Phylogenetic Systematics of the Millipede Family Xystodesmidae
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Insect Systematics and Diversity, (2021) 5(2): 1; 1–26
doi: 10.1093/isd/ixab003
Research
Molecular phylogenetics, phylogenomics, and phylogeography
Phylogenetic Systematics of the Millipede Family
Xystodesmidae
Jackson C. Means,1 Derek A. Hennen,1, Tsutomu Tanabe,2 and Paul E. Marek1,3,
1
Department of Entomology, Virginia Tech, Blacksburg, VA, USA, 2Faculty of Advanced Science and Technology, Kumamoto
University, Kurokami, Kumamoto 860-8555, Japan, and 3Corresponding author, e-mail: paulemarek@gmail.com
Subject Editor: István Mikó
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Received 3 August, 2020; Editorial decision 3 January, 2021
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 biolumin-
escent 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, rec-
ollecting 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 phyl-
ogeny 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.
Key words: Polydesmida, biogeography, aposematic, taxonomy
Millipedes are an ancient terrestrial lineage with members from species of Symphyla and 3,500 species of Chilopoda; although
the Early Devonian (ca. 414 Ma) that breathed atmospheric what major factors led to the greater diversity of millipedes is un-
oxygen (Wilson and Anderson 2004, Suarez et al. 2017) and clear, traits implicated in the radiation include diplosegmentation
later forms that grew to gargantuan body proportions (2 × 0.5 and metachronal gait (thereby fostering burrowing) and chemical
m) sustained by elevated atmospheric oxygen content in the defenses (thus deterring predation). Notably, Geophilomorpha,
Carboniferous (Lucas et al. 2005). Divergence between Diplopoda a centipede order that has independently gained the ability to
and its sister group Pauropoda (Dignatha) has been estimated burrow (through the evolutionary trend of trunk elongation and
using molecular dating to have occurred during the Ordovician leg addition) and produce chemical defenses, is the most species
(Fernandez et al. 2018, but see Szucsich et al. 2020). Although rich non-diplopod myriapod group. As detritivores, millipedes
there are many Paleozoic diplopod fossils, none have been as- feed on decaying plant material, thereby fragmenting detritus
signed to any of the 16 extant orders. Wilson (2006) suggested into smaller pieces fostering later colonization by bacteria and
that diversification accelerated during the Early and mid-Silurian fungi, and some have estimated that through fragmentation
and superordinal stem groups were established by the beginning that certain species [Narceus americanus (Palisot de Beauvois,
of the Devonian. Fossil records during the Mesozoic are gener- 1817), Spirobolida, Spirobolidae], contribute about two tons of
ally scarce, except for many recently discovered Cretaceous (ca. frass per acre to deciduous forests yearly (Coville 1913). This
100 Mya, Burmese) amber fossils (Wesener and Moritz 2018). input aerates and conserves the soil, and the process releases ni-
Thirteen of the 16 extant orders of Diplopoda are represented by trogen, carbon, simple sugars, and other nutrients back into the
amber fossils in Cretaceous (Wesener and Moritz 2018). There are biosphere (Joly et al. 2020). Unfortunately, non-native earth-
830 species of Pauropoda and 12,000 species of Diplopoda—200 worms—released from predation and parasites from their native
© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. 1
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Version of Record, first published online March 1, 2021 with fixed content and layout in compliance with Art. 8.1.3.2 ICZN.
2 Insect Systematics and Diversity, 2021, Vol. 5, No. 2
ranges—have been shown to compete with native millipedes for The central and eastern U.S. families, Eurymerodesmidae Causey,
this seemingly limitless detrital resource (Snyder et al. 2009). 1951 and Euryuridae Pocock, 1909, have been regarded as close re-
Millipedes’ lack of wings, absence of phoresy, and low dispersal latives of the Xystodesmidae for more than 40 yr (Hoffman 1978a,
capability fosters narrow endemism, and many individual species 1990, 1998; Shelley 1989). The family Eurymerodesmidae (Fig. 1E)
are known as short-range endemics (SRE), distributed in areas was considered as a ‘smaller, apparently derivative group’ of
less than 10,000 km2 (Harvey 2002, Harvey et al. 2011). Their Xystodesmidae by Hoffman (1978a, p. 24) and the two families ‘as
narrowly restricted distributions, e.g., the Laurel Creek milli- sister taxa within the Xystodesmoidea’ by Shelley (1989, p. 102).
pede Apheloria whiteheadi (Shelley, 1986) with a global range Although Euryuridae (Fig. 1F) was traditionally suggested to be
of ca. 1 km2, exemplify that they are irreplaceable biodiversity, closely allied to the Platyrhacidae and Aphelidesmidae due to a
which is ultra-susceptible to global extinction due to habitat loss, flattened, broad epiproct (Hoffman 1954, 1980), Hoffman (1998),
and highlights the importance of prioritizing millipede and other upon reexamination of 14 characters, considered the family as
SRE invertebrates in schemes seeking to maximize species diver- closer to Xystodesmidae, in particular close to the xystodesmid sub-
sity. Despite their antiquity and important role as detritivores, family Melaphinae Hoffman, 1980. However, these two taxa were
known millipede species diversity tremendously lags behind es- retained as distinct families due to several characters including the
timated global diversity (Brewer et al. 2012). For example, there ventral mandibular ridge in Eurymerodesmidae and the flattened,
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are more than 50 undescribed species of the twisted-claw milli- broad epiproct and simple gonopods in the Euryuridae (Shelley
pede (Nannaria spp., Polydesmida, Xystodesmidae) in the eastern 1989, Hoffman 1998). A recent study then subsumed the fam-
United States alone, and globally an additional 3,000–80,000 spe- ilies Eurymerodesmidae and Euryuridae under Xystodesmidae
cies of Diplopoda when tropical locales and other poorly sampled and removed Macellolophus rubromarginatus (Lucas, 1846) from
regions are included (Hoffman 1980, Brewer et al. 2012). The Melaphinae, placing it in the family Chelodesmidae Cook, 1895.
lack of basic alpha-taxonomic information on the twisted-claw These higher-level changes were justified solely on similarity of male
millipedes is surprising given their large body size (about 2 cm in genitalic morphology (Shelley and Smith 2018). Nonetheless, as
length) and ubiquity east of the Mississippi River; one new species still remains the case with most taxa of the order Polydesmida since
was even discovered within the bounds of the D.C. Metropolitan Cook 126 yr ago, monophyly of the family is uncertain, inter- and
area. The group exhibits fascinating biological characteristics. intrafamilial relationships are poorly known, and rationale for sys-
The millipede family Xystodesmidae includes notable taxa such as tematic relationships is based on century-old character argumenta-
bioluminescent species in California, Motyxia spp. (Marek et al. tion using overall similarity of a handful of morphological features.
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 Molecular Phylogenetics
m-2 have obstructed trains (Hashimoto et al. 2004); the giant Based on an ordinal-level phylogeny of the Diplopoda inferred using
8-cm long armadillo millipede in Mexico, Rhysodesmus dasypus amino acid data sequenced from transcriptomes of a representative
(Hoffman 1970); and the cherry millipede, Apheloria virginiensis set of familial exemplars, Xystodesmidae is monophyletic and sister
corrugata (Wood, 1864), able to generate hydrogen cyanide in an to Eurymerodesmidae, which as a clade is in turn closely related to
amount 18 times that necessary to be lethal to a pigeon-size bird chelodesmid, sphaeriodesmid, and paradoxosomatid taxa (Rodriguez
(Eisner et al. 2005). 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
Taxonomic History morphological-based hypotheses, including Polydesmida sister to
The family Xystodesmidae Cook, 1895 was first established for Nematophora [i.e., (Chordeumatida, (Stemmiulida, Callipodida) as in
several large-bodied members of the order Polydesmida Pocock, Enghoff 1984; Blanke and Wesener 2014]. Intrafamilial phylogenetic
1887 (Fig. 1). In his pioneering yet insightful classification of systematic relationships of the family have been studied in separate
Diplopoda, Cook (1895) assigned millipede genera to 49 fam- analyses of the eastern and western U.S. xystodesmid taxa (Marek and
ilies and remarked that it was made ‘with some confidence’ in all Bond 2006, 2007; Marek et al. 2011, 2015; Means and Marek 2017).
groups, that is except for those of the Polydesmida. This work However, there has never been a phylogenetic systematic analysis of the
did not specify characters that united polydesmidan genera into group with species from throughout the familial geographical distribu-
their respective families. Later, Cook (1904) provided a diag- tion, and with other ostensibly closely related large-bodied taxa in the
nosis of the family Xystodesmidae and differentiated its mem- Leptodesmidea as outgroups to test the monophyly of the family.
bers from other polydesmidan families by the presence of spines To infer a phylogeny of the Xystodesmidae, we sampled 247 spe-
on the prefemur, bisinuately curved tarsal claws, and the dorsal cies of the family (the largest dataset analyzed in the Diplopoda to
habitus (including wide contiguous paranota that impart a com- date) including taxa spanning the geographical distribution of the
pact appearance). Subsequently, the family was included as part family, and utilized six genes. The phylogeny is used as a basis to up-
of Chelodesmidae Cook, 1895 by Pocock (1910) and Brölemann date the classification of the family, diagnose monophyletic groups,
(1916). These authors’ conception of the family, and later Attems’s and to describe ten new species.
(1926), was broad and inclusive and encompassed an assemblage
of heterogeneous taxa, many of which are now in other families.
Materials and Methods
Later, the taxon was more narrowly diagnosed and set apart from
Chelodesmidae and other large-bodied members of the suborder Fieldwork
Leptodesmidea Brölemann, 1916 by the presence of prefemoral Millipedes were collected in the field from 2014 to 2019 and brought
spines and the shape of the body and antenna (see Marek et al. back alive to the laboratory for DNA preservation and specimen
2014 for a detailed summary of taxonomic history). preparation according to the methods described in Means et al.
Insect Systematics and Diversity, 2021, Vol. 5, No. 2 3
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Fig. 1. Representatives of the millipede family Xystodesmidae (Polydesmida). (A) Apheloria whiteheadi (Shelley, 1986), Virginia, USA. (B) Parafontaria tonominea
(Attems, 1899), Kumamoto, Japan. (C) Parcipromus cooki (Causey, 1955), California, USA. (D) Parafontaria erythrosoma (Takakuwa, 1942), Ibaraki, Japan. (E)
Eurymerodesmus dubius Chamberlin, 1943, Arkansas, USA. (F) Euryurus orestes Hoffman, 1978, Georgia, USA. (G) Thrinaphe hargeri Shelley, 1993, Oregon,
USA. (H) Tubaphe levii Causey, 1954, Washington, USA. (I) Ochthocelata adynata Shelley, 1995, California, USA. (J) Sigmocheir furcata Shelley, 1995, California,
USA. (K) Levizonus takakuwai (Verhoeff, 1941), Hokkaido, Japan. (L) Riukiaria cornuta (Haga, 1968), Kumamoto, Japan.
(2015). For each species, the unique color morphs at a locality were Leptodesmidea. The following taxa were selected as outgroups—
photographed with a Canon EOS 6D digital SLR camera, MT-24EX Xystodesmoidea: Orodesminus Attems, 1929 (Oxydesmidae Cook,
Macro Twin Lite Flash, and a 50 mm lens; a MP-E 65 mm lens was 1895: Mozambique) and an undetermined genus and species of
used for individuals < 20 mm (Canon Inc., Japan). Gomphodesmidae Cook, 1896 (Mozambique); Chelodesmoidea Cook,
1895: Macellolophus rubromarginatus Lucas, 1846 (Chelodesmidae:
Taxon Sampling Spain); Platyrhacoidea Pocock, 1895: Amplinus bitumidus (Loomis,
Based on species occurrence records from revisionary monographs and 1969) (Aphelidesmidae Brölemann, 1916: Mexico); Rhachodesmoidea
other taxonomic literature, we collected material from the field, priori- Carl, 1903: Neoleptodesmus Carl, 1903 (Rhachodesmidae Carl, 1903:
tizing collections from type localities and from within the known spe- Mexico); and Sphaeriodesmoidea Humbert & DeSaussure, 1869: an
cies ranges. We targeted every genus in the family, and searched type undetermined genus and species of Sphaeriodesmidae Humbert &
localities when feasible or nearby localities. Eurymerodesmidae and DeSaussure, 1869 (Mexico).
Euryuridae were recently placed in the Xystodesmidae by Shelley and
Smith (2018), thus we included in our sampling four and six of their DNA Extraction, Amplification, and Sequencing
species. Outgroups were selected in the superfamily Xystodesmoidea Left legs from body rings 8–18 were removed with flame-sterilized
Cook, 1895, and other presumed close relatives in the suborder forceps, immersed in RNAlater (Qiagen) or 100% ethanol, and
4 Insect Systematics and Diversity, 2021, Vol. 5, No. 2
archived at −80°C in the freezer collection of the Virginia Tech Insect literature for identification to species. We refrained from describing
Collection (VTEC, collection.ento.vt.edu) for later DNA extraction. new species and making changes to the classification of the genera
About 50% of the taxa sampled were collected (2014–2019) spe- Apheloria, Cherokia, Rhysodesmus, and Riukiaria because revi-
cifically for this study. Their frozen tissues, archived in the VTEC sions of these taxon are currently underway. We used morpho-
freezer collection, were DNA-extracted and sequenced for additional logical and molecular phylogenetic distinctness (diagnosability) to
genes. We amplified and sequenced six genes (four mitochondrial delimit species as follows. 1) We first used male gonopod morph-
and two nuclear gene fragments): small subunit RNA (12S), tRNA- ology, and if a specimen’s gonopod morphology matched the de-
Valine, large subunit RNA (16S), cytochrome c oxidase subunit scription of a known species then the specimen was ascribed to
I (COI), elongation factor alpha (EF1α), and large subunit RNA that species. However, if the specimen’s gonopod morphology
(28S). Amplification of DNA was carried out as described in Means did not match the known species and was distinct then we estab-
and Marek (2017). DNA amplifications were cleaned, concentra- lished a preliminary new species, Hmorph=yes (e.g., Appalachioria
tions quantified and normalized, and Sanger-sequenced using an ABI n. sp. ‘Clinch Mountain’ Marek and Bond 2006, 2007; Means and
3730 capillary sequencer (Applied Biosystems). Marek 2017). At this moment, we hypothesized what the closest
Previous attempts to sequence the 12S and 16S DNA from rep- relative of the conditional new species would be. New species rep-
resentatives of the genus Nannaria repeatedly failed, so transcrip- resented by a single male (or female) specimen remained prelim-
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tomes of two species of Nannaria collected from Virginia Tech’s inary new species (Hmorph=yes) and were not described until additional
campus were sequenced to develop primers: Nannaria ericacea material can be collected to rule out the possibility of an aberrant
Hoffman, 1949; and Nannaria hokie n. sp. Live specimens were form of a known species. 2) A single male exemplar of a prelim-
frozen with liquid nitrogen, pulverized, and whole-animal RNA was inary new species (Hmorph=yes) from above was sampled for the mo-
extracted from the tissues using a RNeasy kit (Qiagen). A cDNA lecular phylogeny to assess phylogenetic distinctness, Hphy=yes/no?.
library was made using reverse transcriptase PCR and sequenced If the preliminary new species was not sister to its closest relative
using a Kappa prep and Illumina RNA-seq with 200-bp paired-end (hypothesized previously) in the concatenated molecular phylogeny
read sequencing. Raw DNA reads were assembled into transcripts (Hmorph=yes, Hphy=yes), or if it was sister to its closest relative but had
using Trinity RNA-Seq de novo transcriptome assembly (Grabherr overt morphological genitalic differences then we established a new
et al. 2011). The ribosomal genes were identified in the assembled species and described it here (Hmorph=yes, Hphy=no). However, if the pre-
Nannaria transcriptome by comparison with the mitochondrial liminary new species was sister to its closest relative but had slight
genome of Appalachioria falcifera (Keeton, 1959) (Swafford and morphological genitalic differences, when re-assessed within the
Bond 2010). We used a local nucleotide BLAST search in Geneious phylogenetic context, then we did not describe it (Hmorph=no, Hphy=no).
to identify the 12S and 16S transcripts in the Nannaria transcrip- We were optimistic that genitalic (gonopodal) differences track spe-
tome (Altschul et al. 1990). Primers were developed spanning the en- cies boundaries. The lock-and-key theory for genitalic variation
tire 12S–16S region (including the intervening tRNA-Valine), and as has been demonstrated in the group previously (Tanabe and Sota
a primer pair within the 16S ribosomal gene region. We used phred 2008, Wojcieszek and Simmons 2012). Therefore, we are confident
and phrap in the Mesquite module Chromaseq for nucleotide base- in using this species delimitation method implementing primarily
calling, trimming, and quality control of Sanger sequences (Ewing male gonopod morphology because shape difference between male
et al. 1998, Maddison and Maddison 2010). and female genitalia prevent nonspecific matings and imparts strong
reproductive isolation. In addition, a diversity of species remains
Phylogenetic Analyses undescribed (ca. 56 in Nannaria) and we are highly motivated to
We used prank for multiple sequence alignment beginning the document new taxa because many of their habitats are directly
analysis with a neighbor-joining guide tree that was later refined threatened by habitat loss, climate change, and other processes that
after the preliminary alignment step (Löytynoja and Goldman cause land conversion and habitat unsuitability.
2005). Aligned gene sequences were partitioned by gene, codon
position, and intron/exon locations in Mesquite. Partitions were Biogeographical Analysis
assessed in PartitionFinder to determine a best-fit partitioning To infer ancestral ranges of the taxa, we plotted their observed biogeo-
plan and to identify appropriate models of nucleotide evolution graphical ranges on the phylogeny using the Dispersal–Extinction–
(Lanfear et al. 2012). Using the best-fit partitioning scheme and Cladogenesis (DEC) model in BioGeoBEARS (Matzke 2013, 2014).
the aligned sequences in nexus format, we used MrBayes 3.2.5 to Biogeographical ranges of the taxa were recorded based on the terres-
estimate phylogeny (Ronquist et al. 2012). Individual gene trees trial ecoregion in which they are found. Terrestrial ecoregions were
for the six genes were analyzed independently in MrBayes to com- according to Olson et al. (2001). A chronogram was estimated with
pare alternative genealogical histories and compare varying gene BEAST using a normal prior on the date of the root with a mean of
tree resolution. In addition, a concatenated dataset with all of the 1 to generate relative dates (Bouckaert et al. 2014, 2019). Outgroup
genes and best-fit partitioning scheme was analyzed in MrBayes. taxa historically not in the family Xystodesmidae were pruned from
Phylogenetic analyses were run on Virginia Tech’s 173-node super- the tree prior to analysis in BioGeoBEARS. We used relative dates be-
computer, NewRiver, with 16 MCMC chains running in parallel cause there are no fossils of the family Xystodesmidae for calibration
on separate processors. A tree and other parameters were sampled points, spare a single equivocal member of the family preserved in
every 100 of 30 million generations. Chains were monitored for Miocene Chiapas amber (Riquelme and Hernández-Patricio 2018).
convergence using the average standard deviation of split frequen- Due to the limited dispersal capabilities of xystodesmid millipedes,
cies and a threshold of 0.01. species exclusively inhabit single ecoregions except for a few wide-
spread taxa (Means and Marek 2017). As a result, the DEC+J model,
Species Discovery and Delimitation which adds the parameter j for founder-event speciation, was hy-
Male specimens collected in the field were compared to type speci- pothesized to be more likely over the DEC model (Matzke 2014). To
mens (and other material in natural history collections [NHCs]) and test this hypothesis, we compared the likelihoods of the alternative
Insect Systematics and Diversity, 2021, Vol. 5, No. 2 5
models using a likelihood ratio test in Microsoft Excel. We visualized Parariukiaria cucphuongensis Nguyen, 2016 from Cuc Phuong
the phylogeny of Xystodesmidae on a world map and plotted the National Park, Vietnam; Parvulodesmus prolixogonus Shelley, 1983
geographical coordinates of the terminal taxa in a tanglegram using from Abbeville County, South Carolina; Lyrranea persica Hoffman,
the R (version 3.2.2) program phytools (version 0.7–20) (Revell 1963a from Peach County, Georgia; and Lourdesia minuscula Shelley,
2012, R Core Team 2016). 1991 from Coffee County, Alabama were visited but no specimens
were found. The monotypic taxa Cheiropus plancus Loomis, 1944
Taxonomy (U.S.); Ochridaphe albanica (Verhoeff, 1932) (Mediterranean rim);
Material in NHCs was used to describe species. Specimens were and Pamelaphe lacustris (Pocock, 1895) (China) were not sampled
examined for somatic and genitalic characters using a Leica M125 for this analysis. Species of the genera Devillea Brölemann, 1902 (five
stereomicroscope. Male gonopods were dissected and photographed species, Mediterranean rim); Kiulinga Hoffman, 1956 (two species,
as above, but with two 350 nm ultraviolet flashes operated from China); Koreoaria Verhoeff, 1937 (two species, South Korea); and
a Visionary Digital Passport II focal-stacking photography system Stelgipus Loomis, 1944 (two species, United States) were not included
with Helicon Focus according to fluorescent photography tech- in the study.
niques outlined in Marek (2017). The higher-level classification of
DNA Extraction, Amplification, and Sequencing
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Xystodesmidae was modified to reflect the phylogeny. Taxon groups
were selected to be equally divergent, mutually exclusive, and stat- Amplification and direct sequencing of DNA from the six targeted
istically well supported. The classification system is informative, in genes generally worked consistently across taxa sampled. However,
that it provides a hierarchical guide to family diversity, and extensible DNA fragments from certain taxa repeatedly failed to amplify and/
in that it provides a framework to name new species. Changes to the or sequence. In general, rounds of amplification and sequencing were
higher-level classification of Xystodesmidae were made according repeated two to three times for a specimen before a second specimen
to the principle that monophyletic and some paraphyletic groups was substituted or attempts abandoned. The ‘full-taxa’ dataset in-
receive formal names. A paraphyletic taxon retained its preexisting cluded 253 species with reduced occupancy of genes (omitted due to
name when the group was morphologically distinct, and to preserve amplification or sequencing failure), and the ‘full-gene’ dataset with
stability and familiarity of the classification scheme; in some cases, 227 species possessing at least three of the six genes. If sequences of
paraphyletic taxon names were retained because a revision of the the following genes were attained then the specimen was included in
taxon was currently underway. In contrast, the names of polyphyletic the ‘full-taxa’ molecular phylogenetic analysis: 16S and either 12S,
taxa were not retained. If existing, monophyletic taxa were named COI, EF1α, or 28S. Taxa sampled by gene are listed in Supp Table
according to an available name; in contrast if there were no available 1 (online only). RNA-seq of total RNA from Nannaria ericacea re-
names, then a new name was provided. Diagnoses of new higher- sulted in 56,428,924 paired-end reads and Nannaria hokie n. sp. in
level taxa were made in part with nucleotide site substitutions based 56,662,265 paired-end reads. Amplification of DNA from Nannaria
on unique and uniform states identified in Mesquite using the ‘With with primers that we developed using transcriptome sequences de-
State Distinguishing Selected Taxa’ tool (Maddison and Maddison signed to extend through the entire 12S–16S region (including tRNA-
2010). Unique and uniform states were identified from the ‘full-taxa’ Val) consistently failed. However, those primers developed to anneal
alignment (see below ‘Results, DNA extraction, amplification, and and extend through regions within the separate 16S rRNA compo-
sequencing’) and their site numbers are supplied in parentheses (the nent successfully amplified 797 bp of DNA with high sequence iden-
first number is specific to the gene locus and the second is for the tity via BLAST to other 16S fragments of Xystodesmidae in NCBI
whole matrix). Holotypes and other type material were deposited in GenBank (Altschul et al. 1990). The 12S and tRNA-Val genes are
the Virginia Tech Insect Collection and Virginia Museum of Natural hence missing from these fragments, and sequences of Nannaria are
History. Museum abbreviations are as follows: VTEC (Virginia Tech therefore consistently shorter than others.
Insect Collection, Blacksburg, Virginia), VMNH (Virginia Museum
of Natural History, Martinsville, Virginia), and NCSM (North Phylogenetic Analyses
Carolina Museum of Natural History, Raleigh, North Carolina). Multiple sequence alignment in prank and inference of DNA evo-
lution models in PartitionFinder resulted in a 5497 bp concaten-
ated ‘full-taxa’ matrix composed of 221 bp (12S, GTR+G), 103 bp
Nomenclature
(tRNA-Val, GTR+G), 2052 bp (16S, GTR+I+G), 600 bp (COI, pos
This paper and the nomenclatural act(s) it contains have been re- 1 & 3 GTR+I+G and pos 2 GTR+G), 1640 bp (28S, GTR+I+G), and
gistered in Zoobank (www.zoobank.org), the official register of the 886 bp (EF1α, pos 1& 2 K80+I+G, pos 3 SYM+G, intron HKY+G).
International Commission on Zoological Nomenclature. The LSID The ‘full-gene’ matrix composed 203 bp (12S, GTR+G), 77 bp
(Life Science Identifier) number of the publication is: urn:lsid:zoobank. (tRNA-Val, GTR+G), 1766 bp (16S, GTR+I+G), 600 bp (COI, pos
org:pub:A55797AF-B8E6-4E46-8FD2-53B5C79FCEF8. 1 & 3 GTR+I+G and pos 2 GTR+G), 1367 bp (28S, GTR+I+G), and
844 bp (EF1α, pos 1 & 2 K2P+I+G, pos 3 SYM+G, intron HKY+G).
The 16 separate MCMC chains for the ‘full-taxa’ analysis converged
Results
at 15 million generations (‘full-gene’ at 2 million generations), and
Fieldwork and Taxon Sampling when summarized in a consensus tree resulted in a topology with 140
We collected all three nominal subfamilies, 49 of 57 genera, and 247 of of 190 nodes supported with a posterior probability > 0.95 (Figs.
486 species (Supp Table 1 [online only]). Ten of these 247 species are new 2 and 3). The phylogeny indicated that the family Xystodesmidae
and described below. Outgroups were collected from Mozambique: is not monophyletic because the Mediterranean species Melaphe
Orodesminus sp. (Oxydesmidae) and Gomphodesmidae sp.; Spain: vestita (Koch, 1847) is separated from remaining species by two
Macellolophus rubromarginatus (Chelodesmidae); and Mexico: branches: Macellolophus rubromarginatus (Chelodesmidae) and
Sphaeriodesmidae sp., Neoleptodesmus sp. (Rhachodesmidae), the gomphodesmid (Fig. 2). Historical infrafamilial taxa such as the
and Amplinus bitumidus (Aphelidesmidae). The type localities of Apheloriini and others are monophyletic; however, some other taxa
6 Insect Systematics and Diversity, 2021, Vol. 5, No. 2
Xystocheir “Sierra”
Xystocheir modestior
Xystocheir haerens
Xystocheir reducta
Xystocheir stolonifera
Xystocheir stenomacris
Parcipromus tiemanni
Xystocheirini
Parcipromus cooki
Parcipromus gigantoarboricolus
Xystocheir prolixorama
Xystocheir dissecta
Wamokia remota
Wamokia discordis
Wamokia placera
Wamokia sierrae
Wamokia hoffmani
Wamokia falcata
Wamokia dentata
Anombrocheir spinosa
Anombrocheir bifurcata
Xystocheir brachymacris
Xystocheir solenofurcata
Motyxia ollae
Motyxia alia
Motyxia sequoia
Motyxia kerna
Motyxia sequoiae
1
Motyxia tiemanni
Motyxia porrecta
Motyxia tularea
Motyxia pior
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Motyxia monica
Motyxia bistipita
Sigmocheir furcata
Sigmocheir calaveras Sigmocheirini
Sigmocheir maculifer
Riukiaria semicircularis
Riukiaria montana
Riukiaria cornuta
Riukiaria uncata
Riukiaria rosulans
Riukiaria maculata
Xystodesminae 2 Riukiaria puella
Riukiaria hostii
Xystodesmini
Riukiaria falcifera
Yaetakaria neptuna
Riukiaria mundyi
Riukiaria chelifera
Xystodesmus serrulatus
Xystodesmus martensii
Xystodesmus “Tsurukoba”
Orophe unicus
Orophe cabinetus
Isaphe convexa
Thrinaphe hargeri
Selenocheir sinuata
Harpaphe haydeniana
Metaxycheir prolata
Tubaphe levii
Montaphe elrodi
Chonaphe armata
Semionellus placidus
Levizonus thaumasius
Levizonus takakuwai
Parafontaria tonominea
Parafontaria tokaiensis
Parafontaria longa
Parafontaria crenata Parafontariini
Parafontaria ”Tsukuba”
Parafontaria ishiii
Parafontaria erythrosoma
Ochthocelata adynata Ochthocelatini
Macellelophus
Gomphodesmidae rubromarginatus (Chelodesmidae)
Melaphe vestita
Orodesminus “Gorongosa” (Oxydesmidae)
Sphaeriodesmidae
Neoleptodesmus (Rhachodesmidae)
Amplinus bitumidus (Aphelidesmidae)
Fig. 2. Phylogeny estimated using Bayesian inference from the ‘full-taxa’ dataset (using all available data), part 1: subfamily Xystodesminae and outgroups.
Branch colors indicate posterior probability (orange, posterior probability < 0.7; magenta, posterior probability < 0.95; black, posterior probability < 1.0). Tree
notes: 1. Origin of bioluminescence in the genus Motyxia from California. 2. Genus Riukiaria from Japan with 11 taxa represented out of 33 species. Blue text.
Semionellus placidus (Wood, 1864), a markedly disjunct species from eastern North America, is in the western North American (WNA) clade. Magenta text.
Ochthocelata adynata Shelley, 1995, from California, is the sole adelphotaxon to the Xystodesminae. Scale bar: 0.1 expected substitutions per site. (Note: the
whole tree was cut in two parts with part 1 here and part 2 in Fig. 3.)
including the Rhysodesmini and Xystodesmini are not. The status DEC+J model was the best fit given the data (df = 1, P < 0.0001). The
of these taxa with respect to the phylogeny vary. Separate gene trees species Melaphe vestita, which occurred outside of Xystodesmidae
show varying levels of support from deeper relationships in the 28S on the phylogeny, is known from the Aegean and Western Turkey
topology to shallow relationships in COI (Supp Fig. 1 [online only]). sclerophyllous and mixed forests (AWTS). The root node estimated
Data matrices and trees are available for download from the Supp from the BioGeoBEARS was equivocal between this AWTS eco-
Material (online only) (Supp Dataset 1 [online only]). region and the Sierra Nevada forests ecoregion (California, USA).
The root node of the remaining Xystodesmidae, including Japanese
Biogeographical Analysis species, was the Sierra Nevada forests (California). The major clades
Taxa were distributed in 26 ecoregions (Olson et al. 2001), including of Xystodesmidae occur in the following ecoregions: Sierra Nevada
the Hokkaido deciduous forests and Taiheiyo evergreen forests in forests (California), Taiheiyo evergreen forests (Japan), and the
Japan, Tamaulipan mezquital and Sierra de los Tuxtlas in Mexico, Appalachian Blue Ridge forests (Fig. 4).
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 Discovery and Delimitation
Species primarily occurred in broadleaf deciduous forests, but some In most cases, conditional new species (Hmorph=yes) were not sister
taxa were found in evergreen forests and chaparral shrublands. to their presumed closest relatives (hypothesized a priori based
The likelihoods of the DEC and DEC+J models were -666.54 and solely on morphological similarity) in the concatenated ‘full-
-505.65, respectively, and likelihood ratio test indicated that the taxa’ molecular phylogeny (Hphy=yes). Because these conditional
Insect Systematics and Diversity, 2021, Vol. 5, No. 2 7
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Fig. 3. Phylogeny estimated using Bayesian inference from the ‘full-taxa’ dataset (using all available data), part 2: subfamily Rhysodesminae. Branch colors
indicate posterior probability (orange, posterior probability < 0.7; magenta, posterior probability < 0.95; black, posterior probability < 1.0). (A–H) Representative
millipedes in the Rhysodesminae: (A) Brachoria cedra Keeton, 1959. (B) Apheloria virginiensis corrugata (Wood, 1864). (C) Appalachioria calcaria (Keeton, 1959).
(D) Parvulodesmus stephani Shelley, 2001. (E) Rhysodesmus ‘Hobbs Island’. (F) Nannaria hokie n. sp. (G) Nannaria ericacea Hoffman, 1949. (H) Oenomaea
pulchella (Bollman, 1889). Tree notes: 3. The tribe Apheloriini, primarily distributed in the Appalachian Mountains and with about one-third of the family’s species
diversity is a possible crown group taxon. 4. The genus Nannaria includes a substantial number of undescribed species (ca. 56 species) predominantly in the
Appalachian Mountains of the eastern United States, highlighting the region as the global center of undescribed species diversity in Xystodesmidae. 5. The
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.).
8 Insect Systematics and Diversity, 2021, Vol. 5, No. 2
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Fig. 4. Distribution map of Xystodesmidae with its phylogeny and selected ancestral areas of its taxa. Continental locations of the distribution of the subfamily
Xystodesminae (red-filled outline) in East Asia and western North America, the subfamily Rhysodesminae (blue-filled outline) in eastern North America
and southwestern North America, the subfamily Melaphinae (green-filled outline) in the Mediterranean Region and Ethiopia; and Devilleini (yellow-filled
outline) in the Mediterranean Region. Ancestral areas: A = Aegean and Western Turkey sclerophyllous and mixed forests, B = Appalachian Blue Ridge forests,
C = Appalachian mixed mesophytic forests, E = California interior chaparral and woodlands, N = North Central Rockies forests (United States), R = Sierra Nevada
forests, T = Southeastern mixed forests (United States), V = Taiheiyo evergreen forests (Table 1). Terrestrial ecoregions according Olson et al. (2001).
new species were distinct morphologically and phylogenetically updated on the basis of the phylogenetic analysis, is provided in the
(Hmorph=yes, Hphy=yes), they were described as new species. However, Supp Material (online only) (Supp Text 1 [online only], Supp Dataset
four conditional new species were separated from their closest hy- 2 [online only]). The majority of previously described subspecies were
pothesized relatives by a single branch (Hphy=no), possessed slight not found to be conspecifics, e.g., Rudiloria tortua, Appalachioria
morphological genitalic differences (Hmorph=no) and therefore were ethotela, Appalachioria eutypa, Appalachioria hamata, Sigmoria
not described as distinct species: Brachoria ‘Corinth’, Rudiloria intermedia, Sigmoria nigrimontis, Sigmoria latior, Dicellarius fictus,
‘Monongahela’, Pleuroloma ‘Little Stony’, and Rhysodesmus Pachydesmus duplex, and Motyxia tularea. Due to these results, we
‘Hobbs Island’. The following taxa were represented by a single have therefore raised subspecies to full species status.
male specimen: Rudiloria ‘Paddy Knob’, Brachoria platana (plus
female specimen), and Brachoria ‘Dungannon’. Therefore, we de- Xystodesmidae Taxonomy
termined that the evidence supporting these three taxa as new
Class Diplopoda de Blainville in Gervais, 1844
species was lacking compared to that for others; however, we
Infraclass Helminthomorpha Pocock, 1887
described Brachoria platana based on the presence of two speci-
Order Polydesmida Leach, 1815
mens, a male and female.
Family Xystodesmidae Cook, 1895
Subfamily Rhysodesminae Brolemann, 1916
Taxonomy Tribe Apheloriini Hoffman, 1980
The species descriptions of new millipedes in the family Xystodesmidae A comprehensive species list and higher-level classification of the
are provided in Figs. 5–15. Diagnoses of tribes and an identification family Xystodesmidae is provided in the Supp Material (online only)
key are provided with syntheses of identification resources for each (Supp Text 1 [online only], Supp Dataset 2 [online only]).
tribe. A species list and higher-level classification of Xystodesmidae, Genus Appalachioria Marek and Bond, 2006.
Insect Systematics and Diversity, 2021, Vol. 5, No. 2 9
Table 1. List of the terrestrial ecoregions of the world in which the ventromedially at apex, with a distal cingulum, separating it from
taxa of the family Xystodesmidae sampled in this analysis occurs. Apheloria and Rudiloria (Fig. 6). Prefemoral process short and
Ecoregions according Olson et al. 2001. stout, basal zone lacking tubercles. Acropodite at anterior bend
with three stout, triangular dorsal tubercles, separating it from
Biogeographic realm Ecoregion Label
most other Appalachioria species. Distal zone constricted basally,
Palearctic with a medially curved, blunt, uncinate tip. Color. Appalachioria
Aegean and Western Turkey scler- A bondi n. sp. has multiple color morphs, often co-occurring (Fig.
ophyllous and mixed forests 5A–G). Tergites always with white, light-pink, or yellow paranotal
Hokkaido deciduous forests J spots, sometimes also with concolorous metatergal middorsal
Nansei Islands subtropical ever- M
spots. Dark-brown to black background. Collum always with
green forests
concolorous white, light-pink, or yellow anterior and lateral spots,
Taiheiyo evergreen forests V
Ussuri broadleaf and mixed forests X sometimes also with a posterior spot, never with marginal lines
Nearctic connecting the spots.
Appalachian Blue Ridge forests B
Appalachian mixed mesophytic C Description: Supp Table 3 (online only). Based on Holotype (♂)
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forests SPC000282.
Arizona mountains forests D Measurements (mm): BL = 42.0, CW = 8.5, IW = 5.2, ISW = 1.4,
California interior chaparral and E
B11W = 9.9, B11H = 5.8.
woodlands
Central forest-grasslands transition F
Central Pacific coastal forests G Variation: The dorsal tubercles on the anterior bend of the
Central U.S. hardwood forests H acropodite vary in size and number, ranging from 1 to 3
Eastern Cascades forests I (exceptionally up to 10) small to stout triangular tubercles,
Middle Atlantic coastal forests L sometimes serrated or divided apically. The tubercles can vary
North Central Rockies forests N from dorsal to medial in position. The tip of the acropodite may
Ozark mountain forests O be curved medially to ventrally. The prefemoral process ranges
Piney woods forests P
from short and thin to long and stout and varies from small to
Sierra Nevada forests R
medium in size. Appalachioria bondi n. sp. exhibits six unique
Southeastern conifer forest S
Southeastern mixed forests T color patterns: yellow four-spotted collum (Fig. 5A), red two-
Southern Great Lakes forests U spotted, collum with one anterior and two lateral red spots (Fig.
Tamaulipan mezquital W 5B), yellow two-spotted, collum with one anterior and two lateral
Flint Hills tall grasslands Z yellow spots (Fig. 5E), white four-spotted collum (Fig. 5C), white
Neotropic two-spotted, collum with one anterior and two lateral white spots
Sierra de los Tuxtlas Q (Fig. 5F) and red four-spotted collum (Fig. 5D).
Vernacular name: ‘The Appalachian Mimic Millipedes’ Ecology: Individuals of Appalachioria bondi n. sp. have been found
Type species: Brachoria falcifera Keeton, 1959. in mesic deciduous forests of oak, beech, maple, tuliptree, birch,
Taxa included: 11 species, see Supp Material (online only) (Supp buckeye, and sassafras, with small patches of eastern redcedar
Text 1 [online only], Supp Dataset 2 [online only]). nearby.
Distribution: Appalachioria bondi n. sp. is only known from a small
Appalachioria bondi Marek, Means, Hennen, range of about 8 km2 on Clinch Mountain in Russell and Washington
New Species Counties, Virginia.
Vernacular Name: ‘Bond’s Appalachian Mimic
Millipede’ Etymology: This species is named for Dr. Jason Bond of the
University of California, Davis. The specific name is a genitive noun
Figs. 5A–G and 6. derived as a patronym.
Brachoria ‘Clinch’ (Marek and Bond 2006)
Appalachioria ‘n. sp. Clinch Mountain’ (Marek and Bond 2007,
Means and Marek 2017)
Appalachioria sierwaldae Marek, Means,
Hennen, New Species
Material examined: Type specimens. ♂ holotype (VTEC Vernacular Name: ‘Sierwald’s Appalachian Mimic
SPC000282), ♂ paratype (VMNH SPC000280), and ♀ paratypes Millipede’
(VTEC SPC000281, 283) from Virginia, Washington County, Clinch Figs. 5H–I and 7.
Mountain, CR-612 (36.72317°N, -82.2985°W, Elev. 635 m), 28
May 2004, 15:00, tulip, birch, oak, sassafras (Coll: P. E. Marek). Appalachioria n. sp. ‘Foster Falls’ Means and Marek 2017
Material examined listed in Supp Table 2 (online only).
Material examined: Type specimens. ♂ holotype (VTEC
Diagnosis: Adult males of Appalachioria bondi n. sp. are distinct SPC000296), 1 ♂ paratype (VMNH SPC000297), and 2 ♀
from other apheloriine species based on the following combination paratypes (VTEC SPC000294, 295) from Virginia, Wythe County,
of characters: Gonopods. Gonopodal acropodite curving N slope Fosters Falls Mountain, cove near road (36.89021°N,
10 Insect Systematics and Diversity, 2021, Vol. 5, No. 2
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Fig. 5. Color photographs of new species of xystodesmid millipedes and their color morphs. Appalachioria bondi n. sp. (A–G): A Three-spotted yellow morph
with yellow legs, dorsal view, B Two-spotted pink morph with pink legs, dorsal view, C Three-spotted white morph with white legs, D Three-spotted orange
morph with orange legs, E Two-spotted yellow morph with pink legs, F. Two-spotted white morph with white legs, G Three-spotted yellow morph with pink legs,
dorsal view. Appalachioria sierwaldae, n. sp. (H–I): H. Three-spotted morph with yellow paranotal spots and orange medial spots and orange legs. I. Three-
spotted yellow morph with yellow legs. Appalachioria brownae n. sp. (J–K): J. Three-spotted yellow morph with yellow legs. K. Two-spotted yellow morph with
yellow legs. Brachoria camptera n. sp. (L–M): L. Two-spotted orange morph with white legs. M. Two-spotted red morph with red legs. Brachoria cryocybe n. sp.
(N): N. Two-spotted morph with red paranotal spots and white stripe and red legs. Brachoria platana n. sp. (O–P): O. Striped red morph with red legs. P. Three-
spotted yellow morph with yellow legs. Rudiloria charityae n. sp. (Q): Q. Three-spotted morph with yellow paranotal spots and orange medial spots and yellow
legs. Sigmoria beameri n. sp. (R): R. Two-spotted red morph with light red legs. Nannaria aenigma n. sp. (S–T): S. Two-spotted red morph with light red legs.
T. Two-spotted white morph with white legs. Nannaria hokie n. sp. (U): Two-spotted red morph with gray legs.Insect Systematics and Diversity, 2021, Vol. 5, No. 2 11
-80.8376°W, Elev. 657 m), 29 May 2004, tulip, oak, maple, Distribution: Only known from the type locality on Fosters Falls
rhododendron, moist cove (Coll: P. E. Marek). Material examined Mountain in eastern Wythe County, Virginia.
listed in Supp Table 2 (online only).
Etymology: This species is named for Dr. Petra Sierwald of the Field
Diagnosis: Adult males of Appalachioria sierwaldae n. sp. are distinct Museum of Natural History. The specific name is a genitive noun
from other apheloriine species based on the following combination derived as a matronym.
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. Appalachioria brownae Marek, Means,
Post-cingulum area expanded, wider than pre-cingulum area. Distal Hennen, New Species
zone strongly curved medially into a long, uncinate, thin tip. Color.
Vernacular Name: ‘Brown’s Appalachian Mimic
Tergites with yellow paranotal spots and yellow metatergal spots
Millipede’
(Fig. 5I), but sometimes with orange metatergal spots (Fig. 5H). Black
background. Collum with yellow lateral and anterior spots, sometimes Figs. 5J,K and 8.
with yellow or orange posterior spots, or lacking a posterior spot.
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Material examined: Type specimens. ♂ holotype (VTEC
Description: Supp Table 3 (online only). Based on Holotype (♂) MPE01676), 2 ♂ paratypes (VTEC MPE01663, VMNH
SPC000296. MPE01664), and 2 ♀ paratypes (VTEC MPE01675, 1678) from
Measurements (mm): BL = 40.0, CW = 7.0, IW = 4.7, ISW = 1.4, Virginia, Washington County, Jefferson National Forest, south
B11W = 9.4, B11H = 5.4. of Laurel Bed Lake, off Tumbling Creek Rd. (36.93845°N,
-81.8118°W, Elev. 956 m), 13 June 2016, ex maple, oak, big leaf
Variation: No significant variation from the holotype was observed. magnolia woods, quite dry (Coll: J. C. Means, D. A. Hennen).
Material examined listed in Supp Table 2 (online only).
Ecology: Individuals of Appalachioria sierwaldae n. sp. were found
in an Appalachian cove forest that included tulip, oak, maple, and Diagnosis: Adult males of Appalachioria brownae n. sp. are distinct
rhododendron. from other apheloriine species based on the following combination
Fig. 6. Appalachioria bondi n. sp. holotype ♂ left gonopod (VTEC SPC000282). (A) Anterior view; (B) Medial view; (C) Posterior view. Scale bar = 1.0 mm.
Fig. 7. Appalachioria sierwaldae n. sp. holotype ♂ left gonopod (VTEC SPC000296). (A) Anterior view; (B) Medial view; (C) Posterior view. Scale bar = 1.0 mm.12 Insect Systematics and Diversity, 2021, Vol. 5, No. 2
of characters: Gonopods. Gonopodal acropodite strongly curving Genus Brachoria Chamberlin, 1939.
ventromedially and with a strong distal cingulum, separating it from
Vernacular name: ‘The Mimic Millipedes’
Apheloria and Rudiloria (Fig. 8). Prefemoral process short and stout.
Type species: Brachoria initialis Chamberlin, 1939.
Basal zone medially with small triangular tubercles, separating it
Taxa included: 39 species, see Supp Material (online only) (Supp
from other co-occurring Appalachioria species. Post-cingulum area
Text 1 [online only], Supp Dataset 2 [online only]).
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,
Brachoria camptera Means, Hennen, Marek,
midbody tergites with broken yellow caudal line connecting the New Species
paranotal and metatergal spots. Black background. Collum with Vernacular Name: ‘The City Lake Mimic Millipede’
yellow anterior and lateral spots connected by a yellow marginal Figs. 5L,M and 9.
line, sometimes also with a caudal marginal line.
Material examined: Type specimens. ♂ holotype (VTEC
Description: Supp Table 3 (online only). Based on Holotype (♂)
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MPE01398), 6 ♂ paratypes (VTEC MPE01323, 1325, 1399;
MPE01676. VMNH MPE01403, 1410, 1416), and 2 ♀ paratypes (VTEC
Measurements (mm): BL = 44.0, CW = 7.0, IW = 4.8, ISW = 1.5, MPE01405, 1413) from Tennessee, Putnam County, Cookeville,
B11W = 9.1, B11H = 5.2. City Lake Natural Area, along trail out of parking lot (36.13143°N,
-85.4439°W, Elev. 303 m), 21 May 2016, ex beech, maple,
Variation: There is slight variation in the number and position of deciduous woods, tuliptree, oak, umbrella magnolia, hickory, moist
tubercles on the acropodite basal zone, with numbers ranging from litter on hillsides, understory of poison ivy, honeysuckle, mayapple
4 to 6, spread from the base of the basal zone to halfway up the (Coll: J. C. Means, D. A. Hennen). Material examined listed in
acropodite. The prefemoral process ranges from quite small and Suppl Table 2 (online only).
stout to long and thin, reaching to the level of the distal zone of
the acropodite. Appalachioria brownae n. sp. has two known color Diagnosis: Adult males of Brachoria camptera n. sp. are distinct
morphs, 1) three-spotted yellow, with small yellow metatergal spots from other apheloriine species based on the following combination
and a thin, broken caudal line, collum with yellow anterior and lateral of characters: Gonopods. Gonopodal acropodite D-shaped (Fig.
spots connected by a thin yellow marginal line (Fig. 5J) and 2) two- 9A)—not circular as in Apheloria or oval as in Rudiloria species
spotted yellow, collum with yellow anterior and lateral spots with a (Fig. 12). Acropodite (when viewed posteriorly) narrow, one-
thin anterior marginal yellow line connecting the spots (Fig. 5K). third width of tibia on leg pair 9. Acropodite gradually tapered
to acuminate apex, not with a blunt apex as in B. hubrichti.
Ecology: Individuals of Appalachioria brownae n. sp. were found Acropodite distal to anterior bend slightly curved cephalically.
in a deciduous forest of oak, maple, and big leaf magnolia near a Acropodite shaft with cingulum (midlength transverse groove).
stream during dry conditions. Prefemur with curved sickle-shaped prefemoral process (Fig. 9B).
Color. Tergites black with 2 orange (Fig. 5L), or red (Fig. 5M),
Distribution: Appalachioria brownae n. sp. is known only from the paranotal spots, not with purple/purple-gray metatergal stripes as
type locality, situated in bottomland habitat at the base of Brush in B. hubrichti and B. initialis.
Mountain and Redrock Mountain.
Description: Supp Table 3 (online only). Based on Holotype (♂)
Etymology: This species is named for Dr. Ellen Brown of MPE01398.
Fredericksburg, Virginia. The specific name is a genitive noun Measurements (mm): BL = 39.0, CW = 7.6, IW = 5.0, ISW = 1.5,
derived as a matronym. B11W = 10.0, B11H = 5.2.
Fig. 8. Appalachioria brownae n. sp. holotype ♂ left gonopod (VTEC MPE01676). (A) Anterior view; (B) Medial view; (C) Posterior view. Scale bar = 1.0 mm.Insect Systematics and Diversity, 2021, Vol. 5, No. 2 13
Variation: Individuals of Brachoria camptera n. sp. are known (midlength transverse groove). Prefemur with railroad spike-like
to exhibit two color morphs, 1) two-spotted red (Fig. 5M) and prefemoral process (Fig. 10A), not bidentate as in B. divicuma.
2) two-spotted yellow-orange with a faint light blue caudal stripe Color. Tergites black with 2 red paranotal spots, not with yellow
(Fig. 5L). spots as in B. divicuma. Caudal border of tergites with pale white
stripe (Fig. 5N).
Ecology: Individuals of Brachoria camptera n. sp. were collected in
a mesic deciduous forest with dominant tree species of oak, maple, Description: Supp Table 3 (online only). Based on Holotype (♂)
beech, and tuliptree. MPE02644.
Measurements (mm): BL = 42.0, CW = 7.2, IW = 5.2, ISW = 1.5,
Distribution: Known only from the type locality. B11W = 8.9, B11H = 5.3.
Etymology: This species is named for the shape of its acropodite, Variation: No significant variation from the holotype was observed.
specifically the angular nature of its anterior bend. The specific name
is a noun in apposition derived from the Greek kampter, ‘bend, Ecology: Individuals of Brachoria cryocybe n. sp. were collected in
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angle’. 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 cryocybe Hennen, Means, Marek, Brachoria forficata (Shelley, 1986).
New Species
Vernacular Name: ‘The Frozen Head Mimic Distribution: Known only from the type locality.
Millipede’
Figs. 5N and 10. Etymology: This species is named after Frozen Head State Park,
where it was discovered. The specific name is a noun in apposition
Material examined: ♂ holotype (VTEC MPE02644), 3 ♂ paratypes derived from the Greek kryos, ‘icy cold’, and kybe, ‘head’.
(VTEC MPE02582, 2583; VMNH MPE02653), and 1 ♀ paratype
(VTEC MPE02652) from Tennessee, Morgan County, Wartburg, Brachoria platana Means, Hennen, Marek,
Frozen Head State Park, woods below campground (36.13206°N,
New Species
-84.4978°W, Elev. 423 m), 12 May 2017, 18:45-19:38, oak,
ironwood, beech, hemlock, pawpaw woods, moist litter. (Coll: D. A. Vernacular Name: ‘The Carr Creek Mimic Millipede’
Hennen, J. C. Means, V. Wong). Material examined listed in Supp Figs. 5O,P and 11.
Table 2 (online only).
Material examined: Type specimens. ♂ holotype (VTEC MPE03250)
Diagnosis: Adult males of Brachoria cryocybe n. sp. are distinct and ♀ paratype (VTEC MPE03253) from Kentucky, Knott County,
from other apheloriine species based on the following combination Carr Creek State Park, woods behind campground and around
of characters: Gonopods. Gonopodal acropodite smoothly oval- marina (37.23148°N, -83.00081°W, Elev. 329 m), 27 September
shaped (Fig. 10A)—not circular as in Apheloria or tightly oval as 2017, 15:00, dry litter of tulip poplar, sycamore, autumn olive (Coll:
in Rudiloria species (Fig. 12). Acropodite (when viewed posteriorly) J. C. Means and D. A. Hennen). Material examined listed in Supp
narrow, one-third width of tibia on leg pair 9. Acropodite gradually Table 2 (online only).
tapered to acuminate apex—curved one-dimensionally to apex, not
twisted cephalically as in Apheloria species. Acropodal distal zone Diagnosis: Adult males of Brachoria platana n. sp. are distinct
with tooth on posterior margin. Acropodite shaft with cingulum from other apheloriine species based on the following combination
Fig. 9. Brachoria camptera n. sp. holotype ♂ left gonopod (VTEC MPE01398). (A) Anterior view; (B) Medial view; (C) Posterior view. Scale bar = 1.0 mm.You can also read
