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Moll Stud. (1996), 62, 343-357 © The Malacological Society of London 1996
POPULATION GENETICS OF EUROPEAN ANODONTINAE(BIVALVIA: UNIONIDAE)
KARL-OTTO NAGEL*, GUIDO BAD1NO and BRUNO ALESSANDRIADipartimento di Biologia Animate, Universitd di Torino, Via Accadenua Albemna 17,10123 Torino, Italy
(Received 15 August 1995, accepted 5 February 1996)
ABSTRACT
Enzyme electrophoresis was used to study popula-tion genetics and molecular differentiation of Euro-pean Anodontmae. The existence of two genera(Anodonta Lamarck 1799 and Pseudanodonta Bour-guignat 1876) is supported by the number of diag-nostic loa (4) and Net's D > 0.463 in all cases. Inwestern and central Europe there are two species ofAnodonta, A. ananna (Linnaeus 1758) and Acygnea (Linnaeus 1758) while two other taxa of stilluncertain rank were identified in the Mediterraneanarea. An estimated medium level of gene flow andpronounced genetic differentiation between-the taxasupport this view. Data on genetic distances suggestthat the diversification of European Anodontinactook place in the middle-late Pleistocene
INTRODUCTION
The taxonomy of river mussels of the genusAnodonta has puzzled many biologists sincethe days of Linnaeus. In the absence of directobservations of interbreeding, it was unclearfor a long period of time which charactersshould be used to define the species. The pro-posed numbers of Anodonta species in Europeranged from one to over four hundred. Thisconfusion was due to the inappropriate use ofshell characters like contour line, thickness,obesity, or colouring. The role of environmen-tal factors acting upon these characters and theextent to which they determine the individualshell shape was underestimated.
Today the existence of two Anodontaspecies and one species of the genus Pseudan-odonta in centra] and northern Europe iswidely accepted. Although reliable shell char-acters to distinguish them were known sincethe works of von •Gallenstein (1895) it tookanother forty years to settle this debate(Bloomer, 1937; Franz, 1939). Since then anincreasing number of results from comparativestudies in various research disciplines con-• Addrru for corretpoadencc: Dr K.-O Nigel, Sdmrzwaldsti 15. D-79189 B*d Krazmgen. Germany
finned this finding (parasitology: Davids, 1973;protein variability: Kodolova & Logvinenko,1974; larval morphology: Nagel, 1985, 1988,Kinzelbach & Nagel, 1986; soft body morphol-ogy. Nagel, 1988; Dyduch-Falniowska &Koziol, 1989a, 1989b; allozyme variability:Baag0e, Hvilsom & Pedersen, 1985).
However, the status of the Anodontmae ofsouthern Europe is still a matter of debate.Species determination and concepts on phy-logeny were previously based on shell charac-ters alone (Modell, 1945, 1951, 1964;Kinzelbach, 1989; Falkner, 1994). Additionally,it is not yet clear how far the central Europeanspecies spread into the Mediterranean regionand if there are any indigenous species in thisarea. All hypotheses on number and identity ofspecies and their relationships must likewise becorroborated by other independent characters.
The genetic analysis of allozyme patternshas been successfully used to study the differ-entiation and relationships of river mussels(Unionoidea). Phenomena of subspecific dif-ferentiation, sibling species, and hybridizationwere shown in the unionid fauna of NorthAmerica (Davis, Heard, Fuller & Hesterman,1981; Davis, 1983, 1984; Kat, 1983a, 1983b,1986; Kat & Davis, 1984; Hoeh, 1990) andEurope (Badino, Celebrano & Nagel, 1991).These works have added much to our knowl-edge of patterns and processes in the evolutionof river mussels at generic, specific, and sub-specific levels.
Here we present the results of our studies onthe differentiation and relationships of centraland south European Anodontinae. They arebased on the genetics of allozymes. We studieddifferentiation by calculating genetic differ-ences (Nei, 1978) and the probability of geneflow. The latter was done in three differentways. First, we calculated the parameter F{gt)(Wright, 1931,1978; Porter, 1990) as a measureof intergroup differentiation, secondly we usedSlatkin's graphical method (1981), and thirdlywe calculated frequency distributions of single
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344 K.-O. NAGEL ETAL.
Table 1. Collection localities, sample sizes and drainage systems.
Abbreviations of species names and taxa:Aa, Anodonta anatina (Linnaeus 1758)A-l, Italian Anodonta group IA-ll, Italian Anodonta group IIAc, Anodonta cygnea (Linnaeus 1758)Aw, Anodonta woodiana Lea 1834Pc, Pseudanodonta complanata Rossmassler 1835
N(loc), number of locality in Figure 1N(pop), number of population in Tables 2 and 4N(sp), number of specimens
N(loc)
123456789
10111213
141516171819
20
21
22232425
2627
name ofcollection site
OstermuhlenNiederorkeKasselGinsheimCommercySt. MihielDieueMalauceneMontfrinCompsCandes-st. MartinChazeuilToumy
BrehemontVitry-le-FrancoisMigennesZeitlarnHamburgBistagno
Mantova
Viverone
PassignanoPraia de MiraVirellesFonydd
SzarvasGranges-sur-Aube
N(pop)
123456789
1011121331141516171819232024212522262728
2930
species
AaAaAaAaAaAaAaAaAaAaAaAaAaPcAaAaAaAaAaA-lA-llA-lA-llA-lA-llA-lAcAcAc
AwPc
N(sp)
82011497
128
2015171618136
197
142130155
1833
2621132421
339
river or lake
MuhlenbeckOrkeFuldaRheinMeuseMeuseMeuselake of PatyGardonGardonViennaAllierLavaux
IndreSaulxYonneRegenDonauBormida
Mincio
lake ofViveronelake Trasimenolagoon of Mirapond of VirellesPogany-volgyi-viz(Balaton)Kdr6sAube
basin
EiderWeserWeserRheinRheinRheinRheinRhflneRhdneRhoneLoireLoireLoire
LoireSeineSeineDonauDonauPo
Po
Po
Tevere(Atlantic)RheinOonau
DonauSeine
locus similarity (Ayala, Tracey, Hedgecock &Richmond, 1974; Avise, Smith & Ayala, 1975).Our aims were 1) to identify diagnosticcharacters at generic and specific levels, 2) tostudy the relationships of populations andspecies using methods of population genetics,and 3) to draw conclusions regarding the evo-lution and systematics of the Anodontinae inEurope.
MATERIAL AND METHODS
Taxa studied
A classification (tentative for the mediterraneanpopulations) of the animals studied is given inTable 1 together with sample sizes and collec-tion localities. To facilitate biogeographical con-siderations, the collection localities are listedtogether with their respective drainage system.
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ANODONTINAE POPULATION GENETICS 345
Figure L Collection localities. The locality numbers are the N(loc) in Table 1.
Additionally, the collection localities are also shownin Fig. 1
Pseudanodorua complanata (Rossmassler 1835) isa relatively rare species which almost exclusivelylives in rivers and streams. It is difficult to find it inlarger numbers, therefore our samples are small.Anodonta woodiana Lea 1834 is a species from eastand south-east Asia which was introduced into cen-tral Europe in the early sixties, when herbivorousfish species (Ctenopharyngodon tdella, Hypoph-thalmichtys molurix and H. nobila) from China andfrom the USSR were brought to Rumania (Sarkany-Kiss, 1986) and Hungary (Petrd, 1984; Kiss, 1992). Itis now also known to occur in Yugoslavia(Guelmino, 1991, cited from Kiss, 1992) and France(Girardi & Ledoux, 1989). This species most proba-bly will continue to spread through the naturalwaters of Europe.
The shells of all specimens referred to in this arti-cle are kept in the Museum of Natural History,Malacology Section, Turin (Italy).
Electrophorais
A total of seven enzymes (PGI, phosphoglucoiso-merase; PGM, phosphoglucomutase, MDH, raalatedehydrogenase; LAP, leucine aminopeptidase, EST,esterase (alpha- and beta-naphthyl acetate), IDH,
isocitrate dehydrogenase; GOT, glutamate-oxaloac-etate transaminase) and a minimum of ten loci wereanalysed. We used cellulose acetate strips as sup-porting medium Buffers, running conditions andstaining procedures have been published previously(Badino et al, 1991). They are derived from standardmethods (e.g. Brewer, 1970; Richardson, Baverstock& Adams, 1986) and have been modified in our lab-oratory. Three enzymes (PGM, LAP, EST) werebest separated by reverse field electrophoresis. LAPwas analysed from gill tissue while the otherenzymes were analysed from hepatopancreas tissueFurther details on specific modifications will be fur-nished on request.
Analysis of data
AUele frequencies, heterozygosity, percentage ofpolymorphic loci. Nei's genetic distances (Nei 1978).and f(gt)-values were calculated using the BIOSYS-1 software package (Swofford & Selander, 1989). Itwas also used to generate plots of the frequency dis-tributions of single-locus similarity (Avise et al.,1975; Ayala et al., 1974). The NTSYS-pc softwarepackage (Rohlf. 1988) was used to perform clusteranalysis of distance data with the UPGMA algo-rithm.
The use and interpretation of f(gt)-values follows
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Tab
le 2
. A
llele
fre
qu
en
cie
s of
fiv
e p
oly
mo
rph
ic l
oci
fo
r p
op
ula
tio
ns
of E
uro
pe
an
An
od
on
tin
ae
. Lo
cus
ab
bre
via
tio
ns
in t
ext
; p
op
ula
tio
n n
um
be
r is
g-
N(p
op
) in
Ta
ble
1; s
am
ple
siz
es in
pa
ren
the
ses.
Ano
dont
a an
atin
a, w
est
ern
an
d ce
ntr
al E
uro
pe
Locu
s &
P
op
ula
tion
nu
mb
er
alle
le
1 2
3 4
5 6
7 8
9 10
11
12
13
14
15
16
17
18
PG
I (8
) (2
0)
(11)
(4
9)
(7)
(12)
(8
) (2
0)
(15)
(1
7)
(16)
(1
8)
(13)
(1
9)
(7)
(14)
(2
1)
(30)
30 50 90 100
1.00
0 1.
000
1.00
0 1.
000
1.00
0 1.
000
1.00
0 1.
000
0.76
7 1.
000
1.00
0 1.
000
1.00
0 1.
000
1.00
0 1.
000
1.00
0 1.
000
130
150
0.23
3P
GM
(8
) (1
9)
(11)
(4
9)
(7)
(12)
(7
) (1
4)
(15)
(1
7)
(16)
(1
6)
(13)
(1
9)
(7)
(11)
(2
1)
(30)
91
0.14
3 0.
117
100
1.00
0 0.
763
1.00
0 1.
000
1.00
0 1
00
0 0.
571
0.25
0 1.
000
10
00
1.00
0 0.
937
1.00
0 1.
000
1.00
0 1.
000
1.00
0 0.
850
^10
5 0.
237
0.28
6 0.
750
0.06
3 0.
033
510
8 2
110
>M
DH
-1
(7)
(20)
(7
) (4
6)
(7)
(12)
(8
) (2
0)
(15)
(1
7)
(15)
(1
8)
(13)
(1
9)
(6)
(13)
(2
1)
(30)
O
100
0.57
1 0.
143
0.02
2 r
109
0.42
9 1.
000
0 85
7 0.
978
1.00
0 1.
000
1.00
0 1.
000
1.00
0 1.
000
1.00
0 1.
000
10
00
1.00
0 1.
000
1.00
0 1.
000
1.00
0 h,
130
r?14
5 ft
ES
T
(8)
(20)
(1
1)
(47)
(7
) (1
0)
(8)
(20)
(1
3)
(8)
(16)
(1
8)
(13)
(1
9)
(7)
(14)
(2
1)
(30)
80 88 100
1.00
0 1
00
0 1.
000
0.89
4 1.
000
0.95
0 1
00
0 0.
625
0 50
0 0
750
0.06
3 0.
389
0.69
2 0.
079
1.00
0 0.
964
0.23
8 0.
867
102
107
109
0.10
6 0.
050
0.37
5 0.
500
0.25
0 0.
937
0.61
1 0.
308
0.92
1 0.
036
0.76
2 0.
133
115
120
LAP
(8
) (1
0)
(9)
(49)
(7
) (1
2)
(8)
(20)
(1
4)
(11)
(1
3)
(16)
(1
3)
(2)
(6)
(5)
(21)
(3
0)75 90 95
1.
000
0.70
0 0.
667
1.00
0 0.
429
1.00
0 0.
687
1.00
0 0.
071
0.63
6 0.
385
0.43
7 0.
333
0.60
0 0.
095
0.06
710
0 0.
300
0.33
3 0.
571
0.31
3 0.
929
0.36
4 0.
615
0.56
3 1.
000
1.00
0 0
667
0.40
0 0.
905
0.93
310
311
912
4
(co
ntin
ue
d)
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Tab
la 2
.
Locu
s &
alle
le
PG
I 30 50 90 100
130
150
PG
M 91 100
105
108
110
MD
H-1
100
109
130
145
ES
T 80 88 100
102
107
109
115
120
LAP 75 90 95 10
010
311
912
4
(co
ntin
ue
d)
Ital
ian
/ Ano
dont
a, g
rou
p I
Po
pu
latio
n n
um
be
r19 (1
5)
0.03
3
0.83
30.
067
0.06
7(1
5)
0.63
3
0 36
7
(15)
10
00
(15)
1.00
0
(15)
1 00
0
20 (18)
0.02
8
0.94
4
0.02
8(1
8)
0.94
4
0.05
6
(18)
1.00
0
(18)
1.00
0
(18)
1.00
0
21 (3)
1.00
0
(3)
1.00
0
(3)
0.50
00.
500
(3)
1.00
0
(3)
1.00
0
22 (21)
1.00
0
(21)
1.00
0
(21)
1 00
0
(21)
1.00
0
(21)
10
00
Ital
ian
gro
up
23 (5)
1 00
0
(5)
1.00
0
(5)
1.00
0
(5)
1 00
0
(5)
1.00
0
Ano
dont
a,II
24 (3)
0.16
7
0.66
6
0.16
7(2
)
1.00
0
(3)
10
00
(3)
1.00
0
(3)
1.00
0
25 (26)
1 00
0
(18)
1.00
0
(26)
0.80
80
192
(18)
1 00
0
(26)
1.00
0
Ano
dont
a cy
gnea
26 (13)
1.00
0
(13)
1.00
0
(13)
1.00
0
(13)
1.00
0
(13)
1.00
0
27 (23)
1.00
0
(24)
1 00
0
(24)
1.00
0
(16)
1.00
0
(24)
10
00
28 (21)
1.00
0
(20)
0.65
00.
350
(21)
1.00
0
(21)
1.00
0
(21)
1.00
0
Ano
dont
aw
oodi
ana
29 (33)
1.00
(33)
1.00
0
(33)
1.00
0
(21)
0.83
30
167
(32)
0.10
90.
359
0.28
20.
250
Pse
udan
odon
taco
mpl
anat
a
30 (9)
1.00
0
(9)
1.00
0(9
)
1.00
0(9
)0.
056
0.94
4
(9)
1.00
0
31 (6)
1.00
0
(6)
1.00
0(6
)
1.00
0(6
)
1.00
0
(6)
1.00
0
> •z o o z > s I d o o m z m Q
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348 K.-O. NAGEL£T/ l^ -
Porter (1990) and is briefly outlined here. Assumingthe island model of population structure the geneticdifferentiation of populations yields an indirect mea-sure of gene flow, Le. the amount of individualsexchanged between populations in every generation.Genetic differentiation a expressed by parameters Fwhich were originally designed to describe correla-tions of genes in structured populations (Weir &Cockerham, 1984). They relate the observed vari-ance of allele frequencies to the maximum possiblevariance. In the case of one allele A this reads
F{n) = var(st)/(a(l - a ) ) ,
and fixation parameters are related as
1 - f(it) - (1 - FQs)) (1 - F(st) ),
with a: average frequency of allele A among all pop-ulations, letters in parentheses represent i: individu-als, s: populations, C the total set of populations.When a hierarchical arrangement of populations isassumed (in this study: populations within drainagebasins, subspecies, or species) the observed variancecan be partitioned among the various hierarchy lev-els. In this case the parameters F are related as
1 - = (1 - F{is)) (1 - f(sg) ),
with subscript g representing groups of populations.Genetic differentiation between groups of popula-tions (the impetus of this study) is therein expressedby F(gt). Gene flow is then approximately
Nm(gt) - (U/^gt) - 1)11,
with N: effective population size and m: effectiveproportion of migrants between populations. Pan-muus is assumed whenever F-values are negative.Standard errors of F(gi) were obtained after jack-knifing over loci using Tukey's method (Hinkley,1982). The last equation was used to directly calcu-late Mn(gt)-values and their confidence limits.Because of small sample sizes we used Hardy-Wein-berg heterozygosities rather than observed heterozy-gote frequencies.
RESULTS
Allele frequencies, htterozygosity, andpolymorphism
One locus could be identified and interpretedfor each of the enzymes PGI, PGM and LAP,while MDH, IDH and GOT were controlledby two loci. The (unspecific) esterases (EST)snowed up to four loci, but only one was suffi-ciently interpretable in all cases. There werefive monomorphic loci: Mdh-2, Got-1, Got-2,Idh-1, Idh-2 and five polymorphic loci: Pgi,Pgm, Mdh-1, Est and Lap. These results agreewith two previous studies (Baag0e el al, 1985;Hoeh, 1990) with the exception of GOT whereHoeh scored one locus only.
Allele frequencies are shown in Table 2.Each taxon showed some unshared alleles(fixed or co-dominant). The following allelescan be used as diagnostic characters: Pseudan-odonta complanata: Pgi-30, Pgm-110, Mdh-1-145, Est-80, Est-88; Anodonta woodiana:Pgi-90, Est-115, Est-120; Anodonta cygnea:Est-107, Lap-119; central European Anodontaanatina: Est-100, Est-109; Italian Anodontagroup II: Lap-124. Grouping of ItalianAnodonta is done on the basis of their Lapalleles as it will be discussed below in detail.The numbers of unshared alleles for pairwisecomparisons are given in Table 3.
The observed heterozygosity, the percentageof polymorphic loci and the average number ofalleles per locus are generally low. Table 4gives the range of values of these parameters.Twenty-eight polymorphic loci were not in aHardy-Weinberg equilibrium (chi-square test,p > 0.05) due to heterozygote deficiencies,while 16 loci were. With the exception ofAnodonta woodiana the Lap locus never wasin equilibrium. Looking at the larger samplesonly (at least 20 individuals analysed), the pro-portion of loci in disequilibrium is even higher10 out of 12 loci were not balanced.
Genetic similarity and distances
Mean values of genetic distances are given inTable 3; full tables will be furnished onrequest Pseudanodonta complanata generallyexhibits the highest values of genetic distancefrom all other species (mean genetic distanceabove 0.565). At a similar level we findAnodonta woodiana when compared to A.cygnea and the Italian Anodonta group II. Thelevel of mean genetic distance is only slightlylower for Anodonta cygnea compared to theItalian Anodonta group I.
The dendrogram of Fig. 2 gives a clusteranalysis of Nei's D. The populations aregrouped together according to the geographicand hydrologic relationships of the samplinglocalities. Populations from the drainage-basins of the rivers Seine, Rhine, and from theaffluents of the North Sea make up a firstgroup of Anodonta anatina. The Malaucenepopulation of A. anatina links this cluster tothe populations of the rivers Loire, Rhdne,Danube (Donau) and their respective afflu-ents. The Italian Anodonta group I is distantlylinked to the central and western Europeangroups. Italian Anodonta group II, A. woodi-ana, and A. cygnea build up separate groups,as does Pseudanodonta complanata. The den-
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ANODONTINAE POPULATION GENETICS
Table 3. Mean genetic distances and numbers of diagnostic loci.
349
Above the diagonal: numbers of diagnostic loci (no shared alleles);below the diagonal' mean genetic distances (Nei, 1978abbreviations as in Table 1.
Aa
A-l
A-ll
Ac
Aw
Pc
Aa
.064/-(.0OO-.252).221
(.097-.392).417
(.315-506).455
(.348-.507).417
(.361-.545).565
(.463-688)
A-l
1
.108/-(.009-.257).280
(.141-.511).530
(.399-.6931.476
(.456-.513).670
(.647-.693)
A-ll
3
1
.063/-(.003-. 112).395
(.284-.537).624
(.609-.640).675
(.657-.693)
; range of values
Ac
2
3
2
.008/-(.000-.012).632
(.616-.640).682
(.664- 693)
in parentheses);
Aw
3
3
5
5
- / -
.582(.579-.S85)
Pc
4
5
5
5
4
0.000/-
drogram has three mam sections. The branchformed by P complanata separates at the levelof D = 0 604 from the rest, while the branchcontaining A cygnea, A. woodiana, and ItalianAnodonta group II differs at D = 0.392 fromthe A. anatina - Italian Anodonta group Ibranch. The latter is subdivided at the level D= 0.222.
lnterdemic differentiation and gene flow
F(gt)-values for the various groups of taxa ofthe more closely related A. anatina, A. cygneaand Italian Anodonta groups I and II are givenin Table 5. For both central European A.anatina alone (comparison between populationgroups from different drainage basins) andcentral European A. anatina and ItalianAnodonta group I, F(gt)-values are smallerthan 033, the corresponding Mn(gt)-valuesbeing larger than or very close to 0-5. This indi-cates a good probability of gene flow (Porter,1990). If we add the Italian Anodonta group IIpopulations this limit is exceeded (F(gt) =0.390) and gene flow is less likely to occur. Theprobability of gene flow is considerably lowerbetween A. anatina from central Europe andA cygnea (F(gt) = 0.404) than it is betweenthe two Italian Anodonta groups (F(gt) =0347). However, confidence limits of Nm(gt)exceed 05 also in all cases where (presumed)species are involved, indicating even panmixisbetween the two Italian Anodonta groups. Thisresult may be an effect of few polymorphic locias well as of small sample sizes. Both will tendto underestimate genetic differentiation and
bias /Vm-values upward. This calls for somecaution in the interpretation of A/m-values inthis study.
Both species pairs (central European Aanatina - A. cygnea, Italian Anodonta groups Iand II) yielded U-shaped figures when the fre-quency distribution of their single-locus simi-larity was plotted (Fig. 3). This confirms theassumption of an interrupted gene flowbetween them. Comparing A anatina fromcentral Europe to the Italian Anodonta groupII we also obtained a somewhat U-shaped fig-ure. Here, too, gene flow between these taxaseems to be small, or even interrupted.
Finally the graphical method of Slatkin(1981) reveals medium gene flow between theA anatina populations in western and centralEurope. It tends to be smaller between themand Italian Anodonta group I, and distinctlylower when Italian Anodonta group II is added(Fig. 4).
DISCUSSION
The results of this study allow us to give addi-tional characteristics for some of the previ-ously established taxa of the EuropeanAnodontinae. Moreover, they stimulate somespeculations about the existence of additionaltaxa and about the taxonomic diversity ofAnodontinae in Europe.
While mean polymorphism levels are withinthe range of data published for North Ameri-can Anodonta species, mean heterozygosity isvery low. This holds true also in comparison
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350 K.-O.NAGEL ETAL.
Table 4. Genetic variability measures. Standard Error in parentheses.
N(pop), population number (setaverage number of alleles; %P,gosity
N(pop
) Table 1);: n, mean sample size per locus; n(av),% of polymorphic loci; H(o), observed heterozy-
; H(e), expected heterozygosity
) n n(av)
Anodonta anatina, western and <123456789
101112131415161718
Italian19202122
Italian232425
7.9 (.1)18.9(1.0)10.4 (.4)48.5 (.3)7.0 (.0)
11.8 1.2)7.9 1.1)
19.4 (.6)14.7 (.2)15.5(1.0)15.6 1.3)17.6 (.3)13.0 (.0)17.3(1.7)6.8 1.1)
12.7 (.9)21.0 (.0)30.0 (.0)
Anodonta group15.0 (.0)18.0 (.0)3.0 (.0)
21.0 (.0)
Anodonta group5.0 (.0)2.9 1.1)
24.4(1.1)
Anodonta cygnea262728
13.0 (.0)32.1 (.8)20.9 (.1)
Anodonta woodiana29 31.7(12)
1.0961.1291.1121.0281.0961.0101.2091.1481.1711.1461.1031.2011.0741.0171.0801.1001.0781.080
11.1291.0241.1001.000
II1.0001.1001.045
1.0001.0001.083
1.292
Pseudanodonta complanata3031
9.0 (.0)6.0 (.0)
1.0121.000
%P H(o)
:entral Europe102020201010202030202030101010202030
2020100
01010
00
10
20
100
.000 (.000)
.005 (.005)
.000 (.000)
.009 (.009)
.000 (.000)
.010 1.010)
.013 (.012)
.012 (.008)
.056 (.038)
.025 ( 025)
.028 (.019)
.046 (.034)
.046 (.046)
.016 (.016)
.000 (.000)
.007 (.007)
.000 (.000)
.010 1.010)
.040 (.033)
.022 (.015)
.100 1.100)
.000 (.000)
.000 (.000)
.067 (.067)
.000 (.000)
.000 (.000)
.000 (.000)
.030 (.030)
.067 (.062)
.011 (.011)
.000 (.000)
H(e)
.053 (.053)
.081 (.054)
.0731.051)
.024 1.019)
.053 (.053)
.010 (.010)
.107 (.073)
.087 (.058)
.103 (.060)
.088 (.059)
.061 (.049)
.112 (.006)
.044 (.044)
.015 (.015)
.048 ( 048)
.060 (.053)
.055 (.039)
.063 (.034)
.079 (.054)
.022 (.014)
.060 (.060)
.000 (.000)
.000 (.000)
.060 (.060)
.032 (.032)
.000 (.000)
.000 (.000)
.047 (.047)
.101 (.075)
.011 (.011)
.000 (.000)
with other genera of Unionidae both in NorthAmerica and in Europe (Kat, 1983a, 1986; Kat& Davis, 1984; Badino et a/., 1991).
A close intragroup relationship of the Euro-pean Anodontinae is suggested by the fact thatfive out of ten loci were monomorphic for thesame allele in all species. The elevated numberof diagnostic loci for Pseudanodonta com-planata (four) supports the classification of this
species into a separate genus. Morphologicaland anatomical findings such as the shell formand umbonal sculpture of the adults (e.g.Haas, 1969), slight differences in gill morphol-ogy (Clessin, 1876), and some unique charac-ters of the larval shell (Kinzelbach & Nagel,1986) had been put forward in favour of thisinterpretation, but had been judged insuffi-cient by Ortmann (1912). Likewise Anodonta
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ANODONTINAE POPULATION GENETICS 351
0.64I—
0.48— I —
0.32— I —
0.16 0.00
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Aa
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Pc
Figure 2. Dendrogram of Nei (1978) genetic distances. For explanation of abbreviations and numbers seeTable 1, the numbers indicate the N(pop).
cygnea and A woodiana, with two diagnosticloci each, are well distinguished species withinthe genus Anodonta. The present biochemicaldata do not suggest putting A. woodiana into aseparate genus as was done by Modell (1945:Sinanodonta). The two Anodonta species ofcentral and northern Europe have two diag-nostic loci (Lap and Est). All Italian Anodontastudied so far presented a unique Est allele.Furthermore, there were individuals sharingLap alieles with non-Italian A anatina andothers showing a new allele. There were nohybrids between these two types. By analogyto the situation found in central Europe weinterprete these results to be indicative of twodistinct taxa of Anodontinae in Italy.
The probability of genetic exchange acrosspresumed species boundaries can be estimatedfrom studies of inCerdemic differentiation(Porter, 1990). Estimating gene flow from theparameter F(gt) which measures intergroupdifferentiation is done under the assumption ofthe island model of genetic population struc-ture. It is unlikely that natural populations willmeet all the conditions of this specific model.The most important factors to introduce biasesinto gene flow estimation are net balancing
selection and hidden variation. When there areno indications regarding these two factors(which is most often the case for natural popu-lations) the calculated number of individualsexchanged among populations each generation(Nm) is likely to be an upper estimate for theactual gene flow (Porter, 1990). Results fromstudies of natural populations will in principletend to overestimate gene flow rates and hidegenetic differentiation. This bias is probablyaccentuated at least in A cygnea by the exis-tence of hermaphrodites and their capability toself-fertilize (Bloomer, 1940).
The F(gt)-value and the histogram of single-locus allele frequencies show that A anatinafrom western and central Europe and Acygnea behave as good species. Although theA anatina populations come from a widerange of drainage basins, both types of analysisshow results typical of a single species with agood probability of genetic exchange amongpopulations. The Slatkin diagram shows amedium level of gene flow only. This corre-sponds to expectations in view of the distancesbetween sampling sites or drainage basins.When Italian Anodonta group I populationsare added, the F(gt) value rises but remains
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Tab
le 5
. G
ene
flo
w e
stim
ate
s b
etw
ee
n g
rou
ps
of
po
pu
latio
ns
(1)
An
od
on
ta a
na
tina
, w
est
ern
and
ce
ntr
al
Eur
opea
n p
op
ula
tion
s(2
) It
alia
n A
no
do
nta
gro
up
I(3
) It
alia
n A
no
do
nta
gro
up
II
(4)
An
od
on
ta c
ygn
aa
(5)
An
od
on
ta c
ata
ract
a c
ata
ract
a. N
ort
h A
me
rica
n s
peci
es(6
) A
no
do
nta
'c
ata
ract
a' f
rag
ilis,
No
rth
Am
eri
can
spe
cies
F(g
t) a
nd
Nm
(gt)
, see
text
fo
r e
xpla
na
tion
Co
mp
ari
son
(1)
(Ha
nd
(2)
(2)
and
(3)
(1),
(2)
and
(3)
(1)a
nd
(4)
(5)
an
d (
6)
leve
l of
com
pa
riso
n
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ina
ge
bas
ins
? s
ubsp
ecie
s?
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cies
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ubsp
ecie
s/sp
ecie
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sub
spec
ies
nu
mb
er
of l
oci
5 5 4 5 5 6
nu
mb
er
of a
llele
s
11 16 12 17 14 16
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t)
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0.34
70
390
0.40
40.
758
sta
nd
ard
err
or
0.07
490.
1092
0.25
700.
0556
0.09
440.
1344
Nm
igt)
1.18
70.
994
0.47
00.
391
0.36
90.
080
95%
co
nfid
en
ce l
imit
low
er
0.37
10.
229
-0.0
16
0.20
20.
134
-0.0
35
upper
pa
nm
ictlc
pa
nm
ictic
pa
nm
ictic
0.77
21.
709
0.35
3
o m
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ANODONTINAE POPULATION GENETICS 353
bJ 0
Figure 3. Frequency distributions of single-locus sinularity Abbreviations' 1, Anodonta anattna (central andwestern European populations); 2, A. anattna plus Italian Anodonta group I, 3, A. anattna plus ItalianAnodonta groups I and II, 4, A anattna plus A cygnea.
below 033. This indicates that gene flow acrossgroup boundaries is still present or has beeneffective in the near past. Matters change whenAnodonta group II is considered. The compari-son between the two Italian Anodonta groups Iand II gives a F(gt) value which indicates smallor non-existing gene flow. The frequency his-togram is markedly U-shaped, and the Slatkindiagram shows an even lower level of geneflow. These results coincide with the fact thatno pairwise comparison between populationsof Italian Anodonta group II and A anatmaplus Italian Anodonta group I gives geneticdistance values below D = 0.139, a limit pro-posed by Davis (1983) for ailopatric unionidspecies. In the dendrogram (Fig. 2) the ItalianAnodonta group II branch meets the branch ofA anatina and Italian Anodonta group I at thelevel of D = 0.392, typical of different species
of the same genus (Davis, 1983). These inter-pretations agree with previous findings(Kat, 1983a; Hoeh, 1990) on genetic distancesbetween species and genera in the Anodonti-nae. Hoeh (1990) found values of D rangingfrom 0.087 to 0.676 between nominal species(North American only) of the same clade(and up to 12 diagnostic loci out of 24). Hisclades probably represent different generawith a minimum mean genetic distancebetween species from different clades ofD = 0.694.
To compare our results on gene flow to pre-viously published data we calculated the F(gi)-value from allele frequency data of two groupsof the North American Anodonta cataractaSay 1817 (Kat, 1983a). They had been rankedsubspecies before but Kat found morphologi-cal and genetic (also Hoeh, 1990) diversity typ-
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Figure 4. Diagram of gene flow after Slatkin (1981) Abbreviations; p, mean frequency of the allele p in thepopulations where it occurs; i, number of populations in which p occurs; d, total number of populations; high -medium - low, levels of gene flow (Slatkin 1981: 327, figure 2a), 1, Anodoma anattna (central and westernEuropean populations); 2, A. anatina plus Italian Anodonta group I, 3, A anatina plus Italian Anodontagroups I and II.
ical of species. The F(gt)-value of 0.758 sug-gests effective barriers to gene flow, thus sup-porting the hypothesis that these two taxabelong to different species rather than sub-species.
Genetic distances may be used to calculatethe time of divergence between taxa (Nei,1971). Kat (1983b) obtained very reasonableresults in terms of geological events in a groupof North American unionids. Making the sameassumptions on amino acid substitution rates,mean number of amino acids per protein, andproportion of detectable amino acid variation,we got the following results: Pseudanodonta
complanata diverged c. 600,000 years ago fromthe Anodonta stock; c. 450 - 400,000 years agoAnodonta cygnea and the Italian Anodontagroup II separated from the rest (as probablydid A. woodiana, but the database is too smallhere); the remaining stock underwent a majordifferentiation event c. 200,000 years ago whenan Italian branch. Anodonta group I, divergedfrom the rest. Thus the differentiation of gen-era and species presumably took place in themiddle Pleistocene whilst a further differentia-tion might have occurred in the late Pleis-tocene.
Before drawing any phylogenetic conclu-
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ANODONTINAE POPULATION GENETICS 355
sions additional aspects have to be considered.The database at hand is too small to give theItalian Anodonta a certain taxonomic rankbecause of conflicting results from differentanalyses. The F(gt)-value of 0.201 (see Table5) may suggest that Italian Anodonta group I isa subspecies of A. anatina. Because of a moreelevated F(gt)-value, Italian Anodonta groupII may deserve the rank of a species with onediagnostic locus. On the other hand, a cladisticanalysis of the distribution of alleles leads toconflicting cladograms (LAP vs. EST and PGI,P. complanata, A. woodiana and A. cygnea asoutgroups). Here the alternative arrangementof Anodonta groups I and II as sister taxa ismore strongly supported. We were unable tofind constant differences in shell morphologybetween individuals of the two- genotypesgroup I and II. Only the shells of the popula-tion from the central Italian lake Trasimenolook like typical central and northern Euro-pean A. anatina. They belong to our group I.Others have a somewhat different shape(more rounded, small escutcheon). Theseforms seem to be typical of the Po drainagebasin. But as Hoeh (1990) concluded from astudy of 13 North American species, the evolu-tion of conchological characters and allozymediversity seems to be poorly correlated inAnodonta.
The most elaborate ideas on the phylogenyof Anodontinae in Europe come from Modell(1945,1951, 1964). In his view there are threespecies in the area under consideration. Whiletwo of them (A anatina and A cygnea) arefound in the affluents of the Atlantic Oceanand the Black Sea, a third species, A. palustns(for nomenclatural corrections see Falkner,1994), is confined to the Mediterranean area(for recent data see also Kinzelbach, 1989). Acygnea is ranked a 'side' species of A anatinaand is said to have differentiated from the lat-ter during the Pleistocene in lakes near themargins of the glaciers. Dyduch-Falniowska &Koziol (1989b) reach the same conclusionabout the relative age of these two species onmorphological and biological grounds.Recently, Falkner (1994) criticized the hypoth-esis of one widely distributed MediterraneanAnodonta species. Reviewing shell materialFalkner concluded that there are anatina- andcyg/i«a-like animals throughout the Mediter-ranean region, but he encountered very 'aber-rant' forms of uncertain taxonomic rank fromthe three major mediterranean peninsulas. InFalkner's opinion the latter might be eitherspecialized races of the two European species,
or else good species which eventually couldoccur in sympatry with A anatina and Acygnea.
The present results support the specific sta-tus of A cygnea. Molecular data give an esti-mate for the time of divergence of this specieswhich is consistent with Modell's ideas. Thebiochemical characters, too, raise some doubtsabout the hypothesis of only one Anodontaspecies in the Mediterranean area. Two taxamay be identified in continental Italy whoserelationships to other Anodonta species arenot yet clear. The evaluation of genetic dis-tances and gene flow is limited by the lowdegree of polymorphism and by small samplesizes. Additionally, it is impossible in mostcases to quantify the degree of deviance fromthe genetic model. Effective population size isparticularly difficult to assess, not only in thecase of freshwater mussels. Effects introducedby heterogenous population sizes may be animportant source of error, too (Porter 1990).However limited, the present results show thepotential of new approaches to the taxonomyand systematic^ of Unionidae. They hopefullystimulate further researches in the phylogenyof European Anodontinae.
ACKNOWLEDGEMENTS
We would like to thank Drs. L, Castagnolo, G.Celebrano, F. Chiara, M. Gonella, A Kiss,R.M. Iibois, B. Murgi, I. Musko, and H.Girardi for helping to collect animals. Dr. G.Celebrano provided help during electrophore-sis. The manuscript benefitted substantially bycomments from Prof. F. Giusti, Prof. R.Kinzelbach, Dr. J. Heinze, Dr. U. Wirth, andProf. G.M. Davis who served as referee, to allof whom we are most grateful. This researchwas supported, in part, by grants from the Ital-ian Ministero Pubblica Istruzione (60%) (to G.Badino), the German Research Council andthe Evangelisches Studienwerk Villigst (to K.-O. Nagel).
REFERENCES
A VISE, J.C.. SMITH, JJ. & AYALA, FJ. 1975 Adap-tive differentiation with little genie changebetween two native Californian minnows. Evolu-tion, 29- 411-426.
AYAL>, FJ., TRACEY, ML., HEDGECOCK, D &RICHMOND, R.C. 1974. Genetic differentiationduring the speciation process in DrosophUa. Evo-
by guest on February 22, 2013http://m
ollus.oxfordjournals.org/D
ownloaded from
356 K.-O. NAGEL ETAL.
tution, 28:576-592.BAAGOE, P., HVILSOM, M.M. & PEDERSEN, B.V.
1985. The species rank of Anodonta anattna (L.)and A, cygnea (L-), with remarks on Pseudan-odonta complanata (Rossmassler) (Bivalvia,Unionidae). Videnskabelige Meddelelser fra dansknaturhistorisk Forenwg, 146 75-83.
BADINO, G. 1982. Unio elongatulus Pfeiffer(Bivalvia): Variability biometriqu6 et glnltiquedes populations du Piimont (Italic du Nord).Malacologia, 22: 673-677
BADINO, G., CELEBRANO, G. & NAGEL, K.O. 1991.Unio elongatulus and Unio pictorum (Bivalvia:Unionidae): molecular genetics and relationshipsof Italian and Central European populations. Bol-lettirw del Museo Regionale di Scienze Naturali-Torino, 9: 261-274.
BLOOMER, H.H. 1937. On distinguishing the shell ofAnodonta cygnea from that of Anodonta anaana.Journal of Conchology, 20: 321-327.
BLOOMER, H.H. 1940. Experiments on self-fertiliza-tion in Anodonta cygnea (L.). Proceedings of theMalacological Society of London, 24:113-121.
BREWER, G J . 1970. An introduction to isozyme tech-niques. Academic Press, New York.
CLESSIN, S. 1876. Deutsche Excursions-Mollusken-Fauna. NQrnberg.
DAVIDS, C. 1973. The relations between mites of thegenus Unionicola and the mussels Anodonta andUnio. Hydrobiologia, 41: 37-44.
DAVIS, G.M. 1983. Relative roles of moleculargenetics, anatomy, morphometncs, and ecology inassessing relationships among North AmericanUnionidae (Bivalvia). In; Protein polymorphism:adaptive and taxononuc significance (G.S. Oxford& D. Rollinson, eds), 193-222. Systematics Associ-ation Special Volume 24. Academic Press, Londonand New York.
DAVIS, G.M. 1984. Genetic relationships amongsome North American Unionidae (Bivalvia): Sib-ling species, convergence, and cladistic relation-ships. Malacologia, 25: 629-648.
DAVIS, G.M., HEARD, W H., FULLER, S.L.H. & HES-TERMAN, C. 1981. Molecular genetics and specia-tion in Elliptio and its relationships to other taxaof North American Unionidae (Bivalvia). Biologi-cal Journal of the Luinean Society of London, 15:131-150.
DYDUCH-FALNIOWSKA, A. & KOZIOL, R. 1989a.Anatomical and conchological characters in thesysteraatics of the Unionidae of Poland (Bivalvia,Eulamellibranchiata). Malakologische Abhandlun-gen Staatliches Museum fUr Tierkunde Dresden,14: 35-52.
DYDUCH-FALNIOWSKA, A. & KOZIOL, R. 1989b. Onthe anatomical differences between Anodontaanatina (I_, 1758) and Anodonta cygnea (Bivalvia,Eulamellibranchiata: Unionidae). MalakologischeAbhandlungen Staatliches Museum fUr TierkundeDresden, 14:93-102.
FALKNER, G. 1994. Systematik vorderorientalischerNajaden als Vorstudie zur Bearbeitung archdolo-gischer Funde. Forschungen und Berichte zur Vor-
und Frtihgeschichte m Baden-WUrttemberg, 53:BeitrSge zur ArchSozoologie und PrahistorischenAnthropologie (M. Kokabi & J. Wahl, eds), 135-162.
FRANZ, V. 1939. Die Unterscheidung der zwei mit-teleuropaischen Anodonta-Asten cygnea (L.) undpiscinalis (Nilss.) und die Haupttypen derselben.Jenaische Zeitschnft fUr Natwwissenschaft 72: 75-210 (+ plates 8-12)
VON GALLENSTEIN, H. 1895. Die Bivalven- und Ga-stropodenfauna Karntens. I. Die Bivalven Kilm-tens. Jahresbencht des Naturhistonschen MuseumsKarnten, 23:1-67.
GIRARDI, H. St. LEDOUX, J.C 1989. Presence d''An-odonta woodiana (Lea) en France (Mollusques,Lamellibranches, Unionidae). Bulletin mensuel dela Sociiti linnienne de Lyon, 58:286-291.
HAAS, F. 1969. Superfamilia Unionacea. Das Tier-reich, 88. i-x, 1-663.
HINKLEY, D. 1982. Jackknife. In: Encyclopedia ofStatistical Sciences, Vol. 4 (S. Kotz & N.L. John-son, eds), 280-287. John Wiley & Sons, New York.
HOEH, W.R. 1990. Phylogenetic relationships amongeastern North American Anodonta (Bivalvia:Unionidae). Malacological Review, 23:63-82.
KAT, P.W. 1983a. Genetic and morphological diver-gence among nominal species of North AmericanAnodonta (Bivalvia: Unionidae). Malacologia, 23:361-374.
KAT, P.W. 1983b. Morphologic divergence, genetics,and speciation among Lampsius (Bivalvia: Union-idae). Journal of Molluscan Studies, 49:133-145.
KAT, P.W. 1986. Hybridization in a unionid fauna!suture zone. Malacologia, 27:107-125.
KAT, P.W. & DAVIS, G M. 1984. Molecular geneticsof peripheral populations of Nova Scotian Union-idae (Mollusca: Bivalvia). Biological Journal of theUnnean Society of London, 22:157-185.
KINZELBACH, R. 1989. Freshwater mussels (genusAnodonta) from Anatolia and adjacent areas(Bivalvia, Unionidae). Zoology in the Middle East,3:59-72.
KINZELBACH, R. & NAGEL, K.-O. 1986. Redescrip-tion of the gloctudium of Pseudanodonta com-planata (Bivalvia, Unionidae). Verhandlungen desNaturwissenschaftlichen Vereins Hamburg, 28(N J . ) : 65-74.
Kiss, A. 1992. The propagation, growth and biomassof the Chinese huge mussel (Anodonta woodianawoodiana Lea, 1834). University of AgriculturalSciences GOdOUO (Hungary). Internal workreport, 1-33.
KODOLOVA, O.P. & LOGVINENKO, B.M. 1974.Comparison of different populations of bivalvemolluscs of the genus Anodonta (Unionidae) bysystems of myogens and shell morphology. Zoo-logiceskij Zhurnal. 53: 531-545. (in Russian)
MODFI i , H. 1945. Die Anodontinae, Ortm. emend.(Najad., Mollusca). - Eine Studie flber die Zusam-menhange von Klimazonen und Entwicklungs-geschkhte (Klimazonentheone). Jenaische Zeit-schnft fUr Medizm und Natunvissenschaften, 78:58-100.
by guest on February 22, 2013http://m
ollus.oxfordjournals.org/D
ownloaded from
ANODONTINAE POPULATION GENETICS 357
MODELL, H. 1951. Die Najaden Vorderasiens. Revuede la Faculti des Sciences de VUruvenui d' Istan-bul, Ser B, 16: 351-366.
MODELL, H. 1964. Das natUrliche System derNajaden. 3 Archiv fUr Molluskenkunde, 93: 71-126.
NAGEL, K -O 1985 Glochidien und Fortpflanzungs-biologie von Najaden des Rheins (Bivalvia-Union-ldae-AnodonUnae). Marnier Naturwissenschaft-Uches Archiv, Beiheft, 5. 163-174, plates 1 and 2.
NAGEL, K.-O. 1988. Anatonusche, morphologiscbeund biochemische Untersuchungen zur Taxo-nomie und Systematik der europaischenUnionacea (Mollusca: Bivalvia). PhD thesis. Uni-versity of Kassel, 1-100
NEI, M. 1971 Interspecific gene differences and evo-lutionary time estimated from electrophoretic dataon protein identity American Naturalist, 105- 385-398.
NEI, M. 1978. Estimation of average heterozygosityand genetic distance from a small number of indi-viduals. Genetics, 89- 583-590.
ORTMANN, A.E. 1912. Notes upon the families andgenera of the najades. Annals of the CarnegieMuseum, 8: 222-365, plates 18-20.
PETRO, E. 1984. The occurrence of Anodonta woodi-ana in Hungary. Allaaani Kdzlemtnyek, 71 189-191. (in Hungarian)
PORTER, A.H. 1990 Testing nominal species bound-aries using gene flow statistics: the taxonomy oftwo hybridizing admiral butterflies (Ltmenuv.Nymphaiidae). Systematic Zoology, 39: 131-147.
RICHARDSON, B J., BAVERSTOCK, P.R. <t ADAMS, M1986. Alloiyme electrophoresis. Academic Press,Sydney.
ROHLF, FJ. 1988. NTSYS-pc, Numerical taxonomyand mulnvanate analysis system (Manual). ExeterPublishing, New York
SARKANY-KISS, A. 1986. Anodonta woodiana (Lea,1834), a new species in Romania (Bivalvia,Unionacea). Travaux du Mustum d'HistoireNaturelle Gngore Anapa, 28: 15-17
SLATKIN, M. 1981 Estimating levels of gene flow innatural populations. Genetics, 99: 323-335.
SWOFTORD, D.L & SELANDER, R.B. 1989. BIOSYS-1, version 1.7 (Manual), 1-41. Illinois Natural His-tory Survey.
WEIR, B.S. & COCKERHAM, C C. 1984. Estimating F-stanstics for the analysis of population structure.Evolution, 38:1358-1370.
WRIGHT, S. 1931. Evolution in Mendelian popula-tions. Genetics, 16: 97-159
WRIGHT, S. 1978 Evolution and the geneacs of popu-lations. Vol. 4. Variability within and among natu-ral populations University of Chicago Press,Chicago.
by guest on February 22, 2013http://m
ollus.oxfordjournals.org/D
ownloaded from
by guest on February 22, 2013http://m
ollus.oxfordjournals.org/D
ownloaded from
