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Blackwell Publishing Ltd. Molecular phylogeny of Scaphopoda (Mollusca) inferred from 18S rDNA sequences: support for a Scaphopoda– Cephalopoda clade GERHARD STEINER & HERMANN DREYER Accepted: 15 September 2002 Steiner, G. & Dreyer, H. (2003). Molecular phylogeny of Scaphopoda (Mollusca) inferred from 18S rDNA sequences: support for a Scaphopoda–Cephalopoda clade. — Zoologica Scripta, 32, 343–356. The phylogenetic relationships of the Scaphopoda, one of the ‘lesser’ molluscan classes, with the other conchiferan taxa are far from clear. They appear either as the sister-group to the Bivalvia (Diasoma concept) or to a Gastropoda–Cephalopoda clade or to the Cephalopoda alone (helcionellid concept). We compiled a 18S rDNA sequence dataset of 48 molluscan species containing 17 scaphopods to test these hypotheses and to address questions regarding high-level relationships with the Scaphopoda. Both parsimony and maximum likelihood trees show low branch support at the base of the Conchifera, except for the robust clade uniting Scaphopoda and Cephalopoda. This result is corroborated by spectral analysis and likelihood mapping. We also tested alternative topologies which scored significantly worse both in tree length and in likelihood. The 18S rDNA data thus reject the Diasoma in favour of a Scaphopoda– Cephalopoda clade as proposed in the helcionellid concept. When plotted on the molecular tree, the pivotal morphological characters associated with the burrowing life style of the Bivalvia and Scaphopoda, i.e. mantle/shell enclosure of the body and the burrowing foot with true pedal ganglia, appear convergent in these groups. In contrast, the prominent and tilted dorsoventral body axes, multiple cephalic tentacles and a ring-shaped muscle attachment on the shell are potential synapomorphies of Scaphopoda and Cephalopoda. Most of the higher taxa within the Scaphopoda are supported by the molecular data. However, there is no support for the families Dentaliidae and Gadilidae. The basal position of the Fustiariidae within the Dentaliida is confirmed. Gerhard Steiner & Hermann Dreyer, Institute of Zoology, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria. E-mail: gerhard.steiner@univie.ac.at Introduction Scaphopoda is one of the less diverse major groups of conchiferan molluscs, with about 520 Recent species (Steiner & Kabat 2001; Steiner & Kabat 2001) living in all types of unconsolidated marine sediments (Shimek & Steiner 1997). Scaphopoda are adapted to their infaunal habit by a tubular mantle/shell, open at both ends, and a burrowing foot; there are no gills or osphradia (Steiner 1992; Shimek & Steiner 1997). They are generally considered to be microcarnivores collecting their prey items (mainly foraminifers) with numerous captacula — cerebrally innervated cephalic appendages — and processing them with a large radula apparatus (Shimek 1988, 1990; Palmer & Steiner 1998). The phylogenetic relationships of Scaphopoda with the other conchiferan taxa are far from settled. There are two competing basic concepts: (1) the Diasoma–Cyrtosoma (Runnegar & Pojeta 1974; Pojeta & Runnegar 1976) or Loboconcha–Visceroconcha (Salvini-Plawen 1980) concept proposing a Scaphopoda–Bivalvia vs. Gastropoda– Cephalopoda clade; and (2) the ‘helcionellid’ concept (Peel 1991) placing Scaphopoda closer to, or within, the Gastropoda– Cephalopoda lineage (Fig. 1A,B). Neither concept was fundamentally new at the time of proposal. The idea that Scaphopoda and Bivalvia are closely related was introduced by Lacaze-Duthiers (1857−58) who emphasized the similarities in the weakly developed head, pedal morphology and in the formation of mantle and shell. This was further developed by Runnegar & Pojeta (1974; Pojeta & Runnegar 1976, 1979, 1985) proposing the Rostroconchia, a palaeozoic group of laterally compressed, pseudo-bivalved molluscs, as the common stem group of Bivalvia and Scaphopoda and coining the term Diasoma for this lineage. The concept was widely accepted (e.g. © The Norwegian Academy of Science and Letters • Zoologica Scripta, 32, 4, July 2003, pp343–356 343

Blackwell Publishing Ltd.

Molecular phylogeny of Scaphopoda (Mollusca) inferred

from 18S rDNA sequences: support for a Scaphopoda–

Cephalopoda clade

GERHARD STEINER & HERMANN DREYER

Accepted: 15 September 2002

Steiner, G. & Dreyer, H. (2003). Molecular phylogeny of Scaphopoda (Mollusca) inferred

from 18S rDNA sequences: support for a Scaphopoda–Cephalopoda clade. — Zoologica Scripta,

32, 343–356.

The phylogenetic relationships of the Scaphopoda, one of the ‘lesser’ molluscan classes, with

the other conchiferan taxa are far from clear. They appear either as the sister-group to the

Bivalvia (Diasoma concept) or to a Gastropoda–Cephalopoda clade or to the Cephalopoda

alone (helcionellid concept). We compiled a 18S rDNA sequence dataset of 48 molluscan species

containing 17 scaphopods to test these hypotheses and to address questions regarding

high-level relationships with the Scaphopoda. Both parsimony and maximum likelihood trees

show low branch support at the base of the Conchifera, except for the robust clade uniting

Scaphopoda and Cephalopoda. This result is corroborated by spectral analysis and likelihood

mapping. We also tested alternative topologies which scored significantly worse both in tree

length and in likelihood. The 18S rDNA data thus reject the Diasoma in favour of a Scaphopoda–

Cephalopoda clade as proposed in the helcionellid concept. When plotted on the molecular

tree, the pivotal morphological characters associated with the burrowing life style of the

Bivalvia and Scaphopoda, i.e. mantle/shell enclosure of the body and the burrowing foot with

true pedal ganglia, appear convergent in these groups. In contrast, the prominent and tilted

dorsoventral body axes, multiple cephalic tentacles and a ring-shaped muscle attachment on

the shell are potential synapomorphies of Scaphopoda and Cephalopoda. Most of the higher

taxa within the Scaphopoda are supported by the molecular data. However, there is no support

for the families Dentaliidae and Gadilidae. The basal position of the Fustiariidae within the

Dentaliida is confirmed.

Gerhard Steiner & Hermann Dreyer, Institute of Zoology, University of Vienna, Althanstr. 14,

A-1090 Vienna, Austria. E-mail: gerhard.steiner@univie.ac.at

Introduction

Scaphopoda is one of the less diverse major groups of conchiferan

molluscs, with about 520 Recent species (Steiner &

Kabat 2001; Steiner & Kabat 2001) living in all types of

unconsolidated marine sediments (Shimek & Steiner 1997).

Scaphopoda are adapted to their infaunal habit by a tubular

mantle/shell, open at both ends, and a burrowing foot; there

are no gills or osphradia (Steiner 1992; Shimek & Steiner

1997). They are generally considered to be microcarnivores

collecting their prey items (mainly foraminifers) with numerous

captacula — cerebrally innervated cephalic appendages

— and processing them with a large radula apparatus

(Shimek 1988, 1990; Palmer & Steiner 1998).

The phylogenetic relationships of Scaphopoda with the

other conchiferan taxa are far from settled. There are two

competing basic concepts: (1) the Diasoma–Cyrtosoma

(Runnegar & Pojeta 1974; Pojeta & Runnegar 1976) or

Loboconcha–Visceroconcha (Salvini-Plawen 1980) concept

proposing a Scaphopoda–Bivalvia vs. Gastropoda–

Cephalopoda clade; and (2) the ‘helcionellid’ concept (Peel

1991) placing Scaphopoda closer to, or within, the Gastropoda–

Cephalopoda lineage (Fig. 1A,B). Neither concept was

fundamentally new at the time of proposal. The idea that

Scaphopoda and Bivalvia are closely related was introduced by

Lacaze-Duthiers (1857−58) who emphasized the similarities

in the weakly developed head, pedal morphology and in the

formation of mantle and shell. This was further developed by

Runnegar & Pojeta (1974; Pojeta & Runnegar 1976, 1979, 1985)

proposing the Rostroconchia, a palaeozoic group of laterally

compressed, pseudo-bivalved molluscs, as the common stem

group of Bivalvia and Scaphopoda and coining the term Diasoma

for this lineage. The concept was widely accepted (e.g.

Molecular phylogeny of Scaphopoda • G. Steiner & H. Dreyer

Fig. 1 A–D. Competing hypotheses on the

phylogeny of extant conchiferan classes tested

in this study. —A. Diasoma–Cyrtosoma (after

Pojeta & Runnegar 1976) or Loboconcha–

Visceroconcha (Salvini-Plawen 1980, 1990),

with fossil Rostroconchia as stemgroup of

Scaphopoda and Bivalvia. —B. Helcionellid

concept (Waller 1998), with fossil Helcionellida

as stemgroup of Scaphopoda and Cephalopoda;

Scaphopoda–Cephalopoda clade also

according to Grobben (1886). —C. Modified

Visceroconcha concept (Haszprunar 2000).

—D. Scaphopoda–Gastropoda clade according

to Plate (1892) and Simroth (1894).

Engeser & Riedel 1996; Ponder & Lindberg 1997; Reynolds

& Okusu 1999; Salvini-Plawen 1980, 1990; Salvini-Plawen &

Steiner 1996; Wagner 1997) although Steiner (1992, 1996)

pointed out discrepancies in the development of body axes

between Rostroconchia and Scaphopoda.

Connecting Scaphopoda to the Gastropoda–Cephalopoda

line has a similarly long tradition. A close relationship of

Scaphopoda and Gastropoda based on the similarities of

branched head tentacles, prominent dorsoventral body axes

and the occurrence of shell slits was proposed by Plate (1892)

and Simroth (1894) (Fig. 1D). The common derivation of

Scaphopoda and Cephalopoda was favoured by Grobben

(1886) using similar arguments. These hypotheses have

recently gained new support. Waller (1998) elaborated on

and modified the helcionellid concept of Peel (1991) by

deriving the Scaphopoda–Cephalopoda line from helcionellid

monoplacophorans as the sister-group of Gastropoda. In

a recent cladistic analysis of morphological data, Haszprunar

(2000) proposed Scaphopoda as the sister taxon of Gastropoda

and Cephalopoda (Fig. 1C). The alternative to identifying

any of the extant classes of Conchifera as sister-groups

and deriving Scaphopoda directly from an unknown, independent

‘monoplacophoran’ stock was suggested by Edlinger

(1991), while Starobogatov (1974) and Chistikov (1979)

identified the palaeozoic Xenoconchia as the closest relatives

of Scaphopoda. Yochelson (1978, 1979) even considered the

derivation of an un-shelled ancestor.

This variety of competing phylogenetic hypotheses is

partly due to the lack of information provided by the fossil

record. Scaphopoda are the latest to appear in the fossil

record among all conchiferan classes, and there are no obvious

transitional forms connecting them to other molluscs.

The oldest scaphopod reported is the Ordovician Rhytiodentalium

kentuckyesnis Pojeta & Runnegar, 1979, although its

scaphopod nature has been questioned by Engeser & Riedel

(1996), as has that of several Devonian and Carboniferous

scaphopods (Yochelson 1999; Yochelson & Goodison 1999;

Palmer 2001).

It is evident from the competing hypotheses that we

have to deal with convergent morphologies in several organ

systems. The pivotal characters involved (discussed in

Haszprunar 2000; Reynolds & Okusu 1999; Steiner 1992, 1998,

1999a; Waller 1998) are listed in Table 1. Depending on the

topology of the conchiferan phylogenetic tree, at least one of

the sets of similarities must have arisen convergently. If the

Diasoma–Cyrtosoma/Loboconcha–Visceroconcha concept

is favoured, elongation of the dorsoventral body axis (multiple)

cephalic tentacles, and the ring-shaped attachment of

the dorsoventral body muscles (Mutvei 1964; Yochelson et al.

1973) must have evolved convergently in Scaphopoda and the

Cyrtosoma/Visceroconcha. If, however, the ‘helicionellid’

concept is assumed, the ‘ventral’ mantle/shell extension with

similar innervation of the anterior mantle regions, epiatroid

nervous system with fully concentrated pedal ganglia, and

burrowing foot evolved convergently in the Scaphopoda

and Bivalvia. When faced with a tie like that, a morphologyindependent

dataset such as that provided by DNA sequences

may help in addressing this question.

The relationships of the higher taxa within Scaphopoda

are only partly resolved. Cladistic analysis of morphological

344 Zoologica Scripta, 32, 4, July 2003, pp343–356 • © The Norwegian Academy of Science and Letters

G. Steiner & H. Dreyer • Molecular phylogeny of Scaphopoda

Table 1 Potential synapomorphies of

Scaphopoda with Bivalvia, Gastropoda, and

Cephalopoda. Scoring refers to the

presumed ancestral states for each class.

Numbering of characters corresponds to that

of Fig. 7.

Scaphopod character states Bivalvia Gastropoda Cephalopoda

1 Lateroventral extension of mantle-shell enclosing body + – –

2 Burrowing foot with functionally hydraulic component + – –

3 Epiatroid nervous system with true pedal ganglia + – ?

4 Visceral connectives lateral of dorsoventral muscles + – –

5 Prominent dorsoventral body axis with resulting – + +

U-shaped gut

6 More than two cephalic tentacles – – +

7 Ring-shaped attachment of dorsoventral muscles – – +

Fig. 2 Phylogenetic relationships of scaphopod family group taxa

from morphological data after Steiner (1998, 1999a). Taxa in

boldface are represented in the present molecular dataset.

data (Reynolds 1997; Reynolds & Okusu 1999; Steiner 1992,

1998, 1999a) has supported the basic dichotomy separating

the monophyletic Dentaliida and Gadilida and returned

identical topologies within the Gadilida: (Entalinidae

(Pulsellidae (Wemersoniellidae, Gadilidae))) (Fig. 2). In contrast,

relationships of the dentaliid family taxa are less clear

due to the lack of reliable morphological characters for this

systematic level and to uncertain monophyly of certain

families and genera (Reynolds & Okusu 1999; Steiner 1998,

1999a). There is agreement on the basal position of the

Gadilinidae, but the position of the Fustiariidae is controversial.

Reynolds & Okusu (1999) placed it in a derived position

as the sister-group of the Dentaliidae, whereas Steiner (1998,

1999a) recovered it either as a sister-group to the Gadilinidae

or in a position between Gadilinidae and the other dentaliid

families. In addition to these uncertainties, the relationships

of the families Laevidentaliidae, Calliodentaliidae, and Rhabdidae

are completely unresolved.

Having been used only as outgroup taxa in molecular

analyses of other molluscan groups, Scaphopoda are rather

under-represented in molecular data sets. There are three

partial cytochrome-oxidase-I sequences (Hoeh et al. 1998;

Giribet & Wheeler, 2002), two partial engrailed sequences

(Wray et al. 1995), three partial 28S rDNA sequences

(Rosenberg et al. 1997; Giribet & Wheeler, 2002) and five

near-complete 18S rDNA sequences (Winnepenninckx et al.

1996; Giribet et al. 2000; Steiner & Hammer 2000) available

in GenBank. Inadequate representation of the crown-group

conchiferan taxa, at least of Scaphopoda and Cephalopoda,

and/or the use of inadequate molecular markers for resolving

conchiferan relationships (e.g. Rosenberg et al. 1997)

accounts for the lack of information on this question from the

molecular side.

In order to test the morphological data against an independent

data set we obtained near-complete 18S rDNA

sequences of 12 scaphopod species from five families and

aligned them to the existing ones and to a selected set of

gastropods, bivalves and cephalopods, using the available

polyplacophorans as outgroup. The resulting trees provide

a guideline for interpreting the patterns of homology of the

disputed morphological characters and for assessing the

sister-group of the Scaphopoda. Although some of the scaphopod

family group taxa, especially those of the Dentaliida, are

not represented in the present data set, we are able to address

also some of their high-rank internal relationships.

Materials and methods

Most of the specimens were collected by dredging in the

North Atlantic off Trondheim, Norway, and south of Iceland;

Dentalium austini was collected from western Australia and

Fustiaria rubescens from Greece (Table 2).

DNA extraction, amplification and sequencing

Living specimens were fixed in ethanol (96%) or frozen in

liquid nitrogen at −70 °C. Total DNA was isolated from the

entire soft body (small specimens) or pieces of tissue (larger

specimens) of 12 scaphopods (eight Gadilida and four Dentaliida)

with the ‘DNeasy Tissue Kit’ (Qiagen) or with CTAB

(Winnepenninckx et al. 1993a).

Molecular phylogeny of Scaphopoda • G. Steiner & H. Dreyer

Table 2 Species used in the phylogenetic analysis, arranged systematically, with GenBank accession numbers, sources, and sampling location

for those sequenced in this study.

Systematic position Species GenBank no. Authors Sampling locality

Scaphopoda

Dentaliida

Dentaliidae Antalis pilsbry (Rehder, 1942) AF120522 Giribet et al. (2000)

Antalis inaequicostata (Dautzenberg, 1891) AJ389660 Steiner & Hammer (2000)

Antalis vulgaris (Da Costa, 1778) X91980 Winnepenninckx et al. (1996)

Antalis perinvoluta (Boissevain, 1906) AJ389663 Steiner & Hammer (2000)

Dentalium austini (Lamprell & Healy, 1998) AF490594 this study Watering Cove, Burrup

Dampier, NW Australia

Peninsula

Fissidentalium capillosum (Jeffreys, 1877) AF490596 this study BIOICE st. 3188, 62°09.15′–

62°08.73′ N

27°00.32′−27°01.31′ W, 1339–

1338 m

Fissidentalium candidum (Jeffreys, 1877) AF490595 this study BIOICE st. 3172, 60°05.42′–

60°05.68′ N

20°51.30′−20°49.76′ W, 2709 m

Rhabdidae Rhabdus rectius (Carpenter, 1864) AF120523 Giribet et al. (2000)

Fustiariidae Fustiaria rubescens (Deshayes, 1825) AF490597 this study Near Athens, Greece

Gadilida

Entalimorpha

Entalinidae

Entalininae Entalina tetragona (Brocchi, 1814) AF490598 this study Trondheim Fjord, Norway

Hetero- Heteroschismoides subterfissum (Jeffreys, 1877) AF490599 this study

schismoidinae BIOICE st. 3167, 60°54.88′–

60°55.28′ N

22°47.26′−22°47.62′ W, 1897–

1899 m

Gadilimorpha

Pulsellidae Pulsellum affine (M. Sars, 1865) AF490600 this study BIOICE st. 3167, 60°54.88′–

60°55.28′ N

22°47.26′−22°47.62′ W, 1897–

1899 m

Gadilidae

Siphono- Siphonodentalium lobatum (Sowerby, 1860)

dentaliinae AF490601 this study BIOICE st. 3161, 62°37.08′–

62°37.59′ N

23°21.79′−23°21.48′ W, 1230–

1300 m

Polyschides olivi (Scacchi, 1835) AF490602 this study BIOICE st. 3187, 62°09.04′–

62°08.67′ N

27°00.74′−27°01.23′ W, 1327–

1326 m

Gadilinae Cadulus subfusiformis (M. Sars, 1865) AF490603 this study Trondheim Fjord, Norway

Cadulus sp. A AF490604 this study BIOICE st. 3181, 60°52.86′–

60°52.62′ N

26°47.72′−26°48.30′ W, 1543–

1562 m

Cadulus sp. B AF490605 this study BIOICE st. 3173, 60°05.38′–

60°05.59′ N

20°51.23′−20°52.11′ W, 2709–

2710 m

Bivalvia

Protobranchia

Solemyida

Solemyidae Solemya reidi (Bernard, 1980) AF117737 Distel (2000)

Solemya togata (Poli, 1795) AJ389658 Steiner & Hammer (2000)

346 Zoologica Scripta, 32, 4, July 2003, pp343–356 • © The Norwegian Academy of Science and Letters

G. Steiner & H. Dreyer • Molecular phylogeny of Scaphopoda

Table 2 continued.

Systematic position Species GenBank no. Authors Sampling locality

Nuculida

Nuculanoidea

Nuculanidae Nuculana minuta (O. F. Müller 1776) AF120529 Giribet & Wheeler (2002)

Nuculana pella (Linné, 1767) Solemyida AJ389665 Steiner & Hammer (2000)

Yoldiella nana (M. Sars, 1865) AJ389659 Steiner & Hammer (2000)

Neionellidae Neilonella subovata (Verrill & Bush, 1897) AF207645 Giribet & Wheeler (2002)

Pteriomorpha

Arcoidea

Arcidae Arca noae (Linné, 1758) X90960 Steiner & Müller (1996)

Acar plicata (Dillwyn, 1817) AJ389630 Steiner & Hammer (2000)

Mytiloidea

Modiolinae Modiolus auriculatus (Krauss, 1848) AJ389644

Mytilinae Mytilus edulis (Linné, 1758) L33448 Kenchington et al. (1995)

Pterioidea

Pinnidae Pinna muricata (Linné, 1758) AJ389636 Steiner & Hammer (2000)

Atrina pectiniata (Linné, 1767) X90961 Steiner & Müller (1996)

Heteroconchia

Unionida

Unionidae Elliptio complanata (Lightfoot, 1786) AF117738 Distel (2000)

Carditoidea

Carditidae Carditamera floridana (Conrad, 1838) AF229617 Campbell (2000)

Solenoidea

Pharidae Ensiculus cultellus (Linné, 1758) AF229614 Campbell (2000)

Veneroidea

Ungulinidae Diplodonta subrotundata (Issel, 1869) AJ389654 Steiner & Hammer (2000)

Cyamioidea

Sportellidae Basterotia elliptica (Récluz, 1850) AF229616 Campbell (2000)

Gastropoda

Neritopsina

Neritidae Nerita albicilla (Linné, 1758)

Vetigastropoda

Trochidae Monodonta labio (Linné, 1758) X94271 Winnepenninckx et al. (1998)

Fissurellidae Diodora graeca (Linné, 1758) AF120513 Giribet et al. (2000)

Caenogastropoda

Nassariidae Zeuxis siquijorensis (A. Adams, 1852) X94273 Winnepenninckx et al. (1998)

Bursidae Bursa rana (Linné, 1758) X94269 Winnepenninckx et al. (1998)

Calyptraeidae Crepidula adunca (Sowerby, 1825) X94277 Winnepenninckx et al. (1998)

Cephalopoda

Nautiloidea

Nautilidae Nautilus macromphalus (Sowerby, 1848) AJ301606 Bonnaud & Boucher-Rodoni (unpublished.)

Nautilus scrobiculatus (Lightfoot, 1786) AF120504 Giribet & Wheeler (2002)

Coleoidea

Loliginidae Loligo pealei (Lesueur, 1821) AF120505 Giribet & Wheeler (2002)

Sepiidae Sepia elegans (Blainville, 1827) AF120506 Giribet & Wheeler (2002)

Polyplacophora

Ischnochitonina

Chitonidae Liolophura japonica (Lischke, 1873) X70210 Winnepenninckx et al. (1993b)

Acanthochitonina

Acanthochitonidae Acanthochitona crinita (Pennant, 1777) AF120503 Giribet et al. (2000)

Lepidopleurina

Lepidopleuridae Lepidopleurus cajetanus (Poli, 1791) AF120502 Giribet et al. (2000)

Molecular phylogeny of Scaphopoda • G. Steiner & H. Dreyer

Table 3 PCR and sequencing primers used in this study. The NS

primers were designed by White et al. 1990. All other primers were

designed for this project.

Name Position on D. austini Sequence

18A1 −20–0 5′-CCT ACC TGG TTG ATC CTG CCA G-3′

NS3 580–600 5′-GCA AGT CTG GTG CCA GCA GCC-3′

600 r 669–650 5′-CCG AGA TCC AAC TAC GAG CT-3′

NS4 r 1203–1184 5′-CTT CCG TCA ATT CCT TTA AG-3′

NS5 1182–1203 5′-AAC TTA AAG GAA TTG ACG GAA G-3′

1400 f 1473–1495 5′-GAG CAA TAA CAG GTC TGT GAT GC-3′

1400 r 1495–1473 5′-GCA TCA CAG ACC TGT TAT TGC TC-3′

1800 r 1843−1865 5′-ATG ATC CTT CCG CAG GTT CAC C-3′

The complete 18S rRNA gene was amplified in overlapping

fragments using the primer pairs 18A1/600r, NS3/

1800r, NS3/1400r and NS5/1800r (Table 3). The PCR

was performed on a Robocycler 96 (Stratagene) in a 30-µL

reaction mix containing 1.5 mM MgCl 2

, each dNTP at

250 µM, each primer at 0.5 µM, 0.6 units Taq polymerase

(Biotaq Red, Bioline) and the supplied reaction buffer at

1 × concentration. The PCR cycle conditions were as follows:

initial denaturation step of 2 min at 94 °C, 36 cycles of

30 s denaturation at 94 °C, 45 s annealing at 50 °C, and 2 min

primer extension at a 72 °C, followed by a final primer extension

step of 10 min at 72 °C. PCR products were purified

with the Concert Rapid PCR Purification System (Life Technologies)

and sequenced automatically with a range of

primers (Table 3) on an ABI 3700 at VBC-Genomics Bioscience

Research GmbH, Vienna.

Choice of taxa, alignment and phylogenetic analysis

In addition to the five published sequences we obtained 18S

rDNA sequences of 12 species of Scaphopoda resulting in 17

ingroup taxa of a sufficiently wide systematic range to address

major phylogenetic relationships within the group. For the

assessment of the conchiferan relationships we selected 17

bivalve species (six each of the Protobranchia, Pteriomorpha,

and five of the Heteroconchia), seven streptoneuran gastropods,

the four available cephalopod species, and rooted the

trees with three polyplacophoran species.

Sequences were aligned with CLUSTALX 1.8 (Thompson

et al. 1997) applying several combinations of gap penalties

(opening penalty: 10–20; extension penalty: 5–12) and subsequent

manual corrections. The strategy we used was to align

the species of each class first in the ‘multiple alignment mode’

and united these in the ‘profile alignment mode’. The alignment

is available upon request from the corresponding

author (G.S.).

Phylogenetic analyses were performed with PAUP* 4.0b8a

and 4.0b10 (Swofford 1998) on a PC and on the Schrödinger

1 Linux-Cluster at the Central Informatics Service,

University of Vienna. Unweighted heuristic maximum

parsimony (MP) searches were made with 50 random addition

sequences and TBR branch swapping. Bootstrap support

(BS) was assessed by 1000 replicates, each with three random

sequence additions and number of trees limited to 200 per

replicate. Decay indices (DI) (Bremer 1988, 1994) were

calculated using a batch file produced by TREEROT (Sorensen

1996) with 10 random addition sequences, keeping 100 trees

per replicate for each search.

For maximum-likelihood analyses (ML), the most parsimonious

trees (MPTs) were used as starting trees for the

calculation of the model parameters and subsequent branch

swapping. Empirical nucleotide frequencies and the parameters

for the transition/transversion ratio, proportion of invariable

sites, and the gamma shape value were estimated under

the HKY85 model with rate heterogeneity and four categories

of substitution rates following a gamma distribution

(HKY85 + I + Γ model). The resulting values were then set

for subtree-pruning-regrafting (SPR) branch swapping with

rearrangements limited to cross four branches. We tested the

signal in, and the robustness of, the ML tree with the quartetpuzzling

program TREE-PUZZLE 5.0 (Schmidt et al. 2000)

under the same model as the ML analysis and parameters

estimated by the program and with 100.000 puzzling steps.

Four-cluster likelihood-mapping (Strimmer & Haeseler

1997) implemented in TREE-PUZZLE 5.0 was performed with

10 000 randomly chosen quartets to test the relationships of

the four conchiferan classes. Resulting trees were visualized

with TREEVIEW 1.6.1 (Page 1996). Competing alternative

phylogenies were obtained by searching under topological

constraints and subsequently compared with the KH

(Kishino & Hasegawa 1989) and Templeton tests for MP, and

the SH (Shimodaira & Hasegawa 1999) test for ML as implemented

in PAUP* under the RELL option.

The programs PREPARE and HADTREE (Hendy & Penny

1993) were used for spectral analysis using the options for

recoding the data to two-state characters and ‘sum-of-7’ as

in Steiner (1999b) and Steiner & Hammer (2000). As the

number of species is limited to 20 in these programs we

created two data sets, one for the Scaphopoda only and one

with selected molluscan species to address the sisterg-roup

relationships of the Scaphopoda.

Results

The 18S rDNA sequences of scaphopods obtained in this

study range in length from 1808 to 1854 basepairs in the

Dentaliida and from 1915 to 1991 basepairs in the Gadilida.

The increased sequence lengths of the Gadilida are due to

inserts in helices E23_1 and E23_2 to E23_5 of the V4 region

(Table 4), according to the secondary structure model in

Wuyts et al. (2002). Sequence similarity of these inserts is

high and suggests homology. The alignment has 2500

348 Zoologica Scripta, 32, 4, July 2003, pp343–356 • © The Norwegian Academy of Science and Letters

G. Steiner & H. Dreyer • Molecular phylogeny of Scaphopoda

Table 4 Comparison of 18S rDNA sequence

lengths of Scaphopoda with the gastropod

Limicolaria kambeul as reference for the

secondary structure elements. The

representatives of the scaphopod order

Gadilida show extensions in the helices

E23_1 and E23_2 to E23_5 of the V4 region.

Abbreviations: B–E, beginning−end; Dr,

difference to reference.

Species

Length

E23_1

E23_2 to E23_5

B–E Length Dr B–E Length Dr

Reference

Limicolaria kambeul (Gastropoda) 1839 662–713 51 — 714–773 59 —

Dentaliida

Dentalium austini 1842 669–716 47 −4 717–785 68 9

Fissidentalium candidum 1808 649–695 46 −5 696–755 59 0

Fissidentalium capillosum 1812 651–697 46 −5 698–757 59 0

Antalis vulgaris 1865 683–730 47 −4 731–792 61 2

Antalis inaequicostata 1762 635–682 47 −4 683–744 61 2

Antalis perinvoluta 1744 621–668 47 −5 669–727 58 −1

Antalis pilsbryi 1804 648–694 46 −5 695–754 59 0

Rhabdus rectius 1810 649–695 46 −5 696–758 62 3

Fustiaria rubescens 1854 681–727 46 −5 728–793 65 6

Gadilida

Entalina tetragona 1915 675–757 82 31 758–847 89 30

Heteroschismoides subterfissum 1915 675–758 83 32 759–847 88 29

Pulsellum affine 1974 682–774 92 41 775–902 127 68

Siphonodentalium lobatum 1926 677–763 86 35 764–857 93 34

Polyschides olivi 1926 677–763 86 35 764–857 93 34

Cadulus subfusiformis 1986 684–772 88 37 773–915 142 83

Cadulus sp. A 1991 684–776 92 41 777–921 144 85

Cadulus sp. B 1991 685–754 69 18 755–921 166 107

characters, of which 948 are parsimony-informative. Parsimony

analysis returns three MPTs of 3206 steps (CI = 0.522,

RC = 0.3935) (parsimony-uninformative characters excluded).

The strict consensus tree (Fig. 3A and B) is 3220 steps long.

The single ML tree (Fig. 4) has a −ln L = 20050.762 (transition/transversion

ratio = 1.338, proportion of invariable sites

= 0.18, gamma shape parameter = 0.5). Likelihood-mapping

(Fig. 5A) associates 91.1% of all quartets with areas of wellresolved

topologies (tips of the triangle) and 5.1% with the

area of unresolved or star topologies (centre of the triangle).

This indicates a strong phylogenetic signal in the dataset,

although some parts of the trees can be expected to show low

resolution and/or support.

Class relationships

Scaphopoda is a well-supported monophylum in all analyses,

with BS of 92, DI of 9, and quartet-puzzling support (QP) of

74 (Figs 3A, 4 and 6A). There is also high support for the

monophyly of Polyplacophora (BS = 100, QP = 83, DI = 25)

and Cephalopoda (BS = 100, QP = 87, DI = 53). Monophyletic

Gastropoda are not supported by MP (BS = 29) as

the Nerita sequence renders them paraphyletic with regard

to the Cephalopoda and Scaphopoda. However, ML finds

Gastropoda monophyletic with moderate puzzling support

(QP = 63), although the clade appears as sister taxon to the

solemyid bivalves. None of the analyses supports monophyly

of the Bivalvia, which appears as a set of paraphyletic

branches at the base of the conchiferan tree. In general,

branch support and phylogenetic signals in the deep parts of

the tree is low. In contrast to this, the only supported sistergroup

relationship of molluscan classes is that of Scaphopoda

and Cephalopoda (BS = 86, QP* = 51, DI = 10). Parsimony

places Gastropoda as the sister-group of the Scaphopoda–

Cephalopoda clade but with low support (BS = 38, DI = 5).

This topology is not represented in the ML tree where Gastropoda

root within the Bivalvia. However, puzzling support

(QP = 33) for this topology indicates that the signal is also

detected by ML. Spectral analysis results (Fig. 6A) corroborate

the (Gastropoda (Scaphopoda, Cephalopoda)) topology.

The six top-ranked splits are those of the four cephalopod

species, the scaphopod orders Dentaliida and Gadilida, and

the Polyplacophora. The seventh split unites Cephalopoda

and Scaphopoda, whereas the scaphopod split ranks 10th.

Two splits ranking immediately before that of the Scaphopoda

is the first ‘nonsense-split’ uniting the vetigastropod

Monodonta labio with the two coleolids, Loligo pealei and Sepia

elegans (split 8), and a split within the Scaphopoda (split 9).

The 49th split is that uniting the Gastropoda with Cephalopoda

and Scaphopoda. There is also signal for a Gastropoda

and Cephalopoda split which ranks 60th.

The comparison of alternative competing topologies

against the MP and ML trees shows that trees with all conchiferan

classes being constrained as monophyletic is not significantly

worse (Table 5). The latter tree also features a

Scaphopoda–Cephalopoda clade like the unconstrained trees

but with monophyletic Bivalvia. The other trees tested differ

Molecular phylogeny of Scaphopoda • G. Steiner & H. Dreyer

(Scaphopoda + Bivalvia) and (Scaphopoda + Gastropoda)

have 19.5% and 2.3% support, respectively.

Relationships within Scaphopoda

The two ordinal taxa, Dentaliida and Gadilida, are robustly

supported by all analyses (Figs 3A and B, 4, 5B). The monophyly

of the Gadilida is further corroborated by the

homologous extensions in the helices E23_1 and E23_2 to

E23_5; the topology within it is identical in MP and ML trees,

although the internal branches have only moderate to low

support. Note that the puzzling values exceed the bootstrap

values for these branches indicating the ML method being

more efficient in assessing the phylogenetic signal. The basal

dichotomy separating Entalimorpha from Gadilimorpha has

low BS and QP support (Fig. 3B) but takes fifth rank of all

scaphopod splits in the spectral analyses (Fig. 5B). Monophyly

of the Entalinidae and Siphonodentaliidae is fully

supported, whereas the robustness of the Gadilidae is low. The

three Cadulus sequences representing the Gadilidae are set

off together with the single pulsellid species from the Siphonodentaliidae

by a better supported branch (QP = 76). Thus,

Gadilidae and Siphonodentaliidae are not sister taxa.

As in the Gadilida, the dentaliid topology is consistent

between the analyses. However, branch support is considerably

higher for most clades, and bootstrap values tend to be

higher than puzzling values in this subtree. Fustiaria appears

as the first offshoot within the Dentaliida. The monophyly of

the Dentaliidae receives no support, with Rhabdus rectius

(Rhabdidae) emerging from among the dentaliid species.

Furthermore, the genus Antalis represented by four species is

polyphyletic, with only A. inaequicostata and A. vulgaris forming

a clade. Antalis pilsbryi appears closer related to the two

Fissidentalium species than to its congeners.

Fig. 3 A, B. Strict consensus tree (L = 3220, CI = 0.5199, RC = 0.3906)

of three MPT (L = 3206, CI = 0.5221, RC = 0.3935) with support

indices. Bootstrap and puzzling values are above, and decay index

below branches. —A. Subtree showing relationships of Scaphopoda

with the other molluscan taxa. —B. Subtree of Scaphopoda.

in the sister-group relationship of the Scaphopoda and are

significantly worse (P < 0.05) both than the unconstrained

trees and the monophyletic classes trees, both in terms of

tree length and likelihood. The tree with the Diasoma–

Cyrtosoma topology is the least parsimonious and least likely of

those tested. The likelihood-mapping with four clusters represented

by Scaphopoda, Bivalvia, Gastropoda and Cephalopoda

(Fig. 5B), returns the highest support, 51.6%, to the

(Scaphopoda + Cephalopoda) topology. The topologies with

Discussion

Class relationships

Scaphopoda and Cephalopoda are robustly monophyletic in

all analyses. Gastropoda appear as a clade only in the ML

tree, which is likely due to the long branch of the Nerita

sequence and the greater sensitivity to long branch attraction

effects of the MP method. As in previous analyses (Steiner &

Müller 1996, Giribet & Carranza 1999; Steiner 1999b;

Steiner & Hammer 2000), the Bivalvia clade is difficult to

detect, and here they are always paraphyletic at the base of

the conchiferan clade. A likely explanation (see Steiner &

Mueller 1996) posits their low average substitution rates

compared to the other conchiferans. However, the MP and

ML trees found under the constraint for all classes being

monophyletic are insignificantly longer or less likely than the

optimal trees, allowing for assessing their phylogenetic relationships.

Although there are several long-branch taxa — e.g.

the Vetigastropoda (Monodonta labio and Diodora graeca), the

350 Zoologica Scripta, 32, 4, July 2003, pp343–356 • © The Norwegian Academy of Science and Letters

G. Steiner & H. Dreyer • Molecular phylogeny of Scaphopoda

Fig. 4 Maximum likelihood tree of –ln L = 20050.7621 under the

HKY85 + I + Γ model with transition/transversion ratio = 1.3,

proportion of invariable sites = 0.17217, gamma shape parameter =

0.4632, and four categories of substitution rates.

heterodont Bivalvia (except for Carditamera floridana), the

gadilid Scaphopoda, and all four cephalopods — they do not

seem to seriously affect the results as these long branches

do not come together in the trees and do not cluster near the

root. All analyses of the present dataset strongly support the

sister-group relationship of Scaphopoda and Cephalopoda,

whereas there is no apparent signal for a Scaphopoda–

Bivalvia clade. Spectral analysis and likelihood-mapping

using clusters also detect the signal for a Cephalopoda–

Gastropoda clade. It is, however, much weaker in both cases.

This result is further corroborated by the comparison of the

alternative tree topologies (Table 5) showing that they are all

significantly worse than both the best trees and the trees forcing

all classes to be monophyletic. Thus, there is no support

for the Diasoma/Loboconcha concept, its respective topology

being the worst of all alternative trees tested.

In the light of these results, the helcionellid concept as

proposed by Waller (1998) gains considerable support. When

plotted on the topology with monophyletic Bivalvia, the

Fig. 5 A, B. Maximum likelihood mapping using TREE-PUZZLE

5.0. Areas at the corners of the triangle represent one of the three

possible fully resolved four-taxon (quartet) topologies, those along

the edges partly resolved quartets for which it is not possible to

decide between two possible topologies. The central area represents

unresolved quartets. Figures give percentages of 10 000 randomly

chosen quartets in each area. —A. General likelihood mapping

showing 91.1% of all quartets being fully resolved and only 5.1%

unresolved. This indicates high phylogenetic information content of

the 18S rDNA data. —B. Four-cluster likelihood mapping of the

conchiferan classes, testing their phylogenetic relationships. The

corners of the triangle are labelled with the corresponding unrooted

tree topology. The Scaphopoda–Cephalopoda topology receives

greatest support with 51.6% of all quartets, compared to 19.5 and

2.3% for the competing topologies. Note that the topology at the top

corner does not necessarily represent the Diasoma–Cyrtosoma concept

because the root can also be placed at the Bivalvia branch, resulting

in the modified Visceroconcha concept of Haszprunar (2000).

morphological similarities of Scaphopoda and Cephalopoda,

i.e. multiple cephalic tentacles and a ring-shaped dorsoventral

muscle attachment (see Table 1), can be interpreted as

synapomorphies (Fig. 7). The pronounced dorsoventral axis

is already developed in the Gastropoda and therefore

plesiomorphic for this clade. Consequently, the similarities of

Molecular phylogeny of Scaphopoda • G. Steiner & H. Dreyer

Fig. 6 A, B. Spectral analysis. Histogram of signal (positive values on ordinate) and normalized conflict (negative values on ordinate) for the

top 60 splits (abscissa) in the alignment, ranked by net signal (signal minus conflict). —A. Analysis of 20 selected molluscan taxa assessing sistergroup

relationships of Scaphopoda (Polyplacophora: Lepidopleurus cajetanus, Acanthochitona critina; Bivalvia: Solemya togata, Yoldiella nana, Arca

noae, Ensiculus cultellus, Elliptio complanata; Gastropoda: Nerita albicilla, Monodonta labio, Zeuxis siquijorensis; Cephaolopoda: Nautilus

macromphalus, N. scrobiculatus, Loligo pealei, Sepia elegans; Scaphopoda: Antalis perinvoluta, A. pilsbryi, Rhabdus rectius, Entalina tetragona,

Siphonodentalium lobatum, Cadulus subfusiformis). Solid bars represent nodes present in the strict consensus tree. The split uniting Scaphopoda

and Cephalopoda ranks seventh whereas the split uniting Cephalopoda and Gastropoda ranks 60th. —B. Analysis of all scaphopod

species. Solid bars represent nodes present in the unrooted topology of the ML subtree (inset), numbers at branches indicate their rank in

the spectrum.

Scaphopoda and Bivalvia emphasized by the Diasoma/Loboconcha

concept must have arisen convergently. The characters

associated with the burrowing, infaunal habit of these

two groups, i.e. the enclosure of the body by the mantle/shell

and the burrowing foot innervated by fully concentrated

pedal ganglia, are certainly good candidates for convergent

evolution.

More difficult to explain is the derived position of the visceral

connectives median to the dorsoventral muscles shared

by Cephalopoda and Gastropoda, as this is unlikely to be the

result of a similar life-style. However, the shift of nerve position

is not necessarily homologous when the highly concentrated

central nervous system of Cephalopoda is considered.

While the shift in Gastropoda is apparently a result of a

352 Zoologica Scripta, 32, 4, July 2003, pp343–356 • © The Norwegian Academy of Science and Letters

G. Steiner & H. Dreyer • Molecular phylogeny of Scaphopoda

Table 5 Comparison of MP and ML trees with those of competing hypotheses obtained by heuristic searches with topological constraints

enforced. The unconstrained MP and ML trees and the tree with all classes were constrained as monophyletic, showing Scaphopoda and

Cephalopoda as sister-groups. For the MP criterion, tree lengths were tested with KH, for the ML criterion, with SH (see text); the probability

of the null hypothesis (P) is listed. * indicates significant differences (P < 0.05). Note that with reference to the Bivalvia the best trees with all

classes monophyletic are not significantly worse. All trees showing Scaphopoda as not being the sister-group of Cephalopoda are significantly

worse than both the unconstrained and monophyletic trees.

Constraint Tree length Diff. KH-Test (P) –ln L Diff. SH-Test (P)

No [Scaphopoda + Cephalopoda] 3206 — — 20050.762 — —

Monophyletic classes

[Scaphopoda + Cephalopoda] 3213 7 0.3624 20058.367 7.605 0.4328

Scaphopoda + Bivalvia (Diasoma) 3246 40 0.0006* 20078.994 28.228 0.0289*

Scaphopoda + Gastropoda 3236 30 0.0019* 20077.761 26.999 0.0452*

Cephalopoda + Gastropoda 3232 26 0.0079* 20076.278 25.516 0.0452*

Fig. 7 Character-state transitions of characters from Table 1 plotted

on the topology supported by 18S rDNA. Solid bars indicate

nonhomoplastic, empty bars homoplastic characters. 1, lateroventral

extension of mantle-shell enclosing body; 2, burrowing foot; 3, epiatroid

nervous system with true pedal ganglia; 4, visceral connectives lateral

of dorsoventral muscles (change to median of dorsoventral muscles

indicated); 5, prominent dorsoventral body axis with resulting

U-shaped gut; 6, more than 2 cephalic tentacles; 7, ring-shaped

attachment of dorsoventral muscles. Note that the homoplastic

characters 1–3 are associated with infaunal, burrowing life style and,

thus, prone to convergent evolution. Scaphopoda and Cephalopoda

are linked by synapomorphic multiple cephalic tentacles and the

ring-shaped muscle attachment. Both share the prominent

dorsoventral body axis and the U-shaped gut with Gastropoda.

change in developmental timing of muscle and neuronal differentiation,

there are no data on this process for Cephalopoda

(Haszprunar & Wanninger 2000).

The differentiation of a distinct head is yet another problematic

issue. We have not included this character in Table 1

because delimiting character states and subsequent character

coding are not as straightforward as in the other characters.

The head of Scaphopoda, consisting of a movable buccal tube

and a pair of shields from which the captacula arise (Shimek

& Steiner 1997), is clearly separated from foot but not from

the visceral sac or the mantle. It does not protrude from the

shell or directly contact the substratum. Scaphopoda are

thus intermediate between the ‘headless’ Bivalvia and the

Gastropoda and Cephalopoda with their well-developed heads.

They are at a similar level of head differentiation as the

Tryblidia (Recent monoplacophorans) (Haszprunar & Schaefer

1997), with the significant difference that they have preoral

cephalic tentacles like Gastropoda and Cephalopoda. The

assumption of a Scaphopoda–Cephalopoda clade requires a

de-differentiation of the head region in Scaphopoda and the

complete reduction of the (cerebral) eyes which are considered

a potential synapomorphy of Gastropoda and Cephalopoda.

The latter argument is, however, weakened by the

presence of cerebrally innervated eyes on the first ctenidial

filaments of several pteriomorph Bivalvia (e.g. Rosen et al.

1978). Such a de-differentiation of the head and loss of

photoreceptors can, again, be correlated with the acquisition

of an infaunal life style and the anterior elongation of the

mantle/shell. However, as the de-differentiation of the head

region can occur from any level, this character is not very

informative.

Relationships within Scaphopoda

The taxon sampling of the Scaphopoda in this study is not

sufficient to allow definitive conclusions to be drawn on all

relationships of its higher taxa. We have no molecular data

on the dentaliid families Calliodentaliidae, Gadilinidae,

Laevidentaliidae and Omniglyptidae, or on the gadilid deep-sea

family Wemersoniellidae. Moreover, some of the family taxa

are represented by a single species only. Nevertheless, it is

possible to address several important questions and demonstrate

problematic points.

The monophyletic status of the orders Dentaliida and

Gadilida is fully supported, as is that of the Entalimorpha and

Gadilimorpha in the latter taxon. The diphyletic origin of the

Molecular phylogeny of Scaphopoda • G. Steiner & H. Dreyer

Gadilidae was unexpected but is consistent with the analyses

and comparatively well supported. The morphological

character setting Siphonodentaliinae and Gadilinae apart from

all other scaphopods is the anterior constriction of the shell.

Steiner (1992, 1998) noted that not all species of Siphonodentalium

show this constriction. In the light of the present

results, the possibility that this trait evolved independently in

the two groups becomes likely.

The internal branches of the dentaliid clade are generally

shorter than those of the Gadilida but show a similar level of

support. Together with the short-terminal branches in this

subtree, this indicates low substitution rates and/or a recent

series of cladogenetic events. For further phylogenetic

studies on lower systematic levels it seems appropriate to use

faster evolving markers since the genetic distances in the 18S

rDNA become too small within the Dentaliidae. Dentaliidae

itself is at least paraphyletic, with Rhabdus rectius (Rhabdidae)

being part of the quite recent radiation mentioned above.

The addition of more species of the smooth-shelled taxa like

Laevidentaliidae or Calliodentaliidae is likely to make this

even more apparent. The only morphological synapomorphy

of the Dentaliidae is the longitudinally ribbed shell, but

several species of the genus Antalis show this feature transiently

in the juvenile shell only. Moreover, Lamprell & Healy

(1998) demonstrated longitudinal sculpture also in some

Laevidentaliidae. The rather clear separation of Antalis

vulgaris and A. inaequicostata from the other dentaliids could

point to multiple development and/or reduction of shell ribbing.

The present results on dentaliid molecular phylogeny

clearly demonstrate the urgent need of a revision of this

group based on additional morphological and molecular

data.

The position of Fustiaria rubescens (Fustiariidae) at the base

of the Dentaliida is in accordance with the morphological

analyses in Steiner (1998, 1999a). Fustiariidae and Gadilinidae

share a simple anatomy of the posterior mantle edge compared

to the other Dentaliida (Steiner 1991, 1998). However, confirmation

of this character state being plesiomorphic depends

on future analyses including species of the Gadilinidae.

Acknowledgements

We are greatly indebted to the institutions involved in the

BIOICE program, the University of Iceland, the Icelandic

Marine Research Institute, and the Icelandic Institute of

Natural History, and Jon-Arne Sneli (Trondheim Biological

Station, Norway) and Torleiv Brattegard (University of Bergen,

Norway) for the opportunity to join the summer 2000 cruise

where most of the species for this study were collected. We

also wish to thank John Taylor and Emily Glover (Natural

History Museum London), and Kurt Schaefer and Christiane

Todt (University of Vienna) for providing us with specimens.

We are grateful for the discussions with Luitfried Salvini-Plawen

(University of Vienna) and his comments on the manuscript.

The study was partly funded by the Austrian Science Foundation

(FWF), projects P11846-GEN and P14356-BIO.

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