Aliso: A Journal of Systematic and Evolutionary Botany

Volume 19 | Issue 1 Article 5

2000 Phylogenetic Relationships in Phoradendreae () Inferred from Three Regions of the Nuclear Ribosomal Cistron. II. The orN th American Species of Vanessa E. T. M. Ashworth Rancho Santa Ana Botanic Garden; Claremont Graduate University

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Recommended Citation Ashworth, Vanessa E. T. M. (2000) "Phylogenetic Relationships in Phoradendreae (Viscaceae) Inferred from Three Regions of the Nuclear Ribosomal Cistron. II. The orN th American Species of Phoradendron," Aliso: A Journal of Systematic and Evolutionary Botany: Vol. 19: Iss. 1, Article 5. Available at: http://scholarship.claremont.edu/aliso/vol19/iss1/5 Aliso. 19(1) , pp . 41-53 © 2000, by The Rancho Santa Ana Botanic Garden, Claremont, CA 9171 1-3157

PHYLOGENETIC RELATIONSHIPS IN PHORADENDREAE (VISCACEAE) INFERRED FROM THREE REGIONS OF THE NUCLEAR RIBOSOMAL CISTRON. 11. THE NORTH AMERICAN SPECIES OF PHORADENDRON

VANESSA E. T. M. ASHWORTH' Rancho Santa Ana Botanic Garden and Claremont Graduate University Claremont. California 9J7JJ -3J57 USA e-mail: [email protected]

ABSTRACT

A parsimony analysis based on sequences from the ITS region and two partitions of the 26S subunit of nuclear ribosomal DNA was used to infer phylogenetic relationships among the North American species of Phoradendron. A strongly supported clade united all but one of the species typically lacking ca taphylls, a character used previously to distinguish the northern species from those of Central and South America. The divergent placement of P. califo rnicum rel ative to the members of this " northern" clade confirmed the hypothesis that species lacking cataphylls are polyphyletic. Four of five species parasitic on conifers formed a well-supported clade. However. a strongly supported relationship be­ tween P. rhipsa/inum and P. bra chystachyum, the former a parasite of conifers, renders con ifer par­ asitism homoplastic. A sister group relationship between these two species is not apparent from mor­ phological evidence. A clade unit ing P. serotinum, P. tomentosum, and P. velutinum was strongly supported. A broad host range characterized two of the three lineages of the basal trit omy in the northern clade, whereas the third line age united species specialized in parasitism of oaks or conifers. Key words: divergent domains, flowering period, host range, host specificity, internal transcribed spacer (ITS), nuclear ribosomal DNA, Phoradendron, phylogeny, Viscaceae, 26S.

INTRODUCTION cence are arranged in an opposite decussate pattern The two genera Phoradendron Nutt. and (Fig. 1). Inflorescence internodes vary in number from one (Fig. 2) to ca. 10. Each internode consists of two Dendrophthora Eichler together compose the New flower areas on opposite sides of the inflorescence axis World tribe Phoradendreae Engler. Both genera occur (Fig. 1). These flower areas are studded with tiny, ses ­ in Central America, the West Indies, and tropical lat­ sile, mostly trimerous flowers arranged in vertical rows itudes of South America. However, only Phoraden­ (series), the apical flower either remaining the only dron extends into the subtropical and temperate lati­ flower in its row (biseriate inflorescence; Fig. 3) or tudes of North and South America. Phoradendron spe­ crowning a central row (triseriate inflorescence; Fig. cies parasitize a wide range of angiosperms and less 4). The number of series cannot be discerned in spe­ commonly conifers. Trelease (1916) distinguished 240 cies where each side of the inflorescence internode species, but more recent estimates fluctuate between holds an insufficient number of flowers (one to four; some 100 species or less (Kuijt 1982) and over 200 e.g., Fig. 2). Among the Phoradendron species in species (1. Kuijt pers. comm.). This discrepancy is due North America, triseriate inflorescences are the rule, to the continual discovery of new species, but above the biseriate type predominating in species farther all to the difficulty in circumscribing species, reflecting south. an unusual morphology in which few characters can Vegetatively, the North American species of Phor­ be scored as discrete and many cannot be scored at all adendron are characterized by the predominant ab­ in a substantial subset of species. This paper represents sence of cataphylls (Fig. 5), These small leaflike or the second of two contributions on phylogenetic rela­ bractlike structures are inserted in pairs on the first tionships in Phoradendreae, the first dealing with ma­ internode of lateral branches (Fig. 6) and are typical jor clades and the second focusing on Wiens's revision of tropical species. However, occasionally they are (1964) of Phoradendron species in North America. formed in several North American species said to lack cataphylls. Usually, cataphylls are not associated with Morphology floral organs (Fig. 6), but some species produce fertile Phoradendreae are characterized by an articulated cataphylls that subtend inflorescences, Cataphylls dif­ spike, whereby successive internodes of the inflores- fer from foliage leaves by their much reduced size, In some species, however, foliage leaves are reduced to

I Current address: Department of Botany and Sciences-072, scales. The distinction between scalelike foliage leaves University of California, Riverside, Calif. 92521-0124 USA . and cataphylls is then often difficult to diagnose. 42 Ashworth ALISO

flower

3 1 mm 4

fused bracts

2 1 mm

cataphylls inflorescence main axis " . axis ......

flower internode node

bract 5 1 cm 6

main axis median . ~ . foliage leaf O~r;>'; cataphyll bracts

7 lateral branch transverse• main axis • .~ . foliage leaf bract o 0 c~ 1 1 mm lateral 8 branch Fig . 1-8. Morphological characters in Phoradendron.-1-4 Inflorescen ces.-1-2•. Degrees of elaboration•of an articulated spike.-l. Spike consisting of three internodes in opposite decussate orientation. Each internode contains two flower areas on opposite side s of the inflorescence axis . Individual internodes are subtended by paired bracts.-2. Inflorescence in its simplest form, consisting of a single internode with one flower per flower area .-3-4. Inflorescence seriation, showing one flower area of an inflorescence internode and the subtending bract. The number of vertical flower rows (series) determines the inflorescence type. -3. Biseriate inflorescence.--4. Triseriate inflore scence.-5-6. Cataphylls.-5. Cataphylls absent.--6. Cataphylls located near the base of a lateral branch (basal cataphylls).-7-8. Orientation of the lowermost foliar organs on later al branches in relation to the main branch axis (primary orientation).-7. Transectional view of the branch axis just above a node, showing a median orientation of the cataphylls.-8. Tran sectional view, showing a transverse orientation of the foliage leaves . VOLUME 19, NUMBER I North American Phoradendron 43

Primary orientation refers to the orientation of the cent evidence suggests that Phoradendron may not be lowermost (primary) foliar organs (cataphylls, if pre­ monophyletic unless Dendrophthora is included with­ sent, otherwise leaves) on lateral branches in relation in it (Nickrent and Duff 1996; Ashworth 2000). The to the main branch axis . In the majority of tropical 34 Phoradendron taxa include 19 North American rep­ species and several species in North America, the pri­ resentatives of Wiens's and Trelease's treatments, one mary orientation is consistently median (Fig. 7), species (P. rhipsalinum) that was unknown to Trelease whereas most North American species have a consis­ and Wiens, and 14 other species, primarily from Cen­ tently transverse orientation (Fig. 8). Two species, tral and/or South America and the West Indies (Table however, exhibit both orientation types. I). All collected by the author in Mexico were sent to J. Kuijt for identification. For convenience, no­ menclature follows Wiens (1964) for the North Amer­ ican acataphyllous species and Trelease (1916) for the Two taxonomic treatments (Trelease 1916; Wiens remainder, even though some nomenclatural modifi­ 1964) cover the species of Phoradendron in North cations have been made since then (e.g., P. serotinum America. Trelease's monograph of the entire genus is now P. leucarpum; Reveal and Johnston 1989). In places the majority of North American species in " Bo­ most cases, a species is represented by a single se­ reales," an informal group comprising 57 taxa (50 spe­ quence and for multiply sequenced species nucleotide cies). Its members are characterized by the absence of polymorphisms at a particular site are encoded in cataphylls. However, the monophyly of the North terms of standard ambiguity codes. However, multiple American species based on the absence of cataphylls sequences were retained for several species in order to has been questioned, in that certain predominantly aca­ represent separate subspecies (P. bolleanum, P. juni­ taphyllous species occasionally develop cataphylls perinum, P. tomentosum, and P. villosum) and geo­ (Kuijt 1996), while the acataphyllous P. californicum graphic differentiation (P. tomentosum from San Luis shares other morphological traits with southern species Potosi, Mexico, and Texas and P. velutinum from San (Kuijt 1959, 1996; Wiens 1964). Trelease placed P. Luis Potosi and Michoacan, Mexico). californicum, a scale-leaved species, together with two The sequences used in this study comprise the ITS other scale-leaved species in " Aphyllae," a subdivi­ region and two partitions of the 26S nuclear rDNA sion of section Pauciflorae Engler. (Fig. 9). Rapid sequence evolution in Viscaceae (Nick­ Wiens's revision (1964) is confined to the acata­ rent and Franchina 1990; Nickrent and Starr 1994; phyllous species of Phoradendron in North America. Nickrent et aI. 1994; Nickrent and Soltis 1995; Nick­ It covers the same suite of taxa as Trelease's "Bore­ rent and Duff 1996; Molvray et aI. 1999) prompted ales," whereby Wiens's 24 taxa (18 species) are a re­ the use of a composite gene region in which slower­ sult of placing many of Trelease's taxa in synonymy. evolving sequences enabled alignment with outgroups Wiens retained P. californicum in his revision, but and the more divergent ingroup species, while the fast­ placed it in monotypic section Phoradendron. The two er-evolving sequences resolved relationships between taxonomic treatments also differ in the placements of closely related taxa. A detailed account of the rationale P. longifolium, P. robinsonii, and P. scaberrimum in dictating taxon sampling, outgroup selection, and Pluriseriales Engler by Trelease (1916), but in Caly­ choice of an appropriate gene region is given else­ culatae Trelease by Wiens (1964). where (Ashworth 2000). Here, I develop a phylogenetic hypothesis for the Methodology pertaining to DNA extraction fol­ North American species of Phoradendron using mo­ lowed the 2X CTAB buffer protocol by Doyle and lecular sequences of the nuclear rDNA. The phyloge­ Doyle (1987), with DNA extracts diluted to a final netic framework is then used to compare the taxonom­ concentration of 10 ng/f..L1. Double-stranded DNA tem­ ic treatments by Trelease and Wiens in a phylogenetic plate was amplified by the polymerase chain reaction context. The distribution of several morphological and (Mullis et aI. 1986) using amplification conditions de­ life history characters reported in the literature (Wiens tailed in Ashworth (2000). The three DNA regions 1964; Kuijt 1996) on the molecular phylogeny is used were amplified and cycle sequenced using various to infer additional phylogenetic structure. combinations of forward and reverse primers ITS2, ITS3, ITS4, ITS4i, ITS5, ITS5i (White et al. 1990), MATERIALS AND METHODS 1643f (CCGCCCGTCGCTCCTACCG) and ITS4-172 Of the 43 taxa sampled, 34 belong to Phoraden­ (CCCAAACAACCCGACTCACCAACAG) (Ash­ dron, six to Dendrophthora sen su Kuijt (1961) and worth 2000) for the ITS region, and 641 r, 1715f and three are outgroup species belonging to Viscaceae 2134r (D.L. Nickrent), S I' and S2 (Bult and Zimmer (Korthalsella latissima) and (Acanthosyris 1993) for 26S rDNA. The two regions of 26S rDNA asipapote and Thesium carinatum). Dendrophthora (D2 and D8; Fig. 9) are delimited by primer pairs S2 species are considered part of the ingroup because re- + 641r and 1715f + 2134f, respectively. They corre- 44 Ashworth ALISO

Table I. Specimens used in the study. Unless otherwise stated, all are deposited at RSA. Nomenclature follows Wiens (1964) for species from North America and Trelease (1916) for the remainder. All species are Viscaccac, except for Acanthosyris asipapote and Thesium carinatum (Santalaceae).

Taxon Origin Voucher/ specimen GenBank acce ssion number

Acanthosyris asipapote M. Nee Bolivia: Santa Cruz Nickrent 405/, SIU AF181776 (02), AFI81819 (08) Dendrophthora clavata (Benth.) Urban Columbia: Boyaco Nickrent 2/82. SIU AFl78742, AF181770, AF181813 D. costaricensis Urban EI Salvador Villacorta /288, MO AFI78743 (ITS I), AFI78744 (lTS2), AF181771 (02), AF181814 (08) D. domingensis (Spreng.) Eichler Cuba Hermann 7/3/ AF178741 (lTS2), AFl81769 (02). AFI81812 (08) D. guatemalensis Standley Mexico: Chiapas Breedlove 23390 AF 178726, AF 181759, AFl81801 D. opuntioides (L.) Eichler Jamaica Cro sby et al. 285 AFI78739 (ITS I), AFl78740 (ITS2), AF181768 (02), AF181811 (08) D. squamigera (Benth.) Kuntze Costa Rica Schmid /979-/8 AFl78745, AF181772, AFl81815 Korthalsella latissima (Tiegh.) Dans . USA : Hawaii Molvray 309, MO AF181775 (02), AF181818 (08) Phoradendron bolleanum (Seem.) Ei­ Mexico: Sonora Columbus 2753 AFI78708 (ITS) chler subsp. bolleanum P. bolleanum subsp. densum (Torr.) USA: California Ashworth 59 AF 178705, AF 181740. Wiens AFl8l782 P. bolleanum subsp. paucijlorum USA: California Ashworth 42 AFI78706, AF181741, (Torr.) Wiens AFI81783 P. brachystachyum (DC) Nutl. Mexico: Michoacan Ashworth 273 AF178720, AFI81753, AFl8l795 P. brevifolium Oliver Mexico: Puebla Ashworth 282 AFI78724. AFI81757, AFI81799 P. californicum Nutt. USA : Arizona Ashworth 76 AF178728, AFI81761, AFl8l803 P. capitellatum Torr. ex Trel. USA : Arizona Ashworth 84 AFI78702, AFI81737, AF181779 P. carneum Urban Mexico: Michoacan Ashworth 27/ AF178732, AF181765. AFl8l807 P. crassifolium (Pohl ex DC) Eichler Peru Young & Jaramillo 2/76. MO AFI78746 (ITS I). AFI78747 (ITS2), AF 181773 (02), AFI81816 (08) P. forestierae Robinson & Greenman Mexico: Queretaro Ashworth 254 AF178723, AF18l756. AFI81798 P. galeottii Trel. Mexico: Oaxaca Lorence 462/ AF178716, AF181750, AF181792 P. heydeanum Trel. Honduras Forstreuter 9/56, MB AF178729, AFl81762, AF18l804 P. juniperinum Engelm. ex A. Gray USA : Arizona Ashworth 77 AF178703, AFI81738, subsp. juniperinum AFI81780 P. juniperinum subsp. Iibocedri (En­ USA: California Ashworth 37 AF178704, AFI81739, gelm.) Wiens AF181781 P. longifolium Eichler Mexico: Queretaro Ashworth 257 AF178717 (ITS) P. minutifolium Urban Mexico: Puebla Ashworth 280 AFI78707, AFI81742, AFI81784 P. nervosum Oliver Bolivia: La Paz Lewis 8869. MO AF 178734 (ITS I), AF 178735 (ITS2). AF181766 (02), AFI81808 (08) P. piperoides (Kunth) Trel. Puerto Rico Miller & Sherman 6439, MO AF178748 (lTSI), AF178749 (ITS2), AF181774 (02), AF181817 (08) P. reichenbachianum (Seem.) Oliver Mexico: Puebla Ashworth 285 AF178725, AFI81758, AFI81800 P. rhipsalinum Rzed. Mexico: Jalisco Ashworth 270 AF178719, AF181752. AFI81794 P. robinsonii Urban Mexico: San Luis POtOSI Ashworth 260 AF178718, AF181751. AFI81793 P. robustissimum Eichler Honduras Forstreuter 9/35, MB AF 178733 (ITS) VOLUME 19, NUMBER I North American Phoradendron 45

Table I. Continued.

Taxon Origin Voucher/specimen GenBank accession number

P. scaberrimum Trel. Mexico: Chihuahua Ashworth 100 AF17871S, AFI81749, AFI81791 P. serotinum (Raf.) M.e. Johnston USA : Illinois Ashworth 308. SIU AF178711, AF18174S, AFI81787 P. sulfuratum Rizzini Venezuela: Bolivar Steyermark 131692. MO AFI78737 (ITS I), AF178738 (ITS2), AF 181767 (02), AFI81810 (08) P. tamaulipense (Kunth) Krug & Ur­ Mexi co : San Luis Potosf Ashworth 261 AF178730, AF181763, ban AFl8180S P. tomentosum (OC) Engelm. ex A. USA : California Ashworth 58 AFI78709, AFI81743, Gray subsp. macrophyllum (En­ AF18178S gelm.) Wiens P. tomentosum subsp. tomentosum Mexico: San Luis Potosf Ashworth 263 AF17871O. AF181744, AFI81786 P. tomentosum subsp. tomentosum USA : Texas Roa/son 1225 AF178712, AF181746. AFI81788 P. cf. tonduzii Trel. Honduras Forstreuter 9154. MB AF178736, AFI81778, AFl81809 P. trinervium (Lam.) Griseb. Puerto Rico Struwe 1108. NY AF178731 , AFI81764, AFI81806 P. velutinum (OC) NUll. Mexico: San Luis POtOSI Ashworth 262 AF178721, AF1817S4, AFI81796 P. velutinum Mexico: Michoacan Ashworth 275 AFI78722. AF1817SS, AFI81797 P. vernicosum Greenman Honduras Forstreuter 9141, MB AFI78727, AF181760, AFI81802 P. villosum (Nutt.) NUll. subsp. coryae USA : Arizona Ashworth 82 AF178714, AF181748. (Trel.) Wiens AFI81790 P. villosum subsp. villosum USA : California Ashworth 62 AF178713, AF181747. AFI81789 Thesium carinatum A. Oe. South Africa Nickrent 4094, SIU AF181777 (02), AFI81820 (08)

26S nuclear ribosomal DNA ..... ------..... ITS region D2 region D8 region .....------., 18SJ . ~~~~~~~~~~~~~~I-IGS

I 5.8S 02 divergent 08 divergent ITS 1 ITS 2 domain domain

1000 nucleotides Fig . 9. Diagram of the ITS region and 26S large subunit of nuclear rONA drawn to scale. Gene regions sequenced in this study (ITS I and ITS2 spacers, the S.8S portion of the small-subunit nuclear rONA, and the 02 and 08 regions) are printed in bold. The 02 region is bounded by primers S2 and 641 r and the 08 region by primers 171Sf and 2134r. The 02 and 08 divergent domains (light gray) are regions with increased numbers of nucleotide substitution relative to the more conserved flanking sequences of the 26S nuclear rONA. They are located within the 02 and 08 regions (dark gray), respectively. 46 Ashworth ALISO spond to positions 1128-1495 and 2583-2983 in the nucleotides long. All but two are located in the ITS 26S sequence of Arabidopsis thaliana (GenBank region. X52320; Unfried and Gruendler 1990). Figure 10 depicts the strict consensus of four equal­ Sequences were generated on an Applied Biosystems ly most parsimonious trees (MPTs) of 2234 steps. Of 373A automated DNA sequencer using a Sequagel-6 a total of 37 ingroup nodes, 27 have bootstrap values polyacrylamide gel (National Diagnostics). The se­ of ~70%, 28 have decay values of ~3, and 25 have quences were assembled, edited, and combined into a both. A bootstrap value of 88% and a decay value of consensus sequence using Sequencher 3.0 (Gene Codes 10 support a clade, henceforth called the northern Corporation, Inc., Ann Arbor, MI). All sequences are clade (Fig. 10), including P. rhipsalinum and all mem­ available from GenBarIk and accession numbers are bers of Trelease's "Boreales" and Wiens' revision ex­ provided in Table I. Aligned sequences were submitted cept P. californicum. Phoradendron californicum ap­ to parsimony analysis using PAUP* version 4.0 131 pears as sister to a clade that includes not only mem­ (Swofford 1998). Several alternative alignments, in­ bers of the northern clade, but several other species cluding computer-assisted (ClustalW version 1.4; (Dendrophthora guatemalensis, P. brevifolium, P. fo­ Thompson et al. 1994) and visual alignments, were test­ restierae, P. reichenbachianum, and P. vernicosum) ed. The visually aligned matrix is archived in Ashworth that have cataphylls. The precise placement of P. cal­ (1999; Ph.D. thesis) and posted on the Internet at the ifornicum is uncertain owing to weak bootstrap sup­ website: http://www.science.siu.edu/parasitic-plants/ port (67%; Fig. 10) and a long branch (146 nucleotide AlignmentslPhor.align.htrnl. Parsimony analyses used substitutions; Fig. II). Interestingly, the branch decays heuristic searching with accelerated transformation op­ only when all trees more than five steps longer than timization and TBR branch swapping. Starting trees the most parsimonious solution are included in the were obtained by simple addition or 100 cycles of ran­ analysis. dom addition, and branch swapping restricted to the Within the northern clade, several clades receive best trees only. During branch swapping, the STEEP­ strong support (Fig. 10). The first of these combines EST DESCENT option was switched off. All optimal P. brachystachyum and P. rhipsalinum, the second P. trees were saved (MULPARS). serotinum, P. tomentosum, and P. velutinum (hence­ Branch support was calculated using 1000 bootstrap forth the P. serotinum clade), the third P. galeottii and replicates (Felsenstein 1985), as implemented on P. longifolium, the fourth P. villosum subsp. coryae PAUP* ver sion 4.0131 in the "Fast stepwise addition" and P. scaberrimum, and the fifth P. bolleanum subsp. function, and using the decay approach (Bremer 1988; bolleanum to P. capitellatum, seven taxa parasitic on Donoghue et al. 1992). Bootstrap values of at least conifers occurring in western North America (hence­ 70% are taken to indicate strong branch support (Hillis forth the conifer-parasite clade). In the P. serotinum and Bull 1993; but see Efron et al. 1996). Homoplasy clade, P. serotinum and P. tomentosum form a clade of the sequence data was calculated using the consis­ whose sister species is P. velutinum. Within the coni­ tency index (Kluge and Farris 1969; Sanderson and fer-parasite clade, three clades each unite (a) the two Donoghue 1989) and the retention index (Farris 1989). subspecies of P. juniperinum, (b) two of the three sub­ Gaps were coded as missing data. species of P. bolleanum (subspp. den sum and pauci­ fiorum), and (c) P. minutifolium and P. bolleanum RESULTS subsp. bolleanum.

The total length of the aligned data matrix was 1602 DISCU SSION nucleotides, of which 813 nucleotides resided in the Phoradendron californicum ITS region, 382 in the D2 region and 407 in the D8 reg ion. Of 800 variable sites, 594 were potentially par­ The divergent position of P. californicum argues simony-informative. Missing data accounted for strongly against the monophyly of "Boreales" sensu 3.96% of the data matrix. Lengths of sequences of the Trelease (1916) or a monophyletic acataphyllous lin­ ingroup taxa (excluding partially sequenced taxa and eage sensu Wiens (1964) (Fig. II). Instead, it agrees gaps) ranged from 1380 to 1496 nucleotides, with an with morphological evidence (shared biseriate inflo­ average of 1412. rescences) that suggests a closer relationship of P. cal­ Sequence alignment revealed the presence of 36 in­ ifornicum with cataphyllous tropical species (Kuijt dels. Fourteen are relevant in the context of this study. 1959, 1996). Regrettably, P. olae, its putatively closest Eight support branches on the strict consensus tree relative (Kuijt 1997), is not part of this analysis. The (Fig. 10) and six are homoplastic (e, g', n, y, I, J), of data also suggest two independent origins of scale which one (n) is shared by two of three branches in a leaves (Fig. 10). Wiens (1964) placed P. californicum tritomy. Ten of the indels are 1-2 nucleotides in length in a monotypic section Phoradendron. Marked se­ (five each), three are 3 nucleotides long and one is four quence divergence evidenced by the long branch (Fig. VOLUME 19. NUMBER I North American Phoradendron 47

~ scale-leaved taxa I indel

biseriate inflorescences .£. 53 .£. triseriate inflorescences 2

Northern clade -"" 88 10 .£. '" 97 27

75 g' 1 62 J 94 2 11 67 5

83 7 63 5 98 2 15

98 , 18 • 84 I 5 Inflorescences variable 70 3

Fig. 10. Strict consensus tree (CI = 0.56. RI = 0.75) of four equally most parsimonious trees of length 2234 steps from a maximum parsimony analysis of three regions of the nuclear rONA. Bootstrap and decay values are located above and below each branch. respectively. Phoradendron tomentosum and P. velutinum are each represented by two accessions from Texas (TX) and the Mexican states of San Luis POtOSI (SLP) and Michoacan (M). Branches arid species in bold face indicate the members of the northern clade. as defined in the text. Triseriate inflorescences characterize all members of the northern clade and its sister clade. Biseriate inflorescences are found in P. vemi­ cosum, P. califomicum, and the clade that is sister to P. californicum. 48 Ashworth ALISO

2 P. capitellatum 5 P. juniperinum juniperinum 10 3P. juniperinum libocedri 83 P. bolleanum densum 2 P. bolleanum pauciflorum 146 P. minutifolium 7 5 P. bolleanum bolleanum - 4 P. villosum villosum ~~ 84 P. villosum coryae ~~ Pauciflorae ~ Northern 11 P. scaberrimum sensu 35 8 P. galeottii ~ clade Trelease 8 P. longifoliumi1. 12 P. tomentosum macrophyllum ..th I 22 13 P, tomentosum tomentosum SLP ..th 3p . tomentosum tomentosum TX ..th 13 6 P. serotinum ~~~ 39 P. velutinum SLP ~~~ 3 P. velutinum M£ 51 P. robinsonii ~ 43 53 P. rhipsalinum ,I P. brachystachyum ~ 30

58 51 Pluriseriales 146 sensu 55 79 Trelease 73

33 66 32 36 29

18 Pauciflorae sensu Wiens 60 Pluriseriales sensuWiens 44 Calyculataesensu Wiens Phoradendron sensu Wiens

Fig. 11. Phylogram of one of four equally most par simoniou s trees of length 22 34 steps from a maximum parsimony analysis of three reg ion s of the nuclear rONA, show ing the contrasting sect io nal delimitations by Trel ease (1916) and Wien s ( 1964) . Pauciflorae and Pluriseriales se nsu Trele ase are enclosed in a whit e and pale gray box , respe ct ively, wh ile Paucifiorae, Plurise riales, and Caly cularae se nsu Wien s are ind icat ed next to each taxon ins ide a triangle. Taxon nam es of the me mbers of Wiens's section Calycu larae are additionall y italicized and bold ed . Th e northern clade is enclosed in a dark gr ay box. Numbers above branches den ote branch length s. Phoradendron califo rnicum is separated from the rem aining members of Pau ciflorae sensu Trelease. Phoradendron rhipsalinum was not kno wn to eithe r Trelcase or Wiens. Sp ecies outside Trelease's " Borea les " and Wien s's rev ision are printed in a sma ller fon t size. VOLUME 19, NUMBER I North American Phoradendron 49

11) may reflect accelerated sequence evolution, ex­ Phoradendron rhipsalinurn tinctions that eliminated close relatives, or incomplete Pauciflorae sensu Wiens (1964) includes all but one sampling. Incomplete sampling is perhaps the simplest species that parasitize conifers (primarily Cupressa­ explanation, in that this analysis includes less than half ceae s.l.), The discovery of P. rhipsalinum (Calderon of Phoradendron species and ca. 10% of Dendro­ de Rzedowski and Rzedowski 1972) occurred after phthora species. Wiens's revision, but the authors suggested its place­ ment in Pluriseriales sensu Wiens (1964). By contrast, Parasites of Conifers Kuijt (1996) suggested it "may be a distant relative of P. bolleanum." Unlike the members of Paucifiorae, Pauciflorae sensu Wiens (excluding P. californi­ however, P. rhipsalinum has elongated inflorescences cum; Fig. 11) is monophyletic, and its members form with several internodes, occasionally develops cata­ a fairly uniform group in terms of morphology (small phylls (Kuijt 1996), and has both median and trans­ to scalelike leaves and few-flowered spikes), host pref­ verse primary orientation. Both morphological and erence (conifers, usually forming highly specific as­ molecular data thus agree that P. rhipsalinum does not sociations; Fig. 12) and nucleotide sequence data neatly fit into Pauciflorae. (bootstrap 96%, decay of 9). Strongly supported sister Far more surprising is the strongly supported rela­ group relationships for three species pairs suggest that tionship between P. rhipsalinum and P. brachystach­ (1) P. juniperinum is monophyletic, but (2) P. bol­ yum, the latter a parasite of angiosperms (Fig. 12). leanum is not. Further evidence for the polyphyly of Three indels (f, x, and C) are uniquely shared by these P. bolleanum comes from indel data. Although node two species (Fig. 10). Interestingly, P. brachystachyum support is weak (bootstrap of 67%, decay of 4), the also occasionally produces cataphylls (Kuijt 1996). Phoradendron rhipsalinum is an endemic of central clade uniting the three P. bolleanum subspecies and P. Mexico where it is found exclusively on Taxodium minutifolium is supported by insertion b. Insertion d mucronatum Ten. Like P. juniperinum subsp. liboced­ unites P. bolleanum subspp. densum and pauciflorum rio it appears to mimic the pendulous branches of its but not P. bolleanum subsp. bolleanum (Fig. 10, 12). host by means of long internodes and , in this case, Phoradendron bolleanum is monophyletic only if P. linear leaves. Little is known about the biology of this minutifolium is placed in synonymy or subspp. densum species. Phoradendron brachystachyum, by contrast, is and pauciflorum are treated as species. Interestingly, widely distributed in Mexico, grows on a diverse array plants presumed to have originated by hybridization of angiosperms (especially Fabaceae) and exhibits between P. bolleanum subsp. densum and P. ­ considerable variation in leaf shape, without apparent inum resemble P. minutifolium (Vasek 1966; Wiens host mimicry. Vegetative mimicry (leaves) is well doc­ and DeDecker 1972). umented among certain Australian Loranthaceae (Bar­ The sister group relationship between the two sub­ low and Wiens 1977). species of P. juniperinum is strongly supported. Phor­ adendron juniperinum subsp. libocedri differs from Parasites of Angiosperms the typical subspecies by a more pendulous habit and Within the P. serotinum clade, P. serotinum and P. longer internodes that mimic the morphology of its tomentosum are sister species (Fig. 10). They share host, Calocedrus decurrens (Torr.) Florin. The typical medium-sized (ca. 25-30 mm long X 12-20 mm variety is a parasite of Juniperus and Cupressus spe­ wide), orbicular to obovate leaves having somewhat cies. Three MPTs (not shown) support a sister group obtuse apices and inconspicuous venation and some­ relationship between P. juniperinum and P. capitella­ times a tomentum that tends to diminish or disappear tum. The latter is the most divergent within Pauciflo­ with maturity (Wiens 1964). They parasitize a wide rae, by virtue of numerous autapomorphic nucleotide range of angiosperms, including Betulaceae, Fabaceae, substitutions (Fig. 11) and a deletion of 44 nucleotides Fagaceae, Oleaceae, and Platanaceae. Two unique syn­ in the ITS 1 region. Phoradendron capitellatum is also apornorphic insertions (j and E) support the sister the only species in the conifer-parasite clade to flower group relationship between P. serotinum and P. to­ in winter (December-February; Wiens 1964), although mentosum (Fig. 10 and 12). Sister species to this clade reports of summer flowering do exist (1. Kuijt pers. is P. velutinum. This species differs from the preceding comm.). Only one of the four MPTs (not shown) two by a yellower color and more elongate leaves with shows P. capitellatum as sister to a clade composed acute tips and tapered bases. Both P. velutinum spec­ of the three P. bolleanum subspecies and P. minuti­ imens sampled were found growing on Rosaceae and folium. Fosberg (1941) placed P. capitellatum as sub­ not oak, the primary host reported by Wiens (1964), species under P. bolleanum whilst retaining P. minu­ although a survey of herbarium specimens suggests tifolium at the species level. that P. velutinum ha s a wide host range. A patristic 50 Ashworth ALISO

P. capitellatum ~ WI Cupressaceae a P. juniperinum juniperinum ~ S Cupressaceae t a

P.juniperinum libocedri ~ S Cupressaceae I t a P. bolleanum densum ~ S Cupressaceae a b P. bolleanum pauciflorum ~ SPinaceae a

P. minutifolium • S Cupressaceae I t a I . P. bolleanum bolleanum ~ S I Cupr./Encaceae a r--'- n P. villosum villosum ~ S oaks a P. villosum coryae ~ S oaks a P. scaberrimum ~ S oaks a n I P. galeottii • S ,oaks I a P. longifolium • W oaks a --, P. tomentosum mac. ~ WI many a eljE I P. tomentosum tom . SLP ~ W I many a P. tomentosum tom. TX ~ W I many a P. serotinum ~ F many a

P. velutinum SLP • W many' m aIe l y P. velutinum M• W many' m ale i P. robinsonii • G)w many' 1 m ale f xC e yg' J P. rhipsalinum ~ G)yr Cupressaceae' P. brachystachyum • G) wL..Dlany' P. forestierae ~ Oleaceas' m e g'J P. brevifolium • ~:fc . many' m e P. reichenbachianum. Ic ? oaks' m e D. guatemalensis •

Fig. 12. Excerpt of the strict consen sus tree from a maximum parsimony analysis of three regions of nuclear rDNA. Geographic distribution. indel s, morphological traits (Kuijt 1996). life histor y characters (Wiens 1964). and preferred host s (Wiens 1964 and personal observations) are indica ted for the me mbers of the northern clade (gray box ) and adja cent parts of the tree . Thickened branches have weak bootstrap support «70%). Onl y indel s pert aining to species in the northern clade are mapped onto the tree . Homoplastic indels are indicated in bold. Abbreviations: fc: fertile cataphylls: W: winter flowering, S: summer flowering, F: fall flowering. yr: flowering year-round; t: transverse orientation, m: median orientation, mit: both types of orientation; a: cataphylls absent, c: ca taphylls present, ale: cataphylls sometimes present. Asterisks denote host ranges gathered from surveys of herbarium specimens; all other host ranges are from Wiens (1964). Geographic distribution is indi cated by a boxed grid. in which one to three blackened squares respectively indicate a distribution that extends no farth er south than 26°, 22°, and 18° N latitude and four blackened squares represent a distribution extending south of--or occ urring exclusively south of-18° N latitude. All distribution ranges are from Wiens ( 1964). The three latitudes correspond approximatel y to northern Sinaloa, central Agu ascalientes. and southern Puebla, Mexico. VOLUME 19. NUMBER 1 North American Phoradendron 51 distance of 42 nucleotide substitutions between the event within the northern clade may have involved the two P. velutinum sequences (Fig. II) perhaps reflects divergence of the P. brachystachyum clade first, fol­ a high degree of polymorphism of this species (Tre­ lowed by the P. serotinum clade with its sister species lease 1916). P. robinsonii. The strongly supported association between P. sea­ Flowering period suggests a switch from predomi­ berrimum and P. villosum subsp. coryae renders P. nant winter (or fall) flowering in members of the P. villosum polyphyletic. lndel 0 further strengthens the brachystachyum and P. serotinum clades and in P. ro­ sister group relationship between these two species binsonii, to summer flowering in the poorly supported (Fig . 10). Indel n is present in P. galeottii, P. longi­ clade (bootstrap value of 53%, decay value of 2; Fig. folium, P. scaberrimum, and P. villosum subspp. vil­ 10) that includes conifer and oak parasites. Exceptions losum and coryae that are almost exclusively parasites are the winter-flowering P. capitellatum and P. lon­ of oaks (Fig. 12). Insertion I uniting P. villosum subsp. gifolium. Calder6n de Rzedowski and Rzedowski villosum with the P. serotinum clade is homoplastic. (1972) report year-round flowering and fruiting for P. Sister species P. galeottii and P. longifolium share rhipsalinum, a common feature of species in Central the same hosts, have overlapping distribution ranges, and South America. Host specificity shows a correla­ attain a fairly large size, and have a pendulous habit. tion with flowering period, in that most of the winter­ Their leaves are linear-(ob)lanceolate, although those flowering taxa form generalist host associations, of P. longifolium are longer (a mean of 52 rnm , com­ whereas summer flowering tends to accompany narrow pared with 29 mm in P. galeottii; Wiens 1964) How­ host specificity. Phoradendron bolleanum subsp. bol­ ever, they differ in time of anthesis (Fig. 12), P. gal­ leanum is unusual by parasitizing not only Cupressa­ eottii sharing summer flowering (ca. July to Septem­ ceae s.l. but also Ericaceae. Conversely, P. rhipsalin­ ber; Wiens 1964) with members of the conifer-parasite um is highly host-specific, despite its phylogenetic clade (except P. capitellatum) and with P. villosum, proximity to a host generalist. whereas P. longifolium flowers from December to Feb­ ruary (Wiens 1964). Wiens (1964) placed P. galeottii CONCLUSIONS in Pluriseriales Engler, a section including species with shorter internodes and smaller leaves than section The molecular sequence data presented in this study Calyculatae. However, P. galeottii often has flattened supports Kuijt's (1959, 1996) view that cataphyll pres­ and twisted stems with dilated nodes, characteristic of ence/absence and the orientation of the lowermost fo­ section Calyculatae (Wiens 1964). Because members liar organs should not be used to circumscribe two of Pluriseriales sensu Wiens (P. galeottii and P. vil­ major lineages in Phoradendron. However, a record of losum subsp. coryae) each are sister species to a mem­ the variability for these two characters in some species ber of Calyculatae sensu Wiens (P. longifolium and P. (Kuijt 1996) has here been used to infer additional scaberrimum, respectively), Pluriseriales and Calycu­ structure along some of the weakly supported branches latae are polyphyletic. Pluriseriales sensu Wiens and of the molecular phylogeny. The role of temperature Trelease is paraphyletic also because Paucifiorae is or day length in influencing cataphyll formation cannot nested within it (Fig. 11). Trelease's section Calycu­ be ruled out, in that acataphyllous species, including latae is monotypic and its sole representative, P. ca­ P. califomicum, are typically found at higher latitudes. lyculatum, was not included in this study. A latitudinal influence on host specificity and flow­ ering period is likely, in that almost all tropical species Phylogenetic Structure Within the Northern Clade are known to be host generalists with year-round flow­ ering and fruiting. However, within the northern clade, Figure 12 shows a portion of the strict consensus host specificity seems to follow a phylogenetic pattern tree, with geographic distribution, morphological char­ of distribution. acters, flowering period and host range (Wiens 1964; Interestingly, the host -specific associations among Kuijt 1996) indicated for each member of the northern the conifer parasites are almost always associated with clade. Several members of the clade occasionally ex­ vegetative mimicry. Diagnosing leaf mimicry is sub­ hibit morphological characters characteristic of species jective in that it is more readily recognized in mistle­ occurring farther south. Specifically, P. brachystach­ toes growing on hosts with distinctive (scalelike or yum, P. rhipsalinum, P. robinsonii, and P. velutinum needlelike or linear) leaf shapes (i.e., conifers) than on occasionally develop cataphylls and have median ori­ hosts having (ob)ovate leaves. Barlow and Wiens entation of the lowermost foliar organs. 'Fertile cata­ (1977) hypothesize that high host specificity is a con­ phylls and indel y are shared by P. brachystachyum, dition for mimicry. The adaptive role of mimicry is P. rhipsalinum, and P. robinsonii. Indels g' and J are speculative, although camouflage from extant of an­ shared by P. brachystachyum, P. rhipsalinum, and P. cient (vertebrate) herbivores is likely. A preference for brevifolium. This suggests that the initial branching has been documented for white-tailed deer 52 Ashworth ALISO feeding on a range of plants including Phoradendrori Proceedings of the 4th International Symposium on Parasitic spp. (Gallina 1988), and for other vertebrate herbi­ Flowering Plants. Marburg, Germany. DONOGH UE, M. J., R. G. OLMSTEAD. J. E SMITH , AND J. D. PALMER. vores (e.g., Choate et al. 1987). Regardless of mim­ 1992. Phylogenetic relationships of Dipsacales based on rbcL se­ icry, high host specificity is thought to be an advantage quences. Ann. Missouri BOI. Gard. 79: 333 -345. in open habitats dominated by one or only a few host DOYLE, J. J., AND J. L. DOYLE. 1987. A rapid DNA isolation pro­ species, as is true of many conifer habitats (e.g., pin­ cedure for small quantities of fresh leaf tissue. Phytochem. Bull. yon-juniper woodland). 19: 11-15. EFRO N, B., E. HALLORAN. AND S. HOLMES. 1996 . 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