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J.-K. PARK AND D. O´ FOIGHIL

hensive cladistic analysis of Bermudan and Floridian La- saea mitochondrial lineages to test their adherence to rafting hypothesis predictions. In addition to three Bermudan pop- ulations, four populations of Lasaea were sampled along 250 km of contiguous southeastern Floridian coastline. A total of 124 specimens of western Atlantic Lasaea were sequenced for a 655-nucleotide (nt) fragment of cytochrome oxidase I, a protein-encoding mitochondrial gene that, for recently diverged lineages, typically accumulates substitu-

´ tions at a faster rate than does 16S (O Foighil et al., 1998).

Twelve western Atlantic haplotypes were obtained and yielded a phylogenetic tree topology that was a mirror image of that predicted by the rafting hypothesis.

Materials and Methods

Specimens of Lasaea were sampled in April 1998 from four southeastern Florida locations (Sebastian Inlet State Park, Brevard County; Fort Pierce Inlet State Recreation Area, St. Lucie County; Phil Foster Park, West Palm Beach; and Bill Baggs Cape Florida State Recreation Area, Key Biscayne) spanning about 250 km of contiguous coastline (see Fig. 1). The bivalves were moderately abundant in the interstices of barnacle tests at three of the sites; however, intensive sampling at the Key Biscayne site yielded only four individuals. Additional collections were taken in May 1998 from intertidal algal tufts and barnacle test interstices at three Bermudan locations (Boaz Island, the main Island at North Shore Road, and St. George’s Island at the Bermuda Biological Station for Research) spanning about 20 km of the archipelago’s length. At each sampling location, clams were taken from a number of microsites on the shore and pooled prior to fixation in 95% ethanol; thus a representa- tive sample of genetic diversity from the study population was obtained.

Extensive sampling of ostensibly suitable intertidal crev- ice habitats elsewhere in the Caribbean by the second author (Lower Florida Keys; Dry Tortugas; New Providence, Ba- hamas; Puerto Rico), and by A. Frias-Martins (Jamaica), yielded no further specimens of Lasaea. Attempts to am- plify the target gene fragment from a single dried museum specimen sampled from Belize (USNM# 841332) and from a small number of preserved specimens sampled from Cabo Tres Puntas, Venezuela (R.C. Bullock, University of Rhode Island, pers. comm.) were also unsuccessful. In the absence of tractable Caribbean outgroup samples, we generated ho- mologous cytochrome oxidase subunit I (CO I) sequences from Sydney samples of Australian direct-develop- ing polyploid Lasaea lineages (O´ Foighil and Thiriot- Quie´vreux, 1999) that are sister to the study populations in

´ global phylogenetic trees (O Foighil and Jozefowicz, 1999;

655-nt fragment of the mitochondrial CO I gene. DNA tem- plates for thermal cycle amplification were individually ex- tracted from entire adult Lasaea specimens using a QIAamp tissue kit (QIAGEN Inc.). The Folmer et al. (1994) CO I primer set (5 -GGTCAACAAATCATAAAGATATTGG-3 ; 5 -TAAACTTCAGGGTGACCAAAAAATCA-3 ) was used to amplify and to sequence the target gene fragment. A nega- tive control (no template) was included in each run of 35 cycles of amplification (1 min 94°C denaturing, 30 s 45°C annealing, 1 min 72°C extension). Double-stranded products were iso- lated on 1% agarose gels, excised under long-wavelength UV light, and extracted using a GeneClean (Bio 101) NaI/glass powder kit. Both strands on the target fragments were cycle- sequenced using a Big Dye DNA sequencing kit (PE Applied Biosystems), and reaction products were electrophoresed on an ABI 377 automated DNA sequencer. Sequence alignments were initially performed by the CLUSTAL option of Sequence Navigator 1.0.1 (Kececioglu and Myers, 1994) and then opti- mized manually. Sequences were analyzed using an un- weighted maximum parsimony approach (PAUP* 4.0b2a; Swofford, 1998), and root probabilities for individual haplo- types were estimated using the Castelloe and Templeton (1994) heuristic technique. Nucleotide mismatch distribution (Schneider et al., 1999) and lineage-through-time (Nee et al., 1996) analyses were respectively performed on the Bermudan lineages using Arlequin 2.0 (beta 2; Schneider et al., 1999) and the Endemic-Epidemic Phylogenetic Process Analysis (Ram- baut et al., 1997).

Results

Alignment of the 655-nt CO I fragment sequences ob- tained from the 124 individuals of western Atlantic Lasaea

FL1

C

C

C

A

A

A

A

A

A

A

FL2

C

C

C

A

A

A

A

A

A

G

BDA1

C

C

C

A

A

A

A

A

G

A

BDA2

C

C

C

A

G

A

A

A

G

A

BDA3

C

C

C

G

A

A

A

A

G

A

BDA4

C

T

C

A

A

A

A

A

G

A

BDA5

C

C

C

A

A

A

G

A

G

A

BDA6

T

C

C

A

A

A

A

A

G

A

BDA7

C

C

C

A

G

A

T

A

G

A

BDA8

C

C

C

A

A

A

A

G

G

A

BDA9

C

C

C

A

A

G

A

A

G

A

1

2

2

2

2

3

3

4

4

5

9

4

6

8

8

0

4

0

7

3

5

5

4

4

7

5

4

7

9

9

Table I

Alignment of the 10 variable sites in the 655 nucleotide fragment of CO I sequenced for 12 western North Atlantic Lasaea COI genotypes (Genbank accession #s: AF152564, AF182733-AF182743)

BDA10 C C T G A A A A G A

Taylor and O´ Foighil, 2000).

Twenty individuals from each of six study populations, and all four specimens from Key Biscayne, were genetically char- acterized by amplifying and directly sequencing a homologous

Positions of the variable sites within the sequenced fragment are pre- sented vertically above the nucleotide columns. The prefixes FL and BDA respectively indicate haplotypes found in Floridian and Bermudan popu- lations, and these show one diagnostic difference (in bold).

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