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ISSN: 1940-1736 (print), 1940-1744 (electronic)
Mitochondrial DNA, Early Online: 1–3
!2015 Informa UK Ltd. DOI: 10.3109/19401736.2015.1043540
MITOGENOME ANNOUNCEMENT
Mitochondrial genomes of the jungle crow Corvus macrorhynchos
(Passeriformes: Corvidae) from shed feathers and a phylogenetic
analysis of genus Corvus using mitochondrial protein-coding genes
Urszula Krzeminska
1,2
, Robyn Wilson
1,2
, Sadequr Rahman
1,2
, Beng Kah Song
1,2
, Sampath Seneviratne
3
,
Han Ming Gan
1,2
, and Christopher M. Austin
1,2
1
School of Science, Monash University Malaysia, Selangor, Malaysia,
2
Genomics Facility Monash University Malaysia, Selangor, Malaysia, and
3
Avian Evolution Node, Department of Zoology, University of Colombo, Colombo, Sri Lanka
Abstract
The complete mitochondrial genomes of two jungle crows (Corvus macrorhynchos) were
sequenced. DNA was extracted from tissue samples obtained from shed feathers collected in
the field in Sri Lanka and sequenced using the Illumina MiSeq Personal Sequencer. Jungle crow
mitogenomes have a structural organization typical of the genus Corvus and are 16,927 bp and
17,066 bp in length, both comprising 13 protein-coding genes, 22 transfer RNA genes,
2 ribosomal subunit genes, and a non-coding control region. In addition, we complement
already available house crow (Corvus spelendens) mitogenome resources by sequencing an
individual from Singapore. A phylogenetic tree constructed from Corvidae family mitogenome
sequences available on GenBank is presented. We confirm the monophyly of the genus Corvus
and propose to use complete mitogenome resources for further intra- and interspecies genetic
studies.
Keywords
Corvus, large-billed crow, mitogenome,
Sri Lanka
History
Received 16 April 2015
Accepted 18 April 2015
Published online 15 June 2015
The family Corvidae contains 117 species that are found across all
continents except Antarctica (Dickinson, 2003). Within the
family, genus Corvus constitutes substantial species diversity
with over 40 described species (Marzluff & Angell, 2005).
Despite this diversity, the taxonomy of Corvidae has mostly been
based on the morphological data (Goodwin, 1976), and only
limited genetic studies have been undertaken to revise the
Corvidae systematics (Ericson et al., 2005; Haring et al., 2007,
2012; Kryukow et al., 2012).
Corvus macrorhynhos (Wagler, 1827), also known as jungle
crow or large-billed crow, is widely distributed and is believed to
originate from the rainforests of the south-east Asian tropics
(Kryukov et al., 2012). As with most crows, C. macrorhynchos is
a predator of small animals, competitively displaces other bird
species, and can be a vector in the transmission of human diseases
(Shirafuji et al., 2008; Tanimura et al., 2006).
There is considerable disputation over the taxonomy of
C. macrorhynchos. Martens & Eck (1995) divided the species
into two separate subspecies: the northern C. m. japonensis and
the southern C. m. levaillantii with each containing several sub-
subspecies. In contrast, Dickinson et al. (2004) considered that the
species should be subdivided into 11 subspecies consistent with
high intraspecific differentiation revealed by RAPD-PCR
(Spiridonova et al., 2003). However, other ornithologists
have taken yet other positions. Rasmussen & Anderton (2005),
for example, recognize three separate subspecies including
C. m. culminatus from India as a separate subspecies.
Haring et al. (2012) using short mitochondrial control region
(CR) sequences observed intraspecific genetic distances ranging
from 3.0 to 4.1% over the geographical range of the
C. macrorhynchos. Based on their study, C. macrorhynchos
samples formed a monophyletic cluster consistent with its
recognition as a valid species under a genealogical species
concept. Jonsson et al. (2012) based on the analysis of nuclear
(ODC, GAPDH) as well as mitochondrial fragments (ND2, ND3)
found samples of C. macrorhynchos to be a paraphyletic, with
Philippine C. macrorhynchos philippinus being sister to a clade
comprising all other C. macrorhyncos subspecies from East Asia.
Studies have consistently identified C. splendens as the sister
species of C. macrorhynchos with divergence levels of approxi-
mately 5.6% based on CR sequences (Haring et al., 2012). Jonsson
et al. (2012) identified C. kubaryi as the sister species to the
C. splendens–C. macrorhynchos clade.
In this study, dried feather samples of C. macrorhynchos were
collected from Kandy District, Sri Lanka; and a C. splendens
sample was obtained from a pulled feather from a bird from
Singapore. Total genomic DNA was extracted from the feather
shaft using DNeasy Blood and Tissue kit (Qiagen, Haldane,
Germany) following the protocol of the manufacturer with slight
modifications; 30 ml of proteinase K and 30 ml of 1 M DTT were
added at the tissue lysis stage followed by overnight incubation.
Mitogenome sequencing, assembly, and annotation were per-
formed as described by Gan et al. (2014). The cytochrorome
oxidase 1 gene of C. macrorhynchos (JF499119) was used as the
initial bait for MITObim assembly. Full mitogenome sequences
for genus Corvus (KP403809, KJ766304, KP161619, KP161620,
and Y18522) and other members of Corvidae family (KJ5986622,
KJ598623, JQ423932, GU592504, JN108020, KF509923,
HQ915867, JN018413, and JQ083495) that are available from
Correspondence: Urszula Krzeminska, School of Science, Monash
University Malaysia, Jalan Lagoon Selatan, Bandar Sunway, 47500
Petaling Jaya, Selangor, Malaysia. E-mail: ukrz1@student.monash.edu
Mitochondrial DNA Downloaded from informahealthcare.com by Monash University on 06/18/15
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the database were included in the phylogenetic analysis performed
using MitoPhAST tool (1000 bootstraps) that generates a
maximum likelihood phylogenetic tree using optimized protein
model (Tan et al., 2015). Five full mitogenome sequences of non-
Corvidae species (JX256246, KJ909190, KP313823, KJ909193,
and JX486029) were included as an out-group. Genetic distances
based on the complete mitogenome nucleotide sequences were
calculated using Geneious v. 7.0 (Symantec Corp., Mountain
View, CA) (Kearse et al., 2012).
The complete C. macrorhynchos mitogenomes are 16,927 bp
(KR057957) and 17,066 bp (KR072661) of length, while
C. splendens mitogenome is 16,927 bp (KP019940) of length.
All sequenced mitogenomes comprise 37 mitochondrial genes
(13 protein-coding genes, 2 ribosomal subunit genes, and 22
transfer RNA genes) and a non-coding control region. The gene
arrangement is consistent with other Corivdae species.
The overall identity between C. macrorhynchos (KR057957)
and other Corvus species for the complete mitogenome sequences
is as follows: C. macrorhynchos and C. frugilegus are 92.5%,
C.macrorhynchos and C. hawaiiensis are 92.4%, C. macro-
rhynchos and both C. cornix and C. brachyrhynchos are 93.3%.
Higher identity, 95.9%, is observed between C. macrorhynchos
and C. splendens, which is almost as high as the greatest
intraspecific divergence in C. macrorhynchos as observed by
Haring et al. (2012).
The phylogenetic tree (Figure 1) confirms the monophyly of
the genus Corvus and places C. macrorhynchos and C. splendens
as sister species. We support a close association between
C. hawaiiensis and C. frugilegus, consistent with Haring et al.
(2012). Another clade consists of C. brachyrhynchos and
C. cornix, two species previously also found to be closely related
(Haring et al., 2012; Jonsson et al., 2012). Due to a small
number of full mitogenome sequences available, only limited
conclusions can be made on intraspecies variability in mitogen-
ome sequences. However, the two mitogenome sequences of
C. macrorhynchos from Kandy differed by 1.1%, which is more
than the differences between two available C. splendens and two
C. hawaiinesis sequences. The newly sequenced C. splendens
mitogenome from Singapore differed only by 0.2% from the
mitogenome from a Malaysian bird (KJ766304). This is consistent
with a close relationship between these populations and may
reflect migration and gene flow or the establishment of these two
feral populations from the same source population. The two
C. hawaiiensis mitogenomes were almost identical, which is
expected for endangered and highly inbred species (Leonard,
2008).
Interestingly we observed that Oriolus chinensis belonging to
the Corvidae family clusters with other species outside the family.
This has been supported by many bootstrapping repeats and may
suggest that O. chinensis taxonomy and relationships should be
reviewed.
Our results indicate the necessity of more extensive evaluation
of the molecular taxonomy of Corvus species based on
comprehensive geographic sampling using full mitochondrial
genome sequencing. Such studies will be facilitated through the
use of DNA extracted from shed feathers and next-generation
sequencing using the methods described in this paper.
Declaration of interest
Funding for this study was provided by the Monash University Malaysia
Tropical Medicine and Biology Multidisciplinary Platform. Samples in
Sri Lanka were collected under the research permit granted by the
Department of Wildlife Conservation, Sri Lanka (Permit no. WL/3/2/41/
14). Samples from Singapore were provided by the AVA Singapore. The
authors report that they have no conflicts of interest. The authors alone
are responsible for the content and writing of the paper.
Figure 1. Phylogenetic tree representing relationships within family Corvidae, inferred from maximum likelihood estimation with bootstrap valuesfor
amino acid sequences from 13 mitochondrial protein-coding genes.
2U. Krzeminska et al. Mitochondrial DNA, Early Online: 1–3
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DOI: 10.3109/19401736.2015.1043540 Mitogenomes Corvus macrorhynchos 3
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