Aliso: A Journal of Systematic and Floristic Botany Aliso: A Journal of Systematic and Floristic Botany

Trilliaceae are plants of North Temperate forests with a holarctic distribution and a high degree of endemism. Molecular phylogenetic analyses are presented in order to examine the tribal, generic, and species-level classification of the family. These molecular studies, and earlier morphological studies, support the placement of the genus Pseudotrillium as basal in the family and sister to the tribes Trillieae and Parideae, which are monophyletic sister groups. Trillidium (Trillium) govanianum provides an unresolved problem: morphologically it is included within Parideae, but molecular data place it within Trillium as sister to T. undulatum. Within tribe Trillieae, clades are noted that correspond to previous taxonomic groups. Within tribe Parideae the separation of Paris s.l. into Daiswa, Kinugasa, and Paris s.s. is strongly supported. In addition, some biogeographic correlations are noted, and phylogenetic distribution patterns are discussed.


INTRODUCTION
Because of their simple and distinctive morphology, Trilliaceae (Table 1) have been easy to circumscribe but difficult to place.Since initial recognition of the family as a unit by Chevallier (1827), its members have been placed in seven orders and as parts of five families (Table 2; Zomlefer 1996;Farmer 2000).Recent studies using a variety of molecular and morphological techniques (see Farmer & Schilling 2002 for summary) agree that Trilliaceae are monophyletic (Chase et a!. 1993(Chase et a!. , 1995a, b;, b;Davis 1995;Kato et a!. l995a, b;Stevenson and Loconte 1995).
The traditional view of generic limits in Trilliaceae has been the classification into the two Linnaean genera based on floral merosity: Trillium is trimerous, whereas Paris is 4to 11-merous.Within Trillium, a major issue is whether the two subgenera (subgen.Trillium having a flower pedicel, subgen.Phyllantherum lacking a pedicel [Freeman 1969[Freeman , 1975]]) are monophyletic (summary in Zomlefer 1996).Two species within Trillium have more recently been segregated as monotypic genera: Pseudotrillium and Trillidium.Pseudotrillium was erected based its broad spotted petals, continuously elongating pedicel, and molecular evidence; Trillidium was described based on its tepaloid inflorescence, extrorse anther dehiscence, and trimerous phyllotaxy (see Table 1).Within Paris, the debate is whether to recognize a single broad genus (Hara 1969;Li 1984Li , 1998) ) or split it into three genera (Takhtajan 1983;Dahlgren et al. 1985;Tamura 1998).The most recent treatment of the family indicates that the two traditional subgenera of Trillium are not monophyletic, but there is morphological and molecular support for splitting Paris s.l.into three genera so that a total of six genera are recognized in the family: Trillium, Trillidium, Pseudotrillium, Paris, Daiswa, and Kinugasa (Farmer and Schilling 2002;see Tamura 1998;Fig. 1-6).
Trilliaceae are plants of North Temperate forests with a holarctic distribution (Takhtajan 1986; Fig. 7) and are thus hypothesized to be Arcto-Tertiary in origin (Tamura 1998), referring to high latitude, Northern Hemisphere areas having an abundance of Tertiary fossils (Bufford and Spongberg 1983; see Engler 1879).Most contemporary botanists agree that this distribution consists of four disjunct areas: eastern North America, western North America, eastern Asia and southeastern Europe-Asia Minor (Wood 1972;Tiffney 1985b).Members of Trilliaceae occur in each of these regions north of Mexico as well as most of Europe.
Within the Tertiary relict disjunctions, the most widely studied of these patterns is that of eastern Asia and North America, particularly eastern North America (Gray 1846;White 1983;Tiffney 1985a;Hong 1993;Wen 1999; see Bufford and Spongberg 1983 for summary).Other patterns of distribution such as that between the eastern North America and the Old World (Fernald 1931) and the southern Appalachians and western North America (Wood 1971) have also been noted.Thorne (1972) and Raven (1972) discuss more general disjunct patterns in plants.Within Trilliaceae there are some interesting distributional patterns: there are no Trillium in Europe; no members of Parideae are found in the New World; polyploids are restricted to the Old World; the only documented hybrids are in Asia; and the only sessile-flowered species are in the New World.In addition, there is only one globally widespread species, Paris quadrifolia (which is found from Ireland to Mongolia and Siberia); there are two widespread genera, Paris and Trillium; but there are three narrowly endemic genera, Pseudotrillium, Kinugasa, and Trillidium.The traditional sister species groups that are often found in Arcto-Tertiary distributions also exist: the Erectum Group (discussed later) exhibits the traditional eastern North America-Asia pattern (Fernald 1931;Raven 1972;Thorne 1972;Wood 1972;Xiang et al. 1998), while both the Grandiflorum Group (discussed later) and the sessile flowered species of Trillium (subgen.Phyllantherum Raf.) exhibit the western North America-eastern North America pattern (Wood 1971 ).
Table I.Generic types of Trilliaceae.Numbers after the genus name refer to numbers of species: National Flora (e.g., Flora of China [Liang and Soukup 2000]; Flora of North America [Case 2002];Tamura 1998;Farmer and Schilling 2002).All species have a single flower subtended by a whorl of leaves on an otherwise naked stem.Liliaceae is assumed to be in Liliales. 1 = Medeola as Gyroomia, 2 = Medeola, 3 = Scoliopus, 4 = Demidovia, 5 = Listed in synonymy for Trillium, but mentioned in text, 6 = Clintonia, 7 = Daiswa, Kinugasa, and Paris s.s., 8 = Trillium and Trillidium, 9 = Pseudotrillium.Note that Watson and Dallwitz (199lb, 1996) refer to DELTA data sets (Watson and Dallwitz 199la).Both subgenera of Trillium have a long history of study (Small 1897;Gleason 1906;Barksdale 1938;Freeman 1969Freeman , 1975 ; ;lhara andIhara 1978, 1982), but the genus has only recently begun to be studied in a phylogenetic context (Kato et al. 1995a, b;Kazempour Osaloo et al. 1999;Kazempour Osaloo and Kawano 1999;Farmer and Schilling 2002).Forma l subgroups have not been proposed for the pedicellate T. subgen.Trillium, but two informal subgroups are used based on texture and aging characteristics of the petals: the Grandiflorum Group (characterized by delicate textured, undulate-margined petals that age to pink) and the Erectum Group (characterized by more coarsely textured petals without undulate margins that age to brown) (Gleason 1906;Barksdale 1938;Ihara andIhara 1978, 1982;Patrick 1984).However, studies that use informal groups include other characters such as pistil morphology (Gleason 1906;Barksdale 1938;Ihara and Ihara 1978).Often considered a part of the Grandiflorum Group, the Delostyli s Group was recognized by Rafinesque ( 1819) to refer to species with an apparent style between the ovary and three slender stigmas .As defined by Rafinesque, this group comprises four species: Trillium catesbaei, T. nivale, T. persistens, and T. pusillum.All of these  Trilliaceae (after Samejima and Samejima [1987] and Li [1998]) .

Date
species are from the southern Appalachians and southeastern United States, with the exception ofT.nivale, which ranges from Pennsylvania westward to Montana, including distributions in South Dakota, Nebraska, and Missouri.

Sampling Strategy
Seventy species were considered in earlier morphological analyses of Trilliaceae (Farmer 2000;Farmer and Schilling 2002).Samejima and Samejima (1987), Li (1984Li ( , 1998)), and Takhtajan (1983) served as primary taxonomic sources although species more recently recognized were also included.The Flora of China (Liang and Soukup 2000) was used as the source for currently recognized Parideae taxa.
The first molecular analysis, using ITS data alone, was based on 38 accessions from GenBan.k sequence data (Table 3).The second analysis, combining ITS and matK data, focused on the Delostylis Group of Trillium.All 54 members of this group were selected (including varieties, geographical variants, and closely related species) (Table 3).
Molecular markers.-DNAsequence information for two gene regions was available from GenBan.k for a subset of taxa used in the morphological analysis (Table 3).The two molecular data sets were the Internal Transcribed Spacer (ITS) region (ITSl, 5.8S, ITS2 sequences) of nuclear ribosomal DNA, and the chloroplast DNA sequences for the maturase (matK) gene.Because ITS and matK sequence data were already available for species within the family, these two regions were chosen for initial sequencing of additional taxa.All ancillary data sets and sequence alignments are available from the author.

DNA Extraction, PCR, and Sequencing Protocols
For the analysis of the Delostylis Group, genomic DNA was extracted from plant material using the DNeasy Plant Kit (QIAGEN Inc., Valencia, California, USA) from frozen, desiccated, or herbarium specimens (Table 3).PCR amplification and sequencing was carried out using the primers in Table 4.The PCR reactions were performed in 20 JJ..I reactions containing 13 .5JJ-.1 sterile water, 2.0 JJ..I lO X PCR buffer, 1 .8JJ-.1 2.5 mM MgC1 2 , 0.4 JJ-.1 0.2 mM dNTPs in equimolar ratio, 1 unit Taq polymerase (Eppendorf North America, Westburg, New York, USA), 0.4 JJ-.1 0.5 mM each primer, 0.4 JJ-.1 BSA and I JJ-.1 genomic DNA.The ITS protocol was described by Sun et al . (1994), used primers ITS4 and ITS5 , and proceeded as follows: initial denaturing at 94°C for 3 min, followed by 35 cycles of a 94°C denaturing step for 60 sec, a 58°C annealing step of 60 sec, and a 72°C extension step for 120 sec followed by an additional extension step for 5 min at 72°C to complete any unfinished DNA strands.The matK protocol (Fuse and Tamura 2000) used primers trnK-710F and matK-8R and proceeded as follows: initial denaturing at 94°C for 2 min, followed by 40 cycles of a 94°C denaturing step for 60 sec, a 50°C annealing step of 60 sec, and a 72°C extension step for 90 sec followed by an additional extension step for 3 min at 72°C to complete any unfinished DNA strands.After PCR, the samples were purified with ExoSAP-IT (USB Corp. , Cleveland, Ohio, USA) before sequencing.In all cases, both strands of the PCR products were sequenced using the ABI PRISM Dye Terminator cycle Sequencing Ready Reaction Kit (Perkin-Elmer Applied Biosystems, Foster City, California, USA) per manufacturer's instructions.To sequence the ITS region, primers ITS4 and 17SE were used; to sequence the matK region primers trnK-7lOF, matK-1470R, matK-1470F, and matK-8R were used.After the sequencing reaction, the samples were purified with Sephadex (Sigma-Aldrich , St. Louis, Missouri , USA) using Centri-Sep tubes (Princeton Separations, Adelphia, New Jersey, USA) and vacuum dried.The dried samples were delivered to the sequencing facility where they were sequenced on an ABI Prism 3100 automated sequencer (Perkin-Elmer Applied Biosystems) at the University of Tennessee Molecular Biology Resource Facility.

Sequence Manipulation and Alignment
The sequences were manually contiged (where necessary) and aligned using BioEdit (Hall 1997(Hall -2005(Hall , 1999)).The ITS sequences obtained from GenBan.k (Kazempour Osaloo and Kawano 1999) consisted of 648 base pairs (of which 199 were variable for Trilliaceae); the matK sequences obtained from GenBank (Kazempour Osaloo et al. 1999) comprised 1578 base pairs, 84 of which were variable for Trilliaceae.The data matrices from the previous analyses were deposited at TreeBase; the final matrices from these analyses will also be deposited at TreeBase at the completion of the project.All aligned data matrices, tree files, and supporting data are also available from the author.

Phylogenetic Analyses
Data sets were analyzed using PAUP* (Swofford 2003) vers.4.0b I 0 with maximum parsimony using heuristic search methods with TBR (tree bisection reconnection) and MULPARS and a simple addition sequence.Random addition with steepest descent was used to check for islands of trees (Maddison 1991).Bootstrap support (Felsenstein 1985) was estimated based on 100,000 FastStep replicates with the same search strategy as simple parsimony.In the initial analyses, gaps were treated as missing.In subsequent analyses, gaps were coded according to Simmons and Ochoterena (2000).

Previous Analyses
In all cases, Paris s.l.(tribe Parideae) and Trillium (tribe Trillieae) were shown to be sister clades with varying degrees of bootstrap support.Within Parideae, Paris and Daiswa were placed as sister clades.Kinugasa was sometimes sister to Daiswa and sometimes outgroup to Paris + Daiswa.
In Trillieae, T. subgen.Phyllantherum was placed in a welldefined, monophyletic clade, but T. subgen.Trillium was not monophyletic.In morphological analyses, Trillidium govanianum was placed with Parideae, but in molecular analyses, it was placed with Trillieae (Farmer and Schilling 2002).Full details of these analyses are available from the author.

Analysis Using ITS Data Alone
New ITS sequence data were deposited by Y. Li of China with GenBank after the previous analyses were published (Farmer and Schilling 2002).Using these new sequence data, parsimony analysis produced two shortest trees of length 355 with a consistency index (CI) of 0.67; the strict consensus tree is shown in Fig. 10.The data set included 659 characters, 202 variable and 83 informative.As in all other analyses, Trillieae and Parideae were placed as sister clades; the basal split into these two clades was poorly supported, but individual clades typically had bootstrap support over 90%.These new data indicated that with the recognition of the segregate genera Daiswa and Kinugasa, Paris s.s.(Fig. 10) could be restricted to those species with a narrow creeping rhizome and without a seed aril.In addition, Daiswa (Paris) polyphylla and its varieties were found to be nonmonophyletic.The topology of Trillieae changed slightly from previous analyses in that the putative group formed by T. undulatum and Trillidium govanianum was not placed in a basal position in this tree and was less well supported (bootstrap = 69% ).

Analysis of Delostylis Group Using Combined ITS and matK Data
Preliminary parsimony analysis of combined ITS and matK sequence data produced 454 shortest trees of length 913 with a CI of 0.59 in multiple islands; the 50% majority rule is shown in Fig. 11.A poorly supported basal dichotomy separated Parideae and Trillieae.Within Parideae, Paris s.s. was supported at 98%, Daiswa at 100% with Kinugasa as basal to Daiswa at 89%.Within Trillieae, the most notable result was the placement of Trillium ovatum var.hibbersonii as not only the most basal taxon in the clade but also not as a diminutive form of T. ovatum var.ovatum (Taylor and Szczawinski 1974).An unresolved clade comprised sister species T. undulatum and Trillidium govanianum (supported at 82%), the Erectum Group (supported at 98%), and all other species of Trillium as successive sister groups (Fig. 11).The monophyletic group, T. subgen.Phyllantherum was supported at 100% and is sister to the rest of the pedicellate species of Trillium.Within the clade containing the remainder of the pedicellate Trillium, the basal T. ovatum var.ovatum (100% bootstrap support) was followed by paired sister species T. grandiflorum and T. nivale supported at 61%, with the Delostylis Group most terminal and supported at 99%.Within the Delostylis Group, T. catesbaei and T. persistens are sister taxa and most basal, leaving the Pusillum Complex most terminal.Within T. pusillum, there were successivesister clades, the most basal of which contains var.texanum and the Georgia (var.indet.GA) populations.The remainder of the successive sister groups were the wetland sessile-flowered varieties (var.virginianum), the montane sessile-flowered variety (var.monticulum), populations from North Carolina (var.indet.)and Mississippi (var.indet.MS), then the Ozarks (var.ozarkanum), with the Alabama populations (var.alabamicum nom ined.)comprising the terminal-most clade.

Trilliaceae
Based on all molecular analyses, Trilliaceae can be classified into the monotypic Pseudotrillium rivale and two monophyletic tribes: Parideae and Trillieae.The distinctiveness of Pseudotrillium rivale from both Paris and Trillium required erection of a new genus (Farmer and Schilling 2002); the basal position of Pseudotrillium is supported from prior molecular studies incorporating outgroups to the family (Fig. 8, 9).Tribe Parideae is composed of Paris, Daiswa, and Kinugasa whereas Trillieae is comprised of Trillium and apparently (in these analyses) the monotypic Trillidium govanianum.However, there are conflicts with the latter hypothesis.Morphologically, Pseudotrillium and Trillium have omniaperturate, spherical pollen grains with helobial endosperm development, features tentatively hypothesized as ancestral given the basal position of Pseudotrillium in the family.In contrast, Trillidium together with Parideae are characterized by ellipsoidal, monosulcate pollen grains and nuclear endosperm development, features apparently apomorphic in the family.The placement of Trillidium (near Trillium undulatum) within Trillieae in the molecular analyses may be an artifact of long-branch attraction (see later discussion).A new key to the genera of Trilliaceae is provided in Appendix 1.

Tribe Parideae
Parideae is monophyletic in all of the analyses (Fig. 10, 11).In addition to the traditionally used trait of merosity, Table 3. Taxa included in the previously published matK and ITS sequencing analysis.Accession numbers are listed with ITS first then matK.Publication notes: (A) published in 00 -!>-Kazempour Osaloo and Kawano (1999); (B) unpublished data by X. Tang, L. Yao, and R. Tang; (C) sequences produced by S. Farmer; populations will be vouchered at TENN and sequences will be deposited with GenBank at the completion of the project.VIU = voucher information unknown; LMC = leaf material collected; PV = population vouchered.Parideae can be distinguished by synapomorphies such as filiform petals, elliptical, monosulcate pollen, and nuclear endosperm (Farmer and Schilling 2002).
The molecular analyses supported the separation of Parideae into two sister clades, Paris and Kinugasa + Daiswa but D. fargesii has an indehiscent rather than a dehiscent berry, and D. thibetica has a partial rather than a complete aril (Li and Noltie 1997).Character-mapped cladograms are available from author.
In traditional, morphological classifications, Kinugasa has always been aligned with Paris subgen.Paris, but morphology now interpreted in light of the current analyses suggests a closer relationship between Kinugasa and Daiswa, with both exhibiting the thick rhizome and arillate seeds in addition to many of the same insertions/deletions (indels) and base-pair changes.One alternative is to lump Kinugasa    3.
with Daiswa.However, because of the unusual morphology of the species (i.e., the octoploid chromosome count, and showy, white sepals), the segregate genus Kinugasa should be retained.
Because of the recognition of Kinugasa as a genus, and because Paris and Daiswa were separated on a molecular level as well as morphologically, Daiswa as distinct from Paris should be recognized.Nomenclatural consequences will be addressed at a later date.

Tribe Trillieae
Trillium ovatum var.hibbersonii and T. ovatum var.ovatum do not together comprise a monophyletic group and occur in separate clades in the combined ITS-matK analysis (Fig. 11).Trillium ovatum var.hibbersonii is placed at a more basal position in the tree than T. ovatum var.ovatum.There are approximately 29 absolute base pair changes and one indel of five base pairs in length that separate these two taxa in the ITS data set; in the matK data set, there are 13 absolute base pair changes within the coding region and nine base pair changes and three indels of length five, six, and 11 base pairs in the 5' spacer region that separate varieties hibbersonii and ovatum, but only two that unite them as distinct from all other Trillium species.There are several morphological characters that separate them as well, most notably the extremely small stature of T. ovatum var.hibbersonii, but also phenology, gynoecial and androecial morphology, as well as a difference in petal coloration (T.ovatum var.hibbersonii flowers pink before fading to white whereas T. ovatum var.ovatum flowers white and fades to pink [Wiley 1969]).Thus, it is suggested that Trillium ovatum var.hibbersonii be elevated to species rank (Farmer in prep.).
With fewer species of Parideae in the analysis shown in Fig. 11, the grouping of T. undulatum and Trillidium govanianum is better supported at 82% (Fig. 11) than in Fig. 10.Due to insufficient sampling (Graybeal 1998), it is possible that the problematic placement of Trillidium (e.g., as sister species to Trillium undulatum, see Fig. 10, 11) is caused by long-branch attraction or lineage sorting.The best solution for eliminating long-branch attraction is to increase taxon sampling (Graybeal 1998).This hypothesis is supported not only by the drop in bootstrap support from 82% (Fig. 11) with only 10 members of Parideae to 69% (Fig. 10) when 20 species of Parideae are included in the analysis, but also the fact that if Trillium undulatum is removed from the analysis, Trillidium is placed as the basal ingroup species (i.e., sister to Trillium and Parideae combined).
The grouping of Trillium nivale and T. grandifiorum together is poorly supported in the analysis shown in Fig. 11 at 61 %; however, results are available from only one accession ofT.nivale.Traditionally, T. grandifiorum and T. ovatum have been placed with members of the Delostylis Group because of their anthocyanin chemistry and petal texture (Gleason 1906;Barksdale 1938;Ihara andIhara 1978, 1982;Patrick 1984); however, they do not share the character states of a fused style and sub-petiolate to petiolate leaves.This analysis does not support a close relationship between T. grandifiorum and T. ovatum, but rather between T. grandijlorum and T. nivale.
The Delostylis Group s.s.(i.e., Rafinesque's 1819 definition of Delostylis minus Trillium nivale) makes up the remainder of the tree shown in Fig. 11.Trillium persistens and T. catesbaei are sister taxa that form the base of this clade with T. pusillum and its varieties monophyletic.In previous analyses, the placement of T. pusillum varied depending on the data set.In the large morphological analysis (Farmer and Schilling 2002), T. pusillum was part of a clade basal to all other taxa.In addition, its placement in the ITS analysis (Fig. 10) was different from that produced by the combined ITS-matK tree (Fig. 11).In the analysis of the relationships among the varieties of T. pusillum, the strict consensus tree of 454 most parsimonious trees (Fig. 11) shows several welldefined and well-represented groups.However, in the FastStep Bootstrap of 100,000 reps, separation into distinct varietal units with bootstrap support was not observed-only a polytomy was produced.
Trillium pusillum var.texanum and the Georgia populations are more closely related to each other than they are to the rest of the pusillum complex.This relationship is supported by the synapomorphy of stomates on the upper surface of the leaves.Based on sampling of other non-coding plastid regions (Shaw et al. 2005), the varieties of T. pusillum are separable on a molecular level.

Biogeography
Distribution and endemism patterns in Trilliaceae.-Each of the Arcto-Tertiary areas of refuge has its own center of diversity.In eastern Asia, which has the highest diversity of this modern Tertiary flora (Tiffney l985a ), the center of diversity is in south central China in the Qinling Mountains (Bufford 1998).In eastern North America the highest diversity is in the southern Appalachians (Bufford 1998;Wen 1999).The Klamath range of northern California and southern Oregon has the greatest diversity in western North America (Wood 1971).In the Europe-Asia Minor refuge, the center of diversity appears to be in the Caucasus Mountains (Wood 1971).This pattern is similar in Trilliaceae with Asia having the greatest diversity (34 species and five known hybrids in five genera).North America is next with 30 species of Trillium in eastern North America while western North America has at least eight species in two genera (Trillium and Pseudotrillium).Europe has the least diversity with only two species of Paris.
In addition to the traditional Arcto-Tertiary distribution patterns, genetics provide another distribution pattern; polyploidy is restricted to the Old World.All North American Trillium are diploid; although there are scattered reports of triploid specimens (Haga 1942;Sparrow and Pond 1950;Kozuka et al. 1964).Of the nine Trillium in Asia, only one is diploid; the rest are triploid, tetraploid, or hexaploid (Samejima and Samejima 1987).Within Paris, the primary European taxon, P. quadrifolia, is tetraploid.Paris incompleta from the Caucasus Mountains is diploid as are most of the other Asian taxa including all members of Daiswa (Li 1998).Kinugasa japonica is octoploid (Tatewaki and Suto 1935).Of the Asian polyploid Trillium, five are recognized as hybrids.Hybridization is believed to occur in North America in the Erectum Group as well as in the Phyllantherum Group (Freeman 1969), but has not been proven (Case and Case 1997).
Phylogenetic distribution patterns.-Ifthe phylogeny based on a morphological analysis of the family (Farmer 2000;Farmer and Schilling 2002) is an accurate representation of the evolutionary history of the various species, then the oldest extant lineage is in the Klamath Mountains of the Pacific Northwest, represented by Pseudotrillium rivale.Given that Parideae and Trillieae are sister groups occupying largely different geographical areas (Eurasia vs. Asia-North America), a taxon-area cladogram based on phylogeny would not be informative; however, some patterns may be noted.Based on the phylogeny, the basal taxa in the two tribes are either Asian or western North American.From the center of origin, the Paris-like species dispersed to Asia and from Asia into Europe.The Erectum Group dispersed to Asia and eastern North America.The American species may have diverged from the Asian species before or after dispersal, and the Asian taxa are likely to be ancestral in this group.The Grandiftorum Group diverged with Trillium ovatum persisting in the Pacific Northwest and with Trillium grandijlorum migrating (or surviving) in the southern Appalachians (Griffin and Barrett 2004).The Phyllantherum Group diverged and also survives in western North America and eastern North America with the ancestral taxa in the Pacific Northwest.

Fig. 11
Fig. 11.-50% majority rule consensus of 454 trees in 57 islands from the ITS-matK analysis of 54 accessions of Trilliaceae taxa.Length = 913 steps; consistency index = 0.86.Percentages below the branches are bootstrap values.Numbers refer to DNA samples in Table3.

Table 2 .
Historical placement and composition of genera associated with Trilliaceae.