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On the Phylogeny of Halichondrid

Erpenbeck, D.J.G.

Publication date 2004

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Citation for published version (APA): Erpenbeck, D. J. G. (2004). On the Phylogeny of Halichondrid Demosponges. Universiteit van Amsterdam.

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Download date:25 Sep 2021 Chapterr 1

AA combined molecular, morphological and biochemical approach onn the phytogeny of halichondrid demosponges (Partt 1: Introduction)

D.. Erpenbeck, J.A.J. Breeuwer and R.W.M. van Soest

11 1 1:: Introduction

"" Indent ich mit meinen spongiologischen Untersuchungen die Saulen des Herkules iiberschreite,iiberschreite, stehe ich vor einer Aufgabe, die in ihrem ganzen Umfange nur mit den Kraften eineseines Heroen bewaltigt werden könnte. " O.E.. Schmidt 1870, Vorwort 2. Kapitel

"While"While I pass with my spongiological work the columns of Hercules, I am facing a task which, to its fullfull extent, can only be performed with the strength of a hero." O.E.. Schmidt 1870, second chapter, preface

Oskarr Schmidt (1870) describes in these few words the difficulties he personally experienced in workingg on the systematics of , caused by the apparent simplicity of these organisms. More thann a century later these uncertainties in systematics are still present at almost all taxonomie levels,, as shown in the following paragraphs of this introduction. Thiss thesis aims to contribute to a phylogeny of the order Halichondrida Gray, a groupp of demosponges that inhabits all world oceans, in almost all latitudes, from the surface down too great depths. The order is named after the genus Halichondria Fleming, and is derived from the translationn from the Greek: "cartilage of the sea" (Hansson 1994), referring to the amorphous shape andd cartilaginous consistency of many taxa. The type species of this order, the "breadcrumb-sponge" (Halichondria(Halichondria panicea Pallas 1766), belongs to the best-studied of all sponges. Despitee recent major advances in sponge systematics (Hooper and Van Soest, 2002), the phylogeneticc systematics of Halichondrida is largely unresolved notwithstanding the fact that the majorr taxa of demosponges are connected with the phylogenetic position of this order. For this reason, iss it important to discuss halichondrid systematics within the context of demosponge systematics, and vicevice versa. Thiss introduction will firstly explore to what extent systematic problems affect the highest taxonomiee level in sponges, then introduce the current demosponge orders and focus on the genera off the order Halichondrida. Finally, the pivotal role of Halichondrida will be explained in the historic contextt of sponge systematics.

1.11 Poriferan taxonomie problems: Phylum level

"Thee phylum Porifera contains those commonly called sponges". Gribble (1998) introduces withh these words the sponges to his readers, probably bearing in mind that the taxon "Porifera" and especiallyy its metazoan () entity is not commonlyy known. Over the history of their systematics (seee below), sponges have been placed in several kingdoms, but now there is no doubt they belong withinn the Metazoa. Independent of the quest for the "Urmetazoan" (Muller et al. 2001), sponges aree regarded amongst the most primitive animals and are the oldest extant animal group. First, fossil demospongee records are dated from around 750 MYA ago and their bauplan is basically unchanged sincee the Late Cambrian (509 MYA, Reitner and Wörheide 2002). The autapomorphies of the Metazoa (ass Porifera + Epitheliozoa / Eumetazoa) are (a) diploid multicellularity, (b) meiosis, (c) oogenesis (off one egg cell and three polar bodies out of one oocyte), (d) spermatogenesis (four identical sperm cellss out of one spermatocyte) and (e) the bauplan of the sperm cell itself (Ax 1995). These characters differentiatee animals clearly from their assumed closest relatives, the Choanoflagellida. Spongess are defined by their biphasic life cycle, filter feeding habit in combination with sessilee adult life form, pinacocytes, aquiferous system, choanocytes and totipotent motile cells (e.g., 12 2 1:: Introduction Axx 1995; Vacelet 1990; Hooper and Van Soest 2002). Several exceptions exist, such as sponges that lackk a mineral skeleton ("keratose" sponges and Chondrosia, Oscarella and Halisarcida), carnivorous spongess (Cladorhizidae) and (fossil and recent) choanocyte-, choanocyte-chamber-, and aquiferous- system-- lacking sponges (Thymosiopsis, Vacelet and Boury-Esnault 1995). Thee possession of (1) belt desmosomes (zonula adhaerens) to form an epithelium, (2) differentiationn of an ectoderm and entoderm and (3) digestive glands, are robust criteria to separate thee more derived animals from the sponges (Ax 1995).

1.22 Poriferan taxonomie problems: Class level

Thee sponge classes are well defined: Calcarea (calcareous sponges) produce limestode "needles" (spicules)) extracellularly, Hexactinellida (glass sponges) possess a syncytium of somatic cells andd triaxonic silica spicules, and Demospongiae produce monaxonic or tetraxonic silica spicules andd secrete spongin (spongin B) via spongioblasts. Intermediate forms, called Sclerosponges (= "corallinee sponges", with a limestone basal skeleton and siliceous spicules) were initially regarded ass a fourth class of sponges, but morphological (Van Soest 1984a; Vacelet 1985) and molecular data (Chombardd et al. 1997; Cao et al. 1998; Cantino et al. 1999; Carvalho and Hajdu 2001; Casiraghi et al.. 2001) showed their polyphyletic, predominantly demosponge, origin. Conversely,, the phylogenetic relationships between the three extant sponge classes are still largelyy undecided, with several competing hypotheses: Boger (1988) divided sponges by their mineral skeletonn type and opposed Calcarea to Hexactinellida and Demospongiae ("Silicacea"). Other authors regardedd the syncytial tissue of the Hexactinellida ("Symplasma") as more discriminating and opposedd them to the non-syncytial Demospongiae and Calcarea ("Cellularia", Reiswig and Mackie 1983;; Mehl and Reitner 1996). Furthermore,, ultrastructural and molecular data provided evidence for a paraphyletic assemblagee of the three classes: Woollacott and Pinto (1995) investigated the ultrastructure of choanocytess and found the flagella bases of Calcarea closer to those of other diploblastic animals thann to Demospongiae and Hexactinellida. Thee theory of metazoan paraphyly found support in various molecular analyses, although theyy failed to provide a congruent picture. The Hsp70 data of Borchiellini et al. (1998) found sponges monophyleticc clustering at the base of the Metazoa. This was similar to the findings of Kim et al. (1999,, 18S data) and Medina et al. (2001, 18S and 28S data), which lacked sufficient support for spongee paraphyly. In Zrzavy et al.'s analysis (1998, 18S data and morphology) sponges clustered paraphyletically,, with siliceous sponges being most primitive. Adam's 18S data (1999) rejected sisterr group relationships of Calcarea to any other demosponge class. Lafay et al. (1992, 28S data) providedd the first molecular indication of a possible Calcarea - "Coelenterata" relationship, which wass subsequently supported by Cavalier-Smith et al. (1996, 18S data) and Schütze et al. (1999, HSP700 data). Collins (1998, 18S data) found Demospongiae and Hexactinellida at the base of the Metazoaa and Calcarea as (an unsupported) sister group of Ctenophora. Borchiellini et al. (2001,18S data)) and Kruse et al. (1998, cPKC data) found Calcarea as the sister group of all other diploblasts, leavingg Demospongiae and the basal Hexactinellida paraphyletic at the base of the metazoan tree. Thee caveats of sponge systematics are not restricted to the higher taxon level. The intra- ordinall relationships of the three classes are basically unresolved as well:

1.33 Poriferan taxonomie problems: Demosponge systematics

Levii (1957) dubbed the Porifera as the last major group of Metazoa in which the orders were still not clearlyy defined. Difficulties are clearly due to the primitive bauplan of this taxon producing a shortage

13 3 1:: Introduction off characters required for a robust phylogenetic reconstruction. Sponges bear only a few different cell typess of which the sclerocytes produce the characteristic (if present) spicules (see glossary). These spiculess are usually arranged to form a distinct skeleton, and can be loosely distributed throughout thee sponge body without order. The size, type, shape, combination of spicules and their skeletal arrangementss are the fundaments of current sponge systematics. Although spicule morphogenesis and evolutionn have been studied (see Dendy 1921; Jones 1997; Uriz et al. 2003), the mechanisms are yet nott fully understood, which prevents an unambiguous phylogenetic interpretation of these characters. Variouss alternative morphological characters such as shape, surface, texture or color can be variable regardingg microhabitat conditions or season, e.g., Jones (1984), Barthel (1991), Schoenberg and Barthell (1997), or are observable only in situ because sponges might shrink and loose their coloration duringg preservation in ethanol. The use of cytological features in sponge systematics has also been examinedd (Boury-Esnault et al. 1994), but these are technically demanding to observe, prone to preparationn artifacts, and their phylogenetic information content is not fully estimated. Biochemicall compounds are a suggested alternative to morphological characters within sponge systematicss (e.g., Bergquist 1978, 1979; Van Soest 1998; Van Soest and Braekman 1999) and the amountt of biochemical data for sponges have increased immensely in the recent literature (Van Soest andd Braekman 1999), after discovery of sponge-produced bioactive and pharmaceutically valuable moleculess e.g. Bergmann and Freeney (1950), Sarma et al. (1993) Faulkner (2000b). Presence and absencee of a particular compound or pathway may be used in systematics and taxonomy. However, bothh Bergquist (1978) and Van Soest and Braekman (1999) draw attention to the importance of a precisee identification, which often suffers from lack of experienced identifiers. Further pitfalls with chemosystematicss are difficulties with homologization of pathways and a strong emphasis on new compoundss in the chemical literature. In contrast with this practice, only reports of similar compounds inn different sponges contain phylogenetic information (potential synapomorphies). Nucleicc acid data provides a new source of characters for (demo)sponge systematics although molecularr systematics is not as established in sponges as it is in other metazoan phyla. To a certain extentt this is due to the difficulties in extracting PCR amplifiable DNA because: (a) various sponge compoundss can have an inhibitory effect on downstream reactions, and (b) their filter-feedingnatur e associatess sponges with a bewildering array of contaminating DNA-templates (endosymbionts, ingestedd micro-organisms), which can inhabit every part of the organism due to the absence of a true epitheliall barrier. Molecularr systematics of higher demosponge taxa has been performed on several taxa such ass "Sclerosponges" (Chombard et al. 1997), Hadromerida (Chombard and Boury-Esnault 1999), "Tetractinellida"" (Chombard 1998), "Lithistida" (Kelly-Borges and Pomponi 1994; Mclnerney et al. 1999),, (Alvarez et al. 2000), Hadromerida (McCormack and Kelly 2002; Kelly-Borges et al.. 1991), Haplosclerida (McCormack et al. 2002) and Calcarea (Manuel et al. 2003) (see Borchiellini ett al. 2000 for an overview). Some of these studies raised more questions than they answered and/or weree incongruent with the morphological classification. The studies of Laf ay et al. (1992), Chombard ett al. (1997), Chombard and Boury-Esnault (1999), McCormack and Kelly (2002) and others suggest thatt several demosponge orders are non-monophyletic or overlapping. This indicates that for all studiess on higher taxa (orders, families), a wider demosponge systematic context must be considered. Forr this reason a brief overview of the current definitions of orders is given here.

1.44 The current classification of demosponges

Thee definition of orders in the most recent revision of the sponges, the "Systema Porifera" (Hooper andd Van Soest 2002), are mostly based on morphological features. The "Systema Porifera" currentlyy accept 16 demosponge orders as valid. Only a few of them are defined by unambiguous

14 4 1:: Introduction synapomorphiess and exceptions are frequent. The orders are listed here under the three subclasses withh a short and idealized description of the diagnostic characters or character combination:

(A)) HOMOSCLEROMORPHA Bergquist (1)) Homosclerophorida Dendy, with spicules, if present, small calthrops and derivates; unique cinctoblastula larvae.

(B)) TETRACTINOMORPHA Levi, Megascleres are tetraxonid and monaxonid, occurring together or separately; microscleres are asterosee forms and derivatives (2)) Spirophorida Bergquist and Hogg, with sigmaspire microscleres (3)) Astrophorida Sollas, with triaene megascleres and asterose microscleres (4)) Hadromerida Topsent, with radiate skeleton of monaxonic spicules and asterose microscleres (5)) Chondrosida Boury-Esnault & Lopes, with marked, collagenous cortex, without megascleres and asterose microscleres (if present) ) (C)) CERACTLNOMORPHA Levi, Spicules are monaxonic or diactinal (oxeas-strongyles) never tetractinal (although modifications to thee ends of some monaxonic spicules), microscleres are diverse but never asterose (6)) Poecilosclerida Topsent, with monaxonal megascleres and meniscoid microscleres (chelae are unique to the order) (7)) Halkhondrida Gray, with confused or plumoreticulate skeleton, monaxonic spicules and no microscleres except microxeas or raphides s (8)) Agelasida Hartmann, with verticillately spined monactine megascleres (9)) Haplosclerida Topsent, with isodictyal reticulation of fibers and/or exclusively diactinic spicules; meniscoid microscleres (never chelae);; microxeas or microstrongyles. This order also comprises the freshwater sponges (10)) Halisarcida Bergquist, without spicule or fibre skeleton, only fibrouscollage n (11)) Verticillitida Steinmann, recent forms are trabecular; no spicules; composed of aragonite (12)) Dictyoceratida Minchin, without a spicule skeleton but an anastomosing spongine fibre skeleton (13)) Dendroceratida Minchin, without spicule skeleton but with a strongly laminated dendritic spongin fibre skeleton (14)) Verongida Bergquist, without spicule skeleton, but with a regular anastomosing spongin fibre skeleton, no distinction between primaryy (ascending) and secondary (connecting) fibers (??)) Additionally there is the "Lithistida" consisting of sponges with a rigid desma skeleton. "Lithistida" is discussed to be a polyphyleticc assemblage (e.g. Kelly-Borges and Pomponi 1994; molecular data), but they are frequently united as an "informal" groupp (Pisera and Levi, 2002). Basedd on the common absence of a mineral skeleton and the presence of a spongin skeleton the orders Dictyoceratida, Dendroceratida andd Verongida (and occasionally Halisarcida) are also called "keratose sponges". However, a combining super-order "Keratosa" is currentlyy rejected due to the disparity of its orders (Bergquist et al., 1998; Hooper and Van Soest 2002:15).

1.55 The order Halichondrida

Thee order Halichondrida lacks clear autapomorphic characters such as chelae, as in Poecilosclerida,, verticillate acanthostyles, as in Agelasida, or incubated cinctoblastula larvae, as inn Homosclerophorida. Van Soest and Hooper (2002) define Halichondrida with a combination of characterss as (ceractinomorph) demosponges with styles, oxeas, strongyles or intermediate spicules off widely diverging sizes, not functionally localized. The skeleton is plumoreticulate, dendritic or confused.. Microscleres are rare and, if present, microxeas, trichodragmata or birotule cladotoxa. Vann Soest and Hooper (2002) distinguish 45 valid genera, which are arranged in five families: Axinellidaee Carter, 1875, Bubaridae Topsent, 1894, Desmoxyidae Halmann, 1917, Vann Soest, Diaz & Pomponi, 1990 and Halichondriidae Gray, 1867. Their definitions predominantly comprisee combinations usually lacking autapomorphies. Overlapping traits are common:

7.5.77 Differences between the families:

Axinellidae,, Bubaridae and Dictyonellidae lack a specialized ectosomal skeleton Bubaridaee in the definition of Alvarez and Van Soest (2002) are of entirely encrusting habit andd bear a skeleton differentiated into a basal layer of interlacing spicules and a perpendicular layer

15 5 1:: Introduction off monactines with bases embedded in the basal skeleton, producing a hispid surface. Four genera aree assigned. AxineUidaee bear ascending spiculo-fibers connected irregulary by loose spicules and short tracts,, or are plumoreticulated with ascending plumose tracts connected by thinner ones or single spiculess They have a velvety or microhispid surface (Alvarez and Hooper 2002). Ten genera are assigned. . Thee Dictyonellidae have a dense organic ectosomal layer, giving the sponge a fleshy appearance.. The choanosomal skeleton is predominantly built of styles and occasionally oxeas or strongyless occur (Van Soest et al. 2002). Ten genera are placed here. Desmoxyidaee sensu Hooper (2002) and Halichondriidae sensu Erpenbeck and Van Soest (2002)) bear an ectosomal skeleton, with the exception of Axinyssa and Laminospongia in the latter. Inn the Desmoxyidae, the ectosomal skeleton consists of smooth or spined, usually centrangulate microxeaa microscleres or modified forms while in Halichondriidae the ectosomal skeleton consists of aa densely confused tangentially arranged crust of oxeas and/or styles. The choanosomal skeleton in bothh families is a confused arrangement of spicules. There is in Desmoxyidae occasionally a widely spacedd reticulation of bundles or tracts of multispicular fibres. In Halichondriidae, only vague tractss of spicules may lead to the surface. Ten genera are assigned to the Desmoxyidae, eleven to the Halichondriidae. .

7.5.22 The family AxineUidae Carter, 1875

Thee type genus Axinetta Schmidt, 1862 has a choanosomal skeleton differentiated in an axial (compressedd or vaguely reticulated) and extra-axial (plumoreticulated) region. Megascleres are styles andd oxeas. Microscleres, if present, are microraphides and trichdragmata. Inn Pararhaphoxya Burton, 1934 the axial skeleton is based on interwoven sinuous strongyles. Thee extra axial skeleton consists of single or plumose tracts of oxeas. Microscleres are absent. Similarr to Pararhaphoxya Phakellia Bowerbank, 1862 has multiple axes of sinuous strongyles,, but they are plumoechinated by styles and connected by secondary tracts of single spicules.. Megascleres are strongyles, strongyloxeas, styles and occasionally oxeas. Thee tubular genus Auletta Schmidt, 1870 has a plumoreticulated skeleton comparable to PhakelliaPhakellia with an axis of sinuous strongyles lining the inner wall of the tube and a nearly isotropic reticulationn of strongyles, plumo-echinated by styles and/or oxeas. Thee skeleton of the cup-shaped Cymbastela Hooper & Bergquist, 1992 is exclusively made off oxeas and differentiated in a compressed or vaguely reticulated axial and a plumoreticulated extra- axiall region. Thee axinellid entity of Dragmaxia Hallmann, 1916 is thought to be uncertain because the choanosomall skeleton is not clearly plumoreticulated, and rhabostyles occur. The choanosomal skeletonn consists of plumose axes of (rhabdo)styles with peripheral, rarely interconnected spicules, whichh point outwards in the periphery. Microscleres are spined raphides and trichodragmata. Inn Dragmacidon Hallmann, 1917 the plumoreticulated choanosomal skeleton is not differentiatedd in an axial- and extra-axial skeleton. Dragmacidon possesses oxeas and styles and occasionallyy trichodragmata. PhycopsisPhycopsis Carter, 1883, Ptilocaulis Carter, 1883 and Reniochalina Lendenfeld, 1888, have a similarr skeletal architecture, similar (but not identical) spiculation and share the possession of distinct (butt not diagnostic, Alvarez et al. 1998) surface processes. Their choanosomal skeletons consist of a compressed,, vaguely reticulated central axis with radiating extra-axial multispicular tracts. Alvarez andd Hooper (2002) remarked that the extra-axial skeleton is not plumo-reticulated as in Axinella, Cymbastela,Cymbastela, or Dragmacidon, but formed of fibres that merge or intricately interconnect in an irregular

16 6 1:: Introduction reticulationn (fibrofascicles). These tracts terminate in surface processes, which are mostly filamentous inn Phycopsis, spatula-shaped in Ptilocaulis and long and flattened in Reniochalina Lendenfeld. Megascleress are strongyles, oxeas-anisoxeas, and styles. In Phycopsis there is a majority of styles inn two size categories with only occasional strongyles and oxeas-anisoxeas, while Reniochalina has oxeas-anisoxeass with microspined tips and only occasionally styles.

1.5.31.5.3 The family Bubaridae Topsent, 1894

Generaa of Bubaridae can be differentiated by the spiculation of the basal skeleton. HymerhabdiaHymerhabdia Topsent, 1892 is the only bubarid genus with monactinal spicules in the basal skeleton:: interlacing rhabdostyles form the basal skeleton with centrotylote and/or bent oxeas out of whichh project bundles of long styloid spicules (styles, subtylostyles and tylostyles). Microscleres are absent. . BubarisBubaris Gray, 1867 has a basal skeleton formed by smooth sinuous or vermicular strongyles orr oxeas, from which bundles or individual styles project perpendicularly to the substrata. Thee basal skeleton of the monotypical genus Cerbaris Topsent, 1898 is formed by acanthose ceroxass (m-shaped oxeas), smooth oxeas, toxostrongyles or strongyles. Monactines (styles, subtylostyless or rhabdostyles) project perpendicularly to the substrata. Microscleres (if present) are raphides. . Stoutt vermicular, smooth tuberculate or annulate strongyles or strongyloxeas form the basal skeletonn of Monocrepidium Topsent, 1898. Single or bundles of styles, subtylostyles and tylostyles projectt perpendicularly to substrata.

1.5.41.5.4 The family Desmoxyidae Hallmann, 1917

Severall desmoxyid genera can be differentiated by the spiculation of their ectosomal skeletons. HigginsiaHigginsia Higgin, 1877, Parahigginsia Dendy, 1924, Halicnemia Bowerbank, 1864 and MicroxistylaMicroxistyla Topsent 1928 share the possession of microxea microscleres that are smooth in the latter andd acanthose in all other genera. HigginsiaHigginsia has ectosomal and subectosomal coarsely spined centrangulate oxeas. Oxeas protrudee tangentially in bundles supported by extra-axial styles. Megascleres are oxeas, strongyles andd styles. Besides microxeas, sometimes raphides occur, singly or in trichodragmata. Inn the monotypical genus Parahigginsia the spined microxeas (in contrast with Higginsia) formm a dense tangentially crust, supported by a dense peripheral choanosomal layer of oxeas. Further inwards,, the choanosomal skeleton becomes an isotropic reticulation of oxeas, which is more compressedd towards the axis Thee encrusting genus Halicnemia has the choanosomal skeleton reduced to a confused reticulatedd basal layer of oxeas and styles. (Tylo-)styles are embedded in the substrate and protrude to thee surface forming an ectosomal skeleton with acanthose microxeas in brushes. Bundless of smooth microxeas form an ectosomal palisade in Microxistyla Topsent 1928, supportedd by plumose tracts of styles. The choanosomal skeleton is a confused reticulation of styles. . AA similar skeleton with different spiculation is found in Myrmekioderma Ehlers, 1870. Here,, the ectosomal skeleton consists of perpendicular brushes of smaller (acanth)oxea megascleres, supportedd by larger (acanth)oxeas (rarely strongyles or styles), which form a choanosomal skeleton inn halichondrid reticulation. The microscleres are raphides in trichodragmata. DidiscusDidiscus Dendy, 1922 bears strong habit similarities to Myrmekioderma, including sinuous surfacee grooves. They have an ectosomal skeleton of smaller oxeas tangential, paratangantial or as

17 7 1:: Introduction palisade,, and a choanosomal skeleton of confused radial tracts of larger oxeas. Didiscus bears, in contrastt to Myrmekioderma, a crust of unique microsclere-derived microrhabds in the ectosome. Thee encrusting genus Julavis de Laubenfels 1936 has an ectosomal skeleton of spiny strongyless and trichodragmata. The choanosome consists of a basal skeleton of spined strongyles, whichh form a halichondrid reticulation, and smooth strongyles and styles singly and in bundles reachingg from the substratum perpendicularly to the surface. Thee encrusting genus Heteroxya Topsent 1898 lacks microscleres. It has a palisade of smaller acanthoxeass as ectosomal skeleton and a halichondrid arrangement of larger acanthoxeas protruding thee ectosome. NegomboNegombo Dendy, 1905 has an tangential surface layer of sanidaster-like acanthose microrhabdss as ectosomal skeleton, which are assumed to be modified microxeas. The choanosomal skeletonn consists of confused tracts of large styles and/or oxeas. Thee desmoxyid nature of the encrusting monotypic genus Acanthoclada Bergquist 1970 is likewisee uncertain. It possesses acanthose cladotoxas and birotuless in a cortex-like ectosomal region consistingg of brushes of long oxeas. The choanosomal skeleton is plumose or hymedesmioid, bearing aa basal skeleton of rhabdostyles of which tracts of styles process perpendicular to surface.

1.5.51.5.5 The family Dictyonellidae Van Soest, Diaz and Pomponi, 1990

Mostt of the members of this family possess long flexuous styles. These styles are the only spicule type inn the genera Dictyonella Schmidt 1868, Scopalina Schmidt, 1862, Phakettia de Laubenfels, 1936, andand Stylissa Hallmann, 1914. Thee type genus Dictyonella has long, curved or flexuous styles in plumose anastomosing and divergingg bundles, which are thinning out towards the surface. PhakettiaPhakettia differs from Dictyonella as its long styles radiate in thick bundles from the dense andd confused central region to and through the surface. ScopalinaScopalina has a grainy choanosomal skeleton with characteristic sharp conules, formed by bundless of thin styles entirely enclosed in spongin and rising dentritically up from a basal spongin plate. . Thee choanosomal skeleton of Stylissa consists of styles arranged in a confused plumose reticulationn with many spicules in confusion. AcanthellaAcanthella Schmidt, 1862, Liosina Thiele, 1899 and Rhaphoxya Hallmann, 1917 share thee possession of oxeote spicules in addition to styles. Rhaphoxya bears anisoxeas, oxeas with telescopicc ends, in combination with styles, which are arranged in loose wavy and anastoming bundles,, ascending towards the surface. Microscleres are smooth and occasionally spined raphides, singlee and in trichodragmata, which are also considered to be long, thin oxeas. Acanthella possesses characteristicc dendritic tracts of interwoven sinuously bent strongyles / strongyloxeas echinated by straightt styles, oxeas and anisoxeas. LipastrotethyaLipastrotethya De Laubefels, 1954 differs from the other dictyonellids by possessing exclusivelyy oxeas. They are arranged in a mass of bundles that project outside and leading to a microhispid,, finely grooved surface. LiosinaLiosina Thiele, 1899, Svenzea Alvarez et al., 2002 and Tethyspira Topsent 1890 are uncertain entitiess of the Dictyonellidae. Liosina lacks definite synapomorphies with either Halichondrida and Poecilosclerida.. The choanosomal skeleton consists of loose bundles of oxeote or stylote spicules. Mudd particles are enclosed in the surface leading to a characteristically muddy surface. Svezea is tentativee as the choanosomal skeleton has a neat (haplosclerid-like) uni- to paucispicular reticulation off styles and sometimes oxeas but possesses granulose cells and huge larvae similar to Scopalina. Thee monotypical genus Tethyspira, Topsent 1890 is a questionable dictyonellid entity as it bears rare

18 8 1:: Introduction spinedd (microtylo)styles auxiliary to parallel bundles of styles in the choanosome.

1.5.61.5.6 The family Halichondriidae Gray, 1867

Fivee of the eleven Halichondriidae genera contain oxeas exclusively as megascleres: Halichondria, Axinyssa,Axinyssa, Epipolasis, Topsentia, and Spongosorites. HalichondriaHalichondria Fleming, 1828 is the type genus of the family and the order Halichondrida Gray. Itt has a tangential reticulation of ectosomal spicules often in combination with large subectosomal spacers,, which provide the sponge a peel-like surface. The choanosomal skeleton consists of confusedlyy arranged ill-defined bundles. Thee ectosomal skeleton of Topsentia Berg, 1899 and Spongosorites Topsent, 1896 is more- or-lesss paratangential and crust-like. The choanosomal skeleton of Topsentia is confused, while in SpongosoritesSpongosorites oxeas form vague tracts. AA specialized ectosomal skeleton of Axinyssa Lendenfeld, 1897 is lacking. Its choanosomal skeletonn consists of perpendicular bundles at periphery, but is otherwise largely confused. EpipolasisEpipolasis de Laubenfels, 1936 has a different poral and oscular side in the type species, whichh is lacking in the other members of the genus. The ectosomal skeleton is a crust of intercrossing spicules,, while the choanosomal skeleton is confused. It is the only genus of this family to contain microscleress (raphides in trichodragmata). HymeniacidonHymeniacidon Bowerbank, 1859 has a skeleton composed entirely of styles and stylotes, whichh are confused with vague ascending bundles in the choanosomal skeleton, and of intercrossing singlee spicules or bundles in the ectosomal skeleton. AmorphinopsisAmorphinopsis Carter, 1887, Ciocalapata de Laubenfels, 1936, and Ciocalypta Bowerbank, 18622 have oxeas in combination with styles. In Amorphinopsis there are small styles and larger oxea, whichh form an ectosomal skeleton of intercrossing megascleres, singly or in tracts, and a confused choanosomall skeleton with irregular tracts of large oxeas. Ciocalapata differs from Amorphinopsis inn the type of styles, possessing larger styles in combination with oxeas in several size categories, formingg a detachable ectosomal skeleton of intercrossing bundles and a choanosomal skeleton off thick trabecular spicule tracts. The choanosomal skeleton of the fistulous genus Ciocalypta Bowerbank,, 1862 consists of a central confused spicular axis of which strong secondary tracts carry thee tangentially reticulated ectosomal skeleton (if present). Thee encrusting and papillate genus Vosmaeria Fristedt, 1885, is not typical for the family as it possessess (sub)tylostyles. They are forming the skeleton of the papillae and, together with oxeas, form aa confused choanosomal skeleton. The tangential ectosomal skeleton consists entirely of oxeas. Thee monotypical genus Laminospongia Pulitzer-Finali, 1983 is laminar and lacks an ectosomall skeleton. It has a wide range of oxeote, strongylote, and stylote spicule forms in a confused ectosomall skeleton. Laminospongia is a not an unambiguous member of Halichondriidae but was placedd here due to the lack of alternative hypotheses.

1.66 History of Demosponge classification and the pivotal role of Halichondrida

Aristoteless was probably the first to describe sponges scientifically (Spongia and "Aplysina" - SarcotragusSarcotragus cf. Schmidt 1862) and among the first to assume that they are animals (cf. Johnston 1842).. Since Aristoteles, the systematic placement of sponges underwent an odyssey through the classificationn of organisms. Belon (1553) for example placed sponges as a new regnum between plantt and animals. Linnaeus (1759) put them in the Algae (Cryptogamae) and later returned them to Animaliaa (1767). Donati (1750) discovered the presence of spicules as skeletal elements in sponges thatt he used for his first classification. It followed pioneering work of Linne, Pallas, Esper, and

19 9 1:: Introduction Montagu.. Lamarck (1813) placed the marine sponges into his "Polypiers polymorphs". Nardo (1833)) separated sponges into three orders: (1) a group comprising horny sponges, (2) sponges with siliceouss spicules, and (3) calcareous sponges. He extended this system in 1849 independently from Hoggg (1842) by two more orders, comprising siliceous-horny and calcareous-horny sponges. Grant (1836)) published, obviously independent from Nardo (cf. Vosmaer 1882-1886), a similar three-order systemm in which sponges were called "Porifera" for the first time. Bowerbankk (1866) adopted Grant's system and named the three orders "Calcarea", "Silicea", andd "Keratosa". Halichondrid genera were widely distributed in Bowerbank's system. Halichondria, Hymeniacidon,Hymeniacidon, and Ciocalypta have been placed in three (out of seven) separate groups of the order "Silicea",, which they shared with members of several other currently valid orders. Phakellia and HalicnemiaHalicnemia were placed with Ciocalypta. Schmidtt (1866) criticized Bowerbanks system sharply for not being genealogical and thereforee unnatural. In his monograph on Adriatic sponges Schmidt (1862) divided all sponges into sixx groups which can roughly be defined as:

(1)) Calcispongiae = calcareous sponges; (2)) Ceraospongiae = horny-fibrous sponges (3)) Gumminaeae = globular siliceous sponges with cortex (4)) Halisarcinae - sponges without skeleton (5)) Corticatae = true siliceous sponges (6)) Halichondriae = Corticatae "with negative characters"

Schmidtt admitted (a century before Hennigian philosophy) that his Halichondriae were based onn negative characters and therefore indefensible. He placed his (Adriatic) samples of Axinella, Acanthella,Acanthella, Scoplaina, Halichondria (as Reniera) in the Halichondriae together with different species off actual Poecilosclerida, Hadromerida, and Haplosclerida. In 1869, he published one of the first phylogeneticc trees of sponges based on a 12-order system (Schmidt 1870), which was later reduced too six in order to equalize their ranks (1880, emended by Zittel). The taxon Halichondriae disappeared inn his system which consisted of: (1)) Hexactinellidae, (2) Calcispongiae, (3) Ceraospongiae, (4) Halisarcinae & Gumminaceae, (5) Tetractinellidaee and (6) Monactinellidae. The latter order ("monaxon" = spicules with only one axis) comprisedd the actual halichondrid taxa among others:

(1)) Renierinae (with reticulate skeleton, with oxeas and strongyles), including Halichondria, Auletta and a mixture of Haploscleridaa and Poecilosclerida); (2)) Chalinopsidinae (with styles, but no chelae and sigma): Axinella, Phakellia, Acanthella and Dictyonellidae, further RaspailiaRaspailia and Clathria (now Poecilosclerida) and others. (3)) Desmacidinae (with chelae, sigma and toxa): mostly Poecilosclerida and Scopalina (4)) Chalinae (with anisotropic skeleton, with oxeas and strongyles) (5)) Suberitinae (with tylote spicules)

Grayy (1867) published a system of "Poriphora calcarea" and "Poriphora silicea" independently andd obviously ignored the work of Schmidt (cf. Vosmaer 1885). He divided the siliceous sponges byy assumed reproductive features ("ovisacs"). Gray founded the family "Halichondriadae" as a mixturee of Halichondriidae (Halichondria, Ciocalypta), and many Haplosclerida, Hadromerida andd Poecilosclerida. His families "Chalinidae" (including Axinella and Acanthella) and the newly erectedd "Phakellidae" (monotypic for Phakellia) were placed closer to keratose sponges than to thee Halichondriidae. Gray's system was sharply criticized by Bowerbank (1874), Schmidt (1868, whoo did not use the taxon Halichondriidae in his work), Haeckel (1872), and Vosmaer (1882-1886). Grayy (1872) published a modified system, which united "Halichondriidae", "Chalinidae", and "Phakellidae"" with six other families in the taxon "Keratospongia".

20 0 1:: Introduction Thee system of Carter (e.g., 1875) comprised eight orders. He erected the taxon "" insidee the "Echinonemata", as sister group to (actual) poecilosclerid taxa:

(1)(1) Carnosa (sponges without skeleton) (2)) Ceratina (keratose sponges without inclusions) (3)) Psammonemata (keratose sponges with inclusions) (4)) Raphidonemata (with only oxea in fibres) (5)) Echinonemata (mostly styles and tylostyles, in fibres), including the taxon Axinellida (6)) Holoraphidota (various spicules, minimum spongin), including Halichondrina (7)(7) Hexactinellida (glass sponges) (8)(8) Calcarea (calcerous sponges)

Thee distinction of siliceous and calcareous sponges was more important for Vosmaer (1886 [1887]). Hee based his system on a dichotomous separation of sponges in "Porifera calcarea" and the "Porifera non-calcarea",, which were organized as follows:

(1)) Hyalospongiae (= Hexactinellida), (2)) Spiculispongiae (no spongine to cement the spicules, spicules loose). Suborders: (A)) Lithistina; (B)) Tetractina (Plakinidae etc.); (C)) Oligosilicina (cf. Chondrosida, Halisarcidae, etc.); (D)) Pseudotetraxonina (cf. Hadromerida). (3)(3) Cornacuspongiae (with spongine to cement the spicules) and the suborders: (A)) Halichondrina (basically every Cornacuspongiae except keratose sponges) (a)) Halichondridae: (with Halichondria, Axitiella, Acanthella, Dictyonella, Phakelia, Auletta, Eumastia andd several Haplosclerida) (b)) Ectyonidae (with Phycopsis, Ptilocaulis, Leucophleus, Ciocalypta, Hymeraphia, Higginsia) (c)) Desmacidonidae (d)) Spongillidae (B)) Ceratina (keratose sponges).

Withh Vosmaer's "Spiculispongiae" and "Cornacuspongiae" a long tradition began of dividing non- hexactinellidd siliceous sponges (= demosponges) in two clades. These two clades were based on differentt definitions by Lendenfeld, Sollas, Hentschel, Dendy and Levi until the recent past (see below),, but their contents were more-or-less comparable. Sollass (1885) introduced the name "Demospongiae" into sponge systematics. He gave calcareouss sponges class-level status and opposed them to non-Calcarea ("Plethospongiae"). Those "Plethospongiae"" were divided into the orders:

(1)) Hexactinellida (glass sponges) (2)) Myxospongiae (all skeleton-lacking sponges) (3)) Demospongiae. (a)) Monaxonidae (spicules with one axis) (b)) Tetractinellidae (spicules with four axes) (c)) Cerospongiae (keratose sponges, assumed to be polyphyletic by Sollas).

Ridleyy and Dendy (1886) divided the Monaxonidae in "Clavulina" (with radiate skeleton and/or cortex)) and "Halichondrina" (with reticulated skeleton and without cortex). Halichondrina were dividedd into:

(1)(1) Homorrhaphidae (with Halichondria) cylindrical spicules (2)(2) Heterororrhaphidae (3)(3) Desmacidonidae (with Agelas) (4)(4) Axinellidae (with Hymeniacidon, Axinella, Phakellia) Vonn Lendenfeld (1887) adopted Sollas' system of opposing Calcarea and non-calcarea ("Silicea"), 21 1 1:: Introduction butt he regarded, in contrast to Vosmaer, the microsclere shape as more valuable character. His classificationn is congruent with Ridley and Dendy (1886; 1887), and divides the "Subclassis Silicea" intoo the following orders:

(1)) Hexactinellida, (2)) Chondrospongiae (no spongin to cement the spicules; asteroid microsleres) (3)) Cornacuspongiae (spongin cements the spicules; meniscoid microscleres) withh the suborder Halichondrina including: (a)) Spongillidae (freshwater sponges) (b)) Homorhaphinae: incl. family Renieridae (with Halichondria, and mostly haplosclerids) (c)) Heterorhaphidiae (d)) Desmacionidae (cf. Ridley&Dendy, 1887) (with Agelas) (e)) Axinellidae: (with Dendropsis (Higginsia), Hymeniacidon, Phakellia, Ciocalypta, Acanthella, Axinella,Axinella, Dragmacidon and Raspailia (now Poecilosclerida)

Withh Vosmaer, Sollas and Lendenfeld a "certain period of stability" (Levi 1957) was reached in spongee systematics. Calcarea were opposed to non-calcarea, and Hexactinellida obtained a specific positionn among the siliceous sponges. Further classificational work in that time was done by Schulze, Ridley,, Dendy, Carter, Thiele, Lundbeck, Kirkpatrick, Wilson, Minchin and Hallman. Their studiess lead to multiple changes of taxon names but were without major impact on the system. Levi (1957)) remarked the only "original contribution" at that time was Dendy's (1922) classification of the siliceouss demosponges, by giving microscleres a higher priority in the classification than megascleres. Dendy'ss system of siliceous sponges is as follows:

(1)) Triaxonida (Hexactinellidae) (2)) Myxospongida (no skeleton) (3)) Euceratosa (horny sponges) (4)) Tetraxonida, which were divided into the following groups based on Hentschel's (1909,1911) suggestions: (A)) Astrotetraxonida (with asterose microscleres, if not secondarily lost) (B)) Sigmatotetraxonida (with sigmatose microscleres, if not secondarily lost) including families: (a)) Lithistidae (b)) Tetillidae (c)) Haploscleridae with Halichondria, and most actual Haplosclerida (d)) Desmacioidae with Bubaris, Axinella, Phakellia, Auletta, Hymeniacidon, Leucophloeus, Higginsia Spongosorites,Spongosorites, Halicnemia and most actual Poecilosclerida (e)) Clavulidae with Didiscus and many actual Hadromerida

Hallmannn (1917 [1916]) erected the taxon "Desmoxyinae" including its actual taxa Halicnemia, HigginsiaHigginsia (Higginsia) and Higginsia (Desmoxya). Comprehensivee classifications were proposed by Hentschel (1923), Topsent (1928) and Dee Laubenfels (1936). Hentschel (1923) kept Vosmaer's and Von Lendenfeld's groups but placed Dendroceratidaa as separate order equivalent to Calcarea, Hexactinellidae, and the demosponge orders.. Additionally he modified the taxon "Bubaridae", which was founded by Topsent in 1894 (containingg the valid genera Bubaris, Cerbaris and Hymerabdia), by adding (poecilosclerid) taxa. Hentschel'ss system was:

(1)(1) Calcarea (2)) Hexactinellidae (3)) Tetraxonida with the suborders: (A)) Homosclerophora, (B)) Sigmatophora, (C)) Astrophora, (D)) Desmophora,

22 2 1:: Introduction

(E)) Astromonaxonellina (most hadromerids) (4)(4) Cornacuspongida with the suborders: (A)) Protorhabdina (microscleres and monactine megascleres) (B)) Poikilorhabdina (various megascleres, often microscleres) including: (a)) Coelosphaeridae with Topsentia, (b)(b) Crellidae with Didiscus (c)) Bubaridae with Bubaris, Hymerabdia, Cerbaris, Monocrepiduim, but also actual Poecilosclerida. (C)) Phthinorhabdina: (simple megascleres, barely microscleres) (a)Axinellidaee (no ectocomal skeleton) with Auletta, Phakellia, Acanthella) (b)Ciocalyptidaee (with ectocomal skeleton) with Halichondria, Eumastia, Axinyssa, Hymeniacidon, Ciocalypta,Ciocalypta, Liosina, Spongosorites and actual Haplosclerida, (D)) Aporhabdina (cf. Dictyoceratida) (5)) Dendroceratida (with spongin spicules)

Topsentt (1928) divided the demosponges into three subclasses based on megasclere morphology. He unitedd most of the halichondrid taxa and re-redefined Hentschel's Bubaridae, including the valid four species: :

(1)) Tetractinellida (four-rayed spicules: orders Lithistida and Choristida with Homosclerophora) (2)) Monaxonellida (one-rayed spicules): (A)Hadromerina:: family Spongosoritidae with Spongosorites (B)Halichondrina,, with the families: (a)) Axinellidae with most modern Halichondriidae: Halichondria, Hymeniacidon, Ciocalypta, Axinella, Ptilocaulis,Ptilocaulis, Phakellia, Dragmacidon, Microxistyla, Topsentia, Higginsia, Ptilocaulis, Dragmacidon,Dragmacidon, Auletta) (b)) Astraxinellidae: (Halicnemia, and some Hadromerida) (c)) Heteroxyidae with Heteroxya (d)) Bubaridae: {Bubaris, Cerbaris, Monocrepidium, Hymerhabdia) (C)) Poecilosclerina with Didiscus (D)) Haplosclerina (3)(3) Ceratellida: (horny sponges, no spicules orders: Dictyoceratina, Dendroceratina

Dee Laubenfels (1936) modified Topsent's system and published a monograph that aimed to unite all knownn sponge genera known at that time. He placed Topsent's Astraxinellidae over various taxa and putt Halichondria and Hymeniacidon in different families. The resulting family Hymeniacidonidae wass based on its fleshy appearance. De Laubenfels' system was as follows:

(1)(1) Keratosa (2)(2) Haplosclerina with (Desmacionidae: Liosina) (3)) Poecilosclerina: with Agelasidae and Phorbasinae (Didiscus, Myrmekioderma, Heteroxya) (4)) Halichondrina (ectosomal skeleton, no specially localized megascleres) with the families (A)) Axinellidae (with hispid ectosome and axial core) which were separated in: (a)) Axinellinae: (no microscleres) Ptilocaulis, Auletta, Axinella, Dragmacidon, Acanthella, Pararhaphoxya,Pararhaphoxya, Phakellia, Phakettia, Phycopis, Spongosorites, Stylissa, Bubaris (b)) Higginsiinae: (with spined micoscleres) Desmoxya, Higginsia, Negombo (B)) Halichondriidae (simple spiculation, smooth spicules, ectosomal skeleton) Ciocalypta, Ciocalapata, Leucophleus,Leucophleus, Halichondria (C)) Semisuberitidae as an Poecilosclerida/Hadromerid assemblage (D)) Hymeniacidonidae: (simple spiculation, smooth spicules, fleshy appearance) Hymeniacidon, Acanthella,Acanthella, Dictyonella (E)) Monanthidae (5)) Hadromerina (6)) Epipolasida (like Hadromerina, but with oxeas and styles instead: Negombata, Negombata, Parahigginsia, Topsentia, Epipolasis,Epipolasis, Axinyssa) (7)) Choristida (8)) Carnosa

Levii (1951) compared Topsent's findings with his own ontogenetic observations. The orders 23 3 1:: Introduction Halisarca,, Poecilosclerida and Haplosclerida were found to be viviparous, Hadromerida and Homosclerophoridaee (Levi 1953a) to be oviparous. The order Halichondrida was according to Levi (1951)) "artificiel et heterogene" and the viviparous species (such as Halichondria and Hymeniacidori) wouldd have to be separated from the oviparous (such as Axinella) Levii stated that three classes Hexactinellida, Calcarea and Demospongiae in their entities were welll defined, but their reciprocal relationships were completely unknown (Levi 1957). He remarked onn the congruence of most previous authors in dividing the demosponges into two groups (see fig.1), butt criticized the poorly internal division, especially of the Cornacuspongiae/Sigmaxinellidae group (cf.. Halichondrida, Poecilosclerida, Haplosclerida and keratose sponges). He found sponges without skeletonss impossible to classify; the position of the Plakinidae (now separated as Homosclerophorida) ambiguous;; and the Halichondrina (sensu Topsent), especially the family Axinellidae, a pitfall in the spongee systematics. Levii regarded Axinellidae and the skeleton lacking Myxospongida (consisting of Halisarca (noww order Halisarcida) and Oscarella, (now order Homoscleromorpha) as intermediate morphological formss connecting the two demosponge groups (Dendroceratida - Dictyoceratida - Haplosclerida - Poecilosclerida)) and (Homosclerophorida - Tetractinellida - Hadromerida - Epipolasida, fig. 1). Basedd on his ontogenetic data, which was subsequently extrapolated for the entire class, Levi dividedd demosponges into two groups (1953b). These groups were primarily defined by reproductive featuress from his own observations. Halichondrida and Myxospongida were split. The first group comprisedd the viviparous "Ceractinomorpha" with their solid, incubated parenchymella larvae, comprisingg the Dendroceratida, Dictyoceratida, Haplosclerida, Poecilosclerida, and Halichondrida s.s.s.s. (without Axinellidae), with Halisarca (now Halisarcida) as the most primitive taxon (fig. 1).. Opposed to the Ceractinomorpha were the oviparous Tetractinomorpha with (the viviparous)

Myxospongida a Oscarella a Halisarca a (noww Homosclerophorida) (noww Halisarcida)

Homosclerophorida a Dendroceratida a '' Spiculispongiae Vosmaer r Cornacuspongiae e Tetractinellida a Chondrospongiae e Lendenfeld d Cornacuspongiae e Dictyoceratida a Tetractinellidae e Sollas s Monaxonida a Hadromerida a Tetraxonia a Hentschel l Cornacuspongiae e Haplosclerida a Astrotetraxonida a Dendy y Sigmatotetraxonida a Epipolasida a \\ Tetractinomorpha Levi i Ceractinomorpha a Poecilosclerida a

Halichondrida a Axinellida a Halichondridaa s.s (Axinellidae,, Bubaridae, Desmoxyidae) (Halichondriidaee and Hymeniacidonidae)

Fig.. 1: Schematic view on historic classification of demosponges and the system of Levi (1953). 24 4 1:: Introduction OscarellaOscarella (now Homosclerophorida) as the most primitive taxon, and Hadromerida, Tetractinellidae, andd Axinellidae separated from Halichondrida. Levii (re-) erected (1955) the order Axinellida, with nine (assumed oviparous) families, and combinedd Axinellida, Hadromerida, and Epipolasida to the superorder Clavaxinellida - assuming theirr close relationship (Levi 1956, 1957). The Homosclerophorida received subclass status in Levi'ss later (1973) classifications equal to Ceractinomorpha and Tetractinomorpha together with the re-discoveredd (Hartman 1969) Sclerospongiae. Levi's system of the Demosponges (1973) was as follows: :

(1)(1) Homosclerophorida (viviparous) (2)) TectractinomOTpha (oviparous) (A)) Astrophorida (B)) Spirophorida (C)) Desmophorida (and other litistid sponges) (D)) Hadromerida (E)) Axinellida (choanosomal skeleton axial condensed and indiscriminate presence of styles and oxea) (a)) Axinellidae: Axinella, Phakellia, Auletta, Ptilocaulis, Acanthella, Dragmacidon (b)) Bubaridae: Bubaris, Monocrepidium (c)(c) Desmoxyidae; Higginsia, Parahigginsia, Halicnema (d)) Trachycladidae (e)) Rhabdermiidae (f)) Sigmaxinellidae (g)) Raspailidae (h)(h) Euryponidae (i)) Hemiasterellidae (3)) Ceractinomorpha (vivipar) (A)) Dictyoceratida (B)) Dendroceratida (C)) Haplosclerida (D)) Poecilosclerida with families Agelasidae (Agelas) and Latrunculidae (Didiscus) (E)) Halichondrida (defined by the ectosomal tangential skeleton and confused choanosomal skeleton) (a)) Halichondriidae: (basically oxeas): Halichondria, Trachyopsis (Topsentia), Ciocalypta, Amorphinopsis (b)) Hymeniacidonidae: (basically styles): Hymeniacidon, Leucophleus (4)(4) Sclerosponges (which obtained class status in 1970, Hartman and Goreau 1970)

Levi'ss system was extended by Bergquist (1978) and Hartman (1982), who explored additional supportingg characters - mostly biochemical, reproductive and ultrahistological. However, Bergquist (1978)) reported her first doubts about the monophyletic entity of Tetractinomorpha. Nevertheless, she placedd the Bubaridae within the Axinellidae and extended the Ceractinomorpha with the new keratose orderr Verongida, which did not possess a viviparous life cycle:

(1)) Homoscleromorpha (2)) Tetractinomorpha: (A)) Choristida, (B)) Spirophorida, (C)) Lithistida, (D)) Hadromerida, (E)) Axinellida with the families Axinellidae, Desmoxyidae (Desmoxya, Myrmekioderma), Agelasidae (Agelas), Raspailidae,, Rhabdermiidae, Sigmaxinellidae, Hemiasterellidae (3)(3) Ceractinomorpha: (A)) Halichondrida (families Halichondriidae, Hymeniacidonidae), (B)) Poecilosclerida, (C)) Haplosclerida, (D)) Dictyoceratida, (E)) Dendroceratida (with Halisarcida) (F)) Verongida

25 5 1:: Introduction Bergquistt (1980a) separated an order "Nepheliospongida" from Haplosclerida based on chemical compounds.. The new order was later renamed "Petrosida" as the resemblance between recent petrosiidss and fossil nepheliospongiids was not strong. Recently (1996) she split a monogeneric order Halisarcidaa from the Dendroceratida. Hartmann (1982) elevated Agelasida in his demosponge classification inside the Tetractinomorphaa at the order level:

1)) Homoscleromorpha: Order Homosclerophorida 2)) Tetractinomorpha: Orders Astrophorida (Choristida), Spirophorida, Lithistida, Hadromerida, Axinellida, and Agelasida a 3)) Ceractinomorpha: Orders Dendroceratida, Dictyoceratida, Verongiida, Haplosclerida, Petrosiida, Poecilosclerida,, Halichondrida

Morphologicall analyses of Vacelet (1985), Van Soest (1984a) and Reitner (1992), as well as molecular (Chombardd et al. 1997) and chemo-taxonomic data (review in Wórheide 1998) unequivocally demonstratedd that the class Sclerospongia was polyphyletic, and its family Astroscleridae closely relatedd to Agelasidae. Vann Soest (1984b) independently from Hooper (1984) pointed out inconsistencies in the currentt classifications in their analyses of Poecilosclerida and Axinellida, respectively. They found, afterr the introduction of cladistic character analyses in sponge systematics (Van Soest 1985), the Levi- Bergquist-Hartmann classification with its distinction between Ceractinomorpha and Tetractinomorpha unparimoniouss and suggested the re-merging of Axinellida in Halichondrida. Van Soest (1987) presentedd evidence for the paraphyly of the two major demosponge subclasses in a cladistic approach. Thee taxon Tetractinomorpha was only based on the absence of primitive reproduction features. Thee placement of Axinellida in Tetractinomorpha was based on solely hemiasterellid asters and

. . in in c c a> a> oo Halichondriidae O O Q Q

dencKJticc tracts ^^ X

axiall condensaticV corrugatfed/ridged ^>L g5jfhigh spicule density ^kk surfacéV^ ^^ 7_^F vague tracts ^^^ ^V ^< JT spicules in confusion

^^^ ^V JB loss of reticulate skeleton

^^^ ^P fleshy ectosome

11 0MT collagenous mesohyle .. - J 3 )^1 plumoreticulate architecture ^Jll sinuous strongyles WW non-localized interchangeable styles and oxea Fig.. 2: The phylogenetic relationships of the halichondrid families as proposed by van Soest et al. (1990).

26 6 1:: Introduction plesiomorphicc ovipary, and therefore problematic. The plumoreticulate architecture would have to be evolvedd twice. In addition, the placement of halichondrids in Ceractinomorpha was problematic as larvall morphology differed from the remaining Ceractinomorpha and different types of larvae were foundd in Halichondrids (Wapstra and Van Soest 1987). Furthermore,, ceractinomorph taxa were found to be oviparous (verongids, agelasids, petrosiids, desmacellids,, e.g., Bergquist 1978, 1980b; Hoppe and Reichert 1987). It was unlikely that vivipary occurredd only once in sponges - it is reported from several other groups such as mollusks and ascidians. Independently,, Hooper (1990, 1991) observed more close relationships of the tetractinomorph family Raspailidaee to the ceractinomorph family Microcionidae than to other axinellid groups, supporting thee concept of de Laubenfels (1936). Consequently (Clav-)Axinellida sensu Levi was abandoned and itss families partially (Axinellidae and Desmoxyidae) merged with Halichondrida (Van Soest et al. 1990).. Van Soest et al. (1990) erected the family Dictyonellidae as a sister group of Halichondriidae too cope with genera of the polyphyletic order Axinellida. In an (unsupported) phylogeny(fig. 2), they placedd the family Dictyonellidae close to Halichondriidae, based on the loss of reticulate skeleton, andd both families in a sister group relationship to Desmoxyidae, based on fleshy ectosome. The combinationn of non-localized interchangeable styles and oxea, sinuous strongyles, plumoreticulate architecture,, and collagenous mesohyle united the three families with Axinellidae into the order Halichondrida.. Diaz et al. showed earlier (published 1991) a series of halichondrid genera with intermediatee forms between an axinellid plumo-reticulated skeleton and a halichondrid confused skeleton.. The division in families based on predominance of styles or oxeas, e.g., Halichondriidae againstt Hymeniacidonidae, was no longer upheld. Additionally they proposed the placement of the desmoxyidd genera Myrmekioderma and Didiscus back in the Halichondrids. Van Soest et al's (1990)

Characterss for fig. 3: 1: Occurrence of aa combination of oxea, styles and their modifications;; 2: sinuous spicules; 3: 22 E choanosomall reticulation; 4: Spon- EE ~ E E a a E E o o >. . ginn enforced perpendicular tracts; 5: o o o o o o x x < < Highh spicule density; 6: Vague spicule tracts;; 7: Haphazard arrangement of spicules;; 8: Finely conulose surface; 9:: Ectosomal skeleton strengthening; 10:: Collagenous mesohyle; 11: Me- anderingg surface grooves; 12: Sinu- ouss trichodragmata; 13: Discorhabds; 14:: Choanosomal tracts of spicules strengthenedd by some spongin running moree or less parralel with surface; 15: Colour-changee at air; 16: Topsentins; 17:: Progressive loss of spongin; 18: Absencee of true styles; 19: presence of desmata;; 20: Trichodragmata draped aroundd the spicules; 21: Ectosomal smalll styles; 22: Bundles of longer oxeotess 'echinated' by small styles; 23:: Axially arranged fistule structure: 24:: Tufted larvae; 25: Vivipary; 26: Styless with tylote swelling

Fig.. 3: The phylogenetic relationships of the family Halichondriidae as proposed by van Soest et al. (1990).

27 7 1:: Introduction classificationn (figs. 2 and 3) comprised the following halichondrid genera:

Halichondriidae:: (high density of spicules in confusion and in ill-defined tracts) Halichondria, Hymeniacidon, AmorphinopsisAmorphinopsis Myrmekiderma, Didiscus, Epipolasis, Spongosorites, Axinyssa Axinellidaee (choanosomal skeleton axialy condensed, extra-axialy plomoreticulated): Axinella, Axinella, Phakellia, Auletta, Bubaris,Bubaris, Monocrepidium, Hymerhabdia, Cerbaris Dictyonellidae:(sponginn enforced tracts, fleshy-conulose surface) Dictyonella, Scopalina, Acanthella, Acanthella, Dactylella, Tethyspira,Tethyspira, Liosina) Desmoxyidaee (reticulate-fasciculate choanosomal skeleton of spongin-enveloped tracts with a fleshy surface: Higginsia,Higginsia, Halicnemia. Ptilocaulis

Vann Soest (1991) supported the new classification with a cladistic analysis. The resulting consensuss tree combined Poecilosclerida and Haplosclerida, Halichondrida (with Axinellida) and the keratosee sponges in a clade separating Tetractinellida and Hadromerida with Homoscleromorpha and Calcarea.. The axial condensation of the skeleton, synapomorphy for the order Axinellida sensu Levi, wass suggested to be homoplasic in the demosponges. This classification gained in following years broaderr acceptance {e.g. Levi 1997). Nevertheless,, morphological and biochemical data indicated that Axinellidae and Desmoxyidaee might not be a homogeneous group and the genera might not be easily differentiated (Bergquistt and Hartman 1969;Sole-Cavaetal. 1991; Hooper and Bergquist 1992; Hooper etal. 1992). Inn subsequent revisions, the genera nominated for Axinellidae were reduced to eleven {Acanthella, Auletta,Auletta, Axinella, Bubaris, Cymbastela, Dragmaxia. Phakellia, Phycopsis, Ptilocaulis, Reniochalina andd Dragmacidon (as Pseudaxinella) (Hooper and Levi 1993; Alvarez de Glasby 1996; Alvarez et al. 1998))) and morphological as well as molecular phylogenies of this family erected (Alvarez and Crisp 1994;; Alvarez et al. 2000). Van Soest and Lehnert (1997) returned Myrmekioderma and Didiscus too Desmoxyidae, together with the genera Higginsia, Halicnemia, Heteroxya, Myrmekioderma, Didiscus,Didiscus, and Julavis. Carballoo et al. (1996) suggested emending the definition of Dictyonellidae, published formally inn the "Systema Porifera" (Hooper and Van Soest 2002) by Van Soest et al. (2002), in combination withh the families Halichondriidae (Erpenbeck and Van Soest 2002), Desmoxyidae (Hooper 2002), Axinellidaee (Alvarez and Hooper 2002), and the Bubaridae (Alvarez and Van Soest 2002), which re-attainedd family status (Van Soest and Hooper 2002). The generic entities of these families are discussedd above. However,, the merging of axinellids with Halichondrida has found support in the recent years. Nucleicc acid and biochemical data indicate the inclusion of agelasids in Halichondrida. Biochemical dataa of Bergquist and Hartman (1969, free amino acids), Braekman et al. (1992, pyrrol-2-carboxylic acidd derivates and cyanoterpenes), Costantino et al. (1996, glycosylceramides) and others indicated aa closer phylogenetic relationship of Axinella species to Agelasida. This is in congruence with the 28SrDNAA data of Lafay et al. (1992), Chombard et al. (1997) and Alvarez et al. (2000). Furthermore, 28SrDNAA data of Chombard and Boury-Esnault (1999) and McCormack and Kelly (2002) indicated aa closer relationship of Halichondriidae to Suberitidae (Hadromerida), giving support to a taxon "Suberitina".. Chombard and Boury-Esnault (1999) proposed to abandon the taxon Halichondrida. Inn fact, the monophyly of the redefined order Halichondrida sensu Van Soest et al. (1990) andd Van Soest and Hooper (2002) has never been confirmed by any comprehensive phylogenetic analysess to date. Except for studies on Axinellidae by Alvarez (1996), and Alvarez et al. (1998,2000) noo cladistic analyses have been attempted for any halichondrid taxa. The support for the proposed phylogeneticc hypothesis of Van Soest et al. (1990) has never been assessed.

28 8 1:: Introduction 1.77 The approach to unravel the phylogeny of Halichondrida

Consequentlyy the aim of this thesis is to test for mono-, para-, or even polyphyly of the order Halichondridaa sensu Van Soest et al. (1990) and Van Soest and Hooper (2002). The approach chosen iss a combination of morphological, biochemical, and molecular (DNA) data with the main focus on thee latter. Three different data sets were sequenced from three independent genes to prevent bias from phenomenaa such as concerted evolution, as observed among the nuclear ribosomal genes (Hillis and Dixonn 1991), or when choosing entirely mitochondrial genes (see Simon et al. 1994). For this purpose (1)) a nuclear ribosomal gene, (2) a gene for a mitochondrial protein, and (3) a gene for a nuclear proteinn were selected as these three types of genes could guarantee independent evolution. The markerss chosen were a fragment of the long subunit of the cytoplasmatic ribosome (LSU, 28SrDNA) ass described in chapters 2 and 3; a fragment of the mitochondrial cytochrome oxidase 1 (COl, chapterss 4, 5 and 6); and a fragment of the elongation factor 1 alpha (chapter 7). These markers are popularr markers for metazoan phylogeny, although two of them have not been established previously inn sponge molecular phylogeny. While 28SrDNA has been used by several authors (Alvarez et al. 2000;; Chombard et al. 1997; Lafay et al. 1992; Mclnerney et al. 1999; McCormack et al. 2002; McCormackk and Kelly 2002), the cytochrome oxidase 1 is employed for the first time in sponges to constructt the first comprehensive mitochondrial gene tree following the pioneering work of Wörheide ett al. (2000) on cytochrome oxidase 2. Two cDNAs of the elongation factor 1 alpha had been used previouslyy in a study of the diploblast relationships (Kobayashi et al. 1996), but its suitability to resolvee sponge phylogenies had not yet been tested. AA synthesis of molecular, morphological and biochemical data is undertaken in chapterr 8. The morphological data set was erected to compare with the molecular tree and to provide dataa for the reconstruction of assumed character evolution. The taxonomie distribution of sponge secondaryy metabolites was extracted from the literature.

(References(References for the introduction are in chapter 8)

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