Caserta province, Italy; A Rio Flumini, Ogliastra Mountains, Nuoro province (the dry specimen was kindly donated by Prof. C. CORTINI PDROTTI, University of ...
P1. Syst. Evol. 216:69-79 (1999)
Plant Systematics and Evolution © Springer-Verlag 1999 Printed in Austria
Patterns of relationships in
Trichostomoideae (Pottiaceae, Musci) V. SPAGNUOLO,P. CAPUTO, S. COZZOLINO, R. CASTALD0, and R DE LUCA
Received May 30, 1997; in revised version March 3, 1998
Key words: Musci, Pottiaceae, Trichostomoideae. - ITS 1 phylogeny, ribosomal DNA. Abstract: Ribosomal DNA sequences were employed to infer relationships among European Pottiaceae. Intragenic spacer I was sequenced for six Trichostomoideae (Eucladium verticillatum, Pleurochaete squarrosa, Tortella flavovirens, T. nitida, T. tortuosa, Trichostomum brachydontium) and seven taxa from the other European subfamilies included in Pottiaceae (Bryoerythrophyllum recurvirostrum, Didymodon vinealis, Microbryum starkeanum, Tortula muralis, Syntrichia ruralis, Weissia controversa and Timmiella cf. barbuloides). Cladistic analysis of sequence data shows close relationships between Pleurochaete, Tortella and Weissia. Tortella appears to be paraphyletic, as it includes Pleurochaete, Weissia and Trichostomum. Weissia, therefore, seems to be better placed in Trichostomoideae than, as traditionally done, in Pottioideae. Neither Merceyoideae nor Pottioideae appear to be monophyletic sensu stricto, at least within the limits of the taxa in analysis. Within the latter subfamily, Syntrichia is clearly separated from Tortula. These results would suggest that taxonomy in Pottiaceae does not depict the pattern of descent, and therefore is in need of revision.
The comparatively simple anatomy and morphology of bryophytes often makes taxa discriminiation and delimitation difficult, especially at low taxonomic circumscriptions. As a consequence, phylogenetic relationships among taxa appear often obscure and confuse. For these reasons DNA techniques, which provide independent data sets for phylogenetic inference, can be profitably used in bryophyte systematics. Among the segments of DNA normally used in molecular systematics, nuclear rDNA genes, which include regions characterized by different degrees of conservation, can be employed in comparing either very distant or closely related taxa, dependent on the segment of DNA chosen (BALDWIN& al. 1995). Although rDNA has been repeatedly employed in order to clarify phylogenetic relationships in bryophytes (HEDDERSON& al. 1996 and references therein, WATERS & al. 1992), only few studies have investigated bryophyte phylogeny below the class level (COLACINO • MISHLER 1996, SPAGNUOLO& al. 1996; SPAGNUOLO~1; al. 1997). In this paper we analyze the sequences of intragenic spacer I in some Pottiaceae ScnI~P. (Pottiales, Musci) and we discuss the relationships between
70
V. SPAGNUOLO• al.:
various genera of Trichostomoideae (BSG) LIMPR. in BROTh. and other Pottiaceae. Trichostomoideae are acrocarpous mosses with lanceolate leaves, hyaline axillary hairs, leaf margin plane or occasionally incurved in the upper part of the lamina. The costa lacks a differentiated dorsal epidermis and shows a well developed dorsal stereid band and a smaller ventral one. These mosses are highly adapted to life in arid climates. The adaptation to such severe selective pressures, however, often causes the presence of parallel or convergent characters which develop as a response to the same environmental stimuli. This is turn complicates both group delimitation and phylogenetic inference founded on morphology. As a consequence, taxonomy of Pottiaceae is very controversial. A source of independent taxonomic evidence is therefore needed in this family. We examined six Trichostomoideae, chosen within the four European genera of this subfamily, and seven other taxa included in Merceyoideae BROTH., Pottioideae (LIMPR.) BROTh. and Timmielloideae ZAND., which are the other European subfamilies of Pottiaceae. Data are discussed in connection with morphologybased taxonomic and floristic evidence available (CORLEY & al. 1982, CRUM & ANDERSON 1981, SAITO 1975, SMITh 1978, ZA~ER 1993). Materials and methods
Specimens of Bryoerythrophyllum recurvirostrum (HEDw.) CHEN, Didymodon vinealis (BRID.) ZAND., EUCLADIUMVERTICILLATUM(BRID.) BSG, Microbryum starckeanum (HEDW.) ZAND., Pleurochaete squarrosa (BRID.) LINDB., Syntrichia ruralis (HEDW.)WEB & MOHR., TimmielIa cf. barbuloides (BRD.) MONK, Tortella flavovirens (BRucH) BROTh., Tortella nitida (LINDB.) BROTH., TortelIa tortuosa (HEDW.) LIMVR., Tortula muralis HEDW.,
Table 1. Origins, accession numbers of the voucher specimens, acronyms, ITS 1 length (bp) and GC content (%) for the taxa of Pottiaceae in study. S Natural Reserve of Valle delle Ferriere, Salerno province, Italy; N Botanical Garden of Naples, Italy; C Natural Reserve of Castelvolturno, Caserta province, Italy; A Rio Flumini, Ogliastra Mountains, Nuoro province (the dry specimen was kindly donated by Prof. C. CORTINIPDROTTI,University of Camerino, Italy). All specimens are deposited at NAP Taxon
Origin
Accession no. Acronym Length (bp) GC%
Bryoerythrophyllum recurvirostrum Didymodon vinealis Eucladium verticillatum Microbryum starckeanum Pleurochaete squarrosa Syntrichia ruralis Timmiella cf. barbuloides Tortella flavovirens Tortella nitida TortelIa tortuosa Tortula muralis Trichostomum brachydontium Weissia controversa
S N S C C C N C S S S A S
VF103/95 OB41/96 VFl18/95 CV132/95 CV86/96 CV74/76 OB106/95 CV101/96 VF108/95 VF97/95 VF22/96 HG346/96 VF88/96
beri didy eucl micr psqu synt timm tfla tnit ttor tumu tric weis
304 282 301 326 323 411 425 323 324 321 256 318 325
52.0 51.1 52.8 51.8 56.3 48.7 55.8 56.3 55.9 55.5 49.6 54.4 56.0
Relationships in Trichostomoideae
71
Trichostomum brachydontium BRUCI-Iin F. MOLL., and Weissia controversa HEDW. were collected in various localities of southern Italy (Table 1). Voucher specimens of the examined individuals are deposited at NAP (Table 1). DNA was extracted from approximately 200-300 mg of fresh gametophytic tissue, by using the protocol by CAPUTO& al. (1991) with the modifications indicated in SPAGNUOLOt~ al. (1997). Amplification of the ITS 1 was carried out by using primers annealing in the 3 / region of the 18S (51-GGAGAAGTCGTAACAAGGTTTCCG-31) and in the 51 region of the 5.8S (51-ATCCTGCAATTCACACCAAGTATCG-3 I) respectively. PCR reaction mixtures (100 ~tl) contained approximately 5 ng genomic DNA or 100 mg fresh tissue (SPAGNUOLO• al. 1997) and Blotto to 2% final volume (DE BOER & al. 1995). DNAs were amplified for 30 cycles in a Perkin-Elmer Cetns 9600 thermocycler by using the following conditions: 1 rain denaturation at 94 °C, 1 min annealing at 55 °C, 45 sec extension at 72 °C. Samples were denatured for 5 rain at 94 °C before the beginning of the first cycle; extension time was increased of 3 sec/cycle; extension was further prolonged for 7 rain at the end of the last cycle. PCR fragments, purified by using Microcon 100 microconcentrators (Amicon, Danvers, MA, USA), were double-strand sequenced in both directions by using a modification of the Sanger dideoxy method (SANGER~; al. 1977) implemented in a double strand DNA cycle sequencing system with fluorescent dyes. Sequence reactions were then loaded into a 373A Applied Biosystems Automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). Some sequencing experiments had to be repeated to solve all uncertainties. In one case (Timmiella) this approach was unsuccessful and the purified PCR product was ligated into a pUC18 vector (Farmacia Biotech, Uppsala, Sweden) and then sequenced in the same manner as above by using universal M13 primers. The sequences were reduced to only ITS 1 by comparing them with the 3/ terminus of 18S and with 5.8S of various sequences available in the literature and then aligned by using Clustal W ver. 1.6 (THOMPSON& al. 1994). The same software was also used to calculate pairwise distances. Various alignment investigations were carried out by modifying the gap opening (PWGAPOPEN and GAPOPEN) and extension (PWGAPEXT and GAPEXT) costs from - 5 to +5 as compared to the default settings. Transitions and transversions were weighted equally and the alignment delay for the most divergent sequences was set to 75%. Each alignment was then converted in numeric format by means of a word-processor macro and the resulting matrix subjected to an exhaustive parsimony analysis (ie) employing the Hennig86 software (FARRIS1988). TimmieIla was used as an outgroup. Gaps were coded as missing data and characters were treated as unordered. For each analysis consistency and retention indices (C.I. and R.I) were recorded. The alignment with highest C.I. and R.I. values was chosen as optimal. The resulting cladograms were examined and manipulated with the Clados software package (NlXON 1993). Bootstrap analysis (FELSENSTEIN 1985) and Permutation Tail Probability (PTP) test (FAm~ & CRANSTON1991) were carried out by using the SEQBOOT (1000 replciates) and DNAPARS programs of the Phylip 3.57 package (FELSENTEIN1993).
Results The lengths and GC contents of ITS 1 for all taxa in study are reported in Table 1. ITS1 length ranges from 256 to 425 bp, and GC content from 48.7 to 56.3%. ITS1 sequence divergence (Tables 2, 3) ranged from 1.2% (pairwise distance between Tortella nitida and T. tortuosa) to 57.7% (pairwise distance between Syntrichia and
72
V. SPAGNUOLO~5 al.:
Table 2. Pairwise distances between the taxa of Pottiaceae in study
timm eucl psqu tfla tnit ttor tric beri didy micr synt tumu weis
timm eucl
psqu
tfla
tnit
ttor
tric
beri
didy
micr
synt
tumu
weis
0.000 0.498 0.514 0.508 0.511 0.509 0.524 0.543 0.527 0.537 0.577 0.514 0.508
0.514 0.117 0.00 0.034 0.056 0.063 0.093 0.349 0.343 0.358 0.432 0.304 0.056
0.508 0.116 0.034 0.000 0.066 0.073 0.116 0.344 0.348 0.349 0.441 0.300 0.069
0.511 0.124 0.056 0.066 0.000 0.012 0.099 0.335 0.331 0.352 0.430 0.304 0.050
0.509 0.128 0.063 0.073 0.012 0.000 0.103 0.332 0.333 0.353 0.434 0.309 0.051
0.524 0.156 0.093 0.116 0.099 0.103 0.000 0.340 0.357 0.346 0.440 0.314 0.070
0.543 0.351 0.349 0.344 0.335 0.332 0.340 0.000 0.354 0.370 0.396 0.353 0.330
0.527 0.333 0.343 0.348 0.331 0.333 0.357 0.354 0.000 0.292 0.405 0.260 0.338
0.537 0.360 0.358 0.349 0.352 0.353 0.346 0.370 0.292 0.000 0.419 0.147 0.344
0.577 0.427 0.432 0.441 0.430 0.434 0.440 0.396 0.405 0.419 0.000 0.324 0.423
0.514 0.310 0.304 0.300 0.304 0.309 0.314 0.353 0.260 0.147 0.324 0.000 0.297
0.508 0.116 0.056 0.069 0.050 0.051 0.070 0.330 0.338 0.344 0.423 0.297 0.000
0.498 0.000 0.117 0.116 0.124 0.128 0.156 0.351 0.333 0.360 0.427 0.310 0.116
Timmiella). The maximum distance within Trichostomoide was 15.6% (pairwise distance between Eucladium and Trichostomum). The minimum distance between any two members of different subfamilies was 5.0% (pairwise distances between Weissia and Tortella nitida). The cladistic analysis carded out on the optimal alignment shown in Table 3 (PWGAPOPEN=GAPOPEN=9, PWGAPEXT=GAPEXT--5, consensus length 442 bp, informative characters 39.1%) yielded one most parsimonious cladogram (649 steps, C.I.=0.79, R.I.=0.64), shown in Fig. 1. The cladogram of Fig, 1 shows that our ingroup is divided into two major clades, one including all Trichostomoideae and Weissia (Pottioideae), and the other including the other representatives of Pottioideae and Merceyoideae. Trichostomoideae are divided into two subclades, one with Eucladium and the other with the rest of the taxa. In this group, Tortella flavovirens and P. squarrosa are sister taxa, and in turn form a sister group to a clade composed of Trichostomum and Weissia and two species of Tortella. The other major clade has Syntrichia (Pottioideae) at the base, followed by a ladderized sequence of Bryoerythrophyllum, Didymodon (both included in Merceyoideae), Tortula and Microbryum (both included in Pottioideae). Bootstrap analysis (bootstrap values > 50% are shown in Fig. 1) generated a majority-rule consensus tree topologically identical to the cladogram in Fig. 1. The majority of the groups have bootstrap values above 50%; within our ingroup, only the clade including Trichostomum, Weissia, Tortella nitida and T. tortuosa is weakly supported. Another group which is below 50% is the clade including Bryoerythrophyllum, Didymodon, Microbryum and Tortula. The evaluation of phylogenetic signal, carried out by the PTP test (FAITH & CRANSTON 1991), showed that no tree obtained from any of the 500 fictitious matrices was comparable in length to the most parsimonious cladograms
Relationships in Trichostomoideae
73
Table 3. Alignment of the sequences of of Pottiaceae in study (an asterisk indicates position identity) timm
CACACACC
eucl
CACACAAAGTTGCAGCAAAGTTGCAGCAAACCCCCTT---GCGAATTTCATGATA
.........
ATTCAGTGGCAGACCATTCA---GCGAACTCTATGATT
micr
CACACACA
.........
psqu
CACACACA
.........
AAGTTGCAGCAAACCCCCTTT--GCGAATTTCATGAAT
tfla
CACACACA
.........
AAGTTGCAGCAAACCCCCTTT--GCGAATTTCATGAAT
AAGTTGCAGCAAACCCCCTT---GCGAATCTTATGAAT
tnit
CACACACA.
,AAGTTGCAGCAAACCCCCTTT--GCGAATTTCATGAAT
ttor
CACACACA
.AA-TTGCAGCAAACCCCCTTT--GCGAATTTCATGAAT
tric
CACACA
beri
CACACACA
...........
didy
CACACAC
synt
CACACACAC--G
tumu
CACACACA
weis
CACACACA
AAGTTGCAGCAAACCCCCTT---GCGAATTTCATCAAT
.........
AAGTTGCAGCAAACCCCCTT---GCGAATTTCGTGATT
.......... ....
........
AAGTTGC--CAGCCAATCTCT--GCGAATCTTACCATT AATGTTGCAGCCAACCCCTTTTCAGCGAACCGTACCATT AAAGTTGCAGCAAACCCCCTTT--GCGAATCTTACCATT .AAGTTGCAGCAAACCCCCTTT--GCGAATTTCATGAAT
timm
GTCCCCCTTGGTTGGCCTGGTTTTGCTTGGCTCTGGCTCTGGCTCTGGCTCTGGCTCTGG
eucl
GTCCCCCTTG
........
TGTTCTCGGGGGCCGGGGG
micr
GTCCAACCCT
........
CGCAA--CAAGTTTGGGG
psqu
GTCCCCCTTG
........
TGTTTTGGGGGGGCTGGGG
......
tfla
GTCCCCCTTG
........
TGTTTTGGGGGG-CTGGGG
......
CATCGTCGTCACTAT--
tnit
GTCCCCCTTG
........
TGTTTCGGGGGGCTGGGGC
......
CATCGTTGTCACTATCT
ttor
GTCCCCCTTG
........
TGTTTCGGGGGGCTGGGGC
......
CATCGTTGTCACTATCT
tric
GTCCCCCTTG
........
TGTTTTGGGGGGCTGGGGCATCAATCGTCGTTGTCACTACTA
beri
GTCCCCCCTTT
.....
GTTGTT--GGAACTTTGGGGG
didy
GTCCCCCTTCG
.....
CTTGGTTTGGGGGGCTGGGGCGCTTTCGACTGCTCGCCAACTAA
.....
AGCACTTTTGTGTTTTGGGGGGGCTGGGGCGCGCCTTCCTCTCA
................ ..........
CGTCGTCGTCACTAC--
GCTGGGACAGT--
synt
GTCCCCTTTTC
tumu
GTCCAAACC
weis
GTCCCCCTTG
timm
CTTGGTTTGGTTGGTTGGTTGGTTGGTGGAGGGGGGACTTAATTGTCCCAGCTATAGCCC
eucl
...............
micr
--TTTCATCCG-A--CG-ATGCTTGGG-CGCGGCGGCACTAGTCGT-CTGCCCCAAGTGT
psqu
....
TCTTG-TGG--TG-GTGGTGGTGGTGTCCCCGGCCTGCCGAA-CACATCCAAGTCC
tfla
....
TCTTG-TGG--TG-GTGGTGGCGGCGTCCC-GGCCTGCCGAA-CACATCCAAGTTC
tnit
....
TCGTGGTGG--TG-GTGGTGGTGGTGTCCC-GGCCTGCCGAA-CACATCCAAGTCC
ttor
....
TCGTGGTGG--TG-GTGGTGGTGGTGTCCC-GGCCTGCCAAA-CACATCCAAGTCC
tric
CTCCTGGTGGTGG--TG-GTGGTGGTGGTGTCCC-GGCCTTCCAAA-CACATCCAACTCC
......... ........
CGCA---CAGGTTTGGGTC
........
TGTTTTGGGGGGCTGGGGCAT
TG-TTGTTGGTG
.....
A.
GGCTGGGATGCC--
....
TGGCCGGGACAC--CCTCGTTGTCACTAC--
CC-GGCCTGCCAAA-CACATCCAAGTCC
beri
...................
TGTTTCTCCT-TGCC---ACTGATTGATCGTT
.... AGTTG
didy
TTGATCAT
TAGTTGGATGCCAACTGACTTGATCCT-CAGT
......
TGG
synt
CCATCCATATGCA--AGTGTACTTCTGCTCTGCCGGAACTACTAGAGCCTAATGTAGTTC ......
TGA
...........
tumu
--TTTCATCC
weis
....
.........
TGCTTGC
....
CGACCG-ACCGATCAT-CGGT
timm
CGTCCAT-CGCCGCCGCCGCCGCCGCCAGCACCAGCACCAGCACCAGCACCAGCACCAGC
TCGTG---G--TG-GTGGCGGTGGTGTCCC-GGCCTGCCAAA-CACATCCAAGTCC
eucl
CCCCC---TTATACGGGGAGATATTTGGGG
.....
micr
TGTT
....
.....
GTTGTTGGGATAGCGTCTTGG
CTCCCTAAACAGG-G
psqu
CCCCA---ATACAATGG--GAGGCTTGGGGC--TCCTCCCTGAACCGG-GTAGGCGGCCC
tfla
CCCCC---ATACAATGGGGGAGGCTTGGGGC--TCCTCCCTAAACCGG-GTAGGCGGCCC
tnit
CCCC
....
ATACAAATGGGAGGGCTTGGGGCT-CCCTCCCTAAACCGG-GTAGGCGGCCC
ttor
CCCC
....
ATACAAATGGGAGGGCTTGGGG---TCCTCCCTAAACCGG-GTAGGCGGCCC
tric
CCCC
....
TCACCATGAGGGAGGGGAGGGG
beri
GCAGG
....
GTGGTTGG--ACGCGTCTCGG
..... ....
CTCC
.................
CCTCCCAAATCCCT
didy
CGTGA---GTATGTTGG--GAGTGTCTCGG
synt
AGCGGTCGTCTTTTAGGTGGCTCCTAACTGACGGCCTTGCAAAGCCGAAGTATAAAGCAT
tumu
CG .......
weis
CCCCCA--ATACAAAGG--GAGGCTTGGGGCTCTCTTCCCTCAACCGG-GTCGGCGGCCC
GGTGTTGGAAT---GTCTCGG
.......
....
......
GCCC
CCTGCCA-AACAGC-CCCCCAAGTCT
.......
CCC ................
CCTCCC
......
GCCC CGCTC GCC-
G .............
74
V. SPAGNUOLO~2 al.:
Table 3 (continued) timm
ACCAGCACCCACCACCCCAATTGAGACTTGGGGGCTCCCATCCATCCCATCCGGATGGAT
eucl
CT---TGTCCCCCTCCCTTTGGGGTGTGACTTGGCA
........
ACCA
micr
CT---CTTCTGGAGG--GACTTGGGTGGTGTTGTTA
........
TCC
............
psqu
CT---TGTCCCCCC---T---TGGGAGGACTTGGTA
........
ACC
......
tfla
CT---TGTCCCCCCCC-T---GGCTGGGACTTGGTA
........
ACC
.............
.............
tnit
CT---TGTCCCCCT
........
GGGAGGACTTGGTA
........
ACC
.............
ttor
CT---TGTCCCCCT
........
GGGAGGACTTGGTA
........
ACC
.............
........
ACCC
tric
CT---TCTCCTCCCTTGA---GGGTGGGGATTGGTA
beri
CT---TTTCGGGGGCGCGCGGGGTACACACTTTTCGGGTGTGTATCCAA---CAAC
didy
......................
synt
ATGGATGTTGGGAGTTTTGGATGATATGCCTTGGCCCCTCCTTCTCCCAACACAAAAGTT
GAGCTGAGTTGTC
....
..........
GACTTG--TG---TTATC
............
..........
CC .............
tumu
.................
weis
CT---TGTCCCCCCT--T---GGGGGGGACTTGGTA
timm
GGGAGTCTCCAGGACCCTCACTTGGTAACAAACCCGATGGCGCAATGCAT---CTCTTCA
eucl
............
AACCGA
...................
TTGCCCAATGCATCAACACTTTC
micr
............
AACCGA
...................
TTGCACTATGCATCAACACCTCC
psqu
............
AACCGA
...................
TTGCGCAATGCATCAACACTTTC
tfla
............
AACCGA
...................
TTGCGCAATGCATCAACACTTTC
tnit
............
AACCGA
...................
TTGCGCAATGCATCAACACTTTC
........
CC ............. ACC
.............
ttor
............
AACCGA
...................
TTGCGCAATGCATCAACACTTTC
tric
............
AACCGA
...................
TTGCGCAATGCATCAACACTTTC
beri
............
GACCAAC
didy
............
AACCGA
........
...............
T-GTTGCACAATGCATCCCCA
...................
GAACCAACA
.....
TTGCACATTGCATCAACACCTCC
synt
GTT
tumu
............
AACCGA
...................
ACCCCAATTGTTGCACTATGCAT-ACCACCAAT TTGCACTCTGCATCAACACCTCC
weis
............
AACCGA
...................
TTGCGCAATGCATCAACACTTTC
timm
AATATGACTGAGTATTACGTGAACCTTGGATGGATGGTGGTTGTAAGAGACGGCCATCCA
eucl
AATATGACTGATTATAAAA-TC
micr
AATATGACTGAGTATAAAAACAAT-GGGGCCACCCACCGGAATTCCGATTCCGGTGGTGG
psqu
AATATGACTGAGTATAAAAATCC---GGGACACTGCCGC---TTGCAAAGG-CGGTGGCG
tfla
AATATGACTGAGTATAAAAATC
....
GGGACACTGCCGC---TTGCAAAG--CGGTGGCG
tnit
AATATGACTGAGTATAAAAATC
....
GGGACACTGCCGC---TTGCAAAGG-CGGTGGCG
....
GGGATGCTGCCGC---TTGCAAAGG-TGGTGGCA
ttor
AATATGACTGAGTATAAAAATC
....
GGGACACTGCCGG---TGGCAAAGG-CGGTGGCG
tric
AATATGACTGAGTATAAAAATC
....
GGGACAATGCCGC---TTGCAAAGG-TGGCGGTG
bezi
AAAATGACTGAGTATAAAAACC
....
GGGACGACCCTCG
didy
AATATGACTGAGTATAAAAACCTT-AGGGCCCACCACCA
synt
AATATGACTGAGTATAAAACCAAACGGGGCCCCTTTCTCTTCTTATGGAAG-AGGGGGGG
tumu
AATATGACTGAGTATAAAAACAAA-AGGGCCTCTAAACC
weis
AATATGACTGAGTATAAAAATC
timm
TCCGGAGTTGATTTTTTGA-AAAAACA
eucl
TCCC
....
CGAGTTGTAAT-A--AACA
micr
TCCT
....
AGAGTTGTAAT-ATAAACA
psqu
TCCC
....
CGAGTTGTGAT-A--AACA
tfla
TCCC
....
CGAGTTGTGAT-A--AACA
tnit
TCCC
....
CGAGTTGTGAT-A--AACA
ttor
TCCC
....
CGAGTTGTGAT-A--AACA
tric
TCCC
....
CGAGTTGTGAT-A--AACA
beri
TCCT
....
CGAGTTGTAAT-A--AACA
didy
TCCT
....
CGAGTTGTAATTAAAAAAA
synt
CCCTGGAGTGAGTTGTAAT-ATAAACA
tumu
TCCT
....
TGAGTTGTAAT-ATAAACA
weis
TCCT
....
CGAGTTGTGAT-A--AACA
....
.......
AAAAG--AGCGAAG
.............
...............
GGGACACTGCCGC---TTGCAAAGG-TGGCGGTG
CCGGAGGG
TAGGGG
Relationships in Trichostomoideae
75
Fig. 1. Single maximum parsimony cladogram otbained for the taxa in study (649 steps, C.I=0.79, R.I.=0.64). The numbers on the right of the branches indicate the number of synapomorphies for each clade (numbers of homoplasious characters are in parentheses); the numbers on the left indicate the number of occurrences (out of 1000) of the clades in a bootstrap analysis
mentioned above. The length of the resulting 1206 cladograms ranged between 780 and 807 steps (median 794, s.d. 3.84), that is 20 to 24% longer than the most parsimonious trees found for the original data set. In such cases, PTP is
