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Phylogenetic relationships among cirrate octopods (Mollusca: Cephalopoda) resolved using mitochondrial 16S ribosomal DNA sequences
MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 27 (2003) 348–353 www.elsevier.com/locate/ympev
Phylogenetic relationships among cirrate octopods (Mollusca: Cephalopoda) resolved using mitochondrial 16S ribosomal DNA sequences Stuart B. Piertney,a,* Cendrine Hudelot,b,1 F.G. Hochberg,c and Martin A. Collinsd a
c
NERC Molecular Genetics in Ecology Initiative, School of Biological Sciences, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen, AB24 2TZ, UK b Laboratoire de Biologie des Invert ebr es Marins et Malacologie, CNRS ESA 8044, Mus eum National dÕHistoire Naturelle, 55 Rue Buffon, 75005 Paris, France Department of Invertebrate Zoology, Santa Barbara Museum of Natural History, 2259 Puesta del Sol Road, Santa Barbara, CA 93105-2936, USA d British Antarctic Survey, NERC, High Cross, Madingley Road, Cambridge, CB3 0ET, UK Received 2 July 2002; revised 21 October 2002
Abstract Phylogenetic relationships among the cirrate octopods (Mollusca: Cephalopoda) were investigated using partial sequences of the 16S rRNA mitochondrial gene. The derived phylogeny supports the traditional separation of cirrate families based on web form. Genera with a single web (Opisthoteuthis, Grimpoteuthis, Luteuthis, and Cirroctopus) are clearly distinct from those with an intermediate or secondary web (Cirroteuthis, Cirrothauma, and Stauroteuthis). The cirrates with a single web are separated into three groups. The first group is represented by Opisthoteuthis species, the second by Grimpoteuthis and Luteuthis, and the third by members of the genus Cirroctopus. There is no support for the isolation of Luteuthis in a separate family (Luteuthidae). There is, however, evidence of two groupings within the genus Opisthoteuthis. The data suggest the following revisions in the systematic classification of the cirrates: (1) Cirrothauma, Cirroteuthis, and Stauroteuthis be united in the Cirroteuthidae; (2) Grimpoteuthis and Luteuthis be placed in the Grimpoteuthidae; (3) Opisthoteuthis in the Opisthoteuthidae, and; (4) Cirroctopus be considered sufficiently distinct from both Opisthoteuthidae and Grimpoteuthidae to warrant placement in a new family. Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction Cirrate octopods (either Order Cirroctopoda Young 1989 or sub-order Cirrini Grimpe, 1916) are deep-water (>500 m) cephalopods characterised by the presence of fins, paired cirri interspersed between a single row of suckers on the arms and an internal cartilaginous shell (fin support). At present 34 cirrate taxa are recognised as valid (Sweeney, in press; Sweeney and Roper, 1998), although new species are actively being added (e.g., OÕShea, 1999; OÕShea and Lu, 2002; Villanueva et al., in press). The systematic classification of the cirrates is * Corresponding author. Fax: +44-1224-272396. E-mail address: [email protected] (S.B. Piertney). 1 Present address: School of Biological Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, UK.
highly confused, and authors have provided widely differing interpretations on the definition of the main genera, and the placement of species both within genera and within families (OÕShea, 1999; Sweeney, in press; Voss, 1988a,b). Such confusion is a consequence of the small number of specimens available (the description of many species are based on a single specimen), the delicate, gelatinous nature of these animals that are damaged in capture and/or distorted when preserved, and the lack of hard parts for comparative studies. Various classifications have been proposed (e.g., OÕShea, 1999; Robson, 1932; Sweeney, in press; Voss, 1988a,b; see Table 1). Prior to Robson (1932) the cirrates were split into two families, the Cirroteuthidae and Opisthoteuthidae. Robson (1932) added a third family, the Stauroteuthidae, into which he placed the genera Stauroteuthis, Cirrothauma, Chunioteuthis, Froekenia,
1055-7903/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S1055-7903(02)00420-7
S.B. Piertney et al. / Molecular Phylogenetics and Evolution 27 (2003) 348–353
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Table 1 Cirrate genera and their familial placement according to different authors Genera
Robson (1932)
Voss (1988a,b)
Sweeney (in press)
OÕShea (1999)
Cirroteuthis Cirrothauma Stauroteuthis Chunioteuthisa Cirroteuthopsisb Froekenia Grimpoteuthis Enigmatiteuthis Luteuthis Opisthoteuthis Cirroctopus
Cirroteuthidae Stauroteuthidae Stauroteuthidae Stauroteuthidae incertae sedis Stauroteuthidae Stauroteuthidae Not described Not described Opisthoteuthidae Stauroteuthidae
Cirroteuthidae Cirroteuthidae Stauroteuthidae Stauroteuthidae Cirroteuthidae Opisthoteuthidae Opisthoteuthidae Not described Not described Opisthoteuthidae Opisthoteuthidae
Cirroteuthidae Cirroteuthidae Stauroteuthidae Stauroteuthidae Cirroteuthidae Cirroteuthidae Opisthoteuthidae Not described Not described Opisthoteuthidae Opisthoteuthidae
Cirroteuthidae Cirroteuthidae Not stated Not stated Not stated Not stated Grimpoteuthidae (in part) Grimpoteuthidae Luteuthidae Opisthoteuthidae Opisthoteuthidae
a b
Synonymous with Stauroteuthis (Collins and Henriques, 2000). Synonymous with Opisthoteuthis (Villanueva et al., in press).
Grimpoteuthis, and Cirroctopus. The Cirroteuthidae contained the genus Cirroteuthis, and Opisthoteuthidae contained Opisthoteuthis. Voss (1988a) re-defined the group, moving Cirrothauma to the Cirroteuthidae, and Froekenia, Grimpoteuthis, and Cirroctopus to the Opisthoteuthidae. Sweeney (in press) moved Froekenia to the Cirroteuthidae, though the genus is known only from the type specimen that is not extant and it was not sufficiently well described to be evaluated further. Most recently, OÕShea (1999) proposed a substantial re-organisation of the cirrates. He erected two new families, Grimpoteuthidae composed of the genera Grimpoteuthis and Enigmatiteuthis, and Luteuthidae, composed of the genus Luteuthis. Hitherto, there have been few attempts to resolve the relationship among the cirrates from DNA data, and these have either not been formally published (Hudelot, 2000), or are likely to contain errors (Carlini et al., 2001). Here we propose a phylogeny for seven of the nine currently recognised cirrate genera (Table 1) based on variation at the 30 end of the mitochondrial 16S rRNA gene. This gene has previously been shown to be informative for determining the relationship between decapod cephalopods at the infra-family level (Bonnaud et al., 1994).
2. Materials and methods 2.1. Sampling Tissue samples of cirrate octopods were obtained from a variety of locations and sources. Repositories, museum catalogue numbers and EMBL accession numbers are provided as Supplementary material. Many of the samples were obtained from material collected by the University of Aberdeen in the NE and SW Atlantic, with additional material obtained from colleagues throughout the world (Bering Sea, China Sea, NE and SW pacific) (see Supplementary material). Morphological characters considered stable and diagnostic from
classical morphological analysis and keys (e.g., Aldred et al., 1983; Collins and Henriques, 2000; Villanueva et al., in press; Voss and Pearcy, 1990) were checked on selected specimens to confirm species identity. A number of species that are recognised as new and/or undescribed also have been included, namely Grimpoteuthis n. sp. D, which is the most abundant cirrate in the Porcupine Seabight (Collins et al., 2001), a new undescribed cirrate species (N. sp.) that displays characters of both Grimpoteuthis and Opisthoteuthis (Collins, unpublished), and several new Opisthoteuthis species from the China Sea (Lu, unpublished) and from the northeastern Pacific off the coast of California (Hochberg, unpublished). Arm tips were removed from freshly caught specimens and stored in 70–80% ethanol at 4 °C. 2.2. DNA extraction and PCR amplification Total genomic DNA was extracted from tissue samples using a modified CTAB procedure (Shaw et al., 1999). A 30 portion of the 16S rRNA gene of the mitochondrial genome was amplified using the polymerase chain reaction (PCR) with primers 16sar (50 -cgc ctg ttt atc aaa aac at-30 ) and 16sbr (50 -ccg gtc tga act ctg atc at30 ) according to Bonnaud et al. (1994), using either Bioline Taq polymerase or ATGC polymerase. PCR products were purified using the Qiagen PCR purification system then forward and reverse sequences resolved either using dye-terminator cycle sequencing (according to the manufacturerÕs instructions) on a Perkin–Elmer ABI 377 automated sequencer, or by cloning into pMosBlue vector (Amersham) with subsequent sequencing using dideoxy chain termination. 2.3. Sequence analysis Sequences were aligned using ClustalX v 1.8 (Thompson et al., 1997), and confirmed by eye. The phylogenetic relationships among the cirrates was inferred from a single representative sequence of each
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species using likelihood, distance and parsimony-based approaches within PAUP* (version 4.0b8; Swofford, 1998). For the likelihood analysis the program Modeltest (Posada and Crandall, 1998) was used to identify the model that best fits the sequence data from 56 different models using the Akaike information criterion (AIC; Akaike, 1974). This model was defined as: base frequencies of A ¼ 0:3848, C ¼ 0:0940, G ¼ 0:1532, and T ¼ 0:3680; rate matrix of A to C ¼ 1:00, A to G ¼ 4:73, A to T ¼ 2:57, C to G ¼ 2:57, C to T ¼ 2:50, and G to T ¼ 1:00; proportion of invariable sites ðIÞ ¼ 0:3770. For the distance analysis the same model of evolution was used to determine the distances between sequences, and the tree was drawn using neighbour-joining. Maximum parsimony analysis was undertaken using a heuristic search with tree-bisection and reconnection (TBR) as the branch-swapping algorithm. In all cases insertions and deletions were removed from the analysis, and confidence in the resultant topologies was assessed using 1000 bootstrap iterations (Felsenstein, 1985). Trees were rooted through Vampyroteuthis infernalis. Sequence variation was also examined within the genus Stauroteuthis, as comparable sequence divergence was apparent within and between the sister taxa S. syrtensis and S. gilchristi. Whilst this does not affect the phylogenetic relationships of the two species with the other cirrates, the relationships between individuals facilitates examination of how sequence variation is geographically apportioned. Relationships among the sequences were examined using likelihood approaches as described previously, and the tree was rooted through Cirroteuthis muelleri. The optimum model of evolution was defined as: base frequency of A ¼ 0:3193, C ¼ 0:1049, G ¼ 0:1816, and T ¼ 0:3942; rate matrix of A to C ¼ 0:82, A to G ¼ 2:38, A to T ¼ 1:91, C to G ¼ 0:1, C to T ¼ 2:38, and G to T ¼ 1; (proportion of invariable sites ¼ 0.8010).
3. Results The genetic relationships among the cirrates are displayed as a ML-derived phylogeny in Fig. 1a. The topology of this phylogeny is consistent with those derived from maximum parsimony and distance derived analyses (not shown), and shows four major groups with high bootstrap support. First, a basal group composed of the genera Cirrothauma, Cirroteuthis, and Stauroteuthis Secondly, a group containing the two Cirroctopus species. Thirdly, a group containing the genera Grimpoteuthis, Luteuthis and the undescribed cirrate species. Fourthly, a group comprising all the Opisthoteuthis species. Within this Opisthoteuthis group, two clusters are apparent, with the samples from the northeastern Pacific (Opisthoteuthis n. sp. 3 and 4) being separate from all other species.
The relationships among the Stauroteuthis samples examined are shown in Fig. 1b. Levels of sequence divergence are small, and parts of the tree are poorly resolved. However, it is apparent that S. syrtensis and S. gilchristi do not form monophyletic groups. The likelihood of the resolved topology was significantly more likely than any tree constrained to be reciprocally monophyletic for the two putative species (Shimodaira– Hasegawa test; p < 0:05). Mean sequence divergence among Stauroteuthis samples is greater than among samples of Grimpoteuthis sp. D or Opisthoteuthis massyae (p distance ¼ 0.34, 0.022, and 0.024, respectively), though the ranges do overlap (0.002–0.059, 0.002–0.037, and 0.002–0.050, respectively).
4. Discussion The main division in the derived phylogeny separates the genera with a complex (secondary) web form (Vecchione and Young, 1997) and extremely long cirri (Cirroteuthis, Cirrothauma, and Stauroteuthis) from those possessing a simple (single) web and short to moderate length cirri (Opisthoteuthis, Grimpoteuthis, Luteuthis, and Cirroctopus). However, beyond that the phylogeny is inconsistent, in a number of ways, from the proposed recent classifications of OÕShea (1999) and Sweeney (in press). Voss (1988a) united Cirroteuthis and Cirrothauma in the Cirroteuthidae, with Stauroteuthis in a separate family. Subsequently, most authors have followed this classification (Collins and Henriques, 2000; Sweeney, in press). From this study however, it is apparent that the genera Cirroteuthis and Cirrothauma do not form a monophyletic group and the tree structure suggests that the genus Stauroteuthis should be included within the Cirroteuthidae. The Stauroteuthis species differ from Cirroteuthis and Cirrothauma species by lacking a complex shell (Collins and Henriques, 2000), however they do share other characters such as web form, long cirri, sepioid gill structure, and modified suckers (in female S. syrtensis). Collins et al. (2001) questioned whether Cirrothauma possessed a true secondary web, but the samples sequenced here were in excellent condition and clearly possessed a secondary web. Within this group, although the Stauroteuthis genus is monophyletic, S. syrtensis from the North Atlantic could not be separated from its congener, S. gilchristi, from the South Atlantic and Southern Oceans. All S. gilchristi were caught at the same location in the Southern Ocean and appear morphologically similar (Collins and Henriques, 2000). They differ from S. syrtensis in the sucker morphology in the females and the location of the enlarged sucker field in the males. It is possible that all the material examined represents a single species and morphological differences are associated with geographic
S.B. Piertney et al. / Molecular Phylogenetics and Evolution 27 (2003) 348–353
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a
b
Fig. 1. Phylogenetic trees obtained from a maximum likelihood analysis of sequence variation at the 30 end of the 16S mitochondrial DNA gene for: (a) 17 putative cirrate species, and (b) 12 Stauroteuthis individuals. The scale bars represents 0.02 and 0.01 nucleotide substitutions per site, respectively. In both cases, the values at nodes are bootstrap values derived from 1000 iterations (maximum parsimony analysis). EMBL accession numbers are given in parentheses.
variation, that identifications are incorrect, or that incomplete lineage sorting of the 16S sequences is preventing true resolution of the relationship between the species. Further study is clearly required to test the validity of S. syrtensis and S. gilchristi as separate species.
Within the simple webbed forms this study identifies three different groups that would define three families— the Opisthoteuthidae (comprising Opisthoteuthis species), Grimpoteuthidae (comprising Grimpoteuthis, Luteuthis and the undescribed new cirrate species) and
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Table 2 Morphological characters diagnostic of cirrate genera examined in this study Genera
Web form
Cirri length
Fin position
Shell shape
Shell lateral wing termination
Posterior salivary glands
Optic nerve(s)
Radula
Cirroteuthis Cirrothauma Stauroteuthis Grimpoteuthis Enigmatiteuthis Luteuthis Opisthoteuthis Cirroctopus
Complex Complex Complex Simple Simple Simple Simple Simple
Long Long Long Medium Medium Medium Short Short
Lateral Lateral Lateral Lateral Lateral Lateral Subterminal Subterminal
Saddle Butterfly U U, narrow U, narrow ‘‘W’’ U, broad V
N/A N/A Broad, truncate Broad, truncate Broad, truncate Broad, truncate Acutely pointed Acutely pointed
N N N Y/N N N N N
1 1 1 1 1 1 3 9
N N N Y/N N Y N N
Cirroctopus. OÕShea (1999) also proposed three families to accommodate the simple webbed cirrates but included both Opisthoteuthis and Cirroctopus genera within the Opisthoteuthidae. Grimpoteuthis and Enigmatiteuthis genera were subsequently placed in the Grimpoteuthidae family. A new family, Luteuthidae, was created to include the single Luteuthis dentatus specimen, and a second species, L. shuishi, was later added (OÕShea and Lu, 2002). OÕSheaÕs (1999) separation of Luteuthis was based on the slight W-shape of the shell, and the presence of both a radula and a bilobed digestive gland. In this study, the Luteuthis sample was closely allied to Grimpoteuthis sp D., which has a Ushaped shell, an entire digestive gland and lacks a radula. Enigmatiteuthis was not included in the present study, but is morphologically close to Grimpoteuthis and should be retained in Grimpoteuthidae. Grimpoteuthidae thus contains Grimpoteuthis, Luteuthis, Enigmatiteuthis, and the undescribed species from the NE Atlantic. Further work is required to determine the relationship between Grimpoteuthis and Luteuthis, but the genera are clearly closely related and should be united at the family level. The Opisthoteuthidae include the species O. massyae, O. hardyi, O. calypso, O. californiana from the Bering Sea and the two undescribed species from the northeastern Pacific. There is evidence of two groups within the Opisthoteuthis genus, with the northeastern Pacific samples being separate. Again, further investigation of a larger number of samples from that area will clarify the relationship with the other Opisthoteuthidae. The genus Cirroctopus forms a distinct monophyletic grouping outside of Grimpoteuthidae and Opisthoteuthidae. OÕShea (1999) resurrected the genus Cirroctopus to include C. glacialis, C. hochbergi, and C. mawsoni (not analysed here). The configuration of the shell and the number of optic nerves in Cirroctopus was sufficiently different from both Opisthoteuthis and Grimpoteuthis (Table 2) to warrant separation. If Grimpoteuthidae and Opisthoteuthidae are accepted as valid families, then the present analysis provides argument that a new family be erected to accommodate the genus Cirroctopus.
There has been considerable debate (OÕShea, 1999; Robson, 1932; Villanueva et al., in press; Voss, 1988a,b) about which morphological characters should be used to define cirrate phylogeny. Clearly the web form is an important character and separates the two main clades seen here. The optic nerve arrangement and shell form separate the three main species-groups of cirrates with a simple web form, namely Opisthoteuthis, Grimpoteuthis, and Cirroctopus. Recent detailed morphological studies have provided a framework for a better understanding of cirrate systematics and new diagnoses of Opisthoteuthis (Villanueva et al., in press) and Grimpoteuthis (Collins, submitted) should enable the correct placement of species in these genera, which has previously been the source of much confusion. In conclusion, based on the results of this study, the cirrates should be separated into the following four families: Cirroteuthidae (including Cirroteuthis, Cirrothauma, and Stauroteuthis); Opisthoteuthidae (Opisthoteuthis); Grimpoteuthidae (Grimpoteuthis, Luteuthis); and a new family for Cirroctopus. Acknowledgments Thanks to the following who provided samples: Louise Allcock (NMSZ, Edinburgh, UK); Renata Boucher-Rodoni (MNHN, Paris, France); Don Cadien (JWPCP, Carson, California, USA); C.C. Lu (National Chung Hsing University, Taichung, Taiwan); Manuel Haimovici (Universitade de Rio Grande, Rio Grande, Brasil); Chingis Nigmatullin (AtlantNIRO, Kaliningrad, Russia); Steve OÕShea (NIWA, Wellington, New Zealand); M. Vecchione (National Museum of Natural History, Smithsonian Institution, Washington DC, USA). Thanks also to Peter Boyle for useful discussions. References Akaike, H., 1974. A new look at statistical model identification. IEEE Transactions on Automatic Control 19, 716–723.
S.B. Piertney et al. / Molecular Phylogenetics and Evolution 27 (2003) 348–353 Aldred, R.G., Nixon, M., Young, J.Z., 1983. Cirrothauma murrayi Chun, a finned octopod. Philosophical Transactions of the Royal Society of London B 301, 1–54. Bonnaud, L., Boucher-Rodoni, R., Monnerot, M., 1994. Phylogeny of decapod cephalopods based on partial 16S rDNA nucleotide sequences. Competes Rendus De L Academie Des Sciences. Paris, Life Science 317, 581–588. Carlini, D.B., Young, R.E., Vecchione, M., 2001. A molecular phylogeny of the Octopoda (Mollusca: Cephalopoda) evaluated in light of morphological evidence. Molecular Phylogenetics and Evolution 21, 388–397. Collins, M.A., submitted. The genus Grimpoteuthis (Octopoda: Grimpoteuthidae) in the north-east Atlantic, with descriptions of three new species. Zoological Journal of the Linnean Society. Collins, M.A., Henriques, C., 2000. A revision of the family Stauroteuthidae (Octopoda: Cirrata) with rediscriptions of Stauroteuthis syrtensis and S. gilchristi. Journal of the Marine Biological Association of the United Kingdom 80, 685–697. Collins, M.A., Yau, C., Allcock, L.A., Thurston, M.H., 2001. Distribution of deep-water benthic and bentho-pelagic cephalopods from the NE Atlantic. Journal of the Marine Biological Association of the United Kingdom 81, 105–117. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Hudelot, C., 2000. La systematique des Octobrachia (Mollusca: Cephalopoda): Une approche moleculaire, Doctoral Dissertation, Museum National dÕHistoire Naturelle, Paris. OÕShea, S., 1999. In: The Marine Fauna of New Zealand: Octopoda (Mollusca: Cepahlopoda). NIWA Research, Wellington, p. 280. OÕShea, S., Lu, C.C., 2002. A new species of Luteuthis (Mollusca: Cephalopoda: Octopoda: Cirrata) from the South China Sea. Zoological Studies 41, 119–126. Posada, D., Crandall, K.A., 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817–818. Robson, G.C., 1932. A monograph of recent cephalopoda. In: The Octopoda. British Museum, London, p. 359.
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Shaw, P.W., Pierce, G.J., Boyle, P.R., 1999. Subtle population structuring within a highly vagile marine invertebrate, the veined squid Loligo forbesi, demonstrated with microsatellite DNA markers. Molecular Ecology 8, 407–417. Sweeney, M.J., in press. Nominal taxa of cirrate octopods. Appendix to the Workshop Report: Systematics, Ecology and Biology of Cirrate Octopods. Bulletin of Marine Science. Sweeney, M.J., Roper, C.F.E., 1998. Classification, type localities and type repositories of recent Cephalopoda. In: Voss, N.A., Vecchione, M., Toll, R.B., Sweeney, M.J. (Eds.), Systematics and Biogeography of Cephalopods. Smithsonian Institution Press, Washington, DC, pp. 561–599. Swofford, D.L., 1998. PAUP*: Phylogenetic Analysis Using Parsimony (and Other Methods), Version 4.0. Sinauer Associates, Sunderland, MA. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The ClustalX windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24, 4876–4882. Vecchione, M., Young, R.E., 1997. Aspects of the functional morphology of cirrate octopods: locomotion and feeding. Vie Et Milieu-Life and Environment 47, 101–110. Villanueva, R., Collins, M.A., Sanchez, P., Voss, N.A., in press. Systematics and distribution in the Atlantic Ocean of the cirrate octopods of the genus Opisthoteuthis (Mollusca, Cephalopoda), with description of two new species. Bulletin of Marine Science. Voss, G.L., 1988a. The biogeography of the deep-sea Octopoda. Malacologia 29, 295–307. Voss, G.L., 1988b. Evolution and phylogenetic relationships of deepsea octopods (Cirrata and Incirrata). In: Clarke, M.R., Trueman, E.R. (Eds.), The Mollusca, Volume 12. Paleontology and Neontology of Cephalopods. Academic Press, San Diego, pp. 253–276. Voss, G.L., Pearcy, W.G., 1990. Deep-water octopods (Mollusca: Cephalopoda) of the northeastern Pacific. Proceedings of the California Academy of Sciences 47, 47–94.
Phylogenetic relationships among cirrate octopods (Mollusca: Cephalopoda) resolved using mitochondrial 16S ribosomal DNA sequences Stuart B. Piertney,a,* Cendrine Hudelot,b,1 F.G. Hochberg,c and Martin A. Collinsd a
c
NERC Molecular Genetics in Ecology Initiative, School of Biological Sciences, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen, AB24 2TZ, UK b Laboratoire de Biologie des Invert ebr es Marins et Malacologie, CNRS ESA 8044, Mus eum National dÕHistoire Naturelle, 55 Rue Buffon, 75005 Paris, France Department of Invertebrate Zoology, Santa Barbara Museum of Natural History, 2259 Puesta del Sol Road, Santa Barbara, CA 93105-2936, USA d British Antarctic Survey, NERC, High Cross, Madingley Road, Cambridge, CB3 0ET, UK Received 2 July 2002; revised 21 October 2002
Abstract Phylogenetic relationships among the cirrate octopods (Mollusca: Cephalopoda) were investigated using partial sequences of the 16S rRNA mitochondrial gene. The derived phylogeny supports the traditional separation of cirrate families based on web form. Genera with a single web (Opisthoteuthis, Grimpoteuthis, Luteuthis, and Cirroctopus) are clearly distinct from those with an intermediate or secondary web (Cirroteuthis, Cirrothauma, and Stauroteuthis). The cirrates with a single web are separated into three groups. The first group is represented by Opisthoteuthis species, the second by Grimpoteuthis and Luteuthis, and the third by members of the genus Cirroctopus. There is no support for the isolation of Luteuthis in a separate family (Luteuthidae). There is, however, evidence of two groupings within the genus Opisthoteuthis. The data suggest the following revisions in the systematic classification of the cirrates: (1) Cirrothauma, Cirroteuthis, and Stauroteuthis be united in the Cirroteuthidae; (2) Grimpoteuthis and Luteuthis be placed in the Grimpoteuthidae; (3) Opisthoteuthis in the Opisthoteuthidae, and; (4) Cirroctopus be considered sufficiently distinct from both Opisthoteuthidae and Grimpoteuthidae to warrant placement in a new family. Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction Cirrate octopods (either Order Cirroctopoda Young 1989 or sub-order Cirrini Grimpe, 1916) are deep-water (>500 m) cephalopods characterised by the presence of fins, paired cirri interspersed between a single row of suckers on the arms and an internal cartilaginous shell (fin support). At present 34 cirrate taxa are recognised as valid (Sweeney, in press; Sweeney and Roper, 1998), although new species are actively being added (e.g., OÕShea, 1999; OÕShea and Lu, 2002; Villanueva et al., in press). The systematic classification of the cirrates is * Corresponding author. Fax: +44-1224-272396. E-mail address: [email protected] (S.B. Piertney). 1 Present address: School of Biological Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, UK.
highly confused, and authors have provided widely differing interpretations on the definition of the main genera, and the placement of species both within genera and within families (OÕShea, 1999; Sweeney, in press; Voss, 1988a,b). Such confusion is a consequence of the small number of specimens available (the description of many species are based on a single specimen), the delicate, gelatinous nature of these animals that are damaged in capture and/or distorted when preserved, and the lack of hard parts for comparative studies. Various classifications have been proposed (e.g., OÕShea, 1999; Robson, 1932; Sweeney, in press; Voss, 1988a,b; see Table 1). Prior to Robson (1932) the cirrates were split into two families, the Cirroteuthidae and Opisthoteuthidae. Robson (1932) added a third family, the Stauroteuthidae, into which he placed the genera Stauroteuthis, Cirrothauma, Chunioteuthis, Froekenia,
1055-7903/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S1055-7903(02)00420-7
S.B. Piertney et al. / Molecular Phylogenetics and Evolution 27 (2003) 348–353
349
Table 1 Cirrate genera and their familial placement according to different authors Genera
Robson (1932)
Voss (1988a,b)
Sweeney (in press)
OÕShea (1999)
Cirroteuthis Cirrothauma Stauroteuthis Chunioteuthisa Cirroteuthopsisb Froekenia Grimpoteuthis Enigmatiteuthis Luteuthis Opisthoteuthis Cirroctopus
Cirroteuthidae Stauroteuthidae Stauroteuthidae Stauroteuthidae incertae sedis Stauroteuthidae Stauroteuthidae Not described Not described Opisthoteuthidae Stauroteuthidae
Cirroteuthidae Cirroteuthidae Stauroteuthidae Stauroteuthidae Cirroteuthidae Opisthoteuthidae Opisthoteuthidae Not described Not described Opisthoteuthidae Opisthoteuthidae
Cirroteuthidae Cirroteuthidae Stauroteuthidae Stauroteuthidae Cirroteuthidae Cirroteuthidae Opisthoteuthidae Not described Not described Opisthoteuthidae Opisthoteuthidae
Cirroteuthidae Cirroteuthidae Not stated Not stated Not stated Not stated Grimpoteuthidae (in part) Grimpoteuthidae Luteuthidae Opisthoteuthidae Opisthoteuthidae
a b
Synonymous with Stauroteuthis (Collins and Henriques, 2000). Synonymous with Opisthoteuthis (Villanueva et al., in press).
Grimpoteuthis, and Cirroctopus. The Cirroteuthidae contained the genus Cirroteuthis, and Opisthoteuthidae contained Opisthoteuthis. Voss (1988a) re-defined the group, moving Cirrothauma to the Cirroteuthidae, and Froekenia, Grimpoteuthis, and Cirroctopus to the Opisthoteuthidae. Sweeney (in press) moved Froekenia to the Cirroteuthidae, though the genus is known only from the type specimen that is not extant and it was not sufficiently well described to be evaluated further. Most recently, OÕShea (1999) proposed a substantial re-organisation of the cirrates. He erected two new families, Grimpoteuthidae composed of the genera Grimpoteuthis and Enigmatiteuthis, and Luteuthidae, composed of the genus Luteuthis. Hitherto, there have been few attempts to resolve the relationship among the cirrates from DNA data, and these have either not been formally published (Hudelot, 2000), or are likely to contain errors (Carlini et al., 2001). Here we propose a phylogeny for seven of the nine currently recognised cirrate genera (Table 1) based on variation at the 30 end of the mitochondrial 16S rRNA gene. This gene has previously been shown to be informative for determining the relationship between decapod cephalopods at the infra-family level (Bonnaud et al., 1994).
2. Materials and methods 2.1. Sampling Tissue samples of cirrate octopods were obtained from a variety of locations and sources. Repositories, museum catalogue numbers and EMBL accession numbers are provided as Supplementary material. Many of the samples were obtained from material collected by the University of Aberdeen in the NE and SW Atlantic, with additional material obtained from colleagues throughout the world (Bering Sea, China Sea, NE and SW pacific) (see Supplementary material). Morphological characters considered stable and diagnostic from
classical morphological analysis and keys (e.g., Aldred et al., 1983; Collins and Henriques, 2000; Villanueva et al., in press; Voss and Pearcy, 1990) were checked on selected specimens to confirm species identity. A number of species that are recognised as new and/or undescribed also have been included, namely Grimpoteuthis n. sp. D, which is the most abundant cirrate in the Porcupine Seabight (Collins et al., 2001), a new undescribed cirrate species (N. sp.) that displays characters of both Grimpoteuthis and Opisthoteuthis (Collins, unpublished), and several new Opisthoteuthis species from the China Sea (Lu, unpublished) and from the northeastern Pacific off the coast of California (Hochberg, unpublished). Arm tips were removed from freshly caught specimens and stored in 70–80% ethanol at 4 °C. 2.2. DNA extraction and PCR amplification Total genomic DNA was extracted from tissue samples using a modified CTAB procedure (Shaw et al., 1999). A 30 portion of the 16S rRNA gene of the mitochondrial genome was amplified using the polymerase chain reaction (PCR) with primers 16sar (50 -cgc ctg ttt atc aaa aac at-30 ) and 16sbr (50 -ccg gtc tga act ctg atc at30 ) according to Bonnaud et al. (1994), using either Bioline Taq polymerase or ATGC polymerase. PCR products were purified using the Qiagen PCR purification system then forward and reverse sequences resolved either using dye-terminator cycle sequencing (according to the manufacturerÕs instructions) on a Perkin–Elmer ABI 377 automated sequencer, or by cloning into pMosBlue vector (Amersham) with subsequent sequencing using dideoxy chain termination. 2.3. Sequence analysis Sequences were aligned using ClustalX v 1.8 (Thompson et al., 1997), and confirmed by eye. The phylogenetic relationships among the cirrates was inferred from a single representative sequence of each
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species using likelihood, distance and parsimony-based approaches within PAUP* (version 4.0b8; Swofford, 1998). For the likelihood analysis the program Modeltest (Posada and Crandall, 1998) was used to identify the model that best fits the sequence data from 56 different models using the Akaike information criterion (AIC; Akaike, 1974). This model was defined as: base frequencies of A ¼ 0:3848, C ¼ 0:0940, G ¼ 0:1532, and T ¼ 0:3680; rate matrix of A to C ¼ 1:00, A to G ¼ 4:73, A to T ¼ 2:57, C to G ¼ 2:57, C to T ¼ 2:50, and G to T ¼ 1:00; proportion of invariable sites ðIÞ ¼ 0:3770. For the distance analysis the same model of evolution was used to determine the distances between sequences, and the tree was drawn using neighbour-joining. Maximum parsimony analysis was undertaken using a heuristic search with tree-bisection and reconnection (TBR) as the branch-swapping algorithm. In all cases insertions and deletions were removed from the analysis, and confidence in the resultant topologies was assessed using 1000 bootstrap iterations (Felsenstein, 1985). Trees were rooted through Vampyroteuthis infernalis. Sequence variation was also examined within the genus Stauroteuthis, as comparable sequence divergence was apparent within and between the sister taxa S. syrtensis and S. gilchristi. Whilst this does not affect the phylogenetic relationships of the two species with the other cirrates, the relationships between individuals facilitates examination of how sequence variation is geographically apportioned. Relationships among the sequences were examined using likelihood approaches as described previously, and the tree was rooted through Cirroteuthis muelleri. The optimum model of evolution was defined as: base frequency of A ¼ 0:3193, C ¼ 0:1049, G ¼ 0:1816, and T ¼ 0:3942; rate matrix of A to C ¼ 0:82, A to G ¼ 2:38, A to T ¼ 1:91, C to G ¼ 0:1, C to T ¼ 2:38, and G to T ¼ 1; (proportion of invariable sites ¼ 0.8010).
3. Results The genetic relationships among the cirrates are displayed as a ML-derived phylogeny in Fig. 1a. The topology of this phylogeny is consistent with those derived from maximum parsimony and distance derived analyses (not shown), and shows four major groups with high bootstrap support. First, a basal group composed of the genera Cirrothauma, Cirroteuthis, and Stauroteuthis Secondly, a group containing the two Cirroctopus species. Thirdly, a group containing the genera Grimpoteuthis, Luteuthis and the undescribed cirrate species. Fourthly, a group comprising all the Opisthoteuthis species. Within this Opisthoteuthis group, two clusters are apparent, with the samples from the northeastern Pacific (Opisthoteuthis n. sp. 3 and 4) being separate from all other species.
The relationships among the Stauroteuthis samples examined are shown in Fig. 1b. Levels of sequence divergence are small, and parts of the tree are poorly resolved. However, it is apparent that S. syrtensis and S. gilchristi do not form monophyletic groups. The likelihood of the resolved topology was significantly more likely than any tree constrained to be reciprocally monophyletic for the two putative species (Shimodaira– Hasegawa test; p < 0:05). Mean sequence divergence among Stauroteuthis samples is greater than among samples of Grimpoteuthis sp. D or Opisthoteuthis massyae (p distance ¼ 0.34, 0.022, and 0.024, respectively), though the ranges do overlap (0.002–0.059, 0.002–0.037, and 0.002–0.050, respectively).
4. Discussion The main division in the derived phylogeny separates the genera with a complex (secondary) web form (Vecchione and Young, 1997) and extremely long cirri (Cirroteuthis, Cirrothauma, and Stauroteuthis) from those possessing a simple (single) web and short to moderate length cirri (Opisthoteuthis, Grimpoteuthis, Luteuthis, and Cirroctopus). However, beyond that the phylogeny is inconsistent, in a number of ways, from the proposed recent classifications of OÕShea (1999) and Sweeney (in press). Voss (1988a) united Cirroteuthis and Cirrothauma in the Cirroteuthidae, with Stauroteuthis in a separate family. Subsequently, most authors have followed this classification (Collins and Henriques, 2000; Sweeney, in press). From this study however, it is apparent that the genera Cirroteuthis and Cirrothauma do not form a monophyletic group and the tree structure suggests that the genus Stauroteuthis should be included within the Cirroteuthidae. The Stauroteuthis species differ from Cirroteuthis and Cirrothauma species by lacking a complex shell (Collins and Henriques, 2000), however they do share other characters such as web form, long cirri, sepioid gill structure, and modified suckers (in female S. syrtensis). Collins et al. (2001) questioned whether Cirrothauma possessed a true secondary web, but the samples sequenced here were in excellent condition and clearly possessed a secondary web. Within this group, although the Stauroteuthis genus is monophyletic, S. syrtensis from the North Atlantic could not be separated from its congener, S. gilchristi, from the South Atlantic and Southern Oceans. All S. gilchristi were caught at the same location in the Southern Ocean and appear morphologically similar (Collins and Henriques, 2000). They differ from S. syrtensis in the sucker morphology in the females and the location of the enlarged sucker field in the males. It is possible that all the material examined represents a single species and morphological differences are associated with geographic
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a
b
Fig. 1. Phylogenetic trees obtained from a maximum likelihood analysis of sequence variation at the 30 end of the 16S mitochondrial DNA gene for: (a) 17 putative cirrate species, and (b) 12 Stauroteuthis individuals. The scale bars represents 0.02 and 0.01 nucleotide substitutions per site, respectively. In both cases, the values at nodes are bootstrap values derived from 1000 iterations (maximum parsimony analysis). EMBL accession numbers are given in parentheses.
variation, that identifications are incorrect, or that incomplete lineage sorting of the 16S sequences is preventing true resolution of the relationship between the species. Further study is clearly required to test the validity of S. syrtensis and S. gilchristi as separate species.
Within the simple webbed forms this study identifies three different groups that would define three families— the Opisthoteuthidae (comprising Opisthoteuthis species), Grimpoteuthidae (comprising Grimpoteuthis, Luteuthis and the undescribed new cirrate species) and
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Table 2 Morphological characters diagnostic of cirrate genera examined in this study Genera
Web form
Cirri length
Fin position
Shell shape
Shell lateral wing termination
Posterior salivary glands
Optic nerve(s)
Radula
Cirroteuthis Cirrothauma Stauroteuthis Grimpoteuthis Enigmatiteuthis Luteuthis Opisthoteuthis Cirroctopus
Complex Complex Complex Simple Simple Simple Simple Simple
Long Long Long Medium Medium Medium Short Short
Lateral Lateral Lateral Lateral Lateral Lateral Subterminal Subterminal
Saddle Butterfly U U, narrow U, narrow ‘‘W’’ U, broad V
N/A N/A Broad, truncate Broad, truncate Broad, truncate Broad, truncate Acutely pointed Acutely pointed
N N N Y/N N N N N
1 1 1 1 1 1 3 9
N N N Y/N N Y N N
Cirroctopus. OÕShea (1999) also proposed three families to accommodate the simple webbed cirrates but included both Opisthoteuthis and Cirroctopus genera within the Opisthoteuthidae. Grimpoteuthis and Enigmatiteuthis genera were subsequently placed in the Grimpoteuthidae family. A new family, Luteuthidae, was created to include the single Luteuthis dentatus specimen, and a second species, L. shuishi, was later added (OÕShea and Lu, 2002). OÕSheaÕs (1999) separation of Luteuthis was based on the slight W-shape of the shell, and the presence of both a radula and a bilobed digestive gland. In this study, the Luteuthis sample was closely allied to Grimpoteuthis sp D., which has a Ushaped shell, an entire digestive gland and lacks a radula. Enigmatiteuthis was not included in the present study, but is morphologically close to Grimpoteuthis and should be retained in Grimpoteuthidae. Grimpoteuthidae thus contains Grimpoteuthis, Luteuthis, Enigmatiteuthis, and the undescribed species from the NE Atlantic. Further work is required to determine the relationship between Grimpoteuthis and Luteuthis, but the genera are clearly closely related and should be united at the family level. The Opisthoteuthidae include the species O. massyae, O. hardyi, O. calypso, O. californiana from the Bering Sea and the two undescribed species from the northeastern Pacific. There is evidence of two groups within the Opisthoteuthis genus, with the northeastern Pacific samples being separate. Again, further investigation of a larger number of samples from that area will clarify the relationship with the other Opisthoteuthidae. The genus Cirroctopus forms a distinct monophyletic grouping outside of Grimpoteuthidae and Opisthoteuthidae. OÕShea (1999) resurrected the genus Cirroctopus to include C. glacialis, C. hochbergi, and C. mawsoni (not analysed here). The configuration of the shell and the number of optic nerves in Cirroctopus was sufficiently different from both Opisthoteuthis and Grimpoteuthis (Table 2) to warrant separation. If Grimpoteuthidae and Opisthoteuthidae are accepted as valid families, then the present analysis provides argument that a new family be erected to accommodate the genus Cirroctopus.
There has been considerable debate (OÕShea, 1999; Robson, 1932; Villanueva et al., in press; Voss, 1988a,b) about which morphological characters should be used to define cirrate phylogeny. Clearly the web form is an important character and separates the two main clades seen here. The optic nerve arrangement and shell form separate the three main species-groups of cirrates with a simple web form, namely Opisthoteuthis, Grimpoteuthis, and Cirroctopus. Recent detailed morphological studies have provided a framework for a better understanding of cirrate systematics and new diagnoses of Opisthoteuthis (Villanueva et al., in press) and Grimpoteuthis (Collins, submitted) should enable the correct placement of species in these genera, which has previously been the source of much confusion. In conclusion, based on the results of this study, the cirrates should be separated into the following four families: Cirroteuthidae (including Cirroteuthis, Cirrothauma, and Stauroteuthis); Opisthoteuthidae (Opisthoteuthis); Grimpoteuthidae (Grimpoteuthis, Luteuthis); and a new family for Cirroctopus. Acknowledgments Thanks to the following who provided samples: Louise Allcock (NMSZ, Edinburgh, UK); Renata Boucher-Rodoni (MNHN, Paris, France); Don Cadien (JWPCP, Carson, California, USA); C.C. Lu (National Chung Hsing University, Taichung, Taiwan); Manuel Haimovici (Universitade de Rio Grande, Rio Grande, Brasil); Chingis Nigmatullin (AtlantNIRO, Kaliningrad, Russia); Steve OÕShea (NIWA, Wellington, New Zealand); M. Vecchione (National Museum of Natural History, Smithsonian Institution, Washington DC, USA). Thanks also to Peter Boyle for useful discussions. References Akaike, H., 1974. A new look at statistical model identification. IEEE Transactions on Automatic Control 19, 716–723.
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