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UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL
INSTITUTO DE BIOCIÊNCIAS
PROGRAMA DE PÓS-GRADUAÇÃO EM GENÉTICA E BIOLOGIA MOLECULAR
ESTUDO FILOGENÉTICO DAS ESPÉCIES DA SEÇÃO TORVA DO GÊNERO
SOLANUM L. (SOLANACEAE) NA REGIÃO SUL DO BRASIL
Dissertação de Mestrado
Rogéria Beatriz Miz
Porto Alegre, 2006
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Universidade Federal do Rio Grande do Sul
Estudo Filogenético das Espécies da Seção Torva do Gênero Solanum L.
(Solanaceae) na Região Sul do Brasil
Rogéria Beatriz Miz
Dissertação submetida ao Programa
de Pós-Graduação em Genética e
Biologia Molecular da UFGRS como
requisito parcial para obtenção do grau
de mestre.
Orientadora: Dra. Tatiana Teixeira de Souza Chies
Porto Alegre, Março de 2006
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3
Instituições
Este trabalho foi desenvolvido no Laboratório de Genética Molecular Vegetal
do Departamento de Genética, Instituto de Biociências, da Universidade Federal do
Rio Grande do Sul – UFGRS.
Órgãos Financiadores:
CNPq.
4
Dedico esta dissertação a todos que
de alguma forma contribuíram para
realização deste lindo trabalho!
5
Agradecimentos
Gostaria de agradecer ao Programa de Pós-Graduação em Genética e
Biologia Molecular da UFRGS, do qual me orgulho muito em fazer parte. A minha
orientadora Tatiana T. de Souza Chies pela oportunidade de realizar este trabalho e
pelos ensinamentos. Aos colegas do Laboratório de Genética Molecular Vegetal pela
amizade, troca de informações, bate papo, pelo apoio nos momentos críticos, pelas
risadas e vários momentos de descontração, o que tornou o ambiente de trabalho
muito mais agradável e produtivo.
São várias as pessoas as quais preciso agradecer imensamente pela
compreensão, pela atenção, pela mão estendida na hora difícil e pela dedicação:
Ao grupo de pesquisa da professora Lílian Mentz, do PPG Botânica da
UFRGS, pela ajuda nas coletas. Em especial à professora Lílian Mentz, pela
identificação das plantas e, por todo apoio e dedicação com esse trabalho.
Ao Dr. João Renato Stehmann da Universidade Federal de Minas Gerais, a
Dra. Maria de Fátima Agra
da Universidade Federal da Paraíba e a Dra. Gloria
Barboza, IMBIV, Córdoba- Argentina, pelas coletas e colaboração.
A Liliana Essi, pelos vários anos de convivência, amizade, e pela imensa
ajuda com os programas de filogenia.
A Lizandra Robe, André Schnorr e Fernanda Cidade, pela ajuda com as
análises e pela amizade.
Ao Elmo e a Silvia por sempre estarem prontos a ajudar e pelos vários anos
de convivência.
Ao Giancarlo Pasquali, do Centro de Biotecnologia da UFRGS, pelo seu
profissionalismo e por ter cedido espaço para as minhas amostras num momento
crítico do trabalho.
Aos amigos que conquistei nos sete anos de UFRGS, os quais levarei no
coração para o resto da minha vida. Aos meus professores da UFRGS, tanto da
época da graduação como os que também contribuíram para o meu conhecimento
na Pós-graduação.
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Um agradecimento especial a minha família, pelo apoio e compreensão pela
minha ausência em certos momentos. A minha mãe pelo zelo, dedicação, amor e
compreensão. Ao Ivan pelo amor, carinho, compreensão e pela imensa ajuda e apoio
que ele me deu durante todo o meu mestrado, especialmente nestes últimos meses.
Obrigada! Amo vocês!
7
Sumário
RESUMO..................................................................................................................... 8
ABSTRACT............................................................................................................... 10
1.- INTRODUÇÃO..................................................................................................... 12
1.1.-
FAMÍLIA SOLANACEAE E O GÊNERO SOLANUM L.................................................. 12
1.2.-
SEÇÃO TORVA ................................................................................................. 13
1.3.-
MARCADORES MOLECULARES EM FILOGENIA DE PLANTAS................................... 20
1.4-
UTILIZAÇÃO DE MARCADORES MOLECULARES DO TIPO ISSR PARA CARACTERIZAÇÃO
GENÉTICA DE ESPÉCIES VEGETAIS
.............................................................................. 23
1.5-
ANÁLISE DOS DADOS......................................................................................... 25
2.- OBJETIVO GERAL .............................................................................................. 28
3.- ARTIGO 1 ............................................................................................................ 29
4.- ARTIGO 2 ............................................................................................................ 78
5.- DISCUSSÃO...................................................................................................... 120
6.- CONCLUSÕES E PERSPECTIVAS .................................................................. 126
7.- REFERÊNCIAS BIBLIOGRÁFICAS (DISSERTAÇÃO)...................................... 129
8. - ANEXOS........................................................................................................... 141
8
Resumo
O gênero Solanum L. (Solanaceae) compreende mais de 1000 espécies,
incluindo táxons de grande interesse econômico por seu valor alimentício e
medicinal. Este gênero é dividido em três subgêneros: Bassovia, Solanum e
Leptostemonum. O subgênero Leptostemonum é dividido em dez seções, e entre
essas destaca-se a seção Torva que possui representantes no sul do Brasil, e cujas
espécies têm amplo interesse por apresentarem substâncias ativas de grande
utilidade farmacológica. Entretanto, dentro dessa seção existem problemas
taxonômicos, inclusive com a presença de indivíduos de morfologia intermediária,
que dificultam sua classificação e, conseqüentemente, o seu melhor aproveitamento.
Nesse trabalho, foram realizados dois estudos de caráter filogenético a fim de
conhecer as relações de parentesco entre as espécies de Solanum seção Torva,
presentes no sul do Brasil, e destas com espécies de outras seções do subgênero
Leptostemonum. Em ambos os estudos foram utilizados quatro marcadores
(genomas nuclear e plastidial): a região ITS (espaçadores internos transcritos do
DNA nuclear ribossomal) incluindo ITS1, ITS2 e o gene 5,8S; o íntron trnL e os
espaçadores intergênicos trnL-trnF e trnS-trnG do DNA plastidial. O marcador ISSR
(Inter Simple Sequence Repeats) foi utilizado para verificar a variabilidade genética
entre as espécies de Solanum seção Torva e testar o grau de polimorfismo de quatro
“primers” dentro dessa seção. As análises realizadas evidenciaram uma origem
monofilética para a seção Torva. Além disso, foi verificada uma relação de
parentesco mais acentuado dessa seção com S. melongena, S. jamaicense e S.
sisymbriifolium. Dentro da seção Torva foram observados agrupamentos que
relacionam a espécie de morfologia intermediária a seus possíveis progenitores S.
paniculatum e S. guaraniticum. Os quatro agrupamentos mais freqüentes observados
dentro da seção foram: a aproximação de S. guaraniticum, S. bonariense e S.
paniculatum X S. guaraniticum; o relacionamento entre S. adspersum e S.
tabacifolium; a interação entre S. paniculatum e a espécie de morfologia
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intermediária; e a aproximação entre S. paniculatum e S. variabile. Este trabalho
contribuiu para o conhecimento evolutivo das espécies dessa complexa seção que
vem levantando interesse de inúmeros pesquisadores.
Palavras chaves: Solanaceae; gênero Solanum; seção Torva; filogenia; marcadores
nucleares e plastidiais; marcadores ISSR.
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Abstract
The genus Solanum L. (Solanaceae) comprises more than 1,000 species,
including taxa with large economic importance for its nutritive and medicinal values.
This genus is divided into three subgenus: Bassovia, Solanum and Leptostemonum.
The subgenus Leptostemonum of Solanum is divided in ten sections and, among
them, the section Torva, that possess species form the south of Brazil, which showed
large interest by its active substance of large pharmacological utility. However, this
section presents some taxonomic problems, including individuals with intermediate
morphology, that difficult its identification and its utilization. In this work, it were
realized two studies of phylogenetic character, in the attempt to understand the
relationship among species of the section Torva of Solanum, and among the section
Torva with species of other sections of the subgenus Leptostemonum. In these
studies it were used four markers (nuclear and chloroplast genome): the nuclear
ribosomal internal transcribed spacer region (ITS) including ITS1, ITS2 and 5,8S
gene; the trnL intron, and trnL-trnF, trnS-trnG intergenic spacers of the cpDNA. The
ISSR (Inter Simple Sequence Repeats) marker was used to examine the genetic
variability among the species of the section Torva of Solanum, and to test the
polymorphism in the section. The analysis showed the section Torva as
monophyletic, and it was found a close relationship of this section with S. melongena,
S. jamaicense and S. sisymbriifolium. The section Torva showed assembly the
species with intermediate morphology with its supposed parents S. paniculatum and
S. guaraniticum. The four relationships more frequently showed within the section
were: the assembly among S. guaraniticum, S. bonariense and S. paniculatum X S.
guaraniticum; the relationship among S. adspersum and S. tabacifolium; the
relationship among S. paniculatum and the intermediate species; and the relationship
among S. paniculatum and S. variabile. This work was helpful to understand the
evolution of the species belonging to this section that have being of high interest of
many researchers.
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Key words: Solanaceae; genus Solanum; section Torva; phylogeny, nuclear and
plastidial markers; ISSR marker.
12
1.- Introdução
Considerações Gerais
1.1.- Família Solanaceae e o gênero
Solanum
L.
A família Solanaceae possui uma grande importância econômica, agrícola e
farmacêutica. Compreende aproximadamente 93 gêneros e 2300 espécies
(Hunziker, 2001). Segundo Hunziker (2001), a família Solanaceae subdivide-se em
seis subfamílias: Cestroideae, Juanulloideae, Solanoideae, Salpiglossoideae,
Schizanthoideae e Anthocercidoideae. Dentre as seis subfamílias, somente as três
primeiras têm representantes nativos na região sul do Brasil. A distribuição da família
mostra-se cosmopolita, com o principal centro de diversidade taxonômica e
endemismo na América do Sul. Várias espécies desta família movimentam grandes
somas de recursos financeiros em todo o mundo, sendo algumas alimentícias, tais
como: Solanum tuberosum L. (batata-inglesa), Solanum melongena L. (berinjela),
Lycopersicum esculentum L. (tomate), Capsicum anuum L. (pimentão), além de
outras de interesse regional e ornamental como as petúnias. Além disso,
compreende um grande número de espécies com propriedades tóxicas e
farmacológicas, desempenhando, desta forma, uma grande importância na medicina
e na produção de drogas terapêuticas (Roddick, 1991). A indústria farmacêutica
utiliza produtos do metabolismo secundário de várias espécies de Solanum como
fonte de substâncias ativas: alcalóides tropânicos, vitanolídeos e alcalóides
esteroidais. Os alcalóides esteroidais encontrados em Solanum são utilizados na
produção de esteróides farmacêuticos e também se mostram comprometidos com
compostos antitumorais e com agentes que auxiliam contra a esquistossomíase
(Roddick, 1991).
[U1] Comentário: Citar nome
do autor
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O gênero Solanum compreende mais de 1000 espécies em todo o mundo
(D’Arcy, 1991). Este gênero pertence à subfamília Solanoideae, tribo Solaneae,
subtribo Solaninae. Solanum é quinto maior gêneros dentre as Angiospermas
(Magnoliophyta), distribuído em todas as regiões tropicais e subtropicais das
Américas, África e Austrália, com um menor número de espécies euroasiáticas.
Dunal (1852) dividiu o gênero Solanum em duas seções, Pachystemonum e
Leptostemonum, os quais foram elevados a subgênero Solanum e Leptostemonum,
respectivamente, por Bitter (1912, 1913, 1919). Segundo Nee (1999), o gênero está
dividido em três subgêneros: Bassovia, Solanum e Leptostemonum. Segundo Mentz
(comunicação pessoal), na região sul do Brasil o gênero Solanum apresenta 93
espécies nativas distribuídas nos três subgêneros: Bassovia, com 13 espécies,
Solanum, com 49 espécies, e Leptostemonum, com 31 espécies.
O subgênero Lepostemonum, que será tratado neste estudo, é dividido em dez
seções (Nee, 1999): Polytrichum, Melongena, Erythrotrichum, Crinitum,
Herposolanum, Micracantha, Torva, Acanthophora, Persicariae e Lasiocarpa. As
espécies pertencentes a este subgênero caracterizam-se principalmente por serem
espinhentas e possuírem tricomas simples e quase sempre estrelados.
1.2.- Seção
Torva
Uma das seções do subgênero Leptostemonum é a seção Torva, criada por
Nees (1834), com base em S. torvum Sw. (Figura 1- F), espécie originária do México,
América Central e norte da América do Sul. A seção Torva é composta por 40
espécies (Nee, 1999), as quais possuem flores na maioria das vezes perfeitas e
relativamente numerosas em grandes inflorescências, sendo pequenos a grandes
arbustos. Estas espécies estão centralizadas nas Américas, com alguns
representantes na África, Nova Guiné e Pacífico. Para o Brasil são citadas oito
espécies sendo quatro pertencentes à Região Sul (Nee, 1999). Além das espécies,
citadas por Nee, Witasek (1910) também menciona S. adspersum, que ocorre no
Brasil, na região litorânea dos estados de São Paulo e Paraná. Portanto, são citadas
14
ao todo para região sul do Brasil cinco espécies pertencentes à seção Torva:
Solanum paniculatum L., Solanum guaraniticum A. St.-Hil., Solanum adspersum
Witasek, Solanum variabile Mart. e Solanum tabacifolium Dunal (Mentz, 2004).
1.2.1- Solanum paniculatum (Figura1-A)
Popularmente é conhecida como jurubeba, jupeba, juribeba, jurupeba,
gerobeba e joá-manso. A Farmacopéia Brasileira reconhece esta espécie como a
verdadeira “jurubeba” (Farmacopéia Brasileira, 1929). Ocorre no Paraguai e, no
Brasil, principalmente, acompanhando a costa Atlântica do Rio Grande do Norte ao
Rio Grande do Sul, em todas as formações vegetais, às vezes também como ruderal
e cultivada. Na Argentina, foi citada pela primeira vez na flora por Schinini e López
(2000).
Esta espécie apresenta plantas perenes reproduzidas por sementes, com
arbustos de até 2,5 metros de altura (ramificado). Caracterizam-se morfologicamente
por possuírem folhas solitárias, apresentarem tricomas estrelados, sésseis nos
ramos apicais, pela presença de acúleos engrossados e alargados na base, pela
inflorescência cimosa com muitas flores e pelo fruto glabro, globoso, de coloração
amarela. Além disso, esta espécie apresenta longos rizomas subterrâneos, dos quais
emergem caules adventícios, formando clones que podem ser bem amplos. Portanto,
as plantas que ocorrem em um determinado local tendem a ser semelhantes, apesar
do grande polimorfismo que existe nesta espécie (Mentz e Oliveira, 2004).
São plantas utilizadas como medicinais e em rituais afro-brasileiros. Na
farmacopéia popular são utilizadas as folhas, frutos e raízes, no preparo de infusões.
Atribui-se à planta efeitos febrífugas, e estimulante das funções digestivas e do
fígado. No Brasil, Solanum paniculatum é comumente utilizada na medicina folclórica
para o tratamento de doenças do fígado e gastrointestinais (Pio Corrêa, 1984).
Estudos recentes com camundongos apresentam uma associação direta da atividade
antiúlcera dos extratos metabólicos de Solanum paniculatum com a atividade anti-
secretora do suco gástrico, validando o uso folclórico da planta para o tratamento de
15
doenças gástricas (Mesia-Vela e cols., 2002). Do ponto de vista químico, são
encontradas saponinas e alcalóides, cujos compostos apresentam algum efeito
tóxico.
Segundo Mentz e Oliveira (2004), é possível que as populações de Solanum
paniculatum presentes no Rio Grande do Sul tenham tido introdução recente, pois
quase todas as exsicatas diferem das populações coletadas da Ilha de Santa
Catarina para o norte. Além disso, foi verificada a existência de possíveis híbridos
entre Solanum paniculatum e Solanum guaraniticum (Figura 1-C), essa constatação
se deve, a maioria das populações, observadas em Porto Alegre e municípios
vizinhos, terem flores brancas, com anteras praticamente desprovidas de grãos de
pólen e raramente produzindo frutos (Mentz e Oliveira, 2004).
1.2.2- Solanum guaraniticum (Figura 1-B)
Solanum guaraniticum é conhecida popularmente como jurubeba ou falsa-
jurubeba. Saint Hilaire nomeou esta espécie provavelmente relacionando a região
dos povos indígenas do sul do Brasil. Esta espécie tem ocorrência no Paraguai,
Uruguai, Argentina e no Brasil, nos estados de Minas Gerais, São Paulo, Rio de
Janeiro, Paraná, Santa Catarina e Rio Grande do Sul. Na região sul ocorre em todas
as formações vegetais, apresentando também comportamento ruderal.
Esta espécie apresenta-se como arbusto ereto, de até 2 metros de altura, com
floração e frutificação durante todo o ano. Caracteriza-se morfologicamente por
apresentar ramos basais glabros e apicais cobertos de tricomas pedicelados,
estrelados, por possuir acúleos aciculares, raramente alargados na base, folhas
solitárias, inflorescência cimosa (5-20 flores) e pelos frutos glabros, globosos,
amarelos a amarelo-alaranjados quando maduros.
Solanum guaraniticum, sob o nome de Solanum fastigiatum, tem sido citada
como responsável por afetar o sistema nervoso central de bovinos, evidenciada no
campo por crises tipo epileptiforme, com perda de equilíbrio, quedas e tremores
musculares (Riet-Correa e cols., 1983; Paulovich e cols., 2002). Existem alguns
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problemas taxonômicos referentes a esta espécie: o nome Solanum fastigiatum
Willd., citado para o sul do Brasil, corresponde a Solanum bonariense L. (Figura 1-
E), espécie da Argentina e Uruguai, enquanto que a variedade descrita para
Solanum fastigiatum, corresponde a Solanum guaraniticum. O tipo de Solanum
fastigiatum Willd. var. acicularium não difere do tipo de Solanum guaraniticum A. St.
Hil., sendo válido este último. Já Solanum guaraniticum difere morfologicamente de
Solanum bonariense apresentando este último, ramos glabros, angulosos, vinosos,
com acúleos curvos, agudos, glabros, brilhantes, folhas com face adaxial quase
glabra, com tricomas estrelados sésseis e face abaxial com tricomas estrelados
esparsos e inflorescência cimoso-paniculada, sem acúleos (Mentz e Oliveira, 2004).
1.2.3- Solanum adspersum
A espécie Solanum adspersum, recebeu este nome provavelmente devido ao
indumento disperso - do latim “adspersus”, espalhado (Mentz e Oliveira, 2004).
Esta espécie tem ocorrência na região sudeste do Brasil, no estado de São
Paulo, e na região sul, no estado do Paraná, em áreas de formações pioneiras
litorâneas, clareiras e orla da mata, bem como em capoeiras.
Esta espécie é um arbusto ereto de até 2,5 metros de altura e possui um
período de floração entre julho e dezembro e de frutificação entre agosto e
dezembro. Caracteriza-se morfologicamente por possuir ramos lenhosos basais
glabros e os apicais cobertos de tricomas sésseis, pela presença de inflorescência
cimosa com 10-25 flores, sendo as 5-8 basais férteis e frutos globosos de cor
amarela quando maduros (Mentz e Oliveira, 2004).
1.2.4- Solanum variabile (Figura 1-D)
A espécie Solanum variabile é conhecida como falsa “jurubeba” no Brasil.
Segundo Smith e Downs (1966), também pode ser chamada de jurubeba-velame.
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Além deste, também é chamada popularmente como velame, velame-de-capoeira,
juveva e jupicanga.
No Brasil esta espécie tem ocorrência nos estados de Minas Gerais, Rio de
Janeiro, São Paulo, Paraná, Santa Catarina e Rio Grande do Sul (Comissão
Geográfica e Geologia, 1972; Leitão Filho, 1972; Pio Côrrea, 1984; Nee, 1999).
Solanum variabile também é encontrada em alguns países vizinhos como Paraguai e
Uruguai (Smith e Downs, 1966; Sacco, 1985).
Solanum variabile é um arbusto ereto ou arvoreta de até 4 metros de altura,
possue um período de floração entre agosto e maio e de frutificação entre setembro
e junho. Esta espécie é bastante diversificada morfologicamente, apresenta desde
folhas inteiras e mais estreitas, até folhas largas e lobadas, sendo que em algumas
plantas ocorrem muitos acúleos, enquanto em outras esses são muito raros.
Caracteriza-se também pelos seus ramos densamente cobertos de tricomas
estrelado-pedicelados, com folhas solitárias, pela inflorescência cimosa e pelos seus
frutos glabros, globosos, alaranjados quando maduros (Mentz e Oliveira, 2004).
Segundo trabalho recente (Antonio e cols., 2004), o extrato etanólico obtido
das partes aéreas de Solanum variabile tem um significante efeito preventivo e
curativo, em úlceras duodenais.
1.2.5- Solanum tabacifolium
Solanum tabacifolium foi mencionada por Smith e Downs (1966), sob o nome
de Solanum asperolanatum Ruiz e Pavón. Esta última ocorre nos países da costa
ocidental da América do Sul, como Colômbia, Equador, Peru e Venezuela (Mentz e
Oliveira, 2004). O nome Solanum tabacifolium, devido às regras de nomenclatura, é
um nome ilegítimo e precisa ser substituído. Segundo Mentz e Oliveira (2004),
Michael Nee (The New York Botanical Garden) está propondo um novo nome, ainda
inédito para esta espécie. Popularmente, a espécie é conhecida como juveva,
jurubeba ou cardo-branco (Smith e Downs, 1966). No Brasil, é encontrada na Região
Sudeste (Minas Gerais e São Paulo), e na Região Sul (Paraná e Santa Catarina), na
18
região das Florestas Ombrófilas Densa e Mista, na região da Floresta Estacional
Semidecidual, em borda de matas e em terrenos alterados.
Essa espécie é uma arvoreta de até 4 metros de altura, possue um período de
floração e frutificação de junho a dezembro. Caracteriza-se morfologicamente por
possuir ramos cobertos de tricomas estrelados, com acúleos pequenos, levemente
alargados na base, folhas solitárias ou geminadas (desiguais no tamanho),
inflorescência cimosa com flores unilaterais em cada ramo e frutos glabros, globosos,
amarelos quando maduros (Mentz e Oliveira, 2004).
São plantas citadas como útil no tratamento de doenças do fígado (Smith e
Downs, 1966).
19
C)
D)
A
)
B)
E)
F)
Figura 1: Foto de algumas espécies de Solanum seção Torva, incluídas neste
trabalho. A) S. paniculatum (detalhe do cálice); B) S. guaraniticum (detalhe do
cálice); C) S. paniculatum X S. guaraniticum; D) S. variabile; E) S. bonariense; F) S.
torvum. As fotos de A – D foram cedidas por Lílian Mentz.
20
1.3.- Marcadores Moleculares em Filogenia de Plantas
As Filogenias moleculares são baseadas nas ferramentas da Biologia Molecular
e da Cladística para acessar o conhecimento das relações evolutivas entre as
espécies. Esta área do conhecimento tem crescido amplamente desde o início dos
anos 1990, devido ao desenvolvimento dos mais rigorosos métodos de construção
de árvores, combinado com a explosão das informações de seqüências de DNA. A
importância e utilização da filogenia molecular têm aumentado, tanto pelo sucesso
na aplicação de reconstruções de árvores, como pelas novas técnicas filogenéticas
que surgem a cada dia com o advento da Bioinformática, para resolver um dos mais
complexos problemas em biologia.
A classificação taxonômica das espécies vegetais baseava-se, até pouco
tempo, principalmente em características morfológicas; mais recentemente,
caracteres moleculares passaram a constituir uma fonte rica de informação
taxonômica.
Com o desenvolvimento de técnicas e reagentes para seqüênciamento direto, o
uso da informação contida nas seqüências de DNA de certos fragmentos tornou-se
viável para estudos envolvendo maior número de táxons. Diferentes fragmentos de
DNA têm sido utilizados como fonte de caracteres para a análise filogenética, sendo
os espaçadores e/ou íntrons os fragmentos ideais para o estudo infragenérico, pois
acumulam mutações mais rapidamente do que regiões codificantes, graças a
pressões de seleção mais baixas atuando sobre eles (Matioli, 2001). Assim, essas
regiões ficam mais livres para variar, fornecendo informações filogenéticas
suficientes para evidenciar eventos evolutivos relativamente recentes (Small e cols.,
1998).
Um dos fragmentos mais utilizados para inferir filogenia são os espaçadores
internos transcritos – ITS (internal transcribed spacers) do DNA ribossomal nuclear
(nrDNA), que correspondem aos espaçadores ITS1 e ITS2, os quais separam os
genes 18S, 5,8S e 26S (Figura 2). Segundo uma pesquisa realizada por Alvarez e
Wendel (2003), no período entre 1998 e 2003, mais de 65% de todos os estudos
21
filogenéticos de plantas incluíram seqüências de ITS. A popularidade deste marcador
se deve principalmente pela facilidade em gerar dados, pois existem primers
universais para a amplificação da região ITS (Baldwin, 1992). Esta vantagem leva à
geração de muitos dados em pouco tempo. Além disso, existem milhares de cópias
do nrDNA por genoma, facilitando a amplificação das mesmas. A região ITS tem
provado ser suficientemente variável para resolver relações filogenéticas em
diferentes grupos de plantas em níveis taxonômicos mais baixos (Baldwin e cols.,
1995), como os infragenéricos, devido a sua alta taxa de substituição nucleotídica
(Baldwin, 1992; Baldwin e cols., 1995).
Figura 2: Representação esquemática das posições e direções dos “primers”
universais usados para amplificar e seqüenciar as regiões intergênicas ITS 1 e ITS 2
do nrDNA.
Ultimamente, a maioria dos trabalhos envolvendo filogenias moleculares tem
utilizado mais de um fragmento para inferir as relações de parentesco entre os
táxons e, a escolha tem sido feita principalmente com fragmentos contidos nas
regiões do DNA de cloroplasto ou plastidial (cpDNA). Um dos motivos da escolha do
cpDNA é, também, a facilidade em se encontrar regiões caracterizadas e com
primers universais descritos. Um outro motivo que tem levado a combinação de
ambos os genomas (nuclear e plastidial) em análises filogenéticas é o tipo de
22
herança envolvido na origem destes genomas. O DNA plastidial geralmente
apresenta herança uniparental materna, em Angiospermas, enquanto o DNA nuclean0lmeDNTc0.0.8(cle)- Gnndt36925e1nt3e538(e)1.1i.a(a, 38(e)1.q( )]TJ-36.82d0s )6(DN)-5.9( 8pe)1.169d( un)-4.8(ipa 11e,-4.c0.491269d(3d(3 Tc01e,-4.um)-1.4(254866 54.768 TD-0.0019 3eH.1io.768 TD)6(DN)453 Twar453 Twar4.8(r)-25re6ar4.8((oç-4.8(pa.seuiae-.)(f568 TDsã53 Twar45.9( )]TJuap53 T)]TJuap53 á(is(DN)-5..0898 72)6(DN)p53 T)]3q( )]TJ 53 T)]Tm572.24 69( )]TJuap53 T)]TJuadãb2 T72)6(DN8d4.7(s)fm,id4cfm,id4côu .74.7(m)-lao0N)-5.9( 54866e6ar3p.lne( 54866-4.8(nnd)-4.7(l)0.2(a)4.um)-1a)4.ufne8iE.2]TJuadãb2 TcEie6arr4.7(l)i67]TJusnns b2 TcEins b2 TcEi[e7]TJõ]TJmi j99if8((ipa 113(N)0.6oTjhoTjlur90031 Tc269i a493p.fm,idd(3 /TT10 un)-44rdv)-7.4(i)Tj53. 8 8 8 8]TJ7t6523)ahEie6arr4.7(l)u a0.0898 7. 98 7. 3p.fm,iTfm,id39d(3 /TT10jm(a)pp.fm,id996523)a5eh9o 8
23
Figura 3: Genoma do cpDNA de Nicotidiana tabacum. As setas mostram as regiões
do íntron trnL e dos espaçadores intergênicos trnL-trnF e trnS-trnG.
1.4- Utilização de Marcadores Moleculares do tipo ISSR para caracterização
genética de espécies vegetais
Uma das técnicas que vem sendo amplamente utilizada para estudos de
variabilidade genética é o ISSR (Inter Simple Sequence Repeats) que foi proposta
pela primeira vez por Zietkiewicz e cols. (1994). O ISSR é um método baseado na
amplificação de DNA por PCR, que envolve a amplificação de fragmentos de DNA
24
presentes em uma distância amplificável entre dois SSRs ou microssatélites
idênticos repetidos (Figura 4), orientados em direções opostas (Reddy e cols., 2002).
Assim sendo, nessa técnica os SSRs (Seqüências Simples Repetidas), também
chamados de microssatélites, são utilizados como “primers” para amplificar
principalmente regiões entre SSRs (Reddy e cols., 2002). A técnica do ISSR-PCR
permite, portanto, a detecção de polimorfismos em locus localizados entre os
microssatélites, utilizando seqüências simples repetidas (SSRs) como “primers”
(Zietkiewicz e cols., 1994; Wu e cols., 1994). A mudança da taxa evolutiva dentro dos
microssatélites é considerada maior que em outras regiões do DNA.
Conseqüentemente, a probabilidade de polimorfismo dessas seqüências é maior. No
caso do ISSR, a fonte de variabilidade pode ser atribuída à amostra do DNA, à
natureza do “primer” utilizado ou ao método de detecção (Reddy e cols., 2002).
Os produtos resultantes da amplificação do tipo ISSR segregam como
marcadores mendelianos dominantes simples (Gupta e cols., 1994; Tsumura e cols.,
1996; Ratnaparkhe e cols., 1998; Wang e cols., 1998). Entretanto, eles também têm
se mostrado como marcadores codominantes em alguns casos, permitindo assim
distinção entre homozigotos e heterozigotos (Wu e cols., 1994; Akagi e cols., 1996;
Wang e cols., 1998; Sankar e Moore, 2001). O polimorfismo é evidenciado quando
há presença e ausência de um fragmento (bandas) após a amplificação e
eletroforese em gel.
Os ISSR têm sido utilizados em análises genéticas, para verificar a
variabilidade intra e intertaxonômicas, associados a outros marcadores como
RAPD e RFLP (Davierwala e cols., 2000; Panda e cols., 2003). Esses marcadores
vêm sendo utilizados igualmente para reconstruções filogenéticas em níveis de
espécies ou acima (Xu e Sun, 2001; Yockteng e cols., 2003). Além disso, são
igualmente aplicados em estudos de “fingerprinting” (Charters e Wilkinson, 2000),
mapeamento genômico (Becker e Heun, 1995), seleção assistida por marcadores
(Ratnaparkhe e cols., 1998; Hussain e cols., 2000), determinação da freqüência de
motivos SSR (Nagaoka e Ogihara, 1997; Blair e cols., 1999; Varghese e cols.,
25
2000) e estudos de populações naturais e especiação (Wolff e Morgan-Richards,
1998).
Figura 4: Representação esquemática da obtenção do produto de PCR
utilizando a técnica de ISSR (esquema retirado do artigo de Zietkiewicz e cols, 1994).
1.5- Análise dos Dados
1.5.1 Reconstrução Filogenética
Atualmente, estão sendo utilizados quatro métodos principais para inferir as
relações evolutivas entre as espécies na busca de árvores filogenéticas: máxima
parcimônia (MP), método de distância (MD), máxima verossimilhança (MV) e
Inferência Bayesiana (IB), sendo os dois últimos métodos probabilísticos.
A máxima parcimônia consiste na busca da solução mais parcimoniosa, ou
seja, a(s) árvore(s) com menor número de mudanças mutacionais, sendo vista como
26
a hipótese mais econômica. Este tipo de análise tem grande vantagem em relação
aos demais métodos de inferência filogenética por ser muito simples, pois escolhe o
caminho evolutivo com menos pressupostos. Um dos algoritmos heurísticos mais
comumente utilizado para a MP, para casos em que se analisa um grande número
de táxons, é o algoritmo branch swapping - TBR (tree bisection and reconnection).
Nesta opção a árvore é dividida em duas subárvores disjuntas, sendo unidas por um
par de ramos diferentes dos originais. Este procedimento é repetido até que todos os
pares possíveis de ramos dessas duas subárvores sejam unidos e a melhor árvore
seja definida.
Com o método de distância pressupõe-se que todos os caracteres evoluem na
mesma taxa e, os dados são estimados como distâncias pareadas entre as
seqüências nucleotídicas. O algoritmo mais utilizado para este método é o Neighbor-
Joining, NJ (Saitou e Nei, 1987), o qual leva em consideração as taxas evolutivas
distintas e faz parte do grupo de métodos de evolução mínima, no qual a árvore com
a menor soma total de ramos é procurada.
Os métodos probabilísticos MV e IB são estimados segundo um modelo
evolutivo pré-determinado. Ambos os métodos tem o objetivo de encontrar uma
topologia de árvore e correlacionar parâmetros associados, que aumentem a
probabilidade de conter um conjunto de dados de acordo com o modelo proposto. A
máxima verossimilhança tem sido amplamente utilizada para inferir filogenia de
espécies vegetais. Nesse método a probabilidade deve ser calculada para todas as
topologias possíveis, e a árvore combinando topologia mais comprimento de ramos
que apresentar a maior verossimilhança (probabilidade) é considerada a melhor
estimativa da filogenia. A Inferência Bayesiana baseia-se em uma metodologia
estatística antiga e parecida com a MV, mas foi pouco utilizada até recentemente. A
sua principal vantagem em comparação à MV é o tempo de análise, uma vez que
permite estimar os parâmetros do modelo pela mesma técnica.
Para testar a confiabilidade das topologias tanto em árvores de distância, como
de parcimônia e até mesmo de verossimilhança, o teste mais comumente utilizado é
o de Bootstrap (Felsenstein, 1985). A base do método consiste de uma simples
27
reamostragem com reposição pseudoaleatória dos dados. A reamostragem é
repetida muitas vezes (geralmente 1000 replicações) e, no final, uma árvore
consenso é gerada a partir das melhores árvores obtidas. Um outro teste que vem
sendo utilizado para avaliar a confiabilidade das relações filogenéticas é o suporte de
Bremer ou índice de decay (Bremer, 1988), o qual determina o número de passos
extras em que cada agrupamento se mantém a partir de uma árvore inicial pré-
estabelecida.
1.5.2 Métodos de medida de variabilidade
A variabilidade intra e inter taxonômica dos táxons baseados em marcadores
do tipo ISSR vem sendo medida verificando-se a similaridade entre os dados
qualitativos, utilizando principalmente o coeficiente de Jaccard e de Dice. Estas
análises geralmente são realizadas com o pacote NTSYS-pc (Rohlf and Marcus,
1993).
28
2.- Objetivo Geral
O presente trabalho tem como objetivo principal investigar sobre as relações
filogenéticas das espécies da seção Torva do gênero Solanum presentes na região
sul do Brasil, a partir de diferentes marcadores moleculares.
2.1 Objetivos Específicos (Artigo 1)
Evidenciar as relações filogenéticas entre os táxons da seção Torva, e a relação
desta com as seções Acanthophora e Lasiocarpa, e demais espécies
pertencentes ao subgênero Leptostemonum, e verificar a monofilia destes
grupos;
Avaliar a utilidade de quatro marcadores moleculares do tipo sequências de DNA
para o gênero Solanum: ITS (rDNA nuclear); íntron do gene trnL e os
espaçadores trnL-trnF, trnS-trnG do cpDNA;
Verificar quanto à ocorrência de híbridos naturais entre Solanum paniculatum e
Solanum guaraniicum;
Contribuir para o conhecimento taxonômico do gênero Solanum.
2.2 Objetivos específicos (Artigo 2)
Evidenciar as relações filogenéticas e medir a variabilidade inter taxonômica entre
os cinco representantes da seção Torva no sul do Brasil, utilizando caracteres do
tipo seqüências de DNA (regiões de DNA do cloroplasto e ITS) e o marcador
ISSR (Inter Simple Sequence Repeat).
29
3.- Artigo 1
30
Phylogenetic Relationships among the Section Torva and Related Sections from “Spiny
Solanums” (Solanum Subgenus Leptostemonum, Solanaceae)
(A ser submetido para publicação em “Molecular Phylogenetics and Evolution”)
31
Phylogenetic Relationships among the Section Torva and Related Sections from “Spiny
Solanums” (Solanum Subgenus Leptostemonum, Solanaceae)
Rogéria B. Miz
a
, Tatiana T. Souza - Chies
a, b
a
Universidade Federal do Rio Grande do Sul, Programa de Pós Graduação em Genética e
Biologia Molecular, Av. Bento Gonçalves, 9500. Caixa Postal 15053, CEP: 91501-970, Porto
Alegre, RS, Brazil. Tel.:+55 51 3316-9830, Fax: + 55 51 3316-7311
b
Universidade Federal do Rio Grande do Sul, Departamento de Botânica, Av. Bento
Gonçalves, 9500. Caixa Postal 15053, CEP: 91501-970, Porto Alegre, RS, Brazil. Tel.:+55 51
3316-7569
, Fax: + 55 51 3316-7686
Corresponding author: Dra. Tatiana T. Souza-Chies
Address for correspondence.
Departamento de Botânica, UFRGS
Prédio 43433
CEP: 91501-970, Porto Alegre, RS, Brazil
Tel.: 55 51 3316-7569
Fax: 55 51 3316-7686
E-mail: [email protected]
Excluído: 9830
Excluído: 7311
32
Abstract
Solanum section Torva is of considerable interest to phytochemists and biological
researchers because its representatives contain various active chemical substances with
important pharmacological properties although some species provoke toxic effects on cattle.
However, the species belonging to the section Torva and called as “jurubeba” present some
taxonomic problems that make difficult their effective pharmacological use. Because of this,
we proposed a study to investigate and elucidate some evolutionary questions related to this
section and its phylogenetic relationships with other sections of subgenus Leptostemonum. In
this study, 80 samples were investigated based on sequence variability of the nuclear
ribosomal DNA internal transcribed spacers (ITS), as well as the chloroplast intron trnL, trnL-
trnF and trnS-trnG spacers. Five different matrixes were analyzed as basis for phylogenetic
approach. The combined data from the chloroplast analyses formed well supported trees and
provided a high index of consistency. Solanum section Torva was proposed as monophyletic
and it is closed to S. melongena, S. jamaicense and S. sisymbriifolium. Furthermore, we agree
with other authors who proposed that the subgenus Leptostemonum is
monophyletic and that S.
wendlandii must be excluded of the subgenus.
Key Words: Solanaceae; Solanum section Torva; subgenus Leptostemonum; ITS; intron trnL
plus trnL-trnF; trnS-trnG; phylogenetic analysis.
33
Introduction
Solanaceae corresponds to a large family of Angiosperms that contains many taxa
having economical, agricultural, and pharmaceutical importance - the family includes about 92
genera and 2,300 species (Hunziker, 2001). The family is widely distributed with the main
centre of taxonomic diversity and endemism located in South America. The genus Solanum L.
comprises more than 1,000 species (D’Arcy, 1991) and it is distributed worldwide. According
to Dunal (1852) Solanum is divided into two sections: Pachystemonum and Leptostemonum,
which were respectively, elevated into the subgenus Solanum and Leptostemonum by Bitter
(1912, 1913, and 1919). However, Nee (1999) considered that the genus Solanum is divided
into three subgenera: Bassovia, Solanum and Leptostemonum. In southern Brazil, the genus
Solanum comprises about 93 native species, distributed in all three subgenera recognized by
Nee (1999): Bassovia with 13 species, Solanum with 56 species and Leptostemonum with 31
species (Mentz, personal communication). The members of the subgenus Leptostemonum are
also referred as “spiny solanums” because the majority of species have epidermal prickles on
stems and/or leaves and most members of this group have stellate hairs and/or simple
trichomes (Whalen, 1984; Mentz et al., 2000). The species from the subgenus Leptostemonum
are distributed within ten sections (Nee, 1999) including Torva Nees, Acanthophora Dunal
and Lasiocarpa (Dunal) D’Arcy, which are considered in this study.
Solanum section Torva was described based on Solanum torvum Sw., a species from
Mexico, Central America and North of South America. Morphological characters that define
this section include small to large shrubs, mostly perfect and relatively numerous flowers in
large inflorescences, young branches and leaves covered by stellate trichomes, and with stem
34
and leaves armed with spiny at least in early stages. Solanum section Torva comprises 40
species (Nee, 1999) in the world (centered in the Americas, with scattered representatives in
Africa, Asia, New Guinea and Pacific) - eight species are found in Brazil and five of them are
present in Rio Grande do Sul State. Some of the species showed taxonomic problems of
species delimitation with a morphologically intermediate taxon among two species that
complicate its identification. The species from section Torva analysed in the present study are
Solanum paniculatum L., Solanum guaraniticum A. St.-Hil. and Solanum variabile Mart..
The species of section Torva are considered close to species of section Acanthophora
because their similar morphological characters. Therefore, in this work we included species
from Acanthophora and Lasiocarpa sections of Solanum, which, according Nee (1999), are
well supported as sister taxa. The main differences are the predominance of simple hairs on
the stems and upper leaf surfaces and the glabrous mature fruits of the section Acanthophora
in contrast to the stellate hairs and pubescent fruits found in the section Lasiocarpa. Levin et
al. (2005, 2006) and Bohs (2004) agree with Nee (1999) that the Acanthophora and
Lasiocarpa sections have strong support as sister taxa. Notwithstanding the above cited papers
including the section Torva, the species belonging to this section are little studied despite their
taxonomic problems.
Thus, the main goal of the present study were investigate about the phylogenetic
relationships of the taxa within the Solanum section Torva and among it and the Lasiocarpa
plus Acanthophora sections and other closely related members of the subgenus
Leptostemonum.
To accomplish this goal, phylogenetic relationships were inferred from DNA sequence
data from four gene regions. These regions include one from the nuclear genome - the nuclear
35
ribosomal internal transcribed spacer region (ITS) plus the 5,8S gene - and three of chloroplast
data from the trnL intron and trnL-trnF (Taberlet et al., 1991) plus trnS-trnG (Hamilton,
1999) spacer regions. In addition, we discuss about the monophyly of the three sections, and
evaluated the degree to which four different regions were helpful in resolving relationships
among closely related Solanum species.
Material and Methods
Taxon sampling
The overall taxon sampling included in the present study encompasses 80 samples. The
distinct data sets were treated with a different number of taxa (Table 1).
ITS matrix - the ITS matrix was analyzed having 60 samples, the other 20 samples
were not analyzed by different reasons including that the sequence for other fragments was get
from genbank and there is not available sequence to that DNA region, or there were sufficient
number of sequenced samples to represent the taxon. The excluded sequences were: Section
Torva - one sample of intermediate morphology species, four of S. guaraniticum, two of S.
paniculatum and one of S. variabile; section Acanthophora - three of S. atropurpureum, two
of S. aculeatissimum and one of S. vaillantii; section Lasiocarpa - S. felinum and S.
pectinatum; and outgroup - S. mauritianum and S. dulcamara.
trnL-trnF plus intron trnL matrix – this matrix was analyzed having 76 samples, the
other four samples were not analyzed by the same reasons explained above. The excluded
sequences were: Section Torva - one sample of S. variabile; section Acanthophora - S.
agrarium and one sample of S. vaillantii; and outgroup - S. concinum.
36
trnS-trnG matrix – the matrix was analyzed having 58 samples, the other 22 samples
were not analyzed by the same reasons above. The excluded sequences were: Section Torva -
one sample of intermediate morphology species, three of S. guaraniticum, one of S.
paniculatum and one of S. variabile; Section Acanthophora - two samples of S.
atropurpureum, two of S. aculeatissimum; Section Lasiocarpa - only four species were
included: S. candidum, S. pseudolulo, S. quitoense and S. stramonifolium. Other sections of the
subgenus Leptostemonum: one sample of S. sisymbriifolium; and outgroup (no-spiny) - S.
mauritianum, S. dulcamara and S. concinnum.
Chloroplast data set combined - the plastid matrix was analyzed with 56 samples, the
other 24 samples were not analyzed by the same reasons above. The excluded sequences were:
Section Torva - one sample of intermediate morphology species, three of S. guaraniticum, one
of S. paniculatum and one of S. variabile; Section Acanthophora - two samples of S.
atropurpureum, two of S. aculeatissimum, one of S. vaillantii and S. agrarium; Section
Lasiocarpa – the same taxon sampling as above. Other sections of the subgenus
Leptostemonum - one sample of S. sisymbriifolium; and outgroup (no-spiny) - S. mauritianum,
S. dulcamara and S. concinnum.
All data sets combined – the combined matrix of the three DNA sequences was
anayzed with 45 samples. The excluded sequences were: Section Torva - two samples of
intermediate morphology species, six of S. guaraniticum, three of S. paniculatum, two of
S.
variabile; Section Acanthophora - four samples of S. atropurpureum, four of S.
aculeatissimum, one of S. vaillantii and S. agrarium; Section Lasiocarpa - the same 8 samples
excluded of the above matrix. Other sections of the subgenus Leptostemonum - one sample of
S. sisymbriifolium; and outgroup (no-spiny) - S. mauritianum, S. dulcamara and S. concinnum.
37
DNA extraction, amplification, and sequencing
Total genomic DNA was extracted from fresh or silica-gel-dried leaves using the CTAB
method (Doyle and Doyle, 1990).
ITS – Amplification of the internal transcribed spacer (ITS) region of nuclear ribosomal
DNA, composed of ITS 1, the 5.8S gene, and ITS 2 (Baldwin, 1992; Baldwin et al., 1995),
was done using primers 92 (5’- AAG GTT TCC GTA GGT GAA C-3’) and 75 (5’ –TAT
GCT TAA ACT CAG CGG G- 3’) described by Desfeux and Lejeune (1996). Polymerase
chain reaction (PCR) conditions were: 1μl of template DNA (30-100 ng), 2.5 μl of reaction
buffer 10x, 0.75 μl of MgCl
2
(50mM), 0.5 μl of dNTP (10mM), 0.25 μl of Taq DNA
polymerase (5U/μl), 0.5μl of each primer 92 (204 pmol/μl) and 75 (202 pmol/μl), 2.5 μl of
DMSO (96%) and H
2
O to a total volume of 25 μl. In the cycle sequencing reactions, DMSO
was included because GC-rich regions, and the reactions were realized in thermal cycler
Applied Biosystems equipment (Gene Amp PCR System 2400). The DNA amplifications
were performed in a thermal cycler using the “Hot Start” PCR method: 94°C for 5min, 72°C
for 6 min; 35 cycles at 94°C for 45 sec, 58°C for 1 min, 72°C for 1 min and 30 sec; with a
final extension at 72°C for 10 min. The amplified material was purified with enzymatic pre-
treatment (Shrimp alkaline phosphatase and exonuclease I, Amersham Biosciences). The
PCR products were direct sequenced in an ABI PRISM 3100 automated sequencer with the
primers 92 and ITS3 (5’-ATC GAT GAA GAA CGT ACG- 3’) also described by Desfeux
and Lejeune (1996).
38
trnS-trnG - The chloroplast intergenic spacer between trnS and trnG was amplified using
primers trn S (5’- GCC GCT TTA GTC CAC TCA GC- 3’) and trn G ( 5’- GAA CGA ATC
ACA CTT TTA CCA C-3’) of Hamilton (1999). Reactions were obtained with 2.5 μl reaction
buffer 10x, 0.5 μl of dNTPs (10mM), 0.8 μl of MgCl
2
(50mM), 0.5 μl of each primer, 0.2 μl
of Taq DNA polymerase (5U/μl), 1 μl of DNA (30 – 50 ng) and H
2
O to complete 25 μl. The
thermal cycler program included an initial denaturing at 94°C for 5 min; 40 cycles at 94°C for
1 min, 49°C for 1 min, 72°C for 1 min; ending with an extension at 72°C for 10 min. PCR
products were cleaned and sequenced as before, using the same primers as for amplification.
trnL-trnF plus trnL intron – Each region was treated separately in the phylogenetic analyses.
However, the non-coding cpDNA regions trnL-trnF spacer plus trnL intron, are adjacent and
were co-amplified as a single contiguous unit. From a cost/benefit perspective, it is best to
amplify and sequence both of these regions together instead of separately by maximizing the
number of characters obtained per two sequencing reactions (Shaw et al., 2005). Primers used
for amplification were ´´c`` and ´´f`` of Taberlet et al. (1991). Reactions of 25 μl were
obtained with 2.5 μl reaction buffer 10x, with 0.5 μl of dNTPs (10mM), 0.75 μl of MgCl
2
(50mM), 0.5 μl of each primer, 0.25 μl of Taq DNA polymerase (5U/μl), 1 μl of DNA, 1 μl of
DMSO (96%) and H
2
O to complete a volume of 25 μl. The thermal cycler program included
an initial denaturing at 94°C for 3 min; 35 cycle at 94°C for 1 min, 55°C for 1min, 72°C for 2
min; ending with an extension at 72°C for 3 min. These two regions were cleaned and
sequenced as before using with the same primers that were used for PCR.
39
Sequence alignment
Sequences were analyzed and edited by Chromas 1.45 version software (McCarthy
1996-1998). The sequence alignments were made using the Clustal X 1.81 program
(Thompson et al., 1997) and manually refined with the help of GeneDoc (Nicholas and
Nicholas, 1997) and BioEdit softwares (Hall, 1999).
Phylogenetic analyses
The separate (ITS, trnS-trnG, and trnL-trnF plus intron trnL) and combined (overall
plastid data sets or ITS plus plastid regions) phylogenetic analyses were executed using three
main methods: (1) maximum parsimony (MP) using heuristic searches with tree-bisection-
reconnection (TBR) branch swapping, gaps were treated as fifth base, and the Maxtrees option
was used to limit the number of trees, the parsimony analyses were conducted in PAUP
4.0b10 version (Swofford, 2002); (2) Bayesian analysis was performed using MrBayes 3.0b4
by Windows (Huelsenbeck and Ronquist, 2001), to generate a posterior probability
distribution using Markov chain Monte Carlo (MCMC) methods, with the evaluation of at
least 1,000,000 generations and a ‘burn-in’ region of 4,000 trees. This analysis follows the
model proposed by Akaike information criterion (AIC) test (Akaike, 1974), and was executed
using the ModelTest program (Posada and Crandall, 1998); (3) for the distance analysis we
used the Neighbor joining (NJ) (Saitou and Nei, 1987) method, according to the model
proposed by the ModelTest, in PAUP.
The relative proportion of homoplasy data in the MP analyses was estimated though
Consistency and Retention Indexes (CI and RI respectively) available as PAUP tools. The g1
statistics (Hillis, 1991) was calculated from 1,000 random trees generated by PAUP, to
40
measure the phylogenetic information content of the data sets. The general frequencies of the
four nucleotides A, C, G, and T were calculated in PAUP. The resampling Bootstrap test (BS)
(Felsenstein, 1985) was performed with 1,000 full heuristic replicates with tree bisection-
reconnection (TBR) for MP and neighbor joining analyses in PAUP. For the Bayesian analysis
the posterior probability of each clade on the 50% majority rule consensus tree was calculated
(Hall, 2001). Data sets congruence was assessed using partition homogeneity (PHT) tests
(essentially the incongruence length difference (ILD) test of Farris et al. (1994, 1995), as
implemented in PAUP. The partition Bremer support (PBS) scores method (Baker et al., 1998;
Bremer, 1988, 1994) was also used to measure the amount of support provided by trnL-trnF
plus intron trnL, trnS-trnG and ITS regions to each node on the total evidence phylogeny. The
PBS values were calculated using PAUP and TreeRot.v2 Sorenson (1999).
Results
ITS regions (nuclear data sets) – The ITS sequences matrix presented an aligned
length of 727 characters, including ITS1, 5.8 S rRNA gene, and ITS2. Of these 727 characters,
228 were parsimony informative (PI) across 60 samples (Table 2). The general frequencies of
the four nucleotides A, C, G, and T were, respectively, 0.17, 0.36, 0.32, and 0.15, showing
high guanine-cytosine (GC) content (68%). The g1 statistic for ITS trees was of - 0.449,
indicating that for this region the data are significantly skewed and therefore has a substantial
phylogenetic signal (Hillis, 1991). In the maximum parsimony analysis, the heuristic search
for ITS data was limited by Maxtrees in 20,000 most parsimonious trees with 1,079
evolutionary steps with a consistency index (CI) of 0.55 and retention index (RI) of 0.80
41
(Table 2). The 50% majority rule consensus tree presents two large groups (Figure 1). The
first group (supported by BS 93) is composed by species of Solanum section Torva and taxa
belonging to other sections of the subgenus Leptostemonum. In this group the section Torva
(highly supported by BS 100) is divided into two subgroups formed by individuals of S.
guaraniticum and individuals recognized as intermediate morphology species (BS 69), the
second subgroup is formed by S. paniculatum and S. variabile species (BS 82). Solanum
torvum is the most basal species of the section Torva. The second group is divided also into
two subgroups, (i) formed by species of the section Acanthophora (BS 83) and (ii) by the
species of the section Lasiocarpa (BS 100). Torva and Lasiocarpa sections proved to be
monophyletic, while the section Acanthophora is not (due to S. agrarium and S. stenandrum
species) in the ITS analyses. Moreover, this tree showed that the species of the subgenus
Leptostemonum are all closely related (BS 97) except for S. wendlandii, which is found
outside the ingroup. The group formed by S. agrarium and S. stenandrum (species of
Acanthophora), S. stagnale and S. robustum is not supported, but in the work of Levin et al.
(2006) these species make part of the Robustum clade (informal name) and are highly
supported. However, Levin et al. (2006) showed only the trees obtained from total evidence
and probably the plastid DNA contributes to support this clade. The selected model for the
Bayesian analyses using ModelTest was GTR+I+G with gamma shape parameters estimated
of 0.5865. The Bayesian tree topology is very similar to the MP trees using the ITS regions.
The distance analysis showed Solanum section Torva close only to S. melongena and S.
jamaicense, and also confirmed the monophyly of the section Torva (BS 98), data not showed.
42
Chloroplast data
trnS-trnG – The trnS-trnG sequences across 58 taxa showed 87 characters being PI with
aligned length of 868 (Table 2). The general frequencies of the four nucleotides A, C, G, and
T were, respectively, 0.33, 0.17, 0.14, and 0.36 (GC = 31%). The g1 statistic for trnS-trnG
trees was of - 0.162. In the maximum parsimony analysis, the heuristic search was limited to
20,000 most parsimonious trees with 319 steps, with CI of 0.72 and RI of 0.86 (Table 2). The
50% majority rule consensus tree presents four groups (data not shown). The first group shows
species of the section Torva closed to the species: S. sysimbriifolium, S. jamaicense and S.
melongena. Solanum section Torva is divided into two subgroups, one showing S. variabile
close to S. paniculatum and the intermediate species (BS 70), and the other showing S.
guaraniticum related to the intermediate species (BS 98), being this latter group very well
supported. The second group is divided into two subgroups where the first one is composed by
the section Acanthophora, and the other by the section Lasiocarpa (BS 98). The third group
strongly supported (BS 100) is composed by S. agrarium and S. stenandrum both of them
from the section Acanthophora. The fourth group is formed by S. mammosum (Solanum
section Acanthophora), S. robustum and S. stagnale. The group formed by the subgenus
Leptostemonum presents high support (BS 91) and showed S. wendlandii in a basal position.
Torva and Lasiocarpa sections proved to be monophyletic while the section Acanthophora is
not monophyletic, due to S. agrarium, S. stenandrum and S. mammosum (outside the group
formed by this section). The evaluative model selected for the Bayesian analyses was
K81uf+G, with gamma shape parameters estimated of 0.3262. The trees obtained from the
Bayesian and distance analyses (data not showed) are divided into two large groups: one
43
including S. melongena, S. jamaicense, and S. sisymbriifolium adjacent to the section Torva
(94% of support in Bayesian analysis), and the other is formed by one subgroup composed by
the section Acanthophora species (except for S. agrarium and S. stenandrum which formed
another subgroup). In these analyses S. mammosum is included in the group formed by the
species of the section Acanthophora while S. robustum and S. stagnale form another subgroup
with the section Lasiocarpa.
trnL-trnF plus intron trnL- The trnL-trnF intergenic spacer combined with the trnL gene
intron presented a total alignment length of 1,270 bp, being 216 (PI) across 76 taxa (Table 2).
The general frequencies of the four nucleotides A, C, G, and T were, respectively, 0.36, 0.19,
0.16, 0.29 (GC = 35%). The g1 statistic for the trees was of -0.218. In the maximum
parsimony analyses the heuristic search was limited to 20,000 (Maxtrees) most parsimonious
trees with 640 evolutionary steps with CI of 0.74 and RI of 0.89 (Table 2). The 50% majority
rule consensus tree presents two large groups. A group is formed by species of Solanum
section Torva (BS 85) and S. melongena. The section Torva was divided into two subgroups
with S. torvum being the most basal species. The first subgroup showed individuals of S.
guaraniticum and S. paniculatum related to the intermediate species; and the second subgroup
showed S. variabile (BS 91) and S. guaraniticum adjacent to the intermediate species (BS 99).
The second group formed in the consensus tree is divided into two subgroups. One of them is
formed by the section Lasiocarpa (BS 53) and the other by the section Acanthophora (BS
100) with S. stenandrum (a species of the section Acanthophora) being the most basal species
of these two subgroups. Moreover, it is possible to visualize in the majority rule consensus
tree that S. wendlandii is outside of the ingroup formed by species of the subgenus
44
Leptostemonum (BS 97). The evolutive model selected to perform the Bayesian analyses was
TVM+I+G, with gamma shape parameters estimated of 0.8590. The Bayesian tree topology
proved to be similar to the trees originated by the parsimony analyzes except for the position
of S. jamaicense that is a sister group of the section Torva with S. melongena. The distance
analyses showed less resolution in the tree than MP and Bayesian analysis for the
45
intermediate morphology species (BS 85). Solanum torvum maintained its position as the most
basal species of the section Torva. The second group is composed by two subgroups, one
formed by the section
46
robust trees. Total chloroplast data plus ITS were combined and included 2,864 characters for
45 taxa (Table 1). Of these characters, 414 were parsimony informative (PI). The general
frequencies of the four nucleotides A, C, G, and T were, respectively, 0.30, 0.23, 0.19, and
0.28 (GC = 42%). The g1 statistic for the trees was of -0.316. In the maximum-parsimony
analyses, the heuristic search resulted in 18 most-parsimonious trees of 1,737 evolutionary
steps with index of consistency (CI) of 0.64 and retention index (RI) of 0.79 (Table 2). The
50% majority rule consensus tree presents two large groups, being 39 clades indicated (Figure
3) with their respectively support (Table 3 (PBS support) and Table 4). The first group is
represented by Solanum section Torva (Clade 13 - Figure 3, Table 3 and 4) plus S. jamaicense,
S. melongena and S. sisymbriifolium as sister species of the section Torva (Clade 16 - Figure 3
Table 3 and 4). The section Torva was divided into two subgroups each supported by BS 81.
One subgroup is composed by the morphology intermediate species and S. paniculatum that is
more adjacent to S. variabile (Clade 6 - Figure 3, Table 3 and 4). The other subgroup is
composed by the morphology intermediate species and S. guaraniticum (Clade 11, Figure 3,
Table 3 and 4). S. torvum showed a basal position within the section Torva. Solanum
wendlandii is outside of the group formed by the subgenus Leptostemonum (Clade 39 - Figure
3, Table 3 and 4). The evolutive model selected for the Bayesian analyses was GTR+I+G,
with gamma shape parameters estimated of 0.6763. Bayesian and distance analysis showed a
similar tree topology to that of the parsimony analysis. These analyses differed from that of
the parsimony analysis by the position of S. robustum, S. stagnale and S. stenandrum, which
are adjacent to the section Torva (BS 99), and of S. sisymbriifolium that is found not related
from the section Torva. The subgenus Leptostemonum presents also strong support in
Bayesian and distance analyses (Clade 39 - Figure 3, Table 3).
47
To assess the relative contribution of each fragment in our combined analysis the PBS
scores for each fragment on each node were calculated. This test confirmed that the
chloroplast region contributes more to the results than the ITS regions (Table 4) once it
provided almost three times more information than ITS and contributed much more to the
formation of clades in the combined analysis (Figure 3 and Table 4). Similarly, the trnS-trnG
intergenic spacer proved be more informative than the combination of the intron trnL plus the
trnL-trnF spacer. Furthermore, the breakdown of the PBS values also indicated substantial
conflict between the chloroplast and ITS data partitions, because 15 of the 39 nodes resolved
in the combined analysis had conflicting PBS values. This pattern may have been established
because of the high homoplastic index in the ITS regions.
Discussion
The section Torva - The analyses realized in this study suggest that Solanum section Torva is
monophyletic and it is in agreement with Levin et al. (2006). The sections Lasiocarpa and
Acanthophora are respectively monophyletic and paraphyletic as it were proposed by Bohs
(2004) and Levin et al. (2005). We had the same results when species from section Torva and
other from section Acanthophora (specifically those from Southern Brazil) were included.
Section Acanthophora is not monophyletic due to the inclusion of S. stenandrum (in all
analyzed matrixes) plus S. agrarium (based on the analyses using ITS and trnS-trnG data).
This agrees with the work by Levin et al. (2005) although the section Acanthophora showed
strongly related with the section Torva by its morphological characters (Mentz, personal
48
communication). Section Acanthophora proved be strongly related to the section Lasiocarpa
in this work, in agreement with the data provided by Bohs (2004) and Levin et al. (2005)
which indicated that they are sister taxa.
Solanum section Torva is divided into two internal groups in all analyses, with the
basal species being S. torvum. The groups of the section Torva clearly form a strongly
relationship among the intermediate morphology species and the possible progenitors (S.
paniculatum and S. guaraniticum). This relation is not evident in the analyses performed from
the ITS data, where no particular relationship between S. paniculatum and the intermediate
morphology species was found. This finding, however, cannot be taken as conclusive because
of the high homoplastic index (HI) shown for ITS marker and by concerted evolution,
responsible by homogenization of DNA sequence en tandem of ribosomal DNA. In the work
realized by Chase et al. (2003), concerning the origin of hybrids in Nicotiana (Solanaceae), the
authors commented that ITS is not a generally reliable tool for detection of hybrids, once that
the homogenization may be directional accordant with a paternal ITS allele (e.g., N. tabacum)
or with a maternal ITS allele (e.g., N. rustica). However, in the present work it may be
suggested that the homogenization of ITS was in direction of the paternal allele, and it would
be indicate S. guaraniticum as the paternal progenitor because all representatives of
intermediate morphology species are close to S. guaraniticum from ITS analysis and related to
S. paniculatum based on plastidial analysis (that is supposed inherited by maternal origin),
Figures 1 and 2.
The section Torva of Solanum proved be very close to the species: S. melongena, S.
jamaicense and S. sisymbriifolium (species from other sections of the subgenus
Leptostemonum), in most of the performed analyses suggesting that these species can be sister
49
of section Torva. This relationship also is showed in the work of Levin et al. (2005) where the
species S. jamaicense, S. torvum, S. melongena and S. sisymbriifolium formed a group
supported by 100 % of bootstrap.
The monophyly of Solanum section Torva showed in this study may not be applicable
to the section Torva worldwide because our sample of non-Brazilian plants was small but is
certainly applicable and correct for the plants which grow in the southern region of Brazil. The
samples of section Torva used here represent 60% of the Southern Brazil species, 50% of the
brazilian species, but only 13% of the Southern American species and 10% of total species of
section Torva in the world (according to Nee, 1999).
Subgenus Leptostemonum – The subgenus Leptostemonum was found non-monophyletic in
all performed analyses in this study. Solanum wendlandii is placed outside the group formed
by the spiny species and adjacent to the non-spiny species of the outgroup. In the work
realized by Bohs (2004), all spiny taxa of Solanum with exception of S. wendlandii formed a
monophyletic group supported by 100% of bootstrap in all analyses. The author further
reported that S. wendlandii was also excluded from the Leptostemonum clade in previous
molecular analyses realized by Bohs and Olmstead (1997, 1999, 2001) and Levin et al. (2005).
In the present work we also observed that S. wendlandii is very well supported outside the
group formed by the species of the subgenus Leptostemonum, where it has been traditionally
included. Solanum wendlandii display simple and glandular trichomes, different from the
Leptostemonum species which show simple and stellate trichomes. The theory that this species
should be included in Leptostemonum is based only on the presence of the spines (Whalen,
1984). Child (1983, 1990) emphasizing the lack of stellate hairs, transferred these plants from
the Leptostemonum to a new position adjacent to the subgenus Potato (G.Don) D’Arcy. Nee
50
(1999), included S. wendlandii within the subgenus Leptostemonum, but also discussed its
proximity with the Dulcamara and Petota sections (based on the morphology of flowers,
leaves and inflorescence). The phylogenetic and morphological evidences discussed here
permit us to propose the exclusion of S. wendlandii from the subgenus Leptostemonum which,
consequently, becomes a monophyletic subgenus. It is also evident in the recent work of Levin
et al. (2006) where it was proposed the exclusion of S. wendlandii group (including S.
wendlandii and S. refractum Hook. & Arn. species) and S. nemorense Dunal group (S.
hoehnei C.V. Morton, S. reptans Bunbury and S. nemorense) of Solanum subgenus
Leptostemonum. The authors commented that these groups are formed by species with prickles
but lack stellate hairs.
Comparison among markers
A partition homogeneity test (PHT) realized from a maximum parsimonious tree
obtained from four fragments, showed significant incongruence between the chloroplast and
ITS fragments. This incongruence can be observed in the tree topology based on the plastid
DNA only, and in the tree derived from the combined chloroplast and ITS data which differ in
few clades. The plastid DNA presents a greater degree of consistency when compared to the
analyses based on the ITS region (Table 4) and showed almost three times the partition
Bremer support (PBS) scores as compared to the ITS region (Table 4). This discrepancy may
be caused by the different mode of inheritance of the markers (chloroplast = maternal
inheritance, nuclear = biparental); by the high rates of homoplasy found in ITS, (due mainly to
the rapid evolution of the region as compared to the other regions of the plastid genome); and
51
also to the greater number of characters obtained with the cpDNA data (2,137 bp) compared to
the data from the ITS region (727 bp) used for the analysis of the four combined fragments.
The nuclear region (ITS) presented a high degree of phylogenetically informative
characters (31.4%) in comparison with other markers. This evidence is also clear in the Hillis
test, in which the ITS data matrix shows an asymmetrical distribution of the tree lengths to the
right (phylogenetic tree with the lowest number of steps) with a smaller interval to the left (g1
= -0.44) than the other matrixes. However, the consistency index was low for ITS in
agreement with the work by Levin et al. (2005, 2006). The analysis based only on ITS with the
greater taxon sampling from Solanum section Lasiocarpa plus species from section Torva
evidenced that section Lasiocarpa is monophyletic as in agreement with the data obtained by
Bohs (2004). In the latter analysis we observed an approximation of the so-called intermediate
species with S. guaraniticum - this difference may be due to the biparental inheritance, or
perhaps to the high homoplastic index of the fragment, which may be concealing more precise
information about the degree of relationship. The analyses realized with the ITS matrix
allowed the separation among the sections Torva, Acanthophora and Lasiocarpa, and
subgenus Leptostemonum, but showed variation in some taxa of section Acanthophora.
Besides this variation it showed not invalidate its use as phylogenetic marker, since all the
individuals from the same species but one (S. aculeatissimum) form a clade with high support.
Therefore, the variation of ITS do not pass the species barrier and showed that it is an efficient
phylogenetic marker. In addition, the problem concerning the monophyly of S. aculeatissimum
was also showed when the plastid regions were used as characters.
The partition homogeneity test (PHT) realized with the cpDNA matrix showed high
congruency among the chloroplast fragments. This was also found in the PBS scores where
52
conflicts were observed in only nine of the thirty-nine clades obtained with the consensus tree
representing the four combined fragments when we considered only the chloroplast data
(Table 4).
The phylogenetic tree obtained from cpDNA presented a greater number of
informative characters in comparison with the individual analyses of the intron trnL + the
trnL-trnF spacer fragments and the trnS-trnG spacer (Table 2). Amongst the individual
cpDNA analyses the fragment which showed the greatest number of information corresponds
to the intron trnL + the trnL-trnF spacer. These results contradict the work by Shaw et al.
(2005), where they found that the trnS-trnG spacer has a higher PI index than the co-amplified
region of the intron trnL + spacer trnL-trnF.
However, using Solanum species and verifying each fragment individually, we found
greater variability in the intron trnL followed by the trnL-trnF spacer than that of the spacer
trnS-trnG.
Many works were realized using cpDNA to infer phylogenies of Solanaceae, including
the genus Solanum in different taxonomic levels (Olmstead and Palmer, 1991; Bohs and
Olmstead, 1999; Olmstead et al., 1999; Bohs, 2004; Clarkson et al., 2004; Levin et al. 2005
and 2006). Generally, the plastid genome typifies conservative evolution and consequently
displays a smaller number of phylogenetically informative characters when compared to
nuclear genome. In the present work, however, the chloroplast regions exhibit a relatively high
degree of divergence, which helps to elucidate the relationships among the taxa of the genus
Solanum. The chloroplast analyses permit the formation of highly supported clades which
showed the relationship among individuals of the same species and taxa of the same section,
and furthermore, draw a distinction among the species armed with stellate hairs and the other
53
species not belonging to the subgenus Leptostemonum. Among the analyses realized, only in
those of the plastid fragments did we find a height consistency index for the isolated analysis
of fragments corresponding to the intron trnL + the spacer trnL-trnF combined, perhaps
because a greater number of these taxa were included in the analysis (76) in comparison to the
other fragments. However, the tree showing a greater resolution is that which combines all the
chloroplast fragments in only one matrix, showing that in addition of the taxon sampling, the
number of characters contributes to a better phylogenetic inference.
Conclusion
In this work, the monophyly of the sections Torva and Lasiocarpa, and the paraphyly
of the section Acanthophora are showed and the latter is shown to be a sister section of the
section Lasiocarpa. Evidence for the considerable consistency of the interaction of the
intermediate species with both of its so-called progenitor species is showed, reinforcing the
suggestion that an intermediate species to both S. guaraniticum and S. paniculatum exist. The
cpDNA fragments were found to be more efficient in clarifying the phylogenetic relationships
between the taxa of the Solanum genus than those of ITS because the latter presented an
elevated percentage of homoplastic characters. The removal of S. wendlandii from its present
position in the Leptostemonum subgenus should be proposed, as this would result in the
subgenus being accepted as monophyletic.
The phylogenetic relationship of Solanum section Torva clarified in the present study
may assist in the search for alternative species from which important secondary metabolites
54
may be obtained for pharmacological production, as well as contributing to sustainable
utilization, and better exploitation of the species.
Acknowledgments
The authors thank to the Dr Lilian Mentz (Programa de Pós-Graduação em Botânica -
UFRGS) for specimens suply and for their taxonomic identification, and suggestions; Dr.
Maria de Fátima Agra of Universidade Federal da Paraíba, Liliana Essi (M. Sc.) of
Universidade Federal do Rio Grande do Sul for the samples supplying; Dr. Giancarlo Pasquali
(Universidade Federal do Rio Grande do Sul) for sequencing facilities. We also thank the
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), for the
grants supplied.
Formatado
55
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61
Figure1: Majority consensus (50%) tree of the 20,000 (Maxtrees) most parsimonious trees
from ITS data. Numbers above branches correspond to bootstrap support values (when higher
than 50%) based on 1,000 replicates.
62
Figure 2: Majority consensus (50%) tree of the 20,000 (Maxtrees) most parsimonious trees
from the plastid data. Numbers above branches correspond to bootstrap support values (when
higher than 50%) based on 1,000 replicates.
Subgenus
Leptostemonum
Section
Torva
Section
Acanthophora
Section
Lasiocarpa
Section Acanthophora
63
Figura 3: Majority consensus (50%) of the 20,000 (Maxtrees) most parsimonious trees from
four fragments combined data. In face of the internal branches there is an arbitrarily defined
number representing the clade that follows and which can be used to interpret Tables 3 and 4.
Section
Torva
Section
Acanthophora
Section
Lasiocarpa
Subgenus
Leptostemonum
Section
A
cantho
p
hora
64
Table 1:
Table 1: List of Solanum species from section Torva (A), Acanthophora (B), Lasiocarpa (C) and other species of the subgenus
Leptostemonum (D) plus species used as outgroup (no spiny species) (E), used in this study; with their respective voucher, location and
GenBank accession numbers for regions ITS, intron trnL plus trnL-trnF spacer, trnS-trnG spacer. The asterisks correspond to the
fragments used for each taxon and N to the missing data. Where there is no data about sampling places the sequences were get from
GenBank.
A)
Taxa Voucher ITS
(GenBank
accession
numbers)
trnL-trnF plus
intron trnL
(GenBank
accession
numbers)
trnS-trnG
(GenBank
accession
numbers)
Sampling Places
Solanum section Torva
1 Solanum paniculatum X Solanum guaraniticum L. A. Mentz, T.T Souza-
Chies and R.B. Miz, 301.
* * * RS, Porto Alegre
2 Solanum paniculatum X Solanum guaraniticum L. A. Mentz, T. T. Souza-
Chies and R. B. Miz, 302.
* * * RS, Porto Alegre
3 Solanum paniculatum X Solanum guaraniticum L.A. Mentz, E.L.C.
Soares, M. Vignoli-Silva,
G.S. Vendruscolo, R.B.
Miz, 309 - ICN
* * * RS, Passo Fundo, Farm
Sementes and Cabanhas
Butiá
4 Solanum paniculatum X Solanum guaraniticum L.A. Mentz, E.L.C.
Soares, M. Vignoli-Silva,
N
* * RS, Viamão, State Park
of Itapuã
65
G.S. Vendruscolo, 305-
ICN
5 Solanum paniculatum X Solanum guaraniticum L.A. Mentz, E.L.C.
Soares, M. Vignoli-Silva,
G.S .Vendruscolo, R.B.
Miz, 311 - ICN
* *
N
RS, Carazinho
6 Solanum paniculatum X Solanum guaraniticum L.A. Mentz, E.L.C.
Soares , R.B. Miz, 344 –
ICN.
* * * RS, Venâncio Aires
7 Solanum guaraniticum A. St.-Hil. T. T. Souza-Chies, 221. * * * RS, Antônio Prado,
Amarilho Road,
8 Solanum guaraniticum A. St.-Hil. T.T. Souza-Chies, 222 N *
N
RS, Monte Bonito,
district of Pelotas
9 Solanum guaraniticum L.A. Mentz, E.L.C.
Soares , R.B. Miz, 349.
* * * RS, Santa Cruz
10 Solanum guaraniticum A. St.-Hil. (spiny leave) L.A. Mentz, E.L.C.
Soares, M. Vignoli-Silva,
G.S. Vendruscolo, R. B.
Miz, 336- ICN.
N * * RS, Fontoura Xavier, BR
386, km 276
11 Solanum guaraniticum A. St.-Hil. (spiny low
stem)
L.A. Mentz, E.L.C.
Soares , R.B. Miz, 345a.
N * * RS, Santa Cruz
12 Solanum guaraniticum A. St.-Hil. L.A. Mentz, E.L.C.
Soares , R.B. Miz, 345b.
* *
N
RS, Santa Cruz
13 Solanum guaraniticum A. St.-Hil. L.A. Mentz, E.L.C.
Soares , R.B. Miz, 345c.
N * * RS, Santa Cruz
14 Solanum guaraniticum A. St.-Hil. L.A. Mentz, E.L.C.
Soares , R.B. Miz, 339.
* *
N
RS, Coronel Barros -
Stop Lajeado do Tigre –
BR285 Km 476
15 Solanum guaraniticum A. St.-Hil. L.A. Mentz, E.L.C.
Soares, M. Vignoli-Silva,
G.S. Vendruscolo, R. B.
Miz, 333.
* * * RS, Santo Ângelo
16 Solanum guaraniticum A. St.-Hil. L.A. Mentz, E.L.C.
Soares , R.B. Miz, 348.
* * * RS, Santa Cruz
17 Solanum paniculatum L. R.B. Miz, 101. N * * RS, Porto Alegre
18 Solanum paniculatum L. L.A. Mentz, E.L.C.
Soares, M. Vignoli-Silva,
N
* * RS, Viamão, State Park
of Itapuã
66
G.S. Vendruscolo, 304-
ICN.
19 Solanum paniculatum L. M. F. Agra, K. Nurit and
I. Basílio, 6311.
* *
N
Paraíba, Municipal
district João Pessoa,
Campus of the Federal
University of the
Paraíba
20 Solanum paniculatum L. L.A. Mentz, E.L.C.
Soares, R. B. Miz, 341-
ICN.
* * * RS, Triunfo
21 Solanum paniculatum L. L.A. Mentz, E.L.C.
Soares, R.B. Miz, 340-
ICN.
* * * RS, Triunfo
22 Solanum variabile Mart. L. Essi
Li 301
* * * RS/SC, Cambará do Sul.
“Serra do Faxinal”
23 Solanum variabile Mart. L. Essi
Li302
* * * SC, Lauro Muller
24 Solanum variabile Mart. L. Essi
Li304
* * * SC, Bom Jardim da
Serra. Mountain of “Rio
do Rastro”
25 Solanum variabile Mart. L. Essi
Li319
N * * SC, Urubici
26 Solanum variabile Mart. L. Essi
Li320
* N
N
SC, Urubici
27 Solanum torvum Sw. _ *
(AF244729)
*
(AY266246)
*
(AY555478)
GenBank
67
B)
Taxa Voucher ITS
(GenBank
accession
numbers)
trnL-trnF plus
intron trnL
(GenBank
accession
numbers)
trnS-trnG
(GenBank
accession
numbers)
Sampling Places
Solanum section Acanthophora
1 Solanum atropurpureum Schrank L.A. Mentz, E.L.C. Soares,
R. B. Miz, 343-ICN.
* * * RS, Santa Cruz
Solanum atropurpureum Schrank .A. Mentz, E.L.C. Soares,
R. B. Miz, 342- ICN.
N
* * RS, Santa Cruz
3 Solanum atropurpureum Schrank L.A. Mentz, E.L.C. Soares,
M. Vignoli-Silva, G.S.
Vendruscolo, R.B. Miz,
325a. ICN.
* * * RS, Passo Fundo, Farm
Sementes and Cabanhas
Butiá
4 Solanum atropurpureum Schrank L.A. Mentz, E.L.C. Soares,
M. Vignoli-Silva, G.S.
Vendruscolo, R.B. Miz.
325b- ICN.
N
*
N
RS, Passo Fundo,
FarmSementes and
Cabanhas Butiá
5 Solanum atropurpureum Schrank L.A. Mentz, E.L.C. Soares,
M. Vignoli-Silva, G.S.
Vendruscolo, R.B. Miz,
68
Vendruscolo, R.B. Miz,
334- ICN.
Butiá
9 Solanum aculeatissimum Jacq. L.A. Mentz, E.L.C. Soares,
M. Vignoli-Silva, G.S.
Vendruscolo, R.B. Miz,
313- ICN.
* * * RS, Passo Fundo, Farm
Sementes and Cabanhas
Butiá
10 Solanum aculeatissimum Jacq. L.A. Mentz, E.L.C. Soares,
M. Vignoli-Silva, G.S.
Vendruscolo, R.B. Miz,
326a- ICN
* * * RS, Passo Fundo, Farm
Sementes and Cabanhas
Butiá
11 Solanum aculeatissimum Jacq. L.A. Mentz, E.L.C. Soares,
M. Vignoli-Silva, G.S.
Vendruscolo, R.B. Miz,
326b- ICN
* *
N
RS, Passo Fundo, Farm
Sementes and Cabanhas
Butiá
12 Solanum viarum Dunal L.A. Mentz, E.L.C. Soares,
R.B. Miz, 346- ICN.
* * * RS, Santa Cruz
13 Solanum vaillantii Dunal L.A. Mentz, E.L.C. Soares,
M. Vignoli-Silva, G.S.
Vendruscolo, R.B. Miz,
310- ICN.
* * * RS, Passo Fundo, Farm
Sementes and Cabanhas
Butiá
14 Solanum acerifolium Dunal _ *
(AY561261)
*
(AY266249)
*
(AY555454)
GenBank
15 Solanum aculeatissimum Jacq. _ *
(AY561262)
*
(AY559236)
*
(AY555455)
GenBank
16 Solanum agrarium Sedtn. _ *
(AY561263)
N
*
(AY555456)
GenBank
17 Solanum atropurpureum Schrank _ *
(AY561264)
*
(AY559237)
*
(AY555457)
GenBank
18 Solanum capsicoides All. _ *
(AY561265)
*
(AY266251)
*
(AY555460)
GenBank
19 Solanum incarceratum Ruiz & Pavon _ *
(AY561266)
*
(AY559239)
*
(AY555461)
GenBank
20 Solanum mammosum L. _ *
(AF244721)
*
(AY559240)
*
(AY555464)
GenBank
21 Solanum myriacanthum Dunal _ *
(AY561267)
*
(AY559240)
*
(AY555466)
GenBank
69
22 Solanum palinacanthum Dunal _ *
(AY561268)
*
(AY266233)
*
(AY555467)
GenBank
23 Solanum platense Diekman _ *
(AY561269)
*
(AY559241)
*
(AY555468)
GenBank
24 Solanum stenandrum Sendtn. _ *
(AY561273)
*
(AY559242)
*
(AY555475)
GenBank
25 Solanum tenuispinum Rusby _ *
(AY561274)
*
(AY266245)
*
(AY555477)
GenBank
26 Solanum vaillantii Dunal _
N N
*
(AY555479)
GenBank
27 Solanum viarum Dunal _ *
(AY561275)
*
(AY559243)
*
(AY555480)
GenBank
70
C)
Taxa Voucher ITS
(GenBank
accession
numbers)
trnL-trnF plus
intron trnL
(GenBank
accession
numbers)
trnS-trnG
(GenBank
accession numbers)
Sampling Places
Solanum section Lasiocarpa
1 Solanum candidum Lindl. _ *
(AF244722)
*
(AY266250)
*
(AY555459)
GenBank
2 Solanum felinum Whalen _
N
*
(AY266252)
N
GenBank
3 Solanum hirtum Vahl _ *
(AY263458)
*
(AY266254)
N
GenBank
4 Solanum hyporhodium A. Braun & Bouché _ *
(AY263461)
*
(AY266238)
N
GenBank
5 Solanum lasiocarpum Dunal _ *
(AY263457)
*
(AY266256)
N
GenBank
6 Solanum pectinatum Dunal _
N
*
(AY266230)
N
GenBank
7 Solanum pseudolulo Heiser _ *
(AY263459)
*
(AY266242)
*
(AY555470)
GenBank
8 Solanum quitoense Lam. _ *
(AY263460)
*
(AY266228)
*
(AY555471)
GenBank
9 Solanum repandum G. Forst. _ *
(AY263466)
*
(AY266234)
N
GenBank
10 Solanum sessiliflorum Dunal _ *
(AY263455)
*
(AY266260)
N
GenBank
11 Solanum stramoniifolium Jacq. _ *
(AY263465)
*
(AY266244)
*
(AY555476)
GenBank
12 Solanum vestissimum Dunal _ *
(AY263467)
*
(AY266264)
N
GenBank
71
D)
Taxa Voucher ITS
(GenBank
accession
numbers)
trnL-trnF plus
intron trnL
(GenBank
accession numbers)
trnS-trnG
(GenBank
accession
numbers)
Sampling Places
Outgroups in subgenus Leptostemonum
1 Solanum sisymbriifolium Lam. L.A. Mentz, E.L.C.
Soares, R.B. Miz,
351-ICN.
* * N RS, Porto Alegre
– Campus of
Vale- UFRGS
2 Solanum melongena L. _ *
(AF244726)
*
(AY266240)
*
(AY555465)
GenBank
3 Solanum robustum Wendl. _ *
(AY561270)
*
(AY266259)
*
(AY555472)
GenBank
4 Solanum sisymbriifolium Lam _ *
(AY561271)
*
(AY266235)
*
(AY555473)
GenBank
5 Solanum stagnale Moric. _ *
(AY561272)
*
(AY266262)
*
(AY555474)
GenBank
6 Solanum wendlandii Hook. f. _ *
(AF244731)
*
(AY266248)
*
(AY555481)
GenBank
7 Solanum jamaicense Mill. _ *
(AF244724)
*
(AY266239)
*
(AY555472)
GenBank
72
E)
Taxa Voucher ITS
(GenBank
accession numbers)
trnL-trnF plus
intro trnL
(GenBank
accession
numbers)
trnS-trnG
(GenBank
accession
numbers)
Sampling Places
Outgroups outside subgenus Leptostemonum (non—
spiny)
1 Solanum mauritianum Scop. L.A. Mentz, E.L.C.
Soares, R.B. Miz,
350-ICN.
N
*
N
RS, Porto Alegre
– Campus of
Vale - UFRGS
2 Solanum abutiloides (Griseb.) Bitter & Líllo _ *
(AF244716)
*
(AY266236)
*
(AY555453)
GenBank
3 Solanum aviculare Forst. F. _ *
(AF244719)
*
(AY559238)
*
(AY555458)
GenBank
4 Solanum luteoalbum Pers. _ *
(AF244715)
*
(AY266257)
*
(AY555463)
GenBank
5 Solanum pseudocapsicum L. _ *
(AF244720)
*
(AY266241)
*
(AY555469)
Genbank
6 Solanum dulcamara L. _
N
*
(AY266231)
N
GenBank
7 Solanum concinnum Schott ex Sendtn. L.A. Mentz, E.L.C.
Soares, M.
Vignoli-Silva, G.S.
Vendruscolo, 388-
ICN.
*
N N
RS, Viamão,
State Park of
Itapuã
73
Table 2: Comparison among the nuclear and plastid data from Solanum.
Statistic parameters ITS trnS-trnG Intron trnL +
trnL-trnF
chloroplast
data matrix
combined
matrix
Taxa number used 60 58 76 56 45
Aligned length 727bp 868bp 1270bp 2137bp 2864bp
Trees length 1079 319 640 865 1737
Variable sites 363bp 206bp 387bp 523bp 832bp
PI sites 228bp 87bp 216bp 221bp 414bp
CI (RC);
RI
0.55 (0.44)
0.80
0.72 (0.62)
0.86
0.74 (0.67)
0.89
0.72 (0.62)
0.85
0.64 (0.51)
0.79
Note: PI= Parsimony – informative; CI = consistency index (RC= rescaled CI); RI= retension index.
[U2] Comentário:
74
Table 3: Bootstrap values presented for each clade shown in Figure 3 (NJ and MP) and
consensus values in Bayesian analysis by the constructed tree using different phylogenetic
reconstruction methods.
Clade D (NJ) MP Bayesiana
1 58 59 92
2 100 100 100
3 99 100 100
4 71 98 87
5 52 95 100
6 - 81 99
7 - - -
8 88 65 83
9 - 83 83
10 58 75 87
11 87 81 100
12 91 100 100
13 99 100 100
14 - 53 -
15 73 - 100
16 - 97 100
17 - - -
18 - 53 -
19 - 95 93
20 92 - -
21 56 73 99
22 91 86 100
23 - 62 93
24 - 69 -
75
25 69 95 100
26 65 86 100
27 - - 98
28 96 74 100
29 100 93 100
30 73 66 96
31 99 100 100
32 - - -
33 80 97 98
34 100 100 100
35 91 95 100
36 - - -
37 - - -
38 - - -
39 97 100 100
76
Table 4: Partition Bremer support (PBS) scores across the simultaneous analysis of
Parsimony consensus tree for each fragment (intron trnL plus trnL-trnF, trnS-trnG and
ITS) partition in each clade presented in Fig 3.
Clade Intron trnL
+ trnL-trnF
trnS-trnG ITS region Decay index
1
-1 0 2 1
2
3.07 0.63 3.31 7
3
-1.06 0.50 5.56 5
4
-1.06 0.50 1.56 1
5
-0.24 -0.05 1.29 1
6
0 0.50 -0.50 0
7
0.07 0 -0.07 0
8
-0.14 0 1.14 1
9
0 0 0 0
10
-0.06 -0.50 2.56 2
11
-0.86 2.50 0.36 2
12
2.14 -0.30 1.16 3
13
2.94 1 9.06 13
14
-0.24 0.55 -0.31 0
15
-0.73 2.30 -0.61 1
16
2.44 1.50 -1.94 2
17
0 0 0 0
18
0 0 0 0
19
1 0 0 1
20
0 0 0 0
21
0 0 2 2
22
0.33 1 3.67 5
23
0 0 1 1
77
24
0 0 0 0
25
0 0 5 5
26
1 1 3 5
27
0 0 2 2
28
3 1 0 4
29
1 1 3 5
30
-1.42 -0.21 3.63 2
31
3.90 2.98 0.12 7
32
0 0 0 0
33
1.63 -0.63 1 2
34
2.94 2.45 10.60 16
35
3.94 0 -1.94 2
36
1.94 1 -2.94 0
37
1.94 1 -2.94 0
38
1.94 1 -2.94 0
39
7.54 4.50 5.96 18
Total PBS
35.95 25.22 54.79 -
Min steps
312 160 605 -
Total
PBS/Min
steps
a
0.11 0.15 0.09 -
a
PBS values summed across the tree and standardized by the minimum number of steps
for each partition.
78
4.- Artigo 2
79
Phylogeny and genetic variation of species of
Solanum section Torva (Solanaceae) from Southern Brazil
(A ser submetido para publicação em “Botanical Journal of the Linnean Society”)
80
Phylogeny and genetic variation of species of
Solanum section Torva (Solanaceae) from Southern Brazil
Rogéria B. Miz
a
, Tatiana T. Souza - Chies
a, b
a
Universidade Federal do Rio Grande do Sul, Programa de Pós Graduação em Genética e
Biologia Molecular, Av. Bento Gonçalves, 9500. Caixa Postal 15053, CEP: 91501-970,
Porto Alegre, RS, Brazil. Tel.:+55 51 3316-9830, Fax: + 55 51 3316-7311
b
Universidade Federal do rio Grande do Sul, Departamento de Botânica, Av. Bento
Gonçalves, 9500. Caixa Postal 15053, CEP: 91501-970, Porto Alegre, RS, Brazil. Tel.:+55
51 3316-7569, Fax: + 55 51 3316-7686
Corresponding author: Dra. Tatiana T. Souza-Chies
Address for correspondence.
Departamento de Botânica, UFRGS
Prédio 43433
CEP: 91501-970, Porto Alegre, RS, Brazil
Tel.: 55 51 3316-7569
Fax: 55 51 3316-7686
E-mail: [email protected]
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Abstract
Worldwide Solanum subgenus Leptostemonum section Torva comprises
approximately 40 species. Five of them are found in southern Brazil. Some of those species
contain active substances which are useful in the treatment of gastrointestinal diseases and
ulcerations. However, these plants present taxonomic problems concerning their
circumscription, and their evolutionary relationships are hard to understand. In addition,
there is a group of plants presenting an intermediate morphology between S. paniculatum x
S. guaraniticum. In this study, three marker types were used (nuclear, plastid and ISSR) to
investigate about the phylogenetic relationships and to evaluate the variation among the
taxa from section Torva. The ISSR markers showed a high degree of polymorphism with
great variation identified both inter and intra specific levels for the studied species. All the
phylogenetic analysis indicated that the intermediate morphology (S. paniculatum x S.
guaraniticum) species is related to their supposed progenitors S. paniculatum and S.
guaraniticum. Furthermore, we observed that S. guaraniticum/S. bonariense, and S.
adspersum/S. tabacifolium are very close. The southern Brazilian species of Solanum
section Torva can be considered as monophyletic.
Key Words: Solanaceae; Solanum section Torva; ITS; intron trnL plus trnL-trnF; trnS-
trnG; phylogenetic analysis; ISSR marker; genetic variation.
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Introduction
Solanum L. presents some species that are of special interest because of their active
elements, which have pharmacological properties of great importance in medicine and to
produce therapeutic drugs. Among the species under investigation S. tuberosum L., S.
stramonifolium Dunal, S. siparunoides Ewan, S. dulcamara L., S. nigrum L. and S. indicum
L. (Frusciante et al., 2000; Syu et al., 2001; Lee et al., 2004) are some examples.
Notwithstanding the importance of this genus for the pharmaceutical industry, many
other lesser known species are intensely used in folk medicine. This intense use could
create a risk of extinction of innumerable native plants resulting in ecological disturbances
and the disappearance of valuable germ plasm of which pharmaceutical and chemical
potential has not yet been established. To preserve these species is indispensable to know
about the biodiversity. Knowledge of the taxonomy and genetic diversity of these species is
fundamental for sustainable utilization of these important natural resources. One of the
Solanum groups which requires taxonomic study to improve understanding of its species,
and whose pharmaceutical potential is of outstanding interest, is the section Torva (Nees,
1984). The species of Solanum section Torva contain active substances for the treatment of
gastrointestinal diseases and ulcerations (Mesia-Vela et al., 2002; Antonio et al., 2004).
Section Torva belongs to the subgenus Leptostemonum (Dunal) Bitter of Solanum
(Solanaceae), and is composed of more than 40 species (Nee, 1999) distributed worldwide
(its dispersion centre is in the Americas, and some species are found in África, Ásia, New
Guinea and in the Pacific). In southern Brazil, five species are known: Solanum
paniculatum L., Solanum guaraniticum A. St.-Hil., Solanum adspersum Witasek, Solanum
variabile Mart. and Solanum tabacifolium Dunal.
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Solanum paniculatum (known as the real “jurubeba”) is a perennial shrub species,
erect and few branched, armed on stems and branches with curved aculeous widened and
flattened at the base. Leaves bicolour, abaxially covered by dense pubescence with sessile
stellate hairs. Calyx covered by dense pubescence, lobes wide with a narrow tip. Corolla
white or pale violet, lobes divided into the half. Fruits are pendent. Its reproduction is made
by seeds. This species is most often found in the eastern cost of Brazil, from the state of
Rio Grande do Norte in the extreme North to the southern state of Rio Grande do Sul.
Sometimes it is also found as cultivated crop or as a ruderal. According to Mentz &
Oliveira (2004), the populations now found in Rio Grande do Sul have been recently
introduced.
Solanum guaraniticum (one of the false “jurubeba”) is a perennial shrub species,
erect and few branched, armed on stems and branches with straight acicular aculeous, of
different lengths, occasionally slightly widened at the base. Populations can be found with
no prickles or nearly unarmed. Leaves almost concolor, covered with stellate trichomes, in
general having a tiny pedicel. Calyx with strait-triangular lobes, nearly glabrous or covered
by stellate trichomes. Corolla white or very occasionally slightly bluish, divided above the
middle. Fruits erect. Generally with reproductive roots, but it also reproduces from seeds.
This species presents ruderal behaviour and is found also in Paraguay and Argentina - in
Brazil is distributed from the state of Minas Gerais to the state of Rio Grande do Sul in the
extreme South Region. In the city of Porto Alegre and environs, some populations can not
be identified exactly as either S. paniculatum, or S.guaraniticum, because they showed an
intermediate morphology between these two species (Mentz & Oliveira, 2004). It was
observed that the anthers of these plants are empty or almost and they rarely produce fruits.
Subterranean system is present in these populations. The inflorescence is branched as in S.
84
paniculatum, but the aculeous are acicular in the entire plant or only in its upper portion
with sporadic curved and widened aculeous in some points. The calyx seems
morphologically intermediate between those two species. Because of this morphological
variation, the samples collected from these populations may be hybrids, and therefore shall
be denominated in this study as “intermediate species”.
Solanum variabile (known as false “jurubeba” and “jurubeba-velame”) is a
perennial shrub and a very polymorphic species, erect and branched, armed on stems and
branches with aculeous widened at the base, or nearly unarmed. The young leaves are
ferruginous, and can be narrow or wide-lobed. Branches and leaves carry stellate trichomes
with a typical pluricellular and pluriseriate pedicel which does not occur in the remaining
species here analysed. Calyx with oval-shaped lobes, pointed at the apex. Corolla is white
and shallowly lobed. Fruits erect. Probably it reproduces by seeds. This species is found in
Paraguay and in Brazil, where it grows from the states of Minas Gerais and São Paulo to
Rio Grande do Sul.
Solanum adspersum (common name not available) is a perennial shrub species,
erect and few branched, armed on stems and branches with straight acicular aculeous and
with slightly base widened aculeous. Its leaves are very similar to those from S.
guaraniticum. Both species differ on the corolla lobes and on the position of its fruits.
Corolla white, divided bellow the middle. Fruits pendant. Solanum adspersum is endemic
and is only found in the Brazilian coastal regions of the states of São Paulo and Paraná, in
the so called “restinga” vegetation.
Solanum tabacifolium (with the common names “cardo-branco”, “jurubeba” and
“juveva”), miscited as S. asperolanatum by Smith & Downs (1966), is a small perennial
tree. Young stems and branches carry very small aculeous, slightly widened at the base.
85
Leaves concolour, abaxially covered by pubescence with two sizes of stellate hairs. Calyx
covered by ferruginous trichomes, lobes wide with a narrow tip. Corolla is white, lobes
deeply divided and two times longer then the calyx, also covered by ferruginous trichomes.
Fruits erect. The reproduction is made by seeds. This species grows in Brazil in the Center
Region (Mato Grosso and Goiás), South East Region (Minas Gerais, Rio de Janeiro and
São Paulo) and in the South Region (Paraná and Santa Catarina).
Solanum bonariense L. (known as “granadillo” and “naranjillo” in Argentina) is a
perennial shrub morphologically close to S. guaraniticum, from which it differs mainly in
the indumentum and in the calyx morphology. Solanum bonariense has leaf trichomes
sessile and porrect- stellate. The calyx lobes are narrow and long and when the corolla fall,
the calyx lobes interweave. This species is found in Argentina and Uruguay and also in the
state of Paraná, Brazil.
The aim of this study was to clarify the phylogenetic relationships and to measure
inter taxonomic variability among some taxa of Solanum section Torva. To achieve these
goals three types of markers were used: the nuclear and plastid markers (cpDNA) of which
we used three genomic regions; the ITS spacers (internal transcribed spacer of the rDNA)
of the nuclear genome (Desfeux & Lejeune, 1996); the trnL intron plus the trnL-trnF
spacer (Taberlet et al., 1991) and trnS-trnG (Hamilton, 1999) spacer of the cpDNA. The
third marker corresponds to ISSR (Inter Simple Sequence Repeat) which was first
described by Zietkiewicz et al. (1994) and is now widely used for genetic diversity studies
(Davierwala et al., 2000; Panda et al., 2003), and even for phylogenetic analysis (Yockteng
et al., 2003). ISSR-PCR permits the detection of polymorphisms in the locus situated
between the microsatellites using simple repeated sequences (SSRs) as primers (Wu et al.,
1994; Zietkiewicz et al., 1994).
86
Materials and Methods
1) Material
Forty-two individuals were used for the phylogenetic analysis, being ten samples
used as outgroup, the latter are classified in other sections of subgenus Leptostemonum
(samples 33-41, Table 1). The distinct data sets were treated with a different number of taxa
due to different reasons including the lack data and convention (Table 2). In analysis of
nuclear and plastid markers were used three matrixes: ITS matrix- the ITS matrix was
analyzed with 34 samples. The excluded sequences were: Section Torva: one sample of
intermediate morphology species, four of S. guaraniticum, two of S. paniculatum and one
of S. variabile. Plastid matrix- this matrix was analysed with 34 samples. The excluded
sequences were: Section Torva: one sample of intermediate morphology species, three of S.
guaraniticum, one of S. paniculatum, one of S. variabile; and outgroup: one of S.
aculeatissimum, and one of S. sisymbriifolium. Combined matrix- 29 samples were
analyzed in the matrix combining the three fragments. The excluded sequences were:
Section Torva: two samples of intermediate morphology species, six of S. guaraniticum,
two of S. paniculatum, one of S. variabile; and outgroup: one of S. aculeatissimum and one
of S. sisymbriifolium.
Twenty-two samples were used to ISSR markers (Table 1), being used the species S.
viarum, S. atropurpureum and S. aculeatissimum as outgroup.
87
2) DNA Isolation and amplification
The extraction of total DNA was realized with dry leaves in silica gel using the
CTAB technique (Doyle & Doyle, 1990) suitably modified.
2.1) Nuclear and Plastid markers
For the phylogenetic analysis of the Solanum section Torva four fragments were
used, of which one fragment corresponded to the ITS1 regions, the gene 5,8S and ITS2, and
three fragments of plastid DNA: the intron of the gene trnL and the intergenic spacers trnL-
trnF plus trnS-trnG.
For amplification of the fragments corresponding to the ITS region we used the “92”
and “75” primers as described by Desfeux & Lejeune (1996); with PCR conditions as
follows 1μl (30-100 ng) of DNA, 2.5 μl of reaction buffer 10x, 0.75 μl of MgCl
2
(50mM),
0.5 μl of dNTP (10mM), 0.25 μl of Taq DNA polymerase (5U/μl), 0.5μl of each primer (92
and 75), 2.5μl of DMSO (96%) and H
2
O to make up 25 μl. The amplifications of this
fragment were performed in the Applied Biosystems thermocycler (Gene Amp PCR
System 2400) using the “Hot Start” PCR Method: 94°C for 5min, 72°C for 6 min; 35
cycles of 94°C, 45 sec., 58°C for 1 min, 72°C for 1 min and 30 sec; finishing with an
extension at 72°C for 10 min. The PCR products were sequenced with the primers 92 and
ITS3 (Desfeux & Lejeune, 1996).
The amplification of trnS-trnG region was realized with the primers “S” and “G”
described by Hamilton (1999) under the following conditions: 2.5 μl of reaction buffer 10x,
with 0.5 μl of dNTPs (10mM), 0.8 μl of MgCl
2
(50mM), 0.5 μl of each primer (10
pmol/μl), 0.2 μl of Taq DNA polymerase (5U/μl), 1 μl of DNA (30-50 ng) and H
2
O to
88
make up a total volume of 25 μl. The conditions under which the amplification of this
fragment was performed included an initial denaturing temperature of 94°C for 5 min; 40
cycles at 94°C for 1 min, 49°C for 1 min, 72°C for 1 min; ending with an extension at 72°C
for 10 min. The PCR product was sequenced using the same primers as were used in the
amplification.
The other two fragments, (intron of trnL gene and trnL-trnF spacer) were co-
amplified and sequenced, using the primers “c” and “f” described by Taberlet et al. (1991)
under the following conditions: 2.5 μl reaction buffer 10x, 0.5 μl of dNTPs (10mM), 0.75μl
of MgCl
2
(50mM), 0.5 μl of each primer (10 pmol/μl), 0.25 μl of Taq DNA polymerase
(5U/μl), 1 μl of DNA (30-50 ng), 1 μl of DMSO (96%) and H
2
O to make up a total volume
25 μl. The amplification was realized with: initial denaturing of 94°C for 3 min; 35 cycles
at 94°C for 1 min, 55°C for 1min., 72°C for 2 min; finishing with an extension at 72°C for
3 min.
2.2) ISSR Markers
To perform this study, four ISSR primers were used (Table 3) to infer the degree of
variation between the different species belonging to the Solanum section Torva. The PCR
reactions were carried out under the following conditions: 5μl of DNA (30-50ng), 2.5 μl
reaction buffer 10x, with 1μl of dNTPs (10mM), 2.3 μl of MgCl
2
(50mM), 1 μl primer(100
pmol/μl), 0.2 μl of Taq DNA polymerase (5U/μl), 1 μl of DMSO (96%) and H
2
O to
complete a volume of 25 μl. The amplifications were performed in the thermocycler cited
above with the following amplification programs: initial denaturing at 94°C for 5 min; 40
cycles at 94°C for 1 min, 50°C or 48°C – varying according to the primer involved- (Table
89
3) for 45sec, 72°C for 2 min; ending with an extension at 72°C for 5 min. As amplification
products of the ISSR type segregate as dominant mendelian markers, the polymorphism
was proven by the presence and absence of bands after the amplification and
electrophoresis in a 1.8% agarose gel, stained with ethidium bromide and visualized and
photographed under UV light. The size of the fragments was estimated by comparison with
the Ladder 100bp (PB-L Produtos Bio-Lógicos).
3) Data analyses
3.1) Nuclear and Plastid markers
For the phylogenetic analysis of the nuclear and plastid markers we used four
phylogenetic methods: Maximum Parsimony (MP), Maximum Likelihood (ML), Distance
Analysis (D) were realized in PAUP 4.0b10 (Swofford, 2002), and Bayesian Inference (IB)
using Mr.Bayes 3.0b4 program for Windows (Huelsenbeck & Ronquist, 2001). Three data
sets were used: 1) ITS matrix (ITS1, 5.8S, ITS2); 2) Plastid matrix (intron of the trnL gene,
trnL-trnF and trnS-trnG) intergenic spacers; 3) Combined Matrix (ITS matrix + plastid
matrix). For all the matrixes MP, D, IB, and ML analysis were performed.
In addition, it was performed a parcimony analysis (gaps as fifth base) using only
the sequence corresponding to the trnS-trnG spacer. In this analysis 38 samples were used
adding sequences from other species from Torva: as S. crinitipes Dunal (AY998401), S.
glutinosum Dunal (AY998418) and S. lanceolatum
Cav. (AY998431).
Parsimony analyses were conducted using heuristic searches with tree-bisection-
reconnection (TBR) branch swapping. Indels (insertion and deletion events) were treated
either as missing data or as fifth character state. The Consistency (CI), Retention (RI),
90
Rescaled (RC) and Homoplasy (HI) indices were also calculated. The strength of support
for individual tree branches was estimated using bootstrap values (BS) (Felsenstein, 1985)
and decay indices (Bremer partition support method - PBS). Bootstrap values were from
1000 full heuristic bootstrap replicates. The Bremer partition support method was used to
estimate how much each data set (nuclear and plastid regions) contributed to the formation
of each branch in the total phylogenetic evidence in the combined matrix. The PBS values
were calculated in PAUP and TreeRot.v2 programs (Sorenson, 1999).
To estimate the information contained for each data set, the g1 statistical test (Hillis,
1991) was realized from 1000 randomly trees generated by PAUP. The GC% content was
also calculated in the same software. To test for congruence between two data partition
(plastid and nuclear partitions), 1000 replicates of the partition homogeneity (PHT) test
(essentially the incongruence length difference (ILD) test of Farris et al., 1994, 1995), as
contained in PAUP 4.0b10, were applied.
The appropriate models of nucleotide substitutions to perform ML and IB analyses
were determined in ModelTest 3.06 program (Posada & Crandall, 1998) and the Akaike
Information criterion (AIC). The models GTR+I+G, for ITS matrix, TVM+I+G, for Plastid
Matrix, and GTR+I+G, for Combined Matrix were selected. The Bayesian analysis was
conducted for 1,000,000 generations using the Markov Chain Monte Carlo method
(MCMC). The ML analysis was estimated by a heuristic search with the as-is option.
Distance Analysis was conducted using the neighbour-joining (NJ) method (Saitou
& Nei, 1987), with the model proposed by the manufacturer, ModelTest. This analysis was
also realized in the same way in PAUP.
3.2) ISSR Markers
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ISSR approach generated different fragment sizes that were treated as presence (1) or
absence (0). Bands of identical size were assumed to be homologous throughout the
species. The 0/1 binary matrix was used to calculate the similarity matrix using Dice and
Jaccard coefficients. The phenogram was constructed with UPGMA using NTSYS 2.10
program (Rohlf & Marcus, 1993).
The Distance Analysis was inferred using the NJ algorithm and it was realized in
PAUP.
Results
Nuclear data sets - ITS
ITS sequences for 34 samples showed an aligned length of 699 (missing data) and
691 (fifth data). The ITS matrix using gaps as fifth base showed a high number of PI = 201
(Table 2). All analyses based on ITS matrix Solanum section Torva appeared as
monophyletic. In these analyses all samples of the intermediate morphology species are
closely related to S. guaraniticum and S. bonariense (BS 74 for D analysis, results not
showed). However, in the trees based on cloroplast and combined data, the intermediate
morphology species is preferentially close to S. paniculatum (Figures 1 and 2).
ITS matrix presented a low index of homoplasy here, being phylogenetically
informative. Among the different data sets analysed in the present work, the ITS matrix
presented the lowest number of PI, and this is probably due to the length of this fragment
which corresponds to a small region (in bases pairs, bp).
Chloroplast data
92
The combined sequences from intron trnL + trnL-trnF + trnS-trnG spacers (plastid
genome) showed an aligned length of 1959 bp (gaps treated as missing data) and 1920 bp
(gaps treated as fifth base). The plastid matrix corresponds to the highest PI= 364 (Table 2)
when gaps were treated as fifth base.
Figure 1 showed the results obtained from IB analysis based on plastid data. In this
figure as well as in other trees obtained from chloroplast data is observed that intermediate
morphology species is close to S. paniculatum and S. guaraniticum, which may be its
ancestors. This is also observed in figure 2 (MP form combined data).
Figure 1 showed also the group formed by S. variabile and S. adspersum that is also
observed in other analyses based on chloroplast data (results not showed), however this
group is not observed on the other analyses. S. tabacifolium does not form any stable group
in analyses based on chloroplast data, appearing sometimes as the basal species of the
section Torva with S. torvum (Figure 1).
In this analysis based on cpDNA data the section Torva appears as monophyletic
supported by BS 91 (Figure 1).
The monophyly is also maintained with a more complete sampling when S.
crinitipes, S. glutinosum and S. lanceolatum where added and a MP analyses was
performed based on the trnS-trnG spacer. This analysis showed an aligned length of 730
characters, with 32 characters being parsimony-informative (PI) and the most parcimonious
trees were produced with 276 steps. This analysis showed a high consistency index (CI =
0.74), and divided the section Torva into three groups: the first one is formed by S.
paniculatum, intermediate morphology species, S. adspersum and S. variabile (supported
by BS 92); the second is formed by S. guaraniticum, intermediate morphology species, S.
bonariense, S. tabacifolium and S. torvum (BS 51); and the third group is formed by S.
93
crinitipes, S. glutinosum and S. lanceolatum (BS 90). The two first groups are closely
related and this groupment is supported by BS 81, being the other three species a sister
group of the remainder species from section Torva (BS 88).
All data sets combined (total evidence)
The most phylogenetic information was provided by MP analysis based on the
combined matrix, this analysis supplied the greater number of PI and the g1 value obtained
by the Hillis test was -0.83.
Figure 2 showed the majority consensus tree from MP obtained from the total
evidence. The combined matrix showed an aligned length of 2,652 bp (using missing data)
and 2,611 bp (gaps as fifth base). The MP analysis performed from this latter matrix
obtained the highest PI = 552 (Table 2).
All analyses based on the total evidence showed that Solanum section Torva was
monophyletic (BS 100, PBS = 11, Figure 2). In all performed analyses a group formed by
S. guaraniticum, S. bonariense and one individual from the intermediate species was
maintained, as in cpDNA and ITS matrixes. The majority consensus tree of all MPT (most
parcimonious trees) (Figure 2) showed that plastid DNA contribute more to its formation
than the ITS region supported by PBS.
Differences on the topology of the trees obtained by MP analyses were observed
when the different indels treatments were considered. Figure 2 showed a clade grouping S.
paniculatum and S. paniculatum x S. guaraniticum (BS 100) using gaps as fifth base or
missing data. Nevertheless, when indels are treated as fifth base (Figure 2), S. tabacifolium
is more close to this group (supported only by BS 78).
94
The position of S. variabile is very affected by the indels treatment, S. variabile
forms a separate group when indels are treated as fifth base (BS100, Figure 2). However,
this species is grouped to S. adspersum and S. tabacifolium when gaps are treated as
missing data.
ML and IB analyses showed similar topology (results not showed). The trees
differed from MP (Figure 2) by the position of S. variabile and S. paniculatum that are
closely related in these analyses and this group is also evidenced by ITS. Other difference
concern the group formed by S. adspersum and S. tabacifolium. In these trees S.
paniculatum x S. guaraniticum appears as an isolated group and closed to S. guaraniticum.
Genetic variability intra and inter-specific in Solanum Section Torva
The ITS matrix was also used to verify the genetic variability of the section Torva.
Variability intra- and inter-specific were analysed when two or more ITS sequences were
used. The intermediate morphology species showed the great number of variation intra-
specific with 110 variable characters, followed by S. variabile with 80, S. guaraniticum
with 57, S. adspersum with 42 and S. paniculatum with 21 variable characters. The size of
ITS sequences varied from 600 to 640 bp within this species. It showed a high intra-specific
variation within this taxon, once it should present the same sequence among all analysed
individuals. This variation may be mainly due to different local origins and to distinct
selection pressures by individuals of a same species. ITS matrix corresponding to the
section Torva showed an alignment of 658 bp, being 377 bp variable, and 313 bp
parsimony informative sites. A high variation inter-specific was showed among the eight
95
species (circa of 54 to 57% of variation among ITS sequences) from section Torva
(including intermediate morphology species).
All primers used to perform ISSR analyses allowed DNA amplification with a clear
identification of the bands and provided information about the five species of Solanum
section Torva used in this study. The present work can be used as a reference to other
works concerning genetic variability in Solanum. The primers generated a total of 82
fragments (bands) for 22 individuals used in this study (Table 1). The fragments varied in
size from 250 bp to more than 1kbp (Table 2). Of the 82 ISSR bands obtained, only one
was present in all individuals and the remainder (99%) were polymorphic. Figure 3 showed
an amplification pattern obtained to the primer 4 for 22 analysed samples.
The minimum and maximum number of fragments generated per primer was 18
(primers 3 and 4) and 27 (primer 2), respectively (Table 4), with an average of 20.5
fragments. Considering all analyzed primers (Table 4) among the species, S. adspersum
presented the highest number of bands (40 bands), while S. aculeatissimum the lowest (17
bands). Twenty-five marker bands (MB) were obtained for 22 samples, of which 21 MB
were exclusively of the section Torva. The species presenting the highest number of marker
bands were S. adspersum (with ten MB, being two diagnostic bands), S. guaraniticum (four
MB) and S. paniculatum x S. guaraniticum (three MB) (Table 4).
Figure 4 showed a dendogram calculated from similarity coefficients of Dice. The
dendogram illustrates genetic similarities among species from section Torva, in which S.
adspersum corresponds to the more distant species from other taxa. There is a large group
in the dendogram including almost all species from the section Torva collected in the
brazilian state of Rio Grande do yu2T 13.9225l228 -2u-30[(br-2.295zTc0.0872 Tw[(Figure16 Tcnarity )5.9(co)5.9(effici).8(colmo)5.6sTj5(ramo)5.9o56(eg(u90019 T)-5.8((e S0.0r-34)]TJ1. The)7.7(r)-4.1(e Tc0hdeera)7.Tf3.5826 0p7( )5.8(2)5.4(1)o Gcg0mmos)5.
96
sample of the intermediate species. This group lies S. paniculatum and the intermediate
morphology species and reveals an intra-specific variation of S. guaraniticum, which
appears dispersed in the dendogram, coming closer both to S. paniculatum and to S.
variabile. The species from section Acanthophora showed more close to the species from
section Torva than to S. adspersum (collected in the brazilian state of Paraná).
The separation of S. adspersum of other species from Torva can be seen in the
analysis of the principal coordinates (Figure 5), and in the dendogram (Figure 4). This is
probably due to the high number of exclusive bands (markers) of this species obtained by
ISSR. In addition, it can be observed that the interaction between the intermediate
morphology species and their supposed progenitors S. paniculatum and S. guaraniticum is
seen in morphological and molecular evidences.
In NJ analysis the section Torva also appears as monophyletic (Figure 6). The
corresponding dendogram presents two large groups: the first one is formed by S.
paniculatum, S. variabile, S. guaraniticum and the intermediate morphology species (S.
paniculatum x S. guaraniticum), all of them are typical representatives of Solanum section
Torva found in the brazilian state of Rio Grande do Sul; and the second group is formed by
S. bonariense (Argentina), S. tabacifolium (Minas Gerais) and S. adspersum (Paraná), plus
one individual of S. guaraniticum that is almost without aculeous in the base (Rio Grande
do Sul, Brazil) - showing an inter-specific variability within Torva.
Discussion
97
Nuclear and Plastid Markers
Solanum section Torva appears as monophyletic in almost all analyses performed in
this study and it is in agreement with Levin et al. (2006). This monophyly is maintaining
even when a more complete sampling from section Torva is analysed.
The most frequent relationships among the species from section Torva were as
follow: (i) the group concerning S. guaraniticum + S. bonariense + a sample of
intermediate morphology – this occurred in 93% of the trees; (ii) S. adspersum + S.
tabacifolium (60%); (iii) S. paniculatum + the intermediate morphology species (46%); and
(iv) S. paniculatum + S. variabile (40%).
Solanum guaraniticum, S. bonariense and “S. paniculatum x S. guaraniticum” are
very close, which appeared as an isolated group in almost all analyses. The clade
concerning “S. paniculatum x S. guaraniticum” plus S. guaraniticum can be explained by
the supposition that this latter species is considered one of the possible progenitors of the
species with intermediate morphology. The morphological traits of “S. paniculatum x S.
guaraniticum” is intermediate between S. paniculatum and S. guaraniticum including
aciculate aculeous even on the apex branches (as in S. guaraniticum), and a calyx with a
intermediate form between those of S. paniculatum and S. guaraniticum
The strong relationship between S. bonariense and S. guaraniticum can be explained
in part by some morphological similarities shown by these species and by six
synapomorphies showed by molecular data. PBS analysis performed from MP tree based
on total evidence showed that the plastid markers (which are in general more conservative
than ITS) contributed more to the formation of this group.
The species collected in the brazilian states of Paraná (S. adspersum) and Minas
Gerais (S. tabacifolium) are grouped in almost all phylogenetic analyses based on both ITS
98
and combined matrixes. However, the analyses based on the plastid matrix this relationship
is not confirmed.
The third most frequent interaction among the species from section Torva is that
between S. paniculatum and the intermediate morphology species. The latter shares
morphological characters with S. paniculatum, which is supposed be one of its progenitors.
This interaction, however, is only observed in the analyses from chloroplast and combined
matrixes. The relationship between the intermediate morphology species and its supposed
progenitors differ according to the matrix used for the phylogenetic analyses – it is
probably due to different types of inheritance of each marker. The plastid genome generally
is inherited as uniparental, being maternal in Angiosperms, while the nuclear genome has
biparental inheritance. However, there are some reports where the plastid DNA can be
inherited by biparental inheritance (Brent & David, 1989, Avni & Edelman, 1991, Shore &
Triassi, 1998; Chen et al., 2002). In such cases, it is easier to understand the position of the
intermediate morphology species with both supposed progenitor found in the analyses
derived from the chloroplast matrix. This interaction is also observed and very well
supported (BS 100) in the analysis of the combined matrix independently of the treatment
of gaps.
In the fourth most frequent relationship S. paniculatum is related to S. variabile, it is
mainly evidenced in the trees obtained from ITS data. ML and IB analyses from the
combined matrix also showed this relationship.
All data matrixes used in this study equally contributed to the elucidation of the
phylogenetic relationships among the species of Solanum section Torva. PBS analyses
showed that both matrixes (nuclear and plastidial) allowed the construction of the
parsimony tree based on the combined matrix. In addition, another PBS test was performed
99
to quantify the contribution of each chloroplast fragment in the formation of the clades.
This test showed that the information of the fragment trnL-trnF plus trnL intron was more
than twice that provided by trnS-trnG spacer. This data confirms MP analyses from each
region, in which trnL intron + trnL-trnF spacer showed a high PI in comparison with trnS-
trnG intergenic spacer. This is probably due to the size of these plastidial sequences
because the CI in both regions was high independently of how the indels were treated.
All performed analyses conducted from the same matrix showed near the same
topology besides some divergences were observed. Concerning the probabilistic analyses
(ML and IB), the corresponding trees presented similar topologies independent of the
matrix used to infer the phylogeny. So, we can conclude that is not necessary to perform
both analyses, mostly when the sampling is very large and the ML is very time consuming.
Nowadays the number of phylogenetic researchers giving preference to Bayesian Inference
over Maximum Likelihood has increased. This is mainly due to the rapidity of the method
(IB) in comparison to ML and the former permits estimate the model of evolution using the
same technique. The present work bears out the importance in use more than one method of
phylogenetic reconstruction. The trees generated by different methods (Probabilistic,
Parsimony and of Distance), using a combined matrix, present considerable statistical
support, although some topology differences can be observed.
Congruency among the markers (nuclear and plastid)
The Partition Homogeneity Test (PHT) disclosed incongruence among nuclear and
plastidial markers. (P = 0.001). This is probably due to differences in size of the two
partitions (chloroplast = 1848 bp; ITS region = 691bp). However, with the PBS analysis
both regions are congruent. This congruency may be observed by the PBS values in which
100
only two divergences are observed in the 27 clades generated (results not showed). The
congruency between these two marker types, in this analysis, is probably due to the high
indexes of CI and PI in both matrixes, to the similar values obtained with the Hillis test, and
to the homogeneity presented by the sequences representing the species from section Torva.
Genetic diversity intra and inter-specific in Solanum Section Torva
The high genetic variation showed among the species from section Torva by both ITS
and ISSR markers must be due the following reasons: This reasons concern the distinct
morphology showed within one same species, different places of collection, and to different
floral biology and sexual shape presented within section Torva. Therefore, although many
species of Solanum has monoclinic typical flowers, some variation is noted in sexual shape,
including andromonoecy, androdioecy and dioecy (Anderson 1979, Coleman & Coleman
1982, Oliveira Filho & Oliveira 1988, Anderson & Symon 1989). Within section Torva
were described already andromonoecious and monoecious species (Symon, 1979). This
difference in floral and reproductive shapes allowing the supposed high variation to the
section Torva mainly referring S. paniculatum as allogamous species (Forni-Martins et al.,
1998). The allogamous species possess a system of gametophytic incompatibility (Pandey
1960, Heslop-Harrison 1975). Within section Torva, the genetic incompatibility is known
only to S. paniculatum, while S. hispidum (Baksh & Iqbal 1978) and S. torvum (Hossain
1973) are auto-compatible. This difference among the species may lead to reproductive
isolations and/or geographic isolation as showed by species from section Torva in this
work.
The ITS region showed be a good marker to verify the genetic variation intra and
inter-specific within the section Torva. The high variation within the intermediate
101
morphology species is mainly due to distinct morphologies observed within this taxon
being sometimes similar to S. guaraniticum or to S. paniculatum. Moreover, this variation
within species from the section must be to different places of collection and to concerted
evolution present by this marker, homogenising the DNA sequence to one supposed
parental (Chase et al., 2003).
ISSR markers showed a high level of polymorphism for the species from Solanum
section Torva. Moreover, its also provided evidence on the considerable inter-taxonomic
variability of the studied species, as can be seen in the dendogram obtained by similarity.
Furthermore, we also confirmed a high degree of intra-taxonomic variation for almost all
species. This intra-taxonomic variation was observed most clearly within S. guaraniticum,
in the five samples analyzed. This species is found dispersed throughout the dendogram
interacting with various species from Solanum section Torva. This dispersion may be
explained by the distinct morphological variations shown by this species mainly regarding
the presence of aculeous. One of the observed S. guaraniticum relationships was with S.
bonariense.
Solanum bonariense (Argentine and Uruguay) and S. guaraniticum (southern
Brazil) contain toxic substances which cause neurological disturbances in cattle (Riet-
Correa, 1982; Riet-Correa et al., 1983). The name S. bonariense was always well defined
and no doubts were ever raised about the identification of this species. However, S.
guaraniticum was generally cited in the literature from southern Brazil under the name of S.
fastigiatum Willd. Two varieties of S. fastigiatum were referenced (Smith & Downs, 1966).
The typical variety, S. fastigiatum var. fastigiatum was characterized by the presence of few
aculeous, while S. fastigiatum var. aciculare has numerous aculeous over all the plant.
Because the work of Smith & Downs (1966) is widely used in southern Brazil these names
102
are generally accepted. However, according to Mentz & Oliveira (2004), S. fastigiatum var.
fastigiatum corresponds to S. bonariense, while S. fastigiatum var. aciculare corresponds to
S. guaraniticum. On the other hand, the present study showed that S. guaraniticum is close
related to S. bonariense bases on the analyses of similarity and phylogeny. This is probably
due to the high degree of similarity concerning the morphology of these taxa.
In addition to the inter and intra-taxonomic variation shown by ISSR markers, the
analysis of similarity separated S. adspersum (collected in the brazilian state of Paraná)
from the large group formed by the species from section Torva (all of them collected in the
brazilian state of Rio Grande do Sul), and placed S. adspersum as an outgroup. This
position is partially due to the large number of exclusive bands generated for this species in
comparison to other taxa, and to different evolutionary processes brought about the
geographical separation.
The great contribution to a better understanding of the species from section Torva
provided by ISSR markers may serve as a starting point for future studies using this marker
to determine the taxonomic variation among other species from section Torva, including
intra-taxonomic level.
Conclusion
Solanum section Torva has been shown monophyletic, at least as far as the species
utilized in this study are concerned which provide an evolutionary history of this section
from southern region of Brazil. We found four most-frequent groups among the species
from section Torva - all of them were with a strong support in all performed analyses. The
knowledge of these relationships will contribute to understand the biology of these species
and assist possible programs of conservation and maintenance of the local biodiversity.
103
ISSR markers were found especially informative and polymorphic among the
studied species from section Torva. We believe that this work will serve as a basis for
future investigations about Solanum section Torva. The nuclear and plastidial markers help
us to the elucidation of taxonomic problems and for the verification of genetic diversity
among the studied taxa. This work stands, therefore, as a starting point to understand the
evolutionary history of the species from Solanum section Torva, contributing to their
recognition as important components of the Brazilian native plants.
Acknowledgments
The authors are grateful to: Dr Lilian Mentz (Programa de Pós-Graduação em
Botânica - UFRGS) for specimens suply and for their taxonomic identification, and for
suggestions; Dr. João Renato Stehmann of the Universidade Federal de Minas Gerais, Dr.
Maria de Fátima Agra of the Universidade Federal da Paraíba and Dr. Gloria Barboza of
the IMBIV, Córdoba- Argentina, for the samples supplying; to Dr. Giancarlo Pasquali
(Universidade Federal do Rio Grande do Sul) for sequencing facilities; to our colleagues
Lizandra J. Robe, Ivan Schüler, Liliana Essi and Fernanda Cidade for the valuable help. We
also thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (Cnpq,
Brazil), the Fundação de Amparo à Pesquisa no Estado do Rio Grande do Sul (FAPERGS,
Brazil) for financial support.
104
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Table 1: List of species from Solanum section Torva used in phylogenetic analysis and ISSR analyses. The
asterisks indicate fragments sequenced (or presence of data in ISSR) for each sample and N to the lacked data. The
samples of number 33-42 in the table comprise the species of other sections from Solanum subgenus
Leptostemonum, which were used as outgroup.
Samples Voucher ITS
(GenBank
accession
numbers)
trnL-trnF
plus intron
trnL
(GenBank
accession
numbers)
trnS-trnG
(GenBank
accession
numbers)
ISSR
Markers
Sampling Places
1 Solanum paniculatum X
Solanum guaraniticum
L. A. Mentz, T.T
Souza-Chies and
R.B. Miz, 301.
*
*
*
*
RS, Porto Alegre.
2 Solanum paniculatum X
Solanum guaraniticum
L. A. Mentz, T. T.
Souza-Chies and R.
B. Miz, 302.
*
*
*
*
RS, Porto Alegre.
3 Solanum paniculatum X
Solanum guaraniticum
L.A. Mentz, E.L.C.
Soares, M. Vignoli-
Silva, G.S.
Vendruscolo, R.B.
Miz, 309 - ICN
*
*
*
*
RS, Passo Fundo,
Fazenda Sementes and
Cabanhas Butiá
4 Solanum paniculatum X
Solanum guaraniticum
L.A. Mentz, E.L.C.
Soares, M. Vignoli-
Silva, G.S.
Vendruscolo, 305-
ICN
N
*
*
N
RS, Viamão, Parque
Estadual of Itapuã
5 Solanum paniculatum X
S. guaraniticum.
L.A. Mentz, E.L.C.
Soares, M. Vignoli-
Silva, G.S
.Vendruscolo, R.B.
Miz, 311 - ICN
*
*
N
N
Estrada
Carazinho/Ihuí – near
of Rio Colorado.
6 Solanum guaraniticum
A. St.-Hil.
T. T. Souza-Chies,
221.
*
*
*
*
RS, Antônio Prado.
Estrada Amarilho.
Propriedade of Sônia
Montanari
7 Solanum guaraniticum
A. St.-Hil.
T.T. Souza-Chies,
222
*
*
N
*
RS, Monte Bonito,
distrito of Pelotas.
Propriedade Águas
Claras.
8 Solanum guaraniticum
A. St.-Hil.
L.A. Mentz, E.L.C.
Soares, R.B. Miz,
344 – ICN.
*
*
*
*
RS, Venâncio Aires
(65km)
9 Solanum guaraniticum
A. St.-Hil. (almost
without spines)
L.A. Mentz, E.L.C.
Soares, R.B. Miz,
349.
*
*
*
*
RS, Santa Cruz
10 Solanum guaraniticum
A. St.-Hil. (plant with
spines in stem and
leaves)
L.A. Mentz, E.L.C.
Soares, M. Vignoli-
Silva, G.S.
Vendruscolo, R. B.
*
*
*
*
RS, Fontoura Xavier,
BR 386, km 276
110
Miz, 336- ICN
11 Solanum guaraniticum
A. St.-Hil. (plant with
spines only in low part
of stem)
L.A. Mentz, E.L.C.
Soares, R.B. Miz,
345a.
*
*
*
N
RS, Santa Cruz
12 Solanum guaraniticum
A. St.-Hil.
L.A. Mentz, E.L.C.
Soares, R.B. Miz,
345b
*
*
N
N
RS, Santa Cruz
13 Solanum guaraniticum
A. St.-Hil.
L.A. Mentz, E.L.C.
Soares, R.B. Miz,
345c.
*
*
*
N
RS, Santa Cruz
14 Solanum guaraniticum
A. St.-Hil.
L.A. Mentz, E.L.C.
Soares, R.B. Miz,
339.
*
*
N
N
Coronel Barros -
Parada Lajeado do
Tigre –BR285 Km
476
15 Solanum guaraniticum
A. St.-Hil.
L.A. Mentz, E.L.C.
Soares, M. Vignoli-
Silva, G.S.
Vendruscolo, R. B.
Miz, 333.
*
*
*
N
RS, Santo Ângelo
16 Solanum guaraniticum
A. St.-Hil.
L.A. Mentz, E.L.C.
Soares, R.B. Miz,
348.
*
*
*
N
RS, Santa Cruz
17 Solanum paniculatum
L.
R.B. Miz, 101.
*
*
*
*
RS, Porto Alegre
18 Solanum paniculatum
L.
L.A. Mentz, E.L.C.
Soares, M. Vignoli-
Silva, G.S.
Vendruscolo, 304-
ICN.
N
*
*
*
RS, Viamão, Parque
Estadual de Itapuã
19 Solanum paniculatum L. M. F. Agra, K. Nurit
and I. Basílio.
6311
*
*
N
*
Paraíba, Município
João Pessoa, Campus
da Universidade
Federal of Paraíba
20 Solanum paniculatum
L.
L.A. Mentz, E.L.C.
Soares, R. B. Miz,
341- ICN.
*
*
*
N
RS, Triunfo
21 Solanum paniculatum
L.
L.A. Mentz, E.L.C.
Soares, R.B. Miz,
340- ICN.
*
*
*
N
RS, Triunfo
22 Solanum variabile
Mart.
L. Essi, 301.
*
*
*
*
RS/SC, Cambará do
Sul. Serra do Faxinal.
23 Solanum variabile Mart. L. Essi, 302 * * * * SC, Mauro Müller.
24
Solanum variabile Mart. L. Essi, 304
*
*
*
*
SC, Bom Jardim da
Serra. Serra do Rio do
Rastro
25 Solanum variabile Mart. L. Esii, 319 * * *
N
SC, Urubici
26 Solanum variabile Mart. L. Essi, 320 * *
N N
SC, Urubici
27 Solanum torvum Sw. _ *
(AF244729)
*
(AY266246)
*
(AY555478)
N
GenBank
28 Solanum bonariense L. G.E. Barboza, E.M.
Fillipa, F. Chiarini,
E. Marini, 1568.
*
*
*
*
Argentina Entre Rios:
Department
Gualeguaychú, Desde
Ceibas rumbo a Villa
Paranacito.
29 S. tabacifolium Dunal I.M. Palhares, J.R. MG, Belo Horizonte
111
Stehmann. 94050 * * * *
30 S. adspersum Witasek
(plant with fruit)
L.A. Mentz , J.R.
Stehmann, 4185
*
*
*
*
Paraná, Potinga
31 S. adspersum Witasek
(plant with flowers and
fruits)
L.A. Mentz, J.R.
Stehmann, 4186a.
*
*
*
*
Paraná, Potinga
32 S. adspersum Witasek
(sterile plant)
L.A. Mentz, J.R.
Stehmann, 4186b.
*
*
*
*
Paraná, Potinga
33 S. atropurpureum
Schrank
L.A. Mentz, E.L.C.
Soares, R. B. Miz,
343-ICN.
*
*
*
*
RS, Santa Cruz
34 S. aculeatissimum Jacq. L.A. Mentz, E.L.C.
Soares, M. Vignoli-
Silva, G.S.
Vendruscolo, R. B.
Miz, 319- ICN.
*
*
*
*
RS, Passo Fundo.
Fazenda Sementes e
Cabanhas Butiá.
35 S.viarum Dunal L.A. Mentz, E.L.C.
Soares, R.B. Miz,
346- ICN.
*
*
*
*
RS, Santa Cruz
36
S. vaillantii Dunal L.A. Mentz, E.L.C.
Soares, M. Vignoli-
Silva, G.S.
Vendruscolo, R.B.
Miz, 310- ICN.
*
*
*
N
RS, Passo Fundo,
Fazenda Sementes and
Cabanhas Butiá.
37 S. sisymbriifolium Lam. L.A. Mentz, E.L.C.
Soares, R.B. Miz,
351-ICN.
*
*
*
N
RS, Porto Alegre –
Campus do Vale -
UFRGS
38 S. sisymbriifolium Lam _ *
(AY561271)
*
(AY266235)
*
(AY555473)
N
GenBank
39 S. melongena L. _ *
(AF244726)
*
(AY266240)
*
(AY555465)
N
GenBank
40 S. robustum Wendl. _ *
(AY561270)
*
(AY266259)
*
(AY555472)
N
GenBank
41 S. jamaicense Mill. _ *
(AF244724)
*
(AY266239)
*
(AY555472)
N
GenBank
42 Solanum stagnale
Moric.
_ *
(AY561272)
*
(AY266262)
*
(AY555474)
N
GenBank
112
Table 2: Comparison among nuclear and plastid data from Solanum.
ITS Matrix Plastidial Matrix Combined Matrix
Number of individual 34 34 29
Hillis test g1 = - 0.77 g1 = - 0.73 g1 = - 0.89
Content of GC (%) 0.70 0.33 0.44
Índels treated with
date
missing fifth base missing fifth base missing fifth base
Aligned lenght
including or excluded
gaps
699 691 1959 1920 2652 2611
Tree lenght 381 607 417 959 773 1562
Number of characters
constants
471 400 1661 1296 2155 1713
PI 126 201 131 364 246 552
CI 0.74 0.69 0.78 0.78 0.75 0.72
HI 0.25 0.31 0.21 0.22 0.24 0.27
RI 0.80 0.80 0.81 0.86 0.77 0.80
RC 0.60 0.55 0.64 0.67 0.58 0.58
PI = Number of parsimony – informative characters.
113
Table 3: ISSR primers, sequences, annealing temperature, number of fragments scored and approximate
size range (in base pairs) of the fragments resulted from each primer in 22 individuals (Table 1).
Table 4: Number of fragments scored for each taxon to each indicated primer and respective markers bands
(fragments) scored in parenthesis.
Primers
Sequence Annealing
temperature
(°C)
Number of
fragments
scored
Number of
polymorphic
fragments
Fragment size
range (bp)
1 5’-CTC TCT CTC TCT CTC TG-3’ 48 19 19 400pb- +1kb
2 5’- ACA CAC ACA CAC ACA CT- 3’ 50 27 27 300pb- +1 Kb
3 5’- GAG AGA GAG AGA GAG AT-3’ 50 18 18 350pb - +1 kb
4 5’- CTC CTC CTC CTC RC-3’ 50 18 17 250pb - + 1kb
TOTAL
- 82 81 -
Taxa Number of
individuals
Primer
1
Primer
2
Primer
3
Primer
4
Total
S. paniculatum x S. guaraniticum 3 8 (0) 13 (3) 5 (0) 5 (0)
31 (3)
S. guaraniticum 5 9 (1) 12 (0) 7 (0) 10 (3)
38 (4)
S. paniculatum 3 9 (0) 8 (0) 4 (0) 5 (0)
26 (0)
S. variabile 3 8 (1) 8 (0) 7 (0) 6 (0)
29 (1)
S. tabacifolium 1 3 (0) 11 (1) 6 (0) 5 (0)
25 (1)
S. adspersum 3 11(4) 13 (3) 9 (1) 7 (2)
40 (10)
S. bonariense 1 3 (1) 8 (0) 7 (1) 3 (0)
21 (2)
S. viarum 1 4 (0) 14 (1) 3 (0) 8 (1)
29(2)
S. atropurpureum 1 3 (0) 4 (0) 5 (1) 9 (1)
21 (2)
S. aculeatissimum 1 3 (0) 5 (0) 3 (0) 6 (0)
17 (0)
Total 22 (7) (8) (3) (7)
(25)
114
Figure 1: Majority consensus tree obtained from Bayesian analysis based on the plastidial
matrix. The posterior probability for each clade is indicated above its respective internal
branch (when higher than 50%).
S. paniculatum X S. guaraniticum
S. paniculatum
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. variabile
S. variabile
S. variabile
74
S. variabile
100
S. adspersum
S. adspersum
92
S. adspersum
96
64
S. paniculatum
59
S. paniculatum
S. paniculatum
71
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. bonariense
S. guaraniticum
100
S. guaraniticum
S. guaraniticum
S. guaraniticum
96
100
62
S. tabacifolium
100
S. torvum
91
S. atropurpureum
S. vaillantii
100
S. viarum
100
S. robustum
S. stagnale
100
97
S. sisymbriifolium
100
S. melongena
S. jamaicense
115
Figure 2: Majority consensus phylogenetic tree from maximum parsimony analysis (indels
treated as fifth base) from the combined matrix. Bootstrap values >50% are shown above
the branches, decay indices below.
116
Figure 3: Inter-simple sequence repeat (ISSR) patterns visualized on agarose gel (1.8%) for
22 samples using the primer 4. Marker = molecular-size marker (100-base pairs ladder, PB-
L Produtos Bio-Lógicos).
117
Figure 4: Dendogram based on the Coefficient of Dice showing genetic relationship among
22 individuals based on ISSR markers. The vertical bar corresponds to the mean similarity.
118
Figure 5: Principal Coordinates Analysis (PcoA) plot 3D based on ISSR data.
119
Figure 6: Neighbor-Joining tree obtained from ISSR Numbers above branches indicate
bootstrap support values.
S. paniculatum X S. guaraniticum
S. guaraniticum
S. paniculatum
S. variabile
S. guaraniticum
S. bonariense
S. adspersum
S. tabacifolium
S. viarum
S. aculeatissimum
S. atropurpureum
0.01 changes
S. paniculatum X S. guaraniticum
S.
guaraniticum
S. paniculatum
S. paniculatum
S. variabile
S. variabile
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. adspersum
S. adspersum
99
68
61
120
5.- Discussão
A execução do presente trabalho possibilitou a inferência das relações
filogenéticas mais freqüentes entre as espécies da seção Torva, como comentado
no artigo 2. Tais inferências são apresentadas nas árvores filogenéticas inseridas
nos artigos 1 e 2, e grande parte delas é ilustrada nos anexos (capítulo 8) desta
dissertação.
As diferentes análises realizadas com base em diferentes marcadores
evidenciam a importância da utilização de mais de um tipo de marcador
(preferencialmente de genomas distintos) e mais de um método de inferência
filogenética. Foi observada na topologia das árvores uma constância na formação
de grupos ao analisarmos filogenias obtidas com o mesmo tipo de marcador, A
comparação de topologias oriundas de diferentes análises filogenéticas leva à
obtenção de conclusões com maior probabilidade de serem as verdadeiras.
Atualmente, trabalhos que buscam o conhecimento sobre a filogenia de
plantas têm utilizado informações oriundas de marcadores nucleares e plastidiais.
O principal motivo desta utilização é a origem distinta de cada marcador, os quais
têm proporcionado uma visão mais ampla da história evolutiva das espécies.
Dentro da seção Torva ficou clara a relação de parentesco entre os indivíduos que
apresentam uma morfologia intermediária (Solanum paniculatum x Solanum
guaraniticum) com seus supostos progenitores S. paniculatum e S. guaraniticum..
Porém, as relações variaram conforme o tipo de marcador utilizado nas análises.
Nas análises filogenéticas realizadas com os marcadores plastidiais observou-se
uma interação dos indivíduos morfologicamente intermediários com ambos os
progenitores (artigos 1 e 2), esta relação também ficou evidenciada utilizando os
marcadores ISSR (artigo 2). Entretanto, nas análises com ITS, apenas S.
guaraniticum apresentou uma relação de parentesco com os indivíduos de
morfologia intermediária. Tais diferenças resultam provavelmente do tipo de
herança dos marcadores nucleares e plastidiais, e ao grau de homologia entre as
seqüências. Pois, freqüentemente o genoma nuclear tem sido descrito como de
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origem bi-parental e o genoma plastidial de origem uni-parental materna para as
Angiospermas. Porém, em nosso trabalho, ambos os progenitores aparecem
relacionados com os indivíduos de morfologia intermediária nas análises de
cloroplasto, o que pode ser explicado por uma possível herança bi-parental do
genoma plastidial. Essa característica bi-parental do genoma plastidial vem sendo
detectada e testada em vários trabalhos. No traballho realizado por Shore e Triassi
(1998), foi detectado um potencial de herança bi-parental do DNA plastidial em
Turnera ulmifolia (Turneraceae). No trabalho de Avni e Edelman (1991), foi
realizado um estudo para seleção direta da herança paterna de cloroplasto na
progênie sexual de Nicotiana tabacum utilizando a sensibilidade e resistência de
tentoxina (clorose) como detector de herança paterna. Além dessas espécies foi
relatada a herança paterna do cloroplasto em Pinus (Brent e David, 1989). Com
base nesta última evidência, foi realizado um trabalho por Chen e cols. (2002), no
qual eles detectaram herança paterna do cloroplasto em híbridos de Pinus
utilizando o espaçador intergênico trnL-trnF e a técnica de PCR–SSCP (single-
strand conformation polymorphism). Considerando o que tem sido relatado na
literatura, nossos dados sugerem a existência de herança paterna do genoma
plastidial nas espécies da seção Torva do gênero Solanum, no entanto um maior
número amostral deve ser utilizado para a comprovação dessa hipótese.
Entretanto, o esperado seria que a relação de parentesco entre o possível híbrido
e os seus progenitores estaria melhor evidenciada por ITS, por se tratar de um
genoma com herança bi-parental. No entanto, essa relação não foi evidenciada,
pois em nenhuma das árvores filogenéticas obtidas com o fragmento ITS,
observa-se a espécie de morfologia intermediária relacionada com ambos os
parentais, apenas com um deles, S. guaraniticum.
Para a região ITS, tem sido relatada uma evolução em concerto que deve
homogenizar as cópias (seqüências) e, juntas, a amplificação seletiva e evolução
em concerto, podem ocultar informações sobre o processo evolutivo de uma
espécie, uma vez que em grande parte das vezes a região ITS homogeniza suas
cópias conforme o padrão materno (N. rustica) ou paterno (N. tabacum) (Chase e
cols., 2003). Isto explicaria a deficiência do marcador ITS para demonstrar as
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interações dos progenitores e os indivíduos de morfologia intermediária neste
trabalho. Freitas e Souza-Chies (2003) comentam que os espaçadores da região
de ITS apresentam altas taxas de substituição nucleotídicas, capazes de produzir
grande variabilidade, porém as divergências observadas entre as linhagens são
afetadas pelo processo de evolução em concerto, o qual é responsável pela
homogeneização das seqüências en tandem do DNA ribossomal.
Um dos debates mais freqüentes entre os sistematas e taxonomistas tem
sido a escolha do critério ótimo, isso é, qual o melhor método para análise
filogenética. Em geral têm sido utilizados três métodos básicos para estimar a
filogenia, incluindo distância, máxima parcimônia e máxima verossimilhança. As
relativas vantagens e desvantagens destes métodos vêm sendo debatidas por
vários autores (Faith, 1985; Swofford e Olsen, 1990; Kunhner e Felsensteins,
1994; Huelsenbeck, 1995; Farris e cols., 1996; Lewis, 1998; Steel e Penny, 2000;
Pol e Siddall, 2001; Goloboff, 2003; Gaucher e Miyamoto, 2005). Geralmente,
mais de um método é empregado nas análises filogenéticas para compensar as
desvantagens de cada método, tornando as evidências evolutivas mais
consistentes. Recentemente, estudos comparativos têm iniciado uma nova
proposta de reconstrução filogenética, que corresponde à análise Bayesiana, que
apesar de ter sido sugerida por Felsenstein em 1968 (ver Hulsenbeck e cols.,
2002), tem se tornado mais amplamente conhecida recentemente, e para a qual
programas computacionais têm sido disponibilizados. Algumas revisões recentes
sobre análise Bayesiana têm sido propostas por Huelsenbeck e Ronquist (2001),
Lewis (2001), Huelsenbeck e cols. (2002), Holder e Lewis (2003), e Ronquist
(2004). No presente trabalho foi observada uma semelhança muito forte entre a
topologia das árvores obtidas a partir das análises de máxima verossimilhança
(maximum likelihood) e inferência Bayesiana, ambas baseadas em métodos
probabilísticos. Segundo Archibald e cols. (2003), quando aplicada à reconstrução
filogenética, a inferência Bayesiana é similar à máxima verossimilhança (MV), a
qual aplica uma função de probabilidade e um modelo determinado de substituição
nucleotídicas, apresentando muito das vantagens e desvantagens da MV. Desta
forma, é desnecessária a utilização de ambos os métodos, podendo fazer a
123
escolha por apenas um deles. Um dos benefícios apresentados pela Inferência
Bayesiana (relativo à máxima verossimilhança) em filogenia, é a rapidez das
análises. Isto se deve à implementação de MCMC (Markov Chain Monte Carlo),
que possibilita estimar a probabilidade posterior da distribuição das árvores, o que
elimina muito dos cálculos complexos e integrações e, se baseia em comparações
de simples estimativas (Huelsenbeck e cols., 2002). Embora a análise Bayesiana
seja mais lenta que a máxima parcimônia, ambas são mais rápidas em
comparação à máxima verossimilhança. No estudo de Reed e cols. (2002) foi
verificada uma diferença de 84 dias entre as análises de Inferência Bayesiana e
máxima verossimilhança, as quais apresentaram a mesma topologia das árvores.
Estas evidências justificam as topologias semelhantes encontradas entre as
árvores probabilísticas no artigo 2 e ressalta as vantagens em se utilizar somente
a análise Bayesiana, sobretudo quando o número de táxons empregados no
estudo é grande.
Outro fator que alterou a topologia das árvores ao se utilizar a mesma
matriz de dados, foi o tratamento dado aos “gaps”, os quais foram tratados como
dados faltantes ou como quinto estado de caractere na análise de parcimônia. No
artigo 2, observa-se um aumento considerável do número de caracteres
filogeneticamente informativos na matriz, que considera os “gaps” como quinto
estado de caractere e, em ambos os casos, há um baixo índice de homoplasia.
Segundo Giribet e Wheeler (1999), os “gaps” originam-se de eventos biológicos
particulares, tais como mutação (deleção/inserção) e, por este motivo, eles podem
conter tanta informação histórica como a que é observada com as mudanças
nucleotídicas. Além disso, os autores comentam que os métodos que não
consideram a informação dos “gaps” podem ser menos explicativos, uma vez que
descartam algumas das informações importantes, dificultando a elucidação da
verdadeira reconstrução filogenética dos táxons.
A utilização de ambos os genomas plastidial e nuclear, assim como a
aplicação de mais de um fragmento por genoma é muito importante para a
reconstrução filogenética e determinação da história evolutiva das espécies.
Porém, existem dúvidas a respeito de combinar ou não as matrizes de dados.
124
Vários autores têm discutido sobre as vantagens e desvantagens da análise
combinada (De Queiroz e cols., 1995; Miyamoto e Fitch, 1995; Farris e cols.,
1995; Huelsenbeck e cols., 1996), enquanto que outros autores comentam que os
testes de congruência nem sempre correspondem a uma evidência definitiva, uma
vez que não se sabe se é apropriado combinar os dados (Sullivan, 1996; Johnson
e Soltis, 1998; Soltis e Soltis, 2000). Nos estudos realizados aqui, foram utilizados
dois diferentes testes para verificar o grau de congruência entre as matrizes
correspondentes aos marcadores nucleares e plastidiais (PHT (partition
homogeneity test ou incongruence lenght test – ILD) e PBS (partition Bremer
suport)). Observou-se que as diferenças nas escalas taxonômicas, tratadas nos
dois artigos, afetaram diretamente o resultado dos testes de congruência entre as
seqüências utilizadas. Em ambos os artigos oi verificada uma significante
incongruência entre os marcadores plastidiais e nucleares quando utilizado o PHT.
Entretanto, estes resultados só foram corroborados com os de PBS no artigo 1,
onde foram incluídas na análise várias espécies oriundas de outras seções do
subgênero Leptostemonum de Solanum. A incongruência observada em ambos os
testes (artigo 1) pode ter sido ocasionada pela falta de homogeneidade nas
seqüências entre os táxons, devido ao grande número de mutações
(deleções/inserções), e pelas altas taxas de homoplasias apresentadas pela
região de ITS. Entretanto, no artigo 2, o teste de PBS não corroborou com os
resultados de PHT, mostrando uma congruência entre as regiões de cloroplasto e
nucleares, com os índices de PBS não mostrando discrepâncias entre eles para
construção dos clados na análise combinada. No artigo 2 foram utilizados apenas
representantes da seção Torva de Solanum no grupo interno, os quais
apresentaram uma homogeneidade nas seqüências, com poucas mutações, e
ambos marcadores apresentaram taxas de homoplasias muito baixas, o que leva
a dar mais credibilidade ao observado com o teste de PBS.
No artigo 2, apesar de ITS mostrar uma das amostras de S. adspersum fora
do grupo formado pelos demais indivíduos dessa espécie, esta variação não
ultrapassou a barreira específica, e portanto não invalida seu uso como marcador
filogenético, uma vez que a maioria dos indivíduos de uma mesma espécie
125
formam clados com alto suporte. A incongruência observada pelo PHT pode ser
igualmente explicada pelo tamanho das partições das regiões de cloroplasto (1848
pb) e nuclear (691pb), como discutido no artigo 2, uma vez que este tipo de teste
pode ser sensível quando os conjuntos de dados diferem muito em relação ao
tamanho (Dowton e Austin, 2002).
Heterogeneidades significantes entre os grupos de dados podem impedir
sua subseqüente combinação; porém, tem sido mostrado que o PHT é
especialmente sensível a mutações silenciosas e diferentes taxas de mutação
entre os grupos de dados, e isto pode representar uma medida tendenciosa e
incorreta de congruência (Barker e Lutzoni, 2002; Darlu e Lecointre, 2002; Dolphin
e cols., 2000). Portanto, em nosso trabalho optamos em realizar análises
separadas e combinadas, e compararmos os resultados das topologias das
árvores filogenéticas. Isto também foi realizado no trabalho de Neves e cols.
(2005) em que eles observaram uma contrastante taxa evolutiva entre as regiões
de ITS e trnT-trnF, optando em combinar os dados mesmo com a incongruência
observada entre as partições, por PHT.
A técnica de ISSR mostrou-se útil para avaliar o grau de divergência
genética entre as espécies da seção Torva de Solanum. Com o uso desses
marcadores foi possível detectar grupos de espécies mais relacionadas nas
análises de distância e de similaridade, em adição aos outros marcadores
utilizados neste trabalho. Os quatro “primers” utilizados para ISSR se mostraram
polimórficos e filogeneticamente informativos para os 22 acessos analisados,
apesar do alto índice de homoplasias apresentado para este tipo de marcador. O
aumento no número de “primers” e de acessos possivelmente melhoraria a
resolução no dendograma, agrupando as espécies da seção Torva em um único
grupo, afastando a hipótese de agrupamento por distribuição geográfica,
levantada no artigo 2. Entretanto, para ISSR o número de “primers” utilizado para
análises não necessariamente necessita ser elevado. Gilbert e cols. (1999) utilizou
apenas dois “primers” para distinguir 37 acessos estudados. Similarmente, quatro
“primers” foram suficientes para distinguir 34 cultivares de batata (Prevost e
126
Wilkinson, 1999) e três “primers” puderam distinguir 16 genótipos de Ribes rubrum
(Lanham e Brennan, 1998).
Entretanto, o potencial oferecido pelos marcadores ISSR depende da
variedade e freqüência dos microssatélites, variando com as espécies e com os
motivos SSR que são utilizados como “primer” (Morgante e Olivieri, 1993;
Depeiges e cols., 1995). Sendo o “primer” um SSR, a freqüência e distribuição dos
microssatélites em diferentes espécies também influenciam a geração de bandas.
Na análise de ISSR, a espécie S. adspersum foi a que apresentou o maior número
de bandas, mostrando-se a mais distante entre as espécies da seção Torva, fato
observado nas análises de similaridade e das coordenadas principais. Isto se
deve, provavelmente, aos “primers” utilizados nesta análise e à freqüência de
microssatélites, apresentada por esta espécie. É provável que um maior número
de “primers” oferecesse uma informação adicional, uma vez que poderia atingir
com maior abrangência o genoma das demais espécies da seção Torva de
Solanum.
6.- Conclusões e Perspectivas
Os estudos realizados nesse trabalho evidenciaram as relações de
parentesco entre as espécies da seção Torva de Solanum e da seção Torva com
outras espécies do subgênero Leptostemonum. Tais dados certamente auxiliarão
em trabalhos futuros a serem realizados com outras espécies pertencentes a esta
seção, sobre a história evolutiva das espécies da seção Torva.
A monofilia da seção Torva evidenciada certamente não pode ser estendida
a toda seção, uma vez que utilizamos apenas amostras presentes principalmente
na região sul do Brasil e uma amostra típica da Argentina e Uruguai (S.
bonariense). Para a confirmação da monofilia da seção Torva seria necessária a
inclusão de representantes da seção presentes em outros pontos de dispersão
pelo mundo, o que poderá vir a ser feito em trabalhos futuros.
127
O número de marcadores utilizados e os métodos de inferência filogenética
aplicados mostraram-se adequados para evidenciar o grau de parentesco entre as
espécies da seção Torva na região sul do Brasil. Entretanto, são necessários
trabalhos mais aprofundados para afirmar a existência de um híbrido natural entre
as espécies S. guaraniticum e S. paniculatum.
Uma das alternativas para trabalhos futuros que auxiliariam a elucidação
deste relacionamento seria a utilização da citogenética em conjunto com a técnica
de citometria de fluxo, as quais têm potencial para avaliar o nível de ploidia das
esp
128
quais vêm sendo amplamente realizados com outras espécies da família
Solanaceae.
Contudo, as diferentes propostas empregadas neste trabalho, certamente
contribuíram para evidenciar a história evolutiva dos representantes analisados da
seção Torva de Solanum. Além disso, auxiliarão pesquisas futuras sobre estes
importantes recursos naturais.
129
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141
8. – Anexos
Excluído: ¶
¶
¶
¶
¶
¶
142
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S.
p
aniculatum X S.
g
uaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. paniculatum
S. paniculatum
S. paniculatum
S. variabile
S. variabile
S. variabile
S. variabile
S. torvum
S. melongena
S. jamaicense
S. sisymbriifolium
S. sisymbriifolium
S. agrarium
S. stenandrum
S. robustum
S. stagnale
S. atropurpureum
S. atropurpureum
S. tenuispinum
S. vaillantii
S. acerifolium
S. capsicoides
S. aculeatissimum
S. aculeatissimum
S. aculeatissimum
S. viarum
S. viarum
S. aculeatissimum
S. myriacanthum
S. incarceratum
S. platense
S. mammosum
S. palinacanthum
S. candidum
S. hirtum
S. pseudolulo
S. hyporhodium
S. quitoense
S. lasiocarpum
S. repandum
S. sessiliflorum
S. stramonifolium
S. vestissimum
S. aviculare
S. luteoalbum
S. wendlandii
S. pseudocapsicum
S. abutiloides
S. concinnum
100
87
99
98
99
94
71
99
99
59
73
99
99
99
97
99
75
99
86
77
90
98
99
99
73
100
88
99
91
94
99
59
87
Anexo 1: Árvore filogenética da análise Bayesiana da matriz de ITS (Artigo1).
143
S.
p
aniculatum X S.
g
uaraniticum
S. paniculatum X S. guaraniticum
S.
g
uaraniticum
S. guaraniticum
S. paniculatum X S. guaraniticum
S. guaraniticum
S.
g
uaraniticum
S. guaraniticum
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. paniculatum
S. paniculatum
S. paniculatum
S. variabile
S. variabile
S. variabile
S. variabile
S. torvum
S. melongena
S. jamaicense
S. agrarium
S. stenandrum
S. sisymbriifolium
S. sisymbriifolium
S. atropurpureum
S. atropurpureum
S. vaillantii
S. acerifolium
S. tenuispinum
S. capsicoides
S. aculeatissimum
S. aculeatissimum
S. aculeatissimum
S. viarum
S. viarum
S. myriacanthum
S. aculeatissimumi
S. incarceratum
S.
p
latense
S. mammosum
S. palinacanthum
S. candidum
S. hirtum
S. pseudolulo
S. lasiocarpum
S. repandum
S. hyporhodium
S. quitoense
S. stramonifolium
S. sessiliflorum
S. vestissimum
S. robustum
S. stagnale
S. luteoalbum
S. wendlandii
S. pseudocapsicum
S. abutiloides
S. concinnum
S. aviculare
0.005 substitutions/site
Anexo 2: Árvore filogenética da análise de distância da matriz de ITS (Artigo 1).
144
S. paniculatum X S. guaraniticum
S.
paniculatum
S.
paniculatum
S.
p
aniculatum X S.
g
uaraniticum
S. variabile
S. variabile
S. variabile
S. paniculatum
S.
p
aniculatum
S.
p
aniculatum X S.
g
uaraniticum
S. variabile
S.
p
aniculatum X S.
g
uaraniticum
S. guaraniticum
S. guaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S.
gua
r
aniticum
S. guaraniticum
S. guaraniticum
Storvum
S melongena
S.
j
amaicense
S. sis
y
mbrii
f
olium
S.
at
r
opu
r
pu
r
eum
S. atro
p
ur
p
ureum
S. atro
p
ur
p
ureum
S. atropurpureum
S. acerifolium
S. vaillantii
S. tenuispinum
S. aculeatissimum
S. aculeatissimum
S.
vaillantii
S. aculeatissimum
S. viarum
S. viarum
S. aculeatissimum
S. aculeatissimumi
S. myriacanthum
S. platense
S. incarceratum
S. capsicoides
S. palinacanthum
S. candidum
S. pseudolulo
S. quitoense
S. stramonifolium
S. agrarium
S. stenandrum
S. robustum
S. stagnale
S. mammosum
S. wendlandii
S. aviculare
S. luteoalbum
S. pseudocapsicum
S. abutiloides
Anexo 3: Árvore consenso da maioria da análise de parcimônia da matriz de trnS-
trnG (Artigo1).
145
Anexo 4: Árvore filogenética da análise Bayesiana da matriz de trnS-trnG
(Artigo1).
S. atropurpureum
S. atropurpureum
S. atropurpureum
S. atropurpureum
S. vaillantii
S. vaillantii
S. aceri
f
olium
S. tenuispinum
S. ca
p
sicoides
S. incarceratum
S. aculeatissimum
S. viarum
S. aculeatissimum
S. viarum
S. m
y
riacanthum
S. aculeatissimum
S. aculeatissimum
S. aculeatissimum
S.
p
latense
S. mammosum
S.
p
alinacanthum
S.
q
uitoense
S. stramonifolium
S. candidum
S. pseudolulo
S. agrarium
S. stenandrum
S. robustum
S. sta
g
nale
S. paniculatum X S. guaraniticum
S.
p
aniculatum
S. paniculatum X S. guaraniticum
S.
p
aniculatum
S.
p
aniculatum X S.
g
uaraniticum
S.
p
aniculatum
S. variabile
S. variabile
S. variabile
S. variabile
S.
p
aniculatum
S. melongena
S. guaraniticum
S.
g
uaraniticum
S. guaraniticum
S. paniculatum X S. guaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S. guaraniticum
S. guaraniticum
S. torvum
S. jamaicense
S. sis
y
mbrii
f
olium
S. wendlandii
S. luteoalbum
S. aviculare
S. pseudocapsicum
S. abutiloides
146
S.
p
aniculatum X S.
g
uaraniticum
S. paniculatum
S. variabile
S. variabile
S. variabile
S.
p
aniculatum X S.
g
uaraniticum
S.
p
aniculatum X S.
g
uaraniticum
S. paniculatum
S. paniculatum
S.
p
aniculatum
S. melongena
S. paniculatum X S. guaraniticum
S. guaraniticum
S.
g
uaraniticum
S. guaraniticum
S.
g
uaraniticum
S. guaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S. variabile
S. torvum
S. jamaicense
S. sisymbriifolium
S. atro
p
ur
p
ureum
S. atro
p
ur
p
ureum
S. atro
p
ur
p
ureum
S. atro
p
ur
p
ureum
S. vaillantii
S. aceri
f
olium
S.
tenuispinum
S. vaillantii
S. aculeatissimum
S. aculeatissimum
S. viarum
S. viarum
S. aculeatissimum
S. aculeatissimum
S. aculeatissimum
S. m
y
riacanthum
S.
inca
r
ce
r
atum
S. ca
p
sicoides
S. platense
S. mammosum
S. palinacanthum
S. agrarium
S. stenandrum
S. candidum
S. quitoense
S. stramonifolium
S.
p
seudolulo
S. robustum
S. stagnale
S.
wendlandii
S. abutiloides
S.
p
seudoca
p
sicum
S. aviculare
S. luteoalbum
0.001 substitutions/site
Anexo 5: Árvore filogenética da análise de distância (NJ) da matriz de trnS-trnG
(Artigo 1).
147
S. atro
p
ur
p
ureum
S. atro
p
u
r
p
ureum
S. atro
p
ur
p
ureum
S. atro
p
ur
p
ureum
S. atro
p
ur
p
ureum
S. atropurpureum
S. vaillantii
S. tenuispinum
S. acerifolium
S. incarceratum
S. aculeatissimum
S. myriacanthum
S. viarum
S. viarum
S. aculeatissimum
S. aculeatissimum
S. aculeatissimum
S. aculeatissimum
S. aculeatissimum
S. aculeatissimum
S. ca
p
sicoides
S.
p
latense
S. mammosum
S. palinacanthum
S. felinum
S. pseudolulo
S. hyporhodium
S. candidum
S. vestissimum
S. repandum
S. quitoense
S. lasiocarpum
S. pectinatum
S. stramonifolium
S. sessiliflorum
S. hirtum
S. ste
nandrum
S.
g
uaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S.
p
aniculatum X S.
g
uaraniticum
S. paniculatum X S. guaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S. variabile
S. variabile
S. variabile
S. variabile
S.
p
aniculatum X S.
g
uaraniticum
S.
p
aniculatum
S.
p
aniculatum
S.
p
aniculatum
S.
p
aniculatum
S.
p
aniculatum X S.
g
uaraniticum
S.
p
aniculatum
S. paniculatum X S. guaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S. torvum
S. melongena
S. sis
y
mbrii
f
olium
S. sisymbriifolium
S. jamaicense
S. stagnale
S. robustum
S. luteoalbum
S. pseudocapsicum
S. wendlandii
S. aviculare
S. dulcamara
S. mauritianum
S. abutiloides
Anexo 6: Árvore consenso da maioria da análise de parcimônia da matriz de trnL –
trnF + íntron trnL (Artigo1).
148
Anexo 7: Árvore filogenética da análise Bayesiana da matriz de trnL– trnF + íntron
trnL (Artigo1).
69
S. paniculatum X S. guaraniticum
S. paniculatum
S. paniculatum X S. guaraniticum
S.
p
aniculatum X S.
g
uaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S.
p
aniculatum X S.
g
uaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. variabile
S. variabile
S. variabile
S. variabile
S. paniculatum X S. guaraniticum
S. paniculatum
S. paniculatum
S. paniculatum
S. paniculatum
S. torvum
S. melongena
S. jamaicense
S. atropurpureum
S. atropurpureum
S. atropurpureum
S. atropurpureum
S. vaillantii
S. atropurpureum
S. atropurpureum
S. tenuispinum
S. acerifolium
S. incarceratum
S. capsicoides
S. aculeatissimum
S. aculeatissimum
S. viarum
S. viarum
S. aculeatissimum
S. m
y
riacanthum
S. aculeatissimum
S. aculeatissimum
S. aculeatissimum
S. aculeatissimum
S. platense
S. mammosum
S. palinacanthum
S. candidum
S. lasiocarpum
S. quitoense
S. repandum
S. vestissimum
S. felinum
S. hyporhodium
S. pseudolulo
S. pectinatum
S. sessiliflorum
S. stramonifolium
S. hirtum
S. stenandrum
S. sisymbriifolium
S. sisymbriifolium
S. stagnale
S. robustum
S. wendlandii
S. pseudocapsicum
S. luteoalbum
S. aviculare
S. dulcamara
S. mauritianum
S. abutiloides
100
95
73
71
99
99
68
100
100
79
99
99
98
54
99
86
84
92
100
68
98
100
80
93
97
53
100
99
74
99
66
95
82
66
100
99
100
51
99
88
149
Anexo 8: Árvore filogenética da análise de distância (NJ) da matriz de trnL–trnF +
íntron trnL (Artigo1).
S.
p
aniculatum X S.
g
uaraniticum
S. paniculatum
S. paniculatum
S. paniculatum
S. paniculatum X S. guaraniticum
S.
p
aniculatum X S.
g
uaraniticum
S. paniculatum
S. paniculatum
S. variabile
S. variabile
S. variabile
S. variabile
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. torvum
S. melongena
S. atropurpureum
S. atro
p
ur
p
ureum
S. atro
p
ur
p
ureum
S. atropurpureum
S. aculeatissimum
S. vaillantii
S. atro
p
ur
p
ureum
S. atro
p
ur
p
ureum
S. tenuispinum
S. capsicoides
S. aculeatissimum
S. aculeatissimum
S. aculeatissimum
S. viarum
S. aculeatissimum
S. viarum
S. aculeatissimum
S. aculeatissimum
S. acerifolium
S. incarceratum
S. m
y
riacanthum
S. platense
S. mammosum
S. palinacanthum
S. candidum
S. vestissimum
S. lasiocarpum
S. repandum
S. quitoense
S. felinum
S. pseudolulo
S. hyporhodium
S. pectinatum
S. stramonifolium
S. sessiliflorum
S. hirtum
S. stenandrum
S. sisymbriifolium
S. sis
y
mbrii
f
olium
S. stagnale
S. robustum
S. wendlandii
S. jamaicense
S. abutiloides
S. aviculare
S. dulcamara
S. pseudocapsicum
S. luteoalbum
S. mauritianum
0.001
150
Anexo 9: Árvore filogenética da análise Bayesiana da matriz combinada de
cloroplastos (Artigo 1).
S. paniculatum X S. guaraniticum
S. paniculatum
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. paniculatum
S. paniculatum
S. paniculatum
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. variabile
S. variabile
S. variabile
S. variabile
S. torvum
S. melongena
S. jamaicense
S. sisymbriifolium
S. atropurpureum
S. atropurpureum
S. vaillantii
S. atropurpureum
S. atropurpureum
S. tenuispinum
S. acerifolium
S. incarceratum
S. capsicoides
S. aculeatissimum
S. viarum
S. aculeatissimum
S. viarum
S. aculeatissimum
S. myriacanthum
S. aculeatissimum
S. aculeatissimum
S. platense
S. mammosum
S. palinacanthum
S. candidum
S. quitoense
S. pseudolulo
S. stramonifolium
S. stenandrum
S. stagnale
S. robustum
S. wendlandii
S. abutiloides
S. aviculare
S. luteoalbum
S. pseudocapsicum
99
99
60
53
91
99
99
96
55
62
82
99
99
97
99
90
78
99
99
59
99
99
94
95
95
53
98
98
98
58
100
79
73
98
100
93
63
57
90
151
Anexo 10: Árvore filogenética da análise de distância da matriz combinada de
cloroplastos (Artigo 1).
S. paniculatum X S. guaraniticum
S. paniculatum
S. paniculatum
S. paniculatum
S. paniculatum X S. guaraniticum
S. paniculatum
S. variabile
S. variabile
S. variabile
S. variabile
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. torvum
S. atropurpureum
S. atropurpureum
S. aculeatissimum
S. vaillantii
S. atro
p
ur
p
ureum
S. atropurpureum
S. tenuispinum
S. aceri
f
olium
S. incarceratum
S. ca
p
sicoides
S. aculeatissimum
S. viarum
S. aculeatissimum
S. viarum
S. aculeatissimum
S. aculeatissimum
S. m
y
riacanthum
S. platense
S. mammosum
S.
p
alinacanthum
S. candidum
S. pseudolulo
S. quitoense
S. stramonifolium
S. stenandrum
S. sisymbriifolium
S. stagnale
S. melongena
S. robustum
S. jamaicense
S. wendlandii
S. abutiloides
S. aviculare
S. pseudocapsicum
S. luteoalbum
0.001
NJ
152
Anexo 11: Árvore filogenética da análise Bayesiana da matriz combinada dos
quatro fragmentos (Artigo 1).
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. paniculatum
S. paniculatum
S. variabile
S. variabile
S. variabile
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. torvum
S. melongena
S. jamaicense
S. sisymbriifolium
S. robustum
S. stagnale
S. stenandrum
S. atropurpureum
S. atropurpureum
S. tenuispinum
S. vaillantii
S. acerifolium
S. capsicoides
S. aculeatissimum
S. viarum
S. viarum
S. aculeatissimum
S. myriacanthum
S. aculeatissimum
S. incarceratum
S. platense
S. mammosum
S. palinacanthum
S. candidum
S. pseudolulo
S. quitoense
S. stramonifolium
S. wendlandii
S. luteoalbum
S. aviculare
S. abutiloides
S. pseudocapsicum
54
83
99
100
99
60
100
100
73
100
100
99
87
100
100
100
92
100
87
83
83
100
100
100
100
100
100
100
85
93
100
98
99
93
100
76
87
96
100
98
153
Anexo 12: Árvore filogenética da análise de distância da matriz combinada dos
quatro fragmentos (Artigo 1).
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. paniculatum
S. paniculatum
S. variabile
S. variabile
S. variabile
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. paniculatum X S. guaraniticum
S. torvum
S. jamaicense
S. melongena
S. sisymbriifolium
S. stenandrum
S. robustum
S. stagnale
S. atropurpureum
S. atropurpureum
S. acerifolium
S. tenuispinum
S. vaillantii
S. capsicoides
S. aculeatissimum
S. viarum
S. viarum
S. aculeatissimum
S. aculeatissimum
S. myriacanthum
S. incarceratum
S. platense
S. mammosum
S. palinacanthum
S. candidum
S. pseudolulo
S. quitoense
S. stramonifolium
S. wendlandii
S. abutiloides
S. aviculare
S. pseudocapsicum
S. luteoalbum
0.005
154
Anexo 13: Uma das quatro árvores da análise de parcimônia obtida com a matriz
de dados de ISSR (Artigo 2).
S paniculatum X S. guaraniticum
S. paniculatum
S. guaraniticum
S. variabile
S. bonariense
S. adspersum
S. adspersum
S. adspersum
S. tabacifolium
S. viarum
S. aculeatissimum
S. atropurpureum
5 changes
S paniculatum X S. guaraniticum
S. guaraniticum
S. paniculatum
155
Anexo 14: Árvore consenso da maioria da análise de parcimônia obtida com a
matriz de dados de ISSR (Artigo 2).
S. paniculatum X S. guaraniticum
S. paniculatum
S. guaraniticum
S. guaraniticum
S. paniculatum
S. paniculatum
S. guaraniticum
S. bonariense
S. adspersum
S. adspersum
S. adspersum
S. tabacifolium
S. atropurpureum
S. paniculatum X S. guaraniticum
S. variabile
S. variabile
S. variabile
S. guaraniticum
S. guaraniticum
S. viarum
S. aculeatissimum
100
50
50
50
50
100
100
75
100
100
100
50
100
100
100
100
100
100
S. paniculatum X S. guaraniticum
156
Anexo 15: Árvore filogenética da análise de máxima verossimilhança da matriz de
ITS (Artigo 2).
0.01
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S.
g
uaraniticum
S. guaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S.
g
uaraniticum
S. bonariense
S. paniculatum X S. guaraniticum
S. variabile
S. variabile
S. variabile
S. variabile
S.
p
aniculatum
S. paniculatum
S.
p
aniculatum
S. adspersum
S. tabaci
f
olium
S. adspersum
S. guaraniticum
S. torvum
S. ads
p
ersum
S. atropurpureum
S. vaillantii
S. aculeatissimum
S. viarum
S. sis
y
mbrii
f
olium
S. sisymbriifolium
S. robustum
S. stagnale
S. melongena
S.
j
amaicense
157
Anexo 16: Árvore filogenética da análise Bayesiana da matriz de ITS (Artigo 2).
S. guaraniticum
S. guaraniticum
67
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. bonariense
S. guaraniticum
61
S. paniculatum X S. guaraniticum
59
S. variabile
S. variabile
97
S. variabile
S. variabile
100
S. paniculatum
S. paniculatum
S. paniculatum
100
89
S. adspersum
S. tabacifolium
78
S. adspersum
100
S. guaraniticum
57
100
S. torvum
S. adspersum
99
100
S. melongena
54
S. atropurpureum
S. vaillantii
100
S. aculeatissimum
S. viarum
95
100
S. robustum
S. stagnale
99
59
S. sisymbriifolium
S. sisymbriifolium
100
99
S. jamaicense
158
Anexo 17: Árvore consenso da maioria da análise de parcimônia da matriz de ITS
com os índels tratados como quinto estado de caractere (Artigo 2).
S. aculeatissimum
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. paniculatum X S. guaraniticum
S. adspersum
S. adspersum
S. tabacifolium
S. bonariense
S. paniculatum
S. paniculatum
S. paniculatum
S. variabile
S. variabile
S. variabile
S. variabile
S. adspersum
S. torvum
S. atropurpureum
S. vaillantii
S. viarum
S. robustum
S. stagnale
S. jamaicense
S. sisymbriifolium
S. sisymbriifolium
S. melongena
10 changes
159
Anexo 18: Árvore consenso da maioria da análise de parcimônia da matriz de ITS
com os índels tratados como dados faltantes (Artigo 2).
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. guaraniticum
S. bonariense
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. variabile
S. variabile
S. variabile
S. variabile
S. adspersum
S. adspersum
S. tabacifolium
S. paniculatum
S. paniculatum
S. paniculatum
S. torvum
S. adspersum
S. atropurpureum
S. vaillantii
S. aculeatissimum
S. viarum
S. sisymbriifolium
S. sisymbriifolium
S. robustum
S. stagnale
S. jamaicense
S. melongena
100
100
100
100
100
100
100
100
100
100
74
74
100
100
100
100
100
100
100
100
100
100
100
100
100
100
160
Anexo 19: Árvore filogenética da análise de distância da matriz de ITS (Artigo 2).
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. bonariense
S. guaraniticum
S. paniculatum
S. paniculatum
S. paniculatum
S. variabile
S. variabile
S. variabile
S. variabile
S. adspersum
S. tabacifolium
S. adspersum
S. torvum
S. adspersum
S. atropurpureum
S. vaillantii
S. aculeatissimum
S. viarum
S. robustum
S. stagnale
S. sisymbriifolium
S. sisymbriifolium
S. melongena
S. jamaicense
0.005
161
Anexo 20: Árvore consenso da maioria da análise de máxima verossimilhança da
matriz de cloroplasto (Artigo 2).
S. paniculatum X S. guaraniticum
S. paniculatum
S. paniculatum
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. paniculatum
S. paniculatum
S. variabile
S. variabile
S. variabile
S. variabile
S. adspersum
S. adspersum
S. adspersum
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. bonariense
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. tabacifolium
S. torvum
S. atropurpureum
S. vaillantii
S. viarum
S. robustum
S. stagnale
S. sisymbriifolium
S. melongena
S. jamaicense
100
100
100
100
100
100
100
100
100
100
100
100
100
80
60
100
100
100
100
100
100
100
100
162
Anexo 21: Árvore consenso da maioria da análise de parcimônia da matriz de
cloroplasto, com os índels tratados como quinto estado de caractere (Artigo 2).
S. paniculatum X S. guaraniticum
S. paniculatum
S. paniculatum
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. paniculatum
S. paniculatum
S. guaraniticum
S. guaraniticum
S. tabacifolium
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. bonariense
S. adspersum
S. adspersum
S. adspersum
S. variabile
S. variabile
S. variabile
S. variabile
S. torvum
S. atropurpureum
S. vaillantii
S. viarum
S. melongena
S. robustum
S. stagnale
S. sisymbriifolium
S. jamaicense
100
100
100
75
75
75
100
60
60
100
100
100
100
100
100
100
100
88
62
100
100
100
100
100
100
100
100
163
Anexo 22: Árvore consenso da maioria da análise de parcimônia da matriz de
cloroplasto, com índels tratados como dados faltantes (Artigo 2).
1
S. paniculatum X S. guaraniticum
S. paniculatum
S. paniculatum X S. guaraniticum
S. paniculatum
S. paniculatum X S. guaraniticum
S. paniculatum
S. paniculatum
S. variabile
S. variabile
S. variabile
S. variabile
S. adspersum
S. ads
p
ersum
S. adspersum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S.
p
aniculatum X S.
g
uaraniticum
S. guaraniticum
S. guaraniticum
S. bonariense
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. tabacifolium
S. torvum
S. atropurpureum
S. vaillantii
S. viarum
S. robustum
S. stagnale
S. sis
y
mbrii
f
olium
S. melogena
S.
j
amaicense
164
Anexo 23: Árvore filogenética da análise de distância obtida com matriz de
cloroplasto (Artigo 2).
0.01
S.
g
uaraniticum
S. bonariense
S. guaraniticum
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S.
g
uaraniticum
S. variabile
S. tabacifolium
S. ads
p
ersum
S. adspersum
S. adspersum
S. variabile
S. variabile
S. variabile
S.
p
aniculatum
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. paniculatum
S.
p
aniculatum
S.
p
aniculatum
S.
p
aniculatum X S.
g
uaraniticum
S. torvum
S. jamaicense
S. atropurpureum
S. vaillantii
S. viarum
S. robustum
S. stagnale
S. sisymbriifolium
S. melon
g
ena
165
Anexo 24: Árvore filogenética da análise Bayesiana da matriz combinada. (Artigo
2).
S. variabile
S. variabile
100
S. variabile
85
S. variabile
100
S. paniculatum
S. paniculatum
S. paniculatum
100
83
S. adspersum
S. tabacifolium
52
S. adspersum
100
S. adspersum
68
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
89
S. guaraniticum
S. guaraniticum
83
S. guaraniticum
S. guaraniticum
85
S. bonariense
S. guaraniticum
93
S. paniculatum X S. guaraniticum
100
100
S. torvum
100
S. jamaicense
S. atropurpureum
S. vaillantii
100
S. viarum
100
S. robustum
S. stagnale
100
100
S. sisymbriifolium
100
S. melongena
166
Anexo 25: Árvore filogenética da análise de máxima verossimilhança da matriz
combinada (Artigo 2).
0.01
S. variabile
S. variabile
S. variabile
S. variabile
S.
p
aniculatum
S. paniculatum
S. paniculatum
S. adspersum
S. tabacifolium
S. adspersum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. bonariense
S. paniculatum X S. guaraniticum
S.
p
aniculatum X S.
g
uaraniticum
S. paniculatum X S. guaraniticum
S. adspersum
S. torvum
S. atropurpureum
S. vaillantii
S. viarum
S. robustum
S. stagnale
S. sisymbriifolium
S. melon
g
ena
S.
j
amaicense
167
Anexo 26: Árvore consenso da maioria da análise de parcimônia da matriz
combinada, com índels tratados como dados faltantes (Artigo 2).
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S.
p
aniculatum
S. paniculatum
S. paniculatum
S.
p
aniculatum X S.
g
uaraniticum
S.
g
uaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. bonariense
S. variabile
S. variabile
S. variabile
S. variabile
S. adspersum
S. adspersum
S. tabacifolium
S. ads
p
ersum
S. torvum
S. atropurpureum
S. vaillantii
S. viarum
S. robustum
S. stagnale
S. sisymbriifolium
S. melongena
S. jamaicense
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
168
Anexo 27: Árvore filogenética da análise de distância da matriz combinada (Artigo
2).
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. paniculatum X S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. guaraniticum
S. bonariense
S. guaraniticum
S. variabile
S. variabile
S. variabile
S. variabile
S. adspersum
S. adspersum
S. tabaci
f
olium
S. adspersum
S. paniculatum
S. paniculatum
S. paniculatum
S. torvum
S. atropurpureum
S. vaillantii
S. viarum
S. robustum
S. stagnale
S. sisymbriifolium
S. melongena
S. jamaicense
0.005 substitutions/site
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