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Mitochondrial DNA, Early Online: 1–4
! 2013 Informa UK Ltd. DOI: 10.3109/19401736.2013.779263
SHORT COMMUNICATION
Mitochondrial cytochrome oxidase I (COI) DNA sequencing
of the ascidians Didemnum granulatum (JQ013198) and
D. psammathodes (JN624758)
N. Sri Kumaran1, S. Bragadeeswaran1, and V. K. Meenakshi2
1
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Faculty of Marine Science, Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai – 608 502, Tamil Nadu, India
and 2A.P.C. Mahalaxmi College for Women, Tuticorin – 628 002, Tamil Nadu, India
Abstract
Keywords
Two colonial ascidians Didemnum granulatum and D. psammathodes were collected from
Tuticorin coastal waters. These ascidians were sequenced at 603 and 576 bp region of the
mitochondrial cytochrome oxidase subunit I gene (COI) for phylogenetic analysis. Barcode
sequences were extracted via FASTA format from NCBI. The genetic distances of submitted
DNA sequences were compared with related ascidian species. Didemnum granulatum
(JQ013198) sequence shows maximum identical 99% with D. vexillum. Didemnum psammathodes (JN624758) sequence submitted at present shows maximum identical 100% with
another D. psammathodes sequence which was already submitted in NCBI. The sequence also
shows maximum identical 90–89% with D. vexillum. From the present study it is concluded that
precise and accurate identification of ascidians could be performed using the barcode
sequences of the mitochondrial DNA (in the COI gene).
AS 2235, AS 2233, colonial, Tuticorin
Introduction
Species are the fundamental unit of comparison in biology, from
anatomy to behavior, development, ecology, evolution, genetics,
molecular biology, paleontology, physiology, systematic and so
forth (De Queiroz, 2005). Thus, the capability to correctly
identify species is crucial in order to minimize ‘‘error cascades’’
resulting from the use of bad taxonomy in science (Bortolus,
2008). Traditionally, for the identification of species, morphological characters are used. However, the development of
molecular biology created a new set of useful tools to identify
species. Many studies have been published using a diverse
assemblage of molecular approaches and markers to identify
species, such as allozymes (Aron & Sole-Cava, 1991; Gusmao
et al., 2000), restriction fragment length polymorphism (Moysés
& Almeida-Toledo, 2002), DNA arrays (Hajibabei et al., 2007),
single nucleotide polymorphism (Shaffer & Thonsom, 2007),
multiplex PCR (Mendonca et al., 2009), DNA sequences (Pook &
McEwing, 2005; Lemer et al., 2007) and many others.
Mitochondrial DNA (mtDNA) analysis has been employed in
the evolutionary study of the animal species for more than 30
years (Avise & Walker, 1999). Recently, the role of mtDNA
sequences in taxonomy and phylogenetic inference has become
contentious and two extreme viewpoints have emerged, one
position criticizes the use of mtDNA because the marker suggests
misleading patterns of variation; specifically, phylogenies that are
inconsistent with those derived from nuclear gene sequences in
Correspondence: Dr S. Bragadeeswaran, Assistant Professor, Marine
Biotoxinology lab, Faculty of Marine Sciences, Centre of Advanced
Study in Marine Biology, Annamalai University, Parangipettai – 608 502,
Tamil Nadu, India. Tel: +91 4144 243223; Ext: 269. Mobile: +91
9894823364. E-mail: [email protected]
History
Received 31 January 2013
Accepted 20 February 2013
Published online 29 July 2013
the context of species relationships among closely related taxa
(Ballard & Whitlock, 2004). Morphology-based tunicate taxonomy is a highly specialized discipline and the misidentification
of species is a frequent problem (Lambert 2009; Geller et al.,
2010). So this study was aimed at exploring the ‘‘barcoding’’ of
mitochondrial cytochrome oxidase I (COI) gene sequences and
phylogenetic status of the ascidians D. granulatum and
D. psammathodes collected from the Tuticorin Coast of India.
Material and methods
Sample collection
Ascidians D. granulatum and D. psammathodes were collected at
3 m depth from the lime rocks of Hare Island, Tuticorin Coast in
the month of September 2010. The ascidians tissue (25 mg) was
removed from each individual using sterile blade and stored in
95% (v/v) ethanol at 20 C. Salt out protocol was adopted for
precise and quick DNA isolation from the ascidian tissues. The
collected specimens were identified by the standard literature
(Cole & Lambert, 2009; Kott, 2001; Rocha & Bonnet, 2009).
Voucher specimen No AS 2235 and AS 2233 have been deposited
in the National Collection of ascidians in the Museum of the
Department of Zoology, A.P.C. Mahalaxmi College for Women,
Tuticorin-628 002.
DNA extraction and mitochondrial COI DNA sequencing
The ascidian tissues were placed in a 1.5 mL eppendorf tube
separately and 500 mL of solution I (50 mM Tris-HCl pH 8,
20 mM EDTA pH 8 and 2% SDS) was added. The tissues were
homogenized with 5 mL of Proteinase K (20 mg mL1) and mixed
using a vortex mixer. The samples were incubated at 55 C in
water bath for 2 h with frequent mixing. After incubation the
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2
N. Sri Kumaran et al.
samples were cooled over ice for 10 min and 250 mL of solution II
(6 M NaCl) was added, mixed well then cooled on ice for 5 min
and centrifuged at 8000 rpm for 15 min. From this, 500 mL of
supernatant was carefully collected in new eppendorf tubes and
twice the volume (i.e. 1 mL) of 100% AR grade ethanol was added
to precipitate the DNA. The precipitate was pellet down at
8000 rpm for 5 min and the supernatant was removed without
disturbing the pellet. The DNA pellet was rinsed with 500 mL
of cold ethanol and centrifuged at 11,000 rpm for 5 min.
The supernatant was carefully removed and the excess liquid
was drained using pipette. The pellet was partially dried (devoid
of Ethanol) with lid off at 55 C on heating block. The pellet was
re-suspended with 50–200 mL of fresh sterile H2O depending on
the size of pellet (100 mL average) by gently pipetting the sample
with wide-bore filter tip until dissolved. This dissolved DNA
acted as a template for polymerase chain reaction (PCR).
The fragment of COI was amplified by Gene Amp PCR system
9700. PCR was carried out in 25 mL volumes [2.5 mL of 10X PCR
buffer, 1.5 mL of MgCl2 (2 mM mL1) 1 mL of DNA template, 1 pL
of each primer (10 pmoles mL1), 2 dNTPs (1 mM mL1), 10 U of
1 mL of Taq polymerase (Bioserve Biotechnologies Pvt Ltd,
Hyderabad, India) and 15 mL of sterile Mill Q water]. LCO1490:
5’-GGTCAACAAATCATAAAGATATTGG-30 and HCO2198:
5’-TAAAC TTCAGGGTGACCAAAAAATCA-30 primers were
employed for COI amplification (Folmer et al., 1994). The
thermocyclic conditions for PCR included the initial denaturation
at 94 C for 1 min, five cycles of 94 C for 30 s, annealing at 45 C
for 40 s and extension at 72 C for 1 min, with a final extension at
72 C for 10 min, followed by indefinite hold at 4 C.
Following PCR, about 10 mL of PCR product with 2 mL of
bromothymol blue were added to 2% agarose gel, prepared with
2.5 mL of 1% ethidium bromide and electrophorized at 90 V until
the dye moved for 6 cm in the gel. The gel was moved to gel the
doc system for viewing the amplicons with the aid of UV transilluminator. Sequencing PCR was carried out using Dye terminator mix v 3.1 and quantified in Euro bio-agarose gel (Eurobio,
France). The samples were loaded onto MegaBace sequencer
(MB 1000) at Bioserve Biotechnologies, Pvt. Ltd (Hyderabad,
India).
Sequence data analysis
The electrophenerogram generated by automated DNA sequencer
was read by Chromas Pro vl.42 (Technelysium Pty Ltd., Tewantin,
Queensland, Australia) and the sequences were carefully checked
for mis-calls and base spacing. Few ascidians sequences were
extracted via FASTA format from NCBI. ClustalX 2.0.6
(www.clustal.org) was used to align the nucleotide sequence
Figure 1. The phylogenetic tree of the
Didemnum granulatum (JQ013198).
Mitochondrial DNA, Early Online: 1–4
(Thomson, 1997). The nucleotide content of collected barcode
was estimated by BioEdit (www.mbio.ncsu.edu/BioEdit/bioedit.
html) sequence alignment editor (Hall, 1999). MEGA 4 was used
to construct phylogenetic trees via the neighborhood joining
method using Kimura 2 parameter and to calculate genetic
distance of the given set of sequences. The ascidian Ciona
intestinalis (HM209056) and Didemnum incanum (JQ692628)
were selected as out groups in phylogenic tree construction.
Results
The PCR amplified products of mitochondrial cytochrome oxidase
subunit I gene (COI) from these ascidians D. granulatum
(JQ013198) and D. psammathodes (JN624758) were sequenced
and have been deposited in NCBI databases for their phylogenetic
analysis. The present D. granulatum sequence is the first sequence
submitted to NCBI database in this species. The final length after
alignment and trimming was of 603 base pairs (bp) and free of
gaps. Following eight ascidian sequences, D. vexillum
(EU742677), D. psammathodes (EU742661), D. psammathodes2
(JN624758), D. Vexillum2 (JF738067), Didemnum sp.
(KC017432), C. intestinalis (HM209056), D. cfalbopunctatum
(KC017444), A. fuscum (AY600975) (collected from NCBI) of
ascidian were selected for analyzing the phylogenetic relationship
of ascidian D. granulatum sequences (Figure 1). Didemnum
granulatum (JQ013198) sequence shows maximum identity of
99% with D. vexillum. The GC and AT contents showed in ascidian
D. granulatum were 26.53% and 73.47%, respectively. The
nucleotide A, C, G and T contents were 30.35%, 14.76%, 11.77%
and 43.12%, respectively. Table 1 shows the genetic distance with
standard error between the selected ascidian sequences.
In the case of D. psammathodes, the final length
after alignment and trimming was of bp and free gaps. For
analyzing the phylogenetic relationship of ascidian D.
psammathodes sequences following seven ascidians sequences
(collected from NCBI) of ascidian were selected, D.
psammathodes (EU742661), D. vexillum (EU7442669),
Didemnum sp. (JQ731747), D. perlucidum (JQ731735), D.
granulatum (JQ013198), D. fulgens (JX846617) and D.
incaunm (KC017439). The phylogenetic tree shows 100%
similarity with already submitted ascidian D. psammathode2
sequences (Figure 2). In the ascidian D. psammathodes
sequences, the GC and AT contents were 24.83% and
75.17%, respectively. The presence of nucleotide bases A, C,
G and T contents in D. psammathodes sequences were
31.77%, 12.15%, 12.67% and 43.40%, respectively. Table 2
shows the genetic distance with standard error between the
selected ascidian sequences.
Mitochondrial COI DNA sequencing of ascidians
DOI: 10.3109/19401736.2013.779263
3
Table 1. The genetic distance of Didemnum granulatum (JQ013198) with standard error between the selected ascidians.
Ascidians
Didemnum sp2
Didemnum sp1
D. granulatum
D. vexillum
D. vexillum2
D. psammatode
D. psammatode2
A. fuscum
C. intestinalis
D. incanum
Didemnum Didemnum
sp2
sp1
D. granulatum D. vexillum D. vexillum2 D. psammatode D. psammatode2 A. fuscum C. intestinalis D. incanum
(0.018)
0.155
0.164
0.128
0.134
0.172
0.179
0.289
0.297
0.952
(0.019)
(0.022)
0.207
0.168
0.175
0.196
0.198
0.314
0.331
1.100
0.034
0.079
0.151
0.160
0.337
0.351
0.992
(0.016)
(0.019)
(0.008)
0.042
0.115
0.124
0.306
0.319
0.950
(0.017)
(0.020)
(0.013)
(0.009)
0.111
0.120
0.317
0.319
0.950
(0.019)
(0.021)
(0.018)
(0.016)
(0.015)
0.009
0.340
0.314
0.983
(0.020)
(0.021)
(0.019)
(0.016)
(0.016)
(0.004)
0.352
0.322
0.990
(0.027)
(0.028)
(0.019)
(0.028)
(0.029)
(0.030)
(0.031)
(0.027)
(0.029)
(0.031)
(0.029)
(0.029)
(0.028)
(0.029)
(0.029)
0.325
1.116
(0.076)
(0.094)
(0.081)
(0.076)
(0.076)
(0.079)
(0.080)
(0.096)
(0.086)
1.026
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Figure 2. The phylogenetic tree of the
Didemnum psammathodes (JN624758).
Table 2. The genetic distance of Didemnum psammathodes (JN624758)) with standard error between the selected ascidians.
Ascidians
D. psammatode
D. psammatode2
Didemnum sp.1
D. vexillum
D. granulatum
D. perlucidum
D. fulgens
Didemnum sp.2
C. intestinalis
D. psammatode D. psammatode2 Didemnum sp.1 D. vexillum D. granulatum D. perlucidum D. fulgens Didemnum sp.2 C. intestinalis
(0.004)
0.011
0.115
0.105
0.160
0.148
0.165
0.190
0.318
0.111
0.099
0.149
0.144
0.156
0.185
0.308
(0.015)
(0.015)
0.096
0.140
0.174
0.165
0.178
0.340
(0.014)
(0.014)
(0.014)
0.074
0.152
0.148
0.167
0.307
Discussion
Search of the Barcode of Life database (BOLD) using the
online ‘‘Identification Engine’’ returned results in complete
agreement with the BLAST searches of GenBank: sequences
D. granulatum and D. psammathodes were identified as being
in agreement with other submitted ascidian sequences. The
BLAST results provided confirmation of our taxonomic identifications. Since there were no other COI sequences available in
GenBank for D. granulatum, our sequences matched 99% most
closely with ascidian D. vexillum. In the case of D. psammathodes, there are only two sequences submitted to NCBI
database and our sequence matched most closely with another
D. psammathodes sequence, showing 100% maximum identical.
Comparative analysis of mitochondrial COI sequences of two
colonial ascidians, D. granulatum and D. psammathodes, revealed
contrasting patterns of genetic structure.
Mitochondrial DNA (mtDNA) analysis has been employed in
the evolutionary study of animal species for more than 30 years
(Avise & Walker, 1999). Its higher mutation rate and lower
effective population size than nuclear DNA make mtDNA a
powerful tool to probe for the evidence of reproductive isolation
(0.018)
(0.018)
(0.017)
(0.012)
0.194
0.187
0.188
0.346
(0.017)
(0.017)
(0.019)
(0.018)
(0.021)
0.137
0.195
0.313
(0.018)
(0.018)
(0.018)
(0.017)
(0.020)
(0.017)
0.208
0.297
(0.020)
(0.020)
(0.019)
(0.019)
(0.020)
(0.017)
(0.021)
(0.028)
(0.027)
(0.029)
(0.027)
(0.030)
(0.028)
(0.027)
(0.027)
0.303
between lineages. This fact provoked a proposal to standardize
DNA-based species identification by analyzing a uniform segment
of the mitochondrial genome. With this approach, a library of
sequences from taxonomically verified voucher specimens serve
as DNA identifiers for species, in short, DNA barcodes (Hebert
et al., 2003). Phylogeographic studies of taxa across biogeographical ranges can contribute to the elucidation of cryptic
species. For example, Tarjuelo et al. (2001) investigated the
genetic structure of an ascidian, Clavelina lepadiformis, in
harbors and rocky reefs with the COI gene. Their results
suggested two distinct clades due to the lack of gene flow
between harbor and rock reef populations and that C. lepadiformis
are cryptic species instead of differentiated populations of the
same species. In another study, a sessile tunicate Pyura sp. (piure
de Antofagasto) was thought to be restricted to Antofagasto Bay,
Chile. However, molecular evidence with COI indicated that it
was an introduced species from Australia. The ‘‘piure de
Antofagasto’’ clustered with the Australian Pyura praeputialis
rather than with the South African, Pyura stolonifera. It was
concluded that the Australian P. praeputialis was introduced to
Chile via ship fouling, ballast water or rafting (Castilla & Guinez,
4
N. Sri Kumaran et al.
2000). With so much external input, it is difficult to assess
whether there is connectivity among ports or if the external input
(overseas) is from the same source. More data are still needed to
identify the source and confirm the mixing of port populations.
Conclusion
The COI sequence in the phylogram constructed clearly clustered
the ascidian species in individual group proving the efficiency
of COI gene in delineating the members of Didemnum to the
species level. Hence, we conclude that COI sequence could be
potentially used to identify the individual of ascidians to species
level.
Acknowledgements
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The authors are thankful to the Dean, Center of Advanced Study in
Marine Biology, Faculty of Marine Sciences, Annamalai University,
Parangipettai, Tamil Nadu, India, for facilities provided.
Declaration of interest
We declare that we do not have conflict of interest.
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