Botaniwl]ournal uj the Limmm Soriflr (2000), 134:93 !29. With lfi figure'
doi: I 0.1 006/bojl.2000.0:lb(), ;n·ailable online at http:! /v,ww.idealibrarc.com on
IDE 1)-l®
Under the microscope: plant anatomy and systematics.
Edited 0 P]. Rudall and P Gasson
Systematics and evolution of Velloziaceae, with
special reference to sieve-element plastids and
rbcL sequence data
H.-DIETMAR BEHNKE*
<:,ellenlehre, Unimsitat Heidelberg, lm Neuenheimer Feld 230, D-69120 Heidelberg, German_y
]ENS TREUTLEIN, MICHAEL WINK
lnstitutfiir Pharma;:_eutische Biologic, Universitat Heidelberg, Im Neuenheimer Feld 364,
D-69120 Heidelberg, German_y
KLAUS KRAl\IER
Botanischer Garten, Cniversitat Heidelberg, Im Neuenheimer reld 340. D-69120 Heidelber;_[',,
Germany
CHRISTIAN SCHNEIDER
<:,ellenlehre, Unic•ersitat Heidelberg, lm Neuenheimer Feld 230, D-69120 Heidelber;_[',, German_y
P. C. KAO
C'hengdu Institute rj"Biology, Academia Sinica, PO. Box 416, Chengdu 610041, Sichuan,
China
Sieve-element plastids and rbcL nucleotide sequences wen· analysed for \'clloziaceae and
all families sometimes discussed as close allies (Aeanthochlamydaceae, Amaryllidact"ae.
Bromcliaceae, Cyclanthaccae, Haemodoraccae, Hypoxidaccae, Pandanaceae. Pcntastemonaccac, Stemonaceac). Velloziaceae (37 taxa inn·stigatcd) contain sieve-element
plastids defined here as sub forms P2c, ,, P2c,h P2c,p, P2c,;L and P2c,pf and distinct from those
of any other monocotyledon by ( l) angular crystals replacing to a large extent the monocotspecific cuneate crystals, (2) additional loosely-packed crystals, and (3) their very small average
diameters. This sie\T-element plastid syndrome is a synapomorphy of Yelloziaceae and
indicates the monophyly and early isolation of the family. Sieve-element plastids of the
*Corresponding author. E-mail: [email protected]
0024 4074/00/0900'l:l + :J7 $3:J.O(J/0
93
© 200(1 The Linne an Societ\ ot" London
H.-D. BEHNKE ET AL.
94
monotypic Acanthochlamydaceae share their small sizes with Velloziaceae, but all their
crystals are cuneate, i.e. loosely-packed crystals were not found. Among the other families
studied the sieve-element plastids of (a) some Bromeliaceae (Ayensua in particular) contain
loosely-packed crystals, (b) several Amaryllidaceae incorporate additional orthogonal crystals,
and (c) Pandanaceae belong to form-Pcf, but cuneate crystals are present throughout and
the plastids are significantly larger than in Velloziaceae. The new information on the rbcL
nucleotide sequences of 24 taxa of the Velloziaceae and of 20 additional taxa from
putatively related families strongly support the monophyly of Velloziaceae and define
Acanthochlamydaceae as its sister family. At the infra-familial level four evolutionary lines
are discovered which correspond to major generic groups and their geographical distribution.
The divergence time for the split between New Word and Old World taxa has been calculated
as 40-70 million years, that between African and Madagascan species maximally 25 million
years. The cladistic analysis requires that the monotypic genera Nanuza and Talbotia be
included within Tfllozia and Xerophyta, respectively. At the ordinal level the analysis of the
rbcL-data strengthens the Pandanales as a monophyletic clade.
© 2000 The Linnean Society of London
ADDITIOJ\iAL KEY WORDS:--Acanthochlamydaceae - Amaryllidaceae - Bromeliaceae
- Haemodoraceae ·- Hypoxidaceae - molecular data - monocotyledons - Pandanales phylogeny -·· ultrastructure.
COl'\TENTS
Introduction .
Systematics and affinities of V clloziaceae
Sieve-element plastids: types, forms, subtypes, subforms, and sizes
Material and methods
Taxon sampling .
Electron microscopy
DNA isolation, PCR, and DNA-sequencing
Data analysis .
Results
~'>ieve-clement plastids of V elloziaccae
Sieve-element plastids of Acanthochlamydaceae .
Sieve-element plastids of families traditionally allied to Velloziaceae
Sieve-element plastids of Pandanales sensu Kubitzki, Rudall & Chase (1998)
Phylogeny reconstructions based on nucleotide sequences of the rbcL gene
Discussion
Subdivision and relationships within Vclloziaceac
Affinities with other monocotvledonous families
Phytogeography .
,
Taxonomic implications
Character evolution in Pandanales
Acknowledgements
References
94
94
97
l 04
l 04
l 04
104
l 05
105
l 05
l 09
l 10
111
111
115
115
115
ll 7
ll 7
119
122
127
I:'-<TRODUCTION
!iJstematics and affinities qf velloziaceae
The monocotyledon family V elloziaceae was established by Endlicher (1341) who
separated the genera Barbacenia Vand. and vellozia Vand. (including Xerophyta]uss.)
from Haemodoraceae where Martius (1823), Sprengel (1827), Schultes & Schultes
(1829) and Lindley (1330) had placed them. Endlicher's (1341) Vellozieae were part of
his 'classis XVII Ensatae' and placed therein after Hydrocharideae, Burmanniaceae,
.'iiE\ E-ELE.\IE:'\T PL\STIDS .\:"iD RBCl. D:\T.\ 1:'\ \'ELLOZI.'I.CJ:.\E
Irideae, Haemodoraceae and followed by Hypoxideae, Amaryllideae, Agavcae and
Bromeliaceac. This 'dassis' contained all families that during the next 150 years
served as candidates for the putative closest ally of Velloziaceae. Howe\-er, these
families were never grouped together subsequently.
Seubert (1847, 1873), Hutchinson (1934), Cronquist (1968, 1981), and Huber
( 1991) recognized Hacmodoraceae as the sister group of the new family. Bentham
and Hooker ( 1883) and Baker ( 1898) reduced it to tribal/ subfamilial status within
their Amaryllidaceae, while Pax (1887, 1930) and Wettstein (1924) retained the
family status and had Amaryllidaceae as the closest family. Takhtajan (1959, 1969,
1973), Melchior (1964), Ayensu (1973), and Thorne (l992a) suggest Hypoxidaceae,
another early segregation from Amaryllidaceae, to be next to V clloziaceae, a
relationship that was first proposed by Don ( 1830).
Jussieu (1 789) placed his Xeropkyta, together with Burmannia, Tzllandsia, Bromelia,
and Agave, into his 'ordo Bromeliae'. Gmelin (1796) and Lamarck (1808) likewise
treat their X madagascariensis J. F. Gmcl. respectively X. pinijolia Lam. as members
of the 'Pineapple family', and later Kunth ( 1822) also discussed relationships between
Radia tubffiora A. Rich. ( = vellozia tubifiora H. B. K.) and Bromeliaceae. Closer affinities
between Bromcliaceae and Velloziaceae were reestablished by Huber (\969, 1977)
and Gilmartin & Brown (1987) and subsequently taken up by R. Dahlgren (1975,
1980), G. Dahlgren (1989), Takhtajan (1987, 1997), Froelich & Barthlott (1988),
and Thorne (l992b). Eventually, the transfer of Vdlozia uaipanensis ~Iaguire (Maguire
& Wurdack, 195 7) as 4Jensua uaipanensis (l\Iaguire) L. B. Sm. to Bromeliaceae (Smith,
1969) seemed to be symptomatic of the close relationship between the two families.
Molecular characters, mainly rbcL sequence data, grouped Velloziaceae together
with Stcmonaccae, Cyclanthaceae and Pandanaccae as 'stemonoids' or 'Pandanales',
far away from the 'Bromeliads' and not close to any of the families previously
discussed as related (Chase et al., 1993, 1995; Dm·all et a!., 1993; Kubitzki et al.,
1998).
The infra-familial classification ofVelloziaceac is still disputed. The family includes
over 200 species in South America, Africa, l\hdagascar and Arabian Peninsula
(South Y emenJ, grouped into eight or nine genera and two subfamilies vvith several
sections, the delimitation and content of which continue to be controversial among
taxonomists (see e.g. Smith & Ayensu, 1976; Menezes, 1980; l\fello-Silva, 1991;
Menezes, Mello-Silva & Mayo, 1994). In the generic treatment this paper follows
Kubitzki (1998) who like Smith & Ayensu (1976) restricted Xerophyta to African,
Arabian and Madagascan and Vellozia to South American species, but like Menezes
(1980) separated Barbacenia, Pleurostima and Burlemarxia from the latter, while Aylthonia
(likewise segregated by Menezes, 1980) was kept within Vellozia (see also Mello-Silva,
1991 ).
The specific ultrastructure of sieve-clement plastids provides characters that are
useful in the delimitation, circumscription, and classification of taxa and have proved
helpful in their assignment to higher categories of flowering plants (e.g. Behnke,
1981 a, 1991 ). P-type sieve-element plastids with cuneate protein crystals (see Fig.
l ), i.e. in ultrathin sections distinctive by their predominantly triangular shape
and classified as subtype-P2 (Behnke, 1977), were found to be common to all
monocotyledons (Behnke 1968, 1969, 1981 b) and used as a synapomorphy to support
the monophyly of the class (Cronquist, 1981; Dahlgren & Clifford, 1982; Dahlgren
& Rasmussen, 1983; Takhtajan, 1997; Kubitzki et al., 1998).
A first broad survey of sieve-element plastids in the monocotyledons listed some
96
H.-D. BEHNKE ET AL.
e
"'(t
P2ccl
lt
~~
P2ccp
P2CcpS
Monocotyledons
Paleoherbs
1t
~
1t
Pl/2Ccp
Figure I. Putative evolution of the forms and subforms of sieve-element plastids in monocotyledons.
Dicotyledon form-PI/2c,p plastids, derived from S-type (form-Ss) via Pies and Pic, are connected to
monocotyledon subform-P2c,p plastids as found in Tofieldiaceae. The loosely-packed polygonal crystal
of subform-P2c,p plastids breaks up into many small loosely-packed crystals of subform-P2cd which are
eventually lost in subform-P2c,. The addition of starch grains and/or protein filaments gives rise to
form-P2cs, -P2cfs, and -P2cf plastids. Rarely, cuneate crystals are completely lost and form-Ss plastids
formed. The major (sub)forms are connected by heavy arrows in the centre and middle right lanes,
additional subforms (connected by light arrows) characterize detours or specific side ends. The subforms
are arranged so as to evolve P2c-(sub)forms in the centre, add starch to the right (P2-subforms
containing 's') and filaments to the top (P2-subforms with 'f'), while form-P2cfs occurs in the junction
between 'cs' and 'cP and S-type in the far right (from Behnke, 2000; modified).
200 species representing 65 families and included a few species of Velloziaceae and
its putative allies (Behnke, 1981 b). Since then special efforts to obtain samples
from all monocotyledon families as well as from groups that were segregated by
morphological, chemical, molecular or other data, have raised the number of species
investigated to presently over 700, all (except Pistia, Araceae) with subtype-P2 sieveelement plastids (Behnke, 1994, 1995, 2000, and unpublished results).
Among the new specimens studied were several Velloziaceae. Their sieve-element
SIL\ E-l:U::\IE'\T PL\STIDS A:\D RBCL D.\T.\ L'\ \'ELLOZL\CL\1:
'17
plastids containt>d loosely-packed crystals that were surprisinglY identical to those
found in 1!vensua (Bromcliaceae), but had no similarity to siew-clcmcnt plastids of
the few species imTstigatcd from the Pandanales, \\herein according to molecular
data (Chase et aL., 1995) Velloziaceae should be included. Therefore the inn:stigation
of sieve-element plastids was extended to coYer as many taxa as available from
Velloziaceae (plus Acanthochlan~J'S, included by Chase et al., 1995, but raised to family
level by Kao, 1989) and all families considered related. At the same time, an analvsis
of their rbcL gene sequences was initiated with almost the same set of taxa in order
to advance further the relationships ofVelloziaceac and its infra-familial classification
and via calculations of genetic distances to contribute towards the discussion vvhether
Velloziaceae originated in the Old or ~ew \\"orld. This paper presents the results
of these efforts and analyses the new sie\T-element plastid and rbcL sequence data
together with re\c,·ant information taken fi·om the literature.
SieN-I'iemmt pLastids: types, forms, sub[rpe.s, mbfomzs. and .1i;:Ps
Sieve-element plastids are characterized by their content of protein crystals (c),
protein filaments (f) and starch grains (s) which can be identified by histochemical
staining and enzymatic digestion (see Behnke, 1972, 1975), but arc usually detected
with the transmission electron microscope (TEM) in ultrathin sections. Different
morphologies and different quantities of protein and starch determine the specific
sieve-element plastid possessed by a taxon. Consequently, if an arbitrary number of
species is screened, definite criteria are necessary to categorize sieve-element plastids
and eventually to support or disprove the putative systematic position of a taxon
proposed by considering other characters (Behnke, 1991 ).
Ijpes of sien'-element plastids are the result of a basic distinction: if protein is
present, a plastid belongs to P-type, absence of protein defines the S-fype (Behnke,
1971). Starch may be present (s) or absent (o) in either type.
Forms of sicn'-clement plastids give an exact notation of their qualitative content
and commonly suffice to describe a taxon. Eight distinct combinations can be formed
by the four characters, all are found among angiosperms: forms Pc Pes, Pcfs, Pcf,
Pfs, Pf, Ss, So (Behnke, 1991)
Certainly, forms represent only a rough approximation to the genetically determined species-specific composition of a sie\·c-element plastid, which is also
characterized by its size as well as by the morphology, number and arrangement of
its protein crystals, protein filaments and starch grains. The establishment of the
subtype and subform categories is meant as a tool to describe taxa more precisely.
Subtypes of sieve-clement plastids explain P-type taxa at ordinal or higher levels
and are marked by numerals, e.g. subtype-P2 includes all sieve-element plastids
containing cuneate crystals, mainly found in monocotyledons (Behnke, 197 7. 1981 b,
2000).
Su~forms of sieve-clement plastids evaluate different morphologies of protein crystals
or other contents (Behnke, 2000) and are recorded by subscripts to the respective
character symbol: e.g. subform-P2catf describes sie\·e-elcment plastids containing
angular C) plus loosely-packed CJ crystals besides protein filaments (f). subform-P2c,"
those with angular and a single loosely-packed polygonal C,) crystaL and subformP2c,o with cuneate (,) and orthogonal U crystals.
Si;:.es of sieve-clement plastids measured as average diameter are of additional
help for the description of families or higher taxa (sec Behnke. 2000).
Vt:llozia variegata Goethart & Henrard
hirsuta Goethart & Henrard (I)
hirsuta Goethart & Henrard (2)
sel/owii Seub,
sp. (I)
sp. (2)
tubiflora (A. Rich.) H. B. K
variabilis Mart. ex Schult. f.
P2c.,,
P2c.,j
P2c.,,
P2c,,,
P2c.,1,
P2c,,
P2c,,
P2c.,~
P2c.,"
P2c.,l
P2c.,"
P2c.,"
P2c.,pf
Vt:llozia af[ caespitosa L. B. Sm. & Ayensu
Vel/ozia crassicaulis Mart. ex Schult. f.
Vt:llozia declinans Goethart & Henrard
vellozia froesii L. B. Sm.
Vt:l/ozia glochidea Pohl
Vt:l/ozia
Vt:l/ozia
Vt:l/ozia
Vt:llozia
Vt:l/ozia
Vt:l/ozia
Vt:/lozia
P2c.,,
P2c.,,
P2c
P2cf
P2c.,;
P2c.,,
P2c,,
P2c.,;
P2c.,;
P2c,,
P2c.,,
P2c,,;
P2c,;
P2c.,;
P2ca/
P2c,1,
Form
29
8
1.19
0.77
1.05
0.92
0.74
0.62
0.90
0.89
0.92
0.68
0.91
0.92
1.28
1.00
0.91
0.91
0.9+
0.53
0.67
0.94
0.68
0.95
1.12
0.91
0.87
0,79
0.71
1.06
0.52
0.75
0.78
0.81
0.75
0.63
0.86
0.96
0.80
0.56
Sicn'-element plastid data
No. spp
Diameter
stem
leaf
BG-SPF, leg. A. Salatino
BG-SPF, leg. A. Salatino
BG-SPF, leg. A. Salatino
BG-BONN 9635
BG-BONN 9646
BG-BONN 4412
Depto. Santa Cruz, Prov. Chiquitos, nearby
Santiago de Chiquitos (BOL), leg. Ibisch &
Nowicki 99.0123
BG-BONN 9651
BG-HEID 101408
Depto. Santa Cruz, Prov. Florida, Cuevas,
vicinity ofAmbor6 Nat. Park, 63°45'vV, l8°13'S
(BOL), leg. Ibisch & Nowicki 98.133
BG-BONN 9741
BG-BONN 9643
BG-SPF, leg. A. Salatino
BG-BONN 9985
BG-BONN 9639
BG-BONN 96+7
BG-SPF, leg. A. Salatino
BG-BONN 9978
BG-B0:\1'\ 1297+
BG-HEID
BG-BONN 565.5
BG-SPF, leg. A. Salatino
BG-BR 1987-0437; BG-SPF, leg. A Salatino
BG-K 1980-2675
BG-SPF, leg. A. Salatino
BG-SPF, leg. A. Salatino
BG-BONN 12786
Origin of material
Treutlein & Wink 397
Treutlein & Wink 425
Treutlein & Wink 455
Treutlein & \\'ink 4-22
Treutlein & Wink 453
Treutlein & Wink 408
(1) Treutlein & Wink 411
(2) Treutlein & Wink 429
Treutlein & Wink 4-18
Treutlein & Wink 4-28
Treutlein & Wink 456
Treutlein & Wink 452
Treutlein & Wink 403
Treutlein & \\'ink 442
Treutlein & Wink 426
Treutlein & Wink 407
Treutlein & Wink 405
rbcL analysis
l. Sieve-element plastid data and origin of material used in the combined ultrastructural and rbrL analysis of\' clloziaceae and related families
Barbarenia .wisea L. B. Sm.
Barbacenia tmnentosa 1\ I art.
Barbareniopsis boliviensis (Baker) L. B. Sm.
Barbareniopsis castil/onii (Hauman) lbisch
Barbaceniopsis vmgasiana (L. B. Sm.) L. B. Sm.
.\imu.::a plirata (:\[art.) L. B. Sm. & Ayensu
}Vimuza plicata (1\lart.) L. B. Sm. & Ayensu
P/eurostima Janniei N. L. Menezes
Pleurostima purpurea Raf.
Pleurostima purpurea Raf.
Pleurostima riparia N. L. Menezes & Mello-Silva
Pleurostima stenophy!la (Goethart & Henrard) N. L.
Menezes
Talbotia elegans (Hook.) Balf.
Vt:l/ozia andina lbisch, Vasquez & Nowicki
\.ELLOZL\f~EAE
Taxon
L<\BLE
r--
---1
::..
~
::"1
z
;;:;
:c
~
t;e
:c
~
:0
'""
H. Pr'rricr
l1<~k{'l
,(/lfedileri !lbkcr t \1. I .. :\lcw; n
<jilmdnr1 IRrndlc:. :\.I .. ~lrll<'/<".'
ril/w,t (Baker! 1.. B. Sm.
.hrtNr
rdim·rti-'
jJirujiJ/ia Lm1.
rks Baker
(Baker) Dur. & Scbinz
~glandulosa
.
I'I!Filinrw Hn·l>
. \f:m. i.HJo j;rir/i(tr~ I ..
( ;a{anllut.l nirah\ [ ~.
!lalnantlm.\ mbns/n., IHnh.r Sweet
flahrant/w, !uhi.,jlrlilw., {L'Hhi t.:· Tr;rulr
llalnrmt/m.1 \'lrittlillll hybrid rLtnL' l L E. .\I (Jon·
1/rmowr!l!!i\ raroliuamo :I,.: Hcrl 1.
r graudtjlolll PLuwh. & l.ind.
/•:utm,fa amanliata (B;IkcT) Jl;t\
datu.\ ..
(:'!?frmthti5 lur~dt'f'\l·rhlw
( ,'Ji!llllll \
(."r/nwu
(,(tfo.llf'! !ii!Ul
.lmmothorii' (OJrmita (1'\_er-(;;nrl.' lit·r/).
!loophom• dt.llidta (L. [) Herb.
llrullwi~;i11 lienei F.l\1. l.ciuln. "' \\.
F. Barker
A~L-\RYIJ,ll)i\(;EAE
.Jmn!ltoddrw{YI hradutl a P. (:. K<1c1
\( ',;\\iTIHH 'Hl .. \\\1\'IJ;\t :E.\ I
Xemj1hrta
Baker
Yar. petlinala ',Baker! H.
I':Z..
1'2<
p:z,
1'2<
P2c
P2t
p~(
p:z,
P:.!r
J'2c
P:?c
1'2.
I''Lc
p~~(
1'2r
1'2<"
1'2r
p:z,
]':?,
1':!<"
p:z,
1'2d
UG-HE ID
II< ;.H Elll
B<;-HElll
I
.Ill
.O:l
B<;-HEII>
IH:-H EIIl
IH :-HEl ll
.II'>
Jl( ;.H Elf)
~J:)l)
B<:-H Elf)
BC-H E!l)
B<;. HElD
BC-H Eill
B<!-H Eill
BC-HE ID
B(;-HE IJl
Brazor ia (
1.:12
.%
.I
I
~ 1.~n
BC-HE lf)
.:II
J:lj
H<:-HEID
BO-HE ID
B< ;.If EID
.T!
I. \H
BC-HEIJ)
.II
l.:l7
..1~1
I ;Ill \ \':
l\11(1
:\f. II. :\l;,dic ld
.\rca, \\'-Sid ntan (China),
L\ . t·s. \ ..
~loulllaill
kg. I'. C. Kao
He11,;duan
1.1 I
1.:21
lt'g.. \.
liC-E "' f\l;~Ja,\r. lq~. B. I Burtt 1>
IH ;.E
Tr.tllSL<,d. lc~. B. L. Burtt 2'.1117
IH ;.fl(
BG-BU NN i'll'l
BG-BO N!\
BC-BO NN 11771
BC-B O"i\ I I 1:2:.'
HG-B Ol\N 13690
BC:-B ONN 13G'l
.:'7
..'J-1
/.'>:l
I.IH
tu,:,
ll.li:,
1'2<
,.~,,
1.(
II
P:.!(
1':.'<
ll.IJ
lUi!
ll.bii
.Ill
27
O.jH
07.1
!.IU
I'C'r
1'2t
p~{"
P2c.,/
P:!c.,1•
P2r.,
0.60
0.80
!sc<· Tahl•·
l'n-rnk ill ,'V. \\'ink I Hi
Trcntlc in & \l-ink
Trcutlc in & \\'ink 1'2'1
Trn11 kin & \\'ink ~I! I
(oll!inur d
Trcutlc iu & \\'ink II()
Treutle in & Wink 40'1
Trelltk in & Wink 412
~
-i
_
::;::
-;::.
s
'"'
'-
2
/
;..-
~
~
::;
~
""'
::>;,
~
:./'.
c
j
:.t:
~
.--
~
~
~
:"':
....
Aechmea purpureorosea (Hook.) \ Vawra
Ananas comosus (L.) 1\Ierr.
Ayensua uaipanensis (Maguire) L. B. Sm.
Billbergia reichardii Wawra
Bmcchinia micrantha (Baker) 1\Iez
Brocchinia reducta Baker
Canistrum Josterianum L. B. Sm.
Deuterocohnia brevispicata Rauh & H. Hrodm.
Disteganthus basilateralis Lam.
PJckia altissima Lind!.
Fosterella caulescens Rauh
Fosterella villosula (Harms) L. B. Sm.
Greigia sp.
Guzmania monostachya (L.) Rushy ex Mez
Guzmania zahnii (Hook. f.), Mez
Navia phelpsiae L. B. Sm.
Neoregelia olens L. B. Sm.
Nidulariwn innocentii Lem.
.Nuiularium procerum Lindm.
Orthophytum saxicola (Ule) L. B. Sm.
Pepinia corollina (Linden & Andre) Varadarajan &
Gilmartin
Pitcaimia sp.
Pitcaimia villetaensis Rauh
Puya vallo-grandensis Rauh
BROMELIACEAE
Pancratium maritimum L.
Pancratium sickenbergeriAsch. & Schweinf. ex Barb.Boiss. & Barbey
Phaedranassa tunguraguae Ravenna
Rauhia perul'iana Traub
Scadoxus multifiorus (Martyn) Raf.
Spreke/ia fimnosissima (L.) Herb.
IVorslrya rayneri (Hook.) Traub & 1\Ioldenke
:(epll)•ranthes candida (Lind.) Herb.
:(ephyranthes grandifiora Lind!.
Taxon
22
0.73
0.76
P2c, 1
P2c
P2c
P2c
1.39
1.09
1.00
0.86
0.94
P2c"
P2c
P2c"
P2c
0.80
0.83
1.30
1.03
0.73
0.91
P2c
P2c"
P2c
P2c, 1
P2c"
P2c, 1
P2c
0.87
0.79
0.86
0.70
1.30
0.87
1.12
1.27
1.02
1.28
1.25
P2c"
P2c
P2c
P2c
P2c
P2c"
1.10
1.19
1.75
1.39
P2c,,
P2c.J
P2c
P2c,,
P2cf
P2c
P2c
1.00
1.32
1.39
BG-HEID
BG-HEID
BG-HEID
BG-HEID
BG-HEID
BG-HEID; BG-BONN; BG-FRP
BG-HEID
BG-HEID
BG-HEID
BG-HEID
BG-HEID
BG-BONN
BG-HEID
BG-HEID
BG-HEID
BG-HEID
BG-HEID
BG-HEID
BG-HEID
BG-HEID
BG-HEID
BG-HEID
BG-BONN
BG-HEID
BG-HEID
BG-HEID
BG-HEID
BG-BONN; BG-HEID
BG-BONN
BG-HEID
BG-HEID
lie de Re (F), leg. H.-D. Behnke
BG-HEID
Origin of material
!-continued
Sieve-element plastid data
Diameter
:'lio. spp
leaf
stem
P2c
P2c
Form
TABLE
&
&
&
&
Wink
Wink
Wink
Wink
437
399
436
440
Treutlein & Wink 415
Treutlein
Treutlein
Treutlein
Treutlein
Treutlein & Wink 414
Treutlein & Wink 438
Treutlein & Wink 398
Treutlein & Wink 439
Treutlein & Wink 413
rbcL analysis
r-
:>...
"'-1
t>:
t'l
"'
z
::r:
t'l
to
p:
~
c
angwti11ima Ridl.
hrrcimtia hank1ii .\. Cunn.
hr)'cinctia haaeriana Endl.
l·i;rcinl'fia dq:,antuia B. ( :. Stone
hncinrtia ncdl!! F. l\lucll.
hnl"inctia .Junimiari, :\!err.
Flqrinetia inn:E;11i.1 Bl.
l·l~rrinetia
I':Zcr
I':Zcr
P:Zcr
P:Zcr
P:Zcr
P:!cr
I':Zcr
P:Zcr
HI
p:z,
PA:"JDA:"JA<:EAF
.II:)
1.2+
I.IIB
1'2c
1.7B
I.HII
It I
l.:lll
I .:;c,
I.'Jii
.. )/
I.IB
I ..)2
.~:-)
.Oil
I':Zc
p:z,
l.l:l
IB
I IB
1'2c
HYPOXIDA< :EAI<.
1'2<
I IB
I 12
Curru!Zt;o rajJilala (I .our.) 1\.untzt·
H)'poxi., h)gmmefnm Labill.
Hypoxi., l't//o,a L.
Rhodo/l)j}()ti., /)()urii (Baker) Nl'i
1.2:l
I. 17
I. I 'I
I.IH
P:Zc
P:Zc
1'2c
I.B7
l.til
1.'> 7
ll.'lli
I. 71i
l.:l'l
I 'Ill
I.IB
1.:-, 7
1.:).-)
l.bl
l.lli
0.7B
1'2c
D. Dun
DC.
mm~Y,Irsii
I':Zc
I':Zc
I':Zc
P:Zc
P:Zc
P:Zc
P~<
1'2c
p:z,
p:z,
I':Zc
P~h
P:Zc
l.+'l
l.li'l
1'2c
P:Zc
Ill
l.ll
P2c
1'2c
hybrid
Conos/)'lis mndimn1 Endl.
fadm~mthr1 ramliniana (I .am.:· I )and~
Wachmdorjia panicuiata L.
Xiphidiwn coerulewn Auhl.
Jln(~o,cantlws
.1m:Y,o.::antho1
.·ln(~;o.~ant/m, Jlm•idu.l
IIAEJ\.IODORAf:EAE
Asplundia goebeiii (\\",·iss & R. \\'ap;ncr n R.
\\'ap;tHTi Harling
. hpiandia sp.
. hf!lundiu .ljJectabi/i, Harling
Cariurlol'im jJaima/a Ruiz & Pas.
Crdanthu' lnjmrtitu.l Po it.
/Jicranoj~Jgiwn mirmrrjJ!wlwn ~~Hook. L; Harling
/Jirranof!Jgium fto(vrrjJfmlum Harli11g
/Jinmwf!Jgiwn sp.
h•odianthu.<.fimijrr (l'oit.) I .indm.
/.,ur/m•ia lanr_zj(J/ia Hrongn.
<:Y(:J,A'\.J"THA<:EAE
Racinea spiculosa (Griscb.) Spmccr & Smith
lhesea giutirw.w LincH.
!'rima hierogiJ•jJhica (Carr.) E. l\lorr.
B<;-r-.uc
B<;-1\I
B<;-K I'Hi'l-lli<):Z7
B<;-K I'Hi'l-:l'rl~>
B<;-HEII>: K(;-KIEI.
B<;-K l'lHB-1'11
B<;-1\1; BG-K
BG-HEII>
BG-HEID
B<;-HEID
B(;-HEII>: IH;-B< )\;\:
BC-BONN
market garden, leg. \\'. Barthlott
B<;-B< >NN
Cull" Co., FL (LIS.\). L. C. Chafin
B<;-BONN
B<;-1\1
BC-HEID
BC-HEm
B(;-BO:\'f\
B<;-HEID
B<;-HEII)
BC-HEII)
B<;-B<>:".'N
BG-HEID
BG-HEID
B<;-BO~f\
B<;-HE]])
BG-HEID
BG-HEID
BG-HEID
++ I
Trcutlein & \\"ink +1.->
Trcutlci11 & \\·ink lT)
Treutlciu & \\.ittk J:l I
·1 n·utkitt & \\"ittk l"lll
Trnttleiu & \\"ink H+
·1 rnttkiu & \\'iuk t:l~
Treutlein & \\'ink
((mtinunl
c
--
~-.
('
;_.
E
:::;
__.
~
:::;_.
::I
s
s;
C/1
s
L.
-::I
-i
C/1
:;..
:s
;
t:
p
~
~
:,.:.;
Croomiajaponica Miq.
Croomia pauciflora Torr.
Sternona tuherosa Lour.
Stichoneuron caudatum Ridl.
STEMONACEAE
Pentasternona egregia (Schott) Steenis
PENTASTEMONACEAE
B. Pres!.
fi~)'cinflia jJ!tiliji/Jinm.sir Hems!.
Fl~winetia r(£?irhjolia Hems!.
Frercinetia scan dew Gaudich.
Pa~1dmmr albijivns B. C. Stone
Pandanus amarrllijiJ!ius Roxb.
Pandanus bajJit.slii \\'arb.
Pandanus bilamellatus 1\Iartelli
Pandanus candelabmm P. BeaU\·.
Pandanus dyckioides Baker
Pandanusjorsteri C. 1\loorc & F. 1\ludl.
Pandanusjurcatus Roxb.
Pandanus houlletii Carr.
Pandanw irregularis Ridl.
Pandanus kirkii Rendle
Pandanus microcarpus Balf. f.
Pandanus montanus Borv
Pandanus pacificus hort.' Veitch
Pandanus papuanus (So1ms) Ridl.
Pandanus pristis B. C. Stone
Pandanus pulcher Martelli & Pic. Serm.
Pandanus purpurascens Thouars
Pandanus pygmaeus Thouars
Pandanus sanderi Mast.
Pandanus sp.
Pandanus sylvestris Bory
Pandanus tectorius Sol. ex Parkinson
Pandanus toinu H. St. John
Pandanus utilis Bory
Pandanus vandermeeschii Balf. f.
Pandanus variegatus Miq.
Pandanus veitchii Mast. & T. Moore
Sararanga philippinensis Merr.
F1~rcinetia jaranica Bl.
Frerrinetia lu~onen.sis K.
'l'axon
1.33
1.35
1.24
1.58
1.14
P2c
P2c
P2c
P2c
P2c
4
1.26
1.26
1.70
2.79
1.82
1.53
3.64
1.9+
1.23
2.37
1.85
1.33
1.57
:n:J
1.6+
l.3B
0.72
1.23
0.98
1.17
1.84
1.25
0.99
1.17
1.78
1.34
1.13
0.99
1.10
1.35
1.39
0.9+
1.03
1.+2
1.0+
1.+9
0.99
1.07
1.19
1.35
1.0 I
BG-M
BG-M
BG-M
BG-M; BG-HEID
BG-SING; BG-M
BG-:\1
BG-BO'\'\: BG-HEID
BG-:\1
BG-B I.J.I-2+-7+-B:l
BG-K 1988-8618
BG-K 197+-866
BG-K 1986-21.11
BG-:\1
BG-:\1
BG-HEID
BG-HEID
BG-K 1972 25 78
BG-l\1
BG-HEID
BG-K 1985-1510
BG-HEID
BG-K 198+-5361
BG-KIEL
BG-HEID
BG-l\I
BG-HEID
BG-B 001-09-80-10
BG-B 193-10-79-83
BG-KIEL
BG-KIEL
BG-HEID ex Madagascar, leg. W. Rauh 21912
BG-B 280-03-80-10
BG-M
BG-M
BG-HEID
BG-B 132-48-74-80
BG-K 1973-20814
BG-HEID
BG-K 1993-3470
Origin of n1alcria1
!-continued
Sicn·-ckment plastid data
'\o. spp
Diamncr
leaf
stcn1
P2c
P2c
P2cf
P2d'
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2c
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2cf
P2c
P2c
Form
TABLE
anal~·sis
Treutlein & Wink 432
Treutlein & \\'ink .J.OO
Trcutlein & \\'ink +I 7
rb(L
~
t"
t>J
"-i
tTl
;;;::
z
::r:
tTl
t;O
;:c
~
~
SIE\ E-EI.DIL\T PL\STJDS .\:'\D RBCL DAL\ I:\ n:UDZL\C L\L
~E
:::::::::
-.=-:::.=
"-f :--t;;!;
,--.;;
t:
..
·-
c
t ; :;., ::;
-
-~ ·~ :i
~
.:::
~~---
~
8
- ~~ =~~
- c
f
-
:~
-
.,..
-
~
:C
'/
~
'-
:-
~
'-
'f
;..:
.--
-·
~ l.~.~
.
,
.
_
"
"' ,- .
~- ~~~~2
~
;:
~~~~..:::
I
'o::":;:'c~
~~J1~
"''
-
'-
-
~·
~
'-
-
" e ~~- "'
.;::
'-
I
1
- << <
'-
~
7."
~
"'
~
-1-
" '~
~
:::: d:
J'.
--
~
;>,
103
104
H.-D. BEHNKE ET AL.
:\IATERIAL AND METHODS
Taxon sampling
The species included in the present analysis of sieve-element plastids and rbcL
sequences were selected both to cover as many species as available of Velloziaceae
and to include taxa from all the families sometimes treated as related to them
(collection data are given in Table 1). Additional rbcL sequences included were
obtained from different sources (see Table 2).
Vouchers containing a sample of the specimen used, an electron micrograph of
a typical plastid, and a block of embedded material are placed in the TEX herbarium.
The rbcL sequences of taxa analysed in this paper will be deposited in GenBank.
Electron microscopy
Living material is a prerequisite for fixing sieve elements and subsequently investigating their plastids with the TEM. Therefore, the majority of species were
prepared for fixation by the first author in botanical gardens, while other samples
were sent to Heidelberg by express mail.
Stem material (vegetative or generative) was cut with a razor blade into thin
longitudinal sections and immediately immersed in the fixation mixture. Additionally,
or alternatively when stems were not available, parts of petioles or leaf veins were
used. Fixation was for three or more hours in a sodium-cacodylate buffered solution
containing 4% formaldehyde and 5% glutaraldehyde adjusted to pH 7 .3. Postfixation
in buffered l% OsO.h stepwise dehydration with acetone, embedding and polymerization in epon-araldite or low viscosity epoxy mixtures were according to
standard procedures. Semithin sections were cut from embedded material and
inspected for phloem, before ultrathin sections were cut with a diamond knife using
a Reichert Ultracut microtome and subsequently stained with aqueous uranyl acetate
and lead citrate. Sections were viewed and photographed with a Philips CM 10 or
a Philips EM 400, at 100 kV.
DNA isolation, PCR, and DNA-sequencing
Total DNA was isolated from fresh or frozen leaf material using a modified
version of the CTAB method (Doyle & Doyle, 1990). Primers used for PCR flank
the beginning and the end of the rbcL gene (rbcLN: 5'ATGTCACCACAAACAGAAACTAAAGC3',
rbcLR:
S'T ATCCATTGCTGGGAATTCAAATTTG 3'). Alternatively for primer rbcLR, reverse primer 1R was used (rbcL 1R:
5'GGGTGCCCTAAAGTTCCTCC 3'). For amplification total DNA was used as
a template, plus l 0 pmol each of primers rbcLN and rbcLR (rbcL l R), 1.5 mM MgCl 2,
0.1 mM of each dNTP, 5 j.!l amplification buffer and 1.5 U Taq DNA Polymerase
(Amersham Pharmacia Biotech) in a total volume of 50 j.!l. After an initial denaturation step (4 min at 94°C) 30 cycles of 30 s at 95°, 30 s at 45°C, and 90s at
72°C were performed on a Biometra thermocycler. After 30 cycles the reaction
temperature was maintained at 72°C for 4 min and than lowered to 4°C for further
storage. The quality of the resulting PCR products was controlled by electrophoresis
SIE\'1:-ELE\IE:\T PL\ST]])S
.~:\"D
RBCL DXI.\ L\ n:LLOZI.\C:E.\E
105
on an 1.5% agarose gel (Qualex Gold Agarose, Fl\IC BioProducts). Depending on
the strength of the band, l to 6 111 of the PCR product were used to pcrf(Jrm direct
cycle sequencing of the PCR products (Thermo Sequenasc cycle sequencing kit,
Amersham Lik Sciences). The follmving cy5 5' labelled sequencing primers were
used to obtain O\Trlapping sequences: Primer Leg7: 74-5 bp reverse:
5'TTCGCATGTACCCGCAGTAGCA3'; Primer Leg 3: 635 bp fl:mvard:
reverse:
4-19 bp
2:
Leg
Primer
5'TGCGTTGGAGAGACCGTTTC3';
1024- bp fcJrward:
5'ATTCGCA.AATCTTCCAGACG3'; Primer Leg 4-:
5'ACTTTAGGYTTTGTTGATTT3'. The cycle sequencing program used was
3 min at 94°C, followed by 25 cycles of 30 s at 94°C; 90s at 50°C. The st>quencing
products were loaded on longranger acrylamide gel (Fl\IC BioProducts) without
further purification and run on an ALF EXPRESS II sequencer (Amersham
Pharmacia Biotech) for automatic fluorescence detection of the nucleotide sequence.
Sequences wert> aligned using MICROSOFT \VORDPAD 4.00.950.
Data analysL1
The diameters of plastids were recorded in metaphloem sieve clements. The sizes
were measured from negatives taken at standard magnifications and a\·erages
calculated after critical evaluation of the set of plastids obtained f()f each species. A
total of some 5000 measurements formed the data set processed for species and
family averages.
Phylogenetic trees \vere reconstructed using character state and distance methods,
such as Maximum Parsimony (MP), ~eighborjoining (l'{J) and l\Iaximum Likelihood
(ML) as provided by the software package PAUP* 4-.0 (Swofford, 1998).
Divergence times between selected taxa vverc estimated using a p-distancc matrix
(Table 3). The splits between liverwort and vascular plants (jJwrhantia po£vmorpha
Xerophyta villosa), between ferns and seed plants (Angiopteris evecta- Xerop~yta l'illosa) and
between cycads and angiosperms :G)cas revoluta-Xerop~yta villosa), 4-40 million years
ago (Mya), 393 .Mya and 285 Mya (Savard et al., 1994), respectin~ly, were taken as
reference distances.
RESLTI'S
Sieve-element plastids
of Velloz_iaceae
The stem and leaf metaphloem of the species of Velloziaceae inwstigated is
composed of sien: tubes, companion cells and phloem-parenchyma cells (Figs 2A,
4A). Sieve elements are comparatively small (diameters of about 311m haw been
measured), closely associated with companion cells, and combine to iorm sicYe tubes
by compound sie\'e plates (Fig. 2A). During their ontogeny, sieve elements undergo
the same selective autophagy as described for many angiosperms (sec Behnke, 1989)
and comprising above all the degeneration of nuclei, breakdown of the tonoplast as
a delimitation between the vacuole and the cytoplasm, and the concomitant loss of
ribosomes, dictyosomes, microtubules and most of the endoplasmic reticulum. The
mature sieve clement retains its structural and functional identitY by the presence
19
20
21
IH
17
!()
10
II
12
13
14
15
li
(J.()(l.J.):i
1).(11102
0.009()1
IUJII41
Xnvpl~r!a dn~rlnimde.\
Xrwpl~rta dm_ylm.01dr1 \·ar. p('{tinata o.o 11 o:~
Talhotia rlrf!a/1\
0.01341
0.0157:l
0.01.172
0.012:i7
0.01:\13
().()1418
(J.(lllfl5
ll.OIIi83
0.01499
0.01540
ll.011i21
ll.IJ14:18
lfllo:;:ia hinu!a (21
0.01.)7.)
I fllo;:;ia andina
0.01971
.Vanuza plica/a
0.0181'!
O.Ol.H7
0.01.170
0.017(-)(:i
0.02079
0.016.)9
O.Olfi.Sl
!Ul!L):-5
11.00877
0.010.)(-j
IUJIIYl
o.oJ:)fN
11.01840
0.01689
0.(JI:l79
0.01712
11.111.1:\B
0.011181
O.OIH69
O.OIIi07
0.0(192-t
0.01.)..\-/
0.0137G
O.IJ1338
0.01300
0.01334
0.01.):13
0.01811
O.OIIili1
0.010!)6
0.00994
0.00912
11.01241
(J.())()(ifl
()J)()())j
0.00000
O.OIJ:l.13
0.00229
0.00:1.1:1
0.002()()
0.00.12H
O.OOHHO
0.007WI
0.0061.)
0.00979
O.OOB7q
0.00700
O.OI.i2:l
0.01321
0.01457
ll.ll1143
0.01338
0.01497
0.0175!
0.01.'1Wl
ll.OOBB2
ll.IJIIIi2
ll.OII48
O.IIOBBII
0.01799
O.OI51J:l
0.015{)()
O.IJI501
0.01435
O.Olli82
0.01854
ll.ll1777
o.OIOYJ
0.002().)
0.007qj
(J.Illl49
0.0092..\0.111.)47
0.01298
O.IJ1411
0.01378
0.01254
O.OH57
0.0171i4
0.01.177
ll.IJII4:l
0.01071
0.00990
(l.OI:\17
().()1144
O.OO(JWI
O.Olfi97
IJ.01841i
0.01827
0.0Jj72
(Ul17B9
0.01.):)()
0.014'18
0.111304
0.0077:~
(l.\)J
lb7
ll.IJI070
0.009~1()
0.008:\H
0.00/(:i()
ll.ll2228
0.01316
0.01.)49
0.0L)21
0.01 :lB2
IJ.Ol:llfl
0,02059
0.02034
().(11783
0.02032
o.o2n2
0.02370
0.0231()
0.011i19
0.01H78
O.ll201i8
0.02222
0.021.50
0.01 ~15()
IUJI44H
0.01{)2.)
().()1411
0.01229
O.ll211ill
O.IJ191JH
11.01489
O.llll :l4
0.01252
O.fJIS:l2
0.017.18
0.01491
0.012~)(_)
0.0077()
11.00)l2
O.OOG~I 1
0.01!/18
Ill
0.00312
(U HJ~ I:1 0
O.OIHB8
O.IJI708
0.01731
0.012:H
0.0\()()3
0.01NJ
0.1)1790
0.01763
II
1).(11703
0.01451
0.014'10
ll.ll11:l7
0.014B'l
0.01.):1.)
1Hlllil2
0.01661
0.0076~)
12
O.OJ:liO
O.OI:m'l
0.01410
0.01407
O.IJ1154
0.01334
0.010.1.)
().()117'1
13
O.IJ05·10
0.00000
0.00().)9
0.00.1.)()
0.00078
0.01011i
0.00676
14
0.0007'1
o.umm
0.00765
0.00495
0.00764
O.O(J..\-99
O.OO.)til
0.00459
j()
0.0040.)
0.00000
0.00349
]j
0.00411
0.00566
0.00726
li.IJ0742
17
0.00473
0.00705
IJ.IJOSOO
lfl
O.IJ0768
O.OOG63
]q
3. Pair-wise genetic distances between the V elloziaceac taxa studied. Distances are given as p-distanccs (1.0 = l 00% nucleotide substitutions)
Ba!ha(flliojJ\i,\
Bmha(flliojJJi\ ho!itiem/,\
XnojJI{rlaeglmululu\a
.\'nopkrta p111_i[ulia
TABLE
0.00908
211
;--
~
'-1
tl'J
t"l
z
;;::
::r::
~
I:C
~
::r::
a;
SIE\ E-ELL\IE'\T PL\STIDS A.'\D RBCL IHT.\ I'\ n:LI.OZI.\CI: \I:
107
of a plasmalemma which delimits the cell against the apoplast and is continuous
through sieve pores and plasmodesmata. Plastids, mitochondria. and some endoplasmatic reticulum arc still present and occupy a parietal position (Fig. 2A). The
characteristic features of the sieve-element plastids are found at an early stage in
the maturation of a sieve element (Fig. 2A, arrowheads) and continue to be present
during the entire life time of the siew clement.
As typical of angiosperms and found in many (but not all) monocotyledons, the
sieve elements of the Velloziaceae contain P(hloem)-protein that is seen as distinct
filaments (e.g. Figs 3E, 5B, 6A,C) and may occlude the sien~ pores (Fig. 2C). Pprotein is synthesized in the cytoplasm ofyoung, nucleate phloem cells (see Behnke,
1989; Evert, 1990), has well-known biochemical properties (Cronshaw & Sabnis,
1990) and a function in sealing the sieve-plate pores of injured sieve tubes. P-protein
must not be mixed up vvith the filaments defining form-Pf, -Pcf, -Pds, and -Pf~
sieve-element plastids (Behnke, 1991) and present in form-P2cf plastids of some
Velloziaceae. The origin (i.e. coded by nuclear DNA, by plastid DNA, or by both),
biochemical composition and function of 'plastid filaments' arc just as unknown as
are those of protein crystals found in sieve-element plastids. Howen.T, both first
appear in young plastids and are never released as long as the sieve element is alive
(see Behnke, 1989, 1991 ).
In Velloziaceae (37 taxa investigated) sieve-element plastids belong to either formP2c or form-P2cf, i.e. to only two of the four forms present in monocotyledons
(compare Figs 1 and 12). However, most of them are distinctive by ( 1) the presence
of crystals with indistinctly angular faces which replace the monocot-specific cuneate
ones, (2) the inclusion of a single loosely-packed polygonal crystal or several looselypacked crystals of different morphologies, and (3) the very small average' diameters
of sieve-element plastids recorded in both stem and leaf phloem (seC' Table I).
Although one or other of these features may occasionally he present in other
monocotyledons, their combination is unique and accentuated here as the Velloziaceae (sieve-element plastid) syndrome. The presence of angular crystals is used
to redefine the sieve-element plastids of V clloziaccae as belonging to subforms P2c" "
P2cah P2c"1" P2cJ, P2c,,/ (Fig. 12, but see previous nomenclature' in Behnke, 2000)
and thereby distinguish them from others containing cuneate crystals, e.g. subformP2cr~ as in Ayensua (Fig. 10 A,C) and other taxa (see Table 1 and Behnke, 2000).
The six species of Barbacenia and Pleurostima investigated contain form-P2c plastids
with many angular, sometimes roundish crystals and several loosely-packed crystals
of which one is comparatively large. However, while most specimens have subformP2ca1 plastids (Fig. 2C,E,F; Table I), Pleurostima stenopkylla has subform-P2c,,1, (Fig.
2B) and in sieve-clement plastids of Barbacenia ( = "-!J;lthonia) tomentosa neither polygonal
nor other loosely-packed crystals were found (subform-P2c" ,; Fig. 2D).
Form-P2c and form-P2cfsieve-clement plastids are present within ~'ello;:.ia: suhformP2catf in V. crassicaulis (Fig. 3A), subform-P2ca/ in V glodzidea and T~ hirsuta (2),
subform-P2ca1 in V. variabili.s (Fig. 3B) and V. variegata (Fig. 3C,F), and suhform-P2caf>
in V. aff. caespitosa (Fig. 3D), V. declinans (Fig. 3G), V Jroesii, T~ sellrneiz, and T~ tubiflom
(Fig. 3E). The loosely-packed crystals in subform-P2ca 1 plastids are often rod-shaped
or orthogonal (Fig. 3B,C,F). V. hirsuta (I) and the new Bolivian T~ andina (Fig. 3H,I;
see Ibisch et al., in press) have subform-P2c", plastids. The two different forms of
sieve-element plastids present in V hirsuta Goethart & Hem·arcl were found in
specimens that also differed morphologically and add to anatomical and other
characters usC'cl to define three types in this species (see t\lello-Sih·a. 1990).
108
H.-D. BEHNKE ET AL.
SIE\'E-EIE\IE'\T
PI~~STIDS
.\:'\D RBCL DAT.\ !'\ n:LI.UZL\CL.\E
\09
The genus Xeroph)'ta likewise includes all five subforms found in the family. The
African X retineruis (Fig. -1-B) and X villosa (Fig. 4C) contain subform-P2cJ plastids,
the Madagascan X dasylirioides var. pertinata (Fig. 4F), X. eglandulosa (Fig. 4D,E), and
X pinifolia subform- P2cal plastids. However, in the sample investigated of X.
dasylirioides (variety unknown) no filaments could be detected (see Table l). Amongst
the other species, X. schlechteri (Fig. 4H) and X splendens (Fig. 4I) have subform-P2cap
plastids. No loosely-packed crystals were found in X. humilis (subform-P2a,; Fig. 4G).
Sieve-element plastids of X. retineruis (Fig. 4B), X. dasylirioides var. pectinata (Fig. 4F)
and X. splendens (Fig. 4I) contain some tubular structures in addition to crystals (and
filaments). Loosely-packed crystals are rod-shaped/ orthogonal in X. schlechteri (Fig.
4H).
The sieve-element plastids of Barbaceniopsis (three species investigated), including
B. castillonii, sec Ibisch et al., in press) are of subform-P2c"'' but their specific characters
are rather extraordinary. In all samples there are only one or two irregularly shaped
loosely-packed crystals (Fig. 5C-F), and in some plastids it is hard to find them (Fig.
5B). The other (densely-packed) crystals are angular, but in B. vargasiana there is
often only one angular, rather polygonal (Fig. 5A,D,E) or irregular crystal which is
sometimes divided into smaller ones (Fig. 5F).
The monotypic genera Nanu;:,a (Fig. 6C,D) and Talbotia (Fig. 6A,B) have subformP2ca , sieve-element plastids with angular and cuneate crystals. Material of Burlemarxia
species was not available.
Siez'e-element plastids of Acanthochlam_vdaceae
The phloem of the monotypic Acanthoclzlam)'S bracteata (stem, root and leaves were
investigated) is composed of small sieve elements combined to sieve tubes by simple
sieve plates, companion cells and phloem parenchyma cells. Mature sieve elements
contain plastids, mitochondria and P-protein (Fig. 7A-C) and are connected to their
companion cells by sieve pore-plasmodesmata combinations (Fig. 7D). Sieve-element
plastids are among the tiniest in monocots and belong to form-P2c (Fig. 7B-D).
There are many small crystals in a plastid, with the larger ones being distinctly
cuneate. Additional loosely-packed crystals as present in most Velloziaceae were not
found.
Figure 2. Velloziaceac. A, longitudinal ultrathin section through stem metaphloem of Pleurostlma purpurea
showing sieve elements (sc), companion cells (cc), and phloem-parenchyma cells ipc). The lower siew·
element is at an early stage of differentiation and contains cytoplasm with young sicn·-elcment plastids
(arrowheads) and a nucleus (n), while in the upper sieve element the nucleus and vacuole are
degenerated, its cytoplasm with many plastids is delimited to a small wall-adjacent laver, and the
remaining lumen filled with P(hloem)-protein (*1. The upper sin·c element is connected to an immature
one by a sieve plate (between arrows). B, subform-P2c,P plastids with angular raJ and loosely-packed
polygonal (p) crystals in sie,·e clements of Pleurostlma stenoph)'lla; sieve plate between arrows. C, sieve
element of Pleuras lima fanniei with loosely-packed (I) and angular 1a) crystals of broken plastid and with
P-protein (*) partlv occluding sin·C"·platc pores (arrows). D, subform-P2c.,, plastids of Barbawzia tomentosa.
E, F, subform-P2c,1 plastids of Pleurostima jJurpurea (E) and Barbacenia grisea (Fl. Scale bars= l 1.1m.
110
H.-D. BEHNKE ET AL.
Figure 3. Velloziaceae. Sieve-element plastids of ~llo.:;ia: subform-P2cJ plastids in ~llo.:;ia crassicaulis
(A), subform-P2c,1 with rod-shaped loosely-packed crystals in V variabilis (B) and V variegata (C, F),
subform-P2c,P in V aff. caespitosa (D), V tubijlora (E) and V declinans (G). ~llo.:;ia andina has subformP2c,_, plastids with almost cuneate crystals (H, I). a = angular, c =cuneate crystals, f = protein filaments,
I = loosely-packed, p =polygonal, * = P-protein. Scale bar in H = l J.lm. All to same scale.
Sieve-element plastids qf.families traditional!J allied to Vello;::iaceae
The sieve-element plastids of those families long time considered related to the
Velloziaceae also belong to either form-P2c or form-P2cf (i.e. no starch-containing
P2-forms were recorded), but their average diameters are considerably larger than
in Velloziaceae (see Table I and Figs 8- l 0).
SIEH>ELEI'dE:\T PL\ST!DS
~~1\;D
RBC1, DAT\ l:\
\'ELLOZL~CE~\E
Ill
Amaryllidaceae: 29 species investigated, all with form-P2c plastids, except Rauhia
(Fig. SE,F) and Vtonl~ya (Fig. SB) which contain form-P2cf. The sieve-element plastids
of several species contain one to three orthogonal crystals in addition to many
cuneate ones, thus establishing subform-P2c,0 (Fig. SA,C,D,G,J) and subform-P2cJ
(Fig. SE). In ultrathin sections these orthogonal crystals may have rectangular
(including quadrangular), trapezoidal or other outlines (Fig. SC). Like cuneate crystals
they are composed of densely-packed subunits. The average diameter of sieveelement plastids in both stem and leaf phloem is considerably higher than in
Velloziaceae (see Table 1).
Hypoxidaceae: 4 species investigated, all with form-P2c sieve-element plastids
with only cuneate crystals (Fig. 9A). Plastid diameters are similar to those of
Amaryllidaceae.
Haemodoraceae: 7 species investigated. Sieve-element plastids all of form-P2c
(Fig. 9B) but with considerably larger diameter than previous families (Table 1).
Bromeliaceae: 22 species investigated, all with form-P2c plastids, but with subformP2cr~ in several species. The average diameter of their sieve-element plastids is larger
than in Velloziaceae, but among all putatively related families the size of their leaf
phloem sieve-element plastids are the closest to those of Velloziaceae (see Table 1).
Judged by their ultrastructure subform-P2cr~ plastids of .l~yen.sua and Brocchinia (Fig.
lOA,C-D) arc similar to those found in Velloziaceae, but their clear cuneate
crystals do not conform. However, subform-P2c, 1 plastids are also present in some
Laxmanniaceae (formerly Lomandraceae) and different families of Poales (Behnke
2000).
Sieve-element pla.stid.s qf Pandanales sensu Kubit::.ki, Rudall & Chase (1998)
The distinctive sieve-element plastids found in V elloziaceae do not match the
plastids in any of the other families placed in the Pandanales.
Pandanaceae: 41 species from the three genera investigated, almost all with formP2cf plastids. The specific features include very large plastid diameter, a prominent
peripheral ring of protein filaments, and many cuneate crystals (Fig. lll--L, Table
l ).
Cyclanthaceae: l 0 species investigated, all with form- P2c plastids of rather large
diameter (Fig. ll E-H, Table l ). Except for the absence of protein filaments their
characters come close to those of the sieve-element plastids in Pandanaceae.
Pentastemonaceae: the monotypic Pentastemona egregia contains form-P2c sieveelement plastids with many medium-sized cuneate crystals (Fig. ll D); the average
plastid diameter is slightly smaller than in Stemonaceae (Table l ).
Stemonaceae: four species investigated, all with form-P2c sieve-element plastids
of considerably smaller diameter than in Cyclanthaceae (Fig. llA--C, Table l ).
Phylogeny reconstructions based on nucleotide sequences qf the rbcL gene
The phylogenetic analysis focussed on 24 taxa ofVelloziaceac (seven genera) and
10 taxa of related families (Acanthochlamydaceae, Stemonaceae, Pandanaceae,
and Cyclanthaceae) which had already been identified as a monophyletic group
(Pandanales, Chase et al., 1993). Other monocot families, sometimes considered
11 2
H .-D. BEHNKE ET AL.
S IEH>E LE:\I E'\T PL\STIDS A:'-JD RBCL Di\1'.-\ I'\ \ 'ELLOZ I.-\ CL\ E
113
Figure 5. Velloziaceac, sieve-element plastids of Barbaceniopsis. A, longitud inal ultrathin section th rough
sieve element (se) of B. l'argasiana with three plastids. B, two sie,·e clements in stem of B. castillonii
connected by a sieve p late (between arrows) and containing plastids and P-protein (*). C , subformP2c,, plastids of B. holi1•iensis. D , subfonn -P2c,, plastid from leaf vein of B . castillonii. E-F, various aspects
of subform-P2c., p lastids in B. vargasiana. a = densely-packed angular crysta ls, I =loosely-packed crystals.
Scale bars = I 11m .
Figure 4. Vclloziaccac. A, longitudinal ultrathin section through stem melaphloem of Xerophyta retinervis
with several small sieve elements (se), phloem-pa renchyma cells (pc), and la rger xylem clements (x).
B- 1, sieve-element plastids of leaf (B) and stem (C - I) of Xeropf!Jta: subform-P2cJ in X 1~tinervis (B) and
X villosa (C), sublc>rm-P2c,/ in (D , E), X eglandulosa and X dasy lirioide.1 var. pectinata (F), subtorm-P2c, ,
in X humilis (G) and sublorm-P2c.p in X. schlechteri (H) and X 1plendens (I). a = angular crystals, c =
cuneate crystals, f = protein filamems, p = loosely-packed polygonal crystal, t = tubular inclusions. Scale
bars: A = 5 ~-!m; B, C . F = 1 1-!m. D -I to same scale as B.
114
H.-D. BEHNKE ETAL.
Figure 6. Velloziaceae, subform-P2c,, sieve-element plastids in Talbotia elegans (A, B) and .Nanuza plicata
(C, D) with many angular (a) and often cuneate (c) crystals. * = P-protein. Scale bars= l J.lm.
related, were also included in our analysis, and Acarus was chosen as an outgroup.
Distance and character state methods (MP, NJ, ML) produced trees which were
almost congruent with each other in the arrangement of Pandanales (Figs 13-16)
which always cluster as a monophyletic group (85% bootstrap support). Acanthochlamys
is a sister group to Velloziaceae (100% bootstrap). Pandanaceae and Cyclanthaceae
also form a well-supported sister group (94% bootstrap) which apparently shares
ancestry with Stemonaceae. Amaryllidaceae, Bromeliaceae, Haemodoraceae and
Hypoxidaceae, the families that have previously been treated as close allies of
Velloziaceae, are members of different clades, neither closely related to each other
nor to the clade including Velloziaceae. However, their phylogenetic position could
not be established with certainty, as NJ bootstrap (Fig. 13) and MP strict consensus
(Fig. 14) trees do not resolve the underlying bifurcations indicated by the ML
phylogeny (Fig. 15). A more complete sampling would be necessary to resolve their
interfamilial relationships, which was outside the scope of this study.
Within Velloziaceae, at least four evolutionary lines can be discovered by MP,
l'{J, and ML reconstructions which mostly correspond to the major genera:
(1) African and Madagascan species of Xerophyta and the mono typic Talbotia embedded
within Xeropfryta, (2) Barbaceniopsis, (3) Pleurostima and Barbacenia, and (4) Vello:;;ia plus
the monotypic Nanu:;;a. Species of lines 2, 3, and 4 are restricted to South America
(Fig. 14). Genetic distances (Table 3, Fig. 15) between these taxa imply that Old
World and New World members of the Velloziaceae diverged 40-70 Mya (if we
SIE\'E-ELEC\!E'\T PIASTIDS AND RBCL DAT\ I'\ \'ELLOZIACE.'\E
115
equate 0.0389% nucleotide substitutions or 0.0125% non-synonymous nucleotide
substitutions with 1 million years of divergence).
DISCliSSION
Subdivision and relationships within Vello;::iaceae
The infra-familial classification of V elloziaceae is not yet settled. During the last
decade the discussion has concentrated on two proposals that differ not only in size
and composition of subfamilies, but also in circumscription of genera (see MelloSilva, 1991 ).
One of the major disputes is on the treatment of Old World against New
World taxa. Based on entirely different sets of characters, Smith & Ayensu (1976)
incorporated all Old World species, except the monotypic Talbotia, into the genus
Xerophyta, while Menezes (1980) also recognized Xerophyta species in Brazil.
Based mainly on stigma shape and leaf blade anatomy Smith & Ayensu (1976)
assigned the New World species to Barbaceniopsis (with 3 species), their new monotypic
genus Nanuza ( = Vello:::.ia plicata Mart.) and the two major genera Barbacenia and
Vello:::.ia. Menezes (1980) used stamen and anther characters for the delimitation of
genera. She included Barbaceniopsis, Nanu:::.a and Talbotia within Xerophyta and separated
three new genera, Pleurostima Raf., Aylthonia Menezes, and Burlemarxia Menezes, from
Barbacenia. The rbcL tree separates the family into clades which correspond neither
to the subdivisions proposed by Smith & Ayensu (1976) nor to those of Menezes
(1980).
While the distinct sieve-element plastid syndrome is apomorphic to the family,
the distribution of the subforms recognized (Fig. 12) does not reflect any specific
infra-familial classification: all five subforms are found among Vello:::.ia and Xerophyta
species, three in Barbacenial Pleurostirna. The investigated species of Barbaceniopsis have
subform-P2ca1 plastids, and the monotypic Nanu:::.a and Talbotia contain subformP2ca-r· An assessment of the significance of the five subforms to the distinction and
relationship of the taxa studied would require a scoring of the different plastid
contents and their cladistic analysis, which together with other morphological
characters will be carried out separately.
Affinities with other rnonocoryledonous families
Both ultrastructural and molecular analyses substantiate Velloziaceae as a monophyletic family. The sieve-element plastids of Velloziaceae are distinct from those
of all other families by the combination of angular crystals, additional loosely-packed
crystals and comparatively small average diameters. This syndrome is to be added
to the synapomorphies recognized by Menezes et aL (1994), i.e. presence of transfusion
tracheids in the leaf vascular bundles, a reduced inflorescence, and an independent
dehiscence of all four anther locules. · Loosely-packed crystals are present in some
Bromeliaceae, a family that has previously been considered to be closely related, as
well as in some Laxmanniaceae (formerly Lomandraceae) and Poales (Behnke, 2000),
but all these have definite cuneate crystals. The results from our rbcL study, i.e. the
11 6
H.-D. BEHNKE ET AL.
SIE\'E-ELEl\IE:\'T PL\STIDS .-\.c\ID RBCL DAT.\ II'\ \'ELLOZL\CE.\E
117
comparatively great number of character changes and the 100% support in the
bootstrap analysis (Figs 13, 15), put the monophyly of the Velloziaceac as first
established by Chase et al. (1995) and their most likely early isolation (see Kubitzki,
1998) on a broader basis.
Acanthochlamys was raised to family rank by Kao (1989; see also Kao & Kubitzki,
1998) but included in Velloziaceae by Chase et al. (1995) and the Angiosperm
Phylogeny Group (APG, 1998). Our combined results, i.e. small form-P2c plastids
without loosely-packed crystals (Fig. 7), clear distance in Neighbor Joining and strong
bootstrap support (Fig. 13), favour a monotypic family whose closest affinities are
with Velloziaceae, and with no close affinities to Amaryllidaceae where Acanthochlamys
was earlier included (see Kao & Kubitzki, 1998).
Phylogeography
According to our preliminary estimate based on the split between J;farchantia
(Lewis, Mishler & Vilgalys, 1997) and the vascular plants 440 M ya and the split
between cycads and angiosperms 285 Mya (Savard et al., 1994), and using the
greatest distance among the investigated species, New and Old \'Vorld members of
Velloziaceae diverged about 40--70 Mya, ruling out that the disjunction seen today
is a result of plate tectonics. Instead, we have to assume either migration or more
likely, long-distance dispersal either by hurricanes or the sea. Since among the nther
members of the Pandanales there are likewise Old and New \Vorld groups, a clear
decision on where the Velloziaceae originated cannot be made at present. Based
on morphological and chromosome-cytological characters Menezes ( 1980) and Melo
et al. (1997) suggest South America as the centre of origin of the family, while Ayensu
(1973) favoured Madagascar as the origin of dispersal. However, our DNA studies
show that the Xerophyta species from Madagascar (eglandulosa, pinijolia, dasylirioides) arc
separated by up to 1.3% nucleotide substitutions from taxa that occur on the African
mainland, indicating a divergence about 10 to maximally 25 Mya, long after the
isolation of Madagascar, and long after the separation of the African from the South
American species.
Taxonomic implications
The monotypic genus Talbotia from South Africa (mainly Natal to eastern Transvaal) clusters within a clade that is composed of members of the genus Xerophyta,
making this genus paraphyletic (Figs 14, 15 ). It would therefore be plausible to
merge Talbotia with Xerophyta, reinstate Xerophyta elegans (Balf.) Baker (Baker, 1875),
Figure 7. Acanthochlamydaccae, longitudinal ultrathin sections through stem (A, B, D) and transverse
section through root (C) phloem of Aranthochlamy; bracteata. A, sicw· element (se) with plastid (p) and Pprotein (*), adjacent to phlocm-parmchyma cell (pc) with starch-containing plastid (s). B D, form-P2c
plastids in sieve elements containing P-protein (*) and connected to companion cells (cc) by sieve
pore-plasmodesmata combinations (arrowheads). D is serial section to B; c =cuneate crystals, m =
mitochondria. Scale bars= l f.!m.
11 8
H .-D . BEHNKE ET AL.
Sllc\'E-ELE'\IE:'\T PL\STIDS ,\ND RBCL OAT.\ 1:'\ \"El .LOZI.\CL\E
I 19
Figure 9. Form-P2c sieve-element plastids with cuneate crystals. A, Hypoxis villo.w (Hypoxidaceae). B,
lAchnanthe.1 caroliniana (H ae modo race ae). Scale bars= l ~m.
and thus support a treatment that has been proposed already by ~lenezes ( 1980).
Actually, genetic distances between the two genera a re very small (see Table 3).
Nanu;;.a, another monotypic genus, clusters within a strongly supported assemblage
(Figs 14, 15), making the genus Vellozia paraphyletic. Again because of small distances
to the different Vello<.ia species (Table 3), we suggest merging }lanuz:.a with Vello<.ia.
Smith & Ayensu (1976) raised Vello<.ia plicata Mart. to the monotypic Nanu<:,a and
dedicated it to Nanuza L. de Menezes who prefers to treat this species as Xeropfryta
plicata (Mart.) Sprengel (M enezes, 1980). However, Mello-Silva ( 1991 ) on the basis
of stigma and pollen charac ters, opposed its inclusion in either H:ffo.;:ia or Xempfryta.
Barbacenia grisea is closely related to Pleurostima riparia and both are nested within
other Pleurostima species, making the genus paraphyletic. Pleurostima was included
within Barbacenia by Smith & Ayensu ( 1976), but treated as a separate genus by
M enezes (1980). P riparia has been recombined by M ello-Silva (1994) as Barbacenia
riparia. As both genera contain many species (90 or 20, respectively; Kubitzki , 1998),
more species should be studied to corroborate this finding .
Character evolution in Pandanales
W e have taken the phylogenetic trees as a framework to analyse the distribution
of forms of sieve-element plastids. Figure 16 shows a cladogram onto w·hich the
plastid forms have been m apped.
With respect to V elloziaceae, it is evident that (1 ) the so-called Velloziaccae
syndrome (angular crystals together with loosely-packed crystals of any number and
Figure 8. Sieve-element plastids of th e Amaryllidaceae. A, ultra thin sectio n or two siC\T elements or
Eucharis x grandiflora with three subrorm-P2c.., plastids. B, rorm-P2cf plastid of H insl~ra l"i!l'll eri. C, part
or a sieve element of H;·mmocalhf tubiflora with many intac t sie\T-elemcnt plastids and the crys tals of
broken plastids. Orthogonal crystals (o) have difterent shapes. D- .\I, subrorm-P2c,, plastids a re prcscm
in Eucrasia aurantiara (D ), 1-{ymmocallis tubijiora (G) and Galanthus nivalis (J), form-P2c in Pancratium
maritimum (H), Scadoxus multijlorus (I), N erine samiensis (K), Calos/emma purpureum (L) and Boophone disticha
(M), and subform-P2c,,f in Rauhia peruviana (E,F). c =cuneate crystals, r =protein filaments. Scale ba rs
in A and M =I ~m. B- M to sa m e scale.
120
H.-D. BEHNKE ET AL.
Figure 10. Sieve-element plastids of the Bromcliaceae. A-D, subform-P2cd in Ayensua uaipanensis (A, C),
Navia phelpsiae (B), and Brocchinia rnicrantha (D). c =cuneate crystals, I= loosely-packed crystals. Scale
bars= 1 f.tm.
shape and small plastid diameter) figures as a synapomorphy, (2) the addition of
protein filaments, and thus transformation into P2catf- or P2capf-subforms, is a
synapomorphic character to subgroups of both Xerophyta and vellozia, and (3) other
subgroups within the vellozia and Xeroplryta clades (Nanuza, Talbotia, vellozia andina;
see also Table l) have lost the loosely-packed crystals, as has Acanthochlamydaceae,
the sister group of Velloziaceae. The cladogram (Fig. 16) also suggests that in
Velloziaceae the distinction between subform-P2ca1 and -P2cap as well as between
-P2catf -and P2cal sieve-element plastids is only a gradual one and that the transitions
from/to -P2ca1 to/from -P2cap' respectively -P2catf and -P2capf, are gradual. Furthermore, it supports views proposed earlier (Behnke, 2000) that in subtype-P2 sieveelement plastids the addition of protein filaments, defining the P2cf-(sub)forms,
occurred comparatively late.
Within the Pandanales clade, form-P2cf sieve-element plastids (not identical to
the subform-P2ca11/ found in Velloziaceae) are a synapomorphy of Pandanaceae,
Figure II. Sieve-element plastids of Cyclanthaceae (E-H), Pandanaceae (I-L), Pentastemonaceae (D),
and Stemonaceae (A-C). Form-P2c plastids with only cuneate crystals in Stichoneuron caudaturn (A),
Croornia paucijiora (B), Sternona tubijlora (C), Pentasternona egregia (D), Dicranopygiurn sp. (E), Evodianthusfunifer
(F), G)clanthus bipartitus (G), Asplundia spectabilis (H), Sararanga philippinensis (J) and form-P2cf plastids in
Pandanus irregularis (I), Pandanus vandermeeschii (K) and Freycinetia luzonensis (L). f =protein filaments. Scale
bar in E = I flm. All to same scale.
SIE\-E-EI.E:'IIE'\T PL\STII)S .\'\D RBC1. DXL\ 1'\ \TLLO Z I.\CE.\E
D
E
12 1
122
H.-D. BEHNKE ET AL.
Figure 12. The forms and subforms of sieve-element plastids in Velloziaceae. The five subforms
containing angular crystals are a synapomorphy of Velloziaceae and may be superimposed on Figure
I (to the left of subforms P2c,p and P2cc~), but their connection to the other subforms is not yet settled.
while all other taxa contain form-P2c plastids. The presence of subform-P2cci sieveelement plastids in Ayensua and other Bromeliaceae as well as in some Poales (Behnke,
2000) appears as a convergent trait.
ACKNOWLEDGEMENTS
Wilhelm Barthlott initially directed the senior author's attention to the Velloziaceae. We thank him for permitting us to use the splendid Velloziaceae collection
at the Botanical Garden of the University ofBonn and Werner Holler for maintaining
this collection in an excellent condition. We are also grateful to the following for
giving help to our investigations: David E. Bouffard (Herbaria of Harvard University,
Cambridge MA, U.S.A.), Linda G. Chafin (Tallahassee FL, U.S.A.), Pierre L. Ibisch
(Science Dept., F.A.N., Santa Cruz, BOL), Bruce A. Sorrie (Southern Pines NC,
U.S.A.) and Abraham E. van Wyk (Dept. of Botany, University of Pretoria, RSA)
collected live samples in the field. Roselle Andrews (Royal Botanic Gardens, Kew),
Josef Bogner (Botanischer Garten, Universitat Mtinchen, D), Inge Dorr (Botanisches
Institut, Universitat Kiel, D), Ulrich Hecker (Botanischer Garten, Universitat Mainz,
D), Beat Leuenberger (Botanischer Garten, Berlin-Dahlem, D), Wolfram Lobin
(Botanischer Garten, Universitat Bonn, D), John Main (Royal Botanic Garden,
Edinburgh), Elmar Robbrecht (National Botanic Garden, Meise, B), Antonio Salatino
(Inst. Biociencias, Universidade de Sao Paulo, BR) and Hilke Steinecke (Palmengarten, Frankfurt, D) provided samples cultivated in their institutions. Mark W.
Chase (Royal Botanic Gardens, Kew) and Melvin R. Duvall (Northern Illinois
University DeKalb IL, U.S.A.) furnished rbcL sequences of Acanthochlamys, Acarus
and Stemona. Steffi Gold and Brigitte Moraw gave expert help with fixations,
ultramicrotome and photographic work. Early support for this study was provided
by the Deutsche Forschungsgemeinschaft.
SIE\'E-ELE\IE:\T PL\STIDS .\:\"[) RBC1, DAT.\ 1:\ n:LLOZIACE.\L
123
Neighbor Joining
Bootstrap
Hypoxis glabella
r - - - - - Fostere/18 cau/escens
-
Hypoxidaceae
1 - - - - - - Pepinia coral/ina
f - - - - - - Navia phe/psiae
Nidularium innocentii
Canistrum fosterianum
Billbergia reichardii
'------ Neorege/ia olens
Broccllinia micrantha
Ayensua uaipanensis
Vriesea f!ieroglyf?hica
Guzmama zahnu
Prionium serratum
Typha latifolia
f----------------------Lachnan~escarolin~na
. - - - - - - Cyc/anthus bipartitus
Asp/undia spectabilis
Dicranopyg1um p<}lycepha/um
Evodianthus funifer
'------- Carludovica P!Jimata
Pandanus dubius
Freycinetia luzonensis
Stichoneuron caudatum
Stemona japonica
r - - - - - - - - - - Xerophyta schlechteri
Barbaceniopsis vaiJiasiana
Barbaceniopsis bolwiensis
Xerophyt_a eglandulosa
Xerophyta pmifolia
f----~-----l-- XerophY.fa dasylirioides
X dasylirioides var. pectinata
Talbot1a elegans
Xerophyta villosa
XerophyJa retinervis
Pleurostima stenophylla
Pleurostima riparia
Barbacenia grisea
f - - - - - - - - - P/eurostima purpurea
. - - - - - - Vellozia froesii
Vel/ozia glochidea 1
Vellozia glochidea 2
Ve/lozia crassicaulis
'------- Vellozia hirsuta 2
Vellozia sp. 2
Vellozia declinans
1--------- Vellozia andina
'----------- Nanuza p/icata
'------------- Vellozia tubiflora
'--------------------- Acanthoch/amys bracteata
100
Pleea tenuifo/1a
Tofieldia pusilla
Amaryllis belladonna
94
Bowiea vo/ubilis
Kniphofia uvaria
77
Alisma plantago-aquatica
Potamogeton amplifolius
' - - - - - - - - - - - - - - - - - - - Acorus calamus
Bromeliaceae
-
Prioniaceae
Typhaceae
Haemodoraceae
J
Pandanaceae
]
Stemonaceae
JCyol~th.oo~
Velloziaceae
-
J
-
Acanthochlamydaceae
Tofieldiaceae
Amaryllidaceae
Hyacinthaceae
Asphodelaceae
Ahsmataceae
Potamogetonaceae
Outgroup
Figure 13. Molecular phylo~eny and systematics of the Pandanales inferred from nucleotide sequences
of the rbcL gene. The data set was analysed by the J\'eighbor Joinin~ distance method using Kimura
2- parameter as a distance algorithm. The bootstrap cladogram (on 1000 replications) shows a 50%
consensus tree which treats all bifurcations with a bootstrap value lower than 50% as unrcsoked.
124
H.-D. BEHNKE ET AL.
Maximum Parsimony
DISTRIBUTION
Strict
Australia, New Zealand
New World
South Africa
Northern temperate
North America
Tropical
Asia
South Africa/
Madagascar*
South America
China
J
South Africa
J
holarctic
Figure 14. Molecular phylogeny and geographic origin of the Pandanales. Analysis with the Maximum
Parsimony character state method using a heuristic search. The analysis was terminated when I 000
equally parsimonious trees were found. Results are documented as s strict consensus cladogram. Tree
length 956 steps, CI=0.538, Rl=0.777, Hl=0.462.
SIE\ E-ELEl\IE:'>T PL\ST!DS ,\0/D RBCL DAT.\ I'> \'ELLOZIACE.\E
125
Maximum Likelihood
. - - - - - - Hyp_oxis g_labella
Amaryllis belladonna
._______ Bowiea volubilis
L-------- Kniphofia uvaria
Fosterella caulescens
Pepinia coral/ina
Nidu/arium innocentii
Canistrum fosterianum
Billbergia reichardii
Neoregelia olens
Brocchinia micrantha
Ayensua uaipanensis
Vriesea f)ieroglyphica
Guzmama zahn11
Navia phelpsiae
.....---....:.....___;;,___ Prionium serratum
' - - - - - - - Typha latifolia
Lachnanthes caro/iniana
Cyclanthus biparlitus
Asplund1a spectabilis
Dicranopyg1um polycephalum
Carludovica {)t!lmata
Evodianthus funifer
Pandanus dubius
Freycinetia luzonensis
Stichoneuron caudatum
Stemona jap_onica
Xerophyta schlechteri
Xerophyta eulandu/osa
Xerophvta pmifolia
Xerophyta dasylirioides
Xerophyta dasylirioides var. pectinata
Talbotia elegans
XerophY!a vi/losa
Xeroph)'ta retinervis
Barbaceniopsis vargasiana
Barbaceniopsis boliviensis
Pleurostima stenophy/la
Pleurostima riparia
Barbacenia gnsea
P/eurostima purpurea
Vellozia froesii
Velfozia glochidea 1
Vellozia crassicaulis
Vellozia glochidea 2
Ve/lozia nirsuta 2
Ve/lozia sp. 2
Ve/lozia declinans
Nanuza plicata
Ve/lozia andina
Vellozia tubiflora
.___ _ Acanthochlamys bracteata
Pleea tenuifolia
' - - - - - Tofieldia pusilla
L---{=====~===~A~/isma plantago-aquatics
Potamogeton amplifolius
.___ _ _ _ _ _ Acorus calamus
L---[==:
-
0.005 substitutions/site
Figure 15. Maximum Likelihood phylogram of the Pandanales. A substitution rate of2 and a transition/
transversion ratio of2 were assumed. Base abundance: A=0.272, C=0.196, G=0.245, 1'=0.287_ A
single tree was obtained with a score of 7599. Branch lengths are proportional to genetic distances
and thus divergence times.
H.-D. BEHNKE ET AL.
1'26
Maximum Parsimony
Strict
~• • • • • • • • •
•
*
loss of loosely-packed
Hypoxis glabella
Fostere/18 caulescens
Pepinia coral/ina
Nidularium innocentii
Canistrum fosterianum
•••• subform-P2cc/ plastids
Billbergia reichardii
Neorege/ia olens
. E
•• • •
: :
•
t
Velloziaceae syndrome
(angular crystals,
loosely-packed crystals,
small plastid diameter)
• •• • • • ••: • • • Broccflinia micrantha
• • • • Ayensua uaipanensis
:
crystals and of filaments
~~~S,::n'/~e::JI,~fhica
:
+ addition of filaments
: • • • • • • • • • • • · Navia phelpsiae
Prionium serratum
Typha latifolia
t - - - - - - - - - - - Lachnanthes caroliniana
Cyclanthus biparlitus
Asplundia spectabi/is
Dicranopyg1um p_()/ycephalum
Evodianthus funifer
Carludovica pa/mata
Pandanus dubius
¢::=l + filaments
Freyclnetla luzonensis
Sticnoneuron caudatum
Stemona japonica
.----xe rophvt a sch/echteri
~
Xeropliyta eqlandulosa
Xerophyf!l pmifolia
¢::=l +filament s
Xemgh dasyllrioides
JrioldeS var. pectinata
X.
v111asa
~
Xerop
Xeroph"yta retlnfHVis
Talbotia elegans
Barbaceniopsis vaiJiasiana
Barbaceniopsis bo/IViensis
_..----- P/eurostima stenophylla
P/eurostima riparia
Barbacenia grisea
. . . . . .•Pieurostimapu~urea
. . . . . . . Vellozia froesii
Vellozla glochidea 1
Vellozla crassicaulls
¢::=l + filaments
Vellozia glochldea 2
Vellozla hlrsuta 2
Vellozia sp. 2
Vellozia declinans
Vellozia tubiflora
1 - - - - - Vellozia andina
.______ Nanuza plicata
L-------A canthoc hlamys bracteata
Amaryllis belladonna
Bowiea volubilis
Kniphofia uvaria
Pleea tenuifolia
Tofieldia pusil/a
Alisma p/antago-aquatica
Potamogeton amplifolius
L......- ------- ---Aco rus calamus
J
t
J
*.........
mapped
Figure 16. Character state evolution in Velloziaceae . Forms of sieve-clemen t plastids were
sieveonto the strict MP consensus tree. The Velloziaceae clade which is characterize d by the specific
highlighted
are
filaments
additional
with
subgroups
lines,
bold
in
element plastid syndrome is marked
drawn
and marked by an arrow. Branches leading to bromeliacea n taxa with subtype-P2c d plastids are
as dashed lines.
SIE\'E-ELL\IE'\T PIASTIDS .\ND RBCL DAT.\ I:\ \'ELLOZL-\CE.\E
127
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