Randers Fjord and Horsens Fjord. In Sweden, all species were collected ...... In G.oceanicus 26% of 19 loci are polymorphic ac- cording to criterion 1, and 16% ...
Herediras 102: 1-13 (1985)
Genetic studies of Gammarus I. Genetic differentiation of local populations HANS REDLEF SIEGISMUND, VIBEKE SIMONSEN and STEEN KOLDING Institute of Ecology and Genetics, University of Aarhus, Denmark
SIEGISMUND. H. R., SIMONSEN. V. and KOLDING, S. 1985. Genetic studies of Gammarw. 1. Genetic differentiation of local populations. - Herediras 102: 1-13. Lund, Sweden. ISSN 0018-0661. Received March 9. 1984 In five marine species of the amphipod genus Gammarus ( C .duebeni, G.zaddachi, G.salinus, G.oceanicus, and G.locusta) the electrophoretically defined variation at 19 loci in populations from the Baltic Sea and the Kattegat is compared. In four out of the five Gammarw species there is evidence for differentiation among the Baltic and the Kattegat populations. In addition, the phylogenetic relationship of the five Gammarus species and a single species in the genus Chaefogammarus(Cmarinus) is described.
Hans Redlef Siegismund, Institute of Ecology and Genetics, University of Aarhus, N y Munkegade, DK-8000 Arhus C,Denmark
Before 1947 the taxonomy of the marine members G.zaddachi and G.salinus are found. Gammarus of the northwestern European amphipod genus oceanicus and G.locusta occur under the more maGammarus was highly confused, with three recogni- rine conditions. The order in which they occur varized species: G .duebeni, G.zaddachi, and G.locusta. es geographically. In the Limfjord of Denmark, for SEGERSTRALE (1947) and SPOONER (1947) showed that example, G.oceanicus is found at the highest salinitwo additional forms with subspecific status were ties ( F E N C H EKLOLDING ~ ~ ~ 1979), whereas G.locushidden among these three species, and G.zaddachi ta is confined to the more open marine areas in the was divided into three subspecies: G.zaddachi zad- Isefjord (RASMUSSEN 1973). The habitat occurrence dachi, G. zaddachi salinus, and G. zaddachi oceani- is limited both by species specific salinity preferencus. The diagnostic characters are some minor diffe- ces and by competitive interaction among the sperences in the setation of peraeopod 6 and 7, the ur- cies, so the species usually inhabit a smaller range osome, and the mandibular palp. KINNE(1954) of the salinity gradient than is physiologically possiconcluded, on the basis of sterile matings, that these ble (FENCHEL and KOLDING 1979). subspecies should be given species rank. Thus, five In the central part of the Baltic Sea the salinity is Gammarus species are now recognized: G.duebeni rather constant, ranging from 6 to 7 %o, which excluLilljeborg, G.zaddachi Sexton, G.salinus Spooner, des the possibility of habitat selection due to saliniG.oceanicus SegerstrPle, and G,locusta L., and ty. Contrary to expectations, all five species are among these, the three species of the G.zaddachi found in the Baltic Sea (FENCHELand KOLDINC 1979). group are considered to be sibling species. KOLDING This coexistence is explained by avoidance of comand SIMONSEN (1983) described the phylogenetic re- petition through habitat selection and by displacelationships of these Gammarus species, using esti- ment of the breeding periods (KOLDING 1981). The within species differentiation in life cycle and mates of the genetic distance at a number of electrophoretically defined loci. in habitat selection between the Baltic and the KatThe Gammarus species usually show differential tegat suggest that the populations may have diffehabitat selection in salinity gradients (SPOONER 1947; rentiated genetically. To quantify this, three popuDENHARTOG 1964; RASMUSSEN 1973; FENCHEL and lations of each Gammarus species (two from DenKOLDING 1979). Gammarus duebeni is found in the mark and one from the east coast of Sweden) have most freshwater influenced parts of estuaries, in la- been investigated by enzyme electrophoresis. goons, and rockpools. With increasing salinities,
2
Hereditas 102 (I985)
H.R.SlEGlSMUND ETAL.
Material and methods Collections The two collection sites in Denmark were the Limfjord, a sound cutting through the northern part of Jutland, and the east coast of Jutland between Randers Fjord and Horsens Fjord. In Sweden, all species were collected on the island of Asko, south of Stockholm. The localities are shown in Fig. 1 , and the sampling dates are shown in Table 1. Most animals were collected by sweeping a net or a sieve through shallow water vegetation, under stones and in mussel beds, or by shaking algae in a bowl. The only exception to this was G.locusta on Ask& which was collected from algae dredged with a net in about five meters depth. The animals were brought live to the laboratory, sorted, and stored frozen at -70°C until eletrophoretic analysis was performed.
Electrophoresis The animals were homogenized in 50 pl Tris/ maleate buffer (gel buffer used for the analysis of PGM in HARRIS and HOPKINSON (1976). Small animals were homogenized whole, while larger animals were halved. The homogenate was centrifuged at 18,000 rpm for 1.5 min. Paper wicks were dipped into the supernatant and inserted into horizontal starch gels (12% Connaught). Five different buffer systems were used for electrophoresis (numbered from 1 to 5 in Table 2): (1) Tris citrate buffer Bridge: 0.225 M Tris, 0.071 M citric acid, pH7.0.
Fig. 1. Sampling localities, see Table 1 .
Gel: 1/25 dilution of the bridge buffer (NIELSEN and CHAPMAN 1977). (2) Tris citrate buffer Bridge: 0.687 M Tris, 0.143 M citric acid, pH8.0. Gel: 0.023 M Tris, 0.005 M citric acid, pH8.0 (SELANDER et al. 1971). ( 3 ) Amine citrate buffer Bridge: 0.04 M citric acid adjusted to pH 6.1 with 3-Morpholinopropylamine-(1). Gel: 0.002 M citric
Table I . Sampling localities, date of collection and numbering of populations Lirnfjord
EasternJutland
Ask0
G.duebeni
Virksund (2) 22.1V.1981
Kale (3) 16.1.1981
Skutsviken(4) 4.XI.1980
G. zaddachi
0sterild Fjord ( 5 ) 26.X.1981
Randers Fjord (6) 18.X.1981
Skutsviken(7) 7.XI. 1980
Gsalinus
Lendrup(8) 20.1.1981
Kysing Fjord (9) 9.1.1981
Skutsviken(10) 7.XI.1980
G. oceanicw
Feggesund (1 1) 22.1V.1981
Fornzs (12) 14.11.1981
Skutsviken(13) 5 .XI.1980
G. tocusra
outside0sterildFjord (14) 26.X.1981
Vosncs (15) 18.VII.1981
Bjorkholmen (16) 8.XI. 1980
C.marinus
Fornm (1) 4.1V.1981
Herediras 102 (1985)
GENETICSTUDIESOFGAMMARUS. I
acid adjusted to pH 6.1 with 3-Morpholinopropylamine-(1). (CLAYTON and TRETIAK 1972).
3
Results
Staining for the 15 enzymes revealed at least 20 loci with 19 scorable in at least five of the six species. Bridge: 0.3 M boric acid, 0.06 M sodium hydroxide, The allele frequencies at polymorphic loci are given pH 8.2. Gel: 0.076 M Tris, 0.005 M citric acid, pH in Table 4, while the mobility of monomorphic loci 8.7 ( S E L A N D Eal.R 1971). ~~ are given in Table 3. The mobility values are given relative to those of Chaetogammarus marinus which ( 5 ) Tris maleate buffer Bridge: 0.1 M Tris, 0.1 M maleic anhydride adjust- have been defined as 100. Four enzyme stains revealed two loci in all speed to pH 7.2 with 10 N sodium hydroxide. Gel: 1/10 dilution of bridge buffer (HARRIS and HOPKINSONcies: ADA, GOT, MDH, and ME. Among these, the locus that codes for the enzyme with the highest 1976). anodal mobility has been numbered I. The only The gels were run at a current density of 100 mA cathodically moving enzyme was GotZl. Gammarus for 3 h (PGM, PEP, and APK) or 2.5 h (the rest). duebeni had, as the only species, two Alp loci, and After electrophoresis, the gels were sliced into 2 or the fastest moving system was considered homo3 layers and stained. In total, 15staining procedures logous with the systems found in the other species. were employed (see Table 2 for the enzymes studied The second Alp locus in G.duebeni was monomorphic and is not listed in Table 3. In staining for and for references to the staining procedures). The species were compared by the standard gene- DIA a strong activity band and a second weakly tic distances among all populations studied (NEI stained band with a lower mobility appeared in all 1972). Two criteria were used to decide whether a species. Only the fast moving band was considered. locus is polymorhic: (1) the frequency of the most The staining procedure for unspecific esterases common allele was smaller than 0.99; (2) the fre- revealed several bands, but only the fastest moving quency of the most common allelle was smaller than densely stained band was considered. Five polymorphic loci, Apk, Dia, Mpi, Pep, and 0.95. (4) Tris citrate buffer
Table 2. Enzymes studied and buffer systems employed Enzyme
Abbreviation
E.C.No.
Buffer system
Reference to stainingprocedure
Acid phosphatase Adenosine deaminase Alkaline phosphatase Argininephosphatekinase Diaphorase Esterase Glutamic-oxaloacetictransaminase Glucosephosphate isomerase Glutamic-pyruvictransaminase Leucine aminopeptidase Malate dehydrogenase Malic enzyme Mannosephosphateisomerase Peptidase Phosphoglucomutase
ACP ADA ALP APK DIA ES
3.1.3.2 3.5.4.4 3.1.3.1 2.7.3.3 1.6.2.2 3.1.1.1 2.6.1.1 5.3.1.11 2.6.1.2 3.4.1.1 1.1.1.37 1.1.1.40 5.3.1.8 3.4.11/13 2.7.5.1
2 5 4 1 4 2 3 1 1 5 3 2 5 1 1
HARRIS and HOPKINSON (1976) S P E N C E R1968 ~~~I. HARRIS and HOPKINSON (1976) BULNHEIN and SCHOLL(1980) HARRIS and HOPKINSON (1976) SIMONSEN and FRYDENBERG (1972) SCHWARTZeta!. 1963 YNDCAARD (1972) HARRIsand HOPKINSON(1976) SHAW and pRASAD(1970) and SIMONSEN (1973) FRYD~NBERG F R Y ~ ~ N Band E RSIMONSEN G (1973) HARLISand HOPKINSON (1976) SHAWand PRASAD (1970) HARRIS and HOPKINSON (1976)
GOT GPI GPT LAP MDH ME MPI PEP PGM
Table 3. Mobility of alleles in the loci that are monomorphic in all species
Adal Adall A lP Es Lap Mel Mell
C.marinus
G.duebeni
G. zaddachi
G.salinus
G.oceanicus
G.locuta
100 100 100 100 100 100 100
100 100 97 106 95 104 100
100 100 97 104 102 128 121
100 100 97 104 102 128 107
100 100 100 106 102 108 107
100 100 94 113 95 112 121
4
Hewdituc. 102 (198s)
H R SIEGISMUNDETAL.
Pgm, showed two bands in heterozygous individuals and are considered as monomeric enzymes. In seven loci, Acp, GotZ, GotZZ, Gpi, Gpt, MdhZ, and MdhZI, heterozygous individuals had three bands and these enzymes are considered as dimeric enzymes. The remaining loci were monomorphic in all species. In Table 4 the expected heterozygosity under as-
sumption of Hardy-Weinberg proportions has been calculated for every locus and as a mean over loci. Two loci were unscorable in two of the species. The Pep locus could not be scored in G.salinus because of difficulties in interpreting the bands. The Pgm locus could not be scored in G.locusta, as the stain was always very weak (the animals used were small).
Table 4. Allele frequencies in the polymorphic loci. See Table 1 for reference to population number. H is the heterozygosity and N is the sample size. Significant deviations from Hardy-Weinberg proportions are indicated with an asterisk
Locus
Population number
Allele
1
ACP 51
2
3
4
0 0 0 0 0.768 0.232
0 0 0 0 0.973 0.027
5
6
7
8
10
12
13
0
0
0 0 1.O0O 0
0
0 0 1.OOO 0 0
0
0 0 0 1.O00 1.000 0 0 0 0 0
0
0 0 0 0 1.000 0 0 0 0 0 0 1.000
11
14
15
16
0 0
0
0 0 0
0 0
223
0 0 1.0000 0 0 0 0 0 0.750 0 0.250
H
0.000
0.37s 0.357 0.053 0.000 0.000
0.000
0.000
0.000
0 . m 0.000
n.ooo
0.000 0.000 0 . ~ 0 0 0.000
N
SO
46
47
50
49
97
49
97
44
97
49
50
0 0 0.032 0.968
0
0
0
0 0 0 1.000
0 0
0
0
0
100
111 133 155
99
92
l.O001.O0Ol.OOOO 0 0 0 0 0 0 0 0 0 0 1.O00 0 0 0 0 0 0 0 0
9
0
49
50
1.OOO
0
0 1.OOO
APk
97 100 120 140
l.O001.O001.O000 1.0000 0 0 0 0 0 0 0 0.181 0 0 0 0 0.819
H
0.OOO 0.000 0.O0O 0.O00 0.296 0.062 0.031 0.O0O 0.000
N
32
Dia 83
91 93 100
0
49
18
21
94
0.020 0 0 0 0.980 1.O00 1.O00 0 o o o o 1.000 I.OO0 0 0 0 0
0 0
0 0 0.016 0 0.985 1.O00 0
78
97
0
0 0
0
1.000 0
49
0 0 0 0 0 0 1.O00 1.000 1.OOO
0
50
O.OO0 0.O0O 0.000 0.000 0.000 0.000 0.000
50
50
49
0 0.022 0 0 0 0 1.000 1.000 1.000 1.000 0.978 1.000 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 1.O00 1.OOO 1 . N 1.OOO
0
0
SO
45
49
47
0 0
0 0
0
0 0
0
1.000
o
0
1.000 1.000 1.000
n
o
H
0.000 0.039 0.O00 0.OOO 0.000 0.O00 O.OO0 0.OOO 0.000 0.000 0.043 0.O0O 0.OOO 0.000 0.000 O.Oo0
N
50
Gor I
98
100
33
49
50
50
48
50
30
45
50
50
25
48
50
65 83 100 I10
o o o o o o o o o o o o o 0.014 0.011 0 0.911 0.254 0.473 0.010 0.010 0 0.050 0.214 0.025 0 0 0 0.986 0.989 1.O0O 0.075 0 0 0.985 0.990 0.995 0.950 0.786 0.975 1.O0O 0.995 1.ooO 0 0 0 0.014 0.746 0.527 0.005 0 0 0 0.005 0 0 0.005 0 0 0
H
0.000
0.164 0.379 0.498 0.030 0.020 0.010 0.096 0.337 0.049 0.000 0.010 0.000 0.027 0.021
o.ozo
N
45
73
100
continued next page
197
91
98
100
98
99
98
*
100
50
97
49
74
48
0.010 0.990 0 0
Hereditas I02 (1985)
GF.NET1CSTUDlESOFGAMMARUS I
Locus
Population number
Allele
1
2
3
4
0
0
0 0
63 69 loo 108 112
0 0 0 0 1.ooo 0 0
0 0 0 0 0 0 1.OOO
0
9
H
0.000 0.M)O 0.010 0.000 0.010 0.000 0.020 0.Ooo 0.039 0.000 0.020 0.021 O.OO0
N
50
99
63 88 92 100 106 117 124 129 136 147 150
0.070 0 0.010 0.920 0 0 0 0 0 0 0
0 0
H
0.149 0.OOO 0.010 O.Oo0
N
50
48
200
46
0 0
0 0 0 0
0 0 0 0
0
5
6
7
8
9
10
11
0 0
0
0 0 0
12
13
14
15
16
0.0110 0 0 0 0 0 0 0 0.990 0.989 1.000 0 0 0
0 0 1.Ooo 0 0 0
0
0 0 1,000
0 0 0.0100
0 0
0 0
Got I1
55
0
0 0 0.0050 0 0 0 0 0 0 0.0050 0 0 0.995 1.000 0 0 0 0 0 0.995 1.OOO 0 0 0 0 0 197
0
0 0 0 0 0 0.990 1.000 0.010 0 0 0 0 0 0 0 0
0 0 0 0 0 0.980 1.000 0.020 0
0 0
0 0
GPt 37 45 73 84 100 117 132 167 182 236 280 324
0
0
0 0 0 0
0.000 O.Oo0
0.000
46
98
98
98
99
100
66
50
95
49
73
40
89
0 0 0 0 0 1.000 0
0 0 0 0.015 0 0.076 0.010 0.849 0 0 0.050
0 0 0 0 0 0.035 0 0.965 0 0 0
0 0 0
0 0 0
0 0 0
o
u
o
0
0.005 0 0.090 0 0.900 0.005 0
0.010 0 0 0.990 0 0 0 0 0 0
0.066
0 0 0.031 0 0.949 0.020 0 0
0 0 0 0
o
0 0 0 0 0.933 0 0 0.067
0 0 0 0 0 0.119 0 0.814 0.067 0 0
Gpi
0 0 0 0.005 0 0 0 0 1.OOO 0.995 0 0 0 0 0 0 0 0 0 0
0 0
0 1.000 0 0
0 0 0 0
0.021 0 0 0.979 0 0 0 0 0 0 0.605 0.552 0.479 0 0.030 0 0 0 0 0 0 0
0 0 0 0 0 0 0.365 0.448 0.521 0 0 0
0 0 0.924 0 0.010
0 0 0
0
0.271 0.068 0.125 0.098 0.182 0.319 0.500 0.495 0.499 0.041 0.020 0.141 99
100
0 0 0
0 0
97
0 0 0 0 0 0 0 0.035 0.547 0.953 0.953 0 0 0 1.OOo 0 0 0 l.m 1.OOO 0.955 0.100 0 0 0 0 0 0 0 0.320 0.046 0.047 0 0 0 0 0.010 0 0 0 0 0 0.033 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0
0
0 0 0
49
100
97
0 0.011 0 0.280 0 0 0.677 0 0.032 0 0 0
0 0 0 0.239 0 0 0.727 0 0.034 0 0 0
0 0.006 0 0.133 0 0 0.791 0 0.070 0 0 0
100
96
0.021 0 0.191 0 0 0.5.53 0 0 0.234 0.010 0 0 0 0 0 0
0 0.149 0 0 0.536 0 0 0.304
97
48
49
99
0 0 0.163 0 0 0.435 0 0 0.402
0 0
0
0
0
0
0
0
0 0
0 0 0
0 0 0 0 0 0 0 0 0.013 0.01 1 0.980 0.977 0.007 0.011 0 0 0 0
0 0 0 0 0 0 0.979 0.021
H
0.000 0.000 o.Oo0 0.087 0.588 0.089 0.089 0.462 0.413 0.352 0.598 0.602 0.623 0.039 0.045 0.042
N
50
49
100
100
75
43
96
93
44
79
97
0 1.OOO 0 0 0
0.005 0 0.995 0
0.003 0 0.997 0
0 0 1.ooo 0
0.506 0 0.494 0
0 0
0 0
0 0
0
0.540 0 0.460 0 0 0
0.122 0 0.867 0 0.011 0
0.759 0 0.241 0 0 0
0.474 0 0.517 0 0.009 0
0.493 0 0.507 0 0 n
1.000 1.OOO 0 0 0 0 0 0 0 0 o 0
*
41
92
75
44
47
Mdh I
w 100 113 118 132 138
0
0
0 0 0 0.986 1.Ooo 0 0 0.014 0
1.003 0 0 0 0
0
0 0 0
0 0 0 0.994 0
0.006
H
0.000 0.011 0.006 O.Oo0 0.500 0.497 0.233 0.366 0.508 0.500 0.000 0.000 0.000 0.027 0.000 0.012
N
26
93
continued next page
176
46
84
25
94
83
57
73
50
45
10
37
48
81
5
6
Hereditas 102 (198s)
H . R. SIEGISMUNDET AL.
~~
~
~~
Locus
Population number
Allele
1
2
3
4
5
6
7
8
Mdh I1 33 47 100 170 178
0 0 l.W 0
0.994 0 0.006 0 0
0.997 0 0.003 0 0
l.W 0 0 0 0
0 0 0 1.OOO 0
0 0 0 1.OOO 0
0 0 0 1.ooO 0
0 0.0170 0 0 0.983 1.OOO 0 0
H
0.OOO 0.013 0.007 0.W 0.W 0.ooO 0.OOO 0.033 O.Oo0
N
20
Mpi 64 72 76 82 93 100 105 117 122 127 134 144 166
0
80
150
0 0 0.010 0 0 0 0 0 0 0 0 0 0.990 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.020 0 0 0.980 1.Ooo 0 0
0
0
39
49
0 0 0 0 0 0 0 0 0 0 0 0 1.W
0 0 0 0 0 0 0.005
23
0 0 0 0 0 0 0.005 0.184 0.385 0.015 0 0.561 0.595 0.250 0 0 0 0 0
9
0
~-
~~
10
11
12
13
14
15
16
0 0 0 1.OOO 0
0 0 0 l.W 0
0 0 0 1.OOO
0 0 0 1.OOO
0
0
0
0
0
0 0
0
0 0
0
0
0 0 1.000 1.OOO 1.000
0 . W 0.OOO 0 . W 0.OOO 0.OOO 0.000 0.000
66
30
43
45
46
42
10
9
15
8
0 0 0 0 0
0 0 0 0 0 0.010
0 0 0 0 0
0 0 0 0 0 0.020
0
0 0 0 0 0
0 0 0 0 0 0
0 0.015 0.153 0 0.826 0.005
0 0 0.031 0 0.969 0
0 0 0 0.465 0 0.535 0
0 0 0 0 0
0 0 0 0 0 0 0
0.005 0.010 0.144 0.005 0.830 0.005 0 0 0 0
0
0.016 0.193 0 0.787 0.005
0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.230 0.303 0.450 0.165 0.755 0.687 0.530 0.005 0.005 0.010 0 0.825 0 0 0 0.005
0 0 0 0 0.304 0 0.696 0
0
0
0
0 0
H
0.020 0.040 0.W 0.W 0.589 0.498 0.344 0.377 0.436 0.516 0.292 0.423 0.497 0.293 0.060 0.290
N
50
49 0
117 130
0 1.OOO 0 0
H
0.W 0.200 0.479 0.398 0 . W 0.OOO 0 . W
N
50
pep 63 100
100
0.020 0 0.887 0.633 0.112 0.347 0
40
49
46
98
0 0 l.W 0.726 0 0.274 0 0
42
100
96
0 1.Ooo 0 0
0 1.OOO 0 0
100
99
0
48
48
49
0
0.132 0 0.868 0 0
0.744 0 0.256 0 0
0.195 0 0.763 0.042 0
0 0.925 0 0
100
0
100
97
99
98
48
97
0 1.Ooo 0 0
0 1.W 0 0
0 l.W 0 0
0 1.ooO 0 0
0 l.W 0 0
0 1.OOO 0 0
0.OOO 0.OOO O.Oo0
0.OOO o.Oo0 o.oO0
50
50
49
50
0
0
0
47
40
t
e m
100 104 108 114 119
l.W 0 0 0 0
0.600 0.488 0.544 0 0 0 0 0 0 0.390 0.512 0.457 0.0100 0
H
O.Oo0 0.488 0.500 0.496 0.229 0.381 0.379 0.138 0.047 0.064 0.095 0.043 0.063
N
48
50
126
23
19
41
59
0.075 0.024 0.033 0
47
0 0.976 0 0 62
0 0.967 0 0
15
0 0.0140 0.951 0.978 0.035 0.022 0 0 71
45
0 0 0.968 0.032 0 31
Hmean 0.009 0.070 0.092 0.081 0.132 0.085 0.065 0.087 0.109 0.100 0.081 0.084 0.089 0.024 0.008 0.028
Genetic variation within species Chaetogammarus marinus The single population studied in this species has a low genetic variation. Of the 19 loci studied two are polymorphic according to criterion 1 (11%) and only the Gpi locus is polymorphic according to crite-
rion 2 (5%). The average heterozygosity per locus is 0.009.
Gammarus duebeni In G.duebeni 25% of the 20 loci are polymorphic according to criterion 1, and 20% according to crite-
Hereditas 102 (1985)
GENETIC STUDIES OF GAMMARUS I
rion 2. The average heterozygosity is 0.077 per locus. In the three populations studied the same loci are polymorphic according to criterion 2, viz. Acp, GotI, Pep, and Pgm. At three of the four polymorphic loci the populations show considerable differentiation. The Pgrn locus is the only locus where no significant difference is found among the populations. At the Acp locus the population from the Baltic Sea differs significantly from the other two populations, which have about the same allele frequencies. At the Gorllocus all three populations have significantly different allele frequencies. In the Limfjord, the allele 100 is found with a frequency of 0.075, but this allele is not found in the other two populations. The frequencies of the alleles that are common to all populations vary significantly. At the Pep locus the Limfjord population differs significantly from the other two populations, which show similar allele frequencies. Gammarus duebeni shows a rather high, but irregular, differentiation among the populations. There is no evidence of any clinal variation in the loci studied. The population from Arhus can differ more from the other two populations than they differ from each other (e.g., Pep). The genetic distance between the Limfjord and Arhus populations is higher than the genetic distance between the Limfjord and Asko populations, contrary to the geographical separation (Table 5). Gammarus zaddachi
In G.zaddachi 37% of the 19 loci are polymorhic according to criterion 1, and 23% according to crite-
7
rion 2. The average heterozygosity per locus is 0.094. The six loci, Apk, Gpi, Gpt, Mdhl, Mpi, and Pgrn, that are polymorphic according to criterion 2 in at least one of the populations, show a marked heterogeneity among the populations. At the Apk and Gpi loci the Limfjord population deviates significantly from the other two populations. At the Mdhl locus the Asko population differs significantly from the other two populations, which have about the same allele frequencies. At the Mpi locus all three populations differ significantly from each other, e.g., allele 134 is found at a frequency of 0.25 in the Limfjord population, while it is found at a frequency of 0.005 in the Ask0 population and not at all in the Arhus population. The latter two populations differ significantly from each other. The genetic distance between the Limfjord and the Asko populations, which are the populations farthest apart, is again the smallest (Table 5). Gammarus salinus
The G.salinus populations are on the average polymorphic at 37% of the 18 loci according to criterion 1 and at 28% according to criterion 2. The average heterozygosity per locus is 0.099. The population differentiation in G.salinus shows a more regular pattern than in G.duebeni and in G.zaddachi. At Gpi and Gpt the difference in allele frequencies among the Limfjord and Asko populations are in the order of 0.10 to 0.15, while the difference is 0.22 at the Mpi locus. The loci GotZ and Mdhl are polymorphic according to criterion 2 in at least one of the populations, and one of the populations differs from the other two in allele frequency.
Table 5. Genetic identities (above diagonal) and genetic distance (below diagonal) among populations. See Table 1 for reference to the population number
1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1.600 1.641 1.633 1.451 1.341 1.471 1.730 1.786 1.725 1.288 1.288 1.284 1.486 1.495 1.483
2
3
0.202
0.194 0.195 0.234 0.968 0.982 0.225 0.994 0.222 0.006 0.222 1.506 1.506 1.501 1.488 0.035 1.480 1.476 0.021 1.608 1.605 0.164 1.495 1.484 0.163 1.507 1.508 0.158 1.486 1.499 0.500 1.462 1.475 0.487 1.471 1.482 0.476 1.595 1.598 1.236 1.602 1.605 1.244 1.592 1.595 1.234
0.033 0.018 1.494 1.471 1.471 1.578 1.445 1.486 1.475 1.452 1.461 1.432 1.439 1.429
4
5
6
7
0.262 0.230 0.223 0.226 0.966
0.230 0.177 0.230 0.206 0.228 0.200 0.229 0.201 0.979 0.849 0.972 0.796 0.801 0.222 0.207 0.007 0.202 0.010 0.574 0.437 0.558 0.429 0.543 0.422 1.248 1.661 1.256 1.670 1.246 1.659
0.028 0.229 0.236 0.231 0.573 0.561 0.547 1.238 1.246 1.236
8
9
1 0 1 1 1 2 1 3 1 4 1 5 1 6
0.168 0.236 0.224 0.227 0.850 0.790 0.813 0.993
0.178 0.226 0.222 0.221 0.854 0.793 0.817 0.991 0.995
0.005 0.461 0.451 0.443 1.594 1.602 1.591
0.438 0.427 0.415 1.660 1.668 1.657
0.276 0.229 0.226 0.223 0.606 0.564 0.563
0.276 0.234 0.232 0.229 0.614 0.571 0.572 0.646 0.651 0.631 0.637 0.645 0.653 0.998 0.002 0.007 0.003 1.450 1.447 1.458 1.455 1.448 1.445
0.277 0.232 0.230 0.227 0.622 0.579 0.581 0.656 0.642 0.660 0.993 0.997
0.226 0.239 0.203 0.202 0.291 0.290 0.287 0.190
0.203 0.190 0.235 0.235 0.236
1.445 1.453 0.001 1.443 O.OO0
0.224 0.237 0.201 0.201 0.288 0.288 0.285 0.188 0.201 0.189 0.233 0.233 0.234 0.999 0.001
0.227 0.240 0.203 0.203 0.291 0.291 0.288 0.190 0.204 0.191 0.235 0.236 0.236 LOO0 0.999
8
Hereditas 102 (1985)
H R . SltGlSMClND ET A L
Genetic Identity
The more regular pattern found in this species is reflected in the genetic distances among the populations. The populations farthest apart have the largest genetic distance, and the average genetic distance among the three populations is 0.007 (Table 6).
0,’
0,2
0,3
0,L
05
06
07
08
I I
0,9
1 G zoddochi G salmus
G ocemicus
Gammarus oceanicus
C marinus
In G.oceanicus 26% of 19 loci are polymorphic according to criterion 1, and 16% according to criterion 2. The average heterozygosity per locus is 0.085. As in G.salinus, the population differentiation in G .oceanicus is more regular. It shows a marked difference between the Kattegat and the Baltic Sea populations. Like in G.salinus, the more regular population structure is also reflected in the lowest genetic distances among the adjacent populations (Table 5).
G locusla
G duebeni
Rg. 2. UPGMA dendrogram based on the genetic distances for the six species.
marus duebeni and G.zaddachi have the largest differentiation among local populations, while the divergence in the other three Gammarus species is smaller. With respect to within species differentiation of geographically separated populations, the species can be ranked G.locusta < G.oceanicus < G.salinus < G.duebeni < G.zaddachi. The average Gammarus locusta of the genetic distances is shown in Table 6. The G.duebeni and G.zaddachi populations from Gammarus locusta is the Gammarus species with the lowest level of genetic variation. Of the 18 loci the Limfjord seem to be more closely related to the scored 24% are polymorphic according to criterion other species than their conspecific populations 1, and 6% according to criterion 2. The average from Arhus and Ask0 are (Table 5 ) , whereas the remaining three Gammarus species show rather hoheterozygosity is 0.02. Because of the low genetic variation no clear mogeneous genetic distances to the other species. geographic pattern emerges. At the Gpi locus the Table 6 provides the standard genetic distance for Baltic population differs from the other two popu- all pairwise combinations of the six species as the lations, while at the Mpi locus the population from average distance of all populations in the species the Arhus area differs from the other populations. (for the Gammarus species: the arithmetic mean of The low genetic variation found is also reflected nine distances; for the Chaetogammarus-Gammain the low genetic distances among the populations rus comparisons: the average of three distances). (Table 5 ) and a low average genetic distance among Additionally, the mean genetic distance among the populations within species is given in the diagonal. the populations (Table 6). From these genetic distances a dendrogram has been constructed (Fig. 2) using the UPGMA metComparison of species and SOKAL 1973). hod (SNEATH In Table 5 the standard genetic distances and idenThe most closely related among the six species are tities (NEI1972) have been computed for all pairwise those within the Gammarus zaddachi group combinations of the 16 populations studied. Gam- (G.zaddachi, G. salinus, and G.oceanicus).Within
Table 6. Mean genetic distances among species and populations (arithmetic means over populations) C.marinus C.marmus
-
G.duebeni G.zaddachi G.salinus G.oceanrcus G.locustu
1.625 1.421 1.747 1.287 1.488
C.duebeni
G .raddachi
G.salinus
G. oceanicus
G.locus1u
0.019 1.488 1.524 1.474 1.543
0.028 0.201 0.536 1.242
0.007 0.436 1.640
0 004 1.449
0.001
Herrditas 102 (1985)
GENETICSTUDIES O F G A M M A R U J . I
9
Table 7. Diagnostic loci among species. Numbers in parentheses are the number of loci where the species have alleles in common but where the probabilityof not placing an individual in the correct species is less than 0 . 0 1 ~~
C marrnw
G duebenr
this group G.zaddachi and G.salinus are the closest, with a genetic distance of 0.201 and roughly the same distance to G.oceanicus. The remaining three species have about equal genetic distance (about 1.5) among each other and to the G.zaddachi group, giving four equidistant groups: (1) C.marinus, (2) G.duebeni, (3) C.locusta, (4) G.zaddachi group. There is no indication of a larger genetic distance between the single species from the Chaetogammarus genus and the Gammarus species than among the less related species within the Gammarus genus. The relationship of the six species can also be measured by counting the number of diagnostic loci. Table 7 provides two sets of numbers for pairwise comparisons of the species. The first number is the number of loci, where no common allele has been found, and the second number is the number of loci, where the probability of not allocating an individual to the right species is less than 0.001. In any comparison of two species, at least two diagnostic loci exist. This, in addition to the differentiation in loci where the species have alleles in common, clearly, demonstrates the genetic distinctness of the six species studied, and it contradicts the view of RASMUSSEN (1973) that the five Gammarus species are forms of a single species. The picture emerging from the comparison of the number of diagnostic loci (Table 7) generally agrees with the patterns of genetic distances (Table 6 ) . A minor change is that Chaetogammarus marinus has more diagnostic loci (14) than most comparisons within the Gammarus group. In the Gammarus genus, G.locusta has more diagnostic loci (13-14) than any other species (1112 diagnostic loci). The species of the G.zaddachi group are closely related, again with G.zaddachi and G.salinus closest together.
G zaddachr
G salrnus
G oceanicus
crustaceans, (average heterozygosity per locus for the six species is 6.5%, compared to 5 4 % in a study on 44 species of decapods by NELSON and HEDGECOCK (1980)). The variation is lowest in Cmarinus and G.locusta, which live in the upper end of salinity gradients in Danish waters. Chaetogammarus marinus is found intertidally on beaches with pebbles and stones while G .locusta usually is found subtidally. Due to the special habitat demand of Cmarinus, its distribution in Denmark is fairly restricted. It was found for the first time in 1979 on the peninsula of Djursland by FORCHHAMMER (1980). The low genetic variation might thus reflect a founder effect from a recent introduction into this area. However, a comparison with G.locusta suggests that the level of variation found in C.marinus is not exceptionally low. Rather it falls within the range of variation of widely distributed Gammarus species. A comparison of the differentiation of the populations of the five Gammarus species (Table 6) suggests that G.duebeni and G.zaddachi have the most differentiated populations, as expected from their distribution pattern. In Denmark, G.duebeni is confined to habitats like the mouth of rivers, lagoons, the innermost parts of estuaries and brooks running over beaches. In the Baltic it is mainly and KOLDINC 1979; found in rockpools (FENCHEL KOLDING 1981). Habitats like these are usually isolated in occurrence and have a small area. The population sizes will probably be low in the isolated populations, which through random genetic drift could cause differentiation among the populations. Additionally, the migration rate of Gammarus species is probably limited, as the female broods her eggs in a marsupium, where they hatch in a juvenile stage before they are released. Marine animals with a pelagic larval stage have a higher migration potential than animals with a direct development, and they are expected to show Discussion less differentiation among populations. In a study on enzyme polymorphisms in the gastropod genus Variation within species Littorina, BERCER (1973, 1977) found that species The level of genetic variation found in the six with direct development showed a higher differenspecies is comparable to the variation found in other tiation than species with a pelagic stage. A free
10
H. R. SlEGlSMUND ET AL.
swimming stage during the life cycle is not sufficient to prevent diffefentiation among populations. BURTON and FELDMAN (1981) studied the genetic variation at five polymorphic loci in Tigriopus californicus, a harpacticoid copepod living in rockpools along the Californian coast. This species showed a marked differentiation among populations from rockpools separated by sandy beaches, while populations on a single rocky outcrop tended to be genetically more homogeneous. This population structure, where the populations are differentiated despite the fact that animals from time to time are washed into the sea where they can swim, was interpreted by BURTON and FELDMAN (1981) as the effect of a low migration rate. In a study of three polymorphic loci in the freshwater species Gammarus minus in a karst area in the USA, GOOCH and HETRICK (1979) reported a high differentiation among populations. The differentiation found in G.duebeni and the G.zaddachi is not as pronounced as the differentiation described for Tigriopus californicus or Gammarus minus, implying that the migration potential is fairly high in the marine Gammarus species compared with T.ca1ifornicu.sand G.minus. The geographic pattern of the genetic variation in Gammarus duebeni seems to be quite irregular. Further studies would be necessary to give a better picture of the population structure. BULNHEIM and sCHOLL(1981a) have studied two of the polymorphic loci (Apk, Gpi) in G.zaddachi in populations ranging from Brittany to the Baltic Sea and to Tromso in Norway. Based on vertical starch electrophoresis, they found that the frequency of an allele they call Apk 100 (which most likely is Apk 140 in this study) has a high frequency (0.96) in Brittanny, a low frequency (0.04) in northern France, a clinal increase to a frequency of 0.7 western Jutland, and high frequencies (from 0.85 to 1)in the western Baltic and the Baltic proper. The gene frequencies found in the present study fit quite welt those found by BULNHEIM and SCHOLL (1981a). The Gpi locus has a geographic variation similar to the Apk locus (BULNHEIM and SCHOLL 1981a). It has a high frequency of allele 100 in Brittany (this allele is most likely the allele 129in this study) which decreases to 0.7 in the southern part of the North Sea and increases to 0.88 in western Jutland. In the Baltic the frequency is 0.90-0.95. The frequencies of this allele in the present study are well in agreement with BULNHEIM and SCHOLL (1981a). The geographic variation at the remaining three polymorphic loci in G.zaddachi does not seem to have the regular pattern found at the Gpi and Mpi loci, but since only three populations were studied
Hereditus 102 (198.5)
this conclusion might be premature. BULNHEIM and SCHOLL (1981a) conclude that the variation at the Apk and the Gpi loci is heterogeneous, and interpret this as an effect of the relatively low dispersal rate among the isolated populations. The geographic variation at the five polymorphic loci (Gotl, Gpi, Gpt, MdhZ, M p i ) in the three Gammarus salinus populations has a more regular pattern than in G.duebeni and G.zaddachi. Two of the polymorphic loci (Gotl, Gpi) have been studied by BULNHEIM and SCHOLL (1981a) in 18 populations in Europe ranging from the Atlantic coast of France to the Baltic Sea. The more regular pattern might be explained as a result of higher migration rate in this species, since it is found mostly at higher salinities (8-15 %o, FENCHEL and KOLDING 1979; KOLDING 1981). BULNHEIM and SCHOLL (1981a) found that the frequency of their Got 100 allele (most likely allele Gotl 100 of this study) has a high frequency (0.95) in France, at the North Sea coast of the FRG, and in the Baltic proper. At the east coast of Jutland, the frequency is lower and reaches a frequency of 0.69 in southern Jutland from where it increases to a frequency of 1 in the GDR. The frequencies of the Got Z 100 allele found in this study are in accordance with BULNHEIM and SCHOLL'S (1981a) findings. The clinal pattern at the Gpi locus, as suggested in this study, agrees with the results of BULNHEIM and SCHOLL (1981a). The frequency of their allele Pgi 100 (probably allele Gpi 129 in this study) decreases from 0.9 in France to 0.7 in Finland. The remaining three polymorphic loci (Gpt, Mdhl, Mpi) show a pattern like that of the Gpi locus, so the overall pattern of geographic variation of G.salinus is more regular than that of G.duebeni and G.zaddachi. The geographic variation in G.oceanicus is even more regular. This may be explained as the result of a still higher migration potential, as this species is mostly found in more marine habitats, and therefore the populations of this species are not as isolated as the populations of the species that are found at the inner parts of the fjords. The very low degree of genetic variation found in G.locusta does not enable a comprehensible description of the population structure in this species. Excluding Gammarus locusta, the Baltic populations of the Gammarus species are differentiated from the populations in the Kattegat. The differentiation may be irregular, as is found in all species, or the pattern may be clinal. The geological history of the Baltic makes it possible to place an upper bound on the number of generations since the establishment of the Baltic populations. The Baltic Icelake was formed during the withdrawal of the ice
Hereditas 102 ( I 985)
GENETICSTUDIESOFGAMMARUS. I
11
cover after the last glaciation. It was transformed ferentiation between the Baltic and the Kattegat, into the Yoldia Sea in 8000 B.C., and this sea was suggesting that the populations are partially isolacut off from the Kattegat in 7250 B.C. and changed ted. The differentiation observed may have been acinto the freshwater Ancylus lake. In 5100 B.C. the cumulated over 2,000 to 20,000 generations, deconnection to the Kattegat was reestablished and pending on the generation time and on the time of the Ancylus lake changed into the Littorina Sea, colonisation of the Baltic. The only exception is which has existed since that time (MAGAARD and Gammarus locusta, where a very low genetic variRHEINHEIMER ation has been found. This is most regrettable, since 1974). The Gammarus-species that are able to tolerate ecological investigations suggest that the Baltic and the lowest salinities, G.duebeni and G.zaddachi, the Kattegat populations have diverged significantand KOLDING (1979) note that G.locusta have probably been in the Baltic for the longest ly. FENCHEL time. They may either have migrated into the Litto- from the Limfjord shows an increased mortality at rina Sea, or at least G. duebeni may have been in the low salinities, which makes it difficult to to keep aniBaltic during the Ancylus period, since this species mals from these two areas at the same salinity, so it is able to live in freshwater. EKMAN (1967) gives se- is not possible to test reproductive isolation. The differentiation of Baltic and Kattegat popuveral examples of arctic animals that have a relic distribution in the Baltic Sea. The euryhaline arctic lations of the different species may either have been species probably did live in the Baltic during the An- promoted through random genetic drift in isolated cylus lake period, so it may be possible that some populations andtor through the action of natural seof the Gammarus species have been in the Baltic lection. The geographic description of the genetic from the Yoldia time, i.e. for 10,000years, or 10,OOO variation does not by itself provide sufficient inforor 20,000 generations, depending on the number of mation to distinguish between these two possibiliand FRYDENBERG generations per year, (the summer breeding spe- ties. (CHRISTIANSEN 1974). This procies, G.zaddachi, G.salinus, and G.locusta, have blem can probably only be solved by a direct estimtwo generations per year, whereas the winterbree- ation of the possible selective forces acting on the polymorphisms using more direct methods (CHRISTIders have one generation per year (KOLDING 1981)). The genetic differentiations among the Baltic and ANSEN and FRYDENBERG 1973; CHRISTIANSEN et al. the Kattegat populations of the Gammarus species 1973, 1977). are of a similar or smaller magnitude than the differentiations found in other species. In the blues mussel, Mytilus edulis, three polymorphic loci showed Comparison of species a significant differentiation among the Baltic and the Kattegat populations (THEISEN 1978) The Pgi The present work supports the phylogenetic reand the Pgm loci have a frequency of “Baltic” alleles lationship of the five Gammarus species concluded of about 0.95 in the Baltic and (M.15 in the Katte- from investigations of the Limfjord populations gat. At the Lap locus the frequencies are 0.57 and (KOLDING and SIMONSEN 1983). The methods em0.03, respectively. A similar differentiation of Bal- ployed in this study differ from the methods used and SIMONSEN (1983). They group the fitic and Kattegat populations has also been found in by KOLDING the cod, Gadus morrhua. SCHMIDT (1930) was able ve Gammarus species into three groups: (1) G.10to distinguish Baltic and Kattegat races on the basis custa, (2) G.duebeni, (3) G.zaddachi, G.salinus, of morphological characters, and SICK(1965) in a and G.oceanicus, where the species in the third study of a haemoglobin locus found gene frequen- group have about the same genetic distance to each cies of the HbI-1 allele to be 0.61 in Kattegat and other. With the present methods the species G.saliwestern Baltic populations, and 0.03 in populations nus and G.zaddachi are more closely related than from the eastern Baltic. The central Baltic popula- either of these two species to G-oceanicus. tions showed highly variable gene frequencies and The genetic distances among populations may be and other signs of a mixing of isolated Baltic and Katte- compared with recent results of BULNHEIM (1981b). They compared the mobility of the gat populations. A more regular pattern has been SCHOLL found in the eelpout Zoarces viviparus with two pa- most frequent alleles of 10 gammarid species at serallel clines for the loci Hb I and ESTIIZ (HJORTH ven enzyme loci by means of enzyme electrophoreand SIMONSEN 1975). There is no indication of isola- sis. They did not calculate genetic distances, but tion, such as has been observed in the cod and the they observed a general relationship among the speblue mussel. cies which is in agreement with the present results. Of the eight mentioned species seven show a dif- In particular, their results concernilig the five loci
12
H . R . SIEGISMUNI) E T A L .
Herediros 102 (IY8.7)
which have been included in the present study agree the species pairs, there is some suggestion that the Chaetogammarus genus is more separated from the fairly well with our results. The differentiation among the species are within Gammarus genus than the species within the Gamthe ranges that have been reported previously for marus genus are among each other. marine invertebrates (AYALA 1975; HEDGECOCK et The relationships among the three Gammarus al. 1977; AYALA and VALENTINE 1979; MULLEY and species studied by HOLMES (1975) are in accordance LATTER 1980). The distance among the sibling spe- with the present study. Holmes included only one cies G.zaddachi and G.salinus is lower than the dis- species of the G.zaddachi group, but the close relatances that have been reported for other species tionship of the species in this group as found in this (AYALA 1975), while the genetic distances among study and by GOLIKOV and TZVETKOVA (1972) would more distantly related species are in agreement with probably have shown the same close relationship if other findings (MuLLEYand LATTER1980;AYALA and studied by the numerical taxonomic methods emVALENTINE 1979). One exception is HmGEcocKet al. ployed by Holmes. The suggestion by Golikov and (1977), who reported a genetic distance of 0.11 for Tzvetkova that G.zaddachi should be more closely the morphologically distinct species of the Euro- related to G.locusta than to G.duebeni is not suppean and the American species of lobsters (Homa- ported by the present study nor by the study of Holrus gammarus and H . americanus). These allopatric mes. species give viable offspring when they are mated in the laboratory. None of the mating experiments Acknowledgements. -Comments on the manuscript by Drs. F.B. among the Gammarus species carried out by KINNE Christiansen, H.-P. Bulnheim, A.G. Clark, and V. Loeschcke, and the technical assistance by Mrs. K. Petersen are gratefully (1954) gave any offspring. The genetic distances among the Gammarus spe- acknowledged. This study was supported by grant number 11-1713 from the Danish Natural Science Research Council. cies is at variance with previous studies on the taxonomy of the members of the family Gammaridae. The first attempt to reconstruct the phylogenetic reLiterature cited lationship was done by GOLIKOV and TZVETKOVA (1972) on the basis of paleoecological and biogeo- AYALA.F. J. 1975. Genetic differentiation during the speciation PIOC~SS. - Eva[. B i d . 8: 1-78 graphical methods. They conclude that the Chaeto- AYALA. J . W. 1979. Genetic variation in the F. J. and VALENTINE. gammarus group and the Gammarus group split in pelagic environment: a paradox? - Ecology 60 24-29 the lower Paleocene. In the mid Paleocene a species BERGER,E. M. 1973. Gene-enzyme variation in three sympatric species of Lirtorina. - B i d . Bull. 145: 83-90 that later evolved into the present G.duebeni speE. M. 1977. Gene-enzyme variation in three sympatric cies split off the Gammarus line. The line split in BERGER, species of Lirtorina. 11. The Roscoff population with a note on the lower Pliocene into species that evolved into the the origin of North American L.Zt?rorea.- Biol. Bull. 153: 255present G.locusta and a species that in the upper 264 H . P. and SCHOLL. A. 1980. Evidence of genetic diverPliocene split into G. oceanicus, G.salinus, and BULNHEIM. gence between two brackish-water gammaridean sibling species. G .zaddachi plus three other Gammarus species. -Mar. Ecol. Prog. Ser. 3: 165165 GOLIKOV and TZVETKOVA (1972) note that G.salinus BULNHEIM,H. P. and SCHOLL.A. 1981a. Geneticvariation between may have originated first. The opinion that the geographic populations of the amphipods Gammarus zaddachi and G.salinus. -Mar. Biol. 64: 105-115 Chaetogammarus species constitute a group that A. 1981b. Electrophoretic approach H. P. and SCHOLL. should be put into a separate genus is supported by BULNHEIM. to the biochemical systematics of gammarids. - Helgoliinder HOLMES (1975). He made a comparison of seven difMeeresunfers. 34: 391-400 ferent numerical taxonomic methods on 12 species BURTON,R.S . and FELDMAN.M. W . 1981. Population genetics of Tigriopus californicus. 11. Differentiation among neighboring in the genera Gammarus and Chaetogammarus, inpopulations. - Evolufion 35: 1192-1205 cluding G. duebeni, G.zaddachi, G. locusta, and CHRISTIANSEN, F. B. and FRYDENBERG. 0. 1973. Selection compoCmarinus. With regard to the three Gammarus nent analysis of natural polymorphisms using population samples including mother-offspring combinations. - Theor. fop. species, Holmes states that they are about equally Biol. 4: 425-445 related to each other. 0. 1974. Geographical patF. B. and FRYDENBERG, In the present study the Chaetogammarus is not CHRISTIANSEN, terns of four polymorphisms in Zoarces viviparus as evidence of differentiated more from the Gammarus species selection. - Genefics 77: 765-770 B., FRYDENBERG, 0. and SIMGNSEN. V. 1973. Gethan some of the Gammarus species are diffe- CHRISTIANSEN.F. netics of Zoarces populations IV. Selection component analysis rentiated among each other (e.g., G.locusta and of an esterase polymorphism using population samples including G.duebeni versus the other Gammarus species) mother-offspring combinations. - Hereditas 73: 291-304 when compared by genetic distances. When the CHRISTIANSEN. 0.and SIMONSEN. V. 1977. GeF. B., FRYDENBERG. netics of Zoarces populations X. Selection component analysis numbers of diagnostic loci are counted among
Herditas 102 ( I 98S} of the Estlll polymorphism using samples of successive cohorts.
- Hereditas 87: 129-150 J. W. and TRETIAK. D. N. 1972. Amine-citrate buffers CLAYTON, for pH control in starch gel electrophoresis. - J . Fish. Res. Bd. Canada 29: 1169-1172 C. 1964. The amphipods of the deltaic region of the DENHARTOG, rivers Rhine, Meuse and Scheldt in relation to the hydrography of the region. Part 111. The Gammaridae. - Neth. J . Sea Res. 2 : 407457 EKMAN. S. 1967. Zoogeography of the Sea. - Sidgewick and Jackson, London FENCHEL, T. and KOLDING. S. 1979. Habitat selection and distribution pattern of five species of the amphipod genus Gummarus. - Oikos 33: 31&322 FOROHHAMMER, K. 1980. Marinogammarus (Schellenberg) (Crustacea. Amphipoda) in Denmark. (in Danish). -Flora og Fauna 86: 27-32 0. and SIMONSEN. V. 1973. Genetics of Zoarces poFRYDENBERG, pulations V. Amount of protein polymorphism and degree of genic heterozygosity. - Hereditas 75:221-232 GOLIKOV. A. N. and TZVETKOVA. N. L. 1972. The ecological principle of evolutionary reconstruction as illustrated by marine animals. -Mar. Biol. 14: 1-9 GOOCH.J . L. and HETRICK. S. W. 1979. The relation of genetic structure to environmental structure: Gammarw minus in a karst area. - Evolution 33: 192-206 H. and HOPKINSON. D. A. 1976. Handbook of Enzyme HARRIS, Electrophoresis in Human Genetics. - North-Holland Publishing Company, Amsterdam HEDGECOCK,D., NELSON. K., SIMONS,.~. and SHLESER. R. 1977. Genic similarity of American and European species of the lobster Homarus. - Biol. Bull. 152: 41-50 V. 1975. Genetics of Zoarces populaHIOKTH. J. P. and SIMONSEN. tions VIII. Geographic variation common to the polymorphic loci Hbl and EstIIl. - Hereditas 81: 173-184 HOLMES. J. M. C. 1975. A comparison of numerical taxonomic techniques using measurements on the genera Gammarus and Marinogammarus (Amphipoda). - Biol. J . Linn. Soc. 7 183-214 0. 1954. Die Gammarus - Arten der Kieler Bucht. (G.10KINNE. custa, G.oceanicw, G.salinus, G a d d a c h i , G.duebeni.). 2001.Jb. Syst. okol. Geogr. 82: 405-424 S. 1981. Habitat selection and life cycle characteristics KOLDING. of five species of the amphipod genus Gammarus in the Baltic. Oikos 3 7 173-178 S. and SIMONSEN, V. 1983. Phylogenetic relationships of KOLDING. five species of the amphipod genus Cammarus. - Zool. Scr. 12: 215-219 MAGAARD. L. and RHEINHEIMER. G. 1974. Meereskunde der Ostsee. - Springer Verlag, Berlin
GENETICSTUDIES O F GAMMARLIS I
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MULLEY, J. C. and LATTER.B. D. H. 1980. Genetic variation and evolutionary relationship within a group of thirteen species of penaeid shrimps. -Evolution 34: 904-916 NEI,M. 1972. Genetic distances between populations. -Am. Nat. 106: 283-292 NELSON, K. and HEDGECOCK, D. 1980. Enzyme polymorphism and adaptive strategies in the decapod Crustacea. - Am. Nut. 116: 238-280 NIELSEN.J . 7.and CHAPMAN. V. 1977. Electrophoretic variation for X-chromosome linked phosphoglycerate kinase (PGK-1) in the mouse. - Generics 87: 319-325 E. 1973. Systematics and ecology of the Isefjord mariRASMUSSEN, ne fauna. - Ophelia 1 1 : 1-507 SCHMIDT. J. 1930. The Atlantic cod (Gadus callarias L.) and local races of the same. - C. R. Lab. Carlsberg 18: 1-72 SCHWARTZ.M. K., NISSELBAUM.J. S. and B O I l A N S K Y . 0 . 1963. Procedure for staining zones of activity of glutamic oxaloacetic transaminase following electrophoresis with starch gels. - Am. J . Clin. Pafhol. 40: 103-106 S. G. 1947. New observations on the distribution SEGERSTRALE. and morphology of the amphipod, Gammarlls zaddachi Sexton, with notes on related species. -J.Mar. Biol.Ass. U . K . 27: 219244 S. Y., JOHNSON. W. E. and SELANDER, R. K., SMITH. M. H., YANG. J. B. 1971. Biochemical polymorphism and systematics GENTRY, in the genus Peromyscw. I. Variation in the Old-field mouse (Peromyscus polionotus). - Stud. Genet. VI. Univ. Texas Publ. 7103: 49-90 SHAW, C. R. and PRASALI. R. 1970. Starch gel electrophoresis of enzymes - A compilation of recipes. - Biochem. Genet. 4: 297320 SICK, K. 1965. Haemoglobin polymorphism of cod in the Baltic and the Danish Belt Sea. - Hereditas 54: 1 9 4 3 0. 1972. Genetics of Zoarces poSIMONSEN, V. and FRYDEYBERG. pulations 11. Three loci determining esterase isozymes in eye and brain tissue. - Hereditas 70: 235-242 SNEATH. P. H. A. and SOKAL. R. R. 1973. Numerical Taxonomy. - W. H. Freeman and Company, Sun Francisco SPENCER.N., HOPKINSON. D. A. and HARRIS. H. 1968. Adenosine deaminase polymorphism in man. -Ann. Hum. Genet. 32: 9-14 SPOONER. G . M. 1947. The distribution of Gammarus species in estuaries. Part I. - J . Mar. Biol. Ass. U.K . 27: 1-52 B. F. 1978. Allozyme clines and evidence of strong selecTHEISEN, tion in three loci in Mytilus edulis L. (Bivalvia) from Danish waters. - Ophelia 17: 135-142 YNOGAARD. C. F. 1972. Genetically determined electrophoretic variants of phosphoglucose isomerase and 6-phosphogluconate dehydrogenase in Zoarces viviparus L. - Hereditus 71: 151-154
