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Systematics and Phytogeography |
Rancho Santa Ana Botanic Garden, 1500 North College Avenue, Claremont, California 91711-3157 USA
Received for publication May 6, 2005. Accepted for publication February 23, 2006.
ABSTRACT
The large mistletoe family, Loranthaceae, contains 75 genera and approximately 1000 species. The family originated in the Southern Hemisphere and dispersed, apparently early, between fragments of Gondwana. It is now widely distributed on land surfaces of the former supercontinent. The Loranthaceae has three terrestrial, root-parasitic genera—a habit considered ancestral—and 72 genera of aerial, branch parasites. For almost two centuries, the origin of the mistletoe habit has been of interest to biologists. Two main evolutionary pathways have been proposed to explain the transition from terrestrial to aerial parasitism in the family. One theorizes the presence of an intermediate climbing ancestor in the path to the aerial habit. The other proposes a direct transfer from terrestrial to epiphytic growth following the germination of seeds on tree branches. Here we present molecular and morphological evidence that (1) the terrestrial species Nuytsia floribunda is ancestral within the Loranthaceae, (2) aerial parasitism has had multiple origins in the family, (3) the first aerial branch parasites had epicortical roots, and (4) the origin of aerial parasitism in one Old World clade involved the direct transfer from terrestrial to epiphytic growth following the germination of seeds on tree branches. Our results suggest that it is not necessary to evoke a climbing intermediate in the origins of aerial parasitism in the Santalales.
Key Words: aerial parasitism • canopy plants • epicortical roots • haustoria • Loranthaceae • mistletoe • seedling morphology
Parasitism by means of haustorial connections to a host is widespread in angiosperms, having arisen independently 10 or more times (Nickrent et al., 1998
). The Santalales contains five families (APG, 2003
), three of which have aerial parasites known as mistletoes. Malécot et al. (2004)
have proposed a single origin of parasitism within the Santalales. Our research is focused on the largest and most diverse of the mistletoe families, the Loranthaceae. The family is of particular interest for several reasons: (1) it is considered a monophyletic group; (2) it contains both terrestrial, root parasites and aerial, branch parasites; and (3) mistletoes, while only a minor vegetational component in forest and woodland ecosystems, have a disproportionately large impact on species richness and are considered a keystone resource (Watson, 2001
; Shaw et al., 2004
). Our research seeks to understand the pathway(s) by which mistletoes evolved from terrestrial ancestors within this Gondwanan family (Raven and Axelrod, 1974
).
Of the 75 genera comprising the Loranthaceae, 72 are aerial parasites. Four basic haustorial types are recognized in aerial forms—epicortical roots (ERs), clasping unions, wood roses, and bark strands (Calvin and Wilson, 1998
). Epicortical roots, that spread along host branch surfaces and at intervals form haustoria, are regarded as ancestral (Kuijt, 1969
). Taxonomists (Barlow, 1997
) and others (Fineran, 2001
) often designate the remaining haustorial types "solitary unions" because plants appear to have only a single attachment to the host. These presumably derived types are hypothesized to have evolved in relation to increasing aridity (Hamilton and Barlow, 1963
) or other factors (Reid, 1987
; Wilson and Calvin, 2006
).
For almost two centuries, botanists have speculated on the origin of the aerial habit in the Loranthaceae (Meyen, 1846
; Keeble, 1896
). Two main hypotheses have been advanced to explain this transition (Hamilton and Barlow, 1963
). One hypothesis proposed that the aerial habit resulted from a vine-like intermediate plant that began its development on the ground and formed haustorial contacts with host branches through roots produced from its stems (Blakely, 1922
; Fineran, 2001
). The embryonic root of the seedling then became modified into a haustorium, and the plant was wholly aerial. The ability to produce roots along stems was then lost in most taxa. The vine-like growth of some terrestrial Gaiadendron plants (Kuijt, 1963
) and the presence of cauline roots in several aerial genera from the New World (Kuijt, 1981
; Calvin and Wilson, 2006
) support this hypothesis.
The alternative hypothesis suggested that transition to the aerial habit involved a direct transfer from terrestrial to epiphytic growth following the germination of seeds on tree branches (Keeble, 1896
). In this hypothesis, seedling roots that grew along host-branch surfaces formed haustorial attachments, much as seedling roots of the terrestrial Loranthaceae did upon contact with host roots. Soil and organic materials lodged on branches would have facilitated early stages of this transition (Keeble, 1896
). Basal ERs, are present in nearly 60% of aerial genera in the Loranthaceae (Calvin and Wilson, 2006
), including seven of the eight genera hypothesized as ancestral based on floral, cytological, and geographic features (Barlow, 1983
, 1990
). While theoretical evidence has been presented in favor of each pathway to the aerial habit, there has been, until now, little direct scientific evidence in support of either hypothesis.
MATERIALS AND METHODS
Plant collections and selection of taxa
Fifty-one species representing 40 Loranthaceae genera, one genus each from the Opiliaceae and Olacaceae, and two genera from the Santalaceae were collected during 16 field trips between 1988 and 2004 (Appendix). We made multiple collections of one genus within the Loranthaceae, Muellerina, because of its unique seedling and haustorial morphology. Muellerina is an Australian endemic with five species (Downey and Wilson, 2004
) that occur in open and closed forests in mesic regions of southeastern Australia (Barlow, 1984
). Comprehensive collections of M. celastroides and M. eucalyptoides were made in 1991, 1998, 1999, and 2001 near Wisemans Ferry, St. Albans, Kingscliff, Pottsville, and Fingal (New South Wales), and Noosa Heads (Queensland), Australia. We collected materials of all species including leaves, haustorial systems, seedlings (when available), and vouchers of parasite and host. Haustoria were cleaned and air-dried, leaves were dried in silica gel, and seedlings were preserved in alcohol. Vouchers are in our collections at the Rancho Santa Ana Botanic Garden (RSA) and, in some cases, in host country herbaria. Our voucher numbers indicate country, year, and number of collection in sequence during each field trip (Appendix).
Loranthaceae were selected for inclusion in this study based on their geographical distribution and chromosome number. Species representing each of the major areas of distribution (Africa, Australia, New Zealand, Malesia, Mexico, Central America, and South America) and all chromosome numbers (x = 8, 9, 10, 11, and 12) were collected. Chromosome number data were obtained from the Index to Plant Chromosome Numbers (Goldblatt and Johnson, 2003
) and other literature (Wiens 1964
, 1975
; Barlow and Wiens, 1971
; Barlow 1983
, 1990
). Outgroups were selected from each of the other families within the Santalales. Four species, Misodendrum linearifolium DC. (Misodendraceae), Eubrachion gracile Kuijt, Lepidoceras chilense (Molina) Kuijt, and Iodina rhombifolia Hook. & Arn. ex Reissek (Santalaceae), were sequenced but not included in the final study because insertion–deletion (indel) differences and ambiguous base changes produced uncertain alignments of internal transcribed spacer (ITS) sequences of these species with members of the Loranthaceae.
Molecular data and analyses
Dried leaf materials were ground in liquid nitrogen and frozen at –80°C. Extraction of DNA utilized protocols modified from the cetyltrimethyl ammonium bromide (CTAB) method of Doyle and Doyle (1987)
. Modifications from this procedure included RNase treatment and an ethanol precipitation with ammonium acetate following the initial isopropanol precipitation. If coloration remained, it was removed by an ethanol precipitation with sodium acetate.
The 160-bp protein-coding 5.8S gene and the approximately 500-bp flanking ITS 1 and ITS 2 spacer regions (ITS region) were amplified using the following protocol: 97°C for 1 min; 40 cycles of 97°C for 10 s, 48°C for 1 min, 72°C for 20 s; 72°C for 4 min. Primers Leu.1 (Urbatsch et al., 2000
) or ITS5L and ITS4 (White et al., 1990
) were used to amplify the ITS region. Amplification of the trnL gene, trnL intron, and trnL-trnF intergenetic region (trnL-trnF region) followed protocols for the ITS region except that a 52°C annealing temperature was used and 5% dimethyl sulfoxide was added to the amplification mix. The trnL-F region was amplified using primers trna+130L and f (Taberlet et al., 1991
), resulting in a DNA fragment of about 1100 bp or primers trncL and f resulting in a fragment about 700 bp in length. In some cases the primer trnf-10L replaced the f primer. Primers trna+130L, trncL, and trnf-10L were modified from the a, c, and f primers, respectively, of Taberlet et al. (1991)
and ITS5L from ITS5 (White et al., 1990
) for use in the Loranthaceae. Amplification products were visualized on a 1% agarose gel and purified using 30000 normal molecular weight limit (NMWL) Ultrafree centrifugation tubes (Millipore, Bedford, Massachusetts, USA).
Purified amplification products were processed at the Core Facility at Oregon Health Sciences University using a BigDye Terminating (Applied Biosystems, Foster City, California, USA) cycle sequencing reaction and run on an Applied Biosystems 377 automated sequencer following the manufacturer's protocols. Sequencing primers for ITS were ITS4 and ITS2 (White et al., 1990
). For some samples, the internal reverse sequencing primer ITS510L that was designed specifically for the Loranthaceae replaced the ITS2 primer. Sequencing primers for the trnL-trnF region were trncL and f or trnf-10L. Sequences of primers used for PCR amplification and sequencing are listed in Table 1.
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) and bootstrap (Felsenstein, 1985
). One thousand replicates for bootstrap and 500 for the other two analyses were performed using PAUP* version 4.0b10 (Swofford, 2002
) with the heuristic option with random stepwise addition of taxa (10 additions) and branch swapping by tree-bisection-reconnection (TBR). We optimized substitution parameters for ML models using MODELTEST version 3.06 (Posada and Crandall, 1998
) to test model fit across substitution scenarios. Based on MODELTEST analyses ITS, trnL-trnF, and combined data sets utilized the K80 + G, F81 + G, and TIM + G models, respectively. The TIM + G model assumed unequal base frequencies, six substitution models (1.000, 2.0828, 0.7021, 0.7031, 3.6867, 1.000), and a gamma distribution (0.5044) across nucleotide sites. The preference for these models was founded on a comparison of log-likelihood scores to determine whether simpler models with comparable log-likelihood scores existed. Separate ITS, trnL-trnF, and combined data sets were analyzed. Bootstrap percentages greater than 50% are reported. Haustorial types in aerial species were classified as either ERs or solitary unions and optimized on the ML tree using MacClade version 3.4 (Maddison and Maddison, 2002).
Morphological analysis
Seedlings and plants of Muellerina celastroides and M. eucalyptoides growing on host branches were analyzed. We examined more than 150 seedlings and 79 young and mature plants. Specimens were assigned to one of eight octants in a pie diagram based on the angle of their shoot-ER axis relative to the host branch axis (Fig. 1). We then categorized growth of the solitary ER of each seedling as being either acropetal (toward the tip) or basipetal (toward the base) on the host branch. We calculated ER length/infected branch length for 22 representative specimens (10 acropetal and 12 basipetal) as a measure of the sinuosity of ER growth. (Sinuosity is ER length/infected branch length.) A sinuosity of 1 denotes that ER and infected branch lengths are equal. Values >1 reflect the meandering growth of ERs on branch surfaces; the larger the value the greater the degree of sinuosity. We also determined the frequency of both secondary haustoria and rootborne shoots on ERs for the 22 representative specimens as indicators of the intensity of parasitism and vegetative growth of the parasite, respectively. Statistical differences between basipetal and acropetal ER specimens were assessed using a two-tailed Student t test. Significant difference was assigned at the alpha = 0.05 level.
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Molecular analyses
Our aligned, combined data set was 2453 bp, of which the ITS region contributed 983 bp and the trnL-trnF region 1470 bp. The length of the ITS region in all Loranthaceae except Decaisnina species and Macrosolen melintangensis varied from 593 (Dendrophthoe vitellina) to 688 bp (Ligaria cuneifolia). Sequence lengths of the four outgroup taxa (Agonandra racemosa, Ximenia americana, Dendrotrophe varians, and Dendromyza reinwardtiana) were comparable in length to those of most Loranthaceae and ranged between 589 to 666 bp. Sequences of the Decaisnina species and M. melintangensis were longer and ranged from 745 to 851 bp. Of the 758 ITS nucleotide characters analyzed, 58% were variable within the Loranthaceae.
The length of the trnL-trnF region for the Loranthaceae varied from 562 for Cecarria obtusifolia to 878 for Nuytsia floribunda. The N. floribunda sequence was 168 bp longer than sequences from other Loranthaceae species and shared portions of two long insertions with the four outgroup taxa. One insertion was within the trnL intron and exceeded 300 bp in the aligned data set, while the other insertion was within the trnL-trnF intergenetic region and exceeded 100 bp. Outgroup taxa sequences ranged from 872 to 1199 bp in length. Of the 1369 trnL-trnF nucleotide characters analyzed, 22% were variable.
The ITS and trnL-trnF data sets were not significantly incongruent based on the ILD test (P < 1.0). The topology of the ML tree resolved using only ITS data was identical to the tree resulting from an analysis of the combined data set. Two trnL-trnF trees resulted that were less resolved (the consensus tree topology included eight polytomies) than either the ITS or combined data tree. The topology of the consensus trnL-trnF tree, in which bootstrap values exceeded 50%, agreed with the combined data tree.
One ML tree resulted (Fig. 2) with a score of –lnL = 20272. Twenty-nine of the 48 branches had bootstrap values above 50% and 19 branches had values above 70%. Our molecular data determined, with bootstrap support of 89%, that the Loranthaceae was monophyletic with the terrestrial Australian tree, Nuytsia floribunda, basal within the family (Fig. 2). In agreement with the ITS and combined data sets, N. floribunda was resolved as basal within the Loranthaceae using only trnL-trnF data. Three major clades, NW-8, OW-12, and OW-11/9, were resolved (Fig. 3). Each of the three clades had bootstrap values at or above 80%. Approximately 40% of the interior nodes present in these clades had bootstrap values >70% and approximately 30% had values exceeding 90% (Fig. 2).
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Molecular data indicated at least three independent origins of the aerial habit: Notanthera heterophylla, the NW-8 clade, and a clade comprised of both New and Old World taxa (solid arrows, Fig. 3). Epicortical roots were the most common type of haustorium in aerial species and were distributed throughout the tree, while solitary unions mostly occurred on terminal branches (Fig. 3). Optimizing haustorium type on our hypothesized phylogeny indicated that ERs were ancestral for each of the three supported aerial clades (NW-8, OW-12, and OW-11/9). This report focuses on the two Muellerina species at the base of the OW-11/9 clade where bootstrap values are high.
Morphological analyses
Species in the three supported clades (NW-8, OW-12, OW-11/9; Figs. 2, 3) had striking differences in seedling morphology. Species in the NW-8 and OW-12 clades were phanerocotylar (cotyledons exposed after germination). The three terrestrial genera and species in the OW-12 clade had long hypocotyls and small, linear cotyledons (Fig. 4A–C). In the NW-8 clade, in contrast, species had hypocotyls of short-to-intermediate length and longer, wider cotyledons (Fig. 4D). The seedling axes in both the NW-8 and OW-12 clades were erect, and seedling attachment was a two-step process, first by viscin, an adhesive substance derived from the fruit wall (McLuckie, 1923
; Bhatnagar and Johri, 1983
) and then by a large discoidal holdfast (Fig. 4A–D). The holdfast developed at the distal end of the elongate hypocotyl (or hypocotyl-root axis) and was separate from the seed mass (Fig. 4A). Host penetration occurred from the ventral side of the holdfast after the holdfast was securely attached to the host. Cryptocotyly (the retention of cotyledons within the endosperm) occurred throughout the OW-11/9 clade although most, but not all, of the x = 9 taxa had less viscin; larger, more highly developed holdfasts; and orthotropic (upright) shoots.
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In the two Muellerina species a single ER was produced opposite the shoot pole during seedling establishment (Fig. 4F); that is, the shoot–ER axis was bipolar, replicating the bipolar organization of Loranthaceae embryos (Kuijt, 1982
). Significantly, the direction of growth of the ER—acropetal or basipetal—depended on the orientation of the seed on the host branch. When the seedling root axis was oriented more or less basipetally (as in octants 8, 1, and 2; Fig. 1), then ER growth was basipetal, whereas when the seedling root axis was oriented more or less acropetally (octants 4, 5, 6; Fig. 1) ER growth was acroptetal (P < 0.0001). When the shoot–ER axis of seedlings was oriented nearly perpendicular to the branch axis (octants 3 and 7; Fig. 1), their elongating ERs curved toward the host branch and grew either acropetally or basipetally. Of 21 seedlings analyzed in octants 3 and 7, the solitary ERs of 15 grew basipetally, suggesting a preference for basipetal growth.
In the two Muellerina species basipetal ERs grew almost parallel to the host branch, whereas acropetal ERs took a significantly more sinuous path (P < 0.002; Fig. 5A). The meandering growth of acropetal ERs occurred in part because they reached the ends of fruits, stems, or leaves (Fig. 4G–I) where they either reversed growth (Fig. 4H, I) and/or formed ball-like masses (Fig. 4G, H). In some cases ERs formed haustorial connections to host fruits (Fig. 4G), leaves (Fig. 4I), or their own ERs.
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), and acropetally growing ERs typically did not reach branch ends. The ERs of Muellerina formed secondary haustoria laterally along their length and often rootborne shoots, usually in proximity to secondary haustoria (Fig. 4K). The density of secondary haustoria was significantly higher (P < 0.001) for basipetal ERs as compared to acropetal ERs (Fig. 5B). Basipetally growing ERs thus had a greater absorptive area in contact with host vasculature than acropetally growing ERs. The increased contact with host xylem was reflected in a significant increase (P < 0.0002) in the density of rootborne shoots (Fig. 5C). The density of rootborne shoots on basipetally growing ERs was 420% of their density on acropetally growing ERs, while the density of secondary haustoria was 188% of that found on acropetally growing ERs. The increased production of rootborne shoots, beyond what might be predicted given the increased development of secondary haustoria, provides strong evidence that the basipetal growth of ERs is strategic to maximizing vegetative growth.
DISCUSSION
This study, although not primarily a systematic study, provides the first phylogenetic hypothesis for the family since Danser (1933)
placed the approximately 50 genera recognized at that time into three tribes within the subfamily Loranthoideae. Danser assigned genera based on their chromosome number and whether seedlings were crypto- or phanerocotylar. His tribes do not correspond to the major clades resolved in our study. Danser placed all genera with chromosome numbers of x = 12 within Tribe Elytrantheae Danser except the monotypic Tupeia antarctica and Nuytsia floribunda. He considered T. antarctica more closely related to Old World genera with chromosome numbers of x = 9 and placed N. floribunda within a monotypic tribe, Nuytsieae Engl. Danser placed the Old World x = 11 and 9 and New World x = 8 genera within Tribe Lorantheae Engl. Ligaria cuneifolia, with a chromosome number of x = 10, was unknown at that time. Subsequently, Barlow (1964)
elevated the two subfamilies, Loranthoideae and Viscoideae, to family status. Our hypothesized phylogeny represents >50% of the 75 genera within Loranthaceae sensu stricto, including all New World and 60% of Old World x = 12 genera. Although our molecular data is largely congruent with chromosome number data our findings suggest a more complex pattern of evolution than Danser (1933)
proposed.
Nuclear and chloroplast DNA data place the terrestrial Nuytsia floribunda from western Australia basal within the family and indicate that aerial members arose from terrestrial ancestors. The other terrestrial taxa, Gaiadendron punctatum from Central and South America and Atkinsonia ligustrina from southeastern Australia, are placed at the base of the NW-8 clade. Our results indicate that ERs are the ancestral haustorial type for the three major supported aerial clades (NW-8, OW-12, and OW-11/9) and that solitary unions arose later. Solitary haustorial unions are present in species at terminal positions in each of these clades. Notanthera heterophylla from South America, placed basally in our hypothesized phylogeny, has epicortical roots and a chromosome number of x = 12. Although we consider it probable that the presence of ERs is ancestral for each invasion of the canopy, the data is equivocal for the branch leading to Ligaria cuneifolia and OW-12 species as well as for the ancestor of L. cuneifolia, OW-12 species, Tupeia antarctica, Desmaria mutabilis, and Tristerix corymbosus. Because support for these equivocal branches is weak, additional molecular data is required to resolve with confidence the placement of T. antarctica (New Zealand) and the South American species L. cuneifolia, T. corymbosus, and D. mutabilis. Tupeia and Desmaria are monotypic, while Ligaria has two and Tristerix has 11 species. Broader sampling within Tristerix may also clarify relationships, thus providing information on ancestral haustorium types.
Our molecular data indicates that the aerial habit arose several times within the Loranthaceae. This view is supported by seedling establishment patterns that are strikingly different in the OW-12 (see also Docters van Leeuwen, 1954
; Ladley and Kelly, 1997
), NW-8 (see also Kuijt, 1982
; Venturelli, 1981
) and the OW-11/9 (see also Menzies, 1954
; Williams, 1963
; Ladley and Kelly, 1996
) clades. Divergence in seedling morphology between species in the OW-12 and OW-11/9 clades provides evidence that the aerial habit developed independently in the two clades. Our molecular and morphological data collectively indicate that the aerial habit has arisen independently four times, once in each of the three major clades and in Notanthera heterophylla (see arrows, Fig. 3).
Nickrent and Malécot (2001)
proposed five independent origins of the mistletoe habit in Santalales, once in Loranthaceae, once in Misodendraceae, and three times in Santalaceae. Our results suggest that the mistletoe habit has arisen at least eight times within the order. In the Orchidaceae, obligate twig epiphytism has evolved several times (Gravendeel et al., 2004
), and Benzing (1980) suggests multiple transitions to the aerial environment in the Bromeliaceae. These observations suggest that in some lineages colonization of the canopy habitat can occur repeatedly, although it should not be considered a labile character because reversals to the terrestrial habit are not common.
We describe seedling establishment for the two most abundant and widespread Muellerina species, M. celastroides and M. eucalyptoides, and identify a suite of seedling characters that is unique to Muellerina and probably Ileostylus (discussed next). Four characters are of particular interest: (1) a seedling axis that is not erect but instead more or less parallel to the host branch surface; (2) only one ER develops during seedling establishment; (3) the single ER develops opposite the shoot axis (bipolar condition); and (4) the direction of ER growth is tied to the orientation of the shoot–ER axis on the host branch. In other Loranthaceae with ERs, a strikingly different establishment pattern prevails. Shoot axes become erect and multiple ERs form that are perpendicular to the shoot axis. Each ER can extend either acropetally or basipetally. With these innovations, orientation of the seedling axis on the host branch is no longer a factor in determining the direction of ER growth and the consequent extent and vigor of vegetative shoot growth.
Ileostylus, a monotypic New Zealand genus, shares a basic chromsome number of x = 11 with Muellerina (Martin, 1983
). Although we have not examined I. micranthus in detail, its pattern of establishment is similar to that of Muellerina. Seedlings develop within the viscin mass, producing two leaves that emerge early in development (see also Menzies, 1954
; Ladley and Kelly, 1996
). The seedling axis of I. micranthus is not upright but instead is oriented parallel to the host branch and the solitary ER of Ileostylus seedlings grows either acropetally or basipetally. In ERs that grow acropetally, root balls may form at or near branch ends (see Fig. 2 of Menzies, 1954
), much like those formed in Muellerina. Our molecular work provides strong support for a sister relationship between the two genera (Fig. 2). Shared chromosome numbers and seedling morphology further support their sister status.
Both Muellerina species produce a single ER opposite the shoot pole during seedling establishment. The vigor and extent of vegetative growth—a prelude to overall reproductive success—is tied to the direction of ER growth in Muellerina, and the direction of ER growth is determined in large part by the positioning of seeds on host branches by avian vectors. In nearly 40% of seedlings analyzed, the single ERs grew acropetally on host branches. These plants only spread along the limited distal portion of the branch, often appear necrotic, and produce fewer haustorial connections and vegetative shoots, suggesting that the two Muellerina species are poorly adapted to the mistletoe habit relative to other ER-forming genera. We view the two species as living intermediates, relict species confined to residual pockets of mesic forest (Barlow, 1984
, 1990
) and lacking many of the morphological specializations present in other branch-parasitic Loranthaceae. These data provide experimental evidence that the pathway to the aerial habit in the OW-11/9 clade was by the direct transfer of seeds to host branches and the eventual adaptation of their seedlings to branch growth, a process not yet fully perfected in M. celastroides and M. eucalyptoides.
Several lines of evidence argue against the currently preferred hypothesis that aerial parasites necessarily evolved from climbing ancestors. Nuclear and chloroplast data indicate that the two Muellerina species (both x = 11) are basally positioned in a clade with a large number of more specialized x = 9 genera. Calladine (1999)
also considered Muellerina ancestral to the Old World x = 9 species, although he regarded this finding as preliminary because he lacked African and New World taxa. Our results suggest that the x = 11 species are intermediate within a dysploid reduction series from x = 12 to x = 9, and we consider it unlikely that x = 9 ancestors gave rise to x = 11 descendents. Morphological analyses indicate that species in which seedling and ER orientation are uncoupled from seed deposition patterns have enhanced haustorium and vegetative growth potential. In addition, if mistletoes evolved via an intermediate climbing stage one would expect to see transitional morphologies in at least some of the nearly 30 Old World genera with ERs. No transitional morphologies are known. Finally, a persuasive point favoring the direct-transfer pathway in the OW-11/9 clade is the considerably smaller number of steps involved in the transition from terrestrial, root parasite to aerial, branch parasite. The shorter pathway is not only more logical but also more parsimonious.
The greatest concentration of primitive angiosperms occurs today on the Australian Plate, even though angiosperms are hypothesized to have arrived there some 10 million years after their first appearance (Morley, 2003
). It is perhaps not surprising that evidence of the transition from the terrestrial to the aerial habitat is preserved in Australia. The continent is home to two of the three terrestrial genera of Loranthaceae and to aerial genera with a preponderance of ERs (Calvin and Wilson, 2006
). One aerial genus, Muellerina, retains additional morphological features consistent with the earliest colonization of the forest canopy through the deposition of seed on branches and the subsequent development of a parasitic relationship between seedling and host.
Loranthaceae and outgroup species included in this study with voucher and GenBank accession numbers. Vouchers housed at RSA. AR = Argentina, AU = Australia, B = Borneo, CL = Chile, CR = Costa Rica, K = Kenya, MX = Mexico, NZ = New Zealand, T = Tanzania, Y = Yemen.
Species; Voucher; GenBank accession nos.: ITS, trnL-trnF.
Agelanthus sansibarensis (Engl.) Polhill & Wiens; A. Robertson 7365, K; DQ333824, DQ340573. Agonandra racemosa Standl.; Calvin & Wilson MX03-05; DQ333868, DQ340619. Alepis flavida Tiegh.; Calvin & Wilson NZ98-04; DQ333847, DQ340598. Amyema cambagei (Blakely) Danser; Calvin & Wilson AU99-01; DQ333833, DQ340582. A. queenslandica (Blakely) Danser; Calvin & Wilson AU02-22; DQ333834, DQ340583. Amylotheca dictyophleba Tiegh.; Calvin & Wilson AU98-21; DQ333849, DQ340600. Atkinsonia ligustrina F.Muell.; Calvin & Wilson AU00-01; DQ333865, DQ340616. Baratranthus axanthus Miq.; Calvin & Wilson B02-14; DQ333838, DQ340587. Benthamina alyxifolia (Benth.) Tiegh.; Calvin & Wilson AU99-14; DQ333835, DQ340584. Cecarria obtusifolia Barlow; Calvin & Wilson AU02-16; DQ333836, DQ340585. Cladocolea cupulata Kuijt; Calvin & Wilson MX03-08; DQ333861, DQ340612. C. mcvaughii Kuijt; Calvin & Wilson MX03-09; DQ333860, DQ340611.Dactyliophora novae-guineae Danser; Calvin & Wilson AU02-10; DQ333837, DQ340586. Decaisnina brittenii (Blakely) Barlow; Calvin & Wilson AU01-11; DQ333844, DQ340595. D. congesta Barlow; Calvin & Wilson AU02-01; DQ333843, DQ340594. D. signata (Benth.) Tiegh.; Calvin & Wilson AU02-05; DQ333845, DQ340596. Dendromyza reinwardtiana (Korth.) Danser; Calvin & Wilson AU02-11; DQ333870, DQ340621. Dendrophthoe constricta Danser; Calvin & Wilson B02-03; DQ333840, DQ340589. D. vitellina (F. Muell.) Tiegh.; Calvin & Wilson AU98-08; DQ333839, DQ340588. Dendrotrophe varians (Blume) Miq.; Calvin & Wilson B02-15; DQ333871, DQ340622. Desmaria mutabalis (Poepp. & Endl.) Tiegh.; Calvin & Wilson CL03-07; DQ333852, DQ340603. Diplatia grandibractea (F. Muell.) Tiegh.; Calvin & Wilson AU01-05; DQ333832, DQ340581. Englerina ramulosa (Sprague) Polhill & Wiens; A. Robertson 7280, K; DQ333828, DQ340577. Erianthemum dregei Tiegh.; A. Robertson 7366, K; DQ333831, DQ340580. Gaiadendron punctatum G. Don; Calvin & Wilson CR01-08; DQ333866, DQ340617. Helixanthera parasitica Lour.; Calvin & Wilson B02-01; DQ333823, DQ340572. Ileostylus micranthus Tiegh.; Calvin & Wilson NZ00-2; DQ333841, DQ340592. Ixocactus inornus (Robinson & Greenm.) Kuijt; Calvin & Wilson MX03-04; DQ333858, DQ340609. Lepidariaoviceps Danser; Calvin & Wilson B02-19; DQ333851, DQ340602. Ligaria cuneifolia Tiegh.; Calvin & Wilson CL03-01; DQ333853, DQ340604. Lysiana maritima (Barlow) Barlow; Calvin & Wilson AU98-19; DQ333848, DQ340599. Macrosolen melintangensis (Korth.) Miq.; Calvin & Wilson B02-17; DQ333842, DQ340593. Muellerina celestroides Tiegh.; Calvin & Wilson AU99-11; AY572198, DQ340590. M. eucalyptoides (DC.) Barlow; Calvin & Wilson AU99-5; AY572197, DQ340591. Notanthera heterophylla (Ruiz & Pav.) Eichler; Calvin & Wilson CL03-03; DQ333855, DQ340606. Nuytsia floribunda R.Br.; Calvin & Wilson AU01-22; DQ333867, DQ340618. Oncocalyx schimperi (Hochst. ex A.Rich.) M.G. Gilbert.; Calvin & Wilson Y88-02; DQ333825, DQ340574. Oryctanthus occidentalis (L.) Eichler; Calvin & Wilson CR01-11; DQ333862, DQ340613. Peraxilla tetrapetala (L.f.) Tiegh.; Calvin & Wilson NZ98-03; DQ333846, DQ340597. Phragmanthera regularis (Steud. ex Sprague) M.G. Gilbert; Calvin & Wilson Y88-01; DQ333830, DQ340579. Phthirusa pyrifolia Eichler; Calvin & Wilson CR01-03; DQ333857, DQ340608. Plicosepalus curviflorus Tiegh.; Calvin & Wilson Y88–03; DQ333826, DQ340575. Psittacanthus schiedeanus Blume; Calvin & Wilson CR01-09; DQ333859, DQ340610. Scurrula ferruginea (Jack) Danser; Calvin & Wilson B02-02; DQ333827, DQ340576. Struthanthus orbicularis Blume; Calvin & Wilson CR01-02; DQ333856, DQ340607. Taxillus wiensii Polhill; A. Robertson 7364, K; DQ333829, DQ340578. Tripodanthus acutifolius Tiegh.; Calvin & Wilson AR03-04; DQ333864, DQ340615. T. flagellaris Tiegh.; Calvin & Wilson AR03-10; DQ333863, DQ340614. Tristerix corymbosus (L.) Kuijt; Calvin & Wilson CL03-02; DQ333854, DQ340605. Tupeia antarctica Cham. & Schltdl.; Calvin & Wilson NZ98-02; DQ333850, DQ340601. Ximenia americana L.; Calvin & Wilson AU01-13; DQ333869, DQ340620.
FOOTNOTES
1 The authors dedicate this report to Bryan A. Barlow. 
2 Author for correspondence (e-mail: (carol.wilson{at}cgu.edu
)
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