Description of a new species of Penicillate Millipede from the genus Monographis (Diplopoda: Polyxenidae) found in Vietnam Author Huynh, Cuong Author Veenstra, Anneke A. text Zootaxa 2015 3964 4 460 474 journal article 10.11646/zootaxa.3964.4.5 c86698a4-d6e3-454e-b175-3ab9beb9a5e1 1175-5326 238043 8A90D889-4C33-401A-A1A8-AB31663402BC Monographis dongnaiensis sp. n. ( Figure 2 ) Holotype . An adult male, stadium VIII with 13 pairs of legs, was collected by the author, Cuong Huynh, from Trang Bom district, (10°54′59″N, 106°59′31″E northeast Ho Chi Minh City); in Dong Nai province, southern Vietnam , 20th December 2012 . Queensland Museum accession number QMS 98960. Paratypes . Two adult males (QMS 98961, 98962), seven adult females (QMS 98963–98969) and sub-adult female (stadium VII—12 pairs of legs) (QMS 98970) are from the type locality (location 1). An adult female (QMS 98971) and a sub-adult male (stadium VII—12 pairs of legs) (QMS 98972) are from location 2. All mounted slides and specimens preserved in 80% ethanol, have been lodged in the Queensland Museum, Brisbane, Queensland, Australia . Etymology. The specific name of Monographis dongnaiensis refers to the name of the province of Dong Nai, southern Vietnam , where this species was first collected. Diagnosis. Monographis dongnaiensis differs from other species of genus Monographis by having 19–22 sensilla, with 3 anterior thick sensilla and 5 internal sensilla, which form a triangular shape on the 6th antennal article. The surface of the labrum is smooth and asetose. Telotarsus bears a posterior process with a curved and enlarged base and is half the length of the claw. A small lateral process is present and the anterior setiform process is equal to the length of the claw. Description. Measurements : Holotype male body length was 3.2 mm , male ( paratypes ) range in length from 2.4–3.2 mm ( n = 10) and females ( n = 10) range from 3.0– 3.4 mm . The caudal bundles of females are approximately 0.9 mm , longer than in the males ( 0.7 mm ). Colouration : The body is yellowish brown in colour and darker on the latero-posterior rosette trichomes, which form 2 dark lateral bands contrasting with a light yellow medial band. Eyes are bright red with two black horizontal bands apparent in the vertex area, which distinguishes the head from the rest of the animal. The last tergite is darkest in colour and extended into the telson region. Here, the caudal bundle trichomes form light silvery bands near the base. The colour of these bands gradually fades to white and reflects light to produce a black-silver colouration towards the end of the telson. The ventral side of the body is white in colour. FIGURE 2 . A live specimen of Monographis dongnaiensis from Dong Nai province, southern Viet Nam. Head : There are 8 ommatidia on each side: 4 located dorsally and 4 in lateral positions (1 anterior, 2 medial and 1 posterior). The vertex has 2 posterior trichome groups, a large gap is present between the 2 groups and each group is with 2 rows. The anterior, oblique row has a variable number of large trichome sockets in the centre. These trichome sockets reduce in size toward both ends. The posterior rows have fewer small trichome sockets. There is a narrow space between the anterior and posterior rows. The holotype male had 13 + 13 anterior row trichome sockets and 3 + 3 posterior row trichome sockets. The paratypes showed that variation is common in this species, regardless of sex (QMS 98962 with 11(L: left) + 12 (R: right) and 4 (L) + 3(R), QMS 98964 12 (L) + 14 (R) and 3 (L) + 7 (R)). Trichobothria (trichobothrium a , internal position, trichobothrium b , intermediate position and trichobothrium c , external position) are typically thin, funicular and cylindrical structures. All these trichobothria (a, b and c) are equal in size and form an isosceles triangle with equal distance between ab and bc ( Figures 3 A & E). Antennae : These typically consist of 8 articles and four sensory cones, which is characteristic of Polyxenidae . The 6th antennal article (Figures 4C & D) has 20 bacilliform sensilla in males and 19–22 in females. The holotype has 20 sensilla, including 3 thick bacilliform sensilla and 17 thin bacilliform sensilla. Two adult female paratypes , QMS 98969 from Giang Dien, QMS 98971 from Dinh Quan, both have a greater number of sensilla (22) on the 6th antennal article. The typical 3 thick bacilliform sensilla: anterior a (Tbs a ), intermediate i (Tbs i) and posterior p (Tbs p ) are located in the anterior portion. These thick bacilliform sensilla combine with 11–14 thin bacilliform sensilla (tbs) forming a triangular shape, surrounding an inner group of 5 thin bacilliform sensilla (Figure 4B). A conical sensillum is absent. The 7th antennal article has 2 thick bacilliform sensilla (Tbs), the anterior one being slightly longer than the one located posteriorly, with one setiform sensillum (s) between them and a conical sensillum (c) locates in the posterior position (Figure 4A). This pattern of sensilla on the 7th article is commonly seen in all species of Monographis ( Huynh & Veenstra 2013 ) . Clypeo-labrum : 9 setae as long as the width of the labrum are present. Setae on the paratypes range from 8–11 in both sexes. The labrum surface on the holotype is smooth, with a line of minute setae along the posterior portion and the anterior margin asetose. Some paratypes have a few setae on the labrum surface. A 1 + 1 lamellae is associated with lateral lamellae on each side of the median cleft ( Figure 3 F). Lateral palp of gnathochilarium : This is 1.25 times the length of the medial palp. There are 11 conical sensilla on the lateral palp and medial palp with 22 slender conical sensilla, found in all paratypes and the holotype . ( Figure 3 G). FIGURE 3 . Holotype male (QMS 98960), A . Head capsule of Monographis dongnaiensis showing ommatidia pattern, 2 posterior vertex trichomes and 3 trichobothria: trichobothrium a , b and c . B . Collum and lateral protuberances with pattern of trichome sockets (all trichomes were removed). C . Trichome socket arrangements from tergite 2. D . Tergite 10 with densely packed smaller sockets. ( A, B, C, D drawn to the same scale). E . Structure of trichobothria: Trichobothrium a (internal position), Trichobothrium b (intermediate position), Trichobothrium c (external position), sockets and their funicular structures. F . Structure of clypeo-labrum with 2 lateral lamellae, 2 lamellae and a median cleft. G . Structures of 2 lateral palps (a conical sensillum was missed on each lateral palp) and medial palps from gnathochilaria (Lp: lateral palp, Mp: medial palp). Trunk : This consists of 10 segments, 9 pleural projections, excluding the telson and caudal bundle; 13 pairs of legs. The collum is the only tergite with lateral protuberances bearing 3–6 trichomes on each side. All other tergites have a pair of pleural projections located antero-laterally. The tergal trichome socket arrangements typically have 2 broad oval shapes, slightly enlarged laterally, and a posterior row slightly upwardly curved toward the centre with a large gap between these trichome socket rows. The collum is considered to be tergite 1 (smallest tergite) with 57 (Left: L), 57 (Right: R) trichome sockets and 6(L) / 6(R) in lateral protuberance on holotype ( Figure 3 B). The numbers varied in paratype males, range 43–56; and paratype females, range 51–66 trichome sockets. Tergite 2 has the same structural pattern with the posterior row slightly longer and with 58(L) / 57(R) trichome sockets ( Figure 3 C). For Tergites 3–9, the patterns are very similar with the characteristic large gaps. Tergite 10 is an exception where the trichome sockets are smaller and denser. There is almost no space between the lateral rosette trichome sockets and the posterior row ( Figure 3 D). Legs : Leg segments are named following Manton (1956) . Legs 1 and 2 are without trochanter, leg 1 also lacks tarsus 1. Chaetotaxy as follows: coxa 1: 2 sensilla, coxa 2: 2 sensilla, coxae 3–13: 1–4 sensilla; all pre-femurs, postfemurs, tibia with 1 sensillum, except femurs with 2–4 sensilla, tarsus 1 and tarsus 2 with a spine ( Figure 5A ). Coxa, pre-femur and femur have a ridged biarticulate, funicular cylindrical sensillum ( Figure 5 B). There are also 2–3 smaller biarticulate, funicular cylindrical sensillum present on the posterior end of the femur and some on the penis ( Figure 5A and 5 C). At their distal edge, post-femur and tibia are with a setiform sensillum ( Figure 5 D). The spine on tarsus 2 is much larger, and sharply pointed ( Figure 5 F) compared to a spine in the antero-sternal position of tarsus 1 ( Figure 5 E). Two to four ridged biarticulate, funicular cylindrical sensilla are found on the posterior edge of the last sternite. These sensilla are similar to those present on the coxa. The number of sensilla varies; 4 present on the holotype male and 2–4 on the paratypes ( Figure 5 G). FIGURE 4 . A . Arrangement of sensilla on 7th antennal article, a conical sensillum ( c ), thick bacilliform sensillum ( Tbs ) and a setiform sensillum ( s ). B . Arrangement of sensilla of 6th article, showing position of 3 thick bacilliform sensilla ( Tbs ): anterior a , intermediate i and posterior p ; 17 thin bacilliform sensilla ( tbs ) included 5 inner sensilla (arrows). C . Left side antenna and its article. D . Scanning Electron Microscopic (SEM) image showed articles 6th, 7th and 8th with 4 sensory cones (A, B, and C were from the holotype male (QMS 98960), D was an antenna from a female). Claw -Telotarsus : This is a slender structure comprised of a posterior process that is curved with an enlarged base, half as long as the claw. An anterior process is absent. A tiny lateral process is present. The anterior setiform process is almost equal in length to the claw and a common lamella process is present ( Figure 5 H). Sex organs in male : A pair of penes is present on the 2nd coxa and 2 pairs of coxal glands located on coxal plates of 8th–9th legs. Telson : The dorsal ornamental trichome sockets are arranged almost symmetrically on each side, with 16 trichome sockets a in the holotype male. The paratypes have 8–23 trichome sockets a in both sexes. Trichome sockets a form 2 rows; top row with small sockets and bottom row with larger trichome sockets. The two largest holders are situated near trichome b . Three large protruding base trichome sockets c : c1, c2 and c3 , form a triangular shape. Circular indentation d is apparent near the exterior side of trichomes c ( Figure 5 I). FIGURE 5 . A . Leg segments and chaetotaxy of the second leg and penis ( p : penis, co : coxa, pre.fe : pre-femur, fe : femur, post.fe : post-femur, tib : tibia, T1 : tarsus 1, T2 : tarsus 2). Three types of seta present: 1) Ridged biarticulate funicular cylindrical sensilla ( B ) were found in coxa, pre-femur, femur and the last sternite. 2) A similar small form ( C ) found in the posterior position on femur, sex organs—penis or vulva. 3) Setiform seta in post-femur and tibia ( D ). E & F . Spines found on Tarsus 1 and 2. G . Last sternite, with 3 sensilla and a socket without sensillum. H . Telotarsus structure with processes: c : claw, l : lamella process, lp : lateral process, p : posterior process, s : anterior setiform process. I . Dorsal ornamental trichome sockets: trichome sockets a : a 1 to a 16; trichome socket b ; trichome sockets c : c 1, c 2 and c 3. Circular indentation d . J . Nest trichome. K . Caudal trichome. (A, B, C, D, E, F, G, H & I were from the holotype male (QMS 98960), J & K were from a female). FIGURE 6 . Genus Monographis divided into 2 groups of species based on the arrangement of sensilla on the 6th antennal article ( Huynh & Veenstra 2013 ); Monographis dongnaiensis was placed in Group 2, which has sensilla forming a triangular shape. The drawings of sensilla observed on Monographis kraepelini and M. tamoyoensis were from Nguyen Duy-Jacquemin & Condé (1967) . Caudal bundles : In both males and females of Monographis dongnaiensis , these are similar in appearance to those of M. queenslandicus ( Huynh & Veenstra 2013 ) . In males, the caudal bundle is uniform in structure and the caudal trichome sockets are in similar sizes. The caudal trichomes have 2–4 hooks commonly with 2–3 hooks facing ventrally. The hooks are connected with distal facing, small barbates in the middle section and join with a larger barbate stem, forming 3–4 rings of 3 spines, distally facing, at the base of this trichome stem ( Figure 5 K). Few larger barbate trichome sockets are present; commonly 2–3 rows, and form slightly uneven lateral rows that extend toward the centre of the telson. The barbate trichomes (which are used to prevent hooks on trichomes becoming tangled) are large with their length exceeding the longest caudal trichomes in the bundle. In females, the caudal bundle has 2 distinguishing structures: the caudal trichomes, which are the main dorsal structures and are similar to the male caudal bundle, and 2 latero-sternal bundles of nest trichomes ( Figure 5 J). These nest trichomes have small, fine trichome sockets, with trichomes half the length of those on the main caudal trichomes. They differ from caudal trichomes in having smaller, slender stems with double facing spines connected by 3–5 hooks. These nest trichomes are used for nest building to provide protection for egg clusters ( Huynh & Veenstra 2014 ). Comparison of Monographis dongnaiensis to other species in Monographis . In addition to the common characteristics of the genus Monographis , M. dongnaiensis has morphological characteristics similar to a group of Monographis species, which have the sensilla forming a triangular shape on the 6th antennal article ( Huynh & Veenstra 2013 ) (Figure 6). When comparing the sensilla structures on the 6th antennal article between M. yunnanensis and M. dongnaiensis , M. yunnanensis has 17 sensilla with 3 inner sensilla, a conical sensillum ( Ishii & Yin 2000 ) and M. dongnaiensis has 20 sensilla with no conical sensillum but with 5 inner sensilla (Figure 7). These are obviously different species of Monographis occurring in the same region. FIGURE 7 . Comparison of the number of sensilla observed on the 6th antennal article of M. yunnanensis ( A ) and Monographis dongnaiensis ( B ). The pattern of sensilla of M. yunnanensis is referred from Ishii & Yin (2000) . Small arrows indicate the inner sensilla, a large arrow indicates the location of the conical sensillum. Not to scale. FIGURE 8 . Comparison of the number of sensilla between Monographis tamoyoensis ( A ), M. queenslandicus ( B ) and M. dongnaiensis ( C ). Sensilla pattern of M. tamoyoensis is referred from Nguyen Duy-Jacquemin & Condé (1967), and sensilla pattern of M. queenslandicus is from Huynh & Veenstra (2013). Arrows indicate the positions of the conical sensillum. Not to scale. M. tamoyoensis has 18 sensilla with 5 inner sensilla ( Nguyen Duy-Jacquemin & Condé 1967 ) and M. queenslandicus has the same arrangement, but with 19 sensilla ( Huynh & Veenstra 2013 ). M. tamoyoensis has a conical sensillum located in the inner group of sensilla; M. queenslandicus is observed to have a conical sensillum located in the outer group of sensilla, whereas M. dongnaiensis is observed to have 20 sensilla with 5 inner sensilla and the absence of a conical sensillum ( Figures 8 & 9 ). This suggests that M. dongnaiensis is a species that should be included in this group. FIGURE 9. The Scanning Electron Microscopic images showed the similarity of the arrangement of sensilla between M. queenslandicus ( A ) and Monographis dongnaiensis ( B ). But M. dongnaiensis is different in lacking a conical sensillum. Small arrows indicate the inner sensilla, a large arrow indicates the position of the conical sensillum. Not to scale. FIGURE 10 . Comparison between claw structures of M. tamoyoensis ( A ), M. queenslandicus ( B ) and Monographis dongnaiensis ( C ). A claw of M. tamoyoensis is referred from Nguyen Duy-Jacquemin & Condé (1967), and a claw of M. queenslandicus is from Huynh & Veenstra (2013). Arrows indicate the length of the anterior setiform processes. The dot lines compared the length of anterior setiform processes to the claws. a : anterior process, l : lamella process, lp : lateral process, p : posterior process, s : anterior setiform process. Not to scale. In this study, other structures such as the claws were used to distinguish between species in this group. M. dongnaiensis has slender claws as does M. queenslandicus and differs from M. tamoyoensis , which has robust claws and short anterior setiform process. M. dongnaiensis differs from M. queenslandicus in that the length of the anterior setiform process is equal to the claw, the anterior process is absent; while the anterior setiform process is longer than the claw in M. queenslandicus ( Figures 10 & 11 ). DNA analysis. A partial sequence with 1277 base pairs linear DNA was obtained from the DNA of five adult specimens of M. dongnaiensis (Genbank accession number KP255446 (accepted 12 January 2014 )). This truncated sequence excluded the first 27 base pairs and the base pairs from 1332 onward. Based on the results of a BLAST search (http://www.ncbi.nlm.nih.gov), the partial sequences of 18S ribosomal RNA gene from six closely-related penicillate millipedes were chosen for comparison with the sequence of M. dongnaiensis ( KP255446 ): Monographis sp. from China (AY596371.1), M. queenslandicus (KF147166.1), two species from genus Polyxenus ( P. lagurus (EU368619.1) and P. fasciculatus (AF173235.1)) and two millipedes are used as the outgroup ( Zoosphaerium neptunus (FJ409972.1) & Paradoxosomatidae sp. (DQ666179.1)) in the phylogenetic analysis. The rooted consensus tree of M. dongnaiensis was generated by the bootstrap test with 1000 repetitions which yielded a strongly supported phylogenetic tree. There were four clear clades: M. dongnaiensis and M. queenslandicus were a sister group, forming an independent clade. This clade branched with Monographis sp. (AY596371.1) forming a monophyletic group. This group of Monographis species was separated from the two Polyxenus species, as was the outgroup ( Figure 12 ).