Contribution to the Willowsia species having body scales of the long basal rib type : four new species and a redescription of W .

Abstract. Four new species of the genus Willowsia Shoebotham, 1917 are described from China: W. fascia Zhang & Pan sp. nov., W. pseudoplatani Zhang & Pan sp. nov., W. pseudobuskii sp. nov., and W. similis sp. nov. Willowsia qui Zhang, Chen & Deharveng, 2011 is re-described based on types and fresh material. All five species possess the pointed body scales with basal ribs longer than distal ones. These scales are absent on antennae, legs, ventral tube and furca. Colour pattern and dorsal chaetotaxy are the main diagnostic characters for these species. DNA barcodes of four species are also provided, with genetic distances compared. A key to the Willowsia species having scales of the long basal rib type is given.


Introduction
Willowsia Shoebotham, 1917 is the largest scaled Entomobryinae genus, comprising 27 species worldwide (Bellinger et al. 1996(Bellinger et al. -2016)).Its members have body scales mostly pointed, with rough sculpture of very short spinules to long ribs (Zhang et al. 2011).Yoshii & Suhardjono (1989) questioned the monophyly of Willowsia, as well as of Willowsiini Yoshii & Suhardjono, 1989, whose polyphyly was recently demonstrated by molecular phylogeny (Zhang et al. 2014a(Zhang et al. , b, 2015)).Zhang et al. (2011) divided the taxa of Willowsia into four species groups based on the scale sculpture: spinulate type, short rib type, long rib type, and uninterrupted ribbed type.Eleven species, including the generic type W. nigromaculata (Lubbock, 1873), possess scales of the long rib type.In this study, four new species of this group from China are described.Willowsia qui Zhang, Chen & Deharveng, 2011 is re-described based on types and fresh material because of the incorrect designation of one paratype in the original description.A key to the species of Willowsia that have scales of the long basal rib type is given.

Material and Methods
Specimens were mounted in Hoyer's solution after clearing in Nesbitt's fluid, and were studied using a Nikon 80i phase contrast microscope.Photographs were taken with a Nikon DS-Fi1 camera mounted on Nikon SMZ1000 and 80i microscopes and were enhanced with Photoshop CS2 (Adobe Inc.).Dorsal cephalic chaetae are named after Jordana & Baquero (2005), interocular chaetae after Mari-Mutt (1986), labial chaetae after Gisin (1967), and tergal S-chaetae after Zhang & Deharveng (2015).Tergal chaetae are designated following Szeptycki (1979).The number of macrochaetae is given by half-tergite in the descriptions.Material is deposited in the collections of the Department of Entomology, College of Plant Protection, Nanjing Agricultural University (NJAU), Nanjing, China, the School of Life Sciences, Taizhou University (TZU), Zhejiang, China, and the Muséum national d'Histoire naturelle (MNHN), Paris, France.
To obtain DNA barcodes (mitochondrial COI fragment), DNA extraction, amplification, purification and sequencing followed Zhang et al. (2014c).Sequences were assembled in Sequencher 4.5 (Gene Codes Corporation, Ann Arbor, USA), and deposited in GenBank.A final 658 bp alignment was obtained.Genetic distances were calculated in MEGA 5.0 (Tamura et al. 2011), with the Kimura-2 parameter substitution model and pairwise deletion for gaps.

Results
The following characters are shared by the five species included in this paper and are not repeated in the descriptions: Ant.II distally with a small rod;

Etymology
Named after the dark bands on the abdomen.

Description
Body length up to 1.70 mm.
Ground colour yellow in alcohol.Ant.I−IV gradually darker.Transverse bands on Abd.II-III (Fig. 1A).Scales brown, pointed, heavily striate with basal ribs very long (Fig. 1B), and only present dorsally on head and terga.Partial chaetae of the row along the posterior margin of each tergum transformed into scales and larger than other scales.

Ecology
In decomposing leaves.

Etymology
Named after the great similarities between the new species and W. platani (Nicolet, 1842).

Description
Body length up to 1.74 mm.
Ground colour pale in alcohol.Ant.I−IV gradually darker.A transverse band between the eye patches.Dark blue stripe on lateral margin of mesothorax.Metathorax and Abd.II-IV dark blue.Abd.V posteriorly with a transverse band (Fig. 4A).Scales narrow, pointed, heavily striate with basal ribs longer than distal ones (Fig. 4B), and only present dorsally on head and terga.

Ecology
Found in litter, debris and rotten logs of evergreen broad-leaved forest.

Remarks
Willowsia pseudoplatani sp.nov. is characterized by the unique colour pattern and the thoracic chaetotaxy.The new species is closest to W. platani (Nicolet, 1842) in most features except several characters.W. pseudoplatani sp.nov.has pale Abd.V and longer smooth part of dens.In chaetotaxy, mac m5 on Th.II and mac a2, a5, m5i on Th.III are present in W. pseudoplatani sp.nov.but absent in W. platani; on Abd.III, a3 is mic in the new species and mac in W. platani.

Etymology
Named after the great similarities between the new species and W. buskii (Lubbock, 1869).

Description
Body length up to 1.50 mm.Ground colour pale in alcohol.Ant.I−IV gradually darker.A transverse band between the eye patches.Dark stripe on lateral margin of Th.II-Abd.IV.Transverse bands on Th.III, posterior margin of Abd.II, and Abd.III.Irregular longitudinal stripes on lateral part of Abd.IV.Abd.V and Abd.IV posteriorly dark.Base of legs, distal femora, and tibiotarsi pigmented (Fig. 7A-C).Scales brown, pointed, heavily striate with basal ribs longer than distal ones, and only present dorsally on head and terga.Partial chaetae of the row along the posterior margin of each tergum transformed into scales and larger than other scales (Fig. 7E-H).

Ecology
In decomposing litter of Oryza sativa Linnaeus, 1753.

Etymology
Named after the similarity to Willowsia fascia Zhang & Pan sp.nov.

Paratypes
CHINA: 3 ♀♀ on slides and 3 in alcohol, same data as holotype.One paratype on slide (slide # 4381_4) deposited in MNHN,3 in alcohol in TZU,

Description
Body length up to 1.83 mm.
Ground colour yellow in alcohol.Ant.I−IV gradually darker.Transverse bands on Th.III and Abd.II-III (Fig. 10A-B).Scales brown, pointed, heavily striate with basal ribs very long, and only present dorsally on head and terga.Partial chaetae of the row along the posterior margin of each tergum transformed into scales and larger than other scales (Fig. 10D).

Description
Body length up to 1.8 mm.
Ground colour pale in alcohol.Ant.I−II with distal patches and III-IV dark.A transverse band between eye patches.Narrow stripes present on lateral margin of Th.II-Abd.I. Narrow transverse bands on posterior margin of Abd.II-III.Small patches posteriorly on Abd.IV-VI (Fig. 13A).Scales pointed, heavily striate with basal ribs longer than distal ones, and only present dorsally on head and terga.Some chaetae of the row along the posterior margin of each tergum transformed into scales and larger than other scales (Fig. 13B).

Discussion
Willowsia has the most diversified scale morphology (shape and sculpture) in Collembola, with four basic scale types recognized within the genus (Zhang et al. 2011).However, scale morphology, size and distribution across different terga and different areas of a tergum remain largely undocumented.In this study, some of the chaetae of the row along the posterior margin of each tergum were found to be transformed into scales and larger than other scales, differing from all scales along posterior margin in Lepidocyrtinae and Seirinae.Further examination confirmed that this phenomenon also occurred in many species with scales of the long basal rib type, the short rib type, and the uninterrupted type, although information for some species cannot be traced.In scale distribution, the species with scales of the long basal rib type and the uninterrupted type usually have no scales on the antennae and manubrium (Willowsia guangdongensis Zhang, Xu & Chen and Willowsia mekila Christiansen & Bellinger, 1922 excluded).The above evidence implies scale types are likely of high phylogenetic significance within Willowsia.
Within the species having scales of the long basal rib type, W. samarkandica Martynova, 1972, W. jacobsoni (Börner, 1913) and W. guangdongensis seem to be distant from other species.The former two have labral papillae with multiple denticles rather than one.The small subapical tooth and long basal spine on the mucro in W. samarkandica are unique.Most species of the group have 7+7 (A5 as mesochaeta in W. qui) central mac on Abd.IV, whereas they are numerous in W. samarkandica and 5+5 in W. jacobsoni.W. guangdongensis is the only species having scales on the antennae, legs and ventral side of manubrium.The colour pattern and dorsal chaetotaxy are the main diagnostic characters within the group.Among closely related species, the genetic distances of COI barcodes could provide powerful evidence for species delimitation.

Table 1 .
Comparison between W. similis sp.nov.andW.fasciaZhang & Pan sp.nov.Willowsia fascia sp.nov. is characterized by the two transverse bands on Abd.II-III.The new species is closest to W. similis sp.nov. in the dark Abd.II-III, scale sculpture, clypeal chaetae, labrum, labium, claw, mucro and abdominal chaetotaxy, and differs from the latter in the unpigmented metathorax, mac S 0 present dorsally on the head, and thoracic chaetotaxy (Table1).The great genetic distance (0.200) of the mitochondrial COI also indicates two independent species.