﻿A new jewel-like species of the pill-millipede genus Sphaerobelum Verhoeff, 1924 (Diplopoda, Sphaerotheriida, Zephroniidae) from Thailand

﻿Abstract A new species of the giant pill millipede genus Sphaerobelum is described: Sphaerobelumturcosasp. nov. from the northeastern part of Thailand. Species delimitation is based on morphological characters and COI sequence data. The new species can be clearly discriminated from congeners by its greenish-blue body color, the face mask-like appearance of the thoracic and anal shields jointly when rolled up, and the combination of the following four characters: (1) the coxa of the second leg laterally with a sharp and long process, (2) the tarsi of legs 4–21 with 6–7 ventral spines, (3) the anterior telopods consisting of four conspicuous telopoditomeres, and (4) the immovable, slender (not strongly humped) and distally curved finger of the posterior telopods without a membranous spot. The interspecific COI sequence divergence between the new species and other Sphaerobelum species ranges from 17% to 23% (mean 20%). The intergeneric COI sequence divergence between the new species and Zephronia species ranges from 18% to 21% (mean 20%). The relationships among Sphaerobelum and Zephronia species based on the COI sequence data were not resolved in this study. Sphaerobelumturcosasp. nov. is restricted to limestone habitat in Loei province and is probably endemic for the Thai fauna.

Recent fieldwork conducted in the impressive geological landscape at Phu Pha Lom Forest Park, Loei Province in the northeastern part of Thailand, has unveiled remarkable greenish-blue specimens of a giant pill-millipede which clearly belongs to the family Zephroniidae in the order Sphaerotheriida.Recently, the species diversity of this family in Thailand has gained considerable attention (Likhitrakarn et al. 2021;Rosenmejer et al. 2021;Srisonchai et al. 2021;Bhansali and Wesener 2022).The updated records for Zephroniidae in Thailand contains only two genera with a total of 12 valid species (see Likhitrakarn et al. 2023).The number of species, however, is still lower than in some surrounding countries, e.g., Laos, Vietnam and India (Wesener 2019;Semenyuk et al. 2020).
The external characteristics place the specimens within the genus Sphaerobelum and the present contribution aims at providing the formal description of this new Sphaerobelum species based on morphological and DNA data.

Sample collections
Specimens were hand-collected from limestone habitats in Phu Pha Lom Forest Park, Loei Province, Thailand, by visual spotting on open surfaces in daylight.Photographs of live animals were taken with a Canon 70D digital camera with a Canon EF-S 60 mm f/2.8 Macro USM lens.The specimens were euthanized based on AVMA guidelines for the euthanasia of animals (American Veterinary Medical Association 2020), and then preserved in 75% ethanol for morphological study and 95% ethanol for DNA sequence analysis.
The collecting sites were located by GPS using a Garmin GPSMAP 60 CSx, and all coordinates and elevations were rechecked with Google Earth.The background of the distribution map was downloaded from Elastic Terrain Map (http://elasticterrain.xyz/) (Willett et al. 2015) and the figure was composed using Adobe Photoshop CS6.
This research was conducted under the approval of the Animal Care and Use Committee (Protocol Reviews No. IACUC-KKU-136/64 from Khon Kaen University) and No. 1723018 from Chulalongkorn University.

Morphological study
The specimens were examined and measured under a Nikon SMZ 745T trinocular stereo microscope, equipped with a Canon EOS 5DS R digital SLR camera.For scanning electron microscopy (SEM), the specimens were photographed with a JEOL, JSM-5410 LV microscope using gold-coated samples.Line drawings were based on photographs taken under the stereo microscope equipped with a digital SLR camera.All final images were processed and edited with Adobe Photoshop CS6.
The holotypes, as well as most of the paratypes are housed in the Museum of Zoology, Chulalongkorn University (CUMZ), Bangkok, Thailand.

DNA extraction and phylogenetic study
Total genomic DNA was extracted from legs using the NucleoSpin Tissue kit following the manufacturer's instructions.PCR amplifications and sequencing of the standard mitochondrial COI DNA barcoding fragment (Hebert et al. 2003) were done as described by Pimvichai et al. (2020).The COI fragment was amplified with the primers LCO-1490 and HCO-2198 (Folmer et al. 1994).The new COI nucleotide sequences have been deposited in GenBank under accession numbers OR530087−OR530089.Sample data and voucher codes are provided in Table 1.
The COI data included 31 specimens, representing 19 specimens of the genus Sphaerobelum and 10 specimens of the genus Zephronia (Table 1).Two species of the order Glomerida, viz.Glomeris marginata (Villers, 1789) and Hyleoglomeris japonica Verhoeff, 1910 were used as the outgroups.
CodonCode Aligner (ver.4.0.4,CodonCode Corporation) was used to assemble the forward and reverse sequences and to check for errors and ambiguities.All sequences were checked with the Basic Local Alignment Search Tool (BLAST) provided by NCBI and compared with reference sequences in GenBank.Sequence alignment (660 bp) was done with MUSCLE (ver.3.6, see http://www.drive5.com/muscle; Edgar 2004).MEGA11 (ver.11.0.10,see http:// www.megasoftware.net;Tamura et al. 2021) was used to (1) check for stop codons, (2) translate sequences into amino acids, and (3) calculate uncorrected pairwise p-distances among sequences.Pairwise deletion of missing data was applied and all positions containing 'N's were omitted for each sequenced pair in the analysis.
The best-fit substitution model was implemented using JModelTest2 on XSDXE 2.1.6(Darriba et al. 2012) through CIPRES Gateway.A phylogenetic tree was constructed using maximum likelihood (ML).The shape parameter of the gamma distribution, based on 16 rate categories, was estimated using maximum likelihood analysis.ML trees were inferred with RAxML (ver.8.2.12, see http://www.phylo.org/index.php/tools/raxmlhpc2_tgb.html; Stamatakis 2014) through the CIPRES Science Gateway (Miller et al. 2010) using a GTR+G substitution model and 1000 bootstrap replicates to assess branch support.
Coloration: Live animals yellow with contrasting greenish-blue anterior margins of tergites and darker blue dorsal axial stripe (Fig. 2A-D), thoracic shield with a large, paramedian, greenish-blue band at middle (Fig. 2A-C), anal shield with a large, greenish-blue diamond at axial line (Fig. 2B, C), head, collum and groove of thoracic blackish to dark blue (Fig. 2A), legs, antennae, paratergite depressions and venter light brown to brown, venter brown to yellow brownish; coloration in alcohol, after more than 10 years of preservation, faded to light yellow with contrasting greenish-blue anterior margins of tergites, head, collum and groove of thoracic dark blue to blue, legs, antennae, paratergite depressions and venter brown to yellowish.
Head: trapezoid, anterior part of the head with many long setae, posterior part densely dimpled; anterior margin of labrum with a single tooth.Eyes with 37-63 ocelli (male) or 55-67 (female).Aberrant ocellus located inside antennal groove.
Gnathochilarium: Structure typical of the Sphaerotheriida.Palpi with sensory cones arranged in clusters.

Mandibles: not dissected.
Collum: with glabrous surface, except for anterior and posterior margin with a few isolated and long setae.
Laterotergites: laterotergite 1 strongly projecting into a sharp tip.Laterotergite 2 with a broad, stout projection and a deep notch at lateral margin, like following laterotergites.
Following tergites: surface glabrous, shining, except the groove of paratergite with tiny setae.Tips of paratergites of midbody tergites projecting posteriorly.
Thoracic shield: surface glabrous as in tergites.Shallow grooves with few setae, surface glabrous, no keels.

Endotergum:
Posterior margin (pm) flat, regular (Fig. 5A).Outer area (oa) without setae.Middle area (ma) with a single row, conspicuous, elliptical cuticular impressions (cp); distance between impressions as long as individual diameter (Fig. 5B).Bristles arranged in two rows, tip of the longest bristles not extended beyond posterior margin or not reaching to posterior margin (Fig. 5A).Inner area (ia) without tubercles or setae, but with small pits (Fig. 5D).
Anal shield: slightly sexually dimorphic, in female large and well-rounded (Fig. 2B), in male slightly more rectangular, in both sexes glabrous.Surface similar to that of tergites.Underside with a single, very short, black locking carina, six times shorter than width of last laterotergite.
Legs: leg-pair 1 with 1 or 2 ventral spines, leg-pair 2 with 3 or 4, leg-pair 3 with 5 or 6.First two leg-pairs without an apical spine.Leg pairs 4-21 with 6 or 7 ventral spines and one apical spine.In leg 9, femur 1.6 times, tarsus 3.5 times longer than wide (Fig. 3C).Femur extended mesally into a dentate margin featuring 10-14 teeth.All podomeres densely setose.Coxa with a large and marginally toothed process.Coxal process absent at first leg and sharply projecting at second (Fig. 3B).Prefemur at apical margin with a projection laterally and mesally.Lateral projection triangular and sharply edged, juxtaposed to coxal process (Fig. 3C).
Posterior telopods (Fig. 4D-F): inner horns with sharp-edged tips, slightly curved caudad.Telopodite consisting of four podomeres.First telopoditomere rectangular, slightly longer than wide.Immovable finger (process of telopoditomere 2) as long as movable finger, consisting of telopoditomeres 3 and 4. Immovable finger wide, 2.5 times longer than wide, with a characteristic, distally swollen, clearly rounded apically, apex only slightly wider than base; tip strongly curved when seen in dorsolateral view.Immovable finger in anterior view with a small spine, at middle with sclerotized spot.Telopoditomere 3 long and slender, 2.5 times longer than wide, with a membranous lobe at a excavate inner margin.Telopoditomere 4 very short and slender, only 4.5 times shorter than telopoditomere 3, 1.5 times longer than wide, slightly tapering apically.Telopoditomere 4 with one small, weak spine at margin towards immovable finger.Telopoditomere 1 and 2 at both sides covered by setae.Telopoditomere 3 only basally in anterior view with setae, remaining part, as well as telopoditomere 4 glabrous.
Distribution and habitat.Currently known only from the type locality.All specimens were crawling openly on the bottom of several holes in humid rocks (Fig. 6).The stark bright color invited collectors to pick them up.

Discussion
The new species can be distinguished from congeners by its greenish-blue color in combination with (1) a protruding coxal process, (2) a slender shape of the immovable finger, and (3) the shape of the posterior telopod.Yet, on the basis of morphology, little can be said about the relationships with other putatively closely related species.The overlapping COI p-distances and the phylogenetic tree were unable to resolve the relationships both within and among the genera Sphaerobe- lum and Zephronia.A similar unresolved tree was reported for the genus Sphaerobelum by Wesener (2019).Thus, the phylogenetic relationships of the Sphaerobelum and Zephronia species remain unclear and need further investigations.
The new species exhibits a striking greenish-blue coloration, by which it joins other brightly colored millipedes such as the genus Apheloria (A. polychroma) and the genus Desmoxytes (D. purpurosea, D. rubra and D. aurata).Species of this latter genus are not only very colorful, but they also have long spine-like paraterga.These characteristics are probably aposematic (Enghoff et al. 2007;Srisonchai et al. 2018).
The occurrence of the new species in Phu Pha Lom Forest Park is possibly correlated with the type of habitat/microhabitats.Due to its heterogenous topologies, its strongly irregular geomorphology and the good drainage of the limestone substrate, the type locality of S. turcosa sp.nov.probably provides suitable conditions to harbour a rich soil fauna.
With the discovery of S. turcosa sp.nov., the number of Sphaerobelum species in Thailand increases to four, which is still less than the number of Sphaerobelum species in Laos (10 species) and Vietnam (6 species) (Wesener 2019;Semenyuk et al. 2020), but more than in Cambodia and Myanmar, where hitherto no Sphaerobelum species have been reported (Likhitrakarn et al. 2014(Likhitrakarn et al. , 2015)).

Figure 1 .
Figure 1.The COI gene tree based on maximum likelihood analysis of cytochrome c oxidase I (COI) (660 bp).Numbers at nodes indicate branch support based on bootstrapping.Scale bar: 0.6 substitutions per site.# marks branches with <50% bootstrap support.The colored area marks Sphaerobelum turcosa sp.nov.

Figure 3 .
Figure 3. Sphaerobelum turcosa sp.nov.A-C ♂ holotype D, E ♀ paratype A first left coxa with stigmatic plate B coxa of second leg with gonopore C ninth right leg D coxa and prefemur of second leg with vulva E subanal plate.Scale bars: 0.5 mm.

Figure 5 .
Figure 5. Sphaerobelum turcosa sp.nov.♂ paratype, SEM micrographs of the endoterga on the midbody segment.A all areas of endotergum, posterior margin (pm), outer area (oa), middle area (ma), cuticular impressions (cp) and inner area (ia) B middle area and cuticular impressions C bristles D inner area.

Figure 6 .
Figure 6.Distribution and type locality of Sphaerobelum turcosa sp.nov.A distribution map of Sphaerobelum species in Thailand B, C limestone habitat at the type locality.

Table 2 .
Estimates of cytochrome c oxidase I (COI) sequence divergences (uncorrected p-distances) within and among Zephroniidae species and related taxa (rounded to two decimal places).