INSTITUTO POLITÉCNICO NACIONAL ESCUELA NACIONAL DE CIENCIAS BIOLÓGICAS
SECCIÓN DE ESTUDIOS DE POSGRADO E INVESTIGACIÓN
HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
T E S I S que como uno de los requisitos para obtener el grado de DOCTOR EN CIENCIAS QUIMICOBIOLÓGICAS
PRESENTA:
ENRICO ALEJANDRO RUIZ CASTILLO
MÉXICO, D. F., JUNIO DE 2009.
El presente trabajo se realizó en el Laboratorio de Variación Biológica y Evolución de la Escuela Nacional de Ciencias Biológicas del Instituto Politécnico Nacional bajo la dirección del Dr. Gerardo Zúñiga. La presente investigación formó parte del proyecto "Estructura genética poblacional de tres especies del género Dendroctonus (Coleoptera: Curculionidae: Scolytinae) por medio de marcadores moleculares", CGPI 2004978, CGPI 20051200 y
CONACyT 44887-Q.
El sustentante fue becario del Instituto Politécnico Nacional (Beca Institucional) de
Febrero a Diciembre de 2005; del Consejo Nacional de Ciencia y Tecnología
(CONACyT), de Febrero de 2006 a Diciembre de 2008 y del Programa Institucional
De Formación De Investigadores (PIFI) del Instituto Politécnico Nacional, de
Septiembre de 2006 a Diciembre de 2008. HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
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RESUMEN
El propósito de este estudio fue la estimación de diferentes aspectos de la genética y la biología evolutiva del escarabajo descortezador Douglas-fir, Dendroctonus pseudotsugae: el efecto del aislamiento geográfico sobre la estructura genética; la estimación del status taxonómico de sus subespecies; y la historia demográfica y filogeografía de las poblaciones de D. pseudotsugae. Debido a que las respuestas a estas preguntas derivadas de los tres aspectos a evaluar requieren la implementación de muchos métodos diferentes (que a su vez parten de bases conceptuales diferentes), el presente estudio fue dividido en tres capítulos. Todos ellos estuvieron basados en un fragmento de 550 pb de la Citocromo Oxidasa I (COI) del DNA mitocondrial. Para examinar el efecto del aislamiento geográfico sobre la estructura genética (capítulo I) se analizaron 60 haplotipos del gen COI (172 secuencias de 550 pb). Los resultados de los análisis molecular de varianza (AMOVA y SAMOVA), estadísticos F, y regresiones lineales sugieren que la estructura genética de D. pseudotsugae está fuertemente influenciada por la distancia geográfica. También se encontró que la distancia p está correlacionada con las distancia. Las diferencias genéticas observadas entre el norte (Canadá y USA) y el sur (México) en D. pseudotsugae confirman que estos dos conjuntos de poblaciones corresponden a las subespecies previamente asignadas. En el capítulo II, se estimó la validez de la nueva subespecie D. barragani, debido a que su descripción estuvo basada en una única población conocida en México. Para probar si más poblaciones apoyan la existencia de dos subespecies, se realizó una revaluación taxonómica combinando secuencias de COI, los caracteres morfológicos usados en la descripción original, y caracteres morfológicos recientemente descritos. El análisis de máxima parsimonia con 89 haplotipos confirmó que las poblaciones mexicanas son distintas de aquellas provenientes de las localidades del norte (Canadá y USA). Por
i HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
______otro lado, mientras que al menos siete caracteres de la cabeza, el pronoto y los élitros permiten distinguir consistentemente entre las poblaciones de Canadá y USA por un lado, y las de México por otro. Finalmente, en el capítulo III se evaluó si la distribución actual en Norteamérica de D. pseudotsugae ha sido moldeada por las glaciaciones pasadas del periodo Cuaternario. Por otro lado, se cree que el efecto de dichas glaciaciones no fue tan severo en las Montañas Rocosas del sur y en el norte de México. En este trabajo, se pusieron a prueba distintas hipótesis acerca de la localización de refugios del pasado en el Pacifico Noroeste y en las Montañas Rocosas, y se probó también si las poblaciones del sur de Norteamérica y México son más diversas y han experimentado tamaños poblacionales constantes a lo largo del tiempo, en oposición a aquellas de distribución más boreal. Las hipótesis de expansión demográfica de los haplogrupos (identificados a partir de 136 haplotipos de 331 secuencias del gen COI) fueron examinadas a través de pruebas de neutralidad contra crecimiento poblacional, mismatch distribution, y Bayesian Skyline Plots. Los resultados mostraron que los haplogrupos del noroeste de Norteamérica y la Sierra Madre Occidental han experimentado eventos de expansión demográfica, pero no los haplogrupos del suroeste de Norteamérica y de la Sierra Madre Oriental. Los tiempos de divergencia entre pares de haplogrupos fueron estimados entre el Pleistoceno temprano al Pleistoceno medio, mucho tiempo antes del último periodo glacial. Finalmente, el análisis de clados anidados también aportó evidencia de que los cambios demográficos estuvieron acompañados de eventos de expansión de área continua en el noroeste de Norteamérica, mientras que en las poblaciones de Arizona y el norte de México experimentaron fragmentaciones alopátricas y reducción de flujo génico histórico. Los resultados del presente estudio proveen ahora de una base para probar si existe congruencia entre la variación genética de D. pseudotsugae y la correspondiente a la de su huésped.
ii HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
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ABSTRACT
The purpose of the present study was to assess different aspects of genetics and the evolutionary biology of the Douglas-fir beetle Dendroctonus pseudotsugae: the effect of geographic isolation on genetic structure; the assessment of the taxonomic status of subspecies; and the historical demography and phylogeography of D. pseudotsugae populations. Because answering specific questions raised by all three aspects require many different methods (and therefore, different assumptions), this study was divided in three chapters. All of them are based on a fragment of 550 bp of mtDNA Cytochrome Oxidase I (COI). To examine the effect of geographic isolation on genetic structure (chapter I), we analyzed 60 haplotypes of COI gene (172 sequences). Results of analyses of molecular variance (AMOVA and SAMOVA), F-statistics, and linear regressions suggest that the genetic structure of D. pseudotsugae is strongly influenced by geographic distance. We also found that the p-distance is correlated with geographic distance. The observed genetic differences between north (Canada-USA) and south (Mexico) on D. pseudotsugae confirm that these two sets of populations correspond to previously assigned subspecies. In the chapter II, the validity of a new subspecies of D. pseudotsugae was assessed. This was done because the description of D. p. barragani was based on only one known Mexican population. To test if additional populations support the existence of two subspecies, a taxonomic reassessment combining COI sequences, morphological characters used in the original description, and newly described morphological characters was performed. Maximum Parsimony analysis of 89 haplotypes confirms that the Mexican populations are distinct from those from northern locations (in USA and Canada) sampled. On the other hand, at least seven characters on the head, pronotum, and elytra consistently discriminate among Canada-USA and Mexico populations. Finally, in chapter III we evaluated if contemporary northern distribution
iii HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
______of D. pseudotsugae has been shaped by past glaciations of the Quaternary period. It is hypothesized that past glaciations were not that severe in southern Rocky Mountains and Northern Mexico. In this study, we test hypotheses about location of ancient refugia at the Pacific Northwest and Rocky Mountains, and test if southern North American and Mexican populations are more diverse and experienced constant population sizes trough time than northern ones. In this study, 136 haplotypes out of 331 sequences were identified. Hypothesis of demographic expansion of haplogroups was examined trough neutrality tests against population growth, mismatch distribution, and Bayesian Skyline Plots. Results showed that Northwestern North America and Sierra Madre Occidental haplogroups have experienced demographic expansion events while Southwestern North America and Sierra Madre Oriental haplogroups did not. Divergence times between pairs of haplogroups were estimated from early to middle Pleistocene, long before last glacial maxima. Finally, the nested clade analysis provided evidence that the demographic change were accompanied by continuous range expansion events in northwestern North America, while historically reduced gene flow and allopatric fragmentation was inferred in populations from Arizona and Northern Mexico. Results of the present study provide a basis to test for congruence against the intraspecific variation of its plant host.
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Ruiz Castillo Enrico Alejandro.
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RESUMEN i ABSTRACT iii CONTENIDO v FIGURAS vii TABLAS viii INTRODUCCIÓN GENERAL 1 HIPÓTESIS 7 OBJETIVOS 8 CAPÍTULO I: ¿Es afectada la diferenciación genética de Dendroctonus pseudotsugae (Coleoptera: Curculionidae) por el aislamiento geográfico? 9 Does Geographic Isolation Affect the Genetic Differentiation in Dendroctonus pseudotsugae (Coleoptera: Curculionidae)? 10 Abstract 11 Introduction 12 Materials and Methods 14 Results 17 Discussion 20 Acknowledgments 25 References 26 Tables 32 Figure Captions 36 Figures 37 CAPÍTULO II: Análisis molecular y morfológico de Dendroctonus pseudotsugae (Coleoptera: Curculionidae: Scolytinae): valoración del status taxonómico de las subespecies. 40 Molecular and morphological analysis of Dendroctonus pseudotsugae (Coleoptera: Curculionidae: Scolytinae): an assessment of the taxonomic status of subspecies 41
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Ruiz Castillo Enrico Alejandro.
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Abstract 42 Introduction 43 Materials and Methods 44 Results 48 Discussion 53 Acknowledgments 57 References 58 Tables 61 Figure Legends 67 Figures 68 CAPÍTULO III: Historia demográfica y filogeografía de un escarabajo descortezador especialista, Dendroctonus pseudotsugae Hopkins (Curculionidae: Scolytinae) 76 Historical demography and phylogeography of a specialist bark beetle, Dendroctonus pseudotsugae Hopkins (Curculionidae: Scolytinae) 77 Abstract 78 Introduction 79 Materials and Methods 83 Results 89 Discussion 94 Acknowledgments 101 References 102 Figure Legends 113 Tables 114 Figures 121 APPENDIX I 124 APPENDIX II 131 DISCUSIÓN GENERAL 136 REFERENCIAS GENERALES 149
vi HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
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FIGURAS
Fig. 1.1. Sample localities of Dendroctonus pseudotsugae and results from SAMOVA and Maximum Likelihood (ML) analyses 37 Fig. 1.2. Simple number of differences (p distance) vs corresponding geographic distances among pairs of individuals of D. pseudotsugae 38 Fig. 1.3. Scatter plot showing the relationship between genetic dissimilarities
(estimated as FST / 1-FST) and logarithms of geographical distance of D. pseudotsugae populations 39 Fig. 2.1. Phylogram of D. pseudotsugae individuals inferred from 550 nucleotides of mitochondrial COI gene 68 Fig. 2.2. Relative depth of epicranial suture 69 Fig. 2.3. Median carina on pronotum 70 Fig. 2.4. Margin of strial punctures on elytra 71 Fig. 2.5. Interstria 2 on elytral declivity 72 Fig. 2.6. Frontal region of head 73 Fig. 2.7. Tubercles on lower frons region 74 Fig. 2.8. Cladogram of D. pseudotsugae individuals inferred from 12 morphological characters 75 Fig. 3.1. Sample distribution and haplotype frequency of the four major haplogroups of Dendroctonus pseudotsugae for each region 121 Fig. 3.2. Maximum-likelihood tree reconstruction based on 550 bp of the mtDNA genome of the Dendroctonus pseudotsugae, with the TrN93 + G + I substitution model (D. simplex was used as outgroup) 122 Fig. 3.3 Demographic history of the main four mitochondrial haplogroups inferred in Dendroctonus pseudotsugae 123
vii HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
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TABLAS
Table 1.1. Locations, geographic references, and number of Dendroctonus pseudotsugae specimens analyzed 32 Table 1.2. Haplotype and nucleotide diversities estimated for COI data in D. pseudotsugae populations 33 Table 1.3. Analysis of molecular variance (AMOVA) of COI data from D. pseudotsugae populations, including df, sum of squares, percentage of variation explained, P values and Φ statistics 34 Table 1.4. Fixation indices corresponding to groups of populations inferred by SAMOVA for the D. pseudotsugae populations tested for the mtDNA sequences 35 Table 2.1. Locations, geographic references, number of specimens analyzed and accession numbers in Dendroctonus pseudotsugae 61 Table 2.2. Morphological character state distribution of D. pseudotsugae, as reported by Furniss (2001) and obtained by the present study 64 Table 2.3. Mean percentage of sequence divergence (using the TrN+I+G substitution model) of D. pseudotsugae populations by geographic range 66 Table 3.1. Location, region, geographic references, number of specimens analyzed and accession numbers of mtDNA COI in Dendroctonus pseudotsugae 114 Table 3.2. Nucleotide polymorphism and results of neutrality test for the mtDNA COI (550 bp) of Dendroctonus pseudotsugae 116 Table 3.3. Statistic tests for selective neutrality and population expansion indices based on 550 bp of mtDNA COI of Dendroctonus pseudotsugae 117 Table 3.4. Results of the migration/isolation model and divergence time among haplogroups of Dendroctonus pseudotsugae, obtained using the MDIV software 118 Table 3.5. Summary of inferences regarding the historical demographic events from nested clade phylogeographic analysis 119
viii HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
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INTRODUCCIÓN GENERAL
Las relaciones que guardan entre si todos los seres vivos y la forma en que se han diversificado, han sido exploradas y discutidas formalmente en los últimos 300 años. Durante este tiempo, los esfuerzos por detectar, describir y explicar el mundo biológico han dado como resultado la aceptación de que la clasificación de los organismos debe basarse, ante todo, en la relaciones ancestro-descendiente.
La integración de una base conceptual robusta, y el desarrollo de recursos metodológicos y técnicos, han permitido una mejor estimación de las relaciones genéticas entre individuos de una misma especie (genealogías intra-específicas) y filogenéticas entre especies (filogenia). Estas nuevas metodologías también han permitido estimar cambios en el tamaño poblacional y de distribución geográfica a lo largo de grandes periodos de tiempo, así como inferir los posibles eventos históricos que les dieron origen. En la actualidad, estos aspectos de la evolución son estudiados en disciplinas interrelacionadas: la genética de poblaciones, la sistemática y la filogeografía.
El problema fundamental al que estas disciplinas han tenido que enfrentarse por igual tiene que ver con la búsqueda e identificación de caracteres variables y heredables, que permitan cumplir con los distintos objetivos que persiguen. El
1 HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
______estudio de los ácidos nucleicos ha posibilitado una nueva perspectiva acerca del modo y tiempo de la evolución, al demostrar que es posible estimar filogenias y genealogías correlacionando el grado de diferenciación de los organismos con el tiempo de divergencia de los taxa a los que pertenecen (Hoy, 2003).
De esta manera, los atributos moleculares y las nuevas metodologías para inferir las relaciones entre los organismos a partir de ellos, han mostrado propiedades valiosas no accesibles con los conjuntos de datos tradicionales. Sin embargo, los datos moleculares han debido superar el criticismo derivado de sesgos en cuestiones como: pertinencia y calidad del marcador, constancia de sus tasas de evolución, neutralidad y correcta identificación de homología (Hillis y Moritz, 1990;
Adoutte et al., 2000).
Por otro lado, la determinación de relaciones filogenéticas y genealógicas, así como la elucidación de patrones de distribución geográfica entre poblaciones de una misma especie, pueden tomar ventaja (cuando es el caso) de la íntima relación que tienen con sus huéspedes. Tal es el caso de Dendroctonus pseudotsugae Hopkins.
D. pseudotsugae es un escarabajo descortezador que parasita a árboles de la especie Pseudotsuga menziesii (Mirbel) Franco, el cual constituye su único huésped.
Los primeros ejemplares de esta especie fueron colectados en la localidad de Grant
Pass, Oregon (Hopkins 1909). Posteriormente, fueron también encontrados en las montañas rocosas, en las cadenas montañosas del Pacífico noroeste y más
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Ruiz Castillo Enrico Alejandro.
______recientemente, en las Sierra Madre Occidental y Sierra Madre Oriental en el norte de
México (Wood, 1982; Salinas-Moreno et al., 2004).
A pesar de que los efectos ejercidos por la distancia geográfica y el uso del huésped sobre la estructura genética en otras especies del género Dendroctonus han sido ampliamente estudiados (Namkoong et al. 1979; Stock et al. 1984; Roberds et al. 1987; Six et al. 1999; Zúñiga et al. 2006; Schrey et al. 2008), la evidencia más reciente sugiere que dicha estructuración es un fenómeno mucho más complejo
(Maroja et al. 2007; Mock et al. 2007; Anducho-Reyes et al. 2008). Sin embargo, al ser D. pseudotsugae un insecto fitófago especialista, los posibles efectos de varios huéspedes en su variación genética quedan minimizados.
Por ello, en el capítulo I de la presente tesis se evalúa el posible aislamiento por distancia (i. e. si existe correlación entre la variación genética y la distribución geográfica), en dirección norte-sur, de las poblaciones muestreadas de D. pseudotsugae, mediante la utilización de las secuencias nucleotídicas de un fragmento de la Citocromo Oxidasa I del genoma mitocondrial. Dicho patrón es esperado considerando la distribución original de su huésped y el grado de aislamiento geográfico que han alcanzado sus poblaciones en Norteamérica y
México.
Por otro lado, la observación de D. pseudotsugae más hacia el sur de su área de distribución previamente conocida, en el norte de México, más la observación de diferencias morfológicas de la cabeza, pronoto, élitros y la forma en la que los
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______huevos son depositados en la galería parental (entre otros), llevó a la descripción de una nueva subespecie llamada D. p. barragani (Furniss, 2001), distinta de la especie nominal, D. p. pseudotsugae.
La evidencia reunida a este propósito se basó en una única población, sin considerar que su área de distribución es más amplia, extendiéndose hacia el sur de la Sierra Madre Occidental y hacia el este en la Sierra Madre Oriental (Salinas-
Moreno et al., 2004). Debido a esto y a otros factores (e. g. viabilidad de la progenie más allá de la primera cruza, diferenciación clinal morfológica en un eje norte-sur, etc.), la definición, limites, y área de distribución de ambas subespecies permanece sin esclarecer.
Para contestar estas interrogantes, en el capítulo II se prueba si marcadores moleculares (fragmento de 550 pb de la Citocromo Oxidasa I del DNA mitocondrial), más caracteres morfológicos (los mismos usados por Furniss (2001) más otros propuestos en este estudio), permiten el reconocimiento de grupos monofiléticos que correspondan a las subespecies arriba mencionadas.
Desafortunadamente, a pesar de compartir el conjunto de datos moleculares, los métodos empleados en los capítulos I y II no permiten probar cuáles fueron los procesos históricos y demográficos que han afectado la estructura genética y la distribución actual de D. pseudotsugae. Esto debido a que los modelos de genética poblacional fueron diseñados para ser independientes de la historia y de la geografía
(Epperson, 2003), Además, se requiere un tamaño de la muestra más amplio (tanto
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Ruiz Castillo Enrico Alejandro.
______en número de individuos como en distribución geográfica) y un diseño analítico con diferente base conceptual (Avise, 2004).
Actualmente, se ha reunido una gran cantidad de evidencia que demuestra que la distribución de los organismos en el hemisferio norte cambió rápidamente como resultado del avance de los glaciares en el periodo Cuaternario (Hewitt, 1996;
Hewitt and Ibrahim, 2001). Al cambiar el clima en los periodos interglaciares, los territorios del norte estuvieron disponibles para los colonizadores, lo cual puede ser observado en su menor diversidad genética.
La comparación entre muchos taxa provenientes de una misma área ha permitido la identificación de patrones congruentes de variación genética y de rutas de colonización, lo cual a su vez ha llevado a la inferencia de factores históricos comunes (Soltis et al., 1997; Petit et al., 2003; Avise, 2004; Hewitt, 2004). Sin embargo, a pesar de que las poblaciones de D. pseudotsugae distribuidas en las
Montañas Rocosas del Norte y en la Cordillera de las Cascadas estuvieron bajo la influencia directa de las glaciaciones, no ocurrió lo mismo con aquellas distribuidas en una región más meridional, como las Montañas Rocosas del Sur y el Norte de
México, en las Sierras Madre Oriental y Occidental (Brown, 1985).
Estas características permiten plantearse hipótesis sobre la localización de refugios durante el periodo Plio-Pleistoceno para D. pseudotsugae en el Pacifico noroeste y las Montañas Rocosas (así como las posibles rutas de colonización desde esos refugios). También permiten explorar si las poblaciones del sur de Norteamérica
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Ruiz Castillo Enrico Alejandro.
______y el Norte de México -a diferencia de las del norte- son más diversas y han experimentado tamaños poblacionales constantes a lo largo del tiempo.
Dichas hipótesis son evaluadas en el capítulo III, junto con la estimación de los tiempos de divergencia entre los haplogrupos encontrados y las inferencias filogeográficas que explican los eventos que determinaron su distribución actual.
Para ello, el estudio se basó no solamente en el conjunto de datos moleculares usado en los capítulos anteriores, sino en un conjunto de datos ampliado del mismo gen, el cual aseguró una mejor representación tanto en número de ejemplares como en el área de distribución geográfica de la especie.
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Ruiz Castillo Enrico Alejandro.
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HIPÓTESIS
Si las poblaciones de Dendroctonus pseudotsugae colectadas a lo largo de su distribución geográfica tienen un origen boreal común y son el producto del aislamiento geográfico, entonces:
1) Los valores de divergencia genética, diversidad nucleotídica y diversidad haplotípica deberán reflejar el grado de diferenciación de las poblaciones de D. pseudotsugae.
2) La estructura genética de esta especie deberá ajustarse a un modelo de aislamiento por distancia.
3) La diferenciación genética interpoblacional y los análisis filogenéticos entre las poblaciones del norte (Estados Unidos y Canadá) y el sur (México) deberán ser estadísticamente significativos y reflejar el status taxonómico de las subespecies.
4) Las inferencias histórico-demográficas y filogeográficas entre los haplotipos deberán revelar los procesos que expliquen los actuales patrones de distribución de la especie.
7 HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
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OBJETIVOS
OBJETIVO GENERAL: Determinar la estructura genética poblacional, reevaluar el status taxonómico de las subespecies, y determinar los procesos históricos y demográficos que explican la distribución geográfica actual del escarabajo descortezador Dendroctonus pseudotsugae.
OBJETIVOS PARTICULARES: Estimar el número de sitios polimórficos, el número de haplotipos y los índices de diversidad genética y nucleotídica en las poblaciones de Dendroctonus pseudotsugae. Determinar el grado de diferenciación genética de las poblaciones de Dendroctonus pseudotsugae. Determinar si las poblaciones analizadas de Dendroctonus pseudotsugae describen un modelo de aislamiento por distancia. Estimar el número promedio de diferencias pareadas y el porcentaje estándar de diferencia entre secuencias de DNA, en las poblaciones de Dendroctonus pseudotsugae. Realizar análisis filogenéticos con datos moleculares y morfológicos por métodos de Máxima Parsimonia y Máxima Verosimilitud de los ejemplares de Dendroctonus pseudotsugae. Examinar hipótesis de expansión demográfica de los haplogrupos a través de pruebas de neutralidad contra crecimiento poblacional, mismatch distribution, y Bayesian Skyline Plots. Estimar los tiempos de divergencia entre pares de haplogrupos de Dendroctonus pseudotsugae. Construir una red de haplotipos que permita identificar las relaciones genealógicas entre los individuos de las poblaciones de Dendroctonus pseudotsugae. Realizar un análisis de clados anidados para probar si existen asociaciones estadísticamente significativas entre la red de haplotipos inferida y la distribución geográfica de las poblaciones de Dendroctonus pseudotsugae. Mediante el uso de claves de inferencias, determinar los procesos históricos y demográficos que explican la distribución geográfica actual de Dendroctonus pseudotsugae.
8 CAPÍTULO I
¿Es afectada la diferenciación genética de Dendroctonus pseudotsugae (Coleoptera:
Curculionidae) por el aislamiento geográfico? Ruiz et al.: Genetic differentiation in
Dendroctonus pseudotsugae
Does Geographic Isolation Affect the Genetic Differentiation in Dendroctonus pseudotsugae (Coleoptera: Curculionidae)?
Enrico A. Ruiz1, John E. Rinehart2, Jane L. Hayes3, and Gerardo Zúñiga1
1Escuela Nacional de Ciencias Biológicas-IPN. Laboratorio de Variación Biológica y
Evolución. Departamento de Zoología. Carpio y Plan de Ayala s/n, Col. Santo Tomás, CP
11340 Mexico City, México.
2Biology Program, Eastern Oregon University. 1 University Boulevard, LaGrande, OR 97850
3Forestry and Range Sciences Laboratory, Pacific Northwest Research Station, USDA Forest
Service, 1401 Gekeler Lane, LaGrande, OR 97850
10 Abstract
Genetic structure of phytophagous insects has been widely studied; however, relative influence of the effect of geographic isolation, the host plant or both has been subject of considerable debate. Several studies carried out on bark beetles in the genus Dendroctonus
Erichson evaluated these factors; nonetheless, recent evidence has shown that genetic structuring is a more complex process. Our goal was to examine the effect of geographic isolation on genetic structure of the Douglas-fir beetle Dendroctonus pseudotsugae Hopkins.
We used mtDNA Cytochrome Oxidase I sequences. One hundred-seventy-two individuals were obtained from 17 populations, for which we analyzed 60 haplotypes. Analyses of molecular variance (AMOVA and SAMOVA), F-statistics, and linear regressions suggest that the genetic structure of D. pseudotsugae is strongly influenced by geographic distance. We found that D. pseudotsugae has high intra- and inter-population genetic variation compared with several other bark beetles. Genetic differences of D. pseudotsugae among populations based on COI sequences were correlated with geographic distance. The observed genetic differences between northern (Canada-USA) and southern (Mexico) populations on its host
Pseudotsuga menziesii var. glauca (Beissner) Franco confirm that these two sets of populations correspond to previously assigned subspecies.
11 Introduction
The genetic structure of phytophagous insects has been widely studied (Roderick
1996); however, the factors by which their populations become genetically differentiated have been subject of considerable debate (see Peterson and Denno 1998; Van Zandt and Mopper
1998). Principal among these often-intertwined factors are effects of geographic isolation and host plant influences. Several studies carried out on bark beetles in the genus Dendroctonus
Erickson (Coleoptera: Curculionidae: Scolytinae) evaluated the effect geographic isolation
(Namkoong et al. 1979; Stock et al. 1984; Roberds et al. 1987; Six et al. 1999; Zúñiga et al.
2006; Schrey et al. 2008) and of host plants (Stock and Amman 1980; Sturgeon and Mitton
1986; Langor and Spence 1991; Amman and Stock 1995; Kelley et al. 2000) on the genetic differentiation of their populations. While these studies carried out at both fine and coarse geographic scales documented genetic differences among populations associated with these factors, there is no agreement on the roles geographic isolation or host use have played in genetic differentiation in Dendroctonus species. In addition, recent phylogeographic evidence has shown that genetic structuring of these beetles is a more complex process (Maroja et al.
2007; Mock et al. 2007; Anducho-Reyes et al. 2008).
The distribution of genetic variability appears to be affected by the population dynamics of these insects, the geographical distribution both of insects and their hosts, as well as geomorphologic histories that determine different levels of allopatry or sympatry among populations (Kelley et al. 1999; Maroja et al. 2007; Mock et al. 2007; Anducho-Reyes et al.
2008; Schrey et al. 2008).
Our goal was to examine the effect of geographic isolation on genetic structure of
Dendroctonus pseudotsugae (Douglas-fir beetle). This bark beetle colonizes one single host,
12 Pseudotsuga menziesii (Mirbel) Franco across its geographic range, which has two varieties: a costal variety (var. menziesii) found in the Pacific Northwest, and an interior variety (var. glauca) found throughout Rocky Mountains and North of Mexico (Hermann and Lavender
1990). Perhaps the most intuitive hypothesis of the effect of geographic isolation on genetic structure is the model of isolation by distance (IBD) (Wright 1943), which predicts that genetic differentiation between populations increases with geographic distance. Peterson and
Denno (1998) found by meta-analysis that IBD in phytophagous is less common in highly mobile insects (dispersal > 20 km) compared with those whose vagility is low or moderate.
Nevertheless, it has been shown that a fundamental factor that promotes IBD among populations is the equilibrium between gene flow and drift (Hutchison and Templeton 1999).
Focusing on Douglas-fir beetle found on P. menziesii var. glauca (and thus, reducing the effect of host plant), we assess the distribution of genetic variation and IBD in a north- south direction among populations. Considering the original geographical distribution of this host variety in North America, we expect both strong genetic structuring and IBD among
North American beetle populations and those from Mexico. To address these questions, we examine genetic variation in mtDNA Cytochrome Oxidase I (COI) in D. pseudotsugae samples from across its distribution range.
13 Materials and methods
Samples and DNA extraction. A total of 172 adult insects were collected from 17 geographically distinct populations from Canada, USA and México (Fig. 1.1A; Table 1.1). For comparative purposes, one population was collected from the other variety, P. menziessi, var. menziesii (Mount Hebo, OR). Specimens from northern populations were collected by directly sampling under the bark of recently infested trees or using traps baited with attractant pheromones, whereas almost all individuals from southern populations were collected from recently infested trees. To avoid analysis of genetically related individuals from those populations collected manually, we gathered individuals from six or seven trees per population. All beetles were stored in 100% ethanol.
Mitochondrial COI amplification. PCR amplification of a 550 bp fragment of mtDNA COI was carried out using primers C1-J-2441 (5´ACA GGW ATT AAA ATT TTT AGT TGA
TTA GC 3´) and T12-N-3014 (5´TTC AAT GCA CTA ATC TGC CAT ATT A 3´) (Simon et al. 1994). DNA amplification was performed using a Biometra T Gradient thermocycler
(Biometra GmbH, Göttingen, Germany). Each PCR reaction mixture contained 50 ng of
DNA, 1 μl primer 50 pM, 4 μl dNTP’s 10 mM (1 μl for each dNTP), (dNTPs. InvitrogenTM,
Sao Paulo, Brazil), 6 μl MgCl2 25 mM, 5 μl 1x buffer, and 0.4 μl Taq DNA polymerase (Taq
DNA polymerase, Recombinant, InvitrogenTM, Sao Paulo, Brazil). The final volume was brought to 25 μl with ultrapure water. Cycling parameters were: preheating of samples for 10 min at 95 C, followed by 35 amplification cycles of 5 min at 94 C, 1 min at 51 C, 2 min at
72 C, and a final extension of 5 min at 72 C. Amplification products were separated on 1.5% w/v agarose gel, and stained with ethidium bromide. PCR products were purified using
14 GFXTM PCR DNA and gel band purification kit (Amersham Biosciences, Buckinghamshire,
UK) to remove primers and unincorporated dNTPs prior to sequencing. Cycle sequencing reactions were performed with BigDye fluorescent chemistry reaction (Applied Biosystems,
Inc., Foster City, CA). Both forward and reverse strands were sequenced with an ABI 377 sequencer and contiguous sequences constructed and edited manually using Sequence
Navigator v.1.0.1 (Applied Biosystems Inc., Foster City, CA). Multiple alignments of sequences were assembled with Clustal X v.1.83 (Thompson et al. 1997). Reference sequences of each haplotype were deposited in the GenBank nucleotide sequences database (accession nos. EU043405-EU043464; http://www.ncbi.nlm.nih.gov/Genbank).
Statistical analyses To assess the patterns of molecular diversity of mtDNA sequences, we estimated the haplotype and nucleotide diversity (Nei 1978) using the software DNASP v.4.50.3 (Rozas et al. 2003). To define groups of D. pseudotsugae populations maximally differentiated from each other, we used both a priori (AMOVA) and a posteriori (SAMOVA) approaches (see below). For AMOVA analysis, we performed phylogenetic analyses by maximum likelihood (ML) method. We used MODELTEST 3.7 (Posada and Crandall 2001) to select the best fit model of sequence evolution. The ML analysis was performed with heuristic search and TBR as swapping algorithm. The method was validated through 500 pseudoreplicates by bootstrap analysis. No sister taxa were used as outgroups, because our phylogenetic reconstructions were not performed for the purpose of revealing phylogenetic relationships per se. All reconstructions were carried out using the program PAUP* (Swofford
2002).
Based on recovered groups by ML analysis, we performed AMOVA analysis using
Arlequin v.3.0 (Excoffier et al. 1992; Excoffier et al. 2005) to partition the molecular variance
15 into different hierarchical levels: within sampling sites (populations), among populations within groups, and between groups. The statistical significance of partitioned molecular variance and the associated estimate of -statistics were assessed by conducting 10,000 random permutations of the data (Excoffier et al. 1992). The spatial analysis of molecular variance using SAMOVA 1.0 (Dupanloup et al. 2002) was carried out, despite the fact that sampling points of D. pseudotsugae are not geographically adjacent and populations are not genetically homogeneous in the region under study, as it is assumed by this approach. The method implemented in this programme indirectly detects genetic barriers and defines groups of populations geographically homogeneous and maximally differentiated from each other.
We ran this program repeatedly, changing n (number of groups) from 2 to 16. Significance tests were performed with 1000 permutations.
To determine whether the genetic structure of D. pseudotsugae describes an IBD pattern, two approaches were followed. First, linear regression of least squares between FST /
1-FST (using ST) vs ln-transformed geographic distances among the populations was conducted (Rousset 1997). Second, ordinary linear regression of p-distance was carried out vs geographic distances. In both cases, the Mantel test was used to determine statistical significance (Mantel 1967) after 5000 random permutations using NTSYSpc v2.02j (Rohlf
1998).
16 Results
Genetic diversity. From the 172 mtDNA COI sequences, 60 different haplotypes of 550 bp length were identified. The populations with the highest number of haplotypes were Spring
Creek (SCOR), Missoula (MSMT) and Flagstaff (FLAZ) with nine, eight and eight haplotypes, respectively. The most frequent haplotypes were H4, H6, and H110, which were found in most populations. The remaining haplotypes had a frequency lower than 5%; however, most of them were population-specific (Table 1.1). Mean haplotype and nucleotide diversities were h 0.945 ± 0.009 and π = 0.027 ± 0.001, respectively (Table 1.2). Estimates of diversity ( h and π) were not statistically different among populations, or between northern and southern populations (P > 0.05). Also, there was no correlation between latitude and haplotype diversity per population or between latitude and nucleotide diversity per population
(data not shown).
Genetic structure. The ML topology of the 172 individuals using only mtDNA sequences showed two clearly distinguishable groups: one composed only by northern populations and the other only by Mexican populations (Fig. 1.1B). The results of the AMOVA analysis (- statistics, sum of squares, variation percentage, and probability (P) associated with values) are summarized in Table 1.3. The source of variation between groups of populations (CT) was assessed using the two groups found by phylogenetic analysis. Both types of markers were consistent in describing the partitioning of genetic variation according to the source of variation, and revealed that variation within populations is much greater than between populations or between groups. In particular, the ST showed strong genetic differentiation,
17 and all -statistics were statistically different from zero, indicating that genetic variation was geographically structured.
The results obtained by SAMOVA analysis are shown in Figure 1C and Table 1.4.
This approach detected five groups of populations geographically differentiated (FCT = 0.631), separated from each other by inferred genetic barriers (Fig. 1.1C). Three groups contained only populations from Mexico, while the two remaining groups contained only populations from North America. Mexican groups were composed as follows: group 1, ACOA from
Coahuila; group 2, SNLO, MDGO and EDGO from southern Durango and Nuevo León; and group 3, PDGO, EDGO, LDGO and ECHI from northern Durango and Chihuahua. The two groups from the North America were: group 4, FLAZ from Arizona and group 5, MHOR,
JDOR, SCOR, BCOR, DHOR, EROR, MSMT and RVBC from northwestern USA and southwestern Canada. SAMOVA results showed a clear differentiation due to a genetic barrier between the Mexico and the North America populations. This analysis also suggests that within these two regions genetic barriers may exist between populations of Sierra Madre
Oriental and Sierra Madre Occidental, as well as between those from northwestern and southwestern USA.
Isolation by distance. Regression analyses of FST / 1 - FST vs ln-transformed geographic distances revealed no positive and monotonic relationship between genetic differentiation and geographic distance within southern populations (r Mantel = 0.161, P =
0.216), nor within northern populations (r Mantel = -0.324, P = 0.116) (Fig. 1.2A, B).
However, when all populations sampled in this study were considered, they described an isolation by distance pattern (r Mantel = 0.231, P = 0.005) (Fig. 1.2C). The linear regression
18 of pairwise p-distances and geographic distances also show that the simple number of differences among 172 individuals was positively related with their geographic distance
(rMantel = 0.488, P = 0.0004, Fig. 1.3). In most cases, pairs of geographically close individuals are less differentiated than those separated by longer distances. Therefore, while the effect of geographical isolation on genetic differentiation is insignificant for relatively geographically close populations, northern and southern groups of populations differ significantly.
19 Discussion
Genetic diversity. mtDNA haplotype and nucleotide diversities have been used as estimators of intraspecific genetic diversity of many insects groups. The nucleotide diversity estimate for mtDNA COI in D. pseudotsugae ( = 0.027 ± 0.001) is higher than in D. mexicanus Hopkins (0.011 0.009, Anducho-Reyes et al. 2008), D. rufipennis Kirby (0.018 ±
0.004, Maroja et al. 2007) and D. ponderosae Hopkins ( 0.006 ± 0.003, Mock et al. 2007), but lower than in D. valens LeConte (0.028 ± 0.022, Cognato et al. 2005a) and other Scolytinae
(Langor and Sperling 1997; Stauffer et al. 1997; Kohlmayr et al. 2002; Cognato et al. 2003).
Our data also reveal that D. pseudotsugae is a highly polymorphic species ( h 0.945 ± 0.009), as shown by the high number of different haplotypes and their frequencies.
Comparison of bark beetle mtDNA nucleotide and haplotype diversity can be useful to assess demographic history of populations. High haplotype and low nucleotide diversity found in this study for D. pseudotsugae populations suggest that there has been rapid population growth from small populations, assuming sufficient time for recovery of haplotype variation via mutation but too short for the accumulation of large sequence differences (Grant and
Bowen 1998). The same relationship between haplotype and nucleotide diversity has been observed in other Scolytinae, including Dendroctonus species (Cognato et al. 2005a; Maroja et al. 2007; Mock et al. 2007; Anducho-Reyes et al. 2008).
Independent factors such as length of the sequence, number of populations and individuals analyzed, as well as coalescence time, can bias estimations of genetic diversity.
However, general patterns of variation in diversity indices were homogeneous among
20 populations. These results indicate homogenization of interpopulation genetic diversity by high gene flow through extensive dispersal. Nevertheless, the dissimilar haplotype richness
(number of haplotypes per population) observed among populations and population groups suggest that populations have experienced a recent reduction of their effective population size.
Several studies carried out with European scolytines suggest that reduction and rapid population expansion might be the result of glacial or postglacial events that occurred during the Plio-Pleistocene period in Europe, which promoted contraction and expansion of beetle habitats (hosts) and postglacial migration processes from Plio-Pleistocene refugia (Stauffer et al. 1999; Horn et al. 2006). AMOVA and SAMOVA analyses indicate a strong genetic differentiation between Canada-USA and Mexico populations of this bark beetle, probably as a consequence of fragmentation of distribution range of its host plant (P. menziesii var. glauca) during climatic oscillations of the Plio-Pleistocene period. To infer the effect of glacial and postglacial Pleistocene events on genetic structure of this species, it would be necessary to carry out a phylogeographic analysis, including more populations given that historical inferences are biased by the number of population sampled (Petit et al. 2005).
Genetic structure. Several studies have suggested that specialization in host use may be a very important factor influencing genetic structure and differentiation of phytophagous insects
(see Van Zandt and Mopper 1998 and references therein). The expectation of this hypothesis is that insect species with restricted diet breadth (specialist species) should be more prone to genetic differentiation than a generalist species because the distribution of the specialist’s single host is patchier and less dense than the combined distribution of all generalist’s hosts
(Thompson 2004). This hypothesis has been tested in generalist and specialist species of
Dendroctonus using different molecular markers (Kelley et al 1999; Kelley et al. 2000; Zuñiga
21 et al. 2006). In these studies, generalist species showed slight genetic differences among populations colonizing different hosts in the same locality and cumulative differences among geographically isolated populations. Our results seem to support these expectations, because they reveal a strong pattern of differentiation between geographically separated populations, although it is difficult to know whether this differentiation was solely promoted by host fragmentation (due to both habitat loss and the breaking apart of habitat) or geographic distance.
Early genetic studies of D. pseudotsugae using isozymes suggest that differences between populations from Washington and Idaho (S = 0.63) were enough to consider them as two allopatric populations in the process of speciation (Stock et al. 1979; Bentz and Stock
1986). This conclusion was reinforced because the source populations utilized different varieties of P. menziesii. However, other studies have shown even higher similarity coefficients for different species (e.g., S = 0.83 in D. jeffreyi Hopkins and D. ponderosae)
(Higby and Stock 1982). Our results did not reveal sufficient genetic differentiation among northern populations, which includes at least one population found on P. m. var. menziesii, to support the notion of ongoing speciation. The genetic differentiation that we found between D. pseudotsugae populations from north and south supports the expectation that geographical isolation and habitat fragmentation are primary factors affecting genetic structure. Although host use remains to be tested directly, the limited genetic differentiation among northern D. pseudotsugae populations suggests that host use has little effect on the genetic structure of this species. In addition, the absence of an observable pattern of IBD within each north and south population group of D. pseudotsugae indicate that they have not yet reached gene flow-drift equilibrium, which suggest genetic drift is less influential than gene flow, no matter how long
22 the separation among populations (model III, Hutchinson and Templeton 1999). These data agree with those obtained in other studies using various markers (isozyme, RAPD, mitochondrial) in Conophthorus Hopkins (Cognato et al. 2005b), D. brevicomis (Kelley et al.
1999), D. jeffreyi (Six et al. 1999), D. ponderosae (Kelley et al. 2000), D. mexicanus (Zúñiga et al. 2006), Ips typographus L. (Stauffer et al. 1999), and Tomicus piniperda L. (Carter et al.
1996). Our observations also suggest that the genetic structure of specialist species is more sensitive to IBD than generalist species, possibly because genetic differentiation of generalists is minimized due to different host use throughout its geographical distribution.
Isolation by distance. Linear regressions both of FST and p-distance vs geographic distance have shown a significant increase in amount of genetic differentiation among the populations we sampled, relative to the increase of geographic distance. These results agree with the study of Peterson and Denno (1998), which showed that the balance between dispersal and geographic isolation has had a greater effect over genetic structure and differentiation of phytophagous populations than specialization in the host-use. The genetic differentiation among pairs of populations estimated by FST / 1- FST also suggests that the break in the distribution of genetic variation, leading to isolation between North American and
Mexican populations of D. pseudotsugae, is a result of limited gene flow and the consequent increase in genetic drift (Hutchinson and Templeton 1999).
Unfortunately, the methods used do not allow us to test what historical processes or factors have affected the genetic structure of D. pseudotsugae, because classical models of population genetics were designed to be independent of history (e.g., equilibrium models) and of the geographical landscape (e.g., non-structured island models). In addition, as some population genetic models in which a geographical perspective has been introduced, such as
23 IBD or stepping-stone, the landscape is considered uniform, isotropic, and not linked to the history of the population (Epperson 2003). However, low observed genetic differences within distinct geographical populations of the Douglas-fir beetle, and the finding that a model of
IBD is described by all populations that we analyzed, both support the general premise that geographical isolation has played a fundamental role in the differentiation of its populations.
Finally, our results confirm that D. pseudotsugae populations from North America and those from Mexico on P. menziesii var. glauca are two true subspecies: D. p. pseudotsugae
Hopkins and D. p. barragani, as was reported by Furniss (2001). D. p. barragani was collected from a single population in Chihuahua, Mexico, and described based on morphological characters, gallery differences, and intra- and inter-population mating tests. At the time, this was the only known population of D. pseudotsugae in Mexico, where its host P. menziesii is a relict species occurring in isolated locations in Chihuahua, Durango, and
Coahuila.
24 Acknowledgments
Our thanks to Dr. Jaime Villa, Antonio Olivo, Francisco Bonilla, and Sergio Quiñones from Comisión Nacional Forestal-Mexico (CONAFOR) for field assistance. We thank
Malcolm Furniss, Karen E. Mock, and Anthony I. Cognato, and two anonymous reviewers for their review of the manuscript. The project was funded by Consejo Nacional de Ciencia y
Tecnología (CONACYT 44887) and Secretaría de Investigación y Posgrado-IPN (SIP-
20060421). JLH and JER were supported by the Pacific Northwest Research Station.
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fir beetles (Dendroctonus pseudotsugae) from Idaho and coastal Oregon. – Ann.
Entomol. Soc. Am. 72: 394–397.
Sturgeon, K. B. and Mitton, J. B. 1986. Allozyme and morphological differentiation of
mountain pine beetle Dendroctonus ponderosae Hopkins (Coleoptera: Scolytidae)
associated with host tree. – Evolution 40: 290–302.
Swofford, D. L. 2002. PAUP*. Phylogenetic analysis using parsimony (* and other methods).
Version 4.0b10. – Sinauer Associates.
Thompson, J. D., Gibson, T. J., Plewniak, F. et al. 1997. The ClustalX windows interface:
flexible strategies for multiple sequence alignment aided by quality analysis tools.
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Thompson, N. 2004. The coevolutionary process. Chicago University Press.
Van Zandt, P. A., and Mopper, S. 1998. A meta analysis of adaptative deme formation in
phytophagous insect populations. – Am. Nat. 152: 595–604.
Wright, S. 1943. Isolation by distance. – Genetics 28:114–138.
Zúñiga, G., Cisneros, R., Salinas–Moreno, Y. et al. 2006. Genetic structure of Dendroctonus
mexicanus (Coleoptera: Curculionidae: Scolytinae) in the Trans–Mexican volcanic
belt. – Ann. Entomol. Soc. Am. 99: 945–958.
31 Table 1.1. Locations, geographic references, and number of Dendroctonus pseudotsugae specimens analyzed.
No. Pop. no. Localities Pop. key Latitude Longitude Haplotypes Individuals Southern populations
1 Ejido Cienega de la Vaca, San Dimas, DGO. CDGO 24° 05' 20'' N 105º 31' 00'' W 9 H87, H101, H108, H110(6)
2 Ejido Puentesillas, San Dimas, DGO. PDGO 24° 21' 10'' N 105º 54' 39'' W 11 H103, H104, H106, H109, H110(7)
3 Ejido La Manga, San Dimas, DGO. MDGO 24° 22' 08'' N 105º 58' 15'' W 9 H86(2), H92(2), H94(2), H98(2), H110
4 Ejido Nuñez, San Dimas, DGO. EDGO 24° 22' 29'' N 105º 55' 39'' W 10 H96(3), H97, H98(2), H104(2), H110(2)
5 Santa Rita, Nuevo. León SNLO 25° 09' 12'' N 100º 08' 41'' W 7 H91, H94 (2,), H95, H98(2), H99
6 Arteaga, Coahuila. ACOA 25° 26' 14'' N 100º 42' 30'' W 12 H81, H82(2), H83, H84(3), H85, H107(4)
7 Llano Grande, Guanaseví, DGO. LDGO 26° 04' 16'' N 106º 17' 15'' W 9 H88(4), H98, H102(2), H105, H109
8 Ejido El Nopal, Guadalupe y Calvo, CHI. ECHI 26° 05' 31'' N 107º 02' 11'' W 9 H86, H88, H93(2), H100, H110(4)
Northern populations H21(2), H22, H23(3), H52, H53, H54, H55, 9 Snow Bowl, Flagstaff, AZ. FLAZ 35º 17' 54'' N 111º 42' 54''W 13 H56(3) 10 John Day, OR. JDOR 44º 34' 53'' N 118º 31' 21''W 11 H4(2), H5, H6(2), H8, H9(2), H10(3)
11 Balm Creek Reservoir, OR. BCOR 44º 58' 49'' N 117º 33' 44''W 11 H4(4), H6(5), H18, H19
12 Mount Hebo, OR. MHOR 45º 10' 30'' N 123º 40' 08''W 11 H1, H4(2), H6(6), H27, H28 H4(2), H10, H11, H12, H13, H14, H15, H17, 13 Spring Creek, OR. SCOR 45º 20' 25'' N 118º 18' 50''W 11 H79(2) 14 Mt. Emily Road, OR. EROR 45º 25' 29'' N 118º 08' 32''W 9 H3(3), H4(4), H7(2)
15 Drum Hill, OR. DHOR 45º 27' 53'' N 118º 11' 25''W 10 H2, H6(7), H16, H80
16 Lubrecht Experimental Forest, Missoula, MT. MSMT 46° 53' 10'' N 113º 26' 55''W 11 H4, H6(2), H7(2), H9, H27, H28(2), H78, H79
17 Revelstoke, British Columbia. RVBC 51° 08' 15'' N 118º 16' 26''W 9 H4, H6, H27(3), H28(2), H78, H79 Population specific haplotypes are shown in bold, along with haplotype population frequencies.
32 Table 1.2. Haplotype and nucleotide diversities estimated for COI data in D. pseudotsugae populations.
Pop. Haplotype diversity (SE) Nucleotide diversity (SE) southern populations CDGO 0.583 (0.0023) 0.003 (0.00017)
PDGO 0.618 (0.0022) 0.003 (0.00017))
MDGO 0.889 (0.0014) 0.022 (0.0003)
EDGO 0.867 (0.0014) 0.015 (0.00024)
SNLO 0.905 (0.0017) 0.013 (0.00024)
ACOA 0.848 (0.0015) 0.018 (0.00024)
LDGO 0.806 (0.0019) 0.016 (0.00034)
ECHI 0.806 (0.0019) 0.015 (0.0003) northern populations FLAZ 0.910 (0.0013) 0.027 (0.00024)
JDOR 0.800 (0.0015) 0.007 (0.00017)
BCOR 0.709 (0.0017) 0.008 (0.00024)
MHOR 0.705 (0.002) 0.007 (0.00024)
SCOR 0.964 (0.0012) 0.011 (0.00017)
EROR 0.722 (0.0017) 0.002 (0.00017)
DHOR 0.533 (0.0023) 0.005 (0.00024)
MSMT 0.945 (0.0013) 0.012 (0.0003)
RVBC 0.889 (0.0016) 0.014 (0.00024)
Mean 0.945 (0.00052) 0.027 (0.00017) SE = Standard Error.
33 Table 1.3. Analysis of molecular variance (AMOVA) of COI data from D. pseudotsugae populations, including df, sum of squares, percentage of variation explained, P values and Φ statistics.
Source of variation Percentage of d. f. SS P Φ Statistic variation (%)
Between groups (ΦCT) (north and south) 1 4.292 7.72 0.01 0.077
Between populations (Φ ) SC 15 15.181 12.11 0.01 0.131
Within populations (ΦST) 155 62.102 80.16 0.01 0.198
34 Table 1.4. Fixation indices corresponding to the groups of populations inferred by SAMOVA for the D. pseudotsugae populations tested for the mtDNA sequences.
Number of groups FCT FST FSC
2 0.595** 0.705** 0.272**
3 0.615** 0.689** 0.194**
4 0.624** 0.677** 0.139**
5 0.631** 0.659** 0.076**
6 0.630** 0.656** 0.071**
7 0.621** 0.643** 0.059**
8 0.618** 0.636** 0.047**
9 0.618** 0.635** 0.045**
10 0.614** 0.624** 0.027**
11 0.614** 0.613** -0.001**
12 0.616** 0.612** -0.011**
13 0.619** 0.603** -0.041**
14 0.617** 0.595** -0.057*
15 0.619** 0.593** -0.069*
16 0.622* 0.591** -0.083*
*P < 0.01; **P < 0.001
35 Figure Captions
Fig. 1.1. Sample localities of Dendroctonus pseudotsugae and results from SAMOVA and Maximum Likelihood (ML) analyses. A) The geographic range of Douglas fir beetle corresponds to that of its host, Pseudotsuga menziesii. B) Groups found in
SAMOVA analysis are shown within dashed lines. C) Phylogenetic analysis by ML using the mitochondrial data set of Dendroctonus pseudotsugae. The best fit model of nucleotide substitution was TrN+G+I. Bootstrap values at nodes (500 pseudoreplicates).
Fig. 1.2. Scatter plot showing the relationship between genetic dissimilarities (estimated as FST / 1-FST) and logarithms of geographical distance of D. pseudotsugae populations
A) Southern populations. B) Northern populations. C) All 17 populations. Diamonds and continuous line slope, COI data. Regression equations reported only for significant relationships.
Fig. 1.3. Simple number of differences (p-distance) vs corresponding geographic distances among pairs of individuals of D. pseudotsugae.
36
0.6
0.5
T 0.4 S F -
1
/ 0.3 T S F 0.2
0.1
0 1 2 3 4 5 6 7 A Ln geographical distance (km)
0.7
0.6
0.5 T S F - 0.4 1
/ T S
F 0.3
0.2
0.1
0 1 2 3 4 5 6 7 8 B Ln geographical distance (km)
0.9 0.8
0.7 T
S 0.6 F – 0.5 1
/ y = 0.023x + 0.063 T
S 0.4 F 0.3
0.2 0.1
0 1 2 3 4 5 6 7 8 9 C Ln geographical distance (km)
38 0.2
0.18
0.16
0.14
0.12 e c n a t s i 0.1 d y = 1E-05x + 0.1139 p R² = 0.2388 rMantel= 0.488 P=0.0004 0.08
0.06
0.04
0.02
0 0 500 1000 1500 2000 2500 3000 3500
distance (km)
39 CAPÍTULO II
Análisis molecular y morfológico de Dendroctonus pseudotsugae (Coleoptera:
Curculionidae: Scolytinae): valoración del status taxonómico de las subespecies. Ruíz et al.: Dendroctonus pseudotsugae Enrico A. Ruíz
Escuela Nacional de Ciencias Biológicas-IPN.
Annals ESA Laboratorio de Variación Biológica y
Systematics Evolución. Departamento de Zoología.
Carpio y Plan de Ayala s/n, Col. Santo Tomás,
CP 11340 Mexico City, México.
Phone: 52 (55) 5729-6000 ext 62418
Fax: 52 (55) 5729-6000 ext 62418
E-mail: [email protected]
Molecular and morphological analysis of Dendroctonus pseudotsugae (Coleoptera:
Curculionidae: Scolytinae): an assessment of the taxonomic status of subspecies.
Enrico A. Ruíz1, Javier Víctor1, Jane L. Hayes2, and Gerardo Zúñiga1
1Escuela Nacional de Ciencias Biológicas-IPN. Laboratorio de Variación Biológica y
Evolución. Departamento de Zoología. Carpio y Plan de Ayala s/n, Col. Santo Tomás, CP
11340 Mexico City, Mexico.
2Forestry and Range Sciences Laboratory, Pacific Northwest Research Station, USDA Forest
Service, 1401 Gekeler Lane, La Grande, OR 97850
41 ABSTRACT
Dendroctonus pseudotsugae infests Douglas-fir, Pseudotsuga menziesii, throughout the distribution of that tree species from British Columbia to northern Mexico. The subspecies D. p. barragani was described from the mountains of Chihuahua, Sierra Madre Occidental,
Mexico, whereas the nominal subspecies, D. p. pseudotsugae, occurs north of Mexico. The description of D. p. barragani was based on the only known Mexican population at that time.
More recently, new populations of this beetle have been discovered at 13 additional localities in Chihuahua, Durango, Coahuila, and Nuevo Leon, Mexico. To test if these additional populations support the existence of two subspecies, we performed a taxonomic reassessment combining molecular markers (cytochrome oxidase I), morphological characters used in original description, and newly described morphological characters. Phylogenetic analysis of
89 haplotypes confirms that the Mexican populations are distinct from those from northern locations (in USA and Canada) sampled. Morphological analysis indicates that intraspecific variation is greater than previously considered within Mexican populations. However, at least seven characters on the head, pronotum, and elytra (including three previously undescribed characters of frons sculpture) consistently discriminate among Canada-USA and Mexico populations. The extension of the known distribution of this beetle in Mexico and verification of its subspecific status will aid the management and conservation of Pseudotsuga in Mexico.
KEYWORDS: Pseudotsuga, Dendroctonus pseudotsugae, mtDNA, subspecies
42 Introduction
Dendroctonus pseudotsugae Hopkins infests Douglas-fir, Pseudotsuga menziesii
(Mirb.) Franco, throughout the distribution of that tree species from British Columbia to northern Mexico. This bark beetle was first described from Grants Pass, Oregon (Hopkins
1909). A subspecies, D. p. barragani, was described from a disjunct population discovered in
Chihuahua, Mexico in 1974 (Furniss 2001). The description of D. p. barragani was based on morphological characters of the head, pronotum, and elytra; the manner of oviposition in the parent egg gallery; mating compatibility of beetles from Chihuahua and Idaho; and differences in associated insect fauna (Furniss and Cibrián-Tovar 1980). A recent study on genetic differentiation of D. pseudotsugae (using mitochondrial DNA sequences and nuclear DNA markers) showed that genetic differences were geographically correlated, and both markers also revealed that the genetic structure of D. pseudotsugae was influenced by geographic isolation (Ruíz et al. 2009). Observed genetic differences between northern (Canada-USA) and southern (Mexico) populations were also consistent within the two groups of populations that likely correspond to subspecies designation by Furniss (2001).
Our goal is to test if molecular markers and morphological characters allow recognition of monophyletic groups that correspond to at least two different sets of geographically distinct populations (Canada-USA and Mexico) in D. pseudotsugae. For this study, we acquired adult specimens from 13 new localities in four northern Mexico states (Chihuahua, Durango,
Coahuila, and Nuevo Leon) and compared them with specimens from 14 localities in the western USA and British Columbia.
43 Methods and Materials
Live adult beetles were collected from the bark of infested trees, preserved in absolute ethanol, and stored at -80°C until analysis or pinned for study and saved as vouchers in the
ENCB-IPN entomological collection. Some DNA samples were provided from colleagues without corresponding voucher specimens and were used only in the molecular data analysis.
Similarly, specimens loaned from museums were used only for morphological analyses.
Samples from 18 populations were used for both analyses. A total of 235 specimens from 24 localities were analyzed for molecular genetic markers, whereas 98 specimens from 22 localities were compared morphologically (Table 2.1).
Mitochondrial COI Amplification and Sequencing. Total genomic DNA was extracted and purified using DNeasy® Tissue Kit (QIAGEN GmbH, Hilden, Germany). PCR amplification of a 600 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene was carried out using primers C1-J-2441 and T12-N-3014 (Simon et al. 1994). DNA amplification was performed using a Biometra T Gradient thermocycler (Biometra GmbH, Hilden, Germany).
Samples were preheated for 5 min at 94 C, followed by 35 amplification cycles of 30 s at 94
C, 1 min at 51 C, 2 min at 72 C, and a final extension of 5 min at 72 C. Each PCR reaction mixture contained 50 ng of DNA, 1 μl primer 50 pM, 4 μl dNTP’s 10 mM (1 μl for each one),
6 μl MgCl2 25 mM, 5 μl 1x buffer, and 0.4 μl Taq DNA polymerase. The final volume was brought to 25 μl with ultra-pure water. All PCR reactions included negative controls to detect possible contamination.
PCR products were purified using GFXTM PCR DNA and gel band purification kit
(Amersham Biosciences, Buckinghamshire, UK). Cycle sequencing reactions were performed
44 with BigDye fluorescent chemistry reaction (Applied Biosystems, Inc., Foster City, CA). Both
DNA strands were sequenced in an ABI 377 sequencer, and editing of nucleotide sequences and manual alignment were performed using the software program SEQUENCHERTM v4.0.5
(citation?). Sequences obtained correspond to positions 2428 and 2977 in the Drosophila yakuba mitochondrial genome, and are available in GenBank under accession numbers
EU043405- EU043464 and EU193124-EU193152. The compiled DNA data matrix consists of
89 haplotypes of 550 bp long.
Sequence Analysis. Phylogenies were constructed using parsimony optimality criterion, with
PAUP* v4.0b10 (Swofford 2002) and MacClade v4.08 (Madison and Madison 2003). The
COI sequence of the sibling species Dendroctonus simplex LeConte was included as outgroup
(accession no. AF067985). Cladograms were generated using heuristic tree searches starting with 100 random stepwise addition replicates with the tree bisection and reconnection (TBR) option; no gaps were found along aligned sequences. All other settings were default. Non- parametric bootstrap values were established with 1,000 replicates and default PAUP* settings. Support for individual nodes on the resulting strict consensus cladogram was determined by calculation of Partitioned Bremer Support values (Bremer 1994), using
TREEROT.v2 (Sorenson 1999) in conjunction with PAUP*.
We also performed a maximum likelihood analysis, with a heuristic search strategy,
ACCTRAN, and TBR as branch swapping algorithm. To select the best-fit model(s) of nucleotide substitution that best describe the data, we used both the Akaike Information
Criterion (AIC) and hierarchical likelihood ratio test (hLRT), as implemented in Modeltest v3.7 (Posada and Crandall 1998). Both of them supported the Tamura-Nei model (Tamura and
45 Nei 1993) (TrN) with gamma (G) distributed rate variation and estimated proportion of invariable sites (I). A likelihood ratio test was performed to determine if the trees obtained followed a molecular clock. To test for monophyly, we performed the Shimodaira-Hasegawa test (Shimodaira and Hasegawa 1999). All analyses were performed using PAUP*. The assessment of pairwise sequence divergence (as well as corresponding mean, standard deviation and range), using the Jukes-Cantor model of nucleotide substitution (Jukes and
Cantor 1969), allowed us to test whether standardized percent nucleotide sequence divergence is useful to diagnose and assess subspecies boundaries (Hebert et al. 2003).
Morphological Analyses. Thirteen external morphological characters of specimens were examined at magnifications up to 90x to evaluate the patterns of variation within the species range and their taxonomic utility. These characters included those by Furniss (2001) and three new characters on the frons (Table 2.2). A subset of these specimens was photographed with a
JEOL JSM-5800LV scanning electron microscope.
Twelve of these morphological characters were coded for analysis under a phylogenetic framework (Table 2.2). The characters used by Furniss (2001) were reevaluated in order to code them in a precise and unambiguous way. For example, the sculpture of punctures on pronotum was coded into four character states (categories) according to the number of punctures per 0.01 mm² on the posterior half of the left side. Likewise, the relative size of crenulations on the base of the elytra was expressed as the ratio of the average width of crenulations 2 - 6 to the width of elytron base. Additionally, the relative size of tubercles on interstriae of elytral declivity in females was coded as the average basal width of five tubercles
46 on the second interstria of the declivity divided by the interstrial width, and these ratios were then divided into classes as character states.
Finally, phylogenetic analysis of morphological characters was carried out using a heuristic search strategy, with TBR branch swapping, starting with 1,000 random addition sequences. Non-parametric bootstrap values were established with 1,000 replicates and default
PAUP* settings. Analyses were performed with the PAUP* program.
Reconciling Data Sets. To assess character compatibility of both molecular and morphological data , we used an incongruence length differences test (ILD; Farris et al. 1995) as implemented by the partition homogeneity test in PAUP*. The test was used to find whether the matrices formed from the original separate data sets were significantly less homogeneous than those from randomly partitioned from the combined data set. When statistical significance is found, the distribution of phylogenetic information is not homogeneous between these original data matrices, suggesting incongruence between these data.
47 Results
Molecular Data Analysis. Editing of 235 COI sequences resulted in 89 different haplotypes of 550 bp. The average nucleotide composition was A + T rich (A = 0.29, C = 0.16, G = 0.13
T = 0.42), and the most informative nucleotide sites occupied the third codon positions. The low observed rates of change in first and second positions (nt 1 = 2.2%, nt 2 = 0% and nt 3 =
14.0%) are in agreement with the predicted model of amino acid conservation among insects
(Lunt et al. 1996).
With unweighted parsimony analysis using 550 molecular characters, we found 1,640 equally parsimonious solutions. A strict consensus of them is shown in figure 1.1. The tree shows a good basal resolution (many clades exhibited relatively high branch support values, including almost all internal branches), especially among two well-resolved sister clades which represent monophyletic groups: one that includes haplotypes from Canada and western
USA (clade α), and another including only haplotypes from northern Mexico (clade β). It is unclear which one is the basal clade, as both are placed at the same level and are the sister taxon of each other. Within each of these two clades, relationships among haplotypes were less resolved; however, haplotypes from southwestern USA (clade γ) are more basal than those of northwestern USA and Canada (clade δ). The haplotypes from the Sierra Madre
Oriental are the first from Mexico that arose (clade ε), while clade ζ shows little to no nucleotide variation, is poorly resolved and contains haplotypes from both the Sierra Madre
Oriental and Sierra Madre Occidental. Only individuals of Canada-USA and Mexico are found in clades α and β, respectively, and they correspond to monophyletic groups. Maximum likelihood analysis resulted in a single tree topology of –Ln = 3129.913 (not shown). Although
48 less resolved, the tree recovered the same tree topology and two monophyletic groups than maximum parsimony analysis. The Shimodaira-Hasegawa test supports the monophyly of both clades (P = 0.99).
The overall intraspecific pairwise mean calculated with the Jukes-Cantor model (Jukes and Cantor 1969) is 3.9%, almost twice the 2.0% difference frequently used to estimate species boundaries (Avise 2000, Hebert et al. 2003). This result supports previous suggestions that in many groups standardized percent sequence divergence fails to correctly diagnose species boundaries (Cognato 2006). The TrN + I + G model (with α = 0.533 for the γ- distribution and a proportion of invariable sites of 0.665) allowed us to estimate the corrected degree of sequence divergence among all sequences, which ranged from 0 to 21.2%, with an overall intraspecific pairwise mean of 6.2% (± 3.9%). The subdivision in more specific geographic ranges revealed that populations within northwestern USA (NWUSA) have the lowest mean sequence divergence (2.0%), while the highest mean (4.6%) is found in the Sierra
Madre Oriental (SMOR) (Table 2.3). An analysis of variance (ANOVA) of these corrected distances showed statistically significant differences among all geographic regions (P <
0.001). However, when sequence divergence of northern (Canada and USA) vs southern
(Mexico) populations are compared, no statistical difference was found (P < 0.118), revealing no difference between these regions. On the other hand, when all populations from Mexico
(SMOC and SMOR) are compared either with those from southwestern USA (SWUSA) or northwestern USA (NWUSA), high statistical significance is found (P < 0.001). A similar result is observed when all northern populations (Canada and USA) are compared with populations either from SMOR or SMOC (P < 0.001). Finally, pair-wise comparisons of
49 sequence divergence among geographic regions (SMOR, SMOC, SWUSA and NWUSA) showed high statistical significance (P < 0.001) in all cases but one (SMOR and SWUSA, P <
0.134). These results suggest that there is a stronger relationship of populations from southwestern USA and the Sierra Madre Oriental than between any other geographic ranges.
Morphological Data Analyses. Our analyses of morphological data confirm the diagnostic validity of some characters proposed in the original description of D. p. barragani (Furniss
2001), although other characters proved to be more variable. For example, regarding color, although beetles described by Furniss (2001) were mature and melanistic, beetles collected from other Mexican populations exhibited considerable variability in color; in general, color shows a continuous pattern of variation and varies with maturity of a specimen. Therefore, this character was excluded from the matrix for phylogenetic analysis.
In addition to Furniss’ characters, we identified three other previously undescribed features (Table 2.2: characters 11-13) of the frons sculpture as potentially useful for distinguishing among Canada-USA and Mexican population groups.
Parsimony analysis of the morphological dataset for 98 specimens from 22 populations
(plus D. simplex as outgroup) yielded >10,000 most parsimonious trees. Topology of the consensus tree (Fig. 2.8) allowed us to identify two major clades within the ingroup. One of these consists solely of specimens from Canada and the USA, while the other includes all specimens from Mexico. These two main clades correspond to a northern (Canada-USA) and a southern (Mexico) group of populations, putatively equivalent to D. p. pseudotsugae and D. p. barragani sensu Furniss (2001), respectively. Both clades have a high level of support. In contrast, relationships within each one of these clades are poorly resolved. Character
50 optimization for this phylogenetic scenario shows that seven of the 12 considered morphological characters show unambiguous and consistent differences among Canada-USA and Mexico groups of populations (consistency index = 1.00, Table 2.2). Here, we briefly describe these seven diagnostic characters and their corresponding character states:
Relative depth of epicranial suture: In D. pseudotsugae populations from Mexico, the suture located in the upper part of head, in the region of vertex, is deeply impressed, whereas in northern populations this suture is just shallowly impressed and frequently barely visible
(Fig. 2.2).
Median carina on pronotum: D. pseudotsugae exhibits a median line running longitudinally through pronotum. Nevertheless, in populations from Mexico, this median line arises as an elevated line forming a “keel” or carina separating a shallow depression on each side of pronotum, while in populations from USA and Canada this line passes through the pronotum without elevating or forming such a carina (Fig. 2.3).
Margin of strial punctures on elytra: In northern specimens, punctures forming the striae in elytra have a flat margin with no elevation; in contrast, in Mexican beetles such punctures show a conspicuously elevated margin, particularly in the anterior part (Fig. 2.4).
Relative width of second interestriae on elytral declivity: In northern populations, the second interestria on elytral declivity maintains its width constant through its whole extension, while in the Mexican ones interestria 2 has a distinctive constriction along its extremity, generally near its median portion (Fig. 2.5).
51 Median impression on the frontal region: A median, transverse, impressed line between the middle of the eyes is present and conspicuous in Mexican beetles, whereas it is apparently absent (or barely visible, under SEM examination) in beetles from Canada and USA (Fig. 2.6).
Distribution of crenulations on central area of frons: In Mexican specimens the frontal median line is completely surrounded by crenulate-like granules that form nearly concentric series of ridges around the median area of frons, which also has rather inconspicuous punctures. The sculpture of this area is similar in beetles from Canada and the USA, except that crenulations are absent at the central area of the frons and, therefore, the punctures are more evident (Fig. 2.6).
Relative abundance of tubercles on the lower frons region: Surface of the frons also has tuberculate granules (i.e., knob-like protuberances with rounded tips). In Mexican beetles, these tubercles are present but few in the central area of the frons and become slightly more abundant toward the epistomal region. In beetles from Canada and the USA, these tubercles are clearly more abundant, particularly in the lower middle of the frons, downwards to the epistomal region, where they are the most conspicuous element of the sculpture (Fig. 2.7).
52 Discussion
Molecular Character Analysis. mtDNA phylogenetic analysis supports that D. pseudotsugae subspecies are discrete entities (i.e., mtDNA lineages), each of them corresponding to a monophyletic group (Fig. 2.1): one containing only haplotypes from the USA and Canada
(clade α) and the other containing only haplotypes from Mexico (clade β). Within these monophyletic groups, potential or realized paraphyly was not observed and evidence of well- defined evolutionary entities was lacking. Despite low statistical support, however, all populations from northwestern USA and Canada form a clade δ that clearly arises from a basal clade consisting of southwestern USA haplotypes (Arizona), except Wyoming (clade γ), suggesting that northwestern North America was colonized in a general south-north direction with Wyoming possibly having been colonized later. Many haplotypes from Sierra Madre
Oriental (SMOR, clade ε) are basal with respect to all those of Sierra Madre Occidental
(SMOC, clade ζ), suggesting that the latter mountain system was colonized later. However, given the basal position of both α and β clades, the origin of both groups of populations seems to be related to a very specific past event, such as habitat fragmentation of the host. Non- overlapping intra- and interspecific sequence divergence has been used to establish a standardized percent nucleotide sequence divergence for diagnosing and assessing species boundaries (Hebert et al. 2003). However, this practice has been questioned as sequence divergence (including nuclear and mitochondrial loci) has been found to vary and overlap widely among insects (Cognato 2006).
The observed pattern of sequence divergence is lower within northwestern populations than in southwestern populations (Table 2.3). This may be the result of several interrelated
53 factors: retreat of glaciers in northwestern North America during Pleistocene warming, with subsequent host recolonization; fragmentation of the occurrence of Douglas-fir in their southernmost distribution; and regional differentiation and local demographic density of populations of D. pseudotsugae. At their peak (18,000 - 24,000 BP), glaciers covered most of
British Columbia, and permafrost extended to the Columbia River Gorge and the Cascade
Mountains of Oregon, and the northern Rocky Mountains (Hewitt and Ibrahim 2001). After this period, many conifers, including Douglas-fir, re-colonized the area. Previously, Douglas- fir was distributed throughout most of its current range and considerably farther northward
(Hermann 1985). Lower genetic diversity of present day northwestern D. p. pseudotsugae may be due to their recent colonization of the area, compared to the more stable southern populations. However, the percentage of sequence divergence alone is not appropriate for directly testing any of these possible explanations. An examination of likely causes of divergence would require additional data and analyses, such as methods of statistical phylogeography and nested clade phylogeographic, which are beyond the scope of objectives of this study.
Morphological Character Analyses. Analysis of morphological characters of beetles from the 13 new Mexican localities shows that intraspecific variation within Mexican populations is greater than previously considered (Furniss 2001), particularly due to some differentiation of
Coahuila populations with respect to those from Durango and Chihuahua. In addition, Texas specimens (the southernmost location of USA populations), are more related to beetles from
Canada-USA populations than to Mexican ones. Of the ten morphological characters originally proposed to identify D. pseudotsugae subspecies, four show unambiguous and
54 consistent differences among all Mexican and northern populations. The remaining characters identified in the original description of subspecies tend to show overlapping variation among groups of populations, and therefore are taxonomically more labile. However, we found three new features of frons sculpture that also enable separation of northern and southern populations. This combined set of previously described characters plus those proposed in the present study show a consistent pattern of variation among groups of populations found by the phylogenetic analysis. These seven features therefore represent a set of diagnostic characters that define each of these two main monophyletic groups.
Evolution of Dendroctonus pseudotsugae subspecies. Estimates of relationships among D. pseudotsugae populations revealed a consensus pattern of differentiation. Most striking is that every phylogenetic reconstruction using either molecular or morphological data yielded two well-resolved groups, each containing only individuals from Canada-USA or Mexico. This study enhanced the sampled range in the southern distribution of the species, and our results show that observed differentiation was not an artifact of incomplete sampling, as the inclusion of only one population from Mexico in the original description could have suggested. The molecular divergence of these two groups, and the morphological differences found strongly suggests that both subspecies could be ranked as full species. Other similar studies have suggested the presence of cryptic species in bark and cone pine beetles (DeGroot et al. 1992,
Kelley et al. 1999, Cognato et al. 2005). However, additional evidence from other sources
(chemical ecology, reproductive isolation, and additional molecular and morphological characters) is still necessary to fully determine if the evolutionary process of speciation is ongoing or completed.
55 Implications for Management and Conservation of Pseudotsuga in Mexico. Finally, the results of this study could be relevant to the management and conservation of Douglas-fir stands in Mexico, where Douglas-fir is a threatened species included in the Norma Oficial
Mexicana NOM-059-ECOL-2001 (SEMARNAT, 2002). For example, differences may exist between subspecies in their pheromones and olfactory response as has been shown to exist in geographically distant populations of another wide-spread North American bark beetle, Ips pini (Say) (Lanier et al. 1972). Researchers now have a rational basis for investigating such possibilities.
56 Acknowledgments
We thank Francisco Bonilla, Antonio Olivo, Luis M. Torres, Sergio Quiñones
(Comisión Nacional Forestal, Mexico) for providing field assistance; John Rinehart (Eastern
Oregon University), Gene Paul (Pacific Northwest Research Station, USDA Forest Service),
Brytten Steed (Forest Health Protection, USDA Forest Service) and Tom DeGomez (Northern
Arizona University) provided fresh or alcohol preserved specimens. Museum specimens were loaned by Ma. Eugenia Díaz (Museo de Historia Natural de la Ciudad de México), Ronald W.
Billings (Texas Forest Service, College Station, Texas), Patrice Bouchard (Canadian National
Collection, Ottawa), Christopher Marshall (Oregon State University, Corvallis), and Franck
M. Merickel (University of Idaho, Moscow). The manuscript was reviewed by Malcolm M.
Furniss, Moscow, Idaho, USA. The project was funded by Consejo Nacional de Ciencia y
Tecnología (CONACYT 44887) and Secretaría de Investigación y Posgrado-IPN (SIP-
20060421). JLH was supported by the Pacific Northwest Research Station. This work was part of E. A. Ruiz’s PhD dissertation. He was a CONACYT (176313) and Programa Institucional de Formación de Investigadores del Instituto Politécnico Nacional (PIFI-IPN) fellow.
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60 Table 2.1. Locations, geographic references, number of specimens analyzed and accession numbers in Dendroctonus pseudotsugae.
No. No. Samples Location Key Latitude Longitude Samples GenBank accession no. (Morphology) (DNA data)
Sierra Madre Oriental (SMOR)
Santa Rita, Nuevo León SNLO 25° 09' 12'' N 100° 08' 41'' W - 7 EU043405, EU043409, EU043406, EU043408,
EU043407
Montereal, Arteaga, Coahuila MCOA 25° 14' 32'' N 100° 26' 37'' W 5 10 EU193124, EU193125, EU193126, EU193127,
EU193128, EU193129
Los Lirios, Arteaga, Coahuila LCOA 25° 20' 02'' N 100° 33' 51'' W 3 8 EU193130, EU193131, EU193132
Rancho los Angeles, Arteaga, Coahuila ACOA 25° 26' 14'' N 100° 42' 30'' W 5 12 EU043410, EU043415, EU043411, EU043414,
EU043412, EU043413
El Pilar, Arteaga, Couahila PCOA 25° 17' 12'' N 100° 30' 31'' W 5 - -
La Ciruela, Arteaga, Coahuila CCOA 25° 14' 03'' N 100° 27' 01'' W 5 - -
Sierra Madre Occidental (SMOC)
Ejido Cienega de la Vaca, San Dimas, CDGO 24° 05' 20'' N 105° 31' 00'' W - 9 EU043427, EU043426, EU043425, EU043418
DGO
Ejido Puentesillas, San Dimas, DGO PDGO 24° 21' 10'' N 105° 54' 39'' W 5 11 EU043423, EU043421, EU043424, EU043422,
EU043418
Ejido La Manga, San Dimas, DGO MDGO 24° 22' 08'' N 105° 58' 15'' W 5 9 EU043416, EU043417, EU043409, EU043408,
EU043418
Ejido Nuñez, San Dimas, DGO EDGO 24° 22' 29'' N 105° 55' 39'' W 5 10 EU043419, EU043420, EU043408, EU043421,
EU043418
61 Llano Grande, Guanaseví, DGO LDGO 26° 04' 16'' N 106° 17' 15'' W 5 9 EU043428, EU043408, EU043430, EU043429,
EU043422
Ejido El Nopal, Guadalupe y Calvo, CHI ECHI 26° 05' 31'' N 107° 02' 11'' W 5 9 EU043416, EU043428, EU043431, EU043432,
EU043418
Ejido catedral, Guadalupe y Calvo, CHI CCHI 26° 12' 40'' N 106° 33' 49'' W 2 5 EU193133, EU193134
San Juanito, Bocoyna, CHI JCHI 27° 58' 09'' N 107° 36' 27'' W 5 - -
Southwestern USA (SWUSA)
Coronado NF, AZ CRAZ 32° 50' 54'' N 109° 43' 01'' W 3 10 EU193135, EU193136, EU193137
Apache Sitgreaves NF, AZ APAZ 34° 24' 29'' N 110° 02' 21'' W 4 11 EU193138, EU193139, EU193140, EU193141
Flagstaff, Coconino NF, AZ FLAZ 35° 17' 54'' N 111° 42' 54'' W - 13 EU043436, EU043438, EU043434, EU043435,
EU043437, EU043439, EU043433, EU043440
Peaks, Coconino NF, AZ PKAZ 35° 21' 30'' N 111° 33' 53'' W 5 9 EU193142, EU193143, EU193144, EU193145,
EU193146
Culbertson, TX CBTX 31° 56' 49'' N 104° 51' 07'' W 3 - -
Northwestern USA and Canada
(NWUSA)
Pinedale, WY SBWY 42° 52' 10'' N 109° 52' 05'' W 5 10 EU193147, EU193148, EU193149, EU193150,
EU193151, EU193152
Vinegar-Vincent Drainage, Malheur NF, JDOR 44° 34' 53'' N 118° 31' 21'' W 5 11 EU043445, EU043446, EU043444, EU043447,
OR EU043448, EU043449
Balm Creek Reservoir, WWNF OR BCOR 44° 58' 49'' N 117° 33' 44'' W - 11 EU043445, EU043444, EU043458, EU043457
Mount Hebo Rd, Siuslaw NF, OR MHOR 45° 10' 30'' N 123° 40' 08'' W 5 11 EU043443, EU043445, EU043444, EU043441,
EU043442
Spring Creek, WWNF, La Grande, OR SCOR 45° 20' 25'' N 118° 18' 50'' W 5 11 EU043445, EU043449, EU043455, EU043452,
62 EU043454, EU043450, EU043456, EU043451,
EU043453
Mount Emily Rd, WWNF, La Grande, OR EROR 45° 25' 29'' N 118° 08' 32'' W 3 9 EU043462, EU043445, EU043463
Drumhill Ridge, WWNF, La Grande, OR DRHL 45° 27' 53'' N 118° 11' 25'' W - 10 EU043460, EU043444, EU043459, EU043461
Lubrecht Experimental Forest, Missoula, MSMT 46° 53' 10'' N 113° 26' 55'' W - 11 EU043445, EU043444, EU043463, EU043448,
MT EU043441, EU043442, EU043464. EU043453
Revelstoke, British Columbia RVBC 51° 08' 15'' N 118° 16' 26'' W 5 9 EU043445, EU043444, EU043441, EU043442,
EU043464. EU043453
Haplotypes (accesion numbers) found in only one population are shown in bold.
63 Table 2.2. Morphological character state distribution of D. pseudotsugae, as reported by Furniss (2001) and obtained by the present study. Feature State Furniss (2001) This study
A B A B
1. Color “Elytra and body uniformly dark brown to black; head always darker except all black specimens” x x x
“Elytra often reddish” x x x
2. Relative depth of epicranial suture Deeply impressed x x
Shallowly impressed x x
3. Anterior sutures of pregula Joined almost perpendicularly (at an angle close to 90°) x x x
Joined forming a slight curvature close to their joining point x x x
4.Punctures on pronotum “More widely spaced, generally more coarse” x
“More closely spaced, generally finer” x
11 - 12 punctures on pronotum per 0.01 square mm x x
13 - 14 punctures on pronotum per 0.01 square mm x x
15 - 16 punctures on pronotum per 0.01 square mm x x
17 - 18 punctures on pronotum per 0.01 square mm x x
5. Median line on pronotum Anterior one third forming a carina separating a shallow depression on either side x x
Anterior one third without a carina x x
6. Crenulations on basal margins of elytra “Larger, more uniform in size and shape” x
“Smaller, less uniform in size and shape” x
Ratio of relative size of basal crenulations = 0.060-0.069 x x
Ratio of relative size of basal crenulations = 0.070-0.079 x x
Ratio of relative size of basal crenulations = 0.080-0.089 x x
Ratio of relative size of basal crenulations = 0.090-0.099 x x
64 7. Margin of strial punctures on elytra Distinctly elevated, particularly in the anterior part x x
Flat, with no elevation (margin flush with integument) x x
8. Crenulations on interstriae of elytra “More coarsely rugose” (i.e., bigger and abruptly elevated crenulations) x x x
“More finely rugose” (i.e., small and just slightly elevated crenulations) x x x
9. Interstrial tubercles on elytral declivity (in “Larger, more uniform in size” x females) “Smaller, more variable in size” x
Ratio of width of tubercles and the second interstriae on elytral declivity = 0.36-0.40 x
Ratio of width of tubercles and the second interstriae on elytral declivity = 0.41-0.45 x x
Ratio of width of tubercles and the second interstriae on elytral declivity = 0.46-0.50 x x
Ratio of width of tubercles and the second interstriae on elytral declivity = 0.51-0.55 x
10. Relative width of interestriae 2 on Width constant through its whole extension or uniformly, gently tapered x x elytral declivity Width distinctly constricted along its extremity, generally near its median portion x x
11. Median impression on the frontal region Present and conspicuous x
Absent (or barely visible, with SEM) x
12. Distribution of crenulations on central Forming concentrical series of ridges around the median area of frons x area of frons Absent on the central area at the middle of the frons x
13. Relative abundance of tubercles on the Present but few in the central area of the frons; slightly more abundant toward the epistomal region x lower frons region More abundant, particularly in the lower middle of the frons X
A, D. p. barragani; B, D. p. pseudotsugae. Diagnostic characters are in bold font.
65 Table 2.3. Mean percentage of sequence divergence (using the TrN+I+G substitution model) of D. pseudotsugae populations by geographic range.
Geographic Populations Mean percentage, SD
range (range)
NWUSA SBWY, JDOR, MHOR, SCOR, EROR, 2.0 ± 1.9 (0.0 - 10.8)
RVBC
SWUSA CRAZ, APAZ, FLAZ, PKAZ 4.4 ± 2.4 (0.4 - 11.4)
SMOR MCOA, LCOA, ACOA 4.6 ± 2.4 (1.0 - 11.4)
SMOC PDGO, MDGO, EDGO, LDGO, ECHI, CCHI 2.9 ± 2.8 (0.2 - 16.8)
MEX MCOA, LCOA, ACOA, PDGO, MDGO, 4.0 ± 2.7 (0.2 - 16.9)
EDGO, LDGO, ECHI, CCHI
N. AMERICA CRAZ, APAZ, FLAZ, PKAZ, SBWY, JDOR, 3.9 ± 2.4 (0.0 - 12.0)
MHOR, SCOR, EROR, RVBC
ALL 6.2 ± 3.9 (0.0 - 21.2)
66 Figure Legends
Fig. 2.1. Phylogram of D. pseudotsugae individuals inferred from 550 nucleotides of mitochondrial COI gene. The tree is one of 1,640 most parsimonious trees (TL = 472, CI =
0.35, RI = 0.73, RC = 0.26) found after a heuristic search using PAUP* (see text for details).
Bremer support values (below) and Bootstrap support values (above) are given for each internal branch.
Fig. 2.2. Relative depth of epicranial suture. (A) Shallowly impressed. (B) Deeply impressed.
Fig. 2.3. Median carina on pronotum. (A) Without elevation or carina. (B) Forming a carina on its anterior third.
Fig. 2.4. Margin of strial punctures on elytra: (A) Strial punctures with flat margin. (B) Strial punctures with elevated margin.
Fig. 2.5. Interstria 2 on elytral declivity. (A) Constricted. (B) Uniform in width.
Fig. 2.6. Frontal region of head. (A) Without a median impression, and without crenulations on the central area. (B) With a well defined median impression, surrounded by crenulations on whole median area of frons.
Fig. 2.7. Tubercles on lower frons region. (A) Abundant, particularly towards epistoma. (B)
Scarce, and not covering most of epistoma.
Fig. 2.8. Cladogram of D. pseudotsugae individuals inferred from 12 morphological characters. The tree is a consensus of > 10,000 most parsimonious trees (TL = 40, CI = 0.45,
RI = 0.95, RC = 0.43) found after a heuristic search. Bremer support values (below) and
Bootstrap support values (above) are given for each internal branch.
67 68 A B
Fig. 2.2. Relative depth of epicranial suture
69 A B
Fig. 2.3. Median carina on pronotum
70 A B
Fig. 2.4. Margin of strial punctures on elytra
71 A B
Fig. 2.5. Interstria 2 on elytral declivity
72 A B
Fig. 2.6. Frontal region of head
73 A B
Fig. 2.7. Tubercles on lower frons region
74 75 CAPÍTULO III
Historia demográfica y filogeografía de un escarabajo descortezador especialista,
Dendroctonus pseudotsugae Hopkins (Curculionidae: Scolytinae). Historical demography and phylogeography of a specialist bark beetle, Dendroctonus pseudotsugae Hopkins (Curculionidae: Scolytinae).
Enrico A. Ruiza, John E. Rinehartb, Jane L. Hayesc and Gerardo Zúñigaa
aLaboratorio de Variación Biológica y Evolución, Escuela Nacional de Ciencias Biológicas,
IPN, Prolongación de Carpio y Plan de Ayala s/n, col. Santo Tomás, C.P. 11340, México,
D.F., México.
bBiology Program, Eastern Oregon University. 1 University Boulevard, LaGrande, OR 97850
cForestry and Range Sciences Laboratory, Pacific Northwest Research Station, USDA Forest
Service, 1401 Gekeler Lane, LaGrande, OR 97850
Corresponding Author: Enrico A. Ruiz
Laboratorio de Variación Biológica y Evolución, Escuela Nacional de Ciencias Biológicas-
IPN. Prolongación De Carpio y Plan de Ayala s/n, Col. Santo Tomás, C.P. 11340 México D.
F., México.
Telfax: 52 (55) 5729-6000 ext. 62418
E-mail: [email protected]
77 Abstract
Contemporary distribution of North American species has been shaped by past glaciations of the Quaternary period. However, their effects were not that severe in southern
Rocky Mountains and Northern Mexico. In this study, we test hypotheses about location of ancient refugia at the Pacific Northwest and Rocky Mountains of Dendroctonus pseudotsugae, based on mtDNA COI. We also hypothesized that southern North American and Mexican populations are more diverse and experienced constant population sizes trough time than northern ones. We identified 136 haplotypes out of 331 sequences (550 bp). The phylogenetic analysis yield four well resolved haplogroups corresponding to Northwestern North America
(NNA), Southwestern North America (SNA), Sierra Madre Occidental (SMOC) and Sierra
Madre Oriental (SMOR). Hypothesis of demographic expansion of haplogroups was examined trough neutrality tests against population growth, mismatch distribution, and Bayesian Skyline
Plots. Results showed that NNA and SMOC haplogroups have experienced demographic expansion events while SNA and SMOR did not. Divergence times between pairs of haplogroups were estimated from early to middle Pleistocene, long before last glacial maxima.
Finally, the nested clade analysis provided evidence that the demographic change were accompanied by continuous range expansion events in northwestern North America, while historically reduced gene flow and allopatric fragmentation was inferred in populations from
Arizona and Northern Mexico. Results of the present study provide a basis to test for congruence with the intraspecific variation of its plant host.
Keywords: Dendroctonus pseudotsugae; Past glaciations; Divergence time; Demographic expansion; Range expansion; allopatric fragmentation.
78 Introduction
It has been long suggested that common factors leading to contemporary distribution of north hemisphere species are the past glacial-interglacial cycles of the Quaternary period.
Ancient pollen and fossil record shown that the distribution of organisms changed very rapidly as the ice sheets went back and forth (Hewitt, 1996; Hewitt and Ibrahim, 2001 and references therein). A direct consequence of retracting ice sheets has been a shift in species distribution range: northern territories become available for colonizers, and their genetic diversity tends to be lower than that of southern populations. However, such a pattern of lower genetic diversity in the north and higher genetic diversity in the south also depends on fitness, regional geography and the existence of particular relationships with other organisms, such as prey or host. Comparison among several taxa over the same area allowed the identification of congruent geographical patterns of genetic variation and colonization routs, and lead to the inference of common historical factors (Soltis et al., 1997; Taberlet et al., 1998; Arbogast et al., 2001; Petit et al., 2003; Avise, 2004; Hewitt, 2004).
Many members of bark-beetles and other endemic organisms occur within the temperate rain forest of Pacific Northwest of North America. Despite some of them are codistributed species that actually differ in their phylogeographic patterns, it is likely that common vegetation zones and environments make them respond similarly to past climatic events. Some studies have shown that this ecosystem has been shaped by Pleistocene glacial processes, raising many competing phylogeographic hypotheses regarding the number and location of Pleistocene refugia (Brunsfeld, 2001; Carstens et al., 2005a,b; Steele and Storfer,
2006; Brunsfeld et al., 2007). Specifically, phylogeographic inferences of some Dendroctonus
79 species have also been made. This allowed identifying, as a result of multiple glacial cycles, at least three different refugia (Beringia, Rocky Mountain and northwest United States) in the
Pacific Northwest for D. rufipennis (Maroja et al., 2007); divergent populations bearing molecular signal of recent range-wide expansion to the north in D. ponderosae (Mock et al.,
2007) and continuous dispersion events, allopatric fragmentation and long distance colonization for D. mexicanus Hopkins, with no clear relationships between contemporary distribution and mountains systems or hosts (Anducho-Reyes et al., 2008).
Here, we study another species distributed within this area is the Douglas-fir beetle,
Dendroctonus pseudotsugae Hopkins, which follows the distribution range of its only host,
Douglas-fir (Pseudotsuga menziesii). The distribution range of this beetle matches that of its host in the Pacific Northwest and Rocky Mountains. On the other hand, in their southern most range (Mexico) it is only found on Douglas-fir occurring in Sierra Madre Oriental and Sierra
Madre Occidental, but not on those Douglas-firs occurring in central Mexico.
Recently, a number of studies have defined and supported the existence of two subspecies within D. pseudotsugae: Based on morphological and biological attributes Furniss
(2001) first reported the existence of D. p. pseudotsugae from the USA and Canada and D. p. barragani from a population in northern Mexico; Through the use of mitochondria (COI) and nuclear (RAPD) markers, Ruiz et al. (2009) demonstrated that isolation by distance has been the main factor leading to the genetic differentiation in populations of D. pseudotsugae corresponding to subspecies defined by Furniss (2001); and based on molecular (COI) and morphological characters, phylogenetic analyses allowed recovering two discrete, distinct monophyletic clades that also correspond to D. pseudotsugae subspecies aforementioned
80 (Ruiz et al. unpublished data). Nevertheless, none of these studies were designed to fully resolve the relationships among population lineages.
The purpose of this study was to test hypotheses about location of ancient refugia in the Pacific Northwest and Rocky Mountains (and corresponding expansion routes from these refugia), as well as to explore if southern North American and Mexican D. pseudotsugae populations are more diverse and have experienced constant population sizes trough time than northern ones. This is predicted by rapid shifts due to glacial-interglacial cycles of the
Quaternary period, and the corresponding lack of such events in southern range of the species, respectively (Brunsfeld, 2001; Steele and Storfer, 2006; Aoki et al., 2008). Exact location of supposed refugia is difficult because in the case of Pseudotsuga the pollen is not distinguishable from that of Larix Mill. Therefore, some of the pollen percentages that describe the past distribution of Pseudotsuga may actually represent Larix (Bartlein et al.,
1998). However, south of the ice sheet during last glacial maxima the plant-macrofossil data generally include identification of the taxa present at the species level (e.g. National Climatic
Data Center, http://www.ncdc.noaa.gov/paleo/). Recent observed and simulated data on past distribution of Pseudotsuga only span the last 21 000 years, and shown that at the end of last glacial maxima the distribution range of Pseudotsuga was much reduced than is seen today. In this scenario, Pseudotsuga forests were mainly confined to southern USA and Northern
Mexico, while it was almost absent on the Pacific slope of Washington and Oregon. Later, from 16 000 to 11 000 BP observed distribution continually decreased as temperatures rise and southern USA deserts expanded. From there Pseudotsuga distribution increases to the north, via the pacific coast, until reach its current range (Bartlein et al., 1998).
81 By constructing evolutionary models, along with coalescing simulated data underlying these models, we assessed the probability that observed data were generated by these particular evolutionary scenarios. We also inferred the dynamics of temporal, distributional and demographic history of D. pseudotsugae populations. To these ends, our study was based on current geographic distribution and intraspecific genetic diversity of mtDNA COI. The assessment of phylogeographical patterns allowed us to test whether demographic expansions have occurred within groups of populations, and to reconstruct the past demographic history of
D. pseudotsugae.
82 Materials and methods
Sample collection and DNA amplification
Population sample locations were chosen to cover the entire distribution of D. pseudotsugae in North America and Northern México (Table 3.1; Fig. 3.1). Live adult beetles were collected directly under the bark of different recently infested trees, preserved into 100% ethanol, and stored at -80°C until analyses. Individuals were collected covering their entire distribution range (Fig. 3.1). DNA extractions were carried out using DNeasy® Tissue Kit
(Qiagen), following manufacturer´s protocol. Amplification of mtDNA COI region for 96 individuals was carried out as described in Ruiz et al. (2009), using primers C1-J-2441 and
TL2-N-3014 (Simon et al., 1994). Amplicons were purified using GFXTM PCR DNA and Gel
Band Purification Kit (Amersham Biosciences), and sequenced with an ABI Prism 3100
Genetic Analyzer (Applied Biosystems). We did not observed PCR ghost bands neither found a significant proportion of sequence ambiguities, which suggest that nuclear pseudogenes of mtDNA (Numts) were not amplified (Benasson et al., 2001).
Sequences were edited manually and aligned with ClustalX 1.83 (Thompson et al.,
1997). Accession nos. of the resulting 47 haplotypes are FJ174745- FJ174791. We also included 235 sequences of COI gene from earlier studies (Ruiz et al. 2009; Ruiz et al. unpublished data.), which are also deposited in GenBank. Locations were grouped within regions as described in Table 3.1.
Nucleotide polymorphism
We estimated the number of segregating sites, haplotypes, haplotype diversity, nucleotide diversity and average pairwise nucleotide difference per site for each region and all
83 regions combined using DnaSP v4.50 (Rozas et al., 2003). We also used Tajima's (Tajima,
1989) D statistic, Fu and Li's (1993) D and Fu's (1997) FS to detect departures from the mutation-drift equilibrium that could be indicative of natural selection. The tests were implemented using both DnaSP and Arlequin v3.11 (Excoffier et al., 2005).
Molecular phylogenetic reconstruction
To reconstruct the phylogenetic relationships among mitochondrial DNA haplotypes, we used the maximum-likelihood algorithm implemented in the program aLRT-PHYML
(Guindon and Gascuel, 2003). Before the ML analysis, we determined an appropriate model of
DNA evolution and model parameters using both the Akaike Information Criterion (AIC =
8834.07, -lnL = 4409.04, K = 8) and hierarchical likelihood ratio test (-lnL = 4412.65, K = 7), as implemented in Modeltest v3.7 (Posada and Crandall, 1998). Both of them supported the
Tamura-Nei model (Tamura and Nei, 1993) with gamma (G = 0.51) distributed rate variation and estimated proportion of invariable sites (I = 0.63). These G and I values, along with optimized base frequency and ratio of transition/transversions (under maximum likelihood criterion) were used in aLRT-PHYML. We selected to optimize the topology of the tree, rather than branch length. To estimate the reliability of each node, the approximate likelihood ratio test (aLRT, Anisimova and Gascuel, 2006) with the Shimodaira-Hasegawa-like procedure option was used. Sister species Dendroctonus simplex LeConte was used as outgroup
(accession no AF067985).
Analysis of demographic history
Signatures of population demographic changes were examined following three different approaches. All of them were designed to test the major haplogroups as revealed by
84 the molecular phylogenetic analyses [Northwestern North America (NNA), Southwestern
North America (SSA), Sierra Madre Oriental (SMOR) and Sierra Madre Occidental (SMOC) in figure 3.2]. These approaches include: i) Neutrality tests against population growth, ii) distribution of pairwise differences (mismatch distribution; Rogers and Harpending, 1992), and iii) coalescent-based Bayesian skyline plots (BSP; Drummond et al., 2005). The Tajima's
D statistic, although originally designed to test for selective neutrality, can also be used to infer demographic history. For stable populations at demographic equilibrium, the estimated value of theta (θ = 2Neμ) is the same, based either on the number of segregating sites (θS) or on pairwise nucleotide differences (θπ). Since θπ assesses the frequency of the mutant alleles, it is more sensitive to recent changes in effective population size. In this way, negative values of
Tajima's D statistic can reveal recent demographic expansion. We also used Fu's FS (1997) and the Ramos-Onsins and Rozas's R2 statistics (Ramos-Onsins and Rozas, 2002), given that both tests have been demonstrated to have superior statistical power for small or big sample sizes, for low and high number of mutations and for reasonable population growth parameters
(Ramos-Onsins and Rozas, 2002). To test for significance, we generated 10000 random simulations of the data, under the hypothesis of selective neutrality and population equilibrium. The three tests were implemented using either DnaSP or Arlequin programs (see above).
Second, to test for sudden population expansion we used the mismatch distribution. In this approach, the distribution of pairwise sequence differences is expected to be multimodal when haplotypes are drawn from populations at demographic equilibrium. Conversely, when haplotypes are drawn from populations that have undergone sudden demographic expansion
85 they show unimodal distribution of pairwise differences (Slatkin and Hudson, 1991; Rogers and Harpending, 1992). To reflect a sudden population growth from N0 to N1 at t generations ago, the approach implemented in Arlequin v3.11 assumes a stepwise expansion model. We followed the method of Schneider and Excoffier (1999), and estimated three demographic parameters: θ0 = 2µN0; θ1 = 2µN1 and τ = 2ut, where u is the mutation rate for the whole gene region. The goodness-of-fit of the observed data to a simulated model of expansion was tested with the sum of squared deviations (Excoffier, 2004). We also computed the raggedness index
(rg) of Harpending (1994). When the validity of the model was confirmed, we further investigated the time since the putative expansion event from τ, using a generation time of this insect as 1 year, and a divergence rate of 2.3 % per million years (Brower, 1994). For all these parameters we also obtained confidence intervals (95%) by means of 10000 randomizations of the data.
Finally, we investigated the population history of major groups backwards in time by using the Bayesian Skyline Plots. BSP is a new method for estimating ancestral population dynamics from DNA sequences, and allow taking into account both the error inherent in phylogenetic reconstruction and stochastic error intrinsic to the coalescent process
(Drummond et al., 2005). The method uses a Markov chain Monte Carlo procedure to sample the distribution of generalized skyline plots, given the data and according to corresponding posterior probabilities. Generalized skyline plots produce smoother population size estimates than previous skyline plots (Pybus et al., 2000), as it reduces the number of parameters employed. The plots are combined to assess an estimate (with the corresponding 95% credibility intervals) of effective population size at every point backward in time until the most
86 recent common ancestor is reached. Using previously selected substitution model (see above), we calculated BSPs using program BEAST v1.4.8 (Drummond and Rambaud, 2007). We used the Bayesian skyline coalescent as the tree prior. The MCMC was run for 10 million steps and sampled every 1000 steps. We discarded the first 10% as burnin. This resulted in effective sample sizes (ESS) for the posterior probability of much more than 200 for all four analyses.
Convergence of chains and ESS for each parameter were evaluated using the program
TRACER v1.4 (Drummond and Rambaud, 2007).
Migration and divergence time between D. pseudotsugae populations
To distinguish between models of isolation with and without gene flow, and estimate divergence times between D. pseudotsugae groups of populations inferred by phylogenetic analyses, we used the MDIV program (Nielsen and Wakeley, 2001). The software allows obtaining reliable joint estimates of divergence times and migration rates of two populations.
MDIV estimates M (= Ne m = number of migrants between populations per generation), θ (=
2Ne µ), and T (the divergence time between populations where 1 time unit = Ne generations).
The program was first run using default searching settings and default priors. Then we set our prior value of Mmax and Tmax to 10 and 5, respectively, because they produced consistent and well-behaved posterior distributions. The integrated likelihood function surfaces were obtained using the finite sites model HKY, with 5000 000 generation of Metropolis-Hastings
Markov chain Monte Carlo and a 1000 000 generation burn-in time to explore the solution space. This was repeated three times to ensure convergence upon the same posterior distributions for each of the parameter estimates. The modes of posterior distribution for both
θ and T were used to estimate divergence times between haplogroups, according to the
87 formula: Tpop = [(TpopΘ) /2L] 1/μ (Brito, 2005), where L is the sequence length (550 bp) and μ the mutation rate per site per generation. Here, we used the often cited 1.1% per million years per lineage for arthropod mitochondrial DNA (Brower, 1994).
Nested clade phylogeographic analysis
Recently, controversy about nested clade phylogeographic analysis (NCPA) has questioned its legitimacy (Panchal and Beaumont, 2007; Garrick et al., 2008; Petit, 2008;
Templeton, 2008). Nonetheless, we decided to include it because it is the only method of phylogeographic analysis that has been validated using many data sets of positive controls, covering a wide range of species, evolutionary scenarios, geographic scales, and sampling designs (Templeton, 2008). It is also capable of handle large numbers of locations and individuals, and the nature of inferences made is stronger than those yielded by other methods
(Templeton, 2009). We first constructed a statistical parsimony network (Templeton et al.,
1992) to investigate relationships among haplotypes using the program TCS v1.21 (Clement et al., 2000). Ambiguous loops in the network were resolved following frequency and topological criteria (Pfenniger and Posada, 2002). The nesting procedure of the entire network
(Templeton et al., 1987) was performed using GeoDis v2.5 (Posada et al., 2000). This analysis allowed testing the null hypothesis of no geographical association of mtDNA haplotypes
(Templeton and Sing, 1993; Templeton at al., 1995). Concerns raised about validity of inferences using Templeton's key (as mentioned above) lead us to make inferences with caution. Interpretation of the results was made using the inference key provided along with
GeoDis software.
88 Results
Genetic variation
We sequenced 550 nucleotides of mitochondrial COI, and 143 variable sites were found. We identified 136 haplotypes out of 331 sequences; 70 haplotypes were shared among several individuals while each of remaining 66 haplotypes were private. All populations have at least 1 unique haplotype, not shared with other populations (Table 3.1). The three most common haplotypes were found in northern Rocky Mountains (H004, H006) and in southern
Sierra Madre Occidental (H110), representing 17.82% of all haplotypes. Relative high proportion of haplotypes to individuals sequenced (about 41%) reveals a poor likelihood of having sequenced slower evolving nuclear copies (Sorenson and Fleischer, 1996; Pereira and
Baker, 2004).
All regions analyzed are characterized by high haplotype diversity (Hd = 0.818 –
0.964). The mean nucleotide diversity (π) estimated was 0.034 (SD = 0.0009). Nonetheless, π values vary greatly among regions (Table 3.2): it is higher on the Eastern Coahuila (EC) and
Western Wyoming (WW) regions (both with π = 0.031), followed by Northern Colorado region (π = 0.029); while in Western and Eastern Oregon lower π values were found (π =
0.011 and π = 0.007, respectively). Fu and Li's D-test and Fu's FS test (Table 3.2) shown results of over-abundance of singleton mutations and rare haplotypes in some regions under study; however, none of them (with one exception, in Western Oregon) are statistically significant (P < 0.05), reflecting no departures from the mutation-drift equilibrium. On the other hand, the Tajima's D values were lower than expected in eight regions (SD, WD, SC,
NC, WO, EO, WM and NW), but statistical significance was reached only in WO (P = 0.032).
89 The remaining populations showed Tajima's D values higher than expected (Table 3.2), but no statistical significance was observed. This test also revealed no statistical significance for the total of the geographic samples (D = -0.52339, P > 0.10).
Molecular genealogy of the D. pseudotsugae mtDNA haplotypes
The phylogenetic analysis allowed identifying two well resolved monophyletic clades
(clades I and II; Fig. 3.2). Within these two clades, high resolution was also recovered. Clade I contains two well resolved groups, one with haplotypes from Northwestern North America
(NNA, clade III) and the other with fewer haplotypes from Southwestern North America
(SNA, clade IV). The Clade II also has two well resolved groups, one with haplotypes from
Sierra Madre Occidental (SMOC, clade VI) and the other with fewer haplotypes from Sierra
Madre Oriental (SMOR, clade V). Most of the clades (including the aforementioned) have strong nodal support values (aLRT > 60). However, phylogenetic relationships within these four haplogroups could not be fully resolved. While haplotypes from the SMOC group were found in all four Mexican regions (EC, SD, WD and SC), the haplotypes from the SMOR group were found in all of them except the southern Durango (SD) region (Fig. 3.2). On the other hand, haplotypes from the NNA clade were found in all North American regions (SA,
NA, NC, WW, WO, EO, WM, NI, NW, and BC). Haplotypes from SSA were only found in southern and northern Arizona regions (SA and NA; Fig. 3.2).
Demographic history
The Tajima's D reveals negative values for NNA, SMOR and SMOC haplogroups found in the phylogenetic analysis, but not in SNA (Table 3.3). However, none of them were statistically different from zero (P > 0.05). Negativity of this value is consistent with
90 demographic expansion events. However, both FS, and R2 tests revealed statistical significance only for NNA haplogroup, strongly suggesting that populations from Northwestern North
America have experienced demographic expansion events. On the other hand, the mismatch distribution of observed mitochondrial haplogroups failed to reject the model of sudden demographic expansion in NNA and SMOC (Table 3.3; Fig. 3.3A). In contrast, SNA and
SMOR haplogroups (SSD = 0.0224, P < 0.05; SSD = 0.0494, P < 0.01, respectively) showed a multimodal mismatch distribution, suggesting stable population size trough time (Table 3.3;
Fig. 3.3A). Estimation of time since the putative expansion event from τ (using 2.3% per million years) for NNA and SMOC was dated to 230 000 (95% confidence interval: 137 000 -
539 000) and 115 000 (95% confidence interval: 400 000-522 000) year ago, respectively.
Historical demographic reconstructions using Bayesian skyline plots (BSP) are shown in Fig.
3.3B. Haplogroup NNA appears to have experienced a long continuous population growth, going back from before 6 to 3 Myr ago. After this, population growth continues but a slower rate. SMOC also exhibit population growth, but it was much more rapid and recent (2.7 - 2.3
Myr ago). However, both SMOR and SNA haplogroups seems to experienced a more prolonged phase of demographic stability, with bottleneck events occurred around 100 000 years ago.
Migration and divergence time between D. pseudotsugae populations
Estimates of θ, Ne♀, T, TMRCA, and Tpop among different haplogroups were used to determine if corresponding temporal divergence of those haplogroups was consistent with a
Pleistocene timescale (table 3.4). Assuming one generation per year (Wood, 1982) and the estimate of 1.1% per million years per lineage for mtDNA COI (Brower, 1994), the deepest
91 estimated divergence times between NNA and both SMOC and SMOR were very similar
(1.08 and 1.03 Myr, respectively), around early Pleistocene. As expected, the more recent estimated divergence times were between NNA and SNA (0.47 Myr) and SMOC and SMOR
(0.55 Myr), around middle Pleistocene. It is also noteworthy that both SMOC and SMOR seem to diverge from SNA at almost the same time (0.65 and 0.68 Myr, respectively).
However, these estimates should be taken with caution, as the MDIV software only assumes constant population size (which was demonstrated only for SMOR and SNA haplogroups).
Therefore, violation of this assumption could affect estimates of time since divergence.
Nested clade phylogeographic analysis
The statistical parsimony haplotype network within D. pseudotsugae revealed that the spatial distribution was geographically structured for many of the recognized mtDNA haplotypes. The three networks are very similar to the clades obtained by the tree building method. Due to the high number of private haplotypes, no star-like shape network was observed. As 22 haplotypes could not be unambiguously joined at the 95% connection limit used, they were excluded from the subsequent analyses. Nested clades 6-2 and 6-3 are separated by 20 mutational steps, while the minor haplogroup (composed by only five haplotypes from Arizona) is separated only by 13 mutational steps to the 6-2 clade (see
APPENDIX II). Analysis of the spatial distribution of the nested design using GEODIS program revealed many clades with significant geographic associations, especially at the 4, 5, and 6 levels (APPENDIX I). Interpretation of these results (made with the inference key in
Templeton (1998) and Templeton (2004)) found that the structure of significant clades (Table
3.5) is as follows: Contiguous range expansion (clades 1-50, 3-16, 3-25, 4-2, 4-13 and 5-9) is
92 inferred in most haplotypes from WO, EO, WM, NI, NW and BC regions for D. p. pseudotsugae. This event was also inferred in haplotypes from EC, WD and SC regions for D. p. barragani. Restricted gene flow with isolation by distance (1-65, 3-9, 4-4, 4-5, 4-9, 5-6, 6-1 and 6-5) was inferred in some haplotypes from NA, SA, WO, EO, WM, WW, NW and BC regions. This event was also inferred in haplotypes from EC, SD, WD and SC regions.
Allopatric fragmentation (2-11, 3-3, 3-42, 4-16 and 6-4) was mainly inferred in NA and SA, but with some haplotypes from all other northern regions, as well as in the three regions of the
Sierra Madre Occidental (SC, SD and WD) in Northern Mexico. Other clades showing significant geographical associations were found (4-15, 5-1, 5-2 and 5-3); however, there was insufficient genetic resolution to discriminate between range expansion/colonization and restricted dispersal/gene flow (Table 3.5).
93 Discussion
Ancient vs recent populations inferred by genetic variability
The accumulation of independent genetic differences is expected in populations that evolved in allopatry, and higher genetic variability is also expected in ancient populations than in recent populations, because the latter are frequently a subset that arose from original gene pool. Such differences may be used as genetic signatures to trace expansion routes. In this sense, haplotype and nucleotide diversity allowed identifying what regions in Northwestern
North America and northern Mexico are genetically diverse, suggesting ancient Refugial populations of D. pseudotsugae (see below). First, Pleistocene glaciaciations have lower impact on coniferous forest in Northern Mexico, and no Pleistocene refugia have been identified in the area (Brown, 1985). This can be seen in the higher values of haplotype and nucleotide diversity for D. pseudotsugae populations in Northern Mexico, especially in EC and SD regions. In contrast, lower values of haplotype and nucleotide diversity in WO, EO,
NI, NW and BC regions indicate a possible range expansion northward of North America.
Similar results found in other North American and European bark beetles revealed that many range expansion events are linked to Postglacial colonization from Pleistocene refugia
(Stauffer et al., 1999; Kohlmayr et al., 2002; Cognato et al., 2003; Ritzerow et al., 2004;
Faccoli et al., 2005; Horn et al., 2006; Maroja et al., 2007; Mock et al., 2007;Anducho-Reyes et al., 2008).
Phylogenetic reconstruction and molecular genealogy of D. pseudotsugae
Phylogenetic reconstruction confirms previous studies indicating two extant subspecies
(Ruiz et al., 2009; Ruiz et al., unpublished data), and provided more phylogenetic resolution
94 among haplotypes. In this study, the enhanced sampling design and higher number of haplotypes used allowed recovering two lineages that clearly correspond to D. p. pseudotsugae and D. p. barragani subspecies (Fig. 3.2 and APPENDIX II). Nonetheless, clades I and II have a basal position and similar branch length indicate that they could arise very close in time. This could be the result of a very specific past event, such as habitat fragmentation of his host.
It is noteworthy that while many haplotypes from Cascades and Northern Rocky
Mountains are shared throughout the Pacific Northwest, none of the haplotypes from Southern
Rocky Mountains can be found elsewhere. On the other hand, topological patterns of northern populations (clade I) agree with the hypothesis of ancient vicariance (AV) proposed by
Brunsfeld (2001) to explain the patterns of disjunction in the Pacific Northwest (PNW) mesic forest ecosystem. Based on palebotanical studies (Graham, 1999), this hypothesis assumes that an ancient continuous mesic forest system was split by the Pliocene xerification of the
Columbia basin associated with Cascadian orogeny. Therefore, many taxa persisted in the
Cascades and Northern Rocky Mountains throughout the Pleistocene glacial cycles
(Brunsfeld, 2001), including ancient populations of D. pseudotsugae. Our results also agree with other studies which also found that the AV hypothesis better explains the evolutionary history PNW mesic forest ecosystem: Pinus albicaulis (Richardson et al., 2002), Salix melanopsis (Brunsfeld et al., 2007), the close related parasitic Adelgid that also us Douglas-fir has host (Ahern et al., 2009) and the very host Douglas-fir (Li and Adams, 1989).
Evidence of past demographic changes in D. pseudotsugae
95 Although many of the already mentioned results can be used as signatures of historical demographic change (shallow star-like phylogeny, haplotype and nucleotide diversity, and frequency of older (more divergent) lineages), we specifically test the hypothesis of demographic expansion with three different approaches: neutrality tests against population growth, mismatch distribution, and BSP. First, all neutrality tests (Tajima's D, FS, and R2) suggest that populations from Northwestern North America have experienced demographic expansion events (However, negative values of Tajima's D were not statistically significant).
Second, mismatch distribution of observed mitochondrial haplogroups are consistent with population expansion in NNA, but further revealed that while the SMOC haplogroup also experienced demographic expansion, SNA and SMOR did not. Both Harpending's raggedness index (rg) values of NNA and SMOC are much lower than those corresponding to SNA and
SMOR haplogroups (Table 3.3). Lower rg values are expected under the population growth model (Harpending, 1994). Third, historical demographic reconstructions using Bayesian skyline plots were far more ancient and conservative than last estimations, setting the continuous population growth of NNA and SMOC in middle to late Pliocene. Lastly, the average number of nucleotide differences is almost the same for haplogroups NNA and
SMOC, indicating that the ancestral populations of these two groups expanded at about the same time.
Similar results of all these approaches strongly support the demographic expansion of
NNA and SMOC haplogroups. Estimation times of the putative expansion event from τ for both NNA and SMOC were set in middle to late Pleistocene, probably after a period of strong bottlenecks due to past glaciations. In any case, all three approaches are congruent to Pliocene-
96 Pleistocene demographic expansion in NNA and SMOC, due to the factors discussed above.
Our results also agree with studies performed in other Dendroctonus species of similar distribution (Maroja et al., 2007; Mock et al., 2007). Nonetheless, caution most be taken in interpretation of these results, because a single locus analysis should not exclude different models of selection rather than demographic expansion, as well as incongruence of BSP results. The lack of congruence of BSP results respect to the other two methods could be attributed to the bias of population growth model selected (we choose the simplest constant model as the Bayes factor was < 20 for all pairwise comparisons of the three models).
Divergence time estimates in D. pseudotsugae
Despite that the AV hypothesis also predicts a relatively deep divergence between
Cascade orogeny and Northern Rocky Mountains (1.8 – 2.6 Myr or deeper), estimates of divergence time between NNA and SNA yield only 0.47 Myr, about middle Pleistocene. One possible explanation of this result is that D. pseudotsugae lineages within these two haplogroups have experienced significant divergence but with some intermittent contact trough time or continuous expansion northward to North America long before last glacial maxima Longer divergence time between NNA and all other haplogroups (Table 3.4) could be the result of range expansion in PNW via Rocky Mountains and long term isolation from southernmost populations in Mexico.
On the other hand, Mexican D. pseudotsugae lineages experienced differences in host availability, as a direct consequence of climate change. In this scenario, southern range of D. pseudotsugae host may serve as multiple refugia, while glaciers covered most of its northern range. Similar results have been found in other groups, distributed in non-glaciated areas, such
97 as longhorn cactus beetles (Smith and Farrell, 2005) and the Chinese Hwamei (Li et al., 2009).
However, the stochastic nature of the coalescent process makes necessary to consider more loci in order to overcome potential misleading of one single locus.
Phylogeography
According to Castelloe and Templeton (1994), the more frequent haplotype present in the widest geographic distribution are the most likely root of the haplotype network. The coalescence theory also suggest that a nested clade must be as old (or older) than those nested within it (Templeton, 1998). In this sense, haplotypes H004, H006 and H110 could be considered as the most ancient. The first two are distributed in Northern Rocky Mountains and along the Cascades, while the later is mainly found in southern Sierra Madre Occidental,
Mexico. This could be indicative of Pleistocene refugia within these regions.
The statistical parsimony network recognized the existence of three different lineages within D. pseudotsugae. One contains haplotypes exclusively from Mexico, one with haplotypes from Pacific Northwest and Rocky Mountains, and one with haplotypes exclusively from southern Rocky Mountains. Beyond the two recognized subspecies, no previous taxonomic or molecular studies have reported such divergence of D. pseudotsugae populations. The nesting level, position within the network and analysis of the spatial distribution suggests patterns of historically reduced gene flow and allopatric fragmentation in
Arizona.
Interestingly, our data, similar pattern of differentiation found in the specialist parasitic
Adelgid (Ahern et al., 2009) and other Dendroctonus species (Cognato et al., 2003; Maroja et al., 2007; Mock et al., 2007) are in agreement with the recognition of the southern Rocky
98 Mountains as an important reservoir of genetic diversity. The nested clade analysis (NCA) also seems to corroborate inferences drawn from neutrality tests, mismatch distribution and
BSP, providing evidence that demographic change were accompanied by continuous range expansion events. Such inferences have been made for D. p. pseudotsugae populations in the
Cascades and Northern Rocky Mountains in North America and for D. p. barragani populations in Southern Sierra Madre Occidental in Mexico. In contrast, restricted gene flow with isolation by distance was inferred in the nested clade that contained samples from
Northwestern North America and Southern Rocky Mountains, as well as in the nested clade that contained samples from both Sierra Madre Oriental and Sierra Madre Occidental.
Allopatric fragmentation was also inferred in the southernmost range of D. p. barragani.
Inferences made with the NCA did not allowed to drawn firm conclusions on the possible events that explain why there are three different networks, despite that most of the distribution of D. pseudotsugae range was sampled,. This may be indicative of deep and ancient divergence. Past distribution of North American conifers and the evolutionary history of the host Douglas-fir indicate that the warmer climates of interglacial periods pushed conifers northward of Colorado, New Mexico and Arizona, and therefore, isolation conifers within this area. Specifically, environmental changes and stress reduced the population size of
Douglas-fir and forced fragmentation of distribution range southward into Northern Mexico
(Li and Adams, 1989; Aagaard et al., 1998a,b; Bartlein et al., 1998).
The close relationship between herbivores and host plants has long been observed
(Thompson, 2004). Frequently, phylogenetic congruence has been found, with a strong relationship between genetic variation of herbivores and the host geographic distribution
99 (Brown et al., 1997; Cognato et al., 2005; Aoki et al., 2008). Results of the present study now provide a basis to test whether the genetic variation of this specialist bark beetle (D. pseudotsugae) is congruent with the intraspecific variation of its host Douglas-fir or not.
100 Acknowledgements
We thank Francisco Bonilla, Antonio Olivo, Luis M. Torres, Sergio Quiñones
(Comisión Nacional Forestal, Mexico) and Flor Rivera, Javier Zavala (Centro de Investigación y Estudios Avanzados, Mexico) for providing field assistance; Gene Paul (Pacific Northwest
Research Station, USDA Forest Service), Brytten Steed (Forest Health Protection, USDA
Forest Service) and Tom DeGomez (Northern Arizona University) provided fresh or alcohol preserved specimens. The project was funded by Consejo Nacional de Ciencia y Tecnología
(CONACYT 44887) and Secretaría de Investigación y Posgrado-IPN (SIP-20060421). JLH and JER were supported by the Pacific Northwest Research Station. This work was part of E.
A. Ruiz’s PhD dissertation. He was a CONACYT (176313) and Programa Institucional de
Formación de Investigadores del Instituto Politécnico Nacional (PIFI-IPN) fellow.
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112 Figure legends
Fig. 3.1. Sample distribution and haplotype frequency of the four major haplogroups of
Dendroctonus pseudotsugae for each region: Eastern Coahuila (EC), Soutern Durango (SD),
Western Durango (WD), Southern Chihuahua (SC), Southeastern Arizona (SA), Northern
Arizona (NA), Northern Colorado (NC), Western Wyoming (WW), Western Oregon (WO),
Eastern Oregon (EO), Western Montana (WM), Northern Idaho (NI), Northern Washington,
(NW), Southeastern British Columbia (BC). Sample sizes are indicated within parentheses following the abbreviation of each region. Haplogroup names are as given in Table 3.1.
Fig. 3.2. Maximum-likelihood tree reconstruction based on 550 bp of the mtDNA genome of the Dendroctonus pseudotsugae, with the TrN93 + G + I substitution model (D. simplex was used as outgroup). Branch support values are shown under nodes (or using arrows) with an aLRT values > 60.
Fig. 3.3 Demographic history of the main four mitochondrial haplogroups inferred in
Dendroctonus pseudotsugae: NNA, Northwestern North America; SNA, Southwestern North
America; SMOR, Sierra Madre Oriental; SMOC, Sierra Madre Occidental. A) Mismatch distributions of numbers of pairs of nucleotide differences among individuals within each of the four haplogroups. The observed distributions (dark bars) were compared for the goodness- of-fit to a Poisson distribution under a sudden population expansion model (gray lines). B)
Bayesian skyline plots for the same haplogroups, showing the changes in effective population size through time. Black middle lines are median estimates; gray lines are the 95% high probability density (HPD) for upper and lower limits.
113 Table 3.1. Location, region, geographic references, number of specimens analyzed and accession numbers of mtDNA COI in Dendroctonus pseudotsugae. Locations are sorted by region. Region names are as given in Fig. 1. Accession numbers in bold correspond to sequences taken from previous studies (Ruiz et al., 2009; Ruiz et al., unpublished data). Private haplotypes are underlined. Numbers in parenthesis correspond to haplotype frequencies.
Latitude / No. Localition Region Haplogroup* GenBank accession no. Longitude Samples 25° 09' 12'' N 100° Santa Rita, N. León EC SMOR/SMOC 7 EU043405, EU043406, EU043407, EU043408(2), EU043409(2) 08' 41'' W 25° 14' 32'' N 100° Montereal, Arteaga, Coah EC SMOC 10 EU193124, EU193125(2), EU193126, EU193127(2), EU193128(2), EU193129(2) 26' 37'' W 25° 20' 02'' N 100° Los Lirios, Arteaga, Coah EC SMOC 8 EU193130(2), EU193131(4), EU193132(2) 33' 51'' W 25° 26' 14'' N 100° Rancho los Angeles, Arteaga, Coah EC SMOR/SMOC 12 EU043410, EU043411, EU043412, EU043413(4), EU043414(3), EU043415(2) 42' 30'' W 23° 32' 47'' N 104° La Caldera, Mpio. Pueblo Nuevo, Dgo SD SMOC 8 FJ174774(2), FJ174775(2), FJ174776, FJ174777, FJ174778, FJ174779 49' 45'' W 23° 32' 10'' N 104° Cordon del Solitario, Mpio. Pueblo Nuevo, Dgo SD SMOC 12 FJ174780(7), FJ174787(3), FJ174788(2) 50' 53'' W 23° 31' 07'' N 104° Los Yesqueros, Mpio. Pueblo Nuevo, Dgo SD SMOC 11 FJ174789(6), FJ174790(2), FJ174791(3) 48' 09'' W 23° 30' 04'' N 104° Los Hoyos, Mpio. Pueblo Nuevo, Dgo SD SMOC 10 FJ174781(2), FJ174782, FJ174783(2), FJ174784, FJ174785(2), FJ174786(2) 50' 12'' W 24° 05' 20'' N 105° Ejido Cienega de la Vaca, San Dimas, Dgo WD SMOC 9 EU043418(6), EU043425, EU043426, EU043427 31' 00'' W 24° 21' 10'' N 105° Ejido Puentesillas, San Dimas, Dgo WD SMOC 11 EU043418(7), EU043421, EU043422, EU043423, EU043424 54' 39'' W 24° 22' 08'' N 105° Ejido La Manga, San Dimas, Dgo WD SMOR/SMOC 9 EU043408(2), EU043409(2), EU043416(2), EU043417(2), EU043418 58' 15'' W 24° 22' 29'' N 105° Ejido Nuñez, San Dimas, Dgo WD SMOR/SMOC 10 EU043408(2), EU043418(2), EU043419(3), EU043420, EU043421(2) 55' 39'' W 26° 04' 16'' N 106° Llano Grande, Guanaseví, Dgo SC SMOR/SMOC 9 EU043408, EU043422, EU043428(4), EU043429, EU043430(2) 17' 15'' W 26° 05' 31'' N 107° Ejido El Nopal, Guadalupe y Calvo, Chi SC SMOR/SMOC 9 EU043416, EU043418(4), EU043428, EU043431(2), EU043432 02' 11'' W 26° 12' 40'' N 106° Ejido Catedral, Guadalupe y Calvo, Chi SC SMOC 5 EU193133(3), EU193134(2) 33' 49'' W 32° 50' 54'' N 109° Coronado NF, AZ SA NNA/SNA 10 EU193135(4), EU193136(2), EU193137(4) 43' 01'' W 34° 24' 29'' N 110° Apache Sitgreaves, AZ SA NNA/SNA 11 EU193138(6), EU193139(3), EU193140, EU193141 02' 21'' W 35° 17' 54''N 111° EU043433, EU043434(3), EU043435, EU043436(2), EU043437, EU043438, EU043439, Flagstaff, Coconino NF, AZ NA NNA/SNA 13 42' 54'' W EU043440(3) 35° 21' 30'' N 111° Peaks, Coconino NF, AZ NA SNA 9 EU193142, EU193143, EU193144(2), EU193145, EU193146(4) 33' 53'' W 39° 15' 43'' N 105° Deckers, CO NC NNA 4 FJ174757, FJ174758, FJ174759(2) 14' 02'' W 40° 15 '10'' N 105° Larimer County, CO NC NNA 3 FJ174754, FJ174755, FJ174756 22' 35'' W 42° 52' 10'' N 109° Sublette, Pinedale, WY WW NNA 10 EU193147(2), EU193148, EU193149(2), EU193150(2), EU193151(2), EU193152 52' 05'' W
114 44° 35' 50'' N 108° Shoshone National Reservation, WY WW NNA 8 FJ174769(2), FJ174770, FJ174771, FJ174772, FJ174773(3) 50' 13'' W 42° 24' 38'' N 122° Rogue River NF, OR WO NNA 9 FJ174760, FJ174761, FJ174762(4), FJ174763(3) 20' 03'' W 45° 10’30” N 123° Mount Hebo Rd, Siuslaw NF, OR WO NNA 11 EU043441, EU043442, EU043443, EU043444(6), EU043445(2) 40’ 08” W 44° 34' 53'' N 118° Vinegar-Vincent Drainage, Malheur NF, OR EO NNA 11 EU043444(2), EU043445(2), EU043446, EU043447, EU043448(2), EU043449(3) 31' 21'' W 44° 58' 49'' N 117° Balm Creek Reservoir, WWNF OR EO NNA 11 EU043444(5), EU043445(4), EU043457, EU043458 33' 44'' W 45° 20'25'' N 118° EU043445(2), EU043449, EU043450, EU043451, EU043452, EU043453(2) EU043454, Spring Creek, WWNF, La Grande, OR EO NNA 11 18' 50'' W EU043455, EU043456 45° 25' 29'' N 118° Mount Emily Rd, WWNF, La Grande, OR EO NNA 9 EU043445(4), EU043462(3), EU043463(2) 08' 32'' W 45° 27' 53'' N 118° Drumhill Ridge, WWNF, La Grande, OR EO NNA 10 EU043444(7), EU043459, EU043460, EU043461 11' 25'' W 47° 53' 35'' N 120° Chatter Creek, Wenatchee NF, WA NW NNA 12 FJ174762(2), FJ174764(3), FJ174765(2), FJ174766, FJ174767, FJ174768(3) 53' 30'' W 47° 43' 44'' N 116° FJ174745, FJ174746(5), FJ174747(6), FJ174748, FJ174749(2), FJ174750, FJ174751, Coeur d'Alene, ID NI NNA 19 43' 14'' W FJ174752, FJ174753 46° 53' 10'' N 113° EU043441, EU043442(2), EU043444(2), EU043445, EU043448, EU043453, Lubrecht Experimental Forest, Missoula, MT WM NNA 11 26' 55'' W EU043463(2), EU043464 51° 08' 15'' N 118° Revelstoke, British Columbia BC BC NNA 9 EU043441(3), EU043442(2), EU043444, EU043445, EU043453, EU043464 16' 26'' W
*Haplogroup reflects phylogenetic results from Fig. 2. NNA = Northwestern North America; SNA = Southwestern North America; SMOR = Sierra Madre Oriental; SMOC = Sierra Madre Occidental.
115 Table 3.2. Nucleotide polymorphism and results of neutrality test for the mtDNA COI (550 bp) of Dendroctonus pseudotsugae. Abbreviations of each region are as given in Fig. 1. S, number of segregating sites; Nhap, number of haplotypes; Hd, haplotype diversity; π, nucleotide diversity (SD = standard deviation); Fu and Li's D, statistic of Fu and Li's D-test (1993); Fu's FS, statistic of Fu's FS test (1997); Tajima's D, Tajima's (1989) D statistic. *P < 0.05.
Region S Nhap Hd π (SD) Fu and Li's D Fu's FS Tajima's D
EC 70 20 0.964 0.031 (0.002) 0.735 0.236 0.014
SD 61 18 0.939 0.023 (0.002) -0.263 0.591 -0.431
WD 33 14 0.818 0.014 (0.002) 0.814 0.318 -0.15
SC 53 11 0.913 0.022 (0.004) 0.784 2.132 -0.604
SA 30 7 0.852 0.02 (0.001) 0.8 5.553 1.153
NA 37 13 0.939 0.022 (0.002) 0.469 0.343 0.832
NC 46 6 0.952 0.029 (0.008) -1.125 0.955 -0.853
WW 53 11 0.948 0.031 (0.002) 0.451 1.823 0.382
WO 37 9 0.868 0.011 (0.004) -2.55* 0.56 -1.667*
EO 21 16 0.821 0.007 (0.001) -1.952 -3.691 -0.563
WM 20 8 0.945 0.012 (0.003) -0.328 -0.784 -0.224
NI 23 9 0.848 0.013 (0.002) 0.761 0.909 0.317
NW 23 6 0.879 0.013 (0.002) -0.059 2.143 -0.129
BC 20 6 0.889 0.015 (0.003) 0.018 1.094 0.54
116 Table 3.3. Statistic tests for selective neutrality and population expansion indices based on 550 bp of mtDNA COI of Dendroctonus pseudotsugae. Haplogroup names are as given in Table 1. Tajima's D, Tajima's (1989) D statistic; Fu's FS, statistic of Fu's FS test (1997); R2, Ramos-Onsins and Rozas's R2 statistics (Ramos-Onsins and Rozas, 2002); SSD, sum of squared deviations (Excoffier, 2004); rg, Harpending's raggedness index (Harpending, 1994). Demographic parameters: θ0 = 2µN0; θ1 = 2µN1 and τ = 2ut; CI = confidence interval (see text for details). *P < 0.05; **P < 0.01, ***P < 0.001.
Haplogroup NNA SNA SMOC SMOR ALL
No. of sequences 166 25 112 28 331
Tajima's D -1.254 1.229 -0.694 -0.334 -0.523
Fu's FS -23.907*** 0.76 1.137 -6.738 -23.54**
R2 0.049* 0.172 0.071 0.11 0.062
SSD 0.003 0.022* 0.004 0.049** - rg 0.004 0.035 0.007 0.114*** -
τ (95% CI) 11.635 (6.936-27.273) - 5.813 (2.033-26.424) - -
θ0 (95% CI) 5.031 (0-6.884) - 11.088 (0-23.386) - -
θ1 (95% CI) 24.032 (17.242-65.929) - 43.265 (25.616-166.077 - -
117 Table 3.4. Results of the migration/isolation model and divergence time among haplogroups of Dendroctonus pseudotsugae, obtained using the MDIV software. θ, theta; Ne♀, female effective population size; T, scaled divergence time; TMRCA, time to the most recent common ancestor (measured in units of Ne♀ generations); Tpop population divergence time.
Haplogroup 1 Haplogroup 2 θ Ne♀ T TMRCA Tpop (Myr)
NNA SNA 47 2043478 0.3 0.876 470000
NNA SMOC 59 2565217 0.55 0.763 1081000
NNA SMOR 56 2434782 0.55 0.762 1026000
SNA SMOC 49 2130434 0.4 0.824 653000
SNA SMOR 17 739130 1.2 1.613 680000
SMOC SMOR 50 2173913 0.03 0.802 553000
118 Table 3.5. Summary of inferences regarding the historical demographic events from nested clade phylogeographic analysis. * = Clade level for which inferences could not be made because interior ant tip clades could not be determined.
Clade Chain of inference Demographical/Historical event
1-50 1-2-11-12 NO Contiguous range expansion.
1-65 1-2-3-4 NO Restricted gene flow with isolation by distance (restricted dispersal by distance in non-sexual species).
2-11 1-19 NO Allopatric fragmentation.
2-102 1-2 * Inconclusive outcome.
3-3 1-19 NO Allopatric fragmentation.
3-9 1-19-20-2-3-4 NO Restricted gene flow with isolation by distance (restricted dispersal by distance in non-sexual species).
3-16 1-2-11-12 NO Contiguous range expansion.
3-25 1-2-11-12 NO Contiguous range expansion.
3-33 1-19-20-2 * Inconclusive outcome.
3-39 1-19-20-2 * Inconclusive outcome.
3-42 1-19 NO Allopatric fragmentation.
4-2 1-2-11-12 NO Contiguous range expansion.
4-4 1-2-3-4 NO Restricted gene flow with isolation by distance (restricted dispersal by distance in non-sexual species).
4-5 1-19-20-2-3-4 NO Restricted gene flow with isolation by distance (restricted dispersal by distance in non-sexual species).
4-9 1-2-3-4 NO Restricted gene flow with isolation by distance (restricted dispersal by distance in non-sexual species).
4-13 1-2-11-12 NO Contiguous range expansion.
4-15 1-2-3-5-6-7-8 YES Too few Clades: Insufficient genetic resolution to discriminate between range expansion/colonization and restricted dispersal/gene flow.
4-16 1-19 NO Allopatric fragmentation.
4-17 1-2 * Inconclusive outcome.
4-20 1-2 * Inconclusive outcome.
5-1 1-2-3-5-6-7 YES Too few Clades: Insufficient genetic resolution to discriminate between range expansion/colonization and restricted dispersal/gene flow.
5-2 1-2-3-5-6-7-8 YES Too few Clades: Insufficient genetic resolution to discriminate between range expansion/colonization and restricted dispersal/gene flow. 5-3 1-2-3-5-6-7-8 YES Too few Clades: Insufficient genetic resolution to discriminate between range expansion/colonization and restricted dispersal/gene flow. 119 5-6 1-2-3-4 NO Restricted gene flow with isolation by distance (restricted dispersal by distance in non-sexual species).
5-9 1-2-11-12 NO Contiguous range expansion.
6-1 1-2-3-4 NO Restricted gene flow with isolation by distance (restricted dispersal by distance in non-sexual species).
6-4 1-19 NO Allopatric fragmentation.
6-5 1-2-3-4 NO Restricted gene flow with isolation by distance (restricted dispersal by distance in non-sexual species). Total 1-2 * Inconclusive outcome. Cladogram
120 121 122 123 124 125 126 127 128 129 130 APPENDIX I. Clades showing significant geographical associations as determined by the nested clade analysis.
NESTED CLADES significant ch+A73i-square significantly larger significantly smaller
clade CHI-SQUARE P nested clades int/tip Dc P Dn P 1-33 5.0 NS cnvc101+D77.12 T 0.0 NS 130.9825 NS lngr105.13 T 0.0 NS 102.5503 NS pnts109.11 I 96.4511 NS 98.2691 NS I-T - 96.4511 NS -18.4973 NS
1-44 2.0 NS alen031.32 T 0.0 NS 379.0241 NS mthb001.25 I 0.0 NS 223.4884 NS I-T - 0.0 NS -155.5357 NS
1-50 29.0 0.00001 rgrv059.24 T 245.6929 NS 359.6627 0.0490 mthb006.25 I 222.1997 0.0080 239.0193 0.0456 I-T - -23.4933 NS -120.6433 0.0484
1-65 26.4286 NS drhl002.29 T 0.0 NS 23.5699 NS emrd003.30 T 0.0 0.0360 29.4796 NS mthb004.25 I 168.8898 0.0160 164.1496 0.0160 I-T - 168.8898 0.0105 136.1475 0.0018
1-168 1.3333 NS pnts103.11 I 0.0 NS 1.5742 NS ennz104.10 I 1.2913 NS 1.4758 NS
2-7 6.0 NS 1-8 T 0.0 NS 200.2372 NS 1-96 I 28.7458 NS 48.2507 NS I-T - 28.7458 NS -151.9865 NS
2-11 9.0 0.0278 1-13 T 0.0 NS 4.2357 0.0278 1-161 I 0.0 0.0278 1.3201 0.0278 I-T - 0.0 NS -2.9155 0.0278
2-17 7.0 NS 1-22 T 0.0 NS 3.9216 NS 1-97 I 0.0 NS 0.7189 NS I-T - 0.0 NS -3.2027 NS
2-29 5.0 NS 1-37 T 0.0 NS 256.2671 NS 1-40 T 0.0 NS 1334.967 NS
2-36 6.0 NS 1-49 T 0.0 NS 265.5836 NS 1-119 I 0.0 NS 353.5442 NS I-T - 0.0 NS 87.9606 NS
2-37 5.1724 NS 1-51 T 0.0 NS 364.8636 NS 1-50 I 265.4839 NS 267.2367 NS I-T - 265.4839 NS -97.6269 NS
2-46 3.0 NS 1-63 T 0.0 NS 562.6835 NS 1-44 I 280.5182 NS 981.5658 NS I-T - 280.5182 NS 418.8823 NS
2-48 5.0 NS 1-66 T 0.0 NS 159.4521 NS 1-140 I 196.1819 NS 192.6217 NS I-T - 196.1819 NS 33.1696 NS
2-49 5.0 NS 1-67 T 0.0 NS 266.3191 NS 1-139 I 306.0763 NS 296.3285 NS I-T - 306.0763 NS 30.0094 NS
2-53 8.0 NS 1-71 T 0.0 NS 522.3846 NS 1-73 I 0.0 NS 796.7821 NS I-T - 0.0 NS 274.3975 NS
2-60 4.0 NS 1-21 I 0.0 NS 243.3864 NS 1-106 I 0.0 NS 241.9817 NS
2-62 11.5286 NS 1-88 I 85.3488 NS 90.2608 NS 1-33 I 107.9582 NS 111.851 NS APPENDIX I. Clades showing significant geographical associations as determined by the nested clade analysis.
NESTED CLADES significant chi-square significantly larger significantly smaller
clade CHI-SQUARE P nested clades int/tip Dc P Dn P 2-93 5.0 NS 1-168 I 1.4992 NS 11.3779 NS 1-155 I 0.0 NS 39.1123 NS
2-102 18.0 0.0099 1-182 I 0.0 NS 144.6975 NS 1-181 I 372.8796 0.0078 389.789 0.0078 1-42 I 0.0 0.0115 181.4055 0.0467
3-2 4.0 NS 2-2 T 0.0 NS 22.5681 NS 2-66 I 0.0 NS 43.1237 NS 2-3 I 0.0 NS 22.5681 NS I-T - 0.0 NS 10.2778 NS
3-3 27.0 0.0006 2-4 T 0.0 0.0331 137.9787 NS 2-62 I 93.9499 0.0002 109.3917 0.0002 I-T - 93.9499 NS -28.587 0.0349
3-4 6.0 NS 2-5 T 0.0 NS 196.8608 NS 2-93 I 17.6295 NS 44.8565 NS I-T - 17.6295 NS -152.0044 NS
3-5 14.0 0.0272 2-6 T 0.0 NS 311.1435 NS 2-24 T 0.0 NS 184.648 NS 2-67 I 292.5228 NS 316.9078 NS I-T - 292.5228 NS 47.9295 NS
3-7 6.0 NS 2-10 T 0.0 NS 2.2098 NS 2-18 T 0.0 NS 2.4307 NS 2-19 I 0.0 NS 2.2098 NS I-T - 0.0 NS -0.1656 NS
3-8 8.0 NS 2-12 T 0.0 NS 3.6895 NS 2-17 I 1.2151 NS 1.5 NS I-T - 1.2151 NS -2.1895 NS
3-9 7.0 0.0272 2-13 T 0.0 0.0272 149.4293 0.0272 2-60 I 242.6841 0.0272 242.6739 NS I-T - 242.6841 0.0272 93.2446 NS
3-10 6.0 NS 2-14 T 0.0 NS 161.1641 NS 2-63 I 0.0 NS 161.1641 NS 2-92 I 0.0 NS 324.2019 NS I-T - 0.0 NS 81.5189 NS
3-12 5.0 NS 2-16 T 0.0 NS 47.1754 NS 2-88 I 0.0 NS 18.5164 NS I-T - 0.0 NS -28.659 NS
3-14 4.0 NS 2-22 T 100.9332 NS 207.4542 NS 2-23 I 0.0 NS 439.5377 NS I-T - -100.9332 NS 232.0835 NS
3-16 26.5 0.0072 2-29 T 426.5881 NS 969.3282 0.0024 2-41 T 0.0 NS 211.038 NS 2-102 I 308.2983 0.0076 580.4814 0.0400 I-T - -47.1918 NS -262.4651 0.0378
3-17 5.0 NS 2-30 T 0.0 NS 390.7166 NS 2-94 I 0.0 NS 661.4777 NS I-T - 0.0 NS 270.761 NS
3-18 7.0 NS 2-31 T 0.0 NS 837.13 NS 2-36 I 303.281 NS 356.1273 NS I-T - 303.281 NS -481.0027 NS APPENDIX I. Clades showing significant geographical associations as determined by the nested clade analysis.
NESTED CLADES significant chi-square significantly larger significantly smaller
clade CHI-SQUARE P nested clades int/tip Dc P Dn P 3-20 4.0 NS 2-33 T 0.0 NS 5.188 NS 2-85 I 0.0 NS 10.7718 NS I-T - 0.0 NS 5.5838 NS
3-23 1.875 NS 2-42 T 0.0 NS 51.6109 NS 2-99 I 32.2619 NS 38.7144 NS I-T - 32.2619 NS -12.8964 NS
3-24 7.7 NS 2-44 T 317.6212 NS 389.5483 NS 2-49 T 290.582 NS 333.5339 NS 2-43 I 0.0 NS 286.6822 NS I-T - -304.1016 NS -74.8589 NS
3-25 58.6444 0.0084 2-46 T 715.4673 0.0103 862.7087 0.0099 2-47 T 0.0 NS 984.9896 NS 2-48 T 186.5293 NS 242.682 NS 2-101 I 134.511 0.0199 265.566 0.0257 I-T - -207.6055 NS -266.2702 NS
3-27 5.0 NS 2-51 T 0.0 NS 472.7795 NS 2-97 I 0.0 NS 1108.5445 NS 2-100 I 0.0 NS 472.7795 NS I-T - 0.0 NS 158.9413 NS
3-29 5.8333 NS 2-53 T 633.3113 NS 700.6206 NS 2-54 I 0.0 NS 444.6824 NS I-T - -633.3113 NS -255.9382 NS
3-33 15.0 0.0003 2-11 I 2.0129 0.0003 133.7547 0.0294 2-7 I 73.5818 0.0407 171.2583 NS
3-35 8.0 NS 2-26 I 278.3613 NS 273.0047 NS 2-84 I 0.0 NS 183.9826 NS
3-38 31.0 NS 2-37 I 270.9812 NS 270.2493 NS 2-89 I 0.0 NS 365.239 NS
3-39 14.0 0.0279 2-45 I 209.1297 0.0312 195.2968 0.0312 2-90 I 0.0 0.0287 50.3357 NS 2-77 I 0.0 NS 42.2627 NS
3-40 9.0 NS 2-61 I 0.0 NS 286.2513 NS 2-74 I 0.0 NS 39.9163 NS 2-28 I 0.0 NS 39.9163 NS
3-42 14.0 0.0158 2-40 I 0.0 NS 100.0269 0.0300 I 0.0 NS 74.4928 NS I 0.0 NS 82.3013 NS
4-1 1.1429 NS 3-1 T 0.0 NS 12.0345 NS 3-2 I 29.6341 NS 26.3423 NS I-T - 29.6341 NS 14.3078 NS
4-2 6.4286 NS 3-5 T 299.4329 NS 287.6809 NS 3-15 I 0.0 0.0324 135.7192 0.0324 -299.4329 NS -151.9616 NS
4-4 15.12 0.0027 3-7 T 2.315 0.0093 97.9857 NS 3-33 I 151.2167 NS 143.4155 NS I-T - 148.9017 0.0016 45.4299 NS
4-5 7.0 0.0454 3-11 T 0.0 NS 12.2676 0.0454 3-12 I 26.5997 0.0454 28.6312 0.0454 I-T - 26.5997 0.0454 16.3635 0.0454 APPENDIX I. Clades showing significant geographical associations as determined by the nested clade analysis.
NESTED CLADES significant chi-square significantly larger significantly smaller
clade CHI-SQUARE P nested clades int/tip Dc P Dn P 12.25 NS 3-14 T 277.0793 NS 265.021 NS 3-4 I 72.8172 NS 146.8996 NS 4-8 3-35 I 264.1109 NS 242.7982 NS I-T - -94.9514 NS -63.3222 NS
8.9143 NS 3-18 T 441.0101 0.0056 620.9982 NS 3-17 I 491.4649 NS 668.3905 NS 4-9 I-T - 50.4548 NS 47.3923 NS
15.4355 NS 3-19 T 0.0 NS 296.1799 NS 3-38 I 271.9443 NS 273.6459 NS 4-10 I-T - 271.9443 NS -22.534 NS
4.0 NS 3-21 T 0.0 NS 255.2876 NS 3-37 I 291.009 NS 361.9063 NS 4-11 I-T - 291.009 NS 106.6187 NS
2.0571 NS 3-22 T 0.0 NS 65.8018 NS 3-42 I 88.7552 NS 86.9549 NS 4-12 I-T - 88.7552 NS 21.1531 NS
6.0779 NS 3-24 T 356.2915 0.0164 357.7122 0.0180 3-39 I 110.3361 0.0193 226.2526 0.0178 4-13 I-T - -245.9554 0.0168 -131.4596 0.0175
1.875 NS 3-29 T 586.2012 NS 644.179 NS 3-28 I 0.0 NS 704.7844 NS 4-14 I-T - -586.2012 NS 60.6054 NS
14.0 0.0005 3-30 T 0.0 NS 381.0507 NS 3-31 T 0.0 NS 381.0507 NS 4-15 3-34 I 0.0 NS 381.0507 NS 3-43 I 0.0 0.0091 558.8824 0.0005 I-T - 0.0 NS 106.699 0.0005
28.0 NS 3-3 I 114.9555 NS 113.5246 0.0342 3-41 I 0.0 NS 203.3044 0.0342 4-16 23.4333 0.0005 3-8 I 1.8553 0.0000 142.481 0.0000 3-10 I 215.51 NS 274.8143 0.0279 4-17 3-9 I 206.8106 NS 205.282 NS
14.8662 NS 3-23 I 41.2937 NS 186.1333 NS 3-25 I 409.9207 NS 398.1087 NS 4-18 5.0 NS 3-36 I 0.0 NS 141.923 NS 3-20 I 7.0027 NS 38.1883 NS 4-19 47.6889 0.0000 3-16 I 698.5431 0.0403 848.8002 NS 3-40 I 69.9572 0.0000 865.0038 NS 4-20 3-27 I 664.5181 NS 822.9694 NS
36.1833 0.0001 4-1 T 19.6654 0.0015 340.362 0.0097 4-2 I 247.3427 NS 281.8968 NS 5-1 4-8 I 219.0093 0.0385 265.2371 NS I-T - 209.463 0.0079 -69.175 0.0136
22.4196 0.0090 4-3 T 0.0 0.0241 182.0685 0.0132 4-16 I 117.0231 NS 113.0482 0.0259 5-2 I-T - 117.0231 0.0467 -69.0203 0.0132
52.7227 0.0000 4-4 T 130.4356 0.0089 190.3974 NS 4-5 I 23.9988 0.0254 353.2325 0.0000 5-3 4-17 I 201.9827 NS 200.5558 NS I-T - 98.1564 0.0239 -30.5503 NS APPENDIX I. Clades showing significant geographical associations as determined by the nested clade analysis.
NESTED CLADES significant chi-square significantly larger significantly smaller
clade CHI-SQUARE P nested clades int/tip Dc P Dn P 71.2725 0.0000 4-9 T 642.6777 NS 673.7484 0.0071 4-14 T 655.7229 NS 730.0819 NS 5-6 4-20 I 851.4451 0.0010 868.2921 0.0001 I-T - 201.5201 0.0083 163.2473 0.0000
10.0842 NS 4-11 T 311.4027 NS 374.0151 NS 4-10 I 274.8511 NS 272.0837 NS 5-7 I-T - -36.5516 NS -101.9314 NS
15.9808 NS 4-13 T 308.5591 NS 316.2953 NS 4-18 I 371.8207 NS 342.7691 NS 5-8 I-T - 63.2616 NS 26.4738 NS
18.9378 0.0028 4-15 T 453.1447 0.0199 499.3655 0.0000 4-12 I 82.72 0.0070 279.1317 0.0041 5-9 4-19 I 59.9782 0.0362 266.4894 0.0158 I-T - -378.5468 0.0000 -224.7489 0.0000
135.2517 0.0000 5-2 T 120.2723 0.0004 185.6846 0.0102 5-3 T 219.0494 NS 222.6617 NS 6-1 5-1 I 285.077 0.0019 273.6727 0.0044 I-T - 104.3037 0.0014 65.3397 0.0047
65.0 0.0000 5-7 T 283.262 0.0000 558.0248 0.0000 5-9 I 393.3357 0.0001 777.4565 0.0000 6-4 I-T - 110.0737 NS 219.4318 0.0000
83.6078 0.0000 5-8 T 334.2927 0.0000 470.6992 0.0001 5-6 I 784.7367 0.0000 790.7099 0.0000 6-5 I-T - 450.444 0.0000 320.0107 0.0000
85.419 0.0000 6-4 T 650.4416 NS 638.815 NS 6-5 T 629.7119 NS 644.6904 NS 7-4 386.1503 0.0000 7-1 T 228.1012 0.0000 1338.518 0.0000 7-2 T 0.0 NS 88.5107 0.0000 Total Cladogram 7-4 T 642.5711 0.0000 1049.0704 0.0000 HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
______
DISCUSIÓN GENERAL
Capítulo I
Los resultados obtenidos en el capítulo I muestran, en primer lugar, que existe una alta diversidad de haplotipos y una baja diversidad nucleotídica en las poblaciones de D. pseudotsugae. Este patrón es característico de rápido crecimiento poblacional a partir de poblaciones pequeñas, suponiendo que ha trascurrido tiempo suficiente para el surgimiento de variación de haplotipos pero no para la acumulación de grandes diferencias entre secuencias (Grant y Bowen 1998). Este mismo patrón ha sido observado en otras especies del género Dendroctonus (Cognato et al.,
2005a; Maroja et al., 2007; Mock et al., 2007; Anducho-Reyes et al., 2008).
Aunque estas estimaciones pueden estar sesgadas por diversos factores, que van desde la longitud de la secuencia hasta las estimaciones de los tiempos de coalescencia, pasando por el número de individuos analizados, los patrones de variación genética y los índices de diversidad indican un alto flujo génico entre poblaciones, producto de una amplia capacidad de dispersión.
En segundo lugar, la hipótesis de que la especialización en el uso del huésped por parte de los insectos fitófagos puede ser un factor que lleve a su diferenciación genética (Van Zandt y Mopper 1998; Thompson 2004) es apoyada por: 1) el análisis molecular de variancia (AMOVA) y el análisis molecular de variancia espacial
136 HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
______(SAMOVA), que mostraron que existe una alta diferenciación genética entre las poblaciones de Norteamérica y México; 2) las regresiones lineales de FST / 1- FST y distancia-p contra la distancia geográfica, las cuales mostraron un incremento significativo en la cantidad de variación genética entre todas las poblaciones muestreadas, en relación al incremento de la distancia geográfica.
Estos resultados concuerdan con el estudio de Peterson y Denno (1998), el cual mostró que el efecto combinado de la dispersión y el aislamiento geográfico ha tenido un efecto mayor sobre la estructura genética y la diferenciación en los insectos fitófagos que la especialización en el uso del huésped. También concuerdan con los hallazgos en otros estudios de especies generalistas o especialistas en el género
Dendroctonus (Kelley et al., 1999; Kelley et al., 2000; Zuñiga et al., 2006).
La diferenciación entre todos los pares de poblaciones estimada con FST / 1-
FST también sugiere una discontinuidad en la distribución de la variación genética, la cual llevó al aislamiento de las poblaciones de D. pseudotsugae en Norteamérica por un lado, y por otro en el norte de México. Dicho aislamiento parece ser el resultado de flujo génico limitado, con el consecuente incremento en la deriva génica
(Hutchinson y Templeton 1999).
Todos estos resultados permiten afirmar que el aislamiento por distancia y posiblemente la fragmentación del único huésped (Pseudotsuga menziessi) han sido los principales factores que han afectado la estructura genética poblacional en D. pseudotsugae. Los datos obtenidos en este estudio son consistentes con aquellos
137 HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
______obtenidos de otras especies, aun cuando emplearon conjuntos de marcadores diferentes (Carter et al., 1996; Kelley et al., 1999; Six et al., 1999; Stauffer et al.,
1999; Kelley et al., 2000; Zúñiga et al., 2006). Finalmente, también sugieren que la estructura genética de las especies especialistas son más sensibles al aislamiento por distancia que las especies generalistas, debido a que la diferenciación genética en estas últimas es minimizada por el uso de diferentes huéspedes a lo largo de su distribución geográfica.
Capítulo II
Los análisis filogenéticos realizados con las secuencias del gen Citocromo
Oxidasa I son congruentes y apoyan la pertinencia de la designación de una nueva subespecie para D. pseudotsugae (D. p. barragani), realizada con caracteres morfológicos a partir de ejemplares colectados en la localidad de San Juanito, Chi, en 1974 (Furniss, 2001).
En el árbol filogenético correspondiente (usando el método de máxima parsimonia), se encontraron dos grupos que, sin ninguna ambigüedad, mostraron ser monofiléticos. No se encontraron evidencias de parafília, dado que todos los haplotipos provenientes de localidades de Norteamérica se agruparon juntos en un
único clado. Esto mismo fue observado para todos los haplotipos provenientes de localidades del norte de México.
138 HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
______Así, el grupo monofilético compuesto por los haplotipos de Norteamérica corresponde a la subespecie nominal D. p. pseudotsugae, mientras que el grupo monofilético compuesto por los haplotipos del norte de México corresponde a la subespecie D. p. barragani. Sin embargo, dado que ambos grupos monofiléticos son hermanos y derivados de un mismo nodo, no es posible establecer, con esta sola evidencia, cuáles son sus relaciones de ancestría-descendencia (i. e. si alguno es más antiguo que el otro). Sin embargo, la posición basal de ambos clados sugiere que el origen de ambos grupos de poblaciones pudo producirse como consecuencia de un evento del pasado muy específico, el cual muy probablemente pudo haber sido la fragmentación de su huésped Pseudotsuga menziesii.
Dentro del clado con haplotipos de Norteamérica (aunque con un bajo soporte estadístico), todas las poblaciones del noroeste de Norteamérica y Canadá claramente se derivan de un clado basal, compuesto exclusivamente por haplotipos de Arizona, lo cual sugiere que la colonización de Norteamérica se llevó a cabo en dirección sur-norte (con excepción de Wyoming, que parece haber sido colonizado después). En cuanto a las poblaciones del norte de México, la mayor parte de los haplotipos de la Sierra Madre Oriental es basal respecto de aquellos de la Sierra
Madre Occidental, lo que sugiere que este último sistema montañoso fue colonizado después.
En este punto, cabe señalar que la divergencia de secuencias intra e inter- específicas ha sido utilizada para establecer un porcentaje de divergencia de
139 HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
______secuencias nucleotídicas estandarizado, con objeto de estimar y diagnosticar los límites entre especies (Hebert et al., 2003). Sin embargo, esta práctica ha sido cuestionada, debido a que se ha observado que la divergencia entre secuencias
(tanto de loci nucleares como mitocondriales) se traslapa y varía ampliamente en varios grupos de insectos, incluyendo escarabajos descortezadores (Cognato, 2006).
El alto porcentaje de diferencias promedio entre secuencias encontrado en este trabajo (3.9%) concuerda con esto último, ya que es casi el doble a la diferencia del
2% que había sido frecuentemente usado para definir limites ente especies (Avise,
2004; Hebert et al., 2003).
A pesar de estos inconvenientes, el porcentaje de divergencia entre secuencias, más la topología de la reconstrucción filogenética, permiten hacer varias inferencias histórico-poblacionales. Por ejemplo, la baja divergencia ente las secuencias de las poblaciones del noroeste de Norteamérica puede deberse a la reciente colonización del área respecto del norte de México y las Montañas Rocosas del sur, más antiguas y genéticamente diversas. Esto puede ser el resultado de diversos factores. Entre ellos están los sucesivos retraimientos de los glaciares en
Norteamérica durante el periodo Pleistoceno (Hewitt e Ibrahim 2001). Así, áreas previamente cubiertas por el hielo, como la Columbia Británica, la Garganta del rio
Columbia, la cordillera de las Cascadas (Oregón y Washington) y las Montañas
Rocosas del norte (Idaho, Montana) estuvieron disponibles, permitiendo el avance del huésped hacia el norte. Por otro, el correspondiente cambio climático durante
140 HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
______este periodo provocó la fragmentación del área de distribución del huésped en el norte de México.
Por otro lado, la evidencia reunida a partir del análisis morfológico mostró que de los diez caracteres originalmente propuestos por Furniss (2001), solo cuatro presentaron claras diferencias para identificar a las subespecies de D. pseudotsugae.
El resto de los caracteres tienden a mostrar una variación que se traslapa entre los grupos de poblaciones, haciéndolos taxonómicamente menos útiles. Además, los resultados del estudio permitieron la identificación de otros tres caracteres para la identificación de las subespecies. A través del análisis filogenético, este nuevo conjunto de siete caracteres mostró un patrón de variación morfológica consistente que, al igual que con los datos moleculares, permitió la identificación de dos grupos monofiléticos correspondientes a las subespecies asignadas.
El hecho de que dos conjuntos de caracteres completamente independientes
(moleculares y morfológicos), muestren una alta similitud en análisis filogenéticos también independientes, y que además hayan formado los mismos grupos de poblaciones, sugiere fuertemente que ambas subespecies pudieran ser elevadas de categoría taxonómica, es decir, a especies. Esto es plausible, pues estudios similares realizados en otros escarabajos descortezadores y de conos de pinos han mostrado la posible presencia de especies cripticas (DeGroot et al., 1992; Kelley et al., 1999; Cognato et al., 2005b).
141 HISTORIA DEMOGRÁFICA Y FILOGEOGRAFÍA DE Dendroctonus pseudotsugae HOPKINS (COLEOPTERA: CURCULIONIDAE: SCOLYTINAE).
Ruiz Castillo Enrico Alejandro.
______Sin embargo, para determinar sin lugar a dudas si el proceso evolutivo de especiación es completo o si está aún en marcha, es necesario reunir evidencia adicional (e. g. ecología química, de aislamiento reproductivo, asá como de otros marcadores moleculares y caracteres morfológicos).
Estos resultados también son relevantes para el manejo y la conservación de los stands de Pseudotsuga en México, ya que estas coníferas están consideradas como especie amenazada por la Norma Oficial Mexicana NOM-059-ECOL-2001
(SEMARNAT, 2002). En este sentido, es posible que entre las subespecies de D. pseudotsugae existan diferencias en el uso y la respuesta a las feromonas. Esto ha sido observado en poblaciones geográficamente distantes de Ips pini, otro escarabajo descortezador de amplia distribución en Norteamérica (Lanier et al.,
1972). Los resultados del presente trabajo proveen de una hipótesis para investigar tales posibilidades.
Capítulo III
La observación de una variabilidad genética alta es un indicio de poblaciones antiguas, mientras que en las más recientes, la variabilidad genética tiende a ser más baja. Aunque estas características carecen de una perspectiva histórica y demográfica, permiten de manera sencilla identificar rutas de expansión o colonización.
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______En este sentido, las altas diversidades haplotípicas y nucleotídicas muestran que las poblaciones del este de Coahuila y sur de Durango, así como las del norte de
Colorado y norte de Arizona, son las más antiguas. En contraste, los valores de estos estimadores de la diversidad son más bajos en las poblaciones de las regiones del oeste de Wyoming, este de Oregón, norte de Idaho, norte de Washington y la
Columbia Británica. Este patrón sugiere una posible expansión en dirección sur-norte en Norteamérica.
Resultados similares en otras especies de insectos descortezadores de
Europa y Norteamérica han revelado que muchos eventos de expansión de área de distribución estuvieron relacionados con procesos de colonización posteriores a las glaciaciones del Pleistoceno (Stauffer et al., 1999; Kohlmayr et al., 2002; Cognato et al., 2003; Ritzerow et al., 2004; Faccoli et al., 2005; Horn et al., 2006; Maroja et al.,
2007; Mock et al., 2007;Anducho-Reyes et al., 2008).
El análisis filogenético no solamente recuperó los mismos grupos monofiléticos correspondientes a las subespecies de D. pseudotsugae, sino que también presentó una mayor resolución. De esta manera, se obtuvieron cuatro haplogrupos, correspondientes al noroeste de Norteamérica (NNA), suroeste de
Norteamérica (SNA), Sierra Madre Occidental (SMOC), y Sierra Madre Oriental
(SMOR).
La topología de los haplotipos de Norteamérica (clado I; Fig. 3.2) concuerda con la hipótesis de vicarianza antigua propuesta por Brunsfeld (2001) para explicar
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______los patrones de separación en el Pacífico Noroeste. Esta hipótesis postula la existencia de un sistema de bosques húmedos muy antiguos, los cuales fueron divididos por un proceso de desertificación en la cuenca del Rio Columbia asociado a la orogenia de la cordillera de las Cascadas. De esta manera, muchos taxa persistieron en las Cascadas y en las Montañas Rocosas del norte a lo largo de los ciclos glaciales del Pleistoceno (Brunsfeld, 2001), incluyendo a antiguas poblaciones de D. pseudotsugae.
La topología de la filogenia y las inferencias mencionadas concuerdan con otros estudios que también encuentran que la hipótesis de vicarianza antigua explica mejor la historia evolutiva de los bosques húmedos del Pacífico Noroeste. Ente estos estudios destacan los hechos en Pinus albicaulis (Richardson et al., 2002), Salix melanopsis (Brunsfeld et al., 2007), el parasito Adelgido que también tiene a
Pseudotsuga como huésped (Ahern et al., 2009) e incluso al mismo huésped
Pseudotsuga (Li y Adams, 1989).
Aunque los resultados mencionados hasta ahora pueden ser útiles como evidencias de cambios histórico-demográficos, se realizaron pruebas especificas para expansión demográfica. En primer lugar, todas las pruebas de neutralidad (D de
Tajima, FS, y R2) sugieren que las poblaciones del noroeste de Norteamérica han experimentado eventos de expansión demográfica. En segundo, las mismatch distributions y los Bayesian skyline plots (BSP) de los haplogrupos mitocondriales observados son consistentes con expansión poblacional en NNA y en SMOC, pero
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______no en SNA y SMOR (Aunque la estimación de hecha con BSP es mucho más conservadora, pues los eventos de expansión los sitúa en el Plioceno Superior).
La estimación de los tiempos de los posibles eventos de expansión, a partir de
τ, muestra que para NNA y SMOC pudieron haber ocurrido entre el Pleistoceno medio y el Pleistoceno superior. En cualquier caso, las tres aproximaciones son congruentes con eventos de expansión en NNA y SMOC (pero no en SNA y SMOR), durante el Plioceno-Pleistoceno. Los resultados son semejantes a los obtenidos en otras especies del género Dendroctonus de distribución similar (Maroja et al., 2007;
Mock et al., 2007). Sin embargo, estas interpretaciones deben ser tomadas con cautela, debido a que el análisis de un solo locus no permite excluir otros modelos de selección diferentes al de expansión demográfica, y a la estimación mucho más profunda de los resultados del análisis BSP.
Aunque la hipótesis de vicarianza antigua predice un tiempo de divergencia relativamente profundo entre la Cordillera de las Cascadas y las Montañas Rocosas del norte (1.8 – 2.6 Millones de años), las estimaciones de tiempos de divergencia pareadas entre haplogrupos realizadas con un modelo de coalescencia (usando el programa MDIV) mostraron que la separación entre NNA y SNA ocurrió hace solo
0.47 millones de años, alrededor del Pleistoceno medio. Una posible explicación de este resultado es que los linajes de D. pseudotsugae dentro de esos haplogrupos han experimentado una divergencia significativa pero con un contacto intermitente a
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______lo largo del tiempo, o expansiones de área continua mucho tiempo antes del último periodo glacial.
Por otro lado, el mayor tiempo de divergencia entre NNA y el resto de los haplogrupos (tabla 3.4) parece ser el resultado de la expansión de las poblaciones de
D. pseudotsugae hacia el Pacífico noroeste vía las Montañas Rocosas, mientras las poblaciones del norte de México continuaban un proceso de aislamiento. En este escenario, el área de distribución sureña (SMOC, SMOR, SNA, pero no NNA) pudo haber albergado a múltiples refugios, mientras los glaciares cubrieron la mayor parte de Norteamérica. Estudios en otros grupos, distribuidos en áreas que no fueron afectadas por las glaciaciones de forma directa, revelan procesos similares (Smith y
Farrell, 2005; Li et al., 2009). Sin embargo, la naturaleza estocástica del proceso de coalescencia hace necesario considerar más loci para evitar inferencias erróneas derivadas a partir de un único locus.
El análisis de clados anidados no solamente corroboró las inferencias tanto de las pruebas de neutralidad como de las mismatch distribution y los Bayesian Skyline
Plots; además, permitió la inferencia de eventos históricos y demográficos en regiones específicas. Por ejemplo, además de inferir cambios en el tamaño poblacional, se determinaron también expansiones de área de distribución en la
Cordillera de las Cascadas y en las Montañas Rocosas del norte para las poblaciones de D. pseudotsugae en Norteamérica, mientras que para las poblaciones mexicanas estos eventos fueron inferidos para el sur de la Sierra Madre
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______Occidental. En contraste, para el clado anidado que contiene haplotipos del noroeste de Norteamérica por un lado y haplotipos de las Montañas Rocosas del sur por otro, se infirió flujo génico restringido con aislamiento por distancia. La misma inferencia fue hecha para el clado anidado que contiene haplotipos de la Sierra Madre Oriental y de la Sierra Madre Occidental.
Las inferencias hechas con el análisis de clados anidados no permitieron obtener conclusiones firmes sobre los posibles eventos que expliquen por qué se obtuvieron tres redes de haplotipos diferentes, a pesar de que se obtuvieron ejemplares de la mayor parte del área de distribución de D. pseudotsugae. Esto puede ser indicio de una profunda y antigua divergencia. La distribución antigua de las coníferas en Norteamérica y la historia evolutiva del huésped Pseudotsuga menziesii indican que los climas más cálidos de los periodos interglaciares empujaron a las coníferas hacia el norte de Colorado, Nuevo México y Arizona, y aislaron los bosques de coníferas dentro de esta área. Específicamente, los cambios ambientales y el estrés redujeron el tamaño poblacional de Pseudotsuga menziesii y forzaron la fragmentación de su distribución en dirección sur hacia el norte de México
(Li y Adams, 1989; Aagaard et al., 1998a,b; Bartlein et al., 1998).
En este sentido, la cercana relación desarrollada entre herbívoros y sus plantas huésped ha sido largamente observada (Thompson, 2004). Frecuentemente, se ha encontrado que existe congruencia filogenética entre la variabilidad genética de los herbívoros y la distribución geográfica de sus huéspedes (Brown et al., 1997;
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______Cognato et al., 2005b; Aoki et al., 2008). Los resultados del presente estudio proveen ahora de una base para poner a prueba la hipótesis de que la variación genética del insecto especialista D. pseudotsugae es congruente con la variación intra-específica de su huésped Pseudotsuga menziesii o no.
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______
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155 Hereditas 146: 79Á92 (2009)
Effect of geographic isolation on genetic differentiation in Dendroctonus pseudotsugae (Coleoptera: Curculionidae) ENRICO A. RUIZ1, JOHN E. RINEHART2, JANE L. HAYES3 and GERARDO ZU´ N˜ IGA1 1Escuela Nacional de Ciencias Biolo´gicas-IPN, Laboratorio de Variacio´n Biolo´gica y Evolucio´n, Depto de Zoologı´a, Carpio y Plan de Ayala s/n, Col. Santo Toma´s, Mexico City, Me´xico 2Biology Program, Eastern Oregon University, LaGrande, Oregon, USA 3Forestry and Range Sciences Laboratory, Pacific Northwest Research Station, USDA Forest Service, LaGrande, Oregon, USA
Ruiz, E. A., Rinehart, J. E., Hayes, J. L. and Zu´n˜iga, G. 2009. Effect of geographic isolation on genetic differentiation in Dendroctonus pseudotsugae (Coleoptera: Curculionidae). * Hereditas 146:79Á92. Lund, Sweden. eISSN 1601-5223. Received August 28, 2008. Accepted February 17, 2009
Genetic structure of phytophagous insects has been widely studied, however, relative influence of the effect of geographic isolation, the host plant or both has been subject of considerable debate. Several studies carried out on bark beetles in the genus Dendroctonus evaluated these factors; nonetheless, recent evidence has shown that genetic structuring is a more complex process. Our goal was to examine the effect of geographic isolation on genetic structure of the Douglas-fir beetle Dendroctonus pseudotsugae. We used mtDNA cytochrome oxidase I (COI) sequences and RAPD markers. One hundred- seventy-two individuals were obtained from 17 populations, for which we analyzed 60 haplotypes (among 172 sequences of COI gene, 550 bp long) and 232 RAPD markers (7 primers). Analyses of molecular variance (AMOVA and SAMOVA), F-statistics and linear regressions suggest that the genetic structure of D. pseudotsugae is strongly influenced by geographic distance. We found that D. pseudotsugae has high intra- and inter-population genetic variation compared with several other bark beetles. Genetic differences among populations based on COI and RAPD markers were correlated with geographic distance. The observed genetic differences between northern (CanadaÁUSA) and southern (Mexico) populations on Pseudotsuga menziesii var. glauca confirm that these two sets of populations correspond to previously assigned subspecies.
Gerardo Zu´n˜iga, Escuela Nacional de Ciencias Biolo´gicas-IPN, Laboratorio de Variacio´n Biolo´gica y Evolucio´n, Depto de Zoologı´a, Carpio y Plan de Ayala s/n, Col. Santo Toma´s, CP 11340 Mexico City, Me´xico. E-mail: [email protected]
The genetic structure of phytophagous insects has 2007; MOCK et al. 2007; ANDUCHO-REYES et al. been widely studied (RODERICK 1996), however, the 2008). factors by which their populations become geneti- The distribution of genetic variability appears to be cally differentiated have been subject of consider- affected by the population dynamics of these insects able debate (PETERSON and DENNO 1998; VAN (including eruptive and non-eruptive periods, dispersal ZANDT and MOPPER 1998). Principal among these potential), the geographical distribution both of often-intertwined factors are effects of geographic insects and their hosts, as well as geomorphologic isolation and host plant influences. Several studies histories that determine different levels of allopatry or carried out on bark beetles in the genus Dendroctonus sympatry among populations (KELLEY et al. 1999; Erickson (Coleoptera: Curculionidae: Scolytinae) MAROJA et al. 2007; MOCK et al. 2007; ANDUCHO- evaluated the effect geographic isolation (NAMKOONG REYES et al. 2008; SCHREY et al. 2008). et al. 1979; STOCK et al. 1984; ROBERDS et al. 1987; Our goal was to examine the effect of geographic SIX et al. 1999; ZU´ N˜ IGA et al. 2006; SCHREY et al. isolation on genetic structure of Dendroctonus pseu- 2008) and of host plants (STOCK and AMMAN 1980; dotsugae (Douglas-fir beetle). This bark beetle colo- STURGEON and MITTON 1986; LANGOR and SPENCE nizes one single host, Pseudotsuga menziesii (Mirbel) 1991; AMMAN and STOCK 1995; KELLEY et al. 2000) Franco across its geographic range, which has two on the genetic differentiation of their populations. varieties: a costal variety (var. menziesii) found in the While these studies carried out at both fine and coarse Pacific northwest, and an interior variety (var. glauca) geographic scales documented genetic differences found throughout Rocky Mountains and north of among populations associated with these factors, there Mexico (HERMANN and LAVENDER 1990). Perhaps is no agreement on the roles geographic isolation or the most intuitive hypothesis of the effect of geo- host use have played in genetic differentiation in graphic isolation on genetic structure is the model of Dendroctonus species. In addition, recent phylogeo- isolation by distance (IBD) (WRIGHT 1943), which graphic evidence has shown that genetic structuring of predicts that genetic differentiation between popula- these beetles is a more complex process (MAROJA et al. tions increases with geographic distance. PETERSON
DOI: 10.1111/j.1601-5223.2009.02095.x 80 E. A. Ruiz et al. Hereditas 146 (2009)
TM and DENNO (1998) found by meta-analysis that IBD DNA polymerase, Recombinant, Invitrogen ,Sao in phytophagous is less common in highly mobile Paulo, Brazil). The final volume was brought to 25 ml insects (dispersal 20 km) compared with those whose with ultrapure water. Cycling parameters were: pre- vagility is low or moderate. Nevertheless, it has been heating of samples for 10 min at 958C, followed by 35 shown that a fundamental factor that promotes IBD amplification cycles of 5 min at 948C, 1 min at 518C, among populations is the equilibrium between gene 2 min at 728C, and a final extension of 5 min at 728C. flow and drift (HUTCHINSON and TEMPLETON 1999). Amplification products were separated on 1.5% w/v Focusing on Douglas-fir beetle found on P. menziesii agarose gel, and stained with ethidium bromide. PCR var. glauca (and thus, reducing the effect of host plant), products were purified using GFXTM PCR DNA and we assess the distribution of genetic variation and gel band purification kit (Amersham Biosciences, isolation by distance (IBD) in a northÁsouth direction Buckinghamshire, UK) to remove primers and unin- among populations. Considering the original geogra- corporated dNTPs prior to sequencing. Cycle sequen- phical distribution of this host variety in North cing reactions were performed with BigDye fluorescent America, we expect both strong genetic structuring chemistry reaction (Applied Biosystems, Foster City, and IBD among North American beetle populations CA). Both forward and reverse strands were sequenced and those from Mexico. To address these questions, we with an ABI 377 sequencer and contiguous sequences examine genetic variation in mitochondrial (mtDNA) constructed and edited manually using Sequence cytochrome oxidase I (COI) and random nuclear Navigator ver. 1.0.1 (Applied Biosystems, Foster City, markers (RAPD) in D. pseudotsugae samples from CA). Multiple alignments of sequences were assembled across its distribution range. with Clustal X ver. 1.83 (THOMPSON et al. 1997). Reference sequences of each haplotype were deposited in the GenBank nucleotide sequences database (acces- MATERIAL AND METHODS sion no. EU043405-EU043464; http://www.ncbi.nlm. Samples and DNA extraction nih.gov/Genbank). A total of 172 adult Dendroctonus pseudotsugae were collected from 17 geographically distinct populations RAPD-PCR amplification of Douglas-fir Pseudotsuga menziesii var. glauca from Random amplified polymorphic DNA (RAPD) ana- Canada, USA and Me´xico (Fig. 1A, Table 1). For lysis of nuclear genome samples using a set of single comparative purposes, one population was collected short oligonucleotide primers results in a rapid from the other variety, P. menziessi, var. menziesii production of large amounts of genetic information (Mount Hebo, Oregon). Specimens from northern (WILLIAMS et al. 1990). RAPD-PCR is a versatile populations were collected by directly sampling under method that can generate useful nuclear markers for the bark of recently infested trees or using traps baited genetic structure analysis (PARKER et al. 1998; HOY with attractant pheromones, whereas almost all in- 2003). Criticized for its lack of reproducibility and dividuals from southern populations were collected contamination issues (PE´ REZ et al. 1998), we address from recently infested trees. To avoid analysis of these issues as described below. genetically related individuals from those populations The following primers were selected for RAPD-PCR collected manually, we gathered individuals from six analysis based on their polymorphism, brightness and or seven trees per population. All beetles were stored consistent banding pattern: OPAM-07, OPAM-11, in 100% ethanol. OPAM-13, OPB-10, OPB-01, OPM-01 and OPT-05 (Eurofins MWG Operon, Huntsville, AL). Amplifica- Mitochondrial COI amplification tion, including cycling parameters, followed the meth- PCR amplification of a 600 bp fragment of mtDNA ods described for COI, with the exception that each cytochrome oxidase I (COI) was carried out using PCR reaction contained 20 ng of genomic DNA. primers C1-J-2441 (5?ACA GGWATT AAA ATT TTT RAPD amplification products were separated on 2% AGT TGA TTA GC 3?) and T12-N-3014 (5?TTC AAT w/v agarose gel in TAE buffer (Tris-HCL 10 mM, GCA CTA ATC TGC CAT ATT A 3?)(SIMON et al. glacial acetic acid, 1 mM EDTA pH 8.0) at 120 V for 1994). DNA amplification was performed using a 90 min. Gels were stained as described above and Biometra T Gradient thermocycler (Biometra GmbH, photographed with an Alpha ImagerTM 2200 (Alpha Go¨ttingen, Germany). Each PCR reaction mixture Innotech Corporation, San Leandro, CA). contained 50 ng of DNA, 1 ml primer 50 pM, 4 ml The reproducibility of our RAPD analyses was dNTP’s10mM(1ml for each dNTP), (dNTPs, tested in five individuals randomly selected from the TM Invitrogen , Sao Paulo, Brazil), 6 ml MgCl2 25 mM, complete sample pool. Amplifications were performed 5 ml1 buffer, and 0.4 ml Taq DNA polymerase (Taq twice for each primer in two independent assays, and Hereditas 146 (2009) Genetic differentiation in Dendroctonus pseudotsugae 81
Fig. 1AÁC. Sample localities of Dendroctonus pseudotsugae and results from SAMOVA and maximum likelihood (ML) analyses. (A) The geographic range of Douglas fir beetle corresponds to that of its host, Pseudotsuga menziesii.(B) Groups found in SAMOVA analysis are shown within dashed lines. (C) Phylogenetic analysis by ML using only the mitochondrial data set of Dendroctonus pseudotsugae. The best fit model of nucleotide substitution was TrN G I. Bootstrap values at nodes (500 pseudoreplicates). all tests were conducted in the same laboratory using of bark beetles also contain DNA from microorgan- the same enzyme, reagent brand and thermocycler isms (bacteria and yeast) associated with them, we (PE´ REZ et al. 1998). To determine if the total DNA performed PCR on bark beetle DNA using specific 82 .A uze al. et Ruiz A. E.
Table 1. Locations, geographic references, and number of Dendroctonus pseudotsugae specimens analyzed. Population specific haplotypes are shown in bold, along with haplotype population frequencies.
Pop. no. Localities Pop. key Latitude Longitude No. Haplotypes individuals
Southern populations 1 Ejido Cienega de la Vaca, San Dimas, DGO. CDGO 248 05? 20ƒ N 1058 31? 00ƒ W9H87, H101, H108, H110(6) 2 Ejido Puentesillas, San Dimas, DGO. PDGO 248 21? 10ƒ N 1058 54? 39ƒ W11 H103, H104, H106, H109, H110(7) 3 Ejido La Manga, San Dimas, DGO. MDGO 248 22? 08ƒ N 1058 58? 15ƒ W 9 H86(2), H92(2), H94(2), H98(2), H110 4 Ejido Nun˜ez, San Dimas, DGO. EDGO 248 22? 29ƒ N 1058 55? 39ƒ W10 H96(3), H97, H98(2), H104(2), H110(2) 5 Santa Rita, Nuevo. Leo´n SNLO 258 09? 12ƒ N 1008 08? 41ƒ W7H91, H94 (2,), H95, H98(2), H99 6 Arteaga, Coahuila. ACOA 258 26? 14ƒ N 1008 42? 30ƒ W12 H81, H82(2), H83, H84(3), H85, H107(4) 7 Llano Grande, Guanasev´ı, DGO. LDGO 268 04? 16ƒ N 1068 17? 15ƒ W 9 H88(4), H98, H102(2), H105, H109 8 Ejido El Nopal, Guadalupe y Calvo, CHI. ECHI 268 05? 31ƒ N 1078 02? 11ƒ W 9 H86, H88, H93(2), H100, H110(4) Northern populations 9 Snow Bowl, Flagstaff, AZ. FLAZ 358 17? 54ƒ N 1118 42? 54ƒ W13 H21(2), H22, H23(3), H52, H53, H54, H55, H56(3) 10 John Day, OR. JDOR 448 34? 53ƒ N 1188 31? 21ƒ W 11 H4(2), H5, H6(2), H8, H9(2), H10(3) 11 Balm Creek Reservoir, OR. BCOR 448 58? 49ƒ N 1178 33? 44ƒ W 11 H4(4), H6(5), H18, H19 12 Mount Hebo, OR. MHOR 458 10? 30ƒ N 1238 40? 08ƒ W11 H1, H4(2), H6(6), H27, H28 13 Spring Creek, OR. SCOR 458 20? 25ƒ N 1188 18? 50ƒ W 11 H4(2), H10, H11, H12, H13, H14, H15, H17, H79(2) 14 Mt. Emily Road, OR. EROR 458 25? 29ƒ N 1188 08? 32ƒ W9H3(3), H4(4), H7(2) 15 Drum Hill, OR. DHOR 458 27? 53ƒ N 1188 11? 25ƒ W10 H2, H6(7), H16, H80 16 Lubrecht Experimental Forest, Missoula, MT. MSMT 468 53? 10ƒ N 1138 26? 55ƒ W 11 H4, H6(2), H7(2), H9, H27, H28(2), H78, H79 17 Revelstoke, British Columbia. RVBC 518 08? 15ƒ N 1188 16? 26ƒ W 9 H4, H6, H27(3), H28(2), H78, H79 eeia 4 (2009) 146 Hereditas Hereditas 146 (2009) Genetic differentiation in Dendroctonus pseudotsugae 83 primers for the bacterial 16S gene (DELALIBERA et al. p-distance, which allows performing a simple compar- 2005; VASANTHAKUMAR et al. 2006), and yeast 26S ison of number of differences among individuals using gene (FELL 1993; Zu´n˜iga unpubl.). All PCR tests both data sets. The dendrogram was validated through focused on identifying DNA from bacteria and yeasts a bootstrap analysis with 1000 pseudoreplicates. No were negative, which indicates that the extraction sister taxa were used as outgroups, because our kit and specific method used for obtaining total phylogenetic reconstructions were not performed for DNA decreased the possibility of non-beetle DNA the purpose of revealing phylogenetic relationships per contamination. se. All reconstructions were carried out using the RAPD-PCR amplification products were scored program PAUP* (SWOFFORD 2002). visually; fragment size was calculated using a 100 bp Based on recovered groups by ML and NJ analyses, DNA Ladder (InvitrogenTM), manually coded, and we performed AMOVA analysis using Arlequin ver. analyzed in two different ways: 1) as phenotypic 3.0 for mtDNA data and Winamova ver. 1.55 for characters where RAPD bands of each locus were RAPD data (EXCOFFIER et al. 1992, 2005) to partition registered for analysis as binary data of presence (1) the molecular variance into different hierarchical or absence (0), and 2) as allelic variants having levels: within sampling sites (populations), among a dominant inheritance pattern. Allele frequencies populations within groups, and between groups. The of RAPD bands were estimated according to statistical significance of partitioned molecular var- ZHIVOTOVSKY (1999); we only selected those markers iance and the associated estimate of 8-statistics were whose frequencies in all samples were between 0.05% assessed by conducting 10 000 random permutations and 98% (LYNCH and MILLIGAN 1994). of the data (EXCOFFIER et al. 1992). The spatial analysis of molecular variance using Statistical analyses SAMOVA 1.0 (DUPANLOUP et al. 2002) was per- formed using the mitochondrial data set alone. We To assess the patterns of molecular diversity of carried out this analysis despite the fact that sampling mtDNA sequences, we estimated the haplotype and points of D. pseudotsugae are not geographically nucleotide diversity (NEI 1978) using the software adjacent and populations are not genetically homo- DNASP ver. 4.50.3 (ROZAS et al. 2003). Genetic geneous in the region under study, as it is assumed by diversity of RAPD markers was calculated using three this approach. The method implemented in this methods: Nei’s genetic diversity (h) using allelic programme indirectly detects genetic barriers and frequencies (NEI 1973); expected heterozygosity per defines groups of populations geographically homo- population (H) and average heterozygosity (Hw), using allele frequencies according to Zhivotovsky’s Bayesian geneous and maximally differentiated from each other. We ran this program repeatedly, changing n (number method (ZHIVOTOVSKY 1999); and by Shannon’s index (S), calculated assuming that each phenotypic marker of groups) from 2 to 16. Significance tests were represents a distinct locus (ALLNUTT et al. 1999). performed with 1000 permutations. To define groups of D. pseudotsugae populations To determine whether the genetic structure of maximally differentiated from each other, we used D. pseudotsugae describes an IBD pattern, several both a priori (AMOVA) and a posteriori (SAMOVA) approaches were followed. First, linear regression of approaches. For AMOVA analysis, we performed least squares between FST/1ÁFST (using 8ST)and phylogenetic analyses by maximum likelihood (ML) geographic distances among the populations was method using only the mtDNA data set. We used conducted (ROUSSET 1997). Ordinary linear regres- MODELTEST 3.7 (POSADA and CRANDALL 2001) to sions for mtDNA COI and RAPD were independently select the best fit model of sequence evolution. The carried out with ln-transformed geographic distances. ML analysis was performed with heuristic search and The Mantel test was used to determine statistical TBR as swapping algorithm. The method was vali- significance (MANTEL 1967) after 5000 random per- dated through 500 pseudoreplicates by bootstrap mutations using NTSYSpc ver. 2.02j (ROHLF 1998). In analysis. Additionally, although RAPD data have addition, the pairwise p-distances of the concatenated been considered unreliable in phylogenetic reconstruc- matrix (mtDNA COI sequences plus the RAPD bands tion (BACKELJAU et al. 1995; HOY 2003), we chose to of each individual) and geographical distances were concatenate mitochondrial and RAPD data from each used to plot a linear regression of least squares, and individual. This matrix was used to construct a the non-parametric Mantel test (as described above) dendrogram by neighbour-joining (NJ) with the was also used to determine statistical significance. 84 E. A. Ruiz et al. Hereditas 146 (2009)
RESULTS in the case of h¯ and p from mtDNA COI data, the genetic diversity (S and h) showed no difference Genetic diversity between southern (Mexico) and northern populations From the 172 mtDNA cytochrome oxidase I (COI) (USA and Canada) (P 0.05). No correlation between sequences, 60 different haplotypes of 550 bp length latitude and genetic diversity (S and h) per population, were identified. The populations with the highest or between latitude and expected heterozygosity per number of haplotypes were Spring Creek (SCOR), population, were found (data not shown). Missoula (MSMT) and Flagstaff (FLAZ) with nine, eight and eight haplotypes, respectively. The most Genetic structure frequent haplotypes were H4, H6 and H110, which were found in most populations. The remaining The ML topology of the 172 individuals using only mtDNA sequences showed two clearly distinguishable haplotypes had a frequency lower than 5%, however, groups: one composed only by northern populations most of them were population-specific (Table 1). and the other only by Mexican populations (Fig. 1B). Mean haplotype and nucleotide diversities were The combined data set of 782 characters (COI 550 h¯ 0:94590:009 and p 0.0279 0.001, respectively bp 232 RAPD markers) for 172 individuals analyzed (Table 3). Estimates of diversity (/hand¯ p) were not by NJ (using p-distance) gave a dendrogram similar to statistically different among populations, or between the ML analysis, and recovered the same two groups northern and southern populations (P 0.05). Also, of populations (Appendix 1). there was no correlation between latitude and haplo- The results of the AMOVA analysis using both type diversity per population or between latitude and mtDNA COI and RAPD data (8-statistics, sum of nucleotide diversity per population (data not shown). squares, variation percentage, and probability (P) A total of 232 RAPD markers were obtained from associated with 8 values) are summarized in Table 4. 172 beetles analyzed with seven primers. Size of The source of variation between groups of populations RAPD bands selected for analysis ranged between (8CT) was assessed using the two groups found by 300Á2050 bp, and reproducibility tests showed that phylogenetic analysis. Both types of markers were amplifications were highly consistent and reproducible consistent in describing the partitioning of genetic among randomly selected beetles (Table 2). Nei’s variation according to the source of variation, and genetic diversity, heterozygosity per population (Hj), revealed that variation within populations is much and Shannon’s index are shown in Table 3. Mean Nei’s greater than between populations or between groups. genetic diversity was 0.26 (SE 0.002); the lowest In particular, the 8ST (assessed either with mtDNA or value was found in the Flagstaff (FLAZ) population RAPD data) showed strong genetic differentiation, (0.178, SE 0.002), while the highest occurred in the and all 8-statistics were statistically different from La Manga (MDGO) population (0.319, SE 0.002). zero, indicating that genetic variation was geographi- Mean heterozygosity was 0.323 (SE 0.001); the low- cally structured. est heterozygosity occurred in the Flagstaff (FLAZ) The results obtained by SAMOVA analysis are population (0.197, SE 0.002) and the highest in the shown in Fig. 1C and Table 5. This approach detected La Manga (MDGO) population (0.372, SE 0.002). five groups of populations geographically differen- Mean Shannon’s index was high (I 0.389, SE tiated (FCT 0.631), separated from each other by 0.001); the lowest (0.199, SE 0.0004) and highest inferred genetic barriers (Fig. 1C). Three groups (0.482, SE 0.0004) values were found in the same contained only populations from Mexico, while the two populations (FLAZ and MDGO, respectively). As two remaining groups contained only populations
Table 2. Primers, fragment size, number of polymorphic bands and autosimilarity values with RAPD-PCR markers in D. pseudotsugae populations.
Primer Sequence Fragments (bp) Polymorphic bands SXX OPAM-07 5?-AACCGCGGCA-3? 300-2050 35 0.785 OPAM-11 5?-AGATGCGCGG-3? 300-2050 35 0.868 OPAM-13 5?-ACCGGCACAA-3? 300-2050 32 0.805 OPB-10 5?-CTGCTGGGAC-3? 300-2050 33 0.724 OPB-01 5?-GTTTCGCTCC-3? 300-2050 29 0.701 OPM-01 5?-GTTGGTGGCT-3? 300-2050 35 0.732 OPT-05 5?-GGGTTTGGCA-3’ 300-2050 33 0.863 Hereditas 146 (2009) Genetic differentiation in Dendroctonus pseudotsugae 85
Table 3. Haplotype and nucleotide diversities estimated for COI data, and Nei’s genetic diversity (h), heterozygosity (Hj) and Shannon index estimated for RAPD data in D. pseudotsugae populations. SE standard error.
Pop. Haplotype diversity Nucleotide diversity Nei’s genetic diversity Hj(SE) Shannon index (SE) (SE) (SE) (SE) southern populations CDGO 0.583 (0.0023) 0.003 (0.00017) 0.295 (0.0022) 0.355 (0.00052) 0.444 (0.0012) PDGO 0.618 (0.0022) 0.003 (0.00017)) 0.267 (0.0023) 0.343 (0.00054) 0.403 (0.0014) MDGO 0.889 (0.0014) 0.022 (0.0003) 0.319 (0.0021) 0.372 (0.0017) 0.482 (0.0013) EDGO 0.867 (0.0014) 0.015 (0.00024) 0.255 (0.0024) 0.329 (0.00054) 0.382 (0.0013) SNLO 0.905 (0.0017) 0.013 (0.00024) 0.198 (0.0023) 0.255 (0.00054) 0.225 (0.0014) ACOA 0.848 (0.0015) 0.018 (0.00024) 0.305 (0.0022) 0.358 (0.00054) 0.461 (0.0014) LDGO 0.806 (0.0019) 0.016 (0.00034) 0.303 (0.0022) 0.362 (0.00057) 0.455 (0.0015) ECHI 0.806 (0.0019) 0.015 (0.0003) 0.271 (0.0023) 0.332 (0.00057) 0.412 (0.0014) northern populations FLAZ 0.910 (0.0013) 0.027 (0.00024) 0.178 (0.002) 0.197 (0.0017) 0.199 (0.00038) JDOR 0.800 (0.0015) 0.007 (0.00017) 0.246 (0.0023) 0.308 (0.00052) 0.377 (0.0011) BCOR 0.709 (0.0017) 0.008 (0.00024) 0.285 (0.0023) 0.341 (0.0017) 0.431 (0.0014) MHOR 0.705 (0.002) 0.007 (0.00024) 0.254 (0.0023) 0.326 (0.00052) 0.386 (0.0012) SCOR 0.964 (0.0012) 0.011 (0.00017) 0.268 (0.0023) 0.328 (0.00057) 0.408 (0.0015) EROR 0.722 (0.0017) 0.002 (0.00017) 0.295 (0.0022) 0.349 (0.00052) 0.448 (0.0016) DHOR 0.533 (0.0023) 0.005 (0.00024) 0.235 (0.0024) 0.307 (0.00052) 0.357 (0.0013) MSMT 0.945 (0.0013) 0.012 (0.0003) 0.229 (0.0024) 0.305 (0.00057) 0.345 (0.0013) RVBC 0.889 (0.0016) 0.014 (0.00024) 0.263 (0.0023) 0.332 (0.0014) 0.399 (0.0014) Mean 0.945 (0.00052) 0.027 (0.00017) 0.26 (0.0023) 0.323 (0.00084) 0.389 (0.0013)
from the North America. Mexican groups were as well as between those from northwestern and composed as follows: group 1, ARTG from Coahuila; southwestern USA. group 2, STAR, LMNG and ENNZ from southern Durango and Nuevo Leo´n; and group 3, PNTS, Isolation by distance CNVC, LNGR and ENPL from northern Durango Regression analyses of F /1ÁF versus ln- and Chihuahua. The two groups from the North ST ST transformed geographic distances with COI mtDNA America were: group 4, FLGS from Arizona and and RAPD markers revealed no positive and mono- group 5, MTHB, JHDR, SCLG, BCRK, DRHL, tonic relationship between genetic differentiation and EMRD, MSSL and CBRT from northwestern USA geographic distance within southern populations and southwestern Canada. SAMOVA results showed a (r Mantel 0.161, P 0.216, COI data; r Mantel clear differentiation due to a genetic barrier between 0.244, P 0.194, RAPD data), nor within northern the Mexico and the North America populations. This populations (r Mantel Á0.324, P 0.116, COI data; analysis also suggests that within these two regions r Mantel 0.591, P 0.06, RAPD data) (Fig. 2AÁB). genetic barriers may exist between populations of However, when all populations sampled in this study Sierra Madre Oriental and Sierra Madre Occidental, were considered, they described an isolation by
Table 4. Analysis of molecular variance (AMOVA) of RAPD and COI data from D. pseudotsugae populations, including DF, sum of squares, percentage of variation explained, P-values and F statistics. Upper values, RAPD data. Lower values, COI data.
Source of variation DF SS Percentage of variation (%) P F Statistic
Between groups (FCT) (north and south) 1 98.458 2.01 B0.001 0.020 1 4.292 7.72 0.01 0.077 Between populations (FSC) 15 900.892 19.6 B0.001 0.200 15 15.181 12.11 0.01 0.131 Within populations (FST) 155 2706.932 78.38 B0.001 0.216 155 62.102 80.16 0.01 0.198 86 E. A. Ruiz et al. Hereditas 146 (2009)
Table 5. Fixation indices corresponding to the groups A 0.6 of populations inferred by SAMOVA for the D. pseudotsugae populations tested for the mtDNA 0.5 sequences. *PB0.01; **PB0.001 0.4 ST No. of groups FCT FST FSC 0.3
2 0.595** 0.705** 0.272** / 1 - F
3 0.615** 0.689** 0.194** ST F 4 0.624** 0.677** 0.139** 0.2 5 0.631** 0.659** 0.076** 6 0.630** 0.656** 0.071** 0.1 7 0.621** 0.643** 0.059** 8 0.618** 0.636** 0.047** 9 0.618** 0.635** 0.045** 0 1234567 10 0.614** 0.624** 0.027** Ln geographical distance (km) 11 0.614** 0.613** 0.001** 12 0.616** 0.612** 0.011** B 0.7 13 0.619** 0.603** 0.041** 14 0.617** 0.595** 0.057* 0.6 15 0.619** 0.593** 0.069* 16 0.622* 0.591** 0.083* 0.5 ST 0.4 distance pattern (r Mantel 0.231, P 0.005, COI / 1 - F
ST 0.3 data; r Mantel 0.258, P 0.001, RAPD data) F (Fig. 2C). 0.2 The linear regression of pairwise p-distances (mtDNA COI RAPD data) and geographic dis- 0.1 tances also show that the simple number of differences among 172 individuals was positively related with their 0 12345678 geographic distance (r Mantel 0.488, P 0.0004, Ln geographical distance (km) Fig. 3). In most cases, pairs of geographically close C 0.9 individuals are less differentiated than those separated by longer distances. Therefore, while the effect of 0.8 geographical isolation on genetic differentiation is 0.7 insignificant for relatively geographically close popu- 0.6 lations, northern and southern groups of populations ST differ significantly. 0.5
/ 1 - F 0.4 ST F 0.3 y = 0.017x + 0.162 DISCUSSION 0.2 Genetic diversity 0.1 y = 0.023x + 0.063 mtDNA haplotype and nucleotide diversities have 0 123456789 been used as estimators of intraspecific genetic diver- Ln geographical distance (km) sity of many insects groups. The nucleotide diversity estimate for mtDNA COI in D. pseudotsugae (p Fig. 2. Scatter plot showing the relationship between genetic dissimilarities (estimated as FST/1 FST) and logarithms of 0.02790.001) is higher than in D. mexicanus Hop- geographical distances of D. pseudotsugae populations. (A) kins (0.01190.009, ANDUCHO-REYES et al. 2008), southern populations. (B) northern populations. (C) all 17 D. rufipennis Kirby (0.01890.004, MAROJA et al. populations. Diamonds and continuous line slope, COI data; 2007) and D. ponderosae Hopkins (0.00690.003, open circles and dotted line slope, RAPD data. Regression equations reported only for significant relationships. MOCK et al. 2007), but lower than in D. valens LeConte (0.02890.022, COGNATO et al. 2005a) and ¯ other Scolytinae (LANGOR and SPERLING 1997; D. pseudotsugae is a highly polymorphic species (/h STAUFFER et al. 1997; KOHLMAYR et al. 2002; 0:94590:009); as shown by the high number of COGNATO et al. 2003). Our data also reveal that different haplotypes and their frequencies. Hereditas 146 (2009) Genetic differentiation in Dendroctonus pseudotsugae 87
0.2
0.18
0.16
0.14
0.12
0.1 y = 1E 05x + 0.1139 R2 = 0.2388 p distance 0.08 rMantel= 0.488 P=0.0004
0.06
0.04
0.02
0 0 500 1000 1500 2000 2500 3000 3500 distance (km)
Fig. 3. Simple number of differences (p distance, using concatenated mtDNA COI RAPD data) vs corresponding geographic distances among pairs of individuals of D. pseudotsugae.
Few population genetic studies of curculionids been rapid population growth from small populations, (including Scolytinae) have included RAPD markers assuming sufficient time for recovery of haplotype (COGNATO et al. 1995). However, mean expected variation via mutation but too short for the accumula- heterozygosity (HE)ofD. pseudotsugae was 0.3239 tion of large sequence differences (GRANT and BOWEN 0.001, greater than either Diaprepes abbreviatus 1998). The same relationship between haplotype and L. (BAS et al. 2000) or Tomicus piniperda L. (CARTER nucleotide diversity has been observed in other et al. 1996). RAPD markers have not been studied in Scolytinae, including Dendroctonus species (COGNATO other Dendroctonus species, but studies involving et al. 2005a; MAROJA et al. 2007; MOCK et al. 2007; isozymes have demonstrated that they have a wide ANDUCHO-REYES et al. 2008). intra- and inter-population genetic variation. These Independent factors such as length of the sequence, studies have found higher observed heterozygosity number of populations and individuals analyzed, as than expected for random mating both in geographic well as coalescence time, can bias estimations of populations (STOCK et al. 1979, 1984; SIX et al. 1999; genetic diversity. However, general patterns of varia- ZU´ N˜ IGA et al. 2006), and in populations using tion in diversity indices were homogeneous among different host trees (STOCK and AMMAN 1980; populations. These results indicate homogenization of STURGEON and MITTON 1986; LANGOR and SPENCE interpopulation genetic diversity by high gene flow 1991). High values of Nei and Shannon indices, as well through extensive dispersal. Nevertheless, the dissim- as heterozygosity found in D. pseudotsugae, confirm ilar haplotype richness (number of haplotypes per these observations. In addition, genetic differences population) observed among populations and popula- among populations are correlated geographically, as tion groups suggest that populations have experienced has been suggested for this and other Dendroctonus a recent reduction of their effective population size. species using isozymes (STOCK et al. 1979; 1984; SIX Several studies carried out with European scolytines et al. 1999; ZU´ N˜ IGA et al. 2006). The similar magni- suggest that reduction and rapid population expansion tude of genetic diversity of these indices suggests that might be the result of glacial or postglacial events that genetic differences among populations are not an occurred during the Plio-Pleistocene period in Europe, artifact associated with the sample size analyzed. which promoted contraction and expansion of beetle While Nei’s indices are affected by sample sizes of habitats (hosts) and postglacial migration processes individuals as well as the number of loci analyzed (NEI from Plio-Pleistocene refugia (STAUFFER et al. 1999; 1978), the Shannon index is dependent on loci used HORN et al. 2006). AMOVA and SAMOVA analyses but independent of the number of individuals analyzed indicate a strong genetic differentiation between (HARTL et al. 1994). CanadaÁUSA and Mexico populations of this bark Comparison of bark beetle mtDNA nucleotide and beetle, probably as a consequence of fragmentation of haplotype diversity can be useful to assess demo- distribution range of its host plant (P. menziesii graphic history of populations. High haplotype and var. glauca) during climatic oscillations of the Plio- low nucleotide diversity found in this study for Pleistocene period. To infer the effect of glacial and D. pseudotsugae populations suggest that there has postglacial Pleistocene events on genetic structure of 88 E. A. Ruiz et al. Hereditas 146 (2009) this species, it would be necessary to carry out a isolation by distance within each north and south phylogeographic analysis including more populations population group of D. pseudotsugae indicate that they given that historical inferences are biased by the have not yet reached gene flow-drift equilibrium, which number of population sampled (PETIT et al. 2005). suggest genetic drift is less influential than gene flow, no matter how long the separation among populations Genetic structure (model III, HUTCHINSON and TEMPLETON 1999). These data agree with those obtained in other studies Several studies have suggested that specialization in using various markers (isozyme, RAPD, mitochon- host use may be a very important factor influencing drial) in Conophthorus Hopkins (COGNATO et al. genetic structure and differentiation of phytophagous 2005b), D. brevicomis (KELLEY et al. 1999), D. jeffreyi insects (VAN ZANDT and MOPPER 1998). The expecta- (SIX et al. 1999), D. ponderosae (KELLEY et al. 2000), tion of this hypothesis is that insect species with D. mexicanus (ZU´ N˜ IGA et al. 2006), Ips typographus restricted diet breadth (specialist species) should be L. (STAUFFER et al. 1999), and Tomicus piniperda more prone to genetic differentiation than a generalist L. (CARTER et al. 1996). Our observations also suggest species because the distribution of the specialist’s that the genetic structure of specialist species is more single host is patchier and less dense than the sensitive to IBD than generalist species, possibly combined distribution of all generalist’s hosts because genetic differentiation of generalists is mini- (THOMPSON 2004). This hypothesis has been tested mized due to different host use throughout its geo- in generalist and specialist species of Dendrocto- graphical distribution. nus using different molecular markers (KELLEY et al. 1999, 2000; ZU´ N˜ IGA et al. 2006). In these studies, Isolation by distance generalist species showed slight genetic differences among populations colonizing different hosts in the Linear regressions of both FST and p-distance vs same locality and cumulative differences among geo- geographic distance have shown a significant increase graphically isolated populations. Our results seem to in amount of genetic differentiation among the popu- support these expectations, because they reveal a lations we sampled, relative to the increase of geo- strong pattern of differentiation between geographi- graphic distance. These results agree with the study of cally separated populations, although it is difficult to PETERSON and DENNO (1998), which showed that the know whether this differentiation was solely promoted balance between dispersal and geographic isolation has by host fragmentation (due to both habitat loss and had a greater effect over genetic structure and differ- the breaking apart of habitat) or geographic distance. entiation of phytophagous populations than speciali- Early genetic studies of D. pseudotsugae using zation in the host-use. The genetic differentiation isozymes suggest that differences between populations among pairs of populations estimated by FST/1ÁFST from Washington and Idaho (S 0.63) were enough to (using both markers) also suggests that the break in the consider them as two allopatric populations in the distribution of genetic variation, leading to isolation process of speciation (STOCK et al. 1979; BENTZ and between North American and Mexican populations of STOCK 1986). This conclusion was reinforced because D. pseudotsugae, is a result of limited gene flow and the the source populations utilized different varieties of consequent increase in genetic drift (HUTCHINSON and P. menziesii. However, other studies have shown even TEMPLETON 1999). higher similarity coefficients for different species (e.g. Unfortunately, the methods used do not allow us to S 0.83 in D. jeffreyi Hopkins and D. ponderosae) test what historical processes or factors have affected (HIGBY and STOCK 1982). Our results did not reveal the genetic structure of D. pseudotsugae, because sufficient genetic differentiation among northern po- classical models of population genetics were designed pulations, which includes at least one population found to be independent of history (e.g. equilibrium models) on P. m . var. menziesii, to support the notion of and of the geographical landscape (e.g. non-structured ongoing speciation. The genetic differentiation that island models). In addition, as some population we found between D. pseudotsugae populations from genetic models in which a geographical perspective north and south supports the expectation that geogra- has been introduced, such as isolation by distance or phical isolation, and probably habitat fragmentation, stepping-stone, the landscape is considered uniform, are primary factors affecting genetic structure. isotropic, and not linked to the history of the Although host use remains to be tested directly, the population (EPPERSON 2003). However, low observed limited genetic differentiation among northern genetic differences within distinct geographical popu- D. pseudotsugae populations suggests that host use lations of the Douglas-fir beetle, and the finding that a has little effect on the genetic structure of this species. model of isolation-by-distance is described by all In addition, the absence of an observable pattern of populations that we analyzed, both support the Hereditas 146 (2009) Genetic differentiation in Dendroctonus pseudotsugae 89 general premise that geographical isolation has played tions (Scolytinae: Ips confusus). Á Environ. Entomol. 32: a fundamental role in the differentiation of its 1262Á1270. populations. Cognato, A. I., Sun, J., AnduchoÁReyes, M. A. et al. 2005a. Genetic variation and origin of the red turpentine beetle Finally, our results confirm that D. pseudotsugae (Dendroctonus valens LeConte) introduced to the People’s populations from North America and those from Republic of China. Á Agric. For. Entomol. 7: 87Á91. Mexico on P. menziesii var. glauca are two true Cognato, A. I., Gillette, N. E., CamposÁBolan˜os, R. et al. subspecies: D. p. pseudotsugae Hopkins and D. p. 2005b. Mitochondrial phylogeny of pine cone beetles barragani, as was reported by FURNISS (2001). D. p. (Scolytinae, Conophthorus) and their affiliation with barragani was collected from a single population in geographic area and host. Á Mol. Phylogenet. Evol. 36: 494Á508. Chihuahua, Mexico, and described based on morpho- Delalibera, I., Handelsman, J. and Raffa, K. F. 2005. logical characters, gallery differences, and intra- and Contrasts in cellulolytic activities of gut microorganisms inter-population mating tests. At the time, this was the between the wood borer, Saperda vestita (Coleoptera: only known population of D. pseudotsugae in Mexico, Cerambycidae), and the bark beetles, Ips pini and where its host P. menziesii is a relict species occurring Dendroctonus frontalis (Coleoptera: Curculionidae). in isolated locations in Chihuahua, Durango and Á Environ. Entomol. 34: 541Á547. Dupanloup, I., Schneider, S. and Excoffier, L. 2002. A Coahuila. simulated annealing approach to define the genetic structure of populations. Á Mol. Ecol. 11: 2571Á2581. Acknowledgements Á Our thanks to Dr. Jaime Villa, Antonio Epperson, B. K. 2003. Geographical genetics. Á Princeton Olivo, Francisco Bonilla and Sergio Quin˜ones from Comi- Univ. Press. sio´n Nacional Forestal-Mexico (CONAFOR) for field Excoffier, L., Smouse, P. E. and Quattro, J. M. 1992. assistance. We thank Malcolm Furniss, Karen E. Mock Analysis of molecular variance inferred from metric and Anthony I. 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APPENDIX 1
Fig. A1. Dendrogram constructed by neighbour-joining (NJ) with the p distance using PAUP*. The p distance allowed performing a simple comparison of number of differences among individuals considering both data sets (mitochondrial and RAPD data from each individual were concatenated). Bootstrap values at nodes (1000 pseudoreplicates)