MOLECULAR

Molecular Phylogenetics and Evolution 33 (2004) 671–686

PHYLOGENETICSANDEVOLUTION

www.elsevier.com/locate/ympev

Phylogenetic analysis of the nuclear alcohol dehydrogenase(Adh) gene family in Carex section Acrocystis (Cyperaceae)

and combined analyses of Adh and nuclear ribosomalITS and ETS sequences for inferring species relationships

Eric H. Roalsona,*, Elizabeth A. Friarb

a School of Biological Sciences and Center for Integrated Biotechnology, Washington State University, Pullman, WA 99164-4236, USAb Rancho Santa Ana Botanic Garden, 1500 N. College Ave., Claremont, CA 91711, USA

Received 2 February 2004; revised 20 July 2004

Available online 25 September 2004

Abstract

We analyzed sequence variation for the alcohol dehydrogenase (Adh) gene family in Carex section Acrocystis (Cyperaceae) to

reconstruct Adh gene trees for Acrocystis species and to characterize the structure of the Adh gene family in Carex. Two Adh loci

were included with ITS and ETS sequences in a combined Bayesian inference analysis of Carex section Acrocystis to gain a better

understanding of species relationships in the section. In addition, we comment on how the results presented here contribute to our

knowledge of the birth-death process of the Adh gene family in angiosperms. It appears that the structure of the Adh gene family in

Carex is complex with possibly six loci present in the gene family. Additionally, variation among Acrocystis species within loci is

quite low, and there is little phylogenetic resolution in the individual datasets. Bayesian inference analysis of the combined ITS,

ETS, Adh1, and Adh2 datasets resulted in a moderately well-supported phylogenetic hypothesis of relationships in the section which

is discussed in relation to previous hypotheses of relationships.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Acrocystis; Adh; Alcohol dehydrogenase; Carex; Cyperaceae; ETS; Gene family evolution; ITS

1. Introduction

The genus Carex L. (Cyperaceae), with at least 2000

species (Reznicek, 1990), is one of the largest genera in

the world. The genus is relatively poorly known phylo-

genetically, with few studies and a general lack of reso-

lution among phylogenetic studies to date (Roalson etal., 2001; Starr et al., 1999; Starr et al., 2003; Yen and

Olmstead, 2000). Carex taxonomy is complex with four

subgenera and more than 70 sections recognized (Rez-

nicek, 1990). Phylogenetic studies have called into

1055-7903/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.ympev.2004.08.005

* Corresponding author. Fax: +509 335 3184.

E-mail address: roalson@mail.wsu.edu (E.H. Roalson).

question the monophyly of many subgenera and sec-

tions (Roalson et al., 2001).

Carex section Acrocystis consists of 45–49 taxa rang-

ing across North America and Eurasia, with one species

in Andean South America (Mackenzie, 1935). Previous

studies have provided evidence that a portion of this sec-

tion forms a clade within Carex and includes all of theNorth American members and a few of the Eurasian

members of Acrocystis, although not all non-North

American species in the section were sampled (Roalson

et al., 2001). Relationships within this clade of core-

Acrocystis have been examined using nrDNA internal

transcribed spacer (ITS) and external transcribed spacer

(ETS) sequences (Roalson and Friar, 2004). The ITS/

ETS sequence topology of relationships in Carex section

672 E.H. Roalson, E.A. Friar / Molecular Phylogenetics and Evolution 33 (2004) 671–686

Acrocystis supports a grade of Eurasian species leading

to a grade of western North American species, with

two clades of eastern North American species nested

within the western North American grade (Roalson

and Friar, 2004).

In order to explore phylogenetic relationships in thecore-Acrocystis clade in more detail, we present results

on the phylogenetic utility of the nuclear alcohol dehy-

drogenase (Adh) gene family. The Adh gene family re-

cently has been suggested to be useful for

phylogenetic estimation in a number of organisms

(Charlesworth et al., 1998; Gaut et al., 1999; Sang

et al., 1997; Small et al., 1998). Adh, in addition to sev-

eral other low-copy-number nuclear gene families (phy-tochrome B, Mathews et al., 2000; granule-bound

starch synthase, Mason-Gamer et al., 1998; Miller

et al., 1999; pistillata, Bailey and Doyle, 1999), has

been useful for examining divergence among closely re-

lated species where nuclear ribosomal spacers (ITS/

ETS) and chloroplast spacers (e.g., trnL-F) do not pro-

vide sufficient variation for phylogenetic reconstruction

(Sang et al., 1997; Small et al., 1998).Adh genes have been explored in detail in plants with

numerous studies either characterizing the gene family

in a particular species (Gaut and Clegg, 1993; Millar

and Dennis, 1996; Mitchell et al., 1989; Miyashita,

2001; Perry and Furnier, 1996; Small and Wendel,

2000a,b), or utilizing it for phylogenetic inference

(Charlesworth et al., 1998; Gaut et al., 1999; Koch

et al., 2000; Sang et al., 1997; Small et al., 1998). Amajority of flowering plants has been found to have

two or three Adh loci, each composed of 10 exons and

nine introns (Sang et al., 1997; and references therein),

although more recent studies (Small and Wendel,

2000b) have begun to suggest that the gene family struc-

ture of Adh may be more complex than previously

thought. While the process of gene duplication and dele-

tion in the genome is relatively well known (for review,see Li, 1997), the rate of this birth-death process across

organisms has not been studied nearly as much. Differ-

ent gene families in the angiosperms have shown differ-

ent patterns of duplication and loss of gene family

members, from long term persistence of gene family

members in functionally conserved genes (catalase gene

family: Klotz et al., 1997; CONSTANS LIKE gene fam-

ily: Lagercrantz and Axelsson, 2000) to quick turnoverof gene family copies (Pin2 gene family: Barta et al.,

2002) and concerted evolution homogenizing tandem re-

peats, with persistence of alternated copies rare beyond

the species or species complex level (nuclear ribosomal

DNA tandem repeats: Arnheim et al., 1980; Dover,

1982). For Adh, it has been suggested that there is a

‘‘slow flux’’ of gene duplication and loss across angio-

sperms (Clegg et al., 1997).Here, Carex alcohol dehydrogenase gene family

structure is explored and sequences of Adh are used to

investigate relationships in Carex section Acrocystis.

This study has three primary goals: (1) to characterize

the structure of the Adh gene family in Carex; (2) to

reconstruct Adh gene trees for Acrocystis species to ex-

plore the phylogenetic utility of this region of DNA;

and (3) to analyze a combined ITS, ETS, Adh1, andAdh2 dataset to further explore phylogenetic relation-

ships in the Carex section Acrocystis lineage. In addi-

tion, we comment on how the results presented here

contribute to our knowledge of the birth-death process

of gene family evolution in the Adh gene family in

angiosperms.

2. Materials and methods

2.1. Taxon sampling

Total DNAs were isolated from leaf tissue from

either live plants or dried herbarium specimens using a

modification of the CTAB method (Doyle and Doyle,

1987; Roalson and Friar, 2000). Samples of as manytaxa of Carex section Acrocystis as possible were in-

cluded in this study. Forty-five accessions of 24 ‘‘core-

Acrocystis’’ taxa were variously included in the different

individual and combined analyses. Voucher information

and GenBank accessions for all samples are listed in

Table 1.

2.2. Amplification, cloning, and sequencing

Preliminary studies focused on an approximately

2000 bp region of Adh covering exon 2 to exon 9 that

was amplified using primers Adh2f and Adh9r (Fig. 1;

Table 2). The primers were developed using GenBank

Poaceae sequences (X02915, AF050457, X04049,

AF044307, X16296, X12734, X12733, and AF050456).

All PCR amplifications were hot-started, with 1 unitof Taq DNA polymerase being added after 5 min of

denaturing at 96 �C. Thirty-five cycles of amplification

were carried out in a Stratagene Robocycler 96, with 1

min denaturing at 96 �C, 1 min annealing at 56 �C, and2 min extension at 72 �C. A final 10 min extension at

72 �C followed amplification. The products were verified

with 0.8% agarose gel in TBE buffer.

PCR products were cloned using the PCR-ScriptAmp Cloning Kit (Stratagene) and either miniprepped

using the PERFECTprep Plasmid DNA Preparation

Kit (5 Prime-3 Prime, Inc.) followed by direct sequenc-

ing from the plasmid, or PCR amplified directly from

colonies followed by purification and cycle sequencing.

Sequencing was performed using an Applied Biosystems

Model 373A Automated DNA Sequencing System. Di-

rect cycle-sequencing of purified template DNAs fol-lowed manufacturers specifications, using the PRISM

DyeDeoxy Terminator Kit (Perkin–Elmer).

Table 1

Taxa of Carex (abbreviated ‘‘C.’’) section Acrocystis and outgroups sampled in the Adh and combined analyses and their voucher information

Taxon Abbreviation Location and Voucher GenBank Accession

Nos. (Adh)

GenBank Accession

Nos. (ITS)

GenBank Accession

Nos. (ETS)

C. albicans Willd. ex Spreng. var. albicans 8150 West Virginia, Grant Co.; Reznicek 8150 (BRCH) *AY689017 (8150.2)1;

*AY689016 (8150.1)2*AY325479 *AY325454

1200 Missouri, Barry Co.; Morse 1200 AY689015 (1200.1)1 X X

C. albicans Willd. ex Spreng.

var. australis (L.H.Bailey) J. Rettig

1455 Mississippi, Washington Co.; Rettig 1455 (TAES) AY689018 (1455.1)1 X X

C. brainerdii Mackenzie 8588 California, Shasta Co.; Vincent 8588 *AY689014 (8588.3)2 *AY325485 *AY325460

C. brevicaulis Mackenzie ss Oregon, Lincoln Co.; Wilson s.n. (OSC) *AY688968 (ss5)1;

*AY688967 (ss3)2*AY325471 *AY325446

C. communis L.H.Bailey var. communis 1334 Canada, Quebec; Roalson 1334 *AY688988 (1334.1)1;

AY688966 (1334.3)4*AY325469 *AY325444

1344 Michigan, Alcona Co.; Roalson 1344 AY688993 (1344.3)1;

*AY688992 (1344.1)2X X

C. deflexa Hornem. var. boottii L.H.Bailey cl Oregon, Klamath Co.; Kuykendall s.n. (OSC) *AY689020 (cl.3)1;

*AY689013 (cl.2)2*AY686719 *AY686724

C. deflexa Hornem. var. deflexa 9701 Canada, Alberta; Ford 9701 (WIN) *AY689000 (9701.1)1;

*AY689001 (9701.3)2*AY686720 X

C. flacca Schreb. subsp. serrulata

(Biv.) Greuter [og]

Greece; Hartvig & Franzen 8709 X *AF284982 *AY325429

C. floridana Schw. 1288 Texas, Houston Co.; Roalson 1288 *AY688991 (1288.3)1 X X

6261 Texas, Jasper Co.; Jones 6261 (BRCH) X *AY325482 *AY325457

C. geophila Mackenzie 1408 Arizona, Cochise Co.; Roalson 1408 AY688977 (1408.1)1 X X

1409 Arizona, Cochise Co.; Roalson 1409 *AY688999 (1409.3)1;

*AY688998 (1409.1)2*AY325474 *AY325449

C. globosa Boott 1347 California, Monterey Co.; Roalson 1347 AY688984 (1347.2)1;

AY688981 (1347.6)2X X

12316 California, Marin Co.; Zika 12316 (OSC) *AY688979 (12316.12)1;

*AY688980 (12316.13)2*AY325487 *AY325462

C. inops L.H.Bailey subsp. inops lac Oregon, Klamath Co.; Kuykendall s.n. (OSC) *AY688983 (lac.2)1 *AY686721 X

4237 Oregon, Deschutes Co.; Halse 4237 (BRCH) *AY689006 (4237.1)2 X X

C. laxiflora Lam. [og] 1291 Texas, Upshur Co.; Roalson 1291 AY688971 (1291.3)2;

AY688959 (1291.1)6X X

C. lucorum Willd. ex Link var. lucorum 1327 Pennsylvania, Sullivan Co.; Roalson 1327 *AY688969 (1327.1)1;

*AY688970 (1327.2)2X X

1336 Ohio, Lucas Co.; Roalson 1336 X *AY325464 *AY325436

C. mandshurica Meinsh. [og] Korea, Kangwon Province; Tyson 5044 (POM) X *AF285045 *AY325432

C. novae-angliae Schw. 1333 Canada, Quebec; Roalson 1333 *AY689021 (1333.1)1 *AY325475 *AY325450

C. oxyandra Kudo 15896 Japan; Ikeda 15896 (OKAY) *AY689003 (15896.3)1;

AY688960 (15896.2)3;

AY688961 (15896.1)5

*AF285061 *AY325443

C. pauciflora Lightf. [og] s.n. Austria; Polatschek s.n. AY689022 (s.n.1)2 X X

C. peckii Howe 89-178 Minnesota, Clearwater Co.; McNeilus 89-178 (BRCH) *AY689007 (178.1)1 *AY325483 *AY325458

C. pensylvanica Lam. 1330 Canada, Quebec; Roalson 1330 *AY688995 (1330.3)2;

AY688994 (1330.1)2X X

1341 Michigan, Iosco Co.; Roalson 1341 X *AY686722 X

8142 West Virginia, Pendleton Co.; Reznicek 8142 (BRCH) *AY689004(8142.1)1 X X

(continued on next page)

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Table 1 (continued)

Taxon Abbreviation Location and Voucher GenBank Accession

Nos. (Adh)

GenBank Accession

Nos. (ITS)

GenBank Accession

Nos. (ETS)

C. pilulifera L. 805 Sweden, Uppland; Alm 805 *AY689023 (805.4)1 *AF284975 *AY325438

C. rossii Boott 1411 California, San Bernardino Co.; Roalson 1411 AY688973 (1411.1)2 X X

6864 Oregon, Lane Co.; Wilson 6864 (OSC) AY689011 (6864.1)1 X X

8166 California, Siskiyou Co.; Wilson 8166 (OSC) AY688974 (8166.9)2 X X

C. rossii Boott [1] cw Oregon, Benton Co.; Wilson s.n. (OSC) *AY689009 (cw.2)1;

*AY689010 (cw.4)2*AY325463 *AY325435

C. rossii Boott [2] 8101b California, Siskiyou Co.; Wilson 8101b (OSC) AY688976 (8101b.8)2 *AY325473 *AY325448

8107 California, Siskiyou Co.; Wilson 8107 (OSC) *AY688986 (8107.1)1;

*AY688989 (8107.2)2X X

C. serpenticola P.Zika 6803 California, Del Norte Co.; Wilson 6803 (OSC) *AY688978 (6803.15)2 X X

7631 Oregon, Curry Co.; Wilson 7631 (OSC) *AY688982 (7631.6)1 X X

12319 California, Marin Co.; Zika 12319 (OSC) X *AY325476 *AY325451

C. sp. nov. 3011 North Carolina, Buncombe Co.; Rothrock 3011 *AY689002 (3011.3)1;

AY688963 (3011.2)3;

AY688964 (3011.1)5

*AY325467 *AY325441

C. spissa L.H.Bailey [og] California, San Diego Co.; Tilforth & Wisura 2140 X *AF285040 *AY325431

C. tonsa (Fern.) E.P.Bicknell

var. rugosperma (Mackenzie) Crins

1332 Canada, Quebec; Roalson 1332 AY688996 (1332.1)1;

AY688997 (1332.3)2;

AY688962 (1332.2)5

X X

1340 Ohio, Lucas Co.; Roalson 1340 *AY688987 (1340.1)1;

*AY688990 (1340.3)2*AY686723 X

C. tonsa (Fern.) E.P.Bicknell var. tonsa 7047 Michigan, Van Buren Co.; Jones 7047 (BRCH) AY689008 (7047.3)1 X X

C. turbinata Liebm. [1] 1398 Mexico, Chihuahua; Laferriere 1398 (ARIZ) *AY689019 (1398.1)2 *AY325465 *AY325439

C. turbinata Liebm. [2]

(‘‘leucodonta’’-type)

1224 Arizona, Santa Cruz Co.; Roalson 1224 *AY688975 (1224.2)2 *AF284973 *AY325434

1383 Mexico, Chihuahua; Roalson 1383 AY689012 (1383.3)2 X X

C. umbellata Schkuhr ex Willd. [1] 8963 Michigan, Washtenaw Co.; Jones 8963 (BRCH) *AY689005 (8963.2)1 *AY325486 *AY325461

C. umbellata Schkuhr ex Willd. [2]

(‘‘microrhyncha’’-type)

1307 Texas, Burleson Co.; Roalson 1307 X *AY325472 *AY325447

1308 Texas, Brazos Co.; Roalson 1308 *AY688985 (1308.1)1;

AY688965 (1308.6)4X X

C. umbrosa Host subsp. sabynensis

Less. ex Kunth [og]

U.S.S.R. [Russia], Siberia; Murray et al. 344 X *AF285042 *AY325430

C. vulpinoidea Michx. [og] 1294 Texas, Morris Co.; Roalson 1294 AY689024 (1294.1)1;

AY688972 (1294.6)2X X

C. wahuensis C.A.Mey. subsp. robusta

(Fr. & Sav.) T.Koyama [og]

Japan, Shizuoka Pref.; Amano s.n. X *AF285023 *AY325433

Collections from USA unless otherwise noted. Specimens are deposited in RSA unless otherwise noted. The collection named ‘‘sp. nov.’’ is an individual that does not fit well in current species

circumscriptions and may be an undescribed species. Taxa used as outgroups in the various analyses are denoted by ‘‘[og]’’ following the species authority. Asterisks denote those sequences used in

the combined data Bayesian inference analysis. Numbers in parentheses following GenBank accession numbers are clone numbers from the figures and text and superscripts following the clone

numbers refer to Adh locus number as described in the figures and text.

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Table 2

Primers used for amplification and sequencing of Adh

Primer name Sequence

Adh2f 50-CKG CBG TGG CVT GGG AGG CMG GSA

AGC C-30

Adh2-1f 50-GAY GTC TWC TTY TGG GAA GCY A-30

Adh3f 50-CCW CGG ATC TTT GGT CAT GA-30

Adh3if 50-CTA TAC CCT TBG CTC TTC CA-30

Adh3ir 50-TGG AAG AGC VAA GGG TAT AG-30

Adh4xf 50-CAG AGG AGA GCA ACA TGT GTG A-30

Adh4f 50-TCC CGC TTC TCC ATC AAT GGC A-30

Adh4r 50-TGC CAT TGA TGG AGA AGC GGG A-30

Adh5f 50-TGG CAA TTT TTG GTC TGG GAG C-30

Adh5r 50-GCT CCC AGA CCA AAA ATT GCC A-30

Adh7r 50-YTC CGT GCA RCC AAA TTT CT-30

Adh8xr 50-CTC CAT TAG TCA TCT CAG CAA GA-30

Adh9-1r 50-YAC VCC GAC CAA AAC TGC CA-30

Adh9r 50-AAG TTC ATB GGR TGR GTC KTG AA-30

Refer to Fig. 1 for their annealing sites on the Adh gene.

Fig. 1. Diagram of the Adh gene in Carex. Exons and introns are abbreviated ‘‘ex’’ and ‘‘in,’’ respectively. The lines below the gene represent the two

regions sequenced, the longer fragment being approximately 2000 bp and the shorter approximately 1000 bp. Arrows indicate the locations and

directions of the PCR and sequencing primers. Primer sequences are listed in Table 2. Adh2f and Adh9r were used to amplify the 2000 bp fragment

and Adh4xf and Adh8xr were used to amplify the 1000 bp fragment.

E.H. Roalson, E.A. Friar / Molecular Phylogenetics and Evolution 33 (2004) 671–686 675

Due to difficulties in amplification of the 2000+ bp

fragment and problems placing internal primers withinthe large third intron for complete coverage, new prim-

ers located in exon 4 (Adhx4f) and exon 8 (Adhx8r) were

used for the bulk of the samples in these analyses (Fig. 1;

Table 2). These fragments were treated as described

above for the longer fragments. The large fragments

were sequenced using the plasmid primers T3 and T7

and the internal primers Adh 2-1f, Adh3f, Adh3if, Ad-

h3ir, Adh4f, Adh4r, Adh5f, Adh5r, Adh7r, and Adh9-1r (Table 2). The smaller fragment was sequenced using

the plasmid primers T3 and T7 and the internal primers

Adh5f, Adh5r, and Adh7r. For each individual the en-

tire cloned fragment was sequenced in three to nine

clones. In order to avoid having a large percentage of

missing data, all sequences were truncated to include

only the exon 4 to exon 8 sequence region.

Sequences were edited using Sequencher 3.0 (GeneCodes Corporation, Inc.) and aligned manually. Se-

quences obtained in this study have been assigned Gen-

Bank accession numbers as listed in Table 1.

2.3. Phylogenetic analyses

Coding regions of Adh sequences from throughout

the angiosperms and pines, including a subset of the

Carex Adh sequences, were analyzed using maximum

parsimony (MP), as implemented in PAUP*4.0b10

(Swofford, 2001), to test how the Carex sequences com-

pare to other sequenced angiosperms and infer the root

of the Carex sequences within a broader evolutionary

context. This tree was rooted using mammal and birdAdh sequences and a Lycopersicon esculentum short-

chain dehydrogenase (samples and GenBank accessions

in Table 3). This analysis utilized heuristic searches

(ACCTRAN; gaps treated as missing; starting trees ob-

tained via stepwise addition; addition sequence random

with 100 replicates; TBR branch-swapping; STEEPEST

DECENT). Clade support was estimated using 100 heu-

ristic bootstrap (bs) replicates (10 random addition cy-cles per replicate, 10 trees saved from each addition

cycle, TBR branch swapping, STEEPEST DESCENT;

Felsenstein, 1985; Hillis and Bull, 1993).

Relationships among all sequences of Carex Adh

were also inferred using MP (Swofford, 2001). Rooting

was based on the results of the angiosperm coding se-

quence analysis. This analysis utilized heuristic searches

(ACCTRAN; gaps treated as missing; starting trees ob-tained via stepwise addition; addition sequence random

with 1000 replicates; TBR branch-swapping; steepest de-

cent). Due to the large number of trees of equal length,

the number of trees saved per replicate was set to 500.

This allows a more thorough search of tree space in

the analysis given a limited amount of computer mem-

ory. Clade support was estimated using 100 heuristic

bootstrap replicates (10 random addition cycles per rep-licate, 10 trees saved from each addition cycle, TBR

branch swapping, STEEPEST DESCENT; Felsenstein,

1985; Hillis and Bull, 1993). Sequences considered as

outgroups to the Acrocystis sequences in this analysis

are the accessions of Carex laxiflora, C. pauciflora, and

C. vulpinoidea, all members of other subgenera or sec-

tions of Carex (Roalson et al., 2001).

Bayesian inference analysis of a combined ITS/ETS/Adh1/Adh2 Carex section Acrocystis dataset was per-

formed using MrBayes v.3.0 (Huelsenbeck and Ron-

quist, 2001). The four datasets of 30 taxa in this

analysis include some taxa with partial datasets. Missing

data are: ITS, none; ETS, Carex deflexa var. deflexa, C.

inops subsp. inops, C. pensylvanica, and C. tonsa var.

Table 3

GenBank accessions used in the angiosperm Adh coding sequence analyses

Family Taxa, GenBank Accession Nos.

Arecaceae Calamus usitatus, U58363; Phoenix reclinata A, U58362; Washingtonia robusta A, U65973;

Washingtonia robusta B, U65972.

Asteraceae Helianthus annuus, AX146927.

Brassicaceae Arabidopsis griffithiana, AB015504; Arabidopsis himalaica, AB015503; Arabidopsis korshinskyi, AB015505;

Arabidopsis suecica, AB015507; Arabidopsis thaliana, D63463; Arabidopsis wallichii, AB015506;

Arabis flagellosa, AB015500; Arabis gemmifera, D63459; Arabis glabra, AB015499; Arabis hirsuta, AB015502;

Arabis stelleri, AB015498; Arabis lyrata, AB015501; Brassica oleracea, AB015508; Leavenworthia crassa 1, AF037472;

Leavenworthia crassa 2, AF037510; Leavenworthia crassa 3, AF037559; Leavenworthia stylosa 1, AF037564;

Leavenworthia stylosa 2, AF037558; Leavenworthia stylosa 3, AF037560; Leavenworthia uniflora 1, AF037557;

Leavenworthia uniflora 2, AF037512; Leavenworthia uniflora 3, AF037561.

Fabaceae Glycine max 1, AF079058; Glycine max 2, AF079499; Phaseolus acutifolius 1, Z23171; Pisum sativum, X06281;

Trifolium repens 1, X14826.

Joinvilleaceae Joinvillea ascendens, U91623.

Malvaceae Gossypium barbadense A, AF085821; Gossypium barbadense C (sgA), AF036578;

Gossypium barbadense C (sgD), AF036570; Gossypium darwinii C (sgA), AF036579;

Gossypium darwinii C (sgD), AF036573; Gossypium hirsutum A, AF090164; Gossypium hirsutum C (sgA), AF036575;

Gossypium hirsutum C (sgD), AF036569; Gossypium mustelinum C (sgA), AF036577;

Gossypium mustelinum C (sgD), AF036572; Gossypium raimondii C, AF036568; Gossypium robinsonii C, AF036567;

Gossypium tomentosum C (sgA), AF036576; Gossypium tomentosum C (sgD), AF036571.

Paeoniaceae Paeonia anomala 1a, AF009046; Paeonia anomala 2, AF009064; Paeonia californica 1a, AF009041;

Paeonia californica 2, AF009056; Paeonia lactiflora 1a, AF009049; Paeonia lactiflora 2, AF009068;

Paeonia lutea 1a, AF009042; Paeonia lutea 2, AF009057; Paeonia rockii 1a, AF009045; Paeonia rockii 2, AF009063;

Paeonia suffruticosa subsp. spontanea 1a, AF009043; Paeonia suffruticosa subsp. spontanea 2, AF009060.

Pinaceae Pinus banksiana 1, U48366; Pinus banksiana 2, U48367; Pinus banksiana 3, U48368; Pinus banksiana 4, U48369;

Pinus banksiana 5, U48370; Pinus banksiana 6, U48371; Pinus banksiana 7, U48372.

Poaceae Anomochloa marantoidea 1, U91622; Anomochloa marantoidea 2, U91625; Arundo donax 1, U91619;

Bambusa glaucescens 1, U91626; Eragrostis japonica 1, U91620; Hordeum vulgare subsp. spontaneum 1, AF052664;

Hordeum vulgare 2, X12733; Hordeum vulgare 3, X12734; Lithachne humilis 1, U91624;

Muhlenbergia setarioides 1, U91621; Oryza sativa 1, X16296; Oryza sativa 2, X16297; Pennisetum americanum 1, X16547;

Pennisetum glaucum 1, M59082; Sorghum bicolor 1, AF050456; Tripsacum dactyloides 1, AF045548;

Zea luxurians 1, AF044307; Zea mays 1f, AF050457; Zea mays 1s, X04049; Zea mays 2n, X02915.

Rosaceae Malus domestica, Z48234; Fragaria x ananassa, X15588.

Solanaceae Lycopersicon esculentum 2, M86724; Lycopersicon esculentum 3a, S75487; Nicotiana tabacum, X81853;

Petunia hybrida 1, X54106; Petunia hybrida 2, U25536; Solanum tuberosum 1, M25154; Solanum tuberosum 2, M25153;

Solanum tuberosum 3, M25152.

Vitaceae Vitis vinifera, U36586.

Outgroups

(Mammals and Birds)

Apteryx australis subsp. australis 1, S78778; Homo sapiens 1, X03350; Mus caroli 1, M11307; Papio hamadryas, M25035;

Rattus norvegicus, M15327.

Numbers or letters following species names refer to loci designations from original publication. Comments in parentheses refer to the appropriate

subgenome (sg) in Gossypium.

676 E.H. Roalson, E.A. Friar / Molecular Phylogenetics and Evolution 33 (2004) 671–686

rugosperma; Adh1, C. brainerdii, C. flacca subsp. serru-

lata, C. mandshurica, C. spissa, C. turbinata [1], C. turbi-

nata [2], C. umbrosa subsp. sabynensis, and C. wahuensis

subsp. robusta; and Adh2, C. flacca subsp. serrulata, C.

floridana, C. mandshurica, C. novae-angliae, C. oxyandra,

C. peckii, C. pilulifera,C. sp. nov., C. spissa, C. umbellata

[1],C. umbellata [2],C. umbrosa subsp. sabynensis, andC.

wahuensis subsp. robusta. Some taxa included in thisanalysis include sequences concatenated from different

accessions of the same species (Table 1). Outgroups for

this analysis are based on results from previous analyses

(Roalson and Friar, 2004; Roalson et al., 2001) and in-

clude Carex flacca subsp. serrulata, C. mandshurica, C.

spissa, C. umbrosa subsp. sabynensis, and C. wahuensis

subsp. robusta. Six partitions were set to correspond with

the ITS, ETS, Adh1 exon, Adh1 intron, Adh2 exon, andAdh2 intron datasets. The parameters for each dataset

were allowed to vary independently (‘‘unlinked’’). Priors

for the six molecular dataset partitions included each

dataset with a separate model with variable substitution

types, rates, and invariant sites chosen based on the re-

sults of analysis using DT_ModSel (Minin et al., 2003;

ITS: TrN + I + G; ETS: TrN + G; Adh1 exons:

K80 + I; Adh1 introns: HKY; Adh2 exons: JC + I + G;

Adh2 introns: HKY + I). The DT_ModSel uses a Bayes-

ian information criterion to select a model using branch-length error as a performance measure in a decision the-

ory framework that also includes a penalty for overfitting

(Minin et al., 2003). One hundred million generations

were run with four chains (Markov Chain Monte Carlo),

and a tree was saved every 100 generations. The trees

from theMrBayes analysis were loaded into PAUP*, dis-

carding the samples from the ‘‘burnin’’ of the chain

(Huelsenbeck and Ronquist, 2001; the first 20,000,000generations or the first 200,001 samples) to only include

sample points after stationarity was clearly reached.

E.H. Roalson, E.A. Friar / Molecular Phylogenetics and Evolution 33 (2004) 671–686 677

Examination of likelihood plots suggest that stationarity

was reached for all parameters within the designated bur-

nin (data not shown). Additionally, multiple indepen-

dent runs (three) starting from different random trees

were conducted to determine if convergence and mixing

had occurred. A majority rule consensus tree was madefrom one of the analyses, showing nodes with a posterior

probability of 0.50 or more. Majority rule consensus

trees of the trees sampled in Bayesian inference analyses

yielded probabilities that the clades are monophyletic

(Lewis, 2001).

2.4. Molecular evolution

Tests of molecular evolution focused upon anoma-

lous sequences placed in clades outside of the two major

loci or placed basal to the divergence of all of the other

Adh loci. Eight sequences fell into this category (C. lax-

iflora 1291.1, C. oxyandra 15896.1, C. oxyandra 15896.2,

C. tonsa var. rugosperma 1332.2, C. sp. nov. 3011.1, C.

sp. nov. 3011.2, C. communis var. communis 1334.3,

and C. umbellata [2] 1308.6) and the sequences of thesesamples were compared with the entire Carex Adh data-

set in tests of recombination, using Plato 2.02 (Grassly

and Rambaut, 1998). Plato utilizes a sliding window of

varying size to find regions of sequences that deviate

from the null hypothesis of phylogenetic structure pro-

vided by a maximum likelihood model fit to the data

set (results of Modeltest 2.1 [Posada and Crandall,

1998]; data not shown) and phylogenetic tree. MonteCarlo simulations are implemented to test for significant

deviation from the null hypothesis.

The coding regions of the eight anomalous sequences

were also compared with six typical Acrocystis Adh cod-

ing sequences (C. laxiflora 1291.3, C. brevicaulis ss.3, C.

brevicaulis ss.5, C. lucorum var. lucorum 1327.1, C. luco-

rum var. lucorum 1327.2, and C. vulpinoidea 1294.6).

GCUA 1.1 (McInerney, 1998) was employed to test fordifferences of base composition among these sequences.

A resampling test for codon bias was conducted using

techniques described by Morton (1998). While Morton�stest (1998) explored codon adaptation, it was applied

here to measure strict codon bias. This test uses a ran-

dom codon usage distribution with 500 replicates and

compares the gene to this distribution. If the gene is

within two standard deviations from the mean, then biasis considered to be not significantly different from the

cumulative usage.

3. Results

3.1. Angiosperm Adh phylogenetic analysis

The aligned angiosperm Adh coding sequence data

matrix was 1173 bp long with 951 variable sites, of

which 807 were potentially parsimony-informative.

The sequences varied from 351 to 1170 bp in length.

There were 14 gaps ranging from 3 to 18 bp in length.

The length of the unaligned sequences varied in size be-

cause different regions were sequenced in different stud-

ies. Maximum parsimony analysis of the angiospermAdh coding sequence data set resulted in 120 most-par-

simonious trees. Figs. 2 and 3 show the strict consensus

of the 120 most-parsimonious trees.

This analysis provides a hypothesis of relationships of

Adh loci in angiosperms. While several internal nodes

that group major clades together are not well-supported,

many clades do have high bootstrap support (Figs. 2

and 3). For most angiosperm families, Adh loci coalescewithin the family, with the exception of the Arecaceae

and Solanaceae (Figs. 2 and 3). There is clear incongru-

ence between the angiosperm Adh strict consensus tree

and current hypotheses of relationships of angiosperms

(Soltis et al., 2000). Particularly, the dicot samples form

a grade leading to the monocots, the closely related fam-

ilies Fabaceae and Rosaceae are not placed together,

and the single Asteraceae sample (Helianthus annuus)is placed at the base of the angiosperm clade (Figs. 2

and 3). Carex samples were placed by this analysis in

their expected location as sister to the Poaceae + Join-

villeaceae (Chase et al., 2000), and they form a strongly

supported monophyletic group (bs = 91%; Fig. 3). While

there is clear incongruity of the Adh parsimony phylog-

eny with the expected relationships of angiosperm lin-

eages, we expect that this reflects the gene history, andis not a result of analysis-related issues, such as long

branch attraction. Maximum likelihood analyses of the

Adh dataset using a single model for the entire gene

(chosen by DT_ModSel) gives similar results as the par-

simony topology, as it suggests paraphyly of the eudi-

cots to the monocots and the coalescence of most loci

within families (Roalson, unpubl. data).

This preliminary tree suggests there are two mainclades of Carex Adh sequences, with the sequence C.

laxiflora 1291.1 sister to the rest of the Adh sequences.

If the C. laxiflora 1291.1 sequence was a member of

either of these clades, it would be expected to be near

the other outgroup sequences associated with one of

the two clades. As it does not, it was considered as a po-

tential separate locus sister to the rest of the Carex Adh

sequences and used as the outgroup for the more de-tailed Carex Adh sequence analyses. The C. laxiflora

1291.1 sequence was sister to the rest of the Carex

Adh sequences regardless of which other Carex se-

quences were included (data not shown).

3.2. Carex Adh phylogenetic analysis

The five Adh sequencing primers produced overlap-ping fragments that collectively covered the 50 end of

exon 4 to the 50 end of exon 8 along both strands for

Fig. 2. Maximum parsimony strict consensus (part 1) of 120 most-parsimonious trees for the angiosperm Adh coding sequences analysis

(length = 6531 steps, CI = 0.290, RI = 0.749, RC = 0.217). Numbers above or below the branches are bootstrap percentages. Generic epithets are

abbreviated as follows: A. = Arabidopsis; Ap. = Apteryx; Ar. = Arabis; B. = Brassica; G. = Gossypium; Gl. = Glycine; He. = Helianthus, Ho. =Homo;

L. = Leavenworthia; Ly. = Lycopersicon; Mu. = Mus; N. = Nicotiana; P. = Phaseolus; Pe. = Petunia; Pi. = Pisum; Po. = Papio; Pu. = Pinus;

R. = Rattus; So. = Solanum; and T. = Trifolium.

678 E.H. Roalson, E.A. Friar / Molecular Phylogenetics and Evolution 33 (2004) 671–686

the entire Carex Adh dataset. The aligned data matrix

was 978 bp long with 277 variable sites, of which 121

were potentially parsimony-informative. The length of

the unaligned sequences varied from 822 to 915 bp.

There were 31 gaps ranging from 1 to 93 bp in length.

Maximum parsimony analysis of the entire Carex Adh

dataset resulted in 106,000 most-parsimonious trees.

Fig. 4 is the strict consensus of these trees.

Although there are a large number of most-parsimo-

nious trees, this analysis supports two main clades of

Acrocystis Adh sequences, here referred to as Adh1

and Adh2 (Fig. 4). Each of these clades is defined by

being sister to one or more of the outgroup sequences.

The Adh1 clade is sister to C. vulpinoidea 1294.1 and

the Adh2 clade is sister to C. laxiflora 1291.3 (Fig. 4).

In addition to the two main clades, there are several

Acrocystis sequences placed outside of the outgroups

of these clades. Sister to the Adh1/C. vulpinoidea

1294.1 clade is a clade of two Acrocystis Adh sequences

(C. oxyandra 15896.2 and C. sp. nov. 3011.2). Thesecould potentially represent a third locus of Adh in Car-

ex. Sister to the Adh2/C. laxiflora 1291.3 clade is a clade

of two Acrocystis Adh sequences (C. communis 1334.3

and C. microrhyncha 1308.6). Sister to all of these is a

clade of three more Acrocystis Adh sequences (C. oxyan-

dra 15896.1, C. rugosperma 1332.2, and C. sp. nov.

3011.1). These two clades could potentially represent a

fourth or a fourth and fifth locus. Finally, the C. laxifl-

Fig. 3. Maximum parsimony strict consensus (part 2) of 120 most-parsimonious trees for the angiosperm Adh coding sequences analysis

(length = 6531 steps, CI = 0.290, RI = 0.749, RC = 0.217). Numbers above the branches are bootstrap percentages. Generic epithets are abbreviated

as follows: An. = Anomochloa; Ao. = Arundo; Ba. = Bambusa; C. = Carex; Ca. = Calamus; E. = Eragrostis; F. = Fragaria; H. =Hordeum; J. = Join-

villea; Li. = Lithachne; M. = Muhlenbergia; Ma. = Malus; O. = Oryza; Pa. = Paeonia; Ph. = Phoenix; Pn. = Pennisetum; S. = Sorghum; Tr. = Tripsa-

cum; V. = Vitis; W. = Washingtonia; and Z. = Zea. Specific and subspecific epithets are abbreviated as follows: H. vulgare subsp. spon. = H. vulgare

subsp. spontaneum, and Pa. suff. subsp. spon. = Pa. suffruticosa subsp. spontanea.

E.H. Roalson, E.A. Friar / Molecular Phylogenetics and Evolution 33 (2004) 671–686 679

ora 1291.1 sequence used as the outgroup for this anal-

ysis appears to predate the divergence of any of the

other duplication events, possibly indicating it is a fifth

or sixth locus of Adh in Carex.

The data presented here suggest that there may be

two or three times as many Adh loci in Carex than in

some other groups studied. Given the fact that most

Carex species are thought to be diploid (Davies, 1956),this presents a complex scenario of gene family evolu-

tion in the group. While there is the possibility that addi-

tional sequences within species could be the result of

PCR error, we do not expect this to be likely as all of

the sequence types were found in multiple cloning

events. Verification of Adh locus number will require

Southern blot analysis and localization of loci on genetic

or physical maps. More detailed studies in a broader

phylogenetic context will be necessary to explore the

evolution of the Adh gene family in the Cyperaceae.

Only the Carex Adh1 and Adh2 clades have enough

samples to be used to infer species relationships, so fur-ther discussions will focus on these putative loci. Reso-

lution within the Adh1 and Adh2 clades is very low,

with few nodes within the clades supported in the strict

consensus tree (Fig. 4). The Adh1 locus does resolve sev-

Fig. 4. Maximum parsimony strict consensus of 106,000 most-parsimonious trees for the Carex Adh sequence analysis (length = 459 steps,

CI = 0.697, RI = 0.928, RC = 0.647). Numbers above the branches are bootstrap percentages. All species in the tree are members of the genus Carex.

Numbers in brackets following Carex turbinata and C. umbellata refer to the two different ‘‘types’’ recognized within these species, as noted in Table

1. Numbers following species names not in brackets are collection abbreviations and clone numbers.

680 E.H. Roalson, E.A. Friar / Molecular Phylogenetics and Evolution 33 (2004) 671–686

eral of the species from eastern North America forming

a clade (C. albicans var. albicans, C. albicans var. aus-

tralis, C. floridana, C. lucorum var. lucorum, C. pensylva-nica, C. tonsa var. rugosperma, and C. umbellata) as has

been found previously using nrDNA spacer sequences

(ITS + ETS; Roalson and Friar, 2004). The Adh2 locus

also resolves a clade of eastern North American species

and a clade of some of the western North American spe-cies (eastern: C. albicans var. albicans, C. communis var.

communis, C. deflexa var. deflexa, C. lucorum var. luco-

E.H. Roalson, E.A. Friar / Molecular Phylogenetics and Evolution 33 (2004) 671–686 681

rum, C. pensylvanica, and C. tonsa var. rugosperma; wes-

tern: C. geophila, and C. rossii, and C. turbinata; Fig. 4).

3.3. Molecular evolution

Two primary clades of sequences–putative loci–werefound in Carex, which we refer to as Adh1 and Adh2.

Additionally, several samples were placed outside of

the two putative loci and could represent additional loci.

The introns of the two major loci were alignable, but

had distinctive gaps associated with each locus. Both

loci were not found for all samples, but no distinct pat-

tern of which samples had which loci is evident. More

than half of the species were found with both loci,although sometimes in different samples. This implies

that either there has been random loss of copies of these

loci, or that sampling was not sufficient to find both loci

in all samples. Additionally, eight sequences were found

outside of the two main Adh clades from five Acrocystis

species and one outgroup species. These eight sequences

are further explored in Molecular Evolution Results and

the Discussion.Statistical tests for recombination utilizing Plato 2.11

found no evidence of recombination. This was consis-

tent given different window sizes (5–50 bp) and numbers

of replicates (100–500) for the Monte Carlo simulations.

Estimates of base composition using GCUA 1.1

found no differences in the base composition of the

anomalous and typical sequences in both exon GC con-

tent and intron GC content (Table 4). The sequenceshave approximately 50% GC content in exons and

29% GC content in introns.

The resampling tests applied found all of the gene se-

quences to be statistically homogeneous with respect to

codon usage starting at codon 85 (codons 1–84 were ex-

cluded because they were not represented in all samples).

This suggests that there are no real differences in codon

Table 4

Percent GC content of a subset of Carex Adh sequences including

anomalous sequences (marked with an asterisk)

Sequence Exon GC Intron GC

C. communis var. communis 1334.3* 51.20 30.91

C. sp. nov. 3011.1* 51.60 26.80

C. sp. nov. 3011.2* 52.23 29.34

C. laxiflora 1291.1* 48.20 30.67

C. umbellata 1308.6* 50.62 30.04

C. oxyandra 15896.1* 51.60 27.54

C. oxyandra 15896.2* 51.40 28.92

C. tonsa var. rugosperma 1332.2* 51.40 26.80

C. brevicaulis ss.3 50.62 29.93

C. brevicaulis ss.5 49.38 27.81

C. laxiflora 1291.3 50.62 28.82

C. lucorum var. lucorum 1327.1 49.38 27.59

C. lucorum var. lucorum 1327.2 51.56 29.46

C. vulpinoidea 1294.6 50.93 29.43

usage among the different putative loci and anomalous

sequences and does not support any suggestion of selec-

tion, at least in this segment of the coding region. Addi-

tionally, none of the anomalous sequences was found to

have stop codons, suggesting they may not be

pseudogenes.

3.4. Combined ITS/ETS/Adh1/Adh2 Bayesian analysis

In the Bayesian inference analyses, plots of log-like-

lihood scores and all other parameters reach stationa-

rity prior to generation 20,000,000 in all independent

analyses (plots not shown). The first 200,001 sample

points were thus discarded as burn-in, leaving800,000 samples for construction of a 50% majority

rule consensus tree. Fig. 5 illustrates the 50% majority

rule consensus tree from one of the runs (results from

this run used in all further results and discussions). All

three independent analyses resulted in similar posterior

probability distributions and therefore we expect con-

vergence and mixing is occurring (data not shown).

Fifty-two percent of nodes have a posterior probabilityP95%.

Some taxa included in this analysis are missing por-

tions of the total matrix and some species sequences

are combinations of sequences from different accessions

within the species (see Section 2 and Table 1 for details).

While neither of these conditions are optimal for phy-

logeny reconstruction, we believe they have minimal im-

pact on the phylogenetic analyses presented here. Testanalyses with different combinations of taxa with miss-

ing data excluded did not significantly affect the general

tree topology (data not shown). Combining sequences

from different accessions of the same species is often

done (e.g., Michelangeli et al., 2003; Soltis et al.,

2003), and the most critical issue involved with this prac-

tice is confidence that both accessions truly belong to the

same species. Where sequences from different collectionsof a species were combined, we very carefully verified

species identification, and where species circumscription

is problematic, samples were only combined when their

source populations were similar morphologically and in

close geographic proximity.

While the sampling between this combined ITS/ETS/

Adh1/Adh2 analysis and previously published ITS/ETS

analyses is somewhat different, the trees can be gener-ally compared. The Bayesian inference majority rule

consensus tree is congruent with the maximum parsi-

mony strict consensus phylogeny (Roalson and Friar,

2004). There is some difference in internal node struc-

ture between the previously published maximum likeli-

hood topology and this Bayesian inference phylogeny,

but these differences are associated with very short

branches in the maximum likelihood tree, nodes withlow posterior probability values, or both (Roalson

and Friar, 2004).

Fig. 5. Bayesian inference majority rule tree. Numbers above branches are posterior probability values. Numbers in brackets following Carex

turbinata and C. umbellata accessions refer to the two different ‘‘types’’ recognized within these species, as noted in Table 1 and the text. Numbers in

brackets following Carex rossii samples refer only to two different composite sequences included in this analysis. Boxes to the right of species names

refer to previously defined species complexes or ‘‘orphan’’ species and are abbreviated as follows: D = the deflexa complex; N = the nigromarginata

complex; O = orphan species (see Roalson and Friar, 2004 for discussion); P = the pensylvanica complex; and U = the umbellata complex.

682 E.H. Roalson, E.A. Friar / Molecular Phylogenetics and Evolution 33 (2004) 671–686

4. Discussion

4.1. Evolution of the Adh gene family

These results suggest that there are two major loci of

the Adh gene family, Adh1 and Adh2, in Carex. This is

consistent with the presence of two or three Adh loci in

most angiosperms. Contrary to other phylogenetic stud-

ies of angiosperm groups (Paeonia, Sang et al., 1997;

Leavenworthia, Charlesworth et al., 1998; Gossypium,Small et al., 1998; Poaceae, Gaut et al., 1999), Carex

has additional putative loci that form clades outside of

the two major loci or are placed basal to the divergence

of all other Adh loci (Figs. 2–4). Recent studies of Adh

gene family structure (Gossypium; Small and Wendel,

2000b) have also found a more complex gene family

structure than previously thought, with as many as se-

ven Adh loci present in some diploid Gossypium species.

E.H. Roalson, E.A. Friar / Molecular Phylogenetics and Evolution 33 (2004) 671–686 683

The phylogenetic estimations shown in Figs. 2–4 pro-

vide support that the duplication event leading to the

Adh1 and Adh2 copies predated the diversification of

Carex, but did not predate the divergence of Carex from

other graminoid monocots. Until Adh of other Cypera-

ceae genera are sequenced, it will remain unclear if theduplication is Carex-specific or associated with a broad-

er group of Cyperaceae genera. There is no evidence of

within-locus variation within species (data not shown).

Analysis of angiosperm Adh sequences also provides

some insight into the birth–death process of loci in the

Adh gene family. Some have suggested that there is a

small, stable number of loci in the Adh gene family in

which there are constraints on gene copy number anda slow flux of gene duplication and loss leading to a dy-

namic equilibrium of copy number (Clegg et al., 1997).

The angiosperm Adh analysis provides evidence that loss

and gain of loci might be more common than previously

suggested at least in some groups (Figs. 2 and 3). For in-

stance, the Adh loci of Leavenworthia coalesce after the

divergence of Leavenworthia from Arabis, which are clo-

sely related genera (Al-Shehbaz, 1988). While many ofthe loci appear to be of relatively recent origins and coa-

lesce within genera or families, this is not true for all

loci. Some, particularly those in the Arecaceae and Sola-

naceae, appear to coalesce at deep nodes in the tree, sug-

gesting that there has also been persistence of ancestral

Adh loci in some lineages. Additionally, the incongru-

ence of the Adh phylogeny with well-supported relation-

ships in the angiosperms may suggest additional deepcoalescence of some Adh loci, particularly the placement

of the Asteraceae sample at the base of the angiosperms,

the grade of the dicots leading to the monocots, and the

distant relationship of Adh loci from the closely related

families Rosaceae and Fabaceae (Figs. 2 and 3; Soltis

et al., 2000). These results suggest that there may be a

combination of rapid gene family homogenization as

well as long term persistence of gene family members indifferent lineages of the angiosperms. Alternatively,

other molecular evolutionary processes (e.g., signal satu-

ration) could be acting to obscure the ‘‘true’’ phyloge-

netic signal of the Adh gene family. Furthermore,

analysis of the Carex Adh loci (Fig. 4) suggests that there

may be more than the two or three loci typically found in

angiosperms, as has been found in pines (Perry and Fur-

nier, 1996) and Gossypium (Small and Wendel, 2000b).

4.2. Molecular evolution of Carex Adh loci

The individuals that possess anomalous sequences

(Adh loci 3–6) also have sequences that fall within the

Adh1 and Adh2 loci. Carex oxyandra 15896 and C. sp.

nov. 3011 sequences are found in the Adh3 and Adh5

and the Adh2 locus. Carex rugosperma 1332 has se-quences in the Adh5 clade as well as both the Adh1

and Adh2 loci. If these groups of sequences do represent

additional loci, it is likely that either Carex has more

than the expected two or three Adh loci and copies of

each locus have yet to be sequenced, or there is a com-

plicated pattern of gain and loss of loci in this group.

Since there is no evidence that these anomalous se-

quences are recombining with members of the Adh1and Adh2 loci, and the typical Adh copies form mono-

phyletic groups, the usefulness of the Adh1 and Adh2

loci as a phylogenetic marker merits further discussion.

The studies of molecular evolution of the Carex Adh

sequences support the idea that all sequences studied

here are functional and not under radically different

selection regimes (no evidence of recombination, similar

base composition, similar codon usage biases, and nostop codons). This is different from the results found

studying molecular evolution of Adh genes in some

other groups (e.g., Poaceae; Gaut et al., 1999). Some

have suggested that with gene family expansion there

is likely functional diversification, which can often be

seen in the different genetic structure of divergent loci

(Gaut et al., 1999). While there has apparently been

duplication and divergence of copy number in the CarexAdh gene family, we do not see any evidence of changes

in selective constraints on different loci.

4.3. Phylogenetic relationships in Carex section acrocystis

The analyses of Acrocystis Adh sequences do not pro-

vide well-resolved phylogenetic hypotheses of intra-sec-

tional relationships. As with ITS/ETS (Roalson andFriar, 2004), species with multiple samples do not coa-

lesce, with the exception of the C. globosa Adh1 and

Adh2 sequences and the C. communis Adh1 sequences

(Fig. 4). Non-coalescence of Adh sequences within spe-

cies has been found in other angiosperms as well (Small

et al., 1999). In some cases, this lack of coalescence is

due to a lack of sequence variation among samples

(apparently most cases fall into this category), while inothers there may be a persistance of ancestral polymor-

phisms (particularly, C. pensylvanica, C. tonsa var. rugo-

sperma, and C. umbellata; Fig. 4). There is a clade of at

least some eastern North American species supported by

both the Adh1 and Adh2 analyses, as was found with

ITS/ETS.

While the Adh sequences provide less phylogenetic

signal than might be desired, there is sequence variationand some phylogenetic structure. In order to better ad-

dress species relationships within Acrocystis, the two pri-

mary Adh loci, Adh1 and Adh2, were combined with

sequences from the nuclear ribosomal DNA internal

and external transcribed spacer regions (ITS and ETS).

These four datasets were then analyzed using Bayesian

inference analyses in order to apply model-based ap-

proaches and allow each of the datasets or dataset par-titions to be adequately modeled for the analysis. The

results of this analysis continue to support a similar tree

684 E.H. Roalson, E.A. Friar / Molecular Phylogenetics and Evolution 33 (2004) 671–686

topology as previously supported by ITS and ETS

alone, but with greater resolution and better statistical

support for several nodes (Fig. 5; Roalson and Friar,

2004).

Several species as currently defined appear to be para-

or polyphyletic (Fig. 5), particularly, Carex deflexa, C.rossii, C. turbinata, and C. umbellata. Since most species

are represented by a single exemplar, species boundaries

of all species cannot be addressed here. The Bayesian

analysis presented here suggests that Carex deflexa

may be polyphyletic as currently circumscribed (Crins

and Rettig, 2003). Samples of C. deflexa var. deflexa

and C. deflexa var. boottii are here included in the same

analysis for the first time, and they clearly do not form amonophyletic group (Fig. 5). Additionally, they are sep-

arated by at least one strongly supported node (poster-

ior probability = 99%). At times C. deflexa var. boottii

has been considered a separate species (C. brevipes),

and this might be a more appropriate treatment until

more detailed studies of this group are conducted. Carex

rossii has been previously suggested to possibly be poly-

phyletic (Roalson and Friar, 2004) and those results arenot refuted here with the addition of Adh sequences.

Carex turbinata, as currently circumscribed (Crins

and Rettig, 2003), has often been recognized as two spe-

cies: C. turbinata and C. leucodonta. Accessions repre-

senting both of these types were included here ([1] and

[2]; Table 1) and they do not form a monophyletic group

(Fig. 5). Additionally, they are separated by at least one

moderately well-supported node in this analysis. Simi-larly, the circumscription of Carex umbellata has re-

cently been changed to include C. microrhyncha (Crins

and Rettig, 2003). With the inclusion of C. microrhyncha

in C. umbellata, C. umbellata is made paraphyletic with

regards to C. tonsa var. rugosperma (Fig. 5). As C. tonsa

var. tonsa is not included here, its position cannot be ad-

dressed, but it is clear that species circumscriptions

among these species need to be revisited.Systematic studies of Carex section Acrocystis by sev-

eral authors (Crins and Ball, 1983; Mackenzie, 1913a,;

Rettig, 1990; Rettig and Giannasi, 1990; B. Ford, Uni-

versity of Manitoba, ongoing studies, pers. commun.)

have primarily focused on several species complexes

(the deflexa complex; the nigromarginata complex; the

pensylvanica complex; and the umbellata complex).

Whether these species complexes circumscribe mono-phyletic groups or evolutionary lineages has been re-

cently called into question (Roalson and Friar, 2004),

and the results presented here lend support to the non-

monophyly of many of these informal groupings (Fig.

5). Particularly, the nigromarginata complex appears

to be polyphyletic, with none of the sampled species

from the complex grouping with any other species from

the complex (Fig. 5). Unfortunately, previous systematicstudies of the nigromarginata complex (Rettig, 1990;

Rettig and Giannasi, 1990) have been conducted with-

out sampling any other species in the section, so their re-

sults are difficult to compare to the results presented

here.

Alternatively, many of the taxa considered part of the

deflexa complex form a clade, although to the exclusion

of C. deflexa var. deflexa itself as well as C. geophila

(Fig. 5). Several other species previously referred to as

‘‘orphan species’’ (see Roalson and Friar, 2004) and a

member of the pensylvanica complex (C. inops subsp. in-

ops) are clearly related to these members of the deflexa

complex, and so the circumscription of this informal

group would have to be changed considerably to achieve

monophyly. Carex deflexa var. deflexa is here suggested

to form a clade with C. communis var. communis (or-phan species) and C. peckii (nigromarginata complex;

Fig. 5) – a novel suggestion. While morphologically

these species are quite different (hence their placement

in different informal groupings), they all have similar

geographic distributions (Crins and Rettig, 2003). Carex

deflexa var. deflexa and C. peckii, particularly, have sim-

ilar distributions across the northern latitudes of Can-

ada, the northern midwest US, and northeastern US,and these distributions significantly overlap with C.

communis var. communis in the midwest and northeast-

ern US and eastern Canada (Crins and Rettig, 2003).

An alternative hypothesis to the close relationship of

these three species that should be noted (but cannot be

directly addressed here) is that past or current introgres-

sion of these species could lead to the pattern of generic

similarity seen here. We do not consider this likely,though, as hybridization among these species have never

been documented or suggested (Cayouette and Catling,

1992). The potential relationship of these three species

needs to be further explored.

The pensylvanica complex is here represented by

three of the five taxa currently recognized (Roalson

and Friar, 2004). Our analyses clearly suggest that the

western North American C. inops subsp. inops is not clo-sely related to the eastern North American C. lucorum

var. lucorum and C. pensylvanica (Fig. 5). Previous anal-

yses suggested that C. inops subsp. heliophila was rela-

tively closely related to C. lucorum var. lucorum, and

so the monophyly of C. inopsmay be suspect. This needs

to be further explored with all species from the complex.

Interestingly, C. lucorum var. lucorum and C. pensylva-

nica group closely with C. novae-angliae and C. sp.nov. (posterior probability = 96%; Fig. 5). As a close

relationship among these species has not been previously

suggested, potential morphological characteristics sup-

porting this grouping need to be explored.

Of all of the previously recognized species complexes,

the umbellata complex is the only informal group that

appears to form a monophyletic clade (Fig. 5). As not

all taxa recognized in the complex have been sampled(e.g., C. tonsa var. tonsa), we can not unequivocally pro-

nounce its monophyly. Additionally, as previously dis-

E.H. Roalson, E.A. Friar / Molecular Phylogenetics and Evolution 33 (2004) 671–686 685

cussed, species boundaries are yet problematic in this

group.

5. Conclusions

Exploration of alcohol dehydrogenase gene family

structure in Carex section Acrocystis suggests a more

complicated locus structure than has been found in sev-

eral other groups (Paeonia, Sang et al., 1997; Brassica-

ceae, Charlesworth et al., 1998; Poaceae, Gaut et al.,

1999), with potentially as many as six Adh loci present

in the genus (Fig. 4). Divergence within loci among taxa

in Carex section Acrocystis is relatively low, athoughphylogenetic signal is present. In combination with

nrDNA ITS and ETS sequences analyzed using Bayes-

ian inference techniques, the Adh datasets continue to

support the non-monophyly of some species and species

complexes suggested previously (Roalson and Friar,

2004). More detailed studies of lineages in the section

need to be further explored with more detailed sampling

and more variable markers to conclusively define lin-eages in this group of sedges.

Acknowledgments

The authors thank J. Travis Columbus, Bruce Ford,

Brandon Gaut, Takuji Hoshino, Stanley Jones, Brian

Morton, J. Mark Porter, Tony Reznicek, Julian Starr,Barb Wilson, and Richard Whitkus for helpful discus-

sions, research advice, plant material, and help with field

work; J. Travis Columbus, J. Mark Porter, and two

anonymous reviewers for comments on previous ver-

sions of the manuscript; and RSA, TAES, and BRCH

for allowing access to herbarium material. Financial

support was provided by National Science Foundation

Dissertation Improvement Grant DEB-9801495, Ran-cho Santa Ana Botanic Garden, the A.W. Mellon Foun-

dation, the Hardman Foundation, and the Washington

State University School of Biological Sciences. Portions

of the paper are partial fulfillment of the requirements

for a Doctor of Philosophy degree for EHR, Claremont

Graduate University.

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