mill,ti IN 01- MARIN!-. SCIl'Nt T.. (?(2); Abi -480, 1>?<) 
GENETIC DIFFERENTIATION DESPITE TELEPLANIC LARVAL 
DISPERSAL: ALLOZYME VARIATION IN SIPUNCULANS 
OF THE APIONSOMA MISAKIANUM SPECIES-COMPLEX 
Joseph L. Staton and Mary E. Rice 
ABSTRACT 
We examined the genetic evidence for dispersal in tiie sipunculan Apionsoma 
misakianum (Ikeda 1904) by analysis of allozynies (using a total of nine polymorphic 
loci) in larval and adult samples. Genotypes of adults reared from late-stage larvae col- 
lected from the Florida Current were compared with genotypes of adults from three loca- 
tions: eastern central Florida, the Florida Keys and the Bahamas, Genetic differences 
were found between individuals from the southern localities and the majority of speci- 
mens from the northern locality. The multilocus genotypes of the specimens reared from 
larvae of the Florida Current were characteristic of specimens from the Florida Keys and 
the Bahamas. Despite evidence for larval influx northward into the central-Florida area 
via the Florida Current, the southern genotypes were present in only a few adults col- 
lected (4.5%) at a northern site, with no indication of "hybrids" occurring between north- 
ern and southern genotype based on allozymes (i.e., h?t?rozygotes). Furthermore, ge- 
netic separation of the northern and southern genotypes coincides with a faunal bound- 
ary noted for other species. 
The impact of long-lived, planktotrophic larvae on dispersal and speciation of marine 
invertebrates (Crisp, 1978; Jablonski and Lutz, 1983;Scheltema, 1971, 1975, 1978, 1986; 
Shuto, 1974) has been a central question In the field of larval ecology since Thorson 
(1950). Many studies have inferred that the genetic homogeneity within a marine species 
resulted from high levels of larval transport over large geographic distances (e.g.. Campten 
et al., 1992; Johnson and Black, 1982; 1984ab; Nash et al., 1988; Nishida and Lucas, 
1988; Watts et al., 1990). A few studies of invertebrates have attributed homogeneous or 
heterogeneous genetic patterns over a geographic range to long- or short-dispersing lar- 
vae within each species to a greater (McMillan et al., 1992) or lesser degree (Hellberg, 
1996). To date, however, few data examine the direct effect of teleplanic (far-wandering) 
larvae on the phylogeography within a marine species. 
Sipunculan worms exhibit a great variety of developmental types that might be used to 
test directly the effects of differential larval dispersal. These fypes include: direct devel- 
opment without a pelagic stage, short-duration pelagic development (which in one spe- 
cies is parthenogenic), or long-duration pelagic development (produced by strictly out- 
crossing species) which may include teleplanic larvae (cf Rice, 1981). Teleplanic larvae 
of sipunculans are abundant across entire ocean basins (Scheltema, 1975; Scheltema and 
Rice, 1990). However, teleplanic larvae alone may not prevent genetic isolation and spe- 
ciation from occurring in marine invertebrates. The ultimate effect of variation among 
these developmental types on dispersal can be addressed only through detailed genetic 
studies of these species. Previous population-genetic research on sipunculans has been 
limited to examination of variation within a single population (Balakirev and Manchenko, 
1983). 
One species of sipunculan, Apiomoma misakianum (Ikeda 1904), is reported from both 
the Atlantic and Pacific Oceans (Cutler, 1979; Cutler and Cutler, 1980), although the 
467 
458 BLLLKl r\ 01- MAWM; SCIENCl;, VOL. ?.5. NO. 2, IW9 
species is not abundant at every location. In the Atlantic, this species is reported in the 
Bahamas (Cutler and Cutler, 1980), the Gulf of Mexico (Murina, 1967), east coast of 
Florida as far north as Sebastian (Rice, 1986) and from the Mediterranean (Murina 1964). 
A teleplanic larval stage, identified as A. misakianum (Rice, 1978), has been collected in 
all of the major currents of the Atlantic Ocean (Scheltema, 1975). The adults are particu- 
larly well-studied along the central east coast of Florida, and their larvae have been re- 
ported as common constituents of the north-flowing Florida Current, a component of the 
Gulf Stream (Rice, 1978, 1981, 1986). 
Northward transport of large numbers of teleplanic larvae from the Florida Keys and 
Caribbean could provide recruitment to more northerly populations of benthic adults. 
Although adults collected near central Florida and adults reared from larvae collected in 
the Florida Current were morphologically indistinguishable, early development of labo- 
ratory spawning from the two groups was found to differ in egg length, position of the 
first meiotic metaphase spindle, gut pigmentation of the trochophore, and length of time 
from fertilization to the pelagosphera stage (Rice, 1981). The length of developmental 
period is not precisely known for these worms, as culture through the complete life cycle 
has not yet been possible in the laboratory. After 2 mo, neither pure-bred nor hybrid 
crosses between the two types progressed beyond early developmental phases (Rice, unpubl. 
data). These differences in early development led us to an investigation of the genetic 
variation in adults and larvae o? Apionsoma misakianum in the Florida Current. 
M/V1I:RIALS ANO Minnoos 
COLLECTION OF ADULIS AND CULIURL OF LARVAL.?Adults o? Apionsoma misakianum were col- 
lected from the Sebastian Pinnacles off east-central Florida; Pickles Reef, Key Largo, Florida; and 
Chub Cay, Bahamas (Fig. 1 ; Table 1). Adults of this species are small (approximately 1-2 mm in 
diameter and <4 mm long when contracted) and biurow in scleractinid coral rubble (e.g., Oculina 
spp., Poriles spp.). Pieces of rubble were collected errantly by SCUBA divers from submerged 
reefs of the Florida Keys and Bahamas or by dredge at the Sebastian Pinnacles. Living adults were 
extracted individually from random coral pieces, then frozen at -80?C in 20 |lL of grinding buffer 
(10 mM Tris, 1 mM EDTA, and0.1%(v/v)Tween 80) for subsequent homogenization forallozyme 
electrophoresis. 
Pelagosphera laj'vae, identified as the "Type C" larvae of A. misakianum described by Hall and 
Scheltema (1975), were collected on two dates within 10 d from sites in the Gulf Stream otTFt. 
Pierce, Florida (Fig. 1, Table 1), with a 202-mm mesh plankton net (0.75 m diam opening; 2.25 m 
length). Larvae were then divided into three groups (GSLl-3) as part of another study on delay of 
metamorphosis. Metamorphosis was induced (Rice, 1986) by transfer of larvae to containers with 
organic sediment (dredged from areas near the larval collections) and seawater previously exposed 
to adults collected from the Sebastian Pinnacles locality. In order to increase tissue for allozyme 
analysis, juveniles were reared on the organic sediment for approximately 16 mo at 22?C until they 
attained maturity. These adults, morphologically diagnosed as A. misakianum, were stored at -80?C 
as described above. 
Extracts of whole worms were prepared and subsequently allozymes analyzed by starch-gel elec- 
trophoresis using standard methods (e.g., Richardson et al., 1986; Murphy et al., 1990). Enzyme 
systems screened during this study are listed in Table 2; names and abbreviations for enzymes are 
those of Murphy et al. (1990). All scorable enzymes gave optimal results on Tris-citrate buffer (pH 
8.0). Multiple isozymic loci were numbered sequentially with "1" representing the fastest migrat- 
ing systein. Al?eles were designated with letters; "a" represents the fastest anodally migrating al?ele 
within a system. There were no electromorphs that migrated cathodally in this study. 
STATON ANO RIC'Ii: GiTXITlf VARIATION IN SIIHJNC'CI.AN ADULTS AND ? ARVAI". 46^ 
Sebastian Pinnacles 
Lar\'al Collections 
\\j:::^^=^n^ 
Chub Cav    \ 
80? 79? 
O 
28? 
27? 
25? 
Figure 1. Apionsoma misakianum: Sampling locations of adults and larvae. Black dots are locations 
of adults sampled; open circle is location of larval collections. Dates and precise locality information 
for samples are listed in Table !. 
Table 1. Apionsoma misakianum: data for the number of individuals (n) from each locality and 
the date of collection. 
Locality Name n Position Depth Date 
Adult collections 
Sebastian Pinnacles 
Pickles Reef 
Chub Cay, BeiT>' Is., Bahamas 
Larval collections 
GSL 1?thiee transects 
GSL 2?two transects 
GSL 3 
302 27? 49.23'N 79? 57.66'W 
44 24? 59.20'N 80? 24.90'W 
36  25? 24.50'N  77? 55.00'W 
80 m 24 Feb. 1992 
3.5 m 9 Mar. 1993 
4.5 m  13 May 1993 
228  27? 27.54'N 79? 57.44'W to 
27? 27.64'N 79? 57.24'W       surface   31 Dec. 1991 
27? 27.70'N 79? 57.2'W   to 
27? 27.84'N 79? 56.97'W       surface   3! Dec. 1991 
27?28.18'N 79?57.36'Wto 
27? 28.36'N 79? 57.04'W       0-70 m   31 pec. 1991 
131       27? 28.44'N 79? 54.05'W to 
27? 29.60'N 79? 53.60'W       0-70 m    9 Jan. 1992 
27? 30.02'N 79?53.51'Wto 
27? 30.93'N 79? 53.71'W      0-70 m    9 Jan. 1992 
92    collection data same as for GSL 1 
^^g Bi:i,i.i;nN or- MAR?NI-, srii:N(i:. VOL. fi5, NO. 2. ivn 
Table 2. Apionsoma misakianum: Enzymes examned in (his study. 
E. C. no. Symbol' No. of loci'' 
Acid phosphatase 3.1.3.2 Acp 1 
Alanopine dehydrogenase 1,5.1.17 Alpdh 1 
Alkaline phosphatase 3.1.3.1 Alp 3' 
Arginine kinase 2.7.3.3 Ark 1 
Aspartate aminotransferase 2.6.1.1 Aal 1 = 
Esterase (a-naphthyl acetate substrate) 3.1.1. - Es! 2 = 
Fructose bisphosphate aldolase 4.1.2.13 Fha I 
a-galactosidase 3.2.1.22 a-Gal \ 
?-galactosidase 3.2.1.23 ?-Gal 1 
Glucose-6-phosphate isomcrase 5.3.1.9 Gpi r 
l-[ditol dehydrogenase 1.1.1.14 Iddh 1 
Isoeilrate dehydrogenase 1.1.1.42 hlh 2 
1-Lactate dehydrogenase 1.1.1.27 U/h 1 
Malatc dehydrogenase 1.1.1.37 Mdh 2 
a-mannosidase 3.2.1.24 u-Mun \ 
Peptidase (specific to Lcu-Ala) 3.4.13.11 Pep-LA 3' 
Peptidase (specific to Leu-Gly-GIy) 3.4.11.4 Pep-LGG 2' 
Phosphoglucomutase 5.4.2.2 Pgm \' 
Superoxide dismutase 1.15.1.1 Sod 1 
?'Genelic ;ibbicvialion used in Tiiblc 2. 
''Number of lol;il isozymic loci resolved. 
'Systems with polymoiphic loci 
Enzyme systems were screened using adults collected at Sebastian Pimiacles to identify poly- 
morphic loci. Given their small adult size, only one slab gel (1.3-cm thick) could be run for each 
individual. Seven replicates could be sliced from each run to analyze a total of seven enzyme sys- 
tems for each individual. Given this constraint, seventeen loci that were monomorphic for adults 
from the Sebastian Pinnacles were not studied ftirther. Ten polymorphic loci (in seven enzyme 
systems) were resolvable for adult specimens from Sebastian Pitmacles; one {Alp) was inconsis- 
tently scorable and was omitted from the final data analysis. The remaining nine polymorphic loci 
were analyzed for 117 field-collected adults (36 from Chub Cay, 44 from Pickles Reef and 37 from 
Seba.stian Pinnacles) and 76 adults reared from larvae. Statistics (al?ele frequencies and observed 
and expected heterozygosities) were calculated using standard formulae (Murphy et al., 1990). 
Genetic distances were calculated by the unbiased method of Nei (1978), and variances were calcu- 
lated by the approximate formulae of Nei (1987). 
A principal components analysis (PCA) was performed on al?eles from polymorphic loci as 
described by Liu et al. ( 1991 ). PCA is a multivariate data reduction tool that allows genetic patterns 
to be examined from the perspective of the individual. While measures of population subdivision 
are traditionally calculated using Wright's F-statistics or Nei's G-statistics, these methodologies 
require assumptions (random breeding individuals within subpopulations, no selection or genetic 
drift occurring, etc.) that we could not test for our system. It would be artificial to treat the larval 
pool as a population for genetic comparison to the adult populations in any of these methods as 
there is no a prioi reason to assume that they are propagules from a single, randomly interbreeding 
population (two important assumptions required to be met); therefore, we limited our treatment to 
PCA of the niulti-allelic data for individuals. 
An UPGMA dendrogram was constructed from genetic distances D (Nei, 1978) according to 
procedures outlined by Sneath and Sokal (1973). All statistics were calculated using either SAS 
programming (SAS Institute Inc., 1989, 1992) or the GeneStrut genetics analysis package 
(Constantine et al., 1994). 
SIMON AM) RIC'lv, Cr?NCTIC VARIATION IN SIPL^CLLAN ADLL?S AND LARVAL 47 | 
Table 3. Apionsorna misakkmum: Survivorship data from larval settlement groups. 
Culture ID No. of Larvae Set No. of metamorphs No. of Adults 
(Date) (Date) (Date) 
GSLl 228 120 53 
(23 Jan 1992) (11 Feb 1992) (7 May 1993) 
GSL2 131 106 47 
(23 Jan 1992) (11 Feb 1992) (11 Feb 1993) 
GSL3 92 27 7 
(12Feb 1992) (20 Feb 1992) (20 Feb 1993) 
RE.SULT.S 
Initial metamorphic success of larvae in culture ranged from 80.9 to 29.3%, but survi- 
vorship to adult size was 35.9 to 7.6% (Table 3). Heaviest mortality occurred in GSL 3, 
which was a subset of the same larval collection as GSL 1 but differed only in onset of 
metamorphosis by 1 mo. 
Al?ele frequencies for the nine polymorphic loci which were used in the principal com- 
ponents analysis (PC A) and distance calculations are reported in Table 4. The disparity in 
overall observed and theoretical expected heterozygosities (H^^^^^ and H^,^^) arises from 
rare al?eles within populations. In the case of the Sebastian Pinnacles population, all rare 
al?eles for Pep-LGG-2 and Pep-LA-2 occurred in two individuals that appear reproduc- 
tively isolated from the remainder of the population. A few rare al?eles also occurred in 
the reared larvae that were not seen in the adult populations. After pooling rare al?eles 
within each sample into a single category within each locus, no loci were significantly 
different from Hardy-Weinberg expected frequencies Cf" test, a = 0.05), although smaller 
sample sizes limit potential for deviation from Hardy-Weinberg and discovery of rare 
al?eles. The variation in number of individuals for each locus (n) arises from data omitted 
for individuals possessing ambiguous or uninterpretable allozyme profiles for each lo- 
cus. 
PCA demonstrates that there are two distinct genetic entities based on the 44 combined 
al?eles of the polymorphic loci (Fig. 2). The fh-st and second PCs explain 12.1 % and 5.7% 
of the variation in the data set, respectively, with each subsequent PC explaining <5.5% 
of the remaining variation. The left cluster represents the "tropical" genotype which was 
found in all adults from Chub Cay, Bahamas, and Pickles Reef, Florida Keys, and all 
adults reared from larvae collected in the Florida current just south of Sebastian Pin- 
nacles, the temperate site (Fig. 1). Adults collected from the Sebastian Pinnacles dis- 
played genotypes of the "temperate" type, except two that displayed genotypes in the 
"tropical" cluster (these two individuals overlap completely as a single point in Fig. 2). 
Any "hybrids" (i.e., h?t?rozygotes) between the two types would appear as points scat- 
tered behveen the two clusters. The primary loci contributing to the first principal compo- 
nent were (in order of descending weight): Pep-LGG-2, Pep-LA-2, Esl-2, Pgm, and Gpi. 
Additional individuals which were run in the preliminary screening process did demon- 
strate tropical al?ele types, but were not scored for all al?eles used in the overall PCA and, 
and therefore could not be included. A total of five out of 110 adults (4.5%) froiri Sebastian 
Pinnacles screened for relevant loci (inclusive of those in the PCA) possessed "tropical-" 
genotype al?eles. No "tropical" types present in the "temperate" locality showed any signs 
of interiuediate h?t?rozygotes between "tropical" and "temperate" genotypes. 
472 BI.IJ.irnN O?- VIARIM; SClliNCl:. VOL. (i5. NO. 2. l'?<) 
Table 4. Apioiisoma misakianum: Al?ele frequencies, expected (W?,,) and observed (W??^) 
heterozygosities, and numbers of individuals (n) sampled for the six groups (see Table 1). 
- ChubCa/ 
Adults 
Pickles Reef Sebastian 
Larvae 
Locus GSL 1 GSL 2 GSL 3 
Pinnacles 
Aat a 0.00 0.00 0.00 0.02 0.00 0.00 
b 0.97 0.99 1.00 0.91 1.00 1.00 
c 0.03 0.01 0.00 0.07 0.00 0.00 
^i:xi- 0.05 0.02 0.00 0.17 0.00 0.00 
^OIIS 0.06 0.02 0.00 0.18 0.00 0.00 
n .36 44 37 33 32 7 
Esl-I a 0.11 0.10 0.08 0.03 0.17 0.00 
b 0.25 0.23 0.40 0.27 0.22 0.21 
c 0.36 0.23 0.32 0.30 0.30 0.14 
d 0.26 0.43 0.19 0.32 0.31 0.64 
c 0.02 0.01 0.01 0.08 0.00 0.00 
//,,? 0.74 0.71 0.71 0.74 0.75 0.56 
/^??v 0.63 0.43 0.38 0,70 0.56 0.71 
n 32 44 34 30 32 7 
Est-2 a 0.03 0.10 0.11 0.06 0.16 0.14 
b 0.00 0.00 0,00 0.00 0.00 0.07 
c 0.68 0.43 0.15 0.44 0.56 0.57 
d 0.18 0.42 0.32 0.38 0.26 0.22 
e 0.11 0.05 0.42 0.12 0.02 0.00 
^KXr 0.50 0.63 0.70 0.66 0.60 0.65 
^ons 0.45 0.53 0.39 0.55 0.58 0.57 
n 3! 43 31 33 31 7 
Gpi a 0.00 0.00 0.04 0.00 0.02 0.00 
b 0.01 0.05 0.19 0.01 0.08 0.00 
c 0.64 0.72 0.42 0.82 0.65 0.64 
d 0.34 0.18 0.15 0.16 0.14 0.00 
e 0.01 0.04 0.12 0.01 0.09 0.36 
f 0.00 0.01 0.08 0.00 0.02 0.00 
^ixr 0.49 0.46 0.75 0.31 0.54 0.49 
^OHS 0.50 0.43 0.59 0.25 0.50 0.14 
n 36 44 37 36 32 7 
Pep-LA-2 a 0.01 0.00 0.92 0.11 0.03 0.00 
b 0.00 0.00 0.00 0.00 0.02 0.14 
c 0.92 0.98 0.08 0.89 0.93 0.57 
d 0.07 0.02 0.00 0.00 0.02 0.29 
^nv- 0.16 0.05 0.15 0.20 0.12 0.61 
^im 0.11 0.00 0.05 0.17 0.06 0.00 
n 36 42 37 36 32 7 
Genetic distance (D) was calculated and populations clustered by UPGMA (Sneath 
and Sokal, 1973) for comparison with the PC A and other genetic studies. The UPGMA 
dendrogram again reveals two distinct genetic clusters: the adults at Sebastian Pinnacles 
and the adults and larvae with "tropical" genotypes (Fig. 3). An apparent divergence of 
sr.-vroN AND Ric'i;-. <II;NI-:TIC' VARI.VIION IN SII'I.N?LLAN ADLI.IS AND I ARVAI-. 473 
Table 4. Continued. 
- 
Adults 
Pickics Reef Sebastian 
Pinnacles 
Lan'ae 
Locus Chub Cay GSL 1 GSL 2 GSL 3 
Pep-LA-3 a 0.00 0.02 0.00 0.00 0.00 0.00 
b 0.00 0,00 0,03 0.00 0.00 0.00 
c 0.39 0.16 0.35 0.15 0.12 0.00 
d 0.61 0.73 0.58 0.69 0.72 0.86 
e 0.00 0.09 0.04 0.16 0.16 0.14 
n,.,- 0.48 0.44 0.54 0.49 0.45 0.26 
^OIIS 0.28 0.16 0.19 0.26 0.13 0.00 
n 32 44 37 27 32 7 
Pep-LGG-I a 0.00 0.00 0.00 0.00 0.06 0.00 
b 0.04 0.18 0.05 0.06 0.16 0.00 
c 0.33 0.63 0.14 0.45 0.47 0.85 
d 0.60 0.15 0.35 0.31 0.28 0.14 
e 0.03 0.00 0.46 0.02 0.03 0.00 
f 0.00 0.04 0.00 0.16 0.00 0.00 
W?v- 0.54 0.56 0.65 0.68 0.68 0.26 
^OIIS 0.20 0.15 0.16 0.32 0.06 0.00 
n 35 39 37 31 32 7 
Pep-LGG-2 a 0.00 0.00 0.95 0.00 0.00 0.00 
b 0.87 0.90 0.05 0.83 0.84 0.64 
c 0.13 0.10 0.00 0.17 0.14 0.36 
d 0.00 0.00 0.00 0.00 0.02 0.00 
??. 0.23 0.19 0.10 0.29 0.27 0.49 
^ons 0.09 0.07 0.00 0.11 0.19 0.42 
n 35 43 37 35 32 7 
Pgm a 0.00 0.00 0.02 0.00 0.00 0.00 
b 0.00 0.09 0.24 0.02 0.00 0.00 
c 0.40 0.38 0.44 0.56 0.33 0.28 
d 0.50 0.36 0.27 0.30 0.45 0.28 
e 0.07 0.11 0.03 0.06 0.14 0.37 
f 0.03 0.06 0.00 0.06 0.08 0.07 
f^,:.r 0.59 0.71 0.68 0.60 0.67 0.76 
^ims 0.66 0.70 0.73 0.42 0.47 0.43 
n 35 42 33 33 32 7 
GSL 3 from other larval samples is due in large part to the presence of a single esterase 
al?ele (Est-2h) in one GSL-3 individual. Combined with a small sample size for this group 
(n = 7), this produces larger distances from the other larval samples for GSL 3. The 
omission of "tropical" forms present at the Sebastian Pinnacles did not change the branch- 
ing pattern of the UPGMA dendrogram, but it increased the distance between the Sebastian 
and remaining groups by an average of 0.06. The average genetic distance {D) for all 
populations compared with Sebastian Pinnacles was 0.59. 
474 SULLRTIN Of MARINE SCIENCE. VOL. 65, NO. 2, IWJ 
5-, 
O 
4- 
0 
0 
3- 
G 
? 
A 
2- 
D    O          0?        D 
0 
? 
A 
?nct, A A A 
S8 D 0   DO A      ifc A     A 
0- ^^  uj, iH A 
^?Ocf^           i A A 
a         W      o 0 ^   A 
o QEor?9^    ? o O ? A      A 
.9 J 0 0 A 
? 
o A 
-3- 
? A. 
o o 
-4- 
-5- 
o 
 1 1 ^ [  1                 i  1 ' \ 
0 12 3 4 
First Principal Component 
Figure 2. Apionsoma misakianum: a plot of the first v. second principal components based on 
polymorphic loci for adult populations and larval collections. Individuals from each collection are 
identified by the following symbols: (O) adults from Chub Cay, Bahamas, (D) adults from Pickles 
Reef, Florida, (A) adults from Sebastian Pinnacles, Florida, and (0) adults raised from pelagosphera 
from the Gulf Stream southwest of Sebastian Pinnacles. 
Average Distance (!)?) Between Clusters 
0.60 0.45 0.30 0.15 
h//}//////////y//y////y//? 
Chub Cay, Bahamas O 
G.SL ] O 
1 GSL 2 O Pickles Reef, FL D 
GSL 3 O 
Sebastian Pinnacles, FL ? 
Figure 3. Apionsoma misakianum: a UPGMA dendrogram based on Nei's (1978) distances (D) 
between groups sampled. Benthic adult populations are denoted by locality name, whereas pools of 
reared larvae are identified by GSL lot number (seeTable I ). Note that individuals with the "tropical" 
al?ele type are not included in the Sebastian Pinnacles group. Error bars (in diagonal hatch) are 
calculated as described for allozyme data by Nei et al. (1985). 
STATON AND RICI-:; CiHNHI If VARIATION IN SIPl NCUl.AN ADUI.IS AND l.AHVAl- 475 
DISCUSSION 
Past research on the development of A. misakiamim demonstrated distinct differences 
in patterns of early development between offspring of adults reared from larvae collected 
in the Florida Current and offspring of the benthic adults from central Florida (Rice, 
1981). The present study documented genetic differences between the adults reared from 
larvae in the Florida Current and the benthic adults in the area off central Florida. Al- 
though mortality was high for larvae reared to adulthood, the PCA showed that larvae, 
which survived rearing, were more similar genetically to adults collected 150 km south of 
the larval site than they were to adults collected near the larval sampling locality. 
Results of the PCA identify that adults of the "tropical" genotype do occur, naturally, in 
the northern group. Despite the similarity of adult microhabitat (i.e., the coral fragments), 
the differences in depth of the adult populations could be a factor in the limited recruit- 
ment, as many species are found at limited depth ranges. But this depth difference does 
not exclude recruitment of individuals with the "tropical" genotype to the Sebastian Pin- 
nacles, as evidenced by our study. 
Several hypotheses may account for the absence of "hybrid" adults in our northern 
population sample: (1) competition for coral fragments may relegate migrant individuals 
to previously uncolonized fragments and reduce the probability of interbreeding, (2) dif- 
ferential response to environmental stimuli may produce asynchrony in spawning times 
between migrants and the remaining population (although note that both groups spawn at 
the same time under laboratory conditions. Rice, unpubl. data), (3) post-fertilization bar- 
riers to cross-breeding may occur between cryptic species, (4) "hybrid" larvae might 
occur but completely disperse from the area, (5) the temperate habitat (e.g., annual varia- 
tion in temperature, change in food) on the "tropical" type may result in the formation of 
a "tropical" sterile isolate in the north (as suggested by Bhaud, 1989), or (6) "hybrid" 
larvae may have poor survivorship as compared to the "temperate" or "tropical" types. 
Any of these factors alone, or in combination, would produce limited numbers of hybrids 
in the northern locality and thereby limit our ability to observe them. Recruitment in 
other species of sipunculans has been demonstrated to vary under field conditions, even 
in the absence of any competitors (Hutchings et al., 1992; Rice, unpubl. data), so it is 
possible that one or more of these hypotheses has an effect on the introgression of the 
"tropical" genotype. 
Several factors cannot be assessed by our study. Larvae of the "temperate" or "hybrid" 
types in our samples could have been missed. Their absence is attributable to either trans- 
port away by the predominant south-north current flow or death during the high mortality 
from rearing to adult size. We know only that the surviving adults reared from larvae were 
of the "tropical" genotype (despite the fact that they were reared on mud from the north- 
em population). Also, larvae were collected only twice within 10 d for this study. We 
cannot determine based on this limited sample whether they represent a single, mass 
spawning of a more variable population or two (or more) cohorts of only slightly different 
individuals. However, the presence of rare al?eles in the larval pool that are not present in 
the adult populations is suggestive that there are larvae from other populations present in 
our sample. Clearly, more data is needed on larval and adult populations to fully under- 
stand genetics of these populations. 
However, findings from the metamorphosis and larval-rearing are relevant to the in- 
terpretation of our data on larval genetics. Gulf Stream larvae were competent to meta- 
475 [iUi.LiniN Of MARJM; SfiFNCi;.VOL. 65. S'0.2.1 w; 
morphose (up to 80.9%); they metamorphosed when treated with water conditioned by 
adults from the northern locale (as described in Rice, 1981, 1986), and the surviving 
"tropical-" genotype larvae were successfully reared on sediment from the temperate 
area. Also, a water-soluble factor (a low molecular-weight compound of <500) exuded 
by adults has been suggested to be a species-specific cue for metamorphosis (Rice, 1981). 
These factors in combination suggest that selection of larvae by conditions in the north- 
ern locality is not biased against the "tropical" types, as one might expect given their low 
ft-equency in the northern population. 
Differences between tropical and temperate adults might be attributable to local adap- 
tation to regional climate and habitat. Climatic conditions of the continental shelf are 
influenced by the proximity of the Gulf Stream (especially minimum ocean temperatures 
which affect species' distributions, Bhaud, 1993), and it is within the area of 27?N that the 
Gulf Stream begins to diverge from the coastline of Florida. To the south, reef systems 
and coralline beach sediments predominate, whereas to the north the dominant substrate 
type is quartz sand or mud of salt-marsh estuaries. 
The region along the central coast of Florida has long been recognized as a zone of 
faunal transition (Hayden and Dolan, 1976; Hedgpeth, 1953). Most intraspecific studies 
on genetic markers have focused on peninsular Florida and its role as a barrier to dis- 
persal for marine invertebrate species. Genetic transition for these species has been ana- 
lyzed using nuclear and cytoplasmic genetic markers: allozymes for horseshoe crabs 
(Selander et al., 1970), anemones (McCommas, 1982), oysters (Buroker, 1983), stone 
crabs (Bert, 1986; Bert and Harrison, 1988), oyster drills (Liu et al., 1991), ribbed mus- 
sels (Sarver et al., 1992), marsh and fiddler crabs (Felder and Staton, 1994) and ghost 
shrimps (Staton and Felder, 1996); and mitochondrial DNA (mtDNA) for horseshoe crabs 
(Saunders et al., 1986), oysters (Reeb and Avise, 1990), hermit crabs (Cunningham et a!., 
1992), and hydroids (Cunningham and Buss, 1993). The generalized conclusions of these 
studies is that Gulf and Atlantic populations are genetically distinguishable, if not by 
allozymes, then definitely by more sensitive mtDNA analysis (see reviews by Avise, 1992, 
1994). This type of concordance across species has been argued by Avise (1998) to sug- 
gest shared historical forces shaping the genetic isolation across a region; here the dis- 
juncture occurs between the Atlantic the Gulf coasts of Florida. However the history 
shaping this disjuncture may be far from a single, simple history. 
Several factors suggest that, even though contingency and common history play a role 
in evolutionary processes in coastal Florida, history does not necessarily produce simple 
patterns, as causative impacts on this history may vary in time and space. The American 
oyster is arguably the best-examined case of genetic disjuncture for peninsular Florida: 
its populations have been examined for allozymes (Buroker, 1983), mtDNA (Reeb and 
Avise, 1990), and single-copy nuclear DNA (Karl and Avise, 1992). Even though mtDNA 
haplotypes differ across the same geographic region of eastern Florida (Reeb and Avise, 
1990) as do the allozymes for A. misakianum in our study, allozymes (see reanalysis of 
data from Buroker [1983] by Cunningham and Collins [1994]) and nuclear markers (Karl 
and Avise, 1992) are segregated between populations on the western side of the Florida 
peninsula. Concordance across genetic markers over geographic boundaries (Avise, 1998) 
is not satisfied, demonstrating that there is no simple answer to the phylogeographic pat- 
terns seen here. In short, we cannot be sure if mtDNA haplotypes are "leaking" into 
Atlantic populations (as suggested by Reeb and Avise, 1990) or if nuclear al?eles have 
introgressed into the Gulf population. Indeed, both may have happened at different times 
SIATON AM) Riel-,: (lliM- I IC VARIAI 10\ IN SIPLNCl.'I.AN ADLITS A\D I.ARVAI-, 477 
or other more recent abiotic factors of climate change might contribute to the complexity 
of trans-Floridian species (Felder and Staton, 1994). 
Teleplanic larvae are a part of the complex history connecting even distant populations 
of ^. misakiamim. The formation of sibling species requires a panmictic population that is 
subsequently subdivided either by geographic boundaries or within a region by reproduc- 
tive isolating mechanisms. Even if ancestral populations were distant geographically, 
teleplanic larvae are still a considerable genetic link between populations to be overcome, 
given that a single migrant per generation will prevent genetic differentiation between 
populations, theoretically. Although mtDNA haplotypes can diverge even within species 
(given the clonal nature of their transmission), divergence in nuclear genes is direct evi- 
dence of reproductive isolation. Genetic and developmental differences between northern 
and southern individuals across this zone provide a concordance of data that suggest 
cryptic speciation has occurred m A. misakiamim (see Knowlton, 1993). 
Teleplanic larvae have been hypothesized to affect biogeographic distributions of ma- 
rine invertebrates that possess these larvae. In gastropods, it has been suggested that, on 
average, species with planktotrophic development have larger biogeographic ranges than 
those with direct development (see Scheltema, 1989, for review); however, re-examina- 
tion of these data has shown that a species' range may be dependent upon factors affect- 
ing adult survival (e.g., minimum sea-surface temperature) regardless of developmental 
mode (Bhaud, 1993). There are other reports of molluscan species that have distributions 
that are not correlated to length of larval development (Johannesson, 1988, ? Foighil, 
1989). Our data are consistent with the argument that an assessment of a species' biogeo- 
graphic range by larval ecology and morphology alone may lead to erroneous conclu- 
sions. 
In conclusion, we can state that there is strong reproductive isolation between northern 
and southern populations, and this occurs despite the influx into the northern area of 
larvae that are genetically similar to the southern type. Also, there appears to be reproduc- 
tive isolation between the "temperate" and "tropical" types in the northern population 
that suggests cryptic speciation between the two types. Given these results, we emphasize 
that potential larval dispersal may be quite different from realized larval dispersal. Many 
marine species with teleplanic larvae may have more limited ranges than hypothesized 
given their potential for larval dispersal. Furthermore, potential or realized larval dis- 
persal does not necessarily confer genetic homogeneity across a species with a large range. 
In the ftiture, we will investigate the use of other molecular markers to assess the tem- 
poral and spatial variation in larval genotypes within the lower Gulf Stream for members 
of the A. misakiamim species-complex, as well as sampling other adult populations. It is 
hoped that by expanding this research to larger geographic scales, we can gain a fuller 
understanding of the interaction of genetic and geographic variation and the role of 
teleplanic larvae in biogeography and speciation for a marine benthic invertebrate. 
AcKNOVVI.iaXiMIiN'IS 
We thank the following for assistance in this study; H. Reichardt and W. Lee for larval collec- 
tions, H. Reichardt and J. Piraino for cultiu'e of larvae and metamorphosed adults; S. Reed, I Piraino, 
and H. Reichardt For field assistance in collecting coral rubble; D. Foltz for use of his SAS program 
in data analysis. This paper was improved by discussions with T Collins, G. Harasewych, D. Jacobs, 
J. Maldonado, D, ? Foighil, S. Palumbi, A. Rogers, K. West, C. Young and one anonymous re- 
478 lil.Ll.hriN OF VIAUINJ? .SCIIiNCi;. vol.. 65, NO. 2. I'l'W 
viewer. This material is based upon work supported by a Smithsonian Marine Station Postdoctoral 
Fellowship and by the National Science Foundation under grant DEB - 9303301 awarded in 1993 
to JLS and was written in part during postdoctoral fellowships with W. Brown (Univ. Michigan), D. 
Jacobs (UCLA) and completed during a MarCraig Memorial fellowship at the Museum of Com- 
parative Zoology, Harvard. This article is contribution number 473 from the Smithsonian Marine 
Station at Fort Pierce. 
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DATI: SuuMnTHD: May 28, 1998. DAII: A( ( npTi:i): February 18, 1999. 
ADDRHSS: Smithsonian Marine Station ai Fort Pierce, 70/ Seaway Drive. Fort Pierce. Florida 34949-3140. 
PRHSKNT ADDRFSS (J.L.S.) Belle W. Baruch Institute for Marine Biology and Coastal Research, University of 
South Carolina, Columbia, South Carolina 29208. Tel: (803) 777-394!; E-mail: <jstaton@sc.edu>.