Mikhail Potapov

David Porco
email: david.porco.gm@gmail.com

Louis Deharveng
email: deharven@mnhn.fr

A new member of the genus Isotomurus from the Kuril Islands (Collembola: Isotomidae): returning to the problem of "colour pattern species"

Keywords: the Far East, hydrophilic, colour pattern, barcoding, taxonomy, polymorphism . nov.: 9-10, maxillary head, inner

published or not. The documents may come

A new member of the genus Isotomurus from the Kuril Islands (Collembola: Isotomidae): returning to the problem of "colour pattern species"

Introduction

The term "colour pattern species" was proposed by [START_REF] Yoshii | On some Collembola of New Caledonia, with notes on the "Colour pattern species[END_REF] for species that can be separated only by colour pattern of the body. This concept resulted from the study of the "forms" of the Paronellidae subgenus Pseudoparonella (Oceaniella) [START_REF] Yoshii | On some Collembola of New Caledonia, with notes on the "Colour pattern species[END_REF] in New Caledonia, in which all morphological characters were the same, including macrochaetotaxy of the body, while their very distinct colour patterns indicated several valid species. According to Yoshii the term was just a temporary one reflecting the state of Collembolan taxonomy at that time. In the history of taxonomy of Collembola, the value of colour was used and examined many times. It is a central character in the taxonomy of three large genera of Entomobryidae: Seira Lubbock, 1869 [START_REF] Christiansen | A Survey of the Genus Seira (Hexapoda: Collembola: Entomobryidae) in the Americas[END_REF][START_REF] Mutt | Puerto Rican species of Seira (Collembola: Entomobryidae)[END_REF], Entomobrya Rondani, 1861 and Orchesella Templeton in [START_REF] Templeton | Thysanurae Hibernicae, or Descriptions of such Species of Spring-tailed Insects (Podura and Lepisma, Linn.,) as have been observed in Ireland. Descriptions of the Irish Species of Thysanura[END_REF]. Paclt (1959), for instance, considered the various colour pattern of South African Seira as polymorphism within the boundaries of a single species, while previous and subsequent workers recognized more than 20 different species in the country, each with a more or less characteristic pattern [START_REF] Womersley | On some Collembola-Arthropleona from South Africa and Southern Rhodesia[END_REF][START_REF] Yosii | Collembolan Fauna of the Cape Province, with special reference to the genus Seira Lubbock. Special Publications from the Seto Marine Biological Laboratory[END_REF][START_REF] Janion-Scheepers | Updated list of Collembola species currently recorded from South Africa[END_REF]. In Entomobrya or Orchesella, colour pattern has long been the only character for species discrimination, and it remains the easiest one for identifying most species. In the course of modern taxonomy development, the value of colour has been however challenged. For instance, in Entomobrya, [START_REF] Jordana | A proposal of characters for taxonomic identification of Entomobrya species (Collembola, Entomobryomorpha), with description of a new species[END_REF] recently proposed a new setae notation system, which aimed to, as far as possible, "avoid the use of colour pattern". In the monograph on Palearctic Capbryinae and Entomobryini [START_REF] Jordana | Synopses on Palaearctic Collembola. Volume 7/1. Capbryinae & Entomobryini[END_REF], nevertheless, provided two keys to identify species of Entomobrya, one "partly by colour and morphology" and another by chaetotaxy. In the whole Entomobryoidea, beyond problems of colour variations in some species, the validity of "colour pattern species" was clearly supported in several cases by molecular and morphological data, i.e. several species differing only in colour pattern were validated, in particular among Lepidocyrtinae [START_REF] Soto-Adames | Molecular phylogeny of the Puerto Rican Lepidocyrtus and Pseudosinella (Hexapoda: Collembola), a validation of Yoshii's «color pattern species[END_REF][START_REF] Frias | The European Lepidocyrtus lanuginosus group (Collembola: Entomobryidae), definition and description of a new species from Spain[END_REF] and Entomobryinae [START_REF] Zhang | New insight into the systematics of the Willowsia complex[END_REF].

A recent paper by [START_REF] Katz | Taxonomic review and phylogenetic analysis of fifteen North American Entomobrya (Collembola, Entomobryidae), including four new species[END_REF] combining colour pattern and molecular approaches brings additional insight into this question for Entomobrya, showing both the taxonomic interest of colour pattern and its limitation: in some cases, a same colour pattern was shown to be associated to different Molecular Operational Taxonomic Units (MOTUs)-considered as "species" by the authors. This study also shows that chaetotaxy is affected by variability, as expected for plurichaetotic species, and that its use may be no more reliable than colour pattern in Entomobrya. The main conclusion of this paper is that colour pattern is a species-specific issue, in other words that its value as a taxonomic character has to be established for each species through the study of polymorphism.

The use of colour remains a challenge to taxonomists in Entomobryomorpha and Symphypleona. Taxonomy of other groups is less dependent on this character though similar questions may arise also in Poduromorpha, for example when using pattern in banded species of Pseudachorutinae [START_REF] Oliveira | Deux nouvelles espèces de Pseudachorutes d'Amazonie (Collemboles, Neanuridae)[END_REF][START_REF] Hisamatsu | A new species of the genus Pseudachorutes (Collembola, Pseudachorutidae) from the Shiragami Mountains in Northern Japan[END_REF][START_REF] Weiner | Collembola (Entognatha) from East Africa[END_REF] or in some groups of Lobellini [START_REF] Cassagnau | Collemboles des Philippines. I-Les lobelliens multicolores des montagnes de Luzon[END_REF]. The character was also examined in taxa consisting of superficially very similar species, such as Tomoceridae where delicate differences in small pale spots and iridescence of scales covering were shown to be indicators of specific status [START_REF] Felderhoff | Survey of Pogonognathellus Börner (Collembola: Tomoceridae) in the Southern Appalachians Based on Morphological and Molecular Data[END_REF].

In Isotomidae, the pattern of dark spots and bands on the body is mainly used in modern taxonomy for two large genera, Isotoma [START_REF] Bourlet | Memoire sur les Podures[END_REF]Isotomurus Börner, 1903. The latter genus is one of the most abundant arthropods in temperate wet habitats and exhibits a great diversity of colour patterns. However, the species identification in Isotomurus remains one of the most "problematic" task in the family, in spite of considerable contribution of the taxonomists [START_REF] Deharveng | Remarques sur la morphologie et la taxonomie du genre Isotomurus Börner, 1903 et description de deux espèces nouvelles de France (Collembola: Isotomidae)[END_REF][START_REF] Carapelli | Assessing species boundaries and evolutionary relationships in a group of south-western European species of Isotomurus (Collembola, Isotomidae) using allozyme data[END_REF][START_REF] Fjellberg | The Collembola of Fennoscandia and Denmark. Part. II: Entomobryomorpha and Symphypleona[END_REF]). In the past Isotomurus colour forms were often considered as intraspecific variability [START_REF] Folsom | Nearctic Collembola, or springtails, of the family Isotomidae[END_REF][START_REF] Stach | The Apterygotan Fauna of Poland in Relation to the World-Fauna of this Group of Insects. Family: Isotomidae. Polska Akademia Umiejętności[END_REF][START_REF] Gisin | Collembolenfauna Europas. Museum d'Histoire Naturelle[END_REF][START_REF] Poinsot-Balaguer | Contribution а l'étude de quelques espèces du groupe lsotomurus palustris (Müller) (Collembole Isotomidae)[END_REF]. Conversely, [START_REF] Cassagnau | A propos des types de coloration chez Isotomurus palustris (Müller) (Collemboles)[END_REF] considered that colour pattern had been often neglected or inaccurately analyzed, while it could be a useful character if its fluctuation range is studied on a large number of specimens. Further molecular analysis and ecological observations confirmed that "colour forms" often corresponded to valid species (Carapelli et al. 1995a(Carapelli et al. , 1995b[START_REF] Carapelli | Taxonomic revision of 14 south-western European species of Isotomurus (Collembola, Isotomidae), with description of four new species and the designation of the neotype for I. palustris[END_REF][START_REF] Carapelli | Assessing species boundaries and evolutionary relationships in a group of south-western European species of Isotomurus (Collembola, Isotomidae) using allozyme data[END_REF]. Other morphological characters (details of chaetotaxy) partly supported the value of colour pattern, which, however, remained the leading if not the only diagnostic character, as summarized by [START_REF] Carapelli | Taxonomic revision of 14 south-western European species of Isotomurus (Collembola, Isotomidae), with description of four new species and the designation of the neotype for I. palustris[END_REF]: "The habit of considering the different pigmentation patterns as one of the most useful discriminating features at the species level is confirmed". Two problems remained in Isotomurus: one is that several species can be characterized by similar colour patterns; another one is that intra-specific variability of the pattern exists in many species, making the determination of deviated individuals hardly possible [START_REF] Carapelli | Taxonomic revision of 14 south-western European species of Isotomurus (Collembola, Isotomidae), with description of four new species and the designation of the neotype for I. palustris[END_REF][START_REF] Fjellberg | The Collembola of Fennoscandia and Denmark. Part. II: Entomobryomorpha and Symphypleona[END_REF].

In the course of our study of Collembola from the Far East of Russia we found an interesting material of abundant and highly variable new species of Isotomurus, the description of which is given below. The lack of taxonomic significance of its colour forms is supported by genetic data (COI 5' 'barcoding fragment').

Material and methods

Species description. Vocabulary and setal notation follows mostly [START_REF] Potapov | Synopses on Palaearctic Collembola. Vol. 3. Isotomidae[END_REF]. The following abbreviations are used: Abd.-abdominal segment; Ant.-antennal segment; AO-antennal organ; ms-micro s-seta(e) or mssetae (=microsensillum(a) auct.); PAO-post-antennal organ; s in the text and figures-macro s-setae or s-setae (=sensillum(a) auct.); Th.-thoracic segment.

DNA barcoding and sampling. Twelve specimens of I. festus sp. nov., including 3 pale and 9 banded, as well as specimens of three other Isotomurus species were barcoded (Table 1).

Molecular analyses. DNA was extracted from entire specimens in 30µl of lysis buffer (http://www.ccdb.ca/ docs/CCDB_DNA_Extraction.pdf) and proteinase K incubated at 56°C overnight. DNA extraction followed a standard automated protocol using 96-well glass fibre plates [START_REF] Ivanova | An inexpensive, automation-friendly protocol for recovering highquality DNA[END_REF]. Specimens were recovered after DNA extraction using a specially designed work flow allowing their morphological examination [START_REF] Porco | Coupling non-destructive DNA extraction and voucher retrieval for small soft-bodied Arthropods in a high-throughput context: the example of Collembola[END_REF]. The 5' region of COI used as a standard DNA barcode was amplified using M13 tailed primers LCO1490 and HCO2198 [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF]. Samples that failed to generate an amplicon were subsequently amplified with a pair of internal primers combined with full length ones (C_LepFolF/C_LepFolR) (Ivanova-published on www.boldsystems.org). The standard PCR reaction protocol of the Canadian Center for DNA Barcoding was used for amplifications (http://www.dnabarcodes2011.org/conference/preconference/CCDB-Amplification-animals.pdf), and products were checked on a 2% E-gel 96Agarose (Invitrogen). Unpurified PCR amplicons were sequenced in both directions using M13 tailed primers, with products subsequently purified using Agencourt CleanSEQ protocol and processed using BigDye version 3.1 on an ABI 3730 DNA Analyzer (Applied Biosystems). Sequences were assembled and edited with Sequencher 4.5 (GeneCode Corporation, Ann Arbor, MI, USA). The alignment was obtained using BIOEDIT version 7.0.5.3 [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. Sequences are publicly available on BOLD in the dataset [DS-ISOKU] accessible via through the following DOI (dx.doi.org/10.5883/DS-ISOKU) and on GenBank (GU656452-GU656455, GU656457, KY829286-KY829308).

Data analyses. Distance analyses were performed with MEGA6 [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF], utilizing a Neighbor-Joining [START_REF] Saitou | The neighbor-joining method-a new method for reconstructing phylogenetic trees[END_REF] algorithm with the Kimura-2 parameter model [START_REF] Kimura | A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide-sequences[END_REF] to estimate genetic distances. The robustness of nodes was evaluated through bootstrap re-analysis of 1000 pseudoreplicates. The tree was replotted using the online utility iTOL [START_REF] Letunic | Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation[END_REF]. Description. Body size 1.9-2.3 mm. Body shape as common for the genus, with rather prognathous head (Fig. 1). Brightly orange when alive (probably true only for colour forms with light background, see below). In alcohol background pale yellowish. Eye areas black, connected by broad band on anterior part of head, bases of antennae paler. Posterior part of head with large black medial spot. Antennae and foots diffusely dark, furca paler. Other characteristics of colour pattern of body varies (see below).

8+8 ommatidia of subequal size in typical position. PAO broad elliptical, as long as nearest ommatidium. Maxillary outer lobe with 4 sublobal hairs and bifurcate palp (Fig. 11). Labrum with 4 prelabral setae, labral edge as common for the genus, with four sharp ridges and ventro-apical ciliation. Frontoclypeal area with many setae. Labial palp with all usual papillae and complete set (16) of guards, ~ 9 proximal, 5-6 basomedian and 5 basolateral setae present (Fig. 12). Ventral side of head with 5-9+5-9 postlabial setae (Fig. 12). Maxillary head with lamella 1 slender, not expanded, with one row of stronger cilia along edge, other lamellae with fields of thin denticulation. (Figs 9-10). Antennae about 1.4-1.7 as long as head diagonal. With rather few s-setae on three first antennal segments. Ant. 1 with 5-9 s-setae: 5-3 short and thick ("spine-like") and 2-4 long (Fig. 7), Ant. 3 with few s-setae, "AO area" with two thick inner s-setae, group of slender s-setae in more lateral position, ms-seta and one thick lateral s-seta, with several erect s-setae around (Fig. 6). Ant. 4 with minute secondary branch present on subapical pin-seta (bifurcate pin-seta).

Body with cover of finely ciliated setae unequal in length (Fig. 23), ciliation often visible only under highest magnification. Neither trichobothria nor true macrosetae differentiated. Ratio longest setae to length of Abd. V as 0.3-0.4. The s-setae short (Fig. 22), longer on posterior part of trunk (Fig. 23). Number of s-setae in full grown specimens as 4,4/4,5-7,>14,>15,>14 (Fig. 21), first instar juveniles with somewhat fewer s-setae on posterior tergites (Fig. 24). The ms-setae 1,1/1,1,1 in number. Accp-s-setae in front of p-row in grown specimens, Abd. III-V with asymmetrical arrangement and number of s-setae with some in medial position (unpaired). On Abd. IV and V considerable part of dorsal area covered with as-s-setae (Fig. 21). Legs with slender and long claws, ratio tibiotarsus : outer edge of claw as 0.8-1.0 on Leg 1 and 1.5-2.0 on Leg 3 (Figs 234). Claw with a pair of small lateral tooth, an outer one, and rudimentary inner tooth (often invisible ) (Fig. 5). Empodial appendage without inner tooth. Tibiotarsi in juvenile specimens and first instar with 8 distal setae. Retinaculum with 4+4 teeth and 10-15 setae. Ventral tube with 3+3 laterodistal, 0-5+0-5 anterior and 16-20 posterior setae. Manubrium densely covered by setae. Manubrial thickening simple, without teeth in medial part. Dens rather slender, continuously narrowed, with rather few short setae on posterior side which cover only its basal part (ca 1/5 of its length). One of posterior setae in basal part slightly longer than others. Posterior side of dens crenulated, wrinkles regular in distal half and more disordered in basal half (Fig. 8). Mucro with four teeth (Fig. 13141516) and a seta on outer side. "Apical" tooth large (as large as subapical). Subapical tooth with two lamellae running to mucro base. Middle tooth scoop-like with two lamellae running back, outer lamella wider. Outer proximal tooth of common shape. Ratio manubrium : dens : mucro as 6.8-9.7 : 10.5-16.5 : 1. Ratio mucro : outer edge of claw III as 0.25-0.34. First instar juveniles without ventroapical manubrial setae and seta on mucro. Males are sporadic and thus the population consists mostly of females. Fully adult males have not been seen by us and so presence of modified setae on Abd. III-IV remains to be studied.

Colour forms. Type population consists of several colour forms mixed together. Pale variant is almost without pigmentation on corpus apart from weak diffuse spots on lateral parts of body and bases of legs, dark spot is also often seen on front part of mesothorax (Fig. 18). Most individuals have a broad mid-dorsal band interrupted in intersegmental areas and running from thorax to last abdominal segment. The size of spots forming the band become narrow towards posterior, the spots are much weaker or absent at the end of abdomen (Fig 17). Lateral parts of Th. II-Abd. II have dark irregular spots. In some individuals the main colour pattern is masked by darker background and therefore medial band is hardly visible (Fig. 20). Some specimens are, in fact, with homogeneous diffuse and rather pale colouration (Fig. 19). Colour photos of the described forms and their variability (apart from diffusely dark one) are shown in Figs 25-26. Association of colour pattern with size of the individuals or sex has not been detected.

In the subsequent molecular analysis, the variants with clear medial band were named "banded" (Figs 17, 20), while all others, without such a band, were called "pale" .

Name derivation. The name reflects bright (festal) colour of alive specimens. Distribution and ecology. Known only from the type locality. It was found in mass population forming orange layer in wet mosses sprinkled with running water of mountain spring (Fig. 27).

Discussion. Three species, i.e. I. festus sp. nov., I. chaos [START_REF] Potapov | Synopses on Palaearctic Collembola. Vol. 3. Isotomidae[END_REF][START_REF] Potapov | A new species of Isotomurus (Collembola: Isotomidae) without trichobothria[END_REF]I. antennalis Bagnall, 1940, combine in a compact group which probably deserves the rank of supra-specific taxon. So far we named it as the 'antennalis' group [START_REF] Potapov | Synopses on Palaearctic Collembola. Vol. 3. Isotomidae[END_REF] which is defined by the lack of trichobothria and macrosetae on the abdominal segments. S-chaetotaxy is disproportionate in all mentioned species: tergites of thorax and anterior half of abdomen have few s-setae while posterior half of abdomen possesses many such setae. Thus both I. festus sp. nov. (Fig. 17) and I. chaos (Fig. 1 in Potapov & Babenko 2011) have only 3 accp-s-setae on Th. II, III, and Abd. I and many irregularly scattered s-setae on the last abdominal segments. Isotomurus antennalis is also characterized by a similar s-pattern on body (Fjellberg, pers. comm.). The new species differs from the other two members of the group by a combination of very slender claws, large apical tooth of mucro and weak setal covering of posterior side of the dens. All three characters are uncommon within the genus Isotomurus: slender claws are shared with I. alticolus [START_REF] Carl | Uber schweizerische Collembola[END_REF] (Europe) and I. takahashii [START_REF] Yosii | On some Collembola from Formosa[END_REF] (Japan), large apical tooth with I. annectens Yosii, 1963 (Japan). Isotomurus takahashii was described as a member of Isotoma, the genus having no trichobothria. Afterwards [START_REF] Yosii | On some Collembola of Hindukush, with notes on Isotoma Bourlet and its allies. Results of the Kyoto University Scientific Expedition to the Karakoram and Hindukush[END_REF] wrote that "setae sensuales [=trichobothria, authors' note] were previously overlooked, but they are rather short, filiform and ciliated on all sides" and moved accordingly I. takahashii to the genus Isotomurus. The species belongs to group other than 'antennalis'. Isotomurus annectens differs from I. festus sp. nov. by normal shape of claw and presence of trichobothria.

Results of barcode analysis and general comments concerning the colour forms of Isotomurus

The DNA barcoding results support the specific status for the population described here as I. festus sp. nov.: no genetic difference was detected between its colour forms (Fig. 28). The genetic divergence between the new species and the other species of the genus (ranging from 18.74% to 21.18%: Table 2) is comparable to the divergence found among these described species (ranging from 22.07% to 24.82%: Table 2). Intraspecific divergences were also found similar (ranging from 0% to 1.52%: Table 2). Further study based on a larger taxonomic sampling and additional genes less prone to signal saturation than COI will be necessary to understand the relations between I. festus sp. nov., the members of the 'antennalis' group and morphologically similar taxa such as the two 'austral' forms, Chionobora Greenslade & Potapov, 2015 (Tasmania) and Kaylathalia Stevens & d'Haese, 2016 (Antarctic), which share several essential characters with the 'antennalis' group of Isotomurus [START_REF] Greenslade | Biology, affinity and description of an unusual aquatic new genus and species of Isotomidae (Collembola) from high altitude lakes in Tasmania[END_REF][START_REF] Stevens | Antarctic springtail (Collembola: Isotomidae) misguided by morphology and ecology[END_REF] and make the situation even more complex.

Regarding polychromatism, our molecular results support unambiguously that the two contrasting colour patterns of I. festus sp. nov. represent only a polymorphism within a single species. After analysing several Entomobrya species with contrasted patterns, [START_REF] Katz | Taxonomic review and phylogenetic analysis of fifteen North American Entomobrya (Collembola, Entomobryidae), including four new species[END_REF], concluded that "genetic divergence did not always correlate with colour pattern variation" and so that the diagnostic value of this character was species dependent, requiring its evaluation in each species. The result of the present study leads us to similar conclusions for the genus Isotomurus: colour pattern variations may either correspond to intra-specific polychromatism as in I. festus sp. nov., or represent real diagnostic characters among closely related species as shown by Carapelli et al. (1995) who established the taxonomic value of colour patterns in several species of the genus based on genetic markers.

Given the large number of Isotomurus colour forms described in the first half of the XXth century and many synonymies subsequently proposed without any analysis of polymorphism in colour patterns [START_REF] Gisin | Collembolenfauna Europas. Museum d'Histoire Naturelle[END_REF], summarized in Potapov 2001), as well as the frequent co-occurrence of different colour forms in a same habitat (Carapelli et al. 1995b), the re-assessment of the taxonomy of the genus is unavoidable. Such a task might prove to be a long run one and molecular taxonomy can certainly help achieving it.

FIGURES 1 - 8 .

 18 FIGURES 1-8. Isotomurus festus sp. nov.: 1, habitus; 2-4, distal part of leg 3,2, and 1; 5, distal part of leg 3; 6, s of Ant.3 (area of AO); 7, s of Ant.1; 8, dens, posterior view. s-s-seta, ms-ms-setae, lat-lateral s-setae of AO, i-inner s-seta of AO, echerect s-seta.

  FIGURES 21-24. Isotomurus festus sp. nov., chaetotaxy of body: 21, sensillar chaetotaxy in adult individual; 22, s-seta of Th. II, 23, setae and s-setae of dorsal area of Abd. III, 24, chaetotaxy in first instar (common setae shown as dots). ms-micro-sseta, s-s-seta.

  FIGURES 25-27. Photos of banded and pale specimens in dorsal (25) and lateral (26) views; and the type biotope (27) of I. festus sp. nov.

FIGURE 28 .

 28 FIGURE 28. Neighbour-joining trees (K2P) based on COI for the four Isotomurus species and one colour form. 100% bootstrap support values are shown on branches. For I. festus sp. nov. black squares stand for 'banded' specimens and white squares for 'pales' ones.

TABLE 1 .

 1 List of barcoded specimens. Type material: Holotype, Russian Far East, the Kuril Islands, Kunashir Isl., Kurilsky Reserve, vicinity of Alekhinskaya outpost, behind Sernaya Bay, bank of spring, in moss sprinkled with water, 1.vi.2011. A. Matalin leg. Many paratypes in the same locality. The holotype and 15 paratypes are deposited in the collection of the Department of Zoology & Ecology, Moscow State Pedagogical University. 17 paratypes including 12 vouchers of barcoded specimens are deposited in the collection of the Museum national d'Histoire naturelle (MNHN) in Paris.

	Taxonomy
	Isotomurus festus sp. nov.
	Figs 1-26.

TABLE 2 .

 2 Intraspecific and interspecific genetic divergences among species (based on K2p distances percentages for COI)

Acknowledgements

This study received financial support of RFBR (Russia, research project № 16-54-50068) for Potapov M. We would like to express our sincere thanks to two anonymous reviewers for their effective comments and Andrey Matalin (Moscow) for the valuable material.