Download Lack of significant associations between allozyme heterozygosity

104.
Booth, C.L., D.S. Woodruff and S.J. Gould.
Lack of significant associations between allozyme heterozygosity and phenotypic traits
in the land snail Cerion.
Evolution 44(1):210-213. (1989g).
RESEARCH
ARTICLE
A 104
EIIOI",itIft. ,,,,).'990.
pp. 21G-213
LACK OF SIGNIFICANT
ASSOCIATIONS
BETWEEN ALLOZYME
HETEROZYGOSITY
AND PHENOTYPIC
TRAITS IN
THE LAND SNAIL CHRION
CAREY L. BOOTH.' DAVID S. WOODRUFF.
Dcpanmcn: of Ria/ot.!' & Crlllrr for Mo/rrll/or Gcnaics.
UII;I'rrsil)' of California SOli Virgo. La Jolla, C., 92093
AND
STEPHEN
M,/sr,/m
JAY GOULD
of Comparative F.OO/O[:.!'.llarvard UII;I·rrS;I)'. Cambridge, MA 02 138
Received
April
17. 1989.
The: regulation and significanc:c: of phenotypic variation remain central problems at the interface of molecular genetics with classical morphology
and evolutionary
ecology.
Hence.
the rel:llion~hip
between
genotype and phenotype: is of great interest to evolutionary biologists. and over the laM 15 ye:lrs. m:lny
reports of :llIo/.)'me-heter07Yllute
I"henntYl"ic ~upcri.
ority have been published :lnd reviewed (MiIlOIl and
Grant, 1984; Allendorf and Leary, I 9li6; Zouru~ and
FoJt7~ 1987), Individuals
hC'tC'roJ:ygous at some locus
lire often larger. faster growing, longer-lived,
metabolically more efficient, or more fecund than individuals
homozygous
at that locus. Similarly. individuals
with
more heterozygous
loci arc often superior to individuals with fewer heterozygous
loci, and in some popu-
, Present address: Department
lege, Portland. OR 97202,
of Biologv,
Reed Col-
Accepted
October
13. 1989
lalions. the frcqut'nC")' of ht'Icro7~'!ol"$ increases with
cohort age. Heterozygosity
is also on en pcsitivcly associated with developmental
homeostasis:
individuals
with more h('t('rO/YltoIlS loci may exhibit less phenotypic variability
than individuals
with fewer heiero7ygouS loci. However. there arc reports in which no
rel:"ion~hip was found between h('tero/n!O~ity
and f".
m'ss correlates rnr some loci (Koehn ct al .. I 'IR!!) and
even some reports of hOI1l01YllOIC:5ullCriorily (Allendorf and Leary, 19Rfl). In this note. we report mainly
a lack of signiflC'ant associations
ofheter07.ygosit)·
with
phC'notypic traits and variation in Cerion land snails.
Mechanisms
underlying
positive relationships
between allczyrnc h('tero1ygosity
and fitness correlates in
any given case are usually unknown.
Some possible
mechanisms
are dominance
or ovcrderninance
either
at the loci examined or at other loci that arc in linkage
disequilibrium
with the studied loci (Millon and Grant.
1984; Smouse. 1986; Zouros and Foltz, 1987). Under
211
NOTES AND COMMENTS
the dominance hypothesis, heterozygosity serves to
cover deleterious recessives. The overdorninancc hypothesis implies that heterozygous genotypes arc bcucr
per se (Smouse, 1986). At a given locus, thc hetcrozygote will outperform the homozygmcs. Heterosis is
the reverse of inbreeding depression and can be the
result of either increased heterozygosity at a large number of overdominant loci or decreased expression of a
small number of deleterious recessive alleles (Millon
and Grant, 1984), With associative overdorninance,
the studied loci are not directly affecting the traits, but
they are in linkage disequilibrium with loci that do
(Zouros and Foltz, 1987). Individuals heterozygous at
the studied loci are more likely to be heterozygous at
other loci which may have deleterious recessives;
therefore, heterozygotes will have higher fitness than
homozygotes (Nei, 1987).
In this study, we analyzed the relationships between
an individual's multilocus heterozygosity and measures of the same individual's shell morphology in West
Indian land snails (O'r;oll bellda/h). We sought assoelations between heterozygosity and measures of both
shell morphology and phenotypic variability. Cerion
arc well suited to studies of phenotypic variation, as
their shells bear a complete record of their ontogeny
(Woodruff, 1978). A recurved lip marks the cessation
of shell growth in adults, and as the protoconch is
retained, size and shape at different ontogenetic stages
can be measured. More generally, Cerion are especially
interesting as subjects for studies of phenotypic diversity, as their shells exhibit extremely high interpopulation variability in size, shape, sculpture (ribbing), and
coloration. Such diversity led to the recognition of more
than 600 species, whose validity and evolutionary relationships are now under critical review (Gould and
Woodruff, 1986, 1987; Woodruff and Gould, 1980,
1987),
MATERtALS
AND METHODS
Gould and Woodruff'( 1978) collected and published
the data used for this analysis, and only a brief overview of the methods is repeated here, The morphological and genetic data were originally used to discriminate among two subspecies and their hybrids,
which occur on the islands of Lillie Bahama Bank. The
subset for C. bendalli was used for this analysis, Adult
shells are elongate or pupilliform, typically 24-30 mrn
long, and have 7-9 whorls. Twenty adult snails were
collected in the smallest possible area (typically 1m2)
around a randomly chosen point at each of I 3 different
sites. Each individual's shell was measured for 16 continuous characters and three meristic characters defined in Gould et al. (1974) and Gould and Woodruff
(1978). Here we used only the nine continuous and two
meristic characters that had greatest repeatability of
measurement (K. M. Ligarc's [unpubl.] results discussed in Gould and Woodruff] 1986J; Table I).
Starch-gel electrophoresis of each individual's foot
muscle revealed 20 interpretable loci. Of thc corrcsponding enzymes, six were found to be polymorphic:
esterase-Z, malate dehydrogenase- I, malate dchydrogcnasc-Z,
6-phosphogluconate dehydrogenase- I, asparuue aminotransferase (formerly called glutamic oxalacetate transaminase), and leucine aminopeptidase,
Each individual was heterozygous at 0-5 loci; no individual was heterozygous at all six 10C'i,We used the
number of heterozygous loci per individual (h), without
regard for locus or genotype, as our measure of hetcrozygosity, following Zouros and Foltz (1987).
We did statistical analyses using Stat View and SYS·
TAT software for the Macintosh microcomputer. Snails
were categorized into four heterozygosity classes: h 0, I, 2, and z 3; sample sizes of each class were as
follows: N(h .. 0) - 53, N(h ~ I) = 81, N(h ~ 2) - 82,
N(h 2: 3) .. 44. The ipdividuals with three, four, or
five heterozygous loci were placed into one class due
to the small number of individuals with four (N - 10)
or five (N - I) heterozygous loci, A principal-components analysis reduced the nine continuous characters to three axes, which explained 94.6% of the total
variance. Correlations were calculated to assess the relationship. if any, between the mean scores for the first
three axes and the heterozygosity classes, between the
I I characters and the heterozygosity classes, and among
the characters that showed a significant correlation with
the heterozygosity classes. The sequential Bonferroni
method was used to determine the significance level
for each column of tests (Rice, 1989).
To compare phenotypic variability among the heterozygosity classes, we used Levene's test which is robust to deviations from normality (Schultz, 1983), The
median phenotype: value for each heterozygosity class
was subtracted from each phenotype: value in that class,
and the absolute values of these differences were calculated. Correlations between the differences from the
median and the heterozygosity classes were calculated.
RESULTS
The first axis of the principal-components analysis
explained 85.0%, the second axis explained 6,7%, and
the third axis explained 2,9% ofthc total variance. Shell
length and aperture height loaded most strongly on the
first axis, which was expected, as first axes are usually
size axes for biological measurements of this type. Aperture width and width at the fourth whorl loaded most
strongly on the second axis, There was no significant
correlation between the individuals' axis scores and
the heterozygosity classes for the first, second, or third
axes. The advantage of using a principal-components
analysis is that several variables can be tested simultaneously, thus avoiding the problem of multiple statistical tests.
As there were no significant correlations between the
axis scores and heterozygosity, the individual traits
were also tested, and the sequential 13onferroni method
was used to adjust the cxperimentwise significance level,
The numberofribs on the fourth and sixth whorls were
thc only two traits for which a significant correlation
was found with the heterozygosity classes (Table I).
These two traits were also significantly positively correlated with each other (r = 0.74, P < 0,000 I), and the
number of ribs on the fourth whorl was significantly
negatively correlated with shell width (r - -0,37, P <
0.000 I). Levene's test for average deviations from the
median revealed no significant correlation between deviations from the median for any trait and the heterozygosity classes (Table I),
DISCUSSION
This study meets at least four ofZouros' (1987) five
conditions that favor detection ofa positive correlation
between heterozygosity and phenotype. The degree of
212
NOTES AND COMMENTS
TABLEI. Correlation coefficients (r) between trait values and hctcrozygoslty-ctasscs and between trait deviations from the medians and hcterolygosity.classcs
of Cerion bcndalli.
Cllml.. "'"I1(
Contlalion or .rail ,nil dt'"..iation
Trail
Shell length
Shell width
Protoconch width
Height to whorl 4
Whorl-4 width
Weight
Whorl number
Aperture width
Aperture height
Number ofwhorl-4 ribs
Number of whorl-S ribs
wilh he-1frolYlosily
wnh ht"lrro')"('ISIIy
0.Q7
0.10
-O.QJ
-0,06
-0.02
-0,05
0.09
-0,01
-0,04
-0,27-0,28-
.,. < 0.004' lllIe no..(~""IIili,II;("'_,," kvd • 0.0""
lOr each column).
-0.10
0.04
0.08
0.13
0.04
0.01
0.06
-O.QJ
-0,12
0,00
O.QJ
• 0.004'
would be expected between trait variance and heterozygosity, The negative assoclation could be simply due
to the fact that there arc more homozygote classes than
heterozygote classes. In contrast to the above exeeetations. we found no relationship ror II traits. The
absence of associations may indicate that the traits arc
not afl'ccted by the loci studied, that the loci studied
arc not indicative or the level of heterozygosity of the
entire genome (Chakraborty and Ryman, 1983), or that
heter07.ygosity and developmental homeostasis are not
always related,
In conclusion, we mainly found a lack orassociation
between multilocus hetero7.ylto~ity and various measures or phenotype and phenotypic variance, We believe it is important to report the instances in which
no significant relationship was found, as well as those
few positive and negative associations. It seems that
past researchers have overemphasized the few strong
associations at the expense of the message conveyed
by the majority or cases in which there is none, Biased
reporting or only positive results can severely distort
our view of reality (Begg and Berlin, 1988),
ACKNOWLEDGMENTS
We thank I, Beardmore and T, D. Price: for helpful
conversations. Thc commems ofn. D. Collier, and our
reviewers were also very uscful, We thank an anonymous reviewer ror the suggestion to consider the efl'ccts
of pooling the data rrom the 13 sites, This research was
supported, in part, by NSF grants DEB 7714618 and
8207540 (to D,S,W, and 5,J,O,) and-BSR-SSOO733 (to
D.S, W,) and NSF and US PHS prc:doctoral fellowships
(to C.LIJ,),
hetcrozygosity was measured for a large numhcr ofloci
(20, of which sill were polymorphic); the phenotypic
traits studied are likely to be afl'eeted by a largc number
or loci; nongenetie factors likely to afl'ect these: characters are probably random; and the sample size of 260
is large and representative of the_aene pool The Jifth
condition (steady-state linkage disequilibrium) has not
yet been assessed in Cerion.
We found that only two of the I I traits examined
were significantly correlated with the hetcr07ygo~ity
LITERATURE
CmD
classes, Because these two traits were correlated with
ALLENDORF,F, W.. ANDR, F, LEARY. 1986, Heteroeach other, there was actually only one relationship
7ygosity and fitness in natural populations of aniwith heterozygosity, The significance, if any, of a demals, pp. 57-76, In M, E. Soule (ed.), Conservation
crease: in the number of ribs on the fourth and sixth
Biology, Thc Sciencc orScareity and Diversity, Sinwhorls with an increase in heler07.),Sosit)' is unknown,
aucr, Sundcrland, MA,
The ribbing traits were suspected or being an adaptaBEOO,C, B., AND J, A, DERLtN, 1988, Publication
tion associated with defense: from crushing predators;
bias: A problem in intcrpreting medical data, J,
however, J, Quenson (reported in Oould and WoodrulT
Roy, Slat. Soc, A 151:419-463,
(1978) found that shell size (weight and height) was
CHAKRABORTY,
R" ANDN. RYMAN, 1983, Relationmore important than ribbing in determining shell
ship of mcan and variance of scnotypie values with
strength, Some workers have found allozymc-phenobeterozygosiry per individual in a natural populatype covariation at some loci but not at othcrs (Koehn
tion. Genetics 103:149-152,
et aI., 1988; Pemberton et aI., 1988), so analyses based
GOULD,S. J .. AND D, S, WOODRUFF, 1978, Natural
on multiloeus hetero7.ygosity such as ours may miss
history of Cerion VIII: Little Bahama lJank-A resome single-locus eflects.
vision based on genetics, morphometries, and geoIn pooling the 13 sites, there was a risk of obscuring
graphic distribution. Dull. Mus, Compo Zool. 148:
a significant correlation between heterozygosity and
371-415,
phenotype or or finding ~ignifleant correlations where
--.
19R6, Evolution and svstematics of Ccrion
there were none within the sites. In examining the dis(Mollusca: Pulmonata) on Ne~ Providence Island:
tribution of individuals in different heter07.ygosityA radical revision, Bull, Amer. Mus, Nat, Hist. 182:
classes in the 13 sites, snails in five sites were found
389-490.
to have relatively high hetcro~ygosity, and snails in
--,
1987. Systematics and levels of covariarion
eight sites were found to have lower heterozygosiry.
in Cerion from the Turks and Caieos islands, Bull
After pooling the high-hcterozygosity sites in one group
Mus, Comp. Zool, 151:321-363,
and the low-heterozygosity sites in another group and
GOULD, S, J.. D, S. WOODRUFF,ANDJ, P, MARTIN,
redoing the correlation analyses, no significant corre1974, Gcnetics and morphometries of Cerion at
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Pongo carpet: A new systematic: approach to this
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cnigmatie land snail, S)·5t. Zool, 2.1:5IR-535,
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zyrncs of glycolysi5 and I'rotein C':lIaboli~m to the
213
NOTES AND COMMENTS
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Corresponding
Editor: R. C. Vrijenhoek