Dental scales could occur in all scaled subfamilies of Entomobryidae (Collembola): new definition of Entomobryinae with description of a new genus and three new species Author(s): Feng Zhang, Zhixiang Pan, Jun Wu, Yinhuan Ding, Daoyuan Yu and Beixin Wang Source: Invertebrate Systematics, 30(6):598-615. Published By: CSIRO Publishing URL: http://www.bioone.org/doi/full/10.1071/IS16005
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Dental scales could occur in all scaled subfamilies of Entomobryidae (Collembola): new definition of Entomobryinae with description of a new genus and three new species
Feng Zhang A,E, Zhixiang Pan B, Jun Wu C, Yinhuan Ding A, Daoyuan Yu D and Beixin Wang A,F
ADepartment of Entomology, College of Plant Protection, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People’s Republic of China. BSchool of Life Sciences, Taizhou University, 1139 Shifu Road, Jiaojiang, Taizhou 318000, Zhejiang Province, People’s Republic of China. CNanjing Institute of Environmental Sciences under Ministry of Environmental Protection, 8 Jiangwangmiao Street, Nanjing 210042, People’s Republic of China. DSoil Ecology Lab, College of Resources and Environmental Sciences, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People’s Republic of China. EKey Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Beijing 100101, People’s Republic of China. FCorresponding author. Email: [email protected] and [email protected]
Abstract. Body scales are fundamental in the classification of Entomobryidae at all taxonomical levels. Traditionally, scales on dens were considered to be absent in Entomobryinae, but present in other scaled subfamilies; however, this opinion was strongly challenged by recent morphological advances in tergal specialised chaetae (S-chaetae). A new genus, Lepidodens, is strikingly similar to the scaled Entomobryinae genus Willowsia in having pointed scales with relatively long ribs and 2, 2|1, 2, 2, 8, 3 tergal S-chaetae, but differs from it in having dental scales and a unique position of S-microchaetae on the first abdominal segment. Multilocus phylogeny and topology tests also support this view, the new genus clustering with Entomobryinae rather than Seirinae. Three new species, L. nigrofasciatus, L. similis and L. hainanicus, are described from South China. This study clearly undermines the traditional separation of Entomobryinae and Seirinae/Lepidocyrtinae, and demonstrates that dental scales could occur in all entomobryid subfamilies containing scaled taxa. In this new phylogenetic hypothesis, Entomobryinae has the greatest diversity in scale morphology and distribution among scaled collembolan groups, indicating multiple independent origins of scales.
Additional keywords: Lepidodens, gen. nov., molecular phylogeny, Seirinae, South China, Willowsiini.
Received 20 January 2016, accepted 27 April 2016, published online 13 December 2016
Introduction the family Entomobryidae has the largest diversity in both scaled Collembola (springtails), living in almost all kinds of terrestrial and unscaled groups. ecosystems, represents one of the largest radiations of soil Traditional classification of Entomobryidae was mainly organisms. Entomobryidae Tömösváry, 1882 with ~1800 contributed by Börner (1906, 1913), Yosii (1961), Szeptycki named living species accounts for ~20% of known collembolan (1979), Yoshii and Suhardjono (1989) and Soto-Adames et al. species (Bellinger et al. 1996–2016). Entomobryoidea distinctly (2008). The latter authors divided the family into Capbryinae, differs from other superfamilies and orders in a reduced prothorax, Orchesellinae and Entomobryinae (Entomobryini, Willowsiini, long appendages and elongated fourth abdominal segment. Seirini and Lepidocyrtini). Four taxa within Entomobryinae Relationships between Entomobryidae and other families sensu Soto-Adames et al. were often treated as subfamilies or within Entomobryoidea are still controversial. Closely related tribes by different authors. Zhang et al.(2014a) supported Paronellidae and Cyphoderidae are possibly ingroups of the monophyly of Entomobryinae sensu Szeptycki, Seirinae Entomobryidae based on molecular phylogeny and tergal and Lepidocyrtinae and the non-monophyly of Orchesellinae, specialised chaetae (S-chaetae) (Zhang et al. 2015). In any case, Entomobryini and Willowsiini based on ribosomal markers.
Journal compilation CSIRO 2016 www.publish.csiro.au/journals/is New definition of Entomobryinae and Lepidodens, gen. nov. Invertebrate Systematics 599
Integrating molecular and morphological evidence, Zhang and Materials and methods fi Deharveng (2015) revised the classi cation and erected the Taxa sampling framework of seven subfamilies. fi Distribution and morphology (shape and surface sculpture) of Following classi cation of Zhang and Deharveng (2015), 37 body scales are of great taxonomical values and are fundamental species were selected from Entomobryidae, representing the fi in the classification of Entomobryidae at all levels. Scales occur ve main subfamilies: Orchesellinae (3), Heteromurinae (4), in Heteromurinae, Entomobryinae, Seirinae and Lepidocyrtinae, Lepidocyrtinae (5), Seirinae (4) and Entomobryinae (21). among which only Entomobryinae includes both scaled and Tomocerus ocreatus (Tomoceridae), Folsomia candida and unscaled taxa. All traditional studies accepted the idea of the Folsomia quadriocula (Isotomidae) were chosen as outgroup absence of dental scales in Entomobryinae before molecular taxa. Species names, taxonomical positions, collection locality systematics were applied. Scaled taxa within Entomobryinae and GenBank accession numbers are provided in Appendix 1. All were grouped as Siraeformes by Denis (1941)andas materials were collected by aspirator or Tullgren-Berlese funnels, Willowsiini by Yoshii and Suhardjono (1989). However, Zhang stored in 99% ethanol at 20 C and then morphologically and Deharveng (2015)nolongerseparatedEntomobryinaeinto examined using Nikon SMZ1000, Nikon 80i microscopes and unscaled Entomobryini and scaled Willowsiini in the light of a Hitachi scanning electron microscope. independent origins of body scales within the subfamily (Zhang et al. 2014a, 2014b, 2015), returning largely to Szeptycki’s(1979) DNA extraction and sequencing conception. They even transferred the Lepidosira-group sensu DNA was extracted using an Ezup Column Animal Genomic Yoshii & Suhardjono, 1989 with dental scales from Seirinae to DNA Purification Kit (Sangon Biotech, Shanghai, China) Entomobryinae based on the patterns of tergal bothriotricha following the manufacturer’s standard protocols. PCR and S-chaetae and the mucro structure. The absence/presence of amplification of the four fragments, mitochondrial COI and dental scales in Entomobryinae still appears disputable due to the 16SrRNA (16S) and nuclear 18SrRNA (18S) and 28SrRNA lack of comprehensive evidence from molecular phylogeny. D1–3 (28S), was carried out following Zhang et al.(2014a, Within Entomobryinae, scaled taxa except the Lepidosira- 2014d). All successful PCR products were purified and group, whose dental scales are absent (sensu Willowsiini), sequenced by GenScript (Nanjing, China) on an ABI 3730XL usually have scales mostly pointed with coarse ribs. The most DNA Analyzer (Applied Biosystems, Waltham, MA, USA). characteristic scale form is that of Willowsia, with long basal Sequences were assembled in Sequencher 4.5 (Gene Codes ribs on the surface (Fig. 1). A new genus and three new species Corporation, Ann Arbor, MI, USA), and were submitted to discovered in South China, which have pointed scales with GenBank (Appendix 1). Sequences were blasted in GenBank long basal ribs, remarkably resemble Willowsia species in and checked for possible errors. They were then preliminarily most features. However, the presence of scales on dens recalls aligned using MAFFT v7.149 by the Q-INS-I strategy (Katoh and rather Seirinae or Lepidocyrtinae, while other features point Standley 2013). Alignments were checked and corrected to Entomobryinae, to which we here assign the new genus. manually, with partially ambiguous sites of 16S excluded from To clarify the systematic position of the new genus and all analyses. In the final 4016-bp concatenated alignment, COI, improve the understanding of Entomobryinae, we conducted a 16S, 18S and 28S were 658 bp, 415 bp, 1681 bp and 1259 bp, molecular phylogeny and a detailed morphological comparison respectively. among the above taxa. As the second group with dental scales within Entomobryinae, the new genus was also compared with Phylogenetic inference the Lepidosira-group. A new diagnosis is also provided for The partitioned dataset was analysed by maximum likelihood Entomobryinae. (ML) and Bayesian inferences (BI) using online CIPRES services
Fig. 1. Scanning electron microscopy of body scales in Willowsia neocaledonica Zhang, Bedos & Deharveng, 2014c. Scale bar = 10 mm. 600 Invertebrate Systematics F. Zhang et al.
(Miller et al. 2010). Best-fitting substitution models were Results assessed for partitioning schemes and models available in Phylogenetic inference MrBayes under the Bayesian Information Criterion (BIC) criterion in PartitionFinder v1.1.1 (Lanfear et al. 2012). The The ML and BI analyses generated identical topologies at the fi GTR+I+G, GTR+I+G, GTR+I+G and SYM+I+G models were suprageneric level, separating Entomobryidae into ve main > selected for COI, 16S, 28S and 18S, respectively. Maximum clades with high Bayesian posterior probabilities (BPP 0.99): likelihood trees were reconstructed in RAxML v8.2.4 (Orchesellinae + (Heteromurinae + (Lepidocyrtinae + (Seirinae + (Stamatakis 2014) with the GTRGAMMAI model for each Entomobryinae)))) (Fig. 2). Monophyly of Heteromurinae and partition and 1000 bootstrap replicates. Bayesian inference (Seirinae + Entomobryinae), similar to the ML reconstruction of analyses were conducted in MrBayes 3.2.6 (Ronquist et al. Zhang et al.(2015), was supported by slightly weak ML bootstrap > 2012) with four chains (three heated, one cold). Model values (MLB 60). The new genus, whose monophyly was parameters were unlinked and the model allowed the overall strongly supported (MLB = 0.97, BPP = 1), was located within rate to be different across partitions. To avoid the problem Entomobryinae. The relationships between the new genus and of branch-length overestimation, the compound Dirichlet other Entomobryinae taxa were not resolved. Both likelihood and priors ‘brlenspr = unconstrained: gammadir (1, 1, 1, 1)’ for Bayesian approaches absolutely rejected hypothesis B of the new < > branch length were incorporated (Zhang et al. 2012). Fifty genus belonging to Seirinae (probabilities 0.05 and BFs 5; million generations were performed, with the chain sampled Table 1). every 5000 generations. The burn-in value was 25% and other parameters were set as default options. Convergence and Taxonomy effective sample size were checked in Tracer 1.5 (Rambaut and Drummond 2007). Subfamily ENTOMOBRYINAE Schäffer Two topology hypotheses on constraining monophyly were Entomobryinae Schäffer, 1896: 177. tested under likelihood and Bayesian theory frameworks: (A) best Type genus. Entomobrya Rondani, 1861: 40. trees without any constraints (new genus within Entomobryinae); (B) new genus within Seirinae. Approximately unbiased (AU) Diagnosis – – tests, Shimodaira Hasegawa (SH) and weighted Shimodaira Four antennal segments. Eyes 0–8. Post-antennal organ absent. Hasegawa (WSH) tests were calculated in CONSEL V0.1j Abd. IV more than 2 as long as Abd. III at middle line. (Shimodaira and Hasegawa 2001) with the default settings. Per Trochanteral organ developed usually with more than 10 site log-likelihoods before CONSEL analyses were generated chaetae in adults. Tenaculum with 4+4 teeth and one basal fi by RAxML. Hypotheses with p-values signi cant at the level of chaeta. Mucro bidentate or falcate; mucronal basal spine present 0.05 were rejected. Evaluation of Bayes factors (BFs) was also or absent. Male genital plate papillate. Dental scales present or performed for topology tests. A marginal likelihood estimator absent; if body scales present, scales mostly pointed and variously by stepping-stone sampling (Xie et al. 2011) was calculated in sculpted. Polymacrochaetotic chaetotaxy. Bothriotricha 2, 3, 2 MrBayes for the two hypotheses. Informed topology was strictly on Abd. II–IV. Tergal ms 1, 0|1, 0, 1(0) and sens 2, 2|1, 2, 2. constrained in the prior because standard BF tests of monophyly can be misleading (Bergsten et al. 2013). Markov chain Monte Remarks Carlo (MCMC) processes were the same as previous analyses fi (ngen = 50 000 000 samplefreq = 5000). A logarithm difference Compared with the traditional de nition, the present concept of Entomobryinae accommodates the taxa with dental scales, (logBF1–logBF0) in the range of three to five is considered strong evidence against the null hypothesis, while a difference such as Lepidodens, gen. nov., Lepidosira etc. Entomobryinae value above five provides very strong evidence (Kass and Raftery differs from Seirinae and Lepidocyrtinae in the combination of 1995). scale shape and sculpture, bothriotrichal number, mucro and tergal S-chaetae (Table 2).
Genus Lepidodens Zhang & Pan, gen. nov. Type species. Lepidodens nigrofasciatus Zhang & Pan, sp. nov. Morphological description http://zoobank.org/urn:lsid:zoobank.org:act:F13A8291-550F-4308- The labial chaetae terminology follows Gisin’s system (Gisin 82E6-35B500769075 1967). Dorsal cephalic chaetotaxy are designated following Soto- Adames (2008), tergal chaetae after Szeptycki (1979) and tergal Diagnosis S-chaetae after Zhang and Deharveng (2015). The number of Small to large size (up to 3.5 mm) entomobryid. Scales pointed, macrochaetae is given by half-tergite in the descriptions. All striate heavily with basal ribs longer than distal ones, and present material is deposited in the collections of the Department of on Ant. I, head, body, legs, ventral side of manubrium and dens; Entomology, College of Plant Protection, Nanjing Agricultural scales on dens narrower than those on body; scales in posterior University (NJAU), P. R. China. Abbreviations: Th., thoracic row along margin of each tergum much longer than anterior segment; Abd., abdominal segment; Ant., antennal segment; ones. Polymacrochaetotic chaetotaxy. Four antennal segments. mac, macrochaeta/ae; mic, microchaeta/ae; ms, S-microchaeta/ Smooth spiny microchaetae at base of antennae: three dorsal, ae; sens, ordinary tergal S-chaeta/ae. two ventral on Ant. I, one internal, one external and one ventral on New definition of Entomobryinae and Lepidodens, gen. nov. Invertebrate Systematics 601
Outgroup
Orchesellinae
Heteromurinae
Lepidocyrtinae
Seirinae
Entomobryinae
Fig. 2. Bayesian phylogenyof Entomobryidae based on the molecular dataset. Nodevalues represent maximum likelihood bootstrap and posterior probabilities, respectively, with a ‘–’ indicating nodes not compatible between the analyses. Clade containing Lepidodens, gen. nov. and related terminals are marked as red.
Table 1. Tree topology tests using likelihood and Bayesian approaches Ant II. Ant. III organ with two rods and three guard chaetae. Monophyly constraints: A, new genus within Entomobryinae (best tree); B, Ant. IV apical bulb unilobed. Eyes 8+8, G and H smallest. Labral new genus within Seirinae. Asterisks represents very strong evidence against margin with four conical papillae and U-form intrusion. Prelabral an alternative hypothesis. AU, approximately unbiased test; SH, and labral chaetae 4/ 5, 5, 4; prelabral ones ciliate. Five interocular Shimodaira–Hasegawa test; WSH, weighted Shimodaira–Hasegawa test chaetae. Mandibles with four and five teeth. Three hairs on Hypotheses Likelihood tests Bayes factors (logarithm) sublobal plate of maxillary outer lobe. Tip of lateral process of AU SH WSH Model Difference labial palp thin, not reaching apex of labial papilla. Labial chaetae likelihood of posterior row ciliate. Tibiotarsi distally with 10 ciliate chaetae in a whorl. Unguis with four inner teeth. Unguiculus truncate. – A 1 0.998 0.998 32408.05 0 Tenent hair clavate and subequal to unguis in length. Tenaculum – B 3e-004 0.002* 0.002* 32462.75 54.7* with 4+4 teeth and one large striate chaeta. Dental spines absent. 602 Invertebrate Systematics F. Zhang et al.
Table 2. Comparison of Entomobryinae, Seirinae and Lepidocyrtinae
Characters Entomobryinae Seirinae Lepidocyrtinae Dental scales Present or absent Present Present Scale shape Most or some pointed Some pointed Rounded or truncate Striations on scale surface Coarse Coarse Fine Basal ribs longer than distal ones Yes/no No No Bothriotricha on Abd. II–IV 2, 3, 2 2, 3, 3 2, 3, 2 Accessory mic of bothriotrichal complexes Unmodified or modified Modified Modified Mucronal shape Bidentate or falcate Falcate Bidentate or falcate Mucronal spine Present or absent Absent Present or absent Tergal macrochaetae Abundant Abundant Few Tergal S-chaetae 22|122 11|122 11|011
Table 3. Comparison among Lepidodens, gen. nov., Willowsia and Lepidosira-group
Characters Lepidodens, gen. nov. Willowsia Lepidosira-group Dental scales Present Absent Present Scale shape Pointed Mostly pointed Some pointed Basal ribs longer than distal ones Yes Yes/no No Accessory mic of bothriotrichal complexes Unmodified Unmodified Modified
Mucro bidentate, its basal spine short with tip reaching apex of Lepidodens nigrofasciatus Zhang & Pan, gen. & sp. nov. subapical tooth. Tergal ms 1, 0|1, 0, 1, 0, 0 and sens 2, 2|1, 2, 2, 8, (Figs 3A, 4–6; Table 4) 3; ms on Abd. I anterior to m3. Accessory mic of bothriotrichal complexes on Abd. II IV unmodified. http://zoobank.org/urn:lsid:zoobank.org:act:C0DBF6EA-1CAC-4847- B762-73FD3F6B9687
Distribution Material examined South China. Holotype. , on slide, China: Guangdong, Huizhou, Longmen, Nankunshan Natural Reserve, altitude 497 m, 23 3804.0100N, 113 51015.2500E, Z.-X. Pan Remarks and Y.-T. Ma, 22.viii.2010 (sample number S4144). Paratypes. 1, on slide and one in alcohol, same data as holotype. The new genushas pointed scales with basal ribs longer than distal ones and S-chaetotaxic pattern 2, 2|1, 2, 2, 8, 3, which remarkably Description resembles typical Willowsia species (Entomobryinae), but differs from the latter in having dental scales and in the peculiar position Body length up to 1.30 mm. of S-microchaeta on Abd. I (Table 3). Molecular evidence Ground colour pale in alcohol. Base of Ant. I, whole head, Th. – also supports placing Lepidodens, gen. nov. in Entomobryinae II laterally, and Abd. I III dark. Hind femur with proximal and rather than Seirinae/Lepidocyrtinae. The new genus shares distal patches (Fig. 3A). Scales pointed, striate heavily with basal – dental scales with the Lepidosira-group, which is now placed ribs longer than distal ones (Fig. 4A D); scales on ventral side in Entomobryinae, but it differs from the latter in the pointed of dens narrower than those on body (Fig. 4D). Scales absent on – scales with long basal ribs, the position of S-microchaeta and the Ant. II IV, ventral tube, and dorsal side of manubrium and dens. unmodified accessory mic of bothriotrichal complexes (Table 3). Antenna 1.63 as long as cephalic diagonal. Antennal segment ratio as I : II : III : IV = 1 : 1.86 : 1.57 : 3.00. Antennae with various types of smooth chaetae (Fig. 5A). Smooth spiny Etymology mic at base of antennae: three dorsal, two ventral on Ant. I, one The new genus is named after the scaled dens (lepido + dens). It is internal, one external and one ventral on Ant II. Ant. II distally a masculine word because of ‘dens’. with a rod. Ant. III organ with two rods and three guard chaetae (Figs 4E, 5B). Antennal apical bulb unilobed (Fig. 5C). Eyes 8+8, G and H smallest. Labral margin with four conical Key to species of Lepidodens, gen. nov. papillae. Labral intrusion U-shaped. Prelabral and labral chaetae 4/5, 5, 4; prelabral ones ciliate but appearing smooth at low 1. Body uniformly pale; mesothorax protruded ...... magnification (Fig. 5D). Clypeal chaetae ciliate, their number not ...... hananicus Zhang & Pan, sp. nov. fi Body with distinct colour pattern; mesothorax not protruded...... 2 clearly seen. Dorsal cephalic chaetotaxy with ve antennal (An), 2. Pigment scattered on whole body...... similis Zhang & Pan, sp. nov. four median (M) and four sutural (S) mac; interocular chaetae as Transverse bands on Abd. I–III...... pqrst (Fig. 5E); chaetae along posterior margin short and spiny...... nigrofasciatus Zhang & Pan, sp. nov. Mandibles with four and five teeth. Subapical chaeta of maxillary New definition of Entomobryinae and Lepidodens, gen. nov. Invertebrate Systematics 603
(A) (B)
(C)
Fig. 3. Habitus. (A) Lepidodens nigrofasciatus, gen. & sp. nov. (B) Lepidodens similis, gen. and sp. nov. (C) Lepidodens hainanicus, gen. and sp. nov. Scale bar = 500 mm. outer lobe slightly larger than apical one; three hairs on sublobal Metatrochanteral organ with 8 12 smooth spiny chaetae; plate (Fig. 5F). Tip of lateral process of labial palp thin, not 5 7 in L-shaped arms and 3 5 between arms (Fig. 5I). reaching apex of labial papilla (Fig. 5G). Labial chaetae MEL1L2, Tibiotarsi distally with 10 ciliate chaetae in a whorl, and all ciliate; chaeta R absent. Chaetae posterior to labium ciliate. clavate tenent hair subequal to unguis in length (Fig. 5J). Cephalic groove with 7+7(6) ciliate chaetae (Fig. 5H). Unguis with four inner teeth, distal one very tiny; basal inner 604 Invertebrate Systematics F. Zhang et al.
(A) (B)
((C)) (D)
(E) (F)
(G) (H)
Fig. 4. Lepidodens nigrofasciatus, gen. and sp. nov. (A) Scales on Th. II, (B) scales on Abd. IV, (C) scales on ventral side of manubrium, (D) scales on ventral side of dens, (E) Ant. III organ, (F) Abd. I, (G) Abd. III, (H) Abd. V. Scale bar = 20 mm. New definition of Entomobryinae and Lepidodens, gen. nov. Invertebrate Systematics 605
(B) (G) (A)
(D)
(C)
(F) (E)
(I)
(J)
(H)
(L)
(K)
Fig. 5. Lepidodens nigrofasciatus, gen. and sp. nov. (A) Ventral side of Ant. I, (B) Ant. III organ, (C) Ant. IV apical bulb, (D) labrum, (E) dorsal cephalic chaetotaxy, (F) maxillary outer lobe, (G) lateral process of labial palp, (H) ventral side of head, (I) trochanteral organ, (J) hind claw, (K) manubrial plaque, (L) mucro. Scale bars = 20 mm(A–D, F–L); 50 mm(E). pair with tip reaching 0.30 from base, median one 0.66 and tube anteriorly withat least 6+6ciliate chaetae, 2 3of them larger distal one 0.87 from base. Unguiculus truncate with outer edge than others on each side; posteriorly with eight smooth chaetae; smooth. Abd. IV 4.63 as long as Abd. III along dorsal midline. each lateral flap with eight smooth chaetae (Fig. 6A). Manubrial Tenaculum with 4+4 teeth and one large striate chaeta. Ventral plaque with two pseudopores and 2–4 ciliate chaetae (Fig. 5K). 606 Invertebrate Systematics F. Zhang et al.
(A)
(B)
(D)
(C)
(E)
Fig. 6. Lepidodens nigrofasciatus, gen. and sp. nov. (A) Posterior face and lateral flap of ventral tube. (B–E) Tergal chaetotaxy. (B) Thorax, (C) Abd. I–III, (D) Abd. IV, (E) Abd. V. Scale bars = 20 mm(A); 50 mm(B–E).
Distal smooth part 1.33 1.44 as long as mucro. Mucro and p5 as mac (Figs 4A, 6B). Th. III with 13 mac and two sens; bidentate, its basal spine short with tip reaching apex of a1 and a3 as mic (Fig. 6B). Abd. I with two (m3 4) mac and subapical tooth (Fig. 5L). two S-chaetae; ms anterior to m3 and sens on the lateral side Th. II with three (m1, m2, m2i) medio-medial, three (m4, m4i, (Figs 4F, 6C). Abd. II with three (a2, m3, m3e) central, one (m5) m4p) medio-lateral, 10 posterior mac, one ms and two sens; m5 lateral mac and two sens. Abd. III with three (a2, a3, m3) central, New definition of Entomobryinae and Lepidodens, gen. nov. Invertebrate Systematics 607
Table 4. Morphological comparison among the three new species
Characters L. nigrofasciatus, L. similis, L. hainanicus, sp. nov. sp. nov. sp. nov. Pigment scattered on body No Yes No Abd. I–III with dark pigment Yes Yes No Ant./head 1.63 1.96–2.21 1.91–2.13 Mesothorax protruded No No Yes Mac on dorsal head An 5 5 8–9 S443 Ps2/Pa1/Pm1/Pp1 Present Present Absent Spiny chaetae on trochanteral organ 8–12 8–11 28 Unguiculus outer edge Smooth Smooth Serrate Mac on dorsal manubrium Absent Absent Present Ciliate chaetae on manubrial plaque 2–4 86 Uncrenulate dens/mucro 1.33–1.44 1.20–1.75 1.86–2.00 Mac m2i and m4p on Th. II Present Present Absent a4 on Th. II Mac Mac Mic Mac on Abd. I 2 2 1 Central mac on Abd. III 3 3 2 Central mac on Abd. IV 6–87–13 12–16 four (am6, pm6, p6, m7a) lateral mac, one ms and two sens; a3 as Material examined mic in one specimen (Figs 4G, 6C). Abd. IV with 6 8 central, ~15 Holotype. , on slide, China: Guangdong, Nanling National Natural lateral mac and eight sens; mac A5 rarely present; two lateral sens Reserve, altitude 915 m, 24 550000N, 113 020500E, F. Zhang and Z.-H. Li, (as, ps) obviously shorter than others (Fig. 6D). Accessory mic of 23.viii.2010 (sample number C9641). bothriotrichal complexes on Abd. II IV unmodified (Fig. 4B). Paratypes. 2, on slides and three in alcohol, same data as holotype. Abd. V with three sens (Figs 4H, 6E). Additional material examined. , on slide and one in alcohol, China: Guangdong, Huizhou, Longmen, Nankunshan Natural Reserve, altitude Ecology 620 m, 23 38031.3100N, 113 50038.6800E, Z.-X. Pan and Y.-T. Ma, 23. Under fresh leaf litter of Miscanthus floridulus (Labill.) and viii.2010 (sample number S4148). Pueraria lobata (Willd.). Remarks Description Lepidodens nigrofasciatus, sp. nov. is characterised by its unique Body length up to 3.50 mm. colour pattern. The new species is closest to L. similis, sp. nov. in Ground colour pale in alcohol. Dark blue pigment scattered most features except colour pattern and few minor characters on Ant. I, ventral tube, and whole head and body. Blue patches (Table 4). The most distinct separating character is colouration, also present on coxa, trochanter, proximal and distal femur, and dark transverse bands on Abd. I III in L. nigrofasciatus, sp. nov., sometimes hind tibiotarsus (Fig. 3B). Scales pointed, striate – but pigments scattered on whole body in L. similis, sp. nov. heavily with basal ribs longer than distal ones (Fig. 7A F); Length of antennae is longer in L. similis, sp. nov. (1.96–2.21 vs ventral side of manubrium with scales narrower than those on 1.63). In addition, great K2P genetic distances of 16S (0.0929) body (Fig. 7C); ventral side of dens with extremely narrow scales and 28S (0.0647) indicate two independent species. It is also (Fig. 8A). Scales on anterior face of ventral tube, legs and ventral similar to Willowsia ieti Yosii, 1971 in truncate unguiculus, labral side of dens few in individuals smaller than 2 mm, and abundant chaetae and papillae, lateral process of labial palp, maxillary in larger ones. – outer lobe and macrochaetotaxy of Abd. I II, but differs from Antenna 1.96 2.21 as long as cephalic diagonal. Antennal – – – the latter in colour pattern, labial chaetae R absent, presence of segment ratio as I : II : III : IV = 1 : 1.90 2.00 : 1.75 1.90 : 3.30 3.40. dental scales, smooth part of dens shorter, less spiny chaetae on Antennae with various types of smooth chaetae. Smooth spiny trochanteral organ, and chaetotaxy of thorax and Abd. III and IV. mic at base of antennae: three dorsal, two ventral on Ant. I, one internal, one external and one ventral on Ant II. Ant. III organ with Etymology two rods and three guard chaetae. Antennal apical bulb unilobed. The new species is named after the black transverse bands on Eyes 8+8, G and H smallest. Labral margin with four body. conical papillae. Labral intrusion U-shaped. Prelabral and labral chaetae 4/5, 5, 4; prelabral ones ciliate but appearing fi Lepidodens similis Zhang & Pan, gen. & sp. nov. smooth at low magni cation (Fig. 8B). Dorsal cephalic chaetotaxy with five antennal (An), four median (M) and four (Figs 3B, 7, 8; Table 4) sutural (S) mac; interocular chaetae as pqrst; chaetae along http://zoobank.org/urn:lsid:zoobank.org:act:BFCAA0EA-FFFC-4EF2- posterior margin short and spiny. Mandibles with four and five 8A56-58C5C02587CF teeth. Subapical chaeta of maxillary outer lobe slightly larger than 608 Invertebrate Systematics F. Zhang et al.
(A) (B)
(C) (D)
(E) (F)
Fig. 7. Scales in Lepidodens similis, gen. and sp. nov. (A) Dorsal head, (B) Abd. IV, (C) manubrium, (D) hind coxa, (E) mid-femur, (F) mid-trochanter. Scale bar = 20 mm. apical one; three hairs on sublobal plate. Tip of lateral process of Metatrochanteral organ with 8 11 smooth spiny chaetae; labial palp thin, curved, not reaching apex of labial papilla. Labial 6 8 in L-shaped arms and 2 3 between arms. Tibiotarsi chaetae MEL1L2, all ciliate; chaeta R absent. Chaetae posterior distally with 10 ciliate chaetae in a whorl. Unguis with four to labium ciliate. Cephalic groove with 7+7 ciliate chaetae. inner teeth, distal one very tiny; basal inner pair with tip reaching New definition of Entomobryinae and Lepidodens, gen. nov. Invertebrate Systematics 609
(E) (C)
(A)
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Fig. 8. Lepidodens similis, gen. and sp. nov. (A) Scales on dens, (B) labrum, (C) hind claw, (D) manubrial plaque, (E) mucro. (F–I) Dorsal chaetotaxy. (F) Thorax, (G) Abd. I–III, (H) Abd. IV, (I) Abd. V. Scale bars = 20 mm(A–E); 50 mm(F–I). 610 Invertebrate Systematics F. Zhang et al.
0.35 from base, median one 0.66 and distal one 0.88 from base. margin of Abd. III, posterior margin of Abd. IV and Abd. V Unguiculus truncate with outer edge smooth. Tenent hair clavate (Fig. 3C). Scales pointed, striate heavily with basal ribs longer and subequal to unguis in length (Fig. 8C). Abd. IV 4.86 6.36 than distal ones (Fig. 9A–D); scales on ventral side of dens as long as Abd. III along dorsal midline. Tenaculum with 4+4 narrower than those on body and manubrium (Fig. 9D). Scales teeth and one large striate chaeta. Ventral tube anteriorly with present on Ant. I and II (Fig. 9A), head, body, legs and ventral side at least 8+8 ciliate chaetae, 2 4 of them larger than others on of dens, and absent on ventral tube, tenaculum, and dorsal side of each side; posteriorly with eight smooth chaetae; each lateral manubrium and dens. flap with 8–14 chaetae, at least eight of them smooth. Manubrial Antenna 1.91–2.13 as long as cephalic diagonal. Antennal plaque with two pseudopores and ~8 ciliate chaetae (Fig. 8D). segment ratio as I : II : III : IV = 1 : 1.75 : 1.50 : 3.25. Smooth Distal smooth part of dens 1.20 1.75 as long as mucro. Mucro spiny mic at base of antennae: three dorsal, two ventral on bidentate, its basal spine short with tip reaching apex of subapical Ant. I, one internal, one external and one ventral on Ant tooth (Fig. 7E). II. Ant. II distally with a rod. Ant. III organ with two rods and Th. II with three (m1, m2, m2i) medio-medial, three (m4, m4i, three guard chaetae. Antennal apical bulb unilobed. m4p) medio-lateral, 10 posterior mac; m5 and p5 as mac. Th. III Eyes 8+8, G and H smallest. Labral margin with four conical with 13–14 mac; a1 and a3 as mic; p1i and a2a sometimes as mac; papillae. Labral intrusion U-shaped. Prelabral and labral chaetae p4 as mac in one specimen (Fig. 8F). Thoracic S-chaetae not 4/5, 5, 4, prelabrals ciliate (Fig. 10A). Clypeal chaetae ciliate, but clearly seen. Abd. I with two (m3 4) mac and two S-chaetae; ms their number not clearly seen. Dorsal cephalic chaetotaxy with anterior to m3 and sens on the lateral side. Abd. II with three (a2, 8–9 antennal (An), four (M1–4) median and three (S2, S5, S6) m3, m3e) central, one (m5) lateral mac and two sens. Abd. III with sutural mac; mac S3, Ps2, Pa1, Pm1 and Pp1 absent. Interocular three (a2, a3, m3) central, four (am6, pm6, p6, m7a) lateral mac, chaetae as pqrst (Fig. 10B). Chaetae along posterior margin short one ms and two sens; a3 rarely as mic (Fig. 8G). Abd. IV with and spiny. Mandibles with four and five teeth. Subapical chaeta of 7 13 central, 12–20 lateral mac and eight sens; I, M, A6, B3–6 maxillary outer lobe slightly larger than apical one; three hairs always as mac; two lateral sens (as, ps) obviously shorter than on sublobal plate (Fig. 10C). Tip of lateral process of labial palp others (Fig. 8H). Accessory mic of bothriotrichal complexes on thin, not reaching apex of labial papilla (Fig. 10D). Labial chaetae Abd. II IV unmodified. Abd. V with three sens (Fig. 8I). MREL1 L2, all ciliate; R/M = 0.70. Chaetae posterior to labium ciliate. Cephalic groove with ~12+12 ciliate chaetae (Fig. 10E). Ecology Metathorax protruded overlapping head. Metatrochanteral On barks of Cunninghamia lanceolata (Lamb.). organ with ~28 smooth spiny chaetae (Fig. 10F). Tibiotarsi distally with 10 ciliate chaetae in a whorl. Unguis with four Remarks inner teeth, distal one very tiny; basal inner pair with tip reaching 0.29 from base, median one 0.69 and distal one 0.89 from base. Lepidodens similis, sp. nov. is almost identical to L. nigrofasciatus, Unguiculus truncate with outer edge serrate. Tenent hair clavate sp. nov., but can be easily distinguished from the latter by colour and subequal to unguis in length (Fig. 10G). Abd. IV 4.64–5.10 ‘ ’ pattern. For more detail see above Remarks of L. nigrofasciatus, as long as Abd. III along dorsal midline. Tenaculum with 4+4 sp. nov. teeth and one large striate chaeta. Ventral tube anteriorly with ~11+11 ciliate chaetae, 3–4 of them larger than others (Fig. 11A); Etymology posteriorly not clearly seen; each lateral flap with at least seven The new species is named after the similarity to L. nigrofasciatus, smooth chaetae. Dorsolateral side of manubrium with 4+4 ciliate sp. nov. macrochaetae. Manubrial plaque with two pseudopores and six ciliate chaetae, inner two larger than others (Fig. 11B). Distal Lepidodens hainanicus Zhang & Pan, gen. & sp. nov. smooth part 1.86 2.00 as long as mucro. Mucro bidentate, its (Figs 3C, 9–11; Table 4) basal spine short with tip reaching apex of subapical tooth (Fig. 11C). http://zoobank.org/urn:lsid:zoobank.org:act:23585E78-0349-4CFD- Th. II with three (m1, m2, m2e?) medio-medial, two (m4, m4i) 911A-8C060D100756 medio-lateral, nine posterior mac, one ms and two sens; m5 as mic; p5 as mac. Th. III with 11 mac and two sens; a1 and a3 as mic; Material examined p6, m6, m6i and m6e as mac (Fig. 11D). Abd. I with one (m3) mac Holotype. , on slide, China: Hainan, Qionghai, under coconut trees, and two S-chaetae; ms anterior to m3 and sens on the lateral side. 0 00 0 00 19 9 26.7 N, 110 35 2.08 E, Z.-X. Pan, 28.iii.2013 (sample number S4394). Abd. II with three (a2, m3, m3e) central, one (m5) lateral mac and , Paratype. on slide, same data as holotype. two sens. Abd. III with two (a2, m3) central, four (am6, pm6, p6, m7a) lateral mac, one ms and two sens; a3 as mic (Fig. 11E). Abd. Description IV with 12 16 central, ~15 lateral mac, and eight sens; two lateral Body length up to 2.2 mm. sens (as, ps) obviously shorter than others (Fig. 11F). Accessory Ground colour pale in alcohol. Ant. I–II with distal patches. mic of bothriotrichal complexes on Abd. II IV unmodified. Abd. Basal and distal part of Ant. III pigmented or whole segment V with three sens (Fig. 11G). dark. Ant. IV pigmented. Small patches present on distal femora, proximal and median tibiotarsi; dark on hind leg and weak on other two legs. Body only with minute patches or stripes: lateral Ecology margin of Th. II and Th. III, median part and postero-lateral In litter under Cocos nucifera Linn. trees. New definition of Entomobryinae and Lepidodens, gen. nov. Invertebrate Systematics 611
(A) (B)
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Fig. 9. Scales in Lepidodens hainanicus, gen. and sp. nov. (A) Ant. I, (B) Abd. III, (C) dorso-lateral side of manubrium, (D) ventral side of dens. Scale bar = 20 mm.
Remarks S-chaetotaxy and mucro (Table 2). This study again confirms fi Lepidodens hainanicus, sp. nov. is the only member of the genus the high phylogenetic signi cance of S-chaetotaxy in the with pale body colour, the absence of posterior mac along dorsal systematics of Entomobryoidea (Zhang and Deharveng 2015; midline of head, and 4+4 macrochaetae on dorsolateral side of Zhang et al. 2015). Sens 2, 2|1, 2, 2 from Th. II to Abd. III are manubrium. The new species is similar to the other two species of identical to those in Entomobryinae. The peculiar position of the genus in most characters, but differs from them in colour S-microchaeta on Abd. I is unique in Entomobryoidea and pattern, dorsal cephalic chaetotaxy,labial chaetae, andchaetotaxy could be an important synapomorphy for the genus. of Abd. I, III and IV (Table 4). The relationship of Lepidodens, gen. nov. to other Entomobryinae genera is currently unresolved. Phylogenetic trees reconstructed here, as well as trees in previous studies Etymology (Zhang et al. 2014a, 2015), exhibited a polytomy within The new species is named after the type locality. Entomobryinae (Fig. 2). Lepidodens, gen. nov. resembles some of Willowsia in morphology, particularly the scale shape Discussion and surface sculpture. However, Willowsia has been demonstrated to be polyphyletic and can be separated into Systematic position of the new genus several clades (Yoshii and Suhardjono 1989; Zhang et al. Both morphological and molecular evidence strongly support 2014a, 2014b, 2015). Even Willowsia taxa with typical Lepidodens, gen. nov. clustering within Entomobryinae rather pointed scales (Zhang et al. 2011), such as W. nigromaculata, than Seirinae/Lepidocyrtinae. Although the new genus shares W. guangdongensis and W. japonica, have no clear relationships the presence of dental scales with Seirinae/Lepidocyrtinae, it with the new genus on the trees. This indicates that Lepidodens, could be distinctly separated by scale shape and surface sculpture, gen. nov. possibly has an independent origin from the Oriental 612 Invertebrate Systematics F. Zhang et al.
(A) (B)
(C)
(D)
(F) (E)
(G)
Fig. 10. Lepidodens hainanicus, gen. and sp. nov. (A) Labrum, (B) dorsal cephalic chaetotaxy, (C) maxillary outer lobe, (D) lateral process of labial palp, (E) ventral side of head, (F) trochanteral organ, (G) hind claw. Scale bar = 20 mm. realm; the peculiar position of S-microchaeta on Abd. I and the S-chaetotaxic pattern, more molecular evidence is needed the presence of dental scales strongly support this hypothesis. to assess its position. It possibly evolved from an Entomobrya-like ancestor. As for the Lepidosira-group also having dental scales, it distinctly differs from the new genus in the geographical distribution and Systematics of Entomobryinae morphological characters (Table 3). Scale shape and modified The present study demonstrates that dental scales could occur accessory chaetae of the bothriotricha complex indicate that in Entomobryinae, as they do in Heteromurinae, Seirinae the Lepidosira-group is distant from other Entomobryinae and Lepidocyrtinae. This discovery drastically undermines taxa. Although we here place it in Entomobryinae based on the traditional separation of Entomobryinae and Seirinae/ New definition of Entomobryinae and Lepidodens, gen. nov. Invertebrate Systematics 613
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Fig. 11. Lepidodens hainanicus, gen. and sp. nov. (A) Anterior face of ventral tube, (B) dorsal side of manubrium, (C) mucro. (D–G) Dorsal chaetotaxy. (D) Thorax, (E) Abd. I–III, (F) Abd. IV, (G) Abd. V. Scale bars = 20 mm(A–C); 50 mm (D–G).
Lepidocyrtinae. The distribution of scales (present or absent on Entomobryinae, ventral scales can be completely absent on the dens) is not the key character separating the three subfamilies. furca, or be present on only the manubrium or on both manubrium Entomobryinae has the highest diversity in scale morphology (see and dens. Comprehensive classification at generic level is still far Willowsia-complex; Zhang et al. 2011) and scale distribution from resolved although highly diversified scales are useful among all scaled groups in Collembola. Among scaled taxa in for species diagnosis. Multiple independent origins of body 614 Invertebrate Systematics F. Zhang et al. scales (Zhang et al. 2014a, 2015) may have resulted in the (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and extreme complexity of the taxonomy of Entomobryinae. The model choice across a large model space. Systematic Biology 61, present mosaic classification of Entomobryinae does not match 539–542. doi:10.1093/sysbio/sys029 requirements for modern phylogeny and evolution. Major genera, Schäffer, C. (1896). Die Collembolen der Umgebung von Hamburg und such as Entomobrya (Katz et al. 2015) and Willowsia (Zhang benachtbaren Gebeite. Mittheilungen aus dem Naturhistorischen Musenm 13, 147–216. et al. 2014a, 2015), are polyphyletic and remain incorrectly fi Shimodaira, H., and Hasegawa, M. (2001). CONSEL: for assessing the de ned. Great similarities between scaled and unscaled confidence of phylogenetic tree selection. Bioinformatics 17, taxa often challenge the current generic grouping (Zhang et al. 1246–1247. doi:10.1093/bioinformatics/17.12.1246 2014b). Adequate sampling is needed to provide more evidence Soto-Adames, F. N. (2008). Postembryonic development of the dorsal and allow reliable reconstructions of the phylogeny to improve chaetotaxy in Seira dowlingi (Collembola, Entomobryidae); with an the systematics of Entomobryinae. analysis of the diagnostic and phylogenetic significance of primary chaetotaxy in Seira. Zootaxa 1683,1–31. Acknowledgements Soto-Adames, F. N., Barra, J. A., Christiansen, K. A., and Jordana, R. (2008). Suprageneric classification of Collembola Entomobryomorpha. Annals Thanks should be given to Yitong Ma, who helped to collect some of of the Entomological Society of America 101, 501–513. doi:10.1603/ the material. This study was supported by a project funded by the 0013-8746(2008)101[501:SCOCE]2.0.CO;2 Priority Academic Program Development of Jiangsu Higher Education Stamatakis, A. (2014). RAxML version 8: a tool for phylogenetic analysis Institutions and the Zhejiang Provincial Natural Science Foundation of and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313. 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Appendix 1. Sequenced taxa Taxonomical assignment, collection locality and GenBank accession numbers. Numbers marked with a superscript ‘A’ are newly sequenced in the present study, with others extracted from Zhang et al.(2015)
Group Species name Locality 18S 28S COI 16S Tomoceridae Tomocerus ocreatus China KC236262 KC236303 KM978376 KC236221 Isotomidae Folsomia candida China KC236239 KC236281 KM978353 KC236200 Folsomia qudrioculata France KC236240 KC236280 KM978354 KC236199 Orchesellinae Ochesella cincta France KC236250 KC236290 KM978365 KC236208 Orchesellides sinensis China KC236251 KC236293 KM978363 KC236209 Orchesellides sp. China KC236226 KC236267 KM978364 KC236217 Heteromurinae Heteromurus major France KC236241 KC236282 KM978355 KC236201 Heteromurus nitidus France KC236242 KC236283 KM978356 KC291493 Dicranocentrus wangi China KC236232 KC236273 KM978348 KC236192 Alloscopus sp. China KM978398 KM978335 KM978344 KM978389 Seirinae Seira delamarei China KC236255 KC236292 KM978370 KC236213 Seira barnardi South Africa KC236254 KC236296 KU508096A KC236212 Seira sp. 1 China KC236257 KC236297 KM978371 KC236214 Seira sp. 2 South Africa KC236256 KC236298 KM978372 KC236215 Lepidocyrtinae Pseudosinella abla France KC236253 KC236295 KM978368 KC236211 Pseudosinella tumula China KC236252 KC236294 KM978367 KC236210 Lepidocyrtus sp. 1 China KC236248 KC236289 KM978361 KC236206 Lepidocyrtus sp. 2 China KC236249 KC236291 KM978362 KC236207 Ascocyrtus sp. China KC236228 KC236269 KM978345 KC236190 Entomobryinae Sinhomidia bicolor China KC236260 KC236301 KM978375 KC236220 Willowsia japonica China KC236265 KC236307 KM978378 KC236224 Willowsia guangdongensis China KC236264 KC236306 KM978377 KC236223 Willowsia nigromaculata France KC236263 KC236304 KM978379 KC236222 Willowsia sp. 1 China KC236247 KC236288 KM978380 KC236205 Lepidodens similis China KC236266 KC236305 KM978396 KC236225 Lepidodens nigrofaciatus China – KU508244A – KU508241A Lepidodens hainanicus China – KU508245A KU508095A KU508242A Entomobrya proxima China KC236236 KC236279 KM978351 KC236197 Entomobrya aino China KC236235 KC236279 KM978350 KC236195 Entomobrya multifasciata France KC236237 KC236276 KM978392 KC236196 Entomobrya sp. 1 China KC236234 KC236278 KM978352 KC236194 Entomobrya sp. 2 China KC236238 KC236277 KM978381 KC236198 Homidia sinensis China KC236245 KC236286 KM978359 KC236203 Homidia socia China KC236246 KC236287 KM978360 KC236204 Homidia sichuanensis China KC236244 KC236285 KM978358 KU508243A Sinella curviseta China KC236258 KC236300 KM978373 KC236219 Sinella longisensilla China KC236259 KC236299 KM978374 KC236216 Coecobrya tenebricosa France KC236231 KC236272 KM978347 KC236191 Drepanura sp. China KC236233 KC236274 KM978349 KC236193 Himalanura sp. China KC236243 KC236284 KM978357 KC236202
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