Diversification and species delimitation of lichenized fungi in selected groups of the family ()

Kristiina Mark

Tartu  7.10.2016 Publications

I Mark, K., Saag, L., Saag, A., Thell, A., & Randlane, T. (2012) Testing morphology-based delimitation of 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 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 -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 (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 affects a variety of fields and can have financial ramifications

Lichen taxonomy • 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 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 (45) Nesolechia (1) sp Flavopunctelia (5) Canoparmelia s.str. (35) (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) (10) Myelochroa (30)

Hypotrachyna (260) Parmeliopsis (3) Nipponoparmelia (4)

Parmelia (45)

Notoparmelia (16) Relicinopsis (5) Relicina (54) (11) aleurites Oropogon (40) 12 Sulcaria (5) Psiloparmelia (13) 14 trulla Psiloparmelioid (90) Hypogymnioid Pseudevernia (4) 16 Brodoa (3) Arctoparmelia (5) +Lethariella (17) Tuckermanella (6) (2) Ahtiana (3) Arctocetraria (9) Melanelia culbersonii Tuckermannopsis (9) Flavocetraria nivalis (21) Cetreliopsis (7) Flavocetraria cucullata (2) Cetrarioid Vulpicida (6) (9) 3 (15) Usnocetraria oakesiana Cetraria obtusata 15 Cetrariella (4) Dactylina (2) 6 species Melanelia (6) Esslingeriana idahoensis (10) Omphalodium pisacomense Parmeliaceae Bryoria (51) 11 Alectoria (9) Alectorioid Nodobryoria (3) Pseudephebe (2) Bryocaulon (4) 2 Allantoparmelia (3) 17 Protousnea (8) (10) Anzioid Anzia (34) 10 Pannoparmelia (5) 1 7 Usnea (350) Usneoid 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 preparaon Molecular methods Thin-layer DNA chromatography (TLC) extracon

Polymerase chain reacon (PCR) Agarose gel preparaon

PCR product purificaon

Sequencing reacons

Sequencing on ABI 3730xl DNA Analyzer Sample preparaon

Thin-layer DNA chromatography (TLC) extracon

Polymerase chain reacon (PCR) Agarose gel preparaon

PCR product purificaon

Sequencing reacons

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, disnct bacterial communies

Cross-secon 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 preparaon

Thin-layer DNA chromatography (TLC) extracon

Polymerase chain reacon (PCR) Agarose gel preparaon

PCR product purificaon

Second PCR Sequencing with fusion primers reacons

Purificaon, Cloning in vector Sequencing on quanficaon, and plang 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

Mulspecies coalescent model analyses

Single-locus DNA sequence Sequence Recombinaon evoluonary and Species Species Species detecon alignment models concatenated delimitaon tree validaon gene trees Barcoding data analyses

Consensus sequence approach

Cluster Consensus Tree-based idenficaon Target Quality Clustering sequence = idenficaon Chimera using NCBI taxon and filtering detecon at 95% nucleode barcode alignment confirmaon 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 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 monophylec cetrarioides_CKM_49_RU_Tverskaya cetrarioides_CKM_41_RU_Tverskaya 1 japonica_F016073_South_Korea 1 95 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 polyphylec, 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 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 Characterisc Brish Fossils by Darwin (1853), from hp://www.zmescience.com/science/biology/darwins-origin-species-12112015/