ARTICLE IN PRESS

Organisms, Diversity & Evolution 5 (2005) 59–80 www.elsevier.de/ode

Origin and age of Australian Chenopodiaceae Gudrun Kadereita,Ã, DietrichGotzek b, Surrey Jacobsc, Helmut Freitagd aInstitut fu¨r Spezielle Botanik und Botanischer Garten, Johannes Gutenberg-Universita¨t Mainz, D-55099 Mainz, Germany bDepartment of Genetics, University of Georgia, Athens, GA 30602, USA cRoyal Botanic Gardens, Sydney, Australia dArbeitsgruppe Systematik und Morphologie der Pflanzen, Universita¨t Kassel, D-34109 Kassel, Germany

Received 20 May 2004; accepted 31 July 2004

Abstract

We studied the age, origins, and possible routes of colonization of the Australian Chenopodiaceae. Using a previously published rbcL phylogeny of the Amaranthaceae–Chenopodiaceae alliance (Kadereit et al. 2003) and new ITS phylogenies of the Camphorosmeae and Salicornieae, we conclude that Australia has been reached in at least nine independent colonization events: four in the Chenopodioideae, two in the Salicornieae, and one each in the Camphorosmeae, Suaedeae, and Salsoleae. Where feasible, we used molecular clock estimates to date the ages of the respective lineages. The two oldest lineages both belong to the Chenopodioideae (Scleroblitum and Chenopodium sect. Orthosporum/Dysphania) and date to 42.2–26.0 and 16.1–9.9 Mya, respectively. Most lineages (Australian Camphorosmeae, the Halosarcia lineage in the Salicornieae, Sarcocornia, Chenopodium subg. Chenopodium/Rhagodia, and Atriplex) arrived in Australia during the late Miocene to Pliocene when aridification and increasing salinity changed the landscape of many parts of the continent. The Australian Camphorosmeae and Salicornieae diversified rapidly after their arrival. The molecular-clock results clearly reject the hypothesis of an autochthonous stock of Chenopodiaceae dating back to Gondwanan times. Instead, they indicate that most lineages arrived in Australia via long-distance dispersal. Some lineages (e.g. the Halosarcia lineage) may have used the Indonesian archipelagos as stepping stones. The authors are aware that estimates of diversification times using a molecular clock can be subject to considerable levels of error. Our estimates of the age of Australian chenopod lineages based on three alternative fossils were made independently from any knowledge about shifts in climatic and geographical conditions in Australia during the times of arrival. In most cases, however, the paleoclimatic scenario indicates habitat shifts suitable for the respective chenopod colonizer, which corroborates our findings and provides a plausible scenario. r 2005 Elsevier GmbH. All rights reserved.

Keywords: Australia; Chenopodiaceae; Molecular clock; Migration; Long-distance dispersal; Temperate flora

Introduction

The chenopods of Australia

Chenopodiaceae are particularly successful in dry, ÃCorresponding author. saline or disturbed habitats of temperate and subtropical E-mail address: [email protected] (G. Kadereit). climates in the northern and southern hemisphere. The

1439-6092/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.ode.2004.07.002 ARTICLE IN PRESS 60 G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80

Table 1. Number of native Australian taxa and their distribution among suprageneric groups of Chenopodiaceae

Genera Species

Subfamily Tribe Nat. Intr. World Nat. Intr. World

Betoideae Beteae 0 1 6 0 1 16 Chenopodioideae Chenopodieae I–III* 5 11541 9 250 Atripliceae* 1 0 4 58 2 200 Corispermoideae Corispermeae 0 0 3 0 0 68 Salicornioideae Halopeplideae 0 0 5 0 0 10 Salicornieae 6 01036 0 70 Suaedoideae Bienertieae 0 0 1 0 0 1 Suaedeae 1 0 2 2 3 85 Salsoloideae Camphorosmeae 14 2 20 147 2 182 Salsoleae I–II* 1 0 30 1 0 360

Data on Australian genera and species are based on Wilson (1984), those on other continents critically compiled from different sources;the suprageneric classification refers to Kadereit et al. (2003). The redistribution of some genera from the traditional to putative new tribes (*) is still incomplete. Abbreviations: intr. — introduced; nat. — native. Numbers in boldface — particularly high diversity in Australia. family forms a close alliance withthemainly tropical Some genera of wider distribution are represented in Amaranthaceae. Molecular data used to resolve the Australia by endemic sections. relationship of both families so far are conflicting Australian members are unevenly distributed among (Kadereit et al. 2003; Pratt 2003; Mu¨ ller and Borschin the major chenopod lineages (Tables 1 and 2). Of the six press). Therefore, here we consider the subfamilies and subfamilies of Chenopodiaceae (Kadereit et al. 2003), tribes listed in Table 1 as part of Chenopodiaceae s. str. Betoideae and Corispermoideae are missing. Of the In this circumscription, the Chenopodiaceae comprise remaining subfamilies, tribes Halopeplideae (Salicor- about 1250 species in about 100 genera worldwide nioideae), Bienertieae (Suaedoideae), and Salsoleae II (Table 1), with the centre of diversity in the Old World (one of three major lineages of Salsoloideae, see desert belt from the Canary Islands to Central Asia, Kadereit et al. 2003) are absent as well, or they are where all tribes and nearly two-thirds (approximately represented very poorly suchas, for example, Suaedeae 60%) of the genera are present. The second centre is withtwo and Salsoleae I withone species only. In Australia, followed by NorthAmerica, SouthAmerica, contrast, Australia has more genera and species of and SouthAfrica ( Bunge 1880; Ulbrich1934 ; Ku¨ hnet Camphorosmeae and Salicornieae than any other al. 1993; Zhu 1996). continent or floristic region, and it also has a high In Australia, Chenopodiaceae find particularly fa- species diversity in Atripliceae and in several lineages of vourable conditions because more than three-fifths of Chenopodieae s.l. (Tables 1 and 2). In discussing older continental Australia has a mean annual rainfall of less views of a littoral connection to SE Asia that probably than 500 mm, and one-third less than 300 mm (Nix was used at least by the Salicornieae, Carolin (1981) 1981). Furthermore, salt-affected soils cover about a suggested that the Australian Chenopodiaceae origi- third of continental Australia (Northcote and Skene nated from an old autochthonous stock of Chenopo- 1972). In addition, saline soils are associated with diaceae dating back to Gondwana connections. In estuarine plains, coastal lagoons, and salt pans. Species contrast, Burbidge (1960) raised the hypothesis that of Chenopodiaceae are common and sometimes domi- the elements of the arid zone flora with links to the nant in the vast semidesert areas of the Eremaean northern hemisphere, such as Chenopodiaceae, Amar- province and the interzones (Burbidge 1960), in various anthaceae, Asteraceae, Convolvulaceae, and Brassica- vegetation types; especially in woodland, shrubland, and ceae, radiated from ancestors witha cosmopolitan grassland ecosystems as well as in coastal and inland salt distribution on Cretaceous coastlines. Both hypotheses, marshes (Beadle 1981; Specht 1981, and references however, are not compatible with what is known about therein). Australia’s flora includes 32 genera with 302 the age of the major angiosperm orders (Magallo´ n et al. species of Chenopodiaceae, of which 279 are endemic 1999; Magallo´ n and Sanderson 2001; Wikstro¨ m et al. (Wilson 1984; Tables 1 and 2). The four genera Beta, 2001). The two oldest fossils known for Caryophyllales Cycloloma, Monolepis, and Bassia, withone species date back to the Late Cretaceous. These are seeds found each, are introduced. Of the remaining 27 genera that in southern Sweden (Collinson et al. 1993), and pollen include native species (or 28, if Salsola is counted as found in Canada (Srivastava 1969). Chenopodiaceae, as native, see the discussion), 20 are endemic to Australia. part of the ‘core Caryophyllales’, originated and ARTICLE IN PRESS G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80 61

Table 2. Genera of Chenopodiaceae native to Australia (data from Wilson 1984)

Tribe Genus Species in Species Distribution, for maps see Wilson (1984) Australia worldwide

Atripliceae Atriplex 60; 57a,2b 200 Throughout Au except the tropical zone; one sp. in NZ Camphorosmeae Didymanthus 1a 1 Inland SW Au ( ¼ Sclerolaeneae) Dissocarpus 4a 4 W and C Au Enchylaena 2a 2 E. tomentosa widespread incl. the tropical zone, E. lanata in inland SW Au Eremophea 2a 2 W and western C Au Eriochiton 1a 1 Widespread in W, S and southern C Au Maireana 57a 57 Throughout Au, but mainly C Au, only M. georgei in the tropical zone Malacocera 4a 4 inland S and southeastern C Au Neobassia 2a 2 N. astrocarpa in NW Au, N. proceriflora in eastern C Au Osteocarpum 5a 5 W, C and inland SE Au Roycea 3a 3 Inland SW Au Sclerochlamys 1a 1 C and inland SE Au Sclerolaena 62a 62 Throughout arid Au, a few species entering the tropical zone, e.g. S. glabra Stelligera 1a 1 Inland E and SE Au Threlkeldia 2a 2 T. diffusa in coastal W and S Au, T. inchoata in C Au Chenopodieae Chenopodium 23; 14a,8b 140 Throughout Au but rare in the tropical zone; one sp. in NZ Dysphania 10a 10 C and N Au Einadia 6; 4a 6 E and C Au; two spp. in NZ Rhagodia 11a 11 Throughout Au except the tropical zone Scleroblitum 1a 1 From S Queensland to NW Victoria and eastern S Au Salicornieae Halosarcia 23; 22a 23 Throughout Au, mainly coastal (incl. the tropical zone) and C Au; H. indica subsp. indica is widespread on paleotropical coasts Pachycornia 1a 1 C, SW and SE Au Sarcocornia 3; 2a 16 Coasts of W, S, E Au, tropical NE Au; one sp. in NZ and NC Sclerostegia 5a 5 C, SW, S and SE Au Tecticornia 3; 2a 3 Coastal N Au and inland W Au; one sp. in NG Tegicornia 1a 1 Inland SW Au Salsoleae Salsola 1, partly intr. 100 Throughout Australia Suaedeae Suaeda 5; 1a,3b 85 C Au and coasts including the tropical zone; one sp. on SE Asian coasts

Abbreviations: Au — Australia, NC — New Caledonia, NG — New Guinea, NZ —New Zealand. aEndemic. bIntroduced. diversified after the break-up of Gondwana (Kadereit dating of the Australian Chenopodiaceae beyond et al. 2003). Furthermore, the patchy distribution of estimates of relative age in coherent groups was Australian genera among the higher taxa of Chenopo- hampered by the lack of distinct Australian fossils. diaceae, and the fact that they are nested within larger The possibly oldest record is chenopod-like pollen clades of wider distribution, indicate that the Australian (Chenopodipollis chenopodiaceoides or Polyporina chenopod flora has evolved from different ancestors that chenopodiaceoides) found in Southeast Australia and have arrived either via long-distance dispersal or by dating to the Oligocene (Partridge 1971, cited in migration through Southeast Asia. The diversification of Macphail et al. 1994). However, an identical pollen the different chenopodiaceous immigrants after their type was found in the anthers of the recently discovered arrival certainly was dependent on several factors enigmatic Caryophyllacean taxon Caryophylloflora pa- such as time, the inherent genetic variability, and leogenica from the Eocene of Tasmania (Jordan and the availability of suitable habitats. Until now, any Macphail 2003). ARTICLE IN PRESS 62 G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80

Climatic and geographical history and time also shows a large herbaceous element implying the biogeographical relationships of Australia presence of open vegetation types. There are macro- fossils that indicate the presence of heath-like commu- The adaptation of species of Chenopodiaceae to dry nities from the mid-Miocene (about 12–15 Mya). From and saline conditions made them obvious candidates to the mid-Miocene onwards the continent again started to colonize the vast semi-arid and arid Australian land- become more arid, and open sclerophyll woodland and scapes. To understand when Chenopodiaceae had the grassland became dominant. The full development of opportunity to enter Australia and where they probably the West Antarctic ice cap led to an extreme lowering of came from, we outline the geographical and climatic the sea level in the late Miocene (6 Mya), and a drastic history of the continent, possible migration routes and decrease of precipitation caused drying out of many ‘stepping stones’, and the biogeographical relationships water bodies worldwide. Repeated drying and refilling of the extant flora as it is currently understood. during this time resulted in vast amounts of salt This summary of the geographical and climatic precipitation offering suitable conditions for salt- history has been taken mostly from Greenwood tolerant Chenopodiaceae. During the Pliocene (1994), Quilty (1994), Frakes (1999), Hill et al. (1999), (5.3–2.4 Mya), increasing climatic fluctuations caused and references in these. When Australia started to repeated contractions and expansions of vegetation separate from Antarctica (96–83 Mya) and moved types and a severe selection of species. Except for northwards, its climate, landscapes, and vegetation comparatively small areas in SW, E and S Australia, changed. The mild climate of the Palaeocene favoured closed forests had disappeared. After the formation of temperate forests withgymnosperms and probably some the North Polar ice cap (2.4 Mya), the alternation of Nothofagus forming the canopy, and a diverse under- dry, cool and windy glacials withwarmer and less dry storey of angiosperm shrubs. Further warming during interglacials started and formed the vegetation patterns the early Eocene resulted in the replacement of the found today. temperate forests by tropical rainforests. The middle According to current knowledge of the break-up and late Eocene are characterized by a decrease of history of Gondwana, migration between Australia and temperature while precipitation remained rather high, as Antarctica was possible until the end of the Cretaceous can be concluded from leaf physiognomy of macro- (McLoughlin 2001). Then, from about 96 Mya onwards, fossils suggesting the expansion of mesothermal (Notho- south-western Australia and East Antarctica were fagus) forests. The palynological record, however, shows separated (Veevers and Li 1991). Rifting and sea floor strong regional differences, increased habitat diversity, spreading went on eastwards, and by the early Cenozoic and an extraordinary diversity in certain taxa, e.g. (approximately 65–55 Mya) a shallow seaway existed Acacia and Proteaceae. The aridification of Australia between Australia and Antarctica (Lawver and Gaha- started at the Eocene/Oligocene boundary (approxi- gan 1998). Complete separation of the continents took mately 37 Mya), when the glaciation of Antarctica place about 35.5 Mya ago (Veevers et al. 1991). After began to change the temperature and currents of the that time Australia was isolated from other land masses oceans around Australia. That resulted in a sudden and completely surrounded by oceans. Collisions of the decrease of rainfall, seasonal aridity, and lower tem- northern edge of Australia with the Philippines Sea Plate peratures. The remnants of Gondwanan rainforests started about 25 Mya ago. Later (20–5 Mya), parts of started to retreat into their current refugia in north- the northern Australian continental margin split off, and eastern Queensland, New Guinea, and New Caledonia. some fragments moved westwards as far as Sulawesi They were replaced by seasonal Nothofagus-dominated (Hall 1996). The separation of terrestrial environments forests with sclerophyllous and microphyllous taxa. between the Sunda shelf and Australia (today approxi- Open vegetation types like woodlands, bushlands or mately 1000 km) was significantly reduced by the grassland also became dominant. The oldest possible presence of intervening island arcs that might have chenopod-like pollen date back to this period (Macphail served as stepping stones. Therefore, migration barriers et al. 1994). probably were relatively small; especially in times of low The Miocene (23.7–5.3 Mya) was a period of strong sea levels. During periods of eustatic sealevel lowstands climatic fluctuations exerting strong selection pressure during the Quaternary, the marine separation of SE on all organisms. By the early Miocene (approximately Asia and Australia was reduced to approximately 23 Mya) the northern edge of Australia (today’s New 150 km (McLoughlin 2001), which, however, is still five Guinea) had reached 201S latitude. The temperature times as long as the shortest distance across the English rose, the sea level was significantly higher and, at least in Channel. the South, there was higher rainfall. In this time, many There is evidence that parts of Burma, Thailand, of the palaeobasins and drained valleys were filled with Malaya, and Sumatra also were part of the Australian large freshwater lakes, rivers, and swamps. Temperate Plate. They separated very early in the break-up history rainforests were widespread, but the fossil record of this of Gondwana and probably formed a trail of scattered ARTICLE IN PRESS G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80 63 connecting islands between Asia and Australia dating Material and methods from about 60 Mya (Audley-Charles 1987; Audley- Charles et al. 1988). They also might have served as The rbcL-tree presented in Fig. 1 was taken from stepping stones or intermediate stops for taxa that Kadereit et al. (2003), a study that concentrated on the finally reached Australia. systematics of Chenopodiaceae and Amaranthaceae and The geographical isolation from other continents and the evolution of C4 photosynthesis in these two families. the climatic history of Australia resulted in an extra- Only the rbcL sequences of Microcnemum coralloides ordinarily high degree of endemism (80% for all species, and Kochia densiflora were added and included in the 92% for Chenopodiaceae). The Australian flora has molecular clock analyses. The ITS sequences of Cam- strong affinities to neighbouring islands, e.g. New phorosmeae (40) and Salicornioideae (27) were newly Guinea, New Caledonia, New Zealand, and moderate generated in the present study. See Table 3 for GenBank relations to southern South America and southern accession numbers and information on vouchers. Africa. However, there are also many temperate and tropical elements in the Australian flora that have their closest relatives in Eurasia or Africa (Crisp et al. 1999). Many temperate taxa, suchas Poa, Agrostis, Festuca, Generating rbcL and ITS sequences Euphrasia, Scleranthus, Myosotis, Ranunculus, and Cardamine, and the Australian chenopod lineages show Leaf samples were either acquired as herbarium-, a disjunction between temperate areas of the northern silica-dried or freshmaterial, or were preserved in and southern hemispheres and Australia. Recent mole- saturated NaCl-CTAB solution (Rogstad 1992) supple- cular studies show that the dispersal routes of these taxa mented with200 mM sodium ascorbate ( Thomson are complex and not bound to one-way tracks to 2002). The latter method yielded very good quality Australia or New Zealand (Winkworthet al. 2002b , and and quantity of DNA, especially for succulent taxa. references therein). Extraction of total genomic DNA was performed by using NucleoSpin plant DNA extraction kits (Macher- ey-Nagel; Du¨ ren, Germany) following the manufac- Aims of the present study turer’s specifications. Standard PCR amplification was performed using This study deals with the origin and age of Australian standard reaction mixes (e.g. Kadereit et al. 2003), Chenopodiaceae. The phylogeny proposed by Kadereit including 4% DMSO and approximately1 ng/ml DNA, et al. (2003), based on gene sequence variation in the witha Grant Autogene II-Thermocycler programmed as chloroplast ribulosebisphosphate carboxylase large sub- follows: pre-treatment of 60 s at 94 1C, followed by 35 unit (rbcL), is used to obtain an overview of the origins, cycles of 18 s at 94 1C, 30 s at 55 1C, 60 s at 72 1C, and a age, and diversification of the chenopodiaceous immi- post-treatment of 78 s at 55 1C and 8 min at 72 1C. grants to Australia. That study included 18 Australian Amplification products were checked on 0.8% agarose species representing 15 genera of Chenopodioideae, gels. PCR products in most cases were purified directly Salicornieae, and Camphorosmeae, and 56 non-Austra- with the PCR product purification kit of Macherey- lian species covering all larger groups and most of the Nagel. In cases where the test gel showed a smear, total distribution areas. PCR products were gel-purified using Macherey-Nagel Additional ITS analyses were carried out to increase or QiaGen gel extraction kits. The following primers resolution in the Camphorosmeae and Salicornieae. In were used to amplify the nuclear internally transcribed terms of endemic genera, they are the two most diverse spacer (ITS): ITS-28S (forward): 50-CCG CTT ATT groups of Chenopodiaceae in Australia (Table 1). In KAT ATG CTT AAA-30, ITS-18S (reverse): 50-GTC Camphorosmeae the present study includes 23 Holarctic CAC TGA ACC TTA TCA TTT AGA GG-30. For the and 17 Australian species, covering the entire geogra- rbcL primers compare Kadereit et al. (2003). phical range of Camphorosmeae. In the ITS analysis of Purified, double-stranded PCR products were se- Salicornieae, 20 non-Australian species and seven quenced directly, using the ABI Prism Dye Terminator Australian species are included. Cycle Sequencing Ready Reaction Kit (Perkin Elmer). A molecular clock approachis used to estimate the Five-fold diluted PCR primers were used as sequencing time of arrival of the colonizers that gave rise to the primers. For cycle sequencing, thermocyclers were different Australian lineages. The results will be programmed as follows: pre-heating for 60 s at 96 1C, discussed withrespect to current knowledge of the 27 cycles of 6 s at 96 1C, 12 s at 55 1C, 4 min at 60 1C, and hypothetical climatic and geographical conditions in post-treatment of 18 s at 51.4 1C, 4 min at 60 1C. Australia at times of arrival and afterwards, because Extension products were purified withethanol/sodium these conditions may have had a strong impact on the acetate and electrophoresed on ABI 373 or 377 diversification and expansion of the new lineages. automated sequencers. ARTICLE IN PRESS 64 G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80

Chenopodium desertorum Chenopodium auricomum 78 Rhagodia drummondii 99 Chenopodium sanctae-clarae Chenopodium acuminatum 69 62 Chenopodium frutescens Chenopodieae I Microgynoeceum tibeticum Atriplex coriacea Atriplex rosea 97 75 92 Atriplex spongiosa Atriplex undulata 96 Atriplex halimus Atripliceae 59 Atriplex patula Chenopodioideae 72 Holmbergia tweedii Halimione pedunculata Scleroblitum atriplicinum 82 Chenopodium foliosum Monolepis nuttaliana Chenopodieae II 95 Spinacia oleracea Chenopodium bonus-henricus Chenopodium cristatum 73 Dysphania glomulifera 95 Chenopodium botrys Teloxys aristata Chenopodieae III Axyris prostrata 72 Krascheninnikovia ceratoides Pachycornia triandra Halocnemum strobilaceum 51 Halosarcia indica 56 Salicornia dolichostachya Halosarcia peltata Tecticornia australsasica Salicornieae Arthrocnemum macrostachyum Salicornioideae Sarcocornia utahensis 78 Sarcocornia blackiana 55 Halopeplis amplexicaulis Kalidium caspium Halopeplideae 75 Allenrolfea occidentalis Bienertia cycloptera Bienertieae 54 Borszczowia aralocaspica 80 Suaeda altissima Suaeda crassifolia Suaedeae Suaedoideae 100 Suaeda maritima Bassia sedoides 96 Camphorosma monspeliaca 67 Chenoleoides tomentosa 98 93 Kochia prostrata Camphorosmeae 76 Panderia pilosa 97 Kochia americana Maireana brevifolia 58 Sclerolaena obliquicuspis (Sclerolaeneae) 80 58 Dissocarpus paradoxus Bassia dasyphylla 88 Climacoptera crassa 98 Petrosimonia nigdensis 64 Salsola canescens Salsoleae II Salsoloideae 91 Salsola vermiculata Girgensohnia oppositiflora Noaea mucronata 89 74 Ofaiston monandrum 52 Raphidophyton regelii 59 Salsola laricifolia Salsoleae I Sympegma regelii Salsola kali Salsola genistoides Agriophyllum squarrosum 94 Anthochlamys multinervis Corispermeae Corispermoideae 94 Corispermum filifolium 94 Betoideae Amaranthaceae (incl. Polycnemoideae) Achatocarpaceae Fig. 1. Strict consensus of a rbcL-analysis of Chenopodiaceae taken from the results of a larger analysis that included also Amaranthaceae and representatives from other families of the Caryophyllales (Kadereit et al. 2003); Australian lineages in grey boxes; branchsupport (bootstrap) below branches.

Alignment Camphorosmeae data matrix, the alignment was un- ambiguous. For Camphorosmeae, six informative indels Forward and reverse sequences were compared and were coded in a 0/1 matrix, and Petrosimonia nigdensis, edited, and consensus sequences initially aligned using a member of the Salsoleae, was chosen as outgroup SequencherTM 4.1. The alignment of the ITS sequences based on the phylogenetic realtionships proposed by was difficult due to a number of indels and relatively Kadereit et al. (2003). In the Salicornieae analysis high variability in some parts. However, after manual Kalidium foliatum, a member of the Halopeplideae, correction and exclusion of five nucleotides in the was chosen as outgroup according to the results of a ARTICLE IN PRESS G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80 65

Table 3. Taxa sampled (tribal classification after Kadereit et al. 2003), vouchers, and GenBank accession numbers for gene sequences generated in this study (r ¼ rbcL, all others ¼ ITS); for rbcL sequences taken from GenBank Abbreviations: herbarium acronyms according to Index Herbariorum; Australian regions: NFWP—North Far Western Plains, NSW—New SouthWales, SFWP—SouthFar Western Plains.

Species DNA source: voucher (herbarium/garden); GenBank acc. No. field origin

Camphorosmeae Bassia dasyphylla (Fisch. & C.A. Mey.) Kuntze G. & S. Miehe 96-203-02 (KAS, hb. Miehe,); AY489195 Mongolia: Gobi Altai Bassia hirsuta (L.) Asch. H. Freitag 28.077 (KAS); NW Kasakhstan: AY489196 S of Ural0sk Bassia hyssopifolia (Pall.) Volkens H. Freitag 30.046 (KAS); Uzbekistan: near Tashkent AY489197 Bassia muricata (L.) Asch. M. Al-Dosari 1664 (KTUH, KAS); Kuwait AY489198 Bassia sedoides (Pall.) Asch. H. Freitag 28.035 (KAS); NW Kasakhstan: AY489199 S of Ural0sk Camphorosma monspeliaca L. H. Freitag 28.133 (KAS); NW Kasakhstan: AY489200 W of Gur0yev (Atyrau) Camphorosma songorica Pall. H. Freitag 28.034 (KAS); NW Kasakhstan: AY489201 SW of Ural0sk Chenolea diffusa Thunb. L. Mucina 6914/1 (MJG; KAS); South Africa: AY489202 Western Cape Chenoleoides arabica (Boiss.) Botsch. H. Freitag 15.657 (KAS); Palestine: near Jericho AY489203 Chenoleoides tomentosa (Lowe) Botsch. H. Freitag 27.256 (KAS); Canary Islands: Lanzarote AY489204 Dissocarpus biflorus F. Muell. var. biflorus S. Jacobs 8726 (NSW; Australia: NSW, NFWP, Sturt AY489205 N. P. Dissocarpus paradoxus (R. Br.) Ulbr. S. Jacobs 8712 (NSW); Australia: NSW, SFWP AY489206 Enchylaena tomentosa R. Br. N. Schmalz 214 (MJG); Australia: Northern AY489207 Territory, Uluru N. P. Eriochiton sclerolaenoides (F. Muell.) F. Muell. Ex S. Jacobs 8698 (NSW); Australia: NSW, SFWP, AY489208 A.J. Scott W of Balranald Kirilowia eriantha Bunge K.H. Rechinger 17.705 (W); Afghanistan: Ghazni AY489209 prov , near Okak Kochia americana Wats. S.-W. Breckle 2756 (KAS); USA: Utah, Box Boulder AY489210 Co. Kochia indica Wight H. Freitag 19.623 (KAS); Egypt: Fayum oasis AY489211 Kochia densiflora (Moq.) Aellen H. Freitag 28.321 (KAS); Russia: Saratov prov., AY489212; near Pugachev AY489264 r Kochia krylovii Litv. A. Ebel 18.08.2001 (KAS, hb. Ebel); Mongolia: AY489213 Kobdo prov. Kochia laniflora (S.G. Gmel.) Borb. H. Freitag 28.310 (KAS); Russia: Saratov prov., AY489214 Krasnyi Kut Kochia melanoptera Bunge M. Lomonosova 175/2001 (NS, KAS);E Kazakhstan: AY489215 Zaisan distr. Kochia prostrata (L.) Schrad. H. Freitag 28.254 (KAS); SE Russia: near Volgograd AY489216 Kochia saxicola Guss. Cult. in Kassel (KAS); Italy: Capri AY489217 Kochia scoparia (L.) Schrad. H. Freitag 45/00 (KAS); Uzbekistan: near Tashkent AY489218 Kochia stellaris Moq. H. Freitag 18.911 (KAS); Pakistan: Baluchistan AY489219 prov., Kalat distr. Londesia eriantha Fisch. & C.A. Mey. ( ¼ Bassia K.T. Mathew 2794 (KTUH, KAS); Kuwait AY489220 eriophora (Schrad.) Asch.) Maireana integra (Paul G. Wilson) Paul G. Wilson N. Schmalz 213 (MJG); Australia: Northern AY489221 Territory, Uluru N.P. Maireana erioclada (Benth.) Paul G. Wilson S. Jacobs 8699 (NSW); Australia AY489222 Maireana polypterygia (Diels) Paul G. Wilson. S. Jacobs IE 1801 (NSW); Australia AY489223 Malacocera tricornis (Benth.) R. Anderson S. Jacobs 8706 (NSW); Australia: NSW, SFWP, AY489224 near Wentworth Neobassia proceriflora (F. Muell.) A.J. Scott S. Jacobs 8721 (NSW); Australia: NSW, NFWP, AY489225 ‘Fowlers gap’ ARTICLE IN PRESS 66 G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80

Table 3. (continued )

Species DNA source: voucher (herbarium/garden); GenBank acc. No. field origin

Osteocarpum dipterocarpum (F. Muell.) Volkens S. Jacobs 8731 (NSW); Australia: NSW, NFWP, AY489226 near Tibooburra Panderia pilosa Fisch. & C.A. Mey. H. Freitag & G. Kothe 18.894 (KAS); Pakistan: AY489227 Baluchistan prov., Kalat,. Roycea spinescens C. Gardner L.W. Sage 2385 (PERTH); Australia AY489228 Sclerolaena bicornis Lindley var. bicornis S. Jacobs 8723 (NSW); Australia: NSW, NFWP, AY489229 ‘Fowlers gap’ Sclerolaena cornishiana (F. Muell.) A.J. Scott N. Schmalz 227 MJG); Australia: Northern AY489230 Territory, Kings Canyon Resort Sclerolaena diacantha (Nees) Benth. N. Schmalz 239 (MJG); Australia: Northern AY489231 Territory, Kulgera Sclerolaena longicuspis (F. Muell.) A.J. Scott S. Jacobs 8727 (NSW); Australia: NSW, NFWP, AY489232 Sturt N.P. Sclerolaena obliquicuspsis (R. Anderson) Ulbr. N. Schmalz 85 (MJG); Australia: W Australia, AY489233 Norseman Stelligera endecaspinis A.J. Scott S. Jacobs 8733 (NSW); Australia: NSW, NWP, AY489234 Ford0s bridge Halopeplideae Allenrolfea occidentalis Kuntze M. Piep & S. Long 120 (UTC, KAS); USA: Utah AY489236 Halopeplis amplexicaulis Ung.-Sternb.ex Ces., G. Kadereit et al. 2002/14 (MJG); Spain: Arago´ n, AY489237 Passer.& Gibelli Laguna de Guallar Kalidium foliatum (Pall.) Moq. H. Kuerschner & M. Sonnentag 06.650 (BSB, KAS); AY489238 China: Nei Menggu, Gashun Nor Salicornieae Arthrocnemum macrostachyum (Moric.) K. KochG. Kadereit 2002/7 (MJG); Spain: Tarragona, delta AY489239 del Ebro Arthrocnemum macrostachyum (Moric.) K. KochH. Freitag & N. Adıgu ¨ zel 28.846 (KAS, GAZI); AY489240 Turkey: Seyhan prov., SE Adana Halocnemum strobilaceum (Pall.) M. Bieb. H. Freitag & N. Adıgu¨ zel 28.783, (KAS, GAZI) ; AY489241 Turkey: near Konya Halosarcia indica (Willd.) Paul G. Wilson subsp. M.A. Khan & B. Grul s.n. 10.03.2000 (KUH, KAS); AY489242 indica Pakistan: Baluchistan, Gadani Halosarcia indica (Willd.) Paul G. Wilson subsp. S. Jacobs 8691 (NSW); Australia AY489243 julacea Paul G. Wilson Halosarcia peltata Paul G. Wilson N. Schmalz 184 (MJG); Australia: Western Australia, AY489261 Lake King Halosarcia pergranulata (J. Black) Paul G. Wilson S. Jacobs 8692 (NSW); Australia AY489244 subsp. queenslandica Paul G. Wilson Microcnemum coralloides (Loscos & Parda) Buen G. Kadereit 2002/15 (MJG); Spain: Arago´ n, Laguna AY489245; de Guallar AY489263 r Pachycornia triandra (F. Muell.) J. Black S. Jacobs 8702 (NSW); Australia: NSW, SFWP AY489246 Salicornia europaea L. G. Kadereit 2002/1(MJG); France: Camargue, le AY489247 Crau du Roi Salicornia meyeriana Moss J. Walter & L. Mucina 5050 (MJG); SouthAfrica: AY489248 Western Cape prov. Salicornia perennans Willd. B. Neuffer & H. Hurka 13624 (KAS); Russia: Altai AY489249 Republic Salicornia procumbens Sm. E. Lambracht s.n., May 2002 (MJG, in alcohol); AY489250 Italy: Prov. Venezia, Belvedere Salicornia aff. ramosissima A.-M. Csergo s.n., Nires s.n. 2003 (MJG, in alcohol); AY489251 Romania: northern Transsylvania Salicornia ramosissima J. Woods G. Kadereit s.n. 2001 (MJG, in alcohol); Germany: AY489252 Thuringia, Artern Sarcocornia capensis (Moss) A.J. Scott L. Mucina 7202/2 (MJG, hb. Mucina); South Africa: AY489253 Western Cape prov. Salicornia decumbens (Tolken) A.J. Scott L. Mucina 7205/1 (MJG, hb. Mucina); South Africa: AY489254 Western Cape prov., ARTICLE IN PRESS G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80 67

Table 3. (continued )

Species DNA source: voucher (herbarium/garden); GenBank acc. No. field origin

Sarcocornia fruticosa (L.) A.J. Scott s.l. E. Garcı´ a, St. Beck & R. de Michel 600 (LPB, KAS); AY489255 (-aff. Salicornia peruviana Kunth) Bolivia: Avaroa prov., Oruro, Sarcocornia fruticosa (L.) A.J. Scott G. Kadereit 2002/20 (MJG); Spain: Cantabria, Noja AY489256 Sarcocornia littorea (Moss) A.J. Scott L. Mucina 7209/1 (MJG, hb. Mucina); South Africa: AY489257 Western Cape prov. Sarcocornia perennis (Miller) A.J. Scott G. Kadereit 2002/18 (MJG); Spain: Cantabria, Noja AY489258 Sarcocornia quinqueflora (Bunge ex Ung.-Sternb.) N. Schmalz 32 (MJG); Australia: NSW, Karuah AY489259 A.J. Scott Sarcocornia utahensis (Tidestr.) A.J. Scott D. Pratt 196 (ISC); USA: Utah AY489260 Tecticornia australasica (Moq.) Paul G. Wilson S. Jacobs 8685 (NSW); Australia: Queensland, N of AY489262 Townsville Salsoleae Petrosimonia nigdensis Aellen H. Freitag & N. Adıgu¨ zel 28.730 (KAS, GAZI); AY489194 Turkey: Eskis-ehir prov., SW Polatlı

Previously published rbcL sequences used — Kadereit et al. 2003: Acroglochin chenopodioides Schrad. AY270049, Agriophyllum squarrosum (L.) Moq. AY270051, Allenrolfea occidentalis Kuntze AY270052, Anthochlamys multinervis Rech.f. AY270056, Aphanisma blitoides Nutt. ex Moq. AY270057, Arthrocnemum macrostachyum (Moric.) K. Koch AY270058, Atriplex coriacea Forssk. AY270045, Atriplex halimus L. AY270059, Atriplex spongiosa F. Muell. AY270060, Atriplex undulata (Moq.) D. Dietr. AY270061, Axyris prostrata L. AY270062, Bassia dasyphylla (Fisch. & C.A. Mey.) Kuntze AY270150, Bassia sedoides (Pall.) Asch. AY270063, Beta nana Boiss. & Heldr. AY270064, Beta vulgaris L. subsp. maritima Thell. AY270065, Bienertia cycloptera Bunge AY270066, Borszczowia aralocaspica Bunge AY270068, Camphorosma monspeliaca L. AY270071, Chenoleoides tomentosa (Lowe) Botsch. AY270076, Chenopodium acuminatum Willd. AY270077, Chenopodium auricomum Lindley AY270078, Chenopodium bonus-henricus L. AY270079, Chenopodium botrys L. AY270080, Chenopodium cristatum (F. Muell.) F. Muell. AY270046, Chenopodium desertorum (J. Black) J. Black ssp. anidiophyllum (Aellen) Paul. G. Wilson AY270042, Chenopodium foliosum Asch. AY270081, Chenopodium frutescens C.A. Mey. AY270082, Chenopodium sanctae-clarae Johow AY270043, Climacoptera crassa (M.Bieb.) Botsch. AY270083, Corispermum filifolium C. A. Mey. AY270084, Dissocarpus paradoxus (R. Br.) Ulbr. AY270151, Dysphania glomulifera (Nees) Paul G.Wilson AY270086, Girgensohnia oppositiflora (Pall.) Fenzl AY270087, Hablitzia tamoides M. Bieb. AY270092, Halimione pedunculata (L.) Aellen AY270093, Halocnemum strobilaceum (Pall.) M. Bieb. AY270094, Halopeplis amplexicaulis Ung.-Sternb.ex Ces., Passer. & Gibelli AY270095, Halosarcia indica (Willd.) Paul G. Wilson AY270096, Hemichroa diandra R. Br. AY270098, Holmbergia tweedii Speg. AY270100, Kalidium caspicum Ung.-Sternb. AY270102, Kochia americana Wats. AY270103, Kochia prostrata (L.) Schrad. AY270104, Krascheninnikovia ceratoides (L.) Gueldenst. AY270105, Maireana brevifolia (R. Br.) Paul G. Wilson AY270106, Microgynoecium tibeticum Hook. f. AY270107, Monolepis nuttaliana Greene AY270108, Nitrophila occidentalis S. Wats. AY270109, Noaea mucronata (Forssk.) Asch. & Schweinf. AY270110, Ofaiston monandrum (Pall.) Moq. AY270112, Oreobliton thesioides Durieu & Moq. AY270113, Pachycornia triandra (F. Muell.) J. Black AY270047, Panderia pilosa Fisch. & C.A. Mey. AY270114, Petrosimonia nigdensis Aellen AY270116, Polycnemum majus A. Braun AY270118, Polycnemum perenne Litv. AY270119, Raphidophyton regelii (Bunge) Iljin AY270123, Rhagodia drummondii Moq. AY270124, Salicornia dolichostachya Moss AY270125, Salsola canescens (Moq.) Spach AY270127, Salsola genistoides Juss.ex Poir. AY270128, Salsola kali L. AY270129, Salsola laricifolia Litv. ex Drobov AY270130, Salsola vermiculata L. AY270131, Sarcocornia blackiana (Ulbr.) A. J. Scott AY270149 (324 bp missing), Sarcocornia utahensis (Tidestr.) A.J. Scott AY270126, Scleroblitum atriplicinum (F. Muell.) Ulbr. AY270044, Sclerolaena obliquicuspis (R. Anders.) Ulbr. AY270132, Halosarcia peltata Paul G. Wilson AY270133, Suaeda altissima (L.) Pall. AY270135, Suaeda crassifolia Pall. AY270136, Suaeda maritima (‘‘macrocarpa’’) (L.) Dumort. AY270137, Sympegma regelii Bunge AY270138, Tecticornia australasica (Moq.) Paul G. Wilson AY270139, Teloxys aristata (L.) Moq. AY270140; Hudson et al. 1990: Atriplex patula L. X15925, Atriplex rosea L.; Zurawski et al. 1981: Spinacia oleracea L. more detailed molecular analysis of the Salicornioideae assessed using 1000 bootstrap replicates withTBR (G. Kadereit, unpublished data). swapping, random addition of taxa, and witha limit of 1000 trees kept in eachreplicate. The appropriate model of DNA substitution for the Maximum parsimony (MP) and maximum inference of phylogenetic relationships under ML was likelihood (ML) analyses estimated using Modeltest 3.06 (Posada and Crandall 1998). For Camphorosmeae, the GTR+G+I model The complete matrix was executed in PAUP* 4.0b10 was chosen with gamma distribution set to 0.6511. Base (Swofford 2002) and analysed under MP and ML frequencies were set to A ¼ 0:2775; C ¼ 0:2445; G ¼ criteria. MP analysis was performed using heuristic 0:2203; and T ¼ 0:2577: The rate matrix was set to AC searches with 100 replicates of random taxon addition 1.0, AG 5.33, AT 1.0, CG 1.0, CT 2.45, and GT 1.0. For and tree-bisection-reconnection (TBR) branchswap- Salicornieae, the GTR+G+I model was chosen with ping. For Salicornieae, bootstrap was performed under gamma distribution set to 0.932. Estimated base the same conditions as the heuristic search with 100 frequencies were set to A ¼ 0:2247; C ¼ 0:2953; G ¼ replicates. For Camphorosmeae, node support was 0:2672; and T ¼ 0:2128: The rate matrix was set to AC ARTICLE IN PRESS 68 G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80

1.0, AG 2.38, AT 1.0, CG 1.0, CT 5.67, and GT 1.0. The sated by excellent preservation of the vegetative organs. heuristic search settings were stepwise random addition The second group of fossils (fossil 2 in Table 4) are of taxa and TBR swapping. 0.7 mm long Chenopodium-like seeds from southern Germany dated to the lower Miocene (23.3–16.0 Mya). These resemble seeds of members of subg. Chenopodium. Calibration and application of a molecular clock However, these seeds cannot be assigned to any of the terminal clades, but most likely represent the crown For the rbcL clock, non-synonymous substitutions group of Chenopodieae I (position of the calibrated were excluded from all subsequent analyses because they node see Fig. 4A). Fossil 3b (Table 4) is the oldest record are likely to be non-clocklike (e.g. Xiang et al. 2000). To of pollen belonging to the Chenopodiaceae–Amarantha- this end, the nucleotide sequence was translated into an ceae alliance, and was found in Canada. The pantopo- amino acid sequence, and non-synonymous substitu- rate pollen grains withmore than40 pores per grain tions were identified using MacClade 3.08a (Maddison were dated to the Upper Cretaceous (Maastrichtian, and Maddison 1999). Because a global likelihood ratio 86–65 Mya). A more precise placement within the test did not show rate constancy in Chenopodiaceae alliance is not possible, and the recent discovery of (Kadereit et al. 2003), the application of a molecular similar pollen in flowers of Caryophylloflora (Caryo- clock was restricted to the Chenopodioideae clade and phyllaceae) from the Eocene of Tasmania (see above) the Salsoloideae/Suaedoideae/Salicornioideae clade requires caution. Fossil 3a (Table 4) is a pollen record of (termed the SSS clade hereafter). Chenopodiaceae/Amaranthaceae from the USA, and For the rbcL and ITS data sets relative rate tests (Wu was dated to the Paleocene (65–56.5 Mya; position of and Li 1985) were conducted using the program the calibrated node of fossil 3 see Fig. 4A and B). This K2WuLi (Jermiin 1996) to evaluate rate constancy of fossil supports the conservative assumption chosen here rbcL and ITS sequence evolution, using Beta vulgaris (in that the basal lineages of the Amaranthaceae–Cheno- case of rbcL), K. foliatum and P. nigdensis (in case of podiaceae alliance (suchas Betoideae) date back at least ITS) as the reference sequences. These pairwise compar- to the Cretaceous/Tertiary boundary. isons were used to identify taxa with highly deviating substitution rates. These were removed sequentially from the matrices starting with the taxon with the highest or lowest z-score. Rate constancy among Results lineages was then tested with global likelihood ratio tests (Felsenstein 1988) withconsistently recalculated ITS analysis ML models. This was achieved by calculating log- likelihood scores for trees with and without a molecular The data matrix of Camphorosmeae comprises 683 clock enforced. Non-significance at the 0.01 level characters of which 677 were nucleotides and six were between tree topologies, indicating that a molecular informative gaps coded as 0/1. The matrix contained 406 clock can not be rejected, was assessed withModeltest invariable characters. Of the variable characters 159 3.06 (Posada and Crandall 1998). (23.3%) were parsimony informative. In the MP Fossils that were considered to be sufficiently docu- analysis, 1377 shortest trees of 651 steps with a mented, dated and reliably identified to be used as consistency index (CI) of 0.582 and a retention index calibration points for this analysis are listed in Table 4. (RI) of 0.699 were found. The strict consensus is shown Fossil 1 consists of Salicornia-like stem systems found in in Fig. 2, withbootstrap values above 50% indicated Oligocene deposits (35.4–23.3 Mya) from northern Italy. below the respective branches. The ML tree (best score The absence of flowers or fruits is somewhat compen- 4311.7952, not shown) and the MP consensus tree

Table 4. Macro- and pollen fossils of Chenopodiaceae used for calibrating the molecular clock

Fossil no. Fossil, its age and origin Calibrated node, compare Fig. 4

1. Salicornites massalongoi a,b; stem Oligocene, Stem and crown of Salicornioideae 35.4–23.3 Mya; Italy: Chiavu 2. Parvangula randeckensisc; seeds; Lower Miocene, Crown of Chenopodieae I 23.3–16 Mya; Germany: Randecker Maar, Tu¨ bingen 3a. Chenopodipollis multiplexd, pollen; Paleocene, Used to calibrate the root to 65–56.5 Mya (see also text) 65–56.5 Mya; USA 3b. Polyporina cribariae; pollen; Senon, 86–65 Mya; Canada

References: a Principi (1926); b Collinson et al. (1993); c Gregor (1982); d Nichols and Traverse (1971); e Srivastava (1969). ARTICLE IN PRESS G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80 69 agree in all major branches: (i) a large clade consisting of mony uninformative. In the MP analysis, one shortest all Australian taxa which is sister to the following clade; tree of 532 steps was found witha CI of 0.633 and an RI (ii) a small clade consisting of the Central Asian Bassia of 0.733 (Fig. 3). Major clades are identical in the MP dasyphylla, Kochia krylovii, and K. melanoptera; (iii) a and ML analyses. These are: (i) a clade consisting of clade comprising all C4 species plus Bassia sedoides, that Salicornia and Sarcocornia species which is sister to the is sister to the Australian/Central Asian clade; (iv) a following; (ii) a large clade of Australian Salicornieae lineage consisting of the four species Kochia americana, (Halosarcia, Pachycornia, and Tecticornia); (iii) a clade K. saxicola, Bassia hirsuta, and Chenolea diffusa. These consisting of Arthrocnemum and Microcnemum which is are sister to all other Camphorosmeae. The topology sister to Salicornia and Sarcocornia plus the Australian within the C4 clade differs in the MP and ML trees. Salicornieae; (iv) members of the Halopeplideae The data matrix of Salicornieae contains 684 char- (Halopeplis, Allenrolfea) and Halocnemum form the acters, 447 of which were constant and 140 parsimony basal lineages. The ML tree (not shown) mainly differs informative. The remaining 97 characters were parsi- from the MP tree in the relationship between Salicornia

Petrosimonia nigdensis --- outgroup Kochia americana North America 58 Kochia saxicola Eurasia 90 Bassia hirsuta 88 Chenolea diffusa South Africa Kirilowia eriantha Camphorosma monspeliaca 100 Camphorosma songorica Kochia indica s.l. 100 Kochia laniflora Bassia hyssopifolia 99 Kochia scoparia 97 Kochia densiflora Eurasia Kochia prostrata Chenoleoides arabica 74 Bassia muricata 60 Kochia stellaris Chenoleoides tomentosa 82 Londesia eriantha Panderia pilosa Bassia sedoides Enchylaena tomentosa Dissocarpus biflorus Dissocarpus paradoxus 53 64 Eriochiton sclerolaenoides Maireana polypterygia Maireana erioclada supported by Maireana integra 53 cp DNA data Malacocera tricornis (atpB-rbcL spacer and ndhF) Neobassia proceriflora Australia Osteocarpum dipterocarpum Roycea spinescens Sclerolaena obliquicuspis 53 Sclerolaena diacantha Sclerolaena bicornis Sclerolaena cornishiana 73 Sclerolaena longicuspis Stelligera endicaspinis

Kochia melanoptera Central Kochia krylovii 100 Asia 100 Bassia dasyphylla Fig. 2. Strict consensus of Camphorosmeae based on 41 ITS sequences; bootstrap values below branches. ARTICLE IN PRESS 70 G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80

14 Kalidium foliatum (Halopeplideae) - C Asian

24 Halocnemum strobilaceum - Mediterranean, C Asian

32 Allenrolfea occidentalis (Halopeplideae) - N, S American 5 24 Halopeplis amplexicaulis (Halopeplideae) - S Mediterranean

1 Salicornia europaea - European, Mediterranean

Salicornia ramosissima - European, Mediterranean 3 1 65 96 22 1 Salicornia aff. ramosissima - European, 4 Mediterranean 85 Salicornia perennans - E European, Sibirian, C Asian 24 5 Salicornia meyeriana - S African 100 2 Salicornia procumbens - W European, Mediterranean

14 Sarcocornia quinqueflora ---- Australia 2 31 13 3 Sarcocornia capensis - S Africa 9 100 2 10 Sarcocornia decumbens - S Africa

8 Sarcocornia littorea - S Africa 5 12 Sarcocornia fruticosa s.l. 11 (→ Salicornia aff. peruviana) - S America 94 10 Sarcocornia utahensis - N America 11 6 85 5 Sarcocornia fruticosa - Mediterranean 14 100 2 Sarcocornia perennis - Mediterranean

25 Tecticornia australasica - Australia 6 6 Halosarcia peltata - Australia 8 15 Halosarcia pergranulata - Australia 8 9 5 54 30 Halosarcia indica subsp. julacea - Australia 68 7 17 Halosarcia indica subsp. indica - paleotropical 13 Pachycornia triandra - Australia

Arthrocnemum macrostachyum (Turkey) - Mediterranean, Saharo Sindian 10 100 2 7 Arthrocnemum macrostachyum (Spain) - Mediterranean, Saharo Sindian 78 34 Microcnemum coralloides - Spain, Anatolia, NW Iran Fig. 3. Single most parsimonious tree of Salicornieae based on 27 ITS sequences shown as a phylogram; bootstrap values below branches, numbers of characters above branches; species distributed in Australia in grey boxes. and Sarcocornia. They are not sister groups; Sarcocornia among lineages could be achieved for 23 taxa of is paraphyletic in relation to Salicornia. Chenopodioideae and 26 members of SSS clade, respectively. Three taxa of Chenopodioideae and 17 taxa of the SSS clade had to be removed because of Molecular clock analysis strongly deviating z-scores in the relative rate tests (Table 5). Two alternative fossils were used to calibrate The settings and results of the molecular clock the clock in the rbcL analyses (Fig. 4A and B). For the analyses of rbcL and ITS are summarized in Table 5 Chenopodioideae, calibration with fossils 2 and 3a/b and illustrated in Figs. 4 and 5. (Table 4) resulted in a substitution rate of Seventy changes of amino acids were identified in our 0.31–0.45 109 and 0.28–0.32 109 synonymous Chenopodiaceae rbcL sequences. The corresponding 210 substitutions per site per year, respectively. For the nucleotide sites were excluded from the estimation of SSS clade, calibration withfossils 1 and 3a/b ( Table 4) divergence time. In the rbcL analyses, rate constancy resulted in a substitution rate of 0.24–0.55 109 and ARTICLE IN PRESS G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80 71

Table 5. Results of the molecular-clock analyses

Subfamilies, tribes Chenopodioideae Salsoloideae, Suaedoideae, Camphorosmeae and Salicornioideae (SSS clade)

Result criteria Molecular marker rbcL rbcL ITS Taxa included/excluded 23 of 26 Chenopodioideae; 26 of 43 taxa of the SSS 20 of 43 Camphorosmeae; Teloxys aristata, Ch. bonus- clade; 17 taxa excluded, 3 23 taxa excluded henricus and Ch. foliosum taxa of Chenopodioideae excluded, 2 taxa of the SSS included clade included Number of taxa 26 30 21 Outgroup Beta vulgaris B. vulgaris Chenolea diffusa ML settings (the best-fit HKY85+I+G, nucleotide GTR+G+I, nucleotide GTR+G+I, nucleotide model was selected by frequencies set to A ¼ frequencies A ¼ 0:2679; C ¼ frequencies A ¼ 0:2775; C ¼ Modeltest Version 3.06) 0.2610, C ¼ 0:1916; G ¼ 0:1897; G ¼ 0:2419; T ¼ 0:2445; G ¼ 0:2203; T ¼ 0:2497; T ¼ 0:2977; tr/tv- 0:3005; substitution rate 0:2577; substitution rate ratio ¼ 4.0222, matrix: AC 1.0, AG 3.841, matrix: AC 1.0, AG 5.326, rates ¼ gamma, shape AT 0.191, CG 0.191, CT AT 0.1, CG 0.1, CT 2.451, parameter ¼ 1.0061, 5.705, GT 1.0, shape GT 1.0, shape pinvar ¼ 0.7042 parameter ¼ 0.818 parameter ¼ 0.651 ML scores clock enforced –ln L 2831.66715 –ln L 2931.61156 –ln L 2631.79403 clock not enforced –ln L 2810.18914 –ln L 2908.18197 –ln L 2617.03438 Distance root to tips 0.01808 0.02029 0.0778 Calibration Fossil 2: 23.3–16 Mya Fossil 1: 35.4–23.3–Mya Crown of Camphorosmeae calibrated to 16.2–7.1 Mya calculated from rbcL clock (Fig. 4) (Fossils see Table 4) Fossil 3: 65–56.5 Mya Fossil 3: 65–56.5 Mya Ages of Australian lineages Atriplex spongiosa Pachycornia/Tecticornia/ Sclerolaeneae using alternative fossils Halosarcia Fossil 2: 2.5–1.7 Mya Fossil 1: 10.9–4.8 Mya 8.1–3.6 Mya Fossil 3: 2.8–2.4 Mya Fossil 3: 8.5–7.4 Mya Chenopodium/Dysphania Sarcocornia Fossil 2: 14.5–9.9 Mya Fossil 1: 4.0–1.8 Mya Fossil 3: 16.1–14.0 Mya Fossil 3: 3-1–2.7 Mya Chenopodium/Rhagodia Sclerolaeneae Fossil 2: 4.2–2.9 Mya Fossil 1: 6.5–2.9 Mya Fossil 3: 4.7–4.1 Mya Fossil 3: 5.1–4.4 Mya Scleroblitum Fossil 2: 37.9–26.0 Mya Fossil 3: 42.2–36.7 Mya Substitution rates per site Fossil 2: 0.31–0.45 10–9 Fossil 1: 0.24–0.55 10–9 4.8–11.0 10–9 per year Fossil 3: 0.28–0.32 10–9 Fossil 3: 0.36–0.31 10–9

0.31–0.36 109 synonymous substitutions per site per Discussion year, respectively. With the internal fossils (1 and 2) the root of bothtrees ( B. vulgaris, Fig. 4A and B) was dated Origin of Australian lineages within Chenopodiaceae to an age of 83.6–37.0 and 58.3–40.1 Mya, respectively. (Fig. 6) In the ITS analyses, rate constancy among lineages could be achieved for 20 taxa of Camphorosmeae (Table From the relationships of Australian Chenopodiaceae 5), but not for a sufficient number of taxa in to different tribes of worldwide distribution it is obvious Salicornieae. For Camphorosmeae, the ITS clock was that Australian Chenopodiaceae have derived from calibrated with the age of the basal node determined by more than one colonization event. Evidence from the rbcL clock of the SSS clade (Table 5, Fig. 5). molecular data (Kadereit et al. 2003; Schu¨ tze et al. ARTICLE IN PRESS 72 G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80

Sympegma regelii representatives of the Kalidium caspicum SSS clade 2.8 - 1.7 Mya Atriplex coriacea Atriplex halimus A Atriplex patula Atriplex rosea Atriplex spongiosa Atriplex undulata Halimione pedunculata 4.7 - 2.9 Mya Chenopodium acuminatum Chenopodium frutescens Chenopodium sanctae-clarae Chenopodium desertorum 23.3 - 16 Rhagodia drummondii 2 Mya Chenopodium auricomum 42.2 - 26.0 Mya Microgynoeceum tibeticum 65.0 - 56.5 Holmbergia tweedii Mya Monolepis nuttaliana Spinacia oleracea 3 Scleroblitum atriplicinum Axyris prostrata Krascheninninikovia ceratoides Chenopodium botrys Chenopodium cristatum 16.1 - 9.9 Mya Dyypsphania g lomulifera Beta vulgaris - outgroup 0.001 substitutions/site

10.9 - 4.8 Mya Allenrolfea occidentalis Arthrocnemum macrostachyum Microcnemum coralloides 4.0 - 1.8 Mya Pachycornia triandra Halosarcia peltata B Tecticornia australasica 35.4 - 23.3 Mya Sarcocornia utahensis Sarcocornia blackiana 1 Halopeplis perfoliata Kalidium caspicum 1 Halocnemum strobilaceum Borszczowia aralocaspica Suaeda altissima 6.5 - 2.9 Mya Bassia dasyphylla Dissocarpus paradoxus Sclerolaena obliquicuspis Maireana brevifolia Bassia sedoides Kochia densiflora 65.0 - Kochia americana 56.5 Mya Noaea mucronata Raphidophyton regelii Salsola laricifolia 3 Sympegma regelii Salsola canescens Salsola kali Chenopodium frutescens Holmbergia tweedii Chenopodioideae Krascheninnikovia ceratoides Beta vulgaris - outgroup 0.001 substitutions/site Fig. 4. Clock-enforced ML trees of 23 taxa of Chenopodioideae (A) and 26 taxa of the Salsoloideae/Suaedoideae/Salicornioideae clade (B) withconstant substitution rates; analysis settings and calibrated nodes (circles) are described in Tables 4 and 5; black arrows assign age estimates to Australian lineages (in grey boxes).

2003; present study) indicates that Chenopodiaceae deae, two in the Salicornieae, and one each in the entered Australia at least nine times (see Fig. 1 and Camphorosmeae, Suaedeae, and Salsoleae. However, it below for Suaeda and Salsola). There were at least four should be stressed that these numbers are minimum independent colonization events in the Chenopodioi- estimates due to limited sampling in certain groups. ARTICLE IN PRESS G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80 73

Chenopodioideae (Table 2). Except for Einadia, all genera of native Australian Chenopodioideae are represented in the rbcL The majority of Australian Chenopodioideae belong analysis of Kadereit et al. (2003). However, problems to the two large world-wide genera Atriplex (about 200 arise from the high diversity of the Australian Atriplex species) and Chenopodium (about 140 spp.), which are and Chenopodium species. The native Australian species represented in Australia by 58 and 15 native species, of Atriplex belong to two subgenera (Atriplex, and the respectively. The remaining five genera of native monotypic Theleophyton), and subg. Atriplex is divided Australian Chenopodioideae, Einadia, Dysphania, Rha- into five different sections, three of which are endemic to godia,andScleroblitum, are largely endemic to Australia Australia. Of the 58 native species, only one was sampled. For Chenopodium, three species from three of Chenolea diffusa - outgroup the four endemic sections were included in the sampling. 0.01 substitutions/site Chenoleoides arabica The analysis of Kadereit et al. (2003) showed that Bassia muricata Chenopodium clearly is polyphyletic, which had been Kochia stellaris suspected already by Judd and Ferguson (1999). Chenoleoides tomentosa Chenopodium occurs in three different clades. All three Panderia pilosa clades also include Australian members of Chenopo- Bassia eriantha dieae indicating that they have colonized Australia Kochia densiflora independently. For Atriplex, the data suggest at least Kochia laniflora one colonization independent from other Australian Bassia sedoides Chenopodioideae. Camphorosma monspeliaca The following discussion is based on the rbcL tree Kochia krylovii taken from Kadereit et al. (2003) (Fig. 3): Bassia dasyphylla Dissocarpus biflorus (1) Chenopodium auricomum and C. desertorum, repre- 16.2 Dissocarpus paradoxus senting sections Auricoma and Desertorum of subg. - 7.1 Mya Eriochiton sclerolaenoides Chenopodium, respectively, form a monophyletic Maireana polypterygia group with Rhagodia drummondii. Most likely, Osteocarpum dipterocarpum 8.1 - 3.6 Mya Einadia (not sampled) also belongs to this lineage Sclerolaena bicornis because it can only be distinguished from Chenopo- Sclerolaena diacantha dium sect. Desertorum by its succulent pericarp Malacocera tricornis (Wilson 1983). This Australian clade is sister (BS Fig. 5. Clock-enforced ML tree of 20 taxa of Camphorosmeae 99%) to a clade that consists of species belonging to withconstant substitution rates; analysis settings and cali- sections Chenopodium and Skottsbergia of subg. brated node (circle) are described in Tables 4 and 5; black Chenopodium, which are distributed in Eurasia and arrow assigns age estimate to the Australian Camphorosmeae the Juan Fernandez Islands, respectively. A Eurasian (in grey box). origin of the Australian clade is suggested by the

30¡ 90¡ 150¡ -150¡ -90¡

30¡ 30¡ Salicornieae 10.9 - 4.8 Mya Chenopodium/Dysphania 0¡ ? 16.1 - 9.9 Mya 0¡

Camphorosmeae 8.1 - 2.9 Mya -30¡ ? -30¡ Scleroblitum Sarcocornia 42.2 - 26.0 Mya 4.0 - 1.8 Mya

Atriplex -60¡ ? -60¡ 2.8 - 1.7 Mya Chenopodium/Rhagodia ? 4.2 - 2.9 Mya

30¡ 90¡ 150¡ -150¡ -90¡ Fig. 6. Geographic origins and ages of Australian chenopod lineages; Salsoleae and Suaedeae not included due to lack of time-of- arrival estimates for these groups. ARTICLE IN PRESS 74 G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80

basal position of the Central (hereafter C) Asian Jacobs 2001) which may well represent a mono- Microgynoeceum to bothlineages. phyletic group as suggested by Parr-Smith(1982) . (2) Chenopodium cristatum of sect. Orthosporum, which However, the annual A. australasica of the predo- represents the Australian species of subg. Ambrosia, minantly N hemispheric sect. Teutliopsis and also A. and Dysphania glomulifera form a lineage that is billardierei from the monotypic section Theleophyton sister to Ch. botrys of sect. Botryoides (BS 95%), a might represent independent lineages (compare Parr- Eurasian representative of subg. Ambrosia. Bentham Smith1982 ). Our limited sampling of Chenopodioi- and Hooker (1880) recognized a close relation deae (especially in Atriplex) does not allow to draw between Chenopodium sect. Orthosporum and Dys- final conclusions about the number of Australian phania, which share similar glandular hairs and lineages in Chenopodioideae and about the diversity glomerules of small flowers with4–5 petals and one of the different lineages. A detailed analysis of each stamen. Dysphania has been treated as a section of of the Australian lineages of that subfamily is needed Chenopodium (e.g. Aellen 1930; Black 1924)orasa to trace their origin more precisely. family of its own (Pax 1927). Wilson (1983) followed Bentham and Hooker (1880) but still recognized Dysphania as a distinct genus because of differing Salicornieae seed characters. Recently, Mosyakin and Clemants (2002) used morphological characters to separate Our sampling covers three of five genera present in subg. Ambrosia from Chenopodium, and included it Australia. The monotypic Tegicornia and Sclerostegia (5 in Dysphania. This last view is strongly supported by spp.) are not included. The relationships within Sali- our results. However, as there are four more sections cornioideae are only partly resolved in the rbcL tree of subg. Ambrosia outside Australia which are not (Fig. 1). The ITS tree (Fig. 3), however, clearly shows included in the sampling of Kadereit et al. (2003),no that in Salicornieae at least two separate colonization conclusive statement on the founders of the Aus- events took place. tralian lineage is possible at this stage. This also applies to the C Asian species Teloxys aristata, (1) Sarcocornia consists of about 15 species worldwide, which is sister to the Ambrosia/Dysphania clade. the majority growing in South Africa. The three Teloxys has the same unique multicellular glandular Australian species of Sarcocornia are represented by hairs (type 8 in Carolin 1983). A close relationship S. blackiana in the rbcL tree, and by S. quinquefolia between Chenopodium subg. Ambrosia and Teloxys in the ITS tree. An origin of the Australian had been recognized earlier (Scott 1978; Weber 1985; Sarcocornia species independent of the remaining Simo´ n 1996). Australian Salicornieae is supported by morpholo- (3) A third Australian lineage within Chenopodioideae gical and anatomical data. Sarcocornia differs from is represented by Scleroblitum atriplicinum.This all other members of the Australian Salicornieae in monotypic genus is distinct from other Chenopo- the presence of sclereids in the chlorenchyma, an dioideae in and outside Australia by its three-layered adaxial perianthlobe, two stamens instead of only pericarp (exocarp membranous, mesocarp hardened one, and the absence of perisperm (Wilson 1980). and whitish, endocarp crustaceous and reddish The ITS tree suggests that South Africa might have brown) enclosed in a cartilaginous perianthof four been the geographic origin of the Australian acute petals (Ulbrich1934 , Fig. 188; Wilson 1983). Sarcocornia. The closest relative of Scleroblitum in the rbcL tree is (2) The other Australian taxa included in the rbcL tree the widely distributed Eurasian Chenopodium folio- form a polytomy that includes a clade consisting of sum of subg. or sect. Blitum, which differs by its Salicornia, Halocnemum, and Halosarcia indica (Fig. persistent perianthturning fleshyand red in fruit. To 1). The ITS topology is only partly congruent with settle the relationship of Scleroblitum, further the rbcL topology. It clearly indicates that Tecticor- sampling is required. The monotypic sect. Thellungia nia, Pachycornia, and Halosarcia form a monophy- might be of special importance, because C. antarc- letic group of Australian Salicornieae (hereafter ticum (J.D. Hook.) J.D. Hook. from Argentina and termed Halosarcia clade), which is sister to a clade Chile is very similar to Scleroblitum atriplicinum (for consisting of the two cosmopolitan genera Salicornia details see Wilson 1983; Mosyakin 2003). and Sarcocornia. The morphological uniformity of (4) In the rbcL tree, Atriplex spongiosa, a member of the the Halosarcia clade and the distant position of endemic sect. Spongiocarpus, is sister to the Eurasian Sarcocornia had been recognized already by Wilson A. rosea of the northern hemispheric sect. Scleroca- (1980). It is most parsimonious to assume that lymma. The bulk of Australian Atriplex species is, Eurasia is the ancestral area for this group of like A. spongiosa, perennial or facultatively annual. Salicornieae (Figs. 3 and 6) and that at least the They are C4 plants (for the latter see Osmond 1974; Halosarcia clade entered Australia from there. ARTICLE IN PRESS G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80 75

Suaedeae both groups the leaves are succulent and have a C3 sclerolaenoid anatomy, whereas in the large Eurasian The two native Suaeda species (S. arbusculoides, S. sister group all species are C4, except B. sedoides which is australis) are not sampled here, but were included in the somewhat intermediate. In contrast to the shrubby habit study of Schu¨ tze et al. (2003) based on ITS and two of the Australian species, all three C Asian species are chloroplast markers. The two species form a grade at the annuals. base of the S. maritima subclade in sect. Brezia, Our ITS tree clearly demonstrates that the Camphor- indicating one colonization event. Sectio Brezia is made osmeae are in need of a modern revision. This was up of about 31 species that are predominantly northern pointed out already by Wilson (1984) who regarded the hemispheric in distribution. As S. arbusculoides is delimitation and classification of genera in the Austra- endemic to the tropical coasts of Australia, and S. lian Camphorosmeae solely by fruit characters as australis has a wide distribution along the tropical coasts artificial. The situation in the larger Eurasian genera of SE Asia, immigration most likely has occurred from Bassia and Kochia is even worse, because according to the North by crossing the narrow Torres Strait. our trees they are highly polyphyletic.

Camphorosmeae Salsoleae Camphorosmeae belong to subfamily Salsoloideae and comprise 30–40 non-Australian and about 147 In terms of species numbers, Salsoleae are the most Australian species. Outside Australia the Camphoros- successful tribe of Chenopodiaceae. However, native meae are centred in the Old World desert belt from the species are restricted to Eurasia and Africa, withthe Canary Islands to Asia. A few species grow in N exception of Salsola tragus, which extends to Australia. America and Africa. Within Salsoloideae the tribe is This species is native to a vast area from the western clearly monophyletic (97% BS in the rbcL analysis of Mediterranean to C Asia (Rilke 1999) but in arid Kadereit et al. 2003). The rbcL data indicate that the landscapes it has become a worldwide weed. Most Australian Camphorosmeae ( ¼ Sclerolaeneae) are authors also agree on its anthropochorous introduction monophyletic and have originated from within Eurasian to Australia. There can be no doubt that many Camphorosmeae (Fig. 1). These findings are confirmed Australian populations are offspring from introduc- by the ITS analysis of Camphorosmeae, in which 10 of tions, but the species was already present at the the 14 genera of Australian Camphorosmeae are beginning of the 18th century in Australia and might included (Fig. 2), and only the small genera Eremophea, have settled there just before the arrival of Europeans. Didymanthus, Threlkeldia, and Sclerochlamys are miss- In 1770, it was collected twice by JosephBanks along ing (see Table 2). The comprehensive ITS analysis the east coast, and in 1810 it was described by Brown as indicates (although with moderate BS support) that the S. macrophylla from the east coast and under the name Australian taxa are most closely related to a small clade S. australis from islands to the south of S Australia consisting of B. dasyphylla, Kochia melanoptera, and K. (Brown 1810). The species could have arrived by long- krylovii from C Asia (Figs. 1 and 2). Most likely, distance dispersal from the eastern part of its area, either immigration of the founders of the Australian lineage from C Asia or from Pakistani Baluchistan. took place from that region. A sister-group relationship of the three C Asian and the Australian species is further supported by the results of two other studies on Age of Australian Chenopodiaceae (Fig. 6) phylogenetic relationships in Salsoloideae, based on sequences of the atpB-rbcL spacer, ndhF, and on Our estimates of divergence time of the Australian comparative leaf anatomy (Kadereit and Freitag, lineages ranged from late Pliocene as the youngest to unpublished data). late Eocene/early Oligocene as the oldest, with a clear The coherence of the Australian Camphorosmeae and cumulation of arrivals during the late Miocene and the the C Asian B. dasyphylla clade is rather unexpected, entire Pliocene (Fig. 6). This corresponds to the because both Scott (1978) and Wilson (1975) stressed the palynological data, which show an ever-increasing role peculiar features of the Australian taxa, though Wilson of Chenopodiaceae in the pollen spectra of many did not accept Scott’s proposal to classify them in the Australian regions from the late Miocene onwards separate tribe Sclerolaeneae. However, as far as we (Kershaw et al. 1994). know, none of the unique characters ascribed to the According to our results (Fig. 4A), the monotypic Australian Camphorosmeae is exclusive to them. In genus Scleroblitum (Chenopodioideae) represents the contrast, the close relationship of Australian Camphor- oldest Australian lineage dating back to the late Eocene osmeae to the small group of C Asian species is and Oligocene (42.2–26.0 Mya). During the late Eocene, supported by obvious similarities in leaf structure. In Australia was situated in relatively high latitudes. Its ARTICLE IN PRESS 76 G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80 southern margin was not well separated from Antarc- they are ideally adapted to migrating along coasts and tica. Although a shallow seaway separated the southern even to crossing oceans. Some present-day species, e.g. coastline since the early Cenozoic, a complete separation Halosarcia indica s.l., have corky, buoyant infructes- of the continents took place only approximately cences. The sympatric occurrence of many genera and 35.5 Mya ago (Veevers et al. 1991; McLoughlin 2001). species of Salicornieae in present-day Australia, where Therefore, Scleroblitum may be the only Australian they share similar habitats, is remarkable and suggests Chenopodiaceae deriving from Gondwanan ancestors. If that soon after arrival, in the late Miocene, they started the younger age estimate for Scleroblitum is correct, its to spread into various habitats that probably were later ancestors must have entered Australia via long-distance separated geographically. dispersal, because Australia was isolated from all other The Camphorosmeae arrived in Australia only landmasses after the Eocene/Oligocene boundary. slightly later than the Salicornieae. Divergence from its The period during which Scleroblitum arrived in C Asian sister group is estimated at 6.5–2.9 Mya Australia was marked by a dramatic drop in tempera- according to the rbcL data, and at 8.1–3.6 Mya ture (Kamp et al. 1990; Feary et al. 1991) accompanied according to the ITS data (Table 5). All Australian by changes of the flora (Macphail et al. 1993, 1994). The Camphorosmeae are subshrubs or shrubs that inhabit Gondwanan rainforests contracted and were replaced by mainly the Eremaean area of Australia. Only a few seasonal Nothofagus-dominated forests. More open species grow in the tropical northern part of the vegetation types like shrublands or grassland also continent. Some species have adapted to inland salt- became dominant. Interestingly, the oldest record of or gypsum-containing soils. Other species extend into chenopod-like pollen in Australia dates back to this agricultural lands and coastal habitats where they are period. It was found in onshore sediments of the able to tolerate slightly saline soils, and a few species Gippsland Basin, Victoria (Partridge 1971, cited in prefer non-saline conditions. Macphail et al. 1994). Scleroblitum now grows from Habitats suitable for the majority of Camphorosmeae southern Queensland to northwestern Victoria and have always been largely absent from SE Asia. There- eastern SouthAustralia in heavy,periodically water- fore, it seems more likely that the colonizers arrived via logged, often saline soils (Wilson 1984). The related long-distance dispersal from the temperate semi-arid to Eurasian species of Chenopodium subg. Blitum are arid parts of continental Eurasia. However, a few likewise adapted to semi-humid to semi-arid conditions species of Camphorosmeae that grow in the tropics, in cooler, usually montane areas of the northern e.g. Enchylaena tomentosa, also grow in coastal saline temperate zone. habitats like some taxa of Salicornieae. It is conceivable The second-oldest lineage of Chenopodiaceae in that their founders, as in the Halosarcia clade of Australia is the Chenopodium sect. Orthosporum/Dys- Salicornieae, entered Australia via the coasts of SE phania clade, which we estimate to be 16.1–9.9 Mya old. Asian peninsulas and islands. But as nowadays Cam- At that time, Australia still was further south than today phorosmeae are virtually absent from tropical S and SE (Frakes 1999) but collisions with the Philippines Sea Asia, it seems more likely that the tropical Enchylaena Plate had already begun. The numerous island arcs of species of Australia derive from temperate immigrants SE Asia already present at that time and their movement rather than from tropical ancestors that have gone might have served as ‘stepping stones’. extinct. The only striking morphological difference At that time, most of Australia was covered by between the otherwise similar Kochia dasyphylla clade temperate rainforests, sclerophyllous woodland, heath- and the Australian Camphorosmeae is life form. The like communities, and grassland. Open vegetation types Kochia dasyphylla clade consists of exclusively annual suitable for Chenopodioideae might have existed also as species, whereas all Australian species are perennial. minor components of the vegetation. From the middle From the absence of annual Camphorosmeae in Miocene onwards, rainfall decreased and open vegeta- Australia we infer that the immigrant presumably was tion types started to spread. a subshrub like the majority of Eurasian Camphor- According to our molecular clock, the larger of the osmeae. It appears plausible that the C Asiatic annuals two Australian lineages of Salicornieae, the Halosarcia derived only later, under the pressure of successively clade, reached the continent during the late Miocene colder winters, from shrubby ancestors, which are still (Table 5; Figs. 4 and 6), which coincides with the often present in the Kochia saxicola clade. postulated worldwide salinity crisis in the late Miocene. Unfortunately, the ITS cladogram is largely unre- The Salicornieae are highly adapted to saline conditions solved within the Australian clade and does not give and dominate wet salt marshes worldwide. The ancestor additional arguments for or against a migration through of the Halosarcia clade might have reached Australia by SE Asia. There are 31 (4.5%) potentially parsimony migration through SE Asia, using the emerging archi- informative characters within this clade. However, there pelagos as ‘stepping stones’. As diaspores of Salicor- is no clear signal which of the taxa might branch first. nieae are able to survive in salt water for long periods, This might indicate a fast radiation of Camphorosmeae ARTICLE IN PRESS G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80 77 shortly after their arrival in Australia, which did not Eurasian ancestors to New Zealand and migrated allow for the accumulation of numerous mutations or subsequently from New Zealand to Australia, New complete lineage sorting. The climatic history of Guinea, and SouthAmerica. TheAustralian and New Australia at the time of arrival of Camphorosmeae Guinea Alps may have served as stepping stones for supports this hypothesis, because at that time a strong Myosotis on its way to New Zealand. The genus occurs aridification took place and open vegetation types with naturally in the Australian Alps (M. australis), and the and without saline conditions expanded. high altitude areas of New Guinea, from which it has Sarcocornia (4.0–1.8 Mya) and Chenopodium subg. not been recorded, are still very poorly known. Two Chenopodium/Rhagodia (4.7–2.9 Mya) botharrived dur- other striking examples of long-distance dispersal events ing the Pliocene (Table 5). The ITS tree suggests that withsubsequent diversification in Australasia are South Africa might be the geographic origin of the Microseris, which immigrated from western North Australian Sarcocornia. Considering the excellent float- America to Australia (Vijverberg et al. 1999, 2002), ing abilities of Sarcocornia diaspores, long-distance and Scleranthus (Smissen et al. 2003), which came from dispersal from SouthAfrica to Australia does not seem Europe in the Pliocene and entered New Guinea, SE unrealistic. The geographic origin of the Chenopodium Australia, Tasmania, and New Zealand. subg. Chenopodium/Rhagodia clade, and its mode of entering Australia, are unclear and needs further extended sampling. Note Atriplex spongiosa represents the youngest Australian lineage found in this analysis. It dates back to the late Only after submission of the revised version of our Pliocene (Table 5). manuscript the article of Shepherd et al. (2004) on the radiation of Australian Salicornioideae has been pub- lished. Regarding the two independent colonization Concluding remarks events of Australian species of Salicornioideae, the authors arrived at similar general conclusions. They The Chenopodiaceae have entered Australia at least included more Australian taxa. However, as their nine times. Most of the lineages arrived during the late sampling of non-Australian taxa was rather limited Miocene/Pliocene when aridification and increasing (e.g. S African species are lacking) and estimates of salinity were changing the landscape of many parts of divergence times based on a molecular clock were Australia. The Australian Camphorosmeae and Sali- missing, tracing the origin and age of the colonizers cornieae (except Sarcocornia), which both represent remained fragmentary. large monophyletic groups, and probably Atriplex, diversified rapidly after their arrival. In contrast, other and even older lineages, suchas Scleroblitum and the Chenopodium/Dysphania clade, did not speciate success- Acknowledgements fully, although species of Sect. Orthospermum have For supply of plant material we are indebted to many spread to a wide range of habitats. Australian Cheno- colleagues, in particular to S. Beck, S.-W. Breckle, H. podioideae are clearly polyphyletic. They consist of at Ku¨ rschner, M. Lomonosova, L. Mucina, D. Pratt, and least four independent lineages that arrived in Australia N. Schmalz. We also thank J.W. Kadereit (Mainz), at different times. Thomas Borsch, and Ulf Swenson for useful comments The disjunct distribution of chenopod lineages that on the manuscript. Special thanks are due to A. Horn include Australian branches is largely the result of long- and M. Kever (Mainz) for assistance withlaboratory distance dispersal. Even in Salicornieae withtheir work, and to D. Franke (Mainz) for help with peculiar dispersal mechanism adapted to marine envir- processing of illustrations. Support by grants from the onments, the genus Sarcocornia must have crossed Deutsche Forschungsgemeinschaft to GK (DFG Cl 188/ marine environments of considerable dimensions. 1-1) and HF/KW (DFG We1830/2-1) is gratefully The fact that long-distance dispersal plays a decisive acknowledged. role in the biogeography of temperate taxa in Australia is supported by several other recent molecular studies. For example, in Gentianella (von Hagen and Kadereit References 2001)andCardamine (Bleeker et al. 2002), South American species are most closely related to Australa- Aellen, P., 1930. Die systematische Stellung und Gliederung sian taxa and the time of divergence between the der R. Brownschen Gattung Dysphania. Bot. Jb. Syst. 63, respective lineages strongly indicates long-distance dis- 483–490. persal from SouthAmerica to Australia or vice versa. Audley-Charles, M.G., 1987. Dispersal of Gondwanaland: Myosotis (Winkworthet al. 2002a ) spread from relevance to evolution of the Angiosperms. In: Whitmore, ARTICLE IN PRESS 78 G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80

T.C. (Ed.), Biogeographical Evolution of the Malay Hill, R.S., Truswell, E.M., McLoughlin, S., Dettman, M.E., Archipelago. Clarendon Press, Oxford, pp. 1–25. 1999. The evolution of the Australian flora: fossil evidence. Audley-Charles, M.G., Ballantyne, P.D., Hall, R., 1988. In: Orchard, A.E. (Ed.), Flora of Australia, second ed., vol. Mesozoic–Cenozoic rift–drift sequence of Asian fragments 1. ABRS/CSIRO, Melbourne, pp. 251–320. from Gondwanaland. Tectonophysics 155, 317–330. Jacobs, S.W.L., 2001. Review of leaf anatomy and ultra- Beadle, N.C.W., 1981. The Vegetation of Australia. Fischer, structure in the Chenopodiaceae (Caryophyllales). J. Jena. Torrey Bot. Soc. 128, 236–253. Bentham, G., Hooker, J.D., 1880. Genera Plantarum, Vol. 3. Jermiin, L.S., 1996. K2WuLi, Version 1.0. University of Lovell, Reeve & Co., London. Canberra, Canberra. Black, J.M., 1924. Flora of SouthAustralia. BritishScience Jordan, G.J., Macphail, M.K., 2003. A middle to late Eocene Gild, Adelaide. inflorescence of Caryophyllaceae from Tasmania, Austra- Bleeker, W., Franzke, A., Pollmann, K., Brown, A.H.D., lia. Am. J. Bot. 90, 761–768. Hurka, H., 2002. Phylogeny and biogeography of Southern Judd, W.S., Ferguson, I.K., 1999. The genera of Chenopodia- Hemisphere high-mountain Cardamine species (Brassica- ceae in the southeastern United States. Harvard Pap. Bot. ceae). Austral. Syst. Bot. 15, 575–581. 4, 365–416. Brown, R., 1810. Prodromus Florae Novae Hollandiae et Kadereit, G., Borsch, T., Weising, K., Freitag, H., 2003. Insulae van-Diemen. J. Johnson & Co., London. Phylogeny of Amaranthaceae and Chenopodiaceae and the

Bunge, A., 1880. Pflanzengeographische Betrachtungen u¨ ber evolution of C4 photosynthesis. Int. J. Plant Sci. 164, die Familie der Chenopodiaceen. Me´ m. Acad. Imp. Sci. St.- 959–986. Pe´ tersb. Se´ r. 7, 27 Sci. Nat. 8, St. Petersburg. Kamp, P.J.J., Waghorn, D.B., Nelson, C.S., 1990. Late Burbidge, N.T., 1960. Phytogeography of the Australian Eocene–Early Oligocene integrated isotope stratigraphy region. Austral. J. Bot. 8, 75–209. and biostratigraphy for paleoshelf sequences in southern Carolin, R.C., 1981. A review and critique of studies on the Australia: palaeooceanographic implications. Palaeogeogr. phytogeography of Australia. In: Barker, W.R., Green- Palaeoclimatol. Palaeoecol. 80, 311–323. slade, P.J.M. (Eds.), Evolution of the Flora and Fauna of Kershaw, A.P., Martin, H.A., McEwen-Mason, J.R.C., 1994. Arid Australia. Peacock Publ., Frewville, pp. 119–123. The Neogene: a period of transition. In: Hill, R.S. (Ed.), Carolin, R.C., 1983. The trichomes of the Chenopodiaceae and History of the Australian Vegetation: Cretaceous to the Amaranthaceae. Bot. Jb. Syst. 103, 451–466. Recent. Cambridge University Press, Cambridge, pp. Collinson, M.E., Boulter, M.C., Holmes, P.L., 1993. Magno- 299–327. liophyta (‘Angiospermae’). In: Benton, M.J. (Ed.), Ku¨ hn, U., Bittrich, V., Carolin, C., Freitag, H., Hedge, I.C., The Fossil Record, vol. 2. Chapman & Hall, London, Uotila, P., Wilson, P.G., 1993. Chenopodiaceae. In: pp. 809–841. Kubitzki, K. (Ed.), Families and Genera of Vascular Crisp, M.D., West, J.G., Linder, H.P., 1999. Biogeography of Plants, vol. 2. Springer, Berlin, pp. 253–281. the terrestrial flora. In: Orchard, A.E. (Ed.), Flora of Lawver, L.A., Gahagan, L.M., 1998. Opening of Drake Australia, second ed., vol. 1. ABRS/CSIRO, Melbourne, passage and its impact on Cenozoic ocean circulation. In: pp. 321–368. Crowley, T.J., Burke, K.C. (Eds.), Tectonic Boundary Feary, D.A., Davies, P.J., Pilgram, C.J., Symonds, P.A., 1991. Conditions for Climate Reconstructions, Oxford Monogr. Climatic evolution and control on carbonate deposition in Geol. Geophys., vol. 39, pp. 212–223. northeast Australia. Palaeogeogr. Palaeoclimatol. Palaeoe- Macphail, M.K., Jordan, G.J., Hill, R.S., 1993. Key periods in col. 89, 341–361. the evolution of the Cenozoic flora and vegetation of Felsenstein, J., 1988. Phylogenies from molecular sequences: Tasmania 1: the early middle Pleistocene. Austral. J. Bot. inference and reliability. Annu. Rev. Genet. 22, 521–565. 41, 673–707. Frakes, L.A., 1999. Evolution of Australian environments. In: Macphail, M.K., Alley, N.F., Truswell, E.M., Sluiter, I.R.K., Orchard, A.E. (Ed.), Flora of Australia, second ed., vol. 1. 1994. Early Tertiary vegetation: evidence from spores and ABRS/CSIRO, Melbourne, pp. 163–203. pollen. In: Hill, R.S. (Ed.), History of the Australian Greenwood, D.R., 1994. Palaeobotanical evidence for Tertiary Vegetation: Cretaceous to Recent. Cambridge University climates. In: Hill, R.S. (Ed.), History of the Australian Press, Cambridge, pp. 189–261. Vegetation: Cretaceous to Recent. Cambridge University Maddison, W.P., Maddison, D.R., 1999. MacClade. Sinauer, Press, Cambridge, pp. 44–59. Sunderland, MA. Gregor, H.J., 1982. Die ‘‘Parvangulae’’ und ‘‘Guttulae’’ Magallo´ n, S., Sanderson, M.J., 2001. Absolute diversification Hiltermann & Schmitz 1968 aus dem Randecker rates in angiosperm clades. Evolution 55, 1762–1780. Maar — Samenreste von Centrospermae. Pala¨ ontol. Z. Magallo´ n, S., Crane, P.R., Herendeen, P.S., 1999. Phyloge- 56, 11–18. netic pattern, diversity, and diversification of eudicots. von Hagen, K.B., Kadereit, J.W., 2001. The phylogeny of Ann. Mo. Bot. Gard. 86, 297–372. Gentianella (Gentianaceae) and its colonization of the McLoughlin, S., 2001. The breakup history of Gondwana and southern hemisphere as revealed by nuclear and chloroplast its impact on pre-Cenozoic floristic provincialism. Austral. DNA sequence variation. Org. Divers. Evol. 1, 61–79. J. Bot. 49, 271–300. Hall, R., 1996. Reconstructing Cenozoic SE Asia. In: Hall, R., Mosyakin, S.L., 2003. Taxonomy, phytogeography and Blundell, D.J. (Eds.), Tectonic Evolution of South-east genesis of the family Chenopodiaceae Vent. Summary of Asia. Geol. Soc. London Spec. Publ. 106, 153–184. Thesis, MG. Kholodny Institute of Botany, National ARTICLE IN PRESS G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80 79

Academy of Sciences of Ukraine, Kyiv. (In Ukrainian, with Simo´ n, L.E., 1996. Notas sobre Chenopodium L. subgen. Engl. and Russ. Summaries.) Ambrosia A.J. Scott (Chenopodiaceae). An. Jard. Bot. Mosyakin, S.L., Clemants, S.E., 2002. New nomenclatural Madrid 54, 137–146. combinations in Dysphania R.Br. (Chenopodiaceae): Smissen, R.D., Garnock-Jones, P.J., Chambers, G.K., 2003. taxa occurring in NorthAmerica. Ukr. Bot. Zh.59, Phylogenetic analysis of ITS sequences suggests a Pliocene 380–385. origin for the bipolar distribution of Scleranthus (Caryo- Mu¨ ller, K., Borsch, T., in press. Phylogenetics of Amarantha- phyllaceae). Aust. Syst. Bot. 16, 301–315. ceae based on matK/trnK sequence data-evidence from Specht, R.L., 1981. Major vegetation formations of Australia. parsimony, likelihood, and Bayesian analyses. Ann. Mo. In: Keast, A. (Ed.), Ecological Biogeography of Australia, Bot. Gard. vol. 1. Junk, The Hague, pp. 163–297. Nichols, D.J., Traverse, A., 1971. Palynology, petrology, and Srivastava, S.K., 1969. Assorted angiosperm pollen from the depositional environments of some early Tertiary lignites in Edmonton formation (Maastrichtian), Alberta, Canada. Texas. Geosci. & Man. 3, 37–48. Can. J. Bot. 47, 975–989. Nix, H.A., 1981. The environment of Terra Australis. In: Swofford, D.L., 2002. PAUP*. Phylogenetic Analysis Using Keast, A. (Ed.), Ecological Biogeography of Australia, vol. Parsimony (*and Other Methods). Version 4. Sinauer, 1. Junk, The Hague, pp. 103–133. Sunderland, MA. Northcote, K.H., Skene, J.K.M., 1972. Australian Soils with Thomson, J.A., 2002. An improved non-cryogenic transport Saline and Sodic Properties, Soil Publication No. 27. and storage preservative facilitating DNA extraction from CSRIO, Australia Division of Soils. ‘difficult’ plants collected at remote sites. Telopea 9, Osmond, C.B., 1974. Leaf anatomy of Australian saltbushes in 755–760. relation to photosynthetic pathways. Austral. J. Bot. 22, Ulbrich, E., 1934. Chenopodiaceae. In: Engler, A., Prantl, K. 39–44. (Eds.), Die Natu¨ rlichen Pflanzenfamilien, second ed., vol. Parr-Smith, G.A., 1982. Biogeography and evolution in the 16c. Engelmann, Leipzig, pp. 379–584. shrubby Australian species of Atriplex (Chenopodiaceae). Veevers, J.J., Li, Z.X., 1991. Review of seafloor spreading In: Barker, W.R., Greenslade, P.J.M. (Eds.), Evolution of around Australia. II. Marine magnetic anomaly modelling. the Flora and Fauna of Arid Australia. Peacock Publica- Austral. J. EarthSci. 38, 391–408. tions, Frewville, pp. 291–299. Veevers, J.J., Powell, C.Mc.A., Roots, S.R., 1991. Review Pax, F., 1927. Zur Phylogenie der Caryophyllaceen. Bot. Jb. of seafloor spreading around Australia. 1. Synthesis Syst. 61, 223–241. of the patterns of spreading. Austral. J. Earth Sci. 38, Posada, D., Crandall, K.A., 1998. Modeltest: testing 373–389. the model of DNA substitution. Bioinformatics 14, Vijverberg, K., Mes, T.H.M., Bachmann, K., 1999. Chloro- 817–818. plast DNA evidence for the evolution of Microseris Pratt, D., 2003. Phylogeny and morphological evolution of the (Asteraceae) in Australia and New Zealand after long- Chenopodiaceae–Amaranthaceae alliance. Ph.D. Thesis, distance dispersal from western NorthAmerica. Am. J. Iowa State University, Ames, Iowa. Bot. 86, 1448–1463. Principi, P., 1926. La flora oligocenica de Chiavon e Salcedo. Vijverberg, K., Lie, L., Bachmann, K., 2002. Morphological, In: Ministerio dell0Economia Nazionale R. Ufficio Geolo- evolutionary and taxonomic aspects of Australian and New gico (Ed.), Memorie della Carta Geologica d0Italia, vol. 10. Zealand Microseris (Asteraceae). Austral. J. Bot. 50, Provveditorato Generale dello Stato, Rome. 127–143. Quilty, P.G., 1994. The background: 144 million years of Weber, W.A., 1985. The genus Teloxys (Chenopodiaceae). Australian palaeoclimate and palaeogeography. In: Hill, Phytologia 58, 477–478. R.S. (Ed.), History of the Australian Vegetation: Creta- Wikstro¨ m, N., Savolainen, V., Chase, M.W., 2001. Evolution ceous to Recent. Cambridge University Press, Cambridge, of the angiosperms: calibrating the family tree. Proc. R. pp. 14–43. Soc. London 268, 2211–2220. Rilke, S., 1999. Revision der Sektion Salsola s.l. der Gattung Wilson, P.G., 1975. A taxonomic revision of the genus Salsola (Chenopodiaceae). Bibl. Bot., vol. 149. Schweizer- Maireana (Chenopodiaceae). Nuytsia 2, 2–83. bart, Stuttgart. Wilson, P.G., 1980. A revision of the Australian species of Rogstad, S.H., 1992. Saturated NaCl-CTAB solution as a Salicornieae (Chenopodiaceae). Nuytsia 3, 3–154. means of field preservation of leaves for DNA analysis. Wilson, P.G., 1983. A taxonomic revision of the tribe Taxon 41, 701–708. Chenopodieae (Chenopodiaceae) in Australia. Nuytsia 4, Schu¨ tze, P., Freitag, H., Weising, K., 2003. An integrated 135–262. molecular and morphological study of the subfamily Wilson, P.G., 1984. Chenopodiaceae. In: George, A.S. (Ed.), Suaedoideae Ulbr. (Chenopodiaceae). Plant Syst. Evol. Flora of Australia, vol. 4. Australian Government Publish- 239, 257–286. ing Service, Canberra, pp. 81–330. Scott, A.J., 1978. A revision of the Camphorosmoideae Winkworth, R.C., Grau, J., Robertson, A.W., Lockhart, P., (Chenopodiaceae). Feddes Repert 89, 101–119. 2002a. The origins and evolution of the genus Myosotis L. Shepherd, A., Waycott, M., Calladine, A., 2004. Radiation of (Boraginaceae). Mol. Phylogenet. Evol. 24, 180–193. the Australian Salicornioideae (Chenopodiaceae)—based Winkworth, R.C., Wagstaff, S.J., Glenny, D., Lockhart, P., on evidence from nuclear and chloroplast DNA sequences. 2002b. Plant dispersal N.E.W.S. from New Zealand. TREE Am. J. Bot. 91, 1387–1397. 17, 514–520. ARTICLE IN PRESS 80 G. Kadereit et al. / Organisms, Diversity & Evolution 5 (2005) 59–80

Wu, C.I., Li, W.-H., 1985. Evidence for higher rates of corroborates paleontological estimates. Mol. Phylogenet. nucleotide substitution in rodents than in man. Proc. Natl. Evol. 15, 462–472. Acad. Sci. USA 82, 1741–1745. Zhu, G.L., 1996. Origin, differentiation, and geographic Xiang, Q.Y., Soltis, D.E., Soltis, P.S., Manchester, S.R., distribution of the Chenopodiaceae. Acta Phytotax. Sin. Crawford, D.J., 2000. Timing the Eastern Asian-Eastern 34, 486–504. NorthAmerican floristic disjunction: molecular clock