J Hattori 801. lab. No. 76: 87-95 (Oct. 1994)

PHYLOGENETIC RELATIONSHIPS WITHIN SECTION ROSULATA

1 1 2 ROBERT WYATT , ANN STONEBURNER AND lRENEUSZ J. 0DRZYKOSKI

ABSTRACT. We scored a total of 16 morphological characters for all eight species of the ingroup, Plagiomnium section Rosulata, and for the outgroup, Plagiomnium section Undulata . A formal cladistic analysis was performed on the resulting data matrix using the computer programs MacClade and PAUP. The single most parsimonious tree was 20 steps long and, excluding uninformative charac­ ters, had a consistency index of 0.765 and a homoplasy index of 0.235. The topology of the tree agreed well with Koponen 's ( 1971 ) "assumed phylogeny" of the group, especially with respect to the two major subgroups: (I) a "sharp-toothed" clade consisting of P. ellipticum, P. insigne, P. medium, and P. curvatulum; and (2) a "blunt-toothed" clade consisting of P. affine, P. tezukae, P. ciliare, and P. e/atum. Omitting the two allopolyploid species (P. medium and P. curvatulum) from the analysis had no influence on the overall topology of the tree. Comparing the tree to patterns of relationships based on genetic distance, it was apparent that the species of the "blunt-toothed" subgroup were more strongly divergent inter se. It is speculated that these species may have originated longer ago than those of the "sharp-toothed" subgroup, which seems to be of very recent origin, especially P. medium. In addition, the "blunt-toothed' species may have been more strongly affected by Pleistocene glacia­ tion.

INTRODUCTION Koponen 's (1971) monograph of Plagiomnium section Rosu la ta recognized seven species and one subspecies in a group whose , up to that time, had been very con­ fused. In North America, for example, Andrews's (1940) treatment of the group (within the very broadly conceived genus Minium) recognized only "M. affine" and "M. insigne." Ap­ parently, his concept of "M. affine" included at least three taxa now generally accorded species status and one subspecies (but not including P. affine, which is endemic to Europe)! Many North American bryologists continued to reject Koponen's (1971) taxonomic revi­ sion of section Rosulata (e.g., Crum and Anderson 1981) until recently, when his treatment was finally embraced (Anderson et al. 1990). With respect to interrelationships within the group, Koponen ( 1971) was very clear about his views, although he never published a formal cladistic analysis of Plagiomnium section Rosulata. He recognized two distinct subgroups: (1) P. affine (Funck) T. Kop. , P. tezukae (Sak.) T. Kop., P ciliare (C. Miill.) T. Kop., and P. elatum (B.S.G.) T. Kop. , and (2) P. insigne (Mitt.) T. Kop., P. ellipticum (Brid.) T. Kop. and P. medium (B.S.G.) T. Kop. (in­ cluding subsp. curvatulum (Lindb.) T. Kop.). Within the first subgroup, he considered P. affine and P. tezukae sister species, with P. ciliare more closely allied to these two than was

1 Institute of Ecology, University of Georgia, Athens, GA 30602, U.S.A. 2 Permanent address: Department of Genetics, Institute of Biology, Adam Mickiewicz University, 165-Dabrowskiego, Pozmin 60-594, Poland. 88 J. Hattori Bot. Lab. No. 76 l 9 9 4

Undulata

insigne

medium

ellipticum

curvatulum

elatum

cilia re

affine

tezukae Fig. I. Koponen 's (1971) "assumed phylogeny" of Plagiomnium section Rosulata. This tree was drawn based on verbal statements about relationships within the group.

P. elatum. In the second subgroup, he regarded P. insigne as a close relative of P medium (and possibly as the haploid progenitor of the presumed autopolyploid), as distinct from P. ellipticum. We have attempted to draw a tree that reflects Koponen's (1971) informal "as­ sumed phylogeny" of the group (Fig. 1). Since 1984, we have been studying genetic variation in Plagiomnium section Rosulata (Wyatt 1985, 1992; Wyatt et al. 1987, 1988, 1989, 199la, 199lb, 1992, 1993a, 1993b; Odrzykoski et al. 1993). Our current view of the taxonomy of this group, informed by evi­ dence from isozyme data, agrees very closely with Koponen's (1971) revision. Our work has shown unequivocally that P. medium is an allopolyploid, whose haploid progenitors were indistinguishable from extant P ellipticum and P insigne (Wyatt et al. 1988, 1992). Moreover, P. curvatulum (Lindb.) Wyatt & Stoneburner is an allopolyploid derived from hybridization between taxa close to P el/ipticum and P e/atum (Wyatt et al. l 993a). We therefore differ from Koponen ( 1971) in recognizing P. curvatulum as a distinct species, rather than as a subspecies of P. medium. Otherwise, our genetic evidence strongly supports recognition at the species level of all of the taxa delimited by Koponen ( 1971 ). Moreover, our analyses have not uncovered any additional "morphologically cryptic" genetic units R. WYATI et al.: Phylogenetic relationships within Plagiomnium sect. Rosu/a/a 89

(i .e., sibling species), as have been discovered in some liverworts (e.g., Dewey 1989; Odrzykoski and Szweykowski 1992). The purpose of this paper is to produce a formal cladistic analysis of P/agiomnium section Rosulata. The resulting phylogenetic tree will then be compared and contrasted with Koponen 's ( 1971) "assumed phylogeny" of the group and to the patterns of related­ ness revealed by genetic evidence from isozyme analyses. In addition, we will examine var­ ious morphological characters in detail, with special interest focusing on the two taxa now known to be of hybrid orgin: P. medium and P. curvatulum.

MATERIAL AND METHODS Based on the taxonomic descriptions and discussions in the literature, especially Ko­ ponen 's ( 197 l) monograph, we selected a total of 16 morphological characters (Table l ). These were chosen on the basis of their usefulness in discriminating between taxa. The list includes virtually all of the key characters used in previous taxonomic treatments. Charac­ ter states were defined after careful examination of the literature and of herbarium speci­ mens. Character states were scored for all eight species of the ingroup, Plagiomnium sec­ tion Rosulata, and for the outgroup, Plagiomnium section Undulata (see data matrix in Table 2). Because the sporophyte of P. tezukae has only recently been discovered (a single eroded capsule: H. Akiyama, personal communication), we scored its number of setae as ''unknown.'' The data matrix was entered into the computer program MacClade (Maddison and Maddison 1992). The actual search for phylogenetic trees, however, was done using the computer program PAUP (Swofford 1993). All characters were treated as unordered, and an exhaustive search was conducted. The most parsimonious trees found by PAUP were then returned to MacClade for more detailed interactive analysis of character-state changes and

Table 1. List of Characters and character states used in the cladistic analysis.

1. Stem branching in upper part: absent (0), present (I). 2. Sterile shoots rooting: absent (0), present (1). 3. Leaves: not undulate (0), undulate (1). 4. Leaf apex: mucronate (0), acute ( 1) . 5. Length of decurrencies: long (0), short to absent ( 1) . 6. Width of decurrencies: wide (0), narrow ( 1) . 7. Prominence of teeth: well-developed (0), weakly developed (I). 8. Cell numberofteeth: 1- 2 (0), > 2 (I). 9. Length of teeth: < 100 µm (0), > 100 µ.m (1). I 0. Shape of teeth: sharp (0), blunt (I). 11 . Strength of costa: percurrent or ex current (0), ending below apex (I). 12. Ventral epidermis of costa: not thickened (0), thickened (1). 13. Lamina! cells: not in rows (0), in rows (1). 14. Lamina! cell shape: not elongated (0), elongated (I). 15. Lamina! cell size: < 25 µm (0), > 25 µm (1). 16. Numberofsetae: > I (0), I(!). 90 J. Hattori Bot. Lab. No. 76 l 9 9 4

Table 2. Data matrix for 16 characters scored for the ingroup (8 species of Plagiom- nium section Rosulata) and outgroup (Plagiomnium section Undulata).

Character Tax on 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

affine 0 0 0 0 I 0 0 I l 0 0 ciliare 0 I 0 0 0 0 0 I 0 0 0 I I 0 I curvatulum 0 0 0 I I I I 0 I 0 0 I 0 0 0 0 elatum 0 1 0 0 0 0 0 l I I 0 0 I 0 I ellipticum 0 0 0 0 I 0 0 I 0 0 0 insigne 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 medium 0 0 0 I 0 0 0 0 0 0 0 0 0 0 0 tezukae 0 0 0 0 I 0 I 0 I 0 I 0 ? Undulata 0 0 0 0 0 0 0 0 0 0 0 0 0 effects of including and excluding taxa. For example, particular interest attached to com­ paring and contrasting trees with and without the two known hybrids included. For comparative purposes, we produced a phenogram based on cluster analysis of Nei's (1972) genetic distances between the eight species of Plagiomnium section Rosulata. The isozyme data for the analysis have been published previously (Wyatt et al. 1989, 1992, l 993a). We pooled data across all populations of each species and used the unweighted pair-groups method using arithmetic averages (UPGMA) to produce the phenogram (BIOSYS-1: Swofford and Selander I 981 ).

RESULTS AND DISCUSSION When all eight species of Plagiomnium section Rosulata and the outgroup (Plagiom­ nium section Undulata) are included, there is a single most parsimonious tree (Fig. 2). The tree is 20 steps long and has a consistency index of 0.800 and a homoplasy index of 0.200. Excluding uninformative characters, the consistency index is 0.765 and the homoplasy index is 0.235. The retention index is 0.867. Three characters distinguish section Undulata from the ingroup: upper stem branch­ ing (1), undulate leaves (3), and small cell size (15). The most strongly supported feature of the ingroup topology is the initial major split into two groups, which correspond to Kopo­ nen's (1971) "blunt-toothed" and "sharp-toothed" subgroups (Fig. 1). A synapomorphy for the latter group is the acute leaf apex (4), whereas the former clade is supported by four synapomorphies: rooting sterile shoots (2), 2 or more cells in the teeth (8), blunt teeth ( 10), and elongated lamina! cells (14). Within the "sharp-toothed" clade, lamina) cells in rows (13) is a synapomorphy unit­ ing P. ellipticum, P. curvatulum, and P. medium. Note, however, that this character state arose independently in P. affine. It is also important to note that this character could have evolved only once in this lineage (in the haploid P. ellipticum) and then been passed on to its hybrid derivatives (the allopolyploids P. medium and P curvatulum). This could also ex- R. WYATT et al. : Phylogenetic relationships within Plagiomnium sect. Rosulata 91

insigne

medium

curvatulum

ellipticum

elatum

cilia re

affine

tezukae Fig. 2. The one most parsimonious tree constructed from the data matrix in Table 2, showing phylogenetic relationships of eight species of Plagiomnium section Rosulata. Pla­ giomnium section Undulata was used as the outgroup. This tree is 20 steps long and has a consistency index (excluding uninformative characters) of0.765 . plain the four synapomorphies that unite P. el/ipticum and P. curvatulum: short to absent decurrencies (5), weakly developed teeth (7), narrow decurrencies (6), and short teeth (9). Narrow decurrencies apparently arose independently in both P. affine and P. tezukae, where­ as short teeth evolved in parallel in P. e/atum. Within the "blunt-toothed" clade, P. e/atum and P. ciliare are held together by a synapomorphy involving their usual production of but a single sporophyte per gametophore (16). The other clade, consisting of P. affine and P. tezukae is united by a weak costa (11) and narrow decurrencies (9). Omitting the two allopolyploid species from the analysis has little influence on the overall topology of the tree. The two most parsimonious trees differed only in the place­ ment of P. insigne: either as the sister species of P. ellipticum or as the sister species to a clade consisting of P. el/ipticum plus the four "blunt-toothed" species. Both trees were 20 steps long and had consistency indices of0.800 and homoplasy indices of0.200. Excluding uninformative characters, the consistency indices were 0.733 and the homoplasy indices, 0.267. The retention indices were 0.750. The characters that influenced one tree versus an- 92 J. Hattori Bot. Lab. No. 76 I 9 9 4 other were whether to treat the acute leaf apex as a synapomorphy of P ellipticum and P. in­ signe and cells in rows as a parallelism (tree I) or to treat cells in rows as a synapomorphy of P. ellipticum plus the four "blunt-toothed" species and the acute leaf apex as a paral­ lelism (tree 2). Thus, we find that inclusion of the known hybrids P. medium and P. curvatulum has lit­ tle effect on phylogenetic reconstructions of Plagiomnium section Rosu/ata. This agrees with McDade's ( 1990, 1992) recent studies of the impact of hybrids on cladistic analyses, which concluded that "most analyses with one or more hybrids results in minor or no change in topology." As expected, P medium, which represents a cross between sister species in the "sharp-toothed" clade, had no effect on the topology of the tree. It is a bit more surprising that P curvatulum, which involves hybridization between the two major clades, caused no restructuring. It is interesting to note that, although both allopolyploids are intermediate to their parents in some respects, P. curvatulum tends to be morphological­ ly closer to P e/lipticum than it is to P elatum (Wyatt et al. 1993a) and P. medium is mor­ phologically more similar to P. insigne than it is to P. ellipticum (Koponen 1971 ). Comparing our phylogenetic tree (Fig. 2) directly to Koponen's "assumed phylogeny" (Fig. I), we find many points of agreement beyond the clear split into two major subgroups. Both trees consider P. affine and P. tezukae sister species and place P. insigne and P ellip­ ticum close to each other and to the two allopolyploids. Without the outgroup, our tree is 17 steps long and has a consistency index of 0.76 and a retention index of 0.86. Using MacClade, we rearranged the taxa on this tree to fit Koponen's suggested topology. The re­ sulting tree was 22 steps long, and the consistency index dropped to 0.59 and the retention index, to 0.69. There are a number of interesting contrasts between the phylogenetic tree for the eight species of P/agiomnium section Rosulata and the pattern of relationships based on genetic distance calculated from isozyme data (Fig. 3). Within the "sharp-toothed" group, there is surprisingly little genetic differentiation, with P ellipticum clustering closer to P curvatu­ lum and P insigne clustering closer to P medium. Thus, each allopolyploid is most geneti­ cally similar to the parent that it also is most similar to morphologically. Unlike the clado­ gram, the phenogram does not group P affine with P tezukae and P elatum with P ciliare,

Ell Cu

M Ela Fig. 3. UPGMA phenogram expressing ~------T overall levels of genetic similarity among the ------A eight species of Plagiomnium section Rosula- ~------c1 ta based on Nei's (1972) genetic distance using 17 putative gene loci. The cophenetic correlation coefficient is 0.953. 1.0 0 .5 0.0 Genetic Distance R . WYATT et al.: Phylogenetic relationships within P/agiomnium sect. Rosulata 93 which is the most genetically divergent species in section Rosu/ata. Genetically, the four "blunt-toothed" species are very heterogeneous and much more strongly differentiated amongst themselves than are the "sharp-toothed" species. This may indicate that these species are of more ancient origin and have accumulated more genetic mutations than have P. ellipticum and P. insigne. Why this should be true for these haploid species is unclear, but it was expected that the allopolyploids would be little diverged from their progenitors, especially P. medium. Wyatt et al. (1992) have pointed out that this species has only one al­ lele unique to it as a species (G3p-2b), which occurs in only four heterozygous in one population from northern Michigan. Plagiomnium medium appears to be of very recent origin, whereas P. curvatulum may have arisen as a species longer ago (Wyatt et al. I 993a). Koponen (1971) considered the species of Plagiomnium section Undulata to be the closest relatives of section Rosulata, a view with which we concur: this taxon was used as our outgroup in all cladistic analyses. In Koponen's (1971) view P. affine, P. tezukae, and P. ciliare represent derivatives of the Acrto-Tertiary geoflora (Hulten 1937; Li 1952), temper­ ate forest that once extended continuously across the northern continents. Climate change in the Pleistocene severed this range, so that separate species evolved in eastern North America, eastern Asia, and western Europe. Koponen ( 1971) considered P. e/atum, another European endemic, to have become separated earlier from the former three taxa due to its ecological specialization on fens and other wet habitats. If, in fact, these taxa speciated long ago, the observed high degree of genetic differen­ tiation (Fig. 3) should not be surprising. Moreover, Wyatt et al. (1993a) have speculated that reduced population sizes of P affine and P. e/atum in Europe due to glaciation and a lack of refugia to the south may explain the comparatively low levels of genetic variation in those species. Koponen (1971) did not discuss the phytogeography of speciation in the "sharp-toothed" group of section Rosu/ata. We might speculate that P ellipticum and P. in­ signe are more recent derivatives that were forced south by glaciation, during which time they hybridized and produced the allopolyploid P. medium. The origin of P curvatulum is less clear, but Wyatt et al. (1993a) have speculated that it arose longer ago, perhaps as a cross between the common ancestor of both P. affine and P. elatum and a species virtually identical to extant P. ellipticum. In any event, it is remarkable how much the range of P. el­ lipticum, P. medium, and P curvatulum have expanded in the relatively short time since deglaciation, as most of their present-day ranges were under ice until about 8- 10,000 years B.P.

ACKNOWLEDGEMENTS Much of the research reported here was supported by grants from the United States National Science Foundation (BSR-8409893 l, BSR-880636, and DEB-9220676). We thank G. Derda and D. Grise for laboratory assistance; G. Derda for help with MacClade and PAUP; and a large number of bryologists who have assisted our collecting efforts and who are listed individually in relevant papers. We also thank the organizers of the sympo­ sium on Experimental Biology of Bryophytes: Phylogenetic Aspects, M. Bopp, A. J. Shaw, and K. Nehira, for the opportunity to speak and write about the subject of this paper. 94 J. Hattori Bot. Lab. No. 76 I 9 9 4

LITERATURE CITED Anderson, L. E., H. A. Crum & W. R. Buck. 1990. List of the of North America North of Mexico. Bryologist 93: 448-499. Andrews, A. L. 1940. . In A. J. Grout (ed.), Flora of North America North of Mexico: 242-260. Newfane, Vermont. Crum, H. A. & L. E. Anderson. 1981. Mosses of Eastern North America. Columbia University Press, New York. Dewey, R. M. 1989. Genetic variation in the liverwort Riccia dictyospora (Ricciaceae, Hepaticopsi­ da). Syst. Bot. 14: 155- 167. Hulten, E. 1937. Outline of the History of Arctic and Boreal Biota During the Quaternary Period. Stockholm. Koponen, T. 1971. A monograph of Plagiomnium sect. Rosulata (Mniaceae). Ann. Bot. Fenn. 8: 305- 367. Li, H-L. 1952. Floristic relationships between eastern Asia and eastern orth America. Trans. Amer. Philosoph. Soc. 42: 371-429. McDade, L. A. 1990. Hybrids and phylogenetic systematics. I. Patterns of character expression in hy­ brids and their implications for cladistic analysis. Evolution 44: 1685- 1700. McDade, L. A. 1992. Hybrids and phylogenetic systematics. II. The impact of the hybrids on cladis­ tic analysis. Evolution 46: 1329- 1346. Maddison, W. P. & D. R. Maddison. 1992. MacClade: Analysis of Phylogeny and Character Evolu­ tion. Version 3.0. Sinauer Associates, Sunderland, Massachusetts. Nei, M. 1972. Genetic distance between populations. Amer. Naturalist 106: 283-292. Odrzykoski, I. J., A. Stoneburner & R. Wyatt. 1993. Genetic variation in the East Asian endemic moss Plagiomnium tezukae. J. Hattori Bot. Lab. 73: 139- 146. Odrzykoski, I. J. & J. Szweykowski. 1991. Genetic differentiation without concordant morphological divergence in the thallose liverwort Concepha/um conicum. Pl. Syst. Evol. 178: 135- 151. Swofford, D. L. 1993. PAUP: Phylogenetic Analysis Using Parsimony. Version 3.1. Computer pro­ gram distributed by the Illinois Natural History Survey, Champaign, Illinois. Swofford, D. L. & R. B. Selander. 1981. BIOSYS-1: A Fortran program for the comprehensive analy­ sis of electrophoretic data in population genetics and systematics. J. Hered. 72: 281 - 283 Wyatt, R. 1985. Chemosystematics of the Mniaceae. I. Identities of Lowry's species pairs. Mongr. Syst. Bot. Missouri Bot. Gard. 11 : 187- 194. Wyatt, R. 1992. Conservation of rare and endangered bryophytes: Input from population genetics. Biol. Conserv. 59: 99- 107. Wyatt, R., D. M. Lane & A. Stoneburner. 199la. Chemosystematics of the Mniaceae. H. Flavonoids of Plagiomnium section Rosulata. Bryologist 94: 443-448. Wyatt, R., D. M. Lane & A. Stoneburner. 1991 b. Chemosystematics of the Mniaceae. III. Sources of intraspecific variation in flavonoids. Bryologist 94: 452-456. Wyatt, R., I. J. Odrzykoski & A. Stoneburner. 1987. Electrophoretically detectable genetic variation in Plagiomnium ciliare: A preliminary report. Symp. Biol. Hung. 35: 589-602. Wyatt, R., I. J. Odrzykoski & A. Stoneburner. 1989. High levels of genetic variability in the haploid moss Plagiomnium ciliare. Evolution 43: 1085- 1096. Wyatt, R., I. J. Odrzykoski & A. Stoneburner. 1992. lsozyme evidence of reticulate evolution in mosses: Plagiomnium medium is an alloployploid of P. ellipticum X P. insigne. Syst. Bot. 17: 532- 550. Wyatt, R., I. J. Odrzykoski & A. Stoneburner. l 993a. Isozyme evidence regarding the origins of the R. WYAIT et al.: Phylogenetic relationships within Plagiomnium sect. Rosulata 95

allopolyploid moss Plagiomnium curvatulum. Lindbergia 18: 49- 58. Wyatt, R., I. J. Odrzykoski & A. Stoneburner. 1993b. lsozyme evidence proves that the moss Rhizom­ nium pseudopunctatum is an allopolyploid of R. gracile X R. magnifolium. Mem. Torrey Bot. Club 25 : 21- 35 . Wyatt, R., I. J. Odrzykoski, A. Stoneburner, H. W. Bass & G. A. Galau. 1988. Allopolyploidy in bryophytes: Multiple origins of Plagiomnium medium. Proc. Natl. Acad. Sci. U.S.A. 85: 5601 - 5604.