American Journal of Botany 85(9): 1324–1337. 1998.
CIRCUMSCRIPTION AND PHYLOGENY OF THE
ORTHOTRICHALES (BRYOPSIDA) INFERRED FROM RBCL
SEQUENCE ANALYSES1
BERNARD GOFFINET,2 RANDALL J. BAYER,3
AND
DALE H. VITT
Department of Biological Sciences, The University of Alberta, Edmonton, Alberta, Canada T6G 2E9
The affinities as well as the circumscription of the Orthotrichaceae (Bryopsida), one of the most diverse families of
mosses, have been the focus of a controversy for much of the last century. We obtained rbcL sequences for 37 arthrodontous
mosses, including 27 taxa of the Orthotrichales. The sequences were analyzed using maximum parsimony and neighbor
joining in order to (1) test the monophyly of the Orthotrichales and the Orthotrichaceae; (2) determine their phylogenetic
relationships; and (3) test the current subfamilial classification within the Orthotrichaceae. Both analyses suggest that the
Orthotrichales are polyphyletic. The Erpodiaceae and the Rhachitheciaceae as well as Amphidium and Drummondia, two
genera of the Orthotrichaceae, are shown to be of haplolepideous affinity. The Splachnales, the Bryales sensu lato, and the
Orthotrichales form a monophyletic clade sister to the Haplolepideae. Both neighbor joining and maximum parsimony also
suggest that the Orthotrichaceae are composed of two major lineages dominated either by acrocarpous or cladocarpous taxa.
The monophyly of the family is, however, only well supported by Tamura’s distances. The genera Macrocoma, Macromitrium, Orthotrichum, Ulota, and Zygodon all appear to be artificial assemblages. This study illustrates the contribution of
rbcL sequence data to bryophyte systematics and, particularly, in determining the affinities of taxa lacking a peristome,
whose characters are central to the classification of mosses.
Key words: bryophytes; evolution; mosses; Orthotrichales; Orthotrichaceae; phylogeny; rbcL; systematics.
The central concept in the Hennigian phylogenetic approach is the use of appropriate outgroups, which allows
adequate polarization of character state transformation in
the ingroup. A reconstruction of the evolutionary history
of the Orthotrichaceae, one of the most diverse families
of mosses, based on morphology has been hampered by
the lack of a peristome in certain taxa, the absence of
gametophytic characters that are informative at higher
taxonomic levels, and consequently the absence of a consensus regarding the putative sister group and other outgroups to the order. Within the Bryophyta sensu lato,
phylogenetic reconstructions using nucleotide sequence
data have focused exclusively on the monophyly and the
relationships of the division and its classes (e.g., Mishler
et al., 1992, 1994; Waters et al., 1992; Capesius, 1995;
Bopp and Capesius, 1996; Hedderson, Chapman, and
Rootes, 1996) rather than on the evolutionary history of
arthrodontous mosses.
1 Manuscript received 3 August 1997; revision accepted 26 January
1998.
This study is part of a Ph.D. dissertation presented by the senior
author to the Faculty of Graduate Studies of the University of Alberta.
This study was supported by Grants A-3797 and A-6390 from the Nature Sciences and Engineering Research Council of Canada to RJB and
DHV, respectively. Field work was made possible through a grant by
the National Geographic Society to DHV and BG. We are grateful to
William Buck (NY), Celina Matteri (BA), and Heinar Streimann
(CANB) for kindly sending us fresh material of Cardotiella quinquefaria, Macrocoma papillosa, and Desmotheca apiculata, respectively,
as well as Ross Hastings (PMAE) for the loan of Ptychomitrium gardneri for DNA extraction. Finally we thank G. Zurawski for the set of
sequencing primers.
2 Author for correspondence, current address: Department of Botany,
Box 90339, Duke University, Durham, NC 27708.
3 Present address: CSIRO, Division of Plant Industry, Australian National Herbarium, Centre for Plant Biodiversity Research, Molecular
Systematics Lab, GPO Box 1600, Canberra, ACT, 2601, Australia.
In arthrodontous mosses (Bryopsida; sensu Vitt, Goffinet, and Hedderson, 1997), the peristome teeth that line
the mouth of the capsule and regulate spore dispersal are
made of cell wall remnants. Three concentric layers,
namely the outer (OPL), the primary (PPL), and the inner
(IPL) peristomial layer, contribute to the teeth (Blomquist
and Robertson, 1941). The number and the pattern of cell
divisions occurring in these layers, particularly in the
IPL, are central to the classification of arthrodontous
mosses. Vitt (1984) recognized five types of articulate
peristomes (i.e., four diplolepideous and one haplolepideous type), which characterize the subclasses of the
Bryopsida (see also Edwards, 1979, 1984). Vitt, Goffinet,
and Hedderson (1997) recently argued that morphological
characters used to define these major peristome types are
not phylogenetically informative and that higher level
phylogenetic reconstruction of the Bryopsida will rely on
independent sources of data such as ontogeny and gene
sequences. The development of the funariaceous, bryaceous, and dicranaceous peristome types has been studied
recently (Shaw, Anderson, and Mishler, 1989; Shaw,
Mishler, and Anderson, 1989; Schwartz, 1994), but unless
similar data are available for the orthotrichaceous peristome, polarizing the developmental pathways and thus
defining homologies and monophyletic groups based on
peristome architecture remain impossible.
The peristome of the Orthotrichales is diplolepideous
and is characterized by the following features: ‘‘an endostome with segments that alternate with exostome
teeth; lack of basal membrane and with segments, which
are not or are rarely keeled; and an exostome that has a
thick, outer layer and a thin, inner layer’’ (Vitt, 1982a).
Crosby (1980) and Vitt (1981a) were the first to suggest
that the ancestral arthrodontous peristome was diplolepideous. Crosby (1980) and later Shaw and Rohrer
1324
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ET AL.—ORTHOTRICHALES: CIRCUMSCRIPTION AND PHYLOGENY
(1984) considered that the Bryum type represented the
most primitive among the diplolepideous types and consequently that the orthotrichaceous peristome was derived
through reduction, a hypothesis also supported by Hedenäs (1994). Vitt (1984) argued that ‘‘the evolution of the
peristome has not been uni-directional’’ and that the
bryaceous peristome ‘‘is a totally separate evolutionary
line than either the orthotrichaceous or haplolepideous
divergences’’ from an ancestral funariaceous type (see
also Vitt, 1981a). More recently, Lewinsky (1989) argued
in favor of a distinct orthotrichaceous peristome type that
is ‘‘most likely to have evolved from a peristome with a
formula of 4:2:4 with complete alignment of the cells in
the IPL,’’ thus the funariaceous type, but unlike Vitt
(1984) she considers the orthotrichaceous peristome as
possibly pivotal to the evolution of the dicranaceous and
the bryaceous types.
The Orthotrichaceae sensu Vitt (1984; and not sensu
Churchill and Linares, 1995) include over 500 species,
distributed among 22 genera (see Goffinet, 1997a, b),
with Orthotrichum, and Macromitrium accounting for
more than two-thirds of the species richness (Vitt, 1982a).
The family is cosmopolitan in distribution and is particularly prevalent in tropical montane forests. The Orthotrichaceae are traditionally characterized by a unique peristomial architecture (see above), as well as ‘‘small, papillose upper leaf cells; no differentiated alar cells; large,
usually mitriform calyptrae; terminal perichaetia with additional growth by lateral innovations’’ (Vitt, 1982b).
These characters are, however, by no means constant
within the family, resulting in alternative hypotheses of
familial circumscription. The controversy has focused
particularly on the haplolepideous vs. the diplolepideous
affinity of Amphidium, which lacks a peristome, and has
atypical cell ornamentation (Brotherus, 1909 vs. 1925;
Vitt, 1973 vs. Lewinsky, 1976), or of taxa whose peristome is reduced (Drummondia: Shaw, 1985 vs. Vitt,
1972). The current infrafamilial classification (Vitt,
1982a) reflects the four possible combinations of two
characters, position of the female gametangia and
‘‘shape’’ of the calyptrae, and deviates little from Brotherus’s concept early this century (Brotherus, 1925). In
Vitt’s (1982a) phylogenetic arrangement, the Zygodontoideae (Schimp.) Broth. (acrocarpous and cucullate calyptrae) are basal, followed by the Drummondioideae Vitt
(cladocarpous and cucullate calyptrae), which are sister
to a clade composed of the Orthotrichoideae Broth. and
the Macromitrioideae Broth. (acrocarpous and cladocarpous, respectively, and both with mitrate calyptrae).
Addressing the phylogenetic relationships of the Orthotrichales and its delineation is essential for reconstructing the evolutionary history of orthotrichaceous
genera. Goffinet (1997b) recently excluded the Microtheciellaceae from the Orthotrichales based on their pleurocarpy. Goffinet (1997a) also transfered two genera from
the Orthotrichaceae to the Rhachitheciaceae. The peristomial architecture of the Rhachitheciaceae (Goffinet,
1997a) and of the Erpodiaceae Broth. (Crum, 1972; Edwards, 1979) suggests possible affinities to the Haplolepideae, and particularly the Seligeriales. The monophyly
of the Orthotrichaceae and the Orthotrichales has recently
also been questioned by De Luna (1995) while examining
the systematic affinities of the Hedwigiaceae. Based on a
1325
phylogenetic analysis using morphological characters, De
Luna (1995) concluded that the Orthotrichales merely
represent an evolutionary grade, suggesting that a clade
of predominantly cladocarpous Orthotrichaceae (Macromitrioideae, Drummondioideae) and the putatively related Erpodiaceae Broth., Microtheciellaceae Harrington
and Miller, and Helicophyllaceae Broth.(see below) was
sister to the pleurocarpous mosses, and separated from
the acrocarpous Orthotrichaceae and their possible sister
group, the Rhachitheciaceae Robins. by the Hedwigiaceae. Affinities between the Hedwigiaceae and the Orthotrichaceae have been proposed by other authors (Walther, 1983; Frey et al., 1995). Because of the gymnostomous nature of the Hedwigiaceae and a unique combination of gametophytic characters (acrocarpy, lack of
costa, and presence of pseudoparaphyllia), the affinities
of Hedwigia are unlikely to be resolved with traditional
morphological characters.
Severe reduction trends in morphological characters,
particularly with regard to the peristome, are thus the
source of the controversies regarding the delineation of
the Orthotrichales and its type family, the Orthotrichaceae. Ambiguous homologies between characters defining alternative peristome types hamper resolving the phylogenetic affinities of these taxa. Variation in the nucleotide sequence of the chloroplast gene rbcL, encoding for
the ribulose 1,5 biphosphate carboxylase, has found a
wide application in reconstructing the evolutionary histories within suprageneric taxa of vascular plants (Chase
et al., 1993; Hasebe et al., 1995). The present study aims
at setting the foundations for reconstructing the phylogeny of all orthotrichaceous genera based on morphology
and for addressing the character evolution in this taxonomically and morphologically diverse family. Thus, the
objectives were to use rbcL sequence data (1) to test the
monophyly of the Orthotrichales and the Orthotrichaceae;
(2) to determine the phylogenetic position of the Orthotrichales within the arthrodonteae, and thus identify its
putative sister group; and (3) to test the monophyly and
the phylogenetic relationships of the subfamilies proposed by Vitt (1982a).
MATERIALS AND METHODS
Taxon sampling—Following the exclusion of five genera from the
Orthotrichaceae (Goffinet, 1997a, b) the family now consists of 22 genera. While material has been seen for all taxa except for the recently
described Orthomitrium (Lewinsky-Haapasaari and Crosby, 1996), extractions were not attempted for taxa known only from the type specimen (Ceuthotheca Lewinsky-Haapasaari, Leiomitrium Mitt., Leratia
Broth.), or for which only very little material was available (Stoneobryum Norris and Robinson). DNA extractions were attempted for the
remaining 17 genera, and for the most diverse genera, such as Macromitrium and Orthotrichum, several species belonging to morphologically distinct infrageneric taxa were tentatively included. DNA extractions were also attempted for representatives of all related families in
the Orthotrichales. Furthermore, ten outgroup taxa were chosen, representing the Funariaceae, Splachnaceae, Encalyptaceae, Hedwigiaceae,
the Haplolepideae (Ptychomitriaceae and Rhabdoweisiaceae), and the
ciliate alternate peristomate mosses (Mniaceae and Thuidiaceae).
DNA extraction and PCR-DNA amplification—DNA was extracted,
following a modification of Doyle and Doyle (1987), from apices of
stems and branches, or single sporophytes sampled from dried herbar-
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TABLE 1.
a
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Synthetic primers (59–39) used for sequencing the rbcL gene in mosses.
Forward primers
427a
997
GCTTATTCAAAAACTTTCCAAGGCCCGCC
GGTAAACTTGAAGGAGAACG
Reverse primers
295R
515R
678Ra
895Ra
1081R
CTAATGGGTAAGCAACATAAGC
CATCCTAATAATGGACGACC
GATTTCGCCTGTTTCGGCTTGTGCTTTATAAA
ACCATGATTCTTCTGCCTATCAATAACTGC
CCCAGTCTTGAGTGAAGTAAATACC
Marks primers designed and provided by G. Zurawski; others were designed by authors.
ium or fresh collections. Gametophytic samples were cleaned of contaminating material in distilled water, then air-dried overnight and lyophilized prior to the extraction. One to 10 mg dry mass of each sample
were placed in a spot plate, covered with liquid N2, and ground to a
fine powder once the liquid N2 was completely evaporated. The powder
was then suspended in 250 mL of 2X CTAB (hexadecyltrimethylammonium bromide)-1% beta-mercapto-ethanol extraction buffer heated at
608C and the solution transferred to a microcentrifuge tube. The tubes
were then placed in a 608C water bath. After 45 min, 250 mL of CI
(chloroform-isoamyl 24:1) were added and the tubes gently inverted for
10 min. Cell debris were then isolated by centrifuging the tubes for 10
min at maximum speed (e.g., 14 000 rpm). The aqueous phase was
transferred to a fresh tube (the CI extraction can be repeated if the
aqueous solution is not clear). Then 350 mL of 95% ethanol were added
and the DNA allowed to precipitate at 48C. After at least 2–3 h, the
tubes were centrifuged for 5 min at maximum speed again to pellet the
DNA. The ethanol was discarded and the pellet washed gently by adding 350 mL of 70% ethanol. The tubes were centrifuged again for 5
min at 7000 g. The ethanol was discarded and the pellet dried in a
vacuum centrifuge. The DNA was then suspended in 100 mL of TBE
(Tris-boric acid-EDTA) at 658C for 10 min and kept at 2208C subsequently. The rbcL gene was amplified via the polymerase chain reaction
(PCR) using Taq Polymerase (Promega Corporation, Madison, WI). The
amplification reaction was performed in 50 mL volume including 50
mmol/L KCl, 10 mmol/L Tris-HCl, 0.1% Triton, 5% glycerol, 2.5
mmol/L MgCl2, 20 mmol/L of each dNTP, 0.2 mmol/L of primers Z1
and 1351R (Wolf, Soltis, and Soltis, 1994) and 0.2–30.0 ng of template
DNA. The samples were exposed to the following temperature profiles
using a Grant thermal cycler: one cycle of 948C for 3 min and 858C for
4 min, during which 1 unit of Taq DNA Polymerase would be added
to each tube, and 30 cycles of 948C for 1 min, 528C for 1 min, 728C
for 2 min, and finally one segment of 728C for 10 min. The doublestranded product subsequently served as the template for the amplification of single-stranded target DNA. This second PCR followed the
same profiles as before, but the reaction solution included only one of
the two PCR primers: Z1 for amplification of the forward strand and
1351R for the reverse strand. The single-stranded product was precipitated with 20% PEG/2.5 mol/L NaCl, washed first with 70% EtOH and
then with 95% EtOH, before being suspended in 7 mL of Tris bufferEDTA (Morgan and Soltis, 1993).
Sequencing and alignment—Sequencing the single-stranded template followed the dideoxy chain termination method (Sanger, Nicklen,
and Coulson, 1977) using the Sequenaset version 2.0 T7 DNA polymerase following the manufacturer’s instructions (Amersham, Canada).
The sequence of the primers used is given in Table 1. The sequencing
products were electrophoresed on a 6% polyacrylamide gel (0.4 mm
thickness; 1X TBE buffer), at 2400 V for 4 h. The gels were fixed in
10% acetic acid, washed with distilled water, and dried in an oven at
658C for 30 min and autoradiographed for 36–48 h. The sequences were
entered in MacClade (version 3.03) and aligned manually against available sequences of Sphagnum palustre and Andreaea rupestris (GenBank
accession numbers L13485 and L13473, respectively; Manhart, 1994).
The first and last 30 nucleotide sites, corresponding to the sequences of
the PCR primers, were excluded from parsimony analyses.
Sequence and phylogenetic analysis—RbcL sequence variation was
analyzed by neighbor-joining (NJ; Saitou and Nei, 1987), and maximum
parsimony (MP; Fitch, 1971). Neighbor-joining analyses were performed using MEGA version 1.0 (Kumar, Tamura, and Nei, 1993) using
Tamura’s distance parameter, following the authors’ ‘‘Guidelines for
choosing distance measures,’’ and including both transitions and transversions. A ‘‘bootstrap confidence level’’ for the NJ tree was calculated
over 100 replicates. Fitch parsimony analyses of nucleotide data were
performed with PAUP version 3.1 (Swofford, 1993) on a MacIntosh
PowerPC 7200/90 using the heuristic search with the following options
in effect: keep all characters, multistate taxa interpreted as uncertainties,
tree-bisection-reconnection branch swapping, steepest descent, collapse
zero-length branches, and addition sequence ‘‘as is.’’ In an attempt to
locate additional islands of shortest trees (Maddison, 1991), the search
strategy further followed the steps recommended in Pryer, Smith, and
Skog (1995) except that the search was only replicated 100 times. Bootstrap analysis (Felsenstein, 1985; Hillis and Bull, 1993) was performed
with 100 bootstrap replicates of the heuristic search with the same set
of options in effect. Relative support for branches was further determined by the decay analysis (Bremer, 1988; Donoghue et al., 1992)
following the converse constraint method (Baum, Sytsma, and Hoch,
1994). The ACCTRAN (accelerated transformation optimization) option
of PAUP was applied for calculations of branch lengths. The phylogenetic signal present in the rbcL sequence data was estimated by calculating the g1 statistic of the distribution of tree length of 500 000
random trees produced with PAUP using the ‘‘random tree’’ option
(Hillis and Huelsenbeck, 1992). Consistency (CI) and retention indexes
(RI) and f values were calculated for all MPTs using PAUP. PAUP was
also used to generate a matrix of absolute and mean distance between
sequences. Average unit character consistencies (AUCC) were calculated for competing phylogenies in an attempt to select the tree with
the strongest asymmetric distribution of homoplastic characters in the
matrix (Sang, 1995). Costs in terms of number of additional steps required for alternative phylogenies were explored using the enforce constraint command during heuristic searches (Swofford, 1993). The same
set of options as in the unconstrained searches was applied.
RESULTS
The rbcL gene was successfully sequenced for 22 taxa
of the Orthotrichaceae distributed among ten genera, for
representatives of two of the related families as well as
for all selected outgroup taxa (Table 2). Amplification
products were not obtained for Florschuetziella Vitt, Leptodontiopsis Broth., Muelleriella Dusén, Pleurorthotrichum Broth., and Stenomitrium (Mitt.) Broth., Helicophyllum Brid. The sequences obtained from PCR fragments generated using the PCR primers Z1 and 1351R
August 1998]
TABLE 2.
GOFFINET
ET AL.—ORTHOTRICHALES: CIRCUMSCRIPTION AND PHYLOGENY
1327
Taxa for which the rbcL gene sequence was obtained in this study (all vouchers deposited in ALTA unless otherwise noted).
Taxon
ORTHOTRICHALES
ORTHOTRICHACEAE
Zygodontoideae
Zygodon pungensa C. Müll.
Zygodon obtusifolius Hook.
Zygodon intermedius B.S.G.
Zygodon reinwardtii (Hornsch.) Braun
Amphidium lapponicum (Hedw.) Schimp.
Voucher
GenBank accession number
La Farge-England 8097
Vitt 38301
Vitt 29262
Goffinet 636
Vitt 33854
AF005534
AF005535
AF005532
AF005533
AF005543
Orthotrichoideae
Orthotrichum obtusifolium Brid.
Orthotrichum anomalum Hedw.
Orthotrichum lyellii Hook. & Tayl.
Ulota lutea (Hook. f. & Wils.) Mitt.
Ulota obtusiuscula C. Müll. & Kindb.
Bryodixonia perichaetialis Sainsb.
Vitt 33870
Goffinet 4115
Goffinet 3162
Fife 8042
Goffinet 3161
Fife 8083
AF005537
AF005538
AF005536
AF005540
AF005539
AF005541
Drummondioideae
Drummondia prorepens (Hedw.) Britt.
Vitt 26711
AF005542
Vitt 27485
Schäfer-Verwimp 9686
Schäfer-Verwimp 6902
Buck 26230
Goffinet 2764
Goffinet 1173
Matteri 6521
AF005522
AF005520
AF005521
AF005523
AF005527
AF005526
AF005525
Breedlove 69342
Streimann 49345
Goffinet 656
Goffinet 2648
Vinas 96-4
AF005524
AF005528
AF005529
AF005530
AF005531
ERPODIACEAE
Aulacopilum hodgkinsoniae (C. Müll.) Broth.
Venturiella sinensis (Vent. in Rabenh.) Müll. Hal.
Vitt 28261
Vitt 34842
AF005545
AF005546
RHACHITHECIACEAE
Uleastrum palmicola (C. Müll.) Zander
Vitt 21162
AF005547
Vitt 27234
Priddle 1408
AF005514
AF005513
ENCALYPTACEAE
Encalypta procera Bruch
Vitt 37966
AF005548
HEDWIGIACEAE
Hedwigia ciliata (Hedw.) P. Beauv.
Goffinet 3324
AF005517
MNIACEAE
Mnium thomsonii Schimp.
Vitt 35884
AF005518
PTYCHOMITRIACEAE
Ptychomitrium gardneri Lesq.
Ireland 7038 (PMAE)
AF005549
RHABDOWEISIACEAE
Rhabdoweisia crenulata (Mitt.) Jameson
Vitt 36707
AF005544
SPLACHNACEAE
Tayloria lingulata (Dicks.) Lindb.
Splachnum sphaericum Hedw.
Schofield 98443
Goward 95-1470
AF005515
AF005516
THUIDIACEAE
Abietinella abietina (Hedw.) Fleisch.
Goffinet 4106
AF005519
Macromitrioideae
Schlotheimia brownii Schwaegr.
Schlotheimia tecta Hook. f. & Wils.
Schlotheimia trichomitria Schwaegr.
Cardotiella quinquefaria (Hornsch.) Vitt
Groutiella apiculata (Hook.) Crum & Steere
Groutiella chimborazense (Mitt.) Florsch.
Macrocoma papillosa (Thér.) Vitt
Macrocoma tenuis (Hook. & Grev.) Vitt subsp. sullivantii
(C. Müll.) Vitt
Macromitrium incurvifolium (Hook. & Grev.) Schwägr.
Macromitrium longifolium (Hook.) Brid.
Macromitrium richardii Schwaegr.
Desmotheca apiculata (Dozy & Molk.) Lindb.
OUTGROUP TAXA
FUNARIACEAE
Funaria apophysata (Tayl.) Broth.
Funaria hygrometrica Hedw.
a
This specimen is tentatively identified as Zygodon pungens C. Müll., a species hitherto not known from Africa (Malta 1926), but may represent
a new species.
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TABLE 3. Distribution and frequency (%) of constant and phylogenetically informative sites over all taxa and over the Orthotrichaceae only;
characters with ambiguous or missing data are included.
All taxa
Codon position
Constant
Phylogenetically informative
First
Second
Third
Total
Orthotrichaceae
(total)
368 (83.6)
30 (6.1)
407 (92.5)
18 (4.1)
148 (33.6)
109 (24.7)
923 (69.9)
157 (11.9)
1158 (87.8)
108 (8.2)
were, as expected, 1320 bases long (690% of the total
length of the gene). Alignment of the sequences with
known sequences of Sphagnum pallustre and Andreaea
rupestris did not require the inclusion of gaps. Phylogenetically informative characters are preponderant at the
third codon position followed by the first and the second
codon site (Table 3). RbcL sequence variation yields 108
sites that are potentially phylogenetically informative
within the Orthotrichaceae. Informative characters are
distributed fairly evenly across the sequence (Fig. 1). The
distribution of 500 000 random trees using all characters
had a skewness index g1 of 20.55; excluding the first and
second codon position yielded a similar index (g1 5
20.54), while trees generated solely from characters of
these two first codon sites had a higher skewness index
(g1 5 20.39).
Fig. 1. Distribution of phylogenetically informative (PI) characters,
the average consistency index (CI), and the average number of steps
per phylogenetically informative character along the rbcL sequence
based on tree shown in Fig. 3. Each class contains 50 nucleotide sites,
except for the first class that includes only sites 31–50. Cross-lined bars
5 total number of phylogenetic informative characters; solid bars 5
average CI multiplied by 10; hollow bar 5 total number of steps per
total number of phylogenetically informative characters.
Using the maximum parsimony criterion with Sphagnum and Andreaea as the outgroup yields 39 most parsimonious trees (MPT) distributed among three islands of
size 21, 6, and 12 (Fig. 2). The MPTs are 964 steps long
and have a CI of 0.390 and a RI of 0.624. The f value
of the trees varies between 13 306 and 19 198. Both methods of analysis agree on the polyphyly of the Orthotrichales and place the Erpodiaceae and the Rhachitheciaceae as well as the orthotrichaceous genera Amphidium
and Drummondia in a monophyletic clade with Ptychomitrium and Rhabdoweisia (Figs. 3–4). Constraining the
search for the inclusion of the Erpodiaceae and Rhachitheciaceae in the Orthotrichales results in trees that are
17 and 15 steps longer than the shortest unconstrained
trees, respectively. Including Amphidium and Drummondia in the Orthotrichaceae also increases the length of the
most parsimonious topology (125 steps), and furthermore these two genera remain more closely related to
each other than they do to any other genus of the Orthotrichaceae. The NJ tree agrees with the strict consensus
tree over all MPTs in the following relationships (Figs.
3–4): (1) Funaria occupies a basal position among arthrodontous mosses; (2) the Orthotrichales and the
Splachnales form a monophyletic clade sister to the ciliate mosses; and (3) the Haplolepideae, including Amphidium, Drummondia, Venturiella, and Uleastrum, form
a monophyletic group sister to derived Diplolepideae.
Hedwigia is sister to the two ciliate mosses in 36 MPTs
(Fig. 2) as well as in the NJ tree (Fig. 4) and basal in the
diplolepideous clade (excluding the Funariaceae) in the
remaining three trees (Fig. 2). Compared to MP, NJ provides similar or slightly higher bootstrap values for major
lineages (Haplolepideae, combined Splachnales and Orthotrichales, Orthotrichaceae, and ciliate mosses), but like
MP, NJ fails to yield strong support for their relationships
(Figs. 3, 4).
The remaining genera of the Orthotrichaceae (thus excluding Amphidium and Drummondia) form a strongly
supported monophyletic family in the phylogenetic reconstruction using Tamura’s distance parameter (Fig. 4).
Among the 39 MPTs found in the cladistic analysis (Fig.
2), 15 trees (38%), distributed between island 1 and 3
(each with nine and six trees, respectively), support the
monophyly of the family. The trees of these two islands
differ mainly by the position of Encalypta (Fig. 2). In
island 3 (mean f values: 13 943), Encalypta is sister to
the Haplolepideae (Fig. 2B), while in islands 1 and 2 it
occupies a position basal to the dichotomy between Haplolepideae and the derived Diplolepideae (Fig. 2C; mean
f value overall: 16 634). Comparison of the distribution
of homoplasy among characters from two selected trees
contrasted mainly by the position of Encalypta (island 1
vs. island 3), yields an AUCC value of 0.71 for both,
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suggesting no difference in the distribution of homoplastic characters (Sang, 1995). All other indexes being
equal, the tree with the lowest f value (see Farris, 1972)
is shown (Fig. 3). The Orthotrichaceae are composed of
two major lineages: one including all Macromitrioideae
(Cardotiella, Desmotheca, Groutiella, Macrocoma, Macromitrium, and Schlotheimia) as well as Zygodon obtusifolius, and one uniting the remaining species of Zygodon with the Orthotrichoideae (Bryodixonia, Orthotrichum, and Ulota). Large genera such as Macromitrium,
Orthotrichum, and Zygodon appear paraphyletic, independent of the method of analysis. In the NJ analysis, as
well as in all MPT of island 2, Desmotheca is basal in a
clade centered around Macromitrium, while the other
MPT suggest a derived position, sister to Macromitrium
richardii.
DISCUSSION
RbcL sequence data—RbcL sequence data are routinely used to reconstruct evolutionary histories among
(Chase et al., 1993; Hasebe et al., 1995) or more rarely
within (e.g., Haufler and Ranker, 1995) genera of vascular plants. With 30% variable sites, including 12% of
sites with changes that may be phylogenetically informative, the variation in the nucleotide sequences of the
rbcL gene among moss taxa may yield sufficient characters for reconstructing the evolutionary history of the
taxa included in this study. The distribution of informative characters appears rather uniform (Fig. 1) and is similar to that observed in vascular plants, i.e., with no obvious hot spot detectable (Olmstead and Sweere, 1994).
The distribution of randomly generated trees has a lefthand skewness with a g1 value (20.55) significantly
smaller than the critical value (20.10 or 20.08 for 100
or 250 characters, respectively; P 5 0.01) furnished by
Hillis and Huelsenbeck (1992), suggesting that our rbcL
data set is more structured than a random data set of equal
size and that it may contain significant phylogenetic signal (Hillis and Huelsenbeck, 1992). Unlike Conti, Fischbach, and Sytsma’s (1993) observation that the third codon position introduces ‘‘noise,’’ a lower g1 value is observed when the matrix is restricted to the third codon
position (20.54 vs. 20.39), suggesting that the changes
at the third codon position are more structured. Analyses
of a data set restricted to the third codon positions only
yield topologies congruent with those obtained with the
complete data set, whereas a search excluding changes at
the third codon position results in a consensus tree incongruent with monophyletic concepts of either the ciliate mosses, or the Haplolepideae (results not shown).
This observation would suggest that the first two codon
sites carry more homoplasies than the third position,
which may thus be more informative. Alternatively, the
taxon sample may be too disparate for variability at the
more conserved positions 1 and 2 to be mostly phylogenetically informative. The significance of the difference
between the g1 value based on our data set and that of a
random matrix may, as a result, be due to differences in
the frequency of character states rather than in the congruence among characters in both data sets (Källersjö et
al., 1992). This second hypothesis most likely applies to
our data since our restricted taxon sample represents ma-
1329
jor lineages of arthrodontous mosses with a long evolutionary history (Frey, 1977, 1990).
An indication of the accuracy of our sequences as well
as for the presence of pseudogenes may be obtained from
the comparison of amino acid sequences with the distribution of active sites (Kellog and Juliano, 1997). All amino acid residues of the active site found in spinach by
crystallography (Andersson et al., 1989) are conserved
among studied bryophyte taxa, except for position 404
(amino acid numbering), which is scored as polymorphic
(Arg in addition to the conserved Gly) for six taxa, and
thus excluded from phylogenetic analyses in MP. This
observation may provide some preliminary support for
the accuracy of the sequences obtained.
Circumscriptions of the Orthotrichales—The Orthotrichales are currently composed of four families: the Erpodiaceae, Helicophyllaceae, Orthotrichaceae, and
Rhachitheciaceae (Goffinet, 1997b). RbcL sequence data
analyzed using either the neighbor joining or the maximum parsimony criterion indicate that the Orthotrichales
form a polyphyletic taxon. Both the Erpodiaceae (Aulacopilum hodgkinsoniae, Venturiella sinensis) and the
Rhachitheciaceae (Uleastrum palmicola) are indeed
placed in a clade with haplolepideous taxa (Figs. 3, 4).
This clade also includes the ‘‘orthotrichaceous’’ genera
Amphidium and Drummondia. The monophyly of this
haplolepideous clade is strongly supported (MP: decay
index [DI] of 4, bootstrap value [BV] 5 77%; NJ: BV
5 94%). Constraining these taxa to a relationship within
a monophyletic Orthotrichales is furthermore very costly
in terms of parsimony, requiring up to 25 additional steps.
Variation in the nucleotide sequence of the rbcL gene is
thus congruent with earlier hypotheses based on morphology and cytology, suggesting haplolepideous affinities of these taxa (for Amphidium see Anderson and
Crum, 1958; Vitt, 1970, 1973; Drummondia: Shaw 1985;
Erpodiaceae: Edwards, 1979; Rhachitheciaceae: Goffinet,
1997a). More extensive sampling of haplolepideous taxa
would be needed before ordinal affinities of Amphidium
and Drummondia are elucidated (see also below, affinities of excluded taxa).
De Luna (1995) recently argued that the Orthotrichales
represented an evolutionary grade, reached by two lineages. The cladocarpous lineage (including, e.g., Erpodiaceae, Macromitrioideae) was sister to the Leucodontales and separated from the acrocarpous line (e.g.,
Rhachitheciaceae, Orthotrichoideae) by the Hedwigiaceae. Affinities between the Hedwigiaceae and the Orthotrichaceae have also been proposed by other authors
(Walther, 1983; Frey et al., 1995). Constraining the heuristic search to include Hedwigia ciliata in the Orthotrichaceae (as defined in Figs. 3 and 4, and with no further
relationships specified within this clade) yielded 12 trees
that not only are 16 steps longer than the MPTs in the
unconstrained search, but also share a monophyletic Orthotrichaceae. The incongruence between De Luna’s morphological study (De Luna, 1995) and the present molecular analysis may be due to inconsistent and inadequate taxon sampling with the absence of representatives
of the Leucodontineae from the molecular analysis and
of bryalean taxa from the morphological analysis. The
latter analysis further includes taxa that are here shown
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to be unrelated to the Diplolepideae (i.e., Erpodiaceae,
Rhachitheciaceae) and might be affected by the subsequent misinterpretations of the nature of the peristome of
these families (scored as an exostome instead of an endostome). A closer relationship between the Hedwigiaceae and the Orthotrichaceae cannot be excluded, but if
the Hedwigiaceae were closely related to the Orthotrichaceae, rbcL data suggest that the Orthotrichaceae
would most likely remain a natural group. This phylogenetic hypothesis seems to be supported by 18S sequence data analyses (C. Cox and T. Hedderson, personal
communication, University of Reading, U.K.). The
branch that supports the Hedwigiaceae–cladocarpous Orthotrichales–Leucodontales lineage is supported in De
Luna’s analysis (1995) by three synapomorphies, namely
plagiotropic growth, presence of pseudoparaphyllia, and
differentiated perichaetial leaves. Two of these characters
are reversed in the cladocarpous Orthotrichales, leaving
a single character that actually supports a relationship of
these taxa with the Hedwigiaceae and Leucodontales,
namely plagiotropic growth. This character is, however,
homoplastic among many families and genera of mosses
(Meusel, 1935) and may thus not be truly informative at
the ordinal level.
Analysis using the distance method yields a single tree
(Fig. 4) that shows strong support for the monophyly of
the Orthotrichaceae, a phylogeny congruent with that of
15 of the 39 MPTs found in the cladistic analysis. Käss
and Wink (1995) and Barker, Linder, and Harley (1995)
also used both methods (MP and NJ) for phylogenetic
reconstructions based on rbcL sequence data, and the results of both analyses were fairly congruent. Kim, Rohlf,
and Sokal (1993) critically examined the accuracy of the
NJ method under different constraints on a random data
set and found that this method ‘‘has the highest accuracy
overall.’’ Russo, Takezaki, and Nei (1996) recently compared the efficiency of various tree-building methods in
recovering a known phylogeny and concluded that NJ
‘‘gives as good a result as the more time consuming. . .methods.’’ The congruence between the NJ tree
and 15 MPTs is therefore here interpreted as supporting
the monophyly of the Orthotrichaceae.
With the exclusion of the Erpodiaceae, Rhachitheciaceae, and Hedwigiaceae from the Orthotrichales, the order is now reduced to the Orthotrichaceae and the Helicophyllaceae. In the absence of rbcL data, the phylogenetic relationship between these two families remains dubious due to unique gametophytic characters, such as
reduced ventral and dorsal leaves, and lateral leaves that
are inrolled when dry, and the absence of a peristome in
Helicophyllum torquatum, the sole species of the Helicophyllaceae (Vitt, 1982c). Buck and Vitt (1986) argued
in favor of a close relationship with the Racopilaceae
(ciliate diplolepideae), as suggested by earlier authors
(Brotherus, 1909, 1925; Fleischer, 1920; Dixon, 1932;
Reimers, 1954). The perichaetia are, however, produced
terminally on the main axis (De Luna, 1995), a condition
that is a priori incompatible with a placement in a pleurocarpous lineage (La Farge-England, 1996). Other loci
are currently being tested for addressing the affinities of
the Helicophyllaceae. A putative phylogenetic relationship between the Orthotrichaceae and the Cryphaeaceae
and the monotypic Wardiaceae (Walther, 1983) was not
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[Vol. 85
Fig. 2. Summary consensus trees of most parsimonious Fitch trees
found in a heuristic search using rbcL sequence data with Sphagnum
palustre and Andreaea rupestris as outgroups. (A): strict consensus of
all three islands (39 MPTs; tree 964 steps; CI: 0.390; RI: 0.624); (B):
50% majority rule tree of island 3 (12 MPTs; mean f value: 13 943 6
656); (C): 50% majority rule tree of islands 1 and 2 (27 MPTs; mean f
value: 16 633 6 1 308); (D): 50% majority rule tree of all 15 MPTs
showing the Orthotrichaceae monophyletic (mean f value: 16722 6
2015).
examined in the present study. Wardia hygrometrica
should be excluded from the Orthotrichales based on well
differentiated alar cells and prosenchymateous cells.
Combined with the acrocarpous condition (Welch, 1943),
these characters may indicate a bryalean origin, a hypothesis recently examined using 18S gene sequences
(Hedderson, Cox, and Gibbings, 1997). The Cryphaeaceae have traditionally been placed among the Leucodontineae (Vitt, 1984; Buck and Vitt, 1986). A relationship of this family to the Hedwigiaceae, and thus the
Orthotrichales, has been considered and rejected (De
Luna, 1995), but may need to be reexamined in the light
of the discovery of cladocarpous species of Cryphaea
sensu lato (La Farge-England, 1996).
Ordinal relationship of the Orthotrichales—The affinities of the Orthotrichales, here restricted to the Orthotrichaceae, as indicated by comparisons of the rbcL sequences, are within a diplolepideous lineage including the
Splachnales and the ciliate mosses. This clade shares a
common ancestor with the Haplolepideae, and together
they form a sister group to the Funariales (Figs. 3–4).
This topology is congruent with Vitt’s hypothesis (Vitt,
1981a) that the opposite diplolepideous peristome is
primitive among arthrodontous mosses and that the haplolepideous peristome is derived from such an ancestral
type (Vitt, 1984). Alternative hypotheses proposed by
Lewinsky (1989; Orthotrichales basal to dichotomy between haplolepideae and ciliate diplolepideae) or by
Shaw and Rohrer (1984) and Crosby (1980; ciliate diplo-
August 1998]
GOFFINET
ET AL.—ORTHOTRICHALES: CIRCUMSCRIPTION AND PHYLOGENY
1331
Fig. 3. One of 39 most parsimonious Fitch trees found based on
rbcL sequence data and using Sphagnum palustre and Andreaea rupestris as outgroups. The dotted lines identify branches not present in the
strict consensus tree. Bootstrap values (% of 100 replicates) 50% or
higher are given below the branch, and decay indexes are presented
above the branch. Taxa excluded from the Orthotrichales are in boldface.
Fig. 4. Phylogenetic tree reconstructed using the neighbor-joining
method, with Tamura’s distance parameter including both transitions
and transversions. Bootstrap values (% of 100 replicates) 50% or higher
are plotted on the tree. Taxa excluded from the Orthotrichales are in
boldface. Abbreviations for generic names in the Orthotrichaceae follow
those of Table 4.
lepideae are the most primitive mosses) both require five
additional steps.
The phylogeny obtained by either NJ or MP analysis
(Figs. 3, 4) also agrees with Vitt (1984) with regard to
the monophyly of a lineage composed of the Splachnales,
Orthotrichales and the ciliate mosses. The relationships
among these lineages remain however, ambiguous (Fig.
2). The most parsimonious scenario points toward the
Splachnales and the Orthotrichales being sister groups.
Vitt (1984), by contrast, proposed that the Orthotrichales
are sister to the ciliate mosses, a topology that would only
require one additional step based on our rbcL data set.
Koponen (1977, 1983) considered the genus Brachymitrion to be the most primitive extant member of the
Splachnaceae. Unlike in related genera, the thickening on
the PPL is heavier than on the OPL, and the PPL also
has strong trabeculae (Koponen, 1977, 1982). Both these
features are shared with the Funaria and the Bryum peri-
stome type (Shaw and Rohrer, 1984). If the Orthotrichales and the Splachnales formed a monophyletic group,
both orders would most likely remain natural orders, with
the Orthotrichales defined by thick-walled laminal cells.
The heavier thickening on the OPL could have arisen
independently in both lineages or be a synapomorphy for
both, with subsequent reversal in Brachymitrion. In terms
of peristome evolution, these lineages would form a plesiomorphic sister group to the ciliate mosses, the latter
one being defined by the asymmetric division of the IPL
leading to the development of cilia. If we consider the
alternative scenario where the Splachnales are sister to a
clade composed of the Orthotrichales and the ciliate
mosses, the most parsimonious topology based on our
data suggests that within the latter, the ciliate mosses remain monophyletic. An extensive taxon sampling in the
Bryales is, however, needed before their evolutionary relationship can be addressed more critically. The Ortho-
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trichales and the Bryales sensu lato each share one plesiomorphic state with Brachymitrion (Splachnales): either
completely aligned cell divisions (Orthotrichales; Lewinsky, 1989) or heavier thickening of the PPL and ventral
exostomial trabeculae (typical ciliate mosses). The ciliate
mosses have traditionally been defined by a strongly
asymmetric division in IPL leading to the development
of cilia. While some ‘‘ciliate’’ mosses actually lack cilia
(Buck and Vitt, 1986), have a thickened OPL (Eucamptodontopsis, A. Newton, personal communication, Smithsonian), and others even have opposite peristomes (Garovaglia div. sp.; During, 1977; Nishimura and Watanabe,
1992), none has yet been found to have a first-late symmetric division in the IPL. An asymmetric division is
elsewhere found only among haplolepideous taxa. Considering that the Rhachitheciaceae are here shown to be
of haplolepideous affinity, their peristome with a 2:1 formula most likely results from reduction, through the loss
of the asymmetric first-late division. The asymmetric division of the ciliate mosses may not be homologous to
that of the haplolepideae (Shaw, Mishler, and Anderson,
1989). Assuming that the genetic complexity behind the
asymmetric division in both groups is similar, we cannot
exclude the possibility that a loss of it or a reversal to a
symmetric division may be possible among ciliate mosses
and may even occur in such genera as Mielichhoferia
with a peristome formula of 4:2:4 (Shaw and Crum,
1984; Shaw and Rohrer, 1984). Since the possibility of a
reversal to a symmetric division cannot be excluded, a
bryalean origin of the Orthotrichaceae will need to be
further examined in comparison with a broad sample of
ciliate taxa.
Hedenäs (1994) considered Schlotheimia to be pleurocarpous and, based on this interpretation, suggested that
the Orthotrichaceae ‘‘are rather close to the clade where
most pleurocarpous mosses belong.’’ He further hypothesized that ‘‘the transition to pleurocarpy must have been
gradual,’’ and in this scenario the Orthotrichaceae would
occupy an intermediate position. The Macromitrioideae
differ, however, from typical pleurocarps by several characters and should rather be considered cladocarpous (La
Farge-England, 1996). In genera where both cladocarpy
and acrocarpy occur, the former seems to be restricted to
terminal taxa, suggesting that the trend is from acrocarpy
to cladocarpy with no obvious cases of reversals (La
Farge-England, 1996). Such a general trend may suggest
that in the Orthotrichaceae too, the primitive condition is
acrocarpy. Thus if the Orthotrichales are indeed reduced
ciliate mosses, their putative sister group would most
likely belong to a group of acrocarpous Eubryales.
Subfamilial phylogenetic relationships—The ten orthotrichaceous genera remaining in the present analysis
are distributed among two clades that are moderately to
strongly supported by either method of analysis, with
bootstrap values (BV) ranging from 77 to 94% and decay
indexes (DI) of 2 and 3. The orthotrichoid clade combines the Zygodontoideae (Zygodon sections Zygodon
and Bryoides) and the Orthotrichoideae (Bryodixonia,
Orthotrichum, and Ulota), while the macromitrioid clade
includes all the Macromitrioideae (Cardotiella, Desmotheca, Groutiella, Macrocoma, Macromitrium, and
Schlotheimia) as well as Zygodon obtusifolius (Zygodon
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[Vol. 85
sect. Obtusifolii). Zygodon obtusifolius has retained several characters considered here plesiomorphic, such as
acrocarpy, smooth, cucullate calyptrae, but exhibits also
some derived features reminiscent of some Macromitrioideae (strongly bulging cells, coarse papillae, undifferentiated basal cells, etc.; see Goffinet, 1997c). If Z. obtusifolius is to be retained within the Zygodontoideae, the
unexpected relationship with the Macromitrioideae as
proposed by rbcL sequence data may be an artifact due
to a long branch attraction (Hendy and Penny, 1989) or
an indication of hybridization involving chloroplast capture from a macromitrioid taxon (Soltis and Kuzoff,
1995). In the latter case, the topology obtained here may
represent the ‘‘correct’’ gene tree, but deviates from the
true phylogeny of the taxa (Doyle, 1992). This hypothesis
is currently being tested by comparing sequence data of
the nuclear gene 18S.
With the exclusion of the Drummondioideae, Vitt’s
(1982a) phylogenetic arrangement of subfamilies would
have the Zygodontoideae (cucullate calyptrae) basal to a
dichotomy between the Orthotrichoideae and the Macromitrioideae (both typically or exclusively have large
mitrate calyptrae). The sister group relationship between
the orthotrichoid and the macromitrioid clades proposed
here deviates from Vitt’s (1982a) phylogenetic concept of
the family by the inclusion of the Zygodontoideae in the
former clade, and parsimony would need to be relaxed
by six steps for his concept to be satisfied (without constraining the affinities of Z. obtusifolius). The monophyly
of Zygodon and thus Zygodontoideae is not supported by
rbcL sequence data either, and the relationship of sections
Zygodon and Bryoides to the Orthotrichoideae sensu
stricto remain unresolved. If Zygodon is indeed paraphyletic, section Bryoides would most likely be the most
primitive taxon, given that it shares the plesiomorphic
state of ‘‘smooth laminal cells’’ with either a splachnaceous or a typical bryaceous ancestor. The two sections
of Zygodon differ on average by 40 mutations, while only
28 changes separate section Zygodon from the Orthotrichoideae, compared to 43 changes between section Bryoides and the Orthotrichoideae. Such divergences may be
indicative of the monophyly of a clade composed of section Zygodon and the Orthotrichoideae and support a single origin of papillae that would define this clade.
With regards to the Macromitrioideae, both methods of
analysis yield two strongly supported monophyletic
clades (Figs. 2, 4): one composed of all three species of
Schlotheimia, the other including all remaining Macromitrioideae (i.e., Cardotiella, Desmotheca, Groutiella,
Macrocoma, and Macromitrium). Vitt, Koponen, and
Norris (1993) suggested that Schlotheimia and Cardotiella should be placed in a separate subfamily based on
the smooth and distinctly lobate calyptra. RbcL data do
not support such relationship as ten more steps are needed to unite Schlotheimia and Cardotiella into a monophyletic clade. The species of Schlotheimia differ, on average, from other Macromitrioideae (including Cardotiella) by 47 bases. By contrast, the average distance between these other macromitrioid genera is only 23 bases.
If rates of molecular evolution are assumed to be similar
among these taxa (a reasonable assumption considering
both clades are predominantly phyllodioicous, and epiphytic in tropical montane forests; see Britten, 1986), dif-
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
B. 5 Bryodixonia; C. 5 Cardotiella; D. 5 Desmotheca; G. 5 Groutiella; M. 5 Macrocoma; Mt. 5 Macromitrium; S. 5 Schlotheimia; O. 5 Orthotrichum; U. 5 Ulota; Z. 5 Zygodon.
a
Phylogenetic relationships of Orthotrichaceous genera—Analysis of rbcL sequence using either the MP or
the NJ method suggests that four of the five larger genera,
namely Macromitrium, Orthotrichum, Ulota, and Zygodon, are not monophyletic. The paraphyly of Zygodon
has been addressed earlier. Bryodixonia is a monotypic
genus endemic to New Zealand. Sainsbury (1945) argued
S. tecta
S. trichomitria
S. brownii
C. quinquefaria
M. tenue
M. papillosa
G. chimborazense
G. apiculata
Mt. incurvifolium
Mt. longifolium
Mt. richardii
D. apiculata
Z. intermedius
Z. reinwardii
Z. pungens
Z. obtusifolius
O. lyellii
O. obtusifolium
O. anomalum
U. obtusiuscula
U. lutea
B. perichaetialis
ferences in nucleotide sequences may indicate a relatively
ancient divergence between Schlotheimia and the other
Macromitrioideae. Among the morphological characters
that separate Schlotheimia from other Macromitrioideae,
those related to the calyptrae (shape, anatomy, outline)
are shared only with Cardotiella (Vitt, 1981b), whereas
the remaining ones (cell areolation, color of leaves) are
plesiomorphic or autapomorphic (Goffinet, 1997c) and
thus phylogenetically uninformative. The pleisiomorphic
state of ‘‘smooth laminal cells’’ may, as was argued in
the case of Zygodon sect. Bryoides, be indicative of the
primitive position of Schlotheimia in the evolution of the
Macromitrioideae. Considering both the morphological
and molecular divergences between Schlotheimia and the
remaining Macromitrioideae, and the strong support for
the monophyly of both clades on molecular grounds, accommodating Schlotheimia in its own subfamily may better reflect the evolutionary relationship among these
clades.
Brotherus (1925, as Pseudo-Macromitrioideae), Walther (1983), and Crum (1987) isolated Desmotheca from
other Macromitrioideae on the basis of its dimorphic sterile and fertile branches. Vitt (1990), however, argued that
excluding Desmotheca from the Macromitrioideae would
most certainly result in the paraphyly of the later subfamily. Based on variation in the rbcL sequences, the phylogenetic affinities of Desmotheca clearly lay with Macromitrium sensu lato, but remain ambiguous with regard
to its sister group within this clade. Whether Desmotheca
should be retained in its own subfamily, and placed sister
to the Macromitrioideae (excluding Schlotheimia), or be
inserted within the latter, is not clear. In 33 MPTs (including the 15 that share a monophyletic Orthotrichaceae) Desmotheca occupies a derived position (Fig. 3),
nested between two Macromitrium species. In the remaining six MPTs as well as in the NJ tree (Figs. 3, 4),
Desmotheca is sister to the Macromitrioideae (Schlotheimia excluded). The phenetic distance between Desmotheca and other macromitrioid genera (Table 4; excluding Schlotheimia) are, on average, similar to those
between Orthotrichum and Ulota (20 bases), in the Orthotrichoideae, suggesting that placing Desmotheca in a
distinct subfamily may not be appropriate.
Based on our phylogenetic analyses of the rbcL sequence variation, the Orthotrichaceae could be regarded
as composed of two subfamilies, the Orthotrichoideae
and the Macromitrioideae, with the latter further divided
into two tribes ‘‘Macromitriae’’ and ‘‘Schlotheimiae.’’
Alternatively, the two subfamilies may deserve recognition at the family level as suggested by Churchill and
Linares (1995) and the Macromitriaceae would then be
composed of two subfamilies, the Macromitrioideae and
the ‘‘Schlotheimioideae.’’ The status of the Zygodontoideae, and thus the relationship of the two main subgenera to the Orthotrichoideae, needs further study.
1333
— 0.006 0.012 0.035 0.036 0.035 0.040 0.040 0.036 0.049 0.033 0.031 0.032 0.035 0.045 0.033 0.045 0.039 0.042 0.041 0.047 0.047
8
—
0.012 0.036 0.036 0.033 0.040 0.040 0.038 0.047 0.036 0.033 0.032 0.033 0.041 0.030 0.041 0.034 0.038 0.036 0.042 0.042
16
16
—
0.030 0.031 0.030 0.033 0.033 0.033 0.041 0.031 0.029 0.035 0.035 0.041 0.031 0.041 0.034 0.038 0.038 0.043 0.043
46
48
39
—
0.014 0.015 0.017 0.017 0.017 0.025 0.016 0.018 0.038 0.036 0.045 0.035 0.048 0.039 0.043 0.045 0.049 0.049
48
47
41
19
—
0.012 0.011 0.013 0.015 0.020 0.015 0.017 0.036 0.037 0.042 0.031 0.044 0.033 0.039 0.039 0.044 0.044
46
44
39
20
16
—
0.016 0.017 0.018 0.024 0.020 0.020 0.036 0.036 0.042 0.030 0.044 0.036 0.041 0.039 0.044 0.044
53
53
44
22
15
21
—
0.003 0.014 0.016 0.019 0.020 0.042 0.042 0.045 0.034 0.049 0.040 0.042 0.045 0.049 0.049
53
53
44
22
17
23
4
—
0.017 0.016 0.022 0.020 0.045 0.044 0.045 0.037 0.051 0.042 0.044 0.046 0.049 0.051
48
50
43
23
20
24
19
23
—
0.023 0.014 0.017 0.036 0.034 0.044 0.032 0.047 0.038 0.044 0.042 0.047 0.047
64
62
54
33
26
31
21
21
30
—
0.030 0.029 0.048 0.049 0.050 0.042 0.055 0.045 0.048 0.050 0.053 0.055
44
47
41
21
20
26
25
29
18
40
—
0.013 0.033 0.035 0.045 0.032 0.048 0.037 0.045 0.045 0.050 0.050
41
43
38
24
23
27
26
26
23
38
17
—
0.037 0.035 0.045 0.030 0.045 0.036 0.042 0.041 0.046 0.046
42
42
46
50
48
47
55
59
48
64
43
49
—
0.009 0.031 0.034 0.024 0.018 0.023 0.023 0.026 0.026
46
44
46
47
49
48
56
58
45
65
46
46
12
—
0.030 0.036 0.021 0.014 0.019 0.020 0.023 0.021
60
54
54
60
56
56
59
59
58
66
59
59
41
39
—
0.037 0.036 0.027 0.032 0.033 0.036 0.035
44
40
41
46
41
40
45
49
42
55
42
39
45
47
49
—
0.042 0.035 0.037 0.037 0.042 0.042
60
54
54
63
58
58
65
67
62
72
64
60
32
28
47
55
—
0.015 0.017 0.009 0.012 0.009
51
45
45
52
44
47
53
55
50
60
49
47
24
18
35
46
20
—
0.011 0.017 0.020 0.017
56
50
50
57
52
54
56
58
58
63
60
56
31
25
42
49
22
15
—
0.017 0.018 0.017
54
48
50
59
52
52
59
61
56
66
60
54
30
26
43
49
12
22
22
—
0.011 0.009
62
56
56
65
58
58
65
65
62
70
66
60
34
30
47
55
16
26
24
14
—
0.005
62
56
56
65
58
58
65
67
62
72
66
60
34
28
46
55
12
22
22
12
6
—
ET AL.—ORTHOTRICHALES: CIRCUMSCRIPTION AND PHYLOGENY
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
GOFFINET
TABLE 4. Pairwise comparison of rbcL nucleotide sequences within the Orthotrichaceae. Below diagonal is absolute distance; above diagonal is average distance. Distance values used
for intrageneric comparisons (see text) are in boldface.a
August 1998]
1334
AMERICAN JOURNAL
for a generic distinction from Ulota on the basis of ‘‘the
highly differentiated and conspicuous perichaetial bracts
and the diminutive calyptra’’ and the immersed capsule.
In addition, the perichaetial leaves have prorate basal
laminal cells (Goffinet, 1997c), a feature otherwise unknown from the Orthotrichoideae. Bryodixonia does,
however, share many of the characters found in the genus
Ulota, such as very thick-walled cauline cells, the differentiated marginal cells of the lamina, and the flexuose to
crisped leaves. Bryodixonia may thus be patristically very
derived; however, in cladistic terms it may not deserve
taxonomic recognition at the generic level. RbcL sequences of Ulota lutea and Bryodixonia perichaetialis
differ only by six mutations, a degree of divergence that
is similar to that found between species of Groutiella (4)
or Schlotheimia (8–16), but, moreover, it is less than the
divergence recorded between the two species of Ulota
(14; Table 4). Immersed capsules are characteristic of
many mosses that are taxonomically unrelated but share
a xerophytic habitat (Vitt, 1981a). Within the Orthotrichaceae completely immersed capsules are characteristic
for Schlotheimia sect. Stegotheca, and a shortening of the
setae leading ultimately to an immersed capsule occurs
in Orthotrichum subg. Gymnoporus sect. Leiocarpa,
subg. Pulchella sect. Rivularia, and sect. Diaphana, as
well as in subg. Orthotrichum (Lewinsky, 1993). Considering the low degree of morphological and molecular divergence of Bryodixonia, segregation at the generic level
does not seem appropriate.
The monophyly of Ulota (including B. perichaetialis)
is compromised by O. lyellii in the NJ tree and in 13
MPT (in 13 other MPTs their relationship is not resolved). Strong affinities of O. lyellii for Ulota (MP: 99%
BV and DI of 5) and U. obtusiuscula in particular (13
MPTs) may indicate that among the different lineages of
Orthotrichum, subg. Gymnoporus (Braithw.) Limpr. is the
most closely related to Ulota. Among the characters that
subg. Gymnoporus sect. Leiocarpa (see description in
Lewinsky, 1993) shares with Ulota, only the long flexuose vegetative leaves may be derived within the Orthotrichoideae and thus be indicative of common ancestry.
The genus Ulota (even if including Bryodixonia) is morphologically well defined from Orthotrichum, suggesting
that the paraphyly of Orthotrichum, if confirmed, would
need to be resolved by dividing the genus into discrete
entities rather than broadening the concept of Orthotrichum by including Ulota. The relationships of the subg.
Orthophyllum Delogn. (O. obtusifolium) and subg. Orthotrichum (O. anomalum) are not unambiguously resolved either: they form sister taxa in 13 MPTs as well
as the NJ tree, while in 13 other MPTs, O. obtusifolium
is sister to a clade composed of the remaining Orthotrichoideae. Orthotrichum obtusifolium had been placed together with the related O. gymnostomum Brid. in the genus Nyholmiella (see Lewinsky, 1993, for history), based
on ‘‘the obtuse leaves with plane or incurved leaf margin
and incrassate leaf-cells with a stout central papillae on
each side’’ (Lewinsky, 1993). Patterns in peristome ornamentation are similar to those observed elsewhere in
the genus (Lewinsky, 1993), and it may therefore be more
parsimonious to retain subg. Orthophyllum in Orthotrichum.
Within the macromitrioid clade (Fig. 4) the relation-
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BOTANY
[Vol. 85
ships remain ambiguous too (except for Schlotheimia see
above), either because Macromitrium and Macrocoma
truly are paraphyletic, or because of an insufficient taxon
sample. The genus Macromitrium is, with over 250 species, by far the most speciose genus of the Orthotrichaceae (Vitt, 1982a). Mitten (1869) divided the genus in
four sections, to which Buck (1991) recently added sect.
Reverberatum. Two of these have recently been raised to
the genus level, namely Micromitrium (now Groutiella
Steere) and Macrocoma Grout. The genus Macromitrium
remains, however, morphologically extremely diverse in
terms of size of the plant, degree of differentiation of the
basal cells, shape of the urn, and laminal cell shape, orientation, and ornamentation. The relatively high cost in
terms of parsimony, for a monophyletic Macromitrium
(nine steps) may be seen as just one other indication that
the genus as currently defined is still a heterogeneous
assemblage. Groutiella differs from Macromitrium by the
marginal limbidium of hyaline elongate cells and a short
calyptra covering only the upper portion of the urn. Except for G. tomentosa, Groutiella is restricted to the Neotropics, where ten species occur. The sister species in all
shortest trees is Macromitrium longifolium, a neotropical
endemic, rather than any of two paleotropical taxa (M.
richardii is known from Africa and the Americas [van
Rooy and van Wyk, 1992; Vitt 1993] and belongs to M.
ligulare group of the Old World; M. incurvifolium occurs
throughout the Pacific Ocean [Vitt and Ramsay, 1985]).
Whether these putative affinities of Groutiella for neotropical Macromitria indicate a common ancestry with a
distinct neotropical lineage of Macromitrium needs to be
further investigated.
The genus Macrocoma is composed of two subgenera,
subg. Trachyphyllum (M. papillosa) and subg. Macrocoma (M. tenuis), that differ by a series of characters, but
particularly by the well-developed peristome of the former (Vitt, 1980). Our molecular data suggest that these
two taxa, too, form an artificial group; three additional
steps are needed to restore the monophyly of Macrocoma. Macrocoma papillosa is found in a basal position
among macromitrioid taxa (excluding Schlotheimia) in 33
MPTs (Fig. 4), while in the remaining six it is found in
a more derived position with both species of Groutiella,
Macromitrium longifolium, and Macrocoma tenuis.
While the Macromitrioideae are typically cladocarpous
(i.e., with their perichaetia terminal on lateral branches)
and have dimorphic leaves between stem and branches,
M. papillosa is clado- and acrocarpous (i.e, with perichaetia terminal on lateral branches and on the stem) and
the leaves are not differentiated into stem and branch
leaves (Goffinet, 1997c). True acrocarpy also occurs in
subg. Macrocoma (e.g., M. braziliensis [Mitt] Vitt), while
other taxa of this subgenus are strictly cladocarpous (e.g.,
M. tenuis [Hook. & Grev.] Vitt). Acrocarpy and cladocarpy have not been reported before from the same taxon,
not to mention from the same individual (see La FargeEngland, 1996). The combination of plesiomorphies such
as undifferentiated stem and branch leaves, terminal cauline gametangia, and the complete double peristome may
be a strong indication that subg. Trachyphyllum is a primitive clade not only when compared to subg. Macrocoma
(Vitt, 1982c), but maybe even with regard to the evolution of the Macromitrioideae.
August 1998]
GOFFINET
ET AL.—ORTHOTRICHALES: CIRCUMSCRIPTION AND PHYLOGENY
Affinities of excluded taxa—Critically addressing the
relationships of the taxa here excluded from the Orthotrichaceae is beyond the scope of the present study and
would need a broader sampling of haplolepideous taxa.
A suite of unique characters combined with the lack of
characters that are phylogenetically crucial may always
hamper determining sister group relationships based on
morphology only. Crum (1987) already suggested that
gametophytic characters may not suffice to resolve the
phylogenetic relationship of Drummondia. The same
opinion prevails with regards to Amphidium. Brotherus
(1925), Anderson and Crum (1958), and Vitt (1973,
1982b, 1984) placed Amphidium near Rhabdoweisia. Our
results, though preliminary, do not suggest that these genera are closely related, and future study may need to consider alternative relationships, as for example with Glyphomitrium, due to the overall similarity of Amphidium
lapponicum-Glyphomitrium daviesii.
Presence of a peristome allows for a more explicit hypothesis to be made regarding the systematic position of
the Erpodiaceae and the Rhachitheciaceae. Edwards
(1979), as part of a review of the haplolepideous peristome, examined the peristome architecture of Venturiella
and concluded that the teeth are ‘‘strongly dorsally trabeculate, and also have a rudimentary unthickened basal
exostome,’’ and that ‘‘these characters are of a haplolepideous peristome although not of the dicranaceous type’’
but of a distinct type, the Seligeria type. This peristome
type is characterized by little ventral thickening, strong
dorsal trabeculae, and an exostome reduced to a thin,
smooth membrane adhering to the trabeculae. This combination of characters has also been observed in the
Rhachitheciaceae and has been interpreted as a possible
indication of haplolepideous affinities of the family (Goffinet, 1997a). Molecular data thus tend to confirm these
hypotheses, and consequently the peristome of the
Rhachitheciaceae and the Erpodiaceae should be regarded
as derived, through reduction, from a typical haplolepideous peristome. Though the monophyly of a group of
taxa sharing the Seligeria-type peristome has not been
critically examined, the nearly identical peristomes of
Rhachithecium, Glyphomitrium (Ptychomitriaceae), and
Blindia (Seligeriaceae) may be seen as an indication of
close phylogenetic relationships, despite gametophytic
differences. Alternatively, the Rhachitheciaceae may be
more closely related to Rhabdoweisia in the Dicranales,
considering the similarities in the overall habit, leaf
shape, and cell shape and differentiation.
Phylogenetic conclusions—Sequence data of the chloroplast gene rbcL are useful in circumscribing the Orthotrichales, particularly with regard to the systematic position of taxa lacking peristome features that are central
to the classification of mosses. Analyses of the variation
in the nucleotide sequence using either the parsimony or
the distance method strongly suggest that the Orthotrichales are polyphyletic and that the Erpodiaceae and the
Rhachitheciaceae are of haplolepideous affinities as suggested by their Seligeria-type peristome. The Orthotrichaceae too, are shown to be an artificial assemblage due
to the current inclusion of Amphidium and Drummondia,
two genera better placed among the Haplolepideae. The
Orthotrichaceae are only distantly related to the latter
1335
clade and should rather be considered a member of a
derived diplolepideous clade. The relationship to the
Splachnales and the ciliate mosses remains unsettled, but
at present all three lineages are best considered monophyletic. Molecular data do not support Vitt’s (1982a)
subfamilial phylogeny and instead suggest that the Orthotrichaceae are composed of two lineages. The first
consists of the Zygodontoideae and the Orthotrichoideae,
while the second includes all Macromitrioideae. Putative
basal taxa within these clades may be represented by Zygodon sect. Bryoides and Schlotheimia, respectively,
which are characterized by smooth laminal cells.
The Orthotrichaceae are now composed of 20 genera:
Bryodixonia, Cardotiella, Ceuthotheca, Desmotheca,
Florschuetziella, Groutiella, Leiomitrium, Leptodontiopsis, Leratia, Macrocoma, Macromitrium, Muelleriella,
Orthomitrium, Orthotrichum, Pleurorthotrichum,
Schlotheimia, Stenomitrium, Stoneobryum, Ulota, and
Zygodon. Examination of cladistic relationships and associated phenetic distances suggests that the monotypic
genus Bryodixonia may be better regarded as a patristically derived species of Ulota. Our molecular data furthermore reveal that larger genera such as Macromitrium
and Zygodon may merely represent evolutionary grades.
Gene data obviously have made a significant contribution
in resolving the circumscription of the Orthotrichaceae
and the relationship of the main lineages. Above all, however, this molecular study has laid the foundation for critically reexamining the morphological characters that are
central to the generic concept used in the Orthotrichaceae.
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