Ann. Bot. Fennici 47: 321–329 Helsinki 29 October 2010 ISSN 0003-3847 (print) ISSN 1797-2442 (online) © Finnish Zoological and Botanical Publishing Board 2010 Evolutionary relationships between the diploid Turnera grandiflora and the octoploid T. fernandezii (Turneraceae) Aveliano Fernández1,3, Hebe Rey2,3 & Viviana G. Solís Neffa2,3 1) 2) 3) Facultad de Ciencias Exactas y Naturales y Agrimensura (UNNE), CC 209, 3400 Corrientes, Argentina Facultad de Ciencias Agrarias (UNNE), CC 209, 3400 Corrientes, Argentina Instituto de Botánica del Nordeste (UNNE-CONICET), CC 209, 3400 Corrientes, Argentina Received 6 Mar. 2009, revised version received 19 May 2009, accepted 22 May 2009 Fernández, A., Rey, H. & Solís Neffa, V. G. 2010: Evolutionary relationships between the diploid Turnera grandiflora and the octoploid T. fernandezii (Turneraceae). — Ann. Bot. Fennici 47: 321–329. Aiming to analyze the evolutionary relationships between the diploid species Turnera grandiflora (2n = 2x = 10) and the octoploid T. fernandezii (2n = 8x = 40), interspecific hybrids were recovered by in vitro embryo rescue methods. The full-grown plants obtained were all pentaploids (2n = 5x = 25) confirming their hybrid nature. The chromosome associations observed in the hybrids during meiosis were indicative for an autopentaploid, suggesting that T. fernandezii carries the genome of T. grandiflora (CgCg) but at the octoploid level (CgCg CgCg CgCg CgCg). This fact confirms a close evolutionary relationship between the species and supports the hypothesis that T. grandiflora is the progenitor of T. fernandezii. A tentative hypothesis regarding the autopolyploid origin of T. fernandezii is finally formulated. Key words: autopolyploidy, chromosome pairing, embryo rescue, genetic relationships, karyology Introduction The genus Turnera (Turneraceae) comprises about 100 species classified into nine series (Urban 1883), which are distributed from the southern United States to central Argentina with two species occurring also in Africa. Chromosome numbers are known for 35 Turnera species (Raman & Kesavan 1964, Hamel 1965, Barrett 1978, Arbo & Fernández 1983, Barrett & Shore 1987, Fernández 1987, Solís Neffa & Fernández 1993, 2001, Solís Neffa et al. 2004). The basic chromosome number x = 7 is found in the Salicifolieae, Stenodictyae, Mycrophyllae and Leiocarpae series, x = 5 is found in the Turnera series and x = 13 in series Papilliferae (Fernández 1987). Cytological investigations documented the occurrence of diploid to decaploid populations. Polyploids may be autopolyploids as well as allopolyploids (Raman & Kesavan 1964, Barrett 1978, Arbo & Fernández 1983, Shore & Barrett 1985, Fernández 1987). Cytogenetic studies are particularly detailed in the Turnera series and focus on the taxon- Fernández et al. 322 omy and evolutionary relationships. This series presents the most complex floral structure in the family. The flowers are epiphyllous, solitary and the floral tube shows five nectar pockets formed by marginal adnation of each staminal filament to the petal-claws up to the throat (Arbo 2005). The series comprises about 22 species, which are divided into the subseries Turnera and Umbilicatae based on seminal features (Arbo 2005). Subseries Turnera includes two groups of species, one with yellow flowers and one with blue ones (Fernández & Arbo 1989, Arbo 2005). Distyly and the associated dimorphic self-incompatibility system is widespread in the genus (Barrett & Shore 1987) although self-compatible homostyly arose independently at least three times in the genus (Truyens et al. 2005). To clarify the evolutionary relationship among species of Turnera series, a controlled crossing program has been carried out since 1982. Several interspecific hybrids were obtained and the genomic relationships of several yellow-flowered species and of the diploid blue-flowered species were analyzed (Arbo & Fernández 1987, Fernández & Arbo 1989, 1990, 1993a, 1993b, 1996, 2000a, 2000b, Fernández & Solís Neffa 2004). In this context, our objective here is to investigate the relationships between two blue-flowered species, diploid Turnera grandiflora (2n = 2x = 10) and octoploid T. fernandezii (2n = 8x = 40). The species are morphologically similar and T. fernandezii is included within the geographic distribution range of T. grandiflora. Hence, the latter is more widely distributed, occurring in Brazil (Mato Grosso do Sul), Bolivia (Santa Cruz), Paraguay and northern Argentina; while T. fernandezii is endemic to Mato Grosso do Sul (Brazil) and NE Paraguay. In the overlapping • ANN. BOT. FENNIcI Vol. 47 area, both species often grow at the same sites (Arbo 2005). However, despite that 237 interspecific crossings were made involving two accessions of T. grandiflora and one accession of T. fernandezii (Arbo & Fernández 1987), all crossings completely failed to yield F1 hybrids since the fruits obtained aborted after 12 days, probably due to embryo collapse in the early embryogenesis of the hybrid. In hybrid seeds, there often is a paucity of endosperm tissue or its development is abnormal, and the embryo dies at an early stage of development, although it is potentially capable of normal growth. In these cases, embryo rescue methods have proven very effective, and were utilized for diverse interploid, interspecific and intergeneric crosses (Watanabe 1977, Sharma et al. 1996, Momotaz et al. 1998, Kato et al. 2001, Fratini & Ruiz 2006). In this study, we firstly conducted interspecific crosses in an attempt to recover hybrids between T. grandiflora and T. fernandezii. Moreover, we employed in vitro embryo rescue methods to overcome hybrid sterility and used cytological evidence to evaluate the relationships among both species. Finally, we formulated a tentative hypothesis regarding the autopolyploid origin of T. fernandezii. Material and methods Plant material Two accessions of T. grandiflora and one accession of T. fernandezii were used in the present study (Table 1). Voucher specimens were deposited in the herbarium of the Instituto de Botánica del Nordeste (CTES), Corrientes, Argentina. Table 1. Material studied. Turnera 2n Ploidy grandiflora 10 10* 40* 25* 2x 2x 8x 5x fernandezii fernandezii ¥ grandiflora Locality and collector Argentina, Formosa. Arbo 2696 Argentina, corrientes, capital. Fernández s/n Paraguay, Amambay. Arbo 8882 Argentina, corrientes, capital. Fernández s/n *chromosome counts on additional accessions. L = long-styled, S = short-styled. Floral morph code S S L S G4 G11 G10 ANN. BOT. FENNIcI Vol. 47 • Evolutionary relationships between Turnera grandiflora and T. fernandezii Crossing methods Since both T. grandiflora and T. fernandezii are dystylous (Arbo 2005), the 30 crosses performed consisted of legitimate combinations between long-styled (L) and short-styled (S) plants. Crossings were made according to Fernández and Arbo (1989) under greenhouse conditions to avoid undesired insect pollination. Moreover, although both species are self-incompatible (Shore et al. 2006), open flowers used as females were emasculated prior to pollination with anthers of plants selected as males. Pollinated flowers were individually marked indicating the pollen donor. The number of flowers that were pollinated was different for each parental combination used, depending on the availability of plants and on the chance of simultaneous flowering (Table 2). Establishment of seedlings in vitro Pollination was followed by embryo rescue, i.e. cultivation of immature seeds on Murashighe and Skoog’s (1962) medium (MS). Immature fruits were collected ten days after pollination. The fruits were washed 1 min in 70% ethanol, surface-sterilized for 20 min with sodium hypochlorite solution (1.1% available chlorine) containing 1 ml of Tween 20, and then rinsed three times with sterile distilled water. After surface-sterilization, 85 immature seeds were placed on the surface of MS medium in glass tubes that were covered with Resinite AF50® and, incubated in a conditioned chamber at 27 ± 2 °C with a photoperiod of 14 hrs (116 µmol m–2 s–1). To induce rooting, the germinated seeds were transferred to solid culture medium containing 1/2 strength MS supplemented with 3% sucrose and 1 g l–1 active charcoal (AC). Before the addition of 0.7% agar (Sigma A 1296), the pH was adjusted to 5.8 with KOH and/or HCl. The glass tubes were covered with aluminum foil and sterilized in an autoclave at 0.101 MPa for 20 min. The rooted shoots were cloned in vitro in order to increase the number of hybrid plants obtained. The best regenerated plantlets were also acclimatized to conditions ex vitro in a mixture of soil, sand and pearlite (1:1:1) and maintained 323 during 15 days in a conditioned chamber at 27 ± 2 °C, at a luminance intensity of 336 µmol m2 s–1 and 14 hrs photoperiod. The plants were then transferred to the greenhouse. Cytological studies Chromosome numbers of the accessions G10 and G11 used as parents were obtained from meiosis, while the chromosome number of accession G4 was previously determined (Fernández 1987). Meiotic chromosomes were examined in pollen mother cells (PMC) of young buds, fixed in 5:1 absolute ethanol:lactic acid (Fernández 1973) for 12 hrs at 4 °C and stored in 70% ethanol at 4 °C. Anthers were stained using the Feulgen technique and squashed in 3% aceto-orcein. Slides were made permanent in Euparal using Bowen’s (1956) method. In the diploid parents, microspores at tetrad stage were also examined to explore the production of unreduced gametes. For this analysis, young floral buds were fixed as described above and the anthers were stained with carmine:glycerin. Expected n, 2n and 4n pollen percentages were calculated from tetrad, triad, dyad and monad frequencies, taking into account that tetrads form four n gametes, dyads two 2n gametes, while each monad originates one 4n gamete only. In order to estimate pollen viability, pollen stainability was also estimated using carmine-glycerin 1:1. At least 300 grains per flower were scored. The hybrid character of the plants raised was checked by counting their chromosome number. Furthermore, the analysis of meiotic behavior and pollen stainability of the progeny were also performed according to the methods described for the parents. Table 2. crosses carried out between Turnera grandiflora and T. fernandezii. Parents N Ploidy Morphotypes G10 ¥ G4 G10 ¥ G11 G11 ¥ G10 3 21 6 8x ¥ 2x 8x ¥ 2x 2x ¥ 8x L¥S L¥S S¥L 324 Fernández et al. • ANN. BOT. FENNIcI Vol. 47 Fig. 1. chromosome pairing at MI of the parental species. — A: Turnera grandiflora (2n = 2x = 10) with 5II. — B: T. fernandezii (2n = 8x = 40) with 4II + 2IV + 3VIII. — C: T. fernandezii with 6II + 2IV + 1VI + 2VIII. — D and E: T. fernandezii with 5VIII. Bar = 5 µm. Results Crossings Twenty-nine (96.67%) of the crosses between T. grandiflora and T. fernandezii, yielded no fruit, while the only crossing that gave fruits was T. fernandezii (G10) L ¥ T. grandiflora (G11) S. Establishment of seedlings in vitro Seventy percent of the seeds germinated in culture after 15 days, 45 days later the plantlets gave several shoots. Eighty percent of the shoots cultivated in 1/2 MS + AC 1 g l–1 produced roots. From these shoots, eight plants were obtained and cloned in vitro, obtaining a total of 70 plants. Of these, only 20 gave full-grown plants. All flowering hybrid plants obtained so far were morphologically intermediates of their parents. Cytological studies Chromosome numbers of the parental plants and the F1 hybrids are shown in Table 1. In T. grandiflora, all the PMCs analyzed had 5II (bivalents; Fig. 1A). However, the analysis of 7174 sporads showed, beside tetrads (75.30%, Fig. 2A), the production of dyads (0.04%, Fig. 2B), monads (0.03%, Fig. 2C) and other abnormal sporads (i.e. pentads, hexads and sporads with one or more micronuclei, 24.63%, Fig. 2D). The expected n, 2n and 4n pollen were 99.96%, 0.03% and 0.01%, respectively. In T. fernandezii, up to five VIII (octovalents) were observed in metaphase I (Fig. 2B–D). All 20 plants that were obtained from interspecific crosses proved to be pentaploid hybrids (2n = 5x = 25; Fig. 3). A meiotic analysis of the hybrids revealed 16 different configurations in metaphase I (Table 3), with 5V (34.51%, Fig. 3A and B), 1II + 1III + 4V (19.02%) and 1I + 1IV + 4V (15.49%, Fig. 3F) being the most frequent. Chromosome associations at MI of the F1 hybrids are summarized in Table 4. In anaphase I, laggard chromosomes (Fig. 4A and C) and bridges (Fig. 4B and C) were observed. Pollen fertility of the parents was nearly 95% in T. grandiflora and 93.71% (98.68%–99.80%) in T. fernandezii; while the fertility of the pentaploid hybrid was 59.23% (55.56%–64.40%). Discussion Crossings The analysis performed showed that embryo ANN. BOT. FENNIcI Vol. 47 • Evolutionary relationships between Turnera grandiflora and T. fernandezii 325 Fig. 2. Sporads of Turnera grandiflora. — A: Tetrad. — B: Dyad. — C: Monad. — D: Pentad. Bar = 5 µm. rescue provided an effective means for the production of semi-fertile pentaploid hybrids between diploid T. grandiflora and octoploid T. fernandezii. As a result of the 267 reciprocal experimental crosses between T. fernandezii and T. grandiflora presented in this paper and in a previous one (Arbo & Fernández 1987), fruits were recovered only once, when T. fernandezii was used as female parent. This result agrees with those obtained from interploidy crosses involving other Turnera species (Shore & Barret 1985, Arbo & Fernández 1987, Fernández & Solís Neffa 2004) and species of other genera (Stebbins 1958, Woodell & Valentine 1961, Ockendon 1968, Levin 1971). This could be explained by the ratio between the ploidy level of an embryo and its associated endosperm being a critical factor influencing seed development (Ramsey & Table 3. chromosome configurations at first metaphase of meiosis of the PMcs in F1 hybrids between T. fernandezii ¥ T. grandiflora. configuration N Percentage 5V 1II + 1III + 4V 1I + 1IV + 4V 1I + 2II + 4V 1I + 1II + 1III + 1IV + 3V 2II + 2III + 3V 1I + 3II + 1III + 3V 2I + 1III + 4V 2I + 4II + 2III +1IV +1V 2I + 1II + 2III + 3V 2I + 2IV + 3V 2I + 2II + 3III + 2V 4I + 3II + 3V 4II + 4III + 1V 5I + 3II + 1IV + 2V 5I + 4V 49 27 22 11 8 7 5 4 2 1 1 1 1 1 1 1 34.51 19.02 15.49 7.75 5.64 4.94 3.52 2.82 1.41 0.70 0.70 0.70 0.70 0.70 0.70 0.70 326 Fernández et al. • ANN. BOT. FENNIcI Vol. 47 Fig. 3. chromosome pairing at MI of the F1 hybrid Turnera fernandezii ¥ T. grandiflora (2n = 5x = 35). — A and B: 5V. — c: 2I + 2IV + 3V. — D: 3II + 3III + 2V. — E: 5I + 1II + 1III + 3V. — F: 1I + 1IV + 4V. Bar = 5 µm. Schemske 1998). However, the embryo collapse due to unbalance of the embryo/endosperm rate would eventually be overcome when the ploidy level of maternal parents is the highest. Cytological studies Our chromosome counts confirm the numbers 2n = 2x = 10 and 2n = 8x = 40 previously reported for T. grandiflora and T. fernandezii, respectively (Fernández 1987). The analysis of hybrid chromosome complements of the full-grown plants confirms their hybrid nature, since all of them were pentaploids (2n = 5x = 25). The analysis of chromosome pairing at metaphase I and pollen fertility of hybrids are useful for assessing the evolutionary relationship and genetic divergence between species (de Wet & Harlan 1972). Chromosome pairings and pollen fertility observed in the pentaploid T. fernandezii ¥ T. grandiflora hybrid are indicative of an autopentaploid and suggest a close genetic relationship between T. grandiflora and T. fernandezii. Taking into account that T. fernandezii showed a maximum of three octovalents in MI (Fernandez 1987) and that it shares the same karyotype with 4 metacentric and 1 submetacentric with diploid T. grandiflora for all eight chromosome sets (Solís Neffa & Fernández 1993), ANN. BOT. FENNIcI Vol. 47 • Evolutionary relationships between Turnera grandiflora and T. fernandezii 327 Fig. 4. Anaphase I of the F1 hybrid Turnera fernandezii × T. grandiflora (2n = 5x = 35). — A: Laggard chromosomes. — B: Bridge (arrow). — C: Bridge (arrow) and laggard chromosomes (arrow head). Bar = 5 µm. T. fernandezii was proposed to be an octoploid cytotype of T. grandiflora. Based on this cytological evidence as well as on the morphological similarity between the cytotypes, an autopolyploid origin from diploids was hypothesized to explain the origin of the octoploid cytotype (Fernández 1987). However, considering that no hybrids among the cytotypes had been obtained so far and that the octoploid can be distinguished from diploid T. grandiflora by its height and the leaf-indumentum, Arbo (2005) elevated the auto-octoploid cytotype to a species of its own, T. fernandezii. Therefore, the meiotic analysis of the pentaploid hybrids presented in this paper suggests that T. fernandezii (CgCg CgCg CgCg CgCg) inherited the genome CgCg of T. grandiflora and supports the hypothesis that T. fernandezii originated by autopolyploidy from T. grandiflora. Our results also provide evidence that sexual polyploidization may have been involved in the origin of the auto-octoploid T. fernandezii. Sexual polyploidization involves the fusion of 2n gametes and was considered as the main mechanism of origin and evolution of polyploid plant species (Karpechenko 1927, Darlington 1937, 1956, Harlan & De Wet 1975, De Wet 1980, Bretagnolle & Thompson 1995). The sporad analysis performed showed the presence of dyads indicating that 2n gametes occurred in T. grandiflora and that the formation of 2n pollen has to be expected. Moreover, the presence of monads suggests that 4n pollen may also be formed. However, considering that in the diploid hybrid T. subulata ¥ T. scabra secondary roots with duplicate (2n = 4x = 20) or quadruplicate (2n = 8x = 40) chromosome number were found (Fernández 1987) and that plants of Turnera may often grow from secondary roots, somatic chromosome doubling cannot be discarded as an alternative mechanism of polyploid formation in the genus Turnera. Consequently, octoploid plants of T. fernandezii may have also been formed from secondary roots of the diploid T. grandiflora with quadruplicate chromosome number. Acknowledgments The authors are grateful to Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Secretaría General de Ciencia y Técnica of the Universidad Nacional del Nordeste (UNNE) for financial support. The authors are members of the Carrera del Investigador Científico of CONICET. Table 4. Mean, standard error and range of the number of observed chromosome associations at MI of the F1 hybrids between Turnera fernandezii ¥ T. grandiflora. Mean ± SE Range I II III IV V 0.55 ± 0.07 0–5 0.73 ± 0.08 0–4 0.49 ± 0.06 0–4 0.25 ± 0.04 0–2 4.10 ± 0.07 1–5 328 References Arbo, M. M. 2005: Estudios sistemáticos en Turnera (Turneraceae). III. Series Anomalae y Turnera. — Bonplandia 14: 115–318. Arbo, M. M. & Fernández, A. 1983: Posición taxonómica, citología y palinología de tres niveles de ploidía de Turnera subulata Smith. — Bonplandia 5: 211–216. Arbo, M. M. & Fernández, A. 1987: Cruzamientos intra e interespecíficos en Turnera, Serie Canaligerae. — Bonplandia 6: 23–38. Barrett, S. C. H. 1978: Heterostyly in a tropical weed: the reproductive biology of the Turnera ulmifolia complex (Turneraceae). — Canadian Journal of Botany 56: 1713–1725. Barrett, S. C. H. & Shore, J. 1987: Variation and evolution of breeding systems in the Turnera ulmifolia complex (Turneraceae). — Evolution 41: 340–354. Bowen, C. C. 1956: Freezing by liquid carbone dioxide in making slides permanent. — Stain Technology 31: 87–90. Bretagnolle, F. & Thompson, J. D. 1995: Gametes with the somatic chromosome number: mechanisms of their formation and role in the evolution of autopolyploid plants. — New Phytologist 129: 1–22. Darlington, C. D. 1937: Recent advances in cytology. — Blakinston’s Son & Co. Inc., Philadelphia. Darlington, C. D. 1956: Cytology. — J. & A. Churchill Ltd., London. De Wet, J. M. J. 1980: Origins of polyploids. — In: Lewis, W. H. (ed.), Polyploidy, biological relevance: 3–16. Plenum Press, New York. De Wet, J. M. J. & Harlan, J. R. 1972: Chromosome pairing and phylogenetic affinities. — Taxon 21: 67–70. Fernández, A. 1973: El ácido láctico como fijador cromosómico. — Boletín de la Sociedad Argentina de Botánica 15: 287–290. Fernández, A. 1987: Estudios cromosómicos en Turnera y Piriqueta (Turneraceae). — Bonplandia 6: 1–21. Fernández, A. & Arbo, M. M. 1989: Relaciones genómicas entre cuatro especies diploides de Turnera con flores amarillas (Serie Canaligerae). — Bonplandia 6: 93–109. Fernández, A. & Arbo, M. M. 1990: Gametas no reducidas y relaciones genómicas en tres especies de Turnera (Turneraceae). — Darwiniana 30: 21–26. Fernández, A. & Arbo, M. M. 1993a: Relaciones genómicas entre seis especies de Turnera (Serie Canaligerae) del Paraguay. — Candollea 48: 305–318. Fernández, A. & Arbo, M. M. 1993b: Citogenética de híbridos entre Turnera grandidentata (4x) y T. subulata y T. scabra (2x) (Turneraceae). — Bonplandia 7: 119–127. Fernández, A. & Arbo, M. M. 1996: Relaciones genómicas entre las especies diploides de flores blanco-azuladas de Turnera (serie Canaligerae). — Bonplandia 9: 95–102. Fernández, A. & Arbo, M. M. 2000a: Cytogenetic relationships between Turnera aurelii, T. cuneiformis (2n = 8x = 40) and T. orientalis (2n = 6x = 30) (Turneraceae). — Cytologia 65: 97–102. Fernández, A. & Arbo, M. M. 2000b: Relaciones genómicas entre dos especies hexploides de Turnera, T. orientalis y Fernández et al. • ANN. BOT. FENNIcI Vol. 47 T. velutina, y una diploide, T. grandiflora (Turneraceae, serie Turnera). — Bonplandia 10: 181–187. Fernández, A. & Solís Neffa, V. G. 2004: Genomic relationships between Turnera krapovickasii (2x, 4x) and T. ulmifolia (6x) (Turneraceae, Turnera). — Caryologia 57: 45–51. Frattini, R. & Ruiz, M. L. 2006: Interspecific hybridization in the genus Lens applying in vitro embryo rescue. — Euphytica 150: 271–280. Hamel, J. L. 1965: Le noyau et les chromosomes somatiques de Turnera ulmifolia L. — Mémoires du Muséum National d’Histoire Naturelle Série B 16: 3–8. Harlan, J. R. & de Wet, J. M. J. l975: On Ö. Winge and a prayer: the origins of polyploidy. — Botanical Review 41: 361–390. Kato, J., Ishikawa, R. & Mii, M. 2001: Different genomic combinations in inter-section hybrids obtained from the crosses between Primula sieboldii (section Cortusoides) and P. obconica (section Obconicolisteri) by the embryo rescue technique. — Theoretical and Applied Genetics 102: 1129–1135. Karpechenko, G. D. 1927: The production of polyploid gametes in hybrids. — Hereditas 9: 349–368. Levin, D. A. 1971: The origin of reproductive isolating mechanisms in flowering plants. — Taxon 20: 91–113. Momotaz, A., Kato, M. & Kakihara, F. 1998: Production of intergeneric hybrids between Brassica and Sinapis species by means of embryo rescue techniques. — Euphytica 103: 123–130. Murashige, T. & Skoog, F. 1962: A revised medium for rapid growth and bioassays with tobacco tissue culture. — Physiologia plantarum 15: 473–497. Ockendon, D. J. 1968: Biosystematic studies in the Linum perenne group. — New Phytologist 67: 787–813. Raman, V. S. & Kesavan, P. C. 1964: Meiosis and the nature of polyploidy in Turnera ulmifolia. — Journal of the Indian Botanical Society 43: 495–497. Ramsey, J. & Schemske, D. W. 1998: Pathways, mechanisms, and rates of polyploid formation in flowering plants. — Annual Reviews of Ecology and Systematics 29: 467–501. Sharma, D. R., Kaur, R. & Kumar, K. 1996: Embryo rescue in plants — a review. — Euphytica 89: 325–337. Shore, J. S. & Barrett, S. C. H. 1985: Morphological differentiation and crossability among populations of the Turnera ulmifolia L. complex (Turneraceae). — Systematic Botany 10: 308–321. Shore, J. S., Arbo, M. M. & Fernández, A. 2006: Breeding system variation, genetics and evolution in the Turneraceae. — New Phytologist 171: 539–551. Solís Neffa, V. G. & Fernández, A. 1993: Estudios cromosómicos en especies de Turnera (Turneraceae). — Bonplandia 7: 101–118. Solís Neffa, V. G. & Fernández, A. 2001: Cytogeography of the South American Turnera sidoides L. complex (Turneraceae, Leiocarpae). — Botanical Journal of the Linnean Society 137: 189–196. Solís Neffa, V. G., Panseri, A. F., Reynoso, W. L. & Seijo, J. G. 2004: Variación del color de flores y números cromosómicos en el noroeste del área de distribución ANN. BOT. FENNIcI Vol. 47 • Evolutionary relationships between Turnera grandiflora and T. fernandezii de Turnera sidoides (Turneraceae). — Bonplandia 13: 117–128. Stebbins, G. L. 1958: The inviability, weakness and sterility of interspecific hybrids. — Advances in Genetics 9: 147–215. Truyens, S., Arbo, M. M. & Shore, J. S. 2005: Phylogenetic relationships, chromosome and breeding system evolution in Turnera (Turneraceae): inferences from ITS sequence data. — American Journal of Botany 92: 1749–1758. 329 Urban, I. 1883: Monographie der familie der Turneraceen. — Jahrbuch des Königlichen Botanischen Gartens Berlin 2: 1–152. Watanabe, K. 1977: Succesful ovary culture and production of F1 hybrids and androgenic haploids in Japanese Chrysanthemum species. — The Journal of Heredity 68: 317–320. Woodell, S. R. J. & Valentine, D. H. 1961: Studies in the British Primulas. IX. Seed incompatibility in diploidautotetraploid crosses. — New Phytologist 60: 282–294. This article is also available in pdf format at http://www.annbot.net