Reference: Biol. Bull. 217: 292–305. (December 2009) © 2009 Marine Biological Laboratory Cryptic Species Differentiated in Conus ebraeus, a Widespread Tropical Marine Gastropod THOMAS F. DUDA, JR.1,2*, ALAN J. KOHN3 AND AMBER M. MATHENY3† 1Department of Ecology and Evolutionary Biology and Museum of Zoology, University of Michigan, 1109 Geddes Avenue, Ann Arbor, Michigan 48109; 2Smithsonian Tropical Research Institute, Apartado 0843-03092, Balboa, Anco´n, Republic of Panama; and 3Department of Biology, Box 351800, University of Washington, Seattle, Washington 98195 Abstract. Anomalous mitochondrial and nuclear gene radular tooth characters relative to those of the shell. More- sequences in individuals of the widely distributed tropical over, cryptic species could well be important components of marine gastropod Conus ebraeus that were not distinguish- species richness in Conus specifically and marine molluscan able by shell shape and color pattern characters suggested biodiversity more generally. the presence of a second, cryptic species. We tested this hypothesis by genetic, morphological, and ecological com- Introduction parisons of additional individuals from the site in Okinawa where the two forms co-occurred. Radular tooth size and With some 550 recognized valid extant species, Conus is shape, prey type in nature, and microhabitats utilized dif- likely the largest genus of animals in the sea. In addition to fered markedly between the two forms. Adults with typical contributing substantially to marine biodiversity, this cir- C. ebraeus DNA and radular teeth preyed primarily on cumtropical gastropod genus is important ecologically—up errant polychaetes (Eunicidae); those with anomalous DNA to 36 species co-occur on a single coral reef platform (Kohn, and teeth ate mainly sedentary capitellids. Juveniles (shell 2001); evolutionarily—its net diversification rate is the length Ͻ13 mm) had more similar teeth and ate primarily highest among gastropods (Stanley 2007); and neurobio- syllids. Radular teeth of the anomalous form agreed with logically and medically—because of the many highly spe- those of Conus judaeus, distinguished from C. ebraeus by cific and potent neuropeptides in its venom (Olivera, 2006). Rudolph Bergh in 1895 solely on tooth characters of one Conus ebraeus Linnaeus, the most widely distributed specimen from the Philippines. Samples from other widely species of this hyperdiverse genus, occurs in varied shallow- scattered Pacific localities revealed only typical C. ebraeus water habitats throughout the tropical Indian and Pacific Oceans, its range spanning more than one-fourth of the gene sequences. Both forms occurred in Seychelles (western world’s entire ocean area (Ro¨ckel et al., 1995). It is one of Indian Ocean), where their radular teeth and diets were only three of the more than 300 Indo-West Pacific Conus consistent with the data from Okinawa, but DNA of avail- species that extend to the eastern Pacific region. This un- able material was degraded. Although C. judaeus was long usually broad distribution makes C. ebraeus an excellent dismissed as an aberrant specimen and junior synonym of C. subject for examining patterns of genetic connectivity of ebraeus, our results support its validity as a distinct species. populations and patterns of diversification in the broad These results highlight the importance of molecular and Indo-West Pacific marine realm. In a study to interpret patterns of gene flow, Duda and Received 18 May 2009; accepted 14 September 2009. Lessios (2009) demonstrated a general absence of genetic * To whom correspondence should be addressed, at 1109 Geddes Ave- structure in western and central Pacific populations of C. nue, Ann Arbor, Michigan 48109. E-mail: [email protected] ebraeus by using mitochondrial gene sequences at eight †Current address: Benaroya Research Institute, 1201 9th Avenue, Seat- tle, Washington 98101. widespread locations, from Hawaii west to the Philippines Abbreviations: DFA, discriminant function analysis; PCA, principal and from Okinawa south to American Samoa. Also, in a components analysis; SL, shell length; TL, tooth length. study of the evolution of conotoxins, the small peptides 292 CRYPTIC CONUS SPECIES DIFFERENTIATED 293 expressed in Conus venoms and utilized in prey capture, yses. The specimens from Okinawa are preserved in the Duda and Remigio (2008) obtained mRNA sequences from University of Michigan Museum of Zoology. C. ebraeus to determine conotoxin expression patterns The strikingly distinct mitochondrial and venom geno- among a set of closely related Conus. These two studies led types occurred at Okinawa and the Philippines of the eight to the serendipitous discovery that some individuals from Pacific C. ebraeus populations examined by Duda and Les- Okinawa, Japan, that were identified as C. ebraeus from sios (2009). Because of the lack of Indian Ocean represen- shell characters, possessed very different mitochondrial and tatives in these studies, we also examined samples of puta- conotoxin gene sequences. While the sequences from one tive C. ebraeus from Republic of Seychelles and its group agreed with those obtained from C. ebraeus popula- dependencies, collected by AJK during the Yale Seychelles tions at Okinawa and elsewhere, sequences of the other Expedition in 1957–1958 for a comparative ecological were unique, differing strikingly from other C. ebraeus and study of Conus (Kohn, unpubl). from all of the more than 200 other Conus species whose genes have been sequenced. We determined morphological and ecological correlates DNA sequences of the genetic differences between the two forms at Oki- We extracted DNA from about 25 mg of foot tissue with nawa, and we show that these support the hypothesis that a the E.Z.N.A Mollusc DNA Kit (Omega Bio-Tek, Inc.) ac- co-occurring cryptic species had been misidentified as C. cording to manufacturers’ recommendations, except that we ebraeus. Moreover, analysis of radular tooth morphology slightly modified the protocol by extending centrifugation supports the hypothesis that the cryptic species masquerad- of precipitated DNA to 10 min and reducing the volume of ing as C. ebraeus was described more than a century ago, elution buffer to 40 ␮l to increase DNA concentration. We solely on the basis of its radular tooth characteristics. Thus amplified a region of the mitochondrial 16S rRNA gene an available name exists for it in the literature, in contrast to with universal 16S primers (Palumbi et al., 1991) as de- cryptic species identified in recent years from DNA se- scribed previously (Duda and Kohn, 2005). These primers quence differences in other taxa. We also examined indi- amplify about 500 basepairs (bp) of the 16S gene. PCR viduals from additional Indo-West Pacific locations in an products were cleaned with a QIAquick Multiwell PCR effort to better understand the distributions of the two spe- Purification kit (Qiagen), and sequencing was performed in cies. both directions. We examined the chromatograms with Se- quencher 4.8 (Gene Codes Corporation), aligned sequences Materials and Methods by eye, and constructed a haplotype network using TCS 1.21 (Clement et al., 2000). The sequences recovered com- Definitions prised two distinct sets of haplotypes that exhibited a fixed Cryptic species are “reproductively isolated species that difference within a BamHI restriction site. Thus we per- resemble one another closely and, thus, sometimes remain formed restriction digests on amplification products of ad- unrecognized” (Avise, 2006), “two or more distinct but ditional specimens to infer haplotype identity. morphologically similar species that were classified as a For the large sample collected at Okinawa in 2004, we single species” (Pfenninger and Schwenk, 2007), or “two or followed a double-blind protocol in analyzing DNA se- more distinct species that are erroneously classified (and quences and radular tooth morphometry. AJK numbered hidden) under one species name (Bickford et al., 2007). We each specimen and sent numbered foot tissue slices to TFD, use the term in the sense of these essentially similar recent who extracted and sequenced the DNA at the University of definitions. Michigan. AJK prepared and analyzed the similarly num- bered radular teeth at the University of Washington. After completing the analyses, we exchanged the resulting data. Specimens Most specimens were collected from intertidal habitats in Radular tooth morphometry Okinawa, Japan. AJK collected 21 specimens from various sites in 1981 in a study of habitats and diets in nature. The Morphometric methods generally followed Nishi and initial DNA analyses were carried out on specimens col- Kohn (1999), except that data on continuously varying lected at Sesoko Island, Okinawa, near the Sesoko Station radular tooth characters were initially subjected to principal of the Tropical Biosphere Research Center, University of components analysis (PCA), because individual specimens the Ryukyus (26°38.0ЈN, 127°52.4ЈE) in 2001. Following were not preclassified to species prior to discriminant func- the discovery of disparate 16S sequences in that material, tion analysis (DFA). See Table 2 for a list of the characters AJK revisited Sesoko in July 2004 to obtain a larger sample used. All ratios were arcsin transformed prior to PCA and for ecological, morphological, and molecular genetic anal- DFA. 294 T. F. DUDA, JR., ET AL. Table 1 Microhabitats of Conus ebraeus and C. judaeus in Okinawa and Seychelles Okinawa Seychelles Microhabitat Type C. ebraeus C. judaeus C. ebraeus