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Integrative taxonomy of the new millipede genus Coxobolellus, gen. nov. (Diplopoda : Spirobolida : Pseudospirobolellidae), with descriptions of ten new species

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Pseudospirobolellidae is a poorly known family of spirobolidan millipedes with only two genera and five described species. Yet, the descriptive taxonomy and molecular systematics of this group have been largely neglected. Therefore, the present work presents an integrative taxonomic study of new pseudospirobolellid taxa in Thailand. To this end, two mitochondrial gene fragments (COI and 16S rRNA) combined with morphological characters were used to define the genus Coxobolellus, gen. nov. with 10 new species, viz. C. albiceps, sp. nov., C. compactogonus, sp. nov., C. fuscus, sp. nov., C. nodosus, sp. nov., C. serratus, sp. nov., C. simplex, sp. nov., C. tenebris, sp. nov., C. tigris, sp. nov., C. transversalis, sp. nov. and C. valvatus, sp. nov. The interspecific COI sequence divergences among the new species ranged from 6 to 15%. The intergeneric COI sequence divergence between species of Coxobolellus, gen. nov., Benoitolus birgitae and Pseudospirobolellus sp. ranged from 20 to 23%. Three major morphological differences separate Coxobolellus, gen. nov. from Benoitolus and Pseudospirobolellus, namely (1) the protruding process on the 3rd (and 4th) coxae on male legs, (2) the posterior gonopod telopodite divided into two parts, and (3) a conspicuous opening pore at the mesal margin at the end of the coxal part of the posterior gonopod. Thus, the new genus is well supported by both mtDNA and morphological evidence, while the delimitation of the 10 new species is supported by the congruence between mtDNA and morphological data. Yet, with respect to the relationships of Benoitolus birgitae, morphological data suggest a similarity with Coxobolellus, gen. nov. and Pseudospirobolellus, whereas mtDNA data place this species in the Pachybolidae. Further phylogenetic analyses are needed to explore this apparent incongruence and test the monophyly of Pseudospirobolellidae.
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Integrative taxonomy of the new millipede genus Coxobolellus,
gen. nov. (Diplopoda : Spirobolida : Pseudospirobolellidae),
with descriptions of ten new species
Piyatida Pimvichai
A,F
, Henrik Enghoff
B
, Somsak Panha
C
and Thierry Backeljau
D,E
A
Department of Biology, Faculty of Science, Mahasarakham University, Mahasarakham 44150, Thailand.
B
Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15,
DK-2100 Copenhagen Ø, Denmark.
C
Animal Systematics Research Unit, Department of Biology, Faculty of Science, Chulalongkorn University,
Bangkok 10330, Thailand.
D
Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000 Brussels, Belgium.
E
Evolutionary Ecology Group, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium.
F
Corresponding author. Email: piyatida.p@msu.ac.th
Abstract. Pseudospirobolellidae is a poorly known family of spirobolidan millipedes with only two genera and ve
described species. Yet, the descriptive taxonomy and molecular systematics of this group have been largely neglected.
Therefore, the present work presents an integrative taxonomic study of new pseudospirobolellid taxa in Thailand. To
this end, two mitochondrial gene fragments (COI and 16S rRNA) combined with morphological characters were used to
dene the genus Coxobolellus, gen. nov. with 10 new species, viz. C. albiceps, sp. nov., C. compactogonus, sp. nov.,
C. fuscus, sp. nov., C. nodosus, sp. nov., C. serratus, sp. nov., C. simplex, sp. nov., C. tenebris, sp. nov., C. tigris,sp.
nov., C. transversalis, sp. nov. and C. valvatus, sp. nov. The interspecicCOI sequence divergences among the new
species ranged from 6 to 15%. The intergeneric COI sequence divergence between species of Coxobolellus,gen.nov.,
Benoitolus birgitae and Pseudospirobolellus sp. ranged from 20 to 23%. Three major morphological differences
separate Coxobolellus, gen. nov. from Benoitolus and Pseudospirobolellus, namely (1) the protruding process on the 3rd
(and 4th) coxae on male legs, (2) the posterior gonopod telopodite divided into two parts, and (3) a conspicuous opening
pore at the mesal margin at the end of the coxal part of the posterior gonopod. Thus, the new genus is well supported by
both mtDNA and morphological evidence, while the delimitation of the 10 new species is supported by the congruence
between mtDNA and morphological data. Yet, with respect to the relationships of Benoitolus birgitae, morphological
data suggest a similarity with Coxobolellus,gen.nov.andPseudospirobolellus, whereas mtDNA data place thisspecies
in the Pachybolidae. Further phylogenetic analyses are needed to explore this apparent incongruence and test the
monophyly of Pseudospirobolellidae.
Additional keywords: mitochondrial DNA, new genus, phylogeny, species delineation.
Received 16 April 2020, accepted 6 May 2020, published online 14 August 2020
Introduction
The millipede order Spirobolida is one of the most diverse
groups of Diplopoda, with ~1200 described species worldwide
(Enghoff et al.2015). Yet, this gure is steadily increasing,
particularly with the recent discovery of new species of the
family Pachybolidae in Thailand and Laos (e.g. Pimvichai
et al.2018). The present paper aims to describe another series
of new spirobolidan taxa from south-east Asia. It focuses on
the family Pseudospirobolellidae Brölemann, 1913, a species-
poor and largely neglected taxon that currently comprises only
two genera, namely Pseudospirobolellus Carl, 1912 (two
species) and Benoitolus Mauriès, 1980 (three species)
(Jeekel 2001;Enghoffet al.2015). Several of these
species were described from South-east Asia, a region that
is a hotspot for diplopod diversity, such that each time
taxonomists start working on a particular family, the
numbers of new species rise sky high (e.g. 35 species of
the Harpagophoridae (e.g. Pimvichai et al.2016)and
68 species of the Paradoxosomatidae (e.g. Likhitrakarn
et al.2011; Srisonchai et al.2018a,2018b)). This turns out
to apply also to the Pseudospirobolellidae, as will be illustrated
with this paper in which 10 new species and one new genus of
Journal compilation CSIRO 2020 www.publish.csiro.au/journals/is
CSIRO PUBLISHING
Invertebrate Systematics, 2020, 34, 591617
https://doi.org/10.1071/IS20031
this family are described from Thailand by combining
morphological and DNA sequence data.
Material and methods
Live specimens were hand collected in the eld (for locality data
see Table 1). Some were preserved in 70% ethanol for
morphological studies, whereas the rest were placed in a
freezer at 20C for subsequent DNA studies. Specimens
from the following collections were also examined:
CUMZ, Museum of Zoology, Chulalongkorn University,
Bangkok, Thailand.
NHMW, Naturhistorisches Museum, Vienna, Austria.
NHMD, Natural History Museum of Denmark, University of
Copenhagen, Denmark.
This research was conducted under the approval of the
Animal Care and Use regulations (numbers U1-07304-2560
and IACUC-MSU-037/2019) of the Thai government.
Morphology
Gonopods were photographed with a digital camera manipulated
via the program Helicon Remote (ver. 3.1.1.w). The Zerene
Stacker Pro software was used for image-stacking. Samples for
scanning electron microscopy (SEM) were air-dried directly
from alcohol and sputter-coated for 250 s with gold. Scanning
electron micrographs were taken with an environmental
scanning electron microscope (ESEM)-FEI Quanta 200.
Drawings were made using a stereomicroscope and
photographs. The identication and classication of the
specimens followed Carl (1912), Attems (1936,1953),
Mauriès (1980) and Hoffman (1981). Voucher specimens
were deposited in the collection of CUMZ.
Terminology and abbreviations for the structure of gonopods
and vulvae in Coxobolellus, gen. nov.
*at, telopodite of the anterior gonopod, distinctly seen in
posterior view
*cx, coxal part of anterior gonopod
*oeg, opening of efferent groove
*pcx, coxal part of posterior gonopod
*pt, telopodital part of the posterior gonopod
*op, operculum of female vulvae
DNA extraction, amplication and sequencing
Total genomic DNA was extracted from dissected legs using
the NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany)
following the manufacturers instructions. PCR amplications
were done in 11-mL reaction volumes containing 5.50 mLof
Multiplex PCR Master Mix (Qiagen), 1 mLofeachprimer,
2.5 mL of sterilised distilled water and 1 mL of DNA extract.
Two mitochondrial DNA gene fragments (COI and 16S rRNA)
were amplied with the primers LCO-1490 and HCO-2198
(Folmer et al.1994)forCOI,and16Sar and 16Sbr (Kessing
et al.2004)for16S rRNA. The PCR conditions for the
amplication of COI and 16S rRNA were: initial
denaturation at 95C for 15 min, followed by 36 cycles of
94C for 30 s, 50C for 30 s and 72C for 1 min, and then a nal
extension at 72C for 10 min. PCR amplication success was
evaluated by comparing the amplicon sizes in 5 mLofPCR
product through electrophoresis on 1% (w/v) agaroseTBE
gel, stained with SYBR Safe and visualised by UV trans-
illumination. The remaining portion of each PCR reaction
(5 mL) was puried with the ExoSAP protocol (with 37C
for 15 min and 85C for 15 min). DNA sequencing was
performed with the BigDye Terminator (ver. 1.1) cycle
Sequencing kit (Applied Biosystems, Lennik, Belgium),
using the PCR primers. Sequencing was done with an ABI
3130xl capillary DNA sequencer (Applied Biosystems).
The COI data included 48 specimens, representing 19
genera and 13 nominal species of ingroup taxa
(Table 1). The 16S rRNA data included 42 specimens, i.e.
the same specimens as for COI, plus Litostrophus scaber
(Verhoeff, 1938) and Trachelomegalus cf. hoplurus,but
minus Benoitolus birgitae (Hoffman, 1981), Coxobolellus
albiceps, sp. nov. (Stpl), C. simplex, sp. nov. (TNP) and
C. valvatus, sp. nov. (TCD) in the ingroup and
Paraspirobolus lucifugus (Gervais, 1836) and Narceus
annularis Ranesque, 1820 in the outgroup. Thus, the
combined COI +16S rRNA data included 40 specimens.
Species of the order Spirostreptida, namely Anurostreptus
barthelemyae Demange, 1961 (Harpagophoridae),
Chonecambala crassicauda Mauriès & Enghoff, 1990
(Pericambalidae) and Thyropygus allevatus (Karsch, 1881)
(Harpagophoridae) were used as outgroup for tree
reconstruction. All nucleotide sequences have been
deposited in GenBank under accession numbers
MT328211MT328227 and MT328992MT329012 for the
partial 16S rRNA and COI fragment sequences respectively.
Sample data and voucher codes are shown in Table 1.
Alignment and phylogenetic analysis
CodonCode Aligner (ver. 4.0.4, CodonCode Corporation) was
used to assemble the forward and reverse sequences and to
check for errors and ambiguities. The COI and 16S rRNA
sequences were checked with the Basic Local Alignment
Search Tool (BLAST) provided by NCBI and compared
with reference sequences in GenBank. They were aligned
using MUSCLE (ver. 3.6, see http://www.drive5.com/
muscle; Edgar 2004). The alignments consisted of 660 bp
for COI and 458 bp for 16S rRNA (gaps were excluded by
complete-deletion). The sequences were checked for
ambiguous nucleotide sites, saturation and phylogenetic
signal using DAMBE (ver. 5.2.65. see http://www.dambe.
bio.uottawa.ca/DAMBE/dambe.aspx; Xia 2018). MEGA
(ver. X, see http://www.megasoftware.net; Tamura et al.
2013;Kumaret al.2018) was used to (1) translate COI
protein-coding sequences into amino acids, (2) check for
stop codons, (3) calculate uncorrected pairwise p-distances
among sequences, and (4) evaluate transition/transversion
ratios.
Phylogenetic trees were constructed using maximum
likelihood (ML) and Bayesian inference (BI). The shape
parameter of the gamma distribution, based on 16 rate
categories, was estimated using maximum-likelihood analysis.
592 Invertebrate Systematics P. Pimvichai et al.
Table 1. Specimens from which the COI or 16S rRNA partial DNA sequences were sequenced
CUMZ, Museum of Zoology, Chulalongkorn University, Bangkok, Thailand; NHMD, Natural History Museum of Denmark; NHMW, Naturhistorisches
Museum, Vienna, Austria; NHM, The Natural History Museum, London, United Kingdom. Names of countries are in upper case. Abbreviations after
species names refer to the isolate of each sequence. GenBank accession numbers are indicated for each species; , no sequences were obtained
Voucher code Locality COI 16S rRNA
Genus Apeuthes
A. maculatus Amc NHMW-Inv. No.2395 South Annam, VIETNAM MF187404 MF187360
Genus Atopochetus
A. anaticeps SVL CUMZ-D00091 Srivilai temple, Chalermprakiet, Saraburi,
THAILAND
MF187405
A. dollfusii DOL NHM Cochinchina, VIETNAM MF187412 MF187367
A. helix SPT CUMZ-D00094 Suan Pa Thong Pha Phum, Kanchanaburi,
THAILAND
MF187416 MF187371
A. moulmeinensis TAK CUMZ-D00095 87 km between Tha-Song Yang and Muang, Tak,
THAILAND
MF187417 MF187372
A. setiferus HPT CUMZ-D00097 Hub Pa Tard, Lan-Sak, Uthaithani, THAILAND MF187419 MF187374
A. spinimargo Ton27 ZMUC-00047013 Koh Yo, Songkhla, THAILAND MF187423 MF187377
A. truncatus SML CUMZ-D00101 Koh 8, Similan islands, Phang-Nga, THAILAND MF187424 MF187378
A. uncinatus KMR CUMZ-D00102 Khao Mar Rong, Bangsapan, Prachuapkhirikhan,
THAILAND
MF187425 MF187379
A. weseneri Tos29 ZMUC-00047003 Supar Royal Beach Hotel, Khanom,
Nakhonsrithammarat, THAILAND
MF187431 MF187384
Genus Aulacobolus
A. uncopygus Auc NHMW-Inv. No.2375 Nilgiris, South India, INDIA MF187433 MF187386
Genus Benoitolus
B. birgitae BBG NHMD 621687 Chiang Dao, Chiang-Mai, THAILAND MT328992
Genus Coxobolellus, gen. nov.
C. albiceps, sp. nov. Stpw CUMZ-D00121 Tham Pha Tub, Muang District, Nan Province,
THAILAND (green individual)
MT328994 MT328211
C. albiceps, sp. nov. Stpl CUMZ-D00122 Tham Pha Tub, Muang District, Nan Province,
THAILAND (small, brown individual)
MT328993
C. albiceps, sp. nov. TPB CUMZ-D00123 Wat Tham Bampen Bun, Pan District, Chiang-Rai
Province, THAILAND
MT328996 MT328213
C. albiceps, sp. nov. Stvd CUMZ-D00124 Tham Wang Daeng, Noen Maprang District,
Phitsanulok Province, THAILAND
MT328995 MT328212
C. compactogonus, sp. nov. SKR CUMZ-D00134 Sakaerat Environmental Research Station, Wang
Nam Khiao District, Nakhon Ratchasima Province,
THAILAND
MT328998 MT328215
C. compactogonus, sp. nov. KLC CUMZ-D00135 Khao Look Chang, Pak Chong District, Nakhon
Ratchasima Province, THAILAND
MT328997 MT328214
C. fuscus, sp. nov. HKK CUMZ-D00133 Kroeng Krawia waterfall, Sangkhla Buri District,
Kanchanaburi Province, THAILAND
MT328999 MT328216
C. nodosus, sp. nov. SPW CUMZ-D00126 Chao Por Phawo Shrine, Mae Sot District, Tak
Province, THAILAND
MT329000 MT328217
C. serratus, sp. nov. KKL CUMZ-D00132 Khao Kalok, Pran Buri District, Prachuap Khiri Khan
Province, THAILAND
MT329001 MT328218
C. simplex, sp. nov. TNP CUMZ-D00136 Tham Pha Pha Ngam, Mae Prik District, Lampang
Province, THAILAND
MT329002
C. tenebris, sp. nov. KWP CUMZ-D00119 Wat Khao Wong Phrohm-majan, Ban Rai District,
Uthai Thani Province, THAILAND
MT329003 MT328219
C. tenebris, sp. nov. TPL CUMZ-D00120 Wat Tham Phrom Lok Khao Yai, Sai Yok District,
Kanchanaburi Province, THAILAND
MT329004 MT328220
C. tigris, sp. nov. TKP CUMZ-D00130 Wat Tham Khao Plu, Pathio District, Chumphon
Province, THAILAND
MT329005 MT328221
C. tigris, sp. nov. TYE CUMZ-D00131 Tham Yai I, Pathio District, Chumphon Province,
THAILAND
MT329006 MT328222
C. transversalis, sp. nov. Stpg CUMZ-D00125 Tham Pha Tub, Muang District, Nan Province,
THAILAND
MT329007 MT328223
C. valvatus, sp. nov. TCD CUMZ-D00127 Wat Tham Chiang Dao, Chiang Dao District, Chiang-
Mai Province, THAILAND
MT329009
(continued next page )
Phylogeny and new genus of Pseudospirobolellidae Invertebrate Systematics 593
ML trees were inferred with RAxML (ver. 8.2.12, see
http://www.phylo.org/index.php/tools/raxmlhpc2_tgb.html;
Stamatakis 2014) through the CIPRES Science Gateway
(Miller et al.2010) using a GTR+G substitution model
and 1000 bootstrap replicates to assess branch support.
The COI sequence alignment was partitioned by 1st, 2nd
and 3rd codon position and the concatenated sequence
alignment was partitioned by gene fragment and by 1st,
2nd and 3rd codon position for COI.
BI trees were constructed with MrBayes (ver. 3.1.2, see
http://www.phylo.org/index.php/tools/mrbayes_xsede.html;
Huelsenbeck and Ronquist 2001)forCOI and 16S rRNA
separately, as well as for the combined data. Substitution
models were inferred separately for each gene fragment
using PartitionFinder 2 on XSEDE (ver. 2.1.1, see http://
www.phylo.org/index.php/tools/partitionnder2_xsede.html;
Lanfear et al.2017) through the CIPRES Science Gateway
(Miller et al.2010). BI trees were run for 2 million generations
(heating parameters were 0.05 for COI and 0.07 for 16S rRNA
and combined datasets), sampling every 1000 generations.
Convergences were conrmed by verifying that the standard
deviations of split frequencies were below 0.01. Then the rst
1000 trees were discarded as burn-in, so that the nal
consensus tree was built from the last 3002 trees. The
optimal parameters were assessed by calculating the Bayes
factor using Tracer (ver. 1.6.0, A. Rambaut, M. A. Suchard,
W. Xie, and A. J. Drummond, see http://beast.bio.ed.ac.uk/
Tracer, accessed 11 December 2019). Support for nodes was
assessed by posterior probabilities.
We consider clades with bootstrap values (BV) of 70% to be
well supported (Hillis and Bull 1993) and <70% as poorly
supported. For BI analyses, we considered clades with
Table 1. (continued )
Voucher code Locality COI 16S rRNA
C. valvatus, sp. nov. BRC CUMZ-D00128 Tham Borichinda, Chom Thong District, Chiang-Mai
Province, THAILAND
MT329008 MT328224
C. valvatus, sp. nov. TST CUMZ-D00129 Tham Sam Ta, Muang District, Mae Hong Son
Province, THAILAND
MT329010 MT328225
Genus Leptogoniulus
L. sorornus BTN CUMZ-D00109 Botanical Garden, Penang, MALAYSIA MF187434 MF187387
Genus Litostrophus
L. chamaeleon PPT CUMZ-D00111 Phu Pha terb, Mukdahan, THAILAND MF187436 MF187389
L. saraburensis PKS CUMZ-D00113 Phukhae Botanical Garden, Saraburi, THAILAND MF187438 MF187391
L. segregatus Ls19 NHMD 621686 Koh Kut, Trad, THAILAND MF187440 MF187394
Genus Madabolus
M. maximus Mm4 ZMUC-00047007 de Toliara Province, Parc National de Bermaraha,
South Bank of Manambolo River, Near Tombeau
Vazimba, MADAGASCAR
MF187441 MF187395
Genus Narceus
N. annularis NC_003343.1
Genus Parabolus
P. dimorphus Pd34 ZMUC-00047004 Dar es Salaam, TANZANIA MF187442 MF187396
Genus Paraspirobolus
P. lucifugus AB608779.1
Genus Pelmatojulus
P. tigrinus Pt2 ZMUC-00047008 Southern part of the Comoé N.P., 30 km north of
Kakpin, CÔTE d' IVOIRE
MF187443 MF187397
P. togoensis Pto6 ZMUC-00047006 Biakpa, GHANA MF187444 MF187398
Genus Pseudospirobolellus
Pseudospirobolellus avernus GPG CUMZ-D00117 Gua Pulai, Gua Musang, Kelantan, MALAYSIA MT329011 MT328226
Pseudospirobolellus sp. KCS CUMZ-D00118 Koh Chuang, Sattahip, Chonburi, THAILAND MT329012 MT328227
Genus Rhinocricus
R. parcus Rp49 ZMUC-00047009 Puerto Rico, USA MF187449 MF187403
Genus Trachelomegalus
T. sp. Tr54 ZMUC-00047012 Borneo Sabah, MALAYSIA MF187445
T. cf. hoplurus Th ZMUC-00047002 Borneo Sarawak, Niah, MALAYSIA MF187399
Genus Trigoniulus
T. corallinus Tco15 ZMUC-00047010 Vientiane, LAOS MF187446 MF187400
Outgroup
Genus Anurostreptus
A. barthelemyae Tlb CUMZ-D00003 Thale-Ban N.P., Khuan-Don, Satun, THAILAND KC519469 KC519543
Genus Chonecambala
C. crassicauda Ttp CUMZ-D00001 Ton-Tong waterfall, Pua, Nan, THAILAND KC519467 KC519541
Genus Thyropygus
T. allevatus Bb CUMZ-D00013 BangBan, Ayutthaya, THAILAND KC519479 KC519552
594 Invertebrate Systematics P. Pimvichai et al.
posterior probabilities (PP) of 0.90 (e.g. Shiels et al.2014)to
be well supported and <0.90 as poorly supported.
Species delimitation
Putative species were explored by applying the General Mixed
Yule Coalescent (GMYC) (Fujisawa and Barraclough 2013)
and the Automatic Barcode Gap Discovery (ABGD)
(Puillandre et al.2012) methods to the COI sequence data,
i.e. the standard DNA barcoding fragment (Hebert et al.2003).
GMYC requires an ultrametric gene tree (fully dichotomous
tree) input to derive a BI tree using BEAST (ver. 1.8.2,
see http://www.tree.bio.ed.ac.uk/software/beast/; Drummond
et al.2012). An XML le was made with the BEAUti
(ver. 1.8.2, see http://www.beast.community/) interface
under a lognormal relaxed (uncorrelated) molecular clock
with the GTR substitution model. Putative species were
identied using the single and multiple GMYC thresholds
via the web interface at https://species.h-its.org/gmyc/.
With ABGD, barcode gaps can be observed whenever the
sequence divergence among conspecic specimens is smaller
than the sequence divergence among specimens from different
species (http://wwwabi.snv.jussieu.fr/public/abgd/). COI
alignments were uploaded to the ABGD server at http://
wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html and were
run with the default settings, except for the relative gap
width, which was set at X = 0.98 (0.98 was the highest
value that could be applied because the default value for
relative gap width (X = 1.5) value did not produce a result
for the dataset). COI sequence divergence was estimated with
K2P distances.
Results
The nucleotide frequencies in the aligned COI gene fragment
(660 bp) were: A, 0.284; C, 0.208; G, 0.159; T, 0.349 36.7%
GC content. The uncorrected p-distance between the
sequences ranged from 0.00 to 0.26 (Table 2).
The nucleotide frequencies in the aligned 16S rRNA gene
fragment (458 bp) were: A, 0.325; C, 0.108; G, 0.212; T, 0.355
31.9% GC content. The uncorrected p-distance between the
sequences ranged from 0.00 to 0.29 (Table 3).
The nucleotide frequencies in the COI +16S rRNA dataset
(1118 bp) were: A, 0.302; C, 0.166; G, 0.180; T, 0.352 34.6%
GC content. The uncorrected p-distance between the sequences
ranged from 0.00 to 0.27.
Phylogeny
The ML and BI trees for the separate and combined datasets
(COI,16S rRNA and COI +16S rRNA) were largely
congruent (by visual inspection of the branching pattern).
The single 16S rRNA and the combined (COI +16S rRNA)
trees are presented in the Supplementary material (Fig. S1, S2
respectively), whereas the COI tree is used for further
discussion (Fig. 1). PartitionFinder indicated that the best
substitution models for BI analysis were GTR+I+G for 16S
rRNA and the 1st and 2nd codon positions of COI, and GTR
+G for the 3rd codon position.
Although the COI tree includes representatives of six
Spirobolida families (Pachybolidae, Pseudospirobolellidae,
Rhinocricidae, Spirobolidae, Spirobolellidae and
Trigoniulidae), it provides no meaningful support for the
relationships and monophyly of the family Pachybolidae. The
three trigoniulid genera Apeuthes Hoffman & Keeton, 1960,
Leptogoniulus Silvestri, 1897 and Trigoniulus Pocock, 1894
form a well supported clade (BV = 97; PP = 1.00).
Benoitolus birgitae groups with Litostrophus
(Pachybolidae) and thus is well separated from the genus
Pseudospirobolellus. However, this is based on COI only
and with a fairly long branch, the position of which in the
tree is ambiguously supported. COI sequence divergence
between B. birgitae and (1) Coxobolellus, gen. nov. is
2023% (mean: 22%), (2) Pseudospirobolellus is 23%, and
(3) Pachybolidae genera is 1124% (mean: 21%). The lowest
COI sequence divergence is with Litostrophus (1118%;
mean: 15%).
The sister-group relation between Coxobolellus, gen. nov.
and Pseudospirobolellus is well supported by both ML and BI in
all trees, just as is the sister-group relation of the two
Pseudospirobolellus species.
Clade 1 is well supported and comprises 18 sequences of 10
Coxobolellus, gen. nov. species that are grouped into four well
supported clades (1AD) (except for Clade 1B in the 16S
rRNA tree, Fig. S1).
Clade 1A comprises C. nodosus, sp. nov. and C. valvatus,
sp. nov., two species that are distributed in western and
northern Thailand respectively. Their anterior gonopods are
similar, but they differ in their posterior gonopod telopodite,
and their COI sequence divergence is 67%.
Clade 1B comprises two sympatric species: C. albiceps,sp.
nov. and C. transversalis, sp. nov. They differ in the tip of the
anterior gonopod and the posterior gonopod telopodite, and
show 8% COI sequence divergence.
Clade 1C comprises C. compactogonus, sp. nov. and
C. tenebris, sp. nov., which are distributed in north-eastern
and central to western Thailand respectively. They differ in
both the anterior gonopod and posterior gonopod telopodite,
and their COI sequence divergence is 910%.
Clade 1D comprises C. serratus, sp. nov. and C. tigris,sp.
nov., which have similar anterior gonopods, but differ in their
posterior gonopod telopodite. Both species are distributed in
southern Thailand, and their COI sequence divergence is 12%.
Species delimitation based on COI sequences
GMYC
With both the single and multiple thresholds, the
maximum-likelihood values of the null model (=all
sequences belong to a single species) were lower than the
maximum-likelihood value of the GMYC model. The single
threshold yielded 4 clusters and 11 entities, whereas the
multiple thresholds yielded 4 clusters and 7 entities (Table 4).
ABGD
This method was run with prior intraspecicK2P
divergences ranging from P
min
= 0.001 to P
max
=0.1.The
initial partition of sequences into 11 operational taxonomic
units (OTUs) based on a statistically inferred barcode gap
(Kekkonen et al.2015) was stable over the range of prior
Phylogeny and new genus of Pseudospirobolellidae Invertebrate Systematics 595
Table 2. Estimates of COI sequence divergences within and among Coxobolellus, gen. nov. species and related taxa expressed as uncorrected p-distances (rounded up to two decimal places)
The number of base differences per site between sequences are shown. This analysis involved 48 nucleotide sequences. Codon positions included were 1st+2nd+3rd. There were a total of 660 positions in the nal
dataset. Evolutionary analyses were conducted in MEGA (ver. X, see http://www.megasoftware.net; Kumar et al.2018)
1234567891011121314151617181920212223242526272829303132333435363738394041424344454647
1Apeuthes maculatus Amc
2Atopochetus anaticeps SVL 0.19
3Atopochetus dollfusii DOL 0.20 0.11
4Atopochetus helix SPT 0.21 0.14 0.13
5Atopochetus moulmeinensis TAK 0.22 0.14 0.12 0.15
6Atopochetus setiferus HPT 0.19 0.08 0.09 0.14 0.13
7Atopochetus spinimargo Ton27 0.22 0.15 0.14 0.14 0.16 0.14
8Atopochetus truncatus SML 0.19 0.13 0.10 0.12 0.14 0.12 0.14
9Atopochetus uncinatus KMR 0.200.13 0.14 0.14 0.15 0.13 0.15 0.13
10 Atopochetus weseneri Tos29 0.20 0.14 0.12 0.14 0.13 0.12 0.16 0.10 0.13
11 Aulacobolus uncopygus Auc 0.17 0.18 0.18 0.20 0.21 0.19 0.22 0.19 0.20 0.22
12 Benoitolus birgitae BBG 0.21 0.23 0.21 0.21 0.22 0.21 0.22 0.21 0.22 0.24 0.22
13 Coxobolellus albiceps, sp. nov. Stpl 0.18 0.20 0.22 0.21 0.24 0.22 0.23 0.21 0.22 0.22 0.18 0.22
14 Coxobolellus albiceps, sp. nov. Stpw 0.18 0.20 0.22 0.22 0.24 0.22 0.23 0.21 0.21 0.22 0.18 0.22 0.02
15 Coxobolellus albiceps, sp. nov. Stvd 0.17 0.20 0.22 0.21 0.24 0.22 0.23 0.21 0.22 0.23 0.18 0.21 0.02 0.02
16 Coxobolellus albiceps, sp. nov. TPB 0.18 0.20 0.22 0.21 0.24 0.22 0.23 0.21 0.22 0.22 0.18 0.21 0.02 0.02 0.02
17 Coxobolellus compactogonus, sp. nov.
KLC
0.19 0.20 0.21 0.23 0.24 0.21 0.22 0.22 0.22 0.22 0.18 0.22 0.12 0.11 0.12 0.12
18 Coxobolellus compactogonus, sp. nov.
SKR
0.19 0.21 0.21 0.22 0.24 0.21 0.23 0.21 0.21 0.22 0.19 0.23 0.14 0.14 0.14 0.14 0.05
19 Coxobolellus fuscus, sp. nov. HKK 0.20 0.20 0.22 0.22 0.24 0.20 0.23 0.23 0.20 0.23 0.19 0.22 0.12 0.12 0.12 0.13 0.11 0.13
20 Coxobolellus nodosus, sp. nov. SPW 0.20 0.21 0.20 0.21 0.24 0.21 0.23 0.22 0.22 0.23 0.18 0.21 0.11 0.11 0.10 0.11 0.11 0.13 0.11
21 Coxobolellus serratus, sp. nov. KKL 0.18 0.20 0.21 0.22 0.23 0.20 0.23 0.21 0.22 0.22 0.19 0.22 0.12 0.13 0.12 0.13 0.12 0.14 0.12 0.13
22 Coxobolellus simplex, sp. nov. TNP 0.19 0.21 0.22 0.21 0.23 0.22 0.23 0.23 0.23 0.22 0.20 0.23 0.13 0.13 0.13 0.13 0.11 0.12 0.12 0.12 0.11
23 Coxobolellus tenebris, sp. nov. KWP 0.18 0.21 0.22 0.23 0.25 0.22 0.24 0.23 0.23 0.23 0.19 0.22 0.13 0.13 0.12 0.13 0.09 0.10 0.11 0.12 0.14 0.11
24 Coxobolellus tenebris, sp. nov. TPL 0.18 0.21 0.22 0.23 0.25 0.22 0.24 0.23 0.23 0.23 0.19 0.22 0.13 0.13 0.13 0.13 0.09 0.10 0.12 0.12 0.14 0.11 0.00
25 Coxobolellus tigris, sp. nov. TKP 0.20 0.19 0.22 0.22 0.24 0.21 0.24 0.21 0.21 0.21 0.21 0.22 0.13 0.13 0.13 0.12 0.14 0.14 0.12 0.14 0.12 0.13 0.15 0.15
26 Coxobolellus tigris, sp. nov. TYE 0.20 0.19 0.22 0.22 0.25 0.21 0.24 0.22 0.22 0.22 0.21 0.22 0.13 0.13 0.13 0.12 0.13 0.14 0.12 0.13 0.12 0.13 0.14 0.15 0.02
27 Coxobolellus transversalis, sp. nov.
Stpg
0.18 0.20 0.20 0.20 0.23 0.21 0.21 0.20 0.22 0.22 0.18 0.20 0.08 0.08 0.08 0.08 0.13 0.15 0.12 0.11 0.12 0.12 0.14 0.14 0.13 0.13
28 Coxobolellus valvatus, sp. nov. BRC 0.17 0.20 0.21 0.20 0.24 0.20 0.23 0.22 0.22 0.22 0.17 0.21 0.10 0.10 0.10 0.10 0.11 0.13 0.11 0.07 0.13 0.12 0.12 0.12 0.13 0.13 0.11
29 Coxobolellus valvatus, sp. nov. TCD 0.17 0.20 0.20 0.20 0.23 0.20 0.22 0.22 0.22 0.23 0.16 0.21 0.11 0.11 0.11 0.11 0.11 0.13 0.11 0.06 0.13 0.11 0.11 0.12 0.13 0.12 0.10 0.03
30 Coxobolellus valvatus, sp. nov. TST 0.17 0.20 0.20 0.21 0.23 0.20 0.22 0.21 0.22 0.22 0.17 0.21 0.11 0.11 0.11 0.11 0.11 0.13 0.12 0.06 0.13 0.11 0.12 0.12 0.13 0.12 0.10 0.02 0.02
31 Paraspirobolus lucifugus 0.22 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.24 0.24 0.26 0.24 0.24 0.25 0.24 0.23 0.23 0.23 0.24 0.25 0.25 0.24 0.24 0.25 0.24 0.24 0.23 0.24 0.24
32 Leptogoniulus sorornus BTN 0.13 0.18 0.18 0.19 0.21 0.19 0.20 0.19 0.22 0.22 0.17 0.22 0.20 0.21 0.20 0.20 0.18 0.20 0.20 0.19 0.19 0.18 0.20 0.20 0.20 0.20 0.19 0.19 0.19 0.18 0.24
33 Litostrophus chamaeleon PPT 0.18 0.17 0.16 0.15 0.18 0.16 0.18 0.15 0.17 0.17 0.19 0.18 0.20 0.20 0.20 0.20 0.20 0.21 0.20 0.20 0.20 0.21 0.21 0.21 0.20 0.20 0.21 0.20 0.21 0.20 0.24 0.18
34 Litostrophus saraburensis PKS 0.16 0.16 0.15 0.15 0.17 0.15 0.16 0.14 0.16 0.18 0.18 0.11 0.19 0.18 0.18 0.18 0.20 0.20 0.19 0.19 0.20 0.20 0.20 0.20 0.20 0.20 0.18 0.19 0.18 0.18 0.24 0.18 0.11
35 Litostrophus segregatus Ls19 0.180.13 0.13 0.15 0.16 0.13 0.15 0.14 0.14 0.17 0.18 0.17 0.21 0.21 0.20 0.21 0.22 0.21 0.21 0.21 0.20 0.22 0.21 0.21 0.21 0.21 0.20 0.20 0.20 0.20 0.25 0.18 0.11 0.09
36 Madabolus maximus Mm4 0.20 0.21 0.20 0.19 0.22 0.22 0.21 0.20 0.20 0.22 0.18 0.21 0.21 0.20 0.21 0.20 0.22 0.23 0.22 0.21 0.22 0.22 0.24 0.24 0.22 0.22 0.21 0.20 0.20 0.20 0.24 0.20 0.19 0.18 0.20
37 Narceus annularis 0.21 0.23 0.20 0.21 0.22 0.21 0.20 0.20 0.21 0.21 0.20 0.24 0.22 0.22 0.22 0.22 0.23 0.23 0.21 0.23 0.22 0.22 0.23 0.23 0.22 0.22 0.22 0.22 0.21 0.22 0.21 0.21 0.20 0.20 0.21 0.20
38 Parabolus dimorphus Pd34 0.20 0.18 0.20 0.19 0.22 0.18 0.20 0.21 0.19 0.21 0.19 0.22 0.18 0.18 0.19 0.18 0.20 0.22 0.19 0.18 0.20 0.20 0.20 0.20 0.17 0.17 0.19 0.18 0.18 0.18 0.25 0.21 0.19 0.18 0.20 0.17 0.20
39 Pelmatojulus tigrinus Pt2 0.170.22 0.22 0.20 0.23 0.22 0.23 0.22 0.22 0.22 0.16 0.23 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.21 0.22 0.22 0.22 0.22 0.20 0.20 0.21 0.19 0.19 0.19 0.24 0.19 0.20 0.20 0.20 0.18 0.19 0.19
40 Pelmatojulus togoensis Pto6 0.18 0.21 0.22 0.22 0.22 0.20 0.20 0.21 0.20 0.21 0.17 0.22 0.19 0.19 0.19 0.19 0.19 0.20 0.20 0.19 0.19 0.21 0.20 0.20 0.20 0.20 0.20 0.18 0.18 0.18 0.25 0.18 0.18 0.18 0.19 0.19 0.20 0.18 0.17
41 Pseudospirobolellus avernus GPG 0.20 0.21 0.22 0.22 0.23 0.21 0.23 0.22 0.23 0.23 0.20 0.23 0.21 0.20 0.21 0.20 0.21 0.21 0.20 0.21 0.20 0.21 0.21 0.21 0.20 0.20 0.20 0.21 0.21 0.21 0.22 0.19 0.23 0.21 0.21 0.21 0.22 0.23 0.20 0.21
42 Pseudospirobolellus sp. KCS 0.23 0.23 0.23 0.21 0.23 0.23 0.22 0.22 0.23 0.23 0.22 0.23 0.22 0.22 0.22 0.22 0.22 0.22 0.21 0.21 0.22 0.22 0.23 0.23 0.23 0.22 0.22 0.22 0.22 0.22 0.22 0.21 0.23 0.22 0.22 0.24 0.22 0.21 0.22 0.22 0.14
43 Rhinocricus parcus Rp49 0.230.24 0.21 0.22 0.22 0.24 0.21 0.23 0.22 0.23 0.22 0.25 0.26 0.25 0.25 0.25 0.24 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.24 0.24 0.25 0.24 0.25 0.25 0.22 0.22 0.22 0.23 0.23 0.22 0.20 0.23 0.21 0.22 0.22 0.21
44 Trachelomegalus sp. Tr54 0.20 0.19 0.18 0.17 0.20 0.19 0.18 0.18 0.18 0.18 0.20 0.22 0.21 0.21 0.21 0.21 0.24 0.22 0.24 0.23 0.22 0.22 0.23 0.23 0.23 0.24 0.22 0.23 0.23 0.22 0.24 0.21 0.18 0.17 0.15 0.21 0.21 0.21 0.19 0.21 0.22 0.20 0.22
45 Trigoniulus corallinus Tco15 0.12 0.18 0.19 0.20 0.23 0.19 0.20 0.20 0.21 0.21 0.17 0.20 0.18 0.18 0.17 0.17 0.17 0.18 0.17 0.18 0.19 0.17 0.17 0.18 0.17 0.17 0.17 0.16 0.15 0.15 0.23 0.14 0.18 0.16 0.17 0.18 0.20 0.19 0.18 0.17 0.23 0.22 0.23 0.20
46 Anurostreptus barthelemyae Tlb 0.21 0.22 0.22 0.23 0.24 0.23 0.22 0.24 0.23 0.24 0.20 0.25 0.20 0.19 0.19 0.20 0.20 0.21 0.19 0.19 0.20 0.19 0.20 0.20 0.20 0.19 0.19 0.18 0.18 0.18 0.23 0.22 0.21 0.20 0.22 0.22 0.21 0.21 0.21 0.20 0.22 0.21 0.23 0.23 0.19
47 Chonecambala crassicauda Ttp 0.21 0.24 0.24 0.24 0.23 0.24 0.23 0.24 0.22 0.24 0.22 0.26 0.23 0.23 0.23 0.23 0.22 0.23 0.22 0.23 0.22 0.22 0.23 0.23 0.22 0.22 0.22 0.21 0.22 0.22 0.23 0.21 0.23 0.22 0.24 0.24 0.23 0.21 0.22 0.24 0.23 0.23 0.23 0.22 0.22 0.19
48 Thyropygus allevatus Bb 0.21 0.21 0.21 0.22 0.23 0.23 0.23 0.22 0.21 0.24 0.20 0.23 0.20 0.20 0.20 0.20 0.19 0.19 0.20 0.20 0.19 0.19 0.20 0.20 0.19 0.19 0.20 0.19 0.20 0.19 0.22 0.20 0.21 0.20 0.22 0.21 0.20 0.21 0.23 0.21 0.22 0.22 0.21 0.24 0.20 0.15 0.20
596 Invertebrate Systematics P. Pimvichai et al.
Table 3. Estimates of 16S rRNA sequence divergences within and among Coxobolellus gen. nov. species and related taxa expressed as uncorrected p-distances (rounded up to two decimal places)
The number of base differences per site between sequences are shown. This analysis involved 42 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There
were a total of 458 positions in the nal dataset. Evolutionary analyses were conducted in MEGA (ver. X, http://www.megasoftware.net; Kumar et al.2018)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
1Apeuthes maculatus Amc
2Atopochetus dollfusii DOL 0.19
3Atopochetus helix SPT 0.19 0.05
4Atopochetus moulmeinensis TAK 0.20 0.07 0.06
5Atopochetus setiferus HPT 0.21 0.07 0.06 0.06
6Atopochetus spinimargo Ton27 0.19 0.07 0.06 0.06 0.07
7Atopochetus truncatus SML 0.19 0.05 0.05 0.070.07 0.05
8Atopochetus uncinatus KMR 0.18 0.04 0.04 0.06 0.07 0.06 0.05
9Atopochetus weseneri Tos29 0.20 0.06 0.05 0.07 0.05 0.06 0.03 0.05
10 Aulacobolus uncopygus Auc 0.19 0.16 0.16 0.15 0.15 0.15 0.16 0.15 0.16
11 Coxobolellus albiceps, sp. nov. Stpw 0.280.22 0.23 0.23 0.24 0.22 0.220.23 0.23 0.26
12 Coxobolellus albiceps, sp. nov. Stvd 0.25 0.21 0.21 0.22 0.22 0.21 0.21 0.21 0.22 0.25 0.05
13 Coxobolellus albiceps, sp. nov. TPB1 0.24 0.21 0.22 0.23 0.23 0.21 0.22 0.21 0.22 0.24 0.06 0.05
14 Coxobolellus compactogonus, sp. nov.
KLC
0.23 0.21 0.22 0.22 0.22 0.21 0.22 0.22 0.23 0.22 0.15 0.12 0.12
15 Coxobolellus compactogonus, sp. nov.
SKR
0.25 0.22 0.23 0.23 0.23 0.21 0.22 0.22 0.23 0.23 0.14 0.12 0.12 0.04
16 Coxobolellus fuscus, sp. nov. HKK 0.240.20 0.22 0.220.22 0.21 0.220.21 0.23 0.240.12 0.10 0.100.08 0.09
17 Coxobolellus nodosus, sp. nov. SPW 0.23 0.21 0.23 0.23 0.23 0.21 0.21 0.21 0.22 0.24 0.12 0.10 0.09 0.11 0.12 0.11
18 Coxobolellus serratus, sp. nov. KKL 0.25 0.21 0.22 0.23 0.22 0.21 0.21 0.22 0.22 0.23 0.13 0.10 0.11 0.09 0.10 0.09 0.13
19 Coxobolellus tenebris, sp. nov. KWP 0.240.22 0.23 0.23 0.23 0.22 0.23 0.22 0.23 0.23 0.15 0.12 0.12 0.06 0.07 0.09 0.12 0.10
20 Coxobolellus tenebris, sp. nov. TPL 0.24 0.22 0.23 0.23 0.23 0.22 0.23 0.22 0.23 0.23 0.15 0.12 0.12 0.06 0.07 0.09 0.12 0.10 0.00
21 Coxobolellus tigris, sp. nov. TKP 0.25 0.21 0.23 0.24 0.23 0.21 0.22 0.21 0.24 0.24 0.13 0.11 0.10 0.09 0.09 0.08 0.11 0.07 0.09 0.09
22 Coxobolellus tigris, sp. nov. TYE 0.26 0.21 0.23 0.24 0.23 0.22 0.22 0.22 0.24 0.24 0.13 0.11 0.11 0.09 0.09 0.08 0.12 0.07 0.09 0.09 0.01
23 Coxobolellus transversalis, sp. nov.
Stpg
0.24 0.20 0.21 0.21 0.21 0.21 0.21 0.21 0.22 0.23 0.10 0.07 0.08 0.10 0.11 0.09 0.09 0.11 0.11 0.11 0.11 0.12
24 Coxobolellus valvatus, sp. nov. BRC2 0.23 0.20 0.21 0.22 0.22 0.20 0.20 0.21 0.21 0.23 0.10 0.08 0.08 0.11 0.11 0.09 0.05 0.11 0.11 0.11 0.10 0.11 0.07
25 Coxobolellus valvatus, sp. nov. TST 0.23 0.21 0.22 0.23 0.22 0.21 0.21 0.21 0.22 0.24 0.10 0.09 0.09 0.10 0.11 0.09 0.05 0.11 0.11 0.11 0.10 0.11 0.08 0.02
26 Leptogoniulus sorornus BTN 0.10 0.22 0.21 0.23 0.23 0.22 0.22 0.22 0.22 0.21 0.29 0.27 0.27 0.26 0.27 0.25 0.26 0.25 0.27 0.27 0.25 0.26 0.26 0.25 0.26
27 Litostrophus chamaeleon PPT 0.210.10 0.11 0.12 0.10 0.11 0.11 0.11 0.10 0.14 0.23 0.21 0.220.21 0.22 0.210.22 0.21 0.210.21 0.22 0.220.22 0.22 0.230.22
28 Litostrophus saraburensis PKS 0.21 0.11 0.10 0.11 0.11 0.11 0.11 0.10 0.10 0.150.25 0.22 0.240.23 0.24 0.230.25 0.22 0.240.24 0.23 0.240.22 0.24 0.250.22 0.08
29 Litostrophus scaber Sca3 0.21 0.10 0.11 0.11 0.11 0.10 0.10 0.10 0.10 0.14 0.25 0.22 0.22 0.21 0.22 0.22 0.22 0.21 0.22 0.22 0.22 0.22 0.22 0.22 0.230.23 0.09 0.06
30 Litostrophus segregatus Ls19 0.22 0.12 0.11 0.12 0.11 0.11 0.11 0.11 0.11 0.15 0.25 0.24 0.24 0.23 0.23 0.23 0.24 0.22 0.24 0.24 0.24 0.24 0.22 0.24 0.24 0.23 0.10 0.06 0.08
31 Madabolus maximus Mm4 0.21 0.19 0.18 0.19 0.19 0.17 0.19 0.17 0.20 0.19 0.24 0.22 0.22 0.22 0.23 0.24 0.23 0.25 0.24 0.24 0.25 0.26 0.22 0.22 0.23 0.22 0.19 0.21 0.22 0.20
32 Parabolus dimorphus Pd34 0.21 0.17 0.17 0.16 0.18 0.17 0.18 0.16 0.18 0.18 0.25 0.24 0.23 0.24 0.24 0.25 0.24 0.26 0.26 0.26 0.26 0.26 0.25 0.24 0.24 0.24 0.19 0.21 0.19 0.21 0.11
33 Pelmatojulus tigrinus Pt2 0.23 0.21 0.21 0.20 0.20 0.19 0.20 0.20 0.21 0.20 0.27 0.24 0.24 0.24 0.25 0.26 0.25 0.25 0.26 0.26 0.27 0.27 0.25 0.24 0.25 0.26 0.21 0.21 0.22 0.22 0.12 0.12
34 Pelmatojulus togoensis Pto6 0.21 0.19 0.19 0.190.18 0.17 0.190.17 0.19 0.180.27 0.25 0.240.24 0.25 0.260.25 0.25 0.270.27 0.26 0.270.26 0.25 0.250.24 0.21 0.220.20 0.20 0.110.09 0.12
35 Pseudospirobolellus avernus GPG 0.230.23 0.24 0.24 0.24 0.22 0.24 0.22 0.23 0.220.22 0.19 0.190.19 0.19 0.210.21 0.21 0.210.21 0.21 0.210.19 0.20 0.210.25 0.21 0.240.22 0.25 0.240.24 0.26 0.26
36 Pseudospirobolellus sp. KCS 0.26 0.24 0.25 0.24 0.24 0.22 0.25 0.25 0.24 0.23 0.23 0.22 0.21 0.22 0.23 0.21 0.22 0.22 0.22 0.22 0.22 0.22 0.21 0.21 0.22 0.26 0.23 0.25 0.24 0.26 0.25 0.26 0.25 0.26 0.09
37 Rhinocricus parcus Rp49 0.23 0.22 0.22 0.20 0.22 0.21 0.23 0.22 0.23 0.21 0.29 0.28 0.28 0.26 0.26 0.26 0.28 0.28 0.26 0.26 0.26 0.27 0.27 0.26 0.27 0.25 0.22 0.23 0.20 0.22 0.24 0.24 0.25 0.22 0.26 0.28
38 Trachelomegalus cf. hoplurus Th23 0.19 0.12 0.12 0.14 0.14 0.13 0.13 0.12 0.12 0.15 0.26 0.24 0.25 0.23 0.24 0.250.23 0.24 0.240.24 0.24 0.240.24 0.23 0.240.21 0.13 0.110.11 0.12 0.200.19 0.22 0.210.25 0.22 0.24
39 Trigoniulus corallinus Tco15 0.11 0.20 0.20 0.22 0.21 0.21 0.21 0.20 0.22 0.22 0.27 0.24 0.24 0.25 0.26 0.24 0.24 0.24 0.25 0.25 0.25 0.25 0.23 0.24 0.24 0.13 0.21 0.21 0.21 0.21 0.22 0.21 0.25 0.23 0.27 0.25 0.24 0.20
40 Anurostreptus barthelemyae Tlb 0.21 0.19 0.21 0.22 0.22 0.22 0.20 0.20 0.21 0.20 0.28 0.25 0.25 0.24 0.24 0.24 0.23 0.24 0.24 0.24 0.25 0.26 0.24 0.22 0.23 0.24 0.19 0.19 0.18 0.21 0.24 0.23 0.26 0.24 0.27 0.25 0.26 0.20 0.23
41 Chonecambala crassicauda Ttp 0.22 0.20 0.20 0.21 0.21 0.21 0.19 0.21 0.20 0.19 0.29 0.26 0.27 0.27 0.27 0.28 0.26 0.28 0.27 0.27 0.26 0.26 0.26 0.27 0.27 0.23 0.23 0.22 0.21 0.22 0.230.25 0.24 0.240.26 0.27 0.250.21 0.25 0.22
42 Thyropygus allevatus Bb 0.210.23 0.23 0.24 0.23 0.22 0.23 0.22 0.23 0.200.27 0.25 0.240.24 0.25 0.240.26 0.26 0.250.25 0.26 0.260.25 0.26 0.250.23 0.23 0.240.22 0.24 0.250.23 0.25 0.240.28 0.26 0.280.23 0.20 0.140.23
Phylogeny and new genus of Pseudospirobolellidae Invertebrate Systematics 597
values of intraspecic K2P sequence divergence (0.0010.100),
such that the same 11 OTUs were consistently found (Table 5).
The K2P intraspecicCOI sequence divergences of
Coxobolellus species ranged from 0.00 to 0.05. Therefore, in
this study COI K2P sequence divergences above 0.05,
accompanied by distinct gonopodal characters, are regarded as
indicative of species-level differentiation. As such, congeneric
interspecicCOI K2P sequence divergences ranged from 0.06
to 0.15, with an average of 11% (Table 2), whereas intergeneric
COI K2P sequence divergences between the genera Coxobolellus,
gen. nov. and Pseudospirobolellus ranged from 0.20 to 0.23.
Taxonomic corollary
Coxobolellus, gen. nov. includes 10 well supported new species
which share three synapomorphies:
(1) posterior gonopod telopodite clearly divided into two parts:
coxal part (pcx) and telopodital part (pt);
Anurostreptus barthelemyae Tlb
Pseudospirobolellus avernus GPG
Thyropygus allevatus Bb
Chonecambala crassicauda Ttp
Paraspirobolus lucifugus
Pseudospirobolellus sp. KCS
Coxobolellus nodosus, sp. nov. SPW
Coxobolellus valvatus, sp. nov. TST
Coxobolellus valvatus, sp. nov. BRC
Coxobolellus albiceps, sp. nov. Stvd
Coxobolellus albiceps, sp. nov. TPB
Coxobolellus albiceps, sp. nov. Stpw
Coxobolellus fuscus, sp. nov. HKK
Coxobolellus tigris, sp. nov. TKP
Coxobolellus tigris, sp. nov. TYE
Coxobolellus serratus, sp. nov. KKL
Coxobolellus simplex, sp. nov. TNP
Coxobolellus tenebris, sp. nov. KWP
Coxobolellus tenebris, sp. nov. TPL
Coxobolellus compactogonus, sp. nov. KLC
Coxobolellus compactogonus, sp. nov. SKR
Coxobolellus albiceps, sp. nov. Stpl
Coxobolellus transversalis, sp. nov. Stpg
Coxobolellus valvatus, sp. nov. TCD
Rhinocricus parcus Rr49
Pelmatojulus tigrinus Pt2
Atopochetus weseneri Tos29
Litostrophus chamaeleon PPT
Trigoniulus corallinus Tco15
Apeuthes maculatus Amc
Leptogoniulus sorornus BTN
Litostrophus segregatus Ls19
Litostrophus saraburensis PKS
Benoitolus birgitae BBG
Atopochetus helix SPT
Atopochetus moulmeinensis TA K
Atopochetus dollfusii DOL
Atopochetus setiferus HPT
Atopochetus anaticeps SVL
Atopochetus truncatus SML
Atopochetus spinimargo Ton27
Atopochetus uncinatus KMR
Trachelomegalus sp. Tr54
Aulacobolus uncopygus Auc
Madabolus maximus Mm4
Parabolus dimorphus Pd34
Pelmatojulus togoensis Pto6
Narceus annularis
97/1.00
0.7
75/#
56/0.95
#/#
57/#
52/#
#/-
89/1.00 91/0.99
90/1.00
90/1.00
#/#
#/#
#/#
#/#
58/#
61/#
#/-
#/-
#/-
52/#
72/#
#/-
#/-
93/1.00
76/1.00
87/1.00
87/1.00
99/1.00
95/1.00
93/1.00
87/1.00
87/1.00
#/#
#/0.94
#/-
#/-
93/1.00
76/1.00
93/1.00
98/1.00
55/# 72/#
76/#
95/1.00
54/
0.93
Pseudospirobolellidae
Pachybolidae
1A
1B
1C
1D
Fig. 1. Phylogenetic relationships of Coxobolellus, gen. nov. species (Clade 1) based on maximum likelihood analysis (ML) and Bayesian Inference
(BI) of COI (660 bp). Numbers at nodes indicate branch support based on bootstrapping (ML) or posterior probabilities (BI). Scale bar = 0.7
substitutions per site. # indicates branches with <50% ML bootstrap support and <0.90 Bayesian posterior probability. , branch not shown in the BI
tree. Clade memberships and designations are shown as vertical bars.
Table 4. Number of clusters and entities detected within Coxobolellus, gen. nov. by the General Mixed Yule
Coalescent (GMYC) method applied to the COI dataset
Analysis Clusters
(CI)
Entities
(CI)
Likelihood
null
Likelihood
GMYC
Likelihood
ratio
Threshold
Single 4 (15) 11 (117) 72.3704 73.83690 2.933093 0.01678277
Multiple 4 (15) 7 (111) 72.3704 74.28268 3.824573 0.0669506
0.02501948
598 Invertebrate Systematics P. Pimvichai et al.
(2) coxae of the 3rd (and 4th) leg with extremely large,
protruding process;
(3) opening pore (oeg) at mesal margin at the end of coxal part
(pcx).
These three characters are autapomorphic for Coxobolellus,
gen. nov. In the next section we provide formal descriptions of
these new taxa.
Taxonomy
Class DIPLOPODA de Blainville in Gervais, 1844
Order SPIROBOLIDA Bollman, 1893
Suborder SPIROBOLIDEA Bollman, 1893
Family Pseudospirobolellidae Brölemann, 1913
A family of the order Spirobolida characterised, inter alia, by the
strongly to almost completely reduced anterior gonopod
sternum (Pitz and Sierwald 2010).
Fifteen species are included:
Genus Pseudospirobolellus Carl, 1912: P. avernus (Butler,
1876); P. sigmoides Attems, 1953.
Genus Benoitolus Mauriès, 1980 (=Solaenobolellus Hoffman,
1981): B. avicollis Mauriès, 1980; B. birgitae (Hoffman,
1981); B. siamensis (Attems, 1936) (=Cyclothyrophorus
siamensis Attems, 1936) (see Jeekel 2001, p. 47).
Genus Coxobolellus, gen. nov.: C. albiceps, sp. nov.;
C. compactogonus, sp. nov.; C. fuscus, sp. nov.;
C. nodosus, sp. nov.; C. serratus, sp. nov.; C. simplex, sp.
nov.; C. tenebris, sp. nov.; C. tigris, sp. nov.; C. transversalis,
sp. nov.; C. valvatus, sp. nov.
Pitz and Sierwald (2010) included two undescribed species
of Pseudospirobolellidae from China in their analysis, which
suggests that there remains taxonomic diversity to be discovered
in this family outside Thailand.
Genus Coxobolellus, gen. nov.
(Fig. 2)
Type species:Coxobolellus tenebris, sp. nov.
Diagnosis
Differing from the other genera of Pseudospirobollelidae
by having the coxae of the 3rd male leg-pair with extremely
large, protruding processes (in C. albiceps, sp. nov. and
C. transversalis, sp. nov., also the coxae of the 4th male leg-
pair with extremely large, protruding processes). The 4th5th
leg-pairs with large, triangular coxae (Fig. 2AC). Telson
smooth; preanal ring with a short process protruding as far as
or slightly beyond anal valves (Fig. 2D). Anal valves smooth,
rounded. Subanal scale broadly triangular (Fig. 2E). Posterior
gonopod telopodite divided into a coxal part (pcx) and a
telopodital part (pt); with opening of efferent groove (oeg) at
mesal margin at the end of coxal part (pcx).
General description
Adult males with 4757 podous rings, length ~48 cm, diameter
~3.26.0 mm. Adult females with 4757 podous rings, length
~59 cm, diameter ~4.16.4 mm. No apodous rings in front of
telson, except where noted.
Head capsule smooth. Occipital furrow extending down
between, but not beyond eyes; clypeal furrow reaching level
of antennal sockets. Area below antennal sockets and eyes
impressed, forming part of antennal furrow. Incisura lateralis
open. 2+2 labral teeth, a row of labral setae, 2+2, 3+3 or 4+4
supralabral setae. Diameter of eyes approximately half of
interocular space; 69 vertical rows of ommatidia, 4 or 5
horizontal rows, 2432 ommatidia per eye. Antennae short,
not reaching beyond collum when stretched back,
accommodated in a shallow furrow composed of a horizontal
segment in the head capsule and a vertical segment in the
mandibular cardo and stipes. Antennomere lengths 2 >1>6
>3>4=5>7; antennomere 1 glabrous, 2 and 3 with some
ventral setae, 4, 5 and 6 densely setose; 4 apical sensilla.
Gnathochilarium: each stipes with 2 or 3 apical setae; each
lamella lingualis with 2 setae, one behind the other. Basal part
of mentum transversely wrinkled; basal part of stipes
longitudinally wrinkled.
Collum smooth, with a marginal furrow along lateral part
of anterior margin; lateral lobes narrowly rounded, extending as
far ventrad as the ventral margin of body ring 2.
Body rings 25 ventrally concave, hence with distinct
ventrolateral corners. Body rings very smooth, sides parallel
in dorsal view. Tergo-pleural suture visible on pro- and
mesozona. Prozona smooth; mesozona ventrally with ne
oblique striae, dorsally punctate; metazona ventrally with
ne longitudinal striae, otherwise smooth. Pleural parts of
rings with ne oblique striae. Sterna transversely striate.
Ozopores from ring 6, situated in mesozona, ~1 pore
diameter in front of metazona. Sutures between pro-, meso-
Table 5. Number of putative species delimited by the ABGD method
applied to the COI dataset of Coxobolellus
Abbreviations after species names refer to the isolate of each sequence as
shown in Table 1. +, Molecular Operational Taxonomic Unit supported as a
putative species
Group ABGD
Initial
1. C. valvatus, sp. nov. BRC, TCD and TST +
2. C. fuscus, sp. nov. HKK +
3. C. serratus, sp. nov. KKL +
4. C. compactogonus, sp. nov. KLC +
5. C. tenebris, sp. nov. KWP and TPL +
6. C. compactogonus, sp. nov. SKR +
7. C. nodosus, sp. nov. SPW +
8. C. tigris, sp. nov. TKP and TYE +
9. C. simplex, sp. nov. TNP +
10. C. albiceps, sp. nov. TPB, Stpl, Stpw and Stvd +
11. C. transversalis, sp. nov. Stpg +
Phylogeny and new genus of Pseudospirobolellidae Invertebrate Systematics 599
(A)(B)
(D)(E)
(F)(G)
(C)
1 mm
1 mm
1 mm
1 mm
1 mm
1 mm
1 mm
pad
pad
coxa process
Fig. 2. External morphology of Coxobolellus, gen nov. species. A,C. tenebris, sp. nov., anterior end, ventral
view, arrow indicates the 3rd coxa process (paratype, specimen from Wat Tham Phrom Lok Khao Yai, CUMZ-
D00120-1). B,C. fuscus, sp. nov., anterior end, ventral view, arrow indicates the 3rd coxa process (paratype,
specimen from Wat Tha Khanun, CUMZ-D00137). C,C. transversalis, sp. nov., anterior end, ventral view,
arrow indicates the 3rd and 4th coxae processes (holotype, CUMZ-D00125-1). D,C. tenebris, sp. nov., posterior
end, lateral view, arrow indicates preanal process (paratype, specimen from Weluwan Khiriwong, CUMZ-
D00138). E,C. tenebris, sp. nov., posterior end, ventral view, arrow indicates subanal scale (paratype, specimen
from Weluwan Khiriwong, CUMZ-D00138). F,G,C. tenebris, sp. nov., male legs, lateral view, arrows indicate
coxa process and ventral soft pad on the 3rd leg (F) and the ventral soft pad on the 10th leg (G) (paratype,
specimen from Wat Khao Wong Phrohm-majan, CUMZ-D00119).
600 Invertebrate Systematics P. Pimvichai et al.
and metazona distinct; horizontal suture distinct on pro-, meso-
and metazona.
Telson
Smooth, preanal ring with slightly concave dorsal prole,
with a short process protruding as far as, or slightly beyond, anal
valves. Anal valves smooth, rounded. Subanal scale broadly
triangular.
Legs
Length of midbody legs 5181% of body width in males,
4870% of body width in females. Prefemur basally
constricted and longer than other podomeres. First and
second legs with 1 or 2 prefemoral, 1 femoral, 1
postfemoral, and 2 tibial setae, and 3 ventral and 1 dorsal
apical setae on tarsi, numbers of setae reaching constancy from
pair 3: each leg with 1 tibial seta; tarsi in males with 2 or 3
ventral apical and one dorsal apical seta; females with 1 coxal,
1 prefemoral, 1 femoral, 1 postfemoral, 1 tibial seta, tarsi with
13 ventral and one dorsal apical seta, the apical ventral seta
larger than the more basal one.
Male sexual characters
Prefemur of all legs from third to last pair with large ventral
soft pad (Fig. 2F,G) occupying entire ventral surface. Body
ring 7 entirely fused ventrally, no trace of a suture. Anterior
gonopods without a sternum, tip of anterior gonopods visible
when the animal is stretched out (not when it is rolled up).
Posterior gonopods in situ completely hidden within anterior
ones. Posterior gonopod telopodite divided into a coxal part
(pcx) and a telopodital part (pt); with opening of efferent
groove (oeg) at mesal margin at the end of coxal part (pcx).
Female vulvae
Large, in situ projecting beyond lateral extensions of
coxosternum of 2nd legs. Operculum small, rounded
triangular, situated at laterobasal end of vulva. Shape of
valves variable.
Distribution
Hitherto known only from Thailand
Ecology
Specimens of Coxobolellus gen. nov. are mostly found under leaf
litter. Sometimes specimens are found inside rotten wood or
climbing on trees.
Etymology
The name emphasises the importance of coxal characters in the
diagnosis of the new genus.
Species descriptions
Coxobolellus albiceps, sp. nov.
(Fig. 3,13,14)
Material examined
Holotype. Male, Thailand, Nan Province, Muang District, Tham Pha Tub,
185101700N, 1004401000 E, 9.vi.2019, P. Pimvichai and T. Backeljau leg.
(CUMZ).
Paratypes.Thailand: 5 males, 6 females, same data as holotype.
(CUMZ). 1 male, 2 females, Chiang-Rai Province, Pan District, Wat
Tham Bampen Bun, 194203600N, 994501300 E, 10.i.2008, P. Pimvichai
leg. (CUMZ). 2 males, 2 females, Phrae Province, Rong Kwang District,
Tham Pha Nang Khoi, 182104100N, 1002100300 E, 9.vi.2019, P. Pimvichai
and T. Backeljau leg. (CUMZ). 4 males, 4 females, Phrae Province, Rong
Kwang District, Huai Rong Waterfall, 182603100N, 1002700100 E, 31.
viii.2014, P. Pimvichai leg. (CUMZ). 6 males, 5 females, 10.vi.2019,
P. Pimvichai and T. Backeljau leg. (CUMZ). 5 males, 5 females,
Phitsanulok Province, Noen Maprang, Tham Wang Daeng, 164003700N,
1004103800E, 25.ix.2018, P. Pimvichai and S. Saratan leg. (CUMZ).
Diagnosis
Differing from all other species in the genus by having the tip of
the anterior gonopod coxa obliquely truncated, and by having the
telopodital part (pt) of the posterior gonopod short compared
with the coxal part (pcx).
Description
Adult males with 4853 podous rings. Length ~36 cm, diameter
~3.84.7 mm (3.23.4 mm in small individuals). Adult females
with 4751 podous rings. Length ~36 cm, diameter ~4.34.8
mm (4.1 mm in small individuals).
Colour of living animal dark green. Specimens from Tham
Wang Daeng and some specimens from Tham Pha Tub brown,
metazona dark brown. Antennae, collum, legs and preanal
process beige in all specimens. (Fig. 13AC).
Anterior gonopods (Fig. 3A,B,D,E) with high coxae, apically
obliquely truncated, posterior surface with fairly high ridge
laterally for accommodation of telopodite. Telopodite
overreaching coxa, apically narrowly rounded with pigmented
brown node, with constricted lateral margin at
2
/
3
of its height,
forming a triangular process.
Posterior gonopods (Fig. 3C,FI) very simple, with long,
smooth coxal part (pcx); telopodital part (pt) curving mesad,
attened, concave forming a canopy, with strong transverse
ridge near base, with a short spine protruding from surface
near tip.
Female vulvae (Fig. 3J): valves prominent, of equal size.
DNA barcode
The GenBank accession number of the barcode of the holotype
is MT328994 (voucher code CUMZ-D00121).
Distribution
Nan, Phrae, Chiang-Rai and Phitsanulok provinces, Thailand
(Fig. 14).
Note
This species is polymorphic for both colour pattern and size.
Specimens are dark green, green or brown, but the antennae,
collum, legs and preanal process are always beige. Brown
specimens are smaller both in length and diameter than dark
green or green specimens. The three morphs occur in Tham Pha
Phylogeny and new genus of Pseudospirobolellidae Invertebrate Systematics 601
Tub (Fig. 13AC) and Huai Rong Waterfall, but in Tham Wang
Daeng only the brown morph (length 34 cm, diameter
3.23.4 mm) was found.
Etymology
The specic epithet is a Latin noun in apposition, meaning
white/pale headand referring to the contrastingly pale head
in living specimens (Fig. 13AC).
Coxobolellus compactogonus, sp. nov.
(Fig. 4,14)
Material examined
Holotype. Male, Thailand, Nakhon Ratchasima Province, Wang Nam
Khiao District, Sakaerat Environmental Research Station, 143003600N,
1015505100E, 24.iv.2009, P. Pimvichai leg. (CUMZ).
(A)(B)
(D)
(G)(H)(I)(J)
(E)(F)
(C)
Fig. 3. Coxobolellus albiceps, sp. nov., paratype, gonopods (specimen from Wat Tham Bampen Bun, CUMZ-
D00123-1). A,D, anterior gonopod, anterior view. B,E, anterior gonopod, posterior view. C,F, right posterior
gonopod. G, SEM, left posterior gonopod, posterior-mesal view. H, SEM, tip of posterior gonopod, mesal view.
I, SEM, tip of posterior gonopod, dorsal view. J, SEM, left female vulva, posterior mesal view.
602 Invertebrate Systematics P. Pimvichai et al.
Paratypes.Thailand: 1 male, same data as holotype (CUMZ). 2 males,
2 females, Nakhon Ratchasima Province, Pak Chong District, Khao Look
Chang, 143103400N, 1012103000 E, 24.vi.2008, P. Pimvichai leg. (CUMZ).
Diagnosis
Differing from all other species in the genus by having the
anterior gonopod telopodite (at) with a rounded tip projecting
slightly over coxa (cx), and by having the telopodital part (pt)
of the posterior gonopod short and compact, with three
processes surrounding a deep concavity.
Description
Adult males with 4755 podous rings, 1 specimen from
Sakaerat and 1 specimen from Khao Look Chang with
2 apodous rings. Length ~56 cm, diameter ~4.14.6 mm.
(D)(E)(F)
(G)(H)(I)(J)
Fig. 4. Coxobolellus compactogonus, sp. nov., paratype, gonopods (CUMZ-D00135-1). A,D, anterior gonopod,
anterior view. B,E, anterior gonopod, posterior view. C,F, right posterior gonopod. G, SEM, left posterior gonopod,
posterior-mesal view. H, SEM, tip of posterior gonopod, posterior-mesal view. I, SEM, tip of posterior gonopod,
posterior-lateral view. J, SEM, left female vulva, posterior mesal view.
Phylogeny and new genus of Pseudospirobolellidae Invertebrate Systematics 603
Adult females with 4750 podous rings. Length ~7 cm,
diameter ~4.85.2 mm.
Colour after 10 years in ethanol: head, antenna, collum, legs
and telson light brown; body rings dark brown.
Anterior gonopods (Fig. 4A,B,D,E) with high, triangular
coxae, gradually narrowing towards tip; at base of posterior
surface with relatively high ridge laterally for accommodation
of telopodite. Telopodite projecting slightly over anterior
gonopod coxa (cx), simple, gradually narrow towards tip,
apically rounded, curving mesad.
Posterior gonopods (Fig. 4C,FI) simple, rounded, with
very long, smooth coxal part (pcx); telopodital part (pt) short,
with three processes, basal part and apical part ending in a
rounded lobe, the lateral process with serrate margin. The three
processes forming a deep concavity.
Female vulvae (Fig. 4J): valves prominent, of equal size.
DNA barcode
The GenBank accession number of the barcode of the holotype
is MT328998 (voucher code CUMZ-D00134).
Distribution
Known only from the type locality in Nakhon Ratchasima
Province, Thailand (Fig. 14).
Etymology
The specic epithet is a noun in apposition referring to the
particularly compact posterior gonopod.
Coxobolellus fuscus, sp. nov.
(Fig. 5,13,14)
Material examined
Holotype. Male, Thailand, Kanchanaburi Province, Sangkhla Buri District,
Kroeng Krawia waterfall, 145805100N, 983705700 E, 8.vi.2010, P. Pimvichai
leg. (CUMZ).
Paratypes.Thailand: 1 male, 2 females, same data as holotype
(CUMZ). 3 males, 1 female, Kanchanaburi Province, Thong Pha Phum
District, Wat Tha Khanun, 144403000N, 983801400 E, 24.vii.2016,
P. Pimvichai and T. Backeljau leg. (CUMZ).
Diagnosis
Differing from all other species in the genus by having the tip of
the anterior gonopod coxa ending in an abruptly narrowed, long
process, and by having the tip of the telopodital part (pt) of the
posterior gonopod ending in a coarsely serrate lamella with a
sharp point.
Description
Adult males with 5057 podous rings, 1 specimen from Wat Tha
Khanun with 3 apodous rings. Length ~67 cm, diameter
~3.84.3 mm. Adult females with 5157 podous rings. Length
~7 cm, diameter ~4.54.6 mm.
Colour of living animal: antennae, legs and metazona reddish
brown, collum and prozona dark brown, preanal process
yellowish brown (Fig. 13D).
Anterior gonopods (Fig. 5A,B,D,E) with very high coxae,
gradually narrowing towards tip, apically slender, narrow,
mesal margin straight; at base of posterior surface with fairly
high ridge laterally for accommodation of telopodite. Telopodite
far overreaching coxa, apically abruptly narrow, curving mesad,
with constricted lateral margin at ~
2
/
3
of its height, forming a
triangular process.
Posterior gonopods (Fig. 5C,FI) simple, rounded, with
very long, smooth coxal part (pcx); telopodital part (pt) short,
curving mesad, apically ending in a coarsely serrate lamella
with a sharp point.
Female vulvae (Fig. 5J): valves prominent, of equal size.
DNA barcode
The GenBank accession number of the barcode of the holotype
is MT328999 (voucher code CUMZ-D00133).
Distribution
Kanchanaburi Province, Thailand (Fig. 14).
Etymology
The specic name is a Latin adjective, meaning brownand
referring to the general body colour of living specimens (Fig. 13D).
Coxobolellus nodosus, sp. nov.
(Fig. 6,14)
Material examined
Holotype. Male, Thailand, Tak Province, Mae Sot District, Chao Por
Phawo Shrine, 164001700N, 984100800 E, 17.vii.2008, P. Pimvichai leg.
(CUMZ).
Paratypes.Thailand: 5 males, 3 females, 2 juveniles, same data as
holotype (CUMZ).
Diagnosis
Differing from all other species in the genus by having the
telopodital part (pt) of the posterior gonopod ending in a long,
sharp spine, and by having a attened lamella protruding from the
mesal surface near the tip. Further differing from all other
species, except C. valvatus, sp. nov., by having the tip of the
anterior gonopod coxa concave.
Description
Adult males with 5052 podous rings. Length ~78 cm, diameter
~5.35.5 mm. Adult females with 4951 podous rings. Length
~89 cm, diameter ~5.66.0 mm.
Colour after 11 years in ethanol: head, antenna, legs and telson
reddish brown; body rings dark brown.
Anterior gonopods (Fig. 6A,B,D,E) with high coxae, apically
narrowly concave, mesal margins straight, diverging, delimiting
a V-shaped space between both coxae, posterior surface with
fairly high ridge laterally for accommodation of telopodite.
Telopodite slightly overreaching coxa, apically narrow,
forming a triangular process with a pigmented brown node,
with constricted lateral margin at ~
2
/
3
of its height, forming a
triangular process.
Posterior gonopods (Fig. 6C,FH) simple, with long,
smooth coxal part (pcx); telopodital part (pt) as long as
coxal part, curving mesad, forming a deep concavity, mesal
margin forming a broadly expanded sheet, with serrate mesal
604 Invertebrate Systematics P. Pimvichai et al.
margin; apically ending in a long, sharp spine, with a attened
lamella at base of apical sharp spine, protruding from mesal
surface.
Female vulvae (Fig. 6I): valves prominent, of equal size;
free margins meeting in sigmoid suture.
DNA barcode
The GenBank accession number of the barcode of the holotype
is MT329000 (voucher code CUMZ-D00126).
Distribution
Known only from the type locality in Tak Province, Thailand
(Fig. 14).
Etymology
The specic epithet is a Latin adjective referring to the
pigmented node at the tip of the anterior gonopod
telopodite of this species.
(A)
(D)(E)(F)
(G)(H)(I)(J)
(B)(C)
Fig. 5. Coxobolellus fuscus, sp. nov., holotype, gonopods (CUMZ-D00133-1). A,D, anterior gonopod,
anterior view. B,E, anterior gonopod, posterior view. C, right posterior gonopod. F, left posterior gonopod
drawing. G, SEM, left posterior gonopod, posterior-mesal view. H, SEM, tip of posterior gonopod, posterior-
mesal view. I, SEM, tip of posterior gonopod, posterior-mesal view. J, SEM, left female vulva, posterior mesal
view (specimen from Wat Tha Khanun, CUMZ-D00139).
Phylogeny and new genus of Pseudospirobolellidae Invertebrate Systematics 605
Coxobolellus serratus, sp. nov.
(Fig. 7,14)
Material examined
Holotype. Male, Thailand, Prachuap Khiri Khan Province, Pran Buri District,
Khao Kalok, 122000400 N, 995905000E, 13.x.2008, C. Sutcharit leg. (CUMZ).
Paratypes.Thailand: 1 male, same data as holotype (CUMZ).
Diagnosis
Differing from all other species in the genus by having the
anterior gonopod telopodite (at) far overreaching coxa (cx)
and curving laterad, and by having a lateral serrate margin on
the telopodital part (pt) of the posterior gonopod.
Description
Adult males 52 podous rings, 1 specimen with 2 apodous rings.
Length ~67 cm, diameter ~4.44.9 mm.
Colour after 11 years in ethanol: brown; antenna, legs and
metazona dark brown.
Anterior gonopods (Fig. 7A,B,D,E) with high, triangular
coxae, lateral margin concave at 1/2 of its height, mesal margin
straight; at base of posterior surface with fairly high ridge
(A)
(D)(E)(F)
(G)(H)(I)
(B)(C)
Fig. 6. Coxobolellus nodosus, sp. nov., holotype, gonopods (CUMZ-D00126-1). A,D, anterior gonopod,
anterior view. B,E, anterior gonopod, posterior view. C,F, right posterior gonopod. G, SEM, left posterior
gonopod, posterior-mesal view. H, SEM, left posterior gonopod, posterior-lateral view. I, SEM, left female
vulva, posterior mesal view.
606 Invertebrate Systematics P. Pimvichai et al.
laterally for accommodation of telopodite. Telopodite far
overreaching coxa, apically abruptly narrow, curving laterad.
Posterior gonopods (Fig. 7C,FH) very simple, with long,
smooth coxal part (pcx); telopodital part (pt) as long as pcx,
curving mesad, laterally with serrate margin.
DNA barcode
The GenBank accession number of the barcode of the holotype
is MT329001 (voucher code CUMZ-D00132).
Distribution
Known only from the type locality in Prachuap Khiri Khan
Province, Thailand (Fig. 14).
Etymology
The specic epithet is a Latin adjective referring to the
prominent serration of the telopodital part of the posterior
gonopod.
Coxobolellus simplex, sp. nov.
(Fig. 8,14)
Material examined
Holotype. Male, Thailand, Lampang Province, Mae Prik District, Tham
Pha Pha Ngam, 172804900N, 99100500 E, 7.viii.2008, C. Sutcharit leg.
(CUMZ).
(A)
(D)(E)(F)
(G)(H)
(B)(C)
Fig. 7. Coxobolellus serratus, sp. nov., holotype, gonopods (CUMZ-D00132). A,D, anterior gonopod, anterior
view. B,E, anterior gonopod, posterior view. C,F, right posterior gonopod. G, SEM, left posterior gonopod,
posterior-mesal view. H, SEM, tip of posterior gonopod, posterior-lateral view.
Phylogeny and new genus of Pseudospirobolellidae Invertebrate Systematics 607
Diagnosis
Differing from all other species in the genus by having the
telopodital part (pt) of the posterior gonopod short, with a
attened lamella at base, apically ending in a rounded lobe.
Further differing from the other species, except C. tenebris, sp.
nov., by having the tip of the telopodital part (pt) curving mesad,
rounded, smooth.
Description
Adult male with 52 podous rings. Length ~6 cm, diameter
~4.6 mm.
Colour after 11 years in ethanol: collum and telson orange
brown; prozona grey; body ring and legs light brown.
Anteriorgonopods (Fig. 8A,B,D,E)with high, triangularcoxae,
apically abruptly narrower, pointing mesad, lateral margin concave
at
1
/
2
of its height, mesal margin straight; at base of posterior surface
with fairly high ridge laterally for accommodation of telopodite.
Telopodite far overreaching coxa, apically rounded, with
constricted lateral margin near tip, forming a triangular process.
Posterior gonopods (Fig. 8C,F) simple, rounded, with very
long, smooth coxal part (pcx); telopodital part (pt) short, with a
attened lamella at base, apically ending in a rounded lobe.
DNA barcode
The GenBank accession number of the barcode of the holotype
is MT329002 (voucher code CUMZ-D00136).
Distribution
Known only from the type locality in Lampang Province,
Thailand (Fig. 14).
Etymology
The specic epithet is a Latin adjective referring to the
particularly simple posterior gonopod.
Coxobolellus tenebris, sp. nov.
(Fig. 9,13,14)
Material examined
Holotype. Male, Thailand, Uthai Thani Province, Ban Rai District, Wat
Khao Wong Phrohm-majan, 151800900N, 994501600 E, 8.vii.2009,
P. Pimvichai leg. (CUMZ).
Paratypes.Thailand: 3 males, 2 females, same data as holotype
(CUMZ). 1 male, Kanchanaburi Province, Sai Yok District, Wat Tham
Phrom Lok Khao Yai, 141201600 N, 990705800E, 9.vii.2009, P. Pimvichai
leg. (CUMZ). 2 males, 1 female, Suphan Buri Province, Dan Chang
District, Wat Weluwan Khiriwong, 144600500N, 992603300 E, 8.vii.2009,
P. Pimvichai leg. (CUMZ).
Diagnosis
Differing from all other species in the genus by having the tip
of the anterior gonopod coxa very strongly narrowed and
pointed, and by having a slender triangular lamella curling
(A)
(D)(E)(F)
(B)(C)
Fig. 8. Coxobolellus simplex, sp. nov., holotype, gonopods (CUMZ-D00136). A,D, anterior gonopod, anterior
view. B,E, anterior gonopod, posterior view. C,F, left posterior gonopod.
608 Invertebrate Systematics P. Pimvichai et al.
around the basal part of the telopodital part (pt) of the posterior
gonopod. Further differing from all other species except for
C. simplex, sp. nov. from Tham Pha Pha Ngam in having the tip
of the telopodital part (pt) curving mesad, rounded, and smooth.
Description
Adult males with 5355 podous rings, 1 specimen with 2
apodous rings. Length ~78 cm, diameter ~5.06.0 mm. Adult
females with 4853 podous rings. Length ~89 cm, diameter
~5.66.4 mm.
Colour of living animal: dark greenish grey. Edge of
metazona and preanal process light brown (Fig. 13E).
Anterior gonopods (Fig. 3A,B,D,E) with high, triangular
coxae, apically strongly narrowed, pointed. Telopodite far
overreaching coxa, apically narrowly rounded, with a tiny
triangular laterad denticle near tip.
(A)
(D)(E)(F)
(G)(H)(I)(J)
(B)(C)
Fig. 9. Coxobolellus tenebris, sp. nov., holotype, gonopods (CUMZ-D00120-1). A,D, anterior gonopod,
anterior view. B,E, anterior gonopod, posterior view. C,F, left posterior gonopod. SEM, paratype, specimen
from Wat Khao Wong Phrohm-majan, CUMZ-D00119. G, SEM, left posterior gonopod, posterior-mesal view.
H, SEM, tip of posterior gonopod, posterior-mesal view. I, SEM, left posterior gonopod, posterior-lateral view.
J, SEM, left female vulva, posterior-mesal view.
Phylogeny and new genus of Pseudospirobolellidae Invertebrate Systematics 609
Posterior gonopods (Fig. 3C,FI) very simple, with very
long, smooth coxal part (pcx); telopodital part (pt) curving
mesad, rounded, smooth, with a slender triangular lamella
curling around basal part.
Female vulvae (Fig. 3J): valves prominent, of equal size.
DNA barcode
The GenBank accession number of the barcode of the holotype
is MT329004 (voucher code CUMZ-D00120).
Distribution
Uthai Thani, Kanchanaburi and Suphan Buri provinces, Thailand
(Fig. 14).
Etymology
The specic epithet is a Latin adjective, meaning darkand
referring to the general body colour of living specimens.
Coxobolellus tigris, sp. nov.
(Fig. 10,13,14)
Material examined
Holotype. Male, Thailand, Chumphon Province, Pathio District, Tham Yai
I, 104404500N, 992304500 E, 22.v.2010, P. Pimvichai leg. (CUMZ).
Paratypes.Thailand: 2 males, 4 females, same data as holotype
(CUMZ). 2 males, Chumphon Province, Pathio District, Wat Tham Khao
Plu, 104304800N, 991901500 E, 22.v.2010, P. Pimvichai leg. (CUMZ).
Diagnosis
Differing from all other species in the genus by having the
anterior gonopod telopodite (at) simple, far overreaching coxa
(cx), apically narrow, erect, and by having the telopodital part
(pt) of the posterior gonopod ending in a rounded lamella, with
irregularly serrated margin (Fig. 10H).
Description
Adult males with 53 or 54 podous rings. Length ~56 cm,
diameter ~4.24.5 mm. Adult females with 5153 podous
rings. Length ~56 cm, diameter ~4.54.9 mm.
Colour of living animal yellowish brown. Antennae and
legs reddish brown, dorsal prozona creamy yellow, lateral side
of prozona and metazona dark brown, mid dorsal metazona
with a dark brown small triangular spot, collum and preanal
process light brown (Fig. 13F).
Anterior gonopods (Fig. 10A,B,D,E) with high, triangular
coxae, lateral margin concave at
1
/
2
of its height, apically
abruptly narrowed, pointed; at base of posterior surface with
fairly high ridge laterally for accommodation of telopodite.
Telopodite simple, far overreaching coxa, apically narrow,
erected.
Posterior gonopods (Fig. 10C,FH) very simple, with
short, smooth coxal part (pcx); telopodital part (pt) very
long, curving mesad, forming a deep concavity, apically
rounded, with serrate margin.
Female vulvae (Fig. 10I) simple, valves prominent, the right
valve larger than the left valve.
DNA barcode
The GenBank accession number of the barcode of the holotype
is MT329004 (voucher code CUMZ-D00131).
Distribution
Chumphon Province, Thailand (Fig. 14).
Etymology
The specic epithet is a Latin noun in apposition, meaning
tigerand referring to the colour pattern on living specimens
(Fig. 13F).
Coxobolellus transversalis, sp. nov.
(Fig. 11,14)
Material examined
Holotype. Male, Thailand, Nan Province, Muang District, Tham Pha Tub,
185101700N, 1004401000 E, 9.vi.2019, P. Pimvichai and T. Backeljau leg.
(CUMZ).
Paratypes. Thailand: 5 females, same data as holotype (CUMZ).
Diagnosis
Differing from all other species in the genus by having the tip
the of anterior gonopod coxae transversely truncated, and by
having the telopodital part (pt) of the posterior gonopod fairly
long compared with the coxal part (pcx).
Description
Adult male with 53 podous rings. Length ~6 cm, diameter
~4.4 mm. Adult females with 5052 podous rings. Length
~78 cm, diameter ~4.04.9 mm.
Colour of living animal dark green.
Anterior gonopods (Fig. 11A,B,D,E) with high coxae,
apically truncated, mesal margins straight, diverging,
delimiting a V-shaped space between both coxae, posterior
surface with fairly high ridge laterally for accommodation of
telopodite. Telopodite overreaching coxa, apically narrow,
forming triangular process with pigmented brown node,
with extremely constricted lateral margin at 2/3 of its
height, forming a big triangular process.
Posterior gonopods (Fig. 11C,F) very simple, with long,
smooth coxal part (pcx); telopodital part (pt) fairly long, curving
mesad, attened, concave forming a canopy, with strong
transverse ridge near base, with a short spine protruding from
surface near tip.
Female vulvae (Fig. 11G,H): valves prominent, of equal
size.
DNA barcode
The GenBank accession number of the barcode of the holotype
is MT329007 (voucher code CUMZ-D00125).
Distribution
Known only from the type locality in Nan Province, Thailand
(Fig. 14).
610 Invertebrate Systematics P. Pimvichai et al.
Etymology
The specic epithet is a Latin adjective referring to the
transverse truncation of the anterior gonopod coxa.
Coxobolellus valvatus, sp. nov.
(Fig. 12,14)
Material examined
Holotype. Male, Thailand, Chiang-Mai Province, Chiang Dao District,
Wat Tham Chiang Dao, 192303700N, 985504200 E, 8.i.2008, C. Sutcharit leg.
(CUMZ).
Paratypes.Thailand: 1 male, 1 female, Chiang-Mai Province, Chom
Thong District, Tham Borichinda, 183000300N, 984002000 E, 7.x.2007,
P. Pimvichai, S. Panha and H. Enghoff leg. (CUMZ). 1 male, 1 female,
(A)
(D)(E)(F)
(G)(H)(I)
(B)(C)
Fig. 10. Coxobolellus tigris, sp. nov., holotype, gonopods (CUMZ-D00131-1). A,D, anterior gonopod, anterior
view. B,E, anterior gonopod, posterior view. C,F, right posterior gonopod. G, SEM, left posterior gonopod,
posterior-mesal view. H, SEM, left posterior gonopod, posterior-lateral view. I, SEM, left female vulva, posterior
mesal view.
Phylogeny and new genus of Pseudospirobolellidae Invertebrate Systematics 611
Mae Hong Son Province, Muang District, Tham Sam Ta, 193102300 N,
980502200E, 19.vii.2008, S. Panha leg. (CUMZ).
Diagnosis
Differing from all other species in the genus by having the
outer process of the anterior gonopod coxa broadly rounded,
the inner process protruding higher than the outer one, and by
having the telopodital part (pt) of the posterior gonopod ending
in a rounded margin with a sharp spine protruding from the
mesal surface near the tip. Further differing from all other
species, except C. nodosus, sp. nov., by having the tip of the
anterior gonopod coxa concave.
Description
Adult males with 47 or 48 podous rings. Length ~68 cm,
diameter ~4.44.7 mm. Adult females with 47 or 48 podous
rings. Length ~7 cm, diameter ~5.05.2 mm.
Colour after 1112 years in ethanol: head, antenna and legs
light brown; body rings dark brown.
(A)
(D)(E)(F)
(G)(H)
(B)(C)
Fig. 11. Coxobolellus transversalis, sp. nov., holotype, gonopods (CUMZ-D00125). A,D, anterior gonopod,
anterior view. B,E, anterior gonopod, posterior view. C,F, right posterior gonopod. G, left female vulva, posterior
lateral view. H, left female vulva, posterior mesal view.
612 Invertebrate Systematics P. Pimvichai et al.
Anterior gonopods (Fig. 12A,B,D,E) with high coxae,
apically concave, outer process broadly rounded, inner
process small, triangular; posterior surface with fairly high
ridge laterally for accommodation of telopodite. Telopodite
overreaching coxa, apically narrowly rounded with pigmented
brown node, with constricted lateral margin at ~
2
/
3
of its height,
forming a triangular process.
Posterior gonopods (Fig. 12C,FI) very simple, with
long, smooth coxal part (pcx); telopodital part (pt) ending in a
rounded margin with a sharp spine protruding from mesal surface
near tip.
Female vulvae (Fig. 12J): valves prominent, of equal size;
with one fairly large triangular tooth, valves tting tightly
together.
(A)
(D)(E)(F)
(G)(H)(I)(J)
(B)(C)
Fig. 12. Coxobolellus valvatus, sp. nov., paratype, gonopods (specimen from Tham Sam Ta, CUMZ-D00129-
1). A,D, anterior gonopod, anterior view. B,E, anterior gonopod, posterior view. C,F, right posterior gonopod.
SEM, paratype, specimen from Tham Borichinda, CUMZ-D00128-1. G, SEM, left posterior gonopod, posterior-
mesal view. H, SEM, tip of posterior gonopod, posterior-mesal view. I, SEM, distalmost part of posterior
gonopod, posterior-mesal view. J, SEM, left female vulva, posterior mesal view.
Phylogeny and new genus of Pseudospirobolellidae Invertebrate Systematics 613
DNA barcode
The GenBank accession number of the barcode of the holotype
is MT329009 (voucher code CUMZ-D00127).
Distribution
Known from Chiang-Mai and Mae Hong Son provinces,
Thailand (Fig. 14).
Etymology
The specic epithet is a Latin adjective referring to the
prominently toothed vulva valve.
Key to species of the genus Coxobolellus, gen. nov. (based on
adult males)
1 Tip of anterior gonopod coxa truncated..................................................2
Tip of anterior gonopod coxa concave or bilobed or forming a triangular
process................................................................................................3
2 Tip of anterior gonopod coxa transversely truncated (Fig. 11A,D);
telopodital part (pt) of posterior gonopod long compared to coxal
part (pcx) (Fig. 11C,F) .............................. C. transversalis, sp. nov.
Tip of anterior gonopod coxa obliquely truncated (Fig. 3A,D);
telopodital part (pt) of posterior gonopod short compared to coxal
part (pcx) (Fig. 3C,F) ........................................C. albiceps, sp. nov.
(A)
(D)
(E)(F)
(B)
(C)
Fig. 13. Live Coxobolellus, gen. nov. species from Thailand. A,C. albiceps, sp. nov. (from Tham Pha Tub), male
(paratype, CUMZ-D00121-1). B,C. albiceps, sp. nov. (from Tham Wang Daeng), male (paratype, CUMZ-D00124-
1). C,C. albiceps, sp. nov. (from Tham Pha Tub), male (paratype, CUMZ-D00121-2). D,C. fuscus, sp. nov. (from
Wat Tha Khanun), male (paratype, CUMZ-D00137-1). E,C. tenebris, sp. nov. (from Weluwan Khiriwong), male
(paratype, CUMZ-D00138-1). F,C. tigris, sp. nov. (from Wat Tham Khao Plu), male (paratype, CUMZ-D00130-1).
614 Invertebrate Systematics P. Pimvichai et al.
3 Tip of anterior gonopod coxa concave or bilobed..................................4
Tip of anterior gonopod coxa forming triangular process .....................5
4 Tip of anterior gonopod coxa bilobed, outer process broadly rounded,
inner process triangular, protruding higher than outer process
(Fig. 12A,D); telopodital part (pt) of posterior gonopod ending in a
rounded margin with a sharp spine protruding from mesal surface near
tip (Fig. 12C,FI) ..............................................C. valvatus, sp. nov.
Tip of anterior gonopod coxa concave, forming equal outer and inner
lobes (Fig. 6A,D); telopodital part of posterior gonopod (pt) ending
in a long, sharp spine, with a attened lamella protruding from
mesal surface near tip (Fig. 6C,FH) ........... C. nodosus,sp.nov.
5 Tip of anterior gonopod coxa ending in an abruptly narrowed, pointed,
triangular process...............................................................................6
Tip of anterior gonopod coxa ending in a simple triangular process ....8
6 Tip of anterior gonopod telopodite (at) long, narrow, curving
mesad (Fig. 5B,E); tip of telopodital part (pt) of posterior
gonopod ending in coarsely serrate lamella with a sharp point
(Fig. 5FI)..........................................................C. fuscus,sp.nov.
Tip of anterior gonopod telopodite (at) forming a triangular process ...7
7 Telopodital part (pt) of posterior gonopod with a sharp, curling lamella
at base (Fig. 9C,FH) .................. .................. C. tenebris,sp.nov.
Telopodital part (pt) of posterior gonopod without a sharp, curling
lamellaatbase(Fig.8C,F)............................. C. simplex,sp.nov.
8 Anterior gonopod telopodite (at) projecting slightly over anterior
gonopod coxa (cx), with rounded tip (Fig. 4B,E) .........................
............. ........................ ........................ C. compactogonus,sp.nov.
Anterior gonopod telopodite (at) far overreaching anterior gonopod coxa
(cx), with narrowed tip ......................................................................9
9 Anterior gonopod telopodite (at) directed distad (Fig. 10B,E); telopodital
part (pt) of posterior gonopod ending in a rounded, serrate margin
(Fig. 10FH) ........................................................... C. tigris, sp. nov.
Anterior gonopod telopodite (at) curving laterad (Fig. 7B,E);
telopodital part (pt) of posterior gonopod laterally with serrate
margin (Fig. 7C,FH)....................................C. serratus,sp.nov.
Discussion
The 10 new species described here form a well supported clade
in our mtDNA analysis, sister to a clade consisting of two
species of Pseudospirobolellus. Unfortunately, of the other
allegedly pseudospirobolellid genus, Benoitolus, we have only
aCOI sequence of B. birgitae, but no data for the type species,
B. avicollis. However, Coxobolellus, gen. nov. is well
characterised and supported by morphological characters,
notably the posterior gonopod telopodite divided into two
parts, coxal part (pcx) and telopodital part (pt); coxae of the
3rd (and 4th) with extremely large, protruding process; and
with the opening pore (oeg) at the mesal margin at the end of
the coxal part (pcx). In other pseudospirobolellids, the 3rd leg-
pair has no lobes, but lobes are found on the 5th leg-pair
instead (B. avicollis: H. Enghoff (pers. obs.) on specimens
from Singapore in NHMD; B. birgitae: Hoffman (1981)[as
Solaenobolellus birgitae]; B. siamensis:Attems(1936)[as
Cyclothyrophorus siamensis], P. avernus: H. Enghoff (pers.
obs.) on specimens from Fiji in NHMD; P. sigmoides:
unknown). Moreover, the posterior gonopods of the 10 new
Fig. 14. Distribution of the species of Coxobolellus, gen. nov. in Thailand. Droplets vary in size only to improve
readability.
Phylogeny and new genus of Pseudospirobolellidae Invertebrate Systematics 615
species are clearly different from those of Benoitolus and
Pseudospirobolellus. The posterior gonopod is extremely
slender in Pseudospirobolellus or slender in Benoitolus and
without any process, whereas in the 10 new Coxobolellus,gen.
nov. species the posterior gonopod is broad and clearly divided
into two parts (pcx and pt) and obviously with an opening
of the efferent groove (oeg). Furthermore, the COI
sequence divergence between Coxobolellus,gen.nov.and
Pseudospirobolellus or Benoitolus is 2023%. From this
study, the COI sequence divergence between Coxobolellus,
gen. nov and (1) pachybolid genera is 1625% (mean: 21%),
(2) trigoniulid genera is 1521% (mean: 18%), (3) Rhinocricus
parcus is 2426% (mean: 25%), (4) Narceus annularis is
2123% (mean: 22%), and (5) Paraspirobolus lucifugus is
2325% (mean: 24%). The COI sequence divergence among
pachybolid genera is 1323% (mean: 19%). The COI sequence
divergence between B. birgitae and pachybolid genera is
1124% (mean: 21%). The lowest COI sequence divergence
is with Litostrophus (1118%; mean: 15%). In the order
Spirostreptida, family Spirostreptidae the intergeneric COI
sequence divergences are 6.8326.81% (mean: 18.43%)
(Mwabvu et al.2015). Hence, taken together, the amount of
COI sequence divergence between Coxobolellus,gen.nov.and
other pseudospirobolellid, pachybolid and spirostreptid genera
supports its recognition as a separate genus.
The intraspecicCOI sequence divergence of
Coxobolellus, gen. nov. species ranges from 0 to 5% (mean:
2%), and the interspecic divergence ranges from 6 to 15%
(mean: 11%). These gures correspond well with the amounts
of intra- and interspecicCOI sequence divergence observed
in other millipede genera. In the widespread pill millipede,
Glomeris marginata (Villers, 1789) the maximum intraspecic
COI sequence divergence is 5% and the interspecic
divergences between G. marginata and other Glomeris
species range from 12.9 to 15.9% (Reip and Wesener
2018). The intraspecicCOI sequence divergence of
Bavarian Diplopoda varies from 0 to 6.61% (mean: 0.82)
and the mean interspecic divergence is 14.17% (Spelda
et al.2011). High intraspecicCOI sequence divergences
are also found in the harpagophorid genus Thyropygus
Pocock, 1894 (0 to 9%), whereas interspecic divergences
ranged from 5 to 18% (mean: 14%) (Pimvichai et al.2014).
Our mtDNA analysis assigns Benoitolus birgitae to the
Pachybolidae, i.e. phylogenetically very distant from
Coxobolellus, gen. nov. and Pseudospirobolellus. Conversely,
while the prefemoral pads on legs of male Coxobolellus, gen.
nov. constitute a very unusual character, it is shared with
B. birgitae,B. avicollis and P. avernus, although in the latter
two species, the pads are much less prominent (Hoffman 1981;
P. Pimvichai, pers. obs.). Hence it remains to be decided whether
the morphological similarity of the prefemoral pads on the legs of
male Coxobolellus, gen. nov., Pseudospirobolellus and
Benoitolus represents a synapomorphy contradicting our
mtDNA data (which assign B. birgitae to Pachybolidae), or
involves a case of homoplasy that is consistent with our
mtDNA data. In a similar spirit, Enghoff et al.(2015) already
expressed doubts with respect to the monophyly of the
Pseudospirobolellidae. Hence further phylogenetic analyses
are needed to explore the monophyly and relationships of this
still poorly known family. Moreover, since this study is based on
only two mtDNA gene fragments and fairly few specimens,
future phylogenetic analyses would benet from including
nuclear gene markers and more specimens.
Nevertheless, this rst DNA study of the
Pseudospirobolellidae shows that there still is an
overwhelming amount of hidden taxonomic diversity to be
discovered in the south-east Asian diplopod fauna, even in
supposedly species-poor families such as the
Pseudospirobolellidae.
Conicts of interest
The authors declare that they have no conicts of interest.
Declaration of funding
This research was funded by the Thailand Science Research
and Innovation (TSRI) as a TRF Research Career
Development Grant (20192021; RSA6280051) (to
P. Pimvichai). Additional funding came from the Royal
Belgian Institute of Natural Sciences (RBINS).
Acknowledgements
We thank Chirasak Sutcharit, Pongpun Prasankok and members of the
Animal Systematics Research Unit, Chulalongkorn University for
assistance in collecting material. We are indebted to Nesrine Akkari
(NHMW) for providing specimens, to Julien Cillis (RBINS) for help
with SEM photographs, to Yves Barette (RBINS) for help with gonopod
photographs and to Thita Krutchuen (Chulalongkorn University) for the
excellent drawings.
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Handling editor: Mark Harvey
Phylogeny and new genus of Pseudospirobolellidae Invertebrate Systematics 617
www.publish.csiro.au/journals/is
... Total genomic DNA was extracted from dissected legs using the NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions. PCR amplifications and sequencing were done as described by Pimvichai et al. (2020). Seven specimens were sequenced: Apeuthes maculatus (Attems, 1938) Table 1). ...
... The COI data included 47 specimens, representing 16 genera and 41 species of ingroup taxa ( Table 1). The 16S rRNA data included 43 specimens, i.e. the same specimens as for COI, minus Atopochetus anaticeps Pimvichai, Enghoff, Panha & Backeljau, 2018; Benoitolus birgitae (Hoffman, 1981); Coxobolellus simplex Pimvichai, Enghoff, Panha & Backeljau, 2020; Trachelomegalus sp.; Narceus annularis Rafinesque, 1820 and Paraspirobolus lucifugus (Gervais, 1837), and including Litostrophus scaber (Verhoeff, 1938) and Trachelomegalus cf. hoplurus, representing 13 genera and 37 species of ingroup taxa. ...
... Next to the morphological heterogeneity in Apeuthes, there is also a substantial amount of COI sequence variation and differentiation, with intraspecific COI divergences of 3-7% (mean: 5%), and interspecific COI divergences of 11-16% (mean: 13.7%). These figures are in line with those for the genus Coxobolellus (Pimvichai, Enghoff, Panha & Backeljau, 2020) of the related family Pseudospirobolellidae, which has intraspecific COI divergences of 0-5% (mean: 2%) and interspecific COI divergences of 6-15% (mean: 11%) (Pimvichai et al. 2020). ...
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Hitherto, the millipede genus Apeuthes (family Pachybolidae, subfamily Trigoniulinae) was only known from three species described in Vietnam based on morphological characters. The present study uses two partial mitochondrial gene fragments (cytochrome c oxidase I (COI) and 16S ribosomal RNA) and morphology to define four new Apeuthes species from Malaysia, Thailand and Vietnam: A. fimbriatus, sp. nov., A. longeligulatus, sp. nov., A. pollex, sp. nov. and ?A. spininavis, sp. nov. The intraspecific COI sequence divergence of two Apeuthes species is 3–7% (mean: 5%) and the interspecific divergence of five species is 11–16% (mean: 13.7%). All members of the genus share unique male characters, viz the posterior gonopod telopodite with several dentate, serrate or tuberculate lamellae in a boat-like cavity or a boat-like cavity covered with spines. The delimitation of the four new species is supported by the congruence between mitochondrial DNA and morphological data. However, while the monophyly of Trigoniulinae is well supported, the relationships within this subfamily, and particularly among Apeuthes species, including the monophyly of Apeuthes, lack strong support. Therefore assignment of the four new species, and particularly of ?A. spininavis sp. nov., to the genus Apeuthes is tentative and awaits a comprehensive revision of the group.
... from Wat Tham Inthanin, Mae Sot District, Tak Province, Thailand (CUMZ-D00147) using the NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions. PCR amplifications and sequencing of the standard mitochondrial COI DNA barcoding fragment (Hebert et al. 2003) were done as described by Pimvichai et al. (2020). The COI fragment was amplified with the primers LCO-1490 and HCO-2198 (Folmer et al. 1994). ...
... This research was funded by the Thailand Science Research and Innovation (TSRI) together with Mahasarakham University as a TRF Research Career Development Grant (2019-2022; RSA6280051) (to P. Pimvichai). Additional funding came from the Roy- Table 3. Estimates of COI mean sequence divergences within (on diagonal) and among (below diagonal) pachybolid and pseudospirobolellid genera (range in parentheses) (data based on Pimvichai et al. 2018Pimvichai et al. , 2020Pimvichai et al. , 2022. ...
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A new genus of the millipede family Pachybolidae from Southeast Asia is described: Macrurobolus gen. nov. , with Spirobolus macrurus Pocock, 1893 as type species. This latter species is DNA barcoded (COI) and redescribed based on male morphological characters, which hitherto were unknown. The new genus differs from other pachybolid genera by having (1) the preanal ring process long and protruding beyond the anal valves and (2) the anterior gonopod telopodite distally abruptly narrowed, forming an extremely long, slender, elevated process curved caudad. Given that Macrurobolus gen. nov. is a monotypic genus, it is aphyletic and thus requires further taxonomic revision.
... After the first accidental finds of G. subterranea in samples of macrozoobenthos from a karst spring (collected by J. Grego), we decided to examine these habitats in detail and at several localities, focusing on the frequency of the species' occurrence and distribution in Slovakia, and possible morphological adaptations to the aquatic environment. For a comprehensive analysis of species of Geoglomeris from Slovakia, we chose an integrative (morphological and molecular) approach, which is considered to be the future of taxonomy and has already been successfully applied to millipede taxa (Moritz & Wesener, 2017;Oeyen & Wesener, 2015;Pimvichai et al., 2020;Wilbrandt et al., 2015). Given the fact that G. subterranea is a small millipede with a parthenogenetic population, morphological determination can be challenging, because the morphological features used in diplopod taxonomy are mainly based on the species-specific sexual dimorphism of males. ...
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Geoglomeris subterranea Verhoeff 1908 is a small (2–3 mm), blind, and depigmented representative of the order Glomerida, with a predominantly Western European distribution. Towards Central Europe (the Czech Republic, Austria), the species is found only sporadically, and its occurrence is documented by a small number of individuals. Recently, we documented this species in three orographic units of Slovakia (Slovenské stredohorie mountain range, Slovak Karst, and Pieniny Mountains), always on limestone bedrock. These findings represent the easternmost documented occurrence of the species. Analysis of the mitochondrial COI gene confirmed the identity as the species G. subterranea from Western Europe. The circumstances of the findings in Slovakia were very surprising: At the first two localities, living individuals were repeatedly collected from the bottom of karst springs, together with stygobiont fauna. This expands our knowledge of semiaquatic millipedes and proves to be unique to the order Glomerida. In a subsequent study, we found a close association of this species with the rhizosphere within soil saturated by water at karst springs, in humid to wet habitats. Nonetheless, using detailed morphological study, no morphological adaptations to the aquatic environment were found.
... The millipede fauna of Thailand has received considerable attention in recent years. From the time when the first species were recorded from "Siam" by Karsch (1881) and until the first comprehensive list of Thai millipedes was published by Enghoff (2005), 105 species had been recorded, but due to a massive effort by Professor Somsak Panha and his (now former) students from Chulalongkorn University in Bangkok, in collaboration with foreign specialists, the following 14 years saw a dramatic increase to 228 species (Likhitrakarn et al. 2019), and the number is still growing: with the contributions by Pimvichai et al. (2020), and Likhitrakarn et al. (2021), and including the two species described here, 243 millipede species are now known from Thailand. The increased knowledge of Thai millipedes has mainly concerned the orders Polydesmida Leach, 1815, Spirobolida Bollman, 1893and Spirostreptida Brandt, 1833. ...
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Two new species of giant pill-millipedes, Zephronia viridisoma Rosenmejer & Wesener sp. nov. and Sphaerobelum aesculus Rosenmejer & Wesener sp. nov., are described based on museum samples from southern Thailand. Zephronia viridisoma sp. nov. comes from Khao Lak, while the type locality of S. aesculus sp. nov. is on Phuket Island. Both species are described integratively, combining light microscopy, scanning electron microscopy, multi-layer photography, micro-CT scans and genetic barcoding. Genetic barcoding was successfully conducted for holotypes of both new species, which could be added to a dataset of all published sequences of the family Zephroniidae, including all described species from Thailand, Laos and Cambodia up to 2020. Genetic barcoding of the COI gene revealed another female of S. aesculus sp. nov., 160 km east of the type locality. Both new species are genetically distant from all other Zephroniidae from Thailand and surrounding countries, showing uncorrected p-distances of 16.8–23.1%. A virtual cybertype of a paratype of Z. viridisoma sp. nov. was created and made publically accessible.
... Recent intense collecting efforts made by Thai specialists in collaboration with the Department of National Parks, Wildlife and Plant Conservation across the country have revealed numerous interesting millipedes, especially in limestone areas. From these efforts, several new genera and numerous new species have been recorded and described (Pimvichai et al. 2018(Pimvichai et al. , 2020Srisonchai et al. 2018a, b, c, d;Likhitrakarn et al. 2020Likhitrakarn et al. , 2021. The present contribution provides descriptions of three new species of the genus Zephronia, as well as a redescription of Z. siamensis Hirst, 1907 as based both on topotypes and near-topotypes. ...
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Material of the giant pill-millipede genus Zephronia Gray, 1832 recently collected from Thailand contains three new species: Zephronia enghoffi sp. nov. , Zephronia golovatchi sp. nov. , and Zephronia panhai sp. nov. The first Zephronia species recorded for Thailand, Z. siamensis Hirst, 1907, is also redescribed based on new specimens collected both from the type locality in Chonburi Province and from neighboring areas. Morphological characters of all new species, Z. phrain Likhitrakarn & Golovatch, 2021, and Z. siamensis are illustrated, and a distribution map of the confirmed Zephronia species occurring in Thailand is also provided.
... As regards the sphaerotheriidan fauna of Southeast Asia, it is exclusively represented by the family Zephroniidae which presently contains about 140 species in 15 genera, including the type genus Zephronia Gray, 1832 (Enghoff et al., 2015). Even though progress in diplopodological research in Thailand since the latest catalogue (Enghoff, 2005) has been very rapid and considerable (e.g., Likhitrakarn et al., 2017Likhitrakarn et al., , 2019Likhitrakarn et al., , 2020Srisonchai et al., 2018a-d;Pimvichai et al., 2018Pimvichai et al., , 2020, bringing the millipede fauna of the country to a total of 238 species in 48 genera, 19 families and nine orders, the giant pill-millipede diversity still remains badly understudied. Thus, only three species in two genera of Sphaerotheriida have hitherto been recorded from Thailand (Wongthamwanich et al., 2012a, b). ...
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Two new species of Zephronia sensu stricto, both coming from Chiang Mai Province, northern Thailand, are described: Z. lannaensis, new species and Z. phrain, new species. Both these species seem to be especially similar to Z. laotica Wesener, 2019, but clearly distinct by telopoditomere 2 of the anterior telopods showing a sclerotized process located inside a membranous area, coupled with leg-pair 3 featuring one or two dorso-apical spine(s).
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Nine new species constituting the ‘ spiny ’ group of dragon millipedes are assigned to the new genus Spinaxytes Srisonchai, Enghoff & Panha, gen. n. Seven new species are described from Thailand: S.biloba Srisonchai, Enghoff & Panha, sp. n. and S.palmata Srisonchai, Enghoff & Panha, sp. n. from Surat Thani Province, S.hasta Srisonchai, Enghoff & Panha, sp. n. from Chumphon Province, S.krabiensis Srisonchai, Enghoff & Panha, sp. n. (type species) and S.sutchariti Srisonchai, Enghoff & Panha, sp. n. from Krabi Province, S.uncus Srisonchai, Enghoff & Panha, sp. n. , and S.macaca Srisonchai, Enghoff & Panha, sp. n. from Phang Nga Province; as well as one from Malaysia, S.tortioverpa Srisonchai, Enghoff & Panha, sp. n. , and one from Myanmar, S.efefi Srisonchai, Enghoff & Panha, sp. n. The new genus is endemic to South Myanmar, South Thailand, and Malaysia, and all new species are restricted to limestone habitats. All were exclusively found living on humid rock walls and/or inside small caves. Complete illustrations of external morphological characters, an identification key, and a distribution map are provided.
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The dragon millipede genus Desmoxytes s.l. is split into five genera, based on morphological characters and preliminary molecular phylogenetic analyses. The present article includes a review of Desmoxytes s.s., while future articles will deal with Hylomus Cook and Loomis, 1924 and three new genera which preliminarily are referred to as the ‘ acantherpestes ’, ‘ gigas ’, and ‘ spiny ’ groups. Diagnostic morphological characters of each group are discussed. Hylomus is resurrected as a valid genus and the following 33 species are assigned to it: H.asper (Attems, 1937), comb. n. , H.cattienensis (Nguyen, Golovatch & Anichkin, 2005), comb. n. , H.cervarius (Attems, 1953), comb. n. , H.cornutus (Zhang & Li, 1982), comb. n. , H.draco Cook & Loomis, 1924, stat. rev. , H.enghoffi (Nguyen, Golovatch & Anichkin, 2005), comb. n. , H.eupterygotus (Golovatch, Li, Liu & Geoffroy, 2012), comb. n. , H.getuhensis (Liu, Golovatch & Tian, 2014), comb. n. , H.grandis (Golovatch, VandenSpiegel & Semenyuk, 2016), comb. n. , H.hostilis (Golovatch & Enghoff, 1994), comb. n. , H.jeekeli (Golovatch & Enghoff, 1994), comb. n. , H.lingulatus (Liu, Golovatch & Tian, 2014), comb. n. , H.laticollis (Liu, Golovatch & Tian, 2016), comb. n. , H.longispinus (Loksa, 1960), comb. n. , H.lui (Golovatch, Li, Liu & Geoffroy, 2012), comb. n. , H.minutuberculus (Zhang, 1986), comb. n. , H.nodulosus (Liu, Golovatch & Tian, 2014), comb. n. , H.parvulus (Liu, Golovatch & Tian, 2014), comb. n. , H.phasmoides (Liu, Golovatch & Tian, 2016), comb. n. , H.pilosus (Attems, 1937), comb. n. , H.proximus (Nguyen, Golovatch & Anichkin, 2005), comb. n. , H.rhinoceros (Likhitrakarn, Golovatch & Panha, 2015), comb. n. , H.rhinoparvus (Likhitrakarn, Golovatch & Panha, 2015), comb. n. , H.scolopendroides (Golovatch, Geoffroy & Mauriès, 2010), comb. n. , H.scutigeroides (Golovatch, Geoffroy & Mauriès, 2010), comb. n. , H.similis (Liu, Golovatch & Tian, 2016), comb. n. , H.simplex (Golovatch, VandenSpiegel & Semenyuk, 2016), comb. n. , H.simplipodus (Liu, Golovatch & Tian, 2016), comb. n. , H.specialis (Nguyen, Golovatch & Anichkin, 2005), comb. n. , H.spectabilis (Attems, 1937), comb. n. , H.spinitergus (Liu, Golovatch & Tian, 2016), comb. n. , H.spinissimus (Golovatch, Li, Liu & Geoffroy, 2012), comb. n. and H.variabilis (Liu, Golovatch & Tian, 2016), comb. n.Desmoxytes s.s. includes the following species: D.breviverpa Srisonchai, Enghoff & Panha, 2016; D.cervina (Pocock,1895); D.delfae (Jeekel, 1964); D.des Srisonchai, Enghoff & Panha, 2016; D.pinnasquali Srisonchai, Enghoff & Panha, 2016; D.planata (Pocock, 1895); D.purpurosea Enghoff, Sutcharit & Panha, 2007; D.takensis Srisonchai, Enghoff & Panha, 2016; D.taurina (Pocock, 1895); D.terae (Jeekel, 1964), all of which are re-described based mainly on type material. Two new synonyms are proposed: Desmoxytespterygota Golovatch & Enghoff, 1994, syn. n. (= Desmoxytescervina (Pocock, 1895)), Desmoxytesrubra Golovatch & Enghoff, 1994, syn. n. (= Desmoxytesdelfae (Jeekel, 1964)). Six new species are described from Thailand: D.aurata Srisonchai, Enghoff & Panha, sp. n. , D.corythosaurus Srisonchai, Enghoff & Panha, sp. n. , D.euros Srisonchai, Enghoff & Panha, sp. n. , D.flabella Srisonchai, Enghoff & Panha, sp. n. , D.golovatchi Srisonchai, Enghoff & Panha, sp. n. , D.octoconigera Srisonchai, Enghoff & Panha, sp. n. , as well as one from Malaysia: D.perakensis Srisonchai, Enghoff & Panha, sp. n. , and one from Myanmar: D.waepyanensis Srisonchai, Enghoff & Panha, sp. n. The species can mostly be easily distinguished by gonopod structure in combination with other external characters; some cases of particularly similar congeners are discussed. All species of Desmoxytes s.s. seem to be endemic to continental Southeast Asia (except the ‘tramp’ species D.planata ). Some biological observations (relationship with mites, moulting) are recorded for the first time. Complete illustrations of external morphological characters, an identification key, and distribution maps of all species are provided.
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The Black Pill Millipede, Glomeris marginata , is the best studied millipede species and a model organism for Diplopoda. Glomeris marginata is widespread, with numerous colour morphs occurring across its range, especially in the south. This study investigates whether colour morphs might represent cryptic species as well as the haplotype diversity and biogeography of G. marginata . The results of the COI barcoding fragment analysis include 97 G. marginata , as well as 21 specimens from seven potentially related species: G. intermedia Latzel, 1884, G. klugii Brandt, 1833 ( G. undulata C.L. Koch, 1844), G. connexa Koch, 1847, G. hexasticha Brandt, 1833, G. maerens Attems, 1927, G. annulata Brandt, 1833 and G. apuana Verhoeff, 1911. The majority of the barcoding data was obtained through the German Barcode of Life project (GBOL). Interspecifically, G. marginata is separated from its congeners by a minimum uncorrected genetic distance of 12.9 %, confirming its monophyly. Uncorrected intraspecific distances of G. marginata are comparable to those of other widespread Glomeris species, varying between 0–4.7%, with the largest genetic distances (>2.5 %) found at the Mediterranean coast. 97 sampled specimens of G. marginata yielded 47 different haplotypes, with identical haplotypes occurring at large distances from one another, and different haplotypes being present in populations occurring in close proximity. The highest number of haplotypes was found in the best-sampled area, western Germany. The English haplotype is identical to northern Spain; specimens from southern Spain are closer to French Mediterranean specimens. Analyses (CHAO1) show that approximately 400 different haplotypes can be expected in G. marginata . To cover all haplotypes, it is projected that up to 6,000 specimens would need to be sequenced, highlighting the impossibility of covering the whole genetic diversity in barcoding attempts of immobile soil arthropod species.
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The aim of this study was to use cytochrome c oxidase 1 (CO1) sequences to recover a phylogeny for seven morphologically described spirostreptid millipede taxa from southern Africa, and to evaluate the correspondence between morphological and molecular phylogenies. Genetic p-distance generally increased with taxonomic divergence: inter-specific mean 15.33 % (14.09 % –17.02 %), inter-generic mean 18.43 % (6.83 %–26.81 %) and inter-order mean 24.16 % (range 18.56 %–30.77 %). Congruent Bayesian, maximum parsimony and neighbour-joining analyses of 520 nucleotides of the CO1 gene resolved the orders Spirostreptida, Julida and Callipodida. Members of genera within the Spirostreptidae (Archispirostreptus, Bicoxidens, Cacuminostreptus, Doratogonus, Orthoporoides, Plagiotaphrus and Spirostreptus) formed a single clade within which a sample of Thyropygus (family Harpagophoridae) was paraphyletically nested. Phylogenetic analyses failed to recover support for the genera Doratogonus, Bicoxidens, Archispirostreptus and Spirostreptus, as representatives of these genera were not monophyletic. Samples morphologically identified as the same species (Bicoxidens flavicollis) were part of two different clades, one of which was well supported and otherwise contained members of Doratogonus. This high level of divergence (mean 12.64 %) between morphologically identified spirostreptid millipede sister species could indicate that changes in genital morphology occur rather slowly relative to CO1 sequence substitution, and may underestimate species diversity.