Diversification and species delimitation of lichenized fungi in selected groups of the family Parmeliaceae (Ascomycota)
Kristiina Mark
Tartu 7.10.2016 Publications
I Mark, K., Saag, L., Saag, A., Thell, A., & Randlane, T. (2012) Testing morphology-based delimitation of Vulpicida juniperinus and V. tubulosus (Parmeliaceae) using three molecular markers. The Lichenologist 44 (6): 752−772. II Saag, L., Mark, K., Saag, A., & Randlane, T. (2014) Species delimitation in the lichenized fungal genus Vulpicida (Parmeliaceae, Ascomycota) using gene concatenation and coalescent-based species tree approaches. American Journal of Botany 101 (12): 2169−2182. III Mark, K., Saag, L., Leavitt, S. D., Will-Wolf, S., Nelsen, M. P., Tõrra, T., Saag, A., Randlane, T., & Lumbsch, H. T. (2016) Evaluation of traditionally circumscribed species in the lichen-forming genus Usnea (Parmeliaceae, Ascomycota) using six-locus dataset. Organisms Diversity & Evolution 16 (3): 497–524. IV Mark, K., Randlane, T., Hur, J.-S., Thor, G., Obermayer, W. & Saag, A. Lichen chemistry is concordant with multilocus gene genealogy and reflects the species diversification in the genus Cetrelia (Parmeliaceae, Ascomycota). Manuscript submitted to The Lichenologist. V Mark, K., Cornejo, C., Keller, C., Flück, D., & Scheidegger, C. (2016) Barcoding lichen- forming fungi using 454 pyrosequencing is challenged by artifactual and biological sequence variation. Genome 59 (9): 685–704. Systematics • Provides units for biodiversity measurements and investigates evolutionary relationships • Inadequate and unstable taxonomy affects a variety of fields and can have financial ramifications
Lichen taxonomy • Lichens classified based on the fungal partner of the symbiotic organism • Identification traditionally based on thallus morphology • Incongruence between morphological circumscriptions of species and DNA sequence data of mycobiont The main objectives of the thesis (1) Estimate phylogenetic relationships and delimit species in selected groups of the family Parmeliaceae
(2) Evaluate the utility of traditionally used characters in these lichens
(3) Assess the accuracy of DNA-based species identification in lichenized fungi using next-generation sequencing (NGS) Study groups
I Taxonomic study of genus Vulpicida with emphasis on European taxa
II Taxonomic study of genus Vulpicida in northern hemisphere
III Taxonomic study of genus Usnea sect. Usnea in Europe and North America
IV Taxonomic study of genus Cetrelia in Asia
V NGS barcoding of 100 species from the order Lecanorales in Switzerland Study material
I n=78 II n=70 III n=144 IV n=64 V n=100 Parmotrema (350)
Crespoa (4) Study groups Flavoparmelia (32) 9 Austroparmelina (13) 18 Punctelia (45) Nesolechia (1) Canoparmelia sp Flavopunctelia (5) Canoparmelia s.str. (35) Xanthoparmelia (820) 8 Cetrelia (18) Parmelioid 5 Melanohalea (22)
Melanelixia (15) 18 species Emodomelanelia masonii Montanelia (5) Remototrachyna 6 (19) Bulbothrix (20) 4 Bulbothrix (40) Parmelinella (7) Parmelina (10) Myelochroa (30)
Hypotrachyna (260) Parmeliopsis (3) Nipponoparmelia (4)
Parmelia (45)
Notoparmelia (16) Relicinopsis (5) Relicina (54) Platismatia (11) Imshaugia aleurites Oropogon (40) 12 Sulcaria (5) Psiloparmelia (13) 14 Everniopsis trulla Psiloparmelioid Hypogymnia (90) Hypogymnioid Pseudevernia (4) 16 Brodoa (3) Arctoparmelia (5) Letharia+Lethariella (17) Tuckermanella (6) Kaernefeltia (2) Ahtiana (3) Arctocetraria (9) Melanelia culbersonii Tuckermannopsis (9) Flavocetraria nivalis Nephromopsis (21) Cetreliopsis (7) Flavocetraria cucullata Masonhalea (2) Cetrarioid Vulpicida (6) Allocetraria (9) 3 Cetraria (15) Usnocetraria oakesiana Cetraria obtusata 15 Cetrariella (4) Dactylina (2) 6 species Melanelia (6) Esslingeriana idahoensis Evernia (10) Omphalodium pisacomense Parmeliaceae Bryoria (51) 11 Alectoria (9) Alectorioid Nodobryoria (3) Pseudephebe (2) Bryocaulon (4) 2 Allantoparmelia (3) 17 Protousnea (8) Phacopsis (10) Anzioid Anzia (34) 10 Pannoparmelia (5) 1 7 Usnea (350) Usneoid Cornicularia normoerica 13 Menegazzia (70) Coelopogon (2) Protoparmelia s.str. (10) Gypsoplacaceae ca 350 species Protoparmelia Miriquidica Lecanora 0.06 substitutions per site Photos © University of Tartu Cladia Cladonia, Metus Cladoniaceae 6-locus phylogeny by Divakar et al. 2015 Study groups: Vulpicida
• 6 species worldwide, 3 species with overlapping distributions and habitat, and morphological intermediates
© University of Tartu
Are morphospecies genetically distinct? V. juniperinus? V. tubulosus? V. lesii? Study groups: Usnea sect. Usnea
• Young species complex • Transitional forms and complexity of diagnostic characters
Types of soralia
Photos © University of Tartu
Complicated taxonomy and problematic species delimitation Study groups: Cetrelia
• Morphologically uniform but with a characteristic set of lichen metabolites in each species
‘7 Olivetoric acid Imbricaric acid ‘5
OH OH O O O HO HO O O
O O
© University of Tartu CH3 HO OH ‘3 HO O
‘5
Cetrelia olivetorum Cetrelia monachorum
A continuing debate over chemical species concept in Cetrelia Sample prepara on Molecular methods Thin-layer DNA chromatography (TLC) extrac on
Polymerase chain reac on (PCR) Agarose gel prepara on
PCR product purifica on
Sequencing reac ons
Sequencing on ABI 3730xl DNA Analyzer Sample prepara on
Thin-layer DNA chromatography (TLC) extrac on
Polymerase chain reac on (PCR) Agarose gel prepara on
PCR product purifica on
Sequencing reac ons
Sequencing on ABI 3730xl DNA Analyzer Lichen sequencing
• saprophytic, endophytic, and parasitic fungi live intimately admixed with the lichen mycobiont
EPIPHYTES: free living algae, fungi, bacteria
Algal cells of lichen photobiont
Fungal hyphae of lichen mycobiont
ENDOPHYTES: lichen-associated fungi, dis nct bacterial communi es
Cross-sec on photograph by Bill Malcolm 1031bpNGS applicability to lichen barcoding 500bp • Different gene copies in a PCR mixture can produce low quality 10Sanger ng/µL sequences 4 ng/µL • NGS metabarcoding allows identification of a lichen sample even when contaminants dominate in the PCR mixture 1 ng/µL
How effectively can pyrosequencing be applied to lichen barcoding? Sample prepara on
Thin-layer DNA chromatography (TLC) extrac on
Polymerase chain reac on (PCR) Agarose gel prepara on
PCR product purifica on
Second PCR Sequencing with fusion primers reac ons
Purifica on, Cloning in vector Sequencing on quan fica on, and pla ng on ABI 3730xl DNA pooling into agar plates equimolar pools Analyzer
Sequencing on GS Roche Sequencer Colony-PCR
Sequencing on ABI 3730xl DNA Analyzer Molecular markers
I II III IV V nuRNA ITS ITS ITS ITS ITS cistron IGS IGS IGS Bt protein- MCM7 MCM7 MCM7 MCM7 coding RPB1 RPB1 RPB1 RPB2 mitochondrial mtSSU mtSSU Molecular phylogenetic analyses
Mul species coalescent model analyses
Single-locus DNA sequence Sequence Recombina on evolu onary and Species Species Species detec on alignment models concatenated delimita on tree valida on gene trees Barcoding data analyses
Consensus sequence approach
Cluster Consensus Tree-based iden fica on Target Quality Clustering sequence = iden fica on Chimera using NCBI taxon and filtering detec on at 95% nucleo de barcode alignment confirma on database assignment Key results 1
R1. Incongruences between morphological circumscription of species and DNA based results were found in each study group Ex1.1. Vulpicida Ex1.2. Usnea Ex1.1: Morphospecies Vulpicida juniperinus, V. tilesii and V. tubulosus do not represent genetically distinct species
typical V. juniperinus
typical V. tubulosus
Syn. V. juniperinus V. pinastri (= V. tubulosus) (= V. tilesii) Photos © University of Tartu
Vulpicida 3-locus concatenated tree Usnea cavernosa
Ex1.2: Only 4 of 17 sect. Usnea morphologically circumscribed species from Usnea sect. Usnea were Usnea silesiaca recovered monophyletic in genetic analyses
Usnea wasmuthii
Usnea praetervisa Photos © University of Tartu
0.0030 Usnea 6-locus concatenated tree Ex1.2: Only 4 of 17 sect. Usnea morphologically circumscribed species from Usnea sect. Usnea were recovered monophyletic in genetic analyses
4 clusters of two or more species: • Usnea fulvoreagens – U. glabrescens – U. pacificana • Usnea florida – U. subfloridana • Usnea barbata – U. chaetophora – U. dasopoga – U. diplotypus • Usnea barbata – U. intermedia – U. lapponica – U. substerilis
0.0030 Usnea 6-locus concatenated tree Key results 1
R1. Incongruences between morphological circumscription of species and DNA based results were found in each study group Ex1.1. Vulpicida Ex1.2. Usnea ! The level of incongruence is individual for each group Ex1.3. Cetrelia Ex1.3: Chemical species concept in Cetrelia is justified – chemotypes form monophyletic clades with sub-clades usually correlating to morphotypes
Densitree of 20,000 B/MCMC gene trees Photos © University of Tartu sanguinea Outgroup monachorum alaskana pseudolivetorum olivetorum cetrarioides japo chic braunsian +delavayana +or nica itae ien tali a s m
IMBRICARIC OLIVETORIC R=H: ANZIAIC MICROPHYLLINIC R=H: α-ALECTORONIC R=CH3: PERLATOLIC R=CH3: α-COLLATOLIC
Cetrelia 4-locus concatenated densitree Key results 2
R2. Lichen reproductive mode and thallus gross morphology, traditionally with high diagnostic importance, often do not correlate with genetics Ex2.1. Vulpicida Ex2.2. Usnea Ex2.3. Cetrelia Ex2.1: Morphospecies distinguished mainly by the shape of lobes and development of apothecia in the Vulpicida juniperinus complex are not separated genetically
Vulpicida juniperinus with broad, dorsiventral lobes Vulpicida tubulosus with terete lobes Photos © University of Tartu
Syn. V. juniperinus (= V. tubulosus), (= V. tilesii) Ex2.2: Reproductive mode and thallus sect. Usnea gross morphology do not prove useful for separating some genetic lineages in sect. Usnea
subfloridana subfloridana Sexual species Usnea florida subfloridana florida florida subfloridana subfloridana subfloridana subfloridana subfloridana subfloridana subfloridana subfloridana subfloridana subfloridana florida subfloridana subfloridana subfloridana subfloridana Sorediate species U. subfloridana subfloridana subfloridana subfloridana subfloridana subfloridana subfloridana subfloridana subfloridana florida subfloridana subfloridana subfloridana subfloridana subfloridana
Photos © University of Tartu subfloridana 0.0030 subfloridana Usnea 6-locus concatenated tree Xanthoparmelia_loxodes_XAL_01_Estonia 1 Xanthoparmelia_chlorochroa_Leavitt_55437_USA_North_Dakota 100 1 1 Xanthoparmelia_conspersa_XAC_01_Estonia 100 100 Xanthoparmelia_conspersa_MAF_6793_Spain 1 Arctoparmelia_centrifuga_MAF_6879_Sweden
100 Arctoparmelia_incurva_XAM_01_Estonia Ex2.3: monachorum_CKM_17_RU_Primorye 1 1 monachorum_MAF_15507_China
100 0.92 100 monachorum_CKM_31_RU_Tverskaya monachorum_CKM_09_RU_Kavkaz 0.46 51 1 Reproductive mode in monachorum_CKM_52_RU_Tverskaya - 67 monachorum_24434_Japan
0.99 alaskana_63769_USA_Alaska alaskana_CKM_75_Bhutan 1 100 Cetrelia species might alaskana_CKM_68_China 100 0.98 1 alaskana_CKM_66_China 94 100 alaskana_CKM_72_China alaskana_CKM_74_Bhutan not always be sufficient _CKM_70_China alaskana
1 pseudolivetorum_Hur_030784_South_Korea pseudolivetorum_Hur_060718_South_Korea 1 pseudolivetorum_F007677_South_Korea in delimiting Isidiate species 100 pseudolivetorum_F005458_South_Korea 0.53 1 pseudolivetorum_Hur_050176_South_Korea polyphyletic - 98 pseudolivetorum_Hur_061074_South_Korea genetically isolated pseudolivetorum_MAF_15506_China 1 olivetorum_28230_Japan 100 1 olivetorum_25016_Japan
100 olivetorum_CKM_04_RU_Mordovia fungal lineages olivetorum_CKM_59_Estonia olivetorum_CKM_33_RU_Tverskaya olivetorum_CKM_01_RU_Kavkaz 1 sanguinea_CKM_82_China 100 cetrarioides_MAF_15552_Spain 0.82 1 delavayana_CKM_78_China 48 93 1 Sorediate and 1 100 cetrarioides_CKM_63_RU_Chelyabinsk 99 cetrarioides_CKM_51_RU_Tverskaya 1 apotheciate species cetrarioides_CKM_64_RU_Chelyabinsk 100 0.98 cetrarioides_CKM_13_RU_Primorye 96 form monophyle c cetrarioides_CKM_49_RU_Tverskaya cetrarioides_CKM_41_RU_Tverskaya 1 japonica_F016073_South_Korea 1 95 clade japonica_F016072_South_Korea 1 japonica_F016058_South_Korea
100 japonica_Hur_060828_South_Korea japonica_Hur_040790_South_Korea japonica_F016178_South_Korea japonica_F016207_South_Korea
0.4 1 chicitae_25865_Japan - chicitae_CKM_07_RU_Kavkaz 1 94 Sorediate species orientalis_AT406_RU_Primorye 95 0.48 chicitae_F004688_South_Korea 54 polyphyle c, includes 0.99 chicitae_26063_Japan 1 chicitae_25922_Japan lobulate species 98 0.98 chicitae_CKM_18_RU_Primorye 100 chicitae_CKM_15_RU_Primorye 1 braunsiana_28444_Japan 1 1 braunsiana_CKM_21_RU_Primorye 97 97 1 braunsiana_25985_Japan 0.87 braunsiana_28170_Japan 76 braunsiana_AT626_RU_Primorye braunsiana_F005503_South_Korea braunsiana_F000159_South_Korea Cetreliabraunsiana 4-locus_25877_Japan concatenated tree 0.0040 Key results 3
R3.1. Gene tree do not necessarily represent species phylogeny
Ex3.1. Vulpicida Ex3.1: Vulpicida ITS gene tree is in conflict with species tree
Vulpicida ITS gene tree
2 cryptic species in ITS data, but not supported by any other studied locus Key results 3
R3.1. Gene tree do not necessarily represent species phylogeny
Ex3.1. Vulpicida
R3.2. With conflicting information from different genes, multispecies coalescent model is suggested Ex3.2. Vulpicida Ex3.2: True species phylogeny recovered with less data in species tree analyses
• Concatenating 3 conflicting V. juniperinus (= V. tubulosus) genes resulted in erroneous (= V. lesii) results
• Using the same 3-locus data V. pinastri in multispecies coalescent V. viridis model suggested the V. canadensis phylogeny agreeing to the results of 5-locus data
Vulpicida 5-locus species tree Key results 4
R4.1. Pyrosequencing can be effectively applied to lichen identification and lichenized fungal reference database construction Ex4.1. 454 barcoding Ex4.1: Pyrosequencing in lichen identification and lichenized fungal reference database construction
• The full ITS marker of the lichenized fungus was recovered for 99 samples from 100
different genus and species no target • 69 samples identified correctly to the species ‘uncultured fungus’ level, of which 60 with 4 1 similarity >97%, in 8
GenBank BLAST correct genus 18
• DNA-based misidentifications due to: 69 incomplete reference database polyphyletic species low genetic variation in ITS correct species Key results 4
R4.1. Pyrosequencing can be effectively applied to lichen identification and lichenized fungal reference database construction Ex4.1. 454 barcoding
R4.2. Considering artifactual and biological sequence variation in NGS data, additional data processing steps are needed Ex4.2. 454 barcoding Ex4.2: Artifactual and biological sequence variation in NGS data
• Multiple types of sequencing errors (e.g. homopolymer errors) • Intragenomic and/or intra-mycelial ITS variation (detected in 13 specimens from 99) • Presence of several lichen-forming fungi when sequencing environmental samples In conclusion... Species is a fundamental unit of biodiversity, its definition determines how well we can read the book of biodiversity
Tabular View of Characteris c Bri sh Fossils by Darwin (1853), from h p://www.zmescience.com/science/biology/darwins-origin-species-12112015/