J Hattori Bot. Lab. No. 96: 231- 244 (Aug. 2004)

MOLECULAR DIVERGENCE PATTERNS WITHIN THE GOND­ WANAN LIVERWORT (, HE­ PATICOPHYTINA, BRYOPHYTA). STUDIES IN AUSTRAL TEMPER­ ATE RAIN FOREST 25

FRIEDERlKE SCHAUMANN*, TANJA PFEIFFER AND WOLFGANG FREY

ABSTRACT. For six of the seven species of the genus Jensenia (J pisic% r, J. connivens, J. erythro­ pus, J. d(/Jormis, J. spinosa and J. decipiens [Pallaviciniaceae, Hepaticophytina, Bryophyta]) geomol­ ecular divergence patterns are evaluated by comparison of the cpDNA trnT UGU -trnLUAA 5' exon inter­ genic spacer and trnLuAA intron and nrDNA ITS2 sequences. All studied Jensenia taxa are character­ ized by a low infra- and interspecific sequence variation in the investigated DNA regions. The species are differentiated by a sequence divergence of 0-2.0% (trnT-L spacer) and 0-1.3% (trnL intron and ITS2), respectively. A comparison with the molecular data of five s.str. species supports the delimitation of Jensenia as a di stinct taxon.

K EY WORDS: Gondwanan di stribution pattern, Jensenia, Pallaviciniaceae, cpDNA trnT-trnL spacer, trnL 5' exon, trnLuAA intron, nrDNA ITS2.

INTRODUCTION Jensenia Lindb. (Pallaviciniaceae) consists of about 7-8 species (Grolle 1964, Forrest et a1. 2004) with southern temperate and alpine tropical distribution (Fig. 1.1). The genus is lacking in Laurasia, the family Pallaviciniaceae in general is presumably of Gondwanan origin (cf. Schuster 1982, Frey 1990). Together with Pallavicinia Gray and Hattorianthus R. M. Schust. & Inoue, Jensenia belongs to the subfamily Pallavicinioideae. Morphologically, Jensenia is differentiated from its close relative Pallavicinia s.str. by the dendroid habit with regular 2 or 3(-4)X bifurcate terminal branching of the frond (Fig. 1.2, 3), and the gynoecia, which are strictly confined to frond bifurcations and are always situated above a terminal branch in the lower part of the aerial frond (Grolle & Piippo 1986). In literature the systematic position of Jensenia is discussed controversially. Follow­ ing Gottsche (1864), Lindberg (1868), Stephani (1900) and Verdoorn (1932), Schuster (1963) and Grolle (1964) recognize the dendroid species as a distinct genus. This concept is followed by various authors (e.g., Gronde 1980, Scott 1985, Grolle & Piippo 1986, Engel 1990, Perold 1993, Gradstein et al. 2001) and was recently confirmed by morphological studies (Schuette & Crandall-Stotler 2002). In contrast, Schiffner (1893), Hiissel de Menendez (1961), Schuster (1992) and authors dealing with the New Zealand flora (e.g., AlIison & Child 1975, Campbell et al. 1975, Glenny 1998) do not accept the dendroid species as a separate genus, but classify them as a subgenus of Pallavicinia s.1. (Mittenia

Freie Universitat Berlin, Institut fUr Biologie-Systematische Botanik und Pftanzengeographie-AI­ tensteinstr. 6, D-14195 Berlin, Germany. * e-mail correspondingauthor:[email protected] N '"N @v~

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~ ::r: '" •o g. O:l ~ r ,r'" 0 0 z 0 °0 I 'Cla.. 02 2 Q2 c~ 2 ·O,;

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Fig. 1. World distribution of the genus Jensenia (I J. spinosa, 2 J. pisicolor, 3 J. connivens, 4 J. florschuetzii, 5 J. ery­ N thropus, 6 J. dijJormis, 7 J. decipiens) and habit of South American J. pisicolor (2; voucher W. Frey & F. Schaumann 01-257) o o and Australasian J. connivens (3; voucher W. Frey & T. Pfeiffer 98-T27). .j>. F. SCHAUM ANN ET A L.: Molecular divergence patterns within Jensenia 233

[Gottsche] Schiffn.). In this work, Pallavicinia s.str. and Jensenia are treated as separate genera. Pallavicinia s.str. species are prostrate (to ascending), but never dendroid as the species of Jensenia. Jensenia species prefer terricolous sites, moist earth and clayey soil, in perma­ nently atmospherically humid, more or less shaded habitats in montane and alpine environ­ ments. In the Neotropics Jensenia seems to be primarily alpine; below the forest line the species occur mostly in artificial habitats such as on steep, cut earth banks and along trails (Gradstein et al. 200 I). The latter habitats are also characteristic for the New Zealand J connivens (Colenso) Grolle. Jensenia pisicolor (Hook.f. & Taylor) Grolle has an amphi-southatlantic distribution (South Georgia, Tristan da Cunha, Prince Edward Is., Crozet Is., Amsterdam 1., Kerguelen Is. , Falkland Is., Tierra del Fuego, Patagonia, Valdivian Region north to 40 0 10'S; Frey & Schaumann 2002, Grolle 2002; see Fig. I). Jensenia connivens occurs in New Zealand (Grolle 1964; Ratkowsky 1987 excluded it from Tasmania). Scott (1985) lists the latter species from SE Australia. Jensenia connivens is here not considered to occur in SE Aus­ tralia, since the species' description and photograph by Scott (1985) as well as herbarium specimen (voucher B.Fuhrer & G.Scott, Bryo 236017 in B) determined by Scott belong to Pallavicinia rubristipa Schiffn. Jensenia pisicolor and J connivens are habitually very sim­ ilar if not identical (Scott 1985) and may be regarded as Gondwanan, respectively palaeoaustral taxa, and the presumable progenitor of derived alpine tropical species. These latter alpine tropical species include the neotropical J difformis (Nees) Grolle (incl. J wal­ lisii [IBJack & Steph.] Grolle; which is probably conspecific with J difformis [Grolle 1964], after Hiissel de Menendez [1961], however, conspecific with J erythropus) from the Andes, SE-Brazil, Mt. Roraima, and Costa Rica (new record, Holz et al. 2001), J erythro­ pus (Gottsche) Grolle (with two varities, var. erythropus and var. nobandae Gronde [Gronde 1980]; widespread, paramos of Costa Rica, high Andes from Bolivia to Venezuela, Sierra de ltatiaia; Gronde 1980, Gradstein et al. 2001) and J jiorschuetzii Gronde (paramos of Colombia and northern Ecuador) as well as the Indo-Malayan J decipiens (Mitt.) Grolle (Sri Lanka, Sumatra, Java, Celebes, Sabah, Mindanao, Luzon, Papua New Guinea). The only African representative of the genus, J spinosa (Lindenb. & Gottsche) Grolle occur­ ring from Malawi, Tanzania, Rwanda, Zaire to South Africa, Mauritius, Reunion and St. Helena (Perold 1993), may also be a Gondwanan relict species, with an independent evolu­ tion since the disruption of Africa from the palaeoaustral region ca. 105 mio. years ago (McLoughlin 200 I). DNA sequencing offers a suitable tool to clarify the systematic status of Jensenia and the molecular divergence patterns within the taxon. Sequences of the cpDNA (trnT-trnL spacer, trnL 5'exon, trnL intron) and nrDNA (ITS2) are compared. These are suitable markers for the distinction of Pallaviciniacean species (compare Schaumann et al. 2003 for Symphyogyna). Furthermore, geomolecular divergence patterns of the Gondwanan relict species (J pisicolor, J connivens, J spinosa) and presumable modern taxa from secondary speciation centers in the Neotropics and Indo-Malaysia are assessed. 234 J. Hattori Bot. Lab. No. 96 2 0 0 4

MATERIAL AND M ETHODS material. The collecting data as well as further relevant data (GenBank acces­ sion numbers etc.) of the investigated specimens are listed in Table I. The trnLuAA intron sequence of Jensenia connivens (I) was analyzed by M. Stech. Fresh plant material of J jiorschuetzii was not available. DNA preparation, peR and sequencing. For DNA extraction from herbarium tissues and fresh plant material the method of Doyle & Doyle (1990) was followed with the excep­ tion of using only 76% (v/v) ethanol to wash the pellets after precipitation with cold iso­ propanol. PCR reactions with primers CM/DM for the cpDNA trnLuAA intron, A/Dx for the cpDNA trnT UGU-LUAA intergenic spacer (A, CM' DM slightly modified after Taberlet et al. 1991 , the internal intron primer Ox [5'-GTT TCC TTC GAG TCT CTG CAC-3'] was de­ veloped for liverworts [M . Stech, pers. comm.] and 5.8F/25R for nrDNA internal tran­ scribed spacer (ITS2; after Baldwin 1992; primer sequences are available on request), pu­ rification of PCR products and sequencing follow the method described in Quandt et al. (2001). Alignment and free construction. The alignment of the sequences was created manu­ ally with the alignment editor Align32 (HepperJe 1997). Species of the sister genus Sym­ phyogyna (s. hymenophyllum and S. podophylla) were used as outgroup (subfam. Sym­ phyogynoideae [Trev.] Schust., Schuster 1992). PAUP4.0bl 0 (Swofford 2002) was used for the calculation of molecular trees. Maximum parsimony and likelihood analyses were per­ formed with three different data sets: cpDNA (trnT-trnL spacer, trnL 5'exon, trnL intron), nrDNA (ITS2) and combined investigated cpDNA/nrDNA regions. The first eight positions of the trnT-trnL spacer were excluded due to incomplete sequences. The lengths of the trnT-trnL spacer given in the Results are based on this data set, and values are hence small­ er than the 'real' lengths. Analyses were conducted including and excluding ambiguous se­ quence parts (trnT-trnL spacer: positions 255- 274, trnL intron: positions 111 - 137, ITS2: positions 31 - 59). Heuristic searches were performed with the following options: all charac­ ters unweighted and unordered, multi state characters interpreted as uncertainties, gaps coded as missing data, performing TBR branch swapping, collapse zero length branches, MulTrees option in effect. Heuristic bootstrap searches were performed with 1000 repli­ cates, 1000 random addition replicates per bootstrap replicate, and the same options in ef­ fect. Maximum likelihood analyses were executed using the models and parameters that best fit the data as evaluated by Modeltest v.3.06 (Posada & Crandall 1998) employing the winModeltest front-end v4b (Patti 2002). For the nrDNA regions the TrN + G and GTR +G models, for the cpDNA regions the HKY +G and TrN + I models and for the combined data sets the TrN + G and TrN + I models were performed. Likelihood bootstrap analyses were performed with 500 replicates, 10 random addition replicates per bootstrap replicate, and the same options in effect.

R ESULTS

In the SIX investigated Jensenia species the trnL intron length ranges from 3l4- 3l5bp; the frnT-L spacer has a length of 25l- 254bp. In the studied species of Table I. List of investigated specimens with abbreviations (abbr.), origin, sampling data, herbarium (herb.) in which a voucher is held, and GenBank accession numbers (acc. no.). W. Frey = private herbarium W. Frey, H. Kiirschner = private herbarium H. Kiirschner, both Berlin.

Acc. no. Abbr. Origin Sampling data Herb. ITS2 trnT-L spacer trnL intron ~ V> J: ";,. Jensenia connivens (Colenso) Grolle Cs: I New Zealand, South Island, Westland National Park W. Frey & T. Pfeiffer, 98MoII W. Frey, CHR AY547540 AY007623 ;,. z 2 New Zealand, South Island, Nelson Land District D. Glenny, 8100 CHR AY547526 AY5475 I I z CHR 527432 !:l ;,. Jensenia decipiens (Mitt.) Grolle ,. 1 Papua New Guinea T. Koponen, 31048 B AY547532 AY547516 3: o Bryo 270408 (;- Cl 2 Philippines, Luzon M. Jacobs, B 448 B AY547517 C Bryo 234544 ~ 3 West-Irian P Hiepko & W. Schultze-Motel, 2051 B AY547518 P­;; . <> Bryo 219635 cia <> Jensenia difformis (Nees) Grolle ::l Cl Ecuador, Prov. Zamora-Chinchipe H. Kiirschner, G. Parolly & D. Wagner, 02-619 H. Kiirschner AY547530 AY5475 14 ."," Jensenia erythropus (Gottsche) Grolle var. nobandae Gronde a 1 Peru, Dep. Amazonas J.-P. Frahm, P Geissler, S.R. B AY547529 AY5475 13 :; V>" Gradstein, G. Philippi, W. Schultze-Motel, 1043 Bryo 256348 § 2 Colombia, Boyaca A.M. Cleef, 9780 B AY289159 AY547542 AY289136 s- 5' Bryo 229545 ~ Jensenia pisic% r (Hook. f. & Taylor) Grolle " 1 Kerguelen Archipel E. Aubert de la Rue B AY547528 AY547543 '" '";:So Bryo 03797 " 2 Chile, X. region, Cordillera Pelada W. Frey & F. Schaumann, Mo 01-257 W. Frey AY547527 AY5475 12 3 Chile, X. region, Reserva Nacional de Llanquihue W. Frey & F. Schaumann, 01-382a W. Frey AY289160 AY54754I AY289137 Jensenia spinosa (Lindenb. & Gottsche) Grolle Tanzania, Morogoro District T. Pocs, R. Ochyra & H. Bednarek- BONN AY547531 AY547515 Ochyra, Ser. VllI, No. 197 N w V> N W 0'>

Table I. (Continued).

Acc. no. Abbr. Origin Sampling data Herb. ITS2 frnT-l spaccr fml intron

Pallavicinia longispina Steph. Taiwan Yang, Kao et aI., 1004-8 B AY547533 AY547519 Bryo 03758 Pallavicinia Ivellii (Hook.) Carruth. Rwanda T. Pocs, 6434 W. Frey AY547536 AY547522 ~ ::r: Pallavicinia rllbrisfipa Schiffn. e> Australia, Vic. B. Fuhrer & G. Scott, 4125 B AY547539 AY547525 §. Bryo 236017 o:l Pallavicinia slIbciliafa (Austin) Steph. ~ r- 1 Japan, Fukayabakei H. Inoue, 865 B AY547534 AY547520 e> ". Bryo 26 1112 Z 2 Japan, between Sennin-bashi and H. Inoue, 917 B AY547535 AY54752I !=' '-0 Akanuma Bryo 265793 0'> Pallavicinia xiphoides (Hook.f & Taylor) Trevis. I Chile, X. region, near Valdivia H. Manitz, RCH 322 JE AY547537 AY547523 2 New Zealand, South Island, A.J. Fife, 10324 CHR AY547538 AY547524 Fiordland National Park CHR 542064 Symphvogyna hymenophyllum (Hook.) Mont. & Nees New Zealand, North Island, Urewera T. Pfeiffer, 98-T206 W. Frey, AY289177 AY368645 AY289153 National Park CHR Symphvogyna podophylla (Thunb.) Mont. & Nees Chile, X. region, Parque Nacional Puyehuc W. Frey & F. Schaumann, 01-301 W. Frey AY289 169 AY368644 AY289145

N 0 0... F. SCHAUMANN ET AL.: Molecular divergence patterns within Jensenia 23 7

Pallavicinia s.str., the trnL intron varies from 320 bp to 328 bp and the trnT-L spacer from 242- 252 bp. In taxa of the Symphyogyna outgroup the trnL intron sequences are 326- 331 bp and the trnT-L spacer 249- 250 bp long. The trnL 5' exon comprises 35 bp in all studied taxa. In the cpDNA alignment 189 of the 663 positions are variable (29%). Of the variable positions, 148 (78%, 22% of total positions) are parsimony-informative. ITS2 has a length of 233-234 bp in Jensenia and of 224- 236 bp in Pallavicinia s.str. In the outgroup, ITS2 sequence length varies from 216 to 218 bp. Of the 281 positions 97 (35%) are variable. Of the variable positions, 61 (63%, 22% of total included positions) are parsimony-informative. In the combined cpDNA and nrDNA data set 944 positions were used for tree construction. 284 (30%) of the positions are variable and 209 of the variable positions (74%, 22% of total included positions) are parsimony-informative. The studied cpDNA and nrDNA regions show a low sequence divergence in the stud­ ied species of Jensenia. The trnL 5' ex on is identical in all Jensenia taxa. Interspecific dif­ ferentiation ranges from 0- 5 substitutions in the trnT-L spacer, 0-4 substitutions in the trnL intron and 0-3 substitutions in the ITS2 region (interspecific substitution percentage: trnL intron as well as ITS2 0- 1.3%, trnT-L spacer 0-2.0%). Infraspecific differentiation is also very low (infraspecific substitution percentage: trnT-L spacer 0-0.8%, trnL intron 0-0.3%, ITS2 0-0.4%). In comparison to other genera of the Pallaviciniaceae (Pallavicinia and Symphyogyna, compare also Schaumann et al. 2003), the inter- and infraspecific sub­ stitution percentage in the trnL intron, trnT-L spacer and ITS2 region of Jensenia is very low (Tables 2-4). A geographical differentiation between the species of Jensenia is discernible when re­ garding the percentage of substitutions in the combined data set of the trnL intronltrnT-L spacer and ITS2 sequences (Table 4). According to the molecular data, Jensenia pisicolor is more similar to the northern South American species J difformis and J erythropus as well as the African J spinosa than to the Australasian J connivens and Indo-Malayan J de­ cipiens. Jensenia decipiens is most distant from all other Jensenia species. This geomolec­ ular differentiation is only partially displayed in the calculated molecular trees (see Figs. 2, 3). The maximum parsimony analysis of the combined cpDNA and nrDNA data set re­ sulted in 63 most parsimonious trees; the strict consensus of these trees is shown in Fig. 2 (length 408 steps, CI=0.7990, RI=0.8746). The likelihood analysis of the combined cpDNA and nrDNA calculated with the GTR +G model resulted in the phylogram shown in Fig. 3 (In L= - 3208.74312). Both trees are very similar, support Jensenia as a mono­ phylum and demonstrate three Pallavicinia S.str. clades. A well-supported clade is formed by P fyellii, P fongispina and P subciliata, another solely by P xiphoides samples and a third by P rubristipa. Both dendrograms differ only in the relationship within Pallavicinia s.str. All dendrograms, calculated with the three different data sets and model types as well as with the variable positions excluded (data only partially shown), show Jensenia as a well-supported monophylum supported by high bootstrap values (e.g., Figs. 2, 3). Within Jensenia the trees only show slight differences. Except for dendrograms based solely on cpDNA, Jensenia decipiens and J connivens form one clade (bootstrap values 54- 62%; 238 J. Hattori Bot. Lab. No. 96 2 0 0 4

S. hymenophyllum

S. podophylla

P. /yellii 100 - P. /ongispina 99 P. subciliata 1

""'--- P. subciliata 2 100 - 100 P. xiphoides 1

P. xiphoides 2

P. rubristipa

J. pisic%r 1

J. spinosa 55 '"'--- J. difformis

J. erythropus 1

J. erythropus 2 '-- 100 63 J. pisic%r 2

J. pisic%r 3

58 J. connivens 1

J. connivens 2 54

~ J. decipiens 1 78 J. decipiens 2

"-- J. decipiens 3

Fig. 2. Strict consensus of63 most parsimonious trees (length 408 steps, CI=0.7990, RI=0.8746) inferred from combined cpDNA and nrDNA sequences (trnT-L spacer, trnL 5' exon, trnL intron and ITS2) of 21 specimens of Pallaviciniaceae (compare Table I) by a heuristic search with PAUP 4.0bI0. Symphyogyna hymenophyllum and S. podophylla were used as outgroup. Numbers above branches indicate bootstrap values >50% from 1000 repli­ cates with 1000 random addition replicates per bootstrap replicate. F. SCHAUMANN ET AL.: Molecular divergence patterns within Jensenia 239

- S. hyrnenop h yl/urn

r--- S. podophyl/a

100 r P. xiphoides 1

P. xiphoides 2

P./yellii 100 -Po /ongispina

~ P. s ubciliata 1 100 P. subciliata 2

P. rubristipa

J. erythropus 2

'-- J. pisic%r 1

100 J. spinosa

J. difformis

J. erythropus 1

52 J. pisic%r 2 62 J. pisic%r 3

60~ J. connivens 1

J. connivens 2 62 J. decipiens 1

93 J. decipiens 2

.. 0.1 J. declplens 3

Fig. 3. Maximum li kelihood phylogram (lnL=-3208.743 12) based on combined cpDNA and nrDNA sequences (trnT-L spacer, trnL 5' exon, trnL intron and ITS2) of 21 specimens of Pallaviciniaceae (compare Table I) with PAUP 4.0b 10. Symphyogyna hymeno­ phyllum and S. podophylla were used as outgroup. Numbers above branches indicate boot­ strap values from 500 replicates with 10 random addition replicates per bootstrap replicate. 240 1. Hattori Bot. Lab. No. 96 200 4

Table 2. Percentage of substitutions [%] in the trnT-L spacer and trnL intron within the Pallaviciniaceae. (For values on interspecific substitution percentages in the trnL intron within Symphyogyna compare Schaumann et al. 2003)

Jensenia Pallavicinia Symphyogyna Taxa trnT-L spacer trnL intron trnT-L spacer trnL intron trnT-L spacer trnL intron

Jensenia 0- 2.0 0- 1.3 Pallavicinia 10.9- 19.4 8.9- 12 .7 0.8- 20.6 0- 13.2 Symphyogyna 14.9- 16.7 13.5- 15.0 21.0-25.8 12.9- 16.7 2.4 1.2- 11.2

Table 3. Percentage of substitutions [%] in the ITS2 region within the Pallavicini­ aceae. (For values on interspecific substitution percentages within Symphyogyna compare Schaumann et al. 2003)

Taxa Jensenia Pallavicinia Symphyogyna

Jensenia 0-1.3 Pallavicinia 6.9- 20.6 3.6- 18.4 Symphyogyna 15.3- 17.8 14.7- 24.7 6.3- 15.6

Table 4. Percentage of substitutions [%] in the combined trnT-L spacer, trnL intron and ITS2 sequence within the genus Jensenia. (*: value refers only to cpDNA)

J J J J J J Distribution Taxa pisic%r erylhropus dilfiJrmis .Ipinosa connivens decipiens

Southern South America. J pisic% r 0- 0.2* subantarctic islands Northern South America J ervrhropus 0.2 0.2 Northern South America J dilfi)l'll1is 0.2 0.2 Africa J spinosa 0.2 0.2 0.2 New Zealand J connivens 004- 0.7 004- 0.7 004- 0.7 004- 0. 7 004 Indo-Malaysia J decipiens 0.8 0.7 0. 8 0.7 0.7- 1.1 0.2*- 0.5* see Figs. 2, 3). In maximum likelihood phylograms Jensenia pisicolor from Kerguelen Is­ land is not part of the J pisicolor clade, but is displayed as most distant from all other Jensenia species (see Fig. 3; supported, if at all, by a low bootstrap value 52%). Also J ery ­ fhropus from Colombia is occasionally depicted on an own branch (see Fig. 3). These dendrograms as well as the equally parsimonious trees mostly differ in the topology of Pallavicinia s.str. Especially the position of Pallavicinia rubristipa varies. Maximum parsimony and likelihood analyses conducted with the ITS2 sequence and par­ tially the combined cpDNA and nrDNA data set support an affinity of P rubristipa to F. SCHAUMANN ET AL.: Molecular divergence patterns within Jensenia 241

Jensenia (see also Figs. 2, 3). In the ITS2 maximum parsimony phylogram this is support­ ed by a high bootstrap value (data not shown). In some combined cpDNA and nrDNA as well as the nrDNA maximum likelihood dendrograms, Pallavicinia s.str. forms a monophy­ lum (no bootstrap support).

DISCUSSION The species of Jensenia are morphologically, ecologically and molecularly clearly dif­ ferentiated from Pallavicinia s.str. The taxon forms a well-supported monophylum on the molecular level (Figs. 2, 3). All investigated Jensenia species are characterized by a low se­ quence divergence in the investigated cpDNA (trnT-L spacerltrnL intron) and nrDNA (ITS2) regions. The recognition of a separate taxon is therefore justified. The substitution percentages between Jensenia and Pallavicinia are as high as between other genera of the Pallaviciniaceae (s. Table 2, 3) suggesting a delimitation of Jensenia on generic rank. How­ ever, when Jensenia is accepted as a genus, Pallavicinia s.str. is paraphyletic in several phylograms (cf. Figs. 2, 3). This is mainly due to P rubristipa, which shows similarities to Jensenia taxa in the ITS2 region. Further molecular studies with a broader sampling of Pallaviciniacean species even show polyphyly of Pallavicinia (own unpublished data). Morphologically Jensenia is clearly delimited as a genus (e.g., Grolle 1964, Grolle & Piip­ po 1986, Schuette & Crandall-Stotler 2002) and therefore here retained at this rank. Schuster (1992) places the species of Jensenia in Pallavicinia subgenus Mittenia, since some Pallavicinia species are transitional between Jensenia and Pallavicinia s.str. The androecia in Pallavicinia levieri Schiffn. are situated in ill-demarcated rows and do not stand in two rows as typical for Pallavicinia s.str. Additionally, some species intergrade be­ tween dendroid Jensenia and the prostrate Pallavicinia s.str. species. Jensenia specimens always differentiate into creeping rhizomatous axes and an erect flabellate frond (2 or 3-4)X bifurcate terminal branching). Ascending axes, only 1- 2 (very rarely 3)X bifurcate terminal branching, are known from Pallavicinia s.str. (P longispina Steph., P rubristipa, P ambigua [Mitten] Steph.). On the molecular level Pallavicinia longispina and P rubristi­ pa clearly belong to Pallavicinia s.str. The ascending and dendroid taxa evolved indepen­ dently within the Pallaviciniaceae, e.g., in Jensenia, Pallavicinia s.str. (e.g., P longispina) and Symphyogyna spp. (Schaumann et al. 2003). All species of the genus Jensenia are characterized by a very low percentage of substi­ tutions and no indels in the studied cpDNA and nrDNA regions (see Table 2, 3). As in other bryophytes divergence within the trnT-L spacer is higher than in the trnL intron (Meil3ner et al. 1998, Quandt et al. 2001, Pfeiffer et al. 2004). In liverworts trnL intron di­ vergence values between 0 and 2.4% are common on infraspecific level and between 2.4- 6.1 % on interspecific level (Meil3ner et al. 1998, Pfeiffer et al. 2004). Infraspecific variation ranges in the trnT-L spacer from 0- 3.4% and interspecific variation from 5.4- 9.6% (Meil3ner et al. 1998, Pfeiffer et al. 2004).The substitution percentages of these DNA regions are not constant as often supposed but vary enormously in the studied genera of the Pallaviciniaceae (Jensenia, Paliavicinia, Symphyogyna; for data on Symphyogyna cf. Schaumann et al. 2003). Jensenia belongs to the ancient Pallaviciniaceae (Schuster 1992) and probably has a 242 1. Hattori Bot. Lab. No. 96 2 0 0 4

Gondwanan origin. The long geographical isolation led to the development of morphologi­ cal clearly distinct species on former Gondwanan fragments. The observed high sequence homology of Jensenia species in the cpDNA (trnT-L spacerltrnL intron) and nrDNA (ITS2) regions can either be explained by a stenoevolutive behaviour of the genus or by an evolutionarily young age (which would contradict all previous morpho-ecological assess­ ments).

Interspecijic relationships in Jensenia A geographical divergence between the treated Jensenia species is observable, al­ though the infrageneric molecular divergence is very low (Table 4). Taxa evolved in sec­ ondary distribution areas as the northern Andes and Indo-Malaysia are on the molecular level similar to their relatives in southern South America or New Zealand, respectively. Jensenia pisicolor of southern South America and from the subantarctic islands is closely related to J erythropus and J difformis from northern South America. Furthermore, J pisi­ color stands closer to the African J spinosa than to J connivens from New Zealand. The nearest relative of the Indo-Malayan J decipiens is the latter species. In the phylograms both species form a clade (see Figs. 2, 3). The habitual similarity of J connivens and J pisicolor supposed by Scott (1985), lead­ ing to his conclusion of their possible identity, is probably due to his misidentification of J connivens. Fig. I clearly shows that both species differ in their habit. While J pisicolor thalli possess small teeth, barely visible with a hand-lens, the teeth of J connivens are con­ spicuous. Also the molecular data shows that J pisicolor and J connivens are distinct species.

ACKNOWLEDGEMENTS We wish to express our thanks to the Corporaci6n Nacional Forestal in Chile (CONAF, Chile) and the Conservancies of the Department of Conservation in Taupo/Ton­ gariro, Gisborne, Nelson and Hokitika, New Zealand for the research permits and for their logistic support, to Or H. Nowak-Krawietz (B), Prof. Or J.-P. Frahm (BONN), Or M. Manitz, Or H.-J. Ziindorf (both JE), Or A. Wilton and Or D. Glenny (both CHR), L. van Essen (MPN) and PO Or H. Kiirschner for providing herbarium specimens. Furthermore, we wish to thank B. Giesicke for technical assistance, H. Liinser for drawing Fig. 1, Or M. Stech for determining sequences, for help with the tree construction and proof-reading the manuscript. The research was supported by a grant of the Oeutsche Forschungsgemein­ schaft for the BRYO AUSTRAL project (Fr404/3-2).

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