The Chemistry of the Defensive Secretions of Three Species of Millipedes in the Genus Brachycybe.

Millipedes have long been known to produce a diverse array of chemical defense agents that deter predation. These compounds, or their precursors, are stored in high concentration within glands (ozadenes) and are released upon disturbance. The subterclass Colobognatha contains four orders of millipedes, all of which are known to produce terpenoid alkaloids-spare the Siphonophorida that produce terpenes. Although these compounds represent some of the most structurally-intriguing millipede-derived natural products, they are the least studied class of millipede defensive secretions. Here, we describe the chemistry of millipede defensive secretions from three species of Brachycybe: Brachycybe producta, Brachycybe petasata, and Brachycybe rosea. Chemical investigations using mass spectrometry-based metabolomics, chemical synthesis, and 2D NMR led to the identification of five alkaloids, three of which are new to the literature. All identified compounds are monoterpene alkaloids with the new compounds representing indolizidine (i.e. hydrogosodesmine) and quinolizidine alkaloids (i.e. homogosodesmine and homo-hydrogosodesmine). The chemical diversity of these compounds tracks the known species phylogeny of this genus, rather than the geographical proximity of the species. The indolizidines and quinolizidines are produced by non-sympatric sister species, B. producta and B. petasata, while deoxybuzonamine is produced by another set of non-sympatric sister species, B. rosea and Brachycybe lecontii. The fidelity between the chemical diversity and phylogeny strongly suggests that millipedes generate these complex defensive agents de novo and begins to provide insights into the evolution of their biochemical pathways.

De novo formation of an aggregation pheromone precursor by an isoprenyl diphosphate synthase-related terpene synthase in the harlequin bug.

Insects use a diverse array of specialized terpene metabolites as pheromones in intraspecific interactions. In contrast to plants and microbes, which employ enzymes called terpene synthases (TPSs) to synthesize terpene metabolites, limited information from few species is available about the enzymatic mechanisms underlying terpene pheromone biosynthesis in insects. Several stink bugs (Hemiptera: Pentatomidae), among them severe agricultural pests, release 15-carbon sesquiterpenes with a bisabolene skeleton as sex or aggregation pheromones. The harlequin bug, Murgantia histrionica, a specialist pest of crucifers, uses two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol as a male-released aggregation pheromone called murgantiol. We show that MhTPS (MhIDS-1), an enzyme unrelated to plant and microbial TPSs but with similarity to trans-isoprenyl diphosphate synthases (IDS) of the core terpene biosynthetic pathway, catalyzes the formation of (1S,6S,7R)-1,10-bisaboladien-1-ol (sesquipiperitol) as a terpene intermediate in murgantiol biosynthesis. Sesquipiperitol, a so-far-unknown compound in animals, also occurs in plants, indicating convergent evolution in the biosynthesis of this sesquiterpene. RNAi-mediated knockdown of MhTPS mRNA confirmed the role of MhTPS in murgantiol biosynthesis. MhTPS expression is highly specific to tissues lining the cuticle of the abdominal sternites of mature males. Phylogenetic analysis suggests that MhTPS is derived from a trans-IDS progenitor and diverged from bona fide trans-IDS proteins including MhIDS-2, which functions as an (E,E)-farnesyl diphosphate (FPP) synthase. Structure-guided mutagenesis revealed several residues critical to MhTPS and MhFPPS activity. The emergence of an IDS-like protein with TPS activity in M. histrionica demonstrates that de novo terpene biosynthesis evolved in the Hemiptera in an adaptation for intraspecific communication.

Cache Valley Virus in Aedes japonicus japonicus Mosquitoes, Appalachian Region, United States.

We detected Cache Valley virus in Aedes japonicus, a widely distributed invasive mosquito species, in an Appalachian forest in the United States. The forest contained abundant white-tailed deer, a major host of the mosquito and virus. Vector competence trials indicated that Ae. j. japonicus mosquitoes can transmit this virus in this region.

Discovery of a glowing millipede in California and the gradual evolution of bioluminescence in Diplopoda.

The rediscovery of the Californian millipede Xystocheir bistipita surprisingly reveals that the species is bioluminescent. Using molecular phylogenetics, we show that X. bistipita is the evolutionary sister group of Motyxia, the only genus of New World bioluminescent millipedes. We demonstrate that bioluminescence originated in the group's most recent common ancestor and evolved by gradual, directional change through diversification. Because bioluminescence in Motyxia has been experimentally demonstrated to be aposematic, forewarning of the animal's cyanide-based toxins, these results are contrary to aposematic theory and empirical evidence that a warning pattern cannot evolve gradually in unpalatable prey. However, gradual evolution of a warning pattern is plausible if faint light emission served another function and was co-opted as an aposematic signal later in the diversification of the genus. Luminescence in Motyxia stem-group taxa may have initially evolved to cope with reactive oxygen stress triggered by a hot, dry environment and was repurposed for aposematism by high-elevation crown-group taxa colonizing new habitats with varying levels of predation. The discovery of bioluminescence in X. bistipita and its pivotal phylogenetic location provides insight into the independent and repeated evolution of bioluminescence across the tree of life.

La Crosse Virus in Aedes japonicus japonicus mosquitoes in the Appalachian Region, United States.

La Crosse virus (LACV), a leading cause of arboviral encephalitis in children in the United States, is emerging in Appalachia. For local arboviral surveillance, mosquitoes were tested. LACV RNA was detected and isolated from Aedes japonicus mosquitoes. These invasive mosquitoes may significantly affect LACV range expansion and dynamics.

The millipede family Striariidae Bollman, 1893. VIII. Three new genera and four new species of minute millipedes from Oregon and Washington, USA(Diplopoda, Chordeumatida, Striarioidea).

We describe three new genera and four new species of small, litter-dwelling millipedes from the states of Oregon and Washington, USA: Miniaria ramifera, n. gen., n. sp., Miniaria richarti, n. gen., n. sp., Tigraria oregonensis, n. gen., n. sp., and Kingaria prattensis, n. gen., n. sp. Some of the unusual characters of these species are discussed, including a new type of sensory array on the third tarsus of males and a newly observed mandibular gland.

Reconsidering tympanal-acoustic interactions leads to an improved model of auditory acuity in a parasitoid fly.

Although most binaural organisms locate sound sources using neurological structures to amplify the sounds they hear, some animals use mechanically coupled hearing organs instead. One of these animals, the parasitoid flyOrmia ochracea(O. ochracea), has astoundingly accurate sound localization abilities. It can locate objects in the azimuthal plane with a precision of 2°, equal to that of humans, despite an intertympanal distance of only 0.5 mm, which is less than1/100th of the wavelength of the sound emitted by the crickets that it parasitizes.O. ochraceaaccomplishes this feat via mechanically coupled tympana that interact with incoming acoustic pressure waves to amplify differences in the signals received at the two ears. In 1995, Mileset aldeveloped a model of hearing mechanics inO. ochraceathat represents the tympana as flat, front-facing prosternal membranes, though they lie on a convex surface at an angle from the flies' frontal and transverse planes. The model works well for incoming sound angles less than±30∘but suffers from reduced accuracy (up to 60% error) at higher angles compared to response data acquired fromO. ochraceaspecimens. Despite this limitation, it has been the basis for bio-inspired microphone designs for decades. Here, we present critical improvements to this classic hearing model based on information from three-dimensional reconstructions ofO. ochracea's tympanal organ. We identified the orientation of the tympana with respect to a frontal plane and the azimuthal angle segment between the tympana as morphological features essential to the flies' auditory acuity, and hypothesized a differentiated mechanical response to incoming sound on the ipsi- and contralateral sides that depend on these features. We incorporated spatially-varying model coefficients representing this asymmetric response, making a new quasi-two-dimensional (q2D) model. The q2D model has high accuracy (average errors of under 10%) for all incoming sound angles. This improved biomechanical model may inform the design of new microscale directional microphones and other small-scale acoustic sensor systems.

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

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.

Biodiversity hotspots: rediscovery of the world's leggiest animal.

The millipede species Illacme plenipes comes the closest to having its namesake's mythical 1,000 legs--individuals can bear up to 750 legs. Here we record the rediscovery of this extremely rare species, which has not been reported since its original description some 80 years ago, at a tiny locality of 0.8 km2 in San Benito County, California. Because of the rarity and narrow geographical range of this delicate species, its fragile habitat must be protected at all costs.

A Müllerian mimicry ring in Appalachian millipedes.

Few biological phenomena provide such an elegant and straightforward example of evolution by natural selection as color mimicry among unrelated organisms. By mimicking the appearance of a heavily defended aposematic species, members of a second species gain protection from predators and, potentially, enhanced fitness. Mimicking a preexisting warning advertisement is economical because a potentially costly novel one can be avoided; simultaneously, the addition of more aposematic individuals enhances the overall warning effect. The better-known mimetic systems comprise tropical taxa, but here, we show a remarkable example of color mimicry in 7 species of blind, cyanide-generating millipedes endemic to the Appalachian Mountains of temperate North America. Because these millipedes lack eyes, there is no sexual selection or intraspecific signaling for coloration, providing an ideal system for mimicry studies. We document a Müllerian symbiosis where unrelated species vary in color and pattern over geographical space but appear identical where they co-occur. By using spectral color data, estimations of evolutionary history, and detailed field observations of species abundance, we test 4 predictions of Müllerian mimicry theory and begin to unravel the story of an elaborate mimetic diversification in the forests of Appalachia.

The millipede family Striariidae Bollman, 1893. IV. Amplaria oedipus, n. sp., with a secondary sexual modification of males unique among millipedes (Diplopoda, Chordeumatida, Striarioidea).

A new species, Amplaria oedipus, is described from Oregon, USA. Males of the new species have greatly inflated pyriform tarsi on the 5th and 6th legpairs, which is a secondary sexual modification previously not known from any other millipede.

The milliped family Striariidae Bollman, 1893. V. Stegostriaria dulcidormus, n. gen., n. sp., Kentrostriaria ohara, n. gen., n. sp., and the convergent evolution of exaggerated metazonital crests (Diplopoda, Chordeumatida, Striarioidea).

Two new genera and species, Stegostriaria dulcidormus (Trisariinae) and Kentrostriaria ohara (Striariinae), are described from Oregon and Idaho, respectively. The new species are distinct from any other striariids, and indeed from any other chordeumatidan millipedes, in having the second dorsal crests greatly enlarged, giving them a fanciful resemblance to stegosaurid dinosaurs. In spite of this similarity, the two species are so distinct they cannot be accommodated in the same genus or the same subfamily. The exaggerated metazonital crests are therefore attributed to convergent evolution. We present a key to the genera of the family Striariidae.

A revision of the wilsoni species group in the millipede genus Nannaria Chamberlin, 1918 (Diplopoda, Polydesmida, Xystodesmidae).

Although many new species of the millipede genus Nannaria Chamberlin, 1918 have been known from museum collections for over half a century, a systematic revision has not been undertaken until recently. There are two species groups in the genus: the minor species group and the wilsoni species group. In this study, the wilsoni species group was investigated. Specimens were collected from throughout its distribution in the Appalachian Mountains of the eastern United States and used for a multi-gene molecular phylogeny. The phylogenetic tree recovered Nannaria and the two species groups as monophyletic, with Oenomaeapulchella as its sister group. Seventeen new species were described, bringing the composition of the wilsoni species group to 24 species, more than tripling its known diversity, and increasing the total number of described Nannaria species to 78. The genus now has the greatest number of species in the family Xystodesmidae. Museum holdings of Nannaria were catalogued, and a total of 1,835 records used to produce a distribution map of the species group. Live photographs, illustrations of diagnostic characters, ecological notes, and conservation statuses are given. The wilsoni species group is restricted to the Appalachian region, unlike the widely-distributed minor species group (known throughout eastern North America), and has a distinct gap in its distribution in northeastern Tennessee and adjacent northwestern North Carolina. The wilsoni species group seems to be adapted to mesic microhabitats in middle to high elevation forests in eastern North America. New species are expected to be discovered in the southern Appalachian Mountains.

Myriapods.

Paul Marek and William Shear introduce the arthropod sub-phylum myriapoda, which includes centipedes, millipedes and other multi-legged animals.

Phylogenetic review of the millipede genus Cherokia Chamberlin, 1949 (Polydesmida, Xystodesmidae).

The millipede genus Cherokia Chamberlin, 1949 is a monospecific taxon, with the type species Cherokiageorgiana (Bollman, 1889). The last revision of the genus was made by Hoffman (1960) where he established three subspecies. Here we used molecular phylogenetics to assess the genus and evaluate whether it is a monophyletic group, and if the subspecies are each monophyletic. We included material from literature records and three natural history collections. Newly collected samples were obtained through a citizen science project. Morphological characters underlying subspecies groups-the shape of the paranota, body size, and coloration-were evaluated. A molecular phylogeny of the genus was estimated based on DNA sequences for seven gene loci, and a species delimitation analysis was used to evaluate the status of the subspecies. The documented geographical range of Cherokia in the United States was expanded to include a newly reported state record (Virginia) and about 160 new localities compared to the previously known range. Morphological characters, which included the shape of the paranota and body size that had been historically used to establish subspecies, showed clinal variation with a direct relationship with geographical distribution and elevation, but not with phylogeny. Coloration was highly variable and did not accord with geography or phylogeny. The phylogeny recovered Cherokia as a monophyletic lineage, and the species delimitation test supported the existence of a single species. The subspecies Cherokiageorgianaducilla (Chamberlin, 1939) and Cherokiageorgianalatassa Hoffman, 1960 have been synonymized with Cherokiageorgiana. The molecular and morphological evidence showed that Cherokia is a monospecific genus with the sole species, Cherokiageorgiana, being geographically widespread and highly variable in its morphology.

The millipede family Striariidae Bollman, 1893. VI. Six new genera and thirteen new species from western North America (Diplopoda, Chordeumatida, Striarioidea).

The following new genera and species of Striariidae are described from the states of California, Oregon and Washington: Nototrisaria ornata n. gen., n. sp., Maraplia napa n. gen., n. sp., Maraplia chico n. gen., n. sp., Maraplia schusteri n. gen., n. sp., Lamparia curryensis n. gen., n. sp., Lamparia bentonensis n. gen., n. sp., Lamparia pratensis n. gen., n. sp., Lamparia millicoma n. gen., n. sp., Rampalia cheathamensis n. gen., n. sp., Plaramia arcata n. gen., n. sp., Plaramia johnsonae n. gen., n. sp., Ralampia complexa n. gen., n. sp., and Ralampia filamentosa n. gen., n. sp.. The new species are all very small millipedes, 5.5 mm or less in length and with two or three ommatidia on each side of the head or blind. The majority have the sixth metazonital crest larger than the other crests and extended horizontally, so that the animals have the general appearance of having paranota. A new key to striariid genera is presented.

The millipede family Striariidae Bollman, 1893. VII. Petra sierwaldae, n. gen., n. sp., a minute millipede from Idaho, USA (Diplopoda, Chordeumatida, Striariidae).

Petra sierwaldae, n. gen., n. sp. is described from males and females collected at four localities in Kootenai County, Idaho, USA. The new genus is defined by its unique gonopod and ninth leg anatomy, a notched collum and single ommatidium on each side of the head, and lacking third coxal flasks as well as having modified fifth coxae.

Three new genera and eighteen new species of miniature polydesmid millipedes from the northwestern United States (Diplopoda, Polydesmida, Polydesmidae).

Three new genera, Retrorsioides, Rowlandesmus and Benedictesmus, are described and the polydesmid millipede fauna of North America is briefly reviewed with an emphasis on the genera Bidentogon Buckett Gardner, 1968 and Retrorsia Shelley, 2003. Eighteen new species are described: Bidentogon buttensis, Bidentogon norcal, Retrorsia leonardi, Retrorsia benedictae, Retrorsia richarti, Retrorsia gracilis, Retrorsia simplicissima, Retrorsioides castellum, Retrorsioides linnensis, Retrorsioides kittitas, Retrorsioides bammerti, Retrorsioides arboramagna, Rowlandesmus millicoma, Rowlandesmus dentogonopus, Benedictesmus aureua, Benedictesmus ellenae, Benedictesmus yaquina and Benedictesmus timber. Natural history notes and illustrations are provided of putative commensal fungi, nematodes and a mite found on the millipede specimens.

A revision of the minor species group in the millipede genus Nannaria Chamberlin, 1918 (Diplopoda, Polydesmida, Xystodesmidae).

Millipedes in the family Xystodesmidae (Polydesmida) are often referred to as "colorful, flat-backed millipedes" for their bright aposematic coloration and tendency to form Müllerian mimicry rings in the Appalachian region. However, there are many species of Xystodesmidae that do not display colorful warning patterns, and instead have more cryptic appearances. Perhaps for this reason, groups such as the genus Nannaria have remained understudied, despite containing a large number of undescribed species. Before his death in 2012, R. L. Hoffman worked on a revision of the genus Nannaria, and synthesized material and drawings since 1949. Here the work is continued, inferring a molecular phylogeny of the Nannariini (Nannaria + Oenomaea pulchella), and revealing two clades within the genus. One clade is named the minor species group, and the second is the wilsoni species group. This revision, using a molecular phylogenetic framework, is the basis for descriptions of 35 new species in the minor species group. A multi-gene molecular phylogeny is used to make taxonomic changes in the taxon. Eleven putative species of Nannaria are also illustrated and discussed. Additionally, detailed collection, natural history and habitat notes, distribution maps, and a key to species of the Nannaria minor species group are provided. These items are synthesized as a basis for a revision of the genus, which hopefully will aid conservation and evolutionary investigations of this cryptic and understudied group.

The first true millipede-1306 legs long.

The name "millipede" translates to a thousand feet (from mille "thousand" and pes "foot"). However, no millipede has ever been described with more than 750 legs. We discovered a new record-setting species of millipede with 1,306 legs, Eumillipes persephone, from Western Australia. This diminutive animal (0.95 mm wide, 95.7 mm long) has 330 segments, a cone-shaped head with enormous antennae, and a beak for feeding. A distant relative of the previous record holder, Illacme plenipes from California, it belongs to a different order, the Polyzoniida. Discovered 60 m below ground in a drill hole created for mineral exploration, E. persephone possesses troglomorphic features; it lacks eyes and pigmentation, and it has a greatly elongated body-features that stand in stark contrast to its closest surface-dwelling relatives in Australia and all other members of its order. Using phylogenomics, we found that super-elongation (> 180 segments) evolved repeatedly in the millipede class Diplopoda. The striking morphological similarity between E. persephone and I. plenipes is a result of convergent evolution, probably for locomotion in similar soil habitats. Discovered in the resource-rich Goldfields-Esperance region and threatened by encroaching surface mining, documentation of this species and conservation of its habitat are of critical importance.