The Lejeunea tumida species group is positively polyphyletic (Lejeuneaceae: Jungermanniopsida)

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Australian Systematic Botany, 24, 10–18

The Lejeunea tumida species group is positively polyphyletic (Lejeuneaceae: Jungermanniopsida) Matt A. M. Renner A, Elizabeth A. Brown A,C and Glenda M. Wardle B A

National Herbarium of New South Wales, Mrs Macquaries Road, Sydney, NSW 2000, Australia. School of Biological Sciences, Heydon Laurence Building A08, University of Sydney, Sydney, NSW 2006, Australia. C Corresponding author. Email: [email protected] B

Abstract. A phylogeny based on nrITS1 and trnL–F sequences resolves the Lejeunea tumida species group polyphyletic with individuals belonging in two clades either side of the basal-most node within Lejeunea. It is impossible for the Lejeunea tumida species group to be more polyphyletic and still be attributed to the same genus under the existing generic classification. A simulation-based approach to testing the null hypothesis of group monophyly rejects this at the P < 0.01 level of significance. Bayesian tests find very strong support for polyphyly, given the data. The monophyly of L. tumida s.s. + L. colensoana is fully supported; however, although Lejeunea tumida s.s. is nested within L. colensoana, this position is not supported. Both L. oracola and L. rhigophila are resolved as monophyletic. Whereas there is moderate support for the monophyly of L. rhigophila, there is no support for the monophyly of L. oracola. Neither is the monophyly of L. oracola + L. rhigophila supported in Bayesian or parsimony analysis.

Introduction With a handful of exceptions (Pfeiffer et al. 2002), new liverwort species have been described because they are morphologically distinguishable, in terms of either overall similarity or specific character differences. Although morphological discontinuity is the justified basis of the taxonomic species concept (de Queiroz 1998), during the past decade, concerns have been raised about the ability of morphology to provide conclusive data on both establishing relationships between species and establishing species boundaries (Reiner-Drehwald and Goda 2000; Gradstein et al. 2003; Hienrichs et al. 2004; Ilkiu-Borges 2005). Morphology may confuse and confound endeavours to resolve relationships because of lack of stable morphological boundaries between species (Feldberg and Hienrichs 2006), and variability in gametophytic characters (Hienrichs et al. 2004). Both may result in too much being read into subtle morphological differences that do not reflect phylogenetic groups. In the Lejeuneaceae, both scenarios have been demonstrated. Hartmann et al. (2006) found that five characters that were used to circumscribe species of Bryopteris (Stotler and Crandall-Stotler 1974) were diffusely distributed, and formed no sound basis for identifying morphological groups congruent with molecular clades. In contrast, Hienrichs et al. (2009) found that despite clear genetic separation, there was extensive morphological overlap among phylogenetic entities within Marchenisia brachiata (Sw.) Schiffn. The decoupling of morphological and molecular variation and resultant conflict between morphological and molecular groups observed in the Lejeuneaceae, and a range of other bryophyte lineages (Shaw and Allen 2000; Vanderpoorten 2004), demonstrates that morphological data must be interpreted  CSIRO

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with caution in bryophytes (Hienrichs et al. 2003). A cautious approach to interpretation of morphology has generally resulted in broad morphological-species concepts being applied (Reiner-Drehwald and Goda 2000; Hienrichs et al. 2001, 2004; Zhu and Gradstein 2005; Burghardt and Gradstein 2008), resulting in synonymisation of species justified by the observation of absence of morphological discontinuity. Lejeunea tumida Mitt. was described by Mitten (1855) for plants with acute underleaf lobes, tumid lobules and inflated perianths. Mitten cited three specimens in his protologue, but did not identify a type. The first specimen listed by Mitten, collected by Sinclair near Auckland, was designated lectotype by Grolle (1982). Stephani (1896) described Taxilejeunea colensoana Steph., which also possessed tumid perianths. Both L. tumida and T. colensoana are small pellucid plants, and this aspect, in combination with their shared possession of inflated perianths led Schuster (1963a) to the conclusion that T. colensoana was ‘evidently a synonym’ of L. tumida, a view also accepted by Grolle (1982). The inflated perianths of L. tumida are highly unusual (Reiner-Drehwald and Schäfer-Verwimp 2008), and as L. tumida s.l. was the only regional species with this feature it was arguably New Zealand’s most distinctive Lejeunea species. This distinctiveness led Schuster (1963a) to propose a new monotypic subgenus for L. tumida. Lejeunea subg. Sphaerocolea R.M. Schust. was characterised by its (1) multicellular lobular apical tooth, (2) heavily inflated lobules and (3) inflated ecarinate perianths. Schuster (1963a) noted that the lobules of L. tumida s.l. were ‘quite unique within Lejeunea s.lat., the 2-celled apical tooth is found again in no other Lejeunea known to me, and the almost ovoid-spheroid lobule form is also without any exact 10.1071/SB10047

1030-1887/11/010010

The Lejeunea tumida species group is positively polyphyletic

parallel’. However, these lobule characters agree with neither Mitten’s protologue, nor the lectotype designated by Grolle (1982). Contradictory accounts of L. tumida s.l. morphology implies that (1) L. tumida s.l. is a single monophyletic entity with high morphological variability, (2) L. tumida s.l. is a monophyletic complex of two or more morphological entities or (3) L. tumida s.l. is a polyphyletic aggregate of two or more morphological entities. Here, we test each of these alternatives by constructing a molecular phylogeny based on nrITS1 and trnL–F, including a total of 21 individuals encompassing the diversity of forms described by Mitten (1855), Schuster (1963a) and Grolle (1982). Throughout the present paper, we refer to entities resolved on the basis of morphological characters re-examined in lieu of our phylogeny by the names presented by Renner et al. (2010). We refer to the group of plants formerly attributed to L. tumida as the L. tumida species group. Materials and methods Taxon sampling We collected at a range of sites, covering the northern half of the North Island, New Zealand, in June 2007. Field-collected material was rapidly air-dried and placed onto silica gel. In total, we collected molecular data from 21 individuals belonging to the L. tumida species group. Additional samples obtained were L. drummondii Taylor, L. epiphylla Colenso (non Mitt.), L. exilis (Sw.) Reinw., Blume & Nees, L. helmsiana Steph., L. tasmanica Gottsche and Rectolejeunea sp. from field-collected material; and species of Lejeunea (including those formerly assigned to Neopotamolejeunea) and Taxilejeunea from GenBank where both nrITS1 and trnL–F sequences were available. These taxa were included to increase the severity of the test of monophyly imposed by our dataset. Outgroup sequences were obtained from GenBank, including species of Cololejeuneoideae and Cheilolejeunea, which Wilson et al. (2007) found to be sister groups to the Lejeunea generic complex sensu Schuster (1963b). We rooted our trees on Anoplolejeunea conferta (C.F.W.Meissn. ex Spreng.) A.Evans, as per tree topology in Wilson et al. (2007). We note that although L. tasmanica has been regarded as a synonym of L. drummondii, substantiative morphological differences exist between the type specimens of these species. In particular, L. drummondii is paroicous, whereas L. tasmanica is autoicous. Accessions corresponding to L. drummondii and L. tasmanica do not group together in the molecular phylogeny presented below, and as such, we treat them here as distinct species. A paper formally reinstating L. tasmanica and clarifying the circumscription of L. drummondii is in preparation. Marker selection Two nucleotide regions were employed in our study, one from the chloroplast genome, one from the nuclear genome. The nuclear ribosomal ITS region has been used to reconstruct infra-specific and inter-specific relationships in the Lejeuneaceae as well as other leafy liverworts and bryophytes (Chiang and Schaal 1999; Shaw 2000; McDaniel and Shaw 2003; Hartmann et al. 2006; Wilson et al. 2007). Nuclear ITS sequence data have been included in about two-thirds of all plant phylogenetic studies, the widespread use of this marker is testament to its utility,

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particularly in studies aiming to resolve relationships between closely related species (Álvarez and Wendel 2003). The chloroplast DNA trnL–F region has been used to reconstruct species and family-level relationships within a wide range of plant groups, and within the bryophytes exhibits sequence-length variation (Quandt and Stech 2004). Together, nrITS and trnL–F are the most widely employed markers in molecular phylogenetic studies of bryophytes (Stech and Quandt 2010). Although variable among species, trnL–F displays the desirable characteristic of being largely invariant within species in some groups of higher plants (Woods et al. 2005). We use these molecular markers to inform relationships among individuals. DNA amplification and sequencing A sample weighing 2–5 mg (dry weight) of clean shoot tips was excised from each individual. Total DNA was extracted from air-dried, field-collected material with the DNeasy Plant Minikit (QIAGEN Pty Ltd, Sydney, Australia), following the manufacturers instructions. For all samples, the chloroplast trnL–F region (including the trnL intron and the trnL–trnF intergenic spacer) was amplified and sequenced using the primers A50272 and B49317 (Taberlet et al. 1991) with 55C annealing following the protocol described by Heslewood and Brown (2007). We also amplified and sequenced the nuclear ITS1 region with the primers Bryo18SF and Bryo5.8SR, following the protocol described by Hartmann et al. (2006). Both strands of all PCR products were sequenced with the Big-Dye Terminator v3.1 Sequencing Kit. PCR products were purified with ExoSAP (Amersham Biosciences, Uppsala, Sweden). Because our PCR products were generally weak, 3.5 mL of the ExoSAP product provided enough template for strong signal from sequencing reactions. Sequences were generated on a capillary sequencer and assembled, proofed and edited with Sequencher v. 4.5 (Gene Codes Corp., Ann Arbor, Michigan, USA). All sequences were compared against results of BLAST searches of GenBank to avoid inclusion of a contaminated sequence, particularly for nrITS (Camacho et al. 1997; Álvarez and Wendel 2003). All new sequence data have been deposited in GenBank (Appendix 1). Phylogenetic analysis Length variation was observed in both trnL–F and nrITS1 markers, and sequences were aligned using MUSCLE (Edgar 2004), followed by manual editing in BioEdit (Hall 1999), which was necessary in a few cases to homogenise indels across sequences. The alignment is available from the first author on request. We treated gaps as missing data because contiguous gap characters representing multiple base deletions are likely to be non-independent. The sequences were tested for homogeneity of nucleotide composition among taxa using the c2-test for compositional homogeneity in PAUP*4.0b10 (Swofford 2002), considering only parsimony-informative positions because constant and uninformative sites do not contribute to potential phylogenetic biases (Phillips et al. 2006). Incongruence between partitions was tested using the partition-homogeneity test implemented in PAUP*. Phylogenetic trees were estimated under the maximum likelihood (ML) criterion in GARLI 0.951 (Zwickl 2006). We used the Akaike information criterion to

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determine which of the seven substitution schemes considered by the package jModelTest 0.1.1 (Guindon and Gascuel 2003; Posada 2008) provided the best fit for each region separately and the concatenated data. Model parameters were specified on the basis of an estimated ML optimised tree. Estimated model parameter values were then fixed and heuristic searches run until 10 000 generations had been completed without an improvement in tree likelihood. Ten independent runs yielded the same tree topology. Clade support was estimated by using the ML bootstrap, estimated from 100 non-parametric bootstrap replicates, with each replicate automatically terminating after 5000 generations without improvement in tree likelihood. We also assessed clade support under the parsimony criterion using the non-parametric parsimony bootstrap (Felsenstein 1985). Parsimony bootstrap provides an informative complement to the likelihood bootstrap because it assesses node support from data, assuming an underlying substitution model wherein all transformations are equiprobable. Strong support under both parsimony and likelihood indicates robustness independent of analytical method. We excluded all parsimony-uninformative characters, and performed 500 replicate heuristic searches based on 25 replicate random addition sequence searches, TBR branch swapping, collapsing branches of zero length, no upper limit on MAXTREES (other than that imposed by memory), and one tree held at each step, in PAUP*b4.10 (Swofford 2002). Bayesian estimation of phylogeny for the combined trnL–F plus nrITS1 dataset was completed to estimate clade posterior-probability values and to assess whether more highly parameterised, mixed substitution-model analyses influenced our phylogeny estimation (Lemmon and Moriarty 2004) by using MrBayes 3.1 (Huelsenbeck and Ronquist 2001; Huelsenbeck et al. 2003; Ronquist and Huelsenbeck 2003). In the Bayesian analysis, the data matrix was partitioned by gene, and an unlinked GTR + I + G substitution model was specified for the substitution matrix. The six states, portion of invariant sites, and the six gamma categories were all estimated during the Monte Carlo search of the dataset. This allowed our search to optimise both tree topology and substitution-model parameters simultaneously. Two runs, each with four chains and chain heating of 0.2, were run for 2 million generations and sampled every 1000 generations. Bayesian posterior branch probabilities were obtained by taking the majority consensus of the sampled trees from both runs, excluding the first 45 trees (45 000 generations) as burn-in on the basis of stabilisation of –ln L scores. Testing the significance of polyphyly The significance of the polyphyly of L. tumida species group was established in two ways, in both tests the null hypothesis was that the L. tumida species group was monophyletic. First, within a frequentist framework, we asked whether any observed difference in length between unconstrained trees, and trees with the L. tumida species group constrained to be monophyletic, was significant. For this, we followed a method modified from Huelsenbeck et al. (1996), proposed by Swofford in the Mesquite manual (Maddison and Maddison 2009). First, we enforced monophyly on the L. tumida species group and calculated the most likely tree given this constraint in

M. A. M. Renner et al.

GARLI. We then simulated sequence evolution on the topology of this ML-constrained tree under the substitution model estimated for our concatenated data in the unconstrained analysis. We generated 100 simulated DNA matrices of 1638 characters in MESQUITE v.2.6 (Maddison and Maddison 2009). We then estimated the most parsimonious unconstrained tree, and the most parsimonious tree with monophyly of the L. tumida species group enforced for each simulated matrix in PAUP*b4.10 (Swofford 2002). We used parsimony because it allowed the test to be conducted quickly and easily. Both constrained and unconstrained trees were estimated on the basis of twenty replicate random addition sequence searches, TBR branch swapping, collapsing branches of zero length, no upper limit on MAXTREES (other than that imposed by memory), and one tree held at each step. The difference in length between constrained and unconstrained MP trees for each simulated dataset formed the distribution against which the significance of our observed length difference was assessed. Second, within a Bayesian framework, we compared constrained and unconstrained trees by means of the Bayes factor (Kass and Raftery 1995; Nylander et al. 2004) derived from harmonic mean –ln L scores from analyses run in MrBayes.

Results Alignments for the trnL–F and nrITS1 comprised 770 and 868 positions, respectively, sequence lengths for ingroup taxa (Lejeunea-generic complex) ranged from 411 to 456 bp for trnL–F and from 487 to 621 bp for nrITS1. The concatenated dataset totalled 1638 alignment positions, with 40% (49 135) of the cells in the data matrix scored as missing data for gaps in the sequence alignment. Base compositions were not statistically different across taxa for concatenated data (c2(d.f. = 222) = 229.06, P = 0.358), nor for the individual genes trnL–F (c2(d.f. = 222) = 114.31, P = 1.000) and nrITS1 (c2(d.f. = 222) = 198.80, P = 0.867). The partition-homogeneity test found no significant incongruence between molecular markers (combined lengths longer than original partition in two instances, P = (1 – (2/ 100)) = 0.98). The Akaike information criterion (AIC) selected the GTR + G model for nrITS1 (AIC = 25 300.2) and GTR + I + G model for trnL–F (AIC = 8597.5) and the concatenated data (AIC = 34 579.9); nucleotide frequencies and estimated substitution parameters for each dataset the parameters for each model are shown in Table 1. ML analysis of the concatenated dataset produced a single-most likely tree (–lnL = 17 113.8), within which the L. tumida species group is polyphyletic (Fig. 1). The 21 accessions are divided between two clades. Thirteen accessions group within one clade, with L. tumida s.s. nested within L. colensoana (Steph.) M.A.M. Renner. Although it is clear that L. tumida s.s. and L. colensoana go together, there is no support for L. tumida s.s. nesting within L. colensoana. The remaining eight accessions form a second clade which is subdivided into two clades, one comprising three accessions of L. rhigophila M.A.M.Renner from the central North Island the other comprising five accessions of L. oracola M.A.M.Renner from the north of the North Island. While the monophyly of L. rhigophila receives

The Lejeunea tumida species group is positively polyphyletic

Australian Systematic Botany

Table 1. Data characteristics and estimated substitution-model parameters for trnL–F, nrITS1 and the combined dataset Characteristic Aligned positions Variable sites Tree length (MP) CI/RI Best fit model A frequency C frequency G frequency T frequency A–C A–G A–T C–G C–T G–T a I –ln likelihood

trnL–F

nrITS1

Combined

770 130 614 0.565/0.759 GTR + I + G

868 435 2705 0.406/0.662 GTR + I

1638 565 3341 0.433/0.677 GTR + I + G

0.3853 0.0977 0.1240 0.3932 1.2722 2.0718 0.2383 1.0813 2.8174 1.0000 0.3440 0.0000

0.1872 0.2776 0.3003 0.2350 1.1832 3.5044 1.5393 0.8596 5.7483 1.0000 0.6320 –

0.2625 0.2126 0.2386 0.2863 1.0961 2.6851 0.7791 1.0757 4.6911 1.0000 0.4900 0.0630

–4210.7

–12 475.2

–17 113.8

some support, the monophyly of L. oracola + L. rhigophila is not supported in the Bayesian or parsimony analyses. Significance of polyphyly The most likely tree when monophyly was enforced on the L. tumida species group was 66 steps longer than the most likely unconstrained tree. The distribution of differences in length between most parsimonious constrained and unconstrained trees suggests that differences greater than six steps occur by chance of less than one time in 100. The critical value for P = 0.01 is a difference of six steps for parsimony analyses. If this threshold is extrapolated to the difference observed between our most likely constrained and unconstrained trees, we can reject the hypothesis that L. tumida is monophyletic at the 0.01 level of significance (difference = 66, P < 0.01). The Bayes factor, 2lnBF, is 298.5 for the unconstrained tree against the constrained tree, indicating that the unconstrained tree provides a significantly better fit to the underlying data. A Bayes factor (2lnBF) >10 indicates ‘very strong evidence’ against the null hypothesis (Kass and Raftery 1995). The harmonic mean calculated from post burn-in log-likelihood with monophyly of L. tumida enforced was –ln17 071.95; the harmonic mean without constraint was –ln16 922.70. Discussion Lejeunea tumida was resolved as polyphyletic by two independently sampled, non-coding genes from different genomes, one of which is biparentally inherited, whereas the other is uniparentally inherited, between which genetic transfer is a rare event even on an evolutionary timescale and between which genetic recombination is not known to occur as a recurrent process. Polyphyly of the L. tumida species group provides a significantly better fit for the molecular data than does monophyly, which is strongly and unequivocally contradicted. Despite their similar morphology, and previous placement within a single species, individuals of the L. tumida species group

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comprise two relatively distantly related groups that are polyphyletic across the basal-most node of the Lejeunea generic complex. It is not possible to be any more unrelated and still be attributed to the same genus under the existing generic classification. Although incongruence between morphology and molecules is well known in mosses (Shaw and Allen 2000; Vanderpoorten et al. 2001; Vanderpoorten 2004; Draper et al. 2007; Hedenäs 2008), most molecular tests of morphological species in leafy liverworts have found morphological species to be monophyletic (Hienrichs et al. 2004; Hartmann et al. 2006; Feldberg et al. 2007; Hentschel et al. 2007, 2009). The present study is the first within the Lejeuneaceae to have resolved a morphologically circumscribed species as polyphyletic, rather than paraphyletic (Hienrichs et al. 2009) or monophyletic (Hartmann et al. 2006). The L. tumida species group is among a small but growing group of polyphyletic morphological species identified among leafy liverworts; other examples include Frullania ericoides (Nees) Mont. (Hentschel et al. 2009), Marchesinia brachiata (Sw.) Schiffn. (Hienrichs et al. 2009) and Lophozia ventricosa (Dicks.) Dumort. (De Roo et al. 2007; Vilnet et al. 2008). Polyphyletic bryophyte species typically exhibit incongruence between molecules and morphology (Shaw and Allen 2000; Shaw et al. 2005; Draper et al. 2007), and demonstrated instances of morphological homoplasy conflicting with well supported molecular phylogenies are common (i.e. von Hagen and Kadereit 2002; Kearney and Stuart 2004; Whittall et al. 2006). Within each of the two clades of L. tumida sens. lat. there are two species pairs that are not well supported, L. tumida s.s. plus L. colensoana in one clade, and L. oracola plus L. rhigophila in the other. These four species are morphologically distinctive, although differences are subtle (Renner et al. 2010). There are many possible explanations for incongruence and lack of support for the monophyly of each species from molecular data. These include recent divergence such that gene-tree coalescence points predate speciation events (Avise et al. 1983; Rosenberg 2003), incomplete lineage sorting (Doyle 1992) and retention of ancestral polymorphism (Avise et al. 1987), presence of orthologous genes (Soltis et al. 1992) and ongoing hybridisation (King and Roalson 2008). Paraphyletic species may be common in plants (Rieseberg 1994; Crisp and Chandler 1996), and may be the norm in cases where peripheral isolation, long-distance dispersal, ecological speciation and any other process where a newly derived species starts out with a subset of the variation in the ancestral species (MacNair et al. 1989; Theriot 1992; MacNair and Gardner 1998; Shaw and Allen 2000). Evidence of incomplete lineage sorting may vary in persistence through time for different genes, because different gene lineages coalesce at different rates (Baum 1992). Predictions derived from population-genetic theory suggest that, given various assumptions about sample size, initial allelic frequencies and number of alleles, reciprocal monophyly between two sister species is typically achieved after some millions of generations (Knowles and Carstens 2007). However, incomplete lineage sorting seems an unlikely explanation in this case, given the congruent signals from unlinked nuclear and chloroplast genomes with regards the unsupported nesting of L. tumida s.s. within L. colensoana.

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Fig. 1. The single-most likely tree (–lnL = 17 113.8) resolved from analysis of the concatenated dataset. Values associated with branches are parsimony bootstrap, likelihood bootstrap and Bayesian posterior probability, respectively.

The four species identified within the L. tumida species group may have been treated as one because no morphological discontinuities were identified upon which to segregate morphological groups. In particular, the significance of variation in perianth morphology could be overlooked by a superficial homology assessment (Rieppel and Kearney 2002) of perianth characters. Variation in perianth morphology is usually understood in terms of how many carinae are present (Reiner-Drehwald and Schäfer-Verwimp 2008), and from this perspective, all perianths within the L. tumida species group would be viewed as equivalent, because obvious carinae are lacking from all species. Furthermore, readily accessible characteristics of gross-morphological features such as the size and shape of lobules, lobes and underleaves do not provide

unambiguous morphological gaps among species within the L. tumida species group (Renner et al. 2010). The correlation between morphological differences and species status is not universal in bryophytes; related species are known that share similar or even identical morphologies, e.g. mosses within the Grimmia laevigata (Bridel) Bridel complex (Fernandez et al. 2006) and liverworts such as European species of Porella (Bischler et al. 2006), the Aneura pinguis (L.) Dumort. complex (Wachowiak et al. 2007), Conocephalum (Odrzykoski and Szweykowski 1991) and Australasian Treubia (Pfeiffer et al. 2002). However, distantly related species that share similar, nearly identical morphologies are less well known, although one other instance has been identified within the Lejeuneaceae (Hienrichs et al. 2009).

The Lejeunea tumida species group is positively polyphyletic

Within the L. tumida species group, L. colensoana is more similar to L. rhigophila, a distantly related species, than to the more closely related L. tumida s.s. in its size, its lobule shape, its lobule arch comprising 3–5 cells, the inrolled lobule antical margin, and its inflated perianths lacking ventral carinae (Renner et al. 2010). Interestingly, it is possible to collect wefts comprising a mixture of L. colensoana and L. rhigophila growing together, and syntopic samples of these two species have been included in the present analysis (Accessions AK300044 and AK300044a). Although there are many explanations for the co-occurrence of morphs in mixed collections (Wyatt et al. 1982), instances involving species of the L. tumida species group are explained by the existence of genetically distinct species. This conclusion is contrary to currently accepted interpretations of mixed collections as evidence of polymorphism (i.e. So 2002, 2005a, 2005b). In the North Island, L. colensoana and L. tumida s.s. are frequently sympatric in riparian vegetation and forest margins in lowland forest, whereas L. tumida s.s. and L. oracola are frequently syntopic as trunk epiphytes within successional vegetation. The assumption that single, even relatively small, specimens comprise a single individual does not hold for the L. tumida species group, although this may have contributed to the impression that a single variable species was at hand. This observation calls into question the interpretation of co-occurring morphotypes as different expressions of a single variable species, particularly in those genera of similar size and similar habitat occupancy as Lejeunea, and may have implications or application beyond the current study. Acknowledgements Financial support for the molecular component of this research was provided by The Hansjörg Eichler Research Fund of the Australian Systematic Botany Society, and the Joyce Vickery Research Fund of the Linnean Society of New South Wales; Margaret Heslewood and Carolyn Connelly supervised the molecular work; Drs Peter Weston, Bernard Goffinet and Jon Shaw commented on the effective first draft of this manuscript; the time they gave to providing constructive feedback is greatly appreciated; Endymion Cooper commented on early drafts; four anonymous reviewers and the editors of Australian Systematic Botany provided thoughtful, effective criticism on earlier versions of the manuscript; Jenny Lux and Richard Gillies granted hospitality during the New Zealand fieldtrip collecting material from road reserves and private land for this study; Zonda Erskine (NSW), Ewen Cameron and Mei Nee Lee (AK) attended to herbarium loan material, and the curators of the herbaria AK, BM, CANB, CHR, E, F, G, HO, L, P, W, WELT loaned material, some of which was included in this study.

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Manuscript received 26 October 2010, accepted 18 February 2011

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Appendix 1. Taxa, vouchers, localities and GenBank accession numbers for all sequences analysed Accession numbers are in the following order: trnL–F, nrITS1. Newly generated accession numbers are in bold. Species sequenced for the first time in the present study are also in bold Anoplolejeunea conferta (C.F.W.Meissn. ex Spreng.) A.Evans, Ecuador, Wilson et al. 04–08, DQ987438, DQ987335. Aureolejeunea quinquecarinata R.M.Schust., Ecuador, Schäfer-Verwimp 23299/A, DQ987450, DQ987350. Ceratolejeunea cf. guianensis, Ecuador, Wilson et al. 04–15, DQ987442, DQ987340. Ceratolejeunea cornuta (Lindenb.) Steph., Bolivia, Drehwald 4739, DQ238570, DQ987257. Cheilolejeuna trifaria (Reinw. et al.) Mizut., Bolivia, Gradstein 9951, DQ987389, DQ987275. Cheilolejeunea acutangula (Nees) Grolle, Mexico, Gradstein & Velasquez s.n., DQ987386, DQ987270. Cheilolejeunea clausa (Nees & Mont.) R.M.Schust., Brazil, Schäfer-Verwimp 13212, DQ987399, DQ987293. Cheilolejeunea lineata A.Evans, Guadeloupe, Schäfer-Verwimp 22183, DQ987401, DQ987295. Cheilolejeunea revoluta (Herz.) Gradst. & Grolle, Costa Rica, Dauphin 1990, DQ987454, DQ987354. Cheilolejeunea rigidula (Mont.) R.M.Schust., Suriname, Munoz 98–62, DQ987453, DQ987353. Cheilolejeunea sp. indet., Honduras, Allen 17393, DQ987387, DQ987271. Cololejeunea laevigata (Mitt.) Tilden, New Zealand, von Konrat 81 Herangi 503, DQ238571, DQ987349. Cololejeunea metzgeriopsis (K.I.Goebel) Gradst. et. al., Malaysia, Gradstein et al. 10436, DQ242521, DQ987319. Cololejeunea peculiaris (Herz.) Benedix, Malaysia, Schäfer-Verwimp 18861/A, DQ238572, DQ987280. Cololejeunea vitalana Tixier, Costa Rica, Pócs SV/H-0473/A, DQ238573, DQ987348. Cololejeunea vuquangensis Pócs, Vietnam, Pócs 02102/N, DQ987449, DQ987347. Colura acroloba (Mont. ex. Steph.) Ast, Fiji, Pócs 03261/BK, DQ238586, DQ987306. Colura imperfecta Steph., Fiji, Pócs 03261/BA, DQ238585, DQ987305. Diplasiolejeunea sp., Ecuador, Wilson et al. 04–06, DQ987437, DQ987333. Drepanolejeunea biocellata A.Evans, Ecuador, Gradstein et al. 10053, DQ238578, DQ987276. Drepanolejeunea sp., Malaysia, Ilkiu-Borges et al. 3024, DQ987422, DQ987318. Drepanolejeunea vesiculosa (Mitt.) Steph., Malaysia, Gradstein et al. 10372, DQ987421, DQ987317. Harpalejeunea grandistipula R.M.Schust., Ecuador, Schäfer-Verwimp 24289, DQ987451, DQ987351. Lejeunea cancellata Nees & Mont. ex Mont., Ecuador, Wilson et al. 04–02, DQ987433, DQ987329. Lejeunea catinulifera Spruce I, Ecuador, Gradstein & Mandl 10141, DQ987411, DQ987307. Lejeunea catinulifera II, Ecuador, Wilson et al. 04–01, DQ987432, DQ987328. Lejeunea cavifolia (Ehrh.) Lindb., Germany, Hienrichs 3695, DQ238581, DQ987259. Lejeunea cerina (Lehm. & Lindeb.) Gottsche, Lindenb. & Nees, Ecuador, Wilson et al. 04–13, DQ987441, DQ987339. Lejeunea colensoana (Steph.) M.A.M.Renner, New Zealand, North Island, Kaimai Range, M.A.M.Renner, AK300028, JF308562, JF308533; New Zealand, North Island, Kaimai Range, M.A.M.Renner, AK300030, JF308563, JF308534; New Zealand, North Island, Kaimai Range, M.A.M.Renner, AK300039, JF308564, JF308535; New Zealand, central North Island, Hauhangaroa Range, M.A.M.Renner, AK300044, JF308565, JF308536; New Zealand, North Island, Auckland, Hunua Range, M.A.M.Renner, AK300101, JF308572, JF308543; New Zealand, North Island, Auckland, Hunua Range, M.A.M.Renner, AK300103, JF308574, JF308545; New Zealand, North Island, Auckland, Hunua Range, M.A.M.Renner, AK300104, JF308573, JF308544; New Zealand, North Island, Coromandel Range, M.A.M.Renner, AK300127, JF308578, JF308546; New Zealand, North Island, Coromandel Range, M.A.M.Renner, AK300127a, JF308579, JF308547; New Zealand, North Island, Coromandel Range, M.A.M.Renner, AK300130, JF308580, JF308548; New Zealand, North Island, Coromandel Range, M.A.M.Renner, AK300140, JF308581, JF308549. Lejeunea drummondii Taylor, Australia, Western Australia, Tulbrinup. M.A.M.Renner, NSW872058, JF308584, JF308555. Lejeunea epiphylla Colenso, New Zealand, central North Island, Hauhangaroa Range, M.A.M.Renner, AK300056, JF308568, JF308539. Lejeunea exilis (Reinw et al.) Grolle, Australia, Queensland, Mt Lewis, M.A.M.Renner, NSW872056, JF308583, JF308554. Lejeunea flava (Sw.) Nees, Brazil, Gradstein s.n., DQ987413, DQ987309. Lejeunea helmsiana Steph., New Zealand, central North Island, Hauhangaroa Range, M.A.M.Renner, AK300050, JF308567, JF308538; New Zealand, central North Island, Hauhangaroa Range, M.A.M.Renner, AK300069, JF308569, JF308540; New Zealand, central North Island, Hauhangaroa Range, M.A.M.Renner, AK300069a, JF308570, JF308541. Lejeunea laetevirens Nees & Mont., Dominica, Schäfer-Verwimp 17899, DQ987402, DQ987296. Lejeunea mimula Hürl., Bali, Schäfer-Verwimp 20930, DQ238580, DQ987261. Lejeunea oracola M.A.M. Renner, New Zealand, North Island, Northland, Wainui River, AK299972, M.A.M.Renner, JF308557, JF308528; New Zealand, North Island, Northland, Okarari Stream, M.A.M.Renner, AK300003, JF308559, JF308530; New Zealand, North Island, Northland, Waipoua River, M.A.M.Renner, AK300010, JF308560, JF308531; New Zealand, North Island, Northland, Bream Tail, M.A.M.Renner, AK300012, JF308561, JF308532; New Zealand, North Island, Northland, Cape Rodney, M.A.M.Renner, AK300078, JF308571, JF308542. Lejeunea rhigophila M.A.M. Renner, New Zealand, central North Island, Hauhangaroa Range, M.A.M.Renner, AK300044a, JF308566, JF308537 M.A.M.Renner, AK300147 JF308579, JF308550; New Zealand, central North Island, Hauhangaroa Range, M.A.M.Renner, AK300149, JF308580, JF308551. Lejeunea tasmanica Gottsche et al., New Zealand, North Island, Auckland, Waitakere Range, M.A.M.Renner, NSW872054, JF308581, JF308552. Lejeunea tumida Mitt., New Zealand, North Island, Northland, Herekino Range, M.A.M.Renner, AK299949, JF308556, JF308527; New Zealand, North Island, Northland, Okarari Stream, M.A.M.Renner, AK300002, JF308558, JF308529. Lepidolejeunea eluta I (Nees) R.M.Schust., Bolivia, Drehwald 4833, DQ987379, DQ987257. Lepidolejeunea integristipula (Jack & Steph.) R.M.Schust., Fiji, Pócs 03307/AC, DQ987417, DQ987313. Leucolejeunea xanthocarpa II, Brazil, Costa & Gradstein 3839, DQ987470, DQ987371. Luteolejeunea herzogii (Buchloh) Piippo, Costa Rica, Schäfer-Verwimp & Holz 0294/B, DQ987467, DQ987368. Myriocolea irrorata Spruce, Ecuador, Gradstein et al. 10033, DQ238584, DQ987279. Myriocoleopsis gymnocolea (Steph.) E.Reiner & Gradst., Ecuador, Gradstein et al. 10020, DQ238583, DQ987277. Neopotamolejeunea sp. nov., Ecuador, Gradstein & Jost 10063, DQ987416, DQ987312. Pluvianthus squarrosus (Steph.) R.M.Schust. & Schäfer-Verwimp, Brazil, Schäfer-Verwimp 13376, DQ987446, DQ987344. Pycnolejeunea densistipula (Lehm. & Lindenb.) Steph., Ecuador, Schäfer-Verwimp 23368, DQ987400, DQ987294. Rectolejeunea berteroana (Gottsche) A.Evans, Guadeloupe, Schäfer-Verwimp 22245/A, DQ987444, DQ987342. Rectolejeunea sp., Australia, New South Wales, M.A.M.Renner, NSW872055,JF308582, JF308553. Siphonolejeunea elegantissima (Steph.) Grolle, Australia, Pócs & Brown 0026/AA, DQ987452, DQ987352. Taxilejeunea cf. asthenica (Spruce) Steph., Bolivia, Gradstein 9948, DQ987428, DQ987324. Taxilejeunea cf. isocalycina (Nees) Steph., Ecuador, Wilson et al. 04–04, DQ987435, DQ987331. Taxilejeunea cf. pterigonia (Lehm. & Lindenb.) Schiffn., Bolivia, Gradstein 9964, DQ987429, DQ987325. Taxilejeunea sp., Ecuador, Gradstein 10172, DQ987410, DQ987304. Xylolejeunea crenata (Nees & Mont.) X.-L. He & Grolle, Brazil, SchäferVerwimp 11225, DQ987443, DQ987341.

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