Randomly amplified polymorphism detection (RAPD) reveals high genetic diversity in Thalassia testudinum banks ex König (Turtlegrass)

Aquatic Botany 61 (1998) 269±287

Randomly amplified polymorphism detection (RAPD) reveals high genetic diversity in Thalassia testudinum banks ex KoÈnig (Turtlegrass) Janet H. Kirsten, Clinton J. Dawes, Bruce J. Cochrane* University of South Florida, Department of Biology, 4202 E. Fowler Avenue, LIF 169, Tampa, FL 33620-5150, USA Received 8 October 1997; accepted 16 February 1998

Abstract Populations of Thalassia testudinum (Banks ex KoÈnig) at the northern and southern limits of the west coast of Florida were compared with a Jamaican population using randomly amplified polymorphism detection (RAPD). With the exception of those from Apalachicola Bay, virtually all samples were distinct genetic individuals. Those putative clone mates that were identified often had other genets dispersed between them. Several distinct differences were observed between the northern and southern populations. The southern populations have higher percentages of the total possible number of bands present, of polymorphic bands, and of bands exclusive to a population, as well as a greater number of RAPD phenotypes and a greater mean number of differences between phenotypes. The biological phenomena that may explain these patterns include increased reproductive success, decreased inbreeding or increased population size in the southern populations. A fourth possibility is unidirectional gene flow from north to south. An analysis of molecular variance (AMOVA) was done indicating that approximately 81% of the variation is within beds, suggesting a homogeneous species. Yet the populations were clearly distinguishable from one another at microgeographic ranges. The level of genetic variation observed is characteristic of species with the same life history traits as Thalassia testudinum and was not predicted based on field observations of the species. This study, in conjunction with other molecular seagrass studies done to date, challenges our understanding of seagrass growth, reproduction, and propagation. There does not appear to be any pattern of reproductive traits, such as dioecy, monoecy, vivipary, or seed banking, that can reliably predict levels of genetic variation in a given seagrass species. # 1998 Elsevier Science B.V. All rights reserved Keywords: Seagrass; Thalassia testudinum; RAPD; Genetic variation * Corresponding author. Tel.: 001 8139742087; fax: 001 8139743263; e-mail: [email protected] 0304-3770/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved PII S 0 3 0 4 - 3 7 7 0 ( 9 8 ) 0 0 0 7 0 - 9

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1. Introduction Seagrass beds serve as the basis for a productive grazing and detrital food web in the Gulf of Mexico, with coverage estimated to be between 7.8 and 20.7103 km2 (Zieman, 1982). West Florida coastal waters are particularly rich in seagrass beds, extending 650 km from Apalachicola Bay to Florida Bay (Dawes et al., 1985; Iverson and Bittaker, 1986; Zieman and Zieman, 1989). Thalassia testudinum Banks ex KoÈnig (turtle grass) is ``the most important seagrass along the coasts of the of Caribbean and Gulf of Mexico'' (den Hartog, 1970), dominating in biomass and primary productivity (Dawes et al., 1985; Zieman and Zieman, 1989). Presently, Florida estuaries are under major anthropogenic influences and there have been serious losses in seagrass meadows (Zieman and Zieman, 1989). For example, losses have been estimated at 25,000 out of 31,000 ha in Tampa Bay (Lewis and Estevez, 1988) and 4,000 ha (30%) in Florida Bay (Robblee et al., 1991). In order to restore these and other beds, cultivars that are genetically compatible with the environmental conditions of the beds need to be established in seagrass nurseries. Yet, essentially nothing is known of the genetic variability or population dynamics of turtle grass. Thalassia testudinum is an obligately submerged dioecious marine monocotyledon having separate male and female plants (den Hartog, 1970). The incidence of reproductive short shoots (i.e. flowering shoots) varies greatly, both geographically and temporally (Witz and Dawes, 1995). Observations of seed set and flowering rates increase from north to south, with reports as low as 1% of total shoot abundance in North Florida (Marmelstein et al., 1968) to 3±21% in Central Florida (Moffler et al., 1981; Durako and Moffler, 1985; Witz and Dawes, 1995). However, a high rate of spontaneous fruit abortion observed in Central Florida by Witz and Dawes, 1995 suggests that successful sexual reproduction may be rarer than the relatively high flowering rates indicate. Other factors that may affect successful sexual reproduction in northern and central Florida include low water temperatures (Phillips et al., 1981), inappropriate photoperiod regimes (Phillips et al., 1981), spatial segregation of sexes (Durako and Moffler, 1985) and microbial infection in fruits and seeds (Lewis et al., 1985). In contrast, a high density of seed production has been observed in South Florida and seedling beds have been observed in the Florida Keys (Lewis and Phillips, 1980). This suggests that sexual reproduction may play a significant role in southern Florida while vegetative propagation through clonal, rhizomatious growth may be the primary means of bed expansion and maintenance in North and Central Florida. Evidence that genetic differentiation between beds exists is suggested by a latitudinal gradient of increasing leaf blade width (McMillan, 1978; Durako and Moffler, 1981) and decreasing cold chill tolerance (McMillan, 1979) from north to south. These patterns are maintained by seedlings grown under identical growth chamber conditions (McMillan, 1978; Durako and Moffler, 1981). These studies provide strong evidence for the heritability of the traits and support field transplantation studies (Phillips and Lewis, 1983) that demonstrate low phenotypic plasticity levels. Yet isozyme studies of Thalassia testudinum (McMillan, 1980, 1982) suggested a lack of genetic variation in the species throughout the Gulf of Mexico and Caribbean. The degree of genetic diversity in plants depends strongly on the species' life history characteristics, including the relative proportions of sexual and asexual reproduction

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(Hamrick et al., 1979; Loveless and Hamrick, 1984; Hamrick and Godt, 1990). Obligately, clonal populations are highly susceptible to divergence through genetic drift and the accumulation of unique mutations, resulting in increasing genetic differentiation and subdivision into clonal patches. Those species that facultatively reproduce sexually can counteract the loss of genetic variation and promote high levels of diversity within populations and low interpopulation differentiation, but the outcome is highly dependent on the relative contributions of sexual and asexual reproduction and dispersal capabilities (Hamrick et al., 1979; Loveless and Hamrick, 1984). Species that are late successional, obligately outcrossing and widely distributed with minimal phenotypic plasticity, all characteristics of Thalassia testudinum, are predicted to have high species-level genetic diversity and low interpopulational divergence (Hamrick et al., 1979; Loveless and Hamrick, 1984). However, large biogeographic ranges can result in isolation of populations that become genetically distinct due to genetic drift and variable selection (Jelinski and Cheliak, 1992). Thus, the life history characteristics of T. testudinum suggest a genetically diverse species that may exhibit minimal genetic divergence between beds. However, the field and laboratory observations of apparently monomorphic beds (den Hartog, 1970; McMillan, 1980), limited successful sexual reproduction (Moffler et al., 1981; Zieman and Zieman, 1989; Williams, 1990) and lack of isozyme variation (McMillan, 1980, 1982) suggests highly structured populations with minimal genetic diversity. Recently, restriction fragment length polymorphisms (RFLPs) (Fain et al., 1992) and multilocus minisatellite DNA fingerprinting (Alberte et al., 1994) in Zostera marina and randomly amplified polymorphic DNA (RAPDs) (Waycott, 1995) in Posidonia australis have uncovered unpredicted high levels of genetic diversity in seagrasses with life history characteristics similar to Thalassia testudinum. In this study, RAPDs were used to assess the genetic diversity of T. testudinum within and between beds in northern and southern populations of Florida and a disjunct population from Jamaica. RAPD has been well established in the past few years as a cost-effective means of assessing genetic variation at the DNA sequence level without requiring a priori knowledge of species DNA sequences (Williams et al., 1990; Hadrys et al., 1992; Huff et al., 1993). RAPD markers suffer the drawback of being dominant markers, making the unambiguous scoring of genotypes impossible in diploid species. Thus, standard population genetic statistics based on Hardy-Weinberg analysis cannot be determined. The goal of this study was to obtain an assessment of the level of genetic variation existing in Thalassia testudinum to determine whether the field and allozyme studies indicating minimal genetic variation or the high genetic variation predicted by the species' life history characteristics was correct. 2. Materials and methods 2.1. Population sampling Samples of Thalassia testudinum were collected as complete shoots (leaves plus short shoot) plus attached rhizome from four sites (Table 1): Apalachicola Bay offshore from

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Table 1 Thalassia testudinum: Locations and characteristics of sample collection sites Location

Geographic coordinates

Date

Apalachicola Bay (Ecofina)

3081.320 N 83857.180 W

5/23/94

Fiesta Key (gulf side, KOA bank)

24850.410 N 80847.630 W

3/15/94

Craig Key (SW atlatic side)

24850.010 N 80845.660 W

Jamaica

~178N768W

Number of sample

Collection area (m2)

Water characteristics Temperature (8C)

Salinity Depth (ppt) (m)

99

2,550

24.0

21.2

2.5±3.5

28

700

21.5

31.5

0.2±0.5

31/14/94 100

2,500

21.0

30.5

0.2±0.5

6/30/93

Unk.

Unk.

Unk.

Unk.

38

the Ecofina River, Fiesta Key from the northwest side of the Florida Keys, Craig Key from the southeast side of the Florida Keys, and Jamaica (generously provided by William J. Weibe, Dept. of Microbiology, Univ. of Georgia, Athens). The U.S. collections were made in a gridded transect (700±2550 m2) with samples being separated by 5 m in both directions (Fig. 1) (Fig. 2). Approximately, 100 samples each were collected from the Apalachicola site and the Craig Key site to ensure sufficient sample numbers should a highly clonal structure with minimal diversity be uncovered. Jamaican samples were collected a minimum of 5 m distance from one another. Samples were placed in individual zip-locked bags with seawater and stored on ice until return to the lab. All samples were cleaned of epiphytes by gently scraping with a razor blade and rinsing in filtered, autoclaved seawater. Approximately, 40 mg of the nonphotosynthetic apical portion of the short shoot (region of new growth) was removed, cut into fine pieces, and placed in a 1.5 ml microcentrifuge tube for DNA extraction with the remainder of the shoot and rhizome stored in a 50 ml conical tube for later physical analysis. Both were stored at ÿ708C until tested. 2.2. DNA extractions A miniprep extraction procedure was developed based on the protocols of Edwards et al., 1991 and Milligan, 1992. Samples were ground with sterile sand in 100 ml of extraction buffer (2% SDS, 200 mM Tris, 250 mM NaCl, 100 mM EDTA, pH 8.2) in a 1.5 ml microcentrifuge tube following 5 min. of heating at 658C. A disposable pestle (Kontes, Owens-Illinois) was inserted into the jaws of a motorized homogenizer (Fisher Scientific) to provide sufficient force for leaf maceration. Following grinding, 300 ml of extraction buffer was added, the tube vortexed at medium speed for 20 s and heated for an additional 20±30 min at 658C. Samples were centrifuged at maximum speed for 5 min and 250 ml was transferred to a clean tube. This step was repeated once by adding an additional 250 ml of extraction buffer to the original tube, vortexing, heating, and spinning again for a total of 500 ml of supernatant removal.

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Fig. 1. Thalassia testudinum. Relative position of samples in U.S. collections. Lines are 5 m apart. Dash represents a sample collected but not analyzed. Numbers represent the sample number for each tested short shoot collected at a site. Blank areas are sites where either no collection was made. Putative clonemates are represented by the same shading. (A) Apalachicola Bay ± putative clonemates are samples A2, A7, A17, and A21; samples A5 and A6; samples A9, A12 and A72; (B) Fiesta Key ± no clonemates identified; and (C) Craig Key ± putative clonemates are samples C26 and C37; samples C38 and C41.

After removal, the supernatant was diluted with 250 ml of autoclaved, deionized water and 1/10 volume (75 ml) 5 M potassium acetate was added to precipitate proteins. After gentle inversion for 5 min, the sample was centrifuged for 5 min and 600±650 ml of supernatant was transferred to a clean tube. The supernatant was extracted once with chloroform±isoamyl alcohol (24:1). Polysaccharides were differentially precipitated by addition of ethanol equal to 0.35 of the volume of supernatant removed while gently vortexing (Michaels et al., 1994). The sample was incubated for 10 min at ÿ208C and centrifuged at maximum speed for 10 min, resulting in a large gelatinous pellet of polysaccharides. The supernatant was transferred to a new tube and the DNA was precipitated by addition of 2 volumes of ethanol, incubation at ÿ708C for 20 min, and centrifugation for 5 min. The pellet was washed twice in 70% ethanol, dried in a vacuum and resuspended in 100 ml of TE (10 mM Tris±HCl, 0.1 mM EDTA, pH 8.0). All samples

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Fig. 2. Thalassia testudinum. Sample and band distribution patterns among the four collection, sites, Apalachicola Bay, Fiesta Key, Craig Key, and Jamaica. Percentage of samples tested from each population relative to all samples tested (nˆ38); Mean percentage of bands per sample relative to the total number of bands tested (nˆ29); percentage of bands present in each population relative to the total number of bands tested (nˆ29).

were treated with ribonuclease A either before or after precipitation of the DNA. Quantification of DNA yields was by agarose gel electrophoresis, usually accompanied by a DNA Dipstick (Invitrogen) quantification, which measures nucleotide concentration. For an accurate measurement, the DNA was reprecipitated and resuspended in TE to rid the sample of nucleotides produced during RNA digestion. The extracted DNA was stored at 48C. 2.3. RAPD PCR procedure A Perkin-Elmer DNA Thermal Cycler 480 and Cycler-Mate heated lid (BioLogic Engineering) were used for all DNA amplifications. Initial testing was done with a single primer and replicates of one plant each from Jamaica and Cockroach Bay (a local source in Tampa Bay) varying magnesium concentration from 1.5 to 3.5 mM and PCR annealing temperature from 308C to 458C. Final PCR reactions were carried out in 50 ml volumes containing 5 ml of 10 times magnesium-free PCR reaction buffer supplied by Promega (50 mM potassium chloride, 10 mM Tris±HCl, 0.1% Triton X-100, pH 9.0), 2.0 mM magnesium, 5 mg bovine serum albumin (Boehringer Mannheim), 0.25 mM each dNTP, 1.5 u Taq DNA polymerase (Promega), 56:1 molar ratio of TaqStart Antibody (Clontech), 50 pmol primer and 10-50 ng template DNA (1:l). The cycling protocol was 3 min at 948C, 45 cycles of 1 min at 928C, 1 min at 398C, and 1.5 min at 728C, followed by 10 min at 728C. A blank without DNA template was run with all reactions. All PCR amplifications were run on a 2.5% agarose gel in an EC-350 Midicell gel apparatus (EC Apparatus) with 40 wells in 1TBE (89 mM Tris±HCl, 89 mM boric acid, 2 mM EDTA, pH 8.0). Visualization of bands was by ethidium bromide staining.

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Table 2 Thalassia testudinum: Random decamer primers, their sequences and number of bands amplified Primer

Sequence

Total bands

CA919 OPA-19 OPB-5 OPF-4 OPF-14

CCC ACA ACC C CAA ACG TCG G TGC GCC CTT C GGT GAT CAG G TGC TGC AGG T

6 7 11 14 12

Total

50

2.4. PCR amplifications For this study, 10 samples (short shoots) from Apalachicola, 6 from Fiesta Key, 15 from Craig Key and 7 from Jamaica were examined. Fifty primers from Operon (Series OPA-11±20, OPB and OPF) were screened using one sample each from 2 or 3 sites to identify those primers that produced products. Two additional decamer primers composed of two bases with non-repeating patterns were also tested. Using the 18 primers that produced product, replication studies were done to identify those primers that provided a consistent banding pattern on independent extractions from the same plants from run to run. Five primers with a total of 50 bands were chosen for the final study (Table 2). In all cases, the negative control without template contained no product. 2.5. Statistical analyses Lynch and Milligan, 1994 recommend that only those bands with a frequency of less than 1/3 Nÿ1 be used for statistical analysis of RAPDs to compensate for bias resulting from the use of dominant markers, where N is the number of bands scored. Bands that occurred in only a single individual also were eliminated in an effort to preclude scoring errors resulting from non-replicable bands, as these bands were never tested for reproducibility. For this study, a total of 50 bands were scored originally. After eliminating 11 singleton bands and 10 bands with greater than a 92% frequency, 29 bands remained to be analyzed in the samples. These bands were used to create a distance matrix in the RAPDistance Package (ver. 1.03) from a presence±absence table using Upholt, 1977 metric which considers primer length in its calculations. The distant matrix was square root transformed to achieve homogeneity of variance and then analyzed with the AMOVA (Analysis of Molecular Variance) software package by Excoffier et al., 1992, developed for use with restriction haplotypes of mitochondrial DNA. The AMOVA was first used to analyze RAPD data by Huff et al., 1993 for populations of buffalograss (BuchloeÈ dactyloides (Nutt.) Englem.) and has since been used to analyze populations of a woodpecker (Picoides borealis) (Haig et al., 1994), a woody perennial (Grevillea scapigera) (Rosetto et al., 1995), Eucalyptus globulus (Nesbitt et al., 1995), and nematodes (Meloidogyne spp.) (Guirao et al., 1995).

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Excoffier et al., 1992 defined  (phi) statistics to determine the significance of variability in their AMOVA program. The -statistics are similar to Wright's F-statistics, with st being the correlation of random pairs of haplotypes within populations relative to the species, ct being the correlation of random pairs of haplotypes within a group of populations relative to the species, and sc being the correlation of random pairs of haplotypes within populations relative to the group (Excoffier et al., 1992). To determine the significance of the distribution of variance of the molecular diversity, the results are compared with the mean of many randomly permuted matrices to obtain a null distribution. The significance of the results is determined by comparing the -statistics for the data with the resulting estimated variance of the null distribution. In these analyses, 2000 random matrix permutations were performed to obtain the comparative null distribution. The AMOVA program allows partitioning of the variance components hierarchically into 3 components: among regions, among populations within regions (a group), and among individuals within populations. Several partitioning schemes were analyzed by placing different populations into different regions and groups. These partitions were: (a) Apalachicola vs. Florida Keys vs. Jamaica; (b) U.S. (Apalachicola and Florida Keys) vs. Jamaica; (c) Gulf (Apalachicola and Fiesta Key) vs. Atlantic (Craig Key and Jamaica); and (d) North (Apalachicola) vs. South (Florida Keys and Jamaica). 3. Results 3.1. Band distributions Despite the unequal number of individuals in the populations, the average number of bands per sample from all sites was 13 (45% of the 29 total possible bands), reinforcing the validity of the data. Distinct differences were noted between the northern and southern sites. Apalachicola had fewer bands present than the three southern sites. There was also a lower level of polymorphism (Fig. 3) in the north, with Apalachicola having 5 out of 15 bands (33%) in the population being polymorphic while 80% or more of the bands in the three southern populations were polymorphic. Neither Apalachicola nor Craig Key had any private alleles (bands exclusive to their populations), while Jamaica had 6 (26%) and Fiesta Key had 1 (5%). However, when Craig Key and Fiesta Key are treated as a single location they have 7 private alleles. Thus, Apalachicola lacks private alleles while a minimum of 25% of the alleles in the Florida Keys and Jamaica are private. The greater level of genetic polymorphism existing in the southern populations was also evident from the number of RAPD phenotypes detected (Fig. 3) and from the mean number of band differences between RAPD phenotypes in each population (Fig. 4). Apalachicola had few phenotypic banding patterns, with the 10 samples being distributed among only 4 RAPD phenotypes and only one phenotype being exclusive to a single short shoot (Fig. 1). In contrast, with the exception of two RAPD phenotypic patterns in the Craig Key population, all other patterns were sample-specific in the 3 southern populations sampled. One of the duplicate RAPD phenotypes in Craig Key may be

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Fig. 3. Thalassia testudinum. Band characteristics of the four collection sites, Apalachicola Bay, Fiesta Key, Craig Key, and Jamaica. Percentage of polymorphic bands per population relative to the total number of bands in the population; percentage of private bands relative to the number of bands present in the population; percentage of RAPD phenotypes present in each population relative to the number samples in the population.

Fig. 4. Thalassia testudinum. The mean number of band differences among RAPD phenotypes in each population.

artificial, as the two samples (C26 and C37) were distinguishable by one of the bands eliminated in the analysis for having greater than a 92% frequency. The number of phenotypic (band) differences between samples within the populations had a large range with a mean of 2.7 in Apalachicola and 9.7 in Jamaica (Fig. 4).

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Table 3 Thalassia testudinum: Analysis of molecular variance (AMOVA) for 38 individuals using 29 RAPD bands Source of variation

df

Variance (1000

% Total

p

AP vs. KY vs. JM Populations/groups Individuals/populatons

2 1 34

0.416 0.675 4.714

7.17 11.63 81.20

<0.0005 0.0175 <0.0005

Among groups Within groups

2 35

0.962 4.880

16.64 83.54

<0.0005

Among populations Within populations

3 34

1.019 4.714

17.78 82.22

<0.0005

Band with greater than 92% frequency and singletons were excluded. The 38 samples from 4 population in Apalachicola (AP), Fiesta, and Craig Keys (KY), and jamaica (JM) were compared among these three geographic regions in nested analyses and among and within populations. The p-value is the probability of a more extreme random value by chance based on correlation between the variance and -statistics with a random null distribution.

3.2. Analysis of molecular variance Nested analyses of molecular variance in all grouping schemes revealed that 81% of the total genetic variability was within individuals in the populations (Table 3). When Apalachicola, the two Florida Keys populations, and Jamaica were compared as groups, 7% of the variability was between the three regions and 12% was between the two Keys populations. No other regional distinctions were revealed by these analyses (results not shown). Although significant results were obtained in a number of the other comparisons, Bartlett, 1937 test of homogeneity of variance failed for the U.S. vs. Jamaica and the North vs. South comparisons, invalidating the AMOVAs. It is likely that the within population variance was so high that it masked the ability to detect small regional differences in these groups. The Gulf vs. Atlantic comparison had a negative correlation for populations within groups, indicating higher relatedness between groups than within (Excoffier et al., 1992). Given the close proximity of the two Keys populations and the high likelihood of gene flow between them, this result is not unexpected. Of particular interest was the comparison between the U.S. and Jamaican regions, which failed to reveal significant regional differences, presumably due to the high variation present among and within the three U.S. populations. 3.3. Distance matrix and neighbor joining trees A distance matrix was produced from the st for each population pair (Table 4, lower left) with all pair-wise comparisons significantly different from one another (Table 4, upper right). The null hypotheses of homogeneity of variance was accepted (p<0.05) for all pairs using Bartlett's test (data not shown). An unrooted neighbor joining tree was produced from the st figures in the matrix (Fig. 5) using the NEIGHBOR and DRAWTREE programs in PHYLIP (ver. 3.5). Although the four populations are closely related, they are also significantly distinct from one another.

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Table 4 Thalassia testudinum: Distances between pairs of populations in st (below diagonal)

AP FK CK JM

AP

FK

CK

JM

± 0.3176 0.1910 0.2502

<0.0005 ± 0.0939 0.1565

<0.005 0.0325 ± 0.1524

<0.0005 0.0100 <0.0005 ±

Abbreviation: AP ± Apalachicola Bay; FK. Fiesta Key; CK ± Craig Key; JM ± Jamaica. Above the diagonal is the significance levels as the probability of a random st distance being greater than the observed distance.

Fig. 5. Thalassia testudinum. Unrooted neighbor joining tree for the four sampled populations created from the st distance table produced with the AMOVA (see Table 4). The number on the tree branches are the st distance (see text for explanation).

A Euclidian distance matrix for the individual samples was used to produce an unrooted neighbor joining tree (Fig. 6) for illustrative purposes. Samples with identical RAPD phenotypes were combined as putative clonemates (ramets) arising from a single genetic individual (genet). Although 19% of the total genetic variation was between populations, there is considerable overlap between the genets. All Apalachicola samples are on a single branch (left), but they are grouped with 5 of the 7 Jamaican samples and 2 of the Craig Key samples. The remaining Craig Key samples were split between two branches, with 5 of 6 Fiesta Key samples and the remaining 2 Jamaican samples on one of the branches (top). 4. Discussion The most significant result is the high level of genetic variation existing in a seagrass over local spatial scales, until now, considered to reproduce predominantly by clonal

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Fig. 6. Thalassia testudinum. Unrooted neighbor joining tree of all samples tested and created from the distance matrix produced by RAPDistance using Upholt's metric with a square root transformation. Bands included in the matrix were those present in more than one sample and having <92% frequency for a total of 29 bands. putative clonemates (identical RAPD phenotypes) are grouped together as one sample for tree construction. Sample number are preceded by a letter indicating the sample collection site; Aˆ Apalachiocla Bay; FˆFiesta Key; CˆCraig Key; Jˆjamaica.

propagation. The distribution of variation is unusual because on the macrogeographic scale, only 19% is between populations, indicating a genetically homogeneous species. This is especially evident from the neighbor joining tree of the individual samples (Fig. 6). In addition, microgeographic differentiation does exist. The Fiesta and Craig Key populations are only 2±3 km apart on opposite sides of adjacent Keys, yet are genetically distinguishable. The high diversity suggests that new individuals are being introduced. Further, the meadows themselves are long lasting but lack evidence of the clonal dominance that is predicted for clonal species. Instead, the beds appear to be retaining the old genets, while novel genets are introduced. Considerable differences were found between northern and southern populations. The data in this study suggest the possibility of a north to south cline of increasing genetic variation as represented by the number of alleles (bands) in the populations, increasing polymorphism of the alleles, an increasing number of RAPD phenotypes present in the populations, and an increasing number of differences between those phenotypes within

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the populations. Thus, northern populations are more likely to be clonal with southern populations exhibiting more sexual reproduction. This would be consistent with the flowering and seed set observations (Marmelstein et al., 1968; Moffler et al., 1981; Durako and Moffler, 1985; Witz and Dawes, 1995). Other possibilities include that the Apalachicola population may be suffering from considerable inbreeding that has not yet reached the homozygous state or that gene flow is moving north to south, with Apalachicola being the origination point, and thus, containing the least amount of variation. Alternatively, because Apalachicola is the northern limit of the species range and has changed from a sub-tropical to temperate climate, the variation may be limited due to few genets possessing the adaptive gene complexes required to survive. Considerable future work will be required to determine which of these possibilities explains the patterns revealed here and whether a latitudinal gradient does, in fact, exist. It should be noted that the estimate of levels of genetic variation, based on RAPD phenotypes alone, may be an underestimate of the level of sequence variation present. First, only those sequence differences that affect primer annealing or the distance between priming sites is detected. Second, the analysis is based on the assumption that apparently comigrating bands from different samples are in fact the products of amplification of single homologous DNA sequences. Thus, it is possible that a more detailed analysis of variation in Thalassia testudinum will reveal even more genetic diversity within and between beds than this study has uncovered. 4.1. Seagrass growth and reproduction Observation of high levels of genetic diversity have now been reported in Zostera marina, Posidonia australis, and Thalassia testudinum. In contrast, Procaccini et al., 1996 have found Posidonia oceanica to be almost exclusively clonal throughout the western Mediterranean using both minisatellite and RAPD methods. Also, Waycott et al., 1996 have reported Amphibolis antarctica off the south and west coast of Australia to have no detectable genetic variation using 14 allozyme loci in combination with some 18S RFLPs (restriction fragment length polymorphisms) and M13 DNA fingerprinting. This raises serious questions regarding our understanding of the dynamics of seagrass growth, reproduction, and propagation based on field observations. The mode of sexual reproduction, the ability for seed bank storage, and the ability to produce viviparous seedlings do not appear to be factors in the level of genetic variation observed. Of the three species with high levels of genetic variation, Zostera marina is monoecious and can regenerate from a stored seed bank, Posidonia australis is hermaphroditic, and Thalassia testudinum is dioecious with viviparous seedlings. Of the species lacking genetic variation, Posidonia oceanica is hermaphroditic while Amphibolis antarctica is dioecious with viviparous seedlings. Thus, we have two hermaphroditic species (P. australis and P. oceanica) and two dioecious species with viviparous seedlings (T. testudinum and A. antarctica) displaying diametrically opposed levels of genetic variation while neither the high nor low variation seagrasses exhibit common reproductive traits. Thus, there does not appear to be any pattern of reproductive traits that can reliably predict levels of

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genetic variation in a given seagrass species. These contrasting results from 5 different seagrass species indicate that if one is to devise genetically sound restoration practices, the level of genetic variation, the breeding strategies (sexual vs. asexual; inbreeding vs. outbreeding) and the genetic structure of populations of a given species must be characterized. Regarding species with high genetic variation, Alberte et al., 1994 suggested that sexual reproduction is most likely responsible for the distributions observed in Zostera marina and Ruckelshaus, 1995, 1996 has estimated that the size of the genetic neighborhood, or panmictic unit, is 524 m2 with an effective population number of over 6,000. If this model were applied to Thalassia testudinum, it would be unlikely that our sampling scheme would detect evidence of clonal reproduction, yet putative clonemates were identified as much as 50 m apart in Apalachicola and 15 m apart in Craig Key. These data, combined with the life history data from Central and North Florida, argue that successful sexual reproduction may be rare or absent in these locations. If so, then the population structure inferred for Z. marina may be an inappropriate model for T. testudinum. Waycott, 1995 suggested that either present seedling recruitment is much greater than previously thought in Posidonia australis or that historically the beds were founded by a large number of diverse seedlings that have since been maintained by clonal growth and spread. Both Alberte et al., 1994 and Waycott, 1995 suggest the occurrence of high levels of sexual reproduction at some point in the bed's history. A discovery of much greater sexual recruitment than has been observed in the field would not be unprecedented. Using minisatellite DNA fingerprinting, Coffroth et al., 1992 reported that populations of a coral (Plexaura A) clearly contain sexually derived individuals despite 7 years of field observations, which concluded that clonal fragmentation was the only means of successful recruitment within a reef. However, an alternative to sexual reproduction could account for the genetically variable species. Rhizome fragmentation and re-establishment is common in Zostera marina and Posidonia australis. Tomasko et al., 1991 have shown in mitigation studies that bare rhizomes with 2±4 intact short shoots of T. testudinum have as high as an 85% survival rate following reburial. Rhizomes with similar numbers of intact short shoots are often dislodged in storms and may provide a source for such successful reburials. Thus, rhizome fragmentation could result in dispersal and local mixtures of genotypes in the absence of sex for these species. The widely dispersed geographic patterns of putative clone mates based on RAPD phenotypes in both this study and Waycott, 1995, and Alberte et al., 1994 inability to correlate genetic similarity with geographic distance using multi-locus mini-satellite fingerprints may be a reflection of rhizome breakage and re-establishment. Perhaps this breakage and re-establishment is a more common reproductive mode and means of dispersal by seagrasses than previously believed, particularly in shallow beds during storm or hurricane conditions. Population genetic theory predicts that a single migrant between populations per generation is sufficient to prevent extensive population subdivision, independent of population size, provided gene flow is multi-directional (Hartl and Clark, 1989). This prediction is problematic in clonal species where the generation time of a genet may be measured in hundreds or thousands of years even as individual ramets senesce and die on

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shorter time scales. The timespan of a generation for clonal plants and corals is estimated to approach the mean age of the older genotypes rather than age of first reproduction as is estimated for determinate species (Levin, 1978; Potts, 1984). Thus, a prediction of how many migrants per generation occur between populations of seagrasses is difficult. The contrast between isozyme and DNA sequence variation appears to be greatest in Thalassia testudinum. Of the five seagrass species studied to date, Posidonia australis and Amphibolis antarctica are the only species whose isozyme characteristics are representative of DNA sequence variation, albeit less than would be predicted. Isozyme analyses in Zostera marina (McMillan, 1980, 1982) detected variation between populations but no intrapopulational variation. Further, Fain et al., 1992 failed to detect any restriction fragment length polymorphisms at the Adh locus in Z. marina. In T. testudinum, McMillan, 1980, 1982 detected no alternative electromorphs at 7 different isozyme loci in samples from throughout the Caribbean and Gulf of Mexico, yet we have detected extensive RAPD marker polymorphisms with almost exclusively individualspecific RAPD phenotypes. This discrepancy is probably because isozymes specifically sample coding regions while RAPDs sample the genome without specificity as to coding versus non-coding regions. Thus, it is likely that our sampling of the genome provides a more accurate assessment of the genetic variation present. Based on all molecular DNA studies of seagrasses to date, it is not clear what the relative rates of sexual and asexual reproduction are. Reviews of isozyme comparisons in plants (Hamrick et al., 1979; Loveless and Hamrick, 1984; Hamrick and Godt, 1990) support the optimal genotypic replication theory (Silander, 1985). The theory proposes that clonal organisms with facultative sexual reproduction, such as seagrasses, should be capable of maintaining higher levels of genetic variability than either obligately sexual or clonal species. The theory assumes that competitively superior, coadapted gene complexes maintained by asexual propagation are able to exploit new resources while sexual reproduction restores genetic variability and provides new gene complexes to be tested via natural selection. The relative proportions of sexual to asexual reproduction directly influence the levels of genetic variation observed within populations. At present, however, the increasing pattern in the mean number of phenotypic differences in Thalassia testudinum between samples at each site may reflect increasing levels of sexual reproduction on a southern cline, but this is only one possibility. Thus, this study neither supports nor contradicts Silander's theory. Of the five studies of seagrasses that have explored nuclear DNA variation, two have discovered clonal patches at the scales tested, while three have not. We chose to initially test samples that were more widely spaced than the collection pattern for an overall view of the bed, with some samples that were at the 5 m collection distance included. Duplicate RAPD phenotypes were often widely separated with other genets dispersed between them. It is not possible to tell from these results whether rhizome extension is occurring with shoots only being produced in ``suitable'' microhabitats as a result of a guerilla resource acquisition strategy (Harper, 1985), whether putative clone mates are the result of rhizome fragmentation and re-establishment as suggested here, or if the beds are so old that widespread overlap of individual rhizomes has taken place. Studies at much smaller geographic scales will be required to determine such patterns.

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5. Conclusions Despite the enormous losses of Thalassia testudinum coverage on the west coast of Florida, significant levels of genetic variation still exist. With a deeper understanding of the reproductive habits of this and other seagrasses, more successful conservation and restoration plans should be possible. Mitigation projects with T. testudinum presently employ techniques involving damaging a donor bed in order to restore another. Recovery of T. testudinum even into a propeller scar requires an average of 7.6 years (Dawes et al., 1997, in press), thus, use of donor beds could result in long term damage. Additional complications in restoration projects stem from the possibility that turtle grass beds may be genetically heterogeneous, and/or that beds from different geographic locations are genetically distinct. If heterogeneity within beds exists, then restored beds may have lower levels of diversity and, thus, be more susceptible to other deleterious environmental influences such as shifts in salinity, turbidity, or diseases. If between-locale genetic differentiation is a significant factor, then transplantation may introduce genets to locales in which they are maladapted to environmental conditions. Our data suggest that genetic variation within populations is high and between-locale differentiation is low in southern populations, while northern populations have reduced genetic heterogeneity with the possibility of adapted ecotypes dominating. However, with the minimal sample numbers in this study and a methodology that is unlikely to detect adaptive gene complexes, this conclusion will require considerable future work to confirm. At present, no commercial sources of seagrasses for transplants are available, but seagrass nurseries are expected to be established in Florida and other states in the near future. This study begins the genetic characterization required to establish nurseries of T. testudinum from which cultivars can be obtained for successful restoration projects. Acknowledgements We thank William J. Weibe for the motivation which began this work and his collecting efforts in Jamaica, Christina Uranowski, Angela Baker, and John Andorfer for their assistance in the U.S. collections, Stephen A. Karl for his advice and criticism, and Susan Brandon for technical assistance. Supported by grant number NA36RG-0508 from the National Oceanographic and Atmospheric Administration to B.J.C. and C.J.D.

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