Telopea Journal of Plant Systematics Vo I 12(1) • 2008 Botanic Gardens Trust SYDNEY National Herbarium of New South Wales TELOPEA is published by the National Herbarium of New South Wales, Royal Botanic Gardens Sydney. It covers the field of systematic botany in Australia and the Asia-Pacific region, specialising in the flora of New South Wales. Telopea is published twice yearly. Annual subscription Within Australia* Individuals $66* Institutions $100* * This subscription includes GST. Overseas Individuals $100* Institutions $160* (includes postage, surface mail and handling) Subscriptions start with the first issue of the year. Subscription details, with a cheque or money order made out to the Royal Botanic Gardens Sydney, should be sent to: The Administration Officer Mount Annan Botanic Gardens Mount Annan Drive Mount Annan, NSW 2567, Australia Website: http://plantnet.rbgsyd.nsw.gov.au/Telopea Cover Artwork Telopea speciosissima (Sm.) R. Br., adapted by Helen Stevenson from the original by David Ma Telopea Journal of Plant Systematics 12(1): 1-145 • 2008 CONTENTS Proceedings of the 2006 Australian Systematic Botany Society (ASBS) conference Large indels obscure phylogeny in analysis of chloroplast DNA (frnL-F) sequence data: Pomaderreae (Rhamnaceae) revisited Jurgen Kellermann and Frank Udovicic 1-22 Byhlis guehoi (Byblidaceae), a new species from the Kimberley, Western Australia Allen Lowrie and John G. Conran 23-29 A new species and section of Utricularia (Lentibulariaceae) from northern Australia Allen Lowrie, Ian D. Cowie and John G. Conran 31-46 From populations to communities: understanding changes in rainforest diversity through the integration of molecular, ecological and environmental data Maurizio Rossetto 47-58 Two new species of Commersonia (Malvaceae setisu lato) from south-eastern Australia Carolyn F. Wilkins Lachlan M. Copeland and Barbara A. Whitlock 59-69 The genus Cycas (Cycadaceae) in China K.D. Hill 71-118 The genus Cycas (Cycadaceae) in The Philippines A.J. Lindstrom, K.D. Hill and L.C. Stanberg 119-145 Proceedings of the 2006 Australian Systematic Botany Society (ASBS) conference The first four papers in this issue of Telopea are a sample of those presented at the 2006 Australian Systematic Botany Society (ASBS) conference. This conference, held 13-15 November 2006 at James Cook University (Cairns) was the first ASBS meeting in the tropics for more than a decade. Over 60 local and international delegates heard 40 presentations on a wide range of topics in plant systematics and evolution. This is the first time Telopea has published papers from an ASBS meeting as a group. It is our hope that it is the beginning of a tradition. Telopea 12(1): 1-145 • 2008 Scientific Editors Elizabeth Brown, Darren Crayn Editor of Publications Gary Bridle Typesetting and Production Assistance Debby McGerty and Julia Sideris Other members of Editorial Committee Brett Summerell, Peter Wilson, Joy Everett and Darren Crayn ISSN 0312-9764 Telopea 11(4) was distributed on 29 June 2007. National Herbarium of New South Wales Royal Botanic Gardens Sydney Mrs Macquaries Rd Sydney NSW Australia 2000 Telopea 12(1) 1-22 Large indels obscure phylogeny in analysis of chloroplast DNA (frnL-F) sequence data: Pomaderreae (Rhamnaceae) revisited Jurgen Kellermann1'2 3 and Frank Udovicic2 ‘School of Botany, The University of Melbourne, VIC 3010, Australia 2National Herbarium of Victoria, Royal Botanic Gardens Melbourne, Birdwood Avenue, South Yarra, VIC 3141, Australia 3Current address: State Herbarium of South Australia, Plant Biodiversity Centre, P.O. Box 2732, Kent Town, SA 5071, Australia Author for correspondence: [email protected] Abstract Phylogenetic analysis of 69 ingroup-taxa of Pomaderreae using frnL-F sequences confirm the monophyly of the tribe. The analysis was impeded by a paucity of informative characters and the presence of apparently homoplasious indel characters and base changes within the P8 region of the frnL intron: the strict consensus tree of the triiL-F analysis is less resolved and had fewer supported clades than in a previous ITS analysis (Kellermann et al. 2005). The backbone of the cladogram is not supported and relationships between genera/clades are somewhat uncertain. The genera Cryptandra, Stenanthemum and Polianthion are well supported. Pomaderris groups with Siegfriedia and Trymaliutn, but only individual clades within these genera receive support. Blackallia biloba is related to two atypical species of Stenanthemum and B. connata to Cryptandra, but this grouping depends on the exclusion of homoplasious indel characters. Species of Spyridium only group in one clade when these indels are excluded, otherwise they are located in a polytomy at the base of the cladogram. The results mostly agree with earlier findings using ITS sequence data. Two new genera containing atypical species of Stenanthemum are suggested. A synopsis of the Australian genera of Rhamnaceae is provided. Paper from the Australian Systematic Botany Society Conference held in Cairns, November 2006 © 2008 Royal Botanic Gardens and Domain Trust ISSN0312-9764 2 Telopea 12(1): 2007 Kellermann and Udovicic Introduction Australia has a very rich Rhamnaceae flora with about one quarter of the world’s species (c. 250 out of 950) occurring in the country. The majority of species (over 90%) belong to the tribe Pomaderreae, which is almost endemic to Australia. The remaining 10% of species are mostly from genera that are also widespread in the Malesian or Pacific region, and some species occur in southern Australia. A synopsis of the Australian genera of Rhamnaceae is presented in Table 1. Species of Pomaderreae are found mainly in the temperate to semi-arid southern regions of Australia, but some occur in the tropical north, and arid centre of the continent, and eight taxa are found in New Zealand. There are about 230 species, which are currently classified in eight genera (Table 1). The complex taxonomic history of the major genera in the tribe is reviewed in Kellermann et al. (2005) and Kellermann (2007). The tribe has been the focus of recent and on-going research in the Australian Rhamnaceae. Walsh revised Pomaderris and published an infrageneric classification of the genus (e.g., Walsh 1988, 1990; Walsh & Coates 1997). Rye (1995, 2001) re-instated the genus Stenanthemum and revised species from Western Australia (e.g., Rye 1996b). Table 1. Currently accepted genera of Australian Rhamnaceae. Tribal classification follows Medan & Schirarend (2004) and Richardson et al. (2000b). Six genera of Rhamnaceae are not assigned to a tribe: five of these occur in Australia. Tribe Genus Species in Australia Pauureae Reissek ex Endl. Hovenia Thunb. 1 (introduced) Ziziphus Mill. 4 Colletieae Reissek ex Endl. Discaria Hook. 2 Phyuceae Reissek ex Endl. Noltea Rchb. 1 (introduced) Gouanieae Reissek ex Endl. Gouania Jacq. 2 Pomaderreae Reissek ex Endl. Blackallia C.A.Gardner 2 Cryptandra Sm. c. 55 Polianthion K.R.Thiele 4 Pomaderris Labill. c. 70 Siegfriedia C.A.Gardner 1 Spyridium Fenzl 40-45 Stenanthemum Reissek c. 30 Trymalium Fenzl 13 Rhamneae Hook.f. Dallachya F.Muell. 1 Rhamnus L. 2 (1 native, 1 introduced) Sageretia Brongn. 1 Ventilagineae Hook.f. Ventilago Gaertn. 3 Genera incertae sedis Alphitonia Reissek ex Endl. 5 Colubrina Rich, ex Brongn. 1 Emmenosperma F.Muell. 2 Granitites Rye 1 Schistocarpaea F.Muell. 1 Pomaderreae (Rhamnaceae) revisited Telopea 12(1): 2008 3 An atypical species of Pomaderris was excluded from the tribe and segregated into its own genus, Gtanitites (Rye 1996a). Thiele & West (2004) and Thiele (2007) elucidated the delimitations of the genera Cryptandra, Spyridiutn and Stenanthemum. Bean (2004) published new species of Cryptandra and Stenanthemum for Queensland. Kellermann (2006b, 2007) clarified the position of several Spyridium taxa that were misplaced in other genera. The revision of the south-eastern species of Cryptandra has resulted so far in three publications (Kellermann 2006a, 2006c; Kellermann & Udovicic 2007). Kellermann et al. (2005) published a molecular phylogeny using ITS sequence data, as a result of which a new genus, Polianthion K.R.Thielc, was established (Kellermann et al. 2006), The ITS phylogeny confirmed the monophyly of Pomaderreae, corroborating earlier results by Richardson et al. (2000a) and Fay et al. (2001). The clades found in the strict consensus tree were mostly consistent with the currently accepted genera in the tribe. Some species were clearly misplaced, but re-examination of the morphology of these species confirmed their placement in the molecular phylogeny. The major genera/ clades, except Stenanthemum and Blackallia, received moderate to strong bootstrap and jackknife support. Stenanthemum was split into two well-supported clades with the atypical St. gracilipes inserted in between the two clades. Blackallia biloba and St. grandiflorum were sister taxa, and not allied to any of the remaining genera; B. connata was placed in Cryptandra. This study was initiated to clarify questions that could not be resolved in the analysis of ITS data (Kellermann et al. 2005) and to augment the molecular data-set available for Pomaderreae with sequences from the trnL-F region of cpDNA. In this paper, the resulting phylogenies of the trnL-F analysis are presented and we report on the presence of unforeseen problems relating to the structure of the trnL-F region, which hampered and complicated the cladistic analysis of the data. The results add to the base of knowledge needed for the completion of the Flora of Australia treatment of Rhamnaceae (K.R. Thiele, F. Udovicic, N.G. Walsh & ]. Kellermann, in prep.). Materials & Methods Sixty-nine ingroup taxa were sequenced from all genera of Pomaderreae. The outgroup consisted of five species from related tribes of Rhamnaceae. Voucher and collection details are listed in Appendix 1. Manuscript names of taxa are used as they are listed in FloraBase (http://florabase.calm.wa.gov.au) at the time of writing (Mar. 2007). In this paper, the abbreviations used for the genera Pomaderris, Polianthion, Siegfriedia, Spyridium and Stenanthemum are ‘P.\‘Pol.’, ‘Si.’fSp.’ and ‘St.’. Choice of DNA region The trnL-F region consists of the complete trnL intron, trnL 3’ exon, and the intergenic spacer (IGS) between the trnL and the trnF genes of the chloroplast genome. These genes encode the chloroplast’s transfer RNA for Leucine and Phenylalanine, respectively. Both the trnL intron and the trnL-F IGS are non-coding regions. The trnL intron is the only group I intron in the chloroplast genome and has a conserved secondary structure (Simon et al. 2004). 4 Telopea 12(1): 2007 Kellermann and Udovicic The fr/zL-F region was first used in phylogenetic analyses of Gentiana L. (Gielly & Iaberlet 1994) and Crassulaceae (Ham et al. 1994). Currently, it is applied in studies at all taxonomic levels. Borsch et al. (2003) used the trnT-trnF region, which includes the frnL-F region, to infer a phylogeny of basal angiosperms. Most frequently, however, frzzL-F is used for infrafamilial studies, e.g., in Araliaceae (Plunkett et al. 2004), Gentianaceae (Yuan et a!. 2003), Oxylobium Andrews and related genera (Crisp & Cook 2003), or Acacia Mill. (Murphy et al. 2000). The region has already been employed to examine the relationships of Rhamnaceae with other families (Sytsma et al. 2002; Thulin et al. 1998), to resolve the tribal limits of the family (Fay et al. 2001; Richardson et al. 2000a, b), and in studies on the genera Ceanothus Mill. (Islam & Simmons 2006), Phylica L. (Richardson et al. 2001) and Rhamnus L. s. lat. (Bolmgren 8c Oxelman 2004). DNA isolation and sequencing Genomic DNA was isolated using the method described in Kellermann et al. (2005). A few samples of the fr/zL-F region had to be purified using the QIAquick Gel Extraction Kit (QIAGEN). The fr/iL-F region was amplified using the primers designed by Taberlet et al. (1991). For most species the whole region was amplified with primers C and F with one hold at 95°C for 15 min preceding 30 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, and followed by one hold at 72°C for 5 min. In other species, the trnL intron and the frnL-F IGS had to be amplified separately using primer pairs C/D and E/F, respectively. While the fr/iL-F IGS amplified readily, the annealing temperature frequently had to be lowered to 55°C or 52°C when amplifying the fr/iL intron. Some species with a low yield of genomic DNA, in particular from herbarium specimens, had to be amplified with a semi-nested PCR protocol (Udovicic 8c Murphy 2002) using products from a previous amplification with primers C and F as template for a second round of PCR. In this second round the fr//L intron and the fr/zL-F IGS were amplified using the primer pairs C/D and E/F, respectively, and a lower annealing temperature of 55°C. Amplification with primers C and F in the second round of PCR was unsuccessful, a fact already noted by Richardson et al. (2001) for other species of Rhamnaceae. Phylogenetic analysis Sequences were aligned as outlined in Kellermann et al. (2005) and analysed using the computer program PAUP*, version 4.0bl0 (Swofford 2002). Individual base positions were coded as unordered multistates and gaps were treated as missing data. Insertion/ deletion (indel) characters were coded as single binary characters. Uninformative characters were excluded from the data matrix. A two step search was employed, since the computer ran out of memory when using a more straightforward search strategy (e.g., Kellermann et al. 2005). In the first round, a heuristic search was performed with 1000 replicates using random stepwise addition of taxa and TBR branch swapping. Only five trees were held in each replicate. All shortest trees collected in the 1000 replicates were then used as starting trees for a second round of heuristic search. All trees were swapped to completion, or until a maximum number of 10,000 trees was produced, at which point the search was limited and the 10,000 trees saved were swapped. Strict consensus and majority-rule consensus trees were calculated for the 10,000 equally parsimonious trees. Trees were rooted using the outgroup taxa (Maddison et al. 1984). Pomaderreae (Rhamnaceae) revisited Telopea 12(1): 2008 5 To test the support for nodes in the tree, both bootstrap (Felsenstein 1985) and jackknife (Farris et al. 1996) values were calculated in PAUP*. Bootstrap analysis was carried out with 1,000 replicates, TBR branch swapping and a limit of 1,000 trees per replicate. To calculate jackknife values, the ‘Jac’ emulation as implemented in PAUP* was performed with 100,000 replicates and 37% deletion, using the fast heuristic search option. Results Sequences Sequences were obtained for 69 species of Pomaderreae and five outgroup species from related tribes. Two accessions were obtained for each of six taxa to test infraspecific variation: Cryptandra amara, C. mutila, Siegfriedia darwinioides, Spyridium globulosum, Sp. parvifolium and Trymalium ledifolium. The sequence variation between two sequences of the same species was si.6% in all cases and in some cases, sequences were identical. Because of the low sequence variation, only a single sequence of each species, the first listed in Appendix 1, was used in the analysis of the trnL-F sequence data. Large indels In the alignment of the trnL-F sequences, several large indels were identified. In particular, one deletion of approximately 125 base pairs (indel no. 9) seemed to have occurred in unrelated species, a result revealed in the first analysis (A). Subsequently two more analyses were undertaken to explore the effect of indel no. 9 on the resulting topology of the tree. The following analyses of the fniL-F data-set were carried out: Analysis A included all species and characters; Analysis B excluded two of the three sequences with indel no. 9, namely those of Pomaderris rotundifolia and Cryptandra triplex, but included all characters; Analysis C included all species, but excluded the DNA region in which indel no. 9 occurred, and all potential characters therein (following Quandt et al. 2004; see below for discussion). Characteristics of sequences 8i phylogeny The alignment of the frrcL-F data set had 1145 base positions. Four regions in the alignment were ambiguous and unalignable and therefore excluded from the analysis. This reduced the data-set by 46 characters to 1099 base positions. Twenty-three indels were identified in the alignment and coded separately using the simple indel coding method of Simmons and Ochoterena (2000). When all species and all characters were included in analysis A, the alignment provided 90 parsimony-informative characters (8.2%) and 21 out of 23 indel characters were potentially informative characters. In analysis B, the number of parsimony-informative characters in the alignment was reduced due to the exclusion of two species: 87 base characters (7.9%) and 20 of 23 the indels were potentially informative. Analysis C excluded a stretch of 261 bases from the alignment and reduced the number of 6 Telopea 12(1): 2007 Kellermann and Udovicic characters to 838 base positions; this also eliminated 8 indel characters from the data-set. Analysis C included 67 potentially informative base characters (8.0 %), and 13 parsimony-informative indels. In all three parsimony analyses, the maximum number of 10,000 trees was reached when using the two step search strategy. The trees of analysis A had a CI=0.566 and RI=0.789. The Cl and RI for analysis B were 0.564 and 0.786, respectively. The trees in analysis C had a 0=0.572 and a RI=0.805. The strict consensus tree of analysis A (Fig. 1) showed 30 nodes common to all most parsimonious trees (27 nodes common to the ingroup); 23 nodes had bootstrap support (BS) and 22 nodes had jackknife support (JS) s50%. The strict consensus of analysis B (excluding the sequences containing indel no. 9; Fig. 2), had 28 nodes (25 nodes' in the ingroup), of which 21 had bootstrap support and 20 nodes had jackknife support above 50%. Analysis C had 24 nodes present in the strict consensus tree (22 nodes in the ingroup; Fig. 3), statistical support a50% in the bootstrap and jackknife analyses was obtained for 19 nodes. Cladogram topology The strict consensus trees for analyses A and B are shown in Figures 1 and 2. Tree topology is the same in both cladograms, except that in analysis A the species containing indel no. 9 are grouped in one clade within the genus Trymalium. This clade is indicated in bold in Figure 1. Bootstrap (BS) and jackknife (JS) values differ only slightly between the analyses. The strict consensus in analysis C (Fig. 3) has a similar topology to the previous two trees, but is less resolved. However, the genus Spyridium was resolved in one clade (at node 3) in analysis C, and Blackallia connata grouped with Cryptandra (node 24) and not with Stenanthemumgracilipes, St. grandiflorutn ms and B. biloba (clade at node 22). Only the tree in Figure 2 (analysis B) is discussed in the following sections and Figure 1 and 3 are only referred to when there are differences between the analyses. Monophyly of the tribe Pomaderreae is very strongly supported with 100% bootstrap and jackknife support. Sister to Pomaderreae is either Schistocarpaea johnsonii (not placed in any tribe by Richardson et al. 2000b), Adolphia californica (tribe Colletieae), or a weakly supported clade (BS: 57%; JS: <50%) containing Alphitonia aff. irtcana (unplaced genus), Ceanothus coeruleus (unplaced genus) and Phylica buxifolia (tribe Phyliceae). The backbone of the cladogram lacks bootstrap or jackknife support above 50% and thus the relationships among the main clades (genera) are unresolved. Of the currently accepted genera, only Cryptandra and two clades of Stenanthemum have bootstrap/ jackknife support. The species of Spyridium do not group in a clade in the strict consensus tree in analyses A and B. However, they form a clade in 94% of trees in a majority rule consensus tree (majority rule tree not shown). In analysis C the species of Spyridium are united in a clade, albeit without bootstrap or jackknife support above 50%. Within Spyridium, three species from New South Wales {Sp. scortechinii, Sp. buxifolium and Sp. burragorang) form a weakly supported clade at node 4. The two Tasmanian species included, Sp. ulicinum and Sp. gunnii, are sister taxa (node 6; BS: 61%, JS: 58%). Spyridium mucronatum and Sp. cordatum are strongly supported as sister taxa (node 7), but their relationship with the third Western Australian species included, Sp. globulosum, is unresolved. Spyridium daltonii and S. xramosissimum from the