A new species of Illacme Cook & Loomis, 1928 from Sequoia National Park, California, with a world catalog of the Siphonorhinidae (Diplopoda, Siphonophorida)

Abstract Members of the family Siphonorhinidae Cook, 1895 are thread-like eyeless millipedes that possess an astounding number of legs, including one individual with 750. Due to their cryptic lifestyle, rarity in natural history collections, and sporadic study over the last century, the family has an unclear phylogenetic placement, and intrafamilial relationships remain unknown. Here we report the discovery of a second species of Illacme, a millipede genus notable for possessing the greatest number of legs of any known animal on the planet. Illacme tobini sp. n. is described from a single male collected in a cave in Sequoia National Park, California, USA. After 90 years since the description of Illacme, the species represents a second of the genus in California. Siphonorhinidae now includes Illacme Cook & Loomis, 1928 (two species, USA), Kleruchus Attems, 1938 (one species, Vietnam), Nematozonium Verhoeff, 1939 (one species, South Africa) and Siphonorhinus Pocock, 1894 (eight species, India, Indonesia, Madagascar, Vietnam).


Introduction
The genus Illacme is the sole representative of the Siphonorhinidae in the Western Hemisphere. Its closest known relative, Nematozonium filum , is endemic to the Drakensburg Mountains of South Africa Hoffman 2004, Hamer 1998). The current geographical distribution of the Siphonorhinidae in California, Wallacea, Sundaland, Himalayas, Indo-Burma, and southern Africa likely represents remnants of its former range and an ancient radiation predating the breakup of Pangaea more than 200 million years ago (Marek and Bond 2006). There is molecular phylogenetic evidence for monophyly of its order Siphonophorida (Regier et al. 2005, Brewer and Bond 2013, Fernandez et al. 2015. In contrast, it is unclear whether the Siphonorhinidae is a natural group. Because there are so few species in the Siphonorhinidae and little is known about the family from a basic α-taxonomic and a biological perspective, the discovery of a novel species provides significant new data. Here we describe a new species of the genus Illacme from a marble cave in Sequoia National Park and provide a world catalog and map of species in the family Siphonorhinidae.
The Siphonorhinidae are members of the subterclass Colobognatha that contains the orders Platydesmida, Polyzoniida, Siphonocryptida, and Siphonophorida (Shear 2011). The Colobognatha are diminutive in size, with most individuals less than 30 mm in length, about a millimeter or less in trunk width, and possessing an oval or circular cross-section. The Platydesmida, Polyzoniida, and Siphonocryptida are typically wider than the Siphonophorida, and are dorsoventrally flattened in segmental cross-section. Some taxa possess elongated paranota further adding to the flattened appearance, and appear platyhelminth-like (e.g., Brachycybe, Hirudicryptus, Octoglena, Platydesmus). In contrast, most individuals in the order Siphonophorida are pale thread-like millipedes that are even confused with nematodes by the unaccustomed observer. These millipedes possess the greatest number of leg-bearing trunk segments of any animal. One female specimen of I. plenipes Cook & Loomis, 1928 possesses a superlative 192 diplosegments. The trunk rings are translucent and lightly pigmented and lack the heavy cuticle that many chilognathan diplopods possess. Despite the delicate nature of their exoskeleton, siphonophoridans have a variety of cuticular adornments-including spines, tubercles, and silk-secreting setae (Read and Enghoff 2009, Marek et al. 2012. The rings of colobognath millipedes are uniform in appearance throughout the length of the trunk, except those of some Platydesmida (genus Andrognathus) with anteriorly projecting ozopores on the fifth ring and Platydesmida and Siphonocryptida with color patterns that vary antero-posteriorly (Shear andMarek 2009, Enghoff 2011). The cephalic morphology of colobognaths is generally regarded as highly derived relative to other Diplopoda, making it difficult to draw homology to known structures . The orders Siphonocryptida and Polyzoniida possess a cluster of simple eyes; in Platydesmida and Siphonophorida eyes are absent. However, a few platydesmidan taxa possess pigmented patches on the cuticle where the eyes would normally occur. The Colobognatha (meaning "abbreviated jaw") are characterized by reduced mouthparts. Their heads are often small in relation to the trunk and appear triangular in anterior view, while other millipedes have larger, subspherical heads supporting musculature required for strong chewing action by the mandibles. This modification reaches a pinnacle in the Siphonophoridae, which have heads drawn out into long beaks, and mandibles that are highly simplified and styliform, with gnathochilarial components fused and reduced. The Siphonorhinidae possess mouthparts somewhere in between with components that remain identifiable and able to be homologized with those of other non-colobognath millipedes.
In contrast with their derived cephalic morphology, the order Siphonophorida possesses a primitive trunk architecture for helminthomorph diplopods, including unfused rings composed of a free sternite, pleurite, and tergite. The siphonorhinid mouthparts are presumed to be ancestral to the highly derived siphonophorid beak, and based on these features, siphonorhinids are hypothesized to be a basal sister group to the remaining siphonophoridan taxa. Based on molecular phylogenetics, the Siphonophorida are sister to a clade formed by exemplars of the Polyzoniida and Platydesmida (Regier et al. 2005, Sierwald andBond 2007). However, the family Siphonorhinidae has yet to be sampled by recent phylogenomic estimations of the class Diplopoda (Brewer andBond 2013, Fernandez et al. 2015).
Illacme species and their colobognathan relatives exhibit true anamorphosis (euanamorphosis), whereby six-legged hatchlings develop into adulthood in coordination with the addition of new segments (Enghoff et al. 1993). The addition of new segments lengthens the body and adds legs, which develop shortly after segment formation. This process continues beyond attainment of sexual maturity for an indeterminate amount of time, and imparts high variability and a very large number of segments in euanamorphic taxa. A paratype female of I. plenipes collected by O.F. Cook in 1926 possesses 192 segments and 750 legs . The age of this exceptionally segmented individual is unknown, but likely to be several years. While diplosegmentation and sequentially repeated leg-bearing segments serve to provide force for burrowing, the superlative segment count in I. plenipes seems unwarranted and perhaps serves another function. The role is unclear, and several hypotheses have been suggested including: burrowing in deeper soil, clinging to rocks, or lengthening the gut to digest low nutrient food (Marek et al. 2012). As in the highly elongate geophilomorph centipedes, the long, flexible body may be an adaptation to negotiating narrow, pre-existing spaces in the soil.
In the western U.S. (Arizona, California, Texas), Siphonophorida occur in moist refugia within more arid habitats. However, many tropical siphonophoridans occur in mesic habitats and in rainforests that are continuously wet. The microhabitats of siphonophoridan species are usually within deep substrata and individuals are frequently discovered beneath large stones (e.g., I. plenipes in California) and embedded inside large decaying logs (e.g., Siphonophora species in Central America). Persistence in these microhabitats is consistent with their morphology, including a lack of eyes, depigmented exoskeleton, shortened legs, and an elongate flexible body. The Siphonophorida in Arizona and California are found in relatively mesic oak woodlands in mountain foothills, including those of the Coast Ranges (CA), Sierra Nevada (CA), and Madrean Sky Islands (AZ).

Methods and results
From 2002 to 2004, scientists led formal biological surveys of caves in Sequoia and Kings Canyon National Parks for invertebrates, including arachnids, myriapods, and hexapods. From 2006 to 2009 several follow up visits yielded incidental collections, and among these specimens was one sample of I. tobini sp. n. from Lange Cave, collected by JKK on 9 October 2006. Three years after this discovery, myriapod specialists made three additional expeditions to Lange Cave and surrounding habitats to search for additional material for description of the species. Collecting effort was focused within the cave, and surface searches around the cave entrance were carried out. More intensive searches were conducted at the the confluence of Cave, Yucca, and Cascade creeks-the general area where I. tobini sp. n. was discovered. During field expeditions by PEM from 2010-2012, 63 additional localities in the foothills of the Sierra Nevada from El Dorado National Forest southward to the Tehachapi Mountains were explored for I. tobini sp. n. Using techniques previously developed for I. plenipes (and applied to I. tobini sp. n.), the undersides of large stones were examined. The bases of decaying logs and leaf litter were also searched, albeit an improbable microhabitat since previous collections of U.S. siphonophoridans were rarely encountered in these areas. Live millipedes were collected by hand, or in some cases lifted with a paintbrush or forceps if necessary. In spite of all of this additional effort, field biologists found no additional specimens.
Lange Cave is 240 km east of its congener I. plenipes that occurs in San Benito County California (Fig. 1). The single live specimen was sacrificed and preserved in 80% ethanol. Forty segments from the midbody were removed and preserved in 100% ethanol and archived at -20 °C nine years later in an attempt to preserve DNA. The remnants of the holotype (anterior and posterior sections) were removed, dried at room temperature, and mounted on a standard SEM pin stub mount (Ø12.7 mm × 8 mm pin height) with double sided carbon conductive tape. Specimens were coated with 10 nm of platinum and palladium metals with a Leica EM ACE600 high vacuum coater (Wetzlar, Germany), and stored with silica gel desiccant until ready for examination.
Genomic DNA was extracted and purified from half of the ethanol-preserved tissue from the midbody section using a Qiagen DNeasy tissue extraction protocol. The remaining half of the tissues have been retained in the VTEC frozen tissue collection. Standard kit protocol was followed and the DNA was eluted from the spin column with one round of 50 µL AE buffer. Genomic DNA was archived at -20 °C in the freezer collections at the VTEC. A segment of the cytochrome c oxidase I gene (COI), was amplified using polymerase chain reaction and the thermal cycling steps of Hebert et al. (2003) and with the universal DNA barcoding primers LCO1490 and HCO2198 of Folmer et al. (1994). Polymerase chain reaction of the COI barcoding region of the single I. tobini sp. n. individual and visualization of amplifications on a 12% agarose gel, did not indicate the presence of DNA based on comparison with the negative and positive controls. A second PCR was repeated with the amplification product from the first reaction, in case a low concentration of DNA from I. tobini sp. n. was present. The second reaction showed the same results and a lack of DNA based on comparison with the controls.
Habitat and distribution. Illacme tobini sp. n. is only known from a single in-cave collection, within the upper foothills of the Giant Forest in Sequoia National Park (Fig. 1B). Lange Cave is situated at the base of Yucca Mountain at the boundary of the Sierra Nevada Forest and California Interior Chaparral and Woodlands ecoregions (Fig. 20). A region characterized by a Mediterranean climate with temperature and humidity extremes encompassing cold wet winters (< 0 °C and 700 mm precipita- tion) and hot dry summers (> 40 °C and < 2 mm precipitation) (Tobin et al. 2013). The cave is composed of Jurassic-Triassic marble of a white, coarsely crystalline, and schistose to gneissose composition (Sisson and Moore 1994). The marble cave system is encompassed by biotite-feldspar-quartz schist rocks. The cave is ca. 90% surveyed, and has a total volume of 354.2 m 3 , average diameter of 2.1 m, wall area of 733.3 m 2 , and floor area of 124.6 m 2 . Inside the cave, temperatures range between ca. 6 °C in the winter months (October-May) to ca. 9 °C in the summer months (June-September). The woodland habitat around the cave was primarily composed of California live-oak (Quercus agrifolia), California bay (Umbellularia californica), Giant sequoia (Sequoiadendron giganteum), and Mountain maple (Acer glabrum). Understory flora included Scouringrush horsetail (Equisetum hymale), California wood fern (Dryopteris arguta), and Thimbleberry (Rubus parviflorus). Other organisms encountered in the habitat included millipedes-Parcipromus cooki, Californiulus yosemitensis, Taiyutyla loftinae, Amplaria muiri; arachnids-Yorima sp., Ceratinops inflatus, Nesticus spp., Pimoa spp., Mundochthonius sp., Ortholasma colossus, Calicina sp.; hexapods-Tomocerus sp., Amoebaleria caesia, Heleomyza sp., Hippodamia convergens; and the salamander Ensatina eschscholtzii platensis. Discussion. Illacme species have extremely limited known geographic ranges. This feature suggests a formerly widespread, perhaps ancient, distribution, and/or membership in a larger hidden diversification in California encompassing many undiscovered taxa. Illacme individuals occur in the mesovoid shallow substratum (MSS), a cryptic ecosystem, which are miniscule subterranean microhabitats encompassing fissures and cracks below the soil surface (Ortuño et al. 2013). These subterranean areas are the microcaverns (< 1 mm) and mesocaverns (1 mm-20 cm) described by Howarth (1983). The fauna of the MSS likely represents a considerable fraction of unknown biodiversity, yet the habitat is unexplored and its diversity poorly known. Species discovery from these microhabitats has only recently begun, and recent advances in collecting techniques are uncovering a considerable amount of new taxa. These microhabitats are fundamentally miniature caves and many MSS taxa also include cave-restricted species (Espinasa et al. 2014). As a result, MSS organisms possess some troglomorphic features-e.g., lack of eyes, no pigment-but lack the open-space adaptations of cave animals, including long limbs and elongate sensory structures (e.g., antennae and setae). Frequently MSS taxa possess shorter legs than cave or epigean forms and a covering of thin, delicate setae on the exoskeleton (Espinasa et al. 2014). Albeit anecdotally, Manton (1961, pg. 395) associated the hirsute covering of Siphonophora individuals as an adaptation for maneuverability, including spiraling within narrow crevices, and crawling upside-down on the ceilings of caverns; however, it is unclear precisely how this occurs biomechanically. Illacme plenipes individuals are found exclusively beneath large deep-set stones-a common place of discovery for MSS arthropods (Marek et al. 2012). In contrast, I. tobini sp. n. was documented solely from a marble cave. Considering that I. plenipes individuals were found in the MSS, and that they possess MSS adaptations (including a vestiture of setae and absence of long sensory structures and limbs), the possibility that I. tobini sp. n.-with similar adaptations-is cave-restricted is uncertain. Additional material of I. tobini sp. n. would provide evidence to address this claim. Notwithstanding the paucity of material, the significance of the discovery highlights the importance of the Sequoia caves and MSS as a habitat of distinctive biodiversity.   The species I. tobini sp. n. and I. plenipes are the sole members of the genus and family in the Western Hemisphere. Shared morphological characters indicate the family is monophyletic, yet these features have not been considered within the context of a rigorous phylogenetic systematic framework. The features, including lack of a beak and absence of antennal pits, are broadly distributed across helminthomorph millipedes and are potentially shared ancestral traits and thereby do not indicate monophyly. The species I. tobini sp. n. is closely allied to I. plenipes based on unique shape of the head, consistency in appearance of mouthparts, similarly shaped gonopods, and possession of many legs. However, I. tobini sp. n. differs from I. plenipes in noteworthy characters such as the shape of metazonites, ornamentation of the ozopore, and chaetotaxy and number of articles of the posterior gonopods. The divergence in these traits, considering the usual divergence in morphology between siphonorhinid taxa, suggests generic differences. Our PCR possibly failed because specimen preservation in dilute ethanol degraded the DNA (Vink et al. 2005). Pending discovery of additional individuals suitable for DNA sequencing, and phylogenetic analysis within the context of relatives in the Siphonorhinidae, a new generic designation may be justified.
Our knowledge of the cephalic morphology of the Colobognatha is limited due to their small size and derived anatomy, thereby making the generation of homology hypotheses difficult. In the current study, we revise the morphological assessment of the labrum, gnathochilarium, and mandibles. The labrum of I. tobini sp. n. is apically deeply divided into a slit. The dorsal margins of the slit are lined with sharp upwardsprojecting spines. Moving posteriorly into the head, the labrum is further divided into a tridentate projection with additional upwards projecting spines. The remainder of the labrum posterior to the epistome is covered in a field of ca. 200 pores, half of which possess a secretion seemingly extruded from the pore openings (Fig. 7F). These labral features are not observed in other diplopods, and their homology and function is unclear. The pores appear deep and may open to the buccal cavity. The gnathochilarium of I. plenipes was described as "indistinguishably fused" (Marek et al. 2012, pg. 89). However, we now think the gnathochilaria of both species are composed of a mentum and pair of stipes. Illacme tobini sp. n. has paired lamellae linguales each with a palp (Fig. 14B), but whether this feature is present in I. plenipes is unclear. The mandibles of I. tobini sp. n. lack sharp teeth distally (as in other Chilognatha) and possess fingerlike rounded teeth. The mandibular pectinate lamella are composed of numerous rows of small jagged teeth that project ventrally and nest in a groove in the frontal body of the endochilarium. It is evident that these structures are the mandibles and not the epipharynx based on the articulated nature of the articles and separation between the mandibular gnathal lobe and base. In the description of Kleruchus olivaceus, Attems (1938, pg. 296, fig. 195) similarly illustrates the right mandible of the male holotype. In the drawing, the finger-like mandibular teeth and pectinate lamella are strikingly similar in appearance to the equivalent structures in I. tobini sp. n.
Based on examination of the mouthparts of Illacme and other siphonophoridan species, individuals consume liquid or gelatinous foods. In Siphonophorida (and most Colobognatha), the mouthparts are drawn into a cone with a small aperture distally. The mandibles are reduced and are not divided between the cardo, stipes and galea. A suctorial feeding mode has been suggested previously, and Manton (1961, pg. 386) indicated that species of Siphonophora possess a "suctorial fore-gut", proboscis, skeletomuscular features, and head movement behaviors that strongly implicate suction feeding. Manton did not elaborate upon precise modifications of the foregut for suction, but filtration devices and muscular thickening are potential features to explore in the future. The presence of a coiled hindgut and elongation of the trunk (and thereby also the gut) for processing a nutrient poor diet are consistent with plant sap feeding (Marek et al. 2012). Dissecting individual hindguts of I. plenipes suggested a liquid diet since gut contents were gelatinous and homogenous, and lacking particles completely. The mouthpart morphology of I. tobini sp. n. is peculiar and hypothetically represents a morphology adapted for consuming fungus as they are similar in gross anatomy with some sporophagous beetles (Betz et al. 2003, Lipkow and Betz 2005, Yavorskaya et al. 2014. Specifically, I. tobini sp. n. possesses (1) mandibles with inner brush-like "bristle-trough" structures ( Fig. 14C- While the mouthparts of the Siphonophorida are more derived in morphology and function relative to other helminthomorph millipedes, the gonopods are primitive due to their leg-like structure. In contrast with gonopods of eugnathan millipedes, many of which possess two leg podomeres (coxa and telopodite), colobognaths typically have a greater number of podomeres. Although Marek et al. (2012) counted six gonopodal podomeres in I. plenipes, we have reexamined I. plenipes males and found that they, as with I. tobini sp. n. males, have seven gonopodal podomeres, representing the primitive complement, including a seventh tarsungulum that is the terminal article. As in other colobognaths, the tarsungulum of the posterior gonopod is stylus-like, and the anterior tarsungulum spade-shaped with a deep groove. The groove of the anterior gonopod cups the stylus, which is often observed resting within the recess, and may act as a conductor allowing the posterior gonopod to slide into the cyphopods of the female during copulation. This process may be functionally analogous to the spider embolus (=posterior gonopod in millipedes) and conductor (=anterior gonopod in millipedes).
Several groups of dispersal-limited Californian animals show a distribution in which a Sierra Nevada clade is most closely related to a clade in the Coast Ranges. Examples of this spatial pattern occur in bioluminescent millipedes (genus Motyxia), harvestmen (genus Calicina), mygalomorph spiders (Aliatypus californicus, Aliatypus erebus), and several species of salamanders (Batrachoseps attenuatus, Ensatina eschscholtzii, Aneides lugubris- Jockusch and Wake 2002, Martínez-Solano et al. 2007, Kuchta et al. 2009, Lapointe and Rissler 2005. Most studies that demonstrate this biogeographical pattern among taxa infer directionality of diversification from the Coast Ranges to the Sierra Nevada (reviewed in Emata and Hedin 2016). The phylogenetic studies of salamanders indicate a west-to-east pattern, relatively recent in the mid-late Pleistocene. In contrast, the studies of mygalomorphs and diplopods recover an east-to-west directionality of diversification (Satler et al. 2011, Marek andMoore 2015). Several of these taxa (e.g., Calicina and Batrachoseps) have occurred in California since the Eocene, with a "transvalley" split occurring in the harvestman Calicina during mid-late Miocene (Emata and Hedin 2016). Inferred dates for the east-west splits in other taxa are unknown.
Conservation. Illacme tobini sp. n. is a short-range endemic restricted to the base of Yucca Mountain between the North and Marble forks of the Kaweah River in Sequoia National Park, California. The species is only known to occur in one small cave, though its range is likely to include the MSS. Management of this species should include careful consideration of activities that may impact the surface or subsurface. Actions that include vegetation changes, ground disturbance, or alteration of drainage patterns should be restricted in scope to preserve the soil and moisture of this river basin. The abundance and composition of MSS invertebrates in most global habitats remains uncertain, and further exploration and survey of these systems, thereby building knowledge of this fauna, will help to understand more fully biodiversity that is responsible for supporting healthy forests and ecosystem services. Note. Attems (1938)   Note. With regards to S. angustus and S. pallipes (collected from the same area), Pocock (1894, pg. 336) wrote: "These two species are really so much alike that I am perfectly prepared for fresh specimens to show that the differences pointed out are merely due to individual variation. But at present there is no evidence that such is the case and the analogy of other species of the group lends no support to the view."