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Evolution of distyly breakdown in Palicoureeae Robbr. & Manen and Psychotrieae Cham. & Schltdl. (Rubiaceae)

ABSTRACT

Distyly is a floral polymorphism with reciprocal placement of male and female structures, heteromorphic self-incompatibility, and other ancillary traits. However, breeding system breakdowns and loss of polymorphism are common. Here we traced the diversification of breeding strategies in the type genera of tribes Palicoureeae and Psychotrieae and discussed the evolution of distyly in a phylogenetic framework. We used literature and field information for breeding systems transitions in 46 species of Palicourea and Psychotria. Beyond distyly, we found four additional breeding systems, including monomorphism with herkogamy, homostyly (without herkogamy), monoecy and dioecy. Breeding transitions arose independently and were mostly derived from distyly. Only two species presented monomorphism as an intermediate state into gender specialization. It was not possible to evaluate the origin and evolutionary pathways for distyly in Psychotria and Palicourea as a whole, since distyly seems to be ancestral to their diversification. Breeding transitions in Psychotria and Palicourea appeared to be phylogenetically and biogeographically independent and occurred mostly in islands or isolated forest fragments, with distinct divergence times. Breeding transitions were not related to changes in ploidy. We propose that evolution of breeding transitions in Psychotria and Palicourea represent phylogenetically independent strategies to reproductive assurance in isolated or disturbed habitats.

Keywords:
breeding system evolution; heterostyly; homostyly; monomorphism; plant reproduction

Introduction

Distyly is a genetically controlled floral polymorphism with two morphs which differ in the expression of herkogamy (Ganders 1979Ganders FR. 1979. The biology of heterostyly. New Zealand Journal of Botany 17: 607-635.; Barrett 1992Barrett SCH. 1992. Heterostylous genetic polymorphisms: model systems for evolutionary analysis. In: Barrett SCH (ed.) Evolution and function of heterostyly Monographs on theoretical and applied genetics. Berlin, Springer-Verlag. p. 1- 30. ). Distylous species present flowers with a long-styled morph (pin), with anthers below the stigma (approach herkogamy), and a short-styled morph (thrum), where anthers are placed above the stigma (reverse herkogamy) (Cardoso et al. 2018Cardoso JCF, Viana ML, Matias R, et al. 2018. Towards a unified terminology for angiosperm reproductive systems. Acta Botanica Brasilica 32: 329-348. ). In truly distylous species, female (pistil) and male (stamens) sexual organs of opposite floral morphs are placed at reciprocal height (Barrett 2019Barrett SCH. 2019. ‘A most complex marriage arrangement’: recent advances on heterostyly and unresolved questions. New Phytologist 224: 1051-1067). In addition to the morphological floral syndrome, many distylous plants usually present a diallelic self-incompatibility system, in a way that ovule fertilization occurs only when a flower receives pollen from the opposite morph. Altogether, the morphological variation and incompatibility system are interpreted as mechanisms to promote seed production through cross-pollinations (Ganders 1979Ganders FR. 1979. The biology of heterostyly. New Zealand Journal of Botany 17: 607-635.; Barrett 1992Barrett SCH. 1992. Heterostylous genetic polymorphisms: model systems for evolutionary analysis. In: Barrett SCH (ed.) Evolution and function of heterostyly Monographs on theoretical and applied genetics. Berlin, Springer-Verlag. p. 1- 30. ; Barrett 2019Barrett SCH. 2019. ‘A most complex marriage arrangement’: recent advances on heterostyly and unresolved questions. New Phytologist 224: 1051-1067).

Distyly is, however, an unstable breeding strategy (Barrett 2013Barrett SCH. 2013. The evolution of plant reproductive systems: how often are transitions irreversible? Proceedings of The Royal Society - Biological Sciences 280: 20130913. ; Jiang et al. 2018Jiang XF, Zhu XF, Chen LL, Li QJ. 2018. What ecological factors favor the shift from distyly to homostyly? A study from the perspective of reproductive assurance. Journal of Plant Ecology 11: 645-655. ). The breakdown of distyly may result in loss of the polymorphism and acquisition of self-compatibility (Taylor 1989Taylor CM. 1989. Revision of Palicourea (Rubiaceae) in Mexico and Central America. Systematic Botany Monographs 26: 1-102.; Mast et al. 2006Mast AR, Kelso S, Conti E. 2006. Are any primroses (Primula) primitively monomorphic? The New Phytologist 171: 605-16.; Sakai & Wright 2008Sakai S, Wright SJ. 2008. Reproductive ecology of 21 coexisting Psychotria species (Rubiaceae): when is heterostyly lost? Biological Journal of the Linnean Society 93: 125-134.; Ferrero et al. 2009Ferrero V, Arroyo J, Vargas P, Thompson JD, Navarro L. 2009. Evolutionary transitions of style polymorphisms in Lithodora (Boraginaceae). Perspectives in Plant Ecology, Evolution and Systematics 11: 111-125.; Consolaro et al. 2011Consolaro H, Silva SCS, Oliveira PE. 2011. Breakdown of distyly and pin-monomorphism in Psychotria carthagenensis Jacq. (Rubiaceae). Plant Species Biology 26: 24-32.; Yuan et al. 2017Yuan S, Barrett SCH, Duan T, Qian X, Shi M, Zhang D. 2017. Ecological correlates and genetic consequences of evolutionary transitions from distyly to homostyly. Annals of Botany 120: 775-789.). The breakdown of distyly commonly results into homostyly, with loss of intrafloral herkogamy and reproductive whorls placed at the same height within flowers (Ganders 1979Ganders FR. 1979. The biology of heterostyly. New Zealand Journal of Botany 17: 607-635.; Yuan et al. 2017Yuan S, Barrett SCH, Duan T, Qian X, Shi M, Zhang D. 2017. Ecological correlates and genetic consequences of evolutionary transitions from distyly to homostyly. Annals of Botany 120: 775-789.). Alternatively, the loss of the polymorphism can lead to the occurrence of populations with only one flower morph that resembles either the long-styled or short-styled morph (Ganders 1979Ganders FR. 1979. The biology of heterostyly. New Zealand Journal of Botany 17: 607-635.; Barrett 1992Barrett SCH. 1992. Heterostylous genetic polymorphisms: model systems for evolutionary analysis. In: Barrett SCH (ed.) Evolution and function of heterostyly Monographs on theoretical and applied genetics. Berlin, Springer-Verlag. p. 1- 30. ; Yuan et al. 2017Yuan S, Barrett SCH, Duan T, Qian X, Shi M, Zhang D. 2017. Ecological correlates and genetic consequences of evolutionary transitions from distyly to homostyly. Annals of Botany 120: 775-789.). These are referred also as homostylous (e.g.Yuan et al. 2017Yuan S, Barrett SCH, Duan T, Qian X, Shi M, Zhang D. 2017. Ecological correlates and genetic consequences of evolutionary transitions from distyly to homostyly. Annals of Botany 120: 775-789.), but since they retain herkogamy, they have also been referred to as pin- or thrum monomorphism (Cardoso et al. 2018Cardoso JCF, Viana ML, Matias R, et al. 2018. Towards a unified terminology for angiosperm reproductive systems. Acta Botanica Brasilica 32: 329-348. ). These atypical morphologies in distylous groups may occur in distinct ecological contexts, appearing in flowers of the same individual or different individuals (Sakai & Wright 2008Sakai S, Wright SJ. 2008. Reproductive ecology of 21 coexisting Psychotria species (Rubiaceae): when is heterostyly lost? Biological Journal of the Linnean Society 93: 125-134.), kept only in isolated populations (Consolaro et al. 2011Consolaro H, Silva SCS, Oliveira PE. 2011. Breakdown of distyly and pin-monomorphism in Psychotria carthagenensis Jacq. (Rubiaceae). Plant Species Biology 26: 24-32.), or be spread to the whole geographic distribution of the species (Rodrigues & Consolaro 2013Rodrigues EB, Consolaro H. 2013. Atypical distyly in Psychotria goyazensis Mull. Arg. (Rubiaceae), an intramorph self-compatible species. Acta Botanica Brasilica 27: 155-161.).

Several authors have proposed hypotheses for the evolution of distyly. Ernst (1936Ernst A. 1936. Heterostylie-forschung. Zeitschrift für Induktive Abstammungs-und Vererbungslehre 71: 156-230.), Mather & Winton (1941Mather K, De Winton D. 1941. Adaptation and counter-adaptation of the breeding system in Primula. Annals of Botany 5: 297-311.) and Baker (1966Baker HG. 1966. The evolution, functioning and breakdown of heteromorphic incompatibility systems. I. The Plumbaginaceae. Evolution 20: 349-368.) framed the evolution of heterostyly under a strong genetic perspective, although these hypotheses differ in the temporal sequence of assumptions. Later, and particularly for the family Rubiaceae, Anderson (1973Anderson WR. 1973. A Morphological Hypothesis for the Origin of Heterostyly in the Rubiaceae. Taxon 22: 537-542.) proposed the “Morphological hypothesis” for the origin of distyly. In this model, the ancestral condition to distyly is a protandrous and self-compatible flower with delayed maturation and elongation of the style. The short-styled morph appears as a result of a mutation making the stigma matures below the anthers. The establishment of mutants occurs as the morphology enhances self-pollination and seed output. In Anderson’s (1973)Anderson WR. 1973. A Morphological Hypothesis for the Origin of Heterostyly in the Rubiaceae. Taxon 22: 537-542. scenario, the short-styled flowers of distylous Rubiaceae self-pollinate while the long-styled flowers experience outcross pollination. Therefore, according to this hypothesis, the sex polymorphism evolved prior to the establishment of an incompatibility system. In contrast, the model proposed by Charlesworth & Charlesworth (1979Charlesworth B, Charlesworth D. 1979. The Maintenance and Breakdown of Distyly. The American Naturalist 114: 499.) predicted the evolution of the incompatibility system before the morphological polymorphism. Specifically, under a context of inbreeding depression, the appearance of self-incompatibility would be rapidly selected in an homostylous and self-compatible morphology; then, mutations occurred, and reciprocal placement of anthers and stigma (long-styled and short-styled morphs) would be favored in the population by avoiding self-interference, leading to the promotion of cross-pollination and fixation of distyly. Another model was proposed later by Lloyd & Webb (1992Lloyd DG, Webb CJ. 1992a. The evolution of heterostyly. In: Barrett SCH (ed.) Evolution and function of heterostyly. Monographs on theoretical and applied genetics. Berlin, Springer p. 151-178.a; bLloyd DG, Webb CJ. 1992b. The selection of heterostyly. In: Barrett SCH (ed.) Evolution and function of heterostyly. Monographs on theoretical and applied genetics. Berlin, Springer . p. 179-207.), based on an ecological perspective by comparing the variation of flower morphology in distylous species and their close relatives. They predicted the evolution of distyly from an ancestral flower with approach herkogamy and partially outcrossing. Then, a dominant mutation for short style length would lead to a morph with reverse herkogamy (short-styled morph), which would spread in the population favored by pollinators promoting pollen flow between the two floral morphs. After that, ancillary traits (system of incompatibility and other floral polymorphisms) would evolve in this reciprocal herkogamous population and distyly with self-incompatibility would be finally established. Lloyd & Webb (1992a)Lloyd DG, Webb CJ. 1992a. The evolution of heterostyly. In: Barrett SCH (ed.) Evolution and function of heterostyly. Monographs on theoretical and applied genetics. Berlin, Springer p. 151-178. considered the morphology of homostylous flowers as derived from distyly, caused by linkage breakdown of the supergene that controls the full heterostyly syndrome expression (Lloyd & Web 1992bLloyd DG, Webb CJ. 1992b. The selection of heterostyly. In: Barrett SCH (ed.) Evolution and function of heterostyly. Monographs on theoretical and applied genetics. Berlin, Springer . p. 179-207.). The most widely invoked selective pressure to explain distyly breakdown has been reproductive assurance (Yuan et al. 2017Yuan S, Barrett SCH, Duan T, Qian X, Shi M, Zhang D. 2017. Ecological correlates and genetic consequences of evolutionary transitions from distyly to homostyly. Annals of Botany 120: 775-789.), although the breeding strategy transition would depend on genetic breakdown processes.

Aside from the wide occurrence of heterostyly across plant families, the ancestral state reconstruction has not been well documented outside the Amaryllidaceae (Graham & Barrett 2004Graham SW, Barrett SCH. 2004. Phylogenetic reconstruction of the evolution of stylar polymorphisms in Narcissus (Amaryllidaceae). American Journal of Botany 91: 1007-1021.; Santos-Gally et al. 2012Santos‐Gally R, Vargas P, Arroyo J. 2012. Insights into Neogene Mediterranean biogeography based on phylogenetic relationships of mountain and lowland lineages of Narcissus (Amaryllidaceae). Journal of Biogeography 39: 782-798.), Boraginaceae (Schoen et al. 1997Schoen DJ, Johnston MO, Lheureux AM, Marsolais JV. 1997. Evolutionary history of the breeding system in Amsinckia (Boraginaceae). Evolution 51: 1090-1099.; Ferrero et al. 2009Ferrero V, Arroyo J, Vargas P, Thompson JD, Navarro L. 2009. Evolutionary transitions of style polymorphisms in Lithodora (Boraginaceae). Perspectives in Plant Ecology, Evolution and Systematics 11: 111-125.), Passifloraceae (Truyens et al. 2005Truyens S, Arbo MM, Shore JS. 2005. Phylogenetic relationships, chromosome and breeding system evolution in Turnera (Turneraceae): Inferences from its sequence data. American Journal of Botany 92: 1749-1758.), Primulaceae (Mast et al. 2006Mast AR, Kelso S, Conti E. 2006. Are any primroses (Primula) primitively monomorphic? The New Phytologist 171: 605-16.) and Plumbaginaceae (Costa et al. 2019Costa J, Torices R, Barrett SCH. 2019. Evolutionary history of the buildup and breakdown of the heterostylous syndrome in Plumbaginaceae. New Phytologist 224: 1278-1289. ). These studies allow us to comprehend if heterostyly evolved more than once in those families and which theoretical models for evolution can be inferred (Barrett & Shore 2008Barrett SCH, Shore JS. 2008. New insights on heterostyly: Comparative biology, ecology and genetics. In: Franklin-Tong V (ed.) Self-Incompatibility in Flowering Plants: Evolution, Diversity and Mechanisms. Berlin, Springer-Verlag , p. 3-32.; Barrett 2019Barrett SCH. 2019. ‘A most complex marriage arrangement’: recent advances on heterostyly and unresolved questions. New Phytologist 224: 1051-1067). Interestingly, these studies revealed different underlying mechanisms for the changes in the breeding system. For example, in Passifloraceae (Truyens et al. 2005Truyens S, Arbo MM, Shore JS. 2005. Phylogenetic relationships, chromosome and breeding system evolution in Turnera (Turneraceae): Inferences from its sequence data. American Journal of Botany 92: 1749-1758.) and in Primulaceae (Mast et al. 2006Mast AR, Kelso S, Conti E. 2006. Are any primroses (Primula) primitively monomorphic? The New Phytologist 171: 605-16.) breeding system transitions were attributed to changes in ploidy level and recombination in a supergene. In Narcissus L. (Amaryllidaceae) changes in the breeding system correlated with changes in functional pollinators (Pérez-Barrales et al. 2006Pérez‐Barrales R, Vargas P, Arroyo J. 2006. New evidence for the Darwinian hypothesis of heterostyly: breeding systems and pollinators in Narcissus sect. Apodanthi. New Phytologist 171: 553-567.; Santos-Gally et al. 2012Santos‐Gally R, Vargas P, Arroyo J. 2012. Insights into Neogene Mediterranean biogeography based on phylogenetic relationships of mountain and lowland lineages of Narcissus (Amaryllidaceae). Journal of Biogeography 39: 782-798.). Again, variation in ploidy level and inefficiency in pollination service in marginal habitats in Boraginaceae seemed to have driven evolutionary shifts in distylous breeding system (Schoen et al. 1997Schoen DJ, Johnston MO, Lheureux AM, Marsolais JV. 1997. Evolutionary history of the breeding system in Amsinckia (Boraginaceae). Evolution 51: 1090-1099.; Ferrero et al. 2009Ferrero V, Arroyo J, Vargas P, Thompson JD, Navarro L. 2009. Evolutionary transitions of style polymorphisms in Lithodora (Boraginaceae). Perspectives in Plant Ecology, Evolution and Systematics 11: 111-125.). Another interesting shift in the breeding system of distylous species involves gender specialized flowers, as explained by Beach & Bawa (1980Beach JH, Bawa KS. 1980. Role of pollinators in the evolution of dioecy from distyly. Evolution 34: 1138-1142.). In their model, the origin of dioecy from distyly has been caused by a gradual process, triggered by a disruption in the disassortative pollen flow among the distylous morphs and a shift in pollinator fauna (e.g. long-tongued to short-tongued bees), followed by unidirectional pollen flow and ultimately the selection of unisexual flowers, the female flower from the pin morph and the male flower from the thrum morph.

Rubiaceae is one of the plant families with the largest number of distylous species, in which distyly seems to have evolved and been lost multiple times (Barrett & Shore 2008Barrett SCH, Shore JS. 2008. New insights on heterostyly: Comparative biology, ecology and genetics. In: Franklin-Tong V (ed.) Self-Incompatibility in Flowering Plants: Evolution, Diversity and Mechanisms. Berlin, Springer-Verlag , p. 3-32.; Barrett 2019Barrett SCH. 2019. ‘A most complex marriage arrangement’: recent advances on heterostyly and unresolved questions. New Phytologist 224: 1051-1067). Monomorphism (either pin or thrum) and homostyly are hypothesized to be alternative reproductive strategies derived from distyly in the Rubiaceae (Ganders 1979Ganders FR. 1979. The biology of heterostyly. New Zealand Journal of Botany 17: 607-635.; Hamilton 1990Hamilton CW. 1990. Variations on a distylous theme in mesoamericam Psychotria subgenus Psychotria (Rubiaceae). Memoirs of the New York Botanical Garden 55: 62-75.), although the proper genetic mechanisms are yet to be detailed. The tribe Psychotrieae, holding more than 2,000 species, is traditionally considered to be monophyletic (e.g.Taylor 1996Taylor CM. 1996. Overview of the Psychotrieae (Rubiaceae) in the Neotropics. Opera Botanica Belgica 7: 261-270.; Bremer & Eriksson 2009Bremer B, Eriksson T. 2009. Time tree of Rubiaceae: phylogeny and dating the family, subfamilies, and tribes. International Journal of Plant Sciences 170: 766-793.) and, possibly, presents the largest number of distylous species in the Angiosperms (Naiki 2012Naiki A. 2012. Heterostyly and the possibility of its breakdown by polyploidization. Plant Species Biology 27: 3-29.). Among the genera considered in Psychotrieae, Psychotria and Palicourea present complex relationships. Recent studies supported the division of the genera into two sister tribes (Psychotrieae and Palicoureeae) based on molecular, morphological and chemical differences (Robbrecht & Manen 2006Robbrecht E, Manen JF. 2006. The major evolutionary lineages of the coffee family (Rubiaceae, angiosperms). Combined analysis (nDNA and cpDNA) to infer the position of Coptosapelta and Luculia, and supertree construction based on rbcL, rps16, trnL-trnF and atpB-rbcL data. A new classification in two subfamilies, Cinchonoideae and Rubioideae. Systematics and Geography of Plants 76: 85-145.; Razafimandimbison et al. 2014Razafimandimbison SG, Taylor CM, Wikström N, Pailler T, Khodabandeh A, Bremer B. 2014. Phylogeny and generic limits in the sister tribes Psychotrieae and Palicoureeae (Rubiaceae): Evolution of schizocarps in Psychotria and origins of bacterial leaf nodules of the Malagasy species. American Journal of Botany 101: 1102-1126.; 2017Razafimandimbison SG, Kainulainen K, Wikström N, Bremer B. 2017. Historical biogeography and phylogeny of the pantropical Psychotrieae alliance (Rubiaceae), with particular emphasis on the Western Indian Ocean Region. American Journal of Botany 104: 1407-1423.). Despite the large number of distylous species, few studies have attempted to understand breeding system evolution for the groups. Sakai & Wright (2008Sakai S, Wright SJ. 2008. Reproductive ecology of 21 coexisting Psychotria species (Rubiaceae): when is heterostyly lost? Biological Journal of the Linnean Society 93: 125-134.) studied genetic relationships and breeding system transitions in 21 species of Psychotria in the Barro Colorado Island, Panama. They detected a repeated and independent evolution pattern of breeding system transitions derived from distylous ancestors. In the subfamily Rubioideae, Ferrero et al. (2012Ferrero V, Rojas D, Vale A, Navarro L. 2012. Delving into the loss of heterostyly in Rubiaceae: Is there a similar trend in tropical and non-tropical climate zones? Perspectives in Plant Ecology, Evolution and Systematics 14: 161-167.) pointed that distyly is ancestral to the Psychotrieae and Spermacoceae Alliances, suggesting that more detailed studies would be important to understand breeding system evolution in the Rubiaceae. These breeding transitions in Rubiaceae are sometimes linked to changes in ploidy, but a consistent pattern has not been detected for the family (Naiki, 2012Naiki A. 2012. Heterostyly and the possibility of its breakdown by polyploidization. Plant Species Biology 27: 3-29.).

One decade after the evolutionary studies of Sakai & Wright (2008Sakai S, Wright SJ. 2008. Reproductive ecology of 21 coexisting Psychotria species (Rubiaceae): when is heterostyly lost? Biological Journal of the Linnean Society 93: 125-134.) in the genus Psychotria, here we conducted a literature review and interpreted the breeding system evolution for a larger and worldwide sample of species of Psychotrieae and Palicoureeae, with a focus on their type genera. We particularly aimed to address the following questions: i) what breeding systems do occur in the species and whether are they derived from distyly?; ii) are the shifts in breeding systems associated with ecological conditions (e.g. isolated habitats) or polyploidy? iii) how many times has distyly evolved and been lost in these taxa and whether is there a phylogenetic signal for evolution of these traits?; iv) when, in a paleobotanical context, have breeding system shifts occurred?; v) which of the theoretical models for evolution and breakdown of distyly can be supported for these genera?

Material and methods

Selected species and breeding system data

We selected 46 species of Psychotria and Palicourea species for which breeding system information was known from field and/or herbarium observations and effectively published. The studied species (names and authority in Tab. 1; whenever necessary, Palicourea was abbreviated to P. and Psychotria to Psy.) belong to five biogeographic regions: Neotropical, Panamanian, Sino-Japanese, Oriental and Hawaiian (Holt et al. 2012Holt BG, Lessard JP, Borregaard MK, et al. 2012. An update of Wallace's zoogeographic regions of the world. Science 339: 74-78.; Razafimandimbison et al. 2014Razafimandimbison SG, Taylor CM, Wikström N, Pailler T, Khodabandeh A, Bremer B. 2014. Phylogeny and generic limits in the sister tribes Psychotrieae and Palicoureeae (Rubiaceae): Evolution of schizocarps in Psychotria and origins of bacterial leaf nodules of the Malagasy species. American Journal of Botany 101: 1102-1126.). We used their up-to-date accepted names based on IPNI and World Flora Online (www.ipni.org and http://www.wfo.org) and provided synonyms due to recent combinations (e.g.Delprete & Kirkbride 2016Delprete PG, Kirkbride Jr JH. 2016. New combinations and new names in Palicourea (Rubiaceae) for species of Psychotria subgenus Heteropsychotria occurring in the Guianas. Journal of the Botanical Research Institute of Texas 10: 409-442.). We focused on the type genera (Palicourea and Psychotria) since they include most of the accepted species, and to avoid the ongoing taxonomic reorganization within the sister genera in each tribe.

Table 1
Studied species of Palicourea (P.) and Psychotria (Psy.). The accepted names used through the text is followed by the GeneBank reference; recent synonyms due to new combinations; breeding systems; habitat of breeding system studies, either insular, fragmented or continental and relatively pristine; references for breeding system information; and level of ploidy (chromosome number). Pin-mono = Pin-monomorphism, Thrum-mono = Thrum-monomorphism.

We classified the breeding system of the species based on Ganders (1979Ganders FR. 1979. The biology of heterostyly. New Zealand Journal of Botany 17: 607-635.), considering homostyly when flowers presented no herkogamy and monomorphism when all flowers had a morphology like one of the distylous floral morphs. We also included habitat information for the species, whether they occurred in insular, isolated or disturbed areas, or continental and pristine continuous habitats. Both breeding systems and ecological information were retrieved from literature and direct field observations. We added chromosome number information whenever possible based on CCDB (http://ccdb.tau.ac.il), Correa et al. (2010Corrêa AM, Jung-Mendaçolli SL, Forni-Martins ER. 2010. Karyotype characterisation of Brazilian species of the genus Psychotria L.-subfamily Rubioideae (Rubiaceae). Kew Bulletin 65: 45-52.), and Kiehn & Berger (2020Kiehn M, Berger A. 2020. Neotropical Rubiaceae: synthesis of chromosome data from Costa Rican taxa, with insights on the systematics of the family. Annals of the Missouri Botanical Garden 105: 423-458.), and compared the data with chromosome numbers and ploidy levels observed for the genera and the Rubiaceae as a whole (Naiki 2012Naiki A. 2012. Heterostyly and the possibility of its breakdown by polyploidization. Plant Species Biology 27: 3-29.; Kiehn & Berger 2020Kiehn M, Berger A. 2020. Neotropical Rubiaceae: synthesis of chromosome data from Costa Rican taxa, with insights on the systematics of the family. Annals of the Missouri Botanical Garden 105: 423-458.). We tested if there was a relationship between ploidy levels and breeding system by contingency analysis and chi-square independence test (Sokal & Rohlf 1994Sokal RR., Rohlf FJ. 1994. Biometry. New York, W.H. Freeman & Company.).

Molecular data and analyses

We downloaded all DNA sequences available for species of the genera Psychotria and Palicourea in GenBank (www.ncbi.nlm.nih.gov/genbank/) and maximized the number of species with the same sequences available. As a result, we focused on complete 45S ribosomal DNA (composed by 18S ribosomal RNA, internal transcribed spacer 1, 5.8S ribosomal RNA, internal transcribed spacer 2, and 26S ribosomal RNA). DNA sequences were aligned and edited in Geneious version 11.0 (http://www.geneious.com, Kearse et al. 2012Kearse M, Moir R, Wilson A, et al. 2012. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28: 1647-1649.) using the MAFFT v. 7 algorithms (Katoh & Standley 2013Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular biology and evolution, 30: 772-780.). The nucleotides substitution model was estimated in Mega 7.0 (Kumar et al. 2016Kumar S, Stecher G, Tamura K. 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874.) and the best model was selected using the AIC values. The phylogenetic relationship of the species was estimated through Maximum Likelihood analysis based on the GTR model in Mega 7.0 (Kumar et al. 2016Kumar S, Stecher G, Tamura K. 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874.). Node supports were estimated by performing 1,000 bootstrap replications (Felsenstein 1985Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791.). Branch lengths and divergence times were estimated rooting and calibrating the tree with Faramea multiflora A. Rich. as an outgroup and the estimated age for fossil records of the genus Faramea Aubl. as the Oligocene (Graham 2009Graham A. 2009. Fossil record of the Rubiaceae. Annals of the Missouri Botanical Garden 96: 90-108.). The divergence times of species with shifts in their breeding system were estimated using the fossil-calibrated phylogenetic tree (using ~34 mya for Faramea). Besides Faramea multiflora, we included other species as outgroups to allow a better representation and support to the ancestral state inference (see Tab. 1 for names and authority): a species of another Rubioideae tribe, Coussareeae; three species in the sister Spermacoceae Alliance; two less related species inside the Psychotrieae Alliance itself; and three Rudgea Salisb., a sister genus to Palicourea and Psychotria inside the Psychotrieae Alliance. The availability of DNA sequences and breeding system information was also considered for the selection of the outgroups.

We traced the ancestral states for the breeding system in species of Psychotria and Palicourea (and whenever possible to the outgroups) based on the consensus phylogenetic tree. Breeding system character state reconstruction was built-up using maximum likelihood in Mesquite 2.5 (Maddison & Maddison 2008Maddison WP, Maddison DR. 2008. Mesquite: a modular system for evolutionary analysis. Evolution 62: 1103-1118.). We also used trace characters over trees with maximum likelihood. Breeding system information was categorized as 0 - monomorphism, 1 - distyly, 2 hosmostyly, 3 - dioecy and 4 - monoecy. We used stochastic mapping character reconstruction using continuous Markov’s chain model, which allows trait changes in all possible evolutionary pathways (Nielsen 2002Nielsen R. 2002. Mapping mutations on phylogenies. Systematic biology 51: 729-739.). Independent evolution of the breeding systems of the studied species was calculated using Pagel’s Lambda considering free homoplasy of characters, which calculates likelihoods using a speciation/extinction model reduced from the BiSSE model. This index ranges from zero, which means no phylogenetic signal in the trait, to one meaning strong phylogenetic signal in the trait. Low likelihood values (e.g. closer to zero) indicate independent trait evolution (Maddison et al. 2007Maddison WP, Midford PE, Otto SP. 2007. Estimating a binary character's effect on speciation and extinction. Systematic Biology 56: 701-710.).

Results

Taxa breeding system and distribution

Out of the 46 studied species (Tab. 1), 31 were truly distylous and 15 presented other breeding systems. We recorded ten species with monomorphism, mostly pin-monomorphism with stigmas above the anthers (except Palicourea montivaga, which is thrum-monomorphic), three homostylous species, one dioecious species and one monoecious species. Among the outgroups, most were typically distylous. However, Mussaenda lancipetala is thrum-monomorphic and Mussaenda shikokiana is dioecious. Dioecy is reported for Coussarea, although the studied species is truly distylous. Breeding system transitions were commonly associated with island populations and isolated forest fragments, 11 in 15 transition cases, although those habitats were also more common among the studied species (see Tab. 1).

The chromosome number data obtained was still inconclusive to define ploidy trends. Studied species were either diploid (2n = 2x = 22/24), tetraploid (2n = 4x = 44) or octoploid (2n = 8x = 88/84) with a few aneuploidy cases. For the 21 species which we got chromosome number estimates (Tab. 1), only six showed anomalous distyly and only one was diploid. Among the truly distylous, seven species were diploid and eight species polyploid. A chi-square test for the contingency analysis showed no significant dependence between breeding system and ploidy (p=0.098). Limited data preclude finer analyses for chromosome data.

Phylogenetic inference

The phylogenetic relationships between Psychotria and Palicourea species was inferred using 716bp following the GTR nucleotides substitution model (Fig. 1). We found two groups of species of the two genera which were clearly separated from Rudgea species (84 bootstrap) and from the other outgroups. The first group included exclusively Neotropical species of Psychotria (Heteropsychotria) and Palicourea, all in the tribe Palicoureeae (26 species). The second group was distinct from the first with a support of 49 bootstrap, and included species of heterogeneous origin (Fig. 1) but all from the tribe Psychotrieae (20 species). Although some recent synonyms may indicate otherwise, the accepted names in The World Flora were all congruent with phylogenetic placement.

Figure 1
Phylogenetic consensus tree of Psychotria and Palicourea species (Rubiaceae) inferred by maximum likelihood of rRNA sequences. Numbers at branches indicate bootstrap values. Taxonomic classifications and Biogeographical regions followed Holt et al. (2012Holt BG, Lessard JP, Borregaard MK, et al. 2012. An update of Wallace's zoogeographic regions of the world. Science 339: 74-78.) and Razafimandimbison et al. (2014Razafimandimbison SG, Taylor CM, Wikström N, Pailler T, Khodabandeh A, Bremer B. 2014. Phylogeny and generic limits in the sister tribes Psychotrieae and Palicoureeae (Rubiaceae): Evolution of schizocarps in Psychotria and origins of bacterial leaf nodules of the Malagasy species. American Journal of Botany 101: 1102-1126.). Trace over tree breeding system information and estimated transitions are presented in different colors (gray branches indicated undefined ancestral breeding system). Tree was rooted and calibrated with Faramea fossil record (~34 mya) to infer divergence times. Different colors in species names represent different breeding system strategies. Bars represent standard deviation of divergence times.

Ancestral state inference and breakdown of distyly

The mating system transitions were mostly derived from distyly and were present across the species phylogeny, both in tribe Palicoureeae (eight transitions) and Psychotrieae (six transitions). The divergence time analyses indicated tribe divergence ca. 25 mya (Fig. 1). Dioecy and monoecy appeared only in species of Psychotrieae of the Sino-Japanese regions, and the divergence time analyses indicated they arose up to ca. 20 mya. in Psy. manillensis. Homostyly and monomorphism shifts were recorded in 12 species, mostly in the Neotropics, and occurred mostly in the Pliocene and Pleistocene (less than five mya). However, Psy. racemosa, Psy. brachiata (Palicoureeae) and Psy. mapourioides (Psychotrieae) may have diverged earlier, up to ca. 20 mya. The 14 shifts in breeding system seemed to have evolved independently of the phylogenetic similarities between the studied species (Pagel’s lambda = 0.21.)

Discussion

Our results showed evolutionary transitions in breeding systems in species of Psychotria and Palicourea as derived from distyly. Shifts in the breeding system and anomalous distyly appeared independently multiple times across the phylogenetic analyses, with different types of breeding systems and with at least one possible reversion to a distylous stage. Monoecy and dioecy appeared only in Asian taxa and were of Miocene origin, while homostyly and herkogamous monomorphism occurred mostly in Neotropical taxa and appeared mostly later in the ancestral state reconstruction. These trends are discussed in detail below.

Breeding systems

Most species analyzed here were truly distylous with different flower morphs in the studied populations. We did not finer analyses of distylous species for changes in mating systems or isoplethy, which may precede distyly breakdown (et al. 2016Sá T, Furtado MT, Ferrero V, et al. 2016. Floral biology, reciprocal herkogamy and breeding system in four Psychotria species (Rubiaceae) in Brazil. Botanical Journal of the Linnean Society 182: 689-707.), but most distylous species here are self-incompatible and distyly is functional. Despite our limited sample (possibly less than 10 % of the species of either group) we found in Palicourea and Psychotria species almost all the kinds of breeding transitions described in literature, including classical homostyly without herkogamy, pin- and thrum-monomorphism, monoecy and dioecy (Barrett 2019Barrett SCH. 2019. ‘A most complex marriage arrangement’: recent advances on heterostyly and unresolved questions. New Phytologist 224: 1051-1067). But monomorphism was by far the most common kind of breeding deviation in the species.

The occurrence of monomorphism in species of Psychotria and Palicourea may be due to the variation in self-incompatibility expression in Rubiaceae (Bawa & Beach 1983Bawa KS, Beach JH. 1983. Self-Incompatibility in the Rubiaceae of a Tropical Lowland Wet Forest. American Journal of Botany 70: 1281-1288.). The weakening or breakdown of physiological incompatibility can lead to unbalanced population morph ratio (unisoplethy) and even to the establishment of monomorphic populations (et al. 2016Sá T, Furtado MT, Ferrero V, et al. 2016. Floral biology, reciprocal herkogamy and breeding system in four Psychotria species (Rubiaceae) in Brazil. Botanical Journal of the Linnean Society 182: 689-707.; Barrett et al. 1989Barrett SCH., Morgan M, Husband BC. 1989. The dissolution of a complex polymorphism: the evolution of self-fertilization in tristylous Eichhornia paniculata (Pontederiaceae). Evolution 43: 1398-1416.). Among the species of our study, only P. montivaga showed short-styled, thrum-monomorphism (reverse herkogamy; Taylor 1989Taylor CM. 1989. Revision of Palicourea (Rubiaceae) in Mexico and Central America. Systematic Botany Monographs 26: 1-102.), all the other transitions were pin-monomorphic with approach herkogamy. The exposure of pin stigma and possibly incompatibility breakdown may explain why, most of the time, the long-styled morph is the one fixed in monomorphic populations and species (Arroyo et al. 2002Arroyo J, Barrett SCH, Hidalgo R, Cole WW. 2002. Evolutionary maintenance of stigma-height dimorphism in Narcissus papyraceus (Amaryllidaceae). American Journal of Botany 89: 1242-1249.; Sakai & Wright 2008Sakai S, Wright SJ. 2008. Reproductive ecology of 21 coexisting Psychotria species (Rubiaceae): when is heterostyly lost? Biological Journal of the Linnean Society 93: 125-134.; Barrett 2015Barrett SCH. 2015. Influences of clonality on plant sexual reproduction. Proceedings of the National Academy of Sciences 112: 8859-8866.; Balogh & Barrett 2016Balogh CM, Barrett SCH. 2016. Stochastic processes during invasion: the influence of population size on style-morph frequency variation in Lythrum salicaria (purple loosestrife). International Journal of Plant Sciences 177: 409-418.). It is hypothesized that the approach herkogamy in pin flowers has a better performance in founding populations since they are more likely to receive pollen grains than the stigma in thrum flowers (Baker et al. 2000Baker AM, Thompson JD, Barrett SCH. 2000. Evolution and maintenance of stigma-height dimorphism in Narcissus. I. Floral variation and style-morph ratios. Heredity 84: 502-513.). Moreover, in genetic models of distyly expression in other angiosperms, the long-styled morph genotype is commonly recessive (ss), and the short-styled morph is heterozygote (Ss) (Dulberger 1964Dulberger R. 1964. Flower dimorphism and self-incompatibility in Narcissus tazetta L. Evolution 18: 361-363.; Barrett 2019Barrett SCH. 2019. ‘A most complex marriage arrangement’: recent advances on heterostyly and unresolved questions. New Phytologist 224: 1051-1067). Thus, the allelic frequency (s/S) in distylous population under negative frequency-dependent selection is respectively 3:1, and the odds of losing the S allele is much greater than the odds of losing the s allele. If this model holds for Rubiaceae, this can be the reason why pin-monomorphic populations of Psychotria and Palicourea are more common than thrum-monomorphic populations.

The monomorphism of Psy. hoffmansegianna, Psy. racemosa, Psy. brachiata, Psy. tenuifolia, Psy. micrantha (Sakai & Wright 2008Sakai S, Wright SJ. 2008. Reproductive ecology of 21 coexisting Psychotria species (Rubiaceae): when is heterostyly lost? Biological Journal of the Linnean Society 93: 125-134.), Palicourea guianensis (Taylor 1997Taylor CM. 1997. Conspectus of the Genus Palicourea (Rubiaceae: Psychotrieae) with the description of some new species from Ecuador and Colombia. Annals of the Missouri Botanical Garden 84: 224-262.) and P. montivaga (Taylor 1989Taylor CM. 1989. Revision of Palicourea (Rubiaceae) in Mexico and Central America. Systematic Botany Monographs 26: 1-102.) were reported in island populations studies in Central America. In Psy. carthagenensis the monomorphism was also associated with populations somewhat isolated or disturbed, or in the edge of species distribution range (Consolaro et al. 2011Consolaro H, Silva SCS, Oliveira PE. 2011. Breakdown of distyly and pin-monomorphism in Psychotria carthagenensis Jacq. (Rubiaceae). Plant Species Biology 26: 24-32., E. Rodrigues unpublished studies). All monomorphic populations of Palicourea and Psychotria studied so far are self-fertile and are viewed as examples of the advantage of selfing and reproductive assurance as strategies in colonizing islands or marginal distribution habitats, as predict by Baker’s law (Baker 1967Baker HG. 1967. Support for Baker's law-as a rule. Evolution 21: 853-856.; Pannel et al. 2015Pannell JR, Auld JR, Brandvain Y, et al. 2015. The scope of Baker's law. New Phytologist 208: 656-667.). Evolutionary studies on distyly showed that breeding system variation across populations (distyly and monomorphism with approach herkogamy) were associated with differences in pollinators morphology (short-tongued pollinivorous and long-tongued nectivorous), as in Narcissus papyraceus (Amaryllidaceae) (Pérez-Barrales & Arroyo 2010Pérez-Barrales R, Arroyo J. 2010. Pollinator shifts and the loss of style polymorphism in Narcissus papyraceus (Amaryllidaceae). Journal of Evolutionary Biology 23: 1117-28.). In contrast, the monomorphism in populations of Luculia pinceana (Rubiaceae) seems to be linked to founder effect events and differences in the self-incompatibility of the floral morphs (Zhou et al. 2012Zhou W, Barrett SCH, Wang H, Li D-Z. 2012. Loss of floral polymorphism in heterostylous Luculia pinceana (Rubiaceae): a molecular phylogeographic perspective. Molecular Ecology 21: 4631-4645.). Founder effect may also be responsible for the fixation of monomorphism in species and populations of Palicourea and Psychotria. Among the studied species, there are records of truly distylous populations for Psy. hoffmansegianna, Psy. racemosa and P. guianensis in Brazilian forests (et al. 2016Sá T, Furtado MT, Ferrero V, et al. 2016. Floral biology, reciprocal herkogamy and breeding system in four Psychotria species (Rubiaceae) in Brazil. Botanical Journal of the Linnean Society 182: 689-707., E. Rodrigues unpublished studies), while the same species are monomorphic in island habitats (Sakay & Wright 2008Sakai S, Wright SJ. 2008. Reproductive ecology of 21 coexisting Psychotria species (Rubiaceae): when is heterostyly lost? Biological Journal of the Linnean Society 93: 125-134.). Homostyly without herkogamy was reported in P. macrobotrys, P. alpina and Psy. mapourioides. In P. macrobotrys homostyly occurred in marginal habitats of the species distribution (Coelho & Barbosa 2003Coelho CP, Barbosa AAA. 2003. Biologia reprodutiva de Palicourea macrobotrys Ruiz & Pavon (Rubiaceae): um possível caso de homostilia no gênero Palicourea Aubl. Revista Brasileira de Botânica 26: 403-413.) and in P. alpina in an island population in Jamaica (Tanner 1982Tanner E. 1982. Breeding systems in a tropical forest in Jamaica. Biological Journal of the Linnean Society 18: 263-278.). In these species, homostyly seems to be fixed at the species level, since there are no records of distyly in either species elsewhere (Taylor 1997Taylor CM. 1997. Conspectus of the Genus Palicourea (Rubiaceae: Psychotrieae) with the description of some new species from Ecuador and Colombia. Annals of the Missouri Botanical Garden 84: 224-262.). In Psy. mapouriodes, homostyly occurred in a population in the Brazilian Northeastern region (Parque Estadual Mata do Pau-Ferro, E. Rodrigues, pers. obs.) in a rain forest fragment isolated amid the dry Caatinga vegetation (Veloso et al. 1991Veloso HP, Rangel-Filho ALRR, Lima JCA. 1991. Classificação da vegetação brasileira, adaptada a um sistema universal. Rio de Janeiro, IBGE.). Contrastingly, in forests of the Cerrado region, the species appears to be truly distylous (Tangará da Serra, Mato Grosso; Parque Nacional de Brasília, Distrito Federal Brazil, E. Rodrigues, pers. obs).

Classic homostyly has been considered a result of recombination in the distylous supergene, as in Primula (Conti et al. 2000Conti E, Suring E, Boyd D, Jorgensen J, Grant J, Kelso S. 2000. Phylogenetic relationships and character evolution in Primula L.: The usefulness of ITS sequence data. Plant Biosystems 134: 385-392.; Mast et al. 2006Mast AR, Kelso S, Conti E. 2006. Are any primroses (Primula) primitively monomorphic? The New Phytologist 171: 605-16.), in Turnera (Barrett & Shore 1987Barrett SCH, Shore JS. 1987. Variation and Evolution of Breeding Systems in the Turnera ulmifolia L. Complex. Evolution 41: 340-354.) and in Villarsia albiflora (Menyanthaceae) (Ornduff 1988Ornduff R. 1988. Distyly and incompatibility in Villarsia congestiflora (Menyanthaceae), with comparative remarks on V. capitata. Plant Systematics and Evolution 159: 81-83.). Despite the lack of similar genetic studies with the distylous Psychotria and Palicourea species, homostyly is probably analogous to the accepted for other distylous plants groups (Barrett & Shore 2008Barrett SCH, Shore JS. 2008. New insights on heterostyly: Comparative biology, ecology and genetics. In: Franklin-Tong V (ed.) Self-Incompatibility in Flowering Plants: Evolution, Diversity and Mechanisms. Berlin, Springer-Verlag , p. 3-32.). Recent data has shown that the structure of the purported supergene is more complex (Barrett 2019Barrett SCH. 2019. ‘A most complex marriage arrangement’: recent advances on heterostyly and unresolved questions. New Phytologist 224: 1051-1067) and recombination may be rarer (Cocker et al. 2018Cocker JM, Wright J, Li J, et al. 2018. Primula vulgaris (primrose) genome assembly, annotation and gene expression, with comparative genomics on the heterostyly supergene. Scientific reports 8: 1-13.). However, homostyly occurred independently at least in 45 species of Primula (Mast et al. 2006Mast AR, Kelso S, Conti E. 2006. Are any primroses (Primula) primitively monomorphic? The New Phytologist 171: 605-16.; Barrett 2019Barrett SCH. 2019. ‘A most complex marriage arrangement’: recent advances on heterostyly and unresolved questions. New Phytologist 224: 1051-1067). Breakdown events and homostyly in Psychotria and Palicourea may represent similar events and offer a great model for the study of the evolution of the distyly expression and regulation.

Regardless of the genetic process which leads to distyly breakdown and homostyly, transitions will putatively depend on ecological pressures to be established in populations (Richards 1998Richards AJ. 1998. Lethal linkage and its role in the evolution of plant breeding systems. In: Owens SJ, Rudall PJ. (eds.) Reproductive Biology. Richmond, Royal Botanic Gardens. p. 71-83.). Pollinator-mediated selection processes, as in Exochaenium Griseb. (Gentianaceae; Kisling & Barrett 2013Kissling J, Barrett SCH. 2013. Variation and evolution of herkogamy in Exochaenium (Gentianaceae): Implications for the evolution of distyly. Annals of Botany 112: 95-102.), or founder effect, as in Plumbaginaceae (Costa et al. 2019Costa J, Torices R, Barrett SCH. 2019. Evolutionary history of the buildup and breakdown of the heterostylous syndrome in Plumbaginaceae. New Phytologist 224: 1278-1289. ) and Amsinckia (Boraginaceae; Schoen et al. 1997Schoen DJ, Johnston MO, Lheureux AM, Marsolais JV. 1997. Evolutionary history of the breeding system in Amsinckia (Boraginaceae). Evolution 51: 1090-1099.), may explain homostyly establishment in insular or isolated Rubiaceae populations. Thereby, ecological and biogeographic factors seem to influence the breeding system transitions for both Psychotria and Palicourea species, probably leading to a uniparental colonization or loss of one of the morphs when species colonize islands (Sakai & Wright 2008Sakai S, Wright SJ. 2008. Reproductive ecology of 21 coexisting Psychotria species (Rubiaceae): when is heterostyly lost? Biological Journal of the Linnean Society 93: 125-134.) or when populations are isolated by ecological factors such as disturbance, habitat fragmentation, or reduced pollination services (Consolaro et al. 2011Consolaro H, Silva SCS, Oliveira PE. 2011. Breakdown of distyly and pin-monomorphism in Psychotria carthagenensis Jacq. (Rubiaceae). Plant Species Biology 26: 24-32.; Zhou et al. 2012Zhou W, Barrett SCH, Wang H, Li D-Z. 2012. Loss of floral polymorphism in heterostylous Luculia pinceana (Rubiaceae): a molecular phylogeographic perspective. Molecular Ecology 21: 4631-4645.; Costa & Machado 2017Dulberger R. 1964. Flower dimorphism and self-incompatibility in Narcissus tazetta L. Evolution 18: 361-363.). But other transitions, such as gender distinction, may be more complex and require a sequence of events.

Dioecy and monoecy were the breeding system in Psy. asiatica (Psy. rubra) and Psy. manillensis, respectively. The breeding system transition of both species occurred in the Japanese archipelago (Watanabe et al. 2013Watanabe K, Shimizu A, Sugawara T. 2013. Dioecy derived from distyly and pollination in Psychotria rubra (Rubiaceae) occurring in the Ryukyu Islands, Japan. Plant Species Biology 29: 181-191.). Beach & Bawa (1980Beach JH, Bawa KS. 1980. Role of pollinators in the evolution of dioecy from distyly. Evolution 34: 1138-1142.) predicted the evolution of dioecy from distyly by disruption of disassortative pollen flow between the distylous morphs under a context of shifts in pollinator fauna. Thomson & Barrett (1981Thomson JD, Barrett SCH. 1981. Selection for outcrossing, sexual selection and the evolution of dioecy in plants. American Naturalist 118: 443-449.) pointed out the importance of self-incompatibility ancestor in the evolution and selection of dioecy. However, for Psy. asiatica it is unknown if the species ancestors had self-incompatibility or whether there are distylous populations outside the Japanese island, which hinders the evaluation of possible pathways for the evolution of dioecy from distyly. The monoecious Psy. manillensis also occurs in a Japanese island habitat. Putatively the closest related species of Psy. asiatica, Psy. manillensis is polyploid, suggesting that chromosome doubling might be responsible for the origin of male and female flowers in this species (Watanabe & Sugawara 2015Watanabe K, Sugawara T. 2015. Is heterostyly rare on oceanic islands? AoB Plants 7: plv087). However, our phylogenetic reconstruction (see below) did not support this inference since the species appeared apart and possibly required monomorphism as intermediate stages from a hermaphrodite ancestor (Beach & Bawa 1980Beach JH, Bawa KS. 1980. Role of pollinators in the evolution of dioecy from distyly. Evolution 34: 1138-1142.). A complex pathway to dioecy would also require disruptive selection in male and female sex allocation (Barrett 2002Barrett SCH. 2002. The evolution of plant sexual diversity. Nature Reviews Genetics 3: 274-284.), usually associated with some degree of male sterility and selection of unisexual flowers. Actually, recent studies indicate polygamous populations of Psy. manillensis, with different flower morphologies and breeding behaviors (Watanabe et al. 2020Watanabe K, Shimizu A, Sugawara T. 2020. Polygamous breeding system identified in the distylous genus Psychotria: P. manillensis in the Ryukyu archipelago, Japan. BioRxiv. https://doi.org/10.1101/2020.10.14.334318
https://doi.org/10.1101/2020.10.14.33431...
). Thus, intermediate stages and self-interference may have played important roles on the evolution of unisexual flowers, as previously proposed (Casper & Charnov 1982Casper BB, Charnov EL. 1982. Sex allocation in heterostylous plants. Journal of Theoretical Biology 96: 143-149.; Charlesworth 1989Charlesworth D. 1989. Allocation to male and female function in hermaphrodites, in sexually polymorphic populations. Journal of Theoretical Biology 139: 327-342.; Charlesworth & Morgan 1991Charlesworth B, Morgan MT, Charlesworth D. 1991. Multilocus models of inbreeding depression with synergistic selection and partial self-fertilization. Genetics Research 57: 177-194. ). In insular habitats, like the Japanese Islands, these evolutionary transitions to gender isolation may have ensured cross-pollination and eliminated the risks of interference between sexual functions.

Heterostyly has been postulated as rare or absent in islands (Pailler et al. 1998Pailler T, Humeau L, Figier J, Thompson JD. 1998. Reproductive trait variation in the functionally dioecious and morphologically heterostylous island endemic Chassalia corallioides (Rubiaceae). Biological Journal of the Linnean Society 64: 297-313.) and the breakdown of distyly has been observed during species colonization in oceanic islands (Barrett et al. 1989Barrett SCH., Morgan M, Husband BC. 1989. The dissolution of a complex polymorphism: the evolution of self-fertilization in tristylous Eichhornia paniculata (Pontederiaceae). Evolution 43: 1398-1416.; Sakai & Wright, 2008Sakai S, Wright SJ. 2008. Reproductive ecology of 21 coexisting Psychotria species (Rubiaceae): when is heterostyly lost? Biological Journal of the Linnean Society 93: 125-134.; Barrett & Shore, 2008Barrett SCH, Shore JS. 2008. New insights on heterostyly: Comparative biology, ecology and genetics. In: Franklin-Tong V (ed.) Self-Incompatibility in Flowering Plants: Evolution, Diversity and Mechanisms. Berlin, Springer-Verlag , p. 3-32.; Watanabe & Sugawara, 2015Watanabe K, Sugawara T. 2015. Is heterostyly rare on oceanic islands? AoB Plants 7: plv087). However, this mating system transition seems to be species specific, since there are also truly distylous species in islands, such as Psy. cephalophora and Psy. boninensis (Hayata) Nakai and Psy. serpens in the Japanese archipelago (Sugawara et al. 2014Sugawara T, Yumoto M, Tsuneki S, Watanabe K. 2014. Incompatibility and reproductive output in distylous Psychotria boninensis (Rubiaceae), endemic to the Bonin (Ogasawara) Islands, Japan. Journal of Japanese Botany 89: 22-26.; Watanabe et al. 2015Watanabe K, Aleck-Yang TY, Nishihara C, et al. 2015. Distyly and floral morphology of Psychotria cephalophora (Rubiaceae) on the oceanic lanyu (orchid) Island, Taiwan. Botanical Studies 56: 1-9.; Watanabe et al. 2013Watanabe K, Shimizu A, Sugawara T. 2013. Dioecy derived from distyly and pollination in Psychotria rubra (Rubiaceae) occurring in the Ryukyu Islands, Japan. Plant Species Biology 29: 181-191.), and Psy. deflexa, Psy. chagrensis, Psy. marginata and other Psychotria species in Barro Colorado Island, Panama (Sakai & Wright 2008Sakai S, Wright SJ. 2008. Reproductive ecology of 21 coexisting Psychotria species (Rubiaceae): when is heterostyly lost? Biological Journal of the Linnean Society 93: 125-134.). So, the breakdown of distyly and breeding transitions seems to not be more frequent in those habitats, at least in tribes Psychotrieae and Palicoureeae.

Different ploidy levels were present in typical and anomalous distylous species. No clear relationship among breeding systems and polyploidy was found. This relationship is not clear either for species of Amsinckia (Boraginaceae) (Schoen et al. 1997Schoen DJ, Johnston MO, Lheureux AM, Marsolais JV. 1997. Evolutionary history of the breeding system in Amsinckia (Boraginaceae). Evolution 51: 1090-1099.) and Turneraceae (Shore et al. 2006Shore JS, Arbo MM, Fernández A. 2006. Breeding system variation, genetics and evolution in the Turneraceae. New Phytologist 171: 539-551.). There is also evidence for Rubiaceae species that polyploidization has no clear link with the breakdown of heterostyly (Naiki 2012Naiki A. 2012. Heterostyly and the possibility of its breakdown by polyploidization. Plant Species Biology 27: 3-29.). Although wider sampling may show otherwise, the breeding systems transitions of Palicourea and Psychotria do not seem to be related to chromosome number or polyploidy.

Phylogenetic insights

The phylogenetic reconstruction attempted here was limited to the species of Psychotria and Palicourea with both rRNA sequences and breeding system information, so that it is limited in scope, should be used cautiously, and has no taxonomic intent. However, it resulted in a topology that broadly agrees with recent phylogenetic studies in the Rubiaceae (Razafimandimbison et al. 2008Razafimandimbison SG, Rydin C, Bremer B. 2008. Evolution and trends in the Psychotrieae alliance (Rubiaceae) - a rarely reported evolutionary change of many-seeded carpels from one-seeded carpels. Molecular phylogenetics and evolution 48: 207-23.; 2014Razafimandimbison SG, Taylor CM, Wikström N, Pailler T, Khodabandeh A, Bremer B. 2014. Phylogeny and generic limits in the sister tribes Psychotrieae and Palicoureeae (Rubiaceae): Evolution of schizocarps in Psychotria and origins of bacterial leaf nodules of the Malagasy species. American Journal of Botany 101: 1102-1126.; 2017Razafimandimbison SG, Kainulainen K, Wikström N, Bremer B. 2017. Historical biogeography and phylogeny of the pantropical Psychotrieae alliance (Rubiaceae), with particular emphasis on the Western Indian Ocean Region. American Journal of Botany 104: 1407-1423.; Wikström et al. 2020Wikström N, Bremer B, Rydin C. 2020. Conflicting phylogenetic signals in genomic data of the coffee family (Rubiaceae). Journal of Systematics and Evolution 58: 440-460.). The clear exeption was the position of Rudgea, placed in Palicoureeae Alliance in Razafimandimbison et al. (2014)Razafimandimbison SG, Taylor CM, Wikström N, Pailler T, Khodabandeh A, Bremer B. 2014. Phylogeny and generic limits in the sister tribes Psychotrieae and Palicoureeae (Rubiaceae): Evolution of schizocarps in Psychotria and origins of bacterial leaf nodules of the Malagasy species. American Journal of Botany 101: 1102-1126. and here as a sister group Palicoureeae and Psychotrieae, probably a result of our limited sampling. In any case, the phylogenetic framework for the studied group is an ongoing discussion and may result in further reorganization.

Despite being limited by available breeding system information and rRNA sequences, our sample included species from the two tribes in similar proportions and from different biogeographical regions worldwide; and breeding system transitions appeared in both groups and regions. The multiple and apparently independent shifts were similar to the observed by Sakai & Wright (2008Sakai S, Wright SJ. 2008. Reproductive ecology of 21 coexisting Psychotria species (Rubiaceae): when is heterostyly lost? Biological Journal of the Linnean Society 93: 125-134.) for Barro Colorado Psychotria. Due to this ample distribution, we expect the shifting events will be even more numerous and independent whenever a wider sample of species of the tribes are put together. Numerous breeding systems shifts and distyly breakdown events have been described for Rubiaceae (Ferrero et al. 2012Ferrero V, Rojas D, Vale A, Navarro L. 2012. Delving into the loss of heterostyly in Rubiaceae: Is there a similar trend in tropical and non-tropical climate zones? Perspectives in Plant Ecology, Evolution and Systematics 14: 161-167.) and distylous angiosperms as a whole (Barrett 2019Barrett SCH. 2019. ‘A most complex marriage arrangement’: recent advances on heterostyly and unresolved questions. New Phytologist 224: 1051-1067), and seem to be an homoplasic trait (e.g.Zhong et al. 2019Zhong L, Barrett SCH, Wang XJ, et al. 2019. Phylogenomic analysis reveals multiple evolutionary origins of selfing from outcrossing in a lineage of heterostylous plants. New Phytologist 224: 1290-1303.).

Distyly appeared as ancestral to the diversification of the genera Psychotria and Palicourea as previously proposed for the Psychotrieae Alliance (Ferrero et al. 2012Ferrero V, Rojas D, Vale A, Navarro L. 2012. Delving into the loss of heterostyly in Rubiaceae: Is there a similar trend in tropical and non-tropical climate zones? Perspectives in Plant Ecology, Evolution and Systematics 14: 161-167.). The breeding system ancestral state reconstruction for Psychotria and Palicourea species did not corroborate the model of evolution of distyly proposed by Anderson (1973Anderson WR. 1973. A Morphological Hypothesis for the Origin of Heterostyly in the Rubiaceae. Taxon 22: 537-542.). As far as we could see, there was no protandry as ancestral state or as breeding system deviation in the species of our study. The results of the phylogenetic reconstruction did not agree with Charlesworth & Charlesworth (1979Charlesworth B, Charlesworth D. 1979. The Maintenance and Breakdown of Distyly. The American Naturalist 114: 499.) predictions either. Homostyly was mostly derived from distyly and there was little evidence of homostyly as ancestral breeding system or reversion to distyly from homostyly. Our results corroborate Hamilton (1990Hamilton CW. 1990. Variations on a distylous theme in mesoamericam Psychotria subgenus Psychotria (Rubiaceae). Memoirs of the New York Botanical Garden 55: 62-75.) and Lloyd and Webb (1992Lloyd DG, Webb CJ. 1992a. The evolution of heterostyly. In: Barrett SCH (ed.) Evolution and function of heterostyly. Monographs on theoretical and applied genetics. Berlin, Springer p. 151-178.a; bLloyd DG, Webb CJ. 1992b. The selection of heterostyly. In: Barrett SCH (ed.) Evolution and function of heterostyly. Monographs on theoretical and applied genetics. Berlin, Springer . p. 179-207.) predictions about derived floral morphology from distyly. As they both proposed, homostyly and monomorphism were basically derived from distyly. However, the breeding system phylogenetic reconstruction of our study does not allow inferences for the evolution of distyly using Lloyd and Webb (1992a)Lloyd DG, Webb CJ. 1992a. The evolution of heterostyly. In: Barrett SCH (ed.) Evolution and function of heterostyly. Monographs on theoretical and applied genetics. Berlin, Springer p. 151-178. model. Distyly seems to have been already well established before the diversification of the Psychotrieae Alliance (Ferrero et al. 2012Ferrero V, Rojas D, Vale A, Navarro L. 2012. Delving into the loss of heterostyly in Rubiaceae: Is there a similar trend in tropical and non-tropical climate zones? Perspectives in Plant Ecology, Evolution and Systematics 14: 161-167.). Further studies and addition of more species of Psychotria and Palicourea, or even at the family level, will be required to elucidate the evolution of the floral polymorphism, making clear what is the ancestral condition of distylous Rubiaceae.

The breeding system transitions in Psychotria and Palicourea seemed to have occurred in distinct geological times. The Sino-Japanese species, which distyly breakdown events involved gender specialization, had their estimated diversification before the Miocene (more than 10-13 mya), while the Neotropical species, which distyly breakdown events led to monomorphism, had their estimated diversification mostly less than five mya, in the Pliocene and in the Pleistocene (nine out of 12 species). In the Miocene, under a warmer climate, the Sino-Japanese flora presented extensive humid forests (Tanai 1972Tanai T. 1972. Tertiary history of vegetation in Japan. In: Graham A (ed) Floristics and Paleofloristics of Asia and Eastern North America. Amsterdam, Elsevier. p. 235-255.; Hsu 1983Hsu J. 1983. Late cretaceous and cenozoic vegetation in China, emphasizing their connections with North America. Annals of the Missouri Botanical Garden 70: 490-508.). In the Neotropics, dry and humid forests had multiple expansion cycles during the Pleistocene and even earlier than that (Ratter 1992Ratter JA. 1992. Transition between cerrado and forest vegetation in Brazil. In: Furley PA, Proctor J, Ratter JA (eds.) Nature and Dynamics of the Forest-Savanna Boundaries. London, Chapman & Hall. p. 417-429.; Oliveira-Filho and Ratter 1995Oliveira-Filho AT, Ratter JA. 1995. A study of the origin of central Brazilian forests by the analysis of plant species distribution patterns. Edinburgh Journal of Botany 52: 141-194.; Werneck et al. 2012Werneck FP, Nogueira C, Colli GR, Sites Jr JW, Costa GC. 2012. Climatic stability in the Brazilian Cerrado: implications for biogeographical connections of South American savannas, species richness and conservation in a biodiversity hotspot. Journal of Biogeography 39: 1695-1706.). The species with breakdowns in distyly evolved under climate shifts of the Miocene and Pleistocene that have been seen as trigger mechanisms for species diversification (Tanai 1972Tanai T. 1972. Tertiary history of vegetation in Japan. In: Graham A (ed) Floristics and Paleofloristics of Asia and Eastern North America. Amsterdam, Elsevier. p. 235-255.; Antonelli & Sanmartín 2011Antonelli A, Sanmartín I. 2011. Why are there so many plant species in the Neotropics? Taxon 60: 403-414.). In addition, in these new colonizing areas scenario, distyly breakdowns may have been strategies for reproductive assurance (Yuan et al. 2017Yuan S, Barrett SCH, Duan T, Qian X, Shi M, Zhang D. 2017. Ecological correlates and genetic consequences of evolutionary transitions from distyly to homostyly. Annals of Botany 120: 775-789.).

The diverse breeding systems observed for the studied Psychotria and Palicourea derived from distyly and evolved independently across the species of this study. They were not associated with the tribe phylogenetic divisions either, evolving independently in Psychotria and Palicourea of different origins and possibly at different geological times. Despite limited sampling, transitions did not appear to be linked to ploidy changes either. Nevertheless, breeding system transitions in Psychotria and Palicourea were reported in populations and species that occurred in islands or relatively isolated forest habitats, ecological scenarios where founder effect may have played an important role in establishment of species and populations with breeding systems derived from distyly. Our results indicate that, although distyly is widespread in Psychotria and Palicourea across their Pantropical distribution, these plants repeatedly evolved alternative breeding strategies, possibly to ensure reproductive success in their diversity of habitats.

Acknowledgements

We thank FAPEMIG for a doctoral grant for EBR (Finance code 001). CAPES also provided a sandwich grant to EBR in Portsmouth. The work was supported also by research grants to PEO from FAPEMIG (APQ-02138-15) and CNPq (ref. 306551/2014-4, 474516/2013-0 and 477885/2012-8).

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Publication Dates

  • Publication in this collection
    24 June 2022
  • Date of issue
    2022

History

  • Received
    27 July 2021
  • Accepted
    02 Feb 2022
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