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ZooKeys 1115: 1 17—150 (2022)

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Two new species of the dwarf centipede
genus Nannarrup Foddai, Bonato,

Pereira & Minelli, 2003 (Chilopoda,
Geophilomorpha, Mecistocephalidae) from Japan

Sho Tsukamoto', Satoshi Shimano’, Katsuyuki Eguchi!?

I Systematic Zoology Laboratory, Graduate School of Science, Tokyo Metropolitan University, Minami-
osawa 1-1 Hachioji-shi, Tokyo 192-0397, Japan 2 Science Research Center, Hosei University, Fujimi 2-17-
1 Chiyoda-ku, Tokyo 102-8160, Japan 3 Department of International Health and Medical Anthropology,
Institute of Tropical Medicine, Nagasaki University, Sakamoto 1-12-4, Nagasaki, Nagasaki 852-8523, Japan

Corresponding author: Sho Tsukamoto (esutukamoto153@gmail.com)

Academic editor: Lucio Bonato | Received 17 March 2022 | Accepted 6 July 2022 | Published 1 August 2022
Attps://zoobank. org/AB8 9506 1-8444-4026-B5 93-3733 LE598259

Citation: Tsukamoto S, Shimano S, Eguchi K (2022) Two new species of the dwarf centipede genus Nannarrup
Foddai, Bonato, Pereira & Minelli, 2003 (Chilopoda, Geophilomorpha, Mecistocephalidae) from Japan. ZooKeys
1115: 117-150. https://doi.org/10.3897/zookeys.1115.83946

Abstract

The genus Nannarrup Foddai, Bonato, Pereira & Minelli, 2003 is a monotypic genus established on the
basis of the possibly introduced species NV. hoffmani Foddai, Bonato, Pereira & Minelli, 2003, from New
York, USA. In the present study, in a field survey conducted throughout Japan, Nannarrup-like specimens
were collected from Honshu, Shikoku, and Kyushu. These specimens clearly showed the diagnostic char-
acteristics of the genus but were morphologically distinct from N. hoffmani. Furthermore, morphological
analysis and DNA barcoding revealed that these specimens could be assigned to two distinct undescribed
species. On the basis of these results, NV. innuptus Tsukamoto, sp. nov. and NV. oyamensis Tsukamoto,
sp. nov. are described. The three Nannarrup species can be distinguished from each other on the basis of
the following combination of characteristics: presence or absence of a pair of smooth or weakly areolate
areas along the posterior part of the paraclypeal sutures; the width-to-length ratio of the denticle on the
trochanteroprefemur; the pigmentation of the denticle on the tarsungulum. Moreover, the field survey re-
sulted in the collection of exclusively female specimens of N. innuptus Tsukamoto, sp. nov., which shows

the possibility of parthenogenesis of this species.

Keywords
Description, DNA barcoding, molecular phylogeny, morphology, sex ratio, taxonomy

Copyright Sho Tsukamoto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

118 Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

Introduction

The geophilomorph family Mecistocephalidae Bollman, 1893, is a distinct monophy-
letic group, which is well characterized morphologically by a cephalic capsule and the
forcipular segment that are obviously sclerotized and darker than the remaining trunk
segments, the mandible with a series of pectinate lamellae only, trunk sternites with
an internal apodeme, a mid-longitudinal sulcus, and the intraspecific invariance in the
segment number, except in some species of the genus Mecistocephalus Newport, 1843
(Bonato et al. 2003, 2014; Uliana et al. 2007; Bonato 2011). The family is distributed
mainly in tropical and subtropical regions and especially diversified at the species and
higher phylogenetic levels in East Asia (Uliana et al. 2007; Bonato 2011). The fam-
ily Mecistocephalidae comprises three subfamilies: Mecistocephalinae Bollman, 1893;
Dicellophilinae Cook, 1896; and Arrupinae Chamberlin, 1912.

The subfamily Arrupinae comprises four valid genera: Arrup Chamberlin, 1912;
Agnostrup Foddai, Bonato, Pereira & Minelli, 2003; Partygarrupius Verhoeff, 1939;
Nannarrup Foddai, Bonato, Pereira & Minelli, 2003. Arrupinae has been reported
mainly in East Asia and is diversified at the species level in Japan (Foddai et al. 2003;
Uliana et al. 2007). To date, 21 species are reported in East Asia (18 spp.), Central
Asia (1 sp.), California (1 sp.), New York (1 sp.), and 11 species are reported in Japan
(Bonato et al. 2016).

The genus Nannarrup was established for a single species, N. hoffmani Foddai,
Bonato, Pereira & Minelli, 2003. The genus has peculiar morphological characteristics
(for details, see the section “Taxonomic account”), which likely evolved as a result of
miniaturization. Nannarrup hoffmani was originally described on the basis of speci-
mens from New York City, USA. Foddai et al. (2003) stated that the species has been
definitely introduced from western America or East Asia. However, the native range of
the species and the genus remain unknown.

In field surveys in Honshu, Shikoku and Kyushu, Japan (2017-2022), the authors
of the present study (ST and KE) collected 88 mecistocephalid specimens, which clearly
showed the diagnostic characteristics of the genus Nannarrup. However, these “Nannarrup-
like” specimens can be distinguished from NV. hoffmani by the shape of the denticle on the
forcipular trochanteroprefemur. Therefore, the present study aimed to assign the Japanese
“Nannarrup-like” specimens to the current classification of Arrupinae using an integrative
approach of morphological analysis and DNA barcoding, using the mitochondrial COJ
and 16S ribosomal RNA genes, and the nuclear 28S ribosomal RNA genes.

Materials and methods
Taxon sampling
Eighty-eight “Nannarrup-like” specimens, including 13 juveniles (in which sex

determination is not possible), were collected by hand from Honshu (Aomori, Akita,
Iwate, Yamagata, Fukushima, Niigata, Tokyo, Kanagawa, Shizuoka, Wakayama, Hyogo,

New species of the genus Nannarrup from Japan 119

Okayama, and Yamaguchi prefectures), Shikoku (Kochi and Ehime prefectures), and
Kyushu (Fukuoka, Miyazaki, and Kagoshima prefectures). These specimens were
included as ingroup in the present study. Each specimen was specified by its own
specimen identification number in the form “TSYYYYMMDD-XX,” where TS is an
abbreviation of the first author's name, Tsukamoto Sho; YYYYMMDD designates the
date on which the specimen was collected; XX is the identification number assigned to
each specimen collected on a particular date (e.g., TS20171010-01).

Type specimens of Nannarrup were deposited at the Collection of Myriapoda,
Department of Zoology, National Museum of Nature and Science, Tokyo (NSMT),
and Museum of Nature and Human Activities, Hyogo (MNHAH). See the “Taxo-
nomic account” section for the deposition site of each type specimen. All non-type
voucher specimens of Nannarrup are retained by the first author. The collection sites of
examined specimens are shown in Fig. 1 and Taxonomic account section. The altitude
data provided by AW3D of JAXA (https://www.eorc.jaxa.jp/ALOS/jp/index_j.htm)
and the coastal line provided by the digital nation land information (https://nlftp.mlit.
go.jp/index.html) were used for generating Fig. 1.

40.0

0 10 20 30 40km
a

T
140.0

° } 0 100 200 300 400km
* © A

130.0 135.0 140.0
Figure |. Map of collection sites of specimens examined in the present study. Black circle, Nannarrup
innuptus sp. nov.; black square, the type locality of NV. innuptus sp. nov.; white circle, N. oyamensis sp. nov.;

white square, the type locality of NV. oyamensis sp. nov.

120 Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

Morphological analysis

Cephalic capsule, maxillae, mandibles, the forcipular segment and leg-bearing seg-
ments were made transparent using lactic acid or the Chelex-TE protocol to examine
the anatomy and produce images (Satria et al. 2015). Cephalic capsule and maxillae
of some specimens were mounted in Hoyer’s medium (gum arabic, chloral hydrate,
and glycerol) or Euparal. Multi-focused images of these body parts were produced us-
ing Helicon Focus Pro 6.7.1 (https://www.heliconsoft.com/helicon-focus-history-of-
changes-win/) from a series of source images taken using a Canon EOS Kiss X9 digital
camera attached to a Nikon AZ100 microscope and improved using Adobe Photoshop
Elements 10. Body parts were then measured directly using an ocular micrometer at-
tached to the microscope or by measuring on the basis of the images using Image] soft-
ware (http://image].nih.gov/ij/). The morphological terminology used in the present
study is mainly derived from Bonato et al. (2010). Specimens with fully developed
paired gonopods—that is, evidently bi-articulated in males and touching each other
in females—were determined to be adults, and those with incompletely developed
paired gonopods were determined to be subadults; specimens without gonopods were
determined to be juveniles.

DNA sequencing

Genomic DNA was extracted from one or two legs, the head, or a body segment of
each specimen by following the Chelex-TE-ProK protocol (Satria et al. 2015) with
incubation for 24 h.

PCR amplification was performed in a MiniAmp Thermal Cycler (Thermo
Fisher Scientific, Waltham, Massachusetts, USA) in a 10.5-uL reaction volume
containing 5 wL 2x PCR buffer for KOD FX Neo, 2 pL of 2 mM dNTPs, 0.3
uL of 10 pmol/uL forward and reverse primers, 0.2 pL of 1.0 U/uL DNA poly-
merase KOD FX Neo (TOYOBO KFX-201X5), and 1.0 pL DNA template. The
sequences of primers for the mitochondrial COJ and 16S and nuclear 28S genes
are shown in Table 1. Each PCR product was screened by electrophoresis on 2.0%
agarose gel in 1x TAE.

Amplification conditions for mitochondrial CO/ were as follows: 98 °C for 2 min;
5 cycles of 98 °C for 10 s, 45 °C for 30 s, and 68 °C for 45 s; 40 cycles of 98 °C for
10 s, 48.5 °C for 30 s (annealing step), and 68 °C for 45 s; and 68 °C for 7 min. If the
target fragment of CO/ was not appropriately amplified, the annealing temperature
was changed from 48.5 °C to 50 °C, and PCR was performed again by omitting the
first five cycles of annealing and the extension step.

Amplification conditions for mitochondrial /6S were as follows: 98 °C for 2 min;
35 cycles of 98 °C for 10 s, 45 °C for 30 s (annealing step), and 68 °C for 45 s; and
68 °C for 7 min. If the target fragment of 16S was not appropriately amplified, the an-
nealing temperature was changed from 45 °C to 48 °C, and the number of annealing
cycles was changed from 35 to 45.

New species of the genus Nannarrup from Japan 124

Table |. The list of primers used in the present study.

Genes Primer name Sequence (5' - 3') Source
COI LCO-CH TTT CAA CAA AYC AYA AAG ACA TYG G Tsukamoto et al. (2021)
HCO-CH TAA ACT TCT GGR TGR CCR AAR AAT CA
16S rRNA 16Sa CGC CTG TTT ATC AAA AAC AT Xiong and Kocher (1991)
16Sbi CTC CGG TTT GAA CTC AGA TCA
28S rRNA 28S D1F GGG ACT ACC CCC TGA ATT TAA GCA T Boyer and Giribet (2007)
28S rD4b CCT TGG TCC GTG TTT CAA GAC Edgecombe and Giribet (2006)

Amplification conditions for nuclear 28S were as follows: 98 °C for 2 min; 5 cycles
of 98 °C for 10 s, 42 °C for 30 s, and 68 °C for 1 min; 30 cycles of 98 °C for 10 s,
50 °C for 30 s (annealing step), and 68 °C for 1 min; and 68 °C for 7 min. If the tar-
get fragment of 28S was not appropriately amplified, the annealing temperature was
changed from 50 °C to 48 °C, and the number of annealing cycles was changed from
30 to 40-45 cycles. Furthermore, PCR was performed again by omitting the first five
cycles of annealing and the extension step.

The amplified products were incubated at 37 °C for 30 min and 80 °C for 20 min
with IllustralTM ExoStar (GE Healthcare, Buckinghamshire, UK) to remove any
excess primers and nucleotides. All nucleotide sequences were determined by direct
sequencing using ABI PRISM BigDye Terminator Cycle Sequencing Kit ver. 3.1
(Thermo Fisher Scientific) or BrilliantDyeTM Terminator Cycle Sequencing Kit v. 3.1
(Nimagen, B.V., Nijmegen, Netherlands) with an ABI 3130xl automated sequencer
(Thermo Fisher Scientific). The sequences were assembled using ChromasPro 1.7.6
(Technelysium Pty Ltd., Australia) and deposited in the databases DDBJ, EMBL, and
GenBank under the accession numbers LC715482—LC715706 (Table 2).

Molecular phylogenetic analyses

The sequences obtained using the methods described above were used for phylogenetic
analyses; the CO/, 16S, and 28S sequences of the mecistocephalid species Dicellophilus
carniolensis C.L. Koch, 1847; Mecistocephalus guildingii Newport, 1843; and
Mecistocephalus subgigas (Silvestri, 1919), obtained from GenBank were used as
outgroups (Table 2).

All sequences were aligned using MAFFT v. 7.475 (Katoh and Standley 2013). For
COI, the alignment was performed using the default setting. For 16S and 28S, second-
ary structure alignment was performed using the X-INS-i option.

Maximum-likelihood (ML) trees were created on the basis of the sequence dataset
for each gene using [Q-tree 1.6.12 (Nguyen et al. 2015). In the ML analysis for the
COT dataset, TN + F was selected for the first codon position, TNe + G4 was selected
for the second codon position, and F81 + F was selected for the third codon position
as the optimal substitution model according to the Bayesian information criterion
(BIC). In the ML analysis for the 76S dataset, TIM3 + F + I + G4 was selected as the
optimal substitution model according to BIC. In the ML analysis for the 28S dataset,

122

Table 2. The list of specimens that were used in the phylogenetic analyses.

Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

Species Accession No. Reference
COI 16S 28S
Nannarrup innuptus sp. nov. LC715482— LC715557— LC715632- the present study
LC715554 =LC715629 ~LC715704
Nannarrup oyamensis sp. nov. LC715455— LC715530— LC715605- the present study
bE7IS556-" "Ler lo6a— her15706
Dicellophilus carniolensis KF569305 HM453225 HM453285 Murienne et al. (2010),
(C.L. Koch, 1847) Bonato et al. (2014)
Mecistocephalus guildingii AY288747 = AY288728 HM453283 Edgecombe and Giribet (2004),
Newport, 1843 Murienne et al. (2010)
Mecistocephalus subgigas (Silvestri, 1919) AF370837 AF370862 HM453284 Giribet et al. (2001),

Murienne et al. (2010)

TIM3e + G4 was selected as the optimal substitution model according to BIC. Ultrafast
bootstrap analysis (UFBoot; Hoang et al. 2018) and SH-like approximate likelihood
ratio test (SH-aLRT; Guindon et al. 2010) were performed with 1,000 replicates.

Calculation of genetic distances

Aligned datasets (COJ, 16S, and 285) used for phylogenetic analyses were also used to
calculate the genetic distances. Pairwise p-distances and Kimura-two-parameter (K2P)
distances were calculated for each of the three genes of “Nannarrup-like” specimens
using MEGA X (Kumar et al. 2018) using the setting “pairwise deletion.”

Results

Taxonomic position of “Nannarrup-like” specimens

All 88 “Nannarrup-like” specimens collected in Japan possessed the diagnostic char-
acteristics of the subfamily Arrupinae (Bonato et al. 2003): body tapering backwards;
leg-bearing trunk uniform in color, without dark patches; clypeus with 11-17 clypeal
setae, placed in two lateral areas; internal margin of labral anterior ala reduced to a
pointed end; posterior alae without longitudinal stripes; posterior margin of labral
side-piece sinuous (and with short fringe, in contrast to “diagnosis” of Arrupinae in
Bonato et al (2003)); cerrus absent.

Furthermore, these “Nannarrup-like” specimens possessed the diagnostic charac-
teristics of the genus Nannarrup established by Foddai et al. (2003) on the basis of
N. hoffmani: body length ca 10-mm long in adults, with 41 pairs of legs; frontal line
absent; clypeus with two small plagulae; clypeal ratio ca 1:6—-1:7; cephalic pleurites
with a pair of short stili, without setae and a pair of spicula; side pieces of labrum
subdivided into anterior and posterior alae; mandible with four well-developed pec-
tinate lamellae; first maxillae separated from each other by a longitudinal line at the

New species of the genus Nannarrup from Japan 123

coxosternite; second maxillae not separated from each other at the coxosternite, with
metameric pores close to the posterior margin, without claw; forcipular telopodites not
reaching the anterior margin of the head in the closed position; trochanteroprefemur
with single distal denticle; forcipular femur without teeth; tarsungulum with basal,
well-developed denticle; forcipular tergite without median sulcus; sternites with sul-
cus, which is not anteriorly furcate; last metasternite subtriangular; ventral surface of
each coxopleurite with numerous pores; anal pores present.

These “Nannarrup-like” specimens (and even N. hoffmani) can be distinguished
from the genus Arrup, which is speciose in the Japanese Archipelago (Uliana et al.
2007), by first maxillae separated from each other by a longitudinal line at the coxos-
ternite. In addition, they can be distinguished from Agnostrup and Partygarrupius, that
are also known from Japan, by two small clypeal plagulae (plagulae of Agnostrup and
Partygarrupius covered one-half to most of their clypeus (Foddai et al. 2003; Uliana
et al. 2007)). Therefore, these “Nannarrup-like” specimens were confidently assigned
to Nannarrup.

Morphological comparison between Japanese Nannarrup and N. hoffmani

The Japanese Nannarrup specimens were distinguishable from NV. hoffmani on the basis
of two morphological characteristics: the width-to-length ratio of the denticle of the tro-
chanteroprefemur, which was 1:0.53 in NV. hoffmani (measured from fig. 14 in Foddai et
al. (2003)) but 1:1.3—1.6 in the Japanese specimens; pigmentation of the denticle on the
tarsungulum was less prominent than that of the denticle on the trochanteroprefemur
in NV. hoffmani but equivalent to that in the Japanese specimens (Foddai et al. 2003).

In two specimens from Kanagawa prefecture, in the Kanto Region of Japan
(TS20210217-04 and TS20210725-02), “two additional smooth areas along the pos-
terior part of the paraclypeal sutures” in the clypeus (sensu Foddai et al. (2003)) were
not observed. However, this characteristic was observed in the remaining Japanese
Nannarrup specimens.

Therefore, the Japanese Nannarrup specimens were divided into two morphospe-
cies, namely NV. sp. 1 and WN. sp. 2 (see Table 3), both of which were morphologically
distinct from N. hoffmani.

Table 3. Morphological comparison among three species of the genus Nannarrup.

Species Clypeus Forcipule
Two additional The width to length Pigmentation of the
smooth areas along ratio of the denticle on tooth on the tarsungulum
paraclypeal sutures trochanteroprefemur

Nannarrup innuptus sp. nov. + 1: 1.3-1.6 equal to the denticle on
(= Nannarrup sp. 1) trochanteroprefemur
Nannarrup oyamensis sp. nov. - i alee. equal to the denticle on
(= Nannarrup sp. 2) trochanteroprefemur
Nannarrup hoffmani Foddai, Bonato, + 1: 0.5 slighter than the denticle

Pereira & Minelli, 2003 on trochanteroprefemur

124 Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

Molecular phylogenetic analyses

The COL, 16S, and 28S sequences were successfully determined for 73 specimens of
Nannarrup sp. 1 (except for two specimens from Tokyo, i.e., TS20171010-01 and
TS20180627-01) and both specimens of N. sp. 2.

In the ML phylogenetic trees based on the CO/ dataset (Fig. 2), the clade comprising
N. sp. 1 and N. sp. 2 was moderately supported (UFBoot = 81.2%, SH-aLRT = 94%).
However, NV. sp. 1 and NV. sp. 2 were distinctly separated from each other, and each mor-

phospecies was strongly supported in monophyly (UFBoot = 98.5%, SH-aLRT = 96%

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Figure 2. Maximum-likelihood tree of Nannarrup specimens and outgroups (Mecistocephalus guildingii,
M. subgigas, and Dicellophilus carniolensis) based on the COJ dataset. Nodal values are derived from Ultrafast
bootstrap (UFBoot) and SH-like approximate likelihood ratio test (SH-aLRT). Asterisk (*) indicates values
of 100% in UFBoot and SH-aLRT. A hyphen (-) indicates values <95% in UFBoot and <80% in SH-aLRT.
Nodal values are not shown when both UFBoot and SH-aLRT values are <95% and <80%, respectively.
The unit of evolutionary distance is the number of base substitutions per site. Ingroup specimens are shown
as their specimen identification number. Abbreviations: Ao = Aomori pref.; Ak = Akita pref.; Iw = Iwate
pref.; Ya = Yamagata pref.; Fs = Fukushima pref.; Ni = Niigata pref.; To = Tokyo pref.; Kn = Kanagawa pref.;
Sh = Shizuoka pref.; Wa = Wakayama pref.; Hy = Hyogo pref.; Ok = Okayama pref.; Yu = Yamaguchi pref;
Ko = Kochi pref.; Eh = Ehime pref.; Fo = Fukuoka pref.; Mi = Miyazaki pref.; Kg = Kagoshima pref.

New species of the genus Nannarrup from Japan 125

in NV. sp. 1; UFBoot = 99.7%, SH-aLRT = 100% in WN. sp. 2). The intraspecific phy-
logeographic structure of NV. sp. 1 remains unclear because of low support values. It is
noteworthy that one specimen, 1$20210725-01, had been collected near the collection
site of V. sp. 2, but this specimen was included in the WN. sp. 1 clade (see also Fig. 1).
The ML phylogenetic trees based on the 16S dataset (Fig. 3) showed that the clade
comprising JV. sp. 1 and N. sp. 2 was highly supported (UFBoot = 98.9%, SH-aLRT
= 100%), and N. sp. 1 and XN. sp. 2 were also distinctly separated from each other.
Each morphospecies was also strongly supported in their monophyly, as observed in

the phylogenetic trees based on the CO/ dataset (UFBoot = 92.4%, SH-aLRT = 97%

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Figure 3. Maximum-likelihood tree of Nannarrup specimens and outgroups (Mecistocephalus guildingii,
M. subgigas, and Dicellophilus carniolensis) based on the 16S dataset. Nodal values are derived from Ultrafast
bootstrap (UFBoot) and SH-like approximate likelihood ratio test (SH-aLRT). Asterisk (*) indicates values
of 100% in UFBoot and SH-aLRT. A hyphen (-) shows < 95% in UFBoot and < 80% in SH-aLRT. Nodal
values are not shown when both UFBoot and SH-aLRT values are < 95% and < 80%, respectively. The
unit of evolutionary distance is the number of base substitutions per site. Ingroup specimens are shown
as their specimen identification number. Abbreviations: Ao = Aomori pref.; Ak = Akita pref.; Iw = Iwate
pref.; Ya = Yamagata pref.; Fs = Fukushima pref.; Ni = Niigata pref.; To = Tokyo pref.; Kn = Kanagawa
pref.; Sh = Shizuoka pref.; Wa = Wakayama pref.; Hy = Hyogo pref.; Ok = Okayama pref.; Yu = Yamaguchi
pref.; Ko = Kochi pref.; Eh = Ehime pref.; Fo = Fukuoka pref.; Mi = Miyazaki pref.; Kg = Kagoshima pref.

126 Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

in NV. sp. 1; UFBoot = 99.5%, SH-aLRT = 100% in WN. sp. 2). The intraspecific phy-
logeographic structure of NV. sp. 1 remains obscure because of low support values.
TS20210725-01 is included in the NV. sp. 1 clade as seen in the CO/ trees.

In the ML phylogenetic trees based on the 28S dataset (Fig. 4), the clade comprising
N. sp. 1 and WN. sp. 2 was strongly supported (UFBoot = 98.3%, SH-aLRT = 99%).
The monophyly of NV. sp. 1 was not well supported (UFBoot = 62.6%, SH-aLRT
= 49%), but the monophyly of N. sp. 2 was moderately supported (UFBoot = 91.7%,
SH-aLRT = 97%).

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3 Ak Nannarrup innuptus sp. nov.

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~~ _| | 1S20201024-03 Ok,
98.3/99 ‘TS$20211030-01 Ni

TS$20210503-02 Sh Paratype

peace Soe 06 Sh Furstpe
TS20. ee -09 Sh Holotype

0) Sh Paratype
‘aral e
eo

TS20220301-05 K
99.8/* T$20220301-06 Ko

TS20190702-06 Kg
TS20220302- at Ko

TS ‘| ['$20220301-01 Ko
~~1'7820220307-13 Eh

8303 1021708 Ken 0) Nannarrup oyamensis sp. nov.
= ; Dicellophilus carniolensis
Mecistocephalus subgigas

Mecistocephalus guildingii
0.03

Figure 4. Maximum-likelihood tree of Nannarrup specimens and outgroups (Mecistocephalus guildingii,
M. subgigas, and Dicellophilus carniolensis) based on the 28S dataset. Nodal values are derived from Ultra-
fast bootstrap (UFBoot) and SH-like approximate likelihood ratio test (SH-aLRT). Asterisk (*) indicates
values of 100% in UFBoot and SH-aLRT. A hyphen (-) shows < 95% in UFBoot and < 80% in SH-aLRT.-
Nodal values are not shown when both UFBoot and SH-aLRT values are < 95% and < 80%, respectively.
The unit of evolutionary distance is the number of base substitutions per site. Ingroup specimens are shown
as their specimen identification number. Abbreviations: Ao = Aomori pref.; Ak = Akita pref.; Iw = Iwate
pref.; Ya = Yamagata pref.; Fs = Fukushima pref.; Ni = Niigata pref.; To = Tokyo pref.; Kn = Kanagawa
pref.; Sh = Shizuoka pref.; Wa = Wakayama pref.; Hy = Hyogo pref.; Ok = Okayama pref.; Yu = Yamaguchi
pref.; Ko = Kochi pref.; Eh = Ehime pref.; Fo = Fukuoka pref.; Mi = Miyazaki pref.; Kg = Kagoshima pref.

New species of the genus Nannarrup from Japan 127

DNA barcoding of the Japanese Nannarrup specimens

According to the CO/ dataset of Nannarrup, the minimum divergence between WN. sp. 1
and NV. sp. 2 was 14.13% in p-distance and 15.73% in K2P distance (TS20211101-15
and TS20211101-16 from Nagasaki prefecture vs TS20210217-04 and TS20210725-
02 from Kanagawa prefecture), whereas the maximum internal divergence within
N. sp. 1 was 9.726% in p-distance and 10.58% in K2P distance (TS20211030-01
from Niigata prefecture vs TS20210412-01 from Kanagawa prefecture).

According to the 16S dataset of Nannarrup, the minimum divergence between UN.
sp. land N. sp. 2 was 14.92% in p-distance and 16.68% in K2P distance (TS20220306-
11 from Kochi prefecture and TS20220307-03, TS20220307-04, TS20220307-05,
TS20220307-11, TS20220307-12, TS20220307-15, TS20220307-16, TS20220307-
17 from Ehime prefecture vs TS20210217-04 and TS20210725-02 from Kanagawa
prefecture), whereas the maximum internal divergence within NV. sp. 1 was 7.056% in
p-distance and 7.443% in K2P distance (TS20211030-01 from Niigata prefecture vs
TS20211102-04 from Nagasaki prefecture).

According to the 28S dataset of Nannarrup, the minimum divergence between UN.
sp. land N. sp. 2 was 1.207% in p-distance and 1.218% in K2P distance (TS20220301-
01, TS20220301-04 TS20220302-03, TS20220302-11 from Kochi prefecture,
TS20220307-07, TS20220307-13, TS20220307-14 from Ehime prefecture and
TS20200411-06 from Shizuoka prefecture vs TS20210217-04 and TS20210725-
02 from Kanagawa prefecture), whereas the maximum internal divergence within WN.
sp. 1 was 0.6757% in p-distance and 0.6796% in K2P distance (1S$20220307-11,
TS20220307-12, TS20220307-15, TS20220307-16, TS20220307-17 from Ehime
prefecture vs TS20190913-01 from Tokyo prefecture).

Taxonomic account

Family Mecistocephalidae Bollmann, 1893

Genus Nannarrup Foddai, Bonato, Pereira & Minelli, 2003
New Japanese name: Himejimukade-zoku

Fig. 5
Nannarrup Foddai, Bonato, Pereira & Minelli, 2003: 1255-1256.

Type species. Nannarrup hoffmani Foddai, Bonato, Pereira & Minelli, 2003
Diagnosis. Partly modified from Foddai et al. (2003). Adult body length ca 10 mm
(Fig. 5A). Cephalic plate only slightly longer than wide, with frontal line absent or re-
placed by areolation. Two small clypeal plagulae covering ca one-sixth of the clypeus. Bucca
without setae. Stilus present, relatively short. Spiculum absent. Side-pieces of labrum only
incompletely subdivided into anterior and posterior alae by fragmented line very poorly
marked. Mandible provided with four well-developed pectinate lamellae. Coxosternite of

128 Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

<b>
oe
a

Figure 5. Nannarrup innuptus sp. nov., paratype (TS20210503-02) A habitus (provided by Dr Namiki

Kikuchi) B “death pose” with head capsule detached. Scale bars: 1 mm.

first maxillae medially divided. Coxosternite of second maxillae undivided, without suture
or membranous isthmus. Metameric pore close to posterior margin of coxosternum of
second maxillae, not to lateral ones. Claw of second maxillae only represented by terminal
spine. Forcipular telopodites far behind anterior margin of head in the closed position.
Forcipular trochanteroprefemur with pigmented single distal denticle; femur without teeth;
tarsungulum with basal, well-developed denticle. Forcipular tergite without median sulcus.
Sternal sulcus not anteriorly furcate. Last metasternite subtriangular. Ventral surface of each
coxopleuron with numerous pores. Anal pore present. Forty-one pairs of legs.

Remarks. The following characters included in the diagnosis sensu Foddai et al.
(2003) are different among the three Nannarrup species: presence/absence of a pair
of smooth or areolate areas along the posterior part of the paraclypeal sutures, pres-

New species of the genus Nannarrup from Japan 129

ence/absence of a tubercle on the forcipular tibia, pigmentation of the denticle on the
tarsungulum (Table 3). In total, 74 out of 88 collected specimens of Nannarrup (ca
84%) exhibited a leaning posture or even threw back their head when stored in ethanol
(Fig. 5B). Although such a “death pose” has not been quantitatively investigated in
Geophilomorpha, in the authors’ experience, it is a unique phenomenon in Nannarrup
that may be related to the internal morphological characteristics of the genus.

Nannarrup innuptus Tsukamoto, sp. nov.
https://zoobank.org/D2906856-517E-45BE-B2A0-1FE9CBD4D779
New Japanese name: Kaguya-himejimukade

Figs 5-11

Type material. Holotype. 1 adult female, Yugashima, Izu-shi, Shizuoka prefecture,
Japan (34°51.39'N, 138°55.40'E), 3 May 2021, coll. Mayu Susukida (labeled
as 1S20210503-09), deposited at the Collection of Myriapoda, Department of
Zoology, NSMT.

Paratype. 4 females, Yugashima, Izu-shi, Shizuoka prefecture, Japan (34°51.39'N,
138°55.39'E), 11 April 2021, leg. Katsuyuki Eguchi (labeled as TS20210411-04,
TS20210411-05, TS20210411-06, TS20210411-07, respectively), deposited at the
Collection of Myriapoda, Department of Zoology, NSMT. 5 females, Yugashima,
Izu-shi, Shizuoka prefecture, Japan (34°51.39'N, 138°55.40'E), 3 May 2021, leg.
Mayu Susukida (labeled as TS20210503-02, TS20210503-05, TS20210503-06,
TS20210503-07, TS20210503-10, respectively), deposited at MNHAH.

Non-type specimens. 1 female, Minamiosawa, Hachioji-shi, Tokyo prefecture,
Japan (35°37.02'N, 139°22.73'E), 27 June 2018, leg. Sho Tsukamoto (labeled as
TS20180627-01). 1 female, Hirasawa, Akiruno-shi, Tokyo prefecture, Japan
(35°43.64'N, 139°19.20'E), 10 October 2017, leg. Sho Tsukamoto (labeled as
TS20171010-01). 3 females, Hirasawa, Akiruno-shi, Tokyo prefecture, Japan
(35°43.64'N, 139°19.20'E), 13 September 2019, leg. Sho Tsukamoto (labeled as
TS20190913-01, TS20190913-02, TS20190913-03, respectively). 1 female, Shiroy-
ama, Kagoshima-shi, Kagoshima prefecture, Japan (31°35.88'N, 130°32.98'E), 2 July
2019, leg. Sho Tsukamoto (labeled as TS20190702-06). 1 female, Shibakusa, Hatori,
Ten-ei-mura, Iwase-gun, Fukushima prefecture, Japan (37°14.37'N, 140°03.86'E), 21
September 2020, leg. Katsuyuki Eguchi (labeled as’TS20200921-02). 1 female, Kubo,
Hiranuma, Rokkasho-mura, Kamikita-gun, Aomori prefecture, Japan (40°52.37'N,
141°21.76'E), 9 October 2020, leg. Katsuyuki Eguchi (labeled as TS20201009-01). 1
female, Nakagawara, Nagano, Daisen-shi, Akita prefecture, Japan (39°32.41'N,
140°31.76'E), 13 October 2020, leg. Katsuyuki Eguchi (labeled as TS20201013-03).
1 female, Mukounadaka, Nadaka, Tozawa-mura, Mogami-gun, Yamagata prefecture,
Japan (38°44.96'N, 140°11.15'E), 13 October 2020, leg. Katsuyuki Eguchi (labeled as
TS20201013-04). 1 female, Nakagawa, Kaneyama-machi, Onuma-gun, Fukushima
prefecture, Japan (37°28.25'N, 139°31.81'E), 18 October 2020, leg. Katsuyuki Eguchi

130 Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

(labeled as TS20201018-03). 1 female, Sawanishi, Mizunuma, Kaneyama-machi, On-
uma-gun, Fukushima prefecture, Japan (37°28.87'N, 139°33.48'E), 18 October 2020,
leg. Katsuyuki Eguchi (labeled as TS20201018-04). 1 female, Tai, Yamasaki-cho, Shi-
sou-shi, Hyogo prefecture, Japan (35°02.62'N, 134°33.68'E), 24 October 2020, leg.
Katsuyuki Eguchi (labeled as TS20201024-01). 1 female, Kageishi, Nishiawakura-son,
Aida-gun, Okayama prefecture, Japan (35°10.94'N, 134°20.64'E), 24 October 2020,
leg. Katsuyuki Eguchi (labeled as TS20201024-03). 2 females and 2 juveniles (sex
unknown), ‘Teraodai, Ayase-shi, Kanagawa prefecture, Japan (35°27.76'N,
139°25.13'E), 10 April 2021, leg. Joe Kutsukake (labeled as TS20210410-01 and
TS20210410-02 for females, TS20210410-03 and TS20210410-04 for juveniles, re-
spectively). 2 females, Nebukawa, Odawara-shi, Kanagawa prefecture, Japan
(35°12.00'N, 139°08.22'E), 12 April 2021, leg. Joe Kutsukake (labeled as TS20210412-
01 and TS20210412-02, respectively). 3 juveniles (sex unknown), Nebukawa, Oda-
wara-shi, Kanagawa prefecture, Japan (35°12.23'N, 139°08.43'E), 12 April 2021, leg.
Joe Kutsukake (labeled as TS20210412-03, TS20210412-04 and TS20210412-05,
respectively). 1 female, Shimada, Inami-cho, Hidaka-gun, Wakayama prefecture, Ja-
pan (33°47.36'N, 135°14.06'E), 3 of May 2021, leg. Katsuyuki Eguchi (labeled as
TS20210503-12). 1 female, Kurisugawa, Nakahechi-cho, ‘Tanabe-shi, Wakayama pre-
fecture, Japan (33°47.69'N, 135°30.16'E), 3 of May 2021, leg. Katsuyuki Eguchi (la-
beled as TS20210503-13). 1 female, Futo, Ito-shi, Shizuoka prefecture, Japan
(34°54.58'N, 139°07.72'E), 9 June 2021, leg. Joe Kutsukake (labeled as TS20210609-
03). 1 female, Futo, Ito-shi, Shizuoka prefecture, Japan (34°54.66'N, 139°07.19'E), 9
June 2021, leg. Joe Kutsukake (labeled as 'TS20210609-04). 1 juvenile (sex unknown),
Shishihara, Shimizu-ku, Shizuoka-shi, Shizuoka prefecture, Japan (35°11.94'N,
138°31.28'E), 23 of May 2021, leg. Katsuyuki Eguchi (labeled as TS20210523-04). 1
female, Nishiaraya, Tsuruoka-shi, Yamagata prefecture, Japan (38°38.64'N,
139°49.73'E), 29 May 2021, leg. Katsuyuki Eguchi (labeled as TS20210529-03). 3
females, Den-enchofu, Ota-ku, Tokyo prefecture, Japan (35°35.51'N, 139°39.86'E),
30 June 2021, leg. Joe Kutsukake (labeled as TS20210630-01, TS20210630-02 and
TS20210630-03). 1 subadult female, Oyama, Isehara-shi, Kanagawa prefecture, Japan
(35°25.74'N, 139°14.44'E), 25 July 2021, coll. Sho Tsukamoto (labeled as
TS20210725-01; cephalic capsule lost). 1 female, Hikime, Miyako-shi, Iwate prefec-
ture, Japan (39°37.49'N, 141°49.41'E), 6 September 2021, leg. Katsuyuki Eguchi (la-
beled as TS20210906-01). 3 juveniles (sex unknown), Hiyamizucho, Kagoshima-shi,
Kagoshima prefecture, Japan (31°36.21'N, 130°33.02'E), 22 September 2021, leg. Joe
Kutsukake (labeled as TS20210922-01, TS20210922-02 and TS20210922-03). 1 fe-
male, Natsuocho, Miyakonojo-shi, Miyazaki prefecture, Japan (31°52.49'N,
130°57.50'E), 29 September 2021, leg. Joe Kutsukake (labeled as TS20210929-01). 2
females and 1 juvenile (sex unknown), Minamiosawa, Hachioji-shi, Tokyo prefecture,
Japan (35°37.43'N, 139°23.05'E), 18 October 2021, leg. Joe Kutsukake (labeled as
TS20211018-03, TS20211018-04 for females and TS20211018-05 for the juvenile,
respectively). 2 females and 1 juvenile (sex unknown), Era, Toyotacho, Shimonoseki-

shi, Yamaguchi prefecture, Japan (34°10.56'N, 131°02.48'E), 26 October 2021, leg.

New species of the genus Nannarrup from Japan | fo!

Sho Tsukamoto (labeled as TS20211026-04, TS20211026-10 for females and
TS20211026-05 for the juvenile, respectively). 1 female, Hikosan, Soeda-machi, Taga-
wa-gun, Fukuoka prefecture, Japan (33°29.06'N, 130°55.94'E), 29 October 2021, leg.
Sho Tsukamoto (labeled as TS20211029-04). 1 female, Tomaruhinoe, Tsunan-machi,
Nakauonuma-gun, Niigata prefecture, Japan (37°02.16'N, 138°39.46'E), 30 October
2021, leg. Katsuyuki Eguchi (labeled as TS20211030-01). 1 female, Maeda, Yahatahi-
gashi-ku, Kitakyushu-shi, Fukuoka prefecture, Japan (33°51.35'N, 130°47.72'E), 30
October 2021, leg. Sho Tsukamoto (labeled as TS20211030-10). 2 females and 2
specimens (the lower half of body lost, sex unknown), Nishiyama, Nagasaki-shi, Naga-
saki prefecture, Japan (32°45.89'N, 129°53.00'E), 1 November 2021, leg. Sho Tsuka-
moto (labeled as TS20211101-15, TS20211101-16 for females and ’TS20211101-17,
TS20211101-18 for sex-unknown specimens, respectively). 1 female, Nijigaoka, Na-
gasaki-shi, Nagasaki prefecture, Japan (32°47.73'N, 129°50.00'E), 2 November 2021,
leg. Sho Tsukamoto (labeled as TS20211102-04). 1 female, Murotsu, Muroto-shi,
Kochi prefecture, Japan (33°18.06'N, 134°09.31E), 1 March 2022, leg. Katsuyuki
Eguchi (labeled as TS20220301-01). 1 female, Murotomisakicho, Muroto-shi, Kochi
prefecture, Japan (33°16.87'N, 134°10.65E), 2 March 2022, leg. Joe Kutsukake (la-
beled as TS20220302-03). 1 female, Kamoi, Yokosuka-shi, Kanagawa prefecture, Ja-
pan (35°15.44'N, 139°44.61E), 21 March 2022, leg. Katsuyuki Eguchi (labeled as
TS20220321-03). 1 female, Murotsu, Muroto-shi, Kochi prefecture, Japan
(33°18.07'N, 134°09.31E), 1 March 2022, leg. Joe Kutsukake (labeled as TS20220301-
04). 3 females, Makigawa, Tsushimacho, Uwajima-shi, Ehime prefecture, Japan
(33°05.74'N, 132°35.58E), 7 March 2022, leg. Joe Kutsukake (labeled as TS20220307-
03, TS20220307-04, TS20220307-05, respectively). 2 females, Sunokawa, Ainan-
cho, Minamiuwa-gun, Ehime prefecture, Japan (33°02.50'N, 132°29.18E), 7 March
2022, leg. Joe Kutsukake (labeled as TS20220307-06, TS20220307-07, respectively).
1 female, Matsuo, Tosashimizu-shi, Kochi prefecture, Japan (32°44.16'N, 132°58.57E),
6 March 2022, leg. Joe Kutsukake (labeled as TS20220306-11). 2 females, Makigawa,
Tsushimacho, Uwajima-shi, Ehime prefecture, Japan (33°05.87'N, 132°36.98E), 7
March 2022, leg. Joe Kutsukake (labeled as TS20220307-09, TS20220307-10, respec-
tively). 2 females, Ryoke, Muroto-shi, Kochi prefecture, Japan (33°17.23'N,
134°10.59E), 2 March 2022, leg. Joe Kutsukake (labeled as TS20220302-11,
TS20220302-12, respectively). 2 females and 1 specimen (the lower half of body lost,
sex unknown), Motootsu, Muroto-shi, Kochi prefecture, Japan (33°18.81'N,
134°07.33E), 1 March 2022, leg. Joe Kutsukake (labeled as TS20220301-05,
TS20220301-06 for females and TS20220301-07 for sex-unknown specimens, respec-
tively). 7 females, Iwabuchi, Tsushimacho, Uwajima-shi, Ehime prefecture, Japan
(33°08.81'N, 132°32.99E), 7 March 2022, leg. Joe Kutsukake (labeled as TS20220307-
11, 1S20220307-12, TS20220307-13, TS20220307-14, TS20220307-15,
TS20220307-16, TS20220307-17, respectively).

Etymology. The species name is derived from unmarried in Latin. In the Japanese
population, males of this species have not been discovered despite the wide collection
range in Japan.

132 Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

Diagnosis. Clypeus with a pair of smooth or weakly areolate areas along the poste-
rior part of the paraclypeal sutures; forcipular trochantroprefemur with a large denticle
(longer than wide); tarsungulum with a well-pigmented denticle; metasternite of ulti-
mate leg-bearing segment wider than long.

Description. General features (Fig. 5A, B): Body 7.0—12.0 mm long (holotype
12.0 mm), gradually attenuated posteriorly, almost uniformly very pale yellow, with
head and forcipular segment pale ocher.

Cephalic capsule (Fig. GA—D): Cephalic plate ca 1.4—1.6x as long as wide;
lateral margins more distinctly converging anteriorly than posteriorly; posterior
margin straight; scutes approximately isometric and up to 15 um wide; transverse
suture absent but areolate line present in some individuals (Fig. 6A, C); setae up to
ca 37.5 um long. Clypeus ca 1.4—1.5x as wide as long, with lateral margins com-
plete, almost uniformly areolate, with scutes ca 15 um wide, clypeal areas absent;
clypeus with 11-17 setae, 1+1 postantennal, 1-2+2 median, 3—6+3-—5 prelabral;
clypeal ratio ca 1: 6-1: 7; clypeal plagulae with additional smooth or weak areo-
lation area along posterior part of paraclypeal sutures. Anterior and distolateral
parts of pleurites areolate, without setae. Side-pieces of labrum medially in contact,
only incompletely divided into anterior and posterior alae by weak chitinous line,
without longitudinal stripes on posterior alae, with slightly visible short fringe on
posterior margin of side-pieces; mid-piece as long as wide, converging anteriorly
and posteriorly.

Antenna (Fig. 7A—D): Antenna with 14 articles, when stretched, ca 2.1—2.6x as
long as head length. Intermediate articles slightly longer than wide. Article XIV ca
2.5x as long as wide, ca 1.9—2.4x as long as article XIII, and 1.8—2.4x as long as inter-
mediate articles. Setae on articles VIII-XVI denser than articles I-VI]. Setae gradually
shorter from article VII to XIV, up to 65 um long on article I, up to 25 um long on
article VIII and < 15 um long on article XIV. Article XIV with two types of sensilla;
apical sensilla (arrows in Fig. 7C, D) ca 10 um long, with wide flat ring at mid-length;
club-like (arrowheads in Fig. 7C, D) sensilla ca 15 um long, clustered in the distal
part of the internal and the external sides of the article. Three longitudinal rows, each
consists of ca 9 proprioceptive spine-like sensilla, at bases of antennal articles IH-—
V, VII-IX, approximately dorsal, ventro-internal and ventro-external; the rows each
consisting of 1-3 spine-like sensilla on articles I and VI, and 6 on the article II; 0-1
spine-like sensilla on articles X-XIV. A few pointed sensilla, up to 2.5 um long, on
both dorso-external and ventro-internal position, close to distal margin of articles H,
V, [X and XII.

Mandible (Fig. 8A): At least four pectinate lamellae present; first and second
lamellae with ca 5 elongated teeth. Each tooth ca 2x as long as wide. Ventral sur-
face hairy.

First maxillae (Fig. 8B): Coxosternite medially divided but slightly, without setae,
faintly areolate. Coxal projections well developed and hyaline distally, with 1-2+1-2
setae and 3-4+3—4 small sensilla. Telopodite uni-articulated and hyaline distally, with
one(two) seta(e). No lobes on either coxosternite or telopodites.

New species of the genus Nannarrup from Japan

19

j
s
GPG

Figure 6. Nannarrup innuptus sp. nov. A, B holotype (TS20210503-09) C, D paratype (TS20210411-
04). A, B cephalic plate, dorsal C, D clypeus and clypeal pleurite, ventral. Scale bars: 0.3 mm.

134 Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

A B

Figure 7. Nannarrup innuptus sp. nov., holotype (TS20210503-09) A right part of head and right an-
tenna, dorsal B right part of head and right antenna, ventral C antennal article XIV, dorsal D antennal
article XIV, ventral. Arrows indicate apical sensillum; arrowheads indicate club-like sensillum. Scale bars:

0.1 mm (A, B); 0.05 mm (C, D).

New species of the genus Nannarrup from Japan 135

. 1)
Ls) J
jire—=s em
~ \
4 f % ) YA i : X nN

&

Figure 8. Nannarrup innuptus sp. nov. A, D paratype (TS20210411-05) B, C holotype (TS20210503-
09). A right mandible, dorsal B first maxillae, ventral C, D second maxillae, ventral. Scale bars: 0.05 mm.

Second maxillae (Fig. 8C, D): Coxosternite medially undivided, without suture,
with 2+2 setae along anterior margin, with 6—7 setae behind anterior margin, with
1—2+1-2 sensilla on posterior lateral margin in some individuals. with anterior margin
slightly concave, with metameric pores on posterior part. Telopodites tri-articulate,
reaching medial projections and telopodites of first maxillae in some individuals. Claw
of telopodite virtually absent, represented by short spine only.

Forcipular segment (Fig. 9A—F): Tergite trapezoidal, ca 1.4—1.9x as wide as long,
with lateral margins converging anteriorly, approximately as wide as cephalic plate
and ca 0.7x as wide as following tergites; 1+1 setae of similar length arranged in an
anterior row, and 3+3 setae of similar length arranged in a posterior row; one pair of
longitudinal rows of three tiny setae located between middle and distal setae in poste-
rior row. Mid-longitudinal sulcus of tergite not visible. Exposed part of coxosternite ca

136 Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

AB

o\ a 2: A. r38 9 :
Figure 9. Nannarrup innuptus sp. nov. A, B paratype (TS20210411-05) C, D holotype (TS20210503-
09) E, F paratype (TS20210411-04). A anterior part of body, dorsal B anterior part of body, ventral
C forcipular segment, dorsal D forcipular segment, ventral E denticles on forcipule, dorsal F poison calyx,
dorsal. Arrow indicates tubercle on tibia. Circle indicates poison calyx. Scale bars: 0.5 mm (A, B); 0.3 mm

(C, D); 0.05 mm (E, F).

1.1x as wide as long; anterior margin with shallow medial concavity and with one pair
of denticles; coxopleural sutures complete in entire ventrum, sinuous and diverging
anteriorly; chitin-lines absent. Trochanteroprefemur ca 1.3—1.4x as long as wide; with
a well-developed and strong pigmented denticle at distal internal margin, ca 1.3—1.6x
as long as wide. Intermediate articles distinct, with a tubercle on tibia (arrowed in

New species of the genus Nannarrup from Japan 137

| . AR | ROK ek Se, BE.
Figure 10. Nannarrup innuptus sp. nov. A holotype (TS20210503-09) B, C paratype (TS20210411-05)

A pretarsus of left leg 2, anterolateral B tergite of leg-bearing segment 40, dorsal C sternite of leg-bearing
segment 40, ventral. Scale bars: 0.05 mm (A); 0.1 mm (B, C).

Fig. 9E, some individuals not visible). Tarsungulum with well-pigmented basal den-
ticle; both external and internal margins uniformly curved, except for moderate me-
sal basal bulge; ungulum not distinctly flattened. Elongated poison calyx (circle in
Fig. 9F), ca 9x as long as wide, lodged inside intermediate forcipular articles.

Leg-bearing segments (Figs 9A, B, 10A—C): Forty-one pairs of legs present.
Metatergite 1 slightly wider than subsequent one, with two paramedian sulci visible on
tergites of anterior half of body, with pretergite. No paratergites. Walking legs shorter
than width of trunk; legs of first pair much smaller than following ones; claws simple,
uniformly bent, with 2 accessory spines; anterior spine reaching at most 10% of length
of claw; posterior spine equal in length of the anterior spine. Metasternites slightly
longer than wide. Sternal sulcus visible on a few anterior sternites, represented by
very shallow mid-longitudinal thickening, anteriorly not furcate. No ventral glandular
pores on each metasternite.

Ultimate leg-bearing segment (Fig. 11A—D): Pretergite not accompanied by pleu-
rites but incomplete traces of sutures present at both sides. Metatergite subtrapezoidal,
ca 1.1—-1.3x as wide as long; lateral margins convex and converging posteriorly; poste-
rior margin slightly curved. Coxopleuron ca 1.2—1.7x as long as metasternite; coxal or-
gans of each coxopleuron opening through 5—10 independent pores, placed ventrally.
Metasternite trapezoidal, ca 1.3—1.6x as wide as long, anteriorly ca 1.7—2.2x as wide
as posteriorly; lateral margins slightly convex and converging backward; setae almost
arranged symmetrically, dense on posterior margin. Telopodite ca 9-11x as long as
wide, ca 1.9-2.1x as long and ca 1.5—1.6x as wide as penultimate telopodite, with six
articles; tarsus 2 ca 3.6-4.2x as long as wide and ca 1.2—1.6x as long as tarsus 1; setae
arranged uniformly, < 70 um long; pretarsus represented by small tubercle.

Female postpedal segments (Fig. 11A, B): Two gonopods basally touching, subtri-

angular, without traces of articulation, covered with setae. Anal pore present.

138 Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

Figure I 1. Nannarrup innuptus sp. nov., holotype (TS20210503-09) A ultimate leg-bearing segment
and postpedal segment, dorsal B ultimate leg-bearing segment and postpedal segment, ventral C left ulti-
mate leg, dorsal D left ultimate leg, ventral. Scale bars: 0.1 mm (A, B); 0.3 mm (C, D).

Male postpedal segments unknown (male unknown).

Distribution. Honshu, Shikoku and Kyushu.

Remarks. In pairwise comparisons, NV. innuptus sp. nov. can be distinguished from
N. hoffmani by the presence of a well-developed denticle on the trochanteroprefemur

New species of the genus Nannarrup from Japan 139

(width: length = 1:1.3—1.6) and a well-pigmented denticle on the tarsungulum. In ad-
dition, NV. innuptus sp. nov. is also distinguishable from N. hoffmani by the presence of
a tubercle on the forcipular tibia, but this tubercle is not always visible. No male has
been found so far.

Nannarrup oyamensis Tsukamoto, sp. nov.
https://zoobank.org/1543ADD5-1C03-4471-9B6F-D473E4BBO0F22
New Japanese name: Amefuri-himejimukade

Figs 12-17

Type material. Holotype | adult male, Hinata, Isehara-shi, Kanagawa prefecture, Ja-
pan (35°26.07'N, 139°14.75'E), 17 February 2021, coll. Sho Tsukamoto (labeled
as 1S20210217-04), deposited at the Collection of Myriapoda, Department of Zo-
ology, NSMT. Paratype | subadult male, Hinata, Isehara-shi, Kanagawa prefecture,
Japan (35°26.07'N, 139°14.75'E), 25 July 2021, coll. Sho Tsukamoto (labeled as
TS20210725-02), deposited at MNHAH.

Etymology. The species name is derived from the name of Japanese mountain,
namely Mt. Oyama. The word was further Latinized by adding the Latin masculine
adjective suffix -ensis, to form oyamensis. The last “a” of Oyama and the first “e” of -ensis
are merged into “e.” Examined specimens were collected from Mt. Oyama, an object
of the mountain worship.

Diagnosis. Clypeus without smooth or weakly areolate areas along the posterior
part of the paraclypeal sutures; forcipular trochantroprefemur with a large denticle
(longer than wide); tarsungulum with a well-pigmented denticle; metasternite of ulti-
mate leg-bearing segment wider than long.

Description. General features: Body 8.6 mm long (holotype), gradually attenuate
posterior, almost uniformly very pale yellow, with head and forcipular segment pale ocher.

Cephalic capsule (Fig. 12A, B): Cephalic plate ca 1.5x as long as wide, lateral mar-
gins more distinctly converging anteriorly than posteriorly, posterior margin straight;
scutes approximately isometric and up to ca 15 um wide; transverse suture absent; setae
up to ca 50 um long. Clypeus ca 1.5x as wide as long, with lateral margins complete,
almost uniformly areolate, with scutes ca 10 um wide, a pair of clypeal areas absent;
13 setae in holotype, 1+1 postantennal, 1+1 median, 5+4 prelabral; clypeal ratio ca 1:
7; clypeal plagulae without additional smooth area along posterior part of paraclypeal
sutures; 17 pore-like organs on entire part of clypeus. Anterior and distolateral parts
of pleurites areolate, without setae. Side-pieces of labrum medially in contact, only
incompletely divided into anterior and posterior alae by weak chitinous line, without
longitudinal stripes on posterior alae; with slightly visible short fringe on posterior
margin of side-pieces; mid-piece as long as wide, converging anteriorly and posteriorly.

Antenna (Fig. 13A—D): Antenna with 14 articles, when stretched, ca 2.3x as long
as head length. Intermediate articles slightly longer than wide. Article XIV ca 2.0x
as long as wide, ca 1.9x as long as article XIII, and 1.9-2.1x as long as intermediate

140 Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

Figure 12. Nannarrup oyamensis sp. nov., holotype (TS20210217-04) A cephalic plate, dorsal B clypeus

and clypeal pleurite, ventral. Scale bars: 0.1 mm.

articles. Setae on articles VIII-XVI denser than articles I-VI]. Setae gradually shorter
from article VII to XIV, up to 50 um long on article I, up to 33 pm long on article
VIII and < 18 um long on article XIV. Article XIV with two types of sensilla, apical
sensilla (arrows in Fig. 13C, D) ca 5 um long, with wide flat ring at mid-length; club-
like sensilla (arrowheads in Fig. 13C, D) ca 10 ym long, clustered in the distal parts
of the internal and external sides of the article. Three longitudinal rows consisted of
ca 9 proprioceptive spine-like sensilla at bases of antennal articles II-V, VII-IX, ap-
proximately dorsal, ventro-internal and ventro-external; rows reduced to 1—3 spines on
antennal articles I and VI, and 0-1 spine on antennal articles X—XIV. A few pointed
sensilla, up to 2.5 um long, on both dorso-external and ventro-internal position, close
to distal margin of articles II, V, IX and XII.

Mandible (Fig. 14A). At least four pectinate lamellae, with elongated teeth. Each
tooth ca 2x as long as wide.

First maxillae (Fig. 14B): Coxosternite medially divided but slightly, without se-
tae, faintly areolate. Coxal projections well developed and hyaline distally, provided
with 1+1 setae and 3+4 small sensilla. Telopodite uni-articulated and hyaline distally,
with one (two) seta(e). No lobes on either coxosternite or telopodites.

Second maxillae (Fig. 14B): Coxosternite medially undivided, without suture,
with 2+3 setae along the anterior margin, with 4+4 setae located behind anterior mar-
gin, with anterior margin slightly concave, with metameric pores on posterior part.
Telopodites tri-articulate overreaching medial projections and telopodites of first max-
illae. Claw of telopodite virtually absent, represented by short spine only.

New species of the genus Nannarrup from Japan 141

A | >» B

Figure 13. Nannarrup oyamensis sp. nov., holotype (TS20210217-04) A right antenna, dorsal B right
antenna, ventral C antennal article XIV, dorsal D antennal article XIV, ventral. Arrows indicate apical
sensillum; arrowheads indicate club-like sensillum. Scale bars: 0.1 mm (A, B); 0.05 mm (C, D).

142 Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

——s je Mf C2) ae ys

Figure 14. Nannarrup oyamensis sp. nov., holotype (TS20210217-04) A right mandible, ventral B max-
illae complex, ventral. Scale bars: 0.05 mm.

Forcipular segment (Fig. 15A—D): Tergite trapezoidal, ca 1.9x as wide as long,
with lateral margins converging anteriorly, approximately as wide as cephalic plate
and ca 0.7x as wide as following tergite, 1+1 setae of similar length arranged in an
anterior row, and 3+3 setae of similar length arranged in a posterior row, one pair of
longitudinal rows of three tiny setae located between middle and distal setae in poste-
rior row. Mid-longitudinal sulcus of tergite not visible. Exposed part of coxosternite
ca 1.3x as wide as long; anterior margin with shallow medial concavity and with one
pair of denticles; coxopleural sutures complete in entirely ventrum, sinuous and di-
verging anteriorly; chitin-lines absent. Basal distance between forcipules ca 0.1x of
maximum width of coxosternite. Trochanteroprefemur ca 1.3x as long as wide; with a
well-developed and strong pigmented denticle at distal internal margin, ca 1.3x as long
as wide. Intermediate articles distinct, tubercle on tibia not visible. Tarsungulum with
basal denticle well-pigmented; both external and internal margins uniformly curved,
except for moderate mesal basal bulge; ungulum not distinctly flattened. Elongated
poison calyx (circle in Fig. 15D), ca 6x as long as wide, lodged inside intermediate
forcipular articles.

Leg-bearing segments (Fig. 16A—D): Forty-one pairs of legs present. Metatergite
1 slightly wider than subsequent one, with two paramedian sulci visible on tergites
of anterior half of body, without pretergite. No paratergites. Walking legs shorter
than width of trunk; legs of first pair much smaller than following ones; claws sim-
ple, uniformly bent, with two accessory spines; anterior spine reaching at most 10%
of length of claw; posterior spine shorter than anterior spine. Metasternites slightly
longer than wide. Sternal sulcus visible on a few anterior sternites, represented by
very shallow mid-longitudinal thickening, anterior not furcate. No ventral glandular
pores on each metasternite.

New species of the genus Nannarrup from Japan 143

k
; ms s

Figure 15. Nannarrup oyamensis sp. nov., holotype (1S20210217-04) A forcipular segment, dorsal
B forcipular segment, ventral C denticles on right forcipule, dorsal D left poison calyx, dorsal. Circle
indicates poison calyx. Scale bars: 0.1 mm (A, B); 0.05 mm (C, D).

144 Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

os B

| : Vi
, | ; é
\ ; S \\ \ A Pa) pM . 1
/ A a Bury a a
RP s} \ aS

i Be Fo
/ } a= {
au 4 M9 in os a . =
eee eS Baa
— > = . in
ine —
| Fo

; ee ae

€ : a ry
q

: : 7 —_a7 Fa
— a eS, ae x ~ _ =—_ -
Z 4 5 = = ae
y Y = : =
TA 2 Be4. J

Figure 16. Nannarrup oyamensis sp. nov., holotype (TS20210217-04) A pretarsus of left leg 40, dorsal
B pretarsus of left leg 40, ventral C tergite of leg-bearing segment 40, dorsal D sternite of leg-bearing
segment 40, ventral. Scale bars: 0.05 mm (A, B); 0.1 mm (C, D).

Ultimate leg-bearing segment (Fig. 17A—D): Pretergite not accompanied by pleu-
rites but incomplete traces of sutures present at both sides. Metatergite subtrapezoidal,
almost as wide as long, lateral margins convex and converging posteriorly; posterior
margin slightly curved. Coxopleuron ca 1.2x as long as metasternite; coxal organs of
each coxopleuron opening through five or six independent pores, placed ventrally.
Metasternite subtriangular, ca 1.6 as wide as long, anteriorly ca 3.5x as wide as poste-
riorly; lateral margins slightly convex and converging backward; setae almost arranged
symmetrically, dense on posterior margin. Telopodite ca 11x as long as wide, ca 1.9x
as long and ca 1.3x as wide as penultimate telopodite, with 6 articles; tarsus 2 ca 2.7x
as long as wide and ca 1.5x as long as tarsus 1; setae arranged uniformly, < 50 um long.
Pretarsus represented by spines, up to 5 um.

Male postpedal segments (Fig. 17A, B): Two gonopods, very widely separated
from one another, conical in outline, uni-articulated without any sutures, covered with
setae. Anal pore present.

Female postpedal segments unknown (female unknown).

Distribution. Only known from Mt. Oyama, located in Isehara-shi, Kanaga-
wa prefecture.

Remarks. Nannarrup oyamensis sp. nov. is distinguishable from the two congeners by
the absence of smooth or weakly areolate areas along the posterior part of the paraclypeal
sutures. Specifically, NV. oyamensis sp. nov. can be clearly distinguished from N. hoffmani
by the presence of a well-developed denticle on the trochanteroprefemur (width:

New species of the genus Nannarrup from Japan

y \
f ~ 4
=) —o
q 4
j \
j .
7 ~S i \ |
’ ee |
4 ;
= ¢ ; |
od
| i :
| :
\ |
i y
| -
/
= \ =
\ rd j
i
f
‘
\ C
= /
— | a ee
el Oy + tz — ‘ —.
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/ pon ett
en ~ — — | : a
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"tl a et ==
\ ; im ~ =
aa
rrr
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=~ > ] »
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t= _____ 5] =

-

Figure 17. Nannarrup oyamensis sp. nov. A, B holotype (TS20210217-04) C, D paratype (TS20210725-
02). A ultimate leg-bearing segment and male postpedal segment, dorsal B ultimate leg-bearing segment and
male postpedal segment, ventral C right ultimate leg, dorsal D right ultimate leg, ventral. Scale bars: 0.1 mm.

length = 1:1.3) and the absence of smooth or weakly areolate areas along the posterior part
of the paraclypeal sutures. Furthermore, NV. oyamensis sp. nov. can be distinguished from
N. innuptus sp. nov. by the absence of a pair of smooth or weakly areolate areas along the
posterior part of the paraclypeal sutures (see Table 3 for a comparison of characteristics).

146 Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

Discussion

Species recognition based on morphological analysis and DNA barcoding

All three morphospecies (including NV. hoffmani) were determined to be similar to a
certain extent. Nevertheless, each morphospecies was distinguished from the other
two morphospecies on the basis of the following characteristics: presence or absence
of a pair of smooth or weakly areolate areas along the posterior part of the paraclypeal
sutures, the width-to-length ratio of the denticle of trochanteroprefemur, the pigmen-
tation of the denticle on the tarsungulum. Table 3 shows the comparison among the
three morphospecies regarding the key characters.

The maximum internal genetic divergence in the CO/, 16S, and 28S sequences
within each morphospecies was considerably smaller than the minimum divergence in
the COJ, 16S, and 28S sequences between each pair of the morphospecies, that is, the
DNA barcoding gap was evident.

The presence of a DNA barcoding gap (especially in the nuclear 28S) suggests that
N. sp. 2 forms an independent gene pool that is reproductively isolated from JN. sp. 1.
Together, the morphological and molecular evidence suggests that the three morphos-
pecies are distinct species and that NV. sp. 1 and UN. sp. 2 are two novel species; they are
described under the section “Taxonomic account” as Nannarrup innuptus sp. nov. and
Nannarrup oyamensis sp. nov., respectively.

Distribution of the genus Nannarrup

All examined Nannarrup specimens were collected from Honshu, Shikoku, and Ky-
ushu, but the collectors (ST, KE, and collaborators) did not find Nannarrup in the Ry-
ukyu Archipelago despite intensive and repetitive field surveys. Furthermore, Uliana et
al. (2007) have also reported that there are no records of Nannarrup from the Ryukyus
and Taiwan. The distribution range of Nannarrup is likely confined to the temperate
zones of East Asia, namely Honshu, Shikoku, Kyushu, and surrounding islands. How-
ever, future field surveys in Hokkaido, and continental East Asia may lead to additional
reports of Nannarrup.

Foddai et al. (2003) stated that NV. hoffmani is not native to New York, the type
locality of this species, but has been definitely introduced from western America or
East Asia. In the present study, N. /offmani was not collected in Japan. However, the
existence of two congeners in Japan suggests that Nannarrup is native to East Asia. Ad-
ditional field surveys to investigate the original distribution of N. hoffmani should be
conducted mainly in East Asia.

According to the results of DNA barcoding based on COZ, 16S, and 28S sequenc-
es, LV. innuptus sp. nov. does not show a remarkable genetic structure that is consistent
with the geography of Japan and physical distances among the collection sites in the
present study. Nevertheless, considering this intraspecific genetic diversity, it is reason-
able to assume that NV. innuptus sp. nov. is not a species recently introduced by human
activities but a native species in Japan. The reason for such a low genetic diversity and

New species of the genus Nannarrup from Japan 147

no geographic structuring of diversity in mitochondrial genes remains unclear at pre-
sent, but we propose the following two hypotheses to explain this finding: 1) the rate of
evolution of mitochondrial genes is considerably lower than that in other centipedes,
and 2) the establishment of NV. innuptus sp. nov. as an independent species (either
within the Japanese archipelago or following migration from the Asian continent) is
geologically relatively recent. This question may be answered by examining sufficient
specimens of other congeners (e.g., NV. oyamensis sp. nov.) from East Asia including Ja-
pan and estimating the species divergence time by including other congeners and even
genera belonging to Mecistocephalidae in Asia.

Likelihood of parthenogenesis in Nannarrup innuptus sp. nov.

Remarkably, all 71 adult and subadult specimens of NV. innuptus sp. nov. examined in this
study were females without exception. Furthermore, because different collectors (four) did
the specimen collections, in different seasons (March—-November), and in different habi-
tats (forest, urban green space, and riverbed), the presence of any sampling bias that may
have distorted the sex ratio is unlikely. Although there is no direct evidence, the abovemen-
tioned indirect evidence shows the possibility of parthenogenesis of NV. innuptus sp. nov.
To date, a large part of the northern and eastern European population of Geophilus
proximus C.L. Koch, 1847, the European population of Tygarrup javanicus Attems,
1929, and Schendyla dentata (Brélemann & Ribaut, 1911) have been discussed about
their parthenogenesis (Sograff 1882; Barber and Jones 1999; Minelli 2011; Tuf et al.
2018). Nannarrup innuptus sp. nov. may be the second parthenogenetic species in the
family Mecistocephalidae. In contrast, the Japanese congener, N. oyamensis sp. nov.,
exhibits sexual reproduction because two examined specimens were males, i.e., males
of NV. oyamensis sp. nov. should be common. In addition, the type series of N. hoffmani

involved one juvenile male specimen (Foddai et al. 2003).

Acknowledgements

We are grateful to Ms Mayu Susukida (Tokyo Metropolitan University) and Mr Joe
Kutsukake (Tokyo Metropolitan University) for collecting and providing Nannarrup
specimens. We also would like to thank Dr Namiki Kikuchi (Tokyo Metropolitan
University) for providing a high-quality photograph of the habitus of Nannarrup
innuptus sp. nov., and Dr Keisuke Kawano (The Firefly Museum of Toyota Town) for
guiding in the forests in Yamaguchi prefecture. We sincerely thank two reviewers for
providing valuable comments which significantly improved this paper. We would like
to thank Enago (www.enago.jp) for the English language review. This research was
funded by the following foundations and societies: Asahi Glass Foundation [Leader:
Katsuyuki Eguchi; FY2017—FY2020, and Leader: Satoshi Shimano; FY2020-FY2023]
and ‘Tokyo Metropolitan University Fund for TMU Strategic Research [Leader:
Prof. Noriaki Murakami; FY2020—-FY2022]. This research was also funded by JSPS
KAKENHI Grant Numbers 17K07271, 18K06392 for SS.

148 Sho Tsukamoto et al. / ZooKeys 1115: 117-150 (2022)

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