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Genetic variability of the Metridia lucens complex (Copepoda) in the SouthernOcean

Alexandra N. Stupnikova, Tina N. Molodtsova, Nikolay S. Mugue, Tatyana V.Neretina

PII: S0924-7963(13)00106-1DOI: doi: 10.1016/j.jmarsys.2013.04.016Reference: MARSYS 2367

To appear in: Journal of Marine Systems

Received date: 16 September 2012Revised date: 24 April 2013Accepted date: 27 April 2013

Please cite this article as: Stupnikova, Alexandra N., Molodtsova, Tina N., Mugue,Nikolay S., Neretina, Tatyana V., Genetic variability of the Metridia lucens com-plex (Copepoda) in the Southern Ocean, Journal of Marine Systems (2013), doi:10.1016/j.jmarsys.2013.04.016

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Genetic variability of the Metridia lucens complex (Copepoda) in the Southern Ocean

Alexandra N. Stupnikova1, Tina N. Molodtsova

1, Nikolay S. Mugue

2 and Tatyana V. Neretina

3

1 P.P.Shirshov Institute of Oceanology Russian Academy of Science, 36, Nahimovsky prospect,

Moscow, 117997, RF

2 Russian Federal Research Institute of Fisheries and Oceanography, 17, Krasnoselskaya street,

Moscow, 107140, RF.

3 White Sea Biological Station, Department of Biology Lomonosov Moscow State University,

a/ya 20, Glavpochtamt, Kandalakshsky raion, Murmanskaya oblast, 184042, RF.

Correspondence: Alexandra Stupnikova, Fax: +7(499)1245983, E-mail:

astupnikova@gmail.com

Running title: Genetic variability of Metridia lucens

Abstract

Analysis of the fragment of the mtDNA gene СО1 has revealed two genetically distinct

groups of the Metridia lucens complex in the South part of the Atlantic. While the intragroup

polymorphism was less than 1%, the intergroup difference was about 9.5%. These two groups

may be considered as representing two cryptic species within the M. lucens complex: M. lucens

North and M. lucens South. These forms are found mainly to the North and to the South of the

South Polar Front. The results are confirmed by an analysis of the nuclear rDNA ITS1-5,8S-ITS2

fragment. No hybrids between the two forms were detected. M. lucens North inhabits the waters

> 4°C that allow us to discuss the temperature as putative limiting factor for the distribution.

Keywords: plankton, Copepoda, cryptic species, Metridia lucens, genetic diversity, the Southern

Ocean

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1. Introduction

Spatial irregularities in the distribution of planktonic organisms observed in the Southern

Ocean substantially agree with patterns of fronts separating different Antarctic water masses

(Maslennikov, 1994; Read et al., 2002). According to the modern biogeographic classification of

pelagic ecosystems (Errhif et al., 1997; Longhurst, 1998) the Polar Front is the biogeographical

boundary separating Subantarctic and Antarctic biogeographical provinces. The Polar Frontal

Zone is also viewed as a separate province (Longhurst, 1998) inhabited by species from both

adjacent provinces.

The transfrontal water exchange explains the fact that a number of zooplankters (e.g.,

dominant copepods Calanus simillimus, Ctenocalanus citer, Rhincalanus gigas and Metridia

lucens) inhabit all hydrological zones of the Southern Ocean (South Atlantic Zooplankton, 1999).

However, some reports provide evidence for a restricted gene flow between populations

separated by the fronts and for a maintaining of isolated populations (Bucklin et al., 1996, 2000;

Goetze, 2005).

Genetic methods provide alternative or additional means of identifying known species.

Genetic analysis of the region of mitochondrial cytochrome oxidase I (mtCOI) has been widely

used to discriminate species throughout the animal kingdom (Hebert et al., 2003), including

Crustacea (Costa et al., 2007) and in particularly Copepoda (Bucklin et al., 2010). The nuclear

rDNA fragment internal transcribed spacer (ITS) has also been used as a successful marker for

phylogenetic and population analysis in crustaceans (Chu et al., 2001), including copepods

(Elvers et al., 2006; Ki et al., 2009).

Metridia lucens Boeck, 1865 (Copepoda: Calanoida) is a widely distributed dominant

interzonal species (Vervoort, 1957) characterized by a high morphological variability (Bradford-

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Grieve, 1999). It is shown to be an important member of the zooplankton of all open oceanic

regions of the World Ocean, except for the high Arctic.

The aim of this work is to evaluate the degree of genetic isolation of M. lucens populations

inhabiting different hydrological zones of the Southern Ocean based on mitochondrial (COI) and

nuclear (ITS) DNA fragments.

2. Materials and Methods

2.1. Sample collection

The specimens of the M. lucens complex was collected during the Southern Hemisphere

Summer (December 2009 - January 2010) from seven stations and during the Antarctic Spring

(October-November 2010) from 22 stations along the Eastern and Western (The Drake Passage)

sections of the Atlantic sector of the Southern Ocean (Fig. 1, Table 1). The distance between the

sampling sections was 3-4 thousand kilometers. Each sampling location represented a separate

hydrological zone: the Subantarctic Zone (SAZ), the Polar Frontal Zone (PFZ), the Antarctic

Zone (AZ), with additional sampling in the Subtropical Zone (STZ).

Each sample was collected with a Juday net from the water layer 0-300 m. Immediately

after collection, adults of the M. lucens complex were isolated from the samples. The specimens

were identified by the morphological traits (Bradford-Grieve, 1994; Bradford-Grieve et al.,

1999). In total, 89 individuals were collected.

Water temperature and salinity were measured with CTD SeaВird 911+ probe at the

moment of sampling. Vertical profile of temperature and salinity was used to locate the ACC

hydrological boundaries (Sokolov and Rintoul, 2002, 2009). Exact zone boundaries were

evaluated based on the current velocity registered by SADCP (RDI Ocean Surveyor).

For comparison, four specimens of the M. lucens s.s. Boeck, 1865 from the North Sea have

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also been used in this study. They were collected at Marine Biological Station, Espeland, in the

Raunefjord, 20 km South from Bergen.

2.2. DNA Amplification and Sequencing.

Promega Wizard SV Genomic DNA Purification Kit (Promega Corporation, Madison,

USA) was used for tissue lysis and DNA purification following the manufacturer's protocol.

Polymerase chain reaction (PCR) amplification of nuclear ITS1-5.8S rDNA-ITS2 region

was accomplished with the primers LR1 (GGTTGGTTTCTTTTCCT) and SR6R

(AAGWAAAAGTCGTAACAAGG) (Gardes and Bruns, 1993). This 487bp nuclear region

comprises a partial sequence of the 18S rRNA gene, complete sequences of the ITS1, 5.8S rRNA

and ITS2 and a partial sequence of the 28S rRNA gene. PCR amplification of the 535bp fragment

of the mtCOI gene was performed with the universal primers LCOI 1490

(GGTCAACAAATCATAAAGATATTGG) and HCOI 2198

(TAAACTTCAGGGTGACCAAAAAATCA) (Folmer et al., 1994). Loci were amplified using

Encyclo PCR kit (Evrogen Joint Stock Company, Russia). Amplification was done in a total

volume of 25 ml reaction mix containing 1 X PCR buffer, 1 µL of 10 µM of primer pair mix, 1

µL of the template, 0.2 mM of each dNTP and 0.5 units Taq polymerase. Reactions mixtures

were heated to 94°C for 120 s, followed by 35 cycles of 15 s at 94°C, 30 s at a specific annealing

temperature and 60 s at 72 °C, and then the final extension of 7 min at 72°C on Veriti® Thermal

Cycler. Annealing temperature was set to 45° C for the CO1 and 52°C for the ITS1-5.8S rDNA-

ITS2 primer pairs. The Promega PCR Purification Kit protocol (Promega) was employed to

purify amplification products. Amplification products were sequenced in both directions. Each

sequencing reaction mixture, including 1 мl BigDye3.1 (Applied Biosystems, Perkin-Elmer

Corporation, Foster City, CA), 1 мl of 1 µM primer and 1µL of DNA template, ran for 40 cycles

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at 96°C (15 s), 50°C (30 s) and 60 °C (4 min). Sequences were purified by ethanol precipitation

to remove unincorporated primers and dyes. Products were re-suspended in 12 µl formamide and

electrophoresed in an ABI Prism_ 3500 sequencer (Applied Biosystems).

The obtained sequences were submitted to GenBank (Accession numbers in Table 1).

2.3. Molecular data analysis

Multiple alignments for both genes were made using ClustalW (Wang and Jiang, 1994).

Cladogramms were built by the neighbor-joining method (Saitou and Nei, 1987). Tree node

credibility values were calculated in 1000 iterations of the bootstrap analysis (Felsenstein, 1985).

COI sequences from NCBI GenBank (AF513645 – from the Northeast Pacific Ocean, Vancouver

Island; AF531750 – from the South Atlantic, Brantsfield Strait, 62.35 S, 58.54 W; AF474107 –

from the Southwest Pacific Ocean, off New Zealand; AF474106 – from the North Atlantic, Gulf

of Maine; AF259666 – from the North Atlantic, Georges Bank; AB380018– from the North

Pacific 40.01 N 145.01 E) were used along with the original data. Metridia gerlachei Giesbrecht,

1902 (AF531747 and AF536518) was used as an outgroup.

3. Hydrological features

Water temperature increased from the South to the North from -1.5 to +6ºС for the Drake

Passage and from -2 to +19ºС in the Eastern section during Antarctic spring, when samples were

collected. At the Eastern transect the isotherm +4ºС on depth 0-100m was located between the

Polar Front and the Subantarctic Front. In the Drake Passage the same isotherm at that period was

vertical and matched the Subantarctic Front. During Antarctic summer, at the Eastern section the

+4ºС isotherm at a depth of 100m matched the Polar Front and that at a depth of 300m matched

the Subantarctic Front. In the Drake Passage +4ºС isotherm at that period was vertical and

matched the Subantarctic Front.

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Mean temperatures (Table 2) show that the Drake Passage was colder at any given depth

of the 0-300m layer. In the Drake Passage temperatures above +4ºС were recorded only in the

Subantarctic Zone. In the Eastern cross-section such temperatures were found northward to the

Polar Front – in the Polar Frontal Zone, the Subantarctic Zone and the Subtropical Zone. This is

typical for the Antarctic Spring and Summer seasons.

4. Results

4.1. mtCOI gene polymorphism

A cladogram was generated by aligning partial sequences of COI mitochondrial gene

from 80 originally collected adult specimens of Metridia lucens and 6 partial sequences from

NCBI GenBank (Fig. 2A). Two clades of M. lucens were identified with 8% nucleotide

divergence with maximum divergence within the same clade being less than 1%.

Table 3 shows distribution of specimen from different locations among the clades. The

clade A contained all specimen from the Weddell Sea, all AZ samples from western and eastern

parts of Antarctic sector of the Southern Ocean, all specimen from PFZ of the Drake Passage and

several specimen from PFZ of the Eastern transect, one individual from SAZ of the Drake

Passage, and one individuals from the Bellingshausen Sea. The clade B contained all individuals

from STZ and SAZ, and several specimen from FPZ of the Eastern transect and one individual

from South-West Pacific (South off New Zealand).

The specimens from the Northern Hemisphere are close to the clade B. The divergence is

4% for the Northern Atlantic and 2% for the Northern Pacific.

4.2. ITS region polymorphism

A total of 82 individuals of the M. lucens complex were analysed based on ITS1-5,8S-

ITS2 region of nuclear DNA. Phylogenetic analysis of this dataset produced clades C and D with

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1% divergence (Fig. 2B). ITS1 has four highly variable positions and one slightly variable

position for 215bp and ITS2. It has one highly variable position and two slightly variable

positions for 180bp.

Table 3 shows the distribution of the specimen from different locations among the clades.

Copepods of the “C” clade form a single haplotype block (Table 4). Samples grouped to the “D”

(49 specimens) clade show an extent of the polymorphism in ITS1-5,8S-ITS2 region. This group

includes 8 samples from STZ, 10 from SAZ of the Eastern cross-section, 8 from SAZ of the

Drake Passage and 6 samples from PFZ of the Eastern cross-section.

Table 4 shows the states of 5 variable positions of ITS1 and 3 of ITS2. A considerable

amount of heterozygous states was detected, with maximum seven heterozygous positions in

individual D87 from STZ.

Clades C and D based on the ITS region match clades A and B in a mtCOI-based

cladogram.

5. Discussion

5.1. Taxonomic status

According to recent studies on mtCOI polymorphism for crustaceans (Waugh, 2007;

Costa et al., 2007, Radulovici et al., 2010) the boundary between closely related species is

accepted as 2,7 − 3%. Though, obviously, further studies are needed to clarify the status of the

two groups, the detected substitution level of 8% for mtCOI suggests the presence of two cryptic

species within the M. lucens complex.

Our comparative analysis of fragments of mitochondrial and nuclear DNA has shown that

all individuals with the ITS haplotype СТТСАСТТ belong to the mitochondrial clade A (Table

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4). No exceptions were found, even at those sites where both clades were present (e.g., PFZ of

the Eastern section). Thus, in the areas of coexistence we find no evidence for hybridization.

These two clades may correspond to reproductively isolated species. Analysis of polymorphism

in ITS1 and ITS2 regions reveals (Table 4) that MLS shows no sequence variations and is

homozygous for both ITS regions. MLN has nine polymorphic positions in these regions.

It should be noted that two individuals with high number of heterozygous positions in

ITS1 and ITS2 regions, D87 and D90, could be hybrids between MLN and MLS. However, they

were collected away from the zone where the two forms coexist. We conventionally name the

form represented in the clades A and C MLS (Metridia lucens South), and the form of the clades

B and D is named MLN (Metridia lucens North). Therefore, our results suggest that MLS is

the sister species to MLN, and manifest very low genetic variation, possibly due to younger age,

for smaller population sizes or repeated bottlenecks in more severe high-latitude environment.

Our data indicates the possibility of weak unidirectional gene flow towards North, suggesting that

MLS males may hybridize with MLN females. This conclusion is based on our finding of 4

specimen North from species boundaries with MLN mtDNA and heterozygous in few ITS sites,

which differentiate MLN and MLS.

Metridia lucens from the North Sea is considered as M. lucens s.s. as the species was

described originally near the Norway coast (Boeck, 1865). It is clear from Fig. 2 that both MLN

and MLS are genetically distinctive from M. lucens from the North Sea and cannot be considered

as M. lucens s.s. Currently M. lucens is considered to be widely distributed species with a wide

range of variability in morphology (Bradford-Grieve, 1999). There is a high possibility that

several closely related species were reported under the name M. lucens Boeck, 1865. To

determine and define the species inside the M. lucens complex it is crucial to re-describe M.

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lucens s.s. from the locality type based on extensive freshly collected material with involvement

of molecular and morphology analysis. Only upon the ranges of morphological variability of M.

lucens s.s. are determined it would be possible to describe new species in the M. lucens complex.

5.2. Spatial distribution of MLN and MLS and effects of the environment

The Southern Ocean is characterized by a complex system of water circulation (Belkin,

1993; Sokolov and Rintoul, 2009) dominated by the Antarctic Circumpolar Current (ACC). The

ACC can be divided into three main streams: Polar Front, Subantarctic Front, and Southern Front

(Orsi et al., 1995). Two major mechanisms of water exchange between interfrontal zones within

the ACC are known (Koshlyakov and Sazhina, 1996): surface or subsurface drift currents

(usually directed northward) and transfrontal exchange via gyres.

The habitat of MLN and MLS in the Southern Ocean is shown Fig. 3. MLS inhabits the

Polar Frontal Zone and the Antarctic Zone. An individual from the Bellingshausen Sea also

belongs to MLS. The MLN form inhabits the Subtropic Zone, the Subantarctic Zone and the

Polar Frontal Zone. An individual from South-West Pacific off New Zealand also belongs to

MLN. It is remarkable that the MLN population of the Southern Ocean is genetically close to M.

lucens s.s. of the Northern Hemisphere with 2% divergence for Northern Atlantic and 4% for

Northern Pacific in COI. This indicates that M. lucens s.s. from the Northern Hemisphere and

MLN are similar; MLN appears to be more abundant and more widely distributed than MLS.

In the Eastern section, MLN occurs North of the Polar Front and does not depend in its

distribution on other fronts. Conversely, in the Drake Passage, MLN occurs north of the Subpolar

Front. MLS depends in distribution on the Subantarctic Front on both sides of the Southern

Atlantic and does not depend on other fronts.

We suggest that the ranges of optimal temperature for survival and reproduction of MLN

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and MLS species are different, though overlapping. MLS was found at temperatures between -

2oС and +10

oС, while MLN was recorded between +4

oС and +12

oС (Fig. 4, 5). Thus, both were

detected at temperatures from +4 o

С to +10 o

С. Southward of the Polar Front MLN seems to be

represented by a few individuals. Most probably, MLN, on the contrary to MLS, can not

reproduce in water colder than +4 o

С, and the +4 o

С isotherm can be considered the southern

border of MLN population.

The fronts in the Drake Passage are shifted southward (Sokolov and Rintoul, 2009;

Sprintall, 2003, 2008). Therefore, water temperature of same hydrological zones is lower for the

Drake Passage than for the Eastern section (Table 2). The +4 o

С isotherm in the Drake Passage

matches the Subantarctic Front, but for the Eastern section it lies within the Polar Frontal Zone.

However, in both regions the +4 o

С isotherm matches MLN distribution border during both

spring and summer of the Southern Hemisphere.

In the Drake Passage, the individuals of the M. lucens complex are apparently transferred

through the Subantarctic Front only northward. The transfrontal water exchange in the Drake

Passage is known to be generally directed southward (Koshlyakov and Sazhina, 1996; Golivets

and Koshlyakov, 2004, 2009). However, M. lucens penetrates through the Subantarctic Front in

the opposite direction that may prove ecological nature of barriers: most likely both species of the

M. lucens complex are advected through the Subantarctic Front in both distections but MLN can

not survive and/or reproduce in colder waters south of the Polar Front. Between the Polar Front

and the Subantarctic Front both species co-occur in the same geographic locations. However,

they may be isolated vertically living at different depths in different water masses (Fig. 4, 5).

As it was shown by Stupnikova et al. (2013) Antarctic (South of PF) and Subantarctic

(North of PF) populations of two dominant zooplankters Rhincalanus gigas Brady, 1883 and

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Calanus simillimus Giesbrecht, 1902 (Copepoda: Calanoida) do not show any genetic difference.

Apparently the Polar Front can not represent an unpenetrable barrier for MLS and MLN, due to

active water exchange. Most likely, some ecological factors limit MLS and MLN spatial

distribution, while the effect of frontal zones seems to be indirect. In spite of current co-existence

of MLN and MLS in the Polar Frontal Zone, we cannot exclude significance of this isolation in

the past that let these two cryptic forms of copepods concur an ecological divergence via different

temperature tolerance. If so, the spatial coexistence in these two genetically isolated forms can

be a consequence of hydrological history in this area.

5.3. Cryptic species in marine zooplankton

Existence of cryptic species in marine plankton was suggested rather long ago (Knowlton,

1993). However, the mechanisms involved in speciation are appeared to be species-specific.

Some zooplankters show no considerable genetic difference for populations separated by

distances of about 1000 km: e.g. copepods Calanus finmarchicus (Bucklin and Kocher, 1996;

Bucklin et al., 2000), Nannocalanus minor (Bucklin et al., 1996), Pseudocalanus

acuspes (Holmborn et al., 2011), Calanus simillimus and Rhincalanus gigas (Stupnikova et al.,

2013); and also euphausiids Meganyctiphanes norvegica (Bucklin et al., 1997), Euphausia

superba (Zane et al., 1998; Zane and Patarnello, 2000), Euphausia crystallorophias (Jarman et

al., 2002).

On the other hand, many other species are represented by genetically distinct groups,

often recorded even within the same ocean water masses. In addition to the M. lucens complex

studied in this work, there are examples of other cosmopolitan species: the chaetognath

Eukrohnia hamata (Kulagin et al., 2012), the copepods Pleuromamma xiphias (Goetze, 2011)

and Eucalanus hyalinus (Goetze, 2005).

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Our data also demonstrates that the geographic distance is not critical for the plankton

isolation. The individuals of the M. lucens from the North Sea and the Subantarctic waters were

genetically more close than two groups found in the same area of the Polar Front Zone (Fig. 2).

Acknowledgements

This study was supported by the Russian Foundation for Basic Research (grant 12-05-

31383) and the Ministry of Education and Science of the Russian Federation (grant

11.G34.31.0008, grant 8132, grant 8057).

The authors are grateful to M. Flint, A. Vereshchaka, A. Pasternak (P.P. Sirshov Institute

of Oceanology RAS), A. Azovsky (Lomonosov Moscow State University), A. Kondrashov

(University of Michigan) and three anonymous referees for critical reading of the manuscript and

helpful comments. The authors thank K. Goupalo for helping in English language. We thank the

crew of the R/Vs «Akademik Ioffe» and «Akademik Sergey Vavilov» for assistance in sample

collection. We also greatly acknowledge kind help of A. Lunina, D. Kulagin, V. Gagarin in

collecting and sorting the samples. We thank E. Gorokhova (the Norwegian National Mesocosm

Centre, Espegrend, Universitetet in Bergen, Norway) who kindly provided the North Sea

specimen.

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Fig. 1 Sampling sites and ACC front configuration during sampling. A) 30th

Cruise of R/V

«Akademik Ioffe» B) 31th

Cruise of R/V "Akademik Sergey Vavilov". STF – the Subtropical

Front, SAF – the Subantarctic Front, PF – the Polar Front, SACCF – the Southern ACC Front.

STZ – the Subtropical Zone, SAZ – the Subantarctic Zone, PFZ – the Polar Front Zone, AZ – the

Antarctic Zone.

Fig. 2 Phylogeny of M. lucens based on partial sequences of mtCOI gene (A) and rDNA ITS1-

5.8S -ITS2 region (B) with Metridia gerlachei as an outgroup. Dr – the Drake Passage, E –

Eastern section. STZ – the Subtropical Zone, SAZ – the Subantarctic Zone, PFZ – the Polar Front

Zone, AZ – the Antarctic Zone, WS – the Weddell Sea, SW Pacific – South-West Pacific, N

Pacific – North Pacific, NW Atlantic – North-West Atlantic, Ant Peninsula – Antarctic

Peninsula, Bransfield Strait.

Fig. 3 Distribution of MLN and NLS species in the Southern Ocean. Front position by Orsi et al.,

1995. For abbreviations see Fig. 1.

Fig. 4 Geographical distribution of the species MLN (I) and MLS (II) at the Eastern section (А)

during Antarctic summer (December 2009) and (B) Antarctic spring (October 2010). For

abbreviations see Fig. 1.

Fig. 5 Geographical distribution of the species MLN (I) and MLS (II) at the Drake Passage (А)

during Antarctic summer (December 2009) and (B) Antarctic spring (October 2010). For

abbreviations see Fig. 1. PF 1, PF 2, PF 3 – the Southern Polar Current.

(A) (B)

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Table 1

Samples used. Location in different ACC hydrological zones: the Subantarctic Zone (SAZ), the

Polar Frontal Zone (PFZ), the Antarctic Zone (AZ), and the Subtropical Zone (STZ). NS – the

North Sea

Cru

ise

Dat

a

Zone

Sit

e

Longit

ude

Lat

itude

Lay

er

Vouch

er

GenBank

accession

number

mtCO1

GenBank

accession

number

rDNA

Drake Passage

R/V ASV-31 10.11.2010 SAZ 2276 -65,08 -57,21 0-300 F14

F15

D35

D36

JQ889929

JQ889930

JQ889884

JQ889885

JQ944760

JQ944718

JQ944778

JQ944776

R/V ASV-31 10.11.2010 SAZ 2281 -66,10 -56,66 0-300 F12

F13

D30

D31

JQ889927

JQ889928

JQ889867J

Q889883

JQ944720

JQ944719

JQ944779

R/V ASV-31 11.11.2010 SAZ 2283 -66,49 -56,57 0-300 F05

F6

JQ889921

JQ889922

JQ944716

JQ944715

R/V ASV-31 09.11.2010 PFZ 2271 -64,30 -57,94 0-300 D71

D72

D73

JQ889892

JQ889893

JQ944763

JQ944792

JQ944761

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D74

D75

D76

D77

D78

D79

D80

D81

D82

D83

D84

D85

JQ889894

JQ889895

JQ889896

JQ889897

JQ889898

JQ889899

JQ889900

JQ889901

JQ889902

JQ889903

JQ889904

JQ889905

JQ944768

JQ944788

JQ944762

JQ944767

JQ944791

JQ944764

JQ944733

JQ944765

JQ944769

JQ944770

JQ944793

JQ944774

R/V AI-30 03.01.2010 AZ 2296 -63,97 -61,82 0-300 A10

A22

JN588593

JN588595

JQ944759

JQ944757

R/V AI-30 04.01.2010 AZ 2300 -64,21 -61,17 0-300 A36 JN588598 JQ944754

R/V ASV-31 04.11.2010 AZ 2244 -60,02 -61,69 0-300 D59 JQ889887

R/V ASV-31 04.11.2010 AZ 2246 -60,27 -61,49 0-300 D44 JQ889886 JQ944790

R/V ASV-31 05.11.2010 AZ 2248 -60,55 -61,26 0-300 C23 JQ889874 JQ944783

R/V ASV-31 05.11.2010 AZ 2250 -60,89 -60,98 0-300 F09

F10

JQ889925

JQ889926

JQ944712

JQ944721

R/V ASV-31 06.11.2010 AZ 2258 -62,23 -59,81 0-300 F7

F8

JQ889923

JQ889924

JQ944714

JQ944713

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the Eastern section

R/V ASV-31 08.10.2010 STZ 2176 12,75 -37,13 0-300 D86

D87

D88

D89

D90

D20

D21

D22

D67

D70

JQ889906

JQ889907

JQ889908

JQ889909

JQ889910

JQ889877

JQ889878

JQ889879

JQ889888

JQ889891

JQ944777

JQ944732

JQ944731

JQ944730

JQ944728

JQ944738

JQ944781

JQ944772

JQ944766

R/V AI-30 12.12.2009 SAZ 2240 7,33 -44,43 0-300 A09

A87

A88

A89

JN588604

JN588605

JN588606

JQ944746

JQ944748

JQ944747

JQ944789

R/V ASV-31 12.10.2010 SAZ 2186 10,73 -40,07 0-300 D9 JQ944729

R/V ASV-31 13.10.2010 SAZ 2192 9,42 -41,82 0-300 F16

F17

JQ889931

JQ889932

JQ944775

R/V ASV-31 13.10.2010 SAZ 2192 9,42 -41,82 300-

1000

C18

C19

JQ889870

JQ889871

JQ944744

JQ944743

R/V ASV-31 14.10.2010 SAZ 2195 8,74 -42,69 0-300 F3

C20

JQ889919

JQ889872

JQ944742

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R/V ASV-31 15.10.2010 SAZ 2199 7,79 -43,84 0-300 C92

C93

F04

JQ889920

JQ944740

JQ944782

R/V AI-30 13.12.2009 PFZ 2242 6,81 -44,98 0-300 A32

A33

A82

A83

A84

A85

A86

JN588596

JN588597

JN588599

JN588600

JN588601

JN588602

JN588603

JQ944756

JQ944755

JQ944753

JQ944752

JQ944751

JQ944750

JQ944749

R/V ASV-31 17.10.2010 PFZ 2210 4,97 -46,95 0-300 D24 JQ889880 JQ944737

R/V ASV-31 17.10.2010 PFZ 2210 4,97 -46,95 300-

1000

D25

D26

JQ889881

JQ889882

JQ944736

JQ944780

R/V AI-30 20.12.2009 AZ 2273 0,00 -53,92 0-300 B81

B82

JQ889869 JQ944785

JQ944786

R/V AI-30 21.12.2009 AZ 2277 0,00 -55,24 0-300 A11

B63

JN588594

JQ889868

JQ944758

JQ944787

R/V ASV-31 21.10.2010 AZ 2224 0,82 -50,76 0-300 D04 JQ889875 JQ944735

R/V ASV-31 21.10.2010 AZ 2225 0,49 -51,02 0-300 C22

C82

JQ889873

JQ944741

R/V ASV-31 22.10.2010 AZ 2229 0,00 -52,28 0-300 C21

D96

JQ889913

JQ944784

JQ944726

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D97

D98

D99

F1

F2

JQ889914

JQ889915

JQ889916

JQ889917

JQ889918

JQ944725

JQ944724

JQ944723

JQ944722

JQ944717

R/V ASV-31 23.10.2010 AZ 2235 0,00 -54,25 0-300 D8

D68

D69

JQ889876

JQ889889

JQ889890

JQ944734

JQ944773

JQ944771

R/V AI-30 22.12.2009 AZ 2281 0,00 -56,56 0-300 B88 JQ944745

R/V ASV-31 24.10.2010 AZ 2240 0,00 -55,67 0-300 D91

D92

D15

JQ889911

JQ889912

JQ944727

JQ944739

18.05.2012 NS 60,40

-5,31 0 F80

F81

F82

F84

KC412248

KC412249

KC412250

KC414886

KC414887

KC414888

KC414889

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Table 2

The mean temperature for each hydrological zone (ºС) at a depth of 0/100/200 meters, as

measured at sampling locations

Drake Passage the Eastern section

R/V ASV-31

spring

R/V AI-30

summer

R/V ASV-31

spring

R/V AI-30

summer

Subtropical Zone 15.0/14.3/13.1 17.0/16.4/16.0

Subantarctic Zone 5.1/4.8/4.6 6.3/6.2/6.0 8.6/8.3/8.0 11.0/10.1/8.3

Polar Frontal Zone 3.1/2.1/1.8 3.3/2.7/2.4 4.2/4.2/3.8 6.0/5.0/3.4

Antarctic Zone 0/-0.8/1 1.1/-0.3/0.5 0.9/0.7/1.2 1.8/0.3/1.1

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Table 3

Distribution of samples from different hydrological zones among clades of cladogram from Fig.2

СО1 rDNA ITS1-5,8S-ITS2

Clade A Clade B Clade C Clade D

Without

clade

Drake Passage

Subantarctic Zone 1 9 1 6 2

Polar Frontal Zone 14 15

Antarctic Zone 10 9

African section

Subtropical Zone 10 7 2

Subantarctic Zone 10 1 10

Polar Frontal Zone 4 6 4 6

Antarctic Zone 16 19

Brantsfield Strait 1

South-West Pacific 1

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Table 4

Variable positions of ITS1-5,8S-ITS2 region state for individual samples

Vouch

er Zone

Secti

on

ITS1 ITS2 Clade

mtCO1 59 68 76 123 125 135 411 442 494

A33 PFZ E C T A C G C C T T B

A82 PFZ E . . . Y . . . . . B

A83 PFZ E Y . . Y . . . . . B

A84 PFZ E Y . . Y . . . . . B

D24 PFZ E Y . . Y . . . . . B

D25 PFZ E Y . . Y . . . . . B

D31 SAZ Dr Y . . Y . . . . . B

D36 SAZ Dr Y . . Y . . . . . B

F05 SAZ Dr T . . T . . . . . B

F06 SAZ Dr T . . T . . . W . B

F12 SAZ Dr Y . . Y R . . . W B

F13 SAZ Dr . Y . . R . Y . W B

F14 SAZ Dr T . . Y . . . . . B

F15 SAZ Dr Y . . Y . . . . . B

A87 SAZ E Y . . Y . . . . . B

A88 SAZ E Y . . Y . . . . . B

A89 SAZ E Y . . T . . . - - B

C18 SAZ E Y . . . . . . . . B

C19 SAZ E T . . T . . . . . B

C20 SAZ E Y . . . . . . . . B

F16 SAZ E T . . T . . . . . B

C92 SAZ E T . . T . . . . .

C93 SAZ E T . . T . . . . .

A09 SAZ E Y . . Y . . . . .

D67 STZ E Y . . Y . . . . .

D20 STZ E Y . . Y . . . . . B

D21 STZ E Y . . Y . . . . . B

D70 STZ E Y . . Y . . . . . B

D86 STZ E T . . T . . . . . B

D87 STZ E Y Y W Y R . Y . W B

D88 STZ E Y . . . . . . . T B

D89 STZ E Y . . . . . . . T B

D90 STZ E Y Y . Y R . Y . W B

D92 AZ E . . T . A . . . A A

D15 AZ E . . T . A . . . A

B88 AZ E . . T . A . . . A

A10 AZ Dr . . T . A . . . A A

A22 AZ Dr . . T . A . . . A A

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A36 AZ Dr . . T . A . . . A A

C23 AZ Dr . . T . A . . . A A

D44 AZ Dr . . T . A . . . A A

F07 AZ Dr . . T . A . . . A A

F08 AZ Dr . . T . A . . . A A

F09 AZ Dr . . T . A . . . A A

F10 AZ Dr . . T . A . . . A A

A11 AZ E . . T . A . . . A A

B81 AZ E . . T . A . . . A A

D04 AZ E . . T . A . . . A A

D08 AZ E . . T . A . . . A A

D68 AZ E . . T . A . . . A A

D69 AZ E . . T . A . . . A A

D96 AZ E . . T . A . . . A A

D97 AZ E . . T . A . . . A A

D98 AZ E . . T . A . . . A A

D99 AZ E . . T . A . . . A A

F01 AZ E . . T . A . . . A A

F02 AZ E . . T . A . . . A A

B63 AZ E . . T . A . . . A

B82 AZ E . . T . A . . . A

C21 AZ E . . T . A . . . A

C82 AZ E . . T . A . . . A

D72 PFZ Dr . . T . A . . . A

D71 PFZ Dr . . T . A . . . A A

D73 PFZ Dr . . T . A . . . A A

D74 PFZ Dr . . T . A . . . A A

D75 PFZ Dr . . T . A . . . A A

D76 PFZ Dr . . T . A . . . A A

D77 PFZ Dr . . T . A . . . A A

D78 PFZ Dr . . T . A . . . A A

D79 PFZ Dr . . T . A . . . A A

D80 PFZ Dr . . T . A . . . A A

D81 PFZ Dr . . T . A . . . A A

D82 PFZ Dr . . T . A . . . A A

D83 PFZ Dr . . T . A . . . A A

D84 PFZ Dr . . T . A . . . A A

D85 PFZ Dr . . T . A . . . A A

A32 PFZ E . . T . A . . . A A

A85 PFZ E . . T . A . . . A A

A86 PFZ E . . T . A . . . A A

D26 PFZ E . . T . A . . . A A

D35 SAZ Dr . . T . A . . . A A

D09 SAZ E . . T . A . . . A

F84 North Sea . W Y R Y . . W

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F81 North Sea . W Y R Y . . W F80 North Sea . T . A T . . A F82 North Sea . W Y R Y . . A

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Highlights

The study is based on materials collected in the Atlantic part of the Southern Ocean where the

water masses structure and oceanographic fronts over isolation of populations of abundant

zooplankton species.

These two groups were considered as representing two cryptic species within M. lucens: M.

lucens North and M. lucens South. These forms are found mainly to the north and to the south of

the South Polar Front. No hybrids between the two forms were detected.

We propose that the temperature is the limiting factor for M. lucens North, which inhabits the

waters warmer than 4°C. The frontal zones seem to have no direct effect on distribution.