﻿Review of the pill millipede genus Hyperglomeris Silvestri, 1917 (Diplopoda, Glomerida, Glomeridae) with description of two new species from Laos

﻿Abstract The pill millipede genus Hyperglomeris Silvestri, 1917 is reported from Laos for the first time. Two new species, namely H.bicaudata Likhitrakarn, sp. nov. and H.inkhavilayi Likhitrakarn, sp. nov., from Houaphanh and Khammouane provinces, northern Laos, are described and illustrated based on morphological characters and molecular analyses. Sequences of COI gene were used as DNA barcoding markers, and successfully supported the accurate identification of other Glomeridae species. Interspecific divergence of the COI uncorrected p-distance between these new species and other Hyperglomeris species ranged from 7.84–13.07%, while the intraspecific divergence was 0.45% in H.inkhavilayisp. nov. and 5.3% in H.bicaudatasp. nov. The updated status of Hyperglomeris, a map of its distribution, and identification keys for all species are given.


Introduction
The pill millipede genus Hyperglomeris Silvestri, 1917 belongs to the family Glomeridae, order Glomerida. Members of this family and others in the order are capable of complete volvation, where the head and collum are tucked within the rolled-up body segments or ball-like shape when threatened. For this reason they are commonly referred to as 'pill millipedes'. Prior to this study, the genus Hyperglomeris consisted of only seven species, all narrowly endemic to northern Vietnam (Nguyen et al. 2019). Species of this genus are relatively small, ranging in length from 5.5 to 24 millimeters, and have a distinct body color. However, there is one colorless species, H. depigmentata Golovatch et al., 2013, which was found to inhabit a cave (Golovatch et al. 2013).
Hyperglomeris is classified within the subfamily Haploglomerinae, which has nine genera, the majority of which have only one or two species. The classification of these genera is still complicated due to few distinctive morphological characteristics, such as the presence of trichosteles on the prefemur or femur of the telopods, the number of apical cones on the antennae, and the number of striae on the thoracic shield (Wesener 2015a), and it is uncertain whether these characteristics represent species-level or genus-level distinctions. Therefore, it is essential to incorporate genetic information (as in our case, DNA barcoding based on COI gene sequences) in order to delineate species boundaries within this group. It can also be used as a foundation for further classification of the family.
Laos (or the Lao PDR) is a Southeast Asian country that shares borders with Myanmar and China to the north, Vietnam to the east, Cambodia to the south, and Thailand to the west. Laos is well-known for its beautiful mountains and forests, as well as its diverse ecosystems, which include tropical rainforests, dry lowlands, and hilly regions (ADB 2000). Previously, a total of 34 species of millipedes from 20 genera, 13 families, and seven orders have been recorded from Laos (Likhitrakarn et al. 2014). Among these, the Glomerida has remained represented by only four species of the genus Hyleoglomeris Verhoeff, 1910. In this study, we were fortunate to discover two new species of the genus Hyperglomeris from Laos. These two new species are investigated using an approach of integrative taxonomy, combining both morphological characters and a common DNA barcoding fragment of the COI gene. In addition, we have revised the scope of the genus, also providing its distribution map and an identification key to all nine species.

Morphological studies
Specimens were collected from Laos under the Animal Care and Use Protocol Review No. 1723018. Locations of collecting sites were recorded by GPS using a Garmin GPSMAP 60 CSx based on the WGS 84 datum, and all coordinates and elevations were double-checked with Google Earth. Photographs of live animals were taken using a Nikon 700D digital camera with a Nikon AF-S VR 105 mm macro lens. The specimens collected were euthanized by a two-step method following AVMA Guidelines for the Euthanasia of Animals (AVMA 2013). Specimens were then preserved in 90% ethanol for morphological and molecular studies. After 24 hr, ethanol was replaced with new 95% ethanol to prevent their defensive chemicals from interfering with future DNA extraction.
The holotype and all paratypes are housed in the Chulalongkorn University Museum of Zoology (CUMZ), Bangkok, Thailand. The specimens were examined, measured, and photographed under a Nikon SMZ 745T trinocular stereo microscope equipped with a Canon EOS 5DS R digital SLR camera. The acquired digital photos were processed and modified with Adobe Photoshop CS5. Line drawings were based on photographs taken under the stereo microscope equipped with a digital SLR camera.
The terminology used to describe morphological structures is consistent with the most recent publications (Golovatch et al. 2006;Golovatch 2017;Nguyen et al. 2019

DNA extraction, PCR amplification, and sequencing
Total genomic DNA was extracted from the legs and part of thoracic tissue of the paratype using the DNA extraction kit for animal tissue (NucleoSpin Tissue extraction kit, Macherey-Nagel, Germany), following the standard procedure of the manual. Fragments with size of 660 bp of the mitochondrial cytochrome c oxidase subunit I gene (COI) were amplified using LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3'; Folmer et al. 1994) and HCOoutout (5'-GTAAATATATGRTGDGCTC; Schulmeister et al. 2002) or Nancy (5'-CCCGGTAAAATTAAAATATAAACTTC-3'; Bogdanowicz et al. 1993). The PCR amplification was performed using a T100 thermal cycler (BIO-RAD) with a final reaction volume of 20 μL (15 μL of EmeraldAmp GT PCR Master Mix, 1.5 μL of each primer, 10 ng of template DNA and distilled water up to 20 μL total volume). Thermal cycling was performed at 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, annealing at 42-56 °C (depending on samples and the primer pair) for 60 s, extension at 72 °C for 90 s, and a final extension at 72 °C for 5 min. Amplification of PCR products was confirmed through 1.5% (w/v) agarose gel electrophoresis before purification by MEGAquick-spinTM plus (Fragment DNA purification kit) and sequencing in both directions (forward and reverse) using an automated sequencer (ABI prism 3730XL).
All nucleotide sequences obtained in this study were deposited in the GenBank Nucleotide sequences database under accession numbers OQ661871-OQ661874. The collecting localities and GenBank accession numbers of each nominal species are listed in Table 1.
Two phylogenetic methods were used in this study. Firstly, the maximum likelihood (ML) method was performed using RAxML v. 8.2.10 (Stamatakis, 2014), with GTRGAMMA as the nucleotide substitution model and 1,000 ML bootstrap replicates to assess topology bootstrap support (bp). Secondly, Bayesian Inference (BI) analysis was performed by MrBayes 3.2.6 (Ronquist et al. 2012) using the Markov chain Monte Carlo technique (MCMC), and with the best-fit model of nucleotide substitution as suggested by PartitionFinder2 v. 2.3.4 (Lanfear et al. 2016). The selected best-fit models for the three COI codon positions were SYM+G, F81+I, and GTR+G, respectively. The BI trees were run for ten million generations with a random starting tree. The resultant trees were sampled every 1,000 generations and the values were used to estimate consensus tree topology; bipartition posterior probability (bpp), and branch lengths, after the first 25% of obtained trees were discarded as burn-in. All average effective sample size (ESS) values sampled from the MCMC analysis were greater than 2,000 in all parameters. Both phylogenetic analyses were implemented through the on-line CIPRES Science Gateway (Miller et al. 2010 the COI sequence was evaluated using uncorrected p-distances as implemented in MEGA7 (Kumar et al. 2016). The missing data in pairwise comparison were treated with pairwise deletion method.
Diagnosis. Pill millipedes with four apical cones on the antennae; the caudal margins of the pygidium are sometimes modified into small paramedian lobes, but are mostly emarginate or slightly concave medially; leg-pair 18 devoid of any evident mesal outgrowths on the femur or tibia; the posterior telopods are rather stout, with prefemoral trichosteles reduced or only present as a small cone; and the femoral trichosteles are strongly reduced or absent.
Head and antennae black, only labrum and Tömösváry's organ yellowish. Venter and legs dark brown to brown with a pale yellowish claw and the posterior part of each tarsus; coloration in alcohol faded after eight years of preservation (Fig. 1E-G), body pale black to charcoal, with contrasting pale yellow to whitish bands. Head and antennae grey to blackish. Venter and legs pale brown to brownish.
Remarks. Unique to this species is that the caudal margin of the anal shield shows two more (♂, Figs 1C, E, F, G, 2A) or less (♀, Fig. 2A) pronounced paramedian knobs. That the male is equipped with such modifications is quite usual in various lineages of Glomerida (e.g., Liu and Golovatch 2020), but their presence in the female, albeit not as strongly as in the male, is really striking.
This distinguishing character can be hypothesized as possibly playing an important role in a courtship process or being associated with courtship behavior. Certain male structures dedicated to interactions with females during courtship have often diverged relatively quickly during evolution, causing these features to change into species-specific differences (Eberhard 2004). Noteworthy examples of such characters are antennae, legs and heads in springtails (Collembola: Bourletiellidae) (Kozlowski and Aoxiang 2006) and stridulation organs in giant pill millipedes (Sphaerotheria) (Wesener et al. 2011) that may not be involved directly in sperm transfer but are associated with mating behavior. In order to understand the relationship between these types of traits and their function in the glomerids, it is essential to examine the mating behavior of this species. Name. To honor Dr. Khamla Inkhavilay, the director of the Center of Excellence in Biodiversity at National University of Laos, Vientiane, Laos, who participated in collecting the type series.

Hyperglomeris inkhavilayi
Diagnosis. Although its color pattern seems to be similar to that of H. simplex Nguyen, Sierwald & Marek, 2019(Nguyen et al. 2019, it differs by the coloration of the collum, thoracic and anal shields (mostly pale yellowish to brownish vs. dark brown to blackish), leg-pair 18 (with a simple, subtriangular syncoxital notch (sn) vs. an evident pronounced syncoxital tubercles), coupled with a longer prefemoral trichostele (pt) (more than 2/3 vs. 1/2 of femur).
Coloration in alcohol faded after eight years of preservation (Fig. 4), body mostly yellowish, with contrasting black paramedian spots flanking the midline. Mid-dorsal spots on each of tergites 3-11 usually subtriangular (Fig. 4A, D), or parallel-sided ( Fig. 4E), with smaller and detached patches at caudal edge of tergite 2 and at anterior edge of tergite 12. Lateral sides of each of tergites 2-11 also with a pair of large, sublateral, yellow to marbled blackish spots beside the triangles, normally not reaching the translucent caudal and lateral edges (Fig. 4A, C-E). Head and collum pale yellowish to dark brownish with darker color laterally. Antennae black to dark brown, only tip of antennae yellowish. Legs and venter pale yellowish to pale brown (Fig. 4B).
Collum as usual, with two transverse striae. Thoracic shield with a small hyposchism field not projecting caudad to nearly reaching the tergal margin. Striae 5-7, mostly superficial, only lower 2 or 3 lying above schism, one level with schism, remaining 3 or 4 below schism, with 5 or 6 complete, crossing the dorsum. Terga 3 and 4 broadly rounded laterally (Fig. 4C, F). Following terga in front of anal shield rather clearly concave medially at caudal edge and with 2-4 striae starting above lateral edge. Male anal shield slightly concave medially at caudal edge (Fig. 4D, E).
Remarks. Unfortunately, the claw on the male legs 18 tarsus could not be observed, being broken off in both available male specimens, of which only one leg is available for examining the entire tarsus. Consequently, additional specimens are required to determine tarsal claws. Dorsum with a contrasting and brighter axial line against a darker side background (Fig. 4)  Collum, thoracic and anal shields mostly pale yellowish to brownish (Fig. 4). Leg-pair 18 with a simple, subtriangular syncoxital notch (sn) (Figs 3C, D, 5C). Prefemoral trichostele (pt) of telopod longer than 2/3 femur (Fig. 5E)

Phylogenetic analysis
The COI alignment (Table 1) was 660 bp in length and contained 23 individuals, including 21 taxa from the Glomeridae as ingroup and two taxa from the Zephroniidae as outgroup. All ten pill millipede species from seven genera of the family Glomeridae in this study were retrieved as monophyletic with strong support values (Fig. 7) (77-100% bp for ML and 0.96-1 bpp for BI). However, most relationships among these species were still unresolved (< 80% bootstrap values and < 0.95 bpp for BI).
The COI tree demonstrated that the genus Hyperglomeris is at least paraphyletic, because of the inclusion of Peplomeris magna in the same clade with H. bicaudata sp. nov., Hyperglomeris sp. 1, and H. inkhavilayi sp. nov., although with moderate nodal support; and the exclusion of H. simplex, which was placed distantly at the basal position to all Glomeridae. Each of the three Hyperglomeris species in this study (H. bicaudata sp. nov., H. inkhavilayi sp. nov., and H. simplex) was retrieved as a distinct clade/species with significant support (99-100% for ML; and 0.99 bpp for BI, except 0.58 in H. simplex).
The interspecific divergence based on COI uncorrected p-distance among the glomerid species in this study ranged from 8.81 to 16.45%, with an average of 13.07% (Table 2), and among Hyperglomeris species ranged from 8.81 to 12.48%, with an average of 11.23%. This analysis also demonstrated that the intraspecific divergence for H. bicaudata sp. nov. was 5.30% and for H. inkhavilayi sp. nov. was 0.45%.

Discussion and conclusion
This study has revealed two new species of Hyperglomeris, a genus new to the fauna of Laos. In addition, we have also refined the scope of the genus and the species distributions. These new records have increased the number of species of the order Glomerida in Laos from four (all in Hyleoglomeris) to a total of six. At present, the genus Hyperglomeris comprises nine species, mostly recorded from Vietnam (seven species), now also from Laos (two species) (Fig. 6). All Hyperglomeris species appear to be highly localized and endemic, with H. depigmentata probably a troglobiont, found exclusively in Hang Doi Cave. At two of these localities (Mount Mẫu Sơn and Vạn Mai), a coexistence of two species has been documented (Fig. 6). In addition to reporting these two new species, this study presents the southernmost record of Hyperglomeris in southern Laos. The distribution patterns (Fig. 6) clearly indicate that further new species of the genus can be expected from Laos, southern China and northern and/or eastern Thailand in the future.
The intraspecific distance within the new species ranged from 0.45 to 5.3%. This is comparable to the range of the Vietnamese glomerid species, Peplomeris magna (0.2%) and Rhopalomeris sauda (7.7%) (Nguyen et al. 2021). The mean intraspecific distance of R. sauda was rather high (6.84%) because of its extensive distribution (Nguyen et al. 2021), and the fact that the analyzed samples were gathered from multiple localities, Table 2. Matrix of the average uncorrected p-distance (%) based on 660-bp COI barcoding region between Hyperglomeris species and some related glomerid and sphaerotheriid taxa. Interspecific divergence is below diagonal and intraspecific divergence is in bold.  (Fig. 7). Peplomeris Silvestri, 1917 and Hyperglomeris are closely related genera found in the same country and classified in the same subfamily Haploglomerinae. Morphological characteristics of the genus Peplomeris are extremely similar to those of the genus Hyperglomeris, including the basic structure of posterior  Silvestri, 1917 and H. dirupta (Silvestri, 1917) Crossed circle H. simplex Nguyen, Sierwald & Marek, 2019 Open diamond H. nigra Golovatch, 2017 Inverted triangle H. conspicua Golovatch, 1983 andH. maxima Golovatch, 1983 Circle H. depigmentata Golovatch, Geoffroy & VandenSpiegel, 2013 Triangle H. bicaudata Likhitrakarn, sp. nov. Square H. inkhavilayi Likhitrakarn, sp. nov. telopods with reduced or eliminated prefemoral and femoral trichosteles. However, Hyperglomeris has only four apical cones on their antennae, whereas Peplomeris has numerous apical cones (Wesener 2015a;Nguyen et al 2019). Consequently, it is not surprising that the genetic relationship between the members in these two genera is very close. Despite this, it is premature to make conclusions about the relationships among the two genera based on the results of this study; additional persuasive evidence (i.e., more taxa and genetic markers) is needed to clarify the taxonomic status of both genera.
DNA sequence analysis was previously introduced and has provided a more reliable identification of glomerid species (Nguyen et al. 2019(Nguyen et al. , 2021Liu and Golovatch 2020). The present paper provides a phylogenetic analysis of ten members of seven genera within the family Glomeridae, including three new sequences from two recently discovered species. Unfortunately, the phylogenetic relationships among these genera is still not supported, which is consistent with studies by Liu and Golovatch (2020) and Nguyen et al. (2021). Hence, data from the COI gene alone are not sufficient to confirm the relationship between genera within this millipede family. We recommend including more genes such as 16S, 28S ribosomal RNA or other advanced molecular techniques (i.e.,  transcriptomic and phylogenomic data) in future studies to clarify phylogenetic relationships (Means et al. 2021;Benavides et al. 2023). Nonetheless, our findings regarding Hyperglomeris demonstrate that the sequencing of the COI gene is still beneficial for species delimitation and facilitates accurate identification among glomerid species.