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Complete Mitochondrial Genome and
Phylogenetic Analysis of Gruiformes and
Charadriiformes
Peng Chen1,2, Zuhao Huang3, Chaoying Zhu1, Yuqing Han1, Zhifeng Xu1,
Guanglong Sun1, Zhen Zhang1, Dongqin Zhao4, Gang Ge1 and Luzhang Ruan1*
1School of Life Sciences, State Ministry of Education Key Laboratory of Poyang Lake
Environment and Resource Utilization, Nanchang University, Nanchang, 330031, P.R.
China
2Nanjing Institute of Environmental Sciences, Ministry of Ecology and Environment, 8
Jiang wang miao St., Nanjing 210042, China
3School of Life Sciences, Jinggangshan University, Ji’an, Jiangxi Province, China
4Key Laboratory of Animal Resistance of Shandong Province, College of Life Sciences,
Shandong Normal University, Jinan 250014, Shandong Province, P.R. China
Article Information
Received 03 June 2019
Revised 30 July 2019
Accepted 14 September 2019
Available online 10 January 2020
Authors’ Contribution
PC wrote this article. RL and PC
planned this research. ZH, CZ, YH,
ZX, GS, ZZ and DZ performed
the experiments. GG and RL is the
corresponding author of the article
Key words
Next-generation sequencing,
Mitochondrial genome, Classication
status, Phylogenetic, Divergence time
In this study, we used the next-generation sequencing method to obtain mitochondrial DNA (mtDNA) of
Porzana paykullii, Rallus aquaticus and Gallirallus striatus in Gruiformes, and Hydrophasianus chirurgus
in Charadriiformes, after which we analysed and compared structure, phylogeny, and taxonomic origin
of the Gruiformes and Charadriiformes. Based on sequencing, splicing, and annotating the mtDNA of
four birds, the results showed that the lengths of mtDNA were 16,955 bp in Porzana paykullii, 17,149
bp in Rallus aquaticus, 17,647 bp in Gallirallus striatus and 16,855 bp in Hydrophasianus chirurgus,
respectively. The base compositions were A > C > T > G in 73 species complete mitochondrial sequences
in Gruiformes and Charadriiformes. The total AT content in the 73 species was larger than that of GC.
The start codons in protein-coding genes (PCGs) included ATG, GTG, ATT, ATC and ATA, while
its stop codons included TAA, TAG, AGG, AGA and the incomplete cipher T. In PCGs, the highest
frequency of codon was CTA (Leu). The highest frequency of amino acids was Leu, whereas the lowest
was Cys. In phylogenetic analyses, Gruiformes included Grui and Ralli, and Charadriformes included
Charadrii, Lari and Scolopaci. The genus Porzana was closest to Porphyrio. Gallirallus striatus and
Lewinia muelleri consisted a sister group, while Rallus aquaticus was a separate branch. Hydrophasianus
chirurgus (Charadriiformes: Jacanidae) was closely related to Rostratulidae. According to the estimation
of divergence time corrected by fossil records of related birds and compared with previous studies, the
base divergence time of Gruiformes was 46.33 (58.46~25.60) Ma, the emergence time of the suborders
Grui was about 17.62 (29.76~4.15) Ma, and the emergence time of the suborders of the Ralli was about
32.18 (46.17~19.69) Ma. The origin time of Charadriiformes was about 45.44 (58.21~24.67) Ma. The
origin time of the Charadrii was 44.52 Ma (52.66 ~ 23.55 Ma). The divergence time of Charadrii, Lari
and Scolopaci was about 33.46 (43.44~22.92) Ma, 29.10 (40.67~16.43) Ma and 27.06 (34.83 ~ 12.72)
Ma, respectively.
INTRODUCTION
Aves originated in theropod out of the Jurassic and
belongs to Vertebrata. The direct ancestor of Aves was
the descendant of a small dinosaur (Ostrom and Mcintosh,
1967; Zheng, 2015) which was the most abundant class
of tetrapod vertebrates (Zheng, 2015). In traditional
taxonomy, Aves consists Archaeornithes and Neornithes
* Corresponding author: ruanluzhang@sina.com
0030-9923/2020/0002-0425 $ 9.00/0
Copyright 2020 Zoological Society of Pakistan
(Gemmell et al., 1994; Jarvis et al., 2014; Prum et al.,
2015; Zheng, 2015). Neornithes mainly consists of the
Palaiognathae, which has no ying short wings, and
all Neognathae that can y. The study concluded that
the Artistic depiction of asteroidal impact occurred
approximately 66 million years (Ma) ago. This period
serves as the boundary between cretaceous period and
tertiary (paleogene) (k-pg) (Avise, 2004), during which
a wide range of species extinction events took place,
creating conditions for the differentiation of birds and the
generation of new species (Jarvis et al., 2014; Prum et al.,
2015). After the k-pg period, the ancestors of Neognathae
ABSTRACT
Pakistan J. Zool., vol. 52(2), pp 425-439, 2020. DOI: https://dx.doi.org/10.17582/journal.pjz/20190603010623
426
birds differentiated and formed the largest group of existing
birds, including Galloanseres and Neoaves (Jarvis et al.,
2014; Prum et al., 2015; Zhang et al., 2017). Galloanseres
are included in Galliformes and Anseriformes. Neoaves are
included in Galliformes, Passeriformes, Charadriiformes
and other more than 20 orders (Sibley et al., 1988; Zheng,
2012; Zheng, 2015).
In the face of such a large number of birds with
complex kinship, research scholars were keen to explore
the systematic classication of birds and the origin and
evolution of birds (Zheng, 1979; Cracraft, 1981; Smith
and Clarke, 2015). Early classication of bird systems
were based on epigenetics, morphological features,
geographical distribution, and behavioural differences,
but the results were rather confusing (Jarvis et al.,
2014; Prum et al., 2015). Especially the phylogenetic
analysis for Gruiformes and Charadriiformes is more
chaotic (Jarvis et al., 2014; Prum et al., 2015; García–R
et al., 2014). The current research of Gruiformes and
Charadriiformes mainly focus on macroecology, behavior
and histomorphology (Zheng, 1979; Ripley and Beehler,
1985; Avise, 2004; Zheng, 2012; Smith and Clarke, 2015;
Huang et al., 2017). The use of molecular means can more
effectively solve controversial issues such as the origin
and evolution of birds (Jarvis et al., 2014; Prum et al.,
2015). According to traditional taxonomy, there are 143
bird species in Gruiformes, including 12 families and
40 genera (Ripley and Beehler, 1985). Most Gruiformes
birds have a small distribution range, and many of them
are endangered species, such as Grus japonensis and Grus
leucogeranus of Gruidae (Ripley and Beehler, 1985).
There are great differences in the body types of birds of
Gruiformes, with larger body size of Gruidae species if
compers with Rallidae (Zheng, 2012). Charadriformes is
a complex group, mainly including small and medium-
sized waders, curlew and gulls (Sibley et al., 1988;
Zheng, 2012). Charadriiformes includes 8 suborders, 19
families, 94 genera and 384 bird species (Hu et al., 2017),
which distribute on all continents, from the poles to the
tropics (Sibley et al., 1988). Up to now, more than 300
species of mitochondrial genome sequence information of
birds have been published in Genbank (Gao et al., 2009;
Zhang, 2015; Cheng, 2017), among which 33 species are
Gruiformes (García–R et al., 2014; Chen et al., 2017) and
34 species are Charadriiformes (Smith and Clarke, 2015;
Hu et al., 2017).
There have also been a lot of studies on the
molecular phylogenetic classication of Gruiformes and
Charadriiformes (Fain et al., 2007; Smith and Clarke, 2015;
Chen et al., 2017; Hu et al., 2017). But the phylogenetic
tree constructed at present was not comprehensive enough
to contain species, and the phylogenetic status of many
species was controversial (Sibley et al., 1988; Chen et al.,
2017). Ruan et al. (2012) used mitochondrial COI and Cytb
genes of 15 Gruiformes species to construct phylogenetic
trees, Fain et al. (2007) constructed phylogenetic trees and
estimated analysis using genes such as Cytb, 12S rRNA
and 16S rRNA of 26 Gruiformes species. The time of
disagreement mainly includes the Gruidae, Psophiidae,
Rallidae and Heliornithidae (Fain et al., 2007). Among
them, the Gruidae and Psophiidae were sister group, while
Rallidae and Heliornithidae formed sister group. It was
believed that the divergence time of Rallidae was 21.8
Ma, Rallidae and Heliornithidae was 42.6 Ma. García–R
et al. used 17 PCGs, rRNA and tRNA genes of 17 species
mitochondria of Gruiformes to construct a phylogenetic
tree and estimate the divergence time. It was believed that
the divergence time of Rallidae and Heliornithidae was
52 Ma, and the results were more accurate and reliable
(García–R et al., 2014). Gong et al. used a genome-wide
sequence of 31 species of Gruiformes (excluding CR
sequences) to construct a Bayesian inference (BI) tree.
The results showed that Rallidae and Heliornithidae were
a sister group, and Gruidae belonged to another branch,
but did not estimated the divergence time of Gruiformes
(Gong et al., 2017). Gong et al. (2017) was suggested that
the genus Gallirallus were sister to Lewinia, and these
groups in turn were sister Amaurornis and different from
those of García–R (2014). The genus Fulica and the genus
Gallinula were one branch, the genus Porphyrio and the
genus Coturnicops belong to another branch. while the
García–R (2014) considers the genus Porphyrio, genus
Fulica and Coturnicops were a branch. While the genus
Gallirallus and the genus Lewinia was another branch.
There were also reports on mitochondrial
genome sequencing and phylogenetic development in
Charadriiformes birds. Based on 12 PCGs markers, Hu et al.
(2017) constructed BI and Maximum Likelihood (ML) trees
of 40 species to classify Charadriiformes into Charadrii,
Lari and Scolopaci, which was more comprehensive, but
no divergence time estimate for Charadriiformes. Cheng et
al. (2017) studied phylogenetic trees of 18 Charadriiformes
birds, and also divided Charadriiformes into the same
three suborders. The phylogenetic tree supports the Lari
as a suborder, belonging to Charadriiformes. While
the suborder of Scolopaci was the newly differentiated
Charadriiformes and included Jacanidae and Scolopacidae
(Cheng, 2017). It was believed that Charadrii were included
Recurvirostridea and Charadriidae, while Recurvirostra
avosetta and Haematopus ostralegus was belonged to
Recurvirostridea (Chen, 2003). At present, there are few
studies on the divergence time of the Charadriiformes.
Fain et al. (2007) used the genes of Cytb, 12S rRNA and
16S rRNA of 26 species to construct phylogenetic trees
P. Chen et al.
427
and estimate the divergence time. The results indicated that
the base divergence time of Charadriiformes was 74.3 Ma.
Combined with the previous phylogenetic analysis
of Gruiformes and Charadriiformes. It was found that
due to the lack of molecular data, phylogenetic analysis
of Rallidae of Gruiformes was relatively chaotic, the
accuracy of estimation of divergence time of Gruiformes
was not enough. And the systematic analysis of the
divergence time of Charadriiformes was not carried out.
Therefore, it was necessary to make a more accurate
analysis of phylogenetic development and divergence
time of Gruiformes and Charadriiformes. So as to provide
favourable reference and sufcient molecular evidence for
the improvement of their classication system. This study
has collected samples of Gruiformes and Charadriiformes
and performed mitochondrial sequencing. Including
three species Porzana paykullii, Rallus aquaticus and
Gallirallus striatus from Gruiformes, and another bird
Hydrophasianus chirurgus from Charadriiformes (Taylor
and van Perlo, 1998; Manson and Goldizen, 2000).
Combined with mtDNA sequences of four species and
data of Gruiformes and Charadriiformes retrieved from
GenBank, data of mitochondrial genome of 73 bird species
were collected (Table I). In addition, the structure and base
ratio of the mitochondrial genome of 73 species of birds
of Gruiformes and Charadriiformes were compared, and
the use types of starting and ending codons as well as the
use frequency of codons and amino acids were compared.
Phylogenetic tree was constructed by collecting and
sorting mitochondrial genome sequences of Gruiformes
and Charadriiformes, analysing the taxonomic status of
the four species sequenced in this study, and exploring
the relationship between the species of Gruiformes and
Charadriiformes. Combined with the known fossil record of
birds and the previous research results, this paper estimated
the divergence time between the species of Gruiformes
and Charadriiformes. So as to provide more molecular
data for the study of evolutionary species, conservation
aspects and phylogenetic relationships of birds.
MATERIALS AND METHODS
Ethics statement
The sample collection was strictly conducted under
national ethical guidelines (Regulations for Administration
of Affairs Concerning Experimental Animals, China,
1988) for animal husbandry and humane treatment.
Sample collection and DNA extraction
In this study, blood samples from 4 bird species without
mitochondrial complete genome sequencing were collected
nationwide from Gruiformes and Charadriiformes.
Gruiformes including Porzana paykullii, Rallus aquaticus
and Gallirallus striatus. The sampling points were Bengbu
Anhui, province, Lianyungang Jiangsu, province and
Liuzhou, Guangxi province, respectively. Another one
species blood samples of Hydrophasianus chirurgus of
Charadriiformes collected in Mianyang, Sichuan province.
The blood was stored in a 1 ml centrifuge tube, and 20
μL of anticoagulant (0.5% sodium heparin) was added
and stored in a refrigerator at – 20 °C. Total DNA was
extracted using phenol/chloroform and examined on 1.0%
agarose/TBE gel and used as template for PCR reactions.
DNA extraction and sequencing
The mtDNA of four species of birds was extracted and
sequenced. First, the blood samples were digested. 10 μL
of the sample blood was placed in a 1 mL centrifuge tube,
and 375 μL of TE buffer, 20 μL of 10% SDS solution and
5 μL of 20 mg/L protease were added. The total volume of
K was 400 μL of mixed digest. Mix the digestive juice, and
then bathe in a water bath at 55-65 °C for 10 to 14 hours.
Mix the digestive juice at intervals until the blood tissue
was completely digested and dissolved.
The total DNA from birds was extracted by phenolic
extraction: the cells were rst broken up by protease
K and SDS, and the proteins were digested. Then the
supernatant was extracted by phenolic and phenol-
chlorine. And the supernatant liquid was taken after high
speed centrifugation (Chen et al., 2017). The centrifuged
DNA was washed two to three times with 70% ethanol,
dried, dissolved in 100 μL of sterilized double distilled
water, and stored in a refrigerator at 4 °C until use. Take
4 μL of total DNA solution, mix with 1 μL of 6×Loading
Buffer, and then spot it in agarose gel well. After 120V of
constant pressure electrophoresis for 30 minutes, observe
total DNA in UV detector or portable UV lamp. Extract
the results. The complete sequence of the mitochondrial
genome of the three species were sequenced by Beijing
Jinnuo Ruijieji Technology Co. Ltd. and the CR partial
sequence results of common pheasant and leeches were
incomplete and corrected by one generation sequencing.
Primers were designed based on the alignment of
complete mtDNA sequences of Gallinula chloropus and
Rallina eurizonoides by using Primer 5.00 (PREMIER
Biosoft International) (Chen et al., 2017) and shown
in Supplementary Table I. The complete mitochondrial
genome sequence of four species were deposited in Gen-
Bank with accession numbers MG200164, MH229988,
MH219930 and MH219929, respectively (Table I and
Supplementary Tables II, III, IV and V).
Data analysis
Data acquisition and analysis
Mitochondrial complete genomes of Gruiformes and
Mitochondrial Genome and Phylogenetic Analysis of Gruiformes and Charadriiformes 427
428
Table I. The mitochondrial genome of Gruiformes and Charadriiformes.
Order Family Genus Species All(bp) PCGs(bp) GeneBank
Gruiformes Gruidae Anthropoides Anthropoides paradiseus 16696 11381 NC_020572
A. virgo 16541 11381 NC_020573
Balearica B. pavonina 16786 11375 NC_020570
B. regulorum 16802 11375 NC_020569
Grus carunculatus 16677 11381 NC_020571
Grus G. americana 16651 11381 NC_020576
G. antigone 16549 11381 NC_020581
G. canadensis 16697 11381 NC_020582
G. grus 16649 11381 NC_020577
G. japonensis 16715 11381 NC_020575
G. leucogeranus 16688 11381 NC_020574
G. monacha 16650 11381 NC_020578
G. nigricollis 16646 11381 NC_020579
G. rubicunda 16693 11381 NC_020580
G. vipio 16678 11381 NC_021368
Rallidae Amaurornis Amaurornis akool 16950 11296 NC_023982
A. phoenicurus 17213 11368 NC_024593
Coturnicops Coturnicops exquisitus 17136 11368 NC_012143
Eulabeornis Eulabeornis castaneoventris 17339 11368 NC_025501
Fulica Fulica atra 17029 11368 NC_025500
Gallicrex Gallicrex cinerea 17184 11377 NC_028408
Gallinula Gallinula chloropus 17027 11365 NC_015236
Gallirallus Gallirallus australis 17464 11368 KF701060
G. okinawae 18404 11368 NC_012140
G. philippensis 17359 11368 NC_025507
G. striatus 17647 11368 MH219930
Lewinia Lewinia muelleri 17273 11368 NC_025502
Porphyrio Porphyrio hochstetteri 16988 11368 NC_010092
P. porphyrio 17020 11368 NC_025508
Porzana Porzana fusca 16935 11368 KY009736
P. paykullii 16955 11368 MG200164
P. pusilla 16978 11368 KY009737
Rallus Rallus aquaticus 17149 11368 MH229988
Rallina Rallina eurizonoides 16942 11377 NC_012142
Sarothrura Sarothrura ayresi 16767 11362 NC_034316
Rhynochetidae Rhynochetos Rhynochetos jubatus 16937 11396 NC_010091
Heliornithidae Heliornis Heliornis fulica 17008 11367 NC_025499
Otididae Otis Otis tarda 16849 11397 NC_014046
Charadriiformes Laridae Larus Larus crassirostris 16701 11397 KM507782
L. dominicanus 16746 11399 AY293619
L. vegae 16379 11397 NC_029383
Ichthyaetus relictus 16586 11399 KC760146
Chroicocephalus L. brunnicephalus 16769 11399 JX155863
L. ridibundus 16807 11391 KM577662
L. saundersi 16724 11379 JQ071443
Gelochelidon Gelochelidon nilotica 16748 11397 NC_036344
P. Chen et al.
429 Mitochondrial Genome and Phylogenetic Analysis of Gruiformes and Charadriiformes 429
Sternidae Sterna Sterna albifrons 16357 11397 KT350612
S. hirundo 16707 11397 NC_036345
Alcidae Synthliboramphus Synthliboramphus antiquus 16730 11402 AP009042
S. wumizusume 16714 11397 KT592378
Pinguinus Pinguinus impennis 16784 11397 NC_031347
Stercorariidae Stercorarius Stercorarius maccormicki 16669 11403 KM401546
Scolopacidae Eurynorhynchus Eurynorhynchus pygmeus 16707 11397 KP742478
Arenaria Arenaria interpres 16725 11399 AY074885
Tringa Tringa erythropus 16683 11397 NC_030585
T. ochropus 16906 11397 KX668223
T. semipalmata 16603 11399 NC_036016
Xenus Xenus cinereus 16817 11397 KX644890
Gallinago Gallinago stenura 16899 11400 KY056596
Scolopax Scolopax rusticola 16984 11391 KM434134
Numenius Numenius phaeopus 17091 11396 KP308149
Limosa Limosa lapponica 16732 11399 KX371106
Jacanidae Jacana Jacana jacana 16975 11394 KJ631049
J. spinosa 17063 11397 KJ631048
Hydrophasianus Hydrophasianus chirurgus 16855 11397 MH219929
Charadriidae Vanellus Vanellus cinereus 17074 11399 KM404175
V. vanellus 16795 11397 KM577158
Pluvialis Pluvialis fulva 16854 11391 KX639757
Charadrius Charadrius placidus 16895 11397 KY419888
Recurvirostridae Recurvirostra Recurvirostra avosetta 16897 11397 KP757766
Himantopus Himantopus himantopus 17378 11397 NC_035423
Haematopodidae Haematopus Haematopus ater 16791 11400 AY074886
H. ostralegus 16798 11394 NC_034237
Charadriiformes species were retrieved and downloaded
from GenBank, including 35 Gruiformes species and 34
Charadriiformes species (Table I). Combined with the
mitochondrial complete genomes of 4 birds determined in
this study (Fig. 1), a total of 73 mitochondrial complete
genomes of birds were obtained. We rstly used Clustal
X (Thompson et al., 1997) and MEGA5.0 (Tamura et al.,
2011) and DNASTAR (Burland, 2000) to compare the
mitochondrial genome sequences of 73 birds and analysed
the base content, start codon and termination of the
complete genome and PCGs. Then we also analysed the
codon usage and synonymous codon usage of Gruiformes
and Charadriiformes PCGs. The calculation formula for
base skew was AT skew = (A-T) / (A + T), GC skew =
(G-C) / (G + C) (Perna and Kocher, 1995). We used Adobe
Illustrator CS6 and OriginPro 8 (Wass, 2008) to draw
processing diagrams.
Analysis of phylogeny
A total of 73 birds mitochondrial genome sequences
were collected in Gruiformes and Charadriiformes, and
the PCG and rRNA genes of 15 species of Charadriiformes
were searched in GeneBank (Supplementary Table VI).
The sequences of 12 PCGs, 12S rRNA and 16S rRNA
genes of mitochondria of 88 species. Both 38 species in
19 genera, 5 families of Gruiformes, and 50 species in 34
genera, 17 families of Charadriiformes were sequenced
and what were perform phylogenetic analysis. The
construction of the phylogenetic tree requires the use of
DAMBE (Xia, 2001; Xia and Lemey, 2009) software
to verify the saturation of the selected sequence. The
use of the obtained gene sequence substitution does not
reach saturation before the phylogenetic analysis can be
continued.
ML and BI trees were constructed by using the
optimal parameter model selected by MrModelTest3.06
software (Klaus-J et al., 2004) and PAUP 4.0b10 software
(Swofford, 2003) with Akaike information Criterion
(AIC) to select the model with the minimum statistical
value. Among them, the BI trees of Gruiformes and
Charadriiformes birds were constructed using MrBayes
3.1.2 software (Huelsenbeck and Ronquist, 2001). In the
process of BI tree construction, four Monte Carlo Markov
chains (MCMC) were established. Start with random
430 P. Chen et al.
number as the starting tree, run 3,000,000 generations
in total, and sample once every 100 generations. After
discarding the 25% of burning-in samples, the consistent
tree can be constructed according to the remaining 75%
samples, and the Posterior Probability (PP) of the BI tree
can be calculated nally. The ML tree was constructed by
using the RaxML software to run the les in Phy format
(Stamatakis et al., 2008; Stamatakis, 2014), selected the
best model for MrModelTest3.06 software lter, and
set the ML thorough bootstrap mode, bootstrap reps to
50 and running 10000 times to get the ML tree. Finally,
Treeview32 (Saldanha, 2004) and Figtree 1.4.2 (Rambaut,
2014) software were used to open and annotate the
evolutionary tree diagram. The genetic evolution and
phylogenetic relationship of different species in the two
orders were analysed and the taxonomic status of some
birds was discussed.
Fig. 1. Mitochondrial genomes of four species.
Analysis of divergence time
In this study, the divergence time was calculated based
on the ML tree topology constructed by mitochondrial
genome, and the accurate fossil record was used as the
calibration point. The earliest fossilized Galliformes was
recorded as Gallinuloides wyomingensis (Avise, 2004)
with a minimum time of 51.58 Ma (Graybeal, 1998; Prum
et al., 2015), and the stem sphenisciform Waimanu (Slack
et al., 2006) with a normal distribution of 61.5–65.5 Mya.
Combining the fossil record and comparing the results of
Jarvis et al. (2014) and Prum et al. (2015), the calibration
point of the Neognathae was set as 70.00 Ma, and
Galloanseres and Neoaves calibration point was 65 Ma
(Jarvis et al., 2014; Prum et al., 2015) and set the standard
deviation of 0.5. BEAST 1.4.6 was the software that can
build phylogenetic trees and estimate the time of species
divergence (Drummond and Rambaut, 2007). BEAST
1.4.6 software was used to establish MCMC to estimate the
bifurcation time of species. Yule Prior was selected in the
branching evolution rate (reference?). The data calculation
model setting was consistent with the phylogenetic tree
model. MCMC operation parameters were as follows
that length of chain was 8 × 106 generations, once every
200 generations, and burn-in 10% of the samples. Tree
Annotator 1.6.1 (Drummond and Rambaut, 2007) software
was used to construct the tree with the maximum branch
credibility. The running results were analysed by Tracer v
1.4 (Rambaut and Drummond, 2007), and then Fig Tree
1.4.2 (Rambaut, 2014) was used to open the evolutionary
tree with divergence time and 95% of the highest posterior
density (HPD).
RESULTS
Genome organization and arrangement
The mitochondrial genome sequences of 38 species
(19 genera, 5 families) of Gruiformes and 35 species (35
genera, 9 families) of Charadriformes were analysed in
this study (Table I). The structure and gene arrangement of
mitochondrial genomes of Gruiformes and Charadriiformes
were basically the same, including 37 coding genes and
one CR, and the content range of each base was almost the
same, and the uctuation range was less than 3% (Fig. 2).
The mean base composition of mitochondrial genome
of Gruiformes was T (23.92±0.56%), C (30.74±0.71%),
A (31.82±0.64%) and G (13.52±0.34%). The AT content
ranged from 54.70% to 57.53%, with an average value
of 55.74 ± 0.70%, higher than GC content (Fig. 2). The
sequence of base richness of mitochondrial genome
was mostly A>C>T>G, and only Balearica pavonina,
Rhynochetos jubatus and Otis tarda had base contents of
C>A>T>G. The mean AT skew was 0.14±0.02, and GC skew
was -0.39±0.02, indicating that the nucleotide composition
of mitochondrial complete genome of Gruiformes
had a slight specic bias towards A and C (Fig. 3).
The average base composition of mitochondrial
genome of Charadriiformes was T (24.61 ± 0.76%),
C (30.48 ± 0.69%), A (31.20 ± 0.47%) and G (13.72
± 0.38%). The AT contents ranged from 54.39% to
58.35%, with an average value of 55.81 ± 0.86%, slightly
higher than that of the mitochondrial whole genome
431 Mitochondrial Genome and Phylogenetic Analysis of Gruiformes and Charadriiformes 431
of Gruiformes. The sequence of base abundance of
mitochondrial genome was mostly A > C > T > G (Fig. 2).
As for Larus crassirostris, Larus dominicanus, Ichthyaetus
relictus, Larus brininucephalus, Larus ridibundus, Larus
saundersi, Arenaria interpres, and Pluvialis fulva have a
base content of C > A > T > G. The mean deviation of
AT was 0.12 ± 0.02, and the mean deviation of GC was
-0.38 ± 0.01, indicating that the nucleotide composition of
the mitochondrial whole genome of Charadriiformes birds
was slightly biased towards A and C (Fig. 3).
Fig. 2. Analysis of base content of complete mitochondrial
genome of Gruiformes and Charadriiformes.
Fig. 3. The mitochondrial genome base AT skew and GC
skew of Gruiformes and Charadriiformes.
Analysis of PCGs
The structure of PCGs of Gruiformes and
Charadriiformes were very conservative, including 7
NADH reductase genes, 3 cytochrome oxidase genes,
2 ATP synthase genes (ATPase8 and ATPase6) and a
cytochrome oxidase b gene. Among the Gruiformes, the
shortest PCGs was Amaurornis akool (11,296 bp), while
the longest was Otis tarda (11,397 bp), and the shortest
PCGs of Charadriiformes was the Larus saundersi
(11,379 bp), whereas the longest PCGs was Stercorarius
maccormicki (11,403 bp) (Table I).
Sequence analysis of PCGs
The average base composition of Gruiformes PCGs
was T (25.25 ± 0.55%), C (31.60 ± 0.80%), A (29.82 ±
0.68%) and G (13.33 ± 0.36%). The AT content ranged
from 53.60% to 57.09%, and the average value was (55.07
± 0.82%) higher than the GC content (Fig. 4). The AT
skew was 0.08 ± 0.02, and the GC skew was − 0.41 ± 0.02,
indicating that the nucleotide composition of Gruiformes
PCGs has a slight specic bias for A and C (Fig. 3). The
AT content of the rst codon of PCGs of Gruiformes was
50.51 ± 0.59%, where the AT skew was 0.17 ± 0.01, and the
GC skew was −0.13 ± 0.01 The second codon AT content
was 58.37 ± 0.18%, where AT skew was −0.37 ± 0.01 and
GC skew was −0.39 ± 0.01. Third codon AT content was
56.33 ± 2.02%, where AT skew was 0.47 ± 0.04 and GC
skew was −0.74 ± 0.04 (Fig. 3). The AT content of the
third codon of PCGs of Gruiformes uctuated the most,
and the AC content of the rst and third codon bases was
more than the TG content, while the second codon base
consisted of the TC content more than the AG content.
Fig. 4. Analysis of base content of mitochondrial PCGs of
Gruiformes and Charadriiformes.
The base composition of Charadriiformes PCGs
was T (26.13 ± 0.91%), C (31.36 ± 0.86%), A (29.15 ±
0.51%) and G (13.35 ± 0.40%). The AT content ranged
from 53.75% to 58.31%, and the average value was 55.28
± 0.99%, which was slightly higher than the AT content
of Gruiformes PCGs (Fig. 4). The AT skew was 0.05 ±
0.02 and the GC skew was − 0.40 ± 0.02, indicating that
the nucleotide composition of Charadriiformes PCGs has a
slight specic bias for A and C. The rst codon AT content
of Charadriiformes PCGs was 50.55 ± 0.72%, where the
AT skew was 0.14 ± 0.01 and the GC skew was − 0.11 ±
0.01. The second codon AT content was 58.45 ± 0.23%,
432 P. Chen et al.
where the AT skew was − 0.37 ± 0.01 and the average
value of GC skew was − 0.39 ± 0.01. The AT content of
the third codon was 56.85 ± 2.35%, where the AT skew
was 0.42 ± 0.05 and the GC skew was − 0.75 ± 0.04. 3.3)
(Fig. 3). The AT content of the third codon of the PCGs
of Charadriiformes uctuated the most, and the AC content
of the rst and third codon bases was more than the TG
content, while the second codon base consisted of the TC
content more than the AG content.
Fig. 5. The relative usage of stop codon of Gruiformes and
Charadriiformes.
Fig. 6. The relative usage of start codon of Gruiformes and
Charadriiformes.
Codon usage of PCGs
Most of Gruiformes and Charadriiformes PCGs were
used ATG, GTG, ATT, ATC and ATA as the start codon,
while TAA, TAG, AGG, AGA and incomplete codon T were
used as the stop codons (Fig. 5 and Fig. 6). Incomplete stop
codons were frequently found in the COIII gene in birds and
can be complemented by the acidication of adenosine or
poly (A) at the mRNA3’ during transcriptional processing
Stop codon (TAA). The initiation codons of Gruiformes and
Charadriiformes ATPase8, COIII, Nd4L, Nd4, Nd6 and Cytb
of Gruiformes and Charadriiformes were ATG, whereas the
codons of COI and Nd5 were ATG and GTG. The starting
codons of Gruiformes and Charadriiformes other PCGs
genes were somewhat different. The stop codons of the
mitochondrial genome of Gruiformes and Charadriiformes
were more complicated. Among them, only the stop codons
of ATPase8, ATPase6 and Nd4L were TAA, and the stop
codons of COI were AGG. The stop codons of Gruiformes
and Charadriiformes the other 9 PCGs were different.
In the PCGs encoding process of Gruiformes, the
four codons with the highest frequency average were CTA
(Leu), ATC (Ile), ACA (Thr) and TTC (Phe). The average
number of occurrences of these four codons was separately
312.80, 201.00, 155.80, and 155.30, while the average
value of the Synonymous Codon Usage (RSCU) was 2.85,
1.36, 1.71, and 1.45, respectively. In the PCGs encoding
process of Charadriiformes, codons having a relatively
high frequency average were CTA (Leu), ATC (Ile), TTC
(Phe) and ACA (Thr). The average number of occurrences
of these ve codons was separately 301.20, 200.90, 160.60,
and 149.50, while the average RSCU was 2.73, 1.38, 1.44,
and 1.69, respectively. By counting the frequency of amino
acid use of Gruiformes PCGs, we found that the highest
frequency of use was Leu (17.44%) whereas the lowest was
Cys (0.70%). The results for Charadriiformes PCGs were
similar, with the highest average frequency of use being
Leu (17.48%) and the lowest being Cys (0.76%) (Fig. 7).
Fig. 7. Frequency of mitochondrial PCGs amino acids of
Gruiformes and Charadriiformes.
Analysis of phylogeny and divergence time
By arranging the gene sequences of 12 PCGs, 12S rRNA
and 16S rRNA of mitochondria (Table I; Supplementary
Table VI), the base substitution saturation was detected
based on the F84 model in the DAMBE software. We also
analysed the ratio of nucleotide conversion number (×s)
and nucleotide transversion number (Δv) to the genetic
distance of nucleotide sequences of different species.
The results showed that the sequence ratio was increased
with the increase of genetic distance. The conversion and
433 Mitochondrial Genome and Phylogenetic Analysis of Gruiformes and Charadriiformes 433
Fig. 8. BI and ML trees constructed based on 12 PCGs and 2 rRNA genes of Gruiformes and Charadriiformes. Numbers represent
bootstrap values (MP/ML) and only those > 40% are shown. Asterisks indicate posterior probabilities of 100%.
transversion of nucleotides increased linearly
(Supplementary Fig. 1), which was a linear regression
model. The results also indicated that the substitution of
gene sequences was not saturated, so phylogenetic analysis
could continue. The sequence was screened by Modeltest
and Mrmodeltest. The best model was GTR+G+I (-lnL =
322120.5625, K = 10, AIC = 644261.1250) which was then
used to construct BI and ML trees. Since the constructed
BI and ML tree topologies were identical, only the ML tree
(the number on the branch represents the support rate of the
BI and ML trees) was presented to show the phylogenetic
relationship between Gruiformes and Charadriiformes.
The selected model (GTR+G+I) was the same as the
phylogenetic analysis of the divergence Gruiformes and
Charadriiformes, and based on 12 PCGs, 12S rRNA and
16S rRNA gene of 88 species mitochondria sequence of
Gruiformes and Charadriiformes (Fig. 8 and Fig. 9). The
estimating of the divergence time was performed ML tree
harvested from BEAST software. The results showed that
the differentiation of Neognathae was about 70.22 (71.21 ~
69.22) Ma (Fig. 9), belonging to the Late Cretaceous period.
The boundary between the Cretaceous and Tertiary (Paleo)
strata (K-Pg) was about 66 Ma (Jarvis et al., 2014; Prum et
al., 2015), and the species of the Neoaves was beginning to
differentiate at this time, with a divergence time of 64.84
(65.83 ~ 63.82) Ma (Fig. 9). The birds of Galloanseres were
emerged slightly earlier than the birds of Neoaves, and the
time of divergence was 65.91 (66.89 ~ 64.94) Ma. This
result was basically consistent with the results of studies by
Jarvis et al. (2014) and Prum et al. (2015).
434 P. Chen et al.
Fig. 9. Analysis of divergence time based on 12 PCGs and 2 rRNAs genes of Gruiformes and Charadriiformes.
435
Phylogeny and divergent time of Gruiformes
In phylogenetic analyses, Gruiformes include Grui and
Ralli, Gruidae and Otididae were included in the branch of
Grui, while Rallidae and Heliornithidae were included in
the branch of Ralli (Fig. 8). Rhynochetidae was a separate
one and had a long relationship with other Gruiformes
birds. Grui branches were dominated by Gruidae, the genus
Grus and genus Bugeranus belonged to one branch, while
the genus Balearica was the other. Ralli were dominated by
Rallidae, among which Porzana fusca, Porzana paykullii,
Porzana pusilla sequenced in this study and all belonged to
the genus Porzana. The genus Porzana and genus Porphyrio
belonged to one branch, and then the genus Amaurornis,
genus Fulica and genus Gallinula belong to another branch
(81% support for ML trees and 1.00 support for BI trees). In
addition, Gallicrex cinerea was also included in the genus
Amaurornis. Gallirallus striatus was sister to Lewinia
muelleri, and this groups belonged to a branch with the
genus Gallirallus. Whereas Rallus aquaticus belonged to
the genus Rallus for another branch. (100% support for ML
trees and 1.00 support for BI trees) (Fig. 8). In addition,
Heliornis fulica and Sarothrura ayresi were a sister group
and were separated from other birds of Rallidae to form a
single one (100% support for ML trees and 1 support for
BI trees).
The basal divergence time of Gruiformes was 46.33
(58.46 ~ 25.60) Ma, and the divergence time of Grui and
Ralli was 41.36 (53.10 ~ 24.18) Ma (Fig. 9). The divergence
time of Grui was about 17.62 (29.76 ~ 4.15) Ma, and the
divergence time of Ralli was about 32.18 (46.17 ~ 19.69)
Ma. The divergence time of Porzana fusca, Porzana
paykullii, Porzana pusilla of the genus Porzana was 9.60
(14.81 ~ 5.83) Ma, which were sequenced in this study. The
divergence time of Rallus aquaticus of the genus Rallus was
18.32 (24.99 ~ 8.98) Ma. The divergence time of Gallirallus
striatus of the genus Gallirallus was 4.98 (8.87 ~ 1.93) Ma.
Phylogeny and divergence time of Charadriiformes
In this study, Charadriformes included Charadrii, Lari
and Scolopaci (Fig. 8). The branch of Charadrii mainly
included Charadriidae and Recurvirostridea. The branch
of Lari mainly included Alcidae, Sternidae and Laridae.
The branch of Scolopaci mainly included Jacanidae and
Scolopacidae. While Larus vegae was a separate branch.
Among Charadrii, Charadriidae, Haematopodidae and
Recurvirostridea were one branch (100% support for
ML trees and 1.00 support for BI trees). Chionididae and
Burhinidae was a sister group (100% support for ML
trees and 1.00 support for BI trees). In the Lari, Laridae,
Sternidae and Rhynchopidae belonged to a branch (100%
support for ML trees and 1.00 support for BI trees), and
Alcidae and Stercorariidae was a sister group (81%
support for ML trees and 1.00 support for BI trees). While
Glareolidae and Turnicidae were in separate branches.
In Scolopaci, Scolopacidae belonged to a branch (100%
support for ML trees and 1.00 support for BI trees), while
the relative relationship between Jacanidae, Rostratulidae,
Pedionomidae and Thinocoridae was close (100% support
for ML trees and 1.00 support for BI trees). In addition,
Gelochelidon nilotica in Laridae was closed relationship
with Sternidae (100% support for ML trees and 1.00
support for BI trees). Pluvianellus socialis in Charadriidae
and Chionis minor was a sister group (100% support for ML
trees and 1.00 support for BI trees) and closed relationship
with Chionididae.
The basal divergence time of Charadriiformes was
45.44 (58.21 ~ 24.67) Ma, the divergence time of Charadrii,
Lari and Scolopaci was about 33.46 (43.44 ~ 22.92) Ma,
29.10 (40.67 ~ 16.43) Ma and 27.06 (34.83 ~ 12.72) Ma,
respectively (Fig. 9). The divergence time of Charadriidae
and Scolopacidae was 21.27 (33.19 ~ 8.54) Ma and the
divergence time of Laridae and Sternidae was 17.41 (24.29
~ 6.78) Ma, while the divergence time of Alcidae and
Stercorariidae was 18.31 (27.29 ~ 7.10) Ma. The divergence
time of Scolopacidae was 25.24 (30.96 ~ 13.38) Ma, and
the divergence time of Jacanidae was 6.80 (11.87 ~ 3.39)
Ma (Fig. 9).
DISCUSSION
Complete genome sequence
Studies have shown that the length of the mitochondrial
genome of birds was 16,300 bp to 23,500 bp (Gao et al., 2009;
Zheng, 2015), and in this study the shortest mitochondrial
genome of Gruiformes was Anthropoides virgo (16,541
bp), while the longest was Gallirallus okinawae. (18,404
bp). The shortest mitochondrial genome of Charadriiformes
was Sternula albifrons (16,357 bp), whereas the longest
was Himantopus himantopus (17,378 bp). The structure
and gene arrangement of the mitochondrial genome of
Gruiformes and Charadriiformes were basically the same
as those of most birds (Gao et al., 2009; Zheng, 2015),
also including 37 coding genes and a CR (Zheng, 2015).
The AT content of the mitochondrial genome sequence
of Gruiformes and Charadriiformes mitochondria were
greater than the GC content, and the highest AT content was
Gallinago stenura, which reached 58.35% and was higher
than the AT content of the general birds (50.5% to 57.7%)
(Gao et al., 2009; Cheng et al., 2017). PCGs of Gruiformes
and Charadriiformes mitochondria were very conservative,
and their structure and arrangement were consistent with
previous studies of bird mitochondrial PCGs (Gao et al.,
2009; Zheng, 2015). Studies have shown that most birds
have a rst codon AT content of 47.50% to 52.10%, a
Mitochondrial Genome and Phylogenetic Analysis of Gruiformes and Charadriiformes 435
436
second codon AT content of 50.0% to 60.0%, and a third
codon AT content of 42.1% to 62.0% (Gao et al., 2009).
The results of the AT content of each codon of PCGs in
Gruiformes and Charadriiformes were similar to this result,
and the AT content of the third codon uctuated the most.
Because the rst and second codons face greater selection
pressures, while the third codon and non-coding regions
were subject to the least selection pressure. This difference
can itself cause an imbalance in base distribution (Zhong
et al., 2002). Moreover, the restriction of amino acids on
bases and the frequency of corresponding codon usage were
also an important reason for the local base imbalance in the
genome (Frank and Lobry, 1999). This group of genomic
local bases was uneven, showing differences in nucleotide
base composition in different regions (Belozersky and
Spirin, 1958). Compared with the base composition of the
rst two digits of the codon, the third base of the codon
has a higher mutation rate (Daubin and Perrière, 2003).
Whether this situation was the result of choice or neutral
change was still inconclusive (Necşulea and Lobry, 2006).
In addition, differences in AT and GC content were also
considered to be closely related to genomic features such
as repetitive element distribution, methylation patterns,
and gene density. Most of the PCGs of the genus and the
genus PCGs use ATG, GTG, ATT, ATC and ATA as the start
codon, and TAA, TAG, AGG, AGA and incomplete codon
T as stop codons. In the process of coding mitochondrial
proteins in the order of Gruiformes and Charadriiformes,
the codons with the highest frequency average were CTA
(Leu), and the highest frequency of amino acid use was also
Leu. The frequency of amino acid use was similar to that
of other birds, such as Emberiza chrysophrys (Ren et al.,
2014), but it was very different from Invertebrates, which
may be related to the function of mitochondria and the
utilization efciency of mitochondria in different species
(Zhao et al., 2018).
Phylogeny and divergence time
At present, the classication of the species of Gruiformes
and Charadriiformes was based on the characteristics of the
bird’s morphology, sound, and tracheal evolution (Taylor
and van Perlo, 1998; Yang and Wang, 2004; Zheng, 2012),
as well as the molecular phylogenetic analysis (Ruan et
al., 2012). The species of Gruiformes and Charadriiformes
were relatively clear in the classication level of the family,
but they were very complicated and controversial at the
classication level of the genus (García–R et al., 2014; Hu
et al., 2017).
Most of the studies have concluded that Rallidae and
Heliornithidae were closely related, while Gruidae belongs
to another branch (Fain et al., 2007; García–R et al., 2014).
The classication of birds in Rallidae was more complicated.
The genus Grus and the genus Bugeranus of Gruidae was
sister group, while the genus Balearica was another branch
(Yang and Wang, 2004). In this study, the evolutionary
analysis of Gruidae in Gruiformes was generally consistent
with previous studies (Yang and Wang, 2004; Gong et al.,
2017), but the genetic relationship between the species of
Rallidae was slightly different (García–R et al., 2014; Chen
et al., 2017; Gong et al., 2017). For the classication of
Rallidae birds, the results of García–R et al. (2014) were
consistent with this study, and they were believed that the
genus Porphyrio, the genus Amaurornis, the genus Fulica
and the genus Gallinula were belong to a branch. The genus
Gallirallus and the genus Lewinia was more closely related
and belonged to another branch. Gong et al. (2017) thought
that the genus Gallirallus was closely related to the genus
Lewinia, and then that with the genus Amaurornis, Fulica
and Gallinula were belonged to branch, while the genus
Porphyrio and Coturnicops belonged to another branch. The
differences in these classications were related to different
molecular markers. García–R et al. (2014) used 12 PCGs,
tRNA and rRNA gene sequences to construct phylogenetic
trees, while Gong et al. (2017) used the complete sequences
of mitochondrial genome (The CR sequence was removed)
to construct a phylogenetic tree. Therefore, the accuracy of
the phylogenetic tree can only be compared by the support
rate and the posterior probability, and there was no more
acceptable denition (Zheng, 2015). However, the species
data contained in this study was more comprehensive, and
the sequence information also contains a higher posterior
probability value, which has a higher degree of credibility.
In addition, according to the morphological classication,
Gallicrex cinerea was considered to be a bird of the genus
Gallicrex, and Gallirallus striatus belonged to the genus
Gallirallus (Zheng, 2012). However, this study believes
that Gallicrex cinerea and Amaurornis phoenicurus were
sisters and should be classied as the genus Amaurornis. In
addition, Gallirallus striatus and Lewinia muelleri belong
to sister group, preferring the genus Lewinia. Regarding the
classication of the genus Gallicrex, Ruan et al. (2012) and
Gong et al. (2017) also believed that it should be classied as
the genus Amaurornis, rather than the traditionally believed
the genus Gallicrex. The classication results of Gallirallus
striatus need further research and demonstration.
According to the analysis of this study, the divergence
time of Neoaves was 64.84 (65.83 ~ 63.82) Ma, while the
divergence time of Galloanseres was 65.91 (66.89 ~ 64.94)
Ma and diverged about the same time as Neoaves. This
result was basically consistent with the ndings of Jarvis et
al. (2014) and Prum et al. (2015). The basal divergence time
of Gruiformes was 46.33 (58.46 ~ 25.60) Ma, which was
less than that of Jarvis et al. (2014) (65.00 Ma) and Fain et
al. (2007) (66.40 Ma). The diverging time of 41.36 (53.10
P. Chen et al.
437
~ 24.18) Ma of Ralli was similar to that of García–R et al.
(2014) (40.10 Ma), and was consistent with the fossil record
of two ancient birds of Ralli (Mayr, 2009; García–R et al.,
2014). The reasons for the differences in the divergence
time of Gruiformes include the difference in the amount of
data used and the reference point used for the estimation
of the divergence time. Prum et al. (2015) and Jarvis et al.
(2014) used relatively little data on Gruiformes, and even
used only one species instead of the whole order to estimate
the time difference, which may lead to some deviation in
their results. Fain et al. (2007) used the fossil records of
Rallidae and Heliornithidae and compared the results of
previous studies (Krajewski and Fetzner, 1994; Houde,
2009), and set 43 Ma as the reference base point for the
estimation of Rallidae and Heliornithidae as the reference
base point, which was not authoritative and accurate enough
(García–R et al., 2014).
The evolutionary analysis results of Charadriiformes
were basically consistent with previous studies (Smith and
Clarke, 2015; Cheng, 2017; Hu et al., 2017), including
the three branches of Charadrii, Lari and Scolopaci. Lari
and Scolopaci form a sister group, while Charadrii was a
separate branche. Among them, Hydrophasianus chirurgus
of Jacanidae, which was closely related to Rostratulidae.
Cheng et al. (2017) studied the phylogenetic tree of 18
species of Charadriiforme, which were also divided into
Charadrii, Lari and Scolopaci, and analysed the phylogenetic
evolution of 12 species of Charadriiformes, then concluded
that Recurvirostridea and Charadriidae belonged to
Charadrii (Chen, 2003). The results of the evolution analysis
of Charadriiformes were basically the same as those of
the traditional classication, but there were differences
between Gelochelidon nilotica and Pluvianellus socialis.
Traditionally, Gelochelidon nilotica belonged to Laridae,
and Pluvianellus socialis belonged to Charadriidae (Zheng,
2012). However, this study suggested that Gelochelidon
nilotica belonged to Sternidae as a sister group to Thalasseus
bengalensis and Sterna hirundo. Pluvianellus socialis were
sisters of Chionis minor, belonging to Chionididae. There
were no other molecular methods for classifying the two
species, and the results need to be further conrmed.
The estimated divergence time for Charadriiformes
was 45.44 (58.21 ~ 24.67) Ma, lower than that of Jarvis
et al. (2014) (65 Ma) and Fain et al. (2007) (74.3 Ma),
but consistent with that of Prum et al. (2015) (55 Ma).
According to the time difference of each suborder, Charadrii
species rst began to differentiate, followed by Lari, and
nally Scolopaci, which also conrmed the prediction of
Chen et al. (2003) on the differentiation of the suborder of
Charadriiformes. This study ensured accurate and reliable
estimation of the divergence time of Gruiformes and
Charadriiformes species through fossil record calibration
(Prum et al., 2015) and sufcient molecular data support
ACKNOWLEDGEMENTS
This study was supported by the National Science
Foundation of China (NSFC no. 31260510, no. 30960052
and no. 31960107) and Water Resources Department of
Jiangxi Province Science and Technological Project (no.
KT201537).
Supplementary material
There is supplementary material associated with
this article. Access the material online at: https://dx.doi.
org/10.17582/journal.pjz/20190603010623
Conict of interest statement
The authors report no conicts of interest and were
alone responsible for the content and writing of the paper.
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