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Distinctive Collembola communities in the Mesovoid Shallow Substratum: First data for the Sierra de Guadarrama National Park (Central Spain) and a description of two new species of Orchesella (Entomobryidae)

  • Enrique Baquero ,

    Contributed equally to this work with: Enrique Baquero, Rafael Jordana

    Roles Conceptualization, Investigation, Methodology, Resources, Writing – original draft, Writing – review & editing

    ebaquero@unav.es

    Affiliation Departamento de Biología Animal, Universidad de Navarra, Pamplona, Spain

  • Enrique Ledesma,

    Roles Conceptualization, Investigation, Methodology, Resources, Writing – original draft, Writing – review & editing

    Affiliation Departamento de Ciencias de la Vida, Facultad de Biología Ciencias Ambientales y Química, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain

  • José D. Gilgado,

    Roles Conceptualization, Investigation, Methodology, Resources, Writing – original draft, Writing – review & editing

    Affiliation Departamento de Ciencias de la Vida, Facultad de Biología Ciencias Ambientales y Química, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain

  • Vicente M. Ortuño,

    Roles Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Writing – original draft, Writing – review & editing

    Affiliation Departamento de Ciencias de la Vida, Facultad de Biología Ciencias Ambientales y Química, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain

  • Rafael Jordana

    Contributed equally to this work with: Enrique Baquero, Rafael Jordana

    Roles Conceptualization, Investigation, Methodology, Resources, Writing – original draft, Writing – review & editing

    Affiliation Departamento de Biología Animal, Universidad de Navarra, Pamplona, Spain

Distinctive Collembola communities in the Mesovoid Shallow Substratum: First data for the Sierra de Guadarrama National Park (Central Spain) and a description of two new species of Orchesella (Entomobryidae)

  • Enrique Baquero, 
  • Enrique Ledesma, 
  • José D. Gilgado, 
  • Vicente M. Ortuño, 
  • Rafael Jordana
PLOS
x

Abstract

Two new species of the genus Orchesella Templeton, 1836 have been identified following intensive sampling in the Colluvial Milieu Souterrain Superficiel (Mesovoid Shallow Substratum, or MSS) of the Sierra de Guadarrama using Subterranean Sampling Devices (SSD). The data were obtained from the first extraction of the traps between May and October of 2015. During a study of the Collembola taxon, 32 different genera (61 species) were identified. The highest representative genus presence in almost all traps was Orchesella, with two new species. One of the two species described had been misidentified until this study was carried out, indicating that their preferential habitat had not been sampled; the second species had never been identified. The community of the Orchesella species in the Colluvial MSS was investigated, leading to the conclusion that this environment has its own assemblage of characteristic species. The opportunity to study specimens that belong to five species of the genus Orchesella, including three previously recollected, has allowed for obtaining reliable information regarding their macrochaetotaxy. A part of this chaetotaxy is proposed as a useful diagnostic tool for the species of the genus. In conclusion, it can be affirmed that this study has demonstrated that the Colluvial Mesovoid Shallow Substratum (Colluvial MSS) has its own fauna, and it supports the hypothesis that it constitutes a new biotope, at least for Collembola.

Introduction

The study of subterranean habitats has generally focused on caves, pits and even mines (of anthropogenic origin) [1, 2] although shallow subterranean environments [3] and specifically the Milieu Souterrain Superficiel (MSS), have become relevant in recent years [4]. This habitat was discovered in the 1980s and was described as suitable for hypogean species [5, 6, 7, 8]. The MSS is constituted by the fissure networks and interstices of rocky debris, usually under a soil layer, whose genesis is the product of different lithological processes in various types of rocks, i.e. bedrock, colluvial, volcanic or alluvial MSS [9, 5, 6, 10, 11, 12]. Thus, the MSS is a widely extended subterranean environment, although it has not been investigated sufficiently.

Some of the conditions of the MSS are similar to those usually present in caves (absence of light, high humidity and small variations of temperature) [13, 14] however, its close relation with the surface and the layers of the soil allows for wider temperature oscillations [4] and facilitates the entry of organic matter into the system [15]. The MSS can harbor fauna coming from the surface when external conditions are momentarily adverse [16, 17], but it also contains stable resident fauna. This resident fauna includes stenoic and hygrophilous as well as truly hypogean (or troglobiont sensu Sket [18]) species, i.e. species that are highly specialized and adapted to the subterranean environment and unable to complete their lifecycles outside of it [11, 19, 20, 21, 13, 12]

The most notable studies on the MSS have been carried out in Europe, including in the Iberian Peninsula and the Canary Islands, by several groups of researchers [4]. Although comprehensive lists of the Collembola species in the MSS are scarce, some studies have provided extensive lists of Collembola species [11]; however, few have recorded the presence of the Orchesella species in this environment. This genus was recorded in the MSS in Eastern Europe as Orchesella carpatica Ionesco, 1915 and O. pontica Ionesco, 1915 [22]; O. carpatica, O. pontica and O. alticola (Uzel, 1891) [16]; and O. pontica [17]. In addition, this genus has been recorded twice in the MSS of the Iberian Peninsula as Orchesella bifasciata Nicolet, 1842 from an alluvial MSS in eastern Spain[12], and undetermined species of Orchesella in a Colluvial MSS from that region [14].

In recent years, there has been a series of interesting findings of fauna in the MSS in several mountainous massifs of the Iberian Peninsula [23, 24, 25]. Recent samplings have revealed new species of Orchesella in the Colluvial MSS of Sierra de Guadarrama (Central Spain), which are described in this paper.

The Holarctic genus of collembola Orchesella Templeton, 1835 (Entomobryidae) includes 61 species that are among the largest collembolans [26]. These species inhabit the forest litter and are well-known for their key ecological role of recycling decaying organic matter [27]. Despite their size, the morphological characters of taxonomic importance are reduced mainly due to the disposition of sensorial hairs (chaetotaxy) and to pigmentation patterns [28, 29]. In addition, high levels of intraspecific variability affect both chaetotaxy and pigmentation and are sometimes correlated with their lifecycles [30, 31]. The use of these characteristics in combination with the methodologies used by other authors [32, 33] have proven to be useful in the identification of species that belong to the family of Entomobryidae.

Material and methods

Area of study

The study was conducted in the eastern half of the Central System (Iberian Peninsula) in the mountainous subunit called Sierra de Guadarrama, and more specifically within the limits of the protection area (33,960 hectares). A few years ago, it was declared a national park [34], and it has a belt of 62,687 hectares of Peripheral Protection Area [35] (Fig 1). The lower altitudinal limit is around 1200–1300 m a. s. l. (above sea level) on the slopes near the Puerto de la Morcuera, and the highest limit is located at the 2428 m altitude of the summit of Peñalara [36]. The park extends over three mountain ranges (Fig 1) with numerous peaks that exceed 2000 m a. s. l. Each of these ranges converge in a sector characterised by the presence of two natural passages, the Puerto de Los Cotos and the Puerto de Navacerrada. Both have ski slopes because these areas are outside the limits of the park and ZPP. The cord known as Montes Carpetanos (Fig 1) runs in an N–NE direction and serves as a natural borderline between the provinces of Segovia and Madrid. Its most southern end reaches the Puerto de Los Cotos, which constitutes the foothills of the emblematic Macizo de Peñalara, a maximum expression of the glacier model in the Sierra de Guadarrama. It should be noted that the NE boundary of the park only reaches the vicinity of the Reajo Alto peak. A second range, which includes Siete Picos and La Mujer Muerta (Fig 1), runs from the Puerto de Navacerrada in the NW–N direction, entering the province of Segovia. Finally, a third range that runs by the entire province of Madrid consists of a more complex configuration because it is formed by a set of mountains that are not arranged in line but are grouped in an almost parallel formation (Fig 1). The primary axis is Cuerda Larga, which begins in the vicinity of Puerto de Navacerrada, progressing along 16 km in an E–NE orientation until it reaches the Puerto de la Morcuera. To the south, lying on the mountainous buttress, are the mountains Sierra de los Porrones and Sierra de la Pedriza, and the fields of the Altos de la Morcuera are towards the north. The Sierra de Guadarrama is formed by rocks of different origins, but the lithological substrate of the national park is mostly comprised of gneiss metamorphic rocks [37]. This type of primitive rock has an igneous origin, and thus it is more specifically an orthogneis [3837]. This geological material is the oldest of this orography [39], although there is controversy regarding its isotopic age which is said to be between the lower Ordovician and the middle Cambrian [40]. This type of rock is present in the three mountainous ranges and shows a certain tendency to fracture, and it is the main protagonist of the morrenic and colluvial deposits that originated from glacial events [41] and periglaciares [42], forming most of the scree-talus of the Sierra de Guadarrama. The granite, plutonic rock, which is associated with filonian rocks, also forms part of these reliefs; however, within the national park, it is limited to only a part of Siete Picos and almost all of La Pedriza. The massive character of these materials and their erosion due to disintegration does not favour the formation of scree-talus, and therefore these substrates have been excluded from underground surveys (Fig 2).

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Fig 1. Maps of the Sierra de Guadarrama National Park.

(A) basic orography. (B) habitats that occupy greater surfaces [36]. The spatial location of the sampling points is indicated and numbered.

https://doi.org/10.1371/journal.pone.0189205.g001

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Fig 2. Installation of Subterranean Sampling Device (SSD).

(A) Scree slopes from shadowed areas of La Mujer Muerta (Segovia). (B) Scree slopes from Cancho del Río Peces (Segovia). (C) SSD before being buried. (D) SSDs of 1 m (left) and of 0.5 m (right) in the process of installation. (E) Placing of the pitfall trap inside the cylinder (F–I) SSD used in sampling.

https://doi.org/10.1371/journal.pone.0189205.g002

The park consists of three bioclimatic floors, which respond to the typical model of a mountainous Mediterranean enclave: the supramediterranean, oromediterranean and crioromediterranean floors. On the supramediterranean floor (1300–1700 m a. s. l.), the melojo oak (Quercus pyrenaica Willd.) is the most common species, although it has been depleted in favour of the Scots’ pine (Pinus sylvestris L.), which mostly grows in the upper supramediterranean floor but is also found in the oromediterranean and the crioromediterranean floors of some south-facing slopes [43]. These pine forests, whether natural or naturalised, comprise the second most extensive vegetation unit of the park, forming dense forest masses with little fragmentation [36] (Fig 2). The oromediterranean floor (1700–2200 m a. s. l.) is characterised by the presence of Juniperus communis alpina (Suter) Celak. (creeping juniper) and shrubs of Cytisus oromediterraneus Rivas-Martinez et al. (‘piorno serrano’), although these species can enter the supramediterranean floor, reaching lower levels than is optimum. On the oromediterranean floor, these species include shrubs of timberlines and Adenocarpus hispanicus (Lam.) DC. or Erica arborea L. This vegetation cover alternates between grasslands, rocky outcrops and scree-talus. It is the most extensive vegetation in the Sierra de Guadarrama National Park [36], which forms the characteristic habitat of the summits of this mountain along with the areas covered by scree-talus (Fig 2). Finally, the crioromediterranean floor (2200–2400 m a. s. l.) has a combination of psychoxerophilic grasses and Festuca curvifolia Lag. ex Lange, which plays an important role in soil protection in extremely harsh climatic conditions due to its coverage [44]. In addition, there is an entire cohort of colluvial specialist vegetation [36] as well as large colluvial and morrenic deposits that extend along the oromediterranean floor.

In the Sierra de Guadarrama, the climate is a cold continental Mediterranean with dry and fresh summers. Its marked continentality due to its remoteness from the influence of the Mediterranean Sea and the Atlantic Ocean is not incompatible with the presence of a remarkable variety of microclimates. The microclimatic differences result from the special arrangement of the three mountain ranges, which condition the circulation of the air masses [45]. In addition, the topographic and geographical diversity (orientations, slopes, altitude) of each of these mountains decisively influence meteorological parameters, such as temperature, insolation, precipitation and the accumulation and persistence of snow [46]. The cold period extends beyond winter for about six months, while the dry period does not last longer than one month. The remaining five months are characterised by intense biological activity due to a combination of temperature and humidity that promotes the vegetative period of the plants [47]. Meteorological data of the port of Navacerrada (1894 m a. s. l.) recorded over a 20-year period yielded the following average annual values [48] that can guide the tendency of the main climatological variables of the Sierra de Guadarrama: maximum temperature 10.7°C; minimum temperature 3.1°C; 142 days of frost; and 1223 mm of precipitation over 111.3 days, of which 78 days was snow.

Sampling

The sampling was performed mainly using Subterranean Sampling Devices (SSD) similar to those used in other underground surveys in Iberian enclaves [12, 25, 23, 49, 50, 51, 14]. The SSDs consisted of a PVC cylinder 11 cm in diameter and 1 m in length, which had numerous perforations that were 8 mm in diameter in its lower half (Fig 2F). Once buried vertically in a suitable substrate, the top was covered with the ground surface (Fig 2C–2E). A pitfall trap was slid inside using a nylon thread that precisely matched the tube wide (Fig 2G). Previously, the trap was partially filled with a preservative liquid (1,2-propanediol) and fed with a vial containing strongly odorous cheese (Fig 2G and 2I). Inside some SSDs (SSD-5, SSD-7, SSD-12, SSD-14, SSD-17 and SSD-23), a digital thermometer programmed to record temperature at one-hour intervals (Fig 2G) was installed and was protected by a plastic bell. In the La Mujer Muerta range, SSDs of 0.5 m perforated along their entire length except for the upper 20 cm were installed (Fig 2H) along with the four 1 m SSDs (SSD-1, SSD-2, SSD-3 and SSD-4) (Fig 2F). The purpose of the SSD model of 0.5 m was to collect the fauna of the most superficial MSS and thus allow for comparing the fauna collected with that extracted from the SSD of 1 m. Two pitfall traps (TSPs), which contained the same type of preservative and bait as the SSDs, were buried in a visible Colluvial MSS in a forest talus of the Puerto de Navafría. To achieve this, two horizontal holes of about 80 cm in length were created (Table 1). The coordinates used are Universal Transverse Mercator coordinate system (UTM).

The enclaves sampled were located within the national park (32 localities), and to a lesser extent, within the area declared Peripheral Protection Area (2 localities) (Table 1, Fig 1): a) five locations in Siete Picos and La Mujer Muerta, of which four had double SSDs (1 m and 0.5 m); b) 15 localities in Montes Carpetanos, one of which had two TSPs installed and the remaining 14 an SSD of 1 m in length; and c) 12 localities with SSDs of 1 m in length, located in Cuerda Larga and its associated mountainous complex (eight in Cuerda Larga, two in La Maliciosa, the highest area of the Sierra de los Porrones, and two in the Altos de la Morcuera). The authors of the sampling included a team that consisted of Ortuño V.M., Ledesma E., Gilgado J.D., Jiménez-Valverde A., Pérez-Suárez G. and Baquero E.

Sampling permits for the corresponding national parks were obtained from the following appropriate authorities: Ismael Hernández Fernández, Deputy Director General of Management and Planning of Protected Areas (2015) and José Lara Zabía, Head of Conservation Area of Flora and Fauna (2016) at the General Directorate of Environment of the Community of Madrid, and Jose Ignacio Quintanilla Rubio (2015), and Montserrat de Andrés Boal (2016), General Director of the Natural Environment by delegation of the Head of the Territorial Service of the Environment of the Junta de Castilla y León.

The biological samples (specimens collected from May to October 2015) were transported to the laboratory and were preserved in 70% ethanol until the taxonomic study began.

Mounting and observation

Some specimens were cleared in Nesbitt’s fluid, and after washing for one hour in 70% Ethyl alcohol, they were mounted in Hoyer’s medium for the compound microscope observation in a phase contrast and DIC. Some specimens were decapitated, immersed in a saturated solution of KOH and emptied before being mounted on the slides.

Some specimens were examined using a Scanning Electron Microscope (SEM). Specimens from 70% ethyl alcohol were slowly rehydrated in a decreasing series of concentrations (60%, 50%, 40%, 30%, 20%, 10%, distilled water) over 24 hours. Once in distilled water, they were fixed in 4% glutaraldehyde in a cacodylate buffer for 24 h and then transferred to sucrose 0.25 M for 24 h. The specimens were then dehydrated up to 100% ethyl alcohol. Complete desiccation was achieved using the CO2 critical point technique. Samples were then covered with a 16-nm thin layer of molecular gold using an Emitech K550 sputter coater. Observations were performed using a Zeiss Digital Scanning Microscope 940 A.

Taxonomy

The combined use of color and morphological characters (macrochaetotaxy included) allowed for the identification of the species and provided useful descriptions for species identification. Potapov and Kremenitsa used coloration and macrochaetotaxy (some selected areas), the antennae length, width and shape, the eye structure, leg length, claw complex and manubrial organ (males) [33]. The array (or group) of characteristics proposed by Jordana and Baquero [32] and the chaetotaxy nomenclature of [31], which is based on a constant and generally visible set of morphological characters [52, 53, 54], has proven useful for the identification of species that belong to Entomobryini. It is proposed that some of the areas that conform to the simplified formula of [32] for Entomobrya could also apply to Orchesella, and an area for the head macrochaetotaxy (H6) has been added, which indicates the presence of chaetae S0 and S2 and their accessories.

Results

Fauna associated with the Colluvial MSS

For the identification of the Collembola species examined in the present study, a total of 42,240 specimens (20% belonging to the genus Orchesella) were studied individually. This work was conducted at the Department of Environmental Biology, University of Navarra, after the triage was completed at the University of Alcalá for over 61,038 specimens. This means that 69 per cent of the specimens caught in the total traps belonged to the Collembola taxon.

Throughout the first collection of sampling traps from the MSS of the Sierra de Guadarrama, only for the fauna of Collembola, a total of 32 genera and 61 species were identified. Of these 61 species, 18 are likely new to science. The taxon Entomobryidae, with five genera and 18 species, was the most uniformly distributed throughout the entire study area. The genus Orchesella, represented by two new species, was present in almost all traps (absent only in four). Thus, these two species are considered the most representative of the Colluvial MSS of the Sierra de Guadarrama.

Description of new species

Class COLLEMBOLA Lubbock, 1870

Order ENTOMOBRYOMORPHA Börner, 1913, sensu Soto-Adames et al., 2008

Family ENTOMOBRYIDAE Tömösvary, 1882

Subfamily ORCHESELLINAE Börner, 1906

Tribe ORCHESELLINI Börner, 1906

Genus Orchesella Templeton, 1836

Orchesella mesovoides Baquero and Jordana sp. nov.

urn:lsid:zoobank.org:act:BB75F525-25F4-4C47-AABD-EDFC132141B8

(Figs 3A, 3B, 4A, 4B, 5A, 5E–5G, 6A, 6C, 6D, 6F, 6G, 7A, 7E and 8A)

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Fig 3. Habitus and color pattern of the Orchesella species studied.

(A) Orchesella mesovoides sp. nov., lateral view. (B) Orchesella mesovoides sp. nov., dorsal view. (C) Orchesella eolia, dorsal view. (D) Orchesella colluvialis sp. nov., lateral view. (E) Orchesella villosa, lateral view. (F) Orchesella cincta, lateral view (bar 0.5 mm).

https://doi.org/10.1371/journal.pone.0189205.g003

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Fig 4. Antennae of the Orchesella species studied.

(A–B) Orchesella mesovoides sp. nov. aberrant antenna, with a flagellus instead of the normal third and fourth segments and a normal antenna with some areas enlarged. (C–D) Orchesella colluvialis sp. nov. antenna. (E), Orchesella villosa. (F–G) Orchesella cincta (bar for antennae: 0.25 mm; bar for enlarged areas: 0.05 mm).

https://doi.org/10.1371/journal.pone.0189205.g004

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Fig 5. Some morphological characters of the Orchesella species studied.

(A) Trochanteral organ of Orchesella mesovoides sp. nov. (B) Trochanteral organ of Orchesella colluvialis sp. nov. (C) Trochanteral organ of Orchesella villosa. (D) Trochanteral organ of Orchesella cincta. (E) Tip of the leg III (claw and empodial appendage, anterior view) of Orchesella mesovoides sp. nov. (F) Tip of the dens and mucro of Orchesella mesovoides sp. nov. (G) Tenaculum of Orchesella mesovoides sp. nov. (H) Tip of the leg III of Orchesella colluvialis sp. nov. (I) Tip of the dens and mucro of Orchesella colluvialis sp. nov. (J) Maxilar outer lobe of Orchesella colluvialis sp. nov. (K), Tip of the leg III of Orchesella villosa (the arrow points to the absent tooth in the drawn claw). (L) Tip of the dens and mucro of Orchesella villosa. (M) Tip of the leg III of Orchesella cincta. (N) Tip of the dens and mucro of Orchesella cincta. (O–Q) Macrochaetae from tibiotarsus of leg III of the Orchesella colluvialis sp. nov.: long, special and short (normal size), respectively (bars: A–B: 0.1 mm; E–F, H–I, K–L, M–N: 0.5 mm; G: 0.04 mm; J, O–Q: 0.02 mm).

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Fig 6. SEM photographs.

(A) Eye of the Orchesella mesovoides sp. nov. (bar 0.04 mm). (B) Eye of the Orchesella colluvialis sp. nov. (bar 0.04 mm). (C) Tip of the antennal segment II and the basal part of III of the Orchesella mesovoides sp. nov. (bar 0.09 mm). (D) Detail of the antennal segment III in an area with apparent segmentation of the Orchesella mesovoides sp. nov. (bar 0.006 mm). (E) S-microchaeta from the Abd III of the Orchesella colluvialis sp. nov. (bar 0.004 mm). (F) S-chaeta from the Abd II of the Orchesella mesovoides sp. nov. (bar 0.006 mm). (G) S-chaeta from the Abd IV of the Orchesella mesovoides sp. nov. (bar 0.02 mm).

https://doi.org/10.1371/journal.pone.0189205.g006

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Fig 7. Head chaetotaxy of the Orchesella species studied.

(A) Orchesella mesovoides sp. nov.; (B) Orchesella colluvialis sp. nov. (C) Orchesella villosa. (D) Orchesella cincta. (E) Eye details of the Orchesella mesovoides sp. nov. (F) Main chaetotaxy of Orchesella eolia (bar 0.1 mm except for E, 0.04 mm).

https://doi.org/10.1371/journal.pone.0189205.g007

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Fig 8. Dorsal macrochaetotaxy of Th II to Abd V.

(A) Orchesella mesovoides sp. nov. (B) Orchesella colluvialis sp. nov. (bar 0.2 mm; S-chaetae and S-microchaetae drawing in the lateral x10).

https://doi.org/10.1371/journal.pone.0189205.g008

Type material.—Holotype: female, trap SSD-2 (slide 23), Corrales de la Majada Minguete (30 T 410083 4516653, 1818 m a.s.l.), Mujer Muerta/Montón de Trigo, Sierra de Guadarrama, Segovia, SPAIN, 17.IX.2015, pitfall SSD (since 20.V.2015), Ortuño, et al. leg. Paratypes: SSD-1, 26 in ethanol; SSD-2, 6 in slides (22, 1 female; 24, 1 juvenile; 25, 1 female; 26, 1 male; 27, 1 female), 582 in ethanol; SSD-3, 29 in ethanol; SSD-4, 8 in ethanol (all from La Mujer Muerta/Montón de Trigo, Sierra de Guadarrama); SSD-5, 2 in ethanol; SSD-7, 116 in ethanol; SSD-8, 4 in ethanol; SSD-9, 50 in ethanol; SSD-11, 123 in ethanol; SSD-12, 184 in ethanol; SSD-13, 2 in ethanol; SSD-15, 6 in ethanol; SSD-16, 100 in ethanol; SSD-17, 20 in ethanol; SSD-18, 3 in ethanol; SSD-21, 120 in ethanol; SSD-22, 206 in ethanol; SSD-25, 35 in ethanol; SSD-26, 1 male in slide, 1 in ethanol; SSD-29, 4 in ethanol; SSD-30, 252 in ethanol; SSD-31, 20 in ethanol; SSD-33, 50 in ethanol; TSP-1, 12 in slides (01, 3 males; 02, 2 males; 03, 1 female; 04, 1 male; 05, 1 male; 06, 1 male; 07, 3 males); TSP-2, 1 male on slide (01).

Other material studied.—Collado Alto (Alto del León), mina de wolframio, Coordinates: 40.68660815, -4.16397343, 1 specimen labeled Orchesella eolia, Museo Nacional de Ciencias Naturales, Madrid (MNCN)_Ent 144262; Valsaín, 26-VI-63, 1 specimen labeled Orchesella eolia, MNCN_Ent 144266 (MNCN).

Etymology.—The species name follows the ecological niche. This was the first time it had been studied in Sierra de Guadarrama, where it was found.

All specimens were deposited in Museum of Zoology, University of Navarra, Pamplona, Spain (MZNA) except two paratype slides: TSP-1 01, three males, in MNCN; TSP-1 02, two males, in Muséum d’histoire naturelle de Genève (MHNG).

Description.

Body length 3.70–5.80 mm (mean 4.36 mm, n = 17) (Holotype 5.77 mm) (Fig 3A and 3B).

Ground color of the body pale; eyes black; head with light blue pigment on lateral and posterior head; antennae slightly blue at the end of the second part of the first segment, dark blue in the first part of the second segment and slightly blue for the last two segments; Thorax (Th) II–Abdomen (Abd) II with lateral, dorsolateral and dorsal-central longitudinal dark blue stripes, broadened at the end of each segment; Abd III with a transversal dark blue irregular band; Abd IV characterised by a continuation of the pigment patches of the forwarded segments; Abd V with dorsal and lateral spots; Abd VI without pigment; legs: femur with terminal lateral blue pigment and tibiotarsus homogenously light blue. Juveniles with a similar coloration.

Head. Ratio antennae/head = 4.04 (n = 14). First and second sub-segments of Antennal segment (Ant) I thick, twice as Ant II. Distal part of Ant III and Ant IV annulated and verticillate (Figs 4A and 4B and 6C and 6D), without apical bulb; Ant III sensory organ as in Fig 4B (detail). Labral papillae rounded, with pointed and curved tip, as a hook. Prelabral and labral chaetae all smooth (4/554). Lateral process (or differentiated chaeta) of outer labial palp (E) not reaching the middle of its papilla. Anterior labial row with four smooth and subequal chaetae; posterior labial row internal to chaeta E with 6–8 chaetae per side, all ciliated; chaetae E, L1 and L2 ciliated. Outer lobe of maxillary palp with four smooth chaetae in addition to the two basal chaetae. 8 + 8 ommatidia, G and H smaller (Fig 6A).

Body. Abd IV/III ratio 1.40–2.25 (mean 1.75, n = 17). Trochanteral organ with approximately 100 special chaetae (Fig 5A). Tibiotarsus bent distally, as a sub-segmentation, at 55% of its total length; spiniform chaetae with appressed ciliation (apparently wide, smooth chaetae): 7–10 on leg 1; 10–15 on legs 2 and 3. Claw with one outer tooth, a little more distal than a pair of stout lateral teeth, and four inner teeth (two paired at 50%, a first unpaired at 75% and a minute pair at 90% of the internal edge’s total length). Empodial appendage lanceolated and concave, with the interior (towards claw) very curved and with an outer tooth inserted at 30–40% of its total empodial length; empodial appendage/claw length ratio 0.62 (0.57–0.65, n = 7); smooth chaetae on tibiotarsus similar in length to empodial appendage (0.84–1.09, n = 7); tenent hair similar in length to empodial appendage (n = 7) (Fig 5E). Anterior face of ventral tube with many chaetae (>50), including 2+2 Mc on distal area; posterior face with >50 chaetae, including 1+1 Mc on latero-basal area; apical flaps with >50 smooth and ciliate chaetae. Tenaculum as in Fig 5G.

Furcula. Males with manubrial organs with four small chaetae grouped similar to a brush. The smooth area of distal dens 4 to 5 times the mucronal length; basal spine on mucro reaching or slightly surpassing the basal tooth; teeth of similar size (Fig 5F).

Macro-chaetotaxy (Figs 7A, 7E and 8A, Table 2). Head: H1 area with five to seven An chaetae, some as mc (mesochaetae); H2 area with 2–3 Mc (Macrochaetae) and 2–3 mc; H3 area with S’0 present; H4 area with two Mc; the S series (from S4i to the eye) with 10–13 chaetae, 3–4 as Mc (chaetae of different sizes); H5 area with two Mc; H6 area with four or five Mc (sometimes as mc). Th II: T1 area with 10–12 Mc (n = 4); T2 area with 7–10 Mc, m5 not seen in one of the four specimens studied. Th III with three Mc above pseudoporus, and 12–13 chaetae in total; lateral sensillae not identified. Abd I with five Mc around the pseudoporus (a1, a2, m2i, m2 and m3), and four towards lateral (a3, m4i, m4 and a6); lateral sensillae not identified. Abd II with four Mc in A1 area (a1, a2i, a2 and a3), and four on A2 area (m3, m3ei, m3e and m3e2); sensilla ‘as’ above m3 (between m3 and a3); m2 and a5 as bothriotricha. Abd III with one Mc on A3 area (a1), three on A4 area (a2, a2a and a3) and two on A5 area (m3 and m3e); sensilla ‘as’ above m2 (between m3 and a3); a5, m2 and a5 as bothriotricha, three Mc lateral to external bothriotricha. Abd IV: A6 area without Mc; A7 area with three Mc (A1, B1 and Be2); A8 area with three Mc (A3, B2 and B3) and four mc (A03, Ae3, C1 and E1); A9 area–than included the pseudopore, in a more advanced position than in other species of the family that were closer to A5– with three Mc (unpaired A04, A4 and B4); and A10 area with four Mc (unpaired A05, A5, B5 and A6); and A10 with only one Mc (A6).

Simplified Mc formula: 5(7)-2(5)-1-2(3)-2-4(5)/12-9/4-4/1-3-2/0-3-103(6)-102-103 (10 is an unpaired chaeta; maximum in parentheses).

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Table 2. Comparison of characters among the different species of Orchesella cited in this paper.

https://doi.org/10.1371/journal.pone.0189205.t002

S-chaetae formula: 2, 2/1, 5, 5, n, 13 (n = approximately 10–18, some of them four times the ‘normal’ (short) S-chaetae). The ‘normal’ S-chaetae (Fig 6F) measured 24–26 micrometers, while the ‘long’ S-chaetae on Abd IV (Fig 6G) measured 95–100 micrometers. S-microchaetae formula (like spine, approximately 3.5 micrometers): 1, 0/1, 0, 1, 0, 0 (Fig 8A).

Biology.

The study of the intestinal content of the specimens mounted on the slides allowed for determining that they feed mainly on amorphous organic matter. In addition, in almost every specimen, fungal hyphae and spores were observed. There was also a noticeable quantity of crystals (presumably small sand grains).

Remarks.

The coloration of the species O. mesovoides sp. nov was completely different from the seven species mentioned in the Iberian Peninsula, though it was somewhat similar to the original description of O. eolia (from Stromboli Island, cited from the Sierra de Guadarrama) [55]. Two of these specimens cited by Selga were observed. The first was from Collado de la Mina (or Lijar) in Alto del León (mine of wolframio) (MNCN_Ent 144262). Another specimen from Sierra de Navacerrada was a juvenile that was cleared and mounted sideways and the chaetotaxy is not visible, (MNCN_Ent 144270). The first belonged to the O. mesovoides sp. nov. due to its coloration with a less extended pigment, and the macro-chaetotaxy was also similar.

It was possible to identify differences in the pigment patterns between the new species and O. eolia, especially in the Th II and Abd II-V segments. The ratio Abd IV/III in O. eolia (1.48) was lower than that for the species O. mesovoides sp. nov. (1.40–2.25). The macrochaetotaxy was considerably different between the species O. mesovoides (Figs 7A and 8A) and O. eolia (Fig 7F). O. eolia had more Mc in H2 area, and H3 area was present in O. mesovoides sp. nov. but was absent in O. eolia (Table 2).

Orchesella colluvialis Jordana and Baquero sp. nov.

urn:lsid:zoobank.org:act:3881F811-7E6A-4776-8339-92EFCC34DF3D

(Figs 3D, 4C and 4D, 5B, 5H–5J, 5O–5Q, 6B, 6E, 7B and 8B)

Type material.—Holotype: female, trap SSD-22 (slide 01), Canchal del Alto del Puerto (30 T 430419 4537625, 1995 m), Montes Carpetanos, Sierra de Guadarrama, Madrid, SPAIN, 22.IX.2015, pitfall SSD (since 24.VI.2015), Ortuño et al. leg. Paratypes: SSD-1, 5 in slides (09, 1 female; 10, 1 male; 11, 1 female; 12, 1 female; 13, 1 juvenile;), 467 in ethanol; SSD-2, 187 in ethanol; SSD-3, 4 in slides (01, 1 female; 02, 1 juvenile; 03, 2 juvenile), 425 in ethanol; SSD-4, 279 in ethanol; SSD-5, 96 in ethanol; SSD-6, 389 in ethanol; SSD-7, 216 in ethanol; SSD-8, 168 in ethanol; SSD-9, 163 in ethanol; SSD-10, 10 in ethanol; SSD-11, 117 in ethanol; SSD-12, 115 in ethanol; SSD-13, 443 in ethanol; SSD-14, 40 in ethanol; SSD-15, 68 in ethanol; SSD-16, 201 in ethanol; SSD-17, 132 in ethanol; SSD-18, 75 in ethanol; SSD-19, 55 in ethanol; SSD-20, 57 in ethanol; SSD-22, 184 in ethanol; SSD-25, 173 in ethanol; SSD-26, 1 female in slide (01), 591 in ethanol; SSD-27, 112 in ethanol; SSD-28, 38 in ethanol; SSD-29, 135 in ethanol; SSD-30, 73 in ethanol; SSD-31, 700 in ethanol; SSD-33, 1 female in slide (01), 220 in ethanol.

Etymology.—The species name follows the ecological niche of where it was found (rock detritus and soil accumulated at the foot of a mountain) from Latin colluvium.

All specimens were deposited in MZNA except two paratypes: SSD-1 11a, female, in MNCN; SSD-26 01, male, in MHNG.

Description.

Body length 4.50–6.40 mm (mean 5.50 mm, n = 13) (Holotype 4.53 mm) (Fig 3D).

Ground color of the body uniformly pale; eyes black; head with light blue pigment only lateral to eyes and insertion of Ant I; antennae pigmented on dorsal Ant I b, Ant II a, dorsal Ant II b, and last two segments; only the lateral Th III, Abd II–III, posterior lateral Abd IV, lateral Abd V, and in some specimens, external distal trochanter, femur and tibiotarus, had some pigment.

Head. Ratio antennae/head = 4.30 (n = 11). First and second sub-segments of Ant I thickness, twice that of Ant II. Distal part of Ant III and Ant IV annulated and verticillate (Fig 4C and 4D) without apical bulb; Ant III sensory organ similar to that of O. mesovoides sp. nov. Labral papillae rounded, with pointed and curved tip, as a hook; in some specimens hook asymmetrically bifurcate, sometimes with a bristle, with one to three ramus. Prelabral and labral chaetae all smooth (4/554). Lateral process (or differentiated chaeta) of outer labial palp (E) not reaching the middle of its papilla. Anterior labial row with four smooth and subequal chaetae; posterior labial row internal to chaeta E with 13–15 chaetae per side, all ciliated; chaetae E, L1 and L2 ciliated (E and L1 smooth in some specimens). 8 + 8 ommatidia, G and H smaller; the eye H, seen at SEM, poorly developed externally (Fig 6B). Outer lobe of maxillary palp with four smooth chaetae in addition to the two basal chaetae (Fig 5J).

Body. Abd IV/III ratio 1.43–2.09 (mean 1.69, n = 13). Trochanteral organ with approximately 120 special chaetae with a segregate distribution of the longer chaetae (Fig 5B). Tibiotarsus apparently without sub-segmentation, although in some specimens, it was bent slightly beyond its midpoint; spine-like chaetae with appressed ciliation (apparently wide, smooth chaetae): 5–8 on leg 1; 10–15 on legs 2 and 3. Claw with one outer tooth at the level of a pair of stout lateral teeth and four inner teeth (two paired at 45–50%, a first unpaired at 75% and a minute unpaired at 85% of the internal edge total length). Empodial appendage lanceolate and concave with an outer tooth inserted at 30–35% of its total empodial length; empodial appendage/claw length ratio 0.67 (0.63–0.72, n = 4); smooth chaetae on tibiotarsus similar in length to empodial appendage (0.79–0.95, n = 4); tenent hair similar in length to empodial appendage (n = 4) (Fig 5H). Anterior face of ventral tube with many chaetae (>50), including 2+2 Mc on distal area; posterior face with >50 chaetae, including 1+1 Mc on latero-basal area; apical flaps with >50 smooth and ciliate chaetae.

Furcula. Males with manubrial organs with 3–4 small chaetae grouped similar to a brush. The smooth area of distal dens 2 times the mucronal length; basal spine on mucro reaching or slightly surpassing the basal tooth; teeth of similar size (Fig 5I).

Macrochaetotaxy (Figs 7B and 8B, Table 2). Head: H1 area with two An chaetae and seven more as mc; H2 area with four Mc and three mc; H3 area with S’0 present; H4 area with 2–3 Mc; the S series (from S4i to the eye) with 10–13 chaetae, 1–2 as Mc; H5 area with two Mc.; H6 area with five Mc. Th II: T1 area with 7–9 Mc (n = 6); T2 area with 8 Mc. Th III with 2–3 Mc above pseudopore, and 13–22 chaetae in total (n = 3); lateral sensillae not identified. Abd I with three Mc around the pseudoporus (a1, a2 and m2) and 6–8 towards lateral; lateral sensillae not identified. Abd II with three Mc in A1 area (a1, a2 and another one) and four on A2 area (m3, m3ei, m3e and m3e2); sensilla ‘as’ not identified; m2 and a5 as bothriotricha. Abd III with one Mc on A3 area (a1), three on A4 area (a2, a2a and a3) and three on A5 area (m3, m3e and m3e2; an additional, more external Mc was present in one specimen, male); sensilla ‘as’ above m2 (between m3 and a3); a5, m2 and a5 as bothriotricha, three Mc lateral to external bothriotricha. Abd IV: A6 area with two Mc (A1 and B1); A7 area with two Mc (B2 and C1); A8 area with four Mc (A4, A4a, Ae4 and B4); A9 area with two Mc (A5 and B5); and A10 area with only one Mc (A6).

Simplified Mc formula: 2(9)-4(7)-1-2(3)-2-5/7(9)-8/3-4/1-3-3/2-4-4-2-1.

S-chaetae formula: 2, 2/1, 5, 6, n, 14 (in Th II, in some specimens, acc.p6 duplicate; n = approximately 35, some of them four times the ‘normal’ S-chaetae). The ‘normal’ S-chaetae measured 24–26 micrometers, while the ‘long’ S-chaetae on Abd IV measured 95–100 micrometers. S-microchaetae formula 1, 0/1, 0, 1, 0, 0 (approximately 3.5 micrometers) (Fig 6E), respectively.

Biology.

The study of the intestinal content of the specimens mounted on slides allowed for observing amorphous organic matter, although some pollen grains were also present in some specimens that likely belonged to Pinus sylvestris, which are common in the area.

Remarks.

The coloration, which was an almost completely yellowish-white, of this species was similar to O. flavescens, which has two dorsolateral longitudinal lines from the head (behind the eyes) to the segment Abd VI in most specimens. It was possible to deduce part of the formula proposed in this work for O. flavescens using the information provided by Stach [28]:? -?-?-?-?/3-6/8-4/1-3-3/3-7(8)-3-2-0 (?, unknown), which makes it possible to differentiate it from O. colluvialis sp. nov. (Table 2) (other comparison among the different species of Orchesella cited in this paper is presented in Table 3). Other species recorded in Europe have some specimens from some populations with very little pigmentation, but they are always cited together with specimens with apparent pigmentation. In the case of O. colluvialis sp. nov., which was captured in hundreds of specimens in the sampling of this study, all specimens had pigmentation exclusively on the sides of the Abd VI (as shown in Fig 3D, there are very diffuse spots in other parts of the body, but that does not change the general color pattern of the species).

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Table 3. Comparison of characters among the different species of Orchesella cited in this paper.

https://doi.org/10.1371/journal.pone.0189205.t003

Orchesella villosa Linnaeus, 1767

(Figs 3E, 4E, 5C, 5K and 5L, 7C and 9A)

Studied material.—Cizur Menor, Navarra, SPAIN, 22.IX.2016, Pinus nigra forest (on trunk), coordinates 42,789909,-1,676618, sample MZ-20160922a (slide 01, female; slide 02, juvenile), E. Baquero leg. Pamplona, Navarra, SPAIN, 19.I.2017, Campus of University of Navarra (garden), coordinates 42,804044,-1,663833, sample MZ-20170119a (slide 01, female; slide 02, female), E. Baquero leg.

Description.

Body lengths of the specimens were 3.10–5.20 mm (mean 4.30 mm, n = 4) (Fig 3E).

Head. Ratio antennae/head = 3.05 (n = 2). Coloration is as described for the species [56]. First and second sub-segments of Ant I thick, some less than twice that of Ant II. Distal part of Ant III and Ant IV not annulated or verticillate, perhaps only a hint of annulation in the most distal part (Fig 4E) without an apical bulb. Labral papillae rounded, with pointed and curved tip, as a hook. Prelabral and labral chaetae all smooth (4/554). Lateral process (or differentiated chaeta) of outer labial palp (E) not reaching the middle of its papilla. Anterior labial row with four smooth and subequal chaetae; posterior labial row internal to chaeta E with 11–13 chaetae per side, all ciliated; chaetae E, L1 and L2 ciliated. 8+8 ommatidia, G and H smaller.

Body. Abd IV/III ratio 1.22 and 2.17 in the two specimens studied for this characteristic. Trochanteral organ with 95–100 special chaetae, with the longer situated only in a lateral direction (Fig 5C). Tibiotarsus without sub-segmentation; spiniform chaetae with appressed ciliation (apparently wide, smooth chaetae): 5–10 on leg 1; 10–15 on legs 2 and 3. Claw with one outer tooth, slightly more basal than the level of a pair of stout lateral teeth, and four inner teeth (two paired at 35–40%, a first unpaired at 65% and a last minute unpaired–absent in some specimens–at 85% of the internal edge total length). Empodial appendage lanceolated and concave with an outer tooth inserted at 30–35% of its total empodial length; empodial appendage/claw length ratio 0.60 (0.57–0.63, n = 4); smooth chaetae on tibiotarsus similar in length to empodial appendage (1.00–1.05, n = 4); tenent hair similar in length to empodial appendage (n = 4) (Fig 5K). Anterior face of ventral tube with many chaetae (>50), including 2+2 Mc on distal area; posterior face with >50 chaetae, including 2+2 Mc on latero-basal area; apical flaps with >50 smooth and ciliate chaetae.

Furcula. Males with manubrial organ with some chaetae grouped similar to a brush. The smooth area of distal dens was 1–2 times the mucronal length; basal spine on mucro reaching or slightly surpassing the basal tooth; teeth of similar size (Fig 5L).

Macrochaetotaxy (Figs 7C and 9A, Table 2). Head: H1 area with four An chaetae, some more as mc; H2 area with three Mc and 2–3 mc; H3 area with S’0 present; H4 area with two Mc; the S series (from S4i to the eye) with 10–12 chaetae, 5–6 as Mc; H5 area with three Mc; H6 area with five Mc. Th II: T1 area with seven Mc (n = 4), and 1–2 additional mc; T2 area with 2–3 Mc and 2–5 additional mc. Th III with 2–3 Mc above pseudoporus, and 10–11 additional mc (n = 2); lateral sensillae not identified. Abd I with three Mc around the pseudoporus (a2, a2a and m3), and 2–3 towards lateral direction; lateral sensillae not identified. Abd II with six Mc in A1 area (a1, a2 and probably a3 presents) and five on area A2 (m3, m3ei, m3e, m3e2 and m3eai); sensilla ‘as’ not identified; m2 and a5 as bothriotricha. Abd III with or without Mc on A3 area (a1), three on A4 area (a2, a3 and a3a) and three on A5 area (m3, m3e and m3ea); sensilla ‘as’ above m2 (slightly behind the line joining m3 and a3); a5, m2 and a5 as bothriotricha, and three Mc lateral to external bothriotricha. Abd IV: A6 area without Mc; area A7 with (A2 and B2); A8 area with three Mc (A3, B3, and C1); areas A9 and A10 without Mc, only with some mc.

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Fig 9. Dorsal macrochaetotaxy of Th II to Abd V.

(A) Orchesella villosa. (B) Orchesella cincta (bar 0.2 mm; S-chaetae and S-microchaetae drawing in the lateral x10).

https://doi.org/10.1371/journal.pone.0189205.g009

Simplified Mc formula: 4(8)-3(5)-1-2-3-5/7-5/6-5(6)/0(1)-3-3/0-3-3-2-2.

S-chaetae formula: 2, 2/1, 6, 5, n, 7 (n = 17, five of them four times the ‘normal’ S-chaetae). Th II and Abd III with one S-microchaetae (approximately 3.5 micrometers), respectively. The ‘normal’ S-chaetae measured 24–26 micrometers, while the ‘long’ S-chaetae on Abd IV measured 95–100 micrometers. S-microchaetae formula 1, 0/1, 0, 1, 0, 0 (approximately 3.5 micrometers), respectively (Fig 9A).

Remarks.

This species has a wide distribution in the Northern Hemisphere. The macro-chaetotaxy contributed to this paper.

Orchesella cincta Linnaeus, 1758

(Figs 3F, 4F and 4G, 5D, 5M and 5N, 7D and 9B)

Studied material.—Agüero-Marina de Cudeyo, Cantabria, SPAIN, 12.10.1991, Solaheza cave, coordinates (WGS84) 30T VP 441490 4807564 z = 25 (43.41868123,-3.72276624, 25 m), sample A-88 01 (female), C. González-Luque leg. Prellezo–Val de San Vicente, Cantabria, SPAIN, 25.VII.1992, Cueva del Rojo (Red’s cave), coordinates 30T UP 382743 4804399 z = 70 (43.38330352,-4.44761079, 70 m), sample A-108 (slide 01, female; slide 02, female; slide 03, male), C. González-Luque leg. Escobedo-Camargo, Cantabria, SPAIN, 20.VII.1991, Covachos I de Peñajorao, coordinates 30T VP 425723 4804264 z = 52 (43.38757361,-3.91706365, 52 m), sample A-68 BIS 01 (female), C. González-Luque leg. Lizoáin, Navarra, SPAIN, 25.IX.2014, Erro River (Aizpuru-El Puente), coordinates 42.821200, -1.457666, sample MZ-20140925f (slide 01, female; slide 02 female), E. Baquero leg.

Description.

The body lengths of the specimens studied were 3.65–4.40 mm (mean 4.00 mm, n = 7) (Fig 3F).

Head. Ratio antennae/head = 3.18 (n = 3). Coloration is as described for the species [56]. First and second sub-segments of Ant I wider than Ant II. Distal part of Ant III and Ant IV not annulated or verticillate, perhaps only a hint of annulation in the most distal part (Fig 4F and 4G), without an apical bulb. Labral papillae rounded, with pointed and curved tip, as a hook. Prelabral and labral chaetae all smooth (4/554). Lateral process (or differentiated chaeta) of outer labial palp (E) not reaching the middle of its papilla. Anterior labial row with four smooth and subequal chaetae; posterior labial row internal to chaeta E with 7–9 chaetae per side, all ciliated; chaetae E and L1 ciliated, L2 smooth. 8 + 8 ommatidia, G and H smaller.

Body. Abd IV/III ratio 1.30–2.11 (n = 7). Trochanteral organ with 80–90 special chaetae (Fig 5D). Tibiotarsus without sub-segmentation, although in some specimens, it was bent slightly beyond its midpoint; spiniform chaetae with appressed ciliation (apparently wide, smooth chaetae): 5–10 on leg 1; 10–12 on legs 2 and 3. Claw with one outer tooth, slightly more basal than the level of a pair of stout lateral teeth, and four inner teeth (two paired at 40%, a first unpaired at 70% and a last unpaired, with a significant development at 90% of the internal edge total length). Empodial appendage lanceolated and concave, with an outer tooth inserted at 50% of its total empodial length; empodial appendage/claw length ratio 0.67 (0.64–0.70, n = 4); smooth chaetae on tibiotarsus similar in length to empodial appendage (0.97–1.03, n = 4); tenent hair slightly longer (1,27 times) in length than empodial appendage (n = 1) (Fig 5M). Anterior face of ventral tube with many chaetae (>50), including 2+2 Mc on distal area; posterior face with >50 chaetae, including 1+1 Mc on middle-posterolateral area, and 2+2 ms on basal area; apical flaps with >50 smooth and ciliate chaetae, some of them bigger than other.

Furcula. Males with manubrial organs with four chaetae grouped similar to a brush. The smooth area of distal dens 3–4 times the mucronal length (although in one of the studied specimens, it was only nearly 2); basal spine on mucro reaching or slightly surpassing the basal tooth; distal tooth bigger than basal (Fig 5N).

Macrochaetotaxy (Figs 7D and 9B, Table 2). Head: H1 area with five An Mc; H2 area with three Mc; area H3 without S’0; H4 area with two Mc; the S series (from S4i to the eye) with 10 Mc and two mc; H5 area with one Mc (Ps2); H6 area with two Mc. Th II: area T1 with three Mc (n = 1), and 2–3 additional mc; T2 area with five Mc and four additional mc (n = 1). Th III with seven Mc above pseudoporus, and 6–7 additional mc (n = 1); lateral sensillae not identified. Abd I with three Mc around the pseudoporus (a2 and another two), and 3–4 towards the lateral direction (the difference in size between the insertions of Mc and mc was relative); lateral sensillae not identified. Abd II with three Mc in A1 area (a1, a2 and a3), and three on A2 area (m3, m3ei and m3e); sensilla ‘as’ not identified; m2 and a5 as bothriotricha. Abd III with Mc on A3 area (a1)—almost a mc—two on A4 area (a2 and a3) and three on A5 area (m3, m3e and m3ei); sensilla ‘as’ not identified; a5, m2 and a5 as bothriotricha, and three Mc lateral to external bothriotricha. Abd IV: area A6 without Mc; A7 area with three Mc (A3, B3 and C1); A8 area with one Mc (B4 or B5); A9 area without Mc; and ara A10 with only two mc.

Simplified Mc formula: 5-3-0-2-1b-2/7-7/4-3/1-2-3/0-3-1-0-0(2*) (m5 in Th II is present) (*meaning mesochaetae).

S-chaetae. Formula: 2, 2/1, 5, 5, n, 7 (n = 12, two of them four times the ‘normal’ S-chaetae; the pigmentation of the specimens observed) (the pigmentation of the specimens makes it very difficult to see the S-chaetae in most specimens). The ‘normal’ S-chaetae measured 24–26 micrometers, while the ‘long’ S-chaetae on Abd IV measured 95–100 micrometers. S-microchaetae formula 1, 0/1, 0, 1, 0, 0 (approximately 3.5 micrometers), respectively (Fig 9B).

Remarks.

The specimens studied were identified by color. In this study, the information regarding the macro-chaetotaxy of this species is presented for the first time, which will allow for comparisons when studying the specimens identified by other researchers either in the past or in the future. A wide distribution may be attributed to the species based only on coloration changes when the macro-chaetotaxy is used.

Orchesella eolia Altner, 1961

(Figs 3C, 7F and 10)

Studied material.—Stromboli Island, Italy, five specimens (paratypes) in ethyl alcohol from MHNG, code sample 7412 STR and 7412a STR. In two specimens, the color pattern was visible (Fig 3C). The specimen with more damaged pigmentation was cleared (KOH) and mounted in Hoyer’s medium.

Simplified Mc formula: 5-4(3*)-0-2-1-3/11-8/4-7/0-2-4/0-7-2-2-0 (Figs 7F and 10, Table 2).

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Fig 10. Dorsal macrochaetotaxy of Th II to Abd V of Orchesella eolia (bar 0.2 mm).

https://doi.org/10.1371/journal.pone.0189205.g010

Ecology

The study allowed for obtaining data regarding the microclimatic conditions of the sampled habitat, mainly of temperature, during the sampling period from June to September of 2016. It was observed that the highest monthly average temperatures were recorded in July, with 17° C in the highest altitudes and 23° C in the lowest altitudes, which decreased below 15° C in late August. The temperatures in the MSS never exceeded 26° C and did not decrease below 5° C during the cited sampling period. In addition, a temperature range between 5 and 18° C was observed in the traps located in the surroundings of 2000 m. a. s. l., and in the surroundings of 1500 m. a. s. l., the temperature range was between 10 and 25° C (Fig 11). The presence of the two species along the Sierra de Guadarrama was fairly uniform (Table 4); however, it was observed that O. colluvialis sp. nov. seemed to prefer a range of higher altitudes than O. mesovoides sp. nov. Considering the average number of animals per trap (to weigh the number of traps placed by height), it was observed that the maximum abundance for the species O. colluvialis sp. nov. was between 1600 and 1800 m. a. s. l., while O. mesovoides sp. nov., which was always found in lower abundances, had a maximum abundance between 1800 and 2000 m. a. s. l., indicating a preference for higher altitudes (Fig 12 and inset A). Considering the average number of animals per trap (to weigh the quantity of traps per orientation), it was observed that the vector sum of the abundances for the species O. colluvialis sp. nov. was located in the W-NW orientation, whereas for the species O. mesovoides sp. nov., it was N-NE (Fig 12, inset B).

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Fig 11. MSS temperature.

Temperature records of two of the SSDs, one (SSD-14) was the highest in Peñalara (Majada Hambrienta) (1994 m a. s. l.), and the other (SSD-7) was the lowest in El Purgatorio (1406 m a. s. l.). x-axis: number of measures from June to September; y-axis: temperature in Celsius degrees.

https://doi.org/10.1371/journal.pone.0189205.g011

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Fig 12. Number of specimens of the two Orchesella species described in this paper ordered by altitude.

(A) Geographical orientation of the vectors of abundance of the two species. (B) Abundance of the species in relation to altitude (orange: O. colluvialis sp. nov.; blue: O. mesovoides sp. nov.).

https://doi.org/10.1371/journal.pone.0189205.g012

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Table 4. Captures for the species O. mesovoides sp. nov. and O. colluvialis sp. nov. in all the traps.

https://doi.org/10.1371/journal.pone.0189205.t004

The presence of pollen granules in the species O. colluvialis sp. nov. may be related to its more abundant presence in lower areas of the mountain range where the tree vegetation, Pinus sylvestris, is more abundant.

The two new species described have similarities with respect to their distribution and abundance at different places, but it can be argued that the species O. colluvialis sp. nov. seems to be more adapted to the underground environment, which could be based on several factors: its coloration with little pigment, a greater reduction of the eyes (the G and H eyes was far more reduced in the optical microscope, which was confirmed when observed at SEM) (Fig 6B) and more S-chaetae. O. mesovoides sp. nov. had been referenced from Sierra de Guadarrama (erroneously identified as O. eolia), and given the lack of previous samplings in the subterranean environment, these references suggest that this species has less dependence on the subterranean habitat. Nevertheless, it has never been recorded at the surface.

The abundance by trap varied between a few specimens and at a maximum of 700 (in this case, the species O. colluvialis sp. nov.). For traps with two-level catches, no difference was observed for the two species described. In total, 1956 specimens of O. mesovoides sp. nov. and 5929 specimens of O. colluvialis sp. nov. were caught. The data confirmed that the species are abundant in the massif. They have not been captured previously, with the exception of the capture of the specimens identified as O. eolia by Selga in Collado Alto and Valsaín, which demonstrates their absence on the surface in this area. Based on the findings it can be affirmed that the Orchesella communities of the Colluvial MSS of Sierra de Guadarrama have their own species. Interestingly, other Orchesella species appeared to inhabit the Colluvial MSS in other regions of Europe [16, 17, 22] and even in the Iberian Peninsula [12, 14]. Thus, future works should reveal a significant hidden diversity of species of the genus as well as different species assemblages in the MSSs of different regions.

Discussion

Six species of Orchesella have been referenced for the Iberian Peninsula thus far; however, one (O. eolia) was considered a misidentification after study completion. Only four have been cited in the area of Sierra de Guadarrama and surroundings by conducting surface fauna sampling: O. cincta (Linnaeus, 1758) (San Lorenzo de El Escorial, Madrid [55]), O. flavescens (Sierra de Guadarrama, Cercedilla, Madrid [55]; Valsaín, Sierra de Guadarrama, Madrid [55]), O. quinquefasciata (Navacerrada, Sierra de Guadarrama, Madrid [55]; Cercedilla, Sierra de Guadarrama, Madrid [55]; San Lorenzo de El Escorial, Madrid [55]; Navacepeda de Tormes, oak forest, Sierra de Gredos[57]; Sierra de Gredos, pine forest litter [58]; Montejo de la Sierra, beech forest, Mazizo de Ayllón [59]), O. villosa (Los Cotos and Navacerrada, Sierra de Guadarrama, Madrid [55]).

In addition to the work of the researchers cited above and to Bonet and Acon [60, 61], who collected Collembola from 1929 until recently, and with the exception of two specimens identified as O. eolia found in a gallery of a mine, there have not been any additional registers of the Orchesella species described in this paper. Nevertheless, they have been proven to be abundant in the colluvial MSS.

It was possible to compare the two new species described in this paper with the species O. villosa, O. cincta and O. eolia (Table 3). The macrochaetotaxy of O. flavescens sensu Stach [28] has been studied for comparisons with O. colluvialis. It was not possible to study the species O. quinquefasciata. While comparing the macrochetotaxy and other morphological characters, it was found that with these characteristics and without color, these species can be differentiated. This is of enormous importance because until now, the identification of the genus Orchesella was based almost only on the coloration. Moreover, the sensilar chaetotaxy (S-chaetae and S-microchaetae) was examined in detail, since it has been used for other genera [62], although it is difficult to observe in species with large specimens.

Acknowledgments

The study of the specimens identified as Orchesella eolia (by Selga) from the Museum of Natural Sciences, Madrid (MNCN) was possible thanks to the kindness of Mercedes París. The study of some paratypes of Orchesella eolia from the Muséum d’histoire naturelle de Genève (MHNG) was possible thanks to the kindness of Peter Schwendinger. Carlos González-Luque collected some of the Orchesella cincta specimens studied. This work is part of the results of the project ‘Study of the diversity and distribution of animal species residing in the Surface Subterranean Environment of high mountain enclaves (Parque Natural de la Sierra de Guadarrama)’. We must express our gratitude to the other members of the research team responsible for developing this project: Gonzalo Pérez-Suárez, Alberto Jiménez-Valverde, Alberto Sendra, Pablo Barranco, Alberto Tinaut, Luis Subías and Juan José Hererro-Borgoñón. Finally, it should be recognised that the field work could not have been completed without the valuable collaboration of the authorities responsible for the national park as well as other workers (biologists, forestry agents and staff of the Oficina de Atención a la Población Local–Sector Lozoya–and the Centro de Visitantes–Peñalara). In particular, we extend our thanks to Patricia Riquelme, Pablo Sanjuanbenito, Juan A. Vielva, Javier Donés, Marisol Redondo, Ignacio Granados, Ángel Rubio, César Martín, José Carrillo, Miguel Ángel Palomar, Ángel Velasco, Germán Mato, Manuel Criado, Enrique Calvo, Federico Madejón and Montserrat Sanz. The anonymous reviewers provided excellent comments and suggestions that improved this manuscript.

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