Jasione L. (Campanulaceae). Biogeographic history, evolutionary relationships, species delimitation, and nomenclatural revision

TESE DE DOUTORAMENTO JASIONE L. (CAMPANULACEAE). BIOGEOGRAPHIC HISTORY, EVOLUTIONARY RELATIONSHIPS, SPECIES DELIMITATION, AND NOMENCLATURAL REVISION Luis Miguel Serrano Pérez ESCOLA DE DOUTORAMENTO INTERNACIONAL DA UNIVERSIDADE DE SANTIAGO DE COMPOSTELA PROGRAMA DE DOUTORAMENTO EN BIODIVERSIDADE E CONSERVACIÓN DO MEDIO NATURAL SANTIAGO DE COMPOSTELA 2021 DECLARACIÓN DO AUTOR/A DA TESE D.. Luis Miguel Serrano Pérez Título da tese: Jasione L. (Campanulaceae). Biogeographic history, evolutionary relationships, species delimitation, and nomenclatural revision Presento a miña tese, seguindo o procedemento axeitado ao Regulamento, e declaro que: 1) A tese abarca os resultados da elaboración do meu traballo. 2) De ser o caso, na tese faise referencia ás colaboracións que tivo este traballo. 3) Confirmo que a tese non incorre en ningún tipo de plaxio doutros autores nin de traballos presentados por min para a obtención doutros títulos. 4) A tese é a versión definitiva presentada para a súa defensa e coincide a versión impresa coa presentada en formato electrónico E comprométome a presentar o Compromiso Documental de Supervisión no caso de que o orixinal non estea na Escola. En Santiago de Compostela, 30 de Xullo de 2021 Sinatura electrónica AUTORIZACIÓN DO DIRECTOR/TITOR DA TESE D./Dna. Santiago Ortiz Núñez En condición de: Titor/a e director/a Título da tese: Jasione L. (Campanulaceae). Biogeographic history, evolutionary relationships, species delimitation, and nomenclatural revision INFORMA: Que a presente tese, correspóndese co traballo realizado por D. Luis Miguel Serrano Pérez, baixo a miña dirección/titorización, e a utorizo a súa presentación , considerando que reúne os r equisitos esixidos no R egulamento de Estudos de Doutoramento da USC, e que como director/titor desta non incorre nas causas de abstención establecidas na Lei 40/2015. De acordo co indicado no Regulamento de Estudos de Doutoramento, declara tamén que a presente tese de doutoramento é idónea para ser defendida en base á modalidade de Monográfica con reprodución de publicacións, nos que a participación do doutorando foi decisiva para a súa elaboración e as publicacións se axustan ao Plan de Investigación. En Santiago de Compostela, 30 de Xullo de 2021 Sinatura electrónica 7 This thesis includes the following manuscripts in chapters 6 and 7: Serrano, M., Pereña Ortiz, J.; Becerra Parra, M. 2009. Presencia y estado de conservación de Jasione corymbosa Poir. Ex. Schult. (Campanulaceae) en la Península Ibérica. Acta Botanica Malacitana 34: 284-287. Article used for chapter 6. Contribution of the current author: Conceptualization, molecular and morphological analisis, assessment of conservation status, writing—original draft preparation, and writing—review and editing. Acta Botanica Malacitana, peer-reviewed, open access journal The article is licensed under an open access Creative Commons CC BY 4.0 license. Authorisation link: https://revistas.uma.es/index.php/abm/about/submissions#authorGuidelin es Indexed in DIALNET. Total number of times cited: 3 Sanmartín, P., Gambino, M., Fuentes, E., Serrano, M. 2020. A simple, reliable and inexpensive solution for contact color measurement in small plant samples. Sensors 20(8): 2348. Article used for chapter 7. Contribution of the current author: Conceptualization, methodology, validation, formal analysis, statistical analysis, data interpretation, writing— original draft preparation, and writing—review and editing. Sensors, peer-reviewed, open access journal The article is licensed under an open access Creative Commons CC BY 4.0 license. Authorisation link: https://www.mdpi.com/ethics#10 JCR index (IF) 2020 = 3.576, 14/64 (Percentile: 79, Q1) in Instruments & Instrumentation. CiteScore (2020 Scopus data): 5.8, 13/128 (Percentile: 90, D1/Q1) in Instrumentation. Total number of times cited: 4 LUIS MIGUEL SERRANO PÉREZ 8 This thesis reproduces some images not taken by the author. These images are found in the Introduction section and Chapter1. Documents supporting authorization of use (e.g., emails from the photographs’ authors) can be found in a shared online folder in the URL link: https://drive.google.com/drive/folders/114ac9eG2MvrZ69S57V2gsN7eRpNex1u?usp=sharing Introduction: Quini Escalona, figure I5 images’ author, authorizes its use in the dissertation. Chapter 1: The following paragraph was included in Chapter 1 after the acknowledgements section: The images used in this chapter were either taken specifically for this work by the current author’s collaborator Lyuboslava Dimitrova (figures 1.1 and 1.8), or downloaded from JSTOR database for reproduction in this dissertation, a permitted use for authorized users as the University of Santiago de Compostela (figures 1.2, 1.4,1.6,1.7,1.9,1.11,1.12, 1.13,1. 14, 1.15 and 1.16), or taken under request by the staff of the Conservatoire et Jardin botaniques de la Ville de Genève (figure 1.3), and Museo di Storia Naturale, Università degli Studi di Firenze (figure 1.10), and reproduced with permission. Reproduction policy of JSTOR images for authorized users includes “dissertations, including reproductions of the dissertations” and can be found in https://about.jstor.org/terms/#content-use. The University of Santiago the Compostela is an authorized user since 2012 thanks to an agreement regarding the JSTOR Plants Database, that can be found here: https://www.usc.gal/hsant/doc/2012_GPI_agreements.pdf Emails with image use permission from Lyuboslava Dimitrova (Sofia University), Lauren Loze (Conservatoire et Jardin botaniques de la Ville de Genève) and Anna Donatelli (Università degli Studi di Firenze) can also be found in the shared folder. 9 TABLE OF CONTENTS Resumo .............................................................................................. 17 Resumen ............................................................................................ 27 Summary ........................................................................................... 37 General Introduction: General description of the genus Jasione ............................................................................................... 47 1.The genus Jasione L. ................................................................... 48 1.1 Systematic position ............................................................... 48 1.2. General morphology of Jasione .......................................... 50 1.3. Anatomy .............................................................................. 52 1.4. Chromosome number and ploidy level ................................ 58 1.5. Internal systematics of Jasione ............................................ 59 General Objectives ........................................................................... 63 General Methods .............................................................................. 65 m.1. Methods for typification and taxonomic revision (chapter 1) .................................................................................. 65 m.2. Methods for DNA extraction and amplification (chapters 2, 3, 4 and 5) ............................................................... 66 m.3. Methods for ploidy assessment by chromosome counting (chapters 2, 3, 4 and 5) ................................................ 67 m.4. Methods for ploidy assesment by flow cytometry in silica gel dried material (chapter 2) ............................................ 68 m.5. Methods for species delimitation with ASAP and GMYC (chapter 2) ..................................................................... 69 m.5. Methods for morphological and phenological analyses to support species delimitation (chapter 2) ................................ 73 17 Resumo Esta tese doutoral ten como obxectivo revelar diferentes aspectos da realidade evolutiva do xénero Jasione L. (Campanulaceae), unha liñaxe vexetal distribuída pola conca mediterránea. Leváronse a cabo análises moleculares, citolóxicos, morfolóxicos e ambientais para abordar o seguinte: (i) as relacións filoxenéticas internas en Jasione, así como a súa posición entre outros xéneros de Campanulaceae, (ii) a historia bioxeográfica dun xénero cun notable patrón bioxeográfico mediterráneo este-oeste, (iii) os procesos que conducen á especiación e adaptación a novas contornas, (iv) o estudo dos cambios de nicho ambiental asociados con cambios no nivel de ploidía, (v) os métodos utilizados para delimitar entidades crípticas evolutivamente significativas utilizando diferentes liñas de evidencia, e (vi) tipificación de grupos complexos de Jasione como un paso no establecemento dunha nova organización taxonómica do xénero baseada en relacións evolutivas, entre outros aspectos. A familia Campanulaceae é un grupo evolutivo complexo caracterizado polo conflito entre as clasificacións tradicionais e as filoxenias moleculares xeradas nas últimas décadas, conflitos que se agudizan na subfamilia Campanuloideae. Unha das principais razóns das diferenzas entre as clasificacións premoleculares e os resultados filoxenéticos modernos é a escaseza de caracteres morfolóxicos discriminatorios, o que levou a unha énfase excesiva nos tratamentos taxonómicos tradicionais a algúns trazos reprodutivos, a saber, as morfoloxías de flores e froitos. A evolución paralela dos trazos morfolóxicos aparentemente embazou a sistemática do grupo, dificultando a proposta de delimitacións evolutivas significativas entre e dentro dos xéneros. Neste contexto xeral, Jasione é un dos xéneros de Campanuloideae máis homoxéneos desde o punto de vista morfolóxico. En LUIS MIGUEL SERRANO PÉREZ 18 consecuencia, foi desconcertante con respecto á súa sistemática evolutiva. Existe unha controversia xeneralizada entre os autores en canto ao número de taxones que deben recoñecerse, con ao redor dun centenar de diferentes nomes propostos. Con todo, os tratamentos xerais actuais só recoñecen entre 12 e 16 especies, aínda que este enfoque sintético considerouse demasiado simplista e resulta da dificultade do xénero. En consecuencia, reivindicouse a necesidade dun tratamento moderno con perspectiva evolutiva. O obxectivo desta tese doutoral é lanzar luz sobre a realidade evolutiva e a historia bioxeográfica dun xénero distribuído en Europa e na conca mediterránea, utilizando ferramentas moleculares, citolóxicas e ambientais para lograr este obxectivo. O xénero Jasione L. (Campanulaceae) distribúese pola conca mediterránea e por toda Europa, con dous centros de diversidade nos lados opostos do mar Mediterráneo, un na Península Ibérica e Marrocos e outro nas penínsulas dos Balcáns e Anatolia, en consonancia co patrón bioxeográfico de disxunción do Mediterráneo Occidental-Oriental. O xénero atópase nunha serie de condicións ecolóxicas e substratos xeolóxicos nun amplo rango de altitude, incluídos os hábitats costeiros e alpinos. No Capítulo 1 revísanse todos os nomes publicados de Jasione nas especies, subespecies e rangos de variedades para as poboacións das penínsulas dos Balcáns e Anatolia, incluídos os nomes dos taxones afíns a estes grupos nas áreas circundantes. Abórdanse o estado taxonómico e a tipificación dos nomes. En total, desígnanse once lectotipos, un lectoneotipo e un epítipo. Incluíronse os membros do complexo Jasione orbiculata e, por tanto, tamén se trata ao representante do complexo no sur de Italia. A revisión do capítulo presenta os basionimos en orde alfabética, estruturados como unha discusión de cada nome seguido dos seus sinónimos e tipificación, designando tipos cando é necesario. Proporciónase unha lista de nomes actualmente aceptados en checklists recentes e indícanse os sinónimos heterotípicos. Os grupos con estasis morfolóxica son un marco interesante para abordar o posible recoñecemento de especies crípticas escondidas detrás dos tratamentos taxonómicos tradicionais, particularmente cando os rangos de distribución suxiren patróns bioxeográficos disxuntos e Resumo 19 ambientalmente heteroxéneos. Novas hipóteses de delimitación de unidades evolutivas independentes poden conducir á identificación de diferentes patróns bioxeográficos, sentando as bases para investigar o seu significado histórico e ecolóxico. Jasione (Campanulaceae) é un xénero de plantas cunha distribución centrada na conca mediterránea caracterizada por unha estasis morfolóxica significativa. Jasione sessiliflora s.l. e taxóns afíns teñen rangos de distribución disxuntos dentro da Península Ibérica, ocupando rexións ambientalmente diversas. Sábese que no grupo ocorren polo menos dous niveis de ploidía, diploide e tetraploide. Estes aspectos motivaron a avaliación da variabilidade interna no grupo con ferramentas filoxenéticas para a delimitación de especies. Os resultados das análises GMYC e ASAP compáranse no Capítulo 2 con outras liñas de evidencia, incluíndo morfoloxía, citoloxía e fenoloxía. O axuste dos patróns de distribución das entidades inferidas ás subprovincias corolóxicas utilízase como marco bioxeográfico e ambiental para probar a hipótese das especies. A pesar da escaseza de caracteres morfolóxicos diagnósticos no grupo, a delimitación filoxenética apoia a descrición de polo menos unha especie críptica, unha endémica estreita no NE da Península Ibérica, e a separación de J. sessiliflora dun grupo de poboacións do ambiente termófilo no leste da Península. Aínda que os rangos disxuntos suxeriron a avaliación de grupos de poboación, non todas as poboacións disxuntas apuntaron a historias evolutivas independentes. As diferenzas de ploidía apoian a reordenación sistemática suxerida pola delimitación de especies. A reorganización taxonómica en J. sessiliflora s.l. permitiu interpretacións bioxeográficas dos patróns de distribución que están de acordo coa rexionalización bioxeográfica. Estes resultados suxiren que a diferenciación de especies, xunto co illamento xeográfico e a poliploidización asociouse á adaptación a diferentes ambientes, pasando de condicións máis a menos termofílicas durante a historia evolutiva do grupo J. sessiliflora. Por tanto, o recoñecemento de entidades evolutivas ocultas é fundamental para interpretar correctamente os patróns bioxeográficos en rexións cunha historia xeolóxica e evolutiva complexa como a conca mediterránea. Jasione é un xénero taxonómicamente complexo cun patrón de distribución mediterráneo disxunto este-oeste. Actualmente LUIS MIGUEL SERRANO PÉREZ 20 recoñécense ao redor de 16 especies, aínda que se propuxeron máis de cen nomes taxonómicos. Debe probarse a hipótese de que a intensa estasis morfolóxica oculta a variabilidade evolutiva relevante e dificulta a comprensión correcta das relacións evolutivas no xénero. Aínda que o xénero distribúese en Europa e ao redor da conca mediterránea, ata o de agora non se realizou unha análise filoxenético completo. Os obxectivos do Capítulo 3 consisten en probar a monofilia do xénero e determinar a súa posición entre outras liñaxes basales á tribo Campanuleae, un tema conflitivo non resolvido en diferentes estudos. A investigación tamén tivo como obxectivo establecer as relacións filoxenéticas internas mediante unha mostraxe exhaustiva que inclúe diferentes poboacións e a maioría dos nomes propostos, para evitar a exclusión a priori de liñaxes evolutivas significativas. Tamén se investigaron os roles do contacto secundario e a variación nos niveis de ploidía na configuración da historia evolutiva do xénero. Investigáronse os procesos subxacentes que conducen ao patrón de distribución disxunto do Mediterráneo este-oeste. Para abordar estas preguntas, construíuse unha filoxenia calibrada no tempo de cinco loci con 111 terminais, 93 dos cales correspondían a Jasione. O xénero resolveuse como monofilético, dentro dun clado compartido con outras dúas liñaxes illadas, Hesperocodon e Feeria. Este clado é irmán da tribo Campanuleae. A liñaxe que leva a Jasione orixinouse a finais do Oligoceno (c. 25 Mya) e as liñaxes existentes de Jasione derivan dun evento de diversificación moito máis tardío, que ocorreu en c. 9 Mya no Mioceno, dun antepasado orixinado no sur da Península Ibérica ou xa presente nas rexións do Mediterráneo occidental e oriental. Pódense identificar dous clados principais, un só presente no Mediterráneo occidental e que engloba especies con morfoloxías moi diferentes e outro co patrón de distribución disxunto existente. Un episodio de vicarianza é compatible coa apertura mesiniense tardía do estreito de Xibraltar, aínda que en ambos os clados inferíronse varios eventos posteriores de dispersión ultramarina desde a Península Ibérica ao norte de África. O segundo clado comezou a diversificarse a principios do Pleistoceno, probablemente na Península Balcánica, e polo menos dous eventos de dispersión de leste a oeste identifícanse neste clado, con modelos de nichos que identifican o conservadurismo de nicho no Resumo 21 grupo oriental e a expansión de nichos no grupo occidental. A idoneidade do hábitat non se identificou na maioría das rexións europeas intermedias. O patrón de distribución disyunto este-oeste en Jasione é un proceso bioxeográfico dinámico no que as dúas áreas albergan liñaxes que brindan a oportunidade tanto de permanecer como de dispersarse ás outras áreas, o que aumenta as posibilidades de que a liñaxe perdure. Finalmente, a filoxenia resultante non é consistente coa taxonomía actual que mostra que a estasis morfolóxica en Jasione obstaculizou a comprensión sistemática e suxire a necesidade de máis estudos taxonómicos integradores. Existe unha tendencia xeral ao conservadurismo de nicho entre taxones estreitamente relacionados, aínda que nos complexos poliploides, a diverxencia de nichos pode permitir que os poliploides se establézan fora do rango de distribución dos seus parentes diploides. A poliploidización conduce á aparición inmediata de barreiras evolutivas e especiación. Con todo, os poliploide aparecen dentro das poboacións diploides e, por tanto, están exposto para os efectos da competencia e os procesos dependentes da frecuencia que, en última instancia, conducen á exclusión reprodutiva do citotipo minoritario. A diverxencia ecolóxica permitiría aos poliploides evitar estes procesos, converténdose así nun mecanismo crave na configuración da distribución xeográfica dos citotipos nos complexos poliploides. Con todo, aínda se necesitan probas que apoien esta hipótese, xa que se informaron moitos achados contraditorios. Ademais, queda por determinar se os poliploides diverxen ecoloxicamente en maior grao que os diploides. O estudo de complexos poliploides que abarcan tanto poliploides como taxones diploides estreitamente relacionados proporciona unha oportunidade para comparar o cambio de nicho poliploide ou o conservadurismo en relación co cambio de nicho dentro dun marco evolutivo apropiado. A hipótese do cambio de nicho avalíase no Capítulo 4 nun sistema diploide-tetraploide de taxones estreitamente relacionados do xénero Jasione L. (Campanulaceae) pertencentes ao grupo Jasione crispa (Pourr.) Samp. En dous estudos anteriores, o cambio de nicho hipotético noutros grupos poliploides modelo do xénero Jasione foi rexeitado ou non foi totalmente apoiado polos resultados, e os rangos de distribución actuais deses complexos LUIS MIGUEL SERRANO PÉREZ 22 poliploides deben explicarse por outros factores. Aquí, a distribución a escala fina dos citotipos e os grupos taxonómicos significativos evolutivos determinouse mediante reconto de cromosomas, citometría de fluxo en material secado en xel de sílice e marcadores moleculares do xenoma do plastidio. Aplicáronse técnicas de modelado ambiental e desenvolvéronse modelos de distribución de especies para examinar se se produce un cambio de nicho ambiental ou conservadurismo entre tres taxóns diploides e entre estes e un taxón tetraploide presumiblemente descendente. O proceso de modelado utilizou variables climáticas e edáficas, comparando modelos de distribución de especies con e sen variables edáficas. A inclusión de variables edáficas no modelado deste complexo poliploide proporcionou unha descrición máis precisa da realidade ecoxeográfica dos taxones en estudo. Ademais, os modelos demostraron que o grupo tetraploide experimentou un cambio significativo e expansión de nicho en relación cos parentes próximos diploides, nos que se detecta conservadurismo de nicho. A expansión do nicho cara á tolerancia de ambientes máis fríos deu forma á distribución actual de J. sessiliflora tetraploide, mentres que o conservadurismo do nicho restrinxe os diploides a áreas comparativamente térmicas e subhúmedas no centro suroeste e mediterráneo da Península Ibérica. O conservadurismo de nichos diploides parece revelar certa inercia residual nos nichos ancestrais da liñaxe. En resumo, e á inversa doutros sistemas modelo de Jasione, a poliploidización probablemente causou unha innovación ambiental exitosa en Jasione sessiliflora. A variabilidade edáfica é un factor importante que promove a microevolución nas plantas, o que pode conducir á formación de novas especies a longo prazo. Jasione crispa (Campanulaceae) é un grupo composto maioritariamente por orófitos restrinxidos a solos ácidos. No Capítulo 5, a identificación dunha poboación illada e en perigo de extinción de Jasione no macizo calcáreo do Mont Caro (Tarragona, España) permite o estudo de novas presións ambientais nunha liñaxe acidófilo e os pasos iniciais do camiño de especiación. Esta poboación incluíuse na variabilidade de J. sessiliflora, unha especie tetraploide. O nivel de ploidía da poboación determínase como hexaploide, evidenciando relacións máis estreitas con Jasione crispa subsp crispa. Resumo 23 O fluxo de xenes reducido por illamento xeográfico identifícase mediante procedementos de clonación do marcador ITS do nrDNA, nun marco filoxenético e filoxeográfico que inclúe poboacións de todas as cadeas montañosas ocupadas polo taxón. A interrupción do fluxo de xenes e a exposición a novas limitacións ambientais son os principais promotores da especiación adaptativa. Para avaliar se a poboación de Mont Caro está a experimentar un proceso de especiación ecolóxica, cultiváronse plantas de Mont Caro e de dúas poboacións de zonas montañosas silíceas (Sistema Central e Pireneos) e diferentes proximidades filogenéticas a Mont Caro en tres tipos de solo. As medicións non destrutivas de cor por reflectancia representadas no sistema de cor CIELAB, utilizando os parámetros cromáticos: a* (asociado con cambios no vermello-verde) e b* (asociado con cambios no amarelo-azul), demostraron ser relevantes para avaliar estes diferentes comportamentos, e por tanto, son unha boa variable proxy para os estudos de especiación de plantas neste grupo de modelos. As concentracións de biomasa e Al, Ca, Fe e Mg estimáronse no momento de finalización do experimento. A poboación de Mont Caro mostrou mellores respostas aos solos básicos (é dicir, maior produción de biomasa, baixa concentración de Mg, equilibrio nas concentracións de Ca nas raíces e follas) que as outras dúas poboacións, aínda que peor desempeño en solos ácidos e mal manexo do aluminio a nivel das raíces, o que parece ser unha compensación evolutiva negativa. Estes resultados suxiren a ocorrencia dun proceso de especiación ecolóxica en curso en J. crispa de Mont Caro, o que podería contribuír á descrición desta poboación como un novo taxón. O Capítulo 6 baséase parcialmente nunha breve publicación froito da colaboración coa administración ambiental de Andalucía. Jasione corymbosa Poir. ex Schult., nun sentido amplo, é un polimórfico anual endémico da zona béticomauritana, cuxo hábitat preferido son as dunas e areas costeiras do sur da Península Ibérica e o Magreb, desde o oeste de Alxeria ata a costa atlántica de Marrocos. Considerouse extinto na Península Ibérica debido á alteración xeneralizada do hábitat nas costas mediterráneas, aínda que aínda se atopaban poboacións existentes nas costas atlántica e mediterránea do noroeste de África. O coñecemento do hábitat das poboacións africanas guiou unha intensa LUIS MIGUEL SERRANO PÉREZ 24 mostraxe no Mediterráneo andaluz. Finalmente detectouse unha poboación putativa en hábitats de dunas de area en Manilva, Málaga. A súa identificación como Jasione corymbosa foi controvertida entre os botánicos andaluces, e resolveuse definitivamente co uso de ferramentas moleculares, que recuperaron as plantas de Manilva aniñadas filogenéticamente entre as poboacións norteafricanas de Jasione corymbosa. Por tanto, Jasione corymbosa é unha especie da flora da Península Ibérica en grave perigo de extinción. Unha clasificación seguindo a categorización da UICN levou a consideralo baixo a categoría CR (En Perigo Crítico). Finalmente suxírense diferentes medidas de conservación. Finalmente, o Capítulo 7 está dedicado á medición da cor do xénero Jasione. A cor dos órganos das plantas, en particular das follas e as flores, é un trazo fenotípico utilizado tradicionalmente como indicador visual do estado fisiolóxico da planta. De feito, a tensión ambiental relacionada coa cantidade e calidade da luz, os nutrientes, a temperatura e a seca provocan un cambio de cor da planta. Estas correlacións son tan informativas que a cor é un dos trazos fenotípicos para monitorar o crecemento das plantas en enfoques de clasificación de alto rendemento. Desafortunadamente, a detección e clasificación de cores polo ollo humano son extremadamente pouco confiables debido á súa dependencia da experiencia e a capacidade do observador. A elección dunha cor por parte dun organismo vivo baséase en criterios psicolóxicos e evolutivos ou de supervivencia. Por exemplo, entre as cores que distingue o ollo humano, o verde percíbese con maior facilidade e co maior cambio de matiz que calquera outro cor, debido á percepción combinada de bastóns e conos. Exponse a hipótese de que isto podería representar unha adaptación á contorna dos primates en busca de alimento. A cor pódese describir de forma obxectiva e precisa mediante o uso de espectrofotómetros, colorímetros ou cromómetros que describen a cor reflectida no sistema de cor estandarizada CIELAB. Funcionan por contacto nunha superficie, como outros dispositivos espectrómetros de contacto, e promedian a luz reflectida desde unha abertura na cabeza do dispositivo. A apertura é circular e ten un diámetro de entre 3 mm e 60 mm. As superficies cunha área cunha forma diferente ou máis pequena que a apertura do dispositivo non Resumo 25 reflicten por completo a luz emitida, o que conduce a unha perda constante de luz e a resultados pouco fiables. Para realizar a medición da cor correctamente, a superficie da mostra debe cubrir por completo a apertura do dispositivo para evitar fugas de luz ou interferencias de luz externa. Por esta razón, a cor non se pode medir cando a área obxectiva é menor de 3 mm e / ou non é circular. Para abordar este tema, no Capítulo 7 proponse reducir nun 50% e 70% a área de apertura dun espectrofotómetro portátil con adaptadores de cartón en cores branco e negro, co fin de describir a cor no espazo CIELAB de pequenas mostras biolóxicas, como follas e pétalos. Por tanto, mellórase aínda máis a usabilidade do dispositivo sensor ao estender a súa aplicación a mostras máis pequenas que aquelas para as que foi deseñado. Isto desbloquea formas previamente inexploradas de abordar problemas actuais no campo da vixilancia ambiental, como a resposta da biodiversidade ao cambio climático. Neste marco mídese a cor de follas de especies do xénero Jasione e outras da familia Campanulaceae, validando así esta metodoloxía dentro dun amplo grupo filogenético con especies de diferente ecoloxía e frecuentemente con follas de pequeno tamaño. Este grupo engloba unha serie de especies alpinas, con hábito de densas rosetas con follas pequenas, sendo esta unha característica de adaptación vexetal aos ambientes alpinos. Neste capítulo, o uso de follas o suficientemente grandes para ser medidas coa gran apertura do colorímetro garantiu a confiabilidad desta metodoloxía tamén para follas onde só pódese usar a apertura máis pequena da apertura do colorímetro, como é o caso de moitas especies alpinas. Ademais, inclúense mostras de herbario, onde se conservan tecidos vexetais secos como valiosas evidencias ecolóxicas, sistemáticas e históricas da biodiversidade vexetal. Ao estender a presente metodoloxía a mostras secas conservadas, proponse o uso da colorimetría como ferramenta para describir a cor de follas e pétalos despois dun tempo e monitorear as condicións de conservación destes especímenes. Os resultados mostran que, seguindo criterios colorimétricos, a única configuración que proporciona cores indistinguibles segundo a percepción do ollo humano é o uso dun adaptador de redución do 50% na apertura de 3 mm. Ademais, a análise estatística multivariante, desenvolto polo autor da tese, suxire o uso do adaptador branco. O estudo ofrece unha técnica LUIS MIGUEL SERRANO PÉREZ 32 en un sistema diploide-tetraploide de taxones estrechamente relacionados del género Jasione L. (Campanulaceae) pertenecientes al grupo Jasione crispa (Pourr.) Samp. En dos estudios anteriores, el cambio de nicho hipotético en otros grupos poliploides modelo del género Jasione fue rechazado o no fue totalmente respaldado por los resultados, y los rangos de distribución actuales de esos complejos poliploides deben explicarse por otros factores. Aquí, la distribución a escala fina de los citotipos y los grupos taxonómicos significativos evolutivos se determinó mediante recuento de cromosomas, citometría de flujo en material secado en gel de sílice y marcadores moleculares del genoma del plastidio. Se aplicaron técnicas de modelado ambiental y se desarrollaron modelos de distribución de especies para examinar si se produce un cambio de nicho ambiental o conservadurismo entre tres taxones diploides y entre estos y un taxón tetraploide presumiblemente descendiente. El proceso de modelado utilizó variables climáticas y edáficas, comparando modelos de distribución de especies con y sin variables edáficas. La inclusión de variables edáficas en el modelado de este complejo poliploide proporcionó una descripción más precisa de la realidad ecogeográfica de los taxones en estudio. Además, los modelos demostraron que el grupo tetraploide experimentó un cambio significativo y expansión de nicho en relación con los parientes cercanos diploides, en los que se detecta conservadurismo de nicho. La expansión del nicho hacia la tolerancia de ambientes más fríos ha dado forma a la distribución actual de J. sessiliflora tetraploide, mientras que el conservadurismo del nicho restringe los diploides a áreas comparativamente térmicas y subhúmedas en el centro suroeste y mediterráneo de la Península Ibérica. El conservadurismo de nichos diploides parece revelar cierta inercia residual en los nichos ancestrales del linaje. En resumen, y a la inversa de otros sistemas modelo de Jasione, la poliploidización probablemente causó una innovación ambiental exitosa en Jasione sessiliflora. La variabilidad edáfica es un factor importante que promueve la microevolución en las plantas, lo que puede conducir a la formación de nuevas especies a largo plazo. Jasione crispa (Campanulaceae) es un grupo compuesto mayoritariamente por orófitos restringidos a suelos ácidos. En el Capítulo 5, la identificación de una población aislada y Resumen 33 en peligro de extinción de Jasione en el macizo calcáreo del Mont Caro (Tarragona, España) permite el estudio de nuevas presiones ambientales en un linaje acidófilo y los pasos iniciales del camino de especiación. Esta población se ha incluido en la variabilidad de J. sessiliflora, una especie tetraploide. El nivel de ploidía de la población se determina como hexaploide, evidenciando relaciones más estrechas con Jasione crispa subsp crispa. El flujo de genes reducido por aislamiento geográfico se identifica mediante procedimientos de clonación del marcador ITS del nrDNA, en un marco filogenético y filogeográfico que incluye poblaciones de todas las cadenas montañosas ocupadas por el taxón. La interrupción del flujo de genes y la exposición a nuevas limitaciones ambientales son los principales promotores de la especiación adaptativa. Para evaluar si la población de Mont Caro está experimentando un proceso de especiación ecológica, se cultivaron plantas de Mont Caro y de dos poblaciones de zonas montañosas silíceas (Sistema Central y Pirineos) y diferentes proximidades filogenéticas a Mont Caro en tres tipos de suelo. Las mediciones no destructivas de color por reflectancia representadas en el sistema de color CIELAB, utilizando los parámetros cromáticos: a* (asociado con cambios en el rojo-verde) y b* (asociado con cambios en el amarillo-azul), demostraron ser relevantes para evaluar estos diferentes comportamientos, y por lo tanto, son una buena variable proxy para los estudios de especiación de plantas en este grupo de modelos. Las concentraciones de biomasa y Al, Ca, Fe y Mg se estimaron en el momento de finalización del experimento. La población de Mont Caro mostró mejores respuestas a los suelos básicos (es decir, mayor producción de biomasa, baja concentración de Mg, equilibrio en las concentraciones de Ca en las raíces y hojas) que las otras dos poblaciones, aunque peor desempeño en suelos ácidos y mal manejo del aluminio a nivel de las raíces, lo que parece ser una compensación evolutiva negativa. Estos resultados sugieren la ocurrencia de un proceso de especiación ecológica en curso en J. crispa de Mont Caro, lo que podría contribuir a la descripción de esta población como un nuevo taxón. El Capítulo 6 se basa parcialmente en una breve publicación fruto de la colaboración con la administración medioambiental de Andalucía. LUIS MIGUEL SERRANO PÉREZ 34 Jasione corymbosa Poir. ex Schult., en un sentido amplio, es un polimórfico anual endémico de la zona bético-mauritana, cuyo hábitat preferido son las dunas y arenas costeras del sur de la Península Ibérica y el Magreb, desde el oeste de Argelia hasta la costa atlántica de Marruecos. Se consideró extinto en la Península Ibérica debido a la alteración generalizada del hábitat en las costas mediterráneas, aunque aún se encontraban poblaciones existentes en las costas atlántica y mediterránea del noroeste de África. El conocimiento del hábitat de las poblaciones africanas guió un intenso muestreo en el Mediterráneo andaluz. Finalmente se detectó una población putativa en hábitats de dunas de arena en Manilva, Málaga. Su identificación como Jasione corymbosa fue controvertida entre los botánicos andaluces, y se resolvió definitivamente con el uso de herramientas moleculares, que recuperaron las plantas de Manilva anidadas filogenéticamente entre las poblaciones norteafricanas de Jasione corymbosa. Por tanto, Jasione corymbosa es una especie de la flora de la Península Ibérica en grave peligro de extinción. Una clasificación siguiendo la categorización de la UICN llevó a considerarlo bajo la categoría CR (En Peligro Crítico). Finalmente se sugieren diferentes medidas de conservación. Finalmente, el Capítulo 7 está dedicado a la medición del color del género Jasione. El color de los órganos de las plantas, en particular de las hojas y las flores, es un rasgo fenotípico utilizado tradicionalmente como indicador visual del estado fisiológico de la planta. De hecho, el estrés ambiental relacionado con la cantidad y calidad de la luz, los nutrientes, la temperatura y la sequía provocan un cambio de color de la planta. Estas correlaciones son tan informativas que el color es uno de los rasgos fenotípicos para monitorizar el crecimiento de las plantas en enfoques de clasificación de alto rendimiento. Desafortunadamente, la detección y clasificación de colores por el ojo humano son extremadamente poco confiables debido a su dependencia de la experiencia y la capacidad del observador. La elección de un color por parte de un organismo vivo se basa en criterios psicológicos y evolutivos o de supervivencia. Por ejemplo, entre los colores que distingue el ojo humano, el verde se percibe con mayor facilidad y con el mayor cambio de matiz que cualquier otro color, debido a la percepción combinada de bastones y conos. Se plantea la hipótesis de Resumen 35 que esto podría representar una adaptación al entorno de los primates en busca de alimento. El color se puede describir de forma objetiva y precisa mediante el uso de espectrofotómetros, colorímetros o cromómetros que describen el color reflejado en el sistema de color estandarizado CIELAB. Funcionan por contacto en una superficie, como otros dispositivos espectrómetros de contacto, y promedian la luz reflejada desde una abertura en la cabeza del dispositivo. La apertura es circular y tiene un diámetro de entre 3 mm y 60 mm. Las superficies con un área con una forma diferente o más pequeña que la apertura del dispositivo no reflejan por completo la luz emitida, lo que conduce a una pérdida constante de luz y a resultados poco fiables. Para realizar la medición del color correctamente, la superficie de la muestra debe cubrir por completo la apertura del dispositivo para evitar fugas de luz o interferencias de luz externa. Por esta razón, el color no se puede medir cuando el área objetivo es menor de 3 mm y / o no es circular. Para abordar este tema, en el Capítulo 7 se propone reducir en un 50% y 70% el área de apertura de un espectrofotómetro portátil con adaptadores de cartón en colores blanco y negro, con el fin de describir el color en el espacio CIELAB de pequeñas muestras biológicas, como hojas y pétalos. Por lo tanto, se mejora aún más la usabilidad del dispositivo sensor al extender su aplicación a muestras más pequeñas que aquellas para las que fue diseñado. Esto desbloquea formas previamente inexploradas de abordar problemas actuales en el campo del monitoreo ambiental, como la respuesta de la biodiversidad al cambio climático. En este marco se mide el color de hojas de especies del género Jasione y otras de la familia Campanulaceae, validando así esta metodología dentro de un amplio grupo filogenético con especies de diferente ecología y frecuentemente con hojas de pequeño tamaño. Este grupo engloba una serie de especies alpinas, con hábito de densas rosetas con hojas pequeñas, siendo esta una característica de adaptación vegetal a los ambientes alpinos. En este capítulo, el uso de hojas lo suficientemente grandes para ser medidas con la gran apertura del colorímetro garantizó la confiabilidad de esta metodología también para hojas donde solo se puede usar la apertura más pequeña de la apertura del colorímetro, como es el caso de muchas especies alpinas. Además, se incluyen muestras de herbario, donde se conservan tejidos vegetales LUIS MIGUEL SERRANO PÉREZ 36 secos como valiosas evidencias ecológicas, sistemáticas e históricas de la biodiversidad vegetal. Al extender la presente metodología a muestras secas conservadas, se propone el uso de la colorimetría como herramienta para describir el color de hojas y pétalos después de un tiempo y monitorear las condiciones de conservación de estos especímenes. Los resultados muestran que, siguiendo criterios colorimétricos, la única configuración que proporciona colores indistinguibles según la percepción del ojo humano es el uso de un adaptador de reducción del 50% en la apertura de 3 mm. Además, el análisis estadístico multivariante, desarrollado por el autor de la tesis, sugiere el uso del adaptador blanco. El estudio ofrece una técnica de medición de sonido para recopilar información ecológica del color de hojas, pétalos y otras pequeñas muestras. 37 Summary This PhD thesis dissertation aims to reveal different aspects of the evolutionary reality of the genus Jasione L. (Campanulaceae), a plant lineage distributed around the Mediterranean basin. Molecular, cytological, morphological and environmental analyses were carried out to address the following: (i) the internal phylogenetic relationships in Jasione, as well as its position among other genera of the Campanulaceae, (ii) the biogeographical history of a genus with a remarkable east-west Mediterranean biogeographical pattern, (iii) the processes leading to speciation and adaptation to new environments, (iv) the study of environmental niche shifts associated with changes in ploidy level, (v) the methods used to delimit evolutionarily significant cryptic entities by using different lines of evidence, and (vi) typification of complex groups of Jasione as a step in establishing a new taxonomic organization of the genus based on evolutionary relationships, among other aspects. The family Campanulaceae is a complex evolutionary group characterized by conflict between traditional classifications and the molecular phylogenies generated in the last decades, conflicts that exacerbate in the subfamily Campanuloideae. One of the main reasons of the differences between pre-molecular classifications and modern phylogenetic results is the scarcity of discriminatory morphological characters, that led to overly emphasis given in the traditional taxonomic treatments to some reproductive traits, namely the flower and fruit morphologies. Parallel evolution of morphological traits has apparently clouded the systematics of the group, hampering the proposal of evolutionary significant delimitations between and within the genera. LUIS MIGUEL SERRANO PÉREZ 38 In this general context, Jasione is one the genera of the Campanuloideae more homogeneous from the morphological point of view. Consequently, it has been puzzling regarding its evolutionary systematics. There is a general controversy among authors in the number of taxa that must be recognized, with about one hundred of different proposed names. However, current general treatments only recognize between 12 and 16 species, although this synthetic approach has been considered overly simplistic and resulting from the difficulty of the genus. Consequently, the need of a modern treatment with evolutionary perspective has been claimed. It is the aim of this PhD thesis dissertation to cast light on the evolutionary reality and biogeographic history of a genus distributed in Europe and around the Mediterranean basin, using molecular, cytological, and environmental tools to achieve this objective. The genus Jasione L (Campanulaceae) is distributed around the Mediterranean basin and throughout Europe, with two centres of diversity at the opposite sides of the Mediterranean Sea, one in the Iberian Peninsula and Morocco and another in the Balkan and Anatolian peninsulas, consistent with the West-East Mediterranean disjunction biogeographical pattern. The genus occurs in a number of ecological conditions and geological substrates across a wide elevational range, including coastal and alpine habitats. In the Chapter 1 all the published names of Jasione in the species, subspecies and variety ranks for populations from the Balkan and Anatolian peninsulas are reviewed, including names of taxa allied to these groups in surrounding areas. The taxonomic status and typification of the names are addressed. In total, eleven lectotypes, one lectoneotype and one epitype are suggested. The members of the Jasione orbiculata complex were included, and the representative of the complex in southern Italy is therefore also treated. The review presents basionyms in alphabetical order, structured as a discussion of each name followed by its synonyms and typification, designating types when required. A list of currently accepted names from recent checklists is provided, and the heterotypic synonyms are indicated. Groups with morphological stasis are an interesting framework to address the possible recognition of cryptic species hidden behind Summary 39 traditional taxonomic treatments, particularly when distribution ranges suggest disjunct and environmentally heterogeneous biogeographic patterns. New hypotheses of delimitation of evolutionary independent units can lead to the identification of different biogeographic patterns, laying the foundation to investigate their historical and ecological significance. Jasione (Campanulaceae) is a plant genus with a distribution centered in the Mediterranean basin characterized by significant morphological stasis. Jasione sessiliflora s.l., and allied taxa have disjunct distribution ranges within the Iberian Peninsula, occupying environmentally diverse regions. At least two ploidy level, diploid and tetraploid, are known to occur in the group. These aspects motivated the assessment of the internal variability in the group with phylogenetic tools for species delimitation. The results from GMYC and ASAP analyses are compared in the Chapter 2 with other lines of evidence, including morphology, cytology and phenology. The fitting of distribution patterns of the inferred entities to chorological subprovinces is used as a biogeographical and environmental framework to test species hypothesis. Despite the scarcity of diagnostic morphological characters in the group, phylogenetic delimitation supports the description of at least one cryptic species, a narrow endemic in NE Iberian Peninsula, and the separation from J. sessiliflora of a group of populations from thermophile environment in eastern Iberia. Although disjunct ranges suggested the assessment of population groups, not all disjunct populations pointed to independent evolutionary histories. Ploidy differences support the systematic rearrangement suggested by species delimitation. Taxonomic reorganization in J. sessiliflora s.l. allowed biogeographic interpretations of distribution patterns that are in accordance with biogeographical regionalization. These results suggest that species differentiation, together with geographic isolation and polyploidization was associated to adaptation to different environments, shifting from more to less thermophilic conditions during the evolutionary history of the J. sessiliflora group. Thus, the recognition of concealed evolutionary entities is essential to correctly interpret biogeographical patterns in regions with a complex geologic and evolutionary history such as the Mediterranean basin. LUIS MIGUEL SERRANO PÉREZ 40 Jasione is a taxonomically complex genus with a disjunct east-west Mediterranean distribution pattern. Around 16 species are currently recognized, although more than one hundred taxonomic names have been proposed. The hypothesis that intense morphological stasis is concealing evolutionary relevant variability and hampering correct understanding of the evolutionary relationships in the genus needs to be tested. Although the genus is distributed in Europe and around the Mediterranean basin, no comprehensive phylogenetic analysis has been carried out until now. The aims of the Chapter 3 were to test the monophyly of the genus and determine its position among other lineages basal to the tribe Campanuleae, an unresolved conflicting topic in different studies. The research also aimed to establish the internal phylogenetic relationships by comprehensive sampling including different populations and most of the proposed names, to prevent a priori exclusion of evolutionary significant lineages. The roles of secondary contact and variation in ploidy levels in shaping the evolutionary history of the genus were also investigated. The underlying processes leading to the disjunct east-west Mediterranean distribution pattern were investigated. To address these questions, a five loci time-calibrated phylogeny was constructed with 111 terminals, 93 of which corresponded to Jasione. The genus was resolved as monophyletic, within a clade shared with two other isolated lineages, Hesperocodon and Feeria. This clade is sister to the Campanuleae tribe. The lineage leading to Jasione originated in the late Oligocene (c. 25 Mya) and the extant lineages of Jasione derive from a much later diversification event, occurring in c. 9 Mya in the Miocene, from an ancestor either originated in the southern Iberian Peninsula or already present in the western and eastern Mediterranean regions. Two main clades can be identified, one only present in the western Mediterranean and encompassing species with very different morphologies and another with the extant disjunct distribution pattern. One episode of vicariance is compatible with the late Messinian aperture of the Gibraltar strait, although several posterior overseas dispersal events from the Iberian Peninsula to North Africa were inferred in both clades. The second clade began to diversify at the beginning of the Pleistocene, probably in the Balkan Peninsula, and at least two east to west dispersal Summary 41 events are identified in this clade, with niche modelling identifying niche conservatism in the eastern group and niche expansion in the western group. Habitat suitability was not identified in most of the intermediate European regions. The disjunct east-west distribution pattern in Jasione is a dynamic biogeographic process in which the two areas harbour lineages providing the opportunity both to remain and to disperse to the other areas, increasing the chances of the lineage to endure. Finally, the resulting phylogeny is not consistent with current taxonomy showing that morphological stasis in Jasione has hampered the systematic understanding and suggesting the need for further integrative taxonomic studies. There is a general tendency for niche conservatism among closely related taxa, although in polyploid complexes niche divergence may enable polyploids to become established outside the range of distribution of their diploid relatives. Polyploidization leads to the immediate emergence of evolutionary barriers and speciation. However, the new polyploid appears within diploid populations and is thus exposed to the effects of competition and frequency-dependent processes that ultimately lead to reproductive exclusion of the minority cytotype. Ecological divergence would enable polyploids to avoid these processes, thus becoming a key mechanism in shaping geographical distribution of cytotypes in polyploid complexes. However, evidence supporting this hypothesis is still needed, as many contradictory findings have been reported. Moreover, it remains to be determined whether polyploids diverge ecologically to a greater degree than diploids. Study of polyploid complexes encompassing both polyploids and closely related diploid taxa provides an opportunity to compare polyploid niche shift or conservatism in relation to niche shift within an appropriate evolutionary framework. The niche shift hypothesis is evaluated in the Chapter 4 in a diploid-tetraploid system of closely related taxa of the genus Jasione L. (Campanulaceae) belonging to the Jasione crispa (Pourr.) Samp group. In two previous studies, the hypothesized niche shift in other model polyploid groups of the genus Jasione was either rejected or not fully supported by the results, and current distribution ranges of those polyploid complexes must be explained by other factors. Here, the fine scale distribution of cytotypes LUIS MIGUEL SERRANO PÉREZ 48 Consequently, the need for a modern approach with an evolutionary perspective has been pointed out (Kovanda, 1968, Sales & Hedge, 2001b; Pérez-Espona et al., 2005). It is the aim of this work to cast some light on the evolutionary reality and biogeographical history of a genus distributed in Europe and around the Mediterranean basin, using molecular, cytological and environmental tools to achieve this objective. The following brief synopsis of the genus incorporates information from the studies detailed in the chapters of the Results section together with other information gathered during the research reported in this thesis. 1.THE GENUS JASIONE L. 1.1 Systematic position Jasione montana L. was originally described by Linnaeus (1753) to classify a single species and is therefore the type species of the genus. The first attempt to classify the genus Jasione within the subdivisions of the Campanulaceae was carried out by de Candolle (1830), who included Jasione in an unnamed subtribe, later identified with the name Wahlenbergieae Endl. (Endlicher, 1838). The other genera grouped in the Wahlenbergeae mostly had Asian distribution ranges (e.g. Codonopsis Wall., Platycodon A. DC.) or southern hemisphere distributions ranges (e.g. Wahlenbergia W. Roth., Canarina L., Prismatocarpus L’Her.), which made Jasione quite unusual in the group, given its European and circum-Mediterranean distribution. De Candolle (1930) also created the other subtribe, identified as Campanuleae Dumort, which mainly encompasses genera with northern hemisphere or Mediterranean distribution ranges and is therefore, similar to Jasione in this respect (e.g. Trachelium L. Phyteuma L, Campanula L.). However, de Candolle’s subtribal division was based on the mode of capsule dehiscence, which in Jasione and the members of the Wahlenbergieae is loculicidal, by apical valves, and in the Campanuleae is poricidal, by lateral pores. Schönland (18891894) addressed the internal classification of the family by creating three subtribes and relocating some of the genera of de Candolle’s General Introduction: Synopsis of the genus Jasione 49 Wahlenbergeae in the subtribe now called Campanulinae and a new subtribe Platycodinae. Nevertheless, Jasione was left in the Wahlenberginae. Schöndland’s division was based on aspects like calyx morphology and, again, capsule dehiscence. Fedorov (1957) considered that Jasione was distinct enough to be separated in its own tribe, creating the tribe Jasioneae Fed. The characters that justified this new classification included the flowers reunited in dense globose heads, the corolla dissected nearly to the base, the anthers connate at base, and once again, the capsule dehiscence by two apical valves (Fedorov, 1957). The first molecular phylogenetic approaches published in the new millennium would later dramatically alter the systematic view of the family, with many genera being recovered as polyphyletic or paraphyletic (Eddie et al., 2003; Roquet et al., 2008). Regarding Jasione, the traditional classification within the Wahlenbergieae lost support, although its position remained unresolved, and it was suggested that Jasione may form part of a so-called “transitional” group between the Wahlenbergioid and the Campanuloid taxa (Eddie et al., 2003). Cosner et al. (2004) studied the chloroplast DNA rearrangements in the family and identified a greater number of affinities between Jasione and the Campanuloids than with other groups. These authors also found that the chloroplast genome of Jasione was the most heavily rearranged among all the genera studied. The topological position of Jasione and the different phylogenetic hypotheses regarding its most closely related lineages have been controversial topics in the different phylogenetic works on the Campanulaceae published in the last decades (Haberle et al., 2009; Roquet et al. 2009; Mansion et al., 2012, Olesen et al., 2012; Crowl et al., 2016; Xu & Hong, 2021). The main conflicting points in these studies are reviewed in detail in the Results section (Chapter 3). The phylogenetic analysis conducted in the research reported in this thesis contributes to resolving the different issues that have affected the topological position of Jasione and its closest lineages. In summary, the phylogenetic location of Jasione is consistently established in a clade with two monospecific lineages, Hesperocodon Eddie & Cupido and Feeria Buser. Hesperocodon has traditionally been considered a member of the Wahlenbergioids, under the name Wahlenbergia LUIS MIGUEL SERRANO PÉREZ 50 hederacea (L.) Rchb, while Feeria has been considered a member of the Campanuleae, close to Trachelium (Eddie & Cupido, 2014). This clade would be sister to a clade grouping the remaining Campanuleae, which would also include some of the previously called “transitional” groups. The Jasione and allied genera clade, and the Campanuleae clade would, in turn, form a sister clade of the Wahlenbergioids. Morphological traits with long lasting systematic importance in the Campanulaceae conserve some evolutionary significance in the light of the present results, as the type of capsule dehiscence in Jasione, Feeria and Hesperocodon is apical valvate, which is quite similar to that in the Wahlebergioids (Eddie & Cupido, 2014), although this cannot be used to support a natural group. All of these results emphasize the singularity of Jasione and its allied genera Feeria and Hesperocodon. Among these genera, Jasione was the only one that was able to diversify and produce a number of extant lineages. Nevertheless, the internal relationships and taxonomical organization of these lineages is full of complexities and far from being understood, until now. 1.2. General morphology of Jasione Members of the genus Jasione are annual, biannual or perennial plants with leafy stems and undivided leaves, ranging from glabrous to densely hairy. The stems are decumbent or erect, branched or not. Some species can develop thin, whitish, subterranean stolons (e.g. J. laevis lam.). The leaves are frequently spathulate or lanceolate, sometimes ovate or obovate, normally narrow, sessile or with a petiole-like narrowing at the base, alternate in the stem, in some cases forming sterile rosettes. The leaf margin ranges from entire to crenate, sometimes toothed. Leaf margins have hydathodes, which in one of the two main clades of the genus are modified to become remarkable tubular structures with subapical and submarginal locations (see below). The genus is well characterized by its inflorescence, a capitulum-like globose glomerule of small flowers subtended by a crown of involucral bracts arranged in one to four series. The bract shape can be lanceolate, ovate, obovate, rhomboidal or orbiculate, with entire, crenate or toothed serrate margins. Bract teeth can be aristate General Introduction: Synopsis of the genus Jasione 51 (e.g. Jasione laevis Lam.), and in some cases the teeth and the bract apex end in a hardened awn (e.g. J. heldreichii Boiss & Orph.). The glomerule is normally pedunculate, although in individuals of some species the leaves can reach the inflorescence (e.g. Jasione crispa (Pourr.) Samp.). The flowers can be sessile (as in J. sessiliflora Boiss. & Reut.) or more frequently pedicellate. The calyx has a turbinateovoid calyx tube and five triangular, linear or subulate calyx teeth. The calyx teeth can be glabrous, ciliate or woolly-hairy. The corolla is divided into five linear linear-lanceolate lobes nearly to the base and therefore does not form a tube. The corollas are blue or lilac, or more rarely white, with whitish colours more frequently found in taxa from high mountain or coastal dune habitats, although an important variation in flower colour is always observed within the populations. Nevertheless, Jasione mansanetiana Roselló & Peris consistently has white corollas, sometimes with creamy or lilac shades. Flowering is centripetal, as in the Asteraceae family. Thus, the peripheral flowers mature first with the opening sequence proceeding towards the centre. The stamens have free filaments but a connate anther at base, forming a narrow ring, with one species in the genus having free anthers, i.e. J. bulgarica Stoj. & Stef. The ovary is turbinate-ovoid, with one style longer than the lobed corolla and two short stigmas. The upper half of the style is hairy, with hairs in ten longitudinal rows, to facilitate secondary pollen presentation, once the style has passed through the ring formed by the anthers. (Erbar & Leins, 1989). Pollen bluish, pink or purple, very homogeneous in shape among all the species, 3 (4) porate, ranging from prolate-spheroidal to oblate-spheroidal, with microechinate ornamentation (Figure I.1), varying in size between c. 20 and 25 μm (Çelemli, 2020; M. Serrano, unpublished data). The capsule is ovoid, flattened above, bilocular, with a loculicidal mechanism of dehiscence occurring via two small apical valves, located above the insertion of calyx lobes. Seeds are variable in number, small, shiny, brown, with striate coat ornamentation, oblong-elongate, sometimes sub-orbicular, as in some individuals of Jasione corymbosa Poir. A caruncle is present in seeds of most populations of Jasione crispa subs. mariana (Willk.) Rivas Mart. and more rarely in some populations of Jasione sessiliflora. LUIS MIGUEL SERRANO PÉREZ 52 Figure I.1. SEM photomicrographs of pollen grains of Jasione species in the four main clades of the genus, showing morphological homogeneity: (A) Jasione tmolea Stoj. from Bozdağ, Turkey, (B) Jasione foliosa Cav. subsp. xauenensis Dobignard from Jbel Tissouka, Morocco, (C) Jasione crispa subsp. mesatlantica (Emb. & Maire) Dobignard from Jbel Bou Naceur, Morocco, (D) Jasione blepharodon Boiss & Reut. from Grazalema, Spain. 1.3. Anatomy Bokhari & Sales (2001) were the first authors to conduct a relatively extensive anatomical study of the genus, although they focused on the Iberian taxa. Their findings were consistent with the taxonomic treatment reported by Sales & Hedge (2001a) in Flora General Introduction: Synopsis of the genus Jasione 53 Iberica, supporting some disagreements with Tutin (1976), such as the recognition at the species level of Jasione cavanillesii C. Vicioso or Jasione sessiliflora. (Bokhari & Sales, 2001). These authors identified some anatomical traits that characterized some of the taxa reported by Sales & Hedge (2001a). The most important were epidermal cell shape, differences in adaxial and abaxial epidermal cell sizes, venation of the proximal part of the leaf, presence of stem sclereids in the pith, presence of brachysclereids in the cork of old wood, differentiated or undifferentiated mesophyll and relative distribution of the stomata between abaxial and adaxial surfaces. However, many traits showed variations within the same taxon (e.g. epidermal cell shape or stem sclerification) related to the age of the individual or environmental conditions. Other characters that the authors considered stable and enabled differentiation of species, such as the absence of adaxial stomata in Jasione laevis, are not supported by extensive research by the current author in J. laevis stomata across all ploidy levels (M. Serrano, unpublished data). On completing their study, Bokhari & Sales (2001) conceded the limited usefulness of the anatomical characters for establishing relationships among taxa, or even distinguishing taxa within species. Some of their conclusions are not supported by the phylogenetic results of this thesis. No anatomical differences were found among varieties of Jasione montana, although the varieties encompassed species of distant lineages, such as Jasione montana s.str. and Jasione blepharodon (sub Jasione montana var. bracteosa Willk.). Similarly, Bokhari & Sales (2001) found no differences between what they considered populations of Jasione maritima (Duby) Merino from France and the Iberian Peninsula, a species distinguished by having brachysclereids in the cork of old wood and two layers of palisade tissue on abaxial and adaxial leaf sides. However, the research findings reported in the present thesis show that the aforementioned authors dealt with at three differentiated evolutionary entities, with three ploidy levels (diploid, tetraploid and hexaploidy) and at least partly different origins, suggesting that anatomical similarities were probably related to the environmental constraints of the maritime dune habitat of these populations. There is, however, one anatomical character whose distribution among the species of the genus is correlated with the main LUIS MIGUEL SERRANO PÉREZ 54 phylogenetic divisions of Jasione, i.e. the modified hydathodes. The adaxial side of the leaves of some species of Jasione bears remarkable tubular structures (Figure 2), which have been called “trichoids” (Bokhari & Sales, 2001; Sales & Hedge, 2001a; Pérez-Espona et al., 2005). Figure I.2. Modified hydathodes of Jasione sect. Jasione. SEM photomicrographs of the adaxial leaf surface of (A) Jasione crispa subs. tomentosa (A.DC) Rivas Mart. from Mora, Spain and (B) Jasione sp. nova from Maçanet de Cabrenys, Spain, showing apical and apical and marginal modified hydathodes, respectively. Scale bars = 10 μm. (C) ESEM photomicrograph of a modified hydathode of Jasione carpetana Boiss & Reut. from Miraflores de la Sierra, Spain showing stomata-like pores on the hydathode surface. Scale bar = 20 μm. (D) Fully functional apical and marginal modified hydathodes secreting water by guttation in cultivated Jasione carpetana in early morning exterior conditions. Bokhari & Sales (2001) considered that the function of the “trichoids” was not properly understood, and detailed studies were needed. These authors ruled out the possibility that the “trichoids” could General Introduction: Synopsis of the genus Jasione 55 be hydathodes as they did not find any structural resemblance. Nevertheless, the study of cultivated individuals of different species during the research reported in this thesis revealed that the structures are fully functional hydathodes, allowing guttation, i.e. the release of water in conditions of soil saturation of water and low transpiration conditions (Grundwald et al., 2003) such as under high atmospheric humidity or darkness (Figure 2, D). Anatomical analysis showed that the modified hydathodes are unpigmented multicellular structures of length 100-200 μm, vascularized and connected by tracheids to the xylematic system and with several with stomata-like pores on the surface (Figures 2 and 3). Examination of semi-thin sections revealed that the internal structure is similar to the epithem, i.e. thin-walled cells with abundant intercellular spaces in ordinary hydathodes (Figure 3). Transmission electron microscopy images (Figure 4) show that the epithem has vacuolated parenchyma cells with plastids joined to the outer membrane, large intercellular spaces, xylem vessels with tracheids and granules probably containing reserve material, therefore being internally similar to the hydathodes of other plant families (Cerutti et al., 2017). In Jasione and the Campanulaceae, the reserve material is the polysacharid inulin and not starch as found in most plants (Schmeja, 1931). The epithem is connected to the xylem vascularization of the plant, as vessels and tracheids can be observed (Figure 3). The Campanulaceae is distinguished by presence of laticifers (i.e. latex-containing vessels) in their tissues (Kovanda, 1978). In Jasione, the stem and leaves have anastomosed laticifers (M. Serrano, unpublished data), and leaf latex vessels seem to penetrate the modified hydathodes (Figure 4, C and D). The current author agrees with Bokhari & Sales (2001) that the purpose of this specialized structure is far from being understood, and studies addressed to reveal its functionality and evolutionary role in the genus are of major interest. Nevertheless, the name “trichoid” does not seem appropriate, as structurally and functionally the structures are homologous to the ordinary and relatively inconspicuous hydathodes of some species of Jasione and all remaining members of the Campanulaceae. Thus, until the function of the structure is fully understood, the name “hydathoids” is suggested for these modified hydathodes. LUIS MIGUEL SERRANO PÉREZ 56 Figure I.3. “Hydathoids” of Jasione showing vascularization and epithem organization: (A) Light microscope (x40) photograph of a safranin-stained involucral bract of Jasione gr. cripa from Cardaño de Arriba, Spain. (B,C and D) Toluidine blue stained semi-thin sections of hydathoids and the adjacent leaf area of Jasione carpetana from Miraflores de la Sierra, Spain. Epithem and tracheid vascularisation can be seen in C, D and D, and apparent laticifers in C, with more detail in D. Scale bars = 100,000 nm in B and C, 10,000 nm in D. Interestingly, the hydathoids are tubular structures with subapical and submarginal locations, always oriented inwards to the centre of the leaf. Ordinary hydathodes are oriented outwards to facilitate elimination of water from the plant by guttation. The guttation mechanism may only be one of the functions of hydathoids, and several complementary hypotheses should be tested. Preliminary staining experiments showed that fluids can penetrate the leaf through the hydathoids, with internal redistribution being tracked to nearby leaves (M. Serrano, unpublished data). Hydathoids may be associated with mechanisms of leaf water General Introduction: Synopsis of the genus Jasione 57 uptake from dew or fog precipitation. However, observation of water uptake through hydathoids does not necessarily imply that it the uptake is important enough to affect the internal hydric balance of the plants. Robust experimental tests must be conducted to ascertain the physiological importance of foliar water uptake through hydathoids and whether these structures confer any adaptive evolutionary advantage, by comparing the performance of Jasione lineages with and without hydathoids. Other possible functions include scent emission by release of volatile secondary compounds or formation of part of the different immune layers of the plant by release of protective compounds by guttation, as identified in hydathodes of other plant families (Cerutti et al., 2017). Hydathoids are only found in one of the two main clades of the genus (sect. Jasione), and they are completely absent in the other clade (sect. Phyteumopsis). They are present in leaves and involucral bracts and the number may vary. In most taxa of sect. Jasione there is one apical and between three and four marginal pairs of hydathoids. However, in some Iberian and North African groups, the hydathoid number is reduced, and only the apical hydathoid is found in some taxa. Species from more mesic environments tend to have more hydathoids on average than taxa from drier or chasmophytic habitats, and the reduction in number may therefore be related to control of water loss. 65 General Methods In this thesis numerous samples of Jasione and other representatives of the Campanulaceae family were used for molecular or cytological analysis, among others. Occurrence data obtained in the field by the author or extracted from herbaria or bibliographic sources have been used to inform statistical and modelling analyses. To prevent an inflation of database tables in this document, all this information, including vouchers and geographic information can be found in a online shared spreadsheet. GenBank accession numbers of the DNA sequences used in this thesis will be included in the shared link. Link with database tables: https://docs.google.com/spreadsheets/d/1LVcv9jSMR89TX8hh98 _-_ZRFJ-WolXXvLdWQ8FemsT0/edit?usp=sharing The methods used are structured as the different chapters of the thesis, although similar methods have been used in more than one chapter. The cited bibliography can be referred in the indicated chapters. M.1. METHODS FOR TYPIFICATION AND TAXONOMIC REVISION (CHAPTER 1) Currently accepted names of the Jasione, including homotypic and heterotypic synonyms, those listed in the Med-Checklist (Greuter et al., 1984), the World Checklist and bibliography of the Campanulaceae (Lammers, 2007) and the Euro+Med Plantbase (Castroviejo et al., LUIS MIGUEL SERRANO PÉREZ 66 2010) were considered. Other names were obtained from diverse sources (see below), mainly Stojanov (1926) and Damboldt (1978). The protologues and original material of names were analysed in detail and those names lacking a nomenclatural type were typified in accordance with the International Code of Nomenclature for algae, fungi, and plants (Turland et al., 2018). The following herbaria were consulted: B, BR, E, G, FI, K, MPU, P, SO, SOM, GOET and W. M.2. METHODS FOR DNA EXTRACTION AND AMPLIFICATION (CHAPTERS 2, 3, 4 AND 5) Total genomic DNA was extracted using either a modified cetyltrimethyl ammonium bromide (CTAB) method (Doyle & Doyle, 1990) or a commercial kit Nucleo Spin Plant II (Macherey-Nagel). In both cases 0.6 ul of 2-Mercaptoethanol was added during the first liquid step. Foliar tissue was homogenised either with manual grinder or using Mikro – dismembrator (Sartorius). DNA concentration and quality was assessed with Nanodrop 2000C (Thermo Scientific). In Chapter 2 and 4, four plastid DNA regions were amplified, the psbA-trnH intergenic espacer, the trnL-trnF intergenic spacer, the 3’ end of the intron of the ndhF gene and the rps16-trnQ intergenic spacer. After alignment and comparison with plastome sequences of members of the Campanulaceae in the GenBank it turned out that the conserved regions where the primers are anchored belong at the end of the trnL(UUAtrnT(UGU) spacer and the trnT(UGU) gene and the trnQ gene region. This can be related to the massive rearrangements underwent by plastid genomes in Jasione and in general the Campanulaceae family (Cosner et al., 2004). Therefore, the amplified marker will be denominated trnT(UGU)-trnQ. In Chapter 3 five regions of the chloroplast genome were amplified: the petD region, including the petB-petD intergenic spacer, the petD 5’ exon and the petD intron; the rpl32-trnL(UAG) intergenic spacer; the psbA-trnH intergenic spacer; the 3’extreme of ndhF gene intron; and the trnL-trnF intergenic spacer. The primers used for each marker are listed in Table 3.1, including the relevant Methods 67 references. One internal primer, called “trnL(UAG)-Jasi” was designed in this study for the rpl32-trnL (UAG) intergenic spacer to enable amplification of the region in some Jasione accessions with a huge insertion in the 3’ extreme of the region. In chapter 5 the complete ITS region (ITS-1, 5.8S gene, and ITS2) was amplified using the primers ITS1 and ITS4 (White et al., 1990). PCR reactions were carried out in 25 ul with PuReTaq Ready-To-Go PCR BEADS (GE Healthcare) and 80-150 ng of genomic DNA. Thermocycler was programmed for amplification of the ITS region with an initial denaturation step of 95ºC for 2 min, then followed by 31 cycles of 1 min at 94ºC, 1 min at 4648ºC and 2 min 10 s at 72ºC, with a final extension step of 8 min at 72ºC. PCR products were firstly assessed on 1.5 % agarose electrophoresis gels stained with the nucleic acid staining solution RedSafe (iNtRON biotechnology) and purified with E.S.N.A. Cycle Pure Kit. (Omega). DNA Sanger sequencing was carried out in an 8capillary 3500 analyzer (Applied Biosystems) by the DNA sequencing service of the University of Santiago de Compostela, Spain, or Software Sequencing Analysis 5.2 (Applied Biosystems) by STAB VIDA, Portugal. M.3. METHODS FOR PLOIDY ASSESSMENT BY CHROMOSOME COUNTING (CHAPTERS 2, 3, 4 AND 5) Direct chromosome counting was carried out in metaphases from root apices. Root tips measuring c. 5 mm were excised from cultivated plants grown from field collected individuals or from germinated seeds. All plants and seedlings were acclimated to outdoor conditions and were directly moved from temperatures around 15ºC or lower to the laboratory to conduct immediate root excision, always at first hours in the morning (before 9:00 a.m.) to avoid high laboratory room temperatures. Root tips were washed in distillate water a pre-treated in 2 MM 8-hydroxyquinoleine for 3 hours, followed by a rinsing step in distillate water and then a fixation step in freshly prepared 3:1 96% ethanol - 1 glacial acetic acid solution for 24 hours. Samples were LUIS MIGUEL SERRANO PÉREZ 68 thoroughly rinsed in 70% ethanol to remove glacial acetic acid, since it has been described to interfere with later chromosome staining (Norman et al., 2012). Samples were either stored in 70% ethanol at 4ºC for further use or directly used in the following steps. Root tips were rinsed in distilled water for 5 minutes, followed by an additional 5 minutes rinsing step in fresh distilled water. Samples were hydrolised by plunging the root tips in and Eppendorf tube with HCL 1 N for 6-9 minutes in a water bath at 60ºC. The tubes were pre-heated to bath temperature before root tip plunging. Two rinsing steps 5 minutes each in distillate water followed to eliminate all possible HCL remains. For mitotic analysis, root tips were stained in Schiff’s reagent for 12 hours in dark conditions. Stained root apices were excised under binocular magnifying glass, and immediately squashed on a slide. Cells were observed in a Leica TCS-SP2 confocal laser microscope (LEICA Microsystems Heidelberg GmbH, Mannheim, Germany) under a HCX PL APO CS 63.0x1.40 OIL objective, 580nm-725nm emission band and excitation wavelength of 561 nm with DPS (561 nm) laser diode. M.4. METHODS FOR PLOIDY ASSESMENT BY FLOW CYTOMETRY IN SILICA GEL DRIED MATERIAL (CHAPTER 2) Flow cytometry on silica-gel dried plants was carried in some populations, or in other individuals from populations in which chromosome number had been counted previously in at least one individual. Measurements were conducted on a Cell Lab Quanta Beckman Coulter flow cytometer (Beckman Coulter, USA) with a 100W Mercury arc lamp and excitation line optimized at 366 nm for UV estimations of DNA content with DAPI fluorochrome. Dried samples conserved in silica gel were prepared by chopping in 600 µL of Galbraith's buffer with 100 µg ml−1 RNase A. Pisum sativum ‘Express Long’ (2C = 8·37 pg) was initially used as internal reference, although samples from known diploid level (2n=12) of Jasione montana were used for rapid assessment of polyploid populations in other cases. Filtering was conducted in a 33-μm nylon filter and o 2 µg mL−1 of DAPI fluorochrome was added to the mixture. 10,000 particles were measured in each sample, and the 2Cvalues were calculated. As Methods 69 dehydrated instead of fresh samples were used, precise genome size estimation was ruled out, being the objective to grossly estimate the ploidy level of the sample by comparison of the position of plotted peaks. M.5. METHODS FOR SPECIES DELIMITATION WITH ASAP AND GMYC (CHAPTER 2) Assemble Species by Automatic Partitioning (ASAP) is a molecular method for species delimitation that uses group‐specific ad hoc thresholds without any a priori species hypothesis, that are estimated looking for the barcoding gap in the frequency distribution of nucleotide distances. It represents a transition between intraspecific and interspecific categories. (Puillandre et al., 2021). ASAP is based on a hierarchical clustering algorithm using only pairwise genetic distances from single locus sequence alignments to produce a species partition scheme ranked by a scored system (Puillandre et al., 2021). The current author used a set of plastid regions that are inherited together and therefore can be considered a single locus for Species Delimitation methods (Fujisawa & Barraclough, 2013). ASAP creates successive partitions from all terminals split to all lumped into a single group and assigns them a probability that quantifies the partition chances of being a single species. Them it computes the width of the barcode gap between the previous and the new partition. Probability and gap width are combined into a single asap-score that is used to rank the groupings, the lower the better (Puillandre et al., 2021). ASAP analyses were conducted in the program web-interface (https://bioin fo.mnhn.fr/abi/publi c/asap) with the three available substitution models, Jukes-Cantor (JC69) (Jukes & Cantor, 1969), Kimura 2P (Kimura, 1980), and simple p-distances. The analyses were run from a dataset with 14 sequences, including all detected haplotypes in the populations of study group plus a sequence from a population of the hexaploidy Jasione crispa subsp. crispa from the Pyrenees. Generalized Mixed Yule Coalescent (GMYC) model (Pons et al., 2006) was used to suggest hypothetical species as independent LUIS MIGUEL SERRANO PÉREZ 70 evolutionary units. GNMYC is an exploratory method using singlelocus sequence datasets and a ultrametric phylogenetic tree as input to estimate rates of branching to ascertain which parts of the phylogeny behave following population (coalescent) model, and which parts follow a speciation (Yule) model. The favoured species partition maximizes the likelihood of the transition between branching coalescent rates and branching speciation rates in absolute time, using an ultrametric tree, identifying a threshold between speciation and coalescent rates. This single threshold approach is based on the assumption that species are monophyletic, and therefore clades defined by a Most Recent Commons Ancestor (MRCA) reflect diversification events and can be considered species while branches descending from each of the MRCA nodes reflect coalescent events (Pons et al., 2006; Fujisawa & Barraclough, 2013). The set of nodes with highest Maximum Likelihood is chosen as the best species partition model. There is a version of the methods that allows to infer multiple thresholds, relaxing the assumption that all diversification events must be previous in time than all coalescent events (Monaghan et al., 2009). However, it has been demonstrated that this variant of the method is prone to over-splitting (Fujisawa & Barraclough, 2013; Kekkonen & Hebert, 2014), therefore its use was discarded. From each putative species sequences of at least six individuals have been included in the GMYC analysis. When more than one population was included in the sample, at least two individuals per population has been sequenced (mean 4.1 indiv./population). Fujisawa & Barraclough (2013) observed that increases in the number of samples individuals per species led to better performances of the method. The reason is that greater sampling might increase the detected branching rate and therefore the detection of a threshold in branching. However, greater species sampling, particularly without increasing within species sampling of individuals might reduce the ability of GMYC to detect species. The reason is that species monophyly is more easily inferred if random but incomplete sampling provokes that some close species are not included in the analysis (Bergsten et al., 2012). Hence, with the aim of an earnest procedure and to avoid unrealistic over-split results, it has been sampled the complete geographic range of the taxa investigated, even if it could Methods 71 hamper the detection of non-monophyletic species. As within species geographic structure could affect the accuracy of the method, causing that some relatively isolated populations could be delimited as separate species (Papadopoulo et al., 2008; Lohse, 2009), the sampling design aimed to include not only the main geographic groups by all linking populations. Nevertheless, simulations of the HMYC method showed that if the effective population size remains low relative to species divergence (that is, relatively low within species variation) the threshold is optimized at the correct point of the tree and the effect of geographic structuring is minimal (Fujisawa & Barraclough, 2013). Moreover, Talavera et al. (2013) observed that the remotion of intermediate haplotypes had little effect in the results and did not delimit as different species the most extreme haplotypes. The analyses have been conducted by both including repeated sequences and pruning repeated sequences. GMYC performance is affected by identical sequences because terminal zero-length branches affect the likelihood estimation (Monaghan et al., 2009; Fujisawa & Barraclough, 2013). However, when the tree is produced by a genealogy-based inference, as is the case of BEAST, the program does not assign null lengths to identical terminals, since they are treated as different haplotypes coalescing to a MRCA node, what allows GMYC to handle them. Talavera et al. (2013) did not observe differences in GMYC results when using repeated sequences from a tree inferred with BEAST relative to when they were removed, although they recommend collapsing all repeated sequences to only keep different haplotypes to reduce computational times. Conversely, Michanneau (2015) advocated the use of repeated sequences in GMYC analysis using a tree inferred by BEAST. BEAST could overestimate effective population sizes if identical sequences are removed by inferring higher unrealistic levels of genetic diversity (Michanneau, 2015). Since population sizes affect lineage coalescence, the remotion of identical sequences would increase average branch length, hampering the distinction between divergent and coalescent events. Michanneau (2015) considered that collapsing sequences to haplotypes would cause species over-splitting and higher uncertainty in the analysis. While the latter is a probably undesirable outcome, it could also be that keeping identical sequences LUIS MIGUEL SERRANO PÉREZ 72 from BEAST, with minimal (although not zero) lengths could push to the external nodes the species delimitation threshold. Two datasets have been included, one with all identical sequences collapsed to distinct haplotypes (dataset 1, the same as in ASAP) and other in which were retained different sequences in a proportion similar at how they were found in natural populations across the geographic range of the taxa. When one haplotype was found in only one population, it was represented only once in the dataset, either if it was the only haplotype in the populations or not. The possibility that one species could be represented by only one sequence in the dataset (i.e., a singleton) has no demonstrated negative effects in GMYC analysis (Talavera et al., 2013). GMYC analyses were carried out from trees estimated with BEAST2. The substitution model was HKY + G, with the Gamma count categories prior set four categories, as small number of categories of gamma distributed rates of evolution is enough to capture rate variation in small dataset (Jia et al., 2014). In the first dataset (collapsed to haplotypes) two BEAST priors were modified to test possible effects on the GMYC results. It is expected that variation in priors as branching pattern and rate of molecular evolution would not affect the inferred topologies, but the branch lengths will vary. Two different priors were used for the expected branching, a Yule model, also called pure birth model (Yule, 1925), and a Coalescent model with constant population size (Griffiths & Tavaré, 1994). The former is expected when the tree has a constant speciation rate while the latter describes branching patterns within the same species. For the Yule model analysis, the rate of molecular evolution was set to “constant clock”, meaning that the amount of nucleotidic mutations increases at a constant rate with time, and assumption suitable for small datasets of very related taxa. It was also tested a “relaxed clock” allowing that mutation rates could vary throughout the tree following a log-normal distribution. For the coalescent model with constant population size, it was only used a constant clock prior, as it is expected a within-species behaviour. The impact of prior variation has been studied in several works (Monaghan et al., 2009; Talavera et al., 2013; Michanneau, 2015). It has been indicated that the use of a Coalescent constant population model would Methods 73 be more conservative than the Yule model because GMYC also uses a coalescent model as null model of expected branching (Monaghan et al., 2009). In fact, these authors estimated higher species number with Yule model than with constant coalescent model. However, since this parameter affects branch lengths the prediction of how it would affect GMYC results is not straightforward and could be influenced by the amount of variation in the data (Michanneau, 2015). It was tested the effect of prior variation over GMYC results using the LRT test significance. After that, for the GMYC analysis with the second dataset (with repeated sequences) it was inferred a Bayesian phylogeny with the coalescent constant population branching and constant clock molecular rate of molecular evolution priors. Bayesian phylogenies were inferred in BEAST2, with 10 million generation MCMC lengths and two independent runs. Convergence was checked with Tracer v1.6 and after inspection of ESS values in each run a 10% burn-in and posterior sample combination was carried out in LogCombiner. Finally, a maximum credibility clade tree was summarized with TreeAnnotator for each analysis. In the analysis with relaxed clock model prior parameters were left as default in BEAUti2 and clock rate set to 1.0. BEAST ultrametric topologies and posterior probabilities were inspected in FigTree and exported in newick format, since it is required by the R package splits (Ezard et al., 2009) which was used to apply the GMYC method with single-threshold. Each GMYC analysis was repeated in the GMYC server (https://species.h-its.org/gmyc). Species Delimitations, LRT significances and GMYC (AIC) supports values were compared among the analyses. M.5. METHODS FOR MORPHOLOGICAL AND PHENOLOGICAL ANALYSES TO SUPPORT SPECIES DELIMITATION (CHAPTER 2) The putative species delineated by the GMYC and ASAP methods can be interpreted as hypotheses and to determine whether they are biologically sound other lines of evidence need to be investigated. LUIS MIGUEL SERRANO PÉREZ 80 2008; Frajman & Schneeweiss, 2009; Roquet et al., 2009; Cellinese et al., 2009; Mansion et al., 2012; Crowl et al., 2016). The two types of seeds assigned to the genus Campanula from the Upper Carpathian are notably different. In one case, they are regular Campanuloid seeds with a smooth seed coat (Lancucka-Srodoniowa, 1977). The other type shows a reticulate coat formed by cells divided by thin walls. The latter resembles the seeds of the extant Campanula pyramidalis and is in fact the type specimen of the extinct species Campanula paleopyramidalis, interpreted as an immediate ancestor of C. pyramidalis (LancuckaSrodoniowa, 1979). The fossil seeds occurred in a warm and wet environment dominated by peat bogs and swampy forests of tropicalsubtropical vegetal communities linked to extant south-eastern Asian flora (Lancucka-Srodoniowa, 1979). However, C. pyramidalis and its more closely related species are Italian-Balkan plants occurring in limestone crevices or rocky-garrigue Mediterranean dry habitats (Lakušić et al., 2013). C. carpatica is in the same clade as the C. pyramidalis complex (Mansion et al., 2012), from the Polish Carpathians, it has reticulate seeds (Olesen et al., 2012), although its habitat is upper mountain limestone cliffs. Moreover, reticulate seed ornamentation is not restricted to the C. pyramidalis group in the Campanulaceae. For example, it is not rare within the tribe Wahlenbergeae in the genus Wahlenbergia (Plunkett et al., 2009; Cupido et al., 2011). The tribe Wahlenbergeae is mostly distributed in the southern hemisphere and is almost absent in Europe, although the situation may have been different in the paratropical conditions of the European Miocene. However, the absence of past or current evidence of the Wahlenbergeae in Central Europe makes assignment of the fossil seeds to the genus Campanula more likely. Several works have used the Nowy Sącz fossil seeds to calibrate phylogenies, although with different perspectives. Roquet et al. (2009) and Olesen et al. (2012) set a calibration point of 16 Mya at a node ancestral to the tribes Campanuleae, Wahlebergeae, plus Jasione and its allied lineages. Cellinese et al. (2009) used the same age, but for an much more derived node, the stem node between C. pyramidalis and C. carpatica, arguing that these are the only two species with a seed similar to C. paleopyramidalis. This point was contested by Olesen et al., (2012), Methods 81 who stated that a reticulate coat is found in other Campanula species, such as the American C. scouleri. Mansion et al. (2012) followed the approach of Cellinese et al. (2009). Although in all of these cases, other calibration points were used (e.g. indirect dating of the root or other basal nodes from published angiosperm phylogenies), which nuanced the effects of the fossil seed calibration, the results from the two approaches are very different. For example, with the former approach, the divergence of two core Campanula clades (Campanula s. str. and “Rapunculus”) was estimated between 20 Mya (Roquet et al., 2009) and 13.5 Mya (Olesen et al., 2012), while with the latter approach it was estimated between 60 Mya (Cellinese et al., 2009) and 39 Mya (Mansion et al., 2012). Later, in a work focused on a group of eastern Mediterranean Campanula species, Crowl et al. (2015) still used the Nowy Sącz seeds to calibrate the stem node between C. pyramidalis and C. carpatica. These researchers used the 16 Mya calibration point as an older offset for a lognormal distribution of time with mean around 5 Mya. The approach used in the present work is different from those used in the previously cited studies. Although the literature dealing with the seed morphology of the Campanulaceae is not very extensive, it is known that seeds with reticulate coats appear in more groups than the C. pyramidalis clade (Belyaev, 1984; Shetler & Morin, 1986; Cupido et al., 2011). Within the genus Campanula, reticulate seed coat morphology can be found in either Eurasian species, such as the Anatolian C. olympica (Akcin, 2009), or American species, such as C. divaricata or C. prenanthoides, among other species (Shetler & Morin, 1986), and none of these belong to the C. pyramidalis-C. carpatica clade. Nevertheless, the position of all these species is nested in all published phylogenies within the “Rapunculus” clade of Campanula (Wendling et al., 2011; Mansion et al., 2012). The approach applied in the present work uses all this evidence and the fossilized seeds of Campanula sp. and Campanula paleopyramidalis from the Nowy Sącz Miocene to establish the calibration point within the Rapunculus clade, between the early divergence (in the current author’s data set) that led to the genera Hanabusaya and Adenophora and the lineage that led to other Rapunculus groups (represented in the current author’s data set) by C. LUIS MIGUEL SERRANO PÉREZ 82 pyramidalis and Campanula gr. rotundifolia. A late Burdigalian (around 16 Mya) Miocene scenario with the Rapunculus clade already diversified is plausible and supported by C. paleopyramidalis seeds. However, the scattered occurrence of reticulate seeds in the Rapunculus clade suggests that using this trait to calibrate the derived clade of C. pyramidalis-C. carpatica may be somewhat adventurous. Conversely, placing this calibration point at the base of the Campanuleae as in Roquet et al. (2009) seems too conservative. Other works, apparently unaware of the existence of the Nowy Sącz fossil seeds, and using other lines of evidence, have dated the early divergence of the Rapunculus clade in the late Burdigalian (16.5 Mya). Park et al. (2006) used divergence times inferred in the angiosperm phylogeny of Wikström et al. (2003). This approach was also used by Cano-Maqueda et al. (2008), who established a maximum age of 23 Mya to the Rapunculus clade based on the occurrence of Miocene Campanulaceae pollen records (Benton, 1993). In the current study, a lognormal prior distribution with mean 16 Mya and median 15.3 Mya and a standard deviation of 0.3 to the stem node was established for the node subtending C. rotundifolia and C. pyramidalis lineages. The root was also calibrated, as deep calibration points are needed to capture a larger proportion of the overall genetic variation (Duchêne et al., 2014). The root node represents the divergence between the subfamily Lobelioideae and the lineage leading to the Campanuloideae. Wikström et al. (2003) and Magallón et al. (2015) calculated mean ages for this node of 52 Mya and 46.5 Mya, respectively. Mansion et al. (2015) inferred a mean age of 76.5 Mya, although this inference is strongly influenced by the use of the C. paleopyramidalis seeds to date the split between C. pyramidalis and C. carpatica. Nevertheless, the existence of unassigned Campanulaceae pollen in the Late Cretaceous (72.1-66 Mya, Van Itterbeeck et al., 2007) is not very consistent with the inferred divergence between the Campanulacee and the Rousseaceae calculated to be about the same age by Magallón et al. (2015) and slightly earlier, about 80 Mya, by Wikström et al. (2003). Consequently, the inferred ages for the split between the Lobeliodieae and Campanuloideae calculated by these authors seem excessively recent. Therefore, in this study an age older than the mean age provided by these studies was used Methods 83 to calibrate this node and using instead their older boundaries. A normal prior distribution was established for the root, with mean in 56 Mya, standard deviation of 1 and 95% of the distribution of ages between 54 Mya and 58 Mya. The prior substitution model was established as TVM+G4, with a concatenated five partition scheme as the best-fit model selected by ModelFinder. Although BEAST2 does not include TVM as default model, the Site Model tab of BEAUTI2 can be established by selecting the GTR model and setting the AG rate parameter at 1.0. The analysis was preliminarily run with two Tree Models, a Yule model and Birth-Death model. The second would be more realistic when many terminals are absent, as in a data set with only a few representatives of each Campanulaceae group included. On the other hand, the Yule model demands fewer parameters. Nevertheless, no significant differences between the results of both models were observed. Final analyses were carried out with a birth-death tree prior. Five independent runs, each with four Monte Carlo Markov chains (MCMC) and 10 million generations, were carried out, sampling every 1,000 generations. Convergence as effective sample sizes (ESS) values in all parameters was evaluated with Tracer v1.7 (Rambaut et al., 2018) and burn-in was set to discard the first 25% trees, with the conserved trees combined in LogCombiner and summarized in TreeAnnotator (Drummond & Rambaut, 2007). Support for internal nodes was quantified by Bayesian posterior probabilities and node ages were set to median ages. M.9. METHODS FOR DIVERSIFICATION ANALYSIS (CHAPTER 3) The maximum-credibility clade tree from the BEAST2 time calibrated Bayesian analysis and a random subsample of 750 trees from the posterior distribution of the Bayesian analysis were used to create lineage-through time (LTT) plots to study diversification in Jasione. After the set of randomly sampled trees from the posterior distribution was plotted, a Confidence Interval (CI) was calculated for each tree (CI = (1α)-percent; α = 0.05) on the number of lineages given times, and plotted on the median LTT (Revell, 2012). The trees were cropped to only include Jasione terminals and pruned to conserve differentiated LUIS MIGUEL SERRANO PÉREZ 84 lineages. The LTT representations of the diversification and statistical tests were generated and performed in R v.4.1 (R Core Team, 2021) with the packages ape (Paradis et al., 2004) and phytools (Revell, 2012). The net rate of diversification under a birth-death model (r = λ − μ) was calculated on the LTT from the maximum-clade credibility tree, where λ is the rate at which new species are added through speciation and μ is the rate at which species are lost by extinction. The net rate of diversification and extinction fraction (ε = μ/λ) are key parameters to determine the mean clade diversification over time (Rabosky & Benson, 2021). Pybus and Harvey γ constantrates tests were performed on the LTT from the maximum-clade credibility tree to detect deviations from a pure birth Yule model, with two-tailed p-value for the γ-test (Pybus & Harvey, 2000). Accounting for unsampled lineages is considered essential, as missing terminals can cause the spurious detection of declines in diversification rates (Pybus & Harvey 2000; Morlon et al., 2012). Incomplete lineage sampling could favour the hypothesis of deceleration of diversification rates, leading to early burst interpretations (Fordyce, 2010). To avoid the effects of incomplete lineage sampling, a Monte Carlo constant rate test was performed, where the 0.05 p-value used to reject constant rate was calculated from a distribution of γ for simulated trees including incomplete lineage sampling comparable to the studied tree (Pybus & Harvey, 2000). Complementarily, any important departures in the obtained significance of the γ statistics between the standard Pybus and Harvey γ-test and the Monte Carlo constant rate γ-test will provide information about the completeness (or otherwise) of sampled lineages in the data set. A plot representing the computed Pybus and Harvey γ statistic through time is generated by slicing the tree in 100 points, with the function gtt in phytools, to facilitate the identification of the most influential evolutionary periods in the measured value of γ. M.10. METHODS FOR ANCESTRAL AREA RECONSTRUCTION AND BIOGEOGRAPHICAL ANALYSIS (CHAPTER 3) The ancestral area reconstruction and biogeographical analyses were performed on the cropped and pruned Maximum-Credibility Methods 85 Clade tree from the Bayesian posterior distribution. Two analyses were conducted, one retaining Hesperocodon as an outgroup and other “only Jasione”. As no major difference on the estimation of the ancestral area was detected, only the results of the latter analysis are shown. Six areas were considered: (A) north-western Africa; (B) the southern Iberian Peninsula; (C) northern and Central Iberian Peninsula, including the Pyrenees; (D) intermediate Europe including Italy and all of central and northern Europe; (E) the Balkan Peninsula; and (F) the Anatolian Peninsula (Figure 3.2). The biogeographical analysis was performed in R v.4 (R Core Team, 2021) with the package BioGeoBEARS (Matzke, 2013). This R-package implements six biogeographic models in a common likelihood framework: a likelihood version of DispersalVicariance analysis (DIVALIKE; Ronquist, 1997), LAGRANGE Dispersal and Extinction Cladogensis (DEC model, Ree et al., 2005; Ree & Smith, 2008), a likelihood version of BayArea (Landis et al., 2013), and an alternative version for each of the models that includes founder-event speciation (+J). BioGeoBEARS has two primary advantages over other biogeographical programs: (1) the best model is selected with likelihood ratio tests, and (2) founder-event speciation is included, a process ignored by most other methods. M.11. METHODS FOR ENVIRONMENTAL NICHE MODELLING (CHAPTERS 3 AND 4) In chapter 3, Species distribution modelling (SDM) was performed to compare niche suitability inferred from the potential distribution of the western and eastern groups under current climate conditions. Only taxa diverged from the common ancestor immediately older than the initial divergence leading to the E-W disjunction were included in the analysis. The approach used here pooled the occurrence data into two sets, the eastern and western groups. These groups are phylogenetically and geographically defined. Pooling of closely related species is one of the options to consider when most of the taxa are narrow endemics with small sample sizes (Ahmadi et al., 2021). A number of taxa of the LUIS MIGUEL SERRANO PÉREZ 86 eastern and western groups have < 5 occurrences, once the data are thinned to avoid spatial autocorrelation. To diminish excessive contribution of those taxa with more occurrences (e.g. Jasione sessiliflora in the west or Jasione orbiculata var. bosniaca in the east) no taxon had more than 6 occurrences in the pooled data. Nevertheless, the results should be interpreted with caution as pooling can lead to inflation of the predicted niches in each group (Hernández et al., 2006). The “eastern group” included the Balkan-southern Italian Jasione orbiculata complex, the Anatolian Jasione supina complex and those populations of the Balkan-Anatolian Jasione heldreichii complex, including populations assigned to Jasione jankae that were recovered in the previous clade (see Results). The purely annual populations of J. heldreichii recovered in a different clade are not included in any of the groups. The “western group” included the northern African Jasione cedretorum and the populations assigned to Jasione crispa subsp. lanuginella and Jasione crispa subsp. mesatlantica and all the southwestern European groups of Jasione sect. Jasione, and it excluded the annual-biennial Jasione montana and Jasione maritima, diploid and tetraploid lineages as forms related to the recently evolved J. montana lineage. The entropy algorithm implemented in MAXENT v3.3 (Phillips et al., 2006) was used for suitability modelling. The area of study includes Europe and the Mediterranean region. The occurrence was thinned to 5 km to prevent spatial autocorrelation. Nineteen bioclimatic variables for the area of study with current climatic conditions were downloaded from the WorldClim repository (Hijmans et al., 2005). Seven uncorrelated variables were selected: Bio1 (annual mean temperature), Bio3 (isothermality), Bio4 (temperature seasonality), Bio7 (temperature annual range), Bio9 (mean temperature of the driest quarter), Bio12 (annual precipitation) and Bio 19 (precipitation of the coldest quarter). These variables were used as model calibration predictors in MAXEN. Randomly selected occurrences (75%) from each geographic group were used as training data and the remaining occurrences (25%) were used as test data. In chapter 4, environmental modelling used nineteen layers of WorldClim bioclimatic variables (WorldClim website), in addition to Methods 87 five edaphic layers (Table 4.1) reported in the public database ISRIC world soil information (Hengl et al., 2017), because of the importance of the soil factor in the distribution of plant species belonging to acidophilic lineages. For example, soil pH is used as a proxy for the presence of cations in the region, although the spatial pattern of this variable in the environmental layer used is greatly influenced by precipitation (Hengl et al., 2017). In theory, this does not detract from the ecological significance of the variable, as precipitation tends to facilitate the creation of organic soils and leaching of basic cations. However, the soil pH in water is measured at 100-200 cm depth, relatively close to the bedrock, and is indicative of the nature of the substrate. The apparent density provides information about the water storage capacity of a soil, as well as about the structural conditions and degree of compaction (Mukhopadhyay et al., 2019). The resolution of the climatic and edaphic environmental layers is different (i.e. the pixel size of the data is different), being around 1 km (30'' of arc) in the first case and 250 m2 in the second. In order to combine environmental raster layers in ecological modelling processes, they must have the same resolution, as well as the same geographic extension and cartographic projection. The edaphic layers were adjusted to the resolution of the environmental layer with the R package “raster”. The variables finally chosen were selected for their ecological significance in relation to the taxa and because of the low collinearity between them, as indicated by Pearson's correlation coefficients < 0.8. For this purpose, a correlation matrix was generated in R (R Core Team, 2020) using the “gridExtra”, “rasterVis” and “reshape2” packages. Two sets of variables, i.e. climatic only and climatic plus edaphic variables, were used The “spThin” package (Aiello‐Lammens et al., 2015) reduces the number of presences until there is only one registered point per information pixel, thus eliminating those points for which the distance was less than 5 km in J. crispa subsp. mariana, J. sessiliflora subsp. sessiliflora, and J. crispa subsp. tomentosa. By contrast, in J. sessiliflora subsp. appressifolia, the minimum distance between LUIS MIGUEL SERRANO PÉREZ 88 presences was established at 1 km due to the low number of initial data points. One hundred repetitions were generated for each species, to check whether the number of records remained stable. The models were constructed with the R Biomod2 package and using Generalized Linear Model (GLM), Generalized Additive Models (GAM), MAXENT and Random Forest (RF), thus enabling the species records to be related to climatic variables. For this purpose, 4 validations and 3 replications of 5000 pseudo-absences per algorithm were run, creating a total of 48 models for each species. GLM and GAM are both regression methods that use the presence and absence of data. However, GAM, unlike GLM, can generate a non-linear model. MAXENT and RF are machine learning methods based on the presence of data on a background. MAXENT is based on the principle of maximum entropy for those models with a more uniform distribution (Philips & Dudik, 2008). The models created with the different algorithms for the taxa under study were evaluated with 20% of the data, and the remaining 80% of the data was used for calibration (Guisan & Zimmerman, 2000). The models were evaluated by threshold-independent statistical analysis: true skill statistic (TSS) and receiver operating characteristic (ROC). Models with TSS and ROC values > 0.7 were accepted, as this indicates that the predictions are more realistic than would result by chance. The different individual models were coupled creating consensus models. This assembly technique uses the weighted average and average statistical values to select the prediction that is closest to the actual observed data (Araújo & New, 2007). The coefficient of variation of the ensemble models was also calculated. For the model projections, the program QGIS 3.14 (QGIS Development Team, 2020) was used by loading the raster environmental maps, the study environment and the author’s own data. SDM maps with inferred grading of presence-absence optics were thus created. Niches of the four taxa were visualized in the environmental space using environmental PCAs (PCAenv), an ordination approach Methods 89 calibrated with environmental values of the area of study (Di Cola et al., 2017) using the R package “ecospat” (Broennimann et al., 2012). The PCA computes the occurrence density and environmental factor density for each pixel along environmental axes, maximizing the environmental variance of the distribution ranges of each taxon. PCA scores of the occurrences were projected onto a grid of cells bounded by the maximum and minimum PCA scores, allowing the comparison of niches in terms of breadth and location within the environmental space of the area of study (Di Cola et al., 2017). As a measure of niche overlap, the D value of Schoener (1970) was selected as a metric that varies between 0 (indicating no overlap between niches) and 1 (indicating complete overlap). The D values observed between pairs of species were used to test niche similarity (Warren et al., 2008; Broennimann et al., 2012). The niche similarity test determines whether the observed overlap value (D) differs from the overlap between the observed niche in the distribution of one species and randomly selected niches in the distribution of the other species. In this case, the centre of the mesh density is randomly selected from the available environmental space in the opposite distribution (Broennimann et al., 2012). The significance value was evaluated using 100 randomizations (α = 0.05). Niche similarity was tested for all pairs of taxa with the ecospat package. PCAenv and similarity tests were performed only with climatic variables to avoid enlarging the niche breadth visualization with factors such as pH with low variation across recent temporal scales. M.12. METHODS FOR CLONING THE ITS REGION OF THE NRDNA (CHAPTERS 5) PCR products from between two and four individuals from each population were used for cloning procedures, except in some cases when the source material was an herbarium specimen and only one individual was used. PCR amplification products were ligated into the pCR2.1-TOPO vector and cloned according to instructions of the TOPO-TA cloning kit (Invitrogen, Carlsbad, Germany). Plasmid DNA LUIS MIGUEL SERRANO PÉREZ 96 and SAV to the colorimetric information (L*, a* and b*) was assessed. For each species, data were standardized by subtracting the mean of measurements with no adaptor to preserve the units of deviation from the actual colour and lay out all measurements in the same multivariate space. The measurements were distributed in a 2-dimensional Euclidean ordination space using ggplot2 and Stat R packages (Wickham, 2009). To assess the similarity of the measurements obtained with and without the different tested adaptors, a QDA was performed. The individual measurements were assigned to predefined or actual groups (SAV and MAV with different adaptors or without adaptors), and the adaptor type most alike to SAV and MAV (i.e., those whose observations are significantly misclassified as observations obtained from SAV and MAV with no adaptors) were quantitatively identified from the results of the QDA confusion matrix. The analyses were run separately for SAV and MAV measurements and, in order to compare all species together, data were standardized by subtracting the mean of measurements, respectively from SAV or MAV with no adaptor. A Leave-Out-Out cross-validation was performed after the analysis to validate the QDA. Multivariate normality was assessed with the MVN R package (Korkmaz et al., 2014) and the MASS R package was used for the remaining analysis (Venables & Ripley, 2002). To verify the results pointed out by the QDA, an approach combining k-means and agglomerative clustering was performed on the same data from SAV, treated as for QDA. By k-means cluster analysis, similar colour measurements were categorized by defining clusters to minimize the total intra-cluster variation. The optimal number of kclusters in each species was determined using 30 indices provided by the R package NbClust (Charrad et al., 2014). The agglomerative clustering was conducted by computing a dissimilarity matrix based on Euclidean distances and the Ward.D2 agglomeration method in the Stats R package to produce a hierarchical tree for each species (R Core Team, 2020). The identified k-means groups were then superimposed on the dendrogram and, by means of the pvclust R package (Suzuki et al., 2006), the statistical support of all clusters was calculated with Methods 97 approximately unbiased (AU) p-values by multiscaling bootstrap resampling. Clusters with AU p-value equal to or greater than 95% were considered strongly supported by data. Results 101 CHAPTER 1. Nomenclature and typification of the Jasione L. (Campanulaceae) groups from the eastern Mediterranean basin ABSTRACT In this research all the published names of Jasione in the species, subspecies and variety ranks for populations from the Balkan and Anatolian peninsulas are reviewed, including names of taxa allied to these groups in surrounding areas. The taxonomic status and typification of the names are addressed. In total, eleven lectotypes, one lectoneotype and one epitype are suggested. Important note: This chapter is not an effective work of typification, according to the article 29 of the International Code of Nomenclature for algae, fungi, and plants this PhD dissertation this work is not an effective publication since it will not meet the required criteria. Thus, this work will not be published or printed in paper and distributed (through sale, exchange, or gift) to the general public or to scientific institutions with generally accessible libraries. Similarly, this work will not be distributed as a Portable Document Formant (PDF) in an online publication with an International Standard Serial Number (ISSN) or an International Standard Book Number (ISBN). Finally, and advice is included after all suggested types that the actual designation of types will be published elsewhere in a future effective publication. LUIS MIGUEL SERRANO PÉREZ 102 1. INTRODUCTION The genus Jasione L (Campanulaceae) is distributed around the Mediterranean basin and throughout Europe, with two centres of diversity at the opposite sides of the Mediterranean Sea, one in the Iberian Peninsula and Morocco and another in the Balkan and Anatolian peninsulas, consistent with the West-East Mediterranean disjunction biogeographical pattern (Thompson, 2005; Tarkhnishvili, 2014). The genus occurs in a number of ecological conditions and geological substrates across a wide elevational range, including coastal and alpine habitats. The combination of environmental niche diversity, complex geological history of the Mediterranean basin and ploidy variation, which includes five ploidy levels (Küpfer, 1981; Lammers, 2007; M. Serrano, own data), coupled with relative stasis around a general morphological pattern (i.e. medium-sized or small plants with condensed inflorescences in flower heads surrounded by involucral bracts and small flowers with connate anthers), has produced a large number of polymorphic populations frequently lacking clear morphological distinctiveness. In consequence, many names and different taxonomic ranks have been used for members of the genus (e.g. Sales & Hedge, 2001a; Lammers, 2007; Dobignard & Chatelain, 2011). However, most of these names are of unknown evolutionary significance (Pérez-Espona et al., 2005), leading to difficulties in species delimitation by morphological approaches (Kovanda, 1968). General treatment of the genus thus tends to be overly summarized, with the number of species recognized varying between 12 (Sales & Hedge, 2001b) and 17 (Castroviejo et al., 2010). The evolutionary lineage of Jasione seems to be prone to micro-endemism and cryptospeciation, and many of the historically described forms, despite being disregarded by current taxonomic treatments, may therefore deserve further attention. Thus, a correct nomenclatural understanding of the clutter of names described so far is particularly important in studies dealing with evolutionary relationships, species delimitation and biodiversity conservation in such complex groups. Two important nomenclatural summaries have been provided for the centre of diversity of Jasione in areas of the western Mediterranean region. Dobignard & Chatelain (2011) reviewed the synonym names CHAPTER 1. Nomenclature and typification of the Jasione L. 103 published for North African populations, while Sales & Hedge (2001a) tackled the revision of the published names and nomenclatural types of the taxa occurring in the Iberian Peninsula. Parnell (1987) had previously addressed the nomenclature of Jasione montana L. and other taxa that he considered related, dealing with a huge number of published names associated with the wide range and considerable polymorphism of J. montana, which occurs in north-western Africa and most of Europe (although it is almost absent in the Balkan area). These works were later complemented by nomenclatural precisions in the Iberian-North African Jasione corymbosa poir. ex Schult complex (Ferrer-Gallego et al., 2015). By contrast, there are no recent published revisions for the eastern centre of diversity of the genus. The most recent comprehensive work dealing with Jasione in the Balkan and Anatolian peninsulas is that by Stojanov (1926), with a taxonomic treatment recognizing 13 species and varieties, one from the Italian peninsula. This author addressed the alpine taxa of what he called the Jasione supina Sieb. ex Spreng. group, a number of mostly narrow endemic orophytes occurring across the different mountain ranges in both peninsulas. This group should include the Jasione supina complex, the Jasione orbiculata Griseb. ex Velen. complex and Jasione bulgarica Stoj. & Stef. Nevertheless, the evolutionary coherence of this putative group and its internal relationships remain unknown. Stojanov (1926) did not consider the Jasione heldreichii Boiss. & Orph. complex, the other remaining group occurring in the Balkan and Anatolian peninsulas. Parnell (1987) only partly addressed the nomenclature of this complex, describing one variety occurring on the Turkish coasts. A wider study is required that would encompass the nomenclature of the group, within which diverse names with difficult taxonomic interpretation have been described (Hartvig, 1991). Tutin (1973) subordinated Jasione orbiculata as a subspecies of Jasione laevis Lam. in his treatment of Flora Europaea (Tutin, 1976). This rather questionable view overshadowed all taxa described or recognized by Stojanov (1926) within this species, probably influencing later general treatments that only listed Jasione orbiculata (Greuter et al., 1984; Lammers, 2007; Castroviejo et al., 2010). The fact that these taxa LUIS MIGUEL SERRANO PÉREZ 104 have the rank of variety has also hampered their modern recognition, although some of them have been included in regional floras (e.g. J. orbiculata subsp. balcanica Urum. in Ančev, 2012). Damboldt (1976, 1978) tackled the taxonomic diversity of the Anatolian Jasione orophytes, recognizing two species in this group, i.e. Jasione idaea Stoj. and Jasione supina, the latter of which has four subspecies. He designated nomenclatural types to several names in the Flora of Turkey and the East Aegean Islands (Damboldt, 1978), although in the opinion of the current author a formal typification of some of them is still lacking. The research presented here aims to present a nomenclatural revision of the genus Jasione in the Balkan and Anatolian peninsulas, the eastern centre of diversity of the genus, and complements the current author's studies on taxonomy and evolutionary relationships in the genus Jasione. The nomenclatural situation of the Balkan and Anatolian taxa of Jasione, including the Jasione heldreichii, Jasione supina and Jasione orbiculata taxonomic complexes, are addressed by summarizing synonyms and suggesting types that will be effectively designated in a future valid publication. The application of the many names described from the aforementioned regions plus one from southern Italy is discussed. 2. MATERIAL AND METHODS Currently accepted names, including homotypic and heterotypic synonyms, those listed in the Med-Checklist (Greuter et al., 1984), the World Checklist and bibliography of the Campanulaceae (Lammers, 2007) and the Euro+Med Plantbase (Castroviejo et al., 2010) were considered. Other names were obtained from diverse sources (see below), mainly Stojanov (1926) and Damboldt (1978). The protologues and original material of names were analysed in detail and those names lacking a nomenclatural type were typified in accordance with the International Code of Nomenclature for algae, fungi, and plants (Turland et al., 2018). The following herbaria were consulted: B, BR, E, G, FI, K, MPU, P, SO, SOM, GOET and W. CHAPTER 1. Nomenclature and typification of the Jasione L. 105 3. NOMENCLATURE AND TYPIFICATION OF THE JASIONE COMPLEXES FROM THE EASTERN MEDITERANEAN BASIN All names described for Jasione in the Balkan and Anatolian peninsulas at the species, subspecies and variety ranks were analysed. Thus, the members of the Jasione orbiculata complex were included, and the representative of the complex in southern Italy is therefore also treated here. The review presents basionyms in alphabetical order, structured as a discussion of each name followed by its synonyms and typification, suggesting types when required, that will be designated as types in a future effective and valid publication. A list of currently accepted names from recent checklists (e.g. Lammers, 2007) is provided, and the heterotypic synonyms are indicated (Table 1.1). Inclusion of a name in the first column of Table 1.1 only reflects its situation in current general lists, as recognized names may be organized differently after ongoing evolutionary studies on Jasione. Those names that would deserve recognition in light of such studies are highlighted in colour in the second column. Table 1.1. Names currently accepted in general treatments are ordered alphabetically in the first column and marked in bold, with different basionyms indicated. The second column lists the heterotypic synonyms. The names of evolutionary entities deserving recognition are highlighted in blue. Those names that may represent significant evolutionary entities, but for which more detailed studies are required, are highlighted in green. Accepted name Heterotypic synonym(s) J. bulgarica Stoj & Stef. ≡ Jasionella bulgarica (Stoj. & Stef.). = Jasione orbiculata var. orbelica Vel. J. heldreichii Boiss. & Orph.) ≡ Jasione montana L. f. heldreichii = J. glabra Vel. = J. heldreichii var. microcephala Vel. = Jasione heldreichii var. papillosa J.Parn. = J. montana var. dentata f. microcephala (Vel.) Stoj & Stef. = Jasione jankae Neilr. = Jasione montana var. dentata DC J. idaea Stoj. LUIS MIGUEL SERRANO PÉREZ 112 Schmeja in Beih. Bot. Centralbl. 48(2):33 (1931) – Lectotype (designated by Parnell 1987: 265): Macedonia, 1857, Orph. nº 3130 (G barcode G60078161 [photo!]). Figure 1.3. Lectotype of Jasione heldreichii (G60078161). © Conservatoire et Jardin botaniques de la Ville de Genève CHAPTER 1. Nomenclature and typification of the Jasione L. 113 3.4. Jasione heldreichii var. papillosa Parnell (1987) used this name to encompass some morphologically deviant populations of the group J. heldreichii occurring on the Turkish European coast of the Black Sea. These plants are characterized by densely papillose bracts and leaves with thickened margins, and shorttriangular calyx teeth. The Holotype, held in herbarium E, originates from the T. Uslu nº 2070 collection from Istanbul, Terkos Gölü. The specimen has the E barcode E00913661 and is shown here in Figure 1.4. Although these populations may simply be coastal ecotypes of J. heldreichii, the evolutionary significance must be studied in greater detail as they display some different traits from other dune populations of Jasione heldreichii from the Aegean and Black seas. J. heldreichii var. papillosa is remarkably distinct from the typical J. heldreichii, showing morphological convergence with populations of Jasione montana from sand dune habitats in the western Mediterranean region under similar climatic conditions (i.e. subhumid Mediterranean climate). Indeed, this is the reason for the inclusion of Jasione montana in the Flora of Turkey and the East Aegean Islands (Damboldt, 1978), an enduring incorrect interpretation of these populations (e.g. Çelemli, 2020). Jasione heldreichii var. papillosa J.Parn., Watsonia 16 (3): 266 (1987) - Holotype: (Designated by Parnell 1987: 265): Turkey, Istambul, Terkos Gölü, Uslu nº 2070 (E barcode E00913661 [photo!]). LUIS MIGUEL SERRANO PÉREZ 114 Figure 1.4. Holotype of Jasione heldreichii var. papillosa (E00913661). CHAPTER 1. Nomenclature and typification of the Jasione L. 115 3.5. Jasione idaea This name was published by Stojanov (1926) to describe a narrow endemic species in the Kazdağı, the ancient Mount Ida, in northwestern Anatolia, using one collection of Sintenis (Iter trojanum, 1 Aug. 1993, nº 532). Damboldt (1978) addressed the typification of this name from this collection. However, he designated a holotype by indicating “holo. W! iso. B, M, E!, G! GOET, K! LD”. Many specimens exist from the original collection, and the lectotype should be designated in accordance with article X.X of the International Code. Therefore, the current author suggested one of the many specimens from the original collection as the lectotype, the sheet in herbarium E with barcode E00913653 is shown in the Figure 1.5. Jasione idaea Stoj. in Notizbl. Bot. gart. Berlin 9: 556 (1926) - Lectotype (will be designated in a future effective publication): Turkey, Kazdağı (ancient Mount Ida) 1 August 1883, Sintenis nº532, iter Trojanum (E barcode E00913653 [photo!]). LUIS MIGUEL SERRANO PÉREZ 116 Figure 1.5. Lectorype of Jasione idaea (E00913653). CHAPTER 1. Nomenclature and typification of the Jasione L. 117 3.6. Jasione jankae This name is listed as a synonym of Jasione heldreichii Orph. ex Boiss. in Flora Europaea (Tutin, 1976), the Med-Checklist (Greuter et al., 1984) and in the World Checklist of the Campanulaceae (Lammers, 2007), although it is recognised in the Euro+Med PlantBase (Castroviejo et al., 2010), as well as in different regional floras from the Balkan Peninsula (e.g. Velenovský, 1891; Hayek, 1931; Hartvig, 1991), while other floras consider it a synonym of J. heldreichii (e.g., Stojanov & Stefanov, 1948; Ghişa 1964; Ciocârlan,1990; Ančev, 2012). Neilreich (1870) described this species from material collected in the rocky areas of Treskovac, NW of Svinica, in Romania (current names in Romanian language, Trescovăţ and Sviniţa). In herbarium C (C barcode C10009017) there is a gathering collected by Viktor Janka in “rupestribus m. Treszkovácz inter Drenkova et Svinieza ad Danubium inferioren Banatus', on 4 July 1870 during his Iter Banaticum. The specimen is identified in the sheet as Jasione jankae Neilr. and two manuscript labels dated September 2004 are glued on it (Figure 1.6). One label, by I. C. Hedge, indicates that “the formal lectotypification of J. jankae will be published in due course”, and the other, by S. Prince, indicates “Lectotype of Jasione jankae Neil. = J. heldreichii Boiss & Orph.” The current author has no evidence of any subsequent formal publication and therefore has suggested this specimen as the lectotype. There is no doubt that this material was used by Neilreich to describe the species, as correctly pointed out by I.C. Hedge and S. Prince, as the label clearly conveys the location cited in the protologue and the specimen matches all points in Neilreich’s description. The status of Jasione jankae in relation to Jasione heldreichii has been controversial, even in some of the treatments that recognize it as different from J. heldreichii (Hartvig, 1991). Ciocârlan (2006) addressed the question by adopting the position of differentiated species, but provided little more evidence than the differing morphological features already described in the protologue. Thus, J. jankae may refer to plants similar to J. heldreichii but differing in the perennial habit with sterile rosettes, rather than to annual-biennial habit without rosettes in J. heldreichii, dentate bracts but with teeth half the LUIS MIGUEL SERRANO PÉREZ 118 width of the bract teeth rather than pinnate-pectinate bracts with aristate apex. However, as intermediate individuals are found, a wider concept of Jasione heldreichii, including plants from mountain or northern areas where sterile rosettes are more frequent, may appear to be more consistent with the current state of knowledge. Nevertheless, more detailed studies are needed to ascertain whether the observed variation can be linked to independent evolutionary lines. Different degrees of introgression in J. heldreichii populations from current or past sympatric populations of representatives of the J. orbiculata and J. supina perennial groups should not be ruled out as a source of morphological and ecological differentiation within the Jasione heldreichii complex. Jasione jankae Neilr. Diagn. Aufz. Ungarn. Slavon, Gefässpfl., Nachtr.: 43 (1870) ≡ Jasione heldreichii Janka ex Nyman , Consp. Fl. Eur. 2: 486 (1879) ≡ Jasione montana var. jankae (Neilr.) Stoj. & Stef., Фл. Бълг. изд, 1,2: 1094 (1925) ≡ Jasione montana f. jankae (Neilr.) Schmeja in Beih. Bot. Centralbl. 48: 33 (1931) - Lectotype (will be designated in a future effective publication): Romania, Sviniţa, Trescovat peak. 4th July 1870, V. Janka s.n. (C barcode C10009017 [photo!]). CHAPTER 1. Nomenclature and typification of the Jasione L. 119 Figure 1.6. Lectotype of Jasione jankae (C10009017). LUIS MIGUEL SERRANO PÉREZ 120 3.7. Jasione montana var. dentata DC. De Candolle (1838) described this name from plants collected by P.M.R. Aucher-Eloy in the “Olympo Bithino”, i.e. in the UluDag, Turkey. The G-De Candolle herbarium (GDC) holds a gathering collected by Aucher-Eloy from the type location in 1837, number 1880 (G barcode G00329536), matching the morphology described in the protologue (i.e. spiny-dentate acute bracts) shown here in Figure 1.7. The current author is not aware of any formal description of the type species of this name and has therefore suggested this specimen as the lectotype. Another sheet from the Aucher-Eloy nº 1880 collection in K (K barcode K000781177) would be an isolectotype. This name, and the more frequently recognized Halacsy’s combination (1902) Jasione dentata (DC.) Halacsy has been used for plants from Anatolia and from the Balkan Peninsula (e.g. Halacsy, 1902; Josifović, 1974). Although the most commonly followed current treatments consider this name a synonym of Jasione heldreichii (Tutin, 1976; Ančev, 2012), more detailed evolutionary studies are required to clarify this point, given the complexity of the group. Jasione montana var. dentata DC. in Prodr. VII.:415 (1839) ≡ Jasione dentata (DC) Halacsy, Consp. Fl. Gre. 2: 280 (1902) - Lectotype (will be designated in a future effective publication): Turkey, Bursa province, Uludağ (Olympo Bithino), 1837, P.M.R. Aucher-Eloy 1880 (G barcode G00329536 [photo!]). CHAPTER 1. Nomenclature and typification of the Jasione L. 121 Figure 1.7. Lectotype of Jasione montana var. dentata (G0032953). LUIS MIGUEL SERRANO PÉREZ 128 Figure 1.10. Lectotype of Jasione orbiculata var. italica (FI064103). © Museo di Storia Naturale, FI Herbarium, Università degli Studi di Firenze CHAPTER 1. Nomenclature and typification of the Jasione L. 129 3.12. Jasione orbiculata var. supinoides This name is considered a synonym of Jasione orbiculata s. str. by Lammers (2007) and is not recognized in regional floras or taxonomic lists (Hartwig, 1991; Dimopoulos et al., 2013). Stojanov (1926) described the name from material collected in the Kaimaktsalan massif, on the border between North Macedonia and Greece, and from Smolikas peak in the Pindos mountain range in Epirus, Greece. However, as noted by Hartvig (1991), there is significant variation among the Greek populations, with mat-forming plants with creeping, rooting stems and ovate-rhomboidal bracts in the SW populations and more cespitose plants with narrower lanceolate-linear bracts in the northernmost Greek populations. While the epithet “supinoides” (i.e. resembling J. supina Sieb., a cespitose plant) would be suitable for populations from the mountains on the border between North Macedonia and Greece, the description in the protologue could encompass all of these Greek forms. However, the mention of oblongorhomboidea bracts is more consistent with the plants from the Pindus mountain range. The current author has located two sheets in herbarium P (P barcodes P00270608 and P00270609) of a collection of Antonio Baldacci from Smolikas, one of the three gatherings cited by Stojanov in the protologue. The current author has not found any evidence of previous typification of this name and has therefore suggested one of these sheets as the lectotype (Figure 1.11). Jasione orbiculata var. supinoides Stoj. Notizbl. Bot. Gart. Berlin-Dahlem 9; 554 (1926) - Lectotype (will be designated in a future effective publication): Greece, Epirus, mount Smolika, supra Kerasovo. 18 July 1986, A. Baldacci nº 247 (P barcode P00270608 [photo!]). LUIS MIGUEL SERRANO PÉREZ 130 Figure 1.11. Lectotype of Jasione orbiculata var. supinoides (P00270608). CHAPTER 1. Nomenclature and typification of the Jasione L. 131 3.13. Jasione orbiculata var. orbelica Velenovský (1898) described this name in supplementum I of Flora Bulgarica. In this work he tackled the morphological description of two varieties of Jasione orbiculata Griseb., conveying doubts about which was Grisebach’s plant. He left the first variety unnamed, supposedly because he found it was more similar to the typical J. orbiculata, although this is not certain. Secondly, the var. orbelica was described as a plant with longer, thicker and straight stems, larger denticulate leaves and glabrous acutely denticulate bracts. Stojanov & Stefanov (1921) and Stojanov (1926) noted that the morphological description of this name matched Jasione bulgarica. However, as Velenovský (1898) said nothing about one of the main features of J. bulgarica, i.e. the free anthers, the description could also match some forms of J. orbiculata. Stojanov (1926) thought that this was the reason for Velenovský’s doubts about which of the two varieties that he morphologically described would correspond to Grisebach’s Jasione orbiculata. Nevertheless, the forms of Jasione orbiculata that could match Velenovský’s description do not occur in Bulgaria, where populations that Stojanov (1926) called Jasione orbiculata var. bosniaca are found. Velenovský (1898) cited material collected by Stribrný in Gjumrukcal and Musala peaks, by Reiser in the Demir Kapija-Balkan peak, and by Urumov in the Trojan-Balkan mountain range, without linking these localities to any of the varieties. The current author has located two gatherings from Velenovský’s collection in the PRC, with manuscript labels by this author indicating “J. orbiculata var. orbelica”: one collected by Stribrný in 1897 in Musala (PRC code PRC451250) and the other collected in 1893 by Reiser in Demir Kapija (PRC code PRC451251). The latter only includes plants matching the description in the protologue, while the former includes two different types of plants, with one at the bottom that does not match the description, but corresponds to Jasione orbiculata var. balcanica Urum., and five stems in the upper part of the sheet that match the description of var. orbelica. To avoid any ambiguity, and in the absence of any evidence of previous formal typification of the variety, the current author has suggested Reiser’s sheet annotated by Velenovský LUIS MIGUEL SERRANO PÉREZ 132 as the lectotype (Figure 1.12). In any case, this name is a synonym of Jasione bulgarica. Jasione orbiculata var. orbelica Vel., in Fl. Bulg. Suppl. 1: 188 (1898) - Lectotype (will be designated in a future effective publication): Bulgaria, Demir Kapija peak (Pirin mountain range), 1893, Reiser s.n. (PRC barcode PRC451251 [photo!]). CHAPTER 1. Nomenclature and typification of the Jasione L. 133 Figure 1.12. Lectotype of Jasione orbiculata var. orbelica (PRC451251). LUIS MIGUEL SERRANO PÉREZ 134 3.14. Jasione propullulans Freyn & Brandis (1888) equated this name to Jasione orbicularis (sic) Griseb., evidently meaning Jasione orbiculata Griseb. The species is not recognized in Flora Europaea (Tutin, 1976), the Med-Checklist (Greuter et al., 1984) or the World Checklist of the Campanulaceae (Lammers, 2007) and is not even listed as a synonym. However, it is considered a validly published synonym of Jasione supina Sieb. subsp. supina in Plants of the World Online (POWO, 2021). The description indicates that the plant is from Vranica Planina, in Bosnia. As Jasione supina subsp. supina is a plant from eastern Anatolia, the synonym status in POWO (2021) can be ruled out. The origin of this misinterpretation seems to be based on a comment in the protologue stating that this plant is the same as that collected under the name Jasione supina by Velenovský in 1887 in western Bulgaria. This name could have been given greater consideration if it had been validly published, in which case the populations of the taxon known as Jasione orbiculata var. bosniaca Stoj. would have been given a higher taxonomic rank. However, the indication by Freyn & Brandis (1888) does not include any morphological description, and therefore it was not correctly published according to the International Code of Nomenclature. nom. nud. Jasione propullulans in Freyn Verh. K. K. Zool.- Bot. Ges. Wien 38: 618 (1888). 3.15. Jasione supina This name was published by Sprengel (1825), who recognized Sieber as the author's name and vaguely indicated “Asia minor?” as the type locality. Damboldt (1976) revised the names of the Jasione supina group, adopting De Candolle’s (1838) concept of the species for use in the Flora of Turkey and the East Aegean Islands (Damboldt, 1978). This interpretation of Jasione supina is based on the population collected by Aucher (nº 1881) in 1837 from the “Bithynian Olympus”, i.e. the Uludağ mountain in Bursa province, Turkey. Neither Damboldt nor the current author have located any original material collected by Sieber, CHAPTER 1. Nomenclature and typification of the Jasione L. 135 although it may exist somewhere. Damboldt (1978) addressed the typification of this name with Aucher’s nº 1881 gathering, indicating “Neotype: Turkey, Bursa, Olympus Bithynus (Ulu Da.), 1837, Aucher 1881 G! W!)”. Given that two sheets in different herbaria were designated as neotypes there is some ambiguity regarding the actual type. Therefore, according to the International Code, the current author has suggested the sheet in G (G barcode G00329587) as a lectoneotype (Figure 1.13). Jasione supina Sieber ex Spreng., Syst. Veg. I :1810 (1825) emend. DC., Prodr. 7:416 (1839). Lectoneotype (will be designated in a future effective publication): Turkey, Bithynian Olympus (Uludağ mountains), 1837 Aucher (nº 1881) (G barcode G00329587 [photo!]). LUIS MIGUEL SERRANO PÉREZ 136 Figure 1.13. Lectotype of Jasione supina (G00329587). CHAPTER 1. Nomenclature and typification of the Jasione L. 137 3.16. Jasione supina subsp. akmanii This name and rank are recognized by Greuter et al. (1984) and Lammers (2007), although the close affinity to Jasione supina subsp. pontica (Boiss.) Damboldt has been noted since inception of this name (Damboldt, 1976). This plant was described as a narrow endemic from the volcanic mountain range of Köroglu, in Bolu province (Damboldt, 1976; 1978), differing from subsp. pontica only by the entire bracts instead of dentate bracts. Damboldt only had access to one collection (Akman nº 3051, 14 VII 1975) and in the protologue indicated (holo. ANK), while in Flora of Turkey (Damboldt, 1978) wrote “Type (holo E! iso. ANK). The current author has found one specimen in E (E barcode E00913665) identified as type by a manuscript label by Damboldt. This may imply that the only indication of ANK for the type in the protologue is a typographical error that deleted the reference to “E!”. If this were the case, the fact that at least two specimens from the original material collected by Akman exist (in E and ANK) and the unfortunate putative misprint designating as holotype some material explicitly not seen by Dambold, and therefore, not used by him to prepare the protologue, would leave this name without proper typification. Nevertheless, the absence of the “!” after ANK (Damboldt, 1976; 1978), indicating that this material was unseen by this author, could also be attributed to a typographical error. For the sake of precaution, the current author will be conservative and consider that Damboldt properly designated in the protologue a holotype existing in the herbarium ANK. 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(Campanulaceae) ABSTRACT Groups with morphological stasis are an interesting framework to address the possible recognition of cryptic species hidden behind traditional taxonomic treatments, particularly when distribution ranges suggest disjunct and environmentally heterogeneous biogeographic patterns. New hypotheses of delimitation of evolutionary independent units can lead to the identification of different biogeographic patterns, laying the foundation to investigate their historical and ecological significance. Jasione (Campanulaceae) is a plant genus with a distribution centered in the Mediterranean basin characterized by significant morphological stasis. Jasione sessiliflora s.l., and allied taxa have disjunct distribution ranges within the Iberian Peninsula, occupying environmentally diverse regions. At least two ploidy level, diploid and tetraploid, are known to occur in the group. These aspects motivated the assessment of the internal variability in the group with phylogenetic tools for species delimitation. The results from GMYC and ASAP analyses are compared with other lines of evidence, including morphology, cytology and phenology. The fitting of distribution patterns of the inferred entities to chorological subprovinces is used as a biogeographical and environmental framework to test species hypothesis. Despite the scarcity of diagnostic morphological characters in the group, phylogenetic delimitation supports the description of at least one cryptic species, a narrow endemic in NE Iberian Peninsula, and the separation from J. sessiliflora of a group of populations from thermophile environment in eastern LUIS MIGUEL SERRANO PÉREZ 152 Iberia. Although disjunct ranges suggested the assessment of population groups, not all disjunct populations pointed to independent evolutionary histories. Ploidy differences support the systematic rearrangement suggested by species delimitation. Taxonomic reorganization in J. sessiliflora s.l. allowed biogeographic interpretations of distribution patterns that are in accordance with biogeographical regionalization. These results suggest that species differentiation, together with geographic isolation and polyploidization was associated to adaptation to different environments, shifting from more to less thermophilic conditions during the evolutionary history of the J. sessiliflora group. Thus, the recognition of concealed evolutionary entities is essential to correctly interpret biogeographical patterns in regions with a complex geologic and evolutionary history such as the Mediterranean basin. 1. INTRODUCTION The accelerated global biodiversity decay (Hallmann et al., 2017) urges the need to identify units of conservation representing evolutionary significant entities, being the species the most important recognized one (Richardson & Whittaker, 2010). Species has been defined as independent evolving groups of organisms that are genetically and phenotypically distinct from other such groups, a definition that encapsulates both the pattern of species and the causes of the pattern (Barraclough, 2019). Independent evolution leads to divergence, due to the interruption of cohesive processes related to gene flow. Species would be the most resolved unit of diversity with independent evolution. Although populations could develop in isolation they still are not identified as independently evolving entities on account of insufficient time to accumulate diagnosable signatures (Barraclough, 2019). Accumulation of genetic differences and actual independent evolutionary paths are not always correlated with diagnosable differences in morphological traits, as happens in taxonomic groups with high degree of morphological stasis and, therefore, prone to cryptic speciation. Cryptic species would remain undetected, what hampers ecological approaches studying these CHAPTER 2. Species delimitation in the genus Jasione L. 153 complexes. The reason is that cryptic entities would have evolved without clear morphological differences. However, accumulation of differences in environmental preferences is probable, always within the general evolutionary framework of niche conservatism between sister species (Peterson et al., 1999, Wiens & Graham, 2005). Researchers studying groups with hidden entities inadvertently could mix information that should be treated separately, what could lead to propose meaningless biological hypotheses. Finally, and more importantly, these species are excluded from legal lists and therefore conservation policies will be blind to them. Evolutionary lineages with morphological stasis can be composed by species with few, if any, clearly discriminating morphological characters, although genetic differentiation and interruption of gene flow between the different lineages could have a relatively long evolutionary history. However, other phenotypic traits can be investigated to detect significant differences, among them those related with ecological preferences or habitat (Prada & Helberg, 2013; Michonneau. 2015). The use of molecular information can uncover hidden significant specific divergence supported by Species Delimitation methods. It allows researchers to focus on the detection of other corroborating signatures of speciation, on the knowledge of the expected distribution of discontinuities between populations. The Evolutionary Significant Unit (ESU) concept (Moritz, 1994) defines species as units with reciprocal monophyly in at least one molecular marker plus at least other defining trait, including morphology, distribution or in general reciprocal monophyly in another character (Michonneau, 2015). Although the original ESU concept includes differences in distribution among the defining traits, a direct application of reciprocal monophyly and separated ranges would lead to an inflation of species recognition in many taxa. Nevertheless, it could be advisable to include information from species distribution if it indirectly offers relevant information about the environmental envelope of the putative evolutionary entity, The environmental niche in which a species thrives is ultimately conditioned by intrinsic physiological constraints. Evolved ecological tolerances make up the fundamental niche, that is, the potential environmental space that a species could occupy (Hutchinson, 1957). LUIS MIGUEL SERRANO PÉREZ 256 through the Peninsula, the lineage (J. orbiculata var. italica) could be constrained to thrive in oro-Mediterranean environments just in the interface with the temperate formations. As a final remark, the phylogenetic results suggest the need of a taxonomic reorganization of the genus and the recognition of diverse evolutionary significant units of diversity as species or subspecies. 5. CONCLUSIONS The reconstruction of the biogeographical and evolutionary history of Jasione evinces how its diversification has been configured since the inception of the Miocene by climatic changes and geological events by recurrent range expansions and subsequent range fragmentations, with long Miocene-Pliocene periods of diversification decay of the extant leinages, and a Pleistocene burst of diversification. A western origin of the genus is suggested, with eastern colonization occurring by only one of the two main lineages. Within this clade, several range expansions can be inferred, giving a major role to the eastern area of the Mediterranean as source of lineages, even if higher diversification rates can be found in the western part. This roundtrip model between the west and east sides of the Mediterranean basin would have enhanced the survival opportunities of a lineage with access to two refuge harbours where to endure and, in some cases, to evolve and to adapt to changing conditions. This pattern is responsible for the extant taxonomic richness of Jasione in the Mediterranean basin and if made extensible to other groups it could be and important contributor to the overall richness of the region. The north side of the sea was the only scenario of the Eastwest biogeographic events in Jasione. 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(Campanulaceae) endemic to the Iberian Peninsula ABSTRACT There is a general tendency for niche conservatism among closely related taxa, although in polyploid complexes niche divergence may enable polyploids to become established outside the range of distribution of their diploid relatives. Polyploidization leads to the immediate emergence of evolutionary barriers and speciation. However, the new polyploid appears within diploid populations and is thus exposed to the effects of competition and frequency-dependent processes that ultimately lead to reproductive exclusion of the minority cytotype. Ecological divergence would enable polyploids to avoid these processes, thus becoming a key mechanism in shaping geographical distribution of cytotypes in polyploid complexes. However, evidence supporting this hypothesis is still needed, as many contradictory findings have been reported. Moreover, it remains to be determined whether polyploids diverge ecologically to a greater degree than diploids. Study of polyploid complexes encompassing both polyploids and closely related diploid taxa provides an opportunity to compare polyploid niche shift or conservatism in relation to niche shift within an appropriate evolutionary framework. The niche shift hypothesis is evaluated here in a diploid-tetraploid system of closely related taxa of the genus Jasione L. (Campanulaceae) belonging to the Jasione crispa (Pourr.) Samp group. In two previous studies, the hypothesized niche LUIS MIGUEL SERRANO PÉREZ 288 influenced by precipitation (Hengl et al., 2017). In theory, this does not detract from the ecological significance of the variable, as precipitation tends to facilitate the creation of organic soils and leaching of basic cations. However, the soil pH in water is measured at 100-200 cm depth, relatively close to the bedrock, and is indicative of the nature of the substrate. The apparent density provides information about the water storage capacity of a soil, as well as about the structural conditions and degree of compaction (Mukhopadhyay et al., 2019). Table 4.1: ISRIC edaphic variables evaluated. Variable Description Units ACDWRB Degree to which a sub-soil is acid, e.g. of pH < 5 and low BS, grade Grade BLDFIE Apparent density (fine earth) Kg m-3 ORCDRC Soil organic carbon content Per mile PHIHOX pH index measured in aqueous solution pH SNDPPT Weight percentage of sand particles (0.05 mm -2 mm) Percentage The resolution of the climatic and edaphic environmental layers is different (i.e. the pixel size of the data is different), being around 1 km (30'' of arc) in the first case and 250 m2 in the second. In order to combine environmental raster layers in ecological modelling processes, they must have the same resolution, as well as the same geographic extension and cartographic projection. The edaphic layers were adjusted to the resolution of the environmental layer with the R package “raster”. The variables finally chosen were selected for their ecological significance in relation to the taxa and because of the low collinearity between them, as indicated by Pearson's correlation coefficients < 0.8. For this purpose, a correlation matrix was generated in R (R Core Team, 2020) using the “gridExtra”, “rasterVis” and “reshape2” packages. Two CHAPTER 4. Ecological niche conservation and niche shifts in a … 289 sets of variables, i.e. climatic only and climatic plus edaphic variables, were used The “spThin” package (Aiello‐Lammens et al., 2015) reduces the number of presences until there is only one registered point per information pixel, thus eliminating those points for which the distance was less than 5 km in J. crispa subsp. mariana, J. sessiliflora subsp. sessiliflora, and J. crispa subsp. tomentosa. By contrast, in J. sessiliflora subsp. appressifolia, the minimum distance between presences was established at 1 km due to the low number of initial data points. One hundred repetitions were generated for each species, to check whether the number of records remained stable. The models were constructed with the R Biomod2 package and using Generalized Linear Model (GLM), Generalized Additive Models (GAM), MAXENT and Random Forest (RF), thus enabling the species records to be related to climatic variables. For this purpose, 4 validations and 3 replications of 5000 pseudo-absences per algorithm were run, creating a total of 48 models for each species. GLM and GAM are both regression methods that use the presence and absence of data. However, GAM, unlike GLM, can generate a non-linear model. MAXENT and RF are machine learning methods based on the presence of data on a background. MAXENT is based on the principle of maximum entropy for those models with a more uniform distribution (Philips & Dudik, 2008). The models created with the different algorithms for the taxa under study were evaluated with 20% of the data, and the remaining 80% of the data was used for calibration (Guisan & Zimmerman, 2000). The models were evaluated by threshold-independent statistical analysis: true skill statistic (TSS) and receiver operating characteristic (ROC). Models with TSS and ROC values > 0.7 were accepted, as this indicates that the predictions are more realistic than would result by chance. The different individual models were coupled creating consensus models. This assembly technique uses the weighted average and average statistical values to select the prediction that is closest to the actual observed data (Araújo & New, 2007). The coefficient of variation of the ensemble models was also calculated. LUIS MIGUEL SERRANO PÉREZ 290 For the model projections, the program QGIS 3.14 (QGIS Development Team, 2020) was used by loading the raster environmental maps, the study environment and the author’s own data. SDM maps with inferred grading of presence-absence optics were thus created. Niches of the four taxa were visualized in the environmental space using environmental PCAs (PCAenv), an ordination approach calibrated with environmental values of the area of study (Di Cola et al., 2017) using the R package “ecospat” (Broennimann et al., 2012). The PCA computes the occurrence density and environmental factor density for each pixel along environmental axes, maximizing the environmental variance of the distribution ranges of each taxon. PCA scores of the occurrences were projected onto a grid of cells bounded by the maximum and minimum PCA scores, allowing the comparison of niches in terms of breadth and location within the environmental space of the area of study (Di Cola et al., 2017). As a measure of niche overlap, the D value of Schoener (1970) was selected as a metric that varies between 0 (indicating no overlap between niches) and 1 (indicating complete overlap). The D values observed between pairs of species were used to test niche similarity (Warren et al., 2008; Broennimann et al., 2012). The niche similarity test determines whether the observed overlap value (D) differs from the overlap between the observed niche in the distribution of one species and randomly selected niches in the distribution of the other species. In this case, the centre of the mesh density is randomly selected from the available environmental space in the opposite distribution (Broennimann et al., 2012). The significance value was evaluated using 100 randomizations (α = 0.05). Niche similarity was tested for all pairs of taxa with the ecospat package. PCAenv and similarity tests were performed only with climatic variables to avoid enlarging the niche breadth visualization with factors such as pH with low variation across recent temporal scales. CHAPTER 4. Ecological niche conservation and niche shifts in a … 291 3. RESULTS Once the number of records was reduced to a single point per pixel, 45 points were obtained for J. crispa subsp. tomentosa, 33 for J. crispa subsp. mariana, 17 for J. sessiliflora subsp. appressifolia and 203 for J. sessiliflora subsp. sessiliflora. Maps of 5000 pseudo-absences were created from these data by using the “dplyr” package (Figure 4.1), where pseudo-absences are represented in black and the distribution of each taxon is indicated. The maps show that J. crispa subsp. mariana and tomentosa have a similar geographic distribution, while J. sessiliflora subsp. sessiliflora and appressifolia occupy different geographical spaces. Figure 4.1: Distribution and pseudo-absence maps for J. crispa subsp. mariana (top left), J. sessiliflora subsp. appressifolia (top right), J. sessiliflora subsp. sessiliflora (bottom left) and J. crispa subsp. tomentosa (bottom right). LUIS MIGUEL SERRANO PÉREZ 292 The models created for each taxon with the GLM, MAXENT, RF and GAM algorithms in the subset of climatic variables finally selected (Bio3, Bio7, Bio8, Bio15, Bio18, and Bio19) reflect that the most important variable in the distribution is the annual temperature range, i.e. the difference between the maximum temperature of the warmest month and the minimum temperature of the coldest month (Table 4.2). However, in J. sessiliflora subsp. appressifolia, precipitation during the coldest quarter of the year is of similar importance. In the EM with climatic and edaphic variables (Bio6, Bio12, Bio14, pH, % sand and apparent density), the pH of the soil at a depth of 100-200 cm and the percentage of sand are particularly important. The SDM projections obtained enable visualization of the potential distribution area of each species, estimated from climatic variables (Figure 4.2) and climatic plus edaphic variables (Figure 4.3). In addition, the real presences are observed in Figure 4.2, after elimination of the points between which the distance was less than that established as the threshold (1 km or 5 km), thus confirming the goodness of fit of the models to the known distributions of the species. The potential distribution area in the Iberian Peninsula is represented on the maps by shading, where more intense shading indicates a greater probability of finding a specimen of the species of interest. CHAPTER 4. Ecological niche conservation and niche shifts in a … 293 Table 4.2: Importance of the bioclimatic variables in the models created for J. sessiliflora subsp. sessiliflora, J. crispa subsp. tomentosa, J. crispa subsp. mariana and J. sessiliflora subsp. appressifolia using the GLM, MAXENT, RF and GAM algorithms. GLM MAXENT RF GAM sessiliflora Bio 3 0.259 0.004 0.081 0.286 Bio 7 0.706 0.867 0.771 0.821 Bio 8 0.290 0.254 0.334 0.343 Bio 15 0.645 0.041 0.148 0.652 Bio 18 0.683 0.321 0.263 0.510 Bio 19 0.515 0.241 0.188 0.493 tomentosa Bio 3 0.220 0.033 0.075 0.286 Bio 7 0.744 0.862 0.759 0.832 Bio 8 0.299 0.254 0.334 0.343 Bio 15 0.477 0.046 0.141 0.592 Bio 18 0.639 0.351 0.239 0.499 Bio 19 0.601 0.280 0.188 0.477 mariana Bio 3 0.178 0.004 0.047 0.512 Bio 7 0.866 0.806 0.702 0.772 Bio 8 0.299 0.254 0.334 0.343 Bio 15 0.683 0.024 0.165 0.737 Bio 18 0.477 0.336 0.313 0.602 Bio 19 0.440 0.309 0.194 0.512 appressifolia Bio 3 0.151 0.004 0.030 0.625 Bio 7 0.780 0.849 0.491 0.901 Bio 8 0.299 0.254 0.334 0.343 Bio 15 0.645 0.041 0.148 0.652 Bio 18 0.643 0.321 0.263 0.510 Bio 19 0.852 0.910 0.638 0.821 The potential area of distribution of J. sessiliflora subsp. sessiliflora covers a large part of the interior area of the northern half of the peninsula south of the Ebro river, while J. crispa subsp. mariana and J. crispa subsp. tomentosa share areas in the southwestern part of the peninsula. The potential area of distribution of J. sessiliflora subsp. appressifolia is slightly further away, in the region of Valencia and in the province of Teruel. The areas identified as optimal in the SDM with climatic and edaphic variables are more restricted; the presence points have been deleted to facilitate visualization of these optimal areas (Figure 4.3). LUIS MIGUEL SERRANO PÉREZ 294 Figure 4.2: Potential distribution areas of (A) J. sessiliflora subsp. appressifolia, (B) J. crispa subsp. mariana, (C) J. sessiliflora subsp. sessiliflora and (D) J. crispa subsp. tomentosa, modelled using climatic variables. A B C D CHAPTER 4. Ecological niche conservation and niche shifts in a … 295 Figure 4.3: Potential distribution areas of (A) J. sessiliflora subsp. appressifolia, (B) J. crispa subsp. mariana, (C) J. sessiliflora subsp. sessiliflora and (D) J. crispa subsp. tomentosa, modelled using climatic and edaphic variables. The ecological niche of the taxa is represented in the ecological space in the environmental PCAs (Figure 4.4). The first two axes explained 64.6% of the environmental (climatic) variance, with 34.7% and 29.9% explained by axis 1 and axis 2, respectively. The environmental PCA shows that the niche of J. sessiliflora subsp. appressifolia forms a subset of the multidimensional environmental space occupied by J. sessiliflora subsp. sessiliflora. However, the environmental conditions of J. sessiliflora subsp. appressifolia forms part of the environmental distribution of the tetraploid although there is no overlap in the geographic distributions or in the SDM projection (Figure 4.3). This observation may be explained by the marginal A B C D LUIS MIGUEL SERRANO PÉREZ 296 position within the large environmental space occupied by J. sessiliflora subsp. sessiliflora. Figure 4.4: Environmental PCA of the species J. crispa subsp. mariana (top left), J. sessiliflora subsp. appressifolia (top right), J. sessiliflora subsp. sessiliflora (bottom left) and J. crispa subsp. tomentosa (bottom right). The diploids J. crispa subsp. tomentosa and J. crispa subsp. mariana share a large part of their area of distribution. Both have similar environmental requirements for establishment. These results are correlated with the geographic space projected in the SDM, as confirmed by the results of similarity test performed for this pair of taxa (Figure 4.5). Nevertheless, J. crispa subsp. mariana would tend to require milder environments on average, i.e. slightly warmer and more humid than J. crispa subsp. tomentosa. PC1 PC1PC1 PC1 PC2 PC2 PC2 PC2 Axis 1: 34.69% Axis 2: 29.89% Bio 3 Bio 18 Bio 8 Bio 7 Bio 15 Bio 19 CHAPTER 4. Ecological niche conservation and niche shifts in a … 297 The similarity tests indicated no significant difference in the simulated niches between the observed overlap D between the tetraploid and any of the diploids (Table 4.3). Among the diploids, only the observed overlap D in environmental niches of J. crispa subsp. mariana and J. crispa subsp. tomentosa was significantly different (greater) than the simulated shifted values. Nevertheless, the test revealed markedly lower values (i.e. more similar niches) for all diploid pairs than for any pair of a diploid with the tetraploid. However, no evident overlap between either J. crispa subsp. mariana or J. crispa subsp. tomentosa and J. sessiflora subsp. appressifolia can be seen in Figure 4.4 (shaded areas). These relatively low values in the similarity test could indicate that some marginal overlap values exist and could be explained by other unrepresented axes. Relative similarity between all diploids is expected given their proximity in the environmental space, with all diploids occupying the warmer part of the space in an area positively influenced by variables Bio7 (temperature annual range) and Bio 8 (mean temperature of the wettest quarter). Table 4.3: Significance values for similarity tests between pairs of taxa. Significant similarity in relation to a distribution of D values obtained from randomly assigned environmental values from the occupied areas is indicated with an asterisk. sessiliflora tomentosa mariana appressifolia sessiliflora - 0.2079 0.3528 0.2901 tomentosa - 0.0090 * 0.0967 mariana - 0.0826 appressifolia - Although J. crispa subsp. tomentosa and J. sessiliflora subsp. sessiliflora are not geographically distantly separated in some areas, and there even seems to be a small contact zone, they have a low niche overlap. The similarity test indicates that the niche overlap is less than would be produced by chance (Table 4.3; Figure 4.5). A similar situation was found for J. crispa susp. appressifolia (Table 4.3). LUIS MIGUEL SERRANO PÉREZ 304 to the polyploid populations. Only four, little-differentiated haplotypes were found across all tetraploid populations, all diverging from a common ancestor, while no current diploid haplotypes (which may suggest recurrent formation) have been found in tetraploid populations Overall, the niches of the diploid taxa of the studied complex, in addition to signatures of niche conservatism, may indicate the relict nature of these lineages. Compared to the tetraploid niche, all are narrow, or extremely narrow in the case of J. sessiliflora subps. appressifolia. Narrow niches have been related to reduced geographic distributions (Castro et al., 2019). This explanation may apply to the narrow endemic Jasione sessiliflora subsp. appresssifolia but not to J. crispa subsp. mariana or J. crispa subsp. tomentosa. Both latter taxa are relatively widely distributed throughout most of the quartzite mountain ranges within or surrounding the Tagus and Guadiana basins. Relationships between narrow and broad niches in closely related species in plants and animals (Knouft et al., 2006; Vamosi et al., 2014) have been considered an indication of environmental specialization for the narrower niche. The same interpretation has been suggested for poplyploid complexes with a pattern of broad and narrow niche different cytotypes (Parisod & Broennimann, 2016). This would imply that the species and cytotypes with broader environmental tolerances would fill most of the potential niche, forcing those with narrow niches to specialize in a subset of the existing environmental conditions in which their optimal performance would outcompete other cytotypes. However, the results of this study do not suggest environmental assortment conditioned by specialization between cytotypes in the J. sessiliflora complex. Instead, the narrowness and ecologically closeness of niches in the environmental space of the three diploid taxa occupying mild ecological conditions suggest evolutionary inertia and a relict condition. Thus, the diploids would have remained in warm and subhumid enclaves under Mediterranean climatic conditions. Jasione crispa subsp. mariana and J. crispa subsp. tomentosa would benefit from the maritime Atlantic influence that penetrates deeply in inner south-central Iberian Peninsula. This large environmental area has been biogeographically defined as the Luso-Extremadurense subprovince (Rivas-Martínez et al., 2017, Chapter 2) and harbours important populations of other species considered relict from preglacial CHAPTER 4. Ecological niche conservation and niche shifts in a … 305 periods in the same mountain ranges occupied by the diploids, such as Prunus lusitanica L. (Calleja et al., 2009). Indeed, the LusoExtremadurense quartzite mountain ranges are rich in relict and endemic species, with Jasione crispa subsp. mariana and J. crispa subsp. tomentosa considered among the most characteristic taxa of this biogeographical area (Pérez-Chiscano, 1987). The remaining diploid, J. sessiliflora subsp. appressifolia, occurs in sandstone mountain ranges never further than 40 km from the Mediterranean Sea. Although this area of the Valencia-Castellón coast is characterized by dry conditions in summer, the modest altitudes of the mountain ranges (typically below 1.100 m.a.s.l.) are sufficient to allow retention of clouds formed over the Mediterranean Sea and cause frequent summer orographic fog formation that compensates for the general aridity, even enabling the occurrence of some broad-leaved tree formations (Vilches et al., 2013). Indeed, the amounts of water collected from orographic fog during summer on the mountains close to the Valencian coast can reach more than 4.5 l/m2/day, comparable to values found in relict fog-habitats in the Canary Islands (Estrela et al., 2008). Warmer and subhumid conditions characterize narrow endemic rich communities in these mountains (Merlé Farinós & Ferriol Molina, 2008; Vilches et al., 2013) supporting the relict condition of the environmental niche of the diploid J. sessiliflora subsp. appressifolia. In summary, the study of environmental constraints of the diploids suggests that niche conservatism has favoured the retention of relict environmental characteristics, leading to the current pattern of vicariance in the central SW Iberian Peninsula and the coastal Valencian mountains. The evidence of niche conservatism between the studied diploids underpins the suggested hypothesis that niche conservatism is one of the main drivers of current east-west Mediterranean disjunct biogeographic pattern that characterizes the genus Jasione (Chapter 3). The tetraploid Jasione sessiliflora subps. sessiliflora underwent a striking niche enlargement that enabled the polyploid lineage to expand to colder regions and occupy a wide area with conditions ranging from sub-Atlantic climate in temperate NW Iberian Peninsula to continental climatic tendencies in the Iberian System Mountain range. Conversely to findings in other polyploid complexes in Jasione (Castro et al., 2019, LUIS MIGUEL SERRANO PÉREZ 306 2020), the case of J. sessiliflora complex indicates rapid niche shift and expansion in a polyploid. These results stress the utility of comparing niche dynamics of different close diploid relatives in order to properly characterize the magnitude of polyploid niche evolution, within a phylogenetic framework including more than one diploid lineage. This approach and the results are similar to the findings of the recent study of Baniaga et al. (2020). The breaking-up of phylogenetic inertia and niche conservatism after polyploidization in J. sessiliflora through cold-adaptation may have made an important contribution to establishing the genomic and phenotypic bases that enabled higher ploidy levels of the wider polyploid group of J. crispa to occupy colder high mountain environments and expand latitudinally beyond the Iberian Peninsula, as hexaploid populations of J. crispa reach the French Central massif (Küpfer, 1981; M. Serrano, own data). Niche expansion in the tetraploid may have been an immediate effect of the novel genomic make-up at the appearance of the polyploid (either auto or allopolyploid) or a result of later divergence processes involving adaptation under environmental pressures and the need to overcome the negative effects of minority cytotype exclusion. The tetraploid lineage most probably originated in the second half of the Pleistocene (Chapters 2 and 3). The climatic turmoil of Pleistocene glaciations repeatedly harshened environmental conditions at a fast rate in the Iberian Peninsula (Rull, 2020), providing vacant cold niches in the areas occupied by the diploids. It would be interesting to project the environmental niches to glacial period conditions in a future study. The response could help to explain the beginning of the evolutionary history of the polyploid lineage and the niche differentiation that has enabled the shift in environmental space relative to the relict diploids. It would also be interesting to conduct a thorough review of cases to ascertain whether niche shift in polyploid is more likely when the diploid relatives are characterized by slow niche dynamics. Finally, differences in environmental envelopes support the recognition of functional biodiversity units and taxonomic reorganization of the complex, as suggested in the Species Delimitation analyses in Chapter 2. In the diploid-tetraploid system under study there are scarce differences in niche occupancy and geographical space, particularly CHAPTER 4. Ecological niche conservation and niche shifts in a … 307 when edaphic variables are included in the SDMs. Consequently, there are no significant absences of any taxa of the complex in geographic areas with optimal prediction. This excludes the need to use competitive abilities to explain the pattern (Laport et al., 2013). Each cytotype probably has a unique performance in the different environments and should be considered distinct units of biodiversity (Laport & Ng, 2017). However, internal systematic organization within the diploids should be established using other lines of evidence as niche conservatism may blur the recognition of significant units of biodiversity from environmental information alone. Nevertheless, the relict nature and the narrow optimal conditions of the environmental niches of the diploids suggest the need for detailed study of the internal variability within the diploid group and the predicted responses under the potential threat of global climate change. 5. CONCLUSIONS Polyploid niche shift in the tetraploid J. sessiliflora subsp. Sessiliflora is strongly indicated, with a remarkable expansion to colder and more continental submediterranean conditions than the thermic subhumid Mediterranean environments of the diploid relatives. Examination of three closely related diploid taxa provided a phylogenetic framework for comparison to confidently determine the magnitude and dynamics of the polyploid niche shift. Conversely, niche conservatism, niche narrowness and relict characteristics were identified in the environmental envelope of the diploid taxa. Narrow niches are not necessarily related to narrow distribution ranges, as two of the diploids are relatively widespread in the mountain ranges of the southern Iberian central plateau. Species Distribution Models including climatic and edaphic variables better define the fundamental niche of plant species, at least in calcifuge lineages such as the studied polyploid complex. Overall, this research contributes to accumulating evidence supporting the hypothesis that niche shift is an important mechanism in polyploids, at least when no differential reproductive traits (i.e. perenniality, autogamy) have developed to cope with minority cytotype exclusion processes. In summary, the findings suggest that niche shift LUIS MIGUEL SERRANO PÉREZ 308 is significantly faster in polyploids than in closely related diploids, at least when the latter show relict characteristics. 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LUIS MIGUEL SERRANO PÉREZ 320 with different pool of ribotypes. Reduced effective population sizes would accelerate the intrapopulation impact of concerted evolution so founder effects or population decline could be related with low copy variability. Intragenomic ITS variability can be detected by direct sequencing by analyzing multiple peak patterns in electropherograms. Nevertheless, the occurrence of indels among copies can severely difficult the identification of the different copies. Moreover, copies with relative lower frequency can easily remain unnoticed leading to variability underestimation. Therefore, only after cloning procedures can the ribotypic population be confidently characterized (Rosselló et al., 2007). Although long-term geographic structure is a speciation factor, ecological divergence strength the effects of reduced or absent gene flow, accelerating the process of divergence (Barraclough, 2019). The aim of this work is to combine phylogeographic information with experimental measurements that could inform of quantifiable effects of novel edaphic selective pressures in a plant population from a calcifuge lineage. Thus, soil: plant relationships and plant performance under different conditions were analysed. The objective is to use the Mont Caro population as a model system to unveil signatures of initial steps of adaptation after edaphic niche shift. Alternatively, it could be that a mere case of temporary survival of a marginal population in suboptimal habitat conditions was observed. In the former case, they are expectable soil-associated differential performances during the experiment in phenotypic expression and in functional traits assessed after the experiment, as well as in the management of critical soil elements, as a proxy of differential abilities to regulate cellular concentrations of limiting elements for calcifuge species. An informed phylogenetic context for the experiment to account for phylogenetic charge and to rule out the influence of pre-adaptive mechanisms. Whether signatures of geographic isolation and selective pressures can be identified, the Mont Caro population would be a case of in course edaphic speciation, probably deserving the current legal protection and supporting its formal consideration as an evolutionary significant entity by taxonomic recognition. CHAPTER 5. The edaphic factor in plant speciation… 321 2. MATERIAL AND METHODS 2.1. Plant material This work uses two sources of plant material from Mont Caro population, encompassing material from four individuals in total. It was the know population, although thorough sampling works in the area have elevated to 18 exemplars the number of known effectives. Fresh sample of two individuals were collected, preserved in cool conditions and within two days prepared for their use in in vitro micropropagation. Successful micropropagation protocols were developed (M. Serrano, own data), allowing the obtention of living plants. Cloned plants from these two lines were acclimated to outdoor conditions and cultivated on ground, with successful establishments and root developments. These two cloned lines were the source material for chromosome counting and plant: soil experiments. Leave material sampled in field and immediately dried in silica gel from two additional individuals were used in the genetic analyses. 2.2. Chromosome counting Root tips measuring c. 5 mm were excised from in vitro plants cultivated more than two months in ground pot. All plants were acclimated to outdoor conditions and were directly moved from temperatures around 15ºC or lower to the laboratory to conduct immediate root excision, always at first hours in the morning (before 9:00 a.m.) to avoid high laboratory room temperatures. Root tips were washed in distillate water a pre-treated in 2 MM 8-hydroxyquinoleine for 3 hours, followed by a rinsing step in distillate water and then a fixation step in freshly prepared 3:1 96% ethanol - 1 glacial acetic acid solution for 24 hours. Samples were thoroughly rinsed in 70% ethanol to remove glacial acetic acid, since it has been described to interfere with later chromosome staining (Norman et al., 2012). Samples were either stored in 70% ethanol at 4ºC for further use or directly used in the following steps. Root tips were rinsed in distilled water for 5 minutes, followed by an additional 5 minutes rinsing step in fresh LUIS MIGUEL SERRANO PÉREZ 322 distilled water. Samples were hydrolised by plunging the root tips in and Eppendorf tube with HCL 1 N for 6-9 minutes in a water bath at 60ºC. The tubes were pre-heated to bath temperature before root tip plunging. Two rinsing steps 5 minutes each in distillate water followed to eliminate all possible HCL remains. For mitotic analysis, root tips were stained in Schiff’s reagent for 12 hours in dark conditions. Stained root apices were excised under binocular magnifying glass, and immediately squashed on a slide. Cells were observed in a Leica TCSSP2 confocal laser microscope (LEICA Microsystems Heidelberg GmbH, Mannheim, Germany) under a HCX PL APO CS 63.0x1.40 OIL objective, 580nm-725nm emission band and excitation wavelength of 561 nm with DPS (561 nm) laser diode. 2.3. Phylogenetic and phylogeographic relationships of the Mont Caro population Plant material from Mont Caro and representative material from hexaploid Jasione crispa subsp. crispa populations covering its whole distribution range were sampled to assess nr DNA ITS variation (Table 5.1). Total genomic DNA was mainly extracted from field sampled and immediately silica gel dried leaves. In few cases, herbarium specimens were used for DNA extraction. DNA extraction was performed using either a modified cetyltrimethyl ammonium bromide (CTAB) method (Doyle & Doyle, 1990) or a commercial kit Nucleo Spin Plant II (Macherey-Nagel). In both cases 0.6 ul of 2-Mercaptoethanol was added during the first liquid step. Foliar tissue was homogenised either with manual grinder or using Mikro – dismembrator (Sartorius). DNA concentration and quality was assessed with Nanodrop 2000C (Thermo Scientific). The complete ITS region (ITS-1, 5.8S gene, and ITS2) was amplified using the primers ITS1 and ITS4 (White et al., 1990). PCR reactions were carried out in 25 ul with PuReTaq Ready-To-Go PCR BEADS (GE Healthcare) and 80-150 ng of genomic DNA. Thermocycler was programmed for amplification of the ITS region with an initial denaturation step of 95ºC for 2 min, then followed by 31 CHAPTER 5. The edaphic factor in plant speciation… 323 cycles of 1 min at 94ºC, 1 min at 46-48ºC and 2 min 10 s at 72ºC, with a final extension step of 8 min at 72ºC. PCR products were firstly assessed on 1.5 % agarose electrophoresis gels stained with the nucleic acid staining solution RedSafe (iNtRON biotechnology) and purified with E.S.N.A. Cycle Pure Kit. (Omega). PCR products from between two and four individuals from each population were used for cloning procedures, except in some cases when the source material was an herbarium specimen and only one individual was used. PCR amplification products were ligated into the pCR2.1-TOPO vector and cloned according to instructions of the TOPO-TA cloning kit (Invitrogen, Carlsbad, Germany). Plasmid DNA from 10 separated competent cells colonies from each original PCR product was isolated using the S.N.A.P MiniPrep kit (Invitrogen, Carlsbad, Germany). DNA Sanger sequencing was carried out in at least one direct PCR product from almost all populations and in the then isolated DNAs from each population, with an 8-capillary 3500 analyzer (Applied Biosystems) by the DNA sequencing service of the University of Santiago de Compostela. Sequences were initially aligned in BioEdit using the MUSCLE algorithm (Edgar, 2004), although manual editing performed equally well. Pseudogenes chimeras and recombinant sequences were exclude from the analysis. They were easily identified by comparison with those sequences obtained by direct PCR sequencing. GC content, extreme divergence, within single sequence mix of ribotypes and rarity where used criteria. Only those combinations of polymorphism occurring in at least three cloned sequences, or two cloned sequences plus one direct PCR product, even in the form of double peak, were considered true ribotypes and included in the analyses. 2.4. Phylogenetic and phylogeographic analyses Phylogenetic reconstruction was conducted by a median-jonining network in PopART v.1.7 (Population Analysis with Reticulate Trees) considering the six main Iberian Mountain ranges occupied by Jasione crispa subsp. crispa, to simultaneously represent phylogenetic LUIS MIGUEL SERRANO PÉREZ 324 relationships, ribotypic frequencies and geographic distribution. The considered six main mountain ranges are the Cantabrian-Leonese Mountain range, the Central System, the Moncayo in the Iberian System, the Pyrennes, the Montseny Mountain Range and Mont Caro. DnaSP v6.1 (Rozas et al., 2017) was used to calculate Haplotypic diveristies (Hd), interpreted as ribotypic diversity, and Pi, nucleotide diversity. An Analisis of Molecular Variance (AMOVA) was performed to estimate the relative contribution to genetic variances of different sets of samples (Excoffier et al., 1992). The AMOVA was performed in R version 4.1.0 (R Core Team, 2021) with the packages apex, adegenet, pegas, mmod, poppr, and ade4. The analysis was set to consider differences between regions, that is, the six main mountain ranges, between populations within mountain ranges and within populations, following a stratified approach. Randomization MonteCarlo tests with nreps = 1000 were performed in the R package ade4 to assess the significance of the AMOVA results revealed for the different levels of strata (Grünwald & Hoheseil, 2006). Pairwise genetic differentiation coefficients (GammaSt) were calculated in DnaSP v6.1 to estimate the relative importance of gene flow among the six Mountain ranges. Gene flow was calculated as Nm by the equation Nm = (1/GammaSt−1)/2 (Zhu et al., 2019). The Nst nucleotide-based statistics (Lynch &Crease, 1990) and the net number of nucleotide substitution per site (Da) were calculated in DnaSP v6.1 between every population and the remaining set of populations, to estimate the frequency of gene flow affecting each population. 2.5. Soil: plant experiments Jasione crispa subsp. crispa plants from two Mountain ranges, Pyrenees and Central System, were micropropagated in vitro and outdoor acclimated simultaneously with the Mont Caro plants (M. Serrano, own data), in order to have completely comparable individuals. CHAPTER 5. The edaphic factor in plant speciation… 325 Pyrenees and Central System are representative of siliceous populations and Mont Caro of the new calcicole population. A total of 126 plants were used, 42 individuals representing each Mountain Range, were cultivated for five months on three substrates with different chemical characteristics. The three substrates used were, (1) a soil of basic nature from the limestone outcrops of the Serra do Courel (Lugo), here called “Courel”, (2) a soil formed on a granite substrate from a natural environment in Santiago de Compostela, here called “Pedroso”, and (3) a commercial COMPO SANA® substrate, composed of peat, perlite, an amorphous silicate fertilizer enriched with phosphorus, lime and some nutrient elements, to which an extra calcium and magnesium amount was added in the experiment, here called “Composana”. Natural soils from the original population to avoid adaptation and performances related to factors other than soil acid/basic chemical nature, like soil biota or compounds of organic origin from the surrounding plant communities. Plant phenotypical performance was measured as segmented (root, stem and leaves) final biomass and colour changes measured on leaves of living plants with a colorimetric technique adapted for plants with small leaves developed using Jasione as model group (Sanmartín et al., 2020). Soil analyses consisted of measuring pH in water, organic matter, total elements, exchangeable cations and effective CEC. In plants, the total elements in root, stem and leaves were measured, fractions that were separated after determining the total biomass per surviving individual. All measurements were performed in triplicate. Soils were air dried and passed through a 2-mm sieve before analysis. Soil pH was determined in water and 1M KCl suspensions with 1:2.5 sample: solution ratios. Soil total organic matter was determined by weight loss on ignition of dried ground soil at 450 °C. Total elements from soils and plants were extracted after digestion of dried ground sample with aqua regia (HCl and HNO3 in 3:1 ratio) and the elements were analyzed in the extracts by Inductively Coupled Plasma Spectrometry, ICP-MS (Varian 820-MS), equipped with LUIS MIGUEL SERRANO PÉREZ 326 collision reaction interface (CRI) technology to reduce polyatomic interferences. In soils, exchangeable Al was determined after extraction with 2 N KCl (Bertsch & Bloom, 1996). Cation exchange capacity (CEC) and exchangeable bases were determined after saturation of the soil with 1 N ammonium acetate and washing with 1 N KCl (Pansu & Gautheyrou, 2006). Exchangeable Ca, Mg, Na and K were measured in the ammonium acetate extracts by flame atomic absorption/emission spectrophotometry (Varian FS220). Contact-type colour measurements were made on living leaves using a portable spectrophotometer (Konica Minolta CM-700d) equipped with CMS100w (SpectraMagicTM NX) software. The measurements were taken with the specular component included (SCI) mode using a CIE standard daylight illuminant D65, a small area view (SAV) of 3 mm and an observer angle of 2. A total of 3 measurements per sample were made following Sanmartín et al. (2020). Colour measurements were analysed using the CIELAB colour system (CIE S014-4/E: 2007) which represents each colour by means of three scalar parameters or Cartesian coordinates: L*, lightness, which varies from 0 (absolute black) to 100 (absolute white); a*, associated with changes in redness-greenness (positive a* is red and negative a* is green); and b*, associated with changes in yellowness-blueness (positive b* is yellow and negative b* is blue). 3. RESULTS 3.1. Chromosome counting The number of chromosomes (2n=36) observed in the mitotic metaphases of root tips from plants of the Mont Caro population consistently assign these plants to the hexaploidy level (Figure 5.1). These results definitively separate this population from Jasione sessiliflora, an exclusively tetraploid species. Ploidy level matches information from morphological traits indicating that the Mont Caro population more probably belong to the Jasione crispa subsp. crispa CHAPTER 5. The edaphic factor in plant speciation… 327 group of populations (M. Serrano, own data). Under this assumption, following phylogenetic and phylogeographic studies are conducted with the remaining hexaploidy populations of this taxon. Figure 5.1. Mitotic metaphase of Jasione crispa from Mont Caro with 2n=36 chromosomes. 3.2. Genetic diversity and structure, phylogeographic relationships and comparative gene flow Total length of the aligned ITS region was 710 bp with 27 variable and parsimony informative sites and two indels After discarding recombinant chimeras, pseudogenes or unsuccessful/dirty sequencing a total of 228 sequences were included in the analysis, with 11 different ribotypes confidently identified. Total Ribotype diversity (Rd) was 0.802 and nucleotide diversity (Pi) was 0.015. Regarding Mountain range genetic diversity, Rd ranged from 0.000 to 0.671, and nucleotide diversity ranged from 0.000 to 0.00898 (Table 5.1). The Cantabrian- LUIS MIGUEL SERRANO PÉREZ 328 Leonese has the highest genetic diversity, while there is no genetic diversity in the three ranges where only one haplotype was detected, that is Montseny, Moncayo and Mont Caro. Table 5.1. Mountain ranges genetic diversity and ribotype distribution. Mountain range Rd Pi Ribotypes Cantabrian-Leonese 0.671 0.00898 R1, R2, R4, R6, R10, R11 Pyrenees 0.639 0.00355 R2, R3, R5, R6 Central System 0.520 0.00530 R2, R6, R7, R8, R9, R10 Moncayo 0.000 0.000 R2 Montseny 0.000 0.000 R2 Mont Caro 0.000 0.000 R7 The Analysis of molecular variance (AMOVA) stratified by Mountain ranges and populations identified relatively high differentiation level when all populations are considered (Phi = 0.67214), with somewhat lower values of differentiation for populations within Mountain ranges (Phi = 0.55227) and low differentiation level comparing regions. The main part of the genetic variance is explained by variations between populations within the mountain ranges (40.4 %), followed by variations within the populations (32.8 %), with the lesser part of the variance is explained by variations between mountain ranges (Table 5.2). CHAPTER 5. The edaphic factor in plant speciation… 329 Table 5.2. AMOVA based on ITS sequences of Jasione crispa subsp. crispa Σ % Phi Significance Variations between Mountain ranges 0.2363637 26.8 0.2677348 0.001 Variations between populations within Mountain ranges 0.3570251 40.4 0.5522737 0.001 Variations within populations 0.2894389 32.8 0.6721456 0.001 Total 0.8828278 100 NA NA Randomization tests revealed that the values of variance observed for the three levels of analysis were significantly different than random distributions from the data matrices, meaning that there is significant population structure for the three levels of stratification. (Figure 5.2 and Table 5.2). Therefore, there is clear evidence that these groups are significantly differentiated at the three stratification levels, although the variation between regions is comparatively the less important. LUIS MIGUEL SERRANO PÉREZ 336 Table 5.5. Results of the analysis of the total elements (g kg-1) in the leaves (H), stem (T) and root (R) of the populations of Jasione crispa from the Central System (C), Mont Caro (M) and Pyrenees (P) mountain ranges. Soil Composana Courel Pedroso C M P C M P C M P Ca H 23.0 12.6 14.0 61.3 20.0 21.6 9.5 9.6 10.8 T 6.3 8.6 7.3 13.5 12.3 10.1 4.3 6.1 5.7 R 7.6 7.6 6.8 12.7 23.8 11.7 5.7 4.3 4.9 Mg H 3.2 1.2 2.3 2.4 3.9 4.6 2.6 4.3 2.0 T 1.6 1.0 1.4 1.1 3.1 2.9 1.9 3.8 0.8 R 3.7 1.3 2.9 1.6 12.0 8.6 4.8 9.1 5.0 Al H 0.2 0.5 0.2 12.6 3.9 4.6 2.6 4.3 2.0 T 0.5 0.8 0.3 3.4 3.1 2.9 1.9 3.8 0.8 R 0.4 0.3 0.2 6.4 12.1 8.6 4.8 9.1 5.0 Fe H 0.1 0.4 0.2 1.1 4.8 3.9 1.8 2.7 1.2 T 0.4 0.5 0.2 3.0 3.0 1.9 1.2 2.3 0.6 R 0.3 0.3 0.2 6.0 10.4 6.7 3.2 5.6 2.7 Ca/Mg H 7.0 9.7 6.0 25.5 15.9 11.0 5.2 7.5 4.6 T 3.7 8.3 4.9 12.1 14.2 8.6 3.8 7.0 5.5 R 2 5.8 2.3 7.8 13.2 6.0 4.2 3.7 3.5 In the basic Courel soil, with a high presence of calcium and where the Ca / Mg ratio in the CECe presents extreme values (viz., 122), the amount of calcium in Mont Caro roots doubles the amount of this element present in the roots of Central System and Pyrenees. However, the leaf: root ratio in Mont Caro remains around the unit, while it is two times and six timed greater in Pyrenees and Central System, respectively (Table 5.5). Composana soils is characterized by the greater amount of magnesium in the exchange complex, 6.3 cmol kg-1 compared with 0.3 cmol kg-1 and 0.1 cmol kg-1 in Pedroso and Courel soils, respectively (Table 5.4), The presence of this element in plants cultivated in Composana soils is lower in plants from Mont Caro than in plants from the other two populations, with these consistently observed in leaf, stem and root (Table 5.5). Moreover, plants cultivated in Composana soil reveal differences in Ca/ Mg ratio between Mont CHAPTER 5. The edaphic factor in plant speciation… 337 Caro on one side and plants from Pyrenees and Central System on the other, with much greater Ca/Mg ratio values in all three tissues for the Mont Caro. Regarding the chromatic coordinates (a* and b*) it is observed a differential behaviour of Mont Caro in relation to the silicicole populations, particularly in the siliceous Pedroso soil. Interestingly, the colour values of Mont Caro in Pedroso soil are located over the a* axis, evincing achromatic response in this parameter. Mont Caro values in Pedroso are lower in the b* axis compared to Mont Caro in Courel and Composana soils, around 10 CIELAB units towards blue colour. Conversely, Mont Caro in Courel and Composana shift to more extreme values in both axis, either green or red, but in any case escaping from the achromatic axis. Central System and Pyrenees seems to behave similarly, despite their greater phylogenetic distance. Moreover, under the basic conditions of the calcareous Courel soil, Central system tends to adopt closer or over the achromatic axis (Figure 5.8). LUIS MIGUEL SERRANO PÉREZ 338 Figure 5.8. Changes in the green-red colour component (a*) and blue-yellow colour component (b*) in leaves. All data (A), Courel soil (B), Pedroso soil (C), and Composana soil (D). Yellow Blue RedGreen Yellow Blue RedGreen Yellow Blue RedGreen Yellow Blue Red Green A B C D CHAPTER 5. The edaphic factor in plant speciation… 339 4. DISCUSSION The expansion of a lineage to a new environment is a requisite for ecological speciation. However, this evolutionary process can be only apparent, and the patterns observed in nature can better respond to longterm pre-adaptations than to novel adaptations (Moore & Kadereit, 2013). The identification of features of ecological speciation needs to verify phylogenetic and biogeographic hypothesis by which the origin of the population undergoing novel adaptations under new environmental constraints can be tracked to a lineage not pre-adapted to these putatively new ecological pressures. In this work, the initial steps of ecological speciation are shown in a robust phylogeographic framework and signatures of adaptation to calcareous edaphic niche in a population from an otherwise calcifuge lineage. The singularity of the calcicolous population from Mont Caro is brought to light in this study, as well as its phylogenetic affinities and biogeographical relationships. It is identified for the first time as an hexaploid population. This ploidy level rule out its inclusion withing the variability of Jasione sessiliflora, a tretraploid species. Hexaploids orophile populations of the species are considered Jasione crispa subsp, crispa (Sales & Hedge, 2001). Although morphological floral characters like calyx teeth shape or leave traits as number and location of hydathodes support the inclusion of the Mont Caro population within the variability Jasione crispa subsp, crispa, environmental factors like low elevation of Mont Caro population (c. 1,400 m) compared with other populations of the species, or the calcareous nature of the Mont Caro massif while J. crispa subsp. crispa is calcifugue, could raise doubts about the assignation of Mont Caro populations to this subspecies. The genus Jasione is characterized by pervasive morphological stasis and scarce morphological diagnostic characters to confidently differentiate even distantly related species. For this reason, relying on morphological characters can be deceiving in Jasione, as has been exposed in other parts of this thesis. As a matter of fact, different evolutionary approaches of the current author are in conflict with previous taxonomic treatments of different Jasione groups. LUIS MIGUEL SERRANO PÉREZ 340 Ploidy level and phylogenetic analysis reliably place this population within J. crispa subsp. crispa. Ribotypic distribution of Jasione crispa subsp crispa unveils the phylogeographic history in Jasione crispa subsp. crispa, that would be analysed in more detail elsewhere. As a summary, there is a relatively high ribotypic diversity within J. crispa subsp. crispa (Hd = 0.802), and significant genetic structuring between populations, withing populations of the Iberian Mountain ranges and between the different mountain ranges. However, the latter level of differentiation (i.e., between mountain ranges) is less marked than in the two former levels. Nevertheless, all three main Mountain ranges, the Central System, the Pyrenees and the CantabrianLeonese Mountain range, harbour diverse unique ribotypes. This pattern can be explained by a recurrent history of cycles of vicariance and within Mountain range differentiation followed by expansion periods and secondary contact, fitting in the model of refugia within refugia proposed for the Iberian Peninsula by Nieto-Feliner (2014). Pleistocene stadial and interstadial cycles would range allow expansion in colder periods and range shrinking in hotter ones, with these fluctuant biogeographic model fitiing well in cold-adapted lineages (Rull, 2020). Three narrow range Mountain massifs with isolated populations of hexaploid of Jasione crispa subs. crispa have been included in the study. All three shows low levels of internal genetic variability, with only one ribotype identified in each massif. This low variability agrees with out hypothesis that in small sized and isolated populations the mechanisms of concerted evolution would be more effective homogenizing the pool of ribotypic copies occurring within the mountain range, while the dynamics of ribotypic variability will be more complex in Mountain areas with larger population sizes, where random direction of concerted evolution can be different in each subpopulation, keeping the pool of copies and increasing the variability after gene flow among populations within each mountain range. Interestingly, the inferred amounts of gene flow are different for Moncayo and Montseny on one side, and Mont Caro in the other, with the latter population being outlined as the one with lower values of gene flow of all populations in the Iberian Peninsula (Table 5.3 and Figures 5.3 and 5.4). It is caused for the information given by ribotype fixed in CHAPTER 5. The edaphic factor in plant speciation… 341 each of the three populations and its distribution across other populations in all Mountain ranges. The only ribotype identified in Moncayo and Montsenty (R2) is the most frequent in the taxon and the only one present in all mountain ranges but in Mont Caro. Conversely, the ribotype fixed in Mont Caro population (R7) has its only other occurrence as a minority ribotype in some populations of the Central System. Interestingly, this pattern suggests a surprising history of relationships between Mont Caro and the distant populations of the Central System. Therefore, there is no molecular evidence that the much closer populations of Montseny and Moncayo could have been the source for Mont Caro populations. It could be that the Ebro river valley could have been an effective barrier to dispersal. Ebro river is between Mont Caro and Motseny. Moncayo is South of the Ebro, but distantly upriver and separated from Mont Caro by the lowlands of the Ebro valley where environmental conditions suitable for the orophyte J. crispa subsp. crispa could be absent. In any case, the current author data suggest for Mont Caro an evolutionary history of belonging to a calcifuge lineage only occurring in the Central System what makes unlikely pre-adaptation to calcicole edaphic niche. This pattern is opposite to those recovered in other model groups, like the Alpine calcicole lineages of Minuartia (Moore & Kadereit, 2013). Low variability and ribotype fixation can be a signature not only of absent gene flow and founder effect, as would be plausible given the long distances to other populations, but as well as by environmental filtering to dispersal by the regionally predominant calcareous substrates. Mont Caro shows genetic signatures of increased isolation to gene flow from other populations in a relatively structured phylogeographic context. Gene flow interruption related to marked geographic structure is combined in Mont Caro with environmental (edaphic) distinct conditions, setting one scenario suitable for ecological divergence (Nosil, 2012; Barraclough, 2019). Gene flow, otherwise, would promote genetic exchanges from other populations decreasing genetic differentiation (Slatkin, 1987), and threatening the preservation of acquired abilities to cope with new environmental constraints. Interestingly, the experimental part of this work points to the existence of these acquired abilities, and signatures of novel adaptation to LUIS MIGUEL SERRANO PÉREZ 342 calcareous environment are identified. Nevertheless, it cannot be ruled out that adaptation cold be working in other environmental dimensions, as environmental consequences of the elevation range in Mont Caro (around 1,400 m.o.s.l), lower than the elevation range of other populations. For example, Central System populations are most frequently found around 2,000 m.o.s.l. In summary, there exists in the plants from Mont Caro a greater productivity when are cultivated in calcareous or basic soils than when are cultivated in acid siliceous soils. Conversely, better performances in calcareous soils are not registered in plants from the silicicole population of Central System and the Pyrenees. The performances of the plants from the Central System are more similar to the ones from the Pyrenees, despite Mont Caro and Central System plants are more closely related. Phenotypic colour analysis agrees with the biomass results in a way that colour expression in the different soils works differently in Mont Caro when compared to the silicicole populations. Distinct management of calcium and magnesium, and in aluminium accumulation, have been pointed as the main differences between calcicole and calcifuge plants (Jansen et al., 2002). These differences are here identified between Mont Caro and the s silicicole populations of the Central System and the Pyrenees. These results suggest that the ability to thrive in calcareous environments is newly acquired in process of ecological divergence. It is coupled with an apparent loss of efficacy to cope with conditions occurring in acid substrates, particularly the relationship with toxic disposable elements as aluminium. Therefore, not only new pressures modify the genetic makeup of the new population at its phenotypic expression to cope with them, but also the absence of previous ecological pressures in which the lineage evolved before the expansion to the new environment caused the rapid loss of their associated adaptations as an interesting evolutionary trade-off. Whether if all these abilities rely in the in genetic changes or they can be explained by or in combination with other mechanism, like the epigenetic make up, remains open to new studies. Nevertheless, the results suggest that natural selection is likely shaping the evolutionary fate of the Mont Caro population, suggesting an ongoing speciation process by ecological divergence in condition of gene flow isolation. CHAPTER 5. The edaphic factor in plant speciation… 343 5. CONCLUSIONS The hexaploid populations of the calcifuge taxon Jasine crispa subsp. crispa occurring in the Mountain ranges of the northern half of the Iberian Peninsula shows high global genetic variability and geographic structuring of ITS ribotypes, both across populations and within Mountain ranges. The only calcicole population, located in the coastal Mont Caro massif in Tarragona, is related to the distant populations of the Central System. Mont Caro shows the lower levels of gene flow among all studied populations, evincing clear geographic isolation. This population has signatures of ecological divergence under calcareous edaphic conditions. Experiments in siliceous and calcareous soils evince differential phenotypic performances and biomass production between Mont Caro populations and two silicicole populations. Mont Caro performed clearly better in calcareous soils, conversely to the silicicole populations. The management of cations was also different, with Mont Caro accumulating a notably greater amount of aluminum, iron and magnesium in strongly acidic soil, while in basic substrates (despite the abundance of calcium and magnesium), Mont Caro accumulated significantly less amounts of magnesium and showed a balanced relationship of calcium between root and leaf. These results suggest the existence of an adaptive ecological speciation process in Jasione crispa from Mont Caro, which would contribute to support its differentiated taxonomic recognition. ACKNOWLEDGEMENTS The author is grateful to Celestino Quintela-Sabarís and Patricia Sanmartín for their collaboration and assistance. LUIS MIGUEL SERRANO PÉREZ 344 REFERENCES Álvarez, I., Wendel, J.F. 2003. Ribosomal ITS sequences and plant phylogenetic inference. Molecular Phylogenetics and Evolution 29(3): 417-434. Avise, J. 2000. Phylogeography. The History and Formation of Species. Harvard University Press, Cambridge, MA, USA. 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Aluminum hyperaccumulation in Angiosperms: a review of its phylogenetic significance. The Botanical Review 68: 235–269. Kaveh, A., Kazempour-Osaloo, S., Amirahmadi, A., Maassoumi, A., Schneeweiss, G.M. 2019. Systematics of Onobrychis sect. Heliobrychis (Fabaceae): morphology and molecular phylogeny revisited. Plant Systematics and Evolution 305: 33-48. Lande, R. 2014. Evolution of phenotypic plasticity and environment tolerance of a labile quantitative character in a fluctuating environment. Journal of Evolutionary Biology 27: 866-875. Li, W.H. 1997. Molecular Evolution. Sinauer Associates, Sunderland, MA, USA. Lynch, M., Crease, T.J. 1990. The analysis of population survey data on DNA sequence variation. Molecular Biology and Evolution 7(4): 377-394. Moore, A.J., Kadereit, J.W. 2013. The evolution of substrate differentiation in Minuartia series Laricifoliae (Caryophyllaceae) in the European Alps: in situ origin or repeated colonization? American Journal of Botany 100: 2412–2425. Morales‐Briones, D.F. Tank, D.C. 2019.Extensive allopolyploidy in the neotropical genus Lachemilla (Rosaceae) revealed by PCR‐based target enrichment of the nuclear ribosomal DNA cistron and LUIS MIGUEL SERRANO PÉREZ 352 Figure 6.1. Jasione corymbosa Poir. ex Schult. Malaga: Manilva (MGC 70,000). The results of various phylogenetic analyses (Maximum Likelihood, Bayesian), in the framework of a comprehensive study covering the whole genre, revealed that the amplified DNA sequences from of Manilva are part of a clade also encompassing all samples from North-African populations of J. corymbosa. The inferred relationships had a strong statistical support and suggested that J. corymbosa is the sister group of another species of the southern half of the Iberian Peninsula, J. blepharodon Boiss. & Reut. This phylogenetic affinity between the two taxa had already been pointed out by Pérez-Espona et al. (2005). Due to their morphology, the specimens of J. corymbosa collected in Andalusia can be clearly differentiated from some North African populations, mainly from those of Western Rif. However, studies more detailed are needed to future evaluation of a possible differentiated CHAPTER 6. Presence and conservation status of Jasione corymbosa … 353 entity differentiated, given the wide range of variation existing within the species. 2. DISCUSSION. CONSIDERATIONS ON THE STATE OF CONSERVATION (SERRANO ET AL., 2009) The species occupies in the Manilva dunes around 1.5 ha. The regional government surrounded the occupied are by a perimetral ditch to avoid cars entry. It allowed an increasing in the number of exemplars from 2,000 individuals in 2010 to 6.000 individuals in 2012. The restrictedness of its Iberian distribution, the apparent disappearance of the remaining known localities, as well as the strong regression suffered by the dune communities occupied by J. corymbosa throughout the Mediterranean Andalusian coast, threatened by tourism intensification, suggested the proposal for the Iberian Peninsula population the threat category of Critically Endangered (CR), according to the IUCN classification (2001). According to that, the proposal of the 2008 Red List of the Spanish Vascular Flora (Moreno, 2008) should be changed from “EX, RE” to: CR B1ab (i, ii, iii) +2 ab (i, ii, iii). Besides the in situ conservation measures, germplasm collected in the Manilva population is currently conserved in three germplasm facilities, the Córdoba Botanical Garden (BGVA) seeed bank, the Polytechnic University of Madrid seed bank, and the University of Santiago de Compostela seed bank. The discovery of this plant in Manilva arise the possibility that the species may continue to exist in some other locations, what makes it advisable to keep looking for it in the Andalusian coast. REFERENCES Bokhari, M.H., Sales, F. 2001. Jasione anatomy in the Iberian peninsula and its taxonomic significance. Edinburgh Journal of Botany 58: 405-422. LUIS MIGUEL SERRANO PÉREZ 354 Cabezudo, B., Talavera, S. (Coord.). 2005. Lista roja de la flora vascular de Andalucía. Junta de Andalucía. Sevilla, Spain. Fernández López, C., López Pulido, M., Amezcua Ogayar, C., Casado Ponce, D. 1989. Catálogo bibliográfico de las plantas vasculares de Andalucía. Blancoana 7: 3-68. Galán de Mera, A., Pérez Latorre, A.V., Vicente Orellana, J.A. 2003. Relaciones fitogeográficas entre el suroccidente de la Península Ibérica y el Noroeste de África: una propuesta de sectorización. Lagascalia 23: 27-51. Moreno, J.C. (Coord.). 2008. Lista Roja 2008 de la Flora Vascular Española. Dirección General de Medio Natural y Política Forestal (Ministerio de Medio ambiente, y Medio Rural y Marino) - Sociedad Española de Biología de la Conservación de Plantas. Madrid, Spain. Parnell, J. 1987. Variation in Jasione montana L. (Campanulaceae) and related species in Europe and North Africa. Watsonia 16: 249267. Pereira Coutinho, A.X. 1974. Flora of Portugal: vascular plants. 2nd ed. Bertrand. Lisbon. Pereña Ortiz, J., Becerra Parra, M., Rivas Rangel, A., Serrano, M. 2008. Jasione corymbosa Poir. ex Schult. Location of an extinct species. Environment (Board of Andalusia) 62: 34-35. Pereña Ortiz, J., Becerra Parra, M., Rivas Rangel, A., Serrano, M. 2009. About the presence of Jasione corymbosa Poir. Former Schult. (Campanulaceae) in the Iberian Peninsula. Plant Conservation 13: 14-15. Pérez-Espona, S., Sales, F., Hedge, I., Möller, M. 2005. Phylogeny and species relationships in Jasione (Campanulaceae) with emphasis on the ‘montana-complex’. Edinburgh Journal of Botany 62 (1 & 2): 29-51. Peris, J., Romo, A., Stübing, G. 2002. Jasione. In B. Valdés, M. Rejdali, A. Achhal, S.L. Jury & J.M. Montserrat (eds.). Check list of CHAPTER 6. Presence and conservation status of Jasione corymbosa … 355 vascular plants of northern Morocco with identification keys II: 597-598. Better advice Scientific Research, Madrid. Quézel, P. 1953. Les Campanulacées d’Afrique du Nord. Feddes Repertorium Specierum Novarum Regni Vegetabilis 56: 1–65. Sales, F., Hedge, I.C. 2001. Jasione L. In: Paiva, J., Sales, F., Hedge, I. C., Aedo, C., Aldasoro, J. J., Castroviejo, S., Herrero, A., Velayos, M. (Eds). Flora Iberica: Plantas vasculares de la Península Ibérica e Islas Baleares 14: 153–170. Real Jardín Botánico. Madrid, Spain. Serrano, M., Pereña Ortiz, J.; Becerra Parra, M. 2009. Presencia y estado de conservación de Jasione corymbosa Poir. Ex. Schult. (Campanulaceae) en la Península Ibérica. Acta Botanica Malacitana 34: 284-287. IUCN. 2001. Categories and criteria of the list IUCN Red: Version 3.1. Commission of IUCN Species Survival. Gland,Switzerland and Cambridge, UK. 357 CHAPTER 7. A simple, reliable, and inexpensive solution for contact colour measurements in small plant samples, assessed in plants of the genera Jasione L. and Campanula L. (Campanulaceae) ABSTRACT Correct color measurement by contact-type colour measuring devices requires that the sample surface fully covers the head of the device, so their use on small samples remains a challenge. Here, cardboard adaptors on the two aperture masks (3 and 8 mm diameter measuring area) of a broadly used portable spectrophotometer are proposed to use. Adaptors in black and white to reduce the measuring area by 50% and 70% were applied in this study. Representatives of the family Campanulaceae have been used to test the methodology, given the occurrence of small-leaved. The results of the current author show that, following colorimetric criteria, the only setting providing indistinguishable colours according to the perception of the human eye is the use of a 50%-reducing adaptor on the 3-mm aperture. In addition, statistical analysis suggests the use of the white adaptor. The contribution of the current author offers a sound measurement technique to gather ecological information from the colour of leaves, petals and other small samples. 1. INTRODUCTION The colour of plant organs, particularly of leaves and flowers, is a phenotypic trait traditionally used as a visual indicator of the plant LUIS MIGUEL SERRANO PÉREZ 358 physiological status. Indeed, environmental stress regarding light quantity and quality, nutrients, temperature and drought result in a change of plant colour (Rahaman et al., 2015). These correlations are so informative that colour is one of the phenotypic traits for monitoring plant growth in high-throughput classification approaches (Chen et al., 2014). The colour of leaves and flowers fulfill also an ecological role in plants, since bright colours attract pollinators and seed dispersers (Kevan, 1983). It has been also hypothesized that insects could interpret the colour intensity of plant leaves as a signal of the strength of the plant, thus being attracted or repelled (Archetti, 2000; Yamazaki, 2008). Leaves colour reflects also the accumulation of secondary metabolites with important chromatic components, which can have a great commercial and therapeutic value. For example, the purple colour of bud and leaves in tea cultivars (e.g., Benibana-cha, Sunrouge tea, Zijuan tea and Ziyan tea) is associated with the accumulation of anthocyanins, that have been shown to have a therapeutic role against colorectal carcinoma cells (Hsu et al., 2012) and in reinforcing the brain’s antioxidant capacity in mice (Rashid et al., 2014). In addition, since these metabolites accumulate in response to the plant interaction with soil, the leaves and petals colour is an indicator of the characteristics of the soil. Well known examples are the overexpression of carotenoids and anthocyanins in plants growing in soil with high salt concentration (Borghesi et al., 2011) or the changes in colour of flowers in Hortensia (Hydrangea macrophylla), which varies from blue to pink, according to the soil acidity. Besides, colour transitions and pigment variation in plants also inform about genetic variability within an among populations and taxa, allowing the formulation of evolutionary hypothesis in a phylogenetic framework (Abdelaziz et al., 2014; Valenta et al., 2018). Changes in colour, both in vegetative and reproductive organs (Ougham,et al., 2008), tell about important evolutionary processes like pollinator-mediated reproductive isolation (Yuan et al., 2013). Finally, plant disease, including fungal and bacterial parasites, cause lesions and chromatic changes of leaves and flowers (Barbedo, 2013; Matsunaga et al., 2017). Their early perception is fundamental to act on time, to eradicate the disease and to limit economic loss, or to monitor the interaction of plants with damaging CHAPTER 7. A simple, reliable, and inexpensive solution for … 359 organisms or environmental factors. Given the intimate connection of flower and leaf colour with the physiology of the plant, its quantification by human eye has been traditionally used to guide breeding experiments to accumulate metabolites, but also as a proxy for soil parameters and detection of disease. Unfortunately, colour detection and classification by human eye are extremely unreliable, because of their dependency from the experience and the capacity of the observer. The choice of a colour by a living organism is based on psychological and evolutionary or survival criteria. For example, among the colours distinguished by the human eye, green is perceived more readily and with the largest shift in their hue than any other colour, because of the combined perception of rods and cones (Gordon & Abramov, 1977). It is hypothesized that this could represent an adaptation to the environment of primates in search for food (Nerger et al., 1995). The colour can be described in an objective and precise way through the use of spectrophotometers, colorimeters or chroma-meters that describe the reflected colour in the standardized CIELAB colour system (see e.g., CIE 1986; Fairchild, 1998; Hunt & Pointer, 2011). They work by contact on a surface, like other contact-type spectrometer devices (Lu et al., 2019), and they average the light reflected from an aperture on the head of the device. The aperture is circular and has a diameter between 3 mm and 60 mm (see e.g., Fairchild, 1998; Hunt & Pointer, 2011; Prieto et al., 2010). Surfaces with an area in a different shape or smaller than the aperture of the device do not reflect entirely the emitted light, thus leading to a consistent loss of light and to unreliable results. To perform the colour measurement correctly, the sample surface must cover entirely the aperture of the device to prevent leakage of light or interference from external light. For this reason, the colour cannot be measured when the target area is smaller than 3 mm and/or not circular. To address this issue, it is proposed to reduce by 50% and 70% the aperture area of a portable spectrophotometer with cardboard adaptors in white and black colours, in order to describe the colour in the LUIS MIGUEL SERRANO PÉREZ 360 CIELAB space of small biological samples, such as leaves and petals. Thus, the current author further enhances the usability of the sensor device by extending its application to smaller samples than those for which it was designed. This unlocks previously unexplored ways to tackle current issues in the environmental monitoring field, such as the biodiversity response to climate change. In this frame, the colour of leaves from species of the genus of Jasione and other of the Campanulaceae family is measured, thus validating this methodology within a broad phylogenetic group with species of different ecology and frequently with small-sized leaves (Bokhari & Sales, 2001). This group encompasses a number of alpine species, with a habit of dense rosettes with small leaves, being this a characteristic plant adaptation to alpine environments (Nagy & Grabherr, 2009). In the present work, the use of leaves big enough to be measured with the large colorimeter aperture guaranteed the reliability of this methodology also for leaves where only the smallest aperture of the colorimeter aperture can be used, as is the case of many the alpine species. In addition, herbarium samples, where dry plant tissues are conserved as valuable ecological, systematic and historical evidences of plant biodiversity, are included. By extending the present methodology to conserved dry samples, it is proposed the use of colorimetry as a tool to describe the colour of leaves and petals after some time and monitoring the conservation conditions of these specimens. 2. MATERIAL AND METHODS 2.1. Plant materials The genus Jasione has many representatives occurring in mountain environments (Sales & Hedge, 2001), having spread around the Mediterranean basin with a number of species endemic of the alpine ranges of the region (Pérez-Espona et al., 2005). They belong to the alpine flora: the mountain zone located above tree line and below the permanent snow line and considered a conservation target (Byers, 2005). Alpine plant communities are in one of the most threatened ecosystems in the world by climate change (Rixen et al., 2014). Thus, CHAPTER 7. A simple, reliable, and inexpensive solution for … 361 the alpine flora is currently subject of a number of international monitoring projects to study the response of plants to global temperature increase and other environmental stressors (GLORIA, 2019). Colour variation in the leaves of Jasione could be used as ecological indicator in future monitoring studies on alpine plant species. Nevertheless, the peculiar growth of many alpine plant taxa, forming a cushion of dense rosettes of small leaves as a common evolutionary adaptation (Nagy & Grabherr, 2009), represents a challenge for the use of colour as a proxy of physiological status. For these reasons, the proposed methodology has been tested on six fresh plants (Jasione montana, L. Trachelium caeruleum L., Campanula rotundifolia L., Campanula isophylla Moretti, Hesperodocon hederaceus (L.) Eddie & Cupido., and Jasione laevis Lam, Figure 7.1) and six plants from herbarium (Campanula adsurgens Levier & Leresche, Campanula latifolia L., Campanula mollis L., Campanula glomerata L., Campanula trachelium L., and Campanula versicolor Andrews, Figure 7.2), all belonging to the family of Campanulaceae. The two species of Jasione were selected from populations with wide leaves in order to satisfy the conditions required by the study (i.e. 11 mm of diameter in MAV without adaptors, see Section 2.2.). These plants belong to populations of Jasione laevis subsp. laevis and Jasione montana var. latifolia Pugsley. The herbarium record of University of Santiago de Compostela, Spain (SANT) was accessed to obtain the specimens. Each herbarium specimen was checked for possible misidentification, and the herbarium code, name of the specie, sampling location, collector and year were recorded (Table 7.1). LUIS MIGUEL SERRANO PÉREZ 368 2.4. Statistical analysis For a robust evaluation of the effect of the adaptors on the colour measurements, the collected data have been analyzed with three different multivariate approaches: Principal Coordinates Analysis (PCoA), Quadratic Discriminant Analysis of multiple groups (QDA) and a combined approach based on k-means and agglomerative clustering analyses. PCoA was also applied to the measurements obtained from the four patches (green, foliage, yellow green and bluish green) of the Gretag Macbeth color-checker colour rendition chart. With the PCoA analysis the contribution from each adaptor type (black or white, 70% or 50% reduction, or no adaptor) on both MAV and SAV to the colorimetric information (L*, a* and b*) was assessed. For each species, data were standardized by subtracting the mean of measurements with no adaptor to preserve the units of deviation from the actual colour and lay out all measurements in the same multivariate space. The measurements were distributed in a 2-dimensional Euclidean ordination space using ggplot2 and Stat R packages (Wickham, 2009). To assess the similarity of the measurements obtained with and without the different tested adaptors, a QDA was performed. The individual measurements were assigned to predefined or actual groups (SAV and MAV with different adaptors or without adaptors), and the adaptor type most alike to SAV and MAV (i.e., those whose observations are significantly misclassified as observations obtained from SAV and MAV with no adaptors) were quantitatively identified from the results of the QDA confusion matrix. The analyses were run separately for SAV and MAV measurements and, in order to compare all species together, data were standardized by subtracting the mean of measurements, respectively from SAV or MAV with no adaptor. A Leave-Out-Out cross-validation was performed after the analysis to validate the QDA. Multivariate normality was assessed with the MVN R package (Korkmaz et al., 2014) and the MASS R package was used for the remaining analysis (Venables & Ripley, 2002). CHAPTER 7. A simple, reliable, and inexpensive solution for … 369 To verify the results pointed out by the QDA, an approach combining k-means and agglomerative clustering was performed on the same data from SAV, treated as for QDA. By k-means cluster analysis, similar colour measurements were categorized by defining clusters to minimize the total intra-cluster variation. The optimal number of kclusters in each species was determined using 30 indices provided by the R package NbClust (Charrad et al., 2014). The agglomerative clustering was conducted by computing a dissimilarity matrix based on Euclidean distances and the Ward.D2 agglomeration method in the Stats R package to produce a hierarchical tree for each species (R Core Team, 2020). The identified k-means groups were then superimposed on the dendrogram and, by means of the pvclust R package (Suzuki et al., 2006), the statistical support of all clusters was calculated with approximately unbiased (AU) p-values by multiscaling bootstrap resampling. Clusters with AU p-value equal to or greater than 95% were considered strongly supported by data. 3. RESULTS A colorimetric analysis of the gathered measurement from each species of Campanulaceae, as well as the green patches from the Gretag Macbeth color-checker colour rendition chart, was performed. Partial (∆L*, ∆a*, ∆b*) and total (∆E*ab) colour differences using one of the four adaptors and no adaptor were calculated. The latter are summarized in Table 7.2. The colour parameters most widely varying with the use of the adaptors in plant samples were L* (lightness of colour) and b* (associated with changes in yellowness-blueness). L* varied to a larger extent with the use of the white adaptor, increasing the lightness in the registered colour (data not shown), and a similar observation can be made for the parameter b*: when the black adaptor is used, the blue component increases in the registered colour (data not shown). As expected, widening the diameter of the adaptor from 50% to 70% increases the differences in colour calculated with respect to the measurement without adaptor (Table 7.2). In the green patches similar results were obtained. Furthermore, for the green patch, the parameter a* (associated with changes in redness-greenness) changed the most, LUIS MIGUEL SERRANO PÉREZ 370 closely followed by b*. For the foliage and yellow green patches, L* and b* varied the most, and for bluish green patch, it was L* and a* to be affected, while the change in b* was practically negligible. As in other studies, the threshold of 3 CIELAB units as limit noticeable by an observer with normal colour vision (see Section 2.3.) is adopted. With the SAV aperture and the 50% white or black adaptors, none of the twelve studied plant species or of the four Gretag Macbeth patches crossed that threshold in the total difference of colour (∆E*ab), being 2.9 CIELAB units the highest difference observed (Table 7.2). Remarkably, the 50% white adaptor on the SAV aperture performed slightly better than the black adaptor on specific plant samples (Table 7.2). With all other settings, instead, the differences of colour spanned from 3.8 to 48.3 CIELAB units (mostly between 20-30 CIELAB units) in plant samples and from 6.6 to 57.2 CIELAB units in Gretag Macbeth patches, corresponding to a very noticeable difference respect to the colour measured without adaptor. In the case of plant samples, the impact on colour measurements is extreme if the white 50% adaptor is used on the MAV aperture. In the homogeneous patches (except in bluish green), this occurs when employing the white 70% adaptor on the MAV aperture, and to a minor extent on the SAV aperture. The adaptors did not perform better on fresh plants than on their counterpart from the herbarium. It was on two fresh plants, C. isophylla and H. hederaceus, that measurements with adaptors not only lay within the non-noticeable colour differences (< 3 CIELAB units), but they even come close to 1 CIELAB unit. The contribution from the adaptor type (black or white, 70% or 50%, or no adaptor) on both MAV and SAV to the colorimetric information (L*, a* and b*) was analysed by distributing the measurements in a 2-dimensional Euclidean ordination space with a Principal Coordinates Analysis (PCoA) The colour measurements obtained with an adaptor (black or white) reducing 50% of the SAV opening overlaps accurately with the measurements without adaptors. The first two axes explain the major part of the data variability, both in plants (84.38% PC1, 11.68% PC2) (Figure 7.5) and in the four green Gretag Macbeth patches (59.28 % PC1, 31,81% PC2) (Figure 7.6). CHAPTER 7. A simple, reliable, and inexpensive solution for … 371 Between the two, the 50% white shows a slightly more accurate approximation than black 50%, as visible from the distance centroidsellipse. In the case of the MAV, the best adaptor is the black 50%, but overall it is evident that the measurements obtained by using the remaining adaptors are very far from the real colour. LUIS MIGUEL SERRANO PÉREZ 372 Table 7.2. Colorimetric analysis (total colour differences, ΔE*ab) of the measurements with black 50%, white 50%, black 70% and white 70% adaptors, compared to those obtained with no adaptor. ΔE*ab below the threshold of perception (< 3.0 CIELAB units) are in bold and underlined. Plants marked with an asterisk (*) are dry material from herbarium specimens. Jasione montana Trachelium caeruleum Campanula rotundifolia Campanula isophylla Hesperocodon hederaceus Jasione laevis MAV 50% black 9.5 7.4 4.5 3.8 5.6 5.3 50% white 25.0 26.1 24.1 24.5 22.8 23.9 70% black 22.1 19.1 15.0 12.4 17.4 12.3 70% white 46.4 48.3 46.8 47.8 45.1 47.1 SAV 50% black 2.5 2.4 2.0 0.8 0.7 1.2 50% white 2.1 2.7 1.8 1.1 1.1 1.4 70% black 21.1 19.6 15.9 11.7 16.1 11.0 70% white 34.4 36.1 34.4 35.9 34.6 34.9 C. adsurgens* C. latifolia* C. mollis* C. glomerata* C. trachelium* C. vesicular* MAV 50% black 7.4 8.5 4.1 6.5 9.7 5.4 50% white 14.8 18.6 15.9 20.4 17.1 19.1 70% black 19.9 18.1 17.4 21.0 21.1 19.8 70% white 34.5 40.1 35.2 41.8 37.5 39.0 SAV 50% black 2.1 2.9 1.5 2.4 2.9 2.1 50% white 1.1 2.4 2.8 2.4 2.0 0.7 70% black 18.9 17.6 17.3 18.4 19.5 18.9 70% white 30.6 35.1 26.4 37.2 34.0 33.4 CHAPTER 7. A simple, reliable, and inexpensive solution for … 373 GREEN FOLIAGE YELLOW GREEN BLUISH GREEN MAV 50% black 10.7 6.6 12.7 7.9 50% white 27.1 22.3 26.3 13.1 70% black 34.5 20.4 42.4 29.7 70% white 54.2 45.6 57.2 31.4 SAV 50% black 2.8 1.9 2.9 2.5 50% white 1.3 1.4 2.6 0.5 70% black 29.7 16.5 36.6 23.9 70% white 47.2 38.7 50.9 25.2 LUIS MIGUEL SERRANO PÉREZ 374 Figure 7.5. PCoA representing all the colour measurements of the plants with and without adaptor. Ellipses around each type of adaptor indicate the dispersion of the measurements and circumferences encircle the centroids of the distributions of measurements of each adaptor type. Samples were coloured according to the used adaptor. MAV, medium area view; SAV, small area view; 50B, 50% reduction with black adaptor; 50W, 50% reduction with white adaptor; 70B, 70% reduction with black adaptor; 70W, 70% reduction with white adaptor. CHAPTER 7. A simple, reliable, and inexpensive solution for … 375 Figure 7.6. PCoA representing all the colour measurements of green patches of the Gretag Macbeth chart with and without adaptor. Ellipses around each type of adaptor indicate the dispersion of the measurements and circumferences encircle the centroids of the distributions of measurements of each adaptor type. Samples were coloured according to the used adaptor. MAV, medium area view; SAV, small area view; 50B, 50% reduction with black adaptor; 50W, 50% reduction with white adaptor; 70B, 70% reduction with black adaptor; 70W, 70% reduction with white adaptor. LUIS MIGUEL SERRANO PÉREZ 376 Figure 7.7. Percentage of the measurements that have been assigned to a predicted group after the use of the adaptors on (a) MAV and (b) SAV. Samples were coloured according to the used adaptor. MAV, medium area view; SAV, small area view; 50B, 50% reduction with black adaptor; 50W, 50% reduction with white adaptor; 70B, 70% reduction with black adaptor; 70W, 70% reduction with white adaptor. CHAPTER 7. A simple, reliable, and inexpensive solution for … 377 To assess the similarity of the measurements obtained with and without the different adaptors, a Quadratic Discriminant Analysis (QDA) was performed. The mean squared error (MSE) was 0.338 in the SAV analysis and 0.046 in the MAV analysis. The cross-validation of QDA classifications left misclassification rates almost identical to the original analyses, with 0.317 and 0.047 MSE in SAV and MAV, respectively, validating the model in both analyses. The data obtained with SAV reveal a relatively high misclassification rate (33.8 %) in the confusion matrix that can be used to identify the adaptors with the minor impact on the real colour measured with SAV. The major part of measurements from SAV with white (57%) and black (53%) 50% adaptors are better classified as obtained with SAV without adaptor than within their predefined group. Thus, to measure the colour with these adaptors is as reliable as to measure it without (Figure 7.7). In addition, some mutual misclassification is detected between the measurements obtained with white and black 50% reducing adaptors: 17% of the measurements obtained with the white adaptor are classified as obtained with the black and 16% of the ones obtained with the black are classified as obtained with the white. Very few measurements from the 50% reducing adaptors were assigned to the 70% reducing adaptors (2%) and no measurement from white or black 70% adaptors was misclassified, indicating that measurements from these adaptors are very dissimilar to those obtained without adaptor. On the contrary to SAV, the misclassification rate in the QDA of the data collected with MAV was very low (0.046 %). 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Significant morphological stasis and recent radiation, with a Pleistocene burst of diversification, make Jasione one of the most enigmatic and taxonomically difficult genera of the Campanulaceae. Jasione shows one of the most remarkable biogeographic patterns of the Mediterranean basin, the east-west disjunction. Although the genus originated in western Mediterranean, the history of vicariant and dispersal events that lead to extant lineages of Jasione was as a dynamic roundtrip of lineages between both sides of the Mediterranean. Niche conservatism and adaptation to ecotonal sub-Mediterranean mountain shape the disjunct pattern, as intermediate European and Alpine regions are environmentally unsuitable regions. Several processes have relatively enhanced the niche breath of different groups, as recurrent polyploidization in different Iberian lineages, or evolution of annual habit, that appeared independently three times during the evolution of the genus and allowed widespread expansion in lowland areas, with J. montana as the most striking case. Hydathoid is a charismatic trait exclusive of one of the two main clades of Jasione. Apparently related with the hydric status of the plant, its occurrence seems to impose physiological constraints, and reduction in hydathoid number is positively correlated with habitat aridity. Environmental niche analyses, phylogeographic approaches and integrative taxonomic studies from a combination of lines of evidence in different Iberian taxonomic complexes, revealed the importance of detailed studies to unveil hidden biodiversity and its underlying processes, as niche shift associated to ploidy changes or niche [Document text truncated for crawler view.]