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Single origin and subsequent diversification of
central Andean endemic
Umbilicaria
species
Geir Hestmark
1
Department of Biology, University of Oslo, Oslo, Norway
Jolanta Miadlikowska
Department of Biology, Duke University, Durham,
North Carolina
Frank Kauff
Department of Biology, University of Kaiserslautern,
Kaiserslauten, Germany
Emily Fraker
Katalin Molnar
Franc¸ois Lutzoni
Department of Biology, Duke University, Durham,
North Carolina
Abstract
:We studied an Andean endemic group of
species of the lichen-forming fungal genus
Umbili-
caria
from the subalpine and low-alpine zone, with
their biogeographic center in Bolivia and Peru. A
number of species and varieties have been described
from this element, but apparent instability in several
morphological traits has made it difficult to precisely
delimit taxa. Based on DNA sequences of nuclear ITS,
LSU and mitochondrial SSU from extensive collec-
tions from Argentina, Bolivia, Chile, Colombia,
Ecuador and Peru, we present here a molecular
phylogenetic analysis of this Andean endemic ele-
ment within genus
Umbilicaria
. All analyses (MP, ML
and Bayesian) support a single origin for the element
and a division into two major groups characterized by
different apothecium types: the
Umbilicaria dichroa
group and
U. calvescens
group. Taxa
U. krempelhuberi
,
U. peruviana
and
U. subcalvescens
are nested within
U. calvescens
and are treated as conspecific with the
latter species. The endemic element shares a most
recent common ancestor with the
Umbilicaria vellea
group, which has a worldwide distribution and
contains several asexually reproducing (sorediate)
species. Independent reversals to sexual reproduction
might explain the evolution of two types of apothecia
in this monophyletic endemic lineage. A number of
cosmopolitan, mostly high-alpine, species of
Umbili-
caria
also present in the central Andes are related
only remotely to the endemic element and do not
exhibit speciation into endemics. Because the An-
dean element dominates the
Umbilicaria
habitats of
the low- and subalpine zones we propose that the
founder colonized the Andes at a time when the
mountains had not yet reached their current eleva-
tion while the high-alpine species arrived more
recently.
Key words:
apothecium types, endemism, evolu-
tionary radiation, lichen-forming Lecanoromycetes,
Neotropics, reproductive strategies
INTRODUCTION
The lichen-forming fungal genus
Umbilicaria
(Umbi-
licariales, Lecanoromycetes) has a worldwide distri-
bution and constitutes a major element of the
saxicolous lichen flora in boreal and alpine regions
(Frey 1933, 1936a; Llano 1950; Krog and Swinscow
1986; Wei and Jiang 1993). In an early review of the
biogeography of the Umbilicariaceae the Swiss expert
Eduard Frey (1936a) particularly lamented the
scarcity of collections from the high Andes (subalpine
to alpine) and suggested that this region would
provide the key to a further understanding of the
systematics of the family. This situation did not
substantially improve with Llano’s (1950) monograph
of the Umbilicariaceae in the western hemisphere.
Llano himself had not collected in South America
and had to rely on the scant material found in
herbaria. Umbilicariaceae in several Andean nations
recently have received separate treatments or have
been listed in national surveys or checklists (Sipman
and Topham 1992; Marcano and Morales Mendez
1993; Hestmark, 1997, 2009; Galloway and Quilhot
1998; Calvelo and Liberatore 2002; Feuerer and Thell
2008). These studies reveal that most
Umbilicaria
species found in the alpine zone of the Andes are well
known from other high elevation or arctic areas and
that the diversity of the Andean
Umbilicaria
flora is
fairly low compared to that found in the northern
hemisphere. However one major taxonomic challenge
has remained: the central Andean endemic element.
Since the mid-19th century, when the earliest
collections of
Umbilicaria
lichens from South America
were described, it has been known that low alpine and
subalpine zones of the Andes are the habitats of an
apparently endemic group of
Umbilicaria
species, with
their biogeographic center in Bolivia and Peru
(Nylander 1855, 1859, 1861, 1869; Hestmark 2010).
More recent investigations reported that this element
extends southward into parts of Argentina and Chile
Submitted 14 Jan 2010; accepted for publication 14 Jun 2010.
1
Corresponding author. E-mail: geir.hestmark@bio.uio.no
Mycologia,
103(1), 2011, pp. 45–56. DOI: 10.3852/10-012
#2011 by The Mycological Society of America, Lawrence, KS 66044-8897
45
and northward into southern Ecuador (Frey 1936a,
1949; Llano 1950; Hestmark 1997, 2009). From
extensive field observations and collections made by
the first author in Argentina, Bolivia, Chile, Ecuador
and Peru in the past 15 y it can be ascertained that the
central Andean endemic
Umbilicaria
element domi-
nates the lowermost parts (low alpine to subalpine) of
the altitudinal range of the genus (ca. 2500–4400 m)
in Bolivia, Peru and northern Chile and Argentina.
The variation in several morphological characters of
the endemic Andean
Umbilicaria
taxa however has led
to much confusion, and a number of species and
varieties have been named, often based on scant or
badly preserved material (Frey 1949, Llano 1950)
(TABLE I). Another problem was that several research-
ers, before establishing new species or intraspecific
taxa, did not examine the herbarium material on
which William Nylander based his original descrip-
tions of many of the endemic taxa (Hestmark 2010).
Further contributing to the taxonomical confusion is
the fact that characters that have proved fruitful for
the delimitation of species in many other lichen
genera, such as ascospore size, color and septation as
well as secondary compound chemistry, are identical
over the entire central Andean element; ascospores
are all unicellular, hyaline, and in size, 6–12 310–
18 mm (Nylander 1869, Llano 1950, Frey 1949); the
secondary compound chemistry is uniformly restrict-
ed to gyrophoric and lecanoric acid (Posner et al.
1992, Narui et al. 1996). In addition most members of
the endemic element may look broadly the same in
the field, that is monophyllous, gray-white thalli
usually less than 6 cm diam, and often with abundant
organs of sexual reproduction—black apothecia
producing hyaline ascospores—on the upper surface.
Furthermore they commonly co-occur in the same
locality, sometimes in mixtures on the same rock
surface. This morphological similarity could be an
indication of recent common ancestry. Yet on closer
inspection two groups might be distinguished tenta-
tively within the endemic element with reference to
the morphology of their apothecia: one group with
plane (leiodisc) apothecia and one group with gyrose
(gyrodisc) apothecia (TABLE I). Some taxonomists
(e.g. Scholander 1934, Llano 1950) considered
apothecium type a diagnostic character sufficient to
divide genus
Umbilicaria
into several genera, but this
taxonomic treatment was never widely accepted. Even
if the Andean
Umbilicaria
element is divided into two
groups based on apothecium morphology, the prob-
lem remains that in both groups several species,
varieties and forms have been described. Furthermore
in both groups forms, varieties or even species have
been described to accommodate the identification of
individual thalli that reproduce by symbiotic asexual
propagules known as soredia (containing both the
fungal and algal partners), instead of the more usual
sexual reproduction of the fungal partner, via
apothecia, followed by a re-association with a com-
patible alga.
TABLE I. Species and intraspecific taxa of
Umbilicaria
of the endemic Andean element
Taxa References Diagnostic character states
Species with leiodisc apothecia
Umbilicaria dichroa
Nyl. 1855 lower surface black and verrucose
Umbilicaria haplocarpa
Nyl. 1859 lower surface beige to black with abundant
rhizinomorphs
var.
convexa
Ra¨sanen 1944 apothecia convex
var.
subhirsuta
Frey 1949 isidiate and sorediate
f.
tenuis
Frey 1949 intermediate between the type and var.
subhirsuta
var.
friesii
Llano 1950 upper surface olive
var.
ku¨ hnemannii
Llano 1950 thallus indentions under apothecia
Species with gyrodisc apothecia
Umbilicaria calvescens
Nyl. 1861 no or few rhizinomorphs
var.
subvellea
Nyl. 1869 lower surface pale with abundant
rhizinomorphs
var.
hypomelaena
Nyl. 1869 lower surface black with abundant
rhizinomorphs
Umbilicaria krempelhuberi
Mu¨ ll.Arg. 1883 no or few rhizinomorphs
Umbilicaria leprosa
Zahlbr. 1906 with soredia
Umbilicaria peruviana
Llano 1950 lower side black, no or few rhizinomorphs
Umbilicaria subcalvescens
Sipman and Topham 1992 lower surface black with rhizinomorphs
and thalloconidia
46 MYCOLOGIA
The apparent instability of several thallus charac-
ters has led to the suggestion that these endemic
Andean
Umbilicaria
species ‘‘are still in the process of
development’’ (Llano 1950). Hestmark (2009) sug-
gested that the dominance of the element in the
subalpine and low-alpine zones could indicate that
members are derived from a colonizer taxon arriving
in the Andes when the mountain chain was lower
than today and preceded the colonization of high
alpine
Umbilicaria
species. The early colonizer then
had time for evolutionary differentiation and a
radiation into several niches before further rise of
the mountains took place and the arrival of the high
elevation
Umbilicaria
species, common in arctic and
alpine areas of the world, which today dominate the
high Andes. The high central Andes have been ‘‘high
elevation’’ only since the last major uplift that took
place in the late Pliocene or early Pleistocene, 2–4
MYA (Gregory-Wodzicki 2000). This might explain
why the high alpine
Umbilicaria
flora of the central
Andean region (4400–5600 m) consists of species well
known from other high alpine regions; they are a
recent addition and have had less time for local
differentiation (e.g.
U. africana
,
U. aprina
,
U.
cinereorufescens
,
U. decussata
,
U. nylanderiana
and
U. vellea
) (Hestmark 1997, 2009). A number of
molecular phylogenetic studies have indicated that
members of Umbilicariaceae were among the oldest
groups to evolve within Lecanoromycetideae or
Lecanoromycetes in general (Reeb et al. 2004,
Lutzoni et al. 2004, Miadlikowska et al. 2006). The
distinct phenotypic traits and their early divergence
led to their recognition as a separate suborder
(Umbilicarinae), order (Umbilicariales) and might
require classification as a subclass (Umbilicariomyce-
tidae) within Fungi (Poelt 1974, Miadlikowska et al.
2006, Spatafora et al. 2006, Hibbett et al. 2007). Thus
the entire
Umbilicaria
flora in a young high-elevation
habitat such as the central Andes is a comparatively
recent influx, most probably from comparable arctic
and alpine areas of the northern hemisphere, where
the greatest species diversity is found, including the
other genus in Umbilicariaceae,
Lasallia
, which has
not been reported from South America. This absence
of
Lasallia
suggests it encountered major obstacles,
such as absence of suitable stepping stone habitats in
the dense lowland jungle of the Panamanian land
bridge, to the movement of low elevation taxa
betweenNorthandSouthAmerica.
Umbilicaria
species are found almost exclusively on sun-exposed
igneous or metamorphic rocks.
Traditional studies, based on phenotypic variation,
have failed to resolve taxonomic ambiguities within
the central Andean element. PCR-based methods
have been used to evaluate the status of taxa within
lichen species complexes (Grube and Kroken 2000,
Miadlikowska et al. 2003), including genus
Umbili-
caria
(Ivanova et al. 1999, Ott et al. 2004). In the
present study we provide a molecular analysis of the
central Andean element of
Umbilicaria
. We address
these questions: To what degree are the species or
specimens with the same apothecium type phyloge-
netically related, and are the Andean endemics of the
genus
Umbilicaria
derived from a single origin? To
what degree are the endemics related to the other
Umbilicaria
species found in the central Andean area?
We also examined the phylogenetic placement of
these endemic species within the broader context of
family Umbilicariaceae to shed light on their evolu-
tionary history with regard to mode of reproduction
and the colonization of subalpine and low-alpine
Andes.
MATERIALS AND METHODS
Taxon sampling.—
We compiled extensive collections of
fresh lichen material 1994–2009 from Argentina, Bolivia,
Chile, Colombia, Ecuador and Peru. From this material a
total of 49 specimens of
Umbilicaria
were included in our
molecular study (SUPPLEMENTARY TABLE I). Of these 35
belonged to the presumed endemic element (low alpine to
subalpine) while 14 were from the other
Umbilicaria
species
present in the high alpine zone of the central Andes (
U.
aprina
,
U. africana
,
U. cinereorufescens
,
U. decussata
,
U.
nylanderiana
and
U. vellea
). Specimens in the central
Andean endemic element were selected to represent the
range of morphological variation in thallus traits and
reproductive traits (TABLE I). In addition a number of
northern hemisphere
Umbilicaria
taxa (
U. crustulosa
,
U.
grisea
,
U. hirsuta
and
U. spodochroa
) with morphological
similarities to the endemic Andean taxa were selected to
trace the possible origin of the endemic Andean element.
Together with
U. vellea
and
U. cinereorufescens
, which are
present in the central Andes, these northern hemisphere
taxa form a distinct morphological group that we refer to as
the
U. vellea
group. For comparative purposes we also used
all nuclear ITS, LSU and mitochondrial SSU sequences
from members of Umbilicariaceae (genera
Umbilicaria
and
Lasallia
) in the AFTOL (Assembling the Fungal Tree of
Life, WASABI) database (http://www.aftol.org, cf. Lutzoni
et al. 2004, Miadlikowska et al. 2006, Kauff et al. 2007). In
addition we selected three taxa,
Boreoplaca ultrafrigida
,
Hypocenomyce scalaris
and
Ophiopharma ventosa
, as out-
group to the family, based on the phylogenetic relationships
established by Wedin et al. (2005) and Miadlikowska et al.
(2006).
DNA isolation, sequencing and sequence alignment.—
Geno-
mic DNA was extracted from recently collected specimens
with a protocol modified from Zolan and Pukkila (1986)
with 2%sodium dodecyl sulphate (SDS) as extraction
buffer. Isolated DNA was resuspended in sterile water and
stored at 220 C. When pigments or polysaccharides were
thought to be inhibiting PCR, the DNA isolates were
HESTMARK ET AL.: ANDEAN
U
MBILICARIA
47
cleaned with the E.Z.N.A.HFungal DNA Miniprep Kit
(Omega Biotech). PCR amplification followed a modified
Vilgalys and Hester (1990) procedure with 1.5–3.0 mM of
MgCl
2
, 0.4 mg mL
21
bovine serum albumin (Hillis et al.
1996), Red HotHDNA Polymerase and chemistries from
ABgeneH(ABgene Inc., Rochester, New York). Cloning,
when required, was performed with a TOPO TA CloningH
Kit (Invitrogen
TM
, Life Technologies, Carlsbad, California).
Amplified PCR products were purified with the QIAquick
PCR Purification Kit (QIAGEN, Valencia, California) or
Exo-SAP (exonuclease I and shrimp alkaline phosphatase,
USB Corp., Cleveland, Ohio) before automated sequencing
with Big Dye chemistry with 3700 or 3730xl DNA analyzers
(PE Applied Biosystems, Foster City, California).
We amplified and sequenced these three loci: the nuclear
ITS with primers ITS1F or NS24R and ITS4 (White et al.
1990, Gardes and Bruns 1993, Miadlikowska et al. 2003),
<1.4 kb nuclear LSU with primers LR0R–LR7 (or LR5)
(Vilgalys and Hester 1990) and <0.8 kb mitochondrial SSU
with primers mitSSU1–mitSSU3R (Zoller et al. 1999). PCR
and sequencing conditions followed Hofstetter et al.
(2002). Sequences were assembled and edited with the
software package Sequencher
TM
4.1 (Gene Codes Corp.,
Ann Arbor, Michigan). GenBank accession and identifica-
tion numbers are provided and a total of 178 new sequences
were generated (SUPPLEMENTARY TABLE I). The sequences
were aligned manually with MacClade 4.07 (Maddison and
Maddison 2005). The alignments were carefully inspected
for the presence of ambiguously aligned regions caused by
the insertion of gaps. The unequivocal coding of these
ambiguous regions and the elaboration of symmetric step
matrices for each of these coded characters were generated
with the programs INAASE 2.3b (Lutzoni et al. 2000) or
ARC (Miadlikowska et al. 2003). The nexus file was
deposited in TreeBASE (http://purl.org/phylo/treebase/
phylows/study/TB2:S10614) and is available for download
at http://www.lutzonilab.net/publications.
Phylogenetic analyses.—
Each single-locus alignment was
analyzed separately to detect significant conflicts among
these datasets with ML bootstrap (1000 replicates and
GTRCAT model) as implemented in RAxML-VI-HPC
(Stamatakis et al. 2005). Models of evolution for Bayesian
phylogenetic analyses were estimated with the Akaike
information criterion (AIC) as implemented in MrModel-
test 2.3 (Nylander 2004). A conflict among single-locus
datasets was considered significant if a well supported
monophyletic group (i.e. bootstrap value $75%) was found
to be well supported as nonmonophyletic with a different
locus. Sequences causing these conflicts were removed from
the final phylogenetic analyses. When no further interlocus
conflict was detected single-locus datasets were concatenat-
ed into a single dataset of 73 operational taxonomic units
(OTUs). This concatenated dataset included 4254 sites of
which 1950 were excluded from all analyses because their
alignments were ambiguous. For MP analysis 1921 charac-
ters also were excluded because they were constant.
The combined dataset was analyzed with maximum
parsimony (MP) as the optimization criterion with PAUP*
4.0b10 (Swofford 2003) and with ML with RAxML-VI-HPC.
The Bayesian analysis was performed with MrBayes 3.1.2
(Ronquist and Huelsenbeck 2005). For the weighted MP
analysis unambiguously aligned sites of the ITS-1, 5.8S,
ITS-2, nucLSU and mitSSU were subjected to five step
matrices (one for each data partition), which were
generated with the program STMatrix 2.2 (written by S.
Zoller and available at http://www.lutzonilab.net/
downloads). Ambiguously aligned regions were excluded
or replaced by coded characters with INAASE or ARC. MP
searches were conducted with the five step matrices
generate by STMatrix as well as the INAASE and ARC
characters simultaneously. For ITS six of the excluded
ambiguously aligned regions were coded with INAASE and
six regions, which resulted in more than 32 character states
when using INAASE, were coded with ARC. For nucLSU
and mitSSU four and eight ambiguously aligned regions
respectively were coded with INAASE. In total 158 coded
characters were added to the 4254 sites of the concatenated
dataset. Of this grand total of 4412 characters 637 (450 of
which were parsimony informative) were subjected to MP
analyses. Of these 507 had a weight of 1, 24 had a weight of
0.25, 59 had a weight of 0.10 and 47 had a weight of 0.50
(see Reeb et al. 2004 for the weighting scheme used here
that is associated with ARC characters). MP search was done
with 1000 random addition sequences (RAS) and TBR
swapping. A total of 243 equally most parsimonious trees
of 2710.59 steps were recovered for 408 of 1000 RAS. Based
on this frequency, nonparametric MP bootstrap support
(MP-BS) values were generated with the same settings as for
the analysis on the original combined dataset, except that
eight RAS were implemented, for each of the 1000
bootstrap replicates saving the 100 best trees. To assess
the contribution of ARC characters to MP analysis we
performed an additional MP bootstrap analysis on the same
concatenated data matrix but without ARC characters. No
topological conflicts were detected with and without ARC
characters when the same criterion used to detect among
partition conflicts was implemented as described above.
Maximum likelihood analysis with RAxML was performed
with 1000 replicates and GTRGAMMA model with gamma
distribution, approximated with four categories.
In addition to MP-BS, phylogenetic confidence for
relationships inferred from the combined dataset was
estimated with Bayesian posterior probabilities (PP) and
maximum likelihood bootstrap proportions (ML-BS). The
combined dataset was separated into five partitions
(mitSSU, nucLSU, ITS-1, 5.8S, ITS-2), and the Bayesian
analyses were run with four independent chains 20 000 000
generations, sampling every 500th tree. As estimated by
MrModeltest, a six-parameter model for nucleotide substi-
tution (GTR +I+G, Rodrı´guez et al. 1990) with a gamma
distribution approximated with four categories and a
proportion of invariable sites was used for all data partitions
except the 5.8S, for which a two-parameter model with a
proportion of invariable sites (K80 +I, Kimura 1980) was
implemented. Two independent Bayesian runs were con-
ducted. To ensure that the runs reached stationarity and
converged on the same ln-likelihood score, chains were
examined by eye and with AWTY (http://ceb.csit.fsu.edu/
awty). After discarding the burn-in, the last 20 000 trees of
48 MYCOLOGIA
each run were used to calculate a 50%majority rule
consensus tree. Bootstrap proportions were calculated with
1000 bootstrap pseudoreplicates with RAxML implement-
ing the GTRCAT model with gamma distribution, approx-
imated with four categories. Bootstrap proportions (ML-BS
and MP-BS) equal to or greater than 70%, and posterior
probability values (PP, resulting from MrBayes analyses)
equal to or greater than 95%were considered significant.
Internal branches with significant support values revealed
from at least two out of the three methods implemented
were interpreted as well/strongly supported.
RESULTS
Monophyly of the Andean endemic element and its
phylogenetic placement.—
We provided the strict con-
sensus of 243 equally most parsimonious trees (tree
length 52710.5900 steps) (FIG. 1) and the maximum
likelihood tree (ln likelihood 528201.260234) (SUP-
PLEMENTARY FIG. 1). All results from phylogenetic
analyses of the concatenated dataset were concordant
for the well supported (BS $70%,PP$95%)
portions of their respective topologies. All specimens
of the central Andean complex were recovered as a
monophyletic group (MP-BS 5100, ML-BS 598, PP
5100), sister of the
U. vellea
group (MP-BS 5100,
ML-BS 5100, PP 5100) (FIG. 1). The other central
Andean taxa of
Umbilicaria
—
U. africana
,
U. aprina
,
U. dendrophora
,
U. nylanderiana
—are seen to be more
distantly related to the endemic element, with the
exception of
U. cinereorufescens
and
U. vellea
that are
part of the
U. vellea
group.
Two major endemic groups diverging from a
single origin.—
The overall topology of the trees
suggests a division of the central Andean
Umbilicaria
element into two major groups, corresponding to
apothecium morphology: a group with gyrose (gyro-
disc) apothecia and a group with plane (leiodisc)
apothecia (FIG. 1). We designated the former as the
calvescens
group and the latter as the
dichroa
group,
with reference to the first member described within
each clade. The
calvescens
group is well supported as
monophyletic (MP-BS 5100, ML-BS 5100, PP 5
100) (FIG. 1), as is the monophyly of the
dichroa
group (MP-BS 590, ML-BS 595, PP 5100). Within
the
calvescens
group the longer branches in both the
MP tree (FIG. 1) and the ML tree (SUPPLEMENTAL
FIG. 1) might indicate an increase in the rate of
nucleotide substitution, which could have resulted in
higher genetic variation and structure compared to its
sister
dichroa
group (FIG. 1).
In all analyses the majority of specimens from the
central Andean endemic
Umbilicaria
with leiodisc
apothecia (
U. dichroa
group) sorted into two separate,
highly supported, monophyletic subgroups: H1 (MP-BS
589, ML-BS 587, PP 5100) and H2 (MP-BS 599, ML-
BS 5100, PP 5100) (FIG. 1). The morphology of the
specimensinbothHIandH2correspondstowhathas
been referred to as
Umbilicaria haplocarpa
. There is no
easily observable morphological distinction between
these two subgroups, and H1 and H2 do not correspond
to any of the varieties described previously for
U.
haplocarpa
. Both subgroups comprise specimens with
more or less abundant apothecia, more or less
abundant rhizinomorphs and more or less pronounced
reticulation around the holdfast. The only difference
seems to be that H1 never exhibits a non-black lower
surface. These two subgroups appear to reflect geo-
graphical origin; the first subgroup (H1) comprises
specimens from Peru, Bolivia (Tunari) and Ecuador,
the other (H2) comprises specimens from Chile and
Bolivia. Thus the latter subgroup has a slightly more
southern distribution but both subgroups comprise
specimensfromthewesternaswellaseasternAndean
ranges. Neither of the two subgroups can be distin-
guished by elevation preferences. Outside the two well
supported H1 and H2 subgroups three more individu-
als are part of the
dichroa
-group. There is however no
support for their placement within this group. Two of
these individuals, without apothecia, from the same
locality at Puno, Peru, are identical morphologically to
the type specimen of
Umbilicaria dichroa
.Thethird
individual is distinctly sorediate and isidiate and without
apothecia. The latter specimen corresponds morpho-
logically to the taxon
U. haplocarpa
var.
subhirsuta
(Frey
1949: 449). Because it does not exhibit a black, granular
lower side as
U. dichroa
(TABLE I) it here is labeled
U.
haplocarpa
var.
subhirsuta
, pending further collections
and phylogenetic analyses of more variable loci.
The increase in nucleotide substitution, reported
here for the
U. calvescens
group, resulted in more
phylogenetic structure than observed for the
dichroa
group. The first divergence within the
calvescens
group involves three slow evolving sorediate individ-
uals, corresponding to the taxon
Umbilicaria leprosa
(Zahlbr.) Frey (
U. leprosa
2, 3 and 4) (MP-BS 588,
ML-BS 588, PP 599) (FIG.1). One of these
specimens (
U. leprosa
2) is from the type locality of
U. leprosa
, the volcano Chimborazo, Ecuador (Zahl-
bruckner 1906). The clade further includes a speci-
men from the volcano Cayambe, Ecuador, and one
specimen from western Bolivia (volcano Sajama). The
first divergence detected within the lineage sister of
the
U. leprosa
subgroup, although not supported,
involves a sorediate specimen with some apothecia,
tentatively named
U. leprosa
1, which is sister of a non-
sorediate group of 16 specimens with mostly abun-
dant gyrose apothecia.
Within the group of specimens labeled
U. calvescens
1–15 there is little or no support for most suggested
HESTMARK ET AL.: ANDEAN
U
MBILICARIA
49
FIG. 1. Phylogenetic relationships of central Andean endemic Umbilicariaceae based on combined nuclear ITS, LSU and
mitochondrial SSU. Strict consensus of 243 equally most parsimonious trees. Branch lengths were estimated with the
PHYLOGRAM option of PAUP* with the same settings as for the phylogenetic search. Internal branches are shown as thick lines
when both bootstrap values were $70%and posterior probability was $95%. Boxes with continuous lines circumscribe the
two main monophyletic groups within the central Andean endemic Umbilicariaceae-
calvescens
and
dichroa
groups. The
50 MYCOLOGIA
relationships (FIG. 1). At the bottom of this paraphy-
letic group are two specimens (
U. calvescens
14 and 15),
corresponding to the taxon
U. calvescens
var.
hypome-
laena
, with a black lower side, abundant rhizinomorphs
and sparse gyrose apothecia. The next subgroup (
U.
calvescens
10–13), with some support (MP-BS 584),
consists of specimens from the middle alpine zone that
have pronounced reticulation on the lower side, which
is pitch black and with scant or no rhizinomorphs.
Three of these four specimens are from the same area
in Bolivia, the peak Huayna Potosi, and the fourth is
from Tunari, Bolivia. Among the specimens selected
these come closest in morphology to the synonymous
taxon
U. peruviana
. Two individuals with light lower
sides densely covered with rhizinomorphs, correspond-
ing to the variety
U. calvescens
var.
subvellea
Nyl. (
U.
calvescens
8 and 9), are paraphyletic (not supported).
With the exception of
U. subcalvescens
, all remaining
specimens (
U. calvescens
1–7) correspond to the typical
form, with mostly no or only a few rhizinomorphs. All
phylogenetic analyses (including single-gene analyses
of mitSSU and nucLSU) revealed a nested position of
U. subcalvescens
within the
U. calvescens
group. Because
it is represented in our phylogeny by only one specimen
and the backbone of the
calvescens
group is mostly
without statistical support no formal taxonomic chang-
es are proposed. Two internodes within
U. calvescens
typicum (1–7) exhibit strong support but do not
correspond to clear morphological distinctions. Three
specimens (here labeled
U. calvescens
2, 3, 4) come
from the type locality of the synonymous taxon
U.
krempelhuberi
in Argentina but are not supported as
monophyletic. In the
calvescens
group there appears to
be an evolutionary trend from a mainly sorediate
ancestor with rhizinomorphs to a sexually reproducing
lichen-forming fungus without rhizinomorphs.
DISCUSSION
Results from all phylogenetic analyses conducted in
this study support a single origin of the entire central
Andean endemic element of
Umbilicaria
. This com-
mon ancestry is surprising when considering the
variation in apothecium morphology. Gyrose and
leiodisc apothecia often have been considered two
extremes along an evolutionary line, where leiodisc
was considered the ancestral state and gyrose a
derived state (Scholander 1934, Llano 1950). Howev-
er this was challenged by Frey (1936b, 1949) who
pointed to intermediate apothecial morphologies in
some species and by Henssen (1970) who showed the
ontogeny of the different apothecium types to be
fairly similar. Our results suggest that switches
between these two types of apothecia occurred
repeatedly within family Umbilicariaceae and there-
fore might result from only few genetic changes. This
hypothesis also is supported by phylogenetic relation-
ships of the non-endemic species within Umbilicar-
iaceae; several other clades contain species with both
apothecium types (FIG. 1). Thus, while the molecular
analyses confirm the utility of apothecium morphol-
ogy for taxonomic decisions and determinations at
the species level, the use of this trait to establish
several new genera and reorder the entire family
Umbilicariaceae as proposed by Scholander (1934)
and Llano (1950) is not valid if we want taxonomy to
reflect phylogeny. So far no adaptive function can be
attributed to the various apothecium morphologies of
Umbilicaria
.
The molecular phylogenetic analysis further places
the central Andean endemic
Umbilicaria
element as
sister of the
U. vellea
group, which includes members
with both gyrose and omphalodisc apothecia, as well
as several mainly sorediate taxa.
U. vellea
and its
smaller close relative,
U. cinereorufescens
, both are
present in the central Andes, although not frequently
due to habitat specialization (Hestmark 1997, 2009).
The two species have a worldwide distribution,
occurring in almost all alpine areas with acidic rocks.
This suggests that the endemic element at some time
branched from the ancestral population from which
evolved
U. vellea
group (
U. vellea
,
U. cinereorufescens
,
U. crustulosa
,
U. grisea
,
U. hirsuta
). Future molecular
studies might reveal to what degree the central
Andean endemics are related to putative specimens
of
U. haplocarpa
reported from a few high mountain
localities in Natal, South Africa (Frey 1949, Almborn
1987, Wei and Biazrov 1991).
Another unexpected result came from the phylo-
genetic placement of all included sorediate Andean
specimens within the endemic element. Although
sorediate varieties have been described for both
U.
r
monophyletic sister
U. vellea
group is delimited with a discontinuous line. Character states reported here for members of the
central Andean endemic clade and sister group apply only to individual specimens included in this tree. However for
comparative purposes and completeness we have included literature data on apothecium type for the species for the
specimens lacking apothecia (shown in gray shaded boxes). Capital letters in parentheses refer to country of origin for each
specimen, A 5Argentina, B 5Bolivia, C 5Colombia, CH 5Chile, and P 5Peru. One asterisk follows the name of specimens
traditionally referred to (but unsupported) as ‘
U. krempelhuberi
’. Two asterisks are used for specimens that traditionally were
referred to (but unsupported) as ‘
U. peruviana
’.
HESTMARK ET AL.: ANDEAN
U
MBILICARIA
51
calvescens
and
U. haplocarpa
, the majority of sorediate
specimens from the Andes generally have been
referred to the taxon
Umbilicaria leprosa
(Zahlbruck-
ner 1906), which so far has not been considered to be
associated with either of the two main groups revealed
by our molecular phylogenetic study. Many of these
sorediate individuals appear to have discarded sexual
reproduction completely or almost completely. When
no apothecia are present it is practically impossible to
assign a sorediate specimen to the
calvescens
or
dichroa
group. Because soredia originated multiple
times during the evolution of lichens (Bowler and
Rundel 1975) there is an ongoing discussion about
whether two specimens, which differ only by the
presence or absence of soredia, should be considered
to represent two separate species. For example Ott et
al. (2004) argued that the sorediate taxon
Umbilicaria
kappenii
was indistinguishable from the taxon
U.
antarctica
in a molecular study based on nuclear ITS
and LSU rDNA and mitochondrial LSU rDNA and
accordingly should be reduced to synonymy with the
latter. The anatomy of species in the Andean element,
with a continuous, dense algal layer, well separated
from the lower parts of the thallus by a thick layer of
dense hyphae, might aid the evolution of soredia.
Algae from the photobiont layer may easily detach
from the thallus, carrying with them hyphae of the
upper cortex. The sorediate taxa from the northern
hemisphere,
U. grisea
and
U. hirsuta
, are part of the
U. vellea
group, although morphologically
U. hirsuta
is difficult to distinguish from the endemic
U. leprosa
.
This suggests that all previous reports of
U. hirsuta
from the Andes must be re-evaluated.
The tentative phylogenetic placement of sorediate
individuals and their presence in both clades of the
endemic element, as well as in the sister
U. vellea
group (FIG. 1), indicates that the central Andean
endemic species might have evolved from a sorediate
ancestor. If so we might have a case of long-distance
dispersal via asexual propagules containing both
symbionts, followed by local speciation and evolu-
tionary reversal to sexuality and/or loss of asexual
reproduction via soredia in many of the branches of
the central Andean element. The two putative
independent reversals to sexuality in the central
Andean endemic group could be an explanation for
the evolution of two apothecium types within this
element. Similar reversals from predominantly asex-
ually reproducing to sexually reproducing taxa have
been described in other groups of lichen-forming
fungi (Cornejo et al. 2009, Tehler et al. 2009). This
scenario suggests that the genes for sexual reproduc-
tion were not lost through evolution but only
temporarily repressed. The loss of sexual reproduc-
tion is considered an evolutionary disadvantage for
several reasons in non-obligate symbiotic organisms
(Bell 1982, Zeyl and Otto 2007). However for an
obligate mutualistic system asexual propagules con-
taining both symbionts often have been considered a
more effective means of dispersal than fungal
propagules such as ascospores or thalloconidia alone,
which necessitate the proximity of a compatible
photobiont to enable the establishment of the next
generation of lichens (Hestmark 1991). Although
some evidence supports the hypothesis that species of
Umbilicaria
are not very selective in their require-
ments for a compatible algal partner (Romeike et al.
2002), it nevertheless would have been advantageous
for the first
Umbilicaria
to colonize an emerging
mountain chain, arriving with a propagule containing
both mycobiont and photobiont. To what degree the
Andean
Umbilicaria
species share the same algal
species or strains and their algae are related to algae
found in lichens of the
U. vellea
group versus other
lichens remains to be investigated.
Rapid species diversification linked to adaptive
radiation is a common occurrence when new, largely
unoccupied, isolated habitats are colonized by a few
pioneer species (Schluter 2000) and has been
documented for many genera of flowering plants on
the comparatively recent central Andean ‘‘alpine
island’’ (Hughes and Eastwood 2006). Many of these
genera, such as
Draba
,
Lupinus
,
Quercus
,
Salix
,
Sambucus
,
Valeriana
and
Viburnum
, are thought to
have arrived in the Andes from North America after
the uplift (van der Hammen and Cleef 1986,
Burnham and Graham 1999). In general flowering
plants seem to have much more plasticity to evolve
new life histories, dispersal options, etc. (Hughes and
Eastwood 2006). In contrast Ahti (1992) and Sipman
(1992, 2002) discussed the evolutionary rates of
lichens in the Pa´ ramo vegetation zone and concluded
that lichen endemism there is rare, confirming the
opinion that lichen-forming fungi speciate slowly.
Slow evolutionary rates in part might be ascribed to
the long generation of alpine lichens. A study on
growth and reproduction in several alpine
Umbilicaria
species by Hestmark et al. (2004) demonstrated
generation times much longer than that of most
vascular plants: 50–80 y from establishment to first
reproduction. For
Umbilicaria
lichens the plasticity
and possible morphological and structural changes
also seem restricted. In the
calvescens
group there
seems to be an evolutionary trend toward the
elimination of rhizinomorphs, culminating in the
U.
calvescens
typicum (and its synonym
U. krempelhuberi
).
Species in the
vellea
group usually have abundant
rhizinomorphs, sometimes considered beneficial for
water uptake in the habitats of species within this
group.
U. calvescens
varieties with abundant rhizino-
52 MYCOLOGIA
morphs (var.
subvellea
and var.
hypomelaena
) both
tend to grow in similar habitats, with trickling water,
in the Andes. In contrast the typical
U. calvescens
, with
no or sparse rhizinomorphs, frequently are found on
small and large boulders or on sun-exposed surfaces
that exhibit no persistent water trickle. Thus it could
be that the loss of rhizinomorphs is associated with an
extension of the ancestral niche into drier and more
open habitats. The absence of other
Umbilicaria
and
Lasallia
species in subalpine and low-alpine regions
have left these drier niches open for expansion of the
endemic element. The amplitude of variation in
rhizinomorph presence seen in the
calvescens
com-
plex is similar to that observed in the sorediate
members of the
vellea
group from the richly
rhizinomorphic
U. hirsute
, which means hairy, to
the non-hirsute
U. grisea
.
Genetic differences observed among individuals in
the Andean endemic
Umbilicaria
, notably in the
calvescens
group, explains some of the taxonomic
confusion and the problem of field identification that
has been associated with this group of lichens since its
discovery in the mid-19th century. The phylogenetic
tree presented here does not suggest obvious solu-
tions to these problems, and in general the hypothet-
ical nature of any such tree should always be
emphasized (Hestmark 2000). The many unsupport-
ed relationships in the
calvescens
and
dichroa
groups
indicate that new loci (preferentially fast evolving)
need to be sequenced and a population genetic
approach should be implemented, in addition to
phylogenetics if new species are to be delimited and
described within the central Andean endemic group.
In any case, with morphological traits exhibiting
gradual transitions, it will be difficult to use a
taxonomy based on such genetic markers for the
practical purposes of field and herbarium identifica-
tion if species are found in addition to the two main
species groups defined here for the central Andean
element. Therefore to aid field identification we
suggest that with the exception of the distinctly
sorediate taxon
U. leprosa
all other specimens in the
calvescens
group should be named
U. calvescens
,if
desirable with some indication of variety. The typical
specimens of
U. calvescens
(cf. Hestmark 2010)
correspond to the later described taxon
U. krempel-
huberi
Mu¨ll-Arg., which accordingly should be re-
duced to synonymy with
U. calvescens
. The taxon
U.
peruviana
also is considered best a synonym of
U.
calvescens
. Although the taxon
U. haplocarpa
might
appear to be divided into two fairly distinct genetic
subpopulations (H1 and H2), practical consider-
ations of identification suggest that this taxon should
be kept as one unit, species
U. haplocarpa
. The taxon
U. dichroa
can be maintained for specimens with few
FIGS. 2–5. 2.
Umbilicaria calvescens
.3.
U. leprosa
.4.
U.
dichroa
.5.
U. haplocarpa
. Bars 51 cm.
HESTMARK ET AL.: ANDEAN
U
MBILICARIA
53
or no apothecia, a black lower side with wart-like
protrusions until more data is gathered. Field work by
the first author indicates that
U. dichroa
has a
restricted range around Lake Titicaca. The central
high Andean element thus is seen to be in active,
diverging evolution, potentially resulting in several
new and separate species. (Currently recognized
species are depicted in FIGS. 2–5.)
ACKNOWLEDGMENTS
The authors gratefully acknowledgethe permits for collecting
and exporting granted by the Ministerio de Agricultura et
Ganaderia in the Republic of Ecuador. Support was further
provided by Pontificia Universidad Ca´tolica del Ecuador
(PUCE), Quito, and Profs Henrik Balslev, Simon Leegaard
and Finn Borchsenius of Aarhus University, Denmark. This
study was conducted in cooperation with the Herbario
Nacional de Bolivia at Universidad Major San Marco in La
Paz where we thank Director Stephan Beck and Sub-Directora
Rosa Isola Meneses Quisbert. Also we thank the Ministerio de
Desarollo Sostenible y Planification, Servicio Nacional de
Areas Protegidas, Bolivia, and the Directors of the National
Parks of Sajama and Tunari for permits to collect lichens. For
the work conducted in Peru the authors thank Prof Asuncion
Cano Echevarria and Angel Ramirez at the Museo de Historia
Natural, Universidad Nacional Mayor de San Marcos, Lima,
for their help. Permits to collect in Peru were granted by the
Instituto Nacional de Recursos Naturales (INRENA), Lima.
We also thank Martin Kukwa and Valerie Hofstetter for
agreeing to let us use one of their unpublished ITS sequences
that they generated in the Lutzoni Lab.
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