Characterization of three plant or fungal feeding nematodes (Nematoda, Rhabditida, Tylenchomorpha) from coastal dunes in Spain including the SEM study of these species

Three known species, Aphelenchus avenae, Ditylenchus myceliophagus and Tylenchorhynchus aduncus (Rhabditida, Tylenchomorpha), are described from sand coastal dunes in Spain associated to the rhizosphere of xerophilic plants. A. avenae is characterized by having stylet with 16–22 µm long, pharyngeal dorsal gland overlapping the intestine, postvulval uterine sac about 2.5 times the body diameter, female tail short conoid with rounded tip bearing the phasmid and male bursa well developed. D. myceliophagus is characterized by having lateral field with four incisures, stylet 7–8 µm long, basal bulb irregularly pyriform, tail of both sexes similar with rounded tip and bursa of the male reaching about three-quarters of tail length. T. aduncus is characterized by its lateral field with six incisures, stylet 15–18 μm long, spicules with bifurcate tip and distal ventral crest, and gubernaculum with hook-like manubrium. New morphological, including SEM study, and morphometrical data are included for the three species. Also, phylogenetic analysis based on 18S and 28S rDNA is included for two of the species studied. Additionally, two species belonging to the superfamily Aphelenchoidea are transferred to other genera: Aphelenchus assamensis to the genus Aphelenchoides and Aphelenchoides dhanachandi to the genus Potensaphelenchus.


Introduction
The infraorder Tylenchomorpha De Ley and Blaxter (2002) is a cosmopolitan nematode taxon which occurs in soil and fresh-water habitats. The most of them are plant parasites, mycofagous or rarely zooparasites (Siddiqi 2000;Andrássy 2007). Therefore, tylenchids (including aphelenchid species) are the most diversified group of edaphic nematodes, representing about 15% of the known nematode fauna, being the Iberian tylenchids one of the best known (Peña-Santiago et al. 2012). Frequently, these species are associated to plants of agricultural interest (Prot and van Gundy 1981;Vandegehuchte et al. 2015). Of this widespread group, according the classifications proposed by De Ley and Blaxter (2002) and Doucet et al. (2020), species belonging to three genera, Aphelenchus Bastian, 1865(Aphelenchoidea Fuchs 1937, Ditylenchus Filipjev, 1936(Sphaerularioidea Lubbock 1861 and Tylenchorhynchus Cobb, 1913(Tylenchoidea Örley 1880, are studied in the present work. The genus Aphelenchus, belonging to the family Aphelenchidae Fuchs 1937, was described by Bastian (1865), including fungal-feeding nematodes. The genus contains 13 valid species (Hunt 2008;Jianfeng et al. 2020) being A. avenae the type species and the best characterized species of the genus, however, no complete SEM study was provided for this species. The main characteristics of this genus are the number of lateral lines (4-14), subcylindrical female tail with rounded terminus and phasmids located at terminus (Goodey 1927), and males with well-developed bursa.
The genus Ditylenchus Filipjev, 1936, from the family Anguinidae Nicoll (1935), is a very diverse group, knowing 63 valid species (Hashemi and Karegar 2019) being one of the most difficult genera to identify to species level (Escuer 1998) with very small differences between species . This genus comprises obligate plant parasites with 1 3 high variety of feeding habits (Sturhan and Brzeski 1991) and high phytosanitary importance Moens and Perry 2009). D. dipsaci (Kühn 1857) Filipjev (1936) is the type species of the genus. This genus is characterized by having lateral fields with 3-6 incisures, median bulb with or without valve, glandular bulb variable in length, when long may overlap the intestine for a short or long distance, females monodelphic with post-vulval uterine sac present or absent, and males with bursa peloderan never reaching the tail end.
The genus Tylenchorhynchus, belonging to the family Telotylenchidae Siddiqi, 1960, was described by Cobb in 1913with T. cylindricus Cobb 1913 from California, USA, as type species. Actually, the genus has 113 valid species (Handoo 2000;Geraert 2011;Handoo et al. 2014;Azimi et al. 2016) and is considered very difficult to identify to species level (Azimi et al. 2016;Hosseinvand et al. 2020). Species within this genus are distinguished based on pharyngeal gland pyriform, areolate lateral field, male genitalia and differences in female tail morphology (Jairajpuri and Hunt 1984;Gómez-Barcina et al. 1992;Carta et al. 2010).
The present study re-describes three known plant or fungal feeding species representing those three genera: Aphelenchus avenae Bastian, 1865, Ditylenchus myceliophagus Goodey, 1958and Tylenchorhynchus aduncus de Guiran, 1967. Those specimens were collected from the rhizosphere of several coastal dunes in Spain. Identification was performed based on morphological and morphometrical features, providing light (LM) and scanning electron microscopy (SEM) for these three species. Additionally, phylogenetical studies based on 18S and 28S rDNA gene were provided for two of these species.

Nematode sampling and extraction
Soil samples were collected from the rhizosphere of different plants of sand coastal dunes along the south Atlantic and Mediterranean Spanish coasts (survey conducted between 2018 and 2021 to determine the occurrence and diversity of nematodes in these xeric areas) and processed following several nematode techniques (Abolafia 2022). The nematodes were extracted from soil samples using a modified Baermann's (1917) funnel technique provided of a stainless-steel sieve (100 mm diam., 100 µm mesh) bearing a tissue circle at its bottom, according the Abolafia's (2022) technique, to prevent fine soil from falling through the sieve mesh, killed by heat and fixed in a 4% formalin (37% formaldehyde stock solution, glycerine and water) solution. The nematodes were processed to anhydrous glycerine according to Siddiqi's (1964) method using lactophenol-glycerine solutions, and they were permanently mounted on glass microscope slides with the glycerine-paraffin method (de Maeseneer and d'Herde 1963) somewhat modified using hot liquid paraffin.

Light microscopy (LM)
Observations were made using a Nikon Eclipse 80i (Nikon, Tokyo, Japan) microscope. Measurements were taken using an ocular micrometer or a curvimeter after drawing the corresponding organ or structure attached to an Olympus BH-2 microscope (Olympus, Tokyo, Japan). Demanian indices (de Man 1881) and other ratios were calculated. The micrographs were taken with a Nikon Eclipse 80i (Nikon, Tokyo, Japan) light microscope equipped with differential interference contrast optics (DIC) and a Nikon Digital Sight DS-U1 camera. The micrographs were combined using Adobe® Photoshop® CS (Adobe Inc., San José, USA) and figures mounted using Microsoft® PowerPoint® (Microsoft Corporation, Redmond, USA). The terminology used for the morphology of stoma and spicules-gubernaculum follows the proposals by Baldwin et al. (2004) and Abolafia and Peña-Santiago (2017), respectively.

Scanning electron microscopy (SEM)
The specimens preserved in glycerine were selected and processed for their observation with a SEM according to Abolafia (2015). The nematodes were hydrated in distilled water, dehydrated in a graded ethanol-acetone series, critical point dried, coated with gold, and observed with a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany).

DNA extraction, PCR and sequencing
Nematode DNA was extracted from single fresh individuals using the proteinase K protocol and PCR assays as described by Castillo et al. (2003) somewhat modified (Archidona-Yuste et al., 2016). The specimens were cut in small pieces using a sterilized dental needle on a clean slide with 18 µl of TE (Tris-EDTA) buffer [10 mM Tris-Cl (tris hydrochloride) + 0.5 mM EDTA (ethylenediamine-tetraacetic acid); pH 9.0], transferred to a microtube and adding 2 μl proteinase K (700 μg/ml) (Roche, Basel, Switzerland), and stored to − 80 °C within 15 min (for several days). The microtubes were incubated at 65 °C (1 h), then at 95 °C (15 min). For DNA amplification, 3 μl of the extracted DNA was transferred to a microtube containing: 0.6 μl of each primer (10 mM), 3 μl Master Mix Taq DNA Polymerase (5 × Hot FirePol Blend Master Mix, Solis BioDyne, Tartu, Estonia) and double distilled water (ddH 2 O) to a final volume of 20 μl. The primers used for amplification of the region of 18S rRNA gene were the forward primer 988F (5′-CTC AAA GAT TAA GCC ATG C-3′) and the reverse primer 1912R (5′-TTT ACG GTC AGA ACT AGG G-3′) (Holterman et al. 2006). The primers used for amplification of the D2-D3 region of 28S rRNA gene were the D2A (5'-ACA AGT ACC GTG AGG GAA AGTTG-3') and the D3B (5'-TCG GAA GGA ACC AGC TAC TA-3') primers (Nunn 1992;De Ley et al. 1999). PCR cycle conditions were as follows: one cycle of 94 °C for 15 min., followed by 35 cycles of 94 °C for 45 s + annealing temperature of 55 °C for 45 s + 72 °C for 45 s., and finally one cycle of 72 °C for 5 min. After DNA amplification, 5 μl of product was loaded on a 1% agarose gel in 0.5% Tris-acetate-EDTA (40 mM Tris, 20 mM glacial acetic acid and 2 mM EDTA; pH = 8) to verify the amplification using an electrophoresis system (Labnet Gel XL Ultra V-2, Progen Scientific, London, UK). The bands were stained with RedSafe (20,000x) previously added to the agarose gel solution. The sequencing reactions of the PCR products were performed at Sistemas Genómicos (Paterna, Valencia, Spain) according the Sanger et al. (1977) method. The sequences obtained were submitted to the GenBank database.

Phylogenetic analyses
For phylogenetic relationships, analyses were based on 18S and 28S rDNA fragments. The newly obtained sequences were manually edited using BioEdit 7.2.6 (Hall 1999) and aligned with another 18S or 28S rDNA sequences available in GenBank using ClustalW alignment tool implemented in the MEGA7 (Kumar et al. 2016). Poorly aligned regions at extremes were removed from the alignments using MEGA7. The best-fit model of nucleotide substitution used for the phylogenetic analysis was statistically selected using jMod-elTest 2.1.10 (Darriba et al. 2012). Phylogenetic trees were generated with Bayesian inference method using MrBayes 3.2.6 (Ronquist et al. 2012). Nothacrobeles lanceolatus Abolafia and Peña-Santiago, 2003 (OK042925 for the 18S and OK042914 for 28S rDNA fragments) was chosen as outgroup. The analysis under General Time Reversible Plus Invariant sites plus Gamma distribution (GTR + I + G) model was initiated with a random starting tree and run with the Markov Chain Monte Carlo (MCMC) (Larget and Simon 1999) for 1 × 10 6 generations. The trees were visualized and saved with FigTree 1.4.4 (Rambaut 2018).

Description
Adult: Body 0.65-0.73 mm long, almost cylindrical, slightly ventrally arcuate or straight after fixation. Cuticle 1 µm thickness at midbody with well-developed annuli, 1 µm width. Lateral fields occupying 27-37% of the body diameter and with 8-10 incisures at midbody, reduced in number at the posterior end. Lip region low, rounded, not offset, with lips fused, with six labial papillae surrounding the oral opening and four cephalic papillae at top of the two subdorsal and two subventral lips. Amphidial openings a short slit, located at top of lateral lips. Cephalic framework well sclerotized. Stoma occupying by a long stomatostylet (16-22 µm) with cone (metenchium) very refringent, 38-42% of the stylet length, shaft (telenchium) poorly refringent and basal knobs not developed. Pharynx with procorpus fusiform, median bulb (metacorpus) well developed, ovoid, and with conspicuous valve plates situated centrally, isthmus very thin and ending in a dorsal pharyngeal gland very elongated and overlapped into intestine about three times the body width; dorsal pharyngeal gland orifice opening into lumen of median bulb mid-way between anterior end of metacarpal valve and anterior end of median bulb. Nerve ring surrounding the isthmus to 71-81% of neck (stoma + pharynx) level. Excretory pore located at 66-73% of neck length level. Female: Reproductive system is monodelphic-prodelphic; ovary outstretched with oocytes in one row; oviduct short with swollen spermatheca containing round sperm; uterus thickwalled, 5.4-5.5 times the body diameter; post-vulval uterine sac swollen, 1.5-2.5 times the body width; vagina with thick sclerotized walls; vulva slightly protruding. Rectum 2.3 times the anal body diameter. Anus well developed. Tail subcylindrical with phasmid located on tail tip.
Male: Spicules paired, slender and ventrally arcuated, with rounded and ventrally bent manubrium, elongate straight calamus and ventrally curved lamina with acute tip. Gubernaculum almost straight, with manubrium reduced, rounded, and corpus conoid with acute, thinner tip. Bursa well developed, extending from the proximal region of the spicules to the tail tip and supported by four pairs of bursal papillae, one preanal pair and three pairs near the tail end. Tail short, conoid and ventrally curved, tapering to an acute terminus.

Remarks
The Spanish populations examined now agrees well with the type population described from Pennsylvania by Bastian (1865). However, this description is very poor with not well-detailed illustration of the female, while the male was not described. Later, Bütschli (1873) described some specimens collected from Germany, which agree with the present population, although the morphology of the anterior end is something offset than the present population, while the description of the male was not provided. Goodey (1927), redescribed this species from the type locality which also agrees with the Spanish population although the range of the body length is slightly shorter (0.65-0.73 vs 0.80-0.95 μm). With respect to the population described by Loof (2001) from Germany also agrees although the Spanish material has lower number of incisures at lateral field (8-10 vs 10-14 incisures), stylet slightly smaller (16-22 vs 14-16 μm) and spicules and gubernaculum slightly longer (16-21 vs 28-30 μm and 12 vs 14-16 μm, respectively). From the material studied from Japan by Otsubo et al. (2006), although they did not provide description of this species, their study included SEM illustrations of the lateral field, which number of wings are not clear but it can appreciate about ten wings. From the material described by Andrássy (2007) from Hungary, it agrees with the Spanish population but present also a higher number of incisures in the lateral field (8-10 vs 10-14 incisures) and the stylet is something smaller (16-22 vs 14-16 μm). The populations described by Kumari (2012) from Czech Republic also agree well. Finally, respect to the material described by Azimi (2018) from Iran, it agrees well with the Spanish populations although by having shorter body length range (0.65-0.73 vs 0.58-0.91 μm), number of incisures at lateral fields (8-10 vs 12-14) and spicules (16-22 vs 28-30 μm). With respect to the molecular characterization, this species agrees with the sequences of Aphelenchus avenae available in GenBank (JX421761 and JQ348399), in the studies of Pham and Zheng (unpublished) and Kumari (2012), respectively.

Measurements
See Table 1.

Description
Adult: Body almost straight after fixation, slightly ventrally arcuate when relaxed. Cuticle finely annulated with 2 μm wide. Lateral fields with six incisures, occupying 24-25% of body width. Lip region low, 6-7 μm wide, not offset, with fused lips having six labial papillae in a hexagrammate plate surrounded the oral opening. Amphidial openings form a short and narrow slit at top of the lateral lips. Stoma with a short stylet, 7-8 µm long, with conus (metenchium) slightly longer than the base (telenchium) and three rounded knobs. Dorsal gland opening (DGO) at 3-4 μm posterior to stylet knobs. Pharynx with procorpus tubular, median bulb (metacorpus) oval, not very developed having small valves, isthmus slender, and basal bulb irregularly elongate, offset from the intestine and overlapping it for a short part. Nerve ring surrounding the isthmus to 59-68% of neck length. Excretory pore at basal bulb level, located at 90% to neck length. Deirids at basal bulb level, located at 83% of the neck length. Female: Reproductive system monodelphic-prodelphic: ovary very long, outstretched, with oocytes in one row; oviduct short with swollen spermatheca containing round sperm; uterus with thick-walled tubular distal part, about twice the body diameter and swollen proximal part, as long as the body diameter, with thinner walls, rarely with uterine

Habitat and locality
Specimens collected from Palomares (Almería, Spain), associated to the rhizosphere of Ammophila arenaria.

Remarks
The type population described by Goodey (1958) from mushroom in United Kingdom agrees well with the Spanish population but this author described the stylet length having 14 μm, however, it was drawn with smaller size Fig. 2 Aphelenchus avenae (scanning electron microscopy) a Neck region; b, c Lip region in lateral and frontal views, respectively (arrows pointing the amphids); d Lateral field; e Female tail tip (arrows pointed to phasmids); f, g, h, i Female tail in left lateral, ventral, right lateral and dorsal views, respectively (arrows pointing the phasmids) (8-12 µm), agreeing with the present material (7-8 µm). Later, Escuer (1998) described this species from Spain using the measurements taken by Hesling (1974) which agree with the present study. Fortuner (1982) described this species from Ivory Coast and Burkina Faso and  from Australia and Poland, all of them agreeing with the Spanish material. Finally, the population described by Andrássy (2007) from Hungary agrees with the Spanish material although the body length has a shorter range (0.6-0.8 vs 0.6-1.3 μm), as well the female tail (47-56 vs 40-80 μm).

Measurements
See Table 1.

Description
Adult: Body slightly arcuate after fixation. Cuticle 1 µm thickness at midbody with transversal striae; annuli 1-2 mm Fig. 3 Ditylenchus myceliphagus (light microscopy) a Neck (black arrow pointing the excretory pore, white arrow pointing the deirid); b, f, g, i, l Variability of basal bulb (arrow pointing the excretory pore); c Anterior end; d Vulva and post-vulval uterine sac; e Female reproductive system; h Uterine egg; j Female posterior end, lateral view; k Male posterior end, lateral view width. Lateral fields occupying 37-46% of the body diameter originating at the level of the stylet base and extending up-tail terminus, with four incisures or three bands, areolate and the anterior body region. Anterior end distinctly offset by a constriction, bearing four or five annuli, distinguishing six fused lips, the lateral ones smaller. Labial papillae six surrounding the oral opening located in a hexagrammate area; cephalic papillae not observed. Amphidial openings an elongate pore-like. Labial framework not refractive. Stylet slender, conus (metenchium) 8-9 µm or 44-50% of the total stylet length; basal knobs weak and anteriorly sloping. Dorsal pharyngeal gland orifice (DGO) 1-4 µm behind stylet knobs. Pharynx with narrow tubular procorpus, median bulb (metacorpus) rounded with small valves, isthmus very slender and basal bulb irregularly pyriform. Nerve ring surrounding the isthmus at 66-76% from anterior end. Excretory pore at basal bulb level, at 79-95% from anterior end. Hemizonid 6-7 annuli anterior to excretory pore. Deirids at basal bulb level at 85% from anterior end. Intestine tubular without special differentiations. Female: Reproductive system didelphic-amphidelphic, with ovaries straight, oviducts short with rounded spermatheca filled with globular sperm cells, uteri tubular, about 2.6-3.9 times the body diameter long, vagina occupying 56-60% of body diameter, vulva transverse. Rectum very thin, 0.7-0.9 times anal body diameter. Anus small, Fig. 4 Ditylenchus myceliphagus (scanning electron microscopy) a Neck region; b, c, d, e Lip region in frontal, ventral, subfrontal and lateral views, respectively; f excretory pore; g lateral field; h vulva in lateral view; i vulva in ventral view; j, k female tail in ventral and lateral views, respectively rounded. Tail conoid, with 17-22 annuli, single or double, in ventral side, tail terminus smooth, conoid. Phasmids located at 39-51% of tail length from anus. Male: Reproductive system monorchid. Spicules ventrally curved with quadrangular manubrium, undifferentiated calamus and lamina ventrally curved at posterior region with small distal keel and bifurcate tip. Gubernaculum straight with hook-like manubrium and robust corpus with rounded tip. Tail conoid, ventrally curved with thinner rounded tip. Bursa along the tail lacking genital papillae. Phasmids at 50% of tail length from cloacal opening.

Host and locality
Specimens collected from El Altet (Valencia, Spain) are associated to the rhizosphere of Ammophila arenaria and Carpobrotus edulis (L.) N.E. Br.

Remarks
Tylenchorhynchus aduncus is the type species of the genus Tylenchorhynchus, described by de Guiran (1967) from Moroc. The description of this species agrees in general with the Spanish population having similar morphometry, although the Spanish male specimens are slightly smaller (0.65-0.66 vs 0.68-0.75 mm), slightly shorter stylet (17-18 vs 18-20 µm in females and 18 vs 19-20 µm in males), wider female tail terminus with finely rounded tip (vs narrower with slightly truncated tip) and slightly shorter spicules (21-22 vs 22-25 µm). Volvas and Cham (1981) provided SEM illustrations of specimens of this species found in sand dunes of Italy, which agree well with the Spanish specimens.
With respect to the 18S rDNA sequence obtained, this species agrees with other sequence of T. aduncus available in GenBank (KJ461602) and published by Handoo et al. (2014), having 99.5% similarity. Respect to the 28S rDNA sequence, the Spanish material of T. aduncus shows more difference with other sequences of this species (KJ461530, KJ461531) available in GenBank, having 95.4% similarity.

Discussion
The molecular analyses based on 18S (Fig. 7) and 28S (Fig. 8) rDNA fragments of the species studied and their relatives confirm the phylogenetic position of these taxa, each species belonging to one different superfamily of the five ones considered valid by De Blaxter (2002, 2004) based on molecular data [vs the superfamilies proposed by Siddiqi (2000) and Andrássy (2007) based on morphological data]. These three species, Aphelenchus avenae, Ditylenchus myceliophagus and Tylenchorhynchus aduncus are clearly related with other species of their respective genera. With respect to the obtained phylogenetic trees, these superfamilies are best supported in the 28S tree, while in the 18S tree the arrangement of these superfamilies are not clear, especially the superfamily Tylenchoidea, which appears polyphyletic in both 18S and 28S, the latter agreeing with the classification proposed by Andrássy (2007), or paraphyletic with respect to the superfamily Criconematoidea; also the superfamily Sphaerularioidea appears polyphyletic in the 18S, as proposed by Siddiqi (2000).
Regarding to the three genera studied, the genera Aphelenchus and Tylenchorhynchus are showed monophyletic, while the genus Ditylenchus appears monophyletic in the 18S tree while it is showed paraphyletic in the 28S tree with respect to the genera Anguina Scopoli, 1777 andSubanguina Paramonov, 1967. Additionally, after the morphological analysis, we observed that two species recently described belonging to the superfamily Aphelenchoidea, Aphelenchus assamensis described by Bina Chanu et al. (2016) and Aphelenchoides dhanachandi described by Bina Chanu et al. (2012) do not agree with the genera proposed by their authors. Thus, A. assamensis, characterized by having two incisures at lateral fields, male tail without bursa, spicules anteriorly wide (manubrium and ventral rostrum), gubernaculum absent (something similar to a gubernaculum was drew in the line figure but this is absent in the LM figure), and female tail short conoid, which agrees with the morphology of the genus Aphelenchoides, being transferred to this genus as A. assamensis (Bina Chanu et al. 2016) n. comb., not agreeing with other species of the genus by the presence of females with short conoid tail with rounded tip and males with conoid tail with fine elongate mucro. On the other hand, A. dhanachandi, characterized by having lip region rounded and clearly offset, stylet long and wide lumen, metacorpus well developed with strong valves, very long pharyngeal dorsal gland and post-vulval uterine sac and female tail conoid-elongate, which agree with the diagnostic features of the genus Potensaphelenchus Gu et al. 2021, being transferred to this genus as P. dhanachandi (Bina Chanu et al. 2012) n. comb., species not agreeing with the only species of this genus by having longer female tail (63-85 vs 52-69 µm). With respect to this last species, it is also similar to the species of the genus Anomyctus Allen, 1940 although both genera can be mainly differentiated by the morphology of the female tail, conoid-elongate in Potensaphelenchus and short conoid in Anomyctus.

Fig. 7
Bayesian Inference tree from known and the newly sequenced Aphelenchus avenae and Tylenchorhynchus aduncus based on sequences of the 18S rDNA region. Bayesian posterior probabilities (%) are given for each clade. Scale bar shows the number of substitutions per site Acknowledgements The authors thank the University of Jaén, Spain, for financial support received for the Research Support Plans "PAIUJA 2019/2020: EI_RNM02_2019" and "PAIUJA 2021/2022: EI_RNM02_2021". SEM pictures were obtained with the assistance of technical staff (Amparo Martínez-Morales) and equipment of the "Centro de Instrumentación Científico-Técnica (CICT)" at the University of Jaén.
Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature.

Declarations
Conflict of interest All the authors declare that they have no conflict of interest.
Human and animal rights This article does not contain any studies with human participants or animals performed by any of the authors.
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