Marine Biology
Marine Biology93, 517-525 (1987)
.................
9 Springer-Verlag 1987
Fissiparity and population genetics of Coscinasterias calamaria M. S. J o h n s o n and T. J. ThrelfaU Department of Zoology, University of Western Australia; Nedlands, Western Australia 6009, Australia
Abstract
The effect of asexual reproduction on the population genetics of the fissiparous seastar Coscinasterias calamaria was examined at Rottnest Island and the adjacent mainland, Western Australia, in 1985. Field samples and laboratory observations on growth rate and regeneration showed that fission is common at all 14 sites examined. Electrophoretic analysis of six polymorphic enzymes revealed low genotypic diversity and strong genetic disequilibrium at each site, confirming the highly clonal structure of local populations. Striking variation in clonal composition over distances as short as 50 m emphasizes the very localized subdivision of these populations. In the combined sample from all sites, however, the expected range of multilocus genotypes was found in the proportions expected from random mixing of sexually produced larvae, confirming that clonal diversity results from sexual reproduction. Local genotypic diversity was not correlated with fission, emphasizing the difficulty of determining the short-term roles of asexual and larval recruitment in the maintenance of populations. Subtidal populations, however, appear to have both lower incidence of fission and lower larval recruitment than do intertidal populations.
Introduction
The mixture of asexual and sexual reproduction raises ~ questions concerning both the evolutionary roles and the ecological effects of these contrasting modes of reproduction. Since the spatial and temporal scales of asexual and sexual recruitment generally differ, interpretations of the characteristics of populations should be based on the nature of recruitment. Asexual reproduction by fission is known to occur in 19 species of seastars (Emson and Wilkie, 1980). These include the three species of Coscinasterias: C. tenuispina, C. acutis?ina, and C. calamaria. All of the fissiparous species are also thought to reproduce sexually,
although sexual reproduction has been studied in field populations of only C calamaria (Crump and Barker, 1985) and Nepanthia belcheri (Otteson and Lucas, 1982). The relative contributions of fissiparity and larval recruitment to the maintenance of local populations are not known, although a high incidence of fission characterizing most species (Emson and Wilkie, 1980) indicates a substantial effect of clonal proliferation, and there is evidence for obligatory fissiparity in a population of Stephanasterias albula (Mladenov et al., 1986). The contribution of sexual reproduction to local populations is difficult to determine directly. Although gonadal indices can provide evidence for the occurrence of sexual reproduction, they provide no evidence for larval recruitment to those local populations, because larvae may die or disperse. Because of dispersal, even local populations with no sexual reproduction might receive significant larval recruitment from other local populations. Because of these difficulties, a genetic approach offers useful information on the contribution of asexual and sexual reproduction to the maintenance of local populations. Examples of the contrasting effects of different modes of reproduction on the genetic composition of marine populations are provided by several studies of sea anemones (e.g. Black and Johnson, 1979; Shick et al., 1979; Ayre, 1983; Hoffman, 1986) and corals (e.g. Stoddart, 1983a, 1984; Heyward and Stoddart, 1985; Ayre and Resing, 1986). In the present study, we have combined laboratory and field analyses of the incidence of fission with electrophoretic analysis of the genetic composition of populations of Coscinasterias calamaria, in order to determine the contributions of clonal and sexual reproduction. Materials and methods
Samples Between 19 and 47 seastars, Coscinasterias calamaria, were collected from each of ten sites on Rottnest Island (32~
518
M.S. Johnson and T. J. Threlfall: Fissiparity/population genetics of C. calamaria . I , T., G
2,COTTESLOE
ROC,S K 13" A ]4~,~ ~
--
~
~ .....
',
~rA ~.NANCY COVE
",S,R,C,LAND
~]'~3 4. GARDEN I~.] ISLAND
EAS
BAY
O
k ~
5 10 15 SCALE KM '
I
9
Fig. 1. Map of Rottnest Island and adjacent mainland coast, Western Australia, showing 14 sampling sites of Coscinasterias calamaria
115~ and two sites each on Garden Island and the mainland coast, near Perth, Western Australia (Fig. 1) in 1985. The two sites at Garden Island were approximately 50 m apart, and both were from seagrass beds just below the mean low-water mark. All of the other samples were taken from intertidal rocky platforms, except that most of the specimens from Cape Vlamingh were from subtidal pools within the platform. The three sites at Cathedral Rocks were approximately 50 m apart, and C. calamaria was present in the intervening areas. For most specimens, data were obtained on both form and genotype, but deterioration of either the body or enzymes prevented both types of data being obtained from some individuals.
Size and form For each seastar, size was measured as the radius (R), the length along the oral surface of the longest arm, from the mouth to the tip of the ambulacral spine row. Form was described by recording the number and relative lengths of arms. Three categories of arm length were recognized: "short" (S) refers to arms less than half as long as the maxim u m radius; "intermediate" (I) to arms between 0.5 and 0.9 as long as the maximum radius; and "long" (L) to arms at least 0.9 as long as the maximum. Observations in the laboratory were used to examine growth and the relationship between form and fission. Nineteen seastars from Cottesloe were maintained in the laboratory between February and October. Each individual was isolated in a two-litre plastic container with running seawater. They were fed the littoral gastropod Littorina unifasciata, at the rate of five snails per seastar per week, at weekly or, occasionally, fortnightly intervals. The lengths of all arms of the nineteen seastars were recorded at weekly intervals for a period of 213 d, except for one individual which died after 90 d.
To determine the number of arms which commonly replace a lost arm, and hence to test the hypothesis that the various individuals seen in the field with isolated small arms have not grown these as a result of fission, single arms were removed from twelve of the laboratory specimens, and an additional long arm was later removed from four of these. Removal was accomplished by bending a particular arm until a break appeared at the margin of the disc. Arms removed in this manner continued to move for more than 48 h in some cases, but all subsequently ceased movement, and were discarded. In a second experiment, the effects of food availability and container size on growth were examined, in a factorial design. Three levels of food (5, 10, and 30 snails per seastar per week) and three sizes of container (l, 2, and 22 litres) were tested, with five replicates of each treatment. The snails used in each treatment were selected randomly from a common supply. All seastars used in this experiment were collected within a three-day period, and were starved for 7 to 9 d before the experiment was initiated. Initial measurements for each individual were underwater weight (to 0.01 g) and radius. The reliability of underwater weight as a measure of biomass was confirmed by drying preweighed seastars at 80~ for 24h, and measuring dry weight; the correlation coefficient of the two measures was 0.97. Food was added weekly, the shells of the previous week's feeding were removed, and the number of snails eaten by each seastar was recorded. The experiment was terminated after six weeks.
Electrophoresis Samples of tube feet were prepared for electrophoresis by homogenization in 10% sucrose containing 0.I% mercaptoethanol and 0.1% of bromphenol blue (3 ml per gram of tissue). Electrophoresis was carried out in horizontal
M. S.Johnson and T. J. Threlfall: Fissiparity/population genetics of C. calamaria starch gels (Electrostarch), using a lithium hydroxide (LiOH), tris-EDTA-borate (TEB) or tris-maleate (TM) buffer (buffers 2, 6, and 9, respectively, of Selander et al., 1971). Variation was examined for six polymorphic enzymes: esterase (EC 3.1.1.1; Est locus; LiOH buffer); malate dehydrogenase (EC 1.1.1.37; Mdh locus; TM buffer); leucyltyrosine peptidase (EC 3.4.1.3; Ltp locus; LiOH buffer); phosphoglucose isomerase (EC 5.3.1.9; Pgi locus; TEB buffer); phosphoglucomutase (EC 2.7.5.1; Pgm locus; TM buffer); and superoxide dismutase (EC 1.15.1.1; Sod locus; TEB buffer). Staining recipes were essentially those of Nichols and Ruddle (1973). Alleles at these loci are indicated by superscripts representing the relative electrophoretic mobilities of their allozymes (f= fast; re=medium; s--slow). Variation was assumed to be allelic, as the isozyme patterns observed corresponded with those found for these enzymes in other organisms for which Mendelian inheritance has been confirmed directly. Variation in each sample was summarized as the number of multi-locus genotypes observed and the genotypic diversity. Genotypic diversity was calculated as Go = 1/Zg] (Stoddart, 1983b), where gi is the frequency of the ith genotype. Go is the effective number of genotypes, and its highest possible value, which is the number of genotypes present, occurs when all genotypes are equally common. The expected diversity, Ge, under Hardy-Weinberg equilibrium for a sample with the same allelic composition, was determined by simulation of sampling from a population with random mating, using the average of 100 runs. The computer program used was provided by J. A. Stoddart. The ratio Go: Ge provides a measure of single-locus and multilocus equilibrium, such as expected with sexual reproduction. Values of G0:Ge less than one are typical populations with a highly clonal composition (e.g. Black and Johnson, 1979; Stoddart, 1983 b; Hoffman, 1986). The role of sexual reproduction was evaluated at two levels: in the combined population and in each local population. To test for the mixing of sexually produced larvae among local populations, the genotypic frequencies of clones in the combined samples were compared with those expected under Hardy-Weinberg equilibrium. For this purpose, each genotype-site combination was regarded as a single clone. Because mating would occur within sites, variance in allelic frequencies among sites will reduce the expected proportion of heterozygotes through the Wahlund effect. The reduction in the expected proportion of heterozygotes at a locus with a Wahlund effect is equal to the sum of the variances of allelic frequencies among sites at that locus (Li, 1976). Barring mutation and repeated production of the same genotype through sexual reproduction, the number of different genotypes in a population, n, indicates the genetic contribution of sexual reproduction to that population. The repeated sexual production of the same genotype will render this indicator an underestimate of larval recruitment. The expected number of replicate individuals of a genotype is simply (N-1)gi, where N-1 is the sample size minus
519
the first individual with the genotype, and gi is the frequency of the genotype in the total pool of sexually produced individuals. To obtain a corrected estimate of the sexual input to each local population, the expected number of independent replicate individuals of each genotype was calculated, based on the assumptions of free recombination and random mixing of sexual propagules among populations. The 95% upper confidence limit of this estimate was obtained from the graph in Pearson and Hartley (1958), to give n*, the maximal estimate of the number of clones in the population. The contribution of sexual reproduction to each local sample was then taken as n* :N, the proportion of the sample represented by different clones. Possible associations of characteristics were tested by determining the product-moment correlations among sites for n*:N, Go, G0:Ge, R, CVR (coefficient of variation of size), and the proportion of individuals which divided recently (determined from analysis of form).
Results
Growth and form The most common result of removal of an arm from Coscinasterias calamaria was the appearance of one small arm in the gap (13 out of 16 cases), although the growth of up to four small arms in the gap was observed. Small arms also arose in other regions of disc, but in equal frequency in those individuals with and without an arm removed (4 of 12 individuals and 2 of 7 individuals, respectively). A preliminary analysis of field samples indicated that about 15% of individuals had such small, isolated arms. As this is a lower frequency than observed in the laboratory individuals, these small arms probably represent either regeneration following the loss of an arm, or spontaneous generation, instead of fission. Consequently, such arms were not included in the analysis of form. During the 213 d period of observation, 15 of 19 seastars divided at least once, at times ranging from 9 to 212 d from the initiation of the experiment. Further division occurred in three of the containers; in two of these, each of the individuals resulting from the first division subsequently divided. Three observations of the time elapsed between successive divisions were obtained: 128, 135, and 135 d. Following fission, the time elapsed between appearance of the first new arm and the last new arm ranged from less than 1 wk (1 seastar) to up to 4 wk. For those seastars in which new arms were present at three successive weekly observations, the minimum possible delay betwen onset and completion of addition of new arms ranged from 1 to 3 wk. In 10 of 16 regenerations observed to completion (with the gap in the disc completely filled, and no new arm formation for two successive observations), the final arm was formed in the central portion of the gap; in the other six cases, regeneration was completed at the margin, indicat-
520
M.S. Johnson and T. J. Threlfall: Fissiparity/population genetics of C. calamaria
Table 1. Coscinasterias calamaria. Classes recognized in the analysis of form and regeneration Class
Description
Interpretation Time since last division
No. of divisions
L+ G
All arms of similar length (long), with a large gap in disc, with or without arm buds forming in the gap
=<2 wk
1
L+I+G
Two sets of arms (long and intermediate), large gap in disc, with or without arm buds forming in the gap
_-<2wk
>=2
L+S
Two sets of arms (long and short)
2 to 12 wk
>= 1
L + I+ S
Three sets of arms of different lengths
2 to 12 wk
>=2
L+I
Two sets of arms (long and intermediate)
12 to 25 wk
>=1
L+ L
Two sets of arms of similar length (long)
>=25 wk
>= 1
L
One set of arms (long)
Never divided
None
Table 2. Coscinasterias calamaria. Numbers of transitions between forms occurring during weekly intervals between observations of individuals in the laboratory. Form classes as in Table 1 Form after 1 wk
Initial form L+G
L+G L+I+G L+S L+I+S L+I L+L Split
17 12 . -
Total
29
L+I+G 9 2 .
. 11
,
L+S . 46 13 . 2
L+I+S
L+I
-
2
-
9 1
32
2
9
7 -
12
43
7
.
61
ing that there is not a consistent directionality to the addition of new arms. The average time required for growth of new arms to intermediate length was virtually the same for central and marginal arms: 72__+21 (SD) and 67_+ 19 d, respectively. Thus, time delays explain differences in sizes of central and marginal arms after considerable periods following fission. The delay between the onset of formation of initial a n d final new arms suggests that the best available indicator of time since fission is the radius of the longest regenerated arm. This indicator is apparently i n d e p e n d e n t of initial size of the seastar, as the time for regenerated arms to reach intermediate size was only marginally a n d n o n significantly correlated with initial size (r = 0.34, 24 DF). Based on these results, we recognized seven form classes of Coscinasterias calamaria (Table 1). The relationship of these classes to fission was determined by observation of the pattern of regeneration in the laboratory (Table 2). A m o n g all weekly observations of individuals with Form L + G (recently divided, without regenerated arms), 41% changed to Form L + S (appearance of short arms in the gap), while the remainder did not change form. Of those of Form L + S , 21% changed to L + I , 75% remained unchanged, and 3% divided in the interval between suc-
L+L
.
.
.
cessive weekly observations. None of the divided seastars in the laboratory reached the L + L stage during the 213 d period of the observations, and none of the L + L individuals changed form during that period. The L + I + S form was produced only from L + I + G individuals, which result from a second division. Fission occurred most often in the L + I individuals (65% of observed cases of fission), as 16.5% of L + I individuals divided between successive weekly observations. The proportion o f L + S individuals which divided between successive observations was significantly lower (Z2=6.55, P < 0.05), at 4.6%. The fission o f L + I seastars can produce two forms, d e p e n d i n g on the axis of the division across the centre of the disc: L + G and L + G if the division is in the same plane as that of the preceding division; or, L + I + G a n d L + I + G if the division lies at some angle to the plane of the preceding one. Of the 26 individuals resulting from fission of L + I s eastars in the laboratory, only 4 (15 %) were of Form L + I + G , while 13 (50%) were of Form L + G , indicating that successive divisions tend to occur in the same plane. From these results, it can be seen that the most comm o n single sequence of form change and fission is from L + G to L + S to L + I to two seastars, each L + G.
M. S. Johnson and T. J. Threlfa[l: Fissiparity/population genetics of C. calamaria In the experiments on the effects of amount of food and size of container on growth and fission, 37 of the 45 seastars survived the six-week period, leaving a m i n i m u m of two replicates per treatment, rIlae change in weight (A W) and in size (AR) were calculated from initial and final recordings. There was no significant correlation between A W and initial weight (r=0.15, 35 DF), and two-way analysis of variance of A W showed no effect of food, container size, or interaction between the two variables. Similarly, AR was not significantly correlated with initial size (r=4).35, 35 DF), and analysis of variance showed no significant effects of treatments or their interaction. O f the seastars provided with five snails per week, 62% consumed the possible m a x i m u m of 30 snails over the 6 wk(average consumption = 29 snails). Only 38% of those provided with 10 snails per week consumed the possible m a x i m u m of 60 (average = 54), and none of the seastars allowed 30 snails per week ate the possible 180 (average = 117). This variation indicates that the range of food levels offered is large with respect to the appetite of the seastars. There was no significant correlation, however, between the n u m b e r of snails eaten and A W ( r = 0 . 2 2 , 35 DF). One-way analysis of variance also showed no significant effect of container size on A W (F2,34--2.1). Similarly, the proportion of individuals undergoing fission did not vary with respect to food levels or size of container.
Field populations All of the field samples showed evidence of fission. If we apply the laboratory results to the field samples, the implication is that 25 to 91% of individuals had undergone fission between 0 and 12 wk before collection (Table 3), as indicated by either gaps on the disc or a set of small arms. There was, nevertheless, considerable variation in the apparent incidence of fission. The subtidal sites at Garden Island and the largely subtidal sample from Cape Vlamingh had the lowest proportion of recently (less than 12 wk,
521
based on form) divided individuals, 25 to 39%. These were the only samples with more than 20% of individuals either showing no sign of fission, or with the second set of arms having reached the length of the original set, indicating that division had not occurred within the previous 25 wk. The general importance of fission in the recruitment to all of the populations is supported by the genetic data. The n u m b e r of multi-locus genotypes was generally small, ranging from 2 to 16, and the ratio of observed to expected genotypic diversity was less than 0.6 in all but two samples (Table 4). The individual samples displayed numerous cases of extreme departure from Hardy-Weinberg equilibrium, ranging, for example, from the great preponderance of heterozygotes at the Sod locus in several samples [e.g. Garden Island A and B (Sites 3, 4), Radar Reef (9), and Fishhook Bay (10)] to the nearly complete deficit ofheterozygotes at the same locus in Sample B from Cathedral Rocks (13) (Table 5).
Table4. Coscinasterias calamaria. Genotypic diversity (Go) and numbers of distinct six-locus genotypes (n). Ge: expected diversity; n: genetic contribution of sexual reproduction; n*: maximal estimate of no. of clones; n*:N: contribution of sexual reproduction to sample Sample
N
Go
Go: Ge
n
n* :N
Trigg(1) Cottesloe (2) Garden Island, A(3) Garden Island, B (4) Nancy Cove (5) Green Island (6) Strickland, East (7) Strickland Bay (8) Radar Reef(9) Fishhook Bay (10) Cape Vlamingh (11) CathedralRocks, A (12) Cathedral Rocks, B (13) CathedralRocks, C (14)
19 47 30 44 37 31 36 24 22 36 20 24 36 24
3.57 2.34 1.72 1.10 1.84 3.64 5.68 1.18 1.58 1.41 1.22 2.69 4.61 1.55
0.60 0.34 0.12 0.16 1.21 0.30 0.39 0.88 0.40 0.23 0.33 0.51 0.58 0.40
7 9 2 2 7 13 16 2 3 3 2 7 8 4
0.39 0.21 0.09 0.07 0.19 0.47 0.46 0.12 0.20 0.12 0.14 0.34 0.26 0.23
Table 3. Coscinasterias calamaria. Percentage of individuals inferred to have divided within specified periods prior to sampling. Site numbers as in Fig. 1. CVR : coefficient of variation of size Sampling site and (Site No.) Trigg (1) Cottesloe (2) Garden Island, A (3) Garden Island, B (4) Nancy Cove (5) Green Island (6) Strickland, East (7) Strickland Bay (8) Radar Reef (9) Fishhook Bay (10) Cape Vlamingh (11) Cathedral Rocks, A (12) Cathedral Rocks, B (13) Cathedral Rocks, C (14)
N
20 29 30 44 36 32 36 28 35 35 23 46 61 31
Time since fission (wk)
Radius (ram)
=<2
2-12
12-25
= 25
Mean
CVR
0 3 3 0 0 9 33 21 11 11 0 13 18 3
50 45 37 25 53 50 58 68 60 57 39 59 70 84
40 38 27 41 42 41 6 11 23 31 39 28 11 13
10 14 33 34 6 0 3 0 6 0 22 0 0 0
50.3 27.6 44.7 53.7 41.7 47.3 32.6 24.4 44.1 13.9 50.1 48.8 30.6 21.6
0.25 0.23 0.45 0.27 0.23 0.27 0.57 0.62 0.32 0.33 0.45 0.24 0.42 0.31
522
C. calamaria
M . S . J o h n s o n a n d T. J. T h r e l f a l l : F i s s i p a r i t y / p o p u l a t i o n g e n e t i c s o f
T a b l e 5. Coscinasterias calarnaria. G e n o t y p i c c o m p o s i t i o n o f s a m p l e s . V a l u e s a r e n u m b e r s o f i n d i v i d u a l s . S a m p l e c o d e s as i n Fig. 1 a n d T a b l e 3. f: fast; m : m e d i u m ; s: s l o w Genotype
Mainland
Rottnest Island
Est
Mdh
Ltp
Pgi
Pgm
Sod
1
2
3
4
ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff ff fs fs fs ss ss ff ff ff fs ff ff ff
ss ss fs fs ff ff ff ff ff ff fs fs fs fs ff ff ff ff fs ff ff ff ff ff ff fs fs fs ff ff ff ff ff ff ff fs ff ff fs ff fs fs ff ff ff ff ff ff
ff fs fs ss ss ff fs ff ff fs ff fs ff ss fs ff ff ff ff ss ff ff ff fs fs fs ss ss ss ss fs fs fs fs fs ss ff ff fs ff ss ss ss ss ss ss ss ss
mm mm mm mm mm mm mm fm mm mm mm mm fm mm mm ff ff fm fm fm ff ff mm ff ff fm ff fin ff fm fm ff ff mm mm fm fm mm mm ms ss mm mm fm fm mm fm mm
mm ms ms mm mm mm mm mm mm fm mm mm mm fm mm fm mm mm mm fm ms mm mm mm mm mm mm mm mm mm mm fm mm mm ms mm mm mm mm ff ff mm mm mm mm mm mm fm
ss ss ss ss ss ss ss ss fs ss ss ss fs ss fs ss ff ff ff ss ff ss ff ff fs fs fs ff ss ss ss ss ss ff ss ss ss ss ss fs ff fs fs fs ss ff ff ss
1 2 1 9 1 3 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . 30 2 . 3 1 4 2 3 1 1 . . . . .
. . . 9 -
. . . 2 -
Comparisons generally (Table found between
between
populations
very little overlap
5).
Of
the
at more Garden only
sites. Even
between
In contrast
detected,
site (excluding were
found
. . . . . . . . . . .
. . . . . . . . . .
there
with
were found
the paucity
the
combined
sample
the
possible
genotypic
among
Rocks
sites included among
within nearly the
2
. . .
. . . . .
. . . . .
2
. . . . . . .
(Table
. . . . . 1 1 1 4 1 2 1 1 4 2 1
. . . . .
. . . . .
. . . . . . . . . 22 . . . . . . . . . . .
all of
age deficit among from (Table
8 3
1 -
1
-
-
1 -
19 3 -
. . . . . 2
. . . . . . . . .
. . . . . . . .
. . . . . . . .
. 18
.
. . . . . . . . . . . . 30
. 2 2
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
.
.
.
2
1
across
loci when which For
from
sexual
of clones, there
the loci. This variation
values.
1
.
.
. .
and the expected
structure,
-
.
. . . .
4
be expected
population
1
.
.
. . . . . . . . .
1
.
sample
. .
.
. . . .
. . . . . . . . . .
. .
Rocks,
. .
.
.
. . . . . . . . . . . .
. . .
deficit of heterozygotes
the average
. .
.
. . .
. . .
.
. .
5), a s w o u l d
the observed
.
. . . . . . . .
14
. . . . .
. . . .
4 17
. . . . .
.
. .
.
.
13
14
.
. . . . . . . .
. .
.
. . . . .
. . . .
.
.
.
.
. . . . .
. . . . . . . . . 1 1 1 1 1 1 6 2 2 1
several
six loci
. . . . . . .
.
examining
sites,
.
I
In the combined
among
.
12 . . .
2
.
.
1
. . . . . . .
. -
. . . . .
1
11 . . .
.
.
. . . . . . . . . . .
10 . . .
. 2 . . . . . . .
-
.
9 . . .
1 t 11 4
7 5
. . . . .
8
two
at only one site each.
of genotypes
combinations
only
than
the very close sites at Cathedral
genotypes
is
15 w e r e
Cathedral
. . .
1 27 3 1 1
. . . . . . .
shared
at more
. . .
7
. . . . . . . . . .
. . . . . . .
only
. . .
. 42
. . . . . . . . . . .
. . . . . . . . . .
6
. . . . . . -
composition
those
sites or between
6 of these
.
. . . . . . 21 . . . . .
. . . . . . . . . . .
that
in their genotypic
genotypes
one
Island
sites), and
two abundant
48
than
show
. . . . .
.
5
the
-
-
reproduction.
was a consistent
six loci, although
deficits varied underlines using
5
13 2
both
considerably
the importance
genetic
data
of
to infer
is most likely to be reflected in
Coscinasterias ca&maria,
the aver-
the six loci is very close to that expected
the large variance 6), s u p p o r t i n g
in allelic frequencies
the interpretation
among
that sexual
sites repro-
M. S. Johnson and T. J. Threlfall: Fissiparity/population genetics of C calamaria duction plays an important role in the dispersal of individuals among sites. Except for the proximate sites at Garden Island and at Cathedral Rocks, sexual reproduction is the probable cause of sharing of multilocus genotypes among sites. The most widespread genotype, for example, is the multilocus combination most commonly expected from the observed overall allelic frequencies. Mthough both the morphological and the genetic data indicate a major impact of fission in these populations, variation in the indices of fission and of genetic diversity among sites are not correlated (Table 7). The subtidal sites at Garden Island, which appear to show the lowest prevalence of fission, also show the least genetic diversity, measured either as n*:N or G0:Ge. The genotypic diversity in the Cape Vlamingh sample is also among the lowest observed. Among the intertidal samples, however, there is no apparent relationship between incidence of fission and genetic diversity. There is a negative correlation between the incidence of fission and average size. Examination of the occurrence of fission within samples, however, failed to detect a tendency for smaller individuals to divide more fiequently, except at Garden Island Site A. The association in that sample is complicated by a difference in average size Table 6. Coscinasterias calamaria. Proportional deficits of heterozygotes for six loci in the pooled sample of clones, compared with the variance of allelic frequencies among sites. He, Ho: expected and observed proportion of heterozygotes, respectively; Zs} : variance summed amongst alleles at each locus Locus
He- Ho
Est Mdh Ltp Pgi Pgm Sod
Xs}
0.047
0.006
- 0.001
0.038
0.213 0.136 0.033 0.164 0.099 (0.035)
Average (_ SE)
0.183 0.150 0.001 0.139 0.087 (0.033)
523
between the two genotypes found at that site (Table 8), so it is not clear whether the smaller individuals tend to divide more because of their size or their genotype. In none of the other samples were two genotypes sufficiently common to make such comparisons.
Discussion As is typical of fissiparous asteroids (Emson and Wilkie, 1980), populations of Coscinasterias calamaria show evidence that fission plays an important role. The genetic data strongly suggest the predominance of clonal reproduction. All samples had low genotypic diversity, and the large departures from Hardy-Weinberg equilibrium and linkage equilibrium are typical of predominately asexual populations. Consistent with the genetic data is the observation that nearly all individuals showed physical evidence of fission. The laboratory experiments indicate that growth is relatively rapid. Direct extrapolation of these laboratory growth rates to field samples is precarious, especially since the experimental individuals represent an uncertain number of clones from only one population. Nevertheless, the experiments showed that rate of growth and frequency of fission do not vary with respect to a wide range of food availability or container size, providing some confidence in the cautious application of the laboratory results to the field samples. The implication is that fission in populations of C. calamaria is frequent, with most specimens having divided no more than three months before collection. The importance of asexual reproduction can also be seen in the spatial scale of genotypic variation. Although we do not have estimates of the movements of these seastars, the large differences in clonal composition between sites as little as 50 m apart indicate that mixing of asexually produced individuals does not occur over large distances. This highly localized, clonal structure complicates analysis
Table 7. Coscinasterias calamaria. Correlation coefficients among genetic and morphological characteristics of samples from 14 sites. Significant correlations: ** P < 0.01
Sexual contribution, n* :N Genotypic diversity, Go Disequilibrium, Go:Ge Mean size, R Coefficient of variation, CVR % recent fission
Go:Ge
n*:N
Go
~ 0.83"* 0.07 0.16 - 0.11 0.35
. . . ~ 0.04 ~ 0.13 -0.08 ~ 0.16 0.02 0.39 0.30
R
CVR
. - 0.29 - 0.65 **
- 0.42
Table 8. Coscinasterias ealamaria. Comparison between two clones at Garden Island Site A for recency of fission and size Genotype
Time since fission (wk)
Size (mm)
Est
Mdh
Ltp
Pgi
Pgm
Sod
_-<12
12-25
==.25
<30
30-60
>60
ff ff
ff fs
fs ss
mm mm
mm mm
fs ss
4 8
7 1
10 0
2 7
13 2
6 0
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M.S. Johnson and T.J. Threlfall: Fissiparity/population genetics of C. calamaria
of populations of Coscinasterias calamaria. For example, the tendency for larger individuals to be non-fissiparous was observed in New Zealand populations (Crump and Barker, 1985), but such a difference in the Garden Island Site A is confounded by the difference in size between the two clones at that site. It is not clear whether this represents a genetic difference in growth or fission, or simply a different history of the two clones. A similar problem could well underly the finding of large variation in sex ratio in populations of C. calamaria in New Zealand (Crump and Barker, 1985): highly skewed sex ratios could easily reflect the small number of clones in local populations. The difficulty is that, with clonal reproduction, "individuals" are not independent cases, and understanding of differences in ecological characteristics of populations must be based on an understanding of the clonal composition of those populations. Since the scale of clonal mixing in C. calamaria is quite local, even the characterization of populations and their variation over time must be made with careful attention to the clonal composition. Underlying this localized asexual proliferation is sexual reproduction. This species shows a seasonal cycle of gonadal development in New Zealand (Crump and Barker, 1985). Although we did not examine the sexual cycle in Western Australia, the genetic data provide strong evidence of sexual reproduction. Taking all of our samples together, the expected range of multilocus genotypes was found, and in proportions that correspond with random genetic mixing. This implies that the genotypic diversity in this species is due to sexual reproduction, rather than to the evolution of clonal lineages, because only recombination could randomize the genotypic combinations within and amongst loci. The scale of mixing of sexually produced larvae is much larger than that for the mixing of clones. With one exception, the alMic frequencies of samples of clones are quite similar among sites. The exception is the uncommon Est s allele, which was found in six different clones; five of these were from Strickland East (Sample 7, Table 5), suggesting that sexual reproduction and recruitment can be localized. Nevertheless, the fit of the entire sample of clones to Hardy-Weinberg equilibrium is consistent with considerable mixing of larvae throughout the study area, representing distances up to 40 km. Thus, as is typical of species with mixed modes of reproduction (Williams, 1975; Bell, 1982), the sexually produced propagules disperse, whereas the asexually produced individuals remain near their point of origin. The highly clonal structure of populations of Coscinasterias calamaria indicates that, in the short term, asexual proliferation predominates over larval recruitment. It should be emphasized that n* :N provides an estimate of sexual input over the long term, and the actual contribution of sexual reproduction to population maintenance will be smaller than this proportion. At Garden Island, for example, the number of larvae represented in the combined sample of 77 individuals was only 2. That sample represented less than 10% of the population, probably much less. If we take 6 mo as the average interval between divi-
sions of C. calamaria at Garden Island (the data in Table 3 indicate a minimum average of 17 wk), and assume that no mortality has occurred, it would take at least 4 yr of asexual proliferation for two recruits to produce the existing population. While this is a very rough calculation, it indicates that some local populations may go many years without larval recruitment. There is evidence from other marine species with mixed modes of reproduction that some populations may receive larval recruits very rarely (e.g. Fadlallah, 1982; Ayre, 1984). Without knowing the life-span of clones, however, it is not possible to infer from the genetic data just how small is the actual rate of sexual input. The problem of obtaining a more precise measure of the relative contributions of larval and clonal recruitment is exacerbated by the lack of correlation between genetic diversity and the incidence of fission. Thus, a relatively low incidence of fission does not necessarily mean that larval recruitment is locally more important, as is shown clearly by the low genotypic diversity and low incidence of fission in the subtidal samples from Garden Island and the largely subtidal sample from Cape Vlamingh. Less fission was also found in subtidal populations of Coscinasterias calamaria in New Zealand (Crump and Barker, 1985). In those populations, the lower incidence of fission was associated with larger size and higher gonadal indices, indicating that the subtidal contribution to the pool of larvae is probably disproportionately large (Crump and Barker, 1985).The genetic data from the Western Australian samples, however, indicate that the subtidal populations have low levels of recruitment from both asexual and sexual reproduction, emphasizing the important distinction between sexual reproduction and larval recruitment. Beyond this apparently low rate of both types of recruitment in the subtidal populations, there is no detectable relationship between the levels of asexual and sexual input to local populations. Although there is a negative correlation between average size and the incidence of fission, none of the genetic variables were correlated with these characteristics. Thus, an understanding of the dynamics of populations of C. calamaria will require direct estimates of rates of recruitment and turnover from both modes of reproduction. In addition to the general lack of correspondence of variation in the genetic and morphological characteristics of these populations is the surprising lack of a correlation between Go:Ge and either n*:N or Go. This points to a problem in characterizing the genetic pecularities of populations with varying amounts of asexual reproduction. In the present case, the difficulty is the way in which the expected genotypic diversity is calculated. Since Ge is based on allelic frequencies in each local sample, it will be affected by sampling effects of a small number of clones. Ideally, Ge should be based on the pool of potential sexually produced recruits. In the present case, the evidence for widespread mixing of larvae indicates that this pool would be the same for all sites, so that n*:N or Go provide the more meaningful comparison among sites. Although values of Go:Ge much less than unity characterize many predomi-
M. S. Johnson and T. J. Threlfall: Fissiparity/population genetics of C. calamaria nately asexual populations, variation in Go:Ge has been used in only two previous studies, both on anemones (Ayre, 1984; Hoffman, 1986). In each case, the contrast was between populations with predominately sexual recruitment and those with highly clonal structures. Thus, Go:Ge provides an indication of disequilibrium within populations, but its accuracy as a comparative measure of clonal structure is limited.
Acknowledgements. We thank H. Clapin and J. D i a m o n d for assistance, and R. Black for advice, assistance with collection of specimens, and comments on the manuscript. J. Stoddart kindly provided the computer program for estimating expected genotypic diversity. A portion of the study was included in an honours thesis (by TJT), Department of Zoology, University of Western Australia. Accommodation was provided by the Rottnest Island Biological Research Station. This is a contribution from the Marine Biological Laboratory, University of Western Australia.
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Date of final manuscript acceptance: September 5, 1986. Communicated by G. F. Humphrey, Sydney
