Bull. Natl. Mus. Nat. Sci., Ser. B, 34(2), pp. 75–82, June 23, 2008

A Phylogeny for Two Cycad Families (Stangeriaceae and Zamiaceae)
based on Chloroplast DNA Sequences
Pattamon Sangin1,3, Paul I Forster2, Mingkwan Mingmuang1 and Goro Kokubugata3
1

Department of General Science, Faculty of Sciences, Kasetsart University,
Bangkok, 10900, Thailand
2
Queensland Herbarium, Environmental Protection Agency, Brisbane Botanic Gardens,
Mt Coot-tha Road, Toowong, Queensland, 4066, Australia
3
Department of Botany, National Museum of Nature and Science, 4–1–1 Amakubo,
Tsukuba, 305–0005, Japan
E-mail: gkokubu@kahaku.go.jp

Abstract Sequences of the trnS-trnG noncoding region chloroplast DNA from 40 species of theZamiaceae and Stangeriaceae families and 3 out group species (Cycadaceae) were used to reconstruct phylogenetic trees using distance and parsimony methods. The distance tree was similar in
topology to the parsimony tree and both indicated that Encephalartos had a relationship closer to
Lepidozamia than Macrozamia. Encephalartos, Lepidozamia and Macrozamia comprised a monophyletic tribe, while Dioon was a basal-sister of the subfamily Encephalartoideae clade. These twoanalyses also revealed a close relationship between Microcycas and Zamia with high bootstrap
support. Although Stangeria and Ceratozamia have been placed in different families, they werecloser to each other, rather than Bowenia which is contrary to previous morphologically based classifications. The current study provides new molecular evidence that Stangeria and Bowenia are notsister taxa and reinforces the close relationship between Encephalartos and Lepidozamia.
Key words : Bowenia, Chloroplast DNA, Cycadales, Encephalartos, Lepidozamia, Macrozamia,
Stangeria, Stangeriaceae, Zamia, Zamiaceae.

Introduction
The cycads (Order Cycadales; Stevenson,
1992; Norstog and Nichols 1998; Schneider etal., 2002) are a group of seed plants with ancientorigins that have been often termed as ‘living
fossils’, insomuch as the extant species are of lineages little changed since their first occurrence inthe early Permian (Mamay, 1969; Zhu and Du,
1981; Gao and Thomas, 1989). They are distributed across the subtropical and tropical regionsof the world, i.e., Africa, Asia, Central Americaand Australia with one species extending toJapan. On the basis of morphology, cycads havebeen classified into 3 families (Cycadaceae,
Stangeriaceae and Zamiaceae), with 11 genera(Stevenson, 1992) and over 300 known species(Hill et al., 2007). Whilst Cycadaceae is unequivocally unique (cf. Brenner et al., 2003a,

2003b), the distinction between Stangeriaceae
and Zamiaceae remains unclear when molecularcharacters are analyzed, rather than those purely
from morphology (Hill et al., 2003; Chaw et al.,2005).
In the past decade, chloroplast genes havebeen used extensively to elucidate relationships
in seed plants. Chloroplast genes are the consequences of an endosymbiotic event between aeukaryotic host cell and an ancestor of the
cyanobacteria hence they have a slower mutation
rates in comparison with the nuclear genes (Curtis and Clegg, 1984; Raven and Allen, 2003) with
their conserved nature useful for testing relationships among genera thought to be closely related(Gielly and Taberlet, 1994). In recent studies
using molecular markers such as the chloroplast
matK gene, trnL intron and ITS2 rDNA sequences (Treutlein and Wink, 2002; Hill et al.,

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P. Sangin et al.

2003; Bogler and Francisco-Ortega, 2004; Chaw
et al., 2005) the intrafamilial classification of cycads has been slightly modified, particularly in
the Zamiaceae family, where the genera Encephalartos (endemic to Africa) and Lepidozamia (endemic to Australia) have been found to becloser to each other rather than Macrozamia (endemic to Australia). These molecular phylogenetic trees were not congruent with the Stevenson(1992) classification that was based solely onmorphological characters and which included
Lepidozamia and Macrozamia in the subtribe
Macrozamiinae D.Stevenson and Encephalartos
in the subtribe Encephalartinae Benth. et Hook.f.;both subtribes comprising the tribe Encephalarteae Miq. within the subfamily Encephalartoideae D.Stevenson.
Currently, noncoding sequences of the chloroplast genome have been used as a new major
focus for studying plant molecular evolution.
Shaw et al. (2005) evaluated the relative level ofvariability among 21 noncoding chloroplast
DNA regions in seed plants and the noncoding
regions (Tier 1) that provided the greatest numbers of PICs (Potential Informative Characters),
were identified as trnD-trnT, rpoB-trnC, trnStrnG, trnS-trnfM and trnT-trnL. In the current
study we focus on the use of the trnS-trnG sequence (previously unused with cycads) to reconstruct phylogenetic trees for developing relationship hypotheses for within and between genera in
the Zamiaceae and Stangeriaceae families.
Material and Methods
DNA extraction and PCR
Total genomic DNA of nine genera and 40
cycad species (Table 1) was extracted from plants
cultivated in Tsukuba Botanical Garden. Voucher
specimens for each species are deposited in the
Herbarium of the National Museum of Natureand Science (TNS). Three Cycas species, namely
C. revoluta, C. wadei and C. media, were included in the present analysis to serve as out groups
following Hill et al. (2003) and in recognition of
the basal position of this genus in the Cycadales(Brenner, 2003a).
In the present study, Plant DNeasy Mini Kit(Quigen) was used for extracting DNA following
the manufacturer’s protocol. The trnS-trnG intergenic spacer region was amplified with primer
trnS 5 GCCGCTTAGTCCACTCAGC 3 andtrnG 5 GAACGAATCACACTTTTACCAC 3
(Hamilton, 1998). The polymerase chain reaction(PCR) amplification was performed in 5 m l of thereaction with the following components: 2.5 m l of5AmpDirect (Shimazu), 0.5 unit of Ex taq(Takara), 10 m M of each primer and 1 m l of genomic DNA. Amplifications were made in a
Perkin Elmer 9700 thermocycler with an initialdenaturing step of 5 min at 94°C followed by 35
cycles of 30 sec at 94°C, 30 sec at 60°C, 1 min at
72°C and a final extension of 7 min at 72°C. PCR
products were subjected to 1% agarose gel andDNA bands were visualized by ethidium bromide
staining. The PCR products were purified using
the ExoSAP-IT kit (United States Biochemical).
Purified PCR product were sequenced with the
ABI Big Dye Terminator Cycle Sequencing Kit
V3.1 and run on 3130X/ Genetic Analyzer.
Data analysis
Sequences were edited and assembled using the
program ATGC var. 4 (GENETYX Co.) and were
initially aligned using Clustal W (Thompson et al.,1994). The resulting data was imported into the
GENEDOC 2.6 program (Nicholas et al., 1997)
following by a manual adjustment. Two phylogenetic reconstruction methods (maximum parsimony (MP) and neighbor-joining (NJ) method) were
performed, using the program MEGA 4 (Tamura etal., 2007). The MP analyses were conducted withheuristic searches (close-neighbor interchange)
using the random addition trees. Bootstrap analyses used 1,000 replicates for this method. For the
NJ method, analyses were constructed with the nucleotide substitution model (Maximum Composite
Likelihood Method) with the number of bootstrap
replicates was set to 1,000. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons(Pairwise deletion option).

Cycadaceae

Encephalartoideae

Zamiaaceae

Zamioideae

Stangerioideae
Bowenioideae

Subfamily

Microcycas
Zamia

Zamieae

Cycas

Ceratozamia

Ceratozamioideae

Microcycadinae
Zamiinae

Macrozamia

Macrozamiinae

Lepidozamia

Encephalartos

Encephalartinae

Encephalarteae

Stangeria
Bowenia

Genus

Dioon

Subtribe

DiooeaeTribe

Forty-three cycad species analyzed following Stevenson (1992)

Stangeriaceae

FamilyTable 1.

Stangeria eriopus (Kunze) Baill.
Bowenia serrulata (W. Bull) Chamb.
Bowenia spectabilis Hook. ex Hook. f.
Dioon edule Lindl.
Dioon spinulosum Dyer
Encephalartos altensteinii Lehm.
Encephalartos arenarius R. A. Dyer
Encephalartos barteri Carruth. ex Miq.
Encephalartos ferox Bertol. f.
Encephalartos friderici-guilielmi Lehm.
Encephalartos hildebrandtii A. Braum et Bouche
Encephalartos lehmannii Lehm.
Encephalartos longifolius (Jacq.) Lehm.
Encephalartos manikensis (Gilliland)
Encephalartos natalensis R. A. Dyer et I.Verd.
Encephalartos paucidentatus Stapf et Burtt Davy
Encephalartos trispinosus (Hook.) R. A. Dyer
Encephalartos villosus Lem.
Macrozamia communis L. A. S. Johnson
Macrozamia fawcettii C. Moore
Macrozamia macdonnellii (F. Muell. ex Miq.) A. DC.
Macrozamia miquelii (F. Muell. ex Miq.) A. DC.
Macrozamia moorei F. Muell.
Macrozamia pauli-guilielmi W. Hill et F. Muell.
Macrozamia platyrhachis F. Muell.
Macrozamia reducta K. D. Hill et D. L. Jones
Macrozamia riedlei (Gaudich.) C. A. Gardner
Macrozamia secunda C. Moore
Macrozamia spiralis (Salisb.) Miq.
Lepidozamia hopei Regel
Lepidozamia peroffskyana Regel
Ceratozamia hildae G. P. Landry et M. C. Wilson
Ceratozamia kuesteriana Regel
Ceratozamia mexicana Brongn.
Ceratozamia microstrobila Vovides et J. D. Rees
Ceratozamia norstogii D. W. Stev.
Ceratozamia sp ‘plumosa’Microcycas calocoma (Miq.) A. DC.
Zamia fischeri Miq.
Zamia furfuracea L. f.
Cycas media R. Br.
Cycas revoluta Thunb.
Cycas wadei Merrill

Species
GK 10226
GK 10228
GK 10229
GK 10249
GK 10246
GK 10250
GK 10247
GK 10237
GK 10239
GK 10233
GK 10242
GK 10234
GK 10235
GK 10240
GK 10236
GK 10480
GK 10238
GK 10241
GK 10219
GK 10248
GK 10217
GK 10245
GK 10216
GK 10483
GK 10479
GK 10218
GK 10480
GK 10244
GK 10243
GK 10231
GK 10230
GK 10221
GK 10222
GK 10223
GK 10220
GK 10225
GK 10224—
GK 10482
GK 10481
GK 59
GK 7136
GK 10483

Voucher
AB434424
AB434425
AB434426
AB434427
AB434428
AB434429
AB434430
AB434431
AB434432
AB434433
AB434434
AB434435
AB434436
AB434437
AB434438
AB434439
AB434440
AB434441
AB434442
AB434443
AB434444
AB434445
AB434446
AB434447
AB434448
AB434449
AB434450
AB434451
AB434452
AB434453
AB434454
AB434455
AB434456
AB434457
AB434458
AB434459
AB434460
AB434461
AB434462
AB434463
AB434464
AB434465
AB434466

DDBJ

Phylogeny of two cycad families
77

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P. Sangin et al.
Table 2.

Average base frequencies for trnS-G intergenic spacer of 43 Cycad species

RegiontrnS-G

Alignment
length(base pairs)

Base
A

C

G

T

903

30.3

17.6

20.3

31.9

The sequences obtained were submitted to the
DDBJ/EMBL/GenBank databases (Table 1).
Results and Discussion
Sequence characteristics
The length of the trnS-trnG intergenic spacer
averaged 815 base pairs (bp) and the nucleotide
frequencies were 30.3% (A), 17.6% (C), 20.3%(G) and 31.9% (T). This region showed a low GC
content (37.9%), while it represented a high AT
content (62.2%) which is characteristic of thechloroplast genome (Morton, 1995). The transition(Ts)/transversion (Tv) ratio was 1.32 (Table 2).
The sequence alignment of this region was
903 bp and appeared with 22 indels (insertion/
deletion). These results indicated that the trnStrnG region had a high level of variation amongcycad genera.
Phylogenetic analyses
MP was reconstructed using the close-neighbor-interchange (CNI) with the random addition
tree 100 replications producing 188 equally mostparsimonious trees with trees length of 540. The
consistency index was 0.630556, the retention
index was 0.819048 and the composite index was
0.617319 (0.516455) for all sites and parsimonyinformative sites (in parentheses). There were atotal of 725 positions in the final dataset, out of
which 174 were informative. A consensus ofthese 74 trees is presented in Fig. 1. NJ (Fig. 2)
was reconstructed by the Maximum Composite
Likelihood method and was in the units of thenumber of base substitutions per site. The MP
and NJ trees supported monophyly of each genus
with high bootstrap values. In the MP tree cladesconsisted of ((Stangeria, Ceratozamia), ((Micro-

Transition

Transversion

4.102

3.106

cycas, Zamia), (Bowenia, (Dioon, (Macrozamia,(Encephalartos, Lepidozamia)))))) (Figs. 1, 2).
Both the MP and NJ analyses support the
hypothesis that the tribe Encephalarteae (Encephalartos, Macrozamia and Lepidozamia) andtribe Zamieae (Microcycas and Zamia) are respectively monophyletic with high bootstrap values agreeing with Stevenson (1992), and thenDioon (endemic to Central America) was taken
up as a sister group to this Encephalartoideae
clade with a bootstrap value. The current results
indicate that Encephalartos is more closely related to Lepidozamia than to Macrozamia therefore
disagreeing with Stevenson’s (1992) classification of tribe Encephalarteae based only on morphology but in accordance with previous molecular results (Treutlein and Wink, 2002; Hill et al.,
2003; Bogler and Francisco-Ortega, 2004; Chaw
et al., 2005). These results reinforce the conclusion that Lepidozamia and Encephalartos shared
a common ancestry in Gondwana (200–135
MYA) and separated from each other before
Africa and Australia were isolated (Bogler andFrancisco-Ortega, 2004). As noted by Chaw etal. (2005), the relationship between these two
widely disjunct genera is enigmatic; however, unlike some species of Cycas (Dehgan and Yuen,
1983), no species of Lepidozamia or Encephalartos possess seeds with special adaptations thatenable dispersal for long distances, especially on
the continental scale. As with the allied Macrozamia (Snow and Walter, 2007), all extant speciesof Lepidozamia and Encephalartos had large andheavy seeds that are locally or barely dispersed.
These essentially localized distributions indicate
dispersal limited distribution (Primack and Miao,
1992) not conducive to long range colonization.
Bowenia (endemic to Australia) and Stangeria(endemic to Africa) were classified in the familyPhylogeny of two cycad families

Fig. 1. The consensus tree inferred from 74 most parsimonious trees was shown. Branches corresponding topartitions reproduced in less than 50% trees were collapsed. The percentage of parsimonious trees in which
the associated taxa clustered together were shown next to the branches.

79

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P. Sangin et al.

Fig. 2. Neighbor-Joining tree produced by analysis of trnS-trnG sequence data from Zamiaceae and Stangeriaceae families. The optimal tree with the sum of branch length0.81275044 was shown. The percentage of
replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) wereshown next to the branches ( 50%).

Phylogeny of two cycad families

Stangeriaceae by Stevenson (1992), albeit in separate subfamilies. However, most previous studies have implied that Bowenia and Stangeria
were not closely related, and did not support thefamily Stangeriaceae, and most molecular based
phylogenetic studies have concluded that Stangeria and Zamia were sister taxa (Rai et al., 2003;
Bogler and Francisco-Ortega, 2004; Chaw et al.,2005), rather than Stangeria and Ceratozamia as
found in the current study. Previously it was reported that the chromosome numbers of Stangeria and Ceratozamia are both 2n16, whereas
for Bowenia it is 2n18; and Stangeria and Ceratozamia have similar karyotypes (Kokubugata etal., 2000, 2001, 2004). Furthermore, the florescence in situ hybridization using 45S and 5S ribosomal (rDNA) probes elucidated that Stangeria (S. eriopus) and Ceratozamia (C. hildae, C.
kuesteriana, C. mexicana and C. norstogii) had
similar distribution patterns of 45S and 5S rDNA
sites on the somatic chromosome complements,
with the conclusion that Stangeria could be closer to Ceratozamia than the other cycad genera(Kokubugata and Kondo, 1998; Kokubugata etal., 2002, 2004). The present molecular study
supports the previous cytotaxonomic hypothesis.
Data from the trnS-trnG noncoding region ofchloroplast DNA appears to be highly informative for determining relationship hypotheses in
the Cycadales, which largely colloborate othermolecular studies. On the other hand, there is notenough data to explain intrageneric phylogenies
in each genus, and thus further taxon sampling
and analyses based on the other sequences are required, particularly for the New World genera
such as Ceratozamia and Zamia. Ultimately arigorous classification for the Cycadales should
be based on a dataset that incorporates information from a range of molecular studies, together
with morphological characters and that acknowledges aspects of the pollination biology and dispersal abilities of these plants. Ultimately a rigorous classification for the Cycadales should bebased on a dataset that incorporates informationfrom a range of molecular studies, together withmorphological characters and that acknowledges

81

aspects of the pollination biology and dispersal
abilities of these plants. In spite of their lineage,a general consensus is that many of the extant
species of cycads are relatively recent in derivation, possibly radiating since the Pleistocene(Treutlin and Wink, 2002; Vovides et al., 2007),
albeit with a set of biological characteristics ofputatively ancient origins. Their dependence on
insect pollinators (usually in dependent obligate
mutualisms (cf. Terry et al., 2005)) and limited
ability to disperse due to an apparent loss of dedicated dispersal agents (Snow and Walter, 2007)
all indicate that the extant cycads have limited
options for long range colonization. Molecularand morphological differences between the extant genera of cycads (particularly those that aresister taxa such as Encephalartos and Lepidozamia) are likely to have arisen prior to their having
been separated by continental separation events,
such as the splitting up of Gondwana.
Acknowledgements
The present study was financially supported by
the Bilateral Research Cooperation (BRC) scholarship (PS) and a project of “Integration of Systematics and Molecular Phylogenetics in All
Groups of Organisms” handled by the National
Museum of Nature and Science (GK).
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