The external morphology of Austroplatypus incompertus (Schedl) (Coleoptera, Curculionidae, Platypodinae).

Abstract Previous descriptions of adult Austroplatypus incompertus (Schedl) are completed by the addition of descriptions and illustrations of the adults and, in particular, their maxillary palps. I describe and illustrate the non-adult phases of the life cycle and provide a key to the larval instars. The sexual dimorphism of Austroplatypus incompertus is atypical and includes a latitudinal cline which obeys Bergmann’s rule. The taxonomic position of the genus within the Platypodinae is clarified. Platypus incostatus Schedl is recognised as the male of the species, and hence a new synonym of Austroplatypus incompertus.


Introduction
Studying and accurately describing the external morphology of insects is important because it is the external details that characterise and give indications of the animals' biology, ecology and social behaviour. In addition, insects are often identifi ed solely by external adult morphology and this is refl ected in the formal taxonomic descriptions of most species.
Studies of the external morphology of platypodines have generally concentrated on taxonomic research and consequently have mainly dealt with descriptions of specifi c parts of the external morphology of adults. In contrast, relatively few papers (Hogan 1948, Browne 1961, Roberts 1962, Santoro 1957, 1965, Candy 1990) have described all the developmental stages of a particular species.
Th e lack of comprehensive morphological descriptions within the platypodines is typifi ed by Austroplatypus incompertus (Schedl). Th is species was discovered in the early 1950s and named in 1968. Describing and identifying it has been problematic as specimens are unusually diffi cult to collect. Adult beetles are not attracted to light and the only way to obtain all developmental stages is to cut them from living trees. Consequently there has been a dearth of material for description or comparison. In fact, until the current study, no well-documented collection of both adult and larval A. incompertus material had been made. Th is has no doubt contributed to the small number of papers dealing with the taxonomy and biology of A. incompertus (Schedl 1968, Campbell 1969, Browne 1971, Harris et al. 1973, 1976. Th ere are even fewer studies of the immature stages. No descriptions exist of the eggs or pupae, and although it is generally accepted that there are fi ve larval instars (Harris et al. 1973, Wright andHarris 1974;Harris et al. 1976), only the fi nal instar has been described (Browne 1972). Th ere are no records of any morphological diff erences between instars or any measurements of head capsule widths, an important method for diff erentiating instars.
Th e research reported in this paper had two broad aims. Th e fi rst was to verify and complement existing descriptions of the external morphology of A. incompertus. Th is necessitated descriptions of the egg, larval instars and the pupa as well as a description of adult sexual dimorphism and the adult maxillary palps. In addition immature stages and adults were illustrated with line drawings and scanning electron micrographs.
An integral part of these descriptions were measurements such as head capsule widths and adult body lengths. However, such measurements are problematic if variation exists among populations. Th e increase in mature larval head capsule width of Platypus subgranosus Schedl from north to south (Candy 1990) illustrates the need to take measurements over a wide geographical range where possible. Th us, size variation was also examined for a number of factors, viz. geographical location for larvae and adults and sex and host tree species for adults.
Th e second aim was to integrate the new material presented here with existing taxonomic descriptions and discuss the current taxonomic position of A. incompertus.

Material and methods
Th e source materials for this paper were voucher specimens collected during the study as well as material already held in the Forestry Commission of NSW Insect Collection (FCNI). In their entirety these specimens encompassed the currently known geographical and host tree species range of A. incompertus (Kent 2008a

Specimen preparation -General
All specimens of immature life stages were fi xed to preserve their shape and size and thus ensure that descriptions and measurements were accurate and comparable. Eggs, larvae and pupae were fi xed in KAA [kerosene (7%), glacial acetic acid (16%) and 95% ethanol (77%)] for 5-30 minutes depending on size and developmental stage; eggs and small larvae required the shortest fi xing period and the fi fth instar and pupae the longest. Th e fi xed specimens were then passed through immersion stages of several hours each in 90% and 85% ethanol before eventual permanent storage in 80% ethanol. Adults were killed either by freezing or by immersion in 80% ethanol and then air-dried and mounted on card points.

Specimen preparation -Scanning electron microscopy
For scanning electron microscopy, specimens were fi xed in 2% glutaraldehyde in phosphate buff er (pH 6.9) for 24 hours, transferred through a series of increasing ethanol concentrations and then stored in absolute ethanol until coating. Just before coating, the specimens were placed in either ethyl acetate or acetone and then criticalpoint dried, after which they were sputter-coated with platinum or gold.

Observations
Observations of all life cycle stages of the beetle were made using stereo-dissecting and compound microscopes and a Cambridge S120 scanning electron microscope with a Robinson detector. Th ese observations formed the basis for the descriptions of the external structures. Th e morphological terminology for larval descriptions follows that used by Browne (1961Browne ( , 1972 and Roberts (1962).

Sex determination of adults
Adult beetles were sexed based on external morphological diff erences (Kent 2001): females by the presence of mycangia and a spined elytral declivity; males by the absence of mycangia and presence of simple rounded elytral apices.

Measurements
Both adult and larval measurements were made using a stereo-dissecting microscope fi tted with a scaled graticule. One measurement was recorded for larvae: the width of the head capsule at its widest point. Th ree measurements were recorded for adults: dorsal prothorax length (measured along middle of the prothorax) and width (measured at its widest point, the posterior edge of the femoral emargination), and elytral length. Although total length is commonly reported in taxonomic descriptions, this measurement proved to be unreliable due to post-mortem head defl ection and was therefore not recorded. Some adult beetles could not be measured because of their position on the card mounts.

Statistical analysis
Analysis of variance (ANOVA) was carried out on the measurements of the fi fth instar larval head widths to determine whether there was a diff erence in specimens from diff erent localities (the localities from which study material was derived fell naturally into three well separated latitudinal groups - Fig. 1). Similarly, measurements of adult beetles were analysed using ANOVA to determine whether there were diff erences between sexes, or between adult females from diff erent localities or diff erent host species (there being too few males from the range of localities and hosts to permit analysis). Th e analyses of females were restricted to prothorax length, as this was the most repeatable measurement. Where appropriate, Post hoc Tukey Honest Signifi cant Difference (HSD) multiple comparison tests were performed to determine which means diff ered signifi cantly.
First instar larva (Figs 2, 3a, b) length 1.183 mm ± 0.2 (SD) (n = 5), maximum body diameter much smaller than that of gallery. Maximum width on 5th abdominal segment. Body hyaline, white, shape ovoid and hump-backed dorsally. Head exserted, width of head capsule 0.303 mm ± 0.012 (n = 10), wider than long, greatest width over bulbous antero-lateral margins. Head setae prominent, 1 posteriorly to each anterolateral margin, nearly twice as long as any other. Antennae small, one each side of epicranium in unpigmented portion between mandibular condyles (Fig. 3a). Mandibles lightly sclerotised and with comb-like teeth (Fig. 3a). Maxillary palpi 1-segmented; labial palpi 1-segmented (Fig. 3b). Meso-and metathoracic segments each enlarged into pseudopods. All thoracic segments bearing a single prominent lateral seta on each side. Abdominal tergites 6 and 7 each bear a single prominent seta on a dorsolateral protuberance on each side. All sternites with lateral protuberances, each bearing two setae, those of segments 5-9 enlarged into pseudopods (Fig. 2). Only two pairs of spiracles, one on prothorax and the second on abdominal segment eight.
Second instar larva (Fig. 4) slightly larger than fi rst instar, but same general form. Head more elliptical, less angular than fi rst instar. Head capsule width 0.353 mm ± 0.028 (SD) (n = 11). Mandibles similar. Th e main diff erence between the fi rst two instars is the presence of nine pairs of spiracles, one each on the prothorax and one each on eight abdominal segments.
Th ird instar larva (Fig. 5a, b) slightly larger than the second instar, but still smaller than gallery diameter. Head capsule width 0.581 mm ± 0.077 (SD) (n = 32). Body still hump-backed but more fl attened ventrally and not as translucent as fi rst two instars. Head distinctly narrower than prothorax and more rounded in shape in comparison to the fi rst two instars. Pseudopods no longer as prominent. Spiracles as in second instar. Mouthparts as in Fig. 5a, b.
Fourth instar larva (Fig. 6) body stout, more or less closely fi tting the galleries. Head clearly narrower than width of pronotum. Pseudopods not evident. Head capsule slightly wider than long, width 0.868 mm ± 0.071 (SD) (n = 49). Labrum and mandibles as in Fig. 6. Mandibles similar to third instar, only slightly chitinized and still bearing small teeth along apical and subapical edges. Pronotum lacking any chitinized pattern of ridges (see below).  (Figs 7, 8, 9a, b, c, 10, 11) see Browne (1972) for detailed description. Th e following is supplementary to Browne's description. Head capsule width 1.121 mm ± 0.086 (SD) (n = 547). Figures 8 and 9a, b, c illustrate head with details of labrum, mandible and labium, respectively. Mandibles heavily chitinized, bluntly pointed and lacking teeth. Pronotum pattern with two pairs of setae, the lateral seta of each pair socketed and surrounded by irregular chitinized ridges, the medial seta surrounded by lighter chitinized irregular ridges (Fig. 10). Spiracles (Fig. 11) ovate with single, short, dorsally directed air tube; peritreme surrounded by cuticular wrinkles. Th oracic spiracles larger than abdominal ones.

Fifth instar larva
Five distinct larval instars are morphologically discernible using the following key 1.

3.
Only two pairs of spiracles, both the same size, one pair on prothorax and one on abdominal segment 8, head very broad, transversely oblong (Fig. 2)  Pronotum lacking any brown chitinized pattern; mandibles slightly chitinized with teeth on cutting margin (Fig. 6)  Pronotum with brownish chitinized patterned consisting of two pairs setae, surrounded by irregular ridges (Fig. 10); mandibles heavily chitinized, apex bluntly pointed and free of small teeth (Fig. 9b) (Fig. 12) cuticle white and glabrous, setae coarse, arising laterally from an armed tubercle (Fig. 13), larger and more numerous on head and prothorax than on abdomen. Rostrum not reaching fore coxae. Antennal club smooth. Sex indeterminable until the darkening of the cuticle of the young adult appears through the pupal skin. At this point females can be identifi ed by the appearance of mycangia in the centre of the prothorax.
Adults (Fig. 14a, b, c, d) have the typical elongate cylindrical form of platypodines, with a length of approximately 6 mm and a diameter of approximately 2 mm. All types examined at NMV were females. Th is is in contradiction to Schedl's tentative assignment of them as all male (Schedl 1968). Schedl (1972b) described P. incostatus as a new species closely allied to A. incompertus, from a single specimen of unspecifi ed sex from Dorrigo, N.S.W., collected on 23 March 1954. Following the suggestion of R.A. Beaver (pers. comm.) that the holotype of P. incostatus described by Schedl might in fact be a male A. incompertus I examined the holotype (held by the Natural History Museum, London) and confi rmed that it is a male A. incompertus. Interestingly, the FCNI collection contains a single female A. incompertus collected from the same locality on the same day, but Schedl evidently never saw this specimen. As the name P. incompertus predates that of P. incostatus, the latter becomes a synonym.
Provided one is aware of these problems Schedl's 1968 and1972b descriptions of adult beetles are fairly complete. Since they have already been published do not require repeating in this paper. Inadequacies in the original description of the adult head (Schedl 1968) were remedied by Browne (1971). In addition the mycangia in female A. incompertus are described in detail and illustrated in Kent (2008b).
Maxillary palps (Fig. 15a, b, c) of adult A. incompertus are three segmented. Th is is in contrast to a previous report that they have four segments (Browne 1971). Browne's error appears to have resulted from mistaking part of the palpiger as an additional segment (Zimmerman 1994, Kuschel 1995. Because of the importance this misidentifi cation has played in the taxonomic debate regarding whether platypodines and scolytines should be placed within or outside of Curculionidae (Wood 1973), the palp is illustrated here in detail for the fi rst time. Th is fi gure shows the palp in a se- quence of views, the ventral one showing a superfi cial resemblance to a four segmented condition. Th is resemblance disappears when the palp is rotated to the dorsal view.

Sexual dimorphism
Th e sexes are dimorphic in A. incompertus with the most obvious diff erence being the shape and sculpturing of the elytra. In the female the elytral declivity is abrupt and armed with prominent spines, while in the male the elytral apices are more rounded with only very small spines. Th is diff erence between the sexes is easily discernible with the naked eye and can be used to sex individuals in the fi eld. Additional diff erences between the sexes can be seen using a stereo-dissecting microscope: -Th e presence in the female of mycangia in the centre of the prothorax (Fig. 16) and their absence in the male; -Th e presence in the female of a series of ridges at the base of the elytra (Fig.  17) and their absence in the male. Th e ridges are located between the 3rd and 4th interstices and form a series of backwardly directed ridges, twice as wide at the anterior edge (straddling both the 4th and 3rd interstices) and tapering to half that (just the 3rd) for approximately a sixth of the total length of the elytra.

Larvae
Only fi fth instar larvae were present in suffi cient numbers to allow analysis of variance. Th ere were highly signifi cant diff erences in head capsule width among the diff erent latitudinal groups [P < 0.001, DF = (2, 543)] with the width increasing from north to south (Table 1).

Adults
Between sexes. Although both the prothorax and the elytra were measured, the prothorax proved to be better suited to regular measurement because of its fl atness, especially along its length. Measurements based on the prothorax were also less likely to be subject to error, compared to elytral length, as the latter can be diffi cult to measure if the elytra are opened after the death of the specimen. Even so, the results of the analysis of the three diff erent measurements pooled across all hosts and localities showed that all were highly signifi cantly diff erent between the sexes [prothorax length P < 0.001, DF = (1, 604); prothorax width P < 0.001, DF = (1, 603) and elytral length P < 0.001, DF = (1, 604)], with males being smaller than females.
Because all three measurements were signifi cantly diff erent between the sexes but prothorax length was the most suitable and reliable measurement, subsequent analyses   were restricted to this variable. Males were present in too few numbers across all localities and hosts to permit analysis. Between localities. Th ere was a highly signifi cant size diff erence between female beetles from the diff erent latitudinal groups [P < 0.001, DF = (2, 428)] with prothorax length increasing from north to south (Table 2).
Between hosts. Th ere was also a highly signifi cant diff erence between beetles from the diff erent host tree species [P < 0.001, DF = (8, 418)], and Post hoc Tukey HSD multiple comparisons placed the hosts into three groups (Table 3).

Immature stages
Five distinct larval instars could be distinguished on the basis of their morphology and their head capsule widths, as is the case with other platypodines (Browne 1961, 1972, Hogan 1948, Roberts 1960, 1962, Candy 1990. Th e external morphology of the fi fth instar larval stage of A. incompertus is characterised by the design of the prothorax pattern which separates it from the larva of Dendroplatypus, which it most closely resembles (Brown 1972). Brown (1972) also split Austroplatypus from the majority of platypodines based on his observation that the anterior notches of the labrum are shallow. Th is study found that the notches in fi fth instar larvae were not shallow as illustrated by Browne (1972) but more deeply and narrowly notched (Fig. 8). Th is level of detail is only seen in scanning electron micrographs. Preserved specimens and slide preparations show the labrum with shallow notches because these preparation methods appear to result in the closure of the gaps between the notches (Fig. 9a).

Sexual dimorphism
Sexual dimorphism in A. incompertus is reversed, compared with the situation in other platypodines. Males are signifi cantly smaller than females, females have elytral modifi cations, in the form of an elytral declivity modifi ed for both cleaning and defence, which males do not, and only females possess mycangia. In most platypodines,   the sexes are similar in size or the male is only slightly smaller, males alone possess elytral modifi cations or modifi cations are much more strongly developed in males, and males either have no mycangia or a reduction in the number of mycangia compared with females (Chapius 1865, Strohmeyer 1914, Hogan 1948, Milligan 1979, Roberts 1960, Wood 1993, Beaver and Liu 2007. Wood (1993) linked these external modifications, or lack of them, to the typical Platypodinae monogynous mating system and male initiated gallery system. Th is is consistent with the situation in A. incompertus where the reversal in elytral modifi cations refl ects the diff erent roles undertaken by the sexes in gallery systems with only females initiating the gallery systems and carrying out defence and maintenance activities (Kent 2001).

Size diff erences
Fifth instar larval head capsule widths and all three adult body measurements of A. incompertus display a size variation consistent with Bergmann's rule which states that body size increases at higher latitudes (Blanckenhorn and Demont 2004). Latitudinal clines in body size have been observed in a number of ectotherms (Reeve et al. 2000), including arthropods (Blanckenhorn and Demont 2004). Whilst several authors (Shaw andGroeters 1998, Reeve et al. 2000) have suggested possible mechanisms for the evolution of such clines, research in this fi eld is still in its infancy and no theory has gained ascendancy. As a result of the latitudinal cline observed in A. incompertus, measurements for this species should be treated with caution in taxonomic works.

Host species n Prothorax length (mm)
Host distribution
Platypus incompertus was described from six specimens, which Schedl thought to be male. An examination of the type material of A. incompertus (held in the National Museum of Victoria) revealed that it consisted of females only. P. incompertus was subsequently placed in a new genus, Austroplatypus, by Browne (1971). His paper contains a generic description for A. incompertus and a detailed description of its antenna, maxilla and labial palps. Browne's miscount of the segments of the maxillary palps (four instead of three) caused problems in the taxonomic placement of platypodines within the Curculionoidea (Wood 1973, Kuschel 1995. Armed with the knowledge that A. incompertus displays atypical sexual dimorphism, the discrepancy between the original descriptions of both A. incompertus (Schedl 1968) and P. incostatus (Schedl 1972b) may be explained. Th e presence of mycangia, which in platypodines are usually more developed in females than males, should have suggested to Schedl that his original six specimens were females. However, he may have decided (incorrectly) that his six specimens were males based on the structure of their elytra. Subsequent taxonomic and descriptive work (Campbell 1969, Browne 1971, Schedl 1972a, Harris et al. 1973, 1976 correctly assigned descriptions to the sexes. Schedl (1972b) unfortunately erred again when he described P. incostatus as a new species closely allied to A. incompertus. Schedl distinguished this new species from A. incompertus based mainly on the distinctly smaller size, the absence of mycangia and the reduced elytral modifi cations, all of which are manifestations of the atypical sexual dimorphism in this species. He was also unwilling to assign the specimen a sex.
Because the sexes in most platypodines are either similar in size or the female is slightly larger the distinctly smaller size of the single P. incostatus specimen described by Schedl probably convinced him that he was dealing with two diff erent species. However, the current study has shown that not only is there a signifi cant size difference between the sexes, but also a signifi cant size diff erence between beetles from diff erent parts of their distributional range. Th e original type material that was used to describe A. incompertus came from the southern part of the distribution (Eden, NSW and Woodhouse Creek north of Omeo and near Omeo Victoria), whereas the single type specimen of P. incostatus is from the most northern part of the distribu-   tion (Dorrigo, NSW). Th us, Schedl would have been looking at a male specimen from the smallest end of the size range, compared with female specimens from the larger end of the range. Th is situation would have suggested to him that he was dealing with two species.

Position of Austroplatypus within Platypodinae
Th e position of A. incompertus in the family Platypodinae is still unclear. Browne (1971) placed it in the tribe Platypodini near Dendroplatypus Browne, while Schedl (1972a) assigned it to the subfamily Platytarsilinae together with the monotypic Australian genus Notoplatypus Lea and the Malaysian genus Platytarsulus Schedl. Austroplatypus does not fi t within Schedl's Platytarsilinae, since it does not possess the distin- guishing characteristics of very slender elongate body form and the proboscis-like head shape. In addition, both of the other two genera have reduced antennal segments while Austroplatypus has the four antennal segments characteristic of the rest of the platypodines (Schedl 1972a). Wood (1993) synonymised Austroplatypus under Platypus Herbst and thus also placed it in the Platypodini. He gave no reasons for this action but had possibly detected Browne's (1971) error with respect to the number of maxillary palp segments. If so, it is strange that he did not also synonymise Dendroplatypus under Platypus, as Browne (1955) had similarly miscounted its maxillary palp as having four segments. Zimmerman (1992Zimmerman ( , 1994 and Kuschel (1995) noted Browne's miscount of the maxillary palp segments of Austroplatypus and treated it as a valid genus, but did not comment on its classifi catory position. In his review of the genus Platytarsulus Beaver (1998) also agreed that Austroplatypus was a valid genus. Since A. incompertus diff ers from Platypus not only in its atypical sexual dimorphism, but also in its uniquely modifi ed life-history strategies of mating and reproduction Simpson 1992, Kirkendall et al. 1997), its placement in a separate genus within the Platypodini appears justifi ed.