Four new species of the millipede genus Eutrichodesmus Silvestri, 1910 from Laos, including two with reduced ozopores (Diplopoda, Polydesmida, Haplodesmidae)

Abstract Laos has large areas of primary forest with a largely unexplored fauna. This is evidenced by millipedes, class Diplopoda, with fewer than 60 species being recorded from the country. In the widespread Southeast Asian “Star Millipede” genus Eutrichodesmus Silvestri, 1910 (family Haplodesmidae), only two of 49 recorded species have been found in Laos. Four new species of Star Millipedes are here described from caves in Laos: Eutrichodesmus steineri Liu & Wesener, sp. n., Eutrichodesmus deporatus Liu & Wesener, sp. n., Eutrichodesmus paraster Liu & Wesener, sp. n. and Eutrichodesmus parvus Liu & Wesener, sp. n.. A fifth species, for which only a female is available, remains unnamed. The defensive glands (ozopores) are found to be strongly or entirely suppressed in two of the new species, Eutrichodesmus deporatus Liu & Wesener, sp. n. and Eutrichodesmus paraster Liu & Wesener, sp. n., both troglobionts, which is new to the family. All of the Star Millipedes were collected during Northern Lao-European Cave Project faunal surveys conducted by the Senckenberg Museum, Frankfurt. A key to the six species of Eutrichodesmus currently known to occur in Laos is provided.


Introduction
The documenting of biodiversity and the subsequent taxonomic descriptions of undescribed species have been highlighted as one of the most urgent research programmes of our planet, as indicated by the declaration of the "United Nations Decade on Biodiversity", as well as the signing of the UN "Convention on Biological Diversity" by numerous countries (e.g., Wheeler 2008;Padial et al. 2010;Popescu 2015). Laos, a landlocked, largely montane, tropical country in Southeast Asia, is one of the highly biodiverse Great Mekong countries, which together have yielded more than 2200 new species since 1997 (WWF 2016). Furthermore, in contrast to its neighbours China, Vietnam and Thailand, the forests in Laos still remain relatively intact (STEA 2000). However, this might change in the near future as illegal logging and timber smuggling to Vietnam are being conducted on a large scale (EIA 2011;Smirnov 2015;Gan et al. 2016). The very large amount of still undescribed biodiversity in Laos is especially evident in arthropods, including the large, ecologically important, mostly sylvicolous and mesophilous millipedes, class Diplopoda. Most diplopods are detritivores whose primary habitat is forest litter and topsoil, but many species live in caves, dead wood, suspended soil or even tree canopies (e.g., Golovatch and Kime 2009). Diplopoda are an ancient, diverse and widespread group, with fossils dating back to the Silurian (Edgecombe 2015) and with about 12,000 described species in >3,000 recognized genera, >150 families and 16 orders (Minelli 2015). Since the bulk of global millipede diversity is confined to tropical forest, which is a rapidly shrinking biome, and because diplopods are poor dispersers that are largely confined to forests and woodlands, and are prone to strongly localized endemism (e.g. Wesener 2009;Car and Harvey 2014;Enghoff 2015), the problem of documenting millipede faunas is increasingly acute (Golovatch and Kime 2009). These localized occurrences make millipede species important subjects for biogeographic studies (Stoev and Enghoff 2003;Wesener et al. 2010;Wesener et al. 2011), but also put them at risk of local extinction from human activities such as forest destruction or large-scale mining operations (Wesener and Wägele 2007;Iniesta et al. 2012). This holds especially true for Laos where the pace of forest destruction is alarmingly high (Gan et al. 2016).
Fortunately, fresh collections of Laotian millipedes have encouraged recent taxonomic studies, and since the latest checklist for the country which listed 34 species (Likhitrakarn et al. 2014a), another 23 have been added (Likhitrakarn et al. 2014a(Likhitrakarn et al. , 2014b(Likhitrakarn et al. , 2014c(Likhitrakarn et al. , 2015a(Likhitrakarn et al. , 2015b(Likhitrakarn et al. , 2016a(Likhitrakarn et al. , 2016bGolovatch 2016aGolovatch , 2016bGolovatch et al. 2016aGolovatch et al. , 2016b. Still, the achievement is modest, as the faunas of the adjacent Vietnam, Thailand and southern China comprise from >100 to a few hundred millipede species each (e.g., Enghoff et al. 2004;Enghoff 2005;Golovatch 2015). In addition, only seven of the 16 orders of Diplopoda have been recorded so far in Laos, the most species-rich being the Polydesmida.
Within the Polydesmida, the tiny species of "Star Millipedes", genus Eutrichodesmus Silvestri, 1910 (family Haplodesmidae), with their often conspicuous dorsal projections (e.g., Fig. 1), together with the larger "Dragon Millipedes", genus Desmoxytes Chamberlin, 1923 (family Paradoxosomatidae, see Liu et al. 2014Liu et al. , 2016, are among the most remarkable diplopods in Southeast Asia. Eutrichodesmus is one of the most speciose genera of SE Asian millipedes, presently containing 49 described species (Golovatch et al. 2015(Golovatch et al. , 2016a. The genus is distributed from southern Japan in the north, through southern China and Indochina, to Vanuatu, Melanesia in the south. Most species are strongly localized country endemics, this being especially true of the rather numerous cavernicoles. Laos is situated more or less north-centrally within the distribution range of the genus, but only two Laotian species have been named so far: E. multilobatus VandenSpiegel, 2009, andE. nadan Golovatch, Geoff roy, Mauriès &VandenSpiegel, 2016. Both are highly localized endemics found in caves and are presumed troglobites (Golovatch et al. 2009a.
Below we describe four new species and provide a key to all six members of Eutrichodesmus currently known to occur in Laos. In addition, for the first time in the family we report strongly or completely suppressed ozopores, which is unusual because all previously described Eutrichodesmus seem to show normal pore formulae: 5,7,9,10,12,13,[15][16][17][18][19].

Material and methods
Specimens were collected for the Northern Lao-European Cave Project, and kept in 70% ethanol. The holotypes and a number of paratypes are deposited in the zoological collection of the Senckenberg Research Institute and Natural History Museum (SMF), with some material also to be housed in the Zoological Research Museum A. Koenig (ZFMK).
Observation and dissections were performed using an Olympus SZ51 stereo microscope. The line drawings were prepared with the help of an Olympus BX51 microscope and an attached camera for the scope. SEM micrographs were taken using a ZEISS Sigma 300VP scanning electron microscope (based at the ZFMK). Dry SEM material was coated with gold, removed after study from stubs and returned to alcohol. The photographs were taken with Canon EOS 7D cameras and further processed using Adobe Photoshop CS6 software.
The terminology used here follows that of Golovatch et al. (2009aGolovatch et al. ( , 2009b. Metatergum 4 with a high mid-dorsal projection (Figs 1C,8E). Gonopod with a large, lateral, denticulate, distofemoral process (dp); acropodite with a very small mesal tooth (t) subapically (Fig. 10)   Diagnosis. Differs from other species of the genus in showing laterally 3-lobulated paraterga and the extremely high mid-dorsal projections on metaterga 5-19, the latter character very similar to that observed in E. macclurei Hoffman, 1977, from western Malaysia (Hoffman 1977). However, E. steineri sp. n. is distinct from E. macclurei in the gonopod, which has a long, digitiform, distofemoral process, vs. a short spiniform process in the counterpart. See also Key above.
Mandible with a movable external tooth (et), an internal tooth (it) with four cusps; six pectinate lamellae (pl) consisting of long, smooth teeth; intermediate area (ia) covered with small cuticular scales; a large, stairs-like molar plate (mp) close to anterior fringe with pin-like structures (Fig. 3C).  Collum subtrapeziform (Fig. 2D), slightly broader than head, not covering the latter from above; dorsal surface with six transverse rows of round microvillose tubercles, flattened medially (Fig. 2D); each tubercle crowned by a 2-segmented seta, these setae being mostly abraded. Frontal margin slightly elevated (Fig. 2D).
Vulvae lying inside a membranous sac, each vulva consisting of a large horseshoeshaped operculum (op) and a bursa (b) with several long setae (Fig. 3O). Description. Length of adults of both sexes ca. 7.5-8.0 mm, width 0.6-0.8 mm and 1.6-1.8 mm on midbody pro-and metazona, respectively.
Remarks. The specimens come from the Cave Tham Pathok which is about 100 meters long, and the temperature inside is about 15 °C. The animals were found living at a small waterfall at a distance of 20 meters from the entrance. In addition, Heteropoda spp. (Arachnida) and Glyphiulus sp. (Diplopoda, Cambalopsidae) were found in the cave (Steinmetz 2007).
The pallid body and long legs suggest that E. deporatus sp. n. is most likely a troglobite.  Paratypes. 1 female (SMF), same data as holotype; 1 juvenile (ZFMK MYR6131), same data.

Eutrichodesmus paraster
Etymology. To emphasize the similarity to E. aster Golovatch, Geoffroy, Mauriès & VandenSpiegel, 2009;adjective. Diagnosis. Differs from other species of the genus primarily by the completely reduced ozopores. Superficially very similar to E. aster, but distinguished from the latter through the smaller body, laterally 3-lobulated paraterga, and the relatively complex gonopod showing a large, laterally denticulate, distofemoral process; the acropodite subapically has a very small mesal tooth and an evident, digitiform, dorsal lobule. See also Key above.
Remarks. As this species was collected in a cave, and has a pallid body it appears to be a real troglobite. The absence of ozopores is unique for the family Haplodesmidae. Paratypes. 1 male (SEM), (ZFMK MYR6132), 1 female (ZFMK MYR6128), same data as holotype; 2 females (SMF), same data.
Remarks. These specimens do not fit any of the six Eutrichodesmus described from Laos, base on somatic characters alone. As only one adult female is available, we refrain from naming this species.

Discussion
All attempts to extract the DNA from some of the specimens using the DNeasy Blood & Tissue kit from Qiagen were unsuccessful. One reason might be the specimens had been preserved in low-concentration ethanol for nearly ten years. In addition, it was not at all easy to get enough tissue from legs of these tiny specimens for DNA extractions.
All material of Eutrichodesmus from Laos treated here was collected opportunistically by a research group focusing on Arachnida, headed by Peter Jäger (SMF). Because Eutrichodesmus species hide in the soil and are small and often coiled, it is possible they were missed during searches focused on Arachnida. It can only be hoped that more efforts to investigate and describe the highly unique and diverse diplopod fauna of Laos will be undertaken in the future, before the utilization of natural resources leads to the loss of the existing natural forests, something that has already happened in the last decades in neighbouring nations (Sodhi et al. 2010).