J. Plant Res. 114: 459-464, 2001 Journal of Plant Research 0 by The Botanical Society of JaDan 2001

Molecular Phylogeny of Casuarinaceae Based on rbcL and matK Gene Sequences

Akiko Sogo'*, Hiroaki Setoguchi', Junko Noguchi', Tanguy Jaffre3 and Hiroshi Tobe'

2 Department of Natural Environmental Sciences, Faculty of Integrated Human Studies, Kyoto University, Kyoto, 606-8501 Japan Department of Botany, Graduate School of Science, Kyoto University, Kyoto, 606-8502 Japan

Laboratoire de Botanique, Centre IRD de Noumea, BP A5 Noumea cedex, New Caledonia

We present the first overall molecular phylogenetic study of Casuarinaceae on the basis of sequences of two chloro- plast genes, rbcL (1310 bp) and matK (1014 bp), using 15 species representing the family. The study included ana- lyses of Ticodendron (Ticodendraceae) and three species of Betulaceae as close relatives, and one species each of Juglandaceae and Myricaceae as outgroups. Analyses based on matK gene sequences, which provided a much better resolution than the analyses based on rbcL gene sequences alone, resulted in a single most parsimonious tree whose topology is almost identical with the strict con- sensus tree generated by the combined data set of rbcL and matK gene sequences. Results showed that Casuar- inaceae are monophyletic, comprising four distinct genera, A//ocasuarina, Casuarina, Ceuthostoma and Gymnostoma, which were not recognized until recently. Within the family, Gymnostoma is positioned at the most basal position and sister to the remainder. Within the remainder Ceuthostoma is sister to the A//ocasuarina-Casuarina clade. Mor- phologically the basalmost position of Gymnostoma is supported by plesiomorphies such as exposed stomata in the shallow longitudinal furrows of the branchlets, a basic chromosome number x=8 and the gynoecium composed of two fertile, biovulate carpels. The three other genera, A//ocasuarina, Casuarina, and Ceuthastoma, have invisible stomata in the deep longitudinal furrows of the branchlets, a higher basic chromosome number x=9 or 10-14 (unknown in Ceuthostoma), the gynoecium composed of one fertile and one sterile carpel with a single ovule (unknown in Ceuthastoma). The diversity of infructescence morphology found in the latter three genera suggests that they may have evolved in close association with the elaboration of fruit dispersal mechanisms.

Key words: Casuarinaceae - matK - Phylogeny - rbcL

Casuarinaceae, comprising four genera, Allocasuarina L.A.S. Johnson (59 species), Casuarina L. (17 species), Ceuth- ostoma L.A.S. Johnson (two species), and Gymnostoma

* Corresponding author

L.A.S. Johnson (18 species), are known as Gondwanan ele- ments with the major distribution center in Australia and the Melanesian region of the Pacific (Johnson and Wilson 1993). Gymnostoma was first separated from Casuarina by Johnson in 1980. Subsequently, Allocasuarina was separated from Casuarina (Johnson 1982). Ceuthostoma was removed from Gymnostoma (Johnson 1988). While the family is well char- acterized by the slender wiry articulate branchlets with the leaf laminae reduced to small teeth, the genera are distin- guished from one another by a variety of morphological characters, including (1) whether the stomata in the longitu- dinal furrows of branchlets are exposed or invisible, (2) whether the number of leaves per whorl is four, four to 15, or five to 20, (3) whether mature samaras are gray or yellow brown and dull, or red-brown to black and shinning, (4) whether bracteoles are thin and without protuberances or thickly woody, mostly with some protuberances, and (5) whether the infructescences are borne terminally on green branchlets or axillary on short woody branchlets; and whether the basic chromosome numbers are, 8,9, or 10-14 (Johnson 1988). Based on comparisons of morphological and somatic chromosome characters, Johnson and Wilson (1989,1993) considered Gymnostoma to be the most primitive and Allocasuarina the most specialized.

Concerning relationships within the family based on molecular evidence, Maggia and Bousquet (1994), in a study of symbiotic evolution of host plants and actinomycetes, used rbcl gene sequences of chloroplast DNA to analyze the phylogeny of Allocasuarina, Casuarina and Gymnostoma, and showed relationship (Gymnostoma (Allocasuarina- Casuarina)). The study, however, was based on one species each from the three genera and did not include Ceuthos- toma. Exact relationships within the family are therefore uncertain.

The purpose of this study was to clarify phylogenetic relationships within the family by analyzing a moderate number of species and using more sequence data. We sequenced two chloroplast genes, matK and rbcl, because their analyses have provided good resolution for generic relationships within Betulaceae, which are closely related to Casuarinaceae (Kato et a/. 1998). Based on our results, we will also provide a brief discussion of the evolution of morphological characters within the Casuarinaceae.

460 A. Sogo et a/.

Materials and Methods

Materials We used 15 species in our study, including four species of

Allocasuarina, five species of Casuarina, one species of Ceuthostoma and five species of Gymnostoma. Their col- lection data are presented in Table 1.

Branchlets with small leaves were collected from trees growing in natural habitats or from cultivated trees, and were dried and preserved in silica gel.

Total DNA extraction Dry samples were frozen using liquid nitrogen and pulver-

ized to a fine powder. Before the DNA extraction, the powder was suspended in HEPES buffer (pH8.0) and centrifuged at 13,000 rpm and 20 C for 5 min to remove the sticky polysaccharide (Setoguchi and Ohba 1995). Total DNA was isolated from the collected pellet using the CTAB method of Hasebe and lwatsuki (1990).

Amplification and sequencing of the rbcL and matK gene of cpDNA

The double-stranded DNA of the rbcl gene (ca. 1310 bp.) and matK gene (ca. 1014 bp.) of cpDNA were amplified by 34 cycles of symmetric polymerase chain reaction (PCR). The nucleotide regions amplified of the two genes for Casuar- inaceae coincide with those known for Juglandaceae and Myricaceae, which were used as outgroups. PCR cycle conditions in the first cycle consisted of 1 min at 94C for denaturation, 1 min at 42 C for primer annealing, and 1 min at 72 C for primer extension. Primers for amplification of the rbcl gene were aF and cR of Hasebe et a/. (1994), and those of the matK gene were matK4 and matK9R of Manos and Steel (1997).

The PCR products were purified by electrophoresis in 0.5% agarose gel using 1 XTAE buffer. The gel was stained with ethidium bromide and the DNA was extracted using Gene- clean II (BiolOl, CA, U.S.A.). Purified DNAs were sequenced in both directions by the standard methods of the Taq dye deoxy terminator cycle sequencing kit (Perkin Elmer, CA, U.S.A.) on an Applied Biosystems Model 377 automated sequencer (Applied Biosystems, CA, U.S.A.). For sequence determination of the rbcl gene, aF and cR were used as

Table 1. Taxa used in this study, collection data and GenBank accession numbers for rbcL and matK sequences.

-

Species Collections Gen Bank accession

number

rbcL matK

Allocasuarina decussata (Benth.) L.A.S. Johnson A. lehmanniana (Miq.) L.A.S. Johnson A. littoralis (Salisb.) L.A.S. Johnson

A. torulosa (Aiton) L.A.S. Johnson

Casuarina collina J. Poiss.

C. cunninghamiana Miq. C. equisetifolia L.

C. obesa Miq. c. sp. Ceuthostoma terminale L.A.S. Johnson

Gymnostoma chamaecyparis (J. Poiss.) L.A.S. Johnson G. deplancheanum (Miq.) L.A.S. Johnson

G. nobile (Whitmore) L.A.S. Johnson

G. nodiflorum (Thunb.) L.A.S. Johnson G. sumatranum (Jungh. ex. de Vriese) L.A.S. Johnson Out groups reffered from GenBank

Betula papyrifera Marsh. Cofylus cornuta Marsh. Juglans nigra L. Myrica cerifera L. Ticodendron incognitum Gomez L. & Gomez P.

Australia. WA, Pemberton, Bigrook. Abe s.n. (KYO) Cultivated, University of Western Australia. Abe s.n, (KYO)

AY033855 AY033851

Cultivated, Coffs Harbour Botanical Garden, NSW, Australia. AY033849 Floyd 28224 (CFSHB)

Australia. N.S.W., Dorrigo N.P., Rosewood River. Floyd s.n. (CFSHB) New Caledonia. Noumea, Ouen Tor0 Kark.

Sogo & Setoguchi NC9909 (KYO) Cultivated, University of Western Australia. Abe s.n, (KYO) New Caledonia. Noumea, Anse Vata.

Sogo & Setoguchi NC9977 (KYO) Australia. WA, Matilda Bay Reserve. Abe s.n. (KYO) Australia. WA, Matilda Bay Reserve. Abe s.n. (KYO) Malaysia. Sabah, Kinabalu Park.

New Caledonia. Mont Dore. Jaflre 3375 (NOU) New Caledonia. Province Sud, Route de Yatte.

Cultivated, the Sabah Museum, Kota Kinabalu, Sabah, Malaysia. Sogo & Setoguchi MOO20 (KYO) New Caledonia. Cascade de Ciu. Jaffr6 3364 (NOU) Cultivated, Bogor Botanical Garden. Nanda s.n. (BO)

Sogo & Setoguchi MOO09 (KYO)

Sogo ti Setoguchi NC9903 (KYO)

AYo33850

AY033856

AYo33854 AY033858

AYo33853 AY033852 AYO33860

AYO33861 AY033862

AY033866

AY033867 AY033870

X56617 X56619 u00437

AF119179 AF061197

AYO33834 AY033872 AY033830

AY033831

AYo33835

AY033873 AY033837

AYO33833 AY033832 AY033838

AY033839 AYO33840

AYo33844

AYO33845 AY033848

U92853 U92854 U92851 U92857 U92855

Molecular Phylogeny of Casuarinaceae

primers following Hasebe et al. (1994). Additionally, the primers Cas-bF and Cas-bR were originally designed for sequencing an internal region corresponding to about one third of the whole length. Their sequences are 5’ TAAAC- CTAAATGGGATATCYGCTAAG AA-3 (Cas- bF), and 5’- ACCATATCTCGGCAATAATGAGCCAAACT-3 (Cas- bR).

Likewise, for sequence determination of the mafK gene, rnatK4 and matK9R were used as primers following Manos and Steel (1997). Additionally, originally designed primers Cas-matK5 and Cas-matK7 were used for sequencing an internal region corresponding to about one third of the whole length. Their sequences are 5-TAAATAAATAATGCTCC- CAAGAAGCTCGAT-3 (Cas-matK5), and 5-CATGTATC- GAGCTCTGGGAGCATRT-3 (Cas-matK7R).

Sequence data were aligned manually with the GENETYX program (The Software Development Co., Tokyo).

Phylogenetic analyses Manos and Steel (1997), on the basis of matK gene

sequences, showed that Casuarinaceae (Casuarina) are sister to the Betulaceae-Ticodendraceae clade, with Juglan- daceae or Myricaceae as outgroup. Soltis et al. (2000), using sequence data of three genes, 18s rDNA, rbcl, and at@, further showed that, Casuarinaceae (Casuarina) are sister to Betulaceae (Ticodendron was not analyzed) and that the Betulaceae-Casuarinaceae clade is sister to the Juglandaceae-Myricaceae clade. In fact the Betulaceae- Casuarinaceae clade was supported by a jackknife value of 99%, and the clade comprising Betulaceae, Casuarinaceae, Juglandaceae, and Myricaceae by a jackknife value of 100%. Therefore we added three species of Betulaceae and Ticodendraceae to the data matrix and used one species each from Juglandaceae and Myricaceae as out- groups to obtain rooted trees. Sequences of the five species from the four families, Betulaceae, Ticodendraceae, Myricaceae, and Juglandaceae were obtained from Gen- Bank (Table 1).

Phylogenetic analyses were conducted on sequences of 1310 bp. of the rbcl gene and 1014 bp. of the matK gene in the first step, and then with their combined data. Data sets were analyzed by Wagner parsimony (Farris 1970) using PAUP version 3.1.1 (Swofford 1993). We performed a heuris- tic search under the equal weighting criterion using the Tree Bisection Reconnection (TBR) branch-swapping algorithm with MULPARS on, Steepest Descent on, and with the 100 replicates of random addition sequence option. Accelerat- ed transformation (ACCTRAN) was used for optimization in the analyses. Bootstrap analyses (Felsenstein 1985) of 100 replicates were conducted under the same condition as above, and the decay analysis (Bremer 1988,1994) was conducted without the random addition sequence option to assess the internal support for clades found in each analysis.

Results and Discussion

Phylogenetic analyses based on rbcL gene sequences Out of the 1310 bases analyzed of the rbcl gene, 111 were

variable and 54 (4.1%) informative. Phylogenetic analyses

A

r 99 6.5 -

A. liftoralis A. lehmanniana

6.4 A. torulosa A. decussata Ca. sp. Ca. cunninghamiana Ca. collina

Ca. obesa

-Ez -2k-E Ca. equisetifolia

Ce. terminale G. chamaecyparis G. deplancheanum

G. nodiflorum G. nobile G. sumatranurn

46 1

Allocasuarlna

Casuarlna

Ceuthostoma

Gymnostoma

Ticodendron (Ticodendraceae) 59 Betula (Betulaceae) d=1 Corylus (Betulaceae)

Myrica (Myricaceae)

Juglans (Juglandaceae)

A. littoralis A. lehmanniana A. torulosa A. decussata

d=4 d=3 100 d>5

Ca. sp. Ca. cunninghamiana Ca. collina Ca. equisetifolia Ca. obesa

G. chamaecyparis G. deplancheanum G. noditlorum G. nobile G. sumatranum

I

Allocasuarlna

Casuarlna

Ceuthostoma

Gymnostoma

Ticodendron (Ticodendraceae) Betula (Betulaceae) Corylus (Betulaceae)

Myrica (Myricaceae)

d>5 Juglans (Juglandaceae)

Fig. 1. Phylogenetic trees of Casuarinaceae. A. Strict con- sensus tree of 35 most parsimonious trees based on rbcL gene sequences. Length=154, CI=0.660 (excluding uninformative data), Rl=0.849. B. A single most parsimo- nious tree generated by matK gene sequences. Length= 244, Cl=O.833 (excluding uninformative data), RI=0.93l. The numbers above the branches are bootstrap values expressed with percentages of 100 bootstrap replications, and the numbers below the branches decay indices. Arrows indicate branches collapsing in the strict consensus tree of four most parsimonious trees based on combined analyses using both rbc l and matK sequences.

based on the informative sequences resulted in 35 most Rarsimonious trees of 154 steps with a Consistency Index of 0.660 (excluding uninformative characters) and a Retention Index of 0.849. Their strict consensus tree showed that Casuarinaceae, including Ceuthostoma, are monophyletic with 100% bootstrap support and a decay index of more than five (Fig. 1A). Both Betulaceae (Betula and Corylus) and

462 A. Sogo et a/.

Ticodendraceae (Ticodendron) are positioned outside of Casuarinaceae, but it is not clear which family is more closely related to Casuarinaceae.

Within Casuarinaceae, the rbcl tree showed the distinct- ness of the three genera Allocasuarina, Ceuthostoma and Gymnostoma, but did not support the monophyly of Casuar- ina (Fig. IA). Therefore, generic relationships within the family were not clearly resolved.

Phylogenetic analyses based on matK gene sequences Of the 1014 bases analyzed of the matK gene, 190 were

variable and 101 (10.0%) informative. Analyses based on matK gene sequences resulted in only one most parsimoni- ous tree of 244steps with a Consistency Index of 0.833 (excluding uninformative characters) and a Retention Index of 0.931 (Fig. IB). The matK tree, like the rbcl trees, suppor- ted the monophyly of the Casuarinaceae with 100% boot- strap value and a decay index above five. Outside of Casuarinaceae, Betulaceae (Betula and Corylus) and Ticodendraceae (Ticodendron) formed a common clade, but their relationships are not strong, with a bootstrap value of 71% and a decay index of only one.

The matK tree further supported the distinctness of all four genera of Casuarinaceae which were defined by combina- tions of a variety of morphological characters from the branchlets and infructescences (see synoptic key in Johnson 1980, Johnson and Wilson 1989,1993, Wilson and Johnson 1989, see also Fig. 2). In fact, the monophyly of Allocasuar- ina, Casuarina, and Gymnostoma was supported by bootstrap values of loo%, 85%, and 94%, respectively, and decay indices of more than five, three, and three, respectively. Ceuthostoma is not included in any of those three genera. Consequently the matK tree showed generic relationships to be (Gymnostoma (Ceuthostoma (Casuarina-Allocasuarina))).

Blanchlet

Allocasuarlna I

stomata on Number blanchlet ~~~~1

n Ticodendraceae

U Betulaceae

Combined analyses based on rbcL and matK gene sequences

Combined analyses based on both rbcl and matK sequences resulted in four most parsimonious trees of 401 steps with a Consistency Index of 0.756 (excluding uninfor- mative characters) and a Retention Index of 0.895. Their strict consensus tree yielded almost the same topology as that of the matK tree, but it did not resolve the relationships among Allocasuarina littoralis, A. lehmanniana and A. torulosa and between Betulaceae (Betula and Corylus) and Ticoden- draceae (Ticodendron). Except for those unresolved rela- tionships, the combined data, like the matK tree, supported the distinctness of the four genera of Casuarinaceae and the relationships (Gymnostoma (Ceuthostoma (Casuarina - Allo- casuarina))). They supported more strongly the monophyly of Gymnostoma with a bootstrap value of 99% and a decay index of four.

Relationships within the family and character evolution As mentioned in the introduction, Allocasuarina, Ceuthos-

toma, and Gymnostoma have been recognized as distinct genera only for 'past 20 years. Molecular evidence from matK gene sequences and from the combined data set of the rbcL and matK gene sequences showed that the Casuar- inaceae are composed of four distinct genera, Allocasuarina, Casuarina, Ceuthostoma, and Gymnostoma.

As discussed by Johnson and Wilson (1989,1993), how- ever, comparisons of morphological characters do not clarify generic relationships within the family. Johnson and Wilson (1989,1993) believed Gymnostoma to be the most primitive genus in the Casuarinaceae because of its least specialized morphological characters and Allocasuarina to be the most specialized. Johnson and Wilson (1989,1993) also stated that the evolutionary history since the establishment of the very characteristic features of the family has been complex. The molecular analyses presented in this paper,

Infructescences

Cone- Dorsal Direction of Color of Basic bearing Bract protuberance bracteoles number of blanchlet of bracteoles protruding SamaraS chromosomes

f 2 II Y

Fig. 2. Distributions of morphological features in Casuarinaceae and related families.

Molecular Phylogeny of Casuarinaceae 463

however, showed the relationships within the family to be (Gymnostoma (Ceuthostoma (Casuarina-Allocasuarina))).

Morphologically, Gymnostoma is clearly distinct from the three other genera in having exposed stomata in the shallow longitudinal furrows of the branchlets and a basic chromo- some number of x=8 (Johnson 1980). The exposed stomata are found in the outgroups Betulaceae and Ticodendraceae, and x=8 in the base chromosome number in Betulaceae (Fig. 2). In the other three genera the branch- lets are deeply furrowed. The stomata are embedded in narrow longitudinal furrows and thus invisible. The basic chromosome numbers are higher, i.e., x=9 and 10-14 (un- known in Ceuthostoma) (Fig. 2). All the species of Casuar- inaceae are anemophilous, but at least some species of Gymnostoma occur in moist closed forests, in contrast with species of Allocasuarina, Casuarina, and Ceuthostoma, which usually grow in more open habitats (Johnson and Wilson 1989,1993). The reduced, tiny leaves and deeply embedded stomata in the longitudinal furrows of the branchlets in Allocasuarina, Casuarina, and Ceuthostoma decrease transpi- ration and are thus on adaptation to open, dry habitats.

Beside the aforementioned characters, features of the gynoecium may be archaic in Gymnostoma. Basically, the ovary is bicarpellate in Casuarinaceae (Johnson and Wilson 1993). Both carpels are fertile and each has two ovules in Gymnostoma, although mature fruits are always one-seeded (unpublished data). In Allocasuarina and Casuarina, one of the two carpels is sterile and the carpel has a single ovule (Swamy 1948, Barlow 1958). Ticodendron also has a bicar- pellate gynoecium and each carpel has two fertile ovules (Tobe 1991). Betulaceae have bi- or tricarpellate gynoecia, with each carpel having two ovules (Kubitzki 1993). There- fore, the bicarpellate gynoecium with each carpel biovulate, as in Gymnostoma, is a plesiomorphy in the family. The reduction of a fertile carpel seems to provide an advantage for minor resource investment in female structures. How- ever, since it is uncertain whether all carpels of Ceuthostoma are fertile, it is not yet clear, where the reduction of the fertile carpel occurred, in the common ancestor of Allocasuarina, Casuarina and Ceuthostoma or in the common ancestor of Allocasuarina and Casuarina. The gynoecial structure of Ceuthostoma must be investigated to answer this question.

The other significant diversity in the family is in the morphology of the cone-like compound fruits (i.e., the in- fructescences) (Johnson and Wilson 1989,1993, Wilson and Johnson 1989). A few representative characteristics such as the nature of the cone-bearing branchlets, the shape of the bracts and bracteoles, and the color of the samara have made it possible to provide a synoptic key to the four genera (see also Fig. 2). This means that a major evolution in Allocasuarina, Casuarina, and Ceuthostoma may have proceeded in association with seed dispersal mechanisms acquired in the habitats where their ancestral species were evolved. It is uncertain, however, how ‘distally expanded bracts and antrorsely protruding bracteoles of Ceuthostoma are related to distinctive mechanisms of seed dispersal; is it unclear, either, what function non-expanded bracts of Allocasuarina and Casuarina or the dorsal protuberance of

bracteoles of Allocasuarina has. The red-brown to black, shining samara which is found only in Allocasuarina, implies seed dispersal by birds. Nothing is known about seed dispersal mechanisms in Allocasuarina and the other genera, however. Detailed evolutionary trends and functions of morphological characters of the infructescences must be studied through critical field observations.

We are grateful to Hitofumi Abe, Alex Floyd and Utami Nanda for collecting materials used in the study. The study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 11 691185).

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(Received September 14, 2001; accepted October 12, 2001)