Two new dipluran species unearthed from subterranean habitats of the Canary Islands (Arthropoda, Hexapoda, Entognatha)

Two new dipluran species of the family Campodeidae have been unearthed in the Canary Islands. Remycampa herbanica sp. nov. was found in a highly threatened lava tube on Fuerteventura island. It is related to the soil-dwelling northwest African Remycampa launeyi that also inhabits four of the Canary Islands. The two known Remycampa species are characterized by a torsion of the labial palps. They differ chiefly in the distribution of macrosetae and in the features of cave adaptation of R. herbanica, i.e. elongation of body and appendages, and a higher number of olfactory chemoreceptors with a coniform shape unique within campodeids. Spaniocampa relicta sp. nov. was collected in the mesovoid shallow substratum (MSS) and has been assigned to a formerly monotypic genus that includes the soil-dwelling Spaniocampa prima from the Republic of Guinea. The two species differ in the number of abdominal macrosetae. Females of S. relicta sp. nov. have small setae arranged in groups along the posterior border of the first urosternite. These structures of unknown function have never been described in other campodeid species. Sequencing the COI barcode region of R. herbanica has been produced but it proved insufficient to identify closest relatives. The two new hexapods from subterranean habitats raise the Canarian campodeid fauna to six species. Five of them are living in soil and/or MSS, whereas the cave-adapted R. herbanica is known only from a single, particularly endangered lava tube distant from other caves. Subterranean Biology 34: 39–59 (2020) doi: 10.3897/subtbiol.34.50231 http://subtbiol.pensoft.net Copyright Alberto Sendra et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Subterranean Biology Published by The International Society for Subterranean Biology A peer-reviewed open-access journal


Introduction
With almost 1000 known species, Diplura are the second most diverse Entognatha after Collembola (Deharveng and Bedos 2018). All Entognatha like diplurans are good examples of successful colonizers of hypogean habitats, thriving in all kinds of cryptic environments without light (Condé 1955;Racovitzӑ 1907), including caves reaching the deepest habitats in the continental crust (Sendra et al. 2020). Furthermore, diplurans have a series of regressive adaptive features common to cave-dwelling animals, given their thin and almost completely unpigmented cuticle and absence of external eyes. However, they have remnants of lateral sense organs, each lying below the integument at both sides of the head in latero-ventral position, which presumably have a lightperceptive function (George 1963). Diplurans are divided into ten families, Campodeidae and Japygidae having the lion's share of all species in the group (Paclt 1957;Pagés 1959;Rusek 1982;Sendra 2015). The two aforementioned families and the smaller Parajapygidae have already been recorded on most of the Canary Islands (Paclt andBáez 1990, 1992;Pagés 1993;Sendra 1989Sendra , 1990; Sendra and Báez 1986). So far, a total of four Campodeidae, two Japygidae and one Parajapygidae species have been found mainly in soil habitats of this archipelago. We focused the present study in the Canaries on the lesser known subsurface habitats, i.e. the volcanic caves and the "Milieu Souterrain Superficiel" (hereafter MSS) (Juberthie et al. 1980) rather than the soil itself.
Most of the volcanic cavities are lava tubes, which usually lie a few meters below ground due to their particular origin from surface flowing lavas (Wood and Mills 1977;Wood 1979), therefore considered as part of the Shallow Subterranean Habitats (hereafter SSH) (Culver and Pipan 2014), defined as a set of mixed habitats just below the surface (soil, MSS and lava tubes among the terrestrial habitats). In spite of being relatively shallow, in volcanic terrains both lava tubes and the MSS often hold interesting cave-dwelling fauna comparable to that adapted to deeper continental karstic caves (Howarth 2008;Oromí and Martín 1992). However, no important cave-adapted species of Diplura have been found in either lava tubes or the MSS of the Canaries or Hawaii, the richest volcanic archipelagos for cave animals. Some lava tubes can occasionally be located deeper, covered by several layers of younger lava flows and commonly devoid of fauna due to the difficulty to organic matter reaching such depths. Only a few known cases of really deep tubes are suitable for adapted fauna, like the 14 million years old Cueva de Aslobas, in the south-west of Gran Canaria island . Cave-adapted animals are also absent from most lava tubes in very dry areas, such as in most of the semi-arid eastern Canary Islands, with only two exceptions on Fuerteventura: Cueva del Llano and Cueva de Montaña Blanca (Rando et al. 1993;Naranjo and  Oromí 2011) (Figs 1-3). The Canary Islands lava tubes have no permanent water flow inside, making soil accumulation scarce, which may limit the abundance of diplurans.
Another important SSH just below the edaphic layers (i.e. soil) is the "milieu souterrain superficiel" formerly described by Juberthie et al. (1980) for non-calcareous areas of the French Pyrenees, and later named by Culver and Pipan (2010) as "mesovoid shallow substratum" (MSS). There are different kinds of MSS, depending on the rock composition and geomorphologic origin, defined as a habitat representing the underground network of empty air-filled voids and cracks developing within multiple layers of rock fragments (Mammola et al. 2016;Ortuño et al. 2013). The MSS is usually covered by topsoil, connected with underlying deep rock cracks and caves. Fauna in MSS has been successfully surveyed in the Canaries, mostly in the typical colluvial MSS from talus deposits similar to those in continental non-volcanic terrains (Medina and Oromí 1990;Mammola et al. 2016), and in the peculiar volcanic MSS formed by lava clinker covered by a layer of protective soil (Oromí et al. 1986;Pipan et al. 2010). The latter is very abundant in recent and subrecent terrains (a few hundred thousand years) on most islands of the archipelago, providing a widespread subsurface habitat present in areas with or without lava tubes. The MSS in these islands has turned out to be almost as rich in cave-adapted fauna as the caves themselves. The few unidentified diplurans previously collected in such environments were always in colluvial MSS in the older parts of Tenerife and La Gomera, which is richer in soil and organic matter than the younger volcanic MSS Oromí 1990, Pipan et al. 2010). Further sampling in the MSS of Gran Canaria (Fig. 4) and in an old cave on Fuerteventura has provided the new material of Campodeidae diplurans studied herein.

Sampling and imaging
Specimens from Fuerteventura were collected in Cueva de Montaña Blanca (Figs 1-3) using pitfall traps with propylene glycol as preservative and blue cheese as bait, and sometimes just cheese on the ground to attract them, for live collection. Specimens from Gran Canaria were collected in the MSS at Brezal del Palmital (Fig. 4) using the pitfall traps described by López and Oromí (2010), baited with raw liver or cheese and with propylene glycol as preservative. The individuals were stored in ethanol (70-75%), washed with distilled water, mounted on a slide with Marc André II solution, and examined under a phase-contrast optical microscope (Leica DMLS). The illustrations were made with a drawing tube, and measurements taken with an ocular micrometer. To determine body length, specimens were mounted in toto and measured from the base of the distal macrochaetae on the frontal process to the abdominal supra-anal valve. Two specimens from Cueva de Montaña Blanca coated with palladium-gold were used for SEM photography (Hitachi S-4800) and for measurements of the sensilla.

Morphological study
The morphological descriptions and abbreviations are following Condé (1955). We use the term gouge sensilla for the concavo-convexly shaped sensilla on the anten- nae according to Bareth and Condé (1981). For the position of macrosetae we adopt the abbreviations of Condé (1955): ma, medial-anterior la, lateral-anterior, lp, lateralposterior and post, posterior.

DNA extraction, PCR ampand sequencing
Sequences of the 5' end of the cytochrome c oxidase subunit I (COI), a DNA fragment considered the standard DNA barcode region for Metazoa (Hebert et al. 2003), were generated for one of the specimens collected on Fuerteventura. For this, genomic DNA was extracted using the DNeasy Tissue Kit (Qiagen) following the manufacturer's guidelines. Amplification by PCR was done using the primers LCO1490 and HCO2198 (Folmer et al. 1994) in a 25 μl total PCR volume containing 15.4 μl of purified water, 2.5 μl of 10x NH 4 -based Reaction Buffer, 1.5 μl of MgCl 2 (3mM), 2 μl of 10 mM dNTP (2.5 mM each), 0.5 μl of BSA, 1 μl of each primer (10 μM), 0.1 μl of BIOTAQ™ DNA polymerase, and 1 μl of DNA extract. The PCR was executed with the following protocol: initial denaturing step at 95 °C for 2 min, 40 amplification cycles (94 °C for 30 s, 46 °C for 35 s, 72 °C for 45 s), and a final step at 72 °C for 5 min. PCR success was checked by running products on a 1% TAE agarose gel. Successfully amplified products were cleaned following EXO I/rAP PCR clean-up protocol and outsourced for DNA sequencing by Macrogen Inc. (https://dna.macrogen.com).

Depositories
The material examined is deposited in the following collections:

ASM
Personal collection of Alberto Sendra, Valencia, Spain IPNA-CSIC Invertebrates collection of the Instituto de Productos Naturales y Agrobiología (  Other studied material. Same data as holotype, two specimens mounted on two separate aluminium stages and coated with palladium-gold. Description. Body length 3.8-4.4 mm in males (n = 5), 4.2 mm in females (n = 1) and 2.2 mm in one juvenile (Table 1). Epicuticle smooth under optical microscope but slightly reticulated at high magnifications as irregular polygonal structures of variable size (Fig. 14). Body with scarce short clothing setae with one or two apical barbs on each seta (Fig. 18).
Antennae with 36 antennomeres in one complete intact antenna in the holotype; antennae 0.84× as long as the body length with medial antennomeres 2× longer than wide, as is the apical antennomere. Cupuliform organ with about 21 complex olfactory chemoreceptors arranged in two concentric circles with one in the centre, each apparently with a pile of fused plates forming a coniform structure (Figs 5-9). Distal and central antennomeres with two or three whorls of barbed macrosetae and scattered smooth setae, in addition to a single distal whorl of 8-12 short thick gouge sensilla 10 μm long (Fig. 10). These latter are more abundant on the dorsal side of the antennomere, including one or two very short coniform sensilla. Proximal antennomeres with typical trichobothria, plus a small coniform sensillum on third antennomere in ventral position.
Moderate protrusion of frontal process covered with very slightly tuberculated setae with two to five barbs on distal half (Fig. 11). Three macrosetae along each side of the line of insertion of antennomere and setae x with thin distal barbs; length ratios a/i/p/x as the 29/26/17/24 in female paratype (Fig. 11).
Large mandibulae with at least five teeth, the two posterior ones with a row of small denticles. Atypical labium with slight torsion to the right of the labial palps, slight elongation of the palpiform processes, and a deep groove in the middle of labium from posterior border of anterior lobe to the middle of submentum, without reaching the posterior border of labium (Fig. 12). Suboval labial palps each with small lateroexternal sensillum, three guard setae and up to 68 neuroglandular setae (Fig. 12).   Thoracic macroseta distribution (Figs 13-20): pronotum and mesonotum with 1+1 ma, 1+1 la, 1+1 lp macrosetae; metanotum with 1+1 ma macrosetae. All macrosetae short and slightly thick with short barbs along basal two-thirds of each seta; marginal setae longer and more barbed than clothing setae . Legs elongated, metathoracic legs reaching abdominal segment IX, about 0.5× as long as the body length (Figs 21-26; Table 1). Tibia always longer than femur or tarsus (Table 1). Femorae I-III each with one short thick dorsal macroseta with a few barbs. Calcars with long barbs throughout one side (Fig. 25). Tibiae I-III with two short ventral macrosetae with two to four distal barbs; some paratypes with three sternal tibial macrosetae on the metathoracic leg (Figs 23, 26). Two rows of ventral barbed setae with two lines each of two to five barbs (Figs 21, 25). Three smooth dorsal distal tarsal setae longer than the rest (Fig. 21). Subequal claws with a lateral expansion curved towards the two ventral sides. Smooth laminar telotarsal processes curved along and ending in a slightly wide expansion with a narrow prolongation on one side, a unique shape among diplurans (Figs 21, 22, 25).
Distribution of abdominal macrosetae on tergites (Fig. 27): 1+1 ma on I-III; 1+1 ma, 1+1 la on IV, 1+1 ma, 1+1 la, 1+1 lp 3 on V-VII; 1+1 mp, 3+3 lp 1-3 on VIII; and 1+1 mp, 5+5 lp 1-5 on IX abdominal segment. All tergal abdominal macrosetae short, slightly thick with thin short barbs being ma and mp the shortest. Urosternite I with 6+6 macrosetae (Figs 28, 29); urosternites II to VII with 4+4 macrosetae; urosternite VIII with 1+1 macrosetae; urosternal macrosetae of medium length or longer, with a few long barbs in one single row along the distal half to four-fifths. Stylus with an apical, a subapical and a ventromedial seta with a few long barbs arranged in one row along the distal four-fifths (Fig. 30). Cerci more than 2× as long as the body length, 2.1× as long as the body in the only apparently intact cercus of the holotype; with 27 primary articles, not counting the multi-divided basal article (Table 2). Length of cerci increases very slightly from the proximal to distal articles; they are covered with a whorl of alternate smooth thin macrosetae and smooth thin setae, and a whorl of shorter smooth thin setae at the end of each primary article. These whorls, except the apical one, increase from one to four from the proximal to distal primary articles.  Female urosternite I with slim cylindrical appendages, each bearing up to seven glandular a 1 setae in a distal field (Fig. 29).
Male urosternite I with short coniform appendages, each bearing about 13 glandular a 1 setae in a distal field; posterior edge occupied by a large but narrow field of cramped up to 190 glandular g 1 setae (Fig. 28).
Etymology. Referring to Herbania, the ancient name of Fuerteventura, the only island on which it has been found.
Molecular data. The barcode sequence of one specimen of R. herbanica (code 112BC) has been registered in GenBank with the ascension number MN729498.
Phylogenetic analyses. Available COI barcode sequences of Diplura stored in BOLD were retrieved (search for Diplura on 14 th November 2019 at http://www. boldsystems.org/index.php/) to identify the species closest to R. herbanica. After excluding redundant sequences for several taxa, a total of 46 sequences, representing approximately 28 species from at least 10 genera were retained. They were then aligned with the newly generated R. herbanica sequence using the MAFFT E-INS-I algorithm (Katoh et al. 2002). A preliminary maximum likelihood tree was generated using the Fast Tree 2.1.5 (Price et al. 2009) tool in Geneious 7.1.9 (Kearse et al. 2012) to identify taxa closely related to R. herbanica.
The genetic results do not show well supported relationships of R. herbanica with the other diplurans with barcode sequences in BOLD. Based on this preliminary result we only can confirm genetically that this new species belongs to the family Campodeidae. Description. Body length 3.4 mm (paratype) and 4.1 mm (holotype) in females, and 3.5 mm (paratype) in male. Epicuticle with small microdenticles under optical microscope on dorsal side of nota and legs. Body with smooth clothing setae.

Spaniocampa relicta
Broken antennae on the three types; medial antennomeres (antennomere XII intact) as long as wide, a single distal whorl of 8-10 short and thin gouge sensilla 12 μm long. Proximal antennomeres with typical trichobothria plus a bacilliform sensillum on third antennomere in ventral position.
Plain frontal process with one anterior and three posterior smooth setae; length ratios of a/p as 53/23 in holotype. The three macrosetae along each side of the line of insertion of antennomere with thin distal barbs and length ratios of a/i/p as 17/27/16 in holotype; no x setae observed; Each suboval labial palp has a small latero-external subcylindrical sensillum; two guard setae, up to three simple setae on anterior border and up to 70 neuroglandular setae in holotype.
Non-thoracic macrosetae on pronotum, mesonotum and metanotum; short slightly thick marginal setae with very thin or smooth apical barbs (Fig. 31). Metathoracic legs reaching abdominal segment VII. Femora without dorsal macrosetae. Tibiae I-III without typical ventral barbs but with a short ventral apical one with a few thin distal barbs; calcars with three to six thin barbs along one side. Two dorsal tarsal smooth setae similar to clothing setae, but much longer. Subequal claws, slightly wider at the base and regularly curved. Smooth setiform telotarsal processes overpassing the end of the claws.
Stylus with an apical, a subapical and a ventromedial setae with a few distal thin long barbs, more abundant on the ventromedial seta (Fig. 33). Cerci absent in the studied specimens.
Female urosternite I with short subcylindrical appendages, each bearing up to 14 glandular a 1 setae in a distal field. The posterior border of the urosternite bears three or four groups of small setiform setae with between two and ten units (Fig. 32).
Male urosternite I with short thick appendages, each bearing about 35 glandular a 1 setae in two apparently distal fields; posterior edge slightly enlarged at both sides of the first urosternite with a glandular field of about 140 glandular g 1 setae arranged in up to six rows.
Etymology. The specific epithet relicta refers to two situations affecting this new species: i) it has been discovered in a relict patch of laurel forest on Gran Canaria; ii) it is a relict species of a genus also distributed on the Republic of Guinea with one known extant species.

Phyletic affinities
The substantially cave-adapted Remycampa herbanica sp. nov. is certainly related to the monotypic genus Remycampa Condé, 1952, due to several important taxonomic features including similarities in their atypical labium, secondary sexual characters, lateral telotarsal processes and distribution of macrosetae. The only species known so far, Remycampa launeyi Condé, 1953, has a distribution area in northeast Morocco and some of the Canary Islands (Sendra 1989), has extended to the islands of El Hierro, Tenerife, Gran Canaria, and Lanzarote. R. herbanica has been collected in a volcanic lava tube of Fuerteventura. Being only 11 km from Lanzarote one can postulate that R. launeyi might be present also in soils or MSS of Fuerteventura. Both islands were joined during the last glaciation (Fernández-Palacios et al. 2015) and had and have a similar climate. The most visible differences between R. launeyi and R. herbanica are in the cave-adapted features of the new species, which has a larger, more elongated body and appendages with cerci 2.1× longer than the body length and with 28 articles (Table  2), and with metathoracic tibiae bearing 2-3 sternal macrosetae. Furthermore, each apical antennomere has a large cupuliform organ with quite remarkably for the high number of olfactory chemoreceptors (up to 21) with a unique coniform shape . Other noteworthy morphological differences are: shorter and thicker macrosetae and shorter clothing setae with apical barbs in R. herbanica; differences in the shape of their lateral telotarsal processes, with trapezoidal endings in R. launeyi and round with a thin expansion in R. herbanica (Figs 21, 22, 24);Condé 1953: figures 3C, D and E); absence of lateral posterior macrosetae on metanotum in R. herbanica; absence of 1+1 lateral posterior macrosetae on third and fourth urotergites in R. herbanica; and finally differences between their labial pieces with a strong torsion to the right of labial palps and enlargement of the groove in the middle of the labium in R. launeyi compared with a less pronounced and smaller groove in R. herbanica (Fig. 12).
Remycampa is a peculiar genus with an unclear relation to other genera of Campodeinae, but with certain affinities with the tachycampoid phyletic lineage. It is probably more closely related to the two known cave-adapted tachycampoid genera living in caves of northwest Africa: Jeannelicampa Condé, 1952 from Oran in the Tell Atlas, Algeria, and Tachycampa Silvestri, 1936 from karst areas near Taza in the Middle Atlas, Morocco. Like R. herbanica, these two genera lack some thoracic macrosetae, short thoracic macrosetae and lateral expansions on the claws. Nevertheless, new taxonomic tools are needed to unravel the natural phylogenetic relations within Campodeinae and tachycampoid genera (Sendra et al. 2020).
It is difficult to determine the exact systematic position of Spaniocampa relicta sp. nov., not because of the broken antennae or missing cerci that cannot be described, but rather the lack of fresh specimens of the two closely related genera and their species. We refer to the monospecific Spaniocampa Silvestri, 1933 from Kakoulima massif (Republic of Guinea) and Ombrocampa Paclt, 1957that, according to Paclt (1957, includes the three related species O. dahli Condé, 1956 andO. nyongensis Condé, 1956 from Nyong (Cameroon) and O. depauperata (Silvestri, 1918) from Mount Kenya (Kenya). They are all soil-dwelling, whereas Spaniocampa relicta was found in colluvial MSS. All these species have in common with S. relicta a low number of thoracic and abdominal macrosetae, including no dorsal macrosetae on femorae and no ventral ones on tibiae (with the exception of one short ventral tibial macroseta in Spaniocampa prima Silvestri, 1933). Furthermore, S. prima shares with S. relicta sp. nov. the total absence of notal macrosetae (Fig. 31) and similarities in the distribution of abdominal macrosetae: 2+2 lateral posterior macrosetae on eighth urotergite and ninth abdominal segment in S. prima and 1+1 lateral posterior on eighth urotergite and 3+3 lateral posterior on ninth abdominal segment in S. relicta (Fig. 34). Further differences to S. prima were also found in the number of urosternal setae, with: 8+8 macrosetae on first urosternite (this number could be reduced to 7+7, since Silvestri considered some barbed setae in latero-posterior position as macrosetae) and 4+4 macrosetae on second to seventh urosternites in S. prima compared with only 5+5 and 3+3 macrosetae in S. relicta sp. nov.
It is worth mentioning the presence of small setae arranged in groups on the posterior border of the first urosternite in females; their function is unknown, though apparently non-glandular, and they have never been described in any other species of the campodeid family.

Dipluran fauna and their habitats
The Canary Islands have a wide range of SSH in their volcanic landscapes: soils, MSS, and young and old lava tubes with a rich biodiversity (Oromí 2004). Diplurans had been collected in soil and MSS but not in lava tubes until now (Paclt andBáez 1990, 1992;Pagés 1993;Sendra 1989Sendra , 1990Sendra and Báez 1986). Focusing on Campodeidae, six species of the subfamily Campodeinae are present in the Canary Islands. Two species of the genus Campodea are widespread in the Euromediterranean region and beyond: Campodea (Campodea) fragilis Meinert, 1865 and Campodea (Monocampa) devoniensis Bagnall, 1918. Another two have more limited distribution areas: Podocampa ceballosi (Silvestri, 1932) in the Iberian Peninsula and northwest Africa, Remycampa launeyi Condé, 1952 limited to north-west Africa. The two new species Spaniocampa relicta sp. nov. and Remycampa herbanica sp. nov. are endemic to the Canaries. In relation with their habitats, Campodea fragilis, C. devoniensis, Podocampa ceballosi and Remycampa launeyi are frequently found in soil and are also present in the MSS as Spaniocampa relicta sp. nov. And, Remycampa herbanica sp. nov. is the only species occurring in lava tubes and showing cave-adapted features, also known as troglomorphic traits, as a result of its obligate lifestyle. It has been collected exclusively in Cueva de Montaña Blanca, one of the few lava tubes on Fuerteventura suitable for such adapted fauna (Figs 1-3). The presence of cave-adapted diplurans in other lava tubes around the world is not uncommon. Ferguson (1992) provided many localities from the USA, and Borges and Oromí (1994) reported the presence of one species in Gruta do Esqueleto, São Miguel island, Azores. Sendra et al. (2016) described a spe-cies from Mexican volcanic caves, and an interesting cave-adapted Lepidocampa was reported from Reunion in the Indian Ocean (Sendra et al. 2017).

The special case of Cueva Blanca
Fuerteventura has a maximum sub-aerial age of 22 Ma, an exceptional span for a volcanic island, probably due to its extremely slow subsidence into the sea, compared to other volcanic archipelagos (Fernández-Palacios et al. 2011). For this reason, together with its scarce volcanic activity over the last million years, the island is highly eroded and most of the extant caves are dry and often silted with clay, thus being unsuitable to hold adapted troglobiont fauna. Only two of these lava tubes (Cueva del Llano and Cueva de Montaña Blanca, 27 km apart from each other) have appropriate humidity conditions for this fauna that includes eight troglobiont arthropod species which are all endemic to the island and often with no related species in the archipelago. Only the nicoletiid Zygentoma Coletinia majorensis Molero, Gaju, López, Oromí & Bach, 2013 inhabits both caves, the remaining seven species being exclusive to one or the other (Rambla 1993;Molero et al. 2013). The habitat of both caves is highly threatened. Cueva del Llano is a show cave owned by the local government, and many houses are built on the surface surrounding the cave, in spite of the exclusive presence of the officially protected harvestman Maiorerus randoi Rambla, 1993. Cueva de Montaña Blanca is the only known place where Remycampa herbanica sp. nov. and some undescribed troglobiont invertebrates occur (two weevils, one pseudoscorpion and one spider), and its entrance is within an unfinished abandoned four-story building in a tourist resort. The situation is critical for these cave-dwelling species given that most of the Fuerteventura underground is very dry, there is hardly any area of MSS, and therefore their inhabitable environment is highly limited to small distantly dispersed spots.