The guild of saprobiontic nematodes associated with ants ( Formicoidea )

At least 14 different saprobiontic species of Rhabditida are intimately associated with ants on different continents. These myrmecophilous nematodes belong in particular to Diploscapter and Sclerorhabditis, and to the Oscheius Dolichura group (all "Rhabditidae"). Species of Diplogastridae and Halicephalobus (Panagrolaimidae) are rarely found in association with ants, but some records do exist. Oscheius janeti is argued here to be a separate species. Dauerlarvae of all these species invade the postpharyngeal glands of ants via the mouth (including as a result of trophallaxis) and, after some time and under suitable conditions, leave them the opposite way to complete their life cycle. These species usually propagate in the debris of ants' nests using ants as a means of dispersal (endophoresis). Species which live in certain saprobic biochores (such as slime flux) frequented by ants enter the ants to survive the unfavourable conditions of their habitat. Mostly the infestation rate is very low. Most field-collected ants contain no nematodes, and more than ten specimens are rarely found in one ant. The infestation rate increases in formicaria in the laboratory. There are some indications that dauerlarvae obtain nutrients from the carrier/host, so they may perhaps be viewed as facultatively larval parasitic. Queens, which have the potential to found a new colony, have also been documented to be infested. New nematode records from Solenopsis, from Azteca in domatia of Tococa (Melastomataceae) and Cecropia (Urticaceae), from Crematogaster in domatia of Macaranga (Euphorbiaceae), and from the fungus-growing Acromyrmex and Atta are reported. The nematode-ant relationship and distinctive organ specificity of the dauerlarvae to the postpharyngeal glands of ants has been attained by convergent evolution no less than nine times. It is expected that living together with saprobiontic nematodes in myrmecophilous plants offers some kind of benefit to ants.


Introduction
Parasitic nematodes, most often Mermithidae (several genus-taxa), but also Tetradonematidae (Tetradonema, Myrmeconema), Allantonematidae (Formicitylenchus), Seuratidae (Rabbium), and Physalopteridae (Skrjabinoptera), have been recorded in various groups of ants (Poinar, 2012).The last two (usually) heteroxenous taxa utilise ants as intermediate hosts.It is also relatively easy to infect ants with species of the entomoparasitoids Heterorhabditis and Steinernema (Poinar, 2012).Alongside these parasitic relationships, phoretic or mutualistic relationships also exist between saprobiontic nematodes and ants.The occurrence of nematode dauerlarvae in the tubular postpharyngeal glands of ants completing their life cycles in the detritus of nests was first reported by Janet (1893Janet ( , 1894) ) for Formica rufa and Lasius flavus.The species in Formica rufa was described in Janet (1894) as Pelodera janeti De Lacaze-Duthiers, then re-described in detail by de

Research Article
Man (1894).It was synonymized by Andrássy (1952) with Leptodera dolichura Schneider, 1866 (= Oscheius dolichura), and this was confirmed by Wahab (1962) and Sudhaus (1976).Thus was it demonstrated that this well-known saprobiontic species found in an ecologically wide range of habitats and transported by a number of different beetles exhibits a surprising facultative relationship with ants.Wahab (1962) made a comprehensive study of this peculiar association in the laboratory of H.-J. Stammer and G. Osche (Erlangen, Germany).In the postpharyngeal glands of 16 out of 25 species of ants examined (species of Formica, Lasius, Myrmica, Tetramorium) he found not only O. dolichura, but three more nematode species.Ants of 198 out of a total of 550 nests (= 36%) were infested.The nematodes most commonly found were Diploscapter lycostoma Völk, 1950 (in 127 nests, = 23%) and O. dolichura (in 58 nests, = 10.5%),whereas a species of the Pristionchus Lheritieri complex which Wahab identified as P. lheritieri (Maupas, 1919) was found in just 5 nests (0.9%) and Koerneria histophora (Weingärtner, 1955) was found in just 3 (0.5%).Nine nests and several individual workers, respectively, were inhabited by two different species, yet only 5 (2.5%) of the nests infested displayed a mixed occurrence of the most frequent species D. lycostoma and O. dolichura.Although these species do not specialise in different ant species, they are separated ecologically: O. dolichura was frequent in nests found in forests and D. lycostoma in those found in the open country.The four nematode species shown by Wahab (1962) to be myrmecophilous were already known from a variety of decaying substrates such as mulm (duff), slime flux, compost, and manure, and occasionally they occurred phoretically associated with different beetles.In his study they were also shown to be saprobiontic inhabitants of detritus in the nests of ants, and to exhibit an endophoretic relationship with ants.
A further in-depth analysis of the association between nematodes and ants was conducted by Köhler (2008Köhler ( , 2012) ) in the laboratory of A. Fürst von Lieven and myself in Berlin.Köhler focused on the ant species Lasius brunneus, which is associated with slime flux on deciduous trees.In the heads of 114 of 262 workers (= 43.5%) he found a total of 520 dauerlarvae, of which 287 (= 55%) matured and could then be identified to species level.The most frequent nematode species found in this particular ecological situation was K. histophora (58.5%), followed by D. cf.lycostoma (29.3%),O. dolichura (10.8%) and two newly demonstrated associates of ants, Diplogasteroides spengelii de Man, 1912 andHalicephalobus similigaster (Andrássy, 1952) (both 0.7%, respectively).Most ants were uninfested, and 75% of 114 ants bearing nematodes only had 1-3 dauerlarvae in their postpharyngeal glands.The highest numbers of dauers found in ant specimens collected in the field were 20, 40, 58, and 85, respectively (Köhler, 2012), whereas Wahab (1962) rarely counted more than 20 and at most 43 nematodes per ant.
In supplemental studies the two most frequent myrmecophilous species were also found in ants from North America, namely D. lycostoma in Linepithema (formerly Iridomyrmex) humile in multiple places in California (Markin & McCoy, 1968), and O. dolichura in Camponotus herculeanus and Lasius (Acanthomyops) claviger in Ontario (Nickle & Ayre, 1966).In addition, the description of waving juveniles covered by an "oily secretion" suggests that a species of Diplogastridae was also present in one of these ant species, as the secretion in question is unique to diplogastrids.A phoretic diplogastrid was observed by P. E. Hanson on Azteca sp. in Costa Rica (Poinar, 2011, p. 93+325).In Oregon, Poinar (2012) found dauers of an unknown diplogastrid in the postpharyngeal glands of Formica obscuriventris.The latter diplogastrid was further identified as a member of Pristonchus (Poinar, 2016).The hermaphroditic diplogastrid Pristionchus entomophagus (Steiner, 1929) was isolated from Myrmica rubra found at several sites in Maine, and a similar species was detected "in its native range in England" (Groden & Stock, 2011, Michaud, 2013).Recently, Sclerorhabditis neotropicalis Esquivel et al., 2012 was described from nests of Azteca constructor and A. xanthochroa in Cecropia obtusifolia in Costa Rica (Esquivel et al., 2012), and Diploscapter formicidae Zhao et al., 2013 was found in nests and workers of Prolasius advenus, an ant species endemic to New Zealand (Zhao et al., 2013).Kermarrec (1975) reported dauerlarvae of an undetermined species in the postpharyngeal glands of Acromyrmex octospinosus in Guadeloupe.From the shape of the dauerlarva and the structuring of its cuticle it can be identified from the photos as Pelodera cystilarva (Völk, 1950) or a very closely related species.This record is extremely unusual, as Pelodera cystilarva typically lives in compost, and its dauerlarvae rely for dispersal on sticky secretions from glands in their anterior end which they use to attach themselves to the cuticle particularly of gamasid mites.
My own incidental observations on the association between saprobiontic nematodes and ants span a period of around 40 years (Sudhaus, 2016).They are summarised here and discussed in conjunction with data from the literature (fig.1).5) male tail with genital papillae, spiculum and gubernaculum in lateral view.The first three species are "Rhabditidae", having a cylindrical stoma, a terminal bulb with valves and a bursa in the male.The next three species are Diplogastridae, characterised by a glandular terminal bulb without valves and filiform tails.The last three species belong to the Panagrolaimidae, showing typical counter valves posterior of the valves in the terminal bulb.The figures are not in the same scale.

New records
The results of samples analysed by me are listed in tables 1-5.I found the same saprobiontic associates as mentioned above: Diploscapter sp. and D. cf.lycostoma, species of the Oscheius Dolichura group and the Pristionchus Lheritieri group, Koerneria histophora, cf.Halicephalobus sp., and Sclerorhabditis sp.In addition, Panagrolaimus sp.sometimes occurred, and in all likelihood a species of a new genus (of the Panagrolaimidae?) was discovered.With regard to the ants, I established new records of associations between species of the Oscheius Dolichura group and Atta sp., A. texana, A. vollenweideri, Formica polyctena, Messor sp., Mycetophyllax sp., and Solenopsis invicta; between Diploscapter and Azteca sp., Cladomyrma sp., Crematogaster sp., Mycetophyllax sp., and Solenopsis invicta; between Koerneria and Camponotus herculeanus and Lasius fuliginosus; between Pristionchus and Atta cephalotes, A. texana, and Solenopsis invicta, and between at least two species of Sclerorhabditis and Atta cephalotes, A. sexdens, A. vollenweideri, Cladomyrma sp., Crematogaster sp. and Solenopsis invicta (tables 1-5).Of special interest are those ants living in a symbiotic relationship with myrmecophytic plants in tropical regions.Inside the domatia (specialised chambers which house mutualistic ants) of these plants, as yet unspecified nematodes associated with Azteca have been reported on several occasions (Longino, 1991, Schmidt, 2001, Weng et al., 2007, Mora Pineda, 2013, Huertas, 2014).Diploscapter coronata was reported to live in domatia of Hirtella physophora (Chrysobalanaceae) colonised by Allomerus octoarticulatus in the vicinity of Manaus (Brazil) (Vogel, 2012).In the detritus of domatia of Tococa spp.inhabited by Azteca spp. in the American tropics, I found Diploscapter sp., D. cf.lycostoma, O. cf.dolichura and, as stated before, an unknown species of a new genus-taxon (table 1).Along with some unknown species (her pictures 21-22), Huertas (2014, fig.14) isolated a Diploscapter sp.(characterised by its distinctive tail shape) from Cecropia obtusifolia/Azteca constructor in Costa Rica.An equivalent ant-plant system in South-east Asia is constituted by the various Macaranga trees inhabited by Crematogaster spp., where thousands of nematodes were found to occur in the dumps (Maschwitz et al., 2016).I personally detected Diploscapter sp. and Sclerorhabditis sp.(table 1).Samples from Saraca thaipingensis (Fabaceae) and Crypteronia griffithii (Crypteroniaceae), both associated with ants of Cladomyrma spp., also housed piles of nematodes (Maschwitz et al., 2016).In the domatia of Myrmecodia and Hydnophytum (Rubiaceae) occupied by Iridomyrmex sp. in Papua New Guinea Huxley (1978: 248) regularly found "nematodes, Rhabditis sp., which along with mites were usually present on warted surfaces where a thin lyer of debris, probably ant faecal material, was present".Also in Singapore, "nematodes of the genus Rhabditis" were observed in Hydnophytum or Myrmecodia usually associated with Iridomyrmex (Lok & Tan 2009).Whistling thorn (Acacia drepanolobium, Fabaceae) galls inhabited by Crematogaster sp. which I collected in Kenya between Tsavo and Amboseli (22.3.1978)contained some mites but no nematodes.In other African ant-plant systems nematodes were regularly noticed in fungal patches within the domatia (Blatrix et al., 2009).The authors in question found bacterivorous nematodes of "Pelodera" in domatia of Barteria fistulosa (Passifloraceae) hosting colonies of Tetraponera aethiops, and in domatia of Leonardoxa africana (Fabaceae) settled by Petalomyrmex phylax.
Associations between nematodes and leaf-cutting and fungus-growing ants (Acromyrmex crassispinus and Atta spp.) were unexpected.Like Kermarrec (1975, writing about Acromyrmex octospinosus), I have never found nematodes in fungus gardens themselves, but have detected species of the Oscheius Dolichura group, of Sclerorhabditis sp., Pristionchus sp., and cf.Halicephalobus in refuse piles (tables 1-2).Astonishingly, all these species survived for a long time in laboratory ant colonies (table 2).In addition I was able to isolate a species of Panagrolaimus from workers of Acromyrmex crassispinus and Atta cephalotes (table 5).
New records of mixed infestations, where two or, in rare cases, more than two nematode species were present in the same nest or even the same ant, are found in the tables.Most of the nematode species of mixed infestation are morphologically and taxonomically so different that their ecological demands are supposed also to differ significantly.Only Diploscapter cf.lycostoma and an unknown species (gen.et sp.nov.) found in a nest of Azteca (table 1) appeared to be ecologically very similar.

Remarks on the protagonists
Some unsolved taxonomic problems exist with respect to the saprobiontic nematodes associated with ants.This is because they are often hermaphroditic agamospecies which scarcely possess differentiating characters.As residual males are extremely rare, crossing experiments are not possible.Gene sequence data of isolates from ants do not exist to date, even for the extensively investigated Pristionchus (Kanzaki et al., 2014).It is assumed that most of the ant-associated taxa are complexes of cryptic species that are hard to disentangle.
Although some differences can be observed between species of the Oscheius Dolichura group, they are not clear-cut enough to be distinctive.The only exception is a species which looks like O. dolichura in having a wide stoma with two metastomal teeth on each sector (though these are perhaps not as prominent as in O. dolichura) and a relatively short tail, but which differs considerably in the shape of the tail of the dauerlarva.Rather than a tapering conical tail as found in O. dolichura, the tail tip in the description of O. janeti by de Man (1894, fig. 3) is depicted as drill-shaped.De Man described these peculiar dauerlarvae from the postpharyngeal glands of Formica rufa he obtained from Charles Janet from Beauvais (France).On three occasions I had isolates of Oscheius where the ensheathed third stage juveniles exhibited a characteristic tail tip of this nature (though perhaps slightly different, see fig. 2) and the hermaphrodites displayed the characters of O. dolichura.However, they never came from ants.They were isolated from rotted wood duff and once from the bark of an elm tree exhibiting a certain degree of sap flux.The locations were Corsica (France), Naples (Italy) and Cambridge (Massachusetts).These dauerlarvae survived in a dry substrate for more than two years at room temperature.As well as some differences in the tail tip of the dauerlarvae (fig. 2), the sex ratio was also unlike that of O. janeti.While de Man (1894) had so many males he apparently regarded O. janeti not to be hermaphroditic, I never saw a male in any of my cultures.Janet (1894) also reported lots of males, though adults of female habitus were more frequent.With respect to the synonymisation of O. janeti with the hermaphroditic O. dolichura mentioned above, at this stage of knowledge we must regard O. janeti to be a discrete species which so far has been recorded with certainty only in France as an associate of Formica rufa and probably also of Lasius flavus (Janet, 1893).The dauerlarvae of Oscheius we obtained from ants in Berlin all had a conical tail and therefore should be allocated to O. dolichura.Janet (1894) described two different juveniles from the glands of Formica: the one which became known as O. janeti with an obtuse tail and a mucro-like tip (his fig.4, in accordance with the drawing by de Man), and then a second one of half the length, with a nearly banana shaped body and a tail which tapered to a point (fig.3).Janet interpreted them as two juvenile stages of the same species, postulating that first-stage juveniles entered the ants, obtained nourishment from the glands and doubled in size.This, however, would be most unusual for rhabditids.Moreover, the body shape, conspicuousness of the excretory pore and critical measurements of the small nematode all differ from those of a first-stage juvenile of Oscheius obtained through culture, leading me to conclude that the small nematode depicted by Janet represents a different species (Diploscapter?).
In Diploscapter we are confronted also with cryptic species.Here too, species determination is rarely possible.If rare males, which are more informative than females, can be compared, or the peculiar waving of dauerlarvae can be observed, Diploscapter lycostoma can be identified reasonably reliably (see Wahab, 1962, Markin & McCoy, 1968, Köhler, 2012, and the records from nests and workers of Azteca sp. reported here in tables 1 & 5).However, waving behaviour also exists in D. formicidae (Zhao et al., 2013).The male I found in association with Azteca sp. was compatible with the original description of D. lycostoma.I also isolated this or a very similar species twice from dung near Kassel (Germany) and in Eipomek (New Guinea).In 2007 when I discovered Sclerorhabditis sp. in artificial colonies of Atta spp.and in a nest of Solenopsis invicta in the field, the only known species of this genus-taxon was S. tridentata Ahmad, Shah & Mahamood, 2007.Sclerorhabditis tridentata was described from decaying wood in India, but with no mention of the insects inhabiting this matter (Ahmad et al., 2007).Since then, S. neotropicalis has been discovered in Costa Rica in association with Azteca spp.(Esquivel et al., 2012).In the same country, S. cf.neotropicalis was detected as a member of the symbiotic system of five Azteca species and four Cecropia species and apparently of Azteca pittieri in Cordia alliodora (Boraginaceae) (Mora Pineda, 2013, Huertas, 2014).Recently, I observed perhaps two new species of Sclerorhabditis from the Crematogaster-Macaranga system as well as in association with Cladomyrma sp.(tables 1 & 5).It appears that Sclerorhabditis is a strictly myrmecophilous taxon and was therefore long overlooked due to the scarcity of investigations into nematode-ant associations.The taxon is unknown from Europe.
When studying a batch of nematodes in a colony of Azteca sp.found in a stem of Cecropia sp., I detected some tiny nematodes whose morphology was remarkable.The size, fusiform body shape, conical tail and movement of this unknown species so closely resembled Diploscapter that the specimens were almost undetectable under a dissecting microscope among hundreds of swarming Diploscapter cf.lycostoma.Only the movement of the anterior end during feeding was different, enough to enable me to pick them out with a needle and study them more closely.Most characters are rhabditoid.The cylindrical anisotopic stoma is subdivided into four sclerotised rings.The terminal bulb exhibits the counter valves typical of cephalobids and panagrolaimids but unknown from any rhabditid (fig.3).I found no males to help clarify the taxonomic position of this species.So at the moment I am treating it as a species of a new genus (gen.et sp.nov.) incertae sedis.Though juveniles were produced and did grow for a while in juice prepared from brown algae, it was not possible to successfully establish cultures of this species.

The endophoretic relationship
The term myrmecophilous is applied here to those nematodes which invade ants for dispersal.These species generally live in ants' nests, but may also inhabit a saprobic biochore which is regularly frequented by ants (such as slime flux).In the awareness that more than one species may be represented under one name, we can currently assign the attribute myrmecophilous to at least 14 species (fig.1): Oscheius dolichura, O. janeti, Diploscapter lycostoma, D. formicidae, two species of the Pristionchus Lheritieri complex (gonochoristic and hermaphroditic, the last one perhaps P. entomophagus), Koerneria histophora, Diplogasteroides spengelii, Sclerorhabditis neotropicalis, Sclerorhabditis sp., gen.et sp.nov., Halicephalobus similigaster, Panagrolaimus sp., and Pelodera cf.cystilarva.While some of these phoretic species only facultatively use ants as carriers, the nematodes living in domatia are likely to be more specific.
The only two quantitative investigations to date, by Wahab (1962) and Köhler (2012), show different ratios of nematode species to be associated with Lasius brunneus in Germany (fig. 4 B-C).The most frequent species overall, Diploscapter lycostoma and Oscheius dolichura (fig.4 A), are geographically widespread and associated with a wide range of ant species (fig.5).Three further myrmecophilous nematode species are recorded from more than one ant species (fig.5), and the association appears never to be speciesspecific.
Myrmecophilous nematode species depend on being transported by ants or, in some cases, by a guest species living within the ants' nest.Köhler (2008), for example, found Halicephalobus similigaster attached to the ant-associated beetles Euryusa sinuata (Staphylinidae) and Batrisodes sp.(Pselaphidae), and to the scavenger midge Holoplagia lucifuga (Scatopsidae).Once in contact with ants, the dauerlarvae usually enter through the mouth to invade, exclusively, the postpharyngeal glands of workers, queens and males in a bid for dispersal.The invasion might be facilitated by the typical waving behaviour of dauerlarvae we usually observe in unspecific phoretic relationships: the dauerlarvae climb onto the highest points of the substrate, stand on their tails and wave their anterior ends in order to come into contact with insects.This behaviour has been recorded in most of the myrmecophilous nematodes (Oscheius dolichura, Diploscapter lycostoma, D. formicidae, Halicephalobus similigaster, Pelodera cystilarva, and species of the Pristionchus Lheritieri complex).It is ambiguous if it also exists in Sclerorhabditis (Mora Pineda, 2013).Diploscapter lycostoma, 2) Oscheius dolichura, 3) Koerneria histophora, 4) Pristionchus Lheritieri group, 5) Diplogasteroides spengelii, 6) Halicephalobus similigaster.A) Numbers of workers infested by these species, based on a total of 1770 specimens found to be infested out of 5195 specimens investigated from 16 ant species (data from Wahab, 1962).B) Likewise, based on 77 of 150 workers of Lasius brunneus found to be infested (data from Wahab, 1962).C) Numbers of dauerlarvae of the different nematode species in the 114 of 262 workers of Lasius brunneus from seven locations found to be infested (data from Köhler, 2008Köhler, , 2012)).Note: As the point of reference is different, diagrams B and C are not directly comparable.
The infestation of queens before nuptial flight deserves special attention as queens have the potential to found a new colony.Here, as assumed by various authors until it could actually be shown in D. cf.lycostoma and K. histophora using laboratory-reared worker ants (Köhler, 2012), trophallaxis plays an important role in spreading dauerlarvae from ant to ant.Though the infestation rate was low, I found nematodes in queens of Camponotus herculeanus, Crematogaster sp., Lasius niger, and Solenopsis invicta (tables 3 & 5).Köhler (2008) obtained one Diploscapter dauer from a Lasius umbratus queen, and Wahab (1962, p. 54) reported nematodes in "sexual animals" of four Lasius species.
The dauerlarvae remain in the glands for some time.The precise duration of this stay in the natural environment is unclear, as is the question of whether the nematodes require any particular substances from gland secretions before they leave and are able to complete their life cycle.In this respect it is notable that a large proportion of dauers obtained from decapitated ants did not develop in nutrient fluid (Köhler 2008(Köhler , 2012)), perhaps because a maturation process involving the resorption of certain substances was interrupted.Furthermore, there appeared to be a correlation between size increase by nutrient intake in O. dolichura and the size of the ants or -more precisely -the size of the glands from which they were taken (see Harrison's rule) (Wahab, 1962).Dauerlarvae taken from ants were thus able to mature into adults in pure water using only the reserves they had accumulated (Wahab, 1962).The transport of dauers inside an ant (endophoresis) can, then, be regarded as a first step towards larval parasitism, where the carrier serves as a host at the same time (Sudhaus, 2008).In heavily infested ants (100-240 dauers per worker) obtained in artificial colonies in the laboratory, the postpharyngeal glands were even subject to a certain amount of damage (Wahab, 1962, Markin & McCoy, 1968).In fact, Pristionchus entomophagus actually caused mortality in Myrmica rubra through bacteria adhering to its cuticle (Groden & Stock, 2011, Michaud, 2013).On the other hand, myrmecophilous species can be kept continuously in the laboratory for many years without any form of contact to ants or other insects (my own results).Oscheius dolichura, 3) Pristionchus Lheritieri group, 4) Koerneria histophora, 5) Oscheius janeti, 6) Diplogasteroides spengelii, 7) Halicephalobus similigaster.Data from Janet (1893), Wahab (1962), Nickle & Ayre (1966), Markin & McCoy (1968), Köhler (2012) and this article.
We might speculate that when Lasius feeds on slime flux, dauerlarvae of Diplogasteroides spengelii and Koerneria histophora are triggered to disembark through the ant's mouth.Wahab (1962) once observed the descent directly in an undetermined species.Nest-inhabiting (nidicolous) nematodes can actively leave their carrier once they have reached more favourable surroundings within the nest, or a new nest.After 3 days, 77 of 78 Diploscapter and Halicephalobus had left infested ants on agar in Petri dishes (Köhler, 2012).In the detritus of artificial nests founded by infested Formica rufa or Lasius flavus, populations of Oscheius and Diploscapter, respectively, were established within 25 days (Wahab, 1962).
Occasionally, dauerlarvae may also be attached to the body of an ant (table 4).I think this applies to Diplogastrellus gracilis, Fictor sp. and Mesorhabditis sp.(table 5).I once saw a waving dauerlarva of an unidentified species on an ant's leg.The only specimen(s) of Pristionchus sp. which Köhler (2008) found in his study was (were) on the body of a Lasius brunneus worker.Huertas (2014) found 1-3 nematodes apparently belonging to Sclerorhabditis and Diploscapter in the thorax region and on the legs of Azteca workers.Nearly half of the Azteca queens investigated by Mora Pineda (2013) transported cf.Sclerorhabditis, mostly on the abdomen.And in Dominican amber dating back to around 20 million years ago, Poinar (1982Poinar ( , 2012) ) demonstrated very tiny apparently ectophoretic dauerlarvae which appeared to have abandoned the abdomen of worker ants of †Azteca alpha.Poinar later investigated several pieces of amber which contained 1-3 dauerlarvae adjacent to the abdomen of Azteca workers, "suggesting that they were lodged in the intersegmental membranes" (Poinar, 2011: 93).Without mentioning any decisive characters, he assigned them to Diplogastridae.
Myrmecophilous nematodes may sometimes develop into adults in the heads of dead ants (necromeny) outside the nest, thus enabling them to colonise a different habitat.It is possible that they can also be disseminated by the many ants captured in the pitchers of the carnivorous Nepenthes (Nepenthaceae), where some might actually be able to survive and propagate in fluid which contains prey-digesting enzymes.This could explain the unidentified species of Mesorhabditis, Oscheius and Pristionchus, nematodes that are not part of the infauna, which I occasionally found in some Nepenthes species.Assuming that these observations indicate that saprobiontic nematodes are at the starting point of adaptation to life in this extremely specialised habitat, it will be essential, in the course of evolution, that they develop a phoretic association with an insect inhabitant of the pitchers in order to move from one pitcher to the next.

Evolutionary aspects
Unspecialised nematode species can also live in the decaying matter of ants' nests if they have a chance to get in.A Formica aquilonia nest mound in Finland was found to harbour, besides rhabditids, species of Acrobeloides, Aphelenchoides, Cervidellus, Plectus, Prionchulus, Teratocephalus, and Tylencholaimus which were typical in the surrounding soil (Laakso & Setälä, 1998).In a nest of the leaf-cutting Acromyrmex octospinosus in Guadeloupe, Kermarrec (1975) found species of nematodes from groups including Diploscapter, bunonematids, diplogastrids, and the waving dauerlarvae of rhabditids.My own records of unspecialised nematode species in association with ants can be seen in table 1 (from nests of Mycetophyllax sp., Solenopsis invicta, and an unidentified ant species on 3.10.1970)and in table 5, where nematodes were isolated from a worker of an unidentified species (March 2004) that had contact with the substratum.
There are good reasons to hypothesise, like Wahab (1962), that living together in rotten wood was the starting point of the association between saprobiontic nematodes and ants.According to my list of the 14 myrmecophilous nematode species mentioned above, this must have happened in at least nine independent lineages.It has been ascertained that dauerlarvae of the following eleven species are able to invade the heads of ants and locate the postpharyngeal glands (though not all records of gland location are verified): Oscheius dolichura, O. janeti, Diploscapter lycostoma, D. formicidae, a species of the Pristionchus Lheritieri group, Koerneria histophora, Diplogasteroides spengelii, Sclerorhabditis sp., Halicephalobus similigaster, Panagrolaimus sp. and Pelodera cf.cystilarva.It is possible that other species also invade the postpharyngeal glands, but when nematodes disembark from intact ants it cannot be determined whether they perhaps had been present externally (between the abdominal segments) or inside the abdomen.
The striking organ specificity of the dauerlarvae of nine species which are not closely related but which all invade the postpharyngeal glands of various species of ants needs some explaining.Though we might assume that it is at least partially driven by the anatomy and foraging behaviour of the insects, there must be some prerequisites in the behaviour of the nematodes that make this association non-accidental as it does not happen when saprobiontic nematodes that are adapted in another way come into contact with ants.Wahab (1962), for example, did not succeed in infesting ants in artificial colonies with rhabditids such as Pelodera strongyloides (Schneider, 1860) from manure, Rhabditis maupasi Seurat in Maupas, 1919, the typical associate of earthworms, or Rhabditella typhae (Kiontke, 1999), which Wahab called Rhabditis octopleura, and which is specialised to life in the frass of caterpillars in stalks of cattail (see Maschwitz et al., 2016, p. 6).R. A. Bedding (cited in Nickle, 1984) was not able to infect ants with unnamed rhabditoids either.
As mentioned in the introduction, an ant's head usually only contains one dauerlarva.This is not a problem for autogamous species, because a single nematode can reproduce.But only half of the myrmecophilous species are hermaphroditic or -like Halicephalobus similigaster -parthenogenetic.Only in two hermaphroditic species (Diploscapter lycostoma and a species of the Pristionchus Lheritieri group) were residual males occasionally found to occur.Diplogasteroides spengelii, Koerneria histophora, a species of the Pristionchus Lheritieri group, Sclerorhabditis neotropicalis, Pelodera cf.cystilarva, Panagrolaimus sp.and, apparently, Oscheius janeti are gonochoristic.This mode of reproduction requires a higher rate of infestation -as indeed seen in K. histophora (Köhler, 2012).
The beneficiaries of the ant-nematode association are the nematodes, which usually live as inquilines in ants' nests.Every now and then their dispersal stage (dauerlarva) must be transported by a worker to a more favourable place within the nest, or by a queen to a new nest.It is particularly important for nematodes to infest queens, as these go on to found a new colony.Other nematodes live in an ephemeral biochore such as slime flux (D. spengelii, K. histophora) and find shelter inside the heads of ant workers to bridge the period when the flux temporarily dries up (Köhler, 2012).They thus follow the same strategy as those saprobiontic nematodes living in ephemeral habitats which spend the winter or a dry season in or on an insect.The idea has recently started to emerge, however, that ants too could have an "interest" in this association.Nematodes in the domatia of myrmecophilous plants feed on bacteria growing in the debris, and on the faeces of ants.It is presumed that they in turn are consumed and digested by the ants, which might contribute to the recycling of nutrients (Maschwitz et al., 2016, compare Mora Pineda, 2013).This idea is supported by the discovery of dead nematodes in the intestines of ants from heavily infested artificial nests (Wahab, 1962, p. 64) and by the observation that the ant Pogonomyrmex barbatus feeds its larvae with nematodes (the parasitic Skrjabinoptera phrynosoma, Physalopteridae) (Anderson, 2000, Poinar, 2012).It appears fairly certain that: "Many ant-plant symbioses previously considered to be bipartite will soon likely prove to be multipartite symbiotic communities" (Blatrix et al., 2009: 554).

Figure 1 .
Figure 1.Pictorial key to compare characters of nine myrmecophilous nematode species or representatives: (1) lip region and stoma, (2) median part of pharynx, (3) termianl bulb of pharynx, (4) female anal region and tail (very long tails are cut), (5) male tail with genital papillae, spiculum and gubernaculum in lateral view.The first three species are "Rhabditidae", having a cylindrical stoma, a terminal bulb with valves and a bursa in the male.The next three species are Diplogastridae, characterised by a glandular terminal bulb without valves and filiform tails.The last three species belong to the Panagrolaimidae, showing typical counter valves posterior of the valves in the terminal bulb.The figures are not in the same scale.

Figure 2 .
Figure 2. Comparison of original sketches of ensheathed third-stage juveniles of Oscheius cf.janeti (A, D-F) with drawings based on de Man (1894) of a dauerlarva of O. janeti from the postpharyngeal glands of Formica (B-C) in lateral view.A, B) Anterior ends, showing the distinctive lip region and long, narrow stoma (pharyngeal collar not shown).C, E) Tail ends of the corresponding specimens (A/E, B/C).D, F) Tail ends of two further dauerlarvae.One difference in my specimens was the dorsal notch anterior of the drill-like tail end.

Figure 3 .
Figure 3. Sketches of a female/hermaphrodite of gen.et sp.nov.incertae sedis (lateral view).A) Anterior end, showing jointed stoma and pharynx with median bulb and counter valves in the terminal bulb.B) Anisotopic stoma from the left side.C) Posterior end with lateral lines and phasmid.

Figure 4 .
Figure 4. Frequency distribution of six species of nematode dauerlarvae in the heads of ants.The species are: 1) Diploscapter lycostoma, 2) Oscheius dolichura, 3) Koerneria histophora, 4) Pristionchus Lheritieri group, 5) Diplogasteroides spengelii, 6) Halicephalobus similigaster.A) Numbers of workers infested by these species, based on a total of 1770 specimens found to be infested out of 5195 specimens investigated from 16 ant species (data fromWahab, 1962).B) Likewise, based on 77 of 150 workers of Lasius brunneus found to be infested (data fromWahab, 1962).C) Numbers of dauerlarvae of the different nematode species in the 114 of 262 workers of Lasius brunneus from seven locations found to be infested (data fromKöhler, 2008Köhler, , 2012)).Note: As the point of reference is different, diagrams B and C are not directly comparable.

Table 1 .
Material from ants' nests in the field.

Table 2 .
Material from ants' nests in laboratory colonies.

Table 3 .
Nematodes isolated from heads of ants.

Table 4 .
Nematodes isolated from bodies of ants.

Table 5 .
Nematodes isolated from entire ants (head and body not differentiated).