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(American Journal of Botany. 1999;86:302-305.)
© 1999 Botanical Society of America, Inc.

Three groups of species in Petunia sensu Jussieu (Solanaceae) inferred from the intact seed morphology1

Hitoshi Watanabe 2 , Toshio Ando 2, 5, Eisho Nishino 2 , Hisashi Kokubun 2 , Tatsuya Tsukamoto 2 , Goro Hashimoto 3 and Eduardo Marchesi 4

2Faculty of Horticulture, Chiba University, 648 Matsudo, Chiba 271–8510, Japan; 3Centro de Pesquisas de História Natural, 618 Rua Jaime Ribeiro Wright, Itaquera, São Paulo 08260–070, Brazil; and 4Facultad de Agronomia, Universidad de la República, Garzon 780, Montevideo, Uruguay

Received for publication May 26, 1998. Accepted for publication November 5, 1998.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
The intact seed surface morphology in 45 taxa of Petunia sensu Jussieu native to South America (Petunia sensu Wijsman plus Calibrachoa) was compared under scanning electron microscopy. The existence of three groups of species, differentiated in terms of seed morphology, was revealed as follows: (1) all species of Petunia sensu Wijsman, having coarse wavy middle lamellae and anticlinal walls; (2) Calibrachoa parviflora and C. pygmaea, having fine wavy middle lamellae embedded in straight anticlinal walls; and (3) the other species of Calibrachoa, having straight middle lamellae and anticlinal walls. Close relationships between seed morphology and the other characteristics observable in the groups of species are discussed.

Key Words: Calibrachoa • Calibrachoa parviflora; Calibrachoa pygmaea • Petunia • scanning electron microscopy (SEM); seed surface morphology • Solanaceae


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
In considering the genus Petunia, the recognition of two different definitions, Petunia sensu Jussieu and sensu Wijsman, is necessary. The genus Petunia was established by Jussieu (1803) with the type species P. parviflora and P. nyctaginiflora [= P. axillaris (Lam.) Britton, Sterns & Poggenb.] (syntype), which will be referred to as Petunia sensu Jussieu in the present study. Wijsman and de Jong (1985) studied the species of Petunia sensu Jussieu and distinguished two groups of species within the genus. However, their revision caused undesirable consequences for horticulturists because the garden petunias should have had their name changed according to this revision. The proposal for conserving the name Petunia as the generic name for garden petunias (Wijnands et al., 1986 ) was approved (Nicolson, 1991 ), and Wijsman (1990) separated the genus Calibrachoa La Lave & Lex. (1825) [type; C. parviflora (Juss.) Wijsman] from the genus Petunia (conserved type; P. nyctaginiflora). The latter will be referred to as Petunia sensu Wijsman.

However, Wijsman (1990) hesitated to assign about a dozen species to be either Petunia sensu Wijsman or Calibrachoa at the time of separation, and left them in the genus Petunia sensu Jussieu (Ando, Ueda, and Hashimoto, 1992 ). Thereafter, Stehmann and Semir (1997) determined nine of these remaining species as Calibrachoa, and so the genus Calibrachoa is now considered to comprise 25 species. However, several species have not been reclassified as yet and still remain in the genus Petunia sensu Jussieu even today (Table 1).


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  		Table 1. Voucher specimens of Petunia sensu Wijsman and Calibrachoa taxa examined in this study and the harbaria depositing them. Native taxa, collector's number and herbaria depositing the specimens are shown.a

 
For Petunia sensu Wijsman, seven new species have been added (Ando and Hashimoto, 1993 , 1994 , 1995 , 1996 , 1998 ), and Petunia occidentalis R. E. Fr. has been resurrected (Tsukamoto et al., 1998 ) by us, and thus this genus now comprises 15 species.

Wijsman and de Jong (1985) distinguished species of Calibrachoa and Petunia sensu Wijsman by the following characters: Calibrachoa—2n = 18, small shrubs, flower limb white or purple with yellow or at least pale tube, one valve formed by two corolla lobes covering three corolla lobes (conduplicate aestivation), telomeric heterochromatin present, calyx lobes less deeply incised (pentafid); and Petunia sensu Wijsman—2n = 14, large herbs, flower white or entirely purple, cochlear aestivation, telomeric heterochromatin absent, calyx lobes deeply incised (pentapartite).

The features of the seed surface are known to be of considerable value in plant taxonomy. Even though illustrations (Souèges, 1907 ) and scanning electron microscope (SEM) features (Muszynski, Kocon, and Guzewski, 1986 ) of the seed surface in a few members of Petunia sensu Wijsman (P. nyctaginiflora, P. violacea Lindl., and cultivars of P. hybrida Vilm.) were represented before, no comparison of the seed surface between any species of Petunia sensu Wijsman and Calibrachoa has been done until the work of Bahadur, Venkateshwarlu, and Swamy (1989) , who studied SEM features of the seed surface in eight native species of Petunia sensu Jussieu, as well as those in a cultivar of Petunia hybrida. When synonymous names and subspecies were resolved, and the cultivar was ignored, however, their materials represented Petunia axillaris (= P. nyctaginiflora), P. integrifolia (Hook.) Schinz & Thell. (= P. violacea), Calibrachoa linearis (Hook.) Wijsman [= P. linearis (Hook.) Paxt.], and C. parviflora (= P. parviflora). Their key for identification of species in terms of seed surface features gave a hint of an additional index character discriminating Calibrachoa from Petunia sensu Wijsman: the anticlinal walls of the seed surface in Calibrachoa are straight, whereas those in Petunia sensu Wijsman are wavy.

When Stehmann and Semir (1997) transferred nine species from Petunia sensu Jussieu to Calibrachoa, they referred to this index character without mentioning the original source; they did not cite the work of Bahadur, Venkateshwarlu, and Swamy (1989) . In order to check the validity of the index character mentioned above, in this work we observed the SEM features of the seed surface over the total range of native taxa in Petunia sensu Jussieu.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 LITERATURE CITED
 
Seed samples from a total of 45 taxa, including undescribed ones, in Petunia sensu Jussieu were obtained from the voucher specimens collected from the native habitat in Argentina, Brazil, and Uruguay (Table 1). The taxa of 2n = 14 chromosomes (20 taxa) and of 2n = 18 chromosomes (25 taxa) represented in previous papers (Watanabe et al., 1996 , 1997 ) can be regarded as Petunia sensu Wijsman and Calibrachoa, respectively. Even though several Calibrachoa species are still treated as Petunia sensu Jussieu (Table 1) as mentioned earlier, they will be included in Calibrachoa in the further considerations.

In some Solanaceous plants, such as in Solanum, whose seeds have an apparently smooth surface lacking characteristic features, enzyme treatment of seeds to remove periclinal walls of the seed surface cells is recommended, and the intricate structural patterns beneath the periclinal walls, observable under SEM, are of taxonomic value (Lester and Durrands, 1984 ; Knapp and Helgason, 1997 ). In Petunia sensu Jussieu, however, only the morphology of the intact seed surface seems to have taxonomic importance. Seed surface features described below are observable even in the intact seeds, but they were subjected to a treatment in order to shake off the dust on the surface as follows; mature seeds were soaked in a dilute solution of sodium hypochlorite (active chlorine, 5%) with 1% Tween 20, allowed to stand in an ultrasonic wave bath (model Bransonic 12; Branson Cleaning Equipment Co., Shelton, Connecticut, USA) for 3–5 min, held stationary for five more min, rinsed with distilled water, and then dried on filter papers for 1 wk. After they were mounted on stubs using conductive double-stick tape, the seeds were coated with gold-palladium (model JFC-1100; JEOL Co., Ltd., Akishima, Tokyo, Japan) for 8 min. Observations were made under an SEM (model JSM-T200; JEOL Co., Ltd., Akishima, Tokyo, Japan) at an accelerating voltage of 10 kV.

The terminology used in the present study follows Barthlott (1981) and Bahadur, Venkateshwarlu, and Swamy (1989) . The anticlinal walls referred to here represent the entire structure consisting of two anticlinal walls of adjacent seed surface cells, except where otherwise stated. This definition may be preferable for describing the overall features of the seed surface that are especially observable under lower magnification.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
LITERATURE CITED
 
The overall shape and size of seeds and seed surface cells and the lumen texture of these cells in Petunia sensu Wijsman as well as in Calibrachoa were variable depending on the species, or even within species, as will be reported elsewhere. However, the characteristics described below are stable within the group of species.

The surface of seeds of all species was reticulate. The reticulation, in turn, was caused by the prominently projecting anticlinal walls that separated polygonal seed surface cells from one another. Our observations under light microscopy or under SEM at lower magnification (Figs. 1, 3, 5) seemed to support fully the earlier mentioned key of Bahadur, Venkateshwarlu, and Swamy (1989) : the species of Petunia sensu Wijsman showed wavy anticlinal walls (Fig. 1), in contrast to the Calibrachoa species, which had straight anticlinal walls (Figs. 3, 5). Therefore, we can safely add the shape of anticlinal wall on the seed surface as an index character discriminating Calibrachoa from Petunia sensu Wijsman.



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  		Figs. 1–6. SEM features of seed surface in Petunia sensu Jussieu representing the three groups in this taxon. 1, 2. All species of Petunia sensu Wijsman (Photo: P. axillaris subsp. axillaris), having coarse wavy middle lamellae and anticlinal walls. 3, 4. Calibrachoa parviflora and C. pygmaea (C. pygmaea), having fine wavy middle lamellae embedded in straight anticlinal walls. 5, 6. All other species of Calibrachoa [C. selloviana (Sendtn.) Wijsman], having straight middle lamellae and anticlinal walls. Scale bars = 200 µm for Figs. 1, 3, 5, and 20 µm for 2, 4, 6.

 
In two species of Calibrachoa, C. parviflora and C. pygmaea (R. E. Fr.) Wijsman, however, we detected a substantial difference in the fine structure of the anticlinal walls from that of the other Calibrachoa species, when observed under the SEM at higher magnification (Figs. 2, 4, 6). As shown in Fig. 4, the tops of anticlinal walls in C. parviflora and C. pygmaea were broad and flat, in contrast to the acute tops for other members of Calibrachoa (Fig. 6) as well as for Petunia sensu Wijsman (Fig. 2). In addition, there was a fine wavy or rather ripply middle lamella slightly protruding from the flat top of the anticlinal wall in these two species (Fig. 4). The anticlinal wall of each cell is, strictly speaking, wavy on the middle lamella side and straight on the other side, even though the entire anticlinal wall looks straight. In this respect, these two species should be unique in Petunia sensu Jussieu.

In our opinion, the difference in overall appearance between Calibrachoa parviflora and P. pygmaea, and Petunia sensu Wijsman stems solely from the difference in the degree of middle lamella corrugation and/or of wall thickening on both sides of the middle lamella. In C. parviflora and C. pygmaea, the anticlinal wall was so thick that the fine wavy middle lamella was completely embedded in the wall. In Petunia sensu Wijsman, however, the coarse waviness of the middle lamella and/or less thick wall may make the overall appearance of the anticlinal walls wavy.

Bahadur, Venkateshwarlu, and Swamy (1989) described the anticlinal wall structures of C. parviflora as "anticlinal walls straight with thick-walled ridges, middle lamella conspicuous, serrate and interlocking." Evidently they intended to distinguish two characters, "anticlinal wall" and "middle lamella," but chose "anticlinal wall" as an effective character distinguishing C. parviflora or C. linearis from the species of Petunia sensu Wijsman. They unexpectedly observed the complete set of seed samples representing three different phenotypes existing in the genus Petunia sensu Jussieu. If they had chosen both "anticlinal wall" and "middle lamella" as index characters, species of Petunia sensu Jussieu would have been classified into three groups at that time.

Fries (1911) , the latest reviewer of the genus Petunia sensu Jussieu, classified C. parviflora and C. pygmaea into the group composed of all members of Petunia sensu Wijsman known at that time, not into the group of other Calibrachoa species. He regarded Calibrachoa parviflora as having deeply incised calyx lobes resembling those of Petunia sensu Wijsman, but Wijsman and de Jong (1985) denied such morphology, as mentioned earlier. Actually, our materials of C. parviflora had deeply incised calyx lobes, at least at anthesis. Fries (1911) regarded C. pygmaea as being closely related to P. axillaris, because both species shared characters unique to the genus Petunia sensu Jussieu, i.e., white flowers and filaments affixed to the middle part of the corolla tube.

In previous papers (Watanabe et al., 1996 , 1997 ), we described the interspecific cross compatibility of species in the genus Petunia sensu Jussieu; reciprocal crossings between species of Petunia sensu Wijsman and Calibrachoa completely failed to set fruits. We also showed that Calibrachoa pygmaea was crossable only with C. parviflora. Moreover, Petunia pubescens Spreng. (a species of Petunia sensu Jussieu), which has 2n = 18 chromosomes and shares all other morphological characters with Calibrachoa, can freely be crossed with all members of Calibrachoa other than C. parviflora and C. pygmaea.

We would like to conclude herein that the genus Petunia sensu Wijsman seems to be homogeneous, but Calibrachoa is not so, at least when seed morphology is used as an index character, and that the species of Calibrachoa can be classified into two groups: those whose seed surface has fine, wavy middle lamellae embedded in straight anticlinal walls and those with straight middle lamellae and anticlinal walls.


   FOOTNOTES
 
1 The authors thank Dr. Dan H. Nicolson of the Smithsonian Institution for his authoritative knowledge given to us about the current status of the genera Petunia and Calibrachoa, Dr. Juan H. Hunziker of Instituto de Botanica Darwinion for his useful discussion about Petunia species native to Argentina, and Mr. Masao Udagawa of Montevideo, Uruguay, Mr. Sebastião T. Nagase, Mr. Nobuyuki Hiranaka, Mr. Tomio Koshizawa, Mr. Hideo Ohkubo, and Mr. Roberto H. Ohkubo of São Paulo, Brazil, and Mr. Tsuguyoshi Aoki and Mr. Angel Aoki of Buenos Aires, Argentina, for surveying the native habitats of Petunia and Calibrachoa. Back

5 Author for correspondence (fax: +81-47-363-1497; andot{at}midori.h.chiba-u.ac.jp ). Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
Ando, T., Ueda, and G. Hashimoto. 1992 Historical survey and present status of systematics in the genus Petunia Jussieu (Solanaceae). Technical Bulletin of Faculty of Horticulture, Chiba University 45: 17–26.

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———, and ———. 1995 Petunia guarapuavensis (Solanaceae): new species from planalto of Paraná and Santa Catarina, Brazil. Brittonia 47: 38–334.

———, and ———. 1996 A new Brazilian species of Petunia (Solanaceae) from interior Santa Catarina and Rio Grande do Sul, Brazil. Brittonia 48: 217–223.

———, and ———. 1998 Two new species of Petunia (Solanaceae) from southern Rio Grande do Sul, Brazil. Brittonia 50: 483–492.[CrossRef][ISI]

Bahadur, B., K. Venkateshwarlu, and N. R. Swamy. 1989 SEM studies of seeds in nine species of Petunia Juss. (Solanaceae). Phytomorphology 39: 121–128.

Barthlott, W. 1981 Epidermal and seed surface characters of plants: systematic applicability and some evolutionary aspects. Nordic Journal of Botany 1: 345–355.

Fries, R. E. 1911 Die Arten der Gattung Petunia. Kungl. Svenska Vetenskapsakademiens Handlingar 46: 1–72.

Holmgren, P. K., N. H. Holmgren, and L. C. Barnett. 1990 Index herbariorum. Part I: The herbaria of the world. Eighth edition. New York Botanical Garden, Bronx, NY.

Jussieu, A. L. 1803 Sur le Petunia, genre nouveau de la famille des plantes solanées. Annales du Muséum National d'Histoire Naturelle 2: 214–216.

Knapp, S., and T. Helgason. 1997 A revision of Solanum section Pteroidea: Solanaceae. Bulletin of the Natural History Museum, London (Botany) 27: 31–73.

La Lave, P., and J. J. M. Lexarza. 1825 Calibrachoa. Novorum Vegetabilium Descriptiones 2: 3.

Lester, R. N., and P. Durrands. 1984 Enzyme treatment as an aid in the study of seed surface structures of Solanum species. Annals of Botany 53: 129–131.[Abstract/Free Full Text]

Muszynski, S., J. Kocon, and W. Guzewski. 1986 Scanning electron microscopic analysis of the seed surface in wild and cultivated petunias (Petunia axillaris Juss. and Petunia hybrida hort. Vilm.). Acta Agrobotanica 39: 189–194.

Nicolson, D. H. 1991 General committee report 3. Taxon 40: 461–462.[CrossRef][ISI]

Souèges, M. R. 1907 Développement et structure du tégument séminal chez les Solanacées. Annales des Sciences Naturelles, series 9, Botanique 6: 1–124.

Stehmann, J. R., and J. Semir. 1997 A new species and new combinations in Calibrachoa (Solanaceae). Novon 7: 417–419.

Tsukamoto, T., T. Ando, M. Kurata, H. Watanabe, H. Kokubun, G. Hashimoto, and E. Marchesi. 1998 Resurrection of Petunia occidentalis R. E. Fr. (Solanaceae) inferred from a cross compatibility study. Journal of Japanese Botany 73: 15–21.

Watanabe, H., T. Ando, S. Iida, A. Suzuki, K. Buto, T. Tsukamoto, G. Hashimoto, and E. Marchesi. 1996 Cross compatibility of Petunia cultivars and P. axillaris with native taxa of Petunia in relation to their chromosome number. Journal of the Japanese Society for Horticultural Science 65: 625–634.[ISI]

———, ———, ———, ———, ———, ———, H. Kokubun, G. Hashimoto, and E. Marchesi. 1997 Cross compatibility of Petunia pubescens and P. pygmaea with native taxa of Petunia. Journal of the Japanese Society for Horticultural Science 66: 607–612.

Wijnands, D. O., J. J. Bos, H. J. W. Wijsman, C. D. Brickell, and K. Zimmer. 1986 (856) Proposal to conserve 7436 Petunia with P. nyctaginiflora as typ. cons. (Solanaceae). Taxon 35: 748–749.[CrossRef]

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———, and J. H. de Jong. 1985 On the inter-relationships of certain species of Petunia IV. Hybridization between P. linearis and P. calycina and nomenclatorial consequences in the Petunia group. Acta Botanica Neerlandica 34: 337–349.[ISI]




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T. ANDO, H. KOKUBUN, H. WATANABE, N. TANAKA, T. YUKAWA, G. HASHIMOTO, E. MARCHESI, E. SUAREZ, and I. L. BASUALDO
Phylogenetic Analysis of Petunia sensu Jussieu (Solanaceae) using Chloroplast DNA RFLP
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