UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” INSTITUTO DE BIOCIÊNCIAS – RIO CLARO unesp PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS (BIOLOGIA VEGETAL) ESTUDOS TAXONÔMICOS E BIOSSISTEMÁTICOS DO GÊNERO Dryadella LUER (ORCHIDACEAE: PLEUROTHALLIDINAE) DANIELA CRISTINA IMIG Rio Claro – SP 2022 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” INSTITUTO DE BIOCIÊNCIAS – RIO CLARO unesp PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS (BIOLOGIA VEGETAL) ESTUDOS TAXONÔMICOS E BIOSSISTEMÁTICOS DO GÊNERO Dryadella LUER (ORCHIDACEAE: PLEUROTHALLIDINAE) DANIELA CRISTINA IMIG Tese apresentada ao Instituto de Biociências do Câmpus de Rio Claro, Universidade Estadual Paulista, como parte dos requisitos para obtenção do título de doutor em Ciências Biológicas (Biologia Vegetal). Orientador: Dr. Eric de Camargo Smidt Co-orientadora: Dr a . Viviane da Silva-Pereira Rio Claro – SP 2022 I32e Imig, Daniela Cristina Estudos taxonômicos e biossistemáticos do gênero Dryadella Luer (Orchidaceae: Pleurothallidinae) / Daniela Cristina Imig. -- Rio Claro, 2022 214 f. : il., tabs., fotos, mapas Tese (doutorado) - Universidade Estadual Paulista (Unesp), Instituto de Biociências, Rio Claro Orientador: Eric de Camargo Smidt Coorientador: Viviane da Silva-Pereira 1. Filogenia. 2. Taxonomia. 3. Botânica. 4. Morfometria. 5. Anatomia. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca do Instituto de Biociências, Rio Claro. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. UNIVERSIDADE ESTADUAL PAULISTA Câmpus de Rio Claro ESTUDOS TAXONÔMICOS E BIOSSISTEMÁTICOS DO GÊNERO Dryadella LUER (ORCHIDACEAE: PLEUROTHALLIDINAE) TÍTULO DA TESE: CERTIFICADO DE APROVAÇÃO AUTORA: DANIELA CRISTINA IMIG ORIENTADOR: ERIC DE CAMARGO SMIDT Aprovada como parte das exigências para obtenção do Título de Doutora em CIÊNCIAS BIOLÓGICAS (BIOLOGIA VEGETAL), área: Biologia Vegetal pela Comissão Examinadora: Prof. Dr. ERIC DE CAMARGO SMIDT (Participaçao Virtual) Departamento de Botanica / Centro Politecnico UFPR Universidade Federal do Parana Curitiba PR Prof. Dr. JULIO ANTONIO LOMBARDI (Participaçao Virtual) Departamento de Biodiversidade / UNESP Instituto de Biociencias de Rio Claro SP Profa. Dra. ALESSANDRA IKE COAN (Participaçao Virtual) Departamento de Biodiversidade / UNESP - Instituto de Biociências de Rio Claro - SP Prof. Dr. CECÍLIA OLIVEIRA DE AZEVEDO (Participaçao Virtual) Universidade Estadual Do Sudoeste Da Bahia Profª. Drª. SAMANTHA KOEHLER (Participaçao Virtual) Universidade Estadual de Campinas Rio Claro, 04 de abril de 2022 Instituto de Biociências - Câmpus de Rio Claro - Av. 24-A no. 1515, 13506900 http://ib.rc.unesp.br/#!/pos-graduacao/secao-tecnica-de-pos/programas/biologia-vegetal/apresentacao/CNPJ: 48.031.918/0018-72. AGRADECIMENTOS Estes agradecimentos poderiam render um livro com muitas e muitas páginas, e ainda assim eu não me cansaria de citar nomes e nomes, pois muitos são aqueles que me ajudaram, direta ou indiretamente nesse processo. Foi um momento histórico, uma pandemia que chegou de repente e nos obrigou à resiliência a cada perda de amigos, parentes e conhecidos e até desconhecidos, eram números que cresciam em segundos, mas graças à CIÊNCIA, hoje aos poucos voltamos ao ―novo normal‖ e eu, ao final de uma etapa muito importante da minha vida, viva! Por isso, agradeço em especial ao meu orientador, Prof. Dr. Eric de Camargo Smidt, por me apadrinhar quando fiquei órfã na ciência, por toda a orientação e por estar sempre disposto a me ajudar, além da parceria e do respeito cultivados durante nosso tempo de convívio, sou grata pela oportunidade a essa viagem incrível na ciência das plantas de flores tão pequenas, e digo, ela ainda não acabou. Sou também profundamente grata ao Dr. Antonio L.V. Toscano de Brito que em muitos aspectos representou o papel de co-orientador e um colaborador fundamental, sempre pronto a me ajudar e disponibilizar bibliografias valiosas. Que momentos difíceis hein Toscano? Sou grata por tudo! À Prof. Drª. Viviane da Silva-Pereira, aquela que me fez estabelecer um domínio de pequenos movimentos, ah, logo eu? Sempre desajeitada com tudo, mas no laboratório consegui dar conta de algo que jamais imaginei conseguir, os tais tubinhos intactos! Obrigada por me desafiar! Ah, e falando em laboratório e mil tubinhos de números minúsculos, Anna Victória S. R. Mauad, muito obrigada, você nunca me deixou sozinha (ela me conhece bem), e nem fora do lab. Obrigada Anna, por toda força e por ser minha inspiração. À Prof. Drª. Erika Amano pelo grande auxílio e orientação com o trabalho de anatomia vegetativa e pela nossa amizade. Sou grata ao suporte proporcionado pelo Programa de Pós-Graduação em Ciências Biológicas (Biologia Vegetal) do Instituto de Biociências da UNESP, Campus Rio Claro e a todos os docentes. Estendo este agradecimento aos amigos e colegas que fiz em Rio Claro por tornarem meus dias mais leves e felizes enquanto estive fora de casa cursando as disciplinas. Apesar da minha ausência física na UNESP, tenho um carinho enorme por todos. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), pela bolsa de estudos concedida. Ao IBAMA/SISO e ICMBio O pelo fornecimento das licenças de coleta e aos vários funcionários das unidades de conservação visitadas. Agradeço também a todos os curadores e funcionários dos herbários visitados que permitiram o acesso ao acervo e os empréstimos das exsicatas. Agradeço aos professores, técnicos, amigos e colegas do Departamento de Botânica da UFPR, que me acompanharam desde o mestrado, pelas instruções ao longo destes tantos anos. Agradeço aos orquidófilos que disponibilizaram materiais e fotos de Pleurothallidinae, principalmente a Marcos L. klingelfuss e José Vanderlei Alves Neves pela grande ajuda com materiais, pela amizade e parceria durante os campos. Aos amigos e colegas do Laboratório de Sistemática e Ecologia Molecular de Plantas (LSEMP) da UFPR, de imenso valor foi a ajuda e o convívio por parte dessas pessoas. Sou imensamente grata aos amigos que a Botânica me proporcionou (são muitos e cada um sabe sua importância na minha vida!). Cada um a seu modo, ajudou e ainda ajuda a construir a mim mesma, nossas discussões de horas, nossos debates sociais, políticos e econômicos e claro, nossas discussões botânicas me lapidam e me tornam cada dia melhor. Obrigada a todos!! Aos amigos da vida (alguns também são da Botânica, risos), pelos abraços que me aliviaram por muitas vezes o cansaço e principalmente por entenderem minha ausência. Aos colegas do trabalho e à toda(s) a(s) equipe(s). Sou especialmente grata ao meu amigo João Bento, obrigada por ter me acompanhado nos campos em MG, e por ser meu grande amigo! Aos meus pais, que sempre marejaram os olhos quando eu não podia estar em momentos de família, mas sempre vibraram com minhas conquistas e compreenderam meus momentos de ausência. Essa conquista é de vocês e por vocês. Saibam que meu coração sempre esteve presente! Eu amo vocês! À toda a minha família, meus alunos, meus amigos pelo incentivo e suporte imensurável em toda essa caminhada, foram muitas as mensagens que me lembravam de que eu era forte e conseguiria, mesmo muitas vezes, sem nem terem ideia do que é taxonomia e muito menos o que é Dryadella. Em especial, agradeço minhas primas (irmãs) Deise e Iraci que são essenciais para minha vida e aos meus pequenos Giovana, Giordana, Fabrício, Pedro Zion e Geovane...! Um agradecimento ao Dr. Armando Carlos Cervi (in memoriam) por ter me inspirado a seguir na taxonomia e por ter me fortalecido na Botânica! Obrigada por ter deixado a Raphaella e a família em minha vida! E por último, justamente por saber que quando chegasse aqui meus olhos seriam tomados de lágrimas e eu teria de ser breve, agradeço à minha Joanina que escolheu o meu ventre para evoluir, sua passagem por aqui foi breve, mas meu amor por você não! Aos irmãos dela (Chris e Sofia) e ao pai deles Nelson Fernando H. Selesu, meu parceiro e acima de tudo meu grande amigo, que esteve em um dos momentos mais difíceis que já vivi e suportou a barra, muitas vezes sozinho. Obrigada por segurar a minha mão naquele corredor onde o choro ecoava em dor! Obrigada por tanto! Amo-te! Mais um ciclo se encerra e sou muito grata por todas as histórias vividas, amizades construídas e experiências adquiridas. Obrigada! ―Por vezes sentimos que aquilo que fazemos não é senão uma gota de água no mar. Mas o mar seria menor se lhe faltasse uma gota‖. (Madre Teresa de Calcuta) RESUMO Dryadella é um gênero pertencente à subtribo Pleurothallidinae que contém 61 espécies. As espécies de Dryadella são epífitas ou raramente rupícolas, cespitosas, raramente reptantes ou pendentes. As inflorescências podem apresentar de 1 a 4 flores sucessivas dispostas em um racemo congesto. Nas flores, as sépalas laterais apresentam um calo espesso próximo à base, pétalas multiangulares e o labelo longo unguiculado com lâmina provida de dois lobos basais, geralmente retrorsos. A distribuição desse gênero é neotropical e disjunta, desde a América Central através das florestas úmidas dos Andes, até o norte da Argentina e do sul ao nordeste do Brasil na Floresta Atlântica. As relações filogenéticas de Dryadella foram investigadas usando o espaçados nuclear ITS e a região plastidial matK, analisadas por máxima verossimilhança e máxima parcimônia para inferir as relações evolutivas entre as espécies. Datações moleculares e análises biogeográficas também foram realizadas. Os resultados obtidos indicam que Dryadella é monofilético, com alto suporte, inserido na Afinidade Specklinia, com provável origem no noroeste da América do Sul, no final do Mioceno (~7 Ma). A primeira diversificação de Dryadella ocorreu no início do Plioceno (~5Ma), envolvendo um evento de dispersão e três clados foram identificados. O clado 1 tem origem no noroeste da América do Sul e em seguida dispersão para o Sul da Amazônia e para a Floresta Atlântica. O clado 2 tem origem no noroeste da América do Sul com dispersão para América Central e em seguida dispersão para o sul dos Andes. O clado 3 tem origem no noroeste da América do Sul com dispersão para o Cerrado e para a Mata Atlântica, diversificado em torno de 3 Ma. A elaboração da monografia das espécies brasileiras de Dryadella resultou em uma nova espécie, novos sinônimos e novas ocorrências, resultando em 14 espécies reconhecidas para o país. O tratamento taxonômico é apresentado para as espécies brasileiras, incluindo descrições morfológicas, ilustrações, fotografias, chave de identificação e distribuição geográfica. Devido a dificuldade taxonômica encontrada em D. zebrina, D. edwallii e D. wuerstlei durante a revisão taxonômica, dedicamos nossos esforços para explorar morfometricamente populações destes táxons, levantamos as variáveis minuciosamente e tratamos a partir de análises multivariadas a fim de clarear a taxonomia e apresentar uma nova circunscrição para estes táxons. Investigações da micromorfologia dos órgãos vegetativos a partir da anatomia da raiz, rizoma, ramicaule e folha de representantes de Dryadella foram descritas com achados inéditos, como ráfides de cristal de oxalato, ausência de tricomas ou cicatrizes destes na superfície foliar e superfície cuticular verrucosa em algumas espécies. Palavras-chave: análise filogenética, anatomia vegetativa, taxonomia, monocotiledôneas, nomenclatura, morfometria. ABSTRACT Dryadella is a genus belonging to the subtribe Pleurothallidinae, comprising 61 species. They are epiphytic or rarely rupicolous, cespitose, rarely repent or pending; inflorescence always a single or 2-4 successive flowers in a congested raceme; lateral sepals with thick callus near the base, multi-angular petals and long unguiculated lip with two basal lobes. Exclusively neotropical with disjunct distribution, from Central America through the humid forests of the Andes, to northern Argentina and southern Brazil in the Atlantic Rainforest. Dryadella phylogenetic relationships were investigated using nrITS and the matK plastid region, analysed for Maximum likelihood and Maximum parsimony to infer evolutionary relationships. Molecular dating and biogeographic analyses were also performed. The results obtained provide high support for Dryadella monophyly, inserted in the Specklinia Affinity. with probable origin in northwestern South America, at the end of the Miocene (~7Ma). The first diversification of Dryadella occurred in the early Pliocene (~5Ma), involving a dispersion event and three clades clades were identified. Clade 1 originated in northwestern South America and later dispersal to southern Amazonia and a posterior dispersion event for the Atlantic Rainforest. Clade 2 originates from northwest South America with a dispersion event to Central America and subsequent dispersion to the south of the Andes. Clade 3 originates from northwest South America to the Cerrado and the Atlantic Forest, diversified around 3 My. The elaboration of the monograph of the Brazilian species of Dryadella resulted in a new species, in addition to new synonyms and new occurrences, a taxonomic treatment is presented for the Brazilian species, including morphological descriptions, illustrations, photographs, identification key and geographic distribution. Due to the taxonomic difficulty found in D. zebrina, D. edwallii and D. wuerstlei found during the taxonomic review, we dedicated our efforts to morphometrically explore populations of these taxa, survey the variables in detail and treat from multivariate analyzes in order to clarify the taxonomy and present a new circumscription for these taxa. Investigations of the micro morphology of vegetative organs from the anatomy of the root, rhizome, ramicaule and leaf of representatives of Dryadella were described with unpublished findings. Keywords: phylogenetic analysis, vegetative anatomy, taxonomy, monocots, nomenclature, morphometry. SUMÁRIO INTRODUÇÃO GERAL 11 REFERÊNCIAS 19 CAPÍTULO I / CHAPTER I Evidências filogenéticas e biogeográficas para origem de Dryadella (Orchidaceae) no Mioceno no norte da América do Sul e diversificação recente na Floresta Atlântica/ Phylogenetic and biogeographic evidence for a Miocene origin in north South-America and recent diversification of Dryadella (Orchidaceae) in Atlantic rainforest ..........................................................................................................25 CAPÍTULO II / CHAPTER II O gênero Dryadella (Orchidaceae, Pleurothallidinae) no Brasil/ The genus Dryadella (Orchidaceae, Pleurothallidinae) in Brazil.........................61 CAPÍTULO III / CHAPTER III Atualização taxonômica em Dryadella (Orchidaceae: Pleurothallidinae) baseada em análises morfométricas de três espécies endêmicas da Floresta Atlântica/ Taxonomic update in Dryadella (Orchidaceae: Pleurothallidinae) based on morphometric analyses of three endemic species to the Atlantic Rainforest.......................................................................................................................130 CAPÍTULO IV / CHAPTER IV Uma nova Dryadella Luer (Orchidaceae: Pleurothallidinae) da Floresta Atlântica Sul/ A new Dryadella Luer (Orchidaceae: Pleurothallidinae) from the South Atlantic Rainforest..................................................161 CAPÍTULO V/ CHAPTER V Anatomia vegetativa e sua importância sistemática em Dryadella Luer (Orchidaceae: Pleurothallidinae)/ Vegetative anatomy and its systematic importance in Dryadella Luer (Orchidaceae: Pleurothallidinae)..................................173 11 INTRODUÇÃO GERAL Orchidaceae é uma das maiores e mais diversas famílias de plantas com cerca de 27.800 espécies e cerca de 740 gêneros (POWO, 2022). As orquídeas então distribuídas por quase todo o mundo, com exceção das regiões desérticas e polares (DRESSLER, 1993), com maior abundância e diversidade nas regiões tropicais e subtropicais. No Neotrópico, a região dos Andes é a área mais rica em orquídeas, seguida da Floresta Atlântica brasileira. O Brasil é um dos centros de diversidade das orquídeas, abrigando 251 gêneros, sendo 24 endêmicos e cerca de 2.680 espécies, das quais 1.490 são endêmicas para o país (FLORA DO BRASIL, 2020). A família é monofilética, sustentada tanto por dados morfológicos quanto moleculares (DRESSLER, 1981, 1993; FREUDENSTEIN & RASMUSSEN, 1999; FREUDENSTEIN et al., 2000, 2004; PRIDGEON et al., 1999, 2001, 2003, 2005a, 2009, 2014). Apresenta alta diversidade vegetativa e ainda que a estrutura da flor seja uniforme em número e disposição de peças, há uma grande diversidade de tamanho e detalhes estruturais. Com base em dados moleculares, Orchidaceae é dividida em cinco subfamílias: Apostasioideae, Cypripedioideae, Vanilloideae, Orchidoideae e Epidendroideae, sendo essa última a mais representativa, com aproximadamente 18.000 espécies em 650 gêneros, com distribuição cosmopolita (PRIDGEON et al., 1999, 2001, 2003, 2005a, 2009). Epidendroideae, por sua vez, é subdividida em 16 tribos, entre elas Epidendreae, constituída de 6 subtribos: Bletiinae Bentham, Chysinae Schlechter, Coeliinae Dressler, Laeliinae Bentham, Poneriinae Pfitzer e Pleurothallidinae Lindley (PRIDGEON et al., 2005a; CHASE et al., 2015). A subtribo Pleurothallidinae é exclusivamente Neotropical, com aproximadamente 5.100 espécies distribuídas em 44 gêneros e representa cerca de 20% das espécies de toda a família Orchidaceae (HIGGINS & WILLIAMS, 2009; KARREMANS, 2016). É um grupo monofilético com alto suporte e posicionado na tribo Epidendreae (CHASE et al., 2015), caracterizada por ervas de crescimento simpodial, unifoliadas com o caule secundário (ramicaule) não pseudobulboso, e a inflorescência apresenta uma articulação associada a uma zona de abscisão entre o ovário e o pedicelo (LUER, 1986; DRESSLER, 1993; PRIDGEON, 2005a). Suas flores possuem características de miofilia, como por exemplo, o tamanho reduzido e a forma radial, e ainda o labelo que pode conter manchas, pequenas projeções, fendas e lóbulos fusionados (van der PIJL; 12 DODSON, 1966; LUER, 1986; NEYLAND et al., 1995; PRIDGEON et al., 2005a; KARREMANS et al., 2015). Muitas espécies de Pleurothallidinae apresentam estruturas com função de atração de polinizadores através de secreção de fragrâncias, que podem ser encontradas no labelo, pétalas e/ ou sépalas (DRESSLER, 1993; PRIDGEON; STERN, 1985; SCHIESTL et al., 2003; SCHIESTL & JOHNSON, 2013; CARDOSO- GUSTAVSON et al., 2017) e nectários também já foram identificados dentro da subtribo como atrativos para os polinizadores oferecendo recompensa (BARBOSA et al., 2009; MELO et al., 2010). Em Pleurothallidinae esses estudos demonstraram que a subtribo tem uma das maiores taxas de diversificação de espécies dentro de Orchidaceae, e que se originou no Mioceno Inferior (~20 Ma) (GIVNISH et al., 2015; PÉREZ-ESCOBAR et al., 2017). Uma recente atualização sobre as relações filogenéticas de Pleurothallidinae foi realizada por Karremans (2016), através de um compilado de estudos baseados em filogenia molecular que apontam para a existência de nove grupos ou afinidades. Dryadellla Luer, um dos gêneros reconhecidos em Pleurothallidinae e foco deste estudo, encontra-se posicionado na afinidade Specklinia, que inclui ainda os gêneros Andinia Luer, Muscarella Luer, Platystele Schltr., Scaphosepalum Pfitzer, Specklinia Lindl. e Teagueia Luer (Karremans, 2016; Karremans et al., 2016). Dryadellla foi estabelecido por Luer (1978a), a partir de algumas espécies desmembradas do gênero Masdevallia Ruiz & Pav., porém as primeiras espécies haviam sido descritas no gênero Pleurothallis R. Br. e, em seguida, alocadas no gênero Masdevallia seção Saltatrices Luer, (2005) e posteriormente em Masdevallia subg. Trigonanthe Schltr., seção Rhombopelalae Schltr., exceto M. lilliputiana inserida na seção Floribundae Kraenzl (Luer, 2005). Após o estabelecimento do novo gênero, as espécies de Masdevallia correspondentes foram transferidas por Luer (1978a, 1978b, 2005) e ao longo destes anos, novos táxons foram descritos e novos sinônimos foram propostos, somando atualmente 61 nomes aceitos (IMIG et al., 2021; POWO, 2022) (Tabela 01). A distribuição geográfica de Dryadella é disjunta com duas grandes áreas de ocorrência, a Floresta Atlântica do Brasil e a região Andina (PRIDGEON et al., 2005b). No Brasil, o gênero até o momento está representado por 18 espécies, destas, 15 são endêmicas do país e 14 restritas à Floresta Atlântica. Os outros representantes são encontrados na 13 Amazônia (Dryadella osmariniana e D. cardosoi) e Cerrado (Dryadella ana- paulae) (Pridgeon, 2005b; IMIG et al. 2020; POWO 2022). Tabela 1. Listagem e os países de ocorrência das espécies de Dryadella aceitas antes dos nossos estudos. Espécies Ocorrência Dryadella albicans (Luer) Luer Equador Dryadella ana-paulae V.P.Castro, BPFaria & ADSantana Brasil e Bolívia Dryadella ataleiensis Campacci Brasil Dryadella aurea Luer e Hirtz Peru e Equador Dryadella auriculigera (Rchb.f.) Luer Brasil Dryadella aviceps (Rchb.f.) Luer Brasil e Paraguai Dryadella barrowii Luer Equador Dryadella butcheri Luer Paramá Dryadella cardosoi Campacci e JBFSilva Brasil Dryadella clavellata Luer e Hirtz Equador Dryadella crassicaudata Luer Peru e Colômbia Dryadella crenulata (Pabst) Luer Brasil Dryadella cristata Luer & R.Escobar Colômbia Dryadella cuspidata Luer e Hirtz Colômbia e Equador Dryadella dodsonii Luer Colômbia e Equador Dryadella dressleri Luer Panamá Dryadella edwallii (Cogn.) Luer Brasil Dryadella elata (Luer) Luer Equador Dryadella espirito-santensis (Pabst) Luer Brasil Dryadella fuchsii Luer Costa Rica, El Salvador, Honduras e Nicarágua Dryadella gnoma (Luer) Luer Colômbia, Costa Rica, Equador, Panamá, Peru Dryadella gomes-ferreirae (Pabst) Luer Brasil Dryadella greenwoodiana Soto Arenas, Salazar e Solano México e Guatemala Dryadella guatemalensis (Schltr.) Luer Colômbia, Costa Rica, Guatemala, México, Panamá Dryadella hirtzii Luer Equador Dryadella kautskyi (Pabst) Luer Brasil Dryadella krenakiana Campacci Brasil Dryadella lilliputiana (Cogn.) Luer Bolívia e Brasil Dryadella linearifolia (Ames) Luer Belize, Costa Rica, Guatemala, Honduras, México e Nicarágua 14 Dryadella litoralis Campacci Brasil Dryadella lueriana Carnevali e G.A.Romero Venezuela Dryadella marilyniana Luer Equador Dryadella marsupiata Luer Equador e Peru Dryadella meiracyllium (Rchb.f.) Luer Equador Dryadella minuscula Luer & R.Escobar Colômbia e Peru Dryadella mocoana Luer & R.Escobar Colômbia Dryadella nasuta Luer e Hirtz Equador Dryadella nortonii Luer Bolívia Dryadella odontostele Luer Colômbia, Costa Rica e Panamá Dryadella osmariniana (Braga) Garay & Dunst. Brasil Dryadella pachyrhiza Luer e Hirtz Equador Dryadella perpusilla (Kraenzl.) Luer Peru Dryadella pusiola (Rchb.f.) Luer Colômbia e Equador Dryadella rodrigoi Luer Colômbia Dryadella sapucaiensis Campacci & S.L.X.Tobias Brasil Dryadella simula (Rchb.f.) Luer Colômbia e Peru Dryadella sororcula Luer Panamá Dryadella speculifera Vierling Desconhecido Dryadella sublata Luer & J.Portilla Peru Dryadella summersii (L.O.Williams) Luer Equador Dryadella susanae (Pabst) Luer Brasil Dryadella toscanoi Luer Brasil Dryadella vasquezii Luer Bolívia Dryadella verrucosa Luer & R.Escobar Colômbia Dryadella vitorinoi Luer e Toscano Brasil Dryadella werneri Luer Equador Dryadella wuerstlei Luer Brasil Dryadella xavieriana Campacci e C.R.M.Silva Brasil Dryadella yupanki (Luer & R.Vásquez) Karremans Bolívia Dryadella zebrina (Porsch) Luer Bolívia, Brasil, Colômbia e Peru Dryadella são ervas epifíticas ou raramente litófitas, cespitosas, raramente curtas reptantes, com rizoma muito abreviado ou inconspícuo. Possuem ramicaule abreviado, geralmente ereto e sempre unifoliolados, coberto total ou parcialmente por 2(3) bainhas paleáceas; folhas eretas, raramente adpressas, planas, semi-teretes ou teretes. As inflorescências que emergem lateralmente ao ramicaule, logo abaixo do ponto de abscisão, são racemosas de flor única ou até 4 flores sucessivas, pedúnculos curtos e envoltos por 2(3) brácteas paleáceas, com pedicelos inconspicuos. As flores são ressupinadas com ovário tri-alado; a sépala a dorsal é livre, inflexa ou raramente 15 deflexa; as sépalas laterais são parcialmente conectadas na base formando tubo sepalínico com ápice caudado; as pétalas são geralmente inconspícuas e inseridas no tubo sepalínico, geralmente em formato sagitado; labelo é unguiculado, bilobado na base e articulado com o ápice do pé da coluna, geralmente com 2 calos próximos a base; a coluna é alada a partir da metade distal com 2 políneas (LUER, 1978a; LUER, 2005) (Figura 1 e 2). Figura 1– Terminologias morfológicas gerais da flor de Dryadella. Fonte: LUER (2005). 16 Figura 2: Aspecto morfológico geral de Dryadella. a. Hábito. b-f. Aspectos gerais da flor. g. Aspectos do labelo. h. Aspectos gerais do labelo. Embora uma revisão taxonômica completa para Dryadella tenha sido realizada por Luer (2005), poucas espécies e amostras brasileiras foram analisadas e algumas lacunas na taxonomia do gênero permanecem o que reflete em errôneas identificações ou na falta de identificações em herbários e coleções científicas, em coleções particulares e entre os orquidófilos. Apesar de apresentar distribuição disjunta, as espécies do gênero apresentam elevada similaridade entre os táxons e por vezes, sutil diferenças no tamanho e forma das pétalas, sépalas, labelo e no tamanho das folhas são utilizadas em Luer (2005) para diagnóstico das espécies, no entanto, isso parece não contribuir suficientemente na delimitação de algumas espécies. Por outro lado, nenhuma abordagem filogenética para entender as relações entre as espécies do gênero ou abordagens biossistemáticas que possibilitem a melhor delimitação até o momento foi empregada para gênero. 17 Mesmo com poucos registros fósseis de orquídeas (nenhum deles está relacionado à Pleurothallidinae), existe a possibilidade de datar os eventos de cladogênese que originaram os gêneros da subtribo e também suas respectivas linhagens infragenéricas e havendo as idades estimadas para tais cladogêneses é possível inferir sobre a dinâmica evolutiva de todo um grupo, como também definir possíveis rotas de migração e hipotetizar eventos que propiciaram a diversificação de linhagens (Forest, 2009). Ferramentas estatísticas e consolidação da sistemática filogenética como um método para estabelecer relações de parentesco entre organismos, os estudos em sistemática têm apresentado cada vez mais a abordagem filogenética e evolutiva na interpretação dos padrões morfológicos, de distribuição geográfica e delimitações taxonômicas (Avise, 2000; Felsenstein, 2004) e, quando aliado aos métodos biogeográficos, permite inferir padrões de diversificação das linhagens de plantas atuais e a origem desses padrões (Morrone, 2006; Pennington et al., 2006). Neste sentido, a Floresta Atlântica brasileira apresenta três espécies de alta complexidade taxonômica: D. zebrina (Porsch) Luer, D. edwallii (Cogn.) Luer e D. wuerstlei Luer. A morfometria é uma abordagem que pode indicar variação fenotípica potencial dentro de complexos de espécies (Pinheiro et al., 2018) por meio de medidas de diferenças ou semelhanças morfológicas entre as unidades amostradas (Sokal & Michener 1958). Análises morfométricas multivariadas são frequentemente associadas a estudos populacionais como auxílio na delimitação de espécies ou morfotipos, pois podem revelar a ocorrência de descontinuidades em padrões de variação, a princípio, aparentemente contínuos (Oliveira et al., 2008). Considerando a distribuição disjunta e a similaridade morfológica entre as espécies do gênero, uma abordagem anatômica pode evidenciar caracteres de interesse taxonômico, auxiliando a delimitação das espécies ou grupos disjuntos, e na caracterização anatômica do gênero. Estas características anatômicas podem ser úteis para o reconhecimento de grupos com histórias evolutivas distintas (Aybeke, 2012), corroborando ou não a classificação infragenérica proposta por Luer (2005). Com a finalidade de esclarecer lacunas taxonômicas, as hipóteses filogenéticas do gênero e a identidade de morfotipos endêmicos da Floresta Atlântica, propomos aqui diferentes abordagens biossistemáticas. 18 Para tanto, este trabalho tem como objetivo geral investigar as relações filogenéticas e identificar os padrões biogeográficos do gênero baseado em caracteres moleculares e o estudo da distribuição geográfica e taxonomia do gênero. Precisamente: (i) testar o monofiletismo de Dryadella, utilizando o maior número de espécies possíveis, empregando análises com base em dados moleculares; (ii) dada a distribuição disjunta do gênero entre a região andina e a Floresta Atlântica, caracterizar os padrões de distribuição geográfica e datação molecular de Dryadella; iii) realizar um levantamento florístico e tratamento taxonômico das espécies do gênero para o Brasil (vi) propor sinônimos e/ou novos táxons e novos registros de ocorrência para o Brasil; (v) Abordar morfometricamente táxons de complexidade taxonômica ocorrentes na Floresta Atlântica; (vi) investigar caracteres morfológicos e anatômicos vegetativos e sua importância para a sistemática do gênero. Dessa forma, esta tese está disposta em capítulos e cada um deles segue as normas do periódico em que foi ou será publicado e estão dispostos da seguinte forma: CAPÍTULO I: Evidências filogenéticas e biogeográficas de origem Mioceno no norte da América do Sul e diversificação recente de Dryadella (Orchidaceae) na Floresta Atlântica CAPÍTULO II: O gênero Dryadella (Orchidaceae, Pleurothallidinae) no Brasil CAPÍTULO III: Atualização taxonômica em Dryadella (Orchidaceae: Pleurothallidinae) baseada em análises morfométricas de três espécies endêmicas da Floresta Atlântica CAPÍTULO IV: Uma nova Dryadella Luer (Orchidaceae: Pleurothallidinae) da Floresta Atlântica Sul CAPÍTULO V: Anatomia vegetativa e sua importância sistemática na Dryadella Luer (Orchidaceae: Pleurothallidinae) REFERÊNCIAS AVISE, J.C. Phylogeography: the history and formation of species. Cambridge: Harvard University Press, 2000. 19 AYBEKE, M. 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Coral Gables, FL: University of Miami Press, p. 101–122, 1966. 25 CAPÍTULO I / CHAPTER I Evidências filogenéticas e biogeográficas para origem de Dryadella (Orchidaceae) no Mioceno no norte da América do Sul e diversificação recente na Floresta Atlântica (Artigo formatado seguindo as normas do periódico Botanical Journal of the Linnean Society) https://www.researchgate.net/journal/Botanical-Journal-of-the-Linnean-Society-1095-8339 https://www.researchgate.net/journal/Botanical-Journal-of-the-Linnean-Society-1095-8339 26 Phylogenetic and biogeographic evidence for a Miocene origin in north South- America and recent diversification of Dryadella (Orchidaceae) in Atlantic Rainforest DANIELA CRISTINA IMIG 1,4* (ORCID 0000-0003-4867-5608), ANNA VICTÓRIA SILVÉRIO RIGHETTO MAUAD 2 (ORCID 0000-0002-4821-9138), VIVIANE DA SILVA-PEREIRA 3 (ORCID 0000-0003-4543-178X), ANTONIO L. V. TOSCANO DE BRITO 5,6 (ORCID 0000-0003-4566-3871) & ERIC DE CAMARGO SMIDT 1,3 (ORCID 0000-0002-1177-1682) 1 Universidade Estadual Paulista "Júlio de Mesquita Filho‖ - UNESP, Campus de Rio Claro, Programa de Pós Graduação em Ciências Biológicas (Biologia Vegetal), Av. 24 A, 1515, Bela Vista, Caixa Postal 199, Rio Claro, São Paulo, 13506–900, Brazil. 2. Universidade Federal do Paraná - UFPR, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ecologia e Conservação, Centro Politécnico, Caixa Postal 19031, Curitiba, PR, 81531–970, Brazil. 3 Universidade Federal do Paraná- UFPR, Setor de Ciências Biológicas, Departamento de Botânica, Laboratório de Sistemática e Ecologia Molecular de Plantas, Centro Politécnico, Caixa Postal 19031, Curitiba, PR, 81531–970, Brazil. 4 Centro Universitário Campos de Andrade - UNIANDRADE, Setor de Ciências Biológicas, R. João Scuissiato, 001, Santa Quitéria, Curitiba, PR, 80310-310, Brazil. 5 Marie Selby Botanical Gardens, Sarasota, FL 34236–7726, USA. 6 Orchid Herbarium of Oakes Ames, Harvard University Herbaria, Cambridge, MA 20138, USA. *Author for correspondence: daniela.imig@gmail.com Running title: Phylogenetics of Dryadella *Corresponding author. E-mail: daniela.imig@gmail.com https://orcid.org/0000-0002-4821-9138 https://orcid.org/0000-0003-4543-178X mailto:daniela.imig@gmail.com 27 Abstract Dryadella, a small orchids genus in the subtribe Pleurothallidinae with 61 species, are usually epiphytes, with long-repent or caespitose growth; inflorescence always a single or 2-4 successive flowered in congest raceme. Flowers with a thickened callus close to the base of the lateral sepals, multi-angled petals, and a long unguiculate lip with a blade provided with two basal, usually retrorse, lobes. It is distributed from Central America through the Andes to southeastern Brazil, with a disjunct distribution between coastal forests. Thirty species of Dryadella plus related genera and outgroup taxa were investigated using molecular nrITS and plastid matK, analysed using maximum likelihood and maximum parsimony to infer evolutionary relationships. Molecular dating and biogeographical analyses were also performed. Results obtained provide strong support for a monophyletic Dryadella and origin through the end of the Miocene (~7 Ma) in the Pacific domain, northwest of South America and Central America. The three clades identified by the analysis diversified at the beginning of the Pliocene (~5Ma). The first clade has a Pacific dominion origin with posterior dispersion to South Brazilian dominion and to Paraná dominion (Atlantic Rainforest) in the Quaternary period (~2.2Ma). The second clade also has a Pacific dominion origin, but with a dispersion event to Mexican and Mesoamerica dominion and to the South America Transition zone (south Andes). The last clade has a Pacific dominion origin with posterior dispersion by Chacoan dominion to Atlantic Rainforest. The Atlantic clade is the youngest and diversified around 3 Ma with one recent dispersion event to the Pacific dominion (~1Ma). The findings from this study contribute to a better understanding of the evolution of Dryadella, the non-monophyly of the proposed sections, based mainly on plant size, and the evidence of the historical connection of the coastal forests of South America. Also, for Atlantic Rainforest, two independent lineages of Dryadella diversified in the Quaternary period. ADDITIONAL KEYWORDS: Atlantic Rainforest –Andes – biogeography – dispersion – Neotropical flora – orchid molecular phylogenetics 28 INTRODUCTION Orchidaceae are one of the largest angiosperm families, arranged in five subfamilies and placed in 736 genera with over 27,000 species described (Chase et al., 2015). Occur on all continents except Antarctica and in all biomes, except for true deserts (Givnish et al., 2016). Most species are found in tropical forests, and over 80% are epiphytes (Givnish et al., 2015). For the Americas, Orchidaceae are the most diverse family, including 12,983 species, and are the most diverse epiphyte plants in the tropical Andean countries (Ulloa Ulloa et al., 2017). Adaptation to epiphytic habit, the evolution of pollinaria and the development of different pollination mechanisms are the main factors that promoted the rapid diversification of the family, especially in tropical mountain regions (Tremblay et al., 2004; Givnish et al., 2015). In the American continent, this diversification is related to the uplift of the Andes 15 Mya and subsequent speciation events in Central America and the Atlantic Forest (ARF) (Givnish et al., 2016; Pérez-Escobar et al., 2017; Rodrigues et al., 2017; Salazar et al., 2018; Smidt et al., 2018). The subtribe Pleurothallidinae is the most diverse Neotropical group in all orchid family, including about 20% of all known species (Higgins and Williams, 2009; Karremans et al., 2016). With ca 5100 species currently accepted in 44 genera, the subtribe is the most representative element of the Andean orchid flora (Pridgeon et al., 2009; Kolanowska, 2014). Has about 20 My (millions of years) of age and one of the highest species diversification rates in the Orchidaceae (Givnish et al., 2015; Pérez- Escobar et al., 2017). Several recent phylogenetic studies focused on the Pleurothallidinae have confirmed its monophyly (e.g. Pridgeon et al., 2001; Pridgeon & Chase, 2001; Pridgeon & Chase, 2003; Karremans, 2016; Pérez-Escobar et al., 2017) and informally nine genera ―affinities‖ were proposed by Karremans (2016). However, there is still a large gap of studies to clarify the systematic placement of some lineages within this huge subtribe (e.g. Gutiérrez Morales et al., 2020; Smidt et al., 2020). Dryadella Luer belongs to the Affinity Specklinia together with Andinia Luer, Muscarella Luer, Platystele Schltr., Scaphosepalum Pfitzer, Specklinia Lindl. and Teagueia Luer (Karremans, 2016; Karremans et al., 2016). The genus is characterised by the presence of a callus near the base of the lateral sepals and the unguiculated lip with retroverted basal lobules. These were the main characteristics used by Luer (1978) to propose the genus of some 29 dismembered species from the genus Masdevallia Ruiz & Pav., and some of which were previously treated in Pleurothallis R.Br. Currently Dryadella is composed of about 61 species (Luer, 2005; POWO 2022; Imig et al., 2021) occurring from Mexico to South Brazil. The genus presents distribution is disjunct, especially between the Andean region and the ARF, except D. ana-paulae V.P. Castro, B.P. Faria & A. D. Santana, found in gallery forests of the South American Dry Diagonal, and D. gnoma (Luer) Luer, which occurs in the Amazon Rainforest (Pridgeon, 2005; Imig et al., 2020). The ARF hosts 14 species of the genus, of which only two are not endemic to the biome: D. zebrina (Porsch) Luer and D. lilliputiana (Cogn.) Luer, those also occur in the Andes, where other South American species are restricted (Pridgeon, 2005; Luer, 1978, 2005; Imig et al., 2020). The species delimitation in Dryadella is difficult due to the remarkable morphological similarity between the species, even between groups of disjunct areas (Luer, 1978; Pridgeon, 2005). Luer (2005) proposed the division of the genus into three sections, joining the species with linear leaves ―The needle leaves‖, the species with diminutive habit ―The tiny mites‖, and the other species ―All the rest‖. Recent studies on the leaf anatomy of several species from different biomes showed that the variation found does not support the delimitation of sections (Imig et al., 2020b). Neotropical rainforests are currently not continuous, actually are separated by open vegetation with a diagonal distribution and a drier climate zone (Werneck, 2011). The Seasonally Dry Tropical Forests or Caatinga (northeastern Brazil) and the Cerrado savanna (central Brazil) separate the Atlantic Forest from Amazon; whereas the Chaco (northeastern Argentina, western Paraguay and south-eastern Bolivia) divides the Atlantic Forest from the Andean Forest (Werneck, 2011; Turchetto-Zolet et al., 2012), and this disjunction may contribute as a barrier to gene flow between these rainforest regions. One of the fundamental questions about the biodiversity of a region is how this is distributed across major vegetation types. For this reason, identifying where and how orchid species are distributed could improve our understanding to elucidate the evolutionary history of the family (Hou et al., 2017). For this, the ages available for the subtribe can be used to analyse molecular data on a smaller scale, allowing the study of the evolutionary dynamics of a group of species or genus, hypothesising events that allowed its diversification (Forest, 2009). 30 In this sense, our study mainly focuses on testing the monophyly and phylogenetic relationships of the genus Dryadella, the relationships within Specklinia affinity and whether molecular analyses support the proposed infrageneric relationships based on morphological data. Reconstructing the phylogeny, we seek to apply biogeographic principles and analysis to summarise geographic distribution patterns, their times of diversification, and arrivals to the Atlantic Rainforest. To do this, we collected DNA sequence data from nrITS (ITS1, 5.8S, ITS2) and plastid matK from a large number of taxa of the genus and related groups. MATERIAL AND METHODS TAXON SAMPLING Based on previous phylogenetic studies in the subtribe Pleurothallidinae (Pridgeon et al., 2001; Karremans, 2016; Karremans et al., 2016; Pérez-Escobar et al., 2017), we included all accepted genera of Specklinia affinity: Andinia, Platystele, Scaphosepalum, Specklinia, Phloeophila and Muscarella (Pérez-Escobar et al., 2017), as ingroup to investigate the monophyly of the genus. A sampling of 16 other genera of subtribe Pleurothallidinae, in addition to species from closely related subtribes Bletiinae and Laeliinae [according to the phylogenetic proposal of Chase et al., (2015)], were used to accommodate molecular dating calibrations better. Vouchers were deposited in the Herbarium of the Botany Department at the Federal University of Paraná (UPCB), Herbarium Rioclarense (HRCB) and Marie Selby Botanical Gardens Herbarium (SEL) (acronyms following Thiers 2022). Voucher information and GenBank accessions for the species are given in Table 1. Some species of Dryadella that were used in this study are shown in figure 1. Table 1. Species sampled, GenBank (NCBI) accession numbers and geographical distribution (sensu Morrone 2014): A, Mexican transition zone; B, Mesoamerican dominion; C, Pacific Ocean dominion; D, Boreal Brazilian dominion; E, South Brazilian dominion; F, Paraná dominion; G, South American transition zone and H, Chacoan dominion (Cerrado – Caatinga) and I, Antillean subregion. *indicates new sequences generated for this study. Species ITS matK Biome Anathallis gert- MN3323458 MN332524 F 31 hatschbachii ( Hoehne)Pridgeon & M.W. Chase Anathallis obovata (Lindl.) Pridgeon & M.W. Chase MN332358 MN332534 ABCDFG Andinia pensilis (Schltr.) Luer KP012344 KP012517 C Barbosella australis (Cogn.) Schltr. MF669943.1 MF669949 F Bletia catenulata Ruiz & Pav. MN332374 MN332549 CDEFG Brachionidium restrepioides (Hoehne) Pabst MN332375 KX686526 F Brachionidium valerioi Ames & C. Schweinf. AF262913 AF265488 B Cattleya coccinea Lindl. AY008646 MN332552 Cattleya forbesii Lindl. MN332376 MN332552 F Dilomilis montana (Sw.) Sumemrh. AF262915 AF263765 H Dryadella albicans (Luer) Luer KC425742 KC425863 G Dryadella anapaulae V. P. Castro, B.P. Faria & A. D. Santana D.C. Imig 666* D.C. Imig 666* H Dryadella aurea Luer & Hirtz A.L.V. Toscano de Brito 3817* _ C Dryadella auriculigera (Rchb.f.) Luer D.C. Imig 471* D.C. Imig 471* F Dryadella aviceps (Rchb.f.) Luer D.C. Imig 397* D.C. Imig 397* F Dryadella barrowii Luer D.C. Imig 635* _ G Dryadella catharinensis Imig, Mancinelli & E.C. Smidt D.C. Imig 656 * D.C. Imig 656* F Dryadella crenulata (Pabst) Luer D.C. Imig 667* D.C. Imig 667 * F Dryadella cristata Luer & R. Escobar D.C. Imig 399* D.C. Imig 399* C Dryadella elata (Luer) Luer A. L. V. Toscano de Brito 3022 (SEL) _ C Dryadella fuchsii Luer KY988820 KY988636 AB Dryadella gnoma (Luer) Luer A.Krahl 1477* A.Krahl 1477* CD Dryadella guatemalensis (Schltr.) Luer KC425743 _ ABCD Dryadella hirtzii Luer D.C. Imig 505* D.C. Imig 505* G Dryadella kautskyi (Pabst) Luer A.L.V. Toscano de Brito 3476* A.L.V. Toscano de Brito 3476* F Dryadella krenakiana Campacci D.C. Imig 662* D.C. Imig 662* F Dryadella linearifolia (Ames) Luer A.L.V. Toscano de Brito 3435* A.L.V. Toscano de Brito 3435* AB https://pt.wikipedia.org/wiki/Cogn. https://www.ipni.org/a/22383-1 32 Draydella lilliputiana (Cogn.) Luer D.C. Imig 395* D.C. Imig 395* F Dryadella odontostele Luer - JQ771574.1 C Dryadella pachyrhiza Luer & Hirtz D.C. Imig 507* D.C. Imig 507* C Dryadella pusiola (Rchb.f.) Luer D.C. Imig 360* D.C. Imig 360* CG Dryadella simula (Rchb.f.) Luer D.C. Imig 430* D.C. Imig 430* CG Dryadella summersii (L.O. Williams) Luer D.C. Imig 481* D.C. Imig 481* G Dryadella susanae (Pabst) Luer D.C. Imig 491* D.C. Imig 491* F Dryadella verrucosa Luer & Escobar RinconGonzales 954* RinconGonzales 954* C Dryadella vitorinoi Luer D.C. Imig 506* D.C. Imig 506* F Dryadella toscanoi Luer D.C. Imig 661* D.C. Imig 661* F Dryadella zebrina (Porsch) Luer E.C. Smidt 1011* E.C. Smidt 1011* F Dryadella wuerstley Luer D.C. Imig 408* D.C. Imig 408* F Dryadella yupanki (Luer & R. Vásquez) Karremans KF747776 KP012498 E Epidendrum armeniacum Lindl. MN332381 MN332557 CEFG Epidendrum tridactylum Lindl. MN332382 MN332558 CEFG Lepanthes helicocephala Rchb.f. MN332387 MN332560 CDE Lepanthopsis floripecten ( Rchb.f. ) Ames MN332388 MN332561 BCDFG Madisonia bradei (Schltr.) Toscano & E.C. Smidt M. Bolson 565 M. Bolson 565 F Madisonia carrisii (Brade ) Toscano & ECSmidt M. Bolson 572 M. Bolson 572 F Madisonia kerri (Braga) Luer MN332389 MN332562 D Myoxanthus lonchophyllus (Barb.Rodr.) Luer MN332390 MK642625 F Myoxanthus punctatus (Barb. Rodr.) Luer KX686538 MK642626 F Muscarella catoxys (Luer & Hirtz) Luer KY988912 KY988728 C Muscarella schudelii (Luer & Hirtz) Luer KY988914 KY988730 C Neocogniauxia hexaptera Schltr. AF260148 AF263766 A Octomeria gracilis Lodd. ex Lindl. MN332392 KX686527 F Octomeria grandiflora Lindl. WSM1372 KX686528 F Phloeophila pleurothallopsis ( Kraenzl. ) Pridgeon & M.W. Chase KC425747 KP012496 C Pabstiella mirabilis (Schltr.) Brieger & MN332394 MN332565 F https://www.ipni.org/a/8254-1 https://www.ipni.org/a/9117-1 https://www.ipni.org/a/27855-1 https://www.ipni.org/a/1056-1 https://www.ipni.org/a/27855-1 https://www.ipni.org/a/20001466-2 https://www.ipni.org/a/1062-1 https://www.ipni.org/a/5818-1 https://www.ipni.org/a/5818-1 https://www.ipni.org/a/20347-1 https://www.ipni.org/a/5818-1 https://www.ipni.org/a/5818-1 https://www.ipni.org/a/20347-1 https://www.ipni.org/a/5818-1 https://www.ipni.org/a/5013-1 https://www.ipni.org/a/37810-1 33 Senghas Pabstiella versicolor (Porsh) Luer MN551470 MN577779 F Pabstiella villosisepala L. Kollmann & Fraga MN551471 MN577780 F Pabstiella yauaperyensis (Barb.Rodr.) F. Barros MN332395 MN332566 DE Platystele misera (Lindl.) Garay KF747784 KP012389 CDG Platystele oxyglossa (Schltr.) Garay WSM1333 WSM1333 BCDEFG Pleurothallis isthmica Luer MN332398 MN332569 BC Pleurothallopsis nemorosa (Barb. Rodr.) Porto & Brade MN332400 KX686529 F Platystele stenostachya ( Rchb.f.) Garay KC425758 JQ771571 ABCDEG Restrepiella ophiocephala H. Karst MN332403 MN332574 BC Scaphosepalum bicristatum Luer D.C. Imig 523* D.C. Imig 523* C Scaphosepalum microdactylum Rolfe A.L.V. Toscano de Brito 2957* A.L.V. Toscano de Brito 2957* BC Specklinia corniculata (Sw.) Mutel KF747803 KP012402 ABCI Specklinia grobyi (Bateman ex Lindl.) Pridgeon & M.W.Chase MN332406 MN332577 ABCDEFG Stelis argentata Lindl. MN332408 MN332579 BCDEFG Stelis ciliaris Lindl. MN332409 MN332580 BCEF Stelis grandiflora Lindl. MN332410 MN332581 DF Stelis ruprechtiana Rcbh.f. MN332412 MN332583 F https://www.ipni.org/a/3013-1 https://www.ipni.org/a/8254-1 https://www.ipni.org/a/27408-1 34 Figure 1. Species of Dryadella in this study. A, Dryadella auriculigera. B, Dryadella aviceps. C, Dryadella catharinensis. D, Dryadella crenulata. E, Dryadella cristata. F, Dryadella kautskyi. G, Dryadella krenakiana. H, Dryadella lilliputiana. I, Dryadella pachyrhyza. J. Dryadella summersii. K, Dryadella susanae. L, Dryadella zebrina. Photographs by Eric C. Smidt, except C by Werner Mancinelli and D and F by A.L.V. Toscano de Brito. 35 DNA EXTRACTION, AMPLIFICATION AND SEQUENCING Total genomic DNA was extracted from fresh-frozen leaves or leaves stored in CTAB solution (Rogstad, 1992, adapted) using the 2×CTAB protocols (Doyle & Doyle, 1987), without the addition of RNAse A and scaled to 2 mL microtubes. All total DNA samples were stored in a −80 °C ultra-freezer and amplified through polymerase chain reaction (PCR) for fragments of interest. The nuclear ribosomal internal transcribed spacer (nrITS) was targeted with primer pairs 17SE (ACGAATTCATGGTCCGGTGAAGTGTTCG) + 26SE (TAGAATTCCCCGGTTCGCTCGCCGTTAC) (Sun et al., 1994) and 75 (TATGCTTAAACTCAGCGGG) + 92 (AAGGTTTCCGTAGGTGAA) (Desfeux & Lejeune, 1996). Amplification of nrITS were performed with the Invitrogen Platinum Taq DNA polymerase (Thermo Fisher Scientific) PCR kit following the manufacturer‘s recommendations, adding 0.08 mg/mL of bovine serum-albumin (BSA), 0.08% dimethyl sulphoxide (DMSO), 1M betaine, 0.2 μM of each primer and 20−50 ng of total DNA for a final volume of 20 μL. For plastid DNA regions, the Top Taq Master Mix (Qiagen) PCR kit was used following the manufacturer‘s recommendations, adding 0.2 μM each primer and 20–50 ng of total DNA for a final volume of 20 μL. For both PCR methods, the thermocycling program was: 1 min pre-melt at 94 °C, 40 cycles of 30 s of denaturation at 94 °C, 40 s of annealing at 51 °C (nrITS)/53 °C (plastid DNA) and 30 s of extension at 72 °C, ending with a 5-min final extension at 72 °C. The plastid markers, matK exon, were amplified with the respective primer pairs: 19F (5′ CGTTCT GAC CAT ATT GCA CTA TG 3′) + 881R (5′ TMTTCA TCA GAA TAA GAG Tn3′) (Gravendeel et al., 2001) and 2.1f (5′ CCTATCCATCTGGAAATCTTAG 3′) and matK 5R (5′ GTTCTAGCACAAGAAAGTCG 3′; Ford et al., 2009). The Amplification was performed in 20 μL mixtures containing 1× CoralLoad buffer, 0.2 μM each primer, 1× TopTaq Master Mix and 20–50 ng of genomic DNA using the TopTaq Master Mix kit (QIAGEN Biotechnology). The thermocycler was programmed for an initial pre-melt at 94 °C for 1 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 51 °C (nrITS)/53 °C (matK) for 40 s and extension at 72 °C for 40 s, followed by a final extension for 5 min at 72 °C. All the PCR products were visualised with electrophoresis in agarose gel with GelRed (Crisafuli et al., 2015), purified with polyethylene glycol (PEG 20%) and 80% ethanol (Paithankar & Prasad, 36 1991), and sequenced with Big Dye Terminator version 3.1 (Applied Biosystems, Foster City, CA, USA). DNA SEQUENCES ALIGNMENT AND PHYLOGENETIC ANALYSES Reads were trimmed at their extremities when the error probability limit per base was > 0.05 and were de novo assembled into consensus sequences in Geneious Prime 2019.2.1 program (Kearse et al., 2012, https://www.geneious.com). The sequences with a quality threshold > 60% were aligned with MAFFT v.7.388 implemented in Geneious Prime, using the automatic algorithm and 1PAM scoring matrix (Katoh & Standley, 2013). The alignments were concatenated using SequenceMatrix v.1.8 (Vaidya et al., 2011). The alignment of nrITS and plastid matK combined were used for all subsequent phylogenetic analyses. The indels (insertion/deletion markers) were treated as missing data. Maximum Likelihood (ML), and Maximum Parsimony (MP) were used to explore the results under different methods. All resulting phylogenetic trees were edited in FigTree v.1.4.3 (Rambaut, 2009) and in CorelDRAW v.18.0.0.448 (http://www.coreldraw.com). The maximum-likelihood phylogenetic tree was estimated using IQ-tree v.1.6.12 (Nguyen et al., 2014) with tree search with model selection and bootstrap replicates in a single run (Chernomor et al., 2016; Kalyaanamoorthy et al., 2017). The best-fit models of substitution inferred in the IQ-tree analyses by AICc were TIM3e+I+G4 for ITS and TIM+F+R3 for matK. For the support, we used the 1000 ultrafast bootstrap approximation (Nguyen et al., 2014) with the argument ‗–bnni‘ to reduce the risk of overestimating branch support. Maximum parsimony analyses were performed with Fitch's (1971) parsimony using the software PAUPv.4.0a166 (Swofford, 2002). Analyses included 10 000 random taxon- addition replicates and TBR swapping algorithm, holding ten trees per replication, followed by a second search to explore all topologies from the previous search, limited to 10 000 trees. The support was estimated in 1000 bootstrap replications (BP) (Felsenstein, 1985), simple addition, and TBR algorithm, holding 20 trees per replicate in PAUP, and bootstrap percentages ≥ 50 (BS) were considered. Trees were visualised and edited with FigTree v1.4.3 (Rambaut, 2009). 37 ESTIMATION OF DIVERGENCE TIMES Divergence times of Dryadella were estimated using the concatenated alignment in BEAST v.1.8.4 (Drummond et al., 2012) using the CIPRES server (Miller et al., 2010). We set an uncorrelated relaxed clock model with lognormal distribution and GTR+I+Γ substitution model with empirical base frequencies. The dating analysis was constrained by fixing the topology to the ML tree topology. We tested two speciation models, the Yule process (Yule, 1925; Gernhard, 2008) and the birth-death process (Gernhard, 2008), that were compared on Tracer v.1.6 (Rambaut et al., 2014) using tree likelihood under AIC estimated (AICM; Baele et al., 2012). The tree was secondarily calibrated using the estimates of Givnish et al. (2015) for (1) crown age of Pleurothallidinae [mean 14.2 Mya, 95% highest posterior density interval (HPD): 9.7 ± 19.21 Mya]; (2) stem age of Laeliinae [mean 19.82 Mya, 95% HPD: 14.66 ± 25.32 Mya] and (3) the stem age of Bletiinae [mean 25.51 Mya, 95% HPD: 18.35 ± 31.45 Mya], assuming a normal distribution. MCMC chains ran for 50 million generations, with sampling every 10,000 steps. Convergence and ESS were assessed in Tracer v.1.6, and the maximum clade credibility tree was produced in TreeAnnotator v.1.8.4, with a burn-in of 25%. Visualization and basic tree editing were performed in FigTree v.1.4.3. GEOGRAPHICAL DATA AND ANALYSES To infer the biogeographic history of Dryadella, we gathered information on the current distribution of the species provided in GBIF (www.gbif.org; Jan 2022); POWO (2022), literature and herbarium specimens. Nine biogeographical areas were defined based on literature and distribution patterns observed in other plant studies grounded on the Neotropical regionalisation proposed by Morrone (2014), Table 1: A, Mexican transition zone; B, Mesoamerican dominion; C, Pacific Ocean dominion; D, Boreal Brazilian dominion; E, South Brazilian dominion; F, Paraná dominion; G, South American transition zone and H, Chacoan dominion (Cerrado – Caatinga) and I, Antillean subregion. We used the package BioGeoBEARS (Matzke, 2013) as implemented in RASP v.4.0 (Yu, et al., 2015) to evaluate several biogeographic models to infer the contribution of various evolutionary processes (i.e. range expansion, range extinctions, vicariance, founder-event speciation, speciation despite sympatry) in explaining the current species distribution of Dryadella. The highest AICc_wt value was used to select the biogeographic model best fitting the data. 38 RESULTS PHYLOGENETIC RELATIONSHIPS We analysed 144 DNA sequences, of which 51 (35,42%) are specifically produced for this study (Table 1). Both phylogenetic inferences produced similar topologies for the nrITS and cpmatK datasets and are available in the supplementary material (S1). Only the results from combined datasets are presented with the bootstrap percentages from both ML (MLBP) and MP (MPBP) analyses (Fig. 2), and parsimony statistics for all datasets are given in Table 2. Table 2. Statistics of the individual and combined parsimony analyses of Dryadella plus outgroups. nrITS matK combined Number of taxa 72 72 75 Number of characters 760 766 1526 Constant characters 327 (43%) 539 (70%) 866 (57%) Potentially parsimony informative sites 304 109 413 Number of most parsimonious trees 672 10000 10000 Tree steps 1802 414 2245 Consistency index 0.40 0.66 0.44 Retention index 0.63 0.70 0.63 Percentage of resolved nodes 0.84 0.42 0.75 39 Figure 2. Maximum-likelihood (ML) tree of Dryadella plus outgroups. Numbers on nodes represent ML bootstrap percentages (≥50) and maximum parsimony (MP) bootstrap percentages (≥50). See the Supporting Information for trees with support resulting from the separate analyses. In detail, the ML tree is shown with proportional branch lengths. 40 Disagreements in the phylogenetic positions of some taxa are highlighted in the discussion. The aligned combined matrix for 75 species consisted of 2,245 characters. Of these, 413 (18%) were potentially parsimony informative. The ML topology from the analysis of the combined dataset is presented with ML bootstrap percentages and MP bootstrap percentages above the branches (Fig. 2). The Pleurothallidinae (ML-BP 82, MP-BP 65), and the affinity Specklinia (ML-BP 90, MP-BP <50) are monophyletic. Andinia is strongly supported as a sister of the Specklinia affinity in ML analysis, but in MP is in an unresolved clade with Pleurothallis, Stelis and Pabstiella [the Pleurothallis affinity sensu Karremans (2016)]. Dryadella is monophyletic (ML-BP 99, MP-BP <50) with a grade at the base with successive D. guatemalensis (ML-BP 56, MP-BP <50) that occurs in Central America (AB), Pacific dominion (C) and Boreal Brazilian dominion (D) followed by D. linearifolia (ML-BP 60, MP-BP <50), that occurs Central America (AB), the first two early divergences in the first clade, and as a sister of three clades in ML and only D. guatemalensis in MP analysis as a sister of the remained species. The first clade presents the support of 50 ML-BP/MP-BP <50 and the following two sister clades (clades 1 and 2) with moderate support 66 ML-BP/MP-BP <50 and 85 ML- BP/MP-BP <50. The second clade has early divergent D. fuchsii, occurring in Central America (AB) with (ML-BP 66, MP-BP <50), sister of the other species of this clade, high supports, except in D. barrowii (ML-BP 58, MP-BP <50), this clade is predominantly formed by species from the Pacific dominion (C) and South American Transition Zone (G). The last clade (clade 3) is highly supported (85 ML-BP/MP-BP <50) and is formed almost all by ARF species, except D. ana-paulae, the early divergent and sister of the remained species in this clade (ML-BP 94, MP-BP <50), and D. cristata, endemic to Colombia, in lowlands, (ML-BP 93, MP-BP <50) closely related with D. wuerstlei, endemic from the south of the ARF. None of the clades formed showed support for infrageneric sections sensu Luer (2005). DIVERGENCE TIMES AND BIOGEOGRAPHY The birth-death speciation model was chosen based on the AIC results (Table S1). 41 Slight differences were found between the ancestral area estimations using BAYAREALIKE, DEC and DIVALIKE models, with or without the jump speciation parameter j. We report the estimates using DEC (Ree & Smith, 2008), which had the highest likelihood. DEC was implemented allowing a maximum number of either two or three biogeographic units per ancestral range, but this parameter did not impact the analysis and we present only the results from the three biogeographic units analysis (Fig. 3). As shown in the time-calibrated tree with a birth-death speciation model (Fig. 3), Pleurothallidinae (node 147) probably originated in the Parana dominion (Atlantic Rainforest) [F (0.77); AF (0.22)] in the early Miocene, with an estimated crown age of 16.04 My (95% HPD: 13.17 ± 19.76). The Pleurothallis affinity and Specklinia affinity (Fig. 3; node 142) diversified in the Pacific dominion [C 58.83; CF 41.17], in the late Miocene, with an estimated crown age of 10.07 My (95% HPD: 7.43 ± 12.46). 42 Figure 3. Time-calibrated tree for the Dryadella plus outgroups inferred under uncorrelated lognormal clock model in BEAST. Greybar at nodes indicates 95% highest posterior density intervals. 43 Figure 4. Maximum-likelihood tree of Dryadella plus outgroups with ancestral area optimisations. Pie charts at nodes indicate the most plausible geographic origin according to the DEC model. Coloured circles represent the occurrence of that species in geographical areas, following Morrone (2014) A, Mexican Transition Zone; B, Mesoamerica; C, Pacific dominion; D, Boreal Brazilian dominion; E, South Brazilian dominion; F, Paraná dominion (Atlantic Rainforest); G, South American Transition Zone; H, Chacoan dominion I, Antillean subregion. 44 The crown age estimated by our analyses of Dryadella (Fig. 3; node 139) is 6.96 My (95% HPD: 4.97 ± 7.9 - Fig. 3 shows in detail all ages for all nodes), between late Miocene and early Pliocene, in the Pacific domain as the ancestral area, northwest of South America [C (100)], further dispersion to Central America (AB) (Fig. 4). The biogeographic history of Dryadella is marked by some events of long dispersion, among the main ones we identified that the first important event is c. 6.35 My (95% HPD: 4,45 ± 8,22 - Fig. 3; node 138) [C 70.1; BC 14.9; AC 14.9] and was responsible for the divergence of the lineage of clade 1, in two other lineages (clades 2 e 3). Clade 1 is composed of species occurring in the Mexican Transition Zone and Mesoamerica (A, B), up to Pacific dominion, Boreal Brazilian dominion and South Brazilian dominion (C, D, E respectively), except D. susanae and D. catharinensis, endemic to Paraná dominion (F-Atlantic Rainforest) and that have diverged more recently to 2.28 My, (CF 62.36; CDF 23.81; C 13.83) [(95% HPD: 1.16 ± 3.63] (Fig. 4). A second event estimated at 5.84 My, (95% HPD: 4,09 ± 7,63 - Fig. 03, node 137) [C 100] was responsible for the divergence between lineages of the second and third clades. Then another important dispersion event (Fig. 3, node 136) estimated in c. 5.26 Mya (95% HPD: 3,69 ± 7,02), [C 50.87; CF 25.29; CH 9.98; BCF 6.93; ACF 6.93], was responsible for the formation of predominantly Andean lineages in the Pacific Ocean dominion (C) and Andes South American transition (G) of the species occurring in the Paraná dominion (F-Atlantic Forest) and later c. 5.04 My (95% HPD: 3,35 ± 6,62), D. fuchsii (AB) [C 74.09; BC 12.95; AC 12.95] diverged from the Pacific Dominion species (C ) e South American Transition Zone (G) (Fig. 3, node 124). In c. 4.17 My (95% HPD: 2,61 ± 5,83) [FH 27.79; F 25.65; CF 25.57; CFH 15.43; C 5.56], another important dispersion event was responsible for the divergence between D. anapaulae, the only representative of the Cerrado (H) and the Atlantic Rainforest lineage (Paraná dominion F), and D. cristata (C) (Fig. 4). Other dispersal events in Dryadella were frequent from Pacific dominion to Mexican Central America (northern Andes) and South America Transition zone (south Andes) and also to Amazon (Boreal Brazilian dominion) and Cerrado (H), and more recently spread to Atlantic Rainforest (F), the youngest lineage of the genus (Fig. 4). 45 DISCUSSION PHYLOGENETIC ANALYSES The genus Dryadella is, for the first time, a focus of phylogenetic study with a dense sample, with 50% of the species of the genus, including the type species, species from all proposed infrageneric classification (ex. Pabst & Dungs, 1977; Luer, 2005) as well including all morphological variations and geographic distribution. The results based on nuclear and plastid sequences present evidence of a natural origin of the affinity Specklinia, with Andinia as a sister of the other genera and Dryadella sister of the Muscarella, Specklinia, Scaphosepalum and Platystele, with generally strong support. These results agree with the proposed affinities by Karremans (2016), Karremans et al. (2016), and Pérez-Escobar et al. (2017), except for Phloeophila pleurothallopsis Hoehne & Schltr. which appears in the Pleurothallis affinity, with low support (BPML 54), related to Pleurothallis and Stelis and not as a sister group to the Specklinia and Pabstiella affinities. Previous studies treat Phloeophila as monophyletic based on molecular data Chiron et al., (2016), with nrITS and some samples of cpmatK (Karremans et al., 2016; Pérez-Escobar et al., 2017). However, our results seem to corroborate with Gutiérrez Morales et al. (2020); although the sampling of this genus is reduced to one species, the positioning and monophyly still seem questionable, deserving future studies. Besides the well-supported monophyly of Dryadella, the results show three clades moderated supported with two species forming a grade at the base of the phylogeny. Both early divergent species (D. guatemalensis and D. linearifolia) are from northwest South America and Central America. The monophyly of the genus based on molecular data (nrITS and matK) with only two species included had already been proposed by Pridgeon et al. (2001). Karremans et al. (2016) sampled ten species, recognising Phloeophila yupanki in Dryadella, and Pérez- Escobar et al. (2017) include nine species. In the last two studies, D. guatemalensis is an early divergent species of the genus, a position corroborated in our study Three clades are formed, the first clade includes Dryadella yupanki, a Bolivian species, the sister of the remaining species with predominantly South-America northwest distribution, except by D. catharinensis and D. susanae, both endemic to the Brazilian Atlantic Rainforest. This clade brings representatives of the three sections proposed by Luer (2005): D. catharinensis, D. susanae and D. yupanki, ―the tiny mites‖; D. 46 odontostele in ―The needle leaves‖, and the others of the ―All the rest‖ section. We did not find morphological synapomorphies that could characterise this clade. The following two clades include the most species recognised in the ―All the rest‖ section and have a strong geographical component. The clade of D. fuchsii and relatives are predominant from the Pacific dominion and South American Transition Zone (southern Andes), including D. elata (type of the genus). The exception is the sister species of the clade, D. fuchsii, from Central America (northern Andes). This clade includes all the Andean species that have white flowers (D. barrowii, D. albicans, D. pusiola and D. pachyrhiza), cespitose habit, larger than 5 cm tall, with narrow oblong or obovate leaves with long petiole, long peduncles which in most cases raise the flowers to the height of the plant, except for D. fuchsii which is a smaller plant with flowers between the leaves. Although we did not find morphological synapomorphies that could characterise this clade, these characteristics seem to be common for most species of Dryadella from the Andes. The next clade includes D. ana-paulae, the only species from Cerrado, as a sister species of all the Atlantic Rainforest clade, except by D. cristata, endemic of Colombia. These species are morphologically very variable, making it difficult to characterise this clade by morphological synapomorphies. The representatives of this clade are distributed among the three sections according to Luer (2005), although we disagree that D. wuerstlei belongs to the group of ―The needle leaves‖. ORIGIN AND BIOGEOGRAPHY OF DRYADELLA Our analyses estimated the origin of Pleurothallidinae in c. 19 Mya, early Miocene, which agrees with Givnish et al., (2016), Pérez-Escobaret et al. (2017) and Gutiérrez Morales et al. (2020). Like Gutiérrez Morales et al. (2020), our results suggest the Paraná dominion (Atlantic Rainforest) as an ancestral area. This Atlantic Rainforest domain was also recognised as an ancestral area for other orchid groups, such as the Ornithocephalus clade (Oncidiinae, Smidt et al., (2018)) and Spiranthinae (Cranichideae, Salazar et al., (2018)). The results obtained here suggest recent diversification in Dryadella, estimated for the late Miocene to early Pliocene (~7 My), in the Pacific dominion, after the warm period of the Middle Miocene climatic optimum, which coincides with radiation of several plants in the Neotropics (Taylor, 1991; Antonelli et al., 2009; Gustafsson et al., 2010; 47 Pérez-Escobar et al., 2017), at the same time with the intensification of the uplift of the Andes mountains, estimated between 7-11 My (Taylor, 1991; Hoorn et al., 2010) and the beginning of the formation of open vegetation biomes such as the Cerrado that partially separated the forests of South America (Simon et al., 2009). Currently, the Andean Forest/Amazonian and Atlantic Rainforest are separated by the ‗Dry diagonal‘ including the biomes Chaco, Cerrado and Caatinga (Werneck, 2011; Werneck et al., 2012), causing a disjunct pattern of distribution between many groups of Neotropical forested biomes (Costa 2003; Turchetto-Zolet et al., 2012), this disjunction takes place in Dryadella (Pridgeon et al., 2001, Luer, 2005). However, the forests present in these Biomes showed multiple expansions and contractions throughout their history, related to partial floods and drainage in South America from the mid-Miocene to the beginning of the Pliocene (Cabanne et al., 2019; Kessous et al., 2020; Peres et al., 2020). Therefore, previous links between the Atlantic Rainforest, the Amazon and the Andes, which occurred in different periods and across different regions, are reflected in the patterns of Atlantic Rainforest Biota (Kessous et al., 2020, Peres et al., 2020). Dispersion migration in Pleurothallidinae and other orchids between the Andes and Amazonia has been reported in the extensive biogeographic analyses presented by Pérez-Escobar et al. (2017). These authors concluded that the uplift of the Andes did not act as a major barrier to dispersal of orchids from the subtribes Cymbidieae and Pleurothallidinae. In Dryadella, dispersals were recognized as the most frequent biogeographic events in their historical distribution, and the Andes' uplift did not act as a dispersion barrier for the Northern Andes (Central America) and the Andes of South America. Notably, D. guatemalensis and D. linearifolia and D. fuchsii dispersed to Central America, possibly at the beginning of the formation of the Isthmus of Panama, which was only fully established around 3.5 Ma (Hoorn et al., 2010; O'Dea et al., 2016). At the beginning of Pliocene occurred dispersion between the South American Transition Zone and Paraná dominion (Atlantic Rainforest) forming the most recent lineage of the genus, probably related to the drainage of the Paranaean Sea, the contraction of the forests that connected these two biomes and the formation of the Dry Diagonal (Chaco, Cerrado and Caatinga) (Kessous et al., 2020). 48 In this sense, subsequent dispersions from the Amazon region to the west of the continent through the South of the Andes at the beginning of the Pliocene gave rise to exclusively Andean species and, at the same time, through the most central route of the continent to the Atlantic Rainforest, where they settled and due to the isolation by the dry diagonal, became endemic to this Biome. D. ana-paulae is the sister species of the Atlantic clade and the only one currently found in the Cerrado and the seasonally dry forest lowlands of Bolivia, but related to gallery forests, which reinforces the hypothesis of a connection between tropical forests and the Andes, through gallery forests, as an important route for several plant groups (Oliveira-Filho & Ratter, 1995; Costa, 2003). The fact that D. cristata is close to species exclusive to the Paraná dominion (Atlantic Rainforest) may also be evidence of recent links to the south of the continent, between the Andean Cordillera and the Atlantic Forest, but we recognise that we need to investigate further, to understand the relationship between these taxa better. Recent diversification seems to reflect on the taxonomy of the genus. Dryadella has many homoplastic morphological characters, making the delimitation of taxa very difficult, especially in the Atlantic Rainforest endemic taxa (Imig et al. Cap. III). CONCLUSIONS From the most robust sampling of the genus, the results confirm high support for the monophyly of the genus and its positioning in the Specklinia affinity. Dryadella had its origin estimated at the end of the Miocene (~7 Ma) and the Andes emergence had a fundamental contribution to the current distribution of Dryadella species, which allowed the dispersion to the south of the continent, as well as the Isthmus of Panama, allowing the dispersal of species to Central America. The most recent lineage is located in the Atlantic Forest. Dryadella ana-paulae, which occurs in the gallery forests of the Cerrado, also recorded in the lowlands of Bolivia, is sister species of this clade, making evident the connection of gallery forests between the tropical forests of Brazil to the Andes and that the formation of the dry diagonal was important for the establishment of endemisms of the genus. The recent connection between the tropical forests of Brazil and the Andes through gallery forests can be seen through the biographical history of D. ana-paulae. Taxonomically, proposed sections based on plant and leaf sizes are not supported. The difficult taxonomic delimitation due to the presence of many homoplastic characters 49 may be related to their recent origin, and therefore, the clades do not have strong synapomorphies, which is even more evident among the species of the ARF clade, diversified in the Quaternary period. Future work with the genus should focus on including more species and markers and the study of reproductive biology and population genetics, especially in the Atlantic clade, where the species has a recent diversification, being a good model for isolation studies between these species. ACKNOWLEDGEMENTS We would like to thank Sistema de Autorização e Informação em Biodiversidade/Instituto Chico Mendes de Conservação da Biodiversidade (SISBIO/ICMBio) for the collecting permits. We are also grateful for the financial support of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) with scholarship granted to DCI (168348/2017-9) and ECS (CNPq-Nível 1D-proc. 314642/2020-0 and PDJ proc. 203304/2018-7) and to the MCTI/CNPq Nº 14/2013 - Universal (proc. 405493/2021-5). ALVTB thanks to the CAPES (PVE 88881.065009/2014-0). REFERENCES Antonelli A, Nylander JAA, Persson C, Sanmartín I. 2009. Tracing the impact of the Andean uplift on Neotropical plant evolution. Proceedings of the National Academy of Sciences 106: 9749–9754. Baele G, Lemey P, Bedford T, Rambaut A, Suchard MA, Alekseyenko AV. 2012. Improving the accuracy of demographic and molecular clock model comparison while accommodating phylogenetic uncertainty. 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