AN ABSTRACT OF THE ThESIS OF
John H. McDonald
Oceanography
Title:
for the degree of
presented on
Master of Science
in
December 16, 1983.
Ecological Genetics of Two Polymorphic Enzymes in
Three Species of Megalorchestia (Amphipoda: Talitridae)
Abstract
Redacted for privacy
The enzymes inannose phosphate isomerase (MPI, E.C.
5.3.1.8) and phosphoglucose isomerase (PGI, E.C. 5.3.1.9)
are polymorphic in eight Oregon populations of the
beachhopper Megalorchestia californiana (Amphipoda:
Talitridae) when examined using gel electrophoresis.
Many aspects of the data on these enzymes are consistent
with the hypothesis that the polymorphisms are selectively
neutral:
genotype proportions within populations do not
differ significantly from Hardy-Weinberg expectations;
there is no non-random association of Mpi and Pgi alleles;
allele frequencies do not differ significantly between
February and August samples, between males and females,
or between juvenile and adults; and the geographic patterns
of allele frequency within Oregon are consistent with
random drift.
A series of 12 samples from northern Washington to
southern California displays gradients in allele frequency
at both the Mpi and Pgi loci in M. californiana. Five
samples of another amphipod species, M. benedicti, also
show latitudinal dines in Mpi and Pgi, and two samples
of M. corniculata differ in Pgi allele frequency.
When
these data are combined with information on Pgi dines
reported for other crustacean species, a significant
association of electrophoretically faster PGI allozymes
with lower latitudes is apparent.
This non-random pattern
is evidence that selection is acting on the Pgi locus.
Because of its variation with latitude and its effects on
enzyme function, temperature is suggested as a possible
selective agent.
Ecological Genetics of Two Polymorphic Enzymes
in Three Species of Megalorchestia (Amphipoda: Talitridae)
by
John H. McDonald
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed December 16, 1983
Commencement June 1984
APPROVED:
Redacted for privacy
I*its
in
arge of major
Redacted for privacy
ege
Redacted for privacy
Dean of Graduaè School
Date thesis is presented
December 16, 1983
Typed by researcher for
John H. McDonald
ACKNOWLEDGEMENTS
I would like to thank my parents, Howard and Josephine
McDonald; the members of my committee, Drs. Jeff Gonor, Joe
Siebenaller, Pete Dawson, and Jim Winton; Ric Brodeur and
Jon Shenker, for assistance with transportation; and Bronwyn
Carlton, for assistance in the field.
Special thanks go
to my major professor, Jeff Gonor, for his advice and support,
and to Joe Siebenaller for taking time from his duties as
head of the Newport Institute for Cool Ocean Science to try
to cure me of naive pan-selectionism.
TABLE OF CONTENTS
INTRODUCTION
MATERIALS AND METhODS
Population sampling
Electrophoresis
Test crosses
Data analysis
15
15
17
21
22
RESULTS
24
DISCUSSION
39
BIBLIOGRAPHY
48
LIST OF FIGURES
Figure
Page
1.
Pigment patterns of Megalorchestia pugettensis
from Oregon.
16
2.
Locations of Megalorchestia samples.
18
3.
Schematic diagram of MPI and PGI phenotypes
of Megalbrchestia species.
25
Mpi allele frequency in samples of Megalorchestia
californiana.
32
Pgi allele frequency in samples of Megalorchestia
californiana.
33
Pgi allele frequency in samples of Megalorchestia
benedicti and M. corniculata.
36
Mpi allele frequency in samples of Megalorchestia
benedicti.
38
4.
5.
6.
.
LIST OF TABLES
Table
Page
1. Talitrid ainphipod species collected at the
locations shown in Fig. 2.
19
2. Genotype counts in Megalorchestia californiana
samples.
27
3. Allele frequencies of Megalorchestia californiana
samples.
28
4. Genotypes of offspring from a cross of
Megalorchestia californiana.
29
5. Allele frequencies of Megalorchestia benedicti
and M. corniculata samples.
35
6. Mpi and Pgi latitudinal dines.
45
ECOLOGICAL GENETICS OF TWO POLYMORPHIC ENZYMES IN THREE
SPECIES OF NEGALORCHESTIA GAMPHIPODA: TALITRIDAE)
INTRODUCTION
Natural populations of animals contain large amounts of
genetic variation, whether observed in single-locus traits
or quantitative, multiple-locus traits (Nevo 1978,
Lewontin 1974 p. 86-94).
This variation may be maintained
by balancing selection, by mutation and random drift of
neutral alleles, or by a combination of the two processes.
Estimating the proportion of polymorphic loci maintained
by selection is important for understanding genetic
differences between populations and the genetics of
speciat ion.
In this study gel electrophoresis of two polymorphic
enzymes in three congeneric amphipod species was used to
determine genotype proportions within populations and
allele frequency differences among populations.
The
population sampling was designed to seek several types of
evidence which if found would imply that selection was
affecting the loci.
Evidence was sought for: 1. genotype
frequencies which deviate from Hardy-Weinberg expectations;
2. non-random association of alleles at the two loci;
3. differences of allele frequency among age and sex
classes; 4. seasonal variation of allele frequency;
2
5. repeated patterns of variation along environmental
gradients in several estuaries; and 6. latitudinal
differences of allele frequency repeated in several species.
The species investigated in greatest detail was the
talitrid amphipod Megalorchestiacaliforniana, but additional
data were obtained on the geographical patterns of genetic
variation in M. bênedicti and M. comiculata.
These
ainphipods, formerly included inOrchestoidea (see Bousfield
1982), are commonly known as "beachhoppers" or "sand fleas."
Their natural history and distribution are described by
Bousfield (1956, 1957, 1959, 1982), Bowers (1963, 1964),
Craig (1973), Enright (1961), Fawcett (1969) and McClurkin
(1953).
Megalorchestia spp. are in many ways good subjects for
a study of ecological genetics.
Because these amphipods
are weak swimmers, sink rapidly in water, and only emerge
from their burrows to forage for food when the tide is
low, dispersal is limited between beaches separated by
rocky headlands, river mouths and other barriers.
Such
limited dispersal prevents genetic differentiation from
being swamped by migration, thus increasing the chance
that weak selection will produce detectable differences
among populations.
Megalorchestia spp. are present in
densities of thousands of individuals per linear meter
of shoreline (Bowers 1964); this abundance not only makes
3
large samples easy to capture, it also decreases the
likelihood that random effects will mask differences due
to selection.
M. californiana ranges from southern
California, with a warm, dry coastal climate, to Oregon,.
Washington an& British Columbia, with a cooler, wetter
coastal climate.
This species also inhabits both oceanic
and estuarine beaches.
Such a wide range of environmental
variation among habitats of local populations might
produce genetic differences among these populations.
Since the mid-l960's, when electrophoresis was first
used to detect the widespread occurrence of enzyme
polymorphisms, a number of approaches have been tried to
determine what proportion of these polymorphisms are
maintained by selection.
Various statistical tests using
average heterozygosity (e.g. Weir et al. 1976, Ewens and
Feldman 1976, Watterson 1978, Nei 1983) and comparisons
of heterozygosity among species (Powell 1975, Nevo 1978)
have not provided convincing evidence, in part because
almost any pattern may be explained with either a neutral
or a selective model (Lewontin 1974).
Attention is now
turning to examination of individual loci.
This
locus-by-locus approach will answer the question only if
most loci are found to have a selective basis, because
selection could be too weak to be detected yet still
play an important role in maintaining a polymorphism (Lewontin
4
1972, Clarke 1975).
Method which hayebeen used to
detect selection affecting individual loci are sarized
and critically reviewed below; only some of these approaches
are applied to Megalorchestia, but for completeness all
of the major ones are included.
A polymorphic locus with selectively neutral alleles
is affected by random drift, which in turn depends on
mutation rate, population size and migration rate from
other populations.
The theory of a locus governed only by
drift is well developed (Nei 1975, 1983), providing null
hypotheses for attempts to detect selection.
The most
convincing studies test these neutral hypotheses, rather
than using selection as an ad hoc explanation of some
observed pattern.
Several types of evidence may be used to
test neutrality, including seasonal and long-term variation
of allele frequency, conformance to Hardy-Weinberg
expectations, selection component analysis, and geographic
patterns of allele frequency.
In a large population with no immigration, the
genotype frequencies of a neutral polymorphism should fit
Hardy-Weinberg expectations.
Therefore one simple and
often used method to look for selection is to examine
deviations from Hardy-Weinberg proportions.
Unfortunately,
with reasonable sample sizes only very strong selection can
be detected this way, and some patterns of selection will
produce genotype frequencies in perfect agreement with
5
Hardy-Weinberg expectations (Lewontin and Cockerham 1959).
Deviations from Hardy-Weinberg expectations have been
observed in a number of species.
A deficit of heteirozygotes
occurs at one or more loci in several marine benthic
invertebrates, including the oyster Crassostrea virginica
(Zouros et al. 1980), the lobster Homarus amerIcanus
(Tracey et al. l975b), the sand crab Eerita talpoida
(Corbin 1977), the phoronid Phoronopsisviridis (Ayala et al.
1974b), and the mussels Mytiluscalifornianus (Tracey et al.
1975a, Levinton and Suchanek 1978), Mytilus edulis (Koehn
et al. 1976), and Modiolus demissus (Koehn et al. 1973).
There are several possible explanations for these observations.
Mixing of planktonic larvae from differentiated subpopulations
(the Wahlund effect [Wahlund 1928])is one possibility;
however, only for 14. edulis have large differences of
allele frequencies between subpopulations been found
(Koehn et al. 1976).
Partial assortative mating, for instance
if genotypes breed at different times of the year, is
another possible explanation.
The land isopod Porcellio
scaber exhibits a heterozygote deficit at a lactate
dehydrogenase locus in progeny of wild-caught gravid
females; partial assortative mating due to mate choice
is suggested as the reason (Sassaman 1978).
Selection
against heterozygotes, as suggested for Einerita (Corbin
1977), is unlikely on theoretical grounds because one
allele would be expected to become fixed rather than the
locus remaining polymorphic.
Inadvertent sampling of
two cryptic species (Makela and Richardson 1977) or the
presence of an undetected null allele could also produce
an apparent deficit of heterozygotes, so a heterozygote
deficit cannot always be taken as unequivocal evidence
that selection is affecting a locus.
An excess of heterozygotes, as in the prairie dog
çyomys ludovicianus (Foltz and Hoogland 1983) and the
butterfly Daflaus plexippus (Eanes and Koehn 1978), may
result from selection favoring heterozygotes, different
allele frequencies in males and females, or dissassortative
mating.
An increase in the proportion of heterozygotes
with increasing age of a cohort, as in Colias butterflies
(Watt 1977), the toad Bufo boreas (Samollow and Sou1
1983), the mussel Modiolus demissus (Koehn et al. 1973),
the fish Fundulus heteroclitus
Mitton and Koehn 1975),
and the land isopod Porcellio scaber (Sassaman 1978),
may result from heterozygote advantage.
At a locus with
more than two alleles, certain patterns of mixing of
differentiated populations could produce heterozygote
excess (Milkman 1975); no other random process is known
which increases the proportion of heterozygotes, so
heterozygote increase or excess is generally evidence that
selection of some type is acting (Lewontin 1974, p. 242).
7
A powerful technique for detecting selection is
selection component analysis.
The genotype frequencies of
gravid females, their progeny, adult males and non-gravid
adult females, along with the fecundity of each female
genotype, are used to test for viability, fecundity and
sexual selection.
A neutral polymorphism would exhibit
identical genotype frequencies in each class of the
population and equal fecundities among genotypes, so that
significant differences are evidence of selection (Christiansen
and Frydenberg 1973, Christiansen et al. 1977, Ostergaard
and Christiansen 1981, Nadeau et al. 1981).
The approach
has been little used, probably because of the large number
of mother-offspring pairs required to detect anything
but strong selection (Christiansen and Frydenberg 1973).
The fish Zoarces viviparus exhibits a heterozygote deficit
at an esterase locus in adults; selection component
analysis suggests that it is due to viability selection
(Christiansen et al. 1977).
Opposing gametic and sexual
selection may affect a hemoglobin polymorphism in the
mouse Peromyscus maniculatus (Nadeau and Baccus 1981), but
the results may be an artifact of two-locus control of
hemoglobin (Snyder 1983).
Even if mother-offspring pairs
are not collected, some components of selection can be
analyzed by comparing age and sex classes.
For instance,
allele frequencies at an aminopeptidase locus differ
between newly settled juveniles and adults of the mussel
Mttilusêdulis in parts of Long Island Sound, and this
difference has been observed in each of three years
(Koehn et al. 1980).
Because neutral alleles at polymorphic loci would
be randomly associated, linkage disequilibrium, the non-random
association of alleles at two loci, may indicate that some
sort of selection is acting (Thomson 1977).
Linkage
disequilibrium might also result from mixing of differentiated
subpopulations (Feldman and Christiansen 1975).
Examples
of linkage disequilibrium of allozyme loci in natural
populations are rare (see Barker 1979 for review) and
include two esterase loci in the fish Fundulus heteroclitus
(Mitton and Koehn 1975), two aminopeptidase loci in
Mytilus edulis (Mitton and Koehn 1973), and two esterase
loci in the salamander Plethodon cinereus (Webster 1973).
The sample size required to detect linkage disequilibrium
depends on allele frequency and the intensity of selection,
and is rather large for most reasonable values (Brown 1975).
In a large population affected only by mutation
and random drift, any change of allele frequency should
be very slow (Kimura and Ohta 1971).
A large change of
allele frequencies seasonally or over a few generations is
thus evidence that selection is occurring, with the
important assumption that immigration from differentiated
populations inus.t be xuled out.
Seasonal and long-term
changes in frequencies: of chrômosna1 InversIons
Dobzhansky
1970). and long-term changes in frequency of pigmentation
alleles (Kettlewell 1973) are classic demonstrations of
selection, but there are very few cases of temporal change
in allozyme allele frequency.
In a pond population of
largemouth bass (Micropterus salmoides), after the input
of heated water from a power plant stopped, the frequency
of one malate dehydrogenase allele decreased over seven
years from the high frequencies found in another, still-heated
pond to the lower frequencies of an ambient temperature
pond (Smith et al. 1983).
Many polymorphic loci exhibit geographical differentiation,
often in the form of a dine, a gradual frequency change
with geographic distance.
By itself, a dine usually
cannot be taken as evidence of selection, because the
random drift of isolated populations followed by migration
inay.also produce a dine (Endler 1977).
An exception is any
dine observed over a short distance in a widely dispersing
organism, because migration should rapidly swamp any random
differentiation.
Such dines occur in many benthic marine
invertebrates with long-lived planktonic larvae, which is
evidence that selection is maintaining the differentiation
in spite of migration (see Burton and Feldman 1982,
Burton 1983 for reviews).
10
In some species, certain loci show. large differences
of allele frequency between populations, while other
polymorphic loci are uniform over all populations.
This has
been taken as evidence that selection affects at least some
of the loci; it has been thought that if all loci were
neutral they would either all be geographically uniform,
due to migration between populations, or they would all be
geographically heterogeneous, due to isolation and small
population size cCavalli-Sforza 1966., Christiansen and
Frydenberg 1974, Ayala et al. l974a).
The statistical
treatment of such data tests for heterogeneity of
standardized variances of allele frequency (Lewontin and
Krakauer 1973). The method requires several assumptions
which are not often met (Nei and Maruyama 1975, Robertson
1975) and which therefore limit its use to certain types
of data (Lewontin and Krakauer 1975, Tsakas and Krimbas
1976).
Significant heterogeneity of allele-frequency
variance has been detected with this test in the butterflies
Euphydryas editha and
.
chalcedona (NcKechnie et al. 1975)
and the fruit fly Dacus oleae (Tsakas and Krimbas 1976).
The test does not determine which loci selection is affecting,
only that selection is acting.
Clines are often associated with environmental gradients.
A correlation between an environmental variable and a dine
could be a coincidence.
However, if the pattern of allele
11
frequency difference is repeated inseyeral places,
always associated with the saineènvironmental difference,
the non-random pattern implies that selection is acting.
Given the large number of dines reported in the literature,
it is surprising how few have been compared this way.
At
five of six polymorphic loci in DrOsophila melanogaster,
latitudinal dines with similar patterns of allele frequency
are present in Australia, Europe and North America
(Oakeshott et al. 1981, 1982, 1983).
Alleles at an
aminopeptidase locus in the mussel Mytilus edulis covary
with salinity in two creek estuaries (Boyer 1974) and on
both shores of Long Island Sound (Koehn et al. 1976,
Lassen and Turano 1978).
At an aminopeptidase locus in
the ectoproct Schizoporella errata, the same allele is
in high frequency in the warmer temperatures of the southern
Atlantic coast of North America and in warmer inshore
waters of Massachusetts (Gooch and Schopf 1971).
At a
malate dehydrogenase locus in Pundulus heteroclitus, one
allele is commoner both in southern waters (Powers and
Place 1978) and in an artificially heated pond (Mitton and
Koehn 1975).
A lactate dehydrogenase locus in the sockeye
salmon, Oncorhynchus nerka, exhibits dines in both North
America and the Kamchatka Peninsula; in both areas, the same
allele is commoner in the north (Kirpichnikov and Ivanova
1977).
In contrast is a lactate dehydrogenase locus of the
12
marine fish Anoplarchuspurpurescens.
An allele which is
commoner in the warmer waters of southern Puget Sound
than in the northern Sound (Johnson 1971) becomes less common
towards the south on the outer Pacific coast (Yoshima and
Sassaman 1983).
All of the above approaches may implicate selection,
but they cannot distinguish between selection acting
directly on the locus examined arid selection acting on a
closely linked locus (Thomson 1977).
More convincing
evidence is provided if biochemical differences exist
between the allozymes and if these differences are consistent
with the postulated selective mechanism (Clarke 1975,
Koehn 1978).
If there are similar dines in more than one species,
these comparative data may provide evidence of a role for
The
selection in maintaining the dines (Clarke 1975).
ideal comparison would be of dines in related species
sharing homologous polymorphisms, where each allozyme
in one species was identical in amino acid sequence with
the corresponding allozyme in the other species.
However,
not many groups of species share electrophoretically
identical sets of allozymes, and electrophoretically
undetectable amino acid substitutions can have profound
effects on enzyme function (Siebenaller and Somero 1978).
For the present study a broader definition of similar dines
13
is used:
dines are considered "similar" if, for example,
the allele for the Laster migrating allozyme is commoner
in the same environment for each species.
Such similar
dines can only be expected if differences in net charge
between allozymes, which deteiine relative electrophoretic
mobility, are somehow related to adaptation to environment.
Because dines caused by random differentiation would be
similar only by chance, similar dines would be evidence
of selection.
And because even the tightest linkage
disequilibrium would not be expected to persist in separate
species, similar dines would suggest that selection is
acting directly on the locus examined (Clarke 1975).
Each of the few examples of similar
dines involves
only two species, and thus the similarity might be
coincidental.
Two cryptic species of crickets, Gryllus
velétis and C. pennsylvanicus, have similar frequencies
of electrophoretically identical phosphoglucose isomerase
(Pgi) alleles in sympatric populations (Harrison 1977),
although the geographic variation is not a smooth dine
(Harrison 1979).
A Pgi allele, Pgi-A6, shared by the
fish Menidia peninsulae and M. beryllina shows similar
patterns, of variaton in both species along the shore of
the Gulf of Mexico (Johnson 1974).
PGI-A6 is the faster of
the two common PGI allozymes in N. peninsulae, but is the
slower of the two common aliozymes in N. beryllina
14
(Johnson 1975), and thus the dines are not similar by
the definition used here.
At an esterase locus in the
snails Littorina saxatilis and L.obtusata, the allele
for the slowest allozyme becomes commoner with distance
up an estuary in both species, although the dine is not
very smooth in L. obtusata (Newkirk and Doyle 1979).
In the mussels Mytilus edulis and Modiolus demissus,
the allele for the fastest aminopeptidase allozyme is
commonest at the same location in both species (Mitton and
Koehn 1973).
15
NPTERIALS AND METHODS
pulation sampling
Most of the amphipods used in this study were captured
at night with pitfall traps, plastic cups buried flush with
the sand and with 2 to 3 cm of seawater in the bottom.
A
few locations were sampled during the daytime by digging
under piles of drift seaweed.
Amphipods were kept alive
in the laboratory on crumpled moist paper towels in plastic
containers.
activity.
They were kept at 3 C to greatly reduce their
Mortality during capture, return to the laboratory
and storage of up to several weeks was negligible.
There
was no apparent difference in enzyme phenotype between
freshly caught amphipods and those which had been maintained
in the laboratory for several weeks.
Mêgalorchestia species were first identified using
morphological keys (Bousfield 1975, 1982), after which the
distinctive pigment pattern of each species (Bowers
1963, 1975) was used for routine identification.
M. pugettensis from Oregon
The
are more heavily pigmented
than those from California illustrated by Bowers, and might
be confused with M. benedicti; the pigment pattern of
M.pugettensis from Oregon is given in Fig. 1.
Bousfield
(1959, 1982) notes subtle morphological differences
between southern and northern M. pugettênsis; further
Figure 1. Pigment patterns of Megalorchestia
pugettensis from Oregon. a, dorsal view
of pleonites and pereonites 5, 6 and 7.
b, side view.
I0'
17
investigation is needed to determine w:hether M, '1pugettensis"
represents one or two species.
Locations sampled during this study are given in
Fig. 2, and the species found at each location are listed
in Table 1.
In most cases the objective of sampling was
to collect M. californiana, and only approximate counts
were made of the other Megalorchestia species present.
Individuals from the Siuslaw-Mouth samples were
divided into size and sex classes to determine if there
were differences in allele frequency between these classes.
Each individual was weighed to the nearest 10 mg.
The sex
of amphipods weighing 20 mg or more was easily determined,
using the enlarged second gnathopod of males, and these
individuals are here considered"adults."
Amphipods weighing
less than 20 mg are classified as "juveniles."
Electrophoresis
Vertical polyacrylamide gel electrophoresis was used to
screen for polymorphic enzymes in M. californiana.
The
following enzymes were found to be monomorphic in samples
from Oregon, although in several cases only a few individuals
were examined:
acid phosphatase, alcohol dehydrogenase,
aldehyde oxidase, alkaline phosphatase, glutamate dehydrogenase,
a-glycerophosphate dehydrogenase, isocitrate dehydrogenase,
lactate dehydrogenase, leucine aminopeptidase, malate
dehydrogenase, malic enzyme, octanol dehydrogenase, sorbitol
E:I
4B
k
i'
I
C
A('
b. Yaquina Bay
1 km
c. Siuslaw River
It
6A
1km
d. timpqua Rive
1
7
_35?..
e. Coos Bay
Figure 2. Locations of Megalorchestia samples. Numbers
correspond to names of locations given in Table 1. Stippling
represents areas of sand beach.
19
Table 1. Talitrid amphipod species collected at the locations
shown in Fig. 2.
Counts are approximate. Some samples
from which only one species was kept are not listed.
All dates are 1983 unless otherwise noted. NC: present
in sample but not counted. M.cal: Megalorchestia
californiana. M.col: M. columbiana. M.pug: M.
pugettensis. M.ben: Mbenedicti. M.cor: M. corniculata.
Trask: Traskorchestiaipp.
date
1. Port Townsend, Wash.
Fort Worden S.P.
2. Neskowin, Ore.
3. Boiler Bay
4. Yaquina Bay
A. South Beach
A. South Beach
8.
M.ben
16 July 82
10 Jan
28 Oct 82
340
17
S
101
100's
27
25
6
7Nov
lOJan
lOJan
5 April
20 April
B.
l9Jan
B.
C.
27
12
27
15
Feb
July
Feb
Aug
F.
l9Jan
l9Jan
6. Florence
6. Florence
20 April
12 July
E.
M.pug
93
248
0.
E.
0. Mouth
0. Mouth
M.col
17 July
3 July
6 Sept
C.
5. Siuslaw River
A. North Jetty
A. North Jetty
M.cal
M.cor
Trask
NC
26
70
2
3
86
13
2
9
43
70
4
20
153
663
307
14
3
40
25
30
21
60
100
50
50
20
40
100
6
3
1
1
10
2
1
200
500
20
30
6. Lhspqua River
A.
A.
B. Winchester
7. Coos Bay
A. Bastendorf Beach
B.
C. Empire
D.
0.
E.
F.
G. North Slough
8. Hunboldt Bay, Calif.
B side of N spit
9. Bodega Bay
A. S side of spit
B. N side of spit
10. San Francisco
Esplanade
11. Monterey
N of N jetty
12. Carmel
13. Pismo Beach
14. Santa Barbara
A. L.eadbetter Beach
B. B of wharf
15. Orange County
A. Bolsa hica
B. Reef Point
l9Jan
27 Feb
27 Feb
12
255
6 May
300
2Oct82
18 June
2 Oct 82
6May
2 Oct 82
2 Oct 82
18 June
1 Jan
1
40
200
24
12
NC
200
163
15
26
300
30
1
10
197
10
3
100
30
15
100's
100's
S
300
9
8
31 Dec 82
31 Dec 82
12
11 Aug
29
23 Aug
23 Aug
23 Aug
28
22
1
44
14
6
100
22 Aug
22 Aug
29 Dec 82
29 Dec 82
2
1
5
7
2
15
34
1
3
40
110
19
3
7
3
20
dehydrogenase, and tetrazoliuiu oxidase.
Several other
enzymes which were tried did not resolve well enough for
further use.
Glutamate pyruvate transaminase (GPT,
E.C. 2.6.1.2) was found to be polymorphic using the
buffer system and stain of Burton and Feldman (1983).
While some GPT gels yielded clear one- and three-banded
phenotypes, many others could not be scored due to smearing
or lack of activity, and GPT was not investigated further.
Two enzymes resolved well and were polymorphic in
M. californiana:
mannose phosphate isomerase (MPI, E.C.
5.3.1.8) and phosphoglucose isomerase (PGI, E.G. 5.3.1.9).
These enzymes are commonly polymorphic in other amphipods
(Bulnheim and Scholl 1981, Gooch and Hetrick 1979)
crustaceans (Hedgecock et al. 1982).
and
For MPI, the
continuous tris-borate buffer of Ayala et al. (1973) was
used: gel and electrode 87 mM tris, 8.7 mM boric acid,
pH 9.1 at room temperature.
Poulik's buffer system
(Poulik 1957) was used for PGI: gel 76 mM tris, 5 mM citric
acid, pH 8.65 at room temperature; electrode 300 inN boric
acid, 60 inN NaOH, pH 8.1.
A single leg (or several legs from the smallest
individuals) was pulled off each amphipod and macerated
in one drop of 5 percent sucrose in distilled water.
Each
amphipod used was then maintained alive in an individual
vial with a scrap of moistened paper towel.
These amphipods
21
remained alive for up to a week in the vials, and another
leg was used if there was any ambiguity about the scoring
of the enzyme phenotype of an individual.
Both MPI and
PGI phenotypes were determined for each individual.
For
MPI 5 to 10 p1 of crude homogenate were loaded into each
sample well of the gel, and 1 to 2 p1 samples were used
for PGI.
Pairs of gels 16 by 17 cm, 1.5 mm thick, and containing
7 percent acrylamide monomer were run in a 3 C room with
8 mA of current for 30 minutes followed by 50 mA for 3 hours.
The MPI stain contained 5 mg mannose-6-phosphate, Ba salt;
5 mg NADP; 20 units glucose-6-phosphate dehydrogenase
(G6PDH); 20 units phosphoglucose isomerase; 40 mg MgC12;
10 rng MTT; and 3 mg phenazine methosulfate (PMS) in100ml' .05 M
tris-HC1, pH 8.0 at room temperature.
The PGI stain
contained 30 mg fructose-6-phosphate, disodium salt; 5 mg
NADP; 10 units G6PDH; 80 mg MgCl2; 10 mg MTT; and 3 mg
PMS in 100 nil .05 M tris-HC1, pH 8.0.
All chemicals were from
Sigma (St. Louis) except MgC12, which was reagent grade.
Test crosses
A number of single-pair crosses of M. californiana were
attempted.
An adult male and female of known genotypes
were put in a glass vial with moistened tissue paper and
were kept in the dark at room temperature.
The offspring
which resulted from one of these crosses were ground
whole for.electrophoresis, due to their small size.
Data analysis
Deviations from Hardy-Weinberg proportions of genotypes
were examined using Wright's F statistic (Brown 1970). F is
estimated using equation 1
4a1a3 - a2
(1).
2
(2a1 + a2) (2a3 + a2)
where a1 is the number of homozygotes of one allele, a2 is
the number of heterozygotes, and a3 is the number of homozygotes
of the second allele.
heterozygotes, while a negative
heterozygotes.
indicates a deficit of
A positive
indicates an excess of
Statistical significance is tested using
d = F/i, where x is the number of individuals (Christiansen
et al. 1976). A value of d greater than 2 or less than -2
indicates a significant departure from Hardy-Weinberg
proportions at the pc05 level.
Because both MPI and PGI phenotypes were determined for
each amphipod, it was possible to examine non-random
association of Mpi and Pgi alleles.
The linkage disequilibrium
index D of Hill (1974) was calculated for samples with more
than 30 individuals.
The alleles at one locus are designated
A and a, with frequencies p and (1
p); the alleles at the
second locus are B and b, with frequencies q and (1 - q).
If A and B are randomly associated, the frequency of AB
23
chromosomes, f11, is equal to p times q.
The linkage
disequilibrium index D is given by D = f11 - pq.
Because
the types of chromosomes cannot be counted directly,
is estimated using equation 2
N2211(l-p-q+f11)
2
f
11
+ (p - f11)(q - f11)
+
1l
2N
where
= estimated number of AB chromosomes
X11 = (2 x number of AABB genotypes) + number of AABb
genotypes + number of AaBB genotypes
N22 = number of AaBb genotypes
= estimated frequency of A
q
= estimated frequency of B
N
= number of individuals.
An estimate of
is entered in the equation, and it is
iterated until stable.
D is then found using D
- pq.
Significant non-random association of alleles is indicated
when D > 1.96
(l -
)cl
)
(Brown 1975).
N
Differences in allele frequency between samples were
tested with the G-test (Sokal and Rohif 1981, p.. 705-707).
24
RESULTS
MPI phenotypes were either one- or two-banded in
both males and females (Fig. 3a).
This is consistent
with MPI being a monomeric molecule coded by an autosomal
locus with codominant alleles, as found in other crustaceans
(Hedgecock et al. 1982).
The faster common allozyme in
M. californiana was designated MP110° and the slower common
allozyme MP190.
Alleles for three rare allozymes were
pooled with the allele for the closest allozyme for
statistical purposes.
PGI phenotypes were either one- or three-banded in
both males and females (Fig. 3b)
.
This is consistent with PGI
being a dimeric molecule coded by an autosomal locus
with codominant alleles, as found in other crustaceans
(Hedgecock et al. 1982).
The faster common allozyme in
M. californiana was designated PG110°
allozyme PG195.
and the slower common
Two rare alleles were pooled with the
alleles for the closest allozymes for statistical purposes.
One individual had a two-banded phenotype, which was
repeated in three runs; this pattern implies an inactive
allozyme presumed to have the same mobility as PG195, and
the allele for this null allozynie was pooled with Pgi95.
Accurate scoring of enzyme phenotypes is essential
whenever allele or genotype frequencies are used.
Whenever
one enzyme was repeated due to ambiguity in scoring of an
___
a. MPI
V
+
90
90
90
100
100
100
85
90
96
100
100
105
85
85
80
85
90
90
M. benedicti
Megalorchestia californiana
90
90
M. corniculata
b.PGI
100
100
95
100
95
95
90
100
M. californiana
null
100
100
105
100
100
95
100
90
100
M. benedicti
100
105
100
105
100
100
95
100
M. corniculata
Figure 3. Schematic diagram of MPI and PGI phenotypes of Megalorchestia species. The presumed
Alleles are numbered according to the relative
genotype is given under each phenotype.
The origin is
mobility of their enzyme product under the electrophoretic conditions used.
at the top and the anode at the bottom of each diagram.
(
26
individual, the other enzyme was also repeated
it had been resolved well on the first run.
even though
Thus a number
of phenotypes were determined twice, which provided a
check on the accuracy of scoring.
Of 68 PGI phenotypes
which were scored twice, the second scoring agreed with
the first in every case.
Of 254 repeated MPI phenotypes,
only one was inconsistent; a third scoring of this
individual agreed with the second, and that phenotype
was used.
The
Only one of the crosses produced offspring.
Neither
genotypes of the offspring are given in Table 4.
MPI (G=.l39, p>.9) nor PGI (G=l.79, .5>p>.l) deviates
significantly from the number of phenotypes expected
The array of combined
from simple Mendelian inheritance.
MPI and PGI phenotypes does not differ significantly
from that expected if Mpi and Pgi alleles assorted
independently (G=2.lO, p>.9), so there is no evidence that
the Mpi and Pgi loci are genetically linked.
The results of the electrophoretic survey of M.
californiana are summarized in Tables 2 and 3.
None of
the samples showed a significant departure from Hardy-Weinberg
proportions of genotypes at the Pgi locus (Fpgi in Table 2).
At the Mpi locus, only adult males from the 28 February
Siuslaw-Mouth sample showed a significant deviation, with
fewer than expected heterozygotes (F
in Table 2).
The
Table 2. Genotype counts in Megalorchestia californiana samples. Locations are given in
Fig. 1.
Rare alleles were pooled with the allele for the cl9sest conunon allozyme.
N: number of individuals. D: linkage disequilibrium index. F: estimated inbreeding
coefficient. *: significantly different from F=O at the p<.05 level
Np
90
90
90
90
90
90
90
100
g
95
95
95
100
100
100
95
7
0
19
0
10
29
4
7
24
9
27
17
37
genotypes
,
1.
2.
S.
Port Townsend
Neskowin
Siuslaw River
SA. Siuslaw-North Jetty
90
100
90
100
100
100
100
100
100
100
95
100
100
100
95
95
95
100
100
100
4
61
0
33
53
17
13
0
33
11
11
30
31
7
13
21
19
36
48
29
32
38
99
18
8
10
81
10
16
18
44
7
6
3
7
2
3
12
8
15
29
110
5
9
10
18
9
25
78
68
223
6
79
4
48
8
142
14
23
4
95
N
0
19
93
209
- .016
- .013
24
17
162
.012
.109
-.018
27
27
29
83
18
16
187
191
-.006
-.011
-.063
.027
.088
26
60
260
638
.022
.003
.015
.035
-.086
-.023
8
12
12
32
115
S
10
65
88
102
255
893
.047
-.015
- .001
- .069
.046
.033
-.028
.018
- .133
.005
.003
-.013
-.025
.022
- .023
10
.006
.005
- .057
109
80
1135
.029
- .030
- .019
0
P
Mpi
Pgi
.005
- .022
- .092
-.105
SD. Sius law-Mouth
SD. Siuslaw-P4outh, 28 Feb
adult males
adult females
Juveniles
total, 28 Feb
SD. Siuslaw-Mouth, 15 Aug
adult males
adult females
Juvàniles
total, 15 Aug
SD. Siuslaw-Mouth, total
S.
65.
5G. Siuslaw-Plorence
Siuslaw River, total
(pqua-Winchester
Coos Bay
7A. Coos-Bastendorf
7C. Coos-Fmpire
7G. Coos-North Slough
7. Coos Bay, total
6
(
12
27
39
2
53
71
155
17
21
30
36
.156*
9
3
16
5
11
36
5
13
135
49
19
19
3
93
272
185
59
9
148
10
15
50
28
10
21
10
181
.013
-.029
-.042
24
4
12
14
39
56
33
64
30
12
5
12
10
27
39
8
15
.020
.109
.024
.033
- .017
10
18
284
67
- .005
4
- .180
.030
-.122
62
7
22
82
7.
10.
12.
14.
San Francisco
Car.el
Santa Barbara
40
28
67
37
lOS
65
20
9
20
49
115
53
183
534
2
6
3
7
2
1
0
1
3
6
9
1
8
2
0
15
0
0
0
0
27
28
0
4
0
0
0
0
0
15
3
5
12
-.004
-.030
- .014
-.122
.158
.035
.025
-4
Table 3. Allele frequencies of Megalorchestia californiana samples. Locations are given in
Fig. 2.
Rare alleles are not pooled. N: number of individuals.
Mpi
1. Port Townsend
2. Neskowin
Pgi
85
90
96
100
.005
.253
.419
.002
.747
.574
5. Siuslaw River
5A. Sius law-North Jetty
lOS
null
90
95
100
.978
.433
.022
.567
93
209
162
.481
.519
.003
.392
.605
.455
.469
.SO4
.479
.545
.531
.496
.521
.016
.016
.010
.013
.439
.408
.412
.418
.543
.576
.577
.567
.500
.432
.520
.484
.480
.500
.568
.480
.516
.520
.008
.005
.004
.011
.492
.409
.441
.443
.426
.463
.479
.537
.521
.013
.009
.003
.514
.483
.002
.371
.366
.372
.371
.625
.627
.626
.625
lOS
N
SD. Sius law-Mouth
SD. Siuslaw-l4outh, 28 Feb
adult males
adult females
juveniles
total, 28 Feb
SD. Siuslaw-Mouth, 15 Aug
adult males
adult females
juveniles
total, iS Aug
SD. Siuslaw-Mouth, total
SC. Siuslaw-Florence
5. Siuslaw River, total
6B. tbiapqua- Winches ter
7. Coos
7A.
7C.
7G.
7. Coos
Bay
Coos-Bastendorf
Coos-Empire
Coos-North Slough
Bay, total
8. Humboldt Bay
9 Bodega Bay
10. San Francisco
12. Carmel
14. Santa Barbara
15. Orange County
.001
.037
.196
.704
.500
1.000
.2S9
.304
.003
187
.002
.002
260
638
.S00
.585
.5S4
.551
.562
.001
65
88
102
255
893
.325
.414
.663
.575
.001
80
1135
.008
.467
.525
.005
.470
.537
.475
.482
.523
.463
.525
.516
.500
.417
.444
.304
.500
.S83
.537
.696
.867
1.000
.006
.002
.001
.002
.007
.003
.003
.003
.019
191
181
.002
.001
284
67
183
S34
8
12
27
28
.133
15
4
I'-)
00
Table 4.
Genotypes of offspring from a cross of Megalorchestia californiana.
were Mpi90' '00Pg
Mpi
Pgi
number of offspring
Both parents
9S/ 100
90
90
90
90
90
90
90
90
100
100
90
100
100
100
100
100
100
100
95
95
100
100
100
95
95
95
100
100
100
95
95
95
100
100
100
1
2
1
3
4
2
2
4
2
t'.)
'.0
30
deficit was not observed in males taken at the same location
on 15 August; with 23 samples one significant result
at the p <.05 level would be expected by chance.
None of
the samples exhibited significant non-random association of
Mpi and Pgi alleles (D in Table 2).
The Siuslaw-Mouth site was sampled twice, on 28 February
and 15 August 1983, to determine if allele frequencies
differed seasonally (Table 3).
Neither the Mpi (G=.04,
.9>p>.S) nor the Pgi (G=.44, .9>p>.5) locus differed
significantly in allele frequency between the two dates.
The two Siuslaw-Mouth samples were also divided into
juveniles, adult males and adult females.
None of the
pairwise comparisons of allele frequency between these
classes indicated a significant difference.
Allele frequencies of the sample from Siuslaw-Florence,
which is 6.7 km inside the Siuslaw River (Fig. 2), were
compared with the Siuslaw-North Jetty and Siuslaw-Mouth
samples in order to determine whether differences are present
which might be the result of differential selection along an
estuarine gradient.
The only significant frequency difference
found was between the Siuslaw-Florence and Siuslaw-Mouth
samples at the Pgi locus (G=6.04, .025>p>.Ol).
To determine
whether such a difference exists at another estuary, three
locations at Coos Bay were sampled (Fig. 2).
The Pgi allele
frequencies at Coos-Bastendorf, located outside the estuary,
31
were virtually identical to those at Coos-North Slough,
located 13.7 km inside Coos Bay (Table 3).
There is significant heterogeneity of Mpi allele.
frequency among the eight Oregon population samples
(G=21.14, .00S>p>.001), while Pgi allele frequencies are
not heterogeneous in Oregon (G=lO.49, .5>p>.l).
The
Mpi variation within Oregon does not form an obvious
pattern (Table 3).
On a larger geographical scale, the allele frequencies
of both Mpi (Fig. 4) and Pgi (Fig. 5) exhibit latitudinal
dines in H. californiana.
At the Mpi locus, Mpi100 is
highest in frequency in the northernmost sample, then
decreases gradually toward the south.
Pgi100 is
At the Pgi locus,
lowest in frequency in the northernmost
sample and highest in the two southernmost samples.
Rather
than a gradual dine, however, the greatest changes in
Pgi allele frequency occur between Washington and Oregon
and between southern and central California samples; the
samples from Oregon to central California are all similar
in allele frequency.
Both loci were surveyed with the same electrophoretic
procedures in two additional species, H. benedicti and
M. corniculata, to determine whether similar latitudinal
differences are present.
These two species exhibited
one- or three-banded phenotypes similar to H. californiana
1.
Mpi
4
I.
.4
35')
400
latitude
450
Mpi allele frequency in samples of Me&alorchestia californiana. Vertical bars
represent 95 percent confidence limits (Rohlf and Sokal 1981, Table 23). Mpif: MpilOO.
Figure 4.
(j.1
I'.)
1.00
.80
.60
+
L
Pgjf
.40
+4+
.20
.00
I
I
350
40°
45°
latitude.
Figure 5. Pgi allele frequency in samples of MegalOrchestia californiana.
as in Fig. 4.
.f
Pgi
:
.100
Pgi
Symbols are
34
Two of the common PGI allozymes in M.benedicti
(Fig. 3).
had electrophoretic mobility identical to the common allozymes
in M. californiana, and they were designated PG195 and
PGI
100
.
The slowest al].ozyme in M. benedicti has not been
run on the same gel alongside the rare slowest allozyme
in M. californiana, and its designation here as PG190 is
therefore tentative.
The commonest allozyme in M.
corniculata appeared identical in mobility with PG1100 in
M. californiana and M. benedicti, and it was therefore
named PG1100.
The other two allozymes in M. corniculata
were tentatively named PG195 and PG1105, although they
have not been run alongside the comparable allozymes
in the other species.
Like M. californiana, M. benedicti exhibits a latitudinal
dine in Pgi allele frequency (Table 5, Fig. 6).
14.
corniculata was only sampled at two sites; the frequencies
.105
.
.
.
of Pgi
in the two samples differ significantly (G=9.20,
pcz.00S).
In all three species it is the allele for the
electrophoretically faster allozyme--Pgi100 in M.benedicti
and M. californiana, Pgi105 in M. corniculata--which is
higher in frequency in southern samples.
Each of the M. corniculata individuals from the two
sites had a single banded MPI phenotype with the same
mobility as MPI9° in M. californiana (Fig. 3).
14. benedicti
Table 5. Allele frequencies of Megalorchestia benedicti and P4. corniculata samples. Locations
are given in Fig. 1. Rare alleles are not pooled. N: number of individuals scored for
each enzyme.
Mpi
P4.
4A.
10.
12.
13.
14.
M.
11.
14.
benedicti
Yaquina-South Beach
San Francisco
Carmel
Pismo Beach
Santa Barbara
80
85
Pgi
90
1.000
1.000
1.000
.038
.012
.962
.988
NMPi
90
95
100
21
15
21
.379
.200
.143
.076
.167
.167
.024
.545
.633
.690
.983
.976
.017
1.000
.898
26
42
105
Npgj
33
.017
15
21
30
41
corniculata
Monterey
Santa Barbara
1.000
1.000
33
24
.085
33
59
U'
1.00
1
a. M.benedjctj
f
b. M.cornjculata
.80
.60Pgjf
.40
.20-
.00
I
I
I
I
I
I
I
340
latitude
36°
Figure 6. Pgi allele frequency in samples of Megalorchestia
benedicti and M. corniculat'a.
.f
.100
.f
.105
Symbols are as in Fig. 4.
a, Pgi
Pgi
b, Pgi
Pgi
:
.
:
(A
0\
37
from Carmel, San Francisco and Yaquina-South Beach had
a single allozyme identical in mobility to MPI in M.
californiana, while M. benedicti from Pisino Beach and.
Santa Barbara had allozymes tentatively named MP185 and
(Figs. 3 and 7).
In both M. californiana and
M. benedicti it is the allele for the slower allozyme
which has a higher frequency in southern samples.
1.00
t11]
Mpi
.40
.20
.00
35J
450
latitude
Figure 7. Mpi allele frequency in samples of Megalorchestia benedicti.
Symbols are as in Fig. 4.
Mpi:
Mpi90.
00
39
DISCUSS ION
In many ways the data from M. californiana are consistent
with the hypothesis that the MPI and PGI polymorphisms
are selectively neutral.
Such neutral polymorphisms
would have genotypes in Hardy-Weinberg proportions, while
certain types of selection would produce either fewer or
more heterozygotes than expected.
If differential selection
caused populations of M. californiana to have different
allele frequencies,
any area where those populations
were intermingled would have fewer than expected heterozygotes
(the Wahlund effect).
An excess of heterozygotes could be
caused by selection directly favoring heterozygotes or by
selection which produced different allele frequencies in
males and females.
Non-random mating could produce either
an excess or a deficit of heterozygotes.
At the Mpi and
Pgi loci in N. californiana, there are no consistent
deviations from Hardy-Weinberg proportions (Table 2), and
thus no evidence of selection.
When two polymorphic loci are surveyed, the expectation
is that neutral alleles at these loci will be randomly
associated with each other.
Non-random association
generally indicates that some sort of selection is operating.
The data on Npi and Pgi from N. californiana provide no
evidence of non-random association (Table 2).
This is
not surprising.
Most processes which could produce
non-random association of alleles require either genetic
linkage or mixing of differentiated subpopulations.
The
Mpi and Pgi loci in M. californiana are not tightly
linked (Table 4),. and the absence of a heterozygote
deficit indicates that there is little mixing of subpopulations
differentiated at either locus.
If sufficiently strong selection favored One allele in
juveniles and the other allele in adults, different allele
frequencies in adults and juveniles would result.
Similarly,
strong selection which affected the sexes differently
could produce different allele frequencies in males and
females.
A neutral polymorphism would have identical
allele frequencies in all classes of a single population.
In the two samples of M. californiana divided into
juveniles, adult males and adult females, there were no
significant differences in either Mpi or Pgi allele
frequency among any of these groups (Table 3), and
therefore no evidence of strong single-generation selection.
Environmental differences along an estuarine gradient
might cause differences in allele frequency between populations.
At high tide during the winter there is a large difference
in surface salinity between the mouth of the Siuslaw River,
at approximately 30 parts per thousand, and Florence, at
less than 5 parts per thousand (Utt 1974).
Megalorchestia
41
burrows may be flooded at high tide, and low salinity
causes death in some other marine amphipod species (Werntz
1963, Doyle 1978, Marsden 1980).
MPI and PGI are glycolytic
enzymes and are not directly involved in osmoregulation
However, osmoregulation may require large amounts of
energy (Lockwood 1976), and thus low salinity might have
an effect on the Mpi or Pgi polymorphism.
There is a
significant difference of Pgi allele frequency between
the mouth of the Siuslaw River and Florence.
The difference
is not, however, repeated at another estuary, Coos Bay
(Table 3).
If the difference at the Siuslaw River is
caused by selection, the environmental difference producing
the selection is not effective at Coos Bay.
Random
processes are important in small, isolated populations, and
the population of M. californiana at the Siuslaw-Florence
site appeared small at the times when it was sampled.
The difference of allele frequency between it and the
Siuslaw-Mouth population might be the result of random
drift of isolated populations.
The heterogeneity of Mpi
allele frequencies among Oregon populations could also
result from either selection by an
unknown factor or
from random drift.
The latitudinal dines in both Mpi and Pgi allele
frequencies in M. californiana (Figs. 4 and 5) have two
possible explanations.
One allele may be favored in the
42
A
north, while the other allele is favored in the south.
number of possible selective factors vary with latitude,
including air temperature, ocean temperature, humidity,
rainfall, day length and cloud cover.
A dine may also
exist in selectively neutral alleles if two populations
were once isolated.
Random drift could cause divergence
of allele frequencies between the populations, and after
the isolating mechanisms disappear
migration would
produce a gradual dine.
The pattern of dines in other species may provide
evidence on whether selection is important in causing
the dines.
If dines result from similar adaptations to
some environmental factor, allozymes which are associated
with lower latitudes in several species might share some
common characteristic.
If, however, dines result from
random processes, which allozyme is commoner in lower
latitudes in each species is the result of chance, and
those allozymes would not be expected to have characteristics
in common.
One way to divide allozyme data is by relative
electrophoretic mobility.
Although it is difficult to
envision that protein charge could be directly involved
in environmental adaptation, the isoelectric point of
proteins has been related to intracellular degradation
rate (Dice et al. 1979), and other indirect relationships
between protein charge and functional properties might exist.
43
Latitudinal dines of Mpi were found in M. californiana
and M.benedicti (Figs. 4 and 7), and all three Megalorchestia
species examined had latitudinal differences at the Pgi
locus (Figs. S and 6).
Both Mpi dines have the allele for
the slower migrating allozyme in greater frequency in
lower latitudes, and al.l three Pgi dines have the faster
allozyme commoner in the south.
To reject (p<.OS) the
hypothesis that allozyme mobility is unrelated to latitude,
at least six species are necessary.
Therefore I searched
the literature for other latitudinal dines of Mpi and Pgi
in ectothermic animals.
The contents of a number of
journals were scanned, and surveys of enzyme variation
from four or more locations were examined for dines.
Where
three or more alleles were present in a species, they were
pooled into fast and slow classes based on the mobility
of their allozymes.
Decisions on which patterns of allele
frequency variation are dines were rather subjective, but
an effort was made to be unbiased.
In many papers allele
frequencies are presented in a table or figure, while the
relative mobility of the allozymes is buried in the text;
in these cases decisions were made on which patterns are
dines without knowing the relative mobility of the allozymes.
Many electrophorétic surveys do not include MPI, and
only two additional Mpi dines were found in the literature
44
(Table 6).
In each of these dines the alleles for the
slower allozymes are in greater frequency in lower latitudes.
The four similar Mpi climes are not enough to reject the
hypothesis that they result from random drift (p=.l2), so
surveys of Mpi in more species are needed.
Latitudinal Pgi dines have been reported from a number
of species (Table 6).
Of the dines from vertebrates,
six have the slower allozyme commoner in lower latitudes,
while three have the faster allozyme.
In all seven dines
from invertebrates, the faster allozymes are associated
with low latitudes, a significant departure from the
expectation that relative mobility is random (p=.02).
The
non-randomness of the Pgi dines from the six crustacean
species is also significant (p=.03), and it is a more
natural grouping than all invertebrates.
The similar
dines in different species are evidence that selection
is acting directly on Pgi rather than on a closely linked
locus.
For an enzyme polymorphism to be acted upon directly
by selection, there must be functional differences
between the allozymes.
The similar climes in crustacean
Pgi suggest that the electrophoretically Laster allozymes
share some functional property which is adaptive in lower
latitudes, while slower allozymes are adaptive in higher
latitudes.
No data are available on functional differences
between PGI allozymes in crustaceans which could help
45
Table 6.
Mpi and Pgi latitudinal dines.
allosyme associated
with low latitudes
reference
MPI
galorchestia californiana
Megalorchestia benedicti
Caledia captiva, Torresian
chiämosomal ta'on
(grasshopper)
Metapenaeus macleayi (shrimp)
slow
slow
this study
this study
slow
slow
Mulley and Latter 1981
Daly et al. 1981
PGI
crustaceans
Megalorchestia californiana
Megalorchestia benedicti
Megalorchestia corniculata
Asellus quaticus
(freshwater isopod)
Emerita talpoida (sand crab)
Liinnoria tripunctata
fast
fast
fast
this study
this study
this study
fast
fast
Verspoor 1983
Corbin 1977
fast
Henderson 1983
1etridium senile (anemone)
fast
Uoffmann 1981
Anguilla rostrata (eel)
Pundulus heteroelitus (killifish)
slow
Koehn and Williams 1978
Powers and Place 1978
Smith 1979
Johnson 1975
(wooa-boring isopod)
cnidarian
ypterus blacodes
Menidia beryllina
Pseudomugil signifer
fast
slow
fast
slow
slow
slow
Hadfield et al. 1979
Ambystoma ros acetns
slow
Desmognathus fuscus
fast
Shaffer 1983
Tilley and Schwerdtfeger 1981
Salvelinus fontinalis
(brook trout)
locus 1
locus 2
Stoneking et al. l981...
salamanders
explain the similar dines; one speculation is presented
here to suggest areas for further research.
The most
obvious latitudinal environmental gradient is temperature,
and temperature has profound effects on enzyme function
(Somero 1978).
One theory, based largely on interspecific
comparisons of enzymes, is that greater structural stability
of enzyme molecules is needed for proper ligand binding
at high temperatures.
At lower temperatures more structurally
flexible molecules, with faster substrate turnover, are
favored to maintain proper ligand binding and for metabolic
compensation (Somero 1978).
In interspecific comparisons
an enzyme with greater structural stability, as measured by
resistance to thermal denaturation, often contains more ion
pairs (salt bridges) (Perutz 1978).
A single amino acid
substitution in lactate dehydrogenase results in an additional
ion pair, conferring greater resistance to subunit
disaggregation (Muller 1981).
The evidence for PGI is contradictory on the relationship
of thermal stability to protein charge and adaptation to
warmer environments.
In Colias butterflies the PGI allozymes
with greater thermal stability are commoner in warmer, low
elevation populations and are electrophoretically faster
(Watt 1977).
In contrast, the more thermally stable allozyme
in the anemone Metridium senile is commoner in cooler, northern
populations and is electrophoretically slower (Hoffmann 1983).
47
Thermal denaturation in most organisms is measured at
temperatures much higher than those the species would
encounter in nature, and it may not be a direct measure
of those biochemical differences between allozymes
which are important for adaptation to environmental
temperatures.
Differences in electrophoretic mobility between
allozymes are generally due to charge differences, with
more negatively charged allozymes migrating faster under
the usual electrophoretic conditions.
While there is no
evidence other than the similar latitudinal dines, it
may be that amino acid substitutions in crustacean PGI
which cause more negative net ionic charge, and therefore
faster electrophoretic mobility, also result in more
ion pairs within
the PGI molecule and therefore greater
structural stability.
48
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