Evolution and biogeography of the sweet vernal grasses (Anthoxanthum s.l.) with special emphasis on the South American taxa Lema-Suárez, I.*; Pimentel, M. & Sahuquillo, E. Evolutionary Biology Group (GIBE), Universidade da Coruña. A Coruña, Spain. *irene.lema.suarez@udc.es INTRODUCTION A The subcosmopolitan genus Anthoxanthum s.l. (Poaceae; Pooideae; Aveneae) presents a high morphological and karyological diversity and its distribution reflects common patterns in C3 grasses (Fig. 1). It grows in temperate areas of both hemispheres and in tropical high mountains (sky islands), where it belongs to relict communities prone to be affected by climate change. Three main groups can be distinguished in genus Anthoxanthum s.l. (Fig. 2): genera Anthoxanthum L. and Hierochloë R. Br. s.str. and Anthoxanthum section Ataxia, a putative hybrid between the genera (Fig. 3). Reticulate evolution is common in Anthoxanthum, which explains its difficult taxonomy. Given its distribution, relatively low taxonomic diversity and C3 character, Anthoxanthum s.l. is a good model taxon for other highly speciose C3 Pooideae lineages such as Festuca, Bromus and Poa, with huge ecological and economical importance. 28 Mya 23 11 Cenozoic 5.33 Miocene Oligocene // // // // Fig. 2. Floral structure of Anthoxanthum s.l. A: Anthoxanthum s.str. (A. odoratum). Spikelet with barren lower florets. B: Hierochloë (H. odorata). Spikelet with male lower florets. C: Anthoxanthum section Ataxia (A. hookeri). Spikelet with paleate lower florets. Fig. 1. Global distribution of Anthoxanthum s.l. Green: Anthoxanthum s.str. Blue: Hierochloë. Red: Anthoxanthum section Ataxia. 1.8 Glyceria Melica Secale Dasypyrum Helictotrichon A. angustum_L2 A. hookeri_L2 A. horsfieldii_L2 A. dregeanum_L2 A. tongo_L2 A. ecklonii_L2 A. madagascariensis_L2 H. redolens_L2 H. odorata H. hirta A. gracile A. alpinum A. odoratum CEurope A. ovatum // // Ataxia Nuclear data MATERIALS AND METHODS The starting point of this project is the dated phylogeny of genus Anthoxanthum s.str. published by our research group in 2013 (Pimentel et al., 2013, Annals of Botany, doi: 10.1093/aob/mct170; Fig. 3). We added 274 new mostly Hierochloë sequences to our previous data set, which now comprises 38 of the approx. 54 species of Anthoxanthum s.l. A. amarum A. odoratum W Iberia A. aristatum // Macaronesian taxa A. nivale A. dregeanum_L1 A. ecklonii_L1 A. tongo_L1 A. madagascariensis_L1 A. horsfieldii_L1 A. angustum_L1 H. redolens_L1 // // // C Plio Pt // // B // // // // Ataxia Plastid data Fig. 3. Dated phylogeny of genus Anthoxanthum s.str. Taken from Pimentel et al. (2013) doi: 10.1093/aob/mct170. Section Ataxia plastid and nuclear sequences were labelled in order to reflect the different position occupied by these taxa in both phylogenies Lolium Festuca Poa Puccinellia // Brachypodium Two plastid (trnLF and trnTL) and two nuclear (ETS and ITS) DNA regions were used for phylogenetic analyses: (i) Bayesian phylogenies were built using the software MrBayes 3.2 (Huelsenbeck & Ronquist, 2001) and (ii) we constructed rooted phylogenetic networks to clarify unresolved parts of the phylogeny, with special emphasis in the South American Hierochloë species. Networks were built using the software SplitsTree (Huson & Bryant, 2006). AIMS 1. Contribute to the understanding of the evolutionary relationships between genera Anthoxanthum s.str. and Hierochloë. 2. Unravel the biogeographical patterns in genus Anthoxanthum s.l. as representative of other C3 grasses. 3. Clarify the relationships among the South American Hierochloë species. RESULTS AND DISCUSSION Our results offer a first approximation to the phylogeny of Anthoxanthum s.l. and reveal a complex evolutionary and biogeographic history for this taxon. Hybridisation, introgression and long distance dispersal seem to be common in this genus, many of whose species are not monophyletic (Figs. 4, 5). Our phylogenies largely support previous findings on the relationships among the main groups in Anthoxanthum s.l. (genera Anthoxanthum s.str., including sections Anthoxanthum and Ataxia and genus Hierochloë s.str.). Other relevant aspects observed in the phylogeny are explained on the phylogenetic trees (Numbers in paragraphs correspond with numbers in Figs. 4-7). // Melica // Dasypyrum Festuca // H. altissima 8 Chile H. juncifolia 3 Argentina H. juncifolia 1 Chile H. juncifolia 2 Chile H. quebrada 1 Peru H. redolens 11 Peru H. spicata 1 Chile Hierochloe sp. Argentina H. redolens 8 Argentina H. redolens 12 Argentina H. altissima 1 Chile H. altissima 2 Chile H. altissima 3 Chile H. altissima 4 Chile H. altissima 5 Chile H. altissima 6 Chile H. altissima 7 Chile H. gunckelii 1 Chile H. gunckelii 1 Chile H. redolens 1 Chile H. redolens 2 Chile H. redolens 3 Chile H. redolens 4 Chile H. redolens 5 Chile H. redolens 6 Chile H. utriculata 1 Chile H. utriculata 2 Chile H. utriculata 3 Chile H. utriculata 4 Chile H. utriculata 5 Chile H. redolens 9 Argentina H. redolens 10 Argentina H. redolens 7 Chile H. pusilla 1 Chile H. pusilla 2 Chile H. occidentalis 7 USA H. occidentalis 5 USA H. occidentalis 6 USA H. occidentalis 8 USA H. occidentalis 1 USA H. occidentalis 2 USA H. occidentalis 3 USA H. occidentalis 4 USA H. laxa 3 India H. flexuosa 3 India H. laxa 2 India H. laxa 1 India H. laxa 5 India H. odorata 6 USA H. odorata 7 USA H. odorata 2 Norway H. hirta 6 Norway H. hirta 3 Norway H. hirta 4 Norway H. hirta 5 Norway H. hirta 1 Norway H. hirta 2 Norway H. odorata 4 Norway H. odorata 1 Norway H. odorata 3 Norway H. odorata 5 Canada // H. pauciflora 1 Canada H. flexuosa 2 Nepal 1 1. The three main groups are clearly defined in both phylogenies but their composition varies depending on the analysis. Outgroup 7 North America H. alpina 10 USA H. australis 2 Romania H. australis 1 Finland A. gracile 1 Malta A. gracile 2 Malta A. gracile 7 Greece A. gracile 3 Italy A. gracile 9 Greece A. gracile 8 Greece A. gracile 5 Italy A. gracile 6 Italy A. gracile 10 Greece A. gracile 4 Italy A. dregreanum 2 South Africa A. dregreanum 3 South Africa A. dregreanum 4 South Africa A. eckloni 1 South Africa A. eckloni 3 South Africa A. eckloni 4 South Africa A. eckloni 5 South Africa A. hookeri 1 Nepal A. hookeri 2 Myanmar A. hookeri 5 China Ahors_Taiwan A. hookeri 4 China A. japonicum 1 Japan A. tongo 3 South Africa H. mexicana 2 Venezuela H. mexicana 3 Mexico H. mexicana 4 Guatemala A. tongo 1 South Africa A. tongo 2 South Africa H. mexicana 5 Venezuela H. horsfieldii Papua New Guinea H146 A. japonicum 2 Japan A. angustum 1 Papua New Guinea A. hookeri 6 China A. nivale 3 Uganda ODP_28802 ODP_28803 ODP_29159 ODP_36428 ODP_27894 ODP_28223 ODP_28804 ODP_37272 ODP_37273 ODP_37453 ODP_38122 H. mexicana 1 Venezuela ODP_38611 ODP_38613 ODP_38644 A. dregreanum 1 South Africa A. dregreanum 2 South Africa A. dregreanum 3 South Africa A. eckloni 2 South Africa A. tongo 4 South Africa A. madagascariense 1 Madagascar A. madagascariense 2 Madagascar A. madagascariense 3 Madagascar A. alpinum 3 France A. alpinum 2 Sweden A. odoratum 39 Ireland A. odoratum 38 Ireland A. odoratum 37 Ireland A. odoratum 23 Spain A. odoratum 41 Sweden A. odoratum 26 France A. odoratum 40 Ireland A. odoratum 22 Spain A. odoratum 33 Czech Rep. A. odoratum 42 Spain A. odoratum 44 Costa Rica A. odoratum 1 USA A. odoratum 3 USA A. odoratum 9 Russia A. alpinum 4 France A. odoratum 13 Portugal A. odoratum 14 Portugal A. odoratum 35 Ireland A. odoratum 17 Spain A. odoratum 18 Spain A. odoratum 36 Ireland A. odoratum 19 Spain A. odoratum 43 Spain A. alpinum 5 Czech Rep. A. alpinum 6 Czech Rep. A. odoratum 35 Czech Rep. A. odoratum 5 USA A. odoratum 2 USA Europe Mediterranean Basin Anthoxanthum gracile 5 Central America 4 Ataxia 3 Perennial Anthoxanthum (2x, 4x) Eurasia A. amarum 4 Portugal A. amarum 5 Spain A. amarum 7 Spain A. amarum 2 Spain A. amarum 6 Spain A. aristatum 5 Spain A. aristatum 10 Spain A. aristatum 1 Spain A. aristatum 2 Spain A. aristatum 3 Spain A. ovatum 4 Spain A. odoratum 8 Chile A. odoratum 16 Spain A. odoratum 6 Argentina A. odoratum 20 Spain A. aristatum 6 Spain A. alpinum 1 Sweden Perennial Anthoxanthum (4x) A. amarum 3 Portugal A. amarum 1 Spain A. aristatum 4 Spain A. ovatum 5 Spain A. ovatum 6 Spain A. odoratum 4 USA ODP_43087 Glyceria A. nivale WRZ A. aethiopicum A. nivale 1 Uganda A. nivale 2 Uganda ODP_35547 A. aethiopicum 5 Ethiopia A. aethiopicum 4 Kenya A. aethiopicum 3 Ethiopia E Africa Figure 4. Maximum clade credibility tree from the Bayesian analysis of plastid DNA sequences (trnTL and trnLF) constructed with Mr Bayes. Discontinuous lines represent branches with less than 80 posterior probability support (PP). //, shortened branches. Numbers follow the paragraphs in the Results and Discussion section. H. mexicana Guatemala 5 H. mexicana Venezuela 91.2 H. mexicana Venezuela 99.8 H. glabra China H. glabra China H. alpina Greenland H. occidentalis USA H. redolens New Guinea H. laxa India 99.5 86.4 H. rariflora Australia 90.6 H. redolens 14 Papua New Guinea H. flexuosa 1 Nepal H. laxa 3 India H. alpina 5 Sweden H. glabra 1 Russia H. odorata 9 Russia H. odorata 8 Russia H. glabra 3 China H. glabra 4 China H. wendelboi 1 Pakistan H. alpina 9 Russia H. alpina 7 Sweden H. laxa 6 India H. laxa 5 India H. laxa 4 India H. flexuosa 3 India H. laxa 2 India H. occidentalis 9 USA H. occidentalis 5 USA H. occidentalis 7 USA H. rariflora 5 Australia H. rariflora 3 Australia H. rariflora 1 Australia H. rariflora 4 Australia H. rariflora 2 Australia Australia SE Asia North America SE Asia Europe H. australis 2 Romania H. australis 5 Czech Rep. Hierochloë H. odorata 6 USA H. odorata 5 Canada H. glabra 2 China H. juncifolia 2 Chile H. altissima 8 Chile H. juncifolia 3 Chile H. juncifolia 1 Chile H. utriculata 5 Chile H. utriculata 1 Chile H. glunckelii 2 Chile H. utriculata 3 Chile H. redolens 6 Chile H. redolens 4 Chile H. altissima 7 Chile H. altissima 1 Chile H. altissima 2 Chile H. altissima 3 Chile H. glunckelii 1 Chile H. altissima 6 Chile H. utriculata 2 Chile H. redolens 8 Argentina H. redolens 10 Argentina H. utriculata 4 Chile H. redolens 2 Chile H. altissima 4 Chile H. altissima 5 Chile H. redolens 1 Chile H. redolens 5 Chile H. redolens 3 Chile H. redolens 7 Chile H. redolens 11 Peru H. spicata 1 Chile Hierochloe sp. Argentina H. redolens 9 Argentina H. redolens 12 Argentina H. occidentalis 6 USA H. occidentalis 1 USA H. occidentalis 2 USA H. occidentalis 3 USA 3. Sections Anthoxanthum (excluding the Mediterranean relict A. gracile) and Ataxia are sisters in all analyses. The position of the East African A. nivale specimens, particularly those from the Eastern Rift Zone (ERZ) represents a likely reticulation. South America 7 North America A. dregeanum 1 South Africa A. tongo 5 South Africa A. dregeanum 2 South Africa A. dregeanum 3 South Africa A. tongo 3 South Africa A. tongo 4 South Africa A. eckloni 1 South Africa A. eckloni 7 South Africa A. eckloni 8 South Africa A. eckloni 2 South Africa A. eckloni 9 South Africa A. angustum 1 Papua New Guinea A. horsfieldii 1 Papua New Guinea A. japonicum 1 Japan A. japonicum 2 Japan A. hookeri 8 Nepal A. hookeri 5 China A. hookeri 6 China A. hookeri 4 China A. hookeri 1 Nepal A. hookeri 2 Myanmar A. horsfieldii 2 Taiwan A. ecklonii 10 South Africa A. ecklonii 3 South Africa A. ecklonii 11 South Africa A. ecklonii 6 South Africa A. madagascariense 1 Madagascar A. madagascariense 2 Madagascar A. madagascariense 3 Madagascar A. madagascariense 4 Madagascar H. quebrada 1 Peru H. mexicana 2 Venezuela H. mexicana 4 Guatemala 6 South Africa 4. The phylogeny of the Mediterranean Anthoxanthum lineages is not consistent with taxonomy. Invasive A. odoratum specimens from America are clearly linked with Western European populations. Ataxia E Asia South America 5 Hierochloë mexicana Central America H. mexicana 3 Mexico H. mexicana 6 Venezuela A. gracile 11 Italy A. gracile 6 Italy A. gracile 4 Italy A. gracile 12 Italy A. gracile 9 Greece A. gracile 10 Greece A. gracile 8 Greece A. gracile 3 Italy A. alpinum 3 France A. odoratum 26 Francia A. alpinum 5 Czech Rep. A. alpinum 6 Czech Rep. A. alpinum 4 France A. alpinum 2 Switzerland A. alpinum 1 Switzerland A. odoratum 34 Czech Rep. A. odoratum 33 Czech Rep. Anthoxanthum gracile Mediterranean Basin Perennial 4x, 2x Europe 3 A. nivale ERZ E Africa A. nivale WRZ A. aethiopicum 4 Kenya A. aethiopicum 3 Ethiopia A. aethiopicum 5 Ethiopia A. aristatum 7 Italy A. aristatum 9 Italy A. odoratum 45 Montenegro A. odoratum 10 Spain Anthoxanthum A. odoratum 9 Russia 4 A. odoratum 27 France A. odoratum 28 Switzerland A. odoratum 3 USA A. odoratum 36 Ireland A. odoratum 32 Greece A. odoratum 31 Greece A. odoratum 19 Spain A. maderense 3 Portugal A. ovatum 1 Morocco A. ovatum 4 Spain A. ovatum 7 Spain A. ovatum 3 Spain Anthoxanthum sp. 1 Canary Islands Anthoxanthum sp. 2 Canary Islands A. ovatum 2 Morocco A. odoratum 17 Spain A. odoratum 18 Spain A. odoratum 37 Ireland Perennial 4x Annual 2x A. amarum 1 Spain A. amarum 8 Spain A. amarum 6 Spain A. amarum 3 Portugal A. amarum 4 Portugal A. amarum 5 Spain A. amarum 7 Spain A. odoratum 42 Spain A. aristatum 11 Spain A. odoratum 13 Portugal A. aristatum 5 Spain A. aristatum 4 Spain A. odoratum 14 Portugal A. odoratum 15 Portugal A. odoratum 25 Spain A. ovatum 5 Spain A. odoratum 43 Spain Mediterranean Basin Europe A. odoratum 20 Spain A. odoratum 35 Ireland A. odoratum 11 Spain A. aristatum 6 Spain A. odoratum 16 Spain Glyceria Figure 5. Maximum clade credibility tree from the Bayesian analysis of nuclear DNA sequences (ITS and ETS) constructed with Mr Bayes. Discontinuous lines represent branches with less than 80 posterior probability support (PP). //, shortened branches. Numbers follow the paragraphs in the Results and Discussion section. 5. Hierochloë mexicana joins the Anthoxanthum clade, which is consistent with its morphology. Hierochloë mexicana is the only native American Anthoxanthum taxon, which suggests a long distance dispersal event. H. hirta Norway 6 South America (H. pusilla) W Europe A. nivale WRZ 6 A. odoratum 10 Spain Anthoxanthum Dasypyrum Secale Triticum Eurasia/Greenland Mediterranean Basin A. aristatum 7 Italy A. odoratum 29 Greece A. aristatum 8 Italy A. odoratum 30 Greece A. odoratum 25 Spain A. ovatum 1 Morocco A. ovatum 2 Morocco A. ovatum 3 Spain Anthoxanthum sp. 1 Spain Anthoxanthum sp. 2 Spain A. maderense 2 Portugal A. maderense 1 Portugal A. odoratum 27 France A. ovatum 7 Spain A. odoratum 11 Spain A. amarum 8 Spain A. aristatum 9 Italy A. maderense 3 Portugal A. odoratum 15 Portugal A. odoratum 32 Greece A. odoratum 31 Greece A. odoratum 28 Switzerland A. odoratum 24 Spain A. odoratum 21 Spain 95.9 // Puccinellia Helictotrichon Lolium Festuca H. australis 1 Finland H. australis 3 Czech Rep. H. hirta 4 Norway H. odorata 1 Norway H. odorata 3 Norway H. hirta 1 Norway H. hirta 2 Norway H. hirta 3 Norway H. hirta 5 Norway H. odorata 4 Norway H. odorata 8 Norway H. hirta 8 Norway H. odorata 2 Norway H. hirta 7 Norway H. hirta 6 Norway H. alpina 3 Greenland H. pusilla 1 Chile H. pusilla 2 Chile H. alpina 1 Greenland H. alpina 2 Greenland H. alpina 4 Greenland E Asia South Africa Madagascar 100 // Poa South Africa A. nivale ERZ H. mexicana Mexico Brachypodium // 2. None of the groups as currently defined is monophyletic. Excluding the Central American H. mexicana renders Hierochloë (see 5) monophyletic. North America H. australis 4 Sweden // Europe Eurasia + Australia H. australis 3 Czech Rep. // Melica // H. rariflora 2 Australia H. rariflora 4 Australia H. rariflora 5 Australia H. rariflora 1 Australia H. australis 5 Czech Rep. // Hierochloë SE Asia H. flexuosa 1 Nepal H. glabra 2 China H. redolens 13 Papua New Guinea H. redolens 14 Papua New Guinea H. alpina 7 Sweden H. alpina 8 USA H. alpina 11 USA H. glabra 3 China H. alpina 5 Sweden H. repens 1 Russia H. laxa 4 India H. glabra 4 China H. laxa 6 India H. alpina 9 Russia H. alpina 2 Greenland H. alpina 3 Greenland H. alpina 4 Greenland H. alpina 1 Greenland H. odorata 8 Russia H. odorata 9 Russia H. alpina 6 Sweden H. glabra 1 Russia // Secale South America 6 // Lolium Poa Puccinellia Helictotrichon H. juncifolia Chile H. juncifolia Argentina H. pusilla Chile H. pusilla Chile 87.5 99.8 H. alpina Greenland 99 H. hirta Norway 97.3 H. glabra Russia 71.9 96.9 7 H. altissima Chile (3) 99.8 H. gunckelii Chile (2) H. redolens New Guinea Hierochloë sp Argentina H. utriculata Chile (4) H. redolens Chile (2) H. spicata Chile 97.3 H. redolens Peru H. redolens Argentina H. flexuosa India 100 96.8 H. rariflora Australia 100 96.8 H. quebrada Peru 7 94.0 H. redolens Chile H. redolens Argentina 95.7 Hierochloe sp. Argentina H. utriculata Chile (2) H. spicata Chile H. altissima Chile (2) H. glunckelii Chile H. pusilla Chile H. pusilla Chile 91.3 H. juncifolia Chile 100 H. juncifolia Chile 97.2 H. juncifolia Argentina 88.7 100 H. laxa India 100 6. The North American H. occidentalis is clearly differentiated from the remaining American Hierochloë, being closer to the Asian and European taxa. H. occidentalis USA 6 H. quebrada Peru 100 H. altissima Chile 100 H. redolens Chile (2) 95.5 99.8 H. altissima Chile H. redolens Chile H. glunckelii Chile H. redolens Peru Figure 6. NeighborNet phylogenetic Network based on the plastid alignment (trnLF and trnTL) of 36 Hierochloë specimens. Specimens of all American Hierochloë have been included in the analysis. Numbers in black represent boostrap support (BS) values. Coloured numbers follow paragraph numbers in the Results and Discussion section. 7. South American Hierochloë taxa (excluding H. quebrada from Peru whose position needs to be further studied) are clearly monophyletic and closely related. Only H. pusilla and H. juncifolia are differentiated, reflecting their high ecological separation. H. mexicana Venezuela 99.6 5 91.9 H. mexicana Mexico H. mexicana Guatemala H. mexicana Venezuela Figure 7. NeighborNet phylogenetic Network based on the nuclear alignment (ITS and ETS) of 36 Hierochloë specimens. Specimens of all American Hierochloë have been included in the analysis. Numbers in black represent boostrap support (BS) values. Coloured numbers follow paragraph numbers in the Results and Discussion section.