MOLECULAR PHYLOGENETICS AND EVOLUTION Vol. 5, No. 3, June, pp. 495±510, 1996 ARTICLE NO. 0045 Phylogenetics and Evolution of the Daphnia longispina Group (Crustacea) Based on 12S rDNA Sequence and Allozyme Variation DEREK J. TAYLOR,1 PAUL D. N. HEBERT, AND JOHN K. COLBOURNE Department of Zoology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received January 27, 1995; revised August 28, 1995. ography, and evolution. Historical evidence is needed, Although members of the crustacean genus Daphnia for example, to evaluate the proposed mechanisms of have been the target of much research, there is little cladoceran speciation. The much-studied geographic understanding of the group's evolutionary history. We patterns of morphological diversity in cladocerans also addressed this gap by inferring a phylogeny for one of have little meaning without historical or genetic knowl- the major species groups (longispina) using nucleotide edgeÐmorphological similarity of taxa (among conti- sequence variation of a 525-bp segment of the mito- nents or habitat types) may be due to either conver- chondrial 12S rDNA and allozyme variation at 21 loci. gence, cosmopolitanism, introgression, or shared We identi®ed the major lineages and their relation- ancestry. ships, assessed the phylogenetic utility of the few mor- Where extensive biogeographic information about phological characters in the group, and examined morphological and genetic variation is available, the Daphnia phylogeography. Nuclear and mtDNA phy- long-standing concept of cosmopolitanism has been re- logenies were generally concordant in recognizing the same four species complexes. An exception was the po- jected in favor of endemism and vicariant speciation sition of Daphnia galeata mendotae. The allozyme tree (Frey, 1987; Glagolev and Alonso, 1990; Sergeev, 1990; paired this species with the Daphnia rosea lineage, Hebert and Wilson, 1994). The generality of endemism whereas the mtDNA trees grouped D. g. mendotae with in the Cladocera, however, is unclear as the best known Daphnia galeata galeata. This discordance was con- groups (chydorids, Ctenodaphnia, and Daphniopsis) sistent with the reticulate evolution of nuclear genes may be unrepresentative with respect to vagility. There supporting the hypothesis that D. g. mendotae repre- is an oft overlooked tendency of these groups either to sents a case of homoploid hybrid speciation. Striking af®x or to have their propagules lodged in the substrate morphological stasis in the longispina group was evi- of the waterbody in which they were formed (Frey, denced by its very limited morphological divergence 1987; Sergeev, 1990; Fryer, 1991). This impediment to over an estimated 100 MY, and by the unusual transi- dispersal is certainly not universal in cladocerans. For tional saturation of the conservative 12S rRNA gene example, members of the subgenus Daphnia often pos- within a species group. Phylogenetic inference also sess buoyant resting eggs, which carpet a waterbody provided evidence that similarities in cephalic crest surface increasing exposure to dispersal vectors (e.g., shape likely resulted from convergent or parallel evo- Forbes, 1893; Hobñk and Wolf, 1991). lution among species. Endemism at the continental The genus Daphnia has become a model taxon for level was indicated for previously cosmopolitan spe- studies of experimental ecology, life history evolution, cies, but the estimated times of these divisions were toxicology, and evolution of breeding systems (Peters inconsistent with vicariance events suggesting recent and De Bernardi, 1987), but the genus has remained dispersal among continents. A signi®cant role for di- recalcitrant to the tools of the systematist. The group vergent selection in new habitats during speciation was known to science as early as 1669 when Swammer- was suggested by the neighboringly sympatric distri- dam mistakenly classi®ed it as an aquatic ¯ea (see Ed- butions of four sister species pairs over broad geo- mondson, 1987). Exsanguination of the prey was graphic areas. 1996 Academic Press, Inc. thought to occur via the pointed rostrum (the frequent blood-red appearance of Daphnia due to hemoglobin INTRODUCTION probably fueled this myth). Unlike some other micro- crustaceans (e.g., ostracodes, chydorids, bosminids) A lack of phylogenetic knowledge has severely im- there are few useful exoskeletal fossil records for Daph- peded studies of cladoceran comparative biology, bioge- nia (Frey, 1987), the chromosomes are too small for de- tailed cytogenetic investigation, and few if any phyloge- 1 Current address: Dept. of Biology, University of Michigan, Ann Arbor, MI 48109. netically informative morphological characters have 495 1055-7903/96 $18.00 Copyright 1996 by Academic Press, Inc. All rights of reproduction in any form reserved. 496 TAYLOR, HEBERT, AND COLBOURNE been identi®ed despite 200 years of detailed studies TABLE 1 (see Brooks, 1957a; Dodson, 1981; Benzie, 1986). More- over, individuals of many Daphnia species are too Well-Recognized Species of the Daphnia longispina Group (see Hrba cek 1987; Taylor and Hebert, 1994) small for extensive multilocus allozyme analysis using conventional starch electrophoresis. To compound Habitat Helmet these technical problems, recent studies have found Species Geographic range preferences production that introgression, hybridization, and anthropogenic D. cristata Northern Palearctic Lakes Yes faunal interchanges may have created phylogenetic D. cucullata Northern Palearctic Lakes Yes noise in major Daphnia groups (e.g., Taylor and He- D. dubia Eastern Nearctic Lakes Yes D. galeata galeata Palearctic Lakes Yes bert, 1993 a,b; Hebert and Wilson, 1994). D. galeata mendotae Nearctic Lakes Yes Biogeographical evidence suggests that the genus D. hyalina Palearctic Lakes Yes has existed for at least 70 MY (Hebert, 1978; Benzie, D. laevis Neotropic, Nearctic, and Ponds No Ethiopian 1987), and this conclusion may be reinforced by the dis- D. longiremis Holarctic Lakes Yes covery of fossil Daphnia-type ephippia from the early D. longispina Palearctic Ponds No Cretaceous (ca. 130 MYA, Smirnov, 1992). There is, D. rosea Holarctic (and Ethiopian?) Ponds and small No lakes however, little understanding of the age of extant spe- D. thorata Northwest Nearctic Lakes Yes cies, although there is a general sense that many spe- D. umbra sp. n. Eastern Nearctic Lakes and ponds No cies, especially lacustrine forms, have originated since Note. Lakes refer to waterbodies containing ®sh; ponds are ®shless the Pleistocene (Brooks, 1957a). This lack of historical waterbodies. Helmets describe anterior cephalic extensions. information has prevented assessment of the various processes responsible for speciation. Three distinct speciation models have been proposed bert, 1994). A chromosome count of 2n 5 20 is found for Cladocera. Vicariant speciation (Frey, 1987; Hebert in the longispina group, other Daphnia, and ancestral and Wilson, 1994) occurs when a species distribution daphniids. The 12 well-recognized taxa of the D. longis- is disrupted by a barrier to gene ¯ow (often geological, pina group [i.e., taxa that are recognized both by such as continental drift) and the daughter groups Brooks (1957a) and by HrbaÂcek (1987), or have species evolve independently, eventually resulting in reproduc- boundaries genetically con®rmed] are listed in Table 1. tive isolation. A prediction of this hypothesis is that The group is often further divided into a helmeted com- both the time since divergence and the phylogeography plex and the longispina complex (species incapable of of sister taxa should be concordant with the vicariance producing an anterior cephalic crest or helmet). All event. Lynch (1985) developed a second speciation well-recognized longispina species occur in the Holarc- model for cladocerans that invoked founder effects and tic and only one of these species (Daphnia laevis)is divergent selection during habitat shifts. According to common beyond this area. this model, lake to pond founder effect speciation The recent use of sensitive genetic techniques has should be frequent because (a) lake populations rarely provided insight into D. longispina systematics on a re- produce sexual propagules, (b) there is strong divergent gional scale. Cellulose acetate gel electrophoresis allo- selection for the pond lifestyle, and (c) ephemeral ponds zyme studies of the longispina group have proven use- constrain the founder's effective population size, creat- ful for establishing species boundaries and af®nities in ing sampling drift. A third mode of speciation that has the longispina group in Norway (Hobñk and Wolf, been proposed for Daphnia is hybrid speciation (e.g., 1991) and North America (Taylor and Hebert, 1992, FloÈssner, 1993). In this scenario, hybridization with or 1994). In addition, RAPD markers (Schierwater et without introgression (de®ned here as interspeci®c al., 1994) and nucleotide sequences of PCR-ampli®ed gene ¯ow resulting from backcrossing) leads to the for- cytochrome b genes (Schwenk, 1993) have been used to mation of stabilized recombinants that are eventually infer phylogenetic relationships among three European reproductively isolated from the parent taxa. longispina species. In this paper we address the dif®cult systematics of The purpose of this study is to use mitochondrial the Daphnia longispina, MuÈ ller 1763, species group us- DNA sequence and allozyme variation to (1) identify ing a molecular approach. The group is one of two major and compare the major