,. 'J

"

ICES 'CM 1997ff:24

Genetic structuring of Pandalus borealis in the North Atlantic. 11. RA~D analysis

leiar Martinez, Taran Skjerdal, Bent Dreyer and Salah M. Aljanabi

The genetie variation of Pandalus borealis in the Northeast Atlantic was examined by RAPD

analysis on individuals eaught at 8 different sampling stations in the Bare~ts Sea, 3 off

Spitsbergen, 2 off Jan Mayen and in 2 Northem Norwegian fjords. Between 19 and 31

individuals from eaeh station were analyzed. Seven lO-mer arbitrary primers produced 34

polymorphie markers that were used to assess the genetie population structure using the Analysis

of Molecular Variance (AMOVA) to allocate the variation among individuals, among sampling

stations and among regions. There was considerable RAPD diversity among individuals within

alllocations. Over 98% of the genetie variation between Barents and Spitsbergen was ascribed

to individual diversity, and less than 0.3% to differenees between the two areas. After indusion

of the Jan Mayen andNorthem fjords sampIes in the AMOVA analysis, about 7.% of the total

genetie diversity was assigned to regional differences (region 1, BarC?nts and Spitsbergen; region

2, Jan Mayen and region 3, Northem Norwegian fjords) and about 92% of the genetie variability

remained attributed to individual differences. within sampled stations. Principal component

analysis on the frequeney of each RAPD marker on each sampled station confirmed the presence

of 3 subpopulations: Barents and Spitsbergen, Northem Norwegian fjords and Jan Mayen. Our

results support the previously proposed existence of only one population of pink shrimp in the

Barents Sea and indicate that shrimps from Spitsbergen belong to the Barents population.

Individuals from Jan Mayen and from the Northem Norwegian fjords, on the other hand, showed

some degree of genetie diyergence, both from the main population and from each other. "

~ey Words: Shrimp, Pandal,,!s borealis, RAPD, AMOVA, North Atlantie.

'Ieiar Martinez, Taran Skjerdal and Bent Dreyer: Norwegian Institute of Fisheries andAquaculture, 9005-Tromsp, ,Norway [tel:+47.776 29000; fax: +47.776 '29100; e­

~ail:fiskf~rsk@fiskforsk.norut.no).Salah M. Aljanabi: EMBRAPA-CENARGEN, Sain-Parque

rural WS norte; PO Box 02372-CEP: 70849-970; Brasilia, DF-Brazil)

This paper not to be cited· without prior reference to the authors

International Council for the Exploration of the Sea CM 1997rr:24

GLOBEC (T)

Genetic structuring of Pandallis borealis in the North AtI~ntic.

11. RAPD analysis

by

Iciar Martinez, Taron Skjerdal, Bent Dreyer and Salah Aljanabi

Norwegian Institute of Fisheries and Aquaculture, 9005-Troms~, Norway.

ABSTRACT

The genetic variation of Pandalus borealis in the Northeast Atlantic was examined by RAPD

analysis on individuals caught at 8 different sampling stations in the Barents Sea, 3 off '

Spitsbergen, 2 off Jan Mayen and in 2 Northern Norwegian fjords. Between 19 and 31

individuals from each station were analyzed. Seven lO-mer arbitrary primers produced '34­

polymorphie markers that were used to assess the genetic population structure using the Analysis,

of Molecular Variance (AMOVA) to allocate the variation among individuals, among sampling

stations and among regions. There was considerable RAPD d,iversity among individuals within, 'all 10caÜons. Over 98% of the genetic variation between Barents and Spitsbergen was ascribed

to indiv~dual diversity, and less than 0.3% to differences between the two areas.After inclusion •

of the J30 Mayen 30d Northerri fjords sampies in the AMOVA analysis, about 7% of the total

genetic diversity was assigned to regional differences (region I, Barents and Spitsbergen; region

2, Jan Mayen and region 3, Northern Norwegian fjords) and about 92% of the genetie variability

remained attributed to individual differences within sampled stations. Principal componentanalysis on the frequeney of each RAPD marker on each sampled station confirmed the presence

of the 3 subpopulations: Barents and Spitsbergen, Northern Norwegian fjords and Jan Mayen.Our results support the previously proposed existence of only one population of pink shrimp in

the Barents Sea and indicate that shrimps from Spitsbergen belong to the Barents population.

Individuals from Jan Mayen and from the Northern Norwegian fjords, on the other hand, showedsome degree of genetie divergence. both from the main population and from each other.

•

","

IntroductionThe pink shrimp, also called Northem shrimp, Pandalus borealis (Krpyer, 1838), is a protandric

hermaphrodite species with a circumboreal distribution [25]; that is commercially harvested in

both the Atlantic and the Pacific. The pink shrimp is an important resource for Nonvay, which

harvested about 39 thousand tonnes in 1995 with a commercial value of over 765 millionNorwegian kroner [I].

The main Noiwegian fishing grounds are Barents and Spitsbergen. For stock assessment

and management of this species in the Barents/Spitsbergen area it is important to determine its

gerietie structuring. Kartavtsev [16] studied the population differentiation of the pink shrimp at

4 allozyme loci and found the population of the Barents Sea to be homogeneous, supporiing the

hypothesis previously proposed by Berenboim [4] that all the populations in the Barents Sea may

be considered as one superpopulation. Rasmussen et al. [22] assessed the frequeney distrib.ution

of 2 allozyme loci in populations of pink shrimps from Spitsbergen and 2 Northem Norwegian

fjords and found significant difference.s between the sampIes from Spitsbergen a?d those of the

fjords, hut not among the sa~ples from Spitsbergen or among those of the fjords.

Morphological differences among P. borealis have also been used as a criteria to assume

the existence of population structuring. Thus, Kartavtsev et al. [17] studied the genetie

structuring of P. borealis in Far-Eastem Seas arid found that shrimps sampied at different

loeatioris in the same sea wen~ genetically homogenous, hut there were statistically significant

differences of morphological traits among them. The authors attributed the morphologieal

differences to the effect of local selection at the larval and juvenile developmental stages, and,

accordingly, assumed the existence of a subpopulation structure. Morphologicai differences have

also been recorded among P. borealis in the Barents/Spitsbergen area: Aschan and Nielssen [3]

found that P. borealis in southem Barents grew faster and changed sex earlier thari shrimps 'in

,central and northem Barents.. .·The present s~udy was.undertaken to examine more deeply the possible genet!c structurlng

of P. borealis in the Northeast Atlantie. One part of this study used the frequency differences

at :3 polymorphie allozyme loci, and is report~d separately [7]: The second part, repcirted here,used random amplified polymorphie DNA (RAPD) markers [27,28] to address the same question.

~APD analysis w~s chosen over restriction fragment length polymorphisms and.the use

of minisatellite DNA, because RAPD ean be' performed without any previous knowledge ofspecifie DNA sequences ~f the ~pecies unrler study, it requires smalt amounts of DNA arid it is

faster, less costly and less labour intensive than the other two DNA techniques [5,12]. RAPD

analysis has been successfully used to studygeneiie structuring, among.others, in plants [14,26],

birds [13] and snails [15]. Treatment of RAPD markers by the Analysis of the Molecular

Variance (AMOVA) [10] has been proven to be adequate in order to allocate RAPD genetie

variation among iridividuals/within sampled stations, among stations/within regions and among

regions, as shown by [14] and [26].In this work, we have used AMOVA and principal componeni analysis on 34 genetie

markers obtained by RAPD fingerprint analysis of abo~t 400 pink shrimps eaptured at 15

different locations in the Barents Sea, Spitsbergen, Jan Mayen and 2 Norwegian fjords, ~n order

. to study the genetie structure of the species in the Northeast Atlantie.. ,

2

Materials and Mcthods

Pandalus borealis (Krpyer, 1838) were captured by trawler, between 250 and 500 m of depth,

at the 15 different locations in the North Atlantic schematically represented in figure I. The

exact loeation of the stations and the number of individuals analyzed from each station is shown

in table 1. Immediately after capture, the shrimps were beheaded, immersed in 96% ethanol and

stored at room temperature until the DNA was extracted.

DNA extraetion

About 50-100mg of ethanol embedded muscle were cut and bIotted onto tissue paper to

eliminate as much ethanol as possible. The pieces of muscle were transferred to 2ml Eppendorf

tubes, finely minced with scissors and the rest of the ethanol was allowed to evaporate by

incubating at 55°C for about 2min. To each tube, 400Jll of lysis buffer [21], O.4M NaCl, lOmM

Tris-HCI, pH 8.0 and 2mM EDTA, pH 8.0, were added first, followed by either 36Jll or,lOJll

of 20% SDS and 8111 of 20mglml proteinase K, which gave final concentrations of 1.6%, 0.5%

and 400Jlg/ml respectively. The contents of the tubes were'inixed by inversion several times and

incubated at 55°C for 3h or ?vemight with occasional mixing of their contents.

After the incubation, some of the sampies were let to cool down and 360JlI of 6M •

(saturated) NaCI were added to each tube [21]. The tubes were vortexed for Imin 'at maximum

speed, and centrifuged for 20min at 14,000rpm. Other sampies were incubated at 95°C for 5min,

cooled down on ice and centrifuged at 14,000 for 30min' [18]. In both cases, the supernatants

were recovered, transferred to new tubes and 100% ethanol in a 2:1 (ethanol:sample) ratio was

added to each tube. The tubes were inverted several times, incubated at -20°C for at least one

hour and centrifuged for 30min at 14,000 rpm. The supematants were discarded and the DNA

pellets were washed with 70% ethanol. The pellets were dried at room temperature and

resuspended in 300JlI of sterile double distilled Hp. The amount and quality of the DNA was

'estimated by 1% agarose gel electrophoresis in 0.5xTBE buffer and comparison with known, ' ,

amounts of A. DNA.

Seleetion 0/ primers .

Seven lO-mer primers (table 2) were chosen after screening about 200 of them. The criteria for

selecti~n were that they should consistently, between replicate PCRs and for a DNA

concentration ranging from about I to 5ngllll, prod~ce polymofphic bands, the bands should be •

clear to score and of a size between 300 and 600bp approximately [26].

DNA amplijieation

Arbitrarily primed amplifieations [27,28] were performed in 30JlI volumes. Initially, for each

sampie, 2 dilutions containing 1 and 5ng/Jll DNA were amplified as described below. Once it

was established that both DNA concentrations gave the same RAPD profile [27], only the sampie

containing Ingllll was used. WJlI of the DNA extracts containing Wng and 50ng respectively'

of the template DNA, were added to 20111 of a mixture containing 1x DNA polymerase buffer

(supplied by the manufacturer), looJ..IM each dATP, dCTP, dGTP and dTTP, 0.4J..IM IO-merprimer (Operon Technologies Inc. Alameda, Califomia), and 4mM MgCl2, [8] with 2 ~nits of

the ~toffel fragment of AmpliTaq DNA polymerase (perkin EImer). Tbe reaction mixtures wereoverlaid wilh 20JlI of ChilI Out Wax (MI Research Inc. Watertown, MA) and amplification was

3

performed on a PTC-100 programmable thermal controller (MJ Research Inc. Watertown, MA).

The thermal programm for amplification was 94°C for 2min, followed by 40 cycles of 94°C,

20sec (denaturation), 35°C, 20sec (annealing) and 72°C, Imin (extension). The programm

included a final step of 72°C for 5min and the products were maintained at lO-4°C until readyto load onto the gels.

Agarose gel electrophoresis

Fifteen fll of the products obtained after RAPD analysis were separated in 20x13cm, 2% (1:3)

Nusieve:Seakein LE FMC agarose gels. Gel and electrophoresis buffers were 0,5xTBE [23] andelectrophoresis took place for about 2h at 200V.

After electrophoresis, the gels were stained for 20min in 0,5xTBE buffer containing

0.5flg/ml ethidium bromide, destained for another 20min in the same buffer without ethidium

bromide and photographed under uv light with a Polaroid camera using film type 55.

Genetic structure: Analysis of the Molecular Varianci (AAtOVA)

• The presence (1) or absence (0) of each polymorphic marker was determined for each

individual and a matrix of 1/0 for all individuals and markers was constructed. The analysis of,the molecular variance (AMOVA) [10] was performed by using the program ARLEQUIN [24],

retrieved from the internet (address: http://anthropologie.unige.acasunlme'thods.html). AMOVA

allocates the proportion of the genetie variation attributable to i) individuals within the sampled, ,

stations, ii) sampled stations within the 4 regions (Jan Mayen, Spitsbergen, Barents and Northern

fjords), and iii) among the 4 regions.

••

Principal component analysis

The possible presence of genetically similar clusters of sampled stations was examined by

multivariate data analysis on the average frequeney of each, of the 34 RAPD markers in each of,the 15 stations [6]. Principal component analysis was performed using the program Unscramble

[9] and full cr?ss validation. The 34 variables (RAPD markers) were centered and given a weight .

of 1 each.

Rcsults

RAPD profileAfter excluding markers that were monomorphie for the entire dataset, the 7 primers yielded 34

polymorphie markers (table 2). The frequency of each marker in each station is shown on table

3. Five of the markers (4, 6, 9, 30 and 32; 14,7%) were fixed in some of the sta~ions, while 5

other markers (7, 12, 15, 20 and 24; 14,7%) were not detected in some of the stations.

Estimation of the population genetic structureFirst we wanted to examine whether there was an intra-"geographical regions" strueture or

whether these regions could be eonsidered genetieally homogeneous. The results of the analysis,shown on table 4, revealed that there was praetically no genetie strueturing among the stations

sampled in Barents (B), among the stations sampled in Spitsbergen (S), or among the two fjords

(Fj)" where over 98% of the total genetie variability originated in individuals within the sari1ple~

4

stations. There was however some genetic differentiation (3% of the total' genetic variability)

among the shrimps from the southem and northem regions in Jan Mayen (JM): There was no

detectable genetic structuring between the shrimps collected in Barents and in Spitsbergen (B

vs S). Tbc shrimps from thc Barents-Spitsbergen area were, however, different (5% of thc total

. variation) from the shrimps from Jan Mayen «B+S) vs JM) as weil as from the shrimps from

the fjords «B+S) vs Fj; 8.1 % of the total genetic variability). Tbe sampies from Jan Mayen were

also genetically different (although not significatively ) from the fjord shrimps (JM vs Fj; 7,3%

of the total genetic variability).

Analysis of the three areas: Barents+Spitsbergen, Jan Mayen and the fjords indicated a

highly significant (p<O.OOOI), although small (6.7%) genetic diversity among the threc areas, and

the major component of the total genetic diversity was attributable to individual diversity within

the sampled stations (92%, p<O.OOOI).

Principal component analysis (PCA)

PCA analysis on the average frequency of each marker on each station, confirmed the genetic

. structure indicated by AMOVA. Tbe first and second components explained 29% and 17% of

the total variation present in thc dataset (figure 2). Two clear clusters were observed, onc

corresponding to the sampies from Barents and Spitsbergen, and the second to the sampies from

the fjords. Tbe sampies from Jan Mayen, although clearly segregated from'the other two groups,

did not form a compact cluster, which indicated that individuals from the south and from the

north might not be genetically homogeneous; those from the south of Jan Mayen seemed to be

genetically more similar than those from the north of Jan Mayen, to the 'shrimps from the

Barents-Spitsbergen cluster.

Discussion

Tbe genetic structure· of P. borealis, depicted by this w~rk using RAPD markers and by

Drengstig and Fevolden [7] using allozyme variability in Barents, Spitsbergen, Jan Mayen and

Northem No~egian fjords,. is comparable to that described by Fevolden [11]"of Chlamys

islqndica using allozyme variability in the same areas. As the C. isla.ndica, P. borealis has a

planktonic larval stage that lasts for 2-3 months [25] allowing both species a la~ge potential for

dispersion as they are being carried byth~ currents. -Looking ~t the ~urface current system.'

schematically depicted shown on figure I, it is reasonable to assurne dispersion of laryae from

Barents to Spitsbergen and between Jan Mayen and Spitsbergen. Tbe dispersion of larvae among

the fjords, on the other hand; will be dictated by the intricate coastal current system. Tbe

distance between Malangen, in the south, and Balsfjord, in the north, is about 25km. Tbis short

distance and the current system can explain the genetical similarity between these two sampies

that, according to our results, may be considered as one population. Again, similar results were

obtained by Fevolden [11] on C. islandica sampled at two other Northem Norwegian fjords,

Tromspberg and Andammen, which are about 20 and 80 km respectively further north from

Balsfjord.In contrast to Kartavtsev et al. [17], the differences in morphological traits found in the

Bare~ts/Spitsbergenregion by Aschan and Nielssen [3] can, in our opinion, be explained simplyby differenccs in watcr temperature. Thus, P. borealis. in southern Barents, where thc water

5

•

•

..

••

•

temperaitire is higher, grew faster and changed sex earlier than shrimps in central and northern

Barents, where the water temperature is lower [3]. The same trend was observed over time:

dunng the 80's, when water temperatures were low, the shrimps had slower groWth and changed

sex later than in the 90's, when higher water temperatures were registered [3]. Warmer water

. temperature is known to have an speeding up effect on the development on rriany marine species,

including shrimps [25,2], and ~rctic species of fish, [19,20]. In addition, the water temperature

is very likely to have an effect on the availability, bath quantitative and qualitatively, of food.

In ~Y case, a deeper study on the physiology ~f P. borealis, as weIl as mapping of its genome,

will help to ascertain which are the genetically determined morphometric traits of value to define

the population structuring in this species.

In summary, the genetic similarity among pink shrimps from the Barents Sea found in this

work, supports Berenboim's [4] and Kartavtsev's [16] hypothesis on the existence of only one

populat~on in the Barents Sea. In addition, our data indicate that the pink shrimp from

Spitsbergen belong to the same population. The genetic dissimilarity between pink shrimps from

the Northern Norwegian fjords and Spitsbergen described by Rasmussen et aI. [22], is further

conflrmed by our work. Finally, our results indicate that shrimps from the s~uthern and riorthern, '

coasts of lan Mayen, besides being significantly different from the Barents arid fjord populations,

also might be genetically different from each other.

AcknowledgmentsThe authors wish to thank Dr. Peter E. Smouse (Rutger University, New Jersey) for his·

explanations and help to use the program AMOVA. Dr. Laurent Excoffier (University of

Geneva, Switzerland) for making the programms AMOVA (ftp:\\acasunl.unige.ch) and

ARLEQUIN (http://anthropologie.unige.acastiiJmethods.html) available. via the internet. The

Norwegian Research Council is gratefully acknowledged for the financial support (project .!'Ir..

108151/120).

.. RefcrencesI. Anon. (1996) Statistikk. Norges Fiskerie.r 1995. Fiskets Gang, 2: 22. . '

. 2. Apollonio, S., Stevenson, D.K. & Dunton Jr., E.E. (1986) Effects of temperature on the biology of the. Northern shrimp, Pandalus borealis, in the Gulf of Maine. NOAA Technical Report NMFS 42, Sept.

1986,22 pp.3. Aschan; M. & Nie1ssen, E.M. (1997) Length frequency distribution, growth, maximum length arid size

and age at maturity of shrimp Pandalus borealis in the Barents Sea. lCES CM, 1997.4. Berenboim, B.I. (1982) Reproduction of the populations of the shrimp Pandalus borealis in the Barents

Sea. Okeanologiya, 22, 1: 118-124. (In Russian) as quotedin reference [16], below.5. CaetallO-Anolles, G., Bassam, BJ. & Gresshoff, P.M. (1991) DNA amplification fingerprint: a strategy

for genome analysis. Plant. Mol. Biol. Rep., 9: 294-307.6. Demeke, T. & Adams, R.P. (1994) The use of RAPDs to determine germplasm collection strategies in

the African species Phytolacca dodecandra (Phytolaccaceae). In: Consermtion 01 Plant Genomes. 1/: Utilization 01 Ancient and Modem DNA. Eds. Adams, R.P., Miller, l.S.,. Gole~berg, E.M. &

Adams, J.E. Missouri Botanical Garden. pp: 131-139.7. Drengstig, A. & Fevolden, S.E. (1997) Genetic structuring of Pandalus borealis in the NE-Atlantic. I.

. Allozyme studies. lCES,8. Ellesworth, D.L., Rittenhouse, D. & Honeycutt, R.L. (1993) Artifactual variation in randomly amplified

polymorphie DNA banding patterns; Bio~ee~back, 14: 214-218. .

·6

9. Esbesen, K., Schönkopf, S. & Midtgaard, T. (1994) Multivariate Analysis in Practice. CAMO,Wennbergs Trykkei AS, Trondheim, Norway. ISBN 82-993330-0-8.

10. Excoffier, L., Smouse, P.E. & Quattro, J.M. (1992) Analysis ofmolecular variance inferred from metricdistances arnong DNA haplotypes: application to human mitochondrial restriction siies. Genetics,131: 479-491.

11. Fevolden, S.E. (1992) Allozymic variability in the Icelandic scallop Chlamys islandica: geographicalvariation and lack of growth-heterozygosity correlations. Mar. Ecol. Prog. Sert., 85: 259-268.

12. Hadrys, H., Balick, M. & Schierwater, B. (1992) Applications of random arnplified polymorphic DNA(RAPD) in molecular ecology. Mol. Ecol., 1: 55~63.

13. Haig, S.M., Bowman, R. & MuIlins, T.D. (1996) Population structure ofred-cockaded woodpeckers insouth Florida: RAPDs revisited. Mol. Ecol., 5: 725-734.

14. Huff, D.R., PeakeIl, R. & Smouse, P.E. (1993) RAPD variation within and among natural populationsof outcrossing buffalograss [Buchloe dactyloides (Nutt.) Engelrn.]. Theor. Appl. Genet., 86: 927-934.

15. Jacobsen, R., Forbes, V.E. & Skovgaard, O. (1996) Genetic population structure ofthe posobranch snailPotamopyrgus antipodarum (Gray) in Denmark using RAPD-PCR fingerprints. Proc. R. Soc. Land.,8263: 1065-1070.

16. Kartavtsev, Y.P., Berenboim, BJ. & Zgurovsky, K.1. (1991) Population genetic differentiation of thepink shrimp, Pandalus borealis Krpyer, 1838, from the Barents and Bering Seas. J. Shellfish Res.,10: 333-339.

17. Kartavtsev, Y.P., Zgurovsky, K.A. & Fedina, Z.M. (1993) Spatial structure ofthe pink shrimp Pandalus .borealis Krpyer, 1883, from the Far-Eastern Seas as proved by methods of population genetics andmorphometrics. J. Shellfish Res., 12: 81-87. .

18. Lawrence, L.M., Harvey, J. & Gilmour, A (1993) Development of a random amplification ofpolymorphie DNA typing method for Listeria monocytogenes. Appl. Environ.' Microbiol., 59: 3117­3119.

19. Martinez, 1., Dreyer, B., Agersborg, A., Leroux, A & Boeuf, G. (1995) Effects of T3 and rearingtemperature on growth and skeletal myosin heavy chain isoform transition during early development .in the salmonid Salvelinus alpinus (L.). Comp. Biochem. Physiol., 1128: 717-725. .

20. Martinez, I. & Pedersen, G.W. (1992) Temperature-induced precocious transitions of myosin heavychain isoforms in the white muscle of the Arctic charr, Salvelinus alpinus (L.). BAM: 2: 89-95.

21. Miller, S.A; Dykes, D.D., Polesky, H.F. (1988) A simple salting out procedure for extracting DNA fromhuman nucleated cells. Nucleic Acids Res., 16: 1215. .

" 22. Rasmussen, T., Thollesson, M. & Nilssen, E.M. (1993) Preliminary investigations on the population, genetic differentiation of thedeep water prawn, Pandalus borealis Krpyer, 1838, from Northern

Norway and the Barents Sea. ICES CM 19931K:11. ., 23. Sambrook, J.,' Fritsch, E.F. & Maniatis, T. (1989) Molecular Clonni11g. A Laborato/}: Manual. 2nd

Edition. Cold Spring Harbor Laboratory Press. ' .24. Schneider, S., Kueffer, J.M., Roessli, D. & Excoffier, L. (1996) Arlequin: a software package for

populations genetics. Genetics and Biometry Lab., Dept. Anthropology;University 0 Geneva. •25: Shumway, S.E., Perkins, H.C., Schick, D.F. & Stickney, A.P. (1985) Synopsis of biological data on the .

pink shrimp, Pandalus borealis Krpyer 1838. FAO Fish. Synopsis, 144:·1~157..26. Stewart, C.N. & Excoffier, L. (1996) Assessing population genetic structure and variability with RAPD

data: Application to Vaccinium macrocarpon (American cranberry). J. Eval. Biol, 9: 153-171.27. Welsh, J. & McCleIland, M. (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic

Acids Research, 18: 7213-7218. .28. Williams, J.G.K., Kubelik, AR., Livak, KJ., Rafalski, J.A. & Tingey, S.V. (1990) DNA polymorphisms

amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research, 18: 6531-6535.

7

.e

LEGENDS TO THE FIGURES

Figure 1.- Sehematie representation of the sampling sites of Pandalus borealis. ---->, polareurrents; , ••••>, Atlantic eurrents; ----> eoastal eurrents. The numbers 1 to 15 represent theIoeation of the sampling sites.

Figure 2.- Principal eomponent analysis performed on. the average frequenee of eaeh of 34RAPD markers in 15 sampled sites. PCl and PC2 are prineipal eomponents 1 and 2 respeetively.The Ioeation of the stations (1-15) is shown on table 1. The data were eentered and eaeh variablewas given a weight of 1.

8.

/I / I12, I / I

1 I / II / I

I ( ~III,

11,

I/--

I1,1

//

//

//

/

//

/1

/ ' "-/

/I

I

PC2 Scores0.8 -

0 140.6 - 0 15

0.4 -

0 7

0.2-0 12

0 100 8

0-0 30 11

0 5 5

-0.2 -0 9

0 1 0 134 0

2-0.4 -

PCt

-1.0 -0.5 0 0.5RESULT1 X-exol: 29% 17%

PCA RESULTl 20 Scatter Plot

•

Table 1.- Sampling site loeation and number of individuals (n) analyzed per site.

Sarnpling site

12

34

567

8

9

10

11

12

13

14

15

n

29

31

19

28

27

29

28

26

30

27

19

25

28

26

26

Latitude

69° 28'

69° 25'

71° 23'

72° 27'

73° 53'

73° 54'

74° 35'

73° 23'

76° 21'

75° 47'

80° 02'

80° 11'

80° 10'

70° 47'

71°04'

Longitude

18° 85'

19° .02'

23° 13'

34° 18'

31°48'

31° 53'

27" 16'

17° 26'

32° 46'

14° 44'

10° 41'

10° 12'

10° 05'

8° 03'

9° 31'

Table 2.- Sequenee of t~e lO-mer primers used for RAPD analysis and the number of polymorphiemarkers they generated.

•

Primer

OPH-ll

OPI-lO

OPI-14

OPI-16

OPl-4

OPl-6

OPl-12

Total

Nucleotide sequenee 5' to 3'

CTT-CCG-CAG-T

ACA-ACG-CGA-G

TGA-CGG-CGG-T

. TCT-CCG-CCC-T

CCG-AAC-ACG-G

TCG-TTC-CGC-A

GTC-CCG-TGG-T

7

Number of polymorphie loci

6

3

72

8

3

534

Table 3. Frequeneies of the 34 polymorphie RAPD lo.ci in each of the 15 sampled stations

Primer . locus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15OPH·11 . 1 0,41 0,39 0,37 0,39 0,37 0,41 0,25 0,35 0,37 0,26 0,42 0,20 0,25 0,31 0,38

2 0,45 0,45 0,32 0,61 0,59 0,66 0,39 0,35 0;87 0,59 0,42 0,36 0,71 0,42 0,543 0,10 0,06 0,37 . 0,64 0,41 . 0,48 0,39 0,35 0,37 0,37 0,32 0,44 0,57 0,27 0,234 1 1 1 . 1 0,85 0,90 0,89 0,96 0,83 0,96 1 0,76 0,89 0,73 0,925 0,10 0,19 0,16 0,04 0,26 0,21 0,07 0,04 0,20 0,07 0,16 0,20 0,11 0,12 0,126 0,69 0,77 0,63 1 0,67 . 0,66 0,61 0,69 0,83 0,67 0,68 0,72 0,79 0,62 0,73

OPI·10 7 ° ° 0,47 0,46 0,37 0,41 0,54 0,38 0,33 0,41 0,53 0,60 0,36 0,62 0,658 0,38 0,29 0,32 0,36 0,30 0,34 0,36 0,23 0,43 0,33 0,37 0,40 . 0,36 0,31 0,469 0,97 0,90 1 . 1 0,81 0,93 0,86 0,92 1 0,78 0,84 0,80 0,93 0,42 '0,92

OPI·14 10 0,24 0,23 0,26 0,32 0,19 0,24 0,11 0,15 0,20 0,19 0,37 0,40 0,39 0,08 0,0411 0,72 0,68 0,74 0,89 0,67 0,59 0,61 0,54 0,47 0,63 . 0,68 0,92 0,43 0,81 0,6912 0,14 ° 0,21 ° °. 0,03 0,14 0,23 0,07 0,26 0,11 0,28 ° 0,35 0,3813 0,41 0,35 0,26 0,39 0,41 0,24 0,32 0,19 0,13 0,37 0,21 0,20 0,25 0,23 0,2314 0,38 0,42 0,21 0,29 . 0,41 0,45 0,25 0,27 '0,47 0,30 0,26 0,28 0,25 0,42 0,4215 0,10 0,29 0,05 . 0,21 0,19 ·0,34 0,14 0,19 0,10 0,19 0,16 0,16 0,21 0,15 °16 0,17 0,26 0,37 0,14 0,37 .0,48 0,82 0,46 0,47 0,44 0,37 0,48 0,43 0,58 0,73

OPI·16 17 0,38 0,32 0,42 0,43 0,26 0,45 0,36 0,27 0,27 0,30 0,58 0,44 0,39 0,31 0,4218 0,14 0,32 0,11 0,25 0,30. 0,24 0,29 0,23 0,23 0,19 0,21 0,24 0,25 0,12 0,19

OPJ·4 19 0,17 0,19 0,16 0,21 0,22 0,31 0,11 0,12 0,10 0,22 0,26 0,16 0,14 0,42 0,3820 0,03 0,10 . 0,05 0,04 0,11 0,10 ° 0,04 ° 0,04 ° 0,04 0,04 0,04 °21 0,34 0,39 0,32 0,36 0,41 0,34 0,64 0,58 0,50 0,48 0,37 0,32 0,43 0,38 0,3822 0,76 0,71 0,11 0,04 0,07 0,31 0,11 0,12 0,10 0,15 0,21 0,08 0,14 0,31 0,7323 0,55 0,26 0,32 0,21 0,22 0,28 0,21 0,35 0,27 0,33 0,21 0,28 0,32 0,35 0,3524 0,14 ° ° 0,04 0,04 ° ° .0,08 0,07 ° 0,05 0,12 0,07 ° 025 0,17 0,13 0,21 0,25 0,22 . . 0,24 0,18 0,15 0,17 0,15 0,11 0,08 0,18 0,08 0,0426 0,41 0,55 0,26 0,32 0,33 0,59 0,50 0,19 0,43 0,37 0,32 0,44 0,64 0,50 0,38

OPJ·6 27 0,59 0,58 0,68 0,82 0,78 0,79 0,86 0,88 0,60 0,85 0,47 0,76 0,79 0,69 0,8528 0,34 0,19 0,16 0,25 0,37 0,24 0,14 0,19 0,33 0,19 0,42 0,20 0,07 0,46 0,2729 0,48 0,45 0,47 0,64 0,63 0,59 0,46 0,42 0,70 0,41 0,42 0,52 0,36 0,42 0,46

OPJ·12 30 1 1 1 0,93 0,89. 0,93 0,96 0,88 0,97 0,93 1 0,96 0,96 1 0,9631 0,17 0,19 0,79 0,79 0,59 0,72 0,75 0,88 0,60 0,70 0,95 0,80 0,75 0,50 0,1232 1 1 0,89 0,93 0,93 1 0,96 0,96 0,93 0,96 0,95 0,92 0,93 1 133 0,34 0,52 0,37 0,11 0,26 0,38 0,14 0,19 0,33 0,22 0,37 0,28 0,32 0,23 0,1534 0,52 0,48 0,53 0,54 0,59 0,59 0,50 0,54 0,53 0,56 0,47 0,56 0,39 0,77 0,73

.e

~

•Table 4. Genetic structures tested by the analysis ofthe molecular variance (AMOVA) using 34 RAPDmarkers on the following groups: F, fjords, (stations no. 1 and 2); B, Barents (stations no. 2 to 10);S, Spitsbergen (stations no. 11 to 13); and JM, Jan Mayen (stations no 14 and 15); Sst, samplingstations. Statistics are SSDs, sum of squared deviations; Vc, variance component and p, probabilityof obtaining a more extreme component estimate by chance alone. n.a., not available.

Genetic Source of %Total P-valuestructure variation df SSD Vc variationB Sstlregions .7 56.44 0.08 1.40 < 0.0005

individualsfSst 206 1203.57 5.84 98.60 n.a.S Sstlregions 2 16.17 0.09 1.43 <0.05

individualsfSst 69 . 414.91 6.01 98.57 n.a.BvsS BvsS 1 7.93 -0.00 -0.1 0.5

Sstlregions 9 72.61 0.08 1.4 <0.0001

individualsf8st 275 1618.49 5.88 98.6 <0.0001

JM Sstlregions 1 10.98 0.19 3.22 <0.01

individualsfSst 50 294.58 5.89 96.78 n.a.

e (B+8) vs JM (8+S) vs JM 1 36.54 0.32 5.09 <0.05

Sstlregions 11 91.55 0.09 1.49 <0.0001

individualsf8st 325 1913.06 5.88 93.42 <0.0001

Fj Sstlregions 1 4.85 -0.02 -0.44 0.6

individualsfSst 58 324.36 5.59 100.44 n.a.(8+S) vs Fj (8+S) vs Fj 1 59.48 0.52 8.08 <0.0001

Sstlregions 11 85.42 0.07 1.14 <0.0001

individualsfSst 333 1942.85 5.83 90.78 <0.0001

JM vs Fj JM vs Fj 1 33.28 0.45 7.27 0.6

Sstlregio.ns 2 15.83 0.08 1.25 <0.05

individualsfSst 108 618.94 5.73 91.48 <0.0001

(B+8) vs JM vs Fj (8+8) vs JM vs Fj 2 . 92.10 0.43 6.75 <0.0001

Sstlregions 12 96.40 0.08 1.31 :':0.0001

individualsfSst 383 2237.43 5.84 91.94 <0.0001