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Crandall-Stotler & al. • Thalloid liverwort evolution
Evolutionary trends in the simple thalloid liverworts (Marchantiophyta, Jungermanniopsida subclass Metzgeriidae) Barbara J. Crandall-Stotler, Laura L. Forrest & Raymond E. Stotler Department of Plant Biology, Southern Illinois University, Carbondale, Illinois 62901, U.S.A. crandall@ plant.siu.edu (author for correspondence),
[email protected],
[email protected] The phylogenetic history of and evolutionary trends within the simple thalloid liverworts (Jungermanniopsida, subclass Metzgeriidae) are reconstructed in a combined analysis of molecular and morphological data. The molecular dataset comprises loci from all three genomes, including trnL-F, rps4, rbcL, atpβ and psbA from the chloroplast, SSU rRNA and LSU rRNA from the nucleus, and nad5 from the mitochondrion, and 65 characters are scored in the morphological dataset. An initial analysis of a molecular dataset that included 16 outgroup and 50 ingroup taxa resolved a Haplomitrium/Treubiaceae clade as the earliest diverging lineage of the ingroup. Subsequent analyses of ingroup only datasets, rooted on this clade, resolve Metzgeriidae as paraphyletic, with Blasiaceae sister to Marchantiopsida in all analyses. A combined analysis of morphological and molecular datasets resolves basically the same clades as analyses of the molecular dataset alone, except for the resolution of a weak sister group relationship between Metzgeriineae and the leafy liverworts. Reconstructions of morphological character evolution on the combined analysis topology confirm that there is substantial homoplasy in the morphology dataset, even in characters that have been traditionally considered diagnostic of hierarchial relationships, such as apical cell geometry, calyptral type and capsule wall thickness. Ancestral state reconstructions contradict many prevailing hypotheses of character evolution in hepatics, including the model of the ancestral liverwort prototype as an erect, radially symmetric plant. Instead, a more likely model is a prostrate, bilaterally symmetric plant with the diagnostic features of a simple thalloid liverwort.
KEYWORDS: character evolution, homoplasy, Marchantiophyta, multigene analysis, morphology, simple thalloid liverworts.
INTRODUCTION The liverworts are estimated to contain 6000–8000 species in 380 genera and 74 families and are traditionally partitioned into two morphologically defined classes, Jungermanniopsida Stotl. & Crand.-Stotl. and Marchantiopsida Cronquist, Takht. & W. Zimm. (Crandall-Stotler & Stotler, 2000). The classes are morphologically defined by distinctive patterns of antheridial development, including diagnostic features of spermatid ultrastructure (Garbary & al., 1993), divergent patterns of early embryological development, the number of cell layers in the sporophyte capsule wall, and the presence or absence of sporocyte lobing during meiosis (Crandall-Stotler & Stotler, 2000). Most of the taxonomic diversity of the phylum resides in Jungermanniopsida, which itself is divided into two subclasses, the leafy liverworts or Jungermanniidae Engl. as circumscribed by Crandall-Stotler and Stotler (2000), comprising at least 85% of liverwort species (Schuster, 1984), and the simple thalloid liverworts or Metzgeriidae Barthol.-Began. As the common names denote, the two subclasses differ in growth form, but this is not absolute; i.e., a few mem-
bers of Jungermanniidae have simple thalloid vegetative phases, and several elements of Metzgeriidae have leafy, or nodal, morphology. Although Jungermanniidae are the most speciose of the two groups, Metzgeriidae express more heterogeneity in such fundamental characters as apical cell geometry, gametangial arrangement, androecial structure and sporangial dehiscence patterns. Schuster (1992: 305) has attributed the great morphological diversity of the simple thalloids to their antiquity, with the suggestion that “extant members represent the mere remnants of a once larger evolutionary unit”. Although early molecular studies by Capesius & Bopp (1997), Hedderson & al. (1996, 1998), and Lewis & al. (1997) were equivocal in resolving relationships within these three major groups of liverworts as well as between liverworts and other groups of embryophytes, they all supported the early divergence of Marchantiopsida and Jungermanniopsida. For the most part, however, the relationships predicted by these early molecular analyses were incongruent with existing classifications that were based on subjective hypotheses of morphological character evolution, such as that of Schuster (1984). In 2000, Crandall-Stotler & Stotler conducted a cladistic 299
Crandall-Stotler & al. • Thalloid liverwort evolution
analyses of 61 morphological characters scored for the 34 exemplar taxa that had been included in the molecular studies cited above to test objectively the phylogenetic signal in morphological data. These analyses supported the monophyly of both Marchantiopsida and leafy Jungermanniidae, and resolved Metzgeriidae as paraphyletic, albeit with low Bremer decay values. With the increasing numbers of molecular phylogenetic analyses since 2000 (see Forrest & Crandall-Stotler, 2005, for a complete review), the discordance between phylogenetic reconstructions based on morphology and those based on molecules has become even more apparent. Given the morphological heterogeneity of Metzgeriidae and the antiquity of its fossil record (Krassilov & Schuster, 1984; Oostendorp, 1987), it is reasonable to predict that this group occupies a pivotal position in liverwort phylogeny. In 2004, Forrest & Crandall-Stotler published the first comprehensive molecular phylogeny of Metzgeriidae, based on an analysis of four chloroplast protein-coding genes, namely, atpβ, psbA, rbcL and rps4, and the chloroplast trnL/trnF region, for 48 liverworts, five hornworts and seven mosses. This analysis provided fairly robust resolution of five main clades within the liverworts, including monophyletic complex thalloid and leafy liverwort lineages, which are nested within Metzgeriidae. In Forrest & Crandall-Stotler (2005), a multigenome, 8locus, analysis that included 64 liverworts resolved a similar topology with 93.4% of the nodes recovered supported by bootstrap (BS) values above 50%. The next logical step in our continuing studies of Metzgeriidae is a total evidence analysis of a combined molecular and morphological dataset, as presented in this report. Morphological data often reflect episodic evolutionary changes and can, therefore, help resolve deep-level branches of a phylogeny (Lewis & al., 1997). The addition of morphological data will also allow us to test traditional hypotheses of character evolution, evaluate the degree of homoplasy in morphological characters, and determine whether there are morphological characters that are predictive of phylogenetic position.
MATERIALS AND METHODS Taxon sampling. — The ingroup molecular dataset consists of 50 species of hepatics, distributed as follows: 36 species from 28 genera and 13 families, representing all eight suborders of simple thalloid liverworts; six species from six genera and five families, representing four suborders of complex thalloids; and eight species from eight genera and eight families, representing five suborders of leafy liverworts. This sampling includes the following five taxa that have not been 300
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included in our previous analyses: Apotreubia nana (S. Hatt. & Inoue) S. Hatt. & Mizut., Makinoa crispata (Steph.) Miyake, Pallavicinia rubristipa Schiffn., Pallavicinia xiphoides (Hook. f. & Taylor) Trevis. and Pleurozia purpurea Lindb. Seven species in five genera of hornworts, seven species in seven genera of mosses, and two species in two genera of vascular plants comprise the molecular dataset outgroup. Taxon authors, voucher information and GenBank accession numbers for all taxa are provided in Appendix 3. To identify the earliest diverging lineage of Metzgeriidae, an initial analysis of the molecular sequence data included the 50 ingroup and 16 outgroup taxa. Further molecular, morphological and combined morphology-molecular analyses excluded the outgroup taxa to avoid problems in ascertaining homology in morphological character scoring across the three bryophyte lineages. These analyses were rooted on the clade comprising Haplomitrium Nees and Treubiaceae Verd., based on results of the initial molecular analysis as well as results obtained in previous studies (Forrest & Crandall-Stotler, 2004, 2005; Stech & Frey, 2004). Morphological data. — The 50 liverwort taxa of the molecular ingroup dataset were scored for 65 morphological characters, judged to be independent, heritable and phylogenetically informative (Appendices 1, 2). Of these, 38 are binary and 27 are multistate characters. Nine of the multistate characters were ordered, based on supportive ontogenetic and anatomical evidence extracted from the following publications: character 17 (Buch, 1930; Crandall, 1969; Renzaglia, 1982); characters 26, 28 and 36 (Haupt, 1920; Johnson, 1929; Renzaglia, 1982; Bartholomew-Began, 1991); character 34 (Knapp, 1930; Bartholomew-Began, 1991); characters 37 and 42 (Kienitz-Gerloff, 1874; Leitgeb, 1877; Hy, 1884; Schertler, 1979; Kinser, 2003); character 49 (Tilden, 1894); character 61 (Smith, 1978; Newton, 1983). There is no a priori judgment of polarity associated with the numerical assignments given to the states. Both unknown and inapplicable data were scored as missing. Multiple state scorings represent polymorphisms except for character 20 in Apotreubia nana and character 45 in Riccardia capillacea, which are scored as uncertainties. The data were compiled primarily from personal studies of the taxa, complemented by data from the comprehensive morphological treatments of Leitgeb (1874–1881), Crandall-Stotler (1981), Renzaglia (1982), Schuster (1984, 1992), Bischler (1998) and Crandall-Stotler & Stotler (2000). Karyotype data for characters 61 and 62 were compiled from Fritsch (1991). The data matrix is available in Appendix 2. DNA extraction and sequencing. — The molecular dataset includes loci from three genomic compartments, as follows: rbcL, rps4, trnL/trnF intron, exon
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and spacer, atpβ and psbA from the chloroplast, a portion of the 5' end of the 26S rRNA gene (nrLSU) and the 18S rRNA gene (nrSSU) from the nucleus, and nad5 from the mitochondrion. Novel DNA sequences for these loci were obtained following the extraction and sequencing methods described in Forrest & Crandall-Stotler (2004, 2005). Areas of ambiguous alignment (e.g., most of the nad5 intron and the psbA spacer regions) were excluded from the analyses (for exclusion sets see Forrest & Crandall-Stotler, 2005). Within the ingroup, 52 of the 400 potential sequences (13%) are missing, while in the outgroup 49 of the 128 potential sequences (38%) are missing, giving a total of 19% of sequences missing (Appendix 3). Phylogenetic analyses. — All parsimony analyses were conducted using PAUP* version 4.0b10 (Swofford, 2002). Gaps in the molecular dataset were treated as missing data. An heuristic search strategy was employed, using 1000 random addition replicates with Tree Bisection and Reconnection (TBR) and ACCTRAN character-state optimization, saving no more than ten trees per replicate. Bootstrap support was calculated under a maximum parsimony (MP) criterion, with 1000 bootstrap (BS) replicates, each BS replicate consisting of an heuristic search with five random addition replicates, TBR swapping, and no more than five MPTs saved per step. Separate analyses were conducted on the complete molecular dataset, on the liverwort subset of the molecular dataset, on the morphological dataset alone, and on a combined molecular-morphological matrix. Analyses that included morphological characters were also conducted with the nine ordered characters treated as unordered to determine whether character ordering influenced topology. Reconstructions of character evolution were performed in MacClade 4.03 (Maddison & Maddison, 2002), over one of the three MPTs produced by parsimony analysis of the combined molecular and morphological dataset. The topology selected for reconstruction of character evolution was that which showed greatest similarity to well supported topologies produced in more intensive (more loci and/or more taxon sampling) molecular analyses (Davis, 2004; Forrest & Crandall-Stotler, 2004; He-Nygrén & al., 2004; Forrest & CrandallStotler, 2005). The resolving option to show all most parsimonious states at each node was invoked and character statistics were calculated by implicitly examining all most parsimonious reconstructions (MPRs).
RESULTS Phylogenetic analyses. — The numbers of characters, total numbers of MPTs, and tree statistics for each
Crandall-Stotler & al. • Thalloid liverwort evolution
of the four phylogenetic analyses are given in Table 1. Initial parsimony-based analysis of the complete molecular dataset resolves the mosses as monophyletic (98% BS), a hornwort/vascular plant clade as monophyletic (96% BS), and liverworts as monophyletic (100% BS) (Fig. 1). Metzgeriidae are paraphyletic, with a Haplomitrium/Treubiaceae lineage (Liverworts 1) being the earliest divergence within the liverworts, with 100% BS support. The next diverging lineage (Liverworts 2) comprises Blasiaceae H. Klinggr. and Marchantiopsida, again with 100% BS. The positions of the leafy liverwort Pleurozia Dumort., the clade of other leafy liverworts, and the clade comprising Metzgeriineae R. M. Schust. ex Schljakov lack BS support in the strict consensus tree, but are resolved in a large clade with all the remaining taxa of Metzgeriidae (Liverworts 3), with 100% BS. This large clade is sister to a Blasiaceae/Marchantiopsida lineage. Figure 2 illustrates representatives of these three major lineages. When the outgroup taxa were excluded and the resultant tree was rooted with the Haplomitrium/ Treubiaceae clade, the molecular topology of the liverworts remained the same as that resolved in the broader analysis (Fig. 1). The addition of the 65 morphological characters to the molecular dataset had no affect on clades that received over 60% bootstrap support; these were the same in both the combined and molecular analyses. However, the unsupported position of some lineages do differ between MPTs from the two analyses. Hymenophyton Dumort., which was resolved as sister to Pallaviciniaceae Mig. in the molecular analysis with 60% BS (Fig. 1), is nested without BS support between Moerckioideae R. M. Schust. and the rest of Pallaviciniaceae in the combined analysis (Fig. 3). Pleurozia moves from being sister to a simple thalloid clade in the molecules alone analysis (Fig. 1) to a position sister to Metzgeriineae within a leafy liverwort/Metzgeriineae clade in the combined analysis (Fig. 3). Neither the position of Pleurozia nor the relationship of Metzgeriineae with the larger leafy liverwort clade are supported by significant BS values. On the other hand, there is increased support in the combined analysis for a monophyletic Pelliaceae H. Klinggr., comprising Noteroclada Taylor ex Hook. and Pellia Raddi, and monophyletic Blasiaceae, including Blasia L. and Cavicularia Steph. Support for Sphaerocarpos Boehm. as sister to the rest of Marchantiopsida is also improved, from 65% BS in the molecular analysis (Fig. 1) to 79% BS in the combined analysis (Fig. 3). Apotreubia S. Hatt. & Mizut. is resolved as a sister taxon to Treubia K. I. Goebel, forming monophyletic Treubiaceae. Pallavicinia rubristipa and P. xiphoides are resolved as sister taxa to Jensenia connivens (Colenso) Grolle and Podomitrium phyllanthus (Hook.) Mitt., respectively, rendering the genus 301
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Table 1. Matrix and Tree statistics for analyses of the liverwort data set. TC - total characters, CC - constant characters, PUC - parsimony uniformative characters, PIC - parsimony informative characters, MPTs - most parsimonious trees, TL - tree length, SCT - strict consensus tree, RCI - rescaled consistency index.
No. taxa 66 50 50 50
Data set Molecular Molecular Morphological Combined
TC 8381 8381 0065 8446
CC 4257 4834 0000 4834
PUC 1405 1227 0002 1229
No. MPTs 00 1 00 1 1425 00 3
PIC 2719 2320 0063 2383
97 100 100
97
100
100 85 99
85 100 100
55
100
58
99 99
52 60
99
99
100
100
66
100
100
70
74 81 100 66 76
100 87
99
71
79
100
67
78
85 85
99 99 94 100
99
97
94
100
100
80 88 100 87 76
100
87
71
100
66 63
99
50 99 99
51
99
100 100
100
65
100
100 100
88 66 89 100
97
100
100 99
96 100 100 100
100
100
96 100
100 100 97 91
98 99
TL 18732 14512 00436 15025
No. nodes in SCT n/a (64) n/a (48) 0000 0047
Symphyogyna undulata Xenothallus vulcanicolus Greeneothallus gemmiparus Symphyogyna hymenophyllum Jensenia connivens Pallavicinia rubristipa Pallavicinia xiphoides Podomitrium phyllanthus Pallavicinia lyellii Hattorianthus erimonus Moerckia flotoviana Moerckia blyttii Hymenophyton flabellatum Phyllothallia nivicola Calycularia crispula Austrofossombronia peruviana Fossombronia angulosa Fossombronia foveolata Austrofossombronia australis Petalophyllum ralfsii Allisonia cockaynii Makinoa crispata Noteroclada confluens Pellia epiphylla Pleurozia purpurea Aneura pinguis Lobatiriccardia lobata Verdoornia succulenta Riccardia capillacea Metzgeria conjugata Isotachis multiceps Jungermannia leiantha Scapania nemorea Herbertus alpinus Schistochila appendiculata Jubula hutchinsiae Porella navicularis Monoclea gottschei Riccia huebeneriana Reboulia hemisphaerica Marchantia polymorpha Neohodgsonia mirabilis Sphaerocarpos texanus Blasia pusilla Cavicularia densa Haplomitrium blumii Haplomitrium hookeri Haplomitrium gibbsiae Apotreubia nana Treubia lacunosa Megaceros flagellaris Megaceros aenigmaticus Notothylas breutelii Phaeoceros laevis Anthoceros angustus Anthoceros punctatus Leiosporoceros dussii Equisetum telmateia Psilotum nudum Hypnum lindbergii Mnium hornum Polytrichum pallidisetum Andreaea rothii Andreaeobryum macrosporum Sphagnum palustre Takakia lepidozioides
No. nodes > 50% BS 57 42 8 40
RCI 0.1824 0.1968 0.1794 0.1950
Liverworts 3
Liverworts 2
Liverworts 1
Hornworts
Vascular plants
Mosses
Fig. 1. Single MPT tree generated in a maximum parsimony analysis of the 66-taxon molecular data matrix. Numbers above the branches are bootstrap values above 50% from the 66-taxon analysis, while numbers below the branches are bootstrap values from the 50-taxon, liverworts only, analysis. The positions of Hymenophyton and Pleurozia are highlighted in bold type; grey branches indicate Metzgeriineae and bold branches indicate Jungermanniidae.
302
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Crandall-Stotler & al. • Thalloid liverwort evolution
Fig. 2. Select representatives of the three major clades resolved in the 66-taxon molecular analysis. A, Treubia lacunosa; B, Haplomitrium gibbsiae, a male plant with scattered antheridia protected by the leaves; C, Blasia pusilla intermixed with moss; D, Sphaerocarpos texanus; E, Neohodgsonia mirabilis; F, Verdoornia succulenta, with a female inflorescence marked at the arrow; G, Noterclada confluens; H, Phyllothallia nivicola. Photo credits: B, H by B. Malcolm; G by L. Zhang; A, C–F by B. Crandall-Stotler.
303
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Table 2. Apomorphies for selected nodes of the combined analysis tree shown in Fig. 3. Shared characters listed for node 1 represent the more significant plesiomorphies of the clade. The taxonomic hierarchy used to identify the clades follows Crandall-Stotler & Stotler (2000).
Branch No. Clade 1 Haplomitrium/Treubiaceae (Root lineage) 2 Blasiaceae/Sphaerocarpos/ Marchantiidae 3 Pleurozia/Metzgeriineae 4 5 6 7 8 9 10
Metzgeriineae/ Jungermanniidae Pelliineae Fossombroniales/Phyllothallia/ Pallaviciniineae Makinoa/Fossombroniineae Calycularia/Phyllothallia/ Pallaviciniaceae plus Hymenophyton Pallaviciniaceae plus Hymenophyton Pallaviciniaceae minus Moerckioideae
Apomorphies (character numbers are in bold) 10: lateral branches, intercalary exogenous; 14: ventral appendages, stalked papillae; 37: foot, obconoidal; 49: elaters, bispiral [these features are plesiomorphies] 1: thallus with costa; 9: oil bodies, absent; 15: apical cell, cuneate; 34: true calyptra; 59: gemmae in receptacles 15: apical cell, lenticular; 29: archegonia on special branches; 43: capsule wall epidermal cells similar in size to inner cells 9: oil bodies, granular; 10: lateral branches, terminal exogenous or endogenous; 22: perigonial structures, true bracts 20: monoicous; 37: sporophyte foot, cup-shaped (with an involucellum); 49: elaters with more than 2 thickening bands; 52: precocious germination, present; 53: spore germination endosporic 8: endomycorrhizae present; 41: capsule spheroidal; 48: basal elaterophore in capsule 7: rhizoid pigment, present 20: dioicous, monomorphic 45: inner capsule wall, thickenings absent; 48: elaterophore absent (a reversion); 64: spores < 35 µm 47: capsule valves joined at the apices; 61: base chromosome number of 8
Pallavicinia Gray paraphyletic. Makinoa Miyake is resolved as sister to a clade that comprises Fossombroniaceae Hazsl. plus Allisonia Herzog and Petalophyllum Nees & Gottsche ex Lehm., with 80% BS. In both of the molecular dataset analyses, only a single most parsimonious tree was resolved, while in the combined analysis of molecular and morphological datasets three equally parsimonious trees were resolved (Table 1). Analysis of just the morphological dataset produced a far less resolved topology, with 1,425 equally parsimonious trees recovered. Only seven clades received any measure of BS support and no relationships were resolved in 100% of the MPTs (Fig. 4). The largest supported clade comprises the seven leafy liverworts, including Pleurozia, with 77% BS. The placements of only 15 of the 50 taxa are in agreement between the morphology only and the molecular only analyses; i.e., there is little congruence between the topologies produced by the two datasets. Treating the ordered morphological characters as unordered had no effect on either the number or the topology of the MPTs resolved, but did reduce tree length by 15 steps. Character reconstructions. — Individual reconstructions of each of the 65 morphological characters on the same combined analysis tree (Fig. 3) identified ancestral states for 57 of the 65 characters, as indicated in Appendix 2. Forty-eight characters have individual consistency indices (CI) less than 0.50, suggesting that there is considerable homoplasy in the dataset. Only seven characters show no homoplasy (CI = 1). Of these, characters 38 and 40 define the autapomorphies of an 304
articulated seta in Sphaerocarpos and an internally differentiated seta in Haplomitrium, respectively, and are consequently uninformative above the genus level. The presence of Nostoc Vaucher symbionts (character 3), stolons (character 12), and gemmae in special receptacles (character 59) are synapomorphies of Blasiaceae, Haplomitrium, and the Blasiaceae/complex thalloid clades, respectively. Likewise, the derived states of character 19 are of restricted occurrence, with two strands occurring only in the Moerckia Gottsche/Hattorianthus R. M. Schust. & Inoue clade and three strands only in Cavicularia. The division pattern of young antheridia (character 26) unambiguously defines the three liverwort lineages that were resolved in the complete molecular analysis (Fig. 1). Many of the characters that traditionally have been applied to genus, family, or subordinal level circumscriptions exhibit moderate to high levels of convergent or parallel evolution (Figs. 5, 6). For example, reconstructions of apical cell geometry indicate that cuneate or wedge-shaped apical cells have evolved three times, hemidiscoid types, twice, and lenticular types, four times (Fig. 5B). Similarly, all three derived states of calyptral types display multiple origins, with solid perigynia having evolved independently six times (Fig. 6A). Sporophyte characters (37–50) are no more predictive of higher level relationships than gametophyte characters, as illustrated by the character reconstructions of capsule wall thickness (Fig. 6B) and capsule wall dehiscence (Fig. 6C). A tabulation of the apomorphies of ten branches on or near the backbone of the combined analysis tree (Fig.
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Crandall-Stotler & al. • Thalloid liverwort evolution
93
Symphyogyna undulata
100
Xenothallus vulcanicolus
100
Greeneothallus gemmiparus Symphyogyna hymenophyllum
86 100
10
Jensenia connivens
57
100
Pallavicinia rubristipa Pallavicinia xiphoides
99
Podomitrium phyllanthus
9
Pallavicinia lyellii Hymenophyton flabellatum
100
Hattorianthus erimonus
100 75
100
Moerckia flotoviana
8
Moerckia blyttii Phyllothallia nivicola Calycularia crispula 61
Austrofossombronia peruviana
75
Fossombronia angulosa
100
Fossombronia foveolata
77
6
86
7
100
Austrofossombronia australis Petalophyllum ralfsii
80
5
Allisonia cockaynii Makinoa crispata Noteroclada confluens
91
Pellia epiphylla 100
Herbertus alpinus
*
Scapania nemorea Isotachis multiceps
86
90
Jungermannia leiantha 71
Schistochila appendiculata Jubula hutchinsiae
63
Porella navicularis
4
99
Aneura pinguis
97
Lobatiriccardia lobata
97 100
Verdoornia succulenta
100
Riccardia capillacea
3
Metzgeria conjugata Pleurozia purpurea Monoclea gottschei 98
Riccia huebeneriana
99
Reboulia hemisphaerica
79
Marchantia polymorpha
100
2
Neohodgsonia mirabilis
100
Sphaerocarpos texanus 95
Blasia pusilla Cavicularia densa 96
100
Haplomitrium blumii Haplomitrium hookeri Haplomitrium gibbsiae
1 100
Apotreubia nana Treubia lacunosa
100 changes
Fig. 3. Phylogram of one of the three MPTs generated in a maximum parsimony analysis of the 50-taxon combined molecular and morphological data matrix. Numbers above the branches are bootstrap values above 50%. Numbers in circles on the branches indicate clades listed in Table 2. The positions of Hymenophyton and Pleurozia are highlighted in bold type; taxa currently classified in Jungermanniidae are indicated by bold branches, taxa in Metzgeriineae are indicated in grey, and taxa in Marchantiopsida are indicated by dashed lines. The one branch that collapses in the strict consensus of three MPTs is marked with an asterisk. Note the changes in position of Hymenophyton, Pleurozia and these clades, as compared to Fig. 1.
3) shows some morphological definition to the deep level lineages resolved (Table 2). For example, three signifi-
cant synapomorphies of the Blasiaceae/complex thalloid lineage are cuneate apical cells, true calyptras, and gem305
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98 96
67
94
Marchantia polymorpha Neohodgsonia mirabilis Reboulia hemisphaerica
92
Riccia huebeneriana Sphaerocarpos texanus
83
98 63 94
79
77
Austrofossombronia australis Austrofossombronia peruviana
98
Fossombronia angulosa
61
Fossombronia foveolata
98
Blasia pusilla
98
Cavicularia densa
75
Petalophyllum ralfsii
73
Allisonia cockaynii Phyllothallia nivicola
66 98 64
96
Noteroclada confluens Pellia epiphylla Calycularia crispula Makinoa crispata
58
Moerckia blyttii
98
Moerckia flotoviana Greeneothallus gemmiparus 78 77
Isotachis multiceps Schistochila appendiculata
75
Herbertus alpinus
72
Scapania nemorea
87
Jubula hutchinsiae
78
77
Jungermannia leiantha Pleurozia purpurea
63
Aneura pinguis 77 87
77
94
Lobatiriccardia lobata Riccardia capillacea Metzgeria conjugata
60
Hymenophyton flabellatum Podomitrium phyllanthus 98
51 83
91
81
89
Jensenia connivens Pallavicinia rubristipa Pallavicinia lyellii
73
Pallavicinia xiphoides
58
Hattorianthus erimonus Symphyogyna hymenophyllum
59
Xenothallus vulcanicolus Symphyogyna undulata Porella navicularis Verdoornia succulenta 96 94 92
100
98
Haplomitrium blumii
60
Haplomitrium hookeri Haplomitrium gibbsiae Monoclea gottschei Treubia lacunosa Apotreubia nana
Fig. 4. Majority rule consensus of 1425 MPTs generated in a maximum parsimony analysis of the 50-taxon morphological matrix. Numbers above the branches are the percentage of trees in which each given clade appears. Branches with bootstrap support are highlighted in bold, and bootstrap support values are given below the branches. The 15 taxa in bold agree in placement between the molecular and morphological analyses.
mae, when present, produced in receptacles. The Pleurozia/Metzgeriineae clade is defined by lenticular apical cells, a gynoecium that is restricted to short 306
branches, except in Verdoornia R. M. Schust., and capsule walls in which the epidermal cells are similar in size to the inner cells. None of the apomorphies on branch 4
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B
A
nodal organization, radial nodal organization, bilateral
C
Podomitrium phyllanthus Pallavicinia xiphoides Pallavicinia rubristipa Jensenia connivens Xenothallus vulcanicolus Symphyogyna undulata Greeneothallus gemmiparus Symphyogyna hymenophyllum Pallavicinia lyellii Hymenophyton flabellatum Moerckia flotoviana Hattorianthus erimonus Moerckia blyttii Phyllothallia nivicola Calycularia crispula Fossombronia angulosa Austrofossombronia peruviana Fossombronia foveolata Austrofossombronia australis Petalophyllum ralfsii Allisonia cockaynii Makinoa crispata Pellia epiphylla Noteroclada confluens Jungermannia leiantha Isotachis multiceps Scapania nemorea Herbertus alpinus Schistochila appendiculata Porella navicularis Jubula hutchinsiae Lobatiriccardia lobata Aneura pinguis Verdoornia succulenta Riccardia capillacea Metzgeria conjugata Pleurozia purpurea Riccia huebeneriana Monoclea gottschei Reboulia hemisphaerica Marchantia polymorpha Neohodgsonia mirabilis Sphaerocarpos texanus Cavicularia densa Blasia pusilla Haplomitrium hookeri Haplomitrium blumii Haplomitrium gibbsiae Treubia lacunosa Apotreubia nana
tetrahedral, dorsal
absent
tetrahedral, ventral
thin walled, pitted
lenticular
thick walled, pitted
cuneate
thin walled, not pitted
polymorphic
hemidiscoid
thick walled, not pitted
equivocal
equivocal
equivocal
thalloid, costa and wing thalloid, ecostate
Fig. 5. Examples of reconstructions of gametophyte characters, showing different degrees of homoplasy. A, growth form, 1 of 108 MPRs, CI = 0.31; B, apical cell geometry, 1 of 30 MPRs, CI = 0.36; C, strand cells, 1 of 3 MPRs, CI = 0.67.
are diagnostic for all taxa of the clade, and they do not provide a strong morphological argument for the relationship between Metzgeriineae and the leafy liverworts. Five synapomorphies on branch 5 support the sister relationship of Noteroclada and Pellia and the monophyly of Pelliineae R. M. Schust. ex Schljakov. The three apo-
morphies mapped on branch 6, the branch subtending the most inclusive clade of Metzgeriidae, are not expressed throughout the clade, and are in fact, plesiomorphies of this large clade. Pigmented rhizoids, the only apomorphy of branch 7, are a morphological marker of the unexpected relationship of Makinoa and Fossombroniineae R. 307
Crandall-Stotler & al. • Thalloid liverwort evolution
A
B
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C
Podomitrium phyllanthus Pallavicinia xiphoides Pallavicinia rubristipa Jensenia connivens Xenothallus vulcanicolus Symphyogyna undulata Greeneothallus gemmiparus Symphyogyna hymenophyllum Pallavicinia lyellii Hymenophyton flabellatum Moerckia flotoviana Hattorianthus erimonus Moerckia blyttii Phyllothallia nivicola Calycularia crispula Fossombronia angulosa Austrofossombronia peruviana Fossombronia foveolata Austrofossombronia australis Petalophyllum ralfsii Allisonia cockaynii Makinoa crispata Pellia epiphylla Noteroclada confluens Jungermannia leiantha Isotachis multiceps Scapania nemorea Herbertus alpinus Schistochila appendiculata Porella navicularis Jubula hutchinsiae Lobatiriccardia lobata Aneura pinguis Verdoornia succulenta Riccardia capillacea Metzgeria conjugata Pleurozia purpurea Riccia huebeneriana Monoclea gottschei Reboulia hemisphaerica Marchantia polymorpha Neohodgsonia mirabilis Sphaerocarpos texanus Cavicularia densa Blasia pusilla Haplomitrium hookeri Haplomitrium blumii Haplomitrium gibbsiae Treubia lacunosa Apotreubia nana
true calyptra
unistratose
zero
shoot calyptra
bistratose
one
partial perigynium
multistratose
two
solid perigynium
equivocal
four
equivocal
polymorphic equivocal
Fig. 6. Examples of reconstructions of reproductive and sporophyte characters, showing different degrees of homoplasy. A, calyptra type, 1 of 5 MPRs, CI = 0.13; B, capsule wall thickness, 1 of 2 MPRs, CI = 0.17; C, number of capsule wall dehiscences, 1 of 18 MPRs, CI = 0.27.
M. Schust. ex Stotl. & Crand.-Stotl., and a dioicous, monomorphic sexual condition is the only apomorphy mapped to branch 8, as a very weak morphological sig308
nal of relationship between Calycularia and the Phyllothallia E. A. Hodgs./ Pallaviciniineae R. M. Schust. clade. Apomorphies mapped to branches 9 and
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10 include character states, such as inner capsule wall thickenings absent and capsule valves joined at the apices, frequently considered to be diagnostic of the clades they define.
DISCUSSION Our resolution of the liverworts as monophyletic, which in this 8-locus, multi-genome analysis receives 100% BS support, is consistent with the conclusions of earlier studies by Stech & al. (2000), Davis (2004), Duff & al. (2004), He-Nygrén & al. (2004), Groth-Malonek & al. (2005), and Forrest and Crandall-Stotler (2004, 2005). In addition, the topology that we have resolved is comparable to that presented by Qiu (2003, pers. comm.) for a six locus, multi-genome analysis of nine algae and 171 species of embryophytes, including 47 hepatics. This includes the resolution of a sister group relationship between Treubia and Haplomitrium and placement of this clade as the earliest diverging lineage of the liverworts, with 92% BS support. In Davis (2004) and HyNygrén & al. (2004), the position of Haplomitrium is uncertain, but neither of these analyses included Treubia. In the analyses of Groth-Malonek & al. (2005), three major lineages are resolved within a strongly supported, monophyletic liverwort clade (BS and PP = 100%), and Haplomitrium is weakly supported as the earliest diverging lineage. As demonstrated by Forrest and CrandallStotler (2004) and Stech and Frey (2004), Haplomitrium is ambiguously resolved in both likelihood and parsimony analyses if Treubia is excluded from the dataset, but resolves as the earliest diverging lineage with Treubia when the latter is included, with PP and BS values >90%. Furthermore, if Haplomitrium is excluded from the analysis, Treubia is always resolved as the earliest diverging lineage of the liverworts (Stech & al., 2000; Forrest & Crandall-Stotler, 2004). When the long branch between Haplomitrium and Treubia is decreased by increasing the number of taxa sampled within both, the support value for their relationship increases (Forrest & Crandall-Stotler, 2005). Within Treubiaceae resolution of Apotreubia nana as a sister taxon to Treubia is in agreement with the analysis of Stech & al. (2002) that placed A. hortonae R. M. Schust. & Konstantinova in a similar position based on partial trnL intron sequences of it, three additional accessions of Treubiaceae and 22 other bryophytes. It is not expected that the addition of missing data for A. nana will modify its position. With our taxon sampling a Blasiaceae/Marchantiopsida clade is the second divergence within the liverworts. Analyses that resolve either Blasia alone (HeNygrén & al., 2004) or a Blasia/Marchantiopsida lineage (Davis, 2004) as the earliest divergence do not include
Crandall-Stotler & al. • Thalloid liverwort evolution
critical sampling within Treubiaceae. Indeed, as documented by Soltis & Soltis (2004), adequate sampling, especially of long branch taxa, is essential to resolve early divergences. The traditional classification of Marchantiophyta into two classes or lineages, Jungermanniopsida and Marchantiopsida, is not supported by any of the molecular analyses that include Treubia (Stech & al., 2000; Qiu, 2003; Forrest & CrandallStotler, 2004, 2005). Rather, the resolution of three strongly supported lineages in our analyses, designated as Liverworts 1, 2, and 3 (Fig. 1), suggests that at least three classes, as proposed by Stotler & Crandall-Stotler in 1977, should be recognized if morphologically well defined Marchantiopsida are to be retained. Formal changes in the hierarchial scheme, however, are not proposed here since sampling across the diversity of Jungermanniidae and Marchantiopsida has not been included in these analyses. Although the general relationships resolved are congruent with earlier analyses, there are a few noteworthy differences. Pleurozia is herein included in our analyses for the first time. This leafy liverwort was resolved as sister to a Noteroclada/Metzgeria Raddi clade by HeNygrén & al. (2004), with a Bremer support value of 29, and as sister to a Metzgeriaceae H. Klinggr./Aneuraceae H. Klinggr. clade by Davis (2004), with 96% BS. This latter topology is resolved, without BS support, in one of the MPTs in our combined morphological and molecular analysis (Fig. 3), but is not recovered in either the 66taxon or 50-taxon molecular only analyses. Indeed, the positioning of Metzgeriineae between Pleurozia and the rest of the leafy liverworts is counter to intuitive judgments based on morphology as discussed in Forrest & Crandall-Stotler (2005). Within Jungermanniidae, only Pleurozia possesses lenticular apical cells in its adult shoots, which is one of the three apomorphies mapped on branch 3 (Table 2). Although this character might seem to support the relationship between Pleurozia and Metzgeriineae, our reconstructions of apical cell evolution show that lenticular apical cells have evolved independently four times, sometimes from a tetrahedral ancestral state and sometimes from a cuneate ancestral state (Fig. 5B). That the expression of lenticular apical cells by Pleurozia is homoplastic is suggested by significant differences in merophyte division patterns and thallus or shoot ontogeny between the simple thalloids and Pleurozia (Crandall-Stotler, 1976; Renzaglia, 1982). The two- or three-parted leaf of Pleurozia is formed from two or three, more or less cuboidal superficial leaf initials, comparable to other members of Jungermanniidae (Crandall, 1969), while the wing tissue of all simple thalloids is derived from a single, cuneate initial (Renzaglia, 1982). As to the second apomorphy of branch 3, i.e., gynoecia occurring on short branches, these branches are 309
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of three distinct developmental types, being endogenous, lateral in Pleurozia, exogenous, lateral in Aneuracae, and endogenous, ventral in Metzgeriaceae (coded under characters 10 and 11). Likewise, capsule walls, which are consistent within the branch 3 clade for character 43, are eight cell layers thick in Pleurozia (Thiers, 1993) and only two cell layers thick in Metzgeriineae (character 42). Because of its unique assemblage of gametophyte and sporophyte characters, Pleurozia has long been regarded as a remote, isolated lineage of leafy liverworts (Schuster, 1984; Crandall-Stotler & Stotler, 2000). The equivocal alignment of this genus with Metzgeriineae rather than within the larger leafy liverwort clade may be an artifact of inadequate taxon sampling, especially within the basal leafy liverworts. The relationship resolved between Makinoa and Fossombroniineae is also unexpected on the basis of morphology. Gametophytes of the monotypic genus Makinoa have the facies of a large Pellia, and in fact, M. crispata was initially described as a species of Pellia by Stephani (1897). In contrast to the spheroidal capsules that are diagnostic of Pellia and all taxa of Fossombroniineae, however, sporophytes of Makinoa bear long, cylindric capsules that open by a single longitudinal slit, a dehiscence type shared only with Haplomitrium and Monoclea Hook. (Fig. 6C). Schuster (1984, 1992) hypothesized that the affinities of Makinoa lay with Verdoornia and placed the two in separate subfamilies of Makinoaceae Nakai, of suborder Pallaviciniineae. This placement was repeated in Crandall-Stotler & Stotler (2000). In previous molecular analyses (Forrest & Crandall-Stotler, 2004, 2005) as well as the analyses presented herein, Verdoornia is nested in Aneuraceae, a position that recent morphological studies also support (Crandall-Stotler & Millar, 2004). Indeed, not only are Makinoa and Verdoornia not closely related, neither taxon appears to have affinities with Pallaviciniineae. The position of Calycularia remains equivocal, even with the addition of morphology. The single apomorphy mapped to branch 8 is not a strenuous predictor of relationships and is not expressed in all taxa of the clade. Future studies with expanded taxon sampling focused on this clade hopefully will clarify both the position of Calycularia and the paraphyly of Pallavicinia. Within the Blasiaceae/Marchantiopsida clade, the increased support for Sphaerocarpos being the earliest diverging lineage of Marchantiopsida is noteworthy. In an analysis by Boisselier-Dubayle & al. (2002) of nuclear 26S rRNA for 34 taxa of Marchantiopsida, rooted with Makinoa and Pellia, Neohodgsonia Perss. is resolved as the earliest diverging lineage of the complex thalloids, with Sphaerocarpos nested between Lunularia Adans. and the rest of Marchantiopsida. A similar topology was also resolved in both parsimony and likelihood 310
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analyses of five chloroplast loci by Forrest & CrandallStotler (2004). In later analyses by Forrest & CrandallStotler (2005), as well as all of the analyses herein, however, Sphaerocarpos is resolved as sister to the rest of Marchantiopsida. Additional multi-genome taxon sampling is necessary to unequivocally resolve the earliest diverging lineage of Marchantiopsida. There is limited agreement between traditional postulates of the ancestral liverwort morphotype and the ancestral character states reconstructed in our analysis. In the early part of the twentieth century, the most accepted concept of liverwort evolution viewed erect, isophyllous leafy growth forms as ancestral, and prostrate, bilateral thalloid forms as derived (e.g., Evans, 1939). In 1981 Schuster departed somewhat from this postulate, asserting that the ancestral liverwort consisted of a system of creeping and erect, terete, vascularized leafless axes, from which both leafy and thalloid forms were derived (Schuster, 1981: Fig. 5.2). His tabulation of ancestral characters included tetrahedral apical cells, unicellular slime papillae, unicellular smooth rhizoids, exogenous intercalary branching, widely scattered, naked gametangia, and chlorophyllose sporophytes, bearing massive seta and foot and with capsule walls 8–12 cell layers thick (Schuster, 1981: 134). More recent publications by Schuster (1992, 1999) elaborate a similar viewpoint. Of the 11 ancestral characters cited by Schuster (1981), only four, namely, tetrahedral apical cells, ventral, stalked slime papillae, smooth rhizoids, and lateral intercalary, exogenous branches, are reconstructed as ancestral states in our analyses (Appendix 1). Additional ancestral expressions reconstructed for the gametophyte include: a prostrate habit and bilateral, leafy growth form; small, homogeneous oil bodies in all cells; randomly scattered antheridia that are either protected by thallus lobes or submerged in pits; clustered, naked archegonia; and formation of a shoot calyptra around the developing sporophyte. Neither vascular strands nor asexual propagules are reconstructed as ancestral. Assembling the ancestral states for the sporophyte generation predicts as the plesiomorphic expression a moderate-sized sporophyte, with an obconoidal foot, generalized, undifferentiated seta, and ovoid capsule with bistratose walls and fourvalved dehiscence. Although our reconstructions have been assembled from a dataset directed towards the characters of Metzgeriidae, the general evolutionary trend from anisophyllous, bilateral gametophytes to isophyllous, radial forms can also be applied to Jungermanniidae, as postulated by Davis (2004) and HeNygrén & al. (2004). Specific trends in character evolution within the leafy and complex thalloid liverworts, however, remain to be formally reconstructed.
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GENERAL CONCLUSIONS Metzgeriidae as circumscribed by Crandall-Stotler & Stotler (2000) are a paraphyletic assemblage that includes the earliest diverging lineage of the liverworts, as well as taxa that are sisters to the complex thalloid and the leafy liverwort lineages, respectively. The clade comprising Haplomitrium and the two genera of Treubiaceae is strongly supported as the earliest diverging lineage of the liverworts and, therefore, as a suitable root for phylogenetic analyses within the hepatics. There is little congruence between the topologies recovered in separate molecular and morphological dataset analyses, with morphology alone providing very little resolution of ingroup relationships. Over two thirds of the morphological characters examined are homoplastic, including many that have been used to define genera, families and even suborders. Some of the clades resolved in the combined analysis are supported by several morphological apomorphies, e.g., the Blasiaceae/ Marchantiopsida clade (branch 2), the Pelliineae clade (branch 5), and the Pallaviciniaceae minus Moerckioideae clade (branch 10). In other lineages, however, there are few morphological markers that are predictive of the taxon relationships resolved, e.g., the Makinoa/Fossombroniineae clade (branch 7). In contrast to the most widely accepted theorem of liverwort evolution, the archeotype morphology is that of a prostrate, bilaterally symmetric, leafy plant, with the developmental and reproductive features of a simple thalloid liverwort. Future studies that combine comprehensive sampling in the leafy liverwort and complex thalloid lineages with this broad sampling of the simple thalloids are needed before these phylogenies can be translated into a meaningful classification.
ACKNOWLEDGEMENTS This work was supported by NSF grant No. DEB-997796, which is gratefully acknowledged. Thanks are extended to Sedonia Sipes (SIUC) for allowing us to use her sequencing facilities and to Rachel Murray for assistance with graphics. We also thank David Long (E) and Christine Cargill (CANB) for providing critical collections for sequencing. S. Robbert Gradstein, Jochen Heinrichs and Rosemary Wilson (GOET) are acknowledged for organizing the symposium “Bryophylogeny 2004”, and for the invitation to participate therein.
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Appendix 1. Morphological characters and character state codes. States in bold are reconstructed as ancestral; for equivocal reconstructions, possible ancestral states are indicated. CI - Consistency Index.
No. Character Type1 CI 1 Gametophyte growth form U 0.31 2 Gametophore habit U 0.13 3 Nostoc symbionts U 1.00 4 Air chambers U 0.50 5 Rhizoid distribution U 0.50 6 Rhizoid structure U 0.50 7 Rhizoid pigment U 0.11 8 Endomycorrhizae U 0.11 9 Oil body morphology U 0.27 10 Lateral branch type 11 Ventral branch type
U U
0.38 0.27
12 13 14 15 16 17 18
Stolons Rhizomes (subterranean) Ventral appendages Apical cell geometry Leaf-wing origin Apical cell tilt Strand cells
U U U U U O U
1.00 0.10 0.27 0.36 0.50 0.25 0.67
19 20 21 22
Strand number Sexual condition Antheridial distribution Perigonial structures
U U U U
1.00 0.10 0.24 0.40
23 Androecial paraphyses 24 Antheridial shape 25 Antheridial pigment when mature 26 Division pattern, young antheridium 27 Antheridial jacket cells 28 Antheridial stalk 29 Archeonial distribution 30 Archegonial position 31 Outer perichaetial structure 32 Outer perichaetial position 33 Inner perichaetial structure 34 Calyptra type 35 Gynoecial paraphyses 36 Rows of neck cells 37 Foot shape 38 Seta cell arrangement 39 Seta development 40 Seta internal anatomy 41 Capsule shape 42 Capsule wall thickness 43 Epidermal cell size 44 Epidermal cell thickenings
U U U
0.17 0.50 0.20
States 0 = nodal, radial; 1 = nodal, bilateral; 2 = costa and winged thallus; 3 = multistratose thallus 0 = erect; 1 = prostrate 0 = absent; 1 = present 0 = absent; 1 = present 0 = absent; 1 = fascicled; 2 = scattered 0 = smooth only; 1 = both smooth and pegged 0 = absent; 1 = present 0 = absent; 1 = present 0 = absent; 1 = homogeneous, all cells; 2 = botryoidal, all cells; 3 = granular, all cells; 4 = large, filling special cells 0 = true dichotomy; 1 = terminal, exogenous; 2 = intercalary, exogenous; 3 = endogenous; 4 = absent 0 = absent; 1 = terminal, exogenous; 2 = ventral intercalary, exogenous; 3 = lateral-ventral intercalary; 4 = endogenous 0 = absent; 1 = present 0 = absent; 1 = present 0 = absent; 1 = stalked papillae; 2 = multicellular hair; 3 = mutiseriate scales; 4 = true underleaves 0 = tetrahedral, dorsal; 1 = tetrahedral, ventral; 2 = lenticular; 3 = cuneate; 4 = hemidiscoid 0 = one initial; 1 = two initials 0 = dorsal; 1 = absent; 2 = ventral 0 = absent; 1 = thin-walled, pitted; 2 = thick-walled, pitted; 3 = thin-walled, not pitted; 4 = thick-walled, not pitted 0 = one central; 1 = two lateral; 2 = three 0 = dioicous, monomorphic; 1 = dioicous, male smaller; 2 = monoicous 0 = randomly scattered; 1 = rows, two; 2 = rows, more than two; 3 = clustered; 4 = on special branches 0 = absent; 1 = axillary in thallus lobes; 2 = disciform with leafy bracts; 3 = scales derived from epidermis; 4 = involucral pits from thallus; 5 = pits formed from scale fusion; 6 = true bracts, axillary position [Equivocal, 1 or 4] 0 = absent; 1 = present 0 = long, ellipsoidal; 1 = globose to ovoid 0 = white; 1 = yellow-orange
O
1.00
0 = one-celled, like the archegonium; 1 = two-celled; 2 = four celled [Equivocal, 0 or 1]
U O U U U U U O U O O U U U U O U U
0.25 0.21 0.23 0.10 0.30 0.17 0.33 0.13 0.45 0.43 0.18 1.00 0.33 1.00 0.13 0.17 0.17 0.42
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
U U U U O U U U U U U U U U U U O U U U U
0.58 0.27 0.17 0.40 0.21 0.25 0.20 0.25 0.20 0.50 0.20 0.50 0.50 0.36 1.00 0.25 0.29 0.13 0.25 0.08 0.50
0 = untiered; 1 = tiered 0 = more than 4 cell rows; 1 = quadriseriate; 2 = biseriate; 3 = uniseriate [Equivocal, 1 or 2] 0 = scattered; 1 = clustered; 2 = on special branches 0 = acrogynous; 1 = anacrogynous [Equivocal] 0 = absent; 1 = scales from thallus surface; 2 = bracts and bracteoles; 3 = marchantioid “involucre” 0 = encircling archegonia; 1 = posterior only 0 = absent; 1 = pseudoperianth; 2 = perianth; 3 = marchantioid pseudoperianth 0 = true calyptra; 1 = shoot calyptra; 2 = partial perigynium; 3 = solid perigynium 0 = absent; 1 = uniseriate hairs or papillae; 2 = multiseriate scales [Equivocal, 0 or 1] 0 = four; 1 = five; 2 = six; 3 = more than six 0 = bulbous; 1 = obconoidal; 2 = cup-shaped 0 = nonarticulate; 1 = articulate 0 = general; 1 = cruciate 0 = undifferentiated; 1 = internally differentiated 0 = spheroid; 1 = ovoid-ellipsoid; 2 = long cylindric 0 = unistratose; 1 = bistratose; 2 = multistratose 0 = larger than inner cells; 1 = similar to inner cells 0 = absent; 1 = semiannular; 2 = reticulate; 3 = nodular; 4 = evenly thickened; 5 = longitudinal annular [Equivocal] 0 = absent; 1 = annular; 2 = semiannular; 3 = fenestrate; 4 = nodular; 5 = evenly thickened 0 = zero; 1 = one; 2 = two; 3 = four 0 = free; 1 = attached 0 = basal; 1 = absent; 2 = apical 0 = absent; 1 = one; 2 = two; 3 = more than two 0 = one; 1 = many 0 = smooth; 1 = finely papillate; 2 = highly sculptured 0 = absent; 1 = preent 0 = exosporic; 1 = endosporic 0 = absent; 1 = present 0 = present; 1 = absent 0 = cell mass to cylinder; 1 = flattened plate; 2 = short, unbranched filament 0 = absent; 1 = present 0 = absent; 1 = exogenous, unicellular; 2 = exogenous, multicellular; 3 = endogenous 0 = scattered; 1 = in special receptacles 0 = absent; 1 = present 0 = 8; 1 = nine; 2 = ten 0 = absent; 1 = present 0 = absent; 1 = present [Equivocal] 0 = < 35 µm; 1 = > 40 µm [Equivocal] 0 = absent; 1 = present
Internal cell thickenings Capsule wall dehiscence lines Capsule valve apices Elaterophores Elater thickening bands Sporocyte plastid number Spore ornamentation Precocious germination Spore germination type Germ tube Quadrant stage Primary protonema type Tubers Gemmae Gemmae position Cladia (brood branches) Base chromosome number Polyploid populations Mucilaginous sheath Spore size Permanent tetrads
1Type: U - unordered, O - ordered.
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54 (2) • May 2005: 299–316
Appendix 2. Data matrix for 65 morphological characters; numbers included in ( ) designate multiple states coded as polymorphisms, numbers included in { } designate multiple state uncertainties,? designates either inapplicable or missing data. Exemplars 1–10 11–20 21–30 31–40 41–50 51–60 61–65 Allisonia cockaynii 310020111? 000230?0?0 33010?0?11 11000??000 0201(12)0?13? 20????00?0 ?0010 Aneura pinguis 3100200121 000120?0?1 450101?220 1?01031000 1113230211 1000100200 21000 Apotreubia nana 1100200141 00011000?{12} 0011000201 0?000?0000 11002??121 20????00?0 10100 Austrofossombronia australis 1100201?11 0003?000?1 00011?0101 0?030?0000 020020?13? 2000??00?0 ??000 Austrofossombronia peruviana 1100201?11 2003?000?1 00011?0001 0?030?0000 120020?13? 20000000?0 ??000 Blasia pusilla 2110200000 000330?0?1 0511????10 3?00012000 1203030130 1010000211 11010 Calycularia crispula 2100200110 200340?0?1 3301010111 1103011000 020223002? 20????00?0 10010 Cavicularia densa 2110200000 000330?321 05?0????10 3?00010000 1103030130 1110?00210 10010 Fossombronia angulosa 1100201011 00032000?0 0301110101 0?03010000 011020?121 20000000?0 11010 Fossombronia foveolata 1100201011 00022000?2 3001110101 0?03010000 011040?131 20000000?0 11010 Greeneothallus gemmiparus 21002000?0 0002?0?401 23010?0?11 11020??000 110403013? 2?????00?1 ?0000 Haplomitrium blumii 00000??012 2111000101 3210100110 2001(12)01000 20?5?11120 20000000?0 10100 Haplomitrium gibbsiae (01)0000??012 2111000101 (03)21(01)0011(01)1 2002(12)01000 20?5?311(12)? 2000?000?0 10101 Haplomitrium hookeri 00000??012 2111000101 0111100101 2000(12)01001 20?5?(12)1121 2000?000?0 10100 Hattorianthus erimonus 2100200014 3012200410 2301010211 111101?000 220402112? 20001000?0 00010 Herbertus alpinus 0000100013 4?1411?0?1 3601?10210 202111?000 1213230131 1000?000?0 10000 Hymenophyton flabellatum 2000200131 3012201200 450101?220 110301?000 210303022? 10????00?0 10000 Isotachis multiceps 1100100021 400411?0?0 16010???10 20230?2000 220323012? 10000000?0 10000 Jensenia connivens 2000201031 301220?201 3301110211 1013011000 210402113? 20001000?0 00000 Jubula hutchinsiae 110010003(12) 00041120?2 4601011310 2020011010 010333011? 11101100?0 10000 Jungermannia leiantha 1100200?33 000011?0?2 160101??10 202101?000 110323012? 1000100100 ?1000 Lobatiriccardia lobata 3100200?31 0001?0?0?0 450101?320 1101031000 2113(14)3021? 10????00?0 00000 Makinoa crispata 3100201120 00023020?1 3501?10111 1?02011000 2113211011 10001000?0 10000 Marchantia polymorpha 3101210140 000330?0?0 440002?220 3030020000 10?1?0?120 1001010210 11000 Metzgeria conjugata 2100200001 40012010?2 4001010321 010101?000 1113(24)3011? 1000120(23)01 11000 Moerckia blyttii 2100201120 000230?0?0 23010?0111 1011012000 120453012? 2?????00?0 ?0000 Moerckia flotoviana 2100200010 0002?0?311 23010?0111 1011012000 120402012? 20001000?0 10000 Monoclea gottschei 3100200140 000130?0?0 341002?011 3000120000 20?2?11120 10000100?0 10100 Neohodgsonia mirabilis 3101200040 0003???0?2 440?????20 0030020000 00?1?3012? 2001110210 10000 Noteroclada confluens 1100200111 00021000?2 1401010111 0?03012000 020323003? 21101010?1 10010 Pallavicinia lyellii 2100201121 300220?201 1301010211 1011011000 2104031121 20001000?0 00000 Pallavicinia rubristipa 2000201?32 3012???201 33011???11 101??????? ?????????? ??????00?0 ?00?? Pallavicinia xiphoides 2100200031 3002???200 13010???11 10130??000 210403112? 2?????00?0 ?0000 Pellia epiphylla 3100201110 00014010?2 3401010110 0?11012000 020323003? 11101000?0 11010 Petalophyllum ralfsii 2100200111 00031010?1 0301110111 0?11122000 021320?131 20????10?0 10010 Phyllothallia nivicola 1100200?12 00023000?0 3301010111 1002210000 020320?03? 20????00?0 10110 Pleurozia purpurea 1000200023 00102100?0 460?????20 20210??000 121333012? 2110?000?0 10010 Podomitrium phyllanthus 2100201031 3011201200 4301010220 1?11111000 210402112? 20101000?0 00000 Porella navicularis 1100100011 00041120?1 4601010220 2021011000 020303012? 11100000?0 ??010 Reboulia hemisphaerica 3101210040 200330?0?2 341002??20 3000020000 00?0?0?131 200101?0?0 11010 Riccardia capillacea 3100200131 000120?0?0 4501010320 1102031010 1113{02}3021? 1000110300 21000 Riccia huebeneriana 3101210000 000330?0?2 040?02??01 0?0002???? 00?0?0?1?? 200101?0?0 01010 Scapania nemorea 110020013(13) 40101100?0 1611010310 2021011000 121323012? 0000100100 10000 Schistochila appendiculata 1000101033 001411?0?0 16110?0210 2022??2000 220323012? 10????00?0 10000 Sphaerocarpos texanus 2100200000 000130?0?1 0401121301 0030020110 00?0?0?1?1 20010000?0 00011 Symphyogyna hymenophyllum 2000200031 301130?200 2301011211 1102011000 210403112? 20001000?0 ??000 Symphyogyna undulata 2100201031 3001???200 23010??211 110201?000 210403113? 20????00?0 ??000 Treubia lacunosa 1100200142 00011000?0 31111?0011 1?02131000 1200(24)3012? 20????0200 10100 Verdoornia succulenta 3100200131 00012010?1 0401??0?11 110023?000 111443022? 2?????00?0 ??100 Xenothallus vulcanicolus 2000200034 2011???200 040?????11 11020?1000 110402113? 20????0200 ??000
314
Appendix 3. Voucher information and GenBank accession numbers for taxa utilized in this study. All vouchers unless otherwise indicated are deposited in ABSH. Voucher information GenBank accession numbers Taxon (Ax = axenic culture) NrLSU NrSSU nad5 rbcL rps4 trnL atpβ psbA Ingroup Taxa Apotreubia nana (S. Hatt. & Inoue) S. Hatt. & Mizut. Long 30451, Nepal (E) AY877389 AY877397 Allisonia cockaynii (Steph.) R. M. Schust. Stotler & Crandall-Stotler 4470, New Zealand AY688679 AY688701 AY688742 AY507389 AY507432 AY507518 AY507344 AY507472 Aneura pinguis (L.) Dumort. Hatcher culture #12, U.S.A., Wisconsin (Ax) AY877366 AY688702 AY688744 AY507391 AY507520 AY507346 AY507474 Austrofossombronia australis (Mitt.) R. M. Schust. Stotler & Crandall-Stotler 4610, New Zealand AY688681 AY688703 AY688745 AY507392 AY507434 AY507521 AY507347 AY507475 Austrofossombronia peruviana (Gottsche & Hampe) Stotler & Crandall-Stotler 4187, Venezuela (Ax) AY688682 AY688704 AY877384 AF536230 AY507435 AY507522 AY507348 AY507476 Stotl., Crand.-Stotl. & A. V. Freire Blasia pusilla L. Renzaglia s.n., U.S.A., North Carolina (Ax) AY688683 AY688705 AY688746 AF536232 AY507436 AY507523 AY507349 AY507477 Calycularia crispula Mitt. Furuki s.n., Japan AY688684 AY688706 AY688747 AY507395 AY507437 AY507524 AY507350 AY507478 Cavicularia densa Steph. Nishimura s.n., Japan AY507525 AY507479 Fossombronia angulosa (Dicks.) Raddi U. & E. Drehwald s.n. [ref. no. 371], Teneriffe (Ax) AY688686 AY688709 AY688750 AY507398 AY507440 AY507527 AY507353 AY507481 Fossombronia foveolata Lindb. D. & S. Williams s.n., U.S.A., Massachusetts (Ax) AY688687 AY688710 AY507399 AY507441 AY507528 AY507354 AY507482 Greeneothallus gemmiparus Hässel Long 31831, Argentina, Tierra del Fuego (E) AY877367 AY688780 AY688792 AY688808 AY688818 AY688828 Haplomitrium blumii (Nees) R. M. Schust. Furuki 384, Malaya (Ax) AY877368 AY688711 AY507402 AY507443 AY507531 AY507357 AY507485 Haplomitrium gibbsiae (Steph.) R. M. Schust. Stotler & Crandall-Stotler 4541, New Zealand AY877369 AY688712 AY688753 AY688781 AY688793 AY688809 Haplomitrium hookeri (Sm.) Nees Long 28042, Scotland (E) for psbA; others AY312904 AY330437 U87072 AJ251064 AY007642 AF313555 AY877398 accessed from GenBank Hattorianthus erimonus (Steph.) R. M. Schust. & Inoue Furuki s.n., Japan AY688714 AY688754 AY507403 AY507445 AY507533 AY507359 AY507487 Herbertus alpinus (Steph.) E. A. Hodgs. Stotler & Crandall-Stotler 4580, New Zealand AY688689 AY688715 AY507404 AY507446 AY507534 AY507360 AY507488 Hymenophyton flabellatum (Labill.) Dumort. Stotler & Crandall-Stotler 4511, New Zealand AY877370 AY688716 AY688755 AY507406 AY507448 AY507535 AY507362 AY507489 Isotachis multiceps (Lindenb. & Gottsche) Gottsche Stotler & Crandall-Stotler 3478, Panama (Ax) AY877371 AY877385 AY507407 AY507449 AY507537 AY507363 AY507491 Jensenia connivens (Colenso) Grolle Stotler & Crandall-Stotler 4535, New Zealand AY734736 AY688718 AY688782 AY507450 AY688815 AY688819 AY688829 Jubula hutichinsiae (Hook.) Dumort. ssp. javanica Kodama s.n., Japan (Ax) AY877372 AY507408 AY688794 AY507538 AY507364 AY507492 Steph. Jungermannia leiantha Grolle Stotler & Crandall-Stotler 107, U.S.A., Illinois (Ax) AY688719 AY688756 AY507409 AY507451 AY507539 AY507365 AY507493 Lobatiriccardia lobata (Schiffn.) Furuki Glenny & Kinser CS 4581, New Zealand AY877373 AY688757 AY507421 AY507462 AY507553 AY507378 AY507507 Makinoa crispata (Steph.) Miyake Stotler & Crandall-Stotler 4047, China AY877374 AY877386 AY877390 AY877393 AY877394 AY877395 AY877399 Marchantia polymorpha L. [accessed from GenBank] AF226020 X75521 M68929 U87079 X04465 AF228786 NC001319 NC001319 Metzgeria conjugata Lindb. Henson 1247, U.S.A., Illinois (Ax) AY688690 AJ000703 AY507411 AY507453 AY507541 AY507367 AY507495 Moerckia blyttii (Moerch) Brockm. Wagner s.n., U.S.A., Oregon AY507412 AY507454 AY507542 AY507368 AY507496 Moerckia flotoviana (Nees) Schiffn. Kinser & Schuette 709, U.S.A., New York Y16013 AY688758 AY507413 AY688796 AY507543 AY507369 AY507497 Monoclea gottschei Lindb. ssp. elongata Gradst. Stotler & Crandall-Stotler 89-60, Puerto Rico (Ax) AY688691 AY688721 AY877388 AY507414 AY507455 AY507544 AY507370 AY507498 & Mues Neohodgsonia mirabilis (Perss.) Perss. Stotler & Crandall-Stotler 4542, New Zealand AY688692 AY688722 AY688759 AY507415 AY507456 AY507545 AY877396 AY507499 Noteroclada confluens Taylor ex Hook. & Wilson Forrest 566, Venezuela, AY877376 AY688760 AY688784 AY688797 AY507546 AY507372 AY507500 Pallavicinia lyellii (Hook.) Carruth. Marsh s.n., U.S.A., Arkansas (Ax) AY734742 AY688723 AY688761 AY507416 AY688798 AY507547 AY507373 AY507501 Pallavicinia rubristipa Schiffn. Cargill & Vella 507, Australia AY734744 AY734753 AY734693 AY734702 AY734720 AY734711 Pallavicinia xiphoides (Hook. f. & Taylor) Trevis. Stotler & Crandall-Stotler 4517, New Zealand AY734743 AY734752 AY734692 AY734701 AY734719 AY734710 Pellia epiphylla (L.) Corda Stotler & Crandall-Stotler 4414, U.S.A., North AY688693 AY688724 AY688764 AY688787 AY507457 AY507548 AY688823 AY507502 Carolina (Ax) Petalophyllum ralfsii (Wilson) Nees & Gottsche ex J. Proskauer culture via D. Mueller-TAMU to AY877377 AY688725 AY507417 AY507458 AY507549 AY507374 AY507503 Lehm. ABSH U.K. (Ax) Phyllothallia nivicola E. A. Hodgs. Stotler & Crandall-Stotler 4537, New Zealand AY688694 AY688726 AY688765 AY507418 AY507459 AY507550 AY507375 AY507504 Pleurozia purpurea Lindb. Long 28868, Bhutan (E) AY877391 AY877401 Podomitrium phyllanthus (Hook.) Mitt. Stotler & Crandall-Stotler 4517, New Zealand AY734740 AY688727 AY688766 AY507419 AY507460 AY507551 AY507376 AY507505 Porella navicularis (Lehm. & Lindenb.) Pfeiff. Stotler & Crandall-Stotler 3410, U.S.A., AY688695 AY688728 AY688767 AY507420 AY507461 AY507552 AY507377 AY507506 California (Ax)
54 (2) • May 2005: 299–316 Crandall-Stotler & al. • Thalloid liverwort evolution
315
316 Voucher information (Ax = axenic culture)
Forrest 531, U.S.A., Illinois Forrest 558, Venezuela Stotler & Crandall-Stotler 92-175, U.S.A., Illinois (Ax) Stotler & Crandall-Stotler s.n. [ref. no. 265], U.S.A., Illinois (Ax) Schistochila appendiculata (Hook.) Dumort. ex Trevis. Stotler & Crandall-Stotler 4455, New Zealand Sphaerocarpos texanus Austin Stotler & Crandall-Stotler 1515, U.S.A., Illinois (Ax) Symphyogyna hymenophyllum (Hook.) Nees & Mont. Stotler & Crandall-Stotler 4452, New Zealand Symphyogyna undulata Colenso Stotler & Crandall-Stotler 4463, New Zealand Treubia lacunosa (Colenso) Prosk. Stotler & Crandall-Stotler 4561, New Zealand Verdoornia succulenta R. M. Schust. Stotler & Crandall-Stotler 4602, New Zealand Xenothallus vulcanicolus R. M. Schust. Stotler & Crandall-Stotler 4580, New Zealand Outgroup Taxa Anthoceros angustus Steph. [accessed from GenBank] Anthoceros punctatus L. [accessed from GenBank] Leiosporoceros dussii (Steph.) Hässel [accessed from GenBank] Megaceros aenigmaticus R. M. Schust. [accessed from GenBank] Megaceros flagellaris (Mitt.) Steph. Stotler & Crandall-Stotler 4478, New Zealand Notothylas breutelii (Gottsche) Gottsche [accessed from GenBank] Phaeoceros laevis (L.) Prosk. [accessed from GenBank] Andreaea rothii F. Weber & D. Mohr [accessed from GenBank] Andreaeobryum macrosporum Steere & B. M. Murray [accessed from GenBank] Hypnum lindbergii Mitt. [accessed from GenBank] Mnium hornum Hedw. [accessed from GenBank] Polytrichum pallidisetum Funck [accessed from GenBank] Sphagnum palustre L. [accessed from GenBank] Takakia lepidozioides S. Hatt. & Inoue Schofield 3176, Canada, British Columbia for psbA and atpB; others accessed from GenBank Equisetum telmateia Ehrh. [accessed from GenBank] Psilotum nudum (L.) P. Beauv. [accessed from GenBank]
Taxon Ingroup Taxa Reboulia hemisphaerica (L.) Raddi Riccardia capillacea (Steph.) Meenks & C. De Jong` Riccia huebeneriana Lindenb. ssp. sullivantii (Austin) R. M. Schust. Scapania nemorea (L.) Grolle
Appendix 3 (continued).
AY327827 AY327829 AF478295 AF161128 AF231177 AY312944 AF231902 AY312947
D86545 AY507371 AF313556 AF313557 AY688826
AB087434 AY877400 AY312863 AY312893 AY312908 AY312914 AY312920 AY507513 AJ130749 AF313580 AF313542 AJ012794 AP004638 AP004638 AY241586 AP004638 AP004638
AB087443 AF306952 AF306953 AF143035 AF023796 AF306956 AF231892 AF306950
AY507510 AY507511 AY507512 AY688835 AY507516 AY507517
AF313562 X81963
AB087449 U87063 AY463052 L13481 AY463054 AF244562 AF231060 AF231059 AB029390 AF226820 AY312934 AF231887 AF244565
AY507381 AY507382 AY507383 AY688825 AY507385 AY507387 AY507388
-
AY877387 AY312862 AY312879 AY312884 AY312888 AY312889
AY507556 AY507557 AY507558 AY688814 AY507559 AY507561 AY507562
Y16015 AF126293 AY877382 AF126292 U18491 X99750 AJ275005 AF229922 X80985 AY330421 Y11370 AJ269686
AY507465 AY507466 AY507467 AY688804 AY507468 AY507470 AY507471
AF226035 AY877375 AF197062 AY330426 AY330441 AY330445 AY331451 AF197061
AY507424 AY507425 AY507426 AY688790 AY507428 AY507430 AY507431
AY688770 AY688771 AY688772 AY688773 AY688774 AY688776 AY688777
AY688732 AY688733 AY688735 AY688736 AY688738 AY688740 AY688741
psbA
AY688696 AY688697 AY688698 AY877380 AY688699 AY68700 AY877381
atpβ
AY688769 AY507423 AY507464 AY507555 AY507380 AY507509
GenBank accession numbers rbcL rps4 trnL
AY877379 AY688731
nad5
AY877383 AY688788 AY688801 AF264644 AY8773402 AY688768 AY877392 AY688802 AY688833 AY507422 AY507463 AY507554 AY507379 AY507508
Nr SSU
AF226016 AY688729 AY877378 AF226004 AY688730
Nr LSU
Crandall-Stotler & al. • Thalloid liverwort evolution 54 (2) • May 2005: 299–316
