Marine Biology (2005) DOI 10.1007/s00227-005-0089-z RESEARCH ARTICLE Anna L. Bass Æ Heidi Dewar Æ Tierney Thys J. Todd. Streelman Æ Stephen A. Karl Evolutionary divergence among lineages of the ocean sunfish family, Molidae (Tetraodontiformes) Received: 6 May 2005 / Accepted: 8 July 2005 Ó Springer-Verlag 2005 Abstract Ocean sunfish, family Molidae, are enigmatic ulative. The systematic analysis provided strong sup- members of the epipelagic fauna of all tropical and port for the sister taxa relationship between genera temperate oceans. A study, begun in 1998, initially fo- Masturus and Mola and the basal position of the genus cused on the population genetics of Mola mola Linna- Ranzania within the family, as well as the sister group eus 1758 immediately indicated high levels of genetic relationship of the Tetraodontiform families Tetra- divergence in the d-loop and cytochrome b mitochon- odontidae + Diodontidae to the Molidae. drial genes. This preliminary effort was expanded to include Masturus lanceolatus Lie´nard 1840, Ranzania laevis Pennant 1776, and representative sequences of Introduction other Tetraodontiformes. Analysis of the sequence data confirms that there are two species in the genus Mola, Species in the family Molidae are large, primarily pelagic Mola mola and M. ramsayi Giglioli 1883, with the members of the Tetraodontiformes. Commonly referred latter presumed to be limited to the southern hemi- to as ocean sunfish, the Molidae have a distinctive lat- sphere. There is an indication of inter-ocean subdivi- erally compressed shape and ‘‘chopped off’’ appearance sion within both species originating 0.05–0.32 and 1.55– (Fraser-Brunner 1951; Smith and Heemstra 1986). 4.10 million years ago, respectively. Given limited Relying on median fins for swimming, they lack caudal sample sizes, however, the divergence estimates are bones, ribs, pelvic fins, spines or girdles and have fewer minimums and the isolating mechanisms remain spec- vertebrae than any other fish (Tyler 1980). Metamor- phosis from larva to adult is remarkable in that, unlike most fish, they pass through two distinct larva phases—a Communicated by J.P.Grassle, New Brunswick typical Tetraodon-like larval and another highly trans- formative stage resulting in the complete absorption of A. L. Bass Æ S. A. Karl (&) Department of Biology, SCA 110, the tail (Fraser-Brunner 1951). University of South Florida, Knowledge of the natural history of the Molidae is Tampa, FL 33620, USA sparse, but they are recognized as the most fecund extant E-mail: [email protected] vertebrate with a single female capable of producing as Tel.: +1-808-2367401 8 Fax: +1-808-2367443 many as 3·10 eggs at one time (Schmidt 1921; Parenti 2003). Members of the family are globally distributed in H. Dewar both temperate and tropical waters although there is Tagging of Pacific Pelagics, Stanford University some indication of restricted distributions for M. mola c/o Inter-American Tropical Tuna Commission, La Jolla, 92037 CA, USA (Fraser-Brunner 1951; Smith and Heemstra 1986). Parin (1968) presented a distribution map compiled from T. Thys several sources suggesting that spawning areas for Sea Studios Foundation, Monterey, CA 93940, USA Ma. lanceolatus and R. laevis overlap in the Sargasso Sea but with no clear indication of the spawning regions for J. Todd. Streelman School of Biology, The Georgia Institute of Technology, Mola spp. Five spawning areas have been suggested for 310 Ferst Drive, Atlanta, GA 30332-0230, USA the Molidae in the central gyres in the North and South Atlantic, North and South Pacific, and Indian Oceans. S. A. Karl Molids are reported to feed primarily on jellyfish and The Hawai’i Institute of Marine Biology, University of Hawai’i, Manoa, PO Box 1346, planktonic organisms although a variety of prey such as Kane’ohe, HI 96744, USA brittlestars, flounder, and leptocephalus larvae have been found in their guts (Grassi 1897; Norman and panded our analysis to include the mitochondrial cyto- Fraser 1949) indicating that they must sometimes feed chrome b gene sequences representing the identified on the bottom and among patches of floating seaweeds. divergent groups of M. mola and selected Tetraodonti- Due to their primarily pelagic existence, they are difficult form cyt b sequences from GenBank. Here, we present to observe and information on their natural history and the results of these analyses in the context of evolution behavior is generally lacking. Most frequently, they are of globally-distributed, highly-fecund, pelagic marine sighted basking at the ocean surface (Norman and fishes. Fraser 1938). Recent research indicates that basking may be correlated with diving behavior as an attempt to replenish body heat after deep dives into cold water Materials and methods (2–10°C; personal observation; Cartamil and Lowe 2004). It also appears from our tagging studies, that From 1998 to 2004, tissue samples were taken from 13 individual M. mola and Ma. lanceolatus show no indi- putative M. mola Giglioli 1883, four Ma. lanceolatus cation of ocean basin scale movements and do not Lie´nard 1840, and one R. laevis Pennant 1776. Samples undertake large-scale migrations (unpublished data). were from multiple locations in the Atlantic and Pacific Taxonomically, the family Molidae has a long history Oceans and were intended to represent the systematic in the scientific literature with two of the earliest diversity of the group (Table 1). Samples consisted of fin descriptions in 1758 by Linnaeus and 1766 by Koelre- clips or muscle biopsies and were stored in 95% EtOH. uter (see Parenti 2003). Since that time, numerous genera Initially, genomic DNA was isolated using a standard (19) and species (54) of sunfish have been proposed phenol/chloroform method, precipitated with sodium (Parenti 2003) and the taxonomy of the family has been acetate and 100% EtOH, and resuspended in 1X TE relatively volatile. The most complete taxonomic revi- (10 mM Tris HCl, pH 7.5, 1 mM EDTA acid). Later sion of the family (Fraser-Brunner 1951) distinguished samples were isolated using a non-boiling Chelex method five species in three genera; R. laevis, Ma. lanceolatus, (Walsh et al. 1991) and stored at À20°C. Two regions of Ma. oxyuropterus, M. mola and M. ramsayi. Currently, the mitochondrial DNA genome were amplified: control three species are commonly recognized; R. laevis, Ma. region (d-loop) and cytochrome b(cyt b). The primers A lanceolatus, Mola mola, with a fourth, Mola ramsayi, (5¢-TTCCACCTCTAACTCCCAAA GCTAG-3¢), E (5¢- infrequently mentioned in taxonomic treatments (Smith CCTGAAGTAGGAACCAGATG-3¢)andM(5¢-TAT- and Heemstra 1986; Nelson 1994; Parenti 2003). GCTTTAGTTAAGGCTACG-3¢) of Lee et al. (1995) The relationship among currently recognized genera were used to amplify the d-loop. Initially, primers A and was the focus of two recent studies, one morphological M were paired to amplify the entire d-loop [800 base and the other molecular. Santini and Tyler (2002) ana- pairs (bp)] from six individuals, however, primers A and lyzed 48 morphological characters and found strong E (also from Lee et al. 1995) were used to amplify support for the traditional hypothesis of a sister taxon 400 bp of the 5¢ end of the remainder of the samples. relationship between the genera Masturus and Mola, For the cyt b gene, the forward primer (5¢-GTGACTT- with Ranzania holding the basal position within the GAAAAACCACCGTTG-3¢) of Song et al. (1998) and family. Using complete mitochondrial genome se- reverse primer (5¢-AATAG GAAGTATCATTCGGGT- quences, Yamanoue et al. (2004) also supported the TTGATG-3¢) of Taberlet et al. (1992) were used to traditional hypothesis and the morphological analysis. amplify an approximately 750-bp fragment. Each Identification of the sister-group of the Molidae, how- amplification reaction (25 or 50 ll) of the d-loop region ever, has been controversial and was not resolved even consisted of 1X Promega buffer, 1.25 U of Promega Taq, with the addition of complete mitochondrial genomes 0.5 mM dNTPs, 3 mM MgCl2, 0.5 lM of each primer, (Yamanoue et al. 2004). Multiple hypotheses have been 0.12 lg llÀ1 of bovine serum albumen, and 2 llof presented regarding what family or families are closest template. Each amplification reaction (25 or 50 ll) of the to the Molidae: Diodontidae (Breder and Clark 1947), cyt b gene region consisted of 1X Promega buffer, 1.25 U Tetraodontidae + Diodontidae (Winterbottom 1974; of Enzypol Plus 2000 polymerase, 0.8 mM dNTPs, Tyler 1980; Santini and Tyler 2003), and Diodontidae + 2 mM MgCl2, 0.5 lM of each primer, 1.0 M betaine, Ostraciidae (Leis 1984). 0.12 lg llÀ1 of bovine serum albumen, and 2–4 llof As part of ongoing research into the general ecology, template. The cycling conditions for all primer pairs movements, and distribution of M. mola, we have been consisted of 95° (1 min), 35–45 cycles (95, 30 s; 50, 45 s; using molecular data to examine population structure 72, 1 min), with a final extension at 72(3 min). A tem- and biogeography of this species. We chose to analyze plate-free reaction was always included as a negative the mitochondrial control region (d-loop) because its control. Amplicons were purified using sterile nanopure general fast rate of evolution allows the differentiation of water and 30,000 MW Millipore filters (Millipore Inc., closely related groups and it is commonly used in Bedford, MA, USA). The mass of the amplicons was intraspecific studies (Kocher and Stepien 1997). Our determined by comparing EtBR staining intensity of 2.0– initial results, however, indicated genetic divergence 5.0 ll of each purified reaction relative to a standard levels within this species that were as great as levels mass DNA ladder (Invitrogen Life Technologies, Carls- commonly observed between species. We therefore ex- bad, CA, USA).