Oecologia
Oecologia (Berlin) (1984) 65 : 108-111
9 Springer-Verlag 1984
Population structure of the intertidal copepod Tigriopus californicus as revealed by field manipulation of allele frequencies R.S. Burton 1 and S.G. Swisher a
1 Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA 2 Department of Biological Sciences Stanford University Standford, CA 94305, USA Summary. Allele frequencies in natural T. californicus popu-
lations were perturbed by introduction of copepods from neighboring differentiated populations. In five experiments, the Gpt v allele was introduced into single recipient pools at a frequency of approximately 20%. In each case, the introduced allele declined to low frequencies ( < 3%) in less than one month, apparently due to dilution by residents of other pools on the same outcrop. In a larger scale experiment, the Pgi F was introduced into four pools on a single small rock outcrop; all pools on the outcrop were subsequently monitored. While the allele frequency fell from approximately 40% to 10% during the first six weeks after the transplant, no further change in frequency was observed for the duration of the experiment (16 months). Within six weeks some spread of the allele to non-recipient pools on the same outcrop was observed; by eight months, allele frequencies in all pools on the outcrop were similar. Hence, despite the extensive turnover of subpopulations as single pools evaporate or are washed out, genetic homogeneity and stability of entire outcrops are maintained via extensive inter-pool gene flow; this contrasts sharply with the highly restricted levels of inter-outcrop gene flow.
Populations of the harpacticoid copepod Tigriopus californicus occur in high intertidal and supralittoral rock pools along the western coast of North America. Dethier (1980) has shown that this habitat provides a refuge for T. californicus from a diverse and abundant predator fauna which inhabits lower intertidal pools. However, while serving as a refuge from predation, high intertidal pools present an extreme physical environment; entire T. californicus populations are destroyed when pools dry completely during periods of low tides and warm weather. At the other extreme, larvae and adults are washed out of the pools during periods of extreme wave action. Hence, populations of T. californicus inhabiting single rock pools undergo an irregular cycle of colonization, population expansion and contraction, and extinction, with the frequency of the cycle determined by both the weather and pool location in the intertidal (Vittor 1971). In previous work (Burton et al. 1979; Burton and Feldman 1981, 1982), we have made use of enzyme polymorphisms to investigate the genetic structure of natural populations of T. californicus. These studies have documented Offprint requests to. R.S. Burton
the widespread occurrence of unique alleles at several enzyme coding gene loci which " m a r k " populations from different geographic sites along the California coast. While populations inhabiting pools located on a single rock outcrop tended to be genetically homogeneous, those located on outcrops separated by even short stretches (<500 m) of sandy beach were frequently sharply differentiated at one or more loci. No obvious patterning of allele frequencies could be discerned, but the observed differentiation has been stable through time (some sites have been under study for six or more years, Burton, unpublished). The patchy occurrence of unique alleles in high frequency combined with the long-term stability of this pattern suggest that highly restricted gene flow plays an important role in determining population genetic structure in T. californiCUS.
In the experiments discussed here, we have perturbed allele frequencies in natural populations of T. californicus by transplanting large numbers of individuals "marked" with a high frequency of a particular allele into a habitat occupied by a population in which that allele was rare or absent. The purpose of the experiments was two-fold: (1) to determine the feasibility of manipulating allele frequencies in natural T. californicus populations, and (2) to verify the conclusions we have previously drawn from descriptive studies of population structure in this species (Burton and Feldman 1981). Materials and methods
Populations of T. californicus located between Moss Beach (San Mateo County) and Santa Cruz (Santa Cruz County), California, served as source and recipient sites for the transplant experiments. The number of copepods used in each transplant was crudely estimated by counting the number of adults in two 50 ml subsamples of the transplant samples (typically 4 to 8 1) which were obtained from natural pools with a small fish net. These estimates are intended only to give order of magnitude information. Pools were selected to receive transplants based on their size and shape (volume of 8-12 1 and lacking deep crevices) and general suitability of habitat (as evidenced by healthy resident T. californicus population at the time of the transplant and the likelihood that the pool would not dry completely during summer). An effort was made to use pools that were somewhat isolated from other pools on the same outcrop in order to minimize "dilution" of the transplant by local residents. Recipient pools were siphoned dry and rinsed one or more
109 Table l. Allele frequencies for the Z-site transplant, significance of deviations from Hardy-Weinberg genotypic frequencies (Chi-square test), and heterogeneity of Z-site pools (G-test)
Date
Site
N
Pgiv
Pgi~
Pgis
H-W
12/2/81
Pes Ca Cb
30 30 19
0.483 0.000 0.368
0.517 0.833 0.632
0.000 0.167 0.000
n.s. n.s. n.s.
12/6/81
C~
30
0.383
0.617
0.000
n.s.
12/14/81
C
55
0.382
0.609
0.009
n.s.
1/15/82
C D F L H J K
10 8 10 10 9 10 10 67
0.200 0.063 0.150 0.000 0.055 0.200 0.000 0.097
0.700 0.938 0.850 0.850 0.889 0.750 1.00 0.851
0.100 0.000 0.000 0.150 0.055 0.050 0.000 0.052
P<0.05
Pes N70
83 48
0.515 0.000
0.466 0.905
0.015 0.095
n.s. n.s.
D H J M N O
42 47 29 59 41 38 293
0.131 0.085 0.086 0.140 0.100 0.040 0.105 0.101
0.809 0.860 0.793 0.825 0.790 0.865 0.850 0.829
0.060 0.055 0.121 0.035 0.110 0.095 0.048 0.070
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
59
0.000
0.915
0.085
n.s.
47 32 54 46 25 43 49 296
0.106 0.125 0.138 0.108 0.100 0.093 0.143 0.118
0.819 0.812 0.768 0.814 0.740 0.860 0.714 0.791
0.075 0.062 0.092 0.076 0.160 0.046 0.143 0.091
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
Z-site (sum) 8/27/82
Z Z-site (sum)
12/11/82
N70 A C D F J O Z
Z-site (sum) 5/10/83
Z-site (sum)
42
Pes N70
97 105
0.459 0,014
0.536 0.905
0.005 0.081
n.s. n.s.
A C F J
61 80 61 65 267
0.115 0.131 0.098 0.054 0.101
0.795 0.756 0.844 0.854 0.809
0.090 0.113 0.057 0.092 0.090
n.s. n.s. n.s. n,s. n.s.
Heterogeneity of Z-site pools
P<0.05
n.s.
n.s.
a Sampled prior to transplant introduction b Sampled after introduction of transplants ~ Sampled after second introduction of transplants times with seawater to remove as much of the resident population as possible before introducing the transplant sample; after adding the sample, pools were refilled to their original level with seawater. This procedure left much of the pools' attached flora and associated microfauna intact so that the transplant population was introduced to a relatively undisturbed habitat. Sampling schedules are given along with the results of individual experiments. Electrophoresis of individual adult T. californicus was carried out on vertical acrylamide slab gels following Burton and F e l d m a n (1981). Results
The first set of transplants involved moving T. californicus collected at Santa Cruz (SC) to single cleared pools located
near Pescadero State Beach (PES), Bean Hollow State Beach (BH), and Moss Beach (MB). The genetic marker in these experiments was the Gpt v (glutamate-pyruvate transaminase) allele which occurs at SC but not at the other sites (Burton and F e l d m a n 1981). Each transplanted sample from SC consisted of from 3,000 to 10,000 adult copepods. Initial allele frequencies and trajectories are plotted for each pool in Fig. 1. Allele frequencies are based on samples of 35 to over 100 individuals. The rapid decline in the frequency of Gpt v in the first weeks after the transplants at all three sites (five pools) suggested that the transplanted p o p u l a t i o n was being rapidly diluted by immigration from surrounding pools. The hardy nature of 7". californicus adults and the general similarity of the local environment at the transplant and recipient sites makes the alternate explanation (strong differential
110 .3 84 Zx
GPtF
9
.2-.1 a!.~~~~
J J A S O N D J F Month Fig. 1. Allele frequency trajectories for five single-pool transplant experiments carried out in 1979-1980. Symbols: open square= PES, closed circles= BH1, closed triangle=MB, closed square= BH2, open triangle=BH3. Sampling was terminated when the allele frequency dropped below 1.0% or no copepods could be obtained from the recipient pool
N
"Q"'-'",
f
/
(
....
/
'~l l l O
: vZ.
,"
',.
\ -',.a
" ,~
----.. "-
,-,,\\ \ X \
-xx~ \ \ \
Fig. 2. Relative locations of pools on the Z-site outcrop. Contour lines indicate meters above mean high water
mortality among transplanted genotypes) somewhat unlikely. One problem with using G p t F a s the marker of interest was the lack of source populations with that allele in high frequency. In order to alleviate this problem, the final transplant focussed on the Pgi (phosphoglucose isomerase) polymorphism in the area around PES previously described in Burton, et al. (1979) and Burton and Feldman (1981) and was carried out on a considerably larger scale. The source population was located at PES, where the PgiF allele has had a frequency of approximately 0.5 for over four years. Recipient pools were located on a single outcrop (designated Z-site), approximately 700 m south of PES, where the Pgi v allele has been rare or absent in numerous samplings over the past four years. The relative positions of Z-site pools that were inhabitated by T. californicus sometime during the period from December, 1981, to May, 1983, are shown in Fig. 2. A total of approximately 100,000 adult copepods from PES were introduced into pools lettered
" A " through " D " on December 2 and December 6, 1981. Z-site is typical of Tigriopus habitat in that the pools inhabitated by T. californicus change seasonally. For example, pools " M " and " O " typically harbor large copepod populations during the late summer while pools " A " , " B " , and " C " may be dry; in winter, the latter pools are inhabitated by T. californicus while the former (lower) pools are frequently washed by heavy surf and lack copepod populations. Allele frequencies for pools at Z-site, PES, and a control pool (N70) located approximately 70 m north of Z-site (approx. 630 m south of PES) are presented in Table 1. The pools sampled during each visit generally indicate the distribution of Tigriopus populations on that date. While the January, 1981 (6 weeks after the transplant), data set is extremely limited, it is noteworthy that the Pgiv allele had already spread to some non-recipient pools and that the mean frequency of PgiF in Z-site pools (0.097, N = 67) was essentially identical to that of August, 1982, sample (0.101, N = 293) and the two later samples. Genotypic frequencies of the summed Z-site pools deviate from Hardy-Weinberg expectations only in the January, 1982, sample; this deviation was due to a heterozygote deficiency which is attributable to the Wahlund effect following the introduction of the transplants to Z-site. The animals sampled on this date apparently consisted primarily of original transplants and residents and their progeny (i.e. offspring of pure PES crosses and pure Z-site crosses rather than inter-site hybridizations). This is not unexpected since T. californicus females mate only once (Burton, unpublished results); hence, the transplanted adult females had already been fertilized by males of their own population. Subsequent samples were found to meet Hardy-Weinberg expectations indicating that the gene pools of transplants and recipients had mixed to form a single populaton. Allele frequencies of all Z-site pools from the August, 1982, to May, 1983, sampling are homogeneous (0.1 < P < 0.5, G-test). These observations are of considerable interest in that they indicate genetic homogeneity of the Z-site pools over time despite the extensive turnover of individual subpopulations. Pools that were unavailable as T. ealifornicus habitat during the initial transplant period and subsequently colonized were apparently colonized en masse by residents of existing Z-site pools. Similarly, pools that dried completely and subsequently refilled were also apparently recolonized from existing Z-site pools. Discussion
Previous descriptive studies of the genetic structure of Tigriopus populations (Burton et al. 1979; Burton and Feldman 1981) have indicated that while populations inhabiting pools located on the same rock outcrop tend to be undifferentiated, those located on neighboring outcrops separated by even short stretches of sandy beach often show sharp genetic differentiation. The stability of between outcrop differences seemed especially striking in light of the ephemeral nature of habitat; few, if any, of the pools on many outcrops are suitable Tigriopus habitat year-round. In fact, some low elevation outcrops appear to be completely devoid of habitat in winter (all pools awash), while high elevation ledges sometimes dry completely during summer. Since Tigriopus rapidly fall victim to predation in the former case (Dethier 1980) and lack life stages resistant to complete
111 dessication in the latter, reestablishment of the population in any pool depends on the successful arrival of colonists. Hence, from a natural history point of view, one might expect the evolution of strong dispersal capacities in T. californicus and rather extensive gene flow among geographically separated populations. Burton et al. (1979) and Burton and Feldman (1981, 1982) have pointed out that allele frequency data from natural T. californicus populations are not compatible with such an expectation. Because neighboring populations were found to harbor unique alleles at high frequencies, gene flow among geographically separated populations must be restricted. That populations located on the same rock outcrop seldom showed genetic differentiation was presumably due to high levels of inter-pool (but not inter-outcrop) gene flow. While this latter inference seems reasonable with respect to the seasonal reestablishment of populations in high elevation pools in winter; it is not obvious that copepods founding lower elevation pools in summer are derived from higher pools located on the same outcrop. When high pools are refilled, a seawater film usually covers the distance between already occupied lower pools and the newly refilled pools; this film allows Tigriopus to simply swim to the new pools. The behavioral studies of Bozic (1975) suggest that Tigriopus would readily return to pools previously occupied by conspecifics, presumably in response to some chemical attractant. In contrast, such seawater bridges are not always available during the recolonization of lower pools and it is in their reestablishment that we might expect to see an influx of immigrants from neighboring outcrops. The data obtained from the single pool transplants appear to verify that there is extensive movement of individuals among pools on an outcrop. The rapid decline in frequency of the transplanted allele in every case cannot reasonably be attributed to other causes given the extreme tolerance of Tigriopus adults to environmental changes and the general similarity of the source and recipient sites. We conclude from these results that single pools are not, in themselves, evolutionary genetic entities. Allele frequencies in such pools do not necessarily reflect the evolution of the resident population to local microhabitat; rather, allele frequencies reflect the broader experience of all pools on a given outcrop. This conclusion is strongly supported by the results of the larger scale Z-site transplant. While the transplanted allele rapidly declined in frequency in recipient pools, this decline leveled out at a frequency of about 10%, presumably because a larger number of animals were transplanted, a higher initial frequency was obtained, and a relatively small recipient outcrop was used. Having overcome the initial dilution problem, the Pgiv allele now appears to be established in the gene pool of the Z-site T. californicus population and has spread to all pools on the outcrop. While we have asserted that our results are unlikely
to be due to strong selection, an excellent test of this hypothesis could be achieved by simultaneously introducing unique alleles at two or more loci into a recipient population. By monitoring the rate of allele frequency changes at more than one locus, selective versus population structure hypotheses should be distinguishable: population structure will be expected to have a parallel effect on all loci, while natural selection will likely effect different loci differently. Barker and East (1980) used this approach to infer selection in a natural Drosophila population but used lab-reared flies to perturb allele frequencies in the field. In the work reported here, we wanted to use field collected animals from one natural population to perturb allele frequencies in another natural population, thereby eliminating any artifacts due to lab-rearing. Furthermore, since our primary interest here concerned population structure, we restricted the distance between source and recipient pools so that general climatic factors would be similar. This restriction prevented a multilocus analysis since the natural populations available differed at only single loci. In future studies we plan reciprocal transplants between populations differing at two or more loci.
Acknowledgements. The authors thank M.W. Feldman for encouragement and use of lab facilities at Stanford University and M. Levy for field assistance. This work was supported by NSF grant DEB-8207000 to R.S.B. and NIH grant GM28016 to M.W. Feldman. References
Barker JSF, East PD (1980) Evidence for selection following perturbation of allozyme frequencies in a natural population of Drosophila. Nature 284:166-168 Bozic B (1975) Detection actometrique d'un facteur d'inter-attraction chez Tigriopus (Crustaces, Copepodes, Harpacticoides). Bull Soe Zool Fr 100:305-311 Burton RS, Feldman MW (1981) Population genetics of Tigriopus calfornicus. II. Differentiation among neighboring populations. Evolution 35 : 1192-1205 Burton RS, Feldman MW (1982) Population genetics of coastal and estuarine invertebrates: Does larval behavior influence population structure? In: Kennedy VS (ed) Estuarine Comparisons. Academic Press, New York, p 537-551 Burton RS, Feldman MW, Curtsinger JW (1979) Population genetics of Tigriopus californicus (Copepoda: Harpacticoida). I. Population structure along the central California coast. Mar Ecol Prog Ser 1:29-39 Dethier MN (1980) Tidepools as refuges: predation and the limits of the harpacticoid copepod Tigriopus californicus (Baker). J Exp Mar Biol Ecol 42: 99-111 Vittor A (1971) Effects of the environment on fittness-related life history characters in Tigriopus californicus. Ph.D. dissertation, University of Oregon
Received January 31, 1984
