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The Testaria concept(Polyplacophora + Conchifera) updatedLuitfried v. Salvini-Plawena & Gerhard Steinera

a Integrative Zoologie, Universität Wien, Vienna, AustriaPublished online: 19 Nov 2014.

To cite this article: Luitfried v. Salvini-Plawen & Gerhard Steiner (2014) The Testaria concept(Polyplacophora + Conchifera) updated, Journal of Natural History, 48:45-48, 2751-2772, DOI:10.1080/00222933.2014.964787

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The Testaria concept (Polyplacophora + Conchifera) updated

Luitfried v. Salvini-Plawen*,† and Gerhard Steiner

Integrative Zoologie, Universität Wien, Vienna, Austria

(Received 18 November 2013; accepted 21 August 2014)

Mollusca is the second most speciose phylum of animals and includes a highorganizational diversity grouped in eight classes, with the gastropods andbivalves being most familiar. The phylogenetic relationships among the threeaculiferan molluscan classes Solenogastres (Neomeniomorpha), Caudofoveata(Chaetodermomorpha) and Polyplacophora (Placophora, chitons, Loricata), aswell as the sister-group relationship to Conchifera (all other molluscs) are stillunder discussion. On one side, the three aculiferan groups are regarded as amonophyletic clade, Aculifera, contrasted with its sister taxon Conchifera(Aculifera concept). Recent molecular-phylogenomic analyses appear to supportthis hypothesis. Here we point out the plesiomorphic nature of many morpho-logical similarities among the aculiferan taxa and in relation to Conchifera. Onthe other side, a complex of morphological synapomorphies support RecentPolyplacophora as sister group to Conchifera (Testaria concept). The supposedmonophyletic characters for this clade Testaria (Recent Polyplacophora +Conchifera) include the radula complex, the specific differentiation of the midgutinto regions, the dorsoventral musculature, and the excretory organs. A criticalreview of arguments of both hypotheses in the light of present evidence, alsointegrating ontogenetic and palaeontological data, is presented. Morphologicalanalyses favour the Testaria concept, implying a gradual anagenesis from apla-cophoran to polyplacophoran grades and a monophyletic Testaria. Recentlypublished molecular trees differ conspicuously in the position of Aculifera aswell as in conchiferan relationships and show well-supported but conflictingaculiferan and conchiferan topologies. These discrepancies are discussed in con-nection with outgroup representation, the rooting of Mollusca, and refining ofphylogenomic analysis tools. Although considerably improved, current molecularanalyses of ‘deeper’ relationships within major molluscan taxa appear less reli-able than the morphological Testaria concept.

Keywords: Mollusca; Polyplacophora; Neoloricata; Solenogastres; Caudofoveata;phylogeny

Introduction

Recent Mollusca includes eight major organizational clades (classes) which may bearranged into three organizational configurations: the aplacophoran Solenogastres(Neomeniomorpha) and Caudofoveata (Chaetodermomorpha), the Polyplacophora(Placophora), and the Conchifera (Tryblidia or Monoplacophora + Gastropoda +Bivalvia + Scaphopoda + Cephalopoda). In view of a hypothetical ancestral mollusc(HAM), last common ancestor of molluscs (LCA) or molluscan archetype, earlieropinions referred to a (gastropod-like) conchiferan configuration (Salvini-Plawen

*Corresponding author. Email: luitfried.salvini-plawen@univie.ac.at†Luitfried v. Salvini-Plawen sadly passed away after submitting this paper.

Journal of Natural History, 2014Vol. 48, Nos. 45–48, 2751–2772, http://dx.doi.org/10.1080/00222933.2014.964787

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1991, 2006; Haszprunar 1992), mainly based on the predominance of gastropod andbivalve representatives in coincidence with the fossil record. More recently, increasinginterest has been taken in the organization of the ‘lower’, i.e. non-conchiferan,molluscs contrasted and grouped together in a grade Aculifera (replacement ofAmphineura; Salvini-Plawen 1967, 1972), including the aplacophoran groups andPolyplacophora (Salvini-Plawen 1972, 1985; Scheltema et al. 1994; Todt et al. 2008b).Such focus led to three different hypotheses on the basic configuration of Mollusca.

(1) Comparative evaluation of aculiferan characters led to the hypothesis that thethree groups evolved as monophyletic Aculifera in a sister-group relationshipto Conchifera: this is the Aculifera concept. In this scenario, the common,dorsoventrally flattened molluscan ancestor (HAM, LCA) is considered tohave possessed non-specialized extracellular calcium-carbonate deposition, adorsal cuticle and a ventral ciliated gliding sole (Scheltema 1993). The apla-cophoran groups are regarded as progenetic and monophyletically evolved asa sister group to Polyplacophora (Scheltema 1993, 1996).

(2) New data on fossil molluscs with serial dorsal plates or valves (Paleoloricata)have also led to the hypothesis that Aculifera is a monophyletic sister groupto Conchifera (Vendrasco et al. 2004; Sigwart and Sutton 2007; Sutton et al.2012) but with an alternative topology that suggests the ancestral mollusc(LCA) represented a multivalved organism. This scenario suggested that theaplacophoran groups are regressively derived from such multivalved aculifer-ans (Sigwart and Sutton 2007; Vinther et al. 2011, 2012; Sutton and Sigwart2012).

(3) Comparative evaluation of morphological organization among molluscanclasses resulted in the hypothesis that the aplacophoran, polyplacophoranand monoplacophoran (conchiferan) configurations reflect a continuum ofanagenetic differentiation of evolutionary levels with monophyleticPolyplacophora (Neoloricata) + Conchifera: this is the Testaria concept.The basic organization (HAM, LCA) is accepted to belong to the ancestralaplacophoran grade (mantle cover of cuticle and sclerites) and the Recentaplacophoran groups are supposed to reflect diphyletic clades (Salvini-Plawen1972, 1991; Haszprunar 1992; Salvini-Plawen and Steiner 1996; Haszprunaret al. 2008).

Recent molecular-genetic contributions (Kocot et al. 2011; Smith et al. 2011; Vintheret al. 2011) are conflicting in the detail of recovered topologies and thus are also notreliable in view of the basal molluscan branching pattern. However, these analysescoincide in their interpretation of the Aculifera concept. This needs to be borne inmind and the arguments for the three hypotheses suggest the need for a critical,updated review in the light of present evidence.

Analyses of relationships and archetypes

Outgroup comparison reveals that Mollusca have few characters that may point tothe ancestral organization such as the plesiomorphic organization of the ciliaryapparatus in Aculifera (Lundin and Schander 2001). In contrast, several charactersare obviously autapomorphic and the original condition cannot directly be derived

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and polarized (Haszprunar 2000). In fossils, generally only external characters arepreserved, and these are characterized by the differentiation of the mantle cover in theaplacophorans (Solenogastres, Caudofoveata), polyplacophorans (includingPaleoloricata, Neoloricata) and monoplacophorans (as representative of plesio-morphic Conchifera). Thus, the ancestral molluscan organization and its anageneticchanges have to be evaluated synergistically among the different ingroup conditionsthemselves including consideration of larval development and functional dependence.

In search of the internal relationships among Conchifera (monoplacophoranlevel), morphological analyses generally accept a scenario with Tryblidia (of para-phyletic Monoplacophora) as basalmost offshoot, followed by Bivalvia with retainedperipedal pallial cavity, hydrostatically interacting haemolymph with musculature,and course of the lateral (pleuro-visceral) nerve cords outside the dorsoventralmusculature. The hydrostatic musculature system (muscle–muscle antagonism;Shimek and Steiner 1997), true head retractors and the extension of the dorsoventralbody axis support a common branch of Scaphopoda + Gastropoda + Cephalopoda,whereas the formation of a head with eyes, the restriction of the pallial cavity to theposterior and the course of the lateral (pleuro-visceral) nervous system inside thedorsoventral musculature are regarded as synapomorphies for a cladeGastropoda + Cephalopoda (called Visceroconcha or Cyrtosoma; Salvini-Plawenand Steiner 1996; Haszprunar 2000).

In contrast to morphology, molecular genetic results are controversial. Apartfrom non-monophyletic Conchifera (Wilson et al. 2010) or diphyletic Gastropoda,as well as a sister-group relationship of protobranch + filibranch Bivalvia toPolyplacophora + Tryblidia (Serialia) in another analysis (Giribet et al. 2006), furtherresults propose different sister-group relationships (Haszprunar et al. 2008).

Morphologically, analyses of relationships result in two contradicting interpreta-tions of the ‘Aculifera concept’ and the ‘Testaria concept’ (noted above). Within theAculifera concept, Scheltema (1996, p. 57) stated that ‘whether primitive or secondarilyderived through progenesis, structures of the neomenioid aplacophoran anatomycan serve as models for plesiomorphic molluscan structures’. Indeed, anatomically theaplacophoran level has been predominantly accepted as more basal to Polyplacophoraand Conchifera (Salvini-Plawen and Steiner 1996; Scheltema 1996; Haszprunar 2000).Analyses of fossil records, in contrast, led to the adoption of a regressive evolution of theaplacophoran groups from multi-valved aculiferans (Paleoloricata) or even the deriva-tion of Aculifera from Conchifera (Vinther et al. 2011).

Despite the discordant concepts for the polarity of features of the aculiferanmolluscs, conclusions as to how the molluscan archetype (HAM, LCA) was organizedare fairly similar. The analyses and resultant general organization consider most maincharacters, scarcely demonstrable in fossils, in a fairly general consensus (see Salvini-Plawen 1972, 1991; Haszprunar 1992; Scheltema 1993, 1996; Salvini-Plawen andSteiner 1996; Haszprunar et al. 2008; Salvini-Plawen and García-Álvarez 2014):

(1) the dorsal epithelium secretes a cuticle and calcareous products (mantle/pallium);

(2) the ventral ciliated epithelium serves as a locomotory organ (foot);(3) the dorsoventrally, somewhat flattened body is braced and stabilized by

serial pairs of dorsoventral muscle bundles, the lateral ones intercrossingmidventrally;

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(4) an at least terminolateral, U-shaped groove between mantle and foot (mantle/pallial cavity) houses body outlets, areas of glandular epithelium (mucoustracts, hypobranchial glands) and (?) one pair of ctenidia;

(5) there is a paired terminal chemoreceptive (osphradial) sense organ;(6) the nervous system consists of cerebral ganglia, two pairs of medullary nerve

cords along all body (amphineurous tetraneury) and a bucco-pharyngealinnervation;

(7) one-way gut, foregut with radula;(8) a gono-pericardium with heart as invagination of the pericardial roof and

with (at least) one pair of outlets (pericardioducts) for the primary urinefiltered at the pericardial wall (invaginated heart);

(9) open circulatory system;(10) separate sexes and flagellated sperm (aquasperm);(11) development via lecithotrophic trochophore larvae.

For understanding evolutionary pathways, the significance of (monophyletic)clades based on synapomorphies and of paraphyletic grades (stem lineages, levelsor organization) sharing plesiomorphies should be evident. To this end, the mono-phyly of Conchifera is widely accepted (Haszprunar 2000; Haszprunar et al. 2008),even if the internal relationships are still under debate. It is regarded here as a clade,despite results that we regard as molecular oddities (Giribet et al. 2006; Wilson et al.2010; Stöger et al. 2013).

Polyplacophora (also called Placophora or Loricata) includes molluscs with amantle cover of middorsal serial calcareous plates or valves and a peripheral girdlecovered by a cuticle and sclerites. According to that characterization, the Aculiferaconcept defines Polyplacophora as a clade that encompasses all multi-valved molluscsincluding extinct representatives and is thus contrasted with aplacophorans whichposses a cuticle with sclerites but are shell-less (without valves). If, however, thediagnostic anatomical characters of Recent representatives (multiplied ctenidia, sub-divided midgut, etc.) are addressed, the fossil taxa cannot be compared. In theTestaria concept, Conchifera is regarded as a derived polyplacophoran organizationand, therefore Polyplacophora sensu lato would be a paraphyletic grade.

Polyplacophora is subdivided into Paleoloricata and Neoloricata (Bergenhayn 1955),the latter being a clade monophyletically defined by the presence of a ventral shell layercalled the articulamentum. Paleoloricata, on the other hand, represents a paraphyleticgrade (Sutton and Sigwart 2012; Vinther et al. 2012) including the stem-lineage ofNeoloricata and several extinct groups. Within Paleoloricata, Heptaplacota definespalaeoloricates with seven successive homogeneous valves, such as the recently describedKulindroplax Sutton, Briggs, Siveter, Siveter and Sigwart, 2012, though the eight-valvedpalaeoloricate Septemchiton Bergenhayn, 1955 is a misnomer (Salvini-Plawen 1980, p.253, 1991, p. 19; Rolfe 1981). Multiplacophora is the Paleoloricata sister to Neoloricata(Sigwart and Sutton 2007) with 1–3 (or even more?) plate formations in a seven-foldtransverse arrangement (Vendrasco et al. 2004; Vinther et al. 2012). These groups appearto be integrated within the anagenetic stem-lineage of Neoloricata. This implies thestem-lineage of Recent chitons included Neoloricata as a monophyletic taxon (clade) forwhich Eoplacophora Pilsbry, 1893, could be emended (see Bergenhayn 1955, p. 39).

Ihering (1876) introduced Amphineura (= Aculifera Hatschek, 1891) includingSolenogastres (Neomeniadae) and Caudofoveata (Chaetodermata) as the class

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Aplacophora without dorsal calcareous plates in contrast to the class Polyplacophora(Placophora). Aplacophoran organization is thus sensu nomine clearly characterizedby the absence of dorsal shell plates or valves (mantle cover of cuticle and scleritesonly). It is, therefore, misleading to label fossil representatives with plate formations(Paleoloricata) as ‘armoured aplacophoran’ (Sigwart and Sutton 2007; Sutton et al.2012). Aplacophora means a clade incorporating a monophyletic origin of bothSolenogastres and Caudofoveata (Ihering 1876; Scheltema 1993; 1996). With theseparation of two aplacophoran classes, each with rank equivalent toPolyplacophora, the organization of the common ancestor of Aculifera was consid-ered as having had an aplacophoran-like cuticle and calcareous scales (Salvini-Plawen1972). Accordingly, Aplacophora is regarded as a grade of molluscan organizationwhich possesses mantle sclerites but is devoid of dorsal shell formations; it includesboth the Recent clades, Solenogastres and Caudofoveata, as well as possibly fossils,and refers also to hypothetical forerunners at different organizational levels.

It may be noted that the Aculifera and Testaria concepts are based upon similaror even identical underlying tree topologies that differ only in the position of the root.The former places the root between Polyplacophora and Conchifera; the latter withinthe aplacophoran level. The morphological consequences of the different root posi-tions, i.e. the polarity or ‘reading direction’ of character transition series, are outlinedand evaluated below.

Morphological analyses: Aculifera (concept)

The hypothesis of a sister-group relationship between Aculifera and Conchifera isbased uniquely on the different deposition of calcium carbonate of the mantleepithelium (Scheltema 1993, 1996). The unicellularly produced aragonitic scler-ites, as well as the epidermal papillae and the medullar suprarectal commissure,are regarded synapomorphic for Aculifera, whereas the plates or valves(Polyplacophora) and the shell (Conchifera) (Scheltema 1988), as well as thepaired digestive gland separated from the stomach, are supposed to be indepen-dent, convergent formations.

The eight-fold seriality seen in both Polyplacophora and Tryblidia is accepted asa molluscan plesiomorphy (Scheltema 1993), yet this conflicts with the precedingseven-fold mantle cover seen in fossils and recapitulated in larvae. In contrast to theconditions in a segmented coelomate organization and ‘the Monoplacophora withclear serial replication’ (Scheltema 1993, p. 73), eight-fold replication in Tryblidia isrestricted to the dorsoventral musculature with interdigitating lateropedal nerveconnectives (Haszprunar and Schaefer 1996) and in Polyplacophora to the eightplacophoran valves (Friedrich et al. 2002). However, the serial replication is thoughtto be convergently reduced within higher Conchifera and in basal Aculifera, whichundermines the interpretation of an eight-fold seriality as a molluscan plesiomorphy.Nevertheless, the eight valves are accepted as convergent to the shell.

During myogenesis in Polyplacophora (Wanninger and Haszprunar 2002), theprimitive bodywall musculature (muscular grid), as well as the sequence of a multiple(heptamere to octomere) seriality of the dorsoventral musculature, is recapitulated.Along with the differentiation of other muscle sets (Scherholz et al. 2013), theseconditions demonstrate common plesiomorphies of Recent Aculifera (in contrast toeight-fold seriality as a plesiomorphy of total-group Mollusca).

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The proposed synapomorphies for monophyletic aplacophorans in part reflectplesiomorphies in contrast to the more derived Polyplacophora. This also concernsthe suggested progenetic features in aplacophoran groups, such as a one-way gutwithout paired digestive and oesophageal glands (Scheltema 1993). Accordingly, thisconfiguration could represent an early, more basal (plesiomorphic) level of differen-tiation (Scheltema 1993, item 39) preceding Polyplacophora as well as Conchifera,and it coincides with the differentiation displayed in the midgut common in total-group Testaria (see below).

Both the formation of paired oesophageal glands (pharyngeal diverticula) and thecomplex radula apparatus with vesicles in Recent Polyplacophora and Tryblidia areaccepted as molluscan plesiomorphies (Scheltema 1993, 1996). They are thus sup-posed to have evolved in molluscs de novo without preceding structures and then to bereduced again in aplacophorans. Yet there is no corresponding indication neither inaplacophorans, nor particularly with regard to the synorganized differentiation of thewhole gut in Polyplacophora and Conchifera. In Solenogastres, various membersshow a radula support of vesiculous tissue bolsters (incorporating different turgid, or‘chondroid’ cells). All these consolidations, however, refer to certain taxa (genera,families), and with some few exceptions (such as Tegulaherpia myodoryata Salvini-Plawen; cf. Handl and Salvini-Plawen 2001), these are classified within the mostrecently derived order Cavibelonia (e.g. of Pruvotinidae, Simrothiellidae,Amphimeniidae and Dorymeniidae). In Caudofoveata as well, the paired radularsupport is often provided with chondroid cells (such as in Prochaetoderma Thiele,1902). Apart from an equivalent function to support the radula apparatus, there is nosimilarity (or even homology) to conditions in Polyplacophora and Tryblidia.

The original type of the radula is claimed to be of the bipartite type (Scheltema1993; Scheltema et al. 2003), generally called biserial or distichous. Such assertion,based primarily on some simrothiellids, is in contrast to the presence of a monoserialradula in several and particularly more conservative families (Salvini-Plawen 2003a).Moreover, several species (including also the simrothiellids Plawenia Scheltema andSchander, 2000 and Birasoherpia Salvini-Plawen, 1978) have a (repetitive) anlage ofthe paired radula teeth with a primary interconnection (symphysis) as a monoserialorgan that becomes bipartite by median separation (Salvini-Plawen 1988a, 2003b;Handl and Salvini-Plawen 2001). Such repetition points to the monoserial radula typeas the plesiomorphic condition.

Another result lends support for plesiomorphy of the radulogenesis inSolenogastres and Caudofoveata. In contrast to Polyplacophora and Conchifera, inwhich radula teeth and ribbon are separately produced, in Solenogastres a homo-geneous formation takes place without separation between teeth bases and the sub-jacent material or pre-ribbon (Salvini-Plawen 1988a; Wolter 1992). In Caudofoveata,this plesiomorphic condition is present in the youngest teeth of the proximal sheathonly, whereas older teeth are linked by fibrous connections to a radular membrane(Wolter 1992); this fairly parallels (by analogy) the formation of a true ribbon inTestaria.

There is no synapomorphy of a ‘ganglionated nervous system’ in the aplaco-phoran groups; rather the four nerve cords in Caudofoveata are generally medullaryand in Solenogastres they show serial swellings along with regular to scatteredperikarya (Salvini-Plawen 1978; Todt et al. 2008a; Redl and Salvini-Plawen 2009;Faller et al. 2012). This gives evidence that the plesiomorphic amphineury for all

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Aculifera (and Mollusca) subsequently differentiated separately within the two apla-cophoran clades. The terminal section of the lateral nervous system in Aculiferashows (in most cases) a medullary suprarectal commissure (Faller et al. 2012),which innervates the ctenidia and the paired terminal/osphradial sense organ(s)(Salvini-Plawen 1972, 1981; Haszprunar 1987a). It appears that the subrectal positionof the commissure in Conchifera is correlated to the differentiation of the uniformshell and that the aculiferan situation with suprarectal commissure most likely reflectsa plesiomorphy.

The ‘worm shape’ in the Recent aplacophoran groups cannot be considered asynapomorphy of the two classes and their monophyly cannot be supported(Haszprunar 2000; Faller et al. 2012). In Solenogastres there is a narrowing of theentire body with retention of the ciliary-gliding foot as a pedal groove.Caudofoveata, in contrast, has evolved as a group of sediment burrowers by a‘rolling-in’ of the body ventrally, thus reducing the pedal sole and fusing the mantlerims. This process has led to a terminal shift of the posterior mantle grooves withinversion of the mucous tracts and ctenidia (Hoffman 1949; Salvini-Plawen 2003a).As the configurations of the mantle cavity in Solenogastres and in Caudofoveatadiffer considerably (Figure 1B, C), a derivation from each other is mutually exclu-sive and their common differentiation linked to a worm-shaped body is unlikely(Hoffman 1949; Salvini-Plawen 2003a, 2006). The mantle cavities rather were eachreorganized independently (di-phyletically) from common aplacophoran ancestorswith ciliary gliding foot and a posteriorly peripedal mantle cavity (probably with ashape fairly similar to that retained in Polyplacophora; Figure 1A). The separationof the two Recent clades must have occurred from a basal aculiferan organization(Scherholz et al. 2013) from which (with unresolved relationships) the polyplaco-phoran grade is also derived.

The ancestral aculiferan U-shaped mantle cavity with body outlets and mucoustracts most probably also housed one pair of ctenidia. In this hypothetical fore-runner, the mucous tracts themselves were not originally involved in a spawn-producing function (present also in males), but rather were more likely correlatedto clean water in this area for ventilation/respiration (ctenidia). With the evolu-tionary reorganization of the mantle cavity in Solenogastres (the mucous tractregion becoming internalized), the ctenidia were lost. Although Solenogastresappear to have otherwise retained more ‘plesiomorphic molluscan structures’(Scheltema 1996, p. 57; see also Haszprunar 2000), the conventionally ‘morederived’ Caudofoveata (Scheltema 1993, p. 68) could well represent the earliest-derived clade among Aculifera and molluscs (Salvini-Plawen and Steiner 1996) ifthey subsequently underwent a more rapid anagenetic differentiation. Such a sce-nario would imply the absence of ctenidia in Solenogastres as clearly secondary.Alternatively, the characteristic ctenidia could be synapomorphic for Caudofoveataand Testaria (Polyplacophora + Conchifera).

The absence of excretory organs or emunctoria in both aplacophoran groups(Hoffmann 1937; Salvini-Plawen 1988b) is more likely not a synapomorphy ofAplacophora but rather plesiomorphic when comparing the configuration of themolluscan gono-pericardial system in detail. Whereas the ultrafiltration of primaryurine at the auriculo-pericardial wall is common to all molluscs, there is noadaptation of the pericardioducts towards excretory organs to produce secondaryurine in either Solenogastres or Caudofoveata. The pericardioducts of

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Solenogastres directly open into the internalized section of the mantle cavity(spawning ducts). Those of Caudofoveata open into the so-called glandularducts of unknown homology (Figure 1C) which themselves open into the poster-iorly shifted mantle cavity. Although these latter organs may perhaps have

Figure 1. Relationships of the pericardium, pericardioducts and mantle cavity. (A)Polyplacophora (Neoloricata); (B) Solenogastres; (C) Caudofoveata (female). At left, schematicviews from lateral, at right schematic oblique cross sections (as indicated at left by line x)projected from behind (after Salvini-Plawen 2006). ct, ctenidium; em, emunctorium (excretoryportion of pericardioduct); go, gonad; md, glandular duct (of unknown homology); ml, enrol-ling muscle; mt, mucous tract of pallial groove/mantle cavity (black); pc, pericardium; pd,pericardioduct; re, rectum; so, terminal (osphradial) sense organ.

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attained some excretory function (Salvini-Plawen 2003a), they differ in structuretoo much from the typical testarian emunctoria to be considered homologues. Theexcretory organs with the characteristic cell types more plausibly represent anautapomorphy for Testaria.

The heart in many Solengastres and Caudofoveata still represents a dorsally openinvagination of the pericardial roof (Salvini-Plawen and Bartolomaeus 1995); suchcondition is also recapitulated in the vestigial ventricle of Scaphopoda (Shimek andSteiner 1997). In addition, Solenogastres do not have an aorta, but rather have ananteriorly extended, middorsal sinus. This is evidence that the aplacophoran condi-tion is plesiomorphic for Mollusca.

The gono-pericardium of Mollusca results ontogenetically as a single commonorgan complex, only later on is functionally separated into pericardium andgonad(s) (Raven 1958; Pearse 1979); as such it represents an autapomorphy. Inaplacophorans, this single configuration is regarded as a progenetic, retainedcondition, including the interconnection of gonad(s) and pericardium (Scheltema1993, 1996) and would then reflect a plesiomorphy (or separate plesiomorphies inthe two classes). However, in both aplacophoran groups the gono-pericardioductis generally paired, and is in contrast to the condition in Cephalopoda, severalCaenogastropoda and bivalve species (e.g. Anodonta Lamark, 1799) where there isa single connection of pericardium and gonocoel. Solenogastres and Caudofoveatahave paired ciliated ducts (and not just interconnections). Also, in Solenogastresthe variability in these structures points to a possible former existence of propergonoducts (Hoffman 1949; Salvini-Plawen 1972). In addition, at least the connec-tion of the (true, upper) pericardioducts to the mantle cavity in Solenogastres andin Caudofoveata is evidently different and diphyletic (Salvini-Plawen 1972, 2003a;Figure 1B and C).

A sister-group relationship of monophyletic Aculifera + Conchifera (Aculiferaconcept) is also advanced by palaeontologists (Vendrasco et al. 2004; Sigwart andSutton 2007; Sutton et al. 2012) and even a regressive evolution of Aculifera derivedfrom a conchiferan ancestor has been considered (Runnegar and Pojeta 1974; Vintheret al. 2011). The latter scenario with a break-down of the shell into eight plates orvalves is contradicted by the more primitive structure of the mantle inPolyplacophora as well as the nature of the formation of the calcareous hard parts(Haas 1981). Moreover, there is no indication of the vestigial reduction or loss inaculiferans of other conchiferan characters (statocysts, jaws, style-sac type of sto-mach, subrectal commissure).

In view of the records of differently configured Paleoloricata, and given thedearth of unequivocal aplacophoran fossils, the aplacophoran groups are sup-posed to have evolved by mono- or di-phyletic derivation from palaeoloricates(Sigwart and Sutton 2007; Vinther et al. 2011, 2012; Sutton and Sigwart 2012).This hypothesis has been suggested repeatedly in the context of the narrowedbody and reduced valves in the Recent genus Cryptoplax De Blainville, 1818 (seeNierstrasz 1910) and appears to be paralleled in the footless, eight-plated fossilPhthipodochiton Sutton and Sigwart, 2012. This comparison neglects the specificcomparative anatomical conditions. Despite some adaptive changes in Cryptoplaxdue to its unusual form (Plate 1901, p. 351), its organization, including thealimentary tract, retains the typical polyplacophoran (testarian) configuration(Nierstrasz 1910). Similarly, in cephalopods, even under a radical change of

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nutrition, neither the differentiation of the midgut into regions, nor the conchi-feran style-sac type of stomach is abandoned (Salvini-Plawen 1988a, 2003a, 2006).No such recapitulative configuration of the testarian midgut can be traced inRecent aplacophoran groups. Apart from other postulates (loss in the aplaco-phoran groups of all proposed synapomorphies of Testaria), the development ofmuscle systems (Haszprunar and Wanninger 2000) likewise contradicts such ahypothetical scenario of regression and supports the assumed plesiomorphic con-dition in the aplacophoran groups. Thus, in contrast to common plesiomorphicfeatures in Aculifera such as the dorsal seven-fold arrangement of sclerites and thedifferentiation of comparable muscle sets, a regressive derivation of aplacophor-ans from palaeoloricates appears to be less parsimonious and remains speculative.

Morphological analyses: Testaria (concept)

The Testaria concept (Polyplacophora (Neoloricata) + Conchifera) had beenadvanced as result of comparative analysis of the aculiferan molluscs (Salvini-Plawen 1972). Although the monophyly of Testaria rests predominantly on internal,soft-body characters of Recent molluscs, external hard-parts are likewise considered todisplay traces of anagenetic transformations among Aculifera (including extinct repre-sentatives). Recent members of Polyplacophora, Solenogastres and Caudofoveatapossess a chitinous cuticle and an extracellular calcareous deposition of unicellularlyproduced aragonitic sclerites, with the scaly type common to all three groups (sym-plesiomorphy; Salvini-Plawen 1972; Haas 1981).

The LCA of Testaria has conceptually been assumed to exhibit a homoge-neous arrangement of scales (Salvini-Plawen 1972, 1991; Haszprunar 1992;Salvini-Plawen and Steiner 1996; Haszprunar et al. 2008), corresponding to the‘extracellular CaCO3 deposition’ in Scheltema (1993, 1996). Ontogenetic studies,however, have discovered and confirmed a (presumably derived) transient differ-entiation of seven middorsal transverse rows of sclerites (Figure 2) in

Figure 2. Formation of seven middorsal transverse rows of sclerites during development. (A,B) larva of Caudofoveata, Chaetoderma cf. nitidulum (after Gustafson in Nielsen et al. 2007) in(A) ventral view; (B) dorsal view; (C, D) metamorphosing Solenogastres, Nematomenia banyu-lensis (after Pruvot 1890; Salvini-Plawen 1972) in (C) lateral view; and (D) dorsal view(reconstructed); (E) dorsal view of post-metamorphic Polyplacophora, Lepidochitona corrugata(after Kowalevsky 1883). oc, residary ocellus; sd, dorsal sclerites in seven transverse rows; sg,general mantle sclerites.

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metamorphosing Solenogastres as adjacent slender scales (Pruvot 1890; Salvini-Plawen 1972), in larval Caudofoveata as calcium producing papillae and subse-quent sclerites (Nielsen et al. 2007), as well as in post-metamorphosed stages ofneoloricate Polyplacophora as granules to platelets (Kowalevsky 1883) or as rod-like elements to greater fragments or granules (Kniprath 1980). Whereas inPolyplacophora the seven initial uniform shell plates or valves are formed, theseven-fold pattern of sclerites apparently changes after metamorphosis into ahomogeneous arrangement in Solenogastres (see also the postlarva with seventransverse regions bare of sclerites; Scheltema and Ivanov 2002), as well as inCaudofoveata. The seven-fold seriality in the mantle cover is paralleled by myo-genesis (Scherholz et al. 2013) and could be interpreted as a common aculiferan ormolluscan plesiomorphy in the Aculifera concept (see above). This correspon-dence, therefore, suggests the presence of seven middorsal transverse rows ofsclerites, surrounded by homogeneously arranged scales, in the last commonancestor (LCA) of all Recent taxa; the latter also included a somewhat flattenedbody with a ventral foot for ciliary gliding, two pairs of medullary body nervecords (amphineury, tetraneury) and some other ‘polyplacophoran-like features’(Scherholz et al. 2013). Derived from this LCA, three evolutionary lineages(clades) can be traced, although with yet unresolved relationships.

Solenogastres evolved by narrowing the body and the ciliary-gliding foot to apedal groove and the posterior mantle grooves becoming internalized as the spawningducts (Hoffman 1949; Salvini-Plawen 2003a). The formation of extensive glands isrestricted to the foregut, whereas the midgut in Solenogastres remains undifferen-tiated without regionalization.

Second, Caudofoveata species adapted to their burrowing lifestyle by a terminalshift of the posterior mantle grooves, inversion of mucous tracts and ctenidia, as wellas with the reduction of the pedal sole and ventral fusion of the mantle rims(Hoffman 1949; Salvini-Plawen 2003a). The caudofoveate midgut is homogeneousbut longitudinally subdivided in midbody into a laterodorsal midgut duct and anunpaired, ventral midgut sac.

The anagenesis towards the third lineage, Polyplacophora + Conchifera (i.e.Testaria), was introduced in the common ancestor through altering the differentia-tion of the seven-fold pattern of sclerites and the consolidation of the calcareousdeposits as seven uniform valves or plates (Figure 3A). The seven-valved ancestorof Polyplacophora sensu lato is ontogenetically well-documented by recapitulationin neoloricates (Figure 3) and is also supported by evidence from fossil palaeolor-icates (e.g. Kulindroplax; Sutton et al. 2012), thus representing the extinctHeptaplacota.

In the evolutionary line towards Recent polyplacophorans (Neoloricata),later on an eighth valve was added. At this point at the latest, the organizationof Testaria (below) had been differentiated, which then also gave rise toConchifera by unification of the calcium-carbonate depositing fields to a shellgland (compare coalescence of valves in Taki [1932]). The Testaria conceptunderlines the contrast between the common aplacophoran organization (inparticular that of the entire alimentary tract; Figure 4) and the synapomorphiesin Polyplacophora + Conchifera (above all the radula complex and the midgutdivided into different regions; Figure 5). It remains uncertain, however, at whichevolutionary stage the palaeoloricate mantle configuration was paralleled by or

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correlated with the internal reorganization towards the common testarian condi-tions. Yet, it appears to be obvious that in the organization of Heptaplacota atleast, the serial dorsoventral musculature was already concentrated to seven pairsof bundles (see Wanninger and Haszprunar 2002; Scherholz et al. 2013).Consequently, it may be supposed that further internal reorganization of apla-cophoran conditions towards Testaria was already in progress or even settledduring the seven-plated level.

Transversely, seven-fold, multi-plated fossil polyplacophorans with three-partintermediate valves, Multiplacophora such as Diadeloplax and other representa-tives possessed aesthetes (Vendrasco et al. 2004; Vinther et al. 2012) and shellinsertion plates that may be convergent or not to those in Neoloricata. The three-valve partitions are sometimes recapitulated in neoloricates (Heath 1899, figure57; Christiansen 1954, p. 63; Kniprath 1980; Figure 3B) and differentiation ofaesthetes could hypothetically impair consolidation to a single valve. It is there-fore conceivable that the origin of Multiplacophora correlates to a paedomorphicrestoration of the stage of seven-fold, three-portioned valve formations. Moreover,Multiplacophora obviously evolved after the deviation of the Conchifera clade(Figure 8; Sigwart and Sutton 2007) because conchiferans possess no homologuesto aesthetes (Reindl and Haszprunar 1996); the ‘aesthetes’ in Tryblidium (Knightand Yochelson 1960) have been disproved (Erben et al. 1968; Wingstrand 1985).

Figure 3. Polyplacophora: formation of seven shell valves in early juveniles. (A) Lepidochitonacorrugata (after Kowalevsky 1883); (B) Stenoplax heathiana (Heath 1899, figure 57). oc,residual ocellus; sg, sclerites of periphery mantle/girdle.

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The anagenesis towards a clade of Recent Testaria is supported by commoncharacters present in Neoloricata and Conchifera, which clearly discriminate fromthe plesiomorphic organization of the aplacophorans. This monophyly of Testaria,including Polyplacophora (Neoloricata) + the subsequent evolutionary radiation of

Figure 4. Aplacophoran molluscs: Organizational sketch of the alimentary tract with non-regionated midgut, gono-pericardial system and mantle cavity in Solenogastres andCaudofoveata (after Salvini-Plawen 2003a). cg, cerebral ganglion; go, gonad; md, glandularduct; mg, midgut; mgd, midgut duct; mgs, unpaired midgut sack; mt, mucous tract of mantlegroove (in Solenogastres internalized spawning duct); pc, pericardium; pg, pedal gland; ps,pedal shield; so, dorsoterminal (osphradial) sense organ.

Figure 5. Testaria (Polyplacophora + Conchifera): Configuration of the alimentary tract withaxial differentiation of the midgut into regions (after Salvini-Plawen 2006). ae, anterior oeso-phagus; (cr), crop; dfg, paired salivary gland; eg, paired oesophageal gland; ep, paired oeso-phageal pouch; in, intestine; (j), jaw; mg, paired midgut gland; mo, mouth opening; pe,posterior oesophagus; ph, pharynx; pp, pharyngeal pouch; ras, radula sheath; re, rectum; sro,subradular organ; (ss), style sac; st, stomach; vfg, ventral foregut gland(s). Characters inparentheses are present in Conchifera only.

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Conchifera, is shown by a complex of distinct morphological synapomorphies(Salvini-Plawen 1972, 1988a, 2003a, Wingstrand 1985; Salvini-Plawen and Steiner1996, 2006). These include:

(1) The multicellular, extensive calcareous secretion in two layers as valves orshell (coalescence of secretive plate fields to a shell gland in Conchifera;see also coalescence of plates in Taki [1932]).

(2) Such a condition is correlated to the arrangement of the dorsoventral musclebundles according to the eight plates into 2 × 8 pairs, fused to eight pairs inplesiomorphic Conchifera (Lemche and Wingstrand 1959; Salvini-Plawen1969, 2006; Wingstrand 1985); they are subsequently fused in higherConchifera (Salvini-Plawen 1969; Haszprunar and Wanninger 2000; Nielsenet al. 2007).

(3) The lateral (pleuro-visceral) nerve cords run lateral of the dorsoventral mus-culature, rather than between the bundles as in Solenogastres as well as inCaudofoveata (Salvini-Plawen 1972); in Gastropoda they are medial.

(4) The position of the terminal (osphradial) sense organ inside the mantle cavity,where present (it is presumed to be lost in Tryblidia, scaphopods, coleoidcephalopods and nudibranchs as well as stylommatophoran gastropods).According to the chemoreceptive function of these organs (to coordinatespawning? Haszprunar 1987a, 1987b), their position at the posterior end ofthe body appears to be of advantage. In aplacophorans they are at the dorsalborder of the mantle cavity, whereas the internal position inPlacophora + Conchifera parallels the differentiation of extensive dorsalcalcareous deposits as shell formations (see above); it is presumed here tobe correlated with the formation of the posteriormost (VIII) polyplacophoranvalve. The formation of the subrectal commissure in Conchifera appears to becorrelated with the differentiation of the uniform shell. As the ancestralmollusc (HAM, LCA) is accepted (except by palaeontologists) to have lackedany shell, Polyplacophora show an intermediate condition between aplaco-phorans and Conchifera (Salvini-Plawen 2006).

(5) The presence of a subradular sense organ in Polyplacophora, Tryblidia,Scaphopoda and some lower Gastropoda (Salvini-Plawen 1988a); it is absentin bivalves, which have no buccal apparatus and is also missing in allcephalopods (in Nautilus, the term refers to a homonymous organ).

(6) The polyserial radula with a low number of teeth and stereoglossate opera-tion in Polyplacophora, Tryblidia, Scaphopoda, Cephalopoda andGastropoda-Docoglossa (Salvini-Plawen 1988a); this synapomorphy is incontrast to the predominant biserial (originally monoserial) radula in theaplacophoran groups (see above).

(7) The paired radula support (odontophore) consisting of a large vesicle and twocartilages (Figure 6), as well as the medial/anterior cartilages with insertion ofthe approximator muscle (m. impar) in Polyplacophora, Tryblidia and acmeidgastropod limpets (Wingstrand 1985; Salvini-Plawen 1988a). It is lost inBivalvia (which lack a radula) and Scaphopoda.

(8) The presence of radular diverticula, a pair of flat pouches formed from theanterodorsal radula sheath (Lemche and Wingstrand 1959; Salvini-Plawen1988a).

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(9) A separate true radula membrane or ribbon (in contrast to the plesiomorphiccondition of a pre-ribbon in aplacophoran groups).

(10) The division of the midgut into the oesophagus, stomach and intestine(Figure 5).

(11) The differentiation of the oesophagus into an anterior section with paireddorsal ciliary folds as well as a ventral ciliary tract (food channel) and asimpler posterior section (Salvini-Plawen 1988a, 2006).

(12) The anterior oesophagus has paired, dorsolateral pouches or glands (‘phar-yngeal diverticula’ in Tryblidia; Scheltema 1993, 1996).

(13) A stomach with a paired midgut gland (the stomach in Conchifera is furtherspecialized to the style-sac type; Salvini-Plawen 1988a, 2006).

(14) The axially continuous posterior midgut narrowed to a curved or coiledintestine. This is in contrast to Caudofoveata in which the ciliated ductreflects the longitudinally separated dorsal portion of the midgut with its

Figure 6. Testaria: Radula support (ventral view) in (A) Polyplacophora; (B) Trybidia (afterWingstrand 1985). lc, lateral cartilage; mc, medial cartilage; rv, radula vesicle; va, ventralunpaired horizontal approximator muscle (musculus impar).

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ciliary tract, leaving the midgut sac separated (Nierstrasz 1903; Salvini-Plawen 1988a, 2003a).

(15) The presence of emunctoria (differentiation of the pericardioducts to excre-tory organs producing the secondary urine; Figure 7), non-homologous tometanephridia (Hoffmann 1937; Salvini-Plawen 1988b, 2003a). The so-called glandular ducts in Caudofoveata may perhaps likewise be excretory,but at most representing convergence (Salvini-Plawen 2003a, 2006; seeabove).

(16) If the gono-pericardioducts of aplacophorans represent a progenetic,retained plesiomorphy (see above), the exit of gametes via proper gonoductswould appear to be a synapomorphy for Testaria.

Molecular-genetic issues

Phylogenetic analyses using DNA sequence characters often appear unreliable forunravelling the basal molluscan branching pattern. Trees generated from few, mostlyribosomal rRNA, genes failed to resolve monophyly of Aculifera, but also of themolluscs (e.g. Wilson et al. 2010). With the arrival of mitochondrial genome analysesand, in particular, transcriptome (phylogenomic) methods, at least the problem ofcontamination of solenogaster DNA with that of its prey could be solved. Highexpectations were placed on the phylogenetic information content of mitochondrialgenomes (e.g. Dreyer and Steiner 2004, 2006). However, the plesiomorphic genomeorganization, exemplified by the polyplacophoran Katharina tunicata, seems to bepresent in all major groups. The extensive re-organizations found in various taxaapparently occurred subsequent to the split of these evolutionary lineages (Stöger andSchrödl 2013).

Figure 7. Testaria: scheme of the gono-pericardial system (from Salvini-Plawen 2006). ao,aorta; aob, bulb of aorta; at, heart auricle; em, emunctorium (excretory organ); exd, lowerpericardioduct or excretory duct (‘ureter’); exs, excretory sac; gd, gonoduct; gdg, gonoductgland; go, gonad; gpd, gono-pericardial interconnection; pc, pericardium; pd, upper pericar-dioduct (‘reno’-pericardial canal); ve, heart ventricle. Arrows indicate direction of haemolymphflow.

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The great advantage of phylogenomic approaches over direct sequencing is thehigh number of genes made available for tree inference. Smith et al. (2011), Kocotet al. (2011) and Vinther et al. (2012) presented trees based on mostly independentdatasets. The three published trees agree with high support in resolving mono-phyletic Aculifera. However, their position in the tree varied: Aculifera are sistertaxon to either Conchifera (Kocot et al. 2011; Smith et al. 2011) or Cephalopoda(Vinther et al. 2011). Although the respective topologies are mutually exclusive,each is supported by maximum posterior probabilities and bootstrap valuesgreater than 95. Similarly, the topology of the conchiferan taxa differed, alsowith high bootstrap support and posterior probabilities above 0.95. Kocot et al.(2011) and Vinther et al. (2011) showed Scaphopoda as sister taxon to a cladecomprising Bivalvia and Gastropoda (termed Pleistomollusca by the former).Smith et al. (2011), in contrast, resolved a sister group relationship ofGastropoda and Scaphopoda, whereas other studies connected the Scaphopodawith Cephalopoda (Steiner and Dreyer 2003; Wilson et al. 2010; Stöger et al.2013).

Thus, even in cases where hundreds of genes were analysed, the results showcompeting, i.e. mutually exclusive, clades, each with strong statistical support(Kocot et al. 2011; Smith et al. 2011). These discrepancies make aculiferanmonophyly appear less reliable. Apparently, phylogenomic methods requiresome refinement to overcome such discrepancies. A major limitation of the con-sistency of the results is the patchiness of the character matrices. Gene occupanciesbetween 40% and 50% and character completeness of 26–41% translate to theinclusion of taxa or genes lacking at least half of the data, or as much as 74% ofthe characters. Considering the implications of missing data for tree inference, it isnot surprising that topologies vary. The supplementary data provided for thesestudies demonstrate the variation of topologies and the influence of which set oftaxa/genes are used.

Resolving these discrepancies by improving outgroup representation, rootingof Mollusca, and refining phylogenomic analysis tools will reveal different results(Stöger et al. 2013). No matter how severe or insignificant these uncertainties maybe considered, they are certainly sufficient to wonder about the influence ofoutgroup choice and representation and their influence on the position of theroot. This is particularly important here as the unrooted molluscan ingrouptopology of most of the above-mentioned trees does not exclude the monophylyof either Aculifera or Testaria. The decision if any, or which, of the groups isparaphyletic is a question of moving the rooting point by one node in the trees ofSmith et al. (2011) and Kocot et al. (2011). The tree of Vinther et al. (2011),however, cannot be re-rooted to arrive at monophyletic Testaria without disrupt-ing the taxon Mollusca. The possibility of such a slight shift of the root appearswell within the range of the observed topological variation.

Conclusions

According to the interconnected conditions of several synapomorphies, a sister-group relationship of Polyplacophora (Aculifera) and Conchifera is comprehensibleonly via a common testarian forerunner. The view that all those characters shouldrepresent convergences is highly improbable. The speculative postulate that testarian

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synapomorphies could have already belonged to the common molluscan organiza-tion and that the aplacophoran situation, therefore, is secondarily simplified, lackssupport and is not parsimonious. In contrast, Polyplacophora shares several char-acteristics with the more plesiomorphic aplacophorans, characters that differentiatedprior to the autapomorphic derivation of Polyplacophora. These conditions areevidence against the ‘Aculifera concept’ and underline the subsequent common,monophyletic differentiation of Polyplacophora (Neoloricata) and Conchifera as aclade Testaria (Figure 8).

The highly divergent results from several molecular analyses show that these dataare not yet suited to solve the deeper relationships within Mollusca. The support forAculifera (Aculifera concept) may likewise be influenced by shared basal features inthe gene orders of all molluscs, in parallel to the morphological hypothesis. Suchinterpretation, however, is far-reachingly confounded by plesiomorphies, as revealedby the strong support of synapomorphies and thus the weight of evidence for theclade Testaria.

Figure 8. Cladogram of lower molluscan taxa considering the mantle cover as well as thepresence of aesthetes (Paleoloricata other than Heptaplacota and Multiplacophora areomitted).

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Acknowledgements

This is a contribution to the collection Who are the ‘Aculifera’? in memory of ChristofferSchander.

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