﻿The millipede family Polydesmidae Leach, 1816 (Diplopoda, Polydesmida) from Vietnam, with a description of a new cavernicolous species

﻿Abstract The millipede family Polydesmidae Leach, 1816 is reviewed in the scope of the Vietnamese fauna. The distribution of the species, Polydesmusvietnamicus Nguyen, 2009 is extended northward to Ha Giang Province. A new cavernicolous polydesmid, Pacidesmustuachuasp. nov., is described from two caves in northwestern Vietnam, representing the first record of the genus from Vietnam. Extensive illustrations and DNA barcodes are provided for both species, a revised key is presented to all 12 species of Pacidesmus Golovatch, 1991, as well as a key to all eight genera of Asian Polydesmidae.

The present paper updates the knowledge of the family Polydesmidae in Vietnam, with the description of a new cavernicolous species found in northwestern Vietnam.An updated key to all 12 Pacidesmus species is also presented, as well as a key to all eight genera of Polydesmidae reported so far from Asia.

Materials and methods
Millipede specimens were hand-collected from forests and caves in northern Vietnam and preserved in 85-90% ethanol.Morphological characters were investigated with an Olympus SZX16 stereomicroscope.Gonopods were dissected for morphological examination and photographed.Colour images were taken at various focal planes using a Nikon imaging system (Nikon-Br) coupled with a SMZ800N Nikon stereomicroscope.UV images were taken using a Sony a6000 digital camera attached to the aforementioned SMZ800N Nikon stereomicroscope under the UV flashlight Nichia Convoy.Images were stacked using Helicon Focus version 7.0 and assembled in Adobe Photoshop CS6.Scanning electron microscope (SEM) images were taken using the system Prisma E (ThermoFisher Scientific) in the Institute of Ecology and Biological Resources.
Total DNA was extracted using Qiagen DNeasy Blood and Tissue Kits.A 680-bp fragment of the mitochondrial gene, cytochrome c oxidase subunit I (COI), was amplified and sequenced using a pair of universal primers, LCO1490 and HCO2198 (Folmer et al. 1994).Polymerase chain reaction (PCR) conditions for amplification of the COI gene follow those of (Nguyen et al. 2017).The successfully amplified PCR products were sent to the FirstBase Company (Malaysia) for purification and sequencing.COI sequences were checked and confirmed using BLASTN 2.6.0+search (Zhang et al. 2000) and registered for GenBank accession numbers.
Morphological terminology follows Liu and Golovatch (2020).All specimens reported here, including types, are deposited in the Institute of Ecology and Biological Resources (IEBR), Vietnam Academy of Science and Technology, Hanoi, Vietnam.

Abbreviation
IEBR-Myr Institute of Ecology and Biological Resources, Myriapod collection.Remarks.Polydesmus is certainly the largest genus within the family Polydesmidae, with over 200 species and subspecies which mainly occur in Europe and the Mediterranean, west of the central Caucasus (Hoffman 1980;Djursvoll et al. 2001).A few species have been found in the Oriental Region, these being Polydesmus japonicus Miyosi, 1956, P. miyosii Murakami, 1966, P. tanakai Murakami, 1970, and P. tangonis Murakami, 1973 from Japan; P. moorei Pocock, 1895 and P. paludicola Pocock, 1895 from eastern China; P. liber Golovatch, 1991 from Hong Kong, southern China (Golovatch 1991); and P. vietnamicus Nguyen, 2009, the only species of the genus from northern Vietnam (Nguyen 2009).The two old species of Pocock (1895) from mainland China are only provisionally to be assigned to Polydesmus, as both require revision.The distribution of Polydesmus seems to be amphi-Palaearctic (Golovatch 1991).Diagnosis.A typical polydesmid with 20 body rings and three transverse rows of bosses with setae on metaterga.Gonopodal solenomere rather well developed, conspicuously shaped.Endomere elongate and strongly falcate, directed caudally, starting laterally and basally of recurvature point of seminal groove, set off from femorite by a sulcus, with a pair of strong teeth at about midway (mt).Seminal groove largely mesal, crossing the femorite diagonally, terminal lateral loop relatively short and turning around a distofemoral process (ap).Solenomere (sl) short, but evident and bifid.

Polydesmus vietnamicus
The species differs from the morphologically particularly similar Polydesmus liber Golovatch, 1991 in being larger (33.0-38.4mm vs 21.0-23.0mm in length) and in the gonopod endomere (with a pair of teeth at about its midlength vs with two pairs of moderate teeth at 1/3 and 2/3 of its length).
It is particularly noteworthy that all East and Southeast Asian species undoubtedly belonging to Polydesmus, however few, share the symplesiomorphy of densely setose gonopod coxites, which contrasts with very poorly setose ones observed in the much more numerous western Palaearctic counterparts (Golovatch 1991).
Remarks.This species was previously known from only its type locality, Tam Dao National Park (Nguyen 2009).Currently, its distribution is extended northward to Ha Giang Province.There are no significant morphological variations between the type specimens and those samples collected in Ha Giang.
The species is truly cavernicolous, characterized by white or unpigmented colour and living within a cave.As a troglobiont species, it groups with all 12 troglobiont or troglophile congeners from China (Table 1).However, this species differs from all of these, except P. bifidus from the Hengli Xin Don Cave, Guangxi Province in the absence of an exomere and the gonopod telopodite showing no additional processes; the endomere also has two additional processes, and the tip of the endomere bears two tiny teeth.The new species is similar to P. bifidus in having a troglomorphic appearance, the absence of an exomere, and a bifid tip of the endomere, but it differs in having two tiny teeth at the tip of the endomere tip (vs two long processes in P. bifidus).The new species is assigned to Pacidesmus because of the following characters: the seminal groove starts mesally, then recurves laterad at the base of a particularly prominent endomere branch to enter an accessory seminal chamber that opens on a setose pulvillus; endomere bears additional processes.
Etymology.The specific epithet is treated as a noun in apposition and is based on the "Tua Chua" district where the two caves are located.
Legs generally long and slender, apparently slightly incrassate, approximately 1.7-1.8×as long as midbody height, densely setose, almost all setae simple, poorly branching setae with minute, distal, side branchlets only on slender prefemora, latter devoid of lateral bulges.
DNA barcode.Two COI sequences (661 bp) were uploaded to the GenBank with the accession numbers PP118040 and PP118041.The new species has a close COI identity to Epanerchodus koreanus (NC051495) at 88.72% (query coverage 97%).
Habitat.This species is to be considered a true troglobiont because it shows the typical morphological features of a cave-dweller.It was collected exclusively in the dark zone of the caves as described below.Kho Chua La and Xa Nhe caves are both located close together, approximately 500 m in distance.These caves are at the centre of the Xa Nhe commune, Dien Bien Province, northwestern Vietnam.The two caves are tunnel-like: they are high (15-20 m), wide (15-20 m), and long (1,000-1,500 m).The floor is mainly wet, with clay, and some small pools.Several other millipede species have been found in these caves, including Glyphiulus sp.(Spirostreptida, Glyphiulidae) and Eutrichodesmus sp.(Polydesmida,: Haplodesmidae).The new species was found >1000 m from the entrance.
Kho Chua La and Xa Nhe caves are located on the Tua Chua karst plateau in northeastern Dien Bien Province, northwestern Vietnam.The natural area is about 68,414 ha, and 70% of this area is composed of limestone mountains, which are known for their layers of majestic rugged rock and unique natural landscape.The karst region contains many stunning and well-known caves, such as Kho Chua La, Tham Khem, Hau Chua, Xa Nhe, and Pe Rang Ki (Nguyen et al. 2022).Furthermore, the Tua Chua karst plateau of northwestern Vietnam is close to the   Remarks.While there remains a noticeable geographical gap between the mountainous northern Thailand species and the troglobionts of southern

Identification key to species of the genus Pacidesmus Golovatch, 1991
Modified and updated from Golovatch et al. (2010)

Discussion
Beyond southern China and Southeast Asia, there are eight macropolydesmid genera occurring in Asia, mainly in Central Asia.The main differences between those genera are presented in Table 2 below.Pacidesmus tuachua sp.nov. is distinguished from members of Nipponesmus by having a gonopod endomere; the gonopod telopodite has neither a comb of setae nor slender teeth (vs without endomere, with a conspicuous comb of setae or slender teeth) (Golovatch et al. 2011).The new species differs from members of Gleninea in the absence of a gonopod exomere, the gonopod endomere is with only two additional processes, the absence of an accessory seminal chamber (vs the presence of a gonopod exomere, the distal part of the endomere carrying numerous or several spine-like hairs or strong, sometime curved spines, and the presence of an accessory seminal chamber) (Golovatch and Geoffroy 2014).
The new species is also distinguished from Schizoturanius and Uniramidesmus species by its larger size (16.3 mm vs less than 10.0 mm).The new species could possibly be assigned to the genus Uniramidesmus based on the simple, slender, falcate gonopod; however, Uniramidesmus species are all much smaller (<10.0 mm in length), the gonopods are strongly falcate to coiled caudally and cross each other when in situ; the opening of the seminal groove is subterminal to terminal on a bare to more or less pubescent pulvillus (Mikhaljova 2004).On the contrary, the new species is far larger in size (ca 16.3 mm in length), the gonopods in situ are well separated from each other, and the opening of the seminal groove is on a typical hairy pulvillus.
Compared to Schizoturanius (Mikhaljova 2004), Pacidesmus tuachua sp.nov.differs well-developed paraterga (vs a strongylosomoid, moniliform body with narrow to almost missing paraterga, which are mostly smooth and only seldom laterally incised); also, the gonopod is without an accessory seminal chamber (vs with an accessory seminal chamber) and there is no gonopod femoral process (vs with a characteristic femoral process).Finally, the new species can hardly be assigned to the genus Jaxartes, which is confined to Central Asia, due to its

No. Genus Diagnosis Distribution
1 Schizoturanius Body small, strongylosomoid (= without prominent paraterga), moniliform; paraterga narrow, only seldom incised laterally; gonopods falcate and bifid distally; an accessory seminal chamber present; gonopod femorite carrying a characteristic process (Mikhaljova 2004) Ten species in Central Asia and Ukraine, Asian part of Russia, Kazakhstan, northwestern China (Mikhaljova 2004;Nefediev 2023) 2 Uniramidesmus Body small (usually ca 10 mm long); head covered with dense minute hairs; antennomeres 5-7 each with a small field of tiny bacilli dorsally; metaterga rather convex; paraterga small or medium-sized and set below dorsum, with marginal incisions; metatergal polygonal sculpture ranging from well-developed to poorly-developed; metatergal setae pointed; sphaerotrichs present or absent; gonopods slender, strongly falcate to coiled caudally, relatively simple, in situ crossing each other; seminal groove with a loop parabasally; accessory seminal chamber absent; opening of seminal groove subterminally to terminally on a bare to more or less pubescent pulvillus (Mikhaljova 2004(Mikhaljova , 2017) ) Ten species in central Asia and Asian part of Russia (Mikhaljova 2004(Mikhaljova , 2017) ) 3 Jaxartes Body small (usually ca 10 mm long); metaterga with bosses/tubercles with bacilliform or trichoid setae; paraterga clearly incised laterally; four distal ♂ podomeres with ventral sphaerotrichomes; gonopod coxite without outgrowths other than cannula; the gonotelopodite particularly slender, suberect, with the endomere being considerably longer than a ventrally fringed process (if present at all), also bearing a parabasal tooth and a subtruncate apex, basally with a very evident hairy pulvillus, but no distinct accessory seminal chamber (Antić et al. 2019) Twelve species in Central Asia (Antić et al. 2019) 4 Epanerchodus Gonopod endomere mostly absent, rarely present as only a more or less rudimentary structure, while the seminal groove after the recurvature point still makes a long way basad to debauch into a prominent, simple-haired, accessory seminal chamber placed at the bottom of a profound parabasal cavity in the telopodite (Liu and Golovatch 2018;Golovatch 2021).

Nipponesmus
Body size large (up to 20 mm in length).Paraterga broad.Gonopod endomere with conspicuous comb of setae or slender teeth.The seminal groove running mostly mesally to recurve neatly between exomere and endomere, then to debauch somewhat basally into a prominent hairy pulvillus which also beset with the same peculiar trichome, and is devoid of an accessory seminal chamber (Golovatch et al. 2011) Three species in Japan and Taiwan.(Golovatch et al. 2011) 7

Gleninea
Body small to large size (up to 16 mm in length).The third pair of ♂ legs only slightly thickened.

Polydesmus
Body size medium to large.Paraterga usually wide.Gonopod solenomere absent to rather well developed, sometimes conspicuously shaped.Exomere from short and slightly curved to very long and strongly falcate, mostly uniramous, directed caudally, starting laterally or apically and basally of recurvature point of seminal groove.Seminal groove, largely mesal; terminal laterad loop relatively short and turning around a distofemoral process (Djursvoll et al. 2001) About 200 species distributed mainly in the Mediterranean; a few species in East Asia and northern Vietnam (Djursvoll et al. 2001) larger body size (16 mm long), the slender, strongly falcate gonopods without an accessory seminal chamber.On the contrary, the genus Jaxartes is diagnosed by its small body (usually ca 1 cm long); the metaterga show bosses or tubercles with bacilliform or trichoid setae, the paraterga are clearly laterally incised, there are four distal male podomeres with ventral sphaerotrichomes, the gonopod coxite is without outgrowths apart from the typical cannula, and the gonotelopodite is particularly slender, suberect, and with the endomere being considerably longer than a ventrally fringed process (if present at all); also, the endomere bears a parabasal tooth and has a subtruncate apex, basally with a very evident hairy pulvillus, but there is no distinct accessory seminal chamber (Antić et al. 2019).Pacidesmus tuachua sp.nov.can hardly be placed in Epanerchodus or Polydesmus because its paraterga are relatively narrow, the seminal groove starts mesally, as usual, then is recurved laterad at the base of a particularly prominent endomere branch to enter an accessory seminal chamber that opens on a setose pulvillus, and the endomere bears additional processes.
The strongly sigmoid gonopodal telopodite in P. tuachua sp.nov is somewhat unusual in comparison to that in other Pacidesmus species.This difference may suggest a new genus; however, it currently seems best assigned to Pacidesmus based on the above discussion.It is noteworthy that most Schizoturanius or Polydesmus spp.likewise show only slightly curved gonopod telopodites, but relatively few species in these genera are so strongly sigmoid.
To support further study of Polydesmidae in Vietnam and Southeast Asia, an identification key to macropolydesmid genera occurring in Asia is provided:

Figure 1 .
Figure 1.Records of polydesmid species in Vietnam and of all known Pacidesmus species.

Figure 2 .
Figure 2. Polydesmus vietnamicus Nguyen, 2009 from Ha Giang Province (IEBR-Myr 808) A-C ♂.Anterior part of body A dorsal view B lateral view C ventral view D ♀, anterior part of body, ventral view.Scale bars: 1 mm.

Figure 5 .
Figure 5. Polydesmus vietnamicus Nguyen, 2009 from Ha Giang Province (IEBR-Myr 808) ♂, under the UV light A posterior part of body, dorsal view B posterior part of body, ventral view C telson, dorsal view D telson, ventral view.Scale bars: 1 mm.

Figure 12 .
Figure 12.Pacidesmus tuachua sp.nov., holotype ♂ (IEBR-Myr 951) A anterior part of body, under the UV light, dorsal view B segments 8-10, dorsal view C posterior part of body, dorsal view D posterior part of body, ventral view.Scale bars: 1 mm.

Table 2 .
Morphological diagnoses and distribution of all eight macropolydesmid genera in Asia.