High genetic diversity with Epimeria georgiana (Amphipoda) from the southern Scotia Arc

Abstract

DNA barcoding revealed four well-supported clades among amphipod specimens that keyed out to Epimeria georgiana Schellenberg, 1931, three clades with specimens from the southern Scotia Arc and one clade with specimens from the Weddell Sea. Detailed morphological investigations of sequenced specimens were conducted, through light and scanning electron microscopy. High magnification (500–2,000 fold) revealed features such as comb-scales on the first antenna and trich bearing pits on the fourth coxal plate to be similar for all specimens in the four clades. Consistent microstructure character differences in the Weddell Sea specimens combined with high genetic distances (COI divergence > 20%) allowed the description of Epimeria angelikae, a species new to science. Specimens of E. georgiana in the other three COI clades from the Scotia Arc were morphologically indistinguishable. Representative specimens of clade A are also illustrated in detail. Our results on the high genetic divergences in epimeriid amphipods support the theory of the southern Scotia Arc being a centre of Antarctic diversification.

Full text

ORIGINAL PAPER
High genetic diversity within Epimeria georgiana
(Amphipoda) from the southern Scotia Arc
Anne-Nina Lörz &Peter Smith &Katrin Linse &
Dirk Steinke
Received: 9 February 2011 /Revised: 25 May 2011 / Accepted: 6 July 2011
#Senckenberg, Gesellschaft für Naturforschung and Springer 2011
Abstract DNA barcoding revealed four well-supported
clades among amphipod specimens that keyed out to
Epimeria georgiana Schellenberg, 1931, three clades with
specimens from the southern Scotia Arc and one clade with
specimens from the Weddell Sea. Detailed morphological
investigations of sequenced specimens were conducted,
through light and scanning electron microscopy. High
magnification (500–2,000 fold) revealed features such as
comb-scales on the first antenna and trich bearing pits on
the fourth coxal plate to be similar for all specimens in the
four clades. Consistent microstructure character differences
in the Weddell Sea specimens combined with high genetic
distances (COI divergence> 20%) allowed the description
of Epimeria angelikae, a species new to science. Specimens
of E. georgiana in the other three COI clades from the
Scotia Arc were morphologically indistinguishable. Repre-
sentative specimens of clade A are also illustrated in detail.
Our results on the high genetic divergences in epimeriid
amphipods support the theory of the southern Scotia Arc
being a centre of Antarctic diversification.
Keywords DNA barcoding .Epimeriidae .New species .
Scanning electron microscopy.Scotia Arc .High biodiversity
Introduction
The intensified use of mitochondrial gene sequences for the
identification of morphologically indistinguishable and poly-
morphic taxonomic groups has lead to the discovery of species
complexes in many groups (e.g. Costa and Carvalho 2007;
Radulovici et al. 2010;Wilsonetal.2009;O’Loughlin et al.
2011). Furthermore, species complexes of Antarctic benthic
peracarid crustaceans have been identified by DNA barcoding
(e.g. Leese et al. 2010;Held2003; Raupach and Wagele 2006;
Lörz et al. 2009; Havermans et al. 2010,2011). This method
utilizing a fragment of the mitochondrial cytochrome coxidase
Igene(COI) for species discrimination was successfully
applied among crustaceans in general (e.g. Leese et al. 2008;
Chu et al. 1999) and amphipods in particular thereby already
contributing to the taxonomic knowledge of this group
(Meyran et al. 1997;Wittetal.2006; Radulovici et al. 2009).
DNA barcoding has proven to be most beneficial when
used in an integrative approach together with morphological
or ecological data (Meier 2008). Only in concert with
morphological investigations barcodes allow the establish-
ment of formal species boundaries and species descriptions.
A taxon of special interest is the amphipod family
Epimeriidae Boeck, 1871. The globally distributed family
Epimeriidae Boeck, 1871 belongs to the dominant members
of Antarctic shelf benthos (Coleman 2007;Lörzetal.2009).
Epimeriidae are far more diverse in the Southern Ocean than
anywhere in the world. In temperate and warm oceans, they
A.-N. Lörz (*)
National Institute of Water and Atmospheric Research (NIWA),
Private Bag 14 901, Kilbirnie,
Wellington, New Zealand
e-mail: a.loerz@niwa.co.nz
P. Smith
Museum Victoria,
GPO Box 666, Melbourne Vic 3001, Australia
K. Linse
British Antarctic Survey,
High Cross, Madingley Road,
Cambridge CB3 0ET, UK
D. Steinke
Biodiversity Institute of Ontario, University of Guelph,
50 Stone Road East,
Guelph, ON, N1G2W1, Canada
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DOI 10.1007/s12526-011-0098-8

are only found in deep waters, while they occur in shallower
waters of the continental shelf in Antarctica and in
Scandinavia. Twenty-six species of Epimeriidae are known
from Antarctic waters (Lörz 2011). Recent research on
circum-Antarctic species or species nominally found in both
the Weddell and Ross Seas has shown that they belong to
species complexes and represent sister species (Lörz et al.
2007,2009). Investigations at smaller spatial scales have not
yet been undertaken.
Synopsis of Epimeria georgiana species complex
1903 Walker describes Epimeria inermis new spec from
Cape Adare, Ross Sea, 50 m depth, with the illustration of
one habitus drawing.
1907 Walker illustrates E. inermis: pereopods 5 and 6
with strong hooks.Systematics
1931 Schellenberg describes E. georgiana from
Cumberland Bay, South Georgia (75-310 m) as very
similar to E. inermis, except for shape of basis of
pereopods 5, 6 and 7. No illustrations of the new species
are provided by Schellenberg. No further details were
provided by Schellenberg 1931, except one sentence
“tooth on basis of pereopod 7 blunt 90 degrees corner,
placed further distally than basis P5 and P6”(translation
from the German original by first author)
1932 Barnard describes Epimeria exisipes new spec,
with a photograph, and one figure of the coxa 4–7 with the
bases of pereopods 5–7 from South Shetlands, South
Georgia and the Palmer Archipelago.
1989 Watling and Thurston synonymise Epimeria excisipes
K.H. Barnard, 1932 with Epimeria georgiana Schellenberg,
1931 and draw the habitus, antenna 1, lower lip, maxilliped,
parts of gnathopod 1 and the telson. Watling and Thurston
(1989) do not draw or describe the pereopods.
2007 Coleman presents a synopsis for all Antarctic
Epimeriidae and mentions in his diagnosis for E. georgiana
“Bases 5–7 ridged, with deep notches on posterior margin,
creating prominent teeth”.Coleman(2007) noted that E.
inermis can be differentiated from E. georgiana by the
unsculptured coxa, which has a straight or convex ventral
margin and a linear posterodistal margin. The posterior margin
of the basis of pereopods 5–6 is not developed to a ventrally
directing tooth, as it is the case in E. georgiana, but these
articles have posterobasal rounded lobes showing posteriorly.
2009 Lörz et al. discovered high genetic difference
between four specimens of the Epimeria georgiana group
from the Antarctic Peninsula and the Weddell Sea.
Here we use the concept of integrative taxonomy and
combine DNA barcoding and morphological analyses of
specimens of the Epimeria georgiana complex, from the
Antarctic Peninsula and the eastern Weddell Sea to describe
diversity in this Southern Ocean biota.
Material and methods
Taxon sampling
Epimeriid amphipods were collected during a RV Tangaroa
voyage to the Western Ross Sea (IPY, TAN0802), during RV
Polarstern voyages to the Weddell Sea (BENDEX and
ANDEEP III) and a RRS James Clark Ross voyage to the
Antarctic Peninsula (BIOPEARL I) (Fig. 1). Specimens
were sorted on deck, often photographed alive to record the
colour morphs, fixed in 98% ethanol and later transferred to
70% ethanol.
The amphipod specimens were registered and curated at
the National Institute for Water and Atmospheric Research
(NIWA) in Wellington, New Zealand; the Natural History
Collection London, UK; the British Antarctic Survey (BAS),
Cambridge, UK; and the Zoological Museum Hamburg,
Germany (Table 1). Specimens and especially DNA samples
from marine Antarctic invertebrates are highly valuable.
Morphological description
More than 100 specimens that keyed out to Epimeria
georgiana from the NIWA Invertebrate Collection (New
Zealand), the Zoological Museum Hamburg (Germany) and
the British Antarctic Survey (UK) were morphologically
examined. Additional material examined: Epimeria georgiana
Schellenberg, 1931, one specimen, type material SMNH673,
Stockholm, Swedish Museum of Natural History.
Epimeria georgiana registered as Epimeria exisipes Bar-
nard, 1932, 13 specimens, 1536–1550, Natural History
Museum London. Epimeria inermis Wa l k e r 1907, one speci-
men, BMNH 1903.10.5.19, Natural History Museum London.
Twelve specimens were dissected under a Leica MZ12
stereomicroscope and drawn using a camera lucida. Small
appendages and bodyparts (mouthparts, uropods, telson) were
temporarily mounted in glycerin, examined and drawn using a
Nikon compound microscope fitted with a camera lucida. The
body lengths of specimens examined were measured by
tracing individual mid-trunk lengths (tip of the rostrum to end
of telson) using a camera lucida. All illustrations have been
digitally ‘inked’following Coleman (2003;2009).
Parts of selected specimens (mouthparts, antennae, coxal
plates) were air dried, coated with goldpaladium and
investigated via a Scanning electron microscope LEO1525.
DNA extraction and analyses
DNAwas extracted from sub-samples of muscle tissue from 18
specimens of E. georgiana, three specimens of E. rimicarinata
and one specimen of E. inermis using an automated Glass
Fiber protocol (Ivanova et al. 2006). The 650-bp barcode
region of COI was amplified by polymerase chain reaction
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(PCR) under the following thermal conditions: 1 min at 94°C;
five cycles of 94°C for 40s, 45°C for 40s and 72°C for 1 min,
followed by 35 cycles at 94°C for 40s, 40s at 51°C, and 1 min
at 72°C; and a final step of 72°C for 1 min. The 12.5 μlPCR
reaction mixes included 6.25 μl of 10% trehalose, 2.00 μlof
ultrapure water, 1.25 μl of 10 × PCR buffer [200 mM Tris-HCl
(pH 8.4), 500 mM KCl], 0.625 μlMgCl
2
(50 mM), 0.125 μl
of each primer [0.01 mM, using LCO1490/HCO2198
(Folmer et al. 1994) with M13 tails], 0.062 μlofeachdNTP
(10 mM), 0.060 μl of Platinum Taq Polymerase (Invitrogen),
and 2.0 μl of DNA template. PCR amplicons were visualized
on a 1.2% agarose gel E-Gel (Invitrogen) and bidirectionally
sequenced using sequencing primers M13F or M13R and the
BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied
Biosystems) on an ABI 3730 capillary sequencer following
manufacturer’s instructions. Sequence data are available both
on BOLD (Ratnasingham and Hebert 2007) in the public
project “Epimeria georgiana species complex—EPIME”and
GenBank (accession numbers are given in Table 1).
Sequences were edited in CHROMAS 2.3 (Technely-
sium, Queensland, Australia), and aligned using CLUSTAL
in MEGA v. 4.1 (Tamura et al. 2007). K2P distances among
taxa were estimated in MEGA v. 4.1 (Tamura et al. 2007).
Initial neighbor-joining (NJ) clustering used the BOLD
Management and Analysis System. Maximum likelihood
(ML) trees were built using PAUP* (Swofford 2003), with
heuristic searches, employing tree bisection-reconnection
branch swapping; support for each internode was evaluated
by 1000 bootstrap replications (Felsenstein 1985). Bayesian
phylogenetic analyses were estimated with MrBayes ver-
sion 3.0 (Ronquist and Huelsenbeck 2003). Four simulta-
neous Monte Carlo chains were run for 1 × 10
7
generations,
saving the current tree every 1,000 generations. Consensus
trees with posterior probabilities were created with a burn-
in value equal to 1000 (the first 1,000 trees were discarded).
Nucleotide substitution models were selected in jModeltest
version 0.1.1 (Posada 2008) using both the Akaike
Information Criterion (AIC) and the Bayesian information
criterion (BIC); the transition model TVM + Γmodel was
selected by both criteria. The epimeriid Epimeria rimicar-
inata was used to root the trees because it is closely related
to E. inermis and E. georgiana.
Fig. 1 Map of collections sites
for studied Epimeria specimens.
Key: ♦E. angelikae new spec,
✰E. georgiana type locality, ▲
E. georgiana clade A, ●E.
georgiana clade B, ■E. georgi-
ana clade C, ✚E. inermis
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Table 1 Station locations, vial numbers, individual specimen codes and GenBank accession numbers. BMNH British Museum of Natural History, EI Elephant Island, LI Livingston Island, n
number of specimens, RS Ross Sea, SG South Georgia, SMNH Swedish Museum of Natural History, WS Weddell Sea, ZMH Zoological Museum Hamburg
GenBank Latitute Longitute Depth
Taxon Spec/vial id n COI Station (S) (m) Date Expedition Location
Epimeria angelikae
Holotype ZMH K-42632 1 - PS65/233-1 71° 19.19′013° 57.45′W 844-848 21.12.2003 BENDEX WS
Paratype NIWA 42844 1 FM955305 PS65/232-1 71° 18.37′013° 56.70′W 899-910 21.12.2003 BENDEX WS
Paratype ZMH K-42633 1 FM955299 PS65/233-1 71° 19.19′013° 57.45′W 844-848 21.12.2003 BENDEX WS
BAS KL03-441 3 1 - PS65/232-1 71° 18.37′013° 56.70′W 899-910 21.12.2003 BENDEX WS
BAS KL03-441 2 1 - PS65/232-1 71° 18.37′013° 56.70′W 899-910 21.12.2003 BENDEX WS
BAS KL03-441 4 - PS65/232-1 71° 18.37′013° 56.70′W 899-910 21.12.2003 BENDEX WS
Paratype NIWA 34944 (BAS 03-441-4) 1 - PS65/232-1 71° 18.37′013° 56.70′W 899-910 21.12.2003 BENDEX WS
NIWA 34941 (BAS 03–494 1) 1 - PS65/232-1 71° 18.37′013° 56.70′W 899-910 21.12.2003 BENDEX WS
NIWA 34943 (BAS 03 441 1) 1 - PS65/232-1 71° 18.37′013° 56.70′W 899-910 21.12.2003 BENDEX WS
Epimeria georgiana
Syntype SMNH673 1 - 34 54°11.00′36º18.00′W 252-310 05.06.1902 Swenska Sypolar exp SG
BNHM 1536-1550 13 - ???
Clade A
NIWA 34945 1 JF271115 EI-AGT-2 61°34.52′055º15.38′W 990-976 12.03.2006 BIOPEARL I EI
BAS KL06-378 1 JF271114 EI-AGT-2 61°34.52′055º15.38′W 990-976 12.03.2006 BIOPEARL I EI
BAS KL06-3766 1 JF271111 EI-AGT-2 61°34.52′055º15.38′W 990-976 12.03.2006 BIOPEARL I EI
NIWA 34935 1 JF271112 EI-AGT-2 61°34.52′055º15.38′W 990-976 12.03.2006 BIOPEARL I EI
NIWA 34936 1 JF271113 EI-AGT-3 61°23.16′055º11.60′W 463-483 12.03.2006 BIOPEARL I EI
NIWA 69496 1 JF271110 EI-AGT-2 61°34.52′055º15.38′W 990-976 12.03.2006 BIOPEARL I EI
Berlin 1 AY061802 PS/44-1 61°43.50′055º12.75′W 590 27.11.1996 ANT XIV/2 LI
BAS KL06-376 5 - EI-AGT-2 61°34.52′055º15.38′W 990-976 12.03.2006 BIOPEARL I EI
Clade B
BAS KL06-5742 1 JF271106 PB-AGT-2 61°02.13′046º51.92′W 964-1,014 17.03.2006 BIOPEARL I PB
NIWA 69497 1 JF271105 PB-AGT-2 61°02.13′046º51.92′W 964-1,014 17.03.2006 BIOPEARL I PB
BAS KL06-5743 1 JF271108 PB-AGT-2 61°02.13′046º51.92′W 964-1,014 17.03.2006 BIOPEARL I PB
NIWA 69498 1 JF271109 PB-AGT-2 61°02.13′046º51.92′W 964-1,014 17.03.2006 BIOPEARL I PB
BAS KL06-5741 1 JF271107 PB-AGT-2 61°02.13′046º51.92′W 964-1,014 17.03.2006 BIOPEARL I PB
Clade C
NIWA 69499 1 JF271104 EI-AGT-4 61°20.04′055º11.70′W 199-201 12.03.2006 BIOPEARL I EI
ZMH K-39888 1 AF451341 ANTXVII-3/177-1 61°49.50′060º49.32′W 202 01.05.2000 EASIZ III LI
ZMH K-400520 1 - ANTXVII-3/183-1 62°06.70′060º21.70′W 204 03.05.2000 EASIZ III LI
BAS KL06-4411 1 JF271103 EI-AGT-4 61°20.04′055º11.70′W 199-201 12.03.2006 BIOPEARL I EI
NIWA 34942 1 JF271102 EI-AGT-4 61°20.04′055º11.70′W 199-201 12.03.2006 BIOPEARL I EI
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Results
DNA results
DNA barcodes revealed four well supported clades among
18 specimens that had keyed out to Epimeria georgiana
Schellenberg, 1931 (Fig. 2). The clades are referred to as A,
B, C and new species (= clade D). Clade A consists of
specimens collected on the continental shelf off Elephant
and Livingston Island in 463–990 m depth, clade B of
specimens from the Powell Basin (969–1,014 m), clade C
of specimens collected on the shallow shelf off Elephant
and Livingston Island (199–204 m), while the specimens of
the new species (clade D) were collected in the eastern
Weddell Sea in 844–910 m depth (Table 1). Sequence
divergence (K2P) was low within each of five clades,
including the outgroup E. rimicarinata, ranging from 0 to
0.22% (Table 2). Pairwise inter-clade distances ranged from
2.59 to 22.46%; comparisons including clade D or the
outgroup E. rimicarinata showed the greatest distances
(12.9-22.5%, Table 2).
Clades A, B, and C showed lower inter-clade divergences,
but were well supported by ML and Bayesian analyses, and
the divergences were more than 10-times the intra-clade
divergences (Table 2). It has been suggested that the barcode
“gap”between species should be based on the smallest rather
than mean inter-specific distances (Meier et al. 2008); these
values are shown in parentheses in Table 2. For the Epimeria
COI data set there is little difference between the mean and
smallest values due to the low intra-specific divergences in
all clades with three or more specimens.
Morphology
Based on the genetic results, detailed morphological
examinations on specimens of the four clades were carried
out, including SEM imaging of coxa and antennae micro-
structures (Table 3). No permission was given to take tissue
samples of the type material of E. inermis and as no DNA-
suitable tissue samples from the type locality of E.
georgiana were available, we included type specimens in
the detailed morphological examinations.
Morphological differences between specimens of the
clades A, B, C and specimens of clade D (new species)
were consistent and therefore a new species of Epimeria
from the Weddell Sea is described in detail herein.
The morphological investigation failed to obtain distinct
and significant differences between specimens of the clades
A, B, and C. However, specimens from clade A exhibited
small morphological differences to specimens of clades B
and C (see Table 3) but the selected morphological
characters did not justify new morpho-species descriptions.
Specimens from clade C were morphologically most similar
Table 1 (continued)
GenBank Latitute Longitute Depth
Epimeria inermis
Holotype BMNH 1903.10.5.19 1 - Cape Adare 50 13.05.1903 RS
NIWA 36151 1 JF271116 TAN0802/56 75°37.95′169º51.00′E 525 14.02.2008 TAN0802 RS
NIWA 20164 1 FM955280 TAN0402/ 94 71°31.80′170°06.66′E 220 17.02.2004 TAN0402 RS
NIWA 20169 1 FM955281 TAN0402/ 184c 71°30.03′171°36.42′E 480 27.02.2004 TAN0402 RS
NIWA 20171 1 FM955282 TAN0402/233 67°25.07′163°54.93′E 230 04.03.2004 TAN0402 RS
NIWA 20168 1 FM955292 TAN0402/134 71°38.50′170°09.15′E 65 23.02.2004 TAN0402 RS
NIWA 20162 1 FM955285 TAN0402/33 71°45.28′171°25.02′E 282 10.02.2004 TAN0402 RS
Epimeria rimicarinata
NIWA 35470 1 JF271118 TAN0802/17 73°07.48′174º19.25′E 321 09.02.2008 TAN0802 RS
NIWA 37662 1 GU804288 TAN0802/161 72°04.53′172º54.26′E 535 24.02.2008 TAN0802 RS
NIWA 35469 1 JF271117 TAN0802/17 73°07.48′174º19.25′E 321 09.02.2008 TAN0802 RS
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to the E. georgiana type material. However, since the
specimens of clade A showed a significant genetic
divergence of about 6% to clade C, and the type material
was never fully illustrated, we present detailed illustrations
of one specimen from clade A (Figs. 3,4,5).
Systematics
Order AMPHIPODA Latreille, 1816
Suborder GAMMARIDEA Latreille, 1802
Family EPIMERIIDAE Boeck, 1871
Genus Epimeria Costa,1851
Epimeria angelikae sp. nov.(Figs. 6,7,8,9,10,11,
12.1,12.2)
Lörz and Linse, 2011
Material examined
Holotype: ZMH K-42632, male, 20.2 mm, collected ANT-
XXI/2 via Agassiztrawl station 233, 71°19.19 S, 13º57.45 W,
850 m, 21 Dec 2003.
Paratype: NIWA 42844, male, 25.5 mm, collected ANT-
XXI/2 via an Epibenthic sled, station 232, 71°18.8 S,
13º56.84 W, 870 m, 21 Dec 2003. NIWA 34944, 18.8 mm,
collected same station, GenBank accession number COI
FM955305.
Paratype: ZMH K-42633, 21.2 mm, collected ANT-XXI/
2 via an Epibenthic sled, station 233, as holotype, GenBank
accession number COI FM955299.
Additional material: five further specimens from ANT-
XXI/2 st 232 (NIWA 34941, NIWA 34943, BAS
KL034413, BAS KL034412, BAS KL03441).
Etymology
The species is dedicated to Prof. Dr. Angelika Brandt
in acknowledgement of her devotion to Antarctic
Peracarida.
Diagnosis
Anterior cephalic margin sinuous, lateral cephalic lobe
slightly produced; rostrum as long as the head, reaching
antenna 1 peduncle article 1; eye present, oval, 0.5 × head
height. Pereonite 1 shorter in length than head, pereonite 2
about 0.75×length of 1; pereonites 1–4 lacking mid-dorsal
Within species Between species/clades
Clade A Clade B Clade C Ea Er
Clade A 0.09
Clade B 0.13 2.59 (2.34)
Clade C 0.16 6.23 (5.18) 5.99 (5.86)
Ea 0.00 20.61 20.21 21.42
Er 0.22 12.90 13.42 15.62 22.46
Ei na 18.55 20.44 22.21 23.14 18.92
Ta b l e 2 Nucleotide distances
(K2P) within and between spe-
cies/clades of Epimeria (values
in parentheseslowest inter-clade
nucleotide distances). Epimeria
aff georgiana clades A, B and C,
Epimeria angelikae new spec
(Ea, Clade D), Epimeria
rimicarinata (Er) and Epimeria
inermis (Ei)
Fig. 2 Relationships of COI
sequences from Epimeria speci-
mens. GenBank accession
numbers are given for each
specimen. Numbers at nodes are
Bayesian inference posterior
probability values and ML
bootstrap percentages (>85%)
after 1000 replications; scale
bar represents an interval of the
TVM+ G model. Location
abbreviations, as in Table 1. The
tree has been rooted with
Epimeria rimicarinata
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or dorsolateral processes; pereonite 5–7 dorsolateral carina
developed; coxa 5 laterally broadly produced; pleonites 1–3
with well-developed longitudinal dorsal carina. Epimeron 1
antero- and posteroventral angle rounded; epimeron 2 and 3
posteroventral angle produced. Urosomite 1 with a middor-
sal bump. Urosomite 2 shortest middorsally flat. Urosomite
3 dorsally flat.
Description based on light microscopy of the holotype
Antenna 1 accessory flagellum scale-like; primary flagel-
lum broken off after article 24. Antenna 2 peduncle article 1
with three long processes; article 2 with two slight
processes, slightly shorter than article 1; article 3 longest,
articles 3–5 lacking distal processes; flagellum complete
with 42 articles.
Mandible incisor strongly dentate; molar produced and
triturative; palp article 3 densely setose medially, with long
stout setae distally.
Maxilla 1 medial plate subtriangular, obliquely convex
inner margin with eight plumose setae; lateral plate distal
margin oblique, with 11 medially lobate setae; palp strongly
exceeding outer plate; palp article 1 short, article 2 slightly
curved medially with eight stout setae distomedially.
Table 3 Morphological characterisation of type material of Epimeria angelikae new spec, Epimeria georgiana type material, Epimeria aff
georgiana clade A (NIWA 69496), clade B (BAS KL 574–2), clade C (NIWA 69499) and Epimeria inermis type material
E. angelikae E. georgiana Clade A Clade B Clade C E. inermis
1 Body robust x x x x x x
2 Rostrum longer than first peduncular article of antenna 1 no x x x x x
3 Eyes large x x x x x x
4 Shape of ocular lobe reniform round round round round oval
5 Lateral cephalic lobe produced and pointed rounded rounded rounded rounded rounded
6 Pereonites 2 with short, dorsal posteromarginal protrusions,
not carinate
xxxxxx
7 Pereonites 3 with short, dorsal posteromarginal protrusions,
not carinate
begin of carina x x x x x
8 Pereonites 4–7 with broad, blunt carinae P 3–7 weak carina x x x x x
9 Less posteriorly protruding humps on pleonites 1-2 x x small x x no definite humps
10 Pleonite 3 with 2 shallow dorsal depressions weak x x x x 3 shallow depressions
11 Urosomite 1 with dorsal depression x x x x x x
12 Urosomite 3 dorsolateral ridges, shallow keel drawn out x x x x x x
13 Epimeral plate 1 lateromarginally rounded, posteroventral
angle produced
xxxxxx
14 Epimeral plates 2 and 3 posteroventrally produced x x x x x x
15 Coxae 1–3 with ridged surface x x x x x x
16 Coxa 1 apically rounded more truncate x x x x pointed
17 Coxa 2 with posteroapical angle x x x x x pointed
18 Gnathopods subchelate x x x x x x
19 Gnathopod palm inconspicuously serrate x x x x x x
20 Gnathopod dactylus with short spines on inner curvature x x x x x x
21 Coxa 3 posteroapically truncate truncate (x) (x) (x) pointed
22 Coxa 4 largest x x x x x x
23 Coxa 4 sculptered no x x x x no
24 Coxa 4 apically truncate or weakly excavate no x convex x x x no
25 Coxa 4 posterior and apical margin turned back, creating a
rounded ridge
no x x x x no
26 Coxa 5 laterally produced, dorsal viewing weak x no (x) x weak
27 Coxa 6 rounded posteriorly with produced hump on lateral
surface
xxxxxx
28 Basis pereopod 7 ridged, deep notch on posterior margin,
creating prominent teeth
bit (x) no no no no
29 Basis pereopod 6 ridged, deep notch on posterior margin,
creating prominent teeth
xxxxxno
30 Basis pereopod 5 ridged, deep notch on posterior margin,
creating prominent teeth
x x no x x no
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Maxilla 2 with long, distally crenulate setae distally on
lateral and medial plates.
Maxilliped outer plate, reaching mid length of merus;
row of robust setae; medial plate reaching half the length of
outer plate; palp medial margin strongly setose; merus,
carpus, propodus margins parallel; dactyl with serrate
medial margin.
Gnathopod 1 coxa long and slender, ventral margin
truncate; basis linear, slender, anterior margin with numer-
ous fine setae; merus longer than ischium, anterior margin
very short, distal margin oblique, posterodistal angle acute,
setose; carpus posterior curved margin with long slender
setae; propodus expanded distally, anterior margin naked
except for distal fringe of short setae, palm finely crenulate,
slightly oblique, with cluster of setae defining rounded
distal margin, posterior margin with numerous long setae;
dactylus slender, curved, as long as palm, posterior margin
strongly serrate.
Fig. 3 Epimeria aff georgiana
clade A. NIWA 34935, 37 mm.
aHabitus lateral, blabrum, c
head with epistome, dmaxilla 1,
emandible, ftelson. Scale bars
a: 0.5 mm, b-f:1mm
Mar Biodiv

Gnathopod 2 coxa slightly wider than coxa 1, coxa and
basis very similar to Gnathopod1, broken off after basis.
Pereopod 3 coxa longer than coxa 2, ventral margin
truncate, basis linear, anterior and posterior margin finely
Fig. 4 Epimeria aff georgiana clade A NIWA 34935, 37 mm. aAntenna 1, bantenna 2, cmaxilliped, dmaxilla 2, epleopod 2. Scale bars a-e:1mm
Mar Biodiv

setulose; merus slightly expanded distally, carpus slightly
shorter than merus, anterior margin naked, posterior margin
with six pairs of setae; propodus naked anteriorly, posterior
margin with seven pairs of setae; dactylus stout, curved.
Fig. 5 Epimeria aff georgiana clade A NIWA 34935, 37 mm. aUropod 1, buropod 2, curopod 3, dgnathopod 1, epereopod 3; fgnathopod 2; g
pereopod 4. Scale bars a-e:1mm
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Pereopod 4 coxa longer than 3, very wide, anterior
margin convex, posterior margin with a long posterior lobe
just above mid-length, ventral margin straight, posterodistal
corner rounded; basis to dactylus as for pereopod 3.
Pereopod 5 coxa subrectangular, laterally expanded; basis
expanded midposteriorly with hook, slightly serrated; merus
constricted proximally; carpus slightly widened distally;
propodus linear, posterior margin with five pairs of setae;
dactylus curved, stout, about 0.3×propodus length.
Pereopod 6 coxa anterior half hidden by coxa 5, anterior
margin weakly concave, posterior margin slightly produced;
basis expanded and posteriorly notched, serrated, hook
pointing ventrally; ischium to dactylus as in pereopod 5.
Pereopod 7 coxa small, basis expanded midposteriorly,
slightly serrated, not forming hook; broken off after merus.
Uropod 1 peduncle subequal in length to inner ramus;
medial margin with one robust seta distally, distal margin
with close row of short setae; inner and outer ramus same
length, lateral marins with spaced row of short setae.
Uropod 2 peduncle naked, two distal setae; inner ramus
length 1.5 × outer ramus, lateral margins of rami with
sparse setae.
Fig. 6 Epimeria angelikae new
spec ZMH K-42632, holotype
20.2 mm. aHabitus lateral, b
habitus dorsal, chead with
epistome, dmaxilla 1, ftelson.
Epimeria angelikae new spec
ZMH K-42633, paratype
21.2 mm; eright mandible.
Scale bars 1mm
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Uropod 3 peduncle short, about 0.3×length of inner ramus,
two processes produced; rami with sparse row of short setae.
Telson 1.2 ×longer than wide; V-shaped emargination
0.3×length, lobes triangular, rounded apically.
Additional description based on magnification via scanning
electron microscope, based on Paratype ZMH K-42633
Figs. 9a-d,10,11
Antenna 2 peduncle article 2 shows cuticular scales at the
anterior part (Fig. 9a, b). Each scale has its posterior end
lifted up to form a comb like structure, which show several
pores—up to three pores at the basis of each “tooth of the
comb”(Fig. 9c, d).
Coxa 4 is entirely covered with pits, each pit has one
small setae (Fig. 10a-e).
According to Oshel and Steele (1988)a microtrich is
shorter than 25 μm, a macrotric longer than 25 μm.
The setae in the pits on the surface of the fourth coxa
plate are between 20 and 30 μm in length and
therefore can not be clearly classified as micro- or
macrotrich.
The inner plate of the maxilliped is covered with fine
setae (Fig. 11a). The grinding surface of the molar is
Fig. 7 Epimeria angelikae new
spec ZMH K-42632, holotype,
20.2 mm. aAntenna 1, b
antenna 2, cmaxilliped, d
hypopharynx, E) labrum. Scale
bars 1mm
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slightly convex and consists of 26 lamella (Fig. 11b, c). The
accessory spine row consists of 11 serrated setae, which
each have one slim simple seta at the base. They seem to be
laterally connected at midheight, but this might be an
artefact (Fig. 11d). The palm of gnathopod 1 bears a row of
stout setae which are slightly curved (Fig. 11e, f), the
dactylus is extending the palm with its lateral margin drawn
out into long, curved serration.
Distribution
Off Kapp Norvegia, eastern Weddell Sea, 844–910 m.
Remarks With increasing size of the specimens, the dorsal
carinae on pereonites 4–7 and on the pleonites 1–3 seems to
get more pronounced forming a double carina per segment
(never as pronounced as in E. rimicarinata). The eyes are
Fig. 8 Epimeria angelikae new
spec ZMH K-42632 Holotype
20.2 mm. aUropod 1, buropod
2, curopod 3, dgnathopod 2, e
gnathopod 1, fpereopod 3, g
pereopod 4. Scale bars 1mm
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oval- to kidney-shaped. The colour of the specimen is
crème-white with light-orange eyes (Fig. 121, 2).
Light microscopy
Comparisons of the type material of Epimeria georgiana
from Stockholm museum and the type material of
Epimeria inermis from the London Natural History
Museum with specimens from each of our four clades
revealed a couple of differences which are summarised in
Ta b l e 3. A special focus was given to the pereopods 5–7,
which are illustrated in Fig. 13. Clade A specimens do not
have a ventrally directed tooth on the basis of pereopod 5,
but a rounded lobe showing posteriorly as described for E.
inermis. Clade A specimens have a tooth at the basis of
pereopod 6 pointing ventrally, but in a less distal position
than in clades B or C. The tooth on the basis of pereopod 6
is largest in clade C and the type material of Epimeria
georgiana.
Studies of the type material of E. inermis showed no hooks
on the basis of pereopods 5–7. We assume Walker (1907)
made the drawings of the hook bearing bases of Epimeria
inermis on specimens that actually were Epimeria georgiana.
Electron microscopy
Via scanning electron microscopy, we compared details of
Epimeria angelikae new spec to Epimeria georgiana clade
A, B and C with focus on the second antenna and the fourth
coxa. Antennae 2 showed the same scales at the peduncle in
all clades (see Fig. 9e, f clade A). The antennae of
peracarids are known to have chemosensory organs (e.g.
Kaïm-Malka et al. 1999). The structures observed in
Epimeria angelikae new spec and the E. georgiana clades
are remotely similar to “pseudochaetal formations”described
from the antennal peduncle of Isopoda (Kaïm-Malka et al.
1999). The major difference is that the observed setae-
like structures do not end free, but are connected at
both terminal ends.
Fig. 9 a-dScanning electron
microscope images of Epimeria
angelikae new spec. Paratype
ZMH K-42633 and e-fEpimeria
aff georgiana clade A NIWA
34945. a,e) antenna 2, b,f)
scales on peduncle of antenna 2,
cdetails of scales, dfrontal view
of one scale, showing pores.
Scale bars a: 100 μm, b:20μm,
c:10μm, d:1μm, e: 200 μm
Mar Biodiv

The overall shape of coxa 4 is very similar from
Epimeria angelikae new spec and E. georgiana clade A,
B and C (Figs. 10a,14a, c, e, f). E. angelikae new spec
from the Weddell Sea has a slightly wider coxa 4 than the
Peninsula specimens. Epimeria georgiana clade C shows
the most structured (thick) fourth coxa. While the anterior
margin of coxa 4 of clade C seems straight in lateral view
(Fig 14e), it seems to be concave when the coxa is tilted
slightly (Fig. 14f). We show both views of the same plate
(Fig 14e, f) to stress the influence of the angle how the
features are illustrated and therefore proportional compar-
isons of features amongst the clades are to be treated
carefully. All E. georgiana clades show the same pits
covering the surface of coxa 4 (Figs. 14,15) as described
for E. angelikae new spec (Fig 10a-e). Each pit has a half of
its border thickened, forming a semicircle (e.g. Fig 15a-d).
In the middle of the cuticular semi-circle, a seta of 20–
30 μm is positioned, always at the side of the pit, under the
border. All semicircles of the fourth coxa are positioned the
same direction, in the middle area of the coxa the opening
are to the ventral side. On the distal extension of the fourths
coxa and towards the ventral margin, the semi circles have
their opening towards the posterior end.
Discussion
DNA Barcoding
DNA barcoding began in 2003 with the proposal that
species could be identified by using a short mitochondrial
gene fragment (COI) of a standardized position in the
mitochondrial genome (Hebert et al. 2003a). Since then,
hundreds of barcode projects, covering a very wide range of
taxa, have been undertaken. Most of these projects share
the goal of contributing to an open-access library derived
from referenced (vouchered) specimens that will improve
understanding of biodiversity, highlight cryptic species, and
provide rapid molecular tools for non-taxonomists to
identify species (Ratnasingham and Hebert 2007). The
Fig. 10 Scanning electron
microscope images of Epimeria
angelikae new spec. Paratype
ZMH K-42633. aFourth coxal
plate, blateral posterior corner
of fourth coxal plate, cpits on
surface of fouth coxal plate, d
cuticular pit with macrotrich
seen laterally; eas dseen from
top, flabrum. Scale bars a
200 μm, b,c:20μm, d,e:
10 μm, f: 100 μm
Mar Biodiv

Census of Antarctic Marine Life Barcoding project started
in 2007 (Grant and Linse 2009) and a major output of the
CAML Barcoding Campaign has been a substantial
increase of Antarctic marine species’barcodes, linked with
the discovery of many new fish and invertebrate species, as
well as cryptic species complexes (e.g. Allcock et al. 2011;
Smith et al. 2010; Grant et al. 2011).
While DNA barcoding has its limitations, in particular the
discrimination of recently diverged species that underwent
introgressive hybridisation, the COI barcode region has been
shown to be appropriate for discriminating between closely
related species across diverse animal phyla (Hebert et al.
2003a,b;Wardetal.2005,2009; Bucklin et al. 2011).
Although there is no fixed divergence value that can be
employed as a species criterion, relatively deep divergence
among COI haplotypes within nominal species highlight
potential overlooked species (Shearer and Coffroth 2008;
Ward et al. 2009). Species recognition is based on the
barcode “gap”between intra- and inter-specific variations. In
general, the barcode “gap”has provided a valuable tool for
species identification and for highlighting cryptic species
(Waugh, 2007); although substantial overlap in intra- and
inter-specific variation has been reported in some marine
gastropods (Meyer and Paulay 2005) and in corals (Shearer
and Coffroth 2008).
A sequence divergence value set at 10-times the average
of within species variation has been identified as likely to
be effective for detecting cryptic species in birds (Hebert et
al. 2004) and a similar criterion has been applied in the
identification of cryptic species of marine fishes (Steinke et
al. 2009; Ward et al. 2009; Zemlak et al. 2009). For the E.
georgiana COI data from Elephant Island and the Powell
Basin the inter-clade distances are more than 10-times the
intra-clade distances. There are several reports of cryptic
Antarctic species identified by DNA studies, e.g. in crinoids
(Wilson et al. 2009), ophiuroids (Hunter and Halanych,
2008), pygnogonids (Krabbe et al. 2010; Arango et al.
2011), and fishes (Smith et al. 2010; Smith et al. 2008).
Another potential problem for DNA barcoding is the
occurrence of nuclear copies of COI recently shown for
Fig. 11 Scanning electon
microscop images Epimeria
angelikae new spec, paratype
ZMH K-42633. aMaxilliped, b
mandible, cmolar of mandible,
dlacina mobilis; egnathopod 1,
fdetailed serration of propodus
and dactylus of gnathopod 1.
Scale bars a,b,d: 100 μm, c:
20 μm, e:10μm
Mar Biodiv

crustaceans (Song et al. 2008;Buhay2009), which could
result in erroneous tree topologies mimicking diversity.
However, the discovery of this problem also provided
researchers with a variety of tools that help to identify
pseudogene sequences and to avoid their inclusion
analyses. We found no evidence for psuedogene sequences
in the Epimeria data set.
In the present study, the morphological investigation
of E. georgiana specimens of four barcode clades revealed
one new species with distinct phenotypic differences to
described species of Epimeria. The three remaining clades
require further investigation, e.g. through the use of fast-
evolving nuclear gene regions to confirm their taxonomic
status as they potentially represent cryptic species, based
on the COI data.
Distribution
Recently Pearse et al. (2009) proposed that speciation could
be enhanced in taxa with non-pelagic development.
Glaciation on the Antarctic Continental Shelf subdivided
populations into small isolated units that underwent
speciation. The repeated formation of such refugia during
the glacial-interglacial cycles of the Pliocene-Pleistocene
created a “species diversity pump”. The theory of such an
Antarctic diversity pump predicts the presence of many
closely related often cryptic species around the Antarctic
continent (see Clarke and Crame 1992,2010).
This study further supports this hypothesis, as the
combined DNA and morphological analyses of the Epi-
meria georgiana complex revealed one new species and the
COI results indicated a further three cryptic species
separated by bathymetry, geography, and relatively high
sequence divergences. Epimeria spp are stocky amphipods
unable to swim a long distance. Hence they have a low
capability for dispersion, which results in narrow distribu-
tional ranges of several species.
The Scotia Arc region appears to be unusually diverse
(Barnes et al. 2009; Linse et al. 2007). The high species
richness and high genetic diversity in epimeriid amphi-
pods reported at the Scotia Arc supports the assumption
that the islands of the Scotia Sea might have acted as a
centre of speciation in the Southern Ocean (Linse et al.
2007;Griffithsetal.2011).Ourfindingsofthreecladesof
Epimeria georgiana with less than 7% divergence at
Elephant Island also support the idea of a recent speciation
Fig. 12 Colour photographs
taken from specimens immedi-
ately after sampling. 1,2 Epi-
meria angelikae new spec, 3
Epimeria aff georgiana clade C,
4,5,6 Epimeria aff georgiana
clade A. 1NIWA 34944, 2
BAS_KL034413; 3BAS64411,
4NIWA 34935, 5NIWA 69496,
6NIWA 34936.
Mar Biodiv

among these sympatric populations. We assume that Epimeria
inermis, bearing the smooth basis on pereopods 5–7,
resembles the ancestral morphology of the georgiana-species
complex. Since all three clades of Epimeria georgiana are
found in relative close proximity of Elephant Island and
Powell Basin, all shallower than 1,000 m depth, we assume
that these clades are descendents of a former E. georgiana
population that diverged since the Last Glacial Maximum,
after the Antarctic continental shelf lost its ice shelf cover
(Thatje et al. 2005). We have taken a conservative approach
Fig. 13 Pereopods 5, 6 and 7 (P5, P6, P7) of the different clades of Epimeria aff georgiana,Epimeria angelikae new species, type material of E.
georgiana and E. inermis
Mar Biodiv

and only described the clade from the Weddell Sea as new
species, and not the morphological differences of the clades
from the southern Scotia Arc to the type material collected at
South Georgia and the northern Scotia Arc.
Discussions about species concepts and the level of
interspecific divergence in amphipods are ongoing (e.g. Held
and Leese 2007;Vogler and Monaghan 2007), but high inter-
clade and low intra-clade sequence divergences are indica-
tive of cryptic species, especially when coupled with
significant, consistent, morphological differences (often
microstructure/scale). Sequencing of additional faster evolv-
ing regions of DNA and morphological investigation of inner
structures could assist in resolving the status of these clades:
specimens from clades A (Elephant Island∼987 m), clade B
(Powell Basin in 964 m), and clade C (Elephant Island, but
shallower∼200 m). However, specimens of clade Awere also
collected as shallow as 590 and 463 m on the South Shetland
Islands. A bathymetric tolerance of 500 m is not unusual for
Antarctic epimeriid amphipods. Some species, e.g. E. macro-
donta (Walker 1907)orE. puncticulata Barnard 1932 are
known to have a wide depth distribution over 1,000 m on the
Antarctic shelf (Coleman 2007).
Scanning electron microscopy
Many authors have suggested the use of cuticular structures,
especially setae, to be an important tool for taxonomy of
Peracarida (e.g. Bradbury et al. 1998; Khalaji-Pirbalouty and
Wägele 2010). Zimmer et al. (2009)classified30typesof
setae, four types of microtrichs, different types of pores and
structures formed by setules and denticles of hyalid
amphipods; Halcrow and Bousfield (1987) described surface
microstructures of different amphipod taxa; Kaufmann
(1994) described structure and function of chemoreceptors
in scavenging amphipods. Neither the comb-scale structure
at the peduncle of the second antenna nor the setae bearing
pit with half border thickened of the fourth coxa seem to be
described before.
Fig. 14 Scanning electron mi-
croscopy fourth coxal plate of
the three clades of Epimeria aff
georgiana.a,bclade A NIWA
34945; c,dclade B NIWA
69497; e,fclade C NIWA
34942. Note that eand fare the
same coxal plate pictured from
different angles. Scale bars a,c,
e,f: 1 mm; b,d: 100 μm
Mar Biodiv

The only published scanning electron microscopy
images of epimeriid amphipods are of Paramphithoe
hystrix (Oshel and Steele 1988). Their investigation focused
on the gut content and the modifications of the mouthparts
to allow micropredation on sponges. However, the labrum
and the mandible of Epimeria angelikae new spec
(Figs. 10f,11b-d) bear similar structures of setae as shown
for P. hystrix (Oshel and Steele 1988).
As described above, all semicircles of the fourth coxa are
positioned in the same direction, in the middle area of the
coxa the openings are to the ventral side, on the distal
extension of the fourths coxa and towards the ventral
margin, the semi-circles have their opening towards the
posterior end. One explanation is the current flow. Cuadras
(1982) investigated the morphology and distribution of
microtrichs on the outer surface of different amphipod
species. Gammarus sp., Liljeborgia sp. and Orchomene sp.
are shown to have “peg like microtrichs emerging from
cuticular pits of less than one micron”(Cuadras 1982). The
pits of the Epimeria species are much larger, more than 30
microns in diameter (see Figs. 10b-e and 14b, d), more
complex in structure, they have a semicircle border, and the
setae or trich is positioned at the side, not middle of the pit.
However, the orientation of microtrichs that Cuadras (1982)
showed for Liljeborgia sp. is similar to the orientation of
the trichs in the pits observed for Epimeria. It is assumed
that water flow can lift the peg like units and, therefore,
they have a sensory function, e.g. for slow currents, or the
small cuticular ring surrounding the base of the trich could
register pressure when the trich is pressed against it.
Colour variations
Epimeriid amphipods are known to be very colourful when
alive and to have intraspecific colour morphs (e.g. Schnabel
and Hebert 2003; Coleman 2007;Lörzetal.2009). While
Schnabel and Hebert (2003) noted a colour-specific pattern
for clades of Paramphithoe hystrix separated by COI,28S
rRNA and allozymes differences, Lörz et al. (2007)noted
two different colour patterns, striped and dotted, in Epimeria
Fig. 15 Details of the fourth
coxal plate Epimeria aff
georgiana clade A and clade C.
aClade A NIWA 34945, b-e
clade C NIWA 34942. Scale
bars a,d,e,f:10μm; b,c:
100 μm
Mar Biodiv

schiaparelli specimens belonging to the same COI clade. Lörz
et al. (2009) also found different colour patterns in Epimeria
robusta specimens from the Ross Sea: white specimens with
red dots, specimens light red and specimens light red with
orange patches, all with an intraspecific variation <1%.
Photographs representing in situ colouration have to be
taken immediately after sampling, before the colour fades.
Colour is lost following fixation in ethanol or formaldehyde,
leading to uniformly whitish specimens. We only have colour
records for specimens in clade A and C (Fig. 12). Specimens of
Epimeria georgiana were coloured in different shades of red
that were not clade-specific. On-deck photographs (not
included here) showed specimens of the E.cf.georgiana
clade B to be dark red. Orange patterned Epimeria georgiana
specimens from the Antarctic Peninsula can be separated from
the whitish Epimeria angelikae new spec. based on colour in
live specimens The colour of E. georgiana from the type
locality was not recorded.
Conclusion
DNA barcoding performs well in species and taxa discrimi-
nation of epimeriid amphipods and has highlighted potentially
cryptic species. However, formal descriptions will only be
possible in combination with morphological investigations, as
demonstrated with a new species described here.
Acknowledgements We are grateful to the team at the Canadian
Centre for DNA Barcoding (supported by Genome Canada through the
Ontario Genomics Institute) for providing they sequences for the CAML
project. Angelika Brandt (Hamburg) kindly let the senior author use her
microscope. Renate Walter (Hamburg) helped with the scanning electron
microscopy. Erika Mackay (NIWA) kindly inked the drawings. Niamh
Kilgallen (NIWA) and three anonymous reviewers are thanked for
constructive criticism on an earlier version of the manuscript.
The Natural History Museums of Stockholm and London are thanked
for the loan of type material. The curational help of the NIWA Invertebrate
Collection (NIC) team is highly appreciated. A.N.L and P.S were
supported by research funded by the New Zealand Government under
the NZ International Polar Year-Census of Antarctic Marine Life Project,
and gratefully acknowledge the Ministry of Fisheries Science Team and
Ocean Survey 20/20 CAML Advisory Group (Land Information New
Zealand, Ministry of Fisheries, Antarctica New Zealand, Ministry of
Foreign Affairs and Trade, and National Institute of Water and
Atmospheric Research Ltd.). K.L. was funded by The Natural Environ-
ment Research Council. D.S. was supported by funding of the Alfred P.
Sloan Foundation to MarBOL. This study is part of the British Antarctic
Survey Polar Science for Planet Earth Programme and the NZ
International Polar Year-Census.
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