﻿A new species of Illacme from southern California (Siphonophorida, Siphonorhinidae)

﻿Abstract The millipede fauna inhabiting deep soil are poorly known. They are small and threadlike, slow moving, lacking pigmentation, and rarely encountered due to their obscure underground way of life. One family, the Siphonorhinidae, encompasses four genera and 12 species in a fragmentary distribution in California, southern Africa, Madagascar, the Malay Archipelago, and Indo-Burma. The family is represented in the Western Hemisphere by a single genus, Illacme Cook & Loomis, 1928 from California, with its closest known relative, Nematozoniumfilum Verhoeff, 1939, from southern Africa. A new species of this family is documented from soil microhabitats in the Los Angeles metropolitan area, Illacmesocal Marek & Shear, sp. nov. Based on this discovery and the recent documentation of other endogean millipede species, we show that these grossly understudied subterranean fauna represent the next frontier of discovery. However, they are threatened by encroaching human settlement and habitat loss, and conservation of this species and other subterranean fauna is of high importance.


Introduction
In 1926, American myriapodologist Orator Cook discovered a millipede with 750 legs under a stone in an oak forest near San Juan Bautista, California. As it had more legs than any other animal known at the time, he and Harold Loomis aptly named this millipede Illacme plenipes Cook & Loomis, 1928, meaning "in highest fulfillment of feet" (Cook and Loomis 1928;Marek and Bond 2006). Six years later, Loomis described a 742-legged millipede, Siphonophora millepeda Loomis, 1934 from Tobago and remarked of an unwitting contest among millipedes for the greatest number of legs (Loomis 1934: 9): "The unconscious rivalry between milliped[e]s for the greatest number of segments becomes keener with the finding of this Tobago Island species. Several years ago another species of this order was described from California, one of the specimens having 192 segments [and 750 legs] but the others falling considerably below this number. Although 192 segments is the greatest number thus far known for a milliped [e], it is not unlikely that specimens of the present species or the California one will be found exceeding this number." In 1940, and apparently unaware of the New World taxa, Verhoeff (1940) described the 710-legged millipede Nematozonium elongatissimum Verhoeff, 1940 from Bulwer, South Africa and commented about its unprecedented number of legs (Shelley and Hoffman 2004). Eighty years later, a millipede with a record-breaking 1306 legs, Eumillipes persephone Marek, 2021, was discovered 60 meters underground in Western Australia (Marek et al. 2021). With nearly twice the number of legs of I. plenipes, E. persephone is in a separate order, the Polyzoniida, and in the family Siphonotidae (Marek et al. 2021). Currently, five millipede species are known to have more than 710 legs and possess super-elongated trunks with more than 180 diplosegments (hereafter "rings"): E. persephone (1360 legs, 330 rings), family Siphonotidae; I. plenipes (750 legs, 192 rings), Nematozonium elongatissimum (710 legs, 182 rings), family Siphonorhinidae; and S. millepeda and Siphonacme lyttoni Cook & Loomis, 1928 (742 legs, 190 rings), family Siphonophoridae.
Colobognath millipedes emerge from the egg with at least four leg pairs and incrementally add legged-segments during development, even after attaining sexual maturity, a process known as euanamorphosis (Enghoff et al. 1993;Wong et al. 2020). Although these millipedes have an average maximum ring count of ca. 60 in females, some reach [182][183][184][185][186][187][188][189][190][191][192], and in the case of E. persephone, a superlative 330 rings. Even though euanamorphosis may partly explain why these millipedes have greater than average ring counts, the reason for the discontinuity in the distribution of ring numbers in the Colobognatha remains unclear (Minelli and Edgecombe 2022). So called super-elongation (> 180 rings) has evolved at least twice in the class Diplopoda, in Siphonophorida and Polyzoniida, and these millipedes drastically exceed the leg count of any animal. With significantly more than any other species of diplopod, the great number of legs in these millipedes is linked to a lifestyle burrowing through the soil (Marek et al. 2012(Marek et al. , 2016(Marek et al. , 2021. Adaptations, such as compressible segments due to primitively unfused sclerites, coupled with an extensible and flexible body, allows the millipede to squeeze through narrow underground crevices. The continuous metachronal wave gait and action of concentric tubular rings sliding within one another provide continuous pushing force. In combination with the many legs, longitudinal and oblique muscles pull the rings together, facilitating forward locomotion (Manton 1961). This concertina-like fashion of burrowing resembles that used by earthworms and by centipedes of the order Geophilomorpha, another group of highly elongated myriapods that includes the leggiest centipedes, Gonibregmatus plurimipes Chamberlin, 1920 from Fiji with 191 segments and 382 legs, and Chomatobius bakeri (Chamberlin, 1912) from Los Angeles with 362 legs.
Many species of Siphonophorida await description, especially in the tropics and temperate regions where they often occur deep within the soil (Marek et al. 2016(Marek et al. , 2021. However, their antiquated and fragmentary classification hinders taxonomic work and the primary description of species (Read and Enghoff 2018). Detailed syntheses of the group were published by Verhoeff (1940) and Attems (1951), and later revisited by Hoffman (1980), Jeekel (2001), and Shelley (1996aShelley ( , 1996b. Current catalogs of the order are given in Shelley (1996b, New World Siphonophoridae), Enghoff et al. (2015; list of the genera of Siphonophorida), and Marek et al. (2016;Siphonorhinidae). Although family-level classification is primarily based on characters of the head, genera are mainly defined by male genitalia, specifically the gonopods (ninth and tenth leg pairs modified as sperm transfer organs). Although gonopodal characters appear useful for differentiation of closely related millipede species in a pairwise manner, higher-level groups inferred from them seem inaccurate (Means and Marek 2017). Furthermore, because the male gonopods of Siphonophorida are simple and leg-like (each composed of a primitive complement of seven podomeres) and are relatively conserved in morphology between species and across considerable geographical distances, some taxonomic groups that have been formed on the basis of overall similarity of gonopods may be artificial, overly inclusive, and polyphyletic. The inaccurate classificatory schemes of Siphonophora Brandt, 1837 and Siphonorhinus Pocock, 1894 exemplify this and have accumulated many species. For example, the genus Siphonophora encompasses ca. 80 species, mainly in the Americas, but also with species in Pakistan, Sri Lanka, Vietnam, Myanmar, Malaysia, Indonesia, Philippines, New Caledonia, New Guinea, Australia, Fiji, Solomon Islands, and New Zealand (Shelley 1996a, b;Enghoff et al. 2015). The small size of the gonopods (50 µm wide in Illacme spp.) and their gradual development, which can continuously change shape between stadia leading to adulthood, compounds an already challenging situation. Because there has never been a taxonomic synthesis of the Siphonophorida, including a molecular phylogenetic analysis as an independent data set to test morphological character hypotheses, an informative integrative framework for the placement of species remains unfinished (Marek and Bond 2006;Enghoff 2009, 2018;Marek et al. 2012Marek et al. , 2016. The definition of families by morphology of the head also may need critical reexamination. Based on a phylogeny from Marek et al. (2021), which included a sample of five species of Siphonophorida, the order is monophyletic, but the family Siphonorhinidae is paraphyletic with respect to the Siphonophoridae. These results suggest that the distinguishing characteristics of the Siphonorhinidae, which include a pear-shaped head and elbowed antennae, are shared primitive features, and the distinctive elongate beak of siphonophorids, among other distinguishing features, may have originated more recently in the diversification of the order. Specifically, Siphonorhinidae is a paraphyletic grade with respect to Siphonophoridae. Although uncovering higher level relationships of the order was not an objective of the phylogenomic analysis from Marek et al. (2021), future phylogenetic studies would benefit from an enriched taxon sampling of Siphonophorida (especially Siphonophoridae) to address questions of the systematic relationships of the group. Without informative systematic resources, and the antiquated taxonomic infrastructure of the group, progress on describing diversity in the group is hindered.
Illacme Cook & Loomis, 1928, the sole representative of the family Siphonorhinidae in the Western Hemisphere, is noticeably distinct from other genera in the order Siphonophorida. In combination with its super-elongated trunk, the taxon possesses distinct features that differentiate it from other Siphonophorida, such as its pear-shaped head and lack of a beak (contrasting with other siphonophoridan genera in the Western Hemisphere), elbowed antennae, and small basiconic sensilla in a shallow depression on the antennae. The genus differs from other siphonorhinids in its posterior gonopodal podomere 7 divided into 3-5 branches with one branch spike-like (Marek et al. 2016). Illacme is sister to the monotypic African genus Nematozonium (Shelley and Hoffman 2004;Marek et al. 2016Marek et al. , 2021. Eighty years after the description of I. plenipes, a second species, Illacme tobini Marek, Shear & Krejca, 2016, was discovered in a marble cave in Sequoia National Park, California (Marek et al. 2016). Here we describe a third species of the genus Illacme with 125 rings and 486 legs from the Los Angeles Basin of southern California.

Collections
On 2 April 2018, CL and JB discovered a small white siphonophoridan millipede in Lake Forest, California, and recorded its observation in the citizen science website iNaturalist (Ueda et al. 2019). Then, PEM and CLH collected specimens on 21 December 2018, and based their field work on the geographical coordinates and metadata associated with the original iNaturalist observation. Subsequently, PEM confirmed that the millipede was an undescribed species of Illacme in the family Siphonorhinidae based on the diagnoses in Marek et al. (2012Marek et al. ( , 2016. We selected locations to search for millipedes based on similarity to the habitats and microhabitats of I. plenipes in San Benito County, California. Specifically, we examined oak woodland habitats and microhabitats of the undersides of stones and decaying oak logs (Marek et al. 2012). The stones, detritus (bark and dead leaves), and humus layer were carefully removed, and the underlying soil was gently excavated to a depth of ca. 3 cm. Removed organic material was then replaced in a similar orientation to prevent desiccation. Collecting ethics followed the Insect Collectors' Code (Trietsch and Deans 2018). Due to the very slow locomotion of I. plenipes, its endogean lifestyle, and likeness in appearance to root hairs of plants, any object that was pale, narrow (< 1 mm wide) and longer than 20 mm was carefully examined by eye for more than 2 s to discern movement and identity. Due to their fragility and diminutive size, millipedes were collected by gently prying the middle of the body from beneath with a thin wooden twig and placing them into a 20-mL plastic snap cap vial with soil from their microhabitat to prevent desiccation and for cushioning in transport. In total, ca. 35 millipedes were encountered and 23 individuals were collected: 11 females, 9 males, and 3 juveniles. One female individual was collected from directly beneath a ca. 4-m long oak log (Quercus agrifolia Née, 1801) and the others were found beneath decaying leaves and within the matrix of ca. 3 cm of soil covered by a large pile of downed oak logs. On 18 January 2022, MCB collected ten additional specimens: four males, five females, and one sex-indeterminate juvenile. He recorded his observations on iNaturalist (iNaturalist observation: 105275476). Location data of the sites were recorded on a collection card according to Means et al. (2015). The information was included on specimen labels associated with natural history specimens and was databased in the biodiversity data management system Symbiota Collections of Arthropods Network (http://symbiota4.acis.ufl.edu/scan/portal/).

Photography
Habitat photographs were captured with an iPhone 7 camera and millipede photographs and videos were recorded with a Canon EOS 5D Mark IV digital SLR camera and a 65 mm Canon MPE lens. To record burrowing behavior, a female millipede (MPE04624) and soil from its microhabitat were placed between two pieces of plate glass and mounted vertically in front of a Canon 5D camera on a tripod and illuminated with a GoBe 700-wide LED light (Light & Motion Inc., California). The results of video analysis of movement and locomotion are described below in the species taxonomy section under the heading of 'Behavior'.

Electron microscopy
We used scanning electron microscopy (SEM) to examine morphology of I. socal sp. nov. at 114-12662× magnification. Images were acquired using a FEI Quanta 600 FEG environmental scanning electron microscope (FEI Inc., Hillsboro, Oregon). We prepared a male and female specimen for SEM. Millipedes were live-sectioned transversely into four parts (anterior, two middle trunk sections, posterior) with a flame-sterilized straight-edge razor. Specimens were then ethanol preserved (80%), air-dried, and affixed to a 12.7-mm diameter aluminum SEM stub with double-sided carbon tape (Ted Pella Inc., California), or with graphite conductive adhesive #112 (Electron Microscopy Sciences). Stubs were then plasma-coated under stable argon pressure with a 40-nm thick layer of a mixture of palladium and platinum metals in a Leica EM ACE600 High Vacuum Coater (Leica Microsystems, Wetzlar, Germany). Coated stubs were imaged using a 3.5-spot size and at 5 kV. Micrographs were captured as 16-bit 4096 × 3775-pixel grayscale images and edited in Adobe Photoshop CC and composed as figures in Illustrator CC 5 (Adobe Inc., California). The uncompressed and uncropped scanning electron micrographs of I. socal sp. nov. are deposited in the Dryad Data Repository at https://doi.org/10.5061/dryad.x95x-69pq7 under a public domain CC0 Creative Commons license.

Specimen preparation
After photography and videography, specimens were preserved and deposited in the Virginia Tech Insect Collection (VTEC, https://collection.ento.vt.edu). An approximately 1-cm length of the body trunk from four live specimens (two females and two males) was preserved in 100% ethanol and archived at -80 °C for later extraction of DNA. The remaining body tissue was then preserved in 80% ethanol. Other specimens were preserved directly in 100% ethanol and stored in 8 mL screw cap vials at -20 °C. Three specimens were preserved in 100% methanol for later chemical analysis.

DNA sequencing
From ethanol preserved tissue, genomic DNA was extracted and purified with a Qiagen DNEasy tissue kit following the manufacturer's protocol (Qiagen, Germany). Remaining tissue and genomic DNA stored in Qiagen AE buffer were deposited in the Virginia Tech Insect Collection freezer storage (https://collection. ento.vt.edu). Genomic DNA was used as a template to amplify a fragment of the cytochrome c oxidase subunit I gene (COI) using polymerase chain reaction (PCR) and the Folmer et al. (1994) primers LCO1490 (forward) and HCO2198 (reverse) according to the procedures described in Means and Marek (2017). Amplified DNA was then cleaned, concentration quantified and normalized, and sequenced at the University of Arizona Genetics Core using an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). DNA chromatograms were trimmed, bases called, and overlapping fragments made into a 612-basepair contiguous sequence in Mesquite using phred and phrap in the Chromaseq module (Ewing et al. 1998;Maddison and Maddison 2010). Genomic DNA was sequenced with whole genome sequencing using an Illumina Nextseq platform according to methods described in Marek et al. (2021). Pairwise distance was calculated by dividing the number of nucleotide differences by the total number of nucleotides compared. The diagnosis of I. socal sp. nov. included nucleotide site substitutions that are the unique states of its COI sequence identified in Mesquite using the 'With State Distinguishing Selected Taxa' tool (Maddison and Maddison 2010). The site numbers of the unique states are supplied in parentheses. The unique sites of I. socal sp. nov. are those nucleotides that differ from I. plenipes based on a pair-wise alignment. The site numbers are those from the 612 base-pair COI sequence of I. socal sp. nov. as a reference (U.S. National Center for Biotechnology Information accession numbers for I. socal sp. nov., MPE04622: COI, MT506032; WGS, SRX12626610; I. plenipes, SPC001187: COI, JX962724; WGS, SRX12626606). A corresponding fragment of COI for I. tobini was not available due to degradation of its genomic DNA (Marek et al. 2016).

Materials examined
To compare I. socal sp. nov. with the two other species in the genus Illacme, I. plenipes and I. tobini, we examined material in natural history collections from the Smithsonian Institution (USNM), Florida State Collection of Arthropods (FSCA), Virginia Museum of Natural History (VMNH), and Virginia Tech Insect Collection (VTEC). Examination of specimens at magnifications 8-100× was accomplished with a Leica M125 stereomicroscope illuminated by a Leica LED5000 spotlight illuminator (Wetzlar, Germany). The following dimensions were measured for I. socal sp. nov., (1) body length: measured from anterior margin of labrum to posterior margin of paraprocts, abbreviated BL; (2) head width, HW; (3) head length, HL; (4) interantennal socket width, ISW; (5) antennomere 6 width, AW; (6) collum width, CW; (7) metazonite width at the anterior 1/4 length of body, W1; (8) metazonite length at the anterior 1/4 length of body, L1; (9) metazonite height at the anterior 1/4 length of body, H1; (10) first apodous metazonite width, AS1; (11) anterior gonopod podomere 7 width, A7W; and (12) posterior gonopod podomere 7 width, P7W. These 12 measurements refer to those in Marek et al. (2016), and 1-10, 17, and 18 are used in Marek et al. (2012). Specimen dimensions were measured from specimens using an eyepiece ocular reticule calibrated with a stage micrometer, and from digital scanning electron and light micrographs using the segmented line measurement tool in ImageJ64 (Rasband 2011). Measurements are reported in millimeters and this unit abbreviation is hereafter omitted. The number of rings were counted and legs calculated using the formula l = ((p + a) × 4)-(a × 4)-(10), where l is the number of legs, p is the number of podous rings (each bearing two leg pairs), a is the number of apodous rings (without legs), and 10 is the number to be subtracted because the first ring (the collum) is legless and rings 2-4 (the millipede "thorax") each have one pair of legs (Marek et al. 2016). The identification and terminology of antennal sensilla followed that of Nguyen Duy-Jacquemin (1974) and Chung and Moon (2006), summarized and reviewed by Sombke and Ernst (2014). The terminology of mouthparts is from Silvestri (1903), Koch (2015), and Moritz et al. (2022).
Variation. There is negligible variation in coloration between live specimens. Female specimens are generally larger in size (greater head, ring width) and have more rings and legs. The predominant source of variation between specimens is ring and leg counts (Tables 2, 3). Females have a maximum of 125 rings (486 legs maximum) with a median of 94, and males a maximum of 104 rings (402 legs maximum) with a median of 73. The rings of I. socal sp. nov. (males and females) are uniform in length, width, and height along the trunk, but are slightly taller and more convex in posterior rings.
Ecology. Illacme socal sp. nov. individuals were encountered during the day in a California live oak woodland habitat surrounded by chaparral shrubland (Fig. 5). One female individual was found beneath a dead oak log, and the others were encountered beneath the humus layer and embedded within the underlying soil matrix (Suppl. material 8: figs S28-S31). Co-occurring dominant flora included California live-oak (Quercus agrifolia), California sagebrush (Artemisia californica), California broom (Acmispon glaber), hollyleaf redberry (Rhamnus ilicifolia), Pacific poison-oak (Toxicodendron diversilobum), and Coastal cholla (Cylindropuntia prolifera). Other invertebrates encountered included centipedes (Arenophilus iugans Chamberlin, 1944, Taiyuna isantus (Chamberlin, 1909, Oabius Chamberlin, 1916), beetles (Eleodes osculans, Apsena sp.), and arachnids (Hubbardia sp., Cicurina sp.). Between the collection of the type material by PEM in 2018 and nontype material by MCB in 2022, the Silverado fire (26 October-7 November 2020) burned ca. 12,500 acres including portions of Whiting Ranch Wilderness Park. The impact of this fire on the I. socal sp. nov. population remains unclear, but given their efficient burrowing locomotion (see behavior below), this species likely minimizes fire risk by staying in deeper soils with higher soil moisture.
Behavior. When uncovered, individuals were observed spiraling downward into the soil cavity via a corkscrew-like pattern. Filmed in the laboratory, and within the soil from its microhabitat, the burrowing locomotion of a female I. socal sp. nov. was slow (100 µm/s), undulatory, and continuous (MPE04624, Suppl. ma- terial 12: https://vimeo.com/823446011?share=copy). The sinuous locomotory movement of the millipede appeared to follow a path of low resistance and track the topography within the soil matrix. While burrowing, a single orientation was not continually maintained, and the individual repeatedly turned and continued motion several times in different planes. While passing through a narrow junction, I. socal sp. nov. appeared to squeeze through the crevice by reduction of its body height by ca. 1/2 (Suppl. material 12: https://vimeo.com/823446011?share=copy, at 34 s). After squeezing into the interstice, and through cephalic nodding and dorso-ventral arching of the trunk, the millipede forcibly enlarged the interstice. The body rings did not appear to telescope into one another while passing through narrow junctions. Moving within the soil matrix, the millipede appeared to be positively thigmotactic to contact with the soil. The millipede appeared to navigate by aid of its large antennae, and detection of interstitial voids seemed to be mediated by these appendages. The antennae moved rapidly, and the left and right antennae, each equally and continually, tapped on the soil grains. As it walked, the millipede was observed asymmetrically extending one antenna into the soil lacunae ahead as if the sole appendage was used as a probe by the blind millipede to survey ahead of itself in the confined subterranean space (Suppl. material 12: https://vimeo.com/823446011?share=copy, at 30 s). Distribution. Only known from its type locality at Whiting Ranch Wilderness Park. An observation by CL from Los Angeles County at Eaton Canyon Natural Area in Pasadena, California, appears consistent with I. socal sp. nov. in morphological features. This millipede, a juvenile and of uncertain species identity due to lack of species-diagnostic gonopods, was observed on 10 February 2021; iNaturalist, observation: 69384055.
Etymology. The species name refers to its type locality in Southern California, commonly shortened to SoCal.

Discussion
Our discovery of the millipede Illacme socal sp. nov. (family Siphonorhinidae) from the Los Angeles metropolitan area adds a third species the genus. Two species of Illacme are known from the Coast Ranges (I. socal sp. nov. and I. plenipes) and one is from the Sierra Nevada Range (I. tobini). Although members of the genus are similar in morphological features to one another, I. socal sp. nov. is 19.7% different in the cytochrome c oxidase subunit I (COI) barcode region to I. plenipes from San Benito County (California); 2-3% pairwise distance is the often-cited threshold for metazoan species differentiation (Hebert et al. 2003). The largest female of I. socal sp. nov. has 125 rings and 486 legs, fewer than the 192 rings and 750 legs of the largest I. plenipes female (Cook and Loomis 1928). The many legs and the elongated body of I. socal sp. nov. have been associated to its subterranean soil microhabitat and burrowing behavior (Marek et al. 2012(Marek et al. , 2021. Through video analysis of the locomotion of I. socal sp. nov. in a soil matrix, we found a suite of behaviors and features that appear to facilitate endogean locomotion. These include (1) a sinuous and undulatory 3-dimensional plane of movement; (2) apparent preferential locomotion through preexisting interstitial voids largely mediated by antennation; (3) compressible rings; (4) flexible trunk, achieved through both extensibility and lateral elasticity; and (4) interstice enlargement via cephalic nodding and dorso-ventral arching of the trunk. Manton (1961) analyzed the musculature and locomotion of several members of the Siphonophorida including Siphonophora portoricensis Brandt, 1837 and Siphonophora hartii (Pocock, 1895), species which have similar trunk architecture to I. socal sp. nov. with free tergites, sternites and pleurites. She highlighted the species' dorso-ventral and lateral flexibility, spiraling locomotion, and cephalic nodding behavior, and by careful anatomical dissections, inferred what skeletomuscular features are responsible for these movements (Manton 1961). This is among the first videos analyzed of a siphonophoridan millipede burrowing and moving through small spaces and crevices underground. The locomotory behaviors of I. socal sp. nov. demonstrates the ease with which highly elongated millipedes travel underground, and helps explain the discovery of the 1306-legged E. persephone 60 m below ground. The discovery of I. socal sp. nov. in the Los Angeles Basin, a region with a human population of more than 18 million, shows that future studies of underground fauna, even in well-known locations, can lead to new discoveries of undescribed animal life.
Fortunately, the known occurrences of I. socal sp. nov., which includes the juvenile specimen from Eaton Canyon, are from two parks, which provide some protection from future development. Whiting Ranch Wilderness Park is managed by OCParks and consists of 2,500 acres of riparian and oak woodland canyons, rolling grassland hills and steep slopes of coastal sage scrub and chaparral. This park is open to hikers, mountain bikers, and equestrians. Eaton Canyon Natural Area is 198 acres and is managed by Los Angeles County Parks and Recreation. Eaton Canyon is open to hiking and equestrian use. In order to ensure the viability of I. socal sp. nov. and other subterranean fauna, park managers should still prioritize active conservation measures by focusing on soil preservation. Activities that disturb the soil in any way should be strongly discouraged as such activities could extirpate these populations. Further, both of the known localities are within a few meters of existing trails; therefore, visitors should be restricted from venturing off trail.
It is highly likely that I. socal sp. nov. also occurs in nearby private land holdings as many of these areas contain suitable habitat. Unfortunately, the relentless development pressure that has overwhelmed the greater Los Angeles area for decades will continue into the foreseeable future, putting these areas at high risk for habitat degradation and destruction. Fires, including the 2020 Silverado fire at Whiting Ranch Wilderness Park, also represent an imminent threat to soil fauna as these conserved areas contain abundant fire fuels.