Complete Mitochondrial Genome of Blue-Throated Bee-eater Merops viridis (Coraciiformes: Meropidae) with its Taxonomic Consideration
Complete Mitochondrial Genome of Blue-Throated Bee-eater Merops viridis (Coraciiformes: Meropidae) with its Taxonomic Consideration
Zuhao Huang1, Feiyun Tu2 and Dianhua Ke1*
1School of Life Sciences, Jinggangshan University, Ji’an, 343009, Jiangxi Province, China
2Jiangxi Academy of Forestry, Nanchang, 330013, Jiangxi Province, China
Zuhao Huang and Feiyun Tu contributed equally to this work.
Blue-throated Bee-eater Merops viridis (Coraciiformes: Meropidae) is a bird species of family Meropidae with a very large distribution. The complete mitochondrial genome of Blue-throated Bee-eater M. viridis was determined. The mitogenome is a circular DNA molecule of 18,295 bp and comprises of 13 protein-coding genes, 22 tRNA genes, two rRNA genes and two control regions CR and CCR, which was first reported in the order Coraciiformes. The overall A+T content for the mitogenome is 52%, and the GC and AT skews are -0.400 and 0.108. Unlike to many other birds, no extra base is inserted at certain position relative to ND3. Interestingly, a 484bp repeated sequence appears in both CR and CCR. Genetic distance shows that the differences between M. v. viridis and M. v. americanus is higher than that M. v. viridis vs M. leschenaulti based on Cyt b gene. Morphology, geographical distribution and genetic data suggest the elevation of M. v. americanus to a full species Rufous-crowned Bee-eater M. americanus, supporting the latest taxonomic arrangement by IUCN.
Received 25 July 2016
Revised 09 August 2016
Accepted 21 August 2016
Available online 25 October 2016
ZH, FT and DK designed the study and wrote the arctile. TF analyzed the sequence data and submitted to the Genbank.
Merops viridis, Mitochondrial genome, Cytb, Gene rearrangement, Taxonomic consideration.
* Corresponding author: email@example.com
0030-9923/2017/0001-0081 $ 9.00/0
Copyright 2017 Zoological Society of Pakistan
The bee-eaters (family Meropidae) comprise a group of 25 species of birds with brightly colored, morphologically homogeneous (). They harbor unique feeding habits, and predominantly eat flying insects, especially bees and wasps (). Blue-throated Bee-eater (Merops viridis) is a species of bird in the family Meropidae. The species has a very large distribution ranging from southern China and South-East Asia to the Greater Sundas and the Philippines (; ). Its natural habitat is subtropical or tropical mangrove forests. The species is characterized by a high ornamental value and listed in species checklists of conservation, economic and scientific values.
So far, only partial fragments of its mitochondrial DNA (mtDNA) (Cytb, 12S, ND1 and ND2) were determined. Hitherto, no complete mitogenomic data was reported in the family Meropidae, which has more than 20 species (). Moreover, only six complete mitochondrial genome sequences (Eurystomus orientalis, Halcyon coromanda, H. pileata, H. smyrnensis, Todiramphus sanctus and Upupa epops) were available (; , ; ; ; ) in the order Coraciiformes, which comprises over 250 species ().
Two subspecies (M. v. viridis and M. v. americanus) were generally recognized (, ; ). However, elevated the subspecies M. v. americanus to species Rufous-crowned Bee-eater M. americanus based on the study of , which reported a 3.8% genetic divergence between viridis and americanus based on ND2 gene but made no suggestion for species divergence of two subspecies. Recently, M. americanus was treated as a distinct species by the IUCN (). Therefore, disputes remain as to the species placement of M. americanus (, ; ; ; ). In the study, we tentatively accepted the taxonomy of , .
Herein, we first presented the complete mtDNA (M. viridis) of avian family Meropidae and investigated the placement of M. americanus.
Material And Methods
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.
A specimen of M. viridis was collected from Zhangshan township, Jizhou district, Ji’an city, Jiangxi Province, China (latitude: 27.184o N, longitude: 115.048o E) and the muscle sample and the corresponding voucher specimen (zjbj4) were vouched in the Jinggangshan University.
DNA extraction, amplification and sequencing
Total genomic DNA was extracted from muscle tissue using a standard phenol-chloroform extraction protocol (). Fourteen primers combinations () were designed as reference to an alignment of sequences from published mitochondrial genomes of related species (Todiramphus sanctus and Upupa epops). The overlapping PCR products ranged from 727 to 2,131 bp in length. Primers used for the generation of PCR products and the sizes of the fragments are given in .
PCR protocol and amplification condition were set as follows the study of and made several modifications. All PCR products were examined through electrophoresing on a 1.0% agarose gel and purified with the DNA Agarose Gel Extraction Kit (Omega, Norcross, Georgia, USA) and then directly sequenced with the primer walking method () in an ABI 3730xl sequencer.
All amplified sequences were assembled and edited using the software package SeqMan (DNASTAR 7.1.0) (). The boundaries of protein-coding genes (PCGs) were predicted by homologous sequences of other related species E. orientalis using the software Mega 6.0 (). The transfer RNA (tRNA) genes were identified using tRNA-scan SE 1.21 (). The tRNA-Lys, tRNA-Cys and tRNA-Ser (AGY) genes, which were not found using tRNA-scan SE, were identified by the observation of already proposed secondary rRNA structures (). The maps of the mitochondrial genome and noncoding regions of M. viridis () was drawn by SeqBuilder (DNASTAR 7.1.0).
Subspecies identification and genetic distance analysis
Two mtDNA fragments of ND2 (GenBank No. EU166941) and Cytb (EU167003) were isolated from the same individual, which was treated as M. viridis (). We performed online nblast and tree-based method to delimit the specific subspecies, which was identified as M. viridis in this study and . The nblast () was done based on the complete ND2 gene sequence. The query sequence is assigned to species with 99% of identity. Two methods of building phylogenetic trees consisting of Neighbor joining (NJ) and Bayesian inference (BI) were conducted. NJ tree was used as implemented in Mega 6.0 based on an alignment of 1041bp partial ND2 genes () with Kimura two-parameter (K2P) model (). BI tree was performed using MrBayes 3.1.2 () and following the parameters of .
To investigate the placement of americanus, we retrieved 4 Cytb sequences representing three closely related species of Merops: M. v. viridis (KU821702) (this study), M. leschenaulti (KJ456339), M. orientalis (KJ456340) and M. viridis (EU167003). Genetic distance among pairs of species was calculated using Mega 6.0 with the K2P model.
RESULTS AND DISCUSSION
Genome composition and organization
A comparison analysis performed between the newly sequenced and Coraciiformes mitochondrial genomes indicated that the genomes were similar in many respects (). The total length of the mtDNA genomes varied from 16,562 to 18,295bp (). The nucleotide composition of M. viridis was 28.6% A, 23.4% T, 14.4% G, and 33.6% C, and a A+T bias (52%) was found in this genome, as similar to the A+T rich pattern of other avian (). To investigate the nucleotide bias, skew for a given strand was calculated (). The GC and AT skew values for the mitogenome of were -0.400 and 0.108, respectively, indicating more C and A than G and T. The result was consistent with a bias against the use of G in the vertebrates (; ). The AT skew and GC skew values of mitochondrial genome sequences for Coraciiformes were consistent with the rule that the AT skew was positive while the GC skew was negative in amniote mtDNA ().
|Species||Accession number||Total length (bp)||A(%)||T(%)||G(%)||C(%)||A+T content (%)||AT skew||GC skew||Refer ences|
|Merops viridis||KU82 1702||18295||28.6||23.4||14.4||33.6||52||0.108||-0.400||This study|
|Halcyon coromanda||NC_ 028177||16542||30.2||23.5||13.7||32.5||53.7||0.145||-0.407|
|Halcyon pileata||NC_ 024198||17612||30.6||23.1||13.9||32.4||53.7||0.162||-0.400|
|Todira mphus sanctus||NC_ 011712||17549||31.3||24||13.6||31.2||55.3||0.163||-0.393|
|Eurysto mus orientalis||NC_ 011716||17210||30.1||23.3||14||32.6||53.4||0.146||-0.399|
|Upupa epops||NC_ 028178||16562||31.5||23.9||14||30.6||55.4||0.170||-0.372|
The 13 PCGs of M. viridis contain NADH dehydrogenase genes (Nd1, Nd2, Nd3, Nd4l Nd4, Nd5 and Nd6), cytochrome c oxidase genes (Cox1, Cox2 and Cox3), ATPase (Atp8 and Atp6) and Cytb (). Twelve PCGs are encoded on the H-strand except ND6, which is located on the L-strand. All PCGs initiate with the common start codon ATG, except for Nd3, which begins with ATC (). Five types of stop codons are used by the PCGs, including TAA for Nd1, Cox2, Atp8, Atp6, Nd3, Nd4l and Cytb, TAG for Nd2 and Nd6, AGG for Cox1, AGA for Nd5, and T for Cox3 and Nd4. It is notable that an extra nucleotide is observed at position relative to ND3 sequence in many taxa of bird species (). However, the extra is undetected in the ND3 gene, as similar to some other bird species, e.g. Geococcyx californianus, Lanius collurio ().
tRNA genes and rRNA genes
As similar to most metazoans, the complete mtDNA of M. viridis comprises of 22 tRNAs and two rRNAs (12S rRNA and 16S rRNA) (). The tRNAs sequences range in size from 66 to 84 bp, and all tRNAs have the potential to fold into a complete cover leaf, with the exclusion of tRNA-Ser (AGY), which lacks the DHU stem. The 12S rRNA occurs between tRNA-Phe and tRNA-Val and 16S rRNA locates between tRNA-Val and tRNA-Leu (UUR).
Noncoding regions and gene rearrangement
The CR (1289bp) is located between tRNA-Thr and tRNA-Pro, whereas CCR (1490bp) is flanked by tRNA-Glu and tRNA-Phe (). The CR is comprised of three domains, corresponding to the highly variable peripheral domain I (nt 1-545) and III (nt 986-1289) and to the central conserved domain II (nt 546-985) (). In Domain I, extended termination associated sequences 1 (ETAS1) is observed in position nt 148-225 (). Four conserved sequence boxes (C, D, E and F) are identified in Domain II. CBS1 (nt 1058-1079) occurred in Domain III (). High sequence similarity appears between CR and CCR. A 484bp tandem repeat appears in Part A of CR and CCR (). Interestingly, the 7-bp short sequence motif (CAACAAA) repeats 15 times in Part B (nt 1293-1397) (), and the repeated motif is also found in other species (;). Additionally, two noncoding regions (CR and CCR) are also found in the other voucher specimen (zjbj5) of M. viridis.
The mitochondrial gene order of chicken (Gallus gallus) was treated as typical avian gene order (; ). discovered a new mitochondrial gene rearrangement in Falcon peregrinus, which one more noncoding sequence was found between tRNA-Glu and tRNA-Phe genes except the original control region between tRNA-Thr and tRNA-Pro. In this study, we first discovered the rearrangement of CCR in order Coraciiformes () and the results were similar to several representatives of Passeriformes, Procellariiformes, Cuculiformes, Piciformes and Psittaciformes (; ; ). The sequence of high similarity between CR and CCR found in M. viridis also supporting the duplication hypothesis (), which was also found in the study of Accipiter virgatus (). The short repeated motif (CAACAAA) was found in mitochondrial control regions of Alca torda and H. pileata rather than that of H. coromanda (). However, no relationships were found in these species.
Subspecies identification and genetic distance
|M. v. viridis||M. leschenaulti||M. orientalis||M. v. americanus|
M. v. viridis (1143bp)
M. leschenaulti (1053bp)
M. orientalis (1143bp)
M. v. americanus (1143bp)
Methods of genetic divergence based on mtDNA play an important role in investigating species designation (). In the present study, the genetic distance analysis showed that the intraspecific genetic divergence (viridis and americanus) was higher that the interspecific divergence. Moreover, morphological differentiations were detected between nominotypical viridis and americanus, which had the blue of the throat and upper breast reduced to a slight tinge spreading the malar area (). The nominal subspecies viridis mainly ranges southern China and South-East Asia to the Greater Sundas and the subspecies americanus is confined to the Philippines (; ). Combined the morphological, distribution and genetic divergence data, we confirm that the M. americanus should be treated as full species status, supporting the taxonomic arrangement of americanus by the IUCN ().
The study was supported by National Natural Science Foundation of China (No. 31160423, 31260088, 31560590), Jiangxi Province Talent Project 555, Jiangxi Province Major Disciplines Academic Leaders (20133BCB22010), the Natural Science Foundation of Jiangxi Province (20132BAB204022, 20152ACB21006), and the Science and Technology Foundation of Jiangxi Provincial Department of Education (GJJ13547, GJJ150768).
Conflict of interest statement
The authors declare that there is no conflict of interests regarding the publication of the manuscript.
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