Larval morphology of three species of Hygrobiidae ( Coleoptera : Adephaga : Dytiscoidea ) with phylogenetic considerations

A provisional larval groundplan of the family Hygrobiidae is provided through descriptions of internal and external features of three of six extant species, Hygrobia hermanni (Fabricius, 1775), H. wattsi Hendrich 2001 and H. australasiae (Clark, 1862) and phylogenetic interpretations. Hygrobiidae larvae are morphologically differing dramatically from all other known Adephaga by 20 autapomorphies. Structures involved with feeding, i.e., mouthparts, prepharynx and foregut are highly modified as a result of a specialisation on small tubificid worms and chironomid larvae. A placement of Hygrobiidae within Dytiscoidea is well supported by the reduced condition of the terminal abdominal segments, and the presence of 10 ancestral setae on femur and a clade comprising Hygrobiidae, Amphizoidae, and Dytiscidae by the presence of thin and elongate caudal tentorial arms, a very strong musculus verticopharyngalis and a longitudinally divided adductor tendon of the mandible. A highly modified foregut, reduced terminal spiracles VIII and the presence of tubular gills are features which distinguish hygrobiid larvae from those of other groups of Dytiscoidea (i.e, Amphizoidae, Noteridae, Dytiscidae). A sister-group relationship between Hygrobiidae and Dytiscidae is indicated by a distinctly shortened and transverse prepharynx and a cerebrum and suboesophaeal ganglion shifted to the anterior third of the head. Larvae of the Australian species H. wattsi and H. australasiae share the presence of a bluntly rounded mandible and an apical position of the primary pore MNd in instar I as potential synapomorphies.

Whereas Dytiscoidea is likely a monophyletic group (Beutel & Roughley, 1988;Beutel & Haas, 1996;Ribera et al., 2002), available evidence is still equivocal in regard to the phylogenetic relationships of the dytiscoid lineages, particularly at the most basal levels.In this regard, a study of larval structures undoubtedly would provide valuable data.As different expressions of the same genotype, larval characters help to complement adult characters which have been traditionally the primary basis for the classification of members of the Adephaga.
The larval groundplan of Hygrobiidae is not well known with superficial descriptions available for Hygrobia hermanni only (Bertrand, 1928(Bertrand, , 1972;;Klausnitzer, 1991).The purpose of this study is to provide a detailed analysis of the larval morphology of the Hygrobiidae.More specifically, it aims (1) at describing internal and external structures of larvae of H. wattsi, H. hermanni and H. australasiae; (2) at providing keys and illustrations to facilitate their identification; (3) at identifying ground-plan character states of Hygrobiidae; and (4) at recognizing character states which could be helpful for clarifying the systematic position of the Dytiscoidea lineages (i.e., Noteridae, Amphizoidae, Hygrobiidae, and Dytiscidae).
excluding the epicranial stem.Length of antenna: derived by adding the length of each individual antennomere; comparison among antennomeres was made using the capital letter A with a number corresponding to the article considered (e.g., A1 for antennomere 1); A3' is used as an abbreviation for the lateral elongation of antennomere 3 (= sensorial appendage).Length of maxillary palpus: derived by adding the length of each individual palpomere (e.g., MX1 for palpomere 1).Length of labial palpus: derived by adding the length of each individual palpomere (e.g., LB1 for palpomere 1).Length of legs: derived by adding the length of each individual segment including the longest claw; the length of each segment was taken at the longest point except for the trochanter which includes only the proximal portion (the length of distal portion being included in the femoral length).Dorsal length of last abdominal segment (LLAS): includes the whole sclerite measured dorsally along mid-line from the anterotransverse carina to apex of siphon.
The individual measurements defined above were used in calculating several ratios aiming at characterizing the body shape.

Chaetotaxic analysis
Primary (observed in instar I) and secondary (those added during ontogenetic development) setae and pores were distinguished on the head capsule, head appendages, legs and urogomphi.The setae and pores were coded according to the system developed by Bousquet & Goulet (1984).Setae are coded by two capital letters corresponding to the first two letters of the name of the structure on which the seta is located (ANantenna; CO -coxa; FE -femur; MX -maxilla; LA -labium; TA -tarsus; TI -tibia; TR -trochanter) and a number.Pores are coded in a similar manner except that the number is replaced by a lower case letter.The position of the sensilla is described by adding the following abbreviations: A -anterior; AVanteroventral; D -dorsal; Di -distal; Pr -proximal; PV -posteroventral.Primary setae and pores were subdivided into two categories: ancestral, i.e. those associated with the hypothesized ancestral pattern of Adephaga (generally present on larvae of most families), and additional, i.e. those evolved secondarily in the first instar (generally restricted to a genus, tribe or family).All homologous setae and pores on larvae of Hygrobiidae and on larvae of Dytiscidae, Noteridae, Amphizoidae, Carabidae, Haliplidae, and Gyrinidae, Trachypachidae were considered as part of the ancestral system of the family.
Larvae of Hygrobia are characterized by the presence of a few additional setae on some leg articles.As their number and position vary between species, they were included in the count of secondary setae.

Color
Description of color is given for all species from ethanolpreserved specimens.

Study of internal features
Description of internal features of cephalic capsule and head appendages is based on instar II as these characters occur in a similar condition in instar I and III.Selected specimens of Hygrobia wattsi, H. hermanni and H. australasiae were dissected and were embedded in Historesin; larvae were cut at 3 µm (cross sections) with a Microm Microtome (HM 360) equipped with a glass knife.The sections were stained with methylene blue and Fuchsin.Drawings were made using an ocular grid or a camera lucida (cross sections).Kéler's (1963) muscular nomenclature is applied in the text and the corresponding numbers are used in the illustrations.

Voucher specimens
Voucher specimens are deposited in the research larval collection of Yves Alarie (Laurentian University, Department of Biology, Sudbury, Ontario, Canada) and in the South Australian Museum (North Terrace, Adelaide, SA 5000, Australia, CHS Watts).
THORAX.Small in relation to head and large in relation to abdomen.All tergites sclerotized, slightly explanate laterally, and with an ecdysial suture.Pronotum elliptical dorsally; length of pronotum about twice that of mesonotum; metanotum subequal to mesonotum in length, both as broad as pronotum; thoracic venter semimembranous; spiracular openings absent.
LEGS .Six-segmented (sensu Lawrence, 1991); metathoracic legs longest, 1.20-1.30times length of prothoracic legs, and 1.80-2.30times HW; coxae very elongate and conical, with paired gill tufts arising from their bases; trochanter fairly small; femur and tibia cylindrical, about equally long, tarsus shorter and slender, with two claws, posterior claw slightly shorter than anterior claw on pro-and mesothoracic legs, sllightly longer on metathoracic leg; posterior metathoracic claw 0.67-0.86times as long as metatarsus; spinulae weakly developed along ventral margin of tarsus.Chaetotaxy (Figs 14-19; Tables 2, 3).Coxa with 17 setae (CO1-CO17) and two pores (COa and COd).Trochanter with six setae (TR1, TR3-TR7) and seven pores (TRA-TRg).Femur with 10 setae (FE1-FE10) and two pores (FEa-FEb) and with a variable number of additional setae (Table 3); FE6 elongate and hair-like.Tibia with seven setae (TI1-TI7) and one pore (TIa) and with a variable number of hair-like (= natatory setae) or spine-like additional setae on posterodorsal and anteroventral margin respectively; setae TI1 and TI7 elongate and hair-like, included in the group of additional natatory setae.(TA1-TA7) and six pores (TAa-TAf); the individual pores of the pairs TAc/TAd and TAe/TAf are very difficult to distinguish because they are positioned close together and the ventral margin of the tarsus is generally marked by a pronounced thickening of marginal spinulae; pore TAb is also very difficult to locate because of both its apical position and the presence of setae TA2 and TA7; seta TA2 is generally inserted dorso-apically, and is extremely short.Pretarsus with two short spiniform setae (PT1-PT2).
Biology.Hygrobiids live in still water and prefer waters with fine mud and with little or no plant life.Hygrobiids have a very specialized diet feeding upon tubificid worms and/or chironomid larvae (Cuppen, 2000).
Remarks.Larvae of Hygrobiidae are easily differentiated from those of other Hydradephaga by the very large head (in relation to body size), by the presence of tubular gill tufts ventrally, and by an abdominal tergite VIII forming an elongate median process strongly resembling the terminal filament of Archaeognatha, Zygentoma and Ephemeroptera.Indeed hygrobiids have been characterized by the presence of three "tails threads" covered with hairs (Wichard et al., 2002).This statement is misleading because it suggests the presence of three apical appendages articulated upon the abdominal segment VIII.
LEGS .Metathoracic leg about 1.90 times HW.Chaetotaxy.position and number of additional setae (Table 3).
COLOR.As instar II except body more predominantly brown.
Remarks.Instar I of H. hermanni is characterized by the acute shape of the mandible and by the subapical position of the primary pore MNd.
Distribution.South-western Australia.South of a line from Perth to Albany (Hendrich, 2001).
Remarks.Larvae of H. wattsi closely resemble to those of H. australasiae, the other australian species studied.Both species share the presence of a bluntly rounded mandible of instar I and the apical position of the primary pore MNd (Fig. 9) which are postulated to represent a putative synapomorphies for these species.As instar I of H. wattsi may readily be distinguished from H. australasiae both by the absence of additional spines on coxae, by the absence of natatory setae on tarsi and by the presence of a single row of teeth along inner margin of the mandible (Fig. 9).(Clark, 1862) (Figs 6,8,(14)(15)(16)(17)20,38,42)
Material.The larvae studied (3 instar I, 5 instar II and 2 instars III) were collected in association with adults at the following locality: South Australia: 10 km N Coonwarra, 28.ix.1998.
Remarks.Instar I of H. australasiae are unique among the known larvae of Hygrobia because of the presence of multiple rows of teeth along inner margin of the mandible (Fig. 8), by the presence of additional setae on coxae (Figs 14,15) and by the presence of a row of additional natatory setae on dorsal margin of tarsi (Fig. 17).

CHARACTER ANALYSIS
There now follows a discussion of the phylogenetic importance of selected characters of the larvae of Hygrobiidae, based on the three species described.Essentially, it aims at coding the different manifestations of each individual character into putative plesiomorphies and apomorphies.The congruence of the character states provided in this study is not tested in a formal cladistic analysis as this study is based on a limited character set, i.e. morphological features of larvae.The preliminary polarity assessments and phylogenetic interpretations provided herein may be confirmed or refuted by the results of a future cladistic analysis (Beutel et al., in prep.) of a broad spectrum of morphological characters of different life stages and of molecular data including the new family Aspidytidae (Ribera et al., 2002;Balke et al., 2003;Alarie & Bilton, in prep.).
The polarity rationale in the list of characters presented below is based on the outgroup comparison method (Watrous & Wheeler, 1981).It is carried out individually for each character.The states are coded to reflect the hypothesized polarity, i.e. the presumptive plesiomorphic condition is scored as (0).The outgroup comprises all non-dytiscoid adephagan families (Gyrinidae, Haliplidae, Trachypachidae, Carabidae) and character states found in larvae of Geadephaga and/or at least one of the presumably basal groups Gyrinidae and Haliplidae (Beutel, 1995: Beutel & Haas, 1996) are considered as plesiomorphic.
3. Spiracles VIII: (0) normally sized; (1) enlarged, terminal; (2) absent The spiracles VIII are shifted to a terminal position and distinctly larger than the preceding spiracles in larvae of Amphizoidae, Dytiscidae and Noteridae.This is probably a derived groundplan feature of Dytiscoidea.The spiracles VIII are normally sized and not terminal in larvae of Geadephaga and absent in larvae of Hygrobiidae (see char.below), Haliplidae (Jaboulet, 1960) and Gyrinidae.The complete loss is probably the result of parallel evolution in these taxa.

Autapomorphies of Hygrobiidae
11. Gula: (0) narrow, suture-like, genae closely adjacent; (1) forming a broad and long plate together with the submentum, distinctly delimited laterally by sutures and internal ridges, posterior tentorial grooves widely separated A narrow gula is present in larvae of Gyrinidae and almost all larvae Geadephaga (Beutel, 1993).It is distinctly broadened and laterally limited in the larvae of Hygrobiidae examined (Fig. 22), and as a result of this condition the posterior tentorial grooves are widely separated.The presence of a broad gula and widely separated posterior tentorial grooves in larvae of Haliplidae (Jaboulet, 1960;Beutel, 1986b) and few larval representatives of Carabidae (Loricera, Licinini part; Beutel, 1992) is probably homoplastic.
14. Maxillary base: (0) exposed; (1) strongly retracted into pouch lateral to prementum (Figs 22,27) 15.Cardo: (0) separate sclerite; (1) completely fused with stipes or absent The cardo is generally rather small but well developed in larvae of Adephaga (e.g., Gyrinidae, Haliplidae, phies of Dytiscoidea comprise modifications of the terminal abdominal segments (chars.1-3).Possible synapomorphies of the dytiscoid families excluding Noteridae are the strongly elongated caudal tentorial arms, a modified pharyngeal musculature, the adductor tendon of the mandible completely divided into an upper and a lower branch, and the presence of 10 ancestral setae on the femur (chars.4-7).Exclusion of Haliplidae, however, is contrary to a recent study based on the female reproductive system (Miller, 2001).
The characters listed above also strengthen the case of a sister-group relationship between Hygrobiidae and Dytiscidae which was suggested by Burmeister (1976), Ruhnau (1986) and Beutel (1986a).A distinctly shortened and transverse prepharynx and a cerebrum and suboesophaeal ganglion shifted to the anterior third of the head are likely larval synapomorphies of these families.An elongated coronal suture and an elongate scapus are also presumably derived features found in larvae of Hygrobiidae and many larvae of Dytiscidae.Both character states, however, vary considerably within the latter family.An analysis of 18S rDNA data (Shull et al., 2001) resulted in a placement of Hygrobia as a subordinate dytiscid taxon.However, the implied paraphyly of Dytiscidae appears unlikely as the family is characterized by many derived features of adults (e.g., Baehr, 1979;Beutel, 1995;Miller, 2001) and larvae (e.g., Ruhnau, 1986).
Even though the placement of Hygrobiidae seems to be well established, at least within a clade comprising Dytiscoidea without Noteridae (Beutel & Haas, 1996;Ribera et al., 2002), the beetles (Beutel, 1986a), and especially the larvae differ strongly from those of other dytiscoid taxa.Hygrobiid larvae are characterized by unusual feeding habits and breathing organs, and their locomotor apparatus differs from that of the presumably more basal Noteridae and Amphizoidae (Ribera et al., 2002).As a result of specialized habits Hygrobiidae display more larval autapomorphies than any other group of Adephaga (chars.10-29).
The whole feeding apparatus of the larvae is strikingly modified.The mandibles are long, sickle shaped, and equipped with a serrate edge in instar I.They completely lack a retinaculum, subapical teeth, rows of hairs and other specialisations of the mesal side, and it appears plausible to assume that a second mesal cutting edge was secondarily reduced (char.13).Obviously one of the most important functions of the mandibles is grasping and the serrate edges (char.33) may help to prevent the tubificid worms from escaping.Intensive mechanical treatment by mandibular parts can be excluded as they are widely separated.However, the larvae use the distal parts to press the prey against the surface of the highly modified anterior epi-and hypopharynx (chars. 19, 20;Bertrand, 1972: Fig. 7).At this stage, the enlarged ligula (char.18) is pressed against the rim enclosed by the epipharyngeal lobes (Bertrand, 1972: Fig. 7).Then, simultaneous retraction of the trough-like hypopharynx and the highly specialized maxillae results in a backward movement of one end of the worm towards the posterior part of the short and transverse prepharyngeal tube.The maxillae with their reduced proximal and distal parts (cardo, galea and lacinia absent) and their very unusual insertion in a deep pouch (chars.14-16), strongly resemble antennae .Their moveability is largely restricted to forward and backward movements and they are obviously not involved in catching and manipulating the prey.However, they are probably important for the prey detection and as pointed out above, together with the hypopharynx, for the food transport in the preoral cavity.
One of the most unusual set of apomorphies of hygrobiid larvae is the highly modified pharynx with extremely strong and complex dilators (Fig. 24), longitudinal spiniferous sclerotisations, and a largely reduced ring musculature (chars. 22-26).The contraction of the extremely developed ventral, lateral and dorsal muscles results in a strong dilation of the unusually voluminous pharynx, and thus sucks back the largely intact prey through the anatomical mouth (see also Bertrand, 1972: "jabot aspirateur").The spines of the longitudinal pharyngeal sclerotisations support the backward movement, i.e. prevent the worm from moving in the opposite direction.This is necessary as the food transport is not supported by the contraction of ring muscles, which is a highly unusual feature not known from any other larvae of Coleoptera (e.g., Beutel, 1993;Beutel & Molenda, 1997;Beutel et al., 1998;Beutel & Hörnschemeyer, 2002).The main mechanical breakdown of the food takes place in the proventriculus, which is equipped with a strong ring musculature and sclerotised ridges with spines.Finally, the processed food is digested and absorbed in the midgut.In contrast to other adephagan larvae, preoral digestion can be largely excluded for Hygrobiidae.A groove enclosed by two mesal mandibular edges or a mandibular channel suitable for injection are absent and a prepharyngeal filter apparatus is also lacking.
The specific diet of hygrobiid larvae (and adults) has probably not only resulted in a highly modified feeding apparatus, but also in other structural transformations.It was already pointed out by Bertrand (1972) that the larvae search for their prey at the bottom of their aquatic habitats, and as they may be forced to enter greater depths than most other dytiscoid larvae, good swimming abilities (Bertrand, 1972) are certainly advantageous.The legs are flattened and equipped with dense femoral fringes of swimming hairs (Figs 17,19), but not only very suitable for efficient swimming movements, but apparently also for burrowing in the soft mud or sand in which the tubificid worms and chironomid larvae live (Bertrand, 1972;Cuppen, 2000).
The reduced condition of the abdominal spiracles I-VII (instar I and II) and especially the secondary loss of the large terminal spiracles VIII are almost certainly closely related with the acquisition of the specific type of tubelike gills (char.27).The presence of these breathing organs is probably advantageous for searching prey under water over a longer period of time.The larvae do not have to renew their air supply at the surface like most dytiscid larvae, and this reduces the probability of being caught by predators outside the water.
It is apparent that hygrobiid larvae (and adults) are perfectly adapted to their specific habits.However, the high degree of specialisation, especially in the larval stages, may have led to a situation that is sometimes referred to as an "evolutionary dead end street".Hygrobiidae largely rely on one source of food, much like the closely related Amphizoidae, and like this group they are characterized by an unusual relict distribution, and comprise only few extant species.They are much less successful than their presumptive sister-group, Dytiscidae, which was able to adapt to a wide variety of habitats and food resources and is today widely distributed and represented by nearly 4000 species (Nilsson, 2001).

TABLE 1 .
Position of ancestral setae and pores on the head appendages of instar I of Hygrobiidae.

TABLE 2 .
Position of ancestral setae and pores on legs of instar I of the familiy Hygrobiidae.

TABLE 4 .
Number of secondary setae on the legs of instars II and III of the genus Hygrobia.

TABLE 5 .
Character state matrix.