Evolution, 48(6), 1994, pp. 1986-2001 A SPECIATIONAL HISTORY OF "LIVING FOSSILS": MOLECULAR EVOLUTIONARY PATTERNS IN HORSESHOE CRABS JOHN C. AVISE', WILLIAM S. NELSON', AND HIROAKI SUGITA2 'Department of Genetics, University of Georgia, Athens, Georgia, 30602 2Institute of BiologicalSciences, University of Tsukuba, Ibaraki 305, Japan address correspondence and reprint requests to John C. Avise Abstract.-Horseshoe crabs' exceptional morphological conservatism over the past 150 My has led to their reputation as "living fossils," but also has served to obscure phylogeneticrelationships within the complex. Here we employ nucleotide sequences from two mitochondrial genes to assess molecular evolutionary rates and patterns among all extant horseshoe crab species. The American species Limulus polyphemus proved to be the sister taxon to a clade composed of the Asiatic species Tachypleus gigas, T. tridentatus, and Carcinoscorpius rotundicauda, whose relationships inter se were not resolved definitively. Both absolute and relative rate tests suggest a moderate slowdown in sequence evolution in horseshoe crabs. Nonetheless, dates of the lineage separations remain uncertain primarily because of reservations about molecular-clock calibrations resulting from large rate variances at examined loci across Arthropods and other animal lineages,as inferred in this and prior studies. Thus, ironically, separation dates as estimated by molecular evidence in general may remain most insecure in taxonomic groups for which such information is needed most-those lacking strong biogeographic or fossil benchmarks for internal-clock calibrations. In any event, the current results show that large numbers of molecular characters distinguish even these most morphologically conservative of organisms. Furthermore, comparisons against previ­ ously published mitochondrial sequence data in the morphologically dynamic hermit crab-king crab complex demonstrates that striking heterogeneity in levels of morphotypic differentiation can characterize Arthropod lineages at similar magnitudes of molecular divergence. Key words.-Mitochondrial DNA, molecular clocks, phylogeny. Received September 14, 1993. Accepted March 30, 1994. Evolutionary stasis, the near absence of de­ crabs (Arthropoda; class Merostomata; subclass tectable evolutionary change in some lineages Xiphosura) have been considered the "arche­ over long periods of geological time, has been types of bradytely [extremely slow evolution]" perceived as one of the central challenges for and "a classic example of arrested evolution" modem evolutionary theory (Gould and Eld­ (Fisher 1984). The fossil record indicates that redge 1977; Williams 1992). In some cases, stasis xiphosurans arose and radiated in the early to with regard to morphological appearance also middle Paleozoic, and that by the mid-Mesozoic may extend across speciation events, as in the some taxa had attained a morphological ap­ origin of sibling species. In such lineages and pearance strikingly similar to that ofpresent-day clades, what processes confine long-term mor­ species (Stermer 1955). Thus, with regard to ex­ phological differentiation within boundaries that ternal morphology, these xiphosuran lineages are far narrower than they theoretically could be, have remained remarkably unchanged over 150 given the rapid rates of short-term phenotypic My or more. As a result, the extant horseshoe change commonly observed in most species un­ crabs often are discussed as paradigm examples derartificial ornatural selection? (Williams 1992). of "living fossils" (Eldredge and Stanley 1984) Potential explanations range from molecular(e.g., or "phylogenetic relics" (Selander et al. 1970). low mutation rate; paucity of additive genetic At the molecular level, however, living horse­ variation), to ontogenetic (e.g., developmental shoe crabs appear to be unexceptional with re­ constraint or coherence producing resistance to gard to intraspecific genetic variation and pat­ selection), to ecological (e.g., long-term stability terns ofpopulation differentiation. Thus, in Li­ in environmental selection pressures). Here we mulus polyphemus, the level ofallozyme hetero­ examine patterns of molecular evolution in an zygosity (H = 0.057) proved similar to mean evolutionary clade renowned for long-term stasis estimates for many other animals (Selander et with regard to external morphology. al. 1970); and, levels and patterns ofintraspecific Because of a reputation for extreme conser­ differentiation in mitochondrial DNA (mtDNA) vatism in morphotypic evolution, horseshoe were similar to those of several other inverte- 1986 © 1994 The Society for the Study of Evolution. All rights reserved. PHYLOGENY OF LIVING FOSSILS 1987 brate and vertebrate species inhabiting the same Mitochondrial DNA was isolated from the coastal range in the southeastern United States fresh gill and muscle tissues ofL. polyphemus by (Saunders et al. 1986; Avise 1992). This "nor­ CsCI gradient centrifugation. For the other spe­ malcy" ofgenetic variability within and among cies, phenolic extractions ofgenomic DNA were populations ofL. polyphemus suggests that con­ employed. Mitochondrial sequences were am­ sensus patterns ofmolecular versus morphotypic plified via the polymerase chain reaction (Innis evolution in horseshoe crabs may be conspicu­ et al. 1990; Saiki et al. 1988), using primer pairs ously decoupled (but see Riska [1981] and Shus­ 16sar-L and 16sbr-H for the l6S rRNA gene, ter [1982], who documented variability and geo­ and COlf-L and COla-H for the cytochrome graphic differentiation in the morphological traits oxidase I gene (Palumbi et al. 1991). These pro­ ofL. polyphemus as well). duced fragments ofabout 525 bp and 650 bp in Actually, four extant species ofhorseshoe crabs length, respectively. Fragments were checked for are recognized: L. polyphemus in eastern North correct size on 1% agarose gels and then sepa­ America, and Carcinoscorpius rotundicauda, rated from excess primers and dNTPs with use Tachypleus tridentatus, and T. gigas in Southeast of the Magic PCR Preps system from Promega. Asia. Despite their placement in three genera, to Direct sequencing of heat-denatured, double­ the untrained eye, the differences in morphology stranded amplification products was performed are subtle indeed, consisting ofsuch distinctions either in our laboratory by dideoxy chain ter­ as whether the cross-section of the telson (tail) mination using T7 DNA polymerase and 35S ra­ is subtriangular (C rotundicauda) versus trian­ dioactive labeling (Sanger et al. 1977), or by flu­ gular (other species), and whether the number of orescent-dye sequencing conducted by the Mo­ immovable spines on the midposterior margin lecular Genetics Instrumentation Facility at the of the opisthosomatic carapace is three (T. tri­ University of Georgia. Correctness of DNA se­ dentatus) versus one (other species). As noted by quences was checked by several procedures: first, Selander et al. (1970), generic separation among various portions ofthe l6S rRNA sequence were various living and extinct horseshoe crabs "is a assayed both in our laboratory and in the DNA reflection of the morphological differences be­ sequencing facility and results compared; sec­ tween them, relative to the collection ofall forms ond, both heavy and light strands were se­ in the Xiphosura....since taxonomic lines tend quenced from each individual; third, two indi­ to be drawn relative to the range of variation viduals (A and B) from each species ofhorseshoe specific to the taxon underconsideration." In any crab were sequenced independently. event, one consequence of the general morpho­ The l6S rRNA gene sequences were aligned logical conservatism ofhorseshoe crabs over the using the computer program GeneWorks 2.1.1 past 150 My is that considerable uncertainty ex­ (Intelligenetics), with assigned penalties of 10 and ists overthe evolutionary histories ofthe lineages 4 for opening and extending a gap, respectively. leading to the extant forms. This irresolution of Cytochrome oxidase alignments were unambig­ phylogeny as a result of extreme morphological uous and done by eye. Estimates of genetic di­ stasis, and questions concerning the magnitude vergence were calculated as direct counts ofnu­ ofmolecular differentiation in a clade renowned cleotide sequence differences, and by the "two­ for slow morphological evolution, prompted the parameter" method of correction for multiple current assessment ofmolecular-level evolution­ substitutions at a site (Kimura 1980). Distance ary patterns among all extant horseshoe crab spe­ matrices were clustered by the unweighted pair­ cies. group method with arithmetic means (UPGMA; Sneath and Sokal 1973) and by the neighbor­ MATERIALS AND METHODS joining procedure (Saitou and Nei 1987). Se­ Specimens of Tachypleus gigas and Carcinos­ quences also were analyzed by maximum par­ corpius rotundicauda were collected in the vicin­ simony methods as applied to information coded ity of Bangsaen (Gulf of Siam), Thailand. Spec­ as: (1) nucleotide sequences themselves; (2) pu­ imens of T. tridentatus came from the Bay of rines versus pyrimidines (such that only trans­ Hakata, Fukuoka, Japan. As elaborated below, versions were considered); and (3) for the cyto­ the two samples of L. polyphemus came from chrome oxidase locus, first and second positions genetically distinctive populations along the At­ ofcodons only (such that silent substitutions at lantic and GulfofMexico coastlines in the south­ third positions were disregarded).