Two new Megalothorax species of the minimus group (Collembola, Neelidae).

Two new Megalothorax species, Megalothorax potapovi sp. n. from the Russian Far East and Megalothorax sanguineus sp. n. from the French Pyrénées are described. The two new species have a set of morphological characters (including a smooth mucro) that places them among the minimus group sensu Schneider and D'Haese (2013). Megalothorax potapovi characteristics include dorsal protuberance on forehead, peculiar chaetotaxy of antenna III and strong lanceolate chaetae on body. Megalothorax sanguineus characteristics include strong red pigmentation, large network of integumentary channels on head and elongated apex of the two postero-distal spines of dens. The DNA barcodes (cytochrome oxidase subunit I-COI) of the two species are also provided and analyzed among a broader sampling of the genus in order to support further their specific status. A special focus is given to the labral morphological characteristics. Pseudopores-like elements are reported for the first time in the genus. Positions of the τ-chaetae near the dorsal sensory field of thorax II are compared for several species of the genus.

Introduction

During an expedition organized by Mikhail Potapov in Primorye (Russian Far East) in 2004, a large number of samples yielded a huge diversity of . Several of the most remarkable taxa collected have been recently described, like the new genus with three new species (Pomorski and Sveenkova 2006), a genus that was subsequently retrieved in Northeastern China (Sun et al. 2013) and in other regions of northern Russia (Babenko et al. 2011). The Sino-Korean subfamily was also reported for the first time for Russia (Deharveng et al. 2010) with two new species. Here, we report a morphologically remarkable new species of the genus Willem, 1900 (, ): sp. n. On the other end of the Palaearctis, the faunistical survey of the Pyrenean peatland of Bernadouze yielded another new species of , sp. n. The two new species are described in the present paper, along with their barcode sequences (COI–mitochondrial cytochrome oxidase subunit I). Their genetic divergence levels with other are assessed and discussed. A special focus is given to the labral morphological characteristics. Pseudopores-like elements are reported for the first time in the genus. Positions of the τ-chaetae near the dorsal sensory field of Th. II are compared for several species of the genus.

molecular operational taxonomic units

Material and methods

Sampling

sp. n. The specimens were obtained from a 2000 cm3 sample of forest litter from Primorye, that were processed for fauna extraction in a field laboratory of Anisimovka. The litter sample was dried for 8 days on a Berlese funnel without heating.

sp. n. The two sampled sites are located at medium elevation in Ariège Pyrenees (France): in very humid mossy habitats near the peat-bog of Bernadouze, and from humid litter at Osque du Couret. Samples were processed in the lab on Berlese funnels in the same conditions as above.

Morphology

Specimens were preserved in 95% ethanol then cleared in lactic acid and finally mounted on microscope slides in Marc André II medium. They were examined using a Leica DMLB compound microscope with differential phase contrast optics at magnifications ranging from 250 to 1000. Drawings were made with a drawing tube and vectorized with Inkscape. For observations, specimens were dehydrated in 100% ethanol, before critical point drying (Emitech PageBreakK850) and gold coating (Jeol JFC-1200) and observations were performed with a SEM Jeol 840A. sp. n. was not observed with SEM, however we were able to locate all the positions of the τ-chaetae (trichobothria) with the optical microscope.

Scanning Electronic Microscope

DNA barcode

The standard DNA barcode (658bp of the COI–mitochondrial cytochrome oxidase subunit I gene, Hebert et al. 2003) was sequenced for the 2 new species (Table 4).

Table 4.

GenBank accession number.

Specimen	MOTU name	Barcode GenBank accession number
Megalothorax potapovi 10770C01 RU120	Megalothorax potapovi sp. n.	KR736064
Megalothorax potapovi 10770C02 RU120	Megalothorax potapovi sp. n.	KR736063
Megalothorax potapovi 00620C03 RU120	Megalothorax potapovi sp. n.	KR736067
Megalothorax potapovi 00620C04 RU120	Megalothorax potapovi sp. n.	KR736070
Megalothorax potapovi 00620C05 RU120	Megalothorax potapovi sp. n.	KR736069
Megalothorax potapovi 00620C06 RU120	Megalothorax potapovi sp. n.	KR736068
Megalothorax potapovi 00620C07 RU120	Megalothorax potapovi sp. n.	KR736065
Megalothorax potapovi 00620C08 RU120	Megalothorax potapovi sp. n.	KR736066
Megalothorax sanguineus 6139D02 09761	Megalothorax sanguineus sp. n.	JN298074
Megalothorax sanguineus 6139D03 09761	Megalothorax sanguineus sp. n.	JN298075
Megalothorax sanguineus 6139D04 09761	Megalothorax sanguineus sp. n.	JN298076
Megalothorax sanguineus 6139D05 09761	Megalothorax sanguineus sp. n.	JN298077
Megalothorax sanguineus 6139D06 09761	Megalothorax sanguineus sp. n.	JN298078
Megalothorax cf. interruptus GUF 1	Megalothorax cf. interruptus L1	JN970929
Megalothorax cf. interruptus GUF 2	Megalothorax cf. interruptus L1	JN970928
Megalothorax cf. interruptus GUF 3	Megalothorax cf. interruptus L2	JN970910
Megalothorax granulosus cs110_CHL021	Megalothorax granulosus	KC900204
Megalothorax minimus BEL 1	Megalothorax minimus	JN970925
Megalothorax minimus cs70_Be001	Megalothorax minimus	KC900191
Megalothorax minimus cs71_Be001	Megalothorax minimus	KC900192
Megalothorax minimus cs93_Fr38	Megalothorax minimus	KC900195
Megalothorax nigropunctatus cd345c	Megalothorax nigropunctatus	KC900196
Megalothorax nigropunctatus cs104_CHL102	Megalothorax nigropunctatus	KC900197
Megalothorax nigropunctatus cs118_CHL033	Megalothorax nigropunctatus	KC900198
Megalothorax nigropunctatus cs119_CHL205	Megalothorax nigropunctatus	KC900199
Megalothorax perspicillum cs121_Fr114	Megalothorax perspicillum	KC900200
Megalothorax perspicillum cs122_Fr114	Megalothorax perspicillum	KC900201
Megalothorax perspicillum cs123_Fr114	Megalothorax perspicillum	KC900202
Megalothorax perspicillum cs124_Fr114	Megalothorax perspicillum	KC900203
Megalothorax sp. ARG 1	Megalothorax sp2	JN970916
Megalothorax sp. ARG 2	Megalothorax sp1	JN970926
Megalothorax sp. CHL 1	Megalothorax sp1	JN970927
Megalothorax sp. FRA 8	Megalothorax sp4	JN970913
Megalothorax sp. USA 1	Megalothorax sp3	JN970909
Megalothorax svalbardensis cs40_sva19	Megalothorax svalbardensis	KC900205
Megalothorax willemi ARG 3	Megalothorax willemi L6	JN970918
Megalothorax willemi ARG 4	Megalothorax willemi L6	JN970919
Megalothorax willemi cs91_Be005	Megalothorax willemi L6	KC900193
Megalothorax willemi cs92_Be005	Megalothorax willemi L4	KC900194
Megalothorax willemi FRA 1	Megalothorax willemi L5	JN970912
Megalothorax willemi FRA 2	Megalothorax willemi L6	JN970917
Megalothorax willemi FRA 3	Megalothorax willemi L1	JN970911
Megalothorax willemi FRA 4	Megalothorax willemi L3	JN970920
Megalothorax willemi FRA 5	Megalothorax willemi L3	JN970921
Megalothorax willemi FRA 6	Megalothorax willemi L3	JN970922
Megalothorax willemi FRA 7	Megalothorax willemi L4	JN970915
Megalothorax willemi FRA 9	Megalothorax willemi L2	JN970924
Megalothorax willemi FRA 10	Megalothorax willemi L2	JN970923
Megalothorax willemi FRA 11	Megalothorax willemi L7	JN970914

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 (Ivanova et al. 2006). Specimens were recovered after DNA extraction using a specially designed work flow allowing their morphological examination (Porco et al. 2010). The 5’ region of COI used as a standard DNA barcode was amplified using M13 tailed primers LCO1490 and HCO2198 (Folmer et al. 1994). 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 http://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 (Hall 1999). Sequences are publicly available on GenBank (JN298074-JN298078, JN970909-JN970929, KC900191-KC900205, KR736063-KR736070) and on BOLD at the following doi: 10.5883/DS-MEGAMIN (Table 4).

Data analyses

Forty-nine specimens of dataset representing 14 morphologically recognized species were selected, 36 from Schneider et al. 2011, Schneider and D’Haese 2013 and 13 specimens belonging to the two new species (Table 4).

Distance analyses were performed with MEGA6 (Tamura et al. 2013), utilizing a Neighbor-Joining (Saitou and Nei 1987) algorithm with the Kimura-2 parameter model (Kimura 1980) to estimate genetic distances. The robustness of nodes was evaluated through bootstrap re-analysis of 1000 pseudoreplicates. were defined with the software ‘mothur’ (Schloss et al. 2009).

Molecular Operational Taxonomic Units

Terminology

A nomenclature for the integumentary crests on the labrum is introduced (Fig. 13A–F). Crests are defined as integumentary processes with an apical line of primary grains. The longitudinal crests separating the m-row of chaetae are named ml1–3, when present the transversal crests in posterior position to a chaetae of the m-row are named mt and numbered after the chaetae position (mt2 posterior to chaeta m2), the antero-median transversal crest separating the m-row from the a-row is named amt, and can be further separated in amt0–2 numbered after the chaetae of m-row position. The longitudinal anterior crests separating the a-row are named al1–3. The transversal crest anterior to the a-row is named at—theoretically with the subdivisions at0, at1, at2 though we could only observe the at2 region in Willem, 1900.

Figure 13.

Diagram of the labrum anterior process. sp. n. A dorsal view B frontal view C dorso-lateral view; D dorsal view E frontal view F lateral view. In gray integumentary crests, dotted lines indicate areas not clearly observed.

Head chaetotaxy (Fig. 14) and antenna chaetotaxy (Fig. 15) follow Schneider (in press), trunk chaetotaxy (Fig. 16) follows Schneider and D’Haese (2013). The four swollen chaetae of Ant. III sensory organ are named S1–S4 after Deharveng (1978). We avoid to use the term ‘sensilla’ to designate some chaetae with peculiar shape and light refraction (e.g. Massoud and Ellis 1977), and use instead the following categories defined by Schneider (in press): (i) s-chaetae for the short swollen chaetae of the trunk; (ii) τ-chaetae for the long and thin chaetae of the trunk (shaped as trichobothria in ); (iii) S-chaetae for the swollen chaetae of the antenna; (iv) neosminthuroid chaetae for the special chaetae of Abd. IV sternum as defined in Richards (1968), Betsch (1980) and Schneider and D’Haese (2013). Ordinary chaetae are simply referred as chaetae. Nomenclature of the claw follows Schneider (in press), based on Denis (1948) and Schneider and D’Haese (2013). The presence or absence of specific chaetae is described in reference of the chaetotaxic pattern of . Reference to in this work is always sensu Schneider and D’Haese (2013).

Figure 14.

Diagram of the chaetotaxy and integumentary channels network of the head. sp. n. A dorsal side (pseudopores not represented) B ventral side; sp. n. C dorsal side D ventral side.

Figure 15.

Diagram of antennal III and IV chaetotaxy. sp. n., sp. n. Alternative hypothesis of homology is indicated in parenthesis.

Figure 16.

Diagram of the chaetotaxy of the trunk. A sp. n. B sp. n.

Abbreviations and symbols in text and figures

Crests on the anterior process of the labrum: al2, 3 = anterior longitudinal, ml1–3 = posterior longitudinal, amt0–2 = antero-median transversal, mt2 = posterior transversal. Antenna: Ant. I–IV = antennomere I to IV, S1–S4 = S-chaetae of Ant. III; or = Ant. IV organite; S, Sx, Sy = S-chaetae of Ant. IV. Trunk: Th. I–III = thoracic segment I to III; Abd. I–VI = abdominal segment I to VI; av = chaetae of anal valve; s1, s2 = s-chaetae; sm = special chaetae of male Abd. VI sternum; τ = τ-chaetae; wrc1–wrc8 = free wax rod secretory element 1 to 8. Claw: la, lp, Ba, Bp = auxiliary lamellae and crest of unguis; Ca, Cp = anterior and posterior crests of unguiculal lamella. Misc: dp = dens proximal, dd = dens distal, sf1–6 = sensory field 1 to 6.

Taxonomy

sp. n.

http://zoobank.org/4E6BA360-2EDF-49F3-AF77-54B250A9C490

Figs 1 , 2 , 3 , 4 , 5 , 6 , 7 , 13A–C , 14A, B , 15 , 16A

Figure 1.

sp. n. A Whole body, lateral view B head, dorso-lateral view C integumentary protuberance on forehead, dorso-lateral view D labrum, dorsal view. Scale bars: 100 μm (A, B); 10 μm (C, D).

Figure 2.

sp. n. A sf3 on Th. II tergum, dorsal view B sf5 on Th. III tergum, lateral view C neosminthuroid chaetae on Abd. IV sternum, lateral view D mucro, lateral view. Scale bars: 10 μm (A–D).

Figure 3.

sp. n. Chaetotaxy of head A dorsal side B ventral side; chaetotaxy of antenna C anterior side D posterior side; labrum and maxillary outer lobe E anterior side F posterior side; palp of labium G focused on ventral chaetae H focused on distal chaetae I hypostomal papillate chaeta. * indicates a supplementary chaeta, absent in other specimens.

Figure 4.

sp. n. Chaetotaxy of trunk, dorsal side.

Figure 5.

sp. n. Maxilla A dorsal side B ventral side; mandibula C right mandibula D left mandibula; sensory fields E sf2 on head F sf3 on Th. II G sf4 on Th. II H sf5 on Th. III I sf6 on abdomen J wrc3, 4 and chaetae a5, 6 on Th. III.

Figure 6.

sp. n. Legs chaetotaxy, A leg I B leg II C leg III; claws D claw I posterior side E claw II anterior side F claw III posterior side; unguiculus III G anterior side H posterior side.

Figure 7.

sp. n. A Abd. VI and genital plate, female (B, C) genital plate, male; furca D anterior side E posterior side F tenaculum G ventral tube posterior side.

Material examined.

Holotype: male on slide (MNHN-EA040223), Russia: Primorye: south of Posyet: peninsula facing to the town; 130.8034°E, 42.5709°N; alt=30 m; 28.ix.2004; Berlese extraction, forest litter; Louis Deharveng and Anne Bedos leg (RU-120) [MNHN]. Paratypes: 2 males and 5 females on slides (MNHN-EA040224–229), same data as the holotype [MNHN]; 1 specimen (sex unknown) on mount for SEM (MNHN-EA041012), same data as the holotype [MNHN].

Diagnosis.

Whitish in alcohol. Presence of median integumentary protuberance in front of chaeta a0 on forehead. Presence of chaeta X on Ant. IV. Labium: basomedian fields with 3 + 3 chaetae, basolateral fields with 1 + 1 chaetae. Integumentary channels as a paired tree on posterior part of the head, absent on anterior part, connection of channels with linea ventralis circular. Some chaetae enlarged and lanceolate, of which 5 + 5 dorsal, posterior on head and 2 + 2 on Th. II tergum. Inner chaeta of sensory field 2 slender with blunt apex, all inner chaetae of sensory fields 3–6 short flam-shaped. Dorsal abdominal s-chaetae s2 globular, absence of dorsal abdominal s-chaetae s3. Abd. I to V terga with 18 + 18 ordinary chaetae. Each claw of ordinary morphology, subequal. Tenaculum with 3 + 3 teeth. Abd. IV sternum with 2 + 2 chaetae. Mucro lamellae smooth, thin.

Description.

General aspect. Habitus and segmentation typical of the genus (Fig. 1A). Length from labrum to anus: ~380 μm. Specimens whitish in alcohol. Body chaetotaxy sparse including chaetae, s-chaetae, τ-chaetae as trichobothria, neosminthuroid chaetae, wax rod secretory elements and special swollen chaetae within sf2–6. Length of chaetae ranging from microchaetae [5–9 μm] to mesochaetae [10–14 μm] and macrochaetae [15–24 μm]. Shape of chaetae ranging from simple to lanceolate. Greatest chaetae being macrochaetae a4 and a7 on Th. II tergum (24 μm; Fig. 5F, G).

Integument. Secondary granulation made of the usual dorsal rough granules (e.g. Fig. 2A, B) and of smooth and flat irregular discoid granules near the ventral, post-labial chaetae of head. Integumentary channels extending laterally and dorsally in posterior part of head. Those channels as a pair of trees with five terminal branches (Figs 1A, B, 3A, B, 14A). Cephalic channels connection with linea ventralis circular (Figs 3B, 14B). Thoracic channels simple, restricted to ventral part.

Sensory fields and wax rods. A total of 14 + 14 wax rod secretory crypts (2 + 2 on head, 12 + 12 on body), including the ones inserted in each 6 + 6 sensory fields (Figs 3A, 4, 14A, 16A). sf1 without inner chaeta (Figs 1B, 3A). sf2 with one rather slender, curved inner chaeta with blunt apex (Figs 1B, 5E). Each inner chaeta of sf3–6 short, flam-shaped and curved (Figs 2A, B, 5F–I). sf3 with three inner chaetae (Figs 2A, 5F). sf4 and 5 each with two inner chaetae (Figs 2B, 5G, H). sf6 with one inner chaeta, inner chaeta length : sf6 diameter < 0.5 (Fig. 5I). wrc5 adjoining sf5 border (Figs 2B, 5H).

Labrum. Chaetae (Figs 1D, 3E, F): a- and m-row with rather slender mesochaetae, a2 slightly thicker and longer than m0–2; m0–2 apparently smooth, a1, 2 with one external teeth and with inward tip; m0 almost on the same level than m1. Integumentary crests (Figs 1D, 13A–C): m-row distinctly separated from a-row by the antero-median transversal crest (amt); longitudinal crest ml2 projecting anteriorly, cutting the transversal integumentary crest in two part (amt0, amt2); mt0 distinctly concave; absence of transversal crest posterior to chaetae m2 (pt2); asymmetry present on an least one specimen: ml3 strong and projecting anteriorly on one side, ml3 feeble and not reaching amt2 on the other side. Anterior side of the anterior process with 3 + 3 and one axial integumentary bulge (Fig. 3E). Labrum ridge with two small pikes (Fig. 3E).

Other mouth parts. Oral fold with 2 + 2 mesochaetae. Maxillary outer lobe: palp with subapical mesochaeta and apical papillate macrochaeta, edge of apical papilla with three strong integumentary lobes (Figs 3E, F); sublobal plate with one strong hair (7 μm; Fig. 3E). Basomedian fields of labium with 3 + 3 mesochaetae (Fig. 3B), basolateral fields with 1 + 1 mesochaetae on tubercle. Labial palp chaetal equipment typical of the genus, guard hairs strong in regard of papillate chaetae, hypostomal papillate chaeta flattened laterally in apical part, with subapical enlargement and acuminate apex (Figs 3G–I). Maxilla as in Figs 5A, B. Mandibula each with five apical teeth, right mandibula with a strong tooth between apex and molar plate (Figs 5C, D).

Head chaetotaxy. Trend for posterior chaetae to be longer and stronger than anterior chaetae, with 5 + 5 remarkable posterior lanceolate macrochaetae (up to 18 μm, Figs 1B, 3A). Dorsal anterior area with 11 pairs of chaetae (10 + 10 mesochaetae, 1 + 1 macrochaetae) and two axial mesochaetae (Figs 3A, 14A); with an axial integumentary protuberance in front of chaeta a0, devoid of secondary granules (Figs 1B, C, 3A); with 2 + 2 indistinct pseudopore-like elements between sf1 and insertion of antenna. Lateral anterior area with 1 + 1 mesochaetae (Figs 3A, 14A). Dorsal posterior area with 11 pairs of chaetae (5 + 5 lanceolate macrochaetae, 6 + 6 thickened mesochaetae, Fig. 3A, 14A). Ventral side with three pairs of post-labial mesochaetae (Fig. 3B, 14B).

Antennal chaetotaxy. Illustrated in Figs 3C, D, pattern diagram in Fig. 15 and summarized in Table 1. Ant. I with one mesochaeta. Ant. II with four chaetae: an anterior mesochaetae and three microchaetae. Ant. III with eight chaetae (four mesochaetae, five microchaetae), two long S-chaetae (S1, S4) and two short S-chaetae (S2, S3) in a cupule. S2 and S3 protruding from a shallow cupule but partially covered by a strong integumentary lobe. S1, S4 ornamentation unclear in light microscopy, S2, S3 ornamentation feebly visible. S4 in apical position to S1, on the same level than S2, S3. Tip of S1 rising up to Ant. IV basal whorl of S-chaetae, tip of S4 rising up to Ant. IV apical whorl of S-chaetae. Ant. IV with twelve S-chaetae (10 S, Sy and Sx), six microchaetae (only three chaetae in subapical group including chaeta X), a small organite (Or), two apical and subapical rods (a, sa). S-chaetae S with blunt apex.

Table 1.

Summary of antennal chaetotaxy.

Ant.	I	II	III		IV
	chaetae	chaetae	chaetae	S-chaetae	chaetae	S-chaetae	Organit	Sensory rods
Megalothorax potapovi sp. n.	1	4	8	S1–S4	6 (X incl.)	12 (10 S, Sx, Sy)	Or	2 (a, sa)
Megalothorax sanguineus sp. n.			9		7 (X incl.)

Thoracic terga chaetotaxy. Th. II with 12 + 12 chaetae of variable length, 1 + 1 s-chaetae s1 tubular and curved and 3 + 3 τ-chaetae (Figs 4, 16A). Chaetae including 3 + 3 macrochaetae (length as a4 > a7 > p8), 7 + 7 mesochaetae (a1–3, p1 and p2 thickened, a8 and a9 slender), 2 + 2 microchaetae (p3, p4) (Figs 4, 5F, G). Chaeta p4 postero-lateral PageBreakto sf3 (Figs 4, 5F, 16A). Two τ-chaetae in the periphery of sf3, one in posterior position and next to p2, one in lateral position and 10–11 granules far from p4 (Figs 4, 5F, 16A). Th. III with 10 + 10 chaetae, 6 + 6 free wax-rod generating crypts (wrc1–6) and 5 + 5 τ-chaetae (Figs 4, 16A). Chaetae including 2 + 2 macrochaetae (a9, p7), 7 + 7 mesochaetae (a1, a5, a6, a8, p2, p3, p4) and 1 + 1 microchaetae (a3) (Figs 4, 5H, J). Chaeta p4 moved posteriorly from wrc2 (Figs 4, 16A). Chaeta a6 stronger than a5 (Figs 4, 5J).

Legs chaetotaxy. Legs with ordinary chaetae of variable size as in Fig. 6A–C and summarized in Table 2. Subcoxa 1 I with a mesochaeta, coxa I with a microchaeta. Subcoxa 1, 2 II each with a mesochaeta, coxa II with a macrochaeta. Subcoxa 1, 2 III and coxa III with respectively 2, 1, 1 macrochaetae. Anterior and posterior microchaetae present on each pretarsus.

Table 2.

Summary of leg chaetotaxy.

Leg	I						II						III
Segment	Sc1	Sc2	Cx	Tr	Fe	Ti	Sc1	Sc2	Cx	Tr	Fe	Ti	Sc1	Sc2	Cx	Tr	Fe	Ti
Megalothorax potapovi sp. n.	1	0	1	3	8	12	1	1	1	3	8	12	2	1	1	4	8	11
Megalothorax sanguineus sp. n.

Claws. Ratio unguis length : pretarsus width on leg I–III respectively as 2.2, 2, 1.73, each claw with ordinary morphology, claw III bulkier than claw I and II (Fig. 6A–C). Each claw subequal in unguis length and in ratio unguiculus : unguis (~0.5) (Fig. 6A–C, F). Unguis basal and posterior auxiliary lamellae (la, lp and Bp) well developed, anterior crest (Ba) clear on claw II and III, weaker on claw I (Fig. 6D–F). Each unguiculus with a well developed posterior crest Cp, anterior crest Ca short and in basal position on claw I and II, more developed and not joining the internal border of the unguiculal lamella on claw III, basal tubercle posterior lobe not or feebly protruding (Figs 6D–H). Ratio unguis length : tibiotarsus length on leg I–III respectively as 0.54, 0.60, 0.65.

Abd. I–V terga chaetotaxy. With a total of 18 + 18 chaetae, 1 + 1 τ-chaetae, 2 + 2 free wax-rod generating crypts (wrc7, 8), 1 + 1 globular s-chaetae s2 (Figs 4, 16A). Chaetae including 17 + 17 mesochaetae (the longest ε2, ε3 and ζ2 reaching 15 μm) and 1 + 1 macrochaetae (η3, 17–18 μm). Chaeta α3 close to wrc7, both clearly anterior to β3 (Figs 4, 16A). Chaetae β4 and ε1 missing.

Abd. VI and genital chaetotaxy. Abd. VI: with nine dorsal mesochaetae (Fig. 7A); each anal valve with microchaeta av and several granular crests (Fig. 7A); with 9 PageBreak+ 9 ventral chaetae (Fig. 7A), male with 1 + 1 additional ventral cylindrical swollen chaetae sm (Fig. 7B, C). Genital plate: female with 2 + 2 microchaetae (Fig. 7A); male with 10 + 10 microchaetae (Fig. 7B, C).

Abd. IV sternum and furca. Abd. IV sternum with 2 + 2 neosminthuroid chaetae (Figs 2C, 7A) and 2 + 2 posterior mesochaetae (Fig. 7A). Manubrium with 2 + 2 PageBreakposterior chaetae. Proximal subsegment of dens with one posterior chaeta (Fig. 7E); distal subsegment posteriorly with two basal spines, one median chaeta and two apical spines, anteriorly with three apical spines, spines without elongated apex (Fig. 7D, E). Mucro with a sharp narrowing in the apical 2/5, lamellae edges smooth (Figs 2D, 7D, E). Ratio dp : dd : mucro = 0.75 : 1 : 0.88; ratio mucro width : mucro length ~0.16.

Tenaculum and ventral tube. Tenaculum with 3 + 3 hook-like teeth (Fig. 7F). Ventral tube with two apical pairs of mesochaetae (Fig. 7G).

Affinities.

sp. n. has the characteristics of the group species (Schneider and D’Haese 2013; Papáč and Kováč 2013). Within this group, it shares with Christian, 1998 a median integumentary structure on forehead but differs from it by the presence of the median chaeta a0, the presence of the chaeta X on Ant. IV, the presence of strong lanceolate macrochaetae on head and thorax and the integumentary channels pattern. The absence of a5 on Ant. III and of abdominal pair of chaetae ε1 is a similitude with Schneider and D’Haese, 2013 and Papáč & Kováč, 2013.

The integumentary structure on forehead and the lanceolate macrochaetae clearly separate sp. n. from , Stach, 1951, , Schneider and D’Haese, 2013, Deharveng and Beruete, 1993, Papáč & Kováč, 2013 and . Other peculiar characteristics of the species are shape of hypostomal papillate chaeta, presence of a strong lobe protecting S2, S3 on Ant. III, and S4 in apical position on Ant. III. On the basis of PageBreaklabral features (Fig. 13D–F) differs from sp. n. (Fig. 13A–C) by undivided amt, ml2 not joining amt, presence of mt2. A similar asymmetry was observed in both species (ml3 joining amt on one side, not joining amt on the other side). The morphology of the anterior crests (forming the anterior papillae) could not be comprehensively studied.

Ecology and distribution.

The species was collected in lowland forest litter, and only found so far in the southern part of Primorye.

Etymology.

sp. n. is dedicated to Mikhail Potapov, who led the 2004 field trip in Primorye which allowed to discover the new species.

DNA barcode.

A 658bp fragment of the COI gene was amplified and sequenced from paratype (MNHN-EA040229, voucher 00620C05) and seven other specimens (type locality, specimens lost). 251 bases in 5’ were not readable, thus a final sequence of 407bp is available. The sequences are identical. The sequence is deposited into the GenBank database under accession number KR736069. The base composition of the sequence is A = 26%, C = 23.1%, G = 13.5%, T = 37.4% (A + T = 63.4%).

5’– TAAGTTTTTGACTTCTTCCACCTTCTCTCACCCTTCTACTTTCAAGAGGTCTAGCAGAATCAGGTGCTGGAACAGGTTGAACTGTTTATCCTCCTTTATCTTCAAATATTTCCCATGCAGGAGCCTCTGTCGACTTAACTATTTTCAGTTTACATTTAGCTGGTATGTCATCAATTTTAGGAGCTATTAATTTTATTACAACTATCTTTAACATACGATCCCCAGGAATAACATGAGATCAAACTTCACTATTTATTTGATCTGTTTTAATTACATCAATTTTACTTCTCTTGTCTCTTCCAGTTCTAGCAGGAGCTATCACCATACTTTTAACCGACCGAAATTTAAATACTTCATTTTTTGACCCCGCTGGGGGTGGTGACCCAATTTTATACCAACACCTATTC–3’

http://zoobank.org/DD7E3CB4-26AF-4B47-9788-65E2C11F17F4

Figs 8 , 9 , 10 , 11 , 12 , 14C, D , 15 , 16B

Figure 8.

sp. n. Chaetotaxy of head A dorso-posterior side B dorso-anterior side C ventral side; anterior process of labrum D posterior side E anterior side; maxillary outer lobe F dorsal side G ventral side; labium H focused on ventral chaetae and basolateral field I focused on distal chaetae J hypostomal papilla. Legend: blf = basolateral field of labium, of = oral fold.

Figure 9.

sp. n. Chaetotaxy of trunk, lateral side. Representation of the granulation limited to the sensory fields area.

Figure 10.

sp. n. Maxilla A dorsal side B ventral side; mandibula C left mandibula D right mandibula; chaetotaxy of antenna E ventral side F dorsal side.

Figure 11.

sp. n. Chaetotaxy of legs A leg I B leg II C leg III; claws D claw I anterior side E posterior side F claw II anterior side G posterior side H claw III anterior side I claw III posterior side J unguiculus III interno-posterior side.

Figure 12.

sp. n. A Chaetotaxy of Abd. IV and Abd. VI sterna with furca, female B ventral tube and tenaculum, lateral side C focus on tenaculum D focus on tenaculum axial lobes E chaetotaxy of Abd. IV–VI sterna, male F focus on genital plate and Abd. IV posterior chaetae, male.

Type material. Holotype: female on slide (MNHN-EA040230), France: Midi-Pyrénées: Ariège: Suc-et-Sentenac: Vicdessos: on the edge of the Bernadouze peat-bog, mosses at a spring to the west under beech; lon=1.4220°E; lat=42.8024°N; alt=1360 m; 13.vi.2013; Berlese extraction, mosses, Lorène Marchal and Anne Bedos leg (09-BDZ1306-G03M) [MNHN]. Paratypes: 2 males and 4 females on slides (MNHN-EA040231–236), same data as the holotype [MNHN].

Other material.

4 females on slides (MNHN-EA040237–239]), France: Midi-Pyrénées: Ariège: Saint-Lary: Osque du Couret, forest litter on humid slopes; lon=0.8548; lat=42.8891; alt=1150 m; 28.vii.2010; Berlese extraction, litter, Louis Deharveng and Anne Bedos leg (09-761) [MNHN].

Reddish in alcohol. Absence of median integumentary protuberance in front of chaeta a0 on forehead. Presence of chaeta X on Ant. IV. Labium: basomedian fields with 3 + 3 chaetae, basolateral fields with 1 + 1 chaetae. Integumentary channels as a paired network on posterior part of the head and a simple channel on anterior part, connection of channels with linea ventralis circular. Chaetae on head and trunk with ordinary shape. All inner chaetae of sensory fields 2–6 short flam-shaped. Dorsal abdominal s-chaetae s2 bean-shaped, absence of dorsal abdominal s-chaetae s3. Abd. I to V terga with 20 + 20 ordinary chaetae. Slightly elongated claws. Tenaculum with 3 + 3 teeth. Abd. IV sternum with 2 + 2 chaetae. Mucro lamellae smooth, moderately enlarged.

General aspect. Habitus and segmentation typical of the genus. Length from labrum to anus: ~500 μm. Specimens with pale to deep red pigmentation in alcohol. Body chaetotaxy sparse including chaetae, s-chaetae, τ-chaetae as trichobothria, neosminthuroid chaetae, wax rod secretory elements and special swollen chaetae within sf2–6. Length of chaetae ranging from microchaetae [<6 μm] to mesochaetae [6–10 μm] and macrochaetae [11–15 μm]. Chaetae simple, without any remarkable development.

Integument. Secondary granulation made of the usual dorsal rough granules (Fig. 9) and of smooth and flat irregular discoid granules near the ventral, post-labial chaetae of head (Fig. 8C). Integumentary channels extending laterally and dorsally in anterior and posterior part of head (Fig. 8A, B). Posterior channels as a pair of well developed network. The most detailed observation allowed recognition of at least 10 cycles and 11 terminal branches with unclear tips (Fig. 8A). Anterior channel as a simple branch ending near the lateral edge of sf1, touching lateral chaeta of pra.a-row (Figs 8B, 14C). Cephalic channels connection with linea ventralis circular (Figs 8C, 14D). Thoracic channels simple, restricted to ventral part.

Sensory fields and wax rods. A total of 14 + 14 wax rod secretory crypts (2 + 2 on head, 12 + 12 on body), including the ones inserted in each 6 + 6 sensory fields (Figs 8A, B, 9, 14C, 16B). sf1 without inner chaeta (Fig. 8B). Each inner chaeta of PageBreaksf2–6 flam-shaped and curved (Figs 8A, 9), the biggest in sf6 (Fig. 9). Inner chaeta of sf6 length : sf6 diameter < 0.5. sf2, 6 with one inner chaeta. sf3 with three inner chaetae (Fig. 9). sf4, 5 each with two inner chaetae (Fig. 9). wrc5 adjoining sf5 borders (Fig. 9).

Labrum. Chaetae (Fig. 8D, E): a1, 2 much thicker and longer than chaetae m0–2; m0–2 smooth, a2 with three-four external slender teeth and with inward tip, a1 with three feeble blunt teeth and with outward, flattened tip; m0 almost on the same level than m1. Integumentary crests: m-row distinctly separated from a-row by the antero-median transversal crest (amt); longitudinal crest ml2 apparently not projecting anteriorly. Anterior side of the anterior process with 3 + 3 clear integumentary bulges and one axial, short bulge (Fig. 8E). Anterior process of the labrum not further studied. Ridge of the labrum with three pikes (Fig. 8E).

Other mouth parts. Oral fold with 2 + 2 mesochaetae (Fig. 8C). Maxillary outer lobe: palp with subapical mesochaeta and apical papillate macrochaeta (Fig. 8F, G), edge of apical papilla with weak lobes, sublobal plate with two short hairs (Fig. 8F, G). Basomedian fields of labium with 3 + 3 mesochaetae, basolateral fields of labium with 1 + 1 mesochaetae on tubercle (Fig. 8C, H). Labial palp chaetal equipment typical of the genus, guard hairs strong in regard of papillate chaetae (Fig. 8H–J). Maxilla as in Fig. 10A, B. Left mandibula with five apical teeth (Fig. 10C), right mandibula with six apical teeth and a double tooth between apex and molar plate (Fig. 10D).

Head chaetotaxy. Dorsally and laterally with mesochaetae, posterior and anterior mesochaetae subequal with a slight trend for posterior chaetae to be stronger than anterior chaetae (Fig. 8A, B). Dorsal anterior area with 11 pairs of chaetae and two axial chaetae (Figs 8B, 14C); with 2 + 2 pseudopore-like elements as ovoid, clear rings between sf1 and insertion of antenna (Figs 8B, 14C). Lateral anterior area with 1 + 1 chaetae (Figs 8B, 14C). Dorsal posterior area with 11 pairs of chaetae (Figs 8A, 14C). Ventral side with three pairs of post-labial macrochaetae (Figs 8C, 14D).

Antennal chaetotaxy. Illustrated in Fig. 10E, F, pattern diagram in Fig. 15 and summarized in Table 1. Ant. I with one mesochaeta. Ant. II with four mesochaetae, anterior chaeta longer than the other. Ant. III with nine mesochaetae, two long S-chaetae (S1, S4) and two short S-chaetae (S2, S3) in a cupule. S2 and S3 clearly protruding from a shallow cupule, only weakly covered by a feeble integumentary lobe. S1–S4 ornamentation unclear in light microscopy. Tip of S1 rising slightly above Ant. IV basal whorl of S-chaetae, tip of S4 rising up to Ant. IV basal whorl of S-chaetae. Ant. IV with twelve S-chaetae (10 S, Sy and Sx), seven ordinary microchaetae, a small organite (Or) apically flared, two apical and subapical rods (a, sa). S-chaetae S with blunt apex, rather short (5–6 μm).

Thoracic terga chaetotaxy. Th. II with 12 + 12 chaetae of variable length, 1 + 1 s-chaetae s1 tubular and curved and 3 + 3 τ-chaetae (Figs 9, 16B). Chaetae including 5 + 5 macrochaetae (a4, a7, a8, p1, p8), 5 + 5 mesochaetae (a1, a2, a3, a9, p2) and 2 + 2 microchaetae (p3, p4) (Fig. 9). Chaeta p4 postero-lateral to sf3 (Figs 9, 16B). Two τ-chaetae in the periphery of sf3, one in posterior position next to p2, one in lateral position and 5–6 granules far from p4 (Figs 9, 16B). Th. III area with 10 + 10 chaetae, 5 + 5 τ-chaetae and 6 + 6 free wax-rod generating crypts (wrc1–6; Figs 9, 16B). Chaetae PageBreakincluding 4 + 4 macrochaetae (a6, a8, a9, p7), 4 + 4 mesochaetae (a5, p2, p3, p4) and 2 + 2 microchaetae (a1, a3) (Fig. 9). Chaeta p4 moved posteriorly from wrc2 (Figs 9, 16B). Chaeta a6 slightly bigger than a5 (Fig. 9).

Legs chaetotaxy. Legs with ordinary chaetae of variable size as in Fig. 11A–C and summarized in Table 2. Subcoxa 1 I with a mesochaeta, coxa I with a microchaeta. Subcoxa 1, 2 II each with a mesochaeta, coxa II with a macrochaeta. Subcoxa 1, 2 III and coxa III with respectively 2, 1, 1 macrochaetae. Anterior and posterior microchaetae present on each pretarsus.

Claws. Ratio unguis length : pretarsus width on leg I–III respectively as 3.2, 2, 1.87, claw I with rather slender morphology, claw III bulkier than claw I and II. Claw I with longer unguis and each claw with subequal length of unguiculus, ratio unguiculus : unguis for claw I, II, III as ~ 0.43, 0.5, 0.5 (Fig. 11D–I). Unguis basal and posterior auxiliary lamellae (la, lp and Bp) well developed, anterior crest (Ba) clear on claw II and III (Fig. 11F, H), hardly perceptible on claw I. Each unguiculus with a posterior crest Cp, anterior crest Ca short and in basal position on each claw, joining the internal border of the unguiculal lamella on claw III, basal tubercle with posterior lobe not or weakly protruding (Fig. 11D–J). Ratio unguis length : tibiotarsus length on leg I–III respectively as 1.85, 1.43, 1.47.

Abd. I–V terga chaetotaxy. With a total of 20 + 20 chaetae, 1 + 1 τ-chaetae, 2 + 2 free wax-rod generating crypts (wrc7, 8), 1 + 1 s-chaetae s2 shaped as a bean (Figs 9, 16B). Chaetae including 15 + 15 chaetae rather small and thin (5–7 μm), 5 + 5 stronger chaetae (macrochaetae ε2, ε3 = 11–12 μm, mesochaetae ζ2, η2, η3= 9–10 μm). Chaeta α3 close to wrc7, both clearly anterior to β3 and β4 (Figs 9, 16B).

Abd. VI: with nine dorsal chaetae (6–7 μm) (Fig. 12A); each anal valve with microchaeta av and several granular crests (four paired plus one axial on dorsal valve, four on each ventral valve); with 7 + 7 ventral chaetae (4–8 μm; Fig. 12A), male with 1 + 1 additional ventral cylindrical swollen chaetae sm (Fig. 12E). Genital plate: female with 2 + 2 microchaetae ; male with 9 + 9 microchaetae (Fig. 12E, F).

Abd. IV sternum and furca. Abd. IV sternum with 2 + 2 neosminthuroid chaetae and 2 + 2 posterior mesochaetae (Fig. 12A). Manubrium with 2 + 2 posterior chaetae (Fig. 12A). Proximal subsegment of dens with one posterior chaeta (Fig. 12A); distal subsegment posteriorly with two basal spines, one median chaeta and two apical spines, anteriorly with three apical spines, basal spines without elongated apex, apical spines with elongated apex (longer in posterior spines) (Fig. 12A). Mucro lamellae well developed conferring a slight elliptical shape to the mucro in lateral and dorsal view, with a gradual narrowing in the apical 1/5 (Fig. 12A). Lamellae edges smooth. Ratio dp : dd : mucro = 0.69 : 1 : 75; ratio mucro width : mucro length ~0.23.

Tenaculum and ventral tube. Tenaculum with 3 + 3 hook-like teeth (Fig. 12B–D). Ventral tube with two apical pairs of mesochaetae (Fig. 12B).

sp. n. has the characteristics of the group species (Schneider and D’Haese 2013; Papáč and Kováč 2013). Within this group, it differs clearly from and sp. n. by the absence of a median integumentary structure on forehead. is similar to in terms of chaetotaxic pattern on antenna, legs, and trunk terga (without differences in absence/presence of chaetae). It differs from by the shape of the inner PageBreakchaetae of sf3–7 (some T-shaped in , always flam-shaped in sp. n.), the morphology of chaetae in the dorsal posterior area of head, the integumentary pattern, the morphology of labral chaetae, claw and mucro. The deep red pigmentation of sp. n. might be similar to that of (Salmon, 1946), PageBreakbut the two species differ in dental spines morphology (the four posterior spines with elongated apex in ). sp. n. shares morphological trends with and Schneider & D’Haese, 2013: enlargement of mucro lamellae, developed network of integumentary channels on head and elongation of dental spines apex (Stach 1957, Schneider and D’Haese 2013 and pers. obs.). In term of unguis I length : pretarsus I width ratio, it is surpassed by (epigeic hygrophilous mountains) and Papáč & Kováč, 2013 (troglobiontic), comparable to Deharveng, 1978 (troglobiontic) and Schneider and D’Haese, 2013 (epigeic, deadwood dwelling); it surpasses slightly PageBreak (epigeic hygrophilous) and more significantly , Papáč and Kováč 2013 and (troglobiontic). In term of absolute size of the unguis I, it is similar to the two later species, surpasses and is clearly surpassed by , and sp. n.

The species is known from humid micro-habitats in Pyrenees, though it was absent from the Bernadouze peat-bog itself. Other found in moist mosses in mountains are (1750m in High Tatras Mountains) (Stach 1957) and (up to 1500m in Pyrenees Mountains) (pers. obs.). The combination of morphological features shared with seems to be related to hygrophilous ecology. In that regard, sp. n. would remain less morphologically specialized than but more than . “Red” are present across the whole Pyrenean range (pers. obs.), and might be sp. n., but identification has only been confirmed so far for Ariège and Pyrénées-Atlantique specimens.

sp. n. is named after the deep red pigmentation of the species.

A 658bp fragment of the COI gene was amplified and sequenced from five specimens from the Saint-Lary locality. Specimens were unfortunately lost, sequences identification is based on consistency between: the peculiar pigmentation of the species observed on specimens before destruction, the genetic similarity of the five specimens and the morphological identification of four other specimens with the same pigmentation from the same sample. The sequences are deposited into the GenBank database under accession numbers JN298074–JN298078.

Four sequences are identical (JN298074–JN298077, provided below), base composition is A = 29.6%, C = 17.5%, G = 15.8%, T = 37.1% (A + T = 66.7%). The fifth sequence (JN298078) differs in 11 sites (= 98.3% pairwise identity), base composition is A = 29.5%, C = 17.6%, G = 15.7%, T = 37.2% (A + T = 66.7%).

5’–AACCTTATATTTAATTTTTGGAGTATGATCTGCTATAGTTGGAACAGCATTTAGAGTTTTAATTCGGTTAGAATTAGGACACCCAGGAAGCTTAATTGGAAACGATCAAATCTATAATGTAATAGTTACGGCCCATGCATTTGTAATAATTTTTTTTATAGTAATACCAATAATAATTGGAGGCTTTGGTAATTGATTAGTACCTTTAATAATTGGAGCACCTGATATAGCATTTCCTCGAATAAACAATTTAAGATTCTGACTTTTACCAC

DNA barcoding results

Fig. 17, Table 3.

Figure 17.

Tree inferred from the COI barcode by Neighbor-Joining with Kimura-2 parameter model. Robustness supports (bootstrap) are reported next to the nodes.

Table 3.

Intra and inter MOTUs genetic distances estimated by Neighbor-Joining with Kimura-2 parameter model.

	Intra-MOTU distances
Megalothorax cf. interruptus L1	10,70
Megalothorax cf. interruptus L2	-	22,98
Megalothorax granulosus	-	30,02	27,10
Megalothorax minimus	3,22	38,58	36,01	34,99
Megalothorax nigropunctatus	2,31	28,97	26,91	31,34	29,01
Megalothorax perspicillum	0,00	31,11	30,23	24,91	31,72	26,11
Megalothorax potapovi sp. n.	0,00	29,66	22,89	29,02	27,19	27,91	29,10
Megalothorax sanguineus sp. n.	0,95	29,87	23,49	28,96	33,90	28,19	27,25	25,79
Megalothorax sp1	3,75	35,18	27,46	30,39	25,25	27,74	30,40	23,39	23,36
Megalothorax sp2	-	31,42	26,42	26,02	32,87	29,78	31,61	31,92	32,21	30,97
Megalothorax sp3	-	29,17	27,52	31,81	32,61	31,16	27,84	27,62	27,21	29,64	30,30
Megalothorax sp4	-	28,74	27,23	25,65	27,20	21,90	23,31	24,78	28,56	26,25	23,93	29,61
Megalothorax svalbardensis	-	28,61	26,08	26,72	29,39	26,47	25,67	19,36	22,00	23,83	28,43	29,10	28,38
Megalothorax willemi L1	0,00	27,39	28,66	26,36	27,88	26,69	28,63	22,84	26,89	22,87	32,22	32,80	26,58	23,49
Megalothorax willemi L2	0,00	33,34	32,28	32,22	29,68	32,97	27,64	29,27	29,17	23,80	34,81	33,19	28,69	26,29	21,89
Megalothorax willemi L3	0,52	34,70	35,89	34,40	26,48	31,97	29,36	27,97	34,84	30,04	32,00	37,62	26,26	29,31	24,15	25,08
Megalothorax willemi L4	-	35,46	28,38	37,99	28,84	30,68	32,34	26,23	30,68	24,34	35,00	34,42	27,54	30,71	25,13	24,26	25,45
Megalothorax willemi L5	-	30,24	31,81	32,52	27,27	33,44	29,24	26,12	31,69	26,56	35,48	29,72	30,02	26,05	23,31	26,32	27,08	28,17
Megalothorax willemi L6	0,56	30,64	29,85	28,76	25,65	26,88	26,02	22,39	28,10	21,84	30,24	31,64	27,26	25,06	20,80	25,99	24,20	22,98	20,99
Megalothorax willemi L7	-	32,26	33,03	34,25	26,14	30,37	31,42	23,60	31,72	21,83	31,09	29,61	27,70	26,98	23,08	27,64	26,01	26,68	23,68	20,67

Twenty MOTUs were delineated using a conservative 19.5% threshold based on the higher bound of the barcode gap (not shown). Over the seven species for which several specimens were sequenced, five were represented by a single MOTU (, Schneider & D’Haese, 2013, , sp. n., sp. n.) and two exhibited several discrete MOTUs each (—7 MOTUs, —2 MOTUs). Deep genetic divergences showed among the MOTUs (Fig. 17). The mean genetic divergence among the MOTUs was 28.27% (range: 19.36%–37.99%). The mean intra-MOTU divergence was 0.95% (range: 0%–10.70%). The mean observed divergences between sp. n., sp. n. and the other MOTUs were respectively 26.09% (range: 19.36%–31.92%) and 28.52% (range: 22.00%–34.84%). These ranges of genetic divergences are comparable to those observed among MOTUs included in the dataset as well as with the interspecific variation found among MOTUs corresponding to monophyletic identified species (28.08% ; range: 19.36%–36.47%). This supports further the validity of the specific status for the two new species.

Discussion

DNA barcoding

The two new species sp. n., sp. n. are both supported by differences in morphological and molecular characters. The sequencing of COI for a paratype for sp. n. is critical as it will prevent ambiguities if a case of cryptic diversity is discovered in this species (Porco et al. 2012). Indeed, within the genus, the striking example of exhibiting a high molecular diversity in parallel with morphological stability calls for a dedicated investigation (Schneider and D’Haese 2013). A similar yet less documented diversity is observed in a new species near (Schneider et al. in prep.).

Labrum. Schneider and D’Haese (2013) pointed out the potential of the labrum morphology for taxonomy in reporting the differences between ( group) and (incertus group). Here some differences are described between two species of the group: sp. n. (Fig. 13A–C) and (Fig. 13D–F) , and introduce a nomenclature for the integumentary crests of the anterior process of the labrum. This structure remains unpractical to describe comprehensively: in light microscopy, the integumentary crests can be distinguished but their precise development and connections to each other are hard to assess. SEM allowed the description partially and also the recognition of asymmetry, but the method suffers from several flaws: (i) lack of depth, several shots from different angles would be required; (ii) part of the labrum is generally shadowed; (iii) asymmetry being evidenced, several specimens should be observed to assess intra-specific variation. Those requirements prevent use for regular taxonomic due to the cost and availability of SEM equipment, as well as the need for a significant number of specimens.

Integument. The pairs of pseudopore-like elements at the base of antenna were never reported in the genus but seem to be common features of the genus. We observed them clearly on sp. n. and , they are visible but faint in the following species: sp. n., , , , , and also in French specimens of . They were observed in SEM in and , where the dorsal one consists of a ring of primary grain and a small integumentary duct and the lateral one of a simple ring of primary grain. At the species level, those characters do not yield evident systematics value. They could not be observed in s and but the presence of dermastrons could blur the observation of the integument. In the state of knowledge, it is a putative apomorphy of the genus .

τ-chaetae. Schneider and D’Haese (2013) used the position of the lateral τ-chaetae guarding sf3 as a descriptor, separating from , , and . We now describe more precisely the position of the two τ-chaetae guarding sf3. Position of the most dorsal τ-chaeta: (i) between p1 and p2 and close to p2 (, , , and the two new species); (ii) between p1 and p2 and equidistant to them (, ); (iii) between p1 and p2 and close to p1 (); (iv) between p2 and p3 and close to p2 ().

Position of the most lateral τ-chaeta: (i) between p2 and p3 (), (ii) in lateral position and close to p4 with 2 or less granules between the chaetae (, , , ), (iii) in lateral position and more or less far from p4 with 5 or more granules between the chaetae (the two new species and ). , and were also observed but the τ-chaetae could not be spotted at the exception of the most dorsal τ-chaeta in .

Antenna. The homology of the chaeta directly below S4 (m5) on Ant. III of is ambiguous; an alternative hypothesis is provided in Fig. 15.

Claws. The apparent elongation of the claws in sp. n. called for a comparison with the other species of . The ratio of unguis I length : tibiotarsus I width was used by Papáč and Kováč (2013) as an indicator of troglobiontic adaptation. In practice, the tibiotarsus width is not exactly constant along its whole length and is frequently swollen in slide preparations (with dilatation of the integument). We instead estimated the ratio unguis I length : pretarsus I width and the ratio unguis I length : tibiotarsus I length.