Distribution and Bionomics

The only two extant species are restricted to southwestern Australia (fig. 22B), but Cretaceous fossils are known from France, Spain, Siberia, Lebanon, and Myanmar (Borkent, 2000a; Szadziewski, in press; Szadziewski and Arillo, 2001), proving that the genus was once much more broadly distributed.

The only species in which females have been observed to bite are those of A. mcmillani, which feed on kangaroos and humans. However, the finely serrate mandible and retrorse lacinial teeth of the adult females of A. annettae strongly indicate that these too feed on vertebrates (Borkent, 1995: 129– 132). The morphology of the claws of the female of A. annettae additionally may indicate that these feed on birds (see discussion below under that species). The mouthparts of fossil Austroconops are not visible (Borkent, 2000a), and therefore it is uncertain what they fed upon. The presence, however, of vertebrate blood-feeding in A. mcmillani and the phylogenetic position of the genus as an early lineage within the family where vertebrate blood-feeding is plesiotypic strongly suggest that all extinct species fed on vertebrates (Borkent, 1995, 2000a). Details of male swarming in A. mcmillani are given below.

First-instar larvae of A. annettae and A. mcmillani were both successfully reared to fourth-instar larvae (and A. mcmillani to the pupal stage) in very wet soil, with regular (generally every second day) additions of nematodes and a fecal infusion. All instars of the aquatic larvae were clearly attracted to fresh drops of fecal infusion as shown by the concentrations of larvae directly under these drops and they were rarely seen to feed on nematodes. This phenomenon, and the presence of well developed, finely toothed scopae (figs. 3A, 19) suggest that they are likely associated with feces or concentrated decomposing vegetation in nature (producing an abundance of microorganisms). Some second, nearly all third, and all fourth-instar larvae had pink or red hemolymph, indicating the presence of hemoglobin, which additionally suggests that they may be associated with an oxygen deficient wet habitat. Larvae cannot swim but use a combination of their anterior and posterior prolegs and, especially in later instars, a relatively slow serpentine body motion to move through wet substrate. Further details of behavior and habitat are provided below for each species.

Borkent et al. (1987) suggested that the blue-green pigmentation of live adult A. mcmillani may have indicated that the then unknown larvae were feeding on algae because this coloration has been observed in some Ceratopogonidae such as some Culicoides species of the schulzei species group and some Dasyhelea Kieffer species which feed on algae as larvae. Our observations here show that the relationship between algal feeding and adult color is not substantiated for at least A. mcmillani as larvae matured to adulthood without feeding on algae. Austroconops annettae was also successfully reared to the fourth-instar without algae.

Taxonomic Discussion

We consider the elongate, pale egg of Austroconops species unique within the family. Eggs laid by other Ceratopogonidae are initially pale but soon turn dark. However, of 103 extant genera, eggs have been described for only 18 genera and of those, 4 genera are known only as eggs described from within the female abdomen (so their final color cannot be determined).

We identified the four larval instars of both species based on the following evidence. First-instar larvae are easily identified by their darkly pigmented egg burster (figs. 2H, 7A, B, 8B). Because of the small number of measured second and fourth-instars of reared Austroconops larvae it was initially difficult to identify the instars 2–4. The fourth-instar of A. mcmillani was confidently identified because one of these molted to the pupal stage. Although the head capsule of this individual was not retrieved from the mud and therefore not measured, observations before it pupated showed that it was clearly close to, or within, the range of measurements reported here. Head capsule length increments of all instars followed Dyar's Law, increasing by a factor of 1.32, 1.31, and 1.34 for A. mcmillani and 1.33, 1.32, and 1.25 for A. annettae. The latter value, the factor of change for third to fourth-instar, may be due to the small sample size of fourth-instar A. annettae or perhaps to less than optimal feeding conditions (and hence smaller individuals). These values are similar to those reported for species of Culicoides, which are about 1.4 (Kettle and Lawson, 1952; Kettle and Elson, 1975).

There are significant statistical differences in egg characteristics (table 3) and larval head capsule lengths (table 4) of the different instars between A. mcmillani and A. annettae. Considering, however, that these immatures were obtained from eggs laid by a very few females, and that the larvae were reared under laboratory conditions, the size differences noted here may be artifacts. Otherwise, eggs and larvae of the two species could not be distinguished from one another.

Larvae of Austroconops, when compared to other Ceratopogonidae, are missing head capsule sensilla z, k (sensory pit), and q. Also, there are two additional dorsolateral setae on larval abdominal segment 9 (fig. 3H), here labeled as l₃ and l₄, which have not been reported in other Ceratopogonidae.

The larval antennal sensilla of Ceratopogonidae have never been described in sufficient detail to determine most homologies within and outside the family. Larvae of Austroconops species have a well-defined basal segment bearing 6 apical sensilla and a further second and third segment (figs. 9A, B, 14A, B, 20A). Based on position, relatively large size, and a uniformly porous surface, the most elongate, porous sensillum is homologous to the “large lobe” present in Culicoides (Murphree and Mullen, 1991) and most other Ceratopogoninae (Borkent and Bissett, 1990; Borkent and Craig, 1999). Based on position and some details of structure, the following sensilla are likely homologous to sensilla in Chironomidae: most elongate, porous sensillum = blade; mesal short sensillum = accessory blade; short bilobed sensillum labeled sensillum 3 here = fused style and peg sensillum. The remaining three short sensilla do not have readily apparent homologies with Chironomidae; they are labeled here as sensilla 1, 2, and 4. Sensillum 1 has an open apex bearing a tiny peg. Sensillum 4 has a porous surface.

The labrum of Austroconops shows a number of similarities to other Ceratopogonidae. Sensilla 1–4 are clearly present as a group on the labra of most other Ceratopogonidae (Hribar and Mullen, 1991, labeled as “sensillum styloconicum”). In many Chironomidae the two sensilla SIVA amd SIVB on the anterior portion of the labrum are morphologically very similar to S2 and S4, and these are likely homologous. Chironomidae S1, just dorsal to the labral lamellae, probably is homologous to our S10, and the scopae present in Austroconops and Ceratopogoninae are probably homologous to the labral lamellae (Wiederholm, 1983). Further detailed comparisons require further study of these structures in numerous taxa (both SEM and study of nerves).

Borkent et al. (1987) reported an “M-shaped apodeme” in the female genitalia of A. mcmillani. In further study we are puzzled as to the nature of this structure, which varies in both A. mcmillani and A. annettae from a faint M-shaped sclerotization to the presence of a pair of laterally positioned, pigmented sclerites. Some other Ceratopogonidae also have sclerotized structures anterior to the fused or separated sternite 9 (e.g., some Leptoconops, some Culicoides, Alluaudomyia Kieffer, and others). Careful histological study is needed to better interpret this feature.

Cladistic analysis below indicates that, among extant taxa, Austroconops is the sister group of Leptoconops and that together they form the sister group to all remaining extant Ceratopogonidae. Eight species of Austroconops are now known: two of these are extant and six are Cretaceous fossils:

The genus Jordanoconops Szadziewski was proposed as a monotypic genus to include a Lower Cretaceous Jordanian amber fossil, Jordanoconops weitschati Szadziewski (Szadziewski, 2000). It may actually be a member of Austroconops (see below under Relationships Between Species of Austroconops).

Distribution and Habitat

Austroconops mcmillani is known from three sites in southwestern Western Australia (fig. 22B). A fourth locality from “National Park, Perth” cannot be specifically located (see Taxonomic Discussion below).

Adult A. mcmillani were collected at five sites in Yanchep National Park during 2001– 2002 (fig. 22C: sites A–E). On November 20–21, 2001 adults were extremely abundant at fairway 2 in the golf course (fig. 22C; site A). Biting females were very common along the southern margin of the fairway (fig. 21A) and at times were attacking humans at the rate of more than 50 per minute! Further observations on biting are given below. Swarming males were collected directly southeast of the tee-off area at the southwestern end of the fairway, which was the driest area on the fairway. The southeastern margin of the fairway had some very wet soil about halfway down its length, and the northeastern end included a ditch with open water. The vegetation included the following trees at the western drier end: Agonis flexuosa (Weeping Peppermint; native to Southwest of W.A., but introduced into the Park), Allocasuarina fraseriana (Common Sheoak), Banksia attenuata (Yellow Candle Banksia), Banksia grandis (Bull Banksia), and Banksia menziesii (Firewood Banksia). The shrubs and sedges were the same as listed for the eastern (wet) end as given below, but were sparse. Trees at the wet eastern end of the fairway were Banksia littoralis (Swamp Banksia); shrubs were Acacia saligna (Orange Wattle), Spyridium globulosum (Basket Bush), Templetonia retusa (Cockies Tongues), Xanthorrhoea preissii (Balga or Grass Tree) and sedges were Lepidosperma longitudinale (Pithy Sword-sedge), Lepidosperma striatum (no specific common name; a sword-sedge), and a herb, Centella asiatica (Indian Pennywort). Samples of mud and detritus from the ditch at the east end of fairway and a few wet soil samples from about three-fourths down the fairway failed to produce immatures.

Austroconops mcmillani adults were also observed at the southern margin of Loch McNess (fig. 22C; site B), about 30 meters west of where a small stream enters the lake. The adult population was significantly smaller than at site A, and on November 18–20 females attacked, at best, at the rate of about 1–2/minute. One male was swept from the surrounding vegetation. This wooded area had a narrow innundation zone with very wet soil and mud and some small pools that partially filled after a short rain. The predominant tree at this specific site was Melaleuca rhaphiophylla (Swamp Paperbark) with shrubs of Acacia saligna (Orange Wattle), sedges Carex fascicularis (Tassel Sedge), Lepidosperma effusum (Spreading Sword-sedge), Schoenoplectus validus (Lake Club-rush), and some Typha orientalis and a thick mat of an introduced herbaceous weed Pennisetum clandestinum (Kikuyu Grass). Mud and wet fallen leaves sampled from this site produced no immatures after floating debris and substrate with sugar or sorting by hand.

Two females of A. mcmillani were collected on November 20 at the northeast corner of Loch McNess (fig. 22C; site C). This area was a very wet, but more open, innundation zone.

On November 20 from about 10:50 am to 1:15 pm the first author and his wife walked completely around Loch McNess (fig. 22C), stopping 14 times at more-or-less even intervals around the lake for 3 minutes at a time, breathed deeply (to produce maximum CO₂), and waited for attacking females. The only spots where females were found were at sites B and C (fig. 22C), suggesting that the species is indeed restricted to some specific localities in the park. Females attacked within 5 minutes at site B when arriving at 2:55 pm (November 19, 2001) and within seconds upon arrival at site A at 10 am (November 20, 2001), further indicating the presence of quite localized populations of A. mcmillani.

All sites shared the presence of very wet soils, which corresponds well with the behavior of the reared larvae which actively burrowed through wet mud/detritus.

Additional biting females were collected by Len Zamudio at fairway 3 on January 3 and 9, 2002 by a small creek which traverses the fairway about halfway down its length (fig. 22C; site D) and at fairway 8 on January 9 and 31, 2002 (fig. 22C; site E).

The paratype specimens of A. mcmillani collected at Yanchep N.P. were collected on December 23, 1954 by B. McMillan “at the opening of a small cave to the right of the track leading north from the golf course” (A. L. Dyce, personal commun.) and this would represent another place in the park where A. mcmillani are known. However, the total park area has been progressively drying over the past number of years and it is unknown what impact this might have on the distribution of A. mcmillani in the park since 1954.

Egg Laying, Larval and Pupal Behavior and Development

Eggs were laid by female A. mcmillani in a scattered pattern on the surface of wet mud in a vial or on the sides of the vial just above the mud where moisture had condensed on the vial wall. Three eggs of A. mcmillani that were laid on the sides of the vial were at least somewhat dried after they were laid and had partially collapsed but when immersed in water and rehydrated, they subsequently hatched. The sequence of egg laying, hatching, and larval development is given in table 5.

First-instar larvae burrowed very actively through moderately packed, quite wet mud/ detritus with a serpentine motion. They used different means of propulsion depending on the nature of the substrate and whether they had located food. When larvae were in water, but with substrate to crawl upon, they used their anterior proleg in an anteroposterior motion, dragging the rest of the body along to produce a slightly jerky anterior movement. Larvae that were in very loose detritus and that wanted to move through the water from one clump of detritus to another used their posterior proleg as an anchor, extending the head and length of the body through water to connect with another piece of detritus. In thicker substrate, at least some movement was facilitated by serpentine movements of the anterior half of the body with the abdomen being dragged behind and with the posterior proleg hooks withdrawn into the anus. In dense substrate (such as thick mud/detritus or agar), the head capsule would additionally be extended anteriorly, bent somewhat down to take hold of the substrate and then bent farther anteroventrally, helping to bring the body forward; these larvae also withdrew their anterior proleg into the body in the crease between the cervix and remaining prothorax. The proleg was withdrawn much like the inverting of a shirt sleeve, with the terminal hooks inverting first into the interior of the extended leg and being withdrawn toward the body and the rest of the proleg following suit. This inversion is almost certainly the result of muscles that insert at the apex of the proleg (fig. 4B). Extension of the proleg was the reverse of this sequence. Larvae removed from substrate and placed in a drop of water on a slide never withdrew the anterior proleg as they actively sought a substrate to latch on to, and it is clear that this structure is important to their locomotion.

As larvae were moving through substrate, their head capsules would move rapidly in a flicking motion left and right and dorsoventrally, apparently testing for potential food. The larvae would also periodically stop, at times anchoring themselves with their posterior proleg and poke their heads rapidly here and there among the detritus, again in an apparent search pattern. The larvae rested regularly and most often used their extended posterior proleg hooks as an anchor on the substrate. Such resting larvae were generally in a more-or-less elongate position (with some curves or bends in the body), but occasionally they would assume a tight U-shaped position, with the posterior proleg hooked into detritus.

When first-instar larvae were removed from very wet mud/detritus to water, they immediately became very active, writhing and attempting to find a substrate upon which to take hold. Upon doing so, they immediately burrowed into the substrate and clearly preferred more solid clumps of detritus than fine, looser particles to burrow into. Larvae in detritus that were harassed with a probe quickly escaped with rapid use of the anterior prolegs and, in thick substrate, some undulations of their bodies.

Five first-instar larvae of A. mcmillani were observed feeding on E. coli, and when doing so they had their posterior proleg firmly hooked into the substrate and worked over acceptable substrates with their mouthparts. The head capsule moved back and forth, appressed to the agar, and the mouthparts moved rapidly, with the labrum contracting and apparently moving food into the mouth cavity. The pharyngeal complex periodically moved food into the gut. The gut contents appeared white in these individuals (same color as the E. coli). When feeding, the antennae actively moved independently of one another. After feeding on E. coli for 3 days, all five larvae died.

When drops of the fecal infusion, nematodes, and rotifers were introduced to the larvae in the mud/detritus substrate, further observations were made of feeding. Larvae were obviously attracted by the drops of fecal infusion and were often very concentrated directly underneath these small pools of feces. Occasionally, larvae would be seen lashing at a passing Paramecium or a slower rotifer. A few second-instars were seen to successfully attack and eat passing rotifers but missed most that were nearby. The strong impression of all larval instars was that they could at best only attack slow prey.

Second-instar larvae became increasingly fat and slower and by the third-instar and especially by the fourth-instar, larvae moved more cumbersomely, with a slow, serpentine motion. Serpentine movements of the body were obviously the most important basis for movement as the larvae worked their way through the mud/detritus substrate. To reverse direction, a larva either simply reversed its serpentine movement or, by turning its head capsule and anterior portion of the thorax in a posterior direction, doubled back along the length of its body The anal hooks were withdrawn into the anus when a larva moved and were often (but not always) exserted when the larva rested; in some instances the hooks were attached to substrate, but in more sparsely distributed substrate were simply exserted into the water without being anchored to anything. The anterior proleg also appeared to be rarely used by third- and fourth-instar larvae and apparently was exserted only when the larva had difficultly finding any substrate ahead. Second-, third- and fourth-instar larvae always extended their anterior proleg in a groping motion when placed in a drop of water on a microscope slide and writhed helplessly, very similar to the more ponderous movements of Dasyhelea larvae. Larvae which were harassed with a probe quickly escaped with rapid serpentine undulations of their bodies through the surrounding substrate. When the substrate was shaken and larvae severely disturbed, fourth-instars often assumed a curled-up position (this was not attempted with earlier instars). The darting, probing search pattern of the head capsule continued in these stages. Larvae sometimes moved their antennae and, when ingesting food, moved their mandibles synchronously. Some second-instar and most third-instar larvae took on a pinkish hue. A few third-instar larvae remained unpigmented and translucent. All fourth-instar larvae were pink or reddish.

The day before the single fourth-instar larva pupated, its thorax was strikingly broad, the respiratory organs of the pharate pupa could be clearly seen, and the eyespots had moved posteriorly in the larval head capsule. The pupa, which completely shed the larval exuviae, was initially very pale, with red hemolymph, and, at 22–25°C, was very sluggish, although capable of very slow circular movements of the abdomen. It was buried in the wet substrate at about a 50–60° angle from the surface, with the tip of one respiratory organ just sticking out of the water; later, the tips of both respiratory organs were exposed. Throughout its development the pupa had the apex of either or both respiratory organs exposed to air. After 24 hours, the pupal cuticle appeared nearly transparent (except for the brown respiratory organs), with an inner core of reddish tissue surrounded by completely clear fluid and a few subcutaneous bluish-green patches in the thorax and abdomen. It was virtually motionless, and flooding for 16 minutes (so that the respiratory organs were submerged) did not result in any pupal movement, nor did any very slight jarring of the surrounding substrate. At one point, upon turning on a bright microscope light, the pupa briefly moved its abdomen in small circular motions. After 48 hours, the pupa did not look much different from 24 hours earlier. After 72 hours, the interior of the pupa became somewhat darker. The blue-green patches were also present in the head (this may have been true earlier but the head could not be seen clearly then). The pupa was very lethargic and moved only slightly when bright light was suddenly shone on it. At 84 hours the developing adult eyes became visible. At 96 hours, the developing adult had darkened further, with the blue-green patches more extensively developed. At 108 hours, the dark adult appeared to be more-or-less completely developed and its black head and thoracic cuticle could be clearly seen. Shortly before the adult male emerged, at 120 hours after pupation, the pupa slowly wriggled its way out of its circular burrow and lay on the wet mud surface. The adult emerged during daylight hours (early afternoon) but failed to completely expand its wings (they were entrapped by the wet mud surface); in all respects it appeared normally developed and a hindleg first tarsomere length of 176 μm (males in nature = 178–214 μm, N = 13) indicated that the rearing conditions and food provided to the larva was of sufficient quality to produce a typical adult.

Biting Activity and Behavior

Of those female adults of A. mcmillani biting humans at Yanchep National Park, most bit on the face (fig. 21D, F); less than 15% of individuals fed elsewhere on the body (mostly shoulders, arms, legs). Females on the face clearly favored areas without hair and, of those attacking the face, were primarily close to the eyes and directly on the ears, with fewer on the cheeks and forehead. A few fed among hairs right at the hairline, including the scalp, eyebrows, eyelashes, and the first author's beard. We are unable to suggest the cues used by females to preferentially attack the face. When bare arms were held up and appressed against the face, the face was still preferred; however, in such a position a few females attacked the armpit (no deodorants used), suggesting that secretions of the skin may be involved as an attractant. Slightly elevated heat from these areas cannot be excluded but this seems unlikely, since females were not attracted to handheld cups of warm or hot water, as are females of some species of Culicoides and Culicidae. It is probable that female A. mcmillani were attracted by CO₂. At the south end of Loch McNess (fig. 22C; site B) when the frequency of biting decreased, the number of incoming females could be increased within 3–5 minutes by doing on-the-spot exercises or by intentionally breathing deeply and rapidly.

Females flew about the head for a period before landing. Those landing on the hair nearly always quickly flew off the surface to resume hovering. After landing on skin, nearly all females began feeding immediately (very little walking about on the skin surface).

Kangaroos were closely observed during the first sampling period of October 16–20, 2001, before the emergence period of A. mcmillani. They very rarely scratched or rubbed themselves. When the first author and his wife were experiencing very intense biting on the golf course in Yanchep N.P. on November 20–21, it was clear that the kangaroos were also being severely bothered by midges (figs. 21B). Many rubbed their eyes and scratched at their forelegs and the shins of the hindlegs with their foreleg feet (fig. 22A). Female kangaroos carrying well developed joeys (baby kangaroos) also often scratched at the front of their extended pouches. The midges attacked the eyes and areas with a minimum of hair. The first author chased kangaroos with an aerial net during these observations and collected numbers of A. mcmillani females, but it was uncertain if these specimens were attacking the kangaroos, were attracted to the first author, or were merely free flying in the area.

Females assumed a biting posture similar to those of Leptoconops and Culicoides that the first author has examined, with the body virtually parallel to the skin surface but very slightly elevated posteriorly (fig. 21D, F). Of 27 females studied, total time to complete blood feeding ranged from 1:05 to 4:40 minutes:seconds (mean = 2:42), when ambient air temperatures ranged from 22 to 29°C (taken in shade). There was no significant correlation between temperature and biting time, although all the feeding times longer than 3 minutes (N = 8) were at temperatures at or below 25°C. We found the bite of female A. mcmillani to be more painful than those of other ceratopogonids that we have experienced (Leptoconops, Culicoides), perhaps because they concentrated on the more tender facial area.

Females fed only during the day when temperatures were above at least 21°C. On November 21 females began feeding at fairway 2 (fig. 22C; site A) at 7:50 am, when the ambient temperature was 22°C. On November 18, at the south end of Loch McNess (fig. 22C; site B), they ceased attacking by 5:45 pm as the light was fading and the temperature was 24°C. Females in vials became lethargic at about 18–19°C and at about 16– 17°C were nearly torpid. It appeared that stronger winds dampened activity, although females continued to bite in sheltered areas, such as the leeward sides of trees, a behavior typical of many biting ceratopogonids. High temperatures (generally above 24°C) and increased humidity appear to greatly increase biting rates (L. Zamudio, personal commun.). Cloudy weather and even a light rain did not stop females from attacking, although a steady drizzle did stop biting activity on the afternoon of November 18, 2001.

Adult Seasonality

Intense collecting of Ceratopogonidae in Yanchep National Park showed that no A. mcmillani adults were present October 16– 20, 2001. November 5 produced moderate numbers of females, which slowly increased with large numbers biting on November 18. Biting activity varied, depending on environmental conditions, until at least January 31, 2002, indicating a nearly 3-month period of female adult biting activity. Populations in the 2002–2003 season were substantially less at Yanchep N.P. (possibly due to a very dry season; L. Zamudio, personal commun.) and biting female adult A. mcmillani were collected from November 14, 2002 until January 1, 2003. Two female A. mcmillani were sampled from kangaroo feces on January 21, 2003 (L. Zamudio, personal commun.), indicating that the emergence period was still about 3 months long in the 2002–2003 season.

Records from three years show males have been collected Oct. 22–23 and Nov. 1, 1985; Nov. 19–21, 2001; Nov. 20, Nov. 22, Dec. 6, and Dec. 12, 2002.

Male Swarming

Male swarms were observed on November 21, 2001 on fairway 2 of the golf course at Yanchep National Park (fig. 22C; site A) from 6:45 to 9:15 am. At 6:45 the temperature was only 15°C (in shade, warmer in sun) and calm. At 7:50 am and 22°C a single, small (about 30 cm in diameter), more-or-less spherical swarm was seen about 1 meter above the grass and about 6 meters from the margin of short trees at the edge of the fairway. A light wind began blowing. Several similarly situated swarms were seen during the next 40 minutes. The swarms started to increase in size, became more elongate vertically, and moved to within 3 meters of the leeward side of the trees, just south of the tee-off area. No obvious swarm markers were evident, other than being on the leeward side of the trees. By 8:05 am the bottom of the swarms were 2–3 meters above the ground and larger (about 50 × 100 cm) and withstood some wind, albeit being periodically blown about and then regrouping. Swarming activity ceased in this immediate area at about 8:30 am but continued a short distance farther east along fairway 2 (where it was a bit cooler) and then began in the first area again at 8:37 am for at least 30 minutes. One swarm collected at 9:00 am with a rapidly swept aerial net consisted of 160 males and 10 females (the latter may have been captured because females had begun biting by then). By 9:15 am swarming had ceased and did not resume for the next 30 minutes, at which time observations were terminated.

A male and a female of A. mcmillani were collected in copula at 8:47 am during these observations by sweeping from ground level to about 1 meter in height in the nearby vicinity of the swarms; the pair separated after about 20 seconds while in the net. While still attached the pair stood on the net mesh facing in opposite directions, showing that the male can at least opportunistically rotate his genitalia 180° (they are otherwise generally held at about 90°). Small white sheets (about 1 × 1 m) were placed on the ground under the swarms of males but no mating pairs settled on these. The fact that females captured immediately after they had completed feeding were able to lay fertile eggs proves that mating occurs before feeding.

Other Observations

On November 21, 2002 females did not attack the first author on fairway 2 in Yanchep N.P. before 7:50 am. From 6:15 until 6: 45 am females were sampled by dropping a net over one or more fresh (wet) kangaroo feces and then disturbing the feces with a stick slipped under the margin of the net that was appressed against the grass. Some females were sampled by kicking at the feces and then immediately sweeping briskly over the area. In virtually every patch, some females were collected, with numbers varying from 0 to 8 individuals (58 collected in 30 minutes). Three attempts at collecting females from dry kangaroo feces failed to produce any specimens. This suggests that the females were imbibing nutrients from the wet feces but further observations are required. That reared larvae obtained nutrients from a fecal infusion in the laboratory might suggest the possibility that the females were laying eggs on or near the kangaroo feces, but most of the feces were in an area with normal soils, and the clearly aquatic larvae of A. mcmillani could almost certainly not survive there.

Of 280 preserved male A. mcmillani collected from swarms in Yanchep National Park on November 21, 2001, one specimen had a phoretic female mite intertwined between its legs. The mite belongs to an undescribed species of Blattisocius Keegan (Ascidae) (E. Lindquist, personal commun.). This mite genus is virtually cosmopolitan, and 11 of the 12 known species are, or are likely to be, predators (the twelfth is an ectoparasite in noctuid moth ears). No obvious internal parasites (e.g., no nematode infestation) were seen among the specimens examined.

Female adults clean their antennae with the foretibial spurs. The wings were cleaned by opening the wings very slightly and hooking one hindleg over the anterior margin of the wing and pushing it alongside and under the abdomen where both hindlegs rubbed their tarsi against each corresponding wing surface (so that the left leg rubbed the dorsal surface of the left wing and the right leg rubbed its underside). After wing cleaning, the wings were sometimes left at an angle but shortly after “clicked” back to the typical overlapping position over the abdomen. The hindleg tarsi were cleaned by rubbing them against each other under the body.

Taxonomic Discussion

The adult male and female of A. mcmillani were described in some detail by Borkent et al. (1987). There is some confusion about the exact location of the type locality, recorded on the labels of the type series as “National Park, Perth, W.A., 21-XII-1954”. There is not, and has never been, a national park in Perth, although the renowned 4-km² Kings Park may have been a candidate in the mind of the collector. More likely, the labels may refer to John Forrest National Park, the first national park in Western Australia, located about 22 km northeast of Perth city center. An old “Caltex” roadmap in the possession of the first author, probably from the 1950s or 1960s, indicates John Forrest N.P. merely as “National Park”, perhaps confirming that this is actually the type locality. The original collector, B. McMillan, is deceased (A.L. Dyce, personal commun.). The holotype is housed in the ANIC.

Wirth and Lee (1958) recorded the following original material, all females: holotype and 16 paratypes from “Perth National Park”, 9 paratypes from Yanchep National Park, 11 non-paratypes from 10 miles SE of Darkan and two nonparatypes from Armadale. We were unable to locate 5 paratypes from “Perth National Park”, 1 paratype from Yanchep National Park, and 10 of the specimens from 10 miles SE of Darkan, and these appear to be lost (A.L. Dyce, personal commun.). The specimens we examined from Armadale and 10 miles SE of Darkan are labeled as paratypes, although they were excluded from the type series in the original publication. Furthermore, a specimen from “Perth National Park” (ANIC) was not labeled as a paratype although it almost certainly is one. The slide label is written in W.W. Wirth's handwriting and the specimen is mounted in his typical fashion; we have added a paratype label to indicate this.

The female adults from Yanchep N.P. were larger than most of the specimens from farther south. The following are the ranges and means of wing lengths for the limited specimens available: Yanchep N.P., 0.82–0.93 mm, 0.89 (N = 16); “Perth N.P.”, 0.80–0.83 mm, 0.81 (N = 4); Armadale, 0.71–0.79 (N = 2); Darkan, 0.82 (N = 1). This may indicate the possibility of clinal variation, but further specimens and study are required to determine this.

The pupal dorsal setae are here labeled as i, ii, and iv. Based on their position on the thorax, setae i and iv are likely homologous to those named as such in other Ceratopogoninae. However, seta ii could equally be homologous to seta iii of the Ceratopogoninae.

Specimens Examined

Yanchep National Park, Nov. 18–21, 2001: 442 males, 201 females; Yanchep National Park from females captured Nov. 20, 2001: 10 eggshells, 2 eggs, 1 eggshell with first-instar stuck during emergence, 10 first-instar larvae, 2 second-instar larvae, 8 third-instar larvae, 3 fourth-instar larvae, 1 pupal exuviae and associated male (CNCI); Yanchep National Park, golf course, Dec. 11, 2001– Jan. 31, 2002: 32 females; Yanchep National Park, Oct. 22–Nov. 1, 1985: 3 males (ANIC, CNCI), 5 females (ANIC, CNCI, WAMP); Yanchep National Park, Dec. 23, 1954: 4 female paratypes (BMNH, BPBM, USNM, WAMP); Yanchep National Park, golf course, Nov. 14, 2002–Jan. 1, 2003: 55 males, 1833 females; Armadale, Jan. 4, 1936: 2 females (labeled as paratypes) (ANIC, USNM); Darkan, Jan. 29, 1953: 1 female (labeled as paratype) (USNM); Perth National Park, Dec. 21, 1954: 3 female paratypes, 1 female not labeled but likely a paratype (new paratype label added) (3, USNM; 1, ANIC).

In addition to the above material, more eggs, eggshells, and specimens of the different larval instars were studied but were either left to develop or were subsequently lost. Therefore, the numbers in tables 3 and 4 recording numbers of specimens does not match that listed above.

Derivation of Specific Epithet

The species was named by Wirth and Lee (1958) after the collector of the type series, B. McMillan.

Distribution and Bionomics

Austroconops annettae is known from two sites in southwestern Australia (fig. 22B). The type locality (fig. 22D) is on Loop Road, 5 km SSW of Forest Grove, WA. One male and four females (one lost after being collected) were swept from a very small patch of low vegetation immediately beside a very shallow (less than 3 cm deep) small pool less than a meter in diameter (fig. 21C). The pool was immediately beside and south of the narrow track of Loop Road and was located in a dry, shallow streambed (likely with running water during the winter). Surrounding vegetation was composed of an open Jarrah– Marri forest with thick stands of shrubs (Leucopogon parviflorus was common).

The single female from Augusta (fig. 22B) was collected with a sweepnet and was either collected 1.6 km south of Augusta (most likely) or about 1.6 km east of Jewel Cave (about 7.5 km NW of Augusta) (D. Colless, personal commun.).

Two females found on November 2, 2001 at the type locality were collected between 3: 00 and 4:00 pm when the ambient temperature was 20°C (but substantially warmer in the sun). When the single female retained live in a vial was at 17°C, she became very lethargic (nearly torpid), but when the vial was warmed up, she again became very active. A third female collected on November 4 was lost but was collected at 12:50 pm and at 22°C. One male was collected on November 13 at 1:15 pm at 33°C, and one female was found on the same date and temperature at 3:30 pm. Fervent and repeated sweeping all around this immediate site on each of the above dates failed to produce any further specimens, which suggests that the few adults were indeed concentrated at the small pool that appeared to be the only open water (although very small) in a large area. Samples of mud from the edge and bottom of this small pool failed to produce any immatures.

The distinctive claw of female A. annettae (fig. 1J), with a pronounced inner basal tooth more-or-less situated in the same plane as the rest of the claw, may indicate the type of host upon which the female feeds. Within the Simuliidae, females of species with a similarly shaped claw are restricted to those which feed on birds (Crosskey, 1990). Within the Ceratopogonidae, the only species of vertebrate feeders with variation regarding the presence or absence of an inner basal tooth are those in the genus Leptoconops (Forcipomyia Meigen (Lasiohelea Kieffer) and Culicoides all have simple claws with at most, a small, very slender spicule). Nearly all Leptoconops which bite mammals have a simple claw or a claw with a small basal spicule. Species with a claw with a pronounced inner basal tooth feed either on humans (e.g., L. spinosifrons (Carter) and L. siamensis Carter; Chanthawanich and Delfinado, 1967) or on birds (L. werneri Wirth and Atchley; Wirth and Atchley, 1973), indicating that the shape of the claw may not strictly indicate host type in Leptoconops. Other species which have a claw shape virtually identical to A. annettae are L. freeborni Wirth, L. melanderi Wirth and Atchley and L. patagoniensis Ronderos, but their hosts are unknown. Further research is warranted because we do not have host records for many species of Leptoconops. Lane (1977) has shown that the claw shape of females of species of Forcipomyia (Trichohelea Goetghebuer) are adaptations to cling to the scales of butterfly wings as females feed on butterfly blood (they pierce the wing veins to obtain this). If further study shows a relationship between the claw shape of females of Austroconops and Leptoconops and vertebrate host type, it will provide the basis for interpreting such variation in the fossil record: Lebanese amber species Austroconops gladius and A. gondwanicus, 121 million years old, both have large basal teeth on their claws similar to those of A. annettae (Borkent, 2000a).

The female allotype collected on November 13 laid eggs scattered separately on the surface of wet mud in a vial or on the sides of the vial just above the mud. The sequence of egg laying, hatching, and larval development is given in table 6. Larval behavior was indistinguishable from that of A. mcmillani (see above). In addition to the feeding observations associated with the fecal infusion described under A. mcmillani, one first-instar larva A. annettae was observed to eat a small live nematode whole (in less than 2 seconds). A second larva, upon encountering a somewhat moribund large nematode, pierced it at midlength. This was followed by rapid movement of the pharyngeal complex and ingestion of part of the nematode; it did not complete feeding on the nematode. In spite of these observations, the first- to fourth-instar larvae mostly ignored the large number of nematodes added to the Petri dishes every 2–3 days and, similar to larvae of A. mcmillani, congregated near fresh drops of the fecal infusion, suggesting that they too primarily feed on microorganisms.

Taxonomic Discussion

Austroconops annettae is very similar to A. mcmillani in all its stages (pupa not known) and we were unable to distinguish differences in male or female genitalia, eggs, or the different larval instars (except for some size differences in the immatures, which may have been due to restricted sample size or laboratory rearing conditions; tables 3, 4).

The first-instar larvae are described as having nine undivided segments (fig. 2C). However, in A. mcmillani, early first-instar larvae also appear to have undivided segments, whereas older first-instar larvae have divided segments. The same is likely true for A. annettae.

The female from Augusta was included as a specimen of A. mcmillani in the analysis by Borkent et al. (1987).

Type Material

Holotype, male adult on microscope slide, labeled “HOLOTYPE Austroconops annettae Borkent, 5 km SSW of Forest Grove, Loop Road, WA, Australia, 13-XI-2001, A. Borkent, CD1998” (WAMP); allotype, female adult on microscope slide, labeled as for holotype and “Female which laid eggs, resulting in 1–4 instar larvae” (WAMP); paratypes 3 females, 7 eggshells, 8 first-instar larvae, 6 second-instar larvae, 9 third-instar larvae, 1 fourth-instar larva, 1 terminal portion of abdomen of second- or third-instar larva, all reared from eggs laid by female allotype; 2 females from type locality but collected 2-XI-2001 (CNCI); 1 female from Augusta, WA, 3-X-1970 (ANIC); immatures (CNCI).

In addition to the above material, more eggs, eggshells, and specimens of the different larval instars were studied but were either left to develop further or were subsequently lost. Therefore, the numbers in tables 3 and 4 that record specimens do not match that listed above.

Derivation of Specific Epithet

This species is named after the first author's wife, Annette Borkent. She shared in virtually every aspect of the six-week expedition to study Austroconops, and the results of this paper would not have been possible without her continuous support.

Discussion

This tentative analysis (fig. 24A) indicates that there is a close correspondence between the age of species of Austroconops and the pattern of cladogenesis within the genus. The oldest species in Lebanese amber are also the earliest lineages within the genus. The two extant species are sister species; they are also morphologically very similar, with the male and female genitalia and many other features (including all larval features) being presently indistinguishable. The congruence between fossil lineages and patterns of cladogenesis is similar to that present within the entire family (Borkent, 2000a).

The Jordanian Cretaceous amber fossil Jordanoconops weitschati (Szadziewski, 2000) may actually be a member of Austroconops, as it shares with Austroconops the distinctive wing modification described in character 1 above. The sole feature distinguishing it from Austroconops is the loss of one of the radial cells, and as an autapomorphy, this loss may have occurred as a sister group of Austroconops, but equally well may have occurred within the genus. Jordanoconops weitschati belongs between synapomorphy 1 and 3 on the cladogram (characters 2, 4, and 5 are unknown for this fossil). Recognizing Jordanoconops as a valid genus leaves Austroconops without any defining synapomorphy grouping all extant and extinct species. For the time being, we are considering it as the sister group of Austroconops although there is no logical reason to do so.

Discussion

When Wirth and Lee (1958) first described Austroconops and puzzled over its classification, they stated that “Final disposition of the genus Austroconops would best await the discovery of the male or, perhaps better still, the immature stages.” Our discovery and interpretation of the eggs, larvae, and pupae amply confirm their prediction! Among extant taxa, we demonstrate here that Austroconops and Leptoconops are sister groups. Of these two genera, Leptoconops is overall a highly derived group, while Austroconops is generally plesiomorphic.

In the first cladistic analysis of the Ceratopogonidae, there were two character states proposed by Borkent et al. (1987) to unite Austroconops with all Ceratopogonidae other than Leptoconops. We now consider the first character state, the presence of a medial adult head capsule seta in Austroconops and all Ceratopogonidae other than Leptoconops, to have been secondarily lost in Leptoconops (see character 6 above). The second character state is problematic. Borkent et al. (1987) pointed out that the postoccipital ridge was relatively poorly sclerotized and that the posterior portion of the tentorium was directed in a more posterodorsal angle in adult Leptoconops and that this was also the condition present in Chironomidae (hence plesiomorphic). Austroconops and remaining Ceratopogonidae have a more sclerotized postoccipital ridge and a horizontal tentorium and this was interpreted as evidence of the sister group relationship between these two lineages. This character state is in conflict with the conclusions here and requires further study, especially of the state in further Chironomidae and other Culicomorpha.

Our new analysis also results in significant changes in the interpretation of fossil genera, recognizing for the first time the following as a monophyletic group: the extant Austroconops and Leptoconops and the fossil genera Minyohelea, Jordanoconops, Archiaustroconops, and Fossileptoconops (fig. 25A). Of these, Leptoconops and Minyohelea are now considered as sister groups. There is one previously published character state which suggested different phylogenetic relationships. Borkent (2000a) and Szadziewski (1996) considered a hindleg tarsal ratio/foreleg tarsal ratio of ≥1.40 as derived and evidence for the monophyly of the Austroconopinae, at that time including Austroconops, Archiaustroconops, and Minyohelea. Considered to be a nearly unique feature within the family, Borkent (2000a) noted there was some homoplasy within Forcipomyia. Since then, further instances of homoplasy with values as high as 1.8 have become evident within some extant and some fossil Leptoconops (Szadziewski, in press). We now therefore no longer recognize any synapomorphies for Austroconopinae.

Other relationships shown in figure 24B are those that have been hypothesized earlier (Borkent, 1995), but we have added substantial support through the addition of further characters, especially from the immatures. In particular, the monophyly of Forcipomyiinae and Dasyhelea is confirmed. This is especially important because two characters given by Borkent (1995) have since been shown to be suspect (Borkent, 2000a: 403). In addition, the Ceratopogoninae have further support as a monophyletic group. A recent molecular study of the mitochondrial cytochrome oxidase subunit 2 of a variety of Ceratopogonidae by Beckenbach and Borkent (2003) supported all the relationships shown in figure 24B.

In our analysis here we have excluded the monotypic genus Washingtonhelea Wirth and Grogan, which may form the sister group either of the Forcipomyiinae + Dasyhelea, of the Ceratopogoninae, of Forcipomyiinae + Dasyhelea + Ceratopogoninae, or potentially of a subgroup of the Ceratopogoninae (Borkent, 1995). Female Washingtonhelea frommeri Wirth and Grogan have broadly serrate mandibles, a character state restricted to a subgroup within the Ceratopogoninae, suggesting that the genus may be a member of the Ceratopogoninae. It will be fascinating to examine the features of the immatures of this genus once they are discovered.

It is striking that we have been unable to identify a synapomorphy for Forcipomyia. We suspect that the genus will be found to be paraphyletic in relation to Atrichopogon. There is a wealth of character states in both of these genera, and a cladistic analysis is long overdue. Such an analysis would further test or refine some character states used in our analysis here (characters 2, 26, 39, 45– 48).

Because of their phylogenetic conclusions, Borkent et al. (1987) proposed that Austroconops be placed in the new subfamily Austroconopinae. The discovery here that the extant Leptoconops and Austroconops and the extinct Minyohelea, Jordanoconops, Archiaustroconops, and Fossileptoconops form a monophyletic group negates the necessity of recognizing a separate subfamily Austroconopinae, and we here place all these genera in the subfamily Leptoconopinae (new synonym).

There are several features of ceratopogonids discussed in earlier publications which require some comment. Borkent (1995, 2000a) argued that the presence of an anterior larval proleg must be plesiomorphic within the Ceratopogonidae because it is present in all other Chironomoidea and first-instar Corethrellidae (Borkent and McKeever, 1990). As predicted by Borkent (1995: 103– 104), Austroconops larvae have an anterior proleg. Therefore, within the Ceratopogonidae the anterior proleg has been independently lost by Leptoconops, by Dasyhelea and by the Ceratopogoninae (first-instar larvae of Culicoides species have the feature but it is lost in the second-instar; Jobling, 1953).

Borkent (2000a: char. 17) indicated that the row of palisade setae on the first tarsomere of the hindleg was a synapomorphy of all Ceratopogonidae other than those known as Lower Cretaceous fossils. That was in error and the feature, as discussed here, is restricted to those Ceratopogoninae which form the sister group of the Culicoidini.

The male adults of A. mcmillani and A. annettae have permanently erect antennal plumes. All other male Ceratopogonidae have erect plumes only when they are in a sexually aroused state during swarming periods. Indeed, this is the basis for the name of the family: Ceratopogon Meigen means “bearded horn”, referring to the decumbent male antennal plume. In other Culicomorpha, most families with males possessing a plumose antenna (Chironomidae, Corethrellidae, Chaoboridae) have the plumes permanently erect. Male Anopheles Meigen also have decumbent plumes but other mosquitoes have permanently erect plumes. These outgroup comparisons suggest that the decumbent state has either evolved twice in Ceratopogonidae (in Leptoconops and most Ceratopogonidae) or the plesiomorphic condition has been reacquired by Austroconops. Nijhout and Sheffield (1979) described the mechanism by which male Anopheles erect their plume, and it would be a test of homology to see if the same is present in Ceratopogonidae.

In our analysis we reported only 12 synapomorphies for the genus Leptoconops. In reality, it would be possible to report many more by interpreting each detail of the larval and pupal chaetotaxy and more details of the larval mouthparts (maxillae, buccal cavity, etc.) and other modifications. It is clear that Leptoconops is highly derived in many of its features.

Our proposal that Leptoconops and Minyohelea form a monophyletic group suggests that the extinct Minyohelea had elongate eggs (character 1), larvae with secondarily divided abdominal segments (character 11), and hemolymph with hemoglobin (character 12), as well as those features shared by all Ceratopogonidae (characters 2–4).