Introduction
Lichens reproduce by vegetative dispersal of both symbionts together and/or re-establish by fungal spores (ascospores and/or conidia) that disperse aposymbiotically and subsequently find a compatible photobiont (e.g. Sanders & Lücking Reference Sanders and Lücking2002). Sanders (Reference Sanders2014) showed that in Calopadia puiggari macroconidia and photobiont cells were co-dispersed but that upon germination the fungus might grow away from the algal cells leaving them to form a free-living population. Apart from important but limited field observations, knowledge of lichen-forming fungal reproductive biology is still scarce for two reasons: their inability to successfully execute the complete reproductive cycle under in vitro conditions and the difficulty in tracing the reproductive propagules of most lichen species in nature (Schuster et al. Reference Schuster, Ott and Jahns1985; Ott Reference Ott1987; Sanders Reference Sanders2014; Morando et al. Reference Morando, Favero-Longo, Carrer, Matteucci, Nascimbene, Sandrone, Appolonia and Piervittori2017). However, traceability of lichen spores could now be much easier thanks to the increased sensitivity of new methods developed by Eaton et al. (Reference Eaton, Zúñiga, Czyzewski, Ellis, Genney, Haydon, Mirzai and Yahr2018) combining aerobiological traps with nested PCR protocols.
The aposymbiotic culture of lichens and their symbionts using sexual and asexual propagules has led to a new understanding of the biology of these organisms, including the genetic variability of populations (Dal Grande et al. Reference Dal Grande, Alors, Divakar, Bálint, Crespo and Schmitt2014; Degtjarenko et al. Reference Degtjarenko, Marmor, Torra, Lerch, Saag, Randlane and Scheidegger2016) and horizontal gene transfers (e.g. McDonald et al. Reference McDonald, Dietrich and Lutzoni2012; Beck et al. Reference Beck, Divakar, Zhang, Molina and Struwe2015). Similarly, Molina et al. (Reference Molina, Divakar and González2015) were able to obtain valuable direct information about sexual reproduction such as spore discharge (productivity), germination and ontogenetic development before association with an algal partner (the sexual aposymbiotic phase of the reproduction cycle). Molina et al. (Reference Molina, Stocker-Wörgötter, Türk and Vicente1997) suggested a co-relationship between sexual maturation and size of apothecia on the basis of studies on aposymbiotic cultures. Ascertaining a relationship (should there be one) between size of apothecium and productivity and germination of spores, could provide baseline information for research on reproductive biology in these organisms.
Öckinger et al. (Reference Öckinger, Niklasson and Nilsson2005) demonstrated a relationship between propagule dispersion capacity and the distribution of Lobaria pulmonaria (L.) Hoffm., a relationship that is modulated by environmental factors. Furthermore, Tibell (Reference Tibell1994) noted that species with small spores have a wider global distribution than those with larger spores. Johansson et al. (Reference Johansson, Rydin and Thor2007) established a relationship between the age of trees and the size of spores of epiphytic lichens. Morando et al. (Reference Morando, Favero-Longo, Carrer, Matteucci, Nascimbene, Sandrone, Appolonia and Piervittori2017) investigated the relationship between size and number of pores and spatial structure; they noted that discharge of fewer and larger spores could result in spore aggregation and reduced dispersion. As far as we know, no hypotheses in lichen-forming fungi have been proposed for the relationship between size and number of spores and germination success. However, in plants, relationships between seed size and number of seeds have been proposed to explain the ecological adaptative advantages of two different strategies: 1) having many small seeds; 2) having a small number of large seeds. In general, plants producing large seeds have a competitive advantage over those producing smaller seeds because of their higher germination rates and greater nutrient reserves (Easton & Kleindorfer Reference Easton and Kleindorfer2008a). Nevertheless, small-seeded species also have great success, perhaps due to their dispersive advantage.
Parmelina carporrhizans (Taylor) Poelt & Vêzda and P. quercina (Willd.) Hale were chosen as model organisms for in vitro culture studies. They are phenotypically similar but genetically different so that they are two different species and are phylogenetically unrelated (see e.g. Argüello et al. Reference Argüello, del Prado, Cubas and Crespo2007; Núñez-Zapata et al. Reference Núñez-Zapata, Divakar, Del-Prado, Cubas, Hawksworth and Crespo2011, Reference Núñez-Zapata, Alors, Cubas, Divakar, Leavitt, Lumbsch and Crespo2017). They are foliose epiphytic lichens that reproduce only sexually, and both are heterothallic (Honegger & Zippler Reference Honegger and Zippler2007). Parmelina quercina has a mainly Mediterranean distribution in southern Europe and Morocco, although it is reported from a small number of other sites (Nimis Reference Nimis1993; Clerc & Truong Reference Clerc and Truong2008). Parmelina carporrhizans grows in more oceanic and humid sites, mainly in the Mediterranean but also in the Macaronesian region, and is even occasionally found in Great Britain (Schauer Reference Schauer1965; Argüello et al. Reference Argüello, del Prado, Cubas and Crespo2007; Clerc & Truong Reference Clerc and Truong2008; Hawksworth et al. Reference Hawksworth, Blanco, Divakar, Ahti and Crespo2008; Alors et al. Reference Alors, Dal Grande, Schmitt, Kraichak, Lumbsch, Crespo and Divakar2014). The two species rarely coexist. Recently, P. carporrhizans was confirmed as heterothallic and long-distance dispersal of its ascospores (up to 900 km) was demonstrated using molecular data (Alors et al. Reference Alors, Dal Grande, Cubas, Crespo, Schmitt, Molina and Divakar2017).
In the current study, we aim to investigate some aspects of the reproductive behaviour of these two closely related species, with similar (but slightly different) morphologies, in the light of size and number theory. Specifically, we tested 1) different patterns of spore discharge and germination, 2) new mycelia development and growth in vitro, and 3) the importance of apothecia size in spore ejection and germination.
Materials and Methods
Lichen material
Two subsets of samples were considered. Initially we analyzed three samples of each species, collected in 2012. However, the statistical analyses based on Akaike criterion showed the need to increase the sampling. We therefore also collected further specimens in 2016 and analyzed the number of spores ejected by apothecia and percentage of germination by apothecia from ten thalli of each species. Only the percentage of apothecia able to eject spores, ontogenetic observations and mycelia development are based on the initial samples collected in 2012.
The first subset of samples corresponds to three fresh thalli of Parmelina carporrhizans growing on Castanea sativa Miller collected from Cuevas del Valle, Spain (40°18′28·4″N, 5°00′39·0″W) on 11 October 2012 at 1007 m (Maf-Lich 19190, Maf-Lich 19191), and three thalli of P. quercina collected from Quercus ilex L. in Monfragüe, Spain (39°50′17·47″N, 5°59′36·41″W) on 29 October 2013 at 200 m. Specimen identifications were confirmed using ITS sequences. We used five large and five small apothecia per thallus.
The second subset of samples was collected in October 2016 and consisted of ten thalli of each species; we selected six apothecia from each thallus. Collections were carried out in the same localities as the first subset.
We assessed the possible positive correlation between size of the apothecia (apothecia size range) and the reproductive parameters. Sizes ranged between 3·8–7·0 mm for P. carporrhizans and 1·9–5·9 mm for P. quercina. Apothecia were classified into two size categories: 3·8–4·9 mm (small) and 5–7 mm (large) diam. for P. carporrhizans; and 1·9–3·5 (small) and 3·6–5·9 mm (large) diam. for P. quercina. For each species the two categories were selected such that one was less than and the other greater than the median value of apothecium diameter, discarding very small apothecia without a red hymenial disc, which were considered immature. To test the relationship between the reproductive parameters and apothecium size, data were grouped in discrete intervals of 0·4 mm.
Isolation and culture
Parmelina carporrhizans and P. quercina mycobionts were isolated from discharged ascospores following the inverted Petri dish method of Ahmadjian (Reference Ahmadjian1993). Apothecia were mechanically cleaned and washed following the protocols established by Molina & Crespo (Reference Molina and Crespo2000); the clean ascomata were attached to the inner side of inverted Petri dish lids with petroleum jelly. The plates contained Bold's Basal Medium (BBM, Deason & Bold Reference Deason and Bold1960) which is inorganic and suitable for spore germination (Molina et al. Reference Molina, Stocker-Wörgötter, Türk and Vicente1997), and the ascospores were discharged upwards. The Petri dishes were kept at 18–20 °C in the dark (Molina & Crespo Reference Molina and Crespo2000).
After germination, 90 randomly selected, uncontaminated multispore agar pieces from P. carporrhizans and 35 from P. quercina were subcultured on different media: 3% glucose LBM (w/v) (3G-LBM) according to Lilly & Barnett (Reference Lilly and Barnett1951) as modified by Lallemant (Reference Lallemant1985); 0·2% glucose malt-yeast extract (0·2G-MY) according to Molina et al. (Reference Molina, Divakar, Zhang, González and Struwe2013); and Corn Meal Agar (CMA) following the manufacturer's instructions (Difco Laboratories, Detroit, MI, USA). Cultures were incubated at 20 °C in the dark (Molina & Crespo Reference Molina and Crespo2000). Periodically, mycobionts were examined using a Nikon SMZ800 stereomicroscope and an Olympus CX40 microscope. Photographs were taken with an automatic ring flash system attached to a Canon 450-D camera. The observations were carried out using white light and Nomarski interference contrast.
Ontogenetic parameters measured
The parameters measured to assess the ontogenetic development of these lichen-forming fungi in culture were: spore morphology, volume; germination type; mycobiont aggregate growth over 160 days on different media; the capacity to produce pigments, which was qualitatively assessed.
In the 2012 samples, the fitness parameters studied were: percentage of apothecia able to eject spores (after 24 and 48 h); productivity (number of octet spore packs or plurisporic groups ejected by apothecia in 48 h) of large apothecia (LAP) and small apothecia (SAP); germination (percentage of spore packs with at least one germinated spore) of large apothecia (LAG) and small apothecia (SAG); growth capacity after germination on different media. Growth was analyzed with ImageJ (http://imagej.nih.gov/ij/) using images taken with an Olympus SZ30 binocular microscope. Relative productivity (rprod) and relative germination (rger) of spores were also estimated using the following formulae with values ranging between −1 and 1:
$${\rm rprod} = {\rm} \lpar {{\rm LAP - SAP}} \rpar {\rm /}\lpar {{\rm LAP + SAP}} \rpar $$
$${\rm rger} = {\rm} \lpar {{\rm LAG - SAG}} \rpar {\rm /}\lpar {{\rm LAG + SAG}} \rpar $$In the second collection (2016), a total of 10 thalli and 60 apothecia were analyzed to assess rprod and rger.
To estimate the volume of spores of both species, the length and width of spores were measured, as described by Argüello et al. (Reference Argüello, del Prado, Cubas and Crespo2007), whereby at least 120 spores from six thalli were measured for each species. The mathematical formula for the volume of a spheroid, or ellipsoid of revolution, was used (V = 4/3 πa 2b), since this is the geometric figure that most closely resembles the shape of spores.
Statistical analysis
Generalized linear mixed models (GLMMs) were used to test the hypotheses that spore ejection and germination differ between the two species studied (fixed variable with two levels) and thalli (random variable with three levels). GLMMs were fitted assuming a Gaussian distribution error and, for each response variable, three alternative models were compared: 1) null (or intercept only) model (y = β0); 2) fixed-effect model (y = species); 3) mixed-effect model (y = species + (thallus (species))). The mixed-effect model contained thalli as a random factor nested within species type. In a nested design, each level of the nested predictor is uniquely associated with only one level of the higher-level predictor. With nested data structures, the interaction variance is pooled with the main effect variance of the nested factor (Schielzeth & Nakagawa Reference Schielzeth and Nakagawa2012).
An information theory approach (Burnham & Anderson Reference Burnham and Anderson2002) was used as an alternative to the traditional hypothesis testing approach (Johnson & Omland Reference Johnson and Omland2004) to select the best-fitting models accounting for spore ejection and germination. Models were selected based on the Akaike Information Criterion corrected for small sample sizes (AICc). The AICc rewards goodness of fit (based on the likelihood function), but also penalizes over-fitting (based on a function of the number of estimated model parameters). For each model set, we calculated the difference in AICc between each candidate model and the model with the lowest AICc (ΔAICc). The best model was that with the smallest AICc (Burnham & Anderson Reference Burnham and Anderson2002). All analyses were conducted using R (R Development Core Team 2013). We used the lmer function in the lme4 package and the gls function in the nlme package (Pinheiro et al. Reference Pinheiro, Bates, DebRoy and Sarkar2016) for fitting models; the MuMIn package was used for AICc-based model comparison (Barton Reference Barton2013).
Results
Maturity of apothecia
On the basis of the 2012 study, we found that the spores were ejected mainly in groups or packs of eight (Fig. 1A). Almost all the apothecia ejected spores after 48 hours, except some small apothecia of Parmelina carporrhizans (2·8%). For P. carporrhizans, in the first 24 hours, only 61·1% of large and 33·1% of small apothecia were sexually mature and had ejected spores, while apothecia of all sizes of P. quercina had ejected in that time.
Fig. 1. A– D, Parmelina carporrhizans, spore groups. A, plurisporic aggregate 3 days after discharge; B & C, germination tubes (arrows) in spores 4 days after germination; D, septa (arrows) formed 7 days after germination. E–G, P. quercina, spore groups. E, germination of spores; F, ungerminated spores (asterisks) 10 days after spore ejection; G, degenerate culture dying at 30 days. Scales = 10 μm. In colour online.
Ontogeny
Based on measurements carried out in 2016 on 120 spores of each species, we found that P. carporrhizans spores had a volume of 1600·25 ± 0·20 μm3, while those of P. quercina had a volume of 1418·69 ± 1·35 μm3. The volume of P. carporrhizans spores was 18% greater than that of P. quercina. Spores began to germinate 3–4 days after sporulation in both species. Parmelina carporrhizans produced hyaline meiospores (Fig. 1A), which generated one or two germ tubes growing from the endosporium (Fig. 1B & C). A few days later, the germ tubes developed a clearly defined septum (Fig. 1D). Initially, the development in P. quercina was similar to that of P. carporrhizans but after the germ tube formed, they were often observed to degenerate (Fig. 1E), while many spores remained ungerminated (Fig. 1F), and a substantial number of aborted mycelia were observed after 30 days (Fig. 1G).
Spore productivity
On the basis of the 2012 study (n = 3) of the mean ejection of spore packs, P. quercina had greater productivity than P. carporrhizans (637 ± 347 vs. 221 ± 360 spores), especially in small apothecia (539 ± 179 vs. 95 ± 134 spores) (Fig. 2A). Both species had positive rprod values, indicating that large apothecia eject more spores than smaller ones, and the mean rprod value was higher in P. carporrhizans (Fig. 2B). The mixed-effect model (full model including thalli effect) was best at explaining the patterns of spore ejection (LAP, SAP and rprod) because it showed the lowest values of AICc. These results highlight the need to increase the number of thalli analyzed (Table 1).
Fig. 2. Spore production and germination in Parmelina quercina and P. carporrhizans. A, productivity as absolute production of spores; B, relative productivity of spores (rprod); C, percentage germination; D, relative germination of spores (rger). Closed symbols represent values for each thallus sample (T1–T6). Mean values are plotted as open circles (n = 3). SAP = small apothecium production; LAP = large apothecium production; SAG = small apothecium germination; LAG = large apothecium germination. Error bars represent standard errors (SEM).
Table 1. Generalized Linear Model comparing ejection of spores (production) and germination of spores between species (fixed-effect), between thalli nested within species (mixed-effect) and null effect as an alternative hypothesis, using the Akaike Information Criterion (AIC).
k, degrees of freedom; rprod, relative productivity; productivity (number of octet spore packs or plurisporic groups ejected by apothecia in 48 h) of large apothecia (LAP) and small apothecia (SAP); rger, relative germination; germination (percentage of spore packs with at least one germinated spore) of large apothecia (LAG) and small apothecia (SAG); AICc, Akaike information criterion corrected for small sample size; ΔAICc, difference in AICc.
When ten thalli were analyzed, it was confirmed that that the average productivity per apothecium was higher for P. quercina than for P. carporrhizans (807·64 ± 76·56 vs. 686·13 ± 67·72). In addition, a positive correlation between class size of apothecium and ability to produce and eject spores was observed in P. quercina (Fig. 3A). However, in P. carporrhizans productivity remained constant and independent of apothecium size (Fig. 3C).
Fig. 3. Relationships between spore production, germination and apothecium size class in Parmelina quercina and P. carporrhizans. A & B, P. quercina; A, productivity, with regression line, r 2 = 0.82; B, percentage germination. C & D, P. carporrhizans; C, productivity; D, percentage germination. Mean values are plotted ±1 SEM (n = 10).
Spore germination
In the preliminary results (n = 3), the total percentage germination (disregarding apothecium size class) was much higher in P. carporrhizans (89·0%) than in P. quercina (0·4%) (Fig. 2C). The best model to explain patterns of spore germination (LAG, SAG and rger) was the mixed-effect model (Table 1). The mean rger showed higher positive values for P. quercina, and positive but near-zero values for P. carporrhizans (Fig. 2D). Differences in apothecium size had no effect on percentage germination (Table 1, Fig. 3D).
When the numbers of thalli and apothecia were increased, the percent germination was significantly higher in P. carporrhizans than in P. quercina (72·40 ± 3·49 vs. 14·30 ± 2·87). With increased sampling, the percentage germination was also shown to be independent of the apothecium size in both species (Fig. 3B & D).
Growth in different culture media
The subcultures were transferred to three different culture media to ensure growth until the formation of mycobiont aggregates in both species. The growth success in P. carporrhizans was 100% (n = 90), but was very low in P. quercina at only 7% (n = 35). For this reason, the growth kinetics of P. carporrhizans only were studied. Parmelina carporrhizans grew faster when subcultures were transferred to 0·2G-MY and CMA culture media (mean growth rates of 2·5 ± 0·02 and 1·5 ± 0·01 μm d−1 (n = 30), respectively), although its development was limited in 3G-LBM (Fig. 4).
Fig. 4. Growth kinetics of Parmelina carporrhizans mycobiont aggregates over 160 days in different culture media. Values at 160 days with the same letter are not significantly different (P < 0·05). ▲ = Corn Meal Agar; ● = 0·2% glucose malt-yeast extract; ■ = 3% glucose Lilly and Barnett's Medium. Mean values are plotted ± 1SEM (n = 30).
The mycelium morphology of P. carporrhizans was similar in all media but the appearance of hyphal pigmentation was not. In 3G-LBM the pigmentation appeared after 80 days, whereas in 0·2G-MY media it appeared on day 25 and in CMA full pigmentation had occurred by this point (Fig. 5A–C). The cultures exhibited aerial as well as darkly pigmented hyphae forming the aggregates. After 20–30 days, they also started secreting dark brown liquid that crystallized on the surface. The cultures formed hollow aggregates which produced a halo of enhanced growth restricted to the area in contact with the culture medium. The morphology of P. quercina cultures was similar to those of P. carporrhizans but the growth of P. quercina cultures differed by being comparable in all three culture media (Fig. 5D).
Fig. 5. Morphology and pigmentation of plurisporic aggregates of Parmelina carporrhizans and P. quercina mycobiont growing on Bold's Basal Medium after transferring to three different culture media. A–C, P. carporrhizans at four different times. A, 3% glucose Lilly and Barnett's Medium (3G-LBM); B, 0·2% glucose malt-yeast extract (0·2G-MY); C, Corn Meal Agar (CMA). D, morphology of P. quercina cultures after 160 days on the three culture media. Scales = 1 mm.
Discussion
In Parmelina carporrhizans and P. quercina, ascospores within an ascus are ejected simultaneously as a plurisporic pack or aggregate. This pattern is consistent with those noted in other groups of lichen-forming fungi (Molina & Crespo Reference Molina and Crespo2000; Sangvichien et al. Reference Sangvichien, Hawksworth and Whalley2011; Molina et al. Reference Molina, Divakar, Zhang, González and Struwe2013). However, the patterns of spore ejection differed between the two species including, over time, in the percentage of apothecia actively ejecting and the number of spores ejected by each apothecium. Regarding the time of spore discharge, all apothecia of P. quercina discharged in the first 24 hours while P. carporrhizans required an extra day (48 hours) to expel spores from almost all apothecia.
Spore productivity is a phenotypic trait that can vary yield from a small number of spores to thousands of plurisporic aggregates, depending on several environmental and genetic factors such as species and individual variability, hydration state and collection season (Yamamoto et al. Reference Yamamoto, Kinoshita, Takahagi, Kroken, Kurokawa and Yoshimura1998; Sangvichien et al. Reference Sangvichien, Hawksworth and Whalley2011; Molina et al. Reference Molina, Divakar, Zhang, González and Struwe2013). In the present study it was demonstrated that some apothecia of both P. quercina and P. carporrhizans can produce more than 1000 plurisporic aggregates. Together with Usnea complanata (Müll. Arg.) Motyka (Sangvichien et al. Reference Sangvichien, Hawksworth and Whalley2011) and Myelochroa entotheiochroa (Hue) Elix & Hale (Yamamoto et al. Reference Yamamoto, Kinoshita, Takahagi, Kroken, Kurokawa and Yoshimura1998), these are among the highest spore productivity values recorded so far within Parmeliaceae. In terms of absolute productivity in the two species, P. quercina ejected more plurisporic packs than P. carporrhizans, with significant differences between species. Parmelina quercina showed a positive correlation between productivity and apothecium size, suggesting that the ability to generate and expel spores is acquired as the apothecium develops (Molina et al. Reference Molina, Stocker-Wörgötter, Türk and Vicente1997), perhaps because of the greater surface area of the hymenium. However, in the case of P. carporrhizans no significant differences were observed in the productivity of apothecia of different sizes. In the present study apothecia of P. carporrhizans with a red hymenium smaller than 4 mm in diameter were not found. However, it would be worth collecting new material of a size below this threshold to test the hypothesis of larger apothecia equating to higher productivity in P. carporrhizans.
Germination in P. carporrhizans and P. quercina occurred seven days earlier than that of Parmelia saxatilis (L.) Ach. (Molina & Crespo Reference Molina and Crespo2000). Furthermore, both species showed bipolar germination and radial centrifugal growth of plurisporic aggregates, similar to other parmelioid species (Molina & Crespo Reference Molina and Crespo2000; Armaleo Reference Armaleo1991). Morphology of the mycelia was similar to that described for Parmelina species by Honegger & Zippler (Reference Honegger and Zippler2007). The percentage of germination was much higher in P. carporrhizans than in P. quercina. In both species, this reproductive parameter was independent of apothecium size and it did not show any recognizable pattern. This suggests that percentage germination does not depend on the development of the apothecium (excluding those apothecia which didn't develop a hymenial red disc) but could be taxon specific. This trait probably relies on the quantity of resources that each species expends on reproduction but little is known about this with regard to lichens. In plants, theories about the relationship between seed size and number have been proposed to explain the advantages of having a few large compared to many small seeds. Seedlings from large-seeded species with more nutrients should be able to establish under a range of environmental conditions that would not be tolerated by seedlings from small-seeded species. However, variation in seed size persists, despite the apparent advantages of being large-seeded (Westoby et al. Reference Westoby, Falster, Moles, Vesk and Wright2002). Easton & Kleindorfer (Reference Easton and Kleindorfer2008b), using Frankenia L. species as a model, concluded that smaller-seeded species had lower germination success at medium and high temperatures but greater success at low temperatures. Furthermore, these species delay germination until they have experienced several days of soil-water contact. Our data suggest that P. carporrhizans and P. quercina could be used as models to test the “number-size theory” in lichens. Other studies have shown that species with spore sizes differing by one order of magnitude can have differences in ejection of two orders of magnitude, with those species with a smaller spore size ejecting more spores (Morando et al. Reference Morando, Favero-Longo, Carrer, Matteucci, Nascimbene, Sandrone, Appolonia and Piervittori2017). In contrast to the study of Morando et al. (Reference Morando, Favero-Longo, Carrer, Matteucci, Nascimbene, Sandrone, Appolonia and Piervittori2017), we used two species of the same genus, which indicates that the reproductive strategies of genetically proximal species can differ substantially. Parmelina carporrhizans ejected upward fewer spores of greater size (and therefore with more reserve nutrients) and had a higher level of germination success in several culture media, while P. quercina ejected a greater number of smaller spores with lower germination success. The behaviour of P. quercina might be comparable to that of Frankenia species (Easton & Kleindorfer Reference Easton and Kleindorfer2008b), which release many small seeds with low germination success but which may be delaying germination until appropriate environmental conditions prevail.
Despite presenting results from only one locality per species, differences in spore discharge and spore germination are not considered to be due to variation within populations. Spore discharge and spore germination were also measured in P. quercina samples from La Carlota (Cordoba) and Cuevas del Valle (Avila) collected in autumn 2012 (data not shown), and these yielded similar results of high discharge and very low germination.
The differences observed in spore volume, spore ejection and spore germination between Parmelina species might reflect adaptation to environmental conditions as seen in Frankenia species, since P. carporrhizans inhabits a more stable habitat than P. quercina, especially with respect to humidity. The germination of P. quercina spores could be delayed until triggered by environmental factors such as temperature or humidity.
Finally, P. carporrhizans grew and developed very well on several different media. It has been shown repeatedly that growth rates for lichen-forming fungi, in general, depend on culture media (Cordeiro et al. Reference Cordeiro, Iacomini and Stocker-Wörgötter2004; Brunauer & Stocker-Wörgötter Reference Brunauer and Stocker-Wörgötter2005; Brunauer et al. Reference Brunauer, Hager, Grube, Türk and Stocker-Wörgötter2007; Deduke & Piercey-Normore Reference Deduke and Piercey-Normore2015; Shanmugam et al. Reference Shanmugam, Srinivasan and Hariharan2016). Even closely related species such as P. quercina and P. carporrhizans as well as others (Molina et al. Reference Molina, Crespo, Blanco and Hawksworth2002) might have very different germination and developmental requirements. These results could explain, at least partially, why these closely related and partially sympatric species inhabit different ecological niches, since their nutritional requirements appear to be different. While P. carporrhizans grows very well in the culture media used, the success of development in these same media is lower for P. quercina. This has also been described in other species from the same genus (Molina et al. Reference Molina, Crespo, Blanco and Hawksworth2002). The conditions that are optimal for P. carporrhizans growth may be less favourable for P. quercina. Hence, in nature, it is possible that there are environmental filters that limit the aposymbiotic development of the latter species in favour of the former (or vice versa). After completing the aposymbiotic phase of the cycle, the mycobiont can find a compatible photobiont and establish a successful symbiosis (Marshall Reference Marshall1996; Fedrowitz et al. Reference Fedrowitz, Kuusinen and Snäll2012). The low reproductive success of P. quercina under laboratory conditions contrasts with the distribution and abundance of this species in Europe (Clerc & Truong Reference Clerc and Truong2008), possibly because our laboratory conditions are not optimal for P. quercina (Deduke & Piercey-Normore Reference Deduke and Piercey-Normore2015).
Conclusions
Although both species showed similar development parameters (in vitro) to other species of Parmeliaceae, Parmelina carporrhizans and P. quercina showed significant differences in ontogenetic development during the aposymbiotic phase of their life cycles. Under laboratory conditions, P. carporrhizans took longer to release plurisporic groups and ejected significantly fewer spores upwards than P. quercina, although these spores were larger and had a much higher percentage germination. In P. quercina, the productivity was proportional to the size of the apothecium. However, the percentage germination seems independent of productivity in both species. In this regard, we propose that P. carporrhizans and P. quercina represent a good ‘model system’ to test “number-size theory” in lichens.
We thank Dr Marcos Méndez (URJC) for his insightful comments and Mrs. P. Mason for the language revision of the text. We also want to thank the Managing Editor for her invaluable help in improving this manuscript. This study was supported by the Ministerio de Ciencia e Innovacion, Spain (CGL2013-42498-P) and Santander-Universidad Complutense de Madrid (PR75/18-21605).
