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

Abstract 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 Collembola. Several of the most remarkable taxa collected have been recently described, like the new genus Sensillonychiurus 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 Caputanurininae was also reported for the first time for Russia  with two new species. Here, we report a morphologically remarkable new species of the genus Megalothorax Willem, 1900 (Neelidae, Neelipleona): Megalothorax potapovi sp. n. On the other end of the Palaearctis, the faunistical survey of the Pyrenean peatland of Bernadouze yielded another new species of Megalothorax, M. sanguineus 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 Megalothorax molecular operational taxonomic units (MOTUs) 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.

Sampling
Megalothorax potapovi sp. n. The specimens were obtained from a 2000 cm 3 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.
Megalothorax sanguineus 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 Scanning Electronic Microscope (SEM) observations, specimens were dehydrated in 100% ethanol, before critical point drying (Emitech K850) and gold coating (Jeol JFC-1200) and observations were performed with a SEM Jeol 840A. Megalothorax sanguineus 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.

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).
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 Megalothorax dataset representing 14 morphologically recognized species were selected, 36 from Schneider et al. 2011, Schneider andD'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. Molecular Operational Taxonomic Units (MOTUs) were defined with the software 'mothur' (Schloss et al. 2009).

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 M. minimus Willem, 1900. 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 Megalothorax); (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 M. minimus. Reference to M. minimus in this work is always sensu Schneider and D'Haese (2013).
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, postlabial 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.
Affinities. Megalothorax potapovi sp. n. has the characteristics of the minimus group species (Schneider and D'Haese 2013;Papáč and Kováč 2013). Within this group, it shares with M. sanctistephani 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.  labral features M. minimus (Fig. 13D-F) differs from M. potapovi 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.   Diagnosis. 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.
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.
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 anteromedian 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).
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.
Tenaculum and ventral tube. Tenaculum with 3 + 3 hook-like teeth (Fig. 12B-D). Ventral tube with two apical pairs of mesochaetae (Fig. 12B). Affinities. Megalothorax sanguineus sp. n. has the characteristics of the minimus group species (Schneider and D'Haese 2013;Papáč and Kováč 2013). Within this group, it differs clearly from M. sanctistephani and M. potapovi sp. n. by the absence of a median integumentary structure on forehead. M. sanguineus is similar to M. minimus in terms of chaetotaxic pattern on antenna, legs, and trunk terga (without differences in absence/presence of chaetae). It differs from M. minimus by the shape of the inner but the two species differ in dental spines morphology (the four posterior spines with elongated apex in M. rubidus). Megalothorax sanguineus sp. n. shares morphological trends with M. aquaticus and M. granulosus Schneider & D'Haese, 2013: enlargement of mucro lamellae, developed network of integumentary channels on head and elongation of dental spines apex (Stach 1957, Schneider andD'Haese 2013 andpers. obs.). In term of unguis I length : pretarsus I width ratio, it is surpassed by M. aquaticus (epigeic hygrophilous mountains) and M. draco Papáč & Kováč, 2013 (troglobiontic), comparable to M. massoudi Deharveng, 1978 (troglobiontic) and M. nigropunctatus Schneider and D'Haese, 2013 (epigeic, deadwood dwelling); it surpasses slightly M. granulosus (epigeic hygrophilous) and more significantly M. tuberculatus, M. hipmani Papáč and Kováč 2013 and M. carpaticus (troglobiontic). In term of absolute size of the unguis I, it is similar to the two later species, surpasses M. granulosus and is clearly surpassed by M. nigropunctatus, M. tuberculatus and M. massoudi sp. n.
Ecology and distribution. The species is known from humid micro-habitats in Pyrenees, though it was absent from the Bernadouze peat-bog itself. Other Megalothorax found in moist mosses in mountains are M. aquaticus (1750m in High Tatras Mountains) (Stach 1957) and M. minimus (up to 1500m in Pyrenees Mountains) (pers. obs.). The combination of morphological features shared with Megalothorax aquaticus seems to be related to hygrophilous ecology. In that regard, M. sanguineus sp. n. would remain less morphologically specialized than M. aquaticus but more than M. minimus. "Red" Megalothorax are present across the whole Pyrenean range (pers. obs.), and might be M. sanguineus sp. n., but identification has only been confirmed so far for Ariège and Pyrénées-Atlantique specimens. Etymology. Megalothorax sanguineus sp. n. is named after the deep red pigmentation of the species. DNA barcode. 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.

DNA barcoding
The two new species M. potapovi sp. n., M. sanguineus sp. n. are both supported by differences in morphological and molecular characters. The sequencing of COI for a paratype for M. potapovi 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 M. willemi 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 Megalothorax interruptus (Schneider et al. in prep.).

Morphology
Labrum. Schneider and D'Haese (2013) pointed out the potential of the labrum morphology for taxonomy in reporting the differences between M. minimus (minimus group) and M. nigropunctatus (incertus group). Here some differences are described between two species of the minimus group: M. potapovi sp. n. (Fig. 13A-C) and M. minimus (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 Megalothorax genus but seem to be common features of the genus. We observed them clearly on M. sanguineus sp. n. and M. carpaticus, they are visible but faint in the following Megalothorax species: potapovi sp. n., minimus, granulosus, nigropunctatus, willemi, svalbardensis and also in French specimens of M. sanctistephani. They were observed in SEM in M. perspicillum and M. aquaticus, 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 Neelus murinus and Neelides folsomi 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 Megalothorax.
Position of the most lateral τ-chaeta: (i) between p2 and p3 (M. perspicillum), (ii) in lateral position and close to p4 with 2 or less granules between the chaetae (M. minimus, M. svalbardensis, M. carpaticus, M. granulosus), (iii) in lateral position and more or less far from p4 with 5 or more granules between the chaetae (the two new species and M. nigropunctatus). M. draco, M. tatrensis and M. hipmani were also observed but the τ-chaetae could not be spotted at the exception of the most dorsal τ-chaeta in M. tatrensis.
Antenna. The homology of the chaeta directly below S4 (m5) on Ant. III of M. potapovi is ambiguous; an alternative hypothesis is provided in Fig. 15.
Claws. The apparent elongation of the claws in M. sanguineus sp. n. called for a comparison with the other species of Megalothorax. 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.