1974, 1977, 1979; Luther and Sieff 1991). Most ter 1992 ... Givnish et al. 1990; Terry et al., in press), examina- rently assigned to Tillandsioideae and Pitcairnioi-.
S ~ S ~ P I ~b)ot~!i~!/ I I I ~ (1997), I~ 22(2) pp 333-14: 8 Copyright 1997 by the American Societ! ot Plant Taxonomiits
Phylogenetic Relationships in Subfamily Tillandsioideae (Bromeliaceae) Using ndhF Sequences RANDALL G . TERRY~ and GREGORY K. BROWN Department of Botany, University of Wyoming, Laramie, Wyoming 82071 'Present address: Department of En.rlironmentaland Resource Sciences, University of Nevada, Reno, Nevada 89557
Population, and Organismic Biology, University of Colorado, Department of En.r~iron~nental, Boulder, Colorado 80309 2Present address: Department of Botany, University of Washington, Seattle, Washington 98105 Cornirii~nicating Editor: Ellznbeth A. Kcllogg ABSTRACT. Nucleotide sequences of the plastid locus ndhF are used to examine phylogenetic relationships among 25 representative species of subfamily Tillandsioideae. Genetic divergence among bromeliad taxa is lorv for iidizF sequences, ranging from 2.1'4, between Cntopsis ionngrriniz and Tillnridsiti ivicholrpis, to 0.296 betrveen several species of Ciiziizniiia, Mezobrorrirlzn, and Vriesen. Parsimony analysis produced six shortest trees of 216 steps. These results: 1) suggest Tillandsioideae as traditionally circumscribed is monophyletic; 2) place Catopsis and Glornevopitcniriiin in a clade that is the sister group to the remainder of the subfamily; 3) indicate Vrirsen is a paraphyletic grade from within rvhich Tillniidsia and C~izrriniiiahave arisen, and 4) suggest Mezobromelin is more closely related to Vriesea subg. Vriesea sect. Xzphion than its i n Vrieseti for rvhich traditionally recognized sister-group, Guziizania. All subgenera and sections of T i i i t i ~ ~ f sand at least trvo taxa are sampled are paraphyletic. iidizF sequences support sister-group relationships for Tillnndsin utriculata (subg. Tillandsin) and Vviesrn espiiiosne (subg. Vriesea sect. Vriesea), as well as for Till~iridsz~ifiiiicLiniia (subg. Tillnridsia) and Vi.iesea iiznlziriei (subg. Vviesrn sect. Xiphioii). Other clades well supported by these data include Tillandsioideae sensu stricto (excludes Cntopsis and Glorririopiicnirr~in)a nd a component of Vrieseil subg. Vriesnl sect. Xipiziori. Poor resolution and weak branch support for many clades in the izdhF trees suggests that information from more highly variable sequences or the combining of data sets will be required for a clearer understanding of phylogenetic relationships in Tillandsioideae.
Tillandsioideae, with over 1000 species in six genera (Smith and Downs 1977),is the largest of the three bromeliad subfamilies (Smith and Downs 1974, 1977, 1979; Luther and Sieff 1991). Most species are epiphytic or lithophytic and possess a number of distinctive vegetative features associated with these life-forms. Although several studies have addressed the placement of the subfamily within Bromeliaceae (Pittendrigh 1948; Tc>mlinson 1969; Benzing et al. 1985; Gilmartin and Brown 1987; Clark and Clegg 1990; Ranker et al. 1990; Givnish et al. 1990; Terry et al., in press), examinarelationships within Tillandsitions of phyloge~~etic oideae have been few. Consequently, little is known regarding the monophyly of most taxa in the subfamily (sensu Smith and Downs 1977). Of the tillandsioid genera recognized by Smith and Downs (1977; Cntopsis-20 spp., Glorneropitcnir??in-2 spp., Mezobuornclin-11 spp., G~izrnai~in-200 spp., Tillai~dsin-550 spp., and Vriesca-230 spp.), Catopsis and Glomeropitcaiu~~ia are the most well
defined. Each is delimited by several distinctive features. These include septa1 nectary anatomy and pollen morphology in Cntopsis (BDhme 1988; Halbritter 1992; G. Brown unpubl. data), ovary, fruit, and trichome characteristics in Glomeuopitcninzia (Tomlinson 1969; Smith and Downs 1977; Gilmartin and Brown 1986b), and seed characteristics in both genera (Szidat 1922; Smith and Downs 1977; Gross 1988; Gilmartin et al. 1989). Catopsis was originally described by Grisebach (1864) and placed in the tribe Tillandsieae with several other genera currently assigned to Tillandsioideae and Pitcairnioideae (Smith and Downs 1977, 1979). Harms (1930) emphasized seed characteristics in recognizi~~g Cntopsis as a monotypic tribe in his subfamily Tillandsioideae. All subsequent treatments have followed Harms (1930) in placing Catopsis in Tillandsioideae. Glomeropifcniui~in was first described by Mez (1896) as a subgenus of Pitcnirrzia (Pitcairnioideae). Harms (1930) emphasized the plumose seed appendages characteristic of the
[Volume 22
SYSTEMATIC BOTANY
\
seed appendage
superior ovaries
F
1
mostly epiphyt~c
/ FIG. 1. Implicit relationships in Tillandsioideae according to the classification of Smith and Downs (1977) with the principal diagnostic features. Superior ovaries (except in Glorrirropitcairii~a),c apsular fruits, and seed appendaged with a coma of fine hairs are defining features of the subfamily. 111 addition, nearly all members are epiphytic. The major divisions of the subfamily include the delimitation of the core tillandsioids (Tillaizdsin, Vriesea, Giizrriaiiia, and Mezobrorrirlin) based on basal seed appendages, and the distinction of Tillnlidsin and Vriwea from G~izrrianiti and Mezobrorrirlin based on free or conglutinate petal clarvs, respectively. Both Vliesea and Mezobroiiielin are distinguished from their generic counterparts by the presence of petal appendages.
genus in placing Glorneropitcairnin in the monotypic tribe Glomeropitcairnieae of Tillandsioideae. Mez (1935) and Hutchinson (1973) followed Harms (1930)in maintaining the tribal status of Glomeropitcairniene. Smith and Downs (1977) maintained Gloirieropitcnirnin within Tillandsioideae but did not recognize any tribal-level classification. Cladistic a ~ ~ a l y sof e smorphological features (Gilmartin et al. 1989) and 11dllF sequences (Terry et al., in press) support the inclusion of Glome~opitcniri~ia in Tillandsioideae. Alternatively, parsimony analysis of restriction site variation in the chloroplast genome suggests that Glomeroy7itcniri1ia warrants recognition as a monotypic subfamily (Ranker et al. 1990). In contrast to the distinctiveness of G/omeropitcai~for most of the nin and Catopsis, circumscriptio~~s remainder of Tillandsioideae are based on one or two characters of questio~~able diagnostic utility
(Smith and Downs 1977; Gardner 1986; Evans and Brown 1989; Brown and Terry 1992). Particularly has been reliance on petal appendco~~troversial ages in delimiting genera (Benzing 1980; Beaman 1989; Brown and Terry 1992; see Fig. 1). The unreliability of this feature is underscored by revisions that have concluded that "petal appendages have proven unacceptable as a delimiting character in Bromeliaceae where groups of closely related species are segregated solely on the basis of this character" (Smith and Spencer 1992). Read (1968) has documented variability in the occurrence of petal appendages among Costa Rican vrieseas, and the inconspicuous nature and poor preservational qualities of the character commonly result in misidentifications, particularly between Tillandsin and Vricsea. A study of petal appendage morphology and de.rlelopment suggests that variability in
TERRl ET AL.. NDHF PHYLOGEUY
Ti1landsia Vriesea Catopsis Guzmania Mezobromelia Glomeropitcairnia Aechmea Vellozia FIG.2. Phylogenetic relationships among the genera of Tillandsioideae according to Gilmartin et al. (1989). Length = 36, consistency index = 0.778. The tree was rooted rvith Vel1o:ia (\7elloziaceae), Characters with states are provided along the branches and are as follows: 1-ovaries superior; 2-seed appendage plumose and basally attached, at least in part; 3-spikes or branches mostly distichous flowered; 4-foliar scale disk with wings large and conspicuous; 5-foliar appendages symmetrical; 6-flowers mostly sessile; 7-umbrella-type stigma; 8-petal clarv appendaged; 9-seed coma color brown; 10-sepals always symmetrical; 11-petal claws agglutinated; 12-florvers mostly pedicellate; 13-stamen filaments usually all adhering to petals; 14-gummosis present; 15-stigma type simple-erect and convolute-blade. Figure redrawn from Gilmartin et al. (1989).
this character may be most appropriately used at the species level (Brown and Terry 1992).Similarly, generic pairs separated strictly by the presence or absence of conglutinate petal claws, i.e., Tillandsin/ may be artificial Vuiesen vs. G~izmni~ia/Mezobuornelia, (Smith and Downs 1977).The "core tillandsioids," i.e., Tillnndsia, Vuiesca, Guzmania, and Mezobuomelia, have been delimited using seed appendages that are wholly or largely basal and straight at maturity (Smith and Downs 1977). Figure 1 presents the generic-level classificationof Tillandsioideae (sensu Smith and Downs, 1977)with the principal diagnostic features. Results from the only previous cladistic study of tillandsioid genera (Gilmartin et al. 1989; see Fig. 2) support the monophyly of Tilla~ldsioideae as circumscribed by Smith and Downs (1977). The results were consistent with traditional taxonomies in supporting sister-group relationships for Tillandsia and Vuiesen, and for Guzlrlnnia and Mezobuomelia. Appendaged petal claws, brown seed comas, and symmetrical sepals all supported a clade containing Gloirie~opitcairnia,Glizmai~ia,and Mezobroiriclia, and floral architecture and stigma morphology resolved Cntopsis at the base of this group. Two primary lineages were identified in the subfamily: 1) Tillandsia and Vriesea, and 2) Cntopsis, Gloiric~opitcaiui~ia,Guzmania, and Mczobuolrlelin (Gilmartin et al.
1989; Fig. 2). The monophyly of individual genera was not addressed (Gilmartin et al. 1989). Phylogenetic relatio~lshipsamong the subgenera of Tillandsin and Vuiesca have been addressed using morphological features in tw7o previous studies (Gilmartin 1983; Gilmartin and Brown 1986a). A parsimony analysis of T ~ l l a n d s ~and n Vrzcsca identlfied a clade contaming four subgenera of Tzllandsia Pseudocatopsis, Diapho~anthema, P h y f a ~ u l ~ i z aand , Anoplophyti~m(Gilmartin 1983; see Fig. 3). PhytarDiaphorl~izawas sister-group to a clade contai~li~lg rai~thcmaand Pseudoentopsis, and Anoplopl~ytumwas these three the sister-group of the clade conta~n~ng subgenera Rllands~asubg Tillandsin and T subg Allaudtin, as well as Vulesen subg Vulesca and V subg - Alcai~tauca, were resolved as sister-groups Relatio~lshipsamong the three groups of subgenera identified in Gilmartin (1983) were unresolved (Fig.3),and all terminal units (i.e., subgenera) were assumed to be monophyletic. In contrast to the previous findings, Gilmartin and Brown (1986a) found a sister-group relationship for Pseudocatopsis and a subgroup of Phytnrrhiza. Diaphornntl~eirinand Pseudocatopsis were indicated as monophyletic, and Phytnrrhizn was paraphyletic (Gilmartin and Brown 1986a; see Fig 3). We have used n d l ~ F sequences to examine phylogenetic relationships in Tillandsioideae (Terry
SYSTL\IATIC BOTANY
Vriesea
FIG. 3. Phylogenetic relationships among the subgenera of Tillni!dsin and Vricsrn according to Gilmartin (1983) and Gilmartin and Brown (1986a). A. M'agner unrooted tree showing relationships among the seven subgenera of Tillnnds!~ and the two subgenera of Vriesnl. Redrawn from Gilmartin (1983). B. A tree summarizing relationships among Tillaizdsin subgenera Pseiidocntoysis, Phytnvrliizn, and Dinphoinritiiciiin according to Gillnartin and Bra\\-n (1986a).
and Brown 1996).The ilCil~Fgene in tobacco is 2,200 base pairs (bp) and putatively encodes one of several subunits of a chloroplast NADH dehydrogenase (Sugiura 1992; Olmstead et al. 1993).Comparisons of nucleotide sequences from the chloroplast genome indicate that 11dhF has a substitution rate about two times higher than that of vbcL (Wolfe 1991). Co~~sidering both mutation rate and length, n d h F has the potential to yield three times as many
phylogenetically informative characters as ibcL, an expectation that has been realized in recent studies (e.g., Scotland et al. 1995; Olmstead and Reeves 1995).Moreover, insertion-deletion events are relatively frequent in n d h F , and these have proven to be phylogenetically informative (Scotland et al. 1995; Olmstead and Reeves 1995). Previous studies have used 1zdl1F sequences to resolve phylogenetic relationships in several families of flowering plants
19971
TERRY ET AI.: NDHF PHYLOGENY
337
TABLE1. Sources of chloroplast DNAfor species included in this study.
Bromeliaceae subf. Bromelioideae Aron~elins p Bromeliaceae subf. Pitcairnioideae Broccilirii~?nciimiilni~?L. B. Sm. Nnvin splerideizs L. B. Sm. Bromeliaceae subf. Tillandsioideae Cntopsis ?unn,yeriniiMez & Werckle ex Mez ~1oineropitcn;vriinpendul$orn (Griseb.)Mez Giizinnni~?inoriostnchin (L.) Rusby ex Mez Guzrn~?riinilicnr~?guerisisMez & C . F. Baker ex Mez Giizrn~1riinrhoiiilqfiailn Harms Giizir~niiiasnnguinen (Andre) Andre ex Mez Giiziiinriin spect~?bilis(Mez & Werckle) Utley Giiziri~?iii~? withnnckii (Andre)Andre ex Mez h.Iezobron~elii*pleiosticlln (Griseb.)Utley & H. Luther Tillnnds~~? bevgeri Mez Till~111dsin complnri~?tnBenth. Tillnndsi~?dodsonii L. B. Sm. Tillnr~dsi~?JI.nse,.i Baker Till~?ndsinJiirickinrinBaker Tillnildsin geiniriyor~?Brongn. Tillnildsin seciiridn Humb., Bonpl., & Kunth %ll~?iidsintricilolepis Baker Tillnridsin iitriciilntn L. I7viesen espinosne (L. 8. Sm.) Gilmartin Vriesen glndiolif?orn (H. Wendl) Antoine Vriese~?glutiilosn Lindl. I+iesen i~znlziilriE. Morren I+iesen spleiider~s(Brongn.) Lem. Vriesm virid$ovn (Regel) Wittm. ex Mez I7viesen aiitntn (Mez & Werckle) L. 8. Sm. & Pittendr.
(Olmstead and Sweere 1993; Clark et al. 1995; Kim and Jansen 1995; Olmstead and Reeves 1995; Scotland et al. 1995; Terry et al., in press). In this paper we assess support for the phylogeny presented by Terry and Brown (1996), address in particular the monophyly of Tillandsioideae sensu Smith and Downs (1977), and examine relationships among genera and subgenera in t l ~ esubfamily We then compare our results to taxonomic treatments and morphologically-based cladistic studies of Tillandsioideae.
Twenty-five species representing all tillandsioid genera and seven of the nine recognized subgenera from Tillili7dsia and V ~ i e s e u(Smith and Downs 1977) were sampled in this study (Table 1). A study of phylogenetic relationsl~ipsamong bromeliad subfamilies using n d k F sequences identified three
Avoiiw 3128 (RM) SEL 81-1937 (SEL) SEL 83-02851 (SEL)
Pnlnci 1235 (RM) Giaiiish s.n. (WIS) SEL 82-0225 (SEL) SEL 86-0773 (SEL) SEL 80-1130 (SEL) SEL 80-0572 (SEL) SEL 90-0661 (SEL) SEL 73-0001-038 (SEL) SEL 81-1986 (SEL) B Y O ~ L3217 ' I I (RM) SEL 79-051 9 (SEL) B Y O T V321 ~ I 8 (RM) A Y O ~ L291 ' I I 0 (RM) Avoiiw 3219 (RM) SEL 81-1942 (SEL) B~oiilri2909 (RM) SEL 81-1293 (SEL) B u I ~ L3211 , ~ ~ (RM) A u ~ i i w3207 (RM) SEL 80-0485 (SEL) SEL 86-0303 (SEL) SEL 78-0757 (SEL) SEL 90-0817 (SEL) SEL 90-0282 (SEL) SEL 91-0138 (SEL)
principal lineages in addition to Tillandsioideae (Terry et al., in press). All searches included here were rooted with n d k F sequences of representatives from each of these three lineages (i.e., Brocchii7ii1, Pitcairnioideae, Bromelioideae; see Terry et al., in press). GenBank accession numbers and voucl~er specimens are listed in Table 1. Detailed summaries of methods for DNA extrace chain tion, i7dkF amplification using t l ~ polymerase reaction (PCR), and sequencing have been described (Terry et al., in press). Briefly, genomic DNAs were isolated using either t l ~ emethod of Doyle and Doyle (1987) or t l ~ e organic-stepgradient procedure of R. Wallace (pers. comm.). Extracted DNAs were amplified for singlestranded DNA using a t ~ 7 ostep procedure. First, n d k F was amplified in two partially overlapping segments using t l ~ ePCR and equimolar concentrations of b o t l ~forward and reverse primers. Each of the resulting double-stranded products was used as
338
SYSTEMATIC BOTANY
template in two subsequent PCR amplifications in which one of eacl~of the primers used in the original PCR was limiting (Kaltenboeck 1992).The resulting single-stranded products were sequenced using the dideoxy method of Sanger et al. (1977) and a-35s dATP, Partial sequences were initially obtained for Tilluiidsiu fvflseri and Vriesea z~ittfltu using the primers of Olmstead and Sweere (1994). All other taxa were sequenced using modified versions of previously described primer sequences (Olmstead and Sweere 1993) as well as primers designed specifically for use in Bromeliaceae (see Terry et al., in press). Seq~~ences were aligned by eye and analyzed using PAUP, version 3.1.1 (Swofford 1993). Insertions were encountered in only two sequences relative to the rest, and were not included in cladistic analyses. Sequence between positions 38 and 2,110 was obtained for most taxa providing a total aligned length of 2,073 bp. Searches for most parsimonious trees, bootstrapping, and decay analysis were conducted using the HEURISTIC searcl~ option and the TBR (tree-bisection reconnection) swapping algorithm. All analyses were performed with equally-weighted characters, as recent studies of tzdhF data indicate that differences in substitution probabilities among codon position classes are statistically insignificant for phylogenetically informative sites (Olmstead et al., in press). While transitions are present at a frequency that is about twice that expected at random, a weigl~tingscl~eme based on different probabilities of substitution between transformation classes (i.e., transitions and transoersions) is complicated by lleterogeneity in substitution probabilities within classes (Olmstead et al., in press). Thus, it is our preference not to weight either by codon position or transformation class in phylogenetic analyses of ndhF data. Searcl~esfor islands of most parsimonious trees (Maddison 1991) were conducted using 500 replicates of random order entry addition of taxa, saving all optimal trees. One l~undred replicates of bootstrapping were performed, saving 100 trees per replicate. Due to t l ~ eprohibitively large number of trees, decay analysis included results only from all trees up to two steps longer than the most parsimonious. The stability of individual clades was examined using sequential character removal (SCR)analysis, in which each of the 53 phylogenetically informative positions was successively removed and t l ~ eremainder of the data analyzed (excluding invariant positions; see Davis et al. 1993). Unresolved nodes in the SCR analysis were
(Volume 22
identified by comparing t l ~ estrict consensus of the 53 replicate analyses to that resulting from analysis of the complete matrix (Fig. 4).
Of the 2,073 nucleotide positions obtainable using t l ~ edescribed amplification and sequencing strategy, 97% ( Z 2.8% S.D.) were sequenced for at least one strand, 67% ( Z 13% S.D.) of which were confirmed by sequencing t l ~ e complementary strand. Of the 2073 positions analyzed, 1897 (92%) were invariant, 176 (9%)were variable but phylogenetically uninformative, and 53 (3%)were phylogenetically informative. Variable positions were most frequent in the 3' half of t l ~ egene, with 110 (63%) occurring between positions 1,059 and 2,110. Alignment gaps were encountered in only t ~ 7 osequences relative to the rest, with the same six bp sequence (5'-ATAGAT-'3) inserted in both Tillili~dsiafrilseri and T . d o d s o i ~ i at i position 1,705. While this was not used as cl~aracter evidence for the phylogeny reconstruction, this result is consistent with a single origin of this insertion in t l ~ emost parsimonious trees obtained on t l ~ ebasis of substitional data only (Fig. 4) and increases the strength of the conclusion that the two species are closely related. Sequence divergences were low within Tillandsioideae, ranging from 2.1% between Cntopsis zilaiigeri?~ii and Tillaidsia tricholepis, to 0.2% between several species of Glizmi?i~ii?, Mezobroiil~'lia, and Vriesei?. A maximum divergence of 2.5% ~ 7 a sobserved between Tillaiidsii? tvicholepis and Bronzelin sp. (Bromelioideae). Phylogenetic analysis of tzd1lF sequences produced six most parsimonious trees of 216 steps having a consistency index (CI) of 0.68 (excluding autapomorpl~ies),and a retention index (RI)of 0.79. All shortest-lengtl~trees were from a single island. Figures 3 and 5 show the strict consensus tree and one of the six most parsimonious trees, respectively (see also Terry and Brown 1996). 11d11Fsequences identify two principal groups of Tillandsioideae (see Fig. 4): 1) a clade corresponding to the Tillandsioideae sensu Smith and D o ~ r n s (1977), and 2) a clade corresponding to the core tillandsioids. Tillandsioideae sensu Smith and Downs (1977) and t l ~ ecore tillandsioids are supported by bootstrap values of 90% and 99% respectively, and both are resolved in the strict consensus of all trees two steps longer than the most parsimonious. Otl~ergroups well supported by I I L ~ ~sequences F include Tilliltzdsia f~~i7ckiai7il
19971
TERRY ET AL.: K~~~ P H Y L O G E ~ V
Brocchinia acuminata Navia splendens Bromelia sp.
1
Pitcairnioideae
r] Brornelioideae
Catopsis wangerinii Glomeropitcairnia Guzmania monostachia Guzmania nicaraguensis Guzmania sanguinea Guzmania rhonhofiana Guzmania spectabilis Guzmania wittmackii Tillandsia secunda
7subg, ~
Tillandsia utriculata
7 subg.
l l ~ ~ d t i ~
Tillandsia
2.
v v 7 sect, Vriesea Tillandsia tricholepis 7 subg . Diaphoranthema Tillandsia bergeri 7 subg. Anoplophytum E.
Vriesea espinosae
P,
P'
2'
Tillandsia geminiflora 7 subg . Anoplophytum Tillandsia complanata
7 subg. Allardtia
Tillandsia dodsonii
7subg, phytarrhiza
Tillandsia fraseri Tillandsia funckiana Vriesea malzinei Vriesea splendens
P,
7 subg . Pseudocatopsis 7 subg. Tillandsia 3 sect. Xiphion 7 sect. vriesea
Mezobromel ia Vriesea gladioliflora Vriesea vittata Vriesea viridiflora Vriesea glutinosa
1
sect. Xiphion
-
FIG.4. Strict consensus of six most parsimonious trees of 216 steps (CI = 0.68; RI = 0.79 ). Numbers above and belorv the internodes are bootstrap values and decay indices, respectively. Clades marked by an asterisk are unresolved in the strict consensus tree resulting from sequential character removal analysis. The dark bar indicates the inferred position of the six bp insertion. Subfamilial, generic, subgeneric, and sectional alignments are according to Smith and Dorvns (1977). Sections Xiphiori and Vriesen are both included in Vriesen subg. Vriesm.
(subg. Tillutzdsiu) and Vriesea nzillzitzei (subg. Vvieseil sect. Xiphiotz), Tillui7dsii? tricholepis (subg. Diuphovai7themn) and Tilluizdsii? bergeri (subg. Aizoplophytl~n~), and a component of Vrieseil subg. Vvieseu sect. Xiphi017 ( V . gludiol$loru, V . ~ittfltil,and V . z~iiid@oifl). Bootstrap values for these clades are 96%, 95%, and 98% respectively, and each is resolved in the strict consensus of all trees two steps longer than t l ~ e most parsimonious. A clade containing Tilluizdsii? utvicl~luti? (subg. Tillu?~dsii?)and Vvieseu espinosue (subg. Vvieseil sect. Vvieseil) is supported by a
bootstrap value of 79% and is resolved in t l ~ estrict consensus of all trees one step longer than the most parsimonious. All polytomies in Fig. 3 are due to the absence of data, with the exception of that encompassing the Tillui7dsiu fui7ckii1tzuaVrieseu nzulzi?lei clade. This group is resolved in some of t l ~ e sl~ortesttrees at t l ~ ebase of the polytomy largely consisting of Tilli?i7dsiu and GlLzmi?tzii? (Fig. 5). Parsimony analysis of IILI~IF sequences indicates that Tillu?~dsiu,Viieseu, and Guznli?tzii? (sensu Smith and D o ~ r n s1977) are polypl~yletic.In addition,
340
[Volume 22
SYSTEMATIC BOTANY
Brocchinia acuninata Navia splendens Bromelia s p .
I
,
1
IL
17
Pitcairnioideae
Brornelioideae
Catopsis wangerinii Glomeropi t c a i r n i a Guzmania m o n o s t a c h i a Guzmania n i c a r a g u e n s i s Guzmania s a n g u i n e a
I
-Guzmania r h o n h o f i a n a Guzmania s p e c t a b i l i s Guzmania w i t t m a c k i i
I
r
T i l l a n d s i a secunda
subg . A l l a r d t i a
Tillandsia utriculata
subg . T i l l a n d s i a
Vriesea espinosae
sect. Vriesea
Tillandsia tricholepis
subg . D i a p h o r a n t h e m a
Tillandsia bergeri
subg . A n o p l ophytuni
Tillandsia geminiflora
subg . A n o p l o p h y t u n i
T i l l a n d s i a complanata
subg . A l l a r d t i a
Tillandsia dodsonii
subg . P h y t a r r h i z a
Tillandsia fraseri
subg . P s e u d o c a t o p s i s
T i l l a n d s i a funckiana
subg . T i 1 l a n d s i a
Vriesea malzinei
s e c t . X i p h i on
Vriesea splendens
sect. Vriesea
Vriesea gladioliflora Vriesea v i t t a t a V r i e s e a v i r i d i fl ora
sect.
Vriesea glutinosa Mezobromelia 2
_i
FIG.5. One of the six most parsimonious trees (length = 216, CI = 0.68,RI = 0.79). The number of supporting character state transformations inferred from ACCTRAN optimization is given below the branch. The dark bar indicates the inferred position of the six bp insertion. Subfamilial, generic, subgeneric, and sectional alignments are according to Smith and Downs (1977).Sections Xiphioii and Eiesen are both included in Eiesen subg. E i e s e ~ ? .
none of the subgenera and sections of Tillandsia and V~iesei?,for which more than a single species is sampled, is indicated as monophyletic. These include Tilla?~dsii?subg. Ai70plophyt1~nz, T. subg. Allardtia, and T . subg. Tilla?~dsii?,and sections V~iesei?and Xiphiotz of Vriesea subg. Vriesea. The strict consensus tree indicates sister-group relationships for species of the follo~7ingsubgenera and sections of Tilla?~dsiaand V~ieseasensu Smith and and T . subg. Downs (1977): T , subg. Atzoploph~t~~rrl Diapho~i?i7thenza, T . subg. Allardtia and a clade and T. containing species of T . subg. Atzoplopkyt~~rr~
subg. Diapkora?~tkerrla,T . subg. Pkytarrkizil and T. subg. Pseudocatopsis, and T. subg. Tillatzdsia with a species of sections Xipkio?~and Vrieseil of V . subg. Vriesea.
The study of phylogenetic relationships in Bromeliaceae using molecular metl~odshas been addressed in three previous studies, none of which has produced well supported relationships (Ranker et al. 1990; Clark and Clegg 1990; Terry et al., in
19971
TERRY ET AL.: NDHF PIIYLOGENY
press). Results presented here are consistent with those of previous studies in that the number of phylogenetically informative characters is low compared to other family-level groups in which variability in the same loci or genome has been assessed. A comparison of phylogenetic reconstructions in Bromeliaceae using izdhF and rbcL sequence data indicates that the number of informative characters provided per kilobase (kb) of aligned sequence is nearly identical for these loci (results not presented). A similar result was obtained in phylogenetic studies of Solanaceae (Olmstead and Sweere 1994). Conversely, 11dhF has yielded about twice as many informative characters per kb as rbcL in studies of Acanthaceae (Scotland et al. 1995) and Scrophulariaceae (Olmstead and Reeves 1995). These observations indicate that the number of informative characters provided by ndiiF, and presumably the relative rates of nucleotide substitution, can vary widely among lineages. The low level of variation in ubcL, ndiiF, and restriction site data (Clark and Clegg 1990; Ranker et al. 1990; Terry et al., in press) may indicate a conservative rate of evolution in the bromeliad chloroplast genome, relative to the degree of morphological diversification in the family (also see Gaut et al. 1992). Despite weak support for many branches in the iidhF phylogeny, several relationships presented herein are consistent with traditional perspectives on the taxonomy and phylogeny of Tillandsioideae. Taxonomists have been faced with two primary difficulties in treatments of the group. The first is defining the limits of the subfamily, i.e., should distinctive genera like Gluii~erupitcairniaand Catopsis be included in Tillandsioideae? The second is establishing natural limits for what appears to be a closely related group comprising the core tillandsioids (Tillaiidsia, Vuiesea, Gtrzinania, and Mezobrome-
lia). Harms (1930) was the first to recognize subfamilies in Bromeliaceae, and Tillandsioideae was erected based on the superior ovary, capsular fruit, and appendaged seed, characteristic of most species in the group. Gluineuupitcaiui~ia and Catopsis were accommodated by recognizing each in a monotypic tribe (Harms 1930).Although tribal and generic concepts in Tillandsioideae have changed considerably, the circumscription of the subfamily sensu Harms (1930) has remained stable in subseq ~ e n ttreatments (Smith 1955; Hutchinson 1973; Smith and Downs 1977; Smith 1988). Support for the monophyly of Tillandsioideae sensu Smith and Downs (1977) in the iidhF phylogeny is consistent
341
with: 1) taxonomic treatments that have recognized the subfamily based on superior ovaries, capsular fruits, trichome anatomy, and growth habit (Tomlinson 1969; Smith and D o ~ r n s 1977), and 2) cladistic analysis of morphological features (Gilmartin et al. 1989). Although the monophyly of Tillandsioideae is not supported by the chloroplast restriction site study of Ranker et al. (1990), a study of subfamily relationships in Bromeliaceae using izdhF sequences suggested that this finding is a sampling artifact (Terry et al., in press). If Tillandsioideae sensu Smith and DOM~IIS (1977)is monophyletic, then many of the distinctive features of Gloineropitcairiiia and Catopsis are autapomorphic. The species of Gluineuupitcairnia have partly inferior ovaries, partially indehiscent capsules, and seeds that are appendaged equally both apically and basally. In addition, the trichomes of Gloineropitcaiuiiia have from three to nine stalk cells, a condition considered ancestral in Tillandsioideae (Tomlinson 1969). Catopsis is the only tillandsioid genus that has seed comas that are apical and folded at maturity Moreover, distinctive pollen morphology (Halbritter 1992) and septa1 nectary anatomy (Bollme 1988) have been documented in Catopsis, and the genus is one of a few in Bromeliaceae that possess monoecious taxa (Smith and Downs 1977). The placements of Catopsis and Gluinerupitcairiiia in this study differ from those in a previous study of subfamily relationships using iidhF sequences (Terry et al., in press). In this study, representatives of these genera are resolved as sister groups (Fig. 4), as opposed to being nested at the base of the subfamily (see Terry et al., in press). Moreover, confidence limits around the branch supporting Tillandsioideae (sensu Smith and Downs, 1977) are higher in this study (i.e., bootstrap and decay values of 90% and >2 respectively, as opposed to 64% and 1 in the subfamily study). These results may reflect differences in taxon sampling and outgroup choice between the studies. However, neither hypothesis of relationship for Catopsis and Gloineropitcairiiia is well supported by 11dlzF sequences, and the influence of cladistic error on one or both alternatives may be substantial. Support for a monophyletic Tillandsioideae and strong support for the core tillandsioids in both studies suggests Catopsis and Gluii~erupitcairiiiadiverged early in the phylogenetic history of Tillandsioideae.This contention is consistent with generic-level treatments of the subfamily (Fig. 1) and the morphological distinctiveness of these genera.
342
SYSTEMATIC BOTANY
Although 11dhF sequences strongly support the monophyly of the core tillandsioids (i.e., bootstrap value of 99% and decay index of >2), several relationships within this group are either unresolved or poorly supported. The strict consensus tree (Fig. 4) identifies two principal groups within the core tillandsioids: 1) a clade containing a co~nponent of Vviesea subg. Vviesea sect. Xiphi011, and 2) a clade primarily consisting of Tillaiidsia and Guzrnania, but also containing two species of Vriesea (V. espinosae and V. inalziilei). Wellsupported relationships for Tillandsiafuilckia~aand Vriesea rrialzinei (bootstrap value of 96% and decay index of >2) and, to a lesser extent, Tillai~dsia utriculata and Vriesea espinosae (bootstrap and decay values of 79% and 2 respectively), indicate that both genera are paraphyletic. Generic groups (Tillai~dsia and Vviesea vs. Guzinai~iaand Mezubvuinelia) circumscribed by petal claw characteristics and floral architecture by Smith and Downs (1977; Fig. 1) and maintained in previous phylogenetic studies within Tillandsioideae (Gilmartin et al. 1989; Fig. 2) are not supported by ndkF sequences. Although previous studies have been critical of the use of petal appendages in circumscribing bromeliad genera (Brown and Terry 1992), the distribution of states for this character over the ndhF trees suggests that this feature may be useful, when combined with other characters, in circumscribing groups of genera in Tillandsioideae. Petal appendages are characteristic of Gloinevopitcairnia, Vviesea, and Mezobromelia, and S ~ n i t h and Dom~ns (1977) relied heavily on their presence or absence in distinguishing Vviesea from Tillandsia and Mezobrorrielia from Guziimiiia (Fig. 1). The taxor-tomic distribution of petal appendages over the 11dhF trees is consistent with their origination at the base of Tillandsioideae, and subsequent loss in Catopsis and in the clade consisting largely of Tillandsia and Gtrzmaiiia. The inclusior-t of Vviesea rrialzinei and V. espiiiosae within the Tillandsia-Guzina~iaclade may represent the independent gain of petal appendages in these taxa. Several relationships presented here are consistent with those of studies examining infrageneric phylogeny in Tillandsia and Vviesea (Gilmartin, 1983; Gilmartin and Brown 1986a). Support for a clade containing species of Tillandsia subg. Pseudocatupsis and T. subg. Pkytarrhiza in the ndkF phylogeny substantiates the cor-tter-ttionthat these subgenera are closely related (Gilmartin and B r o n ~ ~ 1986a; Fig. 3). Although the branch supporting this relationship in the ~nolecularphylogeny is weakly sup-
[Volume 22
ported (bootstrap and decay values of 52% and 1 respectively), the presence of a six bp ir-tsertior-t at position 1,705 in Tillai~dsiafraseri (subg. Psetrdocatupsis) and Tillandsia dodsonii (subg. Phytarrkiza) suggests a common ancestry for these species. Strong support in the ndhF phylogeny for a clade cor-ttair-ting Tillandsia tricholepis and Tillandsia bevgeri indicates a close relationship for T. subg. Diaphoraiitkeina and T. subg. Ai~opluphyturnrespectively. This contention is supported by the life-history characteristics of species in these subgenera (i.e.,Ai~oplophyttrin and Diaphovantherria are the only subgenera of Tillandsia that are exclusively xeric; Smith and Downs 1977; Gil~nartin1983). However, a sistergroup relationship for Ai~oplophyttrinand Diaphoraiitkeina was not substantiated by parsi~nonyanalysis of morphological features (Gilmartin, 1983; Gilmartin and Brown 1986a; See Fig. 3). This discrepancy could be attributable to the paraphyly of Ai~oplophyturn, which has been suggested by morphological analysis (Evans and Brown 1989), and is maintained by results presented here (Fig. 4). Smith and Downs (1977) recognized sect. Xiphion of Vviesea subg. Vviesea as having stamens included or equaling the corolla, and floral bracts mostly dull green or brownish. Most of the species traditionally assigned to V. sect. Xiphion and included in this study are resolved as a ~nonophyleticgroup at the base of the core tillandsioids (Fig. 4). A more strongly supported clade in the molecular phylogeny contains three species of sect. Xipkioii (V. gladioliflova, V. oittata, and V. airidiflora; Fig. 4). Although traditional characters provide little fou1-tdation for the strongly supported clade, two of the species included in this group (V. gladioliflova and V, clzrldflora) . have cupulate stigmas (Brown and Gilmartin 1989), while stigma morphology is undocumented in the third member of the clade (V. oittata). In contrast, Vviesea glutii~osahas the cor-tvolute-blade stigma type, and V. inalziiiei has a stig~na ~norphology (simple-erect) which appears to be rare in Vriesea (Brown and Gilmartin 1989). This study is the first to address phylogenetic relationships in Tillandsioideae using molecular data. While the low substitution rate in cpDNA in Bromeliaceae does not provide complete resolution of the tree, the consistency among characters is high (CI = 0.87 for all characters), and some important conclusions are well-supported. These include support for a ~nonophyleticTillandsioideae sensu Smith and Downs (1977), resolution of the clade cor-ttair-ting Catopsis and Glorneropitcairilia as the sister group to the remainder of the subfamily,
19971
343
TEI~RY ET AL.: NDHF PHYLOGENY
support for the monophyly of the core tillandsioids (i.e., Tillai~dsia,Vriesea, Guzrnania, and Mezobroinelia), and resolution of a clade containing a component of Vriesea subg. Vriesea sect. Xiphion. Moreover, well-supported relationships indicate that Tillandsia, Vriesea, subg. Tillandsia and subg. Aiiop1op/zyt~iiz of Tillandsia, and sect. Xiphion of Vriesea subg. Vriesea are polyphyletic as traditionally circumscribed (Smith and Downs 1977). The polyphyly of Gtrzinnnia, as well as close phylogenetic relationships for Tillaiidsia and G ~ ~ z i n a n iMezobromelia a, and a component of Vriesea subg. Vriesea sect. Xiphion, and Catopsis and Gloineropitcairi~ia,are suggested by these results. Resolution among some clades in the trees presented here is precluded by low sequence divergence. As ndhF is among the longest and most highly variable of chloroplast genes (Olmstead and Palmer 1994; Wolfe 1991), it is doubtful that sequence data from any single gene in the chloroplast genome will fully resolve relationships among some Bromeliaceae. Moreover, some relationships between and within all three bromeliad subfamilies were unresolved in a study that used restriction site data from the entire chloroplast genome (Ranker et al. 1990). Thus, greater resolution in phylogenetic studies of Bro~neliaceae likely will require the combining of data from various sources in a total evidence approach (Donoghue and Sanderson 1992; Ol~nsteadand Sweere 1994). We have not combined iidhF and morphological data here because a principal objective of this study was to examine the monophyly of groups delimited by ~norphological features. In addition, the paucity and questionable diagnostic utility of many morphological features used in traditional classifications of Tillandsioideae suggests that their inclusion in this study would add little to our understanding of relationships, as evidenced by discordance between taxonomic and phylogenetic limits for the three largest genera in the subfamily (i.e., Tillaiidsia, Vriesea, and Gtrzinania). This situation underscores the importance of additional data (molecular and morphological) collection and appropriate sampling if phylogenetic reconstructions using combined data are to be realized. ACKNOI\LEDCM~UTS. We thank H. Luther and the Marie Selby Botanical Gardens for providing access to their collections, C. Palaci, Unix7ersity of \\lyoming, for supplying material of Cntoysis, T. Givnish and K. Sytsma, University of Wisconsin, Madison, for pro\.iding Glonzeroj?itcriiriiin DhTA, and 7 . Givnish, an anonymous revieiier,
and Associate Editor Kellogg for many valuable comments on the manuscript. Support of the hrational Science Foundation (BSR-9108268 to GKB and BSR-9107827 to RGO) and the Bromeliad Society (to RGT) is gratefully acknoiiledged.
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