BULLETIN OF MARINE SCIENCE, 48(2): 432-447. 1991 REPRODUCTIVE ISOLATION IN THE POLYDORA LIGNI COMPLEX AND THE STREBLOSPIO BENEDICTI COMPLEX (POL YCHAETA: SPIONIDAE) 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 pOLYCHAETES 433 Polydora ligni Webster, 1879 and Streblospio benedictiWebster, 1879 are among the most opportunistic and widely distributed polychaete 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 Caribbean 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