Abstract
The biochemical genetics of an intertidal bernacle, Tetreclite
rubescens (Darwin, 1354), were examined at four sample sites along
the coast of central California. Examination of the allelic frequencies
at four polymorphic loci indicate that gene flow between
subpopulations is either currentlg high or was so veru recentlu. Not
all sites, however, appear to exchange migrants equallu. The samples
from the Moss Landing site appear to be closelg related to each other.
and other evidence suggests that Moss Landing may be quite isolated
from the more southern populations. Point Conception does not appear
to be a major barrier to gene flow, and migration between Pecific
Grove and Pori Hueneme, three hundred miles awag, appears to be
greater than migration between Pacific Grove and Hoss Landing, onlu
twenty miles awag. A population oi Semibalanus cariasus (Pallas.
1768) was also studied, and found to be almost indistinquishable from
the Tetrsclita populations at all four polymorphic loci scored. This
indicates that strong selection pressures mag be maintaining these
allelic frequencies, and the polumorphisms mag be older than the
species themselves.
Introduction
Since the advent of starch gel electrophoresis in the late 1960s
à great deal of genetic variation has been discovered in countless
populations of orgenisms. Plants, insects, reptiles, mammals.
invertebrstes, birds, and mang other groups have all been studjed and
shown to harbor vast amounts of allelic variation within their
populations. Discovering why this variation exists and its importance
to evolutionary and population biology has been a more difficult and
controversial task. Most explanations can be separated into two
catégories: those that invoke natural selection as a force responsible
for maintaining genetic variation, and those that believe most of the
variation observed is selectivelg neutral. These positions are
commonly known as the selectionist and neutralist theories.
respectivelg. A great deal of time and effort has been spent on truing
to prove or disprove both of these positions.
Hany studies have sought to prove that environmental factors, and
hence natural selection, are responsible for maintaining allelic
polgmorphisms. Hang of these studies have been performed on marine
organisms, and among the more significant are williams, Koehn and
rtton's work on ihe American eel, Anguille restrsts (Williams et al
1972), Koehn, Hilkman, and Mitton's work on the blue mussel, Ftilus
edulis (Koehn et al, 1975), and Power's group's work on a fish
Fundulus beterochitus (Powers, 1985.) All three of these studies
found signiticant changes in allele frequencies over geographic areas,
and all three could correlate these changes with environmental
factors, such as temperature in the case the American eel and
Punoulus, and salinity in the case of vtilus In all three cases
natural selection was shown to be a probable force in creating the
clines, and therefore the polumorphisms.
Thère have been considerably fewer cases where natural selection
could be positivelg identified as a force for mainteining variation
when no clines were present. Werd Wett's work on Colias butterflies
is one such case (Watt, 1984), and Ronald Burton and Harcus Feldman's
work on the copepod Tigriopus is another (Burton and Feldman, 1982.)
Both of these groups have correlated the function of a specific enzume
with differential fitness among individuals cerrying alternate alleles
for the loci studied. These cases are dwarfed by the number studies in
which variation is observed and the forces, if ang, maintaining it have
get to be discovered. Among these are the mang geographic population
studies in which no obvious patterns of allele frequencies were found.
Hang of the projects involving marine organisms on the west coast of
North America fall into this category (see Levinton et al, 1978. Dayis
et al, 1931, Smith et al, 1987, Grant, 1988.)
Altnough there still exists controversy over the meaning and
importance of allelic variation to evolution, this variation has
recentig proven itself useful in the studg of the population dunamics
oi marine organisms. Traditional methods of studging population
subdivision, migration and gene flow by direct observation are
ditticult, if not impossible, to perform on small marine organisms.
The problem is compounded when the species in question have
microscopic, free swimming larva, or release their gametes directlu
into the water. Both of these cases have a high potential for
migration, even if the adult forms are stationarg. Unfortunatelu, it is
impossible to directlg observe the extent of such migration. An
alternative to direct observation is to studg the large scale population
genetics of a species, and infer gene flow and migration rates from
the genetic data. It is possible to estimate both the extent of
population subdivision and migration from allelic frequencies (Slatkin.
1987) The data gained from biochemical population studies is also
useful in that it allows examination of gene flow on a evolutionaru
time scale, instead of simplg examining present trends. Jeff Hitton.
Carl Berg and Katherine Orr used allozyme data to estimate the extent
of gene flow between different subpopulations of the Caribbean Oueen
conch Strombus giges(Mitton et al, 1989), and B.J. Davis et al used
allelic frequencies to estimate gene flow between Pacific Coast
populations of the fish Gylebius pictus (Davis et al 1981) Both
studies found evidence of high gene flow between subpopulations, with
signiticant but unpredicteble differences in local allele frequencies
he purpose of the present studg was twofold: 1) to examine the
biochemical genetics of a common intertidal barnacle, Tetrsclite
rurescens, and use the genetic data to infer migration patterns and
rates, and 2) to attempt to gain additional insight into the
evolutionary importance of allelic variation by also examining the
biocnemical genetics of a related barnacle species, Semibajanus
ceriosus. The study area included the northern end of Tetrsclitss
range and the southern end of Semibalanus s, and encompassed at least
two potential barriers to gene flow: the Honterey Submarine Cangon
and Point Conception, the largest point on the west coast of N.
America.
Haterials and Hethods
Somple collection and storage Ouring the months of April and
Hag, 1990, collected samples of Tetraclite rubescens from jetties
in Port Heuneme, Horro Bag, Hoss Landing, and from a cerent wall in
Pacitic Grove (figure 1). Samples oi Semibalanus csriosus (Pallas.
1738), originallg mistaken for Tetraclita, were collected from a jettu
in Half Moon Bay. In all locations samples were collected throughout
the species' vertical intertidal range. In Pacific Grove the sampled
indiviquels ranged in size from verg small (2 mm basal diameter) to
verg large (50 mm basal diameter). in all other locations ! took onlu
medium to large individuals. I removed the soft tissue from all but
the smallest individuals (Amm besal diemeter or less) with tweezers
and stored each sample in an individual plastic centrifuge tube. The
smallest individuals were simply placed shell and all into individual
tubes. The samples from Half Hoon Bag, Horro Bag, and Port Huenere
were trozen in a cooler of dry ice immediatelg after sampling, and
then stored at -70 degrees C for the remainder of the project. The
samples from Pacific Grove and Hoss Landing were kept on ice for un
to three hours before being stored at -70 degrees C. I analuzed all the
samples within four weeks of sampling time, and the number of
thaw/freeze cycles was kept to a minimum.
Sample pregeration and analysis All semples were diluted with
one to three drops of distilled water, homogenized bi ultre sound, and
then spun down for three to four minutes in a high speed
microcentrifuge. The homogenates were absorbed into whatmans's *1
Tilter paper, and analgzed using horizontal starch gel electrophoresis.
Gel preparation consisted of pouring a mixture of 35 g starch (Sigma)
suspended in 50 ml of cold buffer solution into 220 ml of boiling
butter solution. The final 3ooml volume was mixed vigorouslu.
degassed, and poured into 17 cm by 17 em bg 1 em molds. The gels
were cooled to 4 degrees C before loading the samples. Enzumatic
activitg was detected at the following loci: phosphoglucoisomerase
(Pgi-1 and Pgi-2), phosophoglucomutase (Pgm); tetrazolium oxidase
(To), hexokinase (Hk-1 and Hk-2), malate dehgdrogenase (Mah-1 and
fidh-2), malic enzyme (He), acid phosphotase (Acp), and xanthine
dehydrogensse (Xdh). Onlg Pgi-1, Pgm, To, Hk-2, and Hoh-1 were
scored. The other loci were either not resolved well engugh to be
scorable,or in the case of Hk-1 and Fgi-2, were detectable onlu in
verij small individuals. The buffer systems used are described in
Agals et al (1973) Buffer "C"(Tris, citrate, EDTA,PH 70) was usedto
resolve all scored loci. The enzyme stains used were all from
Eichardson (1956), except that the Göpdh used as a linking enzume in
the stains for Pgm, Pgi, and Hk was NADH, rather than NADEH
dependent
Dsts enslysis: For each sample site, Fst, an inbreeding
coefficient which measures population subdivision, and Nm. an
estimate of the number of migrants per generation was calculated
(Hartl and Clark 1989, chapter 6). Fst-(Ht-Hst)/Ht, and
Nm=17AFst-174, where Ht is the expected heterozugositu if all
samples from all the sampling sites belong to a single panmictic
population, and Hst is the expected heterozygosity of a subpopulation
assuming random mating is occurring within the subpopulation. R. the
coefficient of relation, wes calculated by a computer program (Jeff
Mitton, unpublished) based on the following equation (Queller and
Goodnight, 1939): R=2222(Pi(-j)-Pm)/EZZZ(Pijm-Pm), where i=groups
j-individuals within a group, k-loci, and a-allelic positions within a
locus. All of these statistics are useful in estimating the amount of
subdivision within a population. Fst estimates the loss of
heterozygositg due to population subdivision ( the Wahlund effect). Mm
estimates the amount of migration between subpopulatjons, and R
describes how closely related the individuals in a subpopulation are, in
terms of the probability of shared alleles.
Results
Gecgrephic veriatien:
Ui the five scored loci, Pgi-1, Pgm, To, and Hk were polumorphic
in all populations studied. Näh-2 was monomorphic in all populations
studied. Table shows the allele frequencies for all loci st each
Tetreclite studg site. Alleles are referred to by letter, with A being
the fastest moving allele, and F the slowest. For Pgi, each sample site
has one verj common allele (75-858), one intermediate allele (5-155)
and two or three rare alleles («53). All populations had the same
common allele, but theg differed in the frequencies of the rarer alleles
(see figure 2). In Horro Bay, allele C is the second most common
allele, while in all other populations allele B is the second most:
common. None of the Hoss Landing samples showed allele E. which had
a frequency of 58 in Pacific Grove, only 20 miles awag. The Port
Hueneme samples lacked allele A. The Pgi allele frequencies were
found to differ significantli between sampling locations using a G test
for independence (Sokal and Polhf, 1981.) All the Pgi heterozugotes
displaged a three banded pattern on the gels tupical of a dimeric
enzyme. There were six alleles at the Pam locus, and theu fall into a
pattern similar to Pgi. All populations had one verq common allele
one intermediate allele, and several rare alleles (figure 3). The
gitferences in Pgm allele frequencies were not found to be significant
(pe 1). Tetrazolium oxidase was scored only for Morro Bau and Port
Hueneme, and both locations had verg similar allele frequencies
(tigure 4). Hk was scored for Hoss Landing, Horro Bau and Port
Hueneme, and there were no significent differences in allele
frequencies between these locations (figure 5). Hk, To. and Pam
neterozygotes all displaged a two banded pattern tupical of monomeric
enzymes
lable Il gives the observed and expected heterozugosities, the
inbreeding coefficients, Fst, and Am for each locus. None of the
observed heterozygosities are significantly different from what is
expected under Hardg-weinburg conditions, except for Hk at Hoss
Landing, where there is a significant excess of heterozugotes. The
values for Nm varg considerably between loci, with Pgi, Pgm, and Hk
all giving values between 15 and 25 migrants per generation, but To
giving a value of over 200 migrants per generation. The value for To
13, however, derived from allele frequencies from Horro Bau and Port
Hueneme onlg, while the other estimates are derived from frequencies
from at least three locations. The average values for Nm are 73.2
using all four loci, and 20.2 if To is left out.
Table IIl gives the coefficients of relation for the individuals
sampled at each site. A value of zero indicates no relstionship, and a
value of S would mean the sampled individuals were full sibs.
Although standard errors could not be calculated, ran a computer
Simulation with rändomlg chosen genotupes (expected relationshio
equals zero) and got values ranging from -05 to 01 (average deviation
from zero -03) using 15 individuals and three loci. All of the values
calculated for the sample sites used at least 15 individuals and at
east 3 loci, except for Pacific Grove, which used 40 individuals but
onlg 2 loci. The R values varied considerably from site to site, with
Port Hueneme having a negstive value (less related then would te
expected bi chance) and Hoss Landing having the rather high value of
23. This means that on average ang two individuals sampled at Hoss
Länding share two grandparents. The value of 008 at Pacific Groye
indicates that those individuals are no more related then would be
expect by chance, and the value of 13 at Horro Bay indicates that
those individuals are slightig more related to each other then would be
expected by chance.
Interticsl verietion.
At the Pacific Grove study site individuals were sampled
speciticallg from both the verg bottom and the verg top of their
intertidal range. In addition, verg small individuals were sampled.
again both from the bottom and top of the specie's range. No
signiticant differences in allele frequencies were found between high
and low intertidal samples when only medium to large indiviquals
were sampled, and there were no significant differences in allelic
frequencies between large individuals and small individuals as a group
(tables IVand V) However, there were significant differences in
allele frequency at the Pgi locus between high intertidal and low
intertidal samples among the small individuals alone (p005, table y1)
he small barnacies were estimated to be about two months old
(Kristi Hiller, personal communication.)
Interspecitic veristion:
able VIl gives the alleic frequencies at all four polumorphic loci
scored for the population of Semibslanus cariosus sampled at Half
rioon Bag. Each locus has the same alleles as the Tetraclite
populations, and only at the To locus are the allelic frequencies
signiticantig different from the averaged allelic frequencies of the
letraclite populations. Nei's genetic identity (Nei 1975) between
the pooled Tetraclits populations and the Semibalanus population
equals 9903, a figure normally associsted with two populations
within the same species.
Discussion
Geegrechic veristian
he allele frequencies at each location sampled are clearlu veru
simllar, and the estimated number of migrants per generation between
sample sites is quite high, as might be expected for an intertidal
species with a pelagic larval period. However, the individuals sampled
probablg do not belong to a single panmictic population, and the
results suggest ihat the dispersal patterns for barnscle larva are
quite complex. The Pgi allele frequencies are significantlu different
between subpopulations, indicating that either some subpopulations
ere isolated endugh from each other for genetic drift to occur, or there
are local selection pressures which favor different allelic frequencies
in ditierent places. If local selection is occurring, it is probablu vert
slight, since in general the allele frequencies at esch location sampled
are quite similar.
he coefficients of relation indicate that some subpopulations
probably retain most of their larvae. Moss Landing, with an R of 23
seems to be particularly isolated from the other subpopulations. In
addition to being the northernmost breeding group of Tetrsclits
(Kristi Hiller, personal communication), the Moss Landing
subpopulation is also located right at the mouth of the Montereu Bau
Cangon, which mag act as a barrier to larval dispersal from the more
southern populations. The Horro Bay samples were taken from calm
water deep within a harbor, and their R of 13 suggests that theu mau
also be a fairly isolated subpopulation. The samples from Pacific
Grove were taken right at the southern edge of Honterey Bag, and the
samples from Port Heuneme were taken from the open coast. Neither
of these had large positive R values, indicating that these
subpopulations may recruit individuals from a greater area then either
the Horro Bay or Hoss Landing subpopulations do. it seems quite
possible that the Pacific Srove subpopulstion trades more migrants
witn Port Hueneme, three hundred miles awag, then it does with Hoss
Langing, only twenty miles awag. This idea is further supported bu
the Hoss Landing subpopulation's lack of Pgi allele E, which is present
ai 4-53 at both Pacific Grove and Port Hueneme.
Uf the two potential barriers to gene flow mentioned in the
12
introduction, Point Conception and the Monterey Bag Cangon, the latter
seems to be a more likely candidete as a berrier to Tetractite laryee.
This is not too surprising, considering Honterei Bag is the northern
limit for large Tetraclita populations on the west cosst. It is not
known whether Tetrachite populations do not appear north of Hontereu
bay because they cannot get there, or because they are not capable of
surviving north of the bag. Hy results indicate that Hoss Landing mau
be a very isolated subpopulation, which supports the idea that
Montereg Bay is a berrier to northern migration. The Moss Landing
subpopulation mag be important es a stepping stone to the north, and
mag supply larvae which will eventuallg form populations north of
Monterey Bay. It seems likelg, however, that the barriers which
isolate Hoss Landing from the south will also isolate it from the
north, so this expansion, if it happens, could be a very slow process
Evolutionsry implications and Semibalanus cariosus:
My results have some relevance to the neutralistselectionist
argument about the importance of genetic variation. In brief, the
neutralist position is that most, if not all, natural genetic variation is
selectively neutral and meaningless so far as the individual fitness of
an organism is concerned. The selectionist argument maintains that
much or at least some of the observed genetic variation is adaptive
and maintained by natural selection. My results bear on this argument
in two ways: 1) The values lobtained for Mm varied between loci. I
all the variation observed was selectively neutral, the figation indices
and estimates of Nm should have been nearly identical. Even leaving
out the extremelg high To estimate, there may still be enough
variation in the estimates derived from the other loci to implicate
selection pressure as a force affecting allelic frequencies. 2) The
population of Semibalsnus cariosus sampled at Half Hoon Bau was
almost indistinguishable from the Tetraclits populations at all four
polgmorphic loci studied. It seems unlikely that this is merelu a
coincidence, and there are several alternative possibilities to explain
this result. One possibility is that the two groups are reallu onlu one
species, and there is extensive gene flow between them. This seems
extremelg unlikelg, since there are important morphological
differences between the two groups, and theg brood at opposite times
of the geer (Horris, Abbot, Haderlie, 1980 p517,519). A second
explanation is that there are strong selective forces operating on both
species which maintain the allele frequencies at the same levels. This
nas some plausibilitg; both species occupy the same ecological niche.
the lower intertidal zone, and the species look superficiallu alike.
Their geographical ranges meet up with each other with little overlan
(tigure 1), and it seems likely that they face similar environmental
conditions. In theory, genetic drift can be a powerful force in changing
allele trequencies, even in large populations, so long as the alleles are
selectively neutral and the time period in which change can occur is
great (Hartl and Clark, chapter 2, 1939.) Tetraclits and Semibalanus
nave probablg been reproductively separsted for tens of millions of
gears (Kristi Hiller, personal communication), and this certainly would
have been enough time for their allelic frequencies to have diverged
considerably were the alleles involved selectivelu neutral. If, on the
other hand, the polgmorphism was adaptive for the common ancestral
species of both Tetraclits and Semibalsnus, and the forces
maintsining the allele frequencies remained in place after the tyo
groups diverged, then it is possible that the polumorphisms could
remain and be maintained in the now separate species. Thus, the
polgmorphisms shared by the two species may be older than the
species themselves.
Acknowledgements: would like to thank Denng Powers, Lunna
Hereford, Tobe Cole, Lani West, Simone Sorger, Doug Crawford, Himi
Brown, Rob Rowan, Linda Park, and most especiallu Jeff Mitton for
their kindness, patience, and help with this project.
References:
Agala, F.J., D. Hedgecock, 6. Zumwalt, and J Valentine: Genetic
variation in Tridscne maxims, an ecological analog of some
unsuccessful evolutionary lineages. Evolution 27: 177-191 (1973)
Burton, R.S., and H.W. Feldman: Phgsiological effects of an allozume
polymorphism: glutamate-pyruvate transeminase and response
to hyperosmotic stress in the copepod Tigriepus californicus.
Biochem. Genetics 21 (3/4): 239-251 (1982)
Davis, B.J., E.E. Deflartini, and K. HcGee: Gene flow among population of a
teleost (Painted Breenling, Grylebius pictus from Puget Sound to
Southern California. Harine Biologu 65: 17-23 (1981)
Grant, W.S., and F.M. Utter: Genetic heterogeneitu on different
geographic scales in Nucells lamellase (Prosobranchia, Thaididse)
Halacologia 28 (1-2): 275-237 (1988)
Hartl, D.L., and A.G. Clark: Principles of population genetics, 682 pp.
Sunderland, Hassachusetts: Sinauer 1989
Koehn, R.K., R. Hilkman, and J.B. Mitton: Population genetics of marine
pelecupods. IV. Selection, migration and genetic differentiation
in the Blue Hussel Hytils edulia Evolution 30: 2-32 (1975)
Levinton, J.S. and T.H. Suchanek: Geographic Varistion, Niche Breadth and
Genetic Differentiation at Different Geographic Scales in the
Mussels Mytilus califernienus and M edulis Harine Bioloqu 49;
363-375 (1978)
Mitton, Jefferg B, C.J. Berg, and K.S. Drr: Population Structure, Larval
Dispersal, and Gene Flow in the Queen Conch, Strambus giges, of the
Caribbesn. Siol. Bull 177: 356-362 (1989)
Morris, R. H., D.P. Abbott and E.C. Haderlie: Intertidal invertebrates of
California, 690 pp. Stanford, Californis: Stanford Universitu Press
1950
Nei, H: Holecular population genetics and evolution, 25S pp. New York:
Elesvier 1975
Powers, OA et al: Senetic variation in Fundulus heteractitus
geographic distribution. Amer Zool 26: 131-144 (1986)
Queller, D.C., and Goodnight, K.F.: Estimating relatedness using
genetic markers. Evolution 43 (2): 258-275 (1939)
Richardson, B.J., P.R. Baverstock, and M. Adams: Allozume
electrophoresis, a handbook for animal systematics and population
studies, 410 pp. Sudneg, Australia: Academic Press 1986
Slatkin, Hontgomerg: Sene flow and the geographic structure of natural
populations. Science 236: 757-792 (1937)
Smith, B.L. and D.C. Potts: Clonal and solitarg anemones (Anthepleura of
western North America: population genetics and sustematics.
Harine Biologg 94: 537-546 (1987)
Sokal, R.R., and F.J. Rohlf: Biometry, 859 pp. New York: W.H. Freeman
1951
watt, W.B., P.A. Carter, and S.M. Blower: Adaptation at specific loci. IV
Differential mating success among glycolgtic allozyme genotupes
of Colias butterflies. Genetics 109: 157-175 (1985)
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Evolution 27: 192-204 (1972)
Figure Captions
Figure 1: Map of the study aree and ranges of Tetraclite and Semibalanus
Sample sites are (4) Port Hueneme, (B) Horro Bag, (C) Pacific Grove, (D)
Moss Landing, and (E) Half Hoon Bag. (Hodified from Morris, Abbot and
Haderlie, 1980.)
Figure 2: Geographic distribution of Pgi allele frequencies. Frequencies
are not independent of locstion (P025).
Figure 3: Geographic distribution of Pgm allele frequencies. There are no
signiticent differences in allele frequency between locations.
Figure 4: Tetrazolium oxidase allele frequencies. There are no significant
differences between the two locations
Figure 5: Hexokinase allele frequencies. There are no significant
differences between the three locations.
Table Captions
lable l: Tetraclite allele frequencies at four polymorphic loci. Allele A
is in all cases the fastest moving allele (most anodal), and allele f the
slowest (most cathodal). A — indicates missing data, a means the allele
was not found.
äble II: Expected subpopulation heterozygositg, Hs; observed
heterozygositg, Hi; expected population heterozygositu, Ht; the inbreeding
coefficient, Fis; the subpopulation fixation index, Fst; and the estimated
nümber of migrants per generation, Nm for each of the four Tetreclita
sample sites.
Table III: Coefficients of relation for the four Tetreclite subpopulations.
able IV: Comparison of allelic frequencies between samples from high
and low in the intertidal zone, medium to large barnacles onlu.
lable V. Allele frequencies from samples small, recentlu settled
Tetraclita (less than 4 mm)
Table VI: Comparison of allelic frequencies between samples from high
and low in the intertidel zone, verg small barnacles only. The Pai allele
frequencies are significently different between high and low (05.05).
lable VII: Allelic frequencies at four polgmorphic loci for a population of
Semibolenus coriosus sampled at Half Hoon Bau.
05
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Table 11
1oc1 R
Pgi,Pgm,Hk
Pgi Pgm
0.008
Pgi,Pgm, Hk, To
0.13
Pgi,Pgm, Hk, To
-0.12
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