A phylogenetic analysis of Leucothoe s.l. (Ericaceae; tribe Gaultherieae) based on phenotypic...

A phylogenetic analysis of Leucothoe s.l. (Ericaceae; tribeGaultherieae) based on phenotypic characters

KATHERINE WASELKOV AND WALTER S. JUDD

Department of Botany, University of Florida, 220 Bartram Hall, P. O. Box 118526, Gainesville, FL32611-8526, USA; e-mail: [email protected]

Abstract. Phylogenetic relationships within the genus Leucothoe s.l. (including all eightspecies) and related taxa of the Gaultherieae, Andromedeae, and Vaccinieae wereinvestigated by a cladistic analysis based on phenotypic (external morphology, anatomy,chromosome number, and secondary chemistry) characters. The parsimony analysisresulted in two most parsimonious trees, both very similar, which show Leucothoe s.l. tobe polyphyletic, with its species distributed among three distinct clades. Our resultsindicate that L. racemosa and L. recurva form a strongly supported clade, which is sisterto Chamaedaphne calyculata, and these three species are probably the sister-group ofthe wintergreen clade (consisting of Gaultheria and Diplycosia). Leucothoe axillaris, L.fontanesiana, L. davisiae, L. griffithiana, and L. keiskei, consistently form amonophyletic group corresponding to Leucothoe s.s., which is probably sister to theremaining members of the tribe Gaultherieae. Leucothoe grayana, the final speciestraditionally placed in the genus, belongs to neither of these clades and may be sister toAndromeda. Phenotypic characters provide no support for the monophyly of Leucothoe,instead suggesting that it is polyphyletic, in agreement with preliminary DNA-basedanalyses. Thus, we redefine the genus Leucothoe, placing its species into three genera:1) Eubotrys (the E. racemosa + E. recurva clade), 2) Leucothoe s.s. (the L. axillaris + L.fontanesiana + L. davisiae + L. griffithiana + L. keiskei clade), and 3) Eubotryoides(containing only E. grayana).

Key Words: Ericaceae, Eubotrys, Eubotryoides, Gaultherieae, Leucothoe.

Leucothoe D. Don (eight species, tribeGaultherieae, subfam. Vaccinioideae; see Kronet al., 2002) is a heterogeneous group, themonophyly of which has recently been ques-tioned (Kron et al., 1999a, b, 2002; Powell &Kron, 2001). The characters by which thegenus has traditionally been recognized are theserrate-margined leaves that lack a hypoder-mis; axillary, racemose inflorescences devel-oping from buds formed in the fall (and thuswith meiosis occurring in the fall, and theinflorescences overwintering in an “exposed”condition; but inflorescences emerging in thespring in L. grayana Maxim.); green calyx,turning brown and becoming dry in fruit (i.e.,not becoming colorful and fleshy as inGaultheria Kalm ex L. and DiplycosiaBlume); anthers with two or four minute to

well developed awns (but absent in L.grayana) and disintegration tissue on eachtheca; superior ovaries with numerous ovules;and loculicidal capsules releasing seeds withbulging, flattened marginal cells, which form adistinct to indistinct wing [but a wing lackingin L. racemosa (L.) A. Gray] (Sleumer, 1959;Stevens, 1969, 1971; Luteyn et al., 1996). Allof these features are variable, occurring inother genera of the Gaultherieae, Androme-deae, or Lyonieae. Leucothoe has a broadgeographic distribution, with L. axillaris(Lam.) D. Don and L. racemosa occurringpredominantly in the coastal plain of theeastern United States; L. fontanesiana (Steud.)Sleumer and L. recurva (Buckley) A. Gray inthe Appalachians; L. davisiae Torr. ex A. Grayin the Coast Range, Klamath Mountains, and

Brittonia, 60(4), 2008, pp. 382–397 ISSUED: 1 December 2008© 2008, by The New York Botanical Garden Press, Bronx, NY 10458-5126 U.S.A.

Sierra Nevada from Oregon to California; L.griffithiana C. B. Clarke in the Himalayas andeastward through mountainous areas of south-ern China and Indochina; and L. grayana andL. keiskei Miq. in the mountains of Japan.The early-discovered, temperate species (i.e.,

L. axillaris, L. fontanesiana, L. racemosa andL. recurva) were originally described withinAndromeda L. because they have urceolate-cylindrical flowers with superior ovaries andcapsular fruits. The process of breaking up thisbroadly circumscribed Linnaean genus beganwith D. Don (1834), who described Leucothoe(for L. axillaris), and several other genera nowplaced in the tribes Gaultherieae or Lyonieae.As similar species were discovered, they wereadded to Leucothoe as follows (using theircurrent specific epithets): L. racemosa andL. recurva (in 1856), L. keiskei (in 1863), L.davisiae (in 1868), L. grayana (in 1872), L.griffithiana (in 1882), and L. fontanesiana(in 1959; previously known as L. catesbaei(Walter) A. Gray, 1856), (see Sleumer, 1959;Melvin, 1980). These temperate taxa wereconsistently placed within a broadly circum-scribed Leucothoe from the late 1800s torecent treatments (Hooker, 1876; Gray, 1878;Niedenzu, 1889; Drude, 1897; Sleumer, 1959;Melvin, 1980; Luteyn et al., 1996).Starting with de Candolle (1839), a group

of tropical species was also usually includedin Leucothoe, as sect. Agastia DC. (neotrop-ical species) or sect. Agauria DC. (paleotrop-ical species). The species of these sections arenow recognized as not closely related to thetemperate species of Leucothoe, but insteadare members of the Lyonieae (Kron & Judd,1997; Kron et al., 1999b, 2002); they are hereconsidered generically distinct and placed inAgarista (with two sections: Judd, 1979,1984; Kron et al., 2002; Stevens et al., 2004).The deciduous North American species of

Leucothoe have long been considered distinctfrom the evergreen species. In fact, Nuttall(1843) proposed the genus Eubotrys (for thespecies now known as L. racemosa) on thebasis of its deciduous habit and distinctiveracemose inflorescences (of five-merous cy-lindric-subovate flowers with four-awnedanthers). Britton and Brown (1913) alsorecognized Eubotrys Nutt. as distinct fromLeucothoe, and placed Leucothoe recurva in

this taxon, breaking up Gray’s (1856) morebroadly defined Leucothoe. The genus wassegregated further when Small (1914) recog-nized three genera among the North Ameri-can species of Leucothoe on the basis ofinflorescence position, awned vs. awnlessanthers, calyx shape, and thickness of thecapsule wall. He recognized Eubotrys, con-taining L. racemosa and L. recurva; Leuco-thoe s.s., containing L. axillaris and L.fontanesiana; and a new genus, Oreocallis,containing L. davisiae. Oreocallis Small wasrecognized because of its erect stems, distinc-tive inflorescence position (i.e., towards theend of the branches), and minor differences inbracteole position and shape of calyx lobes.Nakai (1922) proposed sect. Eubotryoides,for L. grayana, and this name was later raisedto generic rank by Hara (1935), based on thenumerous distinctive features of this species(especially its inflorescence form. with avegetative proximal portion bearing leaf-likebracts, urceolate corolla, pubescent filaments,awnless anthers, and capsules with thin-walled valves). Additionally, he noted that ithad deciduous leaves, a condition also char-acteristic of Eubotrys. Finally, L. keiskei wastransferred by Honda (1949) to the newlyerected genus Paraleucothoe, a name basedon sect. Paraleucothoe Nakai (1922), reflect-ing the elongate pedicels, tubular corolla,pubescent filaments, and long, paired antherawns of this species.Sleumer (1959) first treated all the temperate

species and proposed an infrageneric classifi-cation based on leaf persistence, inflorescenceposition and length, bracteole position, antherawn length, and stigma shape. While hemaintained the group as a single genus, hesplit it into six sections: Leucothoe sect.Leucothoe (L. axillaris and L. fontanesiana);sect. Acranthes Sleumer (L. davisiae); sect.Oligarista Sleumer (L. griffithiana and L.tonkinensis Dop, the latter here consideredconspecific with L. griffithiana; see alsoStevens, 1969, and Melvin, 1980); sect. Paral-eucothoe Nakai (L. keiskei); sect. EubotryoidesNakai (L. grayana); and sect. Eubotrys (Nutt.)A. Gray (L. racemosa and L. recurva). Stevens(1969) recognized the same sections of Leu-cothoe, and mentioned many of the morpho-logical and anatomical characters noted by

383WASELKOV & JUDD: PHYLOGENETICS OF LEUCOTHOE2008]

Sleumer, but also added characters discoveredby Palser (1951, 1952), Lems (1962, 1964),and himself. Wood (1961) studied all eightspecies of Leucothoe and concluded that theseparation of the evergreen species into foursections obscured the relationships between theNorth American and Eastern Asian species,and therefore, he combined sects. Leucothoe,Paraleucothoe, Acranthes, and Oligarista intoa single section, Leucothoe. However, hecontinued to recognize sects. Eubotrys andEubotryoides. Melvin (1980) partitioned thespecies into these same three sections based ona division of the group into evergreen anddeciduous species, with a further recognition oftwo deciduous sections due to divergence of L.grayana from L. racemosa and L. recurva.Middleton (1991a) also concurred with Wood’ssubdivisions, based on phenetic considerations.It is noteworthy that Melvin (1980), the mostrecent monographer of this group, retained alleight of Sleumer’s extratropical species withinLeucothoe. A broad circumscription alsohas been followed in several recent floras(Fernald, 1950; Ohwi, 1965; Radford et al.,1968; Godfrey & Wooten, 1981; Hickman,1993; Luteyn et al., 1996; Wunderlin &Hansen, 2003).Molecular (i.e., rbcL and matK sequences)

and morphological/molecular phylogeneticanalyses of the Andromedeae by Kron et al.(1999b) supported Chamaedaphne as sister toL. racemosa and showed L. fontanesiana assister to the other genera of the Gaultheriagroup (now the Gaultherieae). The monophy-ly of Leucothoe (as assessed by inclusion ofonly L. fontanesiana and L. racemosa) wasnot supported in all of the morphology-basedtrees. These results appeared again in analy-ses based on more extensive molecular data(i.e., nrITS, rbcL, atpB-rbcL spacer, matKsequences) in Powell and Kron (2001) andKron et al. (2002), casting the monophyly ofLeucothoe into serious doubt. Relationshipsof the various species of Leucothoe to theother clades of the Gaultherieae are alsounclear, due to the limited sampling in thesestudies.The purpose of the present study is

primarily to test the monophyly of Leucothoe,by inclusion of all eight currently recognizedspecies in a morphology-based phylogenetic

analysis, along with various representativesof Gaultherieae, Andromedeae, and Vacci-nieae, groups suggested as close relatives(Kron et al., 2002). We hope to evaluatehypotheses of relationship within this diversegroup and their relationship with other mem-bers of the Gaultherieae. Morphological andanatomical variation was assessed among alleight species of Leucothoe and carefullyselected potential relatives in neighboringtribes.

Materials and methods

Specimens of the eight currently recognizedspecies of Leucothoe were sampled for thisanalysis employing 73 phenotypic characters,of which 54 were parsimony-informative;autapomorphies were included in the anal-ysis. Character scorings for most of theexternal morphological and anatomical char-acters (Appendix 1, Table I) were derivedfrom the authors’ observations of herbariumspecimens (from A, FLAS, GH) and, wherepossible, living or liquid-preserved material,supplemented by reference to published revi-sionary or floristic studies (Sleumer, 1959;Melvin, 1980; Judd, 1984; Middleton, 1991a,b, 1993; Middleton & Wilcock, 1990a;Luteyn et al., 1996) or other specializedmorphological studies (Palser, 1951, 1952,1958, 1961; Chou, 1952; Lems, 1962, 1964;de Villamil & Palser, 1980). Characters 70 to73, relating to embryology, chromosomenumber, and chemical features, were scoredbased on the literature (Stevens, 1971;Nesom, 1978; Judd, 1984; Middleton &Wilcock, 1990b; Luteyn et al., 1996; inaddition to references cited above and Ap-pendix 1), although presence of methylsali-cylate was also assessed (by smell) whenmaterial was boiled to soften for anatomicalpreparation (and, where possible, in the field).Most morphological characters were readilydivisible into discrete states, thus avoidingarbitrary decisions relating to state delimita-tion (see Stevens, 1991). Infraspecific varia-tion in quantitative characters (see Appendix1) was assessed by means of bar graphs, andthe states were delimited by more or lessdiscrete gaps (e.g., see Figs. 1, 2; representingcharacters #36 and 57). Some characterscould not be included in this analysis because

384 BRITTONIA [VOL 60

TABLEI

CHARACTERSTATEVALUESFORSPECIESUSED

INCLADISTIC

ANALY

SIS.

12

34

56

7Species

1234567890

1234567890

1234567890

1234567890

1234567890

1234567890

1234567890

123

Aga

ristapo

pulifolia

000a0

0000

000

0a00

0000

0000

0000

00a0

0000

000a

0?000

0000

000

000??000

0000

0000

0000

0And

romedaglau

coph

ylla

101020

0102

21?110

0110

0010

011?

01?0

0c11

0011

0?000

0100

001

0010

0101

0000

0001

0001

0Vaccinium

meridiona

le10

1010

0000

0000

0001

?a00

11110

100

1102

110

001

0?000

0000

001

010??110

1100

0011

0000

?Satyriawarszew

iczii

111?10

aa10

0000

1201

?a10

11100

000

1100

10001

10?000

1000

11100

0??110

1000

0011

0000

?Diplycosiaclem

entium

101010

1002

0010

2101

?101111a0001

?000

11111

00?1113

0010

01200??100

0000

0100

0210

0Gaultheriaprocum

bens

201010

0100

1001

0001

?001

0111

01?2

?????10

101

0?120

0100

001

001100

0000

0000

0101

100

Gaultheriadomingensis

201010

0000

0010

0011?1

010111

0100

0000

010

000

0?11a

010

1001

0011100

000

0100

0101

100

Cha

maedaph

necalyculata

101010

0100

a020

001100

00111a1?00

1001

1101

001100

10000

00110

1011

00

0000

1000

01

010

Leucothoe

grayan

a10

1001

010b

000ac001?0

00111a0

100

1011

10001a

0?000

2100

001

000??000

001a00

0a1?

00?

Leucothoe

keiskei

001000

1002

000

a000

111

0011100

000

0000

010

001

100001

0100

01001100

0000

0000

00a?

00?

Leucothoe

griffithiana

001000

1002

0000

000110

0011100

000

a000

010

001

1000000

000

01001100

0000

0000

011?

00?

Leucothoe

davisiae

101020

0000

100a00

01?0

00111a0

a10

1000

0100

01

1000000

000

0110

111001

0010

00011?

00?

Leucothoe

fontan

esiana

001000

1000

0000

000111

0001100

000

1000

1100

aa10

00000

000

0110

111020

0000

100111

000

Leucothoe

axillaris

001000

0000

0000

000111

0001100

000

1000

110

01a

1000000

000

0100111020

0000

100111

000

Leucothoe

recurva

10101101

0120

000001

0000

1111

0100

1102

110

101

1100000

000

0110

1000

0000

0010

011?

011

Leucothoe

racemosa

10101101

0120

000001

0000

1111

0100

1102

1101

0111000

0000

00110

1100

0000

0010

0101

011

a=0/1;

b=1/2;

c=0/2.

?=stateun

know

nor

notapplicable


FIG. 2. Graph of variation in anther awn length (Char. # 57). See Appendix 1 for state delimitations. Species inwhich awns are absent are shown without bars.

FIG. 1. Graph of variation in bracteole width (Char. # 36). See Appendix 1 for state delimitations.


they showed too much infraspecific variationor continuous variation across taxa. Suchcharacters include stem shape, leaf lengthand width, extent of revolution of leafmargin, petiole length, calyx size, inflores-cence axis length, extent of curvature, mostindumentum characters, bract and bracteolelength, calyx lobe length and width, filamentand anther length, anther tubules present vs.absent, extent of impression of style intoovary, extent of capsule lobing, and seed size.All multistate characters were considered tobe unordered (Appendix 1). Unknown char-acter states for particular species were scoredas missing values, as were situations where acharacter was considered “not applicable” ina particular taxon; characters showing infra-taxon variation were coded as variable (seeTable I).Species were employed as terminal taxa, as

discussed in Kron and Judd (1997; see alsoWeins, 1998). The cladistic analysis includedas outgroups Agarista populifolia (Lam.)Judd (Lyonieae), as well as representativesof the tribes Andromedeae (Andromeda glau-cophylla Link) and Vaccinieae (Vacciniummeridionale Sw. and Satyria warszewicziiKlotzsch), based on previous phylogeneticstudies (Kron & Judd, 1997; Kron et al.,1999b, 2002; Powell & Kron, 2001). Inaddition to the eight species of Leucothoe,several other species of the tribe Gaultherieaewere included, i.e., Diplycosia clementiumSleumer, Gaultheria procumbens L., G. dom-ingensis Urb., and Chamaedaphne calyculata(L.) Moench, in order to adequately test themonophyly of Leucothoe s.l. The analysiswas rooted using Agarista populifolia be-cause it is a member of the Lyonieae (Kron etal., 1999b), and previous analyses (Kron &Judd, 1997; Kron et al., 1999b, 2002) haveplaced it outside the clade comprising any ofthe putative relatives of Leucothoe. Thus, wehave provided a test of relationships amongthe related tribes Gaultherieae, Andromedeae,and Vaccinieae (see Kron et al., 2002). Theanalysis utilized the branch-and-bound optionof PAUP* version 4.0b10 (Phylogenetic Anal-ysis Using Parsimony*; Swofford, 2002). Sta-tistical support for clades was assessed usingbootstrap (500 branch-and-bound replicates).Patterns of character evolution were assessed

using MacClade 4.08 (Maddison & Maddison,2005).

Results

The analysis resulted in the recovery oftwo equally parsimonious trees of 155 steps,with a consistency index (CI) of 0.561(including autapomorphic characters) and aretention index (RI) of 0.534. The trees arevery similar, differing only in the placementof the Leucothoe grayana + Andromedaglaucophylla clade, and the Vaccinieae (i.e.,Vaccinium meridionale and Satyria warsze-wiczii) in relation to each other. A represen-tative cladogram with mapped character statechanges (Fig. 3) and the strict consensus treewith bootstrap values (Fig. 4) are presented.The analysis demonstrated that the genus

Leucothoe, as traditionally delimited, is poly-phyletic, and a minimum of eight additionalsteps is required to force the monophyly of thegenus (as assessed in MacClade, 2005). ALeucothoe s.s. clade, containing the evergreenspecies L. axillaris, L. fontanesiana, L. keiskei,L. davisiae, and L. griffithiana, is present in allthe trees, but without bootstrap support. Thisclade is supported by five putative synapo-morphies (see Appendix 1 and Fig. 3), plus theunique feature of fall-formed inflorescences ofthe “L. axillaris-type” (character #42–0 inAppendix 1). Other putative (although homo-plasious) synapomorphies include fine leafvein lengths from 35 to 60 cm/cm2 (#19; butthis may actually be plesiomorphic, based onhigher-level analyses; see Kron & Judd,1997, and Kron et al., 1999b, 2002), inflo-rescence emergence in the fall (#41), antherswith awns (#55), anther awns two per theca(#56), and seeds with a wing of flattened,bulging cells (#69). Anthers with two awnsper theca (#55, 56) are synapomorphic for theentire Gaultherieae on trees where that groupis resolved as monophyletic (see Kron et al.,2002). Thus, the strongest characters diagnos-ing Leucothoe s.s. are the winged seeds(although wings are hypothesized to haveevolved independently in L. grayana andin L. recurva) and the emergence of theracemes (and timing of meiosis) in the fall(an unusual develop-mental pattern that hasevolved independently in the Chamaedaphne +L. racemosa + L. recurva clade; see below). It


is noteworthy that the fall-emergent inflo-rescences of the Leucothoe s.s. clade are ofthe L. axillaris-type, i.e., with the reproductiveshoot having the appearance of an elongatebud, with the individual floral buds coveredby overlapping bracts. In contrast, the fall-emergent inflorescences of Chamaedaphnecalyculata, L. racemosa, and L. recurva areof the L. racemosa-type, i.e., the reproductiveshoot resembles a vegetative shoot and haslong leaf-like bracts not enclosing floral

buds. The internal floral parts are, instead,protected by thick sepals. These structuraldifferences suggest (in agreement with pat-terns of change in character #41) that fall-emergent inflorescences have evolved twicewithin the Gaultherieae. Within Leucothoes.s., the L. axillaris + L. fontanesiana cladehas 88% bootstrap support, and is diagnosedby six synapomorphies (Fig. 3), one of whichis unique, that of stigma shape capitate topeltate (#59–2). The presence of a band of

FIG. 3. Representative most parsimonious cladogram [L=155 steps, CI=0.561, including autapomorphiccharacters, RI=0.534]. Character changes at each node indicated by letters: see Appendix 1 for characterscorresponding to numbers below. Character numbers represent the derived state (i.e., 1) unless otherwise indicated. a=3, 18, 23, 24, 25, 36, 52, 68; b=1, 35; c=8, 26, 28; d=10(2), 39, 47; e=5, 38, 56, 70; f=22, 43, 56, 71; g=8(0), 13,20, 40, 44, 49; h=41, 42, 53, 65, 72; i=6, 10, 11(2), 32, 34(2), 73; j=5, 32, 58, 59, 61, 67; k=19, 41, 55, 56, 69; l=7,10(2); m=57; n=20, 23(0), 35, 59(2), 65, 70. Note: 42 (0) was arbitrarily coded as ancestral, but is most likely derivedand a synapomorphy for Leucothoe s.s. Autapomorphic characters: A. glaucophylla = 5(2), 11(2), 12, 14, 15, 19, 24(0), 25(0), 27, 30, 55, 58, 60, 72; D. clementium = 7, 10(2), 15(2), 16, 28(0), 30, 37, 39, 45, 46(3), 53(2), 55(0), 58,66, 68(0), 70(2); G. domingensis = 1(2), 17, 23(0), 31, 35(0), 38(0), 47, 57, 64; G. procumbens = 1(2), 11, 14, 23(0),30(2), 44(2), 47; C. calyculata = 13(2), 17, 27, 34, 40, 45, 57, 58, 68(0); L. recurva = 69; L. racemosa = 56; L.grayana = 6, 33, 34, 36, 46(2), 63, 69; V. meridionale = 26, 28, 34(2), 54, 62; S. warszewiczii = 2, 9, 15, 16(2), 21, 36(0), 39, 46, 50, 51; L. keiskei = 20, 31(0), 46, 48, 68(0); L. davisiae = 1, 5(2), 11, 29, 53, 60, 63; L. fontanesiana = 7,53; L. axillaris = 39. Where characters are equivocally placed DELTRAN optimization is used.


sclerified cells along the leaf margin (#23–0) isa synapomorphy, although it has also evolvedin Gaultheria. These two species also sharebracts lacking marginal unicellular hairs (#35),a unique synapomorphy within the Leucothoes.s. clade, but present in most other ingrouptaxa. Finally, we note that L. axillaris and L.fontanesiana share the peculiar apomorphy ofhaving a bare spot (i.e., lacking ovules) onthe adaxial surface of the placenta (#65), butthis apomorphy is also homoplasious, occur-ring also in Chamaedaphne calyculata, L.racemosa, and L. recurva. Another groupwith weak support (63% BS) is that of thetwo Asian Leucothoe s.s. species, L. keiskeiand L. griffithiana, which are united by twohomoplasious synapomorphies: leaf apiceslong acuminate (#7) and leaves lacking uni-cellular hairs adaxially (#10–2). We note thatlong acuminate leaves have also evolved in

L. fontanesiana. The western North AmericanL. davisiae is linked (with support less than50%) with the eastern North American L.axillaris + L. fontanesiana clade.A novel clade consisting of Chamae-

daphne calyculata, L. racemosa, and L.recurva is present in all most parsimonioustrees (with 68% bootstrap support). Synapo-morphies of this clade include fall-formedinflorescences (#41) of the “L. racemosa-type” (#42), a unique synapomorphy. Homo-plasious synapomorphies include filamentswith papillae but not unicellular hairs (#53),placenta with a bare spot adaxially (#65), andcampylotropous ovules (#72). The last char-acter is nearly unique within the taxa studied,occurring also only in Andromeda. Withinthis clade, a subclade (henceforth calledEubotrys) containing L. racemosa and L.recurva receives 96% bootstrap support. The

FIG. 4. Strict consensus of the two most parsimonious trees. Bootstrap values are indicated above the lines forclades with bootstrap support above 50%.


Eubotrys clade is supported by two uniquesynapomorphies: leaves with unicellular hairsscattered adaxially (#10) and megagameto-phytes with antipodals dividing (#73). Addi-tional synapomorphies include: deciduousleaves (#6, also evolved in L. grayana),leaves with unicellular hairs scattered abax-ially (#11–2; also in Andromeda), bractsquickly deciduous (#32, also occurring inrepresentatives of the Vaccinieae), and bractsphotosynthetic and elongate, but not equiva-lent to leaves, along the inflorescence axis(#34–2, also in Vaccinium meridionale).These apomorphies make the Eubotrys cladequite distinctive, especially when comparedto the evergreen species of Leucothoe. TheChamaedaphne + Eubotrys clade is sisterto the “wintergreen clade” of the Gaulther-ieae (described in Powell & Kron, 2001)in both most parsimonious trees. This cladeis supported by the bracteole positioned atthe apex of the pedicel (#38) (see alsoFig. 3).The wintergreen clade, represented by

Gaultheria procumbens, G. domingensis,and Diplycosia clementium, does not havebootstrap support in this morphology-basedanalysis. Unique synapomorphies are thepresence of fiber strands in the mesophyll(#22), calyx lobes fleshy and colorful (#43),and methylsalicylate (#71). The presence oftwo awns per theca (#56) is also resolvedhere (see Fig. 3) but is more likely synapo-morphic for the entire Gaultherieae (see Kronet al., 2002). Within this clade, the groupingof Diplycosia and G. domingensis is sup-ported by three unique synapomorphies:multicellular hairs stalked but lacking aglandular head (#13), calyx lobes blue toblack (#44), and corolla with multicellularhairs on the abaxial surface (#49).Another clade present in the strict consensus

tree (Fig. 4) is the Vaccinieae (as representedby Vaccinium meridionale and Satyria wars-zewiczi), which in our analysis has 73%bootstrap support and three distinctive andunique synapomorphies, namely stigma trun-cate with a rim (#59), ovary inferior (#61), andfruit indehiscent (a berry) (#67). The Vacci-nieae is placed as the sister group of a largeclade containing the wintergreen group +Chamaedaphne + Eubotrys in one of the trees

(not shown), but in the other tree (Fig. 3) theyare sister to a wintergreen group + Chamae-daphne + Eubotrys + Andromeda + L.grayana clade.Leucothoe grayana is a highly apomorphic

species (Fig. 3); distinctive autapomorphiesinclude deciduous leaves (#6, evolved inparallel in Eubotrys), the floral bract dis-placed onto the pedicel (#33), bracts of thereproductive shoots large and indistinguish-able from leaves proximally (#34, evolvedalso in Chamaedaphne calyculata), corollastrongly urceolate and short (#46–2), and theovary papillate (#63, also in L. davisiae).Notably, L. grayana never resolved in (oradjacent to) either clade containing the otherspecies of Leucothoe. Instead, it is placed assister to Andromeda glaucophylla in bothtrees. In one tree (Fig. 3) the Leucothoegrayana + Andromeda clade is sister to thewintergreen group + Chamaedaphne + Eubo-trys clade, while in the other tree (not shown)it is sister to all ingroup taxa except for theLeucothoe s.s. clade.We note that the members of the Gaulther-

ieae (i.e., species of Chamaedaphne, Diply-cosia, Gaultheria, and Leucothoe) are notresolved as monophyletic in any of the fourmost parsimonious trees. If the monophyly ofthe tribe is forced (in MacClade), potentialsynapomorphies of Gaultherieae include thepresence of anther awns (#56), two awns pertheca (#57, with ACCTRAN optimization),and chromosome base number, n=11 (#70).In summary, the species of Leucothoe s.l. are

resolved in three clades: the Leucothoe s.s.clade, a group of evergreen species (L. axillaris,L. fontanesiana, L. davisiae, L. griffithiana, andL. keiskei), and two highly apomorphic decid-uous clades, i.e., the Eubotrys clade (L.racemosa and L. recurva) and L. grayana.

Discussion

The results of the present analysis do notsupport the monophyly of the genus Leuco-thoe, as traditionally circumscribed (Sleumer,1959; Stevens, 1969, 1971; Melvin, 1980;Luteyn et al., 1996). Our results support thedelimitation of three different genera forthe species formerly placed in Leucothoes.l. The revised classification is as follows:Eubotrys Nutt., including Eubotrys racemosa


FIG. 5. A–C. Leucothoe fontanesiana. A. Leaf and capsules. B. Arching branch with racemes. C. L. axillaris-typeinflorescences (emergent in the fall). D, E. Eubotrys racemosa. D. Young fruits and E. racemosa-type inflorescences(emergent in fall). E. Flowers, showing non-pubescent filaments and awned anthers. F. Eubotryoides grayana.Racemes with leaf-like bracts and urceolate flowers. Photos A–E by W.S. Judd; photo F by Shu Suehiro.


(L.) Nutt. (= L. racemosa; Fig. 5D, E), andEubotrys recurva (Buckley) Britton (= L.recurva); Leucothoe D. Don, as restrictivelycircumscribed, including L. axillaris, L. fon-tanesiana (Fig. 5A-C), L. davisiae, L. keiskei,and L. griffithiana; and Eubotryoides (Nakai)Hara, including only Eubotryoides grayana(Maxim.) Hara (= L. grayana; Fig. 5F).Our new circumscription is not wholly

unprecedented. Sleumer (1959) was the firstto propose the division of the deciduousspecies of Leucothoe into two different sec-tions, i.e., Eubotrys and Eubotryoides, exactlycorresponding to the deciduous clades thatemerged from this analysis, althoughSleumer’s placement of the evergreen speciesin several different sections is unnecessaryfrom the standpoint of monophyly. Wood(1961), Melvin (1980), and Middleton(1991a), while retaining all eight species inLeucothoe, recognized the three clades result-ing from our analysis as sections within thegenus. Furthermore, the genus Eubotrys wasrecognized as distinct from the evergreenNorth American species of Leucothoe byseveral early workers, including Nuttall(1843), Britton and Brown (1913), and Small(1914, 1933) (see above), and two contempo-rary authors (Gleason & Cronquist, 1991;Stevens et al., 2004). Gleason and Cronquist(1991), in their descriptions of the NorthAmerican species, separated Leucothoe fromEubotrys on the basis of evergreen vs.deciduous leaves, virtually awnless vs. awnedanthers, and the stigma capitate vs. truncate,and all of these characters had some bearing inour cladistic analysis. Stevens et al. (2004), intheir treatment of the Ericaceae in Kubitzki’sThe Families and Genera of Vascular Plants,distinguished Eubotrys from Leucothoe(including L. grayana) on the basis of bracte-ole position. Stevens was likely influencedby the preliminary molecular data of Kronet al. (1999a, b, 2002), which he cites.Eubotryoides has also been considered adistinct genus by Japanese researchers, whogenerally follow Hara (1935), who highlightedseveral of the autapomorphic characters evi-dent in our analysis.A discussion of why Leucothoe s.l. was

maintained as a genus for so long seemswarranted. The strongest characters that ap-

pear to unite Leucothoe s.l. are the presenceof fall-formed inflorescences and wingedseeds. As our analysis indicates (Fig. 3), bothof these characters show parallelisms; in fact,the type of fall-formed inflorescences is verydifferent between Eubotrys and Leucothoes.s. (Fig. 5, compare C & D), and theinflorescences of Eubotryoides are formed inthe spring. The seed with a wing formed byflattened, bulging, marginal cells, althoughdistinctive and quite similar in the threegroups, is apparently homoplasious. Indeed,Eubotrys racemosa and E. recurva, two veryclosely related species, differ in this character,suggesting the genetic switch behind it is notvery complex. Furthermore, symplesiomor-phic similarity between the species of Leuco-thoe also probably factored into previousresearchers’ decisions regarding generic de-limitation, as they did not use cladisticanalyses to test their hypotheses.Several recent molecular phylogenetic

analyses have supported the polyphyly ofLeucothoe (Kron et al., 1999a, b, 2002;Powell & Kron, 2001). Although the genusLeucothoe was not the primary focus of theseprojects, and only E. racemosa and L.fontanesiana were included, their preliminaryresults included the placement of Chamae-daphne sister to Eubotrys and the placementof Leucothoe s.s. as the sister group of therest of the Gaultherieae. Both of these place-ments are upheld by the present analysis.Kron et al. (1999a, b) were the first to placeEubotrys as the sister group of Chamae-daphne calyculata. A probable reason whythis connection was not made earlier is thepresence of many morphological autapomor-phies in Chamaedaphne, the most obvious ofwhich is a dense covering of peltate scales.This genus also differs from Eubotrys in itsevergreen leaves. This second character hasbeen shown to be very homoplasious inrecent phylogenetic analyses (Kron et al.,1999a, b, 2002). Despite minimal taxonsampling from the rest of the Gaultherieae,our results are in agreement with those ofPowell and Kron (2001) in that they supportthe monophyly of the wintergreen group ofthe Gaultherieae. However, the monophylyof the Gaultherieae, supported by molecularor combined molecular and morphological


analyses in Kron et al. (1999a, b), Powell andKron (2001) and Kron et al. (2002), is notapparent in our analysis. The lack of supportfor this tribe (resulting from placements ofAndromeda and Vaccinieae) is not that sur-prising given that morphological charactersalone did not support the monophyly of thistribe in the analyses of Kron et al. (1999b,2002). In addition, our sampling in this part ofthe cladogram is insufficient to properlyaddress this issue. We also note that themonophyly of Gaultherieae was not supportedby analyses based on a single gene (Kron etal., 1999b, 2002; Powell & Kron, 2001). It isonly in combined analyses, i.e., analyses usingseveral genes or several genes and morpholo-gy, that the group is recovered, and the supportfor Gaultherieae in these analyses is relativelyweak compared to that of the related tribesAndromedeae, Vaccinieae, and Lyonieae.The distinctive biogeography of the three

genera, i.e., having a Tertiary relic disjunctionpattern with related species in North Americaand eastern Asia (Li, 1952; Wood, 1972), issimilar to that of several other genera in theEricaceae (e.g., Elliottia Muhl. ex Elliott,Epigaea L., Lyonia Nutt., Pieris, Rhododen-dron L.; Bohm et al., 1978; Judd, 1981, 1982;Kron, 1993; Li, 1952; Wood, 1972), butcontrasts with the circum-Pacific distributionof the species of Gaultheria (Powell & Kron,2001). Superimposing geography on thecladogram of Leucothoe s.s. reveals that thetwo eastern Asian species are sister to a NorthAmerican clade. Within the latter clade, L.davisiae, a species of the western UnitedStates, is sister to the two species of theeastern U.S. This relationship suggests thatthe ancestors of the species of Leucothoe s.s.were once widely distributed in circumborealAsia and North America, and were pushedsouthward as a result of the gradual climaticcooling that has occurred since the mid-Tertiary Period. This southward movementlikely promoted allopatric speciation. Eubo-trys is limited to eastern North America, butits sister group, Chamaedaphne, is circum-boreal. Interestingly, the L. axillaris + L.fontanesiana clade and the Eubotrys clade(with E. racemosa and E. recurva) each havea species in the coastal plain and a sisterspecies in the Appalachians.

Possible future research directions include amolecular analysis of the species examined inthis analysis in order to reevaluate the hypoth-eses presented here. Such analyses are beingplanned by K. A. Kron in connection with hermore extensive systematic investigation of theGaultherieae (Kron, pers. comm.). Molecularresearch may also help in determining theplacement of Eubotryoides grayana: at thepresent time, we are unable to draw any firmconclusions about its relationship to the othergenera.

Acknowledgments

We thank the Arnold Arboretum and GrayHerbarium, of Harvard University, and theherbarium of Florida Museum of NaturalHistory, of the University of Florida, foraccess to herbarium material (743 specimensobserved). Kent Perkins of the University ofFlorida herbarium assisted with specimenprocessing, Barbara Carlsward helped withproduction of the tables, and Kathleen Kron,Terry Lucansky, and Fabián Michelangeliprovided useful comments. Linda and PeterWaselkov helped with field collections of theAppalachian species, and Barbara Carlswardand Michael Heaney assisted in the prepara-tion and evaluation of some anatomicalsections. Gretchen Ionta kindly demonstratedher digital photography technique for micro-scope work. We appreciate the generosity ofShu Suehiro, who allowed use of the photo ofEubotryoides grayana. We would also like tothank the University Scholars Program of theUniversity of Florida for providing fundingfor the research.

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Appendix 1

Morphological characters and characterstates used in the phylogenetic analysis ofspecies of Leucothoe and selected representa-tives of tribes Gaultherieae, Andromedeae,and Vaccinieae.

1. Plant habit: arching (0); shrubby (1); exten-sively rhizomatous (2).

2. Plant substrate: terrestrial (0); epiphytic (1).3. Pith diaphragms: present (0); absent (1).4. Pith lignification: present (0); absent (1).5. Pith type: Calluna-type (0); homogeneous (1);

heterogeneous (2). The Calluna-type pith has

small, thick-walled cells restricted to a sheatharound the periphery of the pith (Stevens, 1971).

6. Leaf persistence: persistent (0); deciduous (1).7. Leaf apex: short-acuminate or slightly acumi-

nate to acute, mucronate, obtuse, or rounded(0); long-acuminate (1).

8. Leaf shape: ovate (rarely a few leaves elliptic)(0); elliptic to obovate (1).

9. Leaf venation: pinnate (0); plinerved (1).10. Unicellular indumentum on adaxial leaf sur-

face: unicellular hairs only on midvein (0);unicellular hairs scattered over the surface (1);lacking unicellular hairs (2).

11. Unicellular indumentum on abaxial leaf sur-face: lacking unicellular hairs (0); unicellularhairs only on midvein (1); scattered unicellularhairs (2).

12. Multicellular hairs: present (0); lacking (1). Tobe considered state “1,” no multicellular hairscould be present on any part of the plant.

13. Form of the multicellular hairs: stalked (usu-ally) with glandular head (0); stalked, alwayslacking glandular head (1); peltate scales (2).

14. Multicellular hairs on abaxial leaf surface:present (0); absent (1).

15. Leaf margin: serrate, at least distally (0);entire, non-ciliate (1); ciliate (i.e., with multi-cellular hairs on an entire margin), at leastdistally (2)

16. Leaf midrib anatomy: unifacial (0); unifacialbut very U-shaped and appearing as a ring (1);bifacial (2).

17. Pattern of lignified cells associated with leafveins (as viewed in cross-section): veinsincluded (without girders) (0); veins excurrent(with girders) (1).

18. Vein reticulum: dense, the secondary andtertiary veins ± equally prominent (0); ± open,the secondary and tertiary veins not equallyprominent (1).

19. Fine leaf vein length: 55 cm/cm2 to 110 cm/cm2

(0); 35 cm/cm2 to 60 cm/cm2 (1). Fine veinlength per unit area are an assessment of areolesize, and are taken from Lems (1964); variationwas assessed on several leaves of a plant,resulting in a clearly differentiation despiteslight overlap in ranges. A subjectiveassessment of areole size would suggest thatthose species scored as “?” are likely state “1”.

20. Lamina thickness: less than 0.28 mm (0);greater than or equal to 0.28 mm (1).

21. Petiole cortex and leaf midrib: lacking sclerids(0); with scattered sclerids (1).


22. Fiber strands in leaf mesophyll: absent (0);present (1). Wandering fibers in the leafmesophyll are very infrequent in Gaultheriaprocumbens.

23. Sclerified band of cells associated with leafmargin: present (0); absent (1).

24. Leaf epidermis: lignified (0); not lignified (1).25. Stomata condition: anomocytic (0); paracytic

(1). The stomata of Agarista populifolia areanomocytic (Judd, 1984); Chamaedaphnecalyculata coded as “1” based on Stevens(1969); stomatal condition of species of An-dromeda, Gaultheria, Diplycosia, Satyria, andVaccinium used in our analysis inferred fromconditions reported for these genera in Stevens(1969).

26. Length of shoot bearing inflorescences usually:greater than 15 cm (0); less than 15 cm (1).

27. Inflorescence position: axillary (0); terminal (1).If the vegetative portion of inflorescence axispersists through the growing season, but lacksbuds in the axils of the leaflike bracts, then thatportion is considered as part of the inflores-cence, and the inflorescence itself is consideredto be axillary (e.g., Leucothoe grayana).

28. Number/distribution of inflorescences: fre-quently numerous (i.e., 4–10) axillary inflor-escences positioned distally to all along shoot(0); usually 1–3 inflorescence axes clustered atthe tip of last year’s shoot, at the distal-mostnodes (1). Species with terminal inflorescenceswere coded as “?”.

29. Inflorescence axis orientation: ± horizontal ordrooping downward (0); rigidly ascending (1).

30. Inflorescence type: raceme (0); fascicle (1);solitary flower (2).

31. If a raceme, number of flowers on eachinflorescence: less than or equal to 9 (0);greater than 9 (1).

32. Floral bract persistence: persistent (0); quicklydeciduous (1).

33. Floral bract position: at pedicel base (0);displaced onto pedicel, at least on flowers atthe apex of the inflorescence (1). It is clear thatin state “1” the structures involved are in factbracts, and not bracteoles, even though theyare on the pedicel, because frequently flowerson the proximal part of the inflorescence havethe bracts in the normal position.

34. Inflorescence/floral bract form: small (to large)non-photosynthetic (0); large, photosynthetic,and indistinguishable from leaves proximally,much smaller distally (1); photosynthetic and

elongate but not equivalent to leaves, alongwhole inflorescence axis (2).

35. Unicellular hairs on margin of floral bracts:present (0); absent (1).

36. Bracteole width: less than 0.8 mm (0); greaterthan 0.8 mm (1).

37. Bracteole fusion: distinct (0); connate (1).38. Bracteole position: at base of pedicel or at least

in lower half of pedicel (0); at apex of pedicel(1).

39. Bracteole margin: with unicellular hairs (0);lacking unicellular hairs (1).

40. Pedicel: with multicellular hairs (0); lackingmulticellular hairs (1).

41. Inflorescence development: inflorescencesemerging in spring, i.e., meiosis occurring inthe spring (0); inflorescences emerging in the fall,i.e., meiosis occurring in fall, but flowers openingin the spring (1). Although the tropical speciesincluded in the analysis occur in habitats withouta distinct spring and fall, they do have flowers inwhich meiosis occurs as the inflorescencesexpand, and thus they were coded as “0”.

42. Inflorescence form: inflorescences formed inthe fall are L. axillaris-type, with shoot like anelongated bud with individual floral budscovered by short overlapping bracts (0);inflorescences formed in the fall are L. race-mosa-type, with shoot more resembling vege-tative shoot, with long, leaf-like bracts notenclosing floral buds, which are protected bysepals (1). Inflorescences emerge in the springthe character is not applicable.

43. Texture of calyx lobes (i.e., when associated withmature fruits): dry (0); fleshy (1). Texture iscorrelated with coloration, so dry fruits arebrown, while fleshy fruits are always colorful(see #44).

44. Color of calyx lobes: green (or light-green tocream), turning brown when fruit matures (0);blue-black, at maturity (1); calyx lobes red, atmaturity (2). In states “1” and “2” the colorfulcalyx assists in attracting dispersal agents.

45. Multicellular hairs on abaxial surface of calyxlobes: absent (0); present (1). Gaultheriadomingensis was coded as 0/1, on the basisof a single hair seen on one sepal.

46. Corolla shape: ± urceolate (0); subtubular (1);strongly urceolate (2); campanulate (3). Corol-la shape is fairly well associated with corollasize, as the strongly urceolate corollas ofLeucothoe grayana are usually less than orequal to 5 mm long, the campanulate corollas


of Diplycosia clementium are ca. 5 mm long,the subtubular corollas of L. keiskei andSatyria warszewiczii are greater than 10 mmlong, and the remaining more or less urceolatecorollas are 5–10 mm long.

47. Unicellular hairs on adaxial surface of corolla:absent (0); present (1).

48. Corolla lobe margins: lacking short unicellularhairs (0); with unicellular hairs (1).

49. Multicellular hairs on abaxial surface ofcorolla: absent (0); present (1).

50. Stamen form, i.e., comparison of stamens in theinner and outer whorls: stamens monomorphicand theca tips never expanded and crown-like(0); stamens dimorphic because of differentialanther lengths, and longer anthers with thecatips expanded and with crown-like (1).

51. Filament fusion: distinct (0); connate (1).52. Filament shape: S-shaped (0); straight (1).53. Filament indumentum: with papillae and uni-

cellular hairs (0); with only papillae (and thesesometimes only at base) (1); glabrous (2). Thefilaments of Leucothoe fontanesiana are al-most always merely papillate; unicellular hairsare present only very rarely, and thus thespecies was coded as “1”.

54. Anther spurs: absent (0); present (1).55. Anther awns: absent (0); present (1).56. Number of anther awns: 1 per theca (0); 2 per

theca (1).57. Awn length: greater than 0.25 mm (0); less

than 0.25 mm (1).58. Disintegration tissue: present (0); absent (1).59. Stigma form: truncate without a rim (0);

truncate with a rim around the edge (1);capitate to peltate (2). Stigmatic conditionwas assessed at anthesis.

60. Style shape: not expanded below stigma (0);expanded below stigma (1).

61. Ovary position: superior (0); inferior (1).62. Ovary locules: not subdivided (0); subdivided,

forming two chambers (1).63. Ovary papillae: absent (0); present (1).64. Ovary indumentum: lacking unicellular hairs

(0); with unicellular hairs (1).65. Placenta form: completely covered with seeds

(0); with a bare spot on the adaxial surface, atleast at anthesis (1).

66. Placenta attachment: to axis centrally (toapically) (0); to axis basally (1).

67. Fruit dehiscence: dehiscent (a capsule) (0);indehiscent (a berry) (1).

68. Shape of testa cells: elongate (0); ± isodiametric(1). Leucothoe grayana is coded as 0/1 inregards to testa cell length because its cells arelong and narrow over the body of the seed, butshort at the apices and on the marginal wing.

69. Seeds: lacking wing of flattened, bulging cellson margin (0); with distinct to discontinuous/indistinct wing of flattened, bulging, marginalepidermal cells (1).

70. Base chromosome number: n=12 (0); n=11(1); n=9 or 18 (2). Chromosome base numbersare taken from Judd (1984), Luteyn et al.(1996), Middleton and Wilcock, (1990b),Nesom (1978), Stevens (1971) with n=12used for Satyria, since this number is charac-teristic of Vaccinieae.

71. Methylsalicylate: absent (0); present (1).72. Ovule: ± anatropous (0); campylotropous (1).

Ovule form is assessed from personal obser-vations and embryological studies (Palser,1952; Chou, 1952; de Villamil and Palser,1980).

73. Antipodal cells of megagametophytes: notdividing (0); dividing (1). Assessed fromreferences listed under #72.


A phylogenetic analysis of Leucothoe s.l. (Ericaceae; tribe Gaultherieae) based on phenotypic...

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