This paper presents the results of a combined analysis of 300 specimens of the genus Cladonia collected in the southwest of province of Pontevedra. The species were classified based on to morphological, chemical (TLC) and genetic (ITS rDNA) characters. A total of 28 species were identified, including is the first record for the Iberian Peninsula of Cladonia portentosa, which contains fumarprotocetraric, perlatolic and usnic acids, as well as Cladonia homosekikaica, new for the Galician flora and Cladonia ciliata, a new record for the province of Pontevedra. It is important to note that morphological characteristics, chemistry and DNA sequences are essential in the taxonomy of the genus.
Resumen
En este trabajo se presentan los resultados de un análisis combinado de 300 ejemplares del género Cladonia recolectados en el suroeste de la provincia de Pontevedra. Las especies se clasificaron en base a caracteres morfológicos, químicos (TLC) y genéticos (ITS rDNA). Se identificaron un total de 28 especies, incluyendo el primer registro para la Península Ibérica de Cladonia portentosa, que contiene ácidos fumarprotocetrárico, perlatólico y úsnico, así como Cladonia homosekikaica, nueva para la flora gallega y Cladonia ciliata, un nuevo registro para la provincia de Pontevedra. Es importante señalar que las características morfológicas, químicas y las secuencias de ADN son esenciales en la taxonomía del género.
Cladonia P. Browne is the genus with the highest number of species in the Cladoniaceae family (STENROOS et al. 2002). Identifying species within this genus is significantly difficult, as morphology varies notably during different stages of development and depending on environmental conditions. A wide variety of lichen substances have been identified in representatives of this genus, many can only be recognized through thin-layer chromatography (TLC) techniques. Although the identification of metabolites can be helpful, doubts and controversies regarding taxonomic status may still arise. Genetic tools, particularly in complex genera such as Cladonia, have significantly advanced the field. Molecular phylogenies were first attempted in the late 20th century, with STENROOS et al. (2002, 2019) conducting the most extensive works on the genus to date. It is important to note that relying solely on the ITS region for taxonomic identification within this group may not always be effective. This is because species within the same clade may have very similar ITS regions, which can lead to misidentifications (STENROOS et al., 2019). Therefore, it should be complemented by morphological and chemical studies or additional genetic markers should be used.
BURGAZ & AHTI (2009) conducted a comprehensive monographic study on the genus Cladonia in the Iberian Peninsula. This was followed by a study that included all species from the Mediterranean Region (BURGAZ et al., 2020). The only previous monographic work on this genus in Galicia was carried out by VALCÁRCEL et al. (1991) in the municipality of A Fonsagrada, Lugo province. BURGAZ (2015) updated the collected material for the volume of the Iberian Lichen Flora, contributing 62 species of the genus Cladonia for Galicia and 46 for Pontevedra. According to BURGAZ (2015), the most frequent and abundant species for Galicia are Cladonia furcata, Cladonia coniocraea, Cladonia rangiformis, Cladonia portentosa, and Cladonia pyxidata.
Materials and Methods
The study collected 300 specimens from various sampling points between September 2020 and March 2022, covering habitat variability. The voucher material has been deposited in the MACB herbarium of the Faculty of Biological Sciences at Complutense University of Madrid. The symbol ** is used to highlight new citations for the province of Pontevedra, * for new citations in the Galician community, and # for new chemotypes for the Iberian Peninsula.
Specimens of Cladonia were collected from various substrates in the municipality of O Rosal (Pontevedra) for a combined study of morphological, chemical, and molecular characters.
The study area is situated in the municipality of O Rosal, south of the province of Pontevedra, near the mouth of the Miño River. It covers an area of 41.13 km2 and consists of a small urban core, with mountainous areas located in two parallel strips along the coast up to 500 m above sea level. These strips delimit the valley through which the Tamuxe River flows. Geologically, the mountainous areas are composed of alkaline granites, which are also present in the valley alongside Holocene deposits. In addition, there are metavolcanic rocks and schists that create acidic substrates (RODRÍGUEZ-GARCÍA 2004). Most of the mountainous areas are covered by plantations of Pinus pinaster, together with others that occupy a smaller area: Pinus radiata and Eucalyptus globulus. However, remnants of natural vegetation remain notably Quercus robur and Q. suber. The area is classified as a semi-hydro-oceanic bioclimate with a temperate thermoclimate according to the Rivas-Martínez climate classification. It is situated in a transition zone between the sub-Mediterranean temperate macroclimate and the typical temperate bioclimate, making it an area of special interest as it allows species from the two climates to coexist (GUITIÁN & RAMIL, 2007; REES et al., 2023). The average annual rainfall is 1,573 mm/m2, and the average annual temperature is 15.0 ºC. The average temperature of the coldest month is 9.5 ºC, whereas the average temperature of the warmest month is 21.1 ºC.
All the sampled localities belong to the municipality of O Rosal, in the province of Pontevedra.
List of sampling localities:
Alto de Campo do Couto, 41º57’32” N 8º51’59” W, 504 m, Pinus radiata and between alkaline granite soil and mosses.
Alto de Os Muíños do Folón, 41º57’48” N 8º50’18” W, 270 m, on alkaline granite soil and mosses.
Alto de Tabagón, 41º56’00” N 8º48’27” W, 117 m, Quercus robur and between schist, metavolcanic rocks, and mosses.
Campo do Couto, 41º57’14” N 8º51’33” W, 332 m, Pinus pinaster, Quercus suber, Salix atrocinerea and between alkaline granite soil and mosses.
Road to Campo do Torroso, 41º56’32” N 8º52’08” W, 165 m, Quercus robur and on alkaline granite soil.
Mirador de Tamuxe, 41º54’58” N 8º49’39” W, 5 m, Quercus robur on alluvial soil.
Muíños do Folón, 41º57’43” N 8º50’26” W, 173 m, Pinus pinaster, Quercus robur, dead wood and on alkaline granite soil and mosses.
Muíños do Picón, 41º57’36” N 8º50’07” W, 211 m, Castanea sativa, Salix atrocinerea and on alkaline granite soil and mosses.
Niño do Corvo, 41º56’55” N 8º48’40” W, 304 m, on schist and metavolcanic soil.
Petroglifos do Ghorghalado, 41º55’52” N 8º47’30” W, 80 m, on alkaline granite soil among mosses.
Playa das Eiras, 41º55’28” N 8º46’46” W, 3 m, Alnus glutinosa, Pinus pinaster and on alluvial soil.
Pozas da Pesqueira, 41º56’17” N 8º49’45” W, 15 m, Pinus pinaster and on alluvial soil.
Río Tamuxe-As Aceñas, 41º55’41” N 8º49’24” W, 17 m, Alnus glutinosa, Quercus suber and on alkaline granite soil and mosses.
Ascent to Alto do Torroso, 41º55’27” N 8º52’15” W, 338 m, Pinus pinaster, P. radiata, Quercus robur, Q. Suber and on alkaline granite soil and mosses.
Valdemiñotos, 41º56’04” N 8º49’27” W, 30 m, Pinus pinaster, Quercus robur and on soil and mosses.
Standard lichenology procedures were used to conduct morphological examination of specimens, using a Nikon SMZ445 stereomicroscope. Species identification primarily followed BURGAZ et al. (2020), with additional reference to other monographic works (BURGAZ & AHTI, 2009; PINO-BODAS et al., 2021) and general keys (CLAUZADE & ROUX, 1985; SMITH et al., 2009). Lichen substances were identified by thin-layer chromatography (TLC) techniques following the standardized method of WHITE & JAMES (1985), using solvents A, B, C and D; for substances not mentioned in this work and some doubtful cases, ORANGE et al. (2001) and SCHUMM & ELIX (2016) were used.
For genetic analysis, several specimens of each identified species were selected based on their morphological and chemical traits, ensuring representation of those with divergences. The NZY PLANT/FUNGI gDNA ISOLATION kit (NZYTech, Portugal) was used for fungal gDNA extraction following the manufacturer's instructions. The resulting DNA was stored at -20 °C until PCR amplification. The ITS region of nrDNA was analyzed for species genetic characterization using the primers ITS1F and ITS4 (PINO-BODAS et al., 2010). The amplification cycle was as follows: initial denaturation at 94 ºC for 5 min; 35 cycles of denaturation at 94 ºC for 30 s, annealing at 54 ºC for 30 s, and extension at 72 ºC for 40 s; and a final extension at 72 ºC for 7 min. Amplifications were visualized on 2% agarose gel with TBE 1X buffer and GreenSafe Premium (NZYTech, Portugal) for DNA visualization, run for 25 min at 100 V. Amplified DNA was sent to the Genomics Service of the Scientific-Technological Research Support Center (University of Vigo) for purification with ExoSap (ThermoFisher) and bidirectional sequencing using the BigDye Terminator v3.1 sequencing kit (Life Technologies) and a SeqStudio Genetic Analyzer capillary sequencer (Applied Biosystems). Sequencer files containing chromatograms were processed using the Geneious Global Alignment algorithm (Geneious, Biomatters) to generate consensus sequences. Initial identification was assigned to each sample using the Basic Local Alignment Search Tool (BLAST) nucleotide search to verify identity. BLASTn search was performed using the nucleotide collection (nr/nt). Sequences analyzed have been deposited in the GenBank genetic sequence database; the corresponding reference for each species is indicated in the database. Table 1 shows the sequences from this study alongside the species with the highest identity percentage after BLAST search, with any discrepancies noted for each species.
Summary of the species and their corresponding codes together with the result of their BLAST search (species with the highest percentage of identity with the one in this study). Comments on some of the percentages obtained are given for each species. For this study, results below 99% in most cases are considered inconclusive.
Species
Code
GenBank
Species with highest %
identity
Code
GenBank
Cladonia caespiticia
OP479892
C. caespiticia (99,54%)
OK332962
C. cervicornis
OP479903
C. cervicornis (100%)
MK179618
C. coccifera
OP479895
C. coccifera (100%)
OM914269
C. coniocraea
OP479897
C. coniocraea (100%)
MN387221
C. cryptochlorophaea
OP479898
C. cryptochlorophaea (99,67%)
MK179507
C. digitata
OP479899
C. digitata (100%)
AF453701
C. diversa
OP479900
C. diversa (99,66%)
FR799158
C. floerkeana
OP479901
C. floerkeana (98,46%)
OQ717815
C. furcata
OP479904
C. furcata (99,75%)
OL625383
C. glauca
OP479905
C. glauca (99,76%)
OQ717816
OP479906
C. glauca (98,76%)
OQ717816
C. humilis
OP479907
C. humilis (99,76%)
OL625557
OP479908
C. humilis (100%)
OL625444
C. mediterranea
OP479910
C. mediterranea (100%)
KP941521
C. merochlorophaea
OP479911
C. merochlorophaea (99,96%)
OQ717818
C. portentosa
OP479913
C. portentosa (99,83%)
FR799168
OP479914
C. portentosa (100%)
KP941519
OP479922
C. portentosa (100%)
FR799167
OP479923
C. portentosa (99,83%)
KP941499
OP479924
C. portentosa (100%)
FR941519
C. pyxidata
OP479915
C. pyxidata (99,75%)
OL625502
OP479925
C. pyxidata (100%)
OL625502
C. ramulosa
OP479916
C. ramulosa (100%)
OK332964
OP479927
C. ramulosa (99,67%)
OK332964
C. rangiformis
OP479917
C. rangiformis (99,87%)
OL625552
C. subcervicornis
OP479918
C. subcervicornis (99,68%)
MK812661
C. subulata
OP479919
C. subulata 4 (99,65%)
FN86857
C. uncialis subsp. biuncialis
OP479921
C. uncialis subsp. biuncialis
(99,47%)
KR019377
Results and Discussion
Of the 300 collected specimens, a total of 28 species of Cladonia were identified. The numbers of the locations correspond to the sampling localities list followed by the herbarium number where the specimens are deposited.
Despite significant human intervention, the study area displays a rich diversity of lichens (FERNÁNDEZ-COSTAS & GARCÍA-MOLARES, 2023). The surveyed area includes 28 species of the 62 Cladonia species cited for Galicia (BURGAZ et al., 2020). Notably, the chemotype of Cladonia portentosa, containing fumarprotocetraric, perlatolic and usnic acids, is recorded for the first time in the Iberian Peninsula. Additionally, C. homosekikaica is a new addition to the Galician flora, and Cladonia ciliata is a new record for the Pontevedra province. The locality with the highest diversity is 'Petroglifos do Ghorghalado' (sample point 10), which is linked to areas with minimal human interference and a high level of moisture.
Our specimens correspond to the chemotype: Pd+ (red), K–, C–, KC–, UV–, containing fumarprotocetraric acid. The specimens were found growing on soil among mosses. It is found in temperate and warm regions of the Northern Hemisphere (GBIF, 2022). The species is widely distributed in the Eurosiberian Region, and has also been observed in other areas of the northern Mediterranean countries, such as southern France, northern Italy, Slovenia, and some isolated locations in northern Africa. In the Iberian Peninsula, it is mainly found in the northernmost areas (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020).
(Fig. 1c) The specimens are: Pd+ (red), K–, C–, KC–, UV–, containing fumarprotocetraric acid. Only two specimens were found on mossy rocks and soil. The species is not very abundant in the study area. It has a cosmopolitan distribution, although it is mainly concentrated in the Northern Hemisphere (GBIF, 2022). In Europe, it is widely distributed, with a higher number of records in western and southern Mediterranean territories. In the Iberian Peninsula, it is also widely spread (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020).
<italic>Cladonia chlorophaea</italic> (Flörke ex Sommerf.) Spreng.
The species is easily confused with other closely related members, such as C. cryptochlorophaea Asah., C. merochlorophaea Asah., C. grayi G. Merr. ex Sandst., C. novochlorophaea (Sipman) Brodo & Ahti, and C. homosekikaica Nuno, due to many morphological similarities. Our specimens match the chemotype: Pd+ (red), K–, C–, KC–, UV–, containing fumarprotocetraric acid. Two specimens were found, on Alnus glutinosa and on Pinus radiata. This species has a subcosmopolitan distribution (GBIF, 2022). It is widely distributed throughout the Mediterranean Region (BURGAZ et al., 2020), with numerous records known from the Iberian Peninsula. However, its presence may be overestimated due to its similarity to other species mentioned above.
Our specimens are compatible with the f. flavicans. The specimens belong to the chemotype: Pd+ (red), K–, C–, KC–, UV–, containing usnic and fumarprotocetraric acids. Several specimens were found directly on mossy soil. The species is distributed throughout the Northern Hemisphere. In Europe, it can be found at practically all latitudes (GBIF, 2022), f. flavicans is more abundant in the Mediterranean Region, where it occupies moister and warmer habitats than f. ciliata (BURGAZ et al., 2020). In the Iberian Peninsula, most records are concentrated in the northern and western regions (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020). This is the first record in Pontevedra province, although it has been cited in Lugo and A Coruña (BURGAZ, 2015).
The specimens are: Pd–, K–, C–, KC+ (yellow), UV–, containing zeorin, usnic and rhodocladonic acids. Cladonia diversa, with very similar chemistry, is distinguished by its densely squamulose-covered podetia. Several specimens were found growing on mossy soil. It is a subcosmopolitan species (GBIF, 2022), common up to the subalpine level of the Eurosiberian Region, while in the Mediterranean Region generally occurs at higher altitudes (BURGAZ et al., 2020). In the Iberian Peninsula, most records are concentrated in the northern third (BURGAZ & AHTI, 2009).
(Fig. 1g) All the specimens are Pd+ (red), K–, C–, KC–, UV–, containing fumarprotocetraric acid. In our case, ochrochlora morphology was observed on the ground and infrequently as an epiphyte, whereas specimens matching the coniocraea morphology were the most abundant, occurring on bark. of different trees (Quercus robur, Q. suber, Pinus pinaster and P. radiata) and occasionally directly on soil. The species has a wide distribution in the Northern Hemisphere, as reported by GBIF (2022). In Europe, it is mainly found in the Eurosiberian Region, but it is also widely distributed in various locations of the Mediterranean Region (BURGAZ et al., 2020). In the Iberian Peninsula, it has been reported several times, but most of the records are concentrated in the north (LLIMONA & HLADUN, 2001; BURGAZ & AHTI, 2009).
Sampling collections: C. coniocraea s. str.: 1 (MACB 117661), 6 (MACB 117660), 7 (MACB 117662, 117663), 10 (MACB 117658, 117659), 15 (MACB 117664). C. coniocraea morphology C. ochrochlora: 10 (MACB 117747), 13 (MACB 117748), 15 (MACB 117749). GenBank accession number: OP479897 for MACB 117662.
<italic>Cladonia cornuta</italic> (L.) Hoffm.
(Fig. 1a). Our specimen is: Pd+ (red), K–, C–, KC–, UV–, containing fumarprotocetraric acid. A single specimen was found growing on soil. The occurrence of this species in the area is remarkable as it is common at higher altitudes (BURGAZ et al., 2020). The molecular taxonomy techniques used to analyse the sequences resulted in a finding that is consistent with C. gracilis (L.) Willd., a species that has been found to be closely related in previous studies (FONTAINE et al., 2010; PINO-BODAS et al., 2012). However, the presence of soredia limited to the upper half of the podetia allows for its clear differentiation from the latter species. The taxon is subcosmopolitan, and more commonly found in cold regions of the Northern Hemisphere (GBIF, 2022). It is also abundant in the Eurosiberian Region and present in some mountainous locations of the Mediterranean Region. In the Iberian Peninsula, the records are concentrated in the northwestern quadrant (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020). It is reported from Ourense (CESPO-PARDO, 2016) and TEIXIDOR (1869) reported this species in Pontevedra.
Our specimens are: Pd+ (orange), K+ (ochre), C± (weak yellow, almost imperceptible), KC+ (wine red), UV+ (pale yellow), containing cryptochloropheaic and fumarprotocetraric acids. Several specimens grow on soil and trunks of Quercus robur, Pinus pinaster and P. radiata. It is the most common species in the study area. The species is mainly distributed in the Eurosiberian Region, but it can also be found in the territories of the Mediterranean Region. It has been reported in various locations in the northern and western areas of the Iberian Peninsula (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020).
<italic>Cladonia cyathomorpha</italic> Stirt. ex Walt. Watson
(Fig. 1b). Our specimen is: Pd+ (red), K–, C–, KC–, UV–, containing fumarprotocetraric and protocetraric acids. It is a rare species in this area. It grows on soil among mosses and on Pinus radiata, in the highest altitude of the municipality (527 m). C. cyathomorpha has a restricted distribution in Western Europe, reaching as far as the Canary Islands. It is probable that it has been disregarded or mistaken for other species within its group. Conversely, it has a broad distribution across the Iberian Peninsula (BURGAZ et al., 2020).
Sampling collections: 1 (MACB 117789, 117790).
<italic>Cladonia digitata</italic> (L.) Hoffm.
The specimen belongs to the chemotype: Pd+ (yellow), K+ (yellow), C–, KC–, UV–, containing thamnolic and rhodocladonic acids. Several specimens were found growing directly on soil, among mosses. The species is distributed across vast territories of the Northern Hemisphere (GBIF, 2022). Its presence is considered a good indicator of forest maturity (PINO-BODAS et al., 2021). In the Iberian Peninsula, records are limited to the northernmost zone (BURGAZ et al., 2020).
<italic>Cladonia diversa</italic> Asperges ex S. Stenroos
Our specimens are: Pd–, K–, C–, KC+ (light yellow), UV–, containing zeorin and usnic acid. It is frequent and was located at different points of the area, developed on soil and rocks. The species has a wide distribution across Europe, Asia, North America and Macaronesia, although its exact distribution is not well-known (AHTI & STENROOS, 2013). In the Iberian Peninsula, the majority of records are concentrated in the northwestern quadrant (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020). STEINOVÁ et al. (2013) have identified it as a polyphyletic taxon.
Our specimen is: Pd+ (reddish-orange), K–, C–, KC–, UV–, containing fumarprotocetraric acid. A single specimen was located growing on soil. The species is cosmopolitan (AHTI & STENROOS, 2013), and is abundant in the Mediterranean Region. It has been widely cited in the Iberian Peninsula (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020).
Sampling collection: 7 (MACB 117680).
<italic>Cladonia floerkeana</italic> (Fr.) Flörke
The specimens are: Pd+ (yellow), K+ (yellow), C–, KC–, UV–, containing barbatic and thamnolic acids, which does not fit with what it is indicated by BURGAZ et al. (2020), although PINO-BODAS et al. (2021) also consider this chemotype. Several specimens were found growing on moss debris on soil and bark of Quercus robur. This species has a subcosmopolitan distribution (GBIF, 2022) and is commonly found in oceanic areas (AHTI & STENROOS, 2013). In the Iberian Peninsula, it is mostly recorded in the northwestern quadrant (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020).
Two chemotypes were found (fumarprotocetraric ± bourgeanic acids) both of them : Pd+ (red), K–, C–, KC–, UV–,. It is abundant in the study area. It was found on soil and occasionally among mosses, as well as on the bark of Salix atrocinerea. This is a subcosmopolitan species (AHTI & STENROOS, 2013; GBIF, 2022) spread across all latitudes and also very common in much of the Mediterranean Region and the Iberian Peninsula (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020).
Our specimens belong to chemotype: Pd–, K–, C–, KC–, UV+ (white), containing squamatic acid, and chemotype: Pd+ (yellow), K+ (yellow), KC±, (yellow), UV–, containing thamnolic and traces of barbatic acids. At once squamatic and thamnolic acids was found in some specimens. It is a very abundant species in some locations of the area of study. It grows on rocks, on soil, usually surrounded by mosses, although it was also found on Pinus radiata bark. According to GBIF (2022), it is mainly distributed in the Northern Hemisphere, particularly in areas with an oceanic tendency (AHTI & STENROOS, 2013). Previous studies have reported its presence in various locations in the western half of the Iberian Peninsula (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020).
(Fig. 1d) Small squamules at the base of the podetia are frequently present. Our specimens belong to the chemotype: Pd–, K–, C–, KC–, UV+ (bluish-white), containing homosekikaic and sekikaic acids. It was found on soil, among mosses and on Pinus radiata bark in the highest altitude locality of the municipality (Alto de Campo do Couto). It is an uncommon species in the Northern Hemisphere and has also been observed in some parts of Australia (GBIF, 2022). In the Iberian Peninsula, it has been found in scattered localities in the northern half (BURGAZ et al., 2020). Our report represents the first record for Galicia.
(Fig. 1e-f) The specimens belong to the chemotype: Pd+ (red), K+ (yellow), C–, KC–, UV–, with atranorin and fumarprotocetraric acid. Several specimens were found growing directly on soil and on various substrates (Alnus glutinosa, Castanea sativa, Pinus radiata, Quercus robur, and Salix atrocinerea), always among mosses. C. humilis has a subcosmopolitan distribution, but it is more widespread in temperate zones (AHTI & STENROOS, 2013; GBIF, 2022). There are numerous records of the species from the Iberian Peninsula (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020).
Figure 1. A, Cladonia cornuta; B, C. cyathomorpha; C, C. cervicornis; D, C. homosekikaica; E-F, C. humilis; G, C. ochrochlora; H-I, C. portentosa. Scales: 5 mm.
<italic>Cladonia macilenta</italic> Hoffm.
Our specimens are: Pd+ (yellow), K+ (yellow-orange), C–, KC–, UV–, containing barbatic, thamnolic and didymic acids. In some specimens, only thamnolic acid has been detected. It is a very abundant species in the area. It grows on soil surrounded by mosses, on stumps and on bark of Pinus pinaster and P. radiata. The species has a wide distribution in both hemispheres, as reported by GBIF (2022). It is found in hills and mountainous zones of the Eurosiberian Region, as well as wet areas of the Mediterranean Region (BURGAZ et al., 2020). The majority of records from the Iberian Peninsula are concentrated in the north and northwestern quadrant (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020).
Our specimen is: Pd–, K–, C–, KC+ (yellow), UV+ (light blue), containing usnic and perlatolic acids. It grows on soil and among mosses. The species is frequently found in humid and warm territories of the hills and montane levels of the Eurosiberian Region. In the Mediterranean basin, its distribution is restricted to pine forests, cork oak groves, and coastal shrub areas (BURGAZ et al., 2020). Its population is currently declining due to the progressive alteration of its habitat. There are numerous records of this species from all over Portugal, the Balearic Islands, Catalonia, and some isolated localities in Galicia (LLIMONA & HLADUN, 2001; CRESPO-PARDO, 2016; BURGAZ et al., 2020).
Our specimens are: Pd+ (orange-red), K–, C+ (reddish), KC+ (wine red), UV+ (pale blue), containing meroclorophaeic and fumarprotocetraric acids. It was found growing on soil and bases of Quercus robur, Q. suber and Pinus pinaster trunks. The taxon is subcosmopolitan, ranging from the Arctic to the temperate regions of the Northern Hemisphere, as well as scattered territories in Australasia and South America (GBIF, 2022). It has also been reported in various locations in the Iberian Peninsula and other countries in the Mediterranean basin, although its presence is likely underestimated due to identification difficulties (BURGAZ et al., 2020).
(Fig. 1h-i) This species is chemically variable. We have detected three chemotypes: 1) Pd–, K–, C–, KC± (pale yellow), UV+ (light blue), containing usnic and perlatolic acids; 2) Pd–, K–, C–, KC± (pale yellow), UV+ (light blue), containing only perlatolic acid; and 3) Pd+ (red), K–, C–, KC± (pale yellow), UV+ (light blue), containing fumarprotocetraric, perlatolic and usnic acids. The first chemotype is more prevalent in the northern half of the Iberian Peninsula, while the second chemotype is limited to a few locations in the western Iberian Peninsula (Galicia, Portugal, Salamanca), and Navarra (BURGAZ & ATHI, 2009). The third chemotype was previously considered a distinct species, Cladonia azorica Ahti, with a Macaronesian distribution. However, PINO-BODAS et al. (2016) concluded that it belongs to C. portentosa. It is common in the study area, growing on soil accompanied by mosses.
The species has a subcosmopolitan distribution, although it is mainly found in the Northern Hemisphere (GBIF, 2022). In Europe, it is more frequently found in the Eurosiberian Region, while in the Mediterranean Region, it is found at higher altitudes (BURGAZ et al., 2020). There are numerous records of this species in northern Spain and Portugal (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020). The third chemotype is the first record for the Iberian Peninsula and the second for the European continent, as it has also been found in the southwest of France (BURGAZ et al., 2020).
Sampling collections: 1 (MACB 11755), 10 (MACB 117750, 117751, 117752, 117753), 4 (MACB 117754), 7 (MACB 117756), 8 (MACB 117757), 9 (MACB 117760), 15 (MACB 117759). GenBank accession numbers: OP479913 for MACB 117759, OP479914 for MACB 117750, OP479922 for MACB 117751, OP479923 for MACB 117757, OP479924 for MACB 117756.
<italic>Cladonia pyxidata</italic> (L.) Hoffm.
Our specimens are: Pd+ (red), K–, C–, KC–, UV–, containing fumarprotocetraric acid. The character to differentiate C. pyxidata from other related taxa is the presence of large, unclustered, corticated podecia, which gradually open to form greenish-grey scyphi; these are covered with microscuamules and flat, peeled plates on the upper part and especially on the inside of the scyphi. Nevertheless, species is highly polymorphic and can be mistaken for both C. chlorophaea and some specimens of C. humilis. Molecular studies have revealed that it is a polyphyletic taxon (STENROOS et al., 2019), indicating the need for further revision. It was collected on soil and rocks growing in association with mosses. The species has a cosmopolitan distribution, as reported by GBIF in 2022, and has frequently cited in the Iberian Peninsula, according to LLIMONA & HLADUN (2001) and BURGAZ et al. (2020).
The specimens are: Pd + (red), K–, C–, KC–, UV–, containing fumarprotocetraric acid. This species is commonly found in the area and can be found on various substrates such as soil, mosses, rocky substrates, and trunks of Quercus suber and Pinus radiata. It has a cosmopolitan distribution, ranging from boreal regions to temperate zones (AHTI & STENROOS, 2013), and is frequently found in the Mediterranean Region. There are numerous records of this species in the Iberian Peninsula (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020).
The specimens are: Pd–, K+ (yellow), C–, KC–, UV–, containing atranorin and rangiformic acid. It is one of the most abundant species in the area. It grows on soil associated with mosses. C. rangiformis has a wide distribution in the Northern Hemisphere, ranging from the Arctic tundra to the Mediterranean Region and Macaronesia (GBIF, 2022). There are numerous records of this species in the Iberian Peninsula (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020).
The specimens collected are: Pd–, K–, C–, KC–, UV+ (white), containing squamatic acid. Molecular phylogenetic studies suggest that it is a polyphyletic taxon (STENROOS et al., 2019). It is relatively abundant in some areas, growing directly on soil and surrounded by mosses, although it has also been found on Pinus radiata. Its distribution is subcosmopolitan (GBIF, 2022), and it prefers areas with an oceanic or suboceanic tendency (AHTI & STENROOS, 2013). The species has been reported in numerous localities throughout the Iberian Peninsula, although it appears to be more abundant in the Eurosiberian Region (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020).
Our specimens are: Pd+ (red), K+ (yellow), C–, KC–, UV–, containing fumarprotocetraric acid and atranorin. It grows on rocks usually accompanied by mosses. The species is distributed in western regions of Europe, although there is evidence of its presence in some localities near the Mediterranean coast. Most of the records are concentrated in the western third of the Iberian Peninsula, as reported by BURGAZ et al. (2020).
The only specimen collected is: Pd+ (red), K–, C–, KC–, UV–, containing fumarprotocetraric acid. It was growing on soil among mosses. C. subulata has a subcosmopolitan distribution (GBIF, 2022) and is prevalent in continental areas of the Mediterranean Region. In the Iberian Peninsula, records are concentrated in the western half (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020).
<italic>Cladonia uncialis</italic> subsp. <italic>biuncialis</italic> (Hoffm.) M. Choisy
Our specimen is: Pd–, K–, C–, KC+ (yellow), medulla UV+ (white), containing usnic and squamatic acids. It is very rare in the area. Only one specimen was found growing on soil and surrounded by mosses. The distribution area of this species covers the European continent and isolated locations in Asia and the eastern coast of North America (GBIF, 2022). In the Mediterranean Region, it is more common than subsp. uncialis (BURGAZ et al., 2020). Records in the Iberian Peninsula are concentrated in the northwestern quadrant (LLIMONA & HLADUN, 2001; BURGAZ et al., 2020).
The following key is presented to identify the Cladonia species in O Rosal:
1a.
Primary thallus developed, secondary thallus absent or very poorly developed
2
1b.
Primary thallus under developed or absent, secondary thallus with dominant
development or equal development of the two parts of the thallus
4
2a.
(1a) Squamules small (<1cm), fragile, with irregular edges
C. caespiticia
2b.
Squamules bigger, not fragile
3
3a.
(2b) Squamules not pruinose, brownish to greyish blue below, occasionally with
some black rizines on the sides of the squamules, usually K–
C. cervicornis
3b.
Squamules elongate (6-10 mm), erect, base at lower side blackened, K+ yellow
to red
C. subcervicornis
4a.
(1b) Primary thallus absent or very reduced, podetia developed, narrow,
generally branched, ascyphose
5
4b.
Primary and secondary thallus present, podetia scyphose or not, simple or little
branched
21
5a.
(4a) Podetia without cortex, surface arachnoid (use hand lens!), without soredia,
richly branched
6
5b.
Podetia with cortex, smooth, sorediate or squamulose, simple, little branched
or richly branched
9
6a.
(5a) Podetia Pd+ red and K–
7
6b.
Podetia Pd–
8
7a.
(6a) Podetia greyish, with apical ramifications dichotomous, deflexed in several
directions
C. ciliata
7b.
Podetia with ultimate branchlets tri-, tetra- or pentachotomous, generally erect
or very little deflexed
C. portentosa
8a.
(6b) Podetial surface with continuous algal layer, thallus forming characteristic
subglobose heads, podetia with ultimate branchlets uniformly dichotomous
C. mediterranea
8b.
Podetial surface without continuous algal layer, thallus not forming
characteristic subglobose heads, podetia with ultimate branchlets tri-, tetra- or
pentachotomous, somewhat curved and podetia with curly appearance in
general
C. portentosa
9a.
(5b) Podetia yellowish
C. uncalis subsp. biuncialis
9b.
Podetia not yellowish
10
10a.
(9b) Podetia corticate over the entire surface, with few squamules, not
sorediate
11
10b.
Podetia corticate at the base or in the lower half, with numerous squamules or
with soredia
13
11a.
(10a) Podetia Pd–, with algal layer discontinuous, richly branched, sometimes
with squamules, tips closed, UV–
C. rangiformis
11b.
Podetia Pd+ red or Pd+ yellow
12
12a.
(11b) Podetia K–, with uniform colour, with continuous algal layer
C. furcata
12b.
Podetia K+ yellow, usually appearing pale or variegated in part, areolate, with
discontinuous algal layer
C. rangiformis
13a.
(10b) Podetia squamulose, with a variable number of squamules, but
distributed throughout the podetia
14
13b.
Podetia with squamules only in the lower half, or without squamules or
sorediate
16
14a.
(13a) Podetia Pd–, UV+ white
C. squamosa
14b.
Podetia Pd+ red or Pd+ yellow, UV–
15
15a.
(14b) Pd+ yellow
C. squamosa
15b.
Pd+ red, podetial squamules > 1 mm long, podetial tips always dichotomously
branched, axils generally open
C. furcata
16a.
(13b) Podetia Pd+ red, Pd+ yellow or yellow later red
17
16b.
Podetia Pd-
21
17a.
(16a) Podetia Pd+ yellow, with red apothecia
20
17b.
Podetia Pd–
18
18a.
(17b) Podetia simple or dichotomously branched, or forming scyphi 1-3 mm
wide, frequently with long marginal subulate proliferation
C. subulata
18b.
Podetia unbranched and subulate, sometimes with narrow scyphi (< 1mm),
without proliferations
19
19a.
(18b) Podetia not corticate or cortex restricted to podetial base, never in scyphi
C. coniocraea
19b.
Podetia clearly corticate at the base and cortical plates often extending
throughout, also to the scyphus
C. coniocraea morph ochrochlora
20a.
(17a) Podetia finely sorediate, with whitish appearance
C. macilenta
20b.
Podetia not finely sorediate, usually granular, without whitish appearance
C. floerkeana
21a.
(16b) Podetia without red apothecia, with a lateral groove that looks like a
depression, surface sorediate
C. glauca
21b.
Podetia with red apothecia
22
22a.
(21b) Podetia finely sorediate, whitish
C. macilenta
22b.
Podetia not finely sorediate, granulose, greyish brown
C. floerkeana
23a.
(4b) Podetia not widened in scyphi, often very narrow
24
23b.
Podetia widened in scyphi
32
24a.
(23a) Podetia squamulose
C. squamosa
24b.
Podetia not squamulose, sorediate or granulose
25
25a.
(24b) Apothecia red
26
25b.
Apothecia of different colour or without apothecia
28
26a.
(25a) Primary thallus with small squamules (1-2 mm long)
27
26b.
Primary thallus with large squamules (3-12 mm long), squamules rounded,
with shallow lobulations, margin finely sorediate
C. digitata
27a.
(26a) Podetia finely sorediate
C. macilenta
27b.
Podetia not finely sorediate, granulose
C. floerkeana
28a.
(25b) Podetia sorediate
29
28b.
Podetia corticate, not sorediate
C. ramulosa
29a.
(28a) Podetia only sorediate in the upper half
C. cornuta
29b.
Podetia sorediate almost throughout
30
30a.
(29b) Podetia unbranched, tips attenuate
C. coniocraea
30b.
Podetia somewhat branched, tips slightly widened or not
Podetia green to grey, unbranched, occasionally narrowly scyphose, corticate
at base and often in patches higher up
C. coniocraea morph ochrochlora
32a.
(23b) Podetia corticate throughout the surface, not sorediate
33
32b.
Podetia sorediate or squamulose, sometimes corticate in the lower half
37
33a.
(32a) Podetia Pd–
C. merochlorophaea
33b.
Podetia Pd+ red
34
34a.
(33b) Podetia without proliferation
35
34b.
Podetia with central or lateral proliferations
36
35a.
(34a) Podetia K+ yellow, primary thallus with white underside and base
blackening
C. subcervivornis
35b.
Podetia K– and C+ weakly red
C. merochlorophaea
36a.
(34b) Scyphi with central proliferations with one to several podetia proliferating
centrally from scyphi, with 1-4 tiers of scyphi, podetia short (7-19 mm)
C. cervicornis
36b.
Scyphi with lateral proliferations, podetial base not much darkened, surface
not arachnoid
C. ramulosa
37a.
(32b) Apothecia red
38
37b.
Apothecia of different colour, or without apothecia
40
38a.
(37a) Secondary thallus more developed than primary thallus
39
38b.
Primary thallus more visible than secondary thallus or equal
C. digitata
39a.
(38a) Podetia corticate, sometimes areolate, with numerous vegetative
propagules
C. coccifera
39b.
Podetia somewhat branched, tips slightly widened or not
C. diversa
40a.
(37b) Podetia Pd–
41
40b.
Podetia Pd+ red or Pd+ yellow
42
41a.
(40a) Podetia not corticate, sorediate or covered with squamules, without real
scyphi and axils perforated
C. squamosa
41b.
Podetia corticate, if sorediate only at the upper part, without verruculose
cortex, sorediate at the upper part
C. homosekikaica
42a.
(29b) Podetia not sorediate, covered by numerous vegetative propagules
like plates
43
42b.
Podetia sorediate, without vegetative propagules like plates
45
43a.
(42a) Squamules of the primary thallus large, veiny below, or even partially
corticate, sometimes margin sorediate
C. cyathomorpha
43b.
Squamules of the primary thallus never partially corticate
44
44a.
(43b) Podetia sorediate or granulate in the margin and inside
the scyphi
C. humilis
44b.
Podetia without soredia, with flat vegetative propagules, sometimes with
squamules
C. pyxidata
45a.
Squamules of primary thallus big, rounded or elongate
46
45b.
Squamules of primary thallus small (< 3 mm)
47
46a.
(45a) Podetia with very short stalks, covered with farinose soredia, generally
corticate in the lower half
C. humilis
46b.
Podetia with longer stalks, covered with granulose soredia, with granulose
soredia, squamules of the primary thallus with many schizidia in the margin,
underside with well veins marked
C. cyathomorpha
47a.
(45b) Podetia with farinose soredia (30-50) μm diameter
C. fimbriata
47b.
Podetia with larger granular soredia (usually > 80 μm diameter)
48
48a.
(47b) Podetia with rugose cortex, with thick rounded granules on the upper
part, UV– or UV+ yellowish
49
48b.
Podetia without verruculose cortex, sorediate on the upper part, UV+ white
C. homosekikaica
49a.
(29b) Podetia UV+ yellowish
C. cryptochlorophaea
49b.
Podetia UV– yellowish, with granular soredia (60-170 μm diameter), pale
greenish brown, without rangiformic acid
C. chlorophaea
Acknowledgments
We thank the council of O Rosal, particularly the Department of the Environment, for granting us permission to conduct this study and for their prompt and courteous assistance throughout.
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