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Int. J. Plant Sci. 167(5):1029–1048. 2006. Ó 2006 by The University of Chicago. All rights reserved. 1058-5893/2006/16705-0012$15.00

PHYLOGENY AND CLASSIFICATION OF CAREX SECTION OVALES (CYPERACEAE) Andrew L. Hipp,1 ,* Anton A. Reznicek,y Paul E. Rothrock,z and Jaime A. Weber§ *Department of Botany, 430 Lincoln Drive, University of Wisconsin, Madison, Wisconsin 53706, U.S.A.; yUniversity of Michigan Herbarium, 3600 Varsity Drive, Ann Arbor, Michigan 48108, U.S.A.; zRandall Environmental Studies Center, Taylor University, Upland, Indiana 46989, U.S.A.; and §Morton Arboretum, 4100 Illinois Route 53, Lisle, Illinois 60532, U.S.A.

Section Ovales is the most species-rich section of the sedge genus Carex in the New World. Phylogenetic analyses of molecular data recover a predominantly New World clade as sister to a solitary east Asian species, C. maackii. Nuclear ribosomal DNA are congruent in the placement of all taxa within the section, with a solitary exception: incongruence between ITS and ETS data in the placement of C. bonplandii and C. roraimensis suggests a hybrid origin for this lineage. Biogeography correlates strongly with phylogeny in the section, but there have been at least two instances of long-range dispersal, one from an eastern North American clade to western North America and one from the New World to Eurasia. Morphological characters studied are all homoplastic. Developing a comprehensive infrasectional classification with a phylogenetic basis would be complicated by the fact that most of the novel morphological characters in the section have evolved within relatively small, independent clades. Keywords: Carex subgenus Vignea, Carex section Ovales, Carex section Stellulatae, hybrid speciation, ancestral character state reconstruction, nuclear ribosomal DNA.

Introduction

marked by vegetative shoots with nodes (‘‘vegetative culms’’ sensu Reznicek and Catling 1986), gynecandrous spikes, and perigynia bearing marginal, epidermal ‘‘wings’’ (Reznicek 1993). This combination of characters makes the section easily recognizable, though distinguishing the species is often difficult. Monophyly of section Ovales has been demonstrated using sequence data from the internal transcribed spacer regions (ITS1 and ITS2) and 5.8S nuclear ribosomal gene (Hipp, forthcoming). Kenneth K. Mackenzie (1931–1935) divided section Ovales into 11 informal species groups based on characters of the perigynia, pistillate scales, leaf sheaths, and vegetative culms (fig. 1). Although some of the species groups have strong ecological, geographical, and morphological homogeneity (e.g., the ‘‘Tribuloideae’’ and ‘‘Alatae’’), most do not, and the groups are not generally viewed as natural (Reznicek 1993). The groups were never described formally. Consequently, Mackenzie’s species group names are in quotation marks throughout this article. In this study, we combine ITS data with sequences from the 59 end of the external transcribed spacer (ETS) region of nrDNA to evaluate biogeographic shifts and patterns of morphological evolution associated with the diversification of Carex section Ovales. We circumscribe major lineages within the section and identify the sister group to the North American clade that makes up the majority of the section. We use tests of topological incongruence to evaluate Mackenzie’s infrasectional classification and the strength of support for alternative placements of key taxa in the ETS and ITS data partitions, providing preliminary phylogenetic and chromosomal evidence for a possible allopolyploid origin of the

The sedge family (Cyperaceae) numbers ca. 5000 species worldwide, making it the third largest family of monocots (Goetghebeur 1998). The genus Carex L. of tribe Cariceae comprises roughly 40% of the family by species, making it one of the largest genera of angiosperms (Reznicek 1990; Mabberley 1997). Carex species are ecologically important members of floodplain forests, dry prairies, alpine meadows, peat lands, swamp forests, sedge meadows, and a wide range of other communities (Reznicek 1990). Molecular work in Cyperaceae tribe Cariceae has mostly focused on generic circumscription or relationships among the subgenera and sections that make up the genus Carex (Starr et al. 1999, 2003, 2004; Yen and Olmstead 2000; Roalson et al. 2001; Hendrichs et al. 2004a, 2004b). There has been little work on fine-scale patterns of morphological diversification within the genus (though see Roalson and Friar 2004). Section Ovales Kunth is the largest section in Carex subgenus Vignea (P. Beauv. ex T. Lestib.) Peterm., containing 72 North American species (Mastrogiuseppe et al. 2002), 15 additional described species endemic to South or Central America, and three species endemic to Europe and Asia (Reznicek 1993), for a total of 90 species worldwide. The section reflects much of the ecological breadth of the entire genus. Morphologically, however, the section is extremely cohesive, 1 Author for correspondence; current address: Morton Arboretum, 4100 Illinois Route 53, Lisle, Illinois 60532-1293, U.S.A.; e-mail [email protected].

Manuscript received September 2005; revised manuscript received April 2006.

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INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 1 Overview of K. K. Mackenzie’s (1940) infrasectional classification of Carex section Ovales, including the six major key characters that Mackenzie used to define the species groups. Biogeographic regions are approximate, and there is overlap in the distribution for several of the species groups indicated as occurring in either western or eastern North America. Perigynium beak cross-sectional shape and perigynium body shape were excluded from analyses presented in this article because herbarium study suggests that they are continuous. Not all key characters are coded for analyses presented in this article as Mackenzie reported them (cf. fig. 5) because of discrepancies between Mackenzie’s keys and more recent studies (Mastrogiuseppe et al. 2002) as well as our own herbarium observations. Per. bk ¼ perigynium beak cross-section; Per. body ¼ perigynium body shape in outline; Pist. scale ¼ pistillate scale; Bract ¼ lowest bract of the entire inflorescence; Veg. culm ¼ vegetative culm; Lf sheath ¼ inner face of the leaf sheath. Illustrations by H. C. Creutzberg, reprinted by permission from the New York Botanical Garden, Bronx, NY (Mackenzie 1940).

Latin American C. bonplandii complex. Finally, we use maximum likelihood to reconstruct the evolution of morphological characters and evaluate the phylogenetic consistency of Mackenzie’s infrasectional classification.

Material and Methods Taxon Sampling Sequences were obtained from 121 individuals representing 18 of ca. 26 recognized sections of subgenus Vignea (which includes sections Ovales and Cyperoideae) and three sections of subgenus Carex (appendix table A1). Sampling includes 75 species and two varieties of Carex section Ovales, representing all continents, in addition to four individuals of indeterminate identity: C. cf. microptera, C. cf. lagunensis, C. cf. brevior from Mexico, and a plant from Arkansas with affinities to C. molesta that is referred to in this article as ‘‘Buffalo River.’’ Sampling outside of section Ovales includes three species of subgenus Vignea known either to reproduce extensively from vegetative shoots (C. chordorrhiza and C. pseu-

docuraica) or to produce true vegetative culms (C. sartwellii), three non-Ovales species with winged perigynia (C. planata, C. brizoides, and C. siccata), and both species from section Cyperoideae (C. bohemica and C. sychnocephala), which has been variously treated as a separate section or as part of section Ovales. Three outgroups are included from subgenus Carex based on their placement in a previous study (Roalson et al. 2001). We follow the taxonomy presented in Flora of North America North of Mexico (FNA; Ball and Reznicek 2002), with two exceptions: (1) we provisionally treat C. teneriformis Mack., which is treated as a synonym of C. subfusca Boott in FNA, as taxonomically distinct based on sequence results and field observations; and (2) we refer to section Cyperoideae G. Don as a species group within section Ovales, following Mackenzie (1931–1935) and previous molecular study in the section (Hipp, forthcoming). The placement of 35 taxa was confirmed using sequences from DNA extracted from an additional individual of each species (table A1). Most ITS sequences were generated for a previous study (Hipp, forthcoming); the remaining ITS and all ETS sequences were generated for this study.

HIPP ET AL.—PHYLOGENY OF CAREX SECTION OVALES

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Table 1 Uncorrected Number of Nucleotide Differences (Minimum and Maximum) between nrDNA Clones for Seven Taxa ETS

Taxon Carex bonplandii complex: C. bonplandii C. roraimensis Section Ovales: C. straminea C. vexans Section Stellulatae: C. echinata C. exilis C. interior All interspecific

ITS þ 5.8S

No. clones

No. unique sequences

Min. diff.

Max. diff.

No. clones

No. unique sequences

Min. diff.

Max. diff.

10 10

8 4

1 1

5 4

9 9

8 6

2 1

15 7

10 9

4 4

1 1

3 6

9 10

2 6

2 1

2 7

10 9 10 68

4 7 5 36

1 1 1 1

6 5 6 39

9 10 10 66

6 7 5 40

1 1 1 3

4 6 4 40

Note. Individuals cloned are the same individuals sequenced for phylogenetic study (table A1). Nine to 10 clones were sequenced per individual. The number of unique sequences recovered for each taxon is closely correlated between ETS and ITS þ 5:8S. Carex bonplandii displays a great deal of interclonal divergence in the ITS þ 5:8S region; sequencing of additional clones would be needed to determine whether sequence types exhibiting this same degree of divergence are present at low levels in all taxa or only in C. bonplandii.

DNA Extraction, PCR, and Sequencing DNA was extracted from live, silica-dried, frozen, or herbarium tissue of single individuals using a modified 6X CTAB method (Doyle and Doyle 1987) and DNeasy kits (QIAGEN, Valencia, CA). The ITS region was amplified using the primers ITS-I (Urbatsch et al. 2000) and ITS4 (White

et al. 1990), and the ETS region was amplified using primers ETS-1F and 18S-R (Starr et al. 2003). PCR was conducted on 50-mL reactions containing 5 mL MgCl2 at 25 mM, 5 mL 10X MgCl2-free Taq buffer, 2 mg BSA, 1.5 mL DMSO, 0.5 mL of each primer at 20 mM, 0.25 mL Taq DNA polymerase (1.25 units), and 1 mL genomic DNA template. PCR conditions

Fig. 2 Unrooted neighbor-joining (NJ) trees of unique clone sequences for seven taxa based on two nrDNA regions: ITS þ 5:8S and ETS. NJ was conducted on GTR distances in PAUP* 4.0b10 (Swofford 2002), with all other settings at default values. NJ bootstraps (1000 replicates) are shown above the branches.

HIPP ET AL.—PHYLOGENY OF CAREX SECTION OVALES for most runs were initial denaturation at 94°C for 5 min; 30 cycles of DNA denaturation at 94°C for 30 s, primer annealing at 48°C for 1 min, and extension at 72°C for 1.5 min; and a 7-min extension at 72°C. Double-stranded PCR products were quantified on 0.8% EtBr-stained agarose and cleaned using Millipore spin columns, QIAquick cleanup kits (QIAGEN), or magnetic beads. Homogenization of nrDNA regions was evaluated by cloning three individuals from section Stellulatae, two from section Ovales, and two members of the C. bonplandii complex (C. roraimensis and two individuals of C. bonplandii). PCR was conducted for these individuals both with and without DMSO, which has been shown to have an effect on the proportion of functional nrDNA sequences recovered in PCR (Buckler et al. 1997). PCR bands were excised and cleaned using the centrifugation protocol of the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI). Each cleaned PCR was cloned using the pGEM-T Easy Vector System (Promega). The manufacturer’s protocol was followed except that some ligation reactions were cut in half, and the corresponding transformation reactions used 4 mL ligation reaction, 25 mL competent cells, and 500 mL SOC medium. Five clones per PCR per individual per nrDNA region were selected and purified using the centrifugation protocol of the Wizard Plus SV Minipreps DNA Purification System (Promega). Cleaned PCR was cycle sequenced in half-reactions (10 mL) using BigDye reaction kits and the primers employed in PCR. Additional ITS sequences from internal primers ITS3B (Baum et al. 1998) and ITS2 (White et al. 1990) were obtained for taxa in which ITS-I and ITS4 sequences did not provide double coverage of a substantial portion of the ITS regions. Plasmid DNA purified from clones was cycle sequenced using primers complementary to the T7 and SP6 promoters. Cycle sequencing products were precipitated in 75% ethanol or cleaned with magnetic beads and sequenced on ABI 377 or ABI 3100 automated sequencers at the University of Wisconsin–Madison Biotechnology Center’s DNA Facility. Clone sequences were analyzed on an ABI 3730 in the Field Museum’s Pritzker Lab (Chicago, IL).

Phylogenetic Analysis Sequences were edited and assembled in Sequencher 3.0 (GeneCodes, 1991–1995) and aligned manually in BioEdit 5.0.9 (Hall 1999). Sequences are deposited in GenBank (table A1), as are all unique clone sequences (ITS: DQ461091– DQ461130; ETS: DQ461055–DQ461090). Sequence alignments were unambiguous except for a few single base pair indels; alternate alignments at these points made no difference in the topology or support levels of final trees. Gaps were coded using the simple indel coding method (Simmons and

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Ochoterena 2000). Indels were analyzed alone to determine whether they recovered topologies that were congruent with sequence data before combined analysis. Ragged ends (74 aligned nucleotides, including seven potentially informative sites) were included in parsimony analyses, where they had a positive effect on resolution of the strict consensus tree, but were excluded from likelihood and Bayesian analyses. Mutational saturation for each sequence partition was estimated by plotting pairwise transition/transversion (Ti/Tv) ratios against Jukes-Cantor distances. A flat or inverse relationship between Ti/Tv ratio and genetic distance was interpreted as evidence of mutational saturation (Halanych et al. 1999; Lee 2000). Maximum parsimony trees were recovered in heuristic searches on equally weighted characters in PAUP* 4.0b10 (Swofford 2002). Searches were conducted using 1000 replicates of random-sequence addition to detect multiple islands of most parsimonious (MP) trees (Maddison 1991), with 100 MP trees saved for each replicate (chuckscore ¼ 1, nchuck ¼ 100, MULTREES ¼ yes) and tree-bisectionreconnection (TBR) branch swapping. The strict consensus was used as a reverse constraint in a second heuristic search under the same parameters, saving only trees not compatible with the consensus. This method is used to ensure that no shorter trees exist and that the strict consensus recovered in this study represents the set of all most parsimonious trees (Catalan et al. 1997). Branch support was estimated by nonparametric bootstrapping, using 1000 heuristic bootstrap replicates of 1000 random-sequence addition replicates each, with 10 trees held at each step during stepwise addition (chuckscore ¼ 1, nchuck ¼ 10, MULTREES ¼ yes) and TBR branch swapping. Data were also analyzed using Metropolis-coupled Markov chain Monte Carlo in MrBayes 3.0b4 (Huelsenbeck and Ronquist 2001). The three nucleotide partitions (ITS, 5.8S, and ETS) were modeled separately, using models selected based on Akaike information criterion (AIC) values calculated in Modeltest 3.6 (Posada and Crandall 1998). Indels for ITS and ETS were modeled separately as two-state characters (data type ¼ standard), with a correction for the fact that only variable characters were scored (coding ¼ variable). For combined data analyses, all parameters except for branch lengths and topology were unlinked across data partitions. Three independent runs of four linked chains were each run for 5 million generations, using the default priors and temperature parameter. Following inspection of run results, trees from the initial 1 million generations were eliminated from analysis to ensure that topologies and support levels represent an unbiased estimate of the posterior probability distribution. Convergence was assessed by comparing post-burn-in tree likelihood and consensus topology for the independent

Fig. 3 Majority-rule consensus trees resulting from separate Markov chain Monte Carlo analyses of each of the nrDNA data sets: ITS þ 5:8S þ ITS indels and ETS þ ETS indels. Topology, branch lengths, and posterior probabilities were estimated from runs of 5 million generations, with the first 1 million generations discarded to ensure that all parameters were estimated while the chain was at stationarity. Numbers above branches are posterior probabilities estimated from 40,000 trees sampled from the post-burn-in trees. Outgroups are excluded from the figure to save space; their resolution provides no new information on relationships in the genus. Position of the Carex bonplandii complex is indicated by the unlabeled boxes.

Fig. 4 Majority-rule consensus of Bayesian trees resulting from Markov chain Monte Carlo analysis of all data. Topology, branch lengths, and posterior probabilities were estimated from a run of 5 million generations, with the first 1 million generations discarded. Substitution model parameters were allowed to vary independently across data partitions. Numbers above branches are posterior probabilities estimated from 40,000 trees sampled from the post-burn-in trees (before the slash) and maximum parsimony (MP) bootstraps (after the slash). The strict consensus of MP

HIPP ET AL.—PHYLOGENY OF CAREX SECTION OVALES runs, but trees and analyses presented in this article are results of a single run.

Tests of Data Partition Congruence and Topological Tests of Phylogenetic Hypotheses The ITS and ETS data partitions were evaluated for congruence using the incongruence length difference (ILD) test (Farris et al. 1994, 1995) in PAUP* 4.0b10. A preliminary set of ILD tests was conducted under a range of relative weighting conditions to estimate the point of balance between data partitions in number of informative characters and overall strength of evidence (Dowton and Austin 2002; Hipp et al. 2004). Indels, 5.8S sequences, and uninformative sites were excluded from ILD analyses to limit the known effects of differences in substitution parameters and data decisiveness on ILD P values (Barker and Lutzoni 2002; Darlu and Lecointre 2002). ILD tests were conducted on 499 randomly partitioned data sets from which indels, 5.8S, and all uninformative sites had been excluded, with heuristic searches conducted using 10 random-sequence addition replicates, with 10 trees held at each replicate (chuckscore ¼ 1, nchuck ¼ 10). The ILD test was then conducted again for the relative weighting of the two partitions that produced the lowest P value. This test was conducted with 25 random-sequence addition replicates, with 20 trees held at each replicate. Alternative phylogenetic hypotheses were evaluated using the Shimodaira-Hasegawa (SH) test and Bonferronicorrected, one-tailed Kishino-Hasegawa (KH) test in PAUP*. Null distribution of the test statistic was simulated using 10,000 bootstrap replicates, likelihoods approximated using the resampling estimated log likelihood (RELL) method of Kishino et al. (1990). Because of the relatively large number of searches required for the topology tests, likelihood searches were conducted using five random-sequence addition replicates, with a limit of 100,000 rearrangements per replicate. In more than 50% of replicates, the rearrangement limit was not reached, and the optimal tree was recovered in at least two replicates in most searches.

Analysis of Morphological and Biogeographic Data Ancestral character states of morphological characters were reconstructed using maximum likelihood. Five characters used by Mackenzie were assessed in the herbarium by measuring individuals from each of 19 taxa that represent a broad morphological and phylogenetic range of the section (table A1). Mean measurements from three individuals per taxon were plotted along with the maximum and minimum, and discontinuities were assessed visually (data available from A. L. Hipp). Of these, two characters were found to be

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continuous and were excluded from analyses presented in this study. A sixth character (production of prominent, leafy vegetative culms) could not be assessed in the herbarium because collections of vegetative shoots are rarely sufficient. This character is nonetheless highly distinctive (Eaton 1959; Reznicek and Catling 1986) and was scored for this study following Mackenzie. Excluding the two continuous characters, the remaining four characters were coded as categorical with two states: scales as long as and effectively concealing the perigynium (1) or perigynia exposed (0); inner face of the leaf sheaths green and nerved to the apex (1) or hyaline, unnerved (0); vegetative culms prominent throughout the year, leaves spreading and distributed along the entire length of the shoot (1) or vegetative culms inconspicuous or prominent mainly late in the season, leaf blades mostly clustered near the apex (0); and inflorescence bracts leaflike and/or conspicuously exceeding the head (1) or setaceous and typically not as long as the head (0). Character states were based primarily on literature reports (Reznicek 1993; Mastrogiuseppe 2002; Mastrogiuseppe et al. 2002), with adjustments based on observation of herbarium material where literature accounts were inadequate or polymorphisms were reported. Character evolution was reconstructed in Mesquite 1.06 (Maddison and Maddison 2005) under the Mk1 (one-parameter) model on both the fully resolved Bayesian ‘‘allcompat’’ tree and a subset of 1000 trees from the Bayesian analysis. Because the placement of C. maackii as sister to the New World clade is strongly supported, while the sister to section Ovales as a whole could not be determined, reconstructions in this study were limited to the New World clade. Biogeography was coded as a four-state character (0: Central and South America; 1: western North America [Rocky Mountains and westward]; 2: eastern North America [east of the Rocky Mountains]; 3: Old World) and, because many taxa are polymorphic, analyzed using parsimony.

Cytology Chromosome analyses of a single C. roraimensis plant (Reznicek 11054) followed the technique of Cooperrider and Morrison (1967), as described by Rothrock and Reznicek (1996). In brief, immature spikes were collected in June and preserved in methanol, chloroform, and propionic acid (6 : 3 : 2). Anthers were dissected from spikes and squashed in 2% lactic-acetic-orcein and viewed using phase contrast at 31000. Seven pollen mother cells (PMCs) were inspected at first meiotic interphase. Drawings, photographs, and voucher specimen have been deposited at the University of Michigan Herbarium (MICH).

trees is almost identical in topology; branches that collapse in the MP strict consensus are indicated with a dash. Geographic distributions follow the most current published accounts. Outgroups are not pictured because the placement of section Ovales within subgenus Vignea is uncertain. Support for the branch that connects Carex maackii to the remainder of the section is based on analysis with outgroups included. Node labels correspond to clades discussed in the text and character reconstructions (table 2). Arrowheads indicate the long-distance dispersal (LDD) events required to explain the geographic distribution of species. LDD events indicated with a question mark are in question either because vicariance cannot be ruled out (in the case of C. interjecta) or because phylogenetic uncertainty makes biogeographic reconstruction at the base of the tree problematic (in the case of C. bohemica).

INTERNATIONAL JOURNAL OF PLANT SCIENCES

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Table 2 Likelihood Reconstructions of Ancestral Character States Major clades within section Ovales New World root (n ¼ 80) Scales concealing perigynia: Single-tree reconstructiona 1000-tree reconstructionb Lowest bracts foliose: Single-tree reconstructiona 1000-tree reconstructionb Leaf sheaths green (inner face): Single-tree reconstructiona 1000-tree reconstructionb Vegetative culms prominent: Single-tree reconstructiona 1000-tree reconstruction

WNA internal (n ¼ 53)

ENA root (n ¼ 39)

Minor clades within section Ovales LFS I (n¼3)

‘‘Ath.’’ þ ‘‘Cyp.’’ (n ¼ 3)

0 (0.7591; ns) 0 (0.9529) 0 (0.9864) 0: 0.459; 1: 0.007 0: 0.713; 1: 0.001 0: 0.937

1 (0.9796) 1: 0.996

0 (0.9983) 0: 1.000

0 (1.000) 0: 1.000

0 (1.000) 0: 1.000

0 (0.9999) 0: 0.992

0 (1.000) 0: 1.000

0 (1.000) 0: 1.000

0 (0.9998) 0: 1.000

1 (0.9933) 1 (0.997)

0 (1.000) 0: 1.000

0 (1.000) 0: 1.000

0 (0.9963) 0: 0.995

0 (0.9753) 0: 0.914

0 (0.6823; ns) 0 (0.9976) 0: 0.047; 1: 0.052 0: 1.000

0 (0.9985) 0: 1.000

1 (0.8128; ns) 1 (0.9622) 1: 0.352 1: 0.842

0 (1.000) 0: 1.000

0 (1.000) 0: 1.000

0 (1.000) 0: 1.000

0 (1.000) 0: 1.000

0 (1.000) 0: 1.000

0 (1.000) 0: 1.000

ENA II (n ¼ 11)

‘‘Trib.’’ (n ¼ 3)

1 (0.9944) 1: 0.989

Note. WNA ¼ western North American; ENA ¼ eastern North American. Other abbreviations correspond to clades labeled in figures 3 and 4. Ancestral character states were reconstructed using a one-parameter model (Mk1) in Mesquite 1.06. Underlined text indicates the reconstruction that is least common for each character among the nodes tested. The major clades are nested and encompass three nodes along the spine of the tree supported at posterior probability ðPPÞ 95%. The minor clades are smaller clades independent of one another and roughly corresponding in size to Mackenzie’s species groups. a Proportional likelihood of the higher-likelihood reconstruction on the Bayesian ‘‘allcompat’’ topology (the fully resolved topology that is compatible with the greatest number of trees encountered after the Markov chain Monte Carlo burn-in). Reconstructions that differ by 0.05 AIC weight have only one or two free parameters. This probably reflects the fact that there is very little information in the 5.8S sequences. The GTR þ I þ G model is the best fit to the ITS regions using AIC (weight ¼

0:6371); the cumulative AIC weight of GTR þ I þ G and GTR þ G is 0.9766, suggesting a good deal of confidence in the model selected. The model selected under the default hLRT (a ¼ 0:01) is TrN þ G, which has an AIC weight of only 0.0002. The GTR þ I þ G model would be selected using the default hLRT at a > 0:097552. Sequences were obtained from nine to 10 clones of each of seven taxa (table 1). The largest number of unique sequences (eight) was found in Carex bonplandii and the lowest number in C. straminea (two). The largest uncorrected interclonal divergence (15 nucleotides) is within C. bonplandii. Clones for each individual are monophyletic except in C. bonplandii, the clones of which are paraphyletic with regard to C. roraimensis (fig. 2).

ETS Data Matrix Aligning the ETS data set required inserting 32 gaps. Unaligned sequences range from 601 to 609 nucleotides long, and the aligned data matrix contains 624 nucleotide positions. Across the entire data set, 224 nucleotide positions are potentially informative. This number drops to 184 without outgroups and to 73 when only Ovales is included (excluding C. illota). Excluding C. roraimensis and C. bonplandii (see ‘‘Congruence and Analysis of Combined Data’’) leaves 57 potentially informative characters. GC content is 52.931%, and homogeneity of base frequencies across taxa cannot be rejected (x2 ¼ 77:247, df ¼ 366, P ¼ 1:000). Genetic distance and Ti/Tv are positively correlated for pairwise comparisons within section Ovales and subgenus Vignea. A nearly flat relationship between Ti/Tv and genetic distance in comparisons between the outgroup and all taxa within subgenus Vignea, combined with Ti/Tv ratio not exceeding 1.6 and averaging

HIPP ET AL.—PHYLOGENY OF CAREX SECTION OVALES 1.19, suggests that a significant percentage of base pair substitutions between the outgroup and ingroup are saturated (Halanych et al. 1999). For Ovales and outgroups, the TVM þ I þ G model has the highest AIC weight (0.2450), with another nine models required to come up to 0.95 cumulative AIC weight. The default hLRT selects the HKY þ G model at a ¼ 0:01 (AIC weight ¼ 0:0217, which falls within the 95% confidence interval). The order of model selection in the default hLRT precludes reaching the TVM þ I þ G model for this data set. For the full data set, the GTR þ G and GTR þ I þ G models have nearly equal AIC weights (0.3927 and 0.3817, respectively), with five models making up 95% of the cumulative AIC weight. The default hLRT (a ¼ 0:01) selects the TrN þ I þ G model, which has an AIC weight of 0.0126 and falls just outside the 99% confidence interval. The default hLRT selects GTR þ I þ G at a > 0:072453. Sequences were obtained from nine to 10 clones of each of seven taxa (table 1). The largest number of unique sequences (eight) was found in C. bonplandii. The lowest number (four) is found in four taxa, including C. roraimensis and members of sections Ovales and Stellulatae. Interclonal uncorrected distances within individuals are similar across taxa (minimum one, maximum three to six). Clones for each individual are monophyletic except in C. interior, the clones of which are paraphyletic with regard to C. echinata (fig. 2).

Congruence and Analysis of Combined Data The ILD test was conducted across a symmetric gradient of relative data partition weights (5 : 1 through 1 : 1), and strongest incongruence was found when partitions are weighted 1 : 1. At that weighting, the ILD P value for the entire data set ¼ 0:002, strongly rejecting the null hypothesis that the data are drawn from a homogeneous pool of characters. Excluding all taxa outside of sections Ovales and Cyperoideae increases ILD P value to 0.040. Excluding C. roraimensis and C. bonplandii in addition to taxa outside of sections Ovales and Cyperoideae increases ILD P value to 0.136, suggesting that these two taxa are a major source of incongruence in the data and that aside from the resolution of these two taxa, there is little if any incongruence between ITS and ETS in the resolution of section Ovales. To evaluate whether the topological difference between ITS and ETS in the position of C. maackii is significant, an additional test was performed with C. maackii and all taxa outside of sections Ovales and Cyperoideae excluded from analysis, but with C. bonplandii and C. roraimensis retained. This test is also significant (P ¼ 0:030), suggesting that C. maackii is not a significant source of incongruence between the data partitions. Because only the ETS data fail to recover a monophyletic section Ovales, the KH and SH tests were used to investigate which specific hypotheses are strongly rejected by the ETS data. Monophyly of section Ovales including C. maackii but excluding C. bonplandii and C. roraimensis is not rejected (SH P ¼ 0:6969). However, the hypothesis that section Ovales is monophyletic including C. bonplandii and C. roraimensis, with C. maackii constrained to be sister to the remainder of the section, is rejected by the ETS data (SH P ¼ 0:0138).

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For purposes of evaluating the support for alternative hypotheses on the combined data using SH and KH tests in PAUP* (which does not currently support separate models for different data partitions), a single nucleotide substitution model was selected for the full data set. The GTR þ I þ G model was selected (AIC weight ¼ 0:9953), though the default hLRT selects the TrN þ I þ G model at a ¼ 0:01 (AIC weight ¼ 2:43 3 105 ).

ITS Phylogenetic Results: All Taxa Heuristic searches of the full ITS data matrix recovered 41,977 unique most parsimonious trees of 658 steps (consistency index [CI] ¼ 0:517, retention index [RI] ¼ 0:754). Because this search was limited to swapping on 1000 trees at each random-addition replicate, many more MP trees are likely to exist, and island structure was not investigated. The strict MP consensus (not shown) resolves somewhat fewer nodes than the Bayesian majority-rule tree (fig. 3), but there are no major points of incongruence between the trees recovered in the MP and Bayesian analyses. Excluding C. illota, monophyly of section Ovales is strongly supported: posterior probability ðPPÞ ¼ 1:00, parsimony bootstrap ðPBÞ ¼ 80%. Carex chordorrhiza, C. pseudocuraica, and C. brunnescens form a strongly supported clade that is weakly supported as sister to Ovales (PP ¼ 0:94, PB < 50%). The largest well-supported clade within section Ovales is an eastern North American (ENA I) clade made up primarily of the eastern members of the ‘‘Festucaceae’’ and including the ‘‘Tribuloideae’’ and two of the ‘‘Alatae’’ (fig. 3, ENA I; PP ¼ 1:00, PB ¼ 69%). This clade derives from a near polytomy of mostly western North American species. Carex maackii resolves as sister to the remainder of section Ovales (PP ¼ 0:99, PB ¼ 61%).

ETS Phylogenetic Results Heuristic parsimony searches of the ETS þ indels data set recovered 72,872 unique trees of 744 steps, CI ¼ 0:569, RI ¼ 0:812. Topology of the strict consensus (tree not shown) is essentially identical to that of the Bayesian analysis (fig. 3), though it is less resolved. Monophyly of section Ovales, with the exception of C. maackii, C. bonplandii, and C. roraimensis, is strongly supported (PP ¼ 1:00, PB ¼ 97%). Carex maackii falls sister to a clade composed of Ovales and several other sections in the Bayesian analysis and sister to C. bromoides (section Deweyanae) in the strict consensus of MP trees but is without strong statistical support for separating it from Ovales in either case. Carex bonplandii and C. roraimensis are placed within section Stellulatae with high support (PP ¼ 1:00, PB ¼ 97%).

Combined Analysis The two nrDNA data partitions support different resolutions for the sister group to section Ovales (fig. 3). Removal of all taxa outside of section Ovales results in a 20-fold decrease in ILD significance (from P ¼ 0:002 to P ¼ 0:040). Combined analysis in this study was conducted using members of section Glareosae and Chordorrhizae as outgroups, but trial analyses using other sections as outgroups recover the same rooting for the section. Carex maackii

HIPP ET AL.—PHYLOGENY OF CAREX SECTION OVALES is conclusively placed as sister to the remainder of the section. Although ITS and ETS recover different topologies, the ILD and KH tests provide no strong evidence of incongruence between the data partitions regarding relationships within Ovales. Likewise, the data partitions are consistent regarding circumscription of the section, with two exceptions. First, the ETS data set, including all taxa, does not place C. maackii at the base of Ovales, as the ITS data set does. However, placement of C. maackii is weakly supported in the ETS data set but relatively strongly supported in the ITS data set. In combination, the position of C. maackii as sister to the remainder of section Ovales is strongly supported. Consequently, this does not appear to be a case of incongruence but rather of inadequate data in the ETS data partition alone to resolve the position of C. maackii. Thus, the only point of real topological incongruence between ITS and ETS regarding the circumscription of section Ovales appears to be the position of C. bonplandii and C. roraimensis. Combined analysis of section Ovales (excluding C. bonplandii, C. roraimensis, and C. illota, and using C. chordorrhiza and C. brunnescens as outgroups) recovers a phylogeny that is largely resolved within section Ovales, with parsimony and Bayesian analysis (fig. 4) and maximum likelihood (ML; not shown) supporting the same well-supported relationships. Maximum parsimony recovers 78,494 unique trees of 459 steps (CI ¼ 0:691, RI ¼ 0:819). The shortest tree recovered using the strict consensus as a reverse constraint is 460 steps (97,900 trees recovered under the same search parameters), suggesting that additional search effort would be highly unlikely to affect the strict consensus. ML analysis recovers a tree of log likelihood ðln LÞ ¼ 4204:82504 (tree not shown). Average ln L of three independent Markov chain Monte Carlo (MCMC) runs is 4464:0671 6 0:3864 standard error. The difference in likelihood scores in the Bayesian and ML searches reflects in part the difference in models used for phylogenetic reconstruction and the inclusion of indels in the Bayesian analyses. Under the GTR þ I þ G model with parameters fixed as in the ML search, ln L of the fully resolved allcompat consensus tree is 4208.23427, which differs insignificantly from the ML topology (one-tailed KH P ¼ 0:1628 under the GTR þ I þ G model, using 10,000 RELL bootstrap replicates). Two major groupings are recovered within the section (fig. 4): a western North American (WNA) grade that contains more than half of the taxa in the section and an eastern North American clade that is subdivided into two smaller clades (ENA I and ENA II). The Asian C. maackii resolves as sister to the remainder of the section.

Character Evolution and Monophyly of Mackenzie’s Species Groups The morphological characters studied, a subset of those used by Mackenzie to define species groups within Ovales,

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are all homoplastic (table 2; fig. 5). Moreover, the ancestors to the larger clades that originate along the spine of the phylogeny share the same most likely set of character states (table 2). The character traits that define the section’s more distinctive species groups—leafy, reproductive vegetative culms, foliose inflorescence bracts, green-nerved inner leaf sheaths, and pistillate scales concealing the perigynia—all evolved at the base of smaller clades embedded within the section. Mackenzie’s species groups are polyphyletic except for the ‘‘Athrostachyae,’’ the two species of which fall sister to C. sychnocephala (fig. 4). The SH test and Bonferronicorrected KH test both indicate that the data fail to reject monophyly of the ‘‘Tribuloideae,’’ ‘‘Cyperoideae,’’ and ‘‘Festivae’’ circumscribed narrowly (table 3). The SH and KH tests also suggest that a section Ovales that excludes C. sychnocephala and C. bohemica (of section Cyperoideae) is not strongly rejected (GTR þ I þ G SH P ¼ 0:2574, corrected KH P ¼ 0:0533). However, optimizing branch lengths on the constrained topology produces a zero-length branch at the root of the section and an exceedingly long branch leading to C. maackii, approximately five times as long as the next longest branch in the section. Trees recovered with C. maackii excluded have branches nearly the same length as those of the unconstrained tree. Consequently, the constrained tree seems at odds with a reasonable phylogenetic reconstruction for the section, despite the fact that it is not rejected in topology tests. Monophyly of the remainder of the Mackenzian species groups is rejected by both tests (P < 0:05).

Cytology of Carex roraimensis Observations of PMCs of C. roraimensis at first meiotic interphase show no indication of monovalents or trivalents (fig. 6), which would suggest aneuploidy as a consequence of chromosome fission. None of the PMCs showed evidence of tetravalents either, which are expected in autopolyploids due to homology between duplicated chromosomes. A chromosome count of n¼62 (2n¼124) was confirmed through inspection of seven PMCs. This is the highest euploid chromosome number known in the genus, excluding aberrant tetraploid cells observed in C. aquatilis 3 C: paleacea (2n¼74; Cayouette and Morisset 1985).

Discussion Data Congruence and the Origin of the Carex bonplandii Complex The ILD, KH, and SH tests suggest that there are two sources of incongruence in the data set presented here: resolution of taxa outside of section Ovales and the placement of the C. roraimensis–C. bonplandii clade (referred to hereafter in this article as the C. bonplandii complex, following Reznicek 1996). There seems to be no incongruence in the

Fig. 5 Maximum likelihood reconstructions of morphological characters on Bayesian ‘‘allcompat’’ consensus (fully resolved tree that is supported by the largest subset of trees sampled using Markov chain Monte Carlo). Circles at the nodes are shaded according to the proportional likelihood of each character state (black denotes character state as described; white denotes the alternative state). Likelihoods at labeled nodes are summarized in table 2. Node abbreviations are the same as in table 2.

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Table 3 Topology Tests of Alternative Hypotheses

Unconstrained (ML tree) Ovales s.s. without ‘‘Cyperoidea’’ ‘‘Alatae’’ Cyperoideae ‘‘Festivae’’ broad ‘‘Festivae’’ narrow ‘‘Festucaceae’’ broad ‘‘Festucaceae’’ narrow ‘‘Fetae’’ ‘‘Foeneae’’ ‘‘Foeneae’’ without C. arapahoensis ‘‘Leporinae’’ ‘‘Specificae’’ ‘‘Tribuloideae’’

ln L

Diff. ln La

KH P value

KH P value, Bonferroni corrected

4204.82504 4238.56830 4291.49387 4225.28575 4273.44220 4244.55044 4370.61901 4345.64365 4258.44594 4290.31199 4266.07341 4300.22860 4306.42435 4211.55587

... 33.74326 86.66883 20.46071 68.61716 39.72540 165.79397 140.81861 53.62090 85.48695 61.24837 95.40356 101.59931 6.73083

... 0.0031 0.0000 0.0186 0.0000 0.0040 0.0000 0.0000 0.0007 0.0000 0.0002 0.0000 0.0000 0.1613

... 0.0403 0.0000 0.2418 0.0000 0.0520 0.0000 0.0000 0.0091 0.0000 0.0026 0.0000 0.0000 1.0000

SH P value ... 0.2050 0.0012 0.5231 0.0057 0.1244 0.0000 0.0000 0.0288 0.0015 0.0178 0.0000 0.0000 0.8558

Note. Kishino-Hasegawa (KH) and Shimodaira-Hasegawa (SH) tests were conducted with the unconstrained maximum likelihood topology and with maximum likelihood trees recovered with constraints indicated. The Bonferroni-corrected P value (P 3 13) is based on the total number of tree comparisons (n  1 ¼ 13; here n ¼ number of trees being compared). Tests were conducted with all taxa indicated in figures 4 and 5. ‘‘ ‘Festivae’ broad’’ includes all taxa labeled ‘‘Festivae’’ and ‘‘Festivae or Festucaceae’’ in table A1; ‘‘ ‘Festivae’ narrow’’ excludes the latter group. Likewise, ‘‘ ‘Festucaceae’ broad’’ includes the taxa in ‘‘Festivae or Festucaceae,’’ while ‘‘ ‘Festucaceae’ narrow’’ excludes them. a Difference in log likelihood between each tree and the unconstrained (ML) tree.

placement of C. maackii. Rather, its placement outside of section Ovales in the ETS data set is due to lack of support, as evidenced by the fact that trees in which C. maackii falls sister to the remainder of section Ovales are supported by both data sets. Notably, the ETS data fail to reject trees constrained so that section Ovales is monophyletic with the inclusion of the C. bonplandii complex and C. maackii. This resolution presumably is not rejected because it does not necessitate breaking up section Ovales; the constrained tree resolves section Stellulatae (including C. roraimensis and C. bonplandii) as paraphyletic with respect to section Ovales, with C. maackii sister to the remainder of section Ovales. However, when the ITS resolution is enforced—C. maackii sister to section Ovales and the C. bonplandii complex embedded within Ovales—the ETS data strongly reject the tree. Incongruence in the placement of the C. bonplandii complex, while strongly supported by all tests employed, appears not to be reflected in intraspecific divergence among ITS sequences or ETS sequences (fig. 2). The raw sequences also show very few double peaks, minimal length polymorphism, almost no variability in the 5.8S region, and no evidence of heterogeneity in base composition between sequences, any of which might suggest that divergent paralogs or pseudogenes are posing a potential problem for phylogenetic reconstruction (Buckler et al. 1997). The clones sequenced in seven taxa recover no pseudogenes and suggest that nrDNA paralogs coalesce subsequent to the origin of the species studied, though paraphyly of ITS þ 5:8S clones in C. bonplandii and ETS clones in C. interior (fig. 2) may be due to retention of

ancestral polymorphisms or ongoing gene flow between close relatives. Laboratory error is not a likely explanation for incongruence between ITS and ETS because both C. bonplandii and C. roraimensis have been sequenced multiple times for both data partitions (separate sequences not shown), and largely unambiguous sequence alignment within both regions rules out the possibility that the difference is an artifact of alignment. A strong positive relationship between Ti/Tv ratio and corrected pairwise genetic distances within subgenus Vignea suggests that substitutional saturation is not an excessive source of noise that could potentially affect both the accuracy of phylogenetic reconstruction and results of the ILD test. The data thus appear to support a hybrid origin for the C. bonplandii complex, with ETS homogenizing to one parental type (section Stellulatae) and ITS to the other (section Ovales) (Okuyama et al. 2005). This hypothesis is consistent with the morphology of the C. bonplandii complex, the members of which are characterized by narrowly and bluntly winged perigynia that resemble those of sections Deweyanae, Remotae, or Stellulatae. In fact, while taxonomists working in the twentieth century have for the most part recognized the C. bonplandii complex as part of section Ovales (Mackenzie 1931–1935; Reznicek 1993), Ku¨kenthal (1909) considered C. bonplandii to be part of section Elongatae Kunth, a conglomerate section that has subsequently been split up into sections Deweyanae, Remotae, Stellulatae, and Glareosae in part (Hipp 2004). Hybridization also is consistent with the habitats and biogeographic distribution of the complex. Carex bonplandii is a conspicuously rhizomatous

HIPP ET AL.—PHYLOGENY OF CAREX SECTION OVALES

Fig. 6 Meiotic figure from one pollen mother cell (PMC) of Carex roraimensis (Reznicek 11054 [MICH]). Based on inspection of seven PMCs from the individual vouchered, the figures shown are interpreted as small bivalents. No tetravalents or other irregular pairing relationships were detected in any of the PMCs inspected, as would be expected in an autopolyploid. Note that chromosome groups on the lower edge of the nucleus are difficult to view in this image, in which abutting bivalents may resemble trivalents. These resolve clearly with through-focusing in this and other cells.

species of paramo and wet meadows at elevations of 1800 to >4500 m, ranging from Bolivia to Mexico. Most species of section Stellulatae occupy wet, sunny, peaty, or mineral soils, the kinds of habitats where C. bonplandii grows, and Mexico, Central America, and northern South America harbor several Stellulatae taxa. While intersectional hybrids are uncommon in the genus, they are not unknown (Cayouette and Morisset 1992), and the combination of molecular and morphological data for a hybrid origin for the C. bonplandii complex is compelling. An intriguing possibility is that the C. bonplandii complex may represent an allopolyploid lineage. Carex roraimensis has one of the highest chromosome counts known in Carex (n¼62; fig. 6), approximately the sum of mean counts in sections Ovales and Stellulatae. Moreover, chromosome figures inspected at first meiotic interphase in numerous PMCs of the C. roraimensis individual studied show only bivalents (fig. 6), suggesting that autopolyploidy is not a likely explanation for the high number found. If confirmed, this would be the first documented case of allopolyploidy in the genus Carex. Ongoing work aims at testing this hypothesis using additional molecular and chromosomal data.

Diversification and the Sister Group to Section Ovales A sister relationship between the New World clade that makes up the core of section Ovales and the east Asian spe-

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cies C. maackii is strongly supported. Assuming that attempts to sample all close relatives of the section have been successful, this finding suggests a high relative rate of diversification within the New World Ovales (assuming 89 taxa within the New World clade and one taxon sister to that clade; P ¼ 0:011 under Slowinski and Guyer’s [1993] test of relative diversification). Increased diversification in this clade is probably not explained by appeal to the distinctive synapomorphies of the section as key innovations, for neither winged perigynia nor vegetative culms have resulted in increased diversification in the other clades in which they occur (Hipp, forthcoming). The role of chromosomal evolution in diversification in the section may be a more reasonable explanation and is under further investigation (Hipp et al. 2006). The sister group to section Ovales as a whole is unresolved in this study (fig. 3). The ITS data recover a clade composed of C. chordorrhiza, C. pseudocuraica, and C. brunnescens (PP ¼ 1:00, PB ¼ 70%) as sister to Ovales (PP ¼ 0:94, PB < 50%), while the ETS data set recovers a clade composed of a wide range of sections (including Chordorrhizae, Phaestoglochin, Remotae, Glareosae, Stellulatae, Deweyanae, Ammoglochin, Holarrhenae, Divisae, and others; PP ¼ 0:96, PB < 50%) as sister to Ovales (PP ¼ 0:96, PB < 50%). This latter clade is also recovered in combined analysis (Ford et al. 2006), but the relationship collapses in strict consensus. Neither of the scenarios supports Savile and Calder’s (1953) hypothesis that section Ovales is sister to section Ammoglochin or Egorova’s (1999) placement of Ovales and Cyperoideae in a lineage with section Boernera (represented in this study by C. duriuscula). Also, none of the nonOvales taxa with winged perigynia (C. remota, C. planata, C. arenaria, C. siccata) fall sister to Ovales under either scenario. Identification of the sister group to section Ovales will require additional sampling of gene regions and an adequate account of the causes of incongruence between the nrDNA regions. While placement of C. illota is also unresolved, the best estimate of its position based on the data presented here is near section Glareosae; this placement is also supported by cpDNA data (A. L. Hipp, unpublished data). Ku¨kenthal (1909) placed C. illota into section Elongatae Kunth. Morphologically, C. illota might be expected to fall most naturally in one of those sections (Whitkus 1988). While inconclusive about the precise placement of the species, the data presented here do not support Mackenzie’s (1931–1935) argument that C. illota’s ‘‘true relationship seems to be with the Ovales, of which it may be regarded as one of the most primitive species’’ (p. 131).

Biogeography Geographic ranges correlate closely with phylogenetic relationships in section Ovales (fig. 4), supporting recent findings in other sections of the genus (Roalson and Friar 2004; Dragon and Barrington, forthcoming). Phylogenetic uncertainty in the placement of C. foenea (widespread northward), C. bohemica (Eurasia), the clade containing C. argyrantha (northeast and northern Great Lakes), and section Ovales

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among the other sections of subgenus Vignea preclude definitive statements about the geographic origins of the section. However, concentration of the western North American taxa in a paraphyletic grade that reaches to the base of the section, accompanied by strong Bayesian support for the eastern North American clade (PP ¼ 1:00, PB ¼ 0:54%), suggests that section Ovales in North America originated in the west and spread eastward. Moreover, parsimony reconstruction of biogeographic regions on the Bayes 1000-tree subsample recovers western North America as the ancestral range for the New World clade at the root of North American clade in 808 trees (eastern North America is reconstructed at the base in 283 trees and the Old World in 680; these sum to >1000 because the figures include equivocal reconstructions). Whether the position of C. maackii as sister to the remainder of the section reflects a Eurasian ancestry for the section or long-distance dispersal from a western North American progenitor cannot be ascertained without an accurate assessment of the section’s relationship to the remainder of the subgenus. In addition to diversification of lineages in situ (e.g., the western North American clades labeled on fig. 4 and clades ENA I and ENA II), there appear to have been at least two cases of more recent long-distance dispersal involved in the diversification of section Ovales: dispersal from eastern North America to the West Coast giving rise to C. feta and the West Coast populations of C. ovalis and dispersal to Eurasia giving rise to the Old World populations of C. ovalis (fig. 4). Additional long-distance dispersal events may be needed to explain C. interjecta (Mexico), C. bohemica (Eurasia), and C. maackii (east Asia), but phylogenetic uncertainty at the base of the tree makes biogeographic history difficult to infer for the latter two taxa, and vicariance cannot be ruled out for C. interjecta.

Character Evolution and the Status of Mackenzie’s Species Groups The morphological characters studied demonstrate a history of parallel evolution in the section, with some morphological innovations (e.g., elongate bracts) arising rarely but others (e.g., pistillate scales that cover the perigynia, green-veined leaf sheaths) evolving repeatedly (fig. 5). This pattern points to the problems with Mackenzie’s infrasectional classification and the difficulties of erecting an alternative system. Despite the fact that none of the morphological characters investigated map cleanly onto the phylogeny, the eastern North American clade and the western North American grade are morphologically distinctive. Indeed, two separate keys are presented in the recent Flora of North America North of Mexico section Ovales treatment for species east of the Rocky Mountains and species of the Rocky Mountains and westward (Mastrogiuseppe et al. 2002), corresponding closely to the western North American grade and the eastern North American clade presented in this study. The eastern and western groups appear not to be defined by a single character but, with some exceptions, do have distinctive appearances, such that species can be placed

with some confidence. Eastern members of the section typically have elongate inflorescences with relatively pale (hyaline to reddish brown) pistillate and staminate scales and flattened, serrulate margined beak apices. Western species frequently have compact headlike inflorescences and darker pistillate and staminate scales, and frequently the apical portion of the beak is terete and smooth. While no single character may turn out to diagnose the eastern North American clade, the look is distinctive enough to suggest that with additional work, characters may be found to distinguish the two.

The Eastern North American Clade: ENA Clade I and ENA Clade II Relationships within clade ENA I based on the nrDNA data are poorly resolved, and several of the sister species relationships implied by the nrDNA data seem improbable in light of the morphology. This could result if radiation in the clade had proceeded at a higher rate than mutation, leaving an unreliable imprint of phylogenetic relationships in the nrDNA sequence data. Phylogeny of clade ENA I has been addressed in detail in a separate study using AFLP data, which provide strong support regarding species relationships in the clade (Hipp et al. 2006). The other major eastern North American clade (ENA II) is composed of members of three species groups: the majority of the ‘‘Alatae,’’ characterized by obovate perigynium bodies and leaf sheaths that are green and veined on the inner face nearly to the apex; the eastern North American members of the ‘‘Fetae,’’ characterized by perigynium bodies that are widest at or below the middle and leaf sheaths green and veined on the inner face nearly to the apex (like the ‘‘Alatae’’); and C. scoparia of the ‘‘Festucaceae,’’ a highly polymorphic species that ranges across much of northern North America but is much more common east of the Rocky Mountains than it is westward. The close relationship between the ‘‘Alatae’’ and ‘‘Fetae’’ is not unexpected (Rothrock et al. 1997): the two share the characteristic of green-veined inner leaf sheaths, several species in each have spikes widely spaced in erect to arching inflorescences, and the distinction between obovate and ovate perigynium bodies is challenging, at best, in several of the taxa. One taxon in this clade (C. interjecta Reznicek) is known only from the type locality, a moist meadow in Morelos (Mexico), and was not known to Mackenzie. The plant is similar in appearance to C. longii, with elliptical to obovate perigynia and leafy vegetative culms, but it has the hyaline inner leaf sheaths, narrow leaf blades, and dark pistillate scales typical of the other Mexican members of the section (Reznicek 1993). The inclusion of C. scoparia within clade ENA II is somewhat surprising because it more closely resembles several taxa within clade ENA I. The exclusion of C. cumulata of the ‘‘Alatae,’’ with its green-nerved inner leaf sheaths and obovate perigynia, is also unexpected. These results are supported, however, by AFLP data (Hipp et al. 2006) as well as by sequences from additional individuals (data not shown).

HIPP ET AL.—PHYLOGENY OF CAREX SECTION OVALES

The Western North American Grade The major species groups of the western North American grade (the‘‘Festivae,’’ ‘‘Leporinae,’’ ‘‘Foeneae,’’ ‘‘Specificae,’’ and ‘‘Cyperoideae’’) are all polyphyletic. Well-supported clades within the western grade fall into two classes: single-speciesgroup clades and clades composed of two to three morphologically similar species groups. In the latter falls the clade containing the ‘‘Athrostachyae’’ and C. sychnocephala, which was discussed in conjunction with monophyly of section Ovales. With ca. 20 recognized species, the ‘‘Festivae’’ is the second largest of Mackenzie’s species groups (after the ‘‘Festucaceae’’). The ‘‘Festivae’’ breaks apart along biogeographic lines into two major clades: one distributed primarily in the coastal ranges and Sierra Nevadas, with two outliers in the Rocky Mountains (‘‘Festivae II’’), and one distributed primarily in the southern Rocky Mountains (‘‘Festivae I’’). ‘‘Festivae II’’ includes the widespread C. macloviana, a bipolar disjunct that bears strong morphological similarity to C. haydeniana of ‘‘Festivae I.’’ It is surprising to find C. macloviana separating from C. haydeniana and C. microptera in this study, though the result has been verified using two accessions, both from the Rocky Mountains. Similarly, C. stenoptila is a distinctive species with narrow, distinctly veined perigynia that is allied morphologically to C. microptera, also of ‘‘Festivae I.’’ Why these species should be related to the coastal/Sierran taxa of ‘‘Festivae II’’ is unclear. These taxa may be products of hybridization that is not revealed in incongruence between the nrDNA data sets. The ITS data alone recover a clade composed of the coastal range species (C. gracilior, C. subbracteata, C. pachystachya, and C. harfordii; PP ¼ 0:91). The ETS data alone recover these taxa as a grade giving rise to the C. macloviana–C. mariposana–C. stenoptila clade. In the combined data, C. harfordii is recovered as part of this clade at PP ; 0:50, sufficiently close to the 50% threshold that it is excluded from the 50% majority-rule tree shown in figure 4. ‘‘Festivae I’’ is part of a larger, well-supported southern Rocky Mountain clade that includes C. wootonii (‘‘Specificae’’) and C. egglestonii (‘‘Festucaceae’’). Two species in this clade, C. microptera and C. haydeniana, extend well into the northern Rocky Mountains and intergrade in portions of their range (Whitkus and Packer 1984). As is the case in ‘‘Festivae I,’’ there is a close relationship between the Rocky Mountain clade and a group of predominantly Sierra Nevadan/Coastal range species, including C. abrupta, C. subfusca, C. integra, C. teneriformis, C. specifica, C. straminiformis, and C. multicostata. These Sierran/Coastal species form a paraphyletic grade from which the Rocky Mountain clade (‘‘Festivae I’’ and allies) arises, suggesting that at least the southern Rocky Mountain ‘‘Festivae’’ may have a Sierran origin. Most of the remaining western taxa fall into three species groups: the ‘‘Specificae,’’ a polyphyletic grouping made up of most of the western species with perigynia >6 mm long; the ‘‘Leporinae,’’ made up of the species with terete perigynium beaks smooth to the tip and pistillate scales that conceal the perigynia; and the ‘‘Foeneae,’’ comprising species with flat, often minutely serrulate perigynium beaks and

1043

pistillate scales that conceal the perigynia. The tendency toward elongate, appressed perigynia nearly concealed by the pistillate scales gives these species a rather sleek look, for want of a better term, and the two clades containing the majority of them (‘‘Leporinae-Foeneae-Specificae’’ [LFS] clades LFS I and LFS II; fig. 4) are morphologically cohesive. The singular exception is C. preslii, a species of the northern Rocky Mountains to Sierra Nevadas and Coastal ranges, with perigynia not concealed by the pistillate scales and perigynia rather ‘‘Festucaceae’’-like in appearance (not slender or terete tipped). The placement of this species is peculiar enough that it bears verification with additional specimens. One of the more interesting finds within the LFS clades is the placement of a specimen identified as C. constanceana in this study, a species previously known only from its type collection (Stacey 1938) and represented in this study by a Nevada County, California, population more than 425 miles from the type locality (Mount Adams, Washington). The specimen is clearly not an accession of C. davyi, to which it falls sister at a patristic distance of 0.00677 mean changes per aligned nucleotide, exceeding, for instance, the genetic distance between C. wootonii and C. peucophila (0.004261) or C. longii and C. vexans (0.003474). While genetic divergence is not sufficient for recognizing separate populations as different species, the combination of genetic distance and morphological divergence suggest that C. constanceana may well be an under-recognized and undercollected member of the western North American flora. An equally plausible alternative is that the collection may represent a local endemic distinct from C. constanceana and perhaps as restricted in distribution as that species. Further investigation of collected material is warranted, as is collecting aimed at finding additional populations of this plant.

Acknowledgments We thank Theodore S. Cochrane (WIS), Phillip E. Hyatt (USFS), and Elizabeth H. Zimmerman (WIS) for providing plant material. We also thank the curators of MICH, MO, RM, RSA, and WIS for allowing us to sample herbarium material. Technical assistance and advice were provided by Eric Roalson, and Julian Starr generously provided ETS primer sequences before their publication. Comments from both Eric and Julian and from Larry Hufford on the first submission of this manuscript improved the paper considerably. The manuscript also benefited from comments by A. L. Hipp’s graduate committee: Paul E. Berry (chair), David A. Baum, Thomas J. Givnish, Carol E. Lee, and Kenneth J. Sytsma. Support for this work was provided by a Lawrence Memorial Fellowship from the Hunt Botanical Institute, an E. K. Allen Fellowship through the University of Wisconsin– Madison Department of Botany, a Karling Graduate Student Research Award from the Botanical Society of America, an American Society of Plant Taxonomists research award, and National Science Foundation Dissertation Improvement Grant 0308975 to A. L. Hipp and P. E. Berry.

Appendix Table A1 Taxa Included in the Study Section/species group Subgenus Carex L.: Acrocystis Dumort. Hymenochlaenae Drej. ex L.H. Bailey Phacocystis Dumort. Subgenus Vignea (P. Beauv. ex T. Lestib.) Peterm. excluding Ovales: Ammoglochin Dumort.

Chordorrhizae Meinsh. Deweyanae Tuckerm.

Divisae Christ ex Ku¨k. Foetidae (Tuck. ex L.H. Bailey) Ku¨k. Glareosae G. Don

1044

Heleoglochin Dumort. Holarrhenae (Doll) Pax Macrocephalae Ku¨k. Multiflorae Kunth Phaestoglochin Dumort.

Physoglochin Dumort. Potosinae Mack. Remotae (Aschers.) C.B. Clarke Stellulatae Kunth

Vulpinae Kunth Section Ovales Kunth: ‘‘Alatae’’

‘‘Athrostachyae’’

Species

Location

Collector

ITS

ETS

pensylvanica Lam. gracillima Schwein. haydenii Dewey

WI (Dane) WI (Dane) WI (Dane)

Hipp 513 (WIS) Hipp 505 (WIS) Hipp 501 (WIS)

AY779137 AY779103 AY779106

DQ461013 DQ460978 DQ460982

a

arenaria L. brizoides L. siccata Dewey a chordorrhiza Ehrh. ex L.f. pseudocuraica Fr. Schmidt bolanderi Olney bromoides Schkuhr ex Willd. a deweyana Schwein. subsp. deweyana praegracilis W. Boott duriuscula C.A. Mey. a vernacula L.H. Bailey a brunnescens (Pers.) Poir. a canescens L. a cusickii Mack. ex Piper & Beattie prairea Dewey ex A.W. Wood sartwellii Dewey a curaica Kunth macrocephala Willd. ex Spreng. a vulpinoidea Michx. cephaloidea (Dewey) Dewey cephalophora Muhl. ex Willd. hoodii Boott muehlenbergii Schkuhr ex Willd. a radiata (Wahlenb.) Small rosea Schkuhr ex Willd. occidentalis L.H. Bailey gynocrates Wormsk. ex Drejer potosina Hemsl. a planata Franch. & Sav. remota L. exilis Dewey interior L.H. Bailey echinata Murray stipata Muhl. ex Willd. var. stipata

USSR Czechoslovakia NM (Taos) WI (Ashland) China CA (Humboldt) WI (Monroe) WI (Menominee) CA (Santa Barbara) MN (Norman) CA (El Dorado) WI (Forest) WI (Jackson) MT (Flathead) WI (Rock) WI (Dane) Siberia, West Sayan, Russia OR (Tillamook) WI (Rock) WI (Trempealeau) WI (Dane) CA (Alpine) WI (Dane) WI (Dane) WI (Dane) NM (Sandoval) MI (Alger) Mexico (Zacatecas) Japan (Honshu) Russia ME (Hancock) WI (Trempealeau) WI (Marquette) WI (Dane)

Pobedimova 5113 (WIS) Bohuslavek 694 (MICH) Hipp 2314 (WIS) Judziewicz 11790 (WIS) Lin 668 (MO) Hipp 480 (WIS) Hipp s.n. (WIS) DeJoode 1543 (WIS) Hipp 216 (WIS) McNeilus 93-927 (WIS) Hipp & Clifton 680 (WIS) Cochrane et al. 6579 (WIS) Hipp et al. 587 (WIS) Schuyler 4989 (WIS) Hipp & Zimmerman 602 (WIS) Hipp 515 (WIS) Krasnoborov et al. 7/21/1976 (MO) Voss 12990 (WIS) Cochrane 13345 (WIS) Hipp & Rothrock 1220 (WIS) Hipp 528 (WIS) Hipp & Clifton 705 (WIS) Hipp 545 (WIS) Hipp 162 (WIS) Hipp 514 (WIS) Hipp et al. 2067 (WIS) Henson 1504 (WIS) Villegas & Garcia s.n. (WIS) Tsugaru & Takahashi 26567 (MO) Novikov 5863 (MO) Little s.n. (WIS) Thompson 399 (WIS) Cochrane 13551 (WIS) Hipp 506 (WIS)

DQ461131 AY779076 DQ461148 AY779087 AY779148 DQ461132 DQ461133 AY779094 AY779143 DQ461136 AY779178 DQ461134 AY779078 DQ461135 AY779144 AY779154 AY779092 DQ461141 AY779180 AY779080 AY779081 ... AY779124 DQ461147 AY779153 AY779128 DQ461140 AY779142 AY779141 AY779150 DQ461139 AY779112 DQ461137 AY779162

DQ460939 DQ460948 DQ461034 DQ460958 DQ461024 DQ460945 DQ460949 DQ460966 DQ461019 DQ460967 DQ461050 DQ460950 DQ460952 DQ460964 DQ461020 DQ461030 DQ460963 DQ460994 DQ461052 DQ460953 DQ460954 DQ460983 DQ461001 DQ461025 DQ461029 DQ461005 DQ460979 DQ461018 DQ461017 DQ461026 DQ460972 DQ460988 DQ460969 DQ461037

b alata Torr. albolutescens Schwein. a cumulata (L.H. Bailey) Mack. b longii Mack. ozarkana P.E. Rothrock & Reznicek vexans F.J. Herm. b athrostachya Olney unilateralis Mack.

GA (Calhoun) OH (Lawrence) ME (Washington) Mexico (Michoacan) AR (Scott) FL (Pasco) CA (Nevada) OR (Benton)

Rothrock 3922 (MICH) McCormac et al. 6807 (MICH) Reznicek 10924 (WIS) Zamudio et al. 11237 (MICH) Hyatt 9357 (MICH) Rothrock 2379 (MICH) Hipp et al. 794 (WIS) Wilson 5882 (MICH)

AY779066 AY779067 AY779091 AY779115 AY779135 AY779179 AY779070 AY779177

DQ460936 DQ460937 DQ460962 DQ460991 DQ461011 DQ461051 DQ460941 DQ461049

Table A1 (Continued ) ‘‘Cyperoideae’’ ‘‘Festivae’’

‘‘Festivae’’ or ‘‘Festucaceae’’

‘‘Festucaceae’’

a

bohemica Schreb. a,b sychnocephala J. Carey abrupta Mack. bonplandii Kunth

1045

cf. microptera Mack.

Austria (Niederosterreich-Zwettl) WI (Waushara) CA (Nevada) Bolivia (La Paz: Nor Yungas Province) Mexico (Durango)

ebenea Rydb. gracilior Mack. harfordii Mack. b haydeniana Olney a illota L.H. Bailey integra Mack. a macloviana D’Urv. mariposana L.H. Bailey a microptera Mack. orizabae Liebm. pachystachya Cham. ex Steud. roraimensis Steyerm. stenoptila F.J. Herm. subbracteata Mack. teneraeformis Mack. multicostata Mack. preslii Steud. a,b peucophila T. Holm subfusca W. Boott b bebbii (L.H. Bailey) Fernald a bicknellii Britton bicknellii a brevior (Dewey) Mack. ex Lunell cf. brevior (Dewey) Mack. ex Lunell

CO (Ouray) CA (Mendocino) CA (Marin) UT (Summit) CA (Alpine) CA (Nevada) WY (Albany) CA (Mariposa) CO (Gunnison) Mexico (Ixtapaluca) UT (Duchesne) Venezuela (Roraima) UT (Summit) CA (Humboldt) CA (El Dorado) CA (El Dorado) MT (Flathead) Mexico (Morelos) NV (Washoe) WI (Dane) WI (Dane) WI (Dane) Mexico (Chiapas)

‘‘Buffalo River’’ crawfordii Fernald egglestonii Mack. b festucacea Schkuhr ex Willdenow hyalina Boott a merritt-fernaldii Mack. a missouriensis P.E. Rothrock & Reznicek a molesta Mack. ex Bright a molestiformis Reznicek & P.E. Rothrock a,b normalis Mack. opaca (F.J. Herm.) P.E. Rothrock & Reznicek oronensis Fernald reniformis (L.H. Bailey) Small scoparia Schkuhr ex Willd. scoparia Schkuhr ex Willd. var. tessellata Fernald & Wiegand a shinnersii P.E. Rothrock & Reznicek b straminiformis L.H. Bailey a tenera Dewey var. echinodes (Fernald) Wiegand a tenera Dewey var. tenera tincta (Fernald) Fernald a,b feta L.H. Bailey b hormathodes Fernald b straminea Willd. ex Schkuhr suberecta (Olney) Britton

AR (Marion) ME (Hancock) CO (Grand) WI (Juneau) MS (Tunica) NH (Strafford) MO (Macon) MS (Bolivar) TN (Jackson) WI (Dane) IL (Washington)

Gonza´lez & Reznicek 10303 (MICH) Hipp 1683 (WIS) Hipp 363 (WIS) Hipp 309 (WIS) Hipp 140.2 (WIS) Hipp & Clifton 700 (WIS) Hipp et al. 774 (WIS) Hipp 1893 (WIS) Hipp & Clifton 644 (WIS) Hipp 1681 (WIS) Rzedowski 36822 (WIS) Goodrich 21180 (RM) Reznicek 11054 (MICH) Hipp 1848 (WIS) Hipp 448 (WIS) Hipp & Clifton 716 (WIS) Hipp & Clifton 714 (WIS) Lesica 7874 (MICH) Gonza´lez & Reznicek 10552 (WIS) Hipp 833 (WIS) Hipp 516 (WIS) Hipp 549 (WIS) Reznicek 10345b (MICH) Gonza´lez & Reznicek 10497 (MICH) Hyatt 10461 (MICH) Reznicek & Reznicek 10918 (WIS) Hipp 1594 (WIS) Hipp et al. 561 (WIS) Rothrock 2947 (MICH) Rothrock 3475 (MICH) Rothrock 3567b (MICH) Bryson 12209 (MICH) Rothrock 3729c (MICH) Hipp 159 (WIS) Reznicek 10856 (MICH)

ME (Penobscot) AR (Dallas) IN (Newton) ME (Washington)

Reznicek et al. 10931 (WIS) Hyatt, P.E. 6996 (WIS) Rothrock 3633b (MICH) Reznicek 10923 (WIS)

AY779131 AY779151 AY779155 AY779156

DQ461008 DQ461027 DQ461031 DQ461032

TX (Delta) NV (Washoe) WI (Ozaukee)

Reznicek 10367 (MICH) Hipp 847 (WIS) Hipp & Rothrock 1188 (WIS)

AY779157 AY779164 DQ461138

DQ461033 DQ461039 DQ460970

OH (Lucas) ME (Hancock) CA (Humboldt) ME (Hancock) WI (Juneau) WI (Rock)

Hipp 1191 (MICH) Rothrock 3734 (MICH) Hipp 457 (WIS) Reznicek 10929 (WIS) Hipp et al. 560 (WIS) Hipp & Zimmerman 598 (WIS)

DQ461149 AY779174 AY779099 AY779108 AY779163 AY779166

DQ461045 DQ461047 DQ460974 DQ460984 DQ461038 DQ461041

a

‘‘Fetae’’

Wallno¨fer 13755 (WIS) Hipp 2578 (WIS) Hipp 799 (WIS) Solomon et al. 18926 (MICH)

AY779073 AY779169 AY779064 AY779074

DQ460944 DQ461043 DQ460934 DQ460946

AY779085

DQ460957

AY779095 AY779102 AY779104 AY779105 AY779110 AY779111 AY779117 AY779118 DQ461142 AY779130 AY779136 AY779152 AY779161 AY779165 AY779172 AY779125 AY779146 DQ461146 AY779167 AY779071 AY779072 AY779075 AY779082

DQ460968 DQ460977 DQ460980 DQ460981 DQ460986 DQ460987 DQ460993 DQ460995 DQ460997 DQ461007 DQ461012 DQ461028 DQ461036 DQ461040 DQ461046 DQ461002 DQ461022 DQ461015 DQ461042 DQ460942 DQ460943 DQ460947 DQ460955

AY779077 AY779089 AY779097 AY779098 AY779109 AY779119 AY779121 DQ461143 DQ461144 DQ461145 AY779129

DQ460951 DQ460960 DQ460971 DQ460973 DQ460985 DQ460996 DQ460998 DQ460999 DQ461000 DQ461004 DQ461006

Table A1 (Continued ) Section/Species group

Species

Location

‘‘Foeneae’’

b

adusta W. Boott arapahoensis Clokey b argyrantha Tuck. ex Dewey foenea Willd. xerantica L.H. Bailey

ME (Washington) CO (Gunnison) ME (Washington) ME (Washington) Ontario (Thunder Bay)

‘‘Fractae’’ ‘‘Leporinae’’

a,b

fracta Mack. leporinella Mack. b ovalis Gooden. b ovalis Gooden. b phaeocephala Piper praticola Rydb.

CA (Mariposa) CA (El Dorado) New Zealand OR (Benton) UT (Summit) Ontario (Rainy River)

tahoensis Smiley constanceana Stacey davyi Mack. petasata Dewey a specifica L.H. Bailey a wootonii Mack. cristatella Britton a,b muskingumensis Schwein. a projecta Mack. tribuloides Wahlenb. var. tribuloides cf. lagunensis M.E. Jones interjecta Reznicek maackii Maxim.

CA (El Dorado) CA (Nevada) CA (Alpine MT (Gallatin) CA (El Dorado) NM (Ruidoso) WI (Rock) WI (Iowa) WI (Adams) WI (Iowa) Mexico (Durango) Mexico (Morelos) Japan (Honshu)

‘‘Specificae’’

‘‘Tribuloideae’’

Ovales, unallied

a b

Sequence has been verified by a second acquisition (data not reported in this study). Taxon was used to evaluate discontinuity in morphological characters analyzed in this article.

Collector Reznicek 10922 (WIS) Hipp 1659 (WIS) Reznicek 10921 (WIS) Reznicek 10928 (WIS) Oldham & Bakowsky 17732 (MICH) Hipp 635 (WIS) Tallent 815 (MICH) Ford 30/98 (MICH) Wilson & Kuykendall 7027 (WIS) Hipp 135 (WIS) Oldham & Bakowsky 21854 (MICH) Hipp 879 (WIS) Hipp et al. 800 (WIS) Hipp 901a (WIS) Morse & Jordan 2082 (MICH) Hipp 861 (WIS) Hyatt 8294 (MICH) Hipp & Zimmerman 606 (WIS) Hipp & Biggs 2009 (WIS) Hipp et al. 1206 (WIS) Hipp 185 (WIS) Gonza´lez et al. 4482 (MICH) Zika 15398 (MICH) Kan 8031 (RSA)

ITS

ETS

AY779065 AY779068 AY779069 AY779100 AY779182

DQ460935 DQ460938 DQ460940 DQ460975 DQ461054

AY779101 AY779114 AY779132 AY779134 AY779139 AY779145

DQ460976 DQ460990 DQ461009 DQ461010 DQ461016 DQ461021

AY779170 AY779088 AY779093 AY779138 AY779160 AY779181 AY779090 AY779126 AY779147 AY779176 AY779084 AY779113 AY779116

DQ461044 DQ460959 DQ460965 DQ461014 DQ461035 DQ461053 DQ460961 DQ461003 DQ461023 DQ461048 DQ460956 DQ460989 DQ460992

HIPP ET AL.—PHYLOGENY OF CAREX SECTION OVALES

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