JOURNAL OF BIOLOGY, 12(3): 335-341, 1992

OSMOREGULATORY CAPACI'IIES IN DISJUNCT WESTERN ATLANTIC POPULATIONS OF THE COMPLEX (: GRAPSIDAE)

Joseph L. Staton and Darryl L. Felder

ABSTRACT Osmotic and ionic regulatorycapacities of a populationof Sesarma reticulatumfrom Bruns- wick, Georgia, on the Atlantic coast of the United States were compared with those of a populationof S. sp. (near reticulatum)from coastal Louisianain the northernGulf of Mexico. Samples of similar-sizedadults from both populationswere subjectedto simultaneous8-day acclimationtreatments over a rangeof salinities from 48-1 ppt. At acclimationsalinities from >5 to <35 ppt, animals from the northernGulf of Mexico hyperosmoregulatedat a lower plateau of blood osmolality than did those from the Atlantic coast. However, from midrange to the lowest acclimationsalinities, hyperosmoticregulation in the Gulf of Mexico population was maintainedwithout measurablereduction of blood osmotic concentration.A major com- ponent of the variationin blood osmolality appearsto be reflectedin differentialregulation of sodium at salinities < 10 ppt. We suggestthat divergencein osmoregulatorypattern of sibling populations is genetically based and is maintained by the absence of gene flow around the peninsula of Florida. This divergencemay reflectdifferential adaptation to habitatsthat vary in salinity structure,tidal effects, and temperature.

Coastal warm-temperatepopulations of Texas, in the Gulf of Mexico (Williams, decapods in the western Atlantic were long 1984). Collections by one of us (DLF) ex- thought to be comprised of widely distrib- tend the southern range of Gulf of Mexico uted species ranging from the Carolinian populations well into Tamaulipas, Mexico. Province of the Atlantic coast into the Priorto studies by Zimmermanand Feld- northern Gulf of Mexico (Felder, 1973; er (1991), work concerning ecology, devel- Powers, 1977; Williams, 1984). However, opment, and physiologyof this complex was recent studies of decapod and focusedalmost exclusively on Atlantic coast other marine invertebrates in this region populations (Crichton, 1960, 1974; Mul- have shown some species complexes to pos- stay, 1975; Foskett, 1977; Seiple, 1979, sess distinct genetic discontinuities around 1981; Seiple and Salmon, 1982, 1987; Sta- the Florida Peninsula (Bert, 1986; Saunders ton and Sulkin, 1991). However,recent work et al., 1986; Reeb and Avise, 1990). This in our laboratoryhas established that Gulf genetic differentiationis evident not only in of Mexico populations,while closely related morphology and ecology of decapod crus- to S. reticulatum,are distinct morphologi- taceans,but may be manifest also in unique cally (Felder and Zimmerman, in prepara- physiologicaladaptations of separatedpop- tion) and genetically (Felder and Staton, in ulations (Williams and Felder, 1986). preparation) from their Atlantic counter- While such physiological variation may parts. In the present paper,we compare the be subtle, it can be evaluated by carefully osmotic and ionic regulatorycapacities of controlledcomparative experimentation. In adult marsh from coastal Louisiana the present study we have undertakensuch in the northern Gulf of Mexico, hereafter a comparativeinvestigation of osmotic and referred to as Sesarma sp. (near reticula- ionic regulatorycapacities in what appear tum), with those of S. reticulatumfrom the to be disjunctsibling populationsof a coast- coast of Georgia on the Atlantic coast. al marsh , separated by the southern extremeof the FloridaPeninsula. Under the MATERIALSAND METHODS label Sesarma reticulatum(Say, 1817), this All specimensused in experimentalstudies were adult species complex has been reportedto range crabs collected during the first week of August 1991. from Massachusetts to Volusia were excavated by shovel at low tide from County, galleriesin vegetatedbanks of tidal streams.Immediate Florida, on the Atlantic coast and from Sar- pre- or postmolt animalsand any animalsinjured dur- asota County, Florida, to Calhoun County, ing collecting were discarded from the samples. All

335 336 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 12, NO. 3, 1992 specimens of Sesarma reticulatumwere taken from a kal and Rohlf, 1981: 226) was used to evaluate the salt marsh(Spartina and Juncus)subject to largetidal level of significancefor any differencebetween means. rangesat Brunswick,Georgia; salinity of adjacentwa- ters at the time of collection rangedfrom 15-18 ppt. All specimensof Sesarma sp. (near reticulatum)were RESULTS collected from brackish to fresh marsh (Sagittaria, Scirpus, and Vigna) subject to low tidal ranges near Both S. reticulatum from Georgia and S. CypermortPoint, Louisiana;salinity of adjacentwaters sp. (near reticulatum) from Louisiana sur- at the time of collection rangedfrom 3-8 ppt. Animals vived all acclimation treatments. Neither were transportedto the laboratoryin bucketsof damp mud and vegetation from the collection site. Simul- population demonstrateda lower or higher taneous acclimation of specimens from both popula- lethal limit at target acclimation salinities tions was begun within 48 h of collection. Specimens during the study. Overall, mortality during from both populations were acclimated side by side acclimation was less than 4% for the Lou- under completelyuniform conditions. isiana and less than 2% for the Initial acclimation attempts with animals of both population populationsproduced high first-daymortality among Georgia population during the final 8 days animals that were groupedtogether without cover in a of acclimation. This rate was substantially common container,usually from aggressive encounters lower than in the first day of early trials between individuals that resultedin crushedappend- when acclimationof animals was attempted ages and blood loss. Thereafter,in order to acclimate animals togetherbut without injury, each individual without use of isolation vials, and mortality was isolated in a perforatedplastic vial for the re- during a single day for animals from both mainingterm of acclimation.Acclimation waters were populations exceeded 12%. well aerated and could flow freely in and out of the Both S. reticulatum and S. sp. (near re- perforatedvials. Animals were maintainedon a 12:12 exhibit in a IV os- h light: darkcycle at a temperatureof 25 ? 0.5?C,and ticulatum) general type were not fed duringthe full course of acclimation.Di- moregulatory curve (sensu Vernberg and lutions of filterednatural sea water were used for all Vernberg,1972) typicalofgrapsid crabs that acclimation salinities from 25-1 ppt. Salinities >25 are strongeuryhaline osmoregulators (Man- ppt were obtained by dilution of a stock 50-ppt sea- tel and Farmer, 1983). Weak hypoosmotic water solution preparedby addition of artificial sea salts to filtered natural sea water. All dilutions were regulation is evident at salinities >25 ppt made with deionized, dechlorinatedwater. Salinities (Fig. 1), with no significantdifference in this of acclimation waters were approximatedwith a re- patternbetween the two populations;at ac- fractometer. climation salinities from 35-48 ppt, blood Animals were introducedinto sea water of 25-ppt osmotic concentration salinity and held there for 36-48 h before acclimation parallels changing transferswere begun. Groups of animals were there- acclimation salinity in both populations. after transferredstepwise in no more than 5-ppt in- However,at midrangesalinities (25-10 ppt), crementsor decrementsper day, until the targetaccli- animals from the Georgiapopulation main- mation salinity was reached. Target acclimation tained blood osmotic concentrationsslight- salinitieswere approximately 48, 35, 25, 15, 10, 5, and < 1 ppt. Crabswere maintainedat the targetacclimation ly but significantly(P 0.001) higher than salinity for at least 8 days before blood was sampled. did those from the Louisiana population. The maximum acclimationperiod (inclusiveof initial Overall, hyperosmotic regulation of the holdingat 25-ppt salinity, stepwisetransfers, and final blood between 25-1 ppt was maintainedon holding at the targetsalinity) was 16 days. Each spec- a stable in both imen was acclimatedto only one targetsalinity in the relatively plateau popula- courseof the experimentand was discardedafter blood tions, though blood osmotic concentration was sampled. decreasedto slightly but significantly(P < Blood was obtained by puncture of the arthrodial 0.01) lower values at the lowest acclimation membranejust proximal to the coxa of the fourth or salinities in the Georgiapopulation. No sig- fifth pereiopod.Replicate 10-,ulsamples of blood and acclimationmedia weretaken for determinationof os- nificant reduction in blood osmolality oc- molalityon a Wescorvapor pressure osmometer (Mod- curredin the Louisianapopulation from 15 el 5100B). When possible, a third 10-ul whole-blood ppt to the lowest acclimation salinity of 1 sample was obtainedand immediatelydiluted for ion ppt. Rather, a very slight but significant(P analysis. Replicate 10-,tl samples of media were also < increase in blood oc- taken and diluted for ion analyses. Chlorideion con- 0.05) osmolality centrationwas determinedwith an Aminco chloride curredwith the decrease of acclimation sa- titrator,and sodium ion concentrationwas determined linity from 15-5 ppt; a similar pattern of with a Coleman (Model 21) flame photometer. significant(P < 0.02) increasein mean blood Replicatedeterminations of any blood sample were ion values also occurredover this reduction averagedto yield a single value. Valueswere thereafter used in calculationof means and 95% confidencein- of acclimation salinity (Figs. 2, 3). tervals (CI) for sets of animals at each acclimation Regulationpatterns for both sodium (Fig. salinity.An unpairedt-test for small sample sizes (So- 2) and chloride (Fig. 3) were very similar to STATON AND FELDER: OSMOREGULATION IN SESARMA 337

1600 700

1400 600 "0 1200 0 50 00 "O 0 1000 0 E 400 m8 800 c)E 300 0 600 E -- 200 400 A Georgia c * Louisiana: A Georgia * Louisiana 200 100

0 0 200 400 600 800 1000 1200 1400 1600 0 100 200 300 400 500 600 700 mOsm, Media mEq/literSodium, Media Fig. 1. Blood osmotic concentration in acclimated Fig. 2. Blood sodium concentration in acclimated specimens of Sesarma reticulatumfrom Georgia (tri- specimens of Sesarma reticulatumfrom Georgia (tri- angles)and Sesarma sp. (near reticulatum)from Lou- angles)and Sesarma sp. (near reticulatum)from Lou- isiana (circles)as a functionof varied osmotic concen- isiana (circles)as a function of varied sodium concen- tration in acclimation media. Each point is mean for tration in acclimation media. Each point is mean for 4-8 animals acclimated at 25 + 0.5?C;vertical bars 3-8 animals acclimated at 25 + 0.5?C;vertical bars indicate 95% CI. Isosmotic line is shown as diagonal. indicate 95% CI. Isoionic line is shown as diagonal. Media osmolalities from lowest to highest correspond Media sodium concentrationsfrom lowest to highest to acclimationsalinities of 1, 5, 10, 15, 25, 35, and 48 correspondto acclimationsalinities of 1, 5, 10, 15, 25, ppt. 35, and 48 ppt. the pattern for overall osmotic concentra- ionic regulatorypatterns reported here for tion. In both cases, blood ion levels at mid- both populations agree in generalwith pat- rangeacclimation salinities in the Louisiana terns of osmotic and ionic regulation re- population were slightlybut significantly(P ported in previous studies of S. reticulatum < 0.01) below those in animals from the by Foskett (1977) and in some studies of Georgiapopulation. However, at the lowest other grapsid crabs (Gross, 1964; Barnes, acclimation salinities (5 and 1 ppt) values 1967; Mantel and Farmer, 1983), the direct for blood sodium and chloride ion concen- comparison of closely related Gulf and At- trationsin animals from the Louisianapop- lantic populations establishes small but ulation recovered to values that approxi- consistent differences in magnitude of re- mated those for animals from the Georgia sponse when animals are acclimated to a population. In the population from Geor- range of estuarine salinities. Our measures gia, concentrations of blood chloride and of osmotic and ionic regulation demon- sodium were both slightly but significantly strate ability of individuals from the Gulf (P < 0.02) lower at the minimum accli- of Mexico to regulateblood homeostatical- mation salinity (1 ppt) than at an acclima- ly, but at a lower plateau than those of the tion salinity of 25 ppt. In animals from the Atlantic. Such differences in osmoregula- Louisiana population, there was no signif- tion are often found with species-level com- icant differencein blood ion concentrations parisonsof physiology(Mantel and Farmer, between animals acclimated to 25 ppt and 1983) and are usually attributed either to those acclimated to 1 ppt. localized environmentalconditioning (phe- notypic compensation) of genetically ho- DISCUSSION mogeneous populations or to genetic diver- The results of this study suggest a subtle gence (adaptation) of isolated populations physiologicaldivergence, measurable in os- (see Vernberg, 1962). moregulatoryability, between Gulf of Mex- Effectsof local, long-term environmental ico and Atlantic populationsof the Sesarma conditioning are difficultto document with reticulatumcomplex. While the osmotic and certainty as such in geographicallyseparat- 338 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 12, NO. 3, 1992 ed populations, especially in the absence of 900 - genetic analyses. In at least some cases, vari- ant physiological responses attributed to . 750- long-term conditioning across latitudinal 0 gradients in temperature or other parame- 600/ / ters may just as readily correlate with mea- 0 / / sures of genetic divergence, once genetic or other systematic data become available. In 0 450 many cases, divergent physiological re- sponses have been portrayed as condition- 300 ing effects without either genetic analyses or ? even rigorous morphological comparison of LU ./ E 150 - / A Georgia the specimen populations under study. 4~/ ~* Louisiana However, this is not to say that climate, nutrition, ontogeny, and other conditioning 750 900 of osmoregulatory response do not occur 0 150 300 450 600 among coastal decapods, and it is probably mEq/literChloride, Media some synergism of these that leads to sea- Fig. 3. Blood chloride concentration in acclimated sonal variation of osmoregulatory capacity specimens of Sesarma reticulatumfrom Georgia(tri- in coastal populations such as grapsid crabs angles)and Sesarma sp. (near reticulatum)from Lou- (Dehnel, 1962), portunid crabs (Mantel, isiana (circles)as a functionof variedchloride concen- Ballard and and cal- tration in acclimationmedia. Each point is mean for 1967; Abbott, 1969), 3-8 animals acclimated at 25 + 0.5?C;vertical bars lianassid mud shrimp (Felder, 1978). To indicate 95% CI. Isoionic line is shown as diagonal. control as much as possible for such effects Media chlorideconcentrations from lowest to highest in the present study, population samples correspondto acclimationsalinities of 1, 5, 10, 15, 25, from Louisiana in the Gulf of Mexico and 35, and 48 ppt. Georgia on the Atlantic coast were taken from within 1? of the same latitude (30- 31 N). As further control, both samples were merman and Felder, 1991); most of these taken at the same time of year, both were populations experience annual mean salin- restricted to the same maturational stages, ities of about 7 ppt. Thus, populations in both were held in the laboratory for the same the Gulf of Mexico appear to center in hab- period of time, and both were acclimated itats of lower mean salinity than those of under identical experimental conditions at the Atlantic, and, given the lower tidal am- the same time. plitude throughout most of the range in the In the case of the S. reticulatum complex, Gulf of Mexico, these populations are less some disparity in the typical habitat be- likely to experience periodic inundation by tween the Atlantic and Gulf of Mexico pop- higher salinity waters carried into upper es- ulations may serve to account for diver- tuaries with high tides. gence in osmoregulatory ability observed in There are also disparities in overall hab- the present study, regardless of whether it itat temperature ranges and means between is environmentally conditioned or geneti- the Atlantic and Gulf of Mexico popula- cally based. The Georgia population, typical tions, and long-term conditioning to these of the Atlantic coast populations in general, differences may influence osmoregulatory is subject to a much higher tidal amplitude patterns beyond the habitat and climate ef- and at least periodically higher salinities than fects that can be controlled for simply by are most populations in the Gulf of Mexico. selecting test samples from similar latitudes Sesarma reticulatum on the Atlantic coast (see above). Assuming that populations on is reported to range from salinities <2 to the Atlantic coast represent one genetically >35 ppt, but it appears to prefer a mean similar clade and that populations in the salinity of about 16 ppt (Seiple, 1979). How- Gulf of Mexico represent another, it is ap- ever, S. sp. (near reticulatum) ranges from parent that mean temperatures of the At- salinities of <1 ppt to (occasionally) >40 lantic range (Massachusetts to Volusia ppt in the Gulf of Mexico, and the largest County, Florida) are presently, and have populations are found from 1-15 ppt (Zim- been historically, lower than within the Gulf STATON AND FELDER: OSMOREGULATION IN SESARMA 339 of Mexico range (SarasotaCounty, Florida, tremes by limiting incursions into low sa- to Tamaulipas,Mexico). Althoughthere are linity waters as might persist during low certain exceptions to the general pattern, tides. Even under some conditions where higher temperaturesappear to both reduce high tides may increaselevels of low salinity optimum blood osmotic concentrationsand waters along inhabited banks of tidal increase the range of tolerance to lowered streams, the tidal range is often so low as salinity in brackishwater animals. This ob- to leave extensive areas of the burrow gal- servationhas been interpretedpreviously as leries unflooded. Thus, the semiterrestrial a physiological reason for adaptation to habits of these animals could serve as a brackishwater being favored in warm water means of avoiding direct and prolongedex- environments (see review by Vernberg, posureto extremelylow salinity under most 1962). The influence of higher temperature circumstances.However, under forced con- on sodium ion transport may also assist tinual exposure to extreme low salinity, as some species in the invasion of more dilute imposed in our acclimationprotocols, com- media (Lockwood, 1960). pensational sodium transport ability may A somewhatdifferent pattern of blood so- be triggered, accounting for the observed dium ion regulation is seen in the Gulf of "recovery"and restabilizationin blood os- Mexico populationfrom that in the Atlantic molality at salinities from 5-1 ppt. In con- population, and this may reflect some fun- trast, S. reticulatumon the Atlantic coast damental differencein response to extreme appearsto saturatesodium transportability low salinities in these two groups. In the at a highersalinity, such as may be expected Louisiana population, a measured increase in a population centered in higher salinity in sodium ion levels at the 5-ppt acclima- habitatsand in localities where moderateto tion salinity cannot be attributed casually high tidal rangesperiodically bathe even up- to artifact, since it is also detected in the per estuarieswith more saline waters. Con- independent determination of total osmo- firmationof the role of sodium transportin lality and appears to be reflected in the in- the divergentpatterns observed will require dependent measure of blood chloride ion. more direct measurementof transportrates Rather, the transition from an acclimation and investigation of possible variations in salinity of 10 ppt to 5 ppt may be coincident factors such as urine production and per- with greatersaturation of the sodium active meability. Acclimation over periods longer transportprocess. This could at least in part than used in the present study may also be account for "recovery" of blood osmotic requiredin order to determine whether the concentrationat low salinities in S. sp. (near two populations maintain divergent pat- reticulatum)to equivalency with that of S. terns of sodium regulation over the long reticulatum.By contrast, saturation of so- term. dium transport could have occurred at a In one of the very few previous compar- higher salinity for S. reticulatum which isons of osmoregulation between Gulf of showed no similar recoverypoint but rather Mexico and Atlantic coast decapod crus- exhibited a tendency for slight attenuation tacean populations, Sharp and Neff (1980) of blood osmotic concentration to lower noted that populations of Clibanarius vit- values as salinity decreased. tatus from coastal Texas were not different A selective advantage of such disparate in osmotic and ionic regulatoryability from patternscould relate to differingsalinity re- those in South Carolina (Young, 1979). gimes of the two populations. In the lower However, despite the wide separationof the salinity habitats of the Gulf of Mexico, S. populations sampled, there is no evidence sp. (nearreticulatum) may achieve some en- to date that those populations are geneti- ergetic advantageby functioning at a lower cally divergent, and the species appears to plateau of blood osmotic concentration;for range fully around the peninsula of Florida example, reserve capacityfor sodium trans- (DLF, unpublished records). The salinity port appearsto be activated only at the low- structureof the typical habitat in C. vittatus est salinity extremes of the typical habitat. does not appear to vary appreciably be- In habitats where salinity may average 3-4 tween the populations evaluated. Rather, ppt, these animals could behaviorallyavoid more typicalof those shallowsubtidal warm- long-duration exposure to low salinity ex- temperate taxa that are not disjunct across 340 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 12, NO. 3, 1992 the Florida Peninsula, C. vittatus exhibits D. L. Felderfor components of LouisianaNSF/EPSCoR some degree of "brackish water submer- Programgrant R 11-8820219 and LouisianaEducation Remane QualitySupport Fund grants86-LUM(2)-084-09 and gence" (sensu and Schlieper, 1958) (1987-1988)-ENH-BS-10. This is contribution No. over its range. That is to say, C. vittatus 30 of the USL Centerfor CrustaceanResearch. occupies extensively the upper littoral en- vironmentsand marshesof coastal estuaries LITERATURECITED on the Atlantic coast, while it is more re- stricted to saltier of Ballard,B. S., and W. Abbott. 1969. Osmotic accom- margins marshes, bay modation in Callinectessapidus Rathbun.-Com- shores, and sublittoralwaters in the north- parativeBiochemistry and Physiology29: 671-687. ern Gulf of Mexico, where lower salinity Barnes, R. S. K. 1967. The osmotic behavior of a waters bathe expansive marshes of upper number of grapsoidcrabs with respect to their dif- estuaries. contrast, brackish water sub- ferentialpenetration of an estuarinesystem. -Jour- By nal of ExperimentalBiology 47: 535-551. mergencewould not be expected in obligate Bert,T. M. 1986. Speciationin westernAtlantic stone intertidal animals such as the Sesarma re- crabs (genus Menippe):the role of geological pro- ticulatumcomplex; rather,a geneticallydis- cesses and climatic events in the formationand dis- tinct counterpart of S. reticulatum (sensu tributionof species.-Marine Biology 93: 157-170. to have become to Crichton,O. W. 1960. Marshcrab, intertidal tunnel- stricto) appears adapted maker and grass eater.-Estuarine Bulletin of the the expansive, low-salinity marshlands of University of Delaware 5: 3-10. the northernGulf of Mexico, perhapsin re- 1974. Caloric studies of Spartina and the sponse to a suite of environmental factors. marsh crab Sesarma reticulatum(Say).--In: C. F. Powers and Place delineated two Diaber, ed., Tidal marshes of Delaware, pp. 142- (1978) 144 (a component chapter In: H. T. Odum, B. J. potential modes of such genetic change in Copeland,and E. A. McMahan,eds., Coastal eco- populations: the "ongoing" model that is logical systems of the United States.Vol. 2, pp. 99- based on adaptation and nonrandom mi- 149). The CoastalConservation Foundation, Wash- grational processes, and the "historical" ington, D.C. model that isola- Dehnel, P. A. 1962. Aspects of osmoregulationin requiresprevious genetic two species of intertidalcrabs. -Biological Bulletin tion to producedifferentiation. Although our 122: 208-227. data do not allow us to argueexclusively for Felder,D. L. 1973. Annotatedkey to crabsand lob- either pattern of differentiation,limitation sters (Decapoda, Reptantia)from coastal waters of of flow in the S. reticulatum the northwesternGulf of Mexico.-Center for Wet- gene complex landResources, Louisiana State University, Sea Grant (as suggested by allozyme differences;our PublicationLSU-SG-73-02: 1-103. unpublisheddata) could allow for allopatric 1978. Osmoticand ionic regulationin several divergence in physiological as well as allo- western Atlantic Callianassidae(Crustacea, Decap- zymic composition of these populations. oda, Thalassinidea).-Biological Bulletin 154: 409- Even the Florida Peninsula serves 429. though Foskett, J. K. 1977. Osmoregulationin the larvae as a well-documentedbiogeographic and ge- and adults of the grapsidcrab Sesarma reticulatum netic boundarybetween coastal Atlantic and Say.-Biological Bulletin 153: 505-526. Gulf of Mexico fauna, the ultimate cause of Gross,W.J. 1964. Trendsin waterand salt regulation differentiationin these faunal among aquatic and amphibious crabs.-Biological regions may Bulletin 127: 447-466. be complex. North American glaciations, Lockwood,A. P. M. 1960. Some effects of temper- pluvial runoff,and expansion of deltaic wet- atureand concentrationof the medium on the ionic lands during the Quaternaryall served to regulation of the isopod Asellus aquaticus (L.).- develop the moder physiographiccharac- Journalof ExperimentalBiology 37: 614-630. ter of estuaries and wetlands in the Gulf of Mantel, L. H. 1967. Asymmetrypotentials, metab- olism and sodium fluxes in gills of the blue crab, Mexico. The moder coastal fauna of the Callinectessapidus. -Comparative Biochemistryand northern Gulf of Mexico, of which S. sp. Physiology 20: 743-753. (near reticulatum) is a part, may demon- ,and L. L. Farmer. 1983. Osmotic and ionic strate a to this regulation.-In: L. H. 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