Photobiont Composition of Some Taxa of the Genera Micarea and Placynthiella (Lecanoromycetes, Lichenized Ascomycota) from Ukraine

Folia Cryptog. Estonica, Fasc. 48: 135–148 (2011)

Photobiont composition of some taxa of the genera Micarea and Placynthiella (Lecanoromycetes, lichenized Ascomycota) from Ukraine

Anna Voytsekhovich, Lyudmila Dymytrova & Olga Nadyeina M. H. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, Kyiv 01601, Ukraine. E-mail: [email protected]

Abstract: The photobionts of 22 specimens of Placynthiella and Micarea genera were identified. The photobionts of Pla- cynthiella dasaea (Stirt.) Khodosovtsev, P. icmalea (Ach.) Coppins & P. James, Micarea melanobola (Nyl.) Coppins and M. misella (Nyl.) Hedl. are reported for the first time. This is also the first report about Elliptochloris reniformis (Watanabe) Ettl & Gärtner, E. subsphaerica (Reisigl) Ettl & Gärtner, Interfilum spp. and Neocystis sp. as the photobionts of lichens. The mycobiont of some taxa of Micarea and Placynthiella can be associated with several algae at the same time. In this case, one is the primary photobiont (common for all lichen specimens of a certain lichen species) while the others are additional algae which vary depending on the substratum or the habitat. Elliptochloris and Pseudococcomyxa species are primary photobionts for the studied taxa of the genus Micarea. The species ofRadiococcuus and Pseudochlorella are primary photobionts of the studied species of Placynthiella. For all investigated lichens the low selectivity level of the mycobiont is assumed. Kokkuvõte: Perekondade Micarea ja Placynthiella (Lecanoromycetes, lihheniseerunud kottseened) mõnede taksonite fotobiondi liigiline koosseis Ukrainas Määrati lihheniseerunud seente Micarea ja Placynthiella 22 eksemplari fotobiondid. Liikide Placynthiella dasaea (Stirt.) Kho- dosovtsev, P. icmalea (Ach.) Coppins & P. James, Micarea melanobola (Nyl.) Coppins ja M. misella (Nyl.) Hedl. fotobiondid identifitseeriti esmakordselt. Samuti on see esimene teade vetikate Elliptochloris reniformis (Watanabe) Ettl & Gärtner, E. subsphaerica (Reisigl) Ettl & Gärtner, Interfilum spp. and Neocystis sp. esinemisest samblike fotobiondina. Perekondade Micarea ja Placynthiella mõned taksonid võivad samaaegselt assotsieeruda mitme vetikaga. Sellisel juhul on üks fotobiont esmane (ühine sama liigi kõigil eksemplaridel), samas kui teised on täiendavad vetikad, mis eksemplariti erinevad, sõltuvalt substraadist ja kasvukohast. Elliptochloris ja Pseudococcomyxa liigid on esmased fotobiondid perekonna Micarea uuritud liikides ning Radiococcuus ja Pseudochlorella liigid – perekonna Placynthiella uuritud liikides. Kõikide uuritud samblike puhul täheldati mükobiondi madalat selektiivsust.

INTRODUCTION For a long time it was considered that the pho- arboricola Puymaly, or with the species which tobiont composition of lichens was constant. are closely related to this alga (T. arboricola Questions concerning the origin and constancy subclade) (Nyati, 2006); investigated species of a photobiont within different lichens ap- of the genus Pertusaria are associated mainly peared about twenty years ago (Friedl, 1987; with Trebouxia potteri Ahmadjian ex Gärtner Ott, 1987; Ihda et al., 1993; Beck et al., 1998; (Ahmadjian, 1993), whereas Umbilicaria spe- Helms, 2003). According to the latest data (Kirk cies associate mainly with Trebouxia simplex et al., 2008) the number of lichen-forming fungi Tscherm.-Woess (Beck, 2002; Romeike et al., average 17 500 to 20 000 species, while the 2002). number of known photobionts does not exceed Some lichen-forming fungi show a middle- 148 species (Voytsekhovich et al., 2011). Sev- level of selectivity. They form permanent asso- eral levels of mycobiont selectivity have been ciations with the different species of the same established (Beck et al., 2002). Thus, majority photobiont genus. This level is known for Cla- of lichen-forming fungi (approximately 40%) is donia species (which form the association with specific to their photobiont and form lichen as- Asterochloris Tscherm.-Woess only – Ahmadjian, sociation with only one algal strain (very high 1993; Piercey-Normore & De Priest, 2001; Yahr level of selectivity) or a certain species (high level et al., 2004), Megalospora (with Dictyochlorop- of selectivity). For instance, Xanthoria parietina sis Geitler em. Tscherm.-Woess – Tschermak- (L.) Th. Fr. forms its thallus with either Trebouxia Woess, 1984), and Collema (with Nostoc Vauch. 136 Folia Cryptog. Estonica ex Born. & Flah. – Degelius, 1954). The same (Romeike et al., 2002) have high and middle selectivity level is probably common within ceph- levels of selectivity. alodial lichens, for instance, Peltigera aphtosa Although, for many lichen species, like e.g., (L.) Willd. which forms symbiodemes with Nostoc Placynthiella Elenkin or Micarea Fr., the data on and Coccomyxa Schmidle (O’Brien et al., 2005). photobiont composition are discrepant and still The lichen-forming fungi that form their in need of further investigations. Several pho- thalli with the photobionts from the same fami- tobionts for the representatives of Placynthiella lies or orders (low selectivity), or even from the have been recorded. For instance, Stigonema groupings of higher taxonomic levels (very low was isolated from Placynthiella arenicola Elenkin selectivity), are characterized by “non-specific” (= P. hyporhoda (Th. Fr.) Coppins & P. James) relations between the lichen bionts. Thus, the (Elenkin, 1912); Gloeocystis sp. (probably, = common examples of low selectivity are the Radiococcus Schmidle) (Oxner, 1974) and Coc- following: Stereocaulon ramulosum Räuschel, cobotrys lecideae Warén (Ettl & Gärtner, 1995) which associates with cyanobionts Gloeocapsa – from Placynthiella uliginosa (Schrad.) Coppins Kütz., Nostoc, Scytonema C. Agardh ex Bornet & P. James; Chlorella lichina Chod. (= Chlo- & Flahault and Stigonema C. Agardh ex Bornet roidium ellipsoideum (Gerneck) Darienko et al.) & Flahault (Lamb, 1951: cit. Tschermak-Woess, and Nostoc sp. – from Placynthiella sp. (Coppins 1989), and lichen-forming fungi of Coenogonia- & James, 1984); Chlorella sp. was reported for ceae, Graphidaceae and Roccellaceae families Placynthiella uliginosa, P. icmalea (Ach.) Coppins that form their thalli with the representatives of & P. James and P. oligotropha (J.R. Laundon) Trentepohliaceae family (Rands, 1933, Santes- Coppins & P. James (Rosentreter et al., 2007). son, 1952, Uyenko, 1965: cit. Tschermak- Besides, Tønsberg (1992) reported several algal Woess, 1989; Meier & Chapman, 1983). The very species for the lichen P. dasaea, but did not low selectivity level is common for Verrucaria indicate their names: the green coccoid photo- Schrad. species that form associations with biont up to 12 μm in diameter and additional Dilabifilum prostratum Broady & Ingerfeld (Ettl algae, which were “2–4-celled, globose or broadly & Gärtner, 1995), Diplosphaera chodatii Bial. ellipsoid or more or less cubic, surrounded by (Geitler, 1960: cit. Tschermak-Woess, 1989), a thick, (3–4 μm wide) gelatinous cap, up to Heterococcus caespitosus Vischer (Tschermak, 15(–17.5) μm in diameter”. 1941; Zeitler, 1954; Sanders, 2004), Petroderma There are various data concerning the maculiforme (Wollny) Kuckuck (Wynne, 1969; photobionts of the genus Micarea. According to Moe, 1997; Sanders, 2004), etc. Some of lichen- Coppins (1983), the photobionts of 45 species ized basidiomycetes can be associated with sev- of Micarea were presented with “three types” eral photobionts at the same time. For instance, of green algae: “micareoid”, “protococcoid” and Multiclavula mucida (Pers.) R.H. Petersen was chlorococcoid. Unfortunately, the author gave associated with Mesotaenium Nägeli, Coccomyxa only descriptions but no species names for and Gloeocystis Nägeli (Geitler, 1955). these algae. The photobiont Pseudochlorella However, the selectivity levels were ascer- pyrenoidosa (Zeitler) Lund was isolated from tained only for 3% of the world lichen diversity Micarea assimilata (Nyl.) Coppins (Zeitler, 1954); (Voytsekhovich et al., 2011), while the photo- Elliptochloris sp. – from M. prasina Fr. (Brunner, bionts of remaining 97% of lichen species are 1985). The cyanobionts Nostoc and Stigonema still unknown. Most of the latest publications were also discovered in cephalodia of some on selectivity of the mycobionts deal with the Micarea species (M. assimilata, M. incrassata representatives of certain families of lichens. It Hedl., M. subviolascens (Magnusson) Coppins) was established that the families Physciaceae (Coppins, 1983). (Bhattacharya et al., 1996; Friedl et al., 2000; The data on photobiont composition often Dahlkild et al, 2001; Helms et al., 2001), Cla- play a valuable role in lichen identification. Cer- doniaceae (Piercey-Normore & DePriest, 2001; tain keys for lichen identification (e.g. Coppins, Yahr et al., 2004), Teloschistaceae (Beck, 2002; 1983; Ahti et al., 1999) require the information Honegger et al., 2004; Nyati, 2006), Graphidace- about the type of the photobiont (i.e. trebouxioid, ae (Nakano, 1988) etc., and genera – Letharia chlorococcoid or micareoid). Thereby��, ��consider- (Th. Fr.) Zahlbr. (Kroken & Taylor, 2000), Le- ing the gap in data concerning the photobionts canora Ach. (Blaha et al., 2006) and Umbilicaria of lichen species of Micarea and Placynthiella, 137 which are often inconsistent, the investigation identified by the fact of its presence in all in- of algal components of these two lichen genera vestigated specimens of certain lichen species. as well as the analysis of the correlation of their For identification, isolated strains of Trebouxia ecological ��characters�� with ��photobiont�� composi- and Asterochloris were compared with cultures tion would be topical and essential, and might of all known species of these genera, obtained be helpful in clarifying of the problems in biont from culture collections (SAG and CCAP). Cul- interactions. ture strains of the isolated photobionts are The aim of the present investigation was maintained in the algal collection of Department the exploration of algal component of the repre- of Lichenology and Bryology of M.G. Kholodnyi sentatives of two lichen genera – Micarea (which Institute of Botany (K). mainly consists of epiphytic bark-growing spe- Epiphytes cies) and Placynthiella (terricolous and lignicol- ous species), as well as a comparison and analy- Epiphytic algae were scrapped from lichen sur- sis of obtained data in respect to the ecological face on agarized medium in Petri dishes with peculiarities of these lichen species. the help of sterile preparation needle. After several weeks of cultivation on agarized Bold’s medium (3N BBM) in standard conditions (Friedl MATERIAL AND METHODS & Büdel, 2008), the isolated strains of epiphytic Lichen samples algae were investigated in all stages of the life cycle using light microscopy techniques and Total 22 specimens belonging to 8 species of compared with photobiont composition of in- Placynthiella and Micarea lichen-forming fungi vestigated lichens. were used in the present investigation. Lichen specimens were collected during 2005–2009 in five different districts of Ukraine (Donetsk, RESULTS Kherson, Kyiv, Luhansk, and Transcarpathian). Photobionts The further information on lichen specimens is given in Table 1. All lichen specimens are depos- The microscopic study revealed that the speci- ited in lichen herbarium of National Academy of mens of Placynthiella dasaea, Micarea prasina Sciences of Ukraine (KW-L). (No. 19, 20) and M. subnigrata contained only one photobiont, while the other investigated li- Photobionts chen species, Placynthiella icmalea, P. uliginosa, Small pieces of lichen thalli were washed in Micarea melanobola, M. misella, M. peliocarpa distilled water. After that the cortical layer of and M. prasina, contained several photobionts the thallus was cut out with a sterile razor at the same time (Fig. 1a). Later the presence of blade. Photobionts were isolated directly from additional photobionts was confirmed with the the photobiont layer according to the micropi- help of the cultural methods. The detailed data pette method (Ahmadjian, 1993) and grown on concerning photobiont composition of inves- agarized Bold’s medium (3N BBM) in standard tigated lichen specimens are given in Table 2. conditions (Friedl & Büdel, 2008): the intensity In seven specimens of Placynthiella uligi- of illumination was 10–30 μmol m-2 s-1 PPFD, at nosa, as well as in two specimens of P. icmalea, 12:12 – light/dark cycle and the temperature the photobiont Radiococcus signiensis was +15±2 °C. After several weeks of cultivation, the discovered (Fig. 2c). The abundance of this algal strains were investigated in all stages of the alga in lichen thalli differed in different speci- life cycle with the help of the light microscopy mens. The cells of Radiococcus signiensis in techniques using the microscope Mikmed-2 the specimen of Placynthiella uliginosa (No. 10) (LOMO, Russia). The photobionts were exam- visually presented more than 80% from a total ined both in the lichenized and cultured state photobiontal mass, while in the specimen of P. by standard light microscopic techniques. Thus, uliginosa (No. 6) – it was approximately 50%, the primary identification of the photobionts on and in P. uliginosa (No. 9) – less than 20%. How- the generic level and their percentage in lichen ever, the mycobiont of some of the investigated thalli was conducted in lichenized state using thalli of Placynthiella icmalea and P. uliginosa light microscopy; five slices from each lichen was associated with other algal species, which thallus were studied. Primary photobiont was usually were presented in photobiont layer in 138 Folia Cryptog. Estonica

Table 1. Original data of investigated lichen specimens

No. Lichen species Locality in Ukraine, date of specimen collection, and the collector(s) 1 Placynthiella dasaea Transcarpathian District, Tiachivsky region, Carpathian Biosphere Reserve, Shy- (Stirt.) Khodosovtsev rokoluzhansky massif, near Posich village, Abies+Fagus woodland, 48˚21.091'N 23�˚��43��.�924��'��E,�� 770�� m alt., �05.10.2009,�� leg. �O.�� Nadyeina,�� L.�� Dymytrova, S.�� Postoy��- alkin & A. Naumovich, det. L. Dymytrova (KW). 2 P. dasaea Transcarpathian District, Tiachivsky region, Carpathian Biosphere Reserve, in the vicinity of Mala Uhol’ka, 48˚16.623'N 23˚37.221'E, 812 m alt., 9.10.2009, leg. & det. L. Dymytrova (KW). 3 P. icmalea (Ach.) Kherson District, Tsuryupynsky region, “Oleshkivski pisky” National Nature Park, Coppins & P. James 46˚38.549'N 33˚01.185'E, 3 m alt., 01.10.2009, leg. & det. A. Khodosovtsev (KW). 4 P. icmalea Transcarpathian District, Tiachivsky region, Carpathian Biosphere Reserve, in the vicinity of Mala Uhol’ka, 48˚16.623'N 23˚37.221'E, 812 m alt., 9.10.2009, leg. & det. L. Dymytrova (KW). 5 P. uliginosa (Schrad.) Luhansk District, Lutugynsky region, in the vicinity of Karl Libkneht’s village, Coppins & P. James sandstone outcrops, 03.05.2005, leg. & det. O. Nadyeina (KW45506). 6 P. uliginosa Luhansk District, Lutugynsky region, in�� the vicinity�� of Verhnya�� Horikhivka village, steppe hills near “Pershozvanivs’ke” water storage, leg. & det. O. Nadyeina (KW45507). 7 P. uliginosa Luhansk District, Lutugynsky region, between Myrne village and Uspenka town, southern slope of “Kryven’ky Yar” gully, 05.05.2005, leg. & det. O. Nadyeina (KW45508). 8 P. uliginosa Luhansk District, Lutugynsky region, between Myrne village and Uspenka town, friable sandstones of “Kryven’ky Yar” gully, 05.05.2005, leg. & det. O. Nadyeina (KW45509). 9 P. uliginosa Luhansk District, Sverdlovsky region, in the vicinity of Provallya village, pasture land, on soil among mosses, 18.07.2005, leg. & det. O. Nadyeina (KW63536). 10 P. uliginosa Luhansk District, Sverdlovsky region, in the vicinity of Provallya village, steppe slopes, 22.07.2005, leg. & det. O. Nadyeina (KW45510). 11 P. uliginosa Kherson District, Tsuryupynsky region, “Oleshkivski pisky” National Nature Park, 46˚38.549'N 33˚01.185'E, 3 m alt., 01.10.2009, leg. & det. L. Dymytrova (KW). 12 Micarea melanobola Transcarpathian�� District��,�� Tiachivsky�� region��,�� Carpathian�� Biosphere�� Reserve��, Shy��- (Nyl.) Coppins rokoluzhansky massif, near Posich village, 48˚21.058'N 23˚43.955'E, 728 m alt., 05.10.2009, leg. & det. L. Dymytrova (KW). 13 M. misella (Nyl.) Hedl. In the vicinity of Kyiv, “Lisnyky” State Botanical Rreserve, 50˚17.53'N 30˚32.35'E, Quercus forest, 02.04.2009, leg. & det. L. Dymytrova (KW). 14 M. peliocarpa (Anzi) Transcarpathian District, Tiachivsky region, Carpathian Biosphere Reser- Coppins & R. Sant. ve, Shyrokoluzhansky massif, near Posich village, Abies-Fagus woodland, 48˚21.091'N 23˚43.924'E, 770 m alt., 05.10.2009, leg. O. Nadyeina, L. Dymytrova, S. Postoyalkin & A. Naumovich, det. L. Dymytrova (KW). 15 M. prasina Fr. Donetsk District, Shakhtars’ky region, in the vicinity of Saurovka village, the dell near Saur-Mohyla, “Donetsky Kryazh” Regional Landscape Park, 17.04.2006, leg. & det. O. Nadyeina (KW63549). 16 M. prasina Donetsk District, Shakhtars’ky region, in the vicinity of Petrivs’ke village, steppe slopes above the dell near tributary of Sevost’yanivka river, 18.04.2006, leg. & det. O. Nadyeina (KW63539). 17 M. prasina Donetsk District, Shakhtars’ky region, in the vicinity of Petrivs’ke village, along the dell near tributary of Sevost’yanivka river, solitary Quercus trees, 18.04.2006, leg. & det. O. Nadyeina (KW63544). 18 M. prasina Donetsk District, Shakhtars’ky region, in the vicinity of Saurivka village, SW di- rection from Saur-Mohyla, Pinus plantation, 19.04.2006, leg. & det. O. Nadyeina (KW63550). 19 M. prasina Donetsk District, Shakhtars’ky region, in the vicinity of Saurivka village, SW di- rection from Saur-Mohyla, Populus+Betula plantation, 19.04.2006, leg. & det. O. Nadyeina (KW63545). 20 M. prasina Donetsk District, Shakhtars’ky region, the dell “Urochysche Hrabove”, 20.04.2006, leg. & det. O. Nadyeina (KW63543). 21 M. prasina Luhansk District, Krasnoluchsky region, the dell along Mius river, 19.07.2006, leg. & det. O. Nadyeina (KW63547). 22 M. subnigrata (Nyl.) Luhansk District, Sverdlovsky region, sandstone between Dar’yino-Yermakovo Coppins & H. Kilias and Astakhovo villages, 22.07.2006, leg. & det. O. Nadyeina (KW 45511). 139 smaller quantity, rather than with the primary The majority of the investigated specimens of photobiont Radiococcus signiensis. The number the genus Micarea contained several photobionts of these algae and their species composition as well. The exceptions were M. prasina (No. varied. In most cases, additionally to Radiococ- 19, 20) and M. subnigrata, which were associ- cus signiensis the thalli of Placynthiella uliginosa ated with one photobiont only. Nine out of ten also contained Elliptochloris subsphaerica (Fig. investigated specimens of Micarea contained the 3c, d) and Interfilum massjukiae (Fig. 2b). Rarely photobionts from the genus Elliptochloris. Thus, the members of Asterochloris and Trebouxia the photobiont Elliptochloris subsphaerica was genera were found. One specimen (No. 10) con- found in thalli of Micarea melanobola, M. prasina tained Leptosira cf. thrombii. However, not all (No. 16) and M. subnigrata. The thalli of Micarea of the investigated species of Placynthiella were misella, and M. prasina (specimens No. 15, 17, associated with Radiococcus. Both specimens of 18, 19, 21) were associated with Elliptochloris bi- P. dasaea were associated with the photobiont lobata (Fig. 3a, b), while M. peliocarpa contained Pseudochlorella sp. (Fig. 1c, 2e). Elliptochloris reniformis (Fig. 3e, f). Furthermore,

Fig. 1. Schematical drawings of cuts of Micarea and Placynthiella thalli: (a) photobiont location in the thallus of Placynthiella uliginosa (scale = 40 μm); (b) Trentepohlia annulata in apothecium of Micarea misella (scale = 40 μm); (c) lichenized cells of Pseudochlorella sp., surrounded by fungal hyphae (scale = 10 μm); (d) lichenized Interfilum sp. in Micarea thallus (scale = 10 μm). 140 Folia Cryptog. Estonica -Woess . (Kütz.) Silva et al. (Reisigl) Ettl & Gärtner Mikhailyuk et al. Bisch. & Bold (Broady) Kostikov et al. Brand Bial.

Tscherm Tscherm.-Woess Epiphytes (J.B.Petersen) Mikhailyuk et al. sp. . flaccidum . flaccidum sp. ovoideus . cf cf cf. sp thrombii thrombii . sp. sp massjukiae sp.

cf. cf. genera compared with literature data. Bracteacoccus giganteus Chlamydomonas Elliptochloris bilobata Radiococcus signiensis Trentepohlia annulata Diplosphaera chodatii Interfilum terricola Elliptochloris bilobata Elliptochloris subsphaerica Pseudococcomyxa Klebsormidium Leptosira Trebouxia Asterochloris Interfilum Trebouxia Asterochloris Diplosphaera chodatii Parietochloris Interfilum massjukiae Leptosira Klebsormidium , - - & - - Placynthiella Ettl Oxner ( . ( and n é sp

sp. (Rosen sp. (Rosen War

, 1995); Coccobotrys Micarea Micarea rtner ä Literature data Green coccoid photo biont and sometimes additional algae (Tøns 1992) berg, Chlorella treter et al., 2007) Gloeocystis 1974); lecideae G Chlorella treter et al., 2007)

Photobiont

Mikhailyuk

11 sp. sp. excentrica signiensis signiensis

incrustata sp. cf. subsphaerica subsphaerica subsphaerica thrombii signiensis signiensis signiensis . sp.

sp massjukiae massjukiae cf.

cf. . al

Original data Pseudochlorella Pseudochlorella Radiococcus signiensis Elliptochloris subsphaerica Radiococcus Elliptochloris Interfilum et Radiococcus signiensis Interfilum massjukiae Radiococcus Elliptochloris subsphaerica Radiococcus Elliptochloris Radiococcus Elliptochloris Interfilum Asterochloris Radiococcus Asterochloris (Archibald) Skaloud & Peksa Elliptochloris subsphaerica Interfilum massjukiae Trebouxia Ahmadjian & Gärtner Radiococcus signiensis Asterochloris Elliptochloris subsphaerica Interfilum massjukiae Leptosira Radiococcus signiensis Interfilum massjukiae Trebouxia -

- - - - tree rock co folia

�� . �� C �� Amandi Cladonia Cladonia Pinus Trapeliop Neofusce ) ) ) near crushed ) and near

( Cladonia �� , (near (near ( covered with covered with with

) pokornyi

Substratum decomposed wood of Abies, mosses decomposed wood of Abies, mosses sand dead wood of (in vicinity of sis flexulosa mosses nea punctata soil mosses mosses fimbriata mosses foliacea lia thalli of niocraea cea sand

No.) The photobiont composition of investigated lichen specimens (No.

icmalea uliginosa uliginosa uliginosa uliginosa uliginosa

. . . . (No. 2) (No. 3) Table 2. Table Lichen (specimen Placynthiella dasaea 1) P. dasaea P. icmalea P (No. 4) P. (No. 5) P (No. 6) P (No. 7) P (No. 8) P. (No. 9) P. uliginosa (No. 10) P. uliginosa (No. 11) 141

Bornet ) tz. ü K ( (Chodat) J.B. Petersen umbrina . cf annulata lobatus

sp. Apatococcus lobatus Trentepohlia Trentepohlia Trebouxia Apatococcus ) sp. «micareoid» type of photobiont (Hedlund, 1882, 1895: cit. Coppins, 1983) «micareoid» type of photobiont (Hedlund, 1882, 1895: cit. Coppins, 1983) Elliptochloris 1985) (Brunner, «micareoid» type of photobiont (Hedlund, 1882, 1895: cit. Coppins, 1983

. . sp

sp sp

bilobata subsphaerica bilobata bilobata bilobata

subsphaerica subsphaerica subsphaerica subsphaerica

...... sp sp sp sp sp sp

Elliptochloris Pseudococcomyxa Elliptochloris Pseudococcomyxa Neocystis Elliptochloris reniformis Ettl & Gärtner (Watanabe) Elliptochloris Elliptochloris bilobata Elliptochloris Interfilum Elliptochloris Interfilum Elliptochloris Elliptochloris Interfilum Elliptochloris Interfilum Elliptochloris bilobata Pseudococcomyxa Elliptochloris Interfilum Elliptochloris subsphaerica ) - - -

Scoli

vitel tree tree tree �� tree tree tree tree tree Acarospora Bellemerea , , Rhizocarpon sp., and

Sarcogyne re Abies Fraxinus Fraxinus Quercus Betula Betula Fraxinus Betula , ) Lecanora hagenii, near ( Candelariella ��

bark of decomposed stub decomposed wood of Abies bark of bark of bark of bark of bark of bark of bark of (near Melanelia ciosporum chlorococcum on lina distinctum cupreoatra fuscata gularis

- -

21) 20) misella subnigra (No. 22) . . �� No. No. Micarea me lanobola (No. 12) M (No.13) M. peliocarpa (No. 14) M. prasina (No. 15) M. prasina (No. 16) M. prasina (No. 17) M. prasina (No. 18) M. prasina (No. 19) M. prasina ( M. prasina ( M �� ta The main photobiont of lichen is in bold. 142 Folia Cryptog. Estonica in the thalli of Micarea melanobola, M. misella additional photobiont of Micarea melanobola, M. and M. prasina (No. 15, 16, 17, 18, 21) several misella and M. prasina (No. 20). The majority of additional photobionts were discovered (see the specimens also contained Interfilum sp. (Fig. Table 2). For instance, Neocystis sp. was found 2a), which differs from Interfilum massjukiae and recognized as an additional photobiont of by the absence of distinct filaments in culture M. misella; Pseudococcomyxa sp. (Fig. 2f) – as conditions.

Fig. 2. Photobionts and epiphytes of Micarea and Placynthiella species in 4-weeks-old cultures: (a) cell packages of Interfilum sp.; (b) the filament ofInterfilum massjukiae; (c) schematical drawing of Radiococcus signiensis (the cells are covered with mucilage), the primary photobiont of Placynthiella icmalea and P. uliginosa; (d) the fragment of filaments of epiphytic alga Trentepohlia umbrina; (e) schematical drawing of Pseudochlorella sp., the primary photobiont of Placynthiella dasaea; (f) schematical drawing of Pseudococcomyxa sp., the primary photobiont of Micarea prasina. Scale = 20 μm. 143

Fig. 3. Micrographs and schematical drawings of primary photobionts of Micarea species in 4-weeks-old cultures: (a, b) Elliptochloris bilobata, photobiont of Micarea misella and M. prasina; (c, d) E. subsphaerica, photobiont of M. melanobola, M. prasina and M. subnigrata; (e, f) E. reniformis, photobiont of M. peliocarpa. Scale = 10 μm.

Epiphytes A total of 17 species out of 14 genera from two the surface of Placynthiella dasaea which grew divisions Chlorophyta and Streptophyta were on decomposed wood (No. 1, 2). In general, the identified as epiphytes of investigated lichens specimens of Placynthiella had more epiphytes (see Table 2). The most frequent of them was than Micarea. Trentepohlia annulata, which was Trebouxia sp., which was found on the surface discovered on the surface of the thallus of M. of three lichen specimens. The highest number misella as an epiphyte at first, was later found of epiphytic algae, five species, was found on in the apothecia of the same lichen (Fig 1b). 144 Folia Cryptog. Estonica

DISCUSSION according to its description cannot be Ellipto- chloris or Pseudococcomyxa, we revealed just Primary photobiont these algae as the primary photobionts of this The alga, which was registered in all specimens lichen species. of a certain lichen species, and associated with Due to relatively high species diversity of the fungal hyphae, is called here the primary photo- photobionts among Micarea and Placynthiella biont. We declare that the primary photobiont of species, we assume a low selectivity of their Placynthiella icmalea and P. uliginosa is Radio- mycobiont toward the algal partner. However, coccus signiensis; the primary photobiont of P. this issue requires further study using lichen dasaea is Pseudochlorella sp. The obtained data samples from a wider geographic area. clarify and add new information to previously known photobiont diversity for investigated Additional photobionts species. Earlier Gloeocystis sp. (probably, = In addition to the primary photobiont, several Radiococcus Schmidle) (Oxner, 1974), Coccobot- non-specific photobionts were found in thalli of rys lecideae Warén (Ettl & Gärtner, 1995) and investigated Micarea and Placynthiella species Chlorella sp. (Rosentreter et al., 2007) have been (see Table 2). The number and species compo- reported for Placynthiella uliginosa. Chlorella sp. sition of these algae varied. These algae were was discovered in P. icmalea (Rosentreter et al., revealed to be associated with fungal hyphae 2007). Unknown green coccoid photobiont up to directly inside the lichen thalli, but not on its 12 μm in diameter was reported for P. dasaea surface, therefore, they are considered as the (Tønsberg, 1992). additional photobionts but not as the epiphytes. In Micarea, ten out of eleven investigated Consequently, our results support the observa- specimens were associated with the primary tions of Tønsberg (1992), who distinguished photobiont from the genus Elliptochloris. One such additional algae from the specimens of specimen of M. prasina contained Pseudococco- Placynthiella dasaea. Unfortunately, the algal myxa sp. as the primary photobiont. According species which Tønsberg (1992) mentioned was to the results of molecular phylogenetic studies not identified; according to his description, it (Beck, 2002), some species of Pseudococcomyxa could be a member of Radiococcaceae (e.g. Ra- are closely related to Elliptochloris bilobata. diococcus signiensis). Our results partly confirm the data of Brunner Some of additional algae presented in this (1985), who reported Elliptochloris sp. as the article are the common lichen photobionts. photobiont of M. prasina. This is the first report For instance, the species of Asterochloris and of Elliptochloris reniformis and E. subsphaerica Trebouxia are well known as obligate photobi- as lichen photobionts. Earlier these two species onts and are associated with more than 55% of were known as free-living terrestrial algae (Ettl all known lichen species (Voytsekhovich et al., & Gärtner, 1995; Kostikov et al., 2001). The 2011). In our opinion, Asterochloris cf. excentrica photobionts of Micarea species were described and Trebouxia cf. incrustata that were discovered as three types of green algae by Coppins (1983): in Placynthiella uliginosa (No. 9) possibly got micareoid, chlorococcoid and “with protococcoid there from the thalli of neighbouring Cladonia type of division”. The most frequent type of pho- foliacea (Huds.) Willd. and Neofuscelia pokornyi tobiont of Micarea species is micareoid, which (Zahlbr.) Essl., respectively (see Table 2). It is is most likely, according to its description (Cop- known that Neofuscelia species form their thalli pins, 1983), referring to Diplosphaera chodatii. with Trebouxia gigantea (Hildreth & Ahmadjian) Description of the second type, chlorococcoid, Gärtner (Ahmadjian, 1993) and Trebouxia in- corresponds to the members of Elliptochloris ge- crustata (Beck, 2002), which are closely related nus. The third type of Micarea photobiont having species according to the molecular phylogenetic a “protococcoid type of division”, corresponds studies (Friedl & Büdel, 2008). The species of to the photobiont of Scoliciosporum umbrinum, Cladonia are associated with Asterochloris spe- which is known as Apatococcus lobatus (Beck, cies (Piercey-Normore & De Priest, 2001). At the 2002). However, these are only our assumptions same time, there is at least one report about that require further confirmation. Thus, despite Placynthiella icmalea being a parasite of other the fact that Coppins ��(1983) reported “mica- lichens (Fedorenko et al., 2006). Unfortunately, reoid” photobiont for Micarea prasina, which the authors did not indicate the species of lichen 145 hosts on which P. icmalea parasitized. There- Thus, the finding of recently described fore, the presence of Asterochloris sp. inside the lithophilous streptophyte algae Interfilum ��mass��-� thallus of P. uliginosa (No. 10) can be explained jukiae and Interfilum sp. (Mikhailyuk et al., by the nearby growth of Cladonia coniocraea 2008) inside lichen thalli was unexpected as the (Flörke) Vainio and C. foliacea. However, the localities where lichen specimens with Interfilum specimens of Micarea (Micarea prasina No. 21 were collected are new for these algae. This is the and M. subnigrata), were not associated with the first report ofInterfilum species, and the second photobionts of neighbouring lichens (see Table of Streptophyte in whole, as the lichen photo- 2). It seems that the entry of the photobiont from bionts. This is also the first report onNeocystis the environment into the thallus has a casual sp. as a lichen photobiont. character. Epiphytes Elliptochloris bilobata, Leptosira thrombi and Pseudococcomyxa sp. are the facultative photo- Most of the investigated epiphytic algae are bionts. It means that these algae exist in both very common terrestrial algae. The species of lichenized and free-living stage. Elliptochloris Apatococcus, Bracteacoccus, Parietochloris and bilobata has been reported as the photobiont Trentepohlia (Fig 2d) are common in aerophytic of Baeomyces rufus, Catolechia wahlenbergii, habitats: tree-bark and rocks (Ettl & Gärtner, Protothelenella corrosa and P. sphinctrioides 1995; Gärtner & Stoyneva, 2003; Mikhailyuk et (Tschermak-Woess, 1980, 1985); Leptosira al., 2003). Radiococcus signiensis is the epibryo- thrombii is known as the photobiont of Throm- phyte (Ettl & Gärtner, 1995). The usual habitat bium epigaeum (Pers.) Wallr. (Tschermak-Woess, of Chlamydomonas, Diplosphaera, Interfilum, 1953, Schiman, 1961: cit. Tschermak-Woess, Leptosira and Pseudococcomyxa is soil, although 1989), and Pseudococcomyxa is known as the they can be found also in aerophytic conditions photobiont of Baeomyces (Pott, 1972, Peveling, (Ettl & Gärtner, 1995; Kostikov et al., 2001; Galun, 1976: cit. Tschermak-Woess, 1989), Li- Mikhailyuk et al., 2008��).�� In contrast, the spe- chenomphalia (Jaag, 1933; Oberwinkler, 1984), cies of Trebouxia and Asterochloris are known Peltigera (Jaag, 1933), Solorina and Icmadophila only as the obligate photobionts of lichens (Ah- (Jaag, 1933). madjian, 1987). The finding of epiphytic algae Alternatively Interfilum spp., Elliptochloris Trentepohlia in lichen apotecium may indicate subsphaerica and Neocystis sp. are known only that the algae that grow in the immediate vicinity in free-living stage and there are no reports of a lichen may be included in its thallus. At the on these species as lichen bionts. We consider moment we do not know whether this alga can that the presence of these algal species in the be considered as a photobiont, and such cases photobiont layer of investigated lichen thalli is require additional investigations. caused by their free-living populations in the Lichens growing-zone of the lichen. The free-living algae The taxonomical status of Micarea melanobola growing in proximity of a lichen can be enveloped which for a long time was considered to be a vari- with the fungal hyphae and gradually become ation or synonym of Micarea prasina (Hedlund, part of the lichen. Lichens with the facultative 1892; V zda & Wirth, 1976), is still unclarified. (non-trebouxioid) photobiont use the pool of free- ě The species, M. melanobola, was described on living algae as the source of their autotrophic the basis of the differences in thallus and epi- component. For instance, the tropical lichen thecium pigmentation, size of apothecia, spores, Strigula sp. often colonizes the free-living Ceph- microconidia and the number of paraphyses in aleuros and uses it as the photobiont (Chapman comparison with M. prasina (Coppins, 1983). & Waters, 2001). In most cases the lichens and Later, these two species were synonymized their free-living algal bionts share the same because of the absence of distinctions except habitat. Thus, the common terrestrial free-living pigmentation of apothecia and pycnidia (Czar- algae from the genera Nostoc��, ��Scytonema��, ��Stigo- nota, 2007). However, three years later, after nema, Myrmecia, Diplosphaera and Stichococcus molecular phylogenetic analysis of the lichens are common photobionts of terricolous lichens from M. prasina-group, it was noticed that the from the families Collemataceae, Psoraceae, Ste- dark-colored morphotypes of M. prasina still reocaulaceae and Verrucariaceae (Tschermak- required an additional critical investigation Woess, 1989; Voytsekhovich et al., 2011). 146 Folia Cryptog. Estonica

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