moments estimator (Baele et al., 2012) as a measure of accuracy of the molecular clock models. The minimum value of the AICM,
corresponding to the combined data set cytb + CHD-Z,
indicates the better model fit. The analyzed models’ accuracy is expressed as the difference between AICM values,
which means that the positive values (+2483.463) indicate
a better model relative fit of the cytb + CHD-Z tree compared to the cytb + myo tree (Table 3). Furthermore, the
AICM values are confirmed by the tree mean posterior
probability (cytb + CHD-Z 0.901, S.E. = 0.078; cytb 0.882,
S.E. = 0.0084; and cytb + myo 0.835, S.E. = 0.119), suggesting a lower level of phylogenetic noise. Furthermore,
the tree topology asymmetry was evaluated in order to assess the combined data set constraining the species’ different potential for speciation (Blum and Francois, 2005).
The Colless index of tree imbalance (Colless, 1982) seems
to be unaffected by data sets, with a value of 0.466. Li et
al. (2007) described a good phylogenetic marker as a gene
with an intermediate substitution rate and high treeness
values, highlighting the importance of the optimal rate and
base composition stationarity in marker suitability. Even if
the CHD-Z base composition is less stationary than myo
or cytb, the low number of variable sites with high base
composition heterogeneity seems to increase the fitness of
Figure 4. Maximum clade credibility trees for three data sets obtained by BI analysis in BEAST v1.8.0. MCC genealogy based on a Yule
speciation process, summarized from the output of a Markov chain Monte Carlo chain run for 10 million iterations and sampled every
1000 iterations.
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CIORPAC et al. / Turk J Zool
Table 3. Combined tree models compared by AICM (S.E. estimated from 1000 bootstrap replicates). The analysis was
performed in Tracer v1.6 using log files from BEAST v1.85 analysis.
Combined tree model
AICM
S.E.
cytb-z_chd-z.log.txt
cytb-z_myo.log.txt
cytb-z_chd-z.log.txt
7513.98
+/– 0.474
-
2483.463
cytb-z_myo.log.txt
9997.443
+/– 0.677
–2483.463
-
this phylogenetic tool. Overall, our new proposed marker
CHD-Z has the greatest phylogenetic signal-to-noise index (treeness index: 0.371), and hence a lower potential
for nonphylogenetic signals to influence phylogeny reconstruction compared to myo (treeness 0.357), which
is diverging faster than CHD-Z (the ratio of myo/CHDZ is 1.15). Due to the coding and noncoding regions, the
CHD-Z marker is able to discriminate both close and distant sylvioid taxa, respectively.
In conclusion, the implications of the CHD gene for
population analysis are substantial, offering a good image
of population dynamics, sex distribution, and population
trends. Additionally, the use of the CHD-Z gene in phylogenic reconstruction gives the most effective results, having a moderate clock rate and being capable of revealing
the relationship between closer and distant taxa. The CHD
genes are reliable tools not just for sex determination in
birds, but also for population and phylogenetic analysis,
providing better results compared to the myoglobin gene,
a frequently used nuclear marker in bird analysis.
Acknowledgment
This work was supported by the project “Doctoral and
Post-doctoral programs of excellence for highly qualified human resources training for research in the field of
Life sciences, Environment and Earth Science”, Grant No.
POSDRU/159/1.5/S/133391, , cofinanced by the European
Social Fund within the Sectorial Operational Program
Human Resources Development 2007–2013, University of
Bucharest, Romania.
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