Highly disjunct and highly infected millipedes – a new cave-dwelling species of Chiraziulus (Diplopoda: Spirostreptida: Cambalidae) from Iran and notes on Laboulbeniales ectoparasites

. Chiraziulus is a highly disjunct, hitherto monotypic genus of cambalid millipedes, geographically isolated in Iran by more than 7000 km from its presumed closest relatives in East Asia and North America. Recent fieldwork in caves of Iran has provided several specimens of this genus, allowing the description of Chiraziulus troglopersicus sp. nov. The intraspecific variability of the type species, C. kaiseri Mauriès, 1983, is illustrated with scanning electron micrographs. Chiraziulus is characterized by exceedingly long microtrichose gonopod flagella which from their insertion points on the posterior face of the anterior gonopod coxites first point distad instead of basad or basad-posteriad as in most other flagelliferous Cambalidea (and Julida), then traverse a groove on the mesal surface of the anterior gonopod coxites, making a full (360°) loop. The same feature is also illustrated for the first time in the genus Cambala . The patterns and prevalence of the infection with a species of ectoparasitic fungus of the genus Rickia (order Laboulbeniales) in the type material of C. kaiseri is described. An updated review of the cave-adapted fauna of Iran is given.


Introduction
The millipede order Spirostreptida is mainly distributed in the tropical and temperate zones of the Southern Hemisphere, and in warm-temperate North America.In the Palaearctic subregion there are some marginal occurrences in East Asia, and very few, disjunct occurrences in the western Palaearctic: Odontostreptus spp. in Morocco, Archispirostreptus spp. in the Middle East, and the so far monotypic genus Chiraziulus Mauriès, 1983, in Iran (Shelley & Golovatch 2011: fig. 29).Whereas Odontostreptus Attems, 1914 andArchispirostreptus Silvestri, 1895 belong to the family Spirostreptidae, suborder Spirostreptidea, Chiraziulus is the only W Palaearctic representative of the suborder Cambalidea (Shelley & Golovatch 2011: fig. 30).
Chiraziulus and its type species, C. kaiseri, were described by Mauriès (1983) who erected it as a subgenus of Nannolene Bollman, 1887.Golovatch (1983) tentatively suggested that full generic status might be warranted for Chiraziulus, and Mauriès (1987) listed it as a full genus.Nannolene, in its current conception (Hoffman 1999), includes numerous nominal species from the western USA and the Hawaiian Islands.Although relationships within Cambalidae are not at all clear, and the delimitation of the family vis-à-vis other cambalidean families is equally uncertain (Enghoff et al. in press), the fact remains that Chiraziulus is a highly disjunct genus, separated from all other Cambalidea by distances of over 2000 km; if its closest relative is really Nannolene, the distance will surpass 7000 km.
We revise the genus Chiraziulus, describe a new species from a cave in Iran and document intraspecific variation of C. kaiseri.New observations on the flagellum in a species of the North American genus Cambala Gray, 1832, are also included.Re-examination of the type material of C. kaiseri led to the discovery of the presence of ectoparasitic fungi of the order Laboulbeniales; we also include information about this infection.

Material and methods
New specimens of Chiraziulus (Fig. 1) were collected in two caves in Iran: Ghar Sarab Cave (Ghar is Persian for "cave"), located at 87 km northwest of Hamadan city, near Sarab village, altitude: 2200 m asl, coordinates 35°14'41.7"N, 48°15'40.6"E, length: 2959.9 m and Neyneh Cave, located at 45 km southwest of Dehdasht city, Kohgiluyeh and Boyerahmad Province, coordinates 30°40'18" N, 50°21'71" E, 770 m asl (Fig. 2).Specimens were studied using a Leica Wild M10 microscope equipped with an ocular micrometer.Parts of specimens were mounted in glycerine on temporary slides for microscope study.Microphotographs were taken using a Leica digital camera M205A mounted on a stereo microscope Leica DFC 420.Images were processed with a Leica Application Suite program, and final stacking was made using ZereneStacker software.For scanning electron microscopy, parts of specimens were critical point dried in a Tousimis Autosamdi 815, series A. Other specimens were transferred to 96% ethanol, then to acetone, air-dried, mounted on aluminium stubs, coated with platinum/palladium and studied in a JEOL JSM-6335F scanning electron microscope.Images were processed with Adobe Photoshop CS6.
Material is deposited in the Zoological Museum, Natural History Museum of Denmark, University of Copenhagen (ZMUC), the Collection of the Biology Department, Zoological Museum of Shiraz University, Shiraz, Iran (ZM CBSU) and the Natural History Museum of Vienna (NHMW).

Diagnosis
A genus of medium-sized blind cambalid millipedes resembling the Nearctic genus Nannolene, with longitudinal crests on the body rings and ozopores placed on prominent tubercles (Fig. 1).Sharing with Nannolene remarkably long gonopodal flagella, which make a complete loop (360°) from their insertion points on the anterior gonopods towards the interior part of the posterior gonopods, differing from Nannolene by having the coxites of the anterior gonopods larger than the telopodites.First pair of male legs not modified.

Distribution
Known from several localities in Iran (Fig. 2).

Intrapecific variability of Chiraziulus kaiseri
In addition to the type locality and a few sites in its vicinity, Chiraziulus kaiseri has been recorded from the south of Iran (Golovatch 1983).Examination of an adult male from an isolated cave (Sarab Cave) in northwestern Iran, far from the type locality, leads us to include it in C. kaiseri as well and to regard the differences in the male gonopods as intraspecific variability (Fig. 7).The anterior gonopods of the specimen from Sarab Cave shows some differences compared to the type material, especially in the shape of the posterior branch of the telepodite (Fig. 7E-J).No remarkable difference was observed in the posterior gonopods (Fig. 7K-L).Some variability was also observed in the chaetotaxy of the gnathochilarial stipes (Fig. 3F).In the specimens from the type locality, Sarab Cave and 19 km W of Shiraz, there are two distolateral and one distomesal setae, whereas in the specimen illustrated by Golovatch (1983: 168, fig. 12), all three setae are distolateral.

Diagnosis
A species of Chiraziulus, differing from C. kaiseri by anterior gonopods not presenting a triangular shape in anterior view, by having equally sized anterior and posterior lobes of anterior gonopod telepodites, and by posterior gonopod having a spine-like process ending in a halberd-like structure.

Etymology
From Greek trōglo: cave, common prefix for terrestrial cave-dwelling animals, and Latin persicus: Iranian.

Habitat
The new species was discovered in the Neyneh Cave (Fig. 10), located in the mountainous Khaeiz protected area, in Zagros Mountains, southwestern Iran.Neyneh Cave is situated in the warmer part of the province where the temperature at the surface reaches to 50°C during the summer.The temperature inside the cave is 25°C; the relative humidity is very high, 95-99.9%,and the CO 2 level is 613-700 ppm.There was no water flow in the cave.This cave is located in cliffs and has more than 700 m of horizontal maze development.It is located in an area with difficult access, thus protected from human pressure; however, there is some destruction due to the activities of treasure hunters.Chiraziulus troglopersicus sp.nov.specimens were found in complete darkness, crawling on guano and cave walls.Some colonies of bats produce enough guano to cover the cave soil, providing an important source of organic matter for this subterranean trophic chain.Species richness in Neyneh Cave is relatively high and includes several arthropod taxa (Acari, Araneae, Oniscidea, Collembola, Orthoptera, Coleoptera), as well as some vertebrate taxa, such as gekkos, bats and hystricid rodents.

Notes on the gonopods of Cambala annulata (Say, 1821)
SEM study of one male of Cambala annulata (USA, NC, Surry Co., Elkin, 12 October 1975, R.M. Shelley leg.) revealed that the anterior gonopod in this species, like that of Chiraziulus (and Nannolene), has a remarkably long flagellum (Fig. 11A) with a similar insertion, and with a similar full-loop course in part involving a groove on the mesal side of the anterior gonopod coxite.The sharktooth-like structure of the mesal gonopodal sternal part is also present (Fig. 11B).Enghoff 2015).These fungi live only on the surface of living animals, and during the revision of the type material of Chiraziulus kaiseri, deposited in ZMUC (Fig. 12), almost all type specimens were found to be infected with an undescribed species of the genus Rickia, which in spite of its small size can be detected due to the dark coloration of the foot and appendages (Fig. 12A).Some Laboulbeniales have a preference for growing on specific parts of the millipede body, and the transmission mechanisms play an important role for the position of the fungus on the host (Enghoff & Santamaria 2015;Reboleira & Enghoff 2015).In the case of Rickia sp. on C. kaiseri, the fungus is randomly distributed along the millipede (Fig. 12): it was found on body rings, legs, head, antennae and surprisingly also on the paraprocts (anal valves), both in adults and juveniles, suggesting that transmission does not exclusively take place during copulation as occurs in other species (Santamaria et al. 2014).
More remarkably, the specimen of Chiraziulus kaiseri from Sarab Cave, despite being part of a disjunct population in the north of Iran, was also infected with the same species of Rickia.

Discussion
Chiraziulus kaiseri presents a considerable degree of intraspecific variation even within the type material.The new population found isolated in a cave in the northwest of Iran is also parasitized by the same Rickia (morpho)species as the type material.These fungi are normally highly host specific (De Kesel 1996), and the presence of the same ectoparasitic species in disjunct populations is herewith recorded for the second time for millipedes, as it was recently reported for Diplopodomyces lusitanipodos Santam., Enghoff & Reboleira and Troglomyces manfrediae Colla (Santamaria et al. 2014).
The highly relict character of the genus Chiraziulus had already been pointed out by Golovatch (1983), due to its remarkable disjunct distribution relative to other Cambalidae, which are distributed mainly in North and Central America, Hawaii and Australia.The closest relative of Chiraziulus may be Nannolene (including Hawaicambala Mauriès, 1983), as suggested by the original classification of C. kaiseri in Nannolene (Mauriès 1983).A synapomorphy could be the exceedingly long gonopodal flagellum which from its insertion point on the posterior face of the anterior gonopod coxites points distad instead of basad or basad-posteriad as in most other flagelliferous Cambalidea (and Julida), and then makes a full  19); also a specimen of Nannolene keiferi Chamberlin, 1943, has flagella of normal length which do not make the 360° loop.On the other hand, we could show that this peculiar type of flagellum is also present in the genus Cambala.Clearly, the genus-level taxonomy and phylogeny of Cambalidae needs a closer study.Mauriès (1983) mentioned the following characters in support of his new subgenus Chiraziulus: 1. Anterior gonopod coxites with much longer parasagittal lobes than in Nannolene s.s. 2. First pair of male legs unmodified (although there is some uncertainty about the first pair of legs in Nannolene s.s.) 3. Lack of eyes 4. Presence of longitudinal crests and grooves on the metaterga 5.The geographical isolation We can support the first and fourth of these characters, although we have re-interpreted the "parties coxales parasagittales" as belonging to the telopodites.The second, third and fifth characters are less convincing: the male of N. keiferi that we have examined has unmodified first legs, several (sub)genera of, e.g., Julidae and Blaniulidae include eyed as well as blind species, and finally, geographical isolation per se should not be used as an argument for creating a new taxon, as this could easily lead to circular argumentation.For the time being, we maintain Chiraziulus as a full genus, following Mauriès (1987).
The anterior gonopod coxite and telopodite in Chiraziulus seem to form a "forceps" with the opposing scaly-rugose edges.This is probably an analogue of the "forceps" formed by the promerite (= anterior gonopod) and mesomerite (part of posterior gonopod) in several Julidae, which has been shown to grip the operculum of the vulva during copulation (Haacker & Fuchs 1970;Tadler 1996; see also Read 1990).Mauriès interpreted both parts of the forceps as belonging to the coxite, but the SEM images clearly show that it is composed of apical parts of the coxite and the anterior branch of the telopodite.
We here add a cave-adapted millipede to the short list of Iranian cave fauna by describing a new species of Chiraziulus, raising to ten the number of cave-adapted species known for the country.

Fig. 9 .
Fig. 9. Chiraziulus troglopersicus sp.nov., ♂, paratype, gonopods.Scanning electron micrographs.A. Anterior gonopods in posterior view.B. Detail of the process on the tip of an anterior gonopod in posterior view.C. Anterior gonopods in anterior view.D. As C, in apical view.E. Tip of the flagellum.F. Posterior gonopods in lateral view.G. Tip of the long spine-like process of the posterior gonopod.H. Posterior gonopods in posterior view.I.As H, in anterior view.J. Detail of the mesal sternal part of the gonopods.Abbreviations: C = coxal process; T = telepodite; f = flagellum; s = setae; dp = distal process.Scale bars: A-D, F, H-I = 10 μm; E, G, J = 1 μm.