Phylogenetic Systematics of the Millipede Family Xystodesmidae

Abstract The millipede family Xystodesmidae includes 486 species distributed primarily in temperate deciduous forests in North America and East Asia. Species diversity of the family is greatest in the Appalachian Mountains of the eastern United States, with 188 species. Although the group includes notable taxa such as those that are bioluminescent and others that display Müllerian mimicry, producing up to 600 mg of cyanide, basic alpha-taxonomy of the group is woefully incomplete and more than 50 species remain undescribed in the Appalachian Mountains alone. In order to establish a robust phylogenetic foundation for addressing compelling evolutionary questions and describing species diversity, we assembled the largest species phylogeny (in terms of species sampling) to date in the Diplopoda. We sampled 49 genera (out of 57) and 247 of the species in the family Xystodesmidae, recollecting fresh material from historical type localities and discovering new species in unexplored regions. Here, we present a phylogeny of the family using six genes (four mitochondrial and two nuclear) and include pivotal taxa omitted from previous studies including Nannaria, Erdelyia, taxa from East Asia, and 10 new species. We show that 6 of the 11 tribes are monophyletic, and that the family is paraphyletic with respect to the Euryuridae and Eurymerodesmidae. Prior supraspecific classification is in part inconsistent with the phylogeny and convergent evolution has caused artificial genera to be proposed. Subspecific classification is likewise incongruent with phylogeny and subspecies are consistently not sister to conspecifics. The phylogeny is used as a basis to update the classification of the family, diagnose monophyletic groups, and to inform species hypotheses.

Millipedes are an ancient terrestrial lineage with members from the Early Devonian (ca. 414 Ma) that breathed atmospheric oxygen (Wilson andAnderson 2004, Suarez et al. 2017) and later forms that grew to gargantuan body proportions (2 × 0.5 m) sustained by elevated atmospheric oxygen content in the Carboniferous (Lucas et al. 2005). Divergence between Diplopoda and its sister group Pauropoda (Dignatha) has been estimated using molecular dating to have occurred during the Ordovician (Fernandez et al. 2018, but see Szucsich et al. 2020). Although there are many Paleozoic diplopod fossils, none have been assigned to any of the 16 extant orders. Wilson (2006) suggested that diversification accelerated during the Early and mid-Silurian and superordinal stem groups were established by the beginning of the Devonian. Fossil records during the Mesozoic are generally scarce, except for many recently discovered Cretaceous (ca. 100 Mya, Burmese) amber fossils (Wesener and Moritz 2018). Thirteen of the 16 extant orders of Diplopoda are represented by amber fossils in Cretaceous (Wesener and Moritz 2018). There are 830 species of Pauropoda and 12,000 species of Diplopoda-200 species of Symphyla and 3,500 species of Chilopoda; although what major factors led to the greater diversity of millipedes is unclear, traits implicated in the radiation include diplosegmentation and metachronal gait (thereby fostering burrowing) and chemical defenses (thus deterring predation). Notably, Geophilomorpha, a centipede order that has independently gained the ability to burrow (through the evolutionary trend of trunk elongation and leg addition) and produce chemical defenses, is the most species rich non-diplopod myriapod group. As detritivores, millipedes feed on decaying plant material, thereby fragmenting detritus into smaller pieces fostering later colonization by bacteria and fungi, and some have estimated that through fragmentation that certain species [Narceus americanus (Palisot de Beauvois, 1817), Spirobolida, Spirobolidae], contribute about two tons of frass per acre to deciduous forests yearly (Coville 1913). This input aerates and conserves the soil, and the process releases nitrogen, carbon, simple sugars, and other nutrients back into the biosphere (Joly et al. 2020). Unfortunately, non-native earthworms-released from predation and parasites from their native Version of Record, first published online March 1, 2021 with fixed content and layout in compliance with Art. 8.1.3.2 ICZN.
Insect Systematics and Diversity, (2021) 5(2): 1; 1-26 doi: 10.1093/isd/ixab003 Research ranges-have been shown to compete with native millipedes for this seemingly limitless detrital resource (Snyder et al. 2009). Millipedes' lack of wings, absence of phoresy, and low dispersal capability fosters narrow endemism, and many individual species are known as short-range endemics (SRE), distributed in areas less than 10,000 km 2 (Harvey 2002, Harvey et al. 2011. Their narrowly restricted distributions, e.g., the Laurel Creek millipede Apheloria whiteheadi (Shelley, 1986) with a global range of ca. 1 km 2 , exemplify that they are irreplaceable biodiversity, which is ultra-susceptible to global extinction due to habitat loss, and highlights the importance of prioritizing millipede and other SRE invertebrates in schemes seeking to maximize species diversity. Despite their antiquity and important role as detritivores, known millipede species diversity tremendously lags behind estimated global diversity (Brewer et al. 2012). For example, there are more than 50 undescribed species of the twisted-claw millipede (Nannaria spp., Polydesmida, Xystodesmidae) in the eastern United States alone, and globally an additional 3,000-80,000 species of Diplopoda when tropical locales and other poorly sampled regions are included (Hoffman 1980, Brewer et al. 2012). The lack of basic alpha-taxonomic information on the twisted-claw millipedes is surprising given their large body size (about 2 cm in length) and ubiquity east of the Mississippi River; one new species was even discovered within the bounds of the D.C. Metropolitan area. The group exhibits fascinating biological characteristics. The millipede family Xystodesmidae includes notable taxa such as bioluminescent species in California, Motyxia spp. (Marek et al. 2011); Müllerian mimics in Appalachia, apheloriine spp. (Marek and Bond 2009); the train millipede in Japan, Parafontaria laminata (Attems, 1909), whose aggregations of 311 individuals m -2 have obstructed trains (Hashimoto et al. 2004); the giant 8-cm long armadillo millipede in Mexico, Rhysodesmus dasypus (Hoffman 1970); and the cherry millipede, Apheloria virginiensis corrugata (Wood, 1864), able to generate hydrogen cyanide in an amount 18 times that necessary to be lethal to a pigeon-size bird (Eisner et al. 2005).

Taxonomic History
The family Xystodesmidae Cook, 1895 was first established for several large-bodied members of the order Polydesmida Pocock, 1887 (Fig. 1). In his pioneering yet insightful classification of Diplopoda, Cook (1895) assigned millipede genera to 49 families and remarked that it was made 'with some confidence' in all groups, that is except for those of the Polydesmida. This work did not specify characters that united polydesmidan genera into their respective families. Later, Cook (1904) provided a diagnosis of the family Xystodesmidae and differentiated its members from other polydesmidan families by the presence of spines on the prefemur, bisinuately curved tarsal claws, and the dorsal habitus (including wide contiguous paranota that impart a compact appearance). Subsequently, the family was included as part of Chelodesmidae Cook, 1895 by Pocock (1910) and Brölemann (1916). These authors' conception of the family, and later Attems's (1926), was broad and inclusive and encompassed an assemblage of heterogeneous taxa, many of which are now in other families. Later, the taxon was more narrowly diagnosed and set apart from Chelodesmidae and other large-bodied members of the suborder Leptodesmidea Brölemann, 1916 by the presence of prefemoral spines and the shape of the body and antenna (see Marek et al. 2014 for a detailed summary of taxonomic history).
The central and eastern U.S. families, Eurymerodesmidae Causey, 1951 andEuryuridae Pocock, 1909, have been regarded as close relatives of the Xystodesmidae for more than 40 yr (Hoffman 1978a(Hoffman , 1990(Hoffman , 1998Shelley 1989). The family Eurymerodesmidae (Fig. 1E) was considered as a 'smaller, apparently derivative group' of Xystodesmidae by Hoffman (1978a, p. 24) and the two families 'as sister taxa within the Xystodesmoidea' by Shelley (1989, p. 102). Although Euryuridae (Fig. 1F) was traditionally suggested to be closely allied to the Platyrhacidae and Aphelidesmidae due to a flattened, broad epiproct (Hoffman 1954(Hoffman , 1980, Hoffman (1998), upon reexamination of 14 characters, considered the family as closer to Xystodesmidae, in particular close to the xystodesmid subfamily Melaphinae Hoffman, 1980. However, these two taxa were retained as distinct families due to several characters including the ventral mandibular ridge in Eurymerodesmidae and the flattened, broad epiproct and simple gonopods in the Euryuridae (Shelley 1989, Hoffman 1998. A recent study then subsumed the families Eurymerodesmidae and Euryuridae under Xystodesmidae and removed Macellolophus rubromarginatus (Lucas, 1846) from Melaphinae, placing it in the family Chelodesmidae Cook, 1895. These higher-level changes were justified solely on similarity of male genitalic morphology (Shelley and Smith 2018). Nonetheless, as still remains the case with most taxa of the order Polydesmida since Cook 126 yr ago, monophyly of the family is uncertain, inter-and intrafamilial relationships are poorly known, and rationale for systematic relationships is based on century-old character argumentation using overall similarity of a handful of morphological features.

Molecular Phylogenetics
Based on an ordinal-level phylogeny of the Diplopoda inferred using amino acid data sequenced from transcriptomes of a representative set of familial exemplars, Xystodesmidae is monophyletic and sister to Eurymerodesmidae, which as a clade is in turn closely related to chelodesmid, sphaeriodesmid, and paradoxosomatid taxa (Rodriguez et al. 2018). Rodriguez et al. (2018) showed that several families of Polydesmida are not monophyletic (including Chelodesmidae and Paradoxosomatidae Daday, 1889) and challenged long-held morphological-based hypotheses, including Polydesmida sister to Nematophora [i.e., (Chordeumatida, (Stemmiulida, Callipodida) as in Enghoff 1984;Blanke and Wesener 2014]. Intrafamilial phylogenetic systematic relationships of the family have been studied in separate analyses of the eastern and western U.S. xystodesmid taxa Bond 2006, 2007;Marek et al. 2011Marek et al. , 2015Means and Marek 2017). However, there has never been a phylogenetic systematic analysis of the group with species from throughout the familial geographical distribution, and with other ostensibly closely related large-bodied taxa in the Leptodesmidea as outgroups to test the monophyly of the family.
To infer a phylogeny of the Xystodesmidae, we sampled 247 species of the family (the largest dataset analyzed in the Diplopoda to date) including taxa spanning the geographical distribution of the family, and utilized six genes. The phylogeny is used as a basis to update the classification of the family, diagnose monophyletic groups, and to describe ten new species.

Fieldwork
Millipedes were collected in the field from 2014 to 2019 and brought back alive to the laboratory for DNA preservation and specimen preparation according to the methods described in Means et al. Insect Systematics andDiversity, 2021, Vol. 5, No. 2 (2015). For each species, the unique color morphs at a locality were photographed with a Canon EOS 6D digital SLR camera, MT-24EX Macro Twin Lite Flash, and a 50 mm lens; a MP-E 65 mm lens was used for individuals < 20 mm (Canon Inc., Japan).

Taxon Sampling
Based on species occurrence records from revisionary monographs and other taxonomic literature, we collected material from the field, prioritizing collections from type localities and from within the known species ranges. We targeted every genus in the family, and searched type localities when feasible or nearby localities. Eurymerodesmidae and Euryuridae were recently placed in the Xystodesmidae by Shelley and Smith (2018), thus we included in our sampling four and six of their species. Outgroups were selected in the superfamily Xystodesmoidea Cook, 1895, and other presumed close relatives in the suborder Leptodesmidea. The following taxa were selected as outgroups-Xystodesmoidea: Orodesminus Attems, 1929(Oxydesmidae Cook, 1895

DNA Extraction, Amplification, and Sequencing
Left legs from body rings 8-18 were removed with flame-sterilized forceps, immersed in RNAlater (Qiagen) or 100% ethanol, and archived at −80°C in the freezer collection of the Virginia Tech Insect Collection (VTEC, collection.ento.vt.edu) for later DNA extraction. About 50% of the taxa sampled were collected (2014-2019) specifically for this study. Their frozen tissues, archived in the VTEC freezer collection, were DNA-extracted and sequenced for additional genes. We amplified and sequenced six genes (four mitochondrial and two nuclear gene fragments): small subunit RNA (12S), tRNA-Valine, large subunit RNA (16S), cytochrome c oxidase subunit I (COI), elongation factor alpha (EF1α), and large subunit RNA (28S). Amplification of DNA was carried out as described in Means and Marek (2017). DNA amplifications were cleaned, concentrations quantified and normalized, and Sanger-sequenced using an ABI 3730 capillary sequencer (Applied Biosystems).
Previous attempts to sequence the 12S and 16S DNA from representatives of the genus Nannaria repeatedly failed, so transcriptomes of two species of Nannaria collected from Virginia Tech's campus were sequenced to develop primers: Nannaria ericacea Hoffman, 1949; and Nannaria hokie n. sp. Live specimens were frozen with liquid nitrogen, pulverized, and whole-animal RNA was extracted from the tissues using a RNeasy kit (Qiagen). A cDNA library was made using reverse transcriptase PCR and sequenced using a Kappa prep and Illumina RNA-seq with 200-bp paired-end read sequencing. Raw DNA reads were assembled into transcripts using Trinity RNA-Seq de novo transcriptome assembly (Grabherr et al. 2011). The ribosomal genes were identified in the assembled Nannaria transcriptome by comparison with the mitochondrial genome of Appalachioria falcifera (Keeton, 1959) (Swafford and Bond 2010). We used a local nucleotide BLAST search in Geneious to identify the 12S and 16S transcripts in the Nannaria transcriptome (Altschul et al. 1990). Primers were developed spanning the entire 12S-16S region (including the intervening tRNA-Valine), and as a primer pair within the 16S ribosomal gene region. We used phred and phrap in the Mesquite module Chromaseq for nucleotide basecalling, trimming, and quality control of Sanger sequences (Ewing et al. 1998, Maddison andMaddison 2010).

Phylogenetic Analyses
We used prank for multiple sequence alignment beginning the analysis with a neighbor-joining guide tree that was later refined after the preliminary alignment step (Löytynoja and Goldman 2005). Aligned gene sequences were partitioned by gene, codon position, and intron/exon locations in Mesquite. Partitions were assessed in PartitionFinder to determine a best-fit partitioning plan and to identify appropriate models of nucleotide evolution (Lanfear et al. 2012). Using the best-fit partitioning scheme and the aligned sequences in nexus format, we used MrBayes 3.2.5 to estimate phylogeny (Ronquist et al. 2012). Individual gene trees for the six genes were analyzed independently in MrBayes to compare alternative genealogical histories and compare varying gene tree resolution. In addition, a concatenated dataset with all of the genes and best-fit partitioning scheme was analyzed in MrBayes. Phylogenetic analyses were run on Virginia Tech's 173-node supercomputer, NewRiver, with 16 MCMC chains running in parallel on separate processors. A tree and other parameters were sampled every 100 of 30 million generations. Chains were monitored for convergence using the average standard deviation of split frequencies and a threshold of 0.01.

Species Discovery and Delimitation
Male specimens collected in the field were compared to type specimens (and other material in natural history collections [NHCs]) and literature for identification to species. We refrained from describing new species and making changes to the classification of the genera Apheloria, Cherokia, Rhysodesmus, and Riukiaria because revisions of these taxon are currently underway. We used morphological and molecular phylogenetic distinctness (diagnosability) to delimit species as follows. 1) We first used male gonopod morphology, and if a specimen's gonopod morphology matched the description of a known species then the specimen was ascribed to that species. However, if the specimen's gonopod morphology did not match the known species and was distinct then we established a preliminary new species, H morph=yes (e.g., Appalachioria n. sp. 'Clinch Mountain Bond 2006, 2007;Means and Marek 2017). At this moment, we hypothesized what the closest relative of the conditional new species would be. New species represented by a single male (or female) specimen remained preliminary new species (H morph=yes ) and were not described until additional material can be collected to rule out the possibility of an aberrant form of a known species. 2) A single male exemplar of a preliminary new species (H morph=yes ) from above was sampled for the molecular phylogeny to assess phylogenetic distinctness, H phy=yes/no? . If the preliminary new species was not sister to its closest relative (hypothesized previously) in the concatenated molecular phylogeny (H morph=yes , H phy=yes ), or if it was sister to its closest relative but had overt morphological genitalic differences then we established a new species and described it here (H morph=yes , H phy=no ). However, if the preliminary new species was sister to its closest relative but had slight morphological genitalic differences, when re-assessed within the phylogenetic context, then we did not describe it (H morph=no , H phy=no ). We were optimistic that genitalic (gonopodal) differences track species boundaries. The lock-and-key theory for genitalic variation has been demonstrated in the group previously Sota 2008, Wojcieszek andSimmons 2012). Therefore, we are confident in using this species delimitation method implementing primarily male gonopod morphology because shape difference between male and female genitalia prevent nonspecific matings and imparts strong reproductive isolation. In addition, a diversity of species remains undescribed (ca. 56 in Nannaria) and we are highly motivated to document new taxa because many of their habitats are directly threatened by habitat loss, climate change, and other processes that cause land conversion and habitat unsuitability.

Biogeographical Analysis
To infer ancestral ranges of the taxa, we plotted their observed biogeographical ranges on the phylogeny using the Dispersal-Extinction-Cladogenesis (DEC) model in BioGeoBEARS (Matzke 2013(Matzke , 2014. Biogeographical ranges of the taxa were recorded based on the terrestrial ecoregion in which they are found. Terrestrial ecoregions were according to Olson et al. (2001). A chronogram was estimated with BEAST using a normal prior on the date of the root with a mean of 1 to generate relative dates (Bouckaert et al. 2014(Bouckaert et al. , 2019. Outgroup taxa historically not in the family Xystodesmidae were pruned from the tree prior to analysis in BioGeoBEARS. We used relative dates because there are no fossils of the family Xystodesmidae for calibration points, spare a single equivocal member of the family preserved in Miocene Chiapas amber (Riquelme and Hernández-Patricio 2018). Due to the limited dispersal capabilities of xystodesmid millipedes, species exclusively inhabit single ecoregions except for a few widespread taxa (Means and Marek 2017). As a result, the DEC+J model, which adds the parameter j for founder-event speciation, was hypothesized to be more likely over the DEC model (Matzke 2014). To test this hypothesis, we compared the likelihoods of the alternative models using a likelihood ratio test in Microsoft Excel. We visualized the phylogeny of Xystodesmidae on a world map and plotted the geographical coordinates of the terminal taxa in a tanglegram using the R (version 3.2.2) program phytools (version 0.7-20) (Revell 2012, R Core Team 2016.

Taxonomy
Material in NHCs was used to describe species. Specimens were examined for somatic and genitalic characters using a Leica M125 stereomicroscope. Male gonopods were dissected and photographed as above, but with two 350 nm ultraviolet flashes operated from a Visionary Digital Passport II focal-stacking photography system with Helicon Focus according to fluorescent photography techniques outlined in Marek (2017). The higher-level classification of Xystodesmidae was modified to reflect the phylogeny. Taxon groups were selected to be equally divergent, mutually exclusive, and statistically well supported. The classification system is informative, in that it provides a hierarchical guide to family diversity, and extensible in that it provides a framework to name new species. Changes to the higher-level classification of Xystodesmidae were made according to the principle that monophyletic and some paraphyletic groups receive formal names. A paraphyletic taxon retained its preexisting name when the group was morphologically distinct, and to preserve stability and familiarity of the classification scheme; in some cases, paraphyletic taxon names were retained because a revision of the taxon was currently underway. In contrast, the names of polyphyletic taxa were not retained. If existing, monophyletic taxa were named according to an available name; in contrast if there were no available names, then a new name was provided. Diagnoses of new higherlevel taxa were made in part with nucleotide site substitutions based on unique and uniform states identified in Mesquite using the 'With State Distinguishing Selected Taxa' tool (Maddison and Maddison 2010). Unique and uniform states were identified from the 'full-taxa' alignment (see below 'Results, DNA extraction, amplification, and sequencing') and their site numbers are supplied in parentheses (the first number is specific to the gene locus and the second is for the whole matrix). Holotypes and other type material were deposited in the Virginia Tech Insect Collection and Virginia Museum of Natural History. Museum abbreviations are as follows: VTEC (Virginia Tech Insect Collection, Blacksburg, Virginia), VMNH (Virginia Museum of Natural History, Martinsville, Virginia), and NCSM (North Carolina Museum of Natural History, Raleigh, North Carolina).

Nomenclature
This paper and the nomenclatural act(s) it contains have been registered in Zoobank (www.zoobank.org), the official register of the International Commission on Zoological Nomenclature. The LSID (Life Science Identifier) number of the publication is: urn:lsid:zoobank. org:pub:A55797AF-B8E6-4E46-8FD2-53B5C79FCEF8.

Fieldwork and Taxon Sampling
We collected all three nominal subfamilies, 49 of 57 genera, and 247 of 486 species (Supp Table 1

DNA Extraction, Amplification, and Sequencing
Amplification and direct sequencing of DNA from the six targeted genes generally worked consistently across taxa sampled. However, DNA fragments from certain taxa repeatedly failed to amplify and/ or sequence. In general, rounds of amplification and sequencing were repeated two to three times for a specimen before a second specimen was substituted or attempts abandoned. The 'full-taxa' dataset included 253 species with reduced occupancy of genes (omitted due to amplification or sequencing failure), and the 'full-gene' dataset with 227 species possessing at least three of the six genes. If sequences of the following genes were attained then the specimen was included in the 'full-taxa' molecular phylogenetic analysis: 16S and either 12S, COI, EF1α, or 28S. Taxa sampled by gene are listed in Supp Table  1 (online only). RNA-seq of total RNA from Nannaria ericacea resulted in 56,428,924 paired-end reads and Nannaria hokie n. sp. in 56,662,265 paired-end reads. Amplification of DNA from Nannaria with primers that we developed using transcriptome sequences designed to extend through the entire 12S-16S region (including tRNA-Val) consistently failed. However, those primers developed to anneal and extend through regions within the separate 16S rRNA component successfully amplified 797 bp of DNA with high sequence identity via BLAST to other 16S fragments of Xystodesmidae in NCBI GenBank (Altschul et al. 1990). The 12S and tRNA-Val genes are hence missing from these fragments, and sequences of Nannaria are therefore consistently shorter than others.

Phylogenetic Analyses
Multiple sequence alignment in prank and inference of DNA evolution models in PartitionFinder resulted in a 5497 bp concatenated 'full-taxa' matrix composed of 221 bp (

Biogeographical Analysis
Taxa were distributed in 26 ecoregions (Olson et al. 2001), including the Hokkaido deciduous forests and Taiheiyo evergreen forests in Japan, Tamaulipan mezquital and Sierra de los Tuxtlas in Mexico, and Sierra Nevada forests and Appalachian Blue Ridge forests in the United States (Fig. 4, Table 1, Supp Figs 2 and 3 [online only]). Species primarily occurred in broadleaf deciduous forests, but some taxa were found in evergreen forests and chaparral shrublands. The likelihoods of the DEC and DEC+J models were -666.54 and -505.65, respectively, and likelihood ratio test indicated that the DEC+J model was the best fit given the data (df = 1, P < 0.0001). The species Melaphe vestita, which occurred outside of Xystodesmidae on the phylogeny, is known from the Aegean and Western Turkey sclerophyllous and mixed forests (AWTS). The root node estimated from the BioGeoBEARS was equivocal between this AWTS ecoregion and the Sierra Nevada forests ecoregion (California, USA). The root node of the remaining Xystodesmidae, including Japanese species, was the Sierra Nevada forests (California). The major clades of Xystodesmidae occur in the following ecoregions: Sierra Nevada forests (California), Taiheiyo evergreen forests (Japan), and the Appalachian Blue Ridge forests (Fig. 4).

Species Discovery and Delimitation
In most cases, conditional new species (H morph=yes ) were not sister to their presumed closest relatives (hypothesized a priori based solely on morphological similarity) in the concatenated 'fulltaxa' molecular phylogeny (H phy=yes ). Because these conditional  Xystodesmidae is paraphyletic with respect to the families Euryuridae (red taxa) and Eurymerodesmidae (green taxa), which are now tribes of the subfamily Rhysodesminae. Light and dark blue text. Genera with species diversity > 40 including Rhysodesmus and Sigmoria (also Nannaria in magenta text) that have not been extensively sampled. Scale bar: 0.1 expected substitutions per site. (Note: the whole tree was cut in two parts with part 2 here and part 1 in Fig. 2.).
new species were distinct morphologically and phylogenetically (H morph=yes , H phy=yes ), they were described as new species. However, four conditional new species were separated from their closest hypothesized relatives by a single branch (H phy=no ), possessed slight morphological genitalic differences (H morph=no ) and therefore were not described as distinct species: Brachoria 'Corinth', Rudiloria 'Monongahela', Pleuroloma 'Little Stony', and Rhysodesmus 'Hobbs Island'. The following taxa were represented by a single male specimen: Rudiloria 'Paddy Knob', Brachoria platana (plus female specimen), and Brachoria 'Dungannon'. Therefore, we determined that the evidence supporting these three taxa as new species was lacking compared to that for others; however, we described Brachoria platana based on the presence of two specimens, a male and female.

Taxonomy
The   Diagnosis: Adult males of Appalachioria bondi n. sp. are distinct from other apheloriine species based on the following combination of characters: Gonopods. Gonopodal acropodite curving ventromedially at apex, with a distal cingulum, separating it from Apheloria and Rudiloria (Fig. 6). Prefemoral process short and stout, basal zone lacking tubercles. Acropodite at anterior bend with three stout, triangular dorsal tubercles, separating it from most other Appalachioria species. Distal zone constricted basally, with a medially curved, blunt, uncinate tip. Color. Appalachioria bondi n. sp. has multiple color morphs, often co-occurring ( Fig.  5A-G). Tergites always with white, light-pink, or yellow paranotal spots, sometimes also with concolorous metatergal middorsal spots. Dark-brown to black background. Collum always with concolorous white, light-pink, or yellow anterior and lateral spots, sometimes also with a posterior spot, never with marginal lines connecting the spots. Table 3 (online only). Based on Holotype (♂) SPC000282.

Variation:
The dorsal tubercles on the anterior bend of the acropodite vary in size and number, ranging from 1 to 3 (exceptionally up to 10) small to stout triangular tubercles, sometimes serrated or divided apically. The tubercles can vary from dorsal to medial in position. The tip of the acropodite may be curved medially to ventrally. The prefemoral process ranges from short and thin to long and stout and varies from small to medium in size. Appalachioria bondi n. sp. exhibits six unique color patterns: yellow four-spotted collum (Fig. 5A), red twospotted, collum with one anterior and two lateral red spots (Fig.  5B), yellow two-spotted, collum with one anterior and two lateral yellow spots (Fig. 5E), white four-spotted collum (Fig. 5C), white two-spotted, collum with one anterior and two lateral white spots (Fig. 5F) and red four-spotted collum (Fig. 5D).
Ecology: Individuals of Appalachioria bondi n. sp. have been found in mesic deciduous forests of oak, beech, maple, tuliptree, birch, buckeye, and sassafras, with small patches of eastern redcedar nearby.
Distribution: Appalachioria bondi n. sp. is only known from a small range of about 8 km 2 on Clinch Mountain in Russell and Washington Counties, Virginia.
Etymology: This species is named for Dr. Jason Bond of the University of California, Davis. The specific name is a genitive noun derived as a patronym.   Table 2 (online only).

Diagnosis:
Adult males of Appalachioria sierwaldae n. sp. are distinct from other apheloriine species based on the following combination of characters: Gonopods. Gonopodal acropodite strongly curving ventromedially and with a strong distal cingulum, separating it from Apheloria and Rudiloria (Fig. 7). Prefemoral process short and stout. Post-cingulum area expanded, wider than pre-cingulum area. Distal zone strongly curved medially into a long, uncinate, thin tip. Color. Tergites with yellow paranotal spots and yellow metatergal spots (Fig. 5I), but sometimes with orange metatergal spots (Fig. 5H). Black background. Collum with yellow lateral and anterior spots, sometimes with yellow or orange posterior spots, or lacking a posterior spot.  of characters: Gonopods. Gonopodal acropodite strongly curving ventromedially and with a strong distal cingulum, separating it from Apheloria and Rudiloria (Fig. 8). Prefemoral process short and stout. Basal zone medially with small triangular tubercles, separating it from other co-occurring Appalachioria species. Post-cingulum area slightly expanded, with a strong twist at acropodite peak, distal zone with a lateral indentation. Acropodite tip curved medially. Color. Tergites with yellow paranotal spots (Fig. 5K), sometimes with small yellow metatergal spots (Fig. 5J). When metatergal spots present, midbody tergites with broken yellow caudal line connecting the paranotal and metatergal spots. Black background. Collum with yellow anterior and lateral spots connected by a yellow marginal line, sometimes also with a caudal marginal line. Table 3  Variation: There is slight variation in the number and position of tubercles on the acropodite basal zone, with numbers ranging from 4 to 6, spread from the base of the basal zone to halfway up the acropodite. The prefemoral process ranges from quite small and stout to long and thin, reaching to the level of the distal zone of the acropodite. Appalachioria brownae n. sp. has two known color morphs, 1) three-spotted yellow, with small yellow metatergal spots and a thin, broken caudal line, collum with yellow anterior and lateral spots connected by a thin yellow marginal line (Fig. 5J) and 2) twospotted yellow, collum with yellow anterior and lateral spots with a thin anterior marginal yellow line connecting the spots (Fig. 5K).

Description: Supp
Ecology: Individuals of Appalachioria brownae n. sp. were found in a deciduous forest of oak, maple, and big leaf magnolia near a stream during dry conditions. Distribution: Appalachioria brownae n. sp. is known only from the type locality, situated in bottomland habitat at the base of Brush Mountain and Redrock Mountain.
Etymology: This species is named for Dr. Ellen Brown of Fredericksburg, Virginia. The specific name is a genitive noun derived as a matronym. Diagnosis: Adult males of Brachoria camptera n. sp. are distinct from other apheloriine species based on the following combination of characters: Gonopods. Gonopodal acropodite D-shaped (Fig.  9A)-not circular as in Apheloria or oval as in Rudiloria species (Fig. 12). Acropodite (when viewed posteriorly) narrow, onethird width of tibia on leg pair 9. Acropodite gradually tapered to acuminate apex, not with a blunt apex as in B. hubrichti. Acropodite distal to anterior bend slightly curved cephalically. Acropodite shaft with cingulum (midlength transverse groove). Prefemur with curved sickle-shaped prefemoral process (Fig. 9B). Color. Tergites black with 2 orange (Fig. 5L), or red (Fig. 5M), paranotal spots, not with purple/purple-gray metatergal stripes as in B. hubrichti and B. initialis. Table 3 (  Variation: Individuals of Brachoria camptera n. sp. are known to exhibit two color morphs, 1) two-spotted red (Fig. 5M) and 2) two-spotted yellow-orange with a faint light blue caudal stripe (Fig. 5L).

Description: Supp
Ecology: Individuals of Brachoria camptera n. sp. were collected in a mesic deciduous forest with dominant tree species of oak, maple, beech, and tuliptree.
Distribution: Known only from the type locality.
Etymology: This species is named for the shape of its acropodite, specifically the angular nature of its anterior bend. The specific name is a noun in apposition derived from the Greek kampter, 'bend, angle'. Diagnosis: Adult males of Brachoria cryocybe n. sp. are distinct from other apheloriine species based on the following combination of characters: Gonopods. Gonopodal acropodite smoothly ovalshaped (Fig. 10A)-not circular as in Apheloria or tightly oval as in Rudiloria species (Fig. 12). Acropodite (when viewed posteriorly) narrow, one-third width of tibia on leg pair 9. Acropodite gradually tapered to acuminate apex-curved one-dimensionally to apex, not twisted cephalically as in Apheloria species. Acropodal distal zone with tooth on posterior margin. Acropodite shaft with cingulum (midlength transverse groove). Prefemur with railroad spike-like prefemoral process (Fig. 10A), not bidentate as in B. divicuma. Color. Tergites black with 2 red paranotal spots, not with yellow spots as in B. divicuma. Caudal border of tergites with pale white stripe (Fig. 5N). Table 3 (online only). Based on Holotype (♂) MPE02644.

Variation: No significant variation from the holotype was observed.
Ecology: Individuals of Brachoria cryocybe n. sp. were collected in a mesic mixed forest with dominant tree species of oak, ironwood, and hemlock. They were sympatric with two other large-bodied xystodesmid species, Apheloria montana (Bollman, 1887) and Brachoria forficata (Shelley, 1986).

Distribution: Known only from the type locality.
Etymology: This species is named after Frozen Head State Park, where it was discovered. The specific name is a noun in apposition derived from the Greek kryos, 'icy cold', and kybe, 'head'.  of characters: Gonopods. Gonopodal acropodite curving dorsomedially at apex, with a distal cingulum, separating it from Apheloria and Rudiloria (Fig. 11). Prefemoral process reduced to small ridge. Distal zone sinuous, curving first ventrally and then dorsally; in anterior view directed posteriorly at 90° from apex. Acropodite tip with lateral and medial flanges forming an envelope-like structure through which runs an elevated ridge carrying the solenomere (Fig. 11A). Color. Brachoria platana n. sp. has two color morphs, co-occurring. Tergites always with red or yellow paranotal spots, sometimes also with concolorous metatergal middorsal spots or stripes. Dark-brown to black background. Collum always with concolorous red or yellow anterior and lateral spots, sometimes also with a posterior spot and concolorous marginal lines connecting the spots. Variation: Individuals of Brachoria platana n. sp. are known to exhibit two color morphs, 1) striped red (Fig. 5O) and 2) threespotted yellow (Fig. 5P).

Brachoria platana Means, Hennen, Marek, New Species
Ecology: Individuals of Brachoria platana n. sp. were found in a mesic deciduous forest of tulip poplar, sycamore and autumn olive by the water's edge of Carr Creek Lake.
Distribution: Brachoria platana n. sp. is known only from the type locality.
Etymology: This species is named after where the holotype was discovered, in a grove of American sycamores (Platanus occidentalis). The specific name is a noun in apposition derived from the Latin genus name of the American sycamore, 'Platanus'.

Distribution:
Daphnedesmus species are known from the mountainous borderlands between North Carolina, Tennesee, and Virginia with Burkes Garden, Virginia as the northern limit and Ashe County, North Carolina as the southern limit.
Etymology: This genus is named after the yellow stripe on the collum that appears as if the millipede is wearing a gold crown, and that mountain laurels (Kalmia latifolia L.) are commonly encountered with members of the genus. The name is a combination of the Greek, daphne, 'laurel' (bay laurel, Laurus nobilis L.) and desma 'band'. The ending '-desmus' is commonly used for genus names in the order. Etymology: This genus is named after where the type was discovered on Mount Ida, Arkansas. The name is a combination of 'Ida' and the Latin -orium, 'place for'. The ending '-oria' is commonly used for genera in the family.  Diagnosis: Adult males of Rudiloria charityae n. sp. are distinct from other apheloriine species based on the following combination of characters: Gonopods. Gonopodal acropodite smoothly oval-shaped (Fig. 12A)-not circular as in Apheloria species. Acropodite (when viewed posteriorly) narrow, onethird width of tibia on leg pair 9. Acropodite gradually tapered to acuminate apex-curved one-dimensionally to apex, not twisted cephalically as in Apheloria species. Acropodite shaft smooth without cingulum (midlength transverse groove) as in Brachoria; without teeth, swellings, joints, or projections as in other Rudiloria species. Prefemur with a nubbin-like prefemoral process, one-tenth length of acropodite (Fig. 12C), not long and scythe-like as in Rudiloria kleinpeteri, Rudiloria trimaculata. Color. Tergites with 3 spots: 1 orange metatergal spot, 2 yellow paranotal spots (Fig. 5Q). Collum with yellow spot anteriorly, gradually fading into orange metatergal spot. Table 3 ( Table 2 (online only).

Variation: No significant variation from the holotype was observed.
Ecology: Sigmoria beameri n. sp. was collected from the riparian area around Toby Creek and was found under oak and maple leaves.

Distribution:
Only known from the type locality, a small wooded area around Toby Creek, located in the Coastal Plain region of South Carolina.
Etymology: This species is named for its discoverer, Dr. David Beamer of Nash Community College. The specific name is a genitive noun derived as a patronym.

Tribe Nannariini Hoffman, 1964
Genus Nannaria Chamberlin, 1918 Vernacular Name: 'The Twisted-Claw Millipedes' Diagnosis: Adult males of Nannaria aenigma n. sp. are distinct from other nannariine species based on the following combination of characters: Gonopods. Gonopodal acropodite long, undivided, gently curving medially at apex, not straight as in N. ericacea 2. Males with long coiled acropodite, forming 1.5 -2 loops, without prefemoral process (Fig. 16N); females with membranous neck of the cyphopod modified into long retractable bellows (Fig. 16O)  3. 3rd pair legs of males with conical coxal process (Fig. 16P), 2nd pair of legs of females with cylindrical coxal process; metatergites occasionally with small bumps (papillae), thereby reducing the glossiness of the cuticle (Figs. 1C and 16Q); not very colorful, usually bimaculate, highly fluorescent when  Gonopod cannula present distally on coxa, separated from coxal cuticle by membrane forming a cannular socket. Gonopods composed of coxa and telopodite. Telopodite composed of a basal prefemur and distal acropodite. Acropodite apex simple, usually ≤ 2 branches. Gonopod aperture transverse relative to trunk axis, elliptical in shape; large, thereby reducing the seventh trunk ring prozonite anteriorly to a narrow cuticular bar. In life, individuals readily emit liquid chemical secretions composed of a strong cherry, almond odor (benzaldehyde byproduct of cyanogenic reaction). Rhysodesminae: colorful (highly variable hues and patterns, Fig.  3A and B) large bodied (> 35 mm) xystodesmid millipedes from North America between meridians 68°W (ca. Maine, USA) and 115°W (ca. New Mexico, USA)*, but with some not very colorful (cryptic with background matching, Fig. 3D-H), small bodied (<35 mm) members. Metatergites usually glossy (Fig. 1A), without small bumps (papillae or tubercles)-papillae or tubercles are typically present in the Xystodesminae. Telopodite usually curved medially ( Fig. 16D and E), twisted cephalically in colorful, large bodied members; telopodite usually sublinear in shape (Fig. 16S) in cryptic, small bodied members.
Apheloriini (ENA): xystodesmid millipedes with telopodites with short or no prefemoral process-not long, acicular as in the Rhysodesmini. If prefemoral process present, it is usually stout, curved-often hook-like. Sternal remnant between gonopodal coxae absent. Telopodite curved medially ( Fig. 16D and E) and twisted cephalically (with torsion)-not linear as in the Rhysodesmini. Paramedial spines absent on midbody sterna. Colorful; highly variable hues and patterns. Large bodied, usually greater than about 35 mm.
Parafontariini (EA): xystodesmid millipedes with membranous neck of the cyphopod modified into long retractable bellows in females (Fig. 16O). Long telopodite with circular apex, forming one and a half to two loops (Fig. 16N). Gonopod without a prefemoral process. Gray, orange, intermediate between gray and orange, occasionally with contrasting stripe on protergite or posterior margin of metatergite. Highly fluorescent when illuminated with ultraviolet light.
Melaphini (Mediterranean Basin and northern Africa): xystodesmid millipedes with the acropodite simple, sickle-shaped (Fig. 16R). Gonopod without a prefemoral process. Colorful, yellow trimaculate-in some species, paranotal spots are red. Lateral carinae of paranota discontinuous, body appearing moniliform with flattened paranota-not compact in general appearance as in other xystodesmids; paranota flat, horizontal-tergites not arched with paranota oriented downwards as in other xystodesmids. Paranotal corners rounded-corners of paranota not sharp (or hooked) as in the Xystodesmini and Xystocheirini. Without spines on the prefemur. Sternal remnant between gonopodal coxae present.

Discussion
The phylogeny of Xystodesmidae recovered mainly western and eastern North American clades, which itself as a monophyletic group is sister to Mediterranean taxa (Fig. 4). The eastern North American (ENA) clade also contains the Madrean genera Rhysodesmus and Stenodesmus, and the western North American (WNA) clade includes species from East Asia (EA); the WNA clade also includes the markedly disjunct species Semionellus placidus from ENA. Noted by Shelley in 1994, and sister to species from the Pacific Northwest, S. placidus is biogeographically noteworthy as the sole ENA member of a uniformly WNA+EA subfamily Xystodesminae (Fig. 4, box 'C' with arrow). The biogeography of the Xystodesmidae illustrates a disjunct temperate fauna that evokes the classic patterns of co-distributed flora in the northern hemisphere (Manos and Meireiles 2015). This pattern is similar to the Old and New World floristic distribution of the hawthorn genus Crateagus (Lo et al. 2009). Prior biogeography of the Xystodesmidae suggested that the 'holarctic group Xystodesmidae, including several mediterranean genera of uncertain status, appears to be the modern representative of older palearctic taxa which also gave rise to the two groups Oxydesmidae and Gomphodesmidae' (Hoffman 1978a, pg. 28). Hoffman's generalization of the family is broadly consistent with our own phylogeny (he even refers to the Eurymerodesmidae as a smaller, apparently derivative group) but omits the Euryuridae, which was later integrated into the family by Shelley and Smith (2018). Another millipede group with molecular phylogenetic data, Brachycybe, is distributed in the northern hemisphere. Also distributed in ENA, WNA, and EA, the biogeographical pattern of the genus is different, with the center of species diversity in WNA, and an ENA species, Brachycybe lecontii Wood, 1864, that is a sister to the EA species Brachycybe nodulosa (Verhoeff, 1935). Although there seems to be congruent disjunctions between EA and NA in unrelated floral and faunal taxa, the variable patterns shown by molecular phylogenetic analyses indicate continuous splitting, expansion, and retraction during the Paleogene mediated by cooling and warming that lead to 'pseudocongruent' distributions (Manos and Meireiles 2015).
The WNA+EA clade, here named the subfamily Xystodesminae, includes five tribes; the ENA clade, named the subfamily Rhysodesminae, encompasses five tribes. Several historical tribes are monophyletic; in contrast, some past tribes in the Xystodesmidae are paraphyletic and include the Rhysodesmini, Sigmocheirini and Xystodesmini. The Chonaphini is polyphyletic and makes up an assemblage of unrelated taxa in the phylogeny. The non-monophyly of these taxa are ostensibly due to the historical (over-)reliance on gonopodal characters for higher-level systematics, which Means and Marek (2017) showed that the majority of which (95%) are homoplasious. The changes to the higher-level classification here, therefore, rely upon phylogeny instead of gonopodal morphology (see 'Materials and Methods, Taxonomy'). The WNA and EA species of the subfamily Xystodesminae were divided into five tribes: Xystodesmini, Parafontariini, Sigmocheirini, Ochthocelatini, and Xystocheirini. All of the WNA species of Chonaphini, Xystodesmini, and Orophini were placed in a restructured, and now monophyletic, Xystodesmini. The EA genera (spare the genus Parafontaria) are retained in the Xystodesmini. The species Ochthocelata adynata Shelley, 1995, from California, is the sole adelphotaxon of diversification in California Moore 2015, Emata andHedin 2016). This mode of speciation in a group of non-mobile moisture dependent organisms may also be commonplace in Appalachian taxa, and centers of clade-level diversity and endemism occur in the Cumberland Mountain region (with the genus Brachoria), the mountains of South Carolina and Georgia (with the genus Sigmoria), and Valley and Ridge Province (with the genus Nannaria).
Of the 10 new species, six are known from only their type localities. In some well sampled taxa, such as Apheloria whiteheadi, the species are micro range endemics (MRE) and are distributed in geographical areas of less then 1,000 km 2 (Means and Marek, 2017). (In the case of A. whiteheadi, a taxon that is extremely limited in distribution, has a global range restricted to less than 1 km 2 .) Nannaria hokie, which persists in forest habitats in Montgomery County, Virginia, occurs in highly fragmented and threatened areas. For example, Stadium Woods, a 4.5-hectare old-growth white oak forest on the Virginia Tech campus, is surrounded by the university football stadium on its western side and residential housing on its eastern side. The species also occurs on the south side of a pond on the university's campus (Duck Pond, 37.2250°N, -80.4276°W).
Though it now persists in several small forest fragments, the species is globally endemic to only 100 km 2 . Because these species are distributed in extremely restricted distributions, which themselves are highly fragmented, the potential for loss of substantial amounts of species diversity is very high. Since human industry is nearly entirely responsible for the fragmentation and loss of habitat, the irrevocable extinction of these species and co-occurring species is not without moral neutrality, and eventual detriment to environmental and human health. Stadium Woods, and other forest fragments in the region, are refuges for migratorial birds, indigenous flora and pollinating insects as well, and they sequester atmospheric carbon, so preservation of these habitats should be highly prioritized.
More than half of the species diversity (247/486 species), and 86% of the genus diversity of the family was sampled. However, a handful of xystodesmid taxa from outlying locations in China, Vietnam, Ethiopia, and the Mediterranean Basin have been omitted from this analysis such as Devillea, Kiulinga, Koreoaria, Ochridaphe, Pamelaphe, Parariukiaria, and the enigmatic Melaphe corrupta Attems, 1944 from the Ethiopian Highlands (Fig. 4). Some groups with species diversity > 40 including Rhysodesmus, Nannaria, and Sigmoria, have not been extensively sampled. Omission of these taxa, especially the Mediterranean species, may later prove to be pivotal for a more thorough understanding of the early diversification of the family. Furthermore, a larger genomic dataset and expanded outgroup selection including representatives of Chelodesmidae, Oxydesmidae, and Gomphodesmidae will help comprehend the bounds of the taxon and build a more developed understanding of the morphological and evolutionary extremes of the family. This will provide an important context for studying the fascinating biological qualities of the group, to document the undescribed species diversity, and to conserve biodiversity.

Supplementary Data
Supplementary data are available at Insect Systematics and Diversity online.