TAXONOMY OF MEXICAN DIPLOID WILD POTATOES (SOLANUM
SECT. PETOTA): MORPHOLOGICAL AND MICROSATELLITE DATA
Sabina I. Lara-Cabrera and David M. Spooner
ABSTRACT. Solanum sect. Petota, the potato and its wild relatives, contains about 200 wild species distributed from the southwestern United States to central Argentinaand adjacent Chile. Most species grow in the Andes, but the United States, Mexico, andCentral America contain about 30 diploid (2n = 24) taxa, as well as tetraploid (2n = 48) and hexaploid (2n = 72) ones. Chloroplast DNA restriction site data showed13 of these 30 taxa to form a clade containing only diploid species, but there was low resolution within the clade. Some of these 13 taxa are similar morphologically, and we questioned whether they were valid species. We analyzedthese and related taxa with morphological and microsatellite data. Morphological datashowed extensive overlap of putative species-specific characters, but most species couldbe supported by multivariate techniques, except S. brachistotrichium and perhaps S. stenophyllidium. Previously mapped nuclear microsatellite markers, developed in S. tuberosum, also were explored for help to define species but performed poorly, showing that these microsatellites have reduced phylogeneticutility to analyze the United States, Mexican, and Central American diploid wild potato species.
Key words: microsatellite, potato, simple sequence repeats, Solanum sect. Petota, SSR.
9
200
A FESTSCHRIFT FOR WILLIAM G. D’ARCY
Solanum sect. Petota Dumort., the potatoand its wild relatives, contains about 200wild species distributed from the south-western United States to central
Argentina and adjacent Chile (Hawkes, 1990;Spooner & Hijmans, 2001). Most species grow inthe Andes, but the United States, Mexico, andCentral America contain about 30 diploid taxa(2n = 24), as well as tetraploid (2n = 48) and hexa-ploid (2n = 72) ones. The United States, Mexico,and Central American diploids are classified inSolanum series Bulbocastana, Morelliformia,Pinnatisecta, Polyadenia, and Tuberosa (Hawkes,1990); see Table 1 for authorities of series, species,and subspecies. For simplicity, we refer to speciesfrom this region as Mexican or primarily Mexicanspecies. Some Mexican diploids appear very simi-lar morphologically and led us to questionwhether they were valid species. In this paper weexplore the use of morphology and microsatel-lites (or simple sequence repeats, SSRs) to test thevalidity of these primarily Mexican diploidspecies.
Hawkes (1989, 1990) divided Solanum sect.Petota into 21 series. Two of these series are non-tuber-bearing, and 19 are tuber-bearing. Hawkes(1989, 1990) placed the two non-tuber-bearingseries in subsection Estolonifera Hawkes, and theremaining 19 tuber-bearing series in subsectionPotatoe Don of Solanum. He divided subsectionPotatoe into two superseries, Stellata Hawkesand Rotata Hawkes, based on the corolla shape.Hawkes (1990) and Hawkes and Jackson (1992)proposed a phylogeny of subsection Potatoe ofSolanum based on data from morphology, bio-geography, ploidy level, and Endosperm BalanceNumber. The Endosperm Balance Number (EBN)relates to a strong isolating mechanism inSolanum sect. Petota and explains success or fail-ure of intra- and interspecific crosses, due to thefunctioning or breakdown of the endospermafter fertilization. The EBNs are hypotheticalgenetic factors independent of ploidy and empir-ically determined, relative to crossing with stan-dard test EBN crossers of known EBN. In potato,these are 2x(1EBN), 2x(2EBN), 4x(2EBN), 4x(4EBN),
and 6x(4EBN). Species can cross within EBN, irre-spective of ploidy (Hanneman, 1994).
Hawkes (1990) and Hawkes and Jackson (1992)informally designated subsets of Solanum specieswithin each superseries with more “primitive” or“advanced” morphological characters, providingfour groups, primitive Stellata, advanced Stellata,primitive Rotata, and advanced Rotata. In thistheory the Mexican diploids are primitive Stellata(in ser. Bulbocastana, Morelliformia, Pinnatisecta,and Polyadenia). All Solanum species in theseseries are 2x(1EBN). Representatives of theseprimitive Stellata in Solanum migrated to SouthAmerica and remained primitive Stellata (ser.Circaeifolia, Commersoniana, Lignicaulia,Olmosiana). These then evolved to advancedStellata, then to primitive Rotata, and finally toadvanced Rotata. The species in Mexico that areadvanced Rotata (S. verrucosum of ser. Tuberosa,and members of ser. Conicibaccata, Demissa, andLongipedicellata) either remigrated back fromSouth America, or are products of hybridizationbetween these remigrants and primitive indige-nous Mexican diploids. For the sake of simplicity,all Solanum species of primitive Stellata in theUnited States, Mexico, and Guatemala will herebe referred to as “primitive Mexican diploids.”
Spooner et al. (1991) investigated the Hawkes(1990) Stellata–Rotata hypothesis using chloro-plast DNA (cpDNA) restriction site analysis inSolanum series Bulbocastana, Conicibaccata,Demissa, Longipedicellata, Morelliformia,Pinnatisecta, Polyadenia, and Tuberosa. Theirresults gave partial support to the Hawkes (1989,1990) and Hawkes and Jackson (1992) hypothe-ses, by supporting most primitive Mexicandiploids as basal, and other Mexican species asadvanced. Some putative primitive Stellata fromSouth America, however, such as Solanum cir-caeifolium (ser. Circaeifolia), were later supportedas derived by further cpDNA restriction site stud-ies (Spooner & Sytsma, 1992; Spooner & Castillo,1997).
Two other cpDNA restriction site studies investi-gated relationships of Mexican potatoes.Spooner and Sytsma (1992) produced a clado-
TAXONOMY OF MEXICAN DIPLOID WILD POTATOES (SOLANUM SECT. PETOTA)
201
1 21
Bu
lboc
asta
naSo
lanu
m b
ulbo
cast
anum
Dun
al
2751
98
Haw
kes
1595
MEX
ICO
. Méx
ico:
Cer
ro d
e Co
coti
tlán
(Ryd
b.) H
awke
ssu
bsp.
bul
boca
stan
um(IB
UG
, MEX
U, P
TIS)
2 21
S.
bul
boca
stan
um27
5184
H
awke
s 15
81
MEX
ICO
. Fed
eral
Dis
tric
t: P
edre
gal d
e Sa
nsu
bsp.
bul
boca
stan
umÁ
ngel
, Ciu
dad
Uni
vers
itar
ia (I
BUG
, IEB
, M
EXU
, PTI
S, W
IS)
3 23
S.
bul
boca
stan
um27
5194
H
awke
s 15
91
MEX
ICO
. Oax
aca:
rui
ns o
f M
onte
albá
n su
bsp.
bul
boca
stan
um(IB
UG
, PTI
S)
4 14
S.
bul
boca
stan
um49
8224
O
choa
141
62
MEX
ICO
. Mic
hoac
án: L
as P
alm
itas
su
bsp.
dol
icho
phyl
lum
(Bit
ter)
Haw
kes
(CIP
, IBU
G, K
, PTI
S, W
IS)
5 21
S.
bul
boca
stan
um54
5752
Ta
rn e
t al
. 244
M
EXIC
O. M
éxic
o: E
of
Coat
epéc
Har
inas
, su
bsp.
dol
icho
phyl
lum
betw
een
San
Fran
cisc
o an
d Po
rfir
io D
íaz
(B, K
, IBU
G, P
TIS)
6 17
S.
bul
boca
stan
um25
5516
G
raha
m 3
00B
MEX
ICO
. Jal
isco
: Cui
dad
Guz
mán
(PTI
S)
subs
p. d
olic
hoph
yllu
m
7 28
S.
bul
boca
stan
um55
8379
Sp
oone
r et
al.
MEX
ICO
. Chi
apas
: 9.4
km
S o
f Rt
. 190
su
bsp.
par
titu
m(C
orre
ll) H
awke
s42
24(f
rom
nea
r Te
opis
ca),
to L
as R
osas
(IB
UG
, IN
IFA
P, P
TIS,
WIS
)
8 29
S.
bul
boca
stan
um27
5200
H
awke
s 17
96
GU
ATE
MA
LA. H
uehu
eten
ango
: rd.
fro
m
subs
p. p
arti
tum
Hue
huet
enan
go t
o Q
ueza
lten
ango
, 5
km S
of
Man
laca
tanc
ito
(C, K
, PTI
S)
Acc
essi
on
Map
Seri
es p
lace
men
t
Tax
on
PI3
Col
lect
or
Loc
alit
yN
umbe
r1Lo
calit
y2by
Haw
kes
(199
0)
TA
BL
E1.
Loca
tion
, ser
ies
affi
liati
on w
ithi
n So
lanu
m, P
I num
ber,
and
colle
ctor
, of
the
acce
ssio
ns e
xam
ined
in t
his
stud
y. H
erba
rium
vou
cher
s ar
e de
posi
ted
at P
TIS,
alon
g w
ith
othe
r kn
own
herb
aria
of
depo
siti
on in
the
loca
lity
colu
mn
(CIP
= In
tern
atio
nal P
otat
o Ce
nter
in L
ima,
Per
u, IN
IFA
P =
the
Mex
ican
Nat
iona
lPo
tato
Pro
gram
in T
oluc
a, M
exic
o).1
Acc
essi
on n
umbe
r co
rres
pond
s to
Fig
s. 2
, 4. 2
The
map
loca
lity
corr
espo
nds
to F
ig. 1
. SA
ref
ers
to S
outh
Am
eric
a an
dU
ref
ers
to lo
calit
ies
wit
h un
know
n lo
cati
on b
eyon
d th
e co
untr
y of
ori
gin.
3U
nite
d St
ates
Dep
artm
ent
of A
gric
ultu
re P
lant
Intr
oduc
tion
Num
bers
.
A FESTSCHRIFT FOR WILLIAM G. D’ARCY
Acc
essi
on
Map
Seri
es p
lace
men
t
Tax
on
PI3
Col
lect
or
Loc
alit
yN
umbe
r1Lo
calit
y2by
Haw
kes
(199
0)
9 27
S.
cla
rum
Corr
ell
5583
82Sp
oone
r et
al.
MEX
ICO
. Chi
apas
: 1.1
km
N o
f to
wn
4216
squa
re o
f El
Por
veni
r on
rd.
to
Silt
epec
guez
(IBU
G, I
NIF
AP,
PTI
S)
10
23
Mor
ellif
orm
iaH
awke
s S.
mor
ellif
orm
eBi
tter
& M
ünch
27
5220
H
awke
s 16
48
MEX
ICO
. Ver
acru
z: K
m 3
05.5
fro
m M
éxic
o on
the
rd.
fro
m L
as V
igas
to
Jala
pa,
Mal
país
de
La J
oya
(C, K
, P, P
TIS)
11
16
S. m
orel
lifor
me
27
5218
H
awke
s 16
13
MEX
ICO
. Méx
ico:
44.
5 km
fro
m t
he m
ain
Tolu
ca–M
orel
ia R
d., t
owar
ds V
alle
de
Brav
o fr
om T
oluc
a (C
, PTI
S, U
S)
12
16
S. m
orel
lifor
me
49
8003
Ta
rn e
t al
. 48
MEX
ICO
. Méx
ico:
abo
ut 1
8.5
km t
owar
ds
Valle
de
Brav
o fr
om T
oluc
a–Te
mas
calt
epec
, Hw
y. 1
34 v
ia R
anch
o Sa
n Ra
món
(PTI
S)
13
5 Pi
nnat
isec
ta(R
ydb.
) S.
bra
chis
totr
ichi
um(B
itte
r) R
ydb.
32
0265
H
awke
s 12
34
MEX
ICO
. Chi
huah
ua: M
ajal
ca, 4
0 m
i. H
awke
sN
W o
f Ch
ihua
hua,
nea
r th
e vi
llage
(C
, IBU
G, P
TIS,
US)
145
S. b
rach
isto
tric
hium
49
8217
O
choa
142
06
MEX
ICO
. Chi
huah
ua: R
t. C
hihu
ahua
–Ci
udad
Juá
rez
(CIP
, IBU
G, P
TIS,
US,
WIS
)
15
6 S.
bra
chis
totr
ichi
um
4979
93
Tarn
et
al. 1
3 M
EXIC
O. C
hihu
ahua
: La
Aur
ora
at 4
1 km
, al
ong
rd. t
o Sa
n Ju
anit
o fr
om t
he L
a Ju
nta
to Y
epac
hic
Hw
y. (P
TIS)
16
8 S.
bra
chis
totr
ichi
um
5458
32
Tarn
et
al. 2
07
MEX
ICO
. Agu
asca
lient
es: f
rom
Rin
cón
de
Ram
os o
n H
wy.
45,
30
km a
long
the
tra
ck
tow
ards
El C
hiqu
ihui
tillo
, Ran
cho
Tier
ra
Colo
rada
(BR,
C, K
, MEX
U, P
TIS)
TA
BL
E1
CO
NT
INU
ED.
202
203
TAXONOMY OF MEXICAN DIPLOID WILD POTATOES (SOLANUM SECT. PETOTA)
17
8 S.
bra
chis
totr
ichi
um
5458
15
Tarn
et
al. 2
15
MEX
ICO
. Agu
asca
lient
es: H
wy.
70,
abo
ut
33 k
m f
rom
Agu
asca
lient
es, 8
.2 k
m a
long
th
e tr
ack
past
Milp
illas
de
Arr
iba
tow
ards
Po
trer
o (P
TIS)
18
8 S.
bra
chis
totr
ichi
um
2555
27
Gra
ham
346
X
MEX
ICO
. Agu
asca
lient
es: b
etw
een
348
Agu
asca
lient
es a
nd C
alvi
llo (P
TIS)
19
22
S. c
ardi
ophy
llum
Lind
l. 34
7759
Ta
rn 2
41D
M
EXIC
O. P
uebl
a: le
ft s
ide
of T
ehua
cán–
subs
p. c
ardi
ophy
llum
Hua
juap
an d
e Le
ón r
d. o
n th
e Pu
ebla
sid
e of
bor
der,
Hw
y. 1
25, a
lmos
t at
bor
der
of
Pueb
la–O
axac
a st
ates
(PTI
S)
20
U
S. c
ardi
ophy
llum
2830
62
Gra
ham
s.n
. M
EXIC
O (P
TIS)
subs
p. c
ardi
ophy
llum
21
U
S. c
ardi
ophy
llum
2830
63
Gra
ham
s.n
. M
EXIC
O (P
TIS)
su
bsp.
car
diop
hyllu
m
22
11
S. c
ardi
ophy
llum
2792
72
Haw
kes
1458
M
EXIC
O. A
guas
calie
ntes
: 7 m
i. fr
om
subs
p. e
hren
berg
iiBi
tter
Agu
asca
lient
es o
n th
e rd
. to
Lore
to,
1.25
mi.
from
the
mai
n A
guas
calie
ntes
to
Zaca
teca
s H
wy.
(C, K
, PTI
S, U
S)
23
12
S. c
ardi
ophy
llum
1847
62
Haw
kes
1086
M
EXIC
O. Q
ueré
taro
: San
Jua
n de
l Río
, su
bsp.
ehr
enbe
rgii
SW o
f th
e to
wn
(ECO
N, P
TIS)
24
8 S.
car
diop
hyllu
msu
bsp.
ehr
enbe
rgii
3412
31
Com
mon
wea
lthM
EXIC
O. J
alis
co: K
m 2
01 f
rom
Po
tato
G
uada
laja
ra o
n th
e rd
. to
San
Luis
Pot
osí
Colle
ctio
n 23
02(P
TIS)
25
12
S. c
ardi
ophy
llum
subs
p. e
hren
berg
ii18
6548
H
awke
s 11
00
MEX
ICO
. Zac
atec
as: 7
26.5
km
fro
m M
éxic
o to
war
ds Z
acat
ecas
(LL,
PTI
S)
26
9 S.
car
diop
hyllu
msu
bsp.
ehr
enbe
rgii
2555
20
Gra
ham
371
M
EXIC
O. S
an L
uis
Poto
sí(P
TIS)
27
12
S. c
ardi
ophy
llum
subs
p. e
hren
berg
ii27
5213
H
awke
s 14
28
MEX
ICO
. Que
réta
ro: 3
km
SSE
of
tow
n (C
, K, P
TIS,
US)
Acc
essi
on
Map
Seri
es p
lace
men
t
Tax
on
PI3
Col
lect
or
Loc
alit
yN
umbe
r1Lo
calit
y2by
Haw
kes
(199
0)
A FESTSCHRIFT FOR WILLIAM G. D’ARCY
Acc
essi
on
Map
Seri
es p
lace
men
t
Tax
on
PI3
Col
lect
or
Loc
alit
yN
umbe
r1Lo
calit
y2by
Haw
kes
(199
0)
28
22
S. h
into
niiC
orre
ll
Spoo
ner
et a
l. M
EXIC
O. M
éxic
o: 6
.5 k
m S
W o
f 40
33Te
mas
calt
epec
–Val
le d
e Br
avo
rd.,
on r
d. t
o Sa
n Pe
dro
Tena
yac,
on
S si
de
of r
d., c
a. 5
0 m
dow
nstr
eam
of
brid
ge
over
rd.
(IN
IFA
P, P
TIS)
29
2 S.
jam
esii
Torr
. 45
8425
U
gent
&
U.S
.A. A
rizo
na: A
pach
e Co
., H
wy.
180
, Ru
hde
16-7
83.
2 km
S o
f Ea
ger
at t
he ju
ncti
on w
ith
Rt. 1
30 (W
IS, P
TIS)
30
2 S.
jam
esii
56
4051
Sa
las
et a
l. 24
U
.S.A
. Ari
zona
: Apa
che
Co.,
near
Nel
son
rese
rvoi
r, tw
o m
i. S
on R
t. 6
66 t
hen
E 1
mi.
on r
d. 2
75 (P
TIS)
31
3 S.
jam
esii
5640
49
Sala
s et
al.
21
U.S
.A. N
ew M
exic
o: C
atro
n Co
., Re
serv
e vi
cini
ty, a
t ab
out
6 m
i. N
E of
Ara
gon
on
Hw
y. 1
2, a
t 33
mi.
mar
ker
(fro
m H
wy.
180
) (P
TIS)
32
1 S.
jam
esii
2751
69
Haw
kes
1176
U
.S.A
. New
Mex
ico:
Gra
nt C
o., S
end
of
the
Big
Burr
o M
ount
ains
, Silv
er C
ity,
17
mi.
from
Lor
dsbu
rg (R
t. 1
80) (
K, P
TIS)
33
1 S.
jam
esii
45
8423
U
gent
&
U.S
.A. N
ew M
exic
o: G
rant
Co.
, Hw
y. 9
0,
Ruhd
e 7-
7843
.5 k
m S
W o
f Si
lver
Cit
y, G
ila N
atio
nal
Fore
st (P
TIS,
WIS
)
34
2 S.
jam
esii
27
5172
H
awke
s 12
07
U.S
.A. A
rizo
na: C
ochi
se C
o., C
hiri
cahu
a M
ount
ains
, Mon
ita
Cano
n, ju
st b
elow
Fa
raw
ay R
anch
at
the
edge
of
Nat
iona
l M
onum
ent
(K, P
TIS)
TA
BL
E1
CO
NT
INU
ED.
204
205
TAXONOMY OF MEXICAN DIPLOID WILD POTATOES (SOLANUM SECT. PETOTA)
35
20
S. ×
mic
hoac
anum
(Bit
ter)
55
8497
Sp
oone
r et
al.
MEX
ICO
. Mic
hoac
án: a
t km
21
mar
ker
Rydb
. (pu
tati
ve o
rigi
n is
40
77al
ong
Rt. 1
20 S
of
Mor
elia
, on
W s
ide
of
S. b
ulbo
cast
anum
×S.
pin
nati
sect
um)
Rt. 1
20 (I
NIF
AP,
PTI
S)
36
7 S.
nay
arit
ense
(Bit
ter)
Ryd
b.
5458
27
Tarn
et
al.
MEX
ICO
. Nay
arit
: nea
r St
a. A
nita
, abo
ut
270A
1.5
km N
E of
Sta
. Ter
esa
(PTI
S)
37
8 S.
nay
arit
ense
54
5820
Ta
rn e
t al
. M
EXIC
O. Z
acat
ecas
: Hw
y. 5
4, 1
0 km
SW
23
4Aof
Jal
pa, 2
6.3
km a
long
the
tra
ck t
owar
d Tl
alte
nang
o (K
, PTI
S)
38
8 S.
nay
arit
ense
54
5825
Ta
rn e
t al
. 225
M
EXIC
O. Z
acat
ecas
: Hw
y. 7
0, 1
2 km
NW
of
Jal
pa, 1
8 km
alo
ng t
he t
rack
SE
tow
ard
Tlac
hich
ila (K
, PTI
S)
39
13
S. p
inna
tise
ctum
Dun
al
2752
34
Haw
kes
1456
M
EXIC
O. J
alis
co: 1
5 m
i. fr
om L
eón
on t
he
rd. t
o A
guas
calie
ntes
(C, I
BUG
, K, M
EXU
, PT
IS, U
S)
40
11
S. p
inna
tise
ctum
18
4764
H
awke
s 10
93
MEX
ICO
. Gua
naju
ato:
Leó
n, 2
km
SE
of t
he
cem
ent
fact
ory
(BR,
IBU
G, K
, LL,
MEX
U,
MPU
, NY,
PTI
S, W
AG
)
41
14
S. p
inna
tise
ctum
27
5233
H
awke
s 14
55
MEX
ICO
. Gua
naju
ato:
2 m
i. fr
om S
iláo
on
the
rd. t
o G
uana
juat
o, E
l Cas
tillo
(C, I
BUG
, K
, MEX
U, P
TIS)
42
13
S. p
inna
tise
ctum
27
5236
H
awke
s 15
05
MEX
ICO
. Jal
isco
: 29
mi.
from
Gua
dala
jara
on
the
rd.
to
Mex
ico
City
, hac
iend
a de
H
uejo
tita
n (C
, IBU
G, K
, MEX
U, P
TIS,
US)
43
12
S. p
inna
tise
ctum
27
5230
H
awke
s 14
24
MEX
ICO
. Que
réta
ro: 3
km
SSE
of
tow
n, b
y th
e sm
all r
eser
voir
(IBU
G, K
, MEX
U, P
TIS)
44
20
S. p
inna
tise
ctum
19
0115
Ba
ldw
in14
374
MEX
ICO
. Mic
hoac
án: M
orel
ia (L
L, N
A, P
TIS)
45
13
S. b
ulbo
cast
anum
Rodr
ígue
z M
EXIC
O. J
alis
co: S
ierr
a de
La
Prim
aver
a ×
S.ca
rdio
phyl
lum
puta
tive
hyb
rid
2592
(PTI
S)
Acc
essi
on
Map
Seri
es p
lace
men
t
Tax
on
PI3
Col
lect
or
Loc
alit
yN
umbe
r1Lo
calit
y2by
Haw
kes
(199
0)
A FESTSCHRIFT FOR WILLIAM G. D’ARCY
Acc
essi
on
Map
Seri
es p
lace
men
t
Tax
on
PI3
Col
lect
or
Loc
alit
yN
umbe
r1Lo
calit
y2by
Haw
kes
(199
0)
46
11
S. ×
sam
buci
num
Rydb
. 59
5478
Ro
dríg
uez
MEX
ICO
. Que
réta
ro: f
oot-
hills
of
“El B
uey”
(p
utat
ive
orig
in is
S. e
hren
berg
ii25
63m
ount
ain,
on
junc
tion
of
road
s of
×
S. p
inna
tise
ctum
)Q
ueré
taro
–San
Lui
s Po
tosí
and
Mex
ico
City
–San
Lui
s Po
tosí
(F, I
BUG
, MIC
H, M
O,
NY,
PTI
S)
47
18
S.×s
ambu
cinu
m60
4209
Ro
dríg
uez
MEX
ICO
. Gua
naju
ato:
La
Purí
sim
a,
2565
mun
icip
alit
y of
San
Die
go d
e la
Uni
ón,
rd. f
rom
Que
réta
ro c
ity
to S
an L
uis
Poto
sí
(MIC
H, P
TIS)
48
10
S. s
teno
phyl
lidiu
mBi
tter
55
8460
Sp
oone
r et
al.
MEX
ICO
. Jal
isco
: rd.
fro
m N
sid
e of
41
04G
uada
laja
ra, p
ast
side
rd.
to
San
Fran
cisc
o Te
sist
án t
o Sa
n Cr
istó
bal d
e la
Bar
ranc
a,
on W
sid
e of
rd.
, 20.
5 km
(by
post
ed k
m
sign
s), N
of
Gua
dala
jara
, abo
ut 0
.5 k
m S
of
Mir
ador
(IN
IFA
P, P
TIS)
49
16
S. t
arni
iHaw
kes
& H
jert
. 49
8048
Ta
rn e
t al
. 79
MEX
ICO
. Hid
algo
: fro
m L
as T
ranc
as,
Hw
y. 8
5, Z
imap
an t
o Ja
cala
(K, P
TIS)
50
16
S. t
arni
i 54
5808
Ta
rn e
t al
. 257
M
EXIC
O. H
idal
go: t
urni
ng o
ff H
wy.
85
(Zim
apan
/Jac
ala)
at
Las
Tran
cas,
go
6.4
km
alon
g rd
. tow
ards
Nic
olás
Flo
res
(C, F
, K,
MEX
U, P
TIS,
WA
G)
51
15
S. t
arni
i 54
5742
Ta
rn e
t al
. 56
MEX
ICO
. Ver
acru
z: a
bout
14
km S
of
Hua
yaco
cotl
a, n
ear
Vib
orill
os, o
n rd
. to
Tula
ncin
go (K
, PTI
S)
52
16
S. t
arni
i 49
8043
Ta
rn e
t al
. 57
MEX
ICO
. Hid
algo
: 28
km S
of
Hua
yaco
cotl
a, 8
km
bef
ore
Agu
a Bl
anca
(C
, K, M
EXU
, PTI
S)
TA
BL
E1
CO
NT
INU
ED.
206
207
TAXONOMY OF MEXICAN DIPLOID WILD POTATOES (SOLANUM SECT. PETOTA)
53
16
S. t
arni
i 57
0641
H
jert
ing
7361
MEX
ICO
. Hid
algo
: 20
km f
rom
Agu
a Bl
anca
tow
ards
Hua
yaco
cotl
a, n
ear
Palo
Be
ndit
o (P
TIS)
54
17
S. t
rifi
dum
Corr
ell
2555
42
Cana
da
MEX
ICO
. Mic
hoac
án: K
m 4
8 on
the
O
ttaw
a 46
0Ca
rapa
n–U
ruap
an H
wy.
(PTI
S)
55
18
S. t
rifi
dum
55
8478
Sp
oone
r et
al.
MEX
ICO
. Mic
hoac
án: o
n rd
. fro
m R
t. 1
20
4283
NW
to
Cher
an, 2
.5 k
m S
E of
Ser
ina
(INIF
AP,
PTI
S, W
IS)
56
17
S. t
rifi
dum
55
8480
Sp
oone
r et
al.
MEX
ICO
. Mic
hoac
án: a
long
mic
row
ave
4089
Bto
wer
rd.
off
of
dirt
rd.
beg
inni
ng a
bout
5
km N
E of
Cap
acua
ro, 5
.9 k
m f
rom
be
ginn
ing
of t
his
mic
row
ave
tow
er r
d.
(INIF
AP,
PTI
S)
57
17
S. t
rifi
dum
28
3104
H
awke
s 15
41
MEX
ICO
. Jal
isco
: NE
slop
e of
Nev
ado
de
Colim
a, 1
3 km
fro
m t
urn
to T
olim
án o
n th
e rd
. fro
m C
iuda
d G
uzm
án, L
os A
lpes
(IB
UG
, K, M
EXU
, PTI
S)
58
U
S. t
rifi
dum
29
2213
U
gent
574
5 M
EXIC
O (P
TIS)
59
26
Poly
aden
iaBu
kaso
v S.
lest
eriH
awke
s &
Hje
rt.
5584
34
Spoo
ner
et a
l.
MEX
ICO
. Oax
aca:
on
W s
ide
of R
t. 1
25,
4155
3.4
km S
of
Rt. 1
90, j
ust
S of
San
Jua
n Te
posc
olul
a (IN
IFA
P, P
TIS,
WIS
)
60
26
S. le
ster
i 44
2694
H
awke
s et
al.
M
EXIC
O. O
axac
a: 2
5.8
km S
of
Mia
huat
lán
1714
on t
he O
axac
a–Pu
erto
Áng
el r
d. (K
, PTI
S)
61
26
S. le
ster
i 55
8435
Sp
oone
r et
al.
M
EXIC
O. O
axac
a: a
long
E s
ide
of R
t. 1
75,
4177
at k
m 1
34.1
, abo
ut 5
0 m
off
rd.
; up
a sl
ope,
S o
f M
iahu
atlá
n de
Por
firi
o D
íaz
(INIF
AP,
PTI
S, W
IS)
Acc
essi
on
Map
Seri
es p
lace
men
t
Tax
on
PI3
Col
lect
or
Loc
alit
yN
umbe
r1Lo
calit
y2by
Haw
kes
(199
0)
A FESTSCHRIFT FOR WILLIAM G. D’ARCY
Acc
essi
on
Map
Seri
es p
lace
men
t
Tax
on
PI3
Col
lect
or
Loc
alit
yN
umbe
r1Lo
calit
y2by
Haw
kes
(199
0)
62
22
S. p
olya
deni
umG
reen
m.
4980
36
Tarn
et
al. 6
5 M
EXIC
O. H
idal
go: H
wy.
85,
Zim
apan
to
Tam
zunc
hale
, at
Las
Tran
cas,
abo
ut 8
km
E
at J
ague
y on
the
tra
ck t
o N
icol
ás F
lore
s (P
TIS)
63
13
S. p
olya
deni
um
5584
50
Spoo
ner
et a
l.
MEX
ICO
. Jal
isco
: on
mic
row
ave
tow
er r
d.
4137
to C
erro
Gra
nde,
SE
of S
ante
Fé,
1.1
km
do
wnh
ill o
f to
p of
tow
n (IN
IFA
P, P
TIS,
WIS
)
64
24
S. p
olya
deni
um
1617
28
Corr
ell 1
4374
M
EXIC
O. M
icho
acán
: nea
r M
atug
eo
(ECO
N, G
AT,
MEX
U, P
TIS)
65
25
S. p
olya
deni
um
3477
69
Tarn
& G
ómez
M
EXIC
O. P
uebl
a: N
ew M
éxic
o–V
erac
ruz,
22
1to
ll rd
., at
km
208
.5 (K
, PTI
S)
66
19
S. p
olya
deni
um
3477
70
Tarn
& G
ómez
MEX
ICO
. Ver
acru
z: n
ear
Puer
to d
el A
ire,
23
4on
old
Ori
zaba
–Méx
ico
rd.,
Rt. 1
50,
betw
een
Puer
to a
nd V
erac
ruz–
Pueb
la
stat
e bo
unda
ry (K
, PTI
S)
67
SA
Circ
aeif
olia
Haw
kes
S. c
irca
eifo
lium
Bitt
er
4981
88
Haw
kes
et a
l.BO
LIV
IA. C
ocha
bam
ba: A
yopa
ya, n
ear
6581
Inde
pend
enci
a, Ic
hupa
mpa
(PTI
S)
68
SA
Com
mer
soni
aBu
kaso
vS.
com
mer
soni
Dun
al
5667
51
Hof
fman
s.n
. A
RGEN
TIN
A (P
TIS)
69
SA
Coni
ciba
ccat
aBi
tter
S.
san
tolla
lae
Varg
as
4733
72
Haw
kes
et a
l.
PERU
. Cuz
co: P
auca
rtam
bo, P
illah
uata
51
03(P
TIS)
70
SA
Lign
icau
liaH
awke
s S.
lign
icau
leVa
rgas
27
5273
E.
Bau
r
PERU
. Cuz
co: C
alca
, Pis
ac (P
TIS)
So
rtim
ent
1884
71
2 Lo
ngip
edic
ella
taS.
fen
dler
isub
sp. f
endl
eriH
awke
s 27
5163
H
awke
s 11
80
U.S
.A. A
rizo
na: C
ochi
se C
o., C
hiri
cahu
a Bu
kaso
vM
ount
ains
, Rus
tler
Par
k (K
, PTI
S)
72
16
Tube
rosa
(Ryd
b.)
S. v
erru
cosu
mSc
hltd
l.27
5260
H
awke
s 16
58
MEX
ICO
. Hid
algo
(AA
, BM
, C, K
, PTI
S)H
awke
s
TA
BL
E1
CO
NT
INU
ED.
208
TAXONOMY OF MEXICAN DIPLOID WILD POTATOES (SOLANUM SECT. PETOTA)
209
gram that divided the primitive Mexican diploidspecies into these clades in Solanum: (1) seriesPinnatisecta, with internested series Polyadenia,Bulbocastana (in part), and series Morelliformia;(2) S. bulbocastanum and S. cardiophyllum; and(3) S. verrucosum and all Mexican polyploidspecies. The second clade was unexpectedbecause all prior hypotheses suggested that S.bulbocastanum and S. cardiophyllum would asso-ciate with the first clade. Solanum verrucosum,on the other hand, was long recognized by mor-phological and crossing data to truly be distinctand to belong in the third clade. Rodríguez andSpooner (1997) reanalyzed the Solanum bulbo-castanum and S. cardiophyllum clade includingmore accessions and all the subspecies of eachspecies. The resulting tree maintained all the sub-species in the same clade except for all the acces-sions of S. cardiophyllum subsp. ehrenbergii. Thissubspecies was placed in the basal Mexicandiploid clade, most closely related to S. brachis-totrichium and S. stenophyllidium.
The basal relationship of the primitive Stellatawithin Solanum sect. Petota, including S. cir-caeifolium of series Circaeifolia, was furtherinvestigated by Kardolus (1998) using amplifiedfragment length polymorphisms (AFLP). Solanumcircaeifolium was shown to be primitive by AFLPsand nested with the Mexican diploids, unlikecpDNA results (Spooner & Castillo, 1997), sug-gesting a “chloroplast capture” event, known tobe common throughout angiosperms (Rieseberg& Brunsfeld, 1992). Its primitive position, howev-er, matched the Stellata/Rotata hypothesis ofHawkes (1989, 1990) and Hawkes and Jackson(1992).
Microsatellites are hypervariable molecular mark-ers (Tautz, 1989; Powell et al., 1996), consisting oftandem repeats of 1 to 6 bp (Provan et al., 1996b;Rubinsztein et al., 1999; Bichara et al., 2000). Theyhave been used in cultivar identification(Smulders et al., 1997; Lin & Walker, 1998),
germplasm analysis (Powell et al., 1996), thedevelopment of quantitative trait loci (QTL)(Arens et al., 1995; Areshchenkova & Ganal, 1999;Winter et al., 1999; Lorieux et al., 2000), to detectgenetic polymorphisms (Russell et al., 1997; Naderet al., 1999; Teulat et al., 2000), and in phyloge-netic studies (Kostia et al., 2000; Petren et al.,1999; Clisson et al., 2000; Raker & Spooner, 2002).In potato they have been used mainly for cultivaridentification (Milbourne et al., 1997; Schneider &Douches, 1997), to develop linkage groups(Milbourne et al., 1998), in germplasm identifica-tion (Provan et al., 1996b), in analysis of intra-spe-cific somatic hybrids (Provan et al., 1996a), and ingenetic analysis of anther-derived haploid pota-toes (Veilleux et al., 1995).
Milbourne et al. (1998) developed microsatelliteprimers and mapped them to all 12 linkagegroups of cultivated potato, S. tuberosum. Wewished to test their use to investigate the validityand interspecific relationships of the primitiveMexican diploids and other primitive andadvanced potatoes, including members ofHawkes’s (1990) primitive Stellata. Raker andSpooner (2002) showed these primers to be of useto distinguish the subspecies of S. tuberosum, butthis was the very germplasm base from whichthese primers were developed. We needed thesedata for our ongoing floristic studies of Solanumsect. Petota in North America, Mexico, andCentral America. This included investigations ofhybrid origins for some of these species. Thesehybrids and putative origins by Hawkes (1990) areS. ×michoacanum (S. bulbocastanum × S. pinnati-sectum), and S. ×sambucinum (S. cardiophyllumsubsp. ehrenbergii × S. pinnatisectum). In addi-tion, Rodríguez and Vargas-P. (1994) postulated anew unnamed hybrid of S. bulbocastanum × S.cardiophyllum (no subspecies designated foreither species). We include South American prim-itive Stellata species to test the Hawkes (1989,1990) and Jackson and Hawkes (1992) hypothesesof relationships in Solanum.
210
A FESTSCHRIFT FOR WILLIAM G. D’ARCY
MATERIALS AND METHODSPLANT MATERIALMore than one accession per taxon was analyzed(when available) from as many geographicallydispersed sites as possible (Table 1, Fig. 1). Theplants were grown in the greenhouse, becausemany of the primitive Mexican diploids do notthrive outdoors in Wisconsin. An average of threeplants per accession of the Mexican diploids weregrown, and one accession per series from theSouth American diploids. Solanum stenophyllidi-um had only one accession available as seeds, andwe analyzed two separate sets of three plants perthe same accession for morphological analysis.
MORPHOLOGY AND DATA ANALYSISMorphological characters were measured whenthe plants were in full flower (Table 2). Significantdifferences in character states for quantitativecharacters were studied by the Tukey-Kramer
test, and qualitative characters by the LikelihoodRatio Chi square test and Pearson Chi square testin JMP statistical software v. 3.1.5 (SAS InstituteInc., 1998). Phenetic analyses were performedwith NTSYS-pc (Rohlf, 1992). An operational tax-onomic unit (OTU) was the average of three indi-viduals per accession. Data were standardized(STAND), and similarity matrices (in SIMINT), aver-age taxonomic distance (DIST), Euclidean distance(EUCLID), Manhattan distance (MANHAT), andproduct-moment correlation (CORR) were gener-ated. Clustering was performed with theunweighted pair-group method, UPGMA (Sokal& Sneath, 1963), and Neighbor Joining, NJ (Saitou& Nei, 1987). Cophenetic correlation coefficients(COPH, in MXCOMP) were used to measure thedistortion between similarity matrices and theresultant four phenograms. Stepwise discriminateanalyses (SDA) were performed by SAS v.7 (SASInstitute Inc., 1998) using Stepwise discriminateanalysis (STEPDISC).
Figure 1. Distribution of wild potato accessions used in this study from the United States, Mexico, and Guatemala. Accessionsfrom South America are not mapped. Numbers correspond to generalized map localities in Table 1.
TAXONOMY OF MEXICAN DIPLOID WILD POTATOES (SOLANUM SECT. PETOTA)
211
TABLE 2.Morphological characters measured. All values are in cm, unless otherwise indicated. The 35 of 56statistically significant characters (P = 0.05, SAS Institute Inc., 1998) distinguishing all taxa (see text) aremarked with an asterisk (*).
STEM CHARACTERS1. Stem pubescence length (mm)*. 2. Stem pubescence posture: pointing up (1), pointing down (2),
pointing out (3)*. 3. Stem diameter (mm)*. 4. Stem wings: present (1), absent (2). 5. Stem shape: angular(1), rounded (2)*. 6. Pubescence type: glabrescent (1), pubescent (2)*.
LEAF CHARACTERS7. Leaf width*. 8. Ratio: leaf length/width*. 9. Number of leaflet pairs. 10. Leaflet position: alternate
(1), opposite (2). 11. Widest leaflet pair: terminal (1), most distal lateral leaflet pair (2), second most distal lateral leaflet pair (3)*. 12. Interstitial leaflets: present (1), absent (2)*. 13. Terminal leaflet width*. 14. Ratio:length/width of terminal leaflet blade*. 15. Length from widest point of terminal leaflet blade to base ofblade*. 16. Ratio: length from widest point of terminal leaflet blade to base of blade/length of terminalleaflet blade. 17. Petiolule length of terminal leaflet*. 18. Most distal lateral leaflet width*. 19. Ratio: lengthof most distal lateral leaflet blade/width of most distal lateral leaflet*. 20. Length from widest point of mostdistal lateral leaflet blade to base of blade*. 21. Ratio: length from widest point of most distal lateral leafletblade to base of blade/length of most distal lateral leaflet blade*. 22. Petiolule length of most distal lateralleaflet*. 23. Second most distal lateral leaflet width*. 24. Ratio: length of second most distal lateral leafletblade/width of second most distal lateral leaflet*. 25. Length from widest point of second most distal later-al leaflet blade to base of blade*. 26. Ratio: length from widest point of second most distal lateral leafletblade to base of blade/length of second most distal lateral leaflet blade. 27. Petiolule length of second mostdistal lateral leaflet*. 28. Abaxial leaf pubescence length (mm)*. 29. Abaxial leaf pubescence posture: point-ing up (1), pointing down (2), pointing out (3). 30. Lateral leaflet decurrency: decurrent (1), not decurrent(2)*. 31. Petiole wings: present (1), absent (2)*. 32. Petiole length*. 33. Shape of pseudostipular leaflet: pinnulate (1), lunate (2)*. 34. Spicy odor: present (1), absent (2)*.
FLORAL CHARACTERS (see Spooner & van den Berg, 1992, for illustrations of characters 49–52).35. Peduncle length. 36. Length from the base to the articulation of the pedicel*. 37. Ratio: pedicel
length/length from base to the articulation of the pedicel. 38. Length of calyx (mm). 39. Ratio: length ofcalyx lobe/length of calyx*. 40. Posture of calyx lobe: straight (1), bent over (2), curled around (3). 41.Symmetry of calyx: symmetrical (1), asymmetrical (2). 42. Length of calyx acumen (mm)*. 43. Ratio: lengthof calyx lobe/length of calyx acumen (mm). 44. Calyx color: green (1), purple (2), green and purple (3). 45.Position of calyx and corolla: alternate (1), opposite (2). 46. Style exsertion (mm). 47. Length of anther(mm). 48. Length of anther filament (mm). 49. Radius of the corolla measured from the center of the corolla to the tip of the corolla lobes (mm). 50. Ratio: Radius of the corolla measured from the center ofthe corolla to the tip of the corolla lobes/distance between the center of the flower and the junction ofthe corolla lobes (mm)*. 51. Width of the corolla lobe measured at the base of the corolla junction (mm)*.52. Ratio: width of the corolla lobe measured at the base of the corolla junction/length from a line drawnacross widest point of corolla lobes (mm). 53. Primary color of the abaxial surface of the corolla: white (1),creamy white (2), purple (3)*. 54. Secondary color of the abaxial surface of the corolla: white (1), creamywhite (2), purple (3). 55. Posture of style: erect (1), curved (2).
FRUIT CHARACTERS56. Fruit shape: globose (1), conical (2)*.
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A FESTSCHRIFT FOR WILLIAM G. D’ARCY
We used stepwise discriminant analysis to discov-er characters that best distinguished the ninemost phenetically similar primitive Mexicandiploid species (Solanum brachistotrichum, S. car-diophyllum subsp. ehrenbergii, S. hintonii, S. jamesii, S. ×michoacanum, S. nayaritense, S. stenophyllidium, S. trifidum, S. tarnii), with allprocedures as above.
DNA EXTRACTION, AMPLIFICATION, AND DNAFRAGMENT ANALYSISDNA was extracted using the protocol in Rakerand Spooner (2002). Forty-five mapped primerswere selected from Milbourne et al. (1998) thatwere designed for S. tuberosum and screened forability to amplify a fragment in these species:stm0001, stm0003, stm0007, stm0010, stm0013,stm0014, stm0017, stm0019, stm0024, stm0025,stm0028, stm0032, stm0037, stm0038, stm0051,stm0052, stm1003, stm1005, stm1008, stm1017,stm1020, stm1021, stm1024, stm1025, stm1029,stm1031, stm1041, stm1049, stm1055, stm1058,stm1064, stm1069, stm1100, stm1102, stm1104,stm1106, stm2005, stm2012, stm2013, stm2020,stm2022, stm2028, stm3009, stm3010, stm3016.Forward primers were labeled with fluorescentdyes FAM (blue), HEX (yellow), and TETRA(green), from PE Biosystems, to amplify individualaccessions of all of the species. Reaction condi-tions were optimized, modifying conditions listedin Provan et al. (1996b). Conditions for a 25 µlreaction included 1X PCR Buffer II (Perkin Elmer),1.5 mM MgCl2, 0.2 mM dNTPs, 0.4 µM of eachprimer pair, 1 unit of AmpliTaq Gold® (PerkinElmer), and 10–20 ng of DNA. The annealingtemperature followed Milbourne et al. (1998) or1ºC to 2ºC below (Table 3). All reactions wereamplified in a 9600 PE thermocycler with the fol-lowing times and temperatures: 1 cycle for 10min. at 94°C, 2 min. to anneal temperature, 5min. at 72°C, 29 cycles of 1 min. at 94°C, 45 sec. toanneal, 5 min. at 72°C, ending with a 72°C holdfor 45 min.
The microsatellite products for each individualwere pooled in dye combinations of 3 µl FAM: 6µl TETRA: 9 µl HEX, or in some cases each product
was analyzed separately. PCR products were sentto the Biotechnology Center at the University ofWisconsin-Madison for fragment separation.Products were processed as follows: 1.5 µlaliquots of PCR product were mixed with 1.8 µlsample loading buffer (80% formamide, 10mg/ml dextran blue, 5 mM EDTA at pH 8.0) and0.45 µl of TAMRA 500 molecular weight standard(PE Biosystems). The mixture was heated for 3min. at 95°C and then placed on ice.Approximately 1 µl of the cold samples wasloaded on a 5% LongRanger™ (FMC Bioproducts)polyacrylamide/6M urea gel in a PE Biosystems377XL DNA sequencing machine. Samples wererun at 3000 V with 2400 scans/hour in 36 cm well-to-read plates. Data were collected using a PEBiosystems DNA Sequencer data collection v. 2.0and analyzed with GeneScan v. 2.1 (PEBiosystems). Data were later imported in toGenotyper v. 2.1 PE Applied Biosystems for allelesizing, both automatically and manually. Peakswere scored as allele sizes in bp and manuallytranslated into binary presence/absence.
MICROSATELLITE DATA ANALYSISThe multistate matrix was imported to Microsat v.1.5d (Minch et al., 1997) to produce distancematrices with two models, absolute differenceand delta mu squared (∂µ)2, both of whichassume a step-wise mutation model (SMM)(Goldstein et al., 1995). New mutations followingthis model are obtained by adding or subtractingrepeats one by one (Goldstein et al., 1995). Thesedistance matrices were later imported into PAUP4.0b3a (Swofford, 1998) to build trees withUPGMA (Sokal & Sneath, 1963) and NJ (Saitou & Nei, 1987).
The binary matrix was imported into PAUP 4.0b3a(Swofford, 1998) to produce the distance matrixof Nei and Li (1979) that applies the infinite allelemodel (IAM), where every mutation produces anew allele of potentially infinite size (Kimura &Crow, 1964). The Nei and Li matrix was used tobuild UPGMA (Sokal & Sneath, 1963) and NJ(Saitou & Nei, 1987) trees.
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TAXONOMY OF MEXICAN DIPLOID WILD POTATOES (SOLANUM SECT. PETOTA)
Prim
er
Tag
Pr
imer
seq
uenc
eT
arge
ted
repe
atSi
ze r
ange
A
nnea
ling
C
hrom
osom
e Pe
rcen
t (b
p)Te
mp.
(°C
)m
issi
ng d
ata
stm
0003
-f
B
gg
a g
aa t
ca t
aa c
aa c
ca g
(a
c)9
(at)
9 10
1–17
0 48
X
II 4%
stm
0003
-r
aat
tgt
aac
tct
gtg
tg
t g
tg
stm
0007
-f
B
gg
a ca
a g
ct g
tg a
ag t
tt a
t (a
c)9
185–
234
52
XII
58%
stm
0007
-r
aat
tga
gaa
ag
a g
tg t
gt
gtg
stm
0014
-f
G
cag
tct
tca
gcc
cat
ag
g
(gt)
5 (a
t)7
(gt)
10
77–2
01
53
I 18
%
stm
0014
-r
taa
aca
atg
gta
gac
aag
aca
aa
stm
0051
-f
Y
tac
ata
cat
aca
cac
acg
cg
(a
c)7…
(ac)
7 (a
t)4
84–1
16
53
X
23%
stm
0051
-r
ctg
caa
ctt
ata
gcc
tcc
a
stm
1005
-f
B
atg
cct
ctt
acg
aat
aac
tcg
g
(gta
)6
160–
187
59
VIII
27
%
stm
1005
-r
cag
cta
acg
tg
g t
tg g
gg
stm
1017
-f
Gg
ac a
cg t
tc a
cc a
ta a
a (a
tt)5
11
5–13
7 48
IX
21
%
stm
1017
-r
aga
aga
ata
gca
aag
caa
stm
1024
-fB
at
a ca
g g
ac c
tt a
at t
tc c
cc a
a (t
tg)6
12
5–15
2 59
V
III
13%
stm
1024
-r
tca
aaa
ccc
aat
tca
atc
aaa
tc
stm
1029
-f
Y
agg
ttc
act
cac
aat
caa
ag
c a
(c)1
2 12
1–16
6 58
I
41%
stm
1029
-r
aag
att
tcc
aag
aaa
ttt
gag
gg
stm
1041
-f
B
gtt
gag
tag
aag
gag
gat
t
(gaa
)5
86–1
01
53
V
13%
stm
1041
-r
cct
ttg
tct
tct
gct
ttt
g
stm
1049
-f
G
cta
cca
gtt
tg
t tg
a tt
g t
gg
tg
(a
ta)6
17
6–21
2 58
I
27%
stm
1049
-r
agg
gac
ttt
aat
ttg
ttg
gac
g
stm
2022
-f
B
gcg
tca
gcg
att
tca
gta
cta
(c
aa)3
…(c
aa)3
16
7–26
6 57
II
16%
stm
2022
-r
ttc
agt
caa
ctc
ctg
ttg
cg
stm
3009
-f
B
tca
gct
gaa
cg
a cc
a ct
g t
tc
(tc)
13
131–
211
63
VII
4%
stm
3009
-r
gat
ttc
acc
aag
cat
gg
a ag
t c
TA
BL
E3
Prim
er p
airs
use
d in
th
e p
rese
nt
stu
dy.
Tag
co
des
ref
er t
o f
luo
resc
ent
dye
s: B
= b
lue
(FA
M),
G =
gre
en (
TETR
A),
Y =
yel
low
(H
EX).
Prim
er s
equ
ence
s,
targ
et r
epea
t, a
nd
lin
kag
e g
rou
p f
rom
So
lan
um
tu
ber
osu
mfr
om
Milb
ou
rne
et a
l. (1
998)
.
213
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A FESTSCHRIFT FOR WILLIAM G. D’ARCY
RESULTSMORPHOLOGYThirty-five of the 56 characters were significantlydifferent among all taxa (Table 2) as determinedby the Tukey-Kramer test and the LikelihoodRatio Chi square test and Pearson Chi square testfor qualitative characters in JMP v. 3.1.5 (SASInstitute Inc., 1998) statistical software. Thecophenetic correlation coefficient determinedthat the UPGMA tree constructed with EUCLIDhad the highest (best) value of r = 0.835, followedby DIST (r = 0.827), MANHAT (r = 0.812), andCORR (r = 0.747). These four similarity matriceswith NJ always provided lower r scores. Theresulting UPGMA dendrogram (Fig. 2) supportedmost of the species. We will concentrate discus-sion of the primitive Mexican diploid speciesbecause this morphological study was designedmainly to search for phenetic support of these.Figure 2 is labeled as clusters A–I for ease of discussion.
Cluster A is well defined and includes all speciesand subspecies in Solanum ser. Bulbocastana(Solanum bulbocastanum subsp. bulbocastanum,subsp. dolichophyllum, subsp. partitum, and S. clarum). Both of these species perfectly supportHawkes’s (1990) concept of this series. However,Solanum bulbocastanum subsp. dolichophyllum isintermixed with subspecies partitum. Cluster Bincludes both species in Hawkes’s seriesPolyadenia (S. lesteri, S. polyadenium), likewiseproviding support for this series.
Clusters C–I include all members of Hawkes’s(1990) series Pinnatisecta, plus other 2x(1EBN)species from South America in series Circaeifolia,Commersoniana, Lignicaulia, and S. santolallae(2x[2EBN], ser. Conicibaccata). Cluster C includes S.cardiophyllum subsp. cardiophyllum and S. ×sam-bucinum (both Solanum ser. Pinnatisecta) and S.circaeifolium (ser. Circaeifolia). Solanum ×sam-bucinum was of putative origin with none of thesebut with S. pinnatisectum and S. cardiophyllumsubsp. ehrenbergii and S. pinnatisectum. Cluster Dincludes S. pinnatisectum and S. lignicaule(ser. Lignicaulia). Cluster E includes species-specificsubclusters S. tarnii and S. trifidum that group
together, the putative hybrid S. ×michoacanum(S. bulbocastanum × S. pinnatisectum), one “mis-placed” or misidentified accession of S. brachisto-trichium, the sole accession of S. hintonii (ser.Pinnatisecta), and S. commersonii (ser. Commer-soniana). Cluster F includes both accessions of S.nayaritense (ser. Pinnatisecta) and the putativehybrid S. bulbocastanum × S. cardiophyllum (con-taining members of both ser. Bulbocastana andPinnatisecta; Rodríguez et al., 1995). Cluster G con-tains all accessions of S. jamesii (ser. Pinnatisecta),and cluster H all accessions of S. cardiophyllumsubsp. ehrenbergii plus one accession of S. brachis-totrichium (ser. Pinnatisecta). Cluster I containsfour of the six accessions of S. brachistotrichiumand both replicate groups of the sole accession of S. stenophyllidium (ser. Pinnatisecta), but they donot group adjacent to each other.
In summary, the primitive Mexican diploid speciesare well supported phenetically except for S.brachistotrichium. Solanum stenophyllidium isproblematic, perhaps due to a single accessionexamined, but groups with most accessions of S.brachistotrichium. The putative hybrid S. ×sam-bucinum groups between its parents, and theputative hybrids S. ×michoacanum and S. bulbo-castanum × S. cardiophyllum away from their par-ents. The principal component analysis (notshown) presents a very similar clustering, or lackof it, regarding S. brachistotrichium.
Stepwise discriminant analysis separated all taxaby the following 21 morphological characters (cf.see Fig. 3), arranged from highest to lowest dis-criminant ability: (1) lateral leaflet decurrency(character 30, Table 2); (2) stem pubescence type(6); (3) spicy odor of leaves (34); (4) presence ofinterstitial leaflets (12); (5) ratio: length fromwidest point of most distal lateral leaflet blade tobase of blade/length of most distal lateral leafletblade (21); (6) shape of pseudostipular leaflet(33); (7) abaxial leaf pubescence length (28); (8)ratio: length of second most distal lateral leafletblade/width of second most distal lateral leaflet(24); (9) presence of stem wings (4); (10) petiolulelength of most distal lateral leaflet (22); (11)widest leaflet pair (11); (12) ratio: length of most
TAXONOMY OF MEXICAN DIPLOID WILD POTATOES (SOLANUM SECT. PETOTA)
215
Figure 2. UPGMA dendrogram (Euclidean similarity option) based on 35 of 56 morphological characters that differ significantlyamong taxa. Species names and accession numbers, as in Table 1 and Figures 1 and 4, and phenetic groups as discussed in the text.
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A FESTSCHRIFT FOR WILLIAM G. D’ARCY
distal lateral leaflet blade/width of most distal lat-eral leaflet (19); (13) terminal leaflet width (13);(14) petiolule length of second most distal lateralleaflet (27); (15) petiole wings (31); (16) numberof leaflet pairs (9); (17) petiolule length of termi-nal leaflet (17); (18) most distal lateral leafletwidth (18); (19) length from widest point of sec-ond most distal lateral leaflet blade to base ofblade (25); (20) length from widest point of theterminal leaflet blade to base of blade (15); (21)leaf width (7).
We also used stepwise discriminant analysis todiscover characters that best distinguished thenine phenetically most similar primitive Mexicandiploid species. This analysis used the 14 morpho-logical characters, arranged from highest to low-est discriminant ability. Nine of these 14 are quan-titative character distributions and are graphical-ly displayed in Figure 3, while 5 character statedistributions are qualitative: (1) fruit shape: glo-bose in all species except S. hintonii and S. tri-fidum, which had conical fruits (56); (2) leafdecurrence: all are laterally decurrent except S.cardiophyllum subsp. ehrenbergii, S. hintonii, andS. tarnii, which are not (30); (3) shape of pseu-dostipular leaf: lunate in all species except pinnu-late in S. jamesii (33); (4) only S. ×michoacanumand S. tarnii lack interstitial leaflets (12); (5) ratio:length of second most distal lateral leafletblade/width of second most distal lateral leaflet(24); (6) abaxial leaf pubescence length (28); (7)length of calyx acumen (42); (8) petiole length(32); (9) width of second most distal lateral leaflet(23); (10) secondary color of abaxial surface ofcorolla: 100% white in S. hintonii, S. stenophyllid-ium, S. tarnii, and S. trifidum, only 42.8% white inS. brachistotrichium (the rest white with tones ofviolet), 16.6% white in S. cardiophyllum subsp.ehrenbergii and S. jamesii (the rest white withtones of violet), and 50% white in S. nayaritense(the rest white with tones of violet) (54); (11) styleexsertion (46); (12) length from widest point ofsecond most distal lateral leaflet blade to base ofblade (25); (13) stem diameter (3); (14) number ofleaflet pairs (9).
Significant differences in character states for thisreduced matrix of nine phenetically most similarprimitive Mexican diploid taxa, as determined bythe Tukey-Kramer test, found 21 characters (allquantitative) best distinguish these taxa. These 21characters include all 9 of the quantitative char-acters mentioned above as determined bySTEPDISC. The means, ranges, and standard devi-ations of these 21 characters are graphically pre-sented in Figure 3. It shows that most of thesecharacters overlap considerably in range, andprovide few species-specific character states use-ful for easy diagnoses of taxa. Some exceptions,however, are low number of lateral leaflet pairsin S. nayaritense (character 9), long petiolules(17), long petioles (32), and wide corolla lobes asmeasured at the base of the corolla junction (51)in S. hintonii. This trend of the sole to predomi-nant presence of overlapping character states todefine taxa is common in Solanum sect. Petota(Spooner & van den Berg, 1992; Spooner &Hijmans, 2001), and termed polythetic support. Apolythetic morphological species concept is onewhere species are defined where they have thegreatest number of shared features, no singlefeature of which is essential for group member-ship or is sufficient to make an organism a mem-ber of a group (Sokal & Sneath, 1963; Stuessy,1990). Stated otherwise, species are distinguishedonly by a complex set of character states thatoverlap in ranges.
MICROSATELLITE PHENETICSOnly 12 of the 45 primers we tried, from 8 linkagegroups as mapped by Milbourne et al. (1998) forSolanum tuberosum, gave useable results. Therewere amplification failures in these 12 rangingfrom 4% in stm0003 and stm3009, to 58% instm0007, with the average across all primers 22%(Table 3). It is frequent in cross-species amplifica-tion to reduce the annealing temperature inorder to obtain amplification (Ezenwa et al.,1998). A range of up to 5ºC less was attempted toachieve amplification when no product otherwisecould be obtained, but with futile results. Weconsidered an amplification successful when thepositive control (S. tuberosum) amplified, as wellas most of the primitive Mexican diploids.
TAXONOMY OF MEXICAN DIPLOID WILD POTATOES (SOLANUM SECT. PETOTA)
217
Figure 3. Means (dot), ranges (length of line), and one standard deviation of the mean (length of box) for 21 of the 35 statistical-ly significant morphological characters of the phenetically most similar diploid wild species from the United States, Mexico, andCentral America. Included here are only the quantitative characters; the number preceding the character corresponds to Table 2.All measurements are in cm except characters 1, 3, 28, 38, 42, 46, 48, and 51, which are in mm. Three-letter species codes fol-low Spooner and Hijmans (2001): bst = S. brachistotrichium; ehr = S. cardiophyllum subsp. ehrenbergii; hnt = S. hintonii; jam = S.jamesii; mch = S. ×michoacanum; nyr = S. nayaritense; stp = S. stenophyllidium; trf = S. trifidum; trn = S. tarnii.
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A FESTSCHRIFT FOR WILLIAM G. D’ARCY
Figure 4. Neighbor-joining dendrogram based on microsatellite data (Nei & Li, 1979, similarity). Species names and accessionnumbers as in Table 1 and Figures 1 and 2, and phenetic groups from Figure 2.
TAXONOMY OF MEXICAN DIPLOID WILD POTATOES (SOLANUM SECT. PETOTA)
219
The trees obtained from the two stepwise muta-tion model (SMM) matrices showed little to noconcordance with the morphology phenogram,that is, they failed to cluster well-defined mor-phological species (trees not presented). The infi-nite allele model (IAM) with UPGMA and NJ clus-tered taxa somewhat better but still very poorlyrelative to the morphology phenogram (Fig. 2).Our first analysis included all taxa, and NJ clus-tered taxa best (shown in Fig. 4), but still only pro-vided weak clustering of some accessions of somespecies (such as a partial clustering of Solanumcardiophyllum subsp. cardiophyllum, S. jamesii,and S. nayaritense, all members of ser.Pinnatisecta), but with failure to completely clus-ter these and most other species. To attempt bet-ter clustering of putatively related taxa, we ana-lyzed just the most phenetically similar primitiveMexican diploids S. brachistotrichium, S. cardio-phyllum subsp. cardiophyllum, subspecies ehren-bergii, S. nayaritense, and S. stenophyllidium (allwithin ser. Pinnatisecta). This reduced analysis(not presented) still intermixed accessions of allspecies except for S. cardiophyllum subsp. cardio-phyllum as in Figure 4; that is, it still showed littleto no species-specific clustering. Microsatellitesalso were of no use to investigate hybrid originsof S. ×michoacanum, S. ×sambucinum, and theputative hybrid S. bulbocastanum × S. cardiophyl-lum (Rodríguez & Vargas-P., 1994). There were noadditive species-specific bands present to testthese hypotheses.
DISCUSSIONCONCORDANCE OF MORPHOLOGICAL DATAWITH PRIOR HYPOTHESES OF RELATIONSHIPSAND CHLOROPLAST DNA DATAOur morphological results show areas of concor-dance and discordance with cpDNA results ofSpooner and Sytsma (1992), Rodríguez andSpooner (1997), and with prior hypotheses ofinterspecific relationships (Hawkes, 1989, 1990;Hawkes et al., 1988; Hawkes & Jackson, 1992). Forexample, all members of Solanum ser.Bulbocastana are well resolved and separatedfrom the rest of the Mexican diploids (Fig. 2, clus-ter A). However, S. bulbocastanum subsp. bulbo-
castanum was the only subspecies relatively wellsupported, not S. bulbocastanum subsp.dolichophyllum and subspecies partitum. Ourmorphological data also unite S. bulbocastanumand S. clarum, supporting Hawkes (1990) whounited them in Solanum ser. Bulbocastana.Chloroplast DNA data (Spooner & Sytsma, 1992;Rodríguez & Spooner, 1997), however, supportedthe sister-taxon relationship of S. bulbocastanumand S. cardiophyllum (Fig. 2, clusters A and C, inpart), not S. bulbocastanum and S. clarum.
Solanum lesteri and S. polyadenium (cluster B)form a good phenetic group, concordant withcpDNA results (Spooner & Sytsma, 1992) andHawkes’s (1990) placement in Solanum ser.Polyadenia. Solanum ×sambucinum clusters withneither of its putative parents (S. cardiophyllumsubsp. ehrenbergii and S. pinnatisectum).Solanum trifidum and S. tarnii (both ser.Pinnatisecta) also form a good phenetic group(cluster E), concordant with cpDNA data, andrecognition of their phenetic similarity by Hawkeset al. (1988). Although not examined for cpDNA,Hawkes (1990) intuitively noted a morphologicalsimilarity of S. hintonii and S. trifidum, concor-dant with our data (cluster E).
The cluster of Solanum nayaritense with theputative hybrid of S. bulbocastanum × S. cardio-phyllum (cluster F) is discordant with morpholog-ical interpretations (Rodríguez et al., 1995) as S.nayaritense has not been suggested as its pro-genitor. The phenetic relationship of S. jamesii(cluster G) and S. cardiophyllum subsp. ehren-bergii (cluster H) has never been suggestedbefore, but is reasonable based on cpDNA resultsthat show them to be part of the same clade(Spooner & Sytsma, 1992; Rodríguez & Spooner,1997). The phenetic separation of S. cardiophyl-lum subsp. ehrenbergii (cluster H) from sub-species cardiophyllum (cluster C) is concordantwith cpDNA data (Rodríguez & Spooner, 1997).The phenetic similarity of S. brachistotrichiumand S. stenophyllidium (cluster I) also has supportwith cpDNA data (Spooner & Sytsma, 1992),which places them on the same clade.
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MICROSATELLITE DATA ANALYSISThere are two divergent hypotheses ofmicrosatellite evolution and its reflection on bestanalytical methods for assessment of genetic dis-tances and phylogeny. Schlötterer and Tautz(1992) have shown slippage during replication,which would support the use of the simple muta-tion model (SMM) for analyses. However, Colsonand Goldstein (1999) studied microsatellite loci inDrosophila and found that many mutation eventsin microsatellites are more complex than assumedby SMM. They advised the use of sequence datato assess the accumulation of imperfections with-in the microsatellite repeat, reducing the numberof consecutive repeats. These imperfections canbe insertions, deletions, or base substitutions,which, along with stepwise mutation drift tosmaller sizes, have contributed to degradation ofmicrosatellite loci over time. The authors cautionagainst the use of microsatellite size alone forpopulation or phylogenetic relationships (Colson& Goldstein, 1999). The SMM has been supportedby phylogeny reconstructions in bovine species(MacHugh et al., 1997), fish (Ruzzante, 1998), andhuman populations (Valdes et al., 1993; Shriver etal., 1995; Kimmel et al., 1996). On the other hand,the infinite allele model (IAM) has been support-ed for data sets with numerous compoundmicrosatellites (Estoup et al., 1995), which wouldbe the case in our data set. In this study, the bestconcordance with morphological data was pro-duced with the IAM and the worst with the SMM,but both were very poor. The IAM was the mostuseful in analyzing subspecies differences in S.tuberosum (Raker & Spooner, 2002).
MICROSATELLITECROSS-SPECIES AMPLIFICATIONMicrosatellites are expensive to develop. One oftheir appealing features is their potential use inrelated groups beyond the mainly economicplants where they were developed. There are several examples of successful amplificationacross species. Microsatellites distinguished twoclosely related species in genus Sitobion(Hemiptera: Aphidoidea) (Figueroa et al., 1999).Microsatellites developed for the tropical treePithecellobium elegans were conserved in several
species of the tribe Ingeae (Dayanandan et al.,1997). Microsatellites from peach successfullyamplified fragments in apple, suggesting thatthey might be useful as well in other fruit crops(Cipriani et al., 1999). In the genus Dianthus,microsatellite loci developed from the EMBLdatabase were tested in 26 species within thegenus, and successful amplification implied thatthe species are closely related or that the primerregions are well conserved (Smulders et al., 2000).Eight of 17 microsatellite primer pairs designedfor Quercus petraea were successful in producingamplifications in the related genus Castanea, and4 of the 17 in Fagus. Twelve of these 136 amplifi-cations were cloned and sequenced and shown tobe homologous to Quercus petraea; nevertheless,the authors recognized a tendency for decreasingability to successfully amplify loci, with increasingevolutionary distance across the family(Steinkellner et al., 1997). The same relationshipbetween an increasing genetic distance anddecreasing success of microsatellite amplificationwas observed in cassava (Manihot esculentaCrantz), through tests with six Manihot wildspecies (Roa et al., 2000).
In addition to problems producing microsatellitefragments across species, there also are problemswith homology of the microsatellite fragmentthat is necessary for correct phylogenetic inter-pretations (Wendel & Doyle, 1998). For successfulamplification of homologous fragments the tar-get sequence for the primers and flankingsequence about the microsatellites must be suffi-ciently conserved (Ezenwa et al., 1998).Knowledge of these criteria is costly as it requiressequencing the PCR fragments.
In our study the primers were developed througha Genbank search of Solanum tuberosum(Milbourne et al., 1998). Chloroplast DNA data(Spooner & Sytsma, 1992) show the primitiveMexican diploids to be the most distantly relatedgroup to S. tuberosum in section Petota, andmicrosatellites performed poorly for phylogenet-ic reconstructions. Similar problems have beenfound in other groups. Sequence variation and/orvariation in the number of repeats has been
TAXONOMY OF MEXICAN DIPLOID WILD POTATOES (SOLANUM SECT. PETOTA)
found by Peakall et al. (1998), who used 31 soy-bean microsatellite loci in wild relatives andfound that cross-species amplification was low,mainly in closely related genera. When cross-species amplification was achieved there wassequence variation in the flanking regions andwithin the repeats (non-homologous amplifica-tion). Westman and Kresovich (1998) tried to useArabidopsis primer pairs to amplify microsatel-lites in the closely related genus Brassica, butmany amplified loci did not hybridize withmicrosatellite probes, and probably did not con-tain repeats. In animals, microsatellites derivedfrom two species of wasps successfully cross-species amplified in different related genera fromthe Vespidae. However, the loci from one specieswere more conserved than those from the other,warning that it is difficult to reach generaliza-tions regarding conservation rates using datafrom single species (Ezenwa et al., 1998). In avianmicrosatellites, 73 primer sets from 16 speciesacross nine families were tested on Queleaspecies. Only 22 of these 73 pairs producedhomologous amplification (Dallimer, 1999).
The issue of cross-species amplification is com-plex. As seen above, it can be achieved acrossgenera within families, or not even occur acrossspecies within a genus. To our knowledge there isno comprehensive summary that addresses thecomparative extent of microsatellite conservationor whether the failure of cross-species amplifica-tion is due to primer design or phylogenetic dis-tance. Clisson et al. (2000) sequenced primatemicrosatellites with primers designed in humansand found that alterations in the base composi-tion of repeats, and insertions and deletions inflanking regions, are important components ofthe variation but introduced homoplasy (samesize but different composition) reducing theirphylogenetic utility.
CONCLUSIONSMost of the species examined in our study weresupported by morphological characters, exceptSolanum brachistotrichium and possibly S. steno-phyllidium. However, microsatellites provided poorto no support for species. This could be caused bynon-homology of repeat length between the
primers, or in our study by not having enough datato provide phylogenetic signal. There continue tobe unanswered questions about microsatellitesregarding mutation processes, proper analyticaltools for phylogeny, and knowledge of the extentof cross-species amplification. Our results showthat microsatellites developed from S. tuberosumhave reduced utility to establish good morpholog-ical species from the phylogenetically distantUnited States, Mexican, and Central Americandiploid wild potato species. Lara-Cabrera andSpooner (2004) addressed the validity of thesespecies and their interrelationships with AFLP data.
NOTE IN PROOFThese results, and those of Lara-Cabrera andSpooner (2004), were instrumental in revising thewild potatoes of North and Central America(Spooner et al., 2004).
ACKNOWLEDGMENTSWe thank John Bamberg and staff of the UnitedStates Potato Genebank for germplasm and local-ity data; Charles Nicolet and staff of theUniversity of Wisconsin Biotechnology Center fortechnical help; Lynn Hummel and staff at WalnutStreet Greenhouse for help in growing plants; labpartners Brian Karas, Iris Peralta, Celeste Raker,and Sarah Stephenson for technical advice; PeterCrump for statistical advice; and RobertGiannattasio for artwork. We gratefully acknowl-edge the long collaboration of Bill D’Arcy, whoshowed great interest and support to us in thisproblem and many other issues in theSolanaceae. This study was supported by CONA-CYT (Mexico) scholarship number 116742 grantedto Sabina I. Lara-Cabrera, and by the UnitedStates Department of Agriculture.
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