J. Hattori Bot. Lab. No. 74: 105- 119 (Nov. 1993)

CLADISTIC ANALYSIS OF THE HORNWORTS (ANTHOCEROTOPHYTA)

JAAKKO HYV0NEN 1 and SINIKKA PIIPPa2

ABSTRACT. A cladistic analysis of the genera of Anthocerotophyta is presented. The analysis was based on forty characters and included all nine genera currently accepted in the group. Only one most parsimonious tree was found. According to the results, the hornworts are treated as two orders: Anthocerotales Limpricht and Notothylales Hyvonen and Piippo, ord. nov. (merely with Notothylas Sull. ). Anthocerotales comprises two families : Anthocerotaceae Dum. (Anthoceros L. emend. Prosk., Folioceros Bharadw., Leiosporoceros Hassel, Mesoceros Piippo, Phaeoceros Prosk., and Sphaerosporoceros Hassel) , and Dendrocerotaceae (Milde) Hassel (with Dendroceros Nees and Megaceros Campbell) .

INTRODUCTION

The relationship of the hornworts (Anthocerotophyta) to other land plants has been a controversial issue (Crandall-Stotler 1984, Schofield 1985, Schuster 1984, 1992, Mishler & Churchill 1985). It has been claimed that they represent a totally indepen­dent lineage from all other living land plants (Schuster 1984, 1992) or they may be included in a clade together with liverworts, mosses and tracheophytes (Mishler & Churchill 1985) . The latter view assumes that they share a common ancestor with the mosses and all other land plants excluding the liverworts. Mishler and Churchill ( 1985) presented a cladistic analysis supporting this view, and their results were chosen as a starting hypothesis for our analysis. Further support for this view has lately come also from nucleotide-sequence data (Mishler et al. 1992).

The monophyly of the hornworts as a whole has generally been accepted, a view supported by several synapomorphies documented by Mishler and Churchill (1985) and Hassel de Menendez (1988). Traditionally the hornworts have been treated under a single family, Anthocerotaceae. Muller (1951 - 58) treated Notothy/as Sull. in its own family . This same traditional trend has been continued by Schuster (1987, 1992) and Hasegawa (1988), Schuster keeping all the genera in the one and same family, but under different subfamilies (see Table 1 ), Hasegawa treating them under three families. Schuster ( 1992) has a reservation about Anthocerotaceae: "The fami ly includes all extant Anthocerotales except (possibly) for Notothylas". Schuster (1987, 1992) did not treat all the genera. In contrast to other workers (see below, and Table 1 ), he continues to treat Anthoceros L. emend. Poosk as Aspiromitus Steph., and Phaeoceros Prosk. as Anthoceros, and he includes Leiosporoceros Hassel under the latter as well. Dendroceros

1 Jaakko Hyvonen, Department of Botany, P. 0 . Box 47 (Hameentie 153B), FIN-00014 University of Helsinki, Finland.

2 Sinikka Piippo, Botanical Museum, P. 0 . Box 47 (Hameentie 153B), FIN-00014 Uni­versity of Helsinki, Finland.

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J. HYVONEN & S. PIIPPO: Cladistic analysis of the hornworts

A Fig. I. A. The cladogram of the genera of Anthocerotae according to Mishler

and Churchill (1985) and B. according Hassel de Menendez (1988) . Data supported the distinction of only four clades, not seven as in the original figure.

107

Nees, Megaceros Campbell, and Notothylas are in Anthocerotaceae but in different subfamilies. Hasegawa (1988) has five genera: Anthoceros (incl. Folioceros Bharadw.), Phaeoceros (incl. Leiosporoceros) and Megaceros Campbell in Anthocerotaceae, and Dendroceros Nees and Notothylas in their own families. The analysis by Mischler and Churchill (1985) included five genera: Anthoceros (including Folioceros), Dendroceros, Megaceros, Notothylas and Phaeoceros (Fig. la). Higher ranking was not discussed by them. Hassel de Menendez (1988) accepted eight genera with Sphaerosporoceros Hassel and Leiosporoceros. Folioceros was treated as a separate genus from Anthoceros (Fig. l b, Table l ). The classification by Hassel de Menendez radically differs from those of the others, because she accepts four different orders for the hornwort genera (see Table l).

The study of the New Guinean material (Piippo 1993) revealed a formerly undescribed taxon. It did not fit well in any of the known genera and thus required description of one more genus in Anthocerotophyta, Mesoceros Piippo (Piippo 1993). The relationship of this new genus to other genera of the hornworts was only briefly discussed by Piippo, and this inspired interest in analysis of the whole group, based on cladistic principles. We were also interested to find out whether the data collected could possibly offer better resolution of the whole group than formerly presented. Cladistic analysis is based solely on characters of the taxa included and totally avoids any a priori assumptions as to the evolution of the group.

MATERIAL AND METHODS

Nine genera currently accepted in the hornworts are included as the terminal taxa

108 J. Hattori Bot. Lab. No. 74 I 9 9 3

in the analysis. Forty characters (see below) were included in the analysis. Originally three more characters (length of capsules, form of the capsule cells, and the size of spores) were tabulated, but we were unable to code their excessive polymorphism for the use in the final analysis. Of the characters, 39 are morphological and one is ecological. Only three characters included three character states, the others being binary. In addition, six synapomorphies for the hornworts or for the clade of the land plants excluding liverworts (Mishler & Churchill 1984, 1985) were putatively included (characters 41 - 47) when the two out-groups were added to the analysis. For further discussion on these characters, see Mishler and Churchill ( 1985) . The main sources for the data were Mishler and Churchill ( 1985), Hassel de Menendez ( 1986, 1988 ), and Hasegawa (1988) . Study of the herbarium material concentrated on Western Mela­nesian specimens and especially on the genera Dendroceros, Folioceros, Megaceros, Mesoceros and Phaeoceros (see Piippo 1993) .

The original data matrix was compiled by S. Piippo.

Characters included in the analysis 1. Thallus strap-shaped (0), radiate ( l ) . Dendroceros, Folioceros and Megaceros have

distinctly strap-shaped thalli, even though species of Dendroceros are often richly branched; the thalli of the other genera are radiate, except Anthoceros and Phaeoceros, which have both forms.

2. Colar light green (0), dark green ( 1 ) . Most of the hornworts are dark-colored, often almost black, except for the light green Dendroceros, green Anthoceros and variable Notothylas.

3. Costa absent (0), present ( 1 ) . Distinct, but variable in width, a costa is present only in Dendroceros, which also has one-celled laminae outside the costa providing many further characters at specific level. The other genera have a pluristratose thallus throughout except perhaps on the thallus edges.

4. Costa/thallus cavernous (0), solid ( 1 ) . The schizogenous cavities usually serve as a good distinguishing character between the genera. The only genus showing polymorphism is Dendroceros.

5. Tubers absent (0), present ( 1 ). Tubers are known to occur only in Phaeoceros. 6. Rhizoids present (0), absent (1 ). Rhizoids usually occur in hornworts, but may

occasionally be absent. In Dendroceros, rhizoids are few or rare. 7. Epidermal cells not distinctive from medium cells (0), two cell types present (1) .

Especially in Megaceros, Mesoceros and Phaeoceros the epidermal cells are smaller than medium cells.

8. Epidermal cells without trigones (0), with trigones ( 1 ). Only species of Dendro­ceros have trigones in epidermal cells, which serve as good specific characters due to their different sizes, possible perforations, and position on laminae.

9. Pyrenoids in chloroplasts absent (0) , present ( 1 ) . Pyrenoids are present in all thallus cells except in Megaceros and a few species of Folioceros (Bharadwaj 1972) .

10. Apical cell of thallus hemidiscoid (0), cuneate (1). All the other hornwort genera have cuneate apical cells, except the epiphytic Dendroceros.

1. HvvONEN & S. PllPPO: Cladistic analysis of the hornworts 109

11 . Thallus does not continue its growth by the apices after fertilization (0), growth continues (I). Only in Notothylas does the thallus cease apical growth, and the developing capsule remains in an apical notch.

12. lnvolucres 2.5- 5mm (0), 1- 2.5 mm (1), 5- lOmm (2). This is a character serving mainly/occasionally at species level.

13. Involucres with smooth surface (0), surface crenulate or Jamellate ( 1 ). As for the preceding character, it is useful only at species level.

14. Growth of sporophyte terminated (0), continuous ( 1 ). A meristematic zone at the base of the sporophyte allows the continuous development of the capsule, except in Notothylas.

15. Sporophyte short (0), elongated ( 1 ). All the other hornworts except Notothylas have an elongated sporophyte. We follow Mishler & Churchill ( 1985) and interpret the fusiform capsule of Notothylas as plesiomorphic.

16. Stomata in capsules present (0), absent (1) . Stomata are lacking in the capsule walls of only Dendroceros and Megaceros plus Notothylas.

17. Stomata well-developed (0), reduced to pores ( 1 ). According to Crandall-Stotler ( 1981 ), there are unspecialized pores in the capsule walls of Megaceros.

18. Columella very small or essentially absent (0), well-developed ( l) or thick (2) . All the genera have fairly well-developed columella in their capsule except Notothylas. Megaceros has a very thick and therefore apparently derived columella (Mishler & Churchill 1985) .

19. Pseudoelaters with a spiral thickening band (0), without (1 ). All the other genera except Dendroceros and Megaceros do not have a spiral thickening band in pseudoelaters. Elaters with a spiral thickening band are similar in these genera and between species, in contrast to non-spiral elaters.

20. Pseudoelaters thick-walled (0), thin-walled ( l ) . Only the pseudoelaters of Folio­ceros and Leiosporoceros are thick-walled.

21. Pseudoelaters (reddish) brown (0), yellow or light brown (1 ). Pseudoelaters can be distinguished between the genera on the basis of their color. The elaters of Folioceros are usually the darkest. Mesoceros is also distinguishable from Anthoce­ros and Phaeoceros on the basis of its darker elaters.

22. Pseudoelaters short and curved, sometimes with oblique thickenings (0), elongate, branched or unbranched, with or without a spiral thickening ( 1). If left unbroken into individual cells, pseudoelaters are not similar in form and size, except those of Notothylas, which are small, narrow and curved.

23. Pseudoelaters with obtuse (0), acute apices ( l ). The pseudoelaters of Folioceros and Leiosporoceros are similar in having acute apices and a stout and straight appearance.

24. If not spiraled, pseudoelaters mainly formed of 1(- 2) cells (0), 3-4 cells (1) . Pseudoelaters often break into cell components which are either short and ovoid, or long. The elaters of Anthoceros have either very short or longer cell compo­nents, as also with Notothylas; all the other genera except Leiosporoceros have variously elongated cell components. Leiosporoceros probably has only one elon-

110 J. Hattori Bot. Lab. No. 74 I 9 9 3

gated cell in its pseudoelaters. 25. Pseudoelaters breakable (0), unbreakable ( 1 ). The spiral pseudoelaters of Den­

droceros and Megaceros are not specifically breakable in contrast to the nonspiral ones of Phaeoceros and Sphaerosporoceros, and those of some species of Anthoceros and Notothylas.

26. Spores originate in a tetrahedral (0), in bilateral alterno-opposite tetrad ( 1 ). According to Hasegawa (1988), spore tetrads are bilateral in Leiosporoceros, with many kinds of intermediate forms between tetragonal and hexahedral tetrads, never tetrahedral tetrads.

27. Spores greenish (0), dark/black ( 1 ), yellow (2). The spores of Folioceros range from light brown to black. The spores of Leiosporoceros and Phaeoceros are yellow and therefore distinct from the darker spores of related genera. The spores of Dendroceros and Megaceros are greenish due to chloroplasts.

28. Spores trilete (0) monolete (1 ). Because of the form of the spore tetrad, the spores of Leiosporoceros have a monolete mark (see character 29) .

29. Trilete mark present (I), absent or indistinct (0). Trilete marks representing the former borders of the other spores in the spore tetrad on the proximal surface of spores are clearly visible in some genera, but absent or indistinct in other genera, depending as well on the species.

30. Cingulum absent (0), present ( 1 ). The spores of Notothylas and Phaeoceros have an equatorial girdle, i.e. cingulum (Hassel de Menendez 1988).

31. Spores ovoid or triangular (0), rounded (1 ) . Spores are usually rounded, except for Leiosporoceros with its ovoid spores. In Dendroceros, spores are various in shape (see character 35) and in one species (D. pedunculatus Steph.) triangular.

32. Spores smooth (0), papillate (1). Only the spores of Leiosporoceros are smooth; in all the other genera spores have different kinds of papillae. Dendroceros species have granular papillae similar among different species. Folioceros has papillae varying the most between different species, ranging from low and blunt to thin and sharp (see Piippo 1993).

33. Proximal spore surface with concavities (0), no concavities (I). Concavities are present in Anthoceros and Mesoceros. In other genera the possible papillae are lower on the proximal surface.

34. Synchronous (0), non-synchronous spore production ( 1 ). Non-synchronose spore production is a general mode with mature spores being detected at distal ends of splitting capsules, and immature spore tetrads at capsule bases. Only Notothylas has synchronous spore maturation.

35. Germination exosporic (0), endosporic ( 1 ). In Dendroceros, spores germinate within the spore coat and therefore are multicellular at maturity.

36. Antheridia in antheridial chamber many (0), one ( 1 ). Only in Dendroceros and Megaceros does merely one antheridium develop in each antheridial chamber. The only exception in another genus is Phaeoceros pearsonii (M. A. Howe) Prosk. (Hassel de Menendez 1988) .

37. Cells of antheridial body irregularly arranged (0), in four rows (1 ). When the

J . HYVONEN & S. PUPPO: Cladistic analysis of the hornworts Ill

antherial body has irregularly arranged cells, the cells are usually small and numerous: if the cells are arranged in four rows, they are fewer and larger, especially on the upper part of the antheridial body.

38. Gemmae absent (0), present (1). Gemmae are rounded or ovoid, composed of a various number of cells, and occurring only occasionally in a few genera.

39. Capsule indehiscent (0), dehiscence usually by two lines (I). 40. Epigeic (0), epiphytic or epiphyllic ( 1 ). Most of the hornworts grow on soil, or on

logs in the tropics. Only Dendroceros is adjusted to grow on tree trunks and leaves. 41. Nostoc symbionts absent (0) , present ( 1 ) . 42. Pseudoelaters absent (0), present ( 1 ). 43. Antheridia exogenous (0), endogenous ( l ). 44. Spermatozoids asymmetric (0), bilaterally symmetric ( 1 ). 45. Archegonium stalk present (0), absent ( 1). 46. Zygote division transversal (0), vertical ( 1 ) . 47. Ability to distinguish D-methionine absent (0), present ( 1 ) .

PARSIMONY A ALYSIS

The parsimony analysis of the data set (see Table 2) was done with the computer program PAUP 3.0s (Swofford 1991) . Further analysis of the character distribution was performed using the program MacClade 3.0 (Maddison & Maddison 1992). All the analyses were done on an Apple Macintosh LC 4/40 microcomputer.

The parsimony analysis was performed by J . Hyvonen.

RESULTS

The primary analysis was performed using an exhaustive search for the unrooted tree, and all the characters were coded as unordered. Only one most parsimonious tree was found with a length of 59 steps (Fig. 2a) . In order to polarize the characters in the ingroup, in the secondary analysis mosses and hepatics were used as outgroups. We were, however, able to assign only some of the characters for the outgroups. Many of them remained equivocal or were totally unapplicable.

The search was done under the initial constraint according to the results of Mishler and Churchill ( 1985), i.e. assuming that the hornworts share a common ancestor with the mosses and all other land plants excluding the liverworts. The new analysis revealed again only one most parsimonious tree, this one with a length of 85 steps (Figs. 2b, 3) and consistency index of 0. 788. Even when all autapomorphies of single genera and inveariant characters (5, 10, 17, 22, 26, 28, 32, 35, 39 and 40-47) are excluded the value for this corrected consistency index, 0.769, is higher than the expected value for 9 taxa (0.719) based on analysis of the 60 published data sets (Sanderson & Donoghue 1989) . The hornwort clade was rooted between Notothylas and other genera of the group, which is in accordance with the results achieved by Mishler and Churchill (1985) . As can be seen, the tree resembles to a great extent their tree, although it appears to be better resolved. This is, however, only due to the clade uniting the genera Anthoceros, Sphaerosporoceros, Folioceros, Leiosporoceros, Mesoceros and Phaeoceros. The only char-

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Fig. 2. A- B. The most parsimonious cladograms achieved in the present analysis (see text for explanations) .

11 3

acter supporting the clade is the presence of stomata (16) . This is, however, removed if we follow Mishler and Churchill (1985) and accept it as a synapomorphy for the clade uniting hornworts with mosses and tracheophytes. It is more parsimonious to assume homoplasy in this character. There is, however, only one step difference in the length of the resulting trees. In both cases, twice we have to accept independent origin or loss of stomata. Detailed anatomical studies of stomata and their ontogeny would probably help to reveal whether the stomata in these groups are really identical.

The monophyly of other genera of Anthocerotophyta excluding Notothylas was supported by several characters already in Mishler and Churchill (1985), and study of the character distribution revealed this here as well . This resolution was supported by the distribution of characters 11, 14, 15, 18 and 34. If we assume that the common ancestor of the clade including the hornworts and mosses + tracheophytes did have stomata, then their absence becomes a synapomorphy for a clade including Notothylas

114 J. Hattori Bot. Lab. o. 74 I 9 9 3

with Dendroceros and Megaceros. However, the resulting tree proved to be four steps longer than the one accepted, because we then have to assume independent evolution of the four synapomorphies supporting the alternative resolution. Three of the characters given above [continuous growth of sporophyte ( 14 ), its exceptional vertical growth (15), non-synchronous spore production (34), and dehiscent capsule (47)] are clear apomorphies of all hornworts excluding Notothylas - as presented already by Mishler and Churchill (1985). They also note that the latter character is probably a complex of several independent apomorphic characters. This will naturally further corroborate the accepted clad e. Characters 11 (growth of thallus after fertilization) and 18 ( columella) are more problematic (Mishler & Churchill 1984) . The most parsimonious solution is to assume that the common ancestor of the hornworts, mosses and tracheophytes had columella. Accordingly, the very small columella becomes an autapomorphy for the genus Notothy/as. However, if we assume that common ancestor of all the hornworts had a sporophyte with this kind of minute columella, it is more parsimonious to treat the better developed columella as further synapomorphy for the clade excluding Notothylas and as having a homoplasious similarity with the mosses as discussed already by Mishler and Churchill (1985). As already discussed above under the stomata, further research is needed here. It is also impossible to decide which one of the character states for character 11 (growth of thallus after fertilization) is plesiomorphic at this level. In the character number 7 (differentation of median cells) we have to assume two later reversals and even new differentation after one reversal.

According to Hassel de Menendez ( 1988), Notothylas shares a common ancestor with Phaeoceros. However, this clade was supported by only one character (30, presence of cingulum), and such a solution would result in a tree five steps longer than the most parsimonious one. Accordingly, it is assumed that this character evolved twice in the hornworts.

The clade uniting Dendroceros and Megaceros has been presented already by Hassel de Menendez (1988) and Mishler and Churchill (1985). It agrees with the distribution of four characters (1, 19, 27, 36), and three of them were presented by Hassel de Menendez (1988) and one (36, antheridia only one per chamber) also by Mishler and Churchill ( 1985). If character 1 (thallus strap-shaped) is accepted as a synapomorphy for this clade, the similarity observed in Folioceros has to be accepted as homoplasious. However, the division of the character might upon further study prove to be too crude, or even misleading. The presence of a spiral thickening band in pseudoelaters and disintegration of the original wall (character 19) is clearly a synapomorphy uniting Dendroceros and Megaceros. The typical green color of the spores (character 27) is here interpreted as an apomorphic state compared to the dark or yellow spores of the other genera of the group.

Mesoceros and Phaeoceros are united by one synapomorphy (7, epidermal cells distinctive from median cells). The other clade with Anthoceros, Folioceros , Leiosporo­ceros and Sphaerosporoceros is supported by three ( 4, 12, 37), and the apical clade uniting Folioceros and Leiosporoceros by four characters (13, 20, 21 and 23). Distinct epidermal cells are present also in Sphaerosporoceros and Megaceros ; thus we have to

J. HvvONEN & S. PIIPPO: Cladistic analysis of the hornworts 115

assume that this feature evolved independently three times. The sister clade including three genera is much better supported. Cavities in costa or thallus are present only in some species of Dendroceros and in some hepatics in addition to Anthoceros, Folioceros, Leiosporoceros and Sphaerosporoceros. Whether all these cavities can be treated as homologous, is, however, not certain. The regularity of the outer wall of the antheridia is found only in Anthoceros, Sphaerosporoceros and Folioceros plus in some groups of hepatics. There was no data available for Leiosporoceros. The height of the involucre is certainly more difficult to interpret. The character shows much variation even within many genera.

Folioceros and Leiosporoceros are distinguished from other genera by their unique pseudoelaters which are reddish brown (21) and have thick walls (20) and acute apices (23) . A crenulate or lamellate surface of the involucre is found also in some species of many other genera, for example in Anthoceros. However, it can still be accepted as valid synapomorphy for Folioceros and Leiosporoceros, because involucres were most proba­bly smooth in the common ancestor which they share with their sister taxon, Sphaero­sporoceros.

On the generic level most of the autapomorphies show parallelism. This might be indicative of the group's "chaotic state of taxonomy" (Schuster 1992), i.e. the genera as currently limited are not natura), inonophyletic groups. However, analysis at species level was beyond the scope of this study, and accordingly the currently accepted nine genera were used as terminal taxa.

Although Notothylas is clearly distinct from other genera of the hornworts, it is fairly difficult to say which of the characters are true apomorphies and which of them show only a plesiomorphic condition. The presence of cingulum on spores (30) is, however, clearly a unique character which is found only in Notothylas and Phaeoceros. As discussed above, accepting this character as a synapomorphy uniting these to genera forces one to accept a phylogeny much less parsimonious than the one presented. Of the other two characters which separate Notothylas from the other hornworts (thallus growth after fertilization, 11 and form of pseudoelaters, 22) it is impossible to say which one of the character states is apomorphic. Mishler & Churchill ( 1985) interpret the long pseudoelaters of the other hornwort genera as an apomorphic state as compared with the short pseudoelaters found in Notothy/as.

The clade including Dendroceros and Megaceros was already well supported by several unique synapomorphies, and this applies to the genera included as well . Genus Dendroceros is characterized by six apomorphies (characters 2, 29, 31, 3, 8, 12), of which the latter three are unique to the genus. Megaceros has three apomorphies (7, 9, 18), with the latter two being unique to the genus. Accordingly, these two genera seem to be fairly well characterized as already presented by Hassel de Menendez ( 1988) and definitely not poorly defined "waste-baskets of left-overs" as Whittemore ( 1987) says. However, as already stated, the current analysis is limited to the generic level and thus inevitably includes generalizations which by further study might prove ill-founded.

The autapomorphies of all other genera include only characters which show parallelism or reversals. The genus Leiosporoceros seem to be fairly well defined, with

116 J. Hattori Bot. Lab. No. 74 1 9 9 3

30

Fig 3. Same as in Fig. 2, only outgroups removed. Solid bars: unique apomorphies; dark-shaded bars: apomorphies with reversals; light bars: apomorphies with parallelism. Number to the right of each clade indicates decay index, i.e. the number of steps that parsimony must be relaxed to remove support for the particular clade (see text for further explanation) .

five autapomorphies of which the short pseudoelaters with only one to two cells are unique to the genus. However, although the genera Notothylas and Anthoceros show polymorphism in this character, all other characters make the clade consisting of these three genera unacceptable. In the beginning of the analysis the position of the new genus, Mesoceros, was of special interest. It is distinguished from Phaeoceros by two apomorphies (21, color of the pseudoelaters and 33, proximal surface of the spores) . Both of these characters show parallelism with Folioceros and Leiosporoceros (character 21) and with Anthoceros (character 33). However, uniting Mesoceros with these groups would produce a tree 2 or 3- 6 steps longer than the one presented.

By calculating the consensus tree including the shorter tree plus all those trees that are one to four steps longer, it is possible to see how much parsimony must be relaxed

J. HvvONEN & S. PIIPPO: Cladistic analysis of the hornworts 117

to remove the support for a particular clade (Donoghue et al. 1992, Mishler et al. 1992). This, so called decay index, is included in Fig. 3. As can be seen, the clade including all genera of the hornworts except Notothylas is best supported by the current data. The clade uniting Dendroceros and Megaceros seem also to be well supported and the next strongest is the one uniting Folioceros and Leiosporoceros. All the other clades are, however, not particularly well corroborated, because in the consensus tree includ­ing all trees one step longer than the one accepted, all these clades are collapsed.

DISCUSSION

On the basis of the cladistic analysis presented above, Notothylas is clearly separated from the other hornwort genera. A separate status has been granted to it by Hasegawa (1988), Schuster (1992) and Piippo (1993) . Hassel de Menendez (1988) placed it in Anthocerotales in family Notothyladaceae together with Phaeoceros, even though she recognized Folioceros, Leiosporoceros, and Dendroceros + Megaceros in their own orders (Fig. 1 b and Table 1) . Although Notothylas has many unique characters as compared to the other hornwort genera, it has not been clearly distinguished from them. Such characters as the terminating growth of the sporophyte, its shape, synchro­nous spore production, and its lack of columella separate it from the other genera. Moreover, the cladistic analysis presented here as well as that by Mishler and Churchill ( 1985) provide strong support for the monophyly of the other genera of the hornworts excluding Notothylas.

Accordingly, we propose a new order: Notothylales Hyvonen & Piippo. Sporophy­ta sine columellam non continue crescentia, sporae totae simul adultae.

The cladistic analysis supports well the previous classification by Hasegawa (1988), Schuster (1992) and - in part - Piippo's (1993) by considering the other genera except Notothylas as fairly closely related. Dendroceros and Megaceros form an entity which must be recognized at the family level on the basis of stomata being absent from capsule walls, of pseudoelaters with a spiral thickening band, and of the antheri­dial chamber with merely one antheridium. The sister clade of this family is supported by only one character, for which application at this level is questionable (see discussion above). Accordingly, the formal recognition of this entity has to be accepted as merely a pragmatic solution, which might well be changed by further study of the group.

Mesoceros and Phaeoceros are closely related, which has already been pointed out by Piippo (1993) . She did not accept the treatment of Hassel de Menendez (1988) which placed Anthoceros and Phaeoceros in different families , because of the intermedi­ate nature of Mesoceros. The present study, however, shows that the separation of Mesoceros and Phaeoceros from the remaining genera is based merely on one character (7, epidermal cells of thallus differentiated from median cells) . Accordingly we hesitate to recognize the groups formally .

The better resolution of the group as compared with earlier analysis of Mishler and Churchill (1985) and Hassel de Menedez (1988) is not very robust as based on the present data. However, some of the earlier results, for example the clade formed by Dendroceros and Megaceros got further corroboration. The monophyly of all other

118 J . Hattori Bot. Lab. No. 74 1 9 9 3

hornwort genera except Notothylas was also confirmed. As compared with Piippo ( 1993) the position of Mesoceros is also better understood after the cladistic analysis.

ACKNOWLEDGEMENTS

We acknowledge Dr. Carol Norris for revising the English and Mr. Krister Karttunen, Phil. Lie., for the Latin translation.

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