Curr Genet (2009) 55:323–337
DOI 10.1007/s00294-009-0249-7
RESEARCH ARTICLE
Loss of all plastid ndh genes in Gnetales and conifers: extent and evolutionary signiWcance for the seed plant phylogeny
Thomas Werner Anthony Braukmann · Maria Kuzmina · Sana StefanoviT
Received: 13 March 2009 / Revised: 22 April 2009 / Accepted: 27 April 2009 / Published online: 18 May 2009© Springer-Verlag 2009
Abstract The exact phylogenetic position of Gnetales, asmall, highly modiWed group of gymnosperms with anaccelerated rate of molecular evolution, is one of the mostchallenging issues for seed plant systematics. Recent resultsfrom entire plastid genome (ptDNA) sequencing revealedthe absence of the entire suite of plastid ndh genes in sev-eral species of Gnetales and the pine family (Pinaceae)potentially highlighting a major structural feature linkingthese two groups—concerted loss of all plastid genes forthe NADH dehydrogenase complex. However, the preciseextent of ndh gene loss in gymnosperms has not been sur-veyed. Using a slot-blot hybridization method, we probedall 11 ndh genes in 162 species from 70 of 85 gymnospermgenera. We Wnd that all ndh genes are absent acrossGnetales and Pinaceae, but not in any other group of gym-nosperms. This feature represents either a major synapo-morphy for a clade consisting of these two lineages or, lesslikely, a convergent loss. Our survey substantially extendsprevious inferences based on more limited sampling and, ifthe former evolutionary interpretation is correct, it provides
additional support for the contentious “gnepine” hypothe-sis, which places Gnetales as sister to Pinaceae.
Keywords Gnetales · Conifers · ndh genes · Phylogeny · Seed plants · Slot-blot hybridization
Introduction
Arguably the most vexing outstanding question for the seedplant phylogeny remains the placement of Gnetales, a smallgroup of gymnosperms currently circumscribed in threegenera (Ephedra, Gnetum, and Welwitschia), despite inten-sive cladistic investigations over past two decades. Multiplepermutations for the relationship of Gnetales to other extantlineages of spermatophytes (otherwise comprising Ginkgo,cycads, Pinaceae, cupressophyte conifers and Xoweringplants) have been proposed by diVerent studies, usingdiVerent types of data and/or diVerent analytical methods(reviewed by Magallón and Sanderson 2002; Soltis et al.2002; Burleigh and Mathews 2004, 2007a; Mathews 2009).However, because of their repeated recovery across manystudies, six particular hypotheses on the placement of Gne-tales stand out as the most frequently invoked ones (Fig. 1).A series of early cladistic analyses based on morphologicaldata and including relevant fossil taxa suggested the place-ment of Gnetales close to angiosperms (Crane 1985; Doyleand Donoghue 1986; Loconte and Stevenson 1990; Nixonet al. 1994), in accordance with the “anthophyte” hypothe-sis (Wettstein 1907). Later, some molecular studies recov-ered the same results, albeit only with weak support(StefanoviT et al. 1998; Rydin et al. 2002). However, mostearly analyses of molecular data resulted in so-called “gne-tales-sister” hypotheses, featuring Gnetales either sister tothe rest of gymnosperms (“gnetales-sister I”; Hasebe et al.
Communicated by R. Bock.
Electronic supplementary material The online version of this article (doi:10.1007/s00294-009-0249-7) contains supplementary material, which is available to authorized users.
T. W. A. Braukmann (&) · M. Kuzmina · S. StefanoviTDepartment of Biology, University of Toronto Mississauga, 3359 Mississauga Rd. N, Mississauga, ON L5L 1C6, Canadae-mail: [email protected]
Present Address:M. KuzminaBiodiversity Institute of Ontario, University of Guelph, 579 Gordon St., Guelph, ON N1G 2W1, Canada
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http://dx.doi.org/10.1007/s00294-009-0249-7
324 Curr Genet (2009) 55:323–337
1992; Goremykin et al. 1996; Samigullin et al. 1999;Frolich and Parker 2000; Mathews and Donoghue 2000) orsister to the rest of seed plants, including angiosperms(“gnetales-sister II”; Hamby and Zimmer 1992; Albertet al. 1994; Rai et al. 2003, 2008). Subsequent analyses,based mainly on sequences from multiple genes andgenomes, suggested a “gnetifer” hypothesis, in which Gne-tales are found as sister to all conifers (Chaw et al. 1997,2000). But one of the most surprising results of molecularplant systematics placed Gnetales within conifers, as sisterto Pinaceae, in what became known as the “gnepine”hypothesis (Bowe et al. 2000; Chaw et al. 2000; Gugerliet al. 2001; Qiu et al. 2007). More recently, another hypoth-esis rendering conifers paraphyletic has gained somemomentum. According to the “gnecup” hypothesis, Gne-tales are also placed within conifers, but as sister tocupressophytes, not Pinaceae (see Nickrent et al. 2000;Rydin and Källersjö 2002; Doyle 2006; Chumley et al.2008).
One major cause of ambiguity for spermatophyte phy-logeny inference can be attributed to the ambiguous root-ing. While these six competing hypotheses seemsigniWcantly diVerent from one another when polarizedwith outgroups (i.e., rooted; Fig. 1a), they fall into only twocategories, each with an identical underlying unrooted tree(Fig. 1b). For example, it becomes apparent that there areno topological diVerences among the Wrst three major phy-logenetic hypotheses (anthophyte and gnetales-sister, I andII; Fig. 1a, 1–3) when the other land plant outgroups (repre-sented by grey arrows in Fig. 1) are pruned oV and theremainder is taken as an unrooted tree. Similarly, the only
diVerence between the remaining three alternative scenar-ios within seed plants (gnetifer, gnepine, and gnecuphypotheses; Fig. 1a, 4–6), is the placement of the root forthe clade comprising conifers and Gnetales. Besides rootingissues, another important source of ambiguity is due to thesubstantial sequence divergence among living lineages ofseed plants and their nearest outgroups (ferns and lyco-pods). In seed plants, this is particularly evident in longbranches leading to angiosperms, Gnetales, and Pinaceae asseen in most molecular trees, regardless of whether derivedfrom plastid, nuclear, or mitochondrial sequences (Chawet al. 1997, 2000; Bowe et al. 2000; Rydin et al. 2002; Gra-ham and Iles 2009). In conjunction, these two phenomenafrequently lead to the strongly supported yet spurious treerooting due to long-branch attraction or related artifacts(Felsenstein 1983; Hendy and Penny 1989). Further com-plicating the issue, diVerent methods of analyses and diVer-ent optimality criteria frequently support alternativetopologies, even though based on the same sequences(Bowe et al. 2000; Rydin et al. 2002; Burleigh andMathews 2004, 2007b). Surprisingly, the removal of mostrapidly evolving sequences or sites was shown to have littleto no eVect on the inferences on higher-order seed plantrelationships (Burleigh and Mathews 2004; Rai et al. 2008),raising the question of the limits of nucleotide data(Mathews 2009).
Molecular evidence is not limited only to primarysequence data. Additional sources of molecular data rele-vant for the number of open questions of seed plant phylog-eny can be sought from so-called “rare genomic markers”(e.g., Raubeson and Jansen 1992; Rokas and Holland 2000;
Fig. 1 Correspondence between competing rooted and unrooted seedplant phylogenetic hypotheses. a Six most prominent rooted hypothe-ses of relationships among major lineages of seed plants. Arrows indi-cate alternative root placements as in b, 1–6 Parsimony reconstructionsof gene losses for the ndh gene suite are mapped (oval) under theassumption of irreversibility. b Unrooted trees showing that the
underlying topologies for each of the two major sets of hypotheses areidentical when their respective outgroups, the other land plants (1–3)or other seed plants (4–6) are excluded. Note that these two unrootedtrees are incompatible. Taxon abbreviations: ANG angiosperms, CUPcupressophytes, CYC cycads, GIN Ginkgo, GNE Gnetales, GYM¡gymnosperms minus Gnetales, PIN Pinaceae
123
Curr Genet (2009) 55:323–337 325
Moreira and Philippe 2000; Gugerli et al. 2001). In thisregard, the plastid (pt) genome seems to be particularlypromising. In seed plants, this genome is highly conservedin size, structure, content, and synteny (Palmer 1991;Downie et al. 1991; Clegg et al. 1994). Because of theirrelatively infrequent evolutionary occurrence, any majorstructural mutation in the pt genome, such as inversions,gene/intron losses, and contractions/expansions of theinverted repeat (IR), are often considered to be more reli-able phylogenetic markers compared to sequences (e.g.,Downie et al. 1991; Downie and Palmer 1992; Raubesonand Jansen 1992; Doyle et al. 1995; Doyle et al. 1996;Bailey et al. 1997; Graham and Olmstead 2000a; Plunkettand Downie 2000; Jansen et al. 2007; but see McPhersonet al. 2004; Palmer et al. 2004 for cautionary views).
The loss of ndh genes from the pt genome of some seedplant represents one such potentially informative structuralchange. The ndh genes encode subunits of the plastidNAD(P)H-dehydrogenase (Ndh) complex, a homologue ofmitochondrial complex I (Shinozaki et al. 1986). In plas-tids, the Ndh complex seems to be primarily involved withtransfer of electrons from stromal reductants to a plastoqui-none pool, a process commonly known as “chlororespira-tion” (Bennoun 2002; Peltier and Cournac 2002). Inaddition, involvement of this complex in photooxidativestress reduction in high light intensity, regulation of photo-synthesis by modulating the activity of cyclic electron Xowaround photosystem I, and/or leaf senescence regulationhave also been suggested (Casano et al. 2001; Bukhov andCarpentier 2004; Zapata et al. 2005; Diaz et al. 2007;Tallon and Quiles 2007; Romeau et al. 2007; Endo et al.2008).
While the precise role of the Ndh complex is still uncer-tain, the ndh genes are known to be widespread in the auto-trophic seed plants and remain highly conserved over largeevolutionary distances, indicating the presence of strongselection pressure for their retention. Results of entire (orextensive) pt genome sequencing from a number of individ-ual studies indicate that the complete suite of ndh genes ispresent in Ginkgo (Leebens-Mack et al. 2005; Jansen et al.2007) and selected representatives of cycads (Wu et al.2007) and cupressophytes (Hirao et al. 2008) as well as in»60 species of diverse angiosperms (summarized mostrecently by Jansen et al. 2007). As suggested by Bungard(2004), the loss of ndh genes in Xowering plants seems tobe conWned only to parasitic plant lineages (dePamphilisand Palmer 1990; Olmstead et al. 2001; StefanoviT andOlmstead 2005; Funk et al. 2007; McNeal et al. 2007). Thislink with heterotrophy is further supported by the loss ofndh genes in a non-photosynthetic liverwort (Wickett et al.2008) and a green but potentially mycotrophic orchid(Chang et al. 2006). The absence of functional ndh genesfrom the pt genomes of fully autotrophic seed plants is
presently reported only from Gnetales (Wu et al. 2007,2009; McCoy et al. 2008) and several genera of Pinaceae(Wakasugi et al. 1994; Cronn et al. 2008; Rai et al. 2008;Wu et al. 2009). If inferred to have happened concurrently,this loss could represent a strong synapomorphy for Gne-tales and Pinaceae (Chaw et al. 2000; Burleigh andMathews 2004; Wu et al. 2007). Despite the potential ofthis rare structural genomic character to bear signiWcantlyon the seed plant relationships and help choose amongalternative phylogenetic hypotheses, the full extent of pres-ence or absence of ndh genes among living gymnosperms isunknown.
In the present study, we gathered data using a compre-hensive slot-blot hybridization survey of the complete suiteof plastid ndh genes with a dense sampling of gymno-sperms, the most extensive data matrix applied to this issueto date, in order to: (1) ascertain the extent and distributionof ndh gene losses across gymnosperms; and (2) assess theutility of these losses as phylogenetic markers for seedplant phylogeny.
Materials and methods
Taxon sampling
In total, 70 of the 85 genera and 162 of the »1,070 speciesof extant gymnosperms were sampled in this study(Table 1), corresponding to 82% of their generic- and 15%of their species-richness, respectively. Our samplingencompasses all four major lineages of living gymno-sperms; however, the percentage of diversity coveragediVers among these groups (compare with Table 1). Ginkgobiloba is the sole living representative of Ginkgoales. Coni-fers, including both Pinaceae and cupressophytes, are rep-resented by a total of 131 species (out of 680; 20%),grouped into 59 genera (out of 70; 85%), from all sevencurrently recognized families. The species-richness ofcycads is represented to a signiWcantly lesser degree, by 14out of 305 species (5%), but our sampling covers 64% ofgenera (7 out of 11). Finally, 16 species of Gnetales aresampled in total (out of the 92 species; 17%), including allthree recognized genera/families. Representatives of fourgenera of autotrophic angiosperms (Table 1), the lastremaining lineage of extant seed plants, were also includedin our surveys as positive controls.
DNA extraction and hybridization
Total genomic DNA was isolated using the modiWed 2£CTAB method (Doyle and Doyle 1987). Fresh leaf materialwas used where available, but approximately two-thirds ofthe samples were either from silica-gel dried tissue or
123
326 Curr Genet (2009) 55:323–337
Tab
le1
See
d pl
ant t
axa
surv
eyed
for
the
pres
ence
/abs
ence
of
plas
tid n
dh(A
-K)
gene
s
Tax
onV
ouch
ernd
hAnd
hBnd
hCnd
hDnd
hEnd
hFnd
hGnd
hHnd
hInd
hJnd
hK
exon
1ex
on 2
exon
1ex
on 2
5� e
nd3�
end
5� e
nd3�
end
Gin
kgoa
les
[1/1
; 100
%]
Gin
kgo
bilo
ba L
.Q
IU-9
4015
(IN
D)
++++
++++
++++
++++
++++
++++
++++
++
Cyc
adal
es [
7/11
;»64
%]
Cyc
as m
edia
R. B
r.N
o vo
uche
r++
++++
++++
++++
++++
++++
+++
++++
Cyc
as r
evol
uta
Thu
nb.
QIU
-940
51 (
IND
)++
++++
++++
++++
++++
++++
++++
++++
Cyc
as ta
iwan
iana
Car
ruth
.SS
-05-
53 (
TR
TE
)++
+++
+++
++++
++++
++++
++
+++
Dio
on s
pinu
losu
m D
yer
ex E
ichl
.SS
-03-
72 (
TR
TE
)++
+++
+++
++++
++++
++++
++
+++
Enc
epha
lart
os a
lten
stei
nii L
ehm
.SS
-05-
57 (
TR
TE
)++
+++
+++
++++
++++
++++
++
+++
Enc
epha
lart
os b
arte
ri C
arru
th. e
x M
iq.
SS-0
5-11
8 (T
RT
E)
++++
++
++++
++++
++++
+++
++
++
Enc
epha
lart
os fe
rox
Ber
tol.
f.SS
-05-
116
(TR
TE
)++
++++
++++
++++
++++
++++
++++
++++
Enc
epha
lart
os lo
ngif
oliu
s L
ehm
.Q
IU-9
5127
(IN
D)
++++
++++
++++
++++
++++
++++
++++
++
Mac
roza
mia
com
mun
is L
.A.S
. Joh
nson
SS-0
5-11
9 (T
RT
E)
++++
++
++++
++++
++++
++++
++
++
Cer
atoz
amia
mex
ican
a B
rong
n.Q
IU-9
4009
(IN
D)
++++
++
++++
++++
++++
++++
++
++
Stan
geri
a er
iopu
s (K
unze
) B
aill.
QIU
-940
78 (
IND
)++
++++
++++
++++
++++
++++
++++
++++
Zam
ia X
orid
ana
A.D
C.
QIU
-950
35 (
IND
)++
++++
++++
++++
++++
++++
++++
++++
Zam
ia fu
rfur
acea
Ait
.SS
-05-
56 (
TR
TE
)++
++++
++++
++++
++++
++++
++++
++++
Zam
ia p
umil
a L
.SS
-05-
58 (
TR
TE
)++
++++
++++
++++
++++
++++
++++
++++
Gne
tale
s [3
/3; 1
00%
]
Eph
edra
ant
isyp
hili
tica
Ber
land
ex
C.A
. Mey
.Q
IU-9
4004
(IN
D)
++
¡¡
+¡
¡+
¡¡
+¡
¡¡
¡E
phed
ra v
irid
is C
ovill
eSS
-05-
21 (
TR
TE
)¡
+¡
¡+
¡¡
+¡
¡+
¡¡
+¡
Eph
edra
tria
ndra
Tul
.SS
-05-
100
(TR
TE
)¡
+¡
¡+
¡¡
¡¡
¡+
¡+
+¡
Eph
edra
sax
atil
is (
Sta
pf)
Roy
le e
x Fl
orin
SS-0
5-18
8 (T
RT
E)
¡+
¡¡
+¡
¡¡
¡¡
+¡
¡¡
¡E
phed
ra m
onos
perm
a J.
G. G
mel
. ex
C.A
. Mey
.SS
-05-
35 (
TR
TE
)+
+¡
¡+
+¡
¡¡
¡+
¡+
+¡
Eph
edra
sin
ica
Stap
fSS
-05-
112
(TR
TE
)+
+¡
¡+
¡¡
¡¡
¡+
¡+
¡¡
Eph
edra
dis
tach
ya L
.SS
-05-
25 (
TR
TE
)¡
+¡
¡+
¡¡
¡¡
¡+
¡+
¡¡
Eph
edra
cam
pylo
poda
C.A
. Mey
.SS
-05-
60 (
TR
TE
)¡
+¡
¡+
¡¡
¡¡
¡+
¡¡
¡¡
Eph
edra
frag
ilis
Fre
sen.
SS-0
5-61
(T
RT
E)
++
¡¡
¡¡
¡+
¡¡
+¡
¡¡
¡E
phed
ra m
inut
a Fl
orin
SS-0
5-18
1 (T
RT
E)
¡¡
¡¡
+¡
¡¡
¡¡
+¡
¡¡
¡E
phed
ra p
roce
ra C
.A. M
ey.
SS-0
5-34
(T
RT
E)
¡+
¡¡
+¡
¡¡
¡¡
+¡
¡¡
¡G
netu
m g
nem
on L
.Q
IU-9
4005
(IN
D)
++
¡+
+¡
¡+
¡¡
+¡
¡¡
¡G
netu
m le
ybol
dii T
ul.
QIU
-940
17 (
IND
)+
+¡
++
¡¡
¡¡
++
¡+
¡¡
Gne
tum
sp.
SS-0
4-11
9 (T
RT
E)
++
¡¡
+¡
¡+
¡¡
+¡
+¡
¡G
netu
m u
la B
rong
n.SS
-06-
214
(TR
TE
)+
+¡
¡+
¡¡
+¡
¡+
¡¡
+¡
123
Curr Genet (2009) 55:323–337 327
Tab
le1
cont
inue
d
Tax
onV
ouch
ernd
hAnd
hBnd
hCnd
hDnd
hEnd
hFnd
hGnd
hHnd
hInd
hJnd
hK
exon
1ex
on 2
exon
1ex
on 2
5� e
nd3�
end
5� e
nd3�
end
Wel
wits
chia
mir
abili
s H
ook.
f.
SS-0
5-20
(T
RT
E)
++
¡¡
++
¡+
¡¡
+¡
+¡
¡
Pina
ceae
[10
/11;
»91
%]
Abi
es b
alsa
mea
(L
.) M
ill.
SS-0
5-20
8 (T
RT
E)
++
¡¡
+¡
¡¡
¡+
+¡
¡¡
+
Abi
es c
onco
lor
(Gor
don
& G
lend
.) H
ildeb
r.SS
-05-
157
(TR
TE
)+
++
¡+
¡¡
+¡
++
¡¡
¡+
Abi
es W
rma
Sieb
old
& Z
ucc.
QIU
-962
69 (
IND
)+
++
¡+
¡¡
+¡
++
¡¡
¡¡
Abi
es h
omol
epis
Sie
bold
& Z
ucc.
QIU
-962
24 (
IND
)+
++
¡?
¡¡
¡¡
¡+
¡¡
¡+
Abi
es k
orea
na E
.H. W
ilson
SS-0
5-39
(T
RT
E)
++
+¡
+¡
¡¡
¡+
+¡
¡¡
+
Abi
es la
sioc
arpa
(H
ook.
) N
utt.
SS-0
5-15
8 (T
RT
E)
++
+¡
+¡
¡+
¡+
+¡
¡¡
+
Ket
elee
ria
davi
dian
a B
eiss
n.Q
IU-9
6248
(IN
D)
++
+¡
+¡
¡¡
¡¡
+¡
¡¡
¡K
etel
eeri
a sp
.SS
-06-
224
(TR
TE
)+
+¡
¡+
¡¡
¡¡
¡+
¡¡
¡¡
Tsu
ga c
anad
ensi
s C
arri
ère
QIU
-940
77 (
IND
)+
++
¡+
¡¡
+¡
++
¡¡
¡++
Pse
udol
arix
am
abil
is R
ehde
rSS
-06-
206
(TR
TE
)+
++
¡+
¡¡
+¡
++
¡¡
¡+
Pse
udot
suga
men
zies
ii (M
irb.
) Fr
anco
SS-0
3-10
8 (T
RT
E)
++
¡¡
+¡
¡¡
¡¡
+¡
¡¡
+
Lar
ix d
ecid
ua M
ull.
QIU
-962
42 (
IND
)+
++
¡+
¡¡
¡¡
++
¡¡
¡++
Lar
ix la
rcin
a (D
u R
oi)
K. K
och
SS-0
5-37
(T
RT
E)
++
+¡
+¡
¡¡
¡+
+¡
¡¡
++
Pic
ea a
bies
(L
.) H
. Kar
st.
SS-0
3-99
(T
RT
E)
++
¡¡
++
¡+
¡+
+¡
¡¡
++
Pic
ea e
xcel
sa L
ink.
SS-0
3-10
5 (T
RT
E)
++
+¡
+¡
¡+
¡+
+¡
¡¡
++
Pic
ea g
lehn
ii M
ast.
SS-0
5-15
5 (T
RT
E)
++
¡¡
+¡
¡+
¡+
+¡
¡¡
++
Pic
ea e
ngel
man
nii P
arry
ex
Eng
elm
.SS
-05-
210
(TR
TE
)+
++
¡+
+¡
+¡
++
¡¡
¡++
Pic
ea g
lauc
a (M
oenc
h) V
oss
SS-0
5-20
9 (T
RT
E)
++
++
++
¡+
¡+
+¡
¡¡
++
Pic
ea p
unge
ns E
ngel
m.
SS-0
3-10
6 (T
RT
E)
++
+¡
++
¡+
¡+
+¡
¡¡
++
Pic
ea m
aria
na B
ritto
n St
erns
& P
ogge
nb.
SS-0
5-15
6 (T
RT
E)
++
+¡
+¡
¡+
¡+
+¡
¡¡
++
Pic
ea o
mor
ika
(Pan
biT)
Pur
k.SS
-03-
96 (
TR
TE
)+
++
¡+
¡¡
+¡
++
¡¡
¡++
Pic
ea o
rien
tali
s (L
.) L
ink
SS-0
3-10
0 (T
RT
E)
++
¡¡
++
¡+
¡+
+¡
¡¡
++
Cat
haya
arg
yrop
hyll
a C
hun
& K
uang
SS-0
6-23
3 (T
RT
E)
++
¡¡
+¡
¡+
¡¡
+¡
¡¡
¡P
inus
ari
stat
a E
ngel
m.
SS-0
5-40
(T
RT
E)
++
¡¡
+¡
¡¡
¡+
+¡
¡¡
+
Pin
us q
uadr
ifol
ia P
arry
ex
Parl
.SS
-06-
222
(TR
TE
)+
+¡
¡+
¡¡
¡¡
++
¡¡
¡++
Pin
us c
embr
a L
.SS
-05-
104
(TR
TE
)+
+¡
¡+
¡¡
¡¡
¡+
¡¡
¡++
Pin
us p
arviX
ora
Sieb
. & Z
ucc.
SS-0
5-42
(T
RT
E)
++
¡¡
+¡
¡+
¡+
+¡
¡¡
++
Pin
us s
trob
us L
.SS
-05-
36 (
TR
TE
)+
+¡
¡+
¡¡
+¡
++
¡¡
¡++
Pin
us w
alli
chia
na A
.B. J
acks
.SS
-03-
97 (
TR
TE
)+
+¡
¡+
¡¡
¡¡
++
¡¡
¡++
Pin
us c
anar
iens
is C
. Sm
.SS
-05-
64 (
TR
TE
)+
+¡
¡+
¡¡
+¡
++
¡¡
¡++
Pin
us h
eldr
eich
ii C
hris
tSS
-03-
98 (
TR
TE
)+
+¡
¡+
¡¡
+¡
++
¡¡
¡+
Pin
us k
esiy
a R
oyle
ex
Gor
don
SS-0
6-22
3 (T
RT
E)
++
¡¡
+¡
¡¡
¡+
+¡
¡¡
¡
123
328 Curr Genet (2009) 55:323–337
Tab
le1
cont
inue
d
Tax
onV
ouch
ernd
hAnd
hBnd
hCnd
hDnd
hEnd
hFnd
hGnd
hHnd
hInd
hJnd
hK
exon
1ex
on 2
exon
1ex
on 2
5� e
nd3�
end
5� e
nd3�
end
Pin
us n
igra
J.F
. Arn
old
SS-0
3-10
7 (T
RT
E)
++
¡¡
+¡
¡+
¡+
+¡
¡¡
++
Pin
us p
inas
ter
Aito
nSS
-05-
65 (
TR
TE
)+
+¡
¡+
¡¡
+¡
++
¡¡
¡++
Pin
us p
inea
L.
SS-0
5-63
(T
RT
E)
++
¡¡
+¡
¡+
¡+
+¡
¡¡
++
Pin
us r
esin
osa
Aito
nSS
-05-
164
(TR
TE
)+
+¡
¡+
¡¡
+¡
++
¡¡
¡++
Pin
us c
onto
rta
Dou
glas
ex
Lou
don
SS-0
5-20
5 (T
RT
E)
++
¡¡
+¡
¡+
¡+
+¡
¡¡
++
Pin
us c
oulte
ri D
. Don
SS-0
5-28
(T
RT
E)
++
¡¡
+¡
¡+
¡+
+¡
¡¡
++
Pin
us r
adia
ta D
. Don
SS-0
5-99
(T
RT
E)
++
¡¡
+¡
¡+
¡+
+¡
¡¡
+
Ced
rus
atla
ntic
a M
anet
tiSS
-03-
101
(TR
TE
)¡
++
¡+
¡¡
¡¡
¡+
¡¡
¡¡
Ced
rus
deod
ara
(Rox
b. e
x L
ambe
rt)
G. D
onQ
IU-9
6265
(IN
D)
¡+
+¡
+¡
¡+
¡¡
+¡
¡¡
¡
Ara
ucar
iace
ae [
3/3;
100
%]
Aga
this
sp.
SS-0
5-66
(T
RT
E)
++++
++
++++
++++
++++
++++
+++
++
Aga
this
rob
usta
(F.
Mue
ll.)
F.M
. Bai
ley
SS-0
6-18
0 (T
RT
E)
++++
++
++++
++++
++++
++++
+++
++
Ara
ucar
ia a
rauc
ana
K. K
och
SS-0
5-11
7 (T
RT
E)
++++
++
++++
++++
++++
++++
+++
++
Ara
ucar
ia b
idw
illi
i Hoo
k.SS
-05-
67 (
TR
TE
)++
++¡
+++
++++
+++
++++
+++
+++
Ara
ucar
ia c
unni
ngha
mii
Aito
n ex
D. D
onSS
-05-
106
(TR
TE
)++
++¡
+++
++++
+++
++++
+?
+++
Ara
ucar
ia h
eter
ophy
lla
(Sal
isb.
) Fr
anco
QIU
-962
20 (
IND
)++
+++
+++
++++
++++
++++
+++
++++
Ara
ucar
ia r
ulei
F. M
uell.
SS-0
5-10
1 (T
RT
E)
++++
++
++++
++++
++++
++++
?++
++
Ara
ucar
ia s
p.SS
-05-
21 (
TR
TE
)++
++¡
+++
++++
++++
++++
++?
++++
Wol
lem
ia n
obil
is W
.G. J
ones
K.D
. Hil
l & J
.M.A
llen
SS-0
5-17
6 (T
RT
E)
++++
¡+
++++
++++
++++
++++
+++
++
Podo
carp
acea
e [1
4/19
;»74
%]
Phy
lloc
ladu
s al
pinu
s H
ook.
f.
SS-0
6-23
1 (T
RT
E)
++++
?+
++++
++++
++++
++++
+++
++
Phy
lloc
ladu
s as
plen
ifol
ius
(Lab
ill.)
Hoo
k.f.
SS-0
6-19
9 (T
RT
E)
++++
??
++++
++++
++++
++++
+++
++
Hal
ocar
pus
bidw
illii
(Hoo
k. f
. ex
T. K
irk)
Qui
nnSS
-06-
201
(TR
TE
)++
+++
+++
++++
++++
++++
+++
++++
Lag
aros
trob
os c
olen
soi (
Hoo
k.)
Qui
nnSS
-06-
187
(TR
TE
)++
+++
+++
++++
++++
++++
+++
++++
Pru
mno
pity
s an
dina
(P
oepp
. ex
End
l.) d
e L
aub.
SS-0
6-18
8 (T
RT
E)
++++
++
++++
++++
++++
++++
+++
++
Sund
acar
pus
amar
a (B
lum
e) C
·N. P
age
SS-0
6-17
7 (T
RT
E)
++++
++
++++
++++
++++
++++
+++
++
Saxe
goth
ea c
onsp
icua
Lin
dl.
QIU
-962
43 (
IND
)++
+++
+++
++++
++++
++++
++++
++++
Mic
rost
robo
s W
tzge
rald
ii (
F.M
uell
.)
J.G
arde
n &
L.A
.S. J
ohns
onSS
-06-
232
(TR
TE
)++
+++
+++
++++
++++
++++
+++
++++
Acm
opyl
e pa
nche
ri P
ilg.
SS-0
6-18
2 (T
RT
E)
++++
??
++++
++++
++++
++?
+?
++
Dac
ryca
rpus
dac
rydi
oide
s (A
.Ric
h.)
de L
aub.
SS-0
6-18
6 (T
RT
E)
++++
¡+
++++
++++
++?
++++
+++
++
Dac
rydi
um c
upre
ssin
um S
ol. e
x L
amb.
SS-0
5-12
0 (T
RT
E)
++++
++
++++
++++
++++
++++
+++
++
Fal
cati
foli
um s
p.SS
-06-
179
(TR
TE
)++
+++
+++
++++
++++
++++
+++
++++
Pod
ocar
pus
cori
aceu
s R
ich.
& A
. Ric
h.SS
-05-
111
(TR
TE
)++
++
+++
++++
++++
++++
+++
++++
123
Curr Genet (2009) 55:323–337 329
Tab
le1
cont
inue
d
Tax
onV
ouch
ernd
hAnd
hBnd
hCnd
hDnd
hEnd
hFnd
hGnd
hHnd
hInd
hJnd
hK
exon
1ex
on 2
exon
1ex
on 2
5� e
nd3�
end
5� e
nd3�
end
Pod
ocar
pus
cost
alis
C. P
resl
QIU
-961
49 (
IND
)++
+++
+++
++++
++++
++++
+++
++++
Pod
ocar
pus
elat
us E
ndl.
SS-0
5-10
7 (T
RT
E)
++++
¡+
++++
++++
++++
++++
++
++
Pod
ocar
pus
falc
atus
(T
hunb
.) M
irb.
SS-0
5-11
4 (T
RT
E)
++++
¡+
++++
++++
++++
++++
++
++
Pod
ocar
pus
fasc
icul
us d
e L
aub.
QIU
-961
48 (
IND
)++
+++
+++
++++
++++
++++
++++
++++
Pod
ocar
pus
gray
ae d
e L
aub.
SS-0
6-20
4 (T
RT
E)
++++
¡+
++++
++++
++++
++++
++
++
Pod
ocar
pus
lati
foli
us R
. Br.
SS-0
5-17
9 (T
RT
E)
++++
¡+
++++
++++
++++
++++
++
++
Pod
ocar
pus
mac
roph
yllu
s (T
hunb
.) D
. Don
QIU
-950
06 (
IND
)++
+++
+++
++++
++++
++++
+++
++++
Pod
ocar
pus
niva
lis
Hoo
k.SS
-05-
103
(TR
TE
)++
+++
+++
++++
++++
++++
+++
++++
Pod
ocar
pus
sp.
SS-0
3-39
(T
RT
E)
++++
¡¡
++++
++++
++++
++++
¡+
++
Pod
ocar
pus
tota
ra G
. Ben
n. e
x D
. Don
SS-0
5-17
8 (T
RT
E)
++++
++
++++
++++
++++
++++
++++
++
Nag
eia
nagi
Bri
tton
& P
. Wils
onQ
IU-9
6146
(IN
D)
++++
++
++++
++++
++++
++++
++++
++
Afr
ocar
pus
grac
ilio
r (P
ilg.)
C·N
. Pag
eSS
-03-
55 (
TR
TE
)++
+++
+++
++++
++++
++++
++++
++++
Ret
roph
yllu
m c
ompt
onii
(B
uchh
.) C
·N. P
age
SS-0
6-18
1 (T
RT
E)
++++
??
++++
++++
??
++?
++?
++
Ret
roph
yllu
m m
inor
(C
arri
ère)
C·N
. Pag
eSS
-06-
178
(TR
TE
)++
++?
?++
++++
++?
?++
?+
?++
Tax
acea
e [3
/5; 6
0%]
Am
ento
taxu
s fo
rmas
ana
H.L
. Li
SS-0
6-21
1 (T
RT
E)
++++
++
++++
++++
++++
++++
+++
++
Tax
us b
acca
ta L
.SS
-05-
109
(TR
TE
)++
+++
+++
++++
++++
++++
+++
++++
Tax
us c
anad
ensi
s M
arsh
all
SS-0
5-41
(T
RT
E)
++++
++
++++
++++
++++
++++
+++
++
Tax
us c
uspi
data
Sie
b. &
Zuc
c.SS
-05-
38 (
TR
TE
)++
+++
+++
++++
++++
++++
++++
++++
Tax
us £
med
ia R
ahde
rQ
IU-9
6218
(IN
D)
++++
++
++++
++++
++++
++++
++++
++
Tor
reya
cal
ifor
nica
Tor
r.SS
-05-
121
(TR
TE
)++
+++
+++
++++
+++
++++
+++
++++
Tor
reya
nuc
ifer
a S
ieb.
& Z
ucc.
SS-0
5-18
0 (T
RT
E)
++++
¡¡
++++
+++
++++
++++
+¡
++
Tor
reya
taxi
folia
Arn
.Q
IU-9
6251
(IN
D)
++++
+++
++++
++++
++++
++++
+++
++
Cep
halo
taxa
ceae
[1/
1; 1
00%
]
Cep
halo
taxu
s ha
rrin
gton
ia K
. Koc
hSS
-03-
102
(TR
TE
)++
+++
+++
++++
++++
++++
+++
++++
Cep
halo
taxu
s fo
rtun
ei H
ook.
SS-0
6-22
1 (T
RT
E)
++++
++
++++
++++
++++
++++
+++
++
Scia
dopi
tyac
eae
[1/1
; 100
%]
Scia
dopi
tys
vert
icill
ata
Sieb
. & Z
ucc.
QIU
-960
87 (
IND
)++
++++
++++
++++
++++
++++
++++
++++
Cup
ress
acea
e s.
l. [2
7/30
; 90%
]
Cun
ning
ham
ia la
nceo
lata
Hoo
k.Q
IU-9
6254
(IN
D)
++++
+++
++++
++++
+++
++++
++++
++
Tai
wan
ia X
ousi
ana
Gau
ssen
QIU
-962
66 (
IND
)++
+++
++++
++++
++++
++++
++++
++++
Ath
rota
xis
sp.
SS-0
6-23
0 (T
RT
E)
++++
?+
++++
++?
++++
++++
++++
++
Sequ
oia
sem
perv
iren
s E
ndl.
QIU
-960
91 (
IND
)++
+++
++++
++++
++++
+++
++++
++++
Sequ
oiad
endr
on g
igan
teum
(Lin
dl.)
J. B
uchh
olz
QIU
-960
89 (
IND
)++
+++
++++
++++
++++
++++
++++
++++
123
330 Curr Genet (2009) 55:323–337
Tab
le1
cont
inue
d
Tax
onV
ouch
ernd
hAnd
hBnd
hCnd
hDnd
hEnd
hFnd
hGnd
hHnd
hInd
hJnd
hK
exon
1ex
on 2
exon
1ex
on 2
5� e
nd3�
end
5� e
nd3�
end
Cry
ptom
eria
japo
nica
D. D
onQ
IU-9
6267
(IN
D)
++++
+++
++++
++++
+++
++++
++++
++
Gly
ptos
trob
us p
ensi
lis
(lin
eatu
s) K
. Koc
hQ
IU-9
6264
(IN
D)
++++
+++
?++
++++
++++
++++
?++
++
Tax
odiu
m d
isti
chum
H·B
. & K
.SS
-05-
113
(TR
TE
)++
+++
++++
++++
++++
+++
++++
++++
Aus
troc
edru
s ch
ilen
sis
(D.D
on) F
lori
n &
Bou
telje
SS-0
6-19
0 (T
RT
E)
++++
++
++++
++++
+++
++++
++++
++
Lib
oced
rus
plum
osa
Dru
ceSS
-06-
196
(TR
TE
)++
+++
+++
++++
++++
+++
++++
++++
Pil
gero
dend
ron
uvif
erum
Flo
rin
SS-0
6-20
2 (T
RT
E)
++++
++
??
?++
?++
++++
?++
++
Act
inos
trob
us a
rena
rius
C.A
. Gar
dner
SS-0
6-22
6 (T
RT
E)
++++
++
++++
++++
+++
++++
+++
++
Cal
litri
s dr
umm
ondi
i (Pa
rl.)
F. M
uell
.Q
IU-9
6250
(IN
D)
++++
++
++++
++++
++++
++++
++++
++
Dis
elm
a ar
cher
i Hoo
k. f
.SS
-06-
203
(TR
TE
)++
+++
+++
++++
++++
++++
++++
++++
Fit
zroy
a cu
pres
soid
es I
.M. J
ohns
t.SS
-06-
200
(TR
TE
)++
++¡
+++
++++
++++
?++
++++
++++
Thu
ja o
ccid
enta
lis L
.Q
IU-9
6223
(IN
D)
++++
++
++++
++++
+++
++++
++++
++
Thu
ja o
rien
tali
s L
.SS
-05-
69 (
TR
TE
)++
+++
+++
++++
++++
+++
++++
++++
Thu
ja p
lica
ta D
onn
ex D
. Don
SS-0
5-21
1 (T
RT
E)
++++
++
++++
++++
+++
++++
++++
++
Thu
jops
is d
olab
rata
Sie
bold
& Z
ucc.
QIU
-962
46 (
IND
)++
+++
+++
++++
++++
+++
++++
++++
Fok
ieni
a ho
dgin
sii A
. Hen
ry &
H·H
. Tho
mas
SS-0
6-19
8 (T
RT
E)
++++
¡¡
++++
++++
+++
+++
++
++
Cha
mae
cypa
ris
obtu
sa S
iebo
ld &
Zuc
c.SS
-05-
187
(TR
TE
)++
+++
+++
++++
++++
+++
++++
++++
Cha
mae
cypa
ris
pisi
fera
(S
iebo
ld &
Zuc
c.)
End
l.SS
-05-
161
(TR
TE
)++
+++
+?
++++
++++
?++
++++
++++
Cha
mae
cypa
ris
thyo
ides
(L
.)
Bri
tton
Ster
ns &
Pog
genb
.SS
-05-
74 (
TR
TE
)++
+++
+++
++++
++++
+++
++++
++++
Tet
racl
inis
art
icul
ata
Mas
t.SS
-05-
72 (
TR
TE
)++
+++
+++
++++
++++
+++
++++
++++
Cal
oced
rus
decu
rren
s (T
orr.
) Fl
orin
QIU
-962
45 (
IND
)++
+++
+++
++++
++++
+++
++++
++++
Mic
robi
ota
decu
ssat
a K
om.
QIU
-962
52 (
IND
)++
+++
++++
++++
++++
+++
++++
++++
Pla
tycl
adus
ori
enta
lis
(L.)
Fra
nco
SS-0
6-19
2 (T
RT
E)
++++
++
++++
++++
+++
++++
++++
++
Cup
ress
us s
empe
rvir
ens
L.
SS-0
5-12
2 (T
RT
E)
++++
++
++++
++++
+++
++++
++++
++
Cup
ress
us c
ashm
eria
na R
oyle
ex
Car
rier
eSS
-06-
01 (
TR
TE
)++
++?
+++
++++
++++
?++
++?
++++
Cup
ress
us d
upre
zian
a A
. Cam
usSS
-05-
70 (
TR
TE
)++
+++
+++
++++
++++
?++
++?
++++
Juni
peru
s ch
inen
sis
L.
SS-0
5-18
3 (T
RT
E)
++++
¡+
++++
++++
++++
++++
?++
++
Juni
peru
s co
mm
unis
L.
SS-0
5-83
(T
RT
E)
++++
+++
++++
++++
+++
++++
++++
++
Juni
peru
s ho
rizo
ntal
is M
oenc
hSS
-05-
160
(TR
TE
)++
+++
++++
++++
++++
+++
++++
++++
Juni
peru
s in
dica
Ber
tol.
QIU
-962
70 (
IND
)++
++++
++++
++++
++++
++++
++++
++++
Juni
peru
s pr
ocum
bens
Sie
bold
SS-0
5-15
9 (T
RT
E)
++++
+++
++++
++++
+++
++++
++++
++
Juni
peru
s sa
bina
L.
SS-0
5-16
3 (T
RT
E)
++++
++
++++
++++
++++
++++
++++
++
Juni
peru
s sc
opul
orum
Sar
g.SS
-05-
162
(TR
TE
)++
+++
++++
++++
++++
+++
++++
++++
Juni
peru
s vi
rgin
iana
L.
SS-0
5-84
(T
RT
E)
++++
+++
++++
++++
++++
++++
++++
++
123
Curr Genet (2009) 55:323–337 331
herbarium specimens. Quality of DNA varied signiWcantly,from high molecular weight to considerably degraded(from some herbarium material). Because of the poor qual-ity of a number of samples, frequently accompanied also bylimited quantity, the standard restriction endonucleasedigestion followed by electrophoretic separation approachto Southern hybridization (Sambrook et al. 1989) could notbe used. Instead, the slot-blot hybridization method wasused, as described in detail by Doyle et al. (1995).
In brief, a slot-blot apparatus (Bio-Rad) was used tomake seven sets of pseudoreplicate Wlter-blots, followingthe manufacturer’s protocol. Approximately 500–800 ng oftotal DNA (per sample and per set) was bound to Immobi-lon-Ny + nylon membrane (Millipore). DNAs from severalspecies with sequenced pt genomes (e.g., Nicotiana, Aco-rus, Amborella, Ginkgo, or Welwitschia) were included oneach membrane as positive or negative controls. Mem-branes were prehybridized and hybridized at 60°C–62°C in5£ standard saline citrate (SSC), 0.1% SDS, 50 mM Tris(pH 8.0), 10 mM EDTA, 2£ Denhardt’s solution, and 5%dextran sulfate. After hybridization, Wlters were washedtwice for 30–45 min in 0.5% SDS and 2£ SSC at thehybridization temperature. Probes were labeled with 32Pusing random oligonucleotide primers (Invitrogen). Autora-diography was carried out using intensifying screens at¡80°C for 18–48 h. Filters were stripped of probe betweenhybridizations by boiling twice for 5–10 min in 0.1% SSC.The absence of carryover signal from previous hybridiza-tions was assured by an overexposure (3–5 days) prior tonew rounds of hybridization.
Hybridization probes for all 11 plastid-encoded ndhgenes and small plastid ribosomal subunit (16S rDNA; usedas a control probe) were derived from tobacco via polymer-ase chain reaction (PCR). Primer names and sequences areprovided in Supplementary Table 1. A total of 17 probeswere constructed and their relative positions are indicatedin Supplementary Fig. 1. For the two ndh genes, usuallyinterrupted by introns (ndhA and ndhB), two probes wereused, each covering one exon. Two additional longer ndhgenes (ndhD and ndhH) were surveyed with two probes sit-uated at the 5�- and 3�-ends, respectively. In addition, toestimate the unspeciWc background hybridization levels, aninitial negative hybridization control was performed underthe same stringency conditions (see above) and the sameamount of 32P, but without probe added.
Results
Interpretation of slot-blots
The slot-blot data ranged from no diminution to completeabsence of signal and were for the most part readilyT
able
1co
ntin
ued
Num
bers
in s
quar
e pa
rent
hese
s fo
llow
ing
taxa
abo
ve g
ener
ic ra
nk in
dica
te n
umbe
r of
gen
era
sam
pled
out
of
the
tota
l num
ber
of g
ener
a in
that
taxo
n an
d th
e ap
prox
imat
e pe
rcen
tage
of c
over
age.
With
inm
ajor
gro
ups
of s
eed
plan
ts, s
peci
es a
re a
rran
ged
acco
rdin
g to
the
ir p
resu
mpt
ive
phyl
ogen
etic
rel
atio
nshi
ps (
see
text
for
det
ails
). B
ased
on
slot
-blo
t re
sults
, a
“++
” sy
mbo
l in
dica
tes
pres
ence
of
full
hybr
idiz
atio
n, “
+”
dim
inis
hed
hybr
idiz
atio
n, “
–” a
bsen
ce o
f hy
brid
izat
ion
sign
al in
com
pari
son
to p
ositi
ve c
ontr
ols,
and
“?”
una
ble
to s
core
. Sel
ecte
d sl
ot-b
lot h
ybri
diza
tion
resu
lts f
or r
epre
sent
ativ
esfr
om a
ll m
ajor
gro
ups
(spe
cies
indi
cate
d in
bol
dfac
e) a
re s
how
n in
Fig
.1. A
bbre
viat
ions
of
herb
aria
fol
low
Ind
ex H
erba
rior
um
Tax
onV
ouch
ernd
hAnd
hBnd
hCnd
hDnd
hEnd
hFnd
hGnd
hHnd
hInd
hJnd
hK
exon
1ex
on 2
exon
1ex
on 2
5� e
nd3�
end
5� e
nd3�
end
Xan
thoc
ypar
is n
ootk
aten
sis
(D. D
on)
Farj
on
& D
.K. H
arde
rQ
IU-9
4088
(IN
D)
++++
+++
++++
++++
+++
++++
++++
++
Cal
litr
opsi
s ar
izon
ica
(gla
bra)
(G
reen
e) D
.P. L
ittle
QIU
-962
54 (
IND
)++
+++
+++
++++
++++
+++
++++
++++
Cal
litr
opsi
s ba
keri
(Je
ps.)
D.P
. Lit
tle
SS-0
5-73
(T
RT
E)
++++
++
++++
++++
+++
++++
+++
++
Cal
litr
opsi
s sa
rgen
tii (
Jeps
.) D
.P. L
ittl
eSS
-05-
190
(TR
TE
)++
++¡
+++
++++
++++
+++
++++
++++
Ang
iosp
erm
s
Am
bore
lla tr
icho
poda
Bai
ll.N
o vo
uche
r++
++++
++++
++++
++++
++++
++++
++++
Vic
tori
a sp
.N
o vo
uche
r++
++++
++++
++++
++++
++++
++++
++++
Aco
rus
gram
ineu
s So
land
.SS
-03-
70 (
TR
TE
)++
++++
++++
++++
++++
++++
++++
++++
Gly
cine
max
(L
.) M
err.
No
vouc
her
++++
++++
++++
++++
++++
++++
++++
++
123
332 Curr Genet (2009) 55:323–337
interpretable. The presence or absence of ndh genes wasdetermined by eye, by comparison of hybridization signalto the corresponding 16S probe, which is used as a controlto establish the presence of signiWcant amounts of ptDNAon the blots. For each blot set and probe combination, thestrength of signal was estimated by comparison with anumber of positive and negative controls; namely, the spe-cies known to contain functional ndh genes (e.g., Ginkgo,Amborella, Acorus, etc.) or to lack these genes (e.g., Wel-witschia, Pinus spp., etc.), based on previously availableentire ptDNA sequence data.
Representative hybridization results, arranged phyloge-netically, are shown in Fig. 2, and the scores for all surveyedspecies and probes are summarized in Table 1. For everyprobe, the relative presence or absence of signal was scoredfor each taxon as showing either full (++), diminished (+),or absent (¡) hybridization in comparison to their respective16S positive controls (Table 1). Full hybridization strengthis assumed to indicate that the gene is present and func-tional. For genes that have two probes (i.e., two exons or5�- and 3�-end) full hybridization to both probes is requiredto indicate that the gene is functional. Diminished signals,where hybridization is weaker than the control but there isdeWnite signal presence, can be interpreted in two diVerentways. It can indicate that the gene is divergent with respectto tobacco but still present and functional or that the gene ispresent but pseudogenized (i.e., rendered nonfunctional).Absence was scored if no detectable hybridization to theprobe was observed. Under our experimental conditions,plants in which a gene has been transferred to the nucleuswould fail to produce a detectable hybridization signal whencompared to a plant that retains the gene in its plastidgenome, due to signiWcant reduction in copy number and anincrease in nucleotide substitution rates (Wolfe et al. 1987).Hence, the absence of signal implies either outright loss ofgenes or their functional transfer to the nucleus. Given thegenerally conservative substitution rates of ptDNA, it is lesslikely that the absence of signal represents a highly diver-gent yet functional gene. Lastly, in certain cases we wereunable to determine the presence or absence of signal andwe scored these taxa as unknown (“?”; see Table 1). Theseambiguities are due to insuYcient amounts or poor qualityof ptDNA for a given pseudoreplicate.
Altogether, these assumptions on the presence orabsence of genes can lead to potential underestimates oroverestimates of gene losses. For example, signals thatappear present could potentially represent relatively recentpseudogenized genes while signiWcantly diminished signalsmight be due to divergent but functional genes. Neverthe-less, despite these caveats, a hybridization approachremains a cost eVective and eYcient method for surveyingnumerous and diverse samples (Doyle et al. 1995; Adamset al. 2002).
Distribution of ndh gene losses
As expected, the full hybridization signal was observed forall taxa used as positive controls. Based on results fromwhole pt genome sequencing, the entire ndh gene suite isknown to be present in autotrophic angiosperms, indicatingthat its presence is the shared ancestral condition for thisgroup of plants (Jansen et al. 2007). The presence of all 11ndh genes is conWrmed here across the representatives cho-sen to span the basal nodes of Xowering plant diversity(Fig. 2; Table 1). Also, for Ginkgo, the hybridization to allprobes derived from the ndh genes was similar in strengthto the control DNA (Fig. 2; Table 1). This was expected aswell, given the known presence of these genes based onextensive sequencing of its pt genome (Leebens-Mack et al.2005; Jansen et al. 2007). In addition, the relative strengthof the hybridization of tobacco-derived probes to Ginkgoillustrates the conserved nature of the ndh genes acrosslarge phylogenetic distances, including the angiosperm-gymnosperm divergence (>325 Mya; Judd et al. 2002;Palmer et al. 2004).
As a group, cycads strongly hybridized to seven of 11ndh genes. For a few taxa (Table 1), the signal was dimin-ished with probes for ndhB (both exons), ndhH 3, ndhI, andndhJ. Although some of the hybridizations are weaker thanthe positive controls, they do not necessarily indicate lossof function of these ndh genes but rather that these genesare divergent to some degree in these taxa. The presence offunctional ndh genes in cycads is expected given that theyare found as open reading frames (ORFs) in the sequencedptDNA of Cycas taitungensis (Wu et al. 2007).
In contrast to the previous lineages, the evidence for lossof ndh genes from pt genomes is widespread in Gnetalesand Pinaceae. In Gnetales, there was no signiWcant hybrid-ization signal for most of the probes (Fig. 2; Table 1). Forsome taxa weak signal was present for ndhA (both exons),ndhC, and ndhH 5�-end. This pattern indicates that the lossof the ndh genes is common to all Gnetales (Fig. 2), inaccordance with the results of entire ptDNA sequencesfrom a small number of select representatives of this group(McCoy et al. 2008; Wu et al. 2009). Similarly, there was agenerally weak to absent hybridization signal observed formost probes across Pinaceae. In particular, ndhI and ndhJare absent from all surveyed taxa. Substantially diminishedsignal was common for probes derived from ndhA (bothexons), ndhD, ndhE, ndhH, and ndhI genes. However,ndhB (most notably, the 5� end), ndhC, and ndhK appearedpresent in many (but not all) taxa. Given what is knownfrom the entire plastid genome sequences of several Pinusspp. (Wakasugi et al. 1994; Cronn et al. 2008), Picea sitch-ensis (Cronn et al. 2008), and Keteleeria davidiana (Wuet al. 2009) species, it can be deduced that the presence ofweak to moderate hybridization signal observed for these
123
Curr Genet (2009) 55:323–337 333
Fig
.2A
utor
adio
grap
hs s
how
ing
slot
-blo
t hy
brid
izat
ion
resu
lts o
f pr
obes
der
ived
fro
m n
dh(A
-K)
gene
s fo
r 31
sel
ecte
d sp
ecie
s re
pres
entin
g se
ed p
lant
(ou
t of
166
sur
veye
d; c
ompa
re w
ithT
able
1) p
rese
nted
in a
phy
loge
netic
con
text
. Sm
all p
last
id r
ibos
omal
sub
unit
(16
S r
DN
A)
was
use
d as
pos
itive
con
trol
(sh
own
here
is o
ne r
epre
sent
ativ
e ou
t of
seve
n W
lter-
sets
). T
he to
polo
gysh
own
is a
com
posi
te tr
ee d
epic
ting
cur
rent
und
erst
andi
ng o
f re
latio
nshi
ps d
eriv
ed f
rom
sev
eral
pub
lishe
d ph
ylog
enet
ic a
naly
ses
(see
text
for
ref
eren
ces)
. Not
e th
at th
e ab
senc
e or
nea
r ab
senc
eof
hyb
ridi
zatio
n fo
r th
e nd
h pr
obes
is r
estr
icte
d on
ly to
the
surv
eyed
mem
bers
of
Gne
tale
s an
d P
inac
eae.
Tax
on a
bbre
viat
ions
are
the
sam
e as
in F
ig.1
123
334 Curr Genet (2009) 55:323–337
ndh genes (Fig. 2; Table 1) corresponds to pseudogenes.Compared to Pinaceae, Gnetales appear to have fewer rem-nants of ndh genes (Fig. 2; Table 1), which is expected,given the highly elevated rates of molecular evolutionobserved in Gnetales plastids generally (Rydin et al. 2002;Burleigh and Mathews 2007a; McCoy et al. 2008).
Unlike Gnetales and Pinaceae, the general trend acrosscupressophytes was a strong hybridization to almost all ndhprobes, indicating that the entire suite of ndh genes is pres-ent and conserved within this group (Fig. 2; Table 1). Thisis fully in agreement with the only published entirelysequenced ptDNA from cupressophytes, Cryptomeriajaponica (Cupressaceae s. lat.; Hirao et al. 2008). However,some members of Araucariaceae, Podocarpaceae, Taxa-ceae, and Cephalotaxaceae hybridized weakly to ndhB (oneor both exons) and ndhI, while Cupressaceae s. lat. exhibitdiminished hybridization signal to ndhG but not ndhI (seeTable 1). In these cases, diminished signal is most likelydue to the elevated sequence divergence of ndh genes, asevidenced by the presence of ndhB and ndhF ORFs in fewrepresentative species from these families that are currentlysequenced (Rai et al. 2008).
Discussion
Implications of ndh losses for spermatophyte relationships
The ndh genes comprise about one-tenth of the »120 genesretained in plastids of most photosynthetic seed plants.Based on numerous entirely sequenced pt genomes ofangiosperms (see Jansen et al. 2007 for the most recentsummary) as well as a limited number of gymnosperms(Wakasugi et al. 1994; Wu et al. 2007, 2009; Cronn et al.2008; McCoy et al. 2008; Hirao et al. 2008), it appearedthat the loss of the ndh genes was restricted to Gnetales(McCoy et al. 2008; Wu et al. 2009) and Pinaceae(Wakasugi et al. 1994; Cronn et al. 2008; Wu et al. 2009).The results of our survey extend the previous inferences ofthe ndh gene absence to be common to all of Gnetales andPinaceae, but not to other gymnosperms (nor to autotrophicangiosperms). While the losses of ndh genes from the plas-tids is rare in autotrophic plants, their absence has beenobserved repeatedly in heterotrophic angiosperms(Olmstead et al. 2001; StefanoviT and Olmstead 2005).Extrapolating from those cases, it seems that the ndh genesare generally lost as a suite (Bungard 2004; Krause 2008).Hence, from a phylogenetic point of view, the entire suiteshould be considered as a single loss (StefanoviT andOlmstead 2005) and not as 11 independent losses.
Given the extent and distribution of presence andabsence of the ndh genes among spermatophytes, the mostparsimonious solution suggests that the loss of these genes
is a synapomorphy for Gnetales and Pinaceae, a sharedderived character inherited from their common ancestor(Fig. 1) supporting the gnepine hypothesis. Each of the Wvealternatives for the Gnetales relationships with the otherseed plants (i.e., anthophyte, gnetales-sister, gnetifer, andgnecup hypthotheses) would require a minimum of twoindependent losses of ndh genes, one in Gnetales and one inPinaceae (Fig. 1). While the possibility that ndh genes werelost more than once in gymnosperms cannot be positivelyexcluded, the low frequency of loss of these genes, and inparticular the near-absence of loss among autotrophic seedplants, argues against such a scenario.
Additional lines of evidence supporting the gnepinehypothesis are provided by a couple of other plastid struc-tural characters, each with putatively the same phylogeneticdistribution among seed plants as that observed for the lossof ndh genes. First, an expansion of the inverted repeat (IR)that includes the duplication of trnI-CAU and a partialduplication of the psbA gene region situated at the end ofthe large single copy (LSC) is found in several sequencedmembers of Gnetales and Pinaceae but is not known fromany other land plant (Wu et al. 2007, 2009; McCoy et al.2008; Hirao et al. 2008). Second, both Gnetales and Pina-ceae appear to share a common loss of rps16, to the exclu-sion of other gymnosperms and basal angiosperms lineagesfrom which the presence of this gene is ascertained (Waka-sugi et al. 1994; Wu et al. 2007, 2009; Jansen et al. 2007;McCoy et al. 2008). However, contrary to the above exam-ples, one particular structural genomic marker does not sup-port the gnepine phylogeny. The loss (or a signiWcantreduction) of the IR is reported from both cupressophytesand Pinaceae, but not from Gnetales (Raubeson and Jansen1992; Wu et al. 2007, 2009; McCoy et al. 2008; Hirao et al.2008). This ptDNA feature favors the phylogenetic inter-pretation according to which the loss of the ndh genesoccurred independently in Gnetales and Pinaceae.
Future directions
Although the preponderance of genomic structural changescurrently supports the gnepine hypothesis, caution is stillwarranted because most of these features have only beenobserved in a subset of exemplar taxa. Hence, their full dis-tribution across seed plants and their evolutionary signiW-cance remains poorly understood. To determine theirrelative importance, all of these underexamined markersrequire further in-depth surveys across a broader taxonsample, as was done here for the ndh genes. In particular,additional ptDNA sequences are needed from the represen-tatives of Araucariaceae and Podocarpaceae, to help trian-gulate the ancestral conditions for many of these potentiallyphylogenetically important characters in cupressophytes. Itwould also be valuable to survey for ndh genes in Parasit-
123
Curr Genet (2009) 55:323–337 335
axus usta (Podocarpaceae), the only known mycohetero-trophic conifer (Feild and Brodribb 2005). We predict thefunctional absence of all ndh genes in this highly derivedpodocarp species (Sinclair et al. 2002), which would repre-sent an independent loss of the Ndh complex in conifers,related to its shift to a fully heterotrophic nutritional mode.
Acknowledgments For providing generous access to their live plantcollections, the authors are grateful to directors/managers of the fol-lowing institutions: Humboldt State University (Acrata, CA) green-house, Indiana University (Bloomington, IN) greenhouse, Universityof Alberta (Edmonton, AB) greenhouse, University of Toronto (Toron-to, ON) greenhouse, University of Washington (Seattle, WA) green-house and Arboretum, University of Guelph Arboretum (Guelph, ON),Devonian Botanic Garden (Devon, AB), Jardin botanique de Montréal(Montréal, QC), University of Belgrade Arboretum (Belgrade, Serbia),Vienna Botanical Garden (Vienna, Austria), and Carleton University(Ottawa, ON) greenhouse. We also thank curators/directors of ALTA,IND, and TRT for supplying plant material. Special thanks are due toSean Graham and two anonymous reviewers for their valuable sugges-tions that improved the earlier versions of the manuscript. Financialsupport from the Natural Sciences and Engineering Research Councilof Canada, Canada Foundation for Innovation and Ontario ResearchFunds to S. StefanoviT is gratefully acknowledged.
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Loss of all plastid ndh genes in Gnetales and conifers: extent and evolutionary signiWcance for the seed plant phylogenyAbstractIntroductionMaterials and methodsTaxon samplingDNA extraction and hybridization
ResultsInterpretation of slot-blotsDistribution of ndh gene losses
DiscussionImplications of ndh losses for spermatophyte relationshipsFuture directions
References
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