Available online at www.sciencedirect.com
Molecular Phylogenetics and Evolution 45 (2007) 693–705
www.elsevier.com/locate/ympev
A phylogeny of Porella (Porellaceae, Jungermanniopsida) based
on nuclear and chloroplast DNA sequences
Jörn Hentschel a, Rui-Liang Zhu b, David G. Long c, Paul G. Davison d,
Harald Schneider a,1, S. Robbert Gradstein a, Jochen Heinrichs a,*
a
Department of Systematic Botany, Albrecht von Haller Institute of Plant Sciences, Untere Karspüle 2, 37073 Göttingen, Germany
b
Department of Biology, East China Normal University, 3663 Zhong Shan North Road, Shanghai 200062, China
c
Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh EH3 5LR, Scotland, UK
d
Department of Biology, University of North Alabama, UNA Box 5232, Florence, AL 35632-0232, USA
Received 1 March 2007; revised 13 April 2007; accepted 7 May 2007
Available online 25 May 2007
Abstract
The cosmopolitan family Porellaceae includes about 60 species in two or three genera: the large genus Porella and the monospecific
Ascidiota and Macvicaria (alternatively Porella subg. Macvicaria). Maximum parsimony, maximum likelihood and Bayesian inference of
phylogeny of a dataset including three markers (rbcL, trnL–trnF region of cp DNA, nrITS region) of 96 accessions resulted in similar
topologies supporting the generic status of Ascidiota. Macvicaria is nested in a subclade of Porella. Relationships among species of Porella are in general well resolved and many terminal nodes achieve good statistical support whereas basal relationships are at best moderately supported. Multiple accessions of single species are usually placed in monophyletic lineages. Accessions of P. platyphylla split into a
European and a North American clade with one accession from North America embedded within the European samples. The Macaronesian endemic P. inaequalis is closely related to the Asian species P. grandiloba. Porella obtusata and P. canariensis cannot be separated
on the basis of the sequence data presented in this study. The molecular topologies indicate a range extension of the Asian P. gracillima
subsp. urogea to Eastern North America and of the Neotropical P. swartziana to South Africa. Current supraspecific classifications of
Porella are not reflected in the molecular topologies with a correlation between genetic variation and the geographical distribution of the
related accessions rather than a correlation between genetic variation and morphology.
Ó 2007 Elsevier Inc. All rights reserved.
Keywords: Liverwort; Jungermanniopsida; Porellales; Porellaceae; Porella; Ascidiota; Macvicaria; Molecular phylogeny; Cryptic speciation
1. Introduction
Porella L. with over 200 published binomials (Hattori,
1989) and an estimated number of 50–60 species (Schuster,
1980) is characterised by bilobed, incubously inserted
leaves with a vestigial, often recurved keel, large underleaves that are similar to the lobules in shape, terminal
gynoecia on short lateral branches, large, flattened perianths, and a capsule that is barely exserted from the peri*
Corresponding author. Fax: +49 551 39 2329.
E-mail address: jheinri@uni-goettingen.de (J. Heinrichs).
1
Present address: Department of Botany, Natural History Museum,
London SW7 5BD, UK.
1055-7903/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2007.05.005
anth and dehisces into numerous irregular valves. Two
monospecific genera have been placed alongside cosmopolitan Porella, the Asian–North American Ascidiota C.Massal. with leaf lobes developed as water-sacs, and the Asian
Macvicaria W.E.Nicholson with crisped undulate leaves
and inflated perianths, the latter sometimes treated as a
subgenus of Porella (Inoue, 1976; Hattori, 1978; Schuster,
1980). Together these genera make up family Porellaceae
(Schuster, 1980).
Porella has not only been studied morphologically (e.g.,
Hattori, 1970, 1978, 1986; Swails, 1970; Schuster, 1980; So,
2002, 2005) but also in terms of secondary metabolite composition (e.g., Asakawa et al., 1978; Buchanan et al., 1996;
Bungert et al., 1998; Hashimoto et al., 2000). As a result,
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J. Hentschel et al. / Molecular Phylogenetics and Evolution 45 (2007) 693–705
Porella species are now well known for their production of
terpenoids and aromatic compounds with antitumor, antimicrobial, and antifungal activity (Asakawa, 1998). Additionally, several studies have been carried out that
utilized DNA fingerprint methods or isozyme data (e.g.,
Boisselier-Dubayle and Bischler, 1994; Boisselier-Dubayle
et al., 1998; Therrien et al., 1998; Freitas and Brehm,
2001; Bischler et al., 2006). These studies pointed at incongruence of molecular and morphological variation (Therrien et al., 1998) and led to the discovery of the allopolyploid
status of P. baueri (Schiffn.) C.E.O.Jensen, which is possibly the interspecific hybrid of P. platyphylla (L.) Pfeiff.
and P. cordaeana (Huebener) Moore (Boisselier-Dubayle
et al., 1998).
Taxonomy of Porella has been regarded as notoriously
difficult. Schuster (1980): 666) notes ‘‘the actual number
of valid taxa is problematical, since the genus seems to be
in an active state of evolution with species boundaries ill
defined and the species showing phenomenal plasticity’’.
Rampant intraspecific variation and extensive lack of stable morphological characters hampers not only species taxonomy but complicates also supraspecific classification.
Schuster proposed the first comprehensive infrageneric
classification as recently as 1980. Based primarily on leaf
lobe and lobule insertion, he (Schuster, 1980) distinguished
three subgenera [P. subg. Macvicaria (W.E.Nicholson)
Inoue, P. subg. Protoporella R.M.Schust, P. subg. Porella]
and six sections. However, Schuster (1980) considered his
subdivision provisional due to high infraspecific variation
and uniformity in basic architecture of Porella. In contrast
to other authors, he treated the genus Macvicaria as a subgenus of Porella.
DNA-based investigations have already been applied in
several studies focussing on various families of liverworts
(e.g., Schaumann et al., 2005; Heinrichs et al., 2006; Hentschel et al., 2006a; Wilson et al., 2007). These studies have
greatly improved our understanding of species and genus
relationships, and often recovered conflicts with previous
morphology-based classifications.
The principal objective of this study is to recover a first
comprehensive phylogeny of Porella based on a worldwide
sampling and nuclear as well as chloroplast DNA markers.
A further goal is to evaluate species concepts by sequencing
of multiple accessions as well as the supraspecific classification of Porella proposed by Schuster (1980).
2. Materials and methods
2.1. Taxon sampling and outgroup selection
Two hundred and eighty-three new sequences generated
from 96 specimens were used in this study, including 96
rbcL sequences, 95 trnL-F sequences as well as 92 nrITS
sequences. Taxa studied are listed in Table 1, with GenBank accession numbers and voucher details. The determinations of the voucher specimens were carefully examined
and original identifications were corrected when necessary.
Ingroup species were selected according to availability
and to represent the morphological variation and geographical distribution of Porellaceae. Multiple accessions
of several species were used to explore intraspecific genetic
variation.
Two datasets were compiled. Dataset I includes representatives of Lepidolaenaceae, Goebeliellaceae, and Porellaceae (Ascidiota, Macvicaria, Porella div. sp.). It was
mainly designed to determine the sister relationships of
Ascidiota and Porella. Dataset II includes only Porellaceae
and allows for a more comprehensive alignment of spacer
regions. Based on the multi-gene, multi-taxon analyses of
Forrest et al. (2006), He-Nygrén et al. (2006) and Heinrichs
et al. (2007) Lepidolaenaceae and Goebeliellaceae were designated as outgroups in dataset I. Based on results of the
phylogenetic analyses of dataset I Ascidiota blepharophylla
C.Massal. was chosen as outgroup of dataset II.
2.2. DNA extraction, PCR amplification and sequencing
Plant tissue from the distal portions of a few shoots was
isolated from up to 48 years old herbarium collections or
specimens dried in silica gel. Total genomic DNA was purified using Invisorb Spin Plant Mini Kit (Invitek, Berlin,
Germany) prior to amplification. Protocols for PCR were
carried out as described in previous publications: trnL-F
region from Gradstein et al. (2006), rbcL from Hentschel
et al. (2006a), and nrITS region from Hentschel et al.
(2006b). Bidirectional sequences were generated using a
MegaBACE 1000 automated sequencing machine using
DYEnamic ET Primer DNA Sequencing Reagent (Amersham Biosciences, Little Chalfont, UK). Sequencing primers were those used for PCR. For all 96 accessions at least
two from three markers were obtained.
2.3. Phylogenetic analyses
All sequences were aligned manually in Bioedit version
7.0.5.2 (Hall, 1999). Ambiguous positions were excluded
from all alignments and lacking data were coded as missing. Alignment of dataset I resulted in a rbcL alignment
with 1324 positions, trnL-F 525, and an nrITS alignment
with 830 putatively homologous sites (dataset I); alignment
of dataset II resulted in a rbcL alignment with 1324 positions, trnL-F 550, and a nrITS alignment with 846
positions.
Maximum parsimony (MP) and maximum likelihood
(ML) analyses were carried out with PAUP* version
4.0b10 (Swofford, 2000).
MP heuristic searches were conducted with the following options implemented: heuristic search mode, 1000 random-addition-sequence
replicates,
tree
bisectionreconnection (TBR) branch swapping, MULTrees option
on, and collapse zero-length branches off. All characters
were treated as equally weighted and unordered. Nonparametric bootstrapping values (BS) were generated as
heuristic searches with 1000 replicates, each with ten
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J. Hentschel et al. / Molecular Phylogenetics and Evolution 45 (2007) 693–705
Table 1
Taxa used in the present study, including information about the origin of the studied material, voucher information, and the herbarium where the voucher
is deposited, as well as GenBank accession numbers
Taxon
Origin
Ascidiota blepharophylla C.Massal. subsp. alaskana
Steere & R.M.Schust. (I)
A. blepharophylla subsp. alaskana (II)
Gackstroemia ljungneri (Herzog) Grolle
Gackstroemia magellanica (Lam.) Trevis.
Gackstroemia schwabei (Herzog) Grolle
Gackstroemia weindorferi (Herzog) Grolle
Goebeliella cornigera (Mitt.) Steph.
Lepidolaena brachyclada (Taylor ex Lehm.) Trevis.
Lepidolaena clavigera (Hook.) Dumort. ex Trevis.
Lepidolaena taylorii (Gottsche) Trevis.
Porella abyssinica var. abyssinica Trevis.
Porella abyssinica var. hoehnelii (Steph.) Pócs
Porella acutifolia (Lehm. & Lindenb.) Trevis.
Porella acutifolia
trnL–F
nrITS
EF545281
EF545376
EF545468
Lewis, 514 (F)
Drehwald & Drehwald, 910052 (JE)
Frahm, 27-2 (GOET)
Frey & Schaumann, 01-372g (JE)
Streimann, 51752 (JE)
von Konrath, 27327 (GOET)
Scott, MUCV 5228 (JE)
Frahm, 22-3 (GOET)
Frahm, 1-34 (GOET)
Pócs et al., 90079/L (JE)
Pócs et al., 9210/V (JE)
Gradstein, 10311 (GOET)
Toia, 212 (JE)
EF545280
EF545277
EF545276
EF547187
EF545275
EF545274
EF547188
EF545278
EF545279
EF545322
EF545323
EF545309
EF545310
EF545375
EF545371
EF545368
EF545369
EF545370
EF545367
EF545372
EF545373
EF545374
EF545417
EF545418
EF545404
EF545405
EF545467
EF545464
EF545463
—
EF545462
—
—
EF545465
EF545466
EF545509
EF545510
EF545496
EF545497
EF545364
EF545363
EF545362
EF545365
EF545366
EF545333
EF545327
EF545459
EF545458
EF545457
EF545460
EF545461
EF545428
EF545422
EF545551
EF545550
EF545549
EF545552
EF545553
EF545520
EF545514
EF545326
EF545318
EF545317
EF545314
EF545311
EF545308
EF545352
EF545421
EF545413
EF545412
EF545409
EF545406
EF545403
EF545447
EF545513
EF545505
EF545504
EF545501
EF545498
EF545495
EF545539
EF545353
EF545448
EF545540
EF545325
EF545284
EF545420
EF545379
EF545512
EF545471
EF545285
EF545283
EF545312
EF545287
EF545286
EF545288
EF545319
EF545320
EF545330
EF545328
EF545344
EF545380
EF545378
EF545407
EF545382
EF545381
EF545383
EF545414
EF545415
EF545425
EF545423
EF545440
EF545472
EF545470
EF545499
EF545474
EF545473
EF545475
EF545506
EF545507
EF545517
EF545515
EF545531
Japan
China
China
Bhutan
Long, 29768 (GOET)
Caspari, 4/16 (GOET)
Eckstein, 2323 (GOET)
Zündorf, 21671 (JE)
Eckstein, 4261 (GOET)
Churchill et al., 22001 (GOET)
Schäfer-Verwimp & Verwimp, 6997
(GOET)
Churchill et al., 23218 (GOET)
Li & Wang, 1301 (GOET)
Huneck, K 86-12 (JE)
Koponen, 46872 (JE)
Long, 10649 (JE)
Long, 34391 (E)
Schäfer-Verwimp & Verwimp, 24763
(GOET)
Schäfer-Verwimp & Verwimp, 24760
(GOET)
Hedderson, 14779 (BOL)
Schäfer-Verwimp & Verwimp, 7960
(GOET)
Hyvönen, 5572 (JE)
Drehwald & Mues, 970001 (GOET)
Gambaryan, VLA-h-1865 (GOET)
Hentschel, Bryo 01730 (GOET)
Long, 34092 (E)
Schofield, 117519 (UBC)
Streimann, 49240 (JE)
Streimann, 27018 (JE)
Churchill et al., 22864 (GOET)
Churchill et al., 23663 (GOET)
Inoue, Bryophyta Selecta Exsiccata
870 (JE)
Ohnishi, 3407 (HIRO)
Long, 35219 (GOET)
Zhu, 20060422-1 (GOET)
Long, 10823 (JE)
EF545343
EF545340
EF545342
EF545346
EF545439
EF545435
EF545437
EF545436
EF545530
EF545527
EF545529
EF545533
Japan
China
Russia
USA
Tanaka, 7339 (HIRO)
Li & Wang, 4285 (GOET)
Gambaryan, VLA-h-1955 (GOET)
Davison, 6719 (GOET)
EF545321
EF545355
EF545356
EF545354
EF545416
EF545450
EF545451
EF545449
EF545508
EF545542
EF545543
EF545541
China
Long, 34500 (E)
EF545358
EF545453
EF545545
(continued on next page)
arboris-vitae (With.) Grolle
arboris-vitae
arboris-vitae
arboris-vitae (I)
arboris-vitae (II)
brachiata (Taylor) Spruce
brasiliensis (Raddi) Schiffn.
Porella
Porella
Porella
Porella
Porella
Porella
Porella
brasiliensis
caespitans var. cordifolia (Steph.) S.Hatt.
caespitans var. cordifolia
caespitans var. nipponica S.Hatt.
campylophylla (Lehm. & Lindenb.) Trevis.
cf. campylophylla
canariensis (F.Weber) Underw. (I)
Bolivia
China
North Korea
China
Bhutan
China
Canary Islands
Porella canariensis (II)
Canary Islands
Porella capensis (Gottsche) Steph.
Porella chilensis (Lehm. & Lindenb.) Trevis. (I)
South Africa
Argentina
Porella densifolia (II)
Porella densifolia (I)
*Porella densifolia (II)
*Porella densifolia var. appendiculata (Steph.)
S.Hatt.
Porella fauriei (Steph.) S.Hatt.
Porella gracillima Mitt.
Porella gracillima
Porella gracillima subsp. urogea (C.Massal.) S.Hatt.
& M.X.Zhang
Porella cf. grollei S.Hatt.
rbcL
Long, 11186 (GOET)
Porella
Porella
Porella
Porella
Porella
Porella
Porella
chilensis (II)
chilensis (III)
chinensis (Steph.) S.Hatt.
cordaeana (Huebener) Moore
cordaeana
cordaeana
cranfordii Steph. (I)
cranfordii (II)
crispata (Hook.) Trevis. (I)
crispata (II)
densifolia (Steph.) S.Hatt. (I)
GenBank accession
Alaska
Alaska
Argentina
Chile
Chile
Australia
New Zealand
Tasmania
New Zealand
New Zealand
Tanzania
Kenya
Indonesia
Papua New
Guinea
British Isles
Madeira
Canary Islands
Germany
Germany
Bolivia
Brazil
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Voucher (Herbarium)
Argentina
Argentina
Russia
Germany
British Isles
Alaska
Australia
Australia
Bolivia
Bolivia
Japan
696
J. Hentschel et al. / Molecular Phylogenetics and Evolution 45 (2007) 693–705
Table 1 (continued)
Taxon
Origin
Porella
Porella
Porella
Porella
Porella
Porella
grandiloba Lindb.
grandiloba
inaequalis Perss.
japonica (Sande Lac.) Mitt.
leiboldii (Lehm. & Lindenb.) Trevis. (I)
leiboldii (II)
North Korea
Japan
Madeira
Japan
Costa Rica
Costa Rica
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Porella
macroloba (Steph.) S.Hatt. & Inoue (I)
macroloba (II)
madagascariensis (Nees & Mont.) Trevis.
navicularis (Lehm. & Lindenb.) Pfeiff. (I)
navicularis (II)
nitens (Steph.) S.Hatt.
nitens
oblongifolia S.Hatt.
obtusata (Taylor) Trevis.
obtusata
obtusata
China
China
Sri Lanka
USA
USA
Bhutan
China
Bhutan
Italy
France
Canary Islands
Porella perrottetiana (Mont.) Trevis.
Porella perrottetiana (I)
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Porella
Porella
perrottetiana (II)
pinnata L.
platyphylla (L.) Pfeiff.
platyphylla
platyphylla
platyphylla (I)
platyphylla (II)
platyphylla (I)
platyphylla (II)
platyphylla (III)
roellii Steph. (I)
roellii (II)
saccata M.L.So
spinulosa (Steph.) S.Hatt.
Bhutan
Japan
Japan
USA
Germany
Bulgaria
Italy
Canada
Canada
USA
USA
USA
Canada
Canada
Ecuador
Japan
Porella squamulifera (Taylor) Trevis.
Porella squamulifera
Bolivia
Ecuador
Porella stephaniana (C.Massal.) S.Hatt.
Japan
Porella subdentata (Mitt.) E.W.Jones
Porella subobtusa (Steph.) S.Hatt.
DR of Congo
Japan
Porella
Porella
Porella
Porella
Porella
Porella
swartziana (F.Weber) Trevis.
swartziana (I)
swartziana (II)
ulophylla (Steph.) S.Hatt.
vernicosa Lindb.
vernicosa
Porella vernicosa
South Africa
Bolivia
Bolivia
China
Japan
Russia
North Korea
Voucher (Herbarium)
Huneck, K 86-30 (JE)
Ohnishi, 4741 (HIRO)
Caspari, 5/18b (GOET)
Deguchi, 36624 (HIRO)
Holz, CR 99-0539 (GOET)
Schäfer-Verwimp & Holz, SV/
H-0021 (GOET)
Koponen, 45262 (JE)
Li & Wang, 4429 (GOET)
Eggers, SL 7,05 (JE)
Whittemore, 4393 (GOET)
Whittemore, 4405 (GOET)
Long, 8482 (JE)
Long, 34415 (GOET)
Long, 10726 (JE)
Eckstein, 4681 (GOET)
Long, 35373 (GOET)
Schäfer-Verwimp & Verwimp, 24754
(GOET)
Long, 10727 (JE)
Deguchi, Bryophytes of Asia 4, 96
(GOET)
Yamaguchi 12583 (HIRO)
Brant 2991 (GOET)
Hentschel, Bryo 01567 (GOET)
Hentschel, Bryo 0763 (GOET)
Eckstein 4632 (GOET)
Williams & Cain, s.n. (GOET)
Schofield, 106589 (UBC)
Worthington, 32690 (GOET)
Holmberg & Darigo, 22 GOET)
Schofield et al., 114561 (UBC)
Schofield, 77156 (GOET)
Schofield, 112212 (UBC)
Frahm et al., 978 (GOET)
Inoue, Bryophyta Selecta Exsiccata
570 (JE)
Churchill et al., 22570 (GOET)
Schäfer-Verwimp et al., 24401
(GOET)
Inoue, Bryophyta Selecta Exsiccata
920 (JE)
Müller, Z 620 (GOET)
Deguchi, Bryophytes of Asia 4, 97
(GOET)
Arts, RSA 21/20 (JE)
Churchill et al., 23302 (GOET)
Churchill et al., 22662 (GOET)
Zhu, 20060122-1, (GOET)
Mori, 678 (HIRO)
Bakalin, Hepaticae Rossicae
Exsiccatae 43 (GOET)
Huneck, s.n. (JE)
GenBank accession
rbcL
trnL–F
nrITS
EF545301
EF545299
EF545298
EF545300
EF545334
EF545335
EF545395
EF545394
EF545393
EF545396
EF545429
EF545430
EF545488
EF545486
EF545485
EF545487
EF545521
EF545522
EF545336
EF545337
EF545315
EF545293
EF545294
EF545338
EF545339
EF545341
EF545351
EF545350
EF545349
EF545431
EF545432
EF545410
EF545388
EF545389
EF545433
EF545434
EF545438
EF545446
EF545445
EF545444
EF545523
EF545524
EF545502
EF545480
EF545481
EF545525
EF545526
EF545528
EF545538
EF545537
EF545536
EF545306
EF545307
EF545401
EF545402
EF545493
EF545494
EF545305
EF545282
EF545289
EF545290
EF545291
EF545296
EF545297
EF545292
EF545295
EF547189
EF545347
EF545348
EF545329
EF545357
EF545400
EF545377
EF545384
EF545385
EF545386
—
EF545391
EF545387
EF545390
EF545392
EF545442
EF545443
EF545424
EF545452
EF545492
EF545469
EF545476
EF545477
EF545478
EF545483
EF545484
EF545479
EF545482
—
EF545534
EF545535
EF545516
EF545544
EF545331
EF545332
EF545426
EF545427
EF545518
EF545519
EF545345
EF545441
EF545532
EF545324
EF545316
EF545419
EF545411
EF545511
EF545503
EF545303
EF545302
EF545304
EF545313
EF545359
EF545361
EF545398
EF545397
EF545399
EF545408
EF545454
EF545456
EF545490
EF545489
EF545491
EF545500
EF545546
EF545548
EF545360
EF545455
EF545547
Herbarium acronyms follow Holmgren et al. (1990). Sequences marked with an asterisk (*) were not included in combined analyses (for details see Section 4).
random-addition replicates. The number of rearrangements was restricted to 10 millions per replicate. Where
more than one most parsimonious tree was found, trees
were summarised in a strict consensus tree. For the BS,
clades were considered robust or strongly supported if
BS P 90%, moderately supported if <90% and P70%,
poorly supported if <70% and P50%, and unsupported
if BS < 50% (Pedersen et al., 2006). In a first step the
three partitions were analysed separately to check for
incongruence. Based on Mason-Gamer and Kellog
J. Hentschel et al. / Molecular Phylogenetics and Evolution 45 (2007) 693–705
(1996) incongruencent accessions were identified by
inspecting bootstrap scores above 70%. Incongruent
accessions were removed from the dataset partitions. Subsequently the partitions were combined.
Modeltest version 3.7 (Posada and Crandall, 1998) was
used to select a model of evolution for the ML analyses,
performing a hierarchical likelihood ratio test (hLRT)
and the Akaike information criterion (AIC). For the ML
analyses of both datasets the TrN model (Tamura and
Nei, 1993) was chosen with proportion of invariable characters (I) and among-site rate heterogeneity modelled as
discrete gamma distribution with four rate categories,
and its estimated parameters (C). The analysis was performed as heuristic search using ten random-sequence
addition replicates, MULTrees option on, collapse zero
length branches off, and TBR branch swapping. The confidence of branching was assessed using 400 non-parametric
bootstrap resamplings generated as heuristic searches using
a neighbor-joining tree as starting tree. The number of
rearrangements per resampling was restricted to 2000.
Bayesian inferences of phylogeny were conducted on
dataset II using a general time reversible model (GTR) as
implemented in MrBayes version 3.1.2 (Huelsenbeck and
Ronquist, 2001). Bayesian runs were performed with two
model schemes: a single model for the whole alignment
and two models with a partitioning according to chloroplast regions (rbcL, trnL-F) and nuclear region (ITS). All
searches were conducted using four simultaneous Markov
chains over ten million generations and sampling every
100th generation. The software tool Tracer version 1.3
(Rambault and Drummond, 2003) was used to examine
the parameters and determine the number of trees needed
to reach stationarity (burn-in) for each run. Bayesian posterior probability confidence values (BPP) were generated
from trees found after this initial burn-in period.
The Bayes factor (B10) was calculated to test if the heterogeneous models were a better fit to the data than was
the homogeneous model by taking the ratio of harmonic
means of the three model likelihoods (Nylander et al.,
2004). Harmonic means of the log-likelihoods were calculated using MrBayes version 3.1.2 (Huelsenbeck and Ronquist, 2001). The log-likelihoods of all models were
compared with each other. Model II was considered a better fit to the data than was the model I if 2loge(B10) > 10
(Kass and Raftery, 1995).
3. Results
3.1. Dataset I
Non-parametric bootstrap analyses under the MP criterion (data not shown) were compared to check for phylogenetic differences between the three marker regions. Since
none were found the datasets were combined. The alignment of 42 ingroup and eight outgroup taxa resulted in a
single most likely tree ( ln L = 14,445.061; Fig. 1) in the
697
ML analysis using the TrN+I+C model of sequence evolution selected by the hLRT and AIC.
The analysis of a selection of Porella species and Ascidiota blepharophylla using a range of genera of closely
related families as outgroups, resulted in a monophyletic
Porellaceae. Long branches separate the outgroup taxa
from Porellaceae. Porellaceae split into two robust main
clades (ML BS = 100), one includes Ascidiota, the other
all investigated species of Porella including Porella [Macvicaria] ulophylla. Ascidiota and Porella are separated from
each other by long branches.
The Porella clade is split into two main subclades, one
receiving moderate bootstrap support (ML BS = 85) and
the other forming an unsupported grade with P. fauriei
(Steph.) S.Hatt. and P. japonica (Sande Lac.) Mitt. in serial
sister relationships to the remainder of the clade. Both
subclades contain a suite of species from throughout the
range of the genus.
3.2. Dataset II
As for dataset I, non-parametric bootstrap analyses
under the MP criterion from each of the three marker
regions were checked for incongruence. Indication for
putative incongruence with BS P 70% was detected
between both chloroplast partitions and the nuclear ITS
region in a clade comprising several accessions of Porella
densifolia (Steph.) S.Hatt., P. densifolia var. appendiculata
(Steph.) S.Hatt., P. oblongifolia S.Hatt., and P. stephaniana
(C.Massal.) S.Hatt. (Fig. 2). For this reason, sequences of
P. densifolia (II) from Japan and P. densifolia var. appendiculata were excluded from subsequent phylogenetic analyses. The incongruence affected only closely related taxa of
one monophyletic lineage and not the global phylogeny of
Porellaceae.
The maximum parsimony analysis resulted in 45,337
equally parsimonious trees of 1547 steps, consistency index
(CI) 0.5036, and retention index (RI) 0.7898. Of a total of
2720 investigated character sites 2124 characters were constant, 120 autapomorphic and 476 parsimony informative
(rbcL 1324 characters, 814 constant, 41 variable but
parsimony uninformative, 469 parsimony informative;
trnL-F 550-435-30-85; nrITS 846-494-63-289). The strict
consensus tree is presented in Fig. 3. Two most likely trees
( ln L = 12,721.816) were found in the ML analysis using
the TrN+I+C model of sequence evolution selected by
the hLRT and AIC. Since the two topologies were congruent, differing only slightly in branch lengths, only one
topology is shown (Fig. 4). The Bayes factor (2loge(B10))
of the harmonic means of the log-likelihoods obtained
from the Bayesian inference suggested that the independent
heterogeneous model ( ln L = 12,769.985) assigned to the
chloroplast (rbcL, trnL-F) and nuclear partition (ITS)
was a significantly better fit (2loge(B10) > 10) to the data
than the homogeneous model ( ln L = 12,867.572;
(2loge(B10) = 195.174). The resulting topologies were identical and similar to the ML and MP trees. They will not be
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J. Hentschel et al. / Molecular Phylogenetics and Evolution 45 (2007) 693–705
Fig. 1. A single most likely phylogram ( ln L = 14,445.061) resulting from maximum likelihood analysis of combined molecular dataset I. BS P 50% is
indicated at branches. Gackstroemia, Lepidolaena, and Goebeliella designated as outgroups.
discussed further but Posterior probabilities (BPP P 0.95)
based on the heterogeneous two model inference are highlighted in the ML tree (Fig. 4).
The MP and ML analyses reveal similar topologies
and divide Porella into two main lineages, although the
MP analysis resolves P. cranfordii Steph. in an unsupported sister relationship to the remainder of Porella.
One main lineage is moderately supported in the BS
analyses (BS MP = 71, ML = 87) and achieves significant
Bayesian support. The other main lineage is unsupported. Whereas the branching within several subclades
is largely resolved, the phylogenetic relationships between
them are mostly unresolved or poorly supported in the
BS analyses. Several sister relationships with at best
moderate BS support achieve significant Bayesian clade
credibility values.
One clade within the moderately bootstrap supported
main lineage contains a strongly supported clade (MP
BS = 98, ML BS = 99) with accessions predominantly
from Asia. Within this clade different accessions of
P. caespitans s.l. are nested in a polyphyletic lineage with
P. ulophylla (Steph.) S.Hatt. [=Macvicaria], P. madagascariensis (Nees & Mont.) Trevis., P. chinensis (Steph.) S.Hatt.,
and P. subobtusa (Steph.) S.Hatt. Another well-supported
clade (MP BS = 97, ML BS = 94) consists of the holarctic
species P. platyphylla, P. cordaeana, and P. navicularis
(Lehm. & Lindenb.) Pfeiff. The accessions of P. platyphylla
split into a European and a North American clade with one
J. Hentschel et al. / Molecular Phylogenetics and Evolution 45 (2007) 693–705
699
Fig. 2. Clade with incongruence evident from chloroplast and nuclear partitions of dataset II. Incongruent accessions have been removed from partitions
of dataset II before combination.
Fig. 3. Rooted strict consensus of 45,337 equally parsimonious trees recovered during heuristic searches of combined molecular dataset II. Subgenus and
section affiliation of Porella species according to Schuster (1980).
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J. Hentschel et al. / Molecular Phylogenetics and Evolution 45 (2007) 693–705
Fig. 4. One of two most likely phylograms ( ln L = 12,721.816) resulting from maximum likelihood analysis of combined dataset II. BS P 50% is
indicated at branches. Support (P0.95) from Bayesian searches is indicated by thickened branches.
accession from North America embedded within the European samples. The relationships between these four subclades are largely unsupported, however, the monophyly
of P. platypylla achieves a MP BS of 73. Another example
for a geographical split between several accessions of one
taxon is the robust P. arboris-vitae (With.) Grolle clade
with a sister relationship of accessions from Macaronesia
and Europe.
One accession of P. swartziana (F.Weber) Trevis. from
South Africa is nested in a robust lineage that contains
two other accessions of P. swartziana from Bolivia. All
other African accessions are resolved in a separate wellsupported lineage. Three lineages containing members of
the P. pinnata–swartziana complex form a grade with poor
bootstrap support. Porella inaequalis Perss. is placed in a
moderately (MP BS = 81) or strongly supported (ML
BS = 91) sister relationship to P. grandiloba Lindb. Porella
pinnata L. is resolved sister to three accessions of P. chilensis (Lehm. & Lindenb.) Trevis. from Argentina (MP
BS = 98, ML BS = 99).
The molecular backbone of the second main clade is largely unresolved and relationships between several subclades as well as the positions of P. fauriei, P. japonica,
and P. cranfordii are uncertain. Several of the taxa sampled
with more than one accession are not monophyletic. Porella spinulosa (Steph.) S.Hatt. is resolved within a robust
J. Hentschel et al. / Molecular Phylogenetics and Evolution 45 (2007) 693–705
lineage of P. gracillima Mitt. accessions. Porella obtusata
(Taylor) Trevis. and P. canariensis (F.Weber) Underw.
form a monophyletic lineage with strong bootstrap
support; both taxa cannot be separated on the basis of
the presented molecular sequence data. Close relationships
between the P. obtusata/canariensis-clade and accessions of
P. roellii Steph. are poorly (MP BS = 59) or moderately
(ML BS = 71) supported. Porella stephaniana and
P. oblongifolia are nested within a strongly supported
(MP and ML BS = 98) clade with several accessions of
P. densifolia s.l.
4. Discussion
4.1. Supraspecific classification concepts and the positions of
Porella [Macvicaria] ulophylla and Ascidiota
blepharophylla
The phylogenetic analyses (Fig. 1) strongly support the
separation of the Asian–North American Ascidiota from
Porella at the genus level, which is also obvious from morphology. Ascidiota is well separated from Porella by the
presence of well-developed water-sacs originating by
infolding of the margins of lobe and lobule bases, and by
having no or very weakly developed cilia on leaf and
underleaf margins (Schuster, 1980). Our study also shows
that the Asian Macvicaria ulophylla is nested in Porella,
in a robust subclade together with P. caespitans s.l.,
P. madagascariensis, P. chinensis and P. subobtusa. Previously, Macvicaria was treated as a monospecific genus
(Hattori, 1978; Bai, 2000), as a subgenus of Porella (Inoue,
1976; Schuster, 1980) or as a section of Madotheca
Dumort. (=Porella) (Schiffner, 1934). Porella ulophylla differs from all other Porellaceae by the inflated perianths and
the often crisped-undulated leaves.
Schuster (1980) proposed the subgenus Protoporella represented solely by Porella fauriei, based on the broad connection of leaf lobule and stem, with the insertion line
pointing towards stem apex, and the long, more or less convex leaf keel in the latter species. In our molecular topologies, however, deeper nodes are poorly resolved and sister
relationships of P. fauriei to other sampled taxa remain
unclear.
Based on leaf and perianth shape as well as distribution
of paraphyllia, Schuster (1980) accepted several sections of
Porella. He erected P. sect. Platyphyllae R.M.Schust. to
include several holarctic and temperate Asian taxa with
decurved-involute, decurrent lobes, and lobules with a Jshaped lobule insertion such as P. platyphylla, P. arborisvitae, and P. obtusata. However, species assigned to P. sect.
Platyphyllae are placed in both main lineages of Porella
(Fig. 3). Schuster (1980) furthermore proposed a section
Paraphyllae R.M.Schust. for neotropical P. squamulifera
(Taylor) Trevis., P. brachiata (Taylor) Spruce, and P. leiboldii (Lehm. & Lindenb.) Trevis., species that frequently
produce stem paraphyllia. The recently described P. saccata M.L.So (So, 2005) also belongs to this complex. These
701
species form a robust monophylum, however, P. crispata
(Hook.) Trevis. that lacks paraphyllia (Swails, 1970) is
nested in the sect. Paraphyllae clade. Porella sect. Porella
is characterised by the flattened, narrowly lingulate lobules
which are not decurrent below the level of the keel, narrow,
flat underleaves, and narrowly ovate to oblong dorsal
lobes. Schuster (1980) included the P. pinnata-swartziana
complex, as well as P. oblongifolia, P. japonica, and P. madagascariensis. Porella nitens (Steph.) S.Hatt. and P. grandiloba were regarded as being related to the P. pinnata–
swartziana complex (Schuster, 1980; Persson, 1955). These
species are placed in several lineages, hence Porella sect.
Porella is polyphyletic. A well-supported subclade in the
presented phylogenies is an assemblage of Asian species
of which the majority are currently assigned to the sections
Acutifoliae R.M.Schust. or Obtusilobae R.M.Schust. The
subclade contains accessions of P. acutifolia, P. campylophylla, P. perrottetiana, and the P. caespitans complex.
The taxa are mainly characterised by acute, pilose, or
rarely subacute lobes, and obliquely patent lobules and
have been regarded closely related based on morphological
similarities (Hattori, 1976, 1979; Shaheen and Srivastava,
1989). Section Acutifoliae is polyphyletic because P. madagascariensis and P. chinensis are currently assigned to sections Porella or Platyphyllae, respectively, and P. ulophylla
to subgenus Macvicaria.
Schuster (1980) stated that his classification is a preliminary one that may be helpful for the separation of
morphologically closely related groups. However, all sections represented by multiple accessions in our study
were either paraphyletic or polyphyletic (Fig. 3), indicating that the distinguishing morphological characters
must be re-evaluated. We assume that characters of
the Porella gametophyte are not suitable to define
monophyletic entities above species level as the result
of putatively rapid transformation of these characters
in response to environmental stresses. It would be a
worthwhile undertaking to investigate sporophyte variation in Porella, however, present knowledge is insufficient for an evaluation of these characters. Problems
in defining supraspecific entities in larger genera were
reported for other leafy liverworts such as Plagiochila
(Dumort.) Dumort., whose sections can at best be
defined by a combination of gametophytical and sporophytical characters, as well as geographical distribution
(Heinrichs et al., 2005, 2006).
4.2. Implications for species taxonomy
Molecular data may allow for a test of the monophyly of
morphological species concepts. In mosses, several recent
studies revealed rampant parallel morphological evolution
and many species were resolved as polyphyletic (Shaw and
Allen, 2000; Werner and Guerra, 2004; Stech and Wagner,
2005; Vanderpoorten and Goffinet, 2006). In liverworts
such high levels of incongruence have not yet been found
(Heinrichs et al., 2005; Schaumann et al., 2005; Hartmann
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J. Hentschel et al. / Molecular Phylogenetics and Evolution 45 (2007) 693–705
et al., 2006; Hentschel et al., 2006b) and Porella does not
seem to make an exception. Sequencing of multiple accessions of morphologically defined species of Porella usually
resulted in robust monophyletic lineages. Morphological
characters appear well suited to discriminate species in
Porella. However, in some cases our study indicates a need
for modification of current species circumscriptions. Additionally, some novel relationships are uncovered which
allow for resolving controversial treatments based on
morphology.
For example, the synonymy of Neotropical P. swartziana and African P. capensis, advocated by Gottsche et al.
(1844–1847) and Swails (1970) but opposed by Jones
(1963), is not supported by our molecular topologies. The
molecular data also show that P. pinnata and P. inaequalis,
considered closely related by Persson (1955) based on morphological evidence but distantly so by Bischler et al.
(2006) using isozyme data besides morphology, are members of different clades and obviously not closely related.
The Asiatic P. spinulosa, P. cf. grollei S.Hatt. and P. vernicosa Lindb. are nested within the P. arboris-vitae complex.
Porella spinulosa was earlier described as forma spinulosa
(Steph.) S.Hatt. of P. vernicosa (Hattori, 1970). The molecular topologies indicate a close relationship of both taxa
but clearly support species rank. Porella vernicosa also
resembles P. fauriei of the monospecific subgenus Protoporella. Several authors regarded P. fauriei as a subspecies or
variety of. P. vernicosa (e.g., Stotler and Crandall-Stotler,
1977). Porella vernicosa is placed sister to P. arboris-vitae
(Figs. 1, 3, and 4) and hence the separation at the species
level is confirmed.
The poorly supported P. arboris-vitae complex is otherwise made up by P. obtusata, P. canariensis, P. roellii and
P. gracillima. Porella canariensis has often been considered
to be conspecific with P. arboris-vitae. In both species the
lobules and underleaves are usually dentate or spinose-ciliate, whereas in P. obtusata the margins are almost entire.
However, a conspecifity of P. canariensis and P. arborisvitae is not supported in our analysis due to the sister relationship of P. roellii and the P. obtusata/canariensis clade.
Within the P. arboris-vitae complex, the sequence similarities of the accessions of P. obtusata and P. canariensis are
remarkable. Recent studies using isozymes or RAPDs
(Random Amplified Polymorphic DNAs) (BoisselierDubayle and Bischler, 1994; Freitas and Brehm, 2001; Bischler et al., 2006) suggested high levels of intraspecific and
interspecific polymorphisms of these taxa. These polymorphisms are not confirmed in our study. Porella obtusata
and P. canariensis cannot be separated on the basis of
the molecular sequence data presented in this study. Boisselier-Dubayle and Bischler (1994) and Bischler et al.
(2006) also showed that P. obtusata and P. canariensis
are morphologically less clearly separated than previously
believed. Our sequence data do not support the separation
of P. obtusata and P. canariensis. Based on the morphological overlap of both taxa (Bischler et al., 2006) and the
extensive sequence similarities we suggest that the taxa
are combined under the name P. obtusata and separated
at the variety level.
Our chloroplast and nuclear datasets are largely congruent and do not provide evidence for frequent hybridisation
in Porella. Putative incongruence has been detected
between the nuclear and chloroplast partitions of a clade
that includes three Porella densifolia accessions, P. macroloba (Steph.) S.Hatt. & Inoue, P. nitens, and members of
the P. arboris-vitae complex (Fig. 2). For this reason,
sequences of P. densifolia (II) from Japan and P. densifolia
var. appendiculata were excluded from phylogenetic analyses of the combined datasets. These data point towards the
need of a broader species concept in this clade rather than
to hybridisation. The putative incongruence may indicate
intraspecific recombination and not interspecific introgression. Hattori (1970) based the separation of P. densifolia,
P. stephaniana and P. oblongifolia on the shape of the
lobes, lobules and underleaves, and their dentition. It is
already known that these characters are often unreliable
(Boisselier-Dubayle and Bischler, 1994; Bischler et al.,
2006).
Although the molecular data seem to support the broad
species concept in Porella advocated by So (2002, 2005)
some results indicate putative speciation processes that
were overlooked until now. Accessions of the widespread
holarctic P. platyphylla [including P. platyphylloidea
(Schwein.) Lindb., Therrien et al. (1998)] split into a European and a North American clade with one accession from
North America embedded within the European samples
(Figs. 3 and 4). The relatively long branches leading to
the tips (Fig. 4) are conspicuous when compared to that
of other Porella species represented by multiple accessions.
High levels of isozyme or RAPDs variation have been
reported for P. canariensis, P. obtusata, P. arboris-vitae,
and P. platyphylla (Therrien et al., 1998; Freitas and Brehm, 2001; Wyatt et al., 2005; Bischler et al., 2006) but this
variation is reflected in notable sequence divergence leading
to long branches only in the last species (Fig. 4). Genetic
differentiation without or with minute morphological differentiation has sometimes been interpreted as cryptic speciation (e.g., Odrzykoski and Szweykowski, 1991;
Fiedorow et al., 2001; McDaniel and Shaw, 2003). It would
be a worthwhile undertaking to extend the accession set of
P. platyphylla and search for correlation of morphological
characters and the observed tree topologies. Since P. platyphylloidea and P. platyphylla have been separated in terms
of elater characters (Schuster, 1980), special attention
should be drawn to populations with sporophytes.
4.3. Implications for biogeography and extensions of range
Several case studies in bryophytes demonstrated morphological homoplasy above species level that is also evident in Porella (see Section 4.1). These studies suggested
a correlation between genetic variation and the geographical distribution of the related accessions rather than
between genetic variation and morphology (Shaw and
J. Hentschel et al. / Molecular Phylogenetics and Evolution 45 (2007) 693–705
Allen, 2000; Stech and Dohrmann, 2004; Grundmann
et al., 2006; Hartmann et al., 2006). This pattern is also
seen in Porella with several supraspecific Neotropical, African, Asian, American or Holarctic clades.
The molecular topologies support several disjunct species ranges. Extensive sequence similarities of Alaskan
and European P. cordaeana as well as of one North American and several European P. platyphylla point to ongoing
or rather recent intercontinental gene flow. On the other
hand the P. arboris-vitae clade shows clear structure and
a subdivision in a Macaronesian subclade and a subclade
from Continental Europe and the British Isles. A similar
intraspecific variation related to a geographical rather than
a morphological pattern has already been demonstrated for
the liverwort genera Bryopteris (Nees) Lindb. (Hartmann
et al., 2006), Herbertus Gray (Feldberg et al., 2007) and
Plagiochila (Heinrichs et al., 2005).
The Asian P. gracillima [subsp. urogea (C.Massal.)
S.Hatt. & M.X.Zhang] has been observed in a single North
American locality within Great Smoky Mountains
National Park for almost 20 years, and has already been
correctly identified based on morphology by the late S.Hattori. The isolated occurrence of P. gracillima in Eastern
North America could be the result of a quite recent range
extension, as indicated by the extensive sequence similarities of the North American and Asian accessions.
The common neotropical species P. swartziana is here
newly reported for Africa based on a specimen from South
Africa. Recent molecular studies have already lent support
to similar disjunct African-Neotropical species ranges
within the leafy liverwort genera Herbertus (Feldberg
et al., 2007), Tylimanthus Mitt. (Stech et al., 2006), and Plagiochila (Heinrichs et al., 2005). The extensive sequence
similarities of the related neotropical and African accessions add to the growing evidence that many Afro-American ranges could be the result of long distance dispersal
(Heinrichs et al., 2005; Schaumann et al., 2005; Hartmann
et al., 2006).
Heinrichs et al. (2006) and Vanderpoorten and Long
(2006) provided molecular evidence for close relationships
of the tropical American and Macaronesian liverwort floras. However, the Macaronesian endemic Porella inaequalis
is closely related to the Asian P. grandiloba rather than to
Neotropical species. Other Porella species occurring in
Macaronesia (P. arboris-vitae, P. cordaeana, P. pinnata,
P. platyphylla, P. obtusata/canariensis) are further on distributed in the Holarctic or Asia. A close relationship of
these species and Neotropical ones is not observed based
on the current data set.
4.4. Porellaceae in a phylogenetic context, and resolution
within the family
Lepidolaenaceae and Goebeliellaceae have been
resolved as closest relatives of Porellaceae in recent molecular studies (Forrest et al., 2006; He-Nygrén et al., 2006;
Heinrichs et al., 2007). Extension of the Porellaceae sam-
703
pling of Forrest et al. (2006) in the present study confirms
the sister relationship of Ascidiota and Porella. The phylogram resulting from the ML analysis of dataset I shows
long branches, and hence numerous mutations, separating
Ascidiota and Porella. This pattern possibly indicates a
long period of separation of these two lineages.
Currently no other taxa beside Ascidiota are known that
may serve as outgroup for Porella and could shorten the
remarkably long branch leading to Porella. Porellales possibly originated in the Early Permian whereas extant species of Porella branch off in the Tertiary (Heinrichs et al.,
2007). A relatively recent origin of extant species of Porella
would be in accordance with the limited resolution and relatively short branches of the Porella crown group (Figs. 1
and 4). The split of Ascidiota and Porella, however, must
date back at least to the Early Tertiary as indicated by
an Eocene Baltic amber fossil described as the extinct P.
subgrandiloba Grolle & M.L.So (Grolle and So, 2004).
No conflicting phylogenetic signals between the investigated molecular markers affected the Porella backbone.
Thus, possibly a series of initial diversification occurred
nearly simultaneously in Porella resulting in subsequent
weak phylogenetic signals. Similar scenarios have been postulated for certain groups of ferns (Schneider et al., 2004),
angiosperms (Fishbein et al., 2001; Pennington et al., 2004;
Hörandl et al., 2005; Pellmyr et al., 2007) and animals (e.g.,
Waits et al., 1999; Poe and Chubb, 2004; Mendelson and
Shaw, 2005).
Acknowledgments
We thank Jan Eckstein (Göttingen), Steffen Caspari (St.
Wendel), Matt von Konrat (Chicago), Támas Pócs (Eger),
and Alfons Schäfer-Verwimp (Herdwangen-Schönach) as
well as the curators and directors of Herbarium Haussknecht, Jena (JE), Bolus Herbarium, Cape Town (BOL),
Field Museum, Chicago (F), University of British Columbia Herbarium, Vancouver (UBC), Hiroshima Museum
Herbarium (HIRO), and Alaska Museum of the North,
Fairbanks (ALA) for the loan of specimens and the permission to extract DNA. Thanks are also due to the curators
of the herbaria of the Far Eastern Branch of the Russian
Academy of Sciences, Vladivostok (VLA), Duke University, Durham (DUKE) and New York Botanical Garden
(NY) for their search of Ascidiota specimens and to Alain
Vanderpoorten (Liège) for comments on the manuscript.
The late Sinske Hattori kindly identified Porella gracillima
subsp. urogea new to USA. Financial support from the
German Research Foundation (DFG Grant HE 3584/2
to J.H., S.R.G., and H.S.) and the National Natural Science Foundation of China (No. 30470142 to R.L.Z.) is
gratefully acknowledged.
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