SUPPLEMENTARY MATERIALS:

Microsatellite analysis reveals genetic differentiation between year-classes in the icefish Chaenocephalus aceratus at South Shetlands and Elephant Island

Chiara Papetti1, Emanuele Susana1, Mario La Mesa3, Karl-Hermann Kock4, Tomaso Patarnello2 And Lorenzo Zane1*

1 Department of Biology - University of Padova, via U. Bassi 58/B, I-35121 Padova (PD), Italy,

2 Department of Public Health, Comparative Pathology and Veterinary Hygiene - University of Padova, Agripolis, via Romea 16, I-35020 Legnaro (Pd), Italy,

3 Institute of Marine Science (CNR-ISMAR) Marine Fishery Section - Largo Fiera della Pesca, 1, 60125 Ancona (An) Italy.

4 Institut für Seefischerei, Bundesforschungsanstalt für Fischerei, Palmaille 9, 22767 Hamburg, Germany

* corresponding author: Dr Zane Lorenzo, Department of Biology - University of Padova, via U. Bassi 58/B, I-35121 Padova (PD), Italy, e-mail , tel. +39 049 8276220, fax +39 049 827609.

Geographic region / Date / Sampling Station / Coordinates position / Depth range / N. ind
Elephant Island / 29.01.02 / 44 / 60° 58,08' S - 55° 06,85' W / 308.00 / 9
60° 58,09' S - 55° 04,05' W / 398.80
29.01.02 / 48 / 61° 09,82' S - 54° 33,40' W / 343.20 / 6
61° 11,16' S - 54° 35,45' W / 277.60
30.01.02 / 51 / 61° 12,01' S - 54° 50,50' W / 94.40 / 2
61° 11,97' S - 54° 53,79' W / 62.40
30.01.02 / 53 / 61° 20,51' S - 55° 28,66' W / 159.20 / 6
61° 20,22' S - 55° 32,15' W / 117.60
31.01.02 / 55 / 61° 16,59' S - 55° 42,72' W / 135.60 / 22
61° 18,04' S - 55° 42,80' W / 171.60
31.01.02 / 60 / 61° 21,34' S - 56° 02,65' W / 355.20 / 3
61° 22,37' S - 56° 00,47' W / 352.80
31.01.02 / 61 / 61° 17,38' S - 56° 12,98' W / 327.20 / 6
61° 16,90' S - 56° 16,12' W / 317.20
31.01.02 / 63 / 60° 51,39' S - 55° 31,35' W / 278.80 / 9
60° 51,85' S - 55° 28,87' W / 291.60
01.02.02 / 69 / 61° 01,41' S - 55° 58,98' W / 338.00 / 8
61° 00,27' S - 55° 57,48' W / 272.40
01.02.02 / 71 / 60° 58,83' S - 55° 49,74' W / 160.00 / 25
60° 58,13' S - 55° 46,85' W / 187.20
02.02.02 / 74 / 61° 02,91' S - 55° 53,76' W / 187.60 / 17
61° 03,74' S - 55° 55,46' W / 190.80
02.02.02 / 75 / 61° 05,78' S - 55° 53,61' W / 139.20 / 15
61° 04,61' S - 55° 50,94' W / 126.80
02.02.02 / 82 / 60° 59,33' S - 55° 49,12' W / 160.00 / 11
61° 00,92' S - 55° 50,40' W / 145.60
02.02.02 / 84 / 60° 59,08' S - 55° 54,16' W / 189.20 / 10
61° 00,02' S - 55° 51,25' W / 161.40
03.02.02 / 86 / 61° 04,87' S - 56° 00,89' W / 229.10 / 7
61° 03,30' S - 55° 59,24' W / 306.50
03.02.02 / 87 / 61° 01,93' S - 55° 51,47' W / 148.50 / 16
61° 03,61' S - 55° 52,02' W / 149.50
04.02.02 / 91 / 60° 58,45' S - 55° 47,65' W / 200.00 / 6
60° 57,66' S - 55° 44,47' W / 201.20
04.02.02 / 95 / 61° 13,38' S - 55° 52,94' W / 144.60 / 7
61° 14,87' S - 55° 54,74' W / 163.20
05.02.02 / 96 / 61° 10,38' S - 55° 56,01' W / 127.40 / 7
61° 11,17' S - 55° 52,69' W / 125.60
05.02.02 / 97 / 61° 09,62' S - 56° 00,97' W / 153.40 / 10
61° 09,09' S - 56° 04,46' W / 171.50
Subtot / 202
South Shetlands / 13.02.02 / 104 / 61° 39,05' S - 57° 44,39' W / 384.30 / 7
61° 39,05' S - 57° 44,39' W / 384.30
14.02.02 / 107 / 61° 50,36' S - 57° 20,91' W / 277.40 / 3
61° 50,36' S - 57° 20,91' W / 277.40
16.02.02 / 111 / 61° 50,75' S - 59° 12,64' W / 269.90 / 15
61° 50,75' S - 59° 12,64' W / 269.90
16.02.02 / 112 / 62° 00,02' S - 59° 09,91' W / 139.30 / 2
62° 00,02' S - 59° 09,91' W / 139.30
16.02.02 / 113 / 62° 01,57' S - 59° 35,95' W / 174.10 / 8
62° 01,57' S - 59° 35,95' W / 174.10
17.02.02 / 115 / 62° 05,20' S - 59° 31,18' W / 111.50 / 4
62° 05,20' S - 59° 31,18' W / 111.50
18.02.02 / 118 / 62° 20,56' S - 60° 32,01' W / 136.60 / 6
62° 20,56' S - 60° 32,01' W / 136.60
Subtot / 45
Tot / 247

Table 1S.Chaenocephalus aceratus sampling. Geographic regions, stations, start and stop trawling coordinates and sampling depth range, and number of individuals for each sampling station.

Locus* / GenBank Acc. Num. / PCR profile / Annealing temp. / Fluorescent dye
Ca21 / EF103946 / Touch down from 60°C,
12 cycles, 0.5°C decrease / 52°C / 6-FAM
Ca26 / EF103948 / Touch down from 60°C,
12 cycles, 0.5°C decrease / 52°C / HEX
Ca40 / EF103950 / Touch down from 58°C,
16 cycles, 0.5°C decrease / 52°C / TAMRA
Ca48 / EF103951 / Touch down from 58°C,
16 cycles, 0.5°C decrease / 50°C / TAMRA
Ca55 / EF103947 / Touch down from 58°C,
16 cycles, 0.5°C decrease / 50°C / HEX
Ca86 / EF103952 / Touch down from 58°C,
16 cycles, 0.5°C decrease / 50°C / 6-FAM
Ca88 / EF103953 / Touch down from 58°C,
16 cycles, 0.5°C decrease / 50°C / 6-FAM
Cr15 / DQ141191 / NO Touch down / 59°C / HEX
Cr127 / DQ141192 / Touch down from 65°C,
12 cycles, 0.7°C decrease / 55°C / TAMRA
Cr171 / DQ141187 / NO Touch down / 57°C / TAMRA
Cr259 / DQ141188 / Touch down from 65°C,
12 cycles, 0.7°C decrease / 55°C / 6-FAM

*Each locus acronym indicates the species (Ca for Chaenocephalus aceratus and Cr for Chionodraco rastrospinosus) and reference colony number (15-259).

Table 2S. Microsatellite loci amplification protocols (For details see Susana et al. 2007 and Papetti et al. 2006, references in the text) and fluorescent labeling dye. PCRs were carried out in 20 μL total volume containing: Taq buffer 1X (Promega, 50 mM KCl, 10 mM Tris-HCl pH9 at 25 ° C, 0.1% TritonX- 100), MgCl2 1 mM , 150 nM of each primer (except for locus Ca88 for which 300 nM of the forward primer was used), 70 μM dNTPs, 0.8 U of Taq polymerase (Promega) and 50 ng of genomic DNA (except for locus Ca88 for which 100 ng of genomic DNA was used). Loci were amplified with a hot-start touchdown PCR profile (except for loci Cr15 and Cr171, differences between profiles for each loci are reported in the table): (i) predenaturation: 94 ° C 3 min; (ii) 12÷16 touchdown cycles: denaturation 94 ° C 40 s, annealing 65÷58 ° C 30 s decreased of 0.5÷0.7 ° C each cycle, extension 72 ° C 1 min; (iii) 30 cycles: denaturation 95 ° C 40 s, annealing 59÷50 ° C 30 s, extension 72 ° C 1 min; (iv) additional extension for 10 min at 72 ° C.

Age class / N / Females / Males / Undet.
I / 37 / 8 / 10 / 19
II / 2 / 2 / 0 / -
III / 6 / 3 / 3 / -
IV / 11 / 9 / 2 / -
V / 21 / 9 / 12 / -
VI / 27 / 13 / 14 / -
VII / 22 / 10 / 12 / -
VIII / 13 / 7 / 6 / -
IX / 9 / 3 / 6 / -
X / 12 / 6 / 6 / -
XI / 6 / 4 / 2 / -
XII / 12 / 6 / 6 / -
XIII / 9 / 5 / 4 / -
XIV / 4 / 4 / 0 / -
XV / 4 / 3 / 1 / -
XVI / 4 / 4 / 0 / -
XVII / 2 / 2 / 0 / -
Tot / 201 / 98 / 84 / 19

Table 3S. Number of individuals analyzed for each age class and sex. Undet.: sex-undetermined individuals.

/
Hypotheses
/ Ca21 / Ca26 / Ca40 / Ca48 / Ca55 / Ca86 / Ca88 / Cr15 / Cr127 / Cr171 / Cr259 / tot
I) / Geographic differentiation / 0.5288 / 0.0029 / 0.4858 / 0.6901 / 0.1154 / 0.4809 / 0.7439 / 0.0450 / 0.7273 / 0.7439 / 0.3979 / 0.06061
II) / Differentiation among sexes / 0.8915 / 0.5103 / 0.5005 / 0.2336 / 0.9961 / 0.7449 / 0.2287 / 0.9228 / 0.5112 / 0.9697 / 0.3500 / 0.95015
III) / Differentiation among all age classes / 0.6852 / 0.2219 / 0.5240 / 0.8729 / 0.5973 / 0.1916 / 0.4780 / 0.1818 / 0.1066 / 0.9511 / 0.8328 / 0.41642
Differentiation among age classes of more than 9 individuals each (age classes: I, IV, V, VI, VII, VIII, IX, X, XII; XIII) / 0.5865 / 0.1936 / 0.5152 / 0.7126 / 0.7116 / 0.1349 / 0.1818 / 0.1007 / 0.0313 / 0.8964 / 0.3763 / 0.06549
Differentiation among age classes of more than 20 individuals each (age classes: I, V, VI, VII) / 0.3011 / 0.0293 / 0.4174 / 0.2454 / 0.6843 / 0.2981 / 0.1369 / 0.1965 / 0.0117 / 0.8749 / 0.1994 / 0.00574

Table 4S. Locus by locus AMOVA probabilities for three a priori hypotheses.