Cladistics
Cladistics 20 (2004) 151–183
www.blackwell-synergy.com
Phylogeny of the Brachytheciaceae (Bryophyta) based
on morphology and sequence level data
Sanna Huttunen* and Michael S. Ignatov
*Botanical Museum and Division of Systematic Biology, PO Box 7 and 65, FIN-00014 University of Helsinki, Finland
Main Botanical Garden of Russian Academy of Sciences, Botanicheskaya 4, Moscow 127276, Russia
Accepted 3 March 2004
Abstract
Brachytheciaceae is often considered a taxonomically difficult group of mosses. For example, morphological variation has led
to difficulty in generic delimitation. We used DNA sequence data (chloroplast psbT-H and trnL-F and nuclear ITS2) together
with morphology (63 characters) to examine the relationships within this family. The combined unaligned length of the DNA
sequences used in the phylogenetic analyses varied between 1277 and 1343 bp. For phylogeny reconstruction we performed
direct optimization, as implemented in POY. Analyses were performed with three different gap costs and the morphological
data partition was weighted both: (1) equal to gap cost, and (2) with a weight of one. The utility of sensitivity analysis has
recently been cast into doubt; hence in this study it was performed only to explore the effects of weighting on homology
statements and topologies and to enable more detailed comparisons between earlier studies utilizing the direct optimization
method. The wide sequence length variation of non-coding ITS2 sequences resulted in character optimizations (i.e.,
‘‘alignments’’) of very different lengths when various gap costs were applied. Despite this variation, the topologies of equally
parsimonious trees remained fairly stable. The inclusion of several outgroups, instead of only one, was observed to increase the
congruence between data sets and to slightly increase the resolution. An inversion event in the 9 bp loop region in the
chloroplast psbT-N spacer in mosses has been postulated to include only uninformative variation, thus possibly negatively
impacting the phylogeny reconstruction. Despite this inversion, its variation within Brachytheciaceae was clearly congruent with
information from other sources, but inclusion of these 9 bp in the analysis had only a minor effect on the phylogenetic results.
In the most parsimonious topology, which was obtained with equal weighting of all data, Meteoriaceae and Brachytheciaceae
were resolved as monophyletic sister groups, which had recently been suggested based on a few shared morphological
characters. Our study revealed some new generic relationships within the Brachytheciaceae, which are discussed in light of the
morphological characters traditionally used for generic delimitation.
The Willi Hennig Society 2004.
Brachytheciaceae includes about 570 species (Crosby
et al., 1999), and constitutes one of the largest families
of mosses. These species belong to the pleurocarpous
mosses, presumably the youngest group of mosses, and
the family Brachytheciaceae is also possibly still rapidly
evolving (McAdam, 1982). Observations of low
sequence variation among pleurocarpous mosses compared with other bryophyte groups support this idea
(Vanderpoorten et al., 2002), although even rough age
estimates are very difficult to give due to the extremely
scattered fossil data. Among the pleurocarpous mosses,
Brachytheciaceae has been suggested to be phylogenetically close to Amblystegiaceae, Leskeaceae, and Thu The Willi Hennig Society 2004
idiaceae (Robinson, 1962) or to Hylocomiaceae and
Ctenidiaceae (Hedenäs, 1989). According to the latest
phylogenetic studies of pleurocarpous mosses, Brachytheciaceae belongs to the order Hypnales (Buck and
Goffinet, 2000), and based on morphological and
molecular data it is closely related to Meteoriaceae
(Buck, 1998; Ignatov, 1999; Buck et al., 2000a; Quandt
et al., 2004b; Huttunen et al., 2004). Phylogenetic analyses by Buck et al. (2000a) of the chloroplast trnL-F
region and rps4 gene, and Tsubota et al.’s (2002) work
on the rbcL gene suggest another family, Plagiotheciaceae (in the s. str., including only Plagiothecium
Schimp.), is very closely related to Brachytheciaceae.
152
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
Despite the many difficulties encountered in the
delimitation of Brachytheciaceae (for details see Ignatov
and Huttunen, 2002), the family is now considered to be
one of the best-defined pleurocarp families (Hedenäs,
1989, 1992; Ignatov, 1998; Ignatov, 1999). Characters
separating it from other pleurocarpous mosses mostly
include those that have been neglected by previous
authors, such as the shape of the stomatal pore,
perichaetial leaf patterns, and the arrangement of
pseudoparaphyllia around the young branch primordia.
Generic delimitation, however, remains very unclear.
The latest worldwide revision of Brachytheciaceae was
undertaken by Brotherus (1925), who borrowed the
concept of the three largest genera of the family from
Bruch et al. (1851–1855). The genera Brachythecium
Schimp., Rhynchostegiella (Schimp.) Limpr., Rhynchostegium Schimp., and Eurhynchium Schimp. are assumed
to be non-monophyletic (Hedenäs, 1992; Ignatov et al.,
1999; many others). This conclusion was, however, only
based on morphological data, and their monophyly has
not been tested from a phylogenetic perspective. Only
one attempt was made to change the classification of
Brachythecium, in which Robinson (1962) split it into
two parts of almost equal size, Brachythecium s. str. and
Chamberlainia Grout. This idea did not receive wide
acceptance and Robinson (1987) himself later denied it.
At the same time almost all revisions of Brachythecium
accepted 5–8 sections introduced by Kindberg (1897),
then used by Brotherus (1905–1909) and most subsequent authors, with relatively minor corrections and
alternations (Takaki, 1955a,b, 1956; Robinson, 1962;
McFarland, 1994; Ignatov, 1998). Robinson (1987) also
attempted to redefine Rhynchostegium by placing tropical species in the genus Steerecleus H. Robinson, a view
not accepted in recent studies (McFarland, 1994; Buck,
1998; Ignatov et al., 1999; Hedenäs, 2002). Many
authors in the early 20th century followed Warnstorf
(1905), who raised the rank of Oxyrrhynchium (Schimp.)
Warnst. (as a group of species centered around Eurhynchium hians (Hedw.) Sande Lac.) to generic level.
However, the generic status of Oxyrrhynchium was not
accepted in the latter half of the 20th century.
The aim of the present study was to determine whether
molecular data can provide further resolution and help in
clarifying the separation of Brachytheciaceae from Meteoriaceae, as well as providing evidence for the generic
classification of Brachytheciaceae. Only a limited attempt
has been made to analyze Brachytheciaceae phylogenetically before this study (Stech and Frahm, 1999), and the
current classification of the family is mainly based on a
rather intuitive view of the relationships among the taxa.
We believe that our study provides a new, or at least a
better-founded hypothesis of phylogenetic relationships,
which may lead to a more natural classification of the
family. Due to the considerable morphological variation,
and presumably their homoplastic evolution, cladistic
analyses based only on morphological data have been
suspected of providing only limited information on
phylogenetic relationships. We use nuclear and chloroplast molecular data, in addition to morphological
evidence. Genomic regions sequenced for this study
include the nuclear Internal Transcripted Spacer 2
(ITS2), the chloroplast psbT-H gene complex, including
psbT, psbN and psbH genes coding small photosystem II
proteins, and the non-coding spacers between them, and
the trnL-F region, consisting of partial ribosome coding
tRNA-Leu gene and the non-coding spacer preceding it.
Since the majority of sequence level data in this study
originated from non-coding genomic regions, we use
direct optimization alignment (Wheeler, 1996) as implemented in POY (Gladstein and Wheeler, 2001). While the
phylogenetic information from insertion and deletion
events (‘‘gaps’’) have traditionally been neglected by
treating them as missing data, the direct optimization
method utilizes this variation in the same way as
information from all other types of substitution events.
Here we attempt to evaluate the effect of indel events on
the resulting hypothesis of relationships within the study
group. The value of information from gaps and how they
should be treated in phylogenetic analyses has been the
focus of several papers (for example, Giribet and Wheeler, 1999; Simmons and Ochoterena, 2000; Bapteste and
Philippe, 2002; Kawakita et al., 2003). In our study, we
attempt to determine if this kind of exploration affects the
hypotheses of phylogenetic relationships. We also examine the effect of differential treatment of short inversion
regions on the hypothesis of relationships. Similar
inversions are expected to be common in plant chloroplast genomes (Kelchner and Wendel, 1996; Graham
et al., 2000), and recent studies (Kelchner and Wendel,
1996; Quandt et al., 2003) have suggested that these
regions may affect phylogeny reconstruction by increasing homoplasy within the data and adding extra weight
for the inversion event in analyses. Although we agree
that differential weighting does not lead to the most
parsimonious results (see Frost et al., 2001; Grant and
Kluge, 2003), we performed the sensitivity analyses (sensu
Wheeler, 1995) in order to be able to compare the results
favored by this method to those obtained from the equal
weighting of all characters. We also aimed to explore the
contribution of different data partitions on the hypothesis of relationships when differential weighting of gaps
and morphology are used in direct optimization analyses.
Materials and methods
Selection of taxa
We included in our study representatives of all
genera of Brachytheciaceae for which we had recently
collected specimens available (Table 1). In those cases
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
153
Table 1
List of specimens used for sequencing. All specimens are held in the Finnish Museum of Natural History (H) if not indicated otherwise. Of all
specimens in the Main Botanical Garden of Russian Academy of Sciences, Moscow (MHA), duplicates are also available in H
Species
trnL-F
psbT-H
ITS2
Voucher specimen for sequences
AY044072
AF417395
AF395634
AF397780
AF417386
AF403619
AF397802
AF417401
AF403628
Ecuador, Loja Prov., D. H. Norris 92175 & M. Bolivar,
December 18, 1997
China, Hunan Prov., T. Koponen, S. Huttunen &
P.-C. Rao 52753, October 9, 1997
USA, California, D. H. Norris 82503, February 21, 1994
AF397864
AF417442
AF403654
USA, North Carolina, L. E. Anderson 24257, March 3, 1983
AF397875
AF397828
AF417444
AF417352
AF403646
AF403582
Finland, Regio aboënsis, J. Pykälä 6775, September 21, 1990
China, Hunan Prov., T. Koponen, S. Huttunen & P.-C. Rao
51827, October 8, 1997
Brachythecium buchananii (Hook.)
A. Jaeger
Brachythecium collinum (Müll. Hal.)
Schimp.
Brachythecium complanatum Broth.
AF397792
AF417348
AF403595
AY184776
AY184757
AY166440
AY184781
AY184762
AY166444
Brachythecium erythrorrhizon
Schimp. ssp. asiaticum Ignatov
Brachythecium falcatulum (Broth.)
Paris
Brachythecium frigidum (Müll. Hal.) Besch.
Brachythecium geheebii Milde
AY184783
AY184763
AY166446
AF397774
AF417468
AF403662
AF397874
AF397776
AF417456
AF417467
AF403638
AF403660
Brachythecium geheebii Milde
–
AY184770
AF503533
Brachythecium glaerosum
(Spruce) Schimp.
Brachythecium glaciale Schimp.
Brachythecium hylotapetum
B. L. Higinb. & N. L. Higinb.
Brachythecium koponenii Ignatov
AF397865
AF417457
AF403656
–
AF397871
AY184766
AF417445
AY166448
AF403653
AF397872
AF417446
AF403657
AY184782
AF397806
–
AF417394
AY166445
AF403584
AY184786
AY184767
AF503534
Brachythecium mildeanum (Schimp.)
Schimp.
Brachythecium plumosum (Hedw.) Schimp
AY184777
AY184758
AY166441
AF397814
AF417359
AF403586
Brachythecium populeum (Hedw.) Schimp.
AF397873
AF417458
AF403640
Brachythecium oedipodium
(Mitt.) A. Jaeger
Brachythecium ornellanum
(Molendo) Venturi & Bott.
Brachythecium reflexum (Starke) Schimp.
Brachythecium rivulare Schimp.
AF397784
AF417388
AF403649
China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo
& P.-C. Rao 53972, September 30., 1998
Russia, Volgograd Prov., M. Ignatov, August 7,
1999 (MHA)
Russian Far East, Upper Bureya River, M. Ignatov 97–172,
August 28, 1997 (MHA)
Russian Far East, Upper Bureya River, l M. Ignatov
97–1120, August 25, 1997 (MHA)
Russia, Altai Mts., M. Ignatov #0 ⁄ 1680, June 26, 1989
(MHA)
USA, California, Duell, April 23., 1981
Russia, Bashkiria, E. Ignatova #15 ⁄ 68, September 13, 1990
(MHA)
Poland, Sudetes Mts., R. J. Wojcicki, August 26, 1986
(Ochyra & Bednarek-Ochyra, Musci Poloniae exs. 983)
Ukraina, Lvov Prov., M. Ignatov, September 13,
1991 (MHA)
Russia, Caucasus, E. Ignatova, August 8, 1986 (MHA)
Canada, British Colombia, R. Love & J. Rogers B27688,
June 15, 1981
Papua New Guinea, Morobe Prov., T. Koponen 32122,
July 6, 1981
USA, Oklahoma, B. Allen 6208, April 23, 1988
Papua New Guinea, Morobe Prov., T. Koponen 28748,
May 20, 1981
Russia, Kola Peninsula, Khibiny Mts., M. Ignatov, July 31,
1998 (MHA)
Russia, Central European part, Kursk Prov., M. Ignatov,
May 20, 1999 (MHA)
China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo &
P.-C. Rao 56777, October 14, 1998
Finland, Regio aboënsis, J. Pykälä 7029, September 30,
1990
Finland, Tavastia australis, S. Huttunen 1439, April 14, 2000
AF397785
AF417354
AF403593
Russia, Altai Mts, M. Ignatov 16 ⁄ 44, July 18, 1991 (MHA)
AF397858
AY044068
AF417459
AF417471
AF403655
AF395627
Brachythecium rivulare Schimp.
Brachythecium ruderale (Brid.) W. R. Buck
Brachythecium rutabulum (Hedw.) Schimp.
Brachythecium salebrosum
(F. Weber & D. Mohr) Schimp.
Brachythecium salebrosum
(F. Weber & D. Mohr) Schimp.
Brachythecium subalbicans Broth.,
nom illeg.
AF397866
AY184784
AF397867
AY184780
AF417460
AY184764
AF417447
AY184761
AF403651
AF503535
AF403644
AY16643
Finland, Savonia australis, S. Huttunen 1195, May 1997
China, Hunan Prov., T. Koponen, S. Huttunen, S.
Piippo & P.-C. Rao 48671, October 4, 1998
Finland, Ostrobottnia kajanensis, A. Parnela, May 19, 1996
Haiti, W. R. Buck 4826, March 23, 1982
Finland, Nylandia, S. Huttunen 1415, April 16, 2000
Russia, Kursk Prov, M. Ignatov, August 15, 1996 (MHA)
AF397857
AF417448
AF403648
Finland, Tavastia australis, S. Laaka 1835, October 18, 1987
AF397788
AF417373
AF403583
China, Hunan Prov., T. Koponen, S. Huttunen & P.-C. Rao
49112, October 1, 1997
Brachytheciaceae
Aerolindigia capillacea (Hornsch.)
M. Menzel
Aerobryum speciosum
(Dozy & Molk.) Dozy & Molk.
Bestia longipes
(Sull. & Lesq.) Broth.
Brachythecium acuminatum (Hedw.)
Austin
Brachythecium albicans (Hedw.) Schimp.
Brachythecium buchananii (Hook.)
A. Jaeger
Brachythecium laetum (Brid.) Schimp
Brachythecium lamprocarpum
(Müll. Hal.) A.Jaeger
Brachythecium latifolium Kindb.
154
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
Table 1
Continued
Species
trnL-F
Brachythecium turgidum
(Hartm.) Kindb.
Brachythecium udum I. Hagen.
AY184785 AY184765 AY166447
psbT-H
ITS2
Russia, Altai Mts, M. Ignatov 31 ⁄ 291, August 1, 1992 (MHA)
AY184778 AY184759 AF503536
Brachythecium velutinum (Hedw.)
Schimp.
Bryhnia novae-angliae (Sull. & Lesq.)
Grout
Bryhnia novae-angliae (Sull. & Lesq.)
Grout
Bryhnia novae-angliae (Sull. & Lesq.)
Grout
Bryoandersonia illecebra (Hedw.)
H. Robinson
Cirriphyllum cirrosum (Schwägr.) Grout
Cirriphyllum cirrosum (Schwägr.) Grout
AF397832 AF417440 AF403667
Russia, Siberia, Yakutiya, M. Ignatov 00–843, August 19,
2000 (MHA)
Finland, Regio aboënsis, M. Kiirikki, August 23, 1988
AF397843 AF417412 AF403665
AF397826 AF417405 AF403588
China, Hunan Prov., T. Koponen, S. Huttunen &
P.-C. Rao 52900, October 11, 1997
Russia, Kostroma Prov., S. Popov 26, May 11, 1999 (MHA)
AY184779 AY184760 AY166442
Russia, Altai Mts., M. Ignatov 34 ⁄ 227 (MHA)
AF397819 AF417365 AF403626
USA, Arkansas, Bowers 22214, May 24, 1999
AF397856 AF417449 AF403641
AY184793 AY184775 AY447036
China, Xinjiang Prov., B. C. Tan 93–846, July 24, 1993
Russia, Yakutia, M. Ignatov 00–26, August 17,
2000 (MHA)
Russia, Caucasus, M. Ignatov, August 23, 1999 (MHA)
Cirriphyllum crassinervium (Taylor)
AF397868 AF417433 AF403668
Loeske & M. Fleisch.
Cirriphyllum flotowianum (Sendtn.) Ochyra AF397869 AF417461 AF403636
Cirriphyllum piliferum (Hedw.) Grout
Cirriphyllum tommasinii (Boulay) Grout
Clasmatodon parvulus (Hampe) Sull.
Eurhynchiadelphus eustegium (Besch.)
Ignatov & Huttunen
Eurhynchium angustirete (Broth.)
T. J. Kop.
Eurhynchium hians (Hedw.) Sande Lac.
Poland, Krakow-Czestochowa Upland, H. & S. Bednarek,
June 8, 1985
AF397799 AF417403 AF403608 Finland, Regio aboënsis, T. Koponen & S. Huttunen 1324,
May 24, 1999
AF397870 AF417464 AF403639* Poland, Western Carpathians, R. Ochyra, August 26, 1982
AF397813 AF417430 AF403614 USA, Missouri, P. L. Redfearn, Jr. & B. Allen, April 23,
1992
AF397790 AF417378 AF403602 China, Jilin Prov., T. Koponen 36592, September 20, 1981
AF397825 AF417436 AF403621
Russia, Moscow Prov., M. Ignatov, July 3, 1998 (MHA)
AF397815 AF417372 AF403603
China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo &
P.-C. Rao 53740, September 28, 1998
Finland, Regio aboënsis, T. Koponen & S. Huttunen 1321,
May 24., 1999
Georgia, Caucasus, M. Ignatov, August 8, 1987 (MHA)
China, Hunan Prov., T. Koponen, S. Huttunen &
P.-C. Rao 51775, October 8, 1997
Russia, Caucasus, Makridin, August 13., 1998 (MHA)
Papua New Guinea, Morobe Prov., T. Koponen 34413,
July 27, 1981
Eurhynchium pulchellum (Hedw.) Jenn.
AY044069 AF417384 AF395635
Eurhynchium pumilum (Wilson) Schimp.
Eurhynchium savatieri Besch.
AY184790 AY184772 –
AF397859 AF417453 AF403659
Eurhynchium striatum (Hedw.) Schimp.
Eurhynchium vagans (A. Jaeger) E. B.
Bartram var. bergmaniae
(E. B. Bartram) Ignatov
Eurhynchium vagans (A. Jaeger)
E. B. Bartram
Homalotheciella subcapillata (Hedw.)
Broth.
Homalothecium aureum (Spruce)
H. Robinson
Homalothecium fulgescens (Mitt.)
Lawton
Homalothecium laevisetum Sande Lac.
AY184788 AY184769 AF503538
AF397862 AF417450 AF403652
Homalothecium
H. Robinson
Homalothecium
H. Robinson
Homalothecium
A. Jaeger
Homalothecium
Schimp.
Homalothecium
Voucher specimen for sequences
lutescens (Hedw.)
AF397827 AF417379 AF403642
megaptilium (Sull.)
AF397842 AF417431 AF403597
China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo &
P.-C. Rao 49717, September 28, 1998
USA, Missouri, B. Allen & P.L. Redfearn, Jr., March 29,
1995 (NY)
Turkmenia, I.B. Sirotina & A. Abramova, November 22,
1983 (MHA)
Canada, British Colombia, H. Streiman 43136, August 20,
1989
China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo &
P.-C. Rao 54066, July 30, 1998
Czech Republic, Central Bohemia, J. Vana & J. Enroth,
Apri.30, 1999
USA, Oregon, A. Newton 5206, May 30, 1992 (BM)
nuttallii (Wilson)
AF397798 AF417399 AF403589
USA, California, Arcata, M. Ignatov, August 5, 1989
philippeanum (Spruce)
AF397863 AF417451 AF403647
Georgia, Caucasus, V. Vasak B-90123, July 31, 1982
sericeum (Hedw.) Schimp.
AF397805 AF417349 AF403587
Finland, Regio aboënsis, T. Koponen & S. Huttunen 1322,
May 24, 1999
Finland, Regio aboënsis, T. Koponen & S. Huttunen 1320,
May 24, 1999
Isothecium alopecuroides (Dubois) Isov.
AF397878 AF417465 AF403645
AF397860 AF417462 AF403658
AF397876 AF417463 AF403650
AF397877 AF417466 AF403637
AF397820 AF417351 AF403616
AY044065 AF417353 AF395636
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
Table 1
Continued
Species
trnL-F
psbT-H
ITS2
Kindbergia praelonga (Hedw.) Ochyra
AF397804
AF417385
AF403591
Kindbergia praelonga (Hedw.)
Ochyra ‘‘serricuspis’’
Kindbergia praelonga (Hedw.) Ochyra
AF397818
AF417381
Lindigia debilis (Mitt.) A. Jaeger
AF397803
AF417376
Meteoridium remotifolium (Müll. Hal.)
Manuel
Myuroclada maximowiczii
(G. G. Borszcz.)
Steere & W. B. Schofield
Palamocladium euchloron (Müll. Hal.)
Wijk & Margad.
Palamocladium leskeoides (Hook.)
E. Britton
Platyhypnidium austrinum (Hook.
& Wilson) M. Fleisch.
Platyhypnidium patulifolium
(Cardot & Thériot) Broth.
Platyhypnidium riparioides (Hedw.) Dixon
AF397833
AF417418
China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo &
P.-C. Rao 50467, October 7, 1998
AF403590 China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo &
P.-C. Rao 53561, September 29, 1998
AY184794 Colombia, Urrao, S. P. Churschill, I. Sastre-de Jesus &
M. Escoba Acosta 13418, July 1, 1985
AF403617 Ecuador, Provincia de Napo, D. H. Norris 90677 & Eduardo
Barahona M., December 5, 1997
AF403601 Mexico, A. Newton 4399 (BM)
AF397779
AF417392
AF403625
China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo &
P.-C. Rao 53505, September 29, 1998
AF397851
AF417416
AF403623
Russia, Caucasus, M. Ignatov, August 23, 1999 (MHA)
AF397791
AF417387
AF403596
AF397850
AF417443
–
AF397797
AF417470
AF403663
AF397854
AF417423
AF403600
Rhynchostegiella macilenta
(Renauld & Cardot) Cardot
Rhynchostegiella tenella (Dicks.) Limpr.
Rhynchostegiella teneriffae (Mont.)
Dirkse & Bouman
Rhynchostegium confertum
(Dicks.) Schimp.
Rhynchostegium murale (Hedw.) Schimp.
AF397781
AF417370
AF403570
AY044070 AF417400
AF397783 AF417368
AF395633
AF403569
AF397837
AF417413
AF403622
AF397817
AF417454
–
Rhynchostegium murale (Hedw.) Schimp.
Rhynchostegium murale (Hedw.) Schimp.
AF397816
AF397775
AF417355
AF417424
AF403624
AF403661
Rhynchostegium pallidifolium (Mitt.)
A. Jaeger
Rhynchostegium psilopodium Ignatov
& Huttunen
Rhynchostegium rotundifolium
(Brid.) Schimp.
Rhynchostegium serrulatum
(Hedw.) A. Jaeger
Rozea subjulacea Besch.
AF397807
AF417371
AF403618
AF397861
AF417452
AF403643
AF397809
AF417396
AF403611
China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo &
P.-C. Rao 53870, October 1, 1998
Russia, Ural Mts., E. Baisheva, July 9, 1993 (MHA)
Russia, Perm Prov., A. G. Bezgodov #222, June 30,
1999 (MHA)
China, Hunan Prov., T. Koponen, S. Huttunen, & P.-C. Rao
51301, October 3, 1997
China, Hunan Prov., T. Koponen, S. Huttunen & P.-C. Rao
51803, October 8, 1997
Russia, Caucasus, V. Onipchenco, 31. August 1999 (MHA)
AF397829
AF417380
AF403620
USA, New Jersey, B. C. Tan, September 12, 1992
AF397796
AF417366
AF403578
Rozea andrieuxii (Müll. Hal.) Besch.
AF397839
AF417409
AF403579
Scleropodium obtusifolium (Mitt.) Kindb.
Scorpiurium circinatum (Brid.)
M. Fleisch. & Loeske
Scorpiurium deflexifolium (Solms.)
M. Fleisch. & Loeske
Squamidium brasiliense (Hornsch.) Broth.
AF397793
AF397834
AF417434
AF417410
AF403615
AF403598
Venezuela, Estado Mérida, Dana Griffin & Manuel Lopez
No. PV-904, February 19, 1985 (NY)
Dominican Republic, Prov. San Juan, W. R. Buck 14205,
April 28, 1982 (NY)
USA, California, M. Ignatov & D. H. Norris, August 13, 1989
Georgia, Caucasus, M. Ignatov, August 8, 1987 (MHA)
AF397844
AF417407
AF403599
AY044063 AF417393
AF395637
Unclejackia longisetula (E. B. Bartram)
Ignatov, T. J. Kop. & D. H. Norris
Zelometeorium patulum (Hedw.) Manuel
AF397794
AF417357
AF395643
AF397787
AF417362
–
Plasteurhynchium striatulum (Spruce)
M. Fleisch.
Pseudoscleropodium purum (Hedw.)
M. Fleisch.
Rhynchostegiella brachypodia M. Fleisch.
AY184795 –
Voucher specimen for sequences
China, Hunan Prov., T. Koponen, S. Huttunen & P.-C. Rao
52596, October 10, 1997
AY184791 AY184773 AY166449 Australia, Victoria, H. Streimann 49544
China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo
& P.-C. Rao 53920, October 1, 1998
AF397847 AF417411 –
China, Hunan Prov., T. Koponen, S. Huttunen, &
P.-C. Rao 51843, October 9, 1997
AY184792 AY184774 AY166450 Georgia, Caucasus, M. Ignatov, August 1, 1987 (MHA)
Czech Republic, Central Bohemia, J. Vana & J. Enroth,
April 30, 1999
Papua New Guinea, Morobe Prov., T. Koponen 33007,
July 11, 1981
Portugal, Madeira, L. Hedenäs, May 13, 1993 (B4503) (S)
Georgia, Caucasus, M. Ignatov, October 2, 1997 (MHA)
Portugal, Madeira, S. Fontinha, L. Hedenäs, M. Nobrega,
June 10, 1991 (B9121; S)
Georgia, Caucasus, M. Ignatov, August 27, 1987 (MHA)
Portugal, Madeira, L. Hedenäs & I. Bisang, November 21,
1999 (B22455; S)
Tanzania, Arusha District, T. Pócs, R. Ochyra & H.
Bednarek-Ochyra with C. N. Lema 88161 ⁄ R, June 29, 1988
Papua New Guinea, Morobe Prov., T. Koponen 32496,
July 8, 1981
Ecuador, Loja Prov., D. H. Norris 91902, M. Bolivar &
M. Aguirre, December 16, 1997
155
156
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
Table 1
Continued
Species
Meteoriaceae
Aerobryidium filamentosum (Hook.)
M. Fleisch.
Barbella flagellifera (Cardot) Nog.
Chrysocladium retrorsum (Mitt.)
M. Fleisch.
Duthiella wallichii (Mitt.) Müll. Hal.
Floribundaria floribunda
(Dozy & Molk.) M. Fleisch.
Meteorium polytrichum Dozy & Molk.
Pseudospiridentopsis horrida (Cardot)
M. Fleisch.
Trachycladiella aurea (Mitt.) M. Menzel
Trachypus bicolor Reinw. & Hornsch.
Lembophyllaceae
Dolichomitriopsis diversiformis (Mitt.)
Nog.
Lembophyllum clandestinum
(Hook. & Wilson) Lindb.
Amblystegiaceae
Amblystegium serpens (Hedw.) Schimp.
Donrichardsia macroneuron (Grout)
H. A. Crum & L. E. Anderson
Campyliaceae
Campylium stellatum (Hedw.)
C. E. O. Jensen
Hylocomiaceae
Hylocomium splendens (Hedw.) Schimp.
Meteoriella soluta (Mitt.) S. Okamura
Rhytidiadelphus triquetrus (Hedw.)
Warnst.
Hypnaceae
Ctenidium molluscum (Hedw.) Mitt.
Hypnum cupressiforme Hedw.
Leskeaceae
Leskea polycarpa Hedw.
Okamuraea brachydiction (Cardot) Nog.
Leucodontaceae
Leucodon sciuroides (Hedw.) Schwägr.
Myriniaceae
Helicodontium capillare (Hedw.) A. Jaeger
trnL-F
psbT-H
ITS2
Voucher specimen for sequences
AF397789 AF417347 AF403613 China, Yunnan Prov., T. Koponen 37908, October 25, 1981
AY044071 AF417360 AF395630 China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo &
P.-C. Rao 56995, October 13, 1998
AY044061 AF417390 AF395626 China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo &
P.-C. Rao 55572, October 6, 1998
AF397782 AF417375 AF403612 China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo &
P.-C. Rao 54168, October 1. 1997
AF397800 AF417428 AF403580 Papua New Guinea, West Sepik Prov., T. Koponen 35719,
August 7, 1981
AY044073 AF417350 AF395629 Australia, Queensland, H. Streimann 57477, October 22, 1995
AF397801 AF417383 AF403571 China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo &
P.-C. Rao 55834, October 11, 1998
AF397778 AF417397 AF403573 China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo &
P.-C. Rao 55508, October 6, 1998
AY044060 AF417369 AF395624 China, Hunan Prov., T. Koponen, S. Huttunen, S. Piippo &
P.-C. Rao 50721, October 2, 1997
AF397777 AF417469 AF403664 Russian Far East, Kunashir Island,V. A. Nedoluzhko,
July 18, 1990 (MHA)
AF397823 AF417374 AF403630 New Zealand, South Island, D. H. Vitt 29644, December 22,
1981
AF397836 AF417420 AF403633 Finland, Nylandia, S. Huttunen 1416, April 16, 2000
AY009848 –
AF167350 USA, Redfearn 27208 (Sequences from GenBank,
Vanderpoorten et al., 2002)
AF397821 AF417358 AF403609 Finland, Regio aboënsis, J. Pykälä 7033, October 30, 1990
AF397840 AF417408 AF403610 Finland, Tavastia australis, S. Huttunen 1441, April 14, 2000
AF397808 AF417389 AF403606 China, Hunan Prov., T. Koponen, S. Huttunen & P.-C. Rao
49610, October 1, 1997
AF397811 AF417425 AF403631 Finland, Tavastia australis, S. Huttunen 1440, April 14, 2000
–
AF417414 AF403632 Finland, Regio aboënsis, J. Pykälä 8706 & J. Nurmi,
August 22, 1991
AF397812 AF417361 AF403607 Finland, Nylandia, S. Huttunen 1438, November 1999
AF397810 AF417367 AF403604 Finland, Regio aboënsis, J. Pykälä 10872, September 15, 1992
AY184789 AY184771 AF503537 China, Hunan Prov., T. Koponen, S. Huttunen, &
P.-C. Rao 48969, September 26, 1997
AF397786 AF417398 AF403634 Russia, Karelian Republic, S. Huttunen & H. Wahlberg
819, June 27, 1997
AF397855 AF417435 –
Colombia, Departamento del Valle, S. Churchill, A. E.
Franco & N. Hollaender, May 4, 1990
Plagiotheciaceae
Plagiothecium denticulatum (Hedw.) Schimp. AF397845 AF417419 AF403635 Finland, Karelia australis, S. Huttunen 1398, May 23, 2000
*ITS3 sequence instead of primers ITS3 and ITS4 primers 5.SR and LC4-R were used for PCR and sequencing.
when a genus exhibited more or less contrasting
infrageneric units (either recognized in the taxonomic
literature or observed by us in previous studies of the
family), we tried to include representatives of each those
entities. Type species of the genera were included
whenever possible, in order to make subsequent taxonomic conclusions better founded. Several samples were
included for morphologically highly variable species:
Brachythecium salebrosum (Web. & Mohr.) Schimp.,
Bryhnia novae-angliae (Sull. & Lesq.) Grout, Eurhynchium vagans (A. Jaeger) E. B. Bartram, Kindbergia
praelonga (Hedw.) Ochyra, Rhynchostegium murale
(Hedw.) Schimp. These samples were usually taken from
different geographic regions. Outgroup species were
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
selected based on earlier analyses of larger data sets
(Huttunen et al., 2004). Based on this study, Meteoriaceae is sister to Brachytheciaceae. Both of these taxa are
nested within a monophyletic Hypnales (Buck et al.,
2000a). In addition to Meteoriaceae species, some
outgroup species were included, representing other
presumed closely related Hypnales families, such as
Amblystegiaceae, Hylocomiaceae, Hypnaceae, Lembophyllaceae, Plagiotheciaceae, and Leucodontaceae
(Table 1). None of the earlier phylogenetic analyses
suggested a close relationship between Brachytheciaceae
and Leskeaceae, and Leskea polycarpa Hedw. was used
as an outgroup in direct optimization analyses.
Morphological data
The morphological data set included 63 characters (48
gametophytic and 15 sporophytic). It was mainly
compiled using the specimens employed in DNA analysis (Table 1), and supplemented with a study of
herbarium material available in the Finnish Museum
of Natural History, Helsinki (H), particularly for the
sporophytic characters. For these characters, data were
supplemented with specimens in areas that are closely
situated geographically to the sequenced specimen. If
additional material was not available, the sporophytic
characters were left unknown. We found most of the
available morphological characters to vary considerably
on the generic level, and in some cases even on the
species level, and hence we doubted the information
content of the morphological data in phylogenetic
analyses of Brachytheciaceae. For morphologically
variable species (Brachythecium salebrosum, Bryhnia
novae-angliae, Eurhynchium vagans, Kindbergia praelonga, Rhynchostegium murale), we coded gametophytic
character states only according to the specimens used
for molecular study. Variability in some characters in a
few specimens made character coding complicated, and
we could not therefore avoid including some polymorphic characters in the data set. Due to difficulties in
including data on these characters in POY analyses, all
polymorphisms were coded as ‘‘?’’. The quantitative
characters used in this data matrix were mostly for
separating extreme cases, such as species with very
narrow leaves (see Appendix 1, character 20) or very
long cells (Appendix 1, character 35).
Molecular data
We used the information from three, mainly noncoding, DNA sequence regions for the phylogeny
reconstruction: chloroplasts psbT-H and trnL-F, and
nuclear ITS2. These were sequenced using primers C
and F (Taberlet et al., 1991) for the trnL-F region,
universal primers ITS3 and ITS4 (White et al., 1990) for
the ITS2 region, and primers psbT and psbH (Hong
157
et al., 1995; Shaw, 2000) for the psbT-H region.
Sequenced psbT-H region consisted of partial psbT
(102 bp) and psbH (108 bp), genes and the whole psbN
gene (132 bp) between them, which are all coding for the
small proteins of photosystem II. Between these genes
there are two non-coding spacers, psbT-N and psbN-H,
whose lengths vary among Bryopsida between 67–95 bp
and 88–105 bp (Quandt et al., 2003). In the trnL-F
region, length variation is concentrated in trnL intron in
mosses, which is situated within the ribosome coding
tRNA-Leu gene. Compared to some other groups of
land plants, in mosses the intron is relatively short. For
example, Stech et al. (2003) reported a mean length of
308 bp (SD 59.4) for a data set including 24 mosses from
different groups of Bryopsida. Some indel events also
occur in the non-coding trnL-F spacer, whose length in
mosses is 82 bp (SD 41.3) according to Stech et al.
(2003). In addition to these two non-coding fragments,
our analyses included the 50 bp long end of the tRNALeu gene between them. The nuclear Internal Transcripted Spacer 2 (ITS2) is situated between the
ribosome coding the 5,8S and 26S genes. For example,
in the data set including 66 mosses from the subclass
Bryidae, where Brachytheciaceae also belongs, the
length of the ITS2 region varied between 251 and
360 bp (mean 282.8, SD 25.41; Quandt et al., 2004b).
DNA extraction, PCR and sequencing
The isolation of DNA was mainly carried out
according to the CTAB-technique described in Doyle
and Doyle (1990), or in some cases using a Nucleospin
Plant DNA Extraction Kit (Machery-Nagel), following
the manufacturer’s instructions. Most of the specimens
used for the DNA extraction were herbarium specimens,
except for some species commonly found in southern
Finland. Polymerase chain reactions and cycle sequencing reactions were performed using PTC-100 and PTC200 Thermocyclers (MJ Research). The 25-lL reactions
contained 1.5 U Taq DNA polymerase (Amplitaq Gold,
Applied Biosystems), 1 · buffer, 2.5 mmol MgCl2,
0.3 mmol dNTPs-mix and 0.4 lmol of each primer.
The PCR products were purified using a GFX PCR
purification kit (Amersham Pharmacia). For the cycle
sequencing, 8-lL reactions were used, containing 2.2 lL
of Big Dye Terminator Cycle Sequencing premix
(Applied Biosystems), 1.3 lL of 10 lmol primer,
3.8 lL H2O and 0.7 lL purified PCR product. The
sequencing products were purified by eluting through
columns filled with 450 lL 5% Sephadex solution
(Amersham Pharmacia). An ABI PRISM 377 automated sequencer was used to analyse the sequencing
products. All PCR products were sequenced using two
primers. All sequences were submitted to GenBank, and
a list of their accession numbers and voucher specimens
is given in Table 1.
158
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
Phylogenetic analyses
Phylogenetic analyses were performed for two data
sets, first for a wider selection of taxa (124 terminals) to
test for the monophyly of the family. Due to the wide
sequence length variation within this large data set, the
effects of possible differences in character optimization
obtained with POY were tested with a smaller data set
(99 terminals) that included only species of Brachytheciaceae and Floribundaria floribunda (Dozy & Molk.)
M. Fleisch. (Meteoriaceae) as the outgroup. Simmons
and Freudenstein (2003) showed how the increasing
genetic distance within data causes errors in alignment if
programs such as ClustalX (Thompson et al., 1997) are
used, but how this possibly affects direct optimization
has not been explored. The ITS2 region has often been
stated to be too variable at higher taxonomic levels, and
in phylogenetic studies on pleurocarpous mosses based
on manual alignments some sequence fractions are often
left out due to ambiguity (e.g., Quandt et al., 2000,
2004a,b).
Direct optimization, as implemented in POY
(Gladstein and Wheeler, 2001), was used. Alignment
and phylogenetic analysis are performed simultaneously
in direct optimization and several different hypotheses
of character transformation were tested during the
analyses by optimizing characters. This implies that
the resulting set of most parsimonious trees may consist
of several equally most parsimonious trees that are each
based on different character optimizations, unlike the
situation in all other phylogenetic methods. As a result
of this, the definitions for homology also differ from
those of traditional methods (Giribet et al., 2002). The
only homology statement made prior to phylogeny
reconstruction is that the submitted sets of sequences are
assumed to represent the same genomic region in all
taxa. In contrast to static alignment methods, the most
parsimonious hypothesis of character transformation
between sequences is obtained as a result of analyses
performed in POY, and those results are presented in the
form of an implied alignment. Hence, if ordinary static
alignments are regarded as primary homology statements sensu De Pinna (1991), implied alignments
obtained from POY describe true synapomorphies and
can thus be considered secondary homology statements
(sensu De Pinna). In static alignments, the nucleotides
that are expected to be homologous are placed in the
same column, and running cladistic analyses tests this
hypothesis of homology. POY implied alignments,
however, result from the analyses, and the synapomorphies that they present between studied sequences have
already been tested in the preceding direct optimization
analyses.
Phylogenetic analyses were performed using a parallel
version of POY with eight processors of the Silicon
Graphics Origin 2000 ⁄ 128 cluster in the Center of
Scientific Computing, Espoo, Finland. Prior to POY
analysis, the sequences were aligned using ClustalV
(Higgins et al., 1991) and then split using Winclada
(Nixon, 1999a) into shorter fragments within conserved
regions to save computation time and memory
(Gladstein and Wheeler, 2001). The command line used
in all POY analyses is given in Appendix 2. Leskea
polycarpa Hedw. was used as an outgroup for larger
data sets and Floribundaria floribunda for smaller sets.
Implied alignments reported by POY were always saved
and checked afterwards to avoid obvious errors in
homology. We found the hypothesis of homology in
POY character optimizations highly improbable in two
coding regions, and hence an extra 6 bp CGCGCG
repeat was added prior to analyses at the beginning of
the psbT-N and trnL-F spacers (see Quandt et al.,
2004b, for more information). We are aware that the
strict consensus trees of POY topologies may include
branches that are only supported by one character
optimization. Unfortunately, checking parsimonious
trees against original data matrices and removing
ambiguously supported branches may be a fairly complicated task if the direct optimization analysis results in
more than one topology and character optimization.
Since the parsimonious trees obtained from analysis
may be based on different character optimizations,
removing these nodes would demand the study of all
POY topologies together with the character optimization they are connected with, and this should be done
using some other program capable of handling substitution matrices, such as PHAST (Goloboff, 1996).
To compare the effects of information from gaps on
topology, POY character optimizations were used as
static alignments and analyzed using the program Nona
(Goloboff, 1994) within the Winclada (Nixon, 1999a)
shell, and unlike in POY, the gaps were treated as
missing data. In general, the use of POY character
optimizations as static alignments suffers from fundamental weaknesses, and this method should not be used
as a standard method for phylogenetic analysis. Despite
the previous extensive discussion on the importance of
information from gaps (for example Giribet and Wheeler, 1999; Simmons and Ochoterena, 2000; Bapteste and
Philippe, 2002; Kawakita et al., 2003), we still wanted to
see if (and to what extent) they affected the topology in
our study. One method for doing this is to simply
compare the topology obtained from POY and Nona.
For Nona, a multi-ratchet option with three sequential
ratchet runs was used (Nixon, 1999b). Each replicate
included 200 iterations, and 20 trees were held in
memory during the iterations. During the ratcheting,
20% of the characters were resampled. Topological
differences were studied by comparing these Nona
topologies with those from the POY. Since each POY
run most often resulted in more than one character
optimization, they all were analyzed in Nona to deter-
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
mine if the parsimonious trees based on these analyses
were of the same length despite the differences in
treatment of the gaps. Only phylogenies with the
shortest tree lengths were examined with ILD metrics
and included in comparisons of tree topology between
the results of POY and Nona analyses, but for comparisons of parsimony informative (pi) characters in data
sets, suboptimal character optimizations were also
included. For the most parsimonious Nona trees, Bremer
support (Bremer, 1994) for clades was calculated in
Nona by saving suboptimal trees in a series of up to 15
steps longer than that of the most parsimonious topology. During the search, up to 10 000 trees were saved
from 300 replications and in each replication 20 different
starting trees were used. Jackknifing was performed with
Nona within the Winclada shell. In jackknifing 1000
replications, including 10 searches, were performed, and
in each replication 20 starting trees were used. These
trees were submitted for additional tree bisection-reconnection (TBR) branch swapping (do max*).
Quandt et al. (2003) showed that the 9 bp short
loop region in the psbT-N spacer region is inverted in
some mosses, and that this inversion appears to have
occurred independently several times in different
lineages. The authors stated that the original and
inverted reverse complement forms cannot be regarded
as homologous positions, and inclusion of this region
in the original form in phylogenetic analyses could
negatively affect phylogeny reconstruction. They further concluded that inclusion of this region in the
original form will also lead to weighting of the
inversion event. We tested the effect of including this
region on phylogeny reconstruction by analyzing data
in three different ways using the 124-terminal data set.
First we included the 9 bp region and treated it as we
did all other sequences (Table 2). Despite the inversion event in some taxa, the loop region appeared as a
homologous character in POY character optimiza-
Table 2
Base composition of 9-bp inverted loop region in psbT-N spacer in
brachytheciacean species and alternative coding method tested in
analyses. Alternative coding includes absence ⁄ presence (0 ⁄ 1) coding
for inversion event in loop region and reverse complement sequence of
original 9 bp in loop region in those terminals with A-rich type of
inversion (Rhynchostegielloideae type)
Common type
Brachythecium type
Sciurohypnum type
Rhynchostegielloideae
type
or
Original
Reverse complement
or original sequence
+0 ⁄ 1 coding
for inversion event
TTTATTTTA
CTCATTTTA
TTCATTTTA
CAAAATAAA
TTTATTTTA 0
CTCATTTTA 0
TTCATTTTA 0
TTTATTTTG 1
TAAAATAAA
TTTATTTTA 1
159
tions. In another analysis the loop positions were
excluded, and in the final analysis the A-rich forms of
the loop region were transformed into reverse complement sequences and included. The information on
the presence ⁄ absence of the inversion event was
included in analyses as a separate character matrix.
The last method removed the effect of the additional
weighting of the inversion event, but still could not
avoid possible erroneous expectations of the positional
homology when both the uninverted and inverted loop
regions are placed in POY character optimizations as
homologous positions.
Sensitivity analyses and weighting of morphological data
Sensitivity analysis (sensu Wheeler, 1995) has become
somewhat a standard way of exploring the data in direct
optimization analyses. Sensitivity analysis aims to
explore the effect of the different substitution costs on
the stability of the obtained hypothesis of relationships
and on congruence among data partitions. These results
identify the most stable tree hypotheses, presenting the
groups shared by all analyses with differing gap costs, or
selecting the optimal topology using congruence as an
optimality criterion (see Giribet, 2003). This method has
recently been criticized (Frost et al., 2001; Grant and
Kluge, 2003). The increased congruence identified with
the ILD or RILD metrics does not make the less
parsimonious results more acceptable (see Grant and
Kluge, 2003), and in this paper we consider the equal
weighting of all data to be the best fitting result. Due to
the common practice of applying sensitivity analyses in
direct optimization analyses, we explored the impact of
weighting gaps and morphology on the output of POY,
and the contribution of data partitions in differentially
weighted analyses. Three different gap costs (1, 2 and 4)
and two weighting methods for morphological data
were used. This also provides for the possibility of
comparing this research to other studies utilizing the
direct optimization method and sensitivity analysis. For
all weighting methods, data were analyzed as combined,
and additionally the morphological data, chloroplast
(psbT-H and trnL-F) and nuclear (ITS2) regions were
analyzed separately. These separate analyses were used
when evaluating the impact of different data partitions
on the topology obtained from combined analyses and
when calculating the ILD metric. The congruence
among data sets in the different analyses was estimated
using ILD metrics (Mickevich and Farris, 1981; Farris
et al., 1995). ILD utilizes tree lengths of combined and
separately analyzed data sets (ILD ¼ (tree length of
combined data set – S tree lengths of individual data
sets) ⁄ tree length of combined data set).
Morphological data were either always weighted as
one or as the same weight as determined for indel events.
In direct optimization analyses (Wheeler, 1996) of
160
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
combined molecular and morphological data, the latter
have often been given the same weight as the indel
events (for example, Giribet et al., 2002; Schulmeister
et al., 2002). This practice follows Wheeler and
Hayashi’s (1998) study of chelicerates, in which they
found that an equal weighting of morphology and indel
events gave the most congruent results. The method is
also said to avoid the possible overwhelming effects of
the molecular data on the usually much smaller
morphological matrix. The effects of two different
weighting methods on morphological data were tested
with ILD metric (Mickevich and Farris, 1981; Farris
et al., 1995). The method in which the morphology was
given a weight of one appeared to lead to lower ILD
values and was therefore used in all analyses testing the
effect of inversion in the psbT-N region.
Results
Sequence variation and information content of data
partitions
The ITS2 region showed the largest sequence length
variation (Table 3). The length of the sequenced ITS2
fragments varied in the 124 terminal data set between
340 and 415 bp, and in the 99 terminal data set between
352 and 406 bp. In the psbT-H region, all the length
variation was concentrated in the psbT-N spacer, where
the length varied between 66 and 94 bp in the large data
set and between 79 and 90 bp in the smaller one. In the
trnL-F region, lengths of the non-coding sequences trnL
intron and trnL-F spacer varied, while the ribosomal
coding trnL and trnF regions showed no length
variation. The trnL intron of 256–288 bp constitutes
the majority of the total length of the trnL-F region. In
the trnL-F spacer the 17 bp and 9 bp long insertions at
the beginning of the spacer in two species, Trachypus
bicolor and Brachythecium frigidum, constitute the
majority of the length variation in this region. If POY
implied alignments from analyses with a gap cost equal
to one are studied as static alignments, and gaps treated
as missing data, the number of pi base positions in both
chloroplast regions together was about the same as in
the nuclear ITS2 region (Tables 3 and 4). In this implied
alignment, 17.8% of positions in the 863 bp long ITS2
were pi, while both in the 558 bp long psbT-H and in the
552 bp long trnL-F proportions of the pi positions were
only 13.8%. However, if the proportions of pi positions
in each non-coding and coding region in trnL-F and
psbT-H are studied alone, the psbT-N spacer (24.4% of
positions pi) and trnL intron (21.1%) are the most
informative regions. If the 9 bp inversion in the psbT-N
is excluded, the proportion of pi positions decreases
from 24.4 to 21.1%.
The high information content of the ITS2 region clearly
affected the resolution when we compared tree topologies
based on separate analyses of the nuclear ITS2 and
chloroplast data. Within most of the genera in the
Brachytheciaceae, the sequence variation of trnL-F and
psbT-H was extremely low, and most of the information
for resolving relationships inside the genera came from
the ITS2 region. Genus- and species-level relationships
were best resolved in the strict consensus trees based on
ITS2, but at the family level Brachytheciaceae was not
resolved as monophyletic. Separate analyses of the
nuclear ITS2, chloroplast psbT-H and trnL-F, and
morphological data resulted in clearly different topologies (not shown). Most of the generic groupings in ITS2
trees closely resembled those obtained from analyses of
combined data. Some problematic placements of species
in combined analyses also appeared to originate from
ITS2 data. The genus Palamocladium Müll. Hal. was
monophyletic in all analyses based on chloroplast data,
while ITS2 analyses resolve P. leskeoides (Hook.) E.
Britton as sister to the Chinese Bryhnia novae-angliae. In
the latter analyses, P. euchloron (Müll. Hal.) Wijk &
Margad. was placed close to Plasteurhynchium striatulum
Table 3
Sequence length variation on chloroplastic psbT-H and trnL-F, and nuclear ITS2 regions in 124- and 99-terminal data set, and number of parsimony
informative (pi) characters when gaps are treated as missing data. In genomic regions marked with asterisk (*) number of pi characters varies between
different POY implied alignments, depending on gap costs and data sets used in POY analyses; see Table 4
psbT-H
trnL-F
psbT coding
psbN
psbH coding
region
psbT-N coding psbN-H region
trnL
(partial)
spacer region spacer (partial)
intron
Length variation
102
—124-terminal data
set (bp)
Number of pi characters
13
Length variation—99-terminal 102
data set (bp)
Number of pi characters
102
trnL
trnF coding
coding trnL-F region
ITS2
region spacer (partial)
66–94
132
97
108
256–288 50
51–80
40
340–415
*
79–90
14
132
12
97
9
109
*
0
256–281 50
*
51–80
1
40
*
352–406
10
9
8
*
*
*
0
0
*
161
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
Table 4
Number of characters and parsimony informative positions (pi, gaps treated as missing data) in POY character optimizations for genomic regions
with variable sequence length. Analyses were performed separately for 124 terminal data set (98 Brachytheciaceae taxa and 26 outgroup species from
other pleurocarpous moss families) and 99 terminal data set (98 Brachytheciaceae taxa and Floribundaria floribunda, Meteoriaceae, as outgroup)
Number
of taxa
Ti ⁄ Tv ⁄ gap
psbT-N loop
1:1
pi
1:1
pi
1:1
pi
1:1
pi
1:1
pi
1:1
pi
1:1
pi
1:1
pi
1:1
pi
1:1
pi
1:1
pi
1:1
pi
:1
included
99
:2
included
99
:4
included
99
:1
included
124
:1
excluded
124
:1
rev.compl. + 0 ⁄ 1 coding
124
:2
included
124
:2
excluded
124
:2
rev.compl. + 0 ⁄ 1 coding
124
:4
included
124
:4
excluded
124
:4
rev.compl. + 0 ⁄ 1 coding
124
(Spruce) M. Fleisch and Eurhynchium. Consensus trees
based on analyses of chloroplast trnL-F and psbT-H
regions were much less resolved than topologies based on
ITS2, especially with regard to the relationships between
genera of Brachytheciaceae.
The analyses of morphological data alone resulted in
a topology with only poor resolution (not shown), and
Brachytheciaceae was not monophyletic. Despite this,
morphology appeared to be important, especially in
cases where taxa within one species or genus showed
wide sequence variation. For example, Rhynchostegium
murale, Kindbergia praelonga, and Palamocladium
always appeared to be polyphyletic in analyses including
only molecular data (trnL-F, psbT-H and ITS2) but they
were monophyletic in at least the most parsimonious
analyses, including morphology (Fig. 1). In some cases
sequence variation did not follow the taxonomic species
concept, which suggests that the delimitation of some
species is in need of re-evaluation. In Bryhnia novaeangliae, even the inclusion of morphological data in
analyses did not resolve this species as a monophyletic
entity (Fig. 1). Sequences of Bryhnia novae-angliae from
southern China were much closer to those of Brachythecium frigidum (Müll. Hal.) Besch., while two other
Bryhnia novae-angliae specimens (from European and
Asian boreal regions of Russia) shared several mutations in both trnL-F and ITS2. This clearly explains the
position of these terminals in cladograms. In all other
ITS2
trnL
intron
trnL-F
spacer
psbT-N
spacer
594 or 596
99 or 101
540–543
108 or 110
524
132
863
154
864–868
161–163
864
151
798
186
764 or 772
185 or 191
797
187
695–697
235
700
233–234
704
236–237
301
30
301
30
291
32
353
52
353
52
353
52
335
52
341
52
335
52
329
53
330
54–55
330
55
94
12
94
12
94
12
109
23
109
23
109
23
109
23
109
23
109
23
109
23
109
23
109
23
104 or 105
25
103
25
102
25
119
29
109
23
109
28
116 or 117
29
107
24
116
28
116
29
108
23
117
28
analyses the Chinese specimen was resolved to the same
clade with Brachythecium frigidum, while both Northern Eurasiatic Bryhnias always formed a clade (Figs 1,
2 and 3).
Effect of inversion in psbT-N spacer loop region
Topologies obtained using direct optimization were in
some details affected by the inversion in the psbT-N
loop, and it had a minor effect on the congruence
between data sets (Table 5). In the most parsimonious
analyses with a gap cost equal to one, the inclusion of
the loop region in original form resulted in the highest
congruence between data partitions. Despite the short
length, 6 out of a total of 9 bp in the loop are pi when
this region is included in analyses in the original form.
Even in reverse-complement form (A-rich inversions
reverse-complemented in T-rich form, see Table 2) five
pi positions remained. These positions appear to be very
informative in some clades within Brachytheciaceae.
Species with rarer A-rich forms of inversion are
concentrated in Rhynchostegielloideae and the Squamidium clade, which includes Squamidium brasiliense
(Hornsch.) Broth., Meteoridium remotifolium (Müll.
Hal.) Manuel, and Zelometeorium patulum (Hedw.)
Manuel. The latter clade appears in some analyses as
sister to Rhynchostegielloideae (Figs 1a, 1c, 2 and 3,
Table 6). When the loop region is excluded, the position
162
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
Table 5
Lengths of the most parsimonious trees for analyses of chloroplastic (trnL-F + psbT-H), nuclear (ITS2), combined molecular (trnL-F +
psbT-H + ITS2), and combined molecular + morphological data from 124 terminals with different gap costs and analysis methods, and congruence
among data partitions (ILD) in these analyses. Nona analyses are based on character optimizations obtained from direct optimization, but gaps are
treated as missing data
Gap cost ⁄ Weight
psbT-N
Morphology
ILD
No. of
spacer
treatment for
Chloroplast Nuclear
Molecular & molecular ILD for for all
terminals loop region of gaps
morphology DNA
DNA Morphology combined combined
molecular data
POY 1 : 1 : 1
1:1:2
1:1:4
99
99
99
POY 1 : 1 : 1 124
1:1:1
124
1:1:1
124
1
1
1
1
:
:
:
:
1
1
1
1
:
:
:
:
2
2
2
2
124
124
124
124
1
1
1
1
:
:
:
:
1
1
1
1
:
:
:
:
4
4
4
4
124
124
124
124
Nona 1 : 1 : 1 124
1:1:1
124
1:1:1
124
1:1:2
124
1:1:2
124
1:1:2
124
1:1:4
124
1:1:4
124
1:1:4
124
included
included
included
1
2
4
1
1
1
536
671
893
767
1078
1592
525
525
525
included
excluded
rev.compl.
+ 0⁄1
coding
included
included
excluded
rev.compl.
+ 0⁄1
coding
included
included
excluded
rev.compl.
+ 0⁄1
coding
1
1
1
1
1
1
879
847
866
1392
1392
1392
799
799
799
2541
3515
3500
3507
0.106
0.126
0.132
0.128
2
2
2
2
1
2
1
1
1102
1102
1067
1090
2156
2156
2156
2156
799
1598
799
799
3508
4486
5415
4428
4458
0.071
0.096
0.103
0.092
0.093
4
4
4
4
1
4
1
1
1466
1466
1404
1413
3195
3195
3195
3195
799
3196
799
799
5035
6076
8836
5992
6038
0.074
0.101
0.111
0.099
0.105
1
636
765
799
2471
0.096
1
606
772
799
2402
0.094
1
617
759
799
2405
0.096
1
643
951
799
2669
0.103
1
615
958
799
2637
0.100
1
623
954
799
2643
0.101
1
644
1268
799
3016
0.101
1
627
1259
799
2963
0.094
1
636
1265
799
2990
0.097
included
missing
data
excluded
missing
data
rev.compl. missing
+ 0⁄1
data
coding
included
missing
data
excluded
missing
data
rev.compl missing
+ 0⁄1
data
coding
included
missing
data
excluded
missing
data
rev.compl. missing
+ 0⁄1
data
coding
2130
2608
3422
0.142
0.128
0.120
Fig. 1. Strict consensus trees from direct optimization analyses of combined morphological and molecular data including 124 terminals with a gap
cost of 1: (a) strict consensus of six most parsimonious trees (L ¼ 5512), 9-bp inversion in psbT-N loop region included in original form (see
Table 2); (b) strict consensus of three most parsimonious trees (L ¼ 3500), psbT-N loop region excluded from analyses, and (c) strict consensus of
A-rich type of 9-bp loop region reverse-complemented and information on presence ⁄ absence of inversion event included in analyses, strict consensus
of four trees (L ¼ 3507). Branches leading to taxa with A-rich type of psbT-N loop region presented with gray color (this region is lacking in
Donrichardsia macroneuron). The character evolution of two characters, seta surface structure (marked with square, 0 ¼ setae smooth, 1 ¼ setae
rough) and operculum shape (triangle, 0 ¼ operculum rostrate, 1 ¼ operculum conic) is presented in (a). For character optimization of these
characters the unambiguous option in Winclada (Nixon, 1999a) was used.
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
of the Squamidium clade switches from Rhynchostegielloideae to basal Brachytheciaceae (Fig. 1b), but otherwise treatment of the inversion region does not have a
major effect on the topology of Brachytheciaceae. All
163
taxa in these clades have an A-rich form of the psbT-N
loop, although for one species (Donrichardsia macroneuron), the structure of the loop region is unknown due
to a lack of the psbT-H sequence. Outside Rhynchos-
164
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
Fig. 1. Continued
tegielloideae and Squamidium only two species of
Brachytheciaceae with A-type are found, both in the
Rhynchostegioideae clade (Fig. 1).
Base compositions in the more common T-rich
forms also contain some phylogenetic information.
The TTCATTTTA form is found only in the Sciuro-
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
165
Fig. 1. Continued
hypnum clade, while occurrences of the CTCATTTTA
form are restricted to all other groups in the Homalothecioideae and Brachythecioideae clades.
Analyses with 99-terminal data set
POY analyses with a smaller data set (99 terminals)
resulted in topologies that were almost identical to those
166
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
Fig. 2. Strict consensus of 20 most parsimonious trees (L ¼ 2130) from direct optimization analyses of combined morphological and molecular data
with gap costs of one. Only 99 terminals are included in analyses and 9-bp loop region of psbT-N spacer in its original form.
obtained with the larger data set. The number of
characters and pi positions in the character optimizations, however, clearly differed (Tables 3 and 4).
Decreasing sequence length, especially in the ITS2
region, resulted in character optimizations with fewer
indel events and the length of the character optimizations decreased by almost 300 bp (Table 4). The
inclusion of a wider selection of outgroups improved
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
167
Fig. 3. Strict consensus of 629 equally parsimonious trees (L ¼ 2471) from Nona analyses of one of six POY character optimizations with a gap cost
of one. Original direct optimization and results from Nona are based on combined analyses of morphological and molecular data and 9-bp loop
region on psbT-N spacer in original form. Support values for clades are indicated below (jackknife value) and above (Bremer support) each node.
Unlike the original direct optimization analyses, gaps in all Nona analyses are treated as missing data.
168
Table 6
Status of groups within Brachytheciaceae under different analysis methods and parameters; black ¼ monophyletic, gray ¼ paraphyletic, white ¼ polyphyletic, patterned ¼
unresolved
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
congruence among the data sets. The ILD value from
analyses of small data sets was higher with all gap costs
compared with those based on larger data sets (Table 5).
Effect of information on indel events and weighting
of gaps and morphological data
Weighting of morphological data as one, despite the
gap cost, led to the most congruent results according to
ILD (Table 5). If in analyses with gap costs two or four,
and morphology with the same weight as the gaps, the
topologies of strict consensus trees, however, were closer
to those of equal weighting (POY 1 : 1 : 1 in Tables 5,
i.e., the most parsimonious results) (Table 6). In this
case, Palamocladium and Kindbergia praelonga appeared
to be monophyletic, also with high gap costs. Based on
these findings it seemed clear that the monophyly of
these taxa were only due to the phylogenetic signal from
morphology.
Topologies from all combined analyses differed only
in some details, and most of the groupings were fairly
stable, regardless of differences in gap costs. A summary
of the major topological differences is shown in Table 6.
Of all combined analyses, only that with a gap cost of
four, and an unaltered psbT-N loop region, resolved the
Brachytheciaceae as non-monophyletic, due only to the
placement of one species, Platyhypnidium riparioides
(Hedw.) Dixon, which in this analysis clustered within
Meteoriaceae (Table 6).
ILD favored analyses with a gap cost of two and the
psbT-N loop region excluded (Table 5). Although the
most parsimonious results (a gap cost of one) are the
least congruent according to ILD, their topology included several details that were more congruent with the
traditional classification of Brachytheciaceae than any of
the POY topologies with higher gap costs (Table 6). This
analysis resolved Palamocladium and Kindbergia praelonga as a monophyletic clade, while Bryhnia novaeangliae always remained polyphyletic. While in analyses
with a gap cost of one, the ILD supported the inclusion
of an unaltered inversion region on the psbT-N spacer,
whereas all other gap costs employed favored its exclusion from the analyses (Table 5).
Increasing gap costs led to more compact implied
alignments, which, especially in the ITS2 region,
increased the number of pi positions if implied alignments were studied as static alignments (Table 4). The
lengths of the psbT-H and trnL-F sequences in the
implied alignments were quite insensitive to differences
in gap costs: the psbT-H region was 556–559 bp and
trnL-F 528–552 bp long. The number of pi positions
also varied, mainly in the ITS2 region (Tables 3 and 4),
with only minor variations in the trnL intron and the
psbT-N spacer. The topologies based on combined
analyses of all data also strongly reflected the phylogenetic signal from the ITS2 region when higher gap
169
costs were applied. In those analyses, topologies were
closer to those obtained from analyses of only ITS2
data. For example, ITS2 never resolved Rhynchostegioideae, Palamocladium, and Kindbergia praelonga as
monophyletic, or with higher gap costs the Squamidium
clade as sister to Rhynchostegielloideae (Table 6).
Since our Nona analyses were based on POY character
optimizations in which gaps were treated as missing data,
differences between a Nona topology (Fig. 3) and a
topology from POY with a gap cost one (Fig. 1a) resulted
from a different treatment of gaps. When character
optimizations obtained with higher gap costs were
analyzed with Nona, some of the topological differences
occurred due to weighting of the gap information in the
POY analysis. We used abbreviations for Nona analyses
based on different POY character optimizations in the
text and tables. For example, Nona 1 : 1 : 1 means that
Nona analyses based on POY character optimization
were obtained with equal weighting for transitions and
transversions, and a gap cost of one, and in Nona
1 : 1 : 2 analyses character optimization was obtained
from POY analyses with equal weighting for transitions
and transversions, and a gap cost of two.
Topologies based on the Nona analyses of POY
character optimizations constantly differed in some
details from those obtained from the original POY
analyses (see Table 6). The differences resulting from
gap treatment were, however, surprisingly small,
considering the large difference in numbers of pi
characters. For example, when gaps were treated as
missing data, POY character optimizations of ITS2
using a gap cost of one had 154 pi characters in the
124 terminal data set (Table 4), but when gaps were
included in analyses as a fifth character state, the
number with the same optimizations was 406. Nona
resolved the Rhynchostegioideae clade (including
Rhynchostegium, Palamocladium, Scorpiurium Schimp.,
Aerobryum speciosum Dozy & Molk., most Eurhynchium species and Platyhypnidium riparioides) most often
as monophyletic in the basal position compared with
other Brachytheciaceae (Fig. 3), which never occurred
in POY analyses. Bryoandersonia illecebra was also
almost always placed within the Rhynchostegioideae
clade in Nona analyses, while POY resolved it most
often in Rhynchostegielloideae.
Based on ILD metrics, POY analyses gave less
congruent results than analyses of POY character
optimizations with Nona (Table 5). Both Nona
1 : 1 : 1 and Nona 1 : 1 : 4 analyses excluding the
psbT-N loop region gave the most congruent results of
all Nona analyses (Table 5). Nona 1 : 1 : 1 analyses also
resulted the hypothesis of relationships that was very
similar to the current classification of the Brachytheciaceae (Table 6). The shortest trees were obtained when
POY character optimizations with a gap cost of one
were used as a static alignment in Nona (Nona 1 : 1 : 1,
170
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
Table 5, Fig. 3). Although the topologies from all Nona
analyses varied mainly in minor details, Nona 1 : 1 : 2
gave results that were closer to the current classification
than any of the other POY or Nona analyses (Table 6).
Because of problems with character independence in this
method of analysis, we, however, did not regard this or
any other Nona topologies as the most parsimonious
result of our research. In POY, the homology statements
in molecular data partitions may be affected by information from other data partitions, and thus in the
implied alignments the independence of information
within each partition is not guaranteed anymore (for
details see Discussion).
Indel events appeared to have an especially strong
influence on the topology in POY analyses with high
indel costs, because in addition to utilizing the information in them, they are also weighted according to gap
cost. This can be estimated when POY topologies with
higher gap costs (two or four) and Nona topologies
based on character optimizations obtained using these
costs are compared with each other (Table 6). Three
specimens of Kindbergia praelonga never formed a
monophyletic group in POY analyses with gap costs
two or four and morphology with a weight one,
although all Nona resolved these specimens as a
monophyletic group basal to Brachythecioideae. Two
Palamocladium species were also included and in Nona
analyses this genus was always monophyletic and
grouped within Rhynchostegioideae, but were polyphyletic in all other analyses. The position of Squamidium
also varied; in POY analyses with gap costs two and
four it appeared in the basal position within Homalothecium, and in the Nona analyses it was most often
resolved as a sister group of Rhynchostegielloideae or all
other species of the entire Brachytheciaceae clade (Nona
1 : 1 : 2). Topologies from all Nona analyses varied
much less than the original topologies from POY, and
this was found for all weighting schemes used (Table 6).
When gaps were treated as missing data in Nona, the
POY character optimizations obtained from one POY
run did not give trees of the same length. Only the lengths
of the shortest trees are shown in Table 5. However, for
example, in the case of Nona 1 : 1 : 4 analyses (psbT-H
loop included in original form and morphology with a
weight of 1), of the total of the obtained eight character
optimizations only two gave the parsimonious result with
a length of 3016 steps, while the remaining six optimizations resulted in trees with 3017 (one optimization), 3018
(one optimization), 3019 (three optimizations), or 3022
steps (one optimization).
Delimitation of Brachytheciaceae and relationships
within the family
The most parsimonious result (Fig. 1a), as well as all
other combined unweighted analyses (Figs 1b,c, 2 and
3), supported a monophyletic Brachytheciaceae +
Meteoriaceae (B+M) clade, leaving Isothecium alopecuroides (Dubois) Isov., Rozea subjulacea Besch.,
R. andrieuxii (Müll. Hal.) Besch., Lindigia debilis (Mitt.)
A. Jaeger and Meteoriella soluta (Mitt.) S. Okamura
outside that group. The branching order within the
outgroup families varied widely in different analyses.
Our sampling outside B+M is, however, so restricted
that we do not want to draw any conclusions about their
phylogeny even if the results of different analyses had
been congruent. The limit between Meteoriaceae and
Brachytheciaceae is always sharp, showing that they
clearly constitute two independent lineages. Species that
were transferred recently to Brachytheciaceae from
Meteoriaceae (Aerolindigia capillacea (Hornsch.)
M. Menzel, Aerobryum speciosum, Meteoridium remotifolium, Squamidium brasiliense, Zelometeorium patulum), Myriniaceae (Clasmatodon parvulus (Hampe) Sull.,
Helicodontium capillare (Hedw.) A. Jaeger), and Symphyodontaceae (Unclejackia longisetula (E. B. Bartram)
Ignatov, T. J. Kop. & D. H. Norris) were always nested
within Brachytheciaceae. On the other hand, Duthiella
Müll. Hal., which was thought to belong to Brachytheciaceae by Noguchi (1991), was always in Meteoriaceae,
lending support to the results by Buck and Goffinet
(2000). Four distinct groups can be seen within the
Brachytheciaceae (Figs 1 and 3): (1) Rhynchostegioideae
includes Rhynchostegium, Eurhynchium striatum (Hedw.)
Schimp. and E. angustirete (Broth.) T. J. Kop.,
Palamocladium, Pseudoscleropodium purum (Hedw.) M.
Fleisch., Aerobryum speciosum, Plasteurhynchium striatulum, Platyhypnidium riparoides and Scorpiurium, (2)
Rhynchostegielloideae includes Rhynchostegiella, Eurhynchium hians and related species, Cirriphyllum piliferum (Hedw.) Grout, and many small epiphytic taxa from
America, (3) Homalothecioideae includes Homalothecium Schimp., Brachythecium subgen. Velutinum Loeske,
Eurhynchium pulchellum (Hedw.) Jenn., and (4) Brachythecioideae includes most species of Brachythecium and
Bryhnia Kaur. The relationships within Brachytheciaceae and the monophyly of some groups, for example the
Rhynchostegioideae clade, varied depending on the
treatment of gaps (POY versus Nona), the number of
outgroup taxa included in the analyses, and the inverted
psbT-N loop region. While the two other major clades,
Brachythecioideae + Homalothecioideae and Rhynchostegielloideae, were constant in almost all analyses,
only Rhynchostegioideae appeared as a paraphyletic
cluster basal to Brachythecioideae in the POY analyses
with higher gap costs. In the most parsimonious analyses
with a gap cost of one, however, and in all Nona
analyses, Rhynchostegioideae was always resolved as
monophyletic (Table 6).
Rhynchostegioideae clade. This taxon includes all
species of Rhynchostegium, Eurhynchium s. str.
(E. striatum and E. angustirete), Plasteurhynchium,
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
Palamocladium, Platyhypnidium riparioides, Scorpiurium, Pseudoscleropodium (Limpr.) M. Fleisch., Aerobryum Dozy & Molk., and in Nona analyses also
Bryoandersonia H. Robinson. This clade was found to
be monophyletic in the most parsimonious results
(Fig. 1a), and in all Nona analyses in which gaps were
included as missing data (Fig. 3). In Nona analyses it is
sister to the other Brachytheciaceae (Fig. 3), while the
most parsimonious analyses resolved it as sister to
Rhynchostegielloideae (Fig. 1a). Eurhynchium striatum
(the type species of the genus) and E. angustirete formed
the monophyletic Eurhynchium within Rhynchostegioideae. Plasteurhynchium (Eurhynchium) striatulum is also
a close relative of Eurhynchium, but our analysis
supports its placement in a genus of its own. Other
species traditionally placed in Eurhynchium were found
to be only distantly related. Some of these have already
been assigned to Oxyrrhynchium, including E. hians,
E. savatieri Schimp., E. vagans, and E. pumilum (Wilson)
Schimp., and Kindbergia, with K. (Eurhynchium) praelongum (Ignatov and Huttunen, 2002; Ignatov et al.,
2004).
Platyhypnidium riparioides was nested within Rhynchostegium in the most parsimonious resolved topology.
Our results suggest that Platyhypnidium species with
rough seta (P. austrinum and P. patulifolium) are not
related to P. riparioides. Palamocladium appeared to be
monophyletic in the most parsimonious topology
(Fig. 1a), where it appeared close to Eurhynchium.
Direct optimization of only ITS2 resolved Palamocladium euchloron in the same position as in the combined
analyses, while P. leskeoides appeared in a rather
surprising position within Brachythecium, as a sister
group of the Chinese Bryhnia novae-angliae. The
weighting of gaps and the stronger phylogenetic signal
from ITS2 was thus the most likely reason for nonmonophyly of the genus in analyses with higher gap
costs (Table 6).
Scorpiurium is resolved as sister to Rhynchostegioideae in the most parsimonious topology (Fig. 1a).
Pseudoscleropodium, Plasteurhynchium, and Aerobryum
occur within the same clade with Eurhynchium s. str.
Bryoandersonia illecebra was found within this clade
only in the Nona analyses (Fig. 3) and analyses of 99
terminal data set (Fig. 2), while direct optimization
analyses of 124-terminal data set resolved it within
Rhynchostegielloideae (Fig. 1).
Rhynchostegielloideae clade. This clade was always
clearly delimited. The composition of the group remains
the same in all analyses, except for the Squamidium
clade, which includes the three pendent epiphytic species
Zelometeorium patulum, Meteoridium remotifolium and
Squamidium brasiliense (Table 6). In the most parsimonious POY analyses it appears as sister to Rhynchostegielloideae (Fig. 1a). However, if the 9 bp inversion on
the psbT-N spacer is excluded, the Squamidium clade is
171
resolved as sister to all other Brachytheciaceae (Fig. 1b).
The following groups are always included in Rhynchostegielloideae:
1. Rhynchostegiella s. str. forms a monophyletic
cluster including three Mediterranean-Macaronesian
species: R. tenella (Dicks.) Limpr., R. teneriffae (Mont.)
Dirske & Bouman, and R. macilenta (Renauld &
Cardot) Cardot.
2. The tropical Malesian Rhynchostegiella brachypodia M. Fleisch. and tropical South American-African
Aerolindigia (Rhynchostegiella) capillacea form a clade
and are fairly distant from Rhynchostegiella s. str. They
are always sister to the three epiphytic American species,
Homalotheciella subcapillacea (Hedw.) Broth., Clasmatodon parvulus, and Helicodontium capillare. The group
of these five species, which in the figures (Figs 1–3) is
labeled as the Aerolindigia clade, is present in all
analyses.
3. The genus Cirriphyllum Grout, as traditionally
delimited (Grout, 1931), appeared to be polyphyletic,
but a smaller monophyletic Cirriphyllum clade could be
found including the type species of genus, C. piliferum
and C. crassinervum (Taylor) Loeske & M. Fleisch.
(Cirriphyllum clade in Figs 1–3). Brachythecium koponenii Ignatov was found within this group in most
parsimonious analyses. Unlike other taxa earlier included in Cirriphyllum, this monophyletic Cirriphyllum (or
Cirriphyllum + Okamuraea) subclade appeared sister to
Rhynchostegiella. Our results support the positioning of
Okamuraea within Brachytheciaceae. This is in agreement with the recent data by Tsubota et al. (2002) and
contradicts its position in Leskeaceae, where it had been
placed in most of the recent publications (for example,
Noguchi, 1991; Crosby et al., 1999).
4. Oxyrrhynchium (Eurhynchium) hians and several
related species are the only members of the traditional
Eurhynchium that are included in Rhynchostegielloideae. They form a rather stable group in all analyses.
Direct optimization most often resolved Oxyrrhynchium as a monophyletic genus, excluding Oxyrrhynchium
(Eurhynchium) pumilum and the Chinese variety of
O. vagans (Figs 1 and 2, Table 6), although in Nona
analyses all Oxyrrhynchium species resolved as a
monophyletic group. Interestingly, the rare aquatic
Chinese species, known as Platyhypnidium patulifolium
and the aquatic North American endemic species
Donrichardsia macroneuron (Donrichardsia clade in
Figs 1–3) were always found adjacent to or even within
this Oxyrrhynchium group. Donrichardsia H.A. Crum
& L.E. Anderson was described as a monospecific
genus from America, and it had earlier been included in
Amblystegiaceae. The position of D. macroneuron
within Brachytheciaceae was also found in the molecular phylogenetic study of Vanderpoorten et al.
(2002), who, however, thought that it was close to
P. riparioides. Another interesting fact is that P. austri-
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S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
num, an aquatic Australian species, was also always
found within Rhynchostegielloideae, but most often in
the basal position of the clade including Cirriphyllum,
Okamuraea, and Brachythecium koponenii (Figs 1
and 2).
Brachythecioideae + Homalothecioideae clade. This
clade usually consists of two rather distinct subclades,
one centered around Homalothecium and the other
around Brachythecium (Figs 1–3). Bryhnia, Kindbergia,
Eurhynchiadelphus eustegium (Besch.) Ignatov, Huttunen & T.J. Kop. are, in most parsimonious topologies,
resolved in a basal position in Brachythecioideae.
Myuroclada maximowiczii is resolved as a sister group of
the Brachythecioideae + Homalothecioideae clade.
Homalothecioideae clade. Our results do not support
the division of Homalothecium into smaller entities, such
as Camptothecium Schimp. (including in our analyses
H. aureum (Spruce) H. Robinson, H. lutescens (Hedw.)
H. Robinson, and H. nuttalii (Wilson) A. Jaeger) and
Trachybryum (Broth.) W. B. Schofield (H. megaptilium
(Sull.) H. Robinson), because they do not form clearly
independent lineages. The closest relatives of Homalothecium appear to be Brachythecium subgen. Velutinum
(the Brachytheciastrum clade), represented in our analysis by three species: Brachythecium velutinum (Hedw.)
Schimp., B. falcatulum (Broth.) Paris, and B. collinum
(Schleich.) Schimp., with Eurhynchium pulchellum as the
next closest relative.
Brachythecioideae clade. The relationships within this
clade are the least supported within Brachytheciaceae
(compare Figs 1a–c and 3). The only group that is
always very homogeneous is one of the small Brachythecium species centered around B. reflexum (Starke)
Schimp. and B. plumosum (Hedw.) Schimp., which are
segregated by Ignatov and Huttunen (2002) as a genus
of their own, Sciurohypnum. The other species of
Brachythecium are found centered around B. rivulare
Schimp. (section Rutabula Broth.) and B. salebrosum
(Chamberlainia). However, these groupings are not very
clear in our analyses, and our results suggest that it
is better to treat Brachythecium in a broad sense.
B. rivulare and B. rutabulum (Hedw.) Schimp. are
always grouped together (B. rivulare clade in Figs
1–3), but otherwise the closest sister group of this clade
differed in various analyses. A peculiar endemic New
Guinean genus, Unclejackia (E.B. Bartram) Ignatov,
T.J. Kop. & D.H. Norris, is always found within
Brachythecium close to B. lamprocarpum (Müll. Hal.)
A. Jaeger and B. buchananii (Hook.) A. Jaeger.
Bryhnia novae-angliae is resolved within a sister group
of Brachythecium, but the three specimens do not form a
monophyletic group. The Bryhnia clade consists of two
specimens from European Russia and Siberia, which are
morphologically closer to the type of B. scabrida
(Lindb.) Kaur. (the type species of the genus). The
specimen from southern China, however, was never in
that clade, but always appeared as sister to Brachythecium frigidum (Müll. Hal.) Besch. The branching order
within Brachythecium appeared to be sensitive to
changes in the treatment of the psbT-N loop region
(Fig. 1). The position of Kindbergia also appeared to
vary (Table 6). Most of the species traditionally included in Cirriphyllum were found within the Brachythecioideae: C. flotovianum (Sendnt.) Ochyra was resolved
within Sciurohypnum and C. cirrosum (Schwägr.) Grout
and C. tommasinii (Boulay) Grout within Brachythecium
(Figs 1–3).
Discussion
Morphological circumscription of Brachytheciaceae
and its relationships to other families
Our results support the monophyly of Brachytheciaceae and Meteoriaceae, following Ignatov (1999). Ignatov’s idea of the close relationships of these two families
was based on the unique structure of the pseudoparaphyllia, which is similar in these families. The young
developing branch primordia in the B+M clade are
covered by three leaf-like structures, the pseudoparaphyllia, that are always situated regularly at a 120 angle
to each other (see Ignatov, 1999, for details). The close
relationship of the B+M group was also supported by
Buck et al. (2000a,b) and Tsubota et al. (2002). However, both of these studies included only very small
numbers of species from these families. In contrast to
the current delimitation of the family (Buck and
Goffinet, 2000), Isothecium Brid. and Rozea Besch. were
not included in Brachytheciaceae (Hedenäs, 1992; Ignatov, 1999). This is also supported by the absence of
typical brachytheciacean pseudoparaphyllia. In addition, Isothecium was resolved as a close relative of
Lembophyllaceae (Quandt et al., 2004a).
Our analyses suggest, however, that few exceptions
occur in pseudoparaphyllia patterns. Lindigia debilis,
Neobarbela comes (Griff.) Nog., and Meteoriella soluta
have all been reported to have brachytheciacean
pseudoparaphyllia patterns, but not all were found
within the monophyletic B+M clade. Although,
according to its morphology, L. debilis appears similar
to Aerolindigia capillacea, DNA sequence data suggest
they are not closely related. Neobarbella comes was not
included in our analyses, but Quandt et al. (2004a)
showed that it belongs to Lembophyllaceae. Meteoriella
soluta was resolved in our analyses in Hylocomiaceae,
which is consistent with the current classification of this
species (Buck and Goffinet, 2000). In studies of the
pseudoparaphyllia of this species we found some
formerly neglected features that appear to link it with
the other Hylocomiaceae and not Meteoriaceae. Trachypus bicolor Reinw. & Hornsch. is also certainly a
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
member of Meteoriaceae, although its pseudoparaphyllia differ from the B+M type. These exceptions are not
discussed here, but will be examined in detail in future
studies.
Despite the fact that Meteoriaceae and Brachytheciaceae are probably close relatives, the limit between the
two families has remained unclear (Buck, 1994;
Ignatov, 1999). Several morphological characters have
been suggested for distinguishing Meteoriaceae from
Brachytheciaceae, such as the presence of papillose and
short leaf laminal cells, strongly modified peristome,
pendent growth, short seta and hairy calyptra. Due to
the wide variation in these characters in Meteoriaceae,
it was hard to find a character combination for
taxonomic delimitation. Our results within the B+M
clade suggest that a single character alone can be used
to delimit these families. All Meteoriaceae have papillose laminal cells, while in Brachytheciaceae they are
smooth. Surprisingly, this very simple character
appeared to be crucial to family level classification,
although at the species and genus level, papillosity
appears to be in some cases very unreliable (Quandt
et al., 2004b). Thus, delimitation of the Brachytheciaceae is becoming easier because all species, with the few
exceptions mentioned above, can be separated from
close relatives within the pleurocarpous mosses using
two characters: the typical brachytheciacean pseudoparaphyllia and non-papillose cells.
Our analyses do not support Plagiothecium as a close
relative of Brachytheciaceae, as was suggested by Buck
et al. (2000a,b) and Tsubota et al. (2002). This may also
be the result of an uneven selection of outgroup species
and a higher sequence variation within the outgroups, as
compared with the ingroup, which may lead to unnatural groupings in the outgroups (Barriel and Tassy,
1998). The branching order of the outgroups was fairly
variable in our analyses.
New insights on relationships of the main genera
of the Brachytheciaceae
As was often suspected (Hedenäs, 1992; Ignatov
et al., 1999; and many others), some of the largest
genera in Brachytheciaceae are unnatural and their
classification should be corrected. Formerly, only those
attempts to clarify classification that have led to the
formation of small, mono- or oligotypic genera were
successful. The few attempts to rearrange the large
genera of Brachytheciaceae, by dividing them into a few,
but still fairly large entities, were all unsuccessful (see
details in Ignatov and Huttunen, 2002). The most
important factor leading to this situation was the
difficulty in finding morphological characters to justify
these new groups. In light of our results, it appears that
practically all the morphological characters used in
generic level classification in Brachytheciaceae have
173
evolved independently several times in many lineages
and thus cannot serve by themselves as diagnostic
characters. Phylogenetic analysis including molecular
data will clearly change the traditional classification,
and some new placements were quite unexpected.
However, we found all the stable new placements
acceptable and, after a re-evaluation of the morphological characters, well founded.
Brachythecium, with 100–150 species, has always been
the largest genus in the family. It has been stated to be
an artificial assemblage by many authors (see Hedenäs,
1992; Ignatov et al., 1999), but at the same time division
into several sections by Kindberg (1897) has been widely
accepted. Robinson’s (1962) attempt to split the genus
into two, Brachythecium (species centered around B. rivulare) and Chamberlainia (species around B. salebrosum)
was, however, not accepted. In Chamberlainia, Robinson combined the sections Salebrosa Broth. and Velutina
De Not., leaving in Brachythecium the sections Rutabula
Broth., Reflexa Broth., and Cirriphyllopsis (Broth.)
Takaki (B. plumosum–B. populeum group). Our analysis
showed that these sectional limits are stable, while their
relationships differed from Robinson’s view (1962).
Section Velutina is clearly distinct from other sections,
and it is closer to Homalothecium than to any Brachythecium species. Two other sections, Cirriphyllopsis and
Reflexa, form a very clearly delimited group. Based on
our results, the latter group, as well as sect. Velutina,
should be excluded from Brachythecium and placed in
genera of their own (Ignatov and Huttunen, 2002).
Sequence variation within Brachythecium is very low,
which is probably one reason for some of the minor
differences which occur in the branching order in
different analyses. The basal taxa centered around B.
rivulare have rough seta (Fig. 1a), and ecologically they
are meso- to hygrophytic, while the apical part of the
clade centered around B. salebrosum includes more xeric
species with smooth seta (Fig. 1a). These two groups
were in most of the analyses somewhat poorly delimited.
Based on the current results, there is not enough support
for segregating the latter part into Chamberlainia, as
Robinson (1962) suggested. Another problem with
Brachythecium s. str. is that it also includes the southern
Chinese specimen of Bryhnia novae-angliae. It is clear
that analyses which include more specimens are needed
in order to resolve the relationships of this species.
The apical part of Brachythecium includes temperate
to tropical species with a slightly reduced sporophyte.
These species often have a strongly reduced annulus and
capsule inclined to suberect, as exemplified by B. buchananii, B. ruderale (Brid.) W. R. Buck, B. lamprocarpum,
B. laetum (Brid.) Schimp., and B. acuminatum (Hedw.)
Austin. This group also includes a peculiar species,
Unclejackia longisetula, which was originally described
as a distinct genus (Ignatov et al., 1999). To redefine the
genus level delimitation for the Brachythecium clade,
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S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
either Unclejackia should be transferred to Brachythecium, or Brachythecium should be divided into several
smaller genera. The latter choice would result in a
situation with distinct phenotypes of Brachythecium
salebrosum being assigned to different genera (Figs 1
and 2). Although this would enable the retention of
Unclejackia as its own genus, it does not appear to be a
reasonable solution to the problem. Inclusion of Unclejackia in Brachythecium, on the other hand, makes the
morphological characterization of Brachythecium extremely broad. U. longisetula is a large, golden-lustrous
moss with strongly concave leaves, very flexuose leaf
acumen, very short costa (0.1–0.2 of leaf length), and
strongly modified peristome. Unclejackia has most likely
been derived from an ancestor resembling species in a
group of tropical Brachythecium. It has probably
evolved rapidly into a morphologically distinct species
occurring in a geographically very limited area.
U. longisetula is found only in the alpine ‘‘savannas’’
of New Guinea, where it has been collected only on
trunks of tree ferns. Unclejackia was recently transferred
to Brachytheciaceae (Ignatov et al., 1999), and this
placement was immediately criticized. For example, Tan
(2000) noted that Unclejackia would be better placed in
Chaetomitrium Dozy & Molk. (Symphyodontaceae).
Rhynchostegium, with 30–70 species is the second
largest genus in the family. It was also considered an
unnatural segregate, but in our analysis it was resolved
as a very homogeneous and stable monophyletic clade.
Our analysis included species showing wide morphological variation: tropical ‘‘typical Steerecleus’’ (R. pallidifolium (Mitt.) A. Jaeger and R. serrulatum (Hedw.) A.
Jaeger) with pale color and large complanately arranged
leaves, R. murale with imbricate leaves and deep green
color, R. confertum which is usually deep green and
often with costa ending in a spine, R. psilopodium
Ignatov & Huttunen with pure green color and narrow
leaves (previously placed in Rhynchostegiella), and R.
rotundifolium (Scop.) Schimp. with exceptionally lax
areolation of leaves and dark green to brownish color.
The position of Platyhypnidium riparioides remains
ambiguous; our analyses resolve it as a close relative
of Rhynchostegium (Figs 1–3), in which it had formerly
been placed (Takaki, 1956; Hedenäs, 1992, etc.). However, a lack of the most informative ITS2 sequence data
in this species may affect its position. Morphologically,
it shares several leaf characters with Rhynchostegium.
Eurhynchium, unlike Rhynchostegium, appeared to be
even more heterogeneous than expected. Species placed
in this genus were found in all major clades, and it
should be segregated into several smaller units and some
of the species placed in other genera. Eurhynchium is the
third largest genus of the family. Ignatov and Huttunen
(2002) retained only two species in Eurhynchium, while
the remainder were transferred to Plasteurhynchium,
Oxyrrhynchium, Eurhynchiastrum Ignatov & Huttunen,
Eurhynchiadelphus, and Kindbergia. Of the species
occasionally included in this genus, Rhynchostegium
(Eurhynchium) serrulatum was confirmed as belonging
to Rhynchostegium, Platyhypnidium (Eurhynchium)
riparioides, Eurhynchium muelleri (A. Jaeger) E.B.
Bartram, Rhynchostegium aquaticum A. Jaeger to Platyhypnidium, and E. macroneuron (Grout) H.A. Crum to
Donrichardsia. Eurhynchium (Platyhypnidium) austrinum
(Hook. & Wilson) A. Jaeger was resolved as sister to
Rhynchostegielloideae. Our results support the recognition of Oxyrrhynchium for E. hians and its close
relatives.
The two most important reasons for the long misunderstanding of this group are probably the enormous
morphological variation in some of the widespread
species of Oxyrrhynchium and the occurrence of many
similarly appearing and highly variable species. For
example, the E. hians group shows a wide morphological
variation that overlaps not only with other Oxyrrhynchium, but also with species in other brachytheciaceaen
genera. Finding morphological differences delimiting it
from other Oxyrrhynchium as well as from other genera
is a real problem.
Most species of Cirriphyllum were originally placed in
Eurhynchium and have frequently been transferred from
one genus to another. Two species of Cirriphyllum, C.
piliferum and C. crassinervum, formed a monophyletic
clade, while other species were mainly resolved within
Brachythecium. The recently described Malesian B.
koponenii, a species with ambiguous affinity, is better
included in Cirriphyllum.
Rhynchostegiella appeared to be polyphyletic, and
Ignatov and Huttunen (2002) suggested that it be split
into three groups of more or less equal size. These
groups have no, or only partial, geographic overlap;
unlike Eurhynchium, all occur within only one of four
major clades within Brachytheciaceae, namely Rhynchostegielloideae. Only four representatives belonging
to two of the three groups given above were included in
this analysis, but since two small genera related to
Rhynchostegiella, Aerolindigia M. Menzel and Helicodontium (Mitt.) A. Jaeger, were also included in phylogenetic analyses, we have suggested a new classification
for the entire genus (see Ignatov and Huttunen, 2002).
Homalothecium. A clear tendency towards a more
reduced peristome structure can be observed within
Homalothecium. All species in the basal part of the clade
have a complete peristome and thus have formerly been
segregated in a genus of their own, Camptothecium. Two
lineages with reduced peristomes can be found at the
crown of the clade, the Eurasian H. philippeanum
(Spruce) Schimp. and H. sericeum (Hedw.) Schimp.,
and the North American H. nuttalii. Our results do not
support the identification of H. megaptilum as a distinct
genus, Trachybryum. While Homalothecium was always
monophyletic in analyses of combined data, the trees
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
based only on chloroplast data resolved H. laevisetum
Sande Lac. within Rhynchostegium, adjacent to Palamocladium leskeoides. H. laevisetum is the most peculiar
member of the genus, with characters resembling some
of those seen in Palamocladium.
Two rather small genera appeared to be polyphyletic.
Three species of Platyhypnidium included in our analyses, P. riparioides, P. austrinum, and P. patulifolium
never formed a monophyletic group and they did not
even appear to be closely related. While the latter two
species were always resolved within Rhynchostegielloideae, P. riparioides appeared in Rhynchostegioideae,
usually close to, or among, Rhynchostegium.
Of three specimens of Bryhnia novae-angliae, two
from Europe and Siberia that were also morphologically
most similar to each other, were found to be closely
related. At the same time, a specimen from central
China was always resolved as sister to Brachythecium
frigidum, instead of sister to the other two Bryhnia
specimens. The Chinese specimen also differs in morphology from the northern specimens, but improvements in species delimitation are hampered by the
inclusion of the East Asiatic Bryhnia novae-angliae
specimens, which show all the transitional morphotypes
from the Chinese to the Siberian.
Evaluation of morphological characters
in the Brachytheciaceae
Almost all morphological characters of Brachytheciaceae show homoplasy to a large extent. Despite this,
two synapomorphies characterizing Brachytheciaceae,
non-papillose leaf cells and the presence of a unique
pseudoparaphyllia pattern, can also serve as diagnostic
characters for separating brachytheciaceaen species
from other pleurocarpous mosses. On the genus level,
few characters appear to be unique for certain genera
within Brachytheciaceae, such as a reduced exostome in
Clasmatodon parvulus, immersed to emergent capsules in
Squamidium (Müll. Hal.) Broth. and Zelometeorium
Manuel, and the presence of paraphyllia in Kindbergia.
In practice, most of the clades found in phylogenetic
analyses cannot be delimited using currently known
morphological characters. Based on our analyses, we
can now reconsider the value of those characters that
were thought to be especially important in earlier studies
of the Brachytheciaceae.
Peristome reduction was thought to be very important
for classifying the Brachytheciaceae up to the late 19th
century. For example, Schimper (1876) did not include
Homalothecium in this family due to its reduced peristome. Later, however, peristome reduction was found to
be a common phenomenon in many lineages of pleurocarpous mosses. In our analyses, lineages with peristome reduction occur in all major clades and many
subclades. In Rhynchostegioideae, Aerobryum and Pal-
175
amocladium are plants showing a clearly reduced peristome structure, and of those taxa not included in the
present analysis, but expected to belong to this group,
Eriodon also exhibits this character state. Most of
the genera in Rhynchostegielloideae have more or
less reduced peristomes, with the exception of Oxyrrhynchium, Cirriphyllum, Platyhypnidium austrinum and
P. patulifolium. In Homalothecioideae, Homalothecium
species have reduced peristomes. The pattern of peristome reductions in various lineages within this genus
varies somewhat and is also different from that of
Unclejackia longisetula and Brachythecium acuminatum,
representatives of the Brachythecioideae with reduced
peristomes.
Interestingly, some species with relatively ‘‘complete’’,
‘‘hypnoid’’ peristomes display hygrocastique hygroscopic movement (for example, Palamocladium, Homalothecium lutescens, Rhynchostegiella tenella), whereas
some tropical taxa with rather modified peristomes with
heavily incrassate teeth covered with branched papillae
display xerocastique movement (Malesian Rhynchostegiella, referred by Ignatov and Huttunen (2002) to
Remyella Müll. Hal.). In the latter case, peristome
modification is associated with a number of other
characters, which might often have evolved together,
such as straight perichaetial leaves and erect capsule.
Hence, the presence of modification appears to be a
more important character than its hygroscopic movement pattern.
Surface structure of the seta appears to be one of the
most stable characters, showing no homoplasy in some
clades (Fig. 1a). This character is frequently used in keys
of Brachythecium, as a character distinguishing sections.
The setae are invariably smooth in all species of
Rhynchostegioideae (without exception!), but rough in
most species of Rhynchostegielloideae. The latter group,
however, has some taxa with smooth setae, such as
Rhynchostegiella tenella, Eurhynchium vagans, Meteoridium remotifolium, and Squamidium brasiliense. Most of
the basal species in Brachythecioideae have rough setae,
while most of the apical groups, with some exceptions,
have smooth setae.
Operculum shape was considered to be one of the
most important characters in Brachytheciaceae over the
last 150 years. Our analysis shows that this character is
relatively stable in some clades, but not in all. Species in
Rhynchostegielloideae invariably have a rostrate
operculum, as do Rhynchostegioideae (except Pseudoscleropodium; Fig. 1a). The operculum shape in Brachythecioideae is more variable than was previously
thought. Two species traditionally placed in Cirriphyllum or Eurhynchium, C. tommasinii and C. flotowianum,
appeared in Brachythecioideae. The shape of the operculum is rather variable in basal species of Brachythecioideae and in Homalothecioideae. Thus, to use this as
a key character for separating Eurhynchium and Brach-
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ythecium has led to unnatural classifications, i.e., the
misplacement of some species.
Phyllodioicy is a rather rare feature in the pleurocarpous mosses. In Brachytheciaceae it has evolved several
times independently in three major clades: (1) in
Rhynchostegioideae (Aerobryum, Eurhynchium s. str.,
Palamocladium), (2) in Homalothecioideae (many
Homalothecium species and Eurhynchium pulchellum),
and (3) Brachythecioideae (Eurhynchium eustegium and
Brachythecium auriculatum A. Jaeger).
Annulus structure is difficult to evaluate if only
herbarium material is available. Hence, it is safe to
discuss only the most contrasting character state, that of
total absence of specialized rows of annulus cells, which
remain after operculum removal at the mouth of the
urn. The annulus is absent in the most derived members
of the Brachythecium clade, which mainly occur in
tropical and temperate areas. Usually these species tend
to have only a slightly curved capsule instead of a
strongly curved, typical brachythecian one. Most have
smooth setae (B. buchananii, B. ruderale, etc.) with only
one exception, Unclejackia. Species centered around
B. salebrosum commonly have an annulus consisting of
only one cell row, which is rather firmly attached and
difficult to take apart. This can be assumed to present a
form that is transitional to the 2–3-rowed annulus
occurring in most species of the family.
Shape of the pseudoparaphyllia is a stable character in
Rhynchostegioideae, which always has a triangular
pseudoparaphyllia. There are several lineages within
Rhynchostegielloideae, and especially Brachythecioideae, which show a transition to a long and narrow
pseudoparaphyllia (Aerolindigia, Remyella, Rhynchostegiella, Homalothecium, p. p. Kindbergia, the Brachythecium buchananii group and part of the B. salebrosum
group, and Unclejackia). In most cases, the shape of the
pseudoparaphyllia correlates with epiphytic growth,
although Kindbergia is an exception. Some other typical
epiphytes, however, have short triangular pseudoparaphyllia (cf. the Squamidium group).
Costa ending in a spine appears to lack value as a
character at the genus level and above, although it is
frequently used in generic keys. In both Rhynchostegium
(for example, R. confertum (Dicks.) Schimp.) and
Brachythecium (for example, some populations of B.
salebrosum) are some specimens with a fairly strong
tooth at the end of the costa, whereas Eurhynchium
hians, which often has a clear spine, may have a smooth
end of the costa in shaded habitats.
Size of alar cells in basal corners of leaves and their
cell wall thickness appear to be good characters at the
species level, whereas their use at higher levels is
doubtful.
Hairy calyptras are never present in Rhynchostegioideae, but some or all species of Squamidium, Zelometeorium, Okamuraea, Brachythecium (B. complanatum
Broth., B. coreanum Cardot, B. auriculatum), and
Homalothecium have sparsely hairy calyptras. This
character has also been reported in Bryhnia trichomitria
Dixon & Thériot, but its position in this genus should be
confirmed. It, however, appears to be a close relative of
the basal species of the Brachythecium clade, especially
B. auriculatum.
Although axillary hairs may have a rather variable
structure, some peculiarities in shape and color were
found to be useful for classification. Species of
Rhynchostegium often have long, multicellular axillary
hairs with pale basal cells and upper cells which are
more intensively colored. In some species axillary hairs
were found to be verrucose, but this is probably not a
stable character, even at the species level. Axillary hairs
in Rhynchostegiella are usually short, consisting of 2–3
cells of the upper cell as in Oxyrrhynchium vesiculateinflated. This pattern can be seen in some, although
not in all, axillary hairs within a single specimen. A
similar pattern was observed in some Rhynchostegium
specimens, as in the Chinese R. inclinatum (Mitt.) A.
Jaeger, which however, was not included in our
analysis. Axillary hairs in Homalothecioideae are often
composed of several relatively short cells, while in
Brachythecium s. str. they are usually with 2–3 cells, the
upper being long and more or less distinctly tapering at
the distal part. The latter character state is less clear in
the group of species centered around B. reflexum and
B. populeum (Hedw.) Schimp. in the Sciurohypnum
clade.
Color of plants is in most groups of Brachytheciaceae
quite indistinctly green, turning with age to yellow- or
brownish-green. Two peculiar groups, however, can be
found within the family. In the ‘‘Chamberlainia’’ group
(Brachythecium albicans (Hedw.) Schimp., B. buchananii, B. ruderale, and Cirriphyllum cirrosum), the plants
never become brownish with age, but instead turn very
pale stramineous. Another peculiar group is Rhynchostegioideae, with plants turning silvery white when they
become older. This pattern is especially noticeable in
tropical Rhynchostegium species, but some silvery white
aspect is often also present in such taxa as Eurhynchium
angustirete, Aerobryum speciosum, and sometimes in
Pseudoscleropodium purum. In some tropical species the
older parts of plants easily become black. This character
is widespread in Meteoriaceae, while in Brachytheciaceae it is found in two groups, which our analysis
suggests are unrelated: the Squamidium clade (including
Squamidium, Zelometeorium, and Meteoridium) and
Aerobryum.
Prorate leaf laminal cells are found in Bryhnia,
Eurhynchiadelphus eustegium and Kindbergia, all of
which were found to be closely related. Plasteurhynchium striatulum has similar distinctly prorate cells but is
not closely related to other species showing this character. Cells are treated as prorate only when the prorae
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
are very distinct and tooth-like, while a small proration
can be traced in a powerful light microscope in many
other species.
Longitudinal leaf plication is characteristic for
Homalothecioideae, Brachythecioideae and Rhynchostegioideae, including, for example Palamocladium and
Eurhynchium, but this character is also very rarely found
in Rhynchostegielloideae (Cirriphyllum, Okamuraea).
Methodological discussion
The combined analyses of molecular and morphological characters revealed a well-resolved phylogeny of
Brachytheciaceae. As expected, morphology alone did
not include enough information to resolve the relationships of closely related species and genera, which may be
the result of the presumably young age of the family.
Most of the clades found in our analyses were very close
to some groupings in earlier classifications of Brachytheciaeceae (Brotherus, 1925; Robinson, 1962; Hedenäs,
1992; Ignatov, 1998). It appears that, although finding
reliable diagnostic characters for delimiting these groups
has been difficult, there is still something in the general
appearance of species that has enabled their classification together. In addition to resolving the phylogenetic
relationships in Brachytheciaceae, our aim was to
determine whether the number of outgroups and the
phylogenetic information in indel events, or in short
inversion region in psbT-N loop, affect the topology of
phylogenetic hypotheses. We showed that: (1) although
the indel events in our study include a large amount of
phylogenetic information, the topologies obtained with
and without these data are only conflicting in two
details, the position of Bryoandersonia illecebra and
Rhynchostegioideae (Figs 1a and 3); (2) in conflict with
results of Quandt et al. (2003), the inversion occurring
in the 9 bp loop region in the psbT-N spacer appeared
highly informative in Brachytheciaceae; and (3) the large
numbers of outgroups and resulting increased sequence
variation did not disturb phylogeny reconstruction in
direct optimization, but improved the congruence
among data partitions, as measured with the ILD.
In POY, the optimization process is affected by both
parameters used, and interactions between included data
sets. Sometimes even fairly small changes in the original
data set, such as the exclusion of the 9 bp in the psbT-N
spacer in this study, may lead to different character
optimization and topology (Fig. 1a–c). Although the
treatment of gaps as a fifth character state remarkably
increased the information available for phylogeny
reconstruction (see Results), this had only a minor
effect on topology (Figs 1a and 3). One reason for this
may be that, due to the high number of indel events in
ITS2, it is possible to optimize this region so that very
little conflict results with phylogenetic signals from other
data sets.
177
It is rather surprising that analyses based on direct
optimization were, according to ILD metrics, always
less congruent than results based on the same POY
implied alignments analyzed with Nona. The POY
topologies obtained were always based on the most
parsimonious optimization of all available characters,
but when the character optimizations from POY are
analyzed in Nona as static alignments, and the gaps
treated as missing data, they may actually appear to be
suboptimal (see also Wahlberg and Zimmerman, 2000).
Recently, character optimizations from POY have often
been used like ordinary alignments in phylogenetic
studies (Wahlberg and Zimmerman, 2000; Cognato and
Vogler, 2001; Belshaw and Quicke, 2002; Quandt et al.,
2004b). Despite the possible suboptimality resulting
from different treatments of gaps (of the previously
mentioned studies, only Cognato and Vogler (2001)
used the same gap cost for succeeding phylogeny
reconstruction), POY character optimizations are still
usually better (in terms of tree length) than static
alignments obtained using other methods. Independence
of characters in gap-containing regions will, however, be
lost, due to the interactions between data partitions
during direct optimization. Hence, the use of character
optimizations as static alignments in phylogenetic analyses violates the basic assumptions of cladistic analyses
and is not recommended as a method for solving
sequence alignment problems. The different treatment
of indel events, as well as the lack of weighting during
analyses also explains the more stable topologies
obtained from Nona with different POY character
optimizations. The higher gap cost in POY also means
a higher weighting of indel events in analyses, while in
Nona, information included in the gaps is totally
ignored. Due to this the effect of gappy ITS2 is clearly
stronger in POY analyses with higher gap costs.
Topologies obtained from POY with gap costs of two
and four share more features with the topology based
only on ITS2, while trees with a gap cost of one more
closely resemble Nona topologies (Table 6).
Analyses with large numbers of outgroup taxa
improved the congruence among data sets. Despite this,
topologies from POY analyses remained fairly similar to
those obtained from analyses with one outgroup (Floribundaria floribunda). It might be expected that if the
sequence variation is high, exclusion of the most
variable outgroup species could make character optimization easier and hence improve the results. For
example, ITS2 has often been avoided in analyses in
many phylogenetic studies of mosses, because this
region has been considered as impossible to align.
However, wider sequence variation within the large data
set (Table 3) increased the congruence between different
data sets (Table 5), the lower sequence variation
decreased the resolution in the analysis of 99-terminal
data set, and cladograms from both analyses differed
178
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
only in minor details (Figs 1a and 3). The advantages of
a wide sequence variation for phylogeny reconstruction
have been observed previously (Källersjö et al., 1999;
Broughton et al., 2000), and recent phylogenetic studies
have reported that adding taxa in analyses increased
their resolution (Pollock et al., 2002; Rydin and
Källersjö, 2002; Stenroos et al., 2002).
The effect of morphological data on our analyses
appeared rather small if the topologies based on only
molecular data and all data are compared. Some genera
(Palamocladium) and species (Kindbergia praelonga,
Rhynchostegium murale, Brachythecium buchananii),
however, were monophyletic only if the morphological
data were included. In Wheeler and Hayashi’s (1998)
study, where they used direct optimization and tested
different weighting schemes for morphological data,
weighting morphology equally with indel cost gave
consistently more congruent results in light of the ILD
compared with those in which morphology alone was
given a weight one. Equal weighting of indel events and
morphology is becoming a common method in combined POY analyses (Giribet et al., 2002; Schulmeister
et al., 2002), and this is often defended by highlighting
the higher value of morphological data compared with
sequence level data. Weighting morphological data does
not, however, necessarily lead to a higher congruence
between data partitions in all analyses. Thus, the
differential weighting of morphological data is only
based on rather subjective views of the value of that data
partition. In our case, for example, the weighting
method where morphology always attained a weight of
one gave the most congruent results, according to the
ILD metric.
Although the short inversion of the psbT-N spacer
loop was only expected to increase homoplasy within
the data set (Quandt et al., 2003), it appeared to be very
informative within Brachytheciaceae (Fig. 1, Table 2).
Similar short inversions are expected to be common in
non-coding chloroplast regions (Kelchner and Wendel,
1996; Graham et al., 2000), although there are few
documented examples of this. These regions are said to
be prone to frequent changes, which has raised the
question of their phylogenetic informativeness and how
they should be treated in phylogenetic analyses (Kelchner and Wendel, 1996; Quandt et al., 2003). In our
study, the exclusion of a psbT-N loop in analyses with
gap costs of two or four increased the congruence
between data sets in POY analyses and some groupings
in parsimonious trees changed. In most parsimonious
analyses, however, any altering of the loop region
decreased congruence. Thus, in our analyses, we can
see no reason to ignore the information in the psbT-N
loop region. It clearly contains some phylogenetic
information in our study groups, and even if ILD
favored the exclusion of the loop positions from the
analyses, we believe that unnecessary exclusions of data
should always be avoided. Our results support Wenzel
and Siddal’s (1999) view that the globally homoplasious
characters may be locally informative, and hence their
value in any specific study cannot be known without
testing. The base composition within the loop region
seems to include phylogenetically important information and, unlike in the Quandt et al. (2003) study, its
inclusion in the analyses did not have major negative
effects on them.
Our final aim was to revise the classification of
Brachytheciaceae, and for this purpose we need to select
one topology to use as the basis of the new arrangement.
Currently, three different methods can be used to select
the topology for taxonomic revisions in studies utilizing
the direct optimization. First, based on sensitivity
analyses, we can regard the most congruent topology
according to ILD as the best solution. A second choice
is to name only those groups that are stable despite the
variation in substitution costs in the direct optimization
analyses. The third possibility is to avoid all a priori
weightings of data partitions, including the differential
weighting of gaps, and to regard the topology showing
the smallest number of evolutionary steps as the optimal
topology. Of these, the first and third are based on
different views of the concept of parsimony in phylogenetic analyses. While the third represents the traditional concept of parsimony in phylogenetic analyses, in
the first one taxonomic congruence is accepted as an
additional criterion.
As discussed by Schulmeister et al. (2002), the first
method—the use of ILD as a measure of optimality—is
based on the idea that congruence among data partitions can be used as an additional criterion for selecting
the most parsimonious results. According to this
method, the parameter set resulting in the least conflict
between data partitions, and giving the most congruent
result, is regarded as the most parsimonious solution. In
our case, ILD supported the direct optimization analyses and the topology that was obtained with a gap cost
of two and morphology with a weight of one. We find it
hard to accept the differential weighting in this analysis,
as it results in a topology which differs from the most
parsimonious hypothesis of relationships (see Grant and
Kluge, 2003). In addition, some details in topology from
this analysis, such as the polyphyly of Palamocladium
and Kindbergia praelonga, seem surprising in light of the
morphological variation within these taxa.
Giribet (2003) suggested the use of stability of clades
under different substitution costs as a criterion for
accepting them as taxonomic units. As with the first
method, this is also based on exploring the effect of
differential weightings of data partitions on direct
optimization analyses. In this case, the instability of
clades under different substitution costs will be detected
using the sensitivity analyses, and only those clades that
appear most stable with different parameters are accep-
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
ted. They can be visualized, for example, by constructing
the stability trees that sum up the results from different
analyses (see for example Schulmeister et al., 2002) or
using the graphic sensitivity plots, ‘‘Navajo rugs’’ (see
Wheeler, 1995). Stability is thus used as a measure of
support. In our study this would lead to taxonomic
conclusions fairly similar to those of the first method.
For example, the polyphyly Palamocladium and Kindbergia praelonga, and the paraphyly of Rhynchostegielloideae are always supported in all other gap costs
except one (Table 6), and thus the use of stability (sensu
Giribet, 2003) would lead discarding them as taxonomic
units.
Although the use of sensitivity analysis (sensu Wheeler, 1995) for testing the effect of different parameters on
topology and congruence between different data sets is
becoming almost a standard method for evaluating
results, this method has also been criticized. For
example, Frost et al. (2001) and Grant and Kluge
(2003) stated that this type of search for additional
criteria is unnecessary and unrelated to parsimony.
According to them, the only truly parsimonious result
would be that obtained using equal weighting (i.e., all
indel and substitution events and morphology with the
same weight) and to avoid testing several indel and
substitution parameters. Thus, the optimal most parsimonious topology to use as the basis for classifications is
the shortest tree from unweighted analyses of all
available data, the third method of those listed above,
and including also the data from 9 bp inversion region
in psbT-N spacer. This alternative is based on the idea
that equal weighting in analyses minimizes the number
of additional assumptions because any differential
weighting schemes including the weighting of indel
events can be regarded as adding unnecessary ad hoc
hypotheses leading to unparsimonious results. The
weighting of gap positions increases the number of
assumed evolutionary events and hence equal weighting
provides the most parsimonious explanation for the
data. Based on our own observations with the present
data, POY analyses with lower gap costs usually lead to
‘‘looser’’ character optimizations allowing, at least in
most variable sequence regions such as ITS2, more
autapomorphic positions. This reduces the number of pi
positions within the data set (Table 4, see also Caterino
and Vogler, 2002; Quandt et al., 2004b).
Thus, we regard the topology obtained with equal
weighting as the only truly parsimonious hypothesis of
relationships in Brachytheciaceae (Fig. 1a). In addition,
this topology also avoids all of the most doubtful
groupings and maintains the need for minimum nomenclatural changes. Although the stability of the nomenclature cannot be used as a desirable goal of
phylogenetic systematics (Dominguez and Wheeler,
1997), it is easy for us to prefer the choice that is both
a parsimonious explanation of the data as well as the
179
most congruent with the nomenclature and traditional
classification of Brachytheciaceae (see also Wiley, 1981).
Acknowledgments
We thank our colleagues for providing specimens of
Helicodontium capillare, Meteoridium remotifolium,
Homalothecium megaptilium (Angela Newton, BM);
Scorpiurium deflexifolium (Solms.) M. Fleisch. &
Loeske, Rhynchostegiella teneriffae, R. macilenta (Lars
Hedenäs, S); Lindigia debilis, Homalotheciella subcapillata, Rozea subjulacea, R. andrieuxii (William R. Buck,
NY). Sequences of South American Kindbergia praelonga were kindly provided us by Dr Dietmar Quandt. We
want to thank Taran Grant, Arnold Kluge and Gonzalo
Giribet for giving access to their unpublished manuscripts. We are thankful to three referees, Drs Vesa
Selonen, Jaakko Hyvönen, and all our colleagues in the
Finnish Museum of Natural History for valuable
comments on manuscript. This work was financially
supported by the Academy of Finland (to Jaakko
Hyvönen’s Bryosphere project and to Timo Koponen’s
Biodiversity of SE Asiatic Bryoflora project), Finnish
Cultural Foundation, Center for International Mobility
(CIMO), SYS Resource researcher exchange program in
the Natural History Museum, London, the Program
‘‘Biodiversity’’ of Russian Academy of Sciences, and
Russian Foundation for Basic Researches.
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Appendix 1
Morphological characters
0. Color of plant: 0—light green, easily turning to pale
stramineous (e.g. Brachythecium albicans); 1—pure green
to dark green or brownish-green (for example, the grayish
green of Helicodontium); 2—light green easily turning to
whitish argent (many tropical species of Rhynchostegium).
1. Black color in older parts of plants: 0—absent;
1—present.
2. Growth: 0—stem prostrate, never ascending, sympodial branches arching (Brachythecium reflexum, Eurhynchium angustirete); 1—stem ascending in apical parts
(Homalothecium ssp., Brachythecium albicans, B. glareosum, etc.); 2—stem pendent (Aerolindigia, Zelometeorium, Meteoridium).
3. Foliage: 0—loose, stem visible almost throughout
except shoot apices, 1—moderately dense, stem only
partially visible, 2—dense, stem not seen in well-developed young shoots.
4. Branch foliage: 0—terete; 1—more or less complanate. In freely branched species this character was
evaluated for secondary axes.
5. Branching: 0—irregularly pinnate; 1—regularly
pinnate with rather densely arranged branches of the
same length (Kindbergia, Pseudoscleropodium, some
Homalothecium, also outgroup taxa).
6. Branches: 0—not curved, 1—regularly curved (cf.
Scorpiurium spp.). Somewhat flexuose and occasionally
curved branches are coded as 0.
7. Stipelike pattern: 0—absent; 1—present. This
character means, that the proximal part of the sympodial branch has remote foliage with much smaller and
strongly modified, usually deltoid leaves that are often
reflexed. The pattern is quite clear when observed at a
certain distance. It is not expressed in all specimens, but
if it was observed in at least some, it was coded as 1.
8. Central strand: 0—present; 1—absent. Among
ingroup taxa only Rhynchostegiella and Homalotheciella
display the total absence of the central strand. This
character was coded as 1 in cases when at least two
transversal sections of well developed shoot showed no
traces of a central strand, although in some other
sections a 1–3-celled strand could be discerned (Zelometeorium, Rhynchostegiella teneriffae, etc.).
9. Stem cortex: 0— 0–4 cell layers thick; 1 to > 5 cell
layers thick. Those cases, in which a thick cortex was
observed in at least some transverse cross-sections were
coded as 1.
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S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
10. Color of upper cells of axillary hair: 0—hyaline;
1—brownish. If brownish axillary hairs were found only
occasionally, this character was still coded as 0.
11. Width of upper cell of axillary hair: 0—essentially
not wider than the cell below; 1—wider and inflated
compared with cell below (Oxyrrhynchium spp.). Species
were coded as 1 even if only at least some of the axillary
hairs had this pattern.
12. Axillary hairs: 0— 2–4-celled; 1—with 5 or more
cells, at least in some well-developed axillary hairs. Note
that not only the upper cells in the axillary hairs were
counted, but also the basal cells, which sometimes differ
in shape and coloration from apical cells.
13. Pseudoparaphyllia of Brachytheciaceae ⁄ Meteoriaceae type: 0—absent; 1—present. Brachytheciaceae and
Meteoriaceae each branch primordia is covered by three
leaf-like pseudoparaphyllia and they are forming unique
regular pattern (see Ignatov, 1999).
14. Pseudoparaphyllia of Brachytheciaceae ⁄ Meteoriaceae type: 0—acute to obtuse or truncate; 1—longtriangular or acuminate at least at some branch initials
(excluding upper 2 mm, where branch initials were
regarded as immature). If pseudoparaphyllia are not of
B ⁄ M type, the character state was coded as inapplicable.
15. Leaves: 0—straight; 1—falcate or homomallous.
Even slightly homomallous cases, as in Platyhypnidium,
were coded as 1.
16. Stem leaves: 0—erecto patent to patent (Brachythecium rutabulum); 1—erect to erect-appressed (Homalothecium spp.); 2—imbricate (B. cirrosum); 3—reflexed to
squarrose (B. reflexum, Zelometeorium patulum).
17. Stem leaf length: 0— 1.1–2.3 mm; 1 to > 2.3 mm; 2
to <1.1 mm. The largest leaves were measured, and hence
species was coded as 1 if at least some leaves were longer
than 2.3 mm and 2 if no leaves were longer than 1.1 mm).
18. Stem leaf length to width ratio: 0 to > 2 : 1; 1 to
< 2 : 1. Species was coded as 1 if no less than half of the
leaves from the optimally developed stems in their apical
part (5 mm) showed a ratio of 2 : 1 or if they were still
shorter.
19. Leaves: 0— > 0.4 mm wide; 1 to < 0.4 mm wide.
The largest leaves were evaluated, and species was coded
as 1, if no leaves were found wider than 0.4 mm.
20. Stem leaf shape: 0—acuminate with acumen no
less than 1 ⁄ 5 of leaf length; 1—leaves obtuse, acute,
apiculate, or very shortly acuminate with acumen less
than 1 ⁄ 5 of leaf length; 2—abruptly piliferous from
rather rounded part; 3—attenuate.
21. Acumen: 0—not distinctly flexuose; 1—distinctly
flexuose (Squamidium, Meteoridium, Zelometeorium,
etc.).
22. Stem leaves: 0—plane; 1—with concave basal part;
2—entire leaf concave; 3—channeled from the base.
23. Stem leaves: 0—not twisted when dry in mid-leaf;
1—twisted in mid-leaf (Platyhypnidium, Rhynchostegium
rotundifolium).
24. Stem leaves: 0—without distinct longitudinal
plicae; 1—with distinct longitudinally plicae (Homalothecium spp., some Brachythecium spp.). The character
is observed under stereo-microscope when leaves are still
attached on plant, as in slide more plicae appear due to
pressing of concave leaves.
25. Stem leaf base: 0—not clasping; 1—clasping.
Species was coded as 1 if the majority of leaves had a
clasping base. Taxa with U-shaped base, e.g. Aerolindigia, with indistinct aspect of clasping in a few leaves
were coded as 0.
26. Stem leaf: 0—not longly and broadly decurrent,
or decurrences lacking; 1—longly and broadly decurrent. These differences are usually explained in literature as leaves decurrent ⁄ non-decurrent. In fact, almost
all pleurocarpous leaves have some decurrencies,
which however rarely remain with leaf after it is
detached. Decurrencies remain with leaf usually when
they (triangulars extending out of line of insertion) are
no less than four cells wide and four cells long, at
least in some leaves. The latter case is that is coded
here as 1.
27. Stem leaf base: 0—not auriculate; 1—auriculate.
28. Margin of stem leaves: 0—serrulate to serrate;
1—slightly serrulate to subentire; 2—serrate, at least
some teeth backward reflexed.
29. Margin of stem leaves toothed: 0—not to the base;
1—almost to the base.
30. Costa in stem leaves: 0—single, including rarely
forking case of Platyhypnidium; 1—costa absent or
double.
31. Costa length in stem leaves: 0— 0.3–0.8 · total
leaf length; 1 to < 0.3; 2 to > 0.8. If costa double or
absent, character was coded as inapplicable. Maximal
values were used, and hence if in at least two leaves from
optimally developed stem costa is longer than 0.8· of
total leaf length, character was coded as 2 and 1 if we
were not able to find two leaves from one stem in which
costa was longer than 0.3· leaf length.
32. Costa width in stem leaves: 0— < 1/4 of leaf
width; 1— > 1/4 of leaf width. Some species in the
Brachytheciaceae, e.g. Donrichardsia macroneuron, have
exceptionally wide costae.
33. Costa in stem leaves ending: 0—without spine;
1—with spine.
34. Laminal cell length in stem leaves: 0—always <
110 lm; 1—at least sometimes > 110 lm.
35. Laminal cell proportion in stem leaves: 0—at least
50% of cells are > 10 : 1; 1—4 to 10 : 1; 2—at least
50% of cells are < 4 : 1. This character was measured in
mid-leaf cells, excluding paracostal area.
36. Laminal cell in stem leaves: 0—thin-walled, lumen
to wall rate > 3 in more than half of the cells; 1—thickwalled, no less than half of the cells in mid-leaf have
lumen to wall rate < 3. This was measured with a Leitz
S. Huttunen & M. S. Ignatov / Cladistics 20 (2004) 151–183
Duallux microscope at 40· magnification from a water
slide. Error margin in measurements was ± 0.5 lm.
37. Laminal cells in stem leaves: 0—smooth; 1—papillose.
38. Laminal cells: 0—weakly prorate; 1—distinctly
prorate. This character refers to distinctly and sharply
exserted upper ends of cells (Bryhnia, Kindbergia, etc.).
Minutely prorate ends, as in some Rhynchostegium
species, are coded as 0. Sometimes this character is
expressed in branch leaves only.
39. Basal laminal cells to the base of stem leaves:
0—slightly wider than laminal cells, more or less
transparent; 1—1.5 times wider than mid-leaf cells in
no less than five rows across the base (at least in two
leaves from one shoot, from apical 5 mm), lax and
transparent; 2—equal to slightly wider than laminal
cells, in opaque group across the base.
40. Alar cells: 0—not different from juxtacostal cells;
1—in group of larger, thin-walled cells; 2—in group of
small, opaque cells. Character state 2 also includes: (A)
rather large, but thick-walled, porose, resulting in
opaque appearance, cf. Meteoridium and (B) a group
that is a widening of the thick-wall celled area across the
base, as in Brachythecium plumosum. This character was
coded according to the most differentiated cases. For
example, in Rhynchostegium species angular cells are
often not differentiated, but when they are, the alar cells
are larger and thin-walled, and hence were coded as 1.
Similarly, Scorpiurium was coded as 2, although there
are sometimes opaque cells throughout the base, and the
angular group is poorly differentiated.
41. Alar group shape: 0—triangular to subquadrate;
1—elongate along the margin.
42. Branch leaf shape: 0—acuminate with acumen no
less than 1 ⁄ 5th of leaf length; 1—leaves obtuse, acute,
apiculate, or very shortly acuminate with acumen less
than 1 ⁄ 5th of leaf length; 2—abruptly piliferous from
rather rounded part; 3—attenuate.
43. Costa in branch leaves ending: 0—without spine;
1—with spine.
44. Costa in branch leaves by the apex: 0—not
toothed; 1—toothed. This character does not include the
single spine at the very apex.
45. Sexual condition: 0—autoicous; 1—dioicous,
including dioicous + rarely autoicous cases (for example Brachythecium rivulare); 2—phyllodioicous, incuding
dioicous + phyllodioicous case; 3—partly synoicous. In
Aerobryum speciosum phyllodioicousity has not been
reported before and this was observed in 3 specimens:
Bhutan, Griffin 462 (BM!); Sikkim, Himalaya, Levier
(BM!).
46. Perichaetial leaves: 0—reflexed; 1—straight.
47. Seta: 0—longer than branches; 1—shorter than
branches, but longer than two urn lengths; 2—shorter
than two urn lengths. Branch length is measured in place
183
where branching is regularly pinnate and branches show
rather stable length.
48. Seta: 0—smooth; 1—rough or at least slightly
rough, including cases such as Clasmatodon.
49. Capsule orientation: 0—inclined to horizontal;
1—erect. In latter case the urn is sometimes plainly
curved in the middle, as occasionally in Leskea polycarpa, but never below mouth. In Homalothecium
lutescens and some other species with capsule orientation varying from almost erect to distinctly inclined (and
curved also below mouth) are coded as 0.
50. Stomatal pore: 1—round; 0—elongate.
51. Operculum: 0—beak > 2· the height of the rest of
the operculum; 1—beak present but < 2· the height of
the rest of the operculum; 2—conic.
52. Annulus: 0—consisting of 2–3 rows of large cells,
partly separated; 1—consisting of 1()2) rows of cells,
almost nonseparated; 2—absent.
53. Exostome: 0—xerocastique; 1—hygrocastique.
Aerolindigia was coded as xerocastique. The exostome
teeth in this species turn straight when wet, but form a
high conus enclosing the capsule mouth, and hence it is
regarded as functionally xerocastique.
54. Basal part of exostome teeth: 0—with red color;
1—without red color.
55. Exostome teeth transitional zone: 0—short (1–4
plates); 1—expanded.
56. Transversal striae between dorsal trabeculae in
proximal part of tooth: 0—present; 1—absent.
57. Striae in lower part of exostome teeth: 0—nonpapillose including sometimes reticulating striae of
Rhytidiadelphus; 1—papillose above striae, giving an
appearance of papillose surface in the basal part under a
light microscope.
58. Basal membrane of endostome: 0—higher than
1 ⁄ 4 of exostome; 1—lower than 1 ⁄ 4 of exostome.
59. Endostome segments: 0—at least some segments
broadly and more or less regularly perforated; 1—narrowly to not perforated; 2—absent or adherent to teeth.
60. Cilia: 0— > 0.8· length of free part of segment;
1— 0.3–0.8; 2— < 0.3.
61. Spores: 0—diameter never > 25 lm; 1—at least
sometimes > 25 lm.
62. Calyptra: 0—naked; 1—at least sparsely hairy.
Appendix 2
Command line used in all POY analyses:
poy -parallel -solospawn 7 -maxtrees 5 -holdmaxtrees
20 -random 15 -multibuild 14 -treefuse -fuselimit 50
-fusingrounds 1 -driftspr -numdriftspr 5 -drifttbr
-numdrifttbr 5 -slop 1 -checkslop 3 -seed -1 -noleading
-fitchtrees -norandomizeoutgroup -indices -impliedalignment