A Monograph of Usnea from Sao Tome and Principe
A thesis submitted to the faculty of % San Francisco State University MS In partial fulfillment of E><0L the requirements for the Degree • U 33
Master of Science In Biology: Ecology and Systematic Biology
by
Miko Rain Abraham Nadel San Francisco, California December 2015 Copyright by Miko Rain Abraham Nadel 2015 CERTIFICATION OF APPROVAL
I certify that I have read Monograph of Usnea from Sao Tome and Principe by Miko Rain Abraham
Nadel, and that in my opinion this work meets the criteria for approving a thesis submitted in partial fulfillment of the requirement for the degree Master of Science in Biology: Ecology and Systematic
Biology at San Francisco State University.
DennisE. DesL6rdin, Ph.D. Professor ofjsiology San Francisco State University
Jose R. de la Torre, Ph.D. Associate Professor of Biology San Francisco State University
Robert C. Drewes, Ph.D. Curator of Herpetology, Emeritus California Academy of Sciences A Monograph of Usnea from Sao Tome and Principe
Miko Rain Abraham Nadel San Francisco, California 2015
A systematic monograph of the lichen genus Usnea from the remote, under-explored islands of
Sao Tome and Principe (ST&P) in tropical West Africa is presented, treating 11 species representing 2 subgenera, Usnea and Eumitria. Ten of the 11 species are reported as new for
ST&P, including U. firmula, U. baileyi, U. pectinata, U. aff. flammea, U. sanguinea, U. picta, U. krogiana and three undetermined, potentially new species, Usnea species A, B and C. Usnea articulata is confirmed for the main island of Sao Tome. Determinations were made using morphological data, chemistry via thin layer chromatography, and a molecular phylogeny based on nDNA Internal Transcribed Spacer (ITS) sequences. Usnea baileyi, U. pectinata, U. articulata and
Usnea species C represent species complexes consisting of two or more morphotypes each.
Comprehensive descriptions, photographs, and a dichotomous key to the species are provided.
correct representation of the content of this thesis
De nnjs E. Desjarefm; Chair, Thesis Committee Date ACKNOWLEDGEMENTS
I’d like to express my deepest appreciation to the many people who supported me on this journey of exploration and education. Thank you to Dr. Bob Drewes for the incredible opportunity to travel to such an amazing place for my collections and to those in ST&P who provided us with all the support in the field. Thanks also to Dr. Dennis Desjardin for agreeing to work with me despite my strange fascination with lichen, as well as Dr. Jose de la Torre for serving on my committee. Thank you to those who were able to give their time, experience and/or resources in helping me learn and become a better scientist, including: James Shevock, Doug Glavich, Dr. Bruce McCune, Dr. Frank
Cipriano, Dr. Yoshihito Ohmura, Dr. Irwin Brodo, and Dr. Felix Schumm. I’m also immensely grateful for my father and the rest of my family, loved ones and friends and especially my grandmother, Ruth “Dolly” Nadel, who taught me to persevere and to believe in myself from a very young age when life was challenging. Thank you to all my colleagues at San Francisco State
University for assistance along the way whether teaching me, or allowing me to learn by teaching you, every little bit helped. Lastly, thank you to the groups that granted me funds towards my research; The California Academy of Sciences, The California Lichen Society, The American
Bryological and Lichenological Society, and The Mycological Society of San Francisco.
v TABLE OF CONTENTS
List of Table...... vii
List of Figures...... viii
List of Appendices...... ix
Introduction...... 1
Usnea in Sao Tome and Principe...... 2
Materials and Method...... 5
Literature Review...... 5
Specimen and Exterior Data Collection...... 5
Morphological Analysis...... 6
DNA Extraction, Amplification and Sequencing...... 7
Sequencing Data Analysis...... 8
Chemical Analysis...... 9
Results...... 11
Phylogenetic Results...... 11
Subgenus Eumitria...... 12
Subgenus Dolichousnea...... 13
Subgenus Usnea...... 13
Thin Layer Chromatography Results...... 15 Taxonomy...... 17
Key to species...... 17
Species descriptions...... 20
Usnea firmula...... 20
Usnea baileyi...... 22
Usnea pectinata...... 24
Usnea aff. flammea...... 27
Usnea sanguinea...... 28
Usnea species A ...... 30
Usnea species B ...... 32
Usnea articulata...... 34
Usnea picta...... 37
Usnea species C...... 38
Usnea krogiana...... 41
Discussion...... 43
References...... 66
Appendices...... 76
vii LIST OF PLATES
Plate Page
1. Usneafirmula...... 45 2. Usnea baileyi...... 46 3. Usnea pectinata 1...... 47 4. Usnea pectinata 2 ...... 48 5. Usnea aff. flammea...... 49 6. Usnea sanguinea...... 50 7. Usnea species A ...... 51 8. Usnea species B ...... 52 9. Usnea articulata...... 53 10. Usnea picta...... 54 11. Usnea species C...... 55 12. Usnea krogiana...... 56 LIST OF FIGURES
Figures Page
1. Usnea phylogeny ...... 57 2. Subgenus Eumitria phylogeny...... 58 3. Subgenus Eumitria locality...... 59 4. Subgenus Eumitria chemistry ...... 60 5. Subgenus Dolichousnea phylogeny...... 61 6. Subgenus Usnea phylogeny...... 62 7. Subgenus Usnea locality...... 63 8. Subgenus Usnea chemistry...... 64 9. Usnea species C Thin Layer Chromatography...... 65 LIST OF APPENDICES
Appendix
A. Table of significant chemical compounds in species groups as revealed by TLC 1
Introduction
The genus Usnea, in the family Parmeliaceae, has historically been considered somewhat of a thorn in the side of lichenologists when it comes to the delineation of species, due, in part, to it’s high degree of variability and the lack of characters to draw from in distinguishing species (Clerc
1998, Ohmura 2001). The genus, with approximately 600 species and a cosmopolitan distribution
(Hawksworth et al. 1995) has remained difficult to work on. A world monograph of Usnea produced by Motyka (1936), uses primarily morphological features to distinguish the different species. The characters chosen by Motyka for his monograph are not consistent with many modern taxonomists list of features used as relevant taxonomic characters (Clerc 1998). Swinscow and Krog (1975,
1979,1986,1988,1991), Clerc (1997,1998) and Ohmura (2001,2012) have published several regional revisions of Usnea with more currently accepted useful characters, including the presence of various organic compounds determined with thin layer chromatography.
The more recent use of molecular phylogenies generated from DNA sequences, has provided even more data to interpret the different species groups in Usnea, although not without challenges. The use of nuclear ribosomal Internal Transcribed Spacer (ITS) sequences, the fungal barcode gene region, while useful, is not on its own sufficient to delineate species in Usnea (Truong et al. 2013c).
While delimiting the genus from other fruticose genera is not controversial, there is some 2
disagreement regarding the infrageneric arrangement of Usnea. For the purposes of this monograph, the synapomorphies of a central cartilaginous axis and presence of usnic acid are accepted as delineating a single genus Usnea, with internal groups Eumitria, Dolichousnea, and
Usnea accepted at the infrageneric rank, rather than distinct genera as recognized by Articus
(2004b).
Usnea taxonomy on the African continent has received far less attention than in Europe and North
America, and as a consequence there are limited data on species diversity and distribution available. Dodge (1956,1957) reported species primarily from Southern Africa based entirely on morphological data, while Swinscow and Krog (1975,1979,1986,1988) provide a limited number of descriptions of Eastern Africa species based on both morphological and chemical analyses.
These resources, along with the monograph by Motyka (1936) were helpful in identifying the Usnea specimens from Sao Tome and Principe.
Usnea in Sao Tome and Principe
West Africa presents particular difficulties for conducting fieldwork by lichenologists and other scientists because of ongoing political and social conflicts, in addition to naturally dangerous conditions in many countries. One country that falls largely outside of these dangerous conditions 3
is the island republic of Sao Tome and Principe, with its relatively stable government and safe environmental conditions. The islands are oceanic in origin, arising from a volcanic hot spot (the
Cameroon volcanic mountain line) and never connected to the mainland. They are situated approximately 250-300 km west of the country of Gabon, Africa in the Gulf of Guinea, very near 0° latitude and 0° longitude. Because of their oceanic origin and old age (Sao Tome at 15.7 my,
Principe at 30.4 my) they show high levels of endemism. Additionally, humans did not begin to populate the islands until the early 16th century and although there have been many species introduced at lower elevations of the islands, higher elevation (>800 m Sao Tome, ^400 m
Principe) have remained primary forest. Combining these factors makes this an ideal location for scientific study. Despite this, there has been very little research on the lichens of ST&P. Collections from the late 19th century account for the vast majority of lichen specimens from the islands.
Adolpho Mueller, Francisco Quintas, and Francisco Newton collected lichen in the 1880's from
ST&P, and sent them to the Finnish botanist William Nylander for identification. Only five species of
Usnea were reported by Nylander (1886), viz., U. articulata, U. ceratina, U. florida, U. longissima, and U. trichodea. Mueller collected them all from the larger island of Sao Tome. Nylander did not have the modern tools of TLC and DNA sequencing to aid his identification and the taxa reported for the most part represent misapplied names. A sixth species, U. speciosa, was added to the list of known Usnea from the islands when Motyka (1936) described it as a new species based on a specimen collected on Sao Tome by Moller and determined by Nylander (1886) as U. articulata.
Hence, prior to this research, only six species of Usnea were known from ST&P. 4
This monograph provides the first comprehensive treatment of the Usnea species from ST&P, including the first ever collections from the island of Principe. Taxonomic determinations are based on morphology, ecology, chemistry (TLC data) and molecular analyses (nDNA ITS sequences). A key to the species is provided, along with species descriptions of eleven taxa, photographs of taxonomically important features, and phylogenetic trees. 5
Materials and Methods
Literature Review
A literature review using Google Search, Google Scholar Search, and OCLW WorldCat was conducted in order to determine the quantity and quality of lichen collection and research performed from the islands of Sao Tome and Principe. The key terms used included the following in various combinations: “Sao Tome and Principe”, “Lichen”, “Usnea”, “Collection”, “Research”, and
“Gulf of Guinea”.
Specimen Collection
The specimen collection took place over two, four to five week long, field collecting trips during
April—May of 2012 and April—May of 2013. The initial 2012 collecting trip consisted of approximately three weeks on the main island of Sao Tome and two weeks on the smaller island of
Principe. The 2013 collecting expedition consisted of approximately three weeks on Principe and one week on Sao Tome. During the time on Sao Tome, Usnea collections were made primarily at elevation between 800 m and the highest peak, which measures 2009 m. The collections from
Principe were primarily collected between 400-715 m in elevation. Usnea specimens were collected along with relevant ecology and substrate data when possible, as well as precise geolocation and altitude measurements using a Garmin Oregon 450 GPS. Attempts to separate 6
species were made in the field with placement into separate “Rite in the Rain” packets, though further separation of mixed collections occurred in the lab upon return. Packets containing Usnea were dried without heat by taking advantage of the dehumidifier affect of the air conditioning in the hotel where field crews were located. After segregating mixed collections there were a total of 132
Usnea collections made over the course of both trips.
Morphological Analysis
Morphological characters were selected based on recent literature of Clerc (1998) and Ohmura
(2001), and observed with a Leica Zoom 2000 stereoscopic dissecting microscope Images were taken using an Olympus SZX12 Stereoscope with an attached 5-megapixel CCD camera. Digital images were used to measure cortex to medulla to axis ratios (CMA) using the program ImageJ
1.48v. The CMA ratios were determined by making a longitudinal cut in the thallus dissecting the three tissue layers, from a main branch that is branching off of the base (Clerc 1984). On specimens where the base is not available or evident, the ratio is made from a cut made on any main branch that appears to be of an average width for the individual. In fertile specimens, ascospore measurements were made using an Olympus CX41 stereoscopic compound microscope and reticle under 1000X oil immersion magnification. 7
DNA Extraction, Amplification and Sequencing
DNA extractions were obtained from all specimens. Approximately 10-15 mg of tissue was cut from the terminal branches and placed in a 1.5 ml microcentrifuge tube with 1000 |jl of purified water and centrifuged for 60 seconds at 1500 RPMs. After centrifugation the samples were transferred to a new microcentrifuge tube where two 1 mm glass beads were added and mechanical disruption of the sample was performed by use of a Retsch Tissuelyser II Ball Mill
Homogenizer for two cycles of 30 seconds each. Extractions were performed using the Omega
E.Z.N.A. HP Plant DNA Mini Kit according to manufactures specifications. To obtain sequences of the internal transcribed spacer (ITS) gene region, Polymerase Chain Reaction (PCR) was performed with the primers USITS4-R (Truong et al. 2013c) and a primer designed for this study using Geneious 7.1.9, named USITS3-F INT (5-TGC GGA AGG ATC ATT ACC GAG-3’). Bioneer
AccuPower® PyroHotStart Taq PCR PreMix tubes were used with 2 |jl of DNA template. Thermal cycling settings used follow those of Truong et al. (2013c): initial denaturation at 94 °C for 5 min; five cycles at 94 °C for 60 s, 56 °C for 60 s, 72 °C for 90 s; 30 cycles at 94 °C for 60 s, 54 °C for
60 s, 72 °C for 90 s; final extension at 72 °C for 10 min. PCR products were visualized using a 1.8
% agarose gel stained with ethidium bromide. Successfully amplified product was purified using 2 pi exosap diluted at 1:5 mixed with 3 pi of template. The same primers used for PCR were used for the cycle sequencing reaction. Following precipitation and re-suspension in Hi-dye, the samples were loaded into an Applied Biosystems ABI Prism® 3100 Genetic Analyzer for sequencing. 8
Sequencing Data Analysis
Raw sequencing data was analyzed using Applied Biosystems Sequence Analyzing Software with
KB basecaller and then inspected manually for any regions that were ambiguous. The regions of the reads that contained greater than a 3% chance of error per base were trimmed from the resultant reads and contigs were assembled using Geneious 7.1.7. Sequences were then manually perused to determine correct assembly and direction. Non-ST&P sequence data was gathered from the National Center for Biotechnology Information (NCBI) website
(http://www.ncbi.nim.nih.gov). These sequences were evaluated on length and the quality of sequences as determined by the amount of ambiguous nucleotides as well as the requisite of publication in a peer-reviewed journal. Geneious software was used to align final sequences using the Muscle alignment with default settings and the resultant alignment was inspected and corrected manually where necessary. Jmodeltest was used to determine the correct substitution model to use for tree building. Alignments were assembled into trees using the phylocluster at California
Academy of Sciences. Randomized Axelerated Maximum Likelihood 7.2.6 was used to produce a maximum likelihood tree with 2000 replicates, and Mr. Bayes was used to produce a Bayesian inference for phylogeny tree with 10,000,000 generations and a burn-in of 25 percent. 9
Chemical Analysis
Chemical data was generated using thin layer chromatography. Solvent systems C, containing 200 ml toluene and 18 ml acetic acid was used, as well as solvent system B’, containing 140 ml hexane, 72 ml butyl-methyl ether, and 18 ml formic acid. The solvent system solutions were mixed in a flask then poured into 25 * 27 * 7 cm glass tanks with weighted lids and allowed to equilibrate for 30 minutes. Small cuttings of each sample were placed onto a microscope slide and mixed with several drops of acetone. The resulting mixture was then spotted onto an Aluminum/Silica Gel 60
F254 TLC Sheet using a glass capillary tube. The acetone mixture was applied 15-20 times per specimen spot, allowing to dry between spotting, in order to increase the quantity of organic compounds transferred to the plate. Parmotrema hypotropum, containing norstictic acid and atranorin, was used as a control. After all plates were spotted, the B’ plates were placed into a pre-
B’ tank with 5 ml of 60% formic acid contained; likewise the C plates were placed into a pre-C tank with 5 ml of glacial acetic acid. The plates were raised out of the liquid on glass blocks inside of the rectangular glass developing tanks during this phase, and left in for five minutes. Directly following this pre-phase the plates were placed into the tanks containing the respective solvent systems and lids were applied with lead bricks to weight them down in order to maintain a tight seal. The reaction was allowed to run approximately 20 minutes until the solvent had visibly run up the plate to approx. 0.5 cm from the top of the plate. When plates were completely dried spots were traced in pencil on the plates by observing them under 365 and 254 nm ultraviolet lights and outlining spots as well as marking any UV reaction that occurred in the spots. Each plate was then coated with a layer of 10% sulfuric acid (H2S04) and baked at 100° C for 3-5 minutes. Completed plates 10
were scanned with a flat bed scanner 5-7 days later in order to preserve images of the color of the spots, as they fade over time (McCune, unpublished). Plates were analyzed using tables of Rf factors (Culberson, unpublished) and spot color to determine the compounds present and quantities were estimated by the amount and brightness of spotting. Compounds are considered major when present in all specimens in high abundance and as minor when present in all specimens in relative low abundance. 11
Phylogenetic Results
From 108 successful ITS sequences, 87 unique sequences were aligned along with 56 sequence records acquired from Genbank. 21 sequences were determined to be identical to other sequences and were not included in the alignment. The Genbank specimens included 27 distinctly named species of Usnea, as well as Platismatia glauca to serve as an out-group. The alignment had 529 total characters of which 169 were parsimony informative.
GTR+I+G was determined to be the best statistical model for phylogenetic analysis with a -In score of 4778.8336 in jModelTest. Maximum Likelihood and Bayesian Inference analyses produced slightly different topologies. The Bayesian tree showed subgenus Dolichousnea sister to subgenus
Usnea with subgenus Eumitria basal, while the ML tree showed subgenus Usnea basal to the others. The Maximum Likelihood analysis produced nine distinct clades or single branches of
ST&P Usnea with varying levels of support (Fig. 1). Three of the clades represent subgenus
Eumitria and the other seven fell within subgenus Usnea. No clades of ST&P Usnea belong to subgenus Dolichousnea. Two clades in subgenus Eumitria contain the species Usnea baileyi and
Usnea pectinata, nested within numerous ST&P specimen sequences. One clade in subgenus
Usnea contains U. articulata nested within ST&P sequences. The other six clades or branches 12
represent only ST&P sequences; no Genbank representative samples were supported within these
clades.
Subgenus Eumitria
Subgenus Eumitria (Fig. 2) received 100% Maximum Likelihood Bootstrap Support (BS) and 1.0
Bayesian Inference Posterior Probability (PP). The subgenus is composed of three well-supported clades, designated Usnea firmula (with 98% BS, 0.96 PP support), the Usnea baileyi Complex
(81% BS, 1.0 PP), and the Usnea pectinata Complex (93% BS, 1.0 PP).
The Usnea firmula clade represents this species based on ST sequences. The Usnea baileyi complex is composed of two internal clades: one clade has 89% BS and 0.99 PP support and contains only ST&P sequences (UB2); the second clade has 82% BS and 0.99 PP support and contains one ST sequence (MN0472) and two Genbank sequences determined as U. baileyi
(AB051050, AB051051) (UB1). The Usnea pectinata Complex comprises a basal grade of numerous ST sequences (UB0), and two well-supported internal clades with two Genbank sequences determined as U. pectinata (AB051655, AB051656) nested within. One internal clade has 99% BS and 1.0 PP support and contains only ST sequences (UP1), while the other internal clade has 98% BS and 1.0 PP support and contains only P sequences (UP2). 13
Subgenus Dolichousnea
There are no specimens from ST&P in subgenus Dolichousnea, though, for the purpose of
comparison, data (n sequences) from each U. diffracta (4), U. trichodeoides (4) and U. longissima
(6) are included in the study (Fig. 5). The subgenus received very high support (97% BS, 1.0 PP).
The separation of the lineages of U. diffracta (100% BS, 1.0 PP) and U. longissima (94% BS, 1.0
PP) was also strongly supported, although U. trichodeoides received limited support.
Subgenus Usnea
The largest subgenus, Usnea (Fig. 6), lacks significant ML support (53% BS, 1.0 PP). Subgenus
Usnea has six internal clades or individual branches containing ST&P sequences and 23 taxa from
Genbank. The clades that do not include ST&P sequences are composed of only Genbank
samples. Usnea glabrescens, U. lapponica, U. subfloridana, U. florida, U. fulvoreagens, and U.
wasmuthiiform one major clade (89% BS, 1.0% PP). Usnea flavocardia forms another clade (84%
BS, 1.0 PP) sister to U. glabrata. Usnea nidifica and U. himalayana form a well-supported clade
(100% BS 1.0 PP). Usnea cornuta forms a clade (99% BS 1.0 PP) with U. rubicunda, U. rubrotincta, and U. mutabilis falling external to it without significant support. Usnea aciculifera. U. dasaea, and U. ceratina make up a polyphyletic clade with weak support. 14
The ST&P collections constitute 3 internal clades and 3 individual branches in subgenus Usnea.
The internal clades include Usnea articulata Complex (73% BS 1.0 PP), Usnea picta (100% BS 1.0
PP), and an unidentified Usnea species C Complex (100% BS 1.0 PP). Of the individual branches all represent ST sequences: Usnea flammea (MN0579) and Usnea sanguinea (MN0264a) are on branches near U. merrillii without significant support, and Usnea krogiana (MN0560) is sister to U. macaronesica (59% BS 0.98 PP). This pair is sister to the strongly supported U. nidificalU. himalayana clade previously noted.
The Usnea articulata complex is composed of only ST sequences, but the topology shows low statistical support. There are three weakly supported internal clades: one clade has 99% BS and
1.0 PP support and represents a pseudocyphellate form of U. articulata; a second clade with 83%
BS and 0.99 PP support contains three ST sequences and one Genbank sequence determined as
U. articulata (the non-pseudocyphellate form) (UA2); and a weakly supported third clade of multiple sequences, several of which (MN0052, MN0053) are morphologically and chemically similar to
Usnea species A. A weakly supported clade composed of three sequences (MN0191, MN0253,
MN0116) is treated as unidentified Usnea species A, and sequence MN0071 is treated as unidentified Usnea species B. Both of these two clades, sister to the U. articulata Complex, are recognized by morphological and chemical distinctions discussed later. Usnea picta (100% BS, 1.0
PP), composed only of P sequences, is sister to an unidentified Usnea species C Complex with
93% BS and 1.0 PP support. The Usnea species C complex with 100% BS and 1.0 PP support is 15
composed of multiple ST&P sequences. Usnea pygmoidea and U. pangiana are on long branches,
sister to U. picta and U. species C with low support.
Thin Layer Chromatography Results
Thin layer chromatography indicated the presence of at least 18 different chemical compounds
present in the thallus of the specimens. Usnic acid was always present, as is expected for the
genus. The other compounds identified were: constictic acid, stictic acid, connorstictic acid,
norstictic acid, salazinic acid, galbinic acid, protocetraric acid, fumaroprotocetraric acid,
menegazziaic acid, barbatic acid, diffractaic acid, zeorin, eumitrin A1, eumitrin A2, eumitrin B,
psoromic acid, and 2’-0-demethyl-psoromic acid. Unidentified compounds were not listed.
There were discernable chemical differences in many of the taxa (Appendix A). In subgenus
Eumitria, U. firmula has protocetraric acid and eumitrins as major substances, U. baileyi has
norstictic acid as a major substance, and U. pectinata has variable chemistry with salazinic,
constictic, protocetraric and barbatic acids each showing up in significant percentages. Within
subgenus Usnea the specimens determined as U. sanguinea contain trace amounts of barbatic
acid, those of U. krogiana have salazinic, connorstictic, and norstictic acids, while Usnea species B contains constictic and stictic acids. The individual specimen making up Usnea aff. flammea was 16
not sampled with TLC. The internal clades of U. articulata have two distinct chemotypes; UA1 containing barbatic acid as a major compound, and UA2 containing psoromic acid and 2’-0- demethy-psoromic acid as major compounds. The clade treated as unidentified Usnea species A contains salazinic acid as a major compound. The Usnea picta clade has very low levels of chemicals present, but did show salazinic acid as a major component in trace amounts.
Unidentified Usnea species C has a very consistent chemistry with constictic, stictic and diffractaic acid as major compounds and menegazziaic acid as a frequently occurring minor component. 17
Taxonomy:
Key to species
1. Central axis fistulose------2
1. Central axis solid or only slightly deteriorating------3
2. Norstictic acid present, protocetraric acid absent; fibrils > 10 mm long— U. baileyi
2. Protocetraric acid present, norstictic acid absent; fibrils < 10 mm long — U. firmula
3. Cortex completely smooth or cracking but not isidiate, sorediate,soraliate or
otherwise ornamented------4
3. Cortex with papillae, tubercles, soralia or isidia, not smooth------6
4. Fibrils numerous on main branches, fishbone pattern------U. pectinata
4. Fibrils very few or lacking on main branches, no fishbone pattern------5
5. Cortex stramineous to yellowish green; axis white------U. articulata
5. Cortex grayish green to dark green; axis translucent faintly yellow------U. species C
6. Isidiomorphs present------7 18
6. Isidiomorphs absent------8
7. Branches very thin, fragile, always terete; axis white------U. krogiana
7. Branches medium thick to very thick, tough, often ridged; axis red------U. pectinata
8. Red pigments present throughout cortex; secondary branches mottled
green and red------U. sanguinea
8. Cortex unpigmented------9
9. Longitudinal or orbicular pseudocyphellae present directly oncortex ------U. articulata
9. Pseudocyphellae punctiform or present only on tubercles------10
10. Punctiform soralia present------11
10. Soralia absent------12
11. Yellowish orange pigment present in medulla near cortex, not in axis------U. flammea
11. Yellow pigment present in axis, not in medulla------U. species B
12. Tubercles common, large, with pseudocyphellae at apices; papillae
absent------U. picta
12. Tubercles infrequent or absent; papillae numerous------13 19
13. Papillae very small, flat; tubercles occasional with pseudocyphellae on apices;
norstictic acid major substance------U. species A
13. Papillae large, pronounced; tubercles absent; norstictic acid absent------U. species C 20
Species Descriptions
1. Usnea firmula (Stirt.) Motyka, Lich. Gen. Usnea Monogr. 1:51 (1936) (Plate 1)
Thallus erect to subpendant, shrubby, with a hollow axis, 50-100 mm in length, drab green in newer growth to dark green or black in age. Base of thallus blackened several cm above base without annular cracking, occasionally pale. Branching sub-isotonic dichotomous. Lateral branches cylindrical at point of attachment, non-constricting. Branches narrow, terete, uninflated: (0.68-)
0.76-0.78-0.92 (-1.16) mm diameter (n=5). Soralia absent. Soredia occasionally produced from eroded tubercles and/or broken fibrils. Isidiomorphs rare. Fibrils numerous, long (10-15 mm), in a fishbone pattern. Tubercles numerous. Papillae absent. Pseudocyphellae present on apices of tubercles. Apothecia common, numerous, terminal on short side branches, fibrils around margin, with a white pruinose covering on apothecial disc, (0.9-) 1.6-2.1-2.9 (-4.3) mm diameter (n=11).
Ascospores 10x7 |jm, oblong, thick walled, hyaline. Cortex mat, very thin, (5-) 5-6-7 (-8)% of diameter (n=5). Medulla very thin, deep red pigment at border of medulla and cortex, (4-) 4-5-5 (-
6)% of diameter. Axis very thick, non-pigmented, fistulose, (72-) 76-79-80 (-81)% of diameter.
Tubular axis (68-) 69-73-74 (-74)% of axis diameter. 21
Chemistry: usnic acid, protocetraric acid (major), salazinic acid (minor), eumitrin A1 (±), eumitrin
A2 (±), eumitrin B (±), zeorin (±).
Specimens examined (bold items included in molecular phylogeny): MN0084 (ST), MN0117 (ST),
MN0550a (ST), MN0550b (ST), MN0581 (ST).
Notes: Usnea firmula is very similar to U. baileyi, as described by Ohmura (2001,2012) and
Swinscow and Krog (1988). Usnea firmula has a slightly larger branch diameter with a thinner cortex and medulla, as well as a thicker axis and a larger tubular percentage of the axis. The fibrils are also longer in Usnea firmula than in U. baileyi. The other primary distinguishing factor is the chemistry with U. firmula containing protocetraric acid as a major compound while U. baileyi contains norstictic acid as a major compound (Fig. 4), consistent with Ohmura’s (2001) analysis of
U. baileyi. Swinscow and Krog (1988) describe Usnea firmula as having protocetraric acid in specimens coming from West Africa as well as from the type specimen described from the neighboring island of Bioko. The slight morphological differences combined with the chemistry and molecular phylogeny (Fig. 2) distinguishes this species primarily occurring in Africa. MN0550b is the only UV+ specimen of the U. firmula samples. It is also slightly isidiate and this may be the cause of the UV reaction due to the eumitrins coming through in the isidiomorphs. Usnea firmula is noted as new for the Island of Sao Tome and was not observed on Principe (Fig. 3). 22
2. Usnea baileyi (Stirt.) Zahlbr., Denkschr. Kaiserl. Akad. Wiss., Math.-Naturwiss. Kl. 83:183
(1909) (Plate 2)
Thallus erect to subpendant, shrubby, with a hollow axis, 60-110 mm in length, light green in newer growth to dark green or black in age. Base of thallus blackened at point closest to attachment, without annular cracking. Branching sub-isotonic dichotomous. Lateral branches cylindrical at point of attachment, non-constricting. Branches narrow, terete, uninflated, (0.50-)
0.62-0.69-0.83 (-1.11) mm diameter (n=9). Soralia absent. Soredia sometimes produced from eroded tubercles. Isidiomorphs rare. Fibrils numerous, short, fishbone pattern. Tubercles numerous, raised. Pseudocyphellae present on apices of tubercles. Papilla sometimes found on smaller branches. Apothecia rare, terminal on short side branches, (3.2-) 4.6-5.1-5.6 (-8.0) mm diameter (n=8). No mature ascospores seen. Cortex mat, thin, (6-) 7-7-8 (-12)% of diameter
(n=9). Medulla thin, deep red pigment at border of medulla and cortex, occasionally lighter pink/peach, (5-) 7-7-7 (-10)% of diameter. Axis very thick, non-pigmented, fistulose, (63-) 67-
72-73 (-75)% of diameter. Tubular axis: (44-) 52-57-66 (-84)% of axis diameter.
Chemistry: usnic acid, norstictic acid (major), salazinic acid (±), eumitrin A1 (±), eumitrin A2 (±), eumitrin B (±), barbatic acid (±). 23
Specimens examined (bold included in molecular phylogeny): MN0179 (ST), MN0413 (P), MN0417
(P), MN0472 (ST), MN0487 (P), MN0535 (P), MN0539 (P), MN0549a (ST), MN0600a (ST)
Notes: Usnea baileyi is a pantropical species in subgenus Eumitria that is distinguished by its distinct fistulose axis and the presence of norstictic acid and eumitrins in the thallus. Usnea firmula has very similar morphology and is primarily distinguished by having different chemistry and slightly longer fibrils. The U. baileyi species group has potentially been misidentified due to this morphological similarity between groups that appear dissimilar in phylogenetic analyses. The consistent presence of norstictic acid and the lack of protocetraric acid set the U. baileyi group apart from U. firmula (Fig.4), as well as the molecular phylogeny (Fig. 2). The specimens included from Ohmura’s Genbank entries (AB051050 and AB051051) are from Wakayama prefecture,
Japan and Maui, Hawaii, United States, respectively, and show a well-supported phylogenetic relationship with MN0472 (Fig. 2 UB1), the only low elevation (215 m) Usnea collected from Sao
Tome. This species complex appears to represent the Usnea baileyi mar. baileyi described in
Swinscow and Krog (1988) although usually without abundant isidia; only one specimen has abundant isidia (MN0600a). This specimen was also UV+, possibly due to the eumitrins being exposed in the isidia (see U. firmula). Clade UB2 represents specimens collected from higher elevation on both Sao Tome (862-1374 m) and Principe (509-581 m) (Fig. 2). 24
3. Usnea pectinata Taylor, London J. Bot. 6:191 (1847) (Plates 3 & 4)
Thallus pendant, 25-500+ mm in length, pale grayish green to greenish yellow in newer growth to brownish black in age. Base of thallus concolorous to brown or black, often no attachment present.
Branching anisotonic dichotomous. Annular cracks common and/or decorticate outer cortex layer.
Morphotype A - non-isidiate (Plate 3): Lateral branches rare, when present, cylindrical to flattened at point of attachment, non-constricting. Morphotype B - isidiate (Plate 4): Lateral branches common, curved upward, and cylindrical with annular cracking at point of attachment. Branches thin to thick, terete to slightly ridged to rarely 5-6 ridged in morphotype B, uninflated, (0.24)—0.36—
0.44—0.64—(1.20) mm diameter (n=33). Soralia absent. Soredia sometimes produced from broken fibrils or along ridges in morphotype B. Isidiomorphs absent in morphotype A to present along ridges, near soredia, in morphotype B. Fibrils numerous, fishbone pattern, dense. Tubercles absent. Papillae occasional, infrequently spaced on main branches. Pseudocyphellae absent.
Apothecia not observed. Cortex glossy when intact, thin, sometimes decorticate, (1-) 9-70-12 (-
14) % of diameter (n=18). Medulla medium thickness, very dense and compact, (9)—12.5—f5—16.5—
(20) % of diameter. Axis very thick, pigmented lightly ochraceous to reddish brown, occasionally slightly eroding in morphotype B, (39)—46—50—59—(63)% of diameter. 25
Chemistry: Usnic acid, protocetraric acid (±), barbatic acid (±), salazinic acid (±), constictic acid
(±).
Specimens examined (bold included in molecular phytogeny): MN0060 (ST), MN0063 (ST),
MN0065 (ST), MN0068b (ST), MN0111 (ST), MN0125 (ST), MN0163 (ST), MN0229 (ST), MN0241
(ST), MN0406 (P), MN0481 (P), MN0482 (P), MN0488 (P), MN0498 (P), MN0524 (P), MN0527 (P),
MN0528 (P), MN0529 (P), MN0534 (P), MN0536 (P), MN0540 (P), MN0542 (P), MN0554 (ST),
MN0556 (ST), MN0567 (ST), MN0575a (ST), MN0578a (ST), MN0583 (ST), MN0584a (ST),
MN0585 (ST), MN0586b (ST), MN0597 (ST), MN0602 (ST).
Notes: Members of the Usnea pectinata complex share the pendant habit and thick, pigmented, typically reddish brown axis but other morphology and chemistry is quite variable. There are two morphotypes present. The primary form of morphotype A (non-isidiate; Fig. 2 UP1 and mixed in
UPO) has a very thin main branch with a decorticate surface. Soredia and Isidiomorphs are few to absent and there is very few to no side branching (Plate 3 D, E, F). It occurs only on Sao Tome and possesses protocetraric acid as a major compound and no barbatic acid (Fig. 4). A slightly variant form of morphotype A (Fig. 2 UP2) was only collected from Principe and has annular cracking in the cortex but is not decorticate and does not have any Isidiomorphs or soredia with few to no side branching (Plate 3 A, B, C). This second variant of morphotype A consistently has 26
salazinic and barbatic acids as major compounds as well as protocetraric acid (Fig. 4). Morphotype
B (isidiate) possesses a thicker main branch often ridged rather than terete, with soredia and
Isidiomorphs erupting from the cortex, as well as many side branches (Plate 4). This morphotype does not have support in the molecular phylogeny though a chemical race with constictic and barbatic acids as major compounds appears to correlate with the ridged thallus, isidiate specimens.
All of these differ significantly from the Ohmura (2001) specimens that all contain norstictic, constictic, stictic, and menegazziaic acids. In fact no ST&P U. pectinata specimens contain norstictic, stictic or menegazziaic acid (Appendix A). The primary morphology seen in the ST&P specimens with thin main branches (Fig. 2 UP1, UP2 and mixed in UPO) fits the description of U. pectinata, excluding the chemistry (Ohmura 2001). The thicker ridged morphotype matches the description of U. himantodes, excluding the chemistry (Ohmura 2001). Each morphotype, as well as the variable chemistry, fit under Swinscow & Krog’s (1988) concept of Usnea gigas. The molecular phylogeny, however, includes these different morphotypes mixed into the U. pectinata complex without any clear delimitation. U. pectinata is reported herein as new for both islands. 27
4. Usnea aff. flammea Stirt., Scott. Natural. 6:102 (1881).(Plate 5)
Thallus erect to subpendant, 30-40 mm in length, whitish/yellowish green when fresh to yellow orange when dried. Base of thallus concolorous, some annular cracking. Branching anisotonic- dichotomous. Lateral branches cylindrical at point of attachment, slightly constricting. Branches narrow, terete, not inflated, 0.60-0.75 mm diameter. Soralia on main branches and secondary branches, numerous, punctiform, irregular, minute, 0.07-0.14 mm diameter. Isidiomorphs occasionally produced from soralia. Fibrils many. Tubercles few, scattered, eroding into soralia.
Papillae frequent on main branches, sparse on lateral branches. Pseudocyphellae absent.
Apothecia not observed. Cortex medium thickness, mat surface, 11% of diameter (n=1). Medulla cottony with a yellow pigment near the cortex, 29% of diameter. Axis thin non-pigmented, 19%.
Chemistry not available.
Specimens examined (bold items included in molecular phylogeny): MN0579 (ST).
Notes: This single observation appears morphologically similar to the Purvis (1992) description of
Usnea flammea as it has an erect to subpendant habit, yellowish brown pigment in the medulla 28
closest to the cortex, and a concolorous base with annular cracking (Plate 5D). Chemistry data was
not available for this specimen and it is therefore tentatively identified as U. flammea. The
molecular phylogeny shows a sister relationship with U. merrillii (Fig. 6). This relationship may
prove significant with additional data as there is a feature associated with U. merrillii described by
Ohmura (2001) as “regenerated globe-shaped secondary cortex between the segments” that is
also found in MN0579 (Plate 5D). Usnea flammea, if confirmed, would represent a new distribution
record for Sao Tome; it was not observed on Principe.
5. Usnea sanguinea Swinscow & Krog, Lichenologist 11: 243 (1979). (Plate 6)
Thallus erect, 25-30 mm in length; main branches reddish brown; secondary branches mottled
dark grayish green with red pigment, becoming faded in storage. Base of thallus blackened at point
closest to attachment with annular cracking. Branching sub-isotonic dichotomous. Lateral branches
cylindrical at point of attachment, non-constricting. Branches narrow, terete, uninflated, 0.4-0.6 mm diameter. Soralia absent. Soredia absent. Isidiomorphs absent. Fibrils numerous, fishbone pattern often broken. Tubercles absent. Papillae few. Annular pseudocyphellae are present on main
branches. Apothecia not seen. Cortex mat, medium-thin, with red pigment sporadically throughout, 29
fO% of diameter (n=1). Medulla medium-thick, cottony, 25% of diameter. Axis medium thickness, non-pigmented, 29% of diameter.
Chemistry: usnic acid, trace barbatic acid.
Specimens examined (bold items included in molecular phylogeny): MN0264a (ST).
Notes: The single specimen representing Usnea sanguinea was collected on the summit of Pico de
Sao Tome. This relatively newly described species (Swinscow & Krog 1979) is distinguished from other red-pigmented Usnea by the pigment location in the cortex rather than the medulla, and a lack of isidia as is present in Usnea rubicunda. There were postulations that U. sanguinea and U. rubicunda are conspecific morphotypes (Swinscow & Krog 1988) but the molecular phylogeny has this specimen well separated from the Genbank sequences of U. rubicunda (Fig. 6). The chemistry of the Sao Tome specimen (MN0264a) is not consistent with the published report of U. sanguinea, type specimen from Tanzania, but the small size of the specimen did not allow for a high quality
TLC sample. The weak barbatic acid presence does indicate that this specimen is at least a different chemical race. The lack of apothecia also indicates a possible discrepancy with the identification as U. sanguinea, though it is not a disqualifying one. As with MN0579, this specimen exhibits the “regenerated globe-shaped” cortical feature (see Usnea aff. flammea) and MN0264a is 30
basal to the sister pair of U. merrillii, and Usnea aff. flammea (Fig. 6). Usnea sanguinea is reported here as new for Sao Tome; it was not observed on Principe.
6. Usnea species A (Plate 7)
Thallus erect to subpendant, 10-65 mm in length, grayish green to yellowish green in age. Base of thallus concolorous to light brown. Branching sub-isotonic dichotomous. Lateral branches cylindrical at point of attachment, constricting. Branches narrow to wide, terete, inflated, (0.66-)
0.69-0.89-1.11 (-1.22) mm diameter (n=4). Soralia occasional on smaller branches, concave.
Soredia sometimes produced from eroded tubercles. Isidiomorphs absent. Fibrils numerous, fishbone pattern. Tubercles occasional, scattered, with apical pseudocyphellae. Papillae numerous, flattened. Apothecia common, terminal on main and on long side branches, (1.1—) 1.6-
2.7-3.0 (-3.6) mm diameter (n=12). Ascospores 12 x 9 pm oblong, hyaline, thick walled. Cortex mat, thin, (6-) 7-7-8 (-10)% of diameter (n=5). Medulla thick, loose cottony, (28-) 28-33-35 (-
36)% of diameter. Axis medium thickness, ochraceous yellow pigmented, (15-) 16-20-25 (-27)% of diameter. 31
Chemistry: usnic acid, norstictic acid (major), salazinic acid (minor), connorstictic acid ±, stictic acid seen in one specimen (MN0191), as well as one specimen lacking norstictic acid and containing protocetraric acid (MN0136).
Specimens examined (bold items included in molecular phylogeny): MN0116 (ST), MN0136 (ST)
MN191 (ST), MN0253 (ST), MN0552 (ST), MN0553 (ST).
Notes: Usnea species A shares many characters with Usnea ochrophora as described by Motyka
(1936), as does the papillate morphotype of Usnea species C. Without chemistry from the type specimen of U. ochrophora or topotypical material (southern Africa) it is not possible to determine which species, A or C, may represent U. ochrophora, if either. An 1885 Moller specimen from Sao
Tome determined of Usnea florida was reported by Motyka (1936) as representing U. ochrophora.
It is possible this is the same species as some of the material collected by Moller identified by
Nylander (1886) as Usnea florida. The molecular phylogeny (Fig. 6) places the Usnea species A group sister to Usnea species B, and together sister to the Usnea articulata complex, all of which are quite distant from the U. florida Genbank sequences, suggesting that none of the specimens from Sao Tome represent U. florida.
The Sao Tome specimens MN0552 and MN0553 which align molecularly with material in the U. articulata complex are morphologically and chemically similar to Usnea species A, and are 32
herein recognized as belonging to the latter taxon. Usnea species A has an erect to subpendant habit, distinctly yellow tinted axis (Plate 7F), and the presence of norstictic and salazinic acids and the absence of barbatic or psoromic acids. In comparison, U. articulata has a long, pendant habit large swollen segments, white or pale pink axis (Swinscow & Krog 1988), and the presence of barbatic or psoromic acids and the absence of norstictic and salazinic acids. Usnea species A is similar morphologically to U. flavocardia, with its yellow axis, but the molecular phylogeny places U. flavocardia distant from Usnea species A (Fig. 6). Usnea species A is reported here as a new distribution for Sao Tome; it was not observed on Principe.
7. Usnea species B (Plate 8)
Thallus erect to subpendant, 20-50 mm in length, drab green to stramineous in age. Base of thallus concolorous to light brown with annular cracking. Branching sub-isotonic dichotomous.
Lateral branches cylindrical at point of attachment, constricting. Branches narrow, terete, slightly inflated, 0.4-0.5 mm diameter (n=2). Soralia common on all branches, punctiform to irregular.
Soredia produced from annular cracks. Isidiomorphs absent. Fibrils few, long. Papillae rare, very small on main branch if present. Apothecia not seen. Cortex glossy, thin to medium, 8-11% of 33
diameter (n=2). Medulla medium thickness, compact cottony, 18-29% of diameter. Axis medium thickness, white with slight yellow tint to ochraceous yellow pigmented, 25-41 % of diameter.
Chemistry: usnic acid, constictic (major), stictic (major).
Specimens examined (bold items included in molecular phylogeny): MN0071 (ST), MN0190 (ST)
Notes: Usnea species B is similar in morphology to Usnea species A, but lacks a papillate cortex
(Plate 8E), and has constictic and stictic acid rather than norstictic and salazinic acid (Fig. 8). The two specimens observed were sterile as well. The molecular phylogeny (Fig.6) shows MN0071 as being on a rather long branch embedded within the U. articulata + Usnea species A clade. It is possible that Usnea species B is a morpho/chemotype of Usnea species A. If Usnea species B is a unique taxon, it represents a new distribution record for Sao Tome; it was not observed on
Principe. 34
8. Usnea articulata (L.) Hoffm., Deutschl. FI., Zweiter Theil (Erlangen): 133 (1796) [1795],
(Plate 9)
Pseudocyphellate Morphotype/chemotype (Fig. 6, UA1): Thallus pendant, 80-300+ mm in length, stramineous yellow-green, becoming darker in storage. Base of thallus concolorous or occasionally blackened at very base near substrate attachment without annular cracking.
Branching isotonic-dichotomous. Lateral branches constricting at point of attachment. Main branches large, terete, inflated, (0.65-) 0.70-0.75-1.14 (-1.58) mm diameter (n=7). Lateral branches smaller. Soralia absent. Isidiomorphs absent. Fibrils sparse to absent. Tubercles absent.
Papillae absent. Pseudocyphellae present, longitudinal to occasionally orbicular or irregular, flattened or raised. Annular cracking rare on main branches. Apothecia rare, when seen cup with few fibrils surrounding cream colored disc, (1.2-) 1.2-2.0-3.2 (-7.1) mm diameter. Ascospores 12 x 8 |_im, ellipsoid, thick walled. Cortex thin, glossy surface, (4-) 4-5-5.75 (-7)% of diameter
(n=10). Medulla loose, cottony, thick, non-pigmented, (28-) 33-35-36 (-38)% of diameter. Axis thin to medium thickness, non-pigmented, (16-) 19.25—2f—22 (-31)% of diameter.
Chemistry: Usnic acid, barbatic acid (major), protocetraric acid ±, fumaroprotocetraric acid ±, galbinic acid ±. 35
Smooth Morphotype/chemotype (Fig. 6, UA2): Thallus pendant, 150-220+ mm in length, stramineous yellow-green, becoming darker in storage. Base of thallus concolorous to light brown near substrate attachment with annular cracking. Branching isotonic-dichotomous. Lateral branches cylindrical to slightly flattened at point of attachment, non-constricting. Branches narrow, terete, uninflated, 0.5—0.7 mm diameter. Soralia absent. Isidiomorphs absent. Fibrils sparse to absent. Tubercles absent. Papillae absent. Pseudocyphellae absent. Annular cracking present on main branches, 6-10 cracks per cm. Apothecia not seen. Cortex medium thickness, glossy surface, (13-) 15-77-18 (-19) % of diameter (n=3). Medulla compact cottony, medium thickness, non-pigmented, (18-) 19-20-20 (-20) % of diameter. Axis medium thickness, non-pigmented, (26-
) 26-26-30 (-34) % of diameter.
Chemistry: Usnic acid, psoromic acid (major), 2’-0-demethylpsoromic acid (minor).
Specimens examined (bold included in molecular phylogeny): MN0067 (ST), MN0068c (ST),
MN0080 (ST), MN0081 (ST), MN0113 (ST), MN0114 (ST), MN0115 (ST), MN0211 (ST), MN0216
(ST), MN0242 (ST), MN0243 (ST), MN0260 (ST), MN0264b (ST), MN0269 (ST), MN0551 (ST),
MN0555 (ST), MN0571 (ST), MN0575b (ST), MN0578b (ST), MN0584b (ST), MN0586a (ST) 36
Notes: Usnea articulata is known to be variable in morphology and chemistry (Ohmura, 2012).
Swinscow and Krog (1988) describe U. articulata as having several separate morphotypes as well as several chemical races, consistent with the findings from ST&P. The specimens examined from
ST&P with a smooth morphotype belong to the same subclade (UA2) as the Genbank specimen determined as U. articulata (KJ406277, Fig. 6). These all share the psoromic acid chemistry (Fig.
8) as well as the thicker axis and smooth, non-pseudocyphellate cortex (Plate 9B). The pseudocyphellate morphology, clade UA1, shows many longitudinal pseudocyphellae on the cortex
(Plate 9E), a feature diagnostic for Usnea speciosa, described from Sao Tome by Motyka (1936).
The pseudocyphellate morphotype has barbatic acid as a major chemical component (Fig. 8), consistent with the type specimen of U. speciosa. this morphotype includes also some specimens with more raised and oblong pseudocyphellae and slight variations in accessory compounds. Two specimens with pseudocyphellate morphology (MN075b, MN086a) show more molecular similarity to the non-pseudocyphellate morphology, while another two specimens (MN0552, MN0553) show more morphological and chemical similarity with Usnea species A, as reported earlier. These results indicate that there is some discordance between morphology, chemistry and ITS sequence similarity. For these reasons, we treat U. articulata as a species complex and tentatively accept U. speciosa as a morphotype. Usnea articulata is confirmed for its occurrence on Sao Tome; it was not observed on Principe. 37
9. Usnea picta (J. Steiner) Motyka, Lich. Gen. Usnea Monogr. 2:325 (1937). (Plate 10)
Thallus erect to subpendant, 25-80 mm in length, grayish green to dark green, remaining dark in age. Base of thallus concolorous to light brown. Branching sub-isotonic dichotomous. Lateral branches tapering, cylindrical at point of attachment, slightly constricting. Branches narrow, terete, slightly inflated, (0.45-) 0.50-0.55-0.59 (-0.63) mm diameter (n=7). Soralia absent. Soredia produced occasionally from eroded tubercles. Isidiomorphs absent. Fibrils few, scattered.
Tubercles common on all branches, numerous, mostly with pseudocyphellae on apex. Papillae absent. Apothecia common, many fibrils surrounding disc and white pruina, (1.0-) 1.5-18-3.4 (-
5.5) mm diameter (n=13). Cortex glossy, thin to medium, (7-) 9-9-10 (-14)% of diameter (n=10).
Medulla medium thickness, compact to compact cottony, (8-) 18-20-23 (-24)% of diameter. Axis thick, non-pigmented, (34-) 37-40-43 (-67)% of diameter.
Chemistry: usnic acid, salazinic acid (major), barbatic acid (±), diffractaic acid (±).
Specimens examined (bold items included in molecular phylogeny): MN0405 (P), MN0407 (P),
MN0452 (P), MN0485 (P), MN0492 (P), MN0525 (P), MN0530 (P), MN0531 (P), MN0537 (P),
MN0538 (P) 38
Notes: Swinscow and Krog (1979) provide a good description of Usnea picta. The species is
characterized by stiff branches with prominent raised tubercles, no papillae, and trace amounts of
salazinic acid (Fig. 8). It is similar to U. submollis, which also has salazinic acid, but the latter
differs in forming smaller tubercles and has numerous papillae. The type specimen for U. picta was
noted as having only usnic acid and salazinic acid as did the majority of ST&P specimens
collected. The molecular phylogeny places this species on a significantly supported branch (100%
BS, 1.0 PP) that is sister to Usnea species C (Fig. 6). Usnea picta is reported here as a new
distribution record for the island of Principe; it was not observed on Sao Tome.
10. Usnea species C (Plate 11)
Thallus erect, shrubby to very long pendant, 25-500+ mm in length, light green in newer growth to dark green or black in age. Base of thallus concolorous when present to rarely slightly darkened at
point closest to attachment with or without annular cracking. Branching sub-isotonic dichotomous to isotonic dichotomous. Lateral branches cylindrical at point of attachment, sometimes constricting. Branches narrow to wide, terete, slightly inflated, (0.48)—0.61—0.69-0.79—(1.22) mm diameter (n=19). Soralia absent. Soredia sometimes produced from annular cracks. Isidiomorphs absent. Fibrils few to many, when present in abundance, fishbone pattern. Papillae absent to 39
numerous, very small. Tubercles absent. Pseudocyphellae absent. Apothecia common, often very abundant, few to many fibrils surrounding disc, (0.6-) 2.2-2.7-3.6 (-6.0) mm diameter (n=39).
Cortex glossy to mat, very thin to thin, (5-) 7-8-9 (-12)% of diameter (n=21). Medulla medium thick to thick, compact cottony to web-like cottony, (12-) 22—26-30 (-34)% of diameter. Axis medium thick to very thick, hyaline with a slight peach pigment, translucent looking, (22-) 26-29-
39 (-52)% of diameter.
Chemistry: usnic acid, constictic acid (major), stictic acid (major), diffractaic acid (±), menegazziaic acid (±), unknown yellow accessory substance in solvent C.
Specimens examined (bold included in molecular phylogeny): MN0066 (ST), MN0070a (ST),
MN0404 (P), MN0409 (P), MN0410 (P), MN0412 (P), MN0414 (P), MN0415 (P), MN0416 (P),
MN0420 (P), MN0432 (P), MN0434 (P), MN0483 (P), MN0484 (P), MN0486 (P), MN0489 (P),
MN0491 (P), MN0493 (P), MN0494 (P), MN0496 (P), MN0497 (P), MN0515 (P), MN0519 (P),
MN0520 (P), MN0521a (P), MN0521b (P), MN0523 (P), MN0526 (P), MN0532 (P), MN0533 (P),
MN0543 (P), MN0544 (P), MN0548 (ST), MN0549b (ST), MN0580 (ST), MN0595 (ST), MN0596
(ST), MN0599 (ST), MN0600b (ST), MN0603 (ST), MN0604 (ST) 40
Notes: The Usnea species C complex is extremely variable, having two very different morphotypes with intermediary morphotypes as well. One morphotypic extreme has a very long pendant thallus with dichotomous branching and a glossy smooth outer cortex with very few fibrils (Plate 11B). The medulla of this morphotype is cottony, but compact (Plate 11C). The alternative morphotypic extreme is shrubby erect to sometimes subpendant, much shorter, with many papillae and/or tubercles present on the mat cortex surface and abundant fishbone pattern fibrils (Plate 11E). The branches are slightly more inflated than the other morphotype due to the web-like cottony medulla
(Plate 11F). Both morphotypes have a similar axis, though varying in thickness, with slight pigment making it look somewhat translucent (Plate 11C,F). On appearance these would seem to be two very distinct species with the smooth form appearing more like U. articulata and the papillate form appearing more like Usnea species A. The papillate form of Usnea species C and Usnea species A may represent what was determined as Usnea ochrophora by Motyka (1936). Despite the morphological differences within Usnea species C, the molecular phylogeny (Fig. 6) supports these disparate morphotypes as being the same species with a ^97% similarity threshold. They occur mixed on the highly supported branch in a way that does not support a separation with some intermediate morphotypes mixed in as well. Moreover, there is a consistent chemical signature for the species consisting of constictic acid, stictic acid, diffractaic acid, and sometimes menegazziaic acid (Fig. 10). The smooth morphotype approaches the morphology of Usnea exasperata but the latter species is described as having a different chemistry (Swinscow & Krog 1988). Usnea species
C is reported as a new distribution record for both islands and was the only species group that did not show separation in the molecular phylogeny based on the island locality (Fig. 7). 41
11. Usnea krogiana P. Clerc, Lichenologist 38(3): 199 (2006) (Plate 12)
Thallus erect to subpendant, 25-50 mm in length, whitish green fresh to grayish green in herbaria.
Base of thallus blackened slightly near substrate attachment, with annular cracking. Branching anisotonic-dichotomous. Lateral branches cylindrical at point of attachment, non-constricting.
Branches narrow, terete, uninflated, 0.5-0.6 mm diameter. Soralia on main branches and secondary branches, numerous, irregular, minute, 0.11-0.15 mm diameter. Isidiomorphs often produced from soralia. Isidiomorphs present on all surfaces, numerous, mainly protruding from soralia as well as directly from cortex. Fibrils numerous to sparse, eroding in age. Papillae absent.
Pseudocyphellae absent. Apothecia absent. Cortex medium thickness, mat surface, 22% of diameter (n=1). Medulla compact, thin, non-pigmented, 6% of diameter. Axis thick, non-pigmented,
43% of diameter.
Chemistry: usnic acid, norstictic acid and constictic acid.
Specimens examined (bold items included in molecular phylogeny): MN0560 (ST). 42
Notes: Usnea krogiana was recently described by Clerc (2006) from material collected in the
Azores. The Sao Tome specimen represents a delicate form of U. krogiana at the extreme end of the size spectrum reported by Clerc. This specimen does not contain stictic acid as a major substance, but does contain constictic acid and norstictic acids (Fig. 8), both listed as minor compounds in Clerc (2006). Additionally, there is note of one specimen containing only usnic and norstictic acid collected in the West Indies. The thick axis giving rise to a rigid thallus and the irregular punctiform soralia often with Isidiomorphs protruding along with the black base confirm the placement in U. krogiana, as opposed to other erect to subpendant, sorediate Usnea species. The proximity to the type locality (The Canary Islands) to the Gulf of Guinea, and the similar island habitats could be a natural range extension of the species. There is also the possibility of human mediated introduction due to the common historical geopolitics of the islands of Madeira and the
Azores, where many specimens have been reported, and ST&P and their associations with
Portugal. The molecular phylogeny places the specimen sister to Usnea macaronesica (Fig. 6), which is also known from the Canary Islands as well as the Azores. This represents a new distribution record for Sao Tome; it was not observed on Principe. 43
Discussion:
A combination of morphology, chemistry and molecular phylogeny was used in this monograph to
delimit eleven species of Usnea from Sao Tome and Principe. Eight of these represent previously
described species, viz., U. articulata, U. baileyi, U. firmula, Usnea aff. flammea, U. krogiana, U. pectinata, U. picta, and U. sanguinea. Usnea articulata was reported previously from Sao Tome
(Motyka 1936), but the remaining seven species represent new distribution records for the
archipelago. Additionally, I recognize three previously unknown taxa that may represent new species, denoted as Usnea species A, B and C. Although all three datasets (morphology, chemistry, molecules) were conclusive regarding species determinations of most taxa, there was a considerable amount of disagreement between them for several species complexes, including U.
articulata, U. pectinata and Usnea species C. The U. articulata complex contains two U. articulata
morphotypes and two unknown Usnea species, A and B. There is a similar problem with U. pectinata where there is molecular support for several internal morphotypes and chemotypes, but
no clear delimitation of all variation groups. The lack of strong molecular support demonstrates either the limitations of sequencing a single gene region, or a very high level of morphological and chemical diversity within certain species of Usnea. Further research is needed, including multi gene analysis, in order to better clarify these taxa.
Attempts to extract ancient DNA from the 1885 Moller collections determined by Nylander
(1889) as U. articulata, U. ceratina, U. florida, U. longissima, and U. trichodea were unsuccessful, 44
making it difficult to compare them to species recognized in this monograph. Several of these
species names have been misapplied to what I recognize as Usnea species A, B and C. These
include U. florida, U. ceratina, and U. ochrophora, a species reported later from Sao Tome (Motyka
1936). In addition, Usnea longissima and U. trichodea were misapplied to the very long pendant
Usnea specimens recognized here as U. pectinata and Usnea species C. It is unusual that U.
firmula and U. baileyi had not been previously reported from ST&P, given their unique Eumitrioid feature of a fistulose central axis. Usnea species A, B and C are likely candidates for new species,
but more data from multiple gene regions is necessary before they are formally described. 45
Plate 1: A) Usnea firmula. B) Main branch with tubercles and apical pseudocyphellae. C) Longitu dinal section with fistulose axis and red pigment in medulla (CMA=6/4/79). D) Apothecia showing fibrils and pruina. E) Apothecia terminal on short side branches. F) Base showing blackening. (MN0084:B,C) (MN0581:A,D,E,F) 46
Plate 2: A) Usnea baileyi, fertile specimen. B) Main branch with papillae and fishbone fibrils. C) Longitudinal section of U. baileyi showing red pigment in medulla and fistulose axis (CMA=7/8/69). D) Usnea baileyi, sterile specimen. E) Longitudinal section of sterile U. baileyi showing pink/peach pigment in medulla and fistulose axis (CMA=7/6/75). F) Blackened base. (MN0417:A,B,C) (MN0472:D,E,F) 47
Plate 3: A) Usnea pectinata, thin corticate morphotype. B) Main branch showing cortex intact. C) Longitudinal section with compact medulla and red pigmented axis (CMA=12/13/50). D) Usnea pectinata, thin decorticate morphotype. E) Main branch showing decorticate cortex and fibrils. F) Longitudinal section with compact medulla and red pigmented axis (CMA=1/17/63). (MN0540:A,B,C) (MN0597:D,E) (MN0602:F) 48
Plate 4: A) Larger, sorediate morphotype of Usnea pectinata. B) Main branch with sub-isotonic dichotomous branching. C) Main branch. D) Longitudinal section of U. pectinata with compact medulla and red pigmented axis with slight cavitation (CMA=9/16/50). E) U. pectinata cortex showing ridges. F) Large main branch showing soredia (1) and isidiomorphs (2). (MN0068b: A,B,E,F) (MN0583: C,D) 49
Plate 5: A) Usnea aff. flammea. B) Main branch with papillae, punctiform soralia and fishbone fibrils. C) Longitudinal section of U. flammea showing yellow pigment in medulla (CMA=13/27/20). D) Concolorous base with annular cracks. E) Sorediate side branch. (MN0579: A,B,C,D,E) 50
Plate 6: A) Usnea sanguinea. B) Main branch with red pigment and fishbone fibrils. C) Longitudi nal section with cottony medulla. (CMA=10/25/29). D) Blackened base. E) Sorediate annular cracking. F) Cortical pigment in main and secondary branches. (MN0264a: a,b,c,d,e,f) 51
Plate 7: A) Usnea species A, main phylogenetic internal clade. B) Main branch with fishbone fibrils and papillae. C) Longitudinal section very loose web-like medulla and yellow pigmented axis (CMA=7/35/16). D) Usnea species A specimen, displaced in phylogenetic tree. E) Main branch with similar fishbone fibrils and papillae. F) Longitudinal section showing similar ratio and yellow pigment in axis. (CMA=7/33/20). (mnoh6:a,b,c)(mno552:d,e,f) 52
Plate 8: A) Usnea species B. B) Main branch with annular cracking. C) Longitudinal section of Usnea species B showing yellow pigment in axis (CMA=11/18/41). D) Slightly brown base. E) Soralia on side branches. F) Close up of soralia. (mnoo71:a,b,c,d,e,f) 53
Plate 9: A) Usnea articulata, smooth morphotype B) Main branch showing articulation with annular cracks C) Longitudinal section of the smooth morphotype (CMA=17/20/26). D) Usnea articulata, pseudocyphellate morphotype E) Main branch with longitudinal pseudocyphellae F) Longitudinal section of the pseudocyphellate morphotype (CMA=4/38/16). (mn0260:a,b,cmmn02ii:d,e,f) 54
Plate 10: A) Usnea picta. B) Main branch with glossy cortex, tubercles with pseudocyphellae. C) Longitudinal section with thick axis and cottony compact medulla (CMA=10/21/37). D) Apothecia attached longitudinally on side branches. E) Apothecia cluster. F) Concolorous to light brown base. (MN0492: A,D,E,F) (MN0537: B,C) 55
A
Plate 11: A) Usnea species C, smooth morphotype B) Main branch showing articulation with annular cracks C) Longitudinal section of the smooth morphotype compact cottony medulla. (CMA=9/26/31). D) Usnea species C papillate morphotype E) Main branch with papillae and fibrils F) Longitudinal section of the papillate morphotype web-like cottony medulla.(CMA=11/26/26). (MN0533: A,B,C) (MN0600b: D.E.F) 56
Plate 12: A) Usnea krogiana. B) Main branch with isidiomorphs and soredia. C) Longitudinal section of U. krogiana (CMA= 14/11/50). D) Blackened base with annular cracking. E) Sorediate side branch with isidiomorphs. F) Eruptant soredia on terminal branch. (mn0560:a,b,c,d,e,f) 57
Subgenus Eumitria See Figures 2-4
Subgenus Dolichousnea See Figure 5
Subgenus Usnea See Figures 6-8
Figure 1: Phylogeny of Usnea, inferred based on ITS 1 and 2, demonstrating the placement of Sao Tome and Principe specimens within the three subgenera, Eumitria, Dolichousnea and Usnea. Maximum likelihood bootstrap support (BS) £ 50 are displayed above or below branch on left of hash mark. Bayesian Inference posterior probabilities (PP) > 0.70 are displayed above or below branch on right of hash mark. Branches with BS s 70 and PP > 0.95 are thickened. 58
MN0117 MN0581 MN0550a Usnea firmula 84/0.97^ H Z MN0550b MN0084 82/099j------MN0472 100/1.0 ^ ^98/1.0 1 AB051050 U. baileyi AB051051 U. baileyi 78/1 .Or MN0535 / M i l Usnea baileyi MN0417 Complex 66/1.0 MN0413 L MN0539 MN0487 B2 89/(155 - MN0600a MN0549a MN0179 74/ MN0068a - MN0125 93/1.0 MN0597 - MN0060 - MN0602 u - MN0567 72/0.99 MN0556 r MN0241 67/0.96* T- MN0585 MN0583 Usnea pectinata - MN0063 Complex - MN0070b I MN0575a MN0163 99/1.0 - MN0584a MN0578a MN0065 rq /1 n j — AB051655 U. pectinata by/l.U-H_ AB051656 U. pectinata f x MN0527 MN0540 98/1.0 - MN0481 MN0542 0.06 substitutions/site
Figure 2: Exert of the phylogeny of Usnea, inferred based on ITS 1 and 2, demonstrating the placement of Sao Tome and Principe specimens within the subgenus Eumitria. Maximum likelihood bootstrap support (BS) £ 50 are displayed above or below branch on left of hash mark. Bayesian Inference posterior probabilities (PP) > 0.70 are displayed above or below branch on right of hash mark. Branches with BS > 70 and PP> 0.95 are thickened. 59
MN0117 U. firmula i t t MN0581 U. firmula o/i/a Q7 L j— MN0550a U. firmula 84/0.97f^L MN0550b U. firmula ST MN0084 U. firmula 82/0.99i------MN0472 U. ^ " 198/1.01 AB051050 U. baile\ AB051051 U. baileyi f i r MN0535 U. baileyi 1 MN0417 U. baileyi I MN0413 U. baileyi - MN0539 U. baileyi P MN0487 U. baileyi MN0600a U. baileyi MN0549a U. baileyi MN0179 U. baileyi 74/10i— MN0068a U. pectinata — MN0125 U. pectinata MN0597 U. pectinata — MN0060 U. pectinata — MN0602 U. pectinata ■ MN0567 U. pectinata MN0556 U. pectinata i- MN0241 U. pectinata ST L MN0585 U. pectinata MN0583 U. pectinata - MN0063 if. pectinata — MN0070b IJ. pectinata MN0575a U. pectinata MN0163 U. pectinata 99/1.0 - MN0584a U. pectinata MN0578a U. pectinata 1— MN0065 U. pectinata j — AB051655 U. pectinata AB051656 U. pectinata MN0527 U. pectinata MN0540 U. pectinata - MN0481 if. pectinata P MN0542 U. pectinata 0.06 substitutions/site
Figure 3: Phylogeny of the subgenus Eumitria depicting the locality of IVIN specimens. ST=Sao Tome, P=Principe complex U. baileyi U. complex U. pectinata U.
0.06 substitutions/site Figure 4: Phylogeny of the subgenus Eumitria with taxonomicaliy significant organic compounds presence/absence groupings. Bar=barbatic acid, Con-constictic acid, Nornorstictic acid, ProC-protocetraric acid, Sal=salazinic acid. * Chemistry not available 61
81/0.9- AB051059 U. diffracta 100/1.0 AB051060 U. diffracta ^ AB051058 U. diffracta 90/0.87I AB051057 U. diffracta 61/0.97j- AB051668 U. trichodeoides - AB051666 U. trichodeoides — AB051672 U. trichodeoides 50/0.77 AB051669 U. trichodeoides 86/ a - AB051648 U. longissima 92/0.99 H - AB051642 U. longissima I AB051643 U. longissima 94/1.0 AB051647 U. longissima — AB051646 U. longissima 94/0 911- AB051645 U. longissima
0.06 substitutions/site
Figure 5: Exert of the phylogeny of Usnea, inferred based on ITS 1 and 2, demonstrating the subgenus Dolichousnea. Maximum likelihood bootstrap support (BS) s 50 are displayed above or below branch on left of hash mark. Bayesian Inference posterior probabilities (PP) ^ 0.70 are displayed above or below branch on right of hash mark. Branches with BS s 70 and PP £ 0.95 are thickened. No Sao Tome and Principe specimens were present in the subgenus. 62
■ AB051049 U. aciculifera - AB051056 U. dasaea ■ KJ406278 U. ceratina ^ U s n e a aff. flammea
Usnea sanguinea
KJ406270 U. subfloridana — AB051662 U. subfloridana AB051664 U. subfloridana ■'■'06268 U. florida AB051663 U. subfloridana AB051638 U. fulvoreagens — AB051673 U. wasmuthii - KJ406281 U. glabrata . ■ KJ406279 U. flavocardia 1JN086295 U. flavocardia 0.06 substitutions/site KJ406280 U. flavocardia Figure 6: Exert of the phylogeny of Usnea, inferred based on ITS sequence data, demonstrating the placement of Sao Tome and Principe specimens within the subgenus Usnea. Maximum likelihood bootstrap support (BS) > 50 are displayed above or below branch on left of hash mark. Bayesian Inference posterior probabilities (PP) ^ 0.70 are displayed above or below branch on right of hash mark. Branches with BS > 70 and PP > 0.95 are thickened. *MN0552 and MN0553 part of Usnea sp. A ** MN0575b and MN0586a part of pseudocyphellate form of Usnea articulata (UA1). 63
AB051049 U. aciculifera AB051056 U. dasaea KJ406278 U. ceratina AB051052 U. ceratina AB051053 U. ceratina 72/1.0 AB051649 U. merrillii MN0579 Usnea aff. t MN0264a U. sanguinea 69/0 701 MN0191 MINI Usnea sp.A 58/0.96, 50/0. MN0253 Usnea sp. A MN0116 Usnea sp. A MN0071 Usnea sp. B — MN0552 Usnea sp. A MN0211 U. aniculata 73/1.0 MN0571 U. articulata ■70.79 - MN0578b U. articulata 50/0.721 - MN0080 U. articulata 70/0.78 Ml MN0242 U. articulata ST r“ P MN0216 U. articulata MN0068C U. articulata L- MN0551 U. articulata 04 ,4 * MN0553 Usnea sp. A 81/LOr- MN0243 U. articulata MN0269 U. articulata MN0260 U. articulata KJ406277 U. articulata 58/0. MN0575b— 5 7 f...... U. articulata MN0586a3 6 a U.I______articulata ST 67/0.79, AB05T&)? U lm m o id ea AB051654 U. nanaiana 53/1.0 < h P ST
isnea sp. C Jsnea sp. C 9 Usnea sp. C >20 Usnea sp. C H2 Usnea sp. C r Usnea sp. C Usnea sp. C P
qq/1 n 61/0.70]- KJ406273 U. cornuta — KJ406272 U. cornuta ^ ------KJ406274 IU. ‘ cornuta AB051659 U. rubicunda ■ AB051661 U. rubrotincta ■ KJ406275 U. rubicunda ■ AB051651 U. mutabilis 98/0.98— 1QQ/1.Q FJ494941 U. nidik FJ494933 >33 U. nidifiCcnlditica AB051640ABC U. himalavana ■ MN0560 . m 2sTUmacaronesica AB051639 U. glabrescens 'n KJ406265 u. lapponica KJ406266 U. lapponica ■ A B 0 5 1 ... i U wasmuthii • AB051674 U. wasmuthii ■ AB051675 (7. wasmuthii — KJ406270 U. subfloridana 1VmV^79 — AB051662 U. subfloridana AB051664 U. subfloridana KJ406268 U. florida 6 7 /0 .9 1 ^ — AB051663 U. subfloridana AB051638 U. fulvoreagens - AB051673 U. wasmuthii (J406281 U. glabrata 406279 U. flavocardia 86295 U. flavocardia 84 /T o1------— KJ406280 U. flavocardia 0.06 substitutions/site
Figure 7: Phylogeny of Usnea subgenus Usnea depicting the locality of MN specimens. ST=Sao Tome, P=Principe 64
AB051049 U. aciculifera AB051056 U. dasaea KJ406278 U. ceratina AB051052 U. ceratina TLC N/A AB051053 U. ceratina AB051649 U. merrillii MN0579 Usnea aff. flammea MN0264a U. sanguinea MN0191------MN0253------t MN0116------— MN0071 Usnea so. B — MN0552------MN0211------73/1.0 MN0571^lr^ -10.79 - MN0578b— 50/0.72 63AJ.99. - MN0080 ----- 70/0.78 fci MN0242------p-lMNQ216 — 04,J-t— MN0553^ 81/10- MN0243------W M f si0269 — — KJ406277 U. articulata 58/0. ' - MN0575b MN0586a^ 67/0.79, AB051657 U. pygmoidea AB051654 U.pangiana r MN0531-*------53/1.0 — MN04921 f - MN0485' 1 nn/1 n “ MN0407- 64/1.0 M N0452- ^ ■ r- MN0405' 74/1 0t- B T MN0537’ '4/I U ^-MN0538 MN0525-— ’ — MN0523 r MN0580' MN0603 MN0595 83/0.97a - MN0599- k - MN0066- *- MN054L 53/0 - MN0526- 52/0 84/ MN0521b- 53/0.92 - MN0543' MN0532 80/0.90 ' - M N0520- 51/0.94 -M N 0 4 1 2 - MMN0544C N 0 5 1 5 ^ 65/0.99 M N 0 4 9 1 ^ - MN0533^ M N 0 4 9 6 ^ - MN0489X - MN0600b‘ QQ/1 n 6i/o 70j— KJ406273 U. comuta KJ406272 U. comuta ^ ------KJ406274 U. cornuta AB051659 U. rubicunda AB051661 U. rubrotincta • KJ406275 U. rubicunda ■ AB051651 U. mutabilis w w r;FJ494941 U. nidifica FJ494933 U. nidifica ^-A BAB051640 O f U. himalayana MN0560 U. kroaiana - 59/0os/u.so 981 ------ABQ5163g u giabrescensKJ406283 iU. macaronesica KJ406265 u. lapponica KJ406266 U. lapDonica - AB051676 U. wasmuthii AB051674 U. wasmuthii AB051675 U. wasmuthii 71/0.91 KJ406270 U. subfloridana — AB051662 U. subfloridana AB051664 U. subfloridana KJ406268 U. florida 67/0.91 — AB051663 U. subfloridana - AB051638 U. fulvoreagens — AB051673 U. wasmuthii • KJ406281 U. glabrata . ■ KJ406279 U. flavocardia * JN086295 U. flavocardia KJ406280 U. flavocardia 0.06 substitutions/site
Figure 8: Phylogeny of the subgenus Usnea with taxonomically significant organic compounds presence/absence groupings. Bar=barbatic acid, Con=constictic acid, Diff=diffractaic acid, Menn=menegazziaic acid, Nor=norstictic acid, ProC=protoce- traric acid, Pso=psoromic acid, SaNsalazinic acid, Sti=stictic acid. GO M 3 ft 21 V 8 « § * t f S « C> ^ , _PS c~~iL £ > «n ! i \ r > - > S <~s. ' ^ , t e a - ^ T JO » _ a * 4 § : n % : ^ “ a - ^ --.*r ,5r a- 5s ^ ► ‘-fsp r Hi.rv : ^r- cj> 3C. 'vSs' S£>. Pa Sm Int Sm Int Int Pa Pa PaSm Pa Sm Pa Pa Int Pa
Figure 9: Compilation of Thin Layer Chromatography plates from specimens representing Usnea species C. Rf values are not accurate due to cropping and samples run on different plates. Comparison shows consistent pattern of stictic acid, constictic acid and diffractaic acid. Morpho- types are designated for select samples: Solvent system B’ is on top, solvent system C is underneath. Sm=smooth morphotype, Pa=papillate morphotype, Int= intermediate morphotype. 66
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Appendix A: Table of significant chemical compounds in species groups as revealed by TLC.
USN=usnic acid, CON=constictic acid, STI=stictic acid, CNO=connorstictic acid, NOR=norstictic acid, SAL=salazinic acid, GAL=galbinic acid, PRC=protocetraric acid, FPRC=fumaroprotocetraric acid, MEN=menegazziaic acid, BAR=barbatic acid, DIF=diffractaic acid, ZE=zeorin, EA1=eumitrin A1, EA2=eumitrin A2, EB=eumitrin B, PSO=psoromic acid, 2PSO=2'-0-demethyl-psoromic acid. *TLC unavailable for Usnea aff. flammea