Trnl–Trnf) Nucleotide Sequences Kelvin M

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Aliso: A Journal of Systematic and Evolutionary Botany

Volume 23 | Issue 1 Article 32

2007 Phylogeny and Biogeography of Endemic Festuca (Poaceae) from New Zealand Based on Nuclear (ITS) and Chloroplast (trnL–trnF) Nucleotide Sequences Kelvin M. Lloyd Landcare Research, Dunedin, New Zealand

Angela M. Hunter University of Otago, Dunedin, New Zealand

David A. Orlovich University of Otago, Dunedin, New Zealand

Suzanne J. Draffin University of Otago, Dunedin, New Zealand

Alan V. Stewart PGG Seeds, Christchurch, New Zealand

See next page for additional authors Follow this and additional works at: http://scholarship.claremont.edu/aliso Part of the Botany Commons, and the Ecology and Evolutionary Biology Commons

Recommended Citation Lloyd, Kelvin M.; Hunter, Angela M.; Orlovich, David A.; Draffin,uza S nne J.; Stewart, Alan V.; and Lee, William G. (2007) "Phylogeny and Biogeography of Endemic Festuca (Poaceae) from New Zealand Based on Nuclear (ITS) and Chloroplast (trnL–trnF) Nucleotide Sequences," Aliso: A Journal of Systematic and Evolutionary Botany: Vol. 23: Iss. 1, Article 32. Available at: http://scholarship.claremont.edu/aliso/vol23/iss1/32 Phylogeny and Biogeography of Endemic Festuca (Poaceae) from New Zealand Based on Nuclear (ITS) and Chloroplast (trnL–trnF) Nucleotide Sequences

Authors Kelvin M. Lloyd, Angela M. Hunter, David A. Orlovich, Suzanne J. Draffin, Alan V. Stewart, and William G. Lee

This article is available in Aliso: A Journal of Systematic and Evolutionary Botany: http://scholarship.claremont.edu/aliso/vol23/iss1/ 32 Aliso 23, pp. 406–419 ᭧ 2007, Rancho Santa Ana Botanic Garden

PHYLOGENY AND BIOGEOGRAPHY OF ENDEMIC FESTUCA (POACEAE) FROM NEW ZEALAND BASED ON NUCLEAR (ITS) AND CHLOROPLAST (trnL–trnF) NUCLEOTIDE SEQUENCES

KELVIN M. LLOYD,1,4 ANGELA M. HUNTER,2 DAVID A. ORLOVICH,2 SUZANNE J. DRAFFIN,2 ALAN V. STEWART,3 AND WILLIAM G. LEE1 1Landcare Research, Private Bag 1930, Dunedin, New Zealand; 2Department of Botany, University of Otago, PO Box 56, Dunedin, New Zealand; 3PGG Seeds, PO Box 3100, Christchurch, New Zealand 4Corresponding author ([email protected])

ABSTRACT We investigated the phylogenetic relationships of the endemic New Zealand (NZ) species of Festuca (Poaceae, Pooideae) by assessing sequence variation from the nuclear internal transcribed spacers (ITS) and a chloroplast intergenic spacer (trnL–trnF) and by measuring DNA content using flow cytometry. The ITS and trnL–trnF data sets were congruent in showing that the NZ species of Festuca have two origins. One group, containing F. coxii, F. luciarum, F. multinodis, and F. ultramafica, is closely related to Festuca sect. Aulaxyper. The other group includes a clade of five endemic species (F. actae, F. deflexa, F. madida, F. matthewsii, F. novae-zelandiae) and one species (F. contracta) with a circum- Antarctic distribution. The North American species F. californica is sister to the latter group in the trnL–trnF phylogeny but not so in the ITS phylogeny. The differentiation of endemic NZ species into two groups is supported by differences in chromosome number and genome size, the latter showing an inverse relationship to ploidy level. We discuss the ecology and biogeography of NZ’s endemic species of Festuca. Origin from Northern Hemisphere ancestors via dispersal to NZ through the American continents is a plausible hypothesis based on current information. Key words: Austrofestuca, biogeography, ecology, Festuca, genome size, ITS, phylogeny, ploidy, Poeae, trnL–trnF.

INTRODUCTION only recently begun to receive attention (Torrecilla and Cat- alań 2002; Torrecilla et al. 2003, 2004; Catalań et al. 2004). New Zealand (NZ) has nine endemic species of Festuca Three groups of fine-leaved Festuca corresponding to sect. (Poaceae, Pooideae), a large (ca. 450 spp.) genus of grasses Aulaxyper, sect. Eskia, and sect. Festuca were identified on that is prominent in temperate regions of the world. The the basis of internal transcribed spacer (ITS) region (nrDNA) phylogenetic relationships of the NZ species have been un- sequence data by Torrecilla and Catalań (2002). More re- known. Morphologically, species of Festuca can be separat- cently, Catalań et al. (2004) have shown that the fine-leaved ed into two major groups: those with broad leaves and those Festuca clade includes a large number of additional, mostly with fine leaves. New Zealand’s indigenous species are all annual, genera (Ctenopsis, Cutandia Willk., Helleria, Micro- fine leaved (Edgar and Connor 2000), most having an erect, pyrum, Narduroides, Psilurus, Vulpia, Wangenheimia), tufted habit but others forming swards. They occur from sea while Torrecilla et al. (2004) showed that Vulpia is poly- level to the alpine zone, and are found in a range of habitats phyletic within the clade of paraphyletic fine-leaved Festuca. including coastal rocks, inland cliffs, ultramafic (serpentine) Northern Hemisphere taxa have dominated previous mo- and calcareous areas, and tall-tussock (dominated by Chion- lecular studies of Festuca. This geographical bias is unfor- ochloa Zotov) and short-tussock (dominated by Festuca/ tunate because Festuca and other members of Pooideae are Poa) grasslands. One species (F. coxii) previously has been also important genera in temperate areas of the Southern placed in Agropyron Gaertn. on account of morphological Hemisphere (Hartley 1961, 1973). Inclusion of species from features not shared with other NZ species of Festuca, and uncertainty has existed regarding its generic position (Edgar both Northern and Southern hemispheres will help to eval- and Connor 2000). uate hypotheses concerning processes of speciation and bio- Molecular evidence has shown that the broad-leaved spe- geography. Indeed, Catalań et al. (2004) have recently found cies of Festuca are related to Lolium and Schedonorus and a close and unexpected relationship between three South that Festuca s.l. does not form a monophyletic group without American species (the fine-leaved Helleria fragilis and inclusion of these genera (Darbyshire and Warwick 1992; broad-leaved F. coromotensis and F. elviae) and Northern Stammers et al. 1995; Charmet et al. 1997; Gaut et al. 2000; Hemisphere species in the fine-leaved Festuca sect. Aulax- Torrecilla and Catalań 2002). Dactylis is either sister to, or yper. nested within, the broad-leaved Festuca/Lolium/Schedonorus While the origin of the grass family is thought to be South group (Hsiao et al. 1995; Charmet et al. 1997; Torrecilla and American (Soreng and Davis 1998; Hsiao et al. 1999), the Catalań 2002). Most analyses place fine-leaved species of center of diversity for Pooideae is Eurasia (Hartley 1961, Festuca (and related ephemerals) as sister to the broad- 1973; Soreng 1990), and Loliinae lineages such as Festuca leaved Festuca/Lolium/Schedonorus complex (Charmet et al. appear to have arisen in the Mediterranean region (Catalań 1997; Gaut et al. 2000; Torrecilla and Catalań 2002), but et al. 2004). Connor and Edgar (1986) assumed that Austra- relationships within the group of fine-leaved Festuca have lasian Festuca and Poa originated from Northern Hemi- VOLUME 23 New Zealand Festuca 407 sphere ancestors that dispersed southward. Chloroplast re- pmol of each primer. The ITS regions were amplified using striction site data for one Australasian species of Poa sup- the primers ITS-1 and ITS-4 from White et al. (1990) and ports this assertion (Soreng 1990), but it is not known wheth- the following amplification protocol: 95ЊC for 5 min, 35 cy- er the NZ species of Festuca have arisen from northern cles of 94ЊC for 45 sec, 48ЊC for 45 sec, 72ЊC for 2 min 10 forms that dispersed southward relatively recently or wheth- sec, followed by a final extension of 72ЊC for 10 min. To er they are the evolutionary products of long isolation in the amplify the spacer between the trnL (UAA) 3Ј exon and trnF Southern Hemisphere. (GAA) gene of chloroplast DNA, we followed the protocol Accordingly, we included all nine endemic NZ species of of Taberlet et al. (1991) using primers ‘‘e’’ and ‘‘f’’ and an Festuca (F. actae, F. coxii, F. deflexa, F. luciarum, F. mad- annealing temperature of 53ЊC. PCR products were purified ida, F. matthewsii, F. multinodis, F. novae-zelandiae, F. ul- using spin column purification (QIAquick PCR Purification tramafica) in this study, as well as one fine-leaved circum- Kit, QIAGEN Pty, Ltd., Clifton Hill, Victoria, Australia). Antarctic species that is indigenous to the NZ region (F. The approximate concentration of each amplification product contracta), and one species of Austrofestuca, an Australasian was determined by electrophoresis with a known amount of genus that was segregated from Festuca by Alexeev (1976) molecular marker (1 kilobase ladder, Invitrogen New Zea- but which has recently been shown to be aligned with Poa land Limited, Auckland, NZ). The purified PCR products (Hunter et al. 2004). Also included were Northern Hemi- were sequenced, using the original amplification primers, by sphere representatives of the following genera: Ctenopsis, the Centre for Gene Research, Department of Microbiology, Dactylis, Festuca, Lolium, Micropyrum, Narduroides, Poa, University of Otago, NZ. Reactions were done in 20 ␮L Vulpia, and Wangenheimia, as well as South American Hel- volumes using Big Dye Terminator vers. 3.0 cycle-sequenc- leria fragilis, weighting our taxonomic sampling toward the ing chemistry (Applied Biosystems) following the manufac- fine-leaved lineages. turer’s protocol. Electropherograms were edited and assem- Our primary aims were to (1) test whether NZ’s endemic bled using Autoassembler vers. 1.3.0 (Applied Biosystems). Festuca taxa form a monophyletic group and examine the Seventy-seven taxa were included in the ITS data set and generic placement of F. coxii, (2) discover the closest rela- 74 in the trnL–trnF data set. Seven species (Festuca dal- tives of these taxa and thus identify their putative origins, matica, F. filiformis, F. heterophylla, F. lemanii, F. pallens, and (3) examine relationships between Southern and North- F. pseudodalmatica, F. rupicaprina) were represented only ern hemisphere Festuca lineages. We present the results of by ITS sequences, while F. ampla, F. caerulescens, F. co- phylogenetic analyses using sequences of the ITS region of romotensis, F. elviae, and the Kaitorete Spit population of nuclear ribosomal DNA and the trnL–trnF intergenic spacer Austrofestuca littoralis were only represented in the trnL– of chloroplast DNA. We also used flow cytometry to mea- trnF data set. The trnL–trnF data set included many se- sure the DNA contents of the endemic NZ species of Fes- quences from GenBank that incorporated the trnL intron. tuca and compared these with chromosome numbers to ex- This region was included in the analysis, although the cor- amine variation in genome size. responding sequences were missing for the species sequenced in this study. Sequences were aligned initially using MATERIALS AND METHODS CLUSTAL࿞X vers. 1.83 (Thompson et al. 1997) followed by manual adjustment using Se-Al vers. 2.0a11 (Rambaut Our study included representatives from six subgenera and 2002). Gaps were treated as missing data and informative nine sections of Festuca, three species of Schedonorus, two insertions/deletions (indels) were coded as binary (or in two species of Vulpia, and a single species each of Austrofestuca, cases, multistate) characters and added to the data matrix. Brachypodium, Ctenopsis, Dactylis, Helleria, Lolium, Mi- We analyzed the ITS and trnL–trnF data sets separately by cropyrum, Narduroides, Poa, Psilurus, and Wangenheimia. maximum parsimony using PAUP* vers. 4.0b10 (Swofford Three populations of Austrofestuca littoralis from NZ and 2002), with Brachypodium distachyon (Brachypodieae) used Australia were included, and all four subspecies of the NZ as the outgroup in each analysis. Within both data sets we endemic, F. matthewsii, resulting in a total of 81 sampled conducted heuristic searches using 1000 random-addition taxa used in the analyses. A list of DNA sequences and their replicates and the tree-bisection-reconnection branch-swap- sources is shown in Table 1. The majority of these were ping algorithm. Branch support was assessed by parsimony obtained from the National Center for Biotechnology Infor- bootstrap analysis with 1000 replicate heuristic analyses us- mation database (GenBank), but we sequenced all of the ing two random-addition replicates in each bootstrap repli- indigenous species of Festuca (13 taxa), as well as F. rubra cate. and Vulpia bromoides, two species that are naturalized in Relative nuclear DNA content was determined for the NZ NZ. Live specimens were obtained from field collections or taxa (Table 2) by flow cytometry (PA-II, Partec, Mu¨nster, grown from seed and propagated outdoors in pots. For F. Germany). Plant nuclei were first isolated by chopping leaf contracta, DNA was extracted from a herbarium specimen. tissue in an extraction buffer using a razor blade. DNA was DNA was extracted from small samples of young leaf- stained with a 4Ј,6Ј-diamidino-2-phenylindole dye and the base material following the method of Carlson et al. (1991). extract was filtered through a 0.30 ␮m sieve prior to anal- RNA was removed by incubation with DNase-free RNase ysis. The nuclear extraction buffer and staining solution were (F. Hoffman La Roche, Ltd., Basel, Switzerland) for 15 min supplied in a kit (Partec CyStain UV Precise T kit) and used at 37ЊC. Polymerase chain reactions were done in 20 ␮L according to the manufacturer’s instructions. All samples volumes, using AmpliTaq Gold PCR Master Mix (Applied were analyzed immediately after isolation and the instrument Biosystems, Inc., Foster City, California, USA) following was aligned daily with flow check beads (Partec) labeled the manufacturer’s instructions. Each reaction contained 8 with a defined fluorescence intensity. An internal standard 408 Lloyd, Hunter, Orlovich, Draffin, Stewart, and Lee ALISO was used for each sample to account for machine drift and/ are included within a weakly supported (bs 68%) clade or differences in the turbidity of the sample flow. Trifolium loosely corresponding to Festuca sect. Aulaxyper. The re- repens L. var. ‘Grasslands Huia’ was used as the standard maining indigenous species (the endemic F. actae, F. de- as it was known to have a 2C-value of 2.38 picograms (pg) flexa, F. madida, F. matthewsii, and F. novae-zelandiae, and (Campbell et al. 1999). For each sample, a leaf segment of circum-Antarctic F. contracta) are resolved together within this reference tissue was co-chopped with the test sample, a well-supported (bs 90%) clade in which F. madida has a and the ratio of the test sample to the standard peak channel sister relationship (bs 74%) to the other ‘‘southern endemic’’ was used to compute the 2C-values for each test sample. taxa, which are unresolved. Three additional lineages of fine- Between 5000 and 10,000 nuclei were analyzed from each leaved taxa are identifiable. The first corresponds to a weak- sample. The coefficient of variation of the 2C peaks used to ly supported (bs 61%) Festuca sect. Festuca and includes a estimate C-values in all samples was below 6%. strongly supported (bs 93%) group of mainly eastern Euro- Festuca madida, listed as tetraploid by de Lange and Mur- pean species (F. dalmatica, F. pseudodalmatica, F. lemanii, ray (2002), had a 2C-value similar to those of hexaploid F. pallens, F. rupicaprina). The second clade (bs Ͻ50%) taxa. Consequently, the chromosome number for F. madida comprises species from Festuca sects. Eskia and Amphige- was re-determined by R. Pickering (Crop and Food Re- nes (F. carpathica, F. dimorpha, F. eskia, F. gautieri, F. search, Lincoln, NZ). Mitotic chromosome preparations quadriflora). The third clade (bs 100%) contains two ephem- were made from root tips that had been pretreated in iced eral species (Psilurus incurvus, Vulpia myuros). water for 24 hr, fixed in 3 : 1 ethanol : acetic acid for 48 hr, hydrolyzed in 1N HCl for 10 min at 60ЊC, and squashed in trnL–trnF Analysis 1% aceto-carmine. Amplification produced a fragment 331–465 bp long, RESULTS which in most species included a portion of the trnL intron, ITS Analysis the trnL3Ј exon, the trnL–trnF intergenic spacer, and a portion of the trnF gene. Among NZ taxa of Festuca, the trnL– In most species, amplification produced a fragment 630– trnF spacer was generally 251–252 bp long, but was slightly 700 base pairs (bp) long that included partial sequences of longer in F. ultramafica (259 bp) and much longer (282 bp) the 18S and 26S genes flanking the ITS region. The ITS-1 in F. deflexa, F. matthewsii subsp. latifundii, and F. mat- region was 216–219 bp long in most taxa, but longer in thewsii subsp. pisamontis. These sequences were aligned Vulpia bromoides (220 bp) and Festuca madida (225 bp). with the longer sequences, incorporating the whole trnL in- The ITS-2 region was generally 211–213 bp long, but short- tron, obtained from GenBank (Table 1). Twenty-eight infor- er in F. madida (209 bp) and longer in F. multinodis (225 mative indel characters (26 binary, 2 multistate) were added bp). Sequences were truncated for F. matthewsii subsp. la- to the aligned matrix to give 1112 characters in total, of tifundii and the Wekakura population of Austrofestuca lit- which 161 were parsimony informative. A heuristic search toralis (ITS-1 only), F. deflexa and F. ultramafica (partial found 6520 equally parsimonious trees of length 416 and ITS-1 and partial ITS-2), and F. contracta (partial ITS-2 with a consistency index of 0.534 excluding uninformative only). Alignment of trimmed ITS sequences and insertion of characters. Figure 2 shows the strict consensus of these trees. gaps yielded a matrix with 660 characters, of which 157 The trnL–trnF tree (Fig. 2) is broadly similar to the ITS (including 19 binary indel characters) were parsimony informative. A heuristic search found 480 equally parsimoni- tree (Fig. 1), with a strongly supported (bs 100%) Austro- ous trees of length 622 and with a consistency index of 0.466 festuca littoralis/Poa pratensis clade again occupying a sis- excluding uninformative characters. ter relationship to the rest of the ingroup, and Dactylis glom- In the ITS strict consensus tree (Fig. 1) the group of erata again sister (bp 75%) to the remaining species. The broad-leaved taxa is well resolved and supported by boot- chief difference surrounds the ‘‘southern endemic’’ clade, strap (bs) values, but resolution and support was generally which here, along with Festuca californica, are sister (bp poor among fine-leaved taxa. A strongly supported (bs Ͻ50%) to the broad-leaved Festuca/Lolium/Schedonorus 100%) Austrofestuca/Poa pratensis clade is sister to the oth- clade. Together, these clades are sister to the large FEVRE er ingroup taxa, which are also well supported (bs 80%) as clade of fine-leaved Festuca and allied genera. Festuca cal- a group. Dactylis glomerata is sister (bs Ͻ50%) to the re- ifornica occupies a weakly supported (bp 67%) sister posi- maining taxa in this group, which are split into two clades tion to the well-supported (bp 80%) ‘‘southern endemic’’ corresponding to broad-leaved Festuca/Lolium/Schedonorus clade. The trnL–trnF data provide some structure within the (bs Ͻ50%), and fine-leaved Festuca and allied genera (the ‘‘southern endemic’’ clade: Festuca deflexa and two subspe- FEVRE group of Catalań et al. 2004 and Torrecilla et al. cies of F. matthewsii (subspp. latifundii and pisamontis) 2004). A feature of the generally well-resolved broad-leaved form a clade that is sister to the other taxa and share a unique clades is the mixing of species representing different sub- 30 bp repeat near the 3Ј-end of the intergenic spacer. genera of Festuca (Table 1). The broad-leaved Festuca/Schedonorus/Lolium subclades The clade of fine-leaved FEVRE (Festuca, Vulpia, and are nested in this analysis, with Festuca font-queri/F. mairei/ Related Ephemerals) taxa is well supported (bs 79%) as a Lolium/Schedonorus occupying a derived position, and F. group, but its higher order branches lack bootstrap support altissima, F. caerulescens, F. drymeja, F. lasto, F. scariosa, and only a few clades are resolved within it. Notably, the and F. triflora unresolved at the base. Each division in the NZ taxa are split into two different groups. Four endemic broad-leaved clade is well supported by a bootstrap value species (F. coxii, F. luciarum, F. multinodis, F. ultramafica) (71–94%), unlike the ITS tree where relationships among the VOLUME 23 New Zealand Festuca 409 F trn L– trn AY528933 AY327796 AY528934 AF478524 AF478524 AF478521 AY327798 AF478525 AF478511 AF533057 AF478523 AF533064 AF478519 AF543516 AF478515 AF478514 AF478517 AF478512 AF478513 AY528950 New Zealand (CHR) GenBank accession number ITS AY327791 AY524824 AF303415 AF478482 AY254371 AJ240160 AF478481 AY327792 AF478483 AY254374 AF171145 AF303419 AF532957 AF303428 AF147177 AF303426 AF147136 AF478479 AF478518 AJ240519 AF478478 AF478477 AF303423 AF478475 AF478476 AF303422 ń (2002) ń (2002) ń (2002) ń (2002) ń (2002) AF303403ń (2002) AF478510 ń (2002) ´ et al. (2004) ń et al. (2004) ń et al. (2004) ń et al. (2004) ń et al. (2004) ń et al. (2004) ń et al. (2004) ń et al. (2004) ń et al. (2004) Hunter et al. (2004) Hunter et al. (2004) Hunter et al. (2004) Torrecilla and Catala Catala Galli et al. (unpubl.Charmet data) et al. (1997) Torrecilla et al. (2003) Hunter et al. (2004) Catala Galli et al. (unpubl.Gaut data) et al. (2000) Torrecilla and Catala Catala Torrecilla and Catala Cataln Torrecilla and Catala Catala Tredway et al. (unpubl.Catala data) Catala Charmet et al. (1997) Torrecilla et al. (2003) Catala Torrecilla and Catala This study Torrecilla et al. (2003) Catala Torrecilla and Catala Forage Germplasm Centre (OTA057896) GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank New Zealand, Margot Forde ˜ez et Ferrero ń Taxon Source/Voucher Reference Krajina ex Domin Fuerte, Ortu St.-Yves Lam. (Hack.) A. Kern. ´gev. ex Anon. Dufour (Hack.) Markgr.-Dann. Elmer (Litard.) Markgr.-Dann. (Hack.) Richt. Reuter Chaub. Boiss Dumort Boiss. Pourr. Mie (Hack.) K. Richt. GenBank Torrecilla and Catala (Labill.) E. B. Alexeev GenBank Boiss. Litard. Bastard Hack. (Hack.) K. Richt. Suter Hack. L. L. Festuca Exaratae E. B. Alexeev Festuca Festuca Aulaxyper L. A. littoralis F. alpina F. clementei F. dalmatica F. filiformis F. frigida F. glacialis F. querana F. idahoensis F. indigesta F. lemanii F. longiauriculata F. ovina F. plicata F. pseudodalmatica F. rupicaprina F. borderei F. ampla F. heterophylla F. juncifolia F. nevadensis F. pyrenaica F. rivularis F. rothmaleri F. rubra F. capillifolia Subsect. Subsect. Sect. Sect. Table 1. Sources of DNA sequences used in the analyses. Voucher numbers cited refer to specimens in the University of Otago (OTA) and Landcare Research Subgen. herbaria. Austrofestuca Festuca 410 Lloyd, Hunter, Orlovich, Draffin, Stewart, and Lee ALISO F trn L– trn AY098996 AY098999 AY099002 AF478508 AF478506 AF478506 AY099003 AY099001 AF519987 AF519985 AF519984 AF533047 AF533049 AF478505 AY098997 AY098998 AY099004 GenBank accession number ITS AF303424 AF519977 AY099007 AF303412 AF303414 AF303413 AY099005 AY099006 AF519982 AF519980 AF519977 AF543514 AF303405 AF303411 AF303411 AF303418 AF303410 ń (2002) ń (2002) ń (2002) ń (2002) ń (2002) ń (2002) AF303406 AF478509 ń (2002) ń (2002) ń (2002) ń (2002) ń et al. (2004) ń et al. (2004) ń et al. (2004) ń et al. (2004)ń et al. (2004) ń et al. (2004) AF533054 ń et al. (2004)ń et al. (2004) ń et al. (2004) AF538362 AF533052 Torrecilla and Catala Torrecilla et al. (2003) Torrecilla and Catala Torrecilla et al. (2003) Catala Torrecilla and Catala Torrecilla and Catala Torrecilla and Catala Catala Catala Torrecilla et al. (2003) Torrecilla and Catala Torrecilla et al. (2003) Torrecilla et al. (2003) Catala Catala Catala Torrecilla and Catala Catala Torrecilla and Catala Torrecilla et al. (2003) This study AY524830 AY528944 Torrecilla and Catala Torrecilla et al. (2003) (CHR385372) GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank GenBank (Hack.) Markgr.- pulchella baetica Taxon Source/Voucher Reference Krechetovich et Bobrov Desf. GenBank Catala Krivot. Hack. Honck. Janka Dietr. L. subsp. Jan (Griseb.) Hack. Guss. Kirk Australia, Maquarie Island Schrad. subsp. Clauson All. (Griseb.) Krivot. Hack. (C. M. Sm. ex Link) Linding. E. B. Alexeev (Lag.) Asch. et Graebn. Mert. et Koch St.-Yves (Hack.) K. Richt. Boiss. GenBank Torrecilla and Catala Desf. GenBank Catala St.-Yves (S. Watson) Cassidy GenBank Ramond ex DC. Boiss Willk. p. p. Dann. Drymanthele Helleria Leucopoa Amphigenes Pseudatropis Scariosae Subbulbosae Eskia Leucopoa F. mairei F. scariosa F. agustinii F. carpathica F. dimorpha F. pulchella F. spectabilis F. durandoi F. paniculata F. caerulescens F. burnatii F. quadriflora F. eskia F. gautieri F. elegans F. triflora F. altissima F. drymeja F. lasto F. contracta F. kingii Sect. Sect. Sect. Sect. Sect. Sect. Table 1. Continued. Subgen. Subgen. Subgen. VOLUME 23 New Zealand Festuca 411 F trn L– trn AF533044 AF543517 AY528939 AF533055 AF533054 GenBank accession number ITS AF303404 AF532952 AF532956 ń (2002) ń (2002) AF303399 AF478500 ń et al. (2004) ń et al. (2004)ń et al. (2004) AF543518 ń et al. (2004) AF478499 AF478537 ń et al. (2004) ń et al. (2004) ń et al. (2004) AF532953 AF533056 This studyThis studyCatala This studyThis studyThis AY524825 studyThis AY524838 study AY528937 This study AY524828 AY528942 This studyThis study AY524836 AY524835 AY528948 AY524837 AY528946 AY524831 AY528945 AY524827 AY528947 AY524832 AY528940 AY528941 Torrecilla and Catala Catala This study AY524829 AY528949 This study AY524826 AY528938 This study AY524833 AY528943 Catala Catala Forsyth (OTA057589) (OTA057956) (OTA057588) gaharuru Range (OTA057621) Valley (OTA057938) Fyffe (OTA057936) Range (OTA057625) (OTA057945) Head (OTA057958) Valley (OTA057939) (OTA057629) Pillar Range (OTA057627) New Zealand, Fiordland, Takahe GenBank GenBank New Zealand, Hawkes Bay, Maun- GenBank GenBank õ GenBank Catala Taxon Source/Voucher Reference Connor New Zealand, Otago, Pisa Range (Hack.) Cockayne New Zealand, Fiordland, Takahe Connor New Zealand, Marlborough, Mt. Connor New Zealand, Otago, Remarkables Bricen (P. Beauv) Peterm. Krivot. õ Connor New Zealand, Nelson, Windy Point Vasey (L.) P. Beauv. GenBank Torrecilla and Catala (Hack.) Cheeseman Petrie et Hack. New Zealand, Wellington, Baring (Tzvelev) E. B. Alexeev (Lag.) Paunero GenBank Catala St.-Yves GenBank Connor Trin. GenBank Catala Connor New Zealand, Otago, Rock and Connor New Zealand, Nelson, Mt. Owen Trin. Bricen Connor New Zealand, Canterbury, Lake aquilonia latifundii pisamontis matthewsii (Petrie) Hack. New Zealand, Chatham Island P. Beauv. Subulatae Schedonorus De Not. Breviristatae subsp. subsp. subsp. subsp. F. actae F. coromotensis F. coxii F. deflexa F. elviae F. luciarum F. madida F. multinodis F. novae-zelandiae F. ultramafica F. matthewsii B. distachyon C. delicatula F. altaica F. californica F. subulata F. font-queri Sect. Table 1. Continued. Incertae sedis Subgen. Subgen. Brachypodium Ctenopsis 412 Lloyd, Hunter, Orlovich, Draffin, Stewart, and Lee ALISO F trn L– trn AY327794 AY327799 AY061957 AF533043 AY098995 AF478503 AY327797 GenBank accession number ITS L36512 AF303401 AF171182 AF303416 AJ240153 AF303421 AJ240162 ń (2002) ń (2002) ń et al. (2004)ń et al. (2004) AF532960 AF533059 ń et al. (2004) AF478494 AF478534 ń et al. (2004) AF478497 AF478535 ń et al. (2004)ń et al. (2004) ń et al. (2004) AF478493 AF478533 ń et al. (2004) AF478498 AF478536 Hsiao et al. (1995) Hunter et al. (2004) Catala Hunter et al. (2004) Gaut et al. (2000) Stoneberg Holt et al. (2004) Torrecilla and Catala Catala Charmet et al. (1997) Torrecilla et al. (2003) Torrecilla and Catala Catala This studyCharmet et al. (1997) Hunter et al. (2004) AY524834 AY528936 Gorge (OTA057652) GenBank GenBank GenBank GenBank GenBank GenBank GenBank Taxon Source/Voucher Reference L. GenBank (L.) Gray New Zealand, Otago, Cromwell (Boiss.) Rouy GenBank Catala (L.) Holub GenBank (L.) Link GenBank Catala L. GenBank (Huds.) P. Beauv. GenBank (Gouan) Schinz et Thell. GenBank Catala L. GenBank (Scop.) Holub GenBank Luces GenBank Catala (L.) C. C. Gmel. GenBank (L.) Trin. GenBank Catala Moench P. Beauv. Rouy Link Vulpia L. Fourn. Trin. L. D. glomerata H. fragilis L. perenne M. tenellum N. salzmanii P. pratensis P. incurvus S. giganteus S. phoenix S. pratensis V. bromoides V. myuros W. lima C. C. Gmel. L. Sect. Table 1. Continued. Dactylis Helleria Lolium Micropyrum Narduroides Poa Psilurus Vulpia Schedonorus Wangenheimia VOLUME 23 New Zealand Festuca 413

Table 2. DNA content, ploidy level, and genome size of endem- for the remaining hexaploids. The octoploid species have an ic New Zealand species of Festuca. average genome size of 1.03 pg.

DNA/ 2C-value genome DISCUSSION Taxon (pg) Ploidy 2n (pg) Phylogeny of Festuca Sensu Lato F. actae 9.4 6x 42b 1.57 We included a wide selection of taxa in our analyses, but F. deflexa 10.45 6x 42b 1.74 F. madida 9.7 6x 42c 1.62 our intention was to provide a broad context in which to F. matthewsii subsp. aquilonia 9.98 6x 42b 1.66 place the NZ taxa rather than to examine relationships F. matthewsii subsp. latifundii 9.89 6x 42a 1.65 among Northern Hemisphere groups, which have been doc- F. matthewsii subsp. matthewsii 10.13 6x 42b 1.69 umented by others, particularly in relation to the broad- F. matthewsii subsp. pisamontis 10.02 6x 42b 1.67 leaved Festuca/Lolium complex (Darbyshire and Warwick F. novae-zelandiae 9.08 6x 42a 1.51 1992; Stammers et al. 1995; Charmet et al. 1997; Gaut et F. coxii 8.74 8x 56a 1.09 al. 2000; Torrecilla and Catalań 2002; Catalań et al. 2004). F. luciarum 8.06 8x 56b 1.01 The relationships we obtained among the broad-leaved taxa a F. multinodis 7.65 8x 56 0.96 were similar to those obtained by Catalań et al. (2004). In F. ultramafica 8.5 8x 56b 1.06 our analyses, the monophyly of Schedonorus was precluded a Edgar and Connor (2000). by either F. font-queri or L. perenne, but wider sampling b Unpublished data. Brian Murray and Peter de Lange, University within this group will be necessary to accurately determine of Auckland, Private Bag 92019, Auckland, New Zealand. the relationships among these three genera and whether any c This study. new nomenclatural combinations are justified. Both ITS and trnL–trnF analyses show a close relationship between Aus- trofestuca littoralis and Poa, supporting the findings of four broad-leaved subclades were not supported by bootstrap Hunter et al. (2004). analysis. The large FEVRE clade of fine-leaved Festuca and allied Phylogenetic Relationships of New Zealand Festuca genera is supported by a bootstrap value of 75%, but basal Among fine-leaved Festuca and related genera, we ob- nodes within the clade lack support. The only well-resolved tained very similar relationships to those of Catalań et al. lineages are a sect. Festuca clade (bs 78%) to which Wan- (2004) and Torrecilla et al. (2004), who denoted the large genheimia is a weakly supported (bs 58%) sister, and a clade fine-leaved clade containing Festuca s.s., Vulpia, and related (bs 75%) comprising the two species of Vulpia (which form ephemerals as FEVRE (Fig. 1, 2). Relationships within the a monophyletic group in this analysis) and Psilurus incurvus. FEVRE clade are described in detail by the authors. The A weakly supported (bs 67%) red fescue clade includes six relationships between the fine-leaved species of NZ Festuca species from sect. Aulaxyper, one species each from sect. and the FEVRE clade differed according to the data set used. Amphigenes (F. agustinii and F. querana, respectively), two In the ITS tree (Fig. 1), all of the NZ taxa are included in broad-leaved South American species (F. coromotensis and the FEVRE clade. The trnL–trnF tree (Fig. 2) shows one F. elviae), Helleria fragilis, and four fine-leaved NZ endem- group of NZ taxa within the FEVRE clade and one outside ics (F. coxii, F. luciarum, F. multinodis, F. ultramafica) and the FEVRE clade and sister to F. californica. subsect. Exaratae. This clade lacks further resolution except The evidence is compelling that NZ’s endemic species of for a weak (bs 63%) relationship between F. juncifolia and Festuca do not form a monophyletic group and have at least F. rothmaleri. two origins. One group has affinities to Festuca sect. Aulax- yper (red fescues). The other species form a group (‘‘south- DNA Content (C-Value) and Chromosome Number in New ern endemic’’ clade) with the circum-Antarctic F. contracta, Zealand Festuca and may have a close relationship to F. californica of the western USA (Fig. 2), but are not well resolved with respect Three ploidy levels have been recognized among NZ en- to any previously recognized clade of fine-leaved Festuca. demic species (Table 2), but the 2n ϭ 28 (tetraploid) count The evidence for two or more origins is congruent between obtained from F. madida by de Lange and Murray (2002) nucleotide sequences from both the nuclear (ITS) and chlo- appears mistaken. Our own chromosome count gave 2n ϭ roplast (trnL–trnF) phylogenies (Fig. 1, 2), and is supported 42 (hexaploid), as in F. actae, F. deflexa, F. matthewsii, and by concordant differences in ploidy level and genome size F. novae-zealandiae. The remaining species (F. coxii, F. lu- (Table 2). ciarum, F. multinodis, F. ultramafica) are octoploids. The hexaploid/octoploid split corresponds with the separation of Aulaxyper-related taxa.—The first association between NZ Festuca into two groups based on ITS and trnL–trnF northern species of Festuca sect. Aulaxyper and southern sequence variation, with F. madida aligning with the hexa- species and relatives of Festuca was reported by Catalań et ploids (Fig. 1, 2). DNA content (2C-value) ranges from 7.65 al. (2004) for three South American species, F. coromoten- pg per cell in F. multinodis to 10.45 pg per cell in F. deflexa. sis, F. elviae, and Helleria fragilis. Our results confirm this Hexaploid taxa have larger genome sizes (2C-value/ploidy relationship and extend it across the South Pacific Ocean to level) than octoploids (Table 2). The genome size of F. mad- four fine-leaved NZ species (Fig. 1, 2). Two of these species ida (1.62 pg), which we measured on the same plant used (F. luciarum, F. multinodis) are strongly rhizomatous/sto- for the chromosome count, matches the average of 1.64 pg loniferous, whereas the other two (F. coxii, F. ultramafica) 414 Lloyd, Hunter, Orlovich, Draffin, Stewart, and Lee ALISO

Fig. 1.—Strict consensus of 480 trees derived from parsimony analysis of ITS sequences. Indigenous New Zealand taxa and branches are shown in bold. Branch support is indicated by bootstrap values above 50%. Consistency index ϭ 0.466, retention index ϭ 0.749, rescaled consistency ϭ 0.349. have a short-tussock (bunchgrass) habit that is shared with Most studies are similar to ours in showing that sect. Au- the remaining NZ endemics. Extravaginal branching is typ- laxyper taxa occupy a derived position among fine-leaved ical of taxa in sect. Aulaxyper (Torrecilla and Catalań 2002), taxa of Festuca and their allies (Darbyshire and Warwick but while three of the endemic Aulaxyper-related species 1992; Charmet et al. 1997; Torrecilla and Catalań 2002; Tor- have extravaginal branching, F. coxii does not (Connor recilla et al. 2003, 2004; Catalań et al. 2004), although Gaut 1998). Festuca coxii, F. luciarum, and F. multinodis have et al. (2000) found the phylogenetic position of the red fes- lax leaves, while those of F. ultramafica are conduplicate cues to be equivocal, depending on the type of analysis or and pungent. Leaves are smooth in all four species, apart whether indels were used as characters. The placement of F. from hairs present on the lower portions. coxii among sect. Aulaxyper taxa confirms its standing with- VOLUME 23 New Zealand Festuca 415

Fig. 2.—Strict consensus of 6520 trees derived from parsimony analysis of trnL–trnF sequences. Indigenous New Zealand taxa and branches are shown in bold. Branch support is indicated by bootstrap values above 50%. Consistency index ϭ 0.534, retention index ϭ 0.798, rescaled consistency ϭ 0.426. in Festuca, despite its atypical morphological features (Con- Catalań 2002; Torrecilla et al. 2003, 2004; Catalań et al. nor 1998). The isolation and exposed nature of the Chatham 2004). This clade contains the endemic species F. actae, F. Island group, which lies 870 km east of the NZ mainland, deflexa, F. madida, F. matthewsii, and F. novae-zelandiae, may have given rise to selective pressures that affect F. coxii as well as circum-Antarctic F. contracta (Fig. 1, 2). In the uniquely among the endemic taxa. trnL–trnF tree (Fig. 2), F. californica is sister to this clade, and F. matthewsii is paraphyletic, in part due to a 30 bp ‘‘Southern endemic’’ taxa.—The ‘‘southern endemic’’ clade repeat that is shared by only two of its four subspecies and is a novel clade that has not been recognized in previous F. deflexa. No morphological features appear to be correlat- molecular analyses involving fine-leaved Festuca (Darby- ed with this split among the subspecies of F. matthewsii. shire and Warwick 1992; Gaut et al. 2000; Torrecilla and Geographically, the distribution of subsp. pisamontis is nest- 416 Lloyd, Hunter, Orlovich, Draffin, Stewart, and Lee ALISO ed within that of subsp. latifundii and these two taxa do not major environmental fluctuations as forests expanded during overlap with subspp. aquilonia or matthewsii (Fig. 3C). interglacials and receded during glacial periods (McGlone et Apart from F. madida, all of the ‘‘southern endemic’’ spe- al. 2001). Subsequent forest destruction caused by anthro- cies have intravaginally branching tillers. Leaves of all taxa pogenic fires that began around 800 years ago allowed grass- are erect and, apart from F. madida and F. matthewsii subsp. lands to expand in lowland and montane areas formerly oc- matthewsii, scabrid. Festuca contracta and F. madida share cupied by forest (Rogers and Leathwick 1996; McGlone et a cleistogamous breeding system, whereas the other taxa are al. 1997). Lloyd et al. (2003) suggested that for nonforest chasmogamous. taxa such fluctuations would have resulted in evolutionary Festuca contracta is restricted to subantarctic islands and divergence toward one of two alternative strategies: (1) spe- occurs on Macquarie Island (Australian territory) in the New cialist—occupying habitats such as coastal rocks that never Zealand Botanical Region, as well as elsewhere in the South- become forested even during interglacials, or (2) general- ern Ocean (Tierra del Fuego, Falkland Islands, South Geor- ist—persisting in nonforest habitats during interglacials, but gia, Kerguelen Islands). It is a hexaploid (Dubcovsky and having traits (e.g., fast growth rate, wide environmental tol- Martıńez 1991), in common with other members of the erance, strong dispersal) that allow expansion into perigla- ‘‘southern endemic’’ clade (Table 2). Phenetic analysis of cial areas that arise during periods of ice advance. morphological characters among several species of Patagon- The endemic sect. Aulaxyper-related species appear to fit ian Festuca placed F. contracta in a clade with Andean taxa the specialist strategy, and largely remain restricted to sites belonging to sect. Festuca (Dubcovsky and Martıńez 1988). that have lacked forest cover through several glacial/inter- These authors doubted such a placement, and their view is glacial cycles. Festuca multinodis is the most widely distrib- confirmed by our molecular analyses, which show F. con- uted of these species (Fig. 3A). This species occurs on rocky tracta to be related to species in the ‘‘southern endemic’’ coasts in central NZ and extends inland on cliffs and bluffs, clade rather than to species in sect. Festuca (Fig. 1, 2). Fes- mostly on limestone substrates (Connor 1998). Festuca lu- tuca californica is sister to the ‘‘southern endemic’’ clade in ciarum is restricted to four small, geographically separate, the trnL–trnF tree (Fig. 2), but is unresolved among the fine- eastern North Island mountain ranges (Fig. 3A) where it in- leaved fescues in the ITS tree (Fig. 1). The species is found habits limestone rocks and cliffs, and adjacent grassland. in dry grassland, shrubland, and woodland habitats in the Festuca coxii is coastal and restricted to the Chatham Island Coast, Cascade, and Sierra Nevada ranges of California and group, 870 km east of the NZ mainland, while F. ultramafica Oregon (Walsh 1994). It shares a bunchgrass (tussock) habit, occurs in a limited area of ultramafic terrain in the northern tall culms, and openly branched panicles with species in the South Island (Fig. 3A). ‘‘southern endemic’’ clade, but unlike them, it is either tet- In contrast to the restricted distributions of the sect. Au- raploid (Aiken et al. 1996) or octoploid (Hickman 1993), laxyper-related species, taxa from the ‘‘southern endemic’’ rather than hexaploid. clade tend to have large geographic ranges and mostly fit the generalist strategy. They occur as subordinate species in DNA Content and Genome Size of Endemic New Zealand tall-tussock (Chionochloa) grasslands, or as the dominants Festuca of short-tussock grasslands in dry inland basins and montane The 2C DNA contents we report (Table 2) correspond river valleys. Species from this clade were among the small closely with those obtained by flow cytometry for Festuca grasses that pollen analysis has shown to have expanded madida and F. novae-zelandiae (Murray et al. 2003), while rapidly following initial anthropogenic deforestation, before the hexaploid taxa have slightly higher DNA contents than eventual domination by tall tussocks (Chionochloa spp.) in those calculated from Feulgen staining of two fine-leaved all but the driest areas (McGlone 2001). Subsequently, spe- hexaploids (Seal 1983). All of our values, however, are low- cies of Festuca from the ‘‘southern endemic’’ clade have er than the flow cytometry values reported by Huff and Pa- become dominant in tall-tussock grasslands that have been lazzo (1998) for hexaploid and octoploid species of fine- degraded by repeated burning and grazing during pastoral leaved Festuca. Nonetheless, all four studies show the same use (Wraight 1963; Connor 1964; Rose and Platt 1992). trend of declining genome size with increasing ploidy level. Festuca deflexa occurs in the alpine zone on extensive Possibly, the differences in reported values between the flow areas of limestone topography in northwestern South Island cytometry studies relate to the different external standards (Fig. 3D). The four subspecies of F. matthewsii are wide- used for calibration of DNA amounts; Huff and Palazzo spread across the South Island, either in alpine areas or in (1998) used animal cells (chicken red blood cells), whereas montane valley floor grasslands (Fig. 3C). Festuca novae- this study and Murray et al. (2003) used plant cells (Trifo- zelandiae dominates short-tussock grasslands in dry inland lium repens and Actinidia chinensis Planchon, respectively). basins and reaches the alpine zone as a component of tall- tussock grasslands. It is widespread in eastern grasslands and Ecology of Endemic New Zealand Species of Festuca occurs on both the North and South islands (Fig. 3B). Fes- For most of the Tertiary, the NZ landmass was largely tuca actae is the exception to these widespread grassland low lying and covered with dense forest (Lee et al. 2001). taxa, being limited to Banks Peninsula, a volcanic area at Habitats for nonforest species would have been limited until the coastal margin of extensive alluvial plains (Fig. 3D). tectonic mountain building in the late Pliocene created ex- Here F. actae occupies coastal rocks and extends upslope tensive mountainous regions, providing widespread areas around rock outcrops on grassy hillsides. Festuca madida is where species of open habitats could persist (Raven 1973). a diminutive grass of wet places in alpine grasslands, and is Glacial activity in the Pleistocene would have resulted in the only species of endemic Festuca that is found on NZ’s VOLUME 23 New Zealand Festuca 417

Fig. 3A–D.—Distributions of the endemic taxa of Festuca on the main islands of New Zealand. Maps were created using data from a range of sources, including major NZ herbaria (AKL, CANU, CHR, OTA, WELT, WELTU), unpublished species lists, records from the literature, and personal observations by one of us (KML). Festuca coxii and F. contracta, which are restricted to two outlying islands, were not mapped.—A. Ⅵ ϭ F. luciarum, ᭝ ϭ F. multinodis, ⅜ ϭ F. ultramfica.—B. ᭝ ϭ F. novae-zelandiae.—C. F. matthewsii, ᭝ ϭ subsp. aquilonia, ⅜ ϭ subsp. matthewsii, ᭡ ϭ subsp. latifundii, Ⅺ ϭ subsp. pisamontis.—D. ᭝ ϭ F. actae, ᭡ ϭ F. deflexa, ⅜ ϭ F. madida (also on Campbell Island, not shown). 418 Lloyd, Hunter, Orlovich, Draffin, Stewart, and Lee ALISO main islands (Fig. 3D) as well as on a subantarctic island morphological variation apparent in geographically separat- (Campbell Island, 52Њ30ЈS; Connor 1998). ed populations of F. luciarum, F. multinodis, and the four subspecies of F. matthewsii (Fig. 3; Connor 1998) reflects Biogeography of Festuca in New Zealand the ongoing nature of this diversification. Molecular information from a wider sampling of the It has been suggested on the basis of ITS sequence data South American lineages would enable a stronger analysis that the grass family arose in what is present-day South of the origins of Festuca in NZ. The possibility of alternative America and that the ancestors of the core pooid grasses of dispersal routes to NZ through Melanesia and Australia Eurasia (tribes Aveneae, Bromeae, Poeae, and Triticeae) mi- should not be discounted, although Soreng (1990) suggested grated northward and diversified, with some lineages sub- that this is a minor pathway in Poa. Neither should the po- sequently dispersing back southward (Hsiao et al. 1999). The tential role of NZ as a source of dispersing taxa be over- restriction of presumably ancestral diploid taxa of Festuca looked, with recent molecular evidence indicating dispersal to the Mediterranean area (Catalań et al. 2004) supports a in several directions from NZ to other Pacific basin land- northern origin for lineages of Festuca in the Southern masses (Winkworth et al. 2002). Besides greater sampling Hemisphere, and a similar relationship is proposed for south- of South American taxa, future work should concentrate on ern Poa on the basis of chloroplast restriction site informa- taxa that occur on other southern landmasses, such as New tion (Soreng 1990). Guinea, Australia, and southern Africa. Our analyses have shown that the endemic NZ species of Festuca have affinities to taxa on the American continents. ACKNOWLEDGMENTS New Zealand shares many genera with South America and north temperate regions (Wardle et al. 2001), among the KML and WGL were funded by the Foundation for Re- monocots including Carex L., Eleocharis R. Br., Festuca, search, Science and Technology. The Miss E. L. Hellaby Juncus L., Poa, Puccinellia Parl., and Schoenoplectus Indigenous Grassland Trust also contributed to research ex- (Rchb.) Palla. The origin of Festuca in NZ is consistent with penses. We thank V. R. Clark (Botany Department, Univer- the movement of northern ancestors southward through the sity of Otago, NZ) for expert technical assistance, Richard Americas before reaching high latitudes in the Southern Pickering (Crop and Food Research, Lincoln, NZ), who pro- Hemisphere. This hypothesis would account for the relat- vided a chromosome count for Festuca madida, and Hugh edness of some NZ and South American Festuca spp. to the Wilson (Hinewai Reserve, Banks Peninsula, NZ) for detailed northern red fescue clade (Fig. 1, 2) and a potential close information on the distribution of F. actae. We are grateful relationship of F. californica to the ‘‘southern endemic’’ to herbarium curators for access to AKL, CANU, CHR, clade (Fig. 2). OTA, WELT, and WELTU, and for use of specimens from Once in southern temperate latitudes, the westerly wind CHR for DNA analysis. drift may have assisted movement of propagules to landmasses in the Southern Ocean and eventual dispersal to NZ. LITERATURE CITED Wardle et al. (2001) suggested that formerly vegetated Ant- arctica may have been an important stepping-stone that as- AIKEN, S. G., M. J. DALLWITZ, C. L. MCJANNET, AND L. L. CONSAUL. sisted transoceanic dispersal of plants to NZ. The subantarc- 1996 onwards. Festuca of North America: descriptions, illustra- tic islands lying to the south of the NZ landmass may also tions, identification, and information retrieval, vers. 12 Sep 2000. have assisted during these dispersal events. The presence of http://biodiversity.uno.edu/delta/ [Now at: http://delta-intkey.com/ Festuca madida on Campbell Island may represent a trace festuca/ Apr 2006.] of one such event. Festuca madida is well supported as a ALEXEEV, E. B. 1976. Austrofestuca (Tzvel.) E. Alexeev comb. member of the ‘‘southern endemic’’ clade, but has morpho- nov.—a new genus of the family Poaceae from Australia. Byull. logical differences (discussed above) from the remaining Moskovsk. Obshch. Isp. Prir., Otd. Biol. 81: 55–60. members of the clade. Its wide yet disjunct geographical BANCROFT, I. 2001. Duplicate and diverge: the evolution of plant genome microstructure. Trends Genet. 17: 89–93. distribution (Fig. 3D) suggests a longer presence in NZ than CAMPBELL, B. D., J. R. CARADUS, AND C. L. HUNT. 1999. Temper- the other endemic taxa, and could indicate an earlier dis- ature responses and nuclear DNA amounts of seven white clover persal event, in keeping with its basal position within the populations which differ in early spring growth rates. New Zea- clade in the ITS tree (Fig. 1). land J. Agric. Res. 42: 9–17. South America contains tetraploid, hexaploid, and octo- CARLSON, J. E., L. K. TULSIERAM, J. C. GLAUBITZ, V. M. K. LUK, ploid lineages of Festuca. The NZ taxa are hexaploid or AND C. R. R. KAUFFELDT. 1991. Segregation of random amplified octoploid, supporting the hypothesis that increased ploidy DNA markers in F1 progeny of conifers. Theor. Appl. Genet. 83: levels have helped Festuca lineages to colonize new areas 194–200. and diversify (Catalań et al. 2004). Rapid genomic change CATALA´ N, P., P. TORRECILLA, J. A. LO´ PEZ-RODR´ıGUEZ, AND R. G. OLM- has been shown to occur in synthetic polyploids (Song et al. STEAD. 2004. Phylogeny of the festucoid grasses of subtribe Lo- 1995), possibly because polyploidy can conserve novel ge- liinae and allies (Poeae, Pooideae) inferred from ITS and trnL–F sequences. Molec. Phylogen. Evol. 31: 517–541. nomic microstructural changes arising from unequal crossing CHARMET, G., C. RAVEL, AND F. BALFOURIER. 1997. Phylogenetic over during meiosis (Bancroft 2001). New genetic variation analyses in the Festuca-Lolium complex using molecular markers resulting from polyploid formation in Festuca may have re- and ITS rDNA. Theor. Appl. Genet. 94: 1038–1046. sulted in traits that allowed ancestral taxa to disperse from CONNOR, H. E. 1964. 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