Eco-evolutionary dynamics and the evolution of phenotypic plasticity
Abstract
A lo largo de esta tesis me he propuesto comprender cómo las interacciones entre el genotipo y el ambiente pueden moldear la variación fenotípica intraespecífica. La variación fenotípica dentro de una especie puede ser debida a una expresión génica diferencial en respuesta distintos estímulos ambientales, o bien estar causada por divergencias genéticas (adaptativas o no). Mi objetivo es conocer cómo la interacción entre factores ecológicos externos y los mecanismos genéticos subyacentes permiten a los organismos expresar un fenotipo adecuado a las condiciones ambientales imperantes. Esta cuestión es apasionante pero compleja, y para abordarla he planteado una serie de preguntas más concretas y he buscado distintos sistemas de estudio apropiados para responderlas. En mi primer capítulo investigo un intrigante caso de marcada variación fenotípica en un carácter de enorme importancia en todos los organismos: el tamaño corporal. En concreto me he enfocado en comprender las causas de un marcado gradiente de tamaño en poblaciones continentales de anfibios, usando como modelo de estudio al sapo corredor, Epidalea calamita, en el suroeste de España. Las poblaciones enanas del Parque Nacional de Doñana son aproximadamente un 30% menores que poblaciones a tan sólo 60 km de distancia, en ausencia de barrera biogeográfica alguna. Para entender la contribución de distintos factores al establecimiento de este escarpado gradiente intraespecífico de tamaño en una escala geográfica tan pequeña, he combinado análisis de tasa metabólica, análisis de isótopos estables, esqueletocronología, análisis bioacústico de la variación en el canto, análisis comportamentales, y análisis genético-poblacionales usando marcadores neutrales. Además, he analizado y descrito en detalle la variación entre las distintas poblaciones en las principales variables bioclimáticas. He encontrado que la clina de tamaño observada tiene lugar en ausencia de una diferenciación genética entre poblaciones, y sin un aislamiento genético aparente, si bien existen diferencias ecológicas entre las poblaciones estudiadas y todo indica que diferencias en humedad y temperatura durante la etapa de crecimiento juvenil postmetamórfico pueda ser decisiva en la formación de este patrón. En los siguientes dos capítulos, uso el sapo de espuelas, Pelobates cultripes, como especie modelo para estudiar la evolución de la interacción entre el genotipo y el fenotipo, es decir la evolución de la plasticidad fenotípica. Para mi segundo capítulo he realizado un análisis comparado de la capacidad de respuesta al riesgo de desecación del medio acuático en larvas de 10 poblaciones de sapo de espuelas, pertenecientes a dos regiones (entorno de Doñana y sierra de Madrid). Para cada una de estas dos regiones, escogí las poblaciones de modo que la mitad provenieran de medios acuáticos de largo hidroperiodo y la mitad de medios de corto hidroperiodo. Para este estudio colecté puestas de las distintas poblaciones y crié las larvas resultantes en condiciones estándar de laboratorio, exponiendo la mitad de ellas a simulación de desecación de la charca reduciendo el nivel de agua. Mi hipótesis inicial fue que estas poblaciones diferirían en su capacidad de respuesta a la desecación del medio mediante aceleración de su desarrollo larvario, y esperaba que hubiesen por tanto divergido en sus normas de reacción fenotípicas y transcriptómicas. Además, puesto que la aceleración en el desarrollo es detonada por el incremento en la secreción de glucocorticoides, esperaba que los niveles corticosterona variaran entre individuos conforme al tratamiento experimental y su población de origen. Como continuación de este experimento, para mi tercer capítulo tomé fotografías del patrón de pigmentación dorsal de individuos de P. cultripes que fueron metamorfoseando. Mediante estas fotografías cuantifiqué la variación en el patrón dorsal de pigmentación para examinar la idea de que este patrón variara conforme a la población de origen y el grado de estrés ambiental experimentado durante el desarrollo larvario. Encontré que los individuos provenientes del tratamiento de bajo nivel de agua, que habían sido inducidos a acelerar su desarrollo (i.e. habían experimentado mayor estrés ambiental), metamorfosearon con un dorso más oscuro y con manchas más continuas, mientras que individuos que habían experimentado condiciones más benignas metamorfosearon con un dorso más claro y manchas más segregadas. El significado ecológico de la pigmentación dorsal en sapos de espuela y otros anfibios debe ser estudiado con más detenimiento en el futuro. Este capítulo es uno de los pocos ejemplos en la literatura que ha estudiado variaciones en la pigmentación dorsal a través de distintas fases ontogenéticas, y he mostrado que se trata de un carácter plástico. En mi cuarto capítulo, me planteo la pregunta de cuáles pueden ser las consecuencias demográficas poblacionales de la plasticidad fenotípica individual. Abordar esta cuestión con anfibios era inviable y recurrí a otro sistema de estudio clásico en la literatura de plasticidad, el de defensas inducidas por depredador en pulgas de agua, Daphnia magna. Este organismo tiene un ciclo reproductivo muy rápido y es fácil de manipular en el laboratorio, lo que me permitió lanzar un experimento a gran escala que comprendiera varias generaciones y poblaciones en un tiempo razonable. Llevé a cabo un experimento con cinco genotipos originados a partir de dos subpoblaciones de Daphnia, en el que la mitad de las réplicas fueron expuestos a pistas de depredador. A continuación, describí cómo los cambios inducidos por la presencia de depredadores en los caracteres de historia de vida de los distintos clones de D. magna alteraron la dinámica demográfica, acortando el tiempo de generación y alcanzando antes la capacidad de carga del sistema.
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! ! ! ! Eco-evolutionary!dynamics!and!the! evolution!of!phenotypic!plasticity! ! ! Ph.D.!Dissertation! Lee,!Hyeun-Ji
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! Recommended citation: Hyeun-Ji, L. (2020) Eco-evolutionary dynamics and the evolution of phenotypic plasticity. Ph.D. Thesis. Universidad de Sevilla, Sevilla, Spain. The photograph on the cover of chapter 1 is photo courtesy of H. Christoph Liedtke. The images on the cover and of the title pages of chapter 2, 3, and 4 are original creations by Paula Martín Art (@paulamartinart). Any form of reproduction, distribution, public communication or transformation of this property without prior authorization of the author is strictly forbidden.
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! Eco-evolutionary!dynamics!and!! the!evolution!of!phenotypic!plasticity! Memoria presentada por la Licenciada en Biología: Lee, Hyeun-Ji, para optar al título de Doctora por la Universidad de Sevilla PhD dissertation – Sevilla 2020 Fdo. Lee, Hyeun-Ji
! University of Sevilla Biological Station of Doñana Department of Wetland Ecology Doctoral programme: Integrative Biology “Eco-evolutionary!dynamics!and!! the!evolution!of!phenotypic!plasticity”! Ph.D. dissertation submitted by Lee, Hyeun-Ji To obtain the PhD degree by the University of Seville Seville, 2020 PhD dissertation supervised by: Supervisor: Prof. Dr. Ivan Gomez-Mestre Tutor: Prof. Dr. Laura Serrano
! Dr. Ivan Gomez-Mestre, Científico Titular en la Estación Biológica de Doñana, Consejo Superior de Investigaciónes Científicas (EBD-CSIC) como director, y Dra. Laura Serrano del Departamento de Biología Animal y Ecología de la Universidad de Sevilla, como tutora CERTIFICAN: Que los trabajos de investigación desarollados en la Memoria de Tesis Doctoral “Eco-evolutionary dynamics and the evolution of phenotypic plasticity” son aptos para ser presentados por la Licenciada Lee, Hyeun-Ji ante el Tribunal que en su día se designe, para aspirar al grado de Doctora por la Universidad de Sevilla. Y para que así conste, y en cumplimiento de las disposiciones legales vigentes, firman el presente documento en Sevilla, a 26 de Noviembre de 2020. Director Fdo. Dr. Ivan Gomez-Mestre Tutora Fdo. Dra. Laura Serrano
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! The work developed by Lee, Hyeun-Ji in this dissertation, including field sampling, as well as her visits to and stays in other research centers, have been funded by the following institutions: Spanish Ministry of Economy and Competitiveness (MINECO), and the Biological Station of Doñana (EBD) (see below for details). - FPI predoctoral fellowship by the Spanish Ministry of Economy and Competitiveness (MINECO), fellowship ID: BES-2015-074463 - Mobility grant awarded by the FPI predoctoral fellowship by the Spanish Ministry of Economy and Competitiveness (MINECO), fellowship ID: BES-2015-074463 – Belgium - Department of Aquatic Biology, Faculty of Science, Catholic University of Leuven, Belgium - Plan Nacional, grant ID: CGL2014-59206-P - Department of Wetland Ecology, Biological Station of Doñana
! 16! genetic!differentiation!across!populations,!while!there!are!ecological!differences! across!the!studied!populations.! From!my!second!to!third!chapter,!I!am!using!the!spadefoot!toad!Pelobates( cultripes!as!model!species!to!study!the!evolution!of!phenotypic!plasticity.!For!my! second! chapter! I! raise!tadpoles! of! P.( cultripes!and! conduct!water! drop! experiments!on!10!populations!of!P.(cultripes!tadpoles.!Half!of!those!populations! originate! from! long! lasting! ponds! and! half! from! short! lasting!ponds.! These! populations!are! predicted! to! have! evolved! a! different! capacity! to! respond! to! pond! desiccation! and! accelerate! development,! thus! the! phenotypic! as! well! as! transcriptomic!reaction!norm!are!expected!to!vary!across!populations.!Further,! since!developmental!acceleration!is!achieved!through!increased!stress!hormone! levels,! corticosterone! levels! are! expected! to! vary! across! individuals! raised! in! different!experimental!treatments.!! In! continuation! of! this! experiment,! for! my! third! chapter,! I! have! taken! photographs! of! the! dorsal! pigmentation! pattern! of! the! P.( cultripes!individuals! originating!from!my!experiment!at!metamorphosis.!The!photographs!were!used! to!test!whether!the!dorsal!pigmentation!of!juvenile!toads!differed!according!to! the! environmental! stress! experienced! across! life! stages.! I! have! found! that! individuals! originating! from! the! low! water! treatment! (i.e.! experienced! environmental! stress)! metamorphosed! with! a! darker! dorsum! with! more! continuous! blotches,! whereas! individuals! raised! under! benign! conditions! metamorphosed! with! a! lighter! dorsum! and! more! segregated! blotches.! The! ecological!significance!of!dorsal!pigmentation!in!spadefoot!toads!requires!further! study.!My!dissertation!is!one!of!the!few!studies!in!literature!that!has!explored!the!
! 17! dorsal!pigmentation!of!amphibians!across!different!life!stages,!in!the!context!of! phenotypic!plasticity.! In!my!fourth!chapter,!I!use!the!waterflea!Daphnia(magna,!a!study!system! that!is!readily!manipulated!in!lab!conditions!due!to!its!fast!reproductive!cycle.! This! allowed! me! to! conduct! large-scale! experiments! comprising! several! generations! and! populations,! a! process! which!was! more! complicated! with! amphibians.!I!conducted!an!experiment!on!five!genotypes!originating!from!two! subpopulations! of! Daphnia,! of! which! only! half! of! the! treatment! received! a! predator! cue.! Subsequently,! I! described! how! the! shifts! in! life-history! traits! experienced!through!phenotypically!plastic!responses!of!D.(magna!have!altered! the!population!dynamics!of!this!species.!! ! ! ! !
! 18! ! Resumen! ! ! ! ! ! A! lo! largo! de! esta! tesis! me! he! propuesto! comprender! cómo! las! interacciones! entre! el! genotipo! y! el! ambiente! pueden! moldear! la! variación! fenotípica!intraespecífica.!La!variación!fenotípica!dentro!de!una!especie!puede! ser!debida!a!una!expresión!génica!diferencial!en!respuesta!distintos!estímulos! ambientales,!o!bien!estar!causada!por!divergencias!genéticas!(adaptativas!o!no).! Mi!objetivo!es!conocer!cómo!la!interacción!entre!factores!ecológicos!externos!y! los!mecanismos!genéticos!subyacentes!permiten!a!los!organismos!expresar!un! fenotipo! adecuado! a! las! condiciones! ambientales! imperantes.! Esta! cuestión! es! apasionante! pero! compleja,! y! para! abordarla! he! planteado! una! serie! de! preguntas!más!concretas!y!he!buscado!distintos!sistemas!de!estudio!apropiados! para!responderlas.! ! En!mi!primer!capítulo!investigo!un!intrigante!caso!de!marcada!variación! fenotípica! en! un! carácter! de! enorme! importancia! en! todos! los! organismos:! el! tamaño!corporal.!En!concreto!me!he!enfocado!en!comprender!las!causas!de!un! marcado!gradiente!de!tamaño!en!poblaciones!continentales!de!anfibios,!usando! como!modelo!de!estudio!al!sapo!corredor,!Epidalea(calamita,!en!el!suroeste!de! España.! Las! poblaciones! enanas! del! Parque! Nacional! de! Doñana! son!
! 19! aproximadamente! un! 30%! menores! que! poblaciones! a! tan! sólo! 60! km! de! distancia,! en! ausencia! de! barrera! biogeográfica! alguna.! Para! entender! la! contribución!de!distintos!factores!al!establecimiento!de!este!escarpado!gradiente! intraespecífico!de!tamaño!en!una!escala!geográfica!tan!pequeña,!he!combinado! análisis! de! tasa! metabólica,! análisis! de! isótopos! estables,! esqueletocronología,! análisis! bioacústico! de! la! variación! en! el! canto,! análisis! comportamentales,! y! análisis! genético-poblacionales! usando! marcadores! neutrales.! Además,! he! analizado!y!descrito!en!detalle!la!variación!entre!las!distintas!poblaciones!en!las! principales! variables! bioclimáticas.! He! encontrado! que! la! clina! de! tamaño! observada! tiene! lugar! en! ausencia! de! una! diferenciación! genética! entre! poblaciones,!y!sin!un!aislamiento!genético!aparente,!si!bien!existen!diferencias! ecológicas! entre! las! poblaciones! estudiadas! y! todo! indica! que! diferencias! en! humedad! y! temperatura! durante! la! etapa! de! crecimiento! juvenil! postmetamórfico!pueda!ser!decisiva!en!la!formación!de!este!patrón.!! !En! los! siguientes! dos! capítulos,! uso! el! sapo! de! espuelas,! Pelobates( cultripes,!como!especie!modelo!para!estudiar!la!evolución!de!la!interacción!entre! el!genotipo!y!el!fenotipo,!es!decir!la!evolución!de!la!plasticidad!fenotípica.!Para! mi! segundo! capítulo! he! realizado! un! análisis! comparado! de! la! capacidad! de! respuesta!al!riesgo!de!desecación!del!medio!acuático!en!larvas!de!10!poblaciones! de!sapo!de!espuelas,!pertenecientes!a!dos!regiones!(entorno!de!Doñana!y!sierra! de!Madrid).!Para!cada!una!de!estas!dos!regiones,!escogí!las!poblaciones!de!modo! que!la!mitad!provenieran!de!medios!acuáticos!de!largo!hidroperiodo!y!la!mitad! de! medios! de! corto! hidroperiodo.! Para! este! estudio! colecté! puestas! de! las! distintas! poblaciones! y! crié! las! larvas! resultantes! en! condiciones! estándar! de! laboratorio,!exponiendo!la!mitad!de!ellas!a!simulación!de!desecación!de!la!charca!
! 20! reduciendo! el! nivel! de! agua.! Mi! hipótesis! inicial! fue! que! estas! poblaciones! diferirían! en! su! capacidad! de! respuesta! a! la! desecación! del! medio! mediante! aceleración! de! su! desarrollo! larvario,! y! esperaba! que! hubiesen! por! tanto! divergido! en! sus! normas! de! reacción! fenotípicas! y! transcriptómicas.! Además,! puesto!que!la!aceleración!en!el!desarrollo!es!detonada!por!el!incremento!en!la! secreción!de!glucocorticoides,!esperaba!que!los!niveles!corticosterona!variaran! entre!individuos!conforme!al!tratamiento!experimental!y!su!población!de!origen.! ! Como! continuación! de! este! experimento,! para! mi! tercer! capítulo! tomé! fotografías!del!patrón!de!pigmentación!dorsal!de!individuos!de!P.(cultripes!que! fueron!metamorfoseando.!Mediante!estas!fotografías!cuantifiqué!la!variación!en! el! patrón! dorsal! de! pigmentación! para! examinar! la! idea! de! que! este! patrón! variara! conforme! a! la! población! de! origen! y! el! grado! de! estrés! ambiental! experimentado! durante! el! desarrollo! larvario.! Encontré! que! los! individuos! provenientes!del!tratamiento!de!bajo!nivel!de!agua,!que!habían!sido!inducidos!a! acelerar! su! desarrollo! (i.e.! habían! experimentado! mayor! estrés! ambiental),! metamorfosearon! con! un! dorso! más! oscuro! y! con! manchas! más! continuas,! mientras!que! individuos!que!habían!experimentado!condiciones!más!benignas! metamorfosearon! con! un! dorso! más! claro! y! manchas! más! segregadas.! El! significado! ecológico! de! la! pigmentación! dorsal! en! sapos! de! espuela! y! otros! anfibios!debe!ser!estudiado!con!más!detenimiento!en!el!futuro.!Este!capítulo!es! uno!de!los!pocos! ejemplos! en!la! literatura!que! ha!estudiado! variaciones!en! la! pigmentación!dorsal!a!través!de!distintas!fases!ontogenéticas,!y!he!mostrado!que! se!trata!de!un!carácter!plástico.!! ! En!mi!cuarto!capítulo,!me!planteo!la!pregunta!de!cuáles!pueden!ser!las! consecuencias!demográficas!poblacionales!de!la!plasticidad!fenotípica!individual.!
! 21! Abordar! esta! cuestión! con! anfibios! era! inviable! y! recurrí! a! otro! sistema! de! estudio! clásico! en! la! literatura! de! plasticidad,! el! de! defensas! inducidas! por! depredador!en! pulgas!de! agua,!Daphnia(magna.! Este!organismo!tiene! un!ciclo! reproductivo! muy! rápido! y! es! fácil! de! manipular! en! el! laboratorio,! lo! que! me! permitió! lanzar! un! experimento! a! gran! escala! que! comprendiera! varias! generaciones! y! poblaciones! en! un! tiempo! razonable.! Llevé! a! cabo! un! experimento!con!cinco!genotipos!originados!a!partir!de!dos!subpoblaciones!de! Daphnia,! en! el! que! la! mitad! de! las! réplicas! fueron! expuestos! a! pistas! de! depredador.! A! continuación,! describí! cómo! los! cambios! inducidos! por! la! presencia!de!depredadores!en!los!caracteres!de!historia!de!vida!de!los!distintos! clones!de!D.(magna!alteraron!la!dinámica!demográfica,!acortando!el!tiempo!de! generación!y!alcanzando!antes!la!capacidad!de!carga!del!sistema.! !
! 22! ! General!Introduction! In! the! ever-changing! environment! in! which! we! live!in,! populations! are! globally! exposed! to! environmental! fluctuations,! and! such! environmental! variation! induces! phenotypic! changes! in! the! individuals! conforming! those! populations.!Organisms! must! express! an! adequate! phenotype! to! survive! and! reproduce! in! the! environment! they! experience,! and! such! fine-tuning! of! the! phenotype! to! the! environment! is! mainly! achieved! by! a! genomic! response! to! ecological!input!modulated!through!the!neuroendocrine!system.!The!extent!to! which!organisms!can!alter!their!phenotype!to!increase!their!fitness!to!match!the! reigning!environmental!conditions!will!depend!on!its!genetic!background,!their! capacity! to! assess! environmental! cues,! cue! reliability,! and! the! magnitude! of! environmental!changes!in!relation!to!their!potential!for!phenotypic!change.!Such! interactions!may!undergo!selection!and!result!in!local!adaptation!(Kawecki!and! Ebert! 2004,! Hereford! 2009,! Sanford! and! Kelly! 2011).! However,! the! extent! to! which! intraspecific! phenotypic! variation!is! adaptive! will! depend! on! the! equilibrium!among!genetic!variation,!gene!flow,!and!selection!(Savolainen!et(al.! 2007).!
! 23! In!highly!variable!environments,!environmentally-sensitive!genes!may!be! differentially!expressed!in!response!to!environmental!variability!and!give!rise!to! “phenotypic!plasticity”,!which!can!be!defined!as!the!ability!of!a!single!genotype!to! produce!various!phenotypes!depending!on!the!environmental!conditions!(West- Eberhard!1989,!West-Eberhard!2003).!The!varying!degrees!of!the!environmental! heterogeneity!may!steer!the!evolution!of!phenotypic!plasticity!in!different!ways;! phenotypic!plasticity!may!persist!if!favored!by!environmental!heterogeneity!and! hence! is! adaptive! (Lind! and! Johansson! 2007,! Lázaro-Nogal! et( al.! 2015),! or! alternatively,!if!the!environment!becomes!homogeneous!selection!may!favor!the! adequate! phenotype! in! that! environment! and! undergo!canalization,! a! process! known! as! genetic! assimilation! (Grether! 2005,! Pigliucci! et( al.! 2006).! Hence,! phenotypic! plasticity! is! not! only! subject! to! evolution,! but! may! actively! act! as! leader!of!the!course!of!evolution!(Price!et(al.!2003,!Schwander!and!Leimar!2011).! In!this!dissertation,!I!do!not!limit!myself!to!a!single!species!or!system,!but! employ!various!study!systems!to!strive!to!understand!the!correlation!between! nature! and! nurture! and! the! evolution! of! phenotypic! plasticity,! choosing! study! systems! in! accord! to! the! scientific! question! posed.! To! do! so,! I! start! with! i)! addressing! a! case! of! steep! variation! in! body! size! across! a! short! geographical! scale!in! the! natterjack! toad! (Epidalea( calamita)! in! southwestern! Spain,! and! describe!various!ecological!factors!contributing!to!this!phenomenon,!as!well!as! the!genetic!structure!of!the!studied!populations.!I!then!go!on!to!ii)!explore!the! phenotypic! plasticity! of! spadefoot! toads! (Pelobates( cultripes)! by! conducting!a! common!garden!experiment!and!analyzing!both!phenotypic!reaction!norms!as! well! as! transcriptomic! reaction! norms,! and! continue! to! iii)! ask! whether! environmental! stress! experienced! during! the! larval! phase! of! P.( cultripes! may!
! 24! have!an!influence!on!the!dorsal!pigmentation!pattern!of!this!species,!and!if!so,!to! what!extent!and!what!the!ecological!consequences!would!be.!Finally,!I!iv)!expand! my!research!onto!the!population!level!to!test!the!demographic!consequences!of! environmentally-induced! individual! shifts! in! phenotype,! conducting! an! intergenerational!experiment!with!the!waterflea!Daphnia(magna.!! ! A"case"of"extreme"dwarfism"in"the"natterjack"toad" ! Size!variation!across!the!animal!kingdom,!ranging!from!some!minute!ants! to!gigantic!blue!whales,!has!long!intrigued!biologists.!Size!not!only!varies!among! species! but!also! within! a! species.! This! is! particularly! informative! because! it! reflects!how!the!combination!of!genotype!and!varying!environments!can!shape! different! phenotypes! within! a! species.! The! main! external! determinants! of! intraspecific! size! variation! include! abiotic! factors! such! as! temperature,! photoperiod,!altitude,!and!latitude!(Hoffmann!1978,!Ashton!2002,!Rodríguez!et( al.!2008),!or!selection!forces!such!as!competition!or!predation!(Lomolino!1985).! Remarkable! differences! in! body! size! within! species! over! short!geographical! distances!have!usually!been!reported!for!islands!or!island-like!systems,!where! effective! geographical! barriers! have! led! populations! to! evolve! independently! from!each!other!(Lomolino!2005,!Keogh!et(al.!2005,!Jaffe!et(al.!2011).!Meanwhile,! in!continental!systems,!comparatively!greater!distances!have!to!be!travelled!to! be! able! to! observe! similar! degrees! in! size! variation! (Brumfield! and! Remsem! 1996,!Cruz!et(al.!2005).!! However,!in!Doñana!National!Park!(southwestern!Spain),!amphibians!are! remarkably! smaller! in! comparison! to! other! populations! in! the! vicinity! while!
! 25! apparently!lacking!an!efficient!geographical!barrier!such!as!a!big!mountain!chain! or!a!river.!In!this!dissertation!I!focus!on!the!natterjack!toad!(Epidalea(calamita)! as!example!of!an!amphibian!species!showing!such!local!reduction!in!body!size.! Natterjack!toad!populations!from!Doñana!experience!a!2.1!fold!change!in!body! mass!in!comparison!to!populations!only!as!little!as!37!km!northwards!from!the! National!Park!(Sierra!Norte!de!Sevilla).!For!this!study,!I!studied!six!populations:! two!dwarf!sized!populations!of!Doñana!National!Park!(Navazo!del!Toro!and!El! Puntal),! two! geographically! intermediate! populations,! whose!individuals! were! already!as!large!as!those!of!the!northern!populations!(Aznalcazar!and!Gerena),! and! finally! two! large! sized! populations! of! the! Sierra! Norte! de! Sevilla! (Constantina!and!El!Pedroso).!! To! explore! whether! the! observed! size! differences! were! due! to! genetic! differentiation! or! ecological! factors,! I! tested! for! genetic! isolation! of!the! dwarf! populations!using! neutral! markers;!whether! they! differ! in! their! trophic! status! analyzing! stable! isotope! traces;! whether! their! metabolism! differs,! comparing! their!standard!metabolic!rate;!whether!populations!differ!in!age!structure!and! age! at! sexual! maturity! employing! skeletochronology! and! telomere! length.! Moreover,! I! have!also! quantified! variation! in! advertisement! calls! across! populations!and!experimentally!tested!for!behavioral!reinforcement!of!the!body! size! gap! through! female! preferences.! Last,! but! not! least,! I! have! described! the! climatic!variables!of!the!regions!to!be!able!to!connect!the!toads’!ecology!with!the! local!climate.!I!found!this!phenomenon!of!local!dwarfism!to!occur!in!the!absence! of! genetic! isolation! and! with! similar! age! structures! and! effective! population! sizes.!Dwarf!populations,!however,!differed!in!trophic!status,!were!found!to!have! lower! metabolic! rate,! and! exposed! to! drier! and! warmer! climatic! conditions,!
! 32! since!all!the!phenotypic!variation!observed!is!plastic,!it!has!a!short!generation! time!and!relatively!large!populations!can!be!kept!under!laboratory!conditions.! This!was!of!huge!advantage!for!my!dissertation,!as!it!was!realistically!not!feasible! to! run! experiments! on! several! generations! and! various! populations! with! amphibians.! The! experimental! design! included!5! clones! for! each! of! two! subpopulations,!all!originating!from!a!single!population,!and!half!of!those!were! exposed! to! predator! cues! (i.e.! fish! kairomone)! and! the! other! half! was! not.! Aquaria! were! inoculated! with! the! same! water! inoculum.! I! expected!faster! development!and!higher!abundance!of!eggs!to!be!produced!by!clones!exposed!to! predator! cues,! and! I! expected! these! changes! to! have! a! strong! impact! on! the! population! demography! of! this! species.! I! explored! how! alternative! environmentally! induced! phenotypes! contributed! to! population! decline! or! growth! by! estimating! population! size,! extinction! risk! and! population! growth! rate.! ! ! ! !
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! 37! ! ! ! ! ! ! !
! 38! ! ! ! ! Chapter!1! Dwarfism"in"close"continental"amphibian"populations"" despite"lack"of"genetic"isolation" " " " " " " " " Published"in:"L.,"Hyeun-Ji,"J.,"Broggi,"G."Sánchez-Montes,"C.,"Díaz-Paniagua," and" I.," Gomez-Mestre." (2020)." Dwarfism" in" close" continental" amphibian" populations"despite"lack"of"genetic"isolation."Oikos,"129:"1243-1256,"doi:" 10.1111/oik.07086" ! !
! 39! RH:!Causes!of!amphibian!dwarfism! Dwarfism" in" close" continental" amphibian" populations" despite" lack" of" genetic"isolation" " Lee!Hyeun-Ji1,!Juli!Broggi1,2,!Gregorio!Sánchez-Montes1,3,!Carmen!Díaz-Paniagua1! and!Ivan!Gomez-Mestre1*! ! 1.!Ecology,!Evolution,!and!Development!Group,!Department!of!Wetland!Ecology,! Doñana! Biological! Station,! Consejo! Superior! de! Investigaciones! Científicas,! 41092!Seville,!Spain! 2.!Department!of!Biology,!Section!of!Evolutionary!Ecology,!University!of!Lund,!S- 223!62!Lund,!Sweden! 3.! Museo! Nacional! de! Ciencias! Naturales,! Consejo! Superior! de! Investigaciones! Científicas,!28006!Madrid,!Spain! *Corresponding!author:![email protected]" " Abstract Ample! variation! in! body! size! is! common! in! vertebrates! over! extensive! geographical!distances,!or!in!isolated!populations,!where!effective!geographical! barriers! may! cause! dwarfism! or! gigantism.! Here! we! study! potential! causes! of! extreme!size!reduction!in!continental!populations!of!amphibians!within!a!short! geographical! distance! and! in! the! absence! of! geographical! barriers.! Natterjack! toads!(Epidalea(calamita)!in!Doñana!National!Park!(Spain)!experience!up!to!2.1- fold! difference! in! body! mass! in! as! little! as! 37! km.! Studying! six! populations! divergent!in!body!size,!we!tested!for!genetic!isolation!of!the!dwarf!populations! using! multilocus! genotypes! (16! microsatellites),! and! explored! whether! populations!differ!in!trophic!status!(through!stable!isotope!analysis),!standard!
! 40! metabolic!rate,!and!growth!pattern,!senescence!and!age!structure!(conducting! telomere!length!assays!and!skeletochronology).!We!also!recorded!advertisement! calls! across! populations! and! experimentally! tested! for! behavioural! reinforcement! of! the! body! size! variation! through! female! preferences.! Local! dwarfism! in! these! populations! occurs! in! the! absence! of! genetic! isolation! and! while!maintaining!relatively!high!effective!population!sizes.!Dwarf!populations,! however,! are! exposed! to! drier! and! warmer! climatic! conditions,! have! different! trophic!status,!show!lower!mass-specific!metabolic!rate,!and!male!advertisement! calls! with! a! higher! dominant! frequency.! Juvenile! growth! differed! among! populations,! reaching! the! adult! stage! at! different! body! sizes.! Altogether,! our! results! suggest! a! significant! influence! of! environmental! conditions! on! the! physiology!and!ecology!of!the!Doñana!E.(calamita!populations,!mainly!affecting! toads!between!metamorphosis!and! sexual! maturity.!Further! experimental!and! genomic!studies!focusing!on!these!early!life!stages!are!necessary!to!dissect!the! relative! roles! of! the! environment! and! adaptive! genetic! differentiation! on! this! phenomenon.! Keywords:" intraspecific" size" variation," continental" dwarfism," body" size," anurans,"Epidalea(calamita" " Introduction Body!size!is!one!of!the!most!important!features!of!organisms!because!it!largely! influences!their!life! history,! generation!time,!metabolism,!and! ecology!(Martin! and!Palumbi!1993,!Woodward!et!al.!2005).!Intraspecific!body!size!variation!over! broad!geographical!distances!within!species!is!partly!shaped!by!abiotic!factors!
! 41! according!to!altitude!and!latitude!associated!with!temperature,!photoperiod,!or! precipitation!(Hoffmann!1978,!Ashton!2002,!Rodríguez!et!al.!2008).!For!instance,! some! taxonomic! groups! grow! to! larger! sizes! in! higher! altitudes! (Bergmann’s! rule),!as!selection!in!colder!climates!favours!a!lower!surface!to!volume!ratio!to! minimize!heat!loss!(Ashton!2002,!Meiri!et!al.!2005).!While!seemingly!valid!for! endotherms! (Meiri! and! Dayan! 2003),! this! pattern! is! not! universal! since! ectotherms!do!not!always!follow!Bergmann´s!rule!(Mousseau!1997,!Adams!and! Church!2008,!Pincheira-Donoso!et!al.!2008;!but!see!Cruz!et!al.!2005).!Further,! ecological!interactions!(i.e.!competition,!predation,!resource!limitation)!are!also! important! selection! forces! giving! rise! to! intraspecific! size! variation! (Lomolino! 1985).!! ! Remarkable! differences! in! body! size! within! species! over! much! shorter! geographical! distances! (as! short! as! 30! km)! than! in! continental! systems! have! been! reported! for! islands! or! island-like! systems! (Lomolino! 2005,! Keogh! et! al.! 2005,!Jaffe!et!al.!2011).!When!islands!are!formed,!colonizing!organisms!adapt!to! local! resources,! competitors! and! predators! or! lack! thereof.! Depending! on! the! relative! importance! of! intraspecific! competition,! ecological! release! from! predators! or! competitors,! or! resource! limitation! (Lomolino! 2005),! island! populations!can!evolve!gigantic!or!dwarf!versions!of!their!mainland!counterparts! (Keogh! et! al.! 2005).! Generally,! island! dwarfism! in! large! species! is! driven! by! resource!limitation!and!island!gigantism!in!small!species!is!due!to!competitive! release! (Lomolino! 1985).! Large! mammals! (i.e.! mammoths,! elephants,! and! ungulates!(Roth!1990,!Raia!and!Meiri!2006,!Herridge!and!Lister!2012))!are!wellknown! examples! of! island! dwarfism,! while! chelonians! (Jaffe! et! al.! 2011)! and! lizards! (Runemark! et! al.! 2015)! exhibit! island! gigantism.! Tiger! snakes! evolved!
! 48! significant!layer!(from!≤50!cm!up!to!≤100!cm)!of!sand!above!the!clay,!thus!the! area!was!very!dry!and!poor!in!nutrients!(World!reference!base!for!soil!resources! 2014).! Soils! in! Aznalcazar! were! calcaric! cambisol! and! calcaric! fluvisol,! with! a! little!bit!of!eutric!planosol.!These!are!fairly!developed!soil!types!with!clay!layers! that!can!retain! water! and!nutrients.!At!Gerena,! soil! consisted!mostly!of!eutric! regosol,! with! partial! presence! of! chromic! vertisol.! Regosol! was! a! very! underdeveloped!soil,!with!almost!no!layers!of!clay!and!mostly!rocky.!However,! eutric!regosol!refers!to!soil!that!has!a!rich!clay!layer!20–100!cm!deep!above!the! rocky!layer.!Chromic!vertisol!has!well!developed!clay!layers!and!is!very!rich!in! nutrients.!Constantina!and!Pedroso!consisted!largely!of!eutric!regosol.!Soil!types! have!been!characterised!according!to!the!World!reference!base!for!soil!resources! (2014).! We!also!obtained!Normalized!Difference!Vegetation!Indices!(NDVI)!from! satellite! images! through! the! Modis! Vegetation! index! MOD13Q1! (Didan! 2015).! The! best! available! pixel! values! from! 16-day! acquisition! periods! each! were! chosen!under!a!resolution!of!250!x!250!m!and!under!criteria!such!as!low!clouds,! low!view!angle,!and!the!highest!NDVI!values.!The!data!were!acquired!on!a!16-day! basis!from!18/02/2000!to!02/02/2016.!! " POPULATION"GENETICS"–"MICROSATELLITE"ANALYSES" Between!20/01/2015!and!23/03/2015!we!collected!a!total!of!180!E.(calamita! adult! males! across! all! populations! (Table! 1).! All! individuals! were! captured! at! night! while! calling,! to! ensure! including! only! sexually! mature! males.! We! measured!snout-to-vent!length!(SVL)!on!graph!paper!to!the!nearest!0.5!mm,!and!
! 49! body! mass! on! a! transportable! scale! to! the! nearest! 0.1! g.! We! then! clipped! the! fourth!toe!of!the!right!hindlimb,!storing!the!samples!in!100%!ethanol!at!-20!°C.! We!used!these!toe!samples!for!both!skeletochronology!and!DNA!extractions!(to! be! used! for! microsatellite! genotyping! and! telomere! analyses).! All! laboratory! procedures!were!conducted!at!Estación!Biológica!de!Doñana!(hereafter,!EBD).! Genomic! DNA! was! extracted! from! toe! clips! using! an! extraction! robot! (Freedom! EVO! 100;! Tecan).! All! DNA! samples! were! genotyped! at! 16! microsatellite! loci! (Sánchez-Montes! et! al.! 2017a)! with! PCR! multiplex! cycles! consisting!of!initial!denaturation!(95!°C,!5!min),!30!cycles!of!denaturation!(95!°C,! 30!s),!annealing!(60!°C,!90!s),!and!extension!(72!°C,!30!s),!followed!by!a!final! extension!step!(60!°C,!30!min).!All!reactions!were!run!in!a!total!volume!of!15!μL,! containing!7.5!μL!of!Type-it!Master!Mix!(Qiagen),!1.2!μL!of!primer!mix,!5.3!μL!of! H2O,! and! 1! μL! of! DNA.! Genotyping! was! performed! on! an! ABI! PRISM! 3130! sequencer! with! the! GeneScan! 500! LIZ! standard! (Applied! Biosystems).! Allele! peaks!were!assigned!manually!in!GeneMapper!v.4.0!(Applied!Biosystems).! We! calculated! basic! genetic! diversity! indexes! (mean! allelic! richness,! observed! and! expected! heterozygosity,! inbreeding! coeficient! FIS)! for! each! population! and! estimated! pairwise! population! differentiation! by! Hedrick’s! GST! (Hedrick!2005)!and!Jost’s!D!(Jost!2008)!using!the!package!diveRsity!(Keenan!et! al.!2013)!in!R!(R!Core!Team!2015).!We!used!GENEPOP!v4.0!(Rousset!2008)!to!test! for! departures! from! Hardy-Weinberg! proportions! (HWP)! and! linkage! disequilibrium,!and!then!re-estimated(FIS!and!both!genetic!differentiation!indices! (GST!and! D)! after! excluding! loci! out! of! HWP.! We! then! tested! for! isolation! by! distance! (IBD)! across! populations! via! Mantel! tests! with! the! package! adegenet!
! 50! (Jombart! 2008).! Geographic! distances! between! all! population! pairs! were! calculated!based!on!the!package!Imap!(Wallace!2012).!! We! estimated! the! effective! size! (Ne)! of! each! population! by! the! sibship! frequency!method!implemented!in!COLONY!v2.0.6.4!(Jones!and!Wang!2010).!We! used!all!available!genotypes!of!each!population!as!offspring,!without!specifying! any!candidate!fathers!or!mothers,!set!weak!paternal!and!maternal!sibship!size! priors!=!1,!and!genotype!error!rates!of!0.05!for!every!marker!(Sánchez-Montes!et! al.! 2017a,b).! We! conservatively! assumed! the! possibility! of! polygamy! in! both! sexes!and!performed!two!runs!per!population!with!“very!long”!run!length!and! “very! high”! precision! (Wang! 2016).! Finally,! we! tested! for! genetic! structure! across! populations! by! means! of! unsupervised! Bayesian! clustering! analyses! in! Structure!(Pritchard!et!al.!2000).!We!ran!10!analyses!for!each!value!of!K!(the! number!of!clusters)!from!one!to!ten!using!an!admixture!model!with!correlated! allele! frequencies! (Falush! et! al.! 2003),! with!1,000,000! burn-in! and! 1,000,000! post!burn-in!iteration!steps.!Ten!additional!analyses!were!run!under!the!same! settings!after!removing!loci!out!of!HWP!from!the!dataset.! " STABLE"ISOTOPES"ANALYSIS" Small!portions!of!52!E.(calamita!egg!clutches!(ca.!150!eggs!each)!were!collected! from!all!6!populations!(7!from!Navazo,!4!from!El!Puntal,!8!from!Gerena,!12!from! Aznalcazar,!11!from! Constantina,! and!10! from!Pedroso)!between!20/01/2015! and! 23/03/2015.! We! brought! the! eggs! from! each! clutch! to! a! walk-in! climatic! chamber!at!EBD!and!kept!them!in!activated!carbon-dechlorinated!tap!water!at! 20!°C,! and!12:12! L:D!photoperiod! until! hatching.! Hatchlings! were!collected! at!
! 51! stage!23!Gosner!(Gosner!1960)!before!they!reached!the!free-feeding!stage.!This! allowed!direct!assessment!of!the!maternal!diet,!in!particular!during!ovulation.! Hatchlings!were!euthanized!with!MS-222,!dried!to!constant!dry!mass!in!the!oven! at! 60! °C,! and! ground! to! a! fine! powder! using! mortar! and! pestle.! We! weighed! samples!to!the!nearest!0.1!mg!and!placed!them!in!tin!capsules!for!δ13C!and!δ15N! determination.!All!samples!were!combusted!at!1,020!ºC!using!a!continuous!flow! isotope-ratio!mass! spectrometry! system!by!means! of! Flash!HT!Plus! elemental! analyzer!coupled!to!a!Delta-V!Advantage!isotope!ratio!mass!spectrometer!via!a! CONFLO!IV!interface!(Thermo!Fisher!Scientific,!Bremen,!Germany).!The!isotopic! composition! is! reported! in! the! conventional! delta! (δ)! per! mil! notation! (‰),! relative! to! Vienna! Pee! Dee! Belemnite! (δ13C)! and! atmospheric! N2! (δ15N).! Replicate!assays!of!standards!routinely!inserted!within!the!sampling!sequence! indicated! analytical! measurement! errors! of! ±0.1‰! and! ±0.2‰! for! δ13C! and! δ15N,! respectively.! The! standards! used! were:! EBD-23! (cow! horn,! internal! standard),! LIE-BB! (whale! baleen,! internal! standard)! and! LIE-PA! (Razorbill! feather,! internal! standard).! These! laboratory! standards! were! previously! calibrated! with! international! standards! provided! by! the! International! Atomic! Energy!Agency!(IAEA,!Vienna).! " STANDARD"METABOLIC"RATE" We! collected! 67! sexually! mature(natterjack! male! toads! from! our! six! study! populations! (16! from! Navazo,! 10! from! El! Puntal,! 11! from! Gerena,! 10! from! Aznalcazar,!10!from! Constantina,! and!10! from!Pedroso)!between!20/01/2015! and! 23/03/2015.! These! were! different! individuals! from! those! sampled! for!
! 52! genetics!analyses,!albeit!being!captured!at!the!same!location.!Individuals!were! captured! after! dusk! and! placed! individually! in! plastic! containers.! Males! were! then!brought!to!the!laboratory!(EBD)!for!overnight!respirometry!measurements.! Toads! were! individually! placed! in! an! air-sealed! chamber! (0.5! L)! inside! an! insulated!Styrofoam!container,!each!chamber!contained!a!piece!of!cloth!soaked! in!water!to!maintain!air-humidity.!Toads!were!allowed!an!acclimatization!time!of! approx.!5!hours,!to!ensure!measuring!Standard!metabolic!rate!(hereafter,!SMR)! at!a!postabsorptive!stage!of!the!individuals.!SMR!was!determined!as!the!average! minimal! oxygen! consumption! throughout! the! night! in! an! open! circuit! respirometer,!at!a!constant!temperature!of!25!ºC.!The!respirometer!consisted!of! two! independent! modules! consisting! of! 4! and! 8! channels! respectively.! Both! modules!had!the!same!components!except!for!the!number!of!channels.!Outdoor! air! was! pushed! towards! each! chamber! through! independent! mass-flow! controllers!(Flow-bar-8),!adjusted!to!80!mL/min.!A!multiplexer!(RM4-8)!valve! system!conducted!outcoming!air!in!10!min!cycles!from!each!chamber!towards! the!CO2-Oxygen!analyzer!(FOXBOX-C!field!gas!analyzer,!Sable!Systems!Int.,!USA)! before! being! released.! Drierite! was! used! to! reabsorb! air! humidity! before! gas! analyses.! SMR! is! considered! the! lowest! rate! of! metabolism,! measured! at! a! particular!temperature,!in!a!resting!and!post-absorptive!ectotherm!according!to! the! Glossary! of! terms! for! thermal! physiology! (International! Union! for! Physiological! Sciences! 2001).! Hence,! the! values! of! oxygen! consumption! (mLO2/min)!were!averaged!for!the!most!stable!10!minutes!of!each!measurement! session! (approx.! 60! min),! calculated! following! Hill! (1972).! All! animals! were! returned!unharmed!to!the!field!at!dawn!after!the!experiment.! "
! 53! METAMORPHIC"SIZE"AND"ADULT"AGE"DETERMINATION" Size! at! metamorphosis! is! a! highly! plastic! trait! in! amphibians,! sensitive! to! key! factors! such! as! temperature,! food! availability,! or! larval! density,! among! many! others.! We! compared! size! at! metamorphosis! across! populations! by! collecting! individuals! in! their! late! stages! of! larval! development! (Gosner! stage! 40-42)! at! their!sites!of!origin;!these!were!brought!to!the!laboratory!and!kept!in!climatic! chambers!under!identical!conditions!(20!°C!and!80!%!air!humidity)!until!they! completed! reabsorbing! the! tail! and! underwent! metamorphosis.! Sample! sizes! ranged!from!20!to!25!individuals!per!population.!Once!the!forelimbs!emerged,! metamorphs! were! housed! in! 10! L! lidded! plastic! containers! with! moist! paper! towels! to! provide! humidity! and! refugium! while! reabsorbing! their! tails.! Amphibians! do! not! feed! during! the! last! metamorphic! stages! and! hence! metamorphs! were! not! fed! before! they! were! measured.! Upon! tail! resorption,! toadlets! were! weighed! on! an! electronic! scale! and! their! snout-vent-length! measured!with!callipers.!" To! estimate! the! age! of! adult! individuals! from! each! population,! we! obtained! cross! sections! of! the! phalanx! of! 180! clipped! toe! samples! (see! POPULATION! GENETICS! –! MICROSATELLITE! ANALYSES! and! Table! 1).! Glass! slides! were! immersed! in! a! solution! of! 1! g! of! glicerol! and! 0.1! g! of! chromium! potassium!sulphate!dissolved!in!200!mL!of!H2O,!heated!up!to!60!ºC!to!facilitate! adhesion!of!the!bone!sections.!After!five!minutes!of!bathing!in!the!solution,!slides! were!dried!in!the!oven!for!24!h!at!60!ºC.!Phalanxes!were!decalcified!in!3%!nitric! acid! overnight,! and! subsequently! cleaned! with! distilled! water! for! 2! h! with! an! agitator.!We!then!embedded!samples!in!OCT!(Optimal!Cutting!Temperature,!Agar!
! 54! Scientific!Ltd.,!UK),!froze!them!at!-20!ºC!in!a!cryostat!(MikroM,!Germany)!and!cut! them! at! a! thickness! of! 12! µm.! Samples! were! then! directly! adhered! to! treated! slides,!and!stained!with!hematoxylin!for!1!h.!Finally,!the!slides!were!washed!with! tap!water!to!remove!hematoxylin!excess!and!mounted!with!a!cover!glass!using! Aquatex!(Sigma-Aldrich,!USA).!We!estimated!the!age!of!the!toads!by!counting!the! lines! of! arrested! growth! (LAG)! observed! on! the! sections! under! a! microscope! (Sinsch! 2015).! Since! all! samples! originated! from! sexually! mature! males,! all! individuals!had!undergone!a!minimum!of!one!resting!period.!Thus,!we!expected! all!samples!to!have!at!least!one!LAG.!! " TELOMERE"LENGTH"ASSESSMENT" Relative! telomere! length! was! estimated! by! q-PCR! following! Criscuolo! et! al.! (2009).! The! single! copy! gene! glyceraldehyde-3-phosphate! dehydrogenase! (GADPH)!was!used!as!control!to!adjust!the!quantity!of!telomere!sequences!to!the! amount! of! DNA! in! the! q-PCR! reaction.! The! final! PCR! volume! was! 20! µL! containing! 10! µL! of! LightCycler! 480! SYBR! Green! I! Master! (Roche! Molecular! Systems,! Inc.,! USA)! and! 1! µL! of! DNA! at! 20! ng/µL! of! DNA.! The! reactions! for! telomeres! and! GADPH! were! done! in! different! plates! due! to! differing! PCR! conditions.!Telomere!PCR!conditions!consisted!of!denaturation!(95°C,!10!min)! followed! by! 40! cycles! of! annealing! (56°C,! 10! s)! and! extension! (72°C,! 40! s).! GADPH!PCR!started!with!denaturation!(95°C,!10!min)!followed!by!50!cycles!of! annealing! (56°C,! 15! s)! and! extension! (95°C,! 60! s).! Both! were! performed! in! a! LightCycler! 480! RT-PCR! System! (Roche! Molecular! Systems,! Inc.,! USA).! The! primer! concentration! was! 5mM! for! GADPH! and! 10mM! for! telomeres.! Each!
! 55! sample!was!run!in!duplicate!and!samples!with!a!coefficient!of!variation!greater! than! 5! %! were! removed! from! analyses.! Each! 96-well! plate! included! serial! dilutions!of!DNA!(120!ng,!40!ng,!10!ng,!2.5!ng,!0.66!ng!of!DNA!per!µL)!from!a! reference! pool! (the! internal! control)! run! in! triplicate,! which! were! used! to! generate!the!standard!curves,!plus!a!blank!control.!Quantification!cycle!values! (Ct)!were!transformed!into!normalized!relative!quantiles!(NRQs)!following!the! procedure!of!Hellemans!et!al.!(2007),!to!control!for!the!amplifying!efficiency!of! each!qPCR.!The!amplification!efficiency!for!telomere!products!ranged!between! 1.873!and! 2.092!and,! for! the!GADPH! products,!between! 1.872! and!2.021.! The! slope!of!the!calibration!curve!ranged!between!-3.669!and!-3.120!for!the!telomere! product!and!between!-3.671!and!-3.273!for!the!GADPH!product.!CV!calculated!for! a!reference!sample!run!in!all!plates!was!1,5%!for!telomere!RT-PCR!and!1,2%!for! the! GADPH! RT-PCR.! The! melting! curves! of! q-PCR! products! confirmed! no! evidence!of!primer!dimer!or!non-specific!amplifications.! " CALL"RECORDINGS"AND"FEMALE"MATING"PREFERENCE"ASSAYS" We! recorded! male! advertisement! calls! between! dusk! and! midnight! with! a! shotgun!microphone!(Sennheiser!ME64!with!K6!powering!modules;!frequency! response!±!2.5!dB!from!40!to!20,000!Hz;!Hanover,!Germany).!The!microphone! was!connected!to!a!flash!memory!recorder!(Zoom!H1,!Japan)!with!a!sampling! rate!of!44.1!kHz!and!a!resolution!of!16!bit.!Unlike!call!characteristics!such!as!note! repetition!rate!and!note!duration,!which!vary!to!a!large!extent!according!to!daily! fluctuations!of!rainfall!and!temperature,!dominant!frequency!is!an!honest!signal! of! body! size! of! male! anurans! (Gerhardt! and! Huber! 2002).! Hence,! dominant!
! 56! frequency! (Hz)! of! each! note! was! analysed! by! plotting! the! spectrograms,! and! averaged! ~! 10! call! recordings! from! 10! individuals! for! each! population.! We! conducted! these! analyses! using! Raven! Pro! version! 1.4! (Cornell! lab! of! Ornithology,!2011).! ! To!test!for!female!preferences!based!on!call!recordings,!we!collected!25! females! from! large! sized! populations! while! in! amplexus! to! ensure! sexual! receptivity! (10! females! from! Aznalcazar,! 5! from! Constantina,! and! 10! from! Pedroso).!Females!were!brought!to!the!laboratory!that!very!night!and!released!at! dawn! where! captured! previously! after! the! experiment.! Preference! tests! were! conducted!in!a!150!x!100!x!25!cm!fiberglass!arena!with!a!wireless!loud!speaker! (UE!Boom,!Ultimate!Ears,!Irvine,!USA)!in!each!of!the! two! far! ends.! We! placed! each!female!in!random!sequence!in!the!centre!of!the!arena,!covered!by!a!meshed! box!for!five!minutes!to!allow!them!to!acclimate.!In!each!trial!we!always!played! back!calls!from!northern!populations!on!one!speaker!and!calls!from!either!one!of! the! Doñana! populations! on! the! other,! while! randomly! alternating! among! our! recorded!calls.!We!then!gently!removed!the!box!covering!the!female!and!gave! her! ten! minutes! to! make! a! choice.! We! considered! that! a! female! showed! preference!for!one!speaker!when!she!approached!it!close,!touched!it!or!circled! around!it.!! " STATISTICAL"ANALYSES( All!statistical!analyses!were!conducted!in!R!v.3.2.1!(R!core!team,!2015).!Climate! data!was!first!analysed!by!means!of!a!principal!component!analysis!including!all! 19! bioclimatic! variables! obtained! from! Worldclim! (S1! in! Supplementary!
! 57! Material)!using!the!function!‘princomp’.!We!then!extracted!the!scores!from!the! two!main!components!and!conducted!general!linear!models!to!determine!their! association!with!body!size!variation!across!populations.!We!checked!these!and! all! other! linear! models! in! the! study! for! parametric! assumptions! using! the! package!lmtest!(Zeileis!and!Horthorn!2002).!We!used!linear!models!to!test!for! differences!among!populations!in!body!size!(SVL,!in!mm),!dominant!frequency!of! advertisement!calls!(Hz),!isotopic!values!and!telomere!length.!In!the!case!of!δ13C! data,! we! first! normalized! the! values! in! order! to! control! for! variation! in! lipid! content! in! the! samples! using! the! equation! δ13Cnormalized!=! δ13Cuntreated!-!1.11! +! 0.37C:N,!following!Caut!et!al.!(2013).!The!linear!model!to!test!for!differences!in! metabolic! rate! (VO2,! mLO2/min)! across! populations! included! body! mass! as! a! covariate.! Differences! in! age! structure! across! populations! were! assessed! by! using!two-sample!Kolmogorov-Smirnov!tests!(ks.test!function),!and!subsequently! adjusting! the! significance! level! for! multiple! comparisons! using! the! false! discovery! rate! approach! (FDR).! Growth! curves! across! populations! were! compared!by!fitting!Von!Bertalanffy!curves!for!each!population!(Von!Bertalanffy! 1938,! Pujol-Buxó! et! al.! 2012),! using! the! ‘vbfr’! function! of! the! R! package! fishmethods((Nelson!2014).!We!obtained!the!parameters!Linf!(mean!asymptotic! SVL),! K! (growth! coefficient),! t0!(hypothetical! age! at! size! 0).! Subsequently,! residual!analyses! were!done! to!test! our!model! fits.! For! the! female!preference! tests,!we!excluded!data!from!unresponsive!females,!and!fit!a!binomial!model!for! 12! trials! on! responsive! subjects."The! model! was! fit! with! an! alternative! hypothesis! that! the! true! probability! of! success! was! greater! than! 0.5! (representing!a!random!choice)!and!a!confidence!level!of!0.95.!! !
! 64! ! Figure!3.#(a)#Natterjack#toad#populations#varied#in#their#isotopic#signature#roughly#following# a# geographical# pattern# of# increased# δ15N# and# decreased# δ13C# in# a# southwest# to# northeast# cline.#(b)#Adjusted#means#for#standard#metabolic#rate#across#populations#corrected#for#body# mass.#Dwarf#Doñana#populations#showed#significantly#lower#standard#metabolic#rates#than# larger# bodied# populations.# Whiskers# indicate# +/-# standard# errors# (a,# b).# (c)#Dominant# 0 5 10 −27 −26 −25 −24 0.05 0.10 0.15 0.20 N (‰) C (‰) SMR (mLO /min) 15 13 δ δ 2 1.4 1.5 1.6 1.7 1.8 1.9 10 20 30 40 50 Bodymass (g) NAV PUN AZN GER PED CON Dominant frequency (kHz) Populations a b c d
! 65! frequency# (kHz)# shows# a# sharp# geographical# cline# across# populations# in# accordance# with# body#size;#higher#(1636-1655#kHz)#dominant#frequency#in#the#dwarf#populations#and#lower# (1441-1550# kHz)# dominant# frequency# in# the# larger# sized# populations.# (d)#Population# variation#in#male#body#mass#(N=30±3#per#population).#Doñana#individuals#are#between#20# and# 30%# lighter# than# those# from# the# other# populations.# Whiskers# indicate# +/-# standard# deviations#(c,#d)# " AGE"STRUCTURE,"GROWTH"PATTERNS"AND"TELOMERE"LENGTH" Size! at! metamorphosis! varied! across! populations! (F5,122!=! 39.67,! p! <! 0.0001),! with! maximum! differences! across! populations! of! 0.305! mg! in! body! mass! and! 1.57!mm!in!SVL,!corresponding!to!35!%!and!14.84!%!of!the!maximum!observed! values!for!body!mass!and!SVL,!respectively.!However,!those!size!differences!did! not! mirror! patterns! of! variation! in! adult! body! size! as! one! of! the! Doñana! populations!(Navazo!del!Toro)!had!the!largest!metamorphs!whereas!one!of!the! Sierra!populations!(Constantina)!had!the!smallest!metamorphs!(Figure!4a,!Table! S7!in!Supplementary!Material).!" Kolmogorov-Smirnov! tests! showed! that! age! structure! differed! significantly!across!populations!(Table!S8!in!Supplementary!Material),!with!one! dwarf!population!from!Doñana!(El!Puntal)!and!one!large!sized!population!from! Sierra!Norte!(Constantina)!showing!a!greater!proportion!of!young!adults!(Figure! 4b).! Growth! trajectories! were! similar! in! shape! across! populations! since! the! growth!coefficient!K!was!homogeneous!across!them!(Figure!4a).!Nevertheless,! the!maximum!size!at!infinity!(Linf)!was!smaller!for!both!Doñana!populations!than! the!rest,!indicating!that!their!growth!curves!plateaued!at!smaller!size!than!the! rest!(Figure! 4a).!Details! of!maximum! size! at! infinity! Linf,!growth! coefficient!K,! hypothetical!age!at!size!0!t0!provided!in!Table!S9!in!Supplementary!Material.!"
! 66! "Telomere! length! showed! a! significant! negative! relationship! with! body! size! (F1,179! =! 4.465,! p! =! 0.035)! and! age! (F1,156! =! 9.991,! p! =!0.0018)! across! individuals! from! all! populations! pooled! together! (Figure!5).! We! also! found! significant! differences! among! populations! in! their! average! telomere! length,! although! it! did! not! match! population! variation! in! body! size! as! both! a! large! bodied!and!a!dwarf!population!showed!longer!telomeres!than!the!rest!(F5,175!=! 5.265,!p!=!0.0001).!However,!the!populations!with!the!highest!mean!telomere! length! were! the! two! populations! with! the! highest! proportion! of! young! individuals!(El!Puntal!and!Constantina).! ! !
! 67! ! Figure!4.#(a)#Von#Bertalanffy#growth#curves#for#each#population#from#metamorphosis#(age#0)# onwards.#The#shape#of#the#growth#curves#is#homogeneous#(no#major#differences#in#growth# coefficient# K),# except# that# the# average# asymptotic# size# Linf#is# smaller# for# the#two# Doñana# populations,#indicating#that#natterjack#toads#from#Doñana#reach#the#plateau#at#a#smaller#size# than# the# rest.# Small# boxplot# shows# the# average# size# of# metamorphs# of# each# population,# showing#that#one#of#the#dwarf#populations#(NAV)#has#the#largest#metamorphs#and#one#of# the# large# sized# populations# (CON)# has# the# smallest# metamorphs.# Differences# among# populations#in#size#at#metamorphosis#therefore#do#not#predict#observed#differences#in#adult# body#size,#indicating#that#broad#adult#size#differences#become#apparent#largely#during#postmetamorphic# growth# in# this# system.# (b)#Age# structure# for# each# population# as# estimated# through#skeletochronology.#Populations#with#different#letters#in#brackets#significantly#differ# in#their#age#distribution.# # # 0123456 20 40 60 80 Age (years) SVL (mm) Navazo del Toro El Puntal Aznalcazar Gerena El Pedroso Constantina Navazo del Toro n. indiv. 0 10 20 1 2 3 4 5 6 (A) (BC) (ABC) El Puntal (C) Aznalcazar (A) Gerena Pedroso Constantina (C) 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 NAV PUN AZN GER PED CON 8 9 10 11 12 Populations SVL (mm) a b
! 68! # Figure!5.#Relative#telomere#length#(telomere#/#single#copy#gene#ratio)#varied#negatively#with# body#length#(a)#and#age#(b).#We#also#observed#significant#differences#among#populations#in# mean# telomere# length# (c).# The# two# populations# with# a# higher# proportion# of# young# adults# (PUN#and#CON)#showed#significantly#longer#telomeres.# " CALL"VARIATION"AND"FEMALE"MATING"PREFERENCE" 50 55 60 65 70 75 80 85 1 2 3 4 SVL 123456 1234 Age NAV PUN AZN GER PED CON 1 2 3 4 Populations Relative telomere length (T/S) Relative telomere length (T/S) Relative telomere length (T/S) a b c
! 69! The! average! dominant! frequency! of! the! calls! showed! significant! differences! among! populations! (F5,64! =! 15,! p! <! 0.001,! Figure! 3c),! in! accordance! with! the! observed!size!differences.!Smaller!males! from! Doñana! (El!Puntal! and! Navazo)! called! on! average! with! 11%! higher! dominant! frequency! than! the! other! populations.!During!mate!choice!tests!13!females!showed!no!preference!towards! either! speaker,! instead! staying! still!or! trying! to! escape.! Of! the! 12! responsive! females,! 11! out! of! 12! showed! a! strong! preference! towards! males! from! large! bodied! populations,! hence! discriminating! against! smaller! Doñana! males! (binomial!test!p!=!0.003).!The!probability!of!females!choosing!calls!of!males!from! larger!populations!was!92%.!! ! Discussion Here!we!present!evidence!for!dwarfism!in!continental!populations!of!natterjack! toads!in!the!absence!of!genetic!isolation!and!without!any!major!barrier!to!gene! flow!among!populations.!Amphibians!in!Doñana!National!Park!are!conspicuously! smaller! than! those! from! neighbouring! populations! (Diaz-Paniagua! et! al! 1996,! Marangoni!et!al.!2008),!and!toads!of!geographically!intermediate!populations!are! as!large!as!those!of!the!northern!populations.!We!have!found!that!i)!there!is!little! genetic! structure! across! our! sampled! populations,! ii)! the! dwarf! Doñana! populations!are!therefore!not!isolated,!iii)!all!six!populations!have!similarly!high! levels! of! genetic! variation! and! low! inbreeding,! in! concordance! with! values! observed!in!other!Iberian!populations!(Oromi!et!al.!2012;!Sánchez-Montes!et!al.! 2017a),!and!iv)!effective!population!sizes!at!both!sites!in!Doñana!are!comparable! to! those! of! large-bodied! populations.! The! four! above! conclusions! are! in!
! 70! accordance! with! previous! genetic! analyses! of! natterjack! populations! (Gomez- Mestre!and!Tejedo!2004;!Rowe!et!al.!2006;!Oromi!et!al.!2012),!especially!given! the! absence! of! barriers! to! gene! flow! and! the! fact! that! natterjack! toads! are! capable!of!migrating!over!long!distances!(Sinsch!1997,!Miaud!et!al.!2000b).!Note! that! one! dwarf! population! (El! Puntal)! showed! a! somewhat! higher! neutral! differentiation!than!the!rest,!but!not!the!other!dwarf!population!from!Doñana! (Navazo!del!Toro).!Moreover,!the!observed!differentiation!was!so!modest!that! Bayesian!clustering!methods!failed!to!detect!any!evidence!of!genetic!structure! (Figure! S5! in! Supplementary! Material).! Furthermore,! our! results! showed! that! most!of!the!differentiation!observed!was!driven!by!loci!departing!from!HWP,!and! if! removed! from! the! analysis! population! differentiation! was! even! lower! (see! Table! 1! for! FIS!and! Table! 2! for! GST,! Jost´s! D).! This! lack! of! genetic! structure! indicated! by! our! microsatellite! analysis! does! not! preclude! the! possibility! that! selection!could!have!resulted!in!population!divergence!in!loci!directly!associated! with! body! size.! Future! genomic! studies! combining! neutral! and! non-neutral! markers!will!shed!light!on!the!potential!adaptive!genetic!differentiation!of!the! dwarf! populations.! At! this! point! however,! we! can! conclude! that! the! size! differences!observed!are!not!the!result!of!genetic!isolation!and!drift.! Dwarfism! in! natterjack! toads! from! Doñana! seems! so! far! to! be! a! consequence!of!different!growing!environments,!with!no!evidence!to!date!of!it! being!adaptive.!We!found!substantial!differences!among!localities!in!key!climatic! and! habitat! variables.! The! Doñana! area! is! drier! and! warmer! than! the! other! localities! and! this! is! accentuated! in! summer,! harshening! the! aestivation! conditions!for!natterjack!toads.!Increased!temperatures!often!accelerate!larval! development!resulting!in!smaller!body!sizes!at!metamorphosis!(Kingsolver!and!
! 71! Huey! 2008),! but! our! natterjack! toad! populations! from! Doñana! did! not! show! reduced! size! at! metamorphosis! compared! to! the! other! study! localities.! A! biogeographical!analyses!of!body!size!variation!across!265!European!and!North! American! amphibian! species! showed! that! potential! evapotranspiration! (PET)! and!mean!annual!temperature!(AET)!are!the!two!main!drivers!of!body!size!in! these! species,! with! anurans! decreasing! in! size! with! increased! PET! and! AET! (Olalla-Tárraga!and!Rodríguez!2007).!This!inverse!relationship!between!ambient! humidity!and!temperature!and!body!size!is!commonly!observed!across!taxa!and! holds!true!for!natterjack!toads!throughout!their!evolutionary!history!(Martínez- Monzón!et!al.!2017).!This!common!phenomenon!in!line!with!the!current!global! warming!is!already!causing!reductions!in!body!size!worldwide!in!diverse!taxa! (Sheridan!and!Bickford!2011,!Caruso!et!al.!2015).!! The!Doñana!populations!experience!a!warmer!and!drier!climate!than!the! other!populations!(Figure!2a,b),!which!in!combination!with!sandy!soil!with!poor! water! retention,! causes! severe! droughts! over! extended! periods! of! time.! This! undoubtedly!affects!the!growth!of!post-metamorphic!juveniles,!since!amphibians! have!permeable!skin.!A!dry!substrate! constrains!juvenile!growth!in!natterjack! toads!by!21.5%!in!terms!of!body!mass!and!by!19.3%!in!body!length!over!just!five! weeks!(Gomez-Mestre! and!Tejedo! 2005).!Interestingly,! juveniles!from! Doñana! suffered! the! same! reduction! in! body! size! than! other! populations,! including! Pedroso,! included! also! in! this! study.! Juvenile! growth! in! amphibians,! and! in! particular! natterjack! toads,! is! critical! and! largely! determines! adult! size! differences!(Sinsch!et!al.!2010).!Hence,!hampered!juvenile!growth!due!to!drier! conditions!in!Doñana,!and!not!size!at!metamorphosis!per(se,!seems!to!be!a!strong! determinant!for!the!reduction!in!adult!size.!!
! 72! ! Throughout! its! range,! delayed! age! at! maturity! usually! correlates! with! larger!body!size!in!natterjack!toads!(Sinsch!et!al!2010),!and!delayed!maturation! is! compensated! with! extended! lifetime! fecundity! due! to! increased! longevity! (Leskovar!et!al.!2006,!Oromi!et!al.!2012).!Within!our!study!area,!growth!patterns! were! similar! across! populations,! except! that! the! dwarf! Doñana! populations! showed!a!downward!shift!in!their!curves,!as!they!matured!at!a!smaller!size!than! the!rest!of!the!populations!(Figure!4a).!We!initially!hypothesized!that!toads!from! dwarf!populations!could!be!maturing!at!an!earlier!age!and!reducing!their!growth! afterwards.!One! of! the!dwarf! populations! indeed!had!its! age!structure!shifted! towards!younger!individuals!(El!Puntal;!Figure!4b).!However,!the!other!dwarf! population! (Navazo! del! Toro)! did! not,! possibly! due! to! the! lack! of! young! individuals,! because! scarce! rainfall! in! the! previous! year! limited! availability! of! breeding!sites,!and!hence!reproduction!of!this!population.!Meanwhile,!one!of!the! Sierra!populations!also!had!an!age!structure!biased!towards!younger!individuals! and!yet!their!average!size!was!normal!(Figure!4b).!Since!size!at!metamorphosis! and! age! at! sexual! maturity! are! not! determinants! of! adult! body! size! and! postmaturation!growth!patterns!are!similar!across!populations,!constrained!growth! during!the!juvenile!phase!seems!to!be!the!strongest!determinant!of!reduced!body! size!in!Doñana!populations,!which!in!turn!seems!associated!with!a!warmer!and! drier! environment.! Reduced! body! size! also! resulted! in! an! average! higher! dominant!frequency!of!the!mating!calls!emitted!by!males.!Female!preference!for! lower!dominant!frequency!is!common!in!anurans!(Gerhardt!and!Huber!2002),! and!we!observed!that!the!differences!in!dominant!frequency!across!the!studied! populations! were! large! enough! to! bias! the! preference! of! large! females! from! neighbouring! populations! against! calls! of! small! Doñana! males.! However,!
! 73! whether!small!Doñana!females!also!prefer!lower!dominant!frequencies!emitted! by!larger!males!remains!to!be!tested.! ! Steep!differences!in!growth!during!early!ontogenetic!stages!may!result!in! differential!erosion!of!telomeric!chromosomal!regions!(Lee!et!al.!2012,!Burraco! et!al.!2017),!which!in!turn!can!translate!into!differences!in!longevity!(Monaghan! et! al.! 2008).! We! observe! a! very! clear! negative! association! between! telomere! length! and! age! and! size! (Figure!5).! Moreover,! differences! in! telomere! length! among!populations!matched!their!age!structure!as!the!two!populations!with!a! greater! proportion! of! young! adults! sampled! also! showed! significantly! longer! telomeres.! Slow! juvenile! growth! could! have! resulted! in! milder! telomere! shortening,! but! only! one! of! the! dwarf! Doñana! populations! showed! longer! telomere!length,!as!did!also!one!of!the!large!bodied!populations!with!presumably! faster! juvenile! growth.! Dynamics! of! telomere! shortening! across! these! populations! are! therefore! likely! affected! by! additional! factors! apart! from! differences!in!growth!rate.! !Metabolic! rate! is! intrinsically! related! to! body! size! (White! et! al.! 2006).! However,!even!accounting!for!differences!in!body!mass!we!found!a!geographical! cline! implying! substantial! physiological! differences! among! those! populations.! Dwarf! Doñana! populations! presented! lowest! mass-specific! SMR! (metabolic! intensity)! as! opposed! to! highest! values! found! in! the! Sierra! Norte! populations! (Figure! 3b).! Increased! aridity! causing! prolonged! water! deprivation! in! Doñana! may!induce!longer!periods!of!inactivity!for!toads,!resulting!in!reduced!metabolic! intensity!but!it!may!also!cause!reduced!metabolic!rate!to!decrease!their!energetic! expenditure,! since! aestivation! results! in! depressed! metabolic! rate! (Withers!
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! 85! 44281e5d53cf8ca78ca731525ea0/?vgnextoid=e87cd8cb9b1ec110VgnVCM100 0001325e50aRCRD&vgnextchannel=48f87d087270f210VgnVCM1000001325e 50aRCRD&vgnextfmt=rediam&lr=lang_es,"Accessed!26th!June!2018! Roldán,!F.!J.!et!al.!Mapa!Geológico!Digital!continuo!E.!1:50.000,!Zonas:!Subbético,!Cuenca! del! Guadalquivier! y! campo! de! Gibraltar.! In! GEODE.! Mapa! Geológico! Digital! continuo! de! España.! [en! línea].[consultado! el! 25/05/2018]! Disponible! en! :! http://info.igme.es/cartografiadigital/geologica/geodezona.aspx?Id=Z2600&la nguage=es! Van!Liedekerke,!M.!et!al.!2006.!ESDBv2!Raster!Library!-!a!set!of!rasters!derived!from!the! European! Soil! Database! distribution! v2.0,! European( Commission( and( the( European(Soil(Bureau(Network,!CD-ROM,!EUR!19945!EN! Wallace,!J.R.!2012.!Imap:!Interactive!Mapping.!R!package!version!1.32.! !!!!!!!https://CRAN.R-project.org/package=Imap! World!reference!base!for!soil!resources!2014,!International!soil!classification!system!for! naming! soils! and! creating! legends! for! soil! maps,! Food! and! Agriculture! Organization!of!the!United!Nations.! ! ! ! !
! 86! Supplementary"materials" ! S1:"Worldclim"bioclimatic"variables" BIO1!=!Annual!mean!temperature! BIO2!=!Mean!diurnal!range!(mean!of!monthly!(max!temp!–!min!temp))! BIO3!=!Isothermality!(BIO2/BIO7)(*100)! BIO4!=!Temperature!seasonality!(standard!deviation*100)! BIO5!=!Max!temperature!of!warmest!month! BIO6!=!Max!temperature!of!coldest!month! BIO7!=!Temperature!annual!range!(BIO5!-!BIO6)! BIO8!=!Mean!temperature!of!wettest!quarter! BIO9!=!Mean!temperature!of!driest!quarter! BIO10!=!Mean!temperature!of!warmest!quarter! BIO11!=!Mean!temperature!of!coldest!quarter! BIO12!=!Annual!precipitation! BIO13!=!Precipitation!of!wettest!month! BIO14!=!Precipitation!of!driest!month! BIO15!=!Precipitation!seasonality!(coefficient!of!variation)! BIO16!=!Precipitation!of!wettest!quarter! BIO17!=!Precipitation!of!driest!quarter! BIO18!=!Precipitation!of!warmest!quarter! BIO19!=!Precipitation!of!coldest!quarter! ! !
! 87! S2:"Factor"loadings"of"Principal"Component"Analysis"(PCA)" " ! PC1! PC2! Bio_12!(Annual!precipitation)! 0.99" 0.00! Bio_19!(Precipitation!of!coldest!quarter)! 0.99" -0.01! Bio_16!(Precipitation!of!wettest!quarter)! 0.99" -0.07! Bio_11!(Mean!temperature!of!coldest!quarter)! -0.99" -0.03! Bio_13!(Precipitation!of!wettest!month)! 0.99" -0.05! Bio_17!(Precipitation!of!driest!quarter)! 0.98" 0.12! Bio_6!(Max!temperature!of!coldest!month)! -0.98" -0.14! Bio_14!(Precipitation!of!driest!month)! 0.95" -0.07! Bio_1!(Annual!mean!temperature)! -0.94! 0.30! Bio_15!(Precipitation!seasonality!(coefficient!of! variation))! -0.94! -0.24! Bio_8!(Mean!temperature!of!wettest!quarter)! -0.91! -0.30! Bio_4!(Temperature!seasonality!(standard! deviation*100))! 0.89! 0.44! Bio_7!(Temperature!annual!range!(BIO5!-! BIO6))! 0.87! 0.48! Bio_18!(Precipitation!of!warmest!quarter)! 0.86! 0.03! Bio_2!(Mean!diurnal!range!(mean!of!monthly! (max!temp!–!min!temp)))! 0.84! 0.52! Bio_9!(Mean!temperature!of!driest!quarter)! -0.13! 0.99" Bio_10!(Mean!temperature!of!warmest! quarter)! -0.27! 0.96" Bio_3!(Isothermality!(BIO2/BIO7)(*100))! 0.44! 0.78! Bio_5!(Max!temperature!of!warmest!month)! 0.66! 0.74! Proportion"of"variation"explained" 0.74" 0.21" ! ! !
! 88! S3:"Mean"NDVI"values"across"study"populations" " ! S3:#Figure#visualizing#NDVI#across#study#populations,#showing#that#on#average#the#Doñana# area#is#less#densely#vegetated#than#the#Sierra#Norte#area.# El Puntal Navazo del Toro Aznalcazar Gerena El Pedroso Constantina
! 89! S4:"Bayesian"clustering"analysis"results,"showing"that"there" is" no"genetic" clustering"among"populations"regardless"of"K" ! ! ! ! !
! 96! ! N! A! %! AR! HO! HE! HWE! FIS! FIS! _Low! FIS! _High! Ecal4.6( 29! 8! 80! 7.69! 0.66! 0.77! 0.67! 0.145! - 0.066! 0.373! Ecal4.14( 28! 10! 71.43! 9.28! 0.64! 0.87! 0.958! 0.259! 0.064! 0.471! Ecal3.29( 29! 5! 62.5! 4.81! 0.48! 0.57! 0.133! 0.158! -0.15! 0.463! Ecal4.2( 29! 21! 43.75! 16.79! 0.83! 0.93! <0.001" 0.109! - 0.027! 0.26! Ecal3.19( 23! 10! 90.91! 8.58! 0.48! 0.85! 0.013! 0.435! 0.191! 0.678! Ecal4.26( 26! 23! 42.59! 17.43! 0.77! 0.93! <0.001" 0.171! - 0.002! 0.357! Ecal4.29( 29! 8! 47.06! 7.41! 0.76! 0.82! 0.973! 0.069! - 0.117! 0.266! Ecal4.3( 28! 10! 62.5! 9.63! 0.79! 0.85! 1! 0.079! - 0.079! 0.26! Ecal4.16( 29! 5! 100! 4.5! 0.45! 0.6! 0.115! 0.252! - 0.024! 0.537! Ecal3.26( 27! 14! 41.18! 11.49! 0.85! 0.85! <0.001" - 0.004! - 0.148! 0.16! Ecal4.8( 29! 17! 47.22! 13.68! 0.83! 0.85! 0.005! 0.029! - 0.115! 0.187! Ecal4.21( 29! 7! 58.33! 6.46! 0.62! 0.74! 0.561! 0.166! - 0.056! 0.389! Ecal3.4( 29! 8! 57.14! 7.12! 0.72! 0.78! 0.081! 0.077! - 0.112! 0.277! Ecal4.20( 29! 21! 87.5! 16.85! 0.93! 0.92! <0.001" -0.01! - 0.094! 0.1! Ecal4.18( 29! 8! 72.73! 7.44! 1! 0.83! 0.2! - 0.203! - 0.259! -0.167! Ecal4.24( 29! 12! 80! 10.68! 0.93! 0.88! 0.437! - 0.057! - 0.147! 0.057! " " " " " "
! 97! S6:"Snout-to-Vent"length"and"body"mass"of"metamorphs" " Population" SVL"(mm)" Body"mass"(g)" Constantina! 9.01! 0.564! El!Puntal! 9.62! 0.673! El!Pedroso! 9.73! 0.784! Gerena! 9.99! 0.893! Aznalcazar! 10.12! 0.817! Navazo!del!Toro! 10.58! 0.870! S7:"Kolmogorov-Smirnov"test"results" " *each!value!indicates!the!p-value!of!the!Kolmogorov-Smirnov!test,!indicating! whether!two!populations!differ!from!each!other!in!age!structure.! " " " S8:"Von"Bertalanffy"growth"curves"parameters" " ! Linf!! K! t0! Navazo!del!Toro! 64.86! 2.88! -0.06! El!Puntal! 60.90! 2.11! -0.08! Aznalcazar! 67.91! 2.45! -0.07! Gerena! 73.79! 1.83! -0.08! Pedroso! 70.26! 1.43! -0.10! Constantina! 68.57! 2.27! -0.06! " (Linf!=!mean!asymptotic!SVL,!K!=!growth!coefficient,!t0!=!hypothetical!age!at!size! 0)! ! ! ! NAV! PUN! AZN! GER! PED! CON! NAV! ! 0.002! 1! 0.006! 0.6! 0.003! PUN! 0.002! ! 0.004! 0.6! 0.05! 0.7! AZN! 1! 0.004! ! 0.01! 0.3! 0.005! GER! 0.006! 0.6! 0.01! ! 0.09! 0.9! PED! 0.6! 0.05! 0.3! 0.09! ! 0.05! CON! 0.003! 0.7! 0.005! 0.9! 0.05! !
! 98! ! !
! 99! ! ! ! ! Chapter!2! Population"divergence"of"developmental"plasticity"in"the" spadefoot"toad"Pelobates(cultripes,"using"RNA-seq" " ! ! ! ! ! ! ( In(prep.( ( (
! 100! RH:!Developmental!plasticity!of!spadefoot!toads! Population" divergence" of" developmental" plasticity" in" the" spadefoot" toad," Pelobates(cultripes" Lee! Hyeun-Ji1,! H.! Christoph! Liedtke1,! Edgardo! M.! Ortiz2,! and! Ivan! Gomez- Mestre1*! 1.!Ecology,!Evolution,!and!Development!Group,!Department!of!Wetland!Ecology,! Doñana! Biological! Station,! Consejo! Superior! de! Investigaciones! Científicas,! 41092!Seville,!Spain! 2.!Technische!Universität!München,!Germany! *Corresponding!author:![email protected]" ! Abstract Environmental!heterogeneity!is!pervasive!in!nature!and!favours!the!evolution!of! phenotypic! plasticity! when! no! single! trait! value! can! maximize! fitness! across! environments.! Phenotypic! plasticity! is! therefore! a! common! characteristic! of! living!organisms!and!is! based! on!the! differential!activation! of!neuroendocrine! pathways! in! response! to! reliable! environmental! cues! that! can! ultimately! also! result!in!differential!gene!expression.!Solid!experimental!and!theoretical!work! indicates! that! adaptive! phenotypic! plasticity! can! foster! rapid! evolutionary! divergence! through! genetic! accommodation,! However,! although! the! pattern! resulting!from!genetic!accommodation!can!sometimes!be!quite!apparent!when! comparing! taxa! that! have! been! independently! evolving! in! contrasting! environments!for!a!long!time,!we!still!lack!a!good!sense!of!how!the!process!is! initiated!and!its!underlying!mechanisms.!In!that!sense,!the!evolution!of!divergent! larval! developmental! rates! among! spadefoot! toad! species! is! highly! congruent!
! 101! with! the! process! of! genetic! accommodation,! but! we! need! to! understand! how! such! divergence! is! initiated.! To! address! this! question,! we! have! conducted! a! common! garden! experiment! on! ten!Pelobates( cultripes!populations! from! two! distinct!regions!within!the!Iberian!Peninsula!and!locally!associated!with!ponds!of! different! duration.! We! have! observed! marked! differences! across! these! populations!in!their!capacity!to!accelerate!development!in!response!to!decreased! water! levels,! and! have! subsequently! conducted! an! RNA-Seq! analysis! to! determine!which!genes!were!being!differentially!expressed!in!their!liver,!given! the! considerable! metabolic! effort! required! for! accelerating! development.! Our! results!show!that!differences!in!both!climate!and!hydroperiod!heterogeneity!may! have!been!responsible! for! observed!regional!divergence!in! adaptive!plasticity,! whereas!no!local!adaptive!divergence!was!observed!associated!with!pond!type! within!each!of!those!two!regions.!Genes!differentially!expressed!in!individuals! subjected! to! reduced! water! levels! were! associated! with! thyroid! hormone,! glucocorticoids,!and!the!urea!cycle,!all!processes!known!to!be!tightly!linked!to! the!regulation!of!metamorphosis.! Keywords:" phenotypic" plasticity," development," genetic" accommodation," differential"gene"expression,"transcriptomics,"RNA-seq,"clima,"hydroperiod," Pelobates(cultripes" ! Introduction Adaptive! phenotypic! plasticity! is! the! capacity! of! a! genotype! to! assess! and! respond!to!heterogeneous!environments!expressing!appropriate!phenotypes!to! the! local! conditions,! hence! enhancing! the! survival! and! fitness! of! organisms! (West-Eberhard! 1989).! Plasticity! will! evolve! given! sufficient! environmental!
! 102! heterogeneity! and/or! gene! flow! among! populations! exposed! to! different! selection! pressures! (Sultan! and! Spencer! 2002).! This! ability! to! adjust! the! phenotype!to!the!local!environmental!conditions!can!give!rise!to!rapid!adaptive! divergences,! especially! when! colonizing! novel! environments! or! facing! rapid! environmental! changes! such! as! the! current! global! change!(Le! Rouzic! and! Carlborg!2008,!Charmantier!et(al.!2008,!Lande!2009;!Pfennig!et(al.!2010).!! Traditionally,! biologists! were! convinced! that! genetic! changes! must! proceed!for!evolution!of!phenotypes!to!happen,!however,!recent!studies!show! that! phenotypes! may! play! a! crucial! role! in! leading! the! course! of! evolution! (Pfennig!et(al.!2010,!Casasa!and!Moczek!2018).!For!instance,!it!has!been!shown! in!North!American!spadefoot!toads!that!selection!acting!on!ancestral!phenotypic! plasticity! resulted! in! morphological! divergence! among! species! (Levis! et( al.! 2018).!Induced!phenotypes!can!be!subject!to!divergent!selection!depending!on! various! selection! regimes,! for! instance! the! degree! of! environmental! heterogeneity.!The!loss!of!environmental!variability!may!result!in!one!alternative! phenotype! being! favoured! by! selection,! and! subsequently! canalized!into! that! phenotype!(Pigliucci!et(al.!2006,!Lande!2009).!Phenotypic!plasticity!has!strong! evolutionary!potential! since!induced! plasticity!lead! ancestral!states! to!diverge! into!a!wide!array!of!reaction!norms,!giving!rise!to!diversified!populations,!and! ultimately,!species!(Pigliucci!and!Murren!2003).!Such!a!process!where!plasticity! undergoes! adaptive! genetic! changes! fuelled! by! selection! is! known! as! genetic! accommodation! (West-Eberhard! 2003;! Pfennig! et( al.! 2010).! Genetic! accommodation! has! been! empirically! demonstrated! across! various! taxa,! for! instance!in!wing!morphology!of!fruitflies!exposed!to!heat!shocks!(Waddington! 1953),!or!in!color!morphs!of!tobacco!hawk!moth!larvae!also!in!response!to!heat!
! 103! shocks!(Suzuki!and!Nijhout!2006).!However,!the!underlying!genetic!mechanisms! for! this! phenomenon! still! remain! a! vast! field! to! be! explored,! although! recent! transcriptomic!approaches!are!beginning!to!shed!light!on!it!(Casasa!et(al.!2020).!! The!advent!of!the!–omics!era!is!boosting!studies!on!genome-wide!changes! and!differential!gene!expression!using!non-model!organisms!(Minelli!and!Fusco! 2010),!as!modern!next-generation-sequencing!techniques!can!identify!the!genes! under!selection!as!well!as!transcriptional!plasticity!(Jones!et(al.!2017,!Jones!and! Robinson!2018).!Genome-wide!changes!can!be!achieved!by!signal!transduction! via! the! neuroendocrine! system!through! changes! in! hormone! level! changes,! enzymes,!or!epigenetic!signals!that!in!turn!are!mediated!by!mechanisms!such!as! DNA!methylation,!histone!modification,!or!interference!by!small!RNAs!(Gilbert! 2005,! Gilbert! and! Epel! 2009).! In! developmentally!plastic! organisms,! however,! genomes!can!be!differentially!expressed!(hereafter,!DE)!according!to!the!varying! environmental!conditions,!without!actual!changes!in!the!sequence!(Aubin-Horth! and!Renn!2009).!Taking!a!transcriptomic!approach!to!developmental!plasticity! can! help! identify! which! DE! genes! are! responsible! for! the! adaptive! plastic! responses! induced! by! the! environment,! and! assess! the!extent! to! which! organisms!can!respond!to!environmental!pressures!at!a!molecular!level!(Aubin- Horth!and!Renn!2009,!Johansson!et(al.!2013).!! Species! groups! wherein! certain! phenotypic! traits! have! evolved! independently!under!divergent!selection!regimes!are!excellent!model!systems!to! explore! the! process! of! genetic! accommodation! (Gomez-Mestre! and! Buchholz! 2006).!One!such!system!is!the!evolution!of!developmental!rate!across!spadefoot! toad!species,!which!is!highly!congruent!with!the!idea!of!evolutionary!divergence! through!genetic!accommodation!of!ancestral!plasticity.!
! 104! Amphibian!larvae!often!have!the!ability!to!accelerate!their!development! to! metamorphose! faster! and! escape! desiccation,! given! rapid! pond! drying! (Denver! 1998,! Richter-Boix! et( al.! 2006).! Spadefoot! toads! comprise! an! ancient! group! of! species! that! have! evolved! markedly! divergent! developmental! rates! (Buchholz!and!Hayes!2002),!of!which!the!ancestral!state!of!development!showed! similar! levels! of! developmental! plasticity! as! today´s! Western! spadefoot! toad,! Pelobates(cultripes((Gomez-Mestre! and! Buchholz! 2006).!The! spadefoot! toad!P.( cultripes!is!a!developmentally!plastic!species!that!adjusts!its!developmental!rate! to!the!environmental!conditions!experienced:!it!grows!large!without!significant! advancements!in!development!under!benign!conditions,!whereas!it!accelerates! its!development!and!metamorphoses!earlier!to!escape!desiccation!when!ponds! begin!to!dry!up!quickly.!Meanwhile,!the!spadefoot!toads!Spea(multiplicata!and! Scaphiopus(couchii!in!North!America!adapted!to!breed!in!much!more!ephemeral! ponds!(Levis!et(al.!2018).! While!S.(multiplicata!retained!a! rather! intermediate! degree!of!plasticity,!development!in!S.(couchii!became!highly!canalized!into!an! accelerated! version! of! ancestral! state! and! has! a! very! short! larval! period! of! approx.! 7-10! days! (Gomez-Mestre! and! Buchholz! 2006).! Developmental! acceleration!is!achieved!through!increased!corticosterone!and!thyroid!hormone! levels,!and! overexpression! of! thyroid!hormone!receptors!(Gomez-Mestre!et(al.! 2013,!Kulkarni!et(al.!2017).!This!comes!at!the!expense!of!a!high!metabolic!cost,! increased! oxidative! stress,! and! morphometric! consequences! (i.e.! metamorphosing!with!a!lower!body!mass,!shorter!snout-to-vent-,!hindlimb-,!and! snout! length;! Gomez-Mestre! et( al.! 2013).! It! has! been! shown! that! P.( cultripes! substantially!increases!its!corticosterone!and!thyroid!hormone!levels!if!exposed! to! desiccation,! while!S.( multiplicata! has! an! intermediate! level! of! hormone!
! 105! expression! and! S.( couchii,! being! canalized! into! a! fast! developmental! rate,! constitutively! expresses! high! levels! of! stress! hormones! and! a! much! higher! metabolic!rate!(Kulkarni!et(al.!2017).! Although!the!pattern!across!spadefoot!toad!species!seems!clear,!it!is!the! result!of!a!long!period!of!independent!evolution!(>!100!my),!which!has!resulted! in!various!other!changes,!like!a!major!divergence!in!their!genome!sizes!(Zeng!et( al.!2014).!Therefore,!it!is!critical!that!we!study!the!mechanisms!leading!to!genetic! accommodation! at! a! finer! scale,! both! geographically! and! temporally,! as! when! populations! within! species! adaptively! diverge!in! their! developmental! responsiveness.!In!that!spirit,!here!we!study!the!potential!divergence!in!adaptive! developmental! plasticity! among! P.( cultripes!populations! from! two! different! regions! within! the! Iberian! Peninsula,! and! within! those! regions,! comparing! populations!breeding!in!either!short-!or!long-lasting!ponds.!! We! conducted! a! common! garden! experiment! to! test!for! population! differences! in! their! ability! to! accelerate! development! and! achieve! an! early! metamorphosis! in! response! to! decreased! water! level.! We! predicted! that! increased!heterogeneity!in!pond!hydroperiod!would!have!favoured!the!evolution! of! plasticity,! resulting! in! steeper! reaction! norms,! and! consequently! genotypes! from! ponds! with! consistently! shorter! hydroperiods! would! be! more! canalized! into!constitutive!fast!development.!In!addition!to!duration!of!the!larval!period,! we! also! recorded! data! on! size! and! morphology! at! metamorphosis,! which! are! known! to! be! affected! by! changes! in! developmental! rate! (Gomez-Mestre! and! Buchholz!2006;!Gomez-Mestre!et!al.!2010).!Moreover,!we!combined!the!common! garden! experiment! with! standardized! sampling! of! liver! tissue! for! RNA-seq! analysis!of!differential!gene!expression!for!the!three!most!plastic!and!the!three!
! 112! effects!and!clutch!nested!within!population!as!random!effect.!The!significance!of! each!term!was!verified!by!running!likelihood!ratio!tests!specifying!a!chi-squared! test! on! each! model! in! comparison! to! another! model! excluding! that! term.! Analyses!of!variation!in!snout!and!hindlimb!length!were!corrected!for!variation! in!body!length!by!including!SVL!as!covariate!in!the!models.! A!principal!component!analysis!was!conducted!on!climatic!data!including! all!19!bioclimatic!variables!obtained!from!Worldclim!using!the!function!‘prcomp’! to!explore!differences!in!climate!across!localities.!When!clustering!by!region!was! confirmed,! we! further! regressed! variables! of! interest! (annual! precipitation,! precipitation! seasonality,! annual! mean! temperature,! temperature! seasonality)! against!plasticity!achieved!in!each!population!(standardized!as!the!difference!of! larval!period!across!water!treatments)." " TRANSCRIPTOMICS"-"SAMPLE"PREPARATION"" Based!on!the!observed!developmental!responses!(see!results!section),!we!chose! the!three!most!plastic!populations!(Llano,!Jabata,!Espajosas;!all!from!southern! Spain)!and!the!three!least!plastic!ones!(Buitrago,!Canencia,!Turrubuelo;!all!from! central!Spain)!and!conducted!RNA-Seq!analyses!on!preserved!liver!tissue!of!their! experimental! tadpoles.! We! haphazardly! chose! one! tadpole! from! each! of! four! sibships! per! population! as! biological! replicates,! resulting! in! 48! samples! (4! replicates!x! 6!populations! x! 2!treatments).! In!preparation! for! RNA!extraction,! each! cryopreserved! liver! sample! was! homogenized! in! 1! mL! of! TRIzol! reagent! (Sigma-Aldrich!Canada!Ltd.),!and!RNA!was!extracted!using!Qiagen!RNeasy!Mini! Kit!(Qiagen,!Germany)!following!the!manufacturer’s!protocol.!The!quantity!and! quality! of! the! extractions! were! assayed! using!a!Bioanalyzer! 2100! (Agilent!
! 113! Technologies,! Waldbronn,! Germany).! Library! preparation! and! sequencing! was! done! by! BGI! (http://www.bgi.com/global/)! on! a! DNBSeqTM! platform! with! PE100.!Each!sequenced!library!had!a!minimum!of!20!million!pair-end!reads.!! SEQUENCING"QUALITY"CONTROL" Raw!sequencing!reads!were!cleaned!in!two!steps!with!the!script!clean_pe_rna.sh( (see! Supplementary! material! S2! for! full! script),!which! is! a! wrapper! around! BBTools!v.38.86.!The!first!step!consists!of!trimming!remaining!DNBSEQ!adaptor! sequences!and!poly-A!stretches!longer!than!3!bp!from!both!ends!of!the!reads.! The! second! step! is! aimed! at! removing! low-quality! sequences,! including! those! from! the! phiX! viral! genome! (a! common! spiking! sequence! used! in! NGS)! and! sequencing! artifacts.! Also,! we! trimmed! low-quality! bases! (PHRED! score!<! 10)! from!the!ends!of!the!reads.!Finally,!reads!with!an!average!PHRED!score!<!12!after! the!trimming!process!were!removed!as!well.! ! READ"MAPPING" We!used!the! mapping-based! mode!of!the!salmon(software!v.1.2.1!(Patro! et(al.! 2017)!to!quantify!RNAseq!reads.!For!this,!we!used!reference!transcripts!derived! from!the!genome!assembly!PECUL23,!a!draft!genome!of!P.(cultripes!available!to! our! research! group,! in! preparation! for! publication.! To! improve! the! mapping! performance,!we!included!a!decoy!that!consisted!of!non-coding!regions!of!the! genome!as!well!as!the!mitochondrial!genome!(mtDNA!onward)!and!a!consensus! of! the! nuclear! ribosomal! cistron! (nrDNA! onward).! The! mitogenome! was! not! assembled!for!PECUL23!and!so!was!assembled!by!mapping!the!clean!reads!of! one!of!the!biological!replicates!of!our!experiment!(Bui1L9)!against!a!published!P.(
! 114! cultripes(mitochondrial!genome!(Genbank!accession!number:!NC_008144.1;!Gissi! et( al.! 2006).! The! nrDNA! was! assembled! following! the! structure:! 5'ETS-- 18S_rRNA--ITS1--5.8S_rRNA--ITS2--26S_rRNA--3'ETS,!using!MEGAHIT!v.1.2.9!(Li! et(al.!2015).!Since!the!nrDNA!represents!a!consensus!of!a!tandem!repeat!it!can!be! represented!as!a!circular!sequence.!Both,!mtDNA!and!nrDNA,!are!usually!overrepresented! in! RNAseq! experiments! (despite! poly-A! enrichment! or! rRNA! depletion),!therefore!they!had!to!be!integrated!in!the!decoy!reference!to!avoid! forced!mapping!artifacts.! ! DIFFERENTIAL"GENE"EXPRESSION"ANALYSIS" The!raw!counts!quantified!by!salmon!across!all!libraries!were!combined!into!a! single!counts!matrix!using!the!R!package!tximport!v.1.101!(Soneson!et(al.!2016).! Raw! count! data! at! the! transcript! level! were! used! for! Differentially! Expressed! Genes!(hereafter,!DEG)!analysis.!The!DEG!analysis!was!run!using!the!R!package! DESeq2! v.1.22.2! (Love! et( al.! 2019:! updated! version! of! Love! et( al.! 2014).! The! count!data!was!transformed!using!Variance!Stabilizing!Transformation!(VST)!in! order!to!normalize!the!data!and!for!removal!of!the!variance’s!dependency!on!the! mean.! The! transformed! data! was! used! for! counts! exploration,! employing! a! principal!component!analysis.!As!strong!clustering!across!localities!(Central!vs.! South)!was!observed!that!may!obscure!treatment!effects,!we!proceeded!to!run! the!DEG!models!on!the!counts!of!each!population!independently,!setting!water! level!as!the!treatment!contrast!for!the!analyses.! By! default,! the! log! fold! change! (LFC)! calculation! uses! a! normal! prior! distribution,!centred!on!zero!with!a!scale!that!is!fit!to!the!data!(Love!et(al.!2014),! and!applies!a!strong!filter!threshold!excluding!genes!with!low!p-value!LFC.!We!
! 115! used!an!alternative!shrinkage!estimator!using!the!R!package!apeglm!(Zhu!et(al.! 2018).! This! allowed! shrinking! low! counts! and! contrasting! them! by! effect! size! ranking!p!values!associated!with!a!null!hypothesis,!keeping!genes!with!a!small!pvalue! and! low! variation! in! expression.! To! test! for! significant! differences! in! expression!between!the!two!treatments!(i.e.!low/high!water!level),!we!estimated! fold!changes!for!each!gene,!which!estimates!the!effect!size,!arbitrarily!treating! tadpoles! originating! from! the! high! water! treatment! as! control,! using! default! settings!of! the! DESeq2!pipeline.!A! diagnostic! check!of!the! model! fit!was!done! using! the! function! “plotDispEsts”! of! the! DESeq2! package,! comparing! the! final! shrunk! estimates! from! gene-wise! assessments! towards! the! fitted! approximations.!We!considered!any!gene!with!a!false!discovery!rate!<!0.05!to!be! DE!between!the!two!water!level!treatments.!! ! FUNCTIONAL"ENRICHMENT"ANALYSIS! To!test!whether!particular!ontology!terms!or!functional!pathways!were! significantly!enriched,!all!DEG!that!had!available!Xenopus(tropicalis!annotations! (blastx! hits! of! the! P.( cultripes! genome! transcripts! against! the! X.( tropicalis! proteome! v.9.1! of! Ensembl( release! 100),! were! further! analyzed! using! the! R! package!gprofiler2!v.0.2.0!(Reimand!et(al.!2019).!This!list!of!DEG!(pooling!up!and! down! regulated! genes)! was! compared! to! a! custom! background! consisting! of! annotations!of!all!X.(tropicalis(annotations!of!the!P.(cultripes!genome.!Three!Gene! Ontology! groups! (Molecular! Function,! Biological! Process,! and! Cellular! Component),! KEGG! (Encyclopedia! of! Genes! and! Genomes),! and! Reactome! classification! systems! were! used! as! domains! for! searching! functional!
! 116! enrichment.!A!threshold!of!p!<!0.05!was!set!as!cut-off!for!significantly!enriched! pathways.!! VARIANT"CALLING"USING"RNAseq"READS" We! aimed! to! identify! single! nucleotide! polymorphisms! (SNPs)! present! in! our! RNA!sequences,!to!verify!genetic!clustering!across!populations.!In!order!to!do!so,! the!reads!were!mapped!to!a!reference!that!included!the!genome!assembly,!the! mtDNA,!and!the!nrDNA!(Pcu23.ss.mt.nr.fa).!We!used!two!splice-aware!mappers,! GMAP!v.2019.09.12!(Wu.!et(al.!2010)!and!STAR!v.2.7.5a!(Dobin!et(al.!2013).!Then! we! proceeded! to! do! variant! calling! on! the! resulting! BAM! files! using! two! commonly!used!variant!callers,!freebayes!v.1.3.2!(Garrison!and!Marth!arXiv)!and! bcftools!(part! of! samtools! v.1.9)! (Li! 2011).! Finally,! SNP! counts! across! method! combinations! (gsnap! +! bcftools,! gsnap! +! freebayes,! star! +! bcftools,! and! star! +! freebayes)!were!compared.! "The!resulting!VCF!files!were!converted!into!phylogenetic!matrices!using! vcf2phylip!v.2.3!(Ortiz!2019).!The!matrices!were!analyzed!in!IQ-TREE!v.2!(Minh! et(al.!2020)!to!obtain!phylogenetic!trees!using!the!model!GTR+ASC!(general!time! reversible!nucleotide!substitution!model!with!ascertainment!bias!correction!to! account! for! only! including! variable! sites! in! the! alignments)! and! support! was! derived!from!5000!ultrafast!bootstraps!(Hoang!et(al.!2018).! " Results" " DEVELOPMENTAL"ACCELERATION"AND"MORPHOLOGICAL"CONSEQUENCES"
! 117! There!was!a!significant!region!effect!on!larval!period!( χ 2!=!6.84,!p!=!0.008,!Table! 1),!and!also!a!highly!significant!interaction!of!water!treatment!x!region!( χ 2!=!44.2,! p!<! 0.001).! On! the! contrary,! pond! type! (short-lasting/long-lasting)! had! no! significant! effect! on! larval! period! ( χ 2! =! 0.02,! p!=! 0.89),! and! neither! did! the! interaction!of!water!treatment!x!pond!type!effect!( χ 2!=!0.07,!p!=!0.78).!Thus,!we! proceeded! with! exploring! the! effects! of! water! treatment! on! developmental! acceleration!and!its!morphological!consequences!within!each!region.! ! Table" 1." Plasticity" achieved" of" each" population,! obtained! by! standardized! differences! of! larval! period! across! water! treatments.! Positive! plasticity! values! indicate!the!number!of!days!accelerated!when!exposed!to!low!water!treatment,! whereas! negative! values! indicate! that! some! populations! had! a! longer! larval! period!in!control!than!in!the!low!water!treatment.! Region" Population" Plasticity" Southern!Spain! Espajosas! 56.11! Southern!Spain! Llano! 42.69! Southern!Spain! Jabata! 55.25! Southern!Spain! Jimenez! 16.42! Central!Spain! Canencia! 6.81! Central!Spain! Buitrago! -5.69! Central!Spain! Turrubuelo! -12.97! Central!Spain! Santo!Tome! -19.77! Central!Spain! Roblellano! 3.27! Central!Spain! Valdemanco! 10! !
! 118! For! the! central! Spain! populations,! reduced! water! level! did! not! cause! a! general! shortening! of! larval! period! ( χ 2! =! 0.04,! p!=! 0.85).! Nevertheless,! a! significant!water!treatment!x!population!interaction!was!found!( χ 2!=!20.99,!p!=! 0.0008),!showing!that!not!all!populations!responded!the!same!way!to!decreased! water! levels.! (Figure! 3b,! Supplementary! material! S3).! Decreased! water! level! decreased!body!mass!upon!metamorphosis!by!21%!on!average!( χ 2!=!212.58,!p!<! 0.001),!and!body!mass!also!showed!a!significant!water!treatment!x!population! interaction! ( χ 2! =! 12.17,! p!=! 0.03),! showing! population! differences! in! growth! under! varying! water! levels! (Figure! 4b).! Body! length! (SVL)! of! the! metamorphosing! toadlets,! and! size-corrected! snout! and! hindlimb! lengths! decreased!in!individuals!exposed!to!reduced!water!level!(SVL:! χ 2!=!183.31,!p!<! 0.001,!8.67!%!decrease!(Figure!4d),!snout:! χ 2!=!3.74,!p!=!0.05,!4.76!%!decrease,! hindlimb:! χ 2! =! 14.19,! p!=! 0.0001,! 10.55! %! decrease! (Figure! 5b,d)).! However,! these!morphological!features!were!not!affected!by!a!water!x!population!effect! (SVL:! χ 2!=!7.24,!p!=!0.2,!snout:! χ 2!=!7.61,!p!=!0.18,!hindlimb:! χ 2!=!2.12,!p!=!0.83).!! The! southern! populations! instead! significantly! accelerated! their! development! in! response! to! decreased! water! level! ( χ 2! =! 45.36,! p!<! 0.001),! achieving! metamorphosis! on! average! 22.8! %!earlier! (Fig.! 3).! We! observed! no! significant! water! x! population! effect! on! time! to! metamorphosis! ( χ 2!=! 3.5,! p!=! 0.32)! (Figure! 3a,! Supplementary! material! S4).! Decreased! water! level! significantly!decreased!body!mass!( χ 2!=!121.34,!p!<!0.001,!36.8!%!decrease)!and! body!length!(SVL)!upon!metamorphosis!( χ 2!=!104.46,!p!<!0.001,!12!%!decrease)! (Figure! 4a,c).! The! interaction! of! water! treatment! and! population! had! a! significant! effect! on! body! length! (SVL)! ( χ 2! =! 9.45,! p!=! 0.02),! while! it! had! a!
! 119! marginally!significant!effect!on!body!mass!upon!metamorphosis!( χ 2!=!7.52,!p!=! 0.05).! Neither! snout! ( χ 2! =! 0.01,! p! =! 0.91)! nor! hindlimb! ( χ 2! =! 0.99,! p!=! 0.32)! lengths!were!significantly!decreased!when!exposed!to!reduced!water!levels!in! these! southern! populations,! and! there! was! no! significant! water! x! population! effect!on!either!snout!( χ 2!=!1.16,!p!=!0.76)!or!hindlimb! lengths!( χ 2!=!1.93,!p!=! 0.59)!of!individuals!of!both!water!treatments!upon!metamorphosis!(Figure!5a,c).! ! " "
! 120! " Figure! 3#Southern#populations#(a)#accelerated# their# development# significantly# shortening# their# larval# period#when# exposed# to# the# low# water# treatment,# whereas# the# central# populations#(b)#did#not#readily#do#so.#Further,#it#is#noteworthy#that#the#southern#populations# had# a# broader# range# of# larval# period# whereas# the# central# populations# were# much# more# canalized#into#fast#development." " " Buitrago Canencia Turrubuelo Roblellano Valdemanco high low 100 150 200 250 300 water level days to metamorphosis high low Espajosas Jabata Jimenez Llano a b Santo Tome
! 121! ! Figure! 4#Individuals# of# both# the# southern# (a,# c)# and# central# (b,# d)# populations# metamorphosed# with# a# lower# body# mass# and# shorter# snout-to-vent# (SVL)# length# when# exposed# to# low# water# treatment.# This# was# true# for# all# populations# regardless# of# whether# they#accelerated#their#development#or#not.## " " 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Bodymass (g) Espajosas Jabata Jimenez Llano Buitrago Canencia Roblellano Valdemanco 20 25 30 35 40 water level SVL (mm) high low Espajosas Jabata Jimenez Llano high low Buitrago Canencia Roblellano Santo Tome Turrubuelo a b c d Santo Tome Turrubuelo Valdemanco
! 128! Espajosas):! XB-GENE-997273! related! to! amine! oxidase,! XB-GENE-5754317! associated! to! adipocyte! plasma! membrane! associated! protein! (APMAP),! XBGENE-482624! associated! to! arginase! 2,! and! XB-GENE-977784! associated! to! argininosuccinate! synthase! 1.! Also,! two! genes! of! interest! directly! linked! to! developmental!acceleration!have!been!identified!among!the!up!regulated!genes! from!the!Llano!population:!XB-GENE-484679,!the!thyroid!hormone!induced!bZip! protein!and!XB-GENE-6071044,!a!thyroid!hormone!receptor!interactor!1.! !
! 129! ! Figure! 9#Number#of#up-regulated#transcripts#in#each#population#(a)#and#number#of#downregulated# transcripts# in# each# population# (b)# in# response# to# low# water# conditions# and# following#developmental#acceleration,#and#corresponding#intersects#across#populations#for# each#(the#bars#connected#by#dots#show#how#many#transcripts#each#population#group#has#in# common,#for#instance,#Llano#and#Espajosas#have#19#up#regulated#transcripts#in#common).## !
! 130! Further,! we! explored! the! association! between! the! total! number! of! DE!! transcripts! in! each! population! with! the! level! of! plasticity! (standardized! difference!of!larval!period!across!water!treatments)!shown!in!each!population! (Figure!10).!No!significant!relationship!between!number!of!DE!transcripts!and! plasticity!was!found!(F1,4!=!0.32,!p!=!0.6).! ! Figure! 10#Lack# of# relationship#between# number# of# DE# transcripts# (sum# of# up# and# down# regulated# transcripts)# and# plasticity# achieved#(the# slope# of# larval# period# across# water# treatments).#The#three#southern#populations#(Espajosas,#Llano,#Jabata)#are#more#plastic#than# the# central#populations# (Canencia,# Buitrago,# Turrubuelo).# However,# the# number# of# DE# transcripts#was#similar#across#most#populations#of#the#two#localities#except#for#Llano,#which# has#an#exceptionally#high#number#of#DE#transcripts.# " FUNCTIONAL"ENRICHMENT" −10 0 10 20 30 40 50 100 200 300 400 500 600 700 plasticity N. of DE transcripts Espajosas Llano Jabata Canencia Buitrago Turrubuelo
! 131! ! The! enrichment! analysis! identified! 34! enriched! terms! in! the! Llano! population!(Figure!11),!of!which!the!majority!were!involved!in!the!metamorphic! process! and! developmental! acceleration,! 1! enriched! term! was! identified! in! Espajosas! (urea!cycle),! and! 2! in! Buitrago! (adipocytokine! signalling! pathway,! regulation! of! macromolecule! metabolic! process).! No! functionally! enriched! pathways!were! identified!in!the!other!three!populations!Jabata,! Canencia,!and! Turrubuelo.! ! Figure! 11#Bubbleplot,# showing#the# list# of# significantly# enriched# functional# pathways# and# corresponding#intersection#sizes#of#the#Llano#population.# ! VARIANT"CALLING"USING"RNA-seq"READS"
! 132! Different!numbers!of!SNPs!have!been!quantified!using!combinations!of!different! methods!(Table!3)! ! Table"3.!Final!tally!of!SNPs!across!methods! Method Min. 4 samples per SNP Min. 24 samples per SNP Min. 24 variable sites gsnap+ bcftools 2,116,637 1,118,245 507,094 gsnap+freebayes 847,805 543,132 373,388 star + bcftools 1,161,453 773,116 470,613 star + freebayes 788,763 522,771 364,351 "Preliminary!phylogenetic!analyses!showed!that!while!the!topology!of!the! clades! Turrubuelo,! Buitrago,! and! Canencia! (central! Spain)! were! similar! and! showed!high!support!across!all!methods,!the!clades!Espajosas,!Llano,!and!Jabata! southern!Spain)!were!more!intermixed!across!methods.!Nevertheless,!there!was! a!consistent!differentiation!across!the!central!and!southern!populations!across! all!methods.!Combining!gsnap!and!freebayes!showed!the!best!support!(Figure! 12;!trees!resulting!from!other!methods!shown!in!Supplementary!material!S7).!
! 133! ! Figure! 12! Phylogenetic# tree# resulting# from# combining# the# methods# gsnap#and# freebayes,# showing# clear# differentiation# across# central# and# southern# populations.# The# central# populations# are# clearly# differentiated# while# the# southern# populations# are# somewhat# intermixed.# ! !
! 134! Discussion We!observed!marked!population!differences!in!their!ability!to!accelerate!their! response!when!facing!reduced!water!levels!indicative!of!pond!drying.!However,! pond! type! (short-lasting! or! long-lasting)! was! not! a! determining! factor! of! population! divergence! in! developmental! plasticity! in! spadefoot! toads,! as! populations! breeding! in! either! type! of! pond! showed! a! similar! capacity! for! developmental!acceleration!in!response!to!reduced!water!level!(Figure!3).!This! lack!of!fine-scale!adaptive!divergence!within!regions!according!to!pond!duration! is!not!entirely!surprising!given!the!broad!interannual!variation!in!hydroperiod! and! pond! characteristics! typical! of! Mediterranean! temporary! ponds! (Gómez- Rodríguez!et(al.!2010).!Such!environmental!heterogeneity!selects!for!increased! phenotypic!plasticity!(Pigliucci!et(al.!1999;!Lind!and!Johansson!2007).!Also,!gene! flow!among!populations!may!have!impeded!local!divergences,!as!it!would!have! homogenized!the!reaction!norms!of!the!different!populations!within!each!region,! especially! for! the! southern! populations.! These! southern! populations! were! geographically! not! too! distant! from! each! other! and! there! were! no! major! geographical! barriers! among! them.! Consequently,! gene! flow! among! these! populations!was!apparent!as!genetic!variation!in!SNPs!showed!lack!of!structure! and!a!high!level!of!intermixture.!! Instead,! we! found! marked! regional! differences! in! developmental! plasticity.! The! populations! from! central! Spain! showed!consistently! faster! developmental! rates! than! the! southern! ones,! and! a! reduced! capacity! for! developmental! acceleration! in! response! to! pond! drying! (Figure! 3).! The! two! regions!are!clearly!differentiated!in!their!climate,!both!in!terms!of!temperature! and!precipitation!(Figure!6),!and!their!divergence!in!the!degree!of!developmental!
! 135! plasticity!was!significantly!correlated!with!such!climatic!divergence.!In!Doñana! National!Park,!where!the!temperature!is!benign!in!winter!and!there!is!no!risk!of! freezing,! spadefoot! toads! may! start! breeding! as! early! as! October! or! postpone! breeding!until!late!March!or!early!April,!as!it!is!entirely!dependent!upon!rainfall! stochasticity!(Díaz-Paniagua!1986;!Díaz-Paniagua!et(al.!2005).!Regardless!of!the! timing!of!breeding,!southern!spadefoot!toad!tadpoles!can!continue!to!grow!until! ponds!dry!up,!which!usually!takes!place!sometime!in!June.!The!breeding!period! of!the!central!populations!(Madrid!and!Segovia)!is!more!constrained!in!time!due! to!the!colder!temperatures.!Even!if!ponds!fill!up!earlier!in!the!season,!spadefoot! toads! will! not! start! breeding! until! temperatures! start! to! rise! and! the! risk! of! freezing!is!lower,!by!mid-!or!late!February!(Salvador!et(al.!1986;!Salvador!and! Carrascal!1990;!Lizana!et(al.!1994).!Hence,!the!southern!populations!dispose!of!a! longer! window! for! adults! to! breed! and! for! larvae!to! grow!(~7! vs.! 3! months),! which!has!allowed!for!longer!larval!periods!to!evolve!compared!to!the!central! populations.! Moreover,! remote! sensing-based! estimates! of! hydroperiod! fluctuations!for!the!last!>20!years!show!that!there!is!higher!heterogeneity!in!the! hydroperiod! regimes! of! the! southern!populations!(Figure! 7).! Taken! together,! longer! duration! of! the! growing! season! and!greater! heterogeneity! in! pond! duration!explain!the!adaptive!divergence!observed!between!regions!so!that!the! southern!ones!experience!longer!larval!periods,!but!with!a!greater!capacity!for! developmental!acceleration!when!facing!risk!of!pond!drying.!! Under!constant!water!conditions,!the!central!populations!attained!similar! sizes!at!metamorphosis!than!the!southern!ones,!but!over!a!shorter!larval!period! (Figure! 3,! 4),! hence! showing! an! overall! faster!growth! rate.! Interestingly,! however,!regardless!of!whether!larval!period!was!effectively!shortened!or!not!in!
! 136! response! to! pond! drying,! individuals! from! both! regions! experienced! similar! consequences!from!exposure!to!low!water.!Developmental!acceleration!comes!at! the!expense!of!reduced!body!mass,!body!length!and!allometric!changes!in!head! shape!and!limb!length!(Gomez-Mestre!and!Buchholz!2006;!Gomez-Mestre!et!al.! 2010).! However,! the! central!populations! experienced! such! detrimental! consequences!of!exposure!to!risk!of!pond!drying!and!to!a!similar!degree!than!the! more! plastic! southern! populations! but! without! the! adaptive! reward! of! accelerated!development!(Figure!4,!5).!! Phenotypic! plasticity! is! ultimately! the! end! product! of! differential! gene! expression.!Indeed,!it!has!been!defined!as!the!“re-programming!of!the!genome!in! response!to!the!environment”!(Aubin-Horth!and!Renn!2009).!Recent!innovations! such! as! transcriptomic! approaches! and! genome-wide-association-studies! (GWAS)!serve!as!powerful!tools!to!identify!the!genes!of!interest!responsible!for! the! observed! phenotypic! expressions! under! given! environmental! pressures! (Lafuente!and!Beldade!2019).!The!main!advantage!of!transcriptomic!approaches! is!that! it!strives! to!identify! a!series! of! up!and! down-regulated! genes,!that! are! likely!to!add!up!to!explain!the!biological!phenotype!observed!(Aubin-Horth!and! Renn!2009),!which!can!be!subsequently!explored!to!understand!the!biological! functions!of!the!genes!and!pathways!involved.! Contrary! to! our! prediction! that! the! more! plastic! southern! populations! would!have!a!higher!number!of!DEG!involved!in!developmental!acceleration!in! comparison!to!the!more!canalized!central!populations,!we!found!that!the!number! of! DEG! was! within! a! similar! range! across! populations! (Table! 2)! with! the! exception! of! Llano.! In! general,! the! degree! of! DEG! is! expected! to! reflect!the! capacity!of!the!organism!to!respond!to!environmental!pressures!(Aubin-Horth!
! 137! and! Renn! 2009),! as! has! been! demonstrated! for! instance! in! the! correlation! between! beetle! morphology! and! DEG! count! in! response! to! different! nutrition! scenarios! (Casasa! et( al.! 2020).! Nevertheless,! during! the! process! of! genetic! accommodation,!environmentally!induced!changes!in!gene!expression!may!differ! across! genes! and! populations,! maintaining! similar! degrees! of! environmental! sensitivity! even! though! trait! responsiveness! is! reduced! or! lost! (Renn! and! Schumer2013).! The! differences! in! developmental! plasticity! observed! in! our! system! while! the! extent! of! differential! gene! expression! is! similar! across! populations! (with! the! exception! of! Llano)! may! be! driven! by! standing! genetic! variation!across!localities,!or!changes!in!the!responsiveness!of!different!sets!of! genes.!Further!weighted!gene!co-expression!network!analysis!(WGCNA)!would! help!elucidate!whether!specific!clusters!of!genes!explain!the!divergence!in!gene! expression! responsible! for! the! observed! region-by-environment! interaction.! Moreover,! genome! wide! association! studies! (GWAS)! aimed! at! identifying! patterns! of! divergent! standing! genetic! variants! across! populations! in! SNPs! associated! with! developmental! rate! would! be! critical,! although! this! would! require!a!larger!sample!size.! ! Differentially"expressed"genes"associated"with"developmental"responses" Two! transcripts! involved! in! thyroid! hormone! expression! have! been! identified!based!on!the!annotations!of!X.(tropicalis!in!the!Llano!population!(XBGENE-484679,! the! thyroid! hormone! induced! bZip! protein! and! XB-GENE- 6071044,! a! thyroid! hormone! receptor! interactor! 1).! These! changes! are! congruent!with!key!mechanisms!known!to!mediate!developmental!acceleration! in! P.( cultripes:! elevated! thyroid! hormone! secretion! and! overexpression! of! the!
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! 150! Supplementary"materials" ! S1."Worldclim"bioclimatic"variables" BIO1!=!Annual!mean!temperature! BIO2!=!Mean!diurnal!range!(mean!of!monthly!(max!temp!–!min!temp))! BIO3!=!Isothermality!(BIO2/BIO7)(*100)! BIO4!=!Temperature!seasonality!(standard!deviation*100)! BIO5!=!Max!temperature!of!warmest!month! BIO6!=!Max!temperature!of!coldest!month! BIO7!=!Temperature!annual!range!(BIO5!-!BIO6)! BIO8!=!Mean!temperature!of!wettest!quarter! BIO9!=!Mean!temperature!of!driest!quarter! BIO10!=!Mean!temperature!of!warmest!quarter! BIO11!=!Mean!temperature!of!coldest!quarter! BIO12!=!Annual!precipitation! BIO13!=!Precipitation!of!wettest!month! BIO14!=!Precipitation!of!driest!month! BIO15!=!Precipitation!seasonality!(coefficient!of!variation)! BIO16!=!Precipitation!of!wettest!quarter! BIO17!=!Precipitation!of!driest!quarter! BIO18!=!Precipitation!of!warmest!quarter! BIO19!=!Precipitation!of!coldest!quarter! ! !
! 151! S2."clean_pe_rna.sh"full"script"used"for"RNA"read"cleaning" " echo!"Usage:"! echo!""! echo!"bash!clean_pe.sh!raw_data_foldername!R1_ending"! echo!""! echo!"*!raw!paired!end!data!has!no!uniform!ending!for!filenames!R1_ending!may! be!_R1.fq.gz,!_R1_.fastq.gz,!_1.fastq.gz,!etc"! echo!""! echo!"First,!adapters!are!removed!with!bbduk"! echo!"Second,!PhiX!and!sequencing!artifacts!are!filtered!out,!then!quality!is! trimmed!using!bbduk.sh"! echo!"bbduk.sh!uses!PHRED!algorithm,!filtering!based!on!error!probability! instead!of!raw!quality!score"! echo!""! echo!""! echo!"#########################################"! echo!"#!STEP!1:!Remove!adapters!with!bbduk.sh!#"! echo!"#########################################"! echo!""! ! #!Create!folder!for!decontaminated!reads! mkdir!01_adapters_removed! ! #!Switch!to!input!folder!given!by!first!argument!$1! cd!$1! ! #!R1!ending!pattern!is!given!in!$2! patternI1=$2! read1="_R1.fq.gz"! readH="_R#.fq.gz"! readS="_SE.fq.gz"! ! #!RAM!for!bbduk.sh! RAM="-Xmx32g"! ! #!Remove!adapters!with!bbduk,!two!consecutive!rounds! for!file!in!*$patternI1;!do! ! echo!"Removing!adapters:!"${file//$patternI1/}! ! time!(!bbduk.sh!$RAM!in=${file//$patternI1/$readH}!out=stdout.fq!int=f! \! ! ref=$HOME/software/bbmap/resources/adapters.fa,$HOME/software/b bmap/resources/polyA.fa.gz!\! ! ktrim=r!k=21!mink=11!hdist=2!tpe!tbo!minlength=21!trimpolya=4!\! ! stats=../01_adapters_removed/${file//$patternI1/.bbduk.adapters_round 1.stats.txt}!\! ! 2>../01_adapters_removed/${file//$patternI1/.bbduk.adapters_round1.l og.txt}!\!
! 152! ! |!bbduk.sh!$RAM!in=stdin.fq!int=f! out=../01_adapters_removed/${file//$patternI1/$readH}!\! ! ref=$HOME/software/bbmap/resources/adapters.fa,$HOME/software/b bmap/resources/polyA.fa.gz!\! ! ktrim=r!k=19!mink=9!hdist=1!tpe!tbo!minlength=21!trimpolya=4!\! ! stats=../01_adapters_removed/${file//$patternI1/.bbduk.adapters_round 2.stats.txt}!\! ! 2>../01_adapters_removed/${file//$patternI1/.bbduk.adapters_round2.l og.txt}!)! !echo!""! ! echo!"After!first!round:"! !grep!"Result:"! ../01_adapters_removed/${file//$patternI1/.bbduk.adapters_round1.log.txt}!|! column!-ts!$'\t'! !echo!""! !grep!-A4!"#Name"! ../01_adapters_removed/${file//$patternI1/.bbduk.adapters_round1.stats.txt}!|! column!-ts!$'\t'! !echo!""! ! echo!"After!second!round:"! !grep!"Result:"! ../01_adapters_removed/${file//$patternI1/.bbduk.adapters_round2.log.txt}!|! column!-ts!$'\t'! !echo!""! !grep!-A4!"#Name"! ../01_adapters_removed/${file//$patternI1/.bbduk.adapters_round2.stats.txt}!|! column!-ts!$'\t'! !echo!""! !echo!""! done! ! #!#!Remove!adapters!with!bbduk,!single!round! #!for!file!in!*$patternI1;!do! #!! echo!"Removing!adapters:!"${file//$patternI1/}! #!! time!bbduk.sh!$RAM!in=${file//$patternI1/$readH}! out=../01_adapters_removed/${file//$patternI1/$readH}!\! #!! ref=$HOME/software/bbmap/resources/adapters.fa!\! #!! ktrim=r!k=21!mink=11!hdist=2!tpe!tbo!minlength=21!\! #! ! stats=../01_adapters_removed/${file//$patternI1/.bbduk.adapters.stats.t xt}!\! #!!2>../01_adapters_removed/${file//$patternI1/.bbduk.adapters.log.txt}! #!!echo!""! #!!grep!"Result:"! ../01_adapters_removed/${file//$patternI1/.bbduk.adapters.log.txt}! #!!echo!""! #!!grep!-A4!"#Name"! ../01_adapters_removed/${file//$patternI1/.bbduk.adapters.stats.txt}! #!!echo!""!
! 153! #!!echo!""! #!done! ! echo! "####################################################### ##############"! echo!"#!STEP!2:!Remove!contaminants!and!quality!filter/trim!with!bbduk.sh!#"! echo! "####################################################### ##############"! ! #!Create!folder!for!clean!reads! cd!..! mkdir!02_clean! cd!01_adapters_removed! ! #!Remove!contaminants!with!bbduk.sh!! for!file!in!*$read1;!do! !echo!"Cleaning:!"${file//$read1/}! ! time!bbduk.sh!$RAM!in1=${file//$read1/$readH}! out=../02_clean/${file//$read1/$readH}!\! !outs=../02_clean/${file//$read1/$readS}!\! ! ref=$HOME/software/bbmap/resources/phix174_ill.ref.fa.gz,$HOME/sof tware/bbmap/resources/sequencing_artifacts.fa.gz!\! ! k=31!hdist=1!qtrim=lr!trimq=10!maq=12!minlength=21!maxns=5! ziplevel=5!\! !stats=../02_clean/${file//$read1/.contaminants.stats.txt}!\! !&>../02_clean/${file//$read1/.cleaning.log.txt}! !echo!""! ! grep!"Result:"!../02_clean/${file//$read1/.cleaning.log.txt}!|!column!-ts! $'\t'! !echo!""! !grep!-A2!"#Name"!../02_clean/${file//$read1/.contaminants.stats.txt}!|! column!-ts!$'\t'! !echo!""! !echo!""! done! ! echo!"######################################"! echo!"#!STEP!3:!Quality!checks!with!fastqc!#"! echo!"######################################"! ! cd!../02_clean! ! #!Run!fastqc! mkdir!00_fastqc_before!01_fastqc_after! cd!../$1! fastqc!-o!../02_clean/00_fastqc_before!--nogroup!-t!6!*q.gz! !