A revision and phylogenetic analysis of the millipede genus Oxidus Cook , 1911 ( Polydesmida , Paradoxosomatidae )

The genus Oxidus Cook, 1911 is revised to contain five species, O. avia (Verhoeff, 1937), O. gigas (Attems, 1953), O. gracilis (C.L. Koch, 1847), O. riukiaria (Verhoeff, 1940), and “species inquirenda” O. obtusus (Takakuwa, 1942). A cosmopolitan species, O. gracilis, is widely found in temperate and sub-tropical regions over the world, but other species have limited distribution in restricted regions, e.g., O. gigas in northern Vietnam, O. riukiaria and O. avia in the Ryukyu Islands (Japan). Four species, O. gracilis, O. riukiaria, O. avia and O. gigas, are confirmed as different from each other in gonopod characters, coloration and body size. The status of the last species, O. obtusus, is still doubtful and requires examination of further fresh material. The phylogenetic relationships among species of Oxidus is analyzed using two fragments of the mitochondrial genes COI (Cytochrome c Oxidase subunit I) and 16S rRNA. Three species of Oxidus are clearly separated from each other; O. gigas and O. gracilis form a monophyletic sister group with O. riukiaria. The genus Oxidus is also monophyletic and more closely related to the genus Tylopus Jeekel, 1968 than to the genera Sellanucheza Enghoff, Golovatch & Nguyen, 2004 or Kronopolites Attems, 1914. In addition, an identification key to species of Oxidus is provided.


Introduction
The genus Oxidus was proposed for the species Fontaria gracilis C.L. Koch, 1847by Cook (1911).However, Attems (1914) also proposed a subgenus, Kalorthomorpha, of the genus Orthomorpha Bollman, 1893 for the same species.Later, Kalorthomorpha was elevated to full generic rank and considered to be a junior synonym of Oxidus (Jeekel 1968).
O. gracilis (C.L. Koch, 1847) is a cosmopolitan species; O. obtusus (Takakuwa, 1942) was known from South Korea, both O. avia (Verhoeff, 1937) and O. riukiaria (Verhoeff, 1940) were reported from South Japan, and lastly O. gigas (Attems, 1953) was recoded from North Vietnam.Except for O. gracilis, all the remaining species have had no further records since their establishment.This paper seeks to answer three questions based on morphological and molecular analyses.Morphological data is used to classify species and molecular data is used to examine their phylogeny.1) How many species are actually in the genus Oxidus?2) What are the relationships among species of Oxidus? 3) Does the genus Oxidus form a monophyletic group with closely related groups, e.g., Tylopus and Sellanucheza?

Taxon sampling, identifi cation and DNA extraction
Examined material was collected by hand in northern Vietnam and southern Japan, or was borrowed from the collections in Kyungpook National University (KNU) (Korea) and the Hungarian Museum of Natural History (HNHM).All were examined under an Olympus SZX10 microscope with a drawing tube.
Total genomic DNA was extracted from leg tissue using the DNAeasy Blood & Tissue Kit (Qiagen TM).

DNA amplifi cation and sequencing
Fragments of two mitochondrial genes, cytochrome c oxidase subunit I (COI) and 16S rRNA, were amplifi ed using polymerase chain reaction (PCR).Universal primers LCO-1490 and HCO-2918 (Folmer et al. 1994) or COI-1F20 (5'-ACT CTA CTA ATC ATA AGG AT-3') and COI-1R19 (5'-TAA ACC TCC GGG TGA CCAA-3') were used to amplify a 680 bp fragment of the COI gene.Primer sets 16Sa (5'-CGC CTG TTT AHC AAA AAC AT-3') -16Sb (5'-CCG GTY TGA ACT CAR ATC CA-3') or 16S-1F19 (5'-CCG GTT TGA ACT CAG ATCA-3') and 16S-1R20 (5'-TGA CTG TTT AGC AAA GAC AT-3') were used to amplify a 550 bp fragment of the 16S rRNA gene.PCR conditions for amplifi cation of the 16S rRNA gene were: an initial denaturation at 95°C for 2 min followed by 36 cycles of 95°C for 20 sec, 45°C for 40 sec and 72°C for 1 min, and a fi nal extension at 72° for 5 min.The PCR conditions for the amplifi cation of COI were: initial denaturation at 94°C for 5 min followed by 38 cycles of 94°C for 45 seconds, 42°C for 45 seconds, 72°C for 90 seconds, and a fi nal extension at 72°C for 5 min.After thermal cycling, 2 μl PCR products were screened for potentially successful amplifi cation of a fragment of 16S or COI through electrophoresis in 1% agarose-TBE 1X.The electrophoresis was performed at conditions of 100 mA and 120 V for 1 hour.
About 20 μl of successfully amplifi ed PCR products were purifi ed using ExosapIT or the QIAquick PCR Purifi cation Kit (Qiagen Inc.).Purifi ed PCR products were sequenced at Solgen, Inc (Korea) in an Applied Biosystems automatic sequencer (ABI3130 XL) using the same primer sets used for initial PCR.
The reliability of the alignment was estimated using distance estimation and model of p-distance.Nucleotide frequencies were statistically calculated using MEGA 6.0.These COI sequences were translated into amino acids for confi rmation using transversion code in MEGA 6.0.
Model test was implemented in MEGA 6.0 to fi nd the most appropriate maximum likelihood substitution model for COI and 16S.Models with the lowest Bayesian Information Criterion (BIC) scores were considered for describing the best substitution pattern for each gene.Codon positions included were: 1 st + 2 nd + 3 rd + Noncoding.The selected model for COI was the Tamura-Nei model + G + I (Tamura & Nei 1993).For the combination of COI and 16S rRNA, the Hasegawa-Kishino-Yano model (Hasegawa et al. 1985) was used.Phylogenic trees were constructed using both maximum likelihood (ML) and Bayesian inference (BI) models.Maximum likelihood bootstrap analysis was conducted using MEGA 6.0 with 1000 replicates.A Bayesian inference (BI) tree was created using MrBayes ver.The COI dataset consisted of 525 bp sequences from 18 taxa.The 16S rRNA dataset included 466 bp sequences from 13 taxa.The combination of 16S and COI genes contained 991 bp sequences from 12 taxa (Table 2).The incongruence length difference (ILD) test showed congruence of genes COI and 16S.Therefore, the analyses were performed for gene COI and the combination of 16S and COI.Species of the genus Tonkinosoma Jeekel, 1953(tribe Tonkinosomatini Jeekel, 1968) were employed as an outgroup.

Abbreviations used in text and fi gures
The terminology for the genus Oxidus follows that used by Likhitrakarn et al. (2010)

Diagnosis
The genus can be distinguished from other genera in the tribe Sulciferini by the combination of characters: paraterga being well-developed; metaterga with one or two rows of setae; legs without tarsal brushes; 5 th sternum without modifi cations.

Remarks
This genus is distributed in the northern part of the Oriental and the eastern part of the Palearctic regions.
It is unlikely that the genus is of tropical origin because it is absent from the tropical regions in the Oriental (Jeekel 1963a).Its center of origin is more likely to be Japan (Jeekel 1968).
The genus Oxidus was placed in Sulciferini in view of the characters of the spiral solenophore completely sheathing the solenomere, the presence of a postfemoral demarcation and of postfemoral processes (Jeekel 1968).Golovatch & Enghoff (1993) recommended that the genus should be placed relatively close to the genus Tylopus Jeekel, 1968, rather

Diagnosis
This species differs from its congeners in the gonopod femorite being strongly expanded distally, not cylindrically slender; postfemoral lamina l rectangular; spine z pointed tuberculiform; process h lamellar, slightly suberect, but bent upwards from midpart, serrated at distolateral part; both bases of process h and spine z clearly separated; mesal lobule of solenophore very well-developed, lamella-shaped, distinctly separated from tip of solenophore; tips of both mesal lobule and solenophore circularly emarginated.

DNA
For Oxidus gracilis, DNA data was collected for three genes (mitochondrial COI gene, nuclear 18S and 28S rRNA genes), but there is no data from the mitochondrial 16S rRNA.In this study, two fragments of COI and 16S were sequenced and deposited in GenBank (see Table 1).

Diagnosis
This species is particularly similar to Oxidus gracilis (C.L. Koch, 1847) in body appearance and gonopod conformation, but differs in larger size and in details of gonopod structure: process h narrow and long, pointed at the end and not serrated at distolateral margin; bases of process h and spine z less distinctly separated.

DNA
COI and 16S barcode data (partial) are deposited in GenBank (Table 1).

Habitats
All material was found under leaf-litter, logs and decaying wood.

Distribution
The species has only been recorded from northern Vietnam (Lao Cai, Ha Giang and Vinh Phuc Provinces).

Remarks
Attems (1953) proposed a new subspecies Kalorthomorpha gracilis gigas with a short note.Enghoff et al. (2004) raised this subspecies to full rank as Oxidus gracilis, and also showed minor differences in size and gonopod process h.The species is fairly similar to O. gracilis, but the molecular data has provided strong evidence to separate the species.Golovatch (1984) misidentifi ed O. gracilis in Vietnam (sample IEBR-H133), which is currently corrected as O. gigas in this paper.

Diagnosis
Oxidus riukiaria can be distinguished from all other species of Oxidus by its smaller size (19.3−20.6 mm in length, width of pro-and metazonae 1.5−1.8mm and 2.1−2.2mm); gonopod coxae more or less stouter and longer than femorite; femorite narrow at base, and strongly expanded towards distal end; gonopod process h hook-like, larger and getting narrower towards pointed tip, spine z small, pointed spiniform; postfemoral lamina l triangularly rounded; mesal lobule not seperated from solenophore tip, which is distinctly emarginated.

DNA
COI and 16S barcode data (partial) are deposited in GenBank (Table 1).

Remarks
This species was originally described by Verhoeff (1940) in the genus Orthomorpha, but was assigned to Oxidus by Jeekel (1963a).The taxonomic position of the species is also well supported by molecular data.

Diagnosis
This species can be distinguished by its gonopod femorite being short, very broad gently distad; process h bent upward at tip, like a lamellar sickle; spine z small, obtuse; solenophore large, broad and simple, especially wide at its base; mesal lobule not separated from rounded solenophore tip (Takakuwa 1942a).

Remarks
This species was described from South Korea (Takakuwa 1942a).Later, Chamberlin & Wang (1953) reported two ♀♀ in the collection of the American Natural History Museum (New York) from Japan.It is possible that the locality was mislabelled because they stated that those specimens were collected and deposited by Dr. Takakuwa Y. Thus, it is believed that these two ♀♀ were collected from the same locality as the species holotype (South Korea).Lim (2001) synonymized O. obtusus with O. gracilis.He argued that the two species differ only in the number of postfemoral branches, three in O. obtusus and four in O. gracilis; in his opinion, such a difference is minor and cannot be used to separate two species.With the two species O. gracilis and O. gigas, those characters are important for species delimitation.It is recommended, therefore, that Oxidus obtusus should be considered a valid species.Examination of further fresh material is needed to confi rm that recommendation.

Distribution
South Korea.

Phylogenetic analyses
Phylogenetic trees were reconstructed for the COI and 16S-COI combination using ML and BI.For ML-analysis, we considered clades with bootstrap values below 65%, between 65 and 89%, or more than 89% to be weakly supported, moderately supported, or strongly supported, respectively (Pimvichai et al. 2014).For BI-analysis, clades with a BI posterior probability less than 0.7 bpp, between 0.7 and 0.95 bpp, or more than 0.95 bpp are considered to be weakly supported, moderately supported, or strongly supported, respectively.
Based on a Bayesian Inference and Maximum Likelihood analysis of a 525 bp fragment of COI from 18 taxa, a phylogenetic tree was reconstructed (Fig. 9).Based on BI and ML analysis of the 991bp combination of the genes 16S rRNA and COI, a phylogenetic tree of 12 taxa was constructed (Fig. 10).As in the COI tree, the genus Oxidus formed a monophylic clade clearly separated from species of Tylopus, with a well supported ML bootstrap value of 100% and a BI posterior probability of 1.00 bpp.The genus Oxidus is placed in the Sulciferini with the typical characters of a spiral solenophore completely sheathing the solenomere, presence of postfemoral demarcation, and postfemoral processes (Jeekel 1968).Until now, no relationship analysis among sulciferine genera has been reported, except that Golovatch & Enghoff (1993) have reported a close relationship among species of Tylopus and Oxidus gracilis.These authors recommended that Oxidus gracilis or the genus Oxidus should be considered as the sister of genus Tylopus because both genera have similar gonopod characters, such as the presence of lamina l, process h, and spine z.Molecular analysis based on the COI gene and the combination of 16S and COI genes provides good supports for the recommendation of Golovatch & Enghoff (1993).In both phylogenetic trees, Oxidus and Tylopus formed a group with well supported bootstrap value and BI posterior probability (Figs 9-10).
The genus Sellanucheza Enghoff, Golovatch & Nguyen, 2004 was originally assigned to the tribe Tonkinosomatini (Enghoff et al. 2004).However, Golovatch (2013) suggested that it was better to place this genus in the tribe Sulciferini.His suggestion is well supported by the molecular data obtained from this study.The genus Sellanucheza is relatively close to the group of Tylopus + Oxidus, with a very high BI value (0.98 bpp) in the phylogenetic tree inferred from the 525 bp fragment of the COI gene (Fig. 10).

Conclusion
The
genus Oxidus Cook, 1911 consists of fi ve species, O. avia, O. gigas, O. gracilis, O. riukiaria, and a doubtful species, O. obtusus.While O. gracilis is a cosmopolitan species, widely found in temperate and sub-tropical regions over the world, the other species have limited distributions in particular regions: O. gigas in northern Vietnam, and O. riukiaria and O. avia in the Ryukyu Islands (Japan).

Fig. 9 .Fig. 10 .
Fig. 9. Phylogenetic tree of the genus Oxidus and some closely related groups based on Maximum Likelihood and Bayesian Inference Analysis of a 525 bp fragment of the COI gene (# = a value less than 65%).

Table 1 .
for the genus Tylopus: Details of specimens analysed for DNA, including GenBank numbers.
than to other sulciferinine genera.The phylogeny of the genus Oxidus is discussed below.
The genus Oxidus formed a monophyletic clade.It was separated from its sister group (genus Tylopus) with an ML bootstrap value of < 65% and a BI posterior probability of 0.76 bpp.Sellanucheza is considered as the sister group of Tylopus plus Oxidus.The separation of Sellanucheza and (Tylopus plus Oxidus) is supported with an ML bootstrap value of <65% and a BI posterior probability of 0.98 bpp.Within Oxidus, the three species formed three clearly distinguished clades.However, O. gigas was more closely related to O. gracilis, with an ML bootstrap value of 74% and a BI posterior probability of 0.86 bpp.O. riukiaria was separated from the group of

Table 2 .
Average base frequencies of two partial genes COI, 16S rRNA and the 16S-COI combination.
O. gigas and O. gracilis with a well supported ML bootstrap value of 98% and a BI posterior probability of 1.00 bpp.

Table 3 .
(Attems, 1953)us species were also separated from each other.O.riukiaria formed a sister group with O. gigas plus O. gracilis, with an ML bootstrap value of 100% and a BI posterior probability of 1.00 bpp.The species O. gigas was separated from O. gracilis with a moderately supported ML bootstrap value of 83% and a BI posterior probability of 0.91 bpp.The species O. gigas was originally proposed as a subspecies of O. gracilis(Attems, 1953).After being raised to full species rank, it is still closer to O. gracilis than to other species of Uncorrected distance of the combination of COI and 16S rRNA genes calculated by MEGA 6.0.The genetic distances among species of Oxidus are highlighted in bold.
Oxidus.This relationship is well supported by a monophyletic clade of the two species, O. gracilis and O. gigas.The other species, O. riukiaria, is considered as the sister species of O. gracilis plus O. gigas.