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.

A total of 38 specimens morphologically identified as Dicellophilus pulcher, hitherto the only known Japanese 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 digital nation land information (https://nlftp.mlit.go.jp/index.html) were used to generate Fig. 1.

Figure 1. 

Map of the collection sites of specimens examined in the present study. White circle, Dicellophilus pulcher; black circle, D. praetermissus sp. nov.

Each specimen was labeled with its unique specimen identification number in the form “TSYYYYMMDD-XX,” where TS is an abbreviation of the first author’s name, Tsukamoto Sho; YYYYMMDD designates the date on which the specimen was collected; and XX is the identification number assigned to each specimen collected 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 Myriapoda, 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 “Taxonomic account” section. All non-type voucher specimens of Dicellophilus pulcher are managed by the first author.

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 series of source images taken using a Canon EOS Kiss X9 digital camera attached to a Nikon AZ100 microscope and improved using Adobe Photoshop Elements 10 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 is, evidently biarticulated in males and touching one another in females, were determined to be adults, and those with incompletely developed paired gonopods were determined to be subadults. Specimens without gonopods were determined to be juveniles based on Uliana et al. (2007). In the present study, 23 adult specimens out of 38 collected were examined morphologically.

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.

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

The amplification conditions for mitochondrial COI 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. If the target fragment of COI was not appropriately amplified, then the annealing temperature was changed from 48.5 °C to 50 °C. PCR was performed again by omitting the first five cycles of annealing and the extension step.

The amplification conditions for mitochondrial 16S 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 16S was not appropriately 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 28S was not appropriately amplified, then the annealing temperature was changed from 50 °C to 48 °C. The number of annealing 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^TM Express (Thermo Fisher Scientific) to remove any excess primers and nucleotides. All nucleotide sequences were determined by direct sequencing using the ABI PRISM BigDye^TM Terminator Cycle Sequencing Kit ver. 3.1 (Thermo Fisher Scientific) or BrilliantDye^TM Terminator Cycle Sequencing Kit v. 3.1 (Nimagen, B.V., Nijmegen, Netherlands) equipped with an ABI 3130xl automated sequencer (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).

The sequences obtained using the abovementioned methods were used for phylogenetic analyses, together with the COI, 16S, and 28S sequences of Dicellophilus carniolensis (C.L. Koch, 1847) and Nannarrup innuptus Tsukamoto in Tsukamoto et al. (2022) obtained from GenBank as outgroups (Table 1). The datasets for COI (658 bp positions), 16S (514 bp positions), and 28S (971 bp positions) were concatenated to form the COI + 16S + 28S dataset for phylogenetic analyses.

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

Maximum-likelihood (ML) trees were created on the basis of the sequence dataset for each gene and concatenated using IQ-tree 1.6.12 (Nguyen et al. 2015). As an optimal substitution model in accordance with BIC, TNe + I + G4 was selected for the first codon position of COI in the concatenated dataset; TNe + G4 for the COI dataset; HKY + F was selected for the second codon position of COI in both datasets; TN + F + G4 was selected for the third codon position of COI in both datasets; HKY + F + I + G4 was selected for 16S 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 likelihood ratio test (SH-aLRT; Guindon et al. 2010) were performed with 1,000 replicates.

Bayesian inference trees were created using ExaBayes 1.4.1 (Aberer et al. 2014) under the default substitution model “GTR + G.” The Markov chain Monte 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 COI, but for 20,000,000 generations for the COI dataset. Trees were sampled every 500 generations, 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 default settings. The effective sampling size of each parameter was confirmed to be 200 using Tracer 1.7.1 (Rambaut et al. 2018).

The aligned COI dataset used for phylogenetic analyses were also used to calculate genetic distances. Kimura two-parameter (K2P) distances were calculated using MEGA X (Kumar et al. 2018) with the setting “pairwise deletion.”

The program “assemble species by automatic partitioning (ASAP)” was used to delimit “provisional” operational taxonomic units (POTUs). ASAP is a species delimitation 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 COI sequence dataset of Dicellophilus (excluding the outgroup) under the “pairwise K2P distance” method.

The present study preliminarily relied on the morphological 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 assumption 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: (I) sorting specimens, which are morphologically conferrable to Dicellophilus, into morphospecies; (II) confirming the monophyly of the morphospecies with the phylogenetic tree inferred by the sequence dataset of three gene markers, mitochondrial COI and 16S, and nuclear 28S; (III) calculating the genetic distance of COI between congeneric morphospecies, which are confirmed to be monophyletic, to define the intermorphospecific threshold; (IV) delimiting POTUs using ASAP (see above) and confirming the most conferrable hypotheses of species-level independence by considering the phylogenetic tree and the intermorphospecific threshold defined in step III. By steps III 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 others with a minimum K2P distance higher than the intermorphospecific 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 intermorphospecific 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 distance 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 morphological 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.

Each putative species recognized by following the abovementioned steps was also labeled in accordance with the study of Tsukamoto (2023), with a unique, permanent, and citable identifier “DI,” such as “0000-0003-3020-8454_XXXX,” in which “0000-0003-3020-8454” shows the author’s ORCID and “XXXX” shows a unique identification number given to each species in the author’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 information necessary to formally describe and name species in the conventional manner of Linnaean Taxonomy (The International Commission on Zoological Nomenclature 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 description and naming of the species (step V) were separated as two methodologically distinct phases.

COI was successfully sequenced for 38 specimens, 16S 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 Dicellophilus 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%, 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 contrary, the monophyly of specimens from western Honshu (Gifu Pref. to Kyoto Pref.) was not supported.

Figure 2. 

Maximum-likelihood tree of Dicellophilus based on the concatenated dataset of COI, 16S, and 28S, with the results of species delimitation by ASAP. Note that specimens whose COI 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.

The ML tree based on the COI 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 deeply separated from clade B (UFBoot = 90.8%, SH-aLRT = 95%, PP = 1.00). In addition, the monophyly of specimens 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.

Figure 3. 

Maximum-likelihood tree of Dicellophilus based on the dataset of COI, 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 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.

The ML tree based on the 16S 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 (UFBoot = 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 phylogenetic relationship among Clade B and other Dicellophilus specimens was not clear due to low support values.

Figure 4. 

Maximum-likelihood tree of Dicellophilus based on the dataset of 16S. 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.

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 deeply 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 phylogenetic relationship among Clade B and other Dicellophilus specimens was not clear due to low support values.

Figure 5. 

Maximum-likelihood tree of Dicellophilus based on the dataset of 28S. 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.

Although there is no consistency of phylogenetic relationship among Clades A, B, and D. carniolensis in four datasets, each topology shows that Clade A is a distinct lineage from other Dicellophilus specimens.

The minimum K2P distance between congeneric morphospecies 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% within 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.).

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.

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).

In addition, the 23 adult specimens, including a specimen from the type locality of D. pulcher (Subashiri, 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; cephalic plate with a markedly convex lateral margin; clypeus with densely scattered setae; palaclypeal suture evidently 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 characteristics: both ends of transverse suture not evidently convex forward; long rather than wide trochanteroprefemur; wide rather than long metasternite (Table 3).

Family Mecistocephalidae Bollman, 1893

Genus Dicellophilus Cook, 1896

 Dicellophilus praetermissus sp. nov.

https://zoobank.org/8A967E03-A5B0-4495-A0E9-6CE1298F3E3D

Figs 6, 7, 8, 9, 10, 11, 12, 13 New Japanese name: Date-hirozujimukade

DI

Dicellophilus sp. 0000-0003-3020-8454_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 Tsukamoto (labeled as TS20201007-02), deposited at the Collection of Myriapoda, Department of Zoology, NSMT.

Paratype. 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 Tsukamoto (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, Taihaku-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 pulcher), 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 trochanteroprefemur longer than wide, with one small distal denticle; 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.

Figure 6. 

Habitus of Dicellophilus praetermissus sp. nov., paratype (TS20201007-05). Photo by Joe Kutsukake.

Cephalic capsule (Fig. 7A, B): Cephalic plate ca 1.3–1.4× 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 μm wide in 50 mm long specimen; both ends of transverse suture uniformly rounded or slightly convex forward; setae up to ca 300 μm long. Clypeus ca 2.3–2.5× as wide as long, with lateral margins complete, anterior part areolate, with scutes ca 30 μm 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 areolate, without setae, non-areolate part extending forwards distinctly beyond labrum. Side-pieces of labrum not in contact, anterior margin not concave posteriorly but horizontally, divided into anterior and posterior alae by chitinous 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.

Figure 7. 

Dicellophilus praetermissus sp. nov., holotype (TS20201007-02) A cephalic plate, dorsal B clypeus, and clypeal pleurite, ventral. Scale bars: 0.5 mm.

Antenna (Fig. 8A–H): Antenna with 14 articles, when stretched, ca 2.7–3.2× as long as head length. Intermediate articles longer than wide. Distal part of article areolate, remaining surface not areolate in article I–XIII. Article XIV ca 2.1–2.5× as long as wide, ca 1.1–1.5× as long as article XIII. Setae on articles VIII–XVI denser than articles I–VII. Setae gradually shorter from article VIII to XIV, up to ca 290 μm long on article I, up to ca 270 μm long on article VIII and < 75 μm long on article XIV. Article XIV with two types of sensilla; apical sensilla (arrows in Fig. 8G, H) ca 25 μm long, with wide flat ring at mid-length; club-like (arrowheads in Fig. 8G, H) sensilla ca 15 μm long, clustered in distal part of internal and external 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 sensilla, up to 7.5 μm long, on both dorso-external and ventro-internal position, close to distal margin of articles II, V, IX and XIII.

Figure 8. 

Dicellophilus praetermissus sp. nov. A–F. Holotype (TS20201007-02) G, H. Paratype (TS20201007-04) A. Antennal 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).

Mandible (Fig. 9A): Five–six pectinate lamellae present; first lamellae with at least 4 elongated teeth. Anterior surface hairy.

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).

First maxillae (Fig. 9B): Coxosternite medially divided but slightly, without setae, without projection on antero-external corners, non-areolate. Coxal projections well developed, with ca 20 setae along internal margin, distal lobe subtriangular. 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 posterior corners, anterior margin concave, with metameric pores on posterior part. Telopodites tri-articulate, reaching medial projections and telopodites of first maxillae. Claw of telopodite present.

Forcipular segment (Fig. 10A–E): Tergite trapezoidal, ca 1.3–1.4× as wide as long, with lateral margins converging anteriorly, areolation mainly along two marginal lateral and anterior bands and two paramedian posterior areas, gradually fading into central non-areolate surface; ca 0.5–0.6× 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 sulcus of tergite not visible. Pleurite 1.8–1.9× 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 coxosternite, 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 denticles; coxopleural sutures complete in entire ventrum, sinuous and diverging anteriorly; chitin-lines absent; condylar processes of forcipular coxosternite well developed. Trochanteroprefemur ca 1.3–1.4× as long as wide; with a pigmented tubercle at distal internal margin. Intermediate articles distinct, with a tubercle on femur and tibia. Tarsungulum with well-pigmented basal tubercle on dorsal surface; both external and internal margins uniformly curved, except for moderate mesal basal bulge; ungulum not distinctly flattened; internal margin of ungulum evidently crenulated, with variably spaced notches. Elongated poison calyx lodged inside intermediate forcipular articles.

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).

Leg-bearing segments (Fig. 11A–D): Forty-one pairs of legs present. Metatergite 1 slightly wider than subsequent one, with two paramedian sulci visible on tergites of anterior half of body, 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.

Figure 11. 

Dicellophilus praetermissus sp. nov., holotype (TS20201007-02). A. Tergite of leg-bearing segment 40, dorsal; B. Sternite 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).

Ultimate leg-bearing segment (Figs 12A–D, 13A, B): Pretergite accompanied by pleurites. Metatergite subtrapezoidal, ca 1.2–1.5× as long as wide; lateral margins converging posteriorly. Coxopleuron ca 1.8–2.3× as long as metasternite; coxal organs of each coxopleuron opening through ca 70 independent pores, placed ventrally; distinctly larger pore (macropore) near center of the ventral side. Metasternite trapezoidal, ca 1.1–1.5× as wide as long, anteriorly ca 3.0–3.6× as wide as posteriorly; lateral margins converging backward straightly; setae almost arranged symmetrically, dense on posterior margin. In male holotype (TS20201004-02), telopodite ca 11.5× as long as wide, ca 1.6× as long, and ca 1.3× as wide as penultimate telopodite, with six articles; tarsus 2 ca 3.3× as long as wide and ca 1.1× as long as tarsus 1; setae arranged uniformly, < 200 μm long; pretarsus without claw. In female paratype (TS20201004-04), telopodite ca 13.5× as long as wide, ca 1.8× as long, and ca 1.3× as wide as penultimate telopodite, with six articles; tarsus 2 ca 5.2× as long as wide and ca 1.3× as long as tarsus 1; setae arranged uniformly, < 300 μm long; pretarsus without claw.

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.

Figure 13. 

Dicellophilus praetermissus sp. nov., holotype (TS20201007-02). A. Left ultimate leg, dorsal; B. Left ultimate leg, ventral. Scale bars: 0.5 mm.

Male postpedal segments (Fig. 12A, B): Two gonopods, 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 gonopods 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, 15, 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), Uliana 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 abruptly converged posteriorly; clypeus with densely scattered setae (Fig. 14B); paraclypeal suture evidently converging posteriorly (Fig. 14B); both ends of transverse suture convexed 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 trapezoidal, longer than wide (Fig. 16B); forty-one pairs of legs.

Figure 14. 

Dicellophilus pulcher (TS20210504-01). A. Cephalic plate, dorsal; B. Clypeus, and clypeal pleurite, ventral. Scale bars: 0.5 mm.

Figure 15. 

Dicellophilus pulcher (TS20210504-01). A. Forcipular segment, dorsal; B. Forcipular segment, ventral. Scale bars: 0.5 mm.

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.

Type locality

The first section of the Subashiri trail of Mt. Fuji, Shizuoka Pref., Japan (Kishida 1928).

Distribution

Honshu (Fukushima Pref. to Hyogo Pref.).

Remarks

See remarks and the diagnosis of D. praetermissus sp. nov. for confirming how to distinguish D. pulcher from D. praetermissus sp. nov.

There are three junior synonyms under D. pulcher, which were synonymized by previous authors based on morphological examination (Takakuwa 1940; Shinohara 1983; Uliana et al. 2007): D. latifrons Takakuwa, 1934; D. japonicus Verhoeff, 1934; Tygarrup monoporus Shinohara, 1961. Dicellophilus latifrons Takakuwa, 1934, which was described in a key to Japanese and Taiwanese species of Mecistocephalidae by Takakuwa (1934a), was later described by Takakuwa (1934b, c) as a new species. Takakuwa (1934a) did not designate a type locality for D. latifrons, and Takakuwa (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.), “Ikao (Gumma)” (= Ikahocho, Shibukawa-shi, Gunma Pref.), “Ōta (Gumma)” (= 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 Verhoeff, 1934, was described based on a specimen from “Tokyo” (Verhoeff 1934) and later regarded as a junior synonym of D. latifrons based on the comparison of diagnostic characteristics (Takakuwa 1940; Shinohara 1983). Considering the geographic distribution of D. japonicus and D. pulcher and the phylogenetic analyses of the present study, including 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 juvenile 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 T. 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 locality and could be identified as T. monoporus, T. monoporus can also be regarded as a junior synonym of D. pulcher.

No POTU delimitation hypotheses proposed by ASAP corresponded 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 IV with the intermorphospecific threshold (21% in COI). Therefore, two putative species were recognized in the 38 D. pulcher specimens examined in steps I–IV, and they can be separately 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 a specimen from the Subashiri trail of Mt. Fuji, Shizuoka Pref. (Kishida 1928). Dicellophilus sp. 0068 involves a specimen 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. pulcher. 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 congeners, including D. pulcher. This new species is described in the "Taxonomic account" section under the name Dicellophilus praetermissus sp. nov. (step V); see also the taxonomic discussion of junior synonyms of D. pulcher.

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 Dicellophilus, i.e., D. pulcher, D. carniolensis and D. praetermissus 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 genetic 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 within 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.

Dicellophilus specimens examined in Japan were collected from 34°33'N to 38°16'N on Honshu within a latitudinal band, which is congeners’ distribution. The authors and their collaborators have collected Dicellophilus exclusively 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., 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 phylogenetic analysis based on the concatenated and COI datasets, respectively. However, the boundary between two populations is still not clear due to the lack of field surveys 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 noteworthy 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.