UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE BIOLOGÍA

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UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE BIOLOGÍA

Departamento de Ecología José Antonio Nováis, 12

28004, Madrid, Spain [email protected]

Tel: (34) 91 394 5123 Fax: (34) 91 394 5081

MIGUEL ANGEL CASADO GONZÁLEZ, Profesor Titular del Departamento de Ecología de la Universidad Complutense de Madrid, director de la presente Tesis Doctoral, HACE CONSTAR: Que el trabajo descrito en la presente memoria, titulado “Restauración ecológica de taludes de carretera en ambiente mediterráneo: Comunidades de suelo y efectos del manejo”, ha sido realizado por Dª Sandra Magro Ruiz, dentro del Programa de Doctorado Ecología. Conservación y Restauración de Ecosistemas, adscrito al Departamento de Ciencias de la Vida, de la Universidad de Alcalá. Esta Tesis Doctoral reúne todos los requisitos propios de este tipo de trabajo: rigor científico, aportaciones novedosas y aplicación de una metodología adecuada y que, por lo tanto, tiene mi Visto Bueno para su presentación.

Madrid, 17 de Octubre de 2014

Miguel Ángel Casado González

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UNIVERSIDAD COMPLUTENSE DE MADRID

FACULTAD DE BIOLOGÍA

Departamento de Biología Vegetal I José Antonio Nováis, 2

28004, Madrid, Spain

LUÍS BALAGUER NÚÑEZ, Profesor Titular en el Departamento de Biología Vegetal I de la Universidad Complutense de Madrid, director de la presente Tesis Doctoral, falleció en Madrid el 19 de marzo de 2014. Por lo tanto, yo, MIGUEL ÁNGEL CASADO GONZÁLEZ como director de la presente tesis me comprometo a supervisar la tesis en su totalidad y asimismo, hago constar que: El profesor Balaguer dirigió hasta el momento de su fallecimiento la presente Tesis Doctoral, participando activamente en todas las fases de la misma. Luis confió en la capacidad de Sandra Magro Ruíz para llevar a cabo cada uno de los trabajos que se desarrollan en ésta tesis y mantuvo la ilusión y la certeza de que ésta tesis alcanzaría la calidad suficiente para superar con éxito la etapa de defensa. Sin duda, ésta tesis no habría sido posible sin su perspectiva de futuro, su ilusión y su constante apoyo científico, por lo que solicito que se mantenga como co-director de la misma.

Madrid,17 de Octubre de 2014


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DEPARTAMENTO DE CIENCIAS DE LA VIDA

Edificio de Ciencias Campus Universitario 28871 Alcalá de Henares

(Madrid) Telf. +34918854927 Fax: +34918854929 E-mail: [email protected]

GONZALO PÉREZ SUÁREZ, Director del Departamento de Ciencias de la Vida de la Universidad de

Alcalá,

HACE CONSTAR:

Que el trabajo descrito en la presente memoria, titulado “Restauración ecológica de taludes de carretera

en ambiente mediterráneo: Comunidades de suelo y efectos del manejo”, ha sido realizado por Dña.

Sandra Magro Ruíz dentro del Programa de Doctorado Ecología. Conservación y Restauración de

Ecosistemas (D330) y reúne todos los requisitos necesarios para su aprobación como Tesis doctoral, por

acuerdo del Consejo de Departamento celebrado el día de de 2014.

Alcalá de Henares, de de 2014

Gonzalo Pérez Suárez

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Departamento de Ciencias de la Vida Departamento de Ecología

Unidad Docente de Ecología

Restauración ecológica de taludes de carretera en ambiente

mediterráneo: Comunidades de suelo y efectos del manejo

Memoria presentada para optar al grado de Doctor por la Universidad de Alcalá

Programa de Doctorado

“Ecología, Conservación y Restauración de Ecosistemas” (D330)

Sandra Magro Ruíz

Directores:


Luís Balaguer Núñez

Alcalá de Henares, XXX 2014

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Esta Tesis Doctoral ha sido financiada por una Beca de

Formación de Profesorado Universitario (FPU–AP2009–

0094), concedida por el Ministerio de Educación, cultura y

Deporte del Gobierno de España. Asimismo, los

experimentos realizados durante el desarrollo de esta tesis

han recibido financiación del proyecto CLEAM AIE-

156/2007 (CENIT 2007/2017) concedido por el Ministerio

de Economía y Competitividad del Gobierno de España; y

del Gobierno Regional de la Comunidad de Madrid, a

través de la Red REMEDINAL2-CM (S2009/AMB-1783)

y el apoyo del proyecto ECONET (CDTI IDI-20120317).

Diseño de Portada: Beatriz Vicente

Dibujos originales:Samdra Magro

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A mi madre, por el calor

A mi padre, por la fuerza

A mi hermano, por la claridad

Y a Luís, obviamente.

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AGRADECIMIENTOS

Intentar ponerle un punto y final a esta tesis es casi igual de difícil que cerrar una maleta

después de un largo viaje. Mientras haces el último esfuerzo por intentar que todo entre y llegar a

tiempo, los recuerdos se agolpan contra las costuras y, a pesar del desgaste, es imposible no

esbozar una sonrisa y exhalar un suspiro por lo que se acaba y lo que no. Estos últimos años

dando pasos por el mundo científico han supuesto para mí una verdadera metamorfosis, un

cambio de fondo y de forma en que se han reorganizado mis estructuras, he descifrado mis

procesos y mis dinámicas y… Lo que en un principio se planteaba como un chapuzón en los

espacios degradados y la Restauración Ecológica, se ha convertido en un proceso de

Restauración interna que se mantiene activo, que fluye sin más. En este tiempo me he enamorado

cientos de veces de esta carrera y he querido dejarla otras cien. Me he visto descubriéndome

capacidades impesables pipeta en mano, con la americana puesta. He reido frenéticamente por

encontrar un resultado y he llorado todas y cada una de las veces que se apagó la luz. Se me han

puesto los pelos de punta al saber que la ciencia es inmensa, que no sabemos nada, que las

preguntas son infinitas pero que no lo son las respuestas, y que al final esto se trata de observar,

de discutir y construir el pensamiento, lentamente, paso a paso, disfrutando de cada idea que

conseguimos poner después de la anterior y sobre todo de poder vivir para contarlo. La sensación

es tan rica que sé que es esto - de alguna u otra manera - a lo que quiero dedicarme, a construir y

contar historias de lo que pasa en esta Tierra. Pero sin duda, lo más importante de este ciclo que

ni acaba ni empieza, es que me ha permitido encontrarme con grandes espejos en los que

mirarme y aprender. A todos ellos, a cada una de las personas que han contribuido a alumbrar

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este camino con su luz está dedicada esta sección. Intentaré no dejarme a nadie olvidado pero no

prometo ser breve.

En primer lugar quiero dar las gracias al Grupo de Investigación de Ecología Evolutiva

y Restauración Ecológica de la Universidad Complutense, por acogerme y enseñarme cómo

funciona la ciencia y cómo no. En especial, quiero dar las gracias a mi director, Miguel Ángel

Casado por haber estado al quite y ser el padre adoptivo de esta tesis. Esto no hubiera salido

adelante si tu ayuda, sin tu ojo crítico y tus ánimos. A Iñaki Mola, por la claridad en todo

momento y por el aliento en las horas bajas. Tu sensibilidad extrema y tu diligencia son las

mejores características que se pueden tener. ¡Eres grande compañero! Mil gracias a Adrián

Escudero, por aparecer en el momento justo, por tu cercanía, por el entusiasmo y por toda la

ciencia que llevas dentro y que espero que podamos seguir compartiendo en un futuro. Gracias

infinitas a los Obviously; a mis hermanos de la ciencia Juanma y Rocío, por el campo, los cafés,

los cigarros (activos o pasivos), las sesiones de pizarra y las manos que siempre llegan a tiempo.

A Adri por compartir alegrías y penas de co-granteds. A Ana por echar una mano siempre, por

gestionarnos la vida más de una vez! A Peri porque los abrazos de pasillo son de ida y vuelta,

por tus iluminaciones estadísticas y la paz que me transmites. Y como no, a Agus, porque

conocerte, amiga, ha sido de las mejores cosas de los últimos tiempos. Gracias por las sesiones

de science in pijamas, por ser y estar.

Quiero dar las gracias a la Estación Experimental de Zonas Áridas de Almería (“El

chumbo”), en especial a Paco Pugnaire y Sara Hortal por dejarme participar de sus

experimentos y su mundo científico. También a Nuria, Meire, Carme y Olga por acogerme

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siempre con una sonrisa y por ser tan espectacularmente buenas científicas. Mucho ánimo

mujeres!! También quiero agradecer al Netherland Institute of Ecology (NIOO KNAW), en

especial a Eiko Kuramae: It was a very big pleasure to work with you, not only during my

internship but also afterwards. You are a really good scientist and a wonderful person. Gracias

Clau, Stef, Kostas, Moli y Paolo por hacer que mi vida en Holanda se haya quedado grabada en

mi ADN para siempre. No sé si hubiera sobrevivido sin vosotros! Mención especial a las Saras

(Sara B y Sara C) por haberos dejado liar tanto, por todo el tiempo y el esfuerzo que habéis

dedicado a esta tesis. Es un gusto haber trabajado con vosotras y veros crecer cada día.

Gracias a mis socios y nuevos compañeros de camino en Creando Redes: Ana, Adri,

Alberto y Jorge. Gracias de verdad por querer construir este proyecto tan bonito que para mi es

vital. Vamos a cambiar el mundo, aunque el resto todavía no lo tenga claro. Pero las gracias se

quedan cortas para expresar mi gratitud hacia Quico Balaguer. Te agradezco infinitamente el

aprendizaje personal y profesional, y el apoyo incondicional. Me alegro mucho de que la vida

nos haya hecho encontrarnos aquí y ahora y que nos mantenga dando pasos en la misma

dirección. Mnhe!

Gracias especiales a mis amigas Viky, Paloma y Bea, por comprenderme siempre

incluso cuando ni yo misma me entiendo, por haber soportado todas mis escapadas de cenicienta

y haberos ajustado a mis agendas de infarto. Por los jueves de arreglar el mundo y los viernes de

arreglarme a mi. Por estar siempre. Porque sois un absoluto tesoro! Os quiero mucho! Gracias

además a Javi, Rodri, Dina, Alba, Sergio, Freiji, Katy, Dani, Irene, Airin, Carmen, Julia,

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Bárbara, Ange, Dudi, Andrés, Bea y a todos los [+1] que de una u otra manera han particpado

en el proceso

A mis padres, gracias por no haber puesto nunca en tela de juicio mis capacidades y mis

sueños. Gracias por apoyarme siempre incluso cuando es difícil de entender. La fuerza, el

positivismo, la perseverancia y la ilusión por hacer que cada día el mundo sea un poco mejor son

los mejores regalos que habéis podido hacerme nunca! Gracias en especial a Edu, por tener

siempre la palabra indicada para cada momento, por la confianza infinita. Es un orgullo tener una

familia asi!

Y todo acaba donde empieza y siento del todo que no estés hoy en primera fila, con tus

ojos vivos y la sonrisa puesta. Te has ido antes de que acabe la función y eso duele, no te

imaginas cuánto. Es dura la sensación de no saber si conseguí transmitirte la importancia

capital que has tenido en mi vida. De verdad que conocerte ha supuesto un antes y un después

en mi manera de ver el mundo, en mi manera de verme a mi. Gracias por haberme enseñado

tanto, por inspirarme, por las sesiones de trabajo, vino y café. Gracias por haberme llevado de

la mano los primeros pasos de este camino que está aún por recorrer. Prometo seguir volando

alto como siempre, como nos gusta. Nos vemos en la próxima, jefe.

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“Y cuando la tormenta de arena haya pasado, tú no comprenderás como has

logrado cruzarla con vida. ¡No! Ni siquiera estarás seguro de que la tormenta

haya cesado de verdad. Pero una cosa si quedará clara. Y es que la persona

que surja de la tormenta no será la misma persona que penetró en ella. Y ahí

estriba el significado de la tormenta de arena”

Haruki Murakami

Kafka en la orilla

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ÍNDICE RESUMEN .......................................................................................................................... 19

SUMMARY ......................................................................................................................... 27

CAPÍTULO I:

Introduction and Objectives ............................................................................................. 35

CAPÍTULO II

Soil functionality at the roadside: Zooming in on a microarthropod community in an anthropogenic soil ......................................................................................................... 75

CAPITULO III

Roadside microbes: factors affecting community structure and composition in urban soils from road embankments ............................................................................. 103

CAPITULO IV

Roadside ecosystems: the effect of management on soil functions and microbial community structure ........................................................................................................ 135

CAPÍTULO V

Community ontogeny at the roadside: Critical life‐cycle events throughout a sequential process of primary colonization ................................................................. 175

CAPÍTULO VI

General discussion ............................................................................................................ 205

CONCLUSIONES ........................................................................................................... 223

CONCLUSIONS .............................................................................................................. 229

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RESUMEN

Restauración ecológica de taludes de carretera en ambiente mediterráneo

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Resumen

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Esta Tesis Doctoral estudia el desarrollo de las comunidades biológicas de taludes en

diferentes carreteras de la comunidad de Madrid, así como los efectos de distintas técnicas de

manejo sobre la composición, la diversidad y los procesos ecológicos responsables de la

funcionalidad ecosistémica. El objetivo principal es aportar nuevos conocimientos que permitan

mejorar la efectividad de los esfuerzos de restauración en estos ambientes, de modo que se asista

a la recuperación de las comunidades de suelo, los procesos edafogénicos y el establecimiento de

comunidades de plantas resilientes.

El Capítulo I, de introducción, revisa el estado actual de las investigaciones llevadas a

cabo sobre los efectos de la construcción de carreteras en la composición de las comunidades, su

diversidad y los procesos ecológicos a escala regional y local, así como las aproximaciones

utilizadas para la compensación de dichos efectos en la cuenca Mediterránea.

Los capítulos II y III analizan las comunidades de microartrópodos y bacterias en suelos

de origen antrópico presentes en terraplenes de carretera. Se ha prestado especial atención a estas

comunidades de suelo ya que están implicadas en procesos ecológicos básicos que determinan el

desarrollo y el mantenimiento a largo plazo de los ecosistemas terrestres. En concreto, el

Capítulo II analiza las comunidades de microartrópodos edáficos en terraplenes de carretera así

como los factores que determinan la variación en la composición de estas comunidades. Además

se evalúa la funcionalidad de suelo utilizando estas comunidades de invertebrados como

indicador. Con este fin, se seleccionaron seis terraplenes construidos en 2004 en los cuales se

extendió tierra vegetal tras su construcción, y que fueron estudiados durante dos años (2009 y

2010). En cada terraplén se tomaron muestras de suelo de las que se extrajeron los

microartrópodos edáficos y en las que se evaluaron las propiedades físico-químicas del suelo.

Además se tomaron sondas de suelo para determinar su estructura. El índice de Calidad


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Biológica del Suelo (QBS) fue calculado para cada muestra usando la información relativa a la

composición de la comunidad de microartópodos.

Nuestros resultados mostraron que estas comunidades están dominadas por Ácaros y

Colémbolos. Sin embargo, observamos que la composición de la comunidad de artrópodos varió

en el espacio y en el tiempo desde comunidades caracterizadas por una mayor abundancia de

ácaros Actinédidos, Gamásidos y Acarídidos, grupos primocolonizadores o de hábitos más

generalistas, hacia otras comunidades dominadas por Oribátidos (Ácaros) y Sinfipleones

(Colémbolos), ambos sensibles a las perturbaciones y ligados a condiciones edáficas estables.

Los cambios en la composición observados durante el periodo de estudio fueron debidos

fundamentalmente a variaciones en el contenido de material orgánica, el contenido en arcillas y

la humedad del suelo. Además, los valores de QBS obtenidos para los suelos de terraplenes de

seis años de antigüedad mostraron que la funcionalidad en estos suelos es, en algunos casos,

comparable a la observada en sistemas Mediterráneos naturales con coberturas de vegetación

leñosa. Este es el resultado principal de este estudio y sugiere que, en paralelo al desarrollo de la

comunidad de plantas, la consolidación de suelos totalmente funcionales en taludes de carretera

es un proceso clave para el mantenimiento de sistemas auto-sostenibles, capaces de albergar la

diversidad local y la provisión de servicios ecosistémicos como el control de la erosión y la

integración estética en zonas urbanas.

El Capítulo III estudia las comunidades bacterianas de terraplenes de carretera así como

los factores que determinan el desarrollo de estas comunidades y su organización espacial. Para

ello se seleccionaron cuatro terraplenes de carretera: dos en Barajas, cerca del aeropuerto

internacional y que fueron construidos en 2004, y dos en El Molar, construidos en 2007 y 2008.

En todos los terraplenes se extendió una capa de tierra vegetal tras su construcción y se aplicaron

Resumen

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mezclas comerciales de hidrosiembra. En cada terraplén, se analizaron las propiedades físico-

químicas y la estructura del suelo, así como la composición y diversidad de las comunidades

bacterianas, usando técnicas de pirosecuenciación. Los resultados mostraron que las

comunidades de bacterias de terraplenes poseen unos niveles de diversidad similares a los

observados en otros suelos de origen antrópico como suelos agrícolas de Europa, tal y como

muestran otros estudios. Estas comunidades de bacterias están dominadas por Actinobacterias,

Acidobacterias, y Planctomicetos, relacionados con la dinámica de la materia orgánica del suelo

e indicadores de condiciones edáficas estables. También se encontraron abundancias relativas

bajas de Cianobacterias, Bacteroidetes, Gemmatimonadetes y Firmicutes, que indican procesos

de sucesión secundaria en estos taludes desde que fueron construidos. La composición de la

comunidad bacteriana varió entre terraplenes y los principales factores relacionados con este

cambio fueron el pH y la textura del suelo (contenido en arcillas y arenas).

Los siguientes capítulos describen los efectos de distintas técnicas convencionales usadas

en la restauración y mantenimiento de taludes de carretera. El Capítulo IV evalúa el efecto de

las siegas, la aplicación de fertilizantes y la adición experimental de biomasa sobre la

composición y la diversidad de las bacterias del suelo, así como sobre la descomposición de la

hojarasca y la disponibilidad de fases minerales de nitrógeno en el suelo. Este experimento fue

desarrollado en los mismos terraplenes estudiados en el Capítulo III, en cada uno de los cuales se

establecieron las parcelas experimentales. Los resultados mostraron que los ecosistemas de

taludes de carretera son sensibles a las perturbaciones, lo que se traduce en una alternación

rápida y significativa tanto de la estructura de las comunidades bacterianas como de los procesos

ecológicos del suelo. Específicamente, la adición de biomasa y la aplicación de fertilizantes

afectaron la diversidad y la composición de las comunidades de bacterias, mientras las siegas


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modificaron las condiciones físico-químicas de suelo y la tasa de descomposición de hojarasca.

El hecho de que tanto la estructura como la función de la comunidad no se vean afectadas

simultáneamente por el mismo tratamiento puede estar relacionado con atributos de la

comunidad del suelo como la redundancia o plasticidad funcional.

Por último, en el Capítulo V, se analizan los procesos de primocolonización y

ensamblaje de comunidades en un desmonte de carretea, de exposición sur, con una pendiente

superior al 40% y donde no se han aplicado técnicas de manejo convencional de manera previa a

nuestro estudio. Los filtros ambientales de tipo abiótico como la disponibilidad de agua y

nutrientes, así como la disponibilidad de lugares aptos para la retención de semillas y el

reclutamiento, son los principales factores que determinan el desarrollo de las comunidades de

plantas en desmontes de carretera. Por ello, en este capítulo se describe un experimento en que

esto filtros han sido manipulados mediante la aplicación de dos tratamientos diferentes:

extendido de tierra vegetal y escarificación de la superficie del talud. Además se establecieron

parcelas control donde no se aplicó ningún tipo de tratamiento. El efecto de los tratamientos se

analizó sobre la disponibilidad de semillas (en el banco de semillas de suelo y en la lluvia de

semillas proveniente de la matriz circundante), sobre la emergencia y supervivencia de plántulas,

así como sobre la riqueza de especies y la cobertura vegetal total durante los años 2010 y 2011.

Los resultados mostraron que el extendido de tierra vegetal facilitó el ensamblaje temprano de la

comunidad de plantas en desmontes mediante la mejora del contenido de nutrientes, el pH, la

textura del suelo y la estabilidad de sustrato. Además, este tratamiento favoreció la emergencia

de un mayor número de especies y la supervivencia de plántulas lo que se tradujo en mayores

niveles de riqueza y cobertura en la comunidad durante los dos años siguientes a la construcción

de la carretera. A pesar de no haber observado un efecto del escarificado sobre la disponibilidad

Resumen

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de semillas, la emergencia o la supervivencia de plántulas, la escarificación de la superficie del

desmonte en combinación con la aplicación de tierra vegetal aumentó la riqueza de especies en la

zona alta del desmonte al final del experimento, en 2011.

En conclusión, nuestros resultados mostraron que las condiciones edáficas locales

relacionadas con la materia orgánica, el pH, la textura y la estructura del suelo son en gran parte

responsables de la organización de las comunidades de suelo y de los procesos de sucesión

secundaria en taludes de carretera. Además, la aplicación de técnicas de manejo convencional

orientadas a la mejora de las condiciones abióticas de desmontes facilitó el re-ensamblaje de la

comunidad vegetal. Sin embargo, otras técnicas como la aplicación de fertilizantes o las siegas

que han sido usadas tradicionalmente para el mantenimiento y restauración de taludes, inducen

cambios significativos en las comunidades de suelo, homogeneizándolas y favoreciendo una

mayor abundancia de grupos asociados con baja disponibilidad de recursos. Asimismo, estas

técnicas afectaron la funcionalidad del suelo acelerando la descomposición de la hojarasca.

Teniendo en cuenta que los cambios en las comunidades de suelo pueden afectar a las

comunidades de plantas y vice versa, nuestros resultados deberían ser considerados durante el

proceso de selección de técnicas de manejo y restauración de taludes de carreteras de cara a

mantener los servicios ecosistémicos que estos sistemas pueden proveer. En base a los resultados

obtenidos, las técnicas de manejo empleadas deben ir orientadas a la mejora de las condiciones

edáficas en los primeros momentos tras la construcción del talud, y después deben asegurar y

promover la estabilidad del suelo que permita que se verifiquen procesos de sucesión secundaria

y el desarrollo de este tipo de ecosistemas.


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SUMMARY

Summary

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This Doctoral Thesis studies the development of biological communities in roadslopes

from different highways in Madrid, as well as the effects of varying management techniques on

the composition, diversity and ecological processes that are in turn responsible for ecosystem

functionality. The goal is to provide new insight and knowhow to improve the effectiveness of

efforts to assist the recovery and reassembly of soilborne biota, pedological processes, and

resilient plant communities in these highly perturbed settings.

Following an introductory chapter, chapters II and III of the thesis analyze

microarthropod and bacterial communities in heavily disturbed, anthropogenic soils, or

Anthrosols in roadside embankments. These topics were given priority in the thesis in view of

the importance of these soilborne communities in basic soil processes that underlie long term

development and resilience of terrestrial ecosystems. Specifically, Chapter II analyzes

microarthropod communities in road embankments as well as factors determining community

composition. Moreover, soil functionality is evaluated using microarthropod communities as an

indicator. In the present study, six road embankments constructed in 2004 were selected and

studied over two years (2009-2010). After construction, all embankments were covered by with a

layer of topsoil that had been collected at the site, and preserved. On each road embankment soil

samples were collected, microarthopods were extracted, and physico-chemical soil properties

were measured. Additionally, soil structure was analyzed by means of extracted soil cores. The

well-known QBS index (Biological Soil Quality) was obtained for each soil sample using

information related to microarthropod community composition.

Our results showed that microarthropod communities from these road embankments were

dominated by Acari and Collembola. However, we observed a transition between pioneer

communities in which Actinenida, Gamasida and Acaridida (Acari) are more abundant to other


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communities characterized by higher abundances of Brachypilina (Oribatida; Acari) and

Symphypleona (Collembola) both indicative of stable soil conditions and sensitive to disturbance.

Changes in soil community composition between sites after two years were mostly explained by

changes in organic matter content, clay content, and soil humidity. Furthermore, QBS index

values obtained from the six-year old road embankments studied showed that soil functionality

in these settings was in some cases comparable to that observed in other, much longer

established Mediterranean natural systems such as woodlands and scrublands. This is a major

result of the study, and suggests that along with plant community assembly, consolidation of a

fully functional soil may be an important key to successful roadside restoration toward self-

sustaining and resilient ecosystems that harbor biodiversity and provide direct ecosystem

services such as erosion control and aesthetic values in urbanized areas.

In Chapter III, studies on bacterial communities in anthrosols from the road

embankments under study are presented, as well a discussion of the factors underlying soil

community development with regards spatial organization and changes in community

composition and diversity over time. To this end, four embankments were selected: two sites

from Barajas constructed in 2004, and two sites from El Molar constructed in 2007 and 2008,

respectively. In all embankments, a topsoil layer was spread after construction. On each

embankment soil samples were studied with respect to physicochemical properties, bacterial

community composition and diversity (using pyrosequencing techniques). Results showed that

bacterial communities from these anthrosols had diversity levels similar to those observed in

other anthropogenic soils, namely European agricultural soils, as reported in other studies. Soil

bacterial communities were dominated by Actinobacteria, Acidobacteria, and Planctomycetes,

all groups related with organic matter dynamics in soil and indicative of soil stable conditions.

Summary

31

Relatively low abundances of Cyanobacteria, Bacteroidetes, Gemmatimonadetes, and Firmicutes

were also observed, which indicates that secondary succession processes are taking place in these

anthrosols since the roadslopes were artificially constructed and then covered with topsoil.

Differences in bacterial community composition were observed between sites. The driving

factors underlying this pattern were found to be soil pH and soil texture (clay and sand content).

The next two chapters of the thesis describe the effects of different commonly used

techniques employed to restore and maintain roadside ecosystems. Chapter IV evaluates the

effect of mowing, fertilizing, and also experimental biomass addition, on the composition and

diversity of soil bacterial communities, and on ecological processes such as rate of litter

decomposition and soil nutrient availability, especially nitrates and ammonium. This experiment

was carried out in the same four road embankments studied in Chapter III, in each of which

experimental plots were set up. Results revealed that roadside ecosystems are sensitive in their

response to treatments with both bacterial community structure and processes being rapidly and

significantly affected. In particular, biomass addition and fertilization affected diversity and

community composition, while mowing affected soil properties and litter decomposition. The

fact that neither structure nor function was affected under the effect of a given treatment may be

related to bacterial community attributes such as functional plasticity or redundancy.

Finally, Chapter V analyzes primary colonization and early community assembly in a

south-oriented roadcut constructed in 2008 with slope greater than 40%, and where no

management techniques were applied prior to our investigations. Abiotic filters such as water

and nutrient availability, as well as the abundance of safe-sites for incoming seeds and

propagules are the main constraints for plant community development in this kind of relatively

harsh environment. Thus, in this chapter we describe an experiment in which we manipulated


32

abiotic filters through the application of two different treatments, namely topsoil spreading and

shallow tillage. We also set up control plots. Specifically, we analyzed the effect of treatments on

seed availability (both soil seed bank and seed rain), seedling emergence, and seedling survival,

as well as their effects on plant community attributes such as species richness and plant cover

from 2010 to 2011. Results showed that topsoil spreading facilitated early community assembly

by improving soil properties in term of nutrients, pH, soil stability, and texture. Additionally, it

favored the emergence of higher species and improved seedling survival, with further

consequences on plant community richness and cover. This treatment also increased species

recruitment over two years after road construction. Although there was no effect of tillage on

seed availability, emergence, and survival, by the end of the experiment in 2011, it appeared that

this treatment had increased plant species richness on the upper-slope zone.

In conclusion, our results showed that local soil conditions mainly related with organic

carbon, pH, soil texture, and structure, were in large part responsible for the organization of soil

communities and secondary succession processes on roadside sites undergoing restoration in our

study area. Moreover, the application of conventional techniques oriented to overcome abiotic

filters through the improvement of soil conditions did facilitate plant community reassembly.

However, other management techniques that have been traditionally used in roadslope

engineering and restoration, such as fertilization and mowing, significantly changed soil

community structure, by increasing evenness and the abundance of certain groups related to low

resource availability; they also affected functionality, by accelerating biodegradation of leaf

litter. Given that changes in belowground communities may also induce changes in aboveground

communities and vice versa, our results should be taken into account when selecting

management techniques in order to ensure the maintenance of the ecosystem services that these

Summary

33

‘young’ or emerging ecosystems are expected to provide. Based on our results, conventional

management techniques applied in roadside systems should be oriented to improve soil

conditions after roadslope building, and aim to preserve and promote soil stability in the

subsequent stages in order to ensure secondary succession processes and ecosystem

development.

35

CAPÍTULO I

Introduction and Objectives

Este capítulo reproduce parcialemente el manuscrito

Understanding Mediterranean roadside ecosystems to increase restoration

success: A review. (En preparación)

Capítulo I

37

Changes in land use due to human activities have been described as one of the main

drivers of ecosystem degradation (Foley et al. 2005). The huge transformation in ecosystem

attributes gave rise to the term Anthropocene (Crutzen 2002), defining a new era beginning 1800

with the industrial revolution. Since the mid 20th century, and as a result of the rapid growth in

the human population, the consequences of human activities on land transformation have

intensified (Steffen et al. 2007). In particular, earth works associated with the development of

road networks, that aim to satisfy the needs of social connection and economic development

(Coffin 2007), is today one of the principal geomorphological agents, even more important than

natural relief-modeling forces (Hooke et al. 2012). Despite the unquestionable benefits of road

network growth from a social perspective, road construction is associated with the

transformation and, in many cases, destruction of natural systems.

The effects of road construction are obvious across-scale, with direct consequences on the

abiotic and biotic components of ecosystems. At a landscape scale, road construction contributes

to fragmentation which, depending on the area of the land fragments, may be associated with a

rapid decrease in habitat quality with dramatic biodiversity loss (Forman & Alexander 1998).

Moreover, fragmentation has a direct effect on landscape connectivity limiting or impeding

dispersal processes, which in turn jeopardizes the persistence of species, populations and

communities in the short term (Marcantonio et al. 2013). This paradigmatic set up is currently

being revised as recent studies have highlighted the existence of parallel positive effects of roads

under specific conditions. For example, roadsides can act as shelter for a number of native

species in severely altered environments (Tikka et al. 2000). They also function as corridors

facilitating the movement of species and connecting different habitat patches (Haddad et al.


38

2003). These areas can play a key role in metapopulation and metacommunity dynamics by

boosting stepping-stone processes (Deckers et al. 2005).

At a local scale, road construction creates new rectilinear landforms much different from

equilibrium reliefs, in which natural drainage networks tend to develop quickly. Thus, during the

first months or years after road construction, geomorphic processes are highly relevant.

Specifically, on roadcuts, steep slopes with smooth and compacted surfaces, coupled with the

absence of vegetation, generate vast areas that are highly exposed to erosion (Andrés et al. 1996),

which results in sediment emission to road drainage systems. On the other hand, road

embankments are constructed by aggregate and foreign materials that confer particular features

to roadslope substrates such as specific granulometry related to project demands (Alfaya 2013),

poor in nutrients with high pH levels (Lehmann & Stahr 2007). These newly created

environments may also favor the establishment of new plant community assemblages, usually

including alien species that more easily access limiting resources and rapidly spread in the

absence of strong competitive processes exerted by native species (Hansen & Clevenger 2005;

Hobbs et al. 2009). Other local effects are derived from road operations such as ditch-cleaning,

seasonal mowing to increase visibility and reduce fire risk, re-grading and other disturbance

regimes, which set back succession to early developmental stages (Forman & Alexander 1998;

Spooner 2005). The effect of these common practices has not been studied in depth despite their

implications in ecosystem structure and functionality. Moreover, road traffic also contributes to

local impacts through roadkills (which in turn affects road security) and the increase in pollutants

(Clavenger et al. 2003; Forman et al. 2002).

Social perception about the consequences of road construction on human well-being has

contributed to the development of lines of research and environmental policies oriented toward

Capítulo I

39

compensating for possible impacts. Regarding landscape scale impacts, the first studies about

fragmentation were carried out in the United States and Europe in the 1970’s. These studies

contributed to prioritizing the construction of animal crossing structures to favor connections

between patches (Coffin 2007 and references there in). At the same time, a huge battery of

techniques aimed to compensate for local effects emerged. These measures were largely

influenced by agronomy and forestry and were principally focused on meeting geotechnical

demands (substrate stabilization and erosion control) by the establishment of a dense vegetation

cover. Hydroseeding, plantings, fertilizing application, mulches, and other so-called

bioengineering techniques have been used to this end.

All of these approaches later arrived in the Mediterranean countries where the urgency

for curbing the impacts of rapid development forced technicians to use the same integration

measures that were developed in northern countries, pursuing the same goals related to

exploitation risks and overcoming geotechnical constraints. However, Mediterranean ecosystems

differ from those in northern Europe. Mediterranean ecosystems have been historically shaped

by human activities such as agriculture, livestock and the effects of fire (Naveh 1974). These

perturbation dynamics along with Mediterranean climatic conditions characterized by summer

drought (Aschman 1973), have given rise to nutrient-poor soils exposed to wind and water

erosion processes (Pausas & Vallejo 1999). The particular climate and the long history of land-

use changes in the Mediterranean Basin have given rise to rich plant communities dominated by

annual species that are highly dynamic over time (Figueroa & Davy 1991). Hence, there is some

uncertainty associated with the application of conventional integration measures, given their lack

of adaptation to the local environment. Moreover, these measures are associated with low costs

of design but high investment in material implementation. Some decades after their first


40

implementation, the new economic and ecological context increased the need to review the

effectiveness of these integration practices under Mediterranean conditions. Furthermore, the

growing field of research developed around road ecology offers a good opportunity to reinterpret

roadside restoration directly tackling causes of degradation, suggesting scientifically-based

approaches oriented to the recovery of ecological processes and ecosystem functionality and

ensuring the efficient environmental integration of transport infrastructures.

The purpose of this introduction is to present the state of art of restoration measures

developed in the Mediterranean Basin, to analyze how conventional solutions perform under

Mediterranean conditions. We attempt to identify new knowledge frontiers to advance a more

ecological interpretation of these newly created environments and thus, more successful

restoration approaches. Conclusions from this review may also be applicable in other

Mediterranean regions, which share not only climatic characteristics but also highly diverse

biological communities that must be considered in the development phase of new road

restoration approaches. Thus, the first section of the introduction provides a synopsis of the

effects of roads at a landscape scale, considering how road construction affects animal behavior

and the effectiveness of defragmentation measures aimed at favoring connectivity, including the

effect of roads in plant dispersal. The second section deals with the available information of local

effects of road construction and the effectiveness of the measures that have been taken to

compensate for them. Since plant cover has been identified as a key element to control erosion

and trigger ecosystem development in these environments, the effects of environmental factors

and processes limiting plant community development in roadside ecosystems are addressed in

this section. We place special emphasis on the importance of seed availability and site limitations

(abiotic and biotic) in which the suitability of current techniques oriented to re-vegetation are

Capítulo I

41

also reviewed. We include other local effects derived from road operations such as roadkills or

the effect of pollutants generated by traffic. The final section discusses the importance of

considering a more ecological point of view in the restoration of roadside ecosystems as well as

the lines of research that would contribute to the recovery of ecosystem functionality and

provision services in these environments. The studies cited in the introduction were selected

using a systematic review of the literature related to roadslopes (roadcuts and road

embankments) or roadverges, specifically looking for those dealing with defragmentation,

connectivity, seed dispersal and restoration measures. Scientific papers published in international

journals related to ecological theory and technical approaches, as well as the very few

dissertation theses developed on roadside restoration, were considered.

Research and approaches to compensate large scale effects

Fragmentation and connectivity

Roads have been described as one of the main barriers to animal movements in

Mediterranean landscapes (Velasco et al. 1995). However, this effect appears to be context

dependent. For instance, Brotons and Hernando (2001) showed a general decrease in forest bird

species with road proximity, although the intensity of this effect varied depending on the

previous degree of fragmentation of forest habitat close to the road (Silva et al. 2012). This effect

of road proximity on bird breeding also depends on the species considered (Peris & Pescador

2004). Moreover, road proximity may affect not only community structure and connectivity but

also the genetic diversity and physiological status of individuals, which compromises their

fitness, as well as the long-term maintenance of populations (Grilo et al. 2012; Navarro-Castilla

et al. 2014).


42

To improve the connectivity between animal populations and compensate for these

negative effects, different crossing structures have been designed and constructed (Rosell &

Velasco 1999). At the same time, a relatively large field of research has developed around the

efficiency of these structures to restore animal movements. The efficiency of wildlife structures

is mainly determined by the species considered, width of the passages, animal behavior and

seasonality (Jacques et al. 1982; Vassant et al. 1993a, 1993b). In this sense, both engineered

wildlife passages (specific crossing structures constructed for animal crossing) and non-wildlife

passages (general structures associated with road construction such as culverts, drainages, etc.)

are used by animals of different size, suggesting that the adaptation of functional structures of the

road could be a cost effective measure to ensure animal connectivity (Mata et al. 2003). Most

species show preferences among crossing structures, and special attention should be paid to those

species that are especially reluctant to use the passages when the crossing areas are designed

(Grilo et al. 2010; Mata et al. 2005; Mata et al. 2008). Seasonal variability is related to an

individual's activity period, microclimatic conditions and plant cover at the entrance of crossing

structures, although traffic intensity of the road can also affects the use of crossing structures

(Mata et al. 2009; Serronha et al. 2013, Villalva et al. 2013).

After ten years of research, it is clear that there is some bias in the analysis of

fragmentation due to the fact that almost all studies are focused on terrestrial vertebrates.

Moreover, most of the studies reviewed showed that the effectiveness of crossing structures are

context- or species-dependent, and this measure does not seem to be useful in counteracting the

effect of roads as barriers for animal movements. Thus, to increase a company's awareness of the

importance of incorporating animal ecology into road plans and design is the first step toward a

Capítulo I

43

more efficient integration of infrastructure that ensures animal population connectivity and

sustainability (Ruiz-Capillas et al. 2013b).

There are also some studies that highlight the positive effect of road verges as shelters or

corridors for fauna and flora in Mediterranean countries. In severely transformed agricultural

lands, road verges tend to preserve remnants of natural vegetation that offer refuge and food

resources, especially for small mammals (Ascensão et al. 2012; Barrientos & Bolonio 2009;

Planillo & Malo 2013; Ruiz-Capillas et al. 2013a; Sabino-Marques et al. 2011) and may serve as

reservoirs for endangered species (Pita et al. 2006). Changes in animal behavior triggered by the

presence of roads can produce a cascade effect leading to the reconfiguration of food webs, in

which some prey are favored and some predators are excluded by direct competition (Ruiz-

Capillas et al. 2013c). The shelter effect was also visible in roadside plant communities, although

the capacity of roadslopes to preserve populations of endangered plant communities seems to be

context dependent (Filibeck et al. 2011).

Road verges also served as corridors for alien plant and animal species. Research

developed in the Canary Islands showed that road construction strongly alters microclimatic

conditions favoring the rapid spread of alien species in the surroundings closest to the road edges

(Arévalo et al. 2008, Delgado et al. 2013), depleting native species richness and homogenizing

community composition at a local scale (Arévalo et al. 2010). The corridor function of roads for

alien species is mostly related to stress gradients, in which resource availability rather than

competitive interactions or exclusion by native ruderal species promotes higher abundances of

alien species (Arévalo et al. 2005). These stress gradients are related to altitude, with invasive

species more likely to dominate plant communities at intermediate elevations (Otto et al. 2014).

However the potential of these linear infrastructures as conduits for these types of species often


44

depend on their proximity to urban areas (Arteaga et al. 2009). Moreover, new plant assemblages

that characterize roadside vegetation also have an effect on mutualistic relationships among

species. In this sense, plant communities modify ecosystem features with further effects on the

relative abundance of other plants and animal communities (Martínez & Wool 2006; Rotholz &

Mandelik 2013). Habitat provided by roadside ecosystems simultaneously affects animal and

plant communities, not only within road verges, but having further effects on surrounding

habitats and diversity. For this reason, roadside restoration plans should take into account this

source/sink and corridor function, orienting restoration efforts to maintain biodiversity, reduce

invasive species establishment and promote connectivity and landscape continuity with adjacent

areas.

Seed dispersal processes

Although the study of large scale effects of roads have primarily focused on animal

communities and their connectivity, recent work has developed on the effects of dispersal

processes in roadside reclamation under Mediterranean climatic conditions. Some studies

highlight the importance of surrounding vegetation as a reservoir for species susceptible to the

colonization of roadside areas (Tormo 2007). Anemochores species are more likely to arrive in

these environments during primary succession. However, to leave roadslope colonization in the

"hands of passive restoration", remnants of natural vegetation in the nearby surroundings are

necessary (Bochet et al. 2007b). Likewise, the availability of these seed sources and the

occurrence of dispersal processes will strongly depend on the spatial structure of surrounding

vegetation as well as the historical land use that had shaped plant dispersal traits and animal

communities, especially for zoochorus species, at a landscape scale (De Torre 2014;

Capítulo I

45

Marcantonio et al. 2013). Although seed dispersal does not seem to be an important process in

constraining roadside revegetation (Mola et al. 2011), dispersal limitations associated with the

quality of the surrounding patches and dispersal vectors may affect plant community

composition in these ecosystems (García-Palacios et al. 2011b).

Research and approaches to compensate for local scale effects

Plant community development

It is well known that the effects of road construction usually exceed the limits of the road

itself (Forman & Alexander 1998). However, most of the studies developed in the Mediterranean

Basin have focused on local scale effects and specifically on the processes affecting plant

community development in these environments. Plant colonization is fundamental to roadside

restoration because it is widely accepted that vegetation plays an important role in the

stabilization of motorway slopes (Andrés et al. 1996; Andrés & Jorba 2000; Cerdá 2007).

Although in the beginning, most work was related to the factors limiting the establishment of an

unspecific plant cover meeting the goal of erosion control, more and more studies attempt to go

further in our understanding of the processes underlying functionality of roadside ecosystems.

By doing this, our capacity to improve ecological restoration and to enhance the environmental

services that these scenarios can provide has increased. In this section, we present the main

processes constraining the development of plant communities in roadslopes and the ecological

filters that species must overcome. The picture is completed with the effect of other factors

derived from road traffic on plant communities.


46

Seed availability

The first barrier to successful plant colonization is in-site seed availability. This limitation

for colonization was traditionally ensured by hydroseeding applications as an easy way to obtain

high vegetation cover in the short term (Merlin et al. 1999). After many years of using this

technique without a clear sense of its efficiency, some lines of research were promoted by private

companies to fill this gap in knowledge (Andrés, Jorba and García-Fayos, Com. Pers). The first

results highlighted that hydroseeded species do not perform better than those from the

surroundings (Matesanz et al. 2006) and even more importantly, that successful hydroseeded

species are usually grasses that provide poor ground cover with little contribution to erosion

control (Tena 2006). This failure is partially associated with the fact that hydroseeding mixtures

commonly include non-native species unadapted to Mediterranean conditions. For this reason,

some studies have tested the suitability of enriching hydroseeding mixtures with native species.

Generally, the inclusion of native species in sowing mixtures significantly improves plant cover

and richness in Mediterranean roadslopes (Bochet et al. 2010b; Katritzidakis et al, 2007; Martin

et al. 2002; Matesanz & Valladares 2007). Moreover, Estaún and colleagues (2007) found that

the performance of hydroseeding mixtures improves with the inoculation with arbuscular

mycorrhizal fungi favoring plant establishment and growth. Unfortunately, and despite the fact

that this technique has been widely used in this region, recent works indicate the low value of

this revegetation technique. On the one hand, it does not improve site conditions or soil

processes (García-Palacios et al. 2010). On the other, and in relation to the capacity of

hydroseeded species to “kidnap” succession, it conditions plant community composition in the

long term and impedes the establishment of other late-arrival species (García-Palacios et al.

2010; Matesanz & Valladares 2007; Zelnik et al. 2010).

Capítulo I

47

Likewise, the topsoil removed during the first stages of linear infrastructure construction

is a valuable resource for roadside restoration as it is expected to preserve original seed and

propagule banks (Balaguer 2002). However, the very few studies considering the positive effect

of spreading topsoil on plant community development in Mediterranean roadslopes, found that

effects were not related with soil seed banks (Mola et al. 2011; Tormo et al. 2007). These

findings are related to the fact that typical topsoil management strongly affects the viability of

seeds and the ability to recruit from seed bank sources (Rivera et al. 2012b). Hence, if seed

availability mostly depends on seed rain and propagules provided by surrounding vegetation,

restoration actions should prioritize the maintenance or recovery of sustainable rich plant

communities near roadslopes. Thus, in order to optimize biodiversity in roadslopes, management

techniques should be oriented to ensuring succession and the establishment of later-arrival

species.

Abiotic filters

In parallel, many works have highlighted the importance of environmental filters as main

constraints to plant community development on roadslopes (Alborch et al. 2003; Mola et al.

2011; Tormo et al. 2006). These filters are especially important in roadcuts and are directly

related to construction features such as soil compaction, steep slopes, smooth surfaces or aspect

(Bochet et al. 2009; Bochet et al. 2010a; Bochet & García-Fayos; 2004; Cano et al. 2002;

Navarro 2002; Tormo et al. 2009). Given that Mediterranean areas are subjected to strong

seasonal variability characterized by torrential rains and summer drought (Romero et al. 1998),

roadslope surfaces are highly exposed to runoff and erosive events, which in turn contribute to

sediment and nutrient loss. Moreover, runoff decreases water permeability and availability for


48

plants, which has been described as the main constraint for seed germination with further effects

on plant community composition and structure (Bochet et al. 2007a; Tormo et al. 2008).

Other studies reinforce the importance of plant-soil relationships in early successional

stages in roadslopes (García-Palacios et al. 2011a; Jiménez et al. 2013), and for this reason recent

work has been developed to test how soil improvements contribute to plant community

establishment and growth in these environments. For instance, topsoil used in restoration projects

increases water retention and improves nutrient contents at the same time that it triggers the

development of a more diverse plant community independent of topsoil layer depth (Mola et al.

2011; Rivera et al. 2014). Although the positive effect of topsoil on critical soil processes such as

litter degradation has not yet been demonstrated (Jáuregui et al. 2012), it has been shown that

this treatment has positive effects on microbial activity (Rivera 2012). In addition to topsoil

spreading, the effect of organic and inorganic amendments on the habitat carrying capacity of

roadside systems has been tested. On the one hand, horse manure improves physico-chemical

soil properties and generates similar plant covers to those with topsoil use, but results in less

diverse plant communities (Rivera 2012). On the other hand, sewage sludge seems to be a useful

amendment to improving soil conditions since it decreases soil pH, increases organic matter

content and maintains heavy metal soil concentrations at acceptable values (Ferrer et al. 2011).

However, this treatment slightly increases plant cover development and its effect is often diluted

and can not be distinguished from topsoil effects. It is worth noting the interesting work

developed by García-Palacios and colleagues (2011a, 2011b and 2012) who demonstrated that

the effect of traditional roadside management techniques such as irrigation or inorganic

fertilization is strongly site-dependent and often driven by dominant plant communities that were

established before any treatment was applied. They then go on to suggest the importance of

Capítulo I

49

testing the effect of management below-ground, probably one of the most forgotten ecosystem

compartments, and found that fertilization enhanced plant compositional shifts that translate into

changes in soil functionality, principally during the first stages of plant community development

and colonization (0 to 5 year old slopes).

The techniques applied in Mediterranean countries in order to overcome abiotic factors

constraining roadside ecosystem development are comparable to those commonly used in

northern countries and show similar results regarding geotechnical goals. However, the most

interesting insight provided by these studies is the importance of soil compartments in the

organization of roadside communities. Hence, further research on how to promote soil

functionality is needed to efficiently restore these environments.

Biotic filters

Mediterranean ecosystems at pioneer stages are dominated by annual plant species with

lower productivity due to abiotic stress. Therefore, competitive relationships among species are

expected to be of less importance than abiotic factors on plant community dynamics (Maestre et

al. 2009). However, under certain circumstances, biotic interactions between plant species may

be critical in Mediterranean roadslopes (García-Palacios et al. 2010, Valladares et al. 2008). In

road embankments, where the colonization process is easier due to gentle slopes and topsoil

spreading, the rapid growth of hydroseeded or non-hydroseeded species leads to competitive

exclusion processes that shape plant community composition in the short term (de la Riva et al

2011). Along these lines, Raevel and colleagues (2013) evaluated plant community assembly

processes in roadcuts in a time span of 80 years and found that early succession was

characterized by a transition between ruderal to long-lived species, in which the former better

compete for light, water and nutrients, and the latter appeared and remained in low abundances


50

until the end of the cronosequence due to their more efficient “water economy”. Some studies

suggest that competitive exclusion may emerge with the co-occurrence of certain functional traits

that were previously filtered by roadside environmental conditions. For instance, plant

assemblages present in road embankments tend to maximize their functional dissimilarities to

avoid competition for the same resources, while in roadcuts plant species tend to share some

functional traits and seem to be more similar than expected by chance (Tena 2006), suggesting

the existence of a more critical abiotic filter in these environments. To manage competition

among species, mowing has been proposed as a useful tool (Simões et al. 2013). This treatment

should be applied twice a year, in spring and in autumn, to ensure road safety and promote the

maintenance of roadside habitat quality.

Besides competition, other types of biotic interactions may be relevant in the

development of roadside ecosystems. In this regard, García-Palacios and colleagues (2011a)

highlighted the importance of soil microbes in the first stages of roadside ecosystem

development as well as the relationships between certain plant species and some microbial

functional groups. However, their results revealed that these interactions tend to disappear with

time, with plants being primarily responsible for ecosystem development in the long term

(García-Palacios et al. 2011b). Management should be oriented to handle biotic relationships

among species to maintain diversity levels in roadside communities, also considering relevant

above/below-ground interactions.

Local effects derived from road operations: roadkills and pollutants

Once the road is opened to traffic, other local impacts such as pollution appear. The

intensity of emissions has been measured in different countries in the Mediterranean Basin.

Pollutants generated by traffic may vary with many surrogates of economic development such us

Capítulo I

51

tourism, way of life, car-to-person ratio and vehicle age (Waked & Afif 2012). Petrol-burning,

break wear, and road risk barriers are principal sources of heavy metals (Scher & Thiery 2005),

being swept along with water runoff and jeopardizing water quality (Vieria et al. 2013). These

pollutants tend to accumulate in the upper layers of roadside topsoil and on plant leaves through

air deposition (Azkoy & Öztürk 1997; Teutsch et al. 2011). Heavy metals also make up a portion

of the fine fraction of particulate matter, while more coarse fractions are made up of soil dust re-

suspended by road traffic (Amato et al. 2011; Amato et al. 2012; Oliveira et al. 2010). Particulate

matter seems to be one of the most important pollutants affecting human health and for this

reason some measures, such as road watering or the application of dust binders, are now

common. However, the efficiency of these measures should be explored in depth (Amato et al.

2014; Karanasiou et al. 2014). Although the effects of pollutants to humans are obvious, very

few studies have been conducted in Mediterranean countries about their effect on wildlife and

ecosystem functionality. Scher & Thyeri (2005) evaluated the side effects of roads on insects and

amphibians in water retention ponds and their results indicated a general decrease in species

richness. A very recent study highlighted that the increase in CO2 due to traffic intensity depletes

photosynthetic rates in plants, decreasing their fitness and increasing bioaccumulation of heavy

metals (De Torre 2014).

On the other hand, the effect of traffic upon animal communities through roadkills is

well-known, and principally affected by species-specific features, habitat type and infrastructure

characteristics. Although there is some debate, the age of individuals seems to affect road

fatalities: depending on the species, adults or juveniles are the most vulnerable individuals

(Gomes et al. 2009; Grilo et al. 2009). Roadkill incidence also varies with life-history

phenological periods, more prone to occur during dispersal, meeting or breeding periods, where


52

females are most susceptible (Carvalho & Mira 2011; Grilo et al. 2009; Medinas et al. 2013).

Behavior also plays a role since species that tend to avoid roads are less likely to be killed

(Battisti et al. 2012; Gomes et al. 2009; Grilo et al. 2009). Moreover, it has been shown that

carnivores are especially vulnerable because of their larger home range (Grilo et al. 2012), and

because road verges tend to concentrate food resources (Barrientos & Bolonio 2009; Barrientos

& Miranda 2012; Carvalho & Mira 2011). Regarding habitat features, patches of suitable habitat

near the roads increase roadkills (Carvalho & Mira 2011), as does the proximity to urban areas in

the case of species linked to human activities (Grilo et al. 2012). Finally, road construction

features, like sinuosity, bridges or other structures adjacent to the road, increase the number of

road fatalities (Barrientos & Bolonio 2009; Barrientos & Miranda, 2012; Gomes et al. 2009).

Likewise, traffic intensity strongly conditions roadkills, although it is not clear if low or high

traffic levels correspond to less or more collisions (Barrientos & Bolonio 2009; Grilo et al. 2009;

Grilo et al. 2012). To increase road safety and to decrease the impacts of traffic upon animal

communities, road planning and design should take into account animal behavior and its spatial

distribution especially during the breeding season.

New frontiers for roadside restoration: towards an ecological approach and new goals for

roadside ecosystems

The Mediterranean region will be impacted by global change in the coming decades (Sala

et al. 2000). However, socio-economic factors may have greater effects than climate as drivers of

changes in land use (Schröter et al. 2005). Road network growth as a consequence of socio-

economic development as well as its effects on terrestrial ecosystems is expected to increase

(Sanderson et al. 2002). There is an increasing need for new approaches that ensure the efficient

integration of road infrastructures minimizing impacts on terrestrial ecosystems and optimizing

Capítulo I

53

the provision of environmental goods and services. Ecological restoration approaches applied to

novel roadside ecosystems is a useful tool to reinitiate ecological processes to ensure self-

organization, sustainability and ecosystem service provision in the long term (Clewell &

Aronson 2013). Nevertheless, after reviewing the literature on restoration (rehabilitation) of

areas affected by road construction in the Mediterranean Basin, it is clear that most studies have

done little to promote ecological processes, focusing on the recovery of ecosystem structure

applying the same approaches used in other countries. It is known that earth works such as those

derived from mining or road construction create new reliefs in which geomorphology and

hydrology is severely altered. These features give rise to stressful abiotic conditions described as

main constraints for ecosystem development (Bochet & García-Fayos 2004; Moreno de las Heras

et al. 2011). The recovery of stable landforms using geomorphic approaches (Martín-Duque &

Bugosh 2013), as well as functional soils that trigger the development of biological communities,

is the first step toward the efficient integration of roads with their environment.

Most research developed in this field is on plant communities probably because they are

considered good surrogates of functionality, and because it is thought that geotechnical risks are

minimized with an adequate vegetation structure. However, in the new context of global change

in which the Mediterranean ecosystem´s diversity is expected to be severely threatened (Sala et

al. 2000), the role of biodiversity maintenance in man-made systems and the provision of high

value ecosystem services, is key to effective land use management (Díaz et al. 2003). Given the

importance of roadside ecosystems as species reservoirs, new goals must be set to preserve the

local species pool. To do this, a more ecological interpretation of plant community dynamics in

roadside systems is needed. Moreover, the vegetation colonization process should be interpreted

under the paradigms of the emergent Coexistence Theory (Götzenberger et al. 2012; Hille Ris


54

Lambers et al. 2012; Temperton et al. 2004;) to identify the rules underlying the reconfiguration

of roadside communities, alternative stable states and thresholds that must be overcome to guide

restoration efforts (Suding & Hobbs 2009).

This introduction sheds light on the importance of soil in the development of roadside

ecosystems. However, most studies carried out in the context of road restoration interpret the soil

as a substrate from an agronomic point of view, and not as a whole system in which essential

ecological processes occur. The importance of soil fauna and microorganisms in key ecosystems

processes such us nutrient cycling are well known, and these are directly linked to the provision

of environmental goods and services (Van der Heijden et al. 2008). Thus, more attention should

be paid to this belowground community compartment as well as its interaction with the

aboveground community (Kardol & Wardle 2010).

Roadside restoration has developed as an entire empirical practice but without a solid

basis in scientific knowledge or theoretical foundation, often lacking clear and ambitious

objectives. Thus, often, success cannot be properly evaluated (Hobbs & Harris 2001). These

facts contribute to a lack of trust in ecological restoration as a solution for efficient

environmental integration of linear infrastructures. Another point to highlight is that novel

ecosystems such as those derived from road construction appear to be somewhat unpredictable

and fragile (Lindenmayer et al. 2008). Roadside ecosystem dynamics are still poorly understood

due to the dearth of large scale or long-term studies, suggesting that to restore these novel

ecosystems we need to understand them more fully. Roadside ecosystems are usually subjected

to different types of management and it is essential to understand how these systems respond to

provide useful guidelines that ensure restoration success.

Capítulo I

55

Objectives

Even though soil is the basis of terrestrial ecosystem development, and most key

processes in regulating services occur in soil, insufficient attention has been paid to this

ecosystem component. Thus, in this Doctoral Thesis, we aim to fulfill some gaps in knowledge

related to soil communities inhabiting roadside anthrosols and their functionality. Specifically,

we aim to examine the factors affecting the spatial organization and temporal variation of

microarthropod and bacterial communities and to ascertain how roadside communities and

ecological processes respond to conventional management practices applied in these scenarios.

These issues are particularly relevant to the ecological restoration of roadside ecosystems.

Firstly, they provide new insight into soil communities that, despite their key role in soil

processes such as nutrient cycling and soil formation, have been overlooked. Secondly, they

interpret roadside ecosystems from a more ecological point of view and provide useful

guidelines to efficiently restore them. The study sites were selected because they are

anthropogenic systems, where physico-chemical conditions have been severely altered and

original biological communities have been eliminated. Thus, these scenarios are a test-bed for

ecological theories and for studying how the system re-configures itself under natural conditions

and under the effect of soil treatments. This thesis is organized into four different chapters in

which general and specific objectives are addressed to provide useful guidelines for practitioners.

Each chapter has been written in a research article format, with a specific introduction including

the theoretical framework, specific methods, results and discussion. After these chapters a

general discussion that integrates main results is also included. Specific objectives for each

chapter are the following:


56

In Chapter II soil microarthropod communities in anthrosols from road embankments as

well as their temporal dynamics are considered. Microarthropods are involved in litter

degradation and organic matter turnover and have been shown to be biological indicators of soil

health and quality, both surrogates of ecosystem functionality. Assessing road ecosystem

functionality is the first step to ensure the maintenance of biological communities and ecosystem

services on the roadside. Specifically, functionality is estimated based on the QBS index (Qualitá

Biologica del Suolo), obtained from microarthropod community composition and their specific

adaptations to the soil environment. In this chapter the following questions are addressed:

1. Are anthropogenic soils from road embankments functional based on their QBS values?

2. If so, which processes determine soil community structure and hence soil quality?

In Chapter III soil bacterial communities in anthrosols from road embankments under

natural conditions are analyzed. Soil bacterial communities are known to play a key role in

nutrient cycling and soil formation, and more often modulate the relationship between

aboveground species. Moreover, bacterial community composition and its spatial organization

may provide valuable information about soil processes and historical effects driving functionality

in roadside ecosystems. Thus, in this chapter the following questions are addressed:

1. What are the main groups of bacteria present in these types of soils?

2. What is the relevant spatial scale that explains changes in bacterial community composition

and diversity and what are the main factors underlying changes in community structure?

In Chapter IV, the response of soil bacterial communities and soil processes to

conventional management applied on roadslopes, such as mowing and fertilization, is analyzed.

Capítulo I

57

Roadside management implies a disturbance that, in terms of community features (e.g.,

resistance, resilience), may affect both structure and functionality with further effects on

environmental services provided by roadside ecosystems. To elucidate the effects of

management on these scenarios, the present chapter addressed the following questions:

1. How does management modify soil factors?

2. How does management modify soil bacterial community composition and diversity?

3. How does management affect soil processes such as litter decomposition and nutrient

availability?

In Chapter V, the early plant community assembly in a sun-exposed roadcut is studied.

Roadcut community assembly is a process usually constrained by abiotic factors. We usually

interpret this process is as fixed, but it is in fact the result of different transitions between life-

history stages occurring at an individual scale and in which every life-stage is precarious. The

present chapter aims to ascertain how an individual’s life-stages respond to different treatments

aimed at loosening environmental filters and how these responses translate to the final

configuration of the plant community. Thus, the following questions are addressed:

1. Does the response to environmental filters change across life-history stages?

2. Is there a threshold at any of the life-history stages throughout the early process of community

assembly? If so, to what extent do these thresholds affect the abundance, richness, and cover of

the emerging community?

3. What are the implications for the current revegetation practices on roadcuts?


58

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75

CAPÍTULO II

Soil functionality at the roadside: Zooming in on a microarthropod community in an anthropogenic soil

S. Magro, M,. Gutiérrez-López, M.A. Casado, M.D. Jiménez, D. Trigo, I. Mola, &

L. Balaguer

Ecological Engineering (2013) 60: 81-87.

Capítulo II

77

Abstract

Earth movements for road construction give rise to nutrient-poor anthrosols. Early onset of soil

processes in these environments has been reported on the basis of plant cover establishment.

Evidences of full soil functionality, however, would reveal the emergence of a self-sustainable

ecosystem on these man-made substrates. The aims of the present study involved (1) assessing

soil functionality on six-year-old road embankments by means of the QBS index, based on

microarthropod communities (2) elucidating soil properties responsible for the composition of

soil microartrhopod communities, and (3) exploring the practical implications of soil quality for

road embankment management. Road embankments were functional with QBS values

comparable to those found in natural systems (>100). Soil quality in these environments

depended on soil organic carbon dynamics. Among the 36 arthropod groups found, Acari and

Collembola dominated the soil community. Variation in microarthropod community composition

was best explained by higher abundances of Brachypilina (Oribatida, Acari) and Symphypleona

(Collembola). These trends in soil community structure were intimately linked to soil organic

carbon content, clay content and humidity. Given its relevance, the acknowledgment of the early

functionality attained by these roadside anthrosols should lead to the revision of current

protocols for roadslope monitoring and management.

Keywords: Anthrosols; Embankments; Mesofauna; QBS index; Soil organic carbon


78

Introduction

In the last two centuries, human activities have become main drivers of ecosystem

transformation (Hooke et al. 2012; Vitousek et al. 1997). These changes have led to the term

‘Anthropocene’ (Crutzen 2002), which describes a new age in which the Earth is undergoing a

rapid human-mediated change, with evident consequences for biodiversity and ecosystem

functioning. This new scenario is giving rise to ecosystems that emerge from within the pre-

existing ones, but which exhibit differential attributes affecting their dynamics and ecological

behavior (Hobbs et al. 2006). Within these emergent ecosystems, recently created substrates,

known as anthropogenic soils (first introduced as a class in FAO 1988), are the basis for pioneer

community development.

Anthropogenic soils or Anthrosols (ISRIC-FAO 2006) have been described as soils

created or profoundly modified by human activities. A particular case of these soils are urban

soils (sensu Lehman & Stahr 2007) characterized by high pH values as well as a large amount of

coarse materials and soil organic matter, which influence porosity dynamics (Nehls et al. 2006).

Among them, soils on road embankments also exhibit high pH values but clearly differ in their

low levels of organic matter as they have been developed from parent materials poor in nutrients

(Jiménez et al. 2013). However, neither the structural characterization nor even partial reports on

nutrient processes account for the degree of functionality of these particular soils.

Soil functionality integrates the interactions between structure and processes (cf. TEEB

2010). Within this integrative approach, soil functionality is also related with the soil quality

concept regarding the soil capability to sustain biotic communities and air and water quality

(Gardi et al. 2002). Recent studies have highlighted the importance of incorporating biological

indicators to evaluate the quality of anthropogenic soils (Hartley et al. 2008). The best indicators

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79

of soil quality will be responsive to land use or management (Doran & Zeiss 2000). Thus, Parisi

and coworkers (2005) proposed a soil-quality indicator focusing on soil microarthropod

communities and tested in different land-use scenarios. Soil microarthropods play a vital role in

the maintenance of soil functions and they have therefore been proposed as suitable indicators of

soil quality for environments impacted by human activities (Parisi et al. 2005; Tsiafouli et al.

2005; van Straalen & Verhoef 1997). Microarthropod communities are involved in pedogenesis,

soil aggregate stability, hydraulic properties, and plant performance (De Deyn et al. 2003;

Lavelle 1996; Liiri et al. 2002; Neher 1999). Particularly, soil microarthropods are always

present in litter and they control soil functions, such as litter decomposition. They resize and

fragment plant residues, increase surface area for microbial attack and the leaching of water-

soluble constituents, and control the structure and activity of the decomposer community

(bacteria and fungi) (Beare et al. 1997; Seastedt 1984). Microarthropods therefore contribute

directly to humus formation and police the soil, playing an important role in mineral turnover

and the dynamics of soil organic matter (Butcher et al. 1971).

Within microarthropod communities, apterygote Acari and Collembola are generally

dominant both numerically and in terms of biomass (Seastedt 1984). Acari are often the most

numerous group in soils with a well-developed O-horizon rich in mor-type humus. In most

ecological studies Acari are traditionally divided into four suborders Oribatida, Gamasida (the

current O. Mesostigmata), Actinedida (the current O. Trombidiformes) and Acaridida (now

taxonomically included within Oribatida but considered functionally different) (following the

classification of Krantz & Walter 2009). Although they all include macrophytophages feeding on

decaying higher plant materials, Oribatida are microphytophages, feeding mainly on bacteria,

yeasts and fungi; Gamasida are mainly predators of small invertebrates; Actinedida exhibit


80

mixed feeding habits and Acaridida are mainly saprophagous. The intimate relationship between

these soil invertebrates and their ecological niches (van Straalen 1998), coupled with the fact that

some (i.e. oribatids) lead sedentary lives characterized by low mobility and, in many cases, low

growth rates make these groups sensitive to disturbance (Maraun et al. 1999). Collembola

community composition has also been observed to be sensitive to human-induced disturbances, a

fact that reduces the presence of rare or restricted distribution groups, mostly Isotomidae and

Onychiuridae, and increases the number of expansive or not so demanding species in terms of

soil moisture and organic matter content, these mainly being Entomobryidae and Neanuridae

(Sousa et al. 2003). Other Apterygota (Protura, Diplura and Thysanura), Micromyriapoda

(Simphyla, Pauropoda and Polyxenidae) or small Diptera larvae and Coleoptera may be locally

important (Lavelle & Spain 2001).

In the present study, we attempt to elucidate to what extent anthropogenic soils from road

embankments are functional or impaired, using microarthropod communities as a soil quality

proxy. Although this crucial feature of ecosystem functioning and dynamics has been studied in

depth in other anthropogenic soils (Arshad & Martin 2002; Giller et al. 1997; Lavelle 1996), it

remains largely unexplored in new soils created by earth movements and urbanization, such as

road embankments. To date, previous studies have addressed soil functionality on roadslopes

through plant cover response (see for instance Ferrer et al. 2011). However, under Mediterranean

conditions, plant cover is only descriptive at best during the first stages after road construction

(Jiménez et al. 2013). In order to tackle roadslope soil functionality from a more accurate

perspective, we applied the QBS index (Quailitá Biologica del Suolo) on six road embankments,

which have been monitored since their construction. The QBS index is an eco-morphological

index based upon the degree of linkage between the microarthropod character syndrome and soil

Capítulo II

81

environment (Parisi 2001). Due to the fact that soil microarthropods are highly heterogeneous in

space and time (Ettema & Wardle 2000) we conducted our study during two consecutive years.

We specifically attempt to address the following questions: 1. Are anthropogenic soils from road

embankments functional based on their QBS values? 2. If so, which processes determine soil

community structure and hence soil quality? Finally, we wished to explore the practical

implications of soil quality for road embankment management.

Material & Methods

Study area

The study area was located on the M-12 and M-13 highways (Barajas International Airport,

Madrid, Central Spain 40º29’N, 03º34’W; Fig. 1). These highways were both built from January

2002 to June 2004. Average annual temperature from 2009 to 2010 was approximately 15°C and

total annual precipitation was 360 mm. Soils in the surrounding area are nutrient-poor,

characterized mainly by silica sands and presenting basal conglomerates, gravels, silts and clays,

corresponding to river floodplains (Blanco-García et al. 2007). For further information see Mola

et al. (2011) and Jiménez et al. (2013).

Experimental design and background of sites

We selected six embankments (T1-T6) constructed in 2004, upon which topsoil was spread.

All of them had similar slope angles (32.1º ± 0.75) and size (12.0 ± 2.5 m). Distances among

road embankments ranged from 1900 to 20 m, with an average distance of 769.12 m ± 236.54

between them. The plant cover of each embankment was visually surveyed every spring from


82

2005 to 2010 by two different observers. Because of the strong influence on soil functionality of

feedbacks between belowground and aboveground communities (Bever et al. 1997), we decided

to set up our study from 2009 to 2010 when plant cover was stabilized (Fig. 2). On each

embankment, four points for soil and microarthropod samplings were established one meter apart

at medium-slope level and separated one meter from one another.

Figure 1. Map of Madrid (Spain) showing the location were the present study was conducted, six road

embankments (T1-T6) in the vicinity of the Barajas airport (aircraft symbol).

Soil properties

On each embankment, we collected four soil samples (12.5 x 12.5 x 5cm) of approximately

500 g weight previously removing the vegetation from on top. Samples were air-dried and sieved

through a 2 mm mesh. In each sample, soil texture was determined following Guitian and

Carballas (1976); as well as percentage of organic carbon by means of micro-plates (adapted

from Anne, 1945) and percentage of nitrogen with the Kjeldahl method, as described by Page et

Capítulo II

83

al. (1982). We also analyzed soil porosity (percentage of pores in soil), soil humidity (percentage

of these soil pores occupied by water) and soil aeration (percentage of soil pores occupied by air)

from four soil cores of known weight and volume collected on each embankment, following

Guitian and Carballas (1976).

Figure 2. Percentages of plant cover surveyed from 2005 to 2010 at the study sites (embankments T1 to

T6). Arrows indicate the years in which the present study conducted.

Microarthropod sampling and QBS index

Four vegetation-free soil samples (25 x 25 x 10 cm) were collected on each embankment for

microarthropod extraction. Microarthropods were extracted from 2 kg of homogenized soil with

the Berlese-Tullgren method (Krantz 1978). They were preserved in Scheerpeltz solution

(alcohol 70ºC, glycerol and acetic acid) until they were counted and identified under a

stereomicroscope (Motic SMZ-168 Series- Zoom 7.5X-50X). To assess soil quality on the

roadslopes, we chose the QBS index (Parisi et al. 2005). This index classifies microarthropods

0

20

40

60

80

100

120

2005 2006 2007 2008 2009 2010

Pla

nt co

ver

(%

)

Time (years)

T-1

T-2

T-3

T-4

T-5

T-6


84

by their morphological features and do not require species level identification. An eco-

morphological score (EMI) from 1 to 20 is assigned to each taxonomical group according to its

adaptation level to soil. This level of adaptation is revealed through some morphological

characters such as: reduction or loss of pigmentation and visual structures; smooth body form,

reduced and more compact appendages (hairs, antennae, legs); reduction or loss of flying,

jumping or running adaptations; reduced water-retention capacity—e.g. thinner cuticle, lack of

hydrophobic compounds on the outer surface (Parisi 1974). Thus, the underlying concept is that

soil quality is positively correlated with the number of microarthropod groups that are well

adapted to soil habitats. The QBS index of each sample is the sum of EMI scores and it was

calculated for each soil invertebrate sample, in both years (2009 and 2010).

Data analysis

To analyze microarthropod community composition, a Detrended Correspondence Analysis

(DCA) was performed with most abundant microarthropod groups (Acari and Collembola).

Pearson correlation analyses were performed between DCA axes, plant cover, and soil properties

in order to test whether soil fauna composition varied in relation to site characteristics. We

analyzed variation of soil quality in space and time using repeated ANOVA (ANOVAR)

measures including embankment as a factor. We also correlated the QBS index with soil

variables and the QBS index in 2009 with the variation in soil quality during the study period

(ΔQBS= QBS 2010 – QBS 2009). Variables were transformed, if necessary, to achieve the

assumptions of normality and homogeneity of variances. For pair-wise comparisons, the Tukey

HSD test was performed. All statistical analyses were conducted using STATISTICA 9.1

software (StatSoft 2009).

Capítulo II

85

Results

Soil characterization

Anthropogenic soils from embankments were sandy-loam and very poor in nutrients, as

shown by their very low levels in soil organic carbon and nitrogen (Table 1). Although soil

carbon remained stable during the study period, total nitrogen content dramatically decreased

toward 2010 (nearly 50%), from normal values to low ones. This decrease had an impact on C/N

ratio, which duplicated its value in 2010, although it remained low.

Table1. Mean values (± SE) of soil variables measured in all roadslopes (T1-T6) during the study period (2009-

2010).

Soil properties (%) 2009 2010

%

%

Humidity 10.24 ± 0.50

9.82 ± 0.39

Porosity 49.97 ± 1.16

48.65 ± 1.14

Aeration 39.73 ± 1.30

38.83 ± 1.25

Sand content 61.56 ± 1.84

66.45 ± 1.47

Loam content 20.94 ± 1.26

17.65 ± 1.06

Clay content 17.50 ± 0.85

15.90 ± 1.02

Organic carbon content 0.33 ± 0.03

0.39 ± 0.03

Nitrogen content 0.16 ± 0.01

0.09 ± 0.01

C/N ratio 2.04 ± 0.72

4.59 ± 0.03

Soil quality

The QBS index was calculated for each sample (Table 2). ANOVAR results showed that soil

quality significantly varied among roadslopes (F [5,36] = 3.806; p < 0.01), embankment T4 being

the one with the highest soil quality, which increased towards 2010 (F [1,36] = 7.377; p < 0.05).

The QBS index in 2009 was negatively correlated with the variation in the index from 2009 to


86

2010 (r = -0.51; p < 0.05; Fig. 3). Across years, the QBS index was positively correlated with the

percentage of organic carbon (r = 0.571; p < 0.001).

Table 2. Mean values (±SE) of QBS index by road embankments both in 2009 and 2010.

Road

Embankment 2009 2010

T1 66.750 ± 7.782 144.000 ± 13.410

T2 90.250 ± 22.462 84.750 ± 19.610

T3 103.000 ± 12.179 123.000 ± 12.049

T4 144.250 ± 12.612 170.000 ± 16.114

T5 80.250 ± 9.286 126.250 ± 28.944

T6 119.500 ± 15.650 116.500 ± 21.941

Figure 3. Pearson correlation analysis between QBS index values in 2009 and the variation of the index during the

study period [ΔQBS= QBS 2010 – QBS 2009].

-100

-50

0

50

100

150

0 50 100 150 200

Δ Q

BS

QBS 2009

r= -0.51; p< 0.05

Capítulo II

87

Microarthropod community structure and dynamics

From the soil invertebrate samples, 36 groups of arthropods were recorded (Table 3).

However, only nine groups of euedaphic microarthropods were considered in the analysis of

community patterns. Groups selected for DCA analysis were as follows: Gamasida, Actinedida,

Acaridida, Oribatida Macropylina and Brachypilina (which includes Poronota and Gymnonota),

Symphypleona (which included the Sminthuridae and Dicyrtomidae families), Poduromorpha

(which included the Neanuridae, Hypogastruridae, Poduridae and Onychiuridae families) and

finally, Entomobriomorpha (which included the Tomoceridae, Isotomidae and Entomobryidae

families).

The first and second axes of the DCA analysis explained 35.1 and 19.3% of the variance,

respectively. The first axis was positively correlated with Gamasida and Acaridida and

negatively with Poronota and Symphypleona abundances. This axis also separated cases from

2009 from those from 2010, which suggests temporal changes at the community level (Fig. 4).

There was, however, certain plot aggregation by embankments which was apparent only in 2009.

As for 2010, we observed a general displacement of plots toward the negative extreme of the

first axis. Differences in plot coordinates from 2009 to 2010 however were not similar in all

embankments, with the highest displacements in the T5 and T6 samples and the lowest ones in

the T4 samples. Correlation between Axis 1 and edaphic and vegetation variables, showed a

significant correlation with percentage of organic carbon (Fig. 5).


88

Table 3. Mean density (number of individuals/kg of soil) (± SE) of different taxonomic groups of soil invertebrates

found on road embankments (T1-T6) for each sampling year (2009 and 2010).

Taxonomical group 2009 2010

individuals / kg.of soil individuals / kg.of soil

SC.Acari*

Gamasida (O. Mesostigmata) 70.112 ± 7.834

66.691 ± 13.033

Actinedida (O. Tombidiformes) 31.362 ± 5.399

246.886 ± 53.690

S.O. Oribatida

Macropylina 3.417 ± 0.957

7.434 ± 2.743

Brachypilina

Gymnonota 12.566 ± 3.227

61.874 ± 15.718

Porononota 30.137 ± 8.318

233.851 ± 63.537

Acaridida (Cohort Astigmatina) 20.071 ± 5.721

5.542 ± 1.928

O. Arachnida 1.375 ± 0.749

1.086 ± 0.219

O. Collembola

Fam. Sminthuridae 0.603 ± 0.246

5.497 ± 2.321

Fam. Dicyrtomidae - - - -

- - - -

0.292 ± 0.252

Fam. Neanuridae - - - -

- - - -

0.682 ± 0.365

Fam. Hypogastruridae 0.298 ± 0.214

0.833 ± 0.437

Fam. Poduridae - - - -

- - - -

0.516 ± 0.347

Fam. Onychiuridae 7.875 ± 4.179

2.090 ± 0.862

Fam. Tomoceridae - - - -

- - - -

0.292 ± 0.175

Fam. Isotomidae 12.320 ± 2.884

17.711 ± 5.195

Fam. Entomobryidae 0.625 ± 0.323

25.950 ± 6.592

O. Protura 0.375 ± 0.261

0.042 ± 0.042

O. Diplura 2.171 ± 0.919

5.287 ± 1.359

O. Thysanura - - - -

- - - -

0.042 ± 0.042

O. Psocoptera - - - -

- - - -

0.208 ± 0.170

O. Isoptera - - - -

- - - -

1.061 ± 0.549

O. Embioptera 0.500 ± 0.458

3.624 ± 1.805

O. Coleoptera 5.913 ± 1.665

9.192 ± 1.904

O. Dictioptera - - - -

- - - -

0.095 ± 0.066

O. Thysanoptera 1.917 ± 0.812

1.826 ± 0.886

O. Diptera 1.635 ± 0.317

4.401 ± 1.226

O. Hemiptera 7.671 ± 1.604

14.605 ± 4.807

O. Hymenoptera 4.402 ± 0.947

11.771 ± 4.835

O. Lepidoptera 0.042 ± 0.042

0.042 ± 0.042

O. Pauropoda 0.638 ± 0.271

0.304 ± 0.212

O. Symphyla - - - -

- - - -

0.919 ± 0.362

O. Polixenida - - - -

- - - -

0.242 ± 0.145

O. Julida 0.208 ± 0.104

0.061 ± 0.061

O. Litobida 1.728 ± 0.697

0.125 ± 0.069

O. Scolopendrida 0.144 ± 0.109

0.455 ± 0.196

O. Isopoda 0.542 ± 0.241

0.417 ± 0.210

Capítulo II

89

Figure 4. Distribution of the sample plots in the space defined by the two first axes of a DCA analysis. Colored

arrows mark the trajectory of the samples from each study road embankment (blue=T1; orange= T2; green=T3;

red=T4; grey=T5 and yellow=T6). Empty dots correspond to samples collected in 2009 while solid ones correspond

to those from 2010.

The second axis was positively correlated with Gymnonota and negatively with

Actinedida. Samples from 2010 also showed a displacement toward the negative extreme of the

second axis in most of the cases (Fig. 4). This axis was significantly correlated with physical

properties of the soil such as percentage of soil humidity and percentage of clay content (Fig. 5).

Gymnonota

Poronota Symphypleona

Gamasida

Acaridida

Entomobryomorpha

Actinedida

Macropylina

Poduromorpha

Axis 1

Axis 2


90

Figure 5. Significant Pearson correlations (p< 0.05) obtained from the analysis of the relationship between DCA

Axes and soil variables.

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

DC

A a

xis

1

% Organic Carbon

0.00 5.00 10.00 15.00 20.00

DC

A A

xis

2

% Humidity

0.00 5.00 10.00 15.00 20.00 25.00 30.00

DC

A A

xis

2

% Clay

r= -0,44; p< 0.01

r= 0.44; p< 0.01

r= 0.535; p<0.001

Capítulo II

91

Discussion

Our results showed that anthropogenic soils on six-years-old road embankments are fully

functional as supported by the values and variation of soil quality indicators (i.e. QBS), and by

the composition of the soil microarthopod community.

Nearly all soils from the road embankments exhibited QBS values above 100 during the

study period, which has previously been interpreted as indicative of well-developed and

functional soils (Parisi et al. 2005). Indeed, the maximum QBS values obtained in the present

study were similar to those found by Parisi and coworkers (2005) in natural systems. Moreover,

QBS spatio-temporal variation was largely accounted for by soil organic carbon content. This is

one of the main results of the present research. In road embankment soils, singled out among

other urban soils by very low levels of organic matter, only a slight increase of approximately

0.6% in organic carbon is enough to trigger a significant change in soil functionality. In these

anthropogenic soils, organic carbon increases with time due to litter input at the early stages of

plant community establishment (Jiménez et al. 2013). However, the observed negative

correlation between the QBS values in 2009 and the QBS increment in 2010 suggest attenuation

in soil quality over time, although two years of data is clearly insufficient with regard to

detecting a representative temporal trend. Strikingly, this attenuation appears to overlap with that

observed in plant cover.

Beyond its contribution to QBS-index definition, microarthropod community

composition also provides further evidence of anthropogenic soil functionality. The presence of

certain groups and the mechanisms regulating their relative abundances are intimately linked to

soil functionality (Cole et al. 2005). During the study period, the soil community on the road

embankments was dominated by Acari and Collembola, groups involved in organic matter


92

decomposition and soil microstructure formation (Palacios-Vargas et al. 2007). Among them,

some Acari groups better explained variation in the microarthropod community during the study

period. The presence of Oribatida Brachypilina (i.e., Poronota and Gymnonota), given their

phytophagus habits, is linked to soil availability in organic matter, particularly plant material and

microflora (Luxton 1972). Acaridida, very abundant in 2009, usually occur sporadically except

in dry soils, such as arable lands and deserts (Wallwork 1970, 1976). It is a generalist group of

microarthropods that easily colonize new habitats because of their facility of dispersion due to

the long hairs that enable them to be phoretic (Norton 1980). Gamasida and many species of

Actinedida are basically predators (Koehler 1997; Lavelle & Spain 2001) and tend to concentrate

in areas where the soil fauna they prey upon is highly abundant (Gutiérrez-López et al. 2003).

Thus, the dominance of these mites in 2009 could be attributed to the availability of Acaridida,

and perhaps to the scarcity of competitors, such as some species of Coleoptera, Diplura or

centipedes. Actinedida is a highly heterogeneous group with regard to ecological requirements

(Kethley 1982), whose meaning in terms of soil functionality has been assessed with respect to

the relative abundance of Oribatida (Loots & Ryke 1967). The oribatid mites Poronota and

Gymnonota, on the other hand, are indicative of more stable soil conditions, as they are very

sensitive to soil disturbance (Wallwork 1970, 1976) owing to their slow development (Maraun et

al. 1999). The intimate dependence of oribatids upon soil conditions relies on their involvement

in the decomposition of organic matter and nutrient cycling and soil formation (Behan-Pelletier

1999). They process litter fragments and resize them for suitable intake (Norton 1980), and

influence soil structure by means of their fecal pellets, increasing the area for microorganism-

mediated nutrient mineralization. Among Collembola, Symphypleona are also indicative of soil

stability. Collembolans play a major role in soil microstructure development at the early stages

Capítulo II

93

of pedogenesis, and in more developed soils contribute to disintegrating macro- and mega-fauna

excrements, accelerating the incorporation of nutrients into the soil (Rusek 1998 and references

therein). Previous studies have found total organic matter to be the most important variable in the

organization of the Oribatida and Collembola communities (Hale 1971; Hasegawa 2001;

Maraun et al. 1999). Different groups respond differently to the type and amount of organic

matter. For example, Oribatida abundances have been correlated with the lightest organic matter

density fraction (Vreeken-Buijs et al. 1998), which is the one that preferably shows an increases

in urban soils (Scharenboch et al. 2005) and the abundances of this microarthropod have also

been correlated with the development of urban soils (Bever et al. 1997).

Conclusions

Changes in microarthropod community composition are usually shaped by land use and

management through their effects on soil properties (Butcher et al. 1971; Gutiérrez-López et al.

2010; Sousa et al. 2003). In our study the microarthopod community transition, from groups less

dependent on soil to those characteristic of more stable soils, was driven by variations in clay

content, soil organic carbon and humidity. Previous studies reported an increase in clay content

in anthropogenic soils (Scharenbroch et al. 2005) related with clay translocation, which is

indicative of soil development (Beyer et al. 1995). Our results showed an increase in clay

content, organic matter and soil humidity resulting in higher abundances of Oribatida. Clays in

soil usually associate with higher levels of organic matter, which in turn ultimately affect soil

porosity (Porta et al. 1999). The habitat created by small but numerous pores sift many

microarthropod species out of the emergent community. In general terms, Oribatida remain

associated with the 6-90 m pore size class (Vreeken-Buijs et al. 1998). In our study, the

Gymnonota observed were most often small-sized oribatids of the Oppiidae family.


94

To our knowledge, the present study is the first to substantiate the occurrence of

functional soils on young road embankments. Soil functioning as ecosystem functioning relays

on nutrient cycling, substrate stability and maintenance of hydrological processes as well

absorption and transfer of energy (Whisenant 1999), which are processess intricately linked with

the activity of microarthropods communities (Lavelle 1996; Liiri et al. 2002; Neher 1999). This

finding is sufficiently relevant to justify revision of current roadslope management and

monitoring practices. Pertinence of roadslope reprofiling actions and drainage works should be

appraised in the light of this emerging ecological capital. To date, roadslope monitoring has

focused on plant cover, while microarthropod community seem to provide a more accurate

assessment of soil processes. The key to successful roadside restoration toward self-sustained

ecosystems probably lies in the consolidation of a fully functional soil.

Acknowledgements

We are indebted to Marga Costa, Ana Buades and Mónica Otero for field and lab support, and to

Mr. Cormac de Brun for the English revision of the text. We also wish to thank the R&D

Department at OHL and Eje-Aeropuerto for their help and permission to conduct the research.

This study was funded by OHL, the Spanish Ministry of Economy and Competitiveness

(ECONECT project: CDTI IDI-20120317), Madrid Regional Government (REMEDINAL-2 S-

2009/AMB-1783), and by an FPU grant program of the Spanish Ministry of Education, Culture

and Sports (FPU-AP2009-0094).

Capítulo II

95

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103

CAPITULO III

Roadside microbes: factors affecting community structure and composition in urban soils from road embankments

S. Magro, E.E. Kuramae & L. Balaguer

Urban Ecosystems (Submitted)

Capítulo III

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Abstract

Human-mediated changes in terrestrial ecosystems give rise to urban soils such as those present

in road embankments. Interest in biological communities associated with these environments has

increased in the last few decades. However, despite the key role of biological communities in

soil processes, little is known about their bacterial community structure. In the present study, we

analyzed bacterial communities in anthrosols from road embankments and determined the

relevant scale for community self-organization. We collected 12 bulk soil samples in four

different road embankments (n=48) and analyzed soil bacteria community composition and

diversity by means of 16S rRNA partial gene sequences, and soil factors (C, N, C:N, texture, soil

structure and pH). Bacterial communities from road embankments present diversity levels

similar to other anthrosols and are dominated by Actinobacteria, Acidobacteria and

Planctomycetes, indicative of stable soil conditions. We also found low abundances of

Bacteroidetes, Firmicutes, Gemmatimonadetes and Cyanobacteria that could be related to

secondary succession pathways. Soil-borne bacterial community structure in these soils is site-

dependent and mainly conditioned by local soil factors as pH and soil texture. Given that plant

community dynamics in road embankments depending on biotic interactions, our results can be

used to guide the management and maintenance of local bacterial community composition,

considering further implications on the goods and services that urban environments may provide.

Keywords: road slopes, anthrosols, microbial ecology, soil factors, 16S rRNA, pyrosequencing


106

Introduction

Changes in land use and land cover significantly affect the structure of terrestrial

ecosystems across biomes (Lambin et al. 2001). Long-term impacts of historical and ongoing

human–mediated changes have altered soils, giving rise to alternative ecosystem configurations,

the so-called anthropogenic soils or anthrosols (ISRIC-FAO 2006). For instance, anthrosols as

those derived from agricultural practices differ in quantity and quality of organic matter

(Eisenhauer et al. 2012), and exhibit a severely altered nutrient storage and soil structure (Guo &

Guifford 2002; Murty et al. 2002). These changes may result in bacterial communities of lower

biomass than those present in natural systems (Gunapala & Scow 1998). Human activities also

shapes soil bacterial community composition (Acosta-Martínez et al. 2008) and its spatial

variability and distribution (Ettema & Wardle 2002). Locally, anthrosols are characterized by

bacterial communities with a simplified composition and low diversity levels, dominated by

specific groups (Buckley & Schmidt 2001; Hartmann & Widmer 2006). Across scale, the spatial

organization of bacterial communities may vary from micrometer to thousands of kilometers

(Nunan et al. 2002; Van der Gucht et al. 2007). This structure may result from both local factors,

such as soil pH (Fierer & Jackson 2006), and landscape and regional processes, such as dispersal

limitations (Van der Gucht et al. 2007).

Urban soils are specific type of anthrosols result of earthworks and infrastructure

construction (Lehmann & Stahr 2007). A particular case of urban soils are those derived from

road construction (Lehmann 2006). The differential characteristics of these anthropogenic soils

emerge from the interaction between their constructive features and the subsequent management,

both aimed to ensure geotechnical stability and plant cover development (Tormo et al. 2007).

Road embankments are constructed by heaping and compacting layers of aggregate materials

Capítulo III

107

that create new reliefs with slopes less than 51 %, and heterogeneous substrates with specific

granulometry that accomplishes with constructive project demands (Alfaya 2013), with very low

organic matter content (under 0.20%) and total nitrogen (around 0.02%) and high pH levels

(above 7.5) (Jiménez et al. 2013). Management of these newly created environments usually

involves the application of topsoil and hydroseeding with mixtures of commercial species

(Balaguer 2002). In Mediterranean regions, the effect of topsoiling coupled with the gentle slope

of embankments boosts primary production that may result in plant cover of around 100 %

during the first growing season after road construction (Mola et al. 2011). This increase in

biomass production leads to high carbon inputs to the soil, from litter fall and root turnover,

which ultimately results in seasonal pulses of organic matter accumulation (Lehmann & Stahr

2007). The specific characteristics of these soils shape the structure and spatial distribution of

the biological communities present on road embankments, giving rise to a particular combination

of plant species and soil microarthropod communities as described in recent studies (García-

Palacios et al. 2011; Magro et al. 2013). Despite the importance of lower levels of soil food webs

in ecosystem functionality (Holtkamp et al. 2011) and the reasonable expectation that specific

characteristics of these anthrosols may result in new assemblages of soil bacteria, little is known

about the community composition and spatial distribution of these organisms on road

embankments.

In the present study, we analyzed soil-borne bacterial communities in anthrosols from

road embankments in order to address the following questions: 1.) What are the main groups of

bacteria present in these soils? In order to answer this question, bacterial community structure

was analyzed by means of 16S rRNA partial gene sequences. We hypothesized that bacterial

communities are low in diversity and show a simplified composition similar to those present in


108

other anthropogenic soils (i.e., agricultural soils) because anthrosols on roadsides are

unstructured substrates; 2.) What is the relevant spatial scale at which to explain changes in

bacterial community composition and diversity and what are the main factors underlying changes

in community structure? We analyzed the spatial variation in bacterial communities considering

distances ranging from meters to kilometers. To elucidate local and regional factors underlying

spatial patterns of community similarity we used a Multi–Model Inference (MMI) (Burnham &

Anderson, 2002) approach. We hypothesized that bacterial communities in nearby embankments

are more similar in composition than those far away because of the influence of regional factors

related to road construction features, surrounding vegetation or roadside management.

Material & Methods

Site description and experimental design

The experiment was conducted in four road embankments located in two highways in

Madrid, central Spain: highway N-1 at El Molar (40º 43' 4.42'' N, 3º 39' 44.28'' W) and highway

M–13 at Barajas, next to the International Airport (40º 28' 49.74'' N, 3º 38' 15.91'' W). The

distance between highways was around 27 km. During the study period, average annual

temperature was approximately 15 ◦C and total annual precipitation was 308 mm

(http://www.tutiempo.net/clima/Madrid_Barajas/82210.htm). Road embankments from El Molar

(M1 and M2) were separated by 4.7 kilometers, and those from Barajas (T1 and T2), separated

by 0.7 km (Table 1). Anthrosols from embankments are artificial and heterogeneous due to the

combined effect of construction materials (aggregates from primary rock, sand and gravels) and

applied topsoil (Hill et al. 2011; Ministerio de Fomento 2002). To capture soil heterogeneity

within sites in our sampling, we constructed twelve experimental plots (2x2m) on each road

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109

embankment. In order to avoid the effect of slope position, all experimental plots where located

in the lower–slope zone, that in road embankments correspond to the further zone to the road that

do not suffer any management such as roadslope re-grading, mowing or herbicide application.

Table 1. Main characteristics and soil properties of the four sites studied. Numerical values are means ± SE (n =

48).(*) Indicates plant cover at the beginning of the experiment.

Soil factor M1 M2 T1 T2

Coordinates 40°45´54´Ń 40°43´53´Ń 40°29´39´Ń 40°29´18´Ń

3°36´21´´W 3°34´12´´W 3°36´13´´W 3°36´04´´W

Motorway A1 A1 M12 M12

Year of

construction 2008 2007 2004 2004

Total lenght (m) 180 165 140 250

Hieght (m) 20 24 14 12.5

Aspect WSW NW W E

Topsoil Spreading Yes Yes Yes Yes

Hydroseeding Yes Yes Yes Yes

Soil type Gneisses Gneisses Coarse sand Coarse sand

Plant cover* 100 100 100 100

C (%) 1,09 ± 0,11 1,38 ± 0,07 0,88 ± 0,02 0,92 ± 0,08

N (%) 0,04 ± 0,01 0,06 ± 0,00 0,05 ± 0,00 0,04 ± 0,00

C/N 31,17 ± 4,84 22,40 ± 1,14 16,73 ± 0,74 22,63 ± 1,08

Sand content (%) 77,06 ± 0,62 71,58 ± 0,85 57,43 ± 3,69 68,27 ± 0,98

Loam content (%) 9,94 ± 0,59 11,95 ± 1,07 26,61 ± 4,73 17,67 ± 1,63

Clay content (%) 13,00 ± 0,19 16,47 ± 0,82 15,95 ± 1,17 14,06 ± 1,32

Humidity (%) 4,48 ± 0,55 4,96 ± 0,52 8,57 ± 1,08 5,18 ± 0,60

Porosity (%) 78,89 ± 2,47 81,37 ± 1,49 68,25 ± 3,82 75,70 ± 1,33

Aeration (%) 74,40 ± 2,26 76,41 ± 1,28 59,68 ± 4,30 70,53 ± 1,30

Bulk density

(g/cm3) 0,69 ± 0,05 0,62 ± 0,03 1,00 ± 0,11 0,72 ± 0,04

pH 7,80 ± 0,15 7,09 ± 0,10 7,95 ± 0,06 8,18 ± 0,08

Soil sampling and physicochemical analyses

Soil samples were collected at the end of the spring (June 2011) agreeing on the principal

activity period in Mediterranean environments. We collected 48 bulk soil samples (1 bulk sample

per experimental plot* 12 experimental plots per embankment* 2 embankments* 2 locations).


110

Bulk samples were composed of four soil subsamples (12.5 x 12.5 x 5cm) from different points

of the experimental plot. From each, a small amount of soil was preserved at –40°C for

molecular analyses. The remaining soil sample was air-dried and sieved through a 2 mm sieve.

Then, texture (percentage of sand, clay and loam) was analyzed following the procedure outlined

by Guitián and Carballas (1976). Soil organic carbon and total nitrogen were analyzed by means

of a basic microanalyzer LECO CHNS–932 (analyses were performed by C.A.I Microanálisis

Elemental UCM). We also measured soil pH of each soil sample in deionized water in a

proportion 1:2.5, with a Crison pH meter. Finally, from each experimental plot, a soil core of

known weight and volume was collected to analyze soil structure (percentages of aeration,

humidity and porosity of soil and bulk density) (Guitián & Carballas 1976).

Nucleic acid extraction and 16S rRNA gene amplification

Soil DNA from samples (n=48) was isolated using the Power Soil kit (MoBio, Carlsbad,

CA), and quantified on an ND–1000 spectrophotometer (Nanodrop Technology, Wilmington,

DE). Region V4 of the 16S rRNA gene was amplified with primers 515F and 806R (Lauber et al.

2009). The 515F primer included the Roche 454-B pyro-sequencing adapter and GT linker,

while 806R included the Roche 454-A sequencing adapter and GG linker. A 12-bp barcode was

included in both primers. The PCR was carried out using 0.2 units of Hot Start Taq Polymerase

(Roche Applied Sciences, Indianapolis, IN, USA), 2.5 ml of 2 mM dNTP and 0.5 mM of each

primer. PCR conditions were an initial denaturation step at 95 °C (5 min), 30 amplification

cycles of denaturation at 95 °C (30 s), annealing at 54 °C (60 s), and extension at 72 °C (60 s),

followed by a final extension at 72 °C (10 min). Negative controls were carried out with water.

Five independent PCR amplifications were performed from each sample and were pooled

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together. Pooled PCR products were purified with the QIAgen PCR purification kit (Qiagen,

Valencia, CA) and reduced to a final volume of 25 µl in an Eppendorf Speedvac Concentrator

5301. Samples were sequenced (Macrogen Inc. Company, South Korea) on a Roche 454

automated sequencer and GS FLX system using titanium chemistry (454 Life Sciences,

Branford, CT).

Data analysis

Sequences and quality information were extracted from the Standard Flowgram Format

(SFF) files using the SFF converter tool in the Galaxy interface (Goecks et al. 2010 ). Only

sequences with a length of 200 to 350 base pairs were accepted and bases with a quality score

lower than 20 were trimmed. Sequences were further analyzed with the Qiime version 1.2.1

scripts (Caporaso et al. 2010), which have been made available in the Galaxy interface. Firstly,

all the sequences with a perfect match to the primer sequences containing no homopolymer run

exceeding six nucleotides and without ambiguous characters were assigned to samples by

matching to barcode sequences. Reverse complementary sequences were put in the forward

orientation and assigned to samples by partially matching to barcode sequences. This step was

confirmed by the V-REVCOMP tool using Hidden Markov Models (HMMs) (Hartmann &

Widmer 2006). Secondly, sequence errors introduced during pyrosequencing were detected using

the option for titanium data in the Denoiser 0.91 program (Reeder & Knight 2010). The

sequences were also checked for PCR chimeras using UCHIME (Edgar et al. 2011). Only

sequences with a length of 200 to 350 base pairs were accepted and bases with a quality score

lower than 20 were trimmed. The low quality sequences were discarded. The obtained high

quality sequences were clustered into Operational Taxonomic Units (OTUs) using UCLUST

version 1.2.21 (Edgar 2010) with a minimum sequence identity cutoff of 97 %. For each OTU


112

the most abundant sequence was selected as a representative for all sequences within an OTU.

Taxonomy was assigned to the representative sequences using the Ribosomal Database Project

(RDP) 2 classifier (release 10.4). Finally, the OTU table was filtered for specific taxonomic

terms using scripts provided by Qiime. Singleton OTUs were removed for community analyses.

The OTU table was rarified considering the lowest number of reads per sample. To explore

variation of bacterial communities from road embankments, a Non–metric Multidimensional

Scaling (NMDS) was performed using Bray–Curtis distance. Shannon diversity index (i.e.,

entropy), Simpson index (i.e., 1–dominance) and Equitability (i.e., Shannon diversity divided by

the logarithm of number of taxa) were calculated per sample using PAST (software v. 2.17c;

Palaeontologia Electronica [http://palaeo–electronica.org/2001_1/past/issue1_01.htm]). General

Linear Models (GLM) were applied to elucidate spatial patterns underlying bacterial community

composition and diversity (Statistica; v. 7.0, StatSoft, 2009). NMDS axis and α–diversity indices

were included as response variables, and location (Barajas and El Molar) and site (embankments

M1, M2, T1 and T2) as fixed factors (Zuur et al. 2009). Due to the hierarchical relationship

between fixed factors, the site factor was nested within location. Pairwise comparison was

performed using the HSD Tukey test. To analyze the contribution of soil factors to microbial

community structure in road slopes we selected the Multi–Model Inference (MMI) (Burnham &

Anderson 2002) approach. MMI is increasingly being used and recommended for the analysis of

observational data collected over ecological gradients (Maestre et al. 2012). This approach may

be of special interest in the analysis of factors affecting bacterial community composition

because soil factors are most often closely correlated with one another and it is difficult to

differentiate the relative contribution of each upon the process we aim to explain. In this sense,

the main advantage of this approach compared with traditional hypothesis testing focused on the

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variables to include versus exclude in a selected model, is that MMI ranks all predictor variables

according to their relative importance (Burnham & Anderson 2002). In MMI analysis, the first

axis of NMDS was included as a response variable and soil characteristics as factors. Loam soil

content was excluded in order to avoid redundant information because it is estimated from sand

and clay content. All models were ranked using Akaike Information Criterion (AIC) (Akaike

1973) applying the correction for small sample sizes (Shono 2000). Best model (with the lowest

AIC value) was selected in order to elucidate the percentage of the variance explained by

environmental factors. If differences in AIC between models was lower or equal to 2, then the

models were considered not to be significantly different (Motulsky & Christopoulos 2004). All

models were fit using GLMs in R (R 2.9.2, R Development Core Team 2008) and associated

packages available in CRAN (http://cran.r–project.org).

Results

Soil bacteria community composition

The total number of 16S rRNA sequences was 48,723 ranging from 1,041 to 10,593

sequences per sample. After rarefaction, sequences were classified into 211 different OTUs

corresponding to 22 phyla. Predominant bacterial phyla in urban soils from road embankments

were Actinobacteria, Planctomycetes and Acidobacteria accounting for 18.80, 16.62 and 11.34%

of the total number of sequences, respectively. The phyla Gemmatimonadetes, Bacteroidetes,

Firmicutes and Cyanobacteria were among the less abundant with relative abundance under 2%.

Archaea relative abundance was very low (< 1.5%) (Fig. 1).

http://cran.r-project.org/


114

Fig

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Spatial variation of soil community composition and diversity

NMDS analysis did not show a clear spatial pattern in soil bacterial communities from

road embankments (Fig. 2). However, GLM analysis performed using the first axis of NMDS as

a response variable revealed significant differences in soil bacterial community composition at a

local scale (F [2,43]=28.421, p site <0.001). Pairwise comparison between sites showed three

homogeneous groups of road embankments regarding bacterial community composition (Fig. 3),

in which there were no differences between sites from Barajas (T1 and T2), at the same time that

those from El Molar (M1 and M2) were different from each other but M1 was similar to T2.

Figure 2. NMDS results showing changes in the structure of soil microbial communities from road embankments

(M1= Open triangles; M2= Solid triangles; T1= Open squares; T2= Solid squares). Circles grouped samples with

95% of similarity regarding microbial community composition.

-0,5 -0,4 -0,3 -0,2 -0,1 0,0 0,1 0,2 0,3 0,4Axis 1 [ R^2 = 0.568]

-0,5

-0,4

-0,3

-0,2

-0,1

0,0

0,1

0,2

0,3

0,4

Axis

2 [

R^

2=

0.2

68

]


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Figure 3. Tukey HSD test results (Mean ±SE) after obtaining significant site effect (p<0.001) upon NMDS Axis

1, which summed up changes in microbial community composition from road embankments. Different letters

indicate significant differences between means at p<0.05.

In El Molar roadlopes, the Shannon index was 3.80±0.06 in M1 and 3.85±0.04 in M2,

while in Barajas roadlopes, this index was slightly higher, 3.88±0.05 in T1 and 3.86±0.03 T2.

Simpson index was 0.95 in M1 and 0.96 in the rest of the embankments (M2, T1 and T2).

Finally, the Equitability was lower in M1 and M2 with an average value of 0.38±0.02 and

0.39±0.01 respectively, while in Barajas the Equitability was 0.41 in both T1 and T2. GLM

analyses did not show any significant variation in diversity indices among sites or locations (F

Shannon [3,43]=0.49. p= 0.69; F Simpson [3,43]= 0.72; p =0.55, F Equitability [3,43]= 1.15, p=0.34).

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

Ax

1

Site

M1 M2 T1 T2

b

bc

c

a

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Soil factors affecting spatial distribution of the bacterial community

Best–fitting models explained over 35% of the variation of bacterial community composition

in road embankments (Table 2). Among the ten soil factors considered in MMI analysis, results

showed that soil pH best explained this variation, followed by factors related to soil texture, soil

structure and finally nutrient content (Fig. 4).

Table 2. Best–fitting models explaining variation in soil microbial community structure from road embankments.

Each column represents a soil factor (predictor variable). Of all 1024 possible models, models with AICc ≤ 2 are

presented. AIC estimates the relative goodness of fit of a given model; the lower its value, the more likely it is that

this model is correct.

Nutrient content Texture Structure

C N C/N Sand Clay Humidity Porosity Aeration Bulk density pH AIC ∆AIC R²

–80,10 0,00 0,360

–78,40 –1,70 0,334

–78,33 –1,77 0,363

–78,14 –1,96 0.361

–78,13 –1,97 0,361

–78,13 –1,97 0,361

–78,10 –2,00 0,360

–78,10 –2,00 0,360

606


118

Figure 4. MMI results illustrating relative contributions of each soil factor explaining changes on the structure of

soil microbial communities from road embankment. Ax1 of NMDS analysis has been used as response variable.

Contribution of each soil factor is calculated depending on the number of models in which the factor appeared,

weighted by the importance of each model.

Discussion

To our knowledge, this is the first study describing bacterial community composition and its

spatial structure in urban soils from road embankments. Our results showed that these

communities have similar diversity and slightly lower equitability values than those observed in

other anthrosols (Øvreås & Torsvik 1998), with a predominance of Actinobacteria,

Acidobacteria and Plactomycetes. Although Actinobacteria has been reported as dominant in

agricultural soils (Buckley & Schmidt 2001), higher abundances of this group have been also

observed in grasslands and abandoned soils (Acosta–Martínez et al. 2008; McCaig et al. 1999).

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Actinobacteria are associated with soils of low organic matter, although they may respond well

to carbon inputs (Bell et al. 2013). They have also been found in latter successional states in soils

with high pH values (Kuramae et al. 2010). Thus, higher abundances of Actinobacteria may

indicate transition to a grassland steady state (Acosta-Martinez et al. 2008). On the other hand,

Acidobacteria are ubiquituous in soil and have been observed as one of the main components of

bacterial communities in grasslands (Will et al. 2010) and managed systems (Jesús et al. 2009;

Navarrete et al. 2013), but tend to decrease in abundance under the effect of pollutants (Ros et al.

2009). Acidobacteria showed oligotrophic habits that enable them to grow and reach high

abundances in soils with scarce resource availability and low carbon mineralization rates (Fierer

et al. 2007). Surprisingly, Planctomycetes showed higher abundances in anthrosols from road

embankments than those previously reported by Whitman and colleagues (1998) in other

anthropogenic systems (15% in road embankments vs. 7% of the total rRNA extracted from

agricultural soils). The presence of Planctomycetes in soil is highly correlated with historical

management practices, such as nutrient and organic amendments (Buckley et al. 2006). The

relative abundance of this group dramatically decreases with changes in plant community

structure (Wakelin et al. 2013) and agricultural practices such as tillage (Buckley & Schmidt

2003), and increases in soils with high values of organic matter content, pH and Ca++

(Buckley

2006). For that reason, Planctomycetes could be ecologically interpreted as an indicator of stable

soil conditions due to the fact that its relative abundance is linked to parameters that take a long

time to recover.

Among less abundant groups, we found Bacteroidetes, Cyanobacteria, Firmicutes and

Gemmatimonadetes, also scarce in other anthropogenic systems (Rodrigues et al. 2013; Shange

et al. 2012). Low abundances of these groups seem to be related with secondary succession


120

processes, at least in regards to Bacteroidetes and Cyanobacteria, which have been described as

highly abundant in intensively managed systems (Kuramae et al. 2011). Bacteroidetes are

copiotrophs that rapidly colonize soil aggregates and easily exploit available forms of carbon

(Acosta-Martinez et al. 2008). Cyanobacteria represents between 6 to 40% of sequences

obtained in different soils (Nemergut et al. 2007). Relative abundance of Cyanobacteria

increases under abiotic stress (Bowker 2007). In that sense, relatively low abundances of

Cyanobacteria observed in anthrosols from road embankments may be linked with the high

productivity of these environments, which improves general soil conditions. Firmicutes and

Gemmatimonadetes are also stress-tolerant (De Bruyn et al. 2011; Kuramae et al. 2010).

Specifically, Firmicutes have the ability to form spores (Wakelin et al. 2013). These

characteristics may explain their presence in anthrosols from road embankments, which usually

exhibit cycles of high temperature and low moisture (Lehmann & Stahr 2007). Still, community

composition results should be taken with caution because of the time of the sampling, having in

mind that bacterial community composition in anthrosols may experiment seasonal variation

(Smit et al. 2001). On the other hand, pyrosequencing approaches allowed us to detect not only

active bacteria but also others that have been taken part of the community in other time point.

Our findings reveal that bacterial communities on road embankments located around 30 km

apart shared a common composition pattern. At regional scales, dispersal limitations are

expected to become increasingly apparent at finer taxonomic resolutions set in the present study

at the phylum level. Despite this methodological limitation, the significant variation found

between sites from the same location suggests a critical role for local environmental factors,

consistent with previous reports on bacterial communities (Van der Gucht et al. 2007). Soil pH

has been described as the first or even the unique factor explaining spatial variation in microbial

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communities across biomes and land uses (Fierer et al. 2012; Lauber et al. 2008). Soil pH is

usually correlated with other soil features, whose effects could be hidden depending on how data

analysis was performed (Kuramae et al. 2011; Navarrete et al. 2013). In this, sense the MMI

approach used in the present study allowed us to identify other factors affecting bacterial

community self-organization and to classify them by their importance. Our results support a

primary role for pH and soil texture as drivers of change in bacterial community composition and

a relatively low importance of nutrients. In anthrosols from road embankments, soil pH is higher

than in other managed systems; likely because of the use of lime as one of the embankment

layers to stabilize the substrate and avoid mass movements (Ministerio de Fomento 2002). These

lime particles are susceptible to vertical movements due to drastic changes in temperature and

evapotranspiration processes, which alkalinizes the soil surface. This alkalization may influence

the uptake of nutrients by microbes in soil, with further effects on bacterial community

composition and diversity (Navarrete et al. 2013). Soil bacteria community composition may

also vary with the shape of soil particles and the type of soil aggregates (Ranjard & Richaume

2001). Higher nutrient contents associated with clay increase bacterial diversity and may trigger

differences in composition, at the same time that the sand fraction may be linked to bacterial

species that are better adapted to nutrient poor conditions and also able to use a wider range of

substrates (Sessitsch et al. 2001). Bacterial community development also interacts with soil

particles through the production of extracellular polysaccharides (Foster 1988), which contribute

to aggregate soil particles and to the construction of soil microstructure. Because of this, there is

an increase in the heterogeneity of resources that also modify community composition (Chenu

1993).


122

However, our results also showed that soil factors just partially explain changes observed in

bacterial community composition and its spatial variation, which suggest that they may be other

factors underlying this process. Roadside ecosystem dynamics at local scale is driven by topsoil

spreading and sowing techniques, and its interaction with the regional pool of species susceptible

to colonize a road embankment, which has been historically shaped by land use (García-Palacios

et al. 2010; Moreno–de las Heras et al. 2008). Moreover, plant community assembly and

vegetation dynamics on road embankments are most often driven by plant-plant interactions (De

la Riva et al. 2011; Valladares et al. 2008), which may adversely affect ecosystem service

provision by roadside landscapes (Balaguer et al. 2011). Hence, plant assemblages from

roadlopes are expected to show different composition at local scale affecting the quantity and

quality of organic matter that is incorporated in soil and influencing microbial functional

diversity with further effects in roadside ecosystem functionality at short term (García-Palacios

et al. 2011). In this sense, differences in the quality of topsoil spread in road embankments or the

differences in seed mixtures applied by hydroseeding, may explain the variation of roadside

bacterial communities we observed at local scale. Due to the critical role of plant-soil feedbacks

on the maintenance of roadside ecosystems, further research is needed about the implications of

management techniques that may alter above-belowground relationships.

Knowing that urban soils perform important functions as buffering temperature and water

variations, as well as nutrient store (De Kimpe & Morel 2000), identifying factors affecting the

development of bacterial communities that are responsible of most of these functions is a key

issue. Moreover, the rising importance of ecosystem services provided by plant communities in

urban environments (Robinson & Lundholm 2012), coupled with the fact that vegetation

dynamics in road embankments also depends on biotic interactions; the local microbiological

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control of road embankments may provide new options for the fine–tuning of the complex

below–above–ground links between plants and soil bacterial communities. This control might be

exerted on the infrastructure construction process or in a more straightforward and efficient way,

during the maintenance practices undertaken by road transport operators and highway

concessionaires. In this sense, our results may provide useful guidelines for practitioners in order

to enhance and maintain the ecosystems services that urban areas can deliver.

Acknowledgements

We thank F. Pugnaire (EEZA–CSIC), S. Hortal (Hawkesbury Institute for the Environment,

Sidney) and A.S. Pijl (NIOO–KNAW) for assistance with molecular work, the research group of

plant ecology and ecological restoration UCM, and S. Bachiller and S. Correa for field and lab

support. We also thank Sarah Young for English editing. This study was funded by OHL, the

Spanish Ministry of Economy and Competitiveness (ECONECT project: CDTI IDI–20120317),

Madrid Regional Government (REMEDINAL–2 S–2009/AMB–1783), and by an FPU grant

program of the Spanish Ministry of Education, Culture and Sports (FPU–AP2009–0094). The

accession number of sequencing data in SRA is PRJEB5308.


124

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135

CAPITULO IV

Roadside ecosystems: the effect of management on soil functions and microbial community structure

S. Magro, E.E. Kuramae, A. Escuero. & L. Balaguer

Journal of Environmental Management (Summited)

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Abstract

Road construction is today one of the main forces of change in terrestrial ecosystems, generating

new reliefs and steep slopes in which hydrology and biological communities are severely altered.

These roadside ecosystems are subjected to conventional management such as mowing or

fertilization in order to reduce fire risk and maintain plant communities to control erosion

processes. However, is not known how this management affects soil microbial communities or

soil processes (i.e., litter decomposition or mineralization) that in turn influence functionality and

the provision of ecosystem services. Thus, the aim of the present study was to elucidate how soil

microbial assemblages in anthrosols from road embankments respond to conventional techniques

applied in roadslope management. To this end, we conducted an experiment on four road

embankments in which we applied different treatments oriented to modify organic matter inputs

and soil fertility. We assessed the microbial community by 16S rRNA gene fragment

pyrosequencing. Our results show that mowing, fertilizing and biomass addition generally

changed ecosystem components such as soil physicochemical parameters, soil microbial

community structure and diversity, and soil processes. However, we did not find a consistent

pattern among treatments in which the variation in soil community translates to changes in soil

processes and vice versa, which suggests that these new ecosystems are fragile and respond to

management in an unpredictable way. Deepening the knowledge about how roadside ecosystems

respond to management is highly relevant in the context of new frontiers in roadside restoration.

Keywords. Pyrosequencing, 16S rRNA, embankment, litter decay, fertilizer, mowing


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Introduction

Earth works constitute one of the main current drivers of global change in terrestrial

ecosystems (Hooke et al. 2012). Construction activities such as road building result in emerging

novel ecosystems characterized by new reliefs generated by compacted layers of aggregate

materials, leading to anthrosols or technosols (ISRIC-FAO 2006). One type of anthrosols is that

in road embankments (Lehmann & Stahr 2007), characterized by simplified substrates, high

spatial heterogeneity, depleted nutrients and high soil pH. These soil features give rise to pioneer

plant communities characterized by particular combinations of fast-growing species that

ultimately affect soil organic matter dynamics and belowground community performance

(Garcia-Palacios et al. 2011; Jiménez et al. 2013). Moreover, roadside ecosystem development

and dynamics are subjected to changes in land use as a consequence of management. To date,

conventional management such as mowing or fertilization is aimed at reducing fire risk and

compensating for low fertility, respectively (Petersen et al. 2004; Spooner 2005). Such practices

also aim to maintain plant communities responsible for controlling erosion processes. However,

they are often applied without sound knowledge of the consequences of these actions on

ecosystem features and function. This is especially critical in relation to the maintenance and

dynamics of microbe soil communities, since it is well known that they are responsible for key

ecosystem processes.

Land use effects on soil bacterial communities have been studied across biomes (Lauber et

al. 2008; Lupatini et al. 2013; Navarrete et al. 2011; Navarrete et al. 2013). Land management

affects the biotic and abiotic conditions with further effects in soil community structure and

functions (Fig. 1). Soil microbial composition and diversity depend on both aboveground

vegetation and soil environments (Wardle et al. 2004). For instance, different plant species may

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favor some bacterial groups within the local microbial diversity pool (Kuramae et al. 2010;

Kuramae et al. 2011; Mendes et al. 2014) through the synthesis of different compounds via

rhizodeposition and litter decay (Kuramae et al. 2013; Singh et al. 2004). However, some studies

revealed that the effect of changes in aboveground vegetation in soil microbial community

composition seems to be weak, and it is unclear if this effect results directly or indirectly from

modifying the quantity and quality of soil resources or providing novel microhabitat conditions

(Kielak et al. 2008; Singh et al. 2008). On the other hand, soil physicochemical factors such as

pH, organic carbon content, total nitrogen, C:N ratio, soil texture and moisture have been

described as determinants of the presence and relative abundance of soil organisms (Drenovsky

et al. 2004; Kuramae et al. 2012; Lauber et al. 2009; Lu et al. 2011). A complex picture emerges

when considering all of these factors: a patent feedback in which soil microbes affect key

ecosystem processes such as nutrient and carbon cycling and soil formation (Van der Heijden et

al. 2008), while such soil conditions -depending on aboveground structure- profoundly affect soil

microbial composition and structure. As a consequence, changes in soil microbial community are

expected to induce changes in ecosystem functionality (Acosta-Martínez et al. 2008). However,

direct links between shifts in soil microbial composition or diversity and its reflection in the

outcomes of soil processes depend on the attributes of soil bacterial assemblages. For instance, if

soil bacterial communities are resistant to disturbance, neither soil community structure nor

processes are expected to change with perturbations (Bowen et al. 2011). Contrarily, if soil

bacterial communities are functionally redundant, the ecosystem perturbation could induce

changes in community structure and composition but not in soil functions (Shade et al. 2011).

Unlike these two opposite scenarios, if soil bacterial communities are sensitive to disturbance

and functionally dissimilar, then both community composition and ecosystem processes may be


140

affected with further consequences in ecosystem functionality and in the ecosystem services they

provide (Allison et al. 2008).

Figure 1. Conceptual framework of the effects of land use in soil bacterial community structure and soil processes,

with further effects on ecosystem services. Land use changes the biotic and abiotic components of ecosystems.

These changes may induce variation in bacterial community diversity and composition. Due to the key role of soil

bacteria in maintaining soil processes, changes in community structure could affect the rates and outputs of these

processes. At the same time, ecosystem services arise from the interaction between structure and processes. In this

sense, if land use has an effect either on bacterial community structure or on soil processes in roadside ecosystems,

environmental services provided by these factors may be jeopardized.

Despite the well documented effects of soil microbes in ecosystem functioning (Torsvik

& Øvreås 2002), very few studies have analyzed the structure and composition of microbial

communities and its relationship to some critical ecosystem processes in roadside ecosystems

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such as plant colonization, soil stability and nutrient cycling (García-Palacios et al. 2011;

Jáuregui et al. 2013). Moreover, to our knowledge, none have addressed how roadside microbial

communities and soil processes (i.e., litter decomposition or mineralization) respond to their

management. This is very important in the context of new frontiers of roadside restoration and

management. Thus, in the present study we attempt to elucidate how soil microbial assemblages

in anthrosols from road embankments respond to conventional techniques applied in roadslope

management. To this end, we conducted an experiment with different treatments aimed at

modifying organic matter inputs in the system. Specifically, we analyzed how management

modifies 1) soil features, 2) soil microbial community composition and diversity and 3) soil

processes such as litter decomposition and nutrient availability.

Material & methods

Site description and experimental design

Since heterogeneous conditions of roadslopes constrain both microbial community assembly

and ecological processes at a fine scale (García-Palacios et al. 2011; Moreno–de las Heras et al.

2008), we selected two areas located in Madrid, central Spain: El Molar (40º 43' 4.42'' N, 3º 39'

44.28'' W) and Barajas (40º 28' 49.74'' N, 3º 38' 15.91'' W), both in dry Mediterranean climates.

Average temperatures were 14.4 ± 0.37 ºC (El Molar) and 15.5 ± 0.39 ºC (Barajas), and total

annual precipitation was 399 mm (Barajas) and 201.5 mm (El Molar) during the study period

(June 2011- June 2012) (www.aemet.es). Lithology in El Molar is glandular gneisses and

amphibolite schists with patches of arkoses, sandy clays and silts, whereas in Barajas, the

substrate is detritic, consisting mainly of coarse sand. Vegetation of the surroundings in both

locations results from agricultural and livestock activities that gave rise to a deforested


142

landscape, with a mosaic of abandoned croplands, annuals-dominated grasslands and scattered

scrublands. At each location, we selected two road embankments (sites: M for El Molar and B

for Barajas): M1 and M2 on highway N–1, separated by 4.7 km, and B1 and B2 on highway M–

13, separated by 0.7 km. Different experimental treatments were applied: Mowing, where the

standing vegetation was cut and removed; Biomass addition, where the cut biomass in mown

plots was spread; Fertilized plots, in which 130 g/plot (32 g/m2) of commercial NPK fertilizer

(Compo©, concentration 12-8-16), commonly used in roadside management in Mediterranean

conditions, was applied; and finally, Control plots (not treated). We sampled 48 plots (2

locations*2 sites*4 treatments*3 replicates per treatment).

Soil sampling and physical-chemical analyses

Soil samples were collected at the end of the spring (June 2011), before treatment application

(t0); and in June 2012 (t1) after treatments. Four soil subsamples (12.5 x 12.5 x 5cm) were

collected randomly on each experimental plot. Soil subsamples of the same plot were then

bulked and mixed, and 40 g of soil was stored at -40 °C for molecular analyses. Organic carbon

and nitrogen contents were analyzed from the remaining fraction by means of a basic

microanalyzer LECO CHNS-932 (analyses were performed by C.A.I Microanálisis Elemental

UCM) and soil pH measured with a Crison pH-meter both in water and KCl (0.1 M) following a

1:2.5 proportion, in order to estimate real and potential pH. Soil texture as the percentage of fine

sand, clay and loam was analyzed following Guitián & Carballas (1976). Finally, a soil cores

from the experimental plots were analyzed for percentages of aeration, humidity and porosity

(Guitián & Carballas 1976).

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Nucleic acid extraction, 16S rDNA gene amplification and pyrosequencing

Total DNA was extracted from ninety-six soil samples (48 samples from t0 and 48 samples

from t1) using Power Soil kits (MoBio, Carlsbad, CA) and DNA was quantified on an ND–1000

spectrophotometer (Nanodrop Technology, Wilmington, DE). Region V4 of the 16S rRNA gene

was amplified with 515F and 806R primers (Lauber et al. 2009). The 515F primer included the

Roche 454-B pyrosequencing adapter and GT linker, while 806R included the Roche 454-A

sequencing adapter and GG linker. A 12-bp barcode was included in the 515F primer. Each PCR

was carried out using 1 µg of DNA, 0.2 units of Hot Start Taq Polymerase (Roche Applied

Sciences, Indianapolis, IN, USA), 2.5 µl of 2 mM dNTP and 0.5 mM of each primer. PCR

conditions were an initial denaturation at 95 °C (5 min), 30 amplification cycles of denaturation

95 °C (30 s), annealing at 54 °C (60 s), and extension at 72 °C (60 s), followed by a final

extension at 72 °C (10 min). Negative controls were carried out with water instead of DNA. Five

independent PCR amplifications for each sample were performed and pooled. Pooled PCR

products/samples were purified with a QIAgen PCR purification kit (Qiagen, Valencia, CA).

Samples were equimolar mixed and sequenced (Macrogen Inc. Company, South Korea) on a

Roche 454 automated sequencer and GS FLX system using titanium chemistry (454 Life

Sciences, Branford, CT). Due to technical issues, two samples (M1-fertilizing treatment (t0) and

M1-mowing treatment (t1)) were discarded. The sequences were deposited at SRA with the

ascension PRJEB5308.


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Soil functional processes

Nutrient availability

Ion exchange resin bags were prepared in nylon mesh (20 µ mesh) with 7 g of cation

exchange resin (IRA-120) and 5 g of anion exchange resin (IRA-402), separately. A pair of

cation and anion exchange resin bags were buried on each experimental plot after treatment

application, in 2011, and then removed at the end of the experiment in 2012. Bags were

maintained at 4 ºC until they were processed and then cleaned with deionized water. Ion

extraction was performed in 50 ml of 2M KCl solution in one hour of continuous agitation (300

rpm). The extracts were then filtered and kept in a freezer until nitrate and ammonium contents

were analyzed. Ion concentration analysis was performed using a SKALAR SAN++ nutrient

analyzer (Nutrilab - URJC, Móstoles, Madrid).

Litter decomposition

Litter mesh bags (2 mm mesh) were placed on the soil surface after treatment application

(September 2011). Each mesh bag had 6 g of standard mix of dry grass plants (Bromus sp.,

Lolium sp., Festuca sp.). Litter organic carbon content was 43% and total nitrogen 0.76%. In

May 2012, the mesh bags were removed from soils and remaining litter in each bag was cleaned

with deionized water. The plant material from each sample was then dried in an oven at 60 ºC

and dry-weight was determined. The plant decomposition rate (K) was estimated by negative

exponential model (Olson 1963): xt/x0 = e –kt

, where x0 is the initial dry litter mass, xt is the

remaining dry litter mass at time t, and k is the daily decay constant expressed in day −1

.

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Data analysis

General Linear Models (GLMs) were applied to analyze the effect of management on soil

properties at the end of the experiment (t1) as well as on their temporal dynamics (t1-t0),

calculating the rates with the standard deviation instead of mean values. In each model, site and

treatment were included as additive fixed factors (Zuur et al. 2009).

Pyrosequence data and quality information were extracted from the Standard Flowgram

Format (SFF) files using the SFF converter tool in the Galaxy interface (Goecks et al. 2010).

Only sequences with a length between 200 and 350 base pairs were accepted and bases with a

quality score lower than 20 were trimmed. Sequences were further analyzed with the Qiime

version 1.2.1 scripts (Caporaso et al. 2010), which have been made available in the Galaxy

interface. Firstly, all the sequences with a perfect match to the primer sequences containing no

homopolymer run exceeding six nucleotides and without ambiguous characters were assigned to

samples by matching them to barcode sequences. Secondly, sequence errors introduced during

pyrosequencing were detected using the option for titanium data in the Denoiser 0.91 program

(Reeder & Knight 2010). The sequences were also checked for PCR chimeras using UCHIME

(Edgar et al. 2011). Only sequences with a length between 200 and 350 base pairs were accepted

and bases with a quality score lower than 20 were trimmed. The low quality sequences were

discarded. The high quality sequences were clustered into Operational Taxonomic Units (OTUs)

using UCLUST version 1.2.21 (Edgar 2010) with a minimum sequence identity cutoff of 97%.

For each OTU the most abundant sequence was selected as a representative for all sequences

within an OTU. Taxonomy was assigned to the representative sequences using the Ribosomal

Database Project (RDP) 2 classifier (release 10.4). Finally, the OTU table was filtered for


146

specific taxonomic terms using scripts provided by Qiime. Singleton OTUs were removed for

community analyses. The OTU table was rarified considering the lowest number of reads in a

sample.

Species richness, Dominance (1-Simpson index) and Evenness of each sample were

calculated at the end of the experiment (t1) and for their temporal dynamics (t1-t0) at the level of

taxonomical order. The same indices were also calculated at the twelve phyla levels,

corresponding to functional groups: Archaea, Acidobacteria, Actinobacteria, Bacterioidetes,

Firmicutes, Planctomycetes, Gemmatimonadetes, Alphaproteobacteria, Betaproteobacteria,

Deltaproteobacteria, Gammaproteobacteria, Verrucomicrobia and other minority groups. In all

models for these taxonomic and functional diversity estimates, the treatments and the sites were

included as additive fixed factors. Redundancy Analysis (RDA) with samples collected at the

end of the experiment (t1) was performed in order to elucidate the main trends of change within

soil microbial community composition. These changes were confirmed by two-way

Permutational Analysis of the Variance (PERMANOVA) by including site and treatment as

additive fixed factors. PERMANOVA post-hoc was performed following Anderson (2001).

Finally, GLM was used to analyze the effect of treatments upon soil processes. K-Olson decay

rate, as a measure of litter decomposition and accumulation rates of ammonia and nitrate

contents in soil, as a measure of nutrient availability were included as response variables and

treatment and site as fixed factors. In order to meet the assumptions of normality and

homogeneity of variance, different models were adjusted and the best model was selected by

Akaike Information Criterion (AIC) (Akaike 1998). All statistical analyses were performed in

the R environment using stats, nlme (Pinheiro et al. 2014) and vegan (Oksanen et al. 2013)

packages.

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Results

Effects of management on soil factors

Table 1. GLM results showing site and treatment effects on soil properties at the end of the experiment (t1) and on

temporal dynamics (t1-t0). Bold numbers indicate significant effects.

Soil factor Main effects (t1) (t1-t0)

Soil organic carbon

(%) df F p value df F p value

Site 3 20.755 0.000 3 1.788 0.165

Treatment 3 0.180 0.909 3 0.694 0.561

Residuals 41

41

Total nitrogen (%) df F p value df F p value

Site 3 13.747 0.000 3 6.480 0.001

Treatment 3 0.047 0.986 3 0.680 0.569

Residuals 40

40

C:N ratio df F p value df Chi p value

Site 3 14.143 0.000 3 59.193 0.000

Treatment 3 0.209 0.890 3 47.004 0.007

Residuals 39

45 131.431

Sand content (%) df F p value df F p value

Site 3 55.707 0.000 3 0.951 0.425

Treatment 3 1.115 0.354 3 0.708 0.553

Residuals 40

40

Loam content (%) df Chi p value df Chi p value

Site 3 25.466 0.000 3 122.560 0.000

Treatment 3 22.614 0.415 3 100.880 0.000

Null 45 51.186 46 153.520

Clay content (%) df F p value df F p value

Site 3 94.348 0.000 3 0.843 0.479

Treatment 3 1.813 0.160 3 0.508 0.679

Residuals 40

41

Humidity (%) df F p value df F p value

Site 3 12.652 0.000 3 5.724 0.000

Treatment 3 0.263 0.852 3 2.326 0.089

Residuals 41

40

Porosity (%) df F p value df F p value

Site 3 3.007 0.041 3 6.915 0.000

Treatment 3 0.676 0.572 3 0.824 0.489

Residuals 41

40

Aeration (%) df F p value df F p value

Site 3 2.940 0.044 3 5.039 0.005

Treatment 3 0.649 0.588 3 1.016 0.395

Residuals 41

41

pH df Chi p value df F p value

Site 44 0.900 0.922 3 1.359 0.269

Treatment 41 0.892 1.000 3 1.181 0.329

Null 47 1.388 41


148

GLMs showed that almost all soil factors varied significantly between sites (Table 1) either

at the end of the experiment (Table 2) or in time (Table 3). Moreover, mowing significantly

increased C:N ratio (Fig. 2A) and loam soil content in time (Fig. 2B).

Table 2. Means (±SE) of soil properties that significantly varied between sites from Barajas (B1, B2) and El Molar

(M1, M2)) at the end of the experiment (t1). Letters by rows show significant differences between site levels after

the HSD Tukey test.

Table 3. Means (±SE) of the temporal dynamics (t1-t0) of soil properties that significantly varied between sites

(Barajas (B1, B2) and El Molar (M1, M2)). Letters by rows show significant differences between site levels after the

HSD Tukey test.

Soil factor

Soil organic carbon (%) 1.078 ± 0.049 b 0.854 ± 0.071 b 0.903 ± 0.041 b 1.488 ± 0.077 a

Total nitrogen (%) 0.083 ± 0.005 b 0.056 ± 0.005 c 0.0717 ± 0.005 bc 0.105 ± 0.014 a

C:N ratio 12.803 ± 0.361 b 15.375 ± 0.305 a 12.839 ± 0.424 b 14.14 ± 0.689 a

Sand content (%) 59.833 ± 1.530 c 68.463 ± 1.576 b 78.498 ± 0.575 a 71.247 ± 0.500 b

Loam content (%) 14.091 ± 1.440 b 16.636 ± 1.589 a 10.833 ± 0.580 c 9.833 ± 0.438 d

Clay content (%) 19.917 ± 0.542 a 15.000 ± 1.137 c 9.583 ± 0.364 d 18.333 ± 0.488 b

Humidity (%) 2.34 ± 0.171 ab 2.069 ± 0.116 b 1.448 ± 0.136 c 2.755 ± 0.170 a

B1 B2 M1 M2

Soil factor

Nitrogen (%) 0.02 ± 0.003 ab 0.009 ± 0.002 b 0.025 ± 0.003 a 0.03 ± 0.009 a

C:N ratio 2.33 ± 0.469 c 4.583 ± 0.749 b 11 ± 3.148 a 5.75 ± 0.705 b

Loam (%) 4.73 ± 3.583 a 3.583 ± 1.235 a 1.417 ± 0.580 b 1.67 ± 0.639 b

Humidity (%) 3.9 ± 0.784 a 2.216 ± 0.401 b 2.019 ± 0.362 b 1.56 ± 0.341 b

Porosity (%) 11.6 ± 1.833 a 10.173 ± 1.504 ab 3.783 ± 0.894 c 10.2 ± 2.142 ab

Aeration (%) 13.5 ± 2.489 a 9.054 ± 1.593 ab 3.848 ± 0.794 b 7.06 ± 1.868 ab

B1 B2 M1 M2

Capítulo IV

149

Figure 2. Means (±SE) of the temporal dynamics (t1-t0) of A) C:N ratio and B) Loam content (%) under the effect of

treatments. Letters show significant differences (p<0.05) between treatment levels after the HSD Tukey test.

Effects of management on microbial diversity and microbial community composition

A total of 458,072 quality sequence reads were obtained in 94 samples. The number of

sequences ranged from 1,041 to 12,245 per sample. Sequences were classified at the taxonomical

order level resulting in 165 different orders. All analyses of soil microbial community and

diversity were performed on a subset of taxonomical orders (Appendix 1).

Biomass addition increased richness of the microbial community whereas fertilization

significantly increased evenness at the end of the experiment (t1). Evenness also varied between

sites with the highest values in M2 (Tables 4. No changes in diversity indices in time were

0

1

2

3

4

5

6

7

8

9

C:N

rat

io

b

bb

a A

0

1

2

3

4

5

6

Control Mowing Biomass

addition

Fertilizing

Lo

am c

on

ten

t (%

)

b

ab

b

a B


150

observed. Moreover, none of the community diversity indices at the phyla level varied between

sites or between treatments or time points (t1-t0) (Table 5).

Table 4. GLM results showing site and treatment effects on microbial community diversity indices calculated with

sequence classified at the taxonomical order level, both at the end of the experiment (t1) and on the temporal

dynamics (t1-t0). Bold numbers indicate significant effects.

(t1) (t1-t0)

Community attribute Main effects df F p value df F p value

Species Richness

Site 3 1.737 0.175 3 0.465 0.708

Treatment 3 3.476 0.025 3 0.052 0.984

Residuals 40 39

Dominance (1-Simpson)

Site 3 1.856 0.154 3 0.591 0.623

Treatment 3 1.702 0.183 3 0.963 0.420

Residuals 37 37

Evenness

Site 3 6.225 0.002 3 0.791 0.506

Treatment 3 4.117 0.013 3 0.406 0.749

Residuals 37 39

Figure 3. Means (±SE) of microbial A) Species richness and B) Evenness at order level at the end of the experiment

(t1). Letters show significant differences (p<0.05) between treatment levels after the HSD Tukey test.

0.780

0.785

0.790

0.795

0.800

0.805

0.810

0.815

0.820

0.825

0.830

0.835


addition

Fertilizing

Ev

en

ness

b

ab

ab

aB

58.00

60.00

62.00

64.00

66.00

68.00

70.00

72.00

74.00

76.00


addition

Fertilizing

Ric

hn

ess [

nu

m.

OT

Us/s

am

ple

] (S

)

ab

ab

a

b

A

Capítulo IV

151

Table 5. GLM results showing site and treatment effects on microbial community diversity indices calculated with

sequence classified at the phylum level, both at the end of the experiment (t1) and on the temporal dynamics (t1-t0).

No significant effects were found.

(t1) (t1-t0)

Community attribute Main effects df F p value df F p value

Dominance (1-Simpson)

Site 3 1.149 0.341 3 2.087 0.118

Treatment 3 0.505 0.681 3 0.172 0.915

Residuals 40 39

Evenness

Site 3 0.877 0.461 3 1.753 0.172

Treatment 3 1.027 0.391 3 0.460 0.712

Residuals 40 39

RDA explained around 14% of the variation in soil microbial composition (RDA1= 10.18%;

RDA2=3.64%). RDA axis 1 and 2 were significantly correlated with soil microbes (F RDA1 [1,40]=

5.16, p< 0,01; F RDA2 [1,40]= 1.84, p<0.05) and with experimental treatments (FTreatment[1,40]=1.83,

p<0.01) (Fig.4). PERMANOVA results confirmed these findings and showed that soil microbial

community composition significantly varied between treatments (F [3,40]= 0.189, p<0.05) and, to

a lesser extent, between sites (F [3,40]=0.121; p<0.05).

PERMANOVA post-hoc showed that, specifically, community composition was significantly

affected by fertilizing treatment (Control-Fertilizing: F[1,22] = 0.231, p<0.01; Mowing-Fertilizing:

F[1,23] = 0.130, p<0.05; Biomass addition-Fertilizing: F[1,22] = 0.240, p<0.01). Other comparisons


152

were not significant. Principal groups of soil microbes Gemmatimonadetes and Chlamydiae

changed with fertilization treatment (Fig. 4).

Figure 4. Redundancy Analysis (RDA) based on Bray Curtis distance calculated from bacterial 16S rRNA gene

sequences. Sites (B1, B2, M1, M2) and treatments (Control = co, Mowing= mo, Biomass addition= db and

Fertilizing= fe) are represented as centroids. References for the name of taxonomic groups are included in Apendix

1.

Effects of management on soil processes

Mowing significantly accelerated litter decomposition (F [3,39]= 9.197, p<0.001, Fig. 5). Both

litter decay and nitrate accumulation rate in soil varied between sites (F K-Olson [3,39]=14.774, p<

0.001; F Nitrate [3,38]=2.964, p<0.05). However, ammonium accumulation rate in soil did not vary

Capítulo IV

153

between sites or between treatments (F Site [3,37]= 0.2486, p> 0.05; F Treatment [3,37]= 0.8892, p>

0.05).

Figure 5. Mean (±SE) of K-Olson values that show the effect of treatments on litter decay rate. (*) Indicates

significant differences (p<0.05).

Discussion

Our results show that soil bacterial communities in road embankments are sensitive and

responsive to management in a very unpredictable way. Roadside management changed

ecosystem components such as physical environment, soil microbial community structure and

soil processes, but we did not find a consistent pattern among treatments. Thus, variations in soil

community do not translate to predictable changes in soil processes or vice versa. Moreover, we

observed that the effect of management on community attributes (diversity and composition) and

processes were very idiosyncratic and also varied at a fine scale.

-0.0265

-0.0260

-0.0255

-0.0250

-0.0245

-0.0240

-0.0235

Control Fertilizing

Biomass

addition Mowing

K-O

lso

n d

ecay

rat

e [g

*d

ay-1

]*


154

Effects of management on soil physicochemical properties

Roadside management affected the abiotic component of the anthrosols from road

ecosystems. In fact, mowing changed nutrient stocks and soil texture, increasing the C:N ratio

and loam content. Both effects can be explained as an indirect consequence of the influence of

this type of management on vegetation. On the one hand, it is known that mowing can favor soil

nitrogen removal by fast growing grasses (Rizand et al. 1989; Robson et al. 2007) and promote

nitrogen leaching from soil with a subsequent decrease in nitrogen availability for the whole

ecosystem (Maron and Jefferies 2001; Roscher et al. 2008). Although some studies have

suggested that mowing must be periodically applied to mobilize nitrogen pools (Jenkinson &

Powlson 1976; Jenkinson & Rayner 1977), in anthrosols from road embankments in which initial

total nitrogen content was very low (around 0.5%) the effect of mowing may be apparent in the

short term with subsequent effects on the C:N ratio. The lack of vegetation generated by mowing

also favored erosion and the loss of the finest particles in soil (clay), translating to a relative

increase in loam content in mown plots compared to plots without this treatment (Castillo et al.

1997). On the contrary, no effects of biomass addition or fertilizer on soil properties were

observed. It is known that plant communities play a key role in edaphic processes on road slopes

(Bochet et al. 2010; Jiménez et al. 2013). In this sense, the lack of response that we found may

be related to the intensity of disturbance exerted by the different types of management (Berga et

al. 2012), with techniques affecting plant communities more likely to affect soil features on road

embankments.

Capítulo IV

155

Effects of management on soil microbial community structure

Our results also show that roadside microbial communities are sensitive to disturbance,

where biomass addition and fertilizing are the main drivers of change in microbial community

diversity and composition. However, the different types of management generated dissimilar

response patterns in community diversity and composition. On the one hand, biomass addition

increased taxonomic diversity. This effect has been tied to new microclimatic conditions created

by surface litter accumulation, buffering temperature fluctuations in soil and increasing moisture

(Holland & Coleman 1987). Moreover, the accumulation of a surface-litter layer serves as a

barrier against drop impacts in soil by reducing soil compaction and preserving microhabitat

with further effects on microbial diversity (Pengthamkeerati et al. 2011). On the other hand, we

observed that evenness was favored by fertilizing, which could be related to the general decrease

in the abundance of almost all taxonomic groups. In this line, many studies have shown that

nitrogen addition has an overall effect of reducing microbial diversity in terrestrial ecosystems

(see for instance Fierer et al. 2012; Lu et al. 2011) at the same time that other studies find that the

effect of nitrogen enrichment in human-impacted environments appears to increase equitability

(Nogales et al. 2010 and references there in). We also observed that fertilization triggered

changes in microbial community composition in soils from road embankments, changing the

relative abundance of certain orders of bacteria. Previous studies have shown that nitrogen

addition induces changes in soil microbial composition and decreases microbial activity by

shifting the metabolic capabilities of soil communities (Fierer et al. 2007; Ramírez et al. 2012).

In concordance with other studies, fertilizing modified the relative abundance of

Gemmatimonadetes (Fa-si et al. 2012; Ge et al. 2008). This group is considered to include


156

oligotrophic bacteria. Changes induced by fertilization indicate slight shifts in the metabolism of

C recalcitrant compounds that may affect carbon stock in road embankment soils in the long

term (Nemergut et al. 2008). Unlike these management tools, mowing did not affect microbial

community structure. These results together indicate that microbial communities from road

embankments are either resistant or sensitive depending on the type of management applied. This

agrees with other studies that have observed that the relationship between disturbance and

community structure is context-dependent (Mackey & Curie 2001) and more often related to

community traits such as growth rate (Haddad et al. 2008).

Effects of management on soil processes

We also found that soil processes from road embankments are sensitive to management, but

the effects are not consistent between treatments and strongly depend on the process considered.

Generally, litter decomposition varies with climatic factors and evapotranspiration regimes (Berg

et al. 1991), with temperature and moisture the most relevant controlling factors. However, the

relative importance of these two factors on decomposition rates is usually modified when the

disturbance effect is considered (Fortunel et al. 2009). In fact, we observed that mowing

accelerated litter decay. The effect of cut vegetation on litter decay may be due to changes in

quantity and quality of organic matter inputs in soil (Zhang et al. 2008). Moreover, changes in

litter decomposition rate under the effect of mowing may be due to photodegradation. Henry and

coworkers (2008) described that in Mediterranean ecosystems, plant litter thickness strongly

influences litter degradation, with the thinner the litter layer the faster the decomposition due to

the higher exposure to sun light. This effect could be intensified under semiarid conditions

(Brandt et al. 2010). On the contrary, we did not observe any changes in nitrogen dynamics

Capítulo IV

157

through experimental management (measured as nitrate and ammonia accumulation rates). In

line with our findings, Maron and Jefferies (2001) described a weak correlation between mowing

and nutrient mineralization because of nitrogen uptake by annual grasses during the growing

season. Moreover, in the same study, it was shown that the effect of mowing on nitrogen

dynamics in soil is expected to take many years to become evident. However, in roadside

grasslands a significant increase of mineralization after mowing has been observed in the short

term (i.e., two weeks, Schaffers 2000). Nitrogen dynamics in soil is expected to change under

the effect of fertilization. Both carbon and nutrients should be more available to soil

microorganisms in more productive sites, and hence nitrogen mineralization rates are expected to

be higher if fertility is improved (Fisk & Fahey 2001 and references therein). In contrast, our

results did not show any effect of this type of management on the accumulation rates of nitrate

and ammonia in soil. This effect may be explained by the fact that fertilizing treatment was

insufficient to increase soil nitrogen (this treatment did not significantly increase nitrogen

content in soil, nor did it improve C:N ratio). Thus, even when nitrogen is sufficient to maintain

mineralization, the process is highly energy-demanding and nitrogen accumulation rates remain

low. A small increase in soil nitrogen increased microbial diversity and its associated

relationships with plants. This results in an increase in nitrogen fixation that also reduced

nitrogen accumulation in soil (Ollivier et al. 2011).

In summary, although the different types of management applied in the present study

partially affected microbial diversity and composition or soil processes, we did not find a clear

link between disturbance and changes in microbial community structure and soil processes

functionality. Previous studies showed a weak or non–existing relationship between changes in

structure and function suggesting that this relationship is context-dependent and highly


158

conditioned by the process considered (Berga et al. 2012; Patra et al. 2008). In our case, mowing

affected litter decomposition without affecting microbial diversity or composition. One possible

explanation for this result could be related to the existence of a high level of functional plasticity

of roadside microbial communities. This would concur with Agrawal`s finding (Agrawal 2001),

who pointed out that changes in ecosystem functionality were not coupled with changes in

community structure. Regarding nitrogen dynamics, fertilizing affected both soil community

diversity and composition without any effects on litter degradation or nutrient availability, which

may indicate that for this novel ecosystem and management approach, the roadside microbial

communities are functionally redundant.

Conclusions and implications for practice

Roadside management today involves the application of conventional techniques such as

mowing or fertilizing. However, to date, the consequences of these standard approaches have not

been evaluated. Our results highlight that the use of these management techniques change

microbial community structure and function at a local scale. Moreover, the way in which

microbial communities from road embankments respond to different types of disturbance,

suggest a certain level of fragility, which in turn may affect ecosystem functionality and the

goods and services that these novel scenarios provide. This is not trivial if we account for the

role that roadslopes play as reservoirs of local biodiversity in severely disturbed landscapes

(Forman & Alexander 1998) or as important patches of habitat that influence connectivity among

populations under a stepping-stone landscape architecture (Kimura & Weiss 1964). Because of

the relevance of roadside ecosystems, there is a need for reviewing current technical approaches

Capítulo IV

159

in order to optimize roadside restoration with the objective of ensuring the maintenance of

ecosystem services.

Acknowledgements

We thank A.S. Pijl (NIOO–KNAW) for assistance with lab and molecular work and the research

group of Evolutionary Ecology and Ecological Restoration (UCM) for field and lab support, and

especially J.M. Arenas. We also thank Sarah Young for English editing. This study was funded

by OHL, the Spanish Ministry of Economy and Competitiveness (ECONECT project: CDTI

IDI–20120317), Madrid Regional Government (REMEDINAL–2 S–2009/AMB–1783), and by

an FPU grant program of the Spanish Ministry of Education, Culture and Sports (FPU–AP2009–

0094).


160

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s B

pb3

Capítulo IV

163

Apendix 1. Subset of orders used to analyze changes in soil bacteria community structure, diversity and

composition. (4 of 4)

Classification ID

Phylum Proteobacteria

Cl.Betaproteobacteria

O. Rhodocyclales Bpb4

Cl. Deltaproteobacteria

O. Bdellovibrionales Dpb1

O. Desulfobacterales Dpb2

O. Desulfurellales Dpb3

O. Desulfuromonadales Dpb4

O. Myxococcales Dpb5

Cl. Gammaproteobacteria

O. Acidithiobacillales Gpb1

O. Alteromonadales Gpb2

O. Enterobacteriales Gpb3

O. Legionellales Gpb4

O. Methylococcales Gpb5

O. Oceanospirillales Gpb6

O. Pseudomonadales Gpb7

O. Xanthomonadales Gpb8

Phylum Verrucomicrobia

Cl. Opitutae

O. Opitutales Ver1

O. Other Ver2

Cl. Spartobacteria

O. Chthoniobacterales Ver3

Cl. Verrucomicrobiae

O. Verrucomicrobiales Ver4


164

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175

CAPÍTULO V

Community ontogeny at the roadside: Critical life‐cycle events throughout a sequential process of primary colonization

S. Magro, M.D. Jiménez, M.A. Casado, I. Mola, J.M. Arenas, J.F. Martín-Duque & L. Balaguer

Applied Vegetation Science (2014), 17 (3): 493- 503.

Capítulo V

177

Abstract

Questions: How does the response to environmental filters change across the life-cycle of

pioneer plants through the early process of community assembly? Is there a threshold at any of

the life-history stages during roadcut primary colonization?

Location: A very steep, sun-exposed, low-fertility and low-water-retention roadcut in a

Mediterranean-continental site in Madrid, central Spain.

Methods: We tracked density of individuals, plant cover, species richness and community

composition throughout the sequential process of primary colonization of a newly-exposed

roadcut surface. We monitored from seed arrival to seedling emergence, seedling survival, and

plant growth across species over two growing seasons. We manipulated the intensity of

environmental filters in twelve experimental plots (10 x 8 m) following a full-factorial design of

two treatments (topsoil spreading and shallow tillage).

Results: The response to environmental filter manipulation varied throughout the individual life-

cycle. Under an equal seed rain, the higher carrying capacity caused by topsoil spreading gave

rise to the emergence of a larger number of species which either persisted or occasionally

appeared in some of the stages of the early community assembly. Then, topsoil spreading

enhanced seedling survival across species, and subsequent plant growth. We therefore detected

two life-history stages acting as thresholds in plant community assembly, due to an ontogenetic

niche shift across species. The first one, at seedling emergence, in response to environmental

cues with lasting consequences in community composition and species richness; and the second

one, at the transition to the adult stage, in response to local resource availability, with

consequences in subsequent plant growth and community cover.


178

Conclusions: During primary colonization, ontogenetic development of pioneers was paralleled

by the action of environmental filters throughout the community assembly process. On roadcuts,

the confluence of both processes gives rise to a community ontogeny marked by two thresholds

determining community richness and cover under Mediterranean conditions. Our findings shed

light on the underlying mechanisms involved in technical solutions, such as topsoil spreading,

and provide a more efficient approach to roadside restoration.

Keywords: ecological restoration; community assembly; environmental filters; life history;

primary succession; threshold points.

Capítulo V

179

Introduction

Primary colonization entails propagule arrival plus the concurrent struggle for survival

throughout pioneers’ life history. On barren surfaces, where a severe disturbance has removed all

vestiges of biological activity, community assembly “starts from scratch” and colonists build up

an initial soil seed bank (cf. Walker & del Moral 2003). Primary colonization is therefore a

sequential process in which stochastic arrival of seeds from the regional species pool is followed

by the development of an ontogenetic sequence whose pace varies across species (see for

instance Walker et al. 1986). From an assembly perspective, the emergent community is shaped

by environmental filters in which both biotic and abiotic factors are intricately linked

(Temperton 2007). In this highly selective process, each life-cycle stage becomes precarious

(Elmarsdottir et al. 2003; Haper 1977). In an attempt to overcome this precariousness, each life-

cycle stage displays a range of responses to the environmental filters. For instance, seedling

emergence may potentially range from negative density dependence (i.e. a decrease in

performance with increasing density), to positive density dependence, or density independence

(Lortie & Turkington 2002 and references therein). At each life-cycle stage, differential

responses of species become accentuated by the hostile environments of early primary

succession (Walker & del Moral 2008). Within a single species, the action of these filters also

changes in magnitude and sign over an individual’s life cycle (Goldberg et al. 2001; Luzuriaga &

Escudero 2008). This phenomenon, known as ontogenetic niche shift, may involve either species

niche contraction or expansion over the individual’s life cycle (Eriksson 2002). It may also

consist of an uncoupling of environmental requirements, or of a change in the biotic interaction

sign among life stages (Eriksson 2002; Miriti 2006). This population-based approach, however,


180

neglects the community-level factors shaping community structure through their effects on

parameters such as birth or death rates (Lortie & Turkington 2002).

At the community scale, it seems reasonable to expect that the overall effect of

environmental filters will give rise to life-history stages that are critical because they act as

thresholds (sensu Suding et al. 2004), which hamper the community assembly process, but which

once crossed, hinder the return to previous states (Hobbs & Norton 2004). During the early

stages of community ontogeny, the response of any of these life-stages to subtle variations in

environmental conditions would eventually lead to large changes in community cover, richness

and/or composition. If so, this would highlight the importance of considering life history traits

and performance for planning restoration programs, as previously recommended (Kleyer et al.

2008; Knevel et al. 2003; Walker & del Moral 2008).

In our study, we explored the occurrence of ontogenetic threshold points during the initial

stages of primary colonization of a roadcut and their implications for ecological restoration. It

has been reported that plant establishment on recently exposed roadcuts follows a process of

primary succession (Jiménez et al. 2013; Mola et al. 2011). Mass earthwork and grading in road

construction deeply transform the geomorphologic, hydrologic, edaphic, and biotic conditions at

the roadsides (Steinfeld et al. 2007). On roadcuts, all the biological structures that could act as

local foci for vegetation development have been lost, and plant colonization and early

community assembly are shaped by the environmental filters resulting from habitat carrying

capacity or microsite limitations (de la Riva et al. 2011; Valladares et al. 2008). Roadcut

environmental harshness arises from slope steepness, soil compactness, and sun exposure

(Bochet & Garcia-Fayos 2004), which in turn result in nutrient shortage and low water capacity

and retention (Jiménez et al. 2013).

Capítulo V

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In this scenario, we wanted to ascertain to what extent the across-species response to

environmental filters during the primary colonization process affects the density and species

richness of the plant community. Specifically, we studied the response of soil seed bank,

seedling emergence, seedling survival, vegetation cover (during the first and the second growing

season) and seed rain, in order to elucidate the coupling between individual life cycle across

populations and plant community early assembly. To this end, we experimentally manipulated

environmental harshness by varying resource levels and recruitment conditions, increasing the

carrying capacity of the system. In an attempt to enhance applicability, these experimental

manipulations were implemented and grounded in an actual road construction process. Thus, we

applied two different treatments (topsoil spreading and shallow tillage), at the early stages of

community assembly on the roadcut. We address three questions: (1) Does the response to

environmental filters change across life-history stages? (2) Is there a threshold at any of the life-

history stages throughout the early process of community assembly? If so, to what extent do

these thresholds affect the abundance, richness, and cover of the emerging community? and (3)

what are the implications for the current revegetation practices on roadcuts?

Material & methods

Study area

The present study was conducted in Torres de la Alameda, Madrid, Central Spain (3° 21'

47.43" W; 40° 24' 22.37" N) from 2009 to 2011. One roadcut was selected on the M-224

highway which was constructed in 2008. It is oriented southwards, and is 150 m wide and

approximately 10 m high, with an average slope exceeding 40°. The climate is Mediterranean


182

with a mean annual temperature of 15.23 ±0.32 °C and total annual precipitation of 340.52

±42.15 mm, recorded during the study period. These values were within the range for the last

two decades (12-16 °C; 200-750 mm, Alcalá de Henares weather station). The relief in the

vicinity of the roadcut was soft, comprising mainly moorlands. Lithological composition consists

of lacustrine clay and fluvial arkosic sands of the Tertiary age (ITGE 1990). Soils at the study

location are clayey and covered with therophytic grasslands, unirrigated croplands with pulses

and plantations of scattered olive trees.

Experimental design

The experimental design was intended to manipulate abiotic filters in order to analyze the

response at different life-history stages. After roadcut construction, in November 2009, two

treatments with two levels each were applied following a full factorial design, which was

replicated three times, giving a total of 12 (4 x 3) experimental plots, grouped following a

spatially-balanced complete block design (van Es et al. 2007). Variables related to plant

community development across life stages were measured in May 2010 and May 2011.

Treatments were applied in consecutive 10 x 8 m plots, and consisted of topsoil spreading (TS:

topsoil spreading; NTS: no topsoil spreading) and a shallow preparation of the roadcut surface

(T: shallow tillage; NT: not tillaged). Topsoil spreading (sensu Rivera et al. 2012) is a common

practice only in embankment restoration because it is assumed that topsoil application on

roadcuts with a slope steepness over 34º increases sediment delivery to the road drainage system

(Ramos 1974; Segura 2002). However, application thereof at our study site was intended to

improve substrate characteristics in terms of nutrient content, texture and water retention, while

shallow tillage aimed at enhancing roughness and safesite availability on the roadcut surface.

Capítulo V

183

Soil properties

After treatment application, six 10 cm deep cores with a diameter of 5 cm were collected

on each experimental plot. To cover the slope gradient, two cores were extracted in the upper-

slope zone, two in the middle one and two in the lower-slope zone. In each sample, we measured

the following set of soil properties: granulometry, calculated as the percentage of sand and silt in

soil (Bouyoucos 1962); organic carbon content using the Walkley-Black procedure (Nelson &

Sommers 1982) then converted to soil organic matter (SOM) by means of the van Bemmelen

factor (Jackson 1958); total nitrogen by means of Kjeldahl and mineral phases of nitrogen

(nitrate and ammonium) with a solid soil sample analyzer SKALAR. Finally, we measured the

pH of each soil sample by means of an ion-selective electrode (Metrohm Ltd., Herisau,

Switzerland), and soil electrical conductivity with a conductivity-meter (CRISOM, model CM

35).

Seed bank sampling

To determine the influence of abiotic filter manipulations on the soil seed bank, we

collected three samples from each experimental plot. The sample dimensions were 12.5 cm x

12.5 cm x 5 cm and were distributed along the slope gradient described above. Samples were

preserved at 4° C until processed. They were spread on aluminum tubs on a mixture of peat and

vermiculite (3:1). The tubs were placed in a germination chamber at 20 °C and 15% RH, with a

12-hour photoperiod during six months. The tubs were well-watered and maintained at field

capacity and rotated in order to avoid the effects of position. We stopped watering tubs from July

2010 to September 2010 to ensure germination conditions for the whole species pool. Finally, all


184

tubs were placed in a greenhouse and watered again. Viable seed density (viable seeds /m2) and

species richness were recorded in each sample.

Field monitoring of seedling emergence and survival

Seedling emergence was recorded in the lower-slope zone of the roadcut by means of

series of three photographs corresponding to an area measuring 0.25 m2 each. Photographs were

taken 2.15 m, 4.25 m and 6.75 m away from the left margin of each experimental plot. We

repeated each series of photographs monthly from April to September 2010. On each

photograph, seedlings were counted, identified at species level when possible and were marked

using Adobe Photoshop Elements (Adobe Systems 2002, San Jose, California, USA). We

determined the phenological stage of individuals through time as well as the density of seedlings

and number of emerged species per photograph. The plant life-cycle stages considered were:

emergence (only with cotyledons), seedling (true leafs), adult (with flowers or fruits), or dead

plants. With the number of emerged seedlings that became adults, we calculated a survival rate

per individual. Likewise, with the number of species that reached the adult state (surviving

species), we constructed a survival rate per species.

Vegetation sampling

Plant cover by species and species richness was visually estimated by the same observers

at the end of the vegetative period (May) both in 2010 and 2011. We used six quadrats (50 x 50

cm) evenly distributed in each experimental plot: 2 quadrats at upper-slope zone, 2 quadrats at

middle-slope zone and 2 quadrats at the lower-slope zone. Each quadrat was placed 1 m away

Capítulo V

185

from the right and left margin of each experimental plot to avoid border effects and also to allow

the observers to move freely along the slope without affecting treatment application.

Seed rain sampling

To determine seed rain input, two funnel traps were placed in each experimental plot in

May 2010. We used funnel traps with 0.38 m2

of effective capture area per trap (22 cm in

diameter). Traps were placed in the upper- and lower-slope zones of the roadcut. Trapped seeds

were collected every two months until May 2011. Seeds were classified within a morphotype

defined by size and dispersal/morphological attributes.

Data analysis

General Linear Models (GLM) were performed to test the effect of filter manipulation on

soil carbon content and soil organic matter (SOM), silt content, nitrate, ph and conductivity as

response variables, including topsoil and shallow tillage as fixed factors and topsoil*shallow

tillage as interaction term. The effect of treatments upon total nitrogen, sand and ammonium soil

content was analyzed by Kruskal Wallis test. GLM were also performed for seedling density and

species richness during emergence, and survival rate per individual and survival rate per species

as response variables, including topsoil and tillage as fixed factors and topsoil*tillage as the

interaction term. Seedling density and survival rate per individual were square-root transformed.

Variation in response to environmental filter manipulation of seedbank, seed rain, and plant

cover in 2010 and 2011 was also tested by means of GLM where treatment (topsoil spreading

and shallow tillage) and slope position (upper-, middle- and lower-slope zones) were included as

fixed factors following Zuur et al. (2009) and position*topsoil, position*tillage and


186

position*topsoil*tillage were included as interaction terms. Plant cover from 2010 and 2011

were also square-root transformed. Pairwise comparisons were performed applying HSD Tukey

test, and sum of squares Type III were used for all the F-statistics. All analyses were performed

with R (R Development Core Team, 2012), with the additional packages “car” (Fox & Weisberg

2011), “sciplot” (Morales 2011) and “multcompView” (Graves et al. 2012).

Results

Experimental manipulation of environmental filters

Topsoil spreading significantly changed soil nutrient content (Kruskal Wallis test H Total

Nitrogen [1,71]=17.430, p < 0.05; F Organic Carbon [1,67]= 8.81, p < 0.01; F SOM= [1,67]= 8.81, p < 0.01;

Kruskal Wallis test H Ammonium [1,71]= 17.238, p < 0.001). It increased total nitrogen content by

over 50%, organic carbon by over 30% and SOM by 35%, and reduced ammonium by around

8%, in comparison with controls. Furthermore, topsoil spreading significantly modified soil

texture, enhancing silt content by 6% (F [1,67]= 8.29, p < 0.01), and marginally increased sand

content by 8% (Kruskal Wallis test H[1,71]= 3.558, p=0.059). Finally, topsoil spreading

significantly reduced other chemical soil conditions such as pH and electrical conductivity (F pH

[1,67]= 24.80, p < 0.001; F Conductivity [1,67]= 14.937, p < 0.001) by 2% and 17%, respectively.

Nitrate content in soil was not affected by treatments (F [1,67] = 0.01, p > 0,05). Shallow tillage

and its interaction with topsoil spreading did not generate significant modifications (p>0.05) in

any soil factors compared to the original substrate (Table 1).

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187

Table 1. Means (± SE) of soil factors significantly affected by treatments (C=control; T= Tillage; TS=Topsoil).

Letters by columns show significant differences between levels of treatments after HSD Tuckey test. (*) Letters in

this case, showed differences between groups after Kruskal-Wallis test. No significant interactions between

treatments were found

.

Soil seed bank

The soil seed bank showed an average density of 46 viable seeds/m2 from nine species

(Fig. 1). GLM results showed that neither seed-bank density (F position[2,36]= 0.69, p > 0.05; F Topsoil

[1,36]=0.72, p > 0.05; F Tillage[1, 36]= 0.20, p > 0.05) nor seed-bank richness (Fposition [2,36]= 0.86, p >

0.05; FTopsoil [1,36]=0.43, p > 0.05; F Tillage [1, 36]= 0.43, p > 0.05.) were affected by treatments or

slope position. In all cases, interactions were not significant (p>0.05).

Field monitoring of seedling emergence and survival

During the study period, 840 individuals from 22 species were monitored at the roadcut

(Fig. 1). At the population and community level, seedling emergence and mortality co-occurred

during this period, although there is a higher incorporation of individuals at the beginning of the

growing season, while mortality increased towards the end of the summer (Fig. 2a).

Silt content Total Nitrogen * Organic carbon

Soil organic

matter Ammonium * pH Conductivity

% % % % % µs

C 33.617 ± 0.858 b 0.026 ± 0.002 b

0.309 ± 0.033 b 0.533 ± 0.057 b

3.399 ± 0.050 a 8.542 ± 0.064 a

110.950 ± 4.943 a

T 33.292 ± 0.742 b 0.030 ± 0.002 b

0.228 ± 0.029 b 0.393 ± 0.049 b

3.352 ± 0.051 a 8.599 ± 0.027 a

114.789 ± 3.850 a

TS 35.482 ± 0.632 a 0.040 ± 0.003 a

0.363 ± 0.032 a 0.626 ± 0.056 a

3.214 ± 0.030 b 8.349 ± 0.047 b

93.256 ± 3.895 b

T*TS 35.714 ± 0.723 a 0.045 ± 0.004 a

0.356 ± 0.027 a 0.614 ± 0.047 b

2.975 ± 0.188 b 8.316 ± 0.046 b

93.924 ± 6.893 b

1

T*TS


188

Figure 1. Species composition across community ontogeny process (SB= Seed Bank; SE= Seedling Emergence; SS

= Seedling Survival; PC10 = Plant Cover in 2010; PC11= Plant Cover in 2011). Established species included all

species found in consecutive life-stages while occasional ones are those that appeared by turns across community

life history. Topsoil spreading treatment included experimental plots TS-T (topsoil spreading- shallow tillage) and

TS-NT (topsoil spreading- not tillaged), and No topsoil spreading treatment, NTS- T (no topsoil spreading- shallow

tillage) and NTS-NT (no topsoil spreading- not tillaged). Species not identified at least at genus level, are not shown.

The effect of shallow tillage was omitted because community composition of TS-T plots (topsoil spreading- shallow

tillage) and TS-NT plots (topsoil spreading – not tillaged) overlapped 52.5 % and of NTS-T plots (no topsoil

spreading- shallow tillage) and NTS- NT plots (no topsoil spreading- not tillaged) overlapped 40%. (*) Epilobium

brachycarpum is the only non native species that appeared at the study site during the study period.

Species SB SE SS PC10 PC11 Species SB SE SS PC10 PC11

Lolium rigidum Bassia scoparia

Papaver rhoeas Papaver rhoeas

Anagallis arvensis Lolium rigidum

Atriplex patula Polygonum aviculare

Avena barbata Aegilops triuncialis

Polygonum aviculare Avena barbata

Salsola kali Bromus diandrus

Bromus diandrus Bromus madritensis

Convolvulus arvensis Carduus pycnocephalus

Fumaria cf. parviflora Carthamus lanatus

Lactuca serriola Chrozophora tinctoria

Vaccaria hispanica Diplotaxis erucoides

Asterolinon linum-stellatum Lactuca serriola

Bromus rubens Vaccaria hispanica

Coronilla scorpioides

Epilobium brachycarpum*

Euphorbia serrata

Rapistrum rugosum

Scabiosa stellata

Sinapis arvensis

Sonchus asper

Tragopogon porrifolius

Amaranthus albus Anagallis arvensis

Avena sterilis Atriplex patula

Bassia scoparia Convolvulus arvensis

Biscutella auriculata Hordeum murinum

Brassica barrelieri Salsola kali

Bromus madritensis Veronica hederifolia

Bromus sp.

Bromus sterilis

Capsella bursa-pastoris

Carthamus lanatus

Cerastium cf. pumilum

Chrozophora tinctoria

Diplotaxis erucoides

Eruca vesicaria

Galium tricornutum

Kickxia lanigera

Papaver argemone

Veronica hederifolia

Topsoil spreading Not Topsoil spreading

Est

ab

lish

ed s

pec

ies

Occ

asi

on

al

spec

ies

Capítulo V

189

Figure 2. Temporal patterns of seedling emergence and survival over the first growing season (2010). (a) Total

density of individuals across treatments and (b) Survival rates on topsoiled vs. no topsoiled plots. Trend lines with

R2 greater than 96% are shown.

Topsoil spreading, switched the survival temporal pattern from convex to concave (Fig.

2b). GLM results showed that topsoil spreading significantly increased the number of emerged

species (F [1,32]= 8.76; p Topsoil < 0.01, Fig. 3b). However, seedling density was not affected by

filter manipulation (Table 2, Fig. 3a). Topsoil spreading also increased the number of individuals

that reached the adult state (F [1,31]= 11.829; p Topsoil <0.01, Fig 3c ) but did not affect community

composition (Fig. 1), or survival rate per species (Table 2, Fig. 3d).

Table 2. GLM results showing the effects of treatments (TS= topsoil spreading; T= tillage) on seedling density

(Seedling/m2); emerged species (number of species/photograph); survival rate per individual (SRI*) (adult

plants/m2) and survival rate per species (SRS*) (surviving species/photograph).

Seedling density Seedling richness SRI* SRS*

nº seedling/m

2

nº

species/photo adult plants/m

2

surviving

species/photo

Treat. df F p df F p df F p df F p

TS 1 0.499 0.485 1 8.758 0.006 1 11.829 0.002 1 2.386 0.132

T 1 0.044 0.835 1 0.757 0.391 1 0.252 0.619 1 1.656 0.208

TS*T 1 0.504 0.483 1 0.015 0.903 1 0.045 0.833 1 0.202 0.656

Resid.

3

2

3

2

3

1 31

0

10

20

30

40

50

60

70

80

90

100

April May June July September

Su

rviv

al

rate

No Topsoil Spreading Topsoil Spreading

0

500

1000

1500

2000

2500

3000

3500

April May June July September

Den

sity

(In

idiv

idu

als

/m2

)Living plants Dead plants a b


190

Figure 3. Means (± SE) of (a) Seedling density, (b) Survival rate per individual (number of adults/ total number of

individuals per m2) (c) Seedling richness and (d) Survival rate per species (number of species that reached the adult

stage/ total number of species found per photograph), under the effects of the treatments ( NTS-NT: no topsoil

spreading - not tillaged; NTS-T: no topsoil spreading- shallow tillage; TS-NT: topsoil spreading - not tillaged; and

TS-T: topsoil spreading combined with shallow tillage ). Seedling density and survival rate per individual were root

transformed in order to achieve normality and homocedasticity. Letters show significant differences (p < 0.05) after

HSD Tukey test.

Vegetation cover, composition and richness

Percentage of plant cover in 2010 varied among treatments and along the slope gradient,

reaching maximum values with topsoil spreading in the lower-slope zone of the roadcut (Table 3,

Fig. 4a). In 2011, differences in plant cover among treatments decreased and the interaction

between topsoil spreading and position interaction disappeared. However, plant cover during this

growing season was also significantly increased in the lower-slope zone and under the effect of

topsoil spreading and tillage separately (Table 3). The number of species increased over the

Capítulo V

191

years (Fig. 1). Plant species richness was higher in the lower-slope zone, specifically in the

topsoiled plots during 2010 (Table 3, Fig. 4b), and influenced by topsoil spreading, lower

position and the interaction between slope position and tillage during 2011 (Table 3, Fig. 5).

Table 3. GLM results showing the effect of treatments (TS=topsoil spreading; T= tillage), the slope gradient (P=

position) and their interactions, on plant cover 2010-2011 (% total plant cover/quadrat), and on species richness

(species/quadrat).

Figure 4. Effect of the interaction of topsoil spreading and slope position on (a) percentage of plant cover sampled

in 2010 and (b) species richness in 2010. TS: topsoil spreading; NTS: no topsoil spreading. Figures showed mean

values grouping shallow tillage treatments because of the null effect of this treatment or its interaction with topsoil

spreading. Vertical bars are ± 1 SE.

Plant cover 2010 Species richness 2010 Plant cover 2011 Species richness 2011

% total plant

cover/quadrat species/quadrat

%total plant

cover/quadrat species/quadrat

Factors df F P df F p df F p df F p

TS 1 4.242 0.044 1 2.085 0.154 1 5.377 0.024 1 6.238 0.015

T 1 0.959 0.331 1 0.927 0.340 1 5.820 0.019 1 3.355 0.072

P 2 7.940 0.000 2 26.525 0.000 2 4.231 0.019 2 11.442 0.000

TS*T 1 0.656 0.421 1 0.724 0.398 1 3.380 0.071 1 1.996 0.163

P*TS 2 5142 0.009 2 14.073 0.001 2 0.777 0.464 2 0.194 0.824

P*T 2 1.203 0.307 2 0.135 0.874 2 2.341 0.105 2 4.057 0.022

P*T*TS 2 0.618 0.543 2 0.087 0.917 2 1.581 0.214 2 1.308 0.278

Residuals 60 60 60 60

1


192

Figure 5. Effect of the interaction of shallow tillage treatment (T: shallow tillage, NT: not tillaged) and slope

position on species richness during 2011. Vertical bars are ± 1 SE.

Seed rain

From July 2010 to May 2011, a total of 144 seed rain traps was analyzed. Therein, we

recorded an average of 5132 seeds/m2

from 42 different morphotypes. We did not find

differences in seed rain density between treatments and slope position (p>0.05). However, seed

rain richness significantly increased toward the lower-slope zone (F [1, 24] = 5.23; p position < 0.05,

Table 4).

Table 4. Means (±SE) seed rain density (seed/m2*year) and morphotype richness (morphotypes/trap). GLM results

indicated a significant effect of slope position (F [1, 24] = 5.23; p position < 0.05) on the number of seed morphotypes

found in the funnel traps.

Slope position Seed rain density Number of morphotypes

seed/m2*year morphotypes/trap

Upper-slope zone 6818.0 ± 1561.145 7.5 ± 0.744

Lower-slope zone 3447.4 ± 972.460 10.1 ± 0.967

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193

Discussion

Our results highlight the relevance of colonization and early community assembly in the

development of practical guidelines for roadside restoration from an applied ecology perspective.

In this scenario, the present study is the first, to our knowledge, to address at the community

level the link between plant ontogenetic progression and the early assembly sequence, critical in

the context of primary succession. Our results showed that the response to microsite limitations

changes throughout community life history, and that this phenomenon results in a colonization

process mediated by response thresholds with pivotal effects on the composition, richness and

cover of the eventual plant community.

Changes in the response to environmental filters throughout life history

Viable seed persistence in topsoil stockpiling is affected by burial depth and diurnal

fluctuations in light and temperature (Rivera et al. 2012; Rokich et al. 2000). After treatment

onset, topsoil spreading and shallow tillage, however, did not affect roadcut seed bank density or

richness, likely due to the lack of differences in seed rain and to the extremely poor seed bank

provided by the topsoil, in consonance with previous reports (Mola et al. 2011). Conversely,

toposoil-mediated changes in soil nutrient availability, soil texture, electrical conductivity, and

pH significantly affected the number of emerged species. That is to say, topsoil enlarged the

recruitment window of a greater number of pioneer species. These pioneers either persisted or

occasionally appeared in some of the stages of the early community assembly. This eventuality

may be a sampling artifact, or more likely reflects that successful colonization is often the result

of repeated arrival events under a strong environmental control (Ejrnæs et al. 2006). The

manipulation of environmental filters as a result of topsoil spreading also increased seedling


194

survival and growth but did not discriminate among species. Across experimental plots, the

sustained effect of filter manipulation on emergence and survival led to a cumulative increase

both in plant abundance and species richness over the years. This is probably due to the typical

low levels of plant cover observed at the early stages of community development on roadcuts.

Even at the lower slope zone, where maximal plant cover values were recorded, they remained

below 50%, and the increase in plant cover along the slope gradient was associated with a

parallel increase in species richness. In Mediterranean grasslands, a unimodal relationship has

been reported between these variables, in which maximum richness is reached at values close to

60% of herbaceous cover (Casado et al. 2004). On road embankments, it has been observed that,

where topsoil spreading provided greater soil resource availability, plant cover exceeded 60%,

which triggered competitive exclusion with drastic effects on species richness (de la Riva et al.

2011). In the light of these previous reports, our results show that the shift of interaction

dynamics does not take place as plants move from seedling to adult life stages, as described

elsewhere (Howard & Goldberg 2001), but rather as plant community structure shifts from

scattered pioneers to densely vegetated grasslands. The absence of strong biotic interactions is

also in consonance with the apparent lack of historical or priority effects (sensu Chase 2003).

The high proportion of species shared between treatments suggest that early community

assembly of roadcut vegetation is controlled by environmental filters rather than by the arrival

order of species, as previously reported in grasslands (Ejrnæs et al. 2006).

Thresholds in early stages of plant community assembly

Our results show that not all life-history stages represent a threshold in the community

assembly process on roadcuts. Manipulation of environmental filters triggered two threshold

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responses. First, at seedling emergence stage, changes in ecosystem carrying capacity due to

topsoil spreading led to significant and sustained changes in community richness and

composition. Seedling emergence has previously been described as a crucial process during

primary colonization on roadcuts (Bochet et al. 2007). Our results showed that beyond its

implications in terms of density of individuals, this stage represents a threshold for the species

composition and richness of the emerging community. This threshold arises because seedling

emergence on roadcuts is a density independent process at the community level, largely

unaffected by neighbors, as described elsewhere (Howard & Goldberg 2001). In primary

succession, species interference is limited to interactions between plants at the same life-history

stage. In that sense, Soliveres and coworkers (2012) have recently highlighted that competition

for resources during the emergence of pioneer seedlings is lower than the observed at subsequent

successional stages, which likely explains the higher number of species found under the topsoil

conditions. Although this neighbor effect seems be consistent across life-history stages

(Goldberg et al. 2001; Howard & Goldberg 2001), our field results suggest variation in density

response to environmental conditions between seedling emergence and survival.

The second threshold that we detected during the early community assembly was related

to the transition from seedling to adult stage. The observed overlapping between seedling

emergence and survival suggest that the described threshold has not a temporal but an

ontogenetic meaning. In other words, it does not occur abruptly in time but sequentially through

the plant community development. This transition seems to occur earlier under the effect of

topsoil spreading (shift from convex to concave temporal patterns). When the emergent

community crosses the threshold of seedling survival under suitable conditions for plant growth,

a sudden shift in community structure occurs, which will become apparent in terms of plant


196

cover. At the individual level, this threshold change represents the transition from the juvenile to

reproductive phases of most herbaceous species, which will ultimately enable community

persistence during the following years. At the community level, this threshold response is

accompanied by an ontogenetic niche shift across community species, in which seedlings

respond to windows of opportunity in temperature, light and water pulses (Bazzaz 1979), while

habitat suitability for adult plants depends on resource availability such as soil stability, or

nutrient and water (Erikson & Erikson 1998). At the ecosystem level, crossing this threshold

likely leads to biological feedbacks, such as enhanced litterfall or new habitats for colonizing

species, and to physical feedbacks, such as the effects of the larger vegetation patches on the

capacity to retain rainfall and foster water infiltration (Tongway & Ludwig 2010).

Implications for practice

Current guidelines advise against topsoil spreading on roadcuts, given their average

steepness of over 34º and the consequent risk of sediment delivery to the road drainage system

(Ramos 1974; Segura 2002). Although questioned by experimental evidence (Mola et al. 2011),

this recommendation has been widely followed in the context of construction in Spain. Our

findings show, however, that when the abiotic filters in this scenario are not modified by topsoil

spreading, the seedling emergence threshold can hardly be overcome and the pioneer community

will invariably be limited to very few species at most. Under these circumstances, the emerging

community becomes highly vulnerable in the face of the subsequent survival/growth threshold.

Failure to overcome this second critical threshold leads to a strong dependence on the arrival of

propagules of a limited number of species. Consequently, our results indicate that a roadcut

under harsh Mediterranean conditions likely remains as a ‘sink’ habitat (sensu Pulliam 1988),

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that serves as a net importer of individuals. Conversely, topsoil spreading provided suitable

conditions for the establishment of a self-sustained richer plant community that may act over

seasons as a source of propagules from a larger number of species. This process is expected to be

particularly active on the bottom half of the slope, where the filter manipulation effect was found

to be more effective. Based on our results, we recommend shallow tillage to extend this effect to

topsoil pockets higher up on the slope. In conclusion, we suggest topsoil spreading on those

roadcut segments where a longer distance between the slope and the drainage system reduces the

risk of sediment deposition in the road ditch. This practice would give rise to a mosaic of sink

and source vegetation patches with functional advantages. First, it would minimize seed dispersal

distance, which is critical at the roadside as seed rain density decreases as a function of distance

from the parent plants (Bochet et al. 2007; Mola et al. 2011). Second, the presence of propagule

sources on the roadcut itself reduces the dependence on remnant vegetation patches located

outside the road boundaries and subject to uncertain disturbances. Finally, it limits the topsoil use

to scattered patches, optimizing the allocation of this scarce resource.

Conclusion

In primary succession, the interplay between seedling colonization, plant ontogeny, and

early community assembly results in a process that can be called ‘community ontogeny’. This

process involves a developmental shift in the perception of and response to local environmental

filters across individuals and species. Analysis of the mechanisms underlying community

ontogeny on roadcuts reveals two sequential thresholds that regulate species richness, and

community composition and cover. A better understanding of these mechanisms should prompt

the reappraisal of current road reclamation practices and help to implement an ecological


198

restoration approach, as well as promoting an alternative perspective based on processes such as

seed dispersal, plant recruitment or environmental filter manipulation, rather than on agronomic

principles, such as fertilization, species selection, or biomass yield.

Acknowledgements

We are indebted to Marga Costa, Ana Buades and Silvia Murillo for their field and lab support,

and to Mr. Cormac de Brun for revision of English. We also wish to thank OHL’s R&D

Department for their help and permission to conduct this research, and all the referees that highly

improve this manuscript with their comments and suggestions. Finally, we would like to thank

Rocío de Torre, Adrián G. Escribano Rocafort, Carlos Granado-Yela and Agustina Ventre

Lespiauq for further discussion of ideas during this process. Soil analyses were performed by

Nutrilab (URJC). This study was funded by OHL, the Spanish Ministry of Economy and

Competitiveness (ECONECT project: CDTI IDI-20120317), the Madrid Regional Government

(REMEDINAL-2 S-2009/AMB-1783), and by the FPU grant program of the Spanish Ministry of

Education, Culture and Sports (FPU-AP2009-0094). None of the co-authors have any conflict of

interest regarding to the information contained in the manuscript

Capítulo V

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CAPÍTULO VI

General discussion

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207

This Doctoral Thesis broadens our knowledge of the relatively unknown features of

roadside ecosystems that are of great relevance in guiding the science and practice of ecological

restoration efforts in this kind of setting. In this chapter, we discuss the importance of soil

variables in the organization of key soil communities such as microarthropods and

microorganisms. Moreover, we analyze how different types of management can affect soil

community structure and functionality as well as early phases of plant community assembly.

The soil system: variables affecting soil community development along the roadside

Soil is the basis of terrestrial ecosystem development due to the fact that most ecological

processes that give rise to regulation services such as nutrient cycling, water holding capacity

and carbon storage, occurs underground (de Groot et al. 2002). Despite its importance,

insufficient attention has been paid to this ecosystem compartment in the context of roadside

engineering and restoration. The recovery of functional soils is especially important in areas

affected by road construction where relief and hydrology been severely altered during the

construction process, and all vestiges of biological communities have been eliminated (Balaguer

2002). For this reason, roadside ecosystems must acquire a minimum carrying capacity to

support biological communities that, once established, trigger feedback processes in which these

communities can contribute to soil formation and stability (Odum 1969). Plant and soil

organisms live in an intimate relationship with the soil environment in which they increase soil

complexity through root development, microarthropod movements, bacterial growth etc., and

help create new conditions for other species with further synergistic effects on biodiversity and

soil functionality (Ettema & Wardle 2002). Thus, in order to maintain and restore soil processes


208

in roadslopes, the first step is to acquire and refine our knowledge of the soil characteristics

affecting the communities intimately involved in soil functionality.

Our results show that physicochemical soil variables determine the organization and

variation of edaphic communities (e.g., microarthropods and bacteria) in roadside ecosystems.

Road embankments are productive and –or should be- nutrient-rich ecosystems where the rapid

development of plant biomass is incorporated into the soil thereby generating organic matter

pulses that, in turn, trigger early edaphogenic processes (Jiménez et al. 2013). We detect that the

increase in soil organic matter facilitates the transition from more generalist/pioneer

microarthropod communities characterized by higher abundances of Gamasida and Actinedida,

to other taxa indicative of more stable states mainly dominated by Oribatid mites (Poronota and

Gymnonota) and Collembola (Symphypleona). This is in line with other studies that show

organic matter content as the best predictor for Oribatida and Collembola community structure

(Hasegawa 2001). Oribatid mites are litter feeders and are involved in the resizing of particulates

that facilitate the subsequent colonization of soil microorganisms, at the same time that they

contribute to fungi and bacteria dispersion (Norton 1990). Due to their specific ecological

requirements and their low mobility, Oribatid mites are highly dependent on stable soil

conditions and hence are commonly used as indicators of soil quality and health (Beham-

Pelletier 1999). Collembolans play an important role in soil organic matter decomposition and

humification through the degradation of recalcitrant compounds by means of the enzymatic

activity in their guts (Saur & Ponge, 1988). Collembolans also contribute to soil microstructure

and the development of topsoil layers through feces deposition and bioturbation processes. Some

species of Collembolans are geophages, whose activity enhances the formation of organo-

mineral complexes in soil (Wolters 2000 and references therein). Owing to the intimate

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relationship between Collembolans and soil conditions, it is expected that community

composition could change under the effect of disturbance. Specifically, Symphypleona are

commonly found in unaltered environments and thus are also good indicators of soil stability

(Badeano et al. 2006).

The incorporation of organic matter into the soil affects ion exchange and decreases soil pH

(Parfitt et al. 1995). However, in road embankments the effect of construction materials coupled

with the strong evapotranspiration processes characterizing Mediterranean environments, tend to

alkalinize the soil (Alfaya 2013). Our results show that soil pH is one of the principal factors

determining bacterial community composition in road embankments. The effect of soil pH on

bacterial community composition and diversity has been previously observed in different types

of soil across biomes and land uses (Fierer et al. 2012; Lauber et al. 2008). Soil pH directly

constrains bacterial physiology, impeding the growth of certain taxa and altering competitive

outcomes that, in turn, affect community composition and diversity (Lauber et al. 2009). More

specifically, previous work shows that some groups of Acidobacteria are negatively correlated

with soil pH, while other groups of the same phylum as well as Actinobacteria, tend to thrive

under higher pH levels (Rousk et al. 2010 and references therein), also in agreement with our

results. However, pH is known to act as an integrative variable that may hide the effect of many

other soil properties (Laubert et al. 2009). This too agrees with our findings revealing that other

variables related to soil texture, such as clay and sand content, are also relevant in the

organization of bacterial communities. Soil bacteria community composition varies with the

shape of soil particles and the type of soil aggregates (Ranjard & Richaume 2001). In this way,

higher nutrient contents associated with clay content increase bacterial diversity and may trigger

differences in composition, at the same time that the sand fraction may be linked to bacterial


210

species that are better adapted to nutrient poor conditions (Sessitsch et al. 2001). Bacterial

community development also interacts with soil particles through the production of extracellular

polysaccharides (Foster 1988), which contributes to aggregate soil particles and to the

development of soil microstructure. Soil heterogeneity and the microenvironments created by the

interaction of microorganisms and soil particles, affect soil structure in terms of porosity and

moisture (Porta et al. 1999, Tisdall &Oades, 1982). The habitat created by small but numerous

pores, leads to many microarthropod species dropping out or being excluded from the emergent

community. For example, and in general terms, Oribatida remains associated with the 6–90 m

pore size class (Vreeken-Buijs et al. 1998). In line with these findings, we determined that

porosity is responsible for changes in community structure, increasing the abundance of small-

sized Oribatids from the Opiidae family.

In light of our results, it is worth noting that some soil variables (mainly nutrient content, pH,

texture, and structure) distinctly change the composition and self-organization of soil

communities. Interestingly, these variables are subject to severe changes during the constructive

processes. For instance, either in road embankments (where foreign aggregate materials are used

during construction) or in roadcuts (where bedrock is exposed), some of these variables (i.e.,

texture and pH) may change drastically. In addition, conventional management techniques such

as the application of topsoil, mowing and organic/inorganic amendments not only have direct

effects on soil conditions but also boost changes in aboveground communities that subsequently

affect the soil environment, especially through the incorporation of soil organic matter. However,

the effect of these types of management on key soil communities is unknown in the context of

roadside restoration. Therefore, in this thesis we have tested the effects of some of these

Capítulo VI

211

treatments on the assembly of different biological communities as well as on relevant ecological

processes.

Managing management: roadside restoration and the maintenance of functionality

Ensuring restoration success it is essential to identify the ecological thresholds that are

constraining an ecosystem´s self-organization (Suding & Hobbs 2009), and then to select the

technique or techniques that facilitate ecosystem recovery with minimum cost and intervention.

The degree of impairment as well as the nature of thresholds that must be overcome, determines

in large part the restoration actions (King & Hobbs 2006). At the same time, the setting of goals

and objectives should be realistic, particularly in the early stages (Ehrenfeld 2000). For instance,

in highly degraded sites and ecosystems, such as roadcuts and road embankments, mine sites,

and elsewhere, where abiotic and biotic components of the local ecosystems have been

drastically altered, full recovery of ecosystem functionality and native biodiversity will be slow,

and will require significant inputs of resources. Thus, project managers should not expect to

achieve very ambitious goals at these sites in the short-term.

In some Mediterranean road embankments, both construction and management

techniques may facilitate plant colonization and establishment, giving rise to high biomass

production. In these scenarios, biotic thresholds derived from competitive exclusion processes

among different species can shape plant community development (De la Riva et al. 2011;

Valladares et al. 2008). Although in older embankments plant species seem to be the decisive

factor in roadside ecosystem organization (García-Palacios et al. 2010), during the earliest stages

of community assembly process the interaction between plant and microbial communities plays a

major role in roadside ecosystem functionality (García-Palacios et al. 2011). Conventional


212

management applied in Mediterranean roadslopes is oriented to the maintenance of plant

communities and does not consider their effects upon soil microbial communities. Some studies

reveal that restoration practices that do not take into account feedback processes among different

ecosystem compartments could give rise to unpredictable outcomes (Suding et al. 2004). Thus,

our results reveal that bacterial community structure as well as soil processes are highly sensitive

to the choice of management practices employed. In particular, fertilization favors community

evenness and alters bacterial community composition. These changes in bacterial community

structure under the application of fertilizers have been observed previously both in natural and

anthropogenic systems (Fierer et al. 2012; Nogales et al. 2010). This effect is related to the fact

that nitrogen addition shifts the metabolic capabilities of soil communities thereby changing

competitive relationships among them with subsequent effects in community composition (Fierer

et al. 2007; Ramírez et al. 2012). Moreover, we observed that biomass addition treatments

increase taxonomical diversity. This could be related to the buffering effect of litter accumulation

in microclimatic soil conditions in road embankments (De Torre 2014), in conjunction with the

fact that the litter layer protects the soil surface against rain drop impacts, preserving soil

microhabitats and increasing bacterial diversity (Pengthamkeerati et al. 2011). Our results also

showed that mowing treatments affect soil properties, thereby altering nutrient stocks and soil

texture, as well as soil processes, while also accelerating litter decay and other basic processes.

These effects may be related mainly to the changes induced in plant communities by mowing

practices. On the one hand, mowing increases niche availability for a large number of fast-

growing plant species that immobilize soil nitrogen (Rizand et al. 1989, Robson et al. 2007),

while on the other hand the relatively low vegetation cover induced by this treatment may favor

leaching processes, decreasing the proportion of nitrogen with further effects on the C:N ratio in

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213

the soil. Moreover, this lack of vegetation can also explain changes in soil texture under the

effect of this treatment. Bare soil exposure to rain events plus the common design of roadslopes

characterized by steep slopes, favor erosion processes that enhance the movement of finer soil

particles (clay) (Castillo et al. 1997), hence increasing the relative content of loam, as we

observed in our experiments. The effect of mowing on plant communities could also be

translated to compositional changes that affect the quantity and quality of organic matter

incorporated into soil with further effects on decomposition rates (Zhang et al. 2008). However,

another plausible explanation for the acceleration of the litter decay rate that we observed is the

lack of vegetation in mown plots, which increases the sun exposure of the soil surface to strong

sunlight, thereby promoting high rates of litter photodegradation. This process has been

described as one of the main factors related to litter decay in Mediterranean climates (Henry et

al. 2008).

Despite the importance of plant cover in the stabilization of roadslope substrates (Andres

& Jorb, 2000), roadside vegetation does not usually recover spontaneously within a time frame

appropriate to an infrastructure construction schedule (Mola et al. 2011). For this reason, some

restoration measures are taken to boost roadside vegetation recovery. Unfortunately, most efforts

in Mediterranean roadslopes have been focused on the development of the plant community

structure, leading to the waste of considerable amounts of money in hydroseeding, fertilizers, or

other plantings with poor results (Tena 2006). For instance, in roadcuts the steep slopes (often

higher that 40%), smooth and compacted surfaces and soils with lower water-holding capacity,

create stressful abiotic conditions and a scarcity of safe-sites that constrain ecosystem recovery

(Bochet & García Fayos 2004). Moreover, the effect of these abiotic filters may have effects

both at an individual species scale, by constraining the transition between different life stages


214

during early community development, and at the community scale by determining the attributes

of the final community. Our results reveal that topsoil spreading helps to overcome some of these

abiotic filters and favors the community assembly processes. Considering the individual life-

cycle of plants, topsoil facilitates the emergence of more species and the transition from seedling

to adult stages. This result may be related to the fact that topsoil induced changes in soil texture

and decreased soil conductivity, which in turn modified osmotic potential in the soil matrix

facilitating water intake by seedlings (Lamsal et al. 1999). This is in line with previous studies

(e.g., Bochet et al. 2007) that point to water availability as the main factor conditioning seed

germination and species segregation in Mediterranean roadslopes. In other words, two life-

history events (emergence and survival) acting as thresholds during community assembly and

with further consequences at community scale were overcome by the spreading of topsoil. This

treatment affects roadside vegetation giving rise to communities with higher species richness and

higher plant cover. Habitat suitability for adult plants mainly depends on resource availability

such as nutrient content and soil stability (Erikson 2002). Thus, the improvement of soil

properties by topsoil spreading, which enhances nitrogen and soil organic matter content,

facilitates an ontogenic niche shift and favors the establishment and growth of more species. On

the contrary, our results show that tillage, which was applied to increase roughness and safe-sites

for recruitment, does not improve recruitment conditions for pioneer plant communities. For

instance, far from enhancing soil heterogeneity, this treatment homogenizes the soil surface and

contributes to substrate instability. This effect may be related to lithological characteristics at the

study site. Substrates there are mainly composed of expansive clays exposed to ‘popcorn’

processes (J.F. Martin-Duque, comm. pers.) derived from cyclic rewetting and desiccation

processes, which create very unstable substrates that usually lead to small mass movements

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215

(Mokthari & Dehghani 2012), reducing plant colonization and establishment. However, a

slightly positive effect of tillage on species richness was observed in the upper part of the roadcut

when combined with topsoil spreading: tillage seems to have had the desirable effect of creating

topsoil pockets on steep slopes.

Implications for practice

Professionals undertaking ecological restoration in areas affected by road construction must

carefully analyze the key ecological processes affected, while techniques they apply to boost

ecosystem recovery should be goal-oriented. Regarding the results obtained in this Doctoral

Thesis, some implications for practice can be drawn:

1. Promote soil stability in road embankments: Microarthropod communities present in

the road embankments studied indicate that 6 years after road construction, these

anthrosols are fully functional. Furthermore, soil bacterial community composition from

road embankments suggests successional processes that give rise to higher abundances of

groups of bacteria implicated in organic matter turnover, indicative of ecosystem stable

states. Assemblages of both soil communities depend mainly on soil, organic carbon, soil

texture and structure. Thus, conventional management affecting soil properties such as

mowing and re-grading should be avoided to prevent the alteration of these soil factors

and to maintain favorable soil conditions that promote ecosystem functionality and

capacity to deliver ecosystem services.

2. Maintain soil cover: Our results indicate that biomass addition increases bacterial

community diversity. However, mowing adversely affects key soil properties responsible

for soil bacterial communities at the same time that it accelerates litter decomposition,


216

with further effects on organic matter dynamics in soil. Given that mowing is a common

practice in conservation activities of road embankments, we recommend not removing

leaf litter from the soil surface to preserve soil conditions that ensure the maintenance of

soil communities and associated processes.

3. Topsoil spreading in roadcuts: Topsoil spreading in roadcuts is not a common practice

because of its possible impacts on road security. However, we demonstrate that topsoil

properties rapidly boost plant community development in these scenarios. The

application of this treatment increases plant cover and promotes the establishment of a

higher numbers of local species. Nevertheless, topsoil spreading should be carefully

planned to optimize the use of this scarce resource and to cope with operational

requirements. One plausible option could be to apply topsoil in thin and discontinuous

layers, in combination with other practices that facilitate its retention, on those roadcut

segments where a greater distance between the slope and the drainage system reduces the

risk of sediment deposition in the road ditch. In this way, ecosystem carrying capacity

can be locally increased, and the recruitment of plant species can be favored without

jeopardizing vehicle drivers’ security.

Capítulo VI

217

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CONCLUSIONES

Conclusiones

225

De los cuatro capítulos experimentales que se presentan en esta tesis doctoral se pueden

extraer las siguientes conclusiones:

1. El cambio en la composición de microartrópodos edáficos desde comunidades dominadas por

grupos generalistas o primocolonizadores a otras asociadas a condiciones edáficas estables,

está relacionado con el carbono orgánico, el contenido en arcillas y la humedad del suelo.

2. Este cambio en la composición se traduce en una mayor abundancia de Oribátidos (Ácaros)

y Sinfipleones (Colémbolos) que están implicados en la degradación de la materia orgánica,

y en el desarrollo de la microestructura del suelo. Dada la estrecha dependencia de estos

organismos con la estabilidad en las condiciones edáficas, son considerados como

indicadores de calidad de suelo.

3. Según el índice de calidad biológica de suelo (QBS), los valores máximos obtenidos en

suelos de terraplenes de carretera de más de 6 años de antigüedad son comparables a los

valores de funcionalidad observados en suelos de ecosistemas naturales.

4. Los terraplenes de carretera presentan una diversidad taxonómica de bacterias baja y similar

a los observados en otros suelos de origen antrópico, dominando unos pocos grupos de

bacterias como son Actinobacteria, Acidobacteria y Planctomycetes. Estos grupos están

implicados en la dinámica de la materia orgánica del suelo y son indicadores de estados

sucesionales estables.

5. Entre los grupos taxonómicos observados en las comunidades bacterianas de terraplenes se

han encontrado también Bacteroidetes, Firmicutes, Gemmatimonadates y Cyanobacteria. La

baja abundancia relativa de estos grupos está asociada con el avance de la sucesión en estos

suelos.


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6. La composición de las comunidades bacterianas de terraplenes varía a escala local y está

relacionada con las características edáficas, especialmente el pH y la textura del suelo.

7. Las técnicas de manejo convencional aplicadas en terraplenes de carretera inducen cambios

en la estructura de las comunidades bacterianas de estos ambientes. En concreto, la

acumulación de biomasa aumenta la diversidad taxonómica al tiempo que la aplicación de

fertilizantes favorece una mayor equitatividad dentro de la comunidad. Además, este último

tratamiento modifica la composición de la comunidad favoreciendo el desarrollo de grupos

como los Gemmatimonadetes relacionados con una baja disponibilidad de nutrientes.

8. La aplicación de siegas en terraplenes de carretera aumenta la relación C:N, incrementa la

proporción de limos en el suelo y acelera la tasa de descomposición de hojarasca. Estos

efectos pueden estar relacionados con los cambios inducidos en la composición de plantas y

con la ausencia temporal de vegetación, que favorece procesos erosivos que arrastran las

partículas más finas (arcillas). Además, la exposición del suelo a la radiación solar, aumenta

la fotodegradación de la hojarasca, proceso típico de ambientes mediterráneos.

9. La aplicación de tierra vegetal en desmontes de carretera permite rebajar el efecto de los

filtros abióticos que condicionan el ensamblaje de la comunidad de plantas en estos

ambientes. En concreto, la aplicación de este tratamiento aumenta el contenido de carbono y

nitrógeno en el suelo, el contenido de limos y arenas, y disminuye el pH y la conductividad

eléctrica.

10. La respuesta de los distintos estadios vitales de las plantas a los tratamientos estudiados

permite detectar dos procesos que actúan como umbrales para el ensamblaje de la comunidad

de taludes: la emergencia y supervivencia de plántulas. La tierra vegetal permite sobrepasar

Conclusiones

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esos umbrales favoreciendo el ensamblaje de la comunidad de plantas en desmontes de

carretera.

11. La tierra vegetal incrementa la ventana de oportunidad para la emergencia de un mayor

número de especies ya que induce mejoras en la estabilidad de sustrato, la retención hídrica y

la captación de agua por parte de las plántulas. Este aumento del número de especies se

traduce en una mayor riqueza a escala de comunidad en los dos años siguientes a la

aplicación del tratamiento.

12. El extendido de tierra vegetal favorece la supervivencia de plántulas y el establecimiento de

individuos adultos, que dependen de una mayor disponibilidad de recursos. Este efecto se

traduce en comunidades con mayor cobertura vegetal en los dos años siguientes a la

aplicación de este tratamiento.

13. La escarificación de la superficie del talud en combinación con la extensión de tierra vegetal

favorece la retención de esta en las partes altas del desmonte y permite el reclutamiento de un

mayor número de especies en esta zona del talud dos años después de la aplicación de los

tratamientos.

14. Las actividades de manejo aplicadas en taludes de carretera deben ir orientadas a aumentar la

capacidad de carga durante los estadios tempranos tras la construcción de la infraestructura y

asegurar la estabilidad de las condiciones edáficas en estadios posteriores de cara a favorecer

los procesos de sucesión secundaria en estos ambientes. En este sentido y en base a los

resultados obtenidos en esta tesis, se recomienda la aplicación de tierra vegetal en desmontes

de carretera para favorecer los procesos de colonización primaria y se desaconsejan las

técnicas convencionales de manejo que impliquen la modificación o desestructuración de los

suelos presentes en terraplenes de carretera.


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229

CONCLUSIONS

Conclusions

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From the four experimental chapters included in this Doctoral Thesis, the following

conclusions can be drawn:

1. The transition observed in microarthropod community composition from

pioneer/generalist components to those associated with more stable soil conditions is

correlated to changes in organic carbon content, clay content, and soil humidity.

2. This successional change in microarthropod community composition is illustrated by

higher abundances of Oribatid mites (Acari) and Symphypleona (Collembola), both

implicated in organic matter turnover and soil formation. Due to the dependence of these

organisms on stable soil conditions, their presence is used as an indicator of soil quality.

3. Regarding the QBS (Biological Soil Quality) index, soils from road embankments are

fully functional and, in some cases, comparable to those found in other soils under natural

land cover, such as Mediterranean woodlands and shrublands.

4. Soil bacterial communities from road embankments are low in species diversity such as

those observed in other anthrosols, and they are dominated by the taxonomic groups such

as Actinobacteria, Acidobacteria, and and Planctomycetes. These groups are involved in

soil organic matter dynamics and are indicative of mature to advanced successional

stages.

5. Soil bacterial communities from road embankments include representatives of

Bacteroidetes, Firmicutes, Gemmatimonadates, and Cyanobacteria. The relatively low

abundance of these groups indicates secondary succession processes in these anthrosols.

6. Changes in soil bacteria community composition observed at the local scale were mainly

driven by changes in pH and soil texture, namely sand and clay content.


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7. Conventional roadside management techniques trigger changes in soil bacteria

community structure. Specifically, biomass addition increases diversity while at the same

time that fertilization increases community equitability or, in other words, this treatment

homogenizes bacterial communities. Notably, fertilization increases the relative

abundance of Gemmatimonadetes, which is a group that is commonly associated with

low nutrient availability.

8. Mowing increases the C:N ratio as well as loam content and accelerated litter

degradation. These effects may be related to changes induced by mowing on plant

community composition or the total lack of vegetation on some slope patches, both of

which favors erosive processes that carry away finer particles along with surface runoff

water. This lack of vegetation also increases sunlight arriving to the soil surface and the

subsequent acceleration in litter decomposition (photodegradation), which is a common

process processes, typical in Mediterranean environments.

9. Topsoil spreading in roadcuts reduces the effect of abiotic filters constraining plant

community assembly in these environments. In particular, this treatment increases

nitrogen, carbon, silt, and sand content, and decreases soil pH and electrical conductivity.

10. Responses in life-history stages of plant communities to the varying management

treatments intended to manipulate environmental filters, allow us to detect two processes

acting as thresholds for plant community assembly, namely seedling emergence and

survival. Topsoil spreading helps to overcome these thresholds and boost the plant

community assembly processes.

11. Topsoil spreading also increases the opportunity for emergence of higher numbers of

plant species through the improvement of soil stability, soil water retention and,

Conclusions

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consequently, improved water uptake by seedlings. The increase in the number of species

at the seedling stage translates into richer plant communities during the first two years

following treatment application.

12. Topsoil spreading favors seedling survival and the establishment of adult plants, which

usually depends on higher nutrient availability. This effect translates into communities

with higher plant cover during the first two years following treatment application.

13. Shallow tillage combined with topsoil spreading facilitates the retention of topsoil in the

upper slope zone, which in turn increases the recruitment of a higher number of species in

the first two years after the application of treatments.

14. Roadside management should aim to improve soil conditions during the early stages of

roadside ecosystem development, and to ensure soil stability in subsequent years to

facilitate secondary succession and increased functionality. Therefore, we recommend the

application of topsoil spreading to trigger plant colonization and the assembly of richer

plant communities seems a valuable intervention. Concurrently, management techniques

that alter or modify soil conditions in roadside embankments should be avoided.

UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE BIOLOGÍA

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