Comparative study of head structures of larvae of Sphindidae and Protocucujidae ( Coleóptera : Cucujoidea )

Selected representatives of Cucujoidea, Cleroidea, Tenebrionoidea, Chrysomelidae, and Lymexylidae were examined. External and internal head structures of larvae of Sphindus americanus and Ericmodes spp. are described in detail. The data were analyzed cladistically. A sister group relationship between Sphindidae and Protocucujidae is suggested by the vertical position of the labrum. The monophyly of Cucujiformia is supported by the reduced dorsal and anterior tentorial arms, fusion of galea and lacinia, and the presence of tube-like salivary glands. Absence of M. tentoriopraementalis inferior and presence of a short prepharyngeal tube are potential synapomorphies of Cleroidea, Cucujoidea and Tenebrionoidea. The monophyly of Cleroidea and Cucujoidea is suggested by the unusual attachment of the M. tentoriostipitalis to the ventral side of the posterior hypopharynx. Cucujoidea are paraphyletic. The families Endomychidae, Coccinellidae and Nitidulidae are more closely related to the monophyletic Cleroidea, than to other cucujoid groups. Separation of the posterior tentorial arms from the tentorial bridge and presence of a maxillolabial complex are synapomorphic features of Cleroidea and these cucujoid families. For a reliable reconstruction of cucujoid interrelation­ ships, further characters and taxa need to be studied.


INTRODUCTION
Sphindidae and Protocucujidae are two small families of Cucujoidea with 61 (McHugh & Lewis, 2000) and seven species, respectively.All sphindids appear to be slime mould feeders as there are no records of larvae in habitats other than spore masses or plasmodia of Myxomycetes (McHugh, 1993).Adults and larvae of Protocu cujidae have been collected from vegetation, but the biology of this group is still unknown (Slipinski, 1998).Both families are considered as primitive by Lawrence (1991).A sister group relationship between them was proposed by Crowson (1954Crowson ( , 1955)), and a cladistic analysis based on 19 adult and eight larval characters of representatives of five families is consistent with this hypothesis (Slipinski, 1998).Illustrations and descrip tions of larvae of Sphindidae are given by Boving & Craighead (1931), Burakowski & Slipinski (1987), Law rence (1991), Chiao & McHugh (in press) and others, and Slipinski (1998) gives a detailed description of larvae of Ericmodes.However, the internal features of immature stages of these groups and of other cucujoid families are virtually unknown.Therefore, the main purpose of this study is to provide anatomical data and more detailed information on external structures of larvae of Sphindidae, Protocucujidae and other groups of Cucuji formia.Characters that are likely to be useful for the clarification of the systematic position of both families are listed, presented as a data matrix, and discussed.A cladistic analysis of character states was carried out, mainly in order to obtain a more reliable assessment of character state polarity.However, it is evident that a much broader spectrum of taxa and characters is needed for a reconstruction of the phylogeny of the cucujoid, tenebrionoid and cleroid families.three-dimensionally on a computer using ALIAS Wavefront software.
V. Keler's (1963) muscular nomenclature is used in the text and the corresponding numbers are used in the illustrations.
Internal skeletal structures  Posterior arms strongly developed, proximal part nearly horizontal, connected by strong tentorial bridge.Dorsal arms strongly flattened but still moderately developed, attached to dorsal wall ofhead capsule by a strong bundle of fibrillar structures.Anterior arms vestigial or absent, not identified in histological sections.Anterior tentorial groove not connected with posterior part of tentorium.
Labrum (Figs 1,14,15) Well developed, but fairly narrow, posteriorly directed, at an angle of approximately 90° to frontoclypeal surface.Anterior-most part semimembranous (Fig. 1).Fits closely with upper part of mandibles and anterior hypopharynx.Distribution of setae as in Figs 1 and 14.
Maxillae (Figs 6,15) Inserted in a well developed fossa maxillaris.Articula tory membrane present.Cardo and stipes connected by a hinge.Separate galea absent.Mala rounded, fairly large, semimembranous, with a fringe of long hairs (Fig. 15).Palp three-segmented inserted on a flat, inconspicuous palpifer.Palpomere I very short, II about as long as broad.Antennomere III longer than II, slightly narrowed apically.
Maxillary musculature (Figs 6, 7, 10): M 15: M. craniocardinalis, O: ventrolaterally from the hind margin of the head capsule, immediately lateral to the posterior tentorial arms, I: laterally on a small condyle of the cardo by means of a tendon; M 17: M. tentoriocardinalis, O: tento rial bridge and posterior tentorial arm, I: mesally on cardo; M 18: M. tentoriostipitalis, composed of three sub components, M 18a, O: tentorial bridge and posterior ten torial arm, I: ventral surface of stipes; M 18b, O: tentorial bridge and posterior tentorial arm, I: mesal edge of stipes; M. 18c, a small bundle, distinctly separated from M 18b, O: posterior edge of hypopharynx, I: laterally on ventral surface of anterior part of stipes; M 19: M. craniolacinialis, one large bundle, O: laterally from head capsule, I: base of lacinia by means of a tendon; M 22, 23: M. stipitopalpalis externus and internus, O: mesal side of stipes, I: base of maxillary palp.
Epipharynx (Figs 5,8) Anterior part of ventral side of labrum completely membranous, with lateral group of short bristles, sepa rated from posterior part by a transverse row of small spines.Posterior epipharynx V-shaped in cross section, 45 -M.frontobuccalis anterior, 46 -M.frontobuccalis posterior, with deep U-shaped median groove, forming narrow lat eral grooves with upper sides of mandibles (Fig. 8).Pos terior part of epipharynx not fused with hypopharynx anterior to anatomical mouth, prepharyngeal tube absent.
A transverse hypopharyngeal muscle connecting poste riorly directed apodemes or the lateral hypopharyngeal walls is absent.
A well developed ring musculature is present over the whole length of the pharynx (Figs 6, 7).

Glands (Figs 7, 10)
Paired gland-like ducts originate in the anterior thorax.The exact distal orifice could not be identified. Fatbody(Fig.10) Fairly extensive parts of fat body located in posterior part ofhead capsule.
Posterior arms arise immediately anterior to foramen occipitale, connected by strongly broadened, plate-like tentorial bridge.Dorsal arms present but very flat and unsclerotized, attached to head capsule by fibrillar struc tures.Anterior arms not identified in histological sections, extremely thin or absent.Anterior tentorial grooves not connected with posterior tentorium.
Labrum (Figs 2,4,16) Fairly broad, almost vertical.Distribution of setae on dorsal surface as in Fig. 16.Anterior margin with a group of four setae in a transverse row, tip with a densely arranged group of shorter setae, and a semicircular dense field of microtrichiae.
A second, thin labial muscle is medially attached to the posterior premental margin.The origin could not be iden tified in the available microtome series.It is likely that it arises from the tentorial bridge, together with M 18b.This would imply homology with M. tentoriopraementalis inferior (M 29).M. 30: M. tentoriopraementalis superior, absent; M 34: Mm. praementopalpalis ext., absent.
Epipharynx (Fig. 9) Anterior epipharynx distinctly V-shaped in cross sec tion.Posterior epipharynx, molar part of mandibles, and posterior part of hypopharynx form an X-shaped preoral chamber.Short, closed prepharyngeal tube formed by lat eral fusion of posteriormost epipharnyx and hypopharynx.
Hypopharynx (Figs 9,16) Anterior hypopharynx with a very dense field of erect stiff bristles.Lateral walls connected by a transverse, extremely strong internal bar.Posterior hypopharynx forms ventral part of preoral chamber and prepharyngeal tube.
The transverse hypopharyngeal muscle is absent.
Pharynx (Fig. 11) Fairly wide in diameter, but lumen very narrow, with narrow ventrolateral, lateral and dorsolateral folds.
Musculature (Fig. 11): M 45: M. frontobuccalis anterior, one moderately sized muscle, O: anterior frontal region, mesal to anteriormost part of M 41, I: dorsolater ally on pharynx, immediately posterior to the frontal gan glion and anatomical mouth; M 46: M. frontobuccalis posterior, several thin and flat parallel bundles, O: poste rior frons, between and lateral to bundles of M 41, I: dor solateral folds of pharynx; M 51: M. verticopharyngalis, absent; M 52: M. tentoriopharyngalis, composed of two subcomponents, M 52a, three pairs of fairly large bundles, O: proximal part of posterior tentorial arms and tentorial bridge, I: ventrolaterally and laterally on poste rior pharynx, M 52b, one pair of muscles, O: tentorial bridge, I: ventrally on anterior pharynx, immediately pos terior to anatomical mouth.
A well developed series of ring muscles is present over the whole length of the pharynx.
Cerebrum and suboesophageal ganglion (Fig. 11) Fairly large in relation to head size and distinctly elon gated.Posterior part of cerebrum shifted into prothorax in early instars.
Glands (Fig. 11) Paired gland-like ducts originate in the prothorax.Thin in early instar larvae, rather massive in late instars.Distal orifice not clearly identified in sections, probably located on anteriormost hypopharynx.

Fat body
Almost completely absent from head capsule.

Fore gut
Oesophagus strongly twisted posterior to pharynx.Fairly wide, with thin wall and well developed ring mus cles.Posterior oesophagus with star-shaped constriction with ring muscles and longitudinal muscles.Proventriculus absent.The gula is absent in larvae of Sphindus, Elateroides (Lymexylidae) and Altica (Chrysomelidae).A short, semimembranous gula is present between the posterior tentorial grooves in larvae of Ericmodes (Slipinski, 1998), in the hypognathous larvae of Mycetina (Burakowski, 1997, Fig. 4; pers.obs.) and in larvae of many groups with a subprognathous head (e.g.Cavoganthidae, Hydraenidae, Derodontidae).The gula is short, inserted between the posterior tentorial grooves, sclerotized and fused with the submentum in larvae of Glischrochilus.A quadrangular or elongated, parallel-sided sclerotized gular plate is present in larvae of Calytis, Temnoscheila (Trogossitidae) and Lemidia (Cleridae).It is distinctly present but unpigmented in larvae of Phycosecis and Melyridae.The gula is broad, sclerotized, inserted between gular ridges, and diverging posteriorly in prog nathous larvae of Cucujoidea (e.g.Silvanidae) and Tenebrionoidea (e.g.Pyrochroidae, Colydiidae).It is apparent that this character is highly variable, associated with feeding habits and different degrees of hypo-or prog nathism.

Posterior tentorial arm: (0) connected with tento rial bridge; (1) posterior arms shifted anteriorly, dis connected from tentorial bridge; (2) part of elongated gular ridges, connected with ventral prepharyngeal wall; (3) tentorial bridge shifted anteriorly, gular ridges present posterior to bridge
The proximal and distal parts of the posterior tentorial arms and the tentorial bridge form a continuous structure close to the foramen occipitale in larvae of Sphindus, Ericmodes, Elateroides, Oryzaephilus, Silvanus (Silvani dae) and Altica.The same, presumably plesiomorphic character state is found in larvae of most other groups of Coleoptera (e.g.Beutel, 1993Beutel, , 1995Beutel, , 1996;;Beutel & Molenda, 1997).A tentorium with the posterior arms clearly separated from the tentorial bridge is found in larvae of Trogossitidae, Phycosecidae, Nitidulidae, Endomychidae and Coccinellidae, and also in larvae of most groups of Elateriformia (Beutel, 1995).The posterior ten torial arms are part of strongly elongated gular ridges and anteriorly connected with the prepharynx in larvae of Lemidia.The tentorial bridge and posterior arms are dis tinctly shifted anteriorly in larvae of Pyrochroa and Bitoma.These larvae possess more or less well developed gular ridges (shallow in Pyrochroa) posterior to the pos terior tentorial grooves.

Dorsal tentorial arm: (0) distinct; (1) unsclerotized and strongly flattened; (2) absent
The dorsal tentorial arm is fairly well developed in larvae of Sphindus and Glischrochilus and serves as an attachment area for the antennal muscles.A well devel oped dorsal arm is also found in larvae of most other groups of Coleóptera (Beutel, 1995(Beutel, , 1999;;Beutel & Molenda, 1997;Beutel et al., 1999).The dorsal arm is unsclerotized, flattened and more or less distinctly folded in larvae of Ericmodes, in larvae of most other cucujoid groups, and also in larvae of Pyrochroa, Bitoma and Altica.The dorsal arm is absent or extremely thin and dis connected from the head capsule in larvae of Lymexylidae and Cleroidea.A reduced dorsal arm is also a characteristic feature of larvae of most groups of Elateriformia (Beutel, 1995).As there is a continuum in the reduction of this structure, it is difficult to define it as distinct character states.

Anterior tentorial arm: (0) distinct; (1) absent or at least obsolete and disconnected from posterior ten torium
The anterior tentorial arms are absent or strongly reduced and disconnected from the posterior tentorium in all cucujiform larvae examined.They are never the origin of antennal muscles as in many groups of Staphylinoidea (Beutel & Molenda, 1997).The anterior arms are thin but sclerotized and distinct in larvae of Pyrochroa.However, a connection with the posterior arms is not recognizable and apparently absent.Well developed anterior arms are present in most groups of Coleoptera such as Adephaga (Beutel, 1993), Myxophaga (Beutel et al. 1999), Hydrophiloidea (Beutel 1999), Staphylinoidea (Beutel & Molenda, 1997), Nosodendridae, and Derodontidae (Beutel 1996).

Orientation of labrum: (0) continuous with dorsal wall of head capsule, almost horizontal; (1) distinctly inclined, almost vertical
A labrum, which is posteriorly directed at an angle of about 90° to the frontoclypeal surface, is present in larvae of Sphindus.A similar condition is found in larvae of Ericmodes (Fig. 4), and in those of other groups of Coleoptera such as Derodontus (Derodontidae) and Sepedophilus (Staphylinidae).Especially in larvae of Sphindus and Sepedophilus, labrum, mandibles and hypopharynx together form a preoral working space, which can be tightly closed.A labrum that is more or less horizontal in relation to the clypeus and Irons is present in larvae of other groups of Cucujoidea, Cleroidea and Tenebrionoidea (e.g. Mycetina, Silvanus, Phycosecis, Lemidia, Calytis, Pyrochroa, Bitoma, Altica).

Maxilla and labium: (0) separate or moderately coadapted; (1) maxillolabial complex
A maxillolabial complex with restricted lateral mobility of the maxillae with a poorly developed articulatory membrane is present in larvae of Cleroidea, Nitidulidae, Coccinellidae and Endomychidae.It moves mainly verti cally.A similar condition is found in most larvae of Elateriformia (Beutel, 1995).A maxillolabial complex as defined above is absent in larvae of Sphindidae, Protocu cujidae and larvae of other groups of Cucujiformia (Law rence, 1991).Moderately coadapted ventral mouthparts of some cucujoid groups (e.g.Sphindidae; coded as 0 in this study) are referred to as a maxillolabial complex by Law rence (1991).The phylogenetic significance of this char acter is weakened by the fact that the formation of a max illolabial complex occurs gradually.

Origin of M. tentoriostipitalis: (0) exclusively from posterior tentorial arm and tentorial bridge; (1) anterior subcomponent originates from ventral hypopharynx
The origin of an anterior bundle of M. tentoriostipitalis (M 18c) from the ventral wall of the prephagyngeal tube or from the posterior edge of the hypopharynx is an unusual feature which is found in larvae of Sphindus, Ericmodes, Zeonidicola, Mycetina, Coccinella, Silvanus, Oryzaephilus and Cleroidea.M 18c is thin and isolated from Mm 18a and b in larvae of Sphindus, moderately sized in larvae of Ericmodes, and large in the other larvae listed above.A hypopharyngeal component of M. tentoriostipitalis had previously been described only for lampyridlarvae (Beutel, 1995).

Tentorial bridge: (0) not distinctly broadened; (1) strongly broadened, plate-like; (2) thin and tendon-like
A distinctly broadened, plate-like tentorial bridge is presumably a derived feature found in larvae of Eric modes but not in other larvae of Cucujoidea or Cleroidea.It is an extensive attachment area of Mm. tentoriocardinalis and -stiptalis.The bridge is also distinctly broadened in larvae of Derodontus, whereas it is very thin and tendon-like in larvae of Cleridae and Trogossitidae.

Galea and lacinia: (0) separate; (1) partly fused; (2) absent
Absence of a separate galea is an apomorphic condition found in almost all larvae of Cucujiformia excl.Lymexylidae.Galea and lacinia are very closely attached to each other but still recognizable as separate structures in larvae of Elateroides.A distinctly cleft mala, as present in some larvae of Erotylidae (Lawrence 1991;McHugh pers. comm.: Megalodacninae), is probably not a plesiomorphy but a secondarily derived feature.

Position of cerebrum: (0) within head capsule; (1) completely shifted to thorax; (2) partly shifted to prothorax and strongly asymmetric
The main part of the cerebrum lies within the head cap sule in larvae of Elateroides, Altica,Sphindus (Figs. 7,12,13), Ericmodes and all other larvae of Cucujoidea and Tenebrionoidea.Only a part of the distinctly extended protocerebrum is shifted to the prothorax in early instar larvae of Ericmodes.The cerebrum and suboesophageal ganglion are completely shifted to the thorax in larvae of Cleroidea independent of their size (e.g.Calytis).A simi lar, presumably apomorphic condition is found in preda cious larvae with prognathous head, distinctly extended attachment areas for M. craniomandibularis, and reduced postcerebral pharyngeal dilators (e.g.Hydrophilidae, His teridae, Cantharoidea; Beutel, 1995Beutel, , 1999)).The cerebrum is partly shifted to the prothorax and markedly asym metric in larvae ofNitidulidae and Melyridae.
Tube like salivary glands, resembling Malpighian tubules (Figs 7,10,11), originate in the thorax or poste rior head region in larvae of Cucujiformia.The proximal part is voluminous and grape-shaped in larvae of Altica.This is not coded as a separate character state.The tubes are absent from larvae of other groups such as Staphylinidae, Derodontidae, Nosodendridae and Dermestidae.

ANALYSIS
The preliminary analysis of the interrelationships of cucujiform taxa was based upon 22 larval characters.

Ochthebius
(Hydraenidae) and Derodontus (Derodontidae) were used as outgroups.All characters were weighted equally and not ordered.Question marks in the matrix indicate missing data.
Most parsimonious trees were sought using branch and bound search in PAUP (version 3.1;Swofford, 1991).
The outgroup taxa were treated as all other groups in the analysis (simultaneous analysis; Nixon and Carpenter, 1993).Analysis of character evolution was conducted in MacClade (version 3; Maddison and Maddison, 1992).Additional analyses were carried out after successive reweigthing.A bootstrap search was not conducted.Boot strap values should not be used as confidence intervals (e.g.Carpenter, 1996;Rudolf Meier, pers. comm.).
The sample of taxa was restricted by the availability of larval material suitable for histological techniques.The genus Sphindus is apparently not a basal lineage within Sphindidae (McHugh ,1993;Chiao & McHugh, in press).However, characters that are variable within this family (Chiao & McHugh, in press) are not considered here.

RESULTS OF THE PHYLOGENETIC ANALYSIS
The analysis resulted in 74 trees of a minimal number of 63 steps (CI: 0.571; HI 0.429, RC 0.409).Interrelation ships of cucujoid families were not resolved in a strict consensus tree (Fig. 17).Successive reweighting resulted in four equally parsimonious trees (Fig. 18: tree 1; Fig. 19 strict consensus tree).Unambiguous apomorphies are mapped on one of these trees and listed below for selected nodes.
The main purpose of the analysis was to determine the character state polarity and a preliminary assessment of some phylogenetic affinities.It is well understood that an analysis of features of the larval head of a very limited set of taxa cannot yield a reliable reconstruction of the inter relationships within Cucuiformia.

DISCUSSION
Larvae of Sphindus and Ericmodes show several pre sumably derived character states, which they probably share with all other larvae of Cucujiformia.Tube-like salivary glands, which were interpreted as a possible autapomorphy of this large lineage of Coleoptera, are unmodified in the larvae of Cleroidea, Cucujoidea, Tenebrionoidea, but distinctly extended and vesicular in larvae of Altica (Chrysomelidae).Other derived character states found in larvae of Ericmodes and Sphindus, and those of other cucujiform families, are the single mala and and a partly or completely reduced anterior tentorial arm, which is always disconnected from the posterior tentorium.The dorsal tentorial arm is weakly sclerotized or vestigial in most cucujiform larvae.However, this is more a tendency than a well defined character state.The tendency to shift the origin of the antennal muscles from the tentorium to the head capsule (all cleroid larvae, Elateroides, Altica, Bitoma, Pyrochroa) is probably correlated with this char acteristic.
A monophyletic unit comprising cucujoid, cleroid and tenebrionoid taxa is only weakly supported by one unam biguous apomorphy, the presence of a short prepharyn geal tube.This implies secondary loss in Sphindus.Besides that, a prepharyngeal tube has evolved several times independently, associated with a predacious life style (e.g.Adephaga; Beutel, 1993).
The origin of an anterior component of M. tentoriostipitalis (M.18) from the hypopharynx is an unusual derived character state, which indicates the monophyly of a large group comprising Cucujoidea and Cleroidea.This compo nent is rather thin in Protocucujidae, Sphindidae, Silvanidae and Cavognathidae.A basal position of these groups within a presumptive cucujoid-cleroid lineage is also indicated by the comparatively plesiomorphic condi tion of the tentorium.The posterior arms and the tentorial bridge form a continuous structure, whereas they are separated from each other in larvae of the other groups.Another plesiomorphic feature is the absence of a maxillolabial complex.Restriction of the lateral movements of the maxillae is associated with increase in size of the ver tically oriented hypopharyngeal component of M. 18.
A sister group relationship between Protocucujidae and Sphindidae was suggested by Slipinski (1998), based on the results of a cladistic analysis of larvae and adults of several groups of Cucujoidea (Sphindidae, Hobartiidae, Phoeostichidae, Brachypteridae).This seems to be well supported by shared derived adult features, especially of the male genital apparatus.However, proposed synapomorphic features of larvae are less convincing.The pres ence of only five stemmata (six in Sphindus) and a short ened coronal suture are character states found in many groups of Coleoptera.The larval head structures studied in this investigation do not yield much evidence for a sister group relationship between Protocucujidae and Sphindidae.A distinctly inclined or almost vertical labrum is characteristic of larvae of both families.How ever, this is a gradual modification rather than a distinct character state, and a similar condition is also found in larvae of other groups such as Staphylinidae (Sepedophilus) and Myxophaga (Beutel et al., 1999).Some character states seem to be more advanced in larvae of Ericmodes than in larvae of Sphindus.The head is almost progna thous, whereas it is distinctly inclined in larvae of Sphin dus.They possess a closed prepharyngeal tube, which is absent from larvae of Sphindus, but present in larvae of other families of the cucujoid-cleroid lineage.The hypopharyngeal component of M. tentoriostipitalis is slightly better developed and not separated from the pos terior parts of the muscle.The mandibular prostheca is largely reduced in later instars.The brain is more elon gated and partly shifted to the prothorax.These differ ences are not strong evidence against a sister group rela tionship between Protocucujidae and Sphindidae.Seem ingly primitive characters of Sphindus, such as the absence of a gula, the distinctly inclined head, and the open cibarium, could be associated with their specialized slime mould feeding habits.It is apparent that more evi dence is needed for a reliable interpretation of some char acter states and of the interrelationships between cucujiform groups.
): M 15: M. craniocardinalis, two subcomponents, O: immediately close to posterior margin of head capsule, dorsolaterally and ventrolaterally between attachment areas of M 19, I: small process of cardinal base; M 17: M. tentoriocardinalis, O: broad ten torial bridge and upper part of posterior tentorial arm, I: ventral surface of cardo; M 18: M. tentoriostipitalis, very large muscle, composed of three subcomponents, M 18a, O: broad tentorial bridge and upper part of posterior ten torial arm, mesal to M 17, I: ventral surface of stipes; M 18b, O: tentorial bridge and upper part of posterior tento rial arm, I: anterior mesal edge of stipes; M 18c, fairly large, not clearly separated from M 18b, O: ventrally from posterior hypopharynx, I: ventrolateral surface of anteriormost part of stipes; M 19: M. craniolacinialis, two well developed bundles, O: ventrolaterally from head capsule, separated by ventrolateral subcomponent of M 15, I: base of mala; M 22, 23: M. stipitopalpalis externus and internus, two distinct bundles, O: ventrally and ventromesally from stipes, I: base of maxillary palp.