A conspicuous Tylopus species is described from Northern Vietnam, namely T. helicorthomorphoides sp. nov. The new species is clearly diagnosed by the gonopodal solenophore completely sheathing the solenomere, both being coiled three times, and the absence of spine z and process h of the gonopod. Fragments of the COI and 16S rRNA genes were extracted, and the phylogenetic analysis also supports the new species.

Biodiversity, COI, 16S rRNA, mountain fauna, taxonomy

The genus Tylopus was established by Jeekel (1968) with type species Agnesia sigma Attems, 1953. Jeekel (1965, 1968) diagnosed this genus by having well-developed paraterga, first pair of legs without modifications, the presence of tibial and tarsal brushes and adenostyles (= ventral tubercles on the podonomeres), gonopod with somewhat distally enlarged femorite, a distinctly laterally demarcated postfemoral region, postfemorite with 1–3 processes, and both lamina medialis and lamina lateralis well developed. The genus was extensively revised, and morphological terms for the genus Tylopus were standardized by Golovatch and Enghoff (1993) and updated by Likhitrakarn et al. (2010, 2016).

Tylopus is considered the most species-rich genus within the family Paradoxosomatidae Daday, 1889. Currently, it contains 77 species (Golovatch and Semenyuk 2021; Likhitrakarn et al. 2021; Sierwald and Spelda 2021) distributed from Southern China down to Indonesia and Malaysia, and from Myanmar to Vietnam. They are seemingly dominant in Southeast Asia, especially in Thailand, Laos, and Vietnam, although this may be due to research bias. In Vietnam, 21 species were recorded so far (Attems 1938, 1953; Golovatch 1984; Korsós and Golovatch 1989; Golovatch and Enghoff 1993; Nguyen 2012; Golovatch and Semenyuk 2018, 2021), all of which completely agree with the typical diagnosis for the genus.

As a continuation of our contributions to the millipede fauna of mountainous regions of Vietnam, this work aims to describe a conspicuous Tylopus species from northern Vietnam and to discuss relationships between Vietnamese Tylopus species.

Material was collected from two localities in Northern Vietnam, Tam Dao National Park and Bac Me Natural Reserve (Fig. 1), and preserved in 90% ethanol. Specimens were observed under an Olympus SZX10 microscope. Images were taken at various focal planes using a Nikon imaging system (Nikon-Br) coupled with a SMZ800N Nikon stereomicroscope. Images were stacked using Helicon Focus version 7.0 and assembled in Adobe Photoshop CS6.

Figure 1. 

Distribution of Tylopus helicorthomorphoides sp. nov. Tam Dao (Red triangular); Bac Me (Red circle)

Total DNA was extracted from several midbody legs using the QIAGEN DNeasy Blood & Tissue Kit. Fragments of the mitochondrial cytochrome c oxidase subunit I (COI) and 16S rRNA genes were amplified using two pairs of primers: COI-1F (5’-ACTCTACTAATCATAAGGAT-3’) and COI-1R (5’-TAAACCTCCGGGTGACCAA-3’), 16S-1F (5’-CCGGTTTGAACTCAGATCA-3’) and 16S-1R (5’-TGACTGTTTAGCAAAGACAT-3’). The amplification protocol followed a previously published method by Nguyen et al. (2017). Each successfully amplified and sequenced fragment was assembled using ChromasPro v.2.1.8 and confirmed by BLAST 2.6.0+ searches (Zhang et al. 2000).

All confirmed sequences were aligned using Cluster X ver.2.0 (Larkin et al. 2007), and ambiguous nucleotide sites and gaps were removed using MEGA X (Kumar et al. 2018). The COI sequences were translated into amino acids for confirmation using transversion code in MEGA X. The concatenated dataset (COI+16S) was created from the COI dataset and 16S rRNA dataset using the software GBLOCK 0.91b.

The nucleotide frequencies were statistically calculated using MEGA X. The final dataset contains 956 bp, including 586 bp of the COI gene and 370 bp of the 16S rRNA gene. It has nucleotide frequencies of 26.1, 38.3, 17.5, and 18.0 for A, T, G, and C, respectively. The GC content accounts for 35.5% of total nucleotides. The dataset has 324 parsimony-informative and 403 variable sites.

The phylogenetic tree was reconstructed using the Maximum Likelihood (ML) analysis with the best model resulting the ModelFinder (Kalyaanamoorthy et al. 2017), performed in IQTREE ver.1.6.2 for Windows (Minh et al. 2020). Models with the lowest BIC scores (Bayesian Information Criterion) are considered to describe the best substitution pattern. As a result, the model TIM3+F+G4 was selected (BIC score of 12506.102; -LnL = 6033.252).

All specimens, including holotype and paratypes and DNA vouchers, have been deposited in the Department of Soil Ecology, Institute of Ecology and Biological Resources (IEBR), Vietnam Academy of Science and Technology, Hanoi, Vietnam. A total of 24 new sequences were deposited in GenBank, with accession numbers as presented in Table 1.

Both Jeekel (1965) and Golovatch and Enghoff (1993) agreed that the gonopod solenophores sheath the flagelliform solenomere for its greater part, curving caudad first, then laterad and finally cephalad in Tylopus members. In the new species, the solenophore and solenomere morphology agree with this statement but are completely coiled three times, the gonopod spine z and process h are absent, whereas only postfemoral lamina l is present. Our phylogenetic analysis also supports the taxonomic position of the new species within the genus Tylopus.

The genus Tylopus is, phylogenetically, divided into two clades as reported above. This also agrees with the results presented by Anh et al. (2021) using only a fragment of the COI gene. It is, therefore, suggested that the genus Tylopus needs to be revised accordingly, using both morphological and molecular data. This case may be similar to that of the genus Desmoxytes Chamberlin, 1923, which was recently intensively revised by Srisonchai et al. (2018a, 2018b, 2018c, 2018d). Finally, the genus Hylomus Cook & Loomis, 1924 has been re-validated, with three new genera proposed for other so-called Desmoxytes members (Srisonchai et al. 2018a, 2018b, 2018c, 2018d). Furthermore, the relationship between Tylopus species was very poorly supported (less than 70% bootstrap value), especially within the second lineage. More samples and species are required for a better analysis.

The genus Tylopus has been found in both lower and higher lands in northern Vietnam, but it has only been recorded in higher lands in southern Vietnam, and has never been found in the Mekong delta (Nguyen 2012; Golovatch and Semenyuk 2018, 2021). The distributional pattern of this genus in Vietnam is similar to that in Thailand, where Tylopus species are mostly found in mountainous regions with altitudes of more than 500 m in northern and central Thailand (Likhitrakarn et al. 2010). The highest recorded altitude is 2,300m in Ngoc Linh Mt. (Vietnam) for Tylopus hilaris (Attems, 1937) and T. phanluongi Nguyen, 2012. Furthermore, as mentioned by Nguyen et al. (2019, 2021), the mountainous region of Vietnam harbors a rich biodiversity in Vietnam, but it is far from completely known because access to the region is difficult and intensive surveys are still lacking.

With the new species described herein, the number of Tylopus species known for Vietnam increases to 22. However, this number is still far from representing the true diversity of the genus in Vietnam. More intensive surveys will reveal more new discoveries, especially in the diverse high mountainous regions of Vietnam, which remain underexplored.

This research was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED), under grant number 106.05-2019.320 to Anh D. Nguyen. The work was also supported by the Tokyo Metropolitan University Fund for TMU Strategic Research (Leader: Prof. Noriaki Murakami; FY2020-FY2022).