The circumscription and phylogenetic relationships of Callitropsis and the newly described genus Xanthocyparis (Cupressaceae).
ABSTRACT A new species of conifer was recently discovered in northern Vietnam. In a preliminary phylogenetic analysis of morphological data a possible sister species, Chamaecyparis nootkatensis (D. Don) Spach, was identified; however, because of the presumed phylogenetic remoteness of these two species to the remainder of the Cupressaceae, a new genus-Xanthocyparis-was described to accommodate both species. Here an analysis of ITS (nrDNA), matK, and rbcL sequence data in combination with 58 informative morphological characters was aimed at testing the monophyly of the remainder of Chamaecyparis and evaluating the placement and monophyly of Xanthocyparis. Chamaecyparis, minus C. nootkatensis, was resolved as a monophyletic group, remote from Cupressus and Xanthocyparis. Cupressus, Juniperus, and Xanthocyparis formed a very highly supported monophyletic group. However, Cupressus was not monophyletic. Instead the Old World species sampled were resolved sister to a clade containing a monophyletic Juniperus, a monophyletic Xanthocyparis, and a clade of New World Cupressus species. If both species of Xanthocyparis are to be treated as members of the same genus, then due to the principal of priority they will have to be recognized in the genus Callitropsis. Research is continuing to resolve the status of New World and Old World Cupressus.
- SourceAvailable from: P. Gadek[show abstract] [hide abstract]
ABSTRACT: Parsimony analysis of matK and rbcL sequence data, together with a nonmolecular database, yielded a well-resolved phylogeny of Cupressaceae sensu lato. Monophyly of Cupressaceae sensu stricto is well supported, and separate northern and southern hemisphere subclades are resolved, with Tetraclinis within the northern subclade; there is no support for any of the tribes sensu Li. Taxodiaceae comprise five separate lineages. Chamaecyparis nootkatensis falls within Cupressus, clustering with a robust clade of New World species. Libocedrus Florin is paraphyletic and should incorporate Pilgerodendron. Evolution of several characters of wood and leaf anatomy and chemistry is discussed in light of this estimate of the phylogeny; numerous parallelisms are apparent. A new infrafamilial classification is proposed in which seven subfamilies are recognized: Callitroideae Saxton, Athrotaxidoideae Quinn, Cunninghamioideae (Sieb. & Zucc.) Quinn, Cupressoideae Rich. ex Sweet, Sequoioideae (Luerss.) Quinn, Taiwanioideae (Hayata) Quinn, Taxodioideae Endl. ex K. Koch. The rbcL sequence for Taxodium distichum is corrected, and the implications for a previously published estimate of the minimum rate of divergence of the gene since the Miocene are highlighted.American Journal of Botany 07/2000; 87(7):1044-57. · 2.59 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data, In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.Evolution 01/1985; 39(4):783-791. · 4.86 Impact Factor
Article: Wood Structure of Thuja OccidentalisBotanical Gazette 01/1941; 103(2).
American Journal of Botany 91(11): 1872–1881. 2004.
THE CIRCUMSCRIPTION AND PHYLOGENETIC
RELATIONSHIPS OF CALLITROPSIS AND THE NEWLY
DESCRIBED GENUS XANTHOCYPARIS (CUPRESSACEAE)1
DAMON P. LITTLE,2,6ANDREA E. SCHWARZBACH,3ROBERT P. ADAMS,4
AND CHANG-FU HSIEH5
2L. H. Bailey Hortorium, 228 Plant Science Building, Cornell University, Ithaca, New York 14853 USA;3Department of Biological
Sciences, Kent State University, 256 Cunningham Hall, Kent, Ohio 44242 USA;4Biology Department, Baylor University, P.O. Box
97388, Waco, Texas 76798 USA; and5Department of Botany, National Taiwan University, Taipei 106, Taiwan
A new species of conifer was recently discovered in northern Vietnam. In a preliminary phylogenetic analysis of morphological
data a possible sister species, Chamaecyparis nootkatensis (D. Don) Spach, was identified; however, because of the presumed phylo-
genetic remoteness of these two species to the remainder of the Cupressaceae, a new genus—Xanthocyparis—was described to
accommodate both species. Here an analysis of ITS (nrDNA), matK, and rbcL sequence data in combination with 58 informative
morphological characters was aimed at testing the monophyly of the remainder of Chamaecyparis and evaluating the placement and
monophyly of Xanthocyparis. Chamaecyparis, minus C. nootkatensis, was resolved as a monophyletic group, remote from Cupressus
and Xanthocyparis. Cupressus, Juniperus, and Xanthocyparis formed a very highly supported monophyletic group. However, Cupressus
was not monophyletic. Instead the Old World species sampled were resolved sister to a clade containing a monophyletic Juniperus, a
monophyletic Xanthocyparis, and a clade of New World Cupressus species. If both species of Xanthocyparis are to be treated as
members of the same genus, then due to the principal of priority they will have to be recognized in the genus Callitropsis. Research
is continuing to resolve the status of New World and Old World Cupressus.
Callitropsis; Chamaecyparis; Cupressaceae; Cupressoideae; Cupressus; phylogeny; taxonomy; Xanthocyparis.
Recently, a new species of conifer was found among the
remnants of moist karst forest in northern Vietnam (Avery-
anov et al., 2002; Farjon et al., 2002). The morphological fea-
tures of this new conifer strongly suggested affinity to Cu-
pressaceae—in particular to the subfamily Cupressoideae—but
the placement of this species within the existing genera of
Cupressaceae proved to be problematic. As a result, a new
genus and species were described. Xanthocyparis vietnamensis
is characterized as having the following:
1. Dimorphic leaves: This feature is very common among
members of the Cupressoideae absent only from Junipe-
rus, Microbiota decussata, and some species of Cupres-
2. Small ovulate cones consisting of either two or three
whorls of opposite decussate cone scales: This configu-
ration is also characteristic of C. benthamii, C. nootkaten-
sis, and M. decussata. Additionally, several species in Cu-
pressoideae consistently produce either two or three
whorls of opposite decussate cone scales, but unlike X.
vietnamensis, these species are not polymorphic.
3. Biennial seed maturation: Two-year seed maturation can
also be found in all species of Cupressus and some species
1Manuscript received 27 January 2004; revision accepted 8 July 2004.
The authors wish to thank J. J. Doyle, M. A. Luckow, K. C. Nixon, and
two anonymous reviewers for their constructive comments on earlier drafts
of this manuscript. B. Øllgaard kindly translated a passage from O¨rsted. N.
Franz generously translated several German works. Thanks to M. H. Alford,
J. Bartel, D. K. Harder, T. Hudson, and K. Rushforth for providing plant
materials for this project. Funding from NSF grant DEB 0206092 (to D. P.
L.), NSF grant DEB 0316685 (to A. E. S. and R. P. A.), the American Society
of Plant Taxonomists (to D. P. L.) and the Clausen Fund (to D. P. L.; a gift
to BH by T. J. Cohn) is gratefully acknowledged.
4. Flattened, winged seeds: With the exception of Juniperus,
M. decussata, and Platycladus orientalis, this state occurs
in all species of Cupressoideae.
5. All three leaf developmental phases (juvenile, transition,
and adult; see de Laubenfels, 1953) are regularly found
on branches of mature trees. This trait is uncommon with-
in Cupressoideae, but does characteristically occur in
some species (e.g., Juniperus chinensis L.). In addition,
individuals belonging to species characterized as having
the standard developmental sequence can, on occasion,
display this trait when mechanically damaged. However,
the regular appearance of all three leaf types in X. viet-
namensis appears to be an unusual characteristic.
Farjon et al. (2002) analyzed 54 morphological characters
that placed X. vietnamensis sister to C. nootkatensis within a
paraphyletic Cupressoideae. The results of this analysis
prompted the nomenclatural transfer of C. nootkatensis to Xan-
thocyparis as well as the renaming of three intergeneric hy-
brids. Xiang and Farjon (2003) provided a phenetic analysis
of epidermal characters that placed X. vietnamensis in a cluster
with Chamaecyparis formosensis and C. obtusa to the exclu-
sion of Cupressus nootkatensis. Analysis of this data matrix
with parsimony resulted in a completely unresolved consensus
tree (D. P. Little, unpublished data).
Ironically, C. nootkatensis itself has had a troubled taxo-
nomic history within Cupressoideae, having been placed in
four different genera: Cupressus, Chamaecyparis, Callitropsis,
and Xanthocyparis. The species was first described as a Cu-
pressus by Don in 1824. Upon the description of Chamaecy-
paris (as a segregate of Cupressus) by Spach in 1842 (p. 331),
Cupressus nootkatensis was transferred to Chamaecyparis.
Prior to the middle of the 20th century, most works treated
Chamaecyparis as an infrageneric taxon within Cupressus or
as a synonym of Cupressus—probably because the only mor-
November 2004] 1873LITTLE ET AL.—XANTHOCYPARIS
phological characters available at the time to distinguish some-
what reliably between the genera were ovulate cone size and
Because of the somewhat unusual ovulate cone configura-
tion of C. nootkatensis, the monotypic genus Callitropsis was
created by O¨rsted in 1865 (‘‘1864’’) and promptly sank into
obscurity. O¨rsted hypothesized that Callitropsis was most
closely related to Callitris and Fitzroya, a hypothesis not borne
out by recent analyses (Gadek et al., 2000; Farjon et al., 2002).
As anatomical, embryological, and chemical data accumu-
lated, it became clear that Chamaecyparis nootkatensis (D.
Don) Spach was an extraordinary member of Chamaecyparis,
differing in the duration of seed maturation (Camus, 1914),
seed wing anatomy (Camus, 1914), wood anatomy (Greguss,
1955), wood secondary chemistry (reviewed in Erdtman and
Norin, 1966), fertilization (Owens and Molder, 1975), cutic-
ular micromorphology (Alvin et al., 1980; Oladele, 1983a),
leaf transfusion tracheid pitting (Gadek et al., 2000 contra
Prause, 1909), and low cross-compatibility of microsatellite
primers (Be ´rube ´ et al., 2003). In addition, C. nootkatensis was
found to form spontaneous hybrids when grown in cultivation
with any of several Cupressus species (Jackson and Dallimore,
1926; Mitchell, 1970), while at the same time, no hybrids be-
tween Chamaecyparis nootkatensis and other Chamaecyparis
species have been reported. Of these hybrids, ?Cupressocy-
paris leylandii (A. B. Jacks. & Dallim.) Dallim. is the most
widely cultivated. It has been formed spontaneously multiple
times with both Cupressus macrocarpa and Chamaecyparis
nootkatensis acting as the maternal parent. Notably, fertile off-
spring are produced by F1?Cupressocyparis leylandii (Jack-
son and Dallimore, 1926).
The recognition that Chamaecyparis nootkatensis would
best be accommodated within Cupressus, rather than in Cha-
maecyparis, came with the realization that the similarities in
gross morphology between Cupressus and Chamaecyparis
were largely from a combination of plesiomorphic characters
and parallelism rather than common ancestry (Gadek et al.,
2000). Interestingly, the notion that Cupressus and Chamae-
cyparis were associated on the basis of primitive, rather than
derived, features was first put forth by Masters (1895, p. 313)
and later reiterated by Li (1953).
Recent analyses of chloroplast DNA sequences and mor-
phological data suggest that Cupressus and Chamaecyparis are
rather distantly related within the Cupressoideae and that Cha-
maecyparis as commonly circumscribed (including C. noot-
katensis) was not monophyletic (Gadek et al., 2000). Previous
phylogenetic studies of the Cupressaceae (Hart, 1987; Gadek
and Quinn, 1993; Brunsfeld et al., 1994) had failed to discover
this because either composite terminals were constructed
(Hart, 1987) or C. nootkatensis was not sampled (Gadek and
Quinn, 1993; Brunsfeld et al., 1994).
Cupressus and Chamaecyparis were placed by Li (1953) in
the subfamily Cupressoideae (Appendix 1; see Supplemental
Data accompanying the online version of this article). In the
cladograms presented by Hart (1987) and Farjon et al. (2002),
Cupressoideae sensu Li is paraphyletic. However, reanalysis
of the matrix of Hart (1987) without compartmentalization or
synthetic outgroups results in a monophyletic Cupressoideae
sensu Li (D. P. Little, unpublished data). Recent family-level
molecular analyses (Gadek and Quinn, 1993; Brunsfeld et al.,
1994; Gadek et al., 2000) also support a monophyletic Cu-
pressoideae, but indicate that the monotypic genus Tetraclinis,
which was placed in Callitroideae by Li (and Hart, 1987),
should be included within Cupressoideae sensu Li.
Li (1953) arranged the genera of Cupressoideae in an almost
linear series (there is a single branching point; plate II in Li,
1953) featuring reduction in both the number of cone scales
and the number of seeds. Cupressus was considered the most
primitive and Juniperus the most derived. This tribal classifi-
cation (Appendix 1; see Supplemental Data accompanying the
online version of this article) was largely overturned by the
results of Gadek et al. (2000), but no revised classification has
been put forth.
This study was designed to (1) fully test the monophyly of
Chamaecyparis by sampling all Chamaecyparis species, (2)
evaluate the placement of Xanthocyparis within the Cupres-
soideae, and (3) test the monophyly of Xanthocyparis as cir-
cumscribed by Farjon et al. (2002).
METHODS AND MATERIALS
Taxon sampling—The sampling scheme for the Cupressoideae used by
Gadek et al. (2000) was replicated with four changes: (1) Calocedrus macro-
lepis var. formosana was used in the current study in place of C. macrolepis
Kurz. var. macrolepis; (2) Cupressus arizonica var. arizonica was used in
place of C. arizonica var. glabra (Sudw.) Little; (3) C. lusitanica var. lusi-
tanica was used in place of C. lusitanica var. benthamii (Endl.) Carrie `re; and
(4) Thuja standishii (Gordon) Carrie `re was omitted. In addition, seven taxa—
Chamaecyparis formosensis, C. obtusa var. formosana, C. pisifera, C. thyoi-
des var. henryae, C. thyoides var. thyoides, Cupressus macrocarpa, and Xan-
thocyparis vietnamensis—were added to the sample. Complete sampling and
voucher information can be found in Appendix 1 (see Supplemental Data
accompanying the online version of this article).
Molecular techniques—DNA was isolated from 0.015–0.025 g of silica
dried tissue using the DNeasy Plant Mini kit (Qiagen, Valencia, California,
USA) according to the supplier’s instructions. The polymerase chain reaction
(PCR) was used to amplify ITS (nrDNA), matK (cpDNA), and rbcL (cpDNA).
The ITS was amplified in a volume of 50 ?L in PCR buffer (at the con-
centration recommended by the supplier; Promega, Madison, Wisconsin,
USA) with 0.23 ?mol/mL of each amplification primer (5? GGAAGGAGA-
AGTCGTAACAAGG 3?; 5? CTTTTCCTCCGCTTATTGATATG 3?), 1.5
units Taq polymerase, 2 mmol/L MgCl2, 0.4 mmol/L dNTPs, and ca. 20–50
ng genomic DNA. The reaction mixture was incubated for 60 s at 94?C and
then cycled 38 times (60 s at 94?C, 60 s at 50?C, 60 s at 72?C) with a final
step of 300 s at 72?C.
A 100-?L PCR reaction was used to amplify matK in modified Pa ¨a ¨bo
(1990) buffer (67 mmol/L tris-HCl pH 8.8, 4 ?g/mL [m/v] bovine serum
albumin, 0.2 mmol/L dNTPs) with 2 ?mol/mL of each amplification primer
(Table 2 in Kusumi et al., 2000), 0.25 units Taq polymerase, 1.5 mmol/L
MgCl2, and ca. 25 ng genomic DNA. The reaction mixture was incubated for
180 s at 95?C and then cycled 35 times (30 s at 95?C, 30 s at 55?C, 120 s at
The PCR amplification of rbcL was similar to that of matK except different
primers were used (5? ATGTCACCACAAACAGAAACTAAAGCAAGT 3?;
5? TCACAAGCAGCAGCTAGTTCAGGACTC 3?), the concentration of
primers was reduced to 0.7 ?mol/mL, and the MgCl2concentration was in-
creased to 2.5 mmol/L.
Unincorporated dNTPs and primers were removed from the PCR products
with the QIAquick PCR Purification kit (Qiagen) following the manufacturer’s
instructions. The ITS sequencing reactions were performed in a 10-?L vol-
ume: 2 ?L BigDye Terminator version 3.1 sequencing solution (Applied Bio-
systems, Foster City, California, USA), 2 ?L Half Term dye terminator (Gen-
pak, St. James, New York, USA), 3 ?L (ca. 45 ng) PCR product, 1 ?L primer
(0.29 ?mols/mL; 5? TCGCATTTCGCTACATTCTTC 3?; 5? TGTGTTGGG
TGTTGACACATC 3?; 5? GAAGAATGTAGCGAAATGCGA 3?), and 2 ?L
of water. The cycling program was 25 cycles of 30 s at 96?C, 15 s at 50?C,
1874 [Vol. 91AMERICAN JOURNAL OF BOTANY
240 s at 60?C. Sequencing of matK (primers from Table 2 in Kusumi et al.,
2000) and rbcL (5? TCGCATGTACCTGCAGTAGC 3?; 5? GCGTTGGA-
GAGAYCGTTTCT 3?) followed the recommendations accompanying the
BigDye Terminator cycle sequencing kit. Sequencing reactions were run on
an ABI 3700 sequencer (Applied Biosystems).
Sequence alignment and manipulation—Sequences were assembled using
Sequencher 4.1 (Gene Codes, Ann Arbor, Michigan, USA). Sequences were
aligned with either ClustalX 1.81 (Thompson et al., 1997) or Sequencher and
then adjusted manually. Inferred insertion/deletion events (indel) in ITS and
matK were coded using the ‘‘simple gap coding’’ method of Simmons and
Ochoterena (2000) as implemented in GapCoder (Young and Healy, 2003).
The rbcL sequence alignment did not imply any indel events, so no gap coding
was necessary. Sequence alignments can be found in Appendices 2–4 (see
Supplemental Data accompanying the online version of this article).
The published matK sequences, deposited in GenBank by Gadek et al.
(2000), were edited in the following manner: (1) bases archived as ‘‘N’’ were
changed to indels (‘‘?’’) if the position(s) corresponded to one reported as
an indel in the original publication (Table 4 in Gadek et al., 2000), and (2)
three bases archived as ‘‘NNN’’ that were not reported as indels (Table 4 in
Gadek et al., 2000) in the sequences of Thuja plicata and Fokienia hodginsii
were deleted to allow the sequences to properly align to the remaining se-
quences and not count as a potentially synapomorphic indel.
The fuse taxon option of WinClada 0.9.99.88 (Nixon, 2003) was used to
merge (i.e., create the mathematical union) sequences into single terminals
representing individual taxa when multiple accessions were used (Appendix
1; see Supplemental Data accompanying the online version of this article). In
all cases the individual sequences that were later fused were either sister or
paraphyletic to each another in preliminary analyses.
Morphological data—The morphological matrix used in this study (Ap-
pendix 5; see Supplemental Data accompanying the online version of this
article) was based in part on that of Gadek et al. (2000) which was in turn
based on the matrix of Hart (1987). In this case, the terminals used are species
or varieties rather than genera (cf. Hart, 1987) or a mixture of genera and
species (cf. Gadek et al., 2000). The morphology of Xanthocyparis vietna-
mensis was scored from Farjon et al. (2002) and Xiang and Farjon (2003).
Characters are nonadditive (unordered) unless otherwise noted.
Character 0: growth form—This character reflects the natural growth form
of the plant excluding environmental effects: (0) no dominant central stem,
specimens either sprawling and prostrate or a caespitose shrub; (1) single
stemmed (monopodial) tree.
Characters 1–5—Conifers generally have three distinct ontogenetic phas-
es—seedling, sapling, and adult—that are usually correlated with the occur-
rence of different phyllotaxy and leaf types (de Laubenfels, 1953). Because
branching pattern is in part dependent upon phyllotaxy, characteristics of
branching pattern were coded only for those species that produce the adult
(scale-like) leaf type (see characters 7 and 8). In Cupressoideae, adult leaves
are arranged in an opposite or whorled manner.
Character 1: arrangement of ultimate branchlets at the node—The ultimate
branch segments (i.e., branch segments that do not bear any branches) are
arranged on the stem such that either (0) one, or occasionally two, ultimate
segments occur at a given node or (1) never more than one ultimate segment
occurs at a given node.
Character 2: arrangement of ultimate branchlets—Ultimate branchlets can
be arranged on the stem such that (0) the segments more or less alternate on
the stem or (1) the vast majority of the segments occur on one side of the
stem (usually directed towards the apex of the next highest branching order).
Character 3: arrangement of the ultimate segments—During any one sea-
son of growth, the ultimate branch segments can be arranged either (0) all on
one plane or (1) on two planes. In some species (e.g., Cupressus macnabiana),
the branching plane occasionally shifts (by 90?) between growing seasons.
Character 4: arrangement of the penultimate segments—Like the ultimate
segments, the penultimate branch segments can be arranged either (0) all on
one plane or (1) on two planes.
Character 5: arrangement of the antepenultimate segments—(0) All on one
plane or (1) on two or more planes.
Character 6: number of cotyledons (additive)—Very rare (?5%) counts
were considered anomalies and therefore excluded from consideration. This
character was scored from new observations, Camus (1914), Butts and Buch-
holz (1940), Wolf (1948), and Li (1975).
Character 7: needle-like leaves—In Cupressaceae, the transition from seed-
ling to sapling is usually marked by a change in leaf type, but the transition
from sapling to adult—as measured by organism size and reproductive abil-
ity—is not marked in many species. The number of steps in this ontogenetic
series that are realized in each species is recorded in two characters: the
retention of needle-like (character 7) leaves in reproductively mature adults
(the result of skipping the final two steps of the series); and the occurrence
of a distinctive ‘‘transitional’’ (i.e., sapling) leaf type (character 8).
Needle-like leaves (0) produced only in young individuals (not reproduc-
tively mature) or (1) produced as the only leaf type in reproductively mature
Character 8: transition leaf type—(0) Not produced or scale-like, indistin-
guishable from mature leaves or (1) lanceolate, different from the mature leaf
Characters 9–20—These characters refer to adult type (scale-like) leaves.
Species that do not produce adult scale-like leaves were scored as inappli-
Character 9: mature phyllotaxy—(2) Opposite decussate or (4) in whorls
Character 10: internode length—(1) Of approximately uniform lengths or
(2) of two different lengths (alternating long and short).
Character 11: leaves born on adjacent nodes—(0) Leaves of two different
sizes, orientated such that the apices of the leaves at the given nodes are
concurrent, or (1) leaves, of one or two sizes, that do not have concurrent
Character 12: externally dimorphic mature leaves—Leaves were consid-
ered dimorphic if alternating whorls differed significantly in size and/or shape.
The occurrence of resin glands was not considered, nor were differences in
leaf shape caused by bending around a nonradially symmetrical stem (note:
Chamaecyparis lawsoniana and X. nootkatensis have externally dimorphic
leaves, but the dimorphism is extremely subtle). Leaves (0) monomorphic or
Character 13: symmetry of the lamina leaves around the midrib—(0) Sym-
metric or (1) asymmetric. In species with dimorphic leaves, the lateral leaves
Character 14: leaf transfusion tracheid pitting type (additive)—The pitting
of the leaf transfusion tissue can be either (0) simple, circular bordered pits;
(1) slightly thickened with small, non-vermiform bars; or (2) vermiform thick-
enings (i.e., trabeculate). This character was scored from new observations,
Prause (1909), Camus (1914), Gadek and Quinn (1988), and Gadek et al.
Character 15: anastomosis of vermiform thickenings—Generally, the large
vermiform pits on the leaf transfusion tracheids do not fuse to one another
November 2004] 1875LITTLE ET AL.—XANTHOCYPARIS
inside the tracheids (0), but in some cases, the pits are large enough to fuse
to one another (1). This character was coded from original observations and
Gadek and Quinn (1988).
Character 16: leaf epicuticular wax—(0) Straight tubules or (1) curly tu-
bules. This character was scored from Wilhelmi and Barthlott (1997).
Character 17: leaf cuticular crystals—Calcium oxalate crystal tubercles in
the adaxial leaf cuticle (0) absent or (1) present. Coded from data presented
by Oladele (1983a) and Xiang and Farjon (2003).
Character 18: stomatal papillae on leaf adaxial surface—(0) absent or (1)
present. Scored from Oladele (1983b) and Xiang and Farjon (2003).
Character 19: stomatal papillae on abaxial leaf surface—(0) absent or (1)
present. Recorded from data presented by Oladele (1983b).
Character 20: florin ring—The (0) presence or (1) absence of interruptions
in the Florin ring was coded from Oladele (1983b) and Xiang and Farjon
Characters 21–32—Characteristics of Microbiota decussata were gleaned
from Jagel and Stu ¨tzel (2001).
Character 21: ovulate cone scale shape—The shape of the most proximal
fully developed ovulate cone scale can be either (0) apically flattened (length
? width) or (1) elongate (length k width).
Character 22: ovulate cone scale phyllotaxy—(2) Whorls of two or mul-
tiples of two or (3) whorls of 3.
Character 23: ovulate cone scale whorls (additive)—The number of fully
developed whorls of ovulate cone scales.
Character 24: ovulate cone retention—Ovulate cones are either (0) abscised
upon maturity (deciduous) or (1) remain attached to the tree for an extended
period (persistent). Abscission of mature cones was scored from field obser-
vations where possible. In some instances, it had to be inferred from the
presence of large quantities of secondary growth in the cone pedicle.
Character 25: seed cones—Ovulate cones (0) wither and open upon seed
maturation or (1) remain alive and closed after seed maturation (serotinous).
Character 26: resin secretion—Active resin secretion on the outer surface
of the ovulate cone scales (0) absent or (1) present.
Character 27: seeds per scale—The maximum number of seeds per ovu-
liferous scale was recorded as (0) more than one or (1) only one.
Character 28: fertile ovulate cone scales—(0) More than one per cone or
(1) one per cone.
Character 29: terminal ovulate cone scales—The ultimate whorl of ovulate
cone scales (1) may or (0) may not bear seeds. This character was scored as
inapplicable for taxa with only one whorl of cone scales.
Character 30: edges of ovulate cone scales—After pollination but before
seed dispersal the ovulate cone is sealed either by (0) the confluence of elon-
gate cells that interlock when inflated or (1) the irreversible fusion to the
edges of neighboring scales.
Character 31: interlocking cells—The location of the elongate inflated cells
on the fertile scales was recorded as either (0) at edge of the ovulate cone
scale or (1) on the outer face of ovulate cone scales. This character provides
an unambiguous way to distinguish between the concept of ‘‘valvate’’ and
‘‘imbricate’’ ovulate cones put forth by Li (1953)—a concept that has been
criticized as being poorly defined by de Laubenfels (1965) and Gadek et al.
(2000) among others. In several cases, taxa are assigned states that differ from
those implied by Li (1953).
Character 32: seed maturation—Number of years (growing seasons) re-
quired for seeds to mature.
Character 33: seed shape—The cross-sectional seed shape was recorded as
either (0) round or (1) flattened.
Character 34: seed symmetry—The symmetry of the seed along the trans-
verse plane was scored as either (0) symmetric or (1) asymmetric.
Character 35: seed uniformity—Mature seeds can be described as either
(0) more or less uniform in shape or (1) irregular.
Character 36: seed wings—Lateral seed wings were scored as (0) absent
or (1) present. Highly reduced (but still observable) wings in some species
(e.g., Cupressus pigmaea) were recorded as present, even though these species
are frequently described as lacking seed wings.
Character 37: seed wing symmetry—The relative size and shape of the seed
wings was recoded as either (0) equal or (1) unequal.
Character 38: seed coat—Resin pustules in the seed coat (0) absent or (1)
Character 39: pollen at pollination—At the time of pollination, pollen can
be either (0) binucleate (occasionally multinucleate) or (1) uninucleate. This
character was scored from Doak (1937), Owens and Molder (1975), Gadek
et al. (2000), and the literature reviewed in Mehra and Malhotra (1947).
Characters 40–49—Characteristics of stem wood anatomy were obtained
from Peirce (1937), Bannan (1941, 1944), Kaeiser (1953), Greguss (1955),
and Crespo (1976).
Character 40: rays—Rays can be either (0) strictly uniseriate or (1) a mix-
ture of uniseriate and occasionally partially biseriate or even multiseriate.
Character 41: ray cell walls—The tangential (end) walls of the ray cells
can be either (0) smooth or (1) nodular.
Character 42: indentures—Indentations at the transverse/tangential wall
union in the walls of the ray parenchyma are either (0) absent or (1) present.
Character 43: ray composition—Rays composed of (0) tracheids and pa-
renchyma or (1) some, but not all, rays composed of tracheids only.
Character 44: xylem parenchyma—The transverse (end) walls of the xylem
parenchyma can be (0) smooth or (1) nodular.
Character 45: cross field pits—(0) distinctly bordered or (1) borderless.
Characters 46–56—Tropolone characters (46: tropolone backbone, 47: ?-
thujaplicin, 48: ?-thujaplicinol, 49: ?-dolabrinol, 50: pygmaein, 51: ?-thuja-
plicin, 52: ?-dolabrin, 53: ?-thujaplicinol, 54: nootkatin, 55: nootkatinol, and
56: ?-thujaplicin) were coded from data summarized in Zavarin and Anderson
(1956), Zavarin et al. (1959), Hegnauer (1962), Enzell and Krolikowska
(1963), Erdtman and Norin (1966), and Zavarin et al. (1967).
Because the actual biosynthetic pathway for tropolones in Cupressaceae is
largely unknown (Fujita et al., 2000), a character state tree (Fig. 1) was con-
structed in such a way as to minimize the number of inferred steps in the
biochemical pathway. See Barkman (2001) for an explanation as to why it is
desirable to use a character state tree. With few exceptions, the binary de-
composition of the character state tree was equivalent to simply coding each
of the compounds as (0) absent or (1) present.
1876 [Vol. 91AMERICAN JOURNAL OF BOTANY
Fig. 1.Assumed biosynthetic pathway used to code tropolone characters (46–56). See Erdtman and Norin (1966) for chemical structures.
Cupressus goveniana and C. pigmaea were scored as ‘‘?’’ for ?-thujaplicin
because, given the assumed biosynthetic pathway, it would have to be pro-
duced as a precursor to ?-thujaplicinol despite the fact that ?-thujaplicin was
reported to be absent from these taxa—compare Zavarin and Anderson (1956)
to Zavarin et al. (1967). For similar reasons, X. nootkatensis was scored as
‘‘?’’ for ?-thujaplicin (see Erdtman and Norin, 1966).
Characters 57 and 58—Biflavones from leaf tissue (57, cupressuflavone;
58, robustaflavone) were scored as (0) absent or (1) present from the data
presented in Erdtman and Norin (1966), Natarajan et al. (1970), Pelter et al.
(1970), Gadek and Quinn (1983, 1985), Qasim et al. (1985), John et al.
(1989), and Gadek et al. (2000). Although Gadek and Quinn (1985) reported
the presence of cupressuflavone in Platycladus orientalis, it was not detected
by Natarajan et al. (1970), Pelter et al. (1970), and John et al. (1989). There-
fore, Platycladus orientalis was scored as polymorphic.
Because there are only two potentially cladistically informative biflavones,
a character state tree could not be constructed for these characters.
Phylogenetic analysis—All analyses were conducted with parsimony un-
informative characters removed. Individual matrices were combined using
WinClada. NONA 2.0 (Goloboff, 1993) was used to conduct ‘‘heuristic’’
searches of the individual and combined data sets: all sequence characters
were treated as non-additive (unordered). Indels were treated as missing data.
All characters were equally weighted. Ambiguously supported nodes were
collapsed (‘‘amb-’’). Up to 20 trees per random addition replicate were held
(‘‘h/20’’). One thousand random addition sequence replicates, using simple
pruning regrafting (SPR) followed by tree bisection-reconnection (TBR)
swapping (‘‘rs0; mult*1000’’), were conducted for each data set.
WinClada was used to generate a bootstrap (Felsenstein, 1985) procedure
file. Each resampled matrix was searched with NONA using 100 random
addition replicates, up to 20 trees per replicate were held, using SPR and TBR
swapping (‘‘rs0; amb-; h/20; mult*100;’’). The strict consensus of the shortest
trees, found for each iteration, was saved. The frequency of each clade was
mapped onto the strict consensus tree using WinClada.
Partial and full constraints were utilized to evaluate the strength of unex-
pected groupings. Constraints were implemented with weighted group mem-
bership variables (Farris, 1974)—full constraints coded all taxa as either ‘‘0’’
or ‘‘1,’’ while partial constraints coded taxa as either ‘‘0,’’ ‘‘1,’’ or ‘‘?.’’
Constrained matrices were analyzed as described earlier. The statistical sig-
nificance of differences in tree topology between the unconstrained and con-
strained analyses were tested with the Wilcoxon (1945) signed rank test as
described by Templeton (1983). Critical values for the test were obtained from
Zar (1984, p. 563).
All trees were rooted between the Thuja-Thujopsis clade and the remaining
Cupressoideae as suggested by the results of Gadek et al. (Figs. 1, 3, and 5
Matrix properties—Among the three sources of DNA se-
quence data, ITS manifested the greatest quantity in both per-
centage and absolute terms of potentially parsimony infor-
mative variation (41.0%), while rbcL produced the least
(3.7%; Table 1). In addition, the ITS alignment produced the
greatest number of indel characters—more than half of which
were not potentially parsimony informative. Interestingly, of
the molecular data sets, trees derived from ITS sequence had
the highest resolution coupled with the lowest consistency and
retention indices (CI, RI; Table 1).
The partition of homoplasy between indel and sequence
characters varied greatly between ITS and matK. For ITS, the
120 potentially informative indel characters had a lower CI
(0.50) than the 522 potentially informative sequence characters
(0.63), whereas the 10 potentially informative matK indel
characters had a higher CI (0.90) than the 141 potentially in-
formative sequence characters (0.79).
Differences among data sets—The individual data sets dif-
fered greatly in the amount of resolution (Table 1). The ITS
data set differed from the cpDNA data set in the following
ways (Fig. 2): (1) The placement of Fokienia hodginsii varied.
The ITS matrix embedded F. hodginsii within Chamaecyparis,
while the cpDNA data set placed F. hodginsii unresolved in
relation to Chamaecyparis. (2) The relationships of Tetraclinis
articulata differed. The ITS data set resolved T. articulata
sister to a clade containing Calocedrus, Cupressus, Juniperus,
Microbiota, Platycladus, and Xanthocyparis, whereas the chlo-
roplast matrix put T. articulata within a clade containing Cal-
ocedrus, Microbiota, and Platycladus. (3) The topology within
the New World Cupressus clade changed. Although the mono-
phyly of New World Cupressus species was not impugned by
any of the data sets, the topology within this clade differed
markedly between the different data sets. This is reflected in
the low bootstrap frequencies for the combined data set (53–
79%) within the clade.
The morphological data set differed in many ways from
both the ITS and cpDNA data set as well as from the com-
bined molecular data set. Notably, Juniperus was very remote
from Cupressus. In addition, Chamaecyparis was paraphyletic
to Cupressus, Microbiota, and Xanthocyparis.
Simultaneous analysis—The combined data produced a
single most parsimonious tree, 2069 steps long (Fig. 2; Table
In the combined analysis, Chamaecyparis was resolved, al-
beit with low bootstrap support (49%), as a monophyletic
group, distantly removed from Cupressus. Among other char-
acters, the monophyly of Chamaecyparis is supported by the
presence of curly epicuticular wax tubules (character 16) and
two ITS indel characters. Within Chamaecyparis, there does
not appear to be a clear division associated with geographic
distribution as can be seen in other genera of Cupressaceae
(e.g., Cupressus, described later). The New World species
(Chamaecyparis thyoides and C. lawsoniana) were not re-
solved as monophyletic.
November 2004] 1877LITTLE ET AL.—XANTHOCYPARIS
Tree statistics for the different data sets and some of their combinations.
No. collapsed nodesc
ITS 1, 5.8S, and ITS 2
1272 17101419 3129 4401
0 5 (1)
8 (5) 1 (1)
0.61 0.79 0.73 0.760.63
0.81 0.93 0.880.910.83
9.3 4.658.11 9.79
DNA ? morphology
aExcluding indel characters.
bInformative characters only.
cFor 29 terminals, a fully resolved tree has 27 nodes. Numbers in parentheses represent nodes collapsed due to conflict rather than lack of character support.
Cupressus, Juniperus, and Xanthocyparis formed a very
highly supported monophyletic group (bootstrap ? 100%).
Among other characters, they share the same penultimate
branching pattern (character 4; there are some reversals), uni-
formly sized internodes (character 10), two-year seed matu-
ration (character 32; there are some reversals in species of
Juniperus not sampled in the present study), two matK indel
characters, and five ITS indel characters.
In the combined analysis, Cupressus was not resolved as a
monophyletic group. Instead, the two Old World species sam-
pled were resolved sister to a clade containing a monophyletic
Juniperus, a monophyletic Xanthocyparis, and a clade com-
posed of the six species of New World Cupressus sampled.
Support for the monophyly of Juniperus, Xanthocyparis,
and New World Cupressus comes from the non-planar ar-
rangement of the ultimate segments (character 3) and one ITS
indel character. This clade received moderate bootstrap support
(51%). A well-supported, monophyletic Xanthocyparis (boot-
strap ? 93%) was placed in a clade intercalated between a
monophyletic Juniperus and a clade of New World Cupressus.
The monophyly of Xanthocyparis is supported by three ITS
indel characters, the inequilateral arrangement of the ultimate
segments (character 2; a character state also found in Thuja,
Thujopsis, and Chamaecyparis), and the presence of externally
dimorphic mature leaves (character 12). Among other char-
acters, the monophyly of the New World Cupressus is sup-
ported by the presence of multiple branches per node (char-
acter 1), large numbers of cotyledons (character 6), and four
ITS indel characters. This clade received a bootstrap value of
Morphological character evolution—As measured by the
increase in resolution (Table 1), the addition of the morphol-
ogy matrix to the sequence data seems to have had a syner-
gistic effect—producing more resolution than either data type
did individually. The character state tree used for the tropolone
data (Fig. 1) was not invalidated by the combined analysis:
When the tropolone characters were optimized onto the tree
produced by the total evidence analysis none of the hypothet-
ical ancestors had an ‘‘impossible’’ combination of states (i.e.,
a set of states that indicated that a particular compound was
produced without the production of all the assumed chemical
precursors in the biosynthetic pathway, such as producing
nootkatinol without first producing ?-thujaplicin; see Fig. 1).
Li (1953) arranged the genera of Cupressoideae based on
the number of ovulate cone scale whorls (character 23), the
number of ovules per scale (character 27), the number of
scales that bear ovules (character 28), whether or not the ter-
minal whorl is fertile (character 29), the aestivation of the
ovulate cone scales (character 31), winged seeds (character
36), seed wing symmetry (character 37), the ovulate cone scale
shape (this character could not be properly defined for anal-
ysis), and the ovulate cone scale texture (this character could
not be properly defined for analysis). When compared to the
other morphological characters in the combined analysis, these
characters have, on average, a higher CI (0.43 vs. 0.35).
Our data suggest that the ancestral cone of the Cupresso-
ideae had three or four whorls of ovulate cone scales, multiple
seeds per scale, multiple fertile scales, a sterile terminal whorl
of scales, ‘‘valvate’’ cone scales (elongate cells positioned on
the scale edge), and bore seeds with symmetrical wings. This
1878 [Vol. 91AMERICAN JOURNAL OF BOTANY
and the generalized geographic range (OW ? Old World; NW ? New World) is indicated. The unambiguous optimization (the intersection of ACCTRAN and
DELTRAN) of the informative morphological characters is shown. Open squares represent homoplasious character state changes; filled squares represent non-
homoplasious state changes. Unambiguously optimized branch lengths for all parsimony-informative characters are given below each branch. Strict consensus
bootstrap frequencies, where applicable, are indicated above the branches. Cases in which the strict consensus of the cpDNA data conflicted with the combined
data are indicated by arrows with wavy tails showing the placement of taxa in the cpDNA tree. Branches drawn with dashed lines were not resolved in the
strict consensus of the cpDNA data. The ITS subtrees (ITS nodes A and B) indicate portions of the ITS tree that conflict with the combined data. Bootstrap
and branch length values on the ITS subtrees reflect only the ITS data.
Single most parsimonious tree for the combined data set (see Table 1 for tree statistics). The generic name of Cupressus species has been abbreviated
contrasts with the extant species of Cupressus—described as
the ‘‘ancestral type’’ by Li (1953)—in fertility of the terminal
whorl of cone scales.
As previously indicated by Farjon and Ortiz Garcia (2002),
the highly reduced configuration found in Juniperus and Mi-
crobiota arose independently in these two lineages.
Previous studies—An earlier study of ITS variation within
Chamaecyparis (Li et al., 2003) produced the same results as
the present ITS data set. The concordance of independently
derived ITS sequence data may indicate that taxon misidenti-
fication (as suggested by an anonymous reviewer) is not the
cause of discordance between the cpDNA and ITS data sets.
November 2004] 1879LITTLE ET AL.—XANTHOCYPARIS
It may, however, suggest that incomplete homogenization of
nrDNA repeats has resulted in the sampling of paralogous loci.
Alternatively, the discordance may be the result of introgres-
sion. No evidence of sequence heterogeneity was detected in
the process of generating the sequence data, but that does not
eliminate paralogy as a potential source of error. No evidence
for, or against, introgression was collected.
The simultaneous analysis of Gadek et al. (Fig. 5 in 2000)
was based on a combination of chloroplast sequence data and
morphology. That analysis placed Tetraclinis articulata within
a clade containing Calocedrus, Microbiota, and Platycladus,
as does the cpDNA data used in the present study. Because
the ITS sequence data provided the largest number of infor-
mative characters and those data argued for the placement of
T. articulata in a different position (Fig. 2), that placement is
reflected in the simultaneous analysis of this study.
The only previous studies to sample both Old and New
World species of Cupressus showed that Cupressus was either
monophyletic (Figs. 4 and 5 in Gadek et al., 2000), paraphy-
letic (Fig. 1 in Gadek et al., 2000), or polyphyletic (Farjon et
al., 2002). The present combined analysis (Fig. 2) indicates
that Cupressus is polyphyletic (sensu Farris, 1974). When Cu-
pressus (sensu Farjon, 1998) was constrained to be monophy-
letic using the combined data set, the New World clade and
the Old World clade remained monophyletic, but the tree
length increased by six steps (ca. 0.29% of overall length; P
? 0.10). The length increased by one step (ca. 0.05% of over-
all length; P ? 0.25), when Cupressus was constrained to be
either paraphyletic to Xanthocyparis or monophyletic; again
the New and Old World clades remained monophyletic, but
Cupressus was paraphyletic to Xanthocyparis. Though the
combined analysis suggests that Cupressus may be polyphy-
letic, it does not provide strong evidence for polyphyly.
The Old and New World species of Cupressus share a com-
mon ovulate cone and leaf morphology that is apparently due
to the retention of ancestral features rather than to convergent
evolution. The polyphyly of Cupressus is due to the recogni-
tion of two distinctive groups: Juniperus has long been rec-
ognized as a distinctive group because of its unusual cone
morphology that resembles—in both form and function—an
angiospermous drupe. More recently, Xanthocyparis has been
recognized because its morphology is similar to, but not the
same as, that of Cupressus. Many of the morphological dif-
ferences between Cupressus and Xanthocyparis can possibly
be attributed to adaptations to moist vs. arid habitats (e.g.,
dimorphic vs. monomorphic leaves).
Our results agree with those of Gadek et al. (2000), who
found that X. nootkatensis has close affinity with Cupressus.
The topology presented here (Fig. 2) contrasts strongly with
that of Farjon et al. (2002), where both species of Xanthocy-
paris were placed, distant from Cupressus, in an unresolved
clade of Callitroideae (sensu Li, 1953). Because the morpho-
logical matrix of Farjon et al. (2002) was not published, the
source of this discordance cannot be ascertained.
Taxonomic implications: Chamaecyparis—The type spe-
cies of Chamaecyparis is C. thyoides. Therefore, the only
change implied by the current data to the commonly used cir-
cumscription of Chamaecyparis, is to exclude C. nootkatensis,
as previously indicated by several authors (Gadek et al., 2000;
Farjon et al., 2002). Because F. hodginsii is included within
Chamaecyparis in only one partition of the data, it seems un-
wise to add this species to Chamaecyparis. Should additional
data suggest that Fokienia and Chamaecyparis be merged, the
correct name for the genus would be Chamaecyparis.
Taxonomic implications: Cupressus—It appears that Cu-
pressus is not monophyletic. If this pattern persists as new data
are added, Cupressus may have to be divided into two gen-
era—with the Old World species of Cupressus retaining their
current names, while a new generic name will be needed for
the New World species. Currently, this is being further inves-
tigated with more exhaustive sampling of Juniperus and Cu-
Taxonomic implications: Xanthocyparis—As circum-
scribed by Farjon et al. (2002), the genus Xanthocyparis ap-
pears to be monophyletic. However, based on the established
rules for botanical nomenclature, this circumscription unfor-
tunately cannot stand: The genus Callitropsis non Callitropsis
sensu Compton (1922), with C. nootkatensis (D. Don) O¨rest.
designated as its type, was described in 1865. Because Calli-
tropsis is the oldest name, the principle of priority (Greuter et
al., 2000: article 11.3), in combination with the phylogenetic
evidence presented here, dictates that Xanthocyparis cannot
include C. nootkatensis. Either Xanthocyparis vietnamensis
must be transferred to the genus Callitropsis or Xanthocyparis
must remain a monotypic genus.
Because X. vietnamensis and C. nootkatensis are sister taxa
and appear to be relatively closely allied, it is more informa-
tive to recognize this relationship in a single genus rather than
two monotypic sister genera. We therefore transfer X. vietna-
mensis to Callitropsis.
Callitropsis vietnamensis (Farjon and Hiep) D. P. Little
comb. nov. Basionym: Xanthocyparis vietnamensis Farjon and
Hiep in Farjon, Hiep, Harder, Loc, and Averyanov Novon 12:
As now circumscribed, Callitropsis contains two species
with disjunct distribution: C. nootkatensis is native to western
North America from 41? N to 60? N. It is primarily found near
the coast (in the moist fog belt), but more inland populations
are known from the southern portion of the range (e.g., Frog
Lake, Siskiyou County, California). Callitropsis vietnamensis
is native to moist karst forest in northern Vietnam (23? N; see
Averyanov et al., 2002).
Callitropsis is diagnosed with the combination of primarily
apically distributed ultimate branchlets (character 2) and ex-
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