Molecular Phylogenetics and Evolution 47 (2008) 523-537
The first mistletoes: Origins of aerial parasitism in Santalales
Romina Vidal-Russell, Daniel L. Nickrent *
Department of Plant Biology, Southern Illinois University Carbondale, IL 62901-6509 USA
Received 16 April 2007; revised 11 October 2007; accepted 17 January 2008
Abstract
Past molecular phylogenetic work has shown that aerial parasites have evolved five times independently in the
sandalwood order (Santalales), but the absolute timing of these diversifications was not addressed. DNA sequences
from nuclear SSU and LSU rDNA, and chloroplast rbcL, matK and trnL-F from 39 santalalean taxa were obtained.
Separate and combined data partitions were analyzed with maximum parsimony and Bayesian inference. Time estimates
were performed with Bayesian relaxed molecular clock and penalized likelihood methods using published fossil data.
Both methods gave comparable divergence dates for the major clades. These data confirm five origins of aerial
parasitism, first in Misodendraceae ca. 80 Mya and subsequently in Viscaceae (72 Mya), “Eremolepidaceae” (53 Mya),
tribe Amphorogyneae in Santalaceae (46 Mya), and Loranthaceae (28 Mya). The rapid adaptive radiation and speciation
in Loranthaceae coincides with the appearance of savanna biomes during the Oligocene. In all clades except
Misodendraceae, it appears that aerial parasites evolved from ancestors that were polymorphic for either root or stem
parasitism – a condition here termed amphiphagous. Convergences in morphological features associated with the
mistletoe habit have occurred such as the squamate habit, seed attachment structures, unisexual flowers, and loss of
chlorophyll.
Keywords: Misodendraceae, Loranthaceae, Santalaceae, Viscaceae, Eremolepidaceae, phylogeny, Bayesian relaxed
molecular clock
1. Introduction
1.1. Trophic modes in angiosperms
Flowering plants are most often viewed as
autotrophs, i.e. photosynthetic organisms that derive their
mineral nutrients from water taken up by roots. This
stereotypic view ignores the wide diversity of
heterotrophic plants that either supplement or replace
autotrophism with carnivory, nitrogen fixation, mycoheterotrophism, or parasitism. Parasitic plants establish a
direct connection with the xylem or phloem (or both) of
their host via modified roots called haustoria. Haustorial
parasites are either holoparasites (which do not engage in
photosynthesis) or hemiparasites (which do
photosynthesize). Current evidence indicates
________
* Corresponding author. Fax +1 6184533441.
Email address: nickrent@plant.siu.edu (D.L. Nickrent)
that haustorial parasitism evolved independently in 11 or
12 distinct clades of angiosperms representing ca. 270
genera and over 4500 species (Nickrent, 1997).
Most families of parasitic plants attach to host roots,
but others attach to aerial portions of the host. Examples
of stem parasites include Cassytha (Lauraceae), Cuscuta
(Convolvulaceae), Apodanthaceae, some species of
Rafflesia, and mistletoes. Because the term “mistletoe”
refers specifically to an aerial parasite that occurs in the
sandalwood order (Santalales), it is both taxonomic and a
description of plant habit. It has long been accepted that
stem-parasitic sandalwoods evolved from root parasites
(Kuijt, 1969), but there has been disagreement as to the
number of times the mistletoe habit evolved. For
example, 19th century classification systems placed the
mistletoe families Loranthaceae and Viscaceae together,
but more recent works (e.g. Barlow, 1964; Wiens and
Barlow, 1971) recognized that these families are derived
from different ancestors.
1.2. Aerial parasites in Santalales
Santalales includes five clades with aerial parasites
(Nickrent, 2002): Misodendraceae, Loranthaceae,
“Eremolepidaceae”, Santalaceae tribe Amphorogyneae
(Stauffer, 1969; Stearn, 1972), and Viscaceae. All
Viscaceae, Misodendrum, and most Loranthaceae are
mistletoes. Previous work (Nickrent et al., 1998;
Nickrent and Malécot, 2001) showed that the root
parasite Schoepfia is not closely related to members of
Olacaceae but was sister to Misodendrum, an aerial
parasite of southern hemisphere beech trees (Nothofagus)
in Chile and Argentina. This clade is sister to
Loranthaceae whose three root parasitic genera are sister
to the aerial parasites. It is considered unlikely that the
ancestor of this entire clade was a stem parasite and that
taxa such as Nuytsia and Schoepfia “reverted” to the root
parasitic condition (Nickrent, 2002). Thus, the phylogeny
strongly suggests that the mistletoe habit evolved here
twice, once in the ancestor of Misodendrum and once in
the ancestor of all aerially parasitic Loranthaceae.
In agreement with Wiens and Barlow (1971), the
“family” Eremolepidaceae was shown via molecular
phylogenetic methods (Nickrent et al., 1998; Nickrent
and Malécot, 2001) to be a component of Santalaceae,
not sister to Viscaceae (Bhatnagar and Johri, 1983) nor
Loranthaceae (Kuijt, 1968). Phylogenetic work using
only nuclear 18S rDNA sequences (Nickrent, 2002)
showed that a second santalaceous group, tribe
Amphorogyneae (Dendrotrophe and Dufrenoya), was
distinct from the eremolepidaceous mistletoes, however,
resolution within this group was poor owing to low taxon
sampling. All members of Viscaceae are mistletoes and
the monophyly of the family is strongly supported
(Nickrent et al., 1998; Nickrent and Malécot, 2001).
Because this clade emerged from a polytomy involving a
paraphyletic Santalaceae, the Angiosperm Phylogeny
Group (APG, 1998; 2003) subsumed Viscaceae into
Santalaceae. This classification will likely require further
revision given that a resolved phylogeny of
“Santalaceae” is now available (Der and Nickrent, in
press).
Although it now appears clear that the mistletoe
habit arose at least five times independently, the relative
timing of these events has not been addressed. It was
suggested (Nickrent, 2002) that Misodendrum may
represent the first evolutionary “experiment” with the
mistletoe habit, but this hypothesis was based solely on
the topological position of the Misodendrum / Schoepfia
/ Loranthaceae clade between Olacaceae and more
derived families where aerial parasitism occurs
(Santalaceae, Viscaceae). To properly address this issue,
the relative timings of the five independent events that
produced aerial parasites must be directly compared
using time-calibrated (ultrametric) trees.
1.3. Timing of diversifications and fossil evidence in
Santalales
Within the past decade the botanical community has
witnessed the integration of paleobotanical and
phylogenetic data, specifically, the calibration of
molecular phylogenetic trees with dates derived from
fossils. This often provides only minimum ages for nodes
because of the possibility of undiscovered older fossils.
Moreover, placement of fossils on phylogenetic trees is
problematic in that they may be positioned at various
points on the stem and crown groups. With good fossil
data, transforming relative ages into absolute ages for
particular nodes on gene trees would be straightforward
if no rate heterogeneity exists (Soltis et al., 2005).
Although rate heterogeneity does exist in most groups
that have been studied, several methods have been
developed that do not rely upon a constant substitution
rate (Sanderson 1997; Thorne et al. 1998; Huelsenbeck et
al., 2000; Sanderson, 2002).
Fossils (mainly pollen) of Santalales are wellrepresented throughout the Cretaceous and Tertiary. One
of the major pollen types seen in deposits dating from the
Campanian is Anacolosidites. This form-genus is
extremely variable morphologically, and not all types
correspond to Santalales. This was demonstrated in the
recent study by Malécot and Lobreau-Callen (2005) who
used phenetic methods to discern which palynomorphs
were most closely related to modern Olacaceae, tribe
Anacolosoideae. They concluded that this tribe dated to
70 Mya (Maastrichian) which agrees with broad-based
surveys of pollen records for angiosperm orders (Muller,
1984).
Although molecular phylogenetic trees indicate
Misodendraceae diverged relatively early in Santalales,
fossil pollen records of M i s o d e n d r u m (as
Compositoipollenites) extend back only to the early
Oligocene (Macphail and Cantrill, 2006). In contrast, the
form-genus Aquilapollenites is known from Maastrichian
through Eocene aged strata in a number of locations,
although most are from northern hemisphere localities.
This pollen type also likely represents more than one
phylogenetic group. Jarzen (1977) suggested two lines of
development, the isopolar form with a close
morphological relationship with Loranthaceae and the
second heteropolar to subisopolar form allied with
Santalaceae, particularly the genus Arjona. Although
pollen has been examined for 23 genera of extant
Loranthaceae (Feuer and Kuijt 1979; 1980; 1985; Han et
al. 2004), 50 genera remain that have not been studied,
thus a comprehensive view of pollen variation across the
entire family is lacking. This has confounded attempts to
specifically associate form genera such as Accuratipollis,
Cranwellia, Gothanipollis, and Loranthacites with
modern ones (see Couper, 1960; Elsik, 1974; Jarzen,
1978; Menke, 1976; Mildenhall, 1980; Taylor, 1989).
Generally the fossil record for Santalaceae is more recent
than that of Olacaceae and Loranthaceae. Genera such as
Santalum, Osyris, and Antidaphne are known from
various Old and New World Eocene deposits (Darrah
1939; Martin 1982). Viscaceae records exist from
throughout the Tertiary with Eocene records of
Arceuthobium (Muller 1981), Phoradendron (Darrah
1939), and Viscum (Selmeier 1975).
1.4. Goals of this study
The objectives of this study are to 1) generate a
molecular phylogeny for representatives of Santalales
that includes all clades with aerial parasites, 2) utilize
Bayesian and likelihood methods, with calibrations
obtained from published fossil evidence, to obtain
divergence dates for the aerial parasite clades, and 3)
compare and contrast the different aerial parasites with
respect to ancestral types, timing of divergence,
morphological diversity, and convergences.
Standard polymerase chain reactions (PCR) were
used for all genes (Nickrent, 1994). For SSU and LSU
rDNA, 5% (final concentration) DMSO was added to the
reactions. Cycle sequencing reactions were performed
directly on the purified PCR products following standard
protocols using BigDye Terminator Cycle Sequencing
Ready Reaction Kit with AmpliTaq DNA Polymerase
(Applied Biosystems, Foster City, CA) with Better
Buffer (The Gel Company, San Francisco, CA).
Sequencing reactions were run on an ABI 377 automated
DNA sequencer.
2. Materials and Methods
2.3. Alignment and phylogenetic analyses
2.1. Sampling
Sequences were aligned by eye in Se-Al (Rambaut,
2004). For r b c L and m a t K, inferred amino acid
sequences were used to guide the alignment. Ambiguities
present in the trnL-F intergenetic spacer alignment were
discarded from the analysis. MP and BI analyses were
conducted on individual genes as well as on a
concatenate data set. MP and bootstrap analyses (100
replicates) were performed with PAUP* v. 4.0b10
(Swofford, 2003). Tree heuristic searches used the Tree
Bisection Reconnection branch-swapping algorithm
starting with 1000 random addition sequences holding
ten trees at each step. Gaps were coded as missing in all
analyses.
BI was performed using MrBayes 3.1.2
(Huelsenbeck and Ronquist, 2001; Ronquist and
Huelsenbeck, 2003) with a model of molecular evolution
chosen via the likelihood ratio test as implemented by
MrModeltest (Nylander, 2004). The model selected for
each partition was the general time reversible plus a
gamma distribution to account for variation among sites
and a proportion of invariant sites (GTR+I+G). Two
independent analyses with four chains each were
performed for five million generations, but they were
stopped if the average standard deviation of split
frequencies between the runs went below 0.01 (reflecting
convergence in topology between runs). The relative
burn-in used to calculate the standard deviation of split
frequencies between the runs was 25%, thus this fraction
was deleted from the total number of sampled trees.
Trees and parameters were saved every 100 generations.
Model parameters were estimated as part of the analysis;
uniform prior probabilities were assigned to all
parameters except the state frequencies for which a
Dirichlet prior distribution was assigned. When more
than one partition was analyzed, parameter estimations
were unlinked and the rate was set to vary, thus allowing
partitions to evolve at different rates.
Character reconstruction (root versus aerial parasite)
was performed using parsimony and likelihood in
Mesquite (Maddison and Maddison, 2006).
The ingroup comprises 34 taxa, represented by
Viscaceae (three species), Santalaceae including
“Eremolepidaceae” (15 species), Opiliaceae (two
species), Loranthaceae (ten species), Misodendraceae
(two species) and Schoepfia (two species). Five species
of Olacaceae, Anacolosa papuana, Chaunochitanon
kappleri, Heisteria concinna, Olax emirnensis, and
Ptychopetalum petiolatum were used as outgroup for the
maximum parsimony (MP) and Bayesian inference (BI)
analyses. This sampling represents all families of
Santalales and includes all lineages with aerial parasites.
Two taxa were missing in each of the nuclear rDNA,
rbcL, and matK alignments, whereas six taxa were
missing for the trnL-F dataset (See Table 1). Six taxa
(mainly Loranthaceae) were missing ca. 600 bp at the 3’
end of the rbcL gene. Accession and voucher information
for all newly generated sequences (76 total), as well as
GenBank numbers all taxa used, are given in Table 1.
2.2. DNA extraction and sequencing
DNA was obtained from silica-dried tissue using a
standard CTAB method (Nickrent, 1994). Two nuclear
genes, small (SSU rDNA) and large subunit ribosomal
DNA (LSU rDNA), were sequenced as were three
chloroplast genes: rbcL, matK, and the trnL-F spacer,
including the intron between the trnL exons. The SSU
rDNA was amplified and sequenced using the 12
forward (5’–TCC TGC CAG TAS TCA TAT GC– 3’)
and 1769 reverse (5’–CAC CTA CGG AAA CCT TGT
T– 3’) primers. Approximately 950 bp from the 5’ end of
the LSU rDNA was amplified and sequenced using the
primer pair 27 forward (5’–CCC GCT GAG TTT AAG
CAT A– 3’) and the 950 reverse (5’–GCT ATC CTG
AGG GAA ACT TC– 3’). The rbcL gene was amplified
and sequenced with the 1 forward (5’–ATG TCA CCA
CAA ACA GAR AC– 3’) and 3’ reverse (5’–TAG TAA
AAG ATT GGG CCG AG–3’) or 889 reverse (5’–CTA
TCA ATA ACT GCA TGC AT– 3’) primers. The gene
matK was amplified and sequenced with primer 78
forward (5’–CAG GAG TAT ATT TAT GCA CT–3’)
and 1420 reverse (5’–TCG AAG TAT ATA CTT TAT
TCG– 3’). Finally, the trnL-F spacer was amplified and
sequenced using the primers described in Taberlet et al.
(1991).
2.4. Molecular dating
Two methods were used to estimate divergence
time: Bayesian relaxed molecular clock (BRMC, Thorne
et al., 1998) and penalized likelihood (PL, Sanderson,
Table 1
Taxon sampling and GenBank accession information for all sequences used in this study
Name
Collector
DNA
Acc. No.
Source
SSU
rDNA
LSU
rDNA
Alepis flavida (Hook.
f.) Tiegh.
B. Molloy
2743
New Zealand L24139
EF464474*
Anacolosa papuana
Schellenberg
R. Regalado & M. 4247
Q. Sirikolo 692
Solomon
Islands
DQ790104
N/A
DQ790144
EF464516*
N/A
Antidaphne viscoidea
Poeppig &
Endlicher
S. Sargent
2730
Costa Rica
L24080
N/A
L26068
EF464500*
N/A
Arceuthobium
verticilliflorum
Engelm.
D. L. Nickrent &
A. Flores
2065
Mexico
L24042
EF464470*
L26067
N/A
EF464482*
Arjona tuberosa Cav.
J. Puntieri
4131 [s]
4566
Argentina
EF464468*
EF464480*
EF464532*
EF464513*
EF464483*
Atkinsonia ligustrina
(A. Cunn. ex F.
Muell.) F. Muell.
D. Watson
4344
Australia
EF464464*
EF464475*
EF464526*
DQ787444
DQ788714
Buckleya
distichophylla
Torrey
L. J. Musselman
2735
U.S.A
X16598
EF464473*
DQ329180
DQ329191
EF464484*
Chaunochiton
kappleri (Sagot)
Ducke
N. Zamora 1928
3052
Costa Rica
DQ790106
DQ790218
DQ790142
DQ790179
EF464485*
Choretrum
glomeratum (F.
Muell.) Benth.
Lepschi & Craven
4602
4312
Australia
EF464461*
N/A
N/A
N/A
N/A
Choretrum
pauciflorum A.
DC.
B. Lepschi, T. R.
Lally & B.R.
Murray 4237
4222
Australia
N/A
N/A
EF464522*
EF464503*
N/A
Daenikera corallina
Hurlimann &
Stauffer
J. Munzinger 2054 4876
New
Caledonia
EF464462*
EF464472*
EF464523*
EF464504*
N/A
Dendromyza cf.
ledermanii (Pilger)
Stauffer
D. L. Nickrent T.
Kierang, & E.
Sape
4466
Papua New
Guinea
EF464463*
N/A
EF464524*
EF464505*
DQ340621
Dendrotrophe varians
(Blume) Miq.
D. L. Nickrent
2827 [r]
4014 [m]
Australia
L24087
N/A
EF464520*
EF464501*
DQ340622
Desmaria mutabilis
(P. & E.) Jacks.
G. Amico
4510
Chile
EF464465*
EF464476*
EF464527*
EF464509*
EF464486*
Eubrachion
ambiguum (Hooker
& Arnott) Engler.
D. L. Nickrent, D.
Clark & P. Clark
2699
Puerto Rico
L24141
AF389273
L26071
EF464498*
N/A
Gaiadendron
punctatum (Ruiz.
& Pav.) G. Don.
S. Sargent
2729
Costa Rica
L24143
DQ790209
L26072
DQ787445
DQ788715
Heisteria concinna
Standl.
C. Augspurger
2732
Costa Rica
L24146
DQ790230
DQ790161
DQ790197
EF464487*
Lepidoceras chilense
(Molina) Kuijt.
C. Marticorena &
R. Rodríguez
10043
4065
Chile
EF464459*
N/A
EF464519*
EF464499*
N/A
Lepionurus sylvestris
Blume
G. Hambali
2879
Java
DQ790101
DQ790206
DQ790131
DQ790170
AF534673
Leptomeria spinosa
(Miq.) A. DC.
A. Markey
3081
Australia
EF464460*
EF464471*
EF464521*
EF464502*
N/A
Leptomeria aphylla
R. Br.
B. Lepschi & A.
Whalen 4875
4609
Australia
N/A
N/A
N/A
N/A
EF464488*
Ligaria cuneifolia
(Ruiz & Pavón)
Tiegh.
G. Amico
4567
Chile
L24152
EF464477*
EF464528*
EF464510*
DQ442940
rbcL
N/A
matK
EF464508*
trnL-F
EF464481*
Name
Collector
Misodendrum
linearifolium DC
D. E. Bran
G. Amico 136
DNA
Acc. No.
Source
SSU
rDNA
LSU
rDNA
rbcL
matK
trnL-F
2829 [s, r]
Argentina
L24397
DQ790211
L26074
DQ787438
DQ788712
N/A
N/A
EF464531*
DQ787443
DQ788711
a
4591 [l,
m, t]
Misodendrum
punctulatum Banks
ex DC
G. Amico
3031 [r]
4593
Argentina
Moquiniella rubra
(Spreng. f.) Balle
K. Steiner
3042
South Africa AF039078
DQ790207
DQ790132
DQ790171
EF464489*
Notanthera
heterophylla (Ruiz
& Pavón) G. Don.
C. Aedo
G. Amico
4372
4582 [t]
Chile
EF464466*
EF464478*
EF464529*
EF464511*
DQ442939
Nuytsia floribunda
(Labill.) R. Br. ex
G. Don f.
B. Lamont
2747
3080 [t]
W. Australia DQ790103
DQ790210
DQ790134
DQ787446
DQ788716
Olax emirnensis
Baker
G. Schatz et al.
3620
4035
Madagascar
DQ790119
DQ790214
DQ790136
DQ790173
N/A
N/A
N/A
N/A
N/A
AF534674
Olax acuminata Wall.
Opilia amentacea
Roxb.
D. L. Nickrent
2816
2816
2809 [t]
Australia
L24407
DQ790202
L26076
AY042621
EF464495*
Osyris lanceolata
Hochst. & Steud.
Orange Free State
Botanic Garden
2731
South Africa L24409
AF389274
EF464525*
EF464506*
N/A
N/A
N/A
N/A
N/A
AY191142
Osyris wightiana
Wall.
Ptychopetalum
petiolatum Oliver
E. J. Breteler
14745
4212
Gabon
DQ790121
DQ790215
DQ790138
DQ790175
EF464490*
Pyrularia pubera
Michx.
L. J. Musselman
2737
U.S.A
L24415
N/A
DQ329179
EF464507*
N/A
N/A
N/A
N/A
N/A
AF534679
Pyrularia edulis A.
DC.
a
*
Quinchamalium
chilense Lam.
R. Vidal-Russell
4503
Argentina
EF464469*
N/A
EF464533*
EF464514*
EF464491*
Santalum album L.
R. Narayana
2734
India
L24416
AY957453
L26077
AY957453
AY191151
Schoepfia
vacciniflora
Planchon ex
Hemsley.
G. McPherson &
P. M. Richardson
15981
3069
Panama
N/A
N/A
N/A
EF464515*
N/A
Schoepfia fragrans
Wall.
Tsi Zhanhuo 91417 MO 4252063
5009
China
N/A
N/A
N/A
N/A
DQ788718
Schoepfia schreberi
Gmelin
D. L. Nickrent
2599
2599
Bahamas
L24418
AF389261
L11205
DQ787447
DQ788717
Spirogardnera
rubescens Stauffer
S. Patrick
4996
5018B [t]
Australia
EF464458*
N/A
EF464518*
EF464497*
EF464492*
Tristerix corymbosus
(L.) Kuijt
V. Melzheimer
G. Amico
G. Amico
G. Amico
4129 [s,
m]
4572 [l]
4597A [t]
4575E [r]
Chile
Chile
Chile
Argentina
EF464467*
EF464479*
EF464530*
EF464512*
EF464493*
Tupeia antarctica
(Forst. f.) Cham. &
Schlect.
B. Molloy 2575
2742
New Zealand L24425
DQ790208
DQ790133
DQ790172
EF464494*
Viscum album L.
P. Faber
3024
U.S.A
L24426
AF389275
L26078
N/A
AF180540
Viscum articulatum
Burman f.
D. L. Nickrent
2812
Australia
L24427
N/A
EF464517*
EF464496*
AY191131
Between braces genes for which that accession was used (s = SSU rDNA, l = LSU rDNA, r = rbcL, m = matK, t = trnL-F)
This study
2002). To calibrate the Santalales trees, Saxifraga was
used as outgroup. The root age was set to 114 Mya
according to the divergence time estimate of Santalales
from Saxifragales determined by a non-parametric rate
smoothing analysis of a multigene dataset (SSUrDNA,
r b c L , and atpB) of 560 species of angiosperms
(Wikström et al., 2001). To take into consideration the
variance of this time estimate, we repeated the analysis
setting the root to the lower and upper end of the 95%
confidence interval of their estimation i.e. 104 and 124
Mya. Additional Santalales fossils were used as time
constraints for crown groups. The first analysis
included the outgroup and Anacolosidites at 70 Mya
(assumed equivalent to extant Anacolosa as per
Malécot and Lobreau-Callen, 2005). The second
analysis included the outgroup, Anacolosidites,
Cranwellia for the crown group of Loranthaceae at 70
Mya (Mildenhall, 1980), Arjona at 65 Mya (Chlonova,
1962; Jarzen, 1977), Santalum at 65 Mya (Darrah,
1939; Christopher, 1979) and Arceuthobium (as
Spinulaepollis arceuthobioides) at 52 Mya (Krutzsch,
1962).
The BRMC method was implemented utilizing the
software MULTIDIVTIME (Thorne et al., 1998). This
method approximates the mean posterior probabilities
of substitution rates and divergence times with
credibility intervals obtained through the Markov
Chain Monte Carlo algorithm. The protocol from the
manual by Rutschmann (2005) as well as the Readme
files from the program were followed. The topology
used for the analysis was the one obtained with BI
using the five genes, but only matK and rbcL were used
for estimating divergence time. These two genes were
chosen because they had the most complete sampling,
are derived from one linkage group (the chloroplast
genome), and have similar rates of evolution.
Parameters estimated with BASEML (Yang, 1997)
under the F84 model were used to estimate branch
lengths and the variance-covariance matrix in
ESTBRANCHES. The output file from this program
was used as input for MULTIDIVTIME. The Markov
chain was run for 1 million generations sampling every
100 generations. The burn-in was set to be 10% of the
samples (1000 trees). We used 1.14, 1.04 and 1.24
(114, 104 and 124 Mya, respectively) for the expected
number of time units between tip and root with
standard errors of 0.57, 0.50 and 0.62, respectively.
The prior for the rate was obtained by dividing the
median of the branches by the time from the ingroup
root to the tips, resulting in 0.1 substitutions/site/time
unit. The Brownian motion parameter (nu) that controls
the degree of rate autocorrelation along the descending
branches of the tree was set so that the time units from
root to tip multiplied by nu was approximately 1.0, as
recommended in the manual. A total of ten runs with
the same settings, but different seeds, were performed
to check for consistency in the results.
The PL method was implemented with r8s
software (Sanderson, 2003). This approach allows each
branch to have its own average rate, invoking a penalty
function that controls rate variation over time. Branch
lengths used to estimate node ages were obtained using
maximum likelihood in PAUP* under the GTR+I+G
model of sequence evolution. The smoothing parameter
was estimated via cross-validation. The tree root date
was set to 114, 104 and 124 Mya and the dates for the
santalalean fossil lineages (above) were used as time
constraints.
3. Results
3.1. Variability of the nuclear and chloroplast markers
The nuclear SSU rDNA matrix consisted of 1832
aligned sites of which 175 were parsimony
informative. Thirty-two most parsimonious trees were
found, each 720 steps in length. The nuclear LSU
rDNA dataset consisted of 972 aligned sites of which
177 were parsimony informative. Four equally
parsimonious trees were found of length 891 steps.
Because the SSU and LSU rDNA matrices, when
analyzed separately, resolved very few clades, they
were concatenated. This is justified given these regions
occur within the same transcriptional cistron. In this
analysis, 51 most parsimonious trees were found of
1705 steps. The chloroplast gene, rbcL, had 1437
aligned positions of which 267 were parsimony
informative. The second chloroplast gene matK had
1387 aligned positions of which 554 were parsimony
informative. Parsimony analysis yielded nine trees of
length 2379. The trnL-F region consisted of 862
aligned sites of which 210 were parsimony informative
(after exclusion of ambiguous sites mainly from the
spacer between the trnL and trnF genes). The search of
the trnL-F region matrix resulted in 4680 trees of 761
steps.
3.2. Topologies of the separate gene trees
Tree topologies resulting from analyses of the
combined SSU and LSU rDNA and the individual
chloroplast genes are shown in Fig. 1. For the rDNA
data (Fig. 1A), a monophyletic Loranthaceae was
supported (BS = 73, PP = 1.0) which was sister to a
clade composed of Misodendraceae, Schoepfia and
Arjona (BS = 88, PP = 1.0). Two other major clades
were recovered by this analysis, one corresponding to
Opiliaceae (BS = 100, PP = 1.00) and another to
Santalaceae (BS = 66, PP = 0.93) including a
monophyletic Viscaceae. The relationship between
these three main clades was not well supported.
Compared with the rDNA tree, the r b c L partition
resolved additional clades (Fig. 1B). Strong support
(BS = 84, PP = 1.0) was obtained for the clade
composed of a monophyletic Loranthaceae and its
sister clade Misodendraceae plus Schoepfia, Arjona
and Quinchamalium. Support for some clades within
Santalaceae was obtained, e.g. the eremolepidaceous
mistletoes (PP = 0.92), Amphorogyneae (BS = 80, PP
Fig. 1. Gene trees derived from Bayesian analyses of the different partitions. The nuclear SSU and LSU rDNA matrices were
concatenated and analyzed together.
= 1.0), and Viscaceae (BS = 100, PP = 1.0), but
relationships among the major clades were not strongly
supported. The chloroplast gene matK (Fig. 1C)
recovered two major clades, one composed of
Loranthaceae, Misodendraceae, Schoepfia, Arjona and
Quinchamalium (BS = 69, PP = 1.0) and another that
included Opiliaceae, Santalaceae and Viscaceae (BS =
73, PP = 1.0). Strong support was obtained for
Misodendraceae, Loranthaceae, Opiliaceae, and two
clades within Santalaceae, but relationships among
Fig. 2. Majority rule consensus tree from Bayesian analysis of the concatenated five-gene data set. Nodal support is given above the
branches as bootstrap values for parsimony and posterior probabilities, respectively. Branch lengths are represented by mean values
of the trees resulted from Bayesian analysis. Dark branches represent aerial parasites. Clades discussed in the text are labeled A – H.
these families was poorly supported. The trnL-F region
(Fig. 1D) recovered the same two major sister clades
obtained with matK. The first included Loranthaceae,
M i s o d e n d r a c e a e , S c h o e p f i a , A r j o n a , and
Quinchamalium (BS= 79, PP = 1.0). Within the first
clade, Misodendraceae was sister to a clade formed by
Schoepfia, Arjona, and Quinchamalium (BS = 76, PP =
0.65), and Loranthaceae was monophyletic (BS = 86,
PP = 1.0). The other major clade comprised Opiliaceae,
Viscaceae and the remaining genera of Santalaceae (BS
= 78, PP = 1.0). Although, Opiliaceae and Viscaceae
were monophyletic with high support, the relationship
among these three families was not resolved.
3.3. Topology of the concatenated data tree
Trees from the four separate data partitions (Fig.
1A-D) differed mainly in their levels of support for
various clades; no strong support was obtained for
conflicting clades. For this reason, data from all
partitions were concatenated. This matrix had 6490
aligned positions of which 1415 were parsimony
informative. MP analysis yielded three most
parsimonious trees of 6080 steps. The three tree
topologies differed only in relationships among genera
of aerially parasitic Loranthaceae. Relationships among
the major santalalean clades were completely resolved
and each node received high bootstrap support in MP
as well as high BI posterior probabilities. The majority
rule BI consensus tree (Fig. 2) will thus be used to infer
the origin of aerial parasitism in the order.
The BI consensus tree is composed of two major
sister clades, each of which contains root and aerial
parasites (clades of interest labeled A – H on Fig. 2).
The first major clade contains a monophyletic
Loranthaceae (clade C) which is sister to another
composed of Misodendraceae (clade A) and clade B
that contains Schoepfia, Quinchamalium and Arjona.
Within clade B the two species of Schoepfia are sister
to Arjona and this clade is sister to Quinchamalium.
For Loranthaceae, the western Australian root parasitic
tree Nuytsia is sister to the remaining members of the
family. The next two diverging taxa, Gaiadendron
(New World tropics) and Atkinsonia (eastern
Australia), are also root parasites. Atkinsonia is weakly
supported as sister to the remaining seven genera,
which are all aerial parasites. Relationships among the
aerial parasite clades received low MP bootstrap
support.
The second major clade (D-H) contains
representatives of Opiliaceae, Santalaceae and
Viscaceae. Here the two root parasitic genera of
Opiliaceae are monophyletic (clade D) and are sister to
the remaining clades E-H. Clade E is composed of the
root parasites Pyrularia and B u c k l e y a . Clade F
contains five genera, two of which are root parasites
(Osyris and Santalum) and the remaining three are
New World mistletoes previously classified in their
own family Eremolepidaceae. Clade G, representing
tribe Amphorogyneae, is composed of root and stem
parasites as well as a dendroparasite (Dendrotrophe –
see Discussion). Clade H, contains two long-branch
taxa that are typically classified in Viscaceae.
3.4. Chronogram from BRMC
The time estimates derived from both the BRMC
and PL methods (using one or five crown group
calibration points derived from fossils) are shown in
Table 2. Except for Loranthaceae, PL gave more recent
time estimates than the BRMC. The addition of
multiple calibration points did not change the PL
estimations but did affect those from the BRMC
method. Here the differences between estimates for a
taxon derived from the three root node dates were less
when multiple calibrations were used. The BRMC
estimates with multiple calibrations and the 114 Mya
Saxifraga root age were used to make the BI tree
ultrametric. The resulting chronogram (Fig. 3) shows
the relative timing of the evolution of aerial parasitism
in Santalales. Among the extant lineages that contain
aerial parasites, an absolute age of 80 Mya is obtained
for Misodendraceae (clade A) followed by Viscaceae
that arose 72 Mya. It should be pointed out that it is not
known at which point along the stem of these clades
each parasitic lineage acquired the aerial habit.
Moreover, relatively large standard deviations are
associated with these BRMC estimates, thus whether
one or the other family evolved first is statistically
equivocal. At 53 Mya, clade F taxa (Eremolepidaceae)
evolved next followed by the dendroparasites and
mistletoes of Santalaceae tribe Amphorogyneae (46
Mya). Finally, the most recent evolution of aerial
parasitism appears to be in Loranthaceae at 28 Mya.
4. Discussion
The present study confirms the previous finding
(Nickrent, 2002) that aerial parasitism evolved five
times independently in Santalales and, for the first
time, assigns dates to these nodes on the phylogenetic
tree. Informed by the ultrametric tree reported here,
these evolutionary events can be better understood by
examining various parasitic modes seen throughout
Santalales (Fig. 4A-I). In this portion of the Santalales
tree the plesiomorphic state is a root parasitic tree or
shrub (Fig. 4A), a condition found in the outgroup
Olacaceae, as well as three genera of Loranthaceae
(Nuytsia, Atkinsonia, and Gaiadendron), Opiliaceae,
and some Santalaceae. In root parasitic lianas (Fig.
4B), the aerial shoots clamber and twine through the
host tree but no haustoria are formed from aerial parts
of the parasite. Examples include C a n s j e r a
(Opiliaceae) and some Santalaceae (Amphorogyneae),
such as Dendrotrophe varians. Some mistletoes such
as Tripodanthus acutifolius (Loranthaceae) initially
form primary haustorial connections to host stems and
later, by means of adventitious roots, form haustorial
connections to host roots (Fig. 4C). These mistletoes
may also exist only as aerial parasites. We suggest
using the term “amphiphagous” to describe a condition
where individuals of a species feed upon stems, roots
or both simultaneously. The stem parasitic lianas or
dendroparasites (Fig. 4D) first attach to host branches
by means of a radicular primary haustorium. Through
further development, secondary haustoria are produced
from roots that arise from the twining stems. Our
definition of dendroparasite differs from the one given
by Macklin and Parnell (2002) where they equate the
term with mistletoe (aerially parasitic non-twining
shrubs). In some species of Dendromyza (Santalaceae,
Amphorogyneae), it appears the shoots are dimorphic
in that some are non-twining, foliose, and
photosynthetic whereas others are twining, squamate
and form haustoria.
Among mistletoes, a number of different
haustorial systems (Calvin and Wilson, 2006) can be
seen (Fig. 4E-I). Some form a primary haustorial
connection to the host and this remains the sole
connection throughout the life of the parasite (Fig. 4E).
The haustorial endophyte remains localized in the
region of original infection. Examples of this include
Misodendrum, many Loranthaceae (e.g. Englerina,
Lysiana, Psittacanthus, Tapinanthus), Dufrenoya
(Amphorogyneae), and some Viscaceae. In other
mistletoes, a primary haustorium is formed but the
endophyte (bark strand) spreads laterally within the
host cortex distal to the point of original infection (Fig.
4F). Examples of this type in Loranthaceae include
Diplatia, Moquiniella, Tristerix and T u p e i a . A
variation also occurs where the bark strands are
capable of initiating secondary shoots (Fig. 4G) such as
in Agelanthus, Oncocalyx, and Helixanthera (all
capable of forming epicortical roots that “explore” the
host surface and form secondary haustorial connections
Table 2
Bayesian relaxed molecular clock (BRMC) with standard deviations and penalized likelihood (PL) time estimates
(Mya) for divergence of aerial parasite clades in Santalales given standard errors around a 114 Mya age for the
Saxifraga root (Wikström et al. 2001).
Clade letter and name
BRMC
104
PL
114
124
104
114
124
Anacolosa calibration point
A. Misodendraceae
94 (± 21)
99 (± 23)
103 (± 25)
74
81
88
C. Loranthaceae
33 (± 09)
34 (± 10)
36 (± 11)
32
35
38
F. Eremolepidaceae
65 (± 16)
68 (± 17)
71 (± 19)
51
55
60
G. Amphorogyneae
56 (± 10)
58 (± 16)
61 (± 17)
40
44
48
H. Viscaceae
87 (± 19)
91 (± 22)
94 (± 24)
68
75
81
Anacolosa and four other calibration points
A. Misodendraceae
79 (± 9)
80 (± 9)
81 (± 9)
74
81
88
C. Loranthaceae
27 (± 5)
28 (± 6)
28 (± 6)
32
35
38
F. Eremolepidaceae
53 (± 7)
53 (± 7)
54 (± 7)
51
55
60
G. Amphorogyneae
46 (± 7)
46 (± 7)
46 (± 8)
40
44
48
H. Viscaceae
71 (± 8)
72 (± 8)
73 (± 8)
68
75
81
Fig. 3. Chronogram resulting from BRMC analysis. Aerial parasite clades (dark branches) are labeled as in Fig. 1. Pie diagrams
derived from Mesquite represent the probabilities of root (white) or aerial (black) parasitism for that node.
(Calvin and Wilson, 2006). Fig. 4H is meant to represent
all variants of this type in that some epicortical roots arise
from haustoria and some from shoots. Shoots frequently
occur at the site where secondary haustorial connections
are made. Examples include many Loranthaceae such as
Alepis, Desmaria, Notanthera, and Scurrula. The
squamate mistletoes (Fig. 4I) are here recognized as
different from the type shown in Fig. 4G only to
emphasize the presence of scale leaves with lowered
photosynthetic activity. This type is seen in Misodendrum,
Phacellaria and Arceuthobium.
4.1. The Misodendraceae clade
Given the standard deviations associated with the dates of
divergence for Viscaceae and Misodendraceae, it cannot
be unambiguously determined which lineage first evolved
aerial parasitism. We propose, however, that
Misodendraceae most likely represent the first mistletoes
because the divergence date of 80 Mya is older than that
obtained for Viscaceae (72 Mya). Moreover, the Mesquite
analysis gave an 0.6 probability that the ancestor of
Viscaceae was aerial whereas for Misodendraceae the
probability was near 1.0 (Fig. 3). Although fossil pollen
for Misodendrum (as Compositoipollenites) are not known
from strata older than Oligocene, this is a minimum age
and older fossils likely exist but remain undiscovered.
Furthermore, we suggest that divergence times for
Viscaceae on the chronogram (Fig. 3) have been
artifactually inflated owing to among lineage rate
heterogeneity (see below). The difference between the
stem and crown group ages for the Misodendraceae clade
spans 53 million years. As no macrofossils are available
and only nodal dates are estimated, we cannot determine
at which point along this long branch aerial parasitism
appeared. Because Misodendraceae are a small and
apparently old family, and that past extinctions have gone
unrecognized, the timing of diversification may be
underestimated. The phylogenetic history of Misodendrum
must be closely tied to that of its only host, Nothofagus
(southern beech). The fossil record for Nothofagus
suggests a Cretaceous origin for the genus in the early
Campanian and all subgenera were differentiated by 75
mya (Dettmann et al., 1990; Knapp et al., 2005). The time
estimate from molecular data for the divergence of
Nothofagaceae from the remaining Fagales was 93 Mya
(Cook and Crisp, 2005). Given the coincidental timing of
the origin of Nothofagus and Misodendrum, it is possible
that these two taxa codiversified during the Cretaceous.
Fig. 4. Parasitic modes present in Santalales. Arrows for A-D represent haustorial connection points. See text for explanation.
The Misodendraceae clade is sister to one
containing Schoepfia, A r j o n a and Quinchamalium.
Schoepfia has most frequently resided in Olacaceae,
but its position on molecular phylogenetic trees
supports its classification in a separate family,
Schoepfiaceae (Nickrent et al., 1998; Nickrent and
Malécot, 2001). These results support those of Der and
Nickrent (in press) who also included the South
American root parasites Arjona and Quinchamalium in
Schoepfiaceae, not Santalaceae.
4.2. The Loranthaceae clade
As reviewed in the Introduction, pollen associated
with Loranthaceae (loranths) has been described from
numerous locations in strata dated to Late Cretaceous.
This correlates well with the divergence date for the
loranth clade reported here as 81 Mya (Fig. 3). Because
the three root parasitic genera in the family (Nuytsia,
Gaiadendron, and Atkinsonia) are successive sister
taxa to all aerial parasites, it is most parsimonious to
envision the ancestral loranth as a root parasite, not an
aerially parasitic mistletoe. If the ancestral loranth was
a stem parasite, the tree topology requires three
reversions to root parasitism. We consider this scenario
less likely based simply on parsimony (but see below).
The ancestral root parasitic loranth likely existed in
warm tropical to subtropical conditions and may have
been arborescent. This follows from the habit of
Nuytsia which speciated first from the main loranth
lineage during the Eocene. Our analyses indicate that
the acquisition of aerial parasitism in Loranthaceae
occurred once and was probably a more recent event
than any of the other four mistletoe lineages (28 Mya).
Branch lengths between aerially parasitic loranth
genera are very short, suggesting a rapid diversification
during the Oligocene. This epoch marked the start of
global cooling where tropical regions diminished
giving way to temperate deciduous woodlands and
grasslands. Today, savanna biomes of Africa and
Australia support an extremely rich loranth flora, thus
suggesting this habitat type provided the environment
favoring the rapid diversification of these mistletoes.
Are there recorded cases of aerially parasitic
loranths “reverting” to root parasitism? Most species of
the genus Helixanthera, a genus that ranges from
Africa to Indomalaya, are stem parasites. The Chinese
species Helixanthera terrestris and H. scoriarum are
cited as being terrestrial root parasites (Huaxing et al.
2003), thus suggesting evolutionary reversions or
atavism. These observations need to be confirmed
because parasitism of small shrubs, especially ones
with atrophied branches, can be misleading. In the case
of Tripodanthus acutifolius (Fig. 4C), it appears that
the mistletoe is first established on host branches and
then attaches to roots via secondary haustoria derived
from adventitious roots (Kuijt, 1982). With regard to
Gaiadendron, Kuijt (1963) observed terrestrial as well
as epiphytic individuals, but makes the point that the
distinction between these zones is not always
meaningful. It is possible that Gaiadendron starts as a
seedling in the soil, attaches to roots (or rhizomes) and
grows upward into the host tree where it then forms
secondary haustorial connections. Alternately, its seeds
could lodge in crevices between host branches where
humus has accumulated. Following germination, the
seedling could attach to roots of the surrounding
epiphytes or to young host branches.
4.3. The “Eremolepidaceae” clade
Fossil pollen of Antidaphne is known from the mid
Eocene of Chile (Darrah, 1939 and references therein).
This date is ca. 5 my more recent than the molecular
estimation for the divergence time of this genus from
the Lepidoceras and Eubrachion clade. Although only
two root parasites were used to represent this clade
( S a n t a l u m and Osyris), a complete molecular
phylogeny of Santalaceae (Der and Nickrent, in press)
added Colpoon, Rhoiacarpos, Nestronia, Mysoschilos,
and Exocarpos, the latter sister to the entire clade.
Among the 26 species of the genus Exocarpos, at least
two have been reported to parasitize either roots or
stems (Lam, 1945; Kuijt, 1969). This, in conjuction
with a 0.6 probability that the ancestor of the
eremolepidaceous parasites was an aerial parasite (Fig.
3), suggests that the amphiphagous habit provided the
genetic preadaptation for the evolution of stem
parasitism in these mistletoes.
4.4. The Santalaceae – Amphorogyneae clade
Among the paraphyletic Santalaceae, the second
lineage to evolve aerial parasitism are various members
of tribe Amphorogyneae. This remarkable group of
plants, first taxonomically recognized by Stauffer
(1969), includes what might be considered a transition
series of parasitic habits – root parasites (Choretrum
and Leptomeria), amphiphagous parasites (Daenikera
and possibly D e n d r o t r o p h e ) , dendroparasites
(Dendromyza) ,
mistletoes
(D u f r e n o y a ) a n d
hyperparasitic mistletoes (Phacellaria). Daenikera
corallina is typically seen as a root parasite, but some
individuals emerge at the base or even some distance
up the stem of the host tree. It has been suggested that
this taxon is a holoparasite, but as noted by Hürlimann
and Stauffer (1957), there is chlorophyll in the
epidermis but it is masked by red pigments. Moreover,
we report here an rbcL sequence for Daenikera that is
full-length, does not show an increased subsitution rate
relative to other Amphorogyneae, and has no amino
acid replacements that might suggest lack of
functionality. Therefore, this plant may be compared to
other flabellate, red-pigmented, photosynthetic
Santalaceae such as Exocarpos casuarinoides.
The chronogram (Fig. 3) shows that the aerial
parasites of tribe Amphorogyneae (Dendrophthoe and
Dendromyza) evolved at the beginning of the Eocene.
No conclusive fossils exists for any member of this
tribe, possibly because the entemophilous members
produce low amounts of pollen and because the
Australian root parasites occur in dry areas. Moreover,
pollen from extant genera present few distinguishing
features that would allow identification of existing
palynomorphs. Older references to Leptomeria from
Baltic amber (Pilger, 1935) are likely not equivalent to
the modern genus that is endemic to Australia.
Because Amphorogyneae is sister to Viscaceae
(all of which are stem parasitic mistletoes) their
common ancestor could be envisioned as an aerial
parasite. But because Daenikera is sister to the
Amphorogyneae clade, and it is amphiphagous, it
seems reasonable to propose this parasitic mode for the
ancestor. In fact the probability that this ancestor was
aerial is only 0.10, increasing to 0.6 in the lineage
leading to Viscaceae (Fig. 3). Specialization in
Viscaceae was entirely toward the mistletoe habit
whereas in Amphorogyneae, this polymorphism was
maintained in the basal lineages. Mistletoes such as
Dufrenoya and Phacellaria thus evolved independently
from Viscaceae. The genera Choretrum, Leptomeria,
and Spirogardnera are all root parasites, thus
suggesting they lost (or do not express) the ability to
parasitize stems, or they descended from individuals
that were already root parasites. This may be correlated
with their increased ability, as compared with stem
parasites, to survive fire via regeneration from
underground parts, as these plants grow in fire-prone
areas.
4.5. The Viscaceae clade
Viscaceae apparently diverged early (72 Mya)
from the common ancestor it shared with
Amphorogyneae. As discussed above, this date
competes with the one for Misodendraceae as the
oldest lineage of aerial parasite, but we consider the
date inflated owing to rate heterogeneity. Within
Santalales, rate acceleration was first observed for
Viscaceae, particularly Arceuthobium (Nickrent and
Franchina, 1990; Nickrent and Starr, 1994). Viscaceae
show longer branches than the rest of the taxa (Fig. 1)
and rate increases are affecting both nuclear and
chloroplast genomes, in agreement with Nickrent et al.
(1998).
All Viscaceae are mistletoes that form primary
haustorial connections but do not produce epicortical
roots (as in some Loranthaceae). Lateral spread of the
endophyte within the host cortex does occur and new
shoots may arise at positions distal to the original
infection (Fig. 4G). The most elaborate manifestation
of endophytic growth is the production of systemic
(isophasic) “witches’ brooms” in some species of
Arceuthobium where the parasite endophyte grows in
synchrony with the host apical meristem.
4.6. Evolution of the mistletoe habit
The data presented here can now be generalized to
help address questions relating to the evolution of the
mistletoe habit. All five mistletoe clades appear to have
evolved from root parasitic ancestors. In four of these
clades there is evidence for preadaptation to stem
parasitism in amphiphagous taxa (and also assumed
present in ancestral nodes). These are Gaiadendron
(Loranthaceae), Exocarpos (eremolepidaceous clade),
Daenikera (Amphorogyneae), and the common
ancestor of the latter and Viscaceae. The evolutionary
directionality of this character state change is strongly
biased from root to stem parasitism. Cases of potential
reversion to root parasitism, such as in Helixanthera
and Tripodanthus (Loranthaceae), possibly indicate
atavism. That such events are rare suggest that aerial
parasitism is a successful trophic mode under strong
positive selection. The Misodendraceae clade is sister
to a clade with only root parasites (Schoepfia, Arjona
and Quinchamalium), thus the Misodendrum ancestor
presumably was root parasitic. Given the evolutionary
transitions seen in other mistletoe lineages, it is
conceivable that the ancestor of Misodendrum also
passed through an amphiphagous condition prior to
becoming an aerial parasite.
Misodendraceae and Viscaceae were apparently
the first lineages to evolve stem parasitism. They also
have a high degree of specialization in morphological
features. Thus, we hypothesize that during this long
evolutionary time period positive selection for some
traits and relaxation of selection in others resulted in
their highly derived conditions. In Loranthaceae,
whose diversification occurred more recently and
rapidly (see shorter branches on Fig. 2), such profound
changes are not seen. Most genera in Loranthaceae
show a higher number of pleisomorphic characters,
e.g., epicortical roots. These are also present in some
members of the eremolepidaceous lineage, but are
absent in Viscaceae and Misodendraceae that
developed cortical strands through endophytic growth
(Fig. 4F, G). Such cortical strand growth is seen in only
ten of the 73 genera of Loranthaceae.
Mistletoe evolution provides good examples of
convergence in several morphological traits. Leaves
reduced to scales (the squamate condition) as seen in
Arceuthobium (Viscaceae) are strikingly similar to
those in Misodendrum. These squamate mistletoes are
also characterized by reduction in chlorophyll content
and small, unisexual flowers. This suite of features is
present in Eubrachion of the eremolepidaceous lineage
and Phacellaria and Daenikera of Amphorogyneae.
Curiously, the squamate habit has rarely evolved
among the 900 species of Loranthaceae. Although
many genera show a high degree of floral reduction,
Ixocactus hutchisonii represents the only example of a
squamate loranth whose phylloclades (not leaves) are
photosynthetic (Kuijt, 1967). Paucity of this growth
form may mean that this trait is associated with a trend
towards holoparasitism, which is essentially absent in
Loranthaceae. Among the five mistletoe lineages,
different evolutionary “experiments” have taken place
that resulted in attachment structures on the seeds or
fruits. In Misodendrum the achenes bear long, plumose
staminodes that first keep the fruit aloft and then serve
to anchor it onto the host branch. Viscin coated seeds
are seen in Loranthaceae, Viscaceae, the
eremolepidaceous clade, and some members of
Amphorogyneae (Dufrenoya and Phacellaria) but
these attachment structures have different
developmental origins and are thus not homologous.
The phylogenetic tree presented in Fig. 2 has two
major clades that suggest southern (clades A-C) and
northern (clades D-H) hemisphere origins for these
families. Loranthaceae are usually cited as having
arisen on Gondwana whereas Viscaceae evolved on the
Laurasian landmass (Barlow, 1983). As discussed
above, it appears Loranthaceae underwent a major
radiation during the Oligocene, but this was preceded
by a massive dispersal throughout Gondwana as early
as the Cretaceous (Barlow, 1983). Santalaceae and
Viscaceae do not have relictual occurrences on
southern landmasses and thus are likely of northern
origin. Essentially nothing has been written about the
biogeographic history of Santalaceae, thus most
interpretations must derive from the fossil record and
the distributions and molecular phylogenies of extant
taxa. Phylogenetic evidence (Der and Nickrent, in
press) indicates the entirely New World
eremolepidaceous taxa diverged early in the clade,
possibly from a root parasite similar to extant
Myoschilos. The sister to all other members of the
Amphorogyneae clade, D a e n i k e r a , is a New
Caledonian endemic whereas the root parasites are
found in Australia and the dendroparasites and
mistletoes range from Queensland through Indomalaya
to China and southern Asia. Based on morphological
features Stauffer and Hurlimann (1957) considered
Amphorogyne and Daenikera as primitive and relictual,
a position supported by the molecular phylogenetic
topology presented here. This suggests a southern
origin for Amphorogyneae, not northern as seen in
Viscaceae and other clades of “Santalaceae”.
4.7. Reliability of the chronogram and fossil data
Error associated with the dates assigned to the
various nodes on the chronogram derives mainly from
two sources: 1) the robustness of the molecular
phylogeny and 2) the age and identity of the fossil
pollen used for calibrating the tree. With regard to
topology, our multigene tree for Santalales has higher
support for the nodes along the “spine” (i.e.,
interfamilial relationships) than any previously
published one. However, even when the topology of
the tree is reliable, rate heterogeneity will artifactually
inflate divergence times. In our case, the longest
branches on the tree were associated with Viscaceae.
Although the r8s program constrains the rate of change
from ancestral to derived lineages, simulations have
shown that it is difficult to estimate divergence times
with heterogeneous rates (Sanderson, 1997). Despite
this, divergence times estimates from BRMC and PL in
Santalales were comparable, as was observed in a
broader study of all angiosperms (Bell et al., 2005).
The second limitation relates to the microfossil
data. It is difficult to precisely place a fossil taxon with
the stem or crown group because a phylogenetic
(cladistic) analysis must be conducted that
simultaneously includes fossil and extant taxa, after
which diagnostic synapomorphies must be identified.
As pointed out by Magallon and Sanderson (2001) this
has rarely been done with angiosperms. Pollen
represents the vast majority of fossils available for
Santalales, thus these must be used in comparisons
with extant genera to determine affinities. In contrast to
Loranthaceae, pollen from essentially all of the genera
of Santalaceae have been described (Feuer, 1977;
Feuer and Kuijt, 1978; Kuijt, 1988). Despite this, to
date no formal cladistic analysis of these data has been
conducted that would allow integration of the fossil
pollen data. Such studies would help determine
whether the fossil pollen is 1) congeneric with a crown
group member, 2) an extinct member of the crown
group, 3) an extinct stem group representative that is
sister to the crown group, or 4) a member of a clade
sister to the stem group. As with all morphological
data, patterns of variation in pollen are complex and
compromised by intrageneric polymorphism and
convergence. Additional comprehensive studies, such
as the one conducted by Malécot and Lobreau-Callen
(2005) on Anacolosidites, are required to sort out
which pollen types are most likely related to extant
santalalean families and genera.
Acknowledgments
The authors thank the numerous collectors listed in
Table 1 who helped obtain specimens for this project,
Missouri Botanical Garden for allowing a portion of
the Schoepfia fragrans sample to be used for DNA
extraction, J. Der and V. Malécot for use of
unpublished DNA sequences, and S. Sipes for
generously allowing use of her automated DNA
sequencer. Comments from two anonymous reviewers
were helpful in improving this manuscript. Financial
support (to RVR) was provided by a Ph.D. fellowship
from the Fulbright Commission Argentina, SIUC
Graduate School and grants from the National Science
Foundation (to DLN).
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