The tricky lichen genus Vulpicida: phylogeny and species delimitation Kristiina Mark, Lauri Saag, Andres Saag, Tiina Randlane Institute of Ecology and Earth Sciences, University of Tartu, Tartu, Estonia, [email protected] V. juniperinus INTRODUCTION: ITS gene tree • The genus Vulpicida (Parmeliaceae, Ascomycota) belongs to the Branch support posterior probabilities (PP) form BEAST & MrBayes morphological group of “cetrarioid lichens” 1 V. tilesii/juniperinus (Russia; TIL 05) sp. 1 1 V. tilesii/juniperinus (Russia; VSP 16) V. juniperinus (Estonia; JUN 02a) V. juniperinus/ • Characteristic bright yellow colour of medulla is caused by V. juniperinus (Estonia; JUN 04) 0.98 V. juniperinus (Estonia; JUN 07) unique set of secondary metabolites V. juniperinus (Norway; JUN 12B) tubulosus/ 0.7 V. tubulosus (Estonia; JUN 14) V. juniperinus/tubulosus (Austria; TUB 28) CRYPTIC • Distributed in the temperate and arctic regions of northern V. tubulosus (Austria; TUB 51) V. juniperinus (Estonia; VSP 12) V. tilesii 0.99 SPECIES 1 • Gene tree heterogenity could be the result of hemisphere V. tilesii (Russia; TIL 13) 0.99 V. tilesii (USA; TIL 15) 1 V. tilesii (USA; TIL 08) includes incomplete lineage sorting that is most characteristic 0.93 V. tilesii (USA; TIL 03B) • Consists of six species: Vulpicida canadensis, V. juniperinus, 1 V. tilesii (Canada; TIL 18) 1 1 V. juniperinus/tilesii (Austria; TUB 27) V. tilesii to young diverging species complexes V. pinastri, V. tubulosus, V. tilesii and V. viridis 1 1 V. juniperinus (Austria; TUB 37) 1 V. juniperinus/tilesii (Austria; TUB 52) 0.99 1 V. juniperinus (Russia; JUN 18) • Morphological distinction between V. juniperinus, V. tilesii and 1 V. juniperinus (Japan; JUN 22) 1 V. juniperinus (Japan; JUN 20) 1 0.85 V. juniperinus (Japan; JUN 21) V. tubulosus is difficult due to frequent intermediate forms V. pinastri (Estonia; PIN 07) 1 V. pinastri (Austria; PIN 09) sp. 2 0.99 V. pinastri (Russia; PIN 13) • AIM OF THE STUDY — to assess the distinctivness of current Vulpicida V. pinastri (USA; PIN 14) 0.8 V. pinastri (USA; PIN 17) V. tubulosus species & investigate the monophyly of the genus using DNA data 1 V. pinastri (USA; PIN 33) V. pinastri (USA; PIN 34) V. pinastri 1 V. pinastri (Japan; PIN 03) 1 V. pinastri (Japan; PIN 04) 0.99 V. pinastri (Japan; PIN 05) V. pinastri (Japan; PIN 06) 1 V. tubulosus (Estonia; TUB 05) V. tubulosus (Sweden; TUB 10) sp. 3 1 V. tubulosus (Sweden; TUB 12) 1 V. tubulosus (Austria; TUB 33) MATERIAL & METHODS: 1 V. juniperinus/tilesii (Austria; TUB 38) V. juniperinus/ V. juniperinus (Estonia; JUN 13) clade B V. juniperinus (Sweden; JUN 16) tubulosus • 71 specimens for molecular study; additional 35 1 V. tubulosus (Sweden; TUB 08b) V. tubulosus (Sweden; TUB 11) 1 V. tubulosus/juniperinus (Sweden; TUB 14) CRYPTIC V. juniperinus & V. tubulosus specimens for 0.91 V. tubulosus (Estonia; TUB 20B) 0.94 V. juniperinus (Austria; TUB 31) SPECIES 2 V. pinastri morphological study V. juniperinus/tubulosus/tilesii (Austria; TUB 41) V. tubulosus (Estonia; VSP 01) V. juniperinus (Estonia; VSP 06) • 5 loci: ITS, IGS, Mcm7, RPB1, mtSSU 0.96 V. canadensis (USA; CAN 01a) 1 sp. 4 V. canadensis (USA; CAN 01b) 0.94 V. canadensis (USA; CAN 15) • 353 new sequences 1 V. canadensis (USA; CAN 17) V. canadensis V. canadensis (USA; CAN 18) • Cloning of 12 specimens for checking paralogous copies 1 V. viridis (USA; VIR 05) 1 sp. 5 1 V. viridis (USA; VIR 10) V. viridis (USA; VIR 02) V. viridis in ITS 1 V. viridis (USA; VIR 03) V. viridis (USA; VIR 06) 1 1 Allocetraria stracheyi (China; AST 01) • Recombination detection in RDP v.3. and v.4. & GARD 1 Allocetraria stracheyi (China; AST 02) 1 Allocetraria flavonigrescens (China; AFL 01) 1 0.99 Cetraria islandica (Sweden; ISL 01) • Species delimitation in Brownie & BPP 1 Cetraria islandica (Estonia; ISL 04) 1 Cetraria muricata (Estonia; CMU 03) V. canadensis • Gene trees (one locus and concatenated) in BEAST & 1 Cetrariella delisei (Canada; CDE 02) 1 Cetrariella fastigiata (Russia; CFA 02) Cetrariella commixta (Sweden; CCO 01) MrBayes Cetraria kamtczatica (Russia; CKA 01) Cetraria sepincola (Estonia; CSE 01) Usnocetraria oakesiana (Norway; OAK 03) • Species trees in *BEAST, STEM & BEST Flavocetraria cucullata (Estonia; FCU 01) 0.005 Photos by Andres Saag Andres Photos by *BEAST species tree RESULTS: V. viridis Branch annotations: *BEAST, STEM, BEST • Strong conflicts between individual gene trees • On the ITS gene tree, two cryptic species can be detected, but Supported by these are not supported by morphology, other gene trees or species 1 method 94/+/91 V. juniperinus / tubulosus A1 tree methods 2 methods 89/+/93 V. juniperinus / tubulosus B • No paralogous copies of ITS or significant recombination events 98/+/100 V. tilesii were found 3 methods 98/+/98 V. juniperinus / tubulosus A2 • Sensitive BPP detected several evolutionarily independent lineages 100/+/100 V. juniperinus Japan while Brownie was more conservative 100/+/100 V. pinastri 1 • According to species tree and delimitation methods: 95/-/84 V. pinastri Japan - V. canadensis and V. viridis are clearly distinct species 64/-/75 V. canadensis - V. juniperinus, V. tilesii, V. tubulosus and V. pinastri form a V. viridis closely related group of taxa 66/+/63 69/-/- Cetraria kamtzatica 91/+/98 Cetraria sepincola 100/+/100 Cetraria muricata Cetraria islandica 100/+/100 88/-/75 Cetrariella fastigiata 92/+/99 Cetrariella delisei DensiTree 94/-/94 Usnocetraria oakesiana Posterior density of species trees Cetrariella commixta 100/+/100 Allocetraria flavonigrescens from *BEAST Allocetraria stracheyi 0.01 s/s CONCLUSIONS: • Tricky Vulpicida exemplifies how analysis methods can affect the conclusions • Strong phylogenetic signal from ITS tends to dominate over other loci in the concatenated phylogenies • Coalescent based species tree inference needs less markers to produce more consistent outcome • Although the monophyly of the genus is not supported by all species tree methods, there is also no conclusive evidence against it • We propose four instead of current six species in Vulpicida, reducing V. tubulosus and V. tilesii to synonymy under V. juniperinus AKNOWLEDGEMENTS: • We thank the collectors of the specimens, especially Emma Sandler Berlin and Ede Leppik, and the curators of ASU, CANL, GZU, LD, MAF, MIN, NY, OSC, UPS. We are grateful to Arne Thell, Steven Leavitt and Thorsten Lumbsch for discussions. • Most phylogenetic analyses were carried out in the High Performance Computing Center of University of Tartu. = = • The study was financially supported by Estonian Science Foundation (grants JD173 and ETF9109), European Union through the European Regional Development Fund (Center of Excellence FIBIR), Estonian Ministry of Education and Research, and V. juniperinus V. tilesii V. tubulosus Archimedes Foundation..