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Initial Observations on the Pollination of Corybas(Orchidaceae) by Fungus-gnats (Diptera: Sciaroidea)

Rudie H. Kuiter and Mitchell J. Findlater-Smith

Aquatic Photographics

Short Paper 5October, 2017AQUATIC

PHOTOGRAPHICS

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Initial Observations on the Pollination of Corybas(Orchidaceae) by Fungus-gnats (Diptera: Sciaroidea)

Rudie H. Kuiter1* and Mitchell J. Findlater-Smith2

1 Aquatic Photographics, Seaford, Victoria. 2 Woolenook Native Plant Nursery, Maffra, Victoria

*Correspondence: [email protected]

Abstract

Pollinating agents of 5 Victorian Corybas species were studied by us in detail and it was discoveredthat they attracted fungus-gnat taxa of the Mycetophilidae family with a food-rewarding scent, werestrictly species-specific and sexually discriminative in vector selection. A preliminary insight to earlystages of the fungus-gnat taxa is also included. All information provided is based exclusively on ourrigorous observations.

1 Aquatic Photographics, Seaford – Short Paper 5, October 2017

IntroductionCorybas species, also known as helmet-orchids,usually form clonal colonies (Fig 1), often hiddenamong grasses, mosses or leaf litter. Their flowersare small, inconspicuous, and sit low on a green,rounded to heart-shaped procumbent leaf (Fig. 3).Their ground-hugging habit made observations ofthe attracted fungus-gnats very difficult, and withthe column completely obscured by the dorsal sepalit is impossible to see the pollination process insidethe flower, whilst opportunities to visit sites werelimited by the seasonally wet weather conditions.

Victoria’s terrestrial orchids that flower during thecolder seasons rely on diptera members as theirpollinating agents, and each of the Corybas taxaattracted a specific Mycetophilidae fungus-gnat.Due to their evasive nature, you had to be readyand in position near some flowers to photographthem, either on your knees or by laying prostrateon the ground, but they were more approachablewhen preoccupied during courtship or copulation.

The general behaviour may differ between generaof the vector species, but the pollinating procedurewas fundamentally the same for all. They wereattracted by an airborne scent and were observedhoming in on flowers, often returning to the sameone several times. Certain flowers in large colonieswere most popular over several days and both sexeswere observed feeding on the boss, which suggestsa food-related attraction. Virtually nothing wasknown about the Corybas pollinators and primaryliterature to date only offered hypotheses. Based onour findings, the persisting statement in literaturethat ‘Corybas species attract fungus-gnats asputative brood-sites’ is incorrect for the taxa inVictoria. No evidence of ovipositing in flowers wasfound. Females feeding looked gravid and werepresumed to be unfertilised. All individuals lookedfresh with undamaged wings and it was apparentthey had recently hatched.

Fig. 2 Corybas aconitiflorus. Typical habitat in eucalyptforest, often amongst similar sized gum-nuts (~14 mm).

Fig. 1 Corybas fimbriatus. A clonal colony and flowersthat look more like woody fruit fallen out of the trees,randomly scattered over the ground.

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Aquatic Photographics, Seaford – Short Paper 5, October 2017 2

Fig. 3 Corybas diemenicus. The flowers are cryptic, butthe leaves are bright green and show vivid pink under UVlight, which may function as a visual cue in low-light.

Flower Mimicry?It is commonly stated that Corybas flowers mimic orresemble fruiting fungi or toadstools, but in Victoriatheir cryptic appearance relates to habitat structuressuch as voids in the leaf litter (Fig. 2), and not anyBasidiomycetes. When searching for Corybas species,the leaves are usually noticed first. The obviousbright green leaves, that look vivid pink under UVlight (Fig. 3), may function as a visual cue and playa role with some of the attracted fungus-gnats thatfly in low light. However, it seems more likely thiscolour is the most efficient for photosynthesis asmany Corybas taxa grow under shrubs in darkshaded habitats, or are partially covered by leaf litter.Flowers of most taxa are generally dark in colour,but the more open types may show a reddish orwhite boss in relation to sun-exposure and seems tofunction as a form of temperature regulation.

Pollinating agentsThe Corybas taxa observed were found to attractfungus-gnats of the Mycetophilidae family, and onlyof the genus Mycetophila (Fig. 4) or Phthinia.Most of their members were seen flying in low-light,in partly to heavily shaded habitats or late in the day.Corybas taxa were found to be species-specific, butunlike sexually deceptive orchids, both sexes areinvolved. The scent is presumed to be food-related,and it was found that a reward is present. Only onegender performs as the vector because of sexualdimorphism, being different in size or shape. Withthe various Corybas species, flowers have evolvedto favour the smallest gender as their pollinatingagent, which in the Mycetophila genus were malesand in the Phthinia genus were females. Femaleswere found to collect the pollinarium from Corybasflowers with a more accessible column, whilst themales were able to collect pollen from flowers inwhich inverted labellum flanges formed a morerestrictive tubular passage to the column’s food-reward, possibly excluding the females.

During suitable weather conditions, when the pollenand stigma are ripe, the flowers produce a scentedsecretion to attract pollinating agents. A thin layerforms on the boss, which glitters when illuminated,and this leads to the main food reward, the moreconcentrated viscous liquid on the basal mound ofthe column (Fig. 5). Feeding was observed by bothsexes in the flowers of Corybas fimbriatus, inwhich the flowers lean back, are more open and thelabellum boss is clearly visible. The chemical makeup of the secretions is unknown, and as each of theCorybas taxa only attract a single fungus-gnatspecies, they are presumed to relate to unique andspecific fungal compounds.

Fig. 4 A male Mycetophila sp 9, which was recognised asthe pollinating agent of Corybas incurvus from the waythe pollinarium is attached and its yellow colour. Onlymales of this species were observed as vectors.

Fig. 5 Column structure of Corybas incurvus, withobscuring flower parts removed, showing the reward onbasal mound of column, presented as a viscous liquid. Oncool and cloudy days, when fungus-gnats were probablynot active, the mound looked dry, but oozed liquid whengently punctured with a pin.

puncturednatural

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Fig. 8 Copulating Phthinia sp 4. In this genus the malesare considerably larger and have longer antennae.

Food-rewarding winter-orchidsThe Corybas genus is closely allied to a small groupthat also bloom during the coldest part of the year,which includes Acianthus and Cyrtostylis. WithCorybas flowers the column is completely obscuredby the dorsal-sepal, but with the others it is fullyexposed. Vectors could not be observed once insidethe Corybas flowers, but we can speculate on thepollination procedure with confidence from thefeeding observations made on their close relativesand the structures of the columns. When ready forpollination, flowers of Acianthus and Cyrtostylishave fundamentally the same strategy of enticing apotential vector feeding on the labellum towards thecolumn along a trail formed by the secretions (Fig.6). In Corybas flowers secretions form on the boss,but the actual reward is present as a thick liquid onthe mound below the stigma (Fig. 5). Examinationof a column mound in Corybas aconitiflorus wasreported to secrete nectar (Coleman, 1931), butthe secretions are not nectariferous and probablyreplicate fungus like compounds.

Sexual dimorphism in fungus-gnatsDifferences between a male and female fungus-gnatof a taxon are not always obvious, but on the otherhand can vary to such an extent that they appear torepresent two distinct species. With such gender-formsthey were usually linked by observation or whenfound in copulation. Sexual dimorphism is oftendramatic in Sciaridae, and that the different formsattracted to Acianthus pusillus (Fig. 7) belonged tothe same taxon was determined from observations,as males were commonly in pursuit of females.

In Acianthus and Cyrtostylis the distance betweenthe labellum and anther (feeding platform andpollinia) evolved to favour the larger stocky femalein order collect the pollinia (Fig. 7). Both sexeswere seen feeding on the flowers at the sametime, but the males have a low thorax profile anddo not contact the anther. With Acianthus pusillusthe primary pollinator was of the family Sciaridae,and about 10 females were observed with polliniaattached on the thorax. The Cyrtostylis reniformisvector was of the family Mycetophilidae, genusSciophila, but only one female was observed withpollinia on the thorax.

In Mycetophilidae genera associated with Corybasspecies the gender differences were primarily in thebody sizes. In Mycetophila the males were smallestand in Phthinia it was the females. In Phthinia theantennae lengths differed considerably (Fig. 8), withfemales measuring 3.5 times the eye-height and inmales 5 times, whilst the eye was slightly larger inmales. Antennae in the Mycetophila males werenot noticeably longer than those of females.Morphological differences in abdominal shape orantennae length were usually too difficult to use fordetermining the gender from images.

Fig. 7 The mosquito-orchid Acianthus pusillus with amember of the Sciaridae family. Possibly of the Trichosiagenus. Left A heavily loaded female vector. Approximately10 individuals were observed with pollinia over the seasons.Sciaridae females feeding always look heavily gravid prior tomating and do not return after laying their eggs. They livedonly a few days in captivity. Right A male of the samespecies feeding on the labellum, many were seen, but nonewith pollinia attached.

Fig. 6 A male Sciophila fungus-gnat feeding on labellum ofCyrtostylis reniformis. It gradually moves forward, whilefeeding on the secretions along the centre of the labellum.A female has a higher thorax and when coming to the basalmounds it may rise and contact the anther. In the enclosedCorybas flowers, a similar feeding trail may lead a potentialvector to the column and collect the pollinarium. The flowerconstruction of the Corybas taxa are a very precise design,accommodating only one gender of a single species, malesin some and females in others.



Fig. 9 Corybas incurvus. Setting fruit, a few weeks afterpollination, and a highly elevated spent seed-capsule somemonths later in the season.

Fig. 11 Corybas despectans. Column details. Left fresh.Right Pollinia breaking up after about 2 weeks of flower-ing, falling onto the stigma and causing self-pollination.

Corybas pollinationAs part of our research on Victoria’s terrestrial orchidpollinators, Corybas species were checked regularlyfor vectors or evidence of their activities. Fruit-settingwas rarely noticed in the majority of locations andonly found in small numbers when looked for in theCorybas taxa where vectors were observed, butswollen ovaries are not obvious until the capsules areelevated by stem elongation prior to seed dispersal(Fig. 9), almost two months after pollination. Onlyin C. despectans (Fig. 10) seed pods were seen inlarge numbers, but through autogamy (Fig. 11).

Of the 8 Victorian Corybas species, 5 were observedwith their pollinating agent, and each taxon attractedonly a single Mycetophilidae species (Kuiter, 2016).Rigorous observations were made on these 5 taxaover multiple seasons that confirmed them to bespecies-specific, as well as being sexual discriminateof their vector:

C. aconitiflorus – vector Phthinia sp 3, female.C. diemenicus – vector Phthinia sp 4, female.C. fimbriatus – vector Mycetophila sp 8, male.C. incurvus – vector Mycetophila sp 9, male.C. unguiculatus – vector Phthinia sp 2, female (?)

Many populations of Corybas diemenicus wereregularily checked for pollinators in localities nearMelbourne and Wonthaggi over many seasons, butfungus-gnats were not observed on the flowers until2017. A site was found in Mullungdung, Gippsland,where fungus-gnats were observed interacting withflowers of C. diemenicus for the first time. Afterlearning about their behavioural routine, especiallybeing active in the afternoon, further observationswere conducted in the Melbourne region.

A particularily good site was found where numerousindividuals could be observed interacting with theflowers and many were seen with a pollinariumattached. Seemingly unperturbed by our presence,their behaviour and the pollinating procedure couldbe studied in greater detail.

Corybas unguiculatus and their fungus-gnat vectorswere only observed over two seasons. Many wereseen flying out of the flowers with pollinia attached,but only one was photographed, which had landed inthe grass close by. It was identified as Phthinia sp 2,and it was a female, but the sex of the vector needsto be confirmed for this species.

Due to location remoteness Corybas hispidus hasnot yet been observed in detail, whilst C. fordhamiican only be accessed after fire removes obstructionsin their swamp habitats. These taxa are expected toattract very closely related fungus-gnats as thosedocumented here.

Fig. 10 Fruit-setting in Corybas despectans. After about 8weeks from fertilisation when seeds have nearly ripened, itsnormally short stem quickly elevates them well up fromground level, for more efficient dispersal.



Fig. 12 Corybas diemenicus with dorsal sepal lifted. Theboss narrows towards the back, curving into a restrictivetubular structure leading to the column. In some species ithas short but dense downward pointing hair-like structures.

Pollination Process in CorybasThe completely hidden column in Corybas flowersmakes it impossible to view the pollination process,which has led to much speculation and hypotheses bydifferent authors. To get an good understanding ofthe procedure, it took countless hours of observingthe interactions of the pollinating fungus-gnat species,as well as studying the structures of the variousCorybas flowers.

The flower construction shown here is for Corybasdiemenicus, but fundamentally is the same for theother members of the genus. Fungus-gnats usuallyfeed on secretions on the boss for some time, andpotential vectors move in further, directed towardthe column. Fluid on the basal mound (Fig. 14-D)may emit a stronger scent and is thought to providethe main reward. Unable to exit through the narrowopenings at the base, it moves back to leave toflower. In retreating, the thorax collects the stickyviscid disc just above the stigma and the pollinariumbecomes attached on top of the thorax, and in C.diemenicus just posteriorly to the highest point. Toexit the flower it manoeuvres itself upwards, usingthe spiky legs to push its head up to turn aroundand climb up to the dorsal sepal, using the edges ofthe labellum’s inverted flanges. The column in C.incurvus is angled back and there is enough spaceabove the anther for a vector to turn around andclimb out, and the pollinarium is attached moreanteriorly on top of the thorax. Both sexes enteredthe flowers of C. incurvus, but only the males wereseen with a pollinarium. The restrictive passage ofthe tubular tunnel seems to prevent the largerstocky females from reaching the column.

Corybas aconitiflorus, C. diemenicus and C.unguiculatus attracted Phthinia taxa and femalevectors. C. fimbriatus and C. incurvus attractedMycetophila taxa and vectors were male.

Fig. 14 Corybas diemenicus, with dorsal sepal and parts of the labellum cut away. A Labellum forming a tubular tunnelbehind the boss. B Side view of labellum, showing inverted flanges and bottom flanges, under which small openings intothe flower to allow for air movement. C Back view with dorsal sepal removed also showing openings at the bottom. D Sideview of column, showing the proportionally large mound below stigma, glittering from secretions.

Fig. 13 Corybas diemenicus, dorsal sepal removed.Labellum from the back, tubular part evenly narrowing.

A B C D



Fig. 16 Female Phthinia sp 3 on Corybas aconitiflorus.Upper: licking on hood which may have some secretions.Lower: The probing with the abdominal tip may suggestsovipositing, but this behaviour seems to be only a habit orfor positioning itself.

Fig. 15 A female Phthinia sp 3, emerged from Corybasaconitiflorus with pollinarium. It was positioned over thebase of a wing, which made flying difficult.

Corybas aconitiflorusObservations on this Corybas taxon were conductedregularly over several seasons at a few locations inGippsland. The main site comprised colonies withgood numbers of fresh flowers scattered over alarge area and was visited over consecutive days,with sessions from noon for up to 5 hours. Theweather conditions were mild for the time of theyear (June) when vectors were observed and manyother diptera insects were active, including aMycomya sp., which was photographed amongstflowering Corybas on the ground. It showed thewing-venation perfectly, and in the assumption of itbeing the vector species, led to a misidentificationat first (Kuiter, 2016).

Corybas aconitiflorus attracted Phthinia sp 3 andmany individuals were observed, males seeminglyoutnumbered females, but only females were seenwith a pollinarium attached (Fig. 15). A vectorfeeding on the basal mound of the column maycontact the viscid part of the stigma with the thoraxand collect the pollinia as it retreats. To leave theflower it does a upward rotation to turn around andclimbs out, coming out head first. Both sexes wereobserved going into the flowers, but no malesemerged with pollinia and were presumedly toolarge or the wrong shape to reach the column. Atleast 6 females were observed leaving the flowerswith pollinia dorsally attached on the thorax, flyingout without stopping. On a few occasions theylanded in grasses a few metres away and wereattended to within seconds by males, attracted tothe pheromones being released. They would eitherfly away whilst in copulation or disappear in the leaflitter before pictures could be taken.

The legs of fungus-gnats are not designed to walkon and in flight they function as feelers instead ofthe antennae. When positioned upright they usethe spurs on the joints to crawl or stand on, andwhen upside down use the hooked tips to hang on.Abdominal probing by fungus-gnats on Corybasflowers was regularly observed, which could beinterpreted as egg-laying, but it appears to be animpulsive habit and used in positioning itself. Theywere also probing with their abdomen as theywalked on the leaves and on adjacent soil, and thisbehaviour was seen for both sexes. It was apparentthat feeding females had recently hatched, probablyearlier that day, and had not yet mated. They wereobserved on a number of flowers for a considerabletime on each and when finished, feeding matingtook place soon after. Eggs are presumed to be laidonly in suitable habitats for larvae of Phthiniamembers, such as moist rotting logs on the ground,rich with associated saprophytic fungi.



Corybas diemenicusVectors of this Corybas species were found to bethe most illusive of all the helmet-orchids that weremonitored. Numerous colonies were checked overmany seasons in regions of southeastern Victoriaand no pollinating agents were found until 2017.The first fungus-gnats observed interacting withflowers was at a newly discovered site in Gippsland,where large colonies with numerous fresh flowerswere found in coastal eucalypt forest in the shadeof dense shrubs. They were growing in seasonallymoist sandy soil, which was predominately coveredwith mosses and leaf litter.

Once familiar with the Corybas fungus-gnat routineregular visits were made to the same site. A femalewith pollinarium attached was seen feeding on theboss of several flowers, spending some minutes oneach one. The females were seen probing withtheir abdomen (Fig. 18), but this is a habit sharedby many other fungus-gnat taxa. Males appeared tobe patrolling a flowering colony, waiting for femalesto become sexually active and then approach themwith a courtship routine. As found with the otherCorybas vectors, the fungus-gnats copulated afterfeeding, and several pairs were observed matinglater in the day. The males of this Phthinia specieswere considerably larger, but this form of sexualdimorphism appears to be typical for this genus.With restricted access to the column, it seems thatonly the smaller females can reach the column orhave the right morphology to collect a pollinarium.The female’s thorax appears to be more elevatedand slightly shorter (Fig 17-inset) than in the largermale. Males can also be identified by the muchlonger antennae Fig. 22).

Fig. 18 Phthinia sp 4, vector on Corybas diemenicus.

Fig. 17 Corybas diemenicus and its pollinating agent, a female Phthinia sp 4, with a pollinarium collected from anotherflower. Body length is about 3–4 mm. Inset Female. showing the high profile of the thorax.



Fig. 20 This female Phthinia sp 4 was observed exuding adrop of liquid from the mouth and then sucked it back in. Itis not known where it was feeding.

Fig. 21 Phthinia sp 4. Copulating, female has pollinarium.

Fig. 22 Phthinia sp 4. This images shows clearly that themale is much larger than the female. Several more pairswere seen and apparently this is normal in this genus.

Fig. 19 Phthinia sp 4, dorsal view, showing wing-venationand colouration of wings, thorax and abdomen, similar tosp 3, vector of Corybas aconitiflorus.

The fungus-gnat taxon was identified as a member ofthe genus Phthinia using the wing-venation and isreferred to as sp 4. The larvae of this genus werefound to be living in decaying logs on the ground inmoist habitats. They feed on saprophytic fungi, andtwo different species of Phthinia were hatched fromlarvae on some pieces of bark collected in the field.

DiscussionIt had taken multiple seasons to discover a sitewhere the pollinating agent of Corybas diemenicuswas present and could be studied. After discoveringthe conditions and the time of the day when thefungus-gnats would be interacting with the flowers,our first observations on this vector species weremade over many days during 5–6 hours sessions.To fully comprehend the fungus-gnat behaviour andpollinating procedure, it was essential to repeatedlyconduct observations over such long sessions forthe preparation of the initial documentation.

At the Mullungdung site, Corybas diemenicusflower visitation varied from day to day, but the fly-ing times of the fungus-gnats were consistent forthe attracted taxon. Their activities started about1:00 pm, usually peaking for about one hour andcontinued until late on some days. The weatherconditions ranged from unseasonally warm to dayswith frosty mornings, but the fungus-gnats weremost abundant on the warmer days. On the coolerdays only a few fungus-gnats were observed, andusually showed less interest in the flowers. Theywere possibly not hatching during the very coldperiods, and the flowers may only emit a weakscent, and probably have no reward present.



Corybas diemenicus var. KallistaPopulations growing on the trunks of tree-ferns inthe Dandenong Ranges were checked many timesover several seasons for pollinators, and finally in2017 fungus-gnats were observed, including manyvectors with pollinia attached. On the first day,about 10 individuals with pollinia were seen, somemay have been observed twice, but all were female.The taxon was identified as Phthinia sp 4, thesame species as the one found on the colonies ofCorybas diemenicus in Gippsland.

No activity was seen until after 1:00 pm. Femaleswere first noticed, as they were flying with polliniaon their thorax and this made them more obvious.They were observed feeding on the boss of manyflowers, going inside and out of sight for severalminutes in each one, totalling a period of at least30 minutes. Over 6 individuals were seen comingback out of the narrow tubular part of the labellumbehind the boss. In order to collect the pollinariuma vector has to go down the tubular section for thethorax to contact the viscid part above the stigma,obtaining the glue which collects the pollinia as itretreats to leave the flower. It turns around by anupward rotation in the more spaced section of thetubular part above the column (see page 5) andclimbs out, using the edges of the inverted labellumflanges to reach the dorsal sepal. Individuals wereobserved flying out up-side down (Fig. 26) from ahanging position in the flower.

The female vectors with a pollinarium attached wereconcentrated on in order to learn the procedure, andconsequently very few males were photographed (Fig.24), but most importantly included one in copulation. Fig. 26 Phthinia sp 4. Flying away after exiting, and still

up-side down as it has just let-go from the dorsal sepal.

Fig. 23 Corybas diemenicus and a female Phthinia sp 4,with a pollinarium from another flower, flying in to feed.

Fig. 24 Male Phthinia sp 4. Sex was determined by thelong antennae, but it also has the slender anterior abdomen.

Fig. 25 Corybas diemenicus with a female Phthinia sp 4.Probing with abdomen seems to be to position itself.



Fig. 28 Phthinia sp 4, feeding on the boss of Corybasdiemenicus for many minutes, and after feeding emittingpheromones to attract a male for copulation.

Fig. 27 Phthinia sp 4, a close-up showing the pollinarium is well secured on the upper-posterior part of the thorax. Afterabout 30 minutes feeding, she was probably emitting pheromones and waited for about 10 minutes for a male to arrive.

DiscussionThe site is situated in a deep dark gully, dominatedby Mountain Ash forest, with an understory of largetree ferns, on which due to lyrebird disturbances theorchids are relegated to grow. It was apparent thatthe fungus-gnats on the day were newly hatched,had not yet copulated and only entered the flowersto feed. They were observed licking secretions onthe boss of many flowers, reminiscent of feedinghabits witnessed on the related winter orchids.

After feeding, the females flew to a nearby spot toattract males and copulate, waiting while emittingpheromones. In one instance a male took about 10minute to arrive (Fig. 27 & 28). Ovipositing wouldfollow some time later in the day, probably in thedecaying logs on the ground that are broken downby saprophytic fungi. Adult stages of most of thefungus-gnat taxa are thought to be to be short-lived,usually lasting only a few days, probably until egglaying is completed.

Their emergence from the pupa stage appears tobe related to atmospheric conditions. On the daythey were seen flying in large numbers, it was mildand sunny after several cold and wet days. The nextday on a similar, but more windy day, none showedat all. Several more vectors were observed a weeklater on another calm sunny day after 1:30 pm.

Flower colour is typically very dark in the heavilyshaded habitats, and even on a sunny day they werevery difficult to focus on with the camera. To beable to capture images of a vector coming out ofthe tubular tunnel a torch light was needed to see itre-appear, before typically flying straight out.



Corybas fimbriatusThis taxon attracted Mycetophila sp 8 and at least10 individual vectors with a pollinarium attachedwere observed, and were identified as males. Manyfemales were observed feeding on the boss (Fig.30/31), with some entering the flower, often goingout of sight for several minutes, but none came outwith a pollinarium. Females may be too stocky toreach the column through the restrictive tubularpart of the labellum, or their thorax morphology isnot compatible for collecting a pollinarium.

Observations were conducted at sites from aboutnoon until dark and it took many sessions to find thevectors. The first fungus-gnats with a pollinarium ontheir thorax were seen flying over a colony late in theafternoon, but it seemed impossible to get images.Their flying was rapid and erratic, whilst it was gettingmuch too dark to see, and after landing they wouldquickly disappear into a flower, but a female wasphotographed when it landed on the boss in front ofthe lens. It spent some time feeding and was identifiedto a genus based on the wing venation (Fig. 33).Subsequently more identifiable images were takenat different localities, and all represented the samespecies. It seems this taxon primarily flies in lowlight conditions, but observations were made onmild, partly cloudy or sunny days. It’s likely thatthey are active on overcast and rainy days as well,and may prefer heavily shaded bush habitats.

Vectors were mostly observed on clonal colonieswith large flower numbers near decaying logs in wetenvironments. Logs in an advanced state of decayare primary breeding sites for fungus-gnat larvae,where they feed on fungal hyphae. Larvae of taxathat are pollinating agents of many terrestrialorchids were found under bark, including some ofthe Corybas vectors.

Fig. 30 Female Mycetophila sp 8 feeding on the boss ofCorybas fimbriatus and this lasted for about 5 minutes.Inset: close-up of proboscis.

Fig. 29 Male Mycetophila sp 8, with 2 lots of pollinia fromCorybas fimbriatus attached. Inset: The pollinarium isattached on top of the thorax, slightly posteriorly to centre.

Fig. 32 Male Mycetophila sp 8 moving up the labellum togo in towards the column of Corybas fimbriatus.

Fig. 31 Female Mycetophila sp 8, feeding by licking onthe boss of Corybas fimbriatus



Fig. 33 Female Mycetophila sp 8 feeding on the boss ofCorybas fimbriatus, clearly showing the wing-venation,which identifies the genus in the Mycetophilidae family.

Fig. 35 Corybas fimbriatus. Showing typical integral structure of the column, which is finely tunedto the morphology of the fungus-gnat, and works only with the male gender.1/ pollinia pair that looks fused into one. 2/ viscid part at the top of the stigma. 3/ stigma, a discwith a sticky surface. 4/ basal mound that secretes the food-reward when the flower is ready andconditions are suited for pollination.In principal the design is virtually identical in the members of Corybas that were studied, but in thisspecies the features seemed to be more defined.

Fig. 34 Male Mycetophila sp 8 emerged from the flowerof Corybas fimbriatus with pollinarium. It kept flying onthe colony for some time, probably searching for female.

1

2

3

4



Fig. 38 Mycetophila sp 9 on Corybas incurvus. Male,with pollinarium, courting a female which just arrived.

Fig. 36 Mycetophila sp 9 on of Corybas incurvus. Pair about to copulate, male with pollinarium approaching female.

Fig. 37 Pair of Mycetophila sp 9, copulating.

Corybas incurvusObservations were conducted over several seasons atmany sites in various regions in Victoria, but vectorswere observed at only a few sites. Corybas incurvusattracted Mycetophila sp 9, and at one site about 6males were seen with a pollinarium attached. At thissite, both sexes of this Mycetophilidae taxon wereobserved in good numbers, and often males wereseen following a female. Copulating pairs (Fig. 37)were photographed over different seasons, and themale was confirmed as being the vector.

In Corybas incurvus the column is angled backwardsand there is a space directly above in which a vectorcan manoeuvre up and climb out the flower. Whenthe viscid glue is obtained, the pollinarium becomesattached to the frontal curve on top of the thorax. InMycetophila the male is considerably smaller thanthe female, but otherwise sexual dimorphism is notobvious. Females are probably too large or stocky(Fig. 39) to come in contact with the stigma or onlythe males have the correct thorax morphology toobtain the pollinarium.

At 3 other sites this fungus-gnat was only seen insmall numbers, despite many hours of observation.At a Mornington Peninsula site, fungus-gnats wereonly observed on a rainy day with a male on a flowerat 3:00 pm Fig. 42), and another about 30 minuteslater. The colony was checked over 5 days and noothers were seen. None were observed on a colonychecked near Belgrave, but some weeks later someswollen ovaries indicated that pollination had takenplace. At Mullungdung no activities were seen formany days, until a male with a pollinarium was foundand with females arriving, multiple courtships andcopulations were witnessed.



Fig. 39 Mycetophila sp 9 on Corybas incurvus. Female inflower, male with pollinarium.

Fig. 43 Mycetophila sp 9. Genus is readily identified whenthe wing-venations can be seen clearly.

Fig. 42 Male Mycetophila sp 9 on Corybas incurvus.

Fig. 40 Mycetophila sp 9, courtship behaviour.

Fig. 41 Mycetophila sp 9, male with pollinarium attached.

Courtship behaviourThe male shown here with the pollinarium attachedwas observed from about 1:00 pm for 3 hours ona single colony. When a female arrived (Fig. 38) themale was witnessed performing a courtship routineon or near a flower, comprising jump-like movesand short bursts of rapid wing-beats. The femaleseemed submissive, approaching the male to a spotnearby where copulation took place. Soon after shewould return to the flower and feed on the boss.Another female arrived 21 minutes later and againthe courtship and copulation took place by the samemale. Courtship behaviour had been observed theseason before, but due to the presence of severalmales was confusing, and getting images was themain priority at the time. The males were seen alsoperforming with rapid wing flapping and bodymotions (Fig. 40). Courtship behaviour in severalgenera of the Mycetophilidae family is known fromthe northern hemisphere, but it was not observed inPhthinia taxa on the Corybas.

Note 1A presumed fungus-gnat pollinator was reported onthis species, then named as Corybas diemenicus,from Mount Morton, near Belgrave (Jones, 1970). Itwas correctly identified at the time, but examinationof the type specimens in Kew showed the mistakeand led to the subsequent naming of C. incurvus asa distinct species in 1988 (Jones, pers. comm.).The vector showing attachment of pollinarium wasfound dead on a flower, trapped by some spiderweb. The fungus-gnat was illustrated in a verydetailed drawing and looked identical to the maleMycetophila sp 9.



Corybas unguiculatusPopulations in the Melbourne regions of this taxon,commonly known as the Small Helmet-orchid orPelican-orchid, have rapidly declined over the last 2decades. One population in the Langwarrin Flora &Fauna Reserve historically produced seed capsulesin large numbers, but a fire competely destroyed itbefore studies could be done. Another population,comprising about 30 to 40 flowers, was discoveredin a different part of the reserve in 2014, and hasbeen monitored since, but fruit rarely sets.

The fungus-gnat species attracted to this Corybas wasfirst observed in 2016 at a single site in maturecoastal tea-tree habitat, where previously seed podshad been seen. On certain days they were seen flyingin good numbers, but many visits were required tomake the observations and it took countless hours toget sharp images of these very evasive, erratic andquick flying fungus-gnats. Most were seen in theafternoon between 1:00 and 5:00 pm. The taxonwas identified as a member of the Mycetophilidaegenus Phthinia. Several vectors with a pollinariumattached on the thorax were seen flying out of theflowers, but no opportunity was presented to get aphotograph. Based on the many images taken, it wasapparent that both sexes were attracted, but genderof the vector was not determined during that season.

The next season only a very few fungus-gnats wereseen at the same coastal site and it seemed that noneof the flowers were setting fruit. Due to unseasonablydry weather, flowering was poor and the habitat wasdrying out with flowers aborting at the site.

Searching a hinterland eucalypt forest resulted inlocating some excellent populations to work with, andwith flowers already setting fruit it was clearly a goodsite to continue our observations. The site was visitedon many days and fungus-gnats were regularly seen ingood numbers on the flowers, and at this locality theywere also active from early in the afternoon. Themore open grounds with better light made it easier toget images, and finally a vector with pollinariumattached was captured on camera. It was determinedto be a female and most of the individuals seen goingon the flowers appeared this gender. Females weresmaller than males, but more observations need to bemade to confirm the gender of this orchid’s vectorand also if the size disparity is a factor, but it would beconsistent with other Phthinia members.

DiscussionFungus-gnats associated with Corybas unguiculatuswere found to be difficult to locate and observe. At thesame time the fungus-gnats were flying in Gippsland,the Langwarrin site was visited on many days in theafternoon, but failed to see pollinator action. During

Fig. 45 Female Phthinia sp 2. Wing-venation shows witha light background, but needs to be free for a clearer view.

Fig. 46 A female Phthinia sp., showing wing-venation ofthe genus. Very similar taxon to sp 2, in which M-III (1) islonger, reaching to below Rs (2), and has a brown thorax(the obvious difference).

Fig. 44 Female Phthinia sp 2 with pollinarium attached tothe front part of the thorax. Probably waiting for male.

1 2



Fig. 50 Female Phthinia sp 2 about to enter the flower. The opening was measured in several fresh flowers in one of thecolonies at the site, and ranged from about 4 to 4.5 mm .

Fig. 47 Male Phthinia sp 2 has longer antennae and islarger than the female, and shown here is the typical moreslender shape in the anterior sections of the abdomen

Fig. 49 Female Phthinia sp 2 on an exceptionally largeflower. Opening about 5 mm in width.

Fig. 48 Phthinia sp 2, female, attracted to a flower ofCorybas unguiculatus.

observations over about 5 weeks at this site, only 2out of the large number of flowers were settingfruit, but it indicated that the vector was present inthe area.

Corybas unguiculatus needs to be observed overadditional seasons to learn more about the pollinationprocess, the vector activities and their occurrences.Gender of the vector needs to be confirmed and thebehaviour of the fungus-gnat taxon further studied.Based on our rigorous work done with fungus-gnatsand their association with the Pterostylis genus, thegreenhoods and rustyhoods, it seems that many ofthe diptera members have declined in numbers. Theyare threatened due to habitat alterations, especiallyfrom inappropriate fires that dry out their habitats,destroying the larval fungal food-source, as well asfrom the overuse of pesticides and other chemicals.



SummaryIt requires many decades of equally in-depth studies tocomprehend both the terrestrial orchid taxa’s floweringregimes and the irregular occurrences of their pollinatingagents. The Corybas species and their diptera fungus-gnatvectors rely on certain sensitive wet habitats, of whichstability is essential for their long term perpetuation.Regular observations were limited in this often difficulthabitat by the seasonally short days and the wet weatherconditions. Learning about the pollination proceduresrequired many hours of observations over their floweringperiods, but to be able to document the completesequence of events it took several seasons. To observethe vectors interacting with the orchid flowers, one hasto be able to predict when this is likely to occur, whichrequires a good knowledge of the insects. To understandthe symbiotic relationship of Corybas and fungus-gnatsin Victoria, it was necessary to learn about the life-cycleof the fungus-gnats, especially where larvae lived.

To record the behaviour of the pollinating agents and toobtain details of their morphology, required to identifyan attracted species to a genus level and to determinethe gender, a very large number of images were taken.Our findings on the pollinating agents on 5 out of the 8Victorian Corybas taxa, provides a good frame work tobuild on, investigate more species and to elaborate in muchmore detail on their dipteran symbiotic relationships.

Vector routineActivity of the fungus-gnats attracted to Corybas flowerswas generally confined to early and mid afternoon, sug-gesting emergence from their pupal stage earlier in theday. It was apparent that both sexes feed shortly afteremerging, and as soon as they were able to fly. They arepresumed to require nourishment prior to copulation orovipositing, especially for egg development in females. Thefeeding routine of females was usually over periods inexcess of 30 minutes by going from one flower to the next,spending a few minutes in each one. As the femalesseemed to take longer in feeding, anxious males would beready for copulation, and this was observed as soon asfemales began emitting their sex-pheromones. With thefungus-gnats attracted to Acianthus pusilla, copulationwas observed after feeding as well, and the femalesobserved on labella generally looked heavily gravid.

Eggs on Corybas flowersIn light of the hypothesis that Corybas taxa attractingfemale fungus-gnats as a brood-site, commonly statedin publications, we made a special effort to investigate ifthis was true. We found no evidence of ovipositing in theflowers by fungus-gnats, nor convincing proof in supportof the hypothesis in literature. Eggs were found on someCorybas flowers, but these proved to be from theCollembola order and not from Diptera. The jelly-beanshaped eggs from a Sciaridae member (Fig. 51) were

Fig. 51 A Sciaridae sp., female with a string of eggsthat had remained attached to the ovipositor.

Fig. 52 Springtail on Corybas hispidus. Eggs thatmay have attached to the back-end during laying.Their eggs are usually white, round to elongated, anda fraction of a millimetre in diameter.Inset Arthropoda/Collembola larvae, ~1 mm longon C. diemenicus, upper one developing legs.

Fig. 54 Larval stage, Mycomya sp. Length 25 mm.

Fig. 53 Larval stage, Phthinia sp. Length 15 mm.



measured, 0.22 mm long, but sizes and shapes wouldvary in relation to different taxa of the Mycetophilidaefamily. A few Arthropoda adults were observed on someCorybas taxa and one individual was found to lay eggsin a flower (Fig. 52). Some tiny first instar hatchlings,about 1mm long were sometimes found on the boss, andmay resemble small maggots. They are white andappeared to be immobile, but were ruled out being of thediptera order as legs developed after moulting in thenext instar (Fig 52-inset).

Looking for Mycetophilidae larvae Virtually nothing seemed to be known about larval stagesof the Mycetophilidae species in Australia, other thanthem being associated with fungi (Colless, 1970). In thenorthern hemisphere the fungus-gnat families are wellstudied, and information found about early stages ofclosely related species on http://sciaroidea/info and forthe Nearctic zones in the Manual of Nearctic Diptera(J.R. Vocxenor, 1981), revealed that most of the specieswe needed to investigate lived in rotting logs and fed onfungal mycelia.

Some Corybas taxa colonies may occur for many yearsadjacent to rotting logs, and in the shared micro-habitatstheir associated fungus-gnats would not have to fly far tothe flowers after emerging from their pupal stage. Thesearch for larvae (Fig. 53/54) was conducted within theorchid’s habitat during the flowering period, and werereadily found in decomposing logs on the ground thatcontained fungal mycelia.

Hatching of fungus-gnats Small pieces of bark, that harboured larvae,were taken and placed in an aquarium witha glass lid. The larvae that were visibleranged from about 15–25 mm in length andthought to comprise at least three differentspecies. Further specimens were collected afew weeks later and the number of speciesrapidly grew, and as the images show thesewet habitats support a great diversity ofdiptera taxa. From the orchids perspectivethis shows how important it is not to interferewith habitats, especially with inappropriatefire regimes, that causes the drying out andconsequent loss of habitats for the dipteralarval stages of pollinators, on which winterand early spring flowering orchids rely.

The first fungus-gnats, about 3 mm in bodylength, hatched after about two weeks andwere identified as members of the familyMycetophilidae, the genus Phthinia, basedon wing-venation. About 10 individualsemerged over 4 days, probably originatingfrom the smaller larvae (Fig. 53), and wereusually observed in the afternoon. Anothermember of the genus hatched a week later.

Fig. 55 Phthinia sp., the most common hatchling.

Fig. 56. Diadocidia sp., only one hatched.

Fig. 57. Keroplatus sp., two males and one female hatched.

Fig. 58 Mycomya spp. Lengths 5 mm. Several hatched.



Fig. 59 This species fits the description and wing-venation ofParamorganiella adventurosa Tonnoir, 1929 from Tasmania.

Many different taxa hatched, including a speciesof the genus Mycomya (Fig. 58), which wouldhave developed from the 25 mm larvae (Fig. 54),and members of this genus are sexually attractedpollinators of a large number of Pterostylis taxa.It was surprising in having so many species acrossseveral different genera hatching from a smallamount of bark.

An unusual female was tentatively identified asParamorganiella adventurosa (Fig. 59), as itmatched the original description by Tonnoir,1929, and also in wing-venation. This taxon hasmandibles and orange spurs on the legs, and itwas only known from a single male collected onBruny Island, Tasmania.

Distinctive larvae with a black head collected atthe Acianthus pusilla site were assumed to bemembers of the Sciaridae family, as many adultsemerged from the collection. The female shown(Fig. 60) is thought to be of the Trichosia genusand appears to be the commonly observed taxonfeeding on those Acianthus flowers (Fig. 7).

The pollinating agents of all the Corybas andalmost every Pterostylis species are fungus-gnatsof the Mycetophilidae family. All these orchidsdepend on seasonally wet undisturbed-habitats,whilst their diptera pollinator species rely ondecomposing tree branches and logs on theground for their early stages. This natural processof recycling provides nutrients back into theecosystem, converted into the food and suitablehabitats for the orchids and their pollinators.

Literature citedVocxenor, J.R., 1981. Manual of Nearctic Diptera,

section 14, Mycetophilidae. Government PublishingCentre, Quebec, Canada.

Coleman, E., 1931. The pollination of Corysanthesbicalcaratus R. Br.. Victorian Naturalist V. 48, p 95.

Colless, D.H., 1970. The Insect of Australia, chapter34. CSIRO. Melbourne University Press.

Jones, D.L., 1970. The Pollination of Corybas diemenicus(H.M.R.) Rupp and W.H. Nicholls ex H.M.R Rupp.Victorian Naturalist V. 87, pp 372–4.

Kuiter, R.H., 2016. Orchid Pollinators of Victoria 4thEd. Aquatic Photographics, Seaford, Victoria.

Scanlen, Eric, & Ian St George, 2009. Colour fieldguide to the native Orchids of New Zealand. NewZealand Native Orchid Group Inc. Wellington.

Tonnoir, A.L., 1929. Australian Mycetophilidae.Proceedings of the Linnean Society of New SouthWales, Vol. IV, Part 5.

AcknowledgementsSpecial thanks to Steven Kuiter and Bob Lindhe for their

participation in the fieldwork and logistic support. Fig. 60 Sciaridae sp. tentatively identified as a Trichosia.Length of this female 2.5 mm. Many hatched and probablycame from larvae as shown above, about 10 mm long. Fewwere found dead with eggs attached to the abdomen.


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