Zoosyst. Evol. 100 (3) 2024, 821-840 | DOI 10.3897/zse.100.121512 eee BERLIN Integrative taxonomy of Dicellophilus Cook, 1896 (Chilopoda, Geophilomorpha, Mecistocephalidae) in Japan, with a description of a new species Sho Tsukamoto!, Katsuyuki Eguchi! 1 Systematic Zoology Laboratory, Graduate School of Science, Tokyo Metropolitan University, Minami-osawa 1-1 Hachioji-shi, Tokyo 192-0397, Japan 2 Department of International Health and Medical Anthropology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan https://zoobank. org/BOAF 778 B-E394-4C17-910D-FA2BDFB14A4F Corresponding author: Sho Tsukamoto (esutukamotol 53@gmail.com) Academic editor: Pavel Stoev # Received 23 February 2024 # Accepted 7 May 2024 Published 19 June 2024 Abstract The genus Dicellophilus Cook, 1896, is a peculiar genus from the point of view of distribution. Dice/lophilus is distributed in three limited areas that are well separated from one another: central Europe (D. carniolensis), Honshu (D. pulcher), and the southwest- ern part of the USA (D. anomalus and D. limatus). In the present study, in a field survey conducted throughout Japan, specimens belonging to the genus Dice/lophilus were collected from Tohoku to the Kansai region, Honshu. Morphological analysis, molec- ular phylogenetic analysis, and genetic distance among Dicellophilus in Japan and D. carniolensis revealed that specimens from Sendai-shi, Miyagi Pref., could be assigned to an undescribed species. This previously unrecognized species is herein described as D. praetermissus sp. nov. The new species can be distinguished from D. carniolensis and D. limatus by the number of pairs of legs (43 pairs in D. carniolensis and 45 in D. limatus, but 41 in D. praetermissus sp. nov.), from D. anomalus by the lack of a pair of setae on the posteromedian part of the clypeus and variable crenulation on the internal margin of the forcipular tarsungulum, and from D. pulcher based on the following combination of characteristics: both ends of the transverse suture not evidently convex forward; long rather than wide trochanteroprefemur; wide rather than long metasternite. Key Words Cryptic species, DNA barcoding, geophilomorph centipede, molecular, phylogeny Introduction Mecistocephalidae are morphologically characterized by a cephalic capsule and a forcipular segment that are The geophilomorph family Mecistocephalidae Bollman, 1893, is mainly distributed from temperate to tropical regions in both hemispheres, and species diversity is re- markably high in Japan (Uliana et al. 2007; Bonato 2011). To date, approximately 180 species are known world- wide, and 31 species have been recorded from Japan (Uli- ana et al. 2007; Tsukamoto et al. 2019, 2022). Therefore, approximately 20% of all known mecistocephalid species are distributed in Japan. Moreover, Japan is the richest with regard to the number of genera (nine out of 11 gen- era; Uliana et al. 2007; Tsukamoto et al. 2022). evidently sclerotized and darker than the remaining trunk segments (Bonato et al. 2003, 2014; Uliana et al. 2007; Bonato 2011). In addition, the following three features characterize Mecistocephalidae: a mandible with a series of pectinate lamellae only; trunk sternites with an inter- nal apodeme; and a mid-longitudinal sulcus (Bonato et al. 2003). Notably, the segment number of most species of Mecistocephalidae has no intraspecific variation, except for some species of the genus Mecistocephalus Newport, 1843, with a very high number of leg-bearing segments (Bonato et al. 2003, 2014; Uliana et al. 2007; Bonato 2011). Copyright Tsukamoto, S. & Eguchi, K. 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. 822 Among Mecistocephalidae, the genus Dicellophilus Cook, 1896, is a distinct genus from a morphological viewpoint, with the following diagnostic characteristics: a macropore near the center of the coxopleuron and a concave margin of the lateral side pieces of the labrum (Uliana et al. 2007; Bonato et al. 2010a). See table 2 of Bonato et al. (2010a) and table 1 of Dyachkov and Bon- ato (2022) for diagnostic characteristics of the genus Di- cellophilus. To date, four valid species have been known in the genus Dicellophilus, namely, D. carniolensis (C.L. Koch, 1847), D. pulcher (Kishida, 1928), D. anomalus (Chamberlin, 1904), and D. /imatus (Wood, 1862). The distribution of Dicellophilus is limited to three areas that are well separated from one another: central Europe (D. carniolensis), Honshu of Japan (D. pulcher), and the southwestern part of the USA (D. anomalus and D. limatus) (Bonato et al. 2003, 2010a). All three distri- bution areas are located within a narrow latitudinal band, at approximately 35—45°N, and all species inhabit humid litter and soil in forests (Bonato et al. 2010a). Bonato et al. (2010a) performed phylogenetic analyses of the genus Dicellophilus with a morphological dataset consisting of 30 characteristics and indicated that the two American species, viz., D. anomalus and D. limatus, were sister species, and D. pulcher formed a clade with the two American species. As far as extant species are concerned, D. carniolensis is thus the first diverged species in the genus Dicellophilus. For a decade, the combination of morphological ob- servation, molecular phylogenetic analyses, and DNA barcoding (“integrative taxonomy”) has helped detect undescribed species and reveal the genetic structure of the taxa concerned (Dayrat 2005; Padial et al. 2010). For example, some studies using integrative approaches to scolopendromorphs and geophilomorphs have revealed the existence of many cryptic species under one valid- ly named species or distinct morphospecies (Joshi and Karanth 2012; Sirtwut et al. 2015, 2016; Tsukamoto et al. 2021b; Peretti et al. 2022; Bonato et al. 2023). Tsukamoto et al. (2022) also detected the existence of two species of the mecistocephalid genus Nannarrup Foddai, Bonato, Pereira & Minelli, 2003 by using an integrative approach. Inspired by these previous studies, the present study aims to reveal the genetic diversity and confirm wheth- er the morphospecies D. pulcher involves unnoticed and undescribed cryptic species by using an integra- tive taxonomic approach. Materials and methods Taxon sampling Although our ongoing sampling efforts to taxonomically reveal the East Asian mecistocephalid faunas cover the whole of Japan and surrounding areas, the present study focused on Honshu (the largest island of mainland Japan), from which D. pulcher was described. zse.pensoft.net Tsukamoto, S. & Eguchi, K.: Integrative taxonomy of Dicellophilus in Japan A total of 38 specimens morphologically identified as Dicellophilus pulcher, hitherto the only known Japa- nese species of the genus, were collected from Honshu from 2018 to 2021. The detailed collection sites of the examined specimens are shown in Fig. 1, Table 1, and the "Taxonomic account" section. The altitude data provided by AW3D of JAXA (https://www.eorc.jaxa.jp/ALOS/jp/ index _j.htm) and the coastal line provided by the digi- tal nation land information (https://nlftp.mlit.go.jp/index. html) were used to generate Fig. 1. Each specimen was labeled with its unique speci- men identification number in the form “TSYYYYM- MDD-XX,” where TS is an abbreviation of the first au- thor’s name, Tsukamoto Sho; YYYYMMDD designates the date on which the specimen was collected; and XX is the identification number assigned to each specimen col- lected on a particular date (e.g., TS20171010-01). All of the type specimens of Dicellophilus designated in this paper were deposited at the Collection of Myriapo- da, Department of Zoology, National Museum of Nature and Science, Tokyo (NSMT), and the Museum of Nature and Human Activities, Hyogo (MNHAH). The deposition site of each type specimen is shown in the “Taxonom- ic account” section. All non-type voucher specimens of Dicellophilus pulcher are managed by the first author. Morphological examination For dissected specimens, the cephalic capsule, maxillae, mandibles, forcipular segment, and leg-bearing segments were made transparent using lactic acid to examine the anatomy and produce images. Multi-focused images of these body parts were produced using Affinity Photo 1.10.4 (https://affinity.serif.com/ja-jp/photo/) from a se- ries of source images taken using a Canon EOS Kiss X9 digital camera attached to a Nikon AZ100 microscope and improved using Adobe Photoshop Elements 10 and Affinity Designer 1.10.5 (https://affinity.serif.com/ja-jp/ designer/). Then, the body parts were measured directly using an ocular micrometer attached to the microscope. The morphological terminology used in this study is in accordance with Bonato et al. (2010b). Specimens with fully developed paired gonopods, that 1s, evidently biar- ticulated in males and touching one another in females, were determined to be adults, and those with incomplete- ly developed paired gonopods were determined to be sub- adults. Specimens without gonopods were determined to be juveniles based on Uliana et al. (2007). In the present study, 23 adult specimens out of 38 collected were exam- ined morphologically. DNA sequencing Genomic DNA was extracted from one or two legs of each specimen in accordance with the Chelex-TE-ProK protocol described by Satria et al. (2015), with incubation for 4—24 h. Zoosyst. Evol. 100 (3) 2024, 821-840 130.0 135.0 e723 0 100 200 300 400km 140.0 Figure 1. Map of the collection sites of specimens examined in the present study. White circle, Dicellophilus pulcher, black circle, D. praetermissus sp. nov. PCR amplification was performed in a MiniAmp Ther- mal Cycler (Thermo Fisher Scientific, Waltham, Massa- chusetts, USA) in a 10.5-uL reaction volume containing 5 wL of 2x PCR buffer for KOD FX Neo, 2 wL of 2 mM dNTPs, 0.3 wh of 10 pmol/uL forward and reverse prim- ers, 0.2 uL of 1.0 U/uL DNA polymerase KOD FX Neo (TOYOBO KFX-201X5), and 1.0 uL of DNA template. The sequences of primers for mitochondrial cytochrome c oxidase subunit I (CO/), 16S rRNA (J6S), and nuclear 28S rRNA (28S) genes are shown in Table 2. Each PCR product was screened by electrophoresis on a 2.0% aga- rose gel in 1x TAE. The amplification conditions for mitochondrial CO/ were as follows: 98 °C for 2 min; 5 cycles of 98 °C for 10 s; 45 °C for 30 s; and 68 °C for 45 s; 40 cycles of 98 °C for 10 s; 48.5 °C for 30 s (annealing step); 68 °C for 45 s; and 68 °C for 7 min. Ifthe target fragment of CO/ was not appropriate- ly amplified, then the annealing temperature was changed from 48.5 °C to 50 °C. PCR was performed again by omit- ting the first five cycles of annealing and the extension step. The amplification conditions for mitochondrial /6S were as follows: 98 °C for 2 min; 35 cycles of 98 °C for 10 s; 45 °C for 30 s (annealing step); 68 °C for 45 s; and 68 °C for 7 min. If the target fragment of /6S was not ap- propriately amplified, then the annealing temperature was changed from 45 °C to 48 °C. The number of annealing cycles was changed from 35 to 45. The amplification conditions for nuclear 28S were as follows: 98 °C for 2 min; 5 cycles of 98 °C for 10 s; 42 °C for 30 s; and 68 °C for 1 min; 30 cycles of 98 °C for 10 s; 50 °C for 30 s (annealing step); 68 °C for 1 min; and 68 °C for 7 min. If the target fragment of 25S was not appropriately amplified, then the annealing temperature was changed from 50 °C to 48 °C. The number of an- nealing cycles was changed from 30 to 40-45 cycles. In addition, PCR was performed again by omitting the first five cycles of annealing and the extension step. The amplified products were incubated at 37 °C for 4 min and at 80 °C for 1 min using ExoSAP-IT™ Ex- press (Thermo Fisher Scientific) to remove any excess primers and nucleotides. All nucleotide sequences were determined by direct sequencing using the ABI PRISM BigDye™ Terminator Cycle Sequencing Kit ver. 3.1 (Thermo Fisher Scientific) or BrilliantDye™ Terminator Cycle Sequencing Kit v. 3.1 (Nimagen, B.V., Nijmegen, Netherlands) equipped with an ABI 3130xI automated se- quencer (Thermo Fisher Scientific). The sequences were assembled using ChromasPro 1.7.6 (Technelysium Pty Ltd., Australia) and deposited onto the DDBJ, EMBL, and GenBank databases under the accession numbers LC815125— LC815233 (Table 1). Molecular phylogenetic analyses The sequences obtained using the abovementioned meth- ods were used for phylogenetic analyses, together with the COI, 16S, and 28S sequences of Dicellophilus carnio- lensis (C.L. Koch, 1847) and Nannarrup innuptus Tsuka- moto in Tsukamoto et al. (2022) obtained from GenBank as outgroups (Table 1). The datasets for CO/ (658 bp po- sitions), /6S (514 bp positions), and 28S (971 bp posi- tions) were concatenated to form the CO/ + 16S + 28S dataset for phylogenetic analyses. zse.pensoft.net 824 Tsukamoto, S. & Eguchi, K.: Integrative taxonomy of Dicellophilus in Japan Table 1. The list of specimens that were used in the phylogenetic analyses. Geographic coordinates enclosed by parentheses are secondary due to the lack of information in the labels. Specimen ID Geographic coordinate of collection sites Dicellophilus pulcher (Kishida, 1928) TS20220518-01 35°16.45'N, 137°00.59'E (Aichi Pref.) TS20210809-01 34°33.71'N, 136°27.33'E (Mie Pref.) TS20210809-02 34°33.71'N, 136°27.33'E (Mie Pref.) TS20220813-01 34°33.42'N, 136°24.13'E (Mie Pref.) TS20230517-01 35°16.38'N, 137°00.96'E (Aichi Pref.) TS20190413-01 35°25.15'N, 139°10.39'E (Kanagawa Pref.) TS20181214-01 35°37.02'N, 139°22.74'E (Tokyo Pref.) TS20180714-01 35°27.46'N, 139°24.51'E (Kanagawa Pref.) TS20191006-02 35°38.15'N, 139°15.71'E (Tokyo Pref.) TS20200924-01 35°52.42'N, 138°25.96'E (Yamanashi Pref.) TS20200927-01 35°54.95'N, 138°20.55'E (Yamanashi Pref.) T$20201122-01 (35°25.62'N, 135°23.69'E) (Kyoto Pref.) TS20210322-01 35°15.40'N, 139°44.43'E (Kanagawa Pref.) TS20210401-03 35°44.46'N, 139°31.01'E (Tokyo Pref.) TS20210411-08 35°26.84'N, 139°02.82'E (Kanagawa Pref.) TS20210424-07 35°25.88'N, 139°14.15'E (Kanagawa Pref.) TS20210504-01 35°21.91'N, 138°48.44'E (Shizuoka Pref.) TS20210418-01 34°34.94'N, 137°02.13'E (Aichi Pref.) TS20210522-01 35°44.78'N, 138°53.15'E (Yamanashi Pref.) TS20210530-02 35°41.19'N, 138°52.64'E (Yamanashi Pref.) T$20210722-01 35°57.71'N, 139°03.60'E (Saitama Pref.) TS20211004-01 (36°08.54'N, 138°16.92'E) (Nagano Pref.) TS20211030-04 36°51.75'N, 138°46.43'E (Niigata Pref.) TS20211018-02 35°26.99'N, 136°49.95'E (Gifu Pref.) TS20210919-01 34°46.18'N, 135°59.73'E (Kyoto Pref.) TS20210711-15 35°17.08'N, 136°32.74'E (Gifu Pref.) TS20210819-01 35°36.51'N, 139°23.48'E (Tokyo Pref.) TS20210523-01 35°27.82'N, 138°29.66'E (Yamanashi Pref.) T$20210531-01 37°05.59'N, 140°31.72'E (Fukushima Pref.) TS20210523-03 35°11.94'N, 138°31.29'E (Shizuoka Pref.) TS20210718-01 35°56.36'N, 139°15.30'E (Saitama Pref.) TS20210508-02 35°19.27'N, 139°18.72'E (Kanagawa Pref.) TS20210531-02 37°05.59'N, 140°31.72'E (Fukushima Pref.) TS20210728-01 35°14.75'N, 139°01.02'E (Kanagawa Pref.) TS20210728-02 35°13.32'N, 139°03.13'E (Kanagawa Pref.) Dicellophilus praetermissus sp. nov. TS20201007-02 38°16.33'N, 140°32.69'E (Miyagi Pref.) TS20201007-03 38°16.33'N, 140°32.69'E (Miyagi Pref.) TS20201007-04 38°16.33'N, 140°32.69'E (Miyagi Pref.) TS20201007-05 38°16.33'N, 140°32.69'E (Miyagi Pref.) Dicellophilus carniolensis (Koch, 1847) (outgroup) DNA102580/LBv7/92 No data Nannarrup innuptus Tsukamoto in Tsukamoto et al. 2022 (outgroup) TS20210503-09 34°51.39'N, 138°55.40'E (Shizuoka Pref.) Sequence data (Accession no.) Remarks col 16S 28S LCS15129= LCSI5167~ (LE815205 Tomoki Sumino leg. LC815130 LC815168 LC815206 Tomoki Sumino & Fukube Sumino leg. LC815131 LC815169 LC815207 Tomoki Sumino & Fukube Sumino leg. LC815132 LC815170 LC815208 Tomoki Sumino leg. LC815133~ LC8i517/l ~LC815209 Tomoki Sumino leg. LCS15134 -Lesl5172> »£CS15210 Sho Tsukamoto leg. LCSlS135, LEST5173 - Joe Kutsukake leg. LC815136 LCSL5174 - Joe Kutsukake leg. LGS15737. «LGS15175 - Sho Tsukamoto leg. LC815138 - - Joe Kutsukake leg. EGSIS139° \LCBL5176 - Koshi Kawamoto leg. LC815140 LC815177 LC815211 Tatsumi Suguro leg. LC815141 LC815178 - Ryo Miyata leg. LC815142 LC815179 \LC815212 Mayu Susukida leg. LCs15143: , LGst51e0 « "LC815213 Namiki Kikuchi leg. LC815144 LC815181 LC815214 Katsuyuki Eguchi leg. LC815145 LC815182 LC815215 A topotype of D. pulcher,; Sho Tsukamoto leg. LCS151462 LCSi5183. -LC815216 Katsuyuki Eguchi leg. LC815147 + =LC815184 LC815217 Takahiro Yoshida leg. LC815148 LC815185 LC815218 Namiki Kikuchi leg. LCS15149.. LOSl51s6. LE8L5219 Mayu Susukida leg. LC815150") \LC815187 -LG815220 Masaru Nonaka leg. LC815151 + LC815188 LC815221 Katsuyuki Eguchi leg. EG6T5152~ \LCB15189" 1 Les1 5222 Katsuyuki Eguchi leg. Les S153. 'ECSLS190-7 1 LG3l 5223 Katsuyuki Eguchi leg. LC815154 LC815191 LC815224 Katsuyuki Eguchi leg. EGS15155re: LCST5192. LE8l5225 Katsuyuki Eguchi leg. ECS15156=, EC815193° 'LC815226 Katsuyuki Eguchi leg. LC815157 =LC815194 LC815227 Katsuyuki Eguchi leg. - LC815195 LC815228 Katsuyuki Eguchi leg. LC815158 LC815196 LC815229 Katsuyuki Eguchi leg. LC815159 LC815197 LC815230 Katsuyuki Eguchi leg. LCS815160 7" LeSi5198" §L6815231 Katsuyuki Eguchi leg. EGSISI61. LOSi5199" 'bGS15232 Sho Tsukamoto leg. Leésl5162 LC8l5200° »LC815233 Sho Tsukamoto leg. LCST5125:> LEShS163. se LESh520r Sho Tsukamoto leg. [CS15126° -LOS8L5164. LG815202 Sho Tsukamoto leg. LC815127°.kesl5165. "LC615203 Sho Tsukamoto leg. LC815128 LC815166 LC815204 Sho Tsukamoto leg. KF569305 HM453225 HM453285 _ Referred from Murienne et al. (2010), Bonato et al. (2014) LC715530 LC715605 LC715680 _ Referred from Tsukamoto et al. (2022) All sequences were aligned using MAFFT v. 7.475 (Katoh and Standley 2013). For COJ, alignment was performed using the default setting. For /6S and 28S, secondary structure alignment was performed using the X-INS-i option. zse.pensoft.net Maximum-likelihood (ML) trees were created on the basis of the sequence dataset for each gene and concat- enated using [Q-tree 1.6.12 (Nguyen et al. 2015). As an optimal substitution model in accordance with BIC, TNe + 1 + G4 was selected for the first codon position Zoosyst. Evol. 100 (3) 2024, 821-840 Table 2. The list of primers used in the present study. 825 Genes Primer name Sequence (5’ - 3’) Source Col LCO-CH TTT CAA CAA AYC AYA AAG ACA TYG G Tsukamoto et al. (2021a) HCO-CH TAA ACT TCT GGR TGR CCR AAR AAT CA 16S rRNA 16Sa CGC CTG TTT ATC AAA AAC AT Xiong and Kocher (1991) 16Sbi CTC CGG TTT GAA CTC AGA TCA 28S rRNA 28S D1F GGG ACT ACC CCC TGA ATT TAA GCA T Boyer and Giribet (2007) 28S rD4b CCT TGG TCC GTG TTT CAA GAC Edgecombe and Giribet (2006) of CO/ in the concatenated dataset; TNe + G4 for the COI dataset; HK Y + F was selected for the second co- don position of CO/ in both datasets; TN + F + G4 was selected for the third codon position of CO/ in both datasets; HKY + F + I + G4 was selected for /6S of both datasets; and TIM3e + G4 was selected for 28S of both datasets. Ultrafast bootstrap analysis (UFBoot; Hoang et al. 2018) and the SH-like approximate like- lihood ratio test (SH-aLRT; Guindon et al. 2010) were performed with 1,000 replicates. Bayesian inference trees were created using ExaBa- yes 1.4.1 (Aberer et al. 2014) under the default sub- stitution model “GTR + G.” The Markov chain Mon- te Carlo method was used with random starting trees and performed once for each of the four chains (three hot and one cold) for 10,000,000 generations for each dataset except CO/, but for 20,000,000 generations for the CO/ dataset. Trees were sampled every 500 gener- ations, tuning parameters every 100 generations, and the first 25% of the trees were discarded as burn-in. Other parameters were set in accordance with the de- fault settings. The effective sampling size of each pa- rameter was confirmed to be 200 using Tracer 1.7.1 (Rambaut et al. 2018). Calculation of the genetic distances The aligned CO/ dataset used for phylogenetic anal- yses were also used to calculate genetic distances. Kimura two-parameter (K2P) distances were calculat- ed using MEGA X (Kumar et al. 2018) with the setting “pairwise deletion.” Delimitation of “provisional” operational taxonomic units The program “assemble species by automatic partitioning (ASAP)” was used to delimit “provisional” operational taxonomic units (POTUs). ASAP is a species delimita- tion program based on a hierarchical clustering algorithm that only uses pairwise genetic distances (Puillandre et al. 2021; available at https://bioinfo.mnhn. fr/abi/public/ asap/). ASAP was performed for the CO/ sequence data- set of Dicellophilus (excluding the outgroup) under the “pairwise K2P distance” method. Delimitation of putative species and provisional naming of each putative species The present study preliminarily relied on the morpho- logical information provided by Bonato et al. (2010a) as the basis for the monophyly of the genus Dicellophilus. Then, putative species were proposed. Except for the as- sumption of monophyly of the abovementioned genus, the following steps generally followed the workflow “DI-system” proposed by the first author in Tsukamoto (2023) for discriminating and labeling putative species: (1) sorting specimens, which are morphologically confer- rable to Dicellophilus, into morphospecies; (II) confirm- ing the monophyly of the morphospecies with the phy- logenetic tree inferred by the sequence dataset of three gene markers, mitochondrial CO/ and /6S, and nuclear 28S, (Ill) calculating the genetic distance of COI be- tween congeneric morphospecies, which are confirmed to be monophyletic, to define the intermorphospecif- ic threshold; (IV) delimiting POTUs using ASAP (see above) and confirming the most conferrable hypotheses of species-level independence by considering the phy- logenetic tree and the intermorphospecific threshold de- fined in step III. By steps HI and IV, species hypotheses can be established from two viewpoints, viz., phylogeny and clustering based on DNA data. In step IV, putative Species were recognized by considering three species delimitation principles: (1) each clade is regarded as an independent putative species if it diverges from all oth- ers with a minimum K2P distance higher than the inter- morphospecific threshold; (2) a single putative species that satisfies (1) can contain inner lineage(s) diverging extremely from the others unless the maximum distance from the sister inner lineage exceeds the intermorphospe- cific threshold; (3) a single putative species that satisfies (2) cannot contain inner lineage(s) diverging extremely from the sister inner lineage with the minimum K2P dis- tance exceeding the intermorphospecific threshold, and such a lineage must be further considered as a distinct species if it exists. As mentioned above, the present study presupposes the monophyly of Dicellophilus, supported by the morpho- logical evidence (Bonato et al. 2010a). This is because the possibility of a difference in evolutionary rate among genera is important for defining the intermorphospecific threshold in the “DI-system”. “DI-system” is planned to be proposed formally in future studies. zse.pensoft.net 826 Each putative species recognized by following the abovementioned steps was also labeled in accordance with the study of Tsukamoto (2023), with a unique, per- manent, and citable identifier “DI,” such as “OO00-0003- 3020-8454 XXXX,” in which “O000-0003-3020-8454” shows the author’s ORCID and “XX XX” shows a unique identification number given to each species in the au- thor’s life-long research. ORCIDs involved in the species identification codes were omitted except for section titles, figure legends, and tables to avoid redundancy. Depending on the availability of the morphological in- formation necessary to formally describe and name spe- cies in the conventional manner of Linnaean Taxonomy (The International Commission on Zoological Nomen- clature 1999), the putative species labeled with the DI can be described and named (step V). In the present study, Species discrimination (steps I-IV) and formal descrip- tion and naming of the species (step V) were separated as two methodologically distinct phases. 99.3/99/* 1 S20 -/95/- = te OT. 21997 96.9/98/*. -/95/* -/98/0.99 tapeyf -/85/0.98 -/83/0/97 So Bis -/90/0.98 je a 20 [ae a 95.4/98/* 97.7/99/0.99 -/90/- -/92/0.98 99.8/*/0.99 -/89/- -/94/0.99 a hed ha Nannarrup innuptus 0.09 7 J * Dicellophilus carniolensis TS20201007-04 My pj, */*/* |TS20201007-05 My TS20201007-02 My (D.s -/88/1T$20201007-03 My Tsukamoto, S. & Eguchi, K.: Integrative taxonomy of Dicellophilus in Japan Results Molecular phylogenetic analyses COI was successfully sequenced for 38 specimens, /6S for 38 specimens, and 28S for 33 specimens (Table 1). The ML tree based on the concatenated dataset (Fig. 2) and BI tree based on the same dataset (only show the posterior probability in Fig. 2) involving 39 specimens shows that the clade consisting of TS20201007-02, TS20201007-03, TS20201007-04, and TS20201007-05 from Miyagi Pref. (UFBoot = 100%, SH-aLRT = 100%, posterior probability (PP) = 1.00; hereafter referred to as “Clade A”) is deeply separated from the clade consisting of all other Dice/lophi- /us specimens collected in Japan and the European species D. carniolensis (UFBoot = 95.4%, SH-aLRT = 98%, PP = 1.00). The monophyly of the clade, which consists of the remaining Dicellophilus specimens from Japan, was strongly supported (UFBoot = 100%, SH-aLRT = 100%, 27 18 19 28 24 3.04.04.06.0 7.0 No. of POTUs ASAP score Figure 2. Maximum-likelihood tree of Dice/lophilus based on the concatenated dataset of CO/, 16S, and 28S, with the results of species delimitation by ASAP. Note that specimens whose CO/ sequence was not determined were included in the conferred species if they belonged to the same concerning clade to easily understand the result of species delimitation. Nodal values are obtained from the ultrafast bootstrap (UFBoot), SH-like approximate likelihood ratio test (SH-aLRT), and posterior probability (PP). The asterisk (*) indicates 100% in UFBoot, SH-aLRT, and 1.0 in PP. Hyphen (-) indicates lower than 95% in UFBoot, 80% in SH-aLRT, or 0.95 in PP. Nodal values are not shown when UFBoot, SH-aLRT, and PP values are <95%, <80%, and <0.95, respectively. The unit of evolutionary distance is the number of base substitutions per site. A broken square shows that the clade consisted of specimens from eastern Honshu. Abbreviations: Ai = Aichi Pref.; Fs = Fukushima Pref.; Gi = Gifu Pref.; Kn = Kanagawa Pref.; Ky = Kyoto Pref.; Mi = Mie Pref.; My = Miyagi Pref.; Ni = Niigata Pref.; Nn = Nagano Pref.; Sh = Shizuoka Pref.; Si = Saitama Pref.; To = Tokyo Pref.; Yn = Yamanashi Pref. zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 821-840 PP = 1.00; hereafter “Clade B”), and notably, specimens from Eastern Honshu (Fukushima Pref. to Shizuika Pref., and one specimen from Gifu Pref.) formed a clade with a high support value (UFBoot = 100%, SH-aLRT = 85%, PP = 0.91; enclosed by a broken square in Fig. 2). On the con- trary, the monophyly of specimens from western Honshu (Gifu Pref. to Kyoto Pref.) was not supported. The ML tree based on the CO/ dataset (Fig. 3) and the BI tree based on the same dataset (only PP shown in Fig. 3) involving 38 specimens also show Clade A (UFBoot = 99.6%, SH-aLRT = 100%, PP = 1.00). Although Clade A forms a further clade with D. carniolensis (UFBoot = 99.6%, SH-aLRT = 100%, PP = 1.00), Clade A is deep- ly separated from clade B (UFBoot = 90.8%, SH-aLRT = 95%, PP = 1.00). In addition, the monophyly of spec- imens from western Honshu in Clade B (Gifu Pref. to Kyoto Pref.) was moderately supported (UFBoot = 88.2%, SH-aLRT = 88%, PP = 0.98; enclosed by a broken square in Fig. 3), but that of eastern Honshu was not supported. 98.7/*/* T igor 1 S20220518-0 Ai -/85/- TS2023051 7-0 TS20211018-0 TS20201122-01 Ky 95.1/99~ 8° 84 TS20210809-01 Mi = -/85/- la TS20210809-02 Mi TS20220813-01 Mi ia T$20190413-01 K a TS20210411-08 Kn Be TS20210728-01 Kn TS20210728-02 Kn ae [| TS20210504-01 Sh aan al || TS20180714-01 ; | —}-1'}1S20210424-07 Kn 1930.97 +] 'T$20210508-02 Kn “196!- IL TS20181214-0 F-}-1S20191006-0 me, elle TS$20210401-0 ea (eae TS$20210819-0 -/86/- ||| TS20210522-0 sv.2rr——t—_ TT | 'T$20210530-0 or ae ||| “1S20210718-01 TS20210722-01 rr) TS20200924 TS20200927 96.6/99/* -/90/- — | |-7S20210711-15 TS202105 ase TS20210322- yea a TS20211004 99.9r"r" eee TS20211030- 1 TS20210531 TS20210418-01 Ai 827 The ML tree based on the /6S dataset (Fig. 4) and the BI tree based on the same dataset (only PP shown in Fig. 4) involving 38 specimens also show Clade A (UF- Boot = 100%, SH-aLRT = 100%, PP = 1.00). Clade A is deeply separated from all other Dicellophilus specimens, but Clade B is not well supported (UFBoot = 78.2%, SH-aLRT = 63%, PP = 0.91). In addition, the phyloge- netic relationship among Clade B and other Dicellophilus Specimens was not clear due to low support values. The ML tree based on the 28S dataset (Fig. 5) and the BI tree based on the same dataset (only PP shown in Fig. 5) involving 33 specimens also show Clade A (UFBoot = 99.7%, SH-aLRT = 100%, PP = 1.00). Clade A is deep- ly separated from all other Dicellophilus specimens, and Clade B conforms to a further clade with D. carniolensis, like the topology of the concatenated dataset (UFBoot = 93.6%, SH-aLRT = 97%, PP = 0.99). However, the phy- logenetic relationship among Clade B and other Dicello- philus specimens was not clear due to low support values. 27 18 19 28 24 3.04.04.06.0 7.0 No. of POTUs ASAP score Dicellophil rniolensis -/9410.98 is TS202010( 99.6/*/" |TS2020101 TS2020100 Seas TS20201007 Nannarrup innuptus 0.2 Figure 3. Maximum-likelihood tree of Dice/lophilus based on the dataset of CO/, with the results of species delimitation by ASAP. Nodal values are obtained from the ultrafast bootstrap (UFBoot), SH-like approximate likelihood ratio test (SH-aLRT), and poste- rior probability (PP). The asterisk (*) indicates 100% in UFBoot, SH-aLRT, and 1.0 in PP. Hyphen (-) indicates lower than 95% in UFBoot, 80% in SH-aLRT, or 0.95 in PP. Nodal values are not shown when UFBoot, SH-aLRT, and PP values are <95%, <80%, and <0.95, respectively. The unit of evolutionary distance is the number of base substitutions per site. A broken square shows that the clade consisted of specimens from eastern Honshu. Abbreviations: Ai = Aichi Pref.; Fs = Fukushima Pref.; Gi = Gifu Pref.; Kn = Kanagawa Pref.; Ky = Kyoto Pref.; Mi = Mie Pref.; My = Miyagi Pref.; Ni = Niigata Pref.; Nn = Nagano Pref.; Sh = Shizuoka Pref.; Si= Saitama Pref.; To = Tokyo Pref.; Yn = Yamanashi Pref. zse.pensoft.net 828 Tsukamoto, S. & Eguchi, K.: Integrative taxonomy of Dicellophilus in Japan eh TS20211004-01 1 "I TS20210531-( TS20210531- TS202011 TS202104 TS20211018- lt TS20220518- TS2023051 TS2021091 TS2022081 97.8/88/0.99 98.4/*/* -/97/0.99 96.2/99/* Shae -/88/- 95.1/-/- -/80/- -/83/- -/98/0.97- 95.3/*/* -/97/- St 2a ae ee -/99/* Dicellophilus carniolensis 7 7 Ho op) pe) jo) NO a je) (ec) (jo) TS2021080 TS2018121 TS2021081 TS2021040 TS20210522-C Cn eS Es a TS2021072 TS2021072 TS2019041 44 Ox) NON (exe) NON —_— —_ (exe) om ON Nannarrup innuptus 0.4 Figure 4. Maximum-likelihood tree of Dicellophilus based on the dataset of 16S. Nodal values are obtained from the ultrafast boot- strap (UFBoot), SH-like approximate likelihood ratio test (SH-aLRT), and posterior probability (PP). The asterisk (*) indicates 100% in UFBoot, SH-aLRT, and 1.0 in PP. Hyphen (-) indicates lower than 95% in UFBoot, 80% in SH-aLRT, or 0.95 in PP. Nodal values are not shown when UFBoot, SH-aLRT, and PP values are <95%, <80%, and <0.95, respectively. The unit of evolutionary distance is the number of base substitutions per site. A broken square shows that the clade consisted of specimens from eastern Honshu. Ab- breviations: Ai = Aichi Pref.; Fs = Fukushima Pref.; Gi = Gifu Pref.; Kn = Kanagawa Pref.; Ky = Kyoto Pref.; Mi = Mie Pref.; My = Miyagi Pref.; Ni = Niigata Pref.; Nn = Nagano Pref.; Sh = Shizuoka Pref.; Si= Saitama Pref.; To = Tokyo Pref.; Yn = Yamanashi Pref. Although there is no consistency of phylogenetic rela- tionship among Clades A, B, and D. carniolensis in four datasets, each topology shows that Clade A is a distinct lineage from other Dicellophilus specimens. Intermorphospecific threshold and POTU delimitation of Dicellophilus specimens The minimum K2P distance between congeneric morphos- pecies was 21% (D. carniolensis (accession no.: KF569305) vs. D. pulcher TS20230517-01 from Aichi Pref.). Thus, the intermorphospecific threshold induced by this dataset is 21%. However, the maximum K2P distance was 24% with- in D. pulcher (TS20191006-02 from Tokyo Pref. in Clade B vs. TS20201007-04 from Miyagi Pref. in Clade A). The maximum K2P distance within Clade A was 0.6% (TS20201007-04 vs. TS20201007-02 and TS20201007-03), and that within Clade B was 15% (TS20210322-01 from Kanagawa Pref. vs. TS20210523-01 from Yamanashi Pref. ). zse.pensoft.net The best five partitioning hypotheses inferred by the ASAP program are shown in Figs 2, 3, with the following ASAP scores: (1) 27 POTUs with a score of 3.0; (2) 18 POTUs with a score of 4.0; (3) 19 POTUs with a score of 4.0; (4) 28 POTUs with a score of 6.0; and (5) 24 POTUs with a score of 7.0. Morphological examination of Japanese Dicellophilus All 38 specimens of D. pulcher (a combination of Clades A and B) examined in steps I-IV have 41 pairs of legs and can be distinguished from D. carniolensis and D. limatus by the number of pairs of legs (43 pairs in D. carniolensis and 45 in D. limatus). Examined 23 adult specimens can also be distinguished from D. anomalus, which has 41 pairs of legs, by the lack of a pair of setae on the posteromedian part of the clypeus and variable crenulation on the internal margin of the forcipular tarsungulum (Bonato et al. 2010a). Zoosyst. Evol. 100 (3) 2024, 821-840 -/97/0.99 Nannarrup innuptus 0.03 */*10.99 [Ih Dicellophilus carniolensis SMTA G) 829 -194/0.9%] | -/85/- -194/98—_] TS20201007-05 | TS20201007-0 TS20201007-03 TS20201007-04 Figure 5. Maximum-likelihood tree of Dicellophilus based on the dataset of 28S. Nodal values are obtained from the ultrafast boot- strap (UFBoot), SH-like approximate likelihood ratio test (SH-aLRT), and posterior probability (PP). The asterisk (*) indicates 100% in UFBoot, SH-aLRT, and 1.0 in PP. Hyphen (-) indicates lower than 95% in UFBoot, 80% in SH-aLRT, or 0.95 in PP. Nodal values are not shown when UFBoot, SH-aLRT, and PP values are <95%, <80%, and <0.95, respectively. The unit of evolutionary distance is the number of base substitutions per site. A broken square shows that the clade consisted of specimens from eastern Honshu. Ab- breviations: Ai = Aichi Pref.; Fs = Fukushima Pref.; Gi = Gifu Pref.; Kn = Kanagawa Pref.; Ky = Kyoto Pref.; Mi = Mie Pref.; My = Miyagi Pref.; Ni = Niigata Pref.; Nn = Nagano Pref.; Sh = Shizuoka Pref.; Si = Saitama Pref.; To = Tokyo Pref.; Yn = Yamanashi Pref. In addition, the 23 adult specimens, including a spec- imen from the type locality of D. pulcher (Subashir1, Oyama, Suntou-gun, Shizuoka Pref.), had the following diagnostic characteristics of D. pulcher (Uliana et al. 2007; Bonato et al. 2010a): trunk segments without dark patches; head 1.2—1.4 times as long as it is wide; cephal- ic plate with a markedly convex lateral margin; clypeus with densely scattered setae; palaclypeal suture evident- ly converging posteriorly; transverse suture uniformly rounded at center; mandible with 5—7 lamellae; forcipular tarsungulum with evident and variably spaced notches; and 41 pairs of legs. On the other hand, four specimens of Clade A (TS20201007-02, TS20201007-03, TS20201007-04, and TS20201007-05) were morphologically different from the specimens of Clade B based on the following charac- teristics: both ends of transverse suture not evidently con- vex forward; long rather than wide trochanteroprefemur; wide rather than long metasternite (Table 3). Taxonomic account Family Mecistocephalidae Bollman, 1893 Genus Dicellophilus Cook, 1896 Dicellophilus praetermissus sp. nov. https://zoobank. org/8A967E03-A5B0-4495-A 0E9-6CE1298F3E3D Figs 6-13 New Japanese name: Date-hirozujimukade DI. Dicellophilus sp. O000-0003-3020-8454 0069 Type material. Holotype. 1 adult male, Baba, Akiu-machi, Taihaku-ku, Sendai-shi, Miyagi Pref., Japan (38°16.33'N, 140°32.69'E), 7 October 2020, coll. Sho Tsu- kamoto (labeled as TS20201007-02), deposited at the Col- lection of Myriapoda, Department of Zoology, NSMT. Paratype. 1 adult male, Baba, Akiu-machi, Tai- haku-ku, Sendai-shi, Miyagi Pref., Japan (38°16.33'N, 140°32.69'E), 7 October 2020, coll. Sho Tsukamoto zse.pensoft.net 830 Tsukamoto, S. & Eguchi, K.: Integrative taxonomy of Dicellophilus in Japan Table 3. Morphological comparison between D. pulcher and D. praetermissus sp. nov. Putative species identification code Species identified in the present study Both ends of transverse suture ratio of trochanteroprefemur ratio of sternite of ULBS The width to length The width to length Dicellophilus sp. Dicellophilus pulcher evidently convex 1: 0.9-1.1 1: 1.0-1.3 0000-0003-3020-8454 0068 (Kishida, 1928) forward Dicellophilus sp. Dicellophilus not evidently convex 1: 1.3-1.4 1: 0.66-1.0 0000-0003-3020-8454 0069 praetermissus sp. nov. Figure 6. Habitus of Dicellophilus praetermissus sp. nov., paratype (TS20201007-05). Photo by Joe Kutsukake. (labeled as TS20201007-03), 1 adult female, Baba, Akiu-machi, Taihaku-ku, Sendai-shi, Miyagi Pref., Japan (38°16.33'N, 140°32.69'E), 7 October 2020, coll. Sho Tsukamoto (labeled as TS20201007-04), 1 adult male, Baba, Akiu-machi, Tathaku-ku, Sendai-shi, Miyagi Pref., Japan (38°16.33'N, 140°32.69'E), 7 October 2020, coll. Sho Tsukamoto (labeled as TS20201007-05), deposited at MNHAH. Etymology. The species name is a masculine adjective derived from “overlooked” in Latin. Since the description by Kishida (1928) of D. pulcher (as Mecistocephalus pul- cher), this new species has been overlooked for 90 years, despite documentation of its distribution as Dicellophilus in the Sendai-shi, Miyagi Pref. (Takakuwa 1940). Diagnosis. Trunk segments without dark patches; head 1.4 times as long as wide; lateral margin of cephalic plate abruptly converged posteriorly; clypeus with densely scattered setae; palaclypeal suture evidently converging posteriorly; both ends of transverse suture uniformly rounded; mandible with 6 lamellae; forcipular trochan- teroprefemur longer than wide, with one small distal den- ticle; forcipular tarsungulum with evident and variably spaced notches; metatergite subtrapezoidal; metasternite trapezoidal, wide rather than long; forty-one pairs of legs. Description. General features (Fig. 6): Body about 50 mm long (holotype ca 52 mm), gradually attenuated posteriorly, almost uniformly pale yellow, with head and forcipular segment ocher. Cephalic capsule (Fig. 7A, B): Cephalic plate ca 1.3— 1.4x as long as wide; lateral margins markedly convex; posterior margin straight; areolate part visible only at anterior margin; scutes approximately isometric and up to 20 um wide in 50 mm long specimen; both ends of transverse suture uniformly rounded or slightly convex zse.pensoft.net forward; setae up to ca 300 um long. Clypeus ca 2.3—2.5x as wide as long, with lateral margins complete, anterior part areolate, with scutes ca 30 um wide in 50 mm long specimen, clypeal areas absent; clypeus with about 200 setae on most part except lateral and posterior margins; clypeal plagulae undivided by mid-longitudinal areolate stripe. Anterior and distolateral parts of pleurites areo- late, without setae, non-areolate part extending forwards distinctly beyond labrum. Side-pieces of labrum not in contact, anterior margin not concave posteriorly but hor- izontally, divided into anterior and posterior alae by chi- tinous line, with longitudinal stripes on posterior alae, with medial tooth, and short fringe on posterior margin of side-pieces; mid-piece ca 6.2 times as long as wide, lateral margin concaved. Antenna (Fig. 8A—H): Antenna with 14 articles, when stretched, ca 2.7—3.2x as long as head length. Intermedi- ate articles longer than wide. Distal part of article areo- late, remaining surface not areolate in article I—XIII. Arti- cle XIV ca 2.1—2.5x as long as wide, ca 1.1—1.5x as long as article XII. Setae on articles VHI—XVI denser than articles I-VII. Setae gradually shorter from article VUI to XIV, up to ca 290 um long on article I, up to ca 270 um long on article VII and < 75 um long on article XIV. Article XIV with two types of sensilla; apical sensilla (ar- rows in Fig. 8G, H) ca 25 um long, with wide flat ring at mid-length; club-like (arrowheads in Fig. 8G, H) sensilla ca 15 um long, clustered in distal part of internal and ex- ternal sides of article. Rows of spine-like basal sensilla (the ‘sensilla microtrichoidea’ of Ernst 1983, 1997, 2000) absent on antennal article VI and X. A few pointed sen- silla, up to 7.5 um long, on both dorso-external and ven- tro-internal position, close to distal margin of articles II, V, TX and XIII. Zoosyst. Evol. 100 (3) 2024, 821-840 831 Figure 7. Dicellophilus praetermissus sp. nov., holotype (TS20201007-02) A cephalic plate, dorsal B clypeus, and clypeal pleurite, ventral. Scale bars: 0.5 mm. Mandible (Fig. 9A): Five—six pectinate lamellae pres- ent; first lamellae with at least 4 elongated teeth. Anterior surface hairy. First maxillae (Fig. 9B): Coxosternite medially divided but slightly, without setae, without projection on antero-ex- ternal corners, non-areolate. Coxal projections well devel- oped, with ca 20 setae along internal margin, distal lobe sub- triangular. Telopodite uni-articulated and hyaline distally, with 5—6 setae. No lobes on either coxosternite or telopodites. Second maxillae (Fig. 9B): Coxosternite medially undivided, without suture but areolated on isthmus, with 4+4 setae along anterior margin, with about 25 setae on isthmus, with about 15 setae on lateral margin and pos- terior corners, anterior margin concave, with metameric pores on posterior part. Telopodites tri-articulate, reach- ing medial projections and telopodites of first maxillae. Claw of telopodite present. Forcipular segment (Fig. 1OA—E): Tergite trapezoi- dal, ca 1.3—1.4x as wide as long, with lateral margins con- verging anteriorly, areolation mainly along two marginal lateral and anterior bands and two paramedian posterior areas, gradually fading into central non-areolate surface; ca 0.5—0.6x as wide as cephalic plate and ca 0.4—0.5* as wide as tergite 1; 3+2 setae of similar length arranged in an anterior row, and ca 20 setae of similar length arranged symmetrically in a posterior row. Mid-longitudinal sul- cus of tergite not visible. Pleurite 1.8-1.9x as long as the tergite; dorsal ridge sclerotized; anterior tip (scapular point) well behind anterior margin of coxosternite, and only slightly projecting. Cerrus composed of a group of 10—20 setae on each side of anterodorsal surface of cox- osternite, but no paramedian rows of setae. Exposed part of coxosternite ca 1.2 as wide as long; anterior margin with shallow medial concavity and with one pair of denti- cles; coxopleural sutures complete in entire ventrum, sin- uous and diverging anteriorly; chitin-lines absent; condy- lar processes of forcipular coxosternite well developed. Trochanteroprefemur ca 1.3—1.4= as long as wide; with a pigmented tubercle at distal internal margin. Interme- diate articles distinct, with a tubercle on femur and tibia. Tarsungulum with well-pigmented basal tubercle on dor- sal surface; both external and internal margins uniformly curved, except for moderate mesal basal bulge; ungulum not distinctly flattened; internal margin of ungulum ev- idently crenulated, with variably spaced notches. Elon- gated poison calyx lodged inside intermediate forcipular articles. Leg-bearing segments (Fig. 11 A—D): Forty-one pairs of legs present. Metatergite 1 slightly wider than subsequent one, with two paramedian sulci visible on tergites of ante- rior half of body, with pretergite. No paratergites. Legs of first pair much smaller than following ones; claws simple, uniformly bent, with 2 accessory spines; posterior spine shorter than anterior spine; with a subsidiary spine near posterior spine (arrow in Fig. 11D). Metasternites slightly longer than wide. Sternal sulcus evident on segment II, but fading towards posterior segments, anteriorly not furcate. No ventral glandular pores on each metasternite. zse.pensoft.net 832 Tsukamoto, S. & Eguchi, K.: Integrative taxonomy of Dicellophilus in Japan articles I-IV, dorsal; B. Antennal articles I-IV, ventral; C. Antennal articles V—VIII, dorsal; D. Antennal articles V—III, ventral; E. Antennal articles [IX—XIV, dorsal; F. Antennal articles [IX—XIV, ventral; G. Antennal article XIV, dorsal; H. Antennal article XIV, ventral. Arrows indicate apical sensillum; arrowheads indicate club-like sensillum. Scale bars: 0.5 mm (A—F); 0.1 mm (G, H). zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 821-840 833 Figure 9. Dicellophilus praetermissus sp. nov., holotype (TS20201007-02) A. Left mandible, ventral; B. Maxillae complex, ventral. Scale bars: 0.1 mm (A); 0.5 mm (B). ‘~ Figure 10. Dicellophilus praetermissus sp. nov., holotype (TS20201007-02). A. Forcipular segment, dorsal; B. Forcipular segment, ventral; C. Right condylar process of forcipular coxosternite, dorsal; D. Right forcipular tarsungulum, dorsal; E. Poison calyx, dorsal. The arrow indicates the basal tubercle of the forcipular tarsungulum. Scale bars: 0.5 mm (A, B); 0.2 mm (C); 0.3 mm (D); 0.1 mm (E). zse.pensoft.net 834 Tsukamoto, S. & Eguchi, K.: Integrative taxonomy of Dicellophilus in Japan Figure 11. Dicellophilus praetermissus sp. nov., holotype (TS20201007-02). A. Tergite of leg-bearing segment 40, dorsal; B. Ster- nite of leg-bearing segment 40, ventral; C. Pretarsus of left leg 40, anterolateral. D. Pretarsus of left leg 2, posterolateral. The arrow indicates a subsidiary spine. Scale bars: 0.5 mm (A, B); 0.1 mm (C, D). Figure 12. Dicellophilus praetermissus sp. nov. A, B. Holotype (TS20201007-02) C, D. Paratype (TS20201007-04) A, C. Ultimate leg-bearing segment and postpedal segment, dorsal; B, D. Ultimate leg-bearing segment and postpedal segment, ventral. Scale bars: 0.5 mm. zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 821-840 835 Figure 13. Dicellophilus praetermissus sp. nov., holotype (TS20201007-02). A. Left ultimate leg, dorsal; B. Left ultimate leg, ventral. Scale bars: 0.5 mm. Ultimate leg-bearing segment (Figs 12A—D, 13A, B): Pretergite accompanied by pleurites. Metatergite subtrapezoidal, ca 1.2—1.5x as long as wide; lateral mar- gins converging posteriorly. Coxopleuron ca 1.8—2.3x as long as metasternite; coxal organs of each coxopleu- ron opening through ca 70 independent pores, placed ventrally; distinctly larger pore (macropore) near center of the ventral side. Metasternite trapezoidal, ca 1.1—1.5x as wide as long, anteriorly ca 3.0—3.6x as wide as poste- riorly; lateral margins converging backward straightly; setae almost arranged symmetrically, dense on posterior margin. In male holotype (TS20201004-02), telopodite ca 11.5x as long as wide, ca 1.6 as long, and ca 1.3 as wide as penultimate telopodite, with six articles; tarsus 2 ca 3.3x as long as wide and ca 1.1 as long as tarsus 1; setae arranged uniformly, < 200 um long; pretarsus without claw. In female paratype (TS20201004-04), te- lopodite ca 13.5 as long as wide, ca 1.8~ as long, and ca 1.3x as wide as penultimate telopodite, with six arti- cles; tarsus 2 ca 5.2x as long as wide and ca 1.3 as long as tarsus 1; setae arranged uniformly, < 300 um long; pretarsus without claw. Male postpedal segments (Fig. 12A, B): Two gono- pods, very widely separated from one another, conical in outline, bi-articulated with sutures, covered with setae. Anal pore present. Female postpedal segments (Fig. 12C, D): Two gono- pods basally touching, subtriangular, bi-articulated with sutures, covered with setae. Anal pore present. Distribution. Only known from the type locality. Remarks. Dicellophilus praetermissus sp. nov. most closely resembles D. pulcher but is distinguishable by the following combination of characteristics: both ends of transverse suture not evidently convex forward; the longer than wide trochanteroprefemur; the wide rather than long metasternite (Table 3). The record of D. latifrons Takakuwa, 1934 (= D. pulcher) from Sendai, Miyagi Pref. (Takakuwa 1940) requires confirmation of its identification. Dicellophilus pulcher (Kishida, 1928) Figs 14-16 Mecistocephalus pulcher Kishida, 1928: Kishida 1928, 300. Dicellophilus latifrons: Takakuwa 1934a, 707; Takakuwa 1934b, 355; Takakuwa 1934c, 878. Dicellophilus japonicus: Verhoeff 1934, 32. Tygarrup monoporus: Shinohara 1961, 212. Dicellophilus pulcher: Uliana et al. 2007, 27; Bonato et al. 2010, 525. DI. Dicellophilus sp. 0000-0003-3020-8454 0068 Material examined. See Table 1. Diagnosis. Mainly based on Bonato et al. (2010a), Uli- ana et al. (2007), and the present study. Trunk segments without dark patches; head 1.2—1.4 times as long as wide (Fig. 14A, B); lateral margin of cephalic plate abrupt- ly converged posteriorly; clypeus with densely scattered setae (Fig. 14B); paraclypeal suture evidently converging posteriorly (Fig. 14B); both ends of transverse suture con- vexed forward (Fig. 14A); mandible with 5—7 lamellae; forcipular trochanteroprefemur almost as long as wide, with one small distal denticle (Fig. 15A, B); forcipular tarsungulum with evident and variably spaced notches; metatergite subtrapezoidal (Fig. 16A); metasternite trape- zoidal, longer than wide (Fig. 16B); forty-one pairs of legs. zse.pensoft.net 836 Tsukamoto, S. & Eguchi, K.: Integrative taxonomy of Dicellophilus in Japan j Ne . Figure 14. Dicellophilus pulcher (TS20210504-01). A. Cephalic plate, dorsal; B. Clypeus, and clypeal pleurite, ventral. Scale bars: 0.5 mm. _ “7 “a a Figure 15. Dicellophilus pulcher (TS20210504-01). A. Forcipular segment, dorsal; B. Forcipular segment, ventral. Scale bars: 0.5 mm. Type locality. The first section of the Subashiri trail of Remarks. See remarks and the diagnosis of D. prae- Mt. Fuji, Shizuoka Pref., Japan (Kishida 1928). termissus sp. nov. for confirming how to distinguish D. Distribution. Honshu (Fukushima Pref. to Hyogo Pref.). | pulcher from D. praetermissus sp. nov. zse.pensoft.net Zoosyst. Evol. 100 (3) 2024, 821-840 Y a J a ‘ Se eC a . i , —. Pr - ps a - * “i ~e 2 = nk, od 7 5 i 837 E ‘ 4 ‘ a _ < = hi ” J 4 - 4 iv, , ‘ * ‘! aa 7 * ‘ -: 4 A 4 f ‘ * ? P / ’ f a “~ L —— 4 J re ? 7 . # ‘' « 3 a hechd —_ “A Figure 16. Dicellophilus pulcher (TS20210504-01). A. Ultimate leg-bearing segment and postpedal segment, dorsal; B. Ultimate leg-bearing segment and postpedal segment, ventral. Scale bars: 0.5 mm. There are three junior synonyms under D. pulcher, which were synonymized by previous authors based on morpho- logical examination (Takakuwa 1940; Shinohara 1983; Uli- ana et al. 2007): D. latifrons Takakuwa, 1934; D. japonicus Verhoeff, 1934; Zygarrup monoporus Shinohara, 1961. Di- cellophilus latifrons Takakuwa, 1934, which was described in a key to Japanese and Taiwanese species of Mecistcepha- lidae by Takakuwa (1934a), was later described by Taka- kuwa (1934b, c) as a new species. Takakuwa (1934a) did not designate a type locality for D. latifrons, and Takaku- wa (1934b, c) listed the localities: “Kaibara (Hyogo)” (= Tamba City, Hyogo Pref.), “Masudo (bei Tokyo)” (possibly misread of Masuko-mura, currently in Akiruno-shi, Tokyo Pref.), “Komono (Miye)” (= Komono-cho, Mie Pref.), “TIkao (Gumma)’ (= Ikahocho, Shibukawa-shi, Gunma Pref.), “Ota (Gummay)” (= Ota-shi, Gunma Pref. ), “Odawara (Kanagawa)” and “Suwa (Nagano)” (annotated by Jonishi and Nakano 2022). Considering the geographic distribution of D. latifrons and D. pulcher sensu stricto, D. latifrons is a Junior synonym of D. pulcher. Dicellophilus japonicus Ver- hoeff, 1934, was described based on a specimen from “To- kyo” (Verhoeff 1934) and later regarded as a junior synonym of D. latifrons based on the comparison of diagnostic char- acteristics (Takakuwa 1940; Shinohara 1983). Considering the geographic distribution of D. japonicus and D. pulcher and the phylogenetic analyses of the present study, includ- ing TS20181214-01, TS20191006-02, TS20210401-03, and TS20210819-01 from Tokyo Pref., it is not conflicting that D. japonicus is a junior synonym of D. pulcher. Tygarrup monoporus Shinohara, 1961, which is identical to the juve- nile of D. pulcher, according to Uliana et al. (2007), was described based on the specimen from Manazuru-machi, Ashigarashimo-gun, Kanagawa Pref. (Shinohara 1961). Considering the geographic distribution of 7? monoporus and D. pulcher and the phylogenetic analyses of the present study, including TS20210728-02, which was collected at a linear distance of approximately 13 km from the type local- ity and could be identified as 77 monoporus, T: monoporus can also be regarded as a junior synonym of D. pulcher. Discussion Species hypothesis for Japanese Dicellophilus No POTU delimitation hypotheses proposed by ASAP cor- responded well to the principle in steps I-IV. This is because each of those hypotheses involves many POTUs, which were separated from each other with a K2P distance lower than the intermorphospecific threshold. Although Clade B could be divided into many more POTUs, such partitioning hypotheses can be rejected as oversplitting in accordance with the species delimitation criteria in step TV with the intermorphospecific threshold (21% in CO/). Therefore, two putative species were recognized in the 38 D. pulcher specimens examined in steps I-IV, and they can be sep- arately labeled as follows: Dicellophilus sp. 0000-0003- 3020-8454 0068 (= Clade B; hereafter referred to as D. sp. 0068) and D. sp. 0000-0003-3020-8454 0069 (= Clade A; hereafter referred to as D. sp. 0069, Table 3, Figs 2-5). Only one validly named species of Dicellophilus from Japan, D. pulcher, was described on the basis of aspecimen from the Subashiri trail of Mt. Fuji, Shizuoka Pref. (Kishi- da 1928). Dicellophilus sp. 0068 involves a specimen zse.pensoft.net 838 from the type locality (TS20210504-01) and shares the number of pairs of legs, the presence of macropores on the coxopleuron, and a wide forcipular trochanteroprefemur with the original description (including figures) of D. pul- cher. Therefore, D. sp. 0068 is conferrable to D. pulcher. By contrast, D. sp. 0069 can be regarded as a new species based on morphological comparison with other conge- ners, including D. pulcher. This new species is described in the "Taxonomic account" section under the name Di- cellophilus praetermissus sp. nov. (step V); see also the taxonomic discussion of junior synonyms of D. pulcher. Oversplit of the number of POTUs by ASAP As mentioned in the result section, many POTUs were divided from the COI dataset of Dicellophilus examined in the present study. When taking into account the overall genetic diversity of all examined specimens of Dicelloph- ilus, 1.e., D. pulcher, D. carniolensis and D. praetermis- SUS Sp. Nov. (Maximum genetic divergence in K2P: 24%), the genetic diversity within the morphospecies D. pulcher alone is quite high (15%). Possibly, such an oversplit of POTU would have been caused by the algorithm ASAP and the quite high genet- ic divergence of D. pulcher in the dataset. According to Puillandre et al. (2021), ASAP is a hierarchical clustering algorithm. Each subgroup was separated depending on the average pairwise distance between subgroups and with- in the subgroup, sample size, and a coalescent mutation rate. Based on this algorithm, the distribution of genetic distances will affect the result of the number of species (= POTUs). In detail, when there is high genetic diversity within one morphospecies compared to the whole dataset, the morphospecies will be divided into several POTUs, in accordance with the possibility of panmixia (p-value). POTUs are divided by the possibility of panmixia, so it can be expected that each POTU will be a biological species. However, each POTU within the morphospecies D. pulcher detected in the present study is not regarded as a species until morphological evidence is discovered. Distribution of Dicellophilus in Japan Dicellophilus specimens examined in Japan were collect- ed from 34°33'N to 38°16'N on Honshu within a latitudi- nal band, which is congeners’ distribution. The authors and their collaborators have collected Dicellophilus ex- clusively from Miyagi Pref. to Kyoto Pref. but not from other areas, despite a comprehensive field trip in Japan (Fig. 1). Therefore, the distribution of Dicellophilus in Japan should be restricted from Miyagi Pref. to Kyoto Pref. (see Remarks of D. pulcher in this paper; Takakuwa 1940; Uliana et al. 2007). According to the molecular phylogenetic analyses of Dicellophilus specimens in Japan, it is possible that there are two large populations among D. pulcher, viz., zse.pensoft.net Tsukamoto, S. & Eguchi, K.: Integrative taxonomy of Dicellophilus in Japan specimens from Eastern Honshu (Fukushima Pref. to Shizuika Pref. and one specimen from Gifu Pref.) and those from western Honshu (Gifu Pref. to Kyoto Pref.), because the monophyly was supported by the phyloge- netic analysis based on the concatenated and CO/ data- sets, respectively. However, the boundary between two populations is still not clear due to the lack of field sur- veys in the central part of Honshu. In field surveys conducted by the authors in Japan, D. praetermissus sp. nov. was collected only in Sendai-shi, Miyagi Pref. (the northern part of Honshu). It is also note- worthy that D. pulcher has yet to be collected from the northern part of Honshu (from Aomori Pref. to Miyagi Pref). This result of field surveys shows that the distribution of D. praetermissus sp. nov. may segregate from D. pulcher, but further field surveys are needed around Miyagi Pref. Acknowledgments We are grateful to Dr Masaru Nonaka (visiting researcher of Tokyo Metropolitan University), Dr Namiki Kikuchi (Toyo- hashi Museum of Natural History), Dr Takahiro Yoshida (assistant professor of Tokyo Metropolitan University), Mr Joe Kutsukake (Tokyo Metropolitan University), Mr Koshi Kawamoto (Tokyo Metropolitan University), Ms Mayu Su- sukida, Mr Ryo Miyata, Mr Tatsumi Suguro (Keio Yochisha Elementary School), Mr Tomoki Sumino, and Mr Fukube Sumino for collecting and providing Dicellophilus speci- mens. We are further grateful to Dr Namiki Kikuchi and Mr Joe Kutsukake for assisting in collecting and taking photo- graphs of Dicellophilus praetermissus sp. nov., respectively. We thank two reviewers for providing valuable comments and suggestions. 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