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Identification of Pleuragramma antarctica larvae in the Ross Sea via mitochondrial DNA

Authors:
  • Institute Pierre-Simon Laplace
WG-FSA
This paper is presented for consideration by CCAMLR and may contain unpublished data, analyses, and/or
conclusions subject to change. Data in this paper shall not be cited or used for purposes other than the work
of the CAMLR Commission, Scientiic Committee or their subsidiary bodies without the permission of the
originators and/or owners of the data.
Commission for the Conservation of Antarctic Marine Living Resources
Commission pour la conservation de la faune et la lore marines de l’Antarctique
Комиссия по cохранению морских живых pесурсов Антарктики
Comisión para la Conservación de los Recursos Vivos Marinos Antárticos
Original: English
J.A. Caccavo (Italy), C. Brooks (USA), L. Zane (Italy) and J.R. Ashford (USA)
WG-FSA-15/61
Identification of Pleuragramma antarctica larvae in the Ross Sea via
mitochondrial DNA
21 September 2015
1
Identification of Pleuragramma antarctica larvae in the Ross Sea via
mitochondrial DNA
Jilda A. Caccavo1†, Cassandra Brooks2, Lorenzo Zane1, Julian R. Ashford3
1 Department of Biology, University of Padua, Padua 35142 Italy
2 Emmett Interdisciplinary Program in Environment and Resources, Stanford University, Stanford,
California 94305 USA
3 Center for Quantitative Fisheries Ecology, Old Dominion University, Norfolk, VA 23508 USA
Correspondence email: jildaalicia.caccavo@studenti.unipd.it
Abstract
Research into the early life stages of Pleuragramma antarctica is essential to understanding
how oceanographic variation will impact spatial distributions over time. The recent findings of
nursery grounds in Terra Nova Bay have led to added inquiry into larval distribution and life
history traits in the Ross Sea. A report submitted to the CCAMLR-EMM working group last
year provided abundance, length and growth data for larvae found in the western and
eastern Ross Sea during the austral summer of 2013, which were identified as P. antarctica
based on morphological characteristics. We extracted genomic DNA from a sample of these
larvae and, using fish universal primers, amplified part of the 16S rDNA and the D-Loop
region. Despite evidence of DNA degradation, sequencing was nevertheless successful in a
fraction of samples. Sequences were aligned with known GenBank sequences for P.
antarctica and several of related notothenioids, which confirmed the species identity of
larvae in the western Ross Sea as P. antarctica. Consistent with the previous report, D-Loop
sequences also demonstrated that recently hatched larvae sampled from the eastern Ross
Sea were from the same species, suggesting the possibility of another nursery ground for P.
antarctica in the vicinity of the Bay of Whales. This is a novel use of mitochondrial DNA to
test morphological identification when examining spatial distributions of P. antarctica that
depart from expectation.
Introduction
In the continental shelf waters of the Southern Ocean Pleuragramma antarctica is the
dominant pelagic fish connecting higher and lower trophic levels throughout its life history
(Granata et al. 2009, La Mesa and Eastman 2012). P. antarctica dominates the
icthyoplankton of the Ross Sea, comprising up to 99% of total catches in previous studies
(Vacchi et al. 1999, Granata et al. 2002). P. antarctica abundance during early life stages is
closely associated with sea-ice cover (Vacchi et al. 2012, Guidetti et al. 2014), and thus is
vulnerable to changing oceanographic conditions in the Ross Sea, where the annual and
seasonal extent and cover of costal polynyas vary (Smith Jr et al. 2007, La Mesa et al.
2010). Despite a growing body of research on this keystone Antarctic species, there remains
much to be learned about its early life history and distribution in the Ross Sea.
While nurseries have been discovered under the platelet ice of the Terra Nova Bay in the
western Ross Sea (WRS) (Granata et al. 2002, Vacchi et al. 2004, Vacchi et al. 2012),
evidence indicative of a potential nursery in the eastern Ross Sea (ERS) in the Bay of
2
Whales was submitted to the last CCAMLR Working Group on Ecosystem Monitoring and
Management (WG-EMM) (Brooks and Goetz 2014). This reported results from a research
cruise undertaken during late summer/early fall of 2013, in which P. antarctica larvae
comprised 99% of the icthyoplankton community opportunistically sampled throughout the
Ross Sea. Species identification was done based primarily on morphological characteristics
developed by Kellerman (1989). While morphological sorting remains the predominant
method of distinguishing taxa in studies of Antarctic pelagic ecosystems (Vacchi et al. 2004,
Granata et al. 2009, Vacchi et al. 2012), it can be problematic if characteristics are subtle or
hard to interpret, or samples are incomplete or damaged.
Species identification based on DNA barcoding has dramatically increased over the past 10
years as a means of augmenting, and even superseding, morphological bases of taxonomic
distinctions (Taylor and Harris 2012). Mitochondrial sequences such as 16S, a region
encoding the ribosomal RNA of the large subunit of the mitochondrial ribosome and the D-
Loop, which forms the Displacement Loop where mtDNA replication starts, have been widely
used to distinguish closely related species and differentiate populations, including in
notothenioids (Patarnello et al. 2003, Near et al. 2004, Zane et al. 2006).
Figure 1. Adapted from Brooks and Goetz 2014. Bathymetry map of the Ross Sea showing the
location of tows where icthyoplankton was sampled in 2013. Mitochondrial DNA was successfully
sequenced from P. antarctica larvae collected from the tows circled in red.
3
Both 16S rDNA and the D-Loop region were sequenced in larvae collected and identified as
P. antarctica by Brooks and Goetz (2014) on board the RVIB Nathaniel B. Palmer (Fig. 1).
These sequences were used for the first time in P. antarctica to test larval identification by
alignment with established GenBank sequences from both the same species and related
notothenioids.
Methods
Sample collection and DNA extraction
Larvae used in this study were collected in the Ross Sea via Tucker trawl using 700 M nets
during March and February 2013. A detailed description of tow procedures can be found in
Brooks and Goetz (2014). Larvae were sorted by standard length (SL) and frozen. Seventy
larvae were randomly selected for DNA extraction. Forty-four larvae from nine separate tows
ranging in SL from 15 26 mm were selected from the western Ross Sea (WRS). Twenty-
six larvae from the one eastern Ross Sea (ERS) tow in the southern part of the Bay of
Whales were used, ranging in SL from 8 11 mm. Positive controls included genomic DNA
from four adult P. antarctica from a previous study of the Antarctic Peninsula (Agostini et al.
2015), two adult P. antarctica collected from the Weddell Sea and two larval P. antarctica
collected also from the Ross Sea but during a previous research cruise, all having shown
successful amplification of segments in previous experiments.
Genomic DNA was extracted from whole larvae using a standard salting-out protocol.
Extraction success was determined by assessing the concentration of nucleic acids in
solution (ng/L) using NanoDropTM spectrophotometry, as well as agarose gel
electrophoresis. Total genomic DNA was diluted to approximately 100 ng/L for use in PCR
amplification, and stored at -20°C.
DNA amplification and sequencing
The 16S large ribosomal unit and the D-Loop control region of mitochondrial DNA were
amplified by PCR. Forward and reverse fish universal primers 16ar and 16br were used for
the amplification of approximately 528 base pairs (bp) of 16S rDNA (Palumbi 2002). Forward
and reverse primers LPRO2 and HDL1 were used to amplify approximately 349 bp of the D-
Loop region (Patarnello et al. 2003). Singleplex PCRs were performed on 2 L of DNA in a
total volume of 20 L consisting of 0.04 U/L of Taq polymerase (Promega), 1x reaction
buffer (with MgCl2), 0.1 M of each dNTPs, and 0.25 nM of each primer. The thermal profile
for 16S rDNA amplification was: (1) predenaturation: 94°C for 3 min; (2) 6 touchdown cycles:
denaturation at 94°C for 40 s, annealing at 56°C for 1 min decreased by 1°C each cycle,
extension at 72°C for 1 min; (3) 30 cycles: denaturation at 94°C for 40 s, annealing at 50°C
for 1 min and extension at 72°C for 1 min; (4) final extension: 72°C for 5 min. The thermal
profile for D-Loop region amplification did not include a touchdown step, and differed slightly
from the 16S rDNA profile in the temperature and duration of the repeated denaturation,
annealing and extension steps. The D-Loop thermal cycle was: (1) predenaturation: 94°C for
3 min; (2) 30 cycles: denaturation at 94°C for 1 min, annealing at 52°C for 45 s and
extension at 72°C for 45 s; (3) final extension: 72°C for 5 min.
4
Figure 2. PCR results of 16S rDNA and D-Loop region amplification. Yellow circles indicate
samples subsequently submitted for sequencing. A 1 kb ladder was used. Note the smear
signature in the majority of larval PCRs.
b) 16S
a) D-Loop
(+) 113 (+) 114 (+) 115 (+) 115
212 316 189 125
*
CA4 Star96 88 R4 R44
59 93 95 99
2-1 2-2 2-3 2-4 2-5 2-6 3-1 3-2
84 83 86 88 92 88 88 88
3-3 3-4 4-1 4-2 4-3 4-4 4-5 5-1 5-2 5-3 5-4 5-5 10-1 10-2 10-3 10-4
68 76 89 86 81 84 84 80 79 91 84 86 84 84 89 94
11-1 11-2 11-3 11-4 12-1 12-2 12-3 12-4 12-5 12-6 12-7 15-1 15-2 15-3 15-4 15-5
98 96 99 103 91 97 89 94 94 94 100 95 91 99 91 100
16-1 16-2 16-3 16-4 19-1 19-2 19-3 19-4 19-5 19-6 19-7 19-8 19-9 19-10 19-11 19-12
76 91 98 97 145 225 148 346 26 23 28 16 21 25 19 13
19-13 19-14 19-15 19-16 19-17 19-18 19-19 19-20 19-21 19-22 19-23 19-24 19-25 19-26
13 41 34 12 25 13 59 104 29 107 395 185 107 212
(-)
(-)
circled samples sent for sequencing
* indicates DNA concentration as measured by NanoDrop
Positive Controls
DNA from older adult and larval P. ant tissue
DNA from 2014 Ross Sea larval tissue
Negative Controls
* indicates DNA concentration as measured by NanoDrop
(+) 113 (+) 114 (+) 115 (+) 115
212 316 189 125
*
CA4 Star96 88 R4 R44
59 93 95 99
2-1 2-2 2-3 2-4 2-5 2-6 3-1 3-2
84 83 86 88 92 88 88 88
3-3 3-4 4-1 4-2 4-3 4-4 4-5 5-1 5-2 5-3 5-4 5-5 10-1 10-2 10-3 10-4
68 76 89 86 81 84 84 80 79 91 84 86 84 84 89 94
11-1 11-2 11-3 11-4 12-1 12-2 12-3 12-4 12-5 12-6 12-7 15-1 15-2 15-3 15-4 15-5
98 96 99 103 91 97 89 94 94 94 100 95 91 99 91 100
16-1 16-2 16-3 16-4 19-1 19-2 19-3 19-4 19-5 19-6 19-7 19-8 19-9 19-10 19-11 19-12
76 91 98 97 145 225 148 346 26 23 28 16 21 25 19 13
19-13 19-14 19-15 19-16 19-17 19-18 19-19 19-20 19-21 19-22 19-23 19-24 19-25 19-26
13 41 34 12 25 13 59 104 29 107 395 185 107 212
(-)
(-)
circled samples sent for sequencing
Positive Controls
DNA from older adult and larval P. ant tissue
DNA from 2014 Ross Sea larval tissue
Negative Controls
5
PCR products were visualized on a 1.8% agarose gel with a 1 kb ladder to assess yield.
Amplification of mtDNA from the larvae showed evidence of DNA damage that can be seen
in the smear signatures of the majority of larval samples compared to the absence of such
signatures in the positive controls (Fig. 2a,b). Thus, only a portion of the larvae from which
DNA was extracted produced enough amplified mtDNA fragments to proceed with
sequencing. The D-Loop region amplified somewhat more successfully than the 16S rDNA,
possibly due to its shorter length (349 bp versus 568 bp). The damage prevented a series of
detailed population analyses that were initially planned; nevertheless, the species
identification by Brooks and Goetz (2014) could still be tested. Six WRS larvae from six
different tows and five ERS larvae were used that showed clear bands in the post-PCR gel
in both the 16S rDNA and D-Loop region PCRs (Fig. 2a,b). Two of the positive controls (one
adult from the Weddell Sea and one larva from the Ross Sea, Table 1) were also included
for sequencing.
Table 1. Samples sequenced indicating species, year, area collected, and study ID. Accession
numbers from NCBI database for GenBank sequences also included.
16S alignment sequences D-Loop alignment sequences
Species
Year Area ID Species Year Area ID
Pleuragramma larva 2014 WRS 2-2 Pleuragramma larva 2014 WRS 2-2
Pleuragramma larva 2014 WRS 3-4 Pleuragramma larva 2014 WRS 3-4
Pleuragramma larva 2014 WRS 10-1 Pleuragramma larva 2014 WRS 10-1
Pleuragramma larva 2014 WRS 11-1 Pleuragramma larva 2014 WRS 11-1
Pleuragramma larva 2014 WRS 12-2 Pleuragramma larva 2014 WRS 12-2
Pleuragramma larva 2014 WRS 16-1 Pleuragramma larva 2014 WRS 16-1
Pleuragramma larva 1998 Ross R4 Pleuragramma larva 1998 Ross R4
Pleuragramma adult 2007 Weddell 11 3 Pleuragramma adult 2007 Wedde ll 113
Pleuragramma larva 2014 ERS 19-2
Pleuragramma larva 2014 ERS 19-4
Pleuragramma larva 2014 ERS 19-11
Pleuragramma larva 2014 ERS 19-26
Species Species
Pleuragramma antarctica Pleuragramma antarctica
Pleuragramma antarctica Pleuragramma antarctica
Pleuragramma antarctica Pleuragramma antarctica
Pleuragramma antarctica Pleuragramma antarctica
Pleuragramma antarctica Pleuragramma antarctica
Pleuragramma antarctica Pleuragramma antarctica
Pleuragramma antarctica Pleuragramma antarctica
Aethotaxis mitopterix Pleuragramma antarctica
Aethotaxis mitopteryx Pleuragramma antarctica
Dissostichus eleginoides Pleuragramma antarctica
Dissostichus eleginoides Pleuragramma antarctica
Dissostichus eleginoides Pleuragramma antarctica
Dissostichus eleginoides Pleuragramma antarctica
Dissostichus mawsoni Pleuragramma antarctica
Dissostichus mawsoni Pleuragramma antarctica
Dissostichus mawsoni Pleuragramma antarctica
Aethotaxis mitopteryx
Dissostichus eleginoides
Dissostichus eleginoides
Dissostichus eleginoides
Dissostichus eleginoides
Dissostichus mawsoni
Dissostichus mawsoni
EF088420.1
DQ487352.1
GenBank Accession
numbers
DQ487351.1
EF589653.1
EF595242.1
DQ854836.1
DQ854837.1
DQ854838.1
DQ854839.1
DQ487354.1
AB889896.1
DQ854830.1
DQ854831.1
DQ854832.1
DQ854833.1
DQ854834.1
DQ854835.1
DQ854824.1
DQ854825.1
DQ854826.1
DQ854827.1
DQ854828.1
DQ854829.1
EF589650.1
AF145410.1
EF595240.1
AY520111.1
AY520110.1
Z32726.1
NC 015652.1
AF145420.1
JF933905.1
AF145408.1
AY520106.1
AY520109.1
GenBank Accession
numbers
KF713478.1
JX974426.1
AY520108.1
AY520107.1
6
To prepare for Sanger sequencing, unincorporated dNTPs and primers were removed from
PCR products using a pre-sequencing kit (Affymetrix) according to the manufacturers
instructions. Fragments were sequenced using an internal service, BMR Genomics
(www.bmr-genomics.it).
DNA sequence analysis
Prior to alignment, the raw fluorogram sequences were visually inspected and cleaned up
using Chromas LITE Version 2.1.1. Sequences were aligned against 7 16S rDNA and 16 D-
Loop region corresponding sequences of P. antarctica retrieved from the nucleotide
database NCBI (Fig. 3, see Table 1 for GenBank accession numbers). Alignment was then
performed by MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/) using MEGA 6.06
(Tamura et al. 2013). Three closely related notothenioids Aethotaxis mitopteryx,
Dissostichus mawsoni and Dissostichus eleginoides (Near et al. 2012) were included as
outgroups (Fig. 3, see Table 1 for GenBank accession numbers). The proportion of
nucleotide positions at which two sequences differed represented by the p-distance was
used to calculate genetic distance in MEGA. Based on these distances, a neighbour-joining
tree was drawn using the Kimura 2-parameter substitution model, with tree robustness
confirmed by 1000 bootstrap replications.
7
Figure 3. Alignment of 16S rDNA and D-Loop region sequences. Identical bases are shaded in
grey.
a) 16S
....|....|....|....|....|....|....|....|....|....|....|....|.... |....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
Pleuragramma larva WRS 2-2
C
G
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T
Pleuragramma larva WRS 3-4
C
G
G
C
C
G
C
G
G
T
A
T
T
T
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G
A
C
C
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A
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C
A
G
A
A
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C
G
G
G
G
A
T
Pleuragramma larva WRS 10-1
C
G
G
C
C
G
C
G
G
T
A
T
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G
A
C
C
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C
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A
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C
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C
G
G
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G
A
T
Pleuragramma larva WRS 11-1
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
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A
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A
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A
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C
G
G
G
G
A
T
Pleuragramma larva WRS 12-2
C
G
G
C
C
G
C
G
G
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A
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Pleuragramma larva WRS 16-1
C
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C
A
G
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A
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C
G
G
G
G
A
T
Pleuragramma larva Ross R4
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
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A
C
C
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T
C
C
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
G
A
T
Pleuragramma adult Weddell 113
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
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A
A
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A
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G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
C
C
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
G
A
T
P. antarctica KF713478.1
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
C
C
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
G
A
T
P. antarctica JX974426.1
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
C
C
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
G
A
T
P. antarctica AY520108.1
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
C
C
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
G
A
T
P. antarctica AY520107.1
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
C
C
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
G
A
T
P. antarctica NC 015652.1
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
C
C
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
G
A
T
P. antarctica AF145420.1 - - - - -
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
C
C
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
G
A
T
P. antarctica JF933905.1
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
C
C
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
G
A
T
A. mitopterix AF145408.1 - - -
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
T
T
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
A
A
T
A. mitopterix AY520106.1
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
T
T
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
A
A
T
D. eleginoides AY520109.1
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
T
T
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
A
A
T
D. eleginoides EF589650.1
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
T
T
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
A
A
T
D. eleginoides AF145410.1
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
T
T
T
C
G
T
G
C
A
G
A
A
G
C
G
G
G
A
A
T
D. eleginoides EF595240.1
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
T
T
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
A
A
T
D. eleginoides AY520111.1
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
T
T
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
A
A
T
D. mawsoni AY520110.1
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
T
T
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
A
A
T
D. mawsoni Z32726.1
C
G
G
C
C
G
C
G
G
T
A
T
T
T
T
G
A
C
C
G
C
G
C
G
A
A
G
G
T
A
G
C
G
C
A
A
T
C
A
C
T
T
G
T
C
T
C
T
T
A
A
A
T
G
G
A
G
A
C
C
T
G
T
A
T
G
A
A
T
G
G
C
A
T
A
A
C
G
A
G
G
G
C
T
T
A
G
C
T
G
T
C
T
C
C
T
C
C
C
C
T
A
A
G
T
T
A
A
T
G
A
A
A
T
T
G
A
T
C
T
T
T
C
C
G
T
G
C
A
G
A
A
G
C
G
G
G
A
A
T
....|....|....|....|....|....|....|....|....|....|....|....|.... |....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
Pleuragramma larva WRS 2-2
A
C
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
C
C
C
A
G
A
C
G
A
C
A
A
G
G
A
C
C
C
T
G
T
G
A
T
A
A
C
A
G
G
G
C
A
A
G
C
C
A
G
A
G
G
G
G
C
C
-
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
Pleuragramma larva WRS 3-4
A
C
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
C
C
C
A
G
A
C
G
A
C
G
A
G
G
A
C
C
C
T
G
T
G
A
T
A
A
C
A
G
G
G
C
A
A
G
C
C
A
G
A
G
G
G
G
C
C
-
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
Pleuragramma larva WRS 10-1
A
C
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
C
C
C
A
G
A
C
G
A
C
G
A
G
G
A
C
C
C
T
G
T
G
A
T
A
A
C
A
G
G
G
C
A
A
G
C
C
A
G
A
G
G
G
G
C
C
-
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
Pleuragramma larva WRS 11-1
A
C
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
C
C
C
A
G
A
C
G
A
C
G
A
G
G
A
C
C
C
T
G
T
G
A
T
A
A
C
A
G
G
G
C
A
A
G
C
C
A
G
A
G
G
G
G
C
C
-
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
Pleuragramma larva WRS 12-2
A
C
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
C
C
C
A
G
A
C
G
A
C
G
A
G
G
A
C
C
C
T
G
T
G
A
T
A
A
C
A
G
G
G
C
A
A
G
C
C
A
G
A
G
G
G
G
C
C
-
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
Pleuragramma larva WRS 16-1
A
C
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
C
C
C
A
G
A
C
G
A
C
G
A
G
G
A
C
C
C
T
G
T
G
A
T
A
A
C
A
G
G
G
C
A
A
G
C
C
A
G
A
G
G
G
G
C
C
-
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
Pleuragramma larva Ross R4
A
C
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
C
C
C
A
G
A
C
G
A
C
G
A
G
G
A
C
C
C
T
G
T
G
A
T
A
A
C
A
G
G
G
C
A
A
G
C
C
A
G
A
G
G
G
G
C
C
-
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
Pleuragramma adult Weddell 113
A
C
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
C
C
C
A
G
A
C
G
A
C
G
A
G
G
A
C
C
C
T
G
T
G
A
T
A
A
C
A
G
G
G
C
A
A
G
C
C
A
G
A
G
G
G
G
C
C
-
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
P. antarctica KF713478.1
A
C
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
C
C
C
A
G
A
C
G
A
C
G
A
G
G
A
C
C
C
T
G
T
G
A
T
A
A
C
A
G
G
G
C
A
A
G
C
C
A
G
A
G
G
G
G
C
C
-
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
P. antarctica JX974426.1
A
C
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
C
C
C
A
G
A
C
G
A
C
G
A
G
G
A
C
C
C
T
G
T
G
A
T
A
A
C
A
G
G
G
C
A
A
G
C
C
A
G
A
G
G
G
G
C
C
-
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
P. antarctica AY520108.1
A
C
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
C
C
C
A
G
A
C
G
A
C
G
A
G
G
A
C
C
C
T
G
T
G
A
T
A
A
C
A
G
G
G
C
A
A
G
C
C
A
G
A
G
G
G
G
C
C
-
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
P. antarctica AY520107.1
A
C
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
C
C
C
A
G
A
C
G
A
C
G
A
G
G
A
C
C
C
T
G
C
G
A
T
A
A
C
A
G
G
G
C
A
A
G
C
C
A
G
A
G
G
G
G
C
C
-
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
P. antarctica NC 015652.1
A
C
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
C
C
C
A
G
A
C
G
A
C
G
A
G
G
A
C
C
C
T
G
T
G
A
T
A
A
C
A
G
G
G
C
A
A
G
C
C
A
G
A
G
G
G
G
C
C
-
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
P. antarctica AF145420.1
A
C
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
C
C
C
A
G
A
C
G
A
C
G
A
G
G
A
C
C
C
T
G
T
G
A
T
A
A
C
A
G
G
G
C
A
A
G
C
C
A
G
A
G
G
G
G
C
C
-
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
P. antarctica JF933905.1
A
C
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
C
C
C
A
G
A
C
G
A
C
G
A
G
G
A
C
C
C
T
G
T
G
A
T
A
A
C
A
G
G
G
C
A
A
G
C
C
A
G
A
G
G
G
G
C
C
-
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
A. mitopterix AF145408.1
A
A
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
A
C
C
A
G
A
C
T
A
C
A
A
A
G
A
C
C
C
T
C
T
C
A
T
A
A
G
A
G
G
A
C
A
A
A
C
C
A
A
A
A
G
G
C
C
C
A
C
T
A
C
C
C
C
G
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
A. mitopterix AY520106.1
A
A
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
T
A
G
A
C
G
T
A
A
G
G
T
A
G
A
C
C
A
G
A
C
T
A
C
A
A
A
G
A
C
C
C
T
C
T
C
A
T
A
A
G
A
G
G
A
C
A
A
A
C
C
A
A
A
A
G
G
C
C
C
A
C
T
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
D. eleginoides AY520109.1
A
A
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
C
A
G
A
C
G
T
A
A
A
G
T
G
G
A
C
C
A
A
G
C
T
A
C
A
A
A
G
A
C
C
C
C
A
T
G
A
T
A
A
A
G
G
G
A
C
A
A
A
C
C
A
A
A
A
G
A
G
C
C
G
C
C
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
D. eleginoides EF589650.1
A
A
A
C
A
C
A
T
A
A
G
A
C
G
A
G
A
A
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
C
A
G
A
C
G
T
A
A
A
G
T
G
G
A
C
C
A
A
A
C
T
A
C
A
A
A
G
A
C
C
C
C
A
T
G
A
T
A
A
A
G
G
G
A
C
A
A
A
C
C
A
A
A
A
G
A
G
C
C
G
C
C
A
C
C
C
C
A
A
T
G
T
C
T
T
T
G
G
T
T
G
G
G
G
C
G
A
C
C
G
C
G
G
G
G
A
A
A
G
A
A
A
A
A
A
C
D. eleginoides AF145410.1
A
T
A
T
A
C
A
T
A
A
G
A
C
G
G
G
A
G
G
A
C
C
C
T
A
T
G
A
A
G
C
T
T
C