A morphological and anatomical study of Brazilian Lygodium species (Lygodiaceae), focusing on spore and stomata morphology and spore germination (Lygodium ×fayae: abrindo o caminho para uma maior compreensão sobre híbridos de samambaias brasileiras) REBEKAH HELEN LINDSEY Dissertação apresentada ao Instituto de Biociências, Câmpus de Botucatu, UNESP, para obtenção do título de Mestre no Programa de Pós-Graduação em Ciências Biológicas (Botânica), Área de concentração Sistemática Vegetal BOTUCATU – SP 2020 2 UNIVERSIDADE ESTADUAL PAULISTA “Júlio de Mesquita Filho” INSTITUTO DE BIOCIÊNCIAS DE BOTUCATU REBEKAH HELEN LINDSEY ORIENTADORA ANA PAULA FORTUNA-PEREZ CO-ORIENTADOR PEDRO BOND SCHWARTSBURD CO-ORIENTADORA ANA PAULA MORAES Dissertação apresentada ao Instituto de Biociências, Câmpus de Botucatu, UNESP, como parte dos créditos para obtenção do título de Mestre no Programa de Pós-Graduação em Ciências Biológicas (Botânica), Área de concentração Sistemática Vegetal BOTUCATU – SP 2020 Lindsey, Rebekah Helen L753l Lygodium ×fayae: abrindo o caminho para uma maior compreensão sobre híbridos de samambaias brasileiras : um estudo morfológico e anatômico sobre as espécies brasileiras de Lygodium (Lygodieaceae), focado em morfologia de estômatos e esporos, e germinação de esporos / Rebekah Helen Lindsey. -- Botucatu, 2020 30 p. : il., tabs., fotos, mapas Dissertação (mestrado) - Universidade Estadual Paulista (Unesp), Instituto de Biociências, Botucatu Orientadora: Ana Paula Fortuna-Perez Coorientador: Pedro Bond Schwartsburd 1. Botânica. 2. Taxonomia. 3. Samambaias. 4. Lygodieaceae. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca do Instituto de Biociências, Botucatu. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. 3 Sumário ABSTRACT ......................................................................................................................... 5 1. INTRODUCTION ............................................................................................................ 6 2. MATERIAL & METHODS ............................................................................................ 12 3. RESULTS & DISCUSSION ........................................................................................... 14 3.1.1. Analysis of spores ...................................................................................................... 14 3.1.2. Analysis of stomata .................................................................................................... 16 3.1.3. Spore germination ...................................................................................................... 17 3.2. TAXONOMIC TREATMENT ..................................................................................... 17 3.2.1. Lygodium venustum Sw. ............................................................................................ 17 3.2.2. Lygodium volubile Sw. .............................................................................................. 18 4. CONCLUSION ............................................................................................................... 19 5. REFERENCES ................................................................................................................ 25 4 ABSTRACT The family Lygodiaceae has always had a contradictory taxonomy, the number of accepted species in Brazil varying from two to twelve through the decades. Lygodiaceae being an old family, this contradictions are believed to be caused by hybrids. Hybrids arise by the crossing of two or more species, and, usually, they are first identified by its intermediate morphological characteristics among the parental species. Normally, early fern hybrids are detected occurring along with its parental species. The spores of hybrid individuals generally are badly formed and/or aborted, thus, not viable, but the few balanced spores could starts the speciation process of a new species. Considering the overlapping of morphological characteristics, it could have occurred in two species of Lygodium in Brazil, L. venustum and L. volubile and the hypothetical hybrid L. ×fayae. Due to the difficult in identification of these taxa, our studies herein analysed two different characters (spore and stomata size and morphology) through scanning electronic microscopy, we studies the spore germination and new descriptions and an identification key were done to the accepted species to help clearing the taxonomic delimitation of the two taxa of Lygodium and their putative hybrid. According to our results, the species herein classified as L. ×fayae on the Brazilian south-east region is not a hybrid, but a morphological variation of Lygodium venustum. Additional studies on morphometric analysis with the measures of stomata and spore are being carry out to to give greater support to our results. Key words: ferns, Lygodiaceae, morphology, Schizaeales, spores, stomata. 5 1. INTRODUCTION The order Schizaeales Schimp. has a fossil record dating back to the Jurassic period (Mickel 1962) or even possibly from the Carboniferous (Kenrick & Crane 1997). Order Schizaeales (sensu Smith et al. 2008) embraces three families and Anemiaceae Link, with 106 accepted species; Lygodiaceae C. Presi, with 30 species; and Schizaeaceae Kaulf, with 22 accepted species, according to The Plant List (access in 05/13/2020) (Fig. 01). The ferns in order Schizaeales are terrestrial and rupestrial ferns, often found in dry habitats (Tryon & Tryon 1982). Some of the synapomorphies (Fig. 1) for the order are the differentiation between fertile and sterile parts of the laminae since the laminae is born and the piriform sporangia, with a transverse, sub-apical and continuous indusium (Tryon & Tryon 1982; Smith et al. 2008). There are three families within Schizaeales (sensu Smith et al. 2008): Anemiaceae Link, , Lygodiaceae C. Presi, and Schizaeaceae Kaulf (Fig. 01) Fig. 01. The three families belong to order Schizaeales: A-Anemiaceae, showing fronds, and the different reproductive structures; B-Schizaeaceae, showing fronds with sorophores at the distal part; C-Lygodiaceae, showing reproductive fronds. The Lygodiaceae is a monogenetic family, widely spread across the Neotropics, represented by the climbing ferns of the genus Lygodium Sw. (Fig. 3) (Duek 1978; Tryon & Tryon 1982; Moran 1995; Mickel & Smith 2004; Smith et al. 2006; Rabelo & Schwartsburd 2016). It is uncommon for 6 climbing ferns to reach considerable heights, but individuals of Lygodium usually could reach 10 or more meters long (until 30 meters) (Moran 1995). Morphologically, these ferns could be identified by its terrestrial habit, with subterranean rhizome bearing short trichomes and few to many fibrous roots; short to long-creeping stem, frequently branched; the fronds have undetermined growth (Moran 1995) and tend to be climbing, polymorphic, intertwined, with furcate pinnae, widely alternately pinnate, glabrous to pubescent, the pinnae is short-stalked, pseudodichotomously branched with and arrested bud in the axil, each primary pinna-branch palmately or radiately lobed or branched or pinnate. The pinnulae are 2- pinnate, and the second order of pinnulae can be linear or palmate, they can partially dimorphic, with the fertile bearing marginal lobes; the veins are free or rarely anastomosed without include free veinlets (Fig. 02). A B C D Fig. 02. Fronds of Lygodium venustum in different states. A - Sterile frond; B - Sub-apical and continuous indusium; C, D - Details of early fertile fronds. Pictures by A. Simões. 7 A B C D Fig. 03. Different stages of sorophores A, B - closer look on the indusium of L. venustum; C - details of trichome; D - spores near the indusium. Pictures by A. Simões. Lygodium comprises about 30 species with six/eight of them occurring in American tropics (Fig. 04), but just two in Brazil (Tryon & Tryon 1982). The taxonomy of Lygodium is still controversial (Tryon & Tryon 1982; Rabelo & Schwartsburd 2011; 2016). The first account of Lygodium from Brazil was done in “Flora Brasiliensis” (von Martius et al. 1840) presenting 12 species: L. acuminatum J.W. Sturm., L. expansum Desv., L. hastatum (Willd.) Desv., L. hirtum Kaulf., L. lucens Kaulf., L. micans J.W. Sturm, L. mucronatum J.W. Sturm., L. palmatilobum J.W. Sturm., L. pohlianum C. Presl, L. puberulum J.W. Sturm., L.venustum Sw. and L. volubile Sw. However, Sturm never got to see the plants in their natural habitat, characterizing them only by parts that arrived in Europe what may have induced to erroneous circumscriptions.. However, some 8 names are still used today, and his work still holds great importance. A few years later, Fée (1869), in his “Cryptogames vasculaire du Brésil”, revisited the genus taxonomy and reduced the number of species to five: L. hastatum, L. hirtum, L. pohlianum, L. venustum, and L. volubile, of which all would occur in south-eastern Brazil, except for L. hastatum, which would only be found in the north-east Brazil. Almost one hundred years later, in the year of 1974, Aloysio Sehnem published “Flora Ilustrada Catarinense”, in which he described four species of Lygodium distributed in the southern region of Brazil: L. mucrolatum J.W. St., L. polymorphum (Cav.) HBK, L. venustum Sw., and L. volubile Sw. After four years, Duek (1978) published “A taxonomical revision of Lygodium (Filicinae) on America”, a book with a great impact in the Lygodium taxonomy. Duek noticed that, for a genus as plastic and volatile as Lygodium, using solely a morphological analysis