BULLETIN OF MARINE SCIENCE, 48(2): 432-447. 1991

REPRODUCTIVE ISOLATION IN THE POLYDORA LIGNI COMPLEX AND THE STREBLOSPIO BENEDICTI COMPLEX (POL YCHAETA: )

Stanley A. Rice

ABSTRACT Results of experimental crosses between individuals from geographically separated pop- ulations indicate that Tampa Bay Po/ydora /igni are reproductively isolated from their North Carolina and Los Angeles morphological counterparts. The North Carolina and Los Angeles populations of P. /igni are interfertile. The Tampa Bay population of Streb/ospio benedicti is likewise reproductively isolated from the planktotrophic North Carolina population. In both genera, experimental crosses were conducted in the laboratory using individual worms that had been raised in isolation prior to the experimental cross. Female worms from unsuccessful interpopulation crosses were back-crossed to intrapopulation males to ensure female fertility. Inter- and intra population crosses in P. /igni were accomplished via artificial transfer of spermatophores from an isolated male to an isolated female. In Streb/ospio crosses, males and females from appropriate populations were placed together and monitored daily for reproductive activity. The presence of spermatophores with active sperm in Streb/ospio cultures was taken to indicate male fertility. In the P. /igni complex, reproductive breakdown appears to occur at the sperm transfer stage. In interpopulation crosses, females accept sper- matophores from exotic males but the sperm fail to accumulate in the sperm storage organs ofthe female. In the S. benedicti complex, the breakdown is also at the sperm transfer stage. Males produce spermatophores in the presence (or absence) of exotic females but no viable larvae are produced from these crosses. Intrapopulational crosses in both genera are routinely successful. Morphological differences between the reproductively isolated P. /igni populations are inconsistent and variable making description of distinct species difficult. Morphological differences are present, at least in females, from the divergent S. benedicti populations.

The relationship between reproductive isolation and morphological differen- tiation of populations is inconsistent and variable depending upon the species involved. Classic cases include the morphologically similar but biologically dis- tinct Drosophila melanogaster-D. simulans group and the morphologically di- vergent but genetically similar Hawaiian Drosophila (Parsons, 1975; Carson et aI., 1970). The situation is further complicated when populations display high degrees of polymorphism for taxonomically important characteristics. In dealing with cases of suspected speciation (cryptic or sibling species), the documentation of reproductive isolation needs to be established first, followed by a systematic analysis of any morphological, chromosomal, molecular, reproductive or behav- ioral characteristics that might be used to distinguish between these biological species. The use of reproductive characteristics in distinguishing between closely related species has become a valuable and necessary tool in systematic biology (Smith, 1958; Mayr, 1963; Fauchald, 1977). Widely distributed "cosmopolitan" species are frequently being found to consist of distinguishable subspecies or sibling species when the appropriate characteristics are examined in sufficient detail (Mayr, 1948; Ayala, 1975; Reish, 1977; Clark, 1977). This situation may present tech- nical problems for routine identification of preserved material in cases where distinct and consistent morphological differences between sibling species are ab- sent or ill defined. Nevertheless, comprehensive study of such species complexes can lead to a more thorough understanding of the factors contributing to the speciation process in its earliest phases.

432 RICE: REPRODUcrlVE ISOLATION IN SPIONID 433

Polydora ligni Webster, 1879 and Streblospio benedictiWebster, 1879 are among the most opportunistic and widely distributed species known (Grassle and Grassle, 1974; Rice and Simon, 1980). In benthic surveys conducted in Florida alone, one or both of these species has been reported among the dominant fauna in over 33 studies (Mahadevan et al., 1984). These species are likewise abundant in parts of California (Rice, 1975; Light, 1978); New England (Webster and Ben- edict, 1884; Mortenson, 1945; Dean, 1965; Grassle and Grassle, 1974); North Carolina (Day, 1973; Levin and Creed, 1984); throughout western Europe (Soder- strom, 1920; Hartman, 1959; Rasmussen, 1973; Ramberg and Schram, 1982; Mustaquim, 1986); and the and South America (Foster, 1971). For such a wide-spread and ecologically important pair of species, it is imper- ative that we understand the systematic relationships between geographically iso- lated populations, i.e., are we dealing with a single species or a complex of bio- logically distinct sibling species? If these distant populations represent different biological species, then methods for their identification must be developed. In the present study, populations of Polydora ligni from Tampa Bay, North Carolina, and Los Angeles were tested for reproductive compatibility in laboratory crosses. Data were collected for each population on standard morphological char- acteristics, reproductive morphology, and sperm ultrastructure in an attempt to find consistent morphological criteria for identifying reproductively isolated pop- ulations. Populations of Streblospio benedicti from Tampa Bay and North Carolina were likewise tested for reproductive compatibility and analyzed for morpholog- ical and reproductive differences.

METHODS

Laboratory populations of Polydora ligni were established from plankton samples collected at the west end of the Courtney Campbell Causeway, Tampa, Florida (TBC) and at Bogue Sound, North Carolina (NC). Adult P. ligni were collected from Davis Island (included as TBC), Tampa, Florida and from Huntington Harbor, California (LA). Streblospio benedicti were collected as adults from the Tampa Bypass Canal (TBC), Florida and as planktonic larvae from Bogue Sound (NC). All populations were acclimated to 250/00seawater collected from the Gulf of Mexico. Adult worms were maintained in 100 x 25 mm plastic petri dishes with freeze-thaw sediment and phytoplankton (Chlorella sp., Tetrase/mis sp.) added as food. Planktonic larvae were raised in 100 x 25 mm plastic petri dishes with Rotorich (yeast-algae suspension) and phytoplankton added as food. Larvae were maintained on a shaker table until near metamorphosis and then transferred into Coming 24-, 12-, or 6-well tissue culture dishes individually and raised to sexual maturity. For experimental crosses involving P. ligni populations, an isolated female was deemed ready for crossing when she produced her first batch of unfertile eggs as determined by direct observation through the bottom of the culture container with a dissecting microscope. Fresh spermatophores were trans- ferred to the isolated female from an isolated male using a clean micropipette. Behavioral observations were recorded for the female when she first encountered the spermatophores. Following spermatophore transfer, each female was checked for egg production daily until either fertile eggs (and larvae) were produced or until three separate batches of unfertile eggs were deposited inside the tube. Following an unsuccessful interpopulation cross, the female was presented with spermatophores from a male of her own population and the observations were repeated. Three replicate crosses were attempted for each combination of sex and population origin. A high degree of variability in larval growth and survival within populations prevented use of this information for interpopulation comparisons. For Streblosp;o benedict; crosses, one male and one female worm from the same or different pop- ulations were placed together in a single well of a 12-well culture dish and observed daily for sper- matophore production by the male and larval production by the female. The presence of spermato- phores in the dish was taken as an indication of male fertility while active larvae in the dish were taken to indicate a successful cross. Pairs were monitored for 3-6 weeks in each cross. Females from unsuccessful interpopulation crosses were placed together with a male from their own population and monitored for larval production. Female worms could not be observed inside their tubes and removal offemales from their tubes was detrimental to the developing embryos, if present, so no attempt was made to monitor egg deposition for Streblospio. Eighteen replicate crosses were attempted for TBC females x NC males and II replicate crosses between NC females and TBC males were completed. 434 BULLETIN OF MARINE SCIENCE, VOL. 48, NO.2, 1991

Table I. Results of experimental crosses between populations of Polydora ligni: Tampa Bay (TBC), North Carolina (NC), and Los Angeles (LA) (Each cross was attempted in triplicate)

Cross Date Results

NC'? X TBC ~ 7/87 No fertilization NC~ X TBC '? 7/87 No fertilization LA~ X TBC'? 8/87 No fertilization LA'? X TBC~ 9/87 No fertilization LA~ X NC'? 9/87 Fertile eggs, normal larvae LA'? X NC~ 10/87 Fertile eggs, normal larvae TBC5 X TBC'? 7/87 Fertile eggs, normal larvae LA5 X LA'? 9/87 Fertile eggs, normal larvae NC5 X NC'? 8/87 Fertile eggs, normal larvae

Specimens for scanning electron microscope (SEM) studies were fixed in either 10% buffered formalin or 2.5% glutaraldehyde in PO. buffer, postfixed in 2% OsO. in 2.5% NaHCO) and dehydrated in ethanol to acetone for critical point drying. Dried specimens were mounted and coated with gold- palladium and viewed on a Zeiss Novascan 30 SEM. Specimens used for histological studies and transmission electron microscopy (TEM) were fixed in glutaraldehyde and osmium as above, dehy- drated to propylene oxide and embedded in Medcast epoxy. One micron sections were cut on a Reichert-lung Ultracut E ultramicrotome, stained with methylene blue and azure II, and photographed with an Olympus BH2 photomicroscope. Thin sections were cut with a diamond knife, stained with aqueous uranal acetate and lead citrate, and viewed on a Hitachi H500 TEM. Statistical analyses were performed using the Stat View 512 + software for the Macintosh.

RESULTS Polydora ligni Crosses. - The results of crosses between the various populations of Polydora ligni are shown in Table 1. The data indicate that the Tampa Bay population (TBC) is reproductively isolated from both the Los Angeles (LA) and North Carolina (NC) populations. However, the LA and NC populations are not reproductively isolated from each other. It is interesting to note that the populations which have the greatest geographical separation (LA and NC) are reproductively compatible while the TBC population, intermediate in geographic separation, is reproductively isolated from the LA and NC populations. In each of the first four crosses listed in Table I, the female worm was successfully back-crossed to a male from her original population and produced fertilized eggs. Reproductive Morphology of Poly dora ligni Populations.-Data collected on the location of the first gametogenic segment, percentage of gametogenic segments, and the total number of segments were used to statistically compare populations (F-tests). Initial comparisons were made between males and females of the same population and significant differences were found between sexes in one or more populations for all three characteristics examined (Table 2).

Table 2. Means of males (M) and females (F) for location of the first gametogenic segment, percentage of gametogenic segments, and total number of segments. Asterisk (*) indicates significant differences between sexes (P < 0.05). North Carolina (NC), Tampa Bay (TBC), and Los Angeles (LA); sample size in parentheses

Population/Sample 1st Gametogenic segment % Gametogenic size (M, f) (M.f) segments (M, f) Total segments (M, f)

TBC(71,30) 12.52, 14,06* 32.6,34.8 39.10, 56.40* NC (18, II) 13.94,15.91* 3\.6, 34.7 45.22, 58.46* LA (25,15) 14.08, 15.29* 40.8,31.7* 55.63, 58.Q7 RICE: REPRODUCfIVE ISOLATION IN SPIONID POLYCHAETES 435

Table 3. Summary of mean values for total segments, location of first gametogenic segment and percent gametogenic segments (gametogenic segments/total segments) in Polydora /igni males from three populations. Standard deviations below means in parentheses. TBC, Tampa Bay; NC, North Carolina; LA, Los Angeles. Means which have the same letter to the right are not significantly different (F-test, P = 0.05)

Population TBC(N -71) NC(N - 18) LA (N - 24) Total segments 39.10 a 45.22 ab 55.63 b (20.39) (11.57) (12.99) Ist gametogenic segment 12.52 a 13.94 b 14.08 b (1.96) (0.73) (1.29) Percent gametogenic 32.6 a 31.6 a 40.8 b (11.3) (8.4) (10.6)

Due to significant differences between sexes, all interpopulational comparisons are reported separately for each sex. Table 3 presents a summary of the inter- population comparisons for males from the three populations along with statistical differences for pairwise comparisons. The TBC population was significantly dif- ferent from the LA population for total segments, location of the first gametogenic segment, and percent gametogenic segments. The TBC population was signifi- cantly different from the NC population in the location of the first gametogenic segment while the LA and NC populations differed significantly in percent ga- metogenic segments. Reproductive morphology of females from the three populations is presented in Table 4 along with statistical comparisons. For interpopulation comparisons of females, the TBC population differed significantly from both the LA and NC populations in the location of the first gametogenic segment. All other differences for females were not statistically significant. The relationship between total size of an individual (total segments) and the percentage of gametogenic segment was assessed through regression analysis (Ta- bles 5 and 7), and experimental observation (Tables 6 and 8). Populations which were known to be reproductively compatible were grouped together for this anal- ysis. Table 5 presents the results of the regression analysis for total segments vs. percent gametogenic segments. A significant correlation was obtained for all treat- ments except for the LA + NC females. This suggests that for all males and for the Tampa Bay females, the percent gametogenic segments increases as the size

Table 4. Summary of mean values for total segments, location of first gametogenic segment and percent gametogenic segments (gametogenic segments/total segments) in Polydora /igni females from three populations. Standard deviations below means in parentheses. TBC, Tampa Bay; NC, North Carolina; LA, Los Angeles. Means which have the same letter to the right are not significantly different (F-test, P = 0.05)

Population TBC (N = 30) NC(N=ll) LA (N = IS)

Total segments 56.40 a 58.46 a 58.07 a (23.86) (16.18) (13.35) Ist gametogenic segment 14.06 a 15.91 b 15.29 b (1.41) (1.30) (1.26) Percent gametogenic 34.8 a 34.7 a 31.7 a (10.5) (7.2) (6.8) 436 BULLETIN OF MARINE SCIENCE, VOL. 48, NO.2, 1991

Table 5. Regression analysis for the percentage of gametogenic segments vs. total segments. Sample sizes, R' values, and probabilities are listed separately for males and females of each interbreeding population

Population Sample size R2 value Probability 71 0.712 0.0001 & {TBC LA + NC 42 0.371 0.0001 37 0.610 0.0001 2 {TBC LA + NC 26 0.092 0.1314 of the individual increases but for the LA and NC females, the percent of ga- metogenic segments does not significantly change as the worm grows. To test the effect of increasing size (in total segments) on the percent of gametogenic segments, 10 males and 7 females from the TBC population were isolated and monitored for gamete distribution over a 28-day period. These results are presented in Table 6. For the males, a significant increase in percent gametogenic segments occurred over the 28-day period but for females, no significant increase was observed. Males grew from a mean of 34.7 segments at day 0 to a mean of 63.0 segments at day 28. Females increased from 41.3 to 62.4 segments during the same period. The location of the first gametogenic segment was analyzed with respect to total size (total segments) for each interfertile population. The regression analysis for this parameter is presented in Table 7. The data suggest that for each sex and population, the location of the first gametogenic segment may vary with the total size of the individual, being located more posteriorly in larger individuals. To test this hypothesis, the same 10 males and 7 females above were scored for location of first gametogenic segment over a 28-day period. These results are presented in Table 8. Neither males nor females showed a significant change in this feature over the 28-day period. Variation in Standard Morphology. -In an attempt to distinguish between the reproductively isolated populations of Polydora, 11 standard morphological char- acteristics were examined on the SEM and compared between populations. Table 9 lists these characteristics and indicates which were found to be polymorphic in each population. All but three of these characteristics were found to be poly- morphic in one or more of the populations. Across all populations, caruncle lengths varied from the beginning of the second setiger (B2) to the end of the fourth (E4); nuchal antennae were present or absent; fifth spines had 1, 2, 3, or no lateral teeth; fifth companion' setae were brush tipped or pointed; fifth capillaries were present or absent; fifth spines had a lateral flange or not; and capillary neurosetae (post 6th) were present in some. The percent occurrence of these polymorphic char- acteristics by population is presented in Table 10. The only standard morpho- logical characteristic, of the 11 examined, that would appear to have promise in differentiating between reproductively isolated populations of P. ligni is the oc- currence of anterior-dorsal and posterior-ventral capillary setae on the fifth setiger in Tampa Bay worms and their relative absence in North Carolina and Los Angeles

Table 6. Mean percentage of gametogenic segments over a 28-day period. Significant changes in the means (F-test) are denoted by an asterisk (*)

Sex Sample size Mean day 0 Mean day 28

Males 10 31.1 44.2* Females 7 30.5 34.8 RICE: REPRODUCTIVE ISOLATION IN SPIONID POLYCHAETES 437

Table 7. Regression analysis for total segments vs. location of first gametogenic segment. Sample sizes, R2 values, and probabilities are listed separately for males and females of each interbreeding population

Population Sample size R2 value Probability

71 0.380 0.0001 0 {TBC LA + NC 42 0.203 0.0028

<;1 {TBC 37 0.634 0.0001 LA + NC 26 0.203 0.0210

worms. This characteristic, however, was not absolutely consistent within pop- ulations (polymorphic) and is at odds with previous published observations (see Discussion). Streblospio benedicti Experimental Crosses. - Two populations of Streblospio benedicti were tested for reproductive compatibility in the laboratory via recip- rocal crosses. Each population produced planktotrophic larvae in laboratory cul- ture which were raised to metamorphosis and then isolated as individual worms until sexual maturity was reached. In the first series of crosses, one North Carolina (NC) female out of five produced larvae while exposed to a Tampa Bay (TBC) male and one TBC female out of six produced larvae in the presence of an NC male (Table 11). In the case of the NC female paired with the TBC male, only one was produced and this larva was at a late stage of development (about 8 setigers) and appeared 1 day after the pair were placed together. Since females normally brood their embryos for at least 3 days before release, this single large larva was likely not sired by the TBC male. In the case of the TBC female paired with the NC male, the first batch oflarvae appeared 3 days following pairing with a second batch released 2 days later. No larvae were produced during the following 25 days in spite of prolific spermatophore production by the male. It is likely that these "interspecific larvae" were produced due to prior exposure of the female to a conspecific male or the inadvertent transfer of spermatophores via pipette. In a subsequent experiment, none out of 12 TBC females produced larvae in the presence of mature NC males. In routine intrapopulational crosses with mature virgin worms, the mean time to first appearance of larvae in the dishes was 9 days (N = 6) for TBC females and 9.5 days (N = 4) for NC females. During backcrosses of females to conspecific males following unsuccessful interpopulational crosses, the mean time to larval release was 8.6 days (N = 10) for TBC females and 21.5 days (N = 2) for NC females. The shortest time to larval release for intrapopulational crosses of mature virgin worms was 6 days for TBC and 8 days for NC. Morphological Variation between Streblospio Populations. - The most striking morphological variation between the Tampa Bay and the North Carolina popu- lations was in the structure of the dorsal brood-holding appendages in mature females. In planktotrophic females from the North Carolina population, paired

Table 8. Mean location of the first gametogenic segment over a 28 day period in Polydora ligni from Tampa Bay. No significant differences were observed

Sex Sample size Mean day 0 Mean day 28

Males 10 12.00 12.00 Females 7 13.14 13.00 438 BULLETIN OF MARINE SCIENCE, VOL. 48, NO.2, 1991

Table 9. Standard morphological characteristics examined for Polydora populations and occurrence of polymorphism for each (+, polymorphic; -, monomorphic)

Polymorphic per population Characteristic TBC NC LA Caruncle length + + + Nuchal antenna + + 1st notopodium 5th setiger spines (tooth) + + 5th setiger spines (flange) + 5th setiger companion setae + + + 5th capillaries + + Hooded hooks + Capillary neurosetae (post 6th) + Branchia Posterior notosetae dorsal pouches were present between setigers 17 and 45 and averaged 8.2 (N = 17) pairs per female. The Tampa Bay worms possessed long thin dorsal pairs of branchiae between setiger 17 and 25 with an average of 7.1 (N = 7) pairs per female. These differences were consistent between populations with no Tampa Bay worms observed to have pouches and no North Carolina worms observed to have branchiae instead of pouches. The location of the first gametogenic segment in female worms varied between the two populations. Tampa Bay females had a mean of 8 (N = 9) while NC females had a mean of 11.1 (N = 7). The dimensions of the sperm also varied between populations with TBC sperm averaging 38.4 ~m (N = 20) head length while NC sperm averaged 55 ~m (N = 5). Male S. benedicti from all populations examined produced spermatophores in the presence or absence offemale worms. These spermatophores were composed of a sickle-shaped proximal piece and a distal sperm ball. The detailed structure of these spermatophores will be presented in a later paper. Sperm transfer was not observed directly and attempts to artificially transfer spermatophores from an isolated male to an isolated female did not result in larval production.

DISCUSSION Establishment of reproductive isolation between allopatric or sympatric pop- ulations may occur in a number of ways (White, 1978; Templeton, 1981; Paterson, 1985). Modifications in mating behavior may result in premating isolation while alterations in gamete morphology or gamete compatibility may result in post- mating isolation. In either case, the earliest indications of speciation may be expressed as modifications of the reproductive system that can be used taxonom- ically to distinguish between species and evolutionarily to study the process of speciation. In the cases of polydora ligni and Streblospio benedicti populations, the evidence for reproductive isolation is strong yet the degree of morphological differentiation is relatively small. Within the Polychaeta, numerous examples have been reported of speciation in progress (Clark, 1977; Grassle, 1984; Rice and Simon, 1980; Beckwitt, 1982; Akesson, 1984; Guerin and Kerambrun, 1984). The organisms involved consist of sympatric or allopatric populations that can rarely be differentiated using stan- dard morphological criteria but nevertheless display significant reproductive dis- similarities. This lack of morphological difference is usually taken to indicate that divergence of the populations is incipient, as opposed to adaptive convergence of RICE: REPRODUCfIVE ISOLATION IN SPIONID POLYCHAETES 439

Table 10. Percent occurrence of character states for eight polymorphic characteristics as determined by SEM observations on individuals from each population. See text for explanation of character states

Population Character and character states Sample size

Caruncle length B2 M2 E2 B3 M3 E3 B4 M4 E4

NC 9 55 18 18 II LA 14 7 72 7 14 TBC 36 21 12 12 7 5 7 42

Nuchal antenna

Present Absent

NC 100 0 II LA 93 7 14 TBC 95 5 42

Fifth spines (tooth) Absent ltooth 2+ teeth NC 0 100 0 21 LA 6 88 6 16 TBC 6 63 31 48

Fifth spines (flange)

Present Absent

NC 0 100 21 LA 0 100 16 TBC 8 92 49

Companion setae All brush-tip Some pointed NC 52 48 21 LA 53 47 15 TBC 41 59 44

Capillary setae on fifth

Anterio- Posterio- Absent dorsal ventral Both

NC 100 0 0 0 19 LA 88 0 0 12 17 TBC 21 2 2 75 43

Hooded hooks Begin on 7 Begin on 8

NC 100 0 21 LA 100 0 16 TBC 98 2 44

Capillary neurosetae (post-6th) Present Absent

NC 0 100 20 LA 0 100 14 TBC 97 3 39 previously distinct species. As time passes, morphological divergence would be expected to appear, with one of the first systems likely to be affected being the reproductive organs or gametes. The work of Grassle and Grassle (1974) on enzyme polymorphism and life history traits in the pollution indicator polychaete Capitella capitata led to the 440 BULLETIN OF MARINE SCIENCE, VOL. 48, NO.2, 1991

Table II. Streblospio benedicti interpopulational crosses North Carolina (NC) planktotrophs x Tam- pa Bay (TBC) planktotrophs

No. successful Spermatophores Number of Duration of Participants No. of pairs crosses observed larval broods cross (days)

NC'? X TBC d II 1* + I 28 NC'? X NCd 4 4 + 17 40 NC'? X NC dt 5 2 + 2 38 TBC'? X NC d 17 1* + 2 31 TBC'? X TBCd 6 6 + 22 31 TBC'? X TBC dt 16 12 + 22 22 • See text for explanation of these results t These crosses involved the same females that had been previously paired with males from the exotic population without successful larval production

discovery of six sibling species in the Massachusetts area (Grassle and Grassle, 1976) and at least four additional sibling species from other areas (Grassle, 1980; Eckelbarger et aI., 1984). The Capitella sibling species show little variation in extemal morphology yet display significantly different life history paUems in- cluding breeding season, length of larval life, egg size and fecundity (Grassle and Grassle, 1976). Detailed observations at the SEM and TEM levels have docu- mented some consistent morphological differences in egg and sperm morphology that may be of systematic importance (Eckelbarger and Grassle, 1983; 1984; 1987). In the polychaete family Spionidae Sars, 1862, the occurrence of polymorphic populations of apparently the same species has been known for some time. Vari- ability of reproductive mode in spionid populations has been demonstrated in Spio martinensis (Mesnil and Caullery, 1917; Hannerz, 1956), Pygospio elegans (Hannerz, 1956; Soderstrom, 1920; Thorson, 1946; Smidt, 1951) and Spio setosa (Simon, 1967). The phenomenon termed poecilogony (coined by Giard, 1904 and reevaluated by Hoagland and Robertson, 1988) proposes that a species may exhibit two or more developmental patterns at different times of the year or within different areas of their geographic range (Simon, 1968; Thorson, 1950). Simon (1968, p. 510) however, pointed out" ... that any "species" which exhibits more than one reproductivle or developmental pattern should be examined critically to insure that two cryptic species are not involved." The spionid polychaetes mentioned above may represent additional cases of species complexes in the earliest stages of divergence and prior to the appearance of distinct external morphological differences. Levin (1984) reported that Streblospio benedicti displayed different patterns of larval development over its range with some populations producing lecithotrophic larvae and others producing planktotrophic larvae. Planktotrophic and lecitho- trophic populations were shown to be interfertile but females from interpopula- tional crosses tended to produce larvae characteristic oftheir original population. The production of different larval types was not affected by temperature or food availability in S. benedicti populations from North Carolina (Levin and Creed, 1986). Levin (pers. comm.) has successfully crossed the branchiate forms from Copano Bay, TX with the lecithotrophic form from Big Slough, TX. She also found larval production in limited crosses between Ballast Point, FL females and Tar Landing, NC males (pers. comm.). In the present study, the only interpop- ulationallarvae that were produced came from a cross between a Bogue Sound, NC female and a TBC male, and between a TBC female and a NC male. In the first case (NC 2 x TBC 5), only one large larva was produced and this appeared one day after pairing, suggesting that this larva was not the product of this cross. RICE: REPRODUCfIVE ISOLATION IN SPIONID POLYCHAETES 441

In the second case (NC 5 x TBC 9), two batches of larvae were produced, one 3 days aft~r pairing and another 5 days after pairing. In all intrapopulation crosses with virgin worms, the shortest time to larval release was 6 days (N = 22) making it unlikely that these larvae were the result of fertilization between this specific NC male and the TBC female. The possibility still remains that there may be some interfertility between the branchiate and pouched forms of S. benedicti. However, based upon the restricted distribution of the branchiate form, its re- productive isolation from the pouched form (from the present research), and its morphological distinctness, the branchiate form deserves species status. A formal description of this new species will be presented in a subsequent paper (Rice et a1., in prep.). The ultimate challenge in characterizing a species complex is to demonstrate reproductive isolation between populations and then explain the mechanism re- sponsible for such reproductive incompatibility. Experimental crosses between suspected sibling species have been attempted with polychaetes on several oc- casions. Grassle and Grassle (1976) attempted cross-breeding experiments be- tween Capitella sibling species and found a very low incidence of fertilization between different strains. These results helped to confirm the suspected degree of evolutionary divergence in Capitella as indicated by electrophoretic patterns and other data. Akesson (1972; 1975; 1978; 1984) has used crossbreeding experiments to help distinguish between closely related species ofthe genus Ophryotrocha that are often morphologically identical. Guerin (1977) characterized closely related Scolelepis spp. through crossbreeding experiments and Levin (1984) successfully interbred populations of Streblospio benedicti that displayed different modes of larval development. In the genus Polydora few crossbreeding experiments have been attempted in spite of the large number of closely related species. Rice and Simon (1980) crossed P. ligni populations from Tampa Bay with a limited number of worms from California and the east coast of Florida. Intrapopulational crosses were always more successful than interpopulational crosses with some combi- nations completely unsuccessful. In the crosses attempted by Rice and Simon (1980) between Tampa Bay and California worms, few individuals were used and reciprocal crosses were not completed. The results from the present work, however, confirm the preliminary crosses attempted in the past and extend those results to the North Carolina population. Behavioral incompatibility has been documented in the form of mate discrim- ination in Ophryotrocha (Akesson, 1972; 1984) and in the sequence of events leading to synchronized spawning in nereids (Clark, 1977). Rice (1978a; 1978b) suggested that behavioral differences related to spermatophore transfer might be responsible for reproductive isolation between closely related sympatric species of Polydora. Preliminary experiments with interpopulation sperm transfer indicate that the sperm are not reaching the sperm storage organs of the female following artificial spermatophore transfer (Rice, unpub1.). If this indeed is the stage of reproductive breakdown, then it is possible that the sperm are not responding to a chemical attractant that might lead them into the seminal receptacle or alter- natively, the female may have physical control over the opening and not respond to exotic spermatophor~s. In Streblospio benedicti, sperm transfer may also involve some behavioral as- pects. The unsuccessful attempts at artificial spermatophore transfer within pop- ulations suggest that some behavioral or recognition event(s) is involved in this process. The ultimate cause of speciation at both early and late stages of the process is genetic differentiation. Our knowledge of polychaete molecular genetics is pres- 442 BULLETIN OF MARINE SCIENCE, VOL. 48, NO.2, 1991

ently quite limited, with protein electrophoresis constituting the most frequently applied technique. (Grassle and Grassle, 1976; Nicklas and Hoffmann, 1979; Robotti, 1979; Beckwitt and Chakroborty, 1980; Rice and Simon, 1980; Britton- Davidian and Amoureux, 1982; Ekaratne et aI., 1982; Levin, 1982). The rela- tionships between genetic differentiation (from electrophoretic data), morpholog- ical differentiating and speciation in polychaetes remains unclear. Mechanisms that control morphological features such as nuclear condensation and shape of mature spermatozoa may involve few genes and thus may represent directly observable functional manifestations of early genetic differentiation. Gamete morphology, especially at the ultrastructural level, presents a promising approach to both the problem of identifying closely related species and to the explanation of isolating mechanisms (Baccetti and Afzelius, 1976). In the Poly- chaeta, gamete ultrastructure research is principally at the descriptive level. As more information becomes available, comparative studies will become more com- mon (Olive, 1983a; 1983b; Sawada, 1984). Comparative studies of sperm ultrastructure in closely related species are few yet those that have been attempted support the hypothesis that sperm structural changes and reproductive isolation are closely linked. Eckelbarger and Grassle (1984; 1987) have found ultrastructural differences between spermatozoa from the various Capitella siblings while Pfannenstiel et al. (1987) reported significant differences in sperm structure between two members of the P. dumerilii complex. Rice (1981) described spermatogenesis and sperm ultrastructure in three species of Polydora and Streblospio benedicti from Florida and suggested that sperm ultrastructure might be a useful tool for identification of geographic variants in spionid polychaetes. He also discussed ultrastructural differences between sperm from P. ligni and those of P. ciliata from Europe (Franzen, 1974), concluding that these represent two biologically separate species rather than morphological vari- ants of the same species (Rasmussen, 1973). Members of the family Spionidae are recognizable in fossils dating back to the early Cambrian Period. Adaptive radiation and subsequent speciation has resulted in approximately 200 known species, most of which are distinguishable using standard taxonomic characteristics. Many spionids are opportunistic species, in- habiting disturbed environments and able to withstand pollutants and low dis- solved oxygen levels. With the recent increase in polluted and enriched coastal habitat as well as the introduction of novel pollutants into the ocean, a new round of adaptive radiation may be underway in species such as P. ligni and S. benedicti. Spionid polychaetes, in general, have numerous external morphological char- acteristics that may be useful in a taxonomic diagnosis. These include branchiae, modified setae, pigmentation patterns, parapodial arrangements, setal distribu- tions and pygidial appendages among others. This variety of useful characteristics is largely responsible for the rapidly increasing number of described species yet an unusually large number of spionids are suspected of being species complexes that are indistinguishable using external morphology (Table 12). Differences be- tween species in external characteristics such as branchial distribution (a generic characteristic) or shape of modified setae (a specific characteristic) are likely a result of post-speciation adaptive radiation or genetic drift and not the initial cause of speciation. Reproductive characteristics, such as sperm morphology, sperm transfer mechanisms and sperm storage organs, on the other hand, can be envisioned as causative agents in the early stages of speciation and can also serve as taxonomic characteristics for species identification. In order to sort out the taxonomic status of widespread, abundant spionids, such as P. ligni and S. ben- RICE: REPRODUCfIVE ISOLA nON IN SPIONID POL YCHAETES 443

Table 12. Studies suggesting cryptic or sibling species in spionid polychaetes

Species Population Characteristics studied Rererence Polydora Iigni Tampa Bay, FL morphology Rice and Simon Ft. Pierce, FL genetics (1980) California physiology crosses Boccardia Long Beach, CA reproduction King (1976) proboscidea larval devel. Polydora ciliata Whitstable, Eng. reproduction Dorsett (1961) morphology Boccardia hamata Maine larval devel. Dean and Blake California metamorphosis (1966) Spio setosa Massachusetts larval devel. Simon (1968) Polydora Maine larval devel. Blake (1969) quadrilobata Spio Sweden reproduction Hannerz (1956) martinensis larval devel. Malacoceros Atlantic Ocean morphology Guerin and fuliginosus Mediterranean ecology Kerambrun Sea larval devel. (1984) crosses Streblospio California morphology Levin (pers. comm.) benedicti Florida larval devel. N onh Carolina Boccardia California larval devel. Blake and Kudenov proboscidea Australia (1978) Polydora nucha/is California reproduction Wible (1984) larval devel. Pseudopolydora Norway morphology Ramberg and paucibranchiata reproduction Schram (1982) Polydora giardi California reproduction Day and larval deveI. Blake (1979)

edicti, it will be necessary to complement existing taxonomic characteristics with new innovative features that will allow separation of biologically distinct species. Among the reproductive characteristics measured, the location of the first ga- metogenic setiger was significantly different between the Tampa Bay worms and those from NC and LA for both males and females. Statistically significant dif- ferences between population means may be taken to indicate that divergence is occurring; however, these population differences are of limited value in distin- guishing between individual worms from different populations. Among the 11 traditional morphological characteristics examined in this study for P. ligni, only one, the presence or absence of fascicles of capillary setae on the fifth setiger showed any promise for discrimination between reproductively iso- lated populations. This character however, varied within most populations and was monomorphic only for the NC population. This is inconsistent with Blake and Maciolek (1987) who synonymized P. ligni with P. cornuta Bose and described the fifth setiger of Charleston Harbor worms as having notopodial capillaries present or absent and neuropodial capillaries absent. Rice and Simon (1980) found these fifth setiger capillaries to be variably present or absent among populations within Tampa Bay. In their report, the CCC population (from the same collection 444 BULLETIN OF MARINE SCIENCE. VOL. 48. NO.2, 1991 site as the TBC collection in the present study) lacked these capillary setae (N = 25) but nearly 90% of the individuals from the Ballast Point population (also in Tampa Bay) had capillary fascicles on the fifth setiger. Blake and Kudenov (1978) reported ventral inferior fascicles of capillaries invariably present on the fifth setiger in Australian specimens of P. ligni. It appears that traditional morphological characters, even taken collectively, do not provide good discriminators for distinguishing between the Tampa Bay pop- ulation and those from which it is isolated. Even within P. ligni populations, the degree of temporal and spatial variation in most characters is surprising. This may be indicative of a species that is exposed to extreme local selection producing divergent populations or it may be due to a series of founder effect events that may change from year to year. It is hard to imagine what factors would favor the development or retention of capillary setae on the fifth setigers of individuals from one population while these are largely absent in individuals from a nearby population or at the same site at a later time. In Tampa Bay, there may be several morphological variants of P. ligni competing for habitat with one morph colo- nizing a particular site at one time and another morph occupying the same site at another time. With the volume of boat traffic in Tampa Bay, it is possible that populations from other geographic areas have been introduced and these may contribute to the high degree of variability observed within the bay.

ACKNOWLEDGMENTS

The substantial assistance of B. Moffat, C. Doege, and A. Fisher in collection of various portions of these data is gratefully acknowledged. This research was supported by a grant from the National Science Foundation, BSR-8606927 and by a Faculty Development Grant from the University of Tampa.

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DATEACCEPTED: March 19, 1990.

ADDRESS:Division of Science and Mathematics. University of Tampa, 401 W. Kennedy Boulevard. Tampa. Florida 33606-1490.