High diversity of Bacidia ( Ramalinaceae , Lecanorales ) species in the Caucasus as revealed by molecular and morphological analyses

Abstract

During a study of the incompletely known lichen flora of the Caucasus, we analyzed 237 specimens of corticolous Bacidia s. str. collected in the Northern and Southern Caucasus, including Armenia, Azerbaijan, Georgia, and Russia. Of these, 54 specimens belonging to 11 species of Bacidia s. str. were selected for molecular studies, representing the observed morphological variability of the genus. We obtained 142 sequences from three RNA-coding genes (nrITS, nrLSU, and mtSSU) and two protein-coding genes ( RPB 1 and RPB 2). The single and concatenated datasets were complemented with Bacidia s. str. sequences from GenBank and subjected to Bayesian inference and two maximum likelihood analyses (RAxML and IQ-TREE). The resulting trees yielded highly concordant topologies of the groups and corresponded with previous results, supporting two main clades correlating with apothecia pigmentation. Our analyses are the first to reveal the presence of Bacidia heterochroa in the Caucasus. An exceptionally high degree of morphological plasticity was found in the Rubella and Suffusa groups. As a result of morphological examination and phylogenetic results, B. caucasica (Suffusa group) was described as new to science. Furthermore, two putative taxa in the Rubella group, Bacidia inconspicua ined. and B. maritima ined., were introduced. This study furthers our understanding and documentation of the understudied lichen flora of the Caucasus, bringing the total number of Bacidia species for the region to 13.

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High diversity of Bacidia (Ramalinaceae,Lecanorales) species in the
Caucasus as revealed by molecular and morphological analyses
Julia V. Gerasimova1,2,3 , Volker Otte4, Irina N. Urbanavichene5, Gennadii P. Urbanavichus6
and Andreas Beck2,3,7
1
Senckenberg Research Institute and Natural History Museum, 60325 Frankfurt am Main, Germany;
2
Systematics, Biodiversity and Evolution of Plants, Faculty of Biology,
Ludwig-Maximilians-Universität München, 80638 Munich, Germany;
3
Department of Lichenology and Bryology, Botanische Staatssammlung München, SNSB-BSM, 80638
Munich, Germany;
4
Senckenberg Museum of Natural History Görlitz, Member of the Leibniz Association, 02826 Görlitz, Germany;
5
Laboratory of Lichenology and
Bryology, Komarov Botanical Institute RAS, 197376 St Petersburg, Russia;
6
Laboratory of Terrestrial Ecosystems, Institute of North Industrial Ecology Problems, Kola
Science Centre, Russian Academy of Sciences, Murmansk Region, Russia and
7
GeoBio-Center, Ludwig-Maximilians-Universität München, D80333 Munich, Germany
Abstract
During a study of the incompletely known lichen flora of the Caucasus, we analyzed 237 specimens of corticolous Bacidia s. str. collected in
the Northern and Southern Caucasus, including Armenia, Azerbaijan, Georgia, and Russia. Of these, 54 specimens belonging to 11 species
of Bacidia s. str. were selected for molecular studies, representing the observed morphological variability of the genus. We obtained 142
sequences from three RNA-coding genes (nrITS, nrLSU, and mtSSU) and two protein-coding genes (RPB1 and RPB2). The single and con-
catenated datasets were complemented with Bacidia s. str. sequences from GenBank and subjected to Bayesian inference and two maximum
likelihood analyses (RAxML and IQ-TREE). The resulting trees yielded highly concordant topologies of the groups and corresponded with
previous results, supporting two main clades correlating with apothecia pigmentation. Our analyses are the first to reveal the presence of
Bacidia heterochroa in the Caucasus. An exceptionally high degree of morphological plasticity was found in the Rubella and Suffusa groups. As
a result of morphological examination and phylogenetic results, B. caucasica (Suffusa group) was described as new to science. Furthermore, two
putative taxa in the Rubella group, Bacidia inconspicua ined. and B. maritima ined., were introduced. This study furthers our understanding
and documentation of the understudied lichen flora of the Caucasus, bringing the total number of Bacidia species for the region to 13.
Keywords: Bacidia s. str.; crustose lichens; new species; phylogeny; taxonomy
(Accepted 25 May 2023)
Introduction
The crustose lichen genus Bacidia De Not. s. lat. is distributed
worldwide and includes up to 230 species (Lücking et al. 2017;
Wijayawardene et al. 2022). Bacidia diversity is most comprehen-
sively documented in Europe due mainly to the accessibility and
long tradition of lichenology in the region. Species have been
recorded in many European lichen floras, for example, in areas
of Germany, the British Isles, and the Iberian Peninsula (e.g.
Llop 2007; Wirth et al. 2013; Cannon et al. 2021), and Bacidia
diversity is also widely studied in North America (Ekman 1996).
However, the diversity of Bacidia in Asia and the Caucasus remains
largely unknown.
The Caucasus region harbours a rich, relict tertiary flora due to
its unique environmental conditions that have remained stable for
a long time. One of the first lichen checklists for the Caucasus
region was made by Vainio (1899) based on the Caucasian collec-
tion of Déchy and Lojka, which included several specimens of
Bacidia s. lat. In the 20th century, extensive research on the
Caucasus flora was carried out by Barkhalov (1975,1983), and by
Vězda who worked particularly in the Caucasian reserve on the
Black Sea coast and Abkhazia (Vězda 1983); these accounts also
documented several species of Bacidia s. lat. More recently,
Bacidia species have been reported in many lichenofloristic papers
and checklists of the Caucasus covering northern (Urbanavichus &
Urbanavichene 2002,2017b,2018; Urbanavichene & Urbanavichus
2019; Urbanavichus et al. 2021), north-western (Otte 2001,2004,
2007a,b; Blinkova & Urbanavichus 2005;Urbanavichus&
Urbanavichene 2014,2017a), western (Urbanavichene &
Urbanavichus 2016; Urbanavichus et al. 2020), south-western
(Urbanavichene & Urbanavichus 2014), north-eastern
(Urbanavichus & Ismailov 2013), central (Urbanavichene &
Urbanavichus 2018), eastern (Ismailov et al. 2017), and southern
(Harutyunyan et al. 2011; Alverdiyeva & Novruzov 2014;
Gasparyan & Sipman 2016; Inashvili et al. 2022)partsoftheregion.
Urbanavichus (2010) was the first to compile data on lichens
known for the Russian territory (incorporating the Caucasus),
including 18 species of Bacidia s. lat. in the checklist currently
known from the Caucasus. In subsequent studies, nearly half of
these species were transferred to other genera, such as
Aquacidia,Bacidina,Bellicidia, Biatora,Bibbya,Catillaria,
Corresponding author: Julia V. Gerasimova; Email: julia.gerasimova@senckenberg.de
Cite this article: Gerasimova JV, Otte V, Urbanavichene IN, Urbanavichus GP and
Beck A (2023) High diversity of Bacidia (Ramalinaceae,Lecanorales) species in the
Caucasus as revealed by molecular and morphological analyses. Lichenologist 55,
275–296. https://doi.org/10.1017/S0024282923000385
© The Author(s), 2023. Published by Cambridge University Press on behalf of the British Lichen Society
The Lichenologist (2023), 55, 275–296
doi:10.1017/S0024282923000385
https://doi.org/10.1017/S0024282923000385 Published online by Cambridge University Press

Scutula and Toniniopsis, and several new species were later
described or recorded for the region (Urbanavichus &
Urbanavichene 2014; Urbanavichene & Urbanavichus 2016;
Kistenich et al. 2018; Malíček et al. 2018; Cannon et al. 2021;
Gerasimova et al. 2021a). In addition, several species have been
synonymized and/or recognized as belonging to other genera,
such as Arthrorhaphis,Haematomma,Lecania and
Scoliciosporum (Davydov & Printzen 2012; Gerasimova &
Ekman 2017). Yet, the taxonomic position of some species is
still unknown, such as Bacidia freshfieldii (Vain.) Zahlbr., which
appears to be closely related to Catillaria (Gerasimova &
Ekman 2017). As such, at the beginning of our investigation 11
species of Bacidia s. str. were known from the Caucasus:
Bacidia absistens (Nyl.) Arnold, B. albogranulosa Malíček et al.,
B. arceutina (Ach.) Th. Fr., B. biatorina (Körb.) Vain., B. fraxinea
Lönnr., B. herbarum (Stizenb.) Arnold, B. laurocerasi (Delise ex
Dube) Zahlbr., B. polychroa (Th. Fr.) Körb., B. rosella (Pers.)
De Not., B. rubella (Hoffm.) A. Massal. and B. suffusa (Fr.)
A. Schneid. However, a revision of the genus integrating molecu-
lar and morphological analysis was needed to comprehensively
document the diversity of Bacidia in the region. Therefore, our
research aimed to investigate the diversity of Bacidia s. str. in
the Caucasus by applying an integrative approach, including
morphological, anatomical, and molecular analyses.
Material and Methods
Study area and sampling
The Caucasus is located between the Caspian and the Black
Seas and is bounded on the north by Russia (Kumo-Manych
Depression) and on the south by Georgia, Armenia and
Azerbaijan (Brummitt et al. 2001). The climatic conditions of the
Caucasus range from warm and moist in the western Colchic region
to hot and dry (Kura valley) in the east, spanning eight of the ten
oceanity levels of the Northern Hemisphere, compared to only
four observed in the Alps, according to the system of Jäger (1968).
The specimens of Bacidia were mainly collected in the National
Parks and Nature Reserves of the Northern Caucasus (Russia),
namely in and around the Nature Reserve Bol’shoy Tkhach and
the Caucasian Biosphere Reserve (1999–2019), Utrish (2001–
2020), Erzi (2018) and Samursky National Park (2017), but also
in Georgia (2012, 2015), Azerbaijan (2013) and Armenia (2015).
We studied the morphology of 237 specimens of Bacidia s. str.
collected in the Caucasus (Supplementary Material File S1, avail-
able online). Of these, we obtained molecular sequences from 54
specimens belonging to 10 species and two putatively introduced,
provisional taxa (B. inconspicua ined. and B. maritima ined.)
of Bacidia s. str. that were representative of the known species
diversity and inter- and infra-specific morphological variability.
The only exception to this was B. herbarum, which we were not
able to include in the molecular analysis as it is known only
from one herbarium specimen (GLM-L-0054141, collected in
Krasnodarskiy Krai at 2115 m a.s.l.). The specimens sampled
for molecular analysis were collected in the northern part of the
Caucasus (64.2%), Azerbaijan (20.7%), Georgia (9.4%), and
Armenia (5.7%) from the bark of various phorophytes (Table 1).
Morphology
Microscopic observations were made using a Zeiss Axioplan
(Oberkochen, GmbH) light microscope equipped with differential
interference contrast (DIC). Cross sections of apothecia were
made on a Leica Jung Histoslide 2000 Mikrotom (Heidelberg,
GmbH), with a thickness of 8–10 μm. Micrographs of cross-
sections were taken on a Zeiss Axioplan with an attached
AxioCam 512 Color camera, and images were processed with
Zeiss ZEN v. 2.3 (blue edition). Macrographs of external charac-
ters were taken on a Leica Z6 Apo microscope (with a 2.0×
Planapo lens; Leica, Germany) with a Sony Alpha 6400 camera
(Sony, Japan) attached and equipped with a Stack Shot Rail
macro rail (Cognisys, USA). A single image was mounted from
30–40 serial images using Helicon Focus v. 7 (Helicon, USA).
Measurements are given as (min–)x
±SD(–max) (SD = stand-
ard deviation, n
1
= number of all observations, n
2
= number of
specimens observed). We provide a detailed description of speci-
mens using traditional microscopic techniques following Smith
et al.(2009) and the subdivision scheme of the proper exciple
according to Ekman (1996), differentiating the following struc-
tures: rim, lateral part, and medullary part. We used the following
diagnostic characters to delimit the species lineages: 1) thallus
structure; 2) colour of disc and margin of apothecium; 3)
hypothecium colour; 4) colour and structure of exciple; 5)
shape and size of ascospores. The standard reagents were used
to study the colour reaction of the apothecia cross-sections and
crystals solubility: a solution of 10% potassium hydroxide
(KOH) in water, abbreviated K, and 50% solution of nitric acid
(HNO
3
), abbreviated N. Pigment characterizations follow Meyer
& Printzen (2000).
DNA extraction, PCR amplification and DNA sequencing
DNA extraction was carried out using the Stratec Invisorb Spin
Plant Mini Kit (Stratec Molecular GmbH, Berlin) following the
manufacturer’s instructions. Five to eight apothecia were used
from fresh material not older than five years, and thallus frag-
ments were removed to minimize the risk of contamination by,
for example, lichenicolous fungi. The same five target loci
(three RNA-coding genes (nrITS, nrLSU, and mtSSU) and two
protein-coding genes (RPB1 and RPB2)) as in Gerasimova et al.
(2021a) have been selected for PCR; amplification, purification
and sequencing were performed as described in Gerasimova
et al. (2018). Cycling conditions included initial denaturation at
95 °C for 2 min, 5 cycles of 95 °C for 40 s, 54 °C for 60 s,
72 °C for 90 s, 33 cycles of 95 °C for 40 s, 54 °C for 60 s, 72 °C
for 90 s, and a final extension step at 72 °C for 7 min. In cases
where the concentration of PCR product was not sufficient, a
second PCR with a reduced number of cycles was conducted:
denaturation at 95 °C for 2 min, 5 cycles of 95 °C for 40 s, 54 °C
for 60 s and 72 °C for 90 s, 22 cycles of 95 °C for 40 s, 54 °C for
60 s and 72 °C for 90 s, with a final extension step at 72 °C for
7 min. We used five pairs of primers: ITS1F (Gardes & Bruns
1993) and ITS4m (Beck & Mayr 2012), LR0R (Rehner &
Samuels 1994) and LR5 (Vilgalys & Hester 1990), mtSSU1 and
mtSSU3R (Zoller et al. 1999), fRPB2-5F and fRPB2-7cR (Liu
et al. 1999) and newly designed primers gRPB1AFba (GAGTG
YCCGGGACATTTTGG) and fRPB1cRba2 (GSCCRGCAATRT
CGTTATCCA) for Bacidia.
Alignment and phylogenetic analyses
We obtained 142 sequences of Bacidia s. str. from the Caucasus,
augmented the dataset with sequences of Bacidia s. str. from
GenBank (Table 1) and included Sporacestra borbonica comb.
276 Julia V. Gerasimova et al.
https://doi.org/10.1017/S0024282923000385 Published online by Cambridge University Press

Table 1. DNA numbers and specimen information for Bacidia species used in this study, with their respective GenBank Accession numbers. New sequences are in bold.
DNA no.
(JG) Name Country Specimen voucher/isolate
GenBank Accession Number
nrITS nrLSU mtSSU RPB1RPB2
JG123 Bacidia absistens Russia Urbanavichus &Urbanavichene M-0311923 (M) MW523506 MW489423 MW522879
B. absistens Norway Ekman 3223 (BG) AF282085 MG925845 MG926139 MG926229
B. albogranulosa Czech Republic Vondrák 17113 (PRA) MK158339 MK158334
B. albogranulosa Russia Malíček 9622 (hb. Malíček) MK158340 MK158335
B. albogranulosa Czech Republic Vondrák 11888 (PRA) MK158342 MK158332
B. albogranulosa Czech Republic Vondrák 11889 (PRA) MK158341 MK158333
B. albogranulosa Czech Republic Malíček 8013 (hb. Malíček) MK158336
B. albogranulosa Ukraine Vondrák 12235 (PRA) MK158337
B. albogranulosa Czech Republic Vondrák 12865 (PRA) MK158338
JG126 B. arceutina Georgia Gagarina M-0182569 (M) MW523507
JG163 B. arceutina Russia Otte GLM-0048917 (GLM) MW523508 MW489424 MW506364 MW540436 MW522880
B. arceutina Sweden Ekman 3110 (BG) AF282083 MG926041 MG925846 MG926140 MG926230
B. arceutina Switzerland van den Boom (LG DNA 579) JQ796851 JQ796842 JQ796829
B. arceutina United Kingdom EDNA09-01505 FR799125
B. arceutina United Kingdom EDNA09-01507 FR799126
B. arceutina United Kingdom EDNA09-01587 FR799127
JG037 B. areolata Russia Gerasimova M-0182592 (M) MH048614 MW506357 MW540434 MW522875
JG114 B. areolata Russia Davydov 17428 & Yakovchenko (ALTB) MW491455 MW506358
JG006 B. biatorina Georgia Gerasimova M-0182570 (M) MW523509
JG122 B. biatorina Russia Urbanavichene &Urbanavichus M-0311922 (M) MW523510 MW489425 MW506365 MW540437 MW522881
JG164 B. biatorina Russia Otte GLM-0053198 (GLM) MW523511 MW489426 MW506366 MW540438 MW522882
B. biatorina Sweden Knutsson 94-148 (hb. Knutsson) AF282079
JG182 B. caucasica Russia Otte GLM-0048447 (GLM) MW523553 MW489444 MW506388
JG083 B. diffracta USA Wetmore 46555-A (M) MH048620
B. diffracta USA Wetmore 26401 (MIN) AF282090
B. ekmaniana USA Lendemer 33836 (NY) KX151741
B. ekmaniana USA Lendemer 33920 (NY) KX151743
B. ekmaniana USA Lendemer 30488A (NY) KX151746
B. ekmaniana USA Lendemer 31362 (NY) KX151744
B. ekmaniana USA Lendemer 33783 (NY) KX151745
B. ekmaniana USA Lendemer 34000 (NY) KX151742
(Continued)
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Table 1. (Continued)
DNA no.
(JG) Name Country Specimen voucher/isolate
GenBank Accession Number
nrITS nrLSU mtSSU RPB1RPB2
JG007 B. elongata Russia Ezhkin M-0182571 (M) MH048626
JG101 B. elongata Russia Ezhkin M-0182625 (M) MH048627 MW493329 MW506351 MW540430 MW522870
JG102 B. elongata Russia Ezhkin M-0182626 (M) MH048628 MW493330 MW506352 MW522871
JG103 B. elongata Russia Ezhkin M-0182627 (M) MH048629
JG049 B. fraxinea Russia Urbanavichene L-15337 (LE) MW523519
JG170 B. fraxinea Russia Otte GLM-0044145 (GLM) MW523521 MW489428 MW506373 MW540441 MW522886
JG205 B. fraxinea Russia Urbanavichene &Urbanavichus L-15341 (LE) MW523522 MW489429 MW506374 MW522887
B. fraxinea Sweden Johansson 1620 (BG) AF282088
B. fuscopallida Korea KBA-L-0001010 ON352607
B. fuscopallida Korea KBA-L-0001049 ON352608
B. gigantensis Canada Isolate MCM240 MT425199
B. gigantensis Canada Isolate MCM242 MT425200 MT425196
B. hostheleoides United Kingdom Seaward 1996 (priv. hb. no. 108121) AF282081
JG010 B. heterochroa Georgia Gerasimova M-0182575 (M) MW523515 MW506369
JG120 B. heterochroa Georgia Gerasimova M-0182575 (M) XXXXXX
JG128 B. heterochroa Georgia Gagarina L-11635 (LE) MW523516 MW506370
JG167 B. heterochroa Russia Otte GLM-0048909 (GLM) MW523517 MW506371 MW522884
JG168 B. heterochroa Russia Otte GLM-0048864 (GLM) MW523518
B. heterochroa Korea KBA-L-0000386 ON352606
B. heterochroa Korea KBA-L-0002727 ON352612
B. heterochroa Korea KBA-L-0002734 ON352613
JG130 B. inconspicua ined. Russia Urbanavichene &Urbanavichus M-0311925 (M) MW523520 MW489427 MW506372 MW540440 MW522885
B. inconspicua ined. Ukraine Vondrák 12200 (PRA) MK158343 MK158331
JG092 B. kurilensis Russia Ezhkin M-0182620 (M) MH048610 MW493325 MW506348 MW540427 MW522868
JG095 B. kurilensis Russia Ezhkin M-0182621 (M) MH048611 MW493326
JG096 B. kurilensis Russia Ezhkin M-0182622 (M) MH048612
JG091 B. laurocerasi Russia Galanina M-0311952 (M) MH048609
JG211 B. laurocerasi Russia Ezhkin M-0308500 (M) MW491460 MW506349
JG124 B. laurocerasi Russia Urbanavichene &Urbanavichus M-0311924 (M) MW523512 MW506367
JG165 B. laurocerasi Russia Otte GLM-0048211 (GLM) MW523513 MW489426 MW506366 MW540438 MW522882
JG166 B. laurocerasi Russia Otte GLM-0053624 (GLM) MW523514
(Continued)
278 Julia V. Gerasimova et al.
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Table 1. (Continued)
DNA no.
(JG) Name Country Specimen voucher/isolate
GenBank Accession Number
nrITS nrLSU mtSSU RPB1RPB2
B. laurocerasi subsp. laurocerasi USA Wetmore 74318 (MIN) AF282078
B. laurocerasi subsp. laurocerasi Alaska Spribille 26334 (KLGO) MN483106 MN460211 MN508264
B. lutescens USA Ekman L1161 (LD) AF282082
JG131 B. maritima ined. Georgia Gerasimova M-0182578 (M) MW523523
JG172 B. maritima ined. Azerbaijan Otte GLM-0040829 (GLM) MW522890
JG206 B. maritima ined. Russia Urbanavichus M-0311935 (M) MW523528 MW489432 MW506377 MW522893
JG208 B. maritima ined. Russia Urbanavichene &Urbanavichus M-0311937 (M) MW523530 MW489434 MW506379 MW522895
JG139 B. obtecta Russia Ezhkin M-0308498 (M) MW491457 MW493335 MW506362 MW522877
JG140 B. obtecta Russia Ezhkin M-0308497 (M) MW491458 MW493336 MW506363 MW522878
JG141 B. obtecta Russia Ezhkin M-0308496 (M) MW491459
JG136 B. polychroa Russia Urbanavichus &Urbanavichene M-0311928 (M) MW523531 MW489435 MW506380 MW540442
JG137 B. polychroa Russia Urbanavichene &Urbanavichus M-0311929 (M) MW523532 MW489436 MW506381 MW540443 MW522896
JG169 B. polychroa Russia Otte GLM-0034608 (GLM) MW523533 MW489437 MW506382 MW540444 MW522897
JG185 B. polychroa Armenia Otte GLM-0041577 (GLM) MW523534 MW522898
JG186 B. polychroa Russia Otte GLM-0048845 (GLM) MW523535 MW522899
JG187 B. polychroa Russia Otte GLM-0048844 (GLM) MW523536
JG188 B. polychroa Russia Otte GLM-0053439 (GLM) MW523537 MW489438 MW506383 MW522900
JG189 B. polychroa Russia Otte GLM-0053126 (GLM) MW523538 MW489439 MW506384 MW540445 MW522901
JG190 B. polychroa Russia Otte GLM-0048243 (GLM) MW523539 MW522902
JG191 B. polychroa Azerbaijan Otte GLM-0052979 (GLM) MW523540
JG192 B. polychroa Azerbaijan Otte GLM-0053001 (GLM) MW523541
JG193 B. polychroa Azerbaijan Otte GLM-0039239 (GLM) MW523542
JG194 B. polychroa Azerbaijan Otte GLM-0038928 (GLM) MW523543 MW522903
JG195 B. polychroa Azerbaijan Otte GLM-0039238 (GLM) MW523544 MW522904
JG209 B. polychroa Russia Urbanavichus L-15342 (LE) MW523545 MW489440 MW506385 MW522905
JG212 B. polychroa Russia Urbanavichene &Urbanavichus L-15343 (LE) MW523546 MW489441 MW506386
B. polychroa Sweden Knutsson 91-215 (hb. Knutsson) AF282089
JG138 B. rosella Russia Urbanavichene &Urbanavichus L-15339 (LE) MW523556 MW506389 MW522909
B. rosella Sweden Ekman 3117 (BG) AF282086 AY300829 AY300877 AY756412 AM292755
JG085 B. rubella Russia Gerasimova M-0182581 (M) MH048630 MW493331 MW506353 MW540431
JG142 B. rubella Russia Ezhkin M-0308499 (M) MW491456 MW493332 MW506354 MW540432 MW522872
(Continued)
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Table 1. (Continued)
DNA no.
(JG) Name Country Specimen voucher/isolate
GenBank Accession Number
nrITS nrLSU mtSSU RPB1RPB2
JG133 B. rubella Russia Gerasimova M-0308494 (M) MW523524
JG134 B. rubella Russia Gerasimova M-0308495 (M) MW523525 MW489430 MW506375 MW522888
JG171 B. rubella Armenia Otte GLM-0041636 (GLM) MW523526 MW489431 MW506376 MW522889
JG173 B. rubella Russia Otte GLM-0031588 (GLM) MW522891
JG174 B. rubella Armenia Otte GLM-0041554 (GLM) MW523527 MW522892
JG207 B. rubella Russia Urbanavichus M-0311936 (M) MW523529 MW489433 MW506378 MW522894
B. rubella Poland AFTOL-ID 1793 HQ650644 DQ986793 DQ986808 DQ992422
B. rubella Switzerland van den Boom (LG DNA 578) JQ796852 JQ796843 JQ796830
B. rubella Sweden Ekman 3021 (BG) AF282087 AY567723
B. rubella Switzerland van den Boom (LG DNA 581) JQ796831
B. rubella Switzerland LIFU076-16 KX132984
B. rubella Hungary Hur H06122 EU266078
JG082 B. sachalinensis Russia Ezhkin M-0182619 (M) MH048621
JG097 B. sachalinensis Russia Ezhkin M-0182623 (M) MH048622 MW493333 MW506355 MW540433 MW522873
JG098 B. sachalinensis Russia Ezhkin L-12963 (LE) MH048623 MW493334 MW506356 MW522874
JG099 B. sachalinensis Russia Ezhkin L-12964 (LE) MH048624
JG100 B. sachalinensis Russia Ezhkin M-0182624 (M) MH048625
JG014 B. schweinitzii Russia Gerasimova M-0182579 (M) MW491454 MW493327
JG015 B. schweinitzii Russia Gerasimova M-0182580 (M) MH048613 MW493327 MW506350 MW540429 MW522869
B. schweinitzii USA Wetmore 72619 (MIN) AF282080 MG926045 MG926146 MG926235
B. schweinitzii USA AFTOL-ID 642 DQ782850 DQ782911 DQ972998 DQ782830 DQ782872
B. schweinitzii USA AFTOL-ID 4969 KJ766527 KJ766354
B. schweinitzii USA Shaheen (NY1451) MG461696
B. schweinitzii USA Tripp 2614 (NY1448) KX151762 KX151750
B. schweinitzii USA Lendemer 29364 (NY1449) KX151763 KX151751
B. schweinitzii USA Lendemer 31230A (NY1450) KX151766
B. schweinitzii USA Lendemer 31238 (NY1451) KX151764 KX151752
B. schweinitzii USA Lendemer 30548 (NY1452) KX151761 KX151749
B. schweinitzii USA Lendemer 31855 (NY1453) KX151765 KX151753
B. scopulicola Sweden Ekman 3106 (BG) AF282084
B. sipmanii Spain Sérusiaux (LG DNA 361) JQ796853 JQ796844 JQ796832
(Continued)
280 Julia V. Gerasimova et al.
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Table 1. (Continued)
DNA no.
(JG) Name Country Specimen voucher/isolate
GenBank Accession Number
nrITS nrLSU mtSSU RPB1RPB2
B. sorediata USA Lendemer 31692 (NY1389) KX151768 KX151755
B. sorediata USA Lendemer 31527 (NY1397) KX151771 KX151758
B. sorediata USA Lendemer 33702 (NY1539) KX151767 KX151754
B. sorediata USA Lendemer 33787 (NY1544) KX151772 KX151759
B. sorediata USA Lendemer 33869 (NY1546) KX151773 KX151760
B. sorediata USA Lendemer 35031 (NY1747) KX151769 KX151756
B. sorediata USA Lendemer 35386 (NY1748) KX151770 KX151757
B. sorediata USA Lendemer 38909 (NY2294) KX151774
B. sorediata USA Barton 658 (NY2496) KX151775
B. squamulosula Ecuador Kalb SE-314, Lich. Neotropici No. 405 MG925955 MG926051 MG925856 MG926152
B. suffusa USA Wetmore 74771 (MIN) AF282091
JG038 B. suffusa Russia Gerasimova M-0182593 (M) MH048616 MW506359 MW540435
JG039 B. suffusa Russia Gerasimova M-0182594 (M) MH048617 MW506360
JG051 B. suffusa Russia Gerasimova M-0182601 (M) MH048615 MW506361 MW522876
JG080 B. suffusa USA Tucker 17000 (M) MH048618
JG081 B. suffusa USA Wetmore 40219 (M) MH048619
JG176 B. suffusa Azerbaijan Otte GLM-0052934 (GLM) MW523547
JG177 B. suffusa Azerbaijan Otte GLM-0052906 (GLM) MW523548 MW522906
JG178 B. suffusa Azerbaijan Otte GLM-0052950 (GLM) MW523549 MW489442
JG179 B. suffusa Russia Otte GLM-0055113 (GLM) MW523550 MW489443 MW506387 MW522907
JG180 B. suffusa Russia Otte GLM-0048445 (GLM) MW523551 MW522908
JG181 B. suffusa Russia Otte GLM-0048460 (GLM) MW523552
JG183 B. suffusa Russia Otte GLM-0048483 (GLM) MW523554
JG184 B. suffusa Russia Otte GLM-0048464 (GLM) MW523555
B. suffusa USA Lumbsch 19190c (AFTOL-ID 5785) KJ766528 KJ766355 KJ766836
B. suffusa Korea KBA-L-0000359 ON352605
B. suffusa Korea KBA-L-0002776 ON352614
B. suffusa Korea KBA-L-0002835 ON352616
Sporacestra borbonica Reunion Krog &Timdal RE08,12 (isolate 511) MG925988 MG926086 MG925890 MG926184
S. pertexta Cuba Pérez-Ortega s. n. (isolate 1040) MG926000 MG926093 MG925903 MG926194 MG926268
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ined. and S. pertexta (Nyl.) Stapnes & Timdal as outgroup species
based on the results of Kistenich et al. (2018).
BLAST searches in GenBank were performed to detect and
exclude sequences from accessory and lichenicolous fungi and
contaminants. We performed phylogenetic analyses on each
locus separately (‘single-locus’analyses) and on concatenated
alignments of loci. In the concatenated analyses, we assembled
one alignment with a minimum of two out of the five target
loci (herein referred to as the ‘two-locus’analysis) to retain a
higher number of samples (88 samples). A second concatenated
alignment was also assembled to test the effect of decreased miss-
ing data on the analyses; a minimum of three loci were included,
but only 54 samples were retained in this alignment (herein
referred to as the ‘three-locus’analysis). An overview of the taxa
number and newly produced sequences for each genetic marker
and concatenated alignments are summarized in Table 2. Due
to the higher number of samples included, we mainly focus on
the results of the nrITS and two-locus analyses in this paper.
Alignment from the single and concatenated datasets is available
as Supplementary Material File S2 (available online).
Each locus was aligned using MUSCLE v. 3.8.31 using the
default settings (Edgar 2004) implemented in the program
PhyDE-1 v. 0.9971 and optimized manually. Ambiguous regions
of nrITS1 were aligned using MAFFT v. 7.505 (Katoh & Standley
2013). Sites with more than 95% gaps were excluded, and
alignments for the two-locus and three-locus analyses were con-
catenated manually. Substitution models for the concatenated
and single locus datasets were selected using jModelTest v. 2
(Guindon & Gascuel 2003; Darriba et al. 2012) for BI, and
ModelFinder (Kalyaanamoorthy et al. 2017) for ML analyses
using IQ-TREE. Partition models were implemented for the con-
catenated alignments, allowing each partition to have its substitu-
tion model. Identical sequences were excluded from the
subsequent analyses but are listed in Table 1.
We performed Bayesian inference (BI) and maximum likelihood
(ML) analyses in RAxML and IQ-TREE on the single-locus and
concatenated alignments of nrITS, nrLSU, mtSSU, RPB1and
RPB2. Bayesian inference was carried out using the Markov chain
Monte Carlo method (MCMC) using MrBayes v. 3.2.6 (Ronquist
et al. 2012). A GTR substitution model with gamma-distributed
rate variations across sites and a proportion of invariable sites was
selected based on the result of jModelTest. Two parallel runs were
performed (two cold chains), with a single tree saved every 10th
generation for a total of 1 000 000 generations. The convergence
of the Markov chain was examined according to the trends in like-
lihood values; the initial 10% was discarded as burn-in, and the
results were summarized as a 50% majority-rule consensus tree.
ML analysis was performed with RAxML v. 8.2.4 following a
GTRGAMMA model of molecular evolution with bipartitions
drawn onto the most likely tree topology using multiple non-
parametric bootstraps (Stamatakis 2014) on the CIPRES web por-
tal (Miller et al. 2010).
Further tree reconstruction using ML analysis was performed
in IQ-TREE v. 1.6.12 using standard bootstrap approximation
with 1000 bootstraps (Felsenstein 1985; Nguyen et al. 2015).
Substitution models for concatenated (partitioned) and single-locus
datasets were selected using ModelFinder (Kalyaanamoorthy et al.
2017).
The ML trees based on the different substitution models
from single and concatenated datasets were congruent and in
accordance with the Bayesian tree topology. Therefore, only the
RAxML tree for the nrITS and concatenated dataset are shown,
with RAxML bootstrap values (BSr), Bayesian posterior probabilities
(PP), and IQ-TREE bootstrap values (BSi) used. The phylogenetic
trees were visualized using FigTree v. 1.4.2 (Rambaut 2009). Only
clades that received BSr ≥70%, PP ≥0.95 and BSi ≥80% were con-
sidered highly supported and given in bold. The concatenated and
individual gene trees obtained from RAxML, MrBayes and
IQ-TREE are provided in Supplementary Material Figs S1–8(avail-
able online).
Results
Morphology and taxonomy
Revision of the 237 herbarium specimens showed that the most
common species of Bacidia s. str. in our Caucasus collection
is B. polychroa (16.5%), while B. fraxinea (7%), B. arceutina
(5.5%), B. laurocerasi (3.8%), B. absistens (c. 1%) and B. biatorina
(c. 1%) are least frequent. Only one herbarium specimen of B. her-
barum was studied, which was collected in Krasnodarskiy Krai at
2115 m a.s.l. (GLM-L-0054141).
Our examination of herbarium material found the first record
of Bacidia heterochroa (Müll. Arg.) Zahlbr. for Caucasus and
Russia. Based on morphological and anatomical analyses, one
new species, Bacidia caucasica sp. nov. (Suffusa group) was
described (see ‘Taxonomy’), and two putative taxa were defined:
Bacidia inconspicua ined. (Rubella group), and B. maritima
ined. (Rubella group). The systematic revision of the Bacidia spe-
cies list provided by Urbanavichus (2010) and their current taxo-
nomic status are given in Table 3. Observations of the complex
morphology of the Rubella group are summarized in Table 4.
Ecology
All studied specimens were collected mainly in old mixed
coniferous-broad-leaved forest communities in floodplains and
river valleys as well as in the drier habitats such as fruit orchards,
Table 2. Overview of the number of taxa and newly produced sequences for each genetic marker and concatenated alignments (excluding outgroup).
nrITS nrLSU mtSSU RPB1RPB2 2-locus dataset 3-locus dataset
Number of taxa 136 47 83 25 48 85 54
Newly produced sequences 52 23 26 10 31 ––
Length with gaps (bp) 486 861 730 672 1083 3835 3832
Constant sites 219 703 533 337 575 2391 2406
Parsimony informative sites 233 122 177 302 478 1294 1260
Number of distinct site patterns 306 149 260 352 531 1551 1495
282 Julia V. Gerasimova et al.
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deciduous forests without conifers, and recently clearcut young
coppices. The main phorophytes included Abies nordmanniana,
Acer campestre,Carpinus betulus,Quercus spp. (Q. pubescens,Q.
petraea and Q. robur), Juniperus spp. (J. excelsa,J. oxycerdrus
and J. foetidissima), and Pistacia mutica. Due to the proximity
of the Caspian and Black Seas, the dense river system and the
high groundwater level, the average relative humidity in all the
studied areas was very high. The average annual rainfall ranged
from 400 mm (Samursky National Park) and 600 mm (Utrish
Nature Reserve) to 2000–3000 mm in the forest belt of the
Caucasian Reserve. Most of the Bacidia species in the
Caucasus are confined to humid environments, except B. rubella
and B. fraxinea, which occur in a broader range of habitats from
dry and warm to humid subtropics with high levels of sunlight.
While both species could be present in dry areas, Bacidia rosella
is one of the most hygrophilous species. It has been collected in
the western and north-western Caucasus in old-growth forest
habitat with Abies nordmanniana,Acer trautvetteri and Fagus
orientalis at 1060–1600 m a.s.l., the most humid and shaded
areas of all the studied localities due to the predominance of
Abies nordmanniana.
Phylogeny
The BI and ML analyses for the single and concatenated datasets
recovered highly concordant topologies of the phylogenetic
groups (with a few exceptions discussed below). Both two-locus
and three-locus trees resulted in well-supported nodes throughout
the tree (the three-locus tree is provided in Supplementary
Material Fig. S2, available online). In total, we produced 142
new sequences for this study for the various genetic markers.
The nrITS comprised 40% of the alignment of a total dataset of
340 sequences, mtSSU comprised 24.7%, nrLSU 14%, RPB2
14%, and RPB1 7.3%.
Table 3. Revised Bacidia species list based on the checklist of Urbanavichus (2010), including recently found or newly described species.
Former name Current name or related genus Reference
Bacidia absistens Bacidia absistens Urbanavichene & Urbanavichus (2016)
B. albogranulosa Bacidia albogranulosa Malíček et al.(2018)
B. arceutina Bacidia arceutina
B. auerswaldii Scutula effusa Kistenich et al.(2018); reported as Bacidia effusa (Barkhalov 1975)
B. bagliettoana Toniniopsis bagliettoana Cannon et al.(2021)
B. beckhausii Biatora beckhausii Printzen (2014)
B. biatorina Bacidia biatorina
Bacidia caucasica Described as a new species in this study
B. circumspecta Scutula circumspecta Kistenich et al.(2018)
B. coprodes Toniniopsis coprodes Cannon et al.(2021), Urbanavichus & Urbanavichene (2014)
B. fraxinea Bacidia fraxinea
B. freshfieldii Catillaria s. str. Gerasimova & Ekman (2017)
B. friesiana Bacidina friesiana Ekman (2023)
B. herbarum Bacidia herbarum
B. heterochroa Bacidia heterochroa Reported as new to the Caucasus in this study
B. igniarii Scutula igniarii Cannon et al.(2021)
B. illudens Toniniopsis illudens Kistenich et al.(2018)
B. incompta Bellicidia incompta Kistenich et al.(2018)
B. laurocerasi Bacidia laurocerasi
B. notarisiana Bacidia notarisiana Ekman (2014), Urbanavichene & Urbanavichus (2018);
belongs to Bacidia s. lat.
B. polychroa Bacidia polychroa
B. propinqua Bilimbia s. str. Ekman et al. (2021)
B. rosella Bacidia rosella
B. rubella Bacidia rubella
B. subincompta auct. Toniniopsis dissimilis/T. separabilis Kistenich et al.(2018), Gerasimova et al.(2021a)
B. suffusa Bacidia suffusa Otte (2007a)
B. trachona Aquacidia trachona Aptroot et al.(2018)
B. vermifera Bibbya vermifera Kistenich et al.(2018)
B. viridifarinosa Aquacidia viridifarinosa Aptroot et al.(2018)
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Table 4. Main characters separating taxa of Bacidia rubella s. lat. group. Information for Bacidia fraxinea and B. rubella are given by Ekman (1996), Ekman & Nordin (1993), Smith et al. (2009) and Llop (2007); B. iberica and B.
parathalassica are from Llop (2007) and Aragón & Martínez (2003); B. thyrrenica from Llop et al.(2007); B. obtecta and B. elongata are from Gerasimova et al. (2018,2021b). Measurements for Caucasian taxa are newly provided
(see taxa with ‘Caucasus’in brackets after the species name. Measurements from the present study are given as (min–) mean ± SD (–max), and are otherwise are taken from the relative references. Taxa are ordered according to
their morphological similarity.
Thallus structure
Hymenium:
size (μm)
Hypothecium:
colour
Exciple:
size
(μm)
Exciple:
cell
layers
Exciple
cells:
size
(μm)
Crystals: presence
& distribution
Crystals: colour
& solubility
Ascospores
size (μm)
Ascospores:
number of
septa
B. rubella (North
America)
Coarsely granular;
consists of globose or
± flattened, almost
squamulose and
slightly incised
granules; pale grey to
greyish green
(67–)69–86–
97(–109)
Almost
colourless or
pale yellowish
to orange
(59–)79
(–103)
0–1upto
6×6
Sometimes with
radiating clusters
of minute crystals
(up to 1 μm)
Colourless
(reaction is not
provided)
(31–)44–52–
63(–104) ×
(2.1–)2.4–2.7–
3.2(–4.3)
(3–)3.2–
7.1–8.7(–13)
B. rubella
(Europe)
Thinly to richly
granular isidiate to
coralloid; grey to
yellow-green
70–115 Colourless, or
upper part
pale yellow or
orange-straw
- 1 to 3–5 Sometimes with
radiating streaks
of minute crystals
in exciple and
minor crystals in
hymenium
Colourless, K+,
N−
(35–)40–70
(–84) × 2.5–3
(–4)
3–7(–13)
B. rubella
(Caucasus)
As above (64)74.1
±9(–95)
Yellow-brown,
brownish,
orange
(51.5–)
69.15 ±
14.4(–
91)
1–3upto
5.5 × 12
Sometimes with
radiating clusters
of minute crystals
in lateral exciple
Colourless to
pale yellow,
K+, N−
(31–)47.8 ±
6.6(–75) ×
(1.5–)2.7 ± 0.4
(–4)
(1–)5.5 ± 3
(–12)
B. maritima ined.
(Caucasus):
JG206 & 208
Consists of scattered
± globose granules or
± flattened, almost
squamulose warts;
whitish to green
73.5–120 Yellow-brown to
pale brown
50–103 1–3upto
6×13
With radiating
clusters of minute
crystals in lateral
exciple
Colourless,
K+, N−
(34–)48 ± 6.7
(–65) × (2–)
2.65 ± 0.3(–
3.5)
(1–)4.7 ± 2.6
(–10)
B. maritima ined.
(Caucasus):
JG131 & 172
Consists of scattered
or contiguous,
±globose or
flattened, irregular
granules; green
81–91 Yellow-brown 73.5–91 1–3upto
5×11
Not observed - (35–)41.5 ±
6.2(–55) × (2–)
2.65 ± 0.47(–
3)
1–3–6
B. rubella
(Far East)
Coarsely granular,
consists of separate
or contiguous,
±globose granules
(49–)74.2 ±
15.8(–97)
Pale straw to
orange
(61–)
95.8 ±
25(–
122.5)
2-3 up to
6×12
Without or with
clusters or
crystals in lateral
exciple
Yellow,
not observed
(36–)58.8 ±
9.2(–72) ×
(2.5–)3.0 ± 0.3
(–4)
(1–)6 ± 3.7
(–12)
B. iberica
(Spain)
Minutely squamulose,
whitish to greenish
grey; squamules
ascending, with
crenate to
subdigitiform
margins
(50–)63–78(–
94)
Colourless or
pale yellowish
(55–)
65–80
(–97)
1 4.0–5.5 With crystals in
lateral exciple
and medulla
Colourless to
pale yellow,
K−,N+
(42–)45.5–
46–46.7(–
50) × (2.5–)
2.9–3.0–3.1
(–3.5)
5–9
B. fraxinea s. str.
(Europe)
Thin and smooth to
thick and verrucose,
areolate or irregularly
cracked,
grey
76–89–103 Straw to pale
orange
-0–1upto
6×6
Sometimes with
radiating clusters
of minute crystals
in exciple rim and
medulla
Colourless to
pale yellow,
K−,N+
(42–)50–67–
85(–109) ×
(2.5–)2.6–3.0–
3.4(–4.3)
3–17
(Continued)
284 Julia V. Gerasimova et al.
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Table 4. (Continued)
Thallus structure
Hymenium:
size (μm)
Hypothecium:
colour
Exciple:
size
(μm)
Exciple:
cell
layers
Exciple
cells:
size
(μm)
Crystals: presence
& distribution
Crystals: colour
& solubility
Ascospores
size (μm)
Ascospores:
number of
septa
B. fraxinea
(Caucasus):
JG170 & 205
Thick, consists of
irregular warts,
wrinkled; warts
adnate to the surface,
grey-green to dirty
green
70–93 Pale straw to
pale
yellow-brown
80–95 1–3upto
6×7
Clusters of
minute crystals in
lateral exciple
Colourless,
not observed
(35–)50 ± 6.7
(–66) × (2–)
2.7 ± 0.3(–3)
(1–)5 ± 3(–9)
B. elongata
(Far East)
Thin to thick, smooth
to areolate, wrinkled
and warted; grey to
dark grey-green
62.5–92–110 Colourless, pale
yellow to
orange-brown
55–
79.5–
87.5–
110
up to 4
layers
up to
7×20
Not observed - (39–)59 ± 8(–
80) × (2.0–)
2.5 ± 0.5(–4.0)
(2–)5–7–12(–
16)
B. obtecta
(Far East)
Thick, wrinkled,
warted; grey-green to
yellowish green
73.5–108–
135
Orange-brown 86–
94.3–98
up to 4
layers
up to
5×13
Throughout
lateral part of
exciple and upper
hymenium
Colourless,
K+, N−
(47–)61.2 ±
7.0(–79) ×
(2.0–)3.0 ±
0.36(–4.0)
(1–)6 ± 3(–
11)
B. inconspicua
ined. (JG130)
Thin, forming
continuous patches,
greenish to greenish
grey; mostly
endophloeodal
80–103 Pale straw to
yellow-brown
80–90 0 - Minor crystals in
medullar part
and hypothecium
Colourless or
yellowish K−,
N−.
Occasionally
crystals in
lateral exciple,
K−,N+
(45–)59.6 ±
5.5(–68) × (2–)
2.9 ± 0.4(–4)
(3–)8 ± 3(–
13)
B. inconspicua
ined. (Vondrak
12200, PRA)
Thin, mainly consists
of patches of
aggregated warts
103–115 Pale orange 86–103 2–3upto
5×8
Along lateral
exciple, medulla,
and hypothecium
Yellow, K−,N−(43–)56.4 ±
5.3(–65) × (2–)
2.6 ± 0.3(–3.2)
(3–)7 ± 3(–
13)
B. parathalassica
(Spain)
Warted, verruculose-
squamulose, with
individual squamules
attached to the
substratum, and
having rounded
margins
(60–)70(–75) Colourless to
yellowish
- 1 up to
4×10
Clusters in
medulla
Colourless to
pale yellow,
K−,N+
(26–)33.6–
52.2(–59) ×
(1.5–)2.1–2.9
(–3.0)
(3–)5–7(–9)
B. thyrrenica
(Mediterranean)
Continuous to
cracked, surface
warted to areolate
(50)60–75
(85)
Colourless to
yellowish
- - - Evenly distributed
in marginal area,
occasionally
clusters in
medulla, in upper
hymenium
Colourless to
yellowish, K+,
N−;
medulla
K−,N+
(32.8)40–55
(60.8) × 2–3
(3.6)
(3)7(10)
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All the sequences from the Caucasus obtained for this study
nested in the correspondent clades recovered in the last multilo-
cus phylogeny of Bacidia s. str. (Gerasimova et al. 2021a).
Bacidia heterochroa from the Caucasus formed a new group
(Figs 1 &2). The two-locus and nrITS trees were congruent
with the topology of the three-locus phylogeny, but while we
retrieved high support in the nrITS tree for nodes towards the
tips, the backbone in the multilocus phylogenies was better sup-
ported (Figs 1 &2; Supplementary Material Fig. S2).
The main groups obtained from single and concatenated data
in both ML and BI analyses are congruent and correspond to
those obtained from the previous phylogenies (Ekman 2001;
Gerasimova et al. 2018,2021b). Furthermore, similar to previ-
ous results, our phylogenies revealed two highly supported
major clades in the two- and three-locus phylogenies. Clade I
included the Laurocerasi, Schweinitzii, Suffusa, and
Heterochroa groups from the Caucasus (BSr/PP/BSi: 100/1/
100 in both phylogenies). Several sequences of B. heterochroa
from South Korea formed a sister clade to B. laurocerasi in
the nrITS phylogeny but with low support (BSr/PP/BSi: 44/
0.75/43) (see ‘Discussion’). Clade II included the Arceutina
(BSr/PP/BSi: 75/1/87, and 73/1/75), Rubella (BSr/PP/BSi: 85/1/
82, and 86/1/88), Polychroa (BSr/PP/BSi: 100/1/100 in both
phylogenies), and B. rosella groups (BSr/PP/BSi: 100/1/100 in
both phylogenies). Bacidia lutescens Malme (referred to as B.
thiersiana Lendemer by Lendemer (2020)), B. hostheleoides
(Nyl.) Zahlbr., and recently described B. fuscopallida B.G. Lee
& T.I. Heo formed a separate group within Clade II but without
support in the nrITS phylogeny (Fig. 2). All clades in the multi-
locus phylogenies received strong support throughout the tree
(i.e. with BSr ≥70%, PP ≥0.95 and BSi ≥80%). However, in
the multilocus trees poor support was retrieved for the sister
relationship of the Suffusa and Heterochroa groups (BSr/PP/
BSi: 62/0.92/65, and 71/0.94/72), the subclades of the
Schweinitzii group (not present in the Caucasus, discussed in
detail in Gerasimova et al.(2021b)), the position of the
Bacidia rubella s. str. clade (BSr/PP/BSi: 72/0.55/66, and 54/-/
37), the placement of B. inconspicua ined.(BSr/PP/BSi:79/
0.74/73, and 55/-/36), and the retrieval of the B. albogranulosa
clade as sister to B. polychroa (JG188) (BSr/PP/BSi: 77/0.91/56
in the two-locus tree).
The retrieved groups are discussed in detail in previous studies
(Gerasimova et al. 2021b); therefore, we focus on those with
Caucasian representatives below. As phylogenies are congruent
in the topology of the groups, the Results and Discussion are
based on the two-locus and nrITS trees, as those are the most
species-inclusive. Trees from the single and concatenated
phylogenies not shown in the manuscript can be found in
Supplementary Material Figs S1–8 (available online).
Laurocerasi group. In concatenated phylogenies, B. biatorina,B.
laurocerasi and B. kurilensis Gerasimova, A. Ezhkin & A. Beck
(Sakhalin endemic) formed one clade with high support of all
subclades (Fig. 1). Two sequences of B. biatorina from the
Caucasus formed a highly supported clade (BSr/PP/BSi: 100/1/
100), including a sequence from Sweden (Fig. 2).
The sequences from the Caucasus formed a well-supported
clade in the multilocus phylogenies and formed a clade together
with the representatives from the Far East and the USA in the
two-locus and nrITS phylogenies (Fig. 1 &2). In all phylogenies,
the sequence from Alaska (Spribille 26334) was placed as sister to
all others with maximum support in the multilocus phylogenies
(BSr/PP/BSi: 100/1/100) and high support in the nrITS tree
(BSr/PP/BSi: 92/0.99/99).
Bacidia heterochroa clades from South Korea and the
Caucasus. Bacidia heterochroa inhabits tropical and subtropical
areas worldwide (Ekman 1996) and was recently found in South
Korea (Lee & Hur 2022). To include nrITS sequences of B. hetero-
chroa from South Korea and keep the informative part of the nrITS
alignment, we constrained our alignment to Bacidia Clade I
(Supplementary Material Fig. S3, available online). Three sequences
of B. heterochroa from Korea (GenBank ON352606, ON352612
and ON352613) formed a sister clade to B. laurocerasi,butwith
support in RAxML and BI analyses only (BSr/PP/BSi: 74/1/65).
Instead, Bacidia heterochroa sequences from the Caucasus formed
a separate clade sister to the Suffusa and Schweinitzii groups in all
the phylogenies but with uncertain sister relationships (Fig. 2). In
the multilocus phylogenies, it was sister to the Suffusa group
with low support (BSr/PP/BSi: 62/0.92/65, and 71/0.94/72; Fig. 1
& Supplementary Material Fig. S2). In the nrITS tree, it was sister
to the Schweinitzii group on a long branch and with low support
(BSr/PP/BSi: 63/0.91/61; Fig. 2).
Suffusa group. The Bacidia suffusa sequences from the Caucasus
formed a clade together with those from North America (JG080
and JG081; Figs 1 &2) and a sequence from South Korea
(ON352616) in the nrITS phylogeny (Fig. 2). They formed a sister
clade to the representatives from the Far East and South Korea
with strong support values in all phylogenies. The sequences
from the Caucasus differ from the Far East individuals by 3%
(up to 15 nucleotides); however, as we could not observe any
strong morphological differences between the specimens from
these two clades, we chose not to recognize these individuals as
new species.
One sequence from the Caucasus (JG182) formed a separate
clade in all phylogenies with strong support. Based on this phylo-
genetic evidence and its distinct morphology, a new species,
Bacidia caucasica, is described (see ‘Taxonomy’).
Schweinitzii group. The taxa from the Schweinitzii group are
mostly known from North America and the coast of the
Russian Far East (see details in Gerasimova et al. (2021b)) and
have not been found in the Caucasus to date.
Rubella group. The highly supported Rubella group (BSr/PP/BSi:
85/1/82) forms four distinct clades comprising sequences of
Bacidia fraxinea,B. rubella,B. elongata Gerasimova & A. Beck,
B. obtecta Gerasimova et al., B. inconspicua ined. and B. maritima
ined. (Fig. 1). Bacidia rubella sequences from the west coast of the
Caspian Sea (JG172, JG206), and the north-east coast (JG208)
and south-east coast (JG131) of the Black Sea formed a sister
clade to Bacidia rubella s. lat. clades in all reconstructed phyloge-
nies with maximum support; these are provisionally defined as
Bacidia maritima ined. here. However, JG131 and JG172 are
represented by only one sequence: nrITS and RPB2, respectively.
The difference of nrITS sequences of JG206 and JG208 is almost
3% compared to other sequences from the B. rubella s. str. clade;
however, this difference does not seem to be correlated with a dif-
ference in studied morphological characters (see ‘Discussion’,
Table 4, and Fig. 3).
Sequences from the Caucasus (JG130) and Ukraine (Vondrák
12200, PRA; identified and published as Bacidia cf. rubella by
Malíček et al. (2018)) formed a highly supported clade sister to
286 Julia V. Gerasimova et al.
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Figure 1. Maximum likelihood (ML) tree of Bacidia s. str. resulting from a RAxML analysis of the concatenated multilocus dataset with a minimum of two loci included (out of
nrITS, nrLSU, mtSSU, RPB1 and RPB2). RAxML bootstrap values (BSr), Bayesian posterior probabilities (PP) and IQ-TREE bootstrap values (BSi) are indicated. Highly supported
branches with BSr ≥70%, PP ≥0.95, and BSi ≥80% are marked in bold; strongly supported branches with BSr ≥70% and BSi ≥80% are also marked in bold with a dot above
the branch; branches with BSr ≥70%, and/or PP ≥0.95, and/or BSi ≥80% are marked with a white dot. Major groups within clades are indicated, as are species within or
outside groups. New sequences are in bold. For further information about sequences, see Table 1. Single phylogenetic trees resulting from the concatenated multilocus data-
sets from RAxML, BI, and IQ-TREE analyses are in Supplementary Material Figs S1 and S2, (available online). In colour online.
The Lichenologist 287
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B. rubella s. str. in the two-locus phylogeny (BSr/PP/BSi: 90/1/87;
Fig. 1), and are provisionally placed in Bacidia inconspicua ined.
The nrITS sequences alone are insufficient to resolve the species
relationships within B. rubella, thus appearing paraphyletic but
without support for the paraphyly (Fig. 2).
The nrITS sequence of Bacidia fraxinea from Sweden
(Johansson 1620) and one from Caucasus (JG170) were nested
in a clade of B. rubella sequences in the nrITS phylogenies
(Fig. 2), revealing paraphyletic status of the species as two other
B. fraxinea sequences from the Caucasus formed a clade with
high support in the two- and three-locus phylogenies (BSr/PP/
BSi: 85/1/86, and 87/-/73).
Polychroa group. The Polychroa group comprises a highly sup-
ported clade of Bacidia polychroa,B. sachalinensis Gerasimova
et al.,B. diffracta S. Ekman and B. albogranulosa in the two-locus
trees (Figs 1 &2). All Caucasian B. polychroa sequences formed
one clade with high support, including a sequence from Sweden
in the nrITS phylogeny (BSr/PP/BSi: 93/1/98).
One sequence from Caucasus (JG188) was placed as a sister to
the B. albogranulosa clade in the two-gene phylogeny with low
support (BSr/PP/BSi: 77/0.91/56) but with high support in the
nrITS tree (BSr/PP/BSi: 89/0.99/99).
Rosella group. The sequences from the Caucasus (JG138)
and Norway (Ekman 3117) formed a highly supported clade in
all phylogenies. In the two- and three-gene phylogenies, these
sequences are retrieved in Clade II with maximum support,
together with the Polychroa and Rubella groups (BSr/PP/BSi:
100/1/100 in both phylogenies).
Arceutina group. The B. arceutina sequences from the Caucasus
(JG126 and JG163) and Switzerland (AFTOL LG579) formed a
well-supported clade sister to the B. arceutina clade from
Sweden in the two- and three-locus trees (BSr/PP/BSi: 100/1/
100, and 99/1/98). The sequences differ by c. 2.5% (7–8 nucleo-
tides) in nrITS. Bacidia arceutina is distributed in the western
part of the Caucasus (viz. Krasnodarskiy Krai, Republic of
Adygea, Georgia), with its southern-most limit recorded in a val-
ley in the Lenkaran district of Azerbaijan (GLM-L-529756).
Throughout its range, it is found at elevations of up to 1000 m
in warm, well-lit and relatively dry lowlands and habitats with
very high rainfall (from 1500 to 3000 mm per year). The B. absis-
tens sequence from the Caucasus (JG123) was placed with the
sequence from Norway as a sister to B. squamulosula (Nyl.) and
B. gigantensis Lendemer et al., with high support in the multilocus
phylogenies (BSr/PP/BSi: 100/1/99, and 73/1/75). Bacidia absistens
intheCaucasusisrecordedfromshadedhabitatsatelevationsof
700 m a.s.l. with very high humidity (rainfall of c. 1000 mm per
year).
Figure 2. Maximum likelihood (ML) nrITS tree of Bacidia s. str. resulting from a RAxML
analysis. RAxML bootstrap values (BSr), Bayesian posterior probabilities (PP), and
IQ-TREE bootstrap values (BSi) are indicated. Highly supported branches with
BSr ≥70%, PP ≥0.95, and BSi ≥80% are marked in bold; strongly supported
branches with BSr ≥70% and BSi ≥80% are also marked in bold with a dot above
the branch; branches with BSr ≥70%, and/or PP ≥0.95, and/or BSi ≥80% are
marked with a white dot. Major groups within clades are indicated, as are species
within or outside groups. New sequences are in bold. For further information
about sequences, see Table 1. Single phylogenetic trees resulting from the concate-
nated multilocus datasets from RAxML, BI, and IQ-TREE analyses are in
Supplementary Material S4 (available online). In colour online.
288 Julia V. Gerasimova et al.
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Discussion
Two main clades correlated with apothecia pigmentation
Our phylogenies of Bacidia with 142 additional sequences from the
Caucasus were congruent with previous results based on nrITS and
multilocus phylogenies (Ekman 2001; Gerasimova et al. 2018,
2021b). In line with previous studies, we retrieved two large clades:
Clade I includes the Laurocerasi, Schweinitzii and Suffusa groups,
as well as the newly defined Heterochroa group (that consists of
representatives from the Caucasus), and Clade II includes the
Rubella, Polychroa and Arceutina groups. In a previous work, phy-
logenies mainly included specimens from temperate regions
(Gerasimova et al. 2021b), while in this study, we also included
representatives from the subtropics. Only the nrITS phylogeny
included B. lutescens (B. thiersiana), B. hostheleoides, and the
recently described B. fuscopallida (Lee & Hur 2022), which formed
an unsupported group nested in Clade II. The former two taxa are
widespread in south-eastern North America and the Neotropics
(Malme 1935;Ekman1996; Lendemer 2020), while B. fuscopallida
is known only from the Gangwon Province in South Korea (Lee &
Hur 2022), characterized by a moist, warm, temperate climate
(Sayre et al. 2020). Further analyses are necessary to clarify the
phylogenetic position of this group.
Similar to previous results, two main clades can be separated
based on apothecial pigment. Specimens from Clade I have
dark brown, red-brown or green pigments (Laurocerasi-brown
Figure 3. Thallus structure and apothecia variability of the most typical representatives of the Rubella group. A, Bacidia rubella s. str. from Caucasus (JG171, GLM-0041636). B,
B. maritima ined. (JG206, M-0311935). C, B. inconspicua ined. (JG130, M-0311925). D, B. fraxinea from Caucasus (JG170, GLM-0044145). E, B. elongata (JG101, M-0182625). F, B.
obtecta (JG141, M-0308496—holotype). Scales: A–E = 1 cm; F = 0.5 cm. In colour online.
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and Bagliettoa-green), or a combination thereof, in the upper part
of the hymenium and lateral exciple. The herein-defined
Heterochroa group from the Caucasus also supports this differen-
tiation; the Caucasian specimens are characterized by a dark
purplish epithecium corresponding to the type specimen of B.
heterochroa. The specimens from South Korea have a dark
brown epithecium corresponding to the Laurocerasi group,
where South Korean sequences were nested. In contrast, represen-
tatives from Clade II have a mixture of yellow, orange and/or
brown apothecial pigments (Arceutina-yellow, Polychroa-brown
and Rubella-orange) in the upper part of hymenium and lateral
exciple. The specimens from the Caucasus in Polychroa and
Rubella groups also supported the apothecial pigment differenti-
ation. The representatives from the Lutescens-Hostheleoides
group have been characterized by almost colourless or faintly
and diffusely pigmented internal apothecial structures (Ekman
1996; Gerasimova et al. 2021b). In contrast, recently described
B. fuscopallida is characterized by the clearly pigmented
orange-brown to brown hypothecium (Lee & Hur 2022).
However, this clade was inferred to have very long branches in
our analyses, suggesting that their relationship warrants further
investigation in future studies, including additional samples.
The clade containing Bacidia absistens,B. gigantensis and
B. squamulosa is exceptional because it has highly variable
pigmentation. This evidence may be related to the exceptional
diversity of secondary compounds in this group detected by
TLC, such as 4-O-methylcryptochlorophaeic and homosekikaic
acids (Tønsberg et al.1995; Lendemer 2020). This combination
is so far unknown for species in other Bacidia s. str. groups,
which contain atranorin as the main known secondary compound
(Ekman 1996; Gerasimova et al. 2022).
As a result of morphological and/or phylogenetic analyses,
the current list of Bacidia s. str. in Caucasus includes 13 species,
namely B. absistens, B. albogranulosa,B. arceutina,B. biatorina,
B. caucasica,B. fraxinea,B. herbarum,B. heterochroa,B. laurocer-
asi,B. polychroa,B. rosella,B. rubella and B. suffusa, which is
almost 68.4% of the 19 species of Bacidia s. str. known from
Russia (Ekman 2009; Gerasimova et al. 2018).
Laurocerasi group
The known distribution of Bacidia biatorina in Russia includes
the European part, Far East, and Caucasus (Gerasimova 2016;
Gerasimova et al.2018). However, only a small number of herb-
arium specimens were confirmed to belong to the species; there-
fore, its distribution in Russia remains insufficiently studied.
Bacidia laurocerasi has a cosmopolitan distribution, having been
recorded in Russia (Caucasus, Ural, Siberia, and the Far East),
Europe, Macaronesia, Africa, North and South America, Asia,
Australia, and New Zealand (Smith et al. 2009; Urbanavichus
2010). Both B. biatorina and B. laurocerasi are restricted to forests
with high humidity in the Caucasus. Bacidia biatorina is mainly
found in undisturbed, beech-dominated, humid, shaded forests
of the middle mountain forest belt of the north-western
Caucasus, while B. laurocerasi is found in cooler, humid middle
and even upper beech- and fir-dominated mountain forest belts
but also inhabits stream valleys and lowlands.
Suffusa group: several species or different populations?
Bacidia suffusa is mainly known from the eastern temperate
region of North America, the Caucasus and Russian Far East
(Ekman 1996; Otte 2007a; Gerasimova et al. 2018), and it was
also recently recorded in South Korea (Lee & Hur 2022). The
closely related B. areolata Gerasimova & A. Beck is known only
from the Far East and is thus still considered endemic. The find-
ing of B. suffusa in the Caucasus was surprising, as it indicates the
species has a disjunct distribution across eastern North America
and parts of Eurasia, including the Caucasus, but is absent in
Europe (Otte 2007a). The species inhabits lower mountain belts
of oak and beech, indicating it could be restricted to Tertiary relict
floras that are common in parts of the Caucasus.
To confirm the relationship between North American, Far
Eastern and Caucasian representatives, we included several speci-
mens from different parts of the Caucasus in our phylogeny
(Table 1). Our results indicate that sequences of B. areolata and
B. suffusa form a strongly supported clade. In previous work, single-
locus and combined nrLSU, mtSSU and RPB1 phylogenies indicate
that Far Eastern B. suffusa and North American B. suffusa sequences
are not monophyletic (Gerasimova et al. 2021b). Intriguingly, in our
analyses, the newly sequenced Caucasian specimens of B. suffusa
form a clade with the North American individuals that is sister to
the Far Eastern B. suffusa clade. The nrITS sequences in these
two clades differed by 3% (up to 15 nucleotides), suggesting substan-
tial genetic differentiation; however, no significant morphological
differences were found between them. Therefore, we suggest clades
containing sequences from 1) the Far East and South Korea and
2) Caucasus, North America and South Korea are two populations
of B. suffusa on the cusp of divergence into separate species.
Importantly, one of the sequences (JG182) was consistently
retrieved as a sister to B. areolata and B. suffusa subclades in all
phylogenies with the highest support. Morphological examination
indicates clear differentiation from B. areolata and B. suffusa
s. lat., strongly supporting the recognition of this individual as a
new species. As a result, a new species, Bacidia caucasica,is
described below (see ‘Taxonomy’).
Rubella group: the complex morphology of taxa
Bacidia rubella is one of the most widespread species of Bacidia
s. str., occurring in the Holarctic and known from Europe,
Macaronesia, Africa, Asia and North America (Ekman 1996;
Llop 2007; Smith et al. 2009). In the Caucasus, it occurs in habi-
tats with a wide range of humidity levels and light intensities,
from dry, subtropical coastlines with Juniperus, to humid,
dense, mid-mountain forests with Abies nordmanniana. It occurs
in open upper mountain forest belts and has also been recorded
from humid stream valleys, with evidence of natural or anthropo-
genic disturbance. Similar to B. rubella,B. fraxinea occurs across a
wide range of habitats, found in the open, Mediterranean-like,
drought-adapted forest vegetation of the Black Sea coast, in forest
patches throughout agricultural areas of the forest-steppe region,
and in lower mountain forest belts and higher-humidity habitats,
in broad-leaved mesophilic forests. In lower mountain forest belts,
B. fraxinea usually occurs in areas of relatively high humidity
but is especially abundant in logged forests. Interestingly, the
provisionally defined B. inconspicua ined. was also collected in
young, dry, and warm secondary forest. This evidence leads to
the conclusion that species from the Rubella group are most
adapted to dry conditions, contrary to other Bacidia species.
This study included 14 additional specimens of both Bacidia
rubella s. lat. and B. fraxinea s. lat. for sequencing and phylogen-
etic analysis. Whereas in the nrITS phylogeny, the Rubella group
renders paraphyletic (viz. nesting B. rubella,B. fraxinea, and
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B. inconspicua ined.), the two- and three-locus phylogenies reveal
six supported clades: 1) a large B. rubella s. str. clade comprising
sequences from North America, Caucasus, Northern and Central
Europe, and the Far East; 2) a newly defined B. maritima ined.
from Caucasus (JG206 and JG208); 3) a clade of B. fraxinea
from Caucasus (JG170 and JG205); 4) a clade of newly defined
B. inconspicua ined. species from Caucasus (JG130) and
Ukraine (Vondrák 12200, PRA); 5) a clade of endemic B. elongata
from the Far East; 6) a clade of B. obtecta, endemic to the Far East
(Figs 1 &2). As we were not able to study the type material of the
Mediterranean-European specimens of Bacidia rubella s. lat. and
there are no sequence data available, we intend to obtain and
include this data in our future studies. Nevertheless, we discuss
the morphology of the Mediterranean representatives based on
the literature summarized in Table 4.
Morphologically, B. fraxinea was mainly separated from
B. rubella by thallus structure (Ekman & Nordin 1993). This sep-
aration was supported in the first phylogenetic study on
Bacidiaceae, where the two species formed two sister clades
(Ekman 2001), with a 1% (4 nucleotides) difference in the nrITS
sequences. More recent studies of the nrITS2 secondary structure
of B. fraxinea (Johansson 1620) and B. rubella (Ekman 3021)
revealed one hemi-compensatory base change (hemi-CBC) in the
structurally conserved regions of helix III (Gerasimova et al.
2018), also supporting the distinction of the species. However,
subsequent single-gene and multilocus phylogenetic analyses
resulted in a paraphyletic Bacidia rubella clade, where B. fraxinea
(Johansson 1620) was frequently nested within the B. rubella
s. str. clade (Gerasimova et al. 2018,2021b); this result is consistent
with our results from the analysis of an enlarged dataset.
As previously mentioned, the thallus structure is an essential
character for distinguishing species in the Rubella group more
broadly (Table 4). Thus, B. fraxinea is characterized by a
thin, inconspicuous to thick, verrucose, wrinkled, and warted
thallus (Ekman & Nordin 1993). In contrast, B. rubella s. str. is
characterized by a granular thallus with isidia- to coral-like
structures. Further delimitation based on thallus characteristics
in later studies led to a description of two new taxa from the
Mediterranean area: Bacidia iberica Aragón & I. Martínez and
B. parathalassica Llop & Gómez-Bolea (Llop & Gómez-Bolea
1999; Aragón & Martínez 2003). The morphology of B. iberica
and B. parathalassica is similar to B. rubella, but B. iberica has
a thallus formed by the adpressed squamules, and B. parathalas-
sica has a continuous, smooth to warted thallus, somewhat closer
to B. fraxinea (Llop & Gómez-Bolea 1999; Aragón & Martínez
2003).
In addition to the thallus structure, the presence and distribu-
tion of crystals were used by Llop et al.(2007) to separate the taxa
in the Rubella group, leading to the description of a new species,
B. thyrrenica Llop. The authors distinguished two main groups:
the first included B. thyrrenica,B. rosella and B. rubella, and the
second was composed of B. fraxinea,B. iberica and B. parathalas-
sica. The first group have crystals equally distributed in the upper
part of the exciple, dissolving in K. However, sometimes B. rubella
has been observed to have scarce crystals restricted to the upper-
most part of the exciple (Llop et al.2007). A second group have
clusters of colourless to pale yellow crystals in the medullary
part of the exciple that dissolve in acid solution (HNO
3
). In all spe-
cimens, we observed colourless crystals, except for JG142 (Far East,
B. rubella s. str. clade) and B. inconspicua ined. (Vondrák 12200,
PRA) with yellow crystals in the exciple. Bacidia obtecta is also
characterized by having crystals in the upper part of the
hymenium and exciple, but these are colourless. However, as crys-
tals are only occasionally present, we suggest this character is not
consistent enough to differentiate the Rubella group taxa.
In addition to crystals, B. fraxinea,B. iberica,B. rubella and B.
parathalassica are characterized by having one layer of more or
less globose cells along the exciple margin. In the provisionally
defined B. inconspicua ined. specimens, this character varies in
two specimens (Table 4). In contrast, B. elongata and B. obtecta
are characterized by having four layers of enlarged lumina cells
along the exciple margin. Additionally, all species differ based
on characteristics of spore size, hymenium height and apothecia
colour (Table 4).
The newly introduced Bacidia inconspicua ined. This is repre-
sented by two specimens: one collected in the Caucasus (JG130)
and one in Ukraine (Vondrák 12200, PRA). The B. inconspicua
ined. specimen was collected in a young, dry, and warm secondary
forest with pine trees and Cladonia rangiferina undergrowth; the
original primary forest of oak and hornbeam, probably logged
and replaced by ash and pine plantations. Bacidia inconspicua
ined. is characterized by a prosoplectenchymatic exciple without
enlarged cells along the exciple rim (JG130), with sometimes up
to three layers of enlarged lumina cells (Vondrák 12200, PRA),
in contrast to B. iberica,B. fraxinea s. str., B. parathalassica and
B. rubella s. str., which are characterized by one layer of more
or less globose cells (Table 4 and references therein, Fig. 4).
Based on thallus structure and phylogenetic evidence using a mul-
tilocus dataset, it seems that B. inconspicua ined. may represent a
separate species belonging to the Rubella group. However, as only
two specimens were studied and in light of the high plasticity of
the phenotypic characters in the Rubella group, we refrain from
describing a new species and thus define B. inconspicua provi-
sionally. A detailed description of all distinguishing morpho-
logical characters for B. inconspicua ined. is given in Table 4
and Fig. 4.
The newly defined Bacidia maritima ined. This is known from
four specimens: JG131, JG172, JG206, and JG208 (Figs 1 &2);
although, from the first two specimens, a sequence could be
obtained from only one locus (nrITS and RPB2, respectively).
Interestingly, most specimens were collected near the Black and
Caspian Seas coasts, suggesting that the species may be confined
to the maritime zone. In more detail, JG206 was collected on the
west coast of the Caspian Sea, c. 3 km from the coast, in a shaded
forest very rich in epiphytic lichens (82 species in 1 ha). JG208
was collected on a south-east slope, c. 750 m from the Black Sea
coast in a Juniperus-Pistacia forest in dry subtropic conditions,
in a sunny, warm locality but apparently influenced by the prox-
imity of the sea. The fog and high humidity favoured a rich epi-
phytic composition of lichens in this locality (71 species for 10–12
trees). JG131 was collected in a shaded pine forest also close to the
south-east coast of the Black Sea. However, JG172, collected in the
southern Caucasus region of Azerbaijan, was found at a site much
further inland, seemingly without maritime influence, in a pas-
tured, coppice-like mixed forest. However, the number of speci-
mens of Bacidia maritima ined. known to date is too small to
characterize its overall distribution with certainty; it seems that
the affinity for coastal sites is more strongly expressed in the
somewhat harsher climate of the northern Caucasus.
Morphological examination indicates B. maritima ined. has a
similar thallus structure and coloration of the upper and inner
apothecia to B. rubella. However, B. maritima ined. has shorter
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spores than B. rubella, similar to B. iberica and B. parathalassica.
The mean spore size of B. rubella from the Caucasus is 47.8 ±
6.6 μm (up to 75 μm) × (1.5–)2.7 ± 0.4(–4) μm wide, and they
have up to 12 septa. The mean spore size of European B. rubella
individuals is 40–70 μm (up to 84 μm) × 2.5–3(–4) μm wide with
up to 13 septa, and of B. rubella North American individuals is
44–63 μm (up to 104 μm) × (2.1–)2.4–2.7–3.2(–4.3) μm wide
with up to 13 septa (Table 4). On the contrary, the spores of
B. maritima ined. are 48 ± 6.7 μm (up to 65 μm) × (2–)2.65 ±
0.3(–3.5) μm wide with up to 10 septa. Nevertheless, to clearly
distinguish between the taxa, additional specimens are necessary
for a more detailed description and measurement of spores; there-
fore, we refrain from describing a new species here.
Polychroa group: the presence of an unpigmented member
Bacidia polychroa is another widespread species of Bacidia s. str.,
occurring in Europe, North and South America and Asia (Ekman
1996; Llop 2007; Smith et al. 2009). Bacidia albogranulosa is
known from the Czech Republic, Poland, Ukraine and the
Caucasus (Malíček et al. 2018). Conversely, Bacidia diffracta
and B. sachalinensis are known only as endemics from North
America and the Russian Far East, respectively. Bacidia polychroa
is particularly common in stream valleys but also inhabits humid
lower and middle mountain forest belts of the north-western
Caucasus. It is the most abundant Bacidia species in these forest
belts of the north-western Caucasus and is usually found in
shaded places with the young regrowth of coppiced trees.
We included 16 specimens of B. polychroa from the Caucasus in
our phylogeny, encompassing its observed morphological variation.
The nrITS analysis also included a European sequence (Fig. 2)and
resulted in a well-supported clade of B. polychroa in the multilocus
phylogeny. All taxa within the Polychroa group share the pigment
Polychroa-brown in the hypothecium, exciple and hymenium, and
a distinguishing K+ purplish reaction in apothecial cross-sections.
This purple reaction is unknown for B. albogranulosa since it is
known only in its sterile form (Malíček et al. 2018) and was also
not observed in the sister JG188, represented by pale or almost
unpigmented apothecia. The specimen JG188 is characterized by
its warted thallus similar to B. polychroa, but has very bright
apothecia, which do not have a K+ purplish reaction. The lack of
a K+ purplish reaction makes it easily confused with B. fraxinea.
Other specimens of B. polychroa from the large clade also con-
tained pale or unpigmented apothecia lacking the characteristic K
+ purplish reaction. Therefore, despite distinct morphological char-
acters, we did not find enough evidence to support the circumscrip-
tion of a new species in this study.
Taxonomy
Bacidia caucasica Gerasimova, Otte & A. Beck sp. nov.
MycoBank No.: MB 847536
Similar to Bacidia suffusa but differs by abundant colourless
crystals above the exciple edge and upper hymenium, and the
Figure 4. Cross-sections of apothecia and thallus structure of Bacidia inconspicua ined. (A–C, M-0182578; D, J. Vondrák 12200, PRA). A, smooth, inconspicuous thallus with
orange pruinose apothecia. B, cross-section of apothecium with detailed exciple structure. C, cross-section of apothecium viewed using a polarized filter. D, cross-section
of apothecium with yellow clusters of crystals arranged in the lateral part of the exciple viewed using a polarized filter. Scales: A = 1 cm; B–D = 100 μm. In colour online.
292 Julia V. Gerasimova et al.
https://doi.org/10.1017/S0024282923000385 Published online by Cambridge University Press

prominent yellow coloration of the apothecia with darker, almost
black, thinner margin.
Type: Russia, Adygea, Maykopskiy Rayon, im Sachraital,
44.12ʹN, 40.33ʹE, 715 m a.s.l., on the bark of Corylus avellana,
17 September 2015, V. Otte s. n. (GLM-L-0048447—holotype).
(Fig. 5)
Thallus indeterminate, thin, rimose, partly smooth, mainly consist-
ing of single or contiguous, more or less roundish or irregularly
shaped warts. Warts ±flat, adnate to and only slightly raised
above the surface; when spreading on mosses, the thallus is more
or less granular; green-grey to dark green. Prothallus presents as
a black line bordering the thallus. Photobiont chlorococcoid.
Apothecia 0.7–1.1 mm diam. (n
1
=1, n
2
= 8), ±flat or with a
margin slightly above the disc. Disc dark yellow to orange, slightly
pruinose. Margin dark brown to black, covered by thick white
pruina. Hymenium 84–125–150 μm tall (n
1
=1,n
2
= 5), with crys-
tals in the upper part ( from pruina) not dissolving in K and
N. Epithecium pale orange, almost colourless. Hypothecium
straw-coloured, almost colourless. Exciple 37–42.5 μm wide
(n
1
=1, n
2
= 4), without or with minor crystals along the rim
(from pruina) not dissolving in K and N. Rim dark brown,
with two layers of enlarged more or less globose lumina cells,
5–8μm wide and 7–10 μm long (n
1
= 55, n
2
= 10); the lateral
part brown to dark brown, paler closer to hymenium, with crystals
less than 0.5 μmorupto5μm, not dissolving in K and
N. Medullary part under hypothecium pale straw to almost
colourless. Paraphyses simple, 1–2.5 μm wide with apices ±clavate
or not swollen. Ascospores acicular, (48–)57.8 ± 9.6(–71) μm long
and (2–)3.3 ± 0.6(–5) μm wide (n
1
=1,n
2
= 44), with (3–)10 ± 4(–
15) septa (n
1
=1, n
2
= 44).
Chemistry. Hypothecium and exciple K+ yellow; brown parts of
exciple N+ purplish brown.
Pigments. Rubella-orange in epithecium; Laurocerasi-brown in
rim and lateral part of exciple.
Etymology. The epithet ‘caucasica’refers to the locality where the
species was collected.
Habitat and distribution. The specimen was collected in a
moist, mixed forest in a valley on the bark of Corylus avellana.
The species is so far known only from a single collection in the
Caucasus area.
Comments. Bacidia caucasica differs from the closely related
B. suffusa primarily by abundant colourless crystals along the
exciple edge and upper hymenium, the prominent yellow color-
ation of the apothecia with darker, almost black, thinner margin,
and a thallus consisting of single or contiguous, more or less
roundish or irregularly shaped warts (Fig. 5).
Figure 5. Cross-section of apothecia and thallus structure of Bacidia caucasica (GLM-0048447, holotype). A, general overview of apothecia and thallus structure with the distinct
black prothallus. B, detail view of apothecia and thallus structure, yellow apothecia with a dark pruinose margin. C, clusters of crystals in the upper part of hymenium. D,
cross-section of apothecia with detailed exciple structure. E, acicular multiseptate spore. Scales: A = 3 cm; B = 1 cm; C–D = 100 μm; E = 10 μm. In colour online.
The Lichenologist 293
https://doi.org/10.1017/S0024282923000385 Published online by Cambridge University Press

Bacidia heterochroa (Müll. Arg.) Zahlbr.
Bacidia heterochroa is distributed throughout the tropics and
subtropics (Ekman 1996). It is recorded in India, Central and
South America, Thailand, Macaronesia and North America
(Ekman 1996; Breuss 2001,2018; Aptroot et al. 2007; Diederich
&Lawrey2007; Joseph & Sinha 2012; Etayo & Berger 2013;
Aptroot & Spielmann 2020), and it has recently been found in
South Korea (Lee & Hur 2022). Here, we report B. heterochroa
for the Caucasus and Russia for the first time, representing
the northernmost subtropical locality for the species. The
individuals of B. heterochroa from the Caucasus were collected
in the warm-wet Colchic region in a humid floodplain
locality. Interestingly, these individuals were nested in the
clade, sister to the Suffusa and Schweinitzii groups, which are
also particularly abundant in the oceanic and temperate warm
climates of Asia and North America (Ekman 1996; Lendemer
et al. 2016).
Bacidia heterochroa collected in the present study is character-
ized by having one layer of enlarged lumina cells along exciple
edge (up to 6 × 8 μm); a thallus whitish, grey to green-grey, con-
sisting of thin adnate warts, rimose; apothecia black or dark
brown with a margin of the same colour, paler below; epithecium
dark brown, almost black; exciple edge dark red-brown or
brown; lateral part pale brown, yellow-brown, brown to reddish
brown; medullar part yellow to nearly colourless, paler than
hypothecium; hypothecium yellow; hymenium (61.5–)85.6 ±
16.1(–122.5) μm; exciple (49–)68.4 ± 13.4(–88) μm. The charac-
teristics of thallus and apothecia correspond to those provided
by Ekman (1996) for North America. However, the ascospores
are shorter (22–56 × 2.5–4.3 μm) with up to 11 septa compared
to ascospores of B. heterochroa from North America that are
longer (32–73 × 2.5–4.3 μm) with up to 15 septa.
The studied type material of B. heterochroa is characterized by the
dark brown, brown-purplish epithecium with a distinct K+ purplish
reaction. In addition, the dark brown to black apothecia have
brighter brown margins, at least in the lower part towards the
base. These characteristics match those of B. heterochroa from the
Caucasus. The specimens of B. heterochroa from South Korea have
a dark brown epithecium (K+ purplish) and dark brown apothecia
with concolourous margins (Lee & Hur 2022). These characters
resemble the closely related B. laurocerasi s. str. and probably
represent a separate species. Nevertheless, B. heterochroa from the
Caucasus was retrieved on a very long branch, suggesting that add-
itional lineages may be missing from the analysis and that further
detailed examination of specimens is necessary.
Additional specimens examined. Georgia: Ochamchirskiy
Rayon: the Abkhazian Research Forest Experimental Station, 22
viii 2012, J. Gerasimova (LE L-11663, LE L-12975, LE L-12974,
M-0182575); ibid., 22 viii 2012, L. Gagarina (LE L-11635, LE
L-11621, LE L-11625, LE L-11626, LE L-11979); ibid., 23 viii
2012, L. Gagarina (LE L-11980). Gudautskiy Rayon: Ritsa Strict
Nature Reserve, 14 viii 2012, J. Gerasimova (LE L-11656).—
Russia: Krasnodarskiy Krai: Schachetal oberhalb von Solochaul,
24 viii 2016, V. Otte (GLM-L-0048864, GLM-L-0048863); ibid.,
25 viii 2016, V. Otte (GLM-L-0048909).
Acknowledgments. We are grateful to Elizabeth Joyce (LMU, Munich) for
improving the English text. IU and GU thank Nikolay Eskin (Caucasian
Nature Biosphere Reserve), Olga Bykhalova (Utrish Nature Reserve) and
Liza Khaykharoeva (Erzi Nature Reserve) for supporting their field research.
VO is grateful to Vladimir I. Karataev (Maykop) for two decades of field
assistance. JG was supported by a BAYHOST fellowship from the Bayerische
Staatsministerium für Bildung und Kultus, Wissenschaft und Kunst. The herb-
arium work at Senckenberg Museum für Naturkunde Görlitz (GLM) was sup-
ported by the Senckenberg Taxonomy Grant (2018). Molecular work was
supported by a grant to AB from the Bayerische Staatsministerium für
Wissenschaft und Kunst via the Staatliche Naturwissenschaftliche
Sammlungen Bayerns within the ‘SNSBinnovativ’framework. Fieldwork was
supported by the institutional research project ‘Flora and systematics of
algae, lichens, and bryophytes of Russia and phytogeographically important
regions of the world’(no. 121021600184-6) of the Komarov Botanical
Institute RAS to IU, and by a grant from the Russian Foundation for Basic
Research to GU (no. 15-29-02396). The herbarium collections of the Utrish
Reserve (LE) were revised with financial support to IU from the Ministry of
Education and Science (Agreement N 075-15-2021-1056).
Author Contribution. AB and JG designed the study; JG performed the
experiments and wrote the first draft of the manuscript; VO, IU and GU pro-
vided specimens; all authors contributed to the draft and revised the manuscript.
Author ORCIDs. Julia V. Gerasimova, 0000-0002-3212-3596; Irina
N. Urbanavichene, 0000-0002-5492-5215; Gennadii P. Urbanavichus,
0000-0003-3222-5151; Andreas Beck, 0000-0003-2875-4464.
Supplementary Material. The Supplementary Material for this article can
be found at https://doi.org/10.1017/S0024282923000385.
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