Abstract

Introduction

With more than 9,000 living species, Aves is the most diverse class of vertebrates. The huge number of species, complex morphological characters and wide range of ecological behaviors make it difficult to solve the phylogenetic relationship of birds in traditional taxonomy (Bock, 1956Bock WJ (1956) A generic review of the family Ardeidae (Aves). Am Mus Novit 1779:1–49.; Howard and Moore, 1980Howard R and Moore A (1980) A Complete Checklist of the Birds of the World. Oxford University Press, Oxford, pp 1–12.; Monroe and Sibley, 1993Monroe BL and Sibley CG (1993) A World Checklist of Birds. Yale University Press, New Haven, 400 pp.).

The order Ciconiiformes, consisting of more than 110 species of large or medium size waders, has traditionally be classified into five families (Ciconiidae, Threskiornithidae, Ardeidae, Balaenicipitidae and Scopidae) (Howard and Moore, 1980Howard R and Moore A (1980) A Complete Checklist of the Birds of the World. Oxford University Press, Oxford, pp 1–12.; Austin, 1985Austin OL (1985) Families of Birds. Golden Press, New York, pp 7–37.; Gill, 1990Gill FB (1990) Ornithology. WH Freeman Company Press, New York, pp 522–524.; Clements, 2000Clements JF (2000) Birds of the World: A Checklist. 5th edition. Ibis Publishing Company Press, Vista, pp 18–25.; Zheng, 2002Zheng GM (2002) A Checklist on the Classification and Distribution of the Birds of the WorId. Science Press, Beijing, 11 p.). However, there have been various uncertainties regarding the evolutionary relationships of different taxa in this order: (1) The phylogenetic relationships among the five families have been questioned in morphological studies (Kahl, 1972Kahl MP (1972) A revision of the family Ciconiidae (Aves). J Zool 167:451–461.; Cracraft, 1981Cracraft J (1981) Toward a phylogenetic classification of the recent birds of the world (Class Aves). Auk 98:681–714.), (2) the Family Ardeidae was divided into two subfamilies (Ardeinae and Botaurinae) by Bock (1956)Bock WJ (1956) A generic review of the family Ardeidae (Aves). Am Mus Novit 1779:1–49. and Zheng (1997)Zheng ZX (1997) Fauna Sinica Aves. Vol. 1. Science Press, Beijing, pp 138–140., but into four subfamilies (Ardeinae, Nycticoracinae, Botaurinae and Tigrisomatinae) by Payne and Risley (1976)Payne RB and Risley CJ (1976) Systematics and evolutionary relationships among the herons (Ardeidae). Misc Publ Univ Mich Mus Zool 150:1–115., and (3) the phylogenetic status of several species in the traditional classification of the subfamily Ardeinae has been questioned. For example, the great egret was initially placed in an independent genus Casmerodius (Peter, 1931Peter JL (1931) Checklist of Birds of the World. Harvard University Press, Cambridge, 110 pp.), but was put in Egretta by Bock (1956)Bock WJ (1956) A generic review of the family Ardeidae (Aves). Am Mus Novit 1779:1–49. and Ardea by Payne and Risley (1976)Payne RB and Risley CJ (1976) Systematics and evolutionary relationships among the herons (Ardeidae). Misc Publ Univ Mich Mus Zool 150:1–115.. Similarly, the intermediate egret was initially included in Egretta, but then placed in Mesophoyx by Sibley and Monroe (1990)Sibley CG and Monroe BL (1990) Distribution and Taxonomy of Birds of the World. Yale University Press, New Haven and London, pp 302–310.. The taxonomic position of the cattle egret had also changed many times; in early taxonomic literature this species belonged to Bubulcus (Peter, 1931Peter JL (1931) Checklist of Birds of the World. Harvard University Press, Cambridge, 110 pp.), but was subsequently placed in Ardeola by Bock (1956)Bock WJ (1956) A generic review of the family Ardeidae (Aves). Am Mus Novit 1779:1–49. and in Egretta by Payne and Risley (1976)Payne RB and Risley CJ (1976) Systematics and evolutionary relationships among the herons (Ardeidae). Misc Publ Univ Mich Mus Zool 150:1–115..

Genome sequences, which provide direct information on evolutionary history, are perfect markers for phylogenetic studies since the resulting analyses can be used to assess and revise the conclusions of traditional taxonomy. In the last 30 years, molecular investigations have shed new light on the evolutionary history of the Ciconiiformes. Based on DNA hybridization results, Sibley et al. (1988)Sibley CG, Ahlquist JE and Monroe BL (1988) A classification of the living birds of the world based on DNA-DNA hybridization studies. Auk 105:409–423. merged Ciconiiformes and four other orders (Gaviiformes, Podicipediformes, Lariformes and Charadriiformes) into a huge new order. However, recent molecular studies have proposed the paraphyly of Ciconiiformes because the herons and ibises in this group showed a close relationship with Pelecaniformes, whereas the storks were closely related to Sphenisciformes (Hedges and Sibley, 1994Hedges SB and Sibley CG (1994) Molecules vs. morphology in avian evolution: The case of the “pelecaniform” birds. Proc Natl Acad Sci USA 91:9861–9865.; Cracraft et al., 2004Cracraft J, Barker FK, Braun MJ, Harshman J, Dyke GJ, Feinstein J, Stanley S, Cibois A, Schikler P, Beresford P, et al. (2004) Phylogenetic relationships among modern birds (Neornithes). Toward an avian tree of life. In: Cracraft J and Donoghue MJ (eds) Assembling the Tree of Life. Oxford University Press, New York, pp 468–489.; Hackett et al., 2008Hackett SJ, Kimball RT, Reddy S, Bowie RC, Braun EL, Braun MJ, Chojnowski JL, Cox WA, Han KL, Harshman J, et al. (2008) A phylogenomic study of birds reveals their evolutionary history. Science 320:1763–1768.; Pacheco et al., 2011Pacheco MA, Battistuzzi FU, Lentino M, Aguilar RF, Kumar S and Escalante AA (2011) Evolution of modern birds revealed by mitogenomics: Timing the radiation and origin of major orders. Mol Biol Evol 28:1927–1942.). The North American Classification Committee (NACC) has recommended that the families Ardeidae, Threskiornithidae, Balaenicipitidae and Scopidae be merged into Pelecaniformes, and Ciconiiformes was restricted to include only the Ciconiidae.

Molecular studies of the Ardeidae have indicated that day herons and night herons are closely related, and that Nycticoracinae should be merged into Ardeinae, while the tiger herons and boat-billed heron were basal lineages and should be placed in the Tigrisomatinae and Cochleariinae, respectively (Sheldon, 1987Sheldon FH (1987) Rates of single-copy DNA evolution in herons. Mol Biol Evol 4:56–69.; Sheldon and Kinnarney, 1993Sheldon FH and Kinnarney M (1993) The effects of sequence removal on DNA-hybridization estimates of distance, phylogeny, and rates of evolution. Syst Biol 42:32–48.; Sheldon et al., 1995Sheldon FH, McCracken KG and Stuebing KD (1995) Phylogenetic relationships of the zigzag heron (Zebrilus undulatus) and white-crested bittern (Tigriornis leucolophus) estimated by DNA-DNA hybridization. Auk 112:672–679., 2000Sheldon FH, Jones CE and McCracken KG (2000) Relative patterns and rates of evolution in heron nuclear and mitochondrial DNA. Mol Biol Evol 17:437–450.). This four-subfamily classification (Ardeinae, Botaurinae, Tigrisomatinae and Cochleariinae) has been generally accepted. Molecular investigations of the subfamily Ardeinae have shown that the great egret and intermediate egret form a monophyletic lineage that is more closely related to Ardea than to Egretta, indicating that they should not be placed in Egretta (Sheldon, 1987Sheldon FH (1987) Rates of single-copy DNA evolution in herons. Mol Biol Evol 4:56–69.; Sibley and Monroe, 1990Sibley CG and Monroe BL (1990) Distribution and Taxonomy of Birds of the World. Yale University Press, New Haven and London, pp 302–310.; Sheldon and Kinnarney, 1993Sheldon FH and Kinnarney M (1993) The effects of sequence removal on DNA-hybridization estimates of distance, phylogeny, and rates of evolution. Syst Biol 42:32–48.; Sheldon et al., 1995Sheldon FH, McCracken KG and Stuebing KD (1995) Phylogenetic relationships of the zigzag heron (Zebrilus undulatus) and white-crested bittern (Tigriornis leucolophus) estimated by DNA-DNA hybridization. Auk 112:672–679., 2000Sheldon FH, Jones CE and McCracken KG (2000) Relative patterns and rates of evolution in heron nuclear and mitochondrial DNA. Mol Biol Evol 17:437–450.; Chang et al., 2003Chang Q, Zhang BW, Jin H, Zhu LF and Zhou KY (2003) Phylogenetic relationships among 13 species of herons inferred from mitochondrial 12S rRNA gene sequences. Acta Zool Sin 49:205–210 (in Chinese with English abstract).).

In molecular systematics, the topologies of phylogenetic trees vary with the molecular markers used and the number of taxa involved (Zwickl and Hillis, 2002Zwickl DJ and Hillis DM (2002) Increased taxon sampling greatly reduces phylogenetic error. Syst Biol 51:588–598.). Consequently, some phylogenetic uncertainties in the Ardeinae (such as the evolutionary status of the cattle egrets Ardeola and Butorides) have not been resolved (Chang et al., 2003Chang Q, Zhang BW, Jin H, Zhu LF and Zhou KY (2003) Phylogenetic relationships among 13 species of herons inferred from mitochondrial 12S rRNA gene sequences. Acta Zool Sin 49:205–210 (in Chinese with English abstract).; Zhou XP, 2008, PhD thesis, Xiamen University, China).

Mitochondrial DNA (mtDNA), with its intrinsic characteristics (small genome size, simple genome structure, exclusively maternal inheritance, lack of extensive recombination and rapid rate of evolution), has been extensively used in taxonomic and phylogenetic studies of vertebrates (Ingman et al., 2000Ingman M, Kaessmann H, Paabo S and Gyllensten U (2000) Mitochondrial genome variation and the origin of modern humans. Nature 408:708–713.; Sheldon et al., 2000Sheldon FH, Jones CE and McCracken KG (2000) Relative patterns and rates of evolution in heron nuclear and mitochondrial DNA. Mol Biol Evol 17:437–450.; Gentile et al., 2009Gentile G, Fabiani A, Marquez C, Snell HL, Snell HM, Tapia W and Sbordoni V (2009) An overlooked pink species of land iguana in the Galápagos. Proc Natl Acad Sci USA 106:507–511.; Zhang and Wake, 2009Zhang P and Wake DB (2009) Higher-level salamander relationships and divergence dates inferred from complete mitochondrial genomes. Mol Phylogenet Evol 53:492–508.; Pacheco et al., 2011Pacheco MA, Battistuzzi FU, Lentino M, Aguilar RF, Kumar S and Escalante AA (2011) Evolution of modern birds revealed by mitogenomics: Timing the radiation and origin of major orders. Mol Biol Evol 28:1927–1942.; Suzuki et al., 2013Suzuki H, Nunome M, Kinoshita G, Aplin KP, Vogel P, Kryukov AP, Jin ML, Han SH, Maryanto I, Tsuchiya K, et al. (2013) Evolutionary and dispersal history of Eurasian house mice Mus musculus clarified by more extensive geographic sampling of mitochondrial DNA. Heredity 111:375–390.). Compared to individual genes, complete mitogenomes contain more information on an organisms or taxon’s evolutionary history, reduce stochastic errors and minimize the effect of homoplasy in phylogenetic studies (Campbell and Lapointe, 2011Campbell V and Lapointe FJ (2011) Retrieving a mitogenomic mammal tree using composite taxa. Mol Phylogenet Evol 58:149–156.). Phylogenies based on complete mitogenomes are generally consistent with those derived from nuclear genes if appropriate sampling of taxa and analysis are applied (Arnason et al., 2002Arnason U, Adegoke JA, Bodin K, Born EW, Esa YB, Gullberg A, Nilsson M, Short RV, Xu X and Janke A (2002) Mammalian mitogenomic relationships and the root of the eutherian tree. Proc Natl Acad Sci USA 99:8151–8156.; Reyes et al., 2004Reyes A, Gissi C, Catzeflis F, Nevo E, Pesole G and Saccone C (2004) Congruent mammalian trees from mitochondrial and nuclear genes using Bayesian methods. Mol Biol Evol 21:397–403.; Kjer and Honeycut, 2007Kjer KM and Honeycut RL (2007) Site specific rates of mitochondrial genomes and the phylogeny of Eutheria. BMC Evol Biol 7:e8.). Complete mitogenomes have increasingly been used to address the evolution and radiation of birds (Moum et al., 1994Moum T, Johansen S, Erikstad KE and Piatt JF (1994) Phylogeny and evolution of the auks (subfamily Alcinae) based on mitochondrial DNA sequences. Proc Natl Acad Sci USA 91:7912–7916.; Sato et al., 1999Sato A, O’hUigin C, Figueroa F, Grant PR, Grant BR, Tichy H and Klein J (1999) Phylogeny of Darwin’s finches as revealed by mtDNA sequences. Proc Natl Acad Sci USA 96:5101–5106.; Pacheco et al., 2011Pacheco MA, Battistuzzi FU, Lentino M, Aguilar RF, Kumar S and Escalante AA (2011) Evolution of modern birds revealed by mitogenomics: Timing the radiation and origin of major orders. Mol Biol Evol 28:1927–1942.). To date, more than 260 avian mitogenomes have been deposited in GenBank, only four of which involve species belonging to the Ardeidae (Egretta eulophotes, Ardea novaehollandiae, Ixobrychus cinnamomeus and Nycticorax nycticora). The lack of complete mitogenome data is an important limitation in solving the evolutionary puzzles of the Ardeidae and Ciconiiformes.

In this report, we describe the complete mitogenome sequence of the little egret (Egretta garzetta) and provide a comprehensive analysis of its genome characters. Although the phylogenetic status of this species has been well-defined by morphological and molecular studies (Bock, 1956Bock WJ (1956) A generic review of the family Ardeidae (Aves). Am Mus Novit 1779:1–49.; Payne and Risley, 1976Payne RB and Risley CJ (1976) Systematics and evolutionary relationships among the herons (Ardeidae). Misc Publ Univ Mich Mus Zool 150:1–115.; McCracken and Sheldon, 1997McCracken KG and Sheldon FH (1997) Avian vocalizations and phylogenetic signal. Proc Natl Acad Sci USA 94:3833–3836.; Rabosky and Matute, 2013Rabosky DL and Matute DR (2013) Macroevolutionary speciation rates are decoupled from the evolution of intrinsic reproductive isolation in Drosophila and birds. Proc Natl Acad Sci USA 110:15354–15359.), the availability of its complete mitogenome data will provide useful information for molecular phylogenetic studies and conservation biology of the Ardeidae.

Material and Methods

Sample collection and extraction of genomic DNA

One specimen of E. garzetta was collected from Wuyi Mountain, Fujian Province, China. The specimen was identified based on external characteristics, using the system of Sibley and Monroe (1990)Sibley CG and Monroe BL (1990) Distribution and Taxonomy of Birds of the World. Yale University Press, New Haven and London, pp 302–310.. Total genomic DNA was extracted from muscle tissue with a Wizard Genomic DNA purification kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The concentration of extracted DNA was determined using a spectrophotometer and adjusted to 50 ng/μL.

PCR amplification and sequencing

The E. garzetta mtDNA was obtained by polymerase chain reactions (PCR) using 28 primer sets reported by Sorenson et al. (1999)Sorenson MD, Ast JC, Dimcheff DE, Yuri T and Mindell DP (1999) Primers for a PCR-based approach to mitochondrial genome sequencing in birds and other vertebrates. Mol Phylogenet Evol 12:105–114.. The PCR products for each set of primers were < 1,500 bp in size and all fragment sequences overlapped each other by at least 200 bp. PCR amplifications were done with a Mycycler Gradient thermocycler (Bio-Rad) in a final volume of 50 μL, including 5 μL of 10x EXTaq buffer (Mg²⁺-free; Takara Biotech, Dalian, China), 2.5 mM of each dNTP, 75 mM MgCl₂, 10 μM of each primer, 1.5 U of ^EXTaq polymerase (Takara of Biotech, Dalian, China) and approximately 20–50 ng of total genomic DNA. The reaction included an initial denaturation at 94 °C for 3 min, followed by 35 cycles consisting of denaturation at 94 °C for 10 s, annealing at 50–56 °C for 30 s and extension at 72 °C for 2 min, with a final extension at 72 °C for 10 min. There was a negative control in each round of PCR to check for contamination. The products were electrophoresed on 1.5% agarose gels staining with ethidium bromide and visualized by ultraviolet transillumination. The PCR products were purified with a gel extraction kit (Sangon BioMedical, Shanghai, China) and directly sequenced (both directions) with an ABI 3730XL automatic sequencer (Perkin-Elmer) using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit (with AmpliTaq DNA polymerase FS, Applied Biosystems).

Sequence assembly, annotation and analysis

Sequence assembly and annotation were done using the DNASTAR software package (Lasergene version 5.0; Madison, WI, USA). The boundaries of protein-coding genes and rRNA genes were determined by aligning our sequences with the complete mtDNA sequences of A. novaehollandiae (NC_008551) and Gallus gallus (NC_001323; Galliformes: Phasianidae) in GenBank. The boundaries and the cloverleaf secondary structures of tRNAs were identified by tRNAscan-SE v 1.12 with the default settings. The complete nucleotide sequence was submitted to GenBank under accession no. NC_023981 and the blast sequences are submitted to DRYAD (doi:10.5061/dryad.3g604). The base composition for protein-coding genes (PCGs), the codon usage of 13 PCGs and the pairwise distances among mitogenomes of the species studied were calculated with MEGA version 5 (Tamura et al., 2011Tamura K, Peterson D, Peterson N, Stecher G, Nei M and Kumar S (2011) MEGA5: Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739.).

Phylogenetic inference using mitogenomes

The phylogenetic relationships among E. garzetta and four other species in the Ardeidae (A. novaehollandiae, E. eulophotes, I. cinnamomeus and N. nycticorax), four species in the Threskiornithidae (Platalea leucorodia, Platalea minor, Threskiornis aethiopicus and Nipponia nippon) and two species in the Ciconiidae (Ciconia boyciana, Ciconia ciconia) were constructed with complete mtDNA sequences and 12 PCGs (excluding ND6). Two species in the family Anatidae, order Anseriformes (Branta canadensis, NC_007011; Anas platyrhynchos, EU009397) were designated as outgroups. The relevant information for each genome is presented in Table S1.

The program Modeltest version 3.7 (Posada and Crandall, 1998Posada D and Crandall KA (1998) Modeltest: Testing the model of DNA substitution. Bioinformatics 14:817–818.) was used to choose an appropriate substitution model of sequence evolution. The GTR+I+G model was selected as the best fitting model. For the Bayesian procedure, four independent Markov chains were run for 10,000,000 generations by sampling one tree per 1,000 generations and allowing adequate time for convergence. After discarding the first 2,500 trees (25%) as part of a burn-in procedure that was determined by checking for the likelihood of being stationary, we used the remaining 7,500 sampling trees to construct a 50% majority rule consensus tree. Two independent runs were used to provide additional confirmation of the convergence of the Bayesian posterior probabilities (BPP) distribution.

Results and Discussion

Genome organization and base composition

The complete mitogenome of E. garzetta is a circular molecule 17,361 bp in length (Figure 1). This size is intermediate to all available ardeid mitogenomes, which range from 17,180 bp (I. cinnamomeus; Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.) to 17,829 bp (N. nycticorax, NC_015807). The gene organization is identical to that of typical avian mtDNA (Wolstenholme, 1992Wolstenholme DR (1992) Animal mitochondrial DNA: Structure and evolution. Int Rev Cytol 141:173–216.; Boore, 1999Boore JL (1999) Animal mitochondrial genomes. Nucleic Acids Res 27:1767–1780.; Roques et al., 2004Roques S, Godoy JA, Negro JJ and Hiraldo F (2004) Organization and variation of the mitochondrial control region in two vulture species, Gypaetus barbatus and Neophron percnopterus. J Hered 95:332–337.; Gibb et al., 2007Gibb GC, Kardailsky O, Kimball RT, Braun EL and Penny D (2007) Mitochondrial genomes and avian phylogeny: Complex characters and resolvability without explosive radiations. Mol Biol Evol 24:269–280.; Kan et al., 2010Kan XZ, Li XF, Zhang LQ, Chen L, Qian CJ, Zhang XW and Wang L (2010) Characterization of the complete mitochondrial genome of the rock pigeon, Columba livia (Columbiformes, Columbidae). Genet Mol Res 9:1234–1249.; Zhang, et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.; Figure 1). Table 1 shows the various features of this genome. There are six regions in which genes overlapped by 29 bp and 18 intergenic spacer regions comprising a total of 97 bp.

The base composition of the E. garzetta mitogenome revealed a slight bias towards A+T (31.5% A, 23.2% T, 31.8% C and 13.5% G). The A+T content for the whole H-strand, different genes and control regions was estimated for 11 mitogenomes in Ciconiiformes (Table 2). This analysis showed that, except for the first codon of PCGs, other portions of these mitogenomes showed varying degrees of preference for A/T. The equations AT-SKEW= (A−T)/(A+T) and GC-SKEW= (G−C)/(G+C) can be used to calculate the skew for a given strand to investigate nucleotide bias (Perna and Kocher, 1995Perna NT and Kocher TD (1995) Patterns of nucleotide composition at fourfold degenerate sites of animal mitochondrial genomes. J Mol Evol 41:353–358.). The positive AT-skew (0.138) and negative GC-skew (−0.399) for the E. garzetta mitogenome suggested the occurrence of more A and C than T and G, which is consistent with other avian mitogenomes (Haring et al., 2001Haring E, Kruckenhauser L, Gamauf A, Riesing MJ and Pinsker W (2001) The complete sequence of the mitochondrial genome of Buteo buteo (Aves, Accipitridae) indicates an early split in the phylogeny of raptors. Mol Biol Evol 18:1892–1904.; Kan et al., 2010Kan XZ, Li XF, Zhang LQ, Chen L, Qian CJ, Zhang XW and Wang L (2010) Characterization of the complete mitochondrial genome of the rock pigeon, Columba livia (Columbiformes, Columbidae). Genet Mol Res 9:1234–1249.; Yang et al., 2010Yang R, Wu X, Yan P, Su X and Yang B (2010) Complete mitochondrial genome of Otis tarda (Gruiformes, Otididae) and phylogeny of Gruiformes inferred from mitochondrial DNA sequences. Mol Biol Rep 37:3057–3066.; Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.).

Protein-coding genes and codon usage

The total length of 13 PCGs in the E. garzetta mitogenome was 11,225 bp, and most of the PCGs were separated by one or more tRNAs (Figure 1). The gene sizes and structures were not significantly different from those of other avian species (Yamamoto et al., 2000Yamamoto Y, Murata K, Matsuda H, Hosoda T, Tamura K and Furuyama JI (2000) Determination of the complete nucleotide sequence and haplotypes in the D-loop region of the mitochondrial genome in the oriental white stork, Ciconia boyciana. Genes Genet Syst 75:25–32.; Haring et al., 2001Haring E, Kruckenhauser L, Gamauf A, Riesing MJ and Pinsker W (2001) The complete sequence of the mitochondrial genome of Buteo buteo (Aves, Accipitridae) indicates an early split in the phylogeny of raptors. Mol Biol Evol 18:1892–1904.; Yang et al., 2010Yang R, Wu X, Yan P, Su X and Yang B (2010) Complete mitochondrial genome of Otis tarda (Gruiformes, Otididae) and phylogeny of Gruiformes inferred from mitochondrial DNA sequences. Mol Biol Rep 37:3057–3066.; Kan et al., 2010Kan XZ, Li XF, Zhang LQ, Chen L, Qian CJ, Zhang XW and Wang L (2010) Characterization of the complete mitochondrial genome of the rock pigeon, Columba livia (Columbiformes, Columbidae). Genet Mol Res 9:1234–1249.; Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.). There is a C insertion at position 174 in ND3, and this insertion was also found in some species of Palaeognathae, e.g., NC_002784, NC_002778 and NC_002782 (Härlid et al., 1998Härlid A, Janke A and Arnason U (1998) The complete mitochondrial genome of Rhea americana and early avian divergences. J Mol Evol 46:669–679.) and Neognathae, e.g., NC_011307 and NC_010962 (Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.). Other analyses have proposed that the insertion is not C at position 174 but A at position 175, as reported in the mitogenomes of Otis tarda (Gruiformes: Otididae, NC_014046) (Yang et al., 2010Yang R, Wu X, Yan P, Su X and Yang B (2010) Complete mitochondrial genome of Otis tarda (Gruiformes, Otididae) and phylogeny of Gruiformes inferred from mitochondrial DNA sequences. Mol Biol Rep 37:3057–3066.) and Trachemys scripta (Testudoformes: Emydidae) (Russell and Beckenbach, 2008Russell RD and Beckenbach AT (2008) Recoding of translation in turtle mitochondrial genomes: Programmed frameshift mutations and evidence of a modified genetic code. J Mol Evol 67:682–695.). The function of this extra C or A in ND3 and its phylogenetic implications are not well known (Russell and Beckenbach, 2008Russell RD and Beckenbach AT (2008) Recoding of translation in turtle mitochondrial genomes: Programmed frameshift mutations and evidence of a modified genetic code. J Mol Evol 67:682–695.), but the effect of this insertion on gene expression can be removed by RNA alternative splicing or a frameshift (Mindell et al., 1998Mindell DP, Sorenson MD and Dimcheff DE (1998) An extra nucleotide is not translated in mitochondrial ND3 of some birds and turtles. Mol Biol Evol 15:1568–1571.).

The average A+T value of 13 PCGs in E. garzetta is 53.10% (Table 3). Except for ND1, the other PCGs had positive AT-skew (0.016 ∼ 0.563) and negative GC-skew (−0.295 ∼ −0.733), indicating the occurrence of more A and C than T and G (Table 3). The nucleotide compositions of three codons in PCGs were estimated for 11 species (Table 4). The results showed that the smallest and greatest variations occurred in the second (A 0.5%, G 0.3%, C 0.6%, T 0.5%) and third (A 4.4%, G 3.0%, C 5.5%, T 3.7%) codons, respectively. The second codon is generally considered to have undergone maximum selective pressure, followed by the first and third codons and other non-coding regions. Different selective pressures result in different nucleotide variability (Zhong et al., 2002Zhong D, Zhao GJ, Zhang ZS and Xu AL (2002) Advance in the entire balance and local unbalance of base distribution in genome. Hereditas 24:351–355.). Table 4 also shows that the G content of the third codon (only 4.1%) was the smallest of the three codons. A similar phenomenon has also been found in mammalian mitogenomes (Reyes et al., 2004Reyes A, Gissi C, Catzeflis F, Nevo E, Pesole G and Saccone C (2004) Congruent mammalian trees from mitochondrial and nuclear genes using Bayesian methods. Mol Biol Evol 21:397–403.; Gibson et al., 2005Gibson A, Gowri-Shankar V, Higgs PG and Rattray M (2005) A comprehensive analysis of mammalian mitochondrial genome base composition and improved phylogenetic methods. Mol Biol Evol 22:251–264.).

The start and stop codons for the PCGs of the E. garzetta mitogenome are shown in Table 1. COIII and DN4 terminated with an incomplete stop codon (T). The use of an incomplete stop codon (T) is common in avian (Härlid et al., 1998Härlid A, Janke A and Arnason U (1998) The complete mitochondrial genome of Rhea americana and early avian divergences. J Mol Evol 46:669–679.; Haring et al., 2001Haring E, Kruckenhauser L, Gamauf A, Riesing MJ and Pinsker W (2001) The complete sequence of the mitochondrial genome of Buteo buteo (Aves, Accipitridae) indicates an early split in the phylogeny of raptors. Mol Biol Evol 18:1892–1904.; Yang et al., 2010Yang R, Wu X, Yan P, Su X and Yang B (2010) Complete mitochondrial genome of Otis tarda (Gruiformes, Otididae) and phylogeny of Gruiformes inferred from mitochondrial DNA sequences. Mol Biol Rep 37:3057–3066.; Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.) and mammalian (Wolstenholme, 1992Wolstenholme DR (1992) Animal mitochondrial DNA: Structure and evolution. Int Rev Cytol 141:173–216.; Arnason et al., 2002Arnason U, Adegoke JA, Bodin K, Born EW, Esa YB, Gullberg A, Nilsson M, Short RV, Xu X and Janke A (2002) Mammalian mitogenomic relationships and the root of the eutherian tree. Proc Natl Acad Sci USA 99:8151–8156.; Gibson et al., 2005Gibson A, Gowri-Shankar V, Higgs PG and Rattray M (2005) A comprehensive analysis of mammalian mitochondrial genome base composition and improved phylogenetic methods. Mol Biol Evol 22:251–264.; Bi et al., 2012Bi XX, Huang L, Jing MD, Zhang L, Feng PY and Wang AY (2012) The complete mitochondrial genome sequence of the black-capped capuchin (Cebus apella). Genet Mol Biol 35:545–552.; Chen et al., 2012Chen W, Sun Z, Liu Y, Yue B and Liu S (2012) The complete mitochondrial genome of the large white-bellied rat, Niviventer excelsior (Rodentia, Muridae). Mitochondrial DNA 23:363–365.; Song et al., 2012Song GH, Lin Q, Yue WB, Liu TF and Hu SN (2012) Sequence analysis of the complete mitochondrial genome and molecular evolution of Cricetulus griseus. Acta Lab Anim Sci Sin 20:70–75 (in Chinese with English abstract).) mitogenomes, and can form a complete UAA terminal signal by posttranscriptional polyadenylation (Ojala et al., 1981Ojala D, Montoya J and Attardi G (1981) tRNA punctuation model of RNA processing in human mitochondria. Nature 290:470–474.; Boore, 2004Boore JL (2004) Complete mitochondrial genome sequence of Urechis caupo, a representative of the phylum Echiura. BMC Genomics 5:e67.).

The ND6 gene was located in the L-strand and its base composition was very different from the other 12 PCGs (Table 3) so it was excluded from the codon usage analysis. Twelve E. garzetta PCGs consisted of 3,626 codons, excluding termination codons (Table S2). The usage frequencies of 21 amino acids ranged from 0.69% (Cys) to 17.9% (Leu). Except for Leu, the most frequently used amino acids were Ile (11.47%), Thr (9.93%) and Ala (7.73%), which was similar with those of other ardeid species (Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.).

Ribosomal and transfer RNA genes

Animal mitogenomes contain small (srRNA) and large (lrRNA) subunits of rRNA (Wu et al., 2003Wu X, Wang Y, Zhou K, Zhu W, Nie J and Wang C (2003) Complete mitochondrial DNA sequence of Chinese alligator, Alligator sinensis, and phylogeny of crocodiles. Chin Sci Bull 48:2050–2054.; Gibson et al., 2005Gibson A, Gowri-Shankar V, Higgs PG and Rattray M (2005) A comprehensive analysis of mammalian mitochondrial genome base composition and improved phylogenetic methods. Mol Biol Evol 22:251–264.; Kan et al., 2010Kan XZ, Li XF, Zhang LQ, Chen L, Qian CJ, Zhang XW and Wang L (2010) Characterization of the complete mitochondrial genome of the rock pigeon, Columba livia (Columbiformes, Columbidae). Genet Mol Res 9:1234–1249.; Krajewski et al., 2010Krajewski C, Sipiorski JT and Anderson FE (2010) Complete mitochondrial genome sequences and the phylogeny of cranes (Gruiformes, Gruidae). Auk 127:440–452.; Bi et al., 2012Bi XX, Huang L, Jing MD, Zhang L, Feng PY and Wang AY (2012) The complete mitochondrial genome sequence of the black-capped capuchin (Cebus apella). Genet Mol Biol 35:545–552.; Chen et al., 2012Chen W, Sun Z, Liu Y, Yue B and Liu S (2012) The complete mitochondrial genome of the large white-bellied rat, Niviventer excelsior (Rodentia, Muridae). Mitochondrial DNA 23:363–365.; Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.; Gao et al., 2013Gao RR, Huang Y and Lei FM (2013) Sequencing and analysis of the complete mitochondrial genome of Remiz consobrinus. Zool Res 34:228–237 (in Chinese with English abstract).), and E. garzetta was no exception (Figure 1). The A+T content for srRNA and lrRNA was 50.8% and 54.7%, respectively, and these values were relatively small among the 11 mitogenomes (Table 2).

Based on the respective anticodons and secondary structures, 22 tRNA genes were identified and their sizes ranged from 67 bp (tRNA^Cys) to 74 bp (tRNA^Leu UUR, tRNA^Asn, tRNA^Ser UCN, tRNA^Glu). Twenty tRNAs can fold into canonical cloverleaf secondary structures, while tRNA-Val and tRNA-Ser (AGY) lost the DHU (dihydrouracil) arms. The cloverleaf structures of tRNA-Val and tRNA-Ser (AGY) were identified by comparing them with counterparts in the E. eulophotes mitogenome (NC_009736). In vertebrate mitogenomes, tRNA-Ser (AGY) generally cannot fold into the canonical cloverleaf secondary structure (Härlid et al., 1998Härlid A, Janke A and Arnason U (1998) The complete mitochondrial genome of Rhea americana and early avian divergences. J Mol Evol 46:669–679.; Shi et al., 2002Shi Y, Shan X, Li J, Zhang X and Zhang H (2002) Sequence and organization of the complete mitochondrial genome of the Indian muntjac (Muntiacus muntjak). Acta Zool Sin 49:629–636.; Wu et al., 2003Wu X, Wang Y, Zhou K, Zhu W, Nie J and Wang C (2003) Complete mitochondrial DNA sequence of Chinese alligator, Alligator sinensis, and phylogeny of crocodiles. Chin Sci Bull 48:2050–2054.; Yang et al., 2010Yang R, Wu X, Yan P, Su X and Yang B (2010) Complete mitochondrial genome of Otis tarda (Gruiformes, Otididae) and phylogeny of Gruiformes inferred from mitochondrial DNA sequences. Mol Biol Rep 37:3057–3066.; Gao et al., 2013Gao RR, Huang Y and Lei FM (2013) Sequencing and analysis of the complete mitochondrial genome of Remiz consobrinus. Zool Res 34:228–237 (in Chinese with English abstract).). Although the gene sizes and anticodon nucleotides agreed with those described for other vertebrates, there were some atypical pairings in the stem regions, such as A-A, A-C, U-C and U-U wobbles. Generally, the tRNA cloverleaf structure contained 7 bp in the aminoacyl stem, 5 bp in the TΨC and anticodon stems, and 4 bp in the D-stem. However, some tRNAs, e.g., tRNA-Phe, tRNA-Leu (CUN) and tRNA-Ile, lacked one or two bp in the T-stem, anticodon stem or D-stem.

Non-coding regions

The non-coding region (the control region, mtCR) of the E. garzetta mitogenome was determined as1,812 bp in length and located between tRNA^Glu and tRNA^Phe (Table 1, Figure 1). The mtCR controls the replication and transcription of animal mitogenomes (Shadel and Clayton, 1997Shadel GS and Clayton DA (1997) Mitochondrial DNA maintenance in vertebrates. Annu Rev Biochem 66:409–435.; Taanman, 1999Taanman JW (1999) The mitochondrial genome: Structure, transcription, translation and replication. Biochim Biophys Acta 1410:103–123.). Based on the nucleotide composition, the mtCR region of E. garzetta contains three domains: a 5′-peripheral domain (Domain I), a central conserved domain (Domain II) and a 3′-peripheral domain (Domain III), an organization that was similar to that of other birds (Southern et al., 1988Southern SO, Southern PJ and Dizon AE (1988) Molecular characterization of a cloned dolphin mitochondrial genome. J Mol Evol 28:32–42.; Saccone et al., 1991Saccone C, Pesole G and Sbisa E (1991) The main regulatory region of mammalian mitochondrial DNA: Structure-function model and evolutionary pattern. J Mol Evol 33:83–91.; Randi et al., 2000Randi E, Lucchini V, Armijo-Prewitt T, Kimball RT, Braun EL and Ligon JD (2000) Mitochondrial DNA phylogeny and speciation in the tragopans. Auk 117:1003–1015.; Roques et al., 2004Roques S, Godoy JA, Negro JJ and Hiraldo F (2004) Organization and variation of the mitochondrial control region in two vulture species, Gypaetus barbatus and Neophron percnopterus. J Hered 95:332–337.; Wang et al., 2008Wang C, Chen Q, Lu G, Xu J, Yang Q and Li S (2008) Complete mitochondrial genome of the grass carp (Ctenopharyngodon idella, Teleostei): Insight into its phylogenic position within Cyprinidae. Gene 424:96–101.; Yang et al., 2010Yang R, Wu X, Yan P, Su X and Yang B (2010) Complete mitochondrial genome of Otis tarda (Gruiformes, Otididae) and phylogeny of Gruiformes inferred from mitochondrial DNA sequences. Mol Biol Rep 37:3057–3066.; Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.; Figure 2).

In Domain I (nt 1–328), two putative extended termination-associated sequence blocks (ETAS1 and ETAS2) were recognized and two putative termination-associated sequences (TAS, conserved palindromic motifs 5′-TACAT-3′ and 5′-TATAT-3′) that act as a signal to terminate synthesis of the control region (Saccone et al., 1991Saccone C, Pesole G and Sbisa E (1991) The main regulatory region of mammalian mitochondrial DNA: Structure-function model and evolutionary pattern. J Mol Evol 33:83–91.; Randi and Lucchini, 1998Randi E and Lucchini V (1998) Organization and evolution of the mitochondrial DNA control region in the avian genus Alectoris. J Mol Evol 47:449–462.; Yamamoto et al., 2000Yamamoto Y, Murata K, Matsuda H, Hosoda T, Tamura K and Furuyama JI (2000) Determination of the complete nucleotide sequence and haplotypes in the D-loop region of the mitochondrial genome in the oriental white stork, Ciconia boyciana. Genes Genet Syst 75:25–32.; Haring et al., 2001Haring E, Kruckenhauser L, Gamauf A, Riesing MJ and Pinsker W (2001) The complete sequence of the mitochondrial genome of Buteo buteo (Aves, Accipitridae) indicates an early split in the phylogeny of raptors. Mol Biol Evol 18:1892–1904.; Roques et al., 2004Roques S, Godoy JA, Negro JJ and Hiraldo F (2004) Organization and variation of the mitochondrial control region in two vulture species, Gypaetus barbatus and Neophron percnopterus. J Hered 95:332–337.) were found in ETAS1. In some birds and mammals, there is a C structure located close to the 5′-peripheral domain of Domain I that can potentially form a stable goose hairpin structure (Quinn and Wilson, 1993Quinn TW and Wilson AC (1993) Sequence evolution in and around the mitochondrial control region in birds. J Mol Evol 37:417–425.; Douzery and Randi, 1997Douzery E and Randi E (1997) The mitochondrial control region of Cervidae: Evolutionary patterns and phylogenetic content. Mol Biol Evol 14:1154–1166.; Sbisà et al., 1997Sbisà E, Tanzariello F, Reyes A, Pesole G and Saccone C (1997) Mammalian mitochondrial D-loop region structural analysis: Identification of new conserved sequences and their functional and evolutionary implications. Gene 205:125–140.; Randi and Lucchini, 1998Randi E and Lucchini V (1998) Organization and evolution of the mitochondrial DNA control region in the avian genus Alectoris. J Mol Evol 47:449–462.); this structure consists of a stem with seven complementary ‘C’s/‘G’s and a loop containing a TCCC motif (Dufresne et al., 1996Dufresne C, Mignotte F and Gueride M (1996) The presence of tandem repeats and the initiation of replication in rabbit mitochondrial DNA. Eur J Biochem 235:593–600.; Yang et al., 2010Yang R, Wu X, Yan P, Su X and Yang B (2010) Complete mitochondrial genome of Otis tarda (Gruiformes, Otididae) and phylogeny of Gruiformes inferred from mitochondrial DNA sequences. Mol Biol Rep 37:3057–3066.). This structure is speculated to be related to H-strand termination (Dufresne et al., 1996Dufresne C, Mignotte F and Gueride M (1996) The presence of tandem repeats and the initiation of replication in rabbit mitochondrial DNA. Eur J Biochem 235:593–600.). The hairpin structure cannot be formed in any of the available ardeid mitogenomes because the interrupted poly-C sequences in Domain I of four species (A. novaehollandiae NC_008551, E. eulophotes NC_009736, N. nycticora NC_015807 and E. garzetta NC_023981) are not followed by a G stretch and Domain I of I. cinnamomeus has no poly-C sequence (Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.). A sequence block similar to the conserved sequence block (CSB1) was found in Domain I (Figure 2) and similar structures have been observed in other avian mitogenomes (Desjardins and Morais, 1990Desjardins P and Morais R (1990) Sequence and gene organization of the chicken mitochondrial genome: A novel gene order in higher vertebrates. J Mol Biol 212:599–634.; Quinn and Wilson, 1993Quinn TW and Wilson AC (1993) Sequence evolution in and around the mitochondrial control region in birds. J Mol Evol 37:417–425.; Randi and Lucchini, 1998Randi E and Lucchini V (1998) Organization and evolution of the mitochondrial DNA control region in the avian genus Alectoris. J Mol Evol 47:449–462.; Kan et al., 2010Kan XZ, Li XF, Zhang LQ, Chen L, Qian CJ, Zhang XW and Wang L (2010) Characterization of the complete mitochondrial genome of the rock pigeon, Columba livia (Columbiformes, Columbidae). Genet Mol Res 9:1234–1249.; Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.).

In Domain II (nt 329–794), four conserved sequence boxes (F, E, D and C) were detected (Figure 2) after aligning with reported counterparts in birds and mammals (Walberg and Clayton, 1981Walberg MW and Clayton DA (1981) Sequence and properties of the human KB cell and mouse L cell D-loop regions of mitochondrial DNA. Nucleic Acids Res 9:5411–5421.; Southern et al., 1988Southern SO, Southern PJ and Dizon AE (1988) Molecular characterization of a cloned dolphin mitochondrial genome. J Mol Evol 28:32–42.; Desjardins and Morais, 1990Desjardins P and Morais R (1990) Sequence and gene organization of the chicken mitochondrial genome: A novel gene order in higher vertebrates. J Mol Biol 212:599–634.; Quinn and Wilson, 1993Quinn TW and Wilson AC (1993) Sequence evolution in and around the mitochondrial control region in birds. J Mol Evol 37:417–425.; Randi and Lucchini, 1998Randi E and Lucchini V (1998) Organization and evolution of the mitochondrial DNA control region in the avian genus Alectoris. J Mol Evol 47:449–462.; Roques et al., 2004Roques S, Godoy JA, Negro JJ and Hiraldo F (2004) Organization and variation of the mitochondrial control region in two vulture species, Gypaetus barbatus and Neophron percnopterus. J Hered 95:332–337.; Kan et al., 2010Kan XZ, Li XF, Zhang LQ, Chen L, Qian CJ, Zhang XW and Wang L (2010) Characterization of the complete mitochondrial genome of the rock pigeon, Columba livia (Columbiformes, Columbidae). Genet Mol Res 9:1234–1249.; Yang et al., 2010Yang R, Wu X, Yan P, Su X and Yang B (2010) Complete mitochondrial genome of Otis tarda (Gruiformes, Otididae) and phylogeny of Gruiformes inferred from mitochondrial DNA sequences. Mol Biol Rep 37:3057–3066.; Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.).

Domain III (nt 795–1812) comprised a conserved sequence block (CSB-1) that regulates mtDNA replication (Figure 2). A poly(C) sequence located upstream of the CSB1 was assumed to represent the origin of H-strand replication (O_H) (Walberg and Clayton, 1981Walberg MW and Clayton DA (1981) Sequence and properties of the human KB cell and mouse L cell D-loop regions of mitochondrial DNA. Nucleic Acids Res 9:5411–5421.; Figure 2). A poly (T) sequence located downstream of the CSB1 was also observed in the mtCR of other birds (NC_008551, NC_009736; NC_015807; Kan et al., 2010Kan XZ, Li XF, Zhang LQ, Chen L, Qian CJ, Zhang XW and Wang L (2010) Characterization of the complete mitochondrial genome of the rock pigeon, Columba livia (Columbiformes, Columbidae). Genet Mol Res 9:1234–1249.; Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.). The bidirectional light- and heavy-strand transcription promoters (LSP/HSP) described in other birds (L’abbé et al., 1991L’abbé D, Duhaime JF, Lang BF and Morais R (1991) The transcription of DNA in chicken mitochondria initiates from one major bidirectional promoter. J Biol Chem 266:10844–10850.; Randi and Lucchini, 1998Randi E and Lucchini V (1998) Organization and evolution of the mitochondrial DNA control region in the avian genus Alectoris. J Mol Evol 47:449–462.; Ritchie and Lambert, 2000Ritchie PA and Lambert DM (2000) A repeat complex in the mitochondrial control region of Adelie penguins from Antarctica. Genome 43:613–618.; Kan et al., 2010Kan XZ, Li XF, Zhang LQ, Chen L, Qian CJ, Zhang XW and Wang L (2010) Characterization of the complete mitochondrial genome of the rock pigeon, Columba livia (Columbiformes, Columbidae). Genet Mol Res 9:1234–1249.; Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.) also existed in Domain III of E. garzetta. In addition, long tandem repeats were found at the 3′ end of Domain III and could be divided into two regions: the first region (nt 977 to 1399) contained three types of tandem repeats: 5′-TACTTTAAAGCACTAAAA-3′ (6×18 bp), 5′-TTTCATTAAAAATATACTATACCCTTCATGAAC-3′ (5×33 bp), and 5′-TGTATCCTTATATCTTTATGT TACCTTTAC-3′ (4×30 bp) while the second region (nt 1406 to 1804) comprised two types of tandem repeats: 5′-TAAACAA-3′ (26×7 bp) and 5′-CAAACAA-3′ (30×7 bp). The existence of repetitive sequences contributed to the large size of the mtCR and the high content of A. Similar tandem repeats (CAAA or CAAACAA) were found in species of Charadriiformes (NC_003712, NC_003713, NC_007978, NC_018548, NC_017601, NC_024069; Wenink et al., 1994Wenink PW, Baker AJ and Tilanus MG (1994) Mitochondrial control-region sequences in two shorebird species, the Turn-stone and the Dunlin, and their utility in population genetic studies. Mol Biol Evol 11:22–31.) and Gruiformes (Yang et al., 2010Yang R, Wu X, Yan P, Su X and Yang B (2010) Complete mitochondrial genome of Otis tarda (Gruiformes, Otididae) and phylogeny of Gruiformes inferred from mitochondrial DNA sequences. Mol Biol Rep 37:3057–3066.), and in C. boyciana in Ciconiiformes (Yamamoto et al., 2000Yamamoto Y, Murata K, Matsuda H, Hosoda T, Tamura K and Furuyama JI (2000) Determination of the complete nucleotide sequence and haplotypes in the D-loop region of the mitochondrial genome in the oriental white stork, Ciconia boyciana. Genes Genet Syst 75:25–32.). These repetitive sequences have been speculated to result from the pause of H-strand replication and subsequent slipped mispairing (Fumagalli et al., 1996Fumagalli L, Taberlet P, Favre L and Hausser J (1996) Origin and evolution of homologous repeated sequences in the mitochondrial DNA control region of shrews. Mol Biol Evol 13:191–199.). The presence of similar conserved repeat sequences in different animal groups (Douzery and Randi, 1997Douzery E and Randi E (1997) The mitochondrial control region of Cervidae: Evolutionary patterns and phylogenetic content. Mol Biol Evol 14:1154–1166.; Nesbø et al., 1998Nesbø CL, Arab MO and Jakobsen KS (1998) Heteroplasmy, length and sequence variation in the mtDNA control regions of three percid fish species (Perca fluviatilis, Acerina cernua, Stizostedion lucioperca). Genetics 148:1907–1919.) has led some researchers to propose that these tandem repeats may have an important role in regulating mitogenome replication and transcription (Delarbre et al., 2001Delarbre C, Rasmussen AS, Arnason U and Gachelin G (2001) The complete mitochondrial genome of the hagfish Myxine glutinosa: Unique features of the control region. J Mol Evol 53:634–641.; Delport et al., 2002Delport W, Ferguson JW and Bloomer P (2002) Characterization and evolution of the mitochondrial DNA control region in hornbills (Bucerotifoormes). J Mol Evol 54:794–806.).

Phylogenomic relationships of 11 species in Ciconiiformes

Mitochondrial sequences provide valuable information for tracing the history of gene rearrangements and phylogenetic reconstructions (Härlid et al., 1998Härlid A, Janke A and Arnason U (1998) The complete mitochondrial genome of Rhea americana and early avian divergences. J Mol Evol 46:669–679.; Braband et al., 2010Braband A, Cameron SL, Podsiadlowski L, Daniels SR and Mayer G (2010) The mitochondrial genome of the onychophoran Opisthopatus cinctipes (Peripatopsidae) reflects the ancestral mitochondrial gene arrangement of Panarthropoda and Ecdysozoa. Mol Phylogenet Evol 57:285–292.; Oh et al., 2010Oh DJ, Oh BS, Jung MM and Jung YH (2010) Complete mitochondrial genome of three Branchiostegus (Perciformes, Malacanthidae) species: Genome description and phylogenetic considerations. Mitochondrial DNA 21:151–159.; Yang et al., 2010Yang R, Wu X, Yan P, Su X and Yang B (2010) Complete mitochondrial genome of Otis tarda (Gruiformes, Otididae) and phylogeny of Gruiformes inferred from mitochondrial DNA sequences. Mol Biol Rep 37:3057–3066.; Cerasale et al., 2012Cerasale DJ, Dor R, Winkler DW and Lovette IJ (2012) Phylogeny of the Tachycineta genus of New World swallows: Insights from complete mitochondrial genomes. Mol Phylogenet Evol 63:64–71.). The availability of an increasing number of complete avian mitogenomes has allowed the construction of phylogenetic trees with better resolution, the results of which show better agreement with morphological and nuclear marker studies (Zhang and Wake, 2009Zhang P and Wake DB (2009) Higher-level salamander relationships and divergence dates inferred from complete mitochondrial genomes. Mol Phylogenet Evol 53:492–508.; Pacheco et al., 2011Pacheco MA, Battistuzzi FU, Lentino M, Aguilar RF, Kumar S and Escalante AA (2011) Evolution of modern birds revealed by mitogenomics: Timing the radiation and origin of major orders. Mol Biol Evol 28:1927–1942.). The phylogenetic tree that included E. garzetta and ten other species in Ciconiiformes (Table S1) was constructed using complete mitogenome sequences, with A. platyrhynchos (EU009397) and B. canadensis (NC_007011) as outgroups. Since some investigators have preferred to use PCGs in tree construction (Härlid et al., 1998Härlid A, Janke A and Arnason U (1998) The complete mitochondrial genome of Rhea americana and early avian divergences. J Mol Evol 46:669–679.; Gibson et al., 2005Gibson A, Gowri-Shankar V, Higgs PG and Rattray M (2005) A comprehensive analysis of mammalian mitochondrial genome base composition and improved phylogenetic methods. Mol Biol Evol 22:251–264.; Shen et al., 2009Shen YY, Shi P, Sun YB and Zhang YP (2009) Relaxation of selective constraints on avian mitochondrial DNA following the degeneration of flight ability. Genome Res 19:1760–1765.; Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.), we also ran an analysis with 13 PCGs to assess the congruence between these two strategies. The results showed that although several regions (tRNAs, CR, rRNAs and ND6) presented some problems in the analysis, e.g., difficulties in alignment, numerous gaps, potential saturation and heterogeneous base composition (Gardner et al., 2005Gardner PP, Wilm A and Washietl S (2005) A benchmark of multiple sequence alignment programs upon structural RNAs. Nucleic Acids Res 33:2433–2439.; Sullivan and Joyce, 2005Sullivan J and Joyce P (2005) Model selection in phylogenetics. Annu Rev Ecol Evol Syst 36:445–466.; Krajewski et al., 2010Krajewski C, Sipiorski JT and Anderson FE (2010) Complete mitochondrial genome sequences and the phylogeny of cranes (Gruiformes, Gruidae). Auk 127:440–452.; Oh et al., 2010Oh DJ, Oh BS, Jung MM and Jung YH (2010) Complete mitochondrial genome of three Branchiostegus (Perciformes, Malacanthidae) species: Genome description and phylogenetic considerations. Mitochondrial DNA 21:151–159.), the topologies of the phylogenetic trees generated by the two strategies were the same (Figure 3).

The phylogenetic relationships among species/genera within the three families examined here were consistent with the conclusions of previous investigations (Sheldon et al., 2000Sheldon FH, Jones CE and McCracken KG (2000) Relative patterns and rates of evolution in heron nuclear and mitochondrial DNA. Mol Biol Evol 17:437–450.; Chang et al., 2003Chang Q, Zhang BW, Jin H, Zhu LF and Zhou KY (2003) Phylogenetic relationships among 13 species of herons inferred from mitochondrial 12S rRNA gene sequences. Acta Zool Sin 49:205–210 (in Chinese with English abstract).; Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.). The monophyly of the Ardeidae, Threskiorothidae and Ciconiidae was strongly confirmed (posterior probabilities = 1.00; Figure 3). In the Ardeidae, I. cinnamomeus was the basal clade and Egretta more closely related to Ardea than to Nycticorax. In Threskiornithidae, Platalea was more closely related to Threskiornis than to Nipponia. The relationships revealed by the phylogenetic trees were also supported by the pairwise distances among mitogenomes (Table S3).

With regard to the evolutionary relationships among the three families, our results supported a closer relationship between Threskiorothidae and Ciconiidae than between Threskiorothidae and Ardeidae, a conclusion similar to that based on amino acid data from 12 PCGs (Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.), but different from that of Hackett et al. (2008)Hackett SJ, Kimball RT, Reddy S, Bowie RC, Braun EL, Braun MJ, Chojnowski JL, Cox WA, Han KL, Harshman J, et al. (2008) A phylogenomic study of birds reveals their evolutionary history. Science 320:1763–1768. and Pacheco et al. (2011)Pacheco MA, Battistuzzi FU, Lentino M, Aguilar RF, Kumar S and Escalante AA (2011) Evolution of modern birds revealed by mitogenomics: Timing the radiation and origin of major orders. Mol Biol Evol 28:1927–1942.. Since the topologies of molecular phylogenetic trees often vary with the markers and taxa used (Zwickl and Hillis, 2002Zwickl DJ and Hillis DM (2002) Increased taxon sampling greatly reduces phylogenetic error. Syst Biol 51:588–598.), divergent evolutionary relationships have often been suggested for the families of Ciconiiformes (Gibb et al., 2007Gibb GC, Kardailsky O, Kimball RT, Braun EL and Penny D (2007) Mitochondrial genomes and avian phylogeny: Complex characters and resolvability without explosive radiations. Mol Biol Evol 24:269–280.; Hackett et al., 2008Hackett SJ, Kimball RT, Reddy S, Bowie RC, Braun EL, Braun MJ, Chojnowski JL, Cox WA, Han KL, Harshman J, et al. (2008) A phylogenomic study of birds reveals their evolutionary history. Science 320:1763–1768.; Pacheco et al., 2011Pacheco MA, Battistuzzi FU, Lentino M, Aguilar RF, Kumar S and Escalante AA (2011) Evolution of modern birds revealed by mitogenomics: Timing the radiation and origin of major orders. Mol Biol Evol 28:1927–1942.; Zhang et al., 2012Zhang L, Wang L, Gowda V, Wang M, Li X and Kan X (2012) The mitochondrial genome of the Cinnamon Bittern, Ixobrychus cinnamomeus (Pelecaniformes, Ardeidae): Sequence, structure and phylogenetic analysis. Mol Biol Rep 39:8315–8326.; this study). More complete mitogenome data for the Ardeidae (and other families in Ciconiiformes) are urgently needed for detailed molecular systematic analyses in this order. The mitogenome sequence data presented here represent a contribution to this long-term goal.

This work was supported by the Natural Scientific Foundation of China (grant nos. 31171189 and 31371252).

References

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