Unraveling the evolutionary history of the liverworts (Marchantiophyta): multiple taxa, genomes and analyses

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Unraveling the evolutionary history of the liverworts(Marchantiophyta): multiple taxa, genomes and analysesAuthor(s): Laura L. Forrest, E. Christine Davis, David G. Long, Barbara J.Crandall-Stotler, Alexandra Clark, and Michelle L. HollingsworthSource: The Bryologist, 109(3):303-334. 2006.Published By: The American Bryological and Lichenological Society, Inc.DOI: 10.1639/0007-2745(2006)109[303:UTEHOT]2.0.CO;2URL:http://www.bioone.org/doi/full/10.1639/0007-2745%282006%29109%5B303%3AUTEHOT%5D2.0.CO%3B2

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Unraveling the evolutionary history of the liverworts

(Marchantiophyta): multiple taxa, genomes and analyses

LAURA L. FORREST

Department of Plant Biology, University of Southern Illinois,

Carbondale, IL 62901, U.S.A.

e-mail: [email protected]

E. CHRISTINE DAVIS

Department of Biology, Duke University, Box 90338,

Durham, NC 27708-0338, U.S.A.


DAVID G. LONG

Royal Botanic Garden, Edinburgh, 20A Inverleith Row,

Edinburgh, EH3 5LR, Scotland, U.K.


BARBARA J. CRANDALL-STOTLER

Department of Plant Biology, University of Southern Illinois,

Carbondale, IL 62901, U.S.A.


ALEXANDRA CLARK AND MICHELLE L. HOLLINGSWORTH

Royal Botanic Garden, Edinburgh, 20A Inverleith Row,

Edinburgh, EH3 5LR, Scotland, U.K.

e-mails: [email protected]; [email protected]

ABSTRACT. Nucleotide sequence data from three chloroplast genes (rbcL, rps4 and psbA), one

nuclear gene (the ribosomal LSU) and one mitochondrial gene (nad5) were assembled for

173 species in 117 genera of liverworts, making this the largest molecular phylogeny of the

group to date. Analyses of these data provide support for the monophyly of the liverworts,

and for previously resolved backbone relationships within the Marchantiophyta. The earliest

divergence involves the ‘‘simple thalloid’’ taxa of the Haplomitriaceae and Treubiaceae. A

Blasiaceae/complex thalloid clade is resolved as sister to all remaining liverworts. The leafy

liverworts do not resolve as monophyletic. The separation of the Aneuraceae/Metzgeriaceae

from all other simple thalloids and their placement within the ‘‘leafy’’ clade as sister to the

enigmatic leafy genus Pleurozia, as suggested in earlier molecular phylogenies, is also

supported by this far larger data set.

KEYWORDS. Liverworts, Marchantiophyta, multi-genome phylogeny, molecular phylogeny,

rps4, rbcL, pbsA, nad5, LSU.

^ ^ ^

THE BRYOLOGIST 109(3), pp. 303–334

Copyright �2006 by the American Bryological and Lichenological Society, Inc.

0007-2745/06/$3.35/0

Liverworts hold a pivotal position in early land plant

evolution, with growing evidence that they represent

the earliest diverging lineage of embryophytes (e.g.,

Groth-Malonek & Knoop 2005; Groth-Malonek et al.

2005; Pruchner et al. 2002; Qiu et al. 1998;

Steinhauser et al. 1999; Wolf et al. 2005). The group

also has the oldest fossil record of any bryophyte, with

some evidence that it dates to at least 475 million

years before present (Wellman et al. 2003). However,

until the start of this millennium surprisingly few

hypotheses of phylogenetic relationships within this

ancient and morphologically heterogeneous group

had been tested with molecular data (Forrest &

Crandall-Stotler 2005). Recently, enormous leaps in

our understanding of liverwort evolution have

occurred, with the publication of a number of key

multi-gene, multi-taxon analyses (e.g., Crandall-

Stotler et al. 2005; Davis 2004; Forrest & Crandall-

Stotler 2004, 2005; He-Nygren et al. 2004, 2006).

However, all but the last of these studies had their

independent foci on specific clades within the

Marchantiophyta Stotler & Crand.-Stotler, with

Forrest and Crandall-Stotler (2004, 2005) and Cran-

dall-Stotler et al. (2005) focusing on the simple

thalloid liverworts (Jungermanniopsida Stotler &

Crand.-Stotler subcl. Metzgeriidae Barthol.-Began),

and Davis (2004) and He-Nygren et al. (2004)

focusing on the leafies (subcl. Jungermanniidae Engl.).

No one of these multi-gene analyses has included a

broad level of sampling across the entire March-

antiophyta and, further, an entire class, the complex

thalloids (Marchantiopsida Cronquist, Takht. & W.

Zinm.), has effectively been neglected since the single-

locus study of Boisselier-Dubayle et al. (2002).

Estimates of the total number of liverwort species

are in the order of 4500–5000 (http://bryophytes.

plant.siu.edu) in 376 genera and 74 families (Cran-

dall-Stotler & Stotler 2000). The vast majority of

species belong to the Jungermanniopsida subcl.

Jungermanniidae or leafy liverwort group (Schuster

1984). Although the leafy liverworts contain most of

the taxonomic diversity of the phylum, the most

significant morphological diversity is expressed within

the thalloid liverwort groups. Thalloids currently

comprise around 500 species of simple thalloids

(Jungermanniopsida subcl. Metzgeriidae) and 350

species of complex thalloids (Marchantiopsida).

Liverwort taxon sizes are representative of the ‘‘hollow

curve’’ distribution of Willis (1922); of the currently

accepted liverwort genera, 148, i.e., ca. 40%, are

monospecific, and relatively few contain more than

100 species, with the two largest genera being

Frullania Raddi (over 300 species), and Plagiochila

(Dumort.) Dumort. (over 400 species). Likewise,

eight families are monospecific, and 37 contain fewer

than 10 species. There are 14 families with over 100

species, including one far outlier, the Lejeuneaceae

Casares-Gil, which alone is estimated to contain

nearly 1000 species in 91 genera (Gradstein et al.

2003). However, to address whether these disparate

taxon sizes are a natural phenomenom, or if this

‘‘hollow curve’’ distribution is an artificial construct,

as suggested by Clayton (1974) and Walters (1986),

discussion must be framed in terms of clades rather

than in terms of traditional arbitrary taxonomic units

like families and genera. This is particularly important

as evidence mounts that polyphyly and paraphyly are

rife in traditional taxonomies of the group (e.g.,

Forrest et al. 2005b; Long et al. 2000; Schaumann et al.

2005).

Recent phylogenetic studies have identified the

major backbone clades within the group to be 1) the

Haplomitriopsida Stotler & Crand.-Stotler, named by

Stotler and Crandall-Stotler in 1977 and here defined

to include the Haplomitriaceae Dedecek and Treu-

biaceae Verd. [¼ Treubiopsida Stech., J.-P. Frahm,

Hilger & W. Frey as delimited in He-Nygren et al.

(2006)], with 20 recognized species, 2) the March-

antiopsida (defined by He-Nygren et al. (2006) to

include the Blasiales (R. M. Schust.) Stotler & Crand.-

Stotler), with around 350 species, and 3) the

Jungermanniopsida Stotler & Crand.-Stotler, with

around 4000–4500 species (Crandall-Stotler et al.

2005; Davis 2004; Forrest & Crandall-Stotler 2004,

2005; Forrest et al. 2005a; Heinrichs et al. 2005; He-

Nygren et al. 2006).

To provide a broader sampling across the

diversity of liverworts, we have assembled a data

matrix with 189 liverwort accessions representing all

but two of the 32 suborders and including approx-

imately 2.5–4% of all liverwort species. The Brevian-

thineae J. J. Engel & R. M. Schust., containing two

genera (Brevianthus J. J. Engel & R. M. Schust. and

Chonecolea Grolle) and four species, and the mono-

304 the bryologist 109(3): 2006

specific Monocarpineae R. M. Schust., were not

available for sampling. Although our sampling is

focused on resolving the major lineages within the

group, and under-represents diversity in the highly

speciose leafy liverworts, it represents the largest

taxonomic sampling of liverworts to date, with 117 of

the estimated 376 genera (over 30%) represented.

Twenty-five of the 32 genera of complex thalloids

(78%), 30 of the 38 genera of simple thalloids (79%),

and 62 of the 306 genera of leafies (20%) are included.

MATERIAL AND METHODS

Taxon sampling. Sequences were assembled for

189 accessions of hepatics, with representation as

follows: eight species, three genera, both suborders of

the Haplomitriopsida; 36 species, 25 genera, seven of

the eight suborders of the Marchantiopsida; 49

species, 27 genera, all eight suborders of the

Jungermanniopsida subcl. Metzgeriidae; and 80

species, 62 genera, 15 of the 16 suborders of the

Jungermanniopsida subcl. Jungermanniidae. Five

hornwort, ten moss species and two vascular plants

were also included, and initial topologies (Maximum

Parsimony) were rooted using four algal taxa. Non-

liverwort sequences were mostly obtained from

GenBank. For GenBank numbers and voucher

information, see Table 1. Classification throughout

the paper follows Crandall-Stotler and Stotler (2000),

unless explicitly stated.

Molecular methods. Molecular methods differ

according to laboratory, with sequences being pro-

duced from three different labs (see Table 1).

Protocols for sequences generated at Southern Illinois

University follow Forrest and Crandall-Stotler (2004,

2005); methodologies from Duke University follow

Davis (2004), and from the Royal Botanic Garden,

Edinburgh, follow Schill et al. (2004).

Data analyses. Sequences were aligned in PAUP*

version 4.0b10 (Swofford 2002). Regions of ambig-

uous alignment and incomplete data (i.e., the

beginnings and ends of sequenced regions) were

identified and excluded from further analyses. The

data matrix is available in NEXUS format at

TreeBASE (www.treebase.org; study accession num-

ber S1519; matrix accession number M2725).

Missing data.—A total of 137 sequences were

missing, out of 1,050 possible sequences (ca. 13%). Of

these, 29 were from the nr LSU, 34 were from nad5,

29 from rbcL, 24 from rps4 and 21 from psbA (see

Table 1). Also, nad5 sequences generated at Duke

University only included the region between primers

K and Li, while those generated at Southern Illinois

University included the region between primers K2 or

K and L (Beckert et al. 1999). Despite this, the entire

K2-L region was included in the analyses, due to its

usefulness in resolving backbone relationships (Forr-

est et al., unpublished data; Groth-Malonek et al.

2005).

Analyses were conducted at Duke University,

Southern Illinois University and the Royal Botanic

Garden, Edinburgh. Maximum parsimony (MP)

phylogenetic analyses were performed using PAUP*

4.0b10 (Swofford 2002), mounted on an Apple

Macintosh G4 [SIU]. Bayesian inference (BI) analyses

were performed using MrBayes 3.0b4 (Huelsenbeck &

Ronquist 2002) and MrBayes 3.1 (Huelsenbeck &

Ronquist 2005), mounted on an Apple Macintosh G4

[SIU], G5 [E] or Unix running Linux Centos 3

[Duke]. Maximum parsimony analyses were run

under Fitch parsimony, using 1000 random addition

replicates, with TBR, saving 15 trees per replicate. The

most parsimonious trees (MPTs) found were then

input to a second round of TBR, with no limit on the

number of trees saved. Bootstrapping was performed

with 1000 replicates, using an heuristic search strategy

(five random addition replicates, saving five trees per

replicate). Prior to the combined analyses, separate

parsimony analyses were conducted for each of the

five loci individually, to identify and eliminate

problem sequences.

MrModeltest version 1.1b (Nylander 2002) was

used to establish the model of DNA evolution with

the best fit to the data for the combined DNA matrix,

and also for each locus individually, using the

topology of one of the trees resolved by MP analysis

(Table 2). Posterior probabilities for clades were

generated with at least two independent runs per

analysis, using the models described in Table 2, with

one million generations run for the homogeneous

analysis (BI1). The homogeneous analysis used the

General Time Reversible model of nucleotide sub-

stitution (Yang 1994) plus a gamma distribution of

rate variation among sites and invariant sites

(GTRþIþR) as selected by Akaike’s Information

Forrest et al.: Liverwort phylogeny 305

Tab

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Tab

le1.

Co

nti

nu

ed.

Tax

on

Vo

uch

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form

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nN

rL

SU

na

d5

rbcL

rps4

psb

AL

ab

Cep

hal

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vari

ans

(Go

ttsc

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Step

h.

New

Zea

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d,E

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964

(F)

mix

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ith

Jam

eson

iell

aco

lora

ta(L

ehm

.)St

eph

.

AY

6082

22—

DQ

4396

89A

Y60

8074

AY

6079

53D

uke

Cep

hal

ozie

lla

hir

ta(S

tep

h.)

R.M

.Sch

ust

.A

ust

rali

a,St

reim

ann

5979

3(N

Y)

AY

6082

04A

Y60

8271

DQ

4396

82A

Y60

8054

AY

6079

33D

uke

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6082

29A

Y60

8293

—A

Y60

8083

AY

6079

62D

uke

Let

hoc

olea

glos

syph

ylla

(Sp

ruce

)G

roll

eE

cuad

or,

Dav

is25

9(D

UK

E)

AY

6082

30A

Y60

8294

DQ

4396

92A

Y60

8084

AY

6079

63D

uke

Lob

atir

icca

rdia

loba

ta(S

chif

fn.)

Fu

ruki

New

Zea

lan

d,G

len

ny

&K

inse

r45

81;d

et.

Cra

nd

all-

Sto

tler

(AB

SH)

AY

8773

73A

Y68

8757

AY

5074

21A

Y50

7462

AY

5075

07SI

U

Lop

hoco

lea

appa

lach

ian

us

R.M

.Sch

ust

.[C

hilo

scyp

hus

appa

lach

ian

us

(R.M

.Sch

ust

.)J.

J.E

ngel

&R

.M.S

chus

t.

U.S

.A.,

Dav

ison

&H

icks

2934

( DU

KE)

AY

6082

06A

Y60

8273

—A

Y60

8056

AY

6079

35D

uke

Loph

ocol

eahe

tero

phyl

la(S

chra

d.)

Du

mor

t.¼

Chi

losc

yphu

s

prof

undu

s(N

ees)

J.J.

En

gel&

R.M

.Sch

ust

.

USA

,IL

,Sto

tler

&C

ran

dal

l-St

otle

rs.

n.(

AB

SH)

DQ

2688

89D

Q26

8932

DQ

2689

73D

Q26

8987

DQ

2689

99SI

U

Lu

nu

lari

acr

uci

ata

(L.)

Lin

db

.M

exic

o,D

istr

ito

Fed

eral

,Lon

g29

553

(E)

DQ

2657

82D

Q26

8933

DQ

2860

11D

Q22

0685

DQ

2657

55E

Lu

nu

lari

acr

uci

ata

(L.)

Lin

db

.Sc

otl

and

,Eas

tL

oth

ian

,For

rest

520

(AB

SH)

DQ

2688

90D

Q26

8934

AY

6887

83A

Y68

8795

AY

6888

30SI

U

Mak

inoa

cris

pata

(Ste

ph

.)M

iyak

eC

hin

a,St

otle

r&

Cra

nd

all-

Stot

ler

4047

(AB

SH)

AY

8773

74A

Y87

7386

AY

8773

90A

Y87

7393

AY

8773

99SI

U

Man

nia

and

rogy

na

(L.)

A.E

van

sP

ort

uga

l,M

adei

ra,S

chil

l20

(E)

DQ

2657

83D

Q26

8935

DQ

2860

12D

Q22

0686

DQ

2657

56E

Man

nia

frag

ran

s(B

alb

is)

Fry

e&

L.C

lark

Swit

zerl

and

,Sch

ill

34(E

)D

Q26

5784

DQ

2689

36D

Q28

6013

DQ

2206

87D

Q26

5757

E

Mar

chan

tia

chen

opod

aL

.M

exic

o,V

erac

ruz,

Lon

g29

660

(E)

DQ

2657

85—

DQ

2860

14D

Q22

0688

DQ

2657

58E

Mar

chan

tia

infl

exa

Nee

s&

Mo

nt.

Mex

ico

,Ver

acru

z,L

ong

2962

8(E

)D

Q26

8891

DQ

2689

37D

Q26

8974

—D

Q26

9000

SIU

Mar

chan

tia

pale

acea

Ber

tol.

Mex

ico

,Ver

acru

z,L

ong

&G

arci

a29

683

(E)

DQ

2657

86D

Q26

8938

DQ

2860

15D

Q22

0689

DQ

2657

59E

Mar

chan

tia

poly

mor

pha

L.

[acc

esse

dfr

om

Gen

Ban

k]*A

F22

6020

*M68

929

*U87

079

*NC

0013

19*N

C00

1319

Mar

supe

lla

emar

gin

ata

(Eh

rh.)

Du

mo

rt.v

ar.a

quat

ica

(Lin

den

b.)

Du

mo

rt.

Fra

nce

,Fra

hm

90/5

89(F

)—

——

AY

6080

87A

Y60

7966

Du

ke

Mar

supi

diu

mla

tifo

liu

mR

.M.S

chu

st.

Co

sta

Ric

a,D

auph

in29

20(N

Y)

AY

6082

33A

Y60

8298

AY

6080

34A

Y60

8088

AY

6079

67D

uke

Mas

tigo

leje

un

eaau

ricu

lata

(Wil

son

&H

oo

k.)

Sch

iffn

.B

oli

via,

Chu

rch

ill

2127

5[a

cces

sed

fro

mG

enB

ank]

——

*AY

5480

70—

—

Mas

tigo

phor

ad

icla

dos

(Bri

d.e

xF

.Web

er)

Nee

sT

anza

nia

,Poc

s89

119/

V(F

)—

——

AY

6080

89A

Y60

7968

Du

ke

Met

zger

iaco

nju

gata

Lin

db

.U

.S.A

.,Il

lin

ois

,Hen

son

1247

( AB

SH)

AY

6886

90D

Q26

8939

AY

5074

11A

Y50

7453

AY

5074

95SI

U

Met

zger

iafr

uti

cola

Spru

ceE

cuad

or,

Dav

is36

1(D

UK

E)

AY

6082

34—

AY

6080

35A

Y60

8090

AY

6079

69D

uke

Met

zger

iafu

rcat

a(L

.)D

um

ort

.U

.S.A

.,N

ort

hC

aro

lina,

For

rest

&V

illar

real

620

(AB

SH)

DQ

2688

92D

Q26

8940

DQ

2689

75D

Q26

8988

DQ

2690

01SI

U

Met

zger

iali

ebm

ann

ian

aL

ind

enb

.&G

ott

sch

eV

enez

uel

a,M

erid

a,F

orre

st57

5(A

BSH

)D

Q26

8893

DQ

2689

41D

Q26

8976

DQ

2689

89D

Q26

9002

SIU

Moe

rcki

abl

ytti

i(M

oer

ch)

Bro

ckm

.U

.S.A

.,O

rego

n,W

agn

ers.

n.(

AB

SH)

—D

Q26

8942

AY

5074

12A

Y50

7454

AY

5074

96SI

U

Moe

rcki

afl

otov

ian

a(N

ees)

Sch

iffn

.U

.S.A

.,N

ewY

ork

,Kin

ser

&Sc

hu

ette

709

(AB

SH)

DQ

2688

94A

Y68

8758

AY

5074

13A

Y68

8796

AY

5074

97SI

U

Mon

ocle

ago

ttsc

hei

Lin

db

.M

exic

o,S

haw

1015

1(D

UK

E)

AY

6082

35A

Y60

8299

DQ

4396

94A

Y60

8091

AY

6079

70D

uke


Tab

le1.

Co

nti

nu

ed.

Tax

on

Vo

uch

erin

form

atio

nN

rL

SU

na

d5

rbcL

rps4

psb

AL

ab

Mon

ocle

ago

ttsc

hei

Lin

db

.M

exic

o,V

erac

ruz,

Lon

g,G

arci

a&

De

Lu

na

2963

7(E

)D

Q26

5787

DQ

2689

43D

Q28

6016

DQ

2206

90D

Q26

5760

E

Mon

ocle

ago

ttsc

heiL

ind

b.ss

p.e

long

ata

Gra

dst

.&R

.Mu

esP

uer

toR

ico

,Sto

tler

&C

ran

dal

l-St

otle

r89

–60

(AB

SH)

AY

6886

91A

Y87

7388

AY

5074

14A

Y50

7455

AY

5074

98SI

U

Mon

osol

eniu

mte

ner

um

Gri

ff.

Ger

man

y(C

ult

.,G

ott

inge

nB

ota

nic

Gar

den

),

Gra

dst

ein

s.n

.(E)

DQ

2657

88D

Q26

8944

DQ

2860

17D

Q22

0691

—E

Myr

ioco

lea

irro

rata

Spru

ceE

cuad

or,

Gra

dst

ein

etal

.100

33B

[acc

esse

dfr

om

Gen

Ban

k]

——

*AY

5480

73—

—

Nar

dia

inse

cta

Lin

db

.U

.S.A

.,D

avis

432

( DU

KE)

(mix

edco

llec

tio

nw

ith

Lop

hoz

iasp

.in

det

.)

AY

6082

32A

Y60

8296

DQ

4396

93A

Y60

8086

AY

6079

65D

uke

Nar

dia

scal

aris

Gra

yU

.S.A

.,D

avis

438

( DU

KE)

AY

6082

36—

DQ

4396

95A

Y60

8092

AY

6079

71D

uke

Neo

hod

gson

iam

irab

ilis

(Per

ss.)

Per

ss.(

1)N

ewZ

eala

nd

,So

uth

Isla

nd

,Mal

colm

s.n

.(E)

DQ

2657

89D

Q26

8945

DQ

2860

18D

Q22

0692

DQ

2657

61E

Neo

hod

gson

iam

irab

ilis

(Per

ss.)

Per

ss.(

2)N

ewZ

eala

nd

,Sto

tler

&C

ran

dal

l-St

otle

r45

42(A

BSH

)A

Y68

8692

AY

6887

59A

Y50

7415

AY

5074

56A

Y50

7499

SIU

Neo

tric

hoc

olea

biss

etii

(Mit

t.)

S.H

att.

Jap

an,I

nou

es.

n.(

F)

AY

6082

37A

Y60

8300

DQ

4396

96A

Y60

8093

AY

6079

72D

uke

Not

eroc

lad

aco

nfl

uen

s(H

oo

k.f.

&T

aylo

r)Sp

ruce

Arg

enti

na,

Tie

rra

del

Fu

ego

,Lon

g31

768

(E)

DQ

2688

95D

Q26

8946

DQ

2689

77D

Q26

8990

DQ

2690

03SI

U

Not

eroc

lad

aco

nfl

uen

s(H

oo

k.f.

&T

aylo

r)Sp

ruce

Ven

ezu

ela,

Mer

ida,

For

rest

566

(AB

SH)

AY

8773

76A

Y68

8760

AY

6887

84A

Y68

8797

AY

5075

00SI

U

Now

elli

acu

rvif

olia

(Dic

ks.)

Mit

t.U

.S.A

.,R

isk

&G

ross

1222

0(D

UK

E)

AY

6082

38A

Y60

8301

DQ

4396

997

AY

6080

94A

Y60

7973

Du

ke

Od

onto

leje

un

ealu

nu

lata

(Nee

s)Sc

hif

fn.

Bra

zil,

Zar

tman

1235

.2(D

UK

E)

AY

6082

39A

Y60

8302

DQ

4396

98A

Y60

8095

AY

6079

74D

uke

Od

onto

sch

ism

ad

enu

datu

m(N

ees)

Du

mo

rt.

U.S

.A.,

Hor

n18

09(D

UK

E)

AY

6082

40A

Y60

8303

AY

6080

36A

Y60

8096

AY

6079

75D

uke

Pal

lavi

cin

ialy

elli

i(H

oo

k.)

Car

ruth

.U

.S.A

.,A

rkan

sas,

Mar

shs.

n.(

AB

SH)

AY

7347

42A

Y68

8761

AY

5074

16A

Y68

8798

AY

5075

01SI

U

Pal

lavi

cin

iaru

bris

tipa

Sch

iffn

.A

ust

rali

a,C

argi

ll&

Vel

la50

7(A

BSH

)A

Y73

4744

AY

7347

53A

Y73

4693

AY

7347

02A

Y73

4711

SIU

Pal

lavi

cin

iaxi

phoi

des

(Ho

ok.

f.&

Tay

lor)

Tre

vis.

New

Zea

lan

d,N

ort

hIs

lan

d,S

totl

er&

Cra

nd

all-

Stot

ler

4514

( AB

SH)

AY

7347

43A

Y73

4752

AY

7346

92A

Y73

4701

AY

7347

10SI

U

Pel

lia

appa

lach

ian

aR

.M.S

chu

st.

U.S

.A.,

Ala

bam

a,D

avis

on35

79(A

BSH

)D

Q26

8896

AY

6887

62A

Y68

8785

AY

6887

99A

Y68

8831

SIU

Pel

lia

end

ivii

foli

a(D

icks

.)D

um

ort

.Ja

pan

(SIU

gree

nh

ou

se),

Hig

uch

is.

n.(

AB

SH)

DQ

2688

97A

Y68

8763

AY

6887

86A

Y68

8800

AY

6888

32SI

U

Pel

lia

epip

hyll

a(L

.)C

ord

a(1

)U

.S.A

.,N

ort

hC

aro

lin

a,St

otle

r&

Cra

nd

all-

Stot

ler

4414

(AB

SH)

AY

6886

93A

Y68

8764

AY

6887

87A

Y50

7457

AY

5075

02SI

U

Pel

lia

epip

hyll

a(L

.)C

ord

a(2

)U

.S.A

.,R

isk

&G

ross

1223

1( D

UK

E)

AY

6082

42A

Y60

8305

——

—D

uke

Pel

tole

pis

quad

rata

(Sau

t.)

Mu

ll.F

rib

.N

orw

ay,P

rov.

Sogn

og

Fjo

rdan

e,L

ong

etal

.313

60(E

)D

Q26

5790

DQ

2689

47D

Q28

6019

DQ

2206

93—

E

Pet

alop

hyl

lum

ralf

sii

(Wil

son

)N

ees

&G

ott

sch

e

exL

ehm

.

U.K

.,J.

Pro

skau

ercu

ltu

revi

aD

.Mu

elle

r-T

AM

Uto

AB

SH(1

),to

Sarg

ent,

toB

erke

lycu

ltu

reco

llec

tio

n

(2)

( AB

SH)

AY

8773

77(1

)A

Y60

8306

(2)

AY

5074

17(1

)A

Y50

7458

(1)

AY

5075

03(1

)SI

U/D

uke

Ph

yllo

thal

lia

niv

icol

aE

.A.H

od

gs.

New

Zea

lan

d,S

totl

er&

Cra

nd

all-

Stot

ler

4537

(AB

SH)

AY

6886

94A

Y68

8765

AY

5074

18A

Y50

7459

AY

5075

04SI

U

Pla

gioc

has

ma

rups

etre

(J.R

.Fo

rst.

&G

.Fo

rst.

)St

eph

.P

ort

uga

l,M

adei

ra,S

chil

l5

(E)

DQ

2657

91D

Q26

8949

DQ

2860

20D

Q22

0694

DQ

2657

63E

Pla

gioc

has

ma

wri

ghti

iSu

ll.

Mex

ico

,Ver

acru

z,L

ong

etal

.296

36(E

)D

Q26

5792

DQ

2689

50D

Q28

6021

DQ

2206

95D

Q26

5764

E


Tab

le1.

Co

nti

nu

ed.

Tax

on

Vo

uch

erin

form

atio

nN

rL

SU

na

d5

rbcL

rps4

psb

AL

ab

Pla

gioc

hil

aau

stin

iiA

.Eva

ns

U.S

.A.,

Ris

k10

849

(DU

KE)

AY

6082

44—

DQ

4396

99A

Y60

8099

AY

6079

78D

uke

Ple

uro

zia

purp

ure

aL

ind

b.

U.S

.A.,

Sch

ofie

ld10

2815

(DU

KE)

—A

Y60

8307

AY

6080

37A

Y60

8100

AY

6079

79D

uke

Ple

uro

zia

purp

ure

aL

ind

b.

Bh

uta

n,L

ong

2886

8(E

)D

Q26

8899

DQ

2689

51A

Y87

7391

—A

Y87

7401

SIU

Pod

omit

riu

mm

alac

cen

se(S

tep

h.)

Cam

pb

.Si

nga

po

re,T

ans.

n.(

AB

SH)

—D

Q26

8952

DQ

2689

79D

Q26

8991

DQ

2690

05SI

U

Pod

omit

riu

mph

ylla

nth

us

(Ho

ok.

)M

itt.

New

Zea

lan

d,S

totl

er&

Cra

nd

all-

Stot

ler

4517

(AB

SH)

AY

7347

40A

Y68

8766

AY

5074

19A

Y50

7460

AY

5075

05SI

U

Por

ella

nav

icu

lari

s(L

ehm

.&L

ind

enb

.)P

feif

f.U

.S.A

.,C

alif

orn

ia,S

totle

r&

Cra

ndal

l-St

otle

r34

10(A

BSH

)A

Y68

8695

AY

6887

67A

Y50

7420

AY

5074

61A

Y50

7506

SIU

Por

ella

pin

nat

aL

.U

SA,G

offi

net

4744

(DU

KE)

AY

6082

45A

Y60

8308

—A

Y60

8101

AY

6079

80D

uke

Pre

issi

aqu

adra

ta(S

cop

.)N

ees

U.S

.A.,

Sch

ofie

ld10

5579

(DU

KE)

AY

6082

46A

Y60

8309

AY

3129

35A

Y60

8102

AY

6079

81D

uke

Pre

issi

aqu

adra

ta(S

cop

.)N

ees

Sco

tlan

d,S

ou

thE

bu

des

,Lon

g33

268

(E)

DQ

2657

93D

Q26

8953

——

—E

Pti

lid

ium

cili

are

(L.)

Ham

pe

Can

ada,

Sch

ofie

ld10

3486

(DU

KE)

AY

6082

47A

Y60

8310

AY

6080

38A

Y60

8103

AY

6079

82D

uke

Rad

ula

com

plan

ata

(L.)

Du

mo

rt.

Ru

ssia

,Bak

alin

[acc

.8]

(NY)

—A

Y60

8311

—A

Y60

8104

AY

6079

83D

uke

Rad

ula

perr

otte

tii

Go

ttsc

he

exSt

eph

.Ja

pan

,Miz

uta

ni

1503

0(F

)A

Y60

8248

AY

6083

12D

Q43

9700

AY

6081

05A

Y60

7984

Du

ke

Reb

ouli

ah

emis

phae

rica

(L.)

Rad

di

U.S

.A.,

Illi

no

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1( A

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card

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ph

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ng

Ven

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ela,

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ida,

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rest

558

(AB

SH)

AY

8773

78A

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Ric

card

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w.)

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iffn

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151

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KE)

AY

6082

49A

Y60

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—A

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8106

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6079

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uke

Ric

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iam

ult

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ver

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nd

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rest

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ck60

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cia

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(E)

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nd

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ler

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s(L

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a,V

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elt

2046

7( E

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T)

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2657

96D

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DQ

2860

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2657

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lla

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icop

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ory

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on

t.)

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nt.

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lica

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e&

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(BM

)D

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——

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ees)

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ill

64(E

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6025

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U.S

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is12

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UK

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AY

6082

50A

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8314

AY

6080

39A

Y60

8108

AY

6079

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uke

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lle

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nd

all-

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[ref

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lin

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a,Y

un

nan

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g33

706

( E)

DQ

2689

02D

Q26

8961

——

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U

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isto

chil

aap

pen

dic

ula

ta(H

oo

k.)

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mo

rt.e

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s.N

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tler

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ran

dal

l-St

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r44

55(A

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Y68

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6887

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AY

5074

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ind

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.)

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n

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stra

lia,

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iman

n58

554

(NY)

AY

6082

51—

DQ

4397

02A

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8109

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6079

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U.S

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76( D

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E)

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6082

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8110

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6079

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nda

ll-St

otle

r15

15(A

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)A

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tep

h.)

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lle

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agas

car,

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s94

46/A

Q(F

)A

Y60

8253

AY

6083

15—

AY

6081

11A

Y60

7990

Du

ke


Tab

le1.

Co

nti

nu

ed.

Tax

on

Vo

uch

erin

form

atio

nN

rL

SU

na

d5

rbcL

rps4

psb

AL

ab

Sym

phyo

gyn

aas

pera

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h.

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ama,

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larr

eal

668

( PM

A)

DQ

2689

03D

Q26

8962

AY

6887

89A

Y68

8803

AY

6888

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phyo

gyn

abr

ongn

iart

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on

t.E

cuad

or,

Dav

is34

1(D

UK

E)

AY

6082

54—

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Y60

8112

AY

6079

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Sym

phyo

gyn

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m(H

oo

k.)

Nee

s&

Mo

nt.

New

Zea

lan

d,S

totl

er&

Cra

nd

all-

Stot

ler

4452

(AB

SH)

AY

6886

98A

Y68

8772

AY

5074

26A

Y50

7467

AY

5075

12SI

U

Sym

phyo

gyn

au

nd

ula

taC

ole

nso

New

Zea

lan

d,S

totl

er&

Cra

nd

all-

Stot

ler

4463

(AB

SH)

AY

8773

80A

Y68

8773

AY

6887

90A

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8804

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6888

35SI

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Tar

gion

iah

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hyl

laL

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ort

uga

l,M

adei

ra,S

chil

l11

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2657

98D

Q26

8964

DQ

2860

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0700

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2657

69E

Tar

gion

iah

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laL

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alif

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ia,S

totle

r&

Cra

ndal

l-St

otle

r33

97(A

BSH

)—

DQ

2689

65A

Y50

7427

AY

6888

05A

Y50

7514

SIU

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aran

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a(S

tep

h.)

R.M

.Sch

ust

.A

ust

rali

a,St

reim

ann

5955

4(N

Y)

AY

6082

56A

Y60

8316

DQ

4397

04A

Y60

8114

AY

6079

93D

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Tem

nom

apu

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oo

k.)

Mit

t.ex

Bas

tow

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lan

d,B

ragg

ins

92/8

7(F

)A

Y60

8257

AY

6083

17D

Q43

9705

AY

6081

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Y60

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ke

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ralo

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rh.)

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Dav

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UK

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AY

6082

58A

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—A

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6079

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(Co

len

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sk.

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lan

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er&

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nd

all-

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ler

4561

(AB

SH)

AY

6886

99A

Y68

8774

AY

5074

28A

Y50

7468

—SI

U

Tre

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gmae

aR

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chu

st.

New

Zea

lan

d,S

totl

er&

Cra

nd

all-

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ler

4582

(AB

SH)

DQ

2689

05A

Y68

8775

AY

5074

29A

Y50

7469

AY

5075

15SI

U

Tri

and

roph

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ok.

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r)

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lfo

rd&

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cher

Ven

ezu

ela,

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ard

iet

al.9

730/

T(F

)A

Y60

8259

AY

6083

19D

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9706

AY

6081

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Du

ke

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choc

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ento

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w.)

Go

ttsc

he

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ado

r,D

avis

368

(DU

KE)

AY

6082

61A

Y60

8320

AY

6080

40A

Y60

8119

AY

6079

98D

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Tri

tom

aria

quin

qued

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ta(H

ud

s.)

H.B

uch

U.S

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Sch

ofie

ld10

6093

(DU

KE)

AY

6082

61A

Y60

8321

DQ

4397

07A

Y60

8119

AY

6079

98D

uke

Ver

doo

rnia

succ

ule

nta

R.M

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ust

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ewZ

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nd

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tler

&C

ran

dal

l-St

otle

r46

02(A

BSH

)A

Y68

700

AY

6887

76A

Y50

7430

AY

5074

70A

Y50

7516

SIU

Wie

sner

ella

den

uda

ta(M

itt.

)St

eph

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epal

,Lon

g30

337

(E)

DQ

2657

99D

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8966

DQ

2860

27D

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Xen

oth

allu

svu

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chu

st.

New

Zea

lan

d,S

totl

er&

Cra

nd

all-

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ler

4580

(AB

SH)

AY

8773

81A

Y68

8777

AY

5074

31A

Y50

7471

AY

5075

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U

Cha

raco

nn

iven

sSa

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n[a

cces

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ank]

—*A

F40

8200

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0971

6—

—

Chl

amyd

omon

asre

inh

ard

tii

Dan

g.[a

cces

sed

fro

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enB

ank]

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1834

63—

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0005

4—

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357

Cho

rell

avu

lgar

isB

eij.

[acc

esse

dfr

om

Gen

Ban

k]—

—*A

F49

9684

—*A

B00

1684

Nep

hro

selm

isol

ivac

eaF

.Ste

in[a

cces

sed

fro

mG

enB

ank]

—*A

F11

0138

*NC

0009

27*N

C00

0927

*NC

0009

27

An

dre

aea

roth

iiF

.Web

er&

D.M

oh

rU

.S.A

.,Sh

aw11

565

(DU

KE)

AY

3128

61A

Y31

2862

AY

6080

25A

Y31

2866

AY

3128

63D

uke

An

dre

aeob

ryu

mm

acro

spor

um

Stee

re&

B.M

.Mu

rray

[acc

esse

dfr

om

Gen

Ban

k]*A

Y33

0426

—*A

F23

1059

*AF

3069

53*A

Y31

2893

Den

droh

ypop

tery

giu

mar

busc

ula

(Bri

d.)

Kru

ijer

Ch

ile,

Hol

z&

Fra

nza

rin

gC

H00

–80

(NY)

AY

6082

09A

Y60

8276

AY

6080

27A

Y60

8059

AY

6079

38D

uke

Hoo

keri

alu

cen

s(H

edw

.)Sm

.U

.S.A

.,B

uck

3771

4(N

Y)

AY

3304

39*Z

9896

9*A

Y31

2931

AJ2

5131

6A

Y31

2906

Du

ke

Mn

ium

hor

nu

mH

edw

.[a

cces

sed

fro

mG

enB

ank]

*AY

3304

41*A

Y31

2879

*AF

2268

20*A

F02

3796

*AY

3129

08

Pol

ytri

chu

mpa

llid

iset

um

Fu

nck

[acc

esse

dfr

om

Gen

Ban

k]*A

Y33

0445

*AY

3128

84*A

Y31

2934

*AF

3069

56*A

Y31

2914

Spha

gnu

mcu

spid

atu

mE

hrh

.ex

Ho

ffm

.[a

cces

sed

fro

mG

enB

ank]

*AY

3095

06*A

Y30

9560

*AF

2318

86*A

F47

8246

*AY

3096

08

Spha

gnu

mpa

lust

reL

.[a

cces

sed

fro

mG

enB

ank]

*AY

3314

51*A

Y31

2888

*AF

2318

87*A

F23

1892

*AY

3129

20

Tak

akia

cera

toph

ylla

(Mit

t.)

Gro

lle

U.S

.A.,

Ala

ska,

Sch

ofie

lds.

n.[

31A

ug

1994

](A

BSH

)D

Q26

8904

DQ

2689

63—

DQ

2689

93—

SIU

Tak

akia

lepi

dozi

oid

esS.

Hat

t.&

Ino

ue

[acc

esse

dfr

om

Gen

Ban

k]*A

F19

7061

*AY

3128

89*A

F24

4565

*AF

3069

50*A

Y31

2921

An

thoc

eros

angu

stu

sSt

eph

.[a

cces

sed

fro

mG

enB

ank]

——

*AB

0874

49*A

B08

7443

*AB

0874

34


Criterion and hierarchical Likelihood Ratio Tests. For

the heterogeneous analysis data partitions were made

according to gene (five partitions; BI5), and the

analyses were run for two million generations.

Sequences were also partitioned by codon, resulting in

a 14-partition model (BI14); analyses using this model

were run for 5 million generations. The burnin for

each run was determined by visualizing the curve of

likelihood versus generations in Microsoft Excel. Trees

from the burnin were disgarded and a majority rule

consensus of the remaining trees was computed using

PAUP*. Posterior probabilities (PP) for all runs for

each of the Bayesian analyses were compared to check

that convergence was reached. We regard Bayesian

support values as significant when they are 95% (or

0.95) or higher (e.g., Cox et al. 2004), although lower

values have been accepted by some authors (e.g.,

Nickrent et al. 2005, who used a value of 0.90).

RESULTS

Parsimony analysis found 128 MPTs 22,302 steps

long, consistency index 0.2310 (0.2000 excluding

uninformative characters), retention index 0.6717 and

rescaled consistency index 0.1552. Bootstrap support

(BS) values greater than 50% were generated for 181

of the 208 potential nodes (i.e., 87%), while 129 nodes

had BS values of 80% or above.

The results are robust to different sampling

methods; i.e., the topologies resolved by MP and

Bayesian analyses are mostly congruent, although

there are some slight differences in the placements of

Sphaerocarpales Cavers, Neohodgsonia Perss., Cya-

thodium Kunze, Allisonia Herzog, Cryptothallus

Malmb., Verdoornia R. M. Schust. and Jungermannia

L. In most cases the alternate placements have PPs of

less than 95%. (It should be noted that our use of

genus rather than species names here and in the

discussions that follow is simply for convenience and

does not imply monophyly of these genera, a

consideration that in most instances is beyond the

scope of this paper.)

Liverworts are resolved as monophyletic with

100% support in MP BS and all Bayesian analyses

(Figs. 1, 3). Relationships resolved among outgroup

taxa are comparable to those resolved by other

analyses focused on these groups (e.g., see Cox et al.

2004; Duff et al. 2004; Wolf et al. 2005) (Fig. 3). ThreeTab

le1.

Co

nti

nu

ed.

Tax

on

Vo

uch

erin

form

atio

nN

rL

SU

na

d5

rbcL

rps4

psb

AL

ab

An

thoc

eros

pun

ctat

us

L.

[acc

esse

dfr

om

Gen

Ban

k]*A

F22

6035

—*U

8706

3—

—

Lei

ospo

roce

ros

du

ssii

(Ste

ph

.)H

asse

l[a

cces

sed

fro

mG

enB

ank]

—*A

Y89

4803

*AY

4630

52—

—

Meg

acer

osfl

agel

lari

s(M

itt.

)St

eph

.N

ewZ

eala

nd

,Sto

tler

&C

ran

dal

l-St

otle

r44

78(A

BSH

)A

Y87

7375

AY

8773

87*A

Y46

3040

—A

Y87

7400

SIU

Ph

ymat

ocer

osbu

lbic

ulo

sus

(Bro

t.)

Sto

tler

,W.T

.Do

yle

&C

ran

d.-

Sto

tler

U.S

.A.,

Cal

ifo

rnia

,Doy

les.

n.[

14A

pr

2004

](m

ixed

wit

hP

hae

ocer

ospe

arso

nii

(M.H

ow

e)P

rosk

.)

(CA

NB)

DQ

2688

98D

Q26

8948

DQ

2689

78—

DQ

2690

04SI

U

Equ

iset

um

telm

atei

aE

hrh

.[a

cces

sed

fro

mG

enB

ank]

—*A

J130

749

*AF

3135

80—

—

Psi

lotu

mn

ud

um

(L.)

P.B

eau

v.[a

cces

sed

fro

mG

enB

ank]

—*A

J012

794

*AP

0046

38*A

P00

4638

*AP

0046

38


major lineages are supported within the liverworts

(Figs. 1, 2), in agreement with previous multigene

studies (Crandall-Stotler et al. 2005; Davis 2004;

Forrest & Crandall-Stotler 2004, 2005; Forrest et al.

2005a; He-Nygren et al. 2006), as well as some single

locus studies (e.g., Heinrichs et al. 2005). These

correspond to the classes Haplomitriopsida (including

the Haplomitriales Schljakov and Treubiineae Stotler

& Crand.-Stotler) (99% MP BS; 100% BI1, BI5, BI14

PP), Marchantiopsida (including the Blasiales) (100%

MP BS; 100% BI1, BI5, BI14 PP) and Jungerman-

niopsida (100% MP BS; 100% BI1, BI5, BI14 PP).

Table 2. Model selection results for each partition using the hierarchical likelihood ratio test and Akaike’s Information Criterion as

implemented in the programme MrModeltest (Nylander 2002). GTR: General time-reversible, Rodriguez et al. (1990); Base: Base

frequencies (A, C, G); Rmat: Rate matrix (AC, AG, AT, CG, CT); Shape: value of a in gamma distribution; Pinvar: proportion of

invariant characters.

Partition No.

bases

Model -ln likelihood Optimised parameters

26S 914 GTRþIþG 9697.6152 Base¼(0.2622 0.2261 0.3260) Rmat¼(0.6895 2.0192 0.5979 0.6695 7.9856)

Rates¼gamma Shape¼0.4493 Pinvar¼0.3285

nad5 1650 GTRþIþG 19033.2910 Base¼(0.2624 0.2156 0.1950) Rmat¼(2.0648 3.9320 0.4061 1.1277 6.2290)


nad5 codon 1 356 GTRþG 2580.6216 Base¼(0.2835 0.1663 0.2862) Nst¼6 Rmat¼(6.1922 4.1587 0.9291 1.9858 58.6202)

Rates¼gamma Shape¼0.5026 Pinvar¼0





nad5 intron 583 GTRþG 7242.5244 Base¼(0.3030 0.2221 0.2218) Nst¼6 Rmat¼(1.1745 2.0723 0.3858 0.5732 2.0765)


rbcL 1308 GTRþIþG 37593.5625 Base¼(0.3074 0.1426 0.1275) Rmat¼(1.4295 6.5097 0.4708 2.3036 8.0060)


rbcL codon 1 436 GTRþIþG 6400.7085 Base¼(0.1984 0.2228 0.3699) Nst¼6 Rmat¼(3.8412 1.5426 0.8986 1.1002 9.9526)






rps4 554 GTRþIþG 22272.8828 Base¼(0.3950 0.1352 0.1493) Rmat¼(1.5579 7.1590 0.2418 1.8934 5.7926)


rps4 codon 1 185 GTRþIþG 5637.0337 Base¼(0.4084 0.1907 0.1739) Nst¼6 Rmat¼(1.9834 6.5365 0.2095 1.2738 4.2443)






psbA 1072 GTRþIþG 20777.5352 Base¼(0.2586 0.1902 0.1554) Rmat¼(1.0597 11.0235 1.7899 0.8227 17.6730)


psbA codon 1 358 GTRþIþG 2747.6267 Base¼(0.2426 0.1604 0.3071) Nst¼6 Rmat¼(5.6606 7.8798 1.0402 1.3272 36.2863)






combined 5498 GTRþIþG 114648.9922 Base¼(0.2943 0.1837 0.1556) Rmat¼(1.2999 6.6361 0.5492 1.4833 7.0032)



The earliest divergence involves a split between

Haplomitriopsida and all other liverworts. Haplomi-

triopsida comprise three extant genera, with Treubia

and Apotreubia forming a monophyletic Treubiaceae,

sister to Haplomitrium Nees (Fig. 4).

The Blasiaceae H. Klinggr., which include two

monospecific genera, Blasia L. and Cavicularia Steph.,

are sister to all the Marchantiopsida or complex

thalloids (Fig. 5). Within the complex thalloids as

traditionally defined, levels of parsimony bootstrap

support are low, although there are significant

posterior probabilities. The branching order of the

earliest divergences in this lineage (Sphaerocarpales/

Neohodgsonia/Lunularia Adans.) varies according to

which analytical criterion is used, with the Sphaer-

ocarpales involved in the earliest divergence under

MP (Fig. 2) and BI1, while Neohodgsonia is involved

under more model-based approaches (BI5 and BI14)

(Fig. 5). However, support for either topology is low

(55% MP BS; 52% BI1 PP for Sphaerocarpales

diverging first; 93% BI5, 97% BI14 PP for Neo-

hodgsonia first). A Marchantia L./Preissia Corda

lineage is supported in all analyses as sister to all

remaining complex thalloids (93% MP BS; 100% BI1,

BI5, BI14 PP). Monoclea Hook., which is nested

within the crown group, receives varying amounts of

support as sister to Dumortiera Nees (55% MP BS;

100% BI1, BI5, BI14 PP).

The level of molecular evolution within the

complex thalloids is lower than for any other lineage,

with the notable exception of the crown group taxon

Cyathodium, which occurs on a comparatively long

branch (Fig. 2). Cyathodium is resolved as sister to

Exormotheca Mitt./Corsinia Raddi in most analyses

(without MP BS support; 100% PP BI1, 99% BI5), but

is sister to Monosolenium Griff. in the 14-partition

Bayesian analysis (91% PP).

The Jungermanniopsida are split into two

supported lineages (Figs. 1, 2), one comprising most

of the simple thalloid liverworts (Fig. 6), including all

taxa of the Pelliineae Schljakov, Fossombroniineae

Stotler & Crand.-Stotler, Phyllothalliineae R. M.

Schust. and Pallaviciniineae R. M. Schust. except

Verdoornia (99% MP BS; 100% BI1, BI5, BI14), and

the other comprising the leafy liverworts, and the

simple thalloids of the Metzgeriineae Schljakov plus

Verdoornia (89% MP BS; 100% BI1, BI5, BI14). This

leafy/Metzgerialean lineage contains two supported

lineages, one comprising the Metzgeriaceae H.

Klinggr., Aneuraceae H. Klinggr., Verdoornia and the

leafy liverwort Pleurozia Dumort. (Fig. 7) (99% MP

BS; 100% BI1, BI5, BI14), and the other, all remaining

taxa of the Jungermanniidae, i.e., leafy liverworts

(93% MP BS; 100% BI1, BI5, BI14).

Within the Jungermanniidae, several clades are

supported under parsimony, likelihood and Bayesian

approaches. The clade designated as Leafy I in Davis

(2004) is supported as monophyletic and excludes a

Ptilidium Nees/Neotrichocolea S. Hatt. clade in

Bayesian analyses (Fig. 8) (100% PP in BI1, BI5,

BI14). Under a parsimony criterion, the position of

this Ptilidium/Neotrichocolea clade is equivocal (e.g.,

unsupported as sister to Leafy I in Fig. 2). Other

lineages that are not supported as part of Leafy I

under a parsimony bootstrap criterion, although are

part of the clade in the MP strict consensus tree (not

Figure 1. Diagram of relationships of major clades. Numbers above the branches are Maximum Parsimony BS values/homogeneous

Bayesian PPs/5-partition Bayesian PPs/14-partition Bayesian PPs.


Figure 2. Phylogram (one of 128 equally parsimonious trees), with major clades (as in Fig. 1) highlighted. Taxa specifically discussed

in the text are annotated. Thinner branches are absent from the strict consensus tree. This tree is also available in NEXUS format

from TreeBASE (see methods section).


shown), are a Porella L./Ascidiota C. Massal. clade

(100% MP BS, BI1, BI5, BI14 PP), a Jubulopsis R. M.

Schust./Lepidolaena Dumort./Gackstroemia Trevis.

clade (98% MP BS, 100% BI1, BI5, BI14 PP) and

Goebeliella Steph. The rest of the Leafy I taxa form a

large, well-supported monophyletic group comprising

several representatives of the Lejeuneaceae, Jubula

Dumort., Frullania and Radula Dumort. (81% MP

BS; 100% BI1, BI5, BI14).

The remaining leafy liverworts are supported in a

single large clade designated as Leafy II (Davis 2004)

(Figs. 1, 2, 9) (89% MP BS; 100% BI1, BI5, BI14).

Although the Neotrichocolea/Ptilidium clade resolves

with Leafy II in Bayesian analyses, this position is not

strongly supported (88% BI1, 95% BI5, 88% BI14

PP). The first well-supported divergence from the

main clade in Leafy II, then, involves Schistochila

Dumort., with Temnoma Mitt. forming the second

divergence. Trichocolea Dumort., Plagiochila, Lopho-

colea (Dumort.) Dumort., Chiloscyphus Corda, Tri-

androphyllum Fulford & Hatcher, Mastigophora Nees,

Herbertus Gray, Lepicolea Dumort., Telaranea Spruce

ex Schiffn., Lepidozia (Dumort.) Dumort., Bazzania

Gray and Acromastigum A. Evans form a mono-

phyletic group that is referred to herein as Clade A

(Fig. 9) (72% MP BS; 100% BI1, BI5, BI14). Their

sister group (82% MP BS; 100% BI1, BI5, BI14) is

split into two lineages; one, designated as Clade B,

includes Adelanthus Mitt., Odontoschisma (Dumort.)

Dumort., Schiffneria Steph., Nowellia Mitt., Cepha-

lozia (Dumort.) Dumort., Herzogobryum Grolle,

Stenorrhipis Herzog, Cephaloziella (Spruce) Schiffn.,

Tetralophozia (R. M. Schust.) Schjakov, Anastrophyl-

lum (Spruce) Steph., Tritomaria Loeske, Scapania

(Dumort.) Dumort. and Diplophyllum (Dumort.)

Dumort. (55% MP BS; 100% BI1, BI5, BI14), and the

other, Clade C, includes Marsupidium Mitt., Leth-

ocolea Mitt., Balantiopsis Mitt., Isotachis Mitt., Ca-

Figure 3. Outgroup relationships (based on the topology from the 5 partition Bayesian analysis). Numbers above the branches are

Maximum Parsimony BS values/homogeneous Bayesian PPs/5-partition Bayesian PPs/14-partition Bayesian PPs.

Figure 4. Haplomitriopsida (based on the topology from the 5 partition Bayesian analysis). Numbers above the branches are

Maximum Parsimony BS values/ homogeneous Bayesian PPs/5-partition Bayesian PPs/14-partition Bayesian PPs.


lypogeia Raddi, Anthelia (Dumort.) Dumort., Leioco-

lea (Mull. Frib.) H. Buch, Jungermannia, Harpanthus

Nees, Gyrothyra M. Howe, Nardia Gray, Marsupella

Dumort. and Gymnomitrion Corda (58% MP BS;

100% BI1, BI5, BI14).

DISCUSSION

The recent extremely rapid progress in our

understanding of liverwort phylogeny through the

application of molecular systematic methods has

resolved many questions regarding early divergences,

and allowed the reinterpretation of morphological,

ultrastructural and biochemical data in the light of

robust evolutionary hypotheses. By combining data

from some of these pivotal studies, as well as new

data, into a supermatrix, we have been able to solidify

these separate pieces of phylogenetic evidence into

one robust framework. Many terminal clades, how-

ever, still lack internal resolution. Some of the more

noteworthy relationships that were either confirmed

or newly resolved within the major lineages are

discussed individually below.

Confirmation of previous results. Many of the

clades supported in the analyses reported herein have

also been resolved in previous studies (e.g., Boisselier-

Dubayle et al. 2002; Crandall-Stotler et al. 2005; Davis

2004; Forrest & Crandall-Stotler 2004, 2005; Hein-

richs et al. 2005; He-Nygren et al. 2004, 2006; Long et

al. 2000; Wheeler 2000). Some of the relationships

revealed by these earlier studies were unexpected and

Figure 5. Marchantiopsida (based on the topology from the 5 partition Bayesian analysis). Numbers above the branches are



have remained somewhat controversial. For example,

until the studies of Davis (2004) and He-Nygren et al.

(2004), the alliance of Pleurozia with the Metzger-

iineae had never been postulated. Nonetheless, this

unexpected relationship has subsequently been re-

solved in several other analyses (Crandall-Stotler et al.

2005; Forrest et al. 2005a; Heinrichs et al. 2005) and is

further supported in our current analyses (Fig. 7). In

addition, the position of a Treubia and Haplomitrium

clade as the first diverging lineage of the extant

liverworts (Figs. 1, 4), suggested in Forrest and

Crandall-Stotler (2004), cemented in Forrest and

Crandall-Stotler (2005) and Crandall-Stotler et al.

(2005), and taxonomically formalized in Heinrichs et

al. (2005), was almost unprecedented in liverwort

classification. A relationship between Haplomitrium

Figure 6. Simple thalloids clade I (based on the topology from the 5 partition Bayesian analysis). Numbers above the branches are


Figure 7. Simple thalloids clade II (based on the topology from the 5 partition Bayesian analysis). Numbers above the branches are

Maximum Parsimony BS values/homogeneous Bayesian PPs/5-partition Bayesian PPs/14-partition Bayesian PPs.


and Treubia, although not reflected in contemporary

classifications, was first suggested by Goebel (1898).

Upon review, this relationship is supported by several

fundamental morphological and ultrastructural fea-

tures that have been reconstructed as plesiomorphic

for hepatics (Crandall-Stotler et al. 2005). These

include mucilage-secreting epidermal cells, massive

blepharoplasts, scattered gametangia and tetrahedral

apical cells. The assertion of He-Nygren et al. (2006)

that tetrahedral apical cells are a derived feature of

this clade has not been supported here, or in previous

analyses (Crandall-Stotler et al. 2005; Forrest &

Crandall-Stotler 2004, 2005; Heinrichs et al. 2005).

The alliance of the Blasiales with the complex

thalloids, as resolved by Davis (2004), Forrest and

Crandall-Stotler (2004, 2005), Crandall-Stotler et al.

(2005), Forrest et al. (2005a), Heinrichs et al. (2005)

and He-Nygren et al. 2006, although suggested by the

ultrastructural work of Pass and Renzaglia (1995), was

also not indicated in any of the morphologically based

classifications of the group. The nesting of Monoclea

well within the complex thalloids was first suggested

by the analyses of Lewis et al. (1997) and has since

been supported by Wheeler (2000), Boisselier-Du-

bayle et al. (2002), Davis (2004), He-Nygren et al.

(2004, 2006), Forrest and Crandall-Stotler (2004,

2005), Crandall-Stotler et al. (2005) and Heinrichs et

al. (2005) (Fig. 5). This phylogenetic placement is in

sharp contrast to its traditional morphologically based

placement as an isolated lineage at the base of the

complex thalloids (Crandall-Stotler & Stotler 2000;

Schuster 1984, 1992). The large genus Radula, tradi-

tionally postulated to represent an isolated lineage

within the leafy liverworts, is instead nested within the

Porellales (R. M. Schust.) Schljakov emend. Stotler &

Crand.-Stotler (Fig. 8), as also shown in the

phylogenies of Ahonen (2004), Davis (2004) and He-

Nygren et al. (2004, 2006).

At lower taxonomic levels, many genera have

been resolved both here and in earlier studies as

paraphyletic, accentuating the need for substantial

taxonomic revision in many lineages, including

Asterella P. Beauv. in the complex thalloids (Long et

al. 2000), Fossombronia Raddi, Pallavicinia Gray and

Symphyogyna Nees & Mont. in the simple thalloids

(Forrest & Crandall-Stotler 2004, 2005), and Junger-

mannia in the leafy liverworts (this study, Fig. 9).

Paraphyly is also evident in many of the large,

morphologically defined families, including the

Marchantiaceae (Bisch.) Lindl., Pallaviciniaceae Mig.,

Jungermanniaceae Rchb., Geocalycaceae H. Klinggr.

and Lophoziaceae Cavers. Future comprehensive

molecular analyses that sample widely within these

larger genera and families are needed to satisfactorily

resolve their within-group relationships.

Complex thalloids. Based on patterns of short

branch lengths, rates of molecular evolution in loci

from both the chloroplast and the mitochondrion

Figure 8. Leafy clade I (based on the topology from the 5 partition Bayesian analysis). Numbers above the branches are Maximum

Parsimony BS values/ homogeneous Bayesian PPs/5-partition Bayesian PPs/14-partition Bayesian PPs.


appear to be far slower in the complex thalloid

liverworts than in the other lineages (Fig. 2). These

reduced rates, which correspond to a lower number of

variable characters, are probably responsible for the

low numbers of supported nodes in this group.

Studies using only the marchantiopsid taxa rooted on

the Haplomitriopsida (Long et al. 2005, in prep.)

produce congruent topologies, and the morphological

evolution of the group is discussed in more detail

therein. Regarding the relative contributions of the

five different loci to the complex thalloid topology,

the nr LSU region, which was utilized by Boisselier-

Dubayle et al. (2002) and Wheeler (2000), although

contributing many of the parsimony-informative

characters, actually performed comparatively poorly

in resolving the relationships of taxa within the group.

For example, morphology strongly supports the

Ricciaceae Rchb. as a natural taxon (Bischler 1998).

Figure 9. Leafy clade II (based on the topology from the 5 partition Bayesian analysis). Numbers above the branches are Maximum

Parsimony BS values/ homogeneous Bayesian PPs/5-partition Bayesian PPs/14-partition Bayesian PPs.


However, based purely on nr LSU data (Boisselier-

Dubayle et al. 2002: fig. 2) they form a polyphyletic

assemblage, in which Ricciocarpos Corda is well

separated from Riccia L. and sister to Monosolenium.

Only when Boisselier-Dubayle et al. (2002) added

morphological data to the analysis did they recover a

clade of Riccia/Ricciocarpos/Oxymitra Bisch. ex Lin-

denb. (65% MP BS). Data from the rbcL gene alone

(this study; not shown) give an unambiguously

monophyletic Ricciaceae, as does analysis of the five

loci combined (Fig. 5) (84% MP BS; 100% BI1, BI5,

BI14 PP).

Early divergences within the complex thalloid

lineages remain equivocal in our analyses, although

they always include Sphaerocarpos Boehm. and Neo-

hodgsonia. Conflicting results for the branching order

of these early branches, albeit with far lower levels of

taxon sampling, were also apparent in Forrest and

Crandall-Stotler (2004, 2005) and Crandall-Stotler et

al. (2005). Despite similarly equivocal resolution of

these taxa in the analyses of He-Nygren et al. (2006)

these authors nonetheless treat Sphaerocarpos as a

separate order, the Sphaerocarpales, because of its

distinctive morphology, while implying a nested

position for Neohodgsonia in the Marchantiales.

Clearly, further work is needed to resolve the

relationships of these taxa.

Often classified in its own order, the Monocleales

(Nees) A. B. Frank, Monoclea has traditionally been

viewed as the link between the complex thalloid and

simple thalloid liverworts because of its blend of

characters from both (Schuster 1984). Its alliance with

the complex thalloids was deemed remote. Although

its pattern of involucral development (Crandall-

Stotler & Stotler 2000; Johnson, 1904; Leitgeb 1877),

Conocephalum-like antheridial receptacle and march-

antioid antheridial ontogeny (Schuster 1984), idio-

blastic oil cells and monoplastidic meiosis (Renzaglia

et al. 1994) link Monoclea to the complex thalloids,

sporophyte characters in particular suggested a

primitive position for the taxon within the complex

thalloid lineage.

Wheeler (2000) produced the first molecular

phylogeny for the group, which included 16 complex

thalloid genera. He resolved Monoclea as part of a

crown polytomy within the complex thalloids, thereby

supporting Johnson’s (1904) conclusion that the

absence of air chambers and ventral scales in

Monoclea was an example of morphological reduction

due to the plant’s semi-aquatic habitat.

Although there has been some discussion re-

garding the nested position of Monoclea within the

main complex thalloid clade (e.g., Forrest & Crandall-

Stotler 2004), what has not been considered is the

identity of its closest relative. Neither Wheeler (2000)

nor Boisselier-Dubayle et al. (2002) have any support

for a sister relationship between it and any other

genus. Although Forrest and Crandall-Stotler (2005)

and Davis (2004) obtained support for a sister

relationship between Monoclea and Dumortiera, this

was based on very limited sampling of complex

thalloid genera. Only now, with 25 of the 32 complex

thalloid genera included, can we feel reasonable

confidence in the placement of Monoclea as sister to

Dumortiera (Fig. 5) (55% MP BS; 100% PP in BI1,

BI5 and BI14). Despite the rather low support for this

topology in parsimony-based analysis, which may well

correspond to low rates of molecular evolution

providing few parsimony-informative characters

within the complex thalloid lineage, this topology is

resolved in all analyses. In fact, there are morpho-

logical characters that unite these genera. While

Dumortiera has a complex archegoniophore, with an

8–10-lobed receptacle on a long stalk, both taxa have

almost sessile rounded male receptacles. Thallus

anatomy is very similar in the two, with neither

having air chambers. In fact, Gradstein et al. (2001:

214) stated that Monoclea often grows in association

with Dumortiera, and that sterile material of the two

genera can be difficult to separate. More significantly,

both taxa have similar slightly lobed sporocytes and

undergo monoplastidic meiosis (Shimamura et al.

2003). Although monoplastidic meiosis also occurs in

other taxa of the complex thalloid lineage, including

Wiesnerella Schiffn., Lunularia, some species of

Marchantia and Blasia, slightly lobed sporocytes occur

only in Monoclea and Dumortiera (Shimamura et al.

2003). It is interesting that as long ago as 1951,

Proskauer concluded from comparative systematic

studies of the two genera that Monoclea ‘‘may indeed

belong to the dumortieroid line’’ (1951: 265). Our

molecular evidence supports his view.

In general, traditional classifications of the

complex thalloids placed far greater weight on


reproductive characters than on vegetative thallus

morphology; therefore the presence/absence of an

archegoniophore outweighed the shared absence of air

chambers when it came to classifying these genera.

The fact that both genera occupy similar habitats,

even growing intermixed, means that convergence

towards a common morphology in what we have

historically regarded as a directly environmentally

responsive feature seems an obvious explanation.

Although it remains possible that air chambers were

lost independently in Monoclea and Dumortiera,

parsimony suggests a single loss in the common

ancestor of this lineage with subsequent reduction of

the archegoniophore and elaboration of the spor-

ophyte in Monoclea.

Unfortunately the wider relationship of these two

genera is not resolved here, although two of the

Bayesian analyses support a sister grouping with

Targionia L./Wiesnerella (100% BI1, 97% BI14 PP), as

also resolved by Long et al. (in prep.).

Within the ‘‘core’’ Marchantiales, neither Du-

mortiera nor Neohodgsonia resolve within the

Marchantiaceae, where they were placed by Bischler

(1998). Within the Marchantiaceae, Marchantia is

paraphyletic with Preissia nested within it. Both the

Aytoniaceae Cavers and Cleveaceae Cavers resolve as

monophyletic, supporting the traditional view of

them as morphologically well-supported families. In

the Aytoniaceae the results of Long et al. (2000) are

supported in that the largest genus, Asterella, is

paraphyletic. In the Cleveaceae, Peltolepis Lindb.,

which was excluded from the family by Bischler

(1998), falls clearly within it. This follows its tradi-

tional placement based on strong gametophytic and

spore ornamentation similarities.

The remaining crown group of complex thalloid

genera falls out as a major unresolved polytomy (Fig.

5) and further resolution of this clade is a priority for

the future. It includes both carpocephalate (e.g.,

Conocephalum Hill, Wiesnerella) and acarpocephalate

(e.g., Targionia, Riccia) genera, confirming the view

that reduction in gametophytic complexity (partic-

ularly of the reproductive structures) has been rife at

all levels in the complex thalloids. However, within

this polytomy a number of relationships are apparent,

such as that of Monoclea and Dumortiera discussed

above, as well as Corsinia and Exormotheca. These

relationships will be discussed in greater depth in a

forthcoming paper (Long et al., in prep.).

In regard to our treatment of Conocephalum, we

are aware of a recent publication by Szweykowski et al.

(2005) that effectively restricts Conocephalum conicum

(L.) Lindb. to Europe, placing samples from the

U.S.A. and India in a new species, C. salebrosum

Szweykowski, Buczkowska & Odrzykoski. We have,

however, retained the epithet conicum for our Illinois

sample because it does not resolve with C. salebrosum

in parsimony analyses that we have conducted in

conjunction with GenBank psbA accessions (from

Kim et al. 2001, as cited by Szweykowski et al. 2005).

Sterile thalli from Illinois populations match the

morphology assigned to C. salebrosum rather than that

of C. conicum s.str., but they contain a seven base pair

insertion in the trnK-psbA intergenic spacer region

that is missing in all C. salebrosum sequences, but

present in all other sampled C. conicum s.l. accessions.

We feel that the diversity within C. conicum s.l. needs

further investigation. Indeed, Szweykowski et al.

(2005) stated that there are six cryptic species within

this taxon; possibly the Illinois material will fall more

naturally into one of the species/races that has not yet

been addressed taxonomically. On the other hand, the

Indian material fits C. salebrosum both in terms of

morphology and of psbA sequence data.

What is clear from our analyses (Fig. 5) is that the

traditional subdivision of the complex thalloids into

the orders Monocleales, Marchantiales Limpr. and

Sphaerocarpales (Bischler 1998; Grolle 1983; Schuster

1984) is now convincingly demolished; more recent

classifications, such as that of Crandall-Stotler and

Stotler (2000), will require major overhaul. However,

a robust classification of complex thalloids is still

some way off without better resolution of both the

early-diverging lineages and the large crown group.

The monophyly of the Blasiales and its placement

as the earliest divergence from the complex thalloid

lineage are well supported in all analyses (Figs. 1, 5);

given the high degree of morphological similarity

between the two genera (Blasia and Cavicularia), this

is not an unexpected result. Previous results suggest-

ing a paraphyletic Blasiales (Forrest & Crandall-

Stotler 2004; Heinrichs et al. 2005) are due to

problems with DNA contamination, as discussed

further in the section ‘‘Benefits of collaboration.’’


Simple thalloids. The significance of the para-

phyly of the simple thalloid liverworts has been

discussed in several recent publications (e.g., Cran-

dall-Stotler 2005 et al.; Davis 2004; Forrest &

Crandall-Stotler 2004, 2005; Frey & Stech 2005;

Heinrichs et al. 2005; He-Nygren et al. 2004). In

general, there has been agreement that with the

reassignment of Haplomitrium and Treubiaceae to the

Haplomitriopsida and the Blasiales to the March-

antiopsida, the Metzgeriidae comprise two main

clades. These have been designated as Simple Thalloid

I (Fig. 6) and Simple Thalloid II (Fig. 7) (Davis 2004);

the former includes the majority of genera tradition-

ally classified in the Metzgeriidae (Crandall-Stotler &

Stotler 2000) and the latter, the genera of the suborder

Metzgeriineae, Verdoornia and the leafy liverwort,

Pleurozia. He-Nygren et al. (2006) relegated the taxa

of Simple Thalloid I to a new subclass, the Pelliidae

He-Nygren et al., and retain only the taxa of Simple

Thalloid II, excluding Pleurozia, in the Metzgeriidae.

Given the ambiguity that still exists in the resolution

of many lineages within the liverworts, however, we

feel that it is premature to translate any molecular

topology into a classification scheme. For example,

the classification of Pleurozia itself is problematic. He-

Nygren et al. (2006) placed Pleurozia with leafy

liverworts in the subclass Jungermanniidae, despite

the fact that in their Bayesian analysis it is resolved in

the Metzgeriaceae/Aneuraceae clade (PP 100%), in

agreement with our analyses. Only in their NONA

analysis (He-Nygren et al. 2006) is Pleurozia resolved,

with very low support, as sister to the rest of the leafy

liverworts. In our analyses and in those of He-Nygren

et al. (2006), there are no polytomies along the

backbone of the Simple Thalloid I clade, in contrast to

those illustrated in the diagrammatic scheme of

relationships presented in Frey and Stech (2005: fig.

1). A Pellia Raddi/Noteroclada Taylor ex Hook. &

Wilson clade is strongly supported as a member of the

earliest divergence within the Simple Thalloid I group,

with the Fossombroniineae sister to the Pallavicinii-

neae in the remaining lineage. Calycularia Mitt. is

weakly supported as sister to the Fossombroniineae/

Pallaviciniineae lineage, while Allisonia, which is

traditionally classified in the same family as Calycu-

laria (Crandall-Stotler & Stotler 2000), is resolved

within the Fossombroniineae clade. This topology has

also been resolved in previous studies focused on the

Metzgeriidae (Crandall-Stotler et al. 2005; Forrest &

Crandall-Stotler 2005), but differs from that resolved

in Heinrichs et al. (2005), particularly as regards the

earliest divergence in the lineage and the positions of

Makinoa Miyake and Noteroclada. The absence of

Pellia in the analyses of Heinrichs et al. (2005),

however, may be responsible for their resolution of

Makinoa in the earliest divergence of Simple Thalloid

I and the nesting of Noteroclada in the Fossombro-

niineae. It has been demonstrated that the absence of

a long-branch taxon can, in fact, drastically alter the

topology of early divergences (e.g., Crandall-Stotler et

al. 2005; Soltis & Soltis 2004); for example, when

Treubia is excluded, a complex thalloid/Blasia clade is

resolved as the earliest diverging lineage from the

main body of the liverworts and Haplomitrium is

nested in the Simple Thalloid I clade (Forrest &

Crandall-Stotler 2004) or its position on the liverwort

backbone phylogeny becomes highly unstable (Davis

2004). Since the relationships resolved within the

Simple Thalloid I clade have been discussed in detail

in earlier publications (Crandall-Stotler et al. 2005;

Forrest & Crandall-Stotler 2004, 2005), our discus-

sions here will be restricted to taxa whose placements

have been considered problematic (Frey & Stech

2005), namely, Makinoa, Phyllothallia E.A. Hodgs.

and Noteroclada.

In the analyses herein Makinoa is resolved with

strong BS and PP support as sister to a Fossombro-

niineae/Allisonia clade (Fig. 6), in congruence with

previous analyses by He-Nygren et al. (2004) and

Crandall-Stotler et al. (2005). In traditional classi-

fications (Crandall-Stotler & Stotler 2000; Schuster

1992), Makinoa was aligned with the Pallaviciniineae.

This placement was based primarily on shared

features of perichaetial organization and sporophyte

anatomy. As in Symphyogyna, the cluster of arche-

gonia is protected by an overarching, large posterior

perichaetial scale, a pseudoperianth is lacking and the

capsule is cylindric with a bistratose capsule wall. The

sporophyte is enclosed in a fleshy, scale-covered shoot

calyptra and the capsule opens along one or two slits.

Thallus anatomy, on the other hand, suggests a closer

affinity to Allisonia, as also suggested in our analyses

(see also Crandall-Stotler et al. 2005). Thalli in both

taxa lack strands of hydrolyzed cells, and bear


uniseriate, ventral slime hairs that are distributed in

two rows near the thallus apex, and red-brown to

purple rhizoids. Like Makinoa, Allisonia fails to

elaborate a pseudoperianth after fertilization, enclos-

ing the sporophyte only in a calyptra that is subtended

at its base by perichaetial scales; in contrast, however,

sporophytic capsules in Allisonia are globose and

dehiscence is irregular. Anatomical studies of peri-

gonial organization and sporophyte development are

needed to clarify this relationship. As discussed in

previous publications (Forrest & Crandall-Stotler

2004, 2005), Verdoornia, which was originally aligned

with Makinoa by Schuster (1984), is well supported in

both molecular (e.g., Fig. 7) and morphological

analyses (Crandall-Stotler et al. 2005) as a member of

the Aneuraceae (Frey & Stech 2005).

The position of Phyllothallia as sister to the large

clade comprising most of the Pallaviciniineae has been

resolved in both previous (Crandall-Stotler et al. 2005;

Forrest & Crandall-Stotler 2004, 2005; Heinrichs et al.

2005; He-Nygren et al. 2006) and the current analyses

without ambiguity (Fig. 6) (BS¼ 85%, PP for all

Bayesian analyses¼100%). Placed in its own suborder

by Schuster (1967, 1984), the genus was later

tentatively assigned to the Treubiales by Crandall-

Stotler and Stotler (2000). In particular, the occur-

rence of mature spheroidal capsules that dehisce

irregularly in P. fuegiana R. M. Schust. (Hassel de

Menendez 1971) seems discordant with the elongate,

valvate capsules that are diagnostic of the Hymeno-

phytaceae R. M. Schust. and Pallaviciniaceae. Ana-

tomical studies have shown, however, that there are

ontogenetic transformations from spheroidal to

elongate capsule morphologies within this clade

(Forrest & Crandall-Stotler 2004). When these find-

ings are considered in concert with gametophytic

characters of Phyllothallia that are suggestive of

Symphyogyna, the placement of Phyllothallia in a

shared ancestry with the Pallaviciniineae does not

seem problematic (see Forrest & Crandall-Stotler 2004

for further discussion).

Since the morphological studies of Schiffner

(1911), various authors have aligned Noteroclada with

Pellia (e.g., Evans 1939; Schuster 1984; Crandall-

Stotler & Stotler 2000). Despite the differences in

habit, leafy in Noteroclada and thalloid in Pellia, the

taxa share several major anatomical features. These

include antheridia individually sunken into ostiolate

chambers, spheroidal capsules with basal elatero-

phores and similar patterns of endosporic spore

germination. In the 8-locus analyses of Forrest and

Crandall-Stotler (2005) and Crandall-Stotler et al.

(2005) Noteroclada is resolved as sister to Pellia, with

BS support of 70% and 85%, respectively. In contrast,

earlier 5-locus analyses that included an incomplete

rbcL sequence for Noteroclada (AF536228) were

ambiguous for both Noteroclada and Pellia (Forrest &

Crandall-Stotler 2004). The position of Noteroclada in

phylogenetic schemes has been further confounded in

the analyses of He-Nygren et al. (2004), which

resolved it in a clade with Metzgeria Raddi and

Pleurozia, Schaumann et al. (2005), where it is

resolved, without support, in a clade comprising the

Metzgeriineae, and Heinrichs et al. (2005) in which

two GenBank accessions resolve it in separate clades,

the former in the Fossombroniineae and the latter as

sister to Aneura Dumort.

To test these various hypotheses we have

included two geographically separated collections of

this species, one from Chile and one from Venezuela

(Table 1), in our current analyses. They resolve as

sister to the four Pellia collections under MP (63%

BS) and Bayesian (100% BI1, BI5, BI14 PP) criteria

(Fig. 6). In addition, unpublished data for rbcL and

rps4 generated by Forrest place a second Venezuelan

collection [Freire & Crandall-Stotler 4189 (ABSH)], and

a collection from the Ecuadorian Andes [Weiss &

Schwerdtfeger s. n. (ABSH)] as monophyletic with the

Noteroclada collections included herein, while He-

Nygren et al.’s (2004) Noteroclada sequences

(AY462318, AY462377) resolve well within an Aneura

pinguis (L.) Dumort./A. maxima (Schiffn.) Steph.

clade (Forrest & Crandall-Stotler, unpublished anal-

ysis). These results strongly suggest that our reso-

lution of Noteroclada sister to Pellia is the most

reliable topology for this taxon and that its resolution

in the Aneuraceae is based on laboratory errors, as will

be discussed further in the section ‘‘Benefits of

Collaboration.’’ Indeed, He-Nygren et al. subse-

quently (2006) excluded Noteroclada from their

analyses due to uncertainties about its sequence data.

The topology of the Simple Thalloid II clade (Fig.

7) resembles that resolved in several previous analyses

(e.g., Crandall-Stotler et al. 2005; Davis 2004; Forrest


& Crandall-Stotler 2005), with a monophyletic

Metzgeriaceae sister to a monophyletic Aneuraceae as

defined by Frey and Stech (2005) to include

Verdoornia. Increased sampling reveals Metzgeria and

Apometzgeria Kuwah. to be paraphyletic, while the

three species of Riccardia Gray are monophyletic. The

resolution of Lobatiriccardia (Mizut. & S. Hatt.)

Furuki sister to an Aneura/Cryptothallus clade sup-

ports its recognition as a genus distinct from Aneura,

as proposed by Furuki (1991). Sampling within this

clade should be greatly increased in future studies, not

only in the species-rich Metzgeria and Riccardia, but

also in Aneura, which, in fact, shows comparatively

large levels of molecular divergence within A. pinguis

alone (Fig. 2; Forrest, unpublished data).

Leafy liverworts. The separation of the leafy

liverworts into two main clades (Leafy I and II), as

first suggested by Davis (2004) and He-Nygren et al.

(2004), remains supported. Many sister group rela-

tionships within these clades have been newly resolved

or gained further support in our analyses. However,

the position of the Ptilidium group in relation to these

two clades remains unresolved (Figs. 2, 9).

The ‘‘Leafy I’’ clade, first named by Davis (2004),

and also resolved in the single-locus studies of

Heinrichs et al. (2005) and Knoop (pers. comm.), is

also resolved in the present analyses (Figs. 1, 2, 8).

This clade has received varying support in the past,

with a PP of 63% and 97% in the homogeneous

Bayesian analyses of Heinrichs et al. and Knoop,

respectively, but posteriors of 100% (homogeneous

and two heterogeneous partition models) in the more

comprehensive analyses of Davis. However, Leafy I

lacked MP and ML BS support in Davis’ and Knoop’s

studies. Again, in our study, Bayesian analyses

(homogeneous and heterogeneous) support this

clade, while MP and ML BS failed. Likely, these

discrepancies in support values are due to sensitivity

in the placement of Ptilidum within the leafy liverwort

lineage.

Davis (2004) first showed that Ptilidium is

associated with Neotrichocolea; however, the position

of this Ptilidium/Neotrichocolea clade within the leafy

liverwort clade was unresolved in all her analyses. In

the recent analyses of He-Nygren et al. (2006), as well

as in our analyses, the position of the Ptilidium/

Neotrichocolea clade is also unsupported. In parsi-

mony-based analyses it is part of a basal leafy

polytomy; in Bayesian analyses it falls, without (or

with barely) significant PPs (Fig. 9) (88% in BI1 and

BI14, although 95% in BI5), with ‘‘Leafy II,’’ in

agreement with Davis (2004) and Knoop (pers.

comm.). Additional topologies that have been re-

ported, but which were not supported by our analyses,

include resolution of Ptilidium within a Porellales/

Radulales (R. M. Schust.) Stotler & Crand.-Stotler

clade in some of the POY analyses of Ahonen (2004)

and He-Nygren et al. (2004), and as sister to the

‘‘Leafy I’’ clade (however, with non-significant PP) in

Heinrichs et al. (2005) and He-Nygren et al. (2006).

Despite the lack of support for the phylogenetic

placement of Ptilidium, both Heinrichs et al. (2005)

and He-Nygren et al. (2006) proposed recognizing

this ‘‘Leafy IþPtilidium’’ clade as an order, Porellales.

The following characters are cited by Heinrichs et al.

(2005) as justification for this placement: mostly trifid

leaves, absence of ventral branching and production

of pinquisanes as secondary metabolites. According to

He-Nygren et al. (2006: 20) ‘‘the only non-homo-

plasious morphological synapomorphy of the clade . . .

is the endogenous spore germination,’’ which is,

however, absent in Ptilidium. While many of the taxa

resolved in Leafy I have previously been associated on

morphological grounds as Porellales (for discussion

see Crandall-Stotler & Stotler 2000), Radula, the

Lepidolaenaceae Nakai and Ptilidium (which has been

placed with Mastigiophoraceae R. M. Schust. and

Chaetophyllopsidaceae R. M. Schust. in suborder

Ptilidiineae R. M. Schust. (see Ahonen 2004)) have

not previously been taxonomically allied with this

lineage.

The placement of the Ptilidium/Neotrichocolea

clade bears directly on the interpretation of ventral

lobule and water sac evolution in the leafy liverworts.

All taxa resolved in Leafy I in this study (Fig. 8) have

ventral lobules that are smaller than the dorsal lobe of

the leaf, while in Leafy II (Fig. 9), all lobulate taxa

have dorsal lobules that are smaller than the ventral

lobe (e.g., Scapania, Diplophyllum, Schistochila, Now-

ellia). The elaboration of lobules into water sacs can

occur in both lineages although it is more common in

Leafy I (e.g., Nowellia in Leafy II, but all genera of the

Lepidolaenineae and Jubulineae (Spruce) Mull. Frib.

in Leafy I). Neotrichocolea does produce ventral


lobules that are sometimes elaborated into water sacs,

but only on leaves of secondary to quartenary

branches (Evans 1905). The water sacs of Neo-

trichocolea are galeate like those of the Lepidolaeni-

neae and Frullaniaceae of Leafy I, but are elaborated

from the ventralmost lobe of the 3-lobed leaf, rather

than from the middle lobe as is the case in these Leafy

I taxa (Evans 1905; Schuster 1972). If Neotrichocolea is

a lineage within Leafy II, the ventral water sac may be

parsimoniously interpreted as a pleisiomorphy for the

leafy liverworts. Alternatively, as suggested by their

different ontogenetic origins, ventral water sacs in

these two lineages may represent homoplasies in

response to common environmental pressures. It

seems that answering the question of where Ptilidium

and Neotrichocolea truly belong will require more

sampling, both of sequence characters and taxa;

clearly, questions about the direction of morpholog-

ical evolution within the leafy liverwort clades cannot

be fully addressed until the position of this lineage is

resolved.

The unexpected inclusion of the Lepidolaenaceae

and Jubulopsidaceae (Hamlin) R. M. Schust. with

taxa traditionally comprising the Porellales and

Radulales in a well-supported lineage, i.e., Leafy I

(Fig. 8), was first resolved by the analyses of He-

Nygren et al. (2004) and Davis (2004). Several

morphological features of the leafy gametophytes,

such as incubous leaf insertions, ventral lobules or

watersacs on at least some leaves and/or underleaves,

lateral (never ventral) branching, and precocious,

endosporic spore germination, support this relation-

ship (see also Davis 2004; He-Nygren et al. 2004;

Heinrichs et al. 2005). In addition, taxa in this clade

are predominantly epiphytes, lack mycorrhizae-like

mutualistic associations with fungi (Kottke & Nebel

2005; Nebel et al. 2004), and bear their perigonia on

either spicate or capitate lateral branches. There is,

however, significant disparity within the clade in the

organization of the sporophyte and its associated

gametophytic investments, with well-developed peri-

anths and spheroidal to ovoid capsules characterizing

the Porellineae R. M. Schust., but scale-covered

perigynia and long cylindric capsules in Lepidolae-

naceae and Jubulopsidaceae. In the Radulales a partial

stem perigynium-perianth complex encloses the

sporophyte (Schuster 1984: fig. 79) and the capsule is

ovoid to cylindric. The widely separated positions

accorded to these three groups in morphologically

based classifications (e.g., Crandall-Stotler & Stotler

2000; Evans 1939; Schuster 1984) are reflective of

these differences. Indeed, it seems that the assumption

in hepaticology that anatomical features of the

sporophyte and its associated investments are the best

predictors of phylogenetic relationship is not true of

many lineages, as also illustrated by the Phyllothallia/

Pallaviciniineae, Makinoa/Fossombroniineae and

Monoclea/Dumortiera clades. This may hold even

more widely across the bryophytes, as Buck (1991)

reported a similar phenomenom in the mosses.

While the monophyly of the Porellaceae Cavers is

strongly supported, its position relative to other

lineages within the clade is equivocal (Fig. 8). It

resolves sister to a clade comprising the Goebeliella-

ceae Verd., Lepidolaenaceae and Jubulopsidaceae, but

without significant support in most analyses (PP .

95% only under homogeneous Bayesian criterion).

The position of Goebeliella Steph., a genus with

sporophytes and perianths like Porella but distinctive

gametophytes that bear pairs of galeate water sacs on

their leaves, has varied among analyses. It is resolved

as sister to Radula in Heinrichs et al. (2005), nested

between the Porellaceae and a Radula/Frullania/

Jubula/Lejeuneaceae clade in He-Nygren et al. (2006)

and sister to the Lepidolaenaceae/Jubulopsidaceae

clade in Davis (2004), He-Nygren et al. (2004) and

this analysis. The Lejeuneaceae resolve as sister to the

Jubulaceae and comprise the crown group of Leafy I.

The Frullaniaceae are placed sister to the Jubulaceae/

Lejeuneaceae clade, with strong support (Fig. 8).

These three families share the occurrence of spores

with exine rosettes (Slageren 1995; Weis 2001),

sporophyte development entirely within the calyptra,

a reduced foot and fixed elaters (Spruce 1884–1885).

Within the Lejeuneaceae, more focused studies

indicate that some generic circumscriptions may

change when molecular data are taken into account

(e.g., Heinrichs et al. 2005; Wilson et al. 2004).

‘‘Leafy II,’’ first named by Davis (2004), is a well-

supported clade in all our analyses (Figs. 1, 2, 9) (but

see discussion of Ptilidium/Neotrichocolea, above).

The resolution of Schistochilaceae H. Buch as an early

diverging lineage, sister to the remaining taxa of Leafy

II, is also well established, in agreement with Davis


(2004), He-Nygren (2004, 2006) and Heinrichs et al.

(2005). The remaining taxa resolve into three major

clades, A, B, and C (Fig. 9), as designated in Davis

(2004). Clade A is well supported in our analyses and

confirms the alliance of the monophyletic families

Lepidoziaceae Limpr., Lepicoleaceae R.M. Schust. and

Herbertaceae Mull. Frib. (including Mastigophora).

These families are morphologically united by incu-

bously inserted leaves that are mostly 2- or 4-parted,

only rarely trifid, as well as by endogenous branches

(or flagellae). The sister group relationships among

these three families, however, are unsupported in our

own or previously published analyses. The inclusion

of Trichocolea within Clade A is well supported by our

analyses, although its position within it is not; based

on the 12-gene analyses of Davis (2004) it likely is part

of the Lepidoziaceae/Lepicoleaceae/Herbertaceae lin-

eage. The present analyses continue to support the

sister group relationship between these families and

some elements of the succubous-leaved Geocalycaceae

H. Klinggr. [Chiloscyphus and Lophocolea of the subf.

Lophocoleodeae Jorg.] plus Plagiochila clade, as

previously reported in multiple studies. It should be

noted, however, that the Geocalycaceae are para-

phyletic, with Harpanthus Spruce of the subf. Geo-

calycoideae H. Klinggr. resolved sister to Gyrothyra M.

Howe in clade C, albeit with weak support.

Our continued use of the name Lophocolea (e.g.,

Fig. 9, clade A) is in contrast to authors who consider

the genus synonymous with Chiloscyphus (Engel &

Schuster 1985). Although molecular phylogenetics has

been brought to bear on this issue (He-Nygren &

Piippo 2003), and reveals that species from Chiloscy-

phus s.str. nest within Lophocolea, low sampling levels

(with only seven out of over 300 recognized species)

and low clade support values mean that the issue

should not be considered resolved. Indeed, given the

range of morphological variation within Chiloscyphus

s.l. (Engel & Schuster 1984), this appears to be a

lineage in which increased molecular sampling,

coupled with morphological character mapping,

could be highly rewarding in isolating morphologi-

cally quantifiable taxa.

Clade B is solidly supported for the first time in

the present study, and is united by succubously or

transversely inserted leaves. Our analyses confirm the

alliance of Scapaniaceae Mig./Lophoziaceae Cavers as

sister to the Cephaloziellaceae Douin. Herzogobryum,

traditionally classified close to Gymnomitrion in the

Gymnomitriaceae Limpr. based on numerous shared

morphological characters (Schuster 2002), is nested

between this clade and that comprising the Cepha-

loziaceae Mig., thereby rendering the Gymnomitria-

ceae paraphyletic. The monophyly of the

Cephaloziaceae and its sister relationship to the

Scapaniaceae/Lophoziaceae/Cephaloziellaceae clade

are well supported, in agreement with numerous

previous studies. The inclusion of Adelanthus in Clade

B, as suggested by classifications (e.g., Crandall-Stotler

& Stotler 2000) as well as previous phylogenetic

results (Davis 2004; Heinrichs et al. 2005) has good

support in our analyses.

In Clade C the sister group relationship between

Nardia and the Gymnomitriaceae (excluding Herzo-

gobryum) is supported for the first time in the present

study. The four sampled species assigned to Junger-

mannia (s.l.) are polyphyletic within Clade C. Three

of the species form a supported monophyletic group

that is sister to the Nardia/Gymnomitriaceae clade in

all but the 14-partition Bayesian analysis, but

Jungermannia leiantha Grolle is resolved in a separate

clade (Fig. 9). In fact, our analyses suggest a

relationship between J. leiantha and Leiocolea hetero-

colpos (Thed.) H. Buch [[ Lophozia heterocolpa

(Thed.) M. Howe], another gemma-producing taxon

with beaked perianths. The status of J. leiantha has

long been controversial. Some authors (e.g., Muller

1957; Schuster 1969) have recognized Jungermannia

to comprise only J. leiantha and one or two closely

related species that also possess beaked perianths, with

the remaining species comprising the genus Solenos-

toma Mitt. This segregation of Solenostoma from

Jungermannia has not been generally accepted in

recent works (e.g., Grolle 1966; Grolle & Long 2000;

Stotler & Crandall-Stotler 2000; Vana 1996). How-

ever, the resolution of J. leiantha in a separate clade

from the other Jungermannia species supports the

hypothesis of two genera, although this should be

tested with increased sampling from within the genus/

genera. To complicate matters, if two genera are,

indeed, to be recognized, the name Liochlaena Nees

must be applied to the J. leiantha segregate since

Jungermannia has been typified by J. atrovirens

Dumort. (Vana 1973), a species recognized by


Schuster (1969) as a Solenostoma; Solenostoma sensu

Schuster would, in turn, become Jungermannia. These

findings also support the conclusions of Yatsentyuk et

al. (2004) based on trnL-trnF sequences that Leiocolea

should be recognized as distinct from Lophozia

(Dumort.) Dumort., which is resolved nested in the

Scapaniaceae of Clade B in several other analyses

(Heinrichs et al. 2005; He-Nygren et al. 2004, 2006;

Yatsentyuk et al. 2004).

The sister group relationship of Balantiopsida-

ceae H. Buch and Acrobolbaceae E. A. Hodgs.,

suggested in the rbcL and rps4 single locus analyses of

Heinrichs et al. (2005) and Knoop (pers. comm.),

respectively, also has support in some of our analyses.

In contrast to the alignment of Gyrothyra with the

Balantiopsidaceae by Schuster (1972, 1984), however,

Gyrothyra is resolved sister to Harpanthus, a rela-

tionship proposed by Crandall-Stotler (1978) based

on similarities in leaf ontogeny, perigonial organiza-

tion, antheridial anatomy and perigynial develop-

ment. Although no one morphological character

defines all the taxa currently resolved in Clade C, most

have succubous leaves (Calypogeia being a noteable

exception) and some level of pendent perigynium or

marsupium development.

Of the major lineages, leafy liverworts (Figs. 8, 9)

and complex thalloids (Fig. 5) are the groups with the

lowest amount of resolution in these analyses,

containing several polytomies under maximum par-

simony and non-significant nodes in Bayesian anal-

yses. While the low resolution in the complex

thalloids appears to be the result of reduced molecular

evolution, that in the leafy liverworts is more likely

due to the age of the group. Leafies comprise

approximately 4,000 species (http://bryophytes.plant.

siu.edu) and appear to be one of the most recent

lineages in the phylum, with some rapidly evolving

taxa. Thus, taxon sampling sensitivity may be

especially problematic here, and loci that are appro-

priate for the other liverwort lineages may be less

appropriate. By contrast the sister group to the leafy

liverworts, Metzgeriidae (simple thalloid II, Fig. 7),

comprising an estimated 220 species, is fully resolved

into supported nodes. Although this study has

resolved relationships among the major lineages

within the liverworts, much more sampling, both of

taxa and loci in the leafy liverworts, will be required to

satisfactorily resolve a phylogeny that is equally robust

for all lineages.

Benefits of collaboration. During the course of

this collaborative project, we have discovered the

extent to which several problems have confounded

previous studies. Collaborative studies such as these

are extremely important at this stage of synthesis,

when researchers need to assess the state and stage of

data collection in order to proceed with relevant

questions to future studies. These problems became

clear to us when data were compared, specimens were

reexamined, and generally, we obtained a better feel

for liverwort phylogeny. They include misidentifica-

tion of taxa, differential amplification of contami-

nants from mixed specimens/DNA extractions, and

perpetuation of these errors in the GenBank database.

Following are examples and suggestions for remedies.

The cases of Cavicularia and Noteroclada (as

mentioned earlier) are examples of problems that can

occur post-extraction, and perpetuation of errors

through GenBank. Contamination of a DNA sample

meant that some erroneous sequences for Cavicularia

were published in Forrest and Crandall-Stotler (2004),

leading to the erroneous suggestion of a paraphyletic

Blasiales. The problem sequences were later deter-

mined to belong to Pellia. Although this was corrected

in Forrest and Crandall-Stotler (2005), Crandall-

Stotler et al. (2005) and in our analyses here, an rbcL

sequence remained in GenBank long enough for

another study to be published using it (Heinrichs et

al. 2005), thus perpetuating the error. In the case of

Noteroclada, He-Nygren et al. (2004) were not able to

recognize their sequences as problematic because they

included only one accession of this monospecific

genus. Only by comparisons with additional data was

it made apparent that the sequences had the incorrect

name associated with them. These sequences remain

in GenBank and the rbcL sequence has subsequently

been used in another phylogenetic study (Heinrichs et

al. 2005), underlining the importance of early

correction of database errors, and of critical review

and verification of GenBank sequences in future work,

as done in a forthcoming study by Knoop (pers.

comm.).

Mixed specimen collections are a common

feature in hepaticology, and can present serious

problems for phylogenetic reconstruction. For exam-


ple, detailed examination of a collection of Lophozia

sudetica (Nees) Grolle (Davis 432 (DUKE)) by Crandall-

Stotler revealed fragments of Nardia insecta Lindb.,

identified by the presence of small triangular under-

leaves. This contamination resulted in the anomalous

position of Lophozia as sister to Nardia scalaris Gray

in Davis (2004). Comparison with the position of

Lophozia in other analyses (e.g., Heinrichs et al. 2005;

He-Nygren et al. 2004, 2006), however, highlighted

the problem and led to reexamination of our material.

In cases of mixed collections, a number of

scenarios are possible. For example, the unintended

taxon can be pulled for extraction (differentially or

randomly); both taxa can be included in the DNA

extraction but unequal preservation of the material

can mean that the DNA from one is degraded and

fails to amplify in PCR, or cells in one taxon may lyse

more readily to release DNA into solution than in the

other; mutations in primer sites can cause one taxon

to amplify over the other; or both taxa can amplify

(giving rise to multiple peaks in sequencing electro-

pherograms). In many ways the worst problems, at

least theoretically, can be caused by differential

preservation of two taxa in a collection that contains

very few contaminants (as in the Lophozia/Nardia

example)—and this is not an entirely unlikely

scenario given the probability that different taxa have

different levels of drought tolerance (or that one

produces dispersal organs such as gemmae). It is thus

possible that a very small fragment of a contaminant

in a collection can amplify differentially for a whole

series of sequenced loci. Differential primer response,

on the other hand, is not likely to apply to every locus

in a multi-gene analysis, and so can be identified by

incongruencies in the placement of taxa for different

data partitions, while if both taxa amplify, problems

become apparent at the sequencing stage. A very

mixed collection, while presenting opportunity for

errors during the initial DNA extraction, is more

likely to be identified when the voucher is rechecked

after the anomalous phylogenetic placement is

observed. However, even when the correct taxon is

carefully pulled from a mixed collection, there is still

potential for error if the contaminant produces

gemmae, as these can be caught between the speci-

men’s leaves or lamellae. Indeed, PCR of separate

DNA extractions using gemmae and thalli of Xen-

othallus R. M. Schust. is far more successful for the

gemmae (Forrest, unpublished data).

The point we intend to make here is that

herbarium-based methodology, while suitable for

reconciling disparities in morphological observations

and verifying identities, cannot resolve problems that

occur post-DNA extraction. All researchers, careful as

they may be, are vulnerable to these pitfalls, and only

by using a comparative method do problems become

apparent. Thankfully, now that the overall picture of

liverwort phylogeny is emerging, we can better judge

when relationships are anomalous and deserve a

second look.

Herbarium-based methodology is, however, es-

sential in rectifying the other common factor that can

confound phylogenetic estimates: the correct identi-

fication and isolation of the material being studied.

For many liverwort taxa this is not a trivial exercise.

Herbarium material is often fragmentary, and collec-

tions, as noted above, are frequently mixed. Further,

annotations, even by acknowledged experts in the

organisms, are sometimes incorrect or incomplete. In

the Lophozia/Nardia example, the bulk of the

collection is Lophozia sudetica. However, similarities

in gross morphology of the sterile dried material—for

example, both taxa with curving bifid leaves—make it

extremely easy to mistake the mixture for a pure

collection. This stresses the importance for phyloge-

netic studies within the hepatics to be performed in

collaboration with taxonomic experts on the group, to

verify both the initial and the reciprocal-illumination

plant identities.

Future research. A robust phylogeny for the

hepatics, as well as providing a framework for

focusing morphological and developmental studies,

provides potential for the dating of major events in

liverwort evolution and their correlation with major

events in Earth history. There has been speculation,

for example, that the radiation within the leafy

liverworts (perhaps particularly the Lejeuneaecae, a

largely epiphytic/epiphyllous group) correlates with

the expansion of angiosperm forests, and the

associated expansion in niches (e.g., Ahonen et al.

2003). In fact, there is phylogenetic evidence to

support this hypothesis, suggesting that the major

lineages of Lejeuneaceae diversified in the early-mid

Cenozoic (41.34–61.68 Mya) in line with the devel-


opment of complex angiosperm forest habitats

(Newton et al. 2006). The idea that ancient lineages

exploited new epiphytic habitats to diversify alongside

flowering plants, rather than prior to, or in the

shadow of, angiosperms, is an idea gaining support

among plant evolutionary biologists (e.g., Schneider

et al. 2004). To test these hypotheses, phylogenies

must have dated nodes. The study by Newton et al.

(2006) is exciting in that it represents the first

systematic attempt to date major bryophyte lineages.

However, as the focus of their paper is pleurocarpous

mosses, the implications of the dating of liverwort

nodes are not discussed. That said, the paper includes

34 liverwort accessions across all three classes, and

certainly does provide us with a first approximation

for clade ages.

However, more in-depth studies are required,

and are a logical next step from the analyses presented

herein. Liverworts are unique among bryophytes in

having a long fossil record, including well-studied

macrofossils (e.g., Krassilov & Schuster 1984; Oos-

tendorp 1987), as well as the putative liverwort spores

described by Wellman et al. (2003) from 475 million

years ago. This offers real possibilities in terms of

putting date ranges onto nodes within the liverwort

phylogeny instead of using an external date, as is the

case in Newton et al. (2006). However, there is an

important caveat: extreme care must be taken when

considering the evolution of the complex thalloids, a

group that shows very different rates of molecular

evolution from the rest of the liverworts (Fig. 2), and

thus has the potential to confound age estimates.

Earlier in the paper we pointed out that liverwort

taxa fall into a classic ‘‘hollow curve’’ distribution

(Willis 1922), but noted that this may be an artifact of

classification within the group, indicating more about

how taxonomists delimit taxa than about evolu-

tionary pattern (Guyer & Slowinski 1993). Low

sampling levels among the leafy liverworts still do not

allow us to address issues of clade size in any depth.

However, it is apparent that hepatic topology is highly

pectinate, with small lineages progressively sister to far

larger ones (e.g., Haplomitriopsida/all other liver-

worts; Blasiaceae/complex thalloids; Pleuroziales/

simple thalloids II—see Fig. 1). Systematically,

pectinate topologies are explained by differential

evolutionary rates such as those produced by key

innovations and/or extinction rates in different

lineages, leading to the concept of ‘‘success’’ (resist-

ance to extinction or propensity for speciation)

(Pearson 1999). Although not every case of lineage

‘‘success,’’ or lack of it, will have a biological

explanation, we remain hopeful that detailed inves-

tigations of the distribution of morphological char-

acters across our phylogeny will elucidate at least

some of the key factors responsible for the present-day

distribution of diversity within the liverwort lineages.

The ability to use our estimate of liverwort

phylogeny to test hypotheses regarding morphological

evolution, radiations and key innovations, as well as

potential coevolution, for example with fungal

associates, within the group, opens up exciting new

opportunities for hepaticology. We have identified

several key areas of the tree, however, that still need

further phylogenetic focus to stabilize nodes, and

these should become priority areas for further

research.

ACKNOWLEDGMENTS

We thank the following people: Angela Newton (BM) and Neil

Bell (BM) for Riella DNA; Daniela Schill (E) for many of the

complex thalloid collections and DNAs; Raymond E. Stotler

(SIU) for help with literature, vouchers and authorities and for

proofreading the final draft; Dylan Kosma (SIU) for maintenance

of the culture collection; Sedonia Sipes (SIU) for access to

sequencing facilities; Michael Moller (E) for running one of the

Bayesian analyses; Cymon Cox (BM) for assistance with analyses;

Rosemary Wilson and Jochen Heinrichs (GOET) for comments

on an earlier draft; Volker Knoop (BONN) for access to his pre-

publication rps4 manuscript; Juan Carlos Villarreal A. (SIU) and

Bernard Goffinet (CONN) for proofreading and comments on

earlier drafts; Jon Shaw (DUKE) for the invitation to present this

paper at the XVII International Botanical Congress in Vienna,

and for providing the impetus behind the manuscript; Jon Shaw

(DUKE) and John Engel (F) for reviewing this paper; and

definitely not least, the many other people listed as collectors in

Table 1, who made this research possible. Funding sources: ECD

would like to acknowledge grant no. EF-0531730 (to A. J. Shaw).

Sequences generated at Southern Illinois University were funded

by NSF grant no. DEB-997796, those at the Royal Botanic

Gardens, Edinburgh were funded by RBGE Molecular Phylo-

genetics Projects, and those at Duke University were funded by a

plant systematics training grant from the Mellon Foundation.

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Unraveling the evolutionary history of the liverworts (Marchantiophyta): multiple taxa, genomes and analyses

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