Diversification of the newly recognized lichen-forming fungal lineage Montanelia (Parmeliaceae, Ascomycota) and its relation to key geological and climatic events

Abstract

Unlabelled:
•

Premise of the study:
In spite of the recent advances in generic and species circumscriptions and in recognizing species diversity in lichen-forming fungi, the timing of speciation and the factors that promote diversification in lichens remain largely unexplored. We used brown parmelioids as a model to assess the timing of divergence and explore the impact of geological and climatic events on lineage divergence and diversification in lichenized fungi. Additionally, to clarify the phylogenetic position of the species currently placed in Melanelia disjuncta group, we evaluated the taxonomic status and phylogenetic relationships within Parmeliaceae. •

Methods:
Phylogenetic relationships and divergence time estimates were inferred from a four-loci data set. Alternative hypotheses were tested using Shimodaira-Hasegawa and expected likelihood weights tests. •

Key results:
The M. disjuncta group forms a strongly supported, monophyletic lineage independent from Melanelia s.s. The M. disjuncta clade arose ca. 23.1 million years ago (Ma). Our results suggest that most of the lineages within the clade diversified during the Miocene (17.6 to 11.2 Ma). The split of other brown parmelioids, such as Emodomelanelia-Melanelixia occurred ca. 41.70 Ma, and the radiation of Melanelixia began during the Eocene-Oligocene transition (ca. 33.75 Ma). •

Conclusions:
Montanelia is described here as a new genus to accommodate species of the Melanelia disjuncta group. Further, the study indicates that the current species delimitation within the newly described genus requires revision. We provide evidence of lineage divergence of Montanelia at the Oligocene-Miocene boundary. Our results indicate that the diversification during Miocene would have happened during major mountain uplifts.

Full text

2014
American Journal of Botany 99(12): 2014–2026, 2012; http://www.amjbot.org/ © 2012 Botanical Society of America
American Journal of Botany 99(12): 2014–2026. 2012.
Major climatic fl uctuations have occurred periodically through-
out the history of the Earth ( Dynesius and Jansson, 2000 ; Zachos
et al., 2001 a , b ; Jansson and Dynesius, 2002 ; Mosbrugger
et al., 2005 ; Zachos et al., 2008 ). Three of the largest climatic
anomalies occurred at the boundaries of Paleocene–Eocene
(~55 Ma), Eocene–Oligocene (~34 Ma), and Oligocene–Miocene
(~23 Ma) and have had a substantial impact on both marine and
terrestrial biota. The last major Oligocene–Miocene (~23 Ma)
climatic aberration consists of a brief but deep (200 kyr) glacial
maximum known as Mi-1 ( Naish et al., 2001 ; Zachos et al.,
2001b ). The Mi-1 glaciation incident stands out as a rare climatic
aberration, and it specifi cally coincides with a major boundary
characterized by accelerated turnover of several groups of ter-
restrial and marine biota ( Edinger and Risk, 1994 ; Zachos et al.,
2001b ; Williams and Duda, 2008 ; Postigo Mijarra et al., 2009 ).
This climatic change included retraction of boreotropical forests
that became relict fragments and were further isolated by the
rise of grassland systems in the late Miocene (see Millar, 1993 ;
Morley, 2000 ; Prothero, 2004 ; Bacon et al., 2012 ). Subsequently,
in the Mid-Miocene Climatic Optimum, the high latitudes of
the northern hemisphere gradually warmed and the tundra land-
scape was once again replaced by coniferous forests ( Prothero,
2004 ).
Lichenized fungi form obligate symbioses with their photo-
synthetic partners, mainly green algae and or cyanobacteria
( Hawksworth and Honegger, 1994 ). The largest number of
lichenized fungi is found in Ascomycota, a phylum that consists
of over 40% lichenized species ( Kirk et al., 2008 ). Currently, only
limited data are available on species turnover and diversifi ca-
tion events in lichen-forming fungi. Amo de Paz et al. (2011)
have shown that most of the parmelioid lineages diversifi ed
during globally changing climatic conditions of the early
1 Manuscript received 31 May 2012; revision accepted 22 October 2012.
The authors thank Trevor Goward for providing valuable material of two
species. They also thank editors and two anonymous reviewers for valuable
comments and suggestions, which improved the manuscript. This work
was supported by the Spanish Ministerio de Ciencia e Innovación (CGL
2010-21646/BOS) and Ramón y Cajal grant (RYC02007-01576) to PKD,
the Universidad Complutense-Banco Santander (GR 35/10A), the National
Science Foundation (“Hidden diversity in parmelioid lichens”, DEB-0949147),
and by the Swedish Research Council (VR621-2009-5372) to M.W.
Sequencing was performed in the Centro de Genómica y Proteómica del
Parque Científi co de Madrid, where Maria Isabel García Saez is especially
thanked, and in the Laboratory for Molecular Systematics at the Swedish
Museum of Natural History.
6 Author for correspondence (e-mail: pdivakar@farm.ucm.es)
doi:10.3732/ajb.1200258
D IVERSIFICATION OF THE NEWLY RECOGNIZED LICHEN-FORMING
FUNGAL LINEAGE M ONTANELIA (PARMELIACEAE, ASCOMYCOTA)
AND ITS RELATION TO KEY GEOLOGICAL AND CLIMATIC EVENTS 1
P RADEEP K. DIVAKAR 2,6 , R UTH D EL-PRADO 2 , H. THORSTEN L UMBSCH 3 , M ATS W EDIN 4 ,
T HEODORE L. ESSLINGER 5 , S TEVEN D. LEAVITT 3 , AND A NA C RESPO 2
2 Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 28040 Spain;
3 Department of Botany, The Field Museum, 1400 S. Lake Shore Drive, Chicago, Illinois 60605 USA;
4 Cryptogamic Botany,
Swedish Museum of Natural History, P.O. Box 50007 SE-104 05 Stockholm, Sweden; and
5 North Dakota State University,
Department of Biological Sciences 2715, PO Box 6050, Stevens Hall, Fargo, North Dakota 58108-6050 USA
• Premise of the study: In spite of the recent advances in generic and species circumscriptions and in recognizing species diversity
in lichen-forming fungi, the timing of speciation and the factors that promote diversifi cation in lichens remain largely unex-
plored. We used brown parmelioids as a model to assess the timing of divergence and explore the impact of geological and
climatic events on lineage divergence and diversifi cation in lichenized fungi. Additionally, to clarify the phylogenetic position
of the species currently placed in Melanelia disjuncta group, we evaluated the taxonomic status and phylogenetic relationships
within Parmeliaceae.
• Methods: Phylogenetic relationships and divergence time estimates were inferred from a four-loci data set. Alternative hypotheses
were tested using Shimodaira-Hasegawa and expected likelihood weights tests.
• Key results: The M. disjuncta group forms a strongly supported, monophyletic lineage independent from Melanelia s.s. The
M. disjuncta clade arose ca. 23.1 million years ago (Ma). Our results suggest that most of the lineages within the clade diversifi ed
during the Miocene (17.6 to 11.2 Ma). The split of other brown parmelioids, such as Emodomelanelia - Melanelixia occurred ca.
41.70 Ma, and the radiation of Melanelixia began during the Eocene–Oligocene transition (ca. 33.75 Ma).
• Conclusions: Montanelia is described here as a new genus to accommodate species of the Melanelia disjuncta group. Further,
the study indicates that the current species delimitation within the newly described genus requires revision. We provide evidence
of lineage divergence of Montanelia at the Oligocene–Miocene boundary. Our results indicate that the diversifi cation during
Miocene would have happened during major mountain uplifts.
Key words: dated phylogenies; lichens; Mi-1 glaciation; Montanelia ; multilocus phylogenies; new genus; uncorrelated relaxed
molecular clock.

2015
December 2012] DIVAKAR ET AL.—DIVERSIFICATION IN BROWN PARMELIOID LICHENS
Oligocene, Miocene, and early Pliocene, and Leavitt et al. (2012a)
have demonstrated Miocene and Pliocene dominated diversifi -
cation of the genus Melanohalea . The low number of published
studies on the timing of diversifi cation events in lichen-forming
fungi is mainly due to the poor fossil record for fungi in gen-
eral, including lichen-forming groups, and uncertainties in the
interpretation of the few known fossil records ( Taylor and Berbee,
2006 ; Lücking et al., 2009 ; Berbee and Taylor, 2010 ). Advances
in molecular phylogenetics have made it possible to estimate
divergence dates from molecular genetic data with increasing
levels of accuracy; however, these estimates still depend heav-
ily on the paltry fossil evidence ( Welch and Bromham, 2005 ;
Taylor and Berbee, 2006 ; Lücking et al., 2009 ; Berbee
and Taylor, 2010 ; Magallón, 2010 ; Amo de Paz et al., 2011 ;
Sérusiaux et al., 2011 ; Leavitt et al., 2012a ).
Molecular dating of phylogenies complements paleontologi-
cal and geological data in studying the relationship between
biotic diversifi cation and climatic variation ( Benner et al., 2002 ).
Striking examples of major radiation events near the Oligo-
cene–Miocene boundary, a period dominated by dramatic cli-
matic change ( Delsuc et al., 2004 ; Steiner et al., 2005 ; Poux
et al., 2006 ; Suto, 2006 ; Besnard et al., 2009 ; Bacon et al.,
2012 ), have been documented in angiosperm families such as
Oleaceae and Trachycarpeae ( Besnard et al., 2009 ; Bacon et al.,
2012 ), diatoms such as Chaetoceros ( Suto, 2006 ), and mam-
mals such as sloths, armadillos, didelphid marsupials, and cav-
iomorph rodents ( Delsuc et al., 2004 ; Steiner et al., 2005 ; Poux
et al., 2006 ) . The environmental changes driving this burst
of diversifi cation in angiosperms, diatoms, and mammals would
likely have left their mark on other organisms, including fungi,
as well. However, the impact of the dramatic climatic changes
near the Oligocene–Miocene boundary on diversifi cation in
lichen-forming fungi remains unclear.
Molecular studies have revolutionized the generic circum-
scriptions in Parmeliaceae and in lichen forming fungi in
general (reviewed by Printzen, 2010 ; Crespo et al., 2011 ). Par-
meliaceae is one of the largest, most studied and widely distrib-
uted families of lichenized Ascomycota ( Crespo and Cubero,
1998 ; Wedin et al., 1999 ; Crespo et al., 2001 , 2007 , 2010a ;
Blanco et al., 2006 ; Divakar et al., 2006 , 2010a ; Gutierrez et al.,
2007 ; Lumbsch et al., 2008 ; Del-Prado et al., 2010 ). Seven
main monophyletic groups have been circumscribed: parmel-
ioid, cetrarioid, alectorioid, psiloparmelioid, hypogymnioid, and
letharioid ( Crespo et al., 2007 ). Brown parmelioid lichens, in-
cluding Emodomelanelia , Melanelixia , Melanohalea , and the
Melanelia disjuncta group belong to the monophyletic group of
parmelioid lichens (Parmeliaceae, Ascomycota) ( Blanco et al.,
2004 ; Crespo et al., 2007 , 2010a ). Melanelixia and Melano-
halea are recent segregates of Melanelia s.l., based on molecular
and morphological data ( Blanco et al., 2004 ). More recently, a
monotypic genus Emodomelanelia endemic to the Himalayas
was described as new and placed in this group based on molecular
data ( Crespo et al., 2010a ). Similarly, other lineages, e.g., Aus-
troparmelina and Remototrachyna are recent segregates from
Parmelina and Hypotrachyna s.l., respectively ( Crespo et al.,
2010b ; Divakar et al., 2010a ). These studies highlight the utility
of molecular data for robust taxonomic revisions in lichenized
fungi.
The genus Melanelixia includes 15 species occurring mainly
in temperate regions of the northern and southern hemispheres
that grow on bark and wood ( Esslinger, 1977 ; Elix, 1994 ;
Divakar and Upreti, 2005 ; Otte et al., 2005 ; Crespo et al., 2010a ;
Divakar et al., 2010b ). Melanohalea consists of 22 species
mainly distributed in the northern hemisphere and which also
grow primarily on bark and wood ( Esslinger, 1977 ; Divakar
et al., 2001 ; Blanco et al., 2004 ; Otte et al., 2005 ; Zhao et al.,
2009 , Crespo et al., 2010a ; Sun et al., 2010 ; Leavitt et al.,
2012c ). The Melanelia disjuncta group also belongs to the
group of brown parmelioid lichens ( Crespo et al., 2010a ). The
group includes fi ve species ( M. disjuncta , M. panniformis ,
M. predisjuncta , M. sorediata , and M. tominii ) largely restricted
to the alpine or montane regions of the northern hemisphere
( Esslinger, 1977 ; Blanco et al., 2004 ; Crespo et al., 2010a ). The
type species of the genus Melanelia , M. stygia, is closely re-
lated to Cetraria and does not belong to the monophyletic group
of parmelioid lichens ( Blanco et al., 2004 ; Crespo et al., 2007 ,
2010a ; Thell et al., 2009 ; Nelsen et al., 2011 ). In previous stud-
ies, only one species of the M. disjuncta group ( M. disjuncta )
was studied ( Blanco et al., 2004 ; Crespo et al., 2007 ), with the
exception of one study by Crespo et al. (2010a) in which three
species of the group were included. However, the relation-
ships of the group remained unresolved. Furthermore, the
taxonomic status of M. disjuncta group was not resolved in
the phylogenetic generic classifi cation of parmelioids by Crespo
et al. (2010a) .
Our specifi c goals were to (1) evaluate the taxonomic status
of M. disjuncta group and its phylogenetic relationship among
parmelioid lichens and (2) estimate the timing of diversifi cation
for this group, as well for other brown Parmeliaceae to eluci-
date potential factors (i.e., tectonic and climatic events) driving
diversifi cation.
MATERIALS AND METHODS
Taxon sampling — Molecular analyses were based on 61 specimens (50 spe-
cies) gathered from wide geographic regions ( Table 1 ). In this study, the sampling
included the main clades of the monophyletic parmelioid group and four genera
of the monophyletic cetrarioid core (see Crespo et al., 2010a ). Species of the
cetrarioid core were used as the outgroup, since these genera have previously
been shown to be related to the monophyletic parmelioid group ( Crespo et al.,
2010a ). The sampling focused on the brown parmelioid Melanelia disjuncta
group that includes M. disjuncta , M. panniformis , M. predisjuncta , M. sorediata ,
and M. tominii , and we included four of the fi ve described species in this group
( Esslinger, 1977 ; Crespo et al., 2010a ). A total of nine samples of Melanelia
disjuncta group collected from wide geographic regions, including Canada
(2 specimens), India (2), United States (1), Sweden (3), and United Kingdom
(1). Seventeen of the 37 described species of other brown parmelioid genera
( Melanelixia , Melanohalea ) were also included. DNA sequences of nuclear
ribosomal internal transcribed spacer (ITS), nuclear ribosomal large subunit
(nuLSU), mitochondrial small subunit rDNA (mtSSU) and a fragment of the
protein-coding MCM7 gene were assembled for all collections. Details of the
studied material, including GenBank accession numbers are shown in Table 1 .
DNA isolation, PCR amplifi cation, and sequencing — Small samples
(ca. 2 mm
2 ) prepared from freshly collected and frozen specimens were ground
with sterile plastic pestles. Total genomic DNA was extracted using the DNeasy
Plant Mini Kit (Qiagen, Valencia, California, USA) according to the manufac-
turer’s instructions but with slight modifi cations ( Crespo et al., 2001 ). Genomic
DNA (5–25 ng) was used for PCR amplifi cations of the ITS, nuLSU, mtSSU,
and MCM7 regions. Standard PCR amplifi cations were conducted in 50-µL
reaction volumes. In some cases, when standard PCR failed to amplify target
loci, we used Ready-To-Go PCR Beads (GE Healthcare, Little Chalfont, UK)
and the manufacturer’s recommendations with markedly improved success.
Primers, PCR and cycle sequencing conditions for nuclear ITS, nuLSU, and
mtSSU were the same as described previously ( Divakar et al., 2005 ; Crespo
et al., 2007 ). Primers Mcm7-709for and Mcm7-1348rev ( Schmitt et al., 2009 )
were used to amplify the MCM7 marker. PCR amplifi cations were carried
out in a Techne R TC-3000 thermal cycler, following conditions: one initial
heating step of 5 min at 94 ° C, followed by a touchdown step of 6 cycles of 45 s
at 94 ° C, 50 s at 58 ° C, and 1 min at 72 ° C, decreasing 1 ° C each cycle the annealing

2016 AMERICAN JOURNAL OF BOTANY [Vol. 99
T ABLE 1. Specimens used in the study, with location, reference collection detail, and GenBank accessions. Newly obtained sequences for this study are in boldface.
Species Locality Collector(s) Voucher specimens
GenBank accession
ITS mtSSU nuLSU MCM7
Austroparmelina endoleuca Australia: Australian Capital Territory Elix 38802 Herb Elix GU183185 GU183192 GU183178 JX974673
Austroparmelina macrospora Australia: Western Australia Elix 32408 Herb Elix GU183187 GU183194 GU183180 JX974674
Austroparmelina pruinata Australia: Western Australia E. McCrum s.n. MAF-Lich 14270 EF042905 EF025481 EF042914 JX974675
Austroparmelina pseudorelicina Australia: New South Wales Amo de Paz 1159 MAF-Lich 16115 GU183188 GU183195 GU183181 JX974676
Bulbothrix apophysata Costa Rica: San Jose Lücking 16650b F DQ279481 DQ287788 EU562670 GQ272392
Cetraria islandica Sweden: Västerbotten Wedin 15/5/05 UPS AF117995 AY340486 AY340539 JX974677
Cetrariastrum andense Peru: Ancash Lumbsch, Wirtz & Ramirez 19334 F (MAF-Lich 15620) GQ919269 GQ919217 GQ919245 GQ272429
Cetrariastrum dulitens Peru: Ancash Lumbsch, Wirtz & Ramirez 19366 F (MAF-Lich 15621) GQ919270 GQ919218 GQ919246 GQ272427
Cetrariella delisei Sweden: Västerbotten Wedin 6351 UPS DQ980005 DQ923628 DQ923657 JX974679
Everniastrum nepalense India: Uttaranchal Divakar s.n. GUH 02-000924 AY611071 AY611129 AY607783 JX974680
Emodomelanelia masonii 1 India: Uttaranchal Divakar s.n. MAF-Lich 15515 GU994549 GU994640 GU994595
JX974681
Emodomelanelia masonii 2 India: Uttaranchal Divakar s.n. MAF-Lich 17602 JX974653 JX974660 JX974667 JX974682
Flavocetraria nivalis Sweden: Jämtland Wedin 5052 BM DQ980011 DQ923635 DQ923663 JX974683
Flavoparmelia marchantii Australia: Western Australia Elix s.n. MAF-Lich 10492 DQ299905 GU994642 GU994598 GQ272420
Flavoparmelia soredians Spain: Cáceres Crespo et al. s.n. MAF-Lich 10176 AY586562 AY586586 AY584835 JX974684
Melanelia disjuncta 1 Sweden: Lycksele Lappmark Wedin 7143 UPS DQ980015 DQ923638 DQ923666 JX974699
Melanelia disjuncta 2 UK: Scotland Coppins s.n. MAF-Lich 17227 JX974654 JX974661 — JX974700
Melanelia hepatizon Sweden: Västerbotten Wedin 6812 UPS DQ980016 DQ923639 DQ923667 JX974678
Melanelia panniformis 1 Canada: British Columbia Goward 07 Herb Goward JX974655 JX974662 JX974668 JX974701
Melanelia panniformis 2 Sweden: Hälsingland
Wedin 8285 S JX974656 JX974663 JX974669 JX974702
Melanelia panniformis 3 USA: Washington Esslinger 18643 NDA JX974657 JX974664 JX974670 JX974703
Melanelia sorediata 1 India: Uttaranchal Divakar s.n. MAF-Lich 15512 GU994556 GU994645 GU994604 JX974704
Melanelia sorediata 2 Sweden: Vasterbotten Wedin 6862 UPS GU994557 — GU994605 JX974705
Melanelia sorediata 3 Canada: British Columbia Goward 08 Herb Goward JX974658 JX974665 JX974672 JX974706
Melanelia stygia Sweden: Hälsingland Wedin 5080 BM AY611121 DQ923640 AY607835 —
Melanelia tominii India: Uttaranchal Divakar s.n. MAF-Lich 15516 GU994559 GU994647 — JX974707
Melanelixia fuliginosa 1 Spain: La Rioja Crespo et al. s.n. MAF-Lich 10219 AY611086 AY611143 AY607798 JX974685
Melanelixia fuliginosa 2 Spain: La Rioja Blanco s.n. MAF-Lich 10223 AY611089 AY611146 AY607801 JX974686
Melanelixia fuliginosa 3 USA: California Robertson 7140 NDA AY611117 AY611173 AY607831 JX974687
Melanelixia glabra Spain: Salamanca Hawksworth s.n. MAF-Lich 7634 AY582300 AY581064 AY578927 —
Melanelixia glabratuloides New Zealand: Otago Knight s.n. OTA 60606 — GU994652 GU994608 JX974688
Melanelixia pilliferella Australia: Australian Capital Territory Elix 28254 MAF-Lich 15374 GU994563 GU994653 GU994609 JX974689
Melanelixia subaurifera1 USA: California Robertson 7138 NDA AY611118 AY611174 AY607832 JX974690
Melanelixia subaurifera 2 Spain: La Rioja Blanco s.n. MAF-Lich 10216 AY611101 AY611158 AY607813 JX126391
Melanelixia subaurifera 3 UK: England, London Crespo s.n. MAF-Lich 10215 AY611095 AY611156 AY607811 JX126390
Melanelixia subglabra New Zealand: Otago Knight s.n. OTA 60604 GU994564 GU994654 GU994610 JX974691
Melanelixia villosella India: Uttaranchal Divakar s.n. MAF-Lich 15516 GU994565 GU994655 GU994611 JX974692
Melanohalea elegantula 1 Spain: Madrid Crespo s.n. MAF-Lich 10231 AY611094 AY611151 AY607806 JX974693
Melanohalea elegantula 2 Spain: Madrid Crespo & Divakar s.n. MAF-Lich 10224 AY611080 AY611137 AY607792
JX974694
Melanohalea exasperata Spain: Guadalajara Blanco s.n. MAF-Lich 10214 AY611081 AY611138 AY607793 JX974695
Melanohalea exasperatula USA: Oregon Esslinger 16554 NDA AY611119 AY611175 AY607833 JX974696
Melanohalea olivacea Finland: Puolanca Vitikainen 16196 H AY611091 AY611148 AY607811 —
Melanohalea septentrionalis Finland: Keski-Pohjanmaa Athi 60893 H AY611093 AY611150 AY607805 JX974697
Melanohalea subelegantula USA: Oregon Esslinger 16132 NDA AY611115 AY611171 AY607829 —
Melanohalea subolivacea USA: Oregon Esslinger 16555 NDA AY611123 AY611178 AY607837 —
Melanohalea trabeculata Canada: British Colombia Goward 13 Herb Goward JX974659 JX974666 JX974672 JX974698
Myelochroa irrugans China: Yunnan Crespo & al. s.n. MAF-Lich 10207 AY611103 AY611160 AY607815 JX974708
Parmelia saxatilis Sweden: Västerbotten Wedin 7091 UPS AF058037 AF351172 AY300849 JX974709
Parmelia serrana Spain: Madrid Crespo & Divakar s.n. MAF-Lich 9756 AY295109 AY582319 AY578948 JX974710
Parmeliopsis hyperopta Spain: Madrid Blanco s.n. MAF-Lich 10181 AY611109 AY611167 AY607823 JX974711
Parmotrema reticulatum China: Yunnan Crespo, Blanco & Argüello s.n. MAF-Lich 10164 AY586577 AY586599 AY584848 JX974712
Parmotrema subtinctorium India: Uttaranchal Divakar s.n. GUH 02-000696 AY586558 AY586582 AY584830 JX974713

2017
December 2012] DIVAKAR ET AL.—DIVERSIFICATION IN BROWN PARMELIOID LICHENS
temperature, after 34 cycles of 45 s at 94 ° C, 50 s at 52 ° C, and 1 min at 72 ° C.
A fi nal extension step of 5 min at 72 ° C was added, after which the samples were
kept at 4 ° C. PCR products were purifi ed using FavorPrep Gel/PCR Purifi cation
MiniKit (Favorgen R Biotech, Kaohsiung, Taiwan) following the manufacturer’s
instructions. Both complementary strands were sequenced using the ABI Prism
Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems,
Foster City, California, USA) with the same primers used in the amplifi cation
step. Sequencing reactions were electrophoresed on a 3730 DNA analyzer
(Applied Biosystems) at the Unidad de Genómica (Parque Científi co de Madrid).
Sequence alignments — Sequence fragments generated for this study were
assembled and edited using the program SeqMan v.7 (Lasergene R, DNASTAR,
Madison, Wisconsin, USA). Sequence identity was confi rmed using the mega-
BLAST search function in GenBank ( Wheeler et al., 2007 ). We used the pro-
gram MUSCLE ( Edgar, 2004 ) to align DNA sequences of 61 specimens ( Table 1 )
for each data set separately. Although alignments for nuLSU and protein-coding
MCM7 markers were relatively straightforward, the ITS and mtSSU alignments
contained a number of ambiguous regions. The program Gblocks v0.91b
( Castresana, 2000 ; Talavera and Castresana, 2007 ) was used to remove
regions of alignment uncertainty, using options for a “less stringent” selection
on the Gblocks web server (http://molevol.cmima.csic.es/castresana/Gblocks_
server.html).
Phylogenetic analyses — The alignments were analyzed using maximum
parsimony (MP), maximum likelihood (ML), and a Bayesian Markov chain
Monte Carlo approach (B/MCMC).
We examined nodes to identify well-supported (ML bootstrap values >
70%) confl icts among the individual ITS, nuLSU, mtSSU, and MCM7 phylog-
enies before combining the alignments ( Mason-Gamer and Kellogg, 1996 ;
Lutzoni et al., 2004 ). Recent studies have shown that the incongruence length
difference (ILD) test ( Farris et al., 1994 ) has limited utility for detecting incon-
gruence among independent loci ( Barker and Lutzoni, 2002 ; Dowton and Austin,
2002 ). Therefore, we used an MP approach to examine the heterogeneity in
phylogenetic signal among the four loci ( Wiens, 1998 ; Buckley et al., 2002 ;
Divakar et al., 2010a ). Individual gene topologies were reconstructed using MP
in the program PAUP* version 4.0b10.0 ( Swofford, 2003 ). Heuristic searches
with 1000 random taxon addition replicates were conducted with tree-bisec-
tion-reconnection (TBR) branch swapping and MulTrees option in effect, and
all characters were equally weighted and gaps treated as missing data. Nodal
support was assessed with 2000 bootstrap replicates. We compared individual
gene topologies to identify confl icting ( ≥ 70% bootstrap) nodes ( Hillis and Bull,
1993 ; Wiens, 1998 ). If no confl ict was evident, it was assumed that the four data
sets were congruent and could be combined.
A MP analysis of the combined four-loci data set (ITS, nuLSU, mtSSU, and
MCM7) was performed using the program PAUP* version 4.0b10.0 ( Swofford,
2003 ). Heuristic searches with 1000 random taxon addition replicates were
conducted with TBR branch swapping and MulTrees option in effect, all char-
acters were equally weighted and gaps were treated as missing data. Bootstrap-
ping ( Felsenstein, 1985 ) was performed using 2000 pseudoreplicates with random
sequence additions. To assess homoplasy levels, we calculated the consistency
index (CI) and retention index (RI) from each parsimony search.
A ML analysis of the combined four-loci data set was performed using an
online version of the program RaxML v7.0.4 ( http://phylobench.vital-it.ch/
raxml-bb/ ; Stamatakis, 2006 ; Stamatakis et al., 2008 ). We used the GTR-
GAMMA model, which includes a parameter ( Γ ) for rate heterogeneity among
sites and chose not to include a parameter for estimating the proportion of
invariable sites ( Stamatakis, 2006 ; Stamatakis et al., 2008 ). All loci were treated
as separate partitions, and for the protein-coding marker we used a three-partition
approach, using the fi rst, second, and third codon positions as separate model
partitions. Nodal support was evaluated using 1000 bootstrap pseudoreplicates.
The concatenated data sets were also analyzed with Bayesian inference as
implemented in MrBayes v3.1.2 ( Huelsenbeck and Ronquist, 2001 ). Models of
DNA sequence evolution for each locus were selected with the program jMod-
eltest v0.1 ( Posada, 2008 ), using the Akaike information criterion (AIC; Akaike,
1974 ). The protein-coding MCM7 fragment was partitioned as described
already, assuming that the fi rst, second, and third codon positions had the same
overall model as the entire gene, because simulations have shown that there
may be frequent errors supporting more complex models from a sample of lim-
ited characters ( Posada and Crandall, 2001 ). The concatenated four-loci data set
was partitioned as described in the ML analysis, specifying the best fi tting
model, allowing unlinked parameter estimation and independent rate variation.
The Metropolis-coupled Markov chain Monte Carlo (MC
3 ) consisted of two
TABLE 1. Continued.
Species Locality Collector(s) Voucher specimens
GenBank accession
ITS mtSSU nuLSU MCM7
Pleurosticta acetabulum Spain: Guadalajara Crespo et al. s.n. MAF-Lich 9914 AY581087 AY582323 AY578953 —
Relicina sydneyensis Australia: Queensland Lumbsch 19179a F GU994581 GU994675 GU994630 JX974714
Remototrachyna crenata China: Yunnan Crespo, Blanco & Arguello s.n. MAF-Lich 10377 DQ279495 DQ287804 EU562683 JX974715
Tuckermannopsis chlorophylla Sweden: Västerbotten Wedin 6995 UPS DQ980025 DQ923647 DQ923674 JX974716
Xanthoparmelia ovealmbornii South Africa: Matroosberg Crespo et al. s.n. MAF-Lich 14268 EF042901 EF025479 EF042911 JX974717
Xanthoparmelia isidiovagans Spain: Guadalajara Crespo & Divakar s.n. MAF-Lich 9956 AY581094 AY582330 AY578960 JX974718
Xanthoparmelia stenophylla Spain: Guadalajara Crespo et al. s.n. MAF-Lich 9917 AY581093 AY582329 AY578959 JX974719
Xanthoparmelia tinctina Spain: Gerona Llimona s.n. BCN-13862 AY581110 AY582345 AY578978 JX974720
Vulpicida pinastri Sweden: Uppland Mattsson 4004 UPS AF058039 DQ923648 DQ923675 JX974721

2018 AMERICAN JOURNAL OF BOTANY [Vol. 99
Dating analyses were also done using the median age of the fossil rather than
the minimum age. Since the resultant chronogram was identical to the tree
obtained using the minimum age and no signifi cant differences were found in the
divergence times (Appendix S1, see Supplemental Data with the online version of
this article), we selected minimum fossil age for the fi nal analyses. Further, esti-
mating the divergence time using minimum fossil age and bounding the crown
group by uniform prior—estimates taken from previous studies—has been shown
to be the most appropriate method and used in several studies (e.g., Ho and
Phillips, 2009 ; Magallón, 2010 ; Amo de Paz et al., 2011 ; Bacon et al., 2012 ;
De-Nova et al., 2012 ).
We also compared potential difference between using an exponential prior dis-
tribution and a lognormal prior for the analyses. Exploratory analyses provided
similar results between both analyses (results not shown), and we selected the
lognormal prior distribution for fi nal analyses because it has been shown to be the
most appropriate for modeling paleontological information ( Ho, 2007 ; Ho and
Phillips, 2009 ).
Since parmelioid lichens largely lack fossil records, we also performed the
dating analyses using substitution rates. We estimated divergence times under a
gene-tree framework from the concatenated data matrix (i.e., ITS, nuLSU, mtSSU,
and MCM 7). In BEAST, the molecular data set was analyzed with unlinked substi-
tutions models across the loci, a birth–death model was used as a prior for the
node heights, and a relaxed clock model (uncorrelated log normal) for each
partition. We used molecular evolution rates for ITS locus recently reported for
Melanelixia , Parmeliaceae ( Leavitt et al., 2012b ). We used ITS rates (2.43 × 10 −9
substitutions·site
−1 ·yr −1 ) estimated for Melanelixia (Parmeliaceae) to estimate the
time to the most recent common ancestor (MRCA) for all clades. Substitution rates
for the nuLSU, mtSSU, and MCM 7 markers were coestimated along the run under
a uniform prior, relative to the previously published rates for the ITS locus.
The rate in each branch was drawn from either an exponential or lognormal
distribution.
We ran fi ve different dating analyses with different assumptions, and for
each dating analysis, two independent MCMC runs of 10 million generations
were conducted, sampling one tree every 1000 generations. The program Tracer
v1.5 ( Rambaut and Drummond, 2007 ) was used to evaluate each chain, deter-
mine appropriate burn-in cut-off (10% of sampled trees), and obtain the ESS for
each parameter. Convergence was also assessed using the program AWTY
( Nylander et al., 2007 ) to ensure that standard deviations of split frequencies
between runs approached zero and visualize split probabilities. An ESS of 200
or greater was considered appropriate. The two chains were combined (with
LogCombiner v1.6.2 and TreeAnotator v1.6.2; Drummond and Rambaut, 2007 )
to obtain the mean node heights posterior distributions of estimated divergence
dates. Mean node age and 95% highest posterior density (HPD) were mapped
on the maximum clade credibility tree. Only the strongly supported nodes obtained
in phylogenetic analyses ( Fig. 1 ) were considered as a working hypothesis of
divergence time estimates.
Morphological and chemical studies — Thallus morphology was studied using
a Leica Wild M 8 dissecting microscope to measure lobe shape, size and width. All
specimens of M. disjuncta group included in the molecular analysis were studied
(see Table 1 ). Vertical sections of apothecia were cut using a razor blade.
Ascospore sizes were observed in water. Sections obtained were observed by
light microscopy (Nikon Eclipse 80i). Chemical constituents were identifi ed
by thin-layer chromatography using standardized methods ( Orange et al., 2001 ).
RESULTS
Molecular data — In this study, we generated seven new ITS,
six nuLSU, seven mtSSU, and 49 MCM7 sequences ( Table 1 ).
The data sets include 151 sequences from previous publications
by our group ( Blanco et al., 2004 ; Divakar et al., 2006 ; Crespo
et al., 2010a , b ), and 13 downloaded from GenBank. The
complete four-loci aligned data matrix included 61 samples
independent runs of 2 million generations, starting with a random tree and
employing 12 simultaneous chains each, in which one in every 100 trees was
sampled. The outputs of MrBayes were examined with the program Tracer v1.5
( Rambaut and Drummond, 2007 ) to check for convergence of different param-
eters, determine the approximate number of generation at which log likelihood
values stabilized and identify the effective sample size (ESS) for each parame-
ter. Topological convergence in the two independent MCMC runs was checked
with the “compare” plots in the program AWTY ( Nylander et al., 2007 ). Posterior
probabilities (PPs) of clades were obtained from the 50% majority rule consen-
sus of sampled trees after excluding the initial 10% as burn-in. Phylogenetic
trees were drawn using the program FigTree v1.3.1 ( Rambaut, 2009 ).
Hypothesis testing — Since the results of the phylogenetic analyses were
incongruent with the current classifi cation of the genus Melanelia, which pres-
ently includes the M. disjuncta group, M. hepatizon , and M. stygia (the type
species); we tested whether our data were suffi cient to reject the monophyly of
M. disjuncta group + Melanelia s.s. For hypothesis testing, we compared the
ML tree constrained to recover M. disjuncta group + Melanelia s.s. as mono-
phyletic and the unconstrained ML tree. These trees were inferred in the pro-
gram Tree-PUZZLE ( Schmidt et al., 2002 ) employing the GTR+I+G nucleotide
substitution model. We used two methods to compare the different topologies:
the Shimodaira–Hasegawa test (SH; Shimodaira and Hasegawa, 1999 ) and the
expected likelihood weight test (ELW; Strimmer and Rambaut, 2002 ). The SH
and ELW tests were performed using the program Tree-PUZZLE 5.2 ( Schmidt
et al., 2002 ) with the combined data set on a sample of 200 unique trees, the
best trees agreeing with the null hypotheses and the unconstrained ML tree.
Divergence time estimates — The dating analysis was based on the taxon
sampling and sequence data used in phylogeny estimation. Ages were estimated
using an uncorrelated Bayesian relaxed molecular clock model implemented in
the program BEAST v1.6.2 ( Drummond et al., 2006 ; Drummond and Rambaut,
2007 ). We used a user-specifi ed chronogram as the starting tree, rather than a
randomly generated tree ( Amo de Paz et al., 2011 ). To generate the starting
topology, we conducted a ML analysis of the four-loci data set using the pro-
gram Garli 0.96 ( Zwickl, 2006 ). The ML topology was converted to an ultra-
metric tree using nonparametric rate smoothing (NPRS) implemented in the
program TreeEdit v10a10 ( Rambaut, 2007 ), with the divergence of parmelioid
node set at 60.28 Ma (mean age estimates taken from Amo de Paz et al., 2011 )
for the BEAST analyses. In BEAST, the partitioned molecular data set was ana-
lyzed with unlinked substitutions models across the loci, and a relaxed clock
model (uncorrelated lognormal) for each partition. A Yule prior was assigned
to the branching process. DNA sequence evolution model for each locus were
selected with the program jModeltest v0.1 ( Posada, 2008 ), using the AIC. Two
calibration points were used: (C1) prior estimates of the crown group of parme-
lioids of 49.81–73.55 Ma ( Amo de Paz et al., 2011 ), and (C2) the diversifi cation
node of Parmelia was calibrated with fossils from the Dominican amber
( Parmelia ambra , 15–45 Ma; Poinar et al., 2000 ). The height of the root node,
corresponding to the crown parmelioid node, was bounded by a uniform prior
between 49.81 and 73.55 Ma. One internal node was constrained with fossil-
derived minimum age and the lognormal prior distribution was set on the Par-
melia fossil calibration. The lognormal distribution has been shown to be the most
appropriate for modeling paleontological information because lineage origination
should not postdate the fossil occurrence ( Ho and Phillips, 2009 ). The lognormal
mean was equal to (fossil age + 10%) Ma, because a clade’s node age is older that
its oldest fossils, and the zero offset was equal to (fossil age – 1%) Ma, to ensure
that the minimum age falls within the distribution ( Magallón, 2010 ).
Assigning fossils to extant groups is crucial in dating analyses, especially in
lichens with a sparse fossil record. Parmelia ambra is a fossil from the Dominican
amber resembling Parmelia saxatilis and similar species ( Poinar et al., 2000 ). It
has terminal pseudocyphellae, elongate isidia, a plane to concave upper surface,
and simple to dichotomously branched rhizines. Since the dichotomously branched
rhizines are also present in other parmelioid groups, specifi cally the Hypotrachyna
clade, we ran the dating analyses under different scenarios. We assigned it to the
Parmelia clade and in a second analysis to the Hypotrachyna clade.
Fig. 1. Phylogenetic relationships among 50 species (61 samples) of Parmeliacae, representing the main parmelioid clades and including the new
genus Montanelia . Tree is based on the ITS, nuLSU, mtSSU, and MCM7 markers analyzed in a concatenated data matrix. Tree topology depicts the results
of the Bayesian Markov chain Monte Carlo (B/MCMC analysis. Posterior probabilities ≥ 0.95 are given below the branches, and values above the branches
are MP/ML bootstrap values ≥ 70%. Branches that received strong support in any of three analyses (MP, RaxML, and B/MCMC) are in boldface. Asterisk
mark depicts the type species of the genus Melanelia .
→

2019
December 2012] DIVAKAR ET AL.—DIVERSIFICATION IN BROWN PARMELIOID LICHENS

2020 AMERICAN JOURNAL OF BOTANY [Vol. 99
clades can be recognized: (1) clade A, includes three samples
of M. panniformis collected from Canada, Sweden, and North
America and one sample of M. sorediata collected from Sweden;
(2) clade B, contains two specimens of M. sorediata from
Canada and India and two samples of M. disjuncta from Europe.
Melanelia sorediata was thus polyphyletic and M. disjuncta
paraphyletic. The relationships of M. tominii within the group
remained unsupported.
Other morphologically similar brown parmelioid genera
clustered in the Melanohalea clade (see Crespo et al., 2010a ).
Emodomelanelia , Melanelixia , and Melanohalea formed well-
supported monophyletic groups each, and Emodomelanelia was
sister to Melanelixia (pp = 0.97). Melanohalea formed a sister-
group relation with Emodomelanelia + Melanelixia (pp = 0.95).
The Parmotrema clade was sister to the Xanthoparmelia clade,
and the two clades together formed a sister group to the Mel-
anohalea clade.
The SH and ELW tests both signifi cantly rejected monophyly
of M. disjuncta group + Melanelia s.s. ( P < 0.001 for both tests).
Divergence time estimates — The uncorrelated lognormal
dating analyses in BEAST yielded high ESS (>300) for all rel-
evant parameters (e.g., branch lengths, topology, clade posteri-
ors), indicating adequate sampling of the posterior distribution.
The maximum credibility chronograms calibrated from analy-
ses of the concatenated data set (ITS, nuLSU, mtSSU, MCM7 )
is presented in Fig. 2 . The tree is nearly identical to the MrBayes
consensus tree ( Fig. 1 ) in topology and posterior values. The
results obtained from the independent dating analyses that (1)
assigned the Parmelia ambra fossil to the Hypotrachyna clade
and (2) used substitution rates rather than fossil record are
shown in online Appendix S1. Since the obtained chronograms
were the same as Fig. 2 and no signifi cant differences were
obtained in the divergence time, the chronogram obtained from
the dating analyses assigning the Parmelia ambra fossil to the
Parmelia s.s. clade was used as a working hypothesis ( Fig. 2 ).
The nodes mean ages and divergence time estimates ranges
(95% highest posterior density intervals, HPD) for the brown
parmelioid clades are depicted in Table 3 . The split of Melano-
halea from a clade including Emodomelanelia and Melanelixia
is estimated at 46.17 Ma (95% HPD = 36.30–59.17); and the
split of Melanelixia from Emodomelanelia is estimated at 41.70
Ma (95% HPD = 31.29–54.41). The radiations of Melanohalea
and Melanelixia are estimated to be 38.46 Ma (95% HPD =
28.56–50.64) and 33.75 Ma (95% HPD = 23.88-45.24), respec-
tively ( Table 3 ). In the current study, the age of the ‘ Montanelia ’
clade is estimated to be 23.16 Ma (95% HPD = 13.59–34.87).
Results obtained from the combined four-loci data matrix suggest
that diversifi cation of the ‘ Montanelia ’ clade primarily occurred
during the Miocene and to a lesser extent during the Pliocene
( Fig. 2 , Table 3 )
Morphological and chemical studies — Diagnostic morpho-
logical, chemical, and ecological features to distinguish the
‘ Montanelia ’ clade from Melanelia s.s. ( M. stygia ), Emodomel-
anelia , Melanelixia , and Melanohalea are given in Table 4 . The
M. disjuncta group is distinguished from Melanelia s.s. ( M. stygia )
by several morphological features such as having short narrow
lobes, cylindrical to fusiform conidia, and orcinol depsides. The
M. disjuncta group can be distinguished from Melanelixia and
Melanohalea by having short narrow lobes, with plane to con-
vex lobe margins, and fl at, effi gurate pseudocyphellae. Further,
the species in the group are strictly saxicolous and occur in
consisting of 2573 unambiguously aligned nucleotide position
characters in the dataset ( Table 2 , http://treebase.org , TreeBase
study no. 13517). The ITS PCR product obtained ranged between
600 to 800 bp. Differences in size were due to the presence or
absence of insertions of about 200 bp identifi ed as group I
introns ( DePriest and Been, 1992 ; Gutierrez et al., 2007 ) at
the 3 ′ end of the SSU rDNA. We excluded group I introns and
163 bp of the mtSSU, 35 bp of the ITS1, and 20 bp of the ITS2
alignments from the analysis using GBlocks. The number of
unambiguous nucleotide positions in each data set, variations,
and the best-fi t model of evolution selected in jModeltest are
summarized in Table 2 . All sequences generated for this study
have been deposited in GenBank under accession JX974653–
JX974721 ( Table 1 ).
Phylogenetic reconstructions — Testing for topological
incongruence showed no strongly supported confl icts (re-
sults not shown) and hence, the concatenated four-loci data
matrix (ITS, nuLSU, mtSSU, and MCM7 ) was used for all
subsequent phylogenetic analyses. The MP analysis of the
combined data matrix resulted in 15 most parsimonious trees
(tree length = 3758 steps, CI = 0.385, RI = 0.574; trees not
shown). The partitioned ML analysis of the concatenated
data matrix yielded the optimal tree with ln likelihood value =
–22 048. Bayesian MCMC for the four combined loci reached
stationarity at ca. 200 000 generations. The effective sample
sizes (ESS) of all estimated parameters were well above
200. The “compare plot” produced by AWTY indicated that
parallel MCMC runs achieved topological convergence
( Nylander et al., 2007 ; results not shown). The mean LnL
value of the two parallel run of the Bayesian analysis was
–21 673. The 50% majority-rule consensus tree estimated
with Bayesian tree sampling is presented in Fig. 1 . The MP,
ML, and B/MCMC topologies were largely similar and did
not show any supported confl ict (i.e., PP ≥ 0.95 in B/MCMC
analysis and MP, ML bootstrap ≥ 70%), and the consensus
tree topology derived from the Bayesian analysis was used
as a working hypothesis of phylogenetic relationships. Nodal
support from the MP and ML analyses is indicated on the
Bayesian tree ( Fig. 1 ).
All species of the M. disjuncta group formed a statistically
supported, independent monophyletic clade, hereafter called
the ‘ Montanelia ’ clade, which was clustered in the monophyletic
parmelioid core ( Fig. 1 ). However, the type species of the genus
Melanelia s.s. ( M. stygia ) was recovered in the monophyletic
group of cetrarioid core and was sister to M. hepatizon ( Fig. 1 ).
The relationship of the ‘ Montanelia ’ clade among the mono-
phyletic group of parmelioid genera was not recovered with
confi dence. Within the ‘ Montanelia ’ clade, two supported
T ABLE 2. Genetic variability of the loci used in this study, including the
number of specimens ( N ), alignment length (number of base pairs),
variable and parsimony-informative (PI) sites for each sampled locus,
and locus-specifi c model of evolution identifi ed using the Akaike
information criterion in jModeltest.
Locus N
Aligned
length
No. of
variable sites
No. of
PI sites
Model
selected
ITS 60 451 210 167 GTR+I+G
nuLSU 59 826 195 135 GTR+I+G
mtSSU 60 751 263 156 TIM3+G
MCM 7 55 545 277 231 TIM1+I+G
Combined alignment 61 2573 945 689 NA

2021
December 2012] DIVAKAR ET AL.—DIVERSIFICATION IN BROWN PARMELIOID LICHENS
distinguished from Emodomelanelia in having short narrow
lobes, plane to convex, infl ated lobe margins, cylindrical to
fusiform conidia, and orcinol depsides.
montane and Arctic regions. Pseudocyphellae are absent in
Melanelixia , and in Melanohalea they are often restricted to
verrucae or isidial tips. The M. disjuncta group can also be
Fig. 2. Timing of brown parmelioid diversifi cation. Chronogram derived from the maximum clade credibility tree estimated with the uncorrelated
lognormal method in BEAST. Mean ages and their 95% highest posterior density (HPD) bars are shown above nodes. The nodes indicated by (C) represents
the calibration nodes: C1 crown node of parmelioids and C2 crown node of Parmelia s.s.

2022 AMERICAN JOURNAL OF BOTANY [Vol. 99
M. panniformis ). Pseudocyphellae fl at, effi gurate. Upper cortex
covered by nonpored epicortex. Medulla white. Lower surface
smooth to rugulose, black, dark brown at periphery, moderately
rhizinate. Rhizines short, simple, concolorous with the lower
surface. Ascomata apothecial, laminal, sessile to subpedicellate;
discs imperforate, concave and becoming convex with age,
brown, apothecial margin pseudocyphellate; hymenium 40–70 µm
high. Asci Lecanora -type, 8-spored. Ascospores simple, color-
less, mostly ellipsoid, rarely ovoid, 8–12 × 4–7 µm; spore wall
thin (up to 1 µm thick). Conidiomata pycnidial, immersed, laminal.
Conidia cylindrical to fusiform, 4–7.5 × 1 µm long.
Chemistry — Medulla containing orcinol depsides (perlatolic,
stenosporic, or gyrophoric acids).
Observations — Montanelia is characterized by having short,
narrow lobes with plane to convex margins, a nonpored epicor-
tex, fl at, effi gurate pseudocyphellae on upper surface, cylindrical
to fusiform conidia, and a medulla containing orcinol depsides.
The genus as it is now circumscribed includes fi ve species, which
grow on rocks in montane regions of the northern hemisphere
and north into the Arctic. Only a single species ( M. panniformis )
is reported from two localities in the southern hemisphere, the
mountains of Venezuela and central Chile ( Esslinger, 1977 ).
New combinations — Only the basionyms with type citation
and one synonym are given; for additional synonyms, refer to
Esslinger (1977 , 1978 , 1992 ).
Montanelia disjuncta (Erichsen) Divakar, A. Crespo, Wedin &
Essl. comb. nov.
Taxonomy — The results of the phylogenetic analyses and
alternative hypothesis testing together with evidence from
morphological and chemical features ( Table 4 ) support the dis-
tinction of the ‘ Montanelia ’ clade as a separate genus, which is
formally described as Montanelia here.
Genus— Montanelia Divakar, A. Crespo, Wedin & Essl.
gen. nov.
MycoBank no.— MB 801556.
Diagnosis — Thallus foliose, adpressed, lobes narrow, 0.4–3 mm
wide, margins plane to convex, often with fl at, effi gurate,
pseudocyphellae on upper surface, epicortex nonpored. Apothecia
sessile to subpedicellate, apothecial margin pseudocyphellate,
ascospores ellipsoid, thin-walled. Conidia cylindrical to fusiform.
Medulla containing orcinol depsides. Almost strictly saxicolous
(very rarely on old wood).
Type species— Montanelia panniformis.
Etymology— The epithet “montane” refers to the montane
distribution and elia to the last syllable of Melanelia , from
which it is segregated.
Description — Thallus foliose, adpressed to sometimes pulvi-
nate, loosely to moderately adnate. Lobes sublinear, short, narrow,
0.4–3 mm wide, periphery plane to convex, margins eciliate.
Upper surface, tan brown to more commonly dark-brown to
blackish, smooth to rugulose toward inner side, emaculate, often
pseudocyphellate (absent only in M. sorediata, infrequent in
T ABLE 3. Mean and range of divergence time estimations for brown parmelioid clades obtained using partitioned data set (consisting of four loci: ITS,
nuLSU, mtSSU, MCM7 ) BEAST analyses with two calibration points. All estimates are given in millions of years ago (Ma).
Clade Mean age Height 95% HPD Stratigraphic interval
Emodomelanelia + Melanelixia – Melanohalea split 46.17 36.30-59.17 Early Eocene (Ypresian)
Melanelixia–Emodomelanelia split 41.70 31.29-54.41 Middle Eocene (Bartonian)
Melanohalea crown 38.46 28.56-50.64 Late Eocene (Priabonian)
Melanelixia crown 33.75 23.88-45.24 Early Oligocene (Rupelian)
Origin of Montanelia ( Melanelia disjuncta group) 23.16 13.59-34.87 Late Oligocene (Chattian)
MRCA ( M. sorediata – M. panniformis complexes) 17.61 10.61-27.49 Early Miocene (Burdigalian)
M. panniformis Canada 11.24 6.11-18.67 Middle Miocene (Serravallian)
M. disjuncta 3.52 1.64-5.97 Early Pliocene (Zanclean)
Notes: HPD, highest posterior density; MRCA, most recent common ancestor.
T ABLE 4. Major diagnostic features for differentiating genera among genera of brown parmelioid lichens and Melanelia s.s. ( M. stygia ).
Parmelioid core Cetrarioid core
Characters Montanelia Melanelixia Melanohalea Emodomelanelia Melanelia s.s. ( M. stygia )
Lobes Short, narrow Broad Broad Broad Long, narrow to terete
Lobe margins Plane to convex Plane to concave, fl at Plane to concave, fl at Plane to concave, fl at Plane to convex
Epicortex Nonpored Pored or fenestrate Nonpored Nonpored Nonpored
Pseudocyphellae Flat, effi gurate Absent On verrucae or isidial tips,
circular to slightly elliptic
Effi gurate Flat, effi gurate
Ascospore size (µm) 8–12 × 4–7 9–15 × 5–11 6–20 × 4–12 13–16 × 6–9 7–10.5 × 4.5–6
Conidia Cylindrical to fusiform Cylindrical to fusiform Cylindrical to fusiform Bifusiform Bifusiform
Medullary chemistry Orcinol depsides Orcinol depsides Absent or β -orcinol
depsidones
β -Orcinol depsidones,
aliphatic acids
β -Orcinol depsidones,
aliphatic acids
Substrate Strictly saxicolous Mainly corticolous Mainly corticolous Strictly saxicolous Strictly saxicolous
Distribution Montane regions,
northern hemisphere
Lowland regions, northern
and southern hemispheres
Lowland regions, mostly
northern hemisphere
Montane regions,
Himalayan endemic
Montane regions, northern
hemisphere

2023
December 2012] DIVAKAR ET AL.—DIVERSIFICATION IN BROWN PARMELIOID LICHENS
differs morphologically from other brown parmelioid genera,
such as Emodomelanelia , Melanelixia , and Melanohalea by
having short narrow lobes, plane to convex, infl ated lobe mar-
gins, and fl at, effi gurate pseudocyphellae on upper surface, and
containing long side-chain depsides, for example, perlatolic
and stenosporic acids. The phylogenetic relationship of
Emodomelanelia has not been elucidated in previous studies,
and our results indicate that Emodomelanelia is a sister lineage
to Melanelixia ( Fig. 1 ).
In this study, all species that were recovered in the ‘ Montanelia ’
clade were previously accommodated in the genus Melanelia
and before that in a section (sect. Melanoparmelia ) within
Parmelia subgenus Melanoparmelia ( Esslinger, 1977 ), to which
Melanelia stygia also belonged. Nevertheless, the morphologi-
cally similar Melanelia stygia (type species of Melanelia s.s.) is
far apart from Montanelia and belongs to the monophyletic
cetrarioid core. Similar relationships have been found in several
previous studies (e.g., Blanco et al., 2004 ; Crespo et al., 2007 ,
2010a ; Thell et al., 2009 ; Nelsen et al., 2011 ).
Some species in the newly recognized genus Montanelia ,
such as M. disjuncta and M. sorediata , were found to be non-
monophyletic. Additional studies are necessary to clarify the
current species delimitation in this group, which is largely
based on macromorphological and chemical characters. A
detailed investigation evaluating the cryptic diversity in this
group is under progress and will be discussed in a forthcom-
ing paper.
Oligocene–Miocene divergence — Concordance between
lineage appearance and major climatic events in the Oligocene–
Miocene period has been shown for several groups of animals
and plants ( Zachos et al., 2001a , b , 2008 ; Mosbrugger et al.,
2005 ). We have also found this for brown parmelioid lichens,
suggesting an important link between major climatic shifts and
clade divergence. The Mi-1 glaciation (about 23 Ma) is recog-
nized as one of the most remarkable paleoclimatic events at the
Oligocene–Miocene boundary ( Zachos et al., 2001a , b ). Our
data suggest that the initial radiation of the genus Montanelia
occurred at the Oligocene–Miocene boundary during this
intense period of glaciation, with crown node age estimates of
23.2 Ma ( Fig. 2 , Table 3 ). Beginning at 23.2 Ma, the terrestrial
climate became colder, especially during the winter, with
marked thermal seasonality apparent at certain times during the
Miocene ( Zachos et al., 2001a , b ; Mosbrugger et al., 2005 ). We
hypothesize that Mi-1 glaciation would have played an impor-
tant role in the origin of the genus Montanelia . This pattern
is in concordance with those found in vascular plants. Nota-
bly, several groups in plants across the angiosperm phylog-
eny are reported to have pronounced diversifi cation at the
Oligocene–Miocene boundary ( Besnard et al., 2009 ; Bacon et al.,
2012 ; De Nova et al., 2012 ). The split between two gymno-
sperm genera Cupressus and Juniperus is also estimated during
this period ( Magallón, 2010 ).
In addition to the pronounced climate fl uctuations during the
Oligocene–Miocene period ( Zachos et al., 2001a , b ; Mosbrugger
et al., 2005 ), which resulted in the accelerated turnover of
several groups of terrestrial and marine biota ( Vrba, 1993 ;
Williams and Duda, 2008 ; Postigo Mijarra et al., 2009 ); a range of
global geological events also contributed to the increased rates
of dispersal and diversifi cation across many paleogeographic
areas ( Tiffney, 1984 ; Morley, 1998 , 2003 ). The closing of the
Tethys Sea, the closure of the Central American Seaway, the
collision of the Sunda and Sahul plates forming Wallace’s Line,
MycoBank no.— MB 801557.
Basionym — Parmelia disjuncta Erichsen, Ann. Mycol. 37:
78 (1939), nom. Nov. for Parmelia sorediata var. coralloidea
Lynge, Lich. Novaya Zemlya: 200 (1928). Type: Germany,
Bavaria, am Wege von Krottensee nach Neuhaus in der Oberp-
falz. Arnold, Lich. Exs. 743b (DUKE, isotype).
Synonym — Melanelia disjuncta (Erichsen) Essl., Mycotaxon
7: 46 (1978).
Montanelia panniformis (Nyl.) Divakar, A. Crespo, Wedin &
Essl. comb. nov.
MycoBank no.— MB 801558.
Basionym — Parmelia prolixa f. panniformis Nyl., Syn. Meth.
Lich. 1: 397 (1860). Type: Sweden, ad Holmiam (Stockholm),
Nylander 1852 (H-NYL, holotype).
Synonym — Melanelia panniformis (Nyl.) Essl., Mycotaxon
7: 46 (1978).
Montanelia predisjuncta (Essl.) Divakar, A. Crespo, Wedin &
Essl. comb. nov.
MycoBank no.— MB 801559.
Basionym — Parmelia predisjuncta Essl., Journ. Hattori Bot.
Lab. 42: 50 (1977). Type: Japan, Prov. Shinano, Azusayama,
Kawakami-mura, Minami-Saku-gun, 1400–1500 m a.s.l.,
Kurokawa 59241 (TNS, holotype).
Synonym — Melanelia predisjuncta (Essl.) Essl., Mycotaxon
7: 47 (1978).
Montanelia sorediata (Ach.) Divakar, A. Crespo, Wedin &
Essl. comb. nov.
MycoBank no.— MB 801560.
Basionym — Parmelia stygia var. sorediata Ach., Lichenogr.
universalis: 471 (1810). Type: Sweden (H-ACH 1414G,
lectotype).
Synonym — Melanelia sorediata (Ach.) Goward & Ahti,
Mycotaxon 28: 94 (1987).
Montanelia tominii (Oxner) Divakar, A. Crespo, Wedin &
Essl. comb. nov.
MycoBank no. — MB 801561.
Basionym — Parmelia tominii Oxner, Zh. Bio-Bot. Tsyklu,
Kiev 7-8: 171 (1933). Type: R.S.F.S.R.: In Oriente Extreme
Provincia Czita (=Chita). Prope pag. Atamanovka. Ad Saxa, 18
September 1927, Oxner (KW, lectotype)
Synonym — Melanelia tominii (Oxner) Essl., Lichenologist
24: 17 (1992).
DISCUSSION
Phylogenetic reconstructions — The newly described genus
Montanelia (= Melanelia disjuncta group) formed a moderately
to strongly supported independent monophyletic group ( Fig. 1 ).
Previous studies had either included only a single species of the
group ( Blanco et al., 2004 ; Crespo et al., 2007 ) or had the genus
Pleurosticta nested within the group ( Crespo et al., 2010a ). In
our study, Pleurosticta fell outside and grouped with Parmelia
s.s., but without support. Montanelia clustered with Parmeliop-
sis , but again this relationship lacks support, indicating that
additional loci will be necessary to identify the closest relative
of the genus.
In contrast to a previous study ( Crespo et al., 2010a ),
Montanelia did not cluster in the Melanohalea clade. In fact, it

2024 AMERICAN JOURNAL OF BOTANY [Vol. 99
the phylogenetic placement of the new genus within the mono-
phyletic parmelioid clade remains uncertain. Our results sug-
gest that the origin of Montanelia about 23.2 Ma was driven by
the Mi-1 glaciation event at the Oligocene and Miocene bound-
ary, a period dominated by dramatic climatic change. The ma-
jor diversifi cation occurred during the Miocene and was most
likely associated to major mountain uplifts when new habitats
were formed. The radiation of Melanelixia began at ca. 33.7
Ma and is correlated with the Oi-1 glaciation during Eocene–
Miocene transition. The diversifi cation of the Australasian lin-
eage commenced at ca 24. 4 Ma and was most probably due to
the tectonic isolation of the continent, leading to drier and more
seasonal climates.
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and the rise of major mountain ranges such as the Alps, the
Himalayas (proposed to have uplifted in phases one of which
occurred in the middle Miocene), all had dramatic effects on
world climate, global oceanic currents, biotic distributions, and
speciation (e.g., Zachos et al., 2001a , b , 2008 ). Montanelia spe-
cies are distributed in montane regions mainly in the northern
hemisphere ( Esslinger, 1977 ). The radiations of the lineages
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a period dominated by major mountain uplifts. Montanelia pan-
niformis , the M. sorediata species complex, and M. tominii
appear to have largely diversifi ed during the Miocene, while
M. disjuncta diversifi ed during the Pliocene. Recently, Leavitt
et al. (2012a) have shown that most diversifi cation within the
genus Melanohalea occurred during the Miocene and Plio-
cene, linked with the new habitats formed during Miocene
orogeny events, major climatic changes, and global shifts in
vegetation. Our results suggest that a similar scenario is true
for Montanelia .
The genera in the monophyletic group of parmelioid lichens,
originated during Eocene and Oligocene and the radiation
within these at different times from late Oligocene to the early
Pliocene, a period of major climate changes and global shifts in
vegetation ( Zachos et al., 2008 ; Amo de Paz et al., 2011 ; Leavitt
et al., 2012a ). Our estimates are consistent with comparable
diversifi cation patterns for the other brown parmelioid genera
Melanelixia and Melanohalea . Our results show that the
Emodomelanelia-Melanelixia split occurred during middle
Eocene (41 Ma). The radiation of Melanelixia commenced dur-
ing Eocene–Oligocene transition, with crown node age esti-
mates of 33.7 Ma ( Fig. 2 , Table 3 ). This transition is referred as
the Oi-1 glaciation, appears to involve reorganization of the cli-
mate system, global wide shifts in the distribution of terrestrial
and marine biota, and initiation of large continental ice sheets
on Antarctica ( Zachos et al., 2001a , 2008 ). The estimated time
based on our analyses is approximately 7 Myr older than that
estimated in a previous study ( Amo de Paz et al., 2011 ). This
difference is most probably caused by the inclusion of the ear-
ly-diverging Australasian lineage ( M. glabratuloides , M. pilif-
erella ), which was not included in the previous study. The
diversifi cation of the Australasian lineage, including M. glabrat-
uloides , M. piliferella , and M. subglabra, commenced at early
Miocene (ca. 24.4 Ma), and the lineages radiated until the late
Miocene (ca. 10.2 Ma), when the climate of the continent
became drier and more seasonal. This pattern is similar to those
found for the radiation of the sclerophyll fl ora endemic to
Australia, such as Bossiaea , Daviesia , and Pultenaea s.l.
(Mirbelieae + Bossiaeeae), Allocasuarina (Casuarinaceae), and
the two major Banksia lineages, which radiated rapidly during
a period of climate change, ca. 25–10 Ma ( Crisp et al., 2004 ). In
contrast, the Eurasian and North American lineages (see Fig. 1 )
began to diversify much earlier during middle Oligocene (ca.
28.5 Ma, Fig. 2 ). The split Emodomelanelia + Melanelixia
clade – Melanohalea is estimated around 46 Ma. The estimates
obtained here are slightly different than those of previous stud-
ies ( Amo de Paz et al., 2011 ; Leavitt et al., 2012a ), which is
probably due to Emodomelanelia included in our study, which
was not included in the other studies.
Concluding remarks — The present study demonstrates the
necessity of revising the current species delimitations in the
‘ Montanelia ’ clade. Montanelia is described as a new genus based
on multiple DNA loci and morphological evidence. However,

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