Zoosyst. Evol. 98 (2) 2022, 313-326 | DOI 10.3897/zse.98.90032 Ate BERLIN A new genus of Pseudospirobolellidae (Diplopoda, Spirobolida) from limestone karst areas in Thailand, with descriptions of three new species Piyatida Pimvichai', Henrik Enghoff*, Somsak Panha’, Thierry Backeljau** Department of Biology, Faculty of Science, Mahasarakham University, Maha Sarakham 44150, Thailand Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen 0, Denmark Animal Systematics Research Unit, Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000 Brussels, Belgium oF WwW NY PF Evolutionary Ecology Group, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium https://zoobank. org/A 7572415-4926-4A 16-810A-F6DIFS55FI2BE Corresponding author: Piyatida Pimvichai (piyatida.p@msu.ac.th) Academic editor: Luiz Felipe Iniesta # Received 6 July 2022 Accepted 9 August 2022 Published 19 August 2022 Abstract A new genus of the millipede family Pseudospirobolellidae, Si/iquobolellus gen. nov., is described from limestone mountains in Thailand, based on three new species, viz. Siliquobolellus amicusdraconis gen. et sp. nov. from Uthaithani Province, Siliquobolellus constrictus gen. et sp. nov. from Prachuap Khiri Khan Province and Siliquobolellus prasankokae gen. et sp. nov. (type species) from Lampang Province. The descriptions are based on gonopod morphology and mitochondrial DNA data (COI barcodes). The COI barcodes grouped the three new species in a well-supported Siliquobolellus gen. nov. clade. The mean interspecific COI sequence divergence among the three new species was 12% (range: 8-15%). The mean intergeneric COI sequence divergence between Sili- quobolellus gen. nov., Coxobolellus Pimvichai, Enghoff, Panha & Backeljau, 2020, and Pseudospirobolellus Carl, 1912 was 19% (range: 14-23%). Three conspicuous gonopodal synapomorphies differentiate Si/iquobolellus gen. nov. from other pseudospirobol- lellid genera: (1) the telopodital part of the posterior gonopod forms a deep concavity, (2) the telopodite of the anterior gonopod is directed distad and does not reach the tip of the coxal part of the anterior gonopod, and (3) the tip of the anterior gonopod coxa is narrowed, curving mesad. As such, the monophyly of the new genus is well supported by both morphological and mitochondrial DNA data. A distribution map and an identification key to the species are provided. Key Words COI, gonopod, monophyly, synapomorphy Introduction Hitherto, the small SE Asian spirobolidan millipede family Pseudospirobolellidae comprised < 20 species in 2 or 3 genera, viz. Pseudospirobolellus Carl, 1912, Coxobolellus Pimvichai, Enghoff, Panha & Backeljau, 2020, and Benoitolus Mauries, 1980 (Enghoff et al. 2015; Pimvichai et al. 2020). Yet, recent mitochondrial DNA sequence analyses suggested that the genera Pseudospirobolellus and Coxobolellus form a_ well- supported clade, while the genus Benoitolus is nested within the family Pachybolidae (whose monophyly is not well-corroborated), though with limited support and obliterating the otherwise strong support for the genus Litostrophus Chamberlin, 1921 (Pimvichai et al. 2020). Thus, the monophyly and taxonomic composition of the family Pseudospirobolellidae are still unclear and need more attention. In this context, the present study uses gonopod mor- phology and mitochondrial DNA sequence data to de- scribe three new species in a new genus of Pseudo- spirobolellidae from limestone karst areas in Thailand. Copyright Pimvichai, P. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 314 Materials and methods Live specimens were hand-collected and were partly pre- served in 70% ethanol for morphological study and partly placed in a freezer at —20 °C for DNA analysis. This research was conducted under the approval of the Animal Care and Use regulations (numbers U1-07304- 2560 and [ACUC-MSU-037/2019) of the Thai government. Morphology Gonopods were photographed with a digital camera. Samples for scanning electron microscopy (SEM: Hita- chi TM4000Plus) were air-dried directly from alcohol and sputter-coated for 60 s with gold (Hitachi: MC1000). Scanning electron micrographs were taken at the Central Lab of Mahasarakham University. Drawings were made using a stereomicroscope and photographs. Voucher specimens were deposited in the collections of CUMZ. DNA extraction, amplification and sequencing Total genomic DNA was extracted from legs of single specimens of Siliquobolellus amicusdraconis gen. et sp. nov. (Hub Pa Tard, Uthaithani Province; CUMZ-D00149), Siliquobolellus constrictus gen. et sp. nov. (Ban Yang Chum, Prachuap Khiri Khan Province; CUMZ-D00150) and Siliquobolellus prasankokae gen. et sp. nov. (Pha Thai, Lampang Province; CUMZ-D00148) using the Nu- cleoSpin Tissue kit (Macherey-Nagel, Duren, Germany) following the manufacturer’s instructions. PCR ampli- fications 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). The new COI nucleotide sequences have been deposited in GenBank under acces- sion numbers OP174621— OP174623. Sample data and voucher codes are provided in Table 1. Alignment and phylogenetic analysis The COI data included 56 specimens, representing 18 genera and 46 nominal species of ingroup taxa (Table 1). Three species of the order Spirostreptida, viz. Anurostreptus barthelemyae Demange, 1961 (Harpagophoridae), Chonecambala crassicauda Mauries & Enghoff, 1990 (Pericambalidae) and Thyropygus allevatus (Karsch, 1881) (Harpagophoridae) were used as outgroup. CodonCode Aligner (ver. 4.0.4, CodonCode Corpora- tion) was used to assemble the forward and reverse se- quences and to check for errors and ambiguities. All se- quences were checked with the Basic Local Alignment Search Tool (BLAST) provided by NCBI and compared with reference sequences in GenBank. All sequences were zse.pensoft.net Pimvichai, P. et al.: Anew genus of Pseudospirobolellidae aligned using MUSCLE (ver. 3.6, see http://www.driveS. com/muscle; Edgar 2004). The alignments consisted of 660 bp. The sequences were checked for ambiguous nucle- otide sites, saturation and phylogenetic signal using DAM- BE (ver. 5.2.65. see http://dambe. bio.uottawa.ca/DAMBE/ dambe_install_ win.aspx; Xia 2018). MEGA (ver. 11.0.10, see http://www.megasoftware.net; Tamura et al. 2021) was used to (1) check for stop codons, (2) translate sequences into amino acids, and (3) calculate uncorrected pairwise p-distances among sequences by computing the proportion of nucleotide sites by which two sequences differ. 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. ML trees were inferred with RAxML (ver. 8.2.12, see http://www.phylo.org/index.php/tools/raxm- Ihpce2_tgb.html; Stamatakis 2014) through the CIPRES Science Gateway (Miller et al. 2010) using a GIR+G substitution model and 1000 bootstrap replicates to assess branch support. BI trees were constructed with MrBayes (ver. 3.2.7a, see http://www.phylo.org/index.php/tools/ mrbayes_xsede.html; Huelsenbeck and Ronquist 2001). Substitution models were inferred using jModeltest (ver. 2.1.10, see https://www.github.com/ddarriba/jmodelt- est2/releases; Darriba et al. 2012) applying Akaike In- formation Criterion (AIC) weights as selection criterion. This yielded TIM2+ I+G (-InL = 11998.1218, gamma shape = 0.4620) as best model. BI trees were run for 2 million generations (heating parameter: 0.02), sampling every 1000 generations. Con- vergences were confirmed by verifying that the standard deviations of split frequencies were below 0.01. Then the first 1000 trees were discarded as burn-in, so that the final consensus tree was built from the last 3002 trees. Support for nodes was assessed by posterior probabilities. For ML we consider branches with bootstrap values (BV) of = 70% to be well supported (Hillis and Bull 1993) and < 70% as poorly supported. For BI trees, we consider branches with posterior probabilities (PP) of > 0.95 to be well supported (San Mauro and Agorreta 2010) and be- low as poorly supported. Results The uncorrected p-distances between the sequences ranged from 0.00 to 0.26 (Suppl. material 1: Table S1). The mean interspecific sequence divergence within Siliquobolellus gen. nov. was 0.12 (range: 0.08—-0.15). The mean intergeneric sequence divergences between Siliquobolellus gen. nov. and the other putative pseudospirobolellid genera were: 0.17 (range: 0.14—0.20) for Coxobolellus, 0.21 (range: 0.20-0.23) for Pseudospirobolellus, and 0.23 (range: 0.22—0.24) for Benoitolus birgitae (Hoffman, 1981). The mean intergeneric sequence divergence between Siliguobolellus gen. nov., Coxobolellus, and Pseudospirobolellus was 0.19 (range: 0.14—0.23). Zoosyst. Evol. 98 (2) 2022, 313-326 Biko) Table 1. Specimens from which the COI gene fragment was 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 capitals. Abbreviations after species names refer to the isolate of each sequence. GenBank accession numbers are indicated for each species. Voucher code Locality Col Genus Siliquobolellus gen. nov. S. amicusdraconis gen. et sp. nov. CUMZ- D00149 Hub Pa Tard, LanSak, Uthaithani, THAILAND OP174621 S. constrictus gen. et sp. nov. CUMZ- DO0150 Ban Yang Chum, Kui Buri, Prachuap Khiri Khan, THAILAND OP1 74622 S. prasankokae gen. et sp. nov. CUMZ- D00148 Pha Thai, Ngao, Lampang, THAILAND OP1 74623 Genus Apeuthes A. maculatus Amc NHMW-Inv. No.2395 South Annam, VIETNAM MF187404 A. maculatus Am26 NHMD-621697 Nha Trang, Bao Dai Villas Hotel, in garden, VIETNAM MZ567159 A. fimbriatus BMP CUMZ-D00144 Bach Ma Peak, Da Nang, VIETNAM MZ567160 A. longeligulatus TPP CUMZ-D00140 Tham Phet Po Thong, Klong Hard, Sa Kaeo, THAILAND MZ567161 A. pollex SMR CUMZ-D00141 Sra Morakot, Klongthom, Krabi, THAILAND MZ567162 A. pollex SML CUMZ-D00142 Koh 8, Similan islands, Phang-Nga, THAILAND MZ567163 A. pollex WTS CUMZ-D00143 Tham Sue Temple, Muang, Krabi, THAILAND MZ567164 ?A. spininavis ABB CUMZ-D00145 Air Banun, Perak, MALAYSIA MZ567165 Genus Atopochetus A. anaticeps SVL CUMZ- DO0091 Srivilai temple, Chalermprakiet, Saraburi, THAILAND MF187405 A. dollfusii DOL NHM Cochinchina, VIETNAM MF187412 A. helix SPT CUMZ- DO0094 Suan Pa Thong Pha Phum, Kanchanaburi, THAILAND MF187416 A. moulmeinensis TAK CUMZ- D00095 Km 87, Tha Song Yang, Tak, THAILAND MF187417 A. setiferus HPT CUMZ- DO0097 Hub Pa Tard, Lan-Sak, Uthaithani, THAILAND MF187419 A. spinimargo Ton27 NHMD- 00047013 Koh Yo, Songkhla, THAILAND MF187423 A. truncatus SML CUMZ- DO0101 Koh 8, Similan islands, Phang-Nga, THAILAND MF187424 A. uncinatus KMR CUMZ- DO0102 Khao Mar Rong, Bangsapan, Prachuapkhirikhan, THAILAND MF187425 A. weseneri Tos29 NHMD-0004.7003 Supar Royal Beach Hotel, Khanom, Nakhonsrithammarat, THAILAND MF187431 Genus Aulacobolus A. uncopygus Auc NHMW-Inv. No.2375 Nilgiris, South India, INDIA MF187433 Genus Benoitolus B. birgitae BBG NHMD 621687 Chiang Dao, Chiang Mai, THAILAND MT328992 Genus Coxobolellus C. albiceps Stpw CUMZ-D00121 Tham Pha Tub, Muang District, Nan Province, THAILAND (green individual) MT328994 C. albiceps Stpl CUMZ-D00122 Tham Pha Tub, Muang District, Nan Province, THAILAND (small, brown individual) MT328993 C. albiceps TPB CUMZ-D00123 Wat Tham Bampen Bun, Pan District, Chiang-Rai Province, THAILAND MT328996 C. albiceps Stvd CUMZ-D00124 Tham Wang Daeng, Noen Maprang District, Phitsanulok Province, THAILAND MT328995 C. compactogonus SKR CUMZ-D00134 Sakaerat Environmental Research Station, Wang Nam Khiao District, Nakhon MT328998 Ratchasima Province, THAILAND C. compactogonus KLC CUMZ-D00135 Khao Look Chang, Pak Chong District, Nakhon Ratchasima Province, THAILAND MT328997 C. fuscus HKK CUMZ-D00133 Kroeng Krawia waterfall, Sangkhla Buri District, Kanchanaburi Province, THAILAND MT328999 C. nodosus SPW CUMZ-D00126 Chao Por Phawo Shrine, Mae Sot District, Tak Province, THAILAND MT329000 C. serratus KKL CUMZ-D00132 Khao Kalok, Pran Buri District, Prachuap Khiri Khan Province, THAILAND MT329001 C. simplex TNP CUMZ-D00136 Tham Pha Pha Ngam, Mae Prik District, Lampang Province, THAILAND MT329002 C. tenebris KWP CUMZ-D00119 Wat Khao Wong Phrohm-majan, Ban Rai District, Uthai Thani Province, THAILAND MT329003 C. tenebris TPL CUMZ-D00120 Wat Tham Phrom Lok Khao Yai, Sai Yok District, Kanchanaburi Province, THAILAND MT 329004 C. tigris TKP CUMZ-D00130 Wat Tham Khao Plu, Pathio District, Chumphon Province, THAILAND MT329005 C. tigris TYE CUMZ-D00131 Tham Yai |, Pathio District, Chumphon Province, THAILAND MT329006 C. transversalis Stpg CUMZ-D00125 Tham Pha Tub, Muang District, Nan Province, THAILAND MT329007 C. valvatus TCD CUMZ-D00127 Wat Tham Chiang Dao, Chiang Dao District, Chiang-Mai Province, THAILAND MT329009 C. valvatus BRC CUMZ-D00128 Tham Borichinda, Chom Thong District, Chiang-Mai Province, THAILAND MT329008 C. valvatus TST CUMZ-D00129 Tham Sam Ta, Muang District, Mae Hong Son Province, THAILAND MT329010 Genus Leptogoniulus L. sorornus BTN CUMZ- D00109 Botanical Garden, Penang, MALAYSIA MF187434 Genus Litostrophus L. chamaeleon PPT CUMZ- D00111 Phu Pha terb, Mukdahan, THAILAND MF 187436 L. saraburensis PKS CUMZ- D00113 Phukhae Botanical Garden, Saraburi, THAILAND MF187438 L. segregatus Ls19 NHMD 621686 Koh Kut, Trad, THAILAND MF187440 Genus Macrurobolus M. macrurus INT CUMZ- D00147 Wat Tham Inthanin, Mae Sot District, Tak Province, THAILAND MZ905519 Genus Madabolus M. maximus Mm4 NHMD-0004.7007 de Toliara Province, Parc National de Bermaraha, South Bank of Manambolo River, MF187441 Near Tombeau Vazimba, MADAGASCAR Genus Narceus N. annularis NC_003343.1 zse.pensoft.net 316 Voucher code Genus Parabolus Pimvichai, P. et al.: Anew genus of Pseudospirobolellidae Locality col P. dimorphus Pd34 NHMD-00047004 Dar es Salaam, TANZANIA MF187442 Genus Paraspirobolus P. lucifugus AB608779.1 Genus Pelmatojulus P. tigrinus Pt2 NHMD-00047008 Southern part of the Comoé N.P., 30 km north of Kakpin, COTE d'lVOIRE MF187443 P. togoensis Pto6 NHMD-0004 7006 Biakpa, GHANA MF187444 Genus Pseudospirobolellus Pseudospirobolellus avernus GPG CUMZ-D00117 Gua Pulai, Gua Musang, Kelantan, MALAYSIA MT329011 Pseudospirobolellus sp. KCS CUMZ-D00118 Koh Chuang, Sattahip, Chonburi, THAILAND MT329012 Genus Rhinocricus R. parcus Rp49 NHMD-00047009 Puerto Rico, USA MF187449 Genus Trachelomegalus Eesps o4: NHMD-00047012 Borneo Sabah, MALAYSIA MF187445 Genus Trigoniulus T. corallinus Tcol5 NHMD-00047010 Vientiane, LAOS MF187446 Outgroup Genus Anurostreptus A. barthelemyae Tlb CUMZ-DO00003 Thale-Ban N.P., Khuan-Don, Satun, THAILAND KC519469 Genus Chonecambala C. crassicauda Ttp CUMZ-DO0001 Ton-Tong waterfall, Pua, Nan, THAILAND KC519467 Genus Thyropygus T. allevatus Bb CUMZ-D00013 BangBan, Ayutthaya, THAILAND KC519479 The mean intergeneric sequence divergences between Siliquobolellus gen. nov. and various pachybolid genera were: 0.20 (range: 0.18—0.23) for Apeuthes Attems, 1938, 0.23 (range: 0.20-0.26) for Atopochetus Attems, 1953, 0.21 (range: 0.18—0.22) for Litostrophus Chamberlin, 1921, and 0.20 (range: 0.19-0.22) for Pe/matojulus De Saussure, 1860. The mean intergeneric sequence divergences between Siliquobolellus gen. nov. and the other Spirobolida families were: 0.22 (range: 0.21-0.24) for Narceus annularis (Rafinesque, 1820) (Spirobolidae) and 0.24 (range: 0.23—-0.26) for Paraspirobolus lucifugus (Gervais, 1836) (Spirobolellidae). Boththe BI and ML trees (Fig. 1) showed Siliquobolellus gen. nov., Coxobolellus and Pseudospirobolellus as three well-supported clades, which jointly formed a well-supported Pseudospirobolellidae clade (though without Benoitolus birgitae). However, the sister group relationship between Siliquobolellus gen. nov. and Coxobolellus was poorly supported by both BI and ML. Taxonomy Class Diplopoda de Blainville in Gervais, 1844 Order Spirobolida Bollman, 1893 Suborder Spirobolidea Bollman, 1893 Family Pseudospirobolellidae Brélemann, 1913 Genus Siliquobolellus gen. nov. https://zoobank.org/A2CA57E0-53B4-4060-B621-E8A3322EAFB4 Fig. 2 Etymology. From Latin si/igua = pod, and referring to the podlike shape of the gonopod telopodite. Type species. Siliquobolellus prasankokae gen. et sp. nov. zse.pensoft.net Other included species. Si/iquobolellus amicusdra- conis gen. et sp. nov. and Siliquobolellus constrictus gen. et sp. nov. Diagnosis. Siliquobolellus species are characterised by (1) the unique shape of the telopodital part of their posterior gonopod, which forms a deep concavity. In contrast, the telopodital part of the posterior gonopod is extremely slender and sickle-shaped in Pseudospirobolellus, does not form a deep concavity in Coxobolellus, or is slender with a broad apical canopy in Benoitolus; (2) the telopodite of anterior gonopod (at) simple, directed distad, not reaching tip of coxal part of the anterior gonopod (cx). In contrast, the telopodite of anterior gonopod (at) is overreaching coxa in the other three genera; and (3) tip of anterior gonopod coxa narrowed, curving mesad. In contrast the tip of the anterior gonopod coxa directed distad in Coxobolellus, or is rounded in Pseudospirobolellus and Benoitolus. General description. 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 (IL) open (Fig. 2A). 2+2 labral teeth, a row of labral setae, 3+3 supralabral setae. Diameter of eyes ca half of interocular space; 7 vertical rows of ommatidia, 4 horizontal rows, 24—26 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>6>1>5>3=4 > 7 in Siliquobolellus amicusdraconis gen. et sp. nov. and S. constrictus gen. et sp. nov. and2>1>6>5>3=4 > 7 in S. prasankokae gen. et sp. nov.; antennomere 1 glabrous, 2 and 3 with some ventral setae, 4, 5 and 6 densely setose; 4 apical sensilla. Mandibles: stipes (Mst) Zoosyst. Evol. 98 (2) 2022, 313-326 Bir Pseudospii Pseudospirobolellus a Siliquobolellus amicus: Siliquobole Siliquobolellus constrictus gen. et sp 100/1.00 100/1.00 97/1.00 2/100 98/1.00 7 56/0.98 63/# 88/0.99 Olt Hit 82/1.00 Coxobolellus S3/#] _100/1.00 731# 78/4.00 am 100/1.00 99/1.00 70/0.97 Coxobolelius Coxobolellus fuscus HKK Paraspirobolus lucifugus Narceus annularis Rhinocricus parcus Rp49 Pelmatojulus tigrinus Pt2 e. Pelmatojul Hit TAl# 53/# #/1.00 54/# 89/1.00 S5/# 96/1.00 100/1.00 76l#. #l- 78/1.00 99/1.00 Anurostreptus barthelemyae Tib Thyropygus allevatus Bb 0.3 Figure 1. Phylogenetic relationships of Si/iquobolellus gen. nov. and several other spirobolidan millipede taxa based on maximum likelihood analysis (ML) and Bayesian inference (BI) of a 660 bp COI gene fragment. Numbers at nodes indicate branch support based on bootstrapping (ML) / posterior probabilities (BI). Scale bar = 0.3 substitutions/site. # indicates branches with < 50% ML bootstrap support or < 0.95 posterior probability, - indicates non-supported branches. The colored areas mark the families Pseudospirobolellidae (minus Benoitolus) (purple) and Pachybolidae (plus Benoitolus) (yellow). broad at base, apically gradually narrowed, triangular. Gnathochilarium (Fig. 2B): each stipe (Gst) with 3 apical setae; each lamella lingualis with 2 setae, one behind the other. Basal part of mentum (Me) transversely wrinkled; basal part of stipites 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 2-3 ventrally concave, hence with distinct ventrolateral “corners”. Body rings very smooth, parallel-sided in dorsal view. Prozona smooth. ‘Tergo-pleural’ suture visible on pro- and mesozona; mesozona ventrally with fine oblique striae, dorsally punctate; metazona ventrally with fine longitudinal striae, otherwise smooth. “Pleural” parts of rings with fine oblique striae. Sterna transversely striate. Ozopores from ring 6, situated in mesozona, ~1/2 pore diameter in front of metazona. Telson smooth; preanal ring (Pre) with slightly recurved dorsal profile, with short process protruding to vertical tangent to anal valves or slightly beyond (Fig. 2C—E). Anal valves (Av) smooth, rounded (Fig. 2C—E). Subanal scale (Sub) broadly triangular (Fig. 2F). zse.pensoft.net 318 Pimvichai, P. et al.: Anew genus of Pseudospirobolellidae H a s ~ a8 35 5 0) : ee ath athe a (ETTE CECE YS Figure 2. External morphology of Siliquobolellus gen. nov. A-C. S. amicusdraconis gen. et sp. nov. A. Head, lateral view; B. Head, ventral view; C. Posterior end, lateral view; D. Posterior end, lateral view (S. prasankokae gen. et sp. nov.); E. Posterior end, lateral view (S. constrictus gen. et sp. nov.); F—I. S. prasankokae gen. et sp. nov.; F. Posterior end, ventral view; G, H. Male leg, latero-ventral view; I. Body rings, dorsal colour pattern; J. S. constrictus gen. et sp. nov., body rings, dorsal colour pattern. Av = anal valves; Gst = gnathochilarial stipes; IL = incisura lateralis Me = mentum; Mst = Mandibular stipes; Pre = preanal ring; Sub = subanal scale. zse.pensoft.net Zoosyst. Evol. 98 (2) 2022, 313-326 Legs (Fig. 2G, H): length of midbody legs 55-60% of body diameter in males, 40-48% of body diameter in fe- males. Prefemur basally constricted and longer than other podomeres. First and second legs with 2 or 3 prefemoral, 2 or 3 femoral, 2 or 3 postfemoral, and 2-4 tibial setae, and 4 or 5 ventral and 1 dorsal apical setae on tarsi, num- bers of setae reaching constancy from pair 3: in males each podomere from postfemur to tibia with 1 seta; tarsi with lventral apical and 1 dorsal apical seta; in females each leg podomere from coxae to tibia with 1 seta, tarsi with 1-3 ventral and | dorsal apical setae. Claw slender and sharp. Male sexual characters. Apical part of coxa and entire ventral surface of prefemur from third to the last body rings with large ventral soft pad. Body ring 7 entirely fused ven- trally, no trace of a suture. Tip of anterior gonopods visible when the animal is stretched out (not when it is rolled up). Anterior gonopods (Figs 3-5A, B, D, E) with a fairly small triangular mesal sternal process (st). Coxa broad at base, apically gradually narrowed, curving mesad, posterior surface folding over telopodite, for accommodation of te- lopodite. Telopodite gradually narrowed towards tip, direct- ed distad, not reaching tip of anterior gonopod coxa (cx). Posterior gonopods (Figs 3—SC, F) simple, round- ed, with short, smooth coxal part (pcx); with prominent opening of efferent groove (oeg) distomesally of pcx; te- lopodital part (pt) twice the length of the pcx, lateral mar- gin folding mesad, forming a deep concavity. Female vulvae (Figs 3L, 4K, 5K) simple, valves prom- inent. Species descriptions Siliquobolellus amicusdraconis gen. et sp. nov. https://zoobank. org/5D598D47-C70C-438C-8A A B-E083A7E7053B Figs'2.3;627 Material studied. Holotype. 1 male(CUMZ-D00149-1), Thailand, Uthaithani Province, Lan-Sak District, Hub Pa Tard; 15°22'37.13"N, 99°37'49.98"E; 119 m a.s.l.; 26 July 2020; P. Pimvichai, T. Backeljau and P. Prasankok leg. Paratypes. 3: males (CUMZ-D00149-3), 3 females (CUMZ-D00149-2); same data as holotype. Etymology. The species epithet means “friend of the dragon” and refers to the type locality, which is shared with the “shocking pink dragon millipede”, Desmoxytes purpurosea Enghoff, Sutcharit & Panha, 2007. Diagnosis. Differing from other species in the genus by having the tip of anterior gonopod crossing over with tip of opposite side, the two together delimiting a drop- shaped “window”, whereas in the other two species the tips of anterior gonopod are separated from each other; externally differing by its bright orange color on the ante- rior and posterior ends. In contrast, S. constrictus gen. et sp. nov. has a row of triangular dark brown spots middor- sally on its body rings, while S. prasankokae gen. et sp. nov. has a row of rectangular dark brown spots middor- sally on the body rings. 319 Description. Adult males with 41 or 42 podous rings, 1 apodous ring. Length ~3 cm, diameter 2.7—2.9 mm. Adult females with 41-43 podous rings, 1-3 apodous rings. Length ~3 cm, diameter 2.8—3.1 mm. Colour. Living animal mainly dark brown. Head, dorsal part of the first four body rings and telson orange, antenna light brown, middorsal metazona orange (Fig. 6A, B). Anterior gonopods (Fig. 3A, B, D, E) with extreme- ly small triangular process between coxae (st), with high coxae, apically narrow, curving mesad, tip crossing over with tip of opposite side, the two together delimiting a drop-shaped “window”. Telopodite flattened apically rounded, curving backward, directed distad, as a thumb- like process (Fig. 3B, arrow). Posterior gonopods (Fig. 3C, F—K) simple, rounded, with short, smooth coxal part (pcx); telopodital part (pt) twice the length of the pcx, lateral margin folding mesad, forming a deep concavity, the inner lateral margin ex- panded into rounded lamella, apically forming a canopy (Fig. 3J, arrow). Female vulvae (Fig. 3L): simple, valves prominent, the right valve slightly larger than the left valve. DNA barcode. The GenBank accession number of the COI barcode of the paratype is OP174621 (voucher code CUMZ-D00149). Habitat. Found under leaf litter and on rocks. Distribution. Known only from the type locality in Uthaithani Province, Thailand (Fig. 7). Siliquobolellus constrictus gen. et sp. nov. https://zoobank.org/FF7A3A FE-2FBB-469D-9A 29-55F70DDE12F8 Figs 2, 4, 6, 7 Material studied. Holotype. 1 male (CUMZ-D00150-1), Thailand, Prachuap Khiri Khan Province, Kui Buri Dis- trict, Ban Yang Chum; 12°04'19.64"N, 99°42'57.63"E; 149 ma.s.l.; 7 August 2014; C. Sutcharit leg. Paratypes. 1 male (CUMZ-D00150-2), 2 females (CUMZ-D00150-3); same data as holotype. Etymology. The species epithet is a Latin adjective de- rived from the verb constringere = “to tighten” and refers to the constricted anterior gonopod coxa. Diagnosis. Differing from other species in the genus by having the anterior gonopod coxae constricted at middle of lateral margin, while the other two species have no constric- tion at middle of lateral margin of anterior gonopod coxae; externally differing by having a middorsal row of triangular dark brown spots, with dark brown bands on both lateral sides running parallel to the dorsal one. Description. Adult males with 41 or 42 podous rings, 1 apodous ring. Length ~3 cm, diameter 3.6—3.7 mm. Adult females with 42 or 43 podous rings, 1 apodous ring. Length ~3 cm, diameter 4.14.5 mm. Colour. After 8 years in alcohol overall beige with 3 stripes from head to telson: a mid-dorsal row of triangular dark brown spots, with dark brown bands on both lateral side running parallel to the dorsal one (Fig. 2J). zse.pensoft.net 320 Pimvichai, P. et al.: Anew genus of Pseudospirobolellidae Figure 3. Siliquobolellus amicusdraconis gen. et sp. nov., holotype, gonopods (specimen from Hub Pa Tard, CUMZ-D00149-1). A. Anterior gonopod, anterior view; B. Anterior gonopod, posterior view, arrow indicates a thumb-like process; C. Right posterior gonopod, lateral view; D. Anterior gonopod, anterior view; E. Anterior gonopod, posterior view; F. Right posterior gonopod, lateral view; G. SEM, Right posterior gonopod, latero-mesal view; H. SEM, Mesal part of right posterior gonopod, lateral view; I. SEM, Right posterior gonopod, latero-mesal view; J. SEM, Tip of right posterior gonopod, latero-mesal view, arrow indicates a canopy; K. SEM, lateral part of pt, latero-mesal view; L. SEM, left female vulva, posterior mesal view. at = anterior gonopod telopodite; Cx = coxa; oeg = opening of efferent groove; op = operculum of vulva; pcx = coxal part of the posterior gonopod telopodite; pt = telopodital part of the posterior gonopod telopodite; st = sternal process. zse.pensoft.net Zoosyst. Evol. 98 (2) 2022, 313-326 321 Figure 4. Siliquobolellus constrictus gen. et sp. nov., holotype, gonopods (specimen from Ban Yang Chum, CUMZ-D00150-1). A. An- terior gonopod, anterior view; B. Anterior gonopod, posterior view, arrow indicates a thumb-like process, with longitudinal ridge; C. Right posterior gonopod, lateral view; D. Anterior gonopod, anterior view; E. Anterior gonopod, posterior view; F. Right posterior gonopod, lateral view; G. SEM, Left posterior gonopod, lateral view; H. SEM, Left posterior gonopod, latero-mesal view; I. SEM, tip of left posterior gonopod, latero-mesal view; J. SEM, Mesal part of left posterior gonopod, lateral view; K. SEM, left female vulva, posterior mesal view. at = anterior gonopod telopodite; cx = coxa; oeg = opening of efferent groove; op = operculum of vulva; pcx = coxal part of the posterior gonopod telopodite; pt = telopodital part of the posterior gonopod telopodite; st = sternal process. zse.pensoft.net 322 Anterior gonopods (Fig. 4A, B, D, E) with a small triangular process between coxae (st), with high coxae, broad at base, gradually narrowing towards tip, distinct- ly constricted at middle of lateral margin, apical part flattened, curving mesad, mesal margin basally straight, curving towards tip, posterior surface folding cover more than half of telopodite. Telopodite small, directed distad, thumb-like process, with longitudinal ridge from base to tip (Fig. 4B, arrow). Posterior gonopods (Fig. 4C, F—J) simple, rounded, with short, smooth coxal part (pcx); telopodital part (pt) twice the length of the pcx, lateral margin expanded, fold- ing mesad, forming a concavity (not so deep as in the other two species). Female vulvae (Fig. 4K): simple, valves prominent, the left valve slightly larger than the right valve. DNA barcode. The GenBank accession number of the COI barcode of the paratype 1s OP174622 (voucher code CUMZ-D00150). Habitat. Found under leaf litter. Distribution. Known only from the type locality in Prachuap Khiri Khan Province, Thailand (Fig. 7). Siliquobolellus prasankokae gen. et sp. nov. https://zoobank.org/E6927A6E-1532-4746-9679-439 1A 1F4C4C9 Figs: 2,'5;°6, 7 Material studied. Holotype. 1 male (CUMZ-D00148-1), Thailand, Lampang Province, Ngao District, Pha Thai; 18°36'01.05"N, 99°54'01.00"E; 350 m as.l.; 28 July 2020; P. Pimvichai, P. Prasankok and S. Saratan leg. Paratypes. 6 females (CUMZ-D00148-2); same data as holotype. Pimvichai, P. et al.: Anew genus of Pseudospirobolellidae Etymology. The species honors Associate Professor Dr. Pongpun Prasankok, biologist and devoted milli- pede collector. Diagnosis. Differing from other species in the genus by having the posterior gonopod apically with a short, sharp protrusion; externally differing by having a row of rect- angular dark brown spots middorsally on the body rings. Description. Adult male with 41 podous rings, 1 apodous ring. Length ~3 cm, diameter ~3 mm. Adult fe- males with 40 or 41 podous rings, 1 apodous ring. Length ~3 cm, diameter 3.2—3.6 mm. Color of living animal: collum and telson beige; an- tenna and leg light brown; dorsal part of body rings yel- lowish brown, with a row of rectangular dark brown spots middorsally on the body rings; head and lateral part of body rings dark brown (Fig. 21, 6C, D). Anterior gonopods (Fig. 5A, B, D, E) with high coxae, apically narrow, curving mesad, mesal margin sigmoid, posterior surface folding to cover more than half of te- lopodite, with strong ridge laterally. Telopodite with thick process, with strong ridge distomesally (Fig. 5B, arrow). Posterior gonopods (Fig. 5C, F—J) simple, rounded, with short, smooth coxal part (pcx); telopodital part (pt) twice the length of the pcx, lateral margin folding mesad, forming a deep concavity, the inner lateral margin ex- panded into a rounded lamella, apically forming a canopy (Fig. 5I, arrow), apically with a short, sharp protrusion (Fig. 5C, G, arrows). Female vulvae (Fig. 5K): simple, valves prominent, the right valve slightly larger than the left valve. DNA barcode. The GenBank accession number of the COI barcode of the paratype is OP 174623 (voucher code CUMZ-D00148). Habitat. Found under leaf litter and inside a rotten log. Distribution. Known only from the type locality in Lampang Province, Thailand (Fig. 7). Key to Pseudospirobolellidae genera (based on adult males) 1 Telopodital part of the posterior gonopod forming a deep concavity; telopodite of anterior gonopod not reaching tip of coxal part of anterior gonopod; tip of anterior gonopod coxa narrowed, curving mesad............. Siliquobolellus gen. nov. Telopodital part of the posterior gonopod simple, not forming a deep concavity; telopodite of anterior gonopod protrud- Ine above theta Ofecexal Parlier anteniGrCOnGOGGl! 2-2 n.d Mol Nadas Poet tees ld pS asses eel arid 2 The coxae of the 3 male leg-pair with extremely large, protruding PrOCESSES ...........ccccscceceeeccesnecneeessseseeseescceseeeeceesseess at re a A Os romans A Rb Ua abi Ba: 1 Wine OP a eLcen eee Coxobolellus Pimvichai, Enghoff, Panha & Backeljau, 2020 The coxa or stine S*-imalelespair without large PrOCESSeS so hscetn! oeceewen cd centinaweanl eee banslal clan) plamnengrndtebeedlaas pawsctanl nance S The posterior gonopod extremely slender, sickle-SNaped ................:cceceeeceeeeeeeeeeeeeeeeeeeeeaes Pseudospirobolellus Carl, 1912 Thesposterior eonopoea slender: apically with broad Canopy, 2 zsacestSet heed ieee lees nated pr szd Benoitolus Mauriés, 1980 Key to species of the genus Siliguobolellus gen. nov. (based on adult males) 1 Tip of anterior gonopod coxa crossing over with tip of opposite side (Fig. 3A), telopodite of anterior gonopod flattened, apically rounded »curViNne DACKWaArChGRI2: SE nE).. ich thstcetc cia emibs sndearit costenang since tcsmailetens S. amicusdraconis gen. et sp. nov. ~ Tip of anterior gonopod coxa separate from that of the opposite Side .......... Lec ceceeccecceesecceeeeeeceeteeececseesseeceecqecsensens 2 2 Posterior gonopod telopodite apically with a short, sharp protrusion (Fig. 5C, H), mesal margin of anterior gonopod Telopacite witha -StFONS CSS Hie OH Eye, ...x Mie diger anne nathlsts sued Para onau appar ee yn a ddabrecyacens S. prasankokae gen. et sp. nov. — Posterior gonopod telopodite apically without a protrusion, anterior gonopod coxae constricted at middle of lateral GaP ee Le St AD) OO ye, EL en creed eI. OUP ow ebiy on Weve oe Olea stierd yas FI) ie ean sue ieete Neem ra ETS S. constrictus gen et sp. nov. zse.pensoft.net Zoosyst. Evol. 98 (2) 2022, 313-326 323 Figure 5. Siliquobolellus prasankokae gen. et sp. nov., holotype, gonopods (specimen from Pha Thai, CUMZ-D00148-1). A. An- terior gonopod, anterior view; B. Anterior gonopod, posterior view, arrow indicates a a thumb-like process with strong ridge di- stomesally; C. Right posterior gonopod, lateral view, arrow indicates a short, sharp protrusion; D. Anterior gonopod, anterior view; E. Anterior gonopod, posterior view; F. Right posterior gonopod, lateral view; G. SEM, Right posterior gonopod, lateral view, arrow indicates a short, sharp protrusion; H. SEM, Mesal part of right posterior gonopod, lateral view; I. SEM, Right posterior gonopod, mesal view, arrow indicates a canopy; J. SEM, Tip of right posterior gonopod, mesal view; K. SEM, left female vulva, posterior mesal view. at = anterior gonopod telopodite; cx = coxa; oeg = opening of efferent groove; op = operculum of vulva; pcx = coxal part of the posterior gonopod telopodite; pt = telopodital part of the posterior gonopod telopodite; st = sternal process. zse.pensoft.net 324 Pimvichai, P. et al.: Anew genus of Pseudospirobolellidae Figure 6. Live Siliquobolellus gen. nov. species from Thailand. A, B. S. amicusdraconis gen. et sp. nov. (from Hub Pa Tard), female (paratype, CUMZ-D00149-2); C, D. S. prasankokae gen. et sp. nov. (from Pha Thai), male (holotype, CUMZ-D00148-1). e 1 = S. amicusdraconis gen. et sp. nov. 2 = 5S. constrictus gen. et sp. nov. 3 = S. prasankokae gen. et sp. nov. Figure 7. Geographic location of the type localities of Siliquobolellus gen. nov. species. zse.pensoft.net Zoosyst. Evol. 98 (2) 2022, 313-326 Discussion Morphologically, the three new species clearly belong to the Pseudospirobolellidae because they share the unique characters of the family viz., (1) prefemora of male legs with large ventral soft pads, (2) posterior gonopod simple and independent of each other, (3) small and slender species, (4) anterior gonopods sternum slightly fused with coxae or present only by a small inward pleat, and (5) tracheal apodeme is articulated at the base of the coxae (Brolemann 1913; Hoffman 1981). The placement in Pseudospirobolellidae is also suggested by the phylogenetic ML and BI trees, which show a well- supported COI grouping of the three new species with the genera Coxobolellus and Pseudospirobolellus. The creation of the new genus is based on three lines of evidence: (1) the three new species share three conspicuous gonopodal synapomorphies by which they consistently differ from the other pseudospirobolellid genera, viz. (1) the telopodital part of their posterior gonopod forms a deep concavity, (11) the telopodite of anterior gonopod is directed distad and does not reach the tip of coxal part of anterior gonopod, and (111) the tip of the anterior gonopod coxa is narrowed, curving mesad, (2) the three new species form a well-supported, rather deeply diverging clade with respect to the genera Coxobolellus and Pseudospirobolellus, and (3) yet, this clade reveals no well-supported sister group relationship to either of these two genera, suggesting that the three new species are phylogenetically not convincingly linked to Coxobolellus or Pseudospirobolellus. Therefore, it is appropriate to assign the three new species to their own, well-defined genus, viz. Si/iquobolellus gen. nov. While the new pseudospirobolellid genus seems to be well-founded, it does show a remarkably high mean interspecific COI sequence divergence of 12% (range: 8—15%). Yet, this appears to be a common phenomenon in millipedes, with reported mean interspecific COI divergences between congeneric species of, for example, 11% (range: 6-15%) in Coxobolellus (Pimvichai et al. 2020), 14% (range: 9-17%) in Atopochetus Attems, 1953 (Pimvichai et al. 2018), 11% (range: 9-11%) in Litostrophus Chamberlin, 1921 (Pimvichai et al. 2018), 15% (range: 15-16%) in Anurostreptus Attems, 1914 (Pimvichai et al. 2014), 14% (range: S—18%) in Thyropygus Pocock, 1894 (Pimvichai et al. 2014), and 12.9-15.9% in Glomeris Latreille, 1802 (Reip and Wesener 2018). Nevertheless, these high interspecific COI divergences between congeneric species are still substantially lower than many mean intergeneric COI divergences. This is well-illustrated by the four pseudospirobollelid genera whose mean intergeneric COI divergence is 20% (range: 14—24%) with Benoitolus, and 19% (range: 14-23%) without Benoitolus. The new genus and the three new species constitute an important expansion of the family Pseudospirobolel- lidae, but at the same time further expose the problem of the highly divergent position of Benoitolus birgitae within Pachybolidae, instead of Pseudospirobolellidae (Pimvichai et al. 2020, 2022). However, its long branch 325 and disruptive effect on the monophyly of Litostrophus means that the B. birgitae sequence may be suspected to reflect an artefact, caused by insufficient DNA and/or tax- on sampling. Hence future work on pseudospirobolellid phylogeny and taxonomy will have to take this point into account, the more so as new tentative pseudospirobolellid Species are lining up for description, including two unde- scribed species from China (Pitz and Sierwald 2010), and several Coxobolellus-like species from northern India (material in Natural History Museum of Denmark). Acknowledgements This research was funded by the Thailand Science Re- search and Innovation (TSRI) together with Mahasara- kham University as a TRF Research Career Development Grant (2019-2022; RSA6280051) (to P. Pimvichai). Ad- ditional funding was provided by the Royal Belgian Insti- tute of Natural Sciences. We thank Sathit Saratan (Sirind- horn Museum) for assistance in collecting material. We are indebted to Thita Krutchuen (College of Fine Arts, Bunditpatanasilpa Institute) for the excellent drawings. We thank Thomas Wesener (Zoological Research Muse- um Alexander Koenig, Bonn, Germany) for the critical comments and helpful discussions, which have substan- tially improved our manuscript. References Brélemann HW (1913) Un nouveau systeme de Spirobolides [Myriapoda. Diplopoda]. 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Molecular Biology and Evolution 35(6): 1550-1552. https://doi.org/10.1093/molbev/ msy073 Supplementary material | Table SI Authors: Piyatida Pimvichai, Henrik Enghoff, Somsak Panha, Thierry Backeljau Data type: excel file Explanation note: Estimates of COI sequence divergences within and among Siliquobolellus gen. nov. species and related taxa expressed as uncorrected p-distances (rounded off to two decimals). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zse.98.90032.suppl1