Phylogenetic Relationships, Morphological Incongruence, and Geographic Speciation in the Fontinalaceae (Bryophyta)

Molecular Phylogenetics and Evolution Vol. 16, No. 2, August, pp. 225–237, 2000 doi:10.1006/mpev.2000.0786, available online at http://www.idealibrary.com on

Phylogenetic Relationships, Morphological Incongruence, and Geographic Speciation in the Fontinalaceae (Bryophyta) A. Jonathan Shaw* ,1 and Bruce Allen† *Department of Botany, Duke University, Durham, North Carolina 27708-0338; and †Missouri Botanical Garden, St. Louis, Missouri 63166-0299 Received June 15, 1999; revised January 14, 2000

Nuclear ribosomal DNA (internal transcribed spacer region) and chloroplast DNA (trnL-trnF region) were sequenced from 40 samples representing all three genera (Brachelyma, Dichelyma, and Fontinalis) and 18 species of the aquatic moss family, Fontinalaceae. Phylogenetic reconstructions recovered from separate and combined analyses were used to test the hypotheses that Fontinalis and Dichelyma are monophyletic (Brachelyma is monotypic), that groups of species within Fontinalis based on leaf morphology (keeled, concave, plane) form monophyletic groups, and that species delineation based on morphological characters within Fontinalis are congruent with nr- and cpDNA gene trees. Using Brachelyma subulata to root the tree, both Dichelyma and Fontinalis are monophyletic and patristically divergent (each united by >15 synapomorphic mutations). Groups of species within Fontinalis defined by leaf morphology are polyphyletic and it is clear that leaf morphology is labile in the genus. As defined morphologically, species of Fontinalis are nonmonophyletic for both nr- and cpDNA sequences and populations of some morphological taxa are separated in widely divergent clades. Molecular evidence suggests that at least some morphospecies are artificial, defined by convergent leaf forms. The weight of the evidence indicates that F. antipyretica is positively paraphyletic, with European populations more closely related to (i.e., share a more recent common ancestor with) European endemic species than to North American populations that are morphologically conspecific. North American populations are more closely related to North American endemic species. © 2000 Academic Press

INTRODUCTION The Fontinalaceae are a small family of riparian and aquatic mosses that include three genera (Dichelyma, Brachelyma, Fontinalis) and about 26 species. Al1

To whom correspondence should be addressed. Fax: (919) 6607293. E-mail: [email protected].

though a few widespread species extend into the Southern Hemisphere and 1 is presently restricted to South America, the family is essentially northern hemispheric in distribution. In the Northern Hemisphere the Fontinalaceae are concentrated in North America (three genera and 23 species) and are fairly well represented in Europe (two genera and 11 species). In general, mosses occupy moist sites and are abundant in riparian habitats. Nevertheless, strictly aquatic taxa are rare. Brachelyma and Dichelyma, with one and five species, respectively, occupy riparian habitats where they grow on trunks, roots, or rocks near and along streams, ponds, or lakes where seasonal fluctuations in water level submerge them for at least part of the season. Fontinalis has species that occupy similar habitats, but are more commonly found as strict aquatics. The three genera of Fontinalaceae are united by unusual endostomial (peristome) morphology. Unlike that of most other mosses, secondary cell wall thickening in the peristomial layers is relatively heavy and as a result the endostome is firm rather than membranous. There is no division of the endostome into a basal membrane and segments and/or cilia. Instead, the Fontinalaceae endostome resembles a net consisting of 16 vertical filaments that are connected by nearly equidistant horizontal filaments. The entire net-like structure is termed a trellis. Monophyly of the Fontinalaceae is strongly supported by these uniquely shared peristomial characters of Brachelyma, Dichelyma, and Fontinalis. Affinities of the Fontinalaceae are controversial. Traditionally the Fontinalaceae have been associated with the genus Climacium (Lindberg, 1879; Brotherus, 1905, 1925; Fleischer, 1908; Vitt, 1984) in the order Leucodontales (⫽Isobryales). Andrews (1954) rejected such a relationship when he supported the alignment of Climacium with the Hylocomiaceae (Hypnobryales), as did Crum and Anderson (1981). A Leucodontalian affinity for the Fontinalaceae was supported by Robinson (1971) and Walther (1983). Buck and Vitt (1986),

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however, returned the family to the Hypnobryales because of its linear leaf cells, well-developed alar cells, endostomial structure, and distribution. Most recently, Buck and Allen (1997) reestablished a relationship between the Fontinalaceae and the Climaciaceae based on paraphyllia evidence, stem anatomy as related to rhizoidal distribution, and branching pattern. Monophyly of the order Leucodontales (characterized by relatively simple peristome structure among other features) is not supported by DNA sequence data from two chloroplast genes (Buck et al., unpublished), which suggests that traditional leucodontoid families are independently derived from Hypnalian taxa. Fontinalis is one of a relatively few genera whose phylogenetic relationships were inconsistent among analyses that differed in weighting schemes and search methodology (Buck et al., unpublished). Fontinalis, with 20 species, has traditionally been divided into three groups based upon whether the leaves are keeled, concave, or plane (Welch, 1960). Usually the diagnosis is not difficult, but there are a number of taxa for which more than one leaf type can be found on the same plant. In some species the stems and branches have different leaf forms and sometimes a single branch or stem can have more than one leaf type. Correctly placing many of these taxa depends largely upon deciding which type is in the majority. One of the goals of this research was to assess monophyly of these species groups within Fontinalis. The keeled group, with 6 taxa, has triangular apical buds and leaves that are sharply creased down the middle. This morphology is similar to that of Brachelyma and Dichelyma, both of which have leaves sharply keeled along the costa. The concave-leaved group (10 taxa) has terete apical buds and leaves that are concave when wet. Some members of this group always have concave leaves, regardless of the habitat, but others have leaves that are weakly concave or sometimes plane. The degree of concavity appears to be correlated with habitat conditions, i.e., the faster the stream current the more concave the leaves. The planeleaved group (4 taxa) has terete apical buds and leaves that are for the most part plane when wet. It is the least-well-defined group. Species often exhibit leaf size dimorphism, with stem leaves more than twice the length of the branch leaves. Every species within this group can have both plane and concave leaves, with plane dominating. As with members of the concave group, there appears to be a correlation between habitats with fast moving water and degree of leaf concavity. The species of Fontinalis are notoriously difficult to identify, apparently because of phenotypic modification in habitats differing in hydrology (Crum and Anderson, 1981). There is extensive intergradation between slightly differing taxa, and because the environment appears to have a profound effect on leaf

form (as in many other aquatic and semiaquatic groups of mosses) it is conceivable that some named “taxa” represent convergent forms of unrelated species. The goals of this research were framed as hypotheses. Hypothesis 1. Genera of Fontinalaceae are monophyletic. Brachelyma is monotypic. We used Brachelyma to root the phylogeny and could not therefore independently assess its relationship to the other two genera. The specific hypotheses that we were able to test are that Dichelyma and Fontinalis are each monophyletic. Alternatively, the four species of Dichelyma may form a paraphyletic grade or may be nested within Fontinalis (which would then be paraphyletic). Hypothesis 2. The three groups of species within Fontinalis defined by leaf morphology are monophyletic. Hypothesis 3. Gene trees derived from geographic sampling of widespread taxa reflect organismal relationships as inferred from morphological similarity. Can we infer patterns of speciation from populationbased sampling of selected taxa? MATERIALS AND METHODS Taxon sampling. There is a long tradition in bryology of distributing so-called exsiccatae sets of specimens to herbaria around the world. Each herbarium receives a portion of a particular collection (representing one population) and so each has duplicate portions. The purpose of such exsiccatae is to provide a reference collection so that members of the worldwide systematic community can know exactly what names are being applied to which forms. In groups such as mosses, in which individual species are often distributed over intercontinental ranges, exsiccatae sets of North American plants thus allow European botanists, for example, to know how names are being applied by specialists to North American collections. The existence of the Fontinalaceae Exsiccatae (Allen, 1986, 1989, 1991, 1994, 1999) provides a unique opportunity to expand the exsiccatae concept to one that permits a combined worldwide understanding of morphological and molecular variation and the relationship between these two types of information. The Fontinalaceae Exsiccatae that have been distributed to date include a total of 158 collections, representing all three genera and 25 species in the family. All collections utilized in this study were distributed to the following herbaria as the Fontinalaceae Exsiccatae: ALTA, B, BM, C, CHR, CINC, COLO, DUKE, F, FH, G, H, L, LAF, MCTC, MICH, MO, NICH, NY, PAC, PC, PRE, S, TNS, UBC, US, Z. Exsiccatae numbers that identify specimens in the set, taxon identifications, geographic provenance, and GenBank accession numbers are provided in Table 1.

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TABLE 1 Collection Identification, Exsiccate No., Locality Information, and GenBank Accession Nos. for Specimens Included in Molecular Analyses Genus

Species

Exsiccatae No.

Country

Brachelyma Dichelyma Dichelyma Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis

subulatum falcatum uncinatum antipyretica antipyretica antipyretica antipyretica antipyretica antipyretica antipyretica antipyretica antipyretica antipyretica antipyretica antipyretica antipyretica antipyretica var. gracilis antipyretica var. gracilis antipyretica var. gracilis chrysophylla dalecarlica duriaei duriaei duriaei gigantea gigantea hypnoides hypnoides hypnoides hypnoides novae-angliae redfearnii sphagnicola squamosa squamosa sullvantii sullvantii welchiana welchiana

86 92 93 7 9 34 35 61 95 96 97 99 100 101 104 105 36

U.S.A. U.S.A. U.S.A. U.S.A. U.S.A. U.S.A. U.S.A. Finland U.S.A. U.S.A. U.S.A. Kazakstan U.S.A. U.S.A. Spain Spain S. Africa

Florida New York California S. Dakota Colorado California Nova Scotia

U.S.A.

Alaska

Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis Fontinalis

70 103 135 109 67 21 68 8 115 69 118 119 120 125 77 65 112 129 131 132 133 157

Russia (Far East) U.S.A. U.S.A. U.S.A. U.S.A. U.S.A. U.S.A. U.S.A. U.S.A. U.S.A. China Spain U.S.A. U.S.A. U.S.A. Spain Spain U.S.A. U.S.A. U.S.A. U.S.A.

State

California California California Michigan Oregon

GenBank Accession No. AF191503 AF191506 AF191504 AF191522 AF191528 AF191525 AF191523 AF191526 AF191533 AF191531 AF191529 AF191532 AF191530 AF191516 AF191518 AF191519 AF191524 AF191514 AF191517

Oregon New York Minnesota Missouri Missouri Michigan New York Ontario Maine

N. Hampshire Oklahoma Indiana

Maryland Missouri Arkansas Alabama

AF191512 AF191534 AF191537 AF191515 AF191539 AF191527 AF191536 AF191538 AF191509 AF191508 AF191510 AF191540 AF191507 AF191513 AF191535 AF191520 AF191521 AF191511 AF191541 AF191542

Note. Some of the species identifications do not agree with the original exsiccatae labels but represent more recent revisions.

DNA extraction, PCR amplification, and sequencing. Green apical portions (ca. 5–10 mm) were used for DNA extractions following a modification of Doyle and Doyle’s (1987) protocol as in Buck et al. (unpublished). Plant material was ground using a small teflon pestle in 700 ␮L of 2⫻ CTAB (hexadecyltrimethylammonium bromide)– 0.2% beta-mercaptoethanol and incubated for at least 30 min. An equal volume of chloroform– isoamyl (24:1) was added and the emulsified solution was centrifuged for 1 min (6500 rpm). The aqueous phase was added to an equal volume of ice-cold isopropanol and the DNA was precipitated at 0°C (30 – 60

min). Tubes were centrifuged for 10 min (13,000 rpm); the pellet was washed with 70% ethanol and centrifuged for 3 min (13,000 rpm). The pellet was dried in a vacuum centrifuge and suspended in 30 ␮L TE (Tris– EDTA, pH 8.0). Amplifications of the nuclear ribosomal DNA (nrDNA) and chloroplast DNA (cpDNA) were performed in 50-␮l reaction volumes containing 1⫻ PCR buffer (Gibco BRL), 0.2 mM dNTPs in equimolar ratio, 2.5 mM MgCl 2, 5% glycerol, 1.0 unit Taq Polymerase (Gibco BRL), and 0.5 mM each primer. The primers trnC and trnF (Taberlet et al., 1991) were used for

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amplification of the trnL-trnF region (trnL 5⬘ exon – trnF). A quantity, 0.5 ␮l, of stock DNA was added to each reaction and amplification was accomplished using the following temperature profile: 95°C for 1 min, 52°C for 1 min, and 72°C for 3 min. After 30 cycles, a final 7-min extension at 72°C was performed and reactions were held at 4°C until further processing. PCR products were screened on 1% agarose gels and, when successful, were cleaned and concentrated with filter cartridges (30,000 NMWL low-binding regenerated cellulose; Millipore). A quantity, 10 –50 ng, of cleaned PCR product served as template in dRhodamine Dye Terminator Cycle sequencing reactions (Perkin– Elmer), performed according to the manufacturer’s protocol modified for 1/4-size reactions. TrnC and trnF were used as primers in separate sequencing reactions, cycled as recommended by the manufacturer. To remove unincorporated reaction components, resulting 10-␮l products were cleaned using Centri-Sep spin columns (Princeton Separations) containing G-50 Fine Sephadex. Labeled fragments were separated on polyacrylamide gels (Long Range Singel; FMC Bioproducts), using an ABI Prism 373 or 377 automated DNA sequencer (Perkin–Elmer). Sequences obtained were edited using Sequencher 3.0 (Gene Codes Corp.), entered and manually aligned, if necessary, in PAUP* 4.01 (Swofford, unpublished). Amplification of the ITS1-5.8S-ITS2 region was accomplished with a nested PCR design. An initial amplification was performed in a 25-␮l reaction volume, as above, with 0.5 ␮M each BMBC-R and LS4-R primers (sequences given in Shaw, unpublished). A quantity, 0.3 ␮l, of stock DNA was added to each reaction as template. The product of this amplification was used as template in a subsequent amplification with the primers ITS-1 and ITS-4. Then, 50-␮l reactions were accomplished as above, with 0.5 ␮l of the initial reaction product. The PCR used the same temperature profile for both amplifications: 95°C for 1 min, 50°C for 1 min, and 72°C for 45 s plus 5 additional s for each successive cycle. After 30 cycles, a final 7-min extension at 72°C was performed, and reactions were held at 4°C until further processing. ITS-1, ITS-4, 5.8S, and 5.8S-R (Baldwin, 1992) were used as primers in four separate sequencing amplifications, cycled as recommended by the manufacturer. Phylogenetic analyses. Sequence chromatograms were compiled using Sequencher software (vers. 2.0, Gene Codes Corp.) to produce contigs based on nucleotide identifications from both DNA strands. All sequences were aligned by eye, with gaps inserted where needed to preserve nucleotide homology. Regions in which alignment was considered ambiguous were deleted from the analyses. Our nuclear ribosomal (nrDNA) sequences include ITS-1, part of the 5.8S ribosomal RNA gene, ITS-2, a

short exon (9 bp) at the 5⬘ end of the 26S ribosomal RNA gene, and approximately 235 bp of an intron in the 26S gene immediately downstream from the exon (Capesius, 1997). Boundaries of the subregions, including the 26S exon and intron were identified by comparison to available GenBank sequences. Approximately 70 bps near the center of the 5.8S gene were excluded from analyses because of missing data for a number of taxa. Our chloroplast (cpDNA) sequences include an intron and exon in the trnL gene, a spacer separating the trnL and trnF genes, and approximately 65 bp of the trnF gene (Taberlet et al., 1991). Boundaries of the subregions were identified by comparison to GenBank sequences. Phylogenetic analyses based on parsimony were accomplished using PAUP* 4.01. Unambiguous indels were scored as additional characters and parsimony analyses were run separately with indel scores included and excluded. Two indels were scored in ITS-1, two in ITS-2, and three in the intron in the 26S ribosomal RNA gene. One indel was scored in the trnL intron and one in the spacer separating the trnL and trnF genes. In analyses in which indel scores were included, corresponding positions scored as gaps in one or more taxa were excluded. The heuristic searches were run with the following settings: steepest descent off, TBR branch swapping, MULPARS on, 100 randomsequence additions saving ⱕ1000 trees per replicate. Exploratory analyses in which one replicate was run without limiting the number of trees saved, and 1000 replicates with MULPARS off, did not reveal any topologies not included in the consensus obtained from 100 replicates saving up to 1000 trees each. Multiple most-parsimonious trees were summarized using the strict consensus. Bootstrap analyses (Felsenstein, 1985) were accomplished using 300 bootstrap replicates and the same heuristic settings used in the parsimony analyses, except with five random-sequence additions per bootstrap replicate. Tests of data partition incongruence. Incongruence in phylogenetic signal in trnL and ITS data sets was assessed using several approaches. The incongruence length difference test (ILD; Farris et al., 1994) compares tree lengths from the nrDNA and cpDNA data sets to random partitions of equal size. The null hypothesis is that the two data sets do not differ significantly more than do random partitions of the same size, in terms of the distribution of phylogenetically significant characters. PAUP* vers. 4.01 implements this test (partition homogeneity test) using I MF (Farris et al., 1995) as the distance metric (Johnson and Soltis, 1998). Wilcoxon’s signed-rank tests and paired t tests assess topological incongruence between rival trees (Mason-Gamer and Kellogg, 1996) by comparing muta-

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TABLE 2 Genomic Regions and Subregions Included in the Molecular Data Sets and Their Sizes and Levels of Variation in the Fontinalaceae and in Fontinalis Fontinalaceae

ITS-1 5.8S ITS-2 26S Intron ITS trnL trnL Intron trnL Exon trnL/trnF Spacer trnF

Fontinalis

Characters

Autapomorphic

Informative

Autapomorphic

Informative

445 107 159 235 954 478 283 50 56 67

39 14 9 21 85 13 5 0 1 2

35 (8) 6 (6) 23 (15) 32 (14) 93 (10) 46 (10) 22 (8) 0 11 (20) 13 (19)

18 14 7 17 66 8 1 0 0 2

28 (6) 6 (6) 17 (11) 10 (4) 51 (5) 30 (10) 11 (4) 0 7 (17) 12 (18)

Note. Numbers in parentheses are percentages of total characters.

tional steps exhibited by individual characters on the alternative trees. We conducted several permutations of these tests. Homoplasy was compared in consensus nrDNA and cpDNA topologies recovered from separate analyses, measured on the nrDNA data set. In this case, the cpDNA topology is considered the rival tree (Mason-Gamer and Kellogg, 1996). Alternatively, the nrDNA consensus topology was used as a rival tree measured on the cpDNA data set. An additional comparison was made between both the nr- and the cpDNA topologies and the topology recovered from a combined analysis, measured on the combined data set. In this case both the nr- and the cpDNA topologies were considered rival trees (to the combined topology). Likelihood ratio tests were applied to compare likelihoods of different trees, given the same combinations of topologies and data sets, as above. The same comparisons were also made using 20 randomly selected equally most-parsimonious trees (MPT) from separate searches rather than the consensus topologies. The same results were obtained; i.e., when tests based on consensus trees indicated incongruence, all pairwise comparisons among individual MPTs from the data sets also detected incongruence. RESULTS

was included in the data set, there is some variation among members of the Fontinalaceae in this typically conserved gene (Table 2). The trnL intron contained relatively few informative sites, whereas high levels of informative variation occurred in the trnL/trnF spacer and in the trnF gene. Uncorrected sequence divergence between the outgroup (Brachelyma subulata) and Fontinalis samples ranged from 5.8 to 8.4%. Divergence between Brachelyma and Dichelyma samples was 3.3–3.7%. Substantial sequence variation occurred both among and within species of Fontinalis. Each of the five populations of F. hypnoides had a unique genotype with regard to combined nr- and cpDNA sequences. Although most populations of F. antipyretica were unique, several combinations of populations had identical sequences for both regions (Figs. 1, 4, and 6). Our only population of F. chrysophylla, from Oregon, is identical to a Michigan population of F. antipyretica. Two Spanish populations of F. antipyretica are identical, as are three North American populations. Two samples of the European endemic species, F. squamosa, had identical nr- and cpDNA sequences (Figs. 3, 4, and 6). The F. squamosa populations are most similar to populations of F. antipyretica from Spain, Finland, and South Africa.

Sequence Variation

Phylogenetic Relationships

Sizes and levels of variation for the different genomic and subgenomic regions are summarized in Table 2. The most variable regions in the nuclear ribosomal DNA region are ITS-2 and the 26S intron. The former is almost as variable within the genus Fontinalis (11% informative sites) as within the family as a whole (15% informative sites), whereas the 26S intron is much less variable within Fontinalis than within and among (mostly among) the three genera of Fontinalaceae. Although only a portion of the 5.8S ribosomal RNA gene

nrDNA reconstruction. The three genera of Fontinalaceae are highly divergent (Fig. 1). Dichelyma and Fontinalis species differ from the outgroup, B. subulata, by at least 30 and 60 synapomorphies, respectively. Two populations of D. falcata form a clade distinct from D. uncinata by eight mutations (Fig. 1). Fontinalis is monophyletic with 100% bootstrap support. In addition to nucleotide substitutions, all accessions of Fontinalis share two large deletions (44 and 12 bp) in the ITS-1 region (not scored). A smaller deletion

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FIGS. 1 and 2. Phylogenetic relationships in the Fontinalaceae inferred from nrDNA sequences. (1) One of the MPTs (most-parsimonious tree). Integers above branches are branch lengths. (2) Strict consensus tree. Integers above branches are bootstrap percentages. Taxa not shaded have keeled leaves.

(8 –10 bp) in the same region distinguishes the three samples of Dichelyma from B. subulata. Approximately 10 bp downstream from these deletions, all samples of Fontinalis share a 69-bp insertion relative to Dichelyma and Brachelyma. These major indel characters provide very strong support for a monophyletic Fontinalis, already well supported by nucleotide substitutions. Within Fontinalis, F. redfearnii differs by at least 21 mutational steps from other congeners and appears sister to the remaining species, with rather weak support. Two major clades within Fontinalis are resolved with strong support (Fig. 2). Species in these two major clades are divergent by a minimum of 16 mutational steps (Fig. 1). The two clades within Fontinalis are not congruent with infrageneric groupings defined by leaf morphology. Clade A includes the keeled species, F. antipyretica (including the variety gracilis), F. chrysophylla, and F. gigantea, the plane-leaved species, F. hypnoides and two populations of F. duriaei, and the concave-leaved species, F. squamosa (Fig. 2). Clade B

includes F. sullivantii, F. dalecarlica, and F. welchiana with concave leaves, and F. sphagnicola plus the other population of F. duriaei with plane leaves. Of the Fontinalis species for which multiple population samples were sequenced, none are monophyletic with regard to nrDNA genealogy, except for 2 populations of F. squamosa from Spain, which had identical sequences (Fig. 2). The two samples of F. sullivantii are para- or polyphyetic with regard to ITS, and 1 of 3 populations of F. duriaei is widely separated from the others in the two major clades within Fontinalis. Four populations of F. hypnoides comprise a well-supported clade, but 2 of the populations of F. duriaei are nested within it. The 16 populations of F. antipyretica do not form a monophyletic group (Fig. 2). However, when five indel characters are included in the analysis, populations of F. antipyretica form two clades with moderate bootstrap support (Fig. 3). The relationship between these clades is without support but F. antipyretica appears at least paraphyletic in the strict consensus tree (Fig. 3). One of the two F. antipyretica clades

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cpDNA clade B, but in addition, some populations of F. antipyretica (keeled leaves) and F. hypnoides (plane leaves) that are unambiguously placed in nrDNA clade A are included in cpDNA clade B. All of the Old World populations of F. antipyretica are included in cpDNA clade A, as are some of the North America populations, but the remaining North American populations of F. antipyretica are included in cpDNA clade B (Fig. 5). None of the three species of Fontinalis for which multiple population samples were sequenced are demonstrably monophyletic with regard to cpDNA genealogy (Fig. 5). Even populations of F. hypnoides, which comprise a well-marked clade for nrDNA, appear widely polyphyletic for cpDNA. Two of three F. duriaei populations are closely related, but the third is more divergent. Despite generally low resolution, or at least low bootstrap support for phylogenetic relationships based on cpDNA variation, three lineages of F. antipyretica populations are resolved with moderate to high bootstrap values (Fig. 5). The European and African populations form a clade with the European F. squamosa (as for nrDNA). Two North American clades are resolved within F. antipyretica, both containing populations from both the eastern and the western United States. The variety gracilis appears to be polyphyletic.

FIG. 3. Strict consensus tree from parsimony analysis of nrDNA sequences with indel scores included in the analysis. Integers above branches are bootstrap percentages.

consists of European, African, and northern Asian populations, plus European populations of F. squamosa. The other clade consists of F. antipyretica populations from North America and 1 southern Asian population, plus North American populations of F. chrysophylla and F. gigantea. F. antipyretica var. gracilis appears at the very least to be paraphyletic. trnL reconstruction. The three genera of Fontinalaceae are also strongly differentiated with respect to trnL genealogy (Fig. 4). Species of Dichelyma are divergent from B. subulata by a minimum of 13 synapomorphic mutational steps and species of Fontinalis differ from B. subulata by a minimum of 20 steps (Fig. 4). Within Fontinalis, two clades are resolved in the consensus tree, but both lack bootstrap support. Indeed, few relationships within the cpDNA genealogy are supported by bootstrap values of ⬎50% (Fig. 5). The two major clades that are resolved in the trnL consensus tree are not congruent with leaf morphology or nrDNA sequences. Species with concave or plane leaves that are included in nrDNA clade B are also in

Data incongruence. Both parsimony and likelihood tests indicate incongruence between nr- and cpDNA topologies, given the nrDNA data set (Table 3). Similarly, the nrDNA and cpDNA trees are significantly incongruent in the context of cpDNA sequences only. Further evidence of character incongruence comes from the ILD test (P ⱕ 0.01) and a comparison of consensus topologies obtained from analyses of nrDNA only, cpDNA only, and the combined data, given the combined data. Both nr- and cpDNA trees are significantly different from the combined topology, and it is noteworthy that even in the context of the combined data, the consensus tree obtained from the same combined data set has a lower likelihood than either the nr- or the cpDNA topology (Table 3). Reconstruction from combined analyses. Phylogenetic exploration included an analysis of the combined data so that incongruence between topologies recovered from separate and combined analyses could be estimated. Although our tests indicated incongruence, the combined analysis allowed us to visually compare the topologies so that we could assess the extent to which poorly supported relationships in the cpDNA topology contributed to statistical incongruence. The consensus tree obtained from a parsimony analysis of the combined data supports some relationships suggested by the separate data sets, as well as providing novel hypotheses of relationship within Fontinalis (Figs. 6 and 7). Both Dichelyma and Fontinalis are resolved and strongly supported as monophyletic groups by the combined data. Within Fontinalis, two

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FIGS. 4 and 5. Phylogenetic relationships in the Fontinalaceae inferred from cpDNA sequences. (4) One MPT. Integers above branches are branch lengths. (5) Strict consensus tree. Integers above branches are bootstrap percentages. Taxa not shaded have keeled leaves.

clades are resolved that correspond to clades A and B in the nrDNA tree. F. antipyretica is clearly nonmonophyletic, as in both the cpDNA and the nrDNA trees, but the relationships among the populations are unresolved in the combined analysis. Unlike either the cpDNA or the nrDNA data sets alone, the combined data suggest that F. hypnoides (plus two populations of F. duriaei) form a paraphyletic grade that is basal to one group of North American (plus the Kazakstan) populations of F. antipyretica. This relationship is, however, without bootstrap support (Fig. 7). The Old World populations of F. antipyretica form a strongly supported monophyletic group distinct from New World conspecific populations (as defined morphologically). The combined analysis supports a polyphyletic status for F. antipyretica var. gracilis. DISCUSSION Monophyly of the Fontinalaceae is strongly supported by morphological characters, especially those

relating to peristome structure (see above). The three genera are distinguished by a combination of morphological features pertaining to both the sporophyte and the gametophyte generations (Welch, 1960). Fontinalis species, for example, differ from both Dichelyma and Brachelyma species in having ecostate (gametophytic) leaves. The sporophyte of Dichelyma has a relatively long seta, such that the capsule is exserted beyond the sheathing perichaetial leaves, whereas the capsules of Brachelyma and Fontinalis are immersed because of a very short seta. There is no reason to assume that these features could not be subject to convergent evolution, but in terms of molecular characters the three genera of Fontinalaceae are patristically divergent and monophyly of the genera Dichelyma and Fontinalis is strongly supported by both nr- and cpDNA sequences. Unscored indel characters corroborate monophyly of Fontinalis. Monophyly of groups of species within Fontinalis defined by leaf morphology (i.e., keeled, plain, and concave; Welch, 1960) is not supported by either data set,

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FIGS. 6 and 7. Phylogenetic relationships in the Fontinalaceae inferred from combined nrDNA and cpDNA sequences. (6) One MPT. Integers above branches are branch lengths. (7) Strict consensus tree. Integers above branches are bootstrap percentages. Taxa not shaded have keeled leaves.

separately or in combination. Although two major clades are resolved within the genus, these do not correspond to groups based on leaf morphology. One includes species with all three leaf morphologies (clade A) and the other (clade B) includes both plane- and concave-leaved species. Although these different leaf morphologies are highly distinctive at their extremes, molecular data suggest that similar forms have evolved convergently. This is consistent with observations that leaf morphology is strongly affected by environmental factors and that substantial variation can be found on a single stem. Brachylema subulata (the only species of that genus), all five species of Dichelyma, and some species of Fontinalis have strongly keeled leaves, suggesting this as the primitive leaf morphology within Fontinalis. Subsequent evolution of leaf morphology within Fontinalis is not absolutely clear, but it appears likely that plane and concave leaves have evolved several times. One Old World clade that includes populations of F. antipyretica (keeled) and F. squamosa (concave) is strongly supported by both data sets separately and by the combined analysis. The plane leaves of F. hypnoides and F. duriaei likely evolved independently of this group. Moreover, plane- and concave-leaved spe-

cies in the other major clade (B) within Fontinalis appear to represent yet another independent origin of these morphologies. The possibility that concave and/or plane leaves evolved early in Fontinalis, and that it is keeled leaves that appeared convergently in different lineages, cannot be eliminated by our data. Nevertheless, whichever the correct character state reconstruction, it is clear that leaf morphology is labile in the genus and does not define monophyletic groups. Species for which multiple sequences were available do not appear to be monophyletic with regard to either cp- or nrDNA genealogy, nor for the combined data set. Two populations of F. sullivantii form a paraphyletic grade, at best. All of the four populations of F. hypnoides included in our sample belong to one clade with high bootstrap support for nrDNA, but two of three populations of F. duriaei are nested within it. The latter is one of the most variable taxa in Fontinalis, and it has at times been considered synonymous with F. hypnoides (Crum and Anderson, 1981). F. hypnoides is even more clearly para- or polyphyletic with regard to cpDNA. Bootstrap support for cpDNA relationships is weak or nonexistent, but a few clades have higher support, and resolution in the strict consensus is itself at least suggestive. cpDNA sequences indicate that if

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TABLE 3 Tests for Incongruence between nr- and cpDNA Data Sets, and between Topologies Obtained from Separate Analyses versus the Combined Analysis Data set 1. 2. 3. 4. 5. 6. 7. 8.

nrDNA nrDNA cpDNA cpDNA Combined Combined cpDNA cpDNA

nrDNA topology

cpDNA topology

220 2703.8 199 1504.4 419 K 4481.0 K

267 KT 2905.8 K 123 KT 1299.8 K 390 KT 4361.4 K 129 1299.8

1307.1

Combined topology

331 4147.7 123

Note. Either tree length or -In likelihood is given for each topology measured on the specified data set. K ⫽ significant difference (at P ⬍ 0.05) for the Kishino–Hasegawa paired t test or, in the case of likelihood comparisons, for the likelihood ratio test; T ⫽ significant difference using the Templeton (Wilcoxon signed rank) test. For tests measured on the combined data set (Nos. 5 and 6), incongruence was tested between the topologies recovered from the separate data sets and that obtained from the combined analysis. The last tests (Nos. 7 and 8) assess incongruence between the original cpDNA topology and a topology constrained to that obtained from the separate nrDNA analysis, measured on the cpDNA data set. Likelihoods were estimated under the general time-reversible model of evolution. Results of the ILD test, which also indicated significant incongruence between nr- and cpDNA data sets, are not included in this table.

F. hypnoides, the morphological species, is monophyletic, its chloroplast genome is para- or polyphyletic. Whether this transpecific distribution of cpDNA sequences results from lineage sorting or from more recent gene flow from other Fontinalis species cannot be resolved at present. The relationship between geographic provenance and cladistic structure among populations of F. hypnoides is not fully resolved and has little bootstrap support, but possible patterns are tantalizing. Three populations from interior North America form a clade with moderate support in the combined analysis, and a sister group relationship between populations from Maine and Spain is highly supported by nrDNA and combined analyses. A Chinese population is more isolated. These patterns, although faint, raise hypotheses of both vicariance and dispersal that could be tested by additional population sampling. It is clear, however, that the morphotype known as F. duriaei is heterogeneous and without phylogenetic meaning. The three populations included in our analyses occur divergently in the two major clades that are resolved by both nrand cpDNA sequences. Sixteen populations of F. antipyretica reveal a clear geographic pattern. Phylogenetic isolation between Old and New World populations is evident, even if their mutual relationships are ambiguous. Both nrand cpDNA sequences, and the combined data, support vicariance. Moreover, Old World populations of F. an-

tipyretica share a more recent ancestor with the European endemic species, F. squamosa, than they do with New World conspecific populations as defined morphologically. A recent origin of this endemic is indicated by the fact that it differs from European populations of F. antipyretica by only a single substitution. The two populations, both from Spain, are identical across both nrand cpDNA sequences. Indeed, although the clade that includes all the Old World populations of F. antipyretica plus F. squamosa is united by eight synapomorphies, populations within the clade differ by only zero to three mutations. Three Old World populations of F. antipyretica are isogenic for the regions that we sequenced. North American populations of F. antipyretica form one or more clades. Nucleotide substitutions alone do not resolve relationships among North American populations, but inclusion of indel scores in the nrDNA analysis provides evidence for a single clade with moderate bootstrap support. cpDNA sequences suggest heterogeneous origins for the chloroplast genome of F. antipyretica, as in F. hypnoides, but with little or no bootstrap support, also as in F. hypnoides. Nevertheless, both genomes provide substantial evidence that the North American species, F. gigantea and F. chrysophylla, are nested within a clade that includes some or all North American populations of F. antipyretica. Relationships among the three F. antipyretica var. gracilis populations and their precise relationships to other F. antipyretica populations are not resolved, but it does appear that this slender morphotype is not monophyletic. The African population was probably introduced from Europe with trout eggs to establish a South African fishery (Sim, 1926). Indeed, the African plants of F. antipyretica var. gracilis appear more closely related to European populations of the type variety than to Alaskan or Russian populations of the var. gracilis. The weight of the evidence suggests that the morphologically defined species, F. antipyretica, is paraphyletic, that North American and Old World populations comprise cryptic genealogical species, and that vicariant speciation has occurred in both North America and Europe. F. antipyretica is widespread in temperate zones of the Northern Hemisphere, and if keeled leaf morphology is ancestral within Fontinalis, as suggested by outgroup comparisons, our data support the hypothesis that Old and New World populations of this relatively old species gave rise to derivative endemic taxa. The high level of sequence identity between F. squamosa and European F. antipyretica suggests that speciation occurred so recently that subsequent mutations have not occurred in the genomic regions that we sampled. Similarly, the North American derivative species, F. gigantea and F. chrysophylla, differ from most closely related populations of F. antipyretica by only 3 and 1 synapomorphies, respectively.

PHYLOGENY AND GEOGRAPHIC SPECIATION IN FONTINALACEAE

This is in contrast to a minimum of 16 synapomorphies separating Old and New World F. antipyretica. There are many reasons that gene trees may differ from organismal phylogenies (Doyle, 1992, 1997), including interspecific gene flow, transpecific coalescence patterns (lineage sorting), sampling error, and historical differences in genome evolution. The fact that neither nr- nor cpDNA trees correspond to organismal relationships based on morphology indicates a decoupling of morphological and molecular evolution. Inconcongruence is apparent at both the inter- and the intraspecific levels. Conflicts between molecular and morphological patterns in this aquatic, morphologically plastic group may be greater than in many genera of mosses and other plants. Aquatic mosses are notoriously variable in morphology (Crum and Anderson, 1981), and in Fontinalis water velocity is known to cause phenotypic modifications. Some morphotypes may be little more than habitat modifications and it is also apparent from our generic-level phylogeny that leaf morphology is evolutionarily labile. None of the species for which we included multiple population samples are monophyletic for the genomic regions that we sequenced, and although incomplete sampling may be part of the explanation, it is not the whole story. Our analyses included only three populations of F. duriaei, for example, but two of these are highly divergent phylogenetically from the third. This taxon represents a case in which convergent morphotypes appear to have erroneously been given taxonomic recognition at the specific level. Significant incongruence between nr- and cpDNA data sets was suggested by most of the tests using both parsimony and likelihood criteria. Significant topological incongruence can be caused by conflicting relationships that are weakly supported by bootstrap analyses (Mason-Gamer and Kellogg, 1996; Cunningham, 1997; Graham et al., 1998). The nrDNA tree had higher support in general (CI, RI, RC), and individual clades tended to have high bootstrap support. The cpDNA tree was more poorly resolved and had lower support, and poorly supported relationships may have contributed to topological incongruence. This is probably not the complete explanation, however. The fact that the consensus trees from separate nr- and cpDNA analyses are substantially incongruent (i.e., even in the absence of bootstrap support), plus significant incongruence indicated by the ILD test, raises the real possibility that the nuclear and chloroplast genomes of these taxa have had different histories. A consensus tree from all equally most-parsimonious trees saved from separate nr- and cpDNA analyses (not shown) shows no resolution, emphasizing conflict between the two data sets. Moreover, similar patterns of conflicting relationships among populations within F. hypnoides and F. antipyretica recovered from nr- and cpDNA sequences suggest that evolutionary dynamics of the chloroplast ge-

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nome has occurred at odds with both morphological and nrDNA evolution. Perhaps most significant is the fact that both the nrand the cpDNA topologies obtained from separate analyses are significantly incongruent with the topology recovered from the combined analysis. This suggests that the combined topology represents an unsatisfactory compromise between two separate data sets. Indeed, the combined topology has a lower likelihood than either separate topology, even in the context of the combined data. The combined topology resolves the same two major clades within Fontinalis that we recovered from the nrDNA analysis alone, but relationships within these clades are in conflict. The paraphyletic and basal position of F. hypnoides (relative to F. antipyretica) in the combined analysis is not, for example, implied by either data set analyzed alone. This relationship may reflect a statistical compromise rather than phylogenetic history. It appears that the nuclear and chloroplast genomes have had truly divergent histories within Fontinalis. Paraphyletic species may be common in plants (Rieseberg, 1994; Crisp and Chandler, 1996), especially in cases of speciation through peripheral isolation, through founder events, or though various modes of sympatric speciation (e.g., host or ecological shifts; Macnair et al., 1989) in which a derived species starts out with a subset of the variation in the ancestral species. In cases of vicariant (i.e., allopatric) speciation, sister taxa are more likely to be demonstrably monophyletic if synapomorphic mutations have been fixed in both lineages. We view speciation as a coalescence process, recognizing that different genes (i.e., nonrecombining regions of DNA) coalesce at different rates (Baum, 1992; Baum and Shaw, 1995; Maddison, 1995). Coalescence times depend on population size, natural selection, recombination rates, and mutation rates (Pamilo and Nei, 1988). Coalescence times for genes used to infer exclusivity (Baum and Shaw, 1995) are expected to be longer in larger populations characterized by lower rates of recombination, and consequently shifts in population sizes and mating behavior during speciation may mean that one lineage “achieves” exclusivity before the other. Evolutionary “snapshots” of cladistic structure at any one point in time are expected to reveal taxa at various stages in the development of monophyly. The New and Old World clades of F. antipyretica appear to be reciprocally monophyletic (notwithstanding ambiguity in relationships among New World populations). If the vicariance event underlying differences between New and Old World clades was associated with the opening of the Atlantic ocean, a rough time frame puts isolation at some 60 –100 million years ago (Raven and Axelrod, 1974). More recent speciation within the Old and New World clades is

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associated with lower levels of sequence divergence and a lack of evidence for cpDNA coalescence within morphologically defined species. It is noteworthy that the Old World and New World clades of F. antipyretica are divergent at the sequence level but are morphologically indistinguishable (and have therefore been considered conspecific), whereas the endemic segregate taxa are morphologically recognizable but barely differ from other populations at the sequence level. Our data suggest that the current species-level taxonomy, in emphasizing morphology (and we believe necessarily so), is not the most accurate representation of evolutionary history. Demonstrably paraphyletic plant species inferred from DNA data are few, but probable paraphyly has been documented in the green algal genus Eudorina (Nazaki et al., 1997) and in the diatom genus Stephanodiscus (Theriot, 1992). Patterns of nr- and cpDNA relationships in Quercus indicate nonmonophyly of several species (Manos et al., 1999). Many bryophytes (mosses, liverworts, hornworts) have very broad, often intercontinental geographic ranges like that of F. antipyretica, and paraphyly may be common. Cryptic speciation has been documented, mainly from isozymes, in several widespread liverwort taxa that appear to be morphologically uniform over intercontinental ranges (e.g., Conocephalum [Szweykoski and Krzakowa, 1979; Odrzykoski, 1995], Marchantia [Boisselier-Dubayle et al., 1995], Riccia [Dewey, 1988, 1989], Pellia [Zielinski, 1986]). Cryptic speciation, based on isozyme variation, has recently been demonstrated in the mosses Plagiomnium cuspidatum and P. acutum (Wyatt and Odrzykoski, 1998). The widespread but rare moss Mielichhoferia elongata consists of two cryptic species that differ in isozyme frequencies (Shaw and Schneider, 1995) and nrDNA variation (Shaw, 2000). One of the clades is restricted to North America but the other is represented in both North America and Europe. Many morphologically defined moss species that have intercontinental ranges exhibit disjunctions that may reflect ancient vicariance events; for example, eastern North American– eastern Asian patterns (Iwatsuki, 1958; Crum, 1972). It is remarkable that such disjunct populations often appear morphologically indistinguishable. There has been much discussion about the relative contributions of slow morphological evolution vs highly effective gene flow in explaining such patterns (Crum, 1972; Zanten and Po´cs, 1981; Zanten and Gradstein, 1988). Population-based molecular analyses of broad-ranging but disjunct moss species hold new potential for understanding the origins of such disjunctions, and, if they are as old as some argue, for understanding area relationships among major continental areas.

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