Marine Biology (2002) 140: 1059–1066 DOI 10.1007/s00227-002-0780-2 A.M. Young Æ C. Torres Æ J.E. Mack C.W. Cunningham Morphological and genetic evidence for vicariance and refugium in Atlantic and Gulf of Mexico populations of the hermit crab Pagurus longicarpus Received: 23 February 2001 / Accepted: 14 December 2001 / Published online: 2 March 2002 Ó Springer-Verlag 2002 Abstract The number and wide variety of southeastern may represent survivors from a northern refugium dur- United States marine taxa with significant differentiation ing the last glacial maximum. between Gulf of Mexico and Atlantic Ocean populations suggests that these taxa may have experienced major vicariance events, whereby populations were subdivided by geological or ecological barriers. The present study Introduction compared variation in morphology, allozymes, and mtDNA in Gulf of Mexico and western Atlantic popu- A wide variety of marine fauna found in the southeast- lations of the longwrist hermit crab Pagurus longicarpus ern United States, from hydroids to sturgeon, have been Say collected during 1997 and 1998. Combined Atlantic the subject of population genetics studies in the past two populations had significantly fewer denticles on the decades (see reviews by Avise 1992; Cunningham and second segment of the third maxilliped than did Gulf of Collins 1994, 1998; Felder and Staton 1994; and addi- Mexico populations, and the mean ratio of dactyl length tional papers by McCommas 1982; Hoagland 1984; to propodus length was significantly greater in the Cunningham et al. 1991; Staton and Felder 1995; Atlantic crabs than in the Gulf of Mexico crabs. Allo- Foighil et al. 1996; Mangum and McKenney 1996; Tam zyme allele frequencies at three loci showed genetic dif- et al. 1996). Many studies have found significant geo- ferentiation between a Gulf of Mexico population and graphic differentiation between populations in the Gulf two Atlantic populations. Analysis of mtDNA sequence of Mexico and along the Atlantic coast, with a hybrid data revealed a clear reciprocal monophyly between zone in the Cape Canaveral area. Most surprising was Gulf and Atlantic populations, with an estimated the discovery of significant genetic differentiation in taxa divergence age of 0.6 million years ago. This estimated that are continuously distributed around the Florida age of divergence is significantly more recent than an age peninsula, and which showed little or no morphological previously estimated for its congener Pagurus pollicaris variation (e.g. the American oyster, Crassostrea virgini- ( 4 million years ago), suggesting that species with a ca). Perhaps less surprising has been the discovery of similar genetic break between Gulf and Atlantic popu- significant genetic differentiation in taxa that are found lations may not necessarily share an identical history. in the Gulf and the Atlantic, but are disjunct in the Surprisingly, there is evidence of geographic subdivision tropical waters off southern Florida. These disjunct taxa within Atlantic populations of P. longicarpus along the are so numerous that they have been designated the east coast of North America. This differentiation is Carolinian biogeographic fauna (Briggs 1974; Cunn- especially strong between Nova Scotia and southern ingham and Collins 1998). populations, suggesting that the Nova Scotia population The discovery of so many taxa with significant geo- graphic differentiation between the Gulf and the Atlantic suggests that these taxa may have experienced Communicated by J.P. Grassle, New Brunswick major vicariance events, whereby populations were A.M. Young (&) Æ J.E. Mack subdivided by geological or ecological barriers. For Biology Department, Salem State College, example, Reeb and Avise (1990) suggested that lowering Salem, MA 01970, USA of the sea level and concurrent changes in climate during E-mail: [email protected] glacial maxima resulted in a lack of estuarine habitat C. Torres Æ C.W. Cunningham along the southern portion of the Florida peninsula. Department of Biology, Duke University, The longwrist hermit crab, Pagurus longicarpus, has Durham, NC 27708, USA been collected in the western Atlantic from Mahone Bay 1060 on the east coast of Nova Scotia (Young, personal ob- parisons – nuclear (allozyme) and mitochondrial servation) to Hutchinson Island on the east coast of (cytochrome oxidase I mtDNA sequence, COI) data Florida (Camp et al. 1977) and in the Gulf of Mexico were collected from several Gulf of Mexico and Atlantic from the Shark River in southwestern Florida (Rouse populations of P. longicarpus. 1970) to Galveston Bay, Texas (Fotheringham 1976). That is, P. longicarpus has a disjunct distribution in the western Atlantic and the Gulf of Mexico (see Fig. 1). Materials and methods Unlike many other taxa with a similar disjunct distri- bution, Gulf of Mexico and Atlantic populations Sample collection and storage of P. longicarpus exhibit morphological differences Fifty live Pagurus longicarpus Say were obtained between April and (Provenzano 1959). Together with its disjunct distribu- September during 1997 and 1998 from each of ten locations tion around southern Florida, these morphological dif- (identifying letter in parentheses): near Meisner’s Island in Mahone ferences are consistent with distinct evolutionary Bay, Nova Scotia (NS); Damariscotta River, Maine (M); Salem histories for Gulf of Mexico and Atlantic populations. Harbor, Salem, Massachusetts (S); Vineyard Sound, Woods Hole, The only previous study comparing Gulf of Mexico Massachusetts (W) (Marine Biological Laboratory Supply Center); off Beaufort, North Carolina (NC); North Inlet, South Carolina and Atlantic P. longicarpus found very little genetic (SC); New College Bayfront in Sarasota Bay, Florida (F); the east variation. In a study using 16 S mtDNA, Cunningham end of Shell Point, Apalachee Bay, Panacea, Florida (P) (Gulf et al. (1992) found only a single base pair (bp) substi- Specimen Marine Supply); and East Beach, Red Fish Cove in West tution distinguishing Gulf of Mexico and Atlantic indi- Galveston Bay, Texas (T) (see Fig. 1). All specimens other than those from Woods Hole and Apalachee Bay (purchased from viduals (0.2% divergence). This contrasts with eight commercial suppliers) were collected by the authors or colleagues substitutions (2% divergence) in the same gene distin- as listed in the ‘‘Acknowledgements’’. The Mahone Bay population guishing Gulf of Mexico and Atlantic individuals of the represents an extension of the northern limit to the species’ Atlantic hermit crab Pagurus pollicaris, which is also disjunct range, previously reported as Chignecto Bay between New Bruns- wick and Nova Scotia (Williams 1984). around southern Florida (Cunningham et al. 1992). This To place this study in a larger context, four other species were large difference in genetic divergence is consistent with a collected for the purposes of mtDNA analysis. These included hypothesis that these two hermit crab species have samples from Woods Hole, Massachusetts, and Apalachee Bay, experienced vicariance events at different times. Cases in Florida, of Pagurus pollicaris, whose distribution is similar to that which taxa have identical disjunctions, but have expe- of P. longicarpus, as well as three northern species belonging to the bernhardus group of hermit crabs (McLaughlin 1974), which par- rienced vicariance at different times have been called ticipated in the trans-Arctic interchange ca. 3.5 million years ago examples of ‘‘pseudocongruence’’ (Page 1990; Cunn- (Vermeij 1991): Pagurus armatus (Bering Sea), Pagurus acadianus ingham and Collins 1994). (Damariscotta River, Maine), and Pagurus bernhardus (Roscoff, One possibility is that 16 S mtDNA is simply evolv- France). Live crabs were removed from their shells, and approximately ing too slowly to detect the divergence between Gulf of 100–200 ll of crab abdomen tissue was preserved in 1 ml 95% Mexico and Atlantic populations of P. longicarpus, but ethanol for examination of DNA signatures. Right chelipeds and one cannot rule out the possibility that, despite the third maxillipeds were removed and preserved in a mixture of 70% disjunction across southern Florida, there has been ethanol plus glycerol for 2 weeks, transferred to 90% ethanol for 1 week, and then maintained in 100% ethanol for examination by recent gene flow between the Gulf of Mexico and the scanning electron microscopy; two walking legs from each crab Atlantic. In order to distinguish between these possibil- were preserved in 70% ethanol for length and width measurements. ities – and in addition to detailed morphological com- The remainder of each crab was homogenized in 1 ml grinding Fig. 1. Pagurus longicarpus. Distribution (shaded regions) and locations of populations used in the study (NS Mahone Bay, Nova Scotia; M Damaris- cotta River, Maine; S Salem Harbor, Massachusetts; W Woods Hole, Massachusetts; NC Beaufort, North Carolina; SC North Inlet, South Caroli- na; F Sarasota Bay, Florida; P Apalachee Bay, Florida; T Galveston Bay, Texas) 1061 buffer (1.21 g Tris, 0.030 g EDTA, 0.040 g NADP, 0.033 g NAD Perkin-Elmer 9600 thermocycler, in 10 l reactions containing 5 ll per 100 ml distilled water) and centrifuged for 1–2 min. Individual DNA, 1 mM primer, 3 ll Big Dye buffer and 1 ll Big Dye ter- 50 ll aliquots of supernatant were frozen in 1.5 ml Eppendorf minator (Perkin-Elmer), with a profile of 25 cycles of 96°C (10 s)/ tubes at –80°C for allozyme electrophoresis. 50°C (5 s)/60°C (240 s). Unincorporated di-deoxynucleotides were removed