Table Of ContentMadrono, Vol. 55, No. 4, pp. 280-284, 2008
MORPHOLOGICAL AND CYTOLOGICAL EVIDENCE FOR
HOMOPLOID HYBRIDIZATION IN lOCHROMA (SOLANACEAE)
Stacey DeWitt Smith', Vanessa J. Kolberg, and David A. Baum
Department of Botany, University of Wisconsin, 430 Lincoln Drive, Madison, WI 53706
[email protected]
Abstract
Previous phylogenetic and biogeographic studies of lochroma (Solanaceae) suggested that /.
ayabaceuse S. Leiva is a hybrid between /. cyaneiim (Lindl.) M. L. Green and /. lehmannii Bitter.
Chromosomecountsforthesethree taxa demonstrate that all have a haploidchromosome number of
n = \2, and thus that the formation of /. ayabaceuse did not involve changes in ploidy level. A
comparison ofvegetative and floral morphology revealed that /. ayabaceuse demonstrates a striking
intermediacy between /. cyaueinii and /. lehmannii. The results support the conclusion that /.
ayabacense is a homoploid hybrid between /. cyaneimi and /. lehmannii.
Resumen
Estudios filogeneticos y biogeograficos de lochroma han sugerido que /. ayabacense S. Leiva es un
hibridoentre /. cyaneiim(Lindl.)M. L. Greeny/. lehmanniiBitter. Recuentosdecromosomasdefmen
un numero haploide de n = 12 para estas especies y asi indican que la formacion de /. ayabacense no
involucre un cambio en el nivel de ploidia. Una comparacion de la morfologia vegetativa y floral
revelo que /. ayabacense tiene un forma intermedia entre /. cyaneiim and /. lehmannii. Estos resultos
respaldan la conclusion que /. ayabacense es un hibrido homoploide entre /. cyaneiim y I. lehmannii.
Key Words: Amotape-Huancabamba, chromosome number, homoploid hybridization, lochroma,
Solanaceae.
Interspecific hybridization has long been rec- case oflochroma ciyabacense, a species suspected
ognized as an important generator ofdiversity in of hybrid ancestry based on recent molecular
flowering plants (Grant 1981; Rieseberg 1997). phylogenetic studies (Smith and Baum 2006).
Hybrid speciation can occur via two modes: lochroma is a genus of approximately 25
allopolyploidy, in which the hybrid exhibits twice species distributed throughout the northern
the chromosome number of its parental species, Andes, from Colombia to Peru. Based on
and homoploidy, in which the hybrid has the conflicting placement across gene trees. Smith
same ploidy as both parents (Arnold 1997). and Baum (2006) identified three suspected
Allopolyploidy is considered to be the more hybrid taxa in lochroma, of which /. ayabacense
common mode of hybrid speciation because the provided perhaps the most compelling case. In
change in chromosome numbercreates an instant phylogenetic analyses of three nuclear regions
reproductive barrier between the hybrid and its (ITS, LFYmivon II and waxy), I. ayabacensewas
parents, facilitating persistence of the hybrid found to contain "divergent" alleles, which fell
species (Ramsey and Schemske 1998; Chapman either in the /. cyaneum group or in the /.
and Burke 2007). However, theoretical work lehmcmnii-I. squamosum group (Smith and Baum
suggests that homoploid hybridization can also 2006). We consider /. squamosum S. Leiva and
lead to stable hybrid lineages (Buerkle et al. Quipuscoa a synonym of /. lehmannii and will
2000), either though recombinational speciation treat it as such in this paper. This phylogenetic
(Stebbins 1959; Grant 1981) or through ecolog- information together with the geographic distri-
ical selection favoring novel hybrid phenotypes bution of/. ayabacense strongly suggested that it
e(Rxiaemspelbeesrgoeft alh.om19o9p9l).oiWdhilheybtrhiednuspmebceiratoiofncleraer- ilsehmtchmenipiroadnudct/. ofcyahnyeburmi.dizIantiothnisbpeatpweere,nw/e.
mains small (Rieseberg 1997), combining molec- examine the morphological features of /. ayaba-
ular phylogenetic studies with traditional lines of cense relative to its putative parents, and report
evidence holds promise for detecting additional on the results ofchromosome counts undertaken
instances (e.g., Ferguson and Sang 2001; Ho- to determine whether the formation of /.
warth and Baum 2005). Here we apply cytolog- ayabacense involved a change in ploidy level.
ical and morphological evidence to examine the
Methods
' Current address: Department of Biology, Box /. ayabacense occurs exclusively in the environs,
90338, Duke University, Durham, NC 27705. of Ayabaca, Peru, near populations of both /.
2008] SMITH ET AL.: HYBRIDIZATION IN lOCHROMA 281
stained with gelatin fuchsin, and twenty pollen
-80°
grains from each individual were measured to the
nearest 0.1 |j.m using a Zeiss AxioSkop 2
microscope with a 40x objective.
/
0°
Results
N / Anthers amtmroughly two-thirds themirmmature
J length (2.2 form/m. cyaneunu 2.1 for /.
ayahacenseand 3.5 for/. lehmannii) provided
s vx the best material for chromosome counts. Within
a single flower containing anthers at this size, we
^j\f \Peru were ableto observeall thestages ofmeiosis I and
II. Some cells could be counted at prophase I and
II, but themostcountable stage forall specieswas
\ jyj / telophase II. Chromosome counts for all species
{ 0 )(52.5 12(5 250 375 including /. ayahacense were n = 12 (Fig. 2),
consistent with previous counts in lochroma [I.
Fig. 1. Geographic distribution ofstudy taxa. Circles cycmeum, n = 12 (Ratera 1961; Mahadavian 1967;
are sites where /. cyaneum has been collected and Mehra and Bawa 1969) and I.fuchsioides, n = \2
squares where /. lelimanuii has been collected. /. (Ratera 1961)]. Pollen size for /. ayahacense fell
ayahacense is only found in Ayabaca, Peru, indicated within the range of the parents (Table 1),
by the star. consistent with the fact that all three share the
same ploidy level (Gould 1957; Kowal 1975).
cyaneum and /. lehmannii (Fig. 1). Buds for The vegetative and floral features of /.
chromosome counts were collected from individ- ayahacense appeared to be largely intermediate
uals of /. aycibacense and /. lehmannii near between its putative parents (Table 1). The plant
Ayabaca, and buds from /. cyaneum were habit, amount ofpubescence, numbers offlowers
collected from a greenhouse-grown accession. per inflorescence, and the form of the flowers
Vouchers for these sources are givemnmin Appendix (Fig. 2) fell exactly between /. lehmcmnii and /.
1. The buds, ranging from 4-20 in length, cyaneum. Size measurements (e.g., plant height)
were fixed in Camoy's solution (3 parts chloro- for /. ayahacense were within the ranges observed
form: 2 parts ethanol: 1 part acetic acid) for 12 in the putative parents although, for a few traits,
24 hr and then transferred to 70% ethanol for such as leaf length, /. ayahacense exhibited a
storage. Slides of meiocytes stained with aceto- wider range of variation than the parents. Also,
carmine were prepared using the "squash'' in some traits, /. ayahacense favored one of the
technique outlined by Beeks (1955) and modified putative parents. For instance, it largely lacks the
WbyLKowal(1975)andviewedwithaZeissstandard stem scales characteristic of /. lehmcmnii, but its
microscope with a lOOx oil immersion fruits are nearly included in the accrescent
objective. Twelve cells with chromosomes clearly fruiting calyx, resembling /. lehmannii rather than
visible in a single plane were counted for each /. cyaneum.
species. Images of meiotic figures from each Principal components analysis (PCA) provided
species were captured with an Axiocam Hrm additional evidence for the morphological inter-
mounted on a Zeiss Axioplan 2 microscope using mediacy of/. ayahacense. Variation across the14
the lOOX oil immersion objective. floral and vegetative traits (Appendix 2) was
Information on floral and vegetative morphol- largelycapturedbythe first fewcomponents, with
ogy of/. ayahacense and its putative parents was components 1 and 2 accounting for 73% (Fig. 3).
compiled from taxonomic descriptions (Bitter Thecomponentsclearly separatethetreetaxa into
1918; Shaw 1998; Leiva et al. 2003; Leiva and discrete clusters in morphospace with /. ayaha-
Lezama 2005) and observations of all available cense falling between its putative parents (Fig. 3).
specimens (see Appendix 1). Fourteen morpho-
logical traits (see Appendix 2) were scored for the Discussion
available specimens of the three taxa, and these
data were analyzed using principal components This cytological and morphological study,
analysis in JMP 7.0 (SAS Institute Inc., Cary, together with existing phylogenetic and biogeo-
NCW).e also measured pollen grain size, which is hgroampohpilcoiindforhmyabtriiodn,bsehtowwesentha/.t /c.yaaynaehuacmenasnedis/a.
known to be positively correlated with ploidy lehmannii. While the phylogenetic data (Smith
level (Gould 1957; Kowal 1975). Measurements and Baum 2006) provided clear evidence that /.
ofmature pollen grains were taken from individ- ayahacense carries alleles derived from both the /.
uals used for chromosome counts. Pollen was cyaneum lineage and the /. lehmanniilineage, they
c
MADRONO
282 [Vol. 55
Fig. 2. Flowers,meioticfiguresandpollengrainsfrom/. cyaneum,I. ayabacenseand/. lehmannii(toptobottom).
The haploid number of/? = 12 chromosomes is visible in all three taxa.
did not indicate whether /. ayabacense was a sufficient to merit specific recognition (Leiva and
homoploid orpolyploidhybrid. Thechromosome Lezama 2005), it remains to be seen whether the
counts and the pollen measurements presented species can maintain reproductive isolation from
here indicate that /. ayabacense has the same its parents. At present, populations of /. ayaba-
ploidy level as its parents {n = 12). Also, the cense are largely separate from populations of/.
morphological data demonstrate that /. ayaba- cyanewn and /. lehmannii, perhaps due to
cense is intermediate between its two parents in a differences in microhabitat preferences (Smith
wide array offloral and vegetative traits. and Baum 2006). However, extensive searching
Although the morphological distinctiveness during a recent collecting trip revealed one
and cohesiveness of /. ayabacense was deemed apparent backcross individual (/. ayabacense X
Table 1. Morphological Differences Among /. cyaneum, I. ayabacenseand I. lehmannii. ' This range
is larger than that given in the species description, which appears to have been based on immature fruits.
/. cvcmeum I. ayabacense I. lehmannii
Shrub, 1.5-3 m Shrub to small tree, 3^ m Small tree, 4-10 m
Young stems green, densely Young stems green or purplish Young stems purplish and covered in
covered in branched hairs toward the tips, covered in triangular scales
branched hairs, with occasional
small triangular scales
Ovate to elliptic, 10-23 cm by Elliptic to lanceolate, 8-22.5 cm by Elliptic to lanceolate, 4-18.5 cm by
5-10 cm 5-10.5 cm 2.5-10 cm
10-20 flowers per inflorescence 3-8 flowers per inflorescence 2-6 flowers per inflorescence
Calyx 5-9 mm, slighty to Calyx 5 7 mm, slightly inflated in Calyx 5-7 mm, not inflated
markedly inflated in flower flower
Corolla tubular, 2.5-4 cm long, Corolla tubular to funnel-shaped, Corolla funnel-shaped to
deep purple, largely glabrous 2.6-4.0 cm, yellow with purple, campanulate, 2-3.5 cm, yellow-
but with hairs around the interior glabrous, exterior with green, interior glabrous, exterior
mouth ofthe tube few to many hairs pubescent
Filaments 2.5^ cm, hairy toward Filaments 1.6-2.2 cm, with sparse Filaments 1.5-2 cm, glabrous orwith
the base hairs toward the base sparse hairs at the base
mm mm mm
Anthers 3-4 Anthers 3.5-6 Anthers 4-7
Pollen 25-33 |im (mean = 28) in Pollen 25-33 |im (mean = 28) in Pollen 28-33 jam (mean = 30) in
diam. diam. diam.
Style 2^ cm Style 1.8-2.8 cm Style 1.2-1.6 cm
Berry conical, 1.8-2.5 cm long, Berry globose to slightly conical, Berry globose to slightly conical,
bottom third enveloped in 1-1.5 cm long', largely enveloped 1-1.6 cm, largely enveloped in
fruiting calyx in fruiting calyx fruiting calyx
2008] SMITH ET AL.: HYBRIDIZATION IN IOCHROMA 283
Literature Cited
2- Arnold, M. L. 1997. Natural hybridization and
evolution. Oxford University Press, Oxford, UK.
(N « 01 -o BeekTse,chRn.iquMe.fo1r95P5l.antImCphrorvoemmoesnotmses.inAtlhiesoS3q:u1a3s1h-
^ 133.
*-4 -3 -2 -1 1^ 2," 3 4, Bittcearn,aeG.adh1u9c18.genSeorliabnuascefaalesisquadastctroirptaaeu.stRreop-earmteorri--
-1 ium Specierum Novarum Regni Vegetabilis 15:
149-155.
-2 Buerkle, C. a., R. J. Morris, M. A. Asmussen, and
L. H. RiESEBERG. 2000. The likelihood of homo-
-3 - ploid hybrid speciation. Heredity 84:441^51.
Chapman, M. A. and J. M. Burke. 2007. Genetic
Component divergence and hybrid speciation. Evolution 61:
1
1773-1780.
Fig. 3. Principal components analysis ofmorpholog- Ferguson, D. M. and T. Sang. 2001. Speciation
ical measurements from /. cyaneum (solid diamonds), /. through homoploid hybridization between allote-
ayahaceme (open diamonds) and /. lehmannii (solid traploids in peonies (Paeonia). Proceedings of the
squares). Component 1 accounts for 60.7% of the NationalAcademyofSciences, USA98:3915-3919.
variation, and component 2 for 12.7%. HowARTH, D. G. AND D. A. Baum. 2005. Genealog-
ical evidence ofhomoploid hybrid speciation in an
adaptive radiation of Scaevola (Goodeniaceae) in
/. lehmannii), suggesting that introgression re- the Hawaiian Islands. Evolution 59:948-961.
mains possible. Also, /. cyaneum, I. lehmanniiand Grant,V. 1981. Plantspeciation. ColumbiaUniversity
/. ayabacense in Ayabaca are all pollinated by the Press, New York, NY.
same two species of hummingbird, Adelomyia Gould, F. W. 1957. Pollen sizeasrelatedtopolyploidy
melanogenys and Coeligena iris (Smith et al. and speciation in the Andropogon saccharoides-A.
barbinodis complex. Brittonia 9:71-75.
2008), suggesting that pollen flow could occur KowAL, R. R. 1975. Systematics ofSenecio aureus and
between the species when they occur in close allied species on the Gaspe Peninsula, Quebec.
proximity. However, individual hummingbirds Memoirs ofthe Torrey Botanical Club 23:1-113.
appear to exhibit preference for particular Leiva G., S., P. Lezama A., and V. Q. Silvestre.
lochroma species, thereby limiting interspecific 2003. lochroma salpoanum y I. squamosum (Sola-
pollen flow (Smith et al. 2008). naceae: Solaneae) dos nuevas especies Andinas del
The frequency and evolutionary significance of Norte del Peru. Arnaldoa 10:95-104.
interspecific hybridization in lochroma merits and p. Lezama A. 2005. lochroma albianthum
additional investigation. Within the last fifteen e lochroma ayabacense (Solanaceae: Solaneae): dos
years, nine new species of lochroma have been Anurenvaalsdoeasp1e2ci:e7s2-d8e0l. Departamento de Piura, Peru.
described from the area of greatest overlap in Madhavadian, p. 1967. The cytology of lochroma
species distributions, the Amotape-Huanca- tubulosa Benth. Caryologia 20:309-315.
bamba zone of Ecuador and Peru, and several Mehra, p. N. and K. S. Bawa. 1969. Chromosomal
new species have yet to be described (S. Leiva, evolution in tropical hardwoods. Evolution 23:
HAO, pers. comm.). While a hybrid origin has 466-481.
been suggested for some ofthese taxa (Smith and Ramsey, J. and D. W. Schemske. 1998. Pathways,
Baum 2006), at least six of these new (described mechanisms and rates of polyploid formation in
and undescribed) taxa have yet to be included in flowering plants. Annual Review of Ecology and
Systematics 29:467-501.
phylogenetic, morphological and biogeographic Ratera, E. L. 1961. Estudios cariologicos en Solana-
studies. Acknowledging the possibility ofhybrid- ceas. Revista del Instituto Municipal de Botanica
ization between lochroma species, it is imperative 1:61-65.
to consider the geographic distribution, the RiESEBERG, L. H. 1997. Hybridoriginsofplant species.
morphological distinctiveness and the stability Annual Review of Ecology and Systematics
of any newly-found form before granting it 28:359-389.
recognition as a new species. , M. A. Archer, and R. K. Wayne. 1999.
Transgressive segregation, adaptation and specia-
Acknowledgments tion. Heredity 83:363-372.
Shaw, J. M. H. 1998. lochroma - a review. The New
The authorsthank S. Leiva forassistance inthefield, Plantsman 5:154-191.
R. R. Kowal for advice in preparing squashes, and F. Smith, S. D. and D. A. Baum. 2006. Phylogenetics of
Lutzoni, K. Pryer and G. Wray for use of their the florally diverse Andean clade lochrominae
microscopes. We also thank the curators of the (Solanaceae). American Journal of Botany 93:
following herbaria for providing material on loan: 1140-1153.
MO, NY and WIS. S. D. S. gratefully acknowledges , S. J. Hall, P. R. Izquierdo, and D. A.
financial support from the National Science Founda- Baum. 2008. Comparative pollination biology of
tion grant DEB-0309310. sympatric and allopatric Andean lochroma (Sola-
MADRONO
284 [Vol. 55
naceae). Annals ofthe Missouri Botanical Garden lochroma lehmannii
95:59^613. *PERU, Piura, Ayabaca, carretera Ayabaca-Piura,
Stebbins, G. L. 1959. The role of hybridization in 4.6596°S 79.7404°W, S. D. Smith, S. Leiva G. 330
evolution. Proceedings ofthe American Philosoph- (HAO, F, MO, NY, USM, WIS), 14/1/04.
ical Society 103:231-251. PERU, Piura, Ayabaca, Bosque Cuyas. 4.6035°S
79.7110°W, S. D. Smith, S. Leiva G. 339 (HAO, F,
Appendix 1 MO, NY, USM, WIS), 15/1/04.
PERU, Piura, Ayabaca, Bosque Cuyas. S. Leiva G.,
N. W. Sawyer, V. Quipuscoa2028 (F), 6/VI/97.
Herbarium Specimens Used for ECUADOR, Canar, Km 15 Chunchi-Zhud, 2.3480°S
Comparative Morphology 78.9377°W, S. D. Smith, L. Lopez 486 (F, QCNE,
MO, WIS), 8/1/05.
Vouchers for chrorhosome counts and pollen mea- ECUADOR, Canar, Km 20 Chunchi-Zhud, 2.3555°S
surements are indicated with asterisks. 78.9590°W, S. D. Smith, L. Lopez487(QCNE, MO,
NY
WIS), 8/1/05.
lochroma ayabacense ECUADOR, Chimborazo, Carretera Alausi-Baguil-
*PpEG.aRmU3p3,a7,(P4iH.uAr6aO1,4,6°AFS,ya7Mb9Oa.c,7a1,1N8Yc,Wa,rUreSSt.Mer,Da.WIASmSyi)at,bha,1c5aS/.-1/Y0La4e.ciuv-a ECG7U9uA.a0mD3oO3t3Re°,,W,VC.Aa.nZaarAkl,v2aK3r7em5z {2a1MnOdC)hM,u.n1clTh/iiVrI-aIZdIho/u1d918,477.25.4(3M3O3)°,S
PERU, Piura, Ayabaca, 4.6456°S 79.7192"W, S. D. 9/Vn/1995.
Smith, S. J. Hall 351 (HAO, F, MO, NY, USM,
WIS), 16/1/04. Appendix 2
PERU, Piura, Ayabaca, Barrio San Jose Obrero,
4.6455 S 79.7178 W, S. D. Smith 500 (QCNE,
WIS), 30/1/05. Characters Scored for Principal
lochroma cyaneum Components Analysis
*USA, Wisconsin, Madison, Grown from seed from a
plant grown by W. G. D'Arcy at the Missouri Leaflength
Botanical Gardens, original collection likely to be Leafwidth
Plowman 4594 from Azuay, Ecuador, S. D. Smith Pedicel length
265 (WIS), 20/V/2003. Calyx width
PERU, Piura, Ayabaca, carretera Ayabaca-Piura, Calyx length
4.6514 S 79.7362°W, S. D. Smith, S. Leiva G. 329 Corolla width at base
(HAO, F, MO, NY, USM, WIS), 14/1/04. Corolla width at mouth
PERU, Piura, Ayabaca, Yacupampa, 4.6194 S Corolla length
79.7106°W, S. D. Smith, S. Leiva G. 336 (HAO, F, Corolla pigmentation (0 = no purple, 1 = light purple,
MO, NY, USM, WIS), 15/1/04. 2 = deep purple)
PERU, Piura, Ayabaca, Barrio San Jose Obrero, Corolla pubescence (0 = glabrous or few hairs, 1 =
4.644rS 79.7185"W, S. D. Smith 502 (QCNE, pubescent)
WIS), 30/1/05. Style length
ECUADOR, Loja, Cerro Sozoranga, 4.3553°S Filament length
79.7022°W, P. M. Jorgensen, C. Ulloa, H. Vargas Anther length
G. Abendano 628 (MO), 29/IV/1994. Flowers per inflorescence
