Diferenciación y aislamiento reproductivo entre especies de moscas

UNIVERSIDAD VERACRUZANA
INSTITUTO DE NEUROETOLOGIA
POSTGRADO EN NEUROETOLOGIA
Diferenciación y aislamiento reproductivo
entre especies de moscas del grupo suavis
en México
TESIS
Que para obtener el grado de:
DOCTOR EN NEUROETOLOGIA
PRESENTA:
MC. Ramos Eduardo Tadeo Hernández
DIRECTOR:
Juan Antonio Rull Gabayet, PhD.
Xalapa, Veracruz.
Noviembre de 2014.
Dedicada a:
Los Doctores Juan A. Rull Gabayet y
Larissa Guillén Conde, gracias por darme la
oportunidad de conocer y trabajar con el
género Rhagoletis.
ii
COMITÉ
Dr. Juan A. Rull Gabayet
Dra. Laura T. Hernández
Dr. Jorge Morales Mavil
Salazar
Dr. Armando J. Martínez
Dr. Francisco Díaz Fleischer
Chacón
iii
AGRADECIMIENTOS

Al Consejo Nacional de Ciencia y Tecnología (CONACyT), por la beca
otorgada durante el periodo 2011-2014 (con número de Becario 272318), para
cursar el doctorado en Neuroetología en la Universidad Veracruzana.

A la Universidad Veracruzana e Instituto de Neuroetología por la oportunidad
otorgada para realizar este posgrado.

A la red de Manejo Biorracional de Plagas y Vectores del Instituto de Ecología
A.C. por las facilidades brindadas para realizar esta investigación.

A mi director de tesis, Dr. Juan A. Rull Gabayet, Investigador titular de la Red
de Manejo Biorracional de plagas y Vectores, gracias por el apoyo, asesoría y
consejos brindados para la elaboración de este trabajo de investigación.

A los miembros de mi comité evaluador, Dra. Laura T. Hernández Salazar, Dr.
Jorge E. Morales Mávil, Dr. Armando J. Martínez Chacón y Dr. Francisco Díaz
Fleischer, por las aportaciones y consejos aportados para mejorar esta tesis.

A la coordinadora del posgrado en Neuroetología Dra. Laura T. Hernández
Salazar y al personal administrativo (en especial a Ivonne Hernández Rivera y
Anell Higueredo Olgín por el apoyo y facilidades brindadas).

A mis compañeros y amigos Emilio Acosta, Jovita Martínez, Rafael Ortega,
Olinda E. Velázquez, Israel Peralta, Christian Rodríguez y Lizbeth González,
por su apoyo y amistad.

A mi familia por su cariño y apoyo incondicional. Gracias por ser el motor que
me impulsa a seguir adelante cada día.
iv
CONTENIDO
Pag.
RESUMEN……………………………………………………………………………………………
1
I.
INTRODUCCIÓN…………………………………………………………………………................
3
II.
ANTECEDENTES……………………………………………………………………………………
6
2.1. Género Rhagoletis……………………………………………………………………………..
6
2.2. Generalidades del género Rhagoletis………………………………………………………..
6
2.3. Distribución del grupo suavis…………………………………………………………............
7
2.4. Características generales del grupo suavis…………………………………………...........
8
2.5. Género Juglans, hospedero natural del grupo suavis……………………………………
9
2.6. Distribución del grupo suavis en México……………………………………………............
10
2.7. Relaciones filogenéticas entre especies del grupo suavis…………………………………
11
2.8. Aislamiento reproductivo……………………………………………………………………….
12
III.
3. HIPÓTESIS………………………………………………………………………………………..
15
IV.
OBJETIVO GENERAL………………………………………………………………………………
16
4.1. Objetivos específicos……….…………………………………………………………………..
16
REFERENCIAS……………………………………………………………………………………….
17
V. ESTUDIOS REALIZADOS (CAPITULOS)……………………………………………………………
21
Capítulo 1. Alternative Mating Tactics as Potential Prezygotic Barriers to Gene Flow
Between Two Sister Species of Frugivorous Fruit Flies…………………….......................
22
Abstract……………………………………………………………………………………………….
22
Introduction……………………………………………………………………………………………
22
Materials and Methods………………………………………………………………………………
24
Results………………………………………………………………………………………………...
26
Discussion…………………………………………………………………………………………….
30
References……………………………………………………………………………………………
32
Capítulo 2. Reproductive isolation as a means to resolve phylogenetic relationships
among recently derived species of frugivorous fruit flies in the genus Rhagoletis…
Abstract……………………………………………………………………………………………….
35
36
v
Introduction……………………………………………………………………………………………
37
Materials and Methods………………………………………………………………………………
40
Results………………………………………………………………………………………………...
43
Discussion…………………………………………………………………………………………….
47
References……………………………………………………………………………………………
52
Figure Legends……………………………………………………………………………………...
58
Figures………………………………………………………………………………………………...
60
Capítulo 3. Behavioral patterns and relative strength of pre- and postzygotic isolation
between two recently derived species of walnut infesting flies in the highlands of
Mexico……………………………………………………………………………………………..
65
Abstract……………………………………………………………………………………………….
66
Introduction……………………………………………………………………………………………
67
Materials and Methods………………………………………………………………………………
70
Results………………………………………………………………………………………………...
74
Discussion…………………………………………………………………………………………….
78
References……………………………………………………………………………………………
84
Figure Legends……………………………………………………………………………………...
90
Figures………………………………………………………………………………………………...
92
VI
DISCUSIÓN GENERAL……………………………………………………………………………..
96
VII
CONCLUSIONES……………………………………………………………………………………
102
MODELO TEÓRICO…………..…………………………………………………………………….
104
REFERENCIAS………………………………………………………………………………………
105
vi
RESUMEN
El género Rhagoletis Loew ha originado un gran número de estudios de tipo
evolutivo tras el descubrimiento en 1876 de Rhagoletis pomonella Walsh infestando
manzanas (Malus pumilla Mill) en lugar de su hospedero ancestral tejocote (Crataegus
spp.) en el valle del río Hudson en Nueva York. Este evento convirtió a R. pomonella
en el
modelo estandarte de especiación simpátrica por cambio de hospedero.
Recientes estudios han revelado que el surgimiento de las razas de hospedero de R.
pomonella en los Estados Unidos, pudo ser el resultado de episodios de flujo génico
entre poblaciones ancestrales refugiadas en México durante las glaciaciones del
Pleistoceno. Al igual que para el grupo pomonella, poblaciones ancestrales de moscas
en el grupo suavis se refugiaron en México durante el Pleistoceno quedaron también
expuestas a procesos de especiación alopátrica que favorecieron el surgimiento de seis
especies asociadas a plantas en el género Juglans (R. suavis Loew, R. completa
Cresson, R. zoqui Bush, R. boycei Cresson, R. juglandis Cresson y R. ramosae
Hernández-Ortiz), dos de estas endémicas de México (R. zoqui y R. ramosae).
Recientes estudios reportan el descubrimiento de una zona de contacto en el noreste
de México donde coocurren y se hibridan poblaciones de R. completa y R. zoqui sin la
existencia aparente de barreras reproductivas que interrumpan el flujo génico entre
especies. Por otro lado estudios moleculares recientes no han logrado separar
filogenéticamente a tres especies mexicanas del grupo suavis. Este hecho y el
descubrimiento de esta zona de contacto brindan la oportunidad de estudiar procesos
evolutivos y abren la interrogativa de ¿Cuál es el grado de diferenciación y que tanto ha
evolucionado el aislamiento reproductivo entre las especies del grupo suavis en México
para evitar el flujo de genes? Para responder a esta pregunta realizamos una serie de
1
análisis sobre aislamiento reproductivo pre y poscigotico que abarcaron la fenología de
diapausa, pruebas de compatibilidad de apareamiento, pruebas de viabilidad de
híbridos y análisis etológicos entre R. completa, R. zoqui y R. ramosae. Los resultados
obtenidos arrojaron evidencia sobre asincrónia en la emergencia de adultos entre
especies influida por la fenología de fructificación de sus hospederos, distintos grados
de aislamiento sexual que se reflejó en una inferioridad hibrida para algunas de las
cruzas heterópicas y una serie de comportamientos que permiten especular sobre el
posible camino evolutivo que siguieron de las especies del grupo suavis presentes en
México.
2
I. INTRODUCCIÓN
Los miembros del género Rhagoletis (Diptera: Tephritidae) se caracterizan por
ser insectos monófagos muy especializados que ajustan su ciclo de vida a la fenología
de fructificación de sus plantas hospederas (Berlocher y Feder, 2002). Para lograr dicho
ajuste pasan el invierno en
diapausa en forma de pupa (capullo) y sincronizan la
emergencia de adultos con los periodos de fructificación de sus hospederos (AliNiazee,
1988; Aluja et al., 1998; Prokopy y Papaj, 2000). Lo cual junto con otras características
biológicas del género han convertido a varias de estas especies en plagas de
importancia económica, debido a que frecuentemente han
pasado de hospederos
nativos de poca importancia económica a plantas cultivadas con valor comercial
frecuentemente introducidas (Boller y Prokopy, 1976).
En los últimos diez años se ha retomado el estudio de varios grupos de especies
en el género Rhagoletis presentes en territorio Mexicano, debido a que se ha
descubierto que son un eslabón importante para entender los procesos evolutivos y de
especiación que dieron origen a las especies simpátricas de importancia económica en
los Estados Unidos (Michel et al., 2007; Xie et al., 2008). Como ejemplo de ello
tenemos que lo que se consideraba una población de Rhagoletis pomonella en México,
es en realidad un grupo de tres especies cripticas, aisladas geográficamente y
distribuidas en zonas elevadas de la Sierra Madre Oriental, Eje Volcánico Trans
Mexicano y los altos de Chiapas (Michel et al. 2007; Xie et al., 2007; Rull et al., 2010).
Estas poblaciones o especies cripticas presentan variaciones genéticas y cierto grado
de aislamiento reproductivo, además de que se ha encontrado que son la fuente de
origen de la variación genética que permitió la sincronización en la emergencia de
3
adultos para los distintos frutos hospederos que favorecieron el surgimiento de las seis
especies simpátricas del grupo pomonella en los Estados Unidos (Michel et al., 2007;
Xie et al., 2007, 2008).
Otro ejemplo de divergencia dentro del género Rhagoletis es el caso del grupo
cingulata, para el cual luego de un controversial debate sobre la presencia de R.
cingulata
en territorio Mexicano (Bush, 1966; Foote, 1981), se ha descubierto un
complejo de al menos dos poblaciones geográficamente aisladas, localizadas en zonas
templadas de la Sierra Madre Oriental y el eje Volcánico Transversal Mexicano (Rull et
al., 2011). Al igual que para las poblaciones de R. pomonella las poblaciones de R.
cingulata presentan algún grado de incompatibilidad reproductiva e indicios de procesos
de especiación incipiente (Tadeo, datos sin publicar).
Respecto al grupo suavis, aún cuando ha sido poco estudiado, se ha demostrado
recientemente la existencia de híbridos naturales en el noreste de México que al
parecer son el producto de cruzamientos naturales entre individuos de dos especies
(Rhagoletis zoqui y R. completa ). Estas especies al parecer no exhiben
barreras
reproductivas que reflejen alguna forma de inferioridad hibrida en comparación con
cruzas homotípicas de las especies parentales (Rull et al., 2012). Además se ha
descubierto que tres especies del grupo suavis presentes en México (R.zoqui,
R.completa y R.ramosae), a pesar de sus notorias diferencias morfológicas son hasta
ahora genéticamente indistinguibles (Frey et al., 2013; Rull et al., 2013). Este hecho,
junto con los antecedentes que existen del género Rhagoletis en México, nos llevó a
formular la interrogativa de ¿Qué es lo que está pasando dentro del grupo suavis en
4
México? y ¿Qué tan diferenciadas se encuentran estas especies de moscas y como
mantienen su integridad sin haber desarrollado barreras reproductivas que interrumpan
el flujo génico? Ante estas incógnitas en este trabajo se planteó someter individuos
adultos de R. completa, R. zoqui y R. ramosae a un análisis experimental sobre
barreras pre y postcigoticas que comprenden aspectos biológicos y conductuales,
esquemas de cruzas intra e interespecíficos y análisis de viabilidad de híbridos que nos
permitan recopilar información para el esclarecimiento del grado de divergencia y
evolución de barreras reproductivas entre estas tres especies moscas del grupo suavis
presentes en México.
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II. ANTECEDENTES
2.1. Género Rhagoletis.
El género Rhagoletis conocido por la mosca de la manzana (Rhagoletis
pomonella Walsh, especie modelo del proceso de especiación simpátrica por cambio de
hospedero), está compuesto por setenta y cuatro especies descritas, arregladas
filogenéticamente en catorce grupos distribuidos en zonas templadas de Asia, Europa y
América (Bush, 1966; Smith y Bush, 2000; Rull et al., 2013). En el continente
Americano se encuentran treinta y cinco especies, agrupadas en diez grupos con
afinidad morfológica y/o compatibilidad en el uso de plantas hospederas, de los cuales
se sabe que los grupos pomonella (moscas de la manzana y el tejocote), cingulata
(moscas de la cereza), striatella (moscas de las solanáceas) y suavis (moscas del
nogal), se encuentran presentes en territorio Mexicano (Bush, 1966).
2.2. Generalidades del género Rhagoletis.
Los miembros del género Rhagoletis se caracterizan por ser tefrítidos con
metamorfosis completa u holometábola, dividida en cuatro etapas (huevo, larva, pupa y
adulto) (Aluja, 1993). Todas las larvas de las mocas en este género se alimentan de la
pulpa de frutos en desarrollo provenientes de plantas que pertenecen a la misma familia
o género (oligófagas), además de que la mayoría de sus especies se caracterizan por
presentar solo una generación por año (univoltinas) (Boller y Prokopy, 1976). Estas y
otras características han convertido a las moscas del género Rhagoletis en insectos
fitófagos muy especializados, que habitan sitios donde se desarrollan plantas con
periodos de fructificación cortos y bien definidos, lo que ha favorecido que las moscas
6
de este género ajusten su ciclo de vida a la fenología de sus hospederos, pasando el
invierno en diapausa en forma de pupa (Prokopy y Papaj, 2000). Durante esta
diapausa, las moscas reducen su actividad metabólica al mínimo para hacer frente a las
condiciones ambientales adversas (Aluja et al., 1998; AliNiazee, 1988). La duración de
la diapausa está regida por la acumulación de horas frío y regulada mediante las
mismas señales ambientales que regulan la fenología de fructificación de las plantas
hospederas (humedad, temperatura, fotoperiodo, etc.), sincronizando el fin de la
diapausa y la emergencia de adultos (Boller y Prokopy, 1976; Feder, et al., 1997).
El sistema de apareamiento de las especies del género Rhagoletis que han sido
estudiadas también está estrechamente ligado a sus plantas hospederas. Tanto
hembras como machos son atraídos a distancia por los volátiles emitidos por los frutos
en maduración y exhiben un sistema con defensa del recurso, donde el macho se posa
sobre un fruto maduro y lo defiende de machos intrusos en espera de la llegada de una
hembra en busca de sitios de oviposición. Posteriormente el macho resguardante
aborda bruscamente a la hembra visitante por detrás para copular con ella sin cortejarla
(Prokopy y Papaj, 2000).
2.3. Distribución del grupo suavis.
El grupo suavis comprende seis especies neárticas (R. suavis, R. completa, R.
zoqui, R.boycei y R. juglandis), distribuidas en zonas templadas de Estados Unidos y
México (Bush, 1966, 1968; Foote, 1981; Smith y Bush, 2000). Rhagoletis suavis se
encuentra distribuida únicamente en el norte y sureste de Estados Unidos, R. completa
se distribuye en el centro este y costa oeste de Estados Unidos, aunque también se
7
encuentra en el norte de México (Foote, 1981; Berlocher 1984; Rull et al., 2013).
Rhagoletis juglandis se encuentra distribuida desde el norte de Nuevo México al centro
de México y posiblemente más al sur, R. zoqui está distribuida únicamente en territorio
Mexicano, en tanto que R. boycei está restringida a Arizona y suroeste de Nuevo
México, aunque aparentemente ocurre también en el norte de Sonora (Foote, 1981;
Bush, 1968). Por otra parte Hernández (1985), describe e incluye una nueva especie
dentro del grupo suavis, basado en las características descritas por Bush (1966),
especie que denomina R. ramosae, la cual se localiza en el centro oeste de México,
específicamente en Guerrero y Michoacán.
2.4. Características generales del grupo suavis.
Todos los miembros del grupo suavis infestan especies nativas o introducidas de
nogal (Juglans spp), por lo que parecen compartir preferencias similares o idénticas en
cuanto
al
huésped
se
refiere
(Foote,
1981;
Bush,
1968).
Sin
embargo,
morfológicamente difieren notablemente unos de otros, ya que presentan variaciones
consistentes en sus patrones alares, además de que R. suavis, R. completa, y R. zoqui,
presentan dimorfismo sexual y una coloración distintiva en tórax y el abdomen (negro y
marrón), en tanto que para R. juglandis son completamente amarillos y para R. boycei
completamente negros (Bush, 1968). En lo que respecta a R. ramosae, al igual que los
otros miembros del grupo suavis, presenta características particulares en su patrón alar,
y aunque es muy parecida a R. zoqui, se diferencia claramente de esta por la presencia
de una franja negruzca en los márgenes laterales del escudo y una diferencia notable
en tamaño tanto de hembras como de machos (Hernández, 1985).
8
2.5. Género Juglans, hospedero natural del grupo suavis.
La familia Juglandaceae está compuesta por siete géneros y más de sesenta
especies distribuidas en el Norte y Sur de América, el Caribe, sureste de Europa, este
de Asia y Japón (Narave, 1983; Manos y Stone, 2001; Aradhya et al. 2005; Aradhya et
al. 2007). Dentro de la familia Juglandaceae, el género Juglans L. es el que incluye el
mayor número de especies (21 especies), mismas que están ampliamente distribuidas
en zonas templadas del Viejo y Nuevo Mundo (Narave, 1983; Aradhya, et al. 2007).
Para México se han reportado seis especies de Juglans, (Narave, 1983), las cuales
según Manning (1957) y Stone et al., (2009), se distribuyen en territorio mexicano de la
siguiente manera: 1) Juglans pyriformis Liebmann (Veracruz); 2)
Juglans hirsuta
Manning (Nuevo León); 3) Juglans mollis Engelm (Nuevo León, Tamaulipas, San Luis
Potosí, Guanajuato, Hidalgo y Puebla); 4) Juglans major Torr (Sonora, Chihuahua,
Sinaloa y Durango); 4a) Juglans major forma stellata Manning (Sonora, Chihuahua y
Durango); 4b) Juglans major (Torr.) Heller variedad glabrata Manning (Durango, Jalisco,
Michoacán, Edo. México y Guerrero); 5) Juglans microcarpa Berlandier in Berl (Nuevo
León y Coahuila); 5a) Juglans microcarpa Berlandier variedad stewartii (Johnston)
Manning (Coahuila y Chihuahua) y 6) Juglans olanchana Williams & Standley variedad
standleyi Manning (Colima y Jalisco). Por otra parte Stone et al., (2009), además de las
especies anteriores, reportan la presencia de J. steyermarkii Manning, en el estado de
Chiapas, en la frontera entre México y Guatemala, en tanto que Narave (1983) y Stone
et al., (2009), mencionan la presencia de cuatro especies de Juglans distribuidas en el
estado de Veracruz, Juglans regia L, Juglans mollis Engelm, Juglans pyiformis
Liebmann y Juglans olanchana Williams & Standley variedad olanchana. En lo que
9
respecta a las características generales del género Juglans, son árboles monoicos,
caducifolios, de corteza rugosa y escamosa, hojas compuestas, flores unisexuales
(formando una inflorescencia), fruto (pseudodrupa) que consiste en una nuez envuelta
en una cascara más o menos carnosa (según la especie) y gran parte de los miembros
de este género (también conocido como nogal) se consideran de gran importancia
económica por el valor de su madera o frutos (nueces) (Manning, 1957; Narave, 1983;
Stone et al., 2009).
2.6. Distribución del grupo suavis en México.
Bush en 1966 documenta la presencia de R. completa en estados Unidos, con
una población muy cercana a territorio Mexicano localizada en los límites de Texas,
Nuevo México, Chihuahua y Coahuila. En tanto que a R. zoqui la describe y documenta
solo en el centro de México, en el Estado de Hidalgo. Por su parte Foote en 1981 y
Berlocher en 1984, mencionan la presencia de R. completa ya en el norte de
Tamaulipas, México, situándola cerca de la frontera de Estados Unidos, en tanto que el
mismo Foote en 1981 sigue considerando a la población de R. zoqui distribuida solo en
el centro de México. En lo que respecta a R. ramosae, Hernández en 1985 la describe
por primera vez, ubicándola en el centro oeste de México, en los estados de Guerrero y
Michoacán. Sin embargo, actualmente Rull et al. (2013) reportan la presencia de R.
completa en los Estados de Coahuila, Nuevo León y Tamaulipas, a R. zoqui
ampliamente distribuida en el centro de país, cubriendo los estados de Veracruz,
Hidalgo, Tlaxcala, Puebla, Distrito Federal, Querétaro y San Luis Potosí, y a R.
Ramosae en los estados de Guerrero, Michoacán y Estado de México. Estos
antecedentes junto con el reporte de Rull et al., (2012) sobre la presencia de híbridos
10
naturales en la frontera Tamaulipas y San Luis Potosi, hacen pensar que las
poblaciones de moscas del grupo suavis en México se encuentran en contacto, quizás
debido a la introducción de la nuez de castilla Juglans regia, cuya expansión quizás ha
permitido establecer zonas hibridas donde ocurre el cruzamiento entre especies.
2.7. Relaciones filogenéticas entre especies del grupo suavis
Las miembros del grupo suavis han generado un particular interés entre los
evolucionistas debido al predominante origen alopátrico de las especies que lo
componen (Bush, 1966). Esto último contrasta con el famoso proceso de especiación
simpátrica
documentado para las especies del
grupo pomonella (Bush, 1966;
Berlocher, 2000; Xie et al. 2008) y parte del grupo cingulata (Bush, 1966). Análisis
moleculares mitocondriales usando citocromo oxidasa II (COII) para establecer la
relación entre las seis especies del grupo suavis, han permitido estimar procesos
evolutivos que datan de los últimos 2 millones de años, con una tasa aproximada de
especiación de una especie cada 320,000 años (Bush y Smith, 1998). Sin embargo,
esfuerzos adicionales incluyendo análisis moleculares, morfológicos y ecológicos, han
sido necesarios para desarrollar la filogenia que involucra a cinco de las seis especies
incluidas dentro del grupo suavis (Bush y Smith, 1998; Smith y Bush, 1999). Esta
filogenia compuesta por dos ramas, coloca a R. suavis aislada en una de ellas,
agrupando a las cuatro especies restantes dentro la una segunda rama conformada por
tres clados, dos de ellos ocupados por R. juglandis y R. boycei, y un tercero compartido
por R. completa y R. zoqui, consideradas estas últimas como las especies más
cercanamente emparentadas dentro del grupo (Bush y Smith, 1998).
11
Recientes filogenias moleculares entre las especies del grupo suavis han
permitido corroborar algunos resultados previamente obtenidos, colocando a R.
juglandis y R. suavis en clados claramente delimitados. Sin embargo aún queda sin
resolver la posición filogenética definitiva de las cuatro especies restantes, en particular
la de R. ramosae, especie prácticamente desconocida (Smith y Bush, 1997; Smith y
Bush, 2000; Frey et al., 2013;. Rull et al., 2013), El primer reporte de R. ramosae la
relaciona como muy cercana a R. zoqui debido a sus características morfológicas
(Hernández-Ortiz, 1985), no obstante lo anterior,
los análisis moleculares no han
logrado esclarecer la relación filogenética que guarda esta con las otras tres especies
también sin delimitar (Rull et al., 2013). Dos hipótesis respecto al origen de R. ramosae
han sido formuladas con base en nueva información recabada, por un lado la coloca
como un pariente cercano de R. boycei, en tanto que por el otro lado la relaciona como
más cercanamente emparentada con R. zoqui y R. completa (Rull et al., 2013).
2.8. Aislamiento reproductivo
El origen de dos o más especies a partir de un ancestro común es producto de la
evolución del aislamiento reproductivo entre poblaciones y puede manifestarse antes ó
después de la formación del cigoto (pre y postcigótico) (Futuyma, 2003). Dentro de los
mecanismos de especiación que resultan en el surgimiento de aislamiento reproductivo,
destacan dos en particular, la especiación alopátrica que se produce cuando las
poblaciones quedan aisladas físicamente debido al surgimiento de barreras geográficas
(ríos, montañas, etc.) que interrumpen el flujo genético y la especiación simpátrica, que
consiste en la diversificación de las distintas poblaciones de una misma especie que
ocupan un mismo rango de distribución, debido a la aparición de mecanismos de
12
aislamiento (ecológico, etológico, sexual y genético) que cumplen la misma función que
las barreras geográficas (Bush, 1969; Ruiz, 1988). La evolución de las barreras
reproductivas entre poblaciones limita el cruzamiento interespecífico en la naturaleza,
pues de suceder tiende a no producir descendencia o se produce
descendencia
completamente estéril o con algún grado de esterilidad hibrida (Dobzhansky, 1940). La
existencia y grado de intensidad del aislamiento reproductivo puede ser utilizado para
estimar el grado de divergencia entre especies cercanas (Coyne y Orr, 1989; 1997).
Una compilación con 119 especies de Drosophila comparando la distancia genética y
grado de divergencia concluye que el aislamiento precigótico evoluciona más rápido
que el aislamiento postcigótico y que el primero suele ser más fuerte entre especies
simpátricas que entre especies alopátricas (Coyne y Orr, 1989; 1997). La intensidad o
evolución del aislamiento reproductivo puede cuantificarse utilizando una diversidad de
índices con una base estadística que estiman el aislamiento sexual usando las
frecuencias de apareamiento entre adultos maduros (Pérez-Figueroa et al., 2004).
Comparaciones entre distintos métodos han demostrado que el margen de error o
confiabilidad de cada método
depende de factores particulares en cada estudio,
aunque como es de esperarse algunos métodos resultan estadísticamente más
robustos que otros cuando se consideran las variables pertinentes en cada caso
particular (Pérez-Figueroa et al., 2004).
En el caso del grupo suavis los primeros
intentos por esclarecer el grado de aislamiento entre
R. completa y R. zoqui no
pudieron encontrar evidencia de aislamiento precopulatorio basado en la elección o
discriminación de apareamiento entre adultos de poblaciones localizadas fuera de la
zona de contacto (Rull et al., 2012). Análisis prostcopulatorios tampoco apoyaron una
reducción de la fertilidad para los apareamientos híbridos ó F1, aunque estos parecían
13
tener menores tasas de fecundidad en comparación con la progenie producida por
apareamientos homotípicos, además la descendencia F2 tuvo menores tasas de
supervivencia, lo cual indica que al parecer la primera barrera intrínseca para el flujo de
genes entre R. completa y R. zoqui es de tipo postcigótico y se manifiesta en híbridos
de generaciones avanzadas (Rull et al., 2012). Esta evidencia sin embargo contradice
los trabajos de Coyne y Orr (1989; 1997), lo cual abre la necesidad de nuevos estudios
que permitan aportar más información para esclarecer el grado de divergencia y
evolución del aislamiento reproductivo entre R. completa y R. zoqui.
14
III. HIPÓTESIS
Las moscas del grupo suavis, R. completa, R. zoqui y R. ramosae se encuentran
en un proceso de diferenciación incompleto que permite cierto grado de hibridación
debido a la existencia de barreras reproductivas que no interrumpen por completo el
flujo génico entre especies.
Para responder a esta hipótesis se plantean las siguientes predicciones:

Aun cuando los miembros del grupo suavis en México presentan diferencias
fenotípicas notorias, no han evolucionado barreras precigóticas absolutas
encaminadas a evitar la copula.

Entre las especies de R. completa, R. zoqui y R. ramosae no han evolucionado
barreras postcigóticas absolutas que se traduzcan en
incompatibilidad
reproductiva o inferioridad hibrida.

Las barreras reproductivas entre especies filogenéticamente más cercanas serán
más débiles que entre especies más derivadas.
15
IV. OBJETIVO GENERAL
Documentar la existencia y el grado de evolución de las barreras reproductivas
entre tres especies de moscas del grupo suavis presentes en México y contribuir al
esclarecimiento de las relaciones filogenéticas entre estas.
4.1. OBJETIVOS ESPECÍFICOS

Determinar la existencia y el grado de evolución de las barreras precigoticas
encaminadas a evitar la copula entre las especies mexicanas de R. completa,
zoqui y R. ramosae.

Determinar la existencia y grado de evolución de las barreras postcigóticas entre
especies que se traduzcan en algún grado de incompatibilidad reproductiva o
inferioridad hibrida.

Esclarecer la historia evolutiva de las especies Mexicanas del grupo suavis
estudiadas aquí mediante la comparación del grado de aislamiento pre y
postcigótico entre especies.
16
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Xie X, Rull J, Velez S, Forbes A, Michel A, Lobo N, Aluja M, Feder J.L. (2007). The Bio
and phylogeography of Hawthorn-infesting Rhagoletis flies in Mexico and
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Radiation and divergence in the Rhagoletis pomonella species group: inferences
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20
V. ESTUDIOS REALIZADOS
(CAPÍTULOS)
21
CAPITULO 1
22
23
24
25
26
27
28
29
30
31
32
33
34
CAPITULO 2
For: Biological Journal of the Linnean Society
Reproductive
isolation
as
a
means
to
resolve
phylogenetic
relationships among recently derived species of frugivorous fruit flies
in the genus Rhagoletis.
EDUARDO TADEO1,2*, MARTIN ALUJA1 , JUAN RULL1
1
Instituto de Ecología, A.C., Carretera Antigua a Coatepec no. 351, Colonia el Haya,
C.P. 91070 Xalapa, Veracruz, México.
2
Posgrado en Neuroetologia, Instituto de Neuroetología, Universidad Veracruzana, Dr.
Luis Castelazo s/n Col. Industrial Animas, Xalapa, Veracruz, México.
“Corresponding autor
Corresponding
author
information:
Eduardo
Tadeo,
Posgrado
Instituto
de
Neuroetología, Universidad Veracruzana, Dr. Luis Castelazo s/n Col. Industrial Animas,
Xalapa, Veracruz, México e-mail: tadeotkd@hotmail.com
Running head: REPRODUCTIVE ISOLATION IN RHAGOLETIS
35
Pleistocene glacial and postglacial cycles producing contraction and expansion of
temperate habitats have resulted in substantial diversification among several plant and
animal taxa of Nearctic origin undergoing periods of isolation and secondary contact in
high elevation areas of Mexico. One of such groups are walnut infesting Tephritid fruit
flies in the genus Rhagoletis, comprised of six recently derived species among which
phylogenetic relationships have been difficult to unravel using conventional molecular
methods. In order to contribute in establishing the time course of diversification and
speciation, and to resolve phylogenetic relationships in the group. We examined pre and
post zygotic isolation between two genetically similar and morphologically distinct
species which are parapatric in central Mexico. Host plant phenology driven local
adaptation differences between R. zoqui and R. ramosae resulted in allocronic isolation.
Despite the existence of prezygotic mating isolation, there was a substantial number of
hybrid matings. R. zoqui females were more reluctant to mate with R. ramosae males
than with males of their own species. Distinctive behavioral differences were observed
between males during contests and approach to conspecifics on fruit. There was also
some asymmetric post zygotic isolation, with the hybrid combination of R. zoqui males
and R. ramosae females producing lower egg hatch than other mating combinations. We
discuss our results in light of previously formulated hypotheses on the evolutionary
history of the R. suavis species group.
ADITIONAL KEYWORDS: Divergence, prezygotic barriers, sexual selection, allopatric
populations, Rhagoletis suavis
36
INTRODUCTION
Historical climatic variation producing pulses of contraction and expansion of particular
habitats and periods of isolation and secondary contact has been found to play an
important role in divergence, genetic structure and distribution of many Neotropical
species (Avise, 2000; Hewitt, 2000; Hewitt, 2004; Feder et al., 2005). In particular,
Pleistocene glacial and interglacial cycles have played a key role in producing some of
the current species diversity patterns (Huntley & Webb, 1989; Joseph et al., 1995;
Hewitt, 1996; Roy et al., 1996; Willis & Whittaker, 2000). During glacial cycles, species
with affinity for temperate climates were displaced to latitudes where less extreme
climatic conditions allowed survival (Hewitt, 2004; Solis et al., 2006; Provan & Bennett,
2008). Following glaciations, such species took refuge in high elevation areas
surrounded by dry and warm lowland habitats (Haffer, 1969; Burnham & Graham, 1999;
Knowles, 2001; Hooghiemstra & Van der Hammen, 2004; Bush & de Oliveira, 2006).
Isolation in refugia and genetic drift triggered differentiation in ecological islands that
could be reinforced during periods of secondary contact (Fosberg, 1983; Petit et al.,
2003; Zarza, et al,. 2008; De Mello, 2011; Ramírez-Barahona & Eguiarte, 2013)
producing current diversity patterns in many lineages (Shepard & Burbrink, 2009;
Salomon, 2001; Knowles, 2001; Qu et al., 2011).
During geographic isolation, the accumulation of genetic differences due to
disruptive environmental selection and genetic drift producing postzygotic isolation, was
thought to be the most prevalent mode of speciation (Mayr, 1963; Salomon, 2001). After
secondary contact, according to vicariance theory, prezygotic isolation tends to evolve to
37
prevent maladaptive hybridization (Dobzhansky, 1942). Although much evidence has
been gathered supporting the likelihood and prevalence of speciation modes not
involving periods of geographical isolation (Berlocher & Feder, 2002; Mallet, 2008), the
relative strength of prezygotic and postzyotig isolation can shed some light into the
evolutionary history and divergence patterns of particular groups (Coyne & Orr; 1989;
1997; 2004).
Many insect species currently inhabiting North America, evolved in mountainous
areas as a result of Pleistocene isolation and contact cycles (Ross, 1953; Howden,
1969). Mexico has a particularly complex topography that produced a rich complex of
temperate pine-oak ecological islands surrounded by warm dry habitats that underwent
several contractions and expansions (Martin & Harrell, 1957; Howden, 1963; Howden,
1969). A good example of the latter are flies in the genus Rhagoletis (Feder. et al., 2003;
2005; Michel et al., 2007; Xie et al., 2008) and in particular those species comprised in
the walnut infesting suavis species group (Bush, 1966; Foote, 1981; Bush & Smith,
1998). The R. suavis species group is currently formed by five species whose
phylogenetic relationships have been examined (R. suavis Loew, R. completa Cresson,
R. zoqui Bush, R. boycei Cresson and R. juglandis Cresson) (Bush, 1966; 1968; Foote,
1981; Smith & Bush, 2000) and a more recently described species (R. ramosae)
(Hernández-Ortiz, 1985). All species in the group excepting R. suavis, occur in Mexico
(Bush, 1966; Foote, 1981; Foote et al., 1993; Smith & Bush, 2000), while R. zoqui and
R. ramosae are endemic to central Mexico (Bush, 1966; Hernández, 1985; Rull et al.,
2013). Rhagoletis zoqui described by Bush (1966), is distributed in a zone
encompassing mid elevation areas (1000-2000m) in Veracruz, Tlaxcala, Puebla,
Hidalgo and San Luis Potosí, while R. ramosae´s range encompasses Michoacán,
38
Guerrero, Estado de México, Jalisco and Nayarit (Bush, 1966; Foote, 1981; HernándezOrtiz, 1985; Foote et al., 1993; Smith & Bush, 2000; Rull et al., 2013).
Molecular phylogenies on species in the R. suavis group have allowed to place R.
suavis and R. juglandis in clearly delimited clades, grouping the remaining species in a
tight unresolved cluster (Smith & Bush 1997; Smith & Bush 2000; Frey et al., 2013; Rull
et al., 2013). In particular, the relationship between R. ramosae and a group comprising
R. zoqui, R. completa and R. boycei is still unclear (Rull et al., 2013). Two hypotheses
regarding the origin of R. ramosae have been put forth, one placing it as a close relative
of R. boycei and a second one as more related to R. zoqui and R. completa.
In the course of an extensive sampling effort to establish the distribution of walnut
infesting Rhagoletis in Mexico (Rull et al., 2013) the discovery of natural hybrids
between R. zoqui and R. completa led Rull et al. (2012) and Tadeo et al. (2013), to
explore the evolution of postzygotic and prezygotic isolation between these two groups.
Results of reproductive compatibility studies between these two fly species revealed
little pre and postzygotic isolation, suggesting very recent divergence and a close
relationship. Here, we report on a series of similar studies exploring the existence and
strength of pre and postzygotic isolation between R. ramosae and R. zoqui. Comparison
of our results and previous similar studies on other species in the suavis group could
contribute in clarifying phylogenetic relationships that have not been resolved using
molecular methods.
39
MATERIAL AND METHODS
SOURCE OF FLIES
Rhagoletis zoqui was recovered from infested Juglans pyriformis Liebmann fruit,
collected in Xalapa (19°30’45.81”N, 96°56’38.86W, 1342 m) and Coacoatzintla Veracruz
(19°38’48.95”N, 96°56’29.76W, 1440 m) between August and September 2011, while R.
ramosae was obtained from Juglans major (Torr) Heller var glabrata Manning collected
between late September and early October in Taxco Guerrero (18°33’16.9”N,
99°39’31.9”W, 1780 m). Fruit was taken to the laboratory at the Instituto de Ecología
A.C, in Xalapa Veracruz, and processed following methods outlined in Rull et al. (2006)
to recover pupae. Pupae were placed in 200 ml plastic cups lined at the bottom with a 2
cm. vermiculite lair and humidified regularly with a sodium benzoate (C 6H5COONa)
solution at a 3g/L proportion to prevent fungal growth and desiccation. Plastic cups were
capped with perforated lids to allow air flow and kept at room temperature until
emergence of adults the following season. At emergence, the species, number, and sex
of emerged adults was recorded on a daily basis. All emerged adults within five days of
age were separated according to species and sex and placed in 3l. plastic cages
provided with water and food (3:1 sugar: hydrolyzed protein) until sexual maturity (15 to
20 days) when they were used in experiments.
PREZYGOTIC ISOLATION (OBSERVATION CAGE – SEXUAL BEHAVIOR)
A 1.08 long, 1.08m. wide, 1.78 m. high cage was constructed using a 13 mm. PVC pipe
frame wrapped with white tergaline cloth. Within the cage, four 1.5 m high potted oak
40
trees (Quercus spp.) were placed at each corner, along with Juglans pyriformis
branches pinned in circle on the cage walls and ceiling to simulate a tree canopy. Ten
ripe Juglans pyriformis fruits were hung in circle from the cage ceiling using metal wire
long enough to reach the potted tree foliage. Fruit was evenly spaced and labeled with a
3cm2 green colored cardboard number to ease spatial location of activity. The
observation cage was provided with water and food as described above.
At 09:00 hours ten R. zoqui adult couples and ten R. ramosae couples (20 couples in
total) between 20 and 36 days of age, were released in the cage. Each individual fly was
marked on the back of the thorax with a dot of water based paint (Politec ®) using a
single color or two-color combinations. Behavioral observations were performed by a
single experienced observer from 10:00 to 17:00 hours with a combination of scanning
and focal observation of key events (fruit and mate guarding, copulations, egg laying,
etc.) Each fly cohort was observed for two consecutive days. A total of eight two-day
observations (replicates) were performed.
Male-female mating combination, hour of initiation and ending, and spatial location
were recorded for every copulation. Fruit guarding was considered when a male
remained motionless on a fruit for one or more minutes. For male-male encounters, the
species, status (resident-intruder), location, and final outcome (considering the male
remaining on the territory as the winner) of conflicts were recorded. Finally, in the case
of females, fruit visits, egg laying events, and male rejections (brisk movements to
prevent intromission after mounting) were recorded.
41
POSTZYGOTIC ISOLATION (HYBRID MATING FERTILITY)
In order to compare fertility (egg hatch rates) among homotypic and heterotypic
R.
zoqui x R. ramosae crosses, groups of five virgin females and five virgin males between
20 and 38 days old were introduced in 3 liter cages provided with water and food in all
possible male-female mating combinations (r♂r♀, r♂z♀, z♂r♀ and z♂z♀). For each one
of the four mating combinations, couples were allowed to interact freely for 24 hours and
then provided with 2.5 cm diameter agar spheres for egg laying. Spheres were replaced
on a daily basis for a two week period and all eggs extracted and aligned over a dark
piece of cloth placed over a moist piece of cotton within a Petri dish (Rull et al., 2010).
Eggs were incubated at 24°C for six days and observed under a dissecting microscope
to calculate percent egg hatch. The procedure was repeated five times for each mating
combination.
STATISTICAL ANALYSES
Statistical comparisons made on the basis of frequencies were done using the total
number of observations of a particular event per replicate. Frequency and duration in
minutes of copulations for each possible male-female R. zoqui x R. ramosae mating
combination, joint fruit residency by same or different species males, and the outcome of
male-male conflicts (frequency of resident or intruder male remaining on fruit) were
compared by means of one General Nolineal Model (GNM) with univariate design
(ANOVA) and adjusted to a Poisson distribution error. Frequency of fruit guarding, male
42
combat, and percent egg-hatch for different male-female R. zoqui x R. ramosae mating
combinations were compared with a univariate design (ANOVA). The frequency of male
combats according to site (fruit vs. mesh) was compared by means of a t-test, while
frequency of fruit visits, egg laying, and clutch size were compared between species and
rank-transformed when necessary followed by a t-test verified that the assumtions of
normality and homogeneity of variances were met. Analyses were performed using
STATISTICA 7.0 Copyrigth © Statsoft, Inc. 1984-2004 and SigmaPlot 10.0 Software.
Sexual isolation indices between R. zoqui and R. ramosae were calculated using
JMATING software (Carvajal-Rodriguez & Rolan-Alvarez, 2006).
RESULTS
ADULT EMERGENCE
Out of a 1050 R.zoqui pupae recovered from fruit collections in Veracruz a total of 247
adults (133 males and 114 females) were obtained, while in the case of R.ramosae
from Guerrreo out of 725 pupae a total of 130 adults (70 males and 60 females)
emerged. Seasonal adult emergence patterns were clearly distinct for R. zoqui and R.
ramosae (Figure 1). In the case of R. zoqui, the emergence period lasted from April 8th
to June 25th, while for R. ramosae it spanned from July 17th to September 1st. In sum,
we detected a time gap between the end of R. zoqui and the beginning of R. ramosae
adult emergence of about three weeks.
43
PREZYGOTIC ISOLATION
A one way ANOVA adjusted to a Poisson distribution revealed significant differences in
the frequency of copulations among male female R. zoqui x R. ramosae combinations
(X2=19.783, p<0.0001). Homotypic combinations (zz=4.38±1.28, rr=4.75±0.82) and the
“z♂r♀” hybrid cross (5.25±1.03) occurred at similar frequencies, however the “r♂z♀”
hybrid cross was clearly less frequent (0.88±0.48) (figure 2). For duration of copulations
a one way ANOVA adjusted to a Poisson distribution did not reveal significant
differences among pure and hybrid male- female mating combinations
(X2=2.118,
p=0.55). For the z♂z♀ combination copulations lasted on average 14.51±0.92 min, r♂r♀
lasted 15.89±1.28 min, z♂r♀ 17.26±1.24 min and r♂z♀ 13.29±2.98 min. With respect to
mating frequencies according to location (fruit or cage mesh) a two way ANOVA
adjusted to a Poisson distribution did not reveal significant differences among different
R. zoqui x R. ramosae male-female mating combinations (X2=1.06, p=0.79), but
revealed significant differences between mating sites (X2=15.61, p<0.001) with 83.61%
of copulations occurring on fruit and only 16.39% on the cage mesh (figure 3). Overall,
we observed significant levels of sexual isolation between R. zoqui and R. ramosae (Ipsi
= 0.34; P<0.001).
MALE BEHAVIOR
A one way ANOVA did not reveal significant differences y the frequency of fruit guarding
among R. zoqui and R. ramosae male-male combinations (ZZ, RZ, RR) (F1,14=0.38,
p=0.55). In general males from both species exhibited similar fruit guarding frequencies
44
(R. zoqui, 24.13±4.09 and R. ramosae 20.88±3.35). With respect to frequencies of joint
fruit guarding, a one way ANOVA adjusted to a Poisson distribution did not reveal
significant differences among the different male male combinations (X2= 2.2, p=0.53).
On average, guarding frequencies of R. zoqui males sharing fruit with other R. zoqui
males was 4.13±1.25, and with R. ramosae males of 3.0±0.71, while the frequency of R.
ramosae males encountering their conspecifics and R. zoqui on fruit was 2.13±0.72 and
3.63±1.27 respectively.
There were significant differences in the frequency of male contests according to
location (fruit or mesh) (t1,
14
=3.663, p=0.003). On average 87.33% of male-male
encounters occurred on fruit, while the remaining 12.67% occurred on the cage walls or
ceiling (figure 4). A one way ANOVA did not reveal significant differences in frequency
among different male-male species combinations according to resident status
(F3,28=0.57, p=0.64). Male contests for the zz combination occurred at an average
frequency of 16.50±4.71, contests for the zr resident intruder combination at 11.25±2.63,
for the rr combination at 11.75±2.58 and for the rz resident intruder combination
14.75±2.87. Male status (resident/intruder) and species had no significant effect on the
frequency of male-male contest victories (a particular male remaining on fruit after a
contest) after a one way ANOVA adjusted to a Poisson distribution (X2=8.697, p=0.191).
A total of 17.38% of male-male contest victories was for resident R. zoqui, 14.71% for R.
zoqui intruders, 18.18% for resident R. ramosae, 10.71% for intruder R. ramosae,
13.10% for both R. zoqui male types, 7.22% for both R. ramosae male types and
18.72% for two males of different species (R. zoqui and R. ramosae). There were
evident behavioral differences between species during male contests, R. zoqui typically
45
lifted their wings showing their patterns while R. ramosae male were static in an
apparent state of alert (Figure 5). During contests, R. ramosae males lifted their wings in
a 45 degree angle with respect to the thorax while R. zoqui males kept their wings
somewhat folded (figure 6).
FEMALE BEHAVIOR
There were no significant differences between species in the frequency of fruit visits (t1,
91=0.403,
p=0.688) but differences were significant in the frequency of egg-laying (t1, 91
=3.149, p=0.002). R. zoqui females performed a total of 381 visits and 32 eggdepositions, while R. ramosae females visited fruit 456 times and laid eggs on 116
occasions (figure 7).
POSTZYGOTIC ISOLATION
There were significant differences in the number of eggs laid per female per egg laying
bout between species (t1, 266 =-4.06, p<0.001). Female R. zoqui laid from 1 to 11 eggs
with an average of 5 eggs per bout, while R. ramosae females laid from 1 to 22 eggs
with an average of 10. In total R. zoqui laid 617 eggs and R. ramosae laid 1611 eggs,
representing 27.69% and 70.30% across the entire study respectively (Figure 8). A one
way ANOVA revealed significant differences in percent egg hatch among different male
female R. zoqui x R. ramosae mating combinations (r♂r♀, r♂z♀, z♂r♀ and z♂z♀) (F3, 15
=5.1455, p<0.01). The hybrid cross r♂z♀ produced the highest egg hatch and eggs laid
by females from the hybrid cross z♂r♀ hatched in significantly lower proportion (Figure
8).
46
DISCUSSION
In sum we found evidence of host plant phenology driven local adaptation differences
between R. zoqui and R. ramosae that could result in some degree of allocronic
isolation. We also found some degree of prezygotic mating isolation, although there was
a substantial number of hybrid matings, R. zoqui females were more reluctant to mate
with R. ramosae males than with males of their own species. The bulk of reproductive
activities (male guarding, male contests, mating, egg laying) took place on the host fruit,
with no evidence of alternative mating locations being used. Distinctive behavioral
differences were observed between males during contests and approach to conspecifics
on fruit. There was also some asymmetric postzygotic isolation, with the hybrid
combination of R. zoqui males and R. ramosae females producing lower egg hatch than
other mating combinations.
Our results unveiled the existence of pre- and postzygotic barriers to gene flow
between two endemic parapatric species of walnut infesting Rhagoletis in Mexico that
have maintained their integrity (clear morphological differences) despite contact. The
first barrier is constituted by clearly defined adult emergence periods tightly linked to
differences in the fruiting phenology of their respective walnut hosts within their
distributional ranges (R. ramosae/ J. major and R. zoqui/J. pyriformis) (see also Rull et
al., 2013). As a result there is a three week temporal gap when adults of both species do
not overlap from the end of the R. zoqui adult emergence period to the beginning of the
R. ramosae emergence period. It has been proven for other species of Rhagoletis that
diapause duration is genetically determined, highly variable, and that it responds to
selection imposed by host plant fruiting phenology (Feder, et al., 1997; Prokopy & Papaj,
47
2000). Synchronization between host plant phenology and overwintering is a key feature
affecting survival and offspring fitness among specialized phytophagous insects with
affinity for temperate climates (Feder et al., 1997; Prokopy & Papaj 2000; Van Asch &
Vissier, 2007). Host plant phenology can have evolutionary consequences, Feder et al.
(1997; 2003) established that a shift in the fruiting period of host plants R. pomonella
(two to three weeks), in addition to allelic changes in host associated populations,
played an important role in host race formation leading to speciation. Temporal isolation
between host races and closely related species has been documented for several
species of phytophagous insects (Drés & Mallet, 2002; Matsubayashi et al., 2010).
We found evidence of asymmetric sexual isolation (sensu Kaneshiro, 1983)
during behavioral observations with R. zoqui females preferring to mate with
conspecifics than with R. ramosae males. In contrast, R. ramosae females mated
assortatively with males of both species. Asymmetric sexual isolation has been
documented between other closely related species pairs in the suavis group (R. zoqui
and R. completa) (Tadeo et al., 2013) to a lesser degree than what we observed
between R. zoqui and R. ramosae. Sexual selection has been claimed to be one of the
most important forces triggering divergence and speciation between populations with
allopatric history (Ritchie, 2007; Jennings et al., 2011). Additionally prezygotic barriers to
gene flow may have a stronger effect than postzygotic barriers in generating
reproductive isolation (Coyne & Orr, 2004; Cuevas, 2012) since they prevent the waste
of costly gametes in production of hybrids with lower fitness (Kaneshiro, 1976).
Although in general, male behavior of both R. zoqui and R. ramosae adjusted to
generic patterns summarized by Prokopy and Papaj (2000) for Rhagoletis flies (males
defending fruit from rivals to force copulations on females seeking to oviposit), some
48
distinctive behavioral displays and tactics may be useful for comparative studies aiming
to clarify the evolutionary history within the entire suavis group as it has been done for
Hawaiian drosophilids (Kaneshiro, 1976). An example of the later may be the degree of
aggressiveness or tolerance to the presence of other males displayed by R. zoqui and
R. ramosae during fruit guarding, which often resulted in two males occupying the same
territory (fruit). Tadeo et al. (2013) had observed this behavioral pattern for some R.
zoqui males which could constitute an alternative mating tactic used to remain on fruit by
avoiding contests with more aggressive or otherwise superior males and thus maintain
access to females visiting fruit for egg-laying. Our study constitutes the first behavioral
description for R. ramosae, a species for which there is are only published records of
taxonomy, parasitism, distribution, and phenology (Hernández-Ortiz, 1985; Ovruski et
al., 2007; Rull et al., 2013). Other interesting details of mating behavior observed during
our study that may have relevance in understanding species relationships in the suavis
group were the differences in male displays during fruit guarding when facing an intruder
(male or female, conspecific or not). Male R. zoqui almost invariably held their wings
lifted perpendicular to the thorax (figure 5) a display also exhibited by R. completa
(Tadeo et al., 2013), but not by R. ramosae. Such pattern argues against the hypothesis
of a close relationship with R. zoqui, since R. ramosae males remain motionless in an
alert posture with their wings folded (figure 6). Another relevant observed trait is the way
R. ramosae males open their wings during male-male combat, a behavior also regularly
displayed by R. boycei, a member of the suavis group which could also have a common
origin with R. ramosae (Rull et al., 2013). The observed behavioral differences with R.
zoqui and R. completa, and the similarities with R. boycei male behavior lend support to
the idea that R. juglandis, R. boycei and R. ramosae arose from an initial
49
migration/speciation event followed by a second round of migration/differentiation giving
rise to R. zoqui and R. completa and argue against a close relationship with R. zoqui
(Rull et al., 2013). Solving R. ramosae phylogenetic relationships will therefore not only
shed light into the evolutionary history of the suavis group but also it will allow to
determine if some behavioral traits are useful in establishing phylogenetic relationships
among genetically similar species.
We observed significant differences in percent eclosion between eggs laid by R.
zoqui x R. ramosae hybrid mating combinations (rz & zr), yet these were only
numerically inferior to those recorded from eggs recovered from both pure crosses (zz
and rr). In contrast, Rull et al. (2012) found no differences in percent eclosion among
eggs laid by pure and hybrid R. zoqui x R. completa crosses, two species that hybridize
in nature. In the case of R. zoqui x R. ramosae hybrid combinations, the observed
pattern (asymmetric cytoplasmic incompatibility) could be indicative of R. zoqui infection
with Wolbachia (Werren et al., 2008). However, it is interesting to note, that the hybrid
cross yielding the highest levels of egg-hatch was the one that was the least likely to
occur during prezygotic isolation tests, a result contrary to findings for uninfected and
infested sympatric pairs of Drosophila species, where uninfected females were selected
to discriminate against infected males in areas of sympatry (Jaenike et al., 2006).
Tracking F1 and F2 hybrid fertility, as it was done by Rull et al. (2012) for other species
in the suavis group, coupled with screening for Wolbachia infections could contribute to
the understanding of the evolution of reproductive compatibility between R. zoqui and R.
ramosae and open avenues for research focused on a finer understanding of
differentiation among all species in the R. suavis group.
50
Altogether, our results appear to indicate that R. zoqui and R. ramosae display a
higher degree of reproductive incompatibility than the one observed between R. zoqui
and R. completa. We believe that biological differences between these two species
could have arisen during geographic isolation as a result in part to adaptation to the
fruiting phenology of their respective host plants, a pattern documented among other
species in the genus Rhagoletis (Feder et al., 1997: 2003, Prokopy & Papaj, 2000; Xie
et al., 2008). For R. ramosae, exploiting late fruiting J. major (Rull et al., 2013),
reproduction may be temporally restricted to a well-defined fruiting period, while R. zoqui
has been recovered from two native and an introduced species of Juglans (J. mollis, J.
pyriformis and J. regia) that differ in the timing of fruit maturation (Rull et al., 2013)
perhaps favoring the evolution of a longer period of adult emergence. Postzygotic
isolation could have been reinforced during periods of secondary contact producing
patterns documented here. According to Coyne & Orr (1989) both prezygotic and
postzygotic isolation in Drosophila increase with divergence time between taxa, if such
pattern holds true for flies in the genus Rhagoletis, our results and those of Rull et al.
(2012) and Tadeo et al. (2013) would support a closer relationship between R. zoqui and
R. completa than between R. zoqui and R. ramosae. It would be interesting to complete
studies on reproductive compatibility among Mexican species in the R. suavis group by
examining interactions between R. ramosae and R. completa and R. ramosae and R.
boycei, these coupled with results of mass sequencing techniques for a detailed
phylogeny of the suavis group, and screening for Wolbachia infections, may yield a
comprehensive picture of speciation for a taxonomic group other than Drosophila on
which most knowledge on the time course of speciation is based (Coyne & Orr 1997).
51
AKNOWLEDGEMENTS
We are grateful to Emilio Acosta for assistance in fruit collections. This work was partly
funded by the Mexican Campaña Nacional Contra Moscas de la Fruta (Secretaría de
Agricultura,
Ganadería,
Desarrollo
Rural
y
Pesca-Instituto
Interamericano
de
Cooperación para la Agricultura (to M.A.), Consejo Nacional de Ciencia y Tecnologia
(CONACyT) special grant 1100/596/04 C-837-04 (to J.R.), and CONACyT CB 200525889-50008Q (to J.R.).
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FIGURE LEGENDS
Figure 1. Total number of emerged adults over time of Rhagoletis zoqui (dots) from
Veracruz collected from Juglans pyriformis Liebmann between August and
September and Rhagoletis ramosae (triangles) collected in Guerrero from Juglans
major Heller between late September and early October.
Figure 2. Mean ± SEof frequency of copulation for different male-female pure and hybrid
mating combinations between Rhagoletis zoqui (z) and Rhagoletis ramosae (r).
Different letters represent significant differences at the α=0.05 level.
Figure 3. Percent of total copulations according to location in cages (fruit [Black bar] or
mesh [White bar]) among adult Rhagoletis zoqui and Rhagoletis ramosae.
Figure 4. Mean ± SE of number of Rhagoletis zoqui x Rhagoletis ramosae male-male
encounters according to contest location in cages (fruit [Black bar] or mesh [White
bar]). Different letters represent significant differences at the α=0.05 level.
Figure 5. Typical fruit guarding postures by A) and B) Rhagoletis ramosae, and C) and
D) Rhagoletis zoqui males.
Figure 6. Typical wing postures of A) a Rhagoletis zoqui male (folded wings) vs. a
Rhagoletis ramosae male (extended wings), B) two Rhagoletis ramosae males
(both contestants with extended wings) and C) two Rhagoletis. zoqui males (both
contestants with folded wings) during male-male contests on host fruit.
Figure 7. Mean ± SE. Number of egg-laying bouts per female per replicate for
Rhagoletis zoqui and Rhagoletis ramosae. Different letters represent significant
differences at the α=0.05 level.
58
Figure 8. Mean ± SE of number of eggs laid per egg-laying bouts for female Rhagoletis
zoqui or Rhagoletis ramosae. Different letters represent significant differences at
the α=0.05 level.
Figure 9. Mean ± SE of percent egg hatch for pure (zz and rr) and hybrid (zr and rz)
crosses between adult R. zoqui and R. ramosae. Different letters represent
significant differences at the α=0.05 level.
59
FIGURES
Figure 1
Figure 2
60
Figure 3
Figure 4
61
Figure 5
Figure 6
62
Figure 7
Figure 8
63
Figure 9
64
CAPITULO 3
For: Biological Journal of the Linnean Society
Behavioral patterns and relative strength of pre- and postzygotic isolation
between two recently derived species of walnut infesting flies in the highlands of
Mexico.
EDUARDO TADEO1,2*, MARTÍN ALUJA1,JUAN RULL1
1
Instituto de Ecología, A.C., Carretera Antigua a Coatepec no. 351, Colonia el Haya,
C.P. 91070 Xalapa, Veracruz, México.
2
Posgrado Instituto de Neuroetología, Universidad Veracruzana, Dr. Luis Castelazo s/n
Col. Industrial Animas, Xalapa, Veracruz, México.
“Corresponding autor
Corresponding
author
information:
Eduardo
Tadeo,
Posgrado
Instituto
de
Neuroetología, Universidad Veracruzana, Dr. Luis Castelazo s/n Col. Industrial Animas,
Xalapa, Veracruz, México e-mail: tadeotkd@hotmail.com
Running head: REPRODUCTIVE ISOLATION IN RHAGOLETIS
65
Episodes of isolation and secondary contact among populations of insects of Nearctic
origin during Pleistocene glacial/postglacial climatic cycles had a strong evolutionary
influence on diversity of flies in the genus Rhagoletis in mountainous areas of Mexico.
As a series of experiments undertaken to gather support for inconclusive molecular
phylogenetic hypotheses on the origin of three walnut infesting species in the suavis
group, we examined pre and postzygotic isolation between Rhagoletis completa
Cresson and R. ramosae Hernandez-Ortiz. Mating experiments in large enclosures
revealed asymetric sexual isolation between R. completa and R. ramosae. There were
notable differences in male behavior between species. While R. ramosae males mated
exclusively on host fruit, R. completa males used fruit and alternative mating locations.
During fruit guarding and male-male contests male R. completa exhibited behavioral
patterns similar to those of its close relative R. zoqui while behavior of R. ramosae
males was more reminiscent of that typically exhibited by R. boycei. During no choice
crosses in small enclosures, there was a marked reduction of egg hatch for the hybrid
cross of R. completa males x R. ramosae females. In comparison to previous studies on
reproductive isolation between species pairs in the suavis group, both pre and post
zygotic isolation were stronger between R. ramosae and R. completa than between R.
ramosae and R. zoqui and R. zoqui and R. completa. Our results appear to support
common ancestry of R. ramosae and R. boycei rather than R. ramosae and R.
completa/R. zoqui. We discuss the value of gathering comprehensive evidence from
different sources (molecular, behavioral, ecological) when resolving phylogeny of
recently derived species.
66
ADITIONAL KEYWORDS: Ecological speciation, Rhagoletis suavis, allochronic
isolation, Pleistocene
INTRODUCTION
Pleistocene glacial cycles played a key role in giving rise to diversity patterns of current
lineages of insects of Nearctic origin in Neotropical highlands (Knowles, 2001; Salomon,
2001; Shepard & Burbrink, 2009). Populations with affinity for temperate climates took
refuge during glaciations in areas of Southern latitude with less harsh conditions (Hewitt,
2004; Solis et al., 2006; Pravan & Bennet, 2008).
During postglacial cycles, such
populations underwent periods of isolation in high elevation areas of México (Ross,
1953; Martin & Harrell, 1957; Howden, 1963; 1969; Domínguez-Domínguez et al., 2011)
followed by periods of secondary contact, producing gene flow, hybridization and
reinforcement.
Populations
undergoing
isolation-secondary
contact
cycles
can
initiate
differentiation as a result of divergent natural selection between environments (Schluter,
2003; Funk et al., 2006), or trough accumulation of differences that result in production
of hybrids with lower fitness (Dobzhansky, 1936;1937; Muller, 1942). In similar
environments, under a polygenetic drift model, populations evolving independently
under stabilizing selection can undergo allelic changes where a few loci are
incompatible with multiple alleles in the genetic background of hybrids (Fierst & Hansen,
2009). During secondary contact, prezygotic reproductive barriers can arise in response
to direct selection against hybrids with lower fitness (Hoskin et al., 2005). Examining the
existence and relative strength of postzygotic and prezygotic isolation between species
67
pairs may shed some insight into their evolutionary history (Coyne & Orr, 2004) and a
number of indices have been developed to compare between species divergence that
allow to make inferences on the evolutionary history of particular lineages (PerezFigueroa et al., 2005).
A good example of insects undergoing recent differentiation triggered by
Pleistoicenic climatic pulses in the highlands of Mexico are Tephritid fruit flies in the
genus Rhagoletis. In particular, within the pomonella species group, ancestral
populations of hawthorn infesting flies took refuge in the Mexican Altiplano 1.57 million
years ago in isolation from North American populations. Subsequent episodes of gene
flow infused North American populations with inversion polymorphisms affecting key
diapause traits that later allowed flies to adapt to plants with different fruiting phenology
(Feder et al., 2003; Feder et al., 2005). Allochronic isolation, coupled with host plant
specific mating, and host fidelity allowed recent spawning of new taxa giving rise to a
group of six morphologically similar sibling species differing in host plant affiliation (Xie
et al., 2008).
Mexican populations of R. pomonella are also in the process of diversification,
three populations exploiting hosts with different fruiting phenology occur across the
Mexican Trans Volcanic Belt, the Sierra Madre Oriental, and los Altos de Chiapas (Rull
et al., 2006). These populations are genetically distinct (Michel et al., 2007; Xie et al.,
2007) and exhibit some degree of reproductive isolation among themselves (Rull et al.,
2010). Also, flies in the black cherry infesting cingulata species group, also appear to be
undergoing geographical host plant mediated differentiation (Rull et al., 2011), with
populations in the central Altiplano and the Sierra Madre Oriental exhibiting distinct
68
morphological features and displaying some degree of reproductive isolation between
them and with US populations (Tadeo et al., unpublished results).
The walnut infesting suavis group is currently composed of six morphologically
distinct species (Rhagoletis suavis Loew, R. juglandis Cresson, R. boycei Cresson, R.
completa Cresson, R. zoqui Bush and R. ramosae Hernández-Ortiz) though to be the
product of isolation in allopatry (Bush, 1966;1975; Foote, 1981; Bush & Smith, 1998).
Until recently, only five of the six described species in the group had been
phylogenetically arranged (Bush, 1966;1968; Bush & Smith, 1997;1998; Smith & Bush,
2000) with R. zoqui and R. completa always appearing in a tight cluster. More recent
molecular scrutiny of the suavis group has incorporated R. ramosae to this cluster (Frey
et al., 2013; Rull et al., 2013) resulting in an unresolved phylogeny and two mutually
exclusive hypotheses regarding its origin.
The first hypothesis proposes that R.
ramosae arose from an R. boycei/R. juglandis ancestor migrating from the Sierra Madre
Occidental, while the second suggests a R. completa/R. zoqui ancestor from the Sierra
Madre Oriental (Rull et al., 2013).
It has been found that R. completa and R. zoqui can hybridize in nature in a
contact zone in Northeastern Mexico (Rull et al., 2012), and exhibit no clear evidence of
prezygotic and postzygotic isolation, except perhaps a breakdown of F2 hybrids and a
tendency of R. completa to copulate both on fruit and on alternative mating locations
(Rull et al., 2012; Tadeo et al., 2013). Additionally, in the case of R. zoqui and R.
ramosae, R. zoqui females were observed to be more reluctant to mate with R. ramosae
males than with males of their own species and there was a significant reduction of egg
hatch for the hybrid cross of R. zoqui males and R. ramosae females in comparison to
that of other male-female mating combinations (Tadeo et al., unpublished results). In the
69
course of experiments R. zoqui and R. completa males exhibited similar distinctive wing
displays and fighting postures, while R. ramosae male behavior was more similar to that
observed for R. boycei (Tadeo et al., 2013; Tadeo et al., unpublished results).
Here, we examine prezygotic and postzygotic isolation between R. completa and
R. ramosae in order to be able to compare it with previous studies involving other
members of the suavis group and contribute in depicting the evolutionary history of the
group.
MATERIAL AND METHODS
BIOLOGICAL MATERIAL
To recover R. completa, infested Juglans hirsuta Manning fruit were collected in Buena
Vista, Nuevo León (25°23’36.6’’N, 100°18’5.1’’W, 1480 msnm) during September 2011
and 2012. To obtain R. ramosae pupae, infested fruit of Juglans major (Torr) Heller var.
glabrata Manning was collected in Taxco, Guerrero (18°33’16.9”N, 99°39’31.9”W, 1780
msnm) during the months of September and October 2011 and 2012. Collected fruit was
taken to the laboratories of the Instituto de Ecología A.C, in Xalapa Veracruz, and
processed according to methods described in Rull et al. (2006) to obtain pupae.
Recovered pupae were placed in 200 ml plastic cup lined with a 2.5 cm vermiculite layer
and regularly moistened with a sodium benzoate (C6H5COONa) 3g/L solution to prevent
desiccation and fungal growth. Cups were sealed with perforated plastic lids to allow air
flow and kept in a sheltered laboratory at room temperature until emergence of
overwintering adults the following season. All adults emerging at five days intervals were
separated according to species and sex and placed in 3 L. plastic cages provided with
70
water and food (a 3:1 sugar; hydrolyzed protein mixture) until sexual maturity (15 to 20
days) when they were used in experiments. Daily records of adult emergence were kept
for pupae from Veracruz and Guerrero.
POSTDIAPAUSE ADULT EMERGENCE
Seasonal patterns of adult emergence were compared between species by recording
daily emergence for lots of pupae collected in 2011. The duration of emergence post
dipause was calculated in days from fruit collection date to emergence of adults. For R.
completa a 300 pupae lot collected on August 30th 2011 was used, while for R.
ramosae emergence was recorded for a 371 pupae lot collected on September 30 of the
same year.
PREZYGOTIC ISOLATION (EXPERIMENTAL CAGE-MATING BEHAVIOR)
An experimental rectangular 1.08 x 1.08 m 1.78 m high cage was built using a 13mm
PVC pipe frame wrapped with white tergaline cloth. Within the cage, four 1.5 m. potted
oak trees (Quercus spp.) were placed at each corner along with walnut (J. pyriformis)
fresh foliage pinned on the upper part of the cage walls and ceiling attempting to mimic
a tree canopy. To serve as mating arenas, ten ripe, evenly spaced, J. pyriformis fruit
were hung in circle from the cage ceiling using copper wires long enough to reach the
potted oak foliage. Each copper wire received an individually numbered 3cm 2 green
cardboard label to ease recording of activity. The cage was provided with water and
adult food as described above. At 09:00 hours ten adult sexually mature (between 26
71
and 41 d old) R. completa and ten adult sexually mature R. ramosae couples (20
couples in total) were released in the experimental cage. Each fly was individually
marked on the back of the thorax with one or two dots of water based paint (Politec ®).
Behavioral observations were performed by a single experienced observer from 10:00 to
17:00 hours for two consecutive days using a combination of scanning and focal
observation of key behavioral events. A total of five 2-day replicates were performed
foror each replicate, the type of male-female combination, duration in minutes, and
location (fruit or mesh) were recorded for all copulations. For male fruit guarding
(considered when a male remained motionless on a particular fruit for more than one
minute) the identity and species of guarding males was recorded for every labeled fruit.
For male-male contests, the identity (species), status (resident-intruder), location (fruitmesh), and outcome (considering the male remaining on the contest site as the winner
and the one leaving the site as the loser) were recorded. Finally, in the case of females,
all fruit visits, egg laying bouts, and rejection of copulation attempts (vigorous
movements preventing intromission after mounting) were recorded.
POSTYGOTIC ISOLATION
To compare fertility among all possible R. completa x R. ramosae pure and hybrid
mating combinations [R. completa ♂ x R. completa ♀, R. completa ♂ x R. ramosae ♀;
R. ramosae ♂ x R. completa ♀; and R. ramosae ♂ x R. ramosae♀ (hereafter cc, cr, rc,
and rr respectively)], groups of five virgin females and five virgin males between 20 and
38 days of age were introduced into 3L, plastic cages provided with water and food as
described above. Flies in cages were allowed to interact for 24 h and offered two 2.5 cm
72
diameter agar spheres (1,350 ml water, 44.70 gr bacterial agar BDBioxon® and 1.5 ml
of McCORMICK® green food colour) for egg laying (Rull et al., 2010). Spheres were
replaced daily for 4 days and dissected under a microscope (Celestron®) to recover
eggs. Eggs were aligned over a piece of black cloth placed over a piece of cotton
(ZUUM®) moistened with water inside a Petri dish. Eggs were counted and incubated at
24°C for six days. After incubation, eggs were observed under the microscope and egg
hatch recorded. The entire procedure was repeated 5 times.
STATISTICAL ANALYSES
The length of dipause was compared between species using a General Nolineal Model
(GNM) twoway ANOVA adjusted to a Poisson distribution error. Frequency of copulation
per replicate was compared among different male-female R. completa x R. ramosae
pure and hybrid mating combinations by means of a oneway ANOVA, mating frequency
according to sex was compared with a oneway ANOVA adjusted to a Poisson
distribution. Copulation frequency according to mating location was compared among
male-female R. completa x R. ramosae pure and hybrid mating combinations with a
twoway ANOVA on ranked data. For male behavior, the frequency of fruit guarding and
contests was compared between species using t-tests on Log10 transformed data. In
the case of guarding frequency according to
male-male potential combinations
(including individual and joint guarding) and male permanency on fruit after contests
frequencies were compared using twoway ANOVAS adjusted to Poisson distribution.
For females, the frequency of fruit visits and egg laying bouts was compared between
species using t-tests, with egg-laying frequencies ranked. Finally, the total number of
73
eggs laid and percent egg hatch were compared among mating combinations using
oneway ANOVAs. Tests were run using STATISTICA 7® and SigmaPlot 10.0®. Sexual
isolation indices for mating frequencies were calculated using JMATING software
(Calvajal-Rodriguez & Rolan-Alvarez, 2006).
RESULTS
POSTDIAPAUSE ADULT EMERGENCE
We recorded 22% of adult emergence for both R. completa and R. ramosae. In the case
of R. completa 45 % of these adults were females and 55% males, while for R. ramosae
48% were females and 52% males. A twoway ANOVA revealed significant differences in
dipause duration between species (X2=39.39, p<0.001) but not between sexes (X2
=1.72, p=0.19). The interaction between species and sex was not significant (X2 =0.86,
p=0.77). Adult R. ramosae emerged on average 302.253 ± 4.189 (S.E) days after fruit
collection, while adult R. completa did so after 245.66±8.05 days (Figure 1). This four
week time lag roughly coincided with seasonal differences in the fruting phenology of the
respective walnut host plants.
PREZYGOTIC ISOLATION
A oneway ANOVA revealed significant differences in the frequency of copulation among
different male-female R. completa x R. ramosae mating combinations (F3,
16=11.67,
p<0.001). The cc pure cross occurred in higher frequency (12.80±1.63 de S.E), followed
by the hybrid cross cr (7.80±2.63 de S.E) and the pure cross rr (3.0±0.95 S.E), while the
74
hybrid cross rc occurred in the lowest frequency (0.2±0.2 de S.E) (Figure 2). A two way
ANOVA adjusted to a Poisson distribution revealed significant differences in individual
mating frequency among males and females of both species (X2=7.03, p=0.008). R.
completa males mated an average of 3.27±0.39 times per replicate and R. completa
females 2.31±1.29 times. In the case of R. ramosae, males mated an average of
1.33±0.14 times, and females 2.25±0.51 (Figure 3). A two way ANOVA on ranked data
revealed a significant interaction between mating combination and mating location (F 3,
32=6.82,
p<0.001). For mating combinations involving R. completa males copulations
occurred both on fruit and the cage mesh, while R, ramosae males mated more often on
fruit (Figure 4). Overall, there was a significant degree of mating isolation between R.
completa and R. completa (Ipsi=0.53, SD=0.08, t=6.68) as revealed by calculations
using JMATING on a total of 114 pure and hybrid copulations.
Other observed behaviors that were not analyzed due to the low frequency of
occurrence were failed copulation attempts and male-male copulation attempts. Five R.
ramosae males unsuccessfully attempted to copulate with R. completa females, while
three R. completa males failed to copulate with R. ramosae females. It is worth noting
that these last males made up to seven consecutive attempts to mate. With respect to
female rejection behavior (pushing with hind legs to dislodge mounting males) six R.
completa males were rejected by R. ramosae females and only one by a female of its
own species. Twelve R. completa males attempted to copulate with R. ramosae males,
while only two R. ramosae attempted copulation with males of R. completa.
75
MALE BEHAVIOR
There was no significant difference in the frequency of fruit guarding between R.
completa (23.2±7.69) and R. ramosae (22.8±6.75 de S.E) males. However, when
comparing the frequency of individual and joint fruit guarding a one way ANOVA
adjusted Poisson distribution revealed significant differences among male and malemale (joint) guarding combinations (X2= 85.56, p<0.001) (Figure 5). R. ramosae males
guarded fruit at greater frequencies (14.2± 3.46 de S.E), followed by lone R. completa
males (10.0±1.97 de S.E), while joint conspecific and heterospecific guarding occurred
at lower frequencies (between 1.4±0.6 and 4.4 ±2.06 S.E.).
There were no significant differences in log 10 transformed frequencies of malemale contests between species (t1,63=1.63, p=0.109). R. completa males participated in
an average of 9.158±1.11 contests per replicate and R. ramosae males in 6.373±0.958.
A twoway ANOVA adjusted to a Poisson distribution revealed significant differences in
the frequencies of contestants remaining of fruit (winner) between species (X 2=5.91,
p=0.015) and according to status (resident, invader, both) (X2=8.82, p=0.012). Male R.
completa remained on contested fruit more frequently (5.2±0.99 de S.E) than R.
ramosae males (2.83±0.51 de S.E) while permanence of both males was more frequent
(4.8±1.09 S.E) than permanence of residents (2.1±0.69 de S.E) or invaders (1.6±0.4 de
S.E) alone (Figure 6). Some noteworthy behavioral observations include contest
avoidance and postures adopted by different species during guarding and contests.
Some R. ramosae males preferred to retreat or abandon fruit rather than engaging in
contests with R. completa males. Males of both species exhibited marked behavioral
differences during guarding and contests across the entire study, while R. completa
76
guarded fruit with both wings raised perpendicular to the thorax R. ramosae remained
with their wings folded and a slight extension of the front legs (Figure 5). During combat
R. ramosae males extended and rotated their wings as if to show wing patterns to rivals
while R. completa males kept their wings folded (Figure 6).
FEMALE BEHAVIOR
There were no significant differences in ranked frequencies of female fruit visits between
species (t1,58=-0.66, p=0.26). Female R. completa visited fruit at an average frequency of
7.47±1.44 times while R. ramosae did so at an average frequency of 8.2±1.8 times per
replicate. We also failed to detect significant differences in transformed egg-laying
frequencies (t1,34=-1.39, p=0.195). Female R. completa laid eggs at an average of 2.37±
0.29 times per replicate while R. ramosae did so at an average frequency pf 3.53±0.56
times per replicate.
POSTZYGOTIC ISOLATION
A one way ANOVA revealed no significant differences in the number of eggs laid by
females among different male-female, R. completa x R. ramosae mating combinations
(F3, 24 =2.29, p=0.107). Females in no choice 5-couple cages of the cc pure cross laid
an average of 92.71±11.71 eggs, females from the cr hybrid cross laid 33.57±15.94
eggs, females from the pure cross rr laid 76.86±23.72 eggs, and females from the hybrid
rc cross laid an average of 71.57±11.94 eggs. In contrast, a oneway ANOVA revealed
significant differences in egg hatch for eggs laid by females from different mating
combinations (F3,24 =5.94, p=0.004). Mean percent hatch for eggs laid by females in cc
77
cages was significantly greater than egg hatch for eggs laid in cages with other malefemale combinations, and egg hatch for eggs laid by females in the cr combination cage
was statistically lower than egg hatch for eggs recovered from both the rr and rc mating
combinations, between which there were no statistical differences (figure 7).
DISCUSSION
We detected asymetric sexual isolation between R. completa and R. ramosae, with R.
completa females being more reluctant to mate with R. ramosae males than with
conspecific males. R. completa males outcompeted R. ramosae in gaining access to
females of both species. While R. ramosae males exclusively mated on host fruit R.
completa males used alternative mating locations. R. completa males won more malemale contests than R. ramosae. During fruit guarding and male-male contests, male R.
completa exhibited behavioral patterns similar to those of R. zoqui reported elsewhere
(Tadeo et al., unpublished results) while behavior of R. ramosae males was reminiscent
of that typically exhibited by R. boycei.
Females of both species visited fruit and
attempted egg laying in similar frequencies. There was a marked reduction of egg hatch
for the hybrid cross of R. completa males x R. ramosae females. In comparison to
previous studies of reproductive isolation between species pairs in the suavis group,
both pre and post zygotic isolation were stronger between R. ramosae and R. completa
than between R. ramosae and R. zoqui and R. zoqui and R. completa.
According to classical allopatric speciation models championed by Mayr (1963),
speciation processes almost invariably initiate when geographically isolated populations
evolve in different environments. During ecological speciation reproductive isolation
78
arises as a consequence of divergent natural selection on populations that may be
allopatric or sympatric (Schluter, 2001; Rundle & Nosil, 2005; Via, 2009; Cocroft et al.,
2010). R. completa and R. ramosae in Mexico are geographically isolated, R. completa
range encompasses the Sierra Madre Oriental, from San Luis Potosí to the Texas border
in Coahuila, while R. ramosae occurs west of Mexico city on the Eje volcanico Trans
Mexicano from Morelos to Jalisco (Hernández-Ortiz, 1985; Rull et al., 2013). R.
completa and R. ramosae occupy environments that differ in humidity and mean
temperature (more arid and cool in the Northeast and warm and moist in Western
Mexico), and develop in fruit of different species of plants in the genus Juglans (Rull et
al., 2013). Rhagoletis completa exploits J. hirsuta, J. microcarpa and J. mollis while R.
ramosae has only been reported on J. major (Hernández-Ortiz, 1985; Rull et al., 2013).
Annual peaks in precipitation and the onset of cool temperatures during winter produce
differences in the fruiting phenology of Northeastern and Western walnut tree species,
which in turn influence diapause regulation of their fly parasites and result in allochronic
isolation between R. completa and R. ramosae (see Rull et al., 2013). Divergence
among phytophagous insect host plants is known to produce reproductive isolation
among host races (Berlocher & Feder, 2002; Nosil, 2003; Cocroft et al., 2010),
especially when mating occurs on the host plant where the offspring develops. Flies in
the genus Rhagoletis are generally specialized on fruit of plants in the same family or
genus where they mate and develop (Boller & Prokopy, 1976; Prokopy & Papaj, 2000),
additionally for flies in the pomonella species group, differences in the fruiting phenology
of host plants have been found to favor or strengthen divergence between host races
eventually resulting in speciation (Berlocher, 2000; Berlocher & Feder, 2002; Feder et
al., 2003; Xie et al., 2008).
79
Asymetric prezygotic isolation was manifest through a marked tendency of R.
completa but not R. ramosae females to mate with males of their own species, a
common outcome among recently diverged insect species (Kaneshiro, 1983; Arnqvist et
al., 2000; Jennings et al., 2011). Asymetric sexual isolation had also been found
between other closely related species pairs in the suavis group (R. completa & R. zoqui
and R. zoqui & R. ramosae) (Tadeo et al., 2013; Tadeo et al., unpublished results). If as
it has been found for drosophila by Coyne & Orr (1997), prezygotic reproductive
isolation in the suavis group increases between taxa with divergence time, then R.
completa and R. zoqui appear to be more closely related between them than with R.
ramosae [R. completa & R. zoqui Ipsi=0 (Tadeo et al., 2013); R. zoqui & R. ramosae con
Ipsi=0.34 (Tadeo et al., Unpublished results); and R. completa & R. ramosae Ipsi=0.53
here]. Such an outcome does not favor one of the hypotheses put forth by Rull et al.
(2013) regarding the origin of R. ramosae where this species would be most closely
related to a common ancestor of R. zoqui and R. completa than to R. boycei.
Divergence between ancestors of R. completa and R. ramosae could have arisen
as a result of natural selection in different environments (Nosil et al., 2002; Mckinnon et
al., 2004) favored in part by physical separation of their respective host plants J. major
vs. J. hirsuta/J. mollis, which could have evolved among other things, differences in
fruiting phenology in response to climatic conditions. Periods of interruption of gene flow,
drift, and differential selection could have resulted in differences in behavioral patterns or
morphological traits related to reproduction (Kaneshiro, 1976, Jennings et al., 2011)
which can become fixed within populations and reinforced during periods of secondary
contact (Kaneshiro, 1983; Ritchie, 2007, Perez et al., 2011). As opposed to flies in the
pomonella species group, there are clearly distinctive morphological differences in wing
80
patterns and body coloration among all known species in the suavis group (Smith &
Bush, 2000), although these differences were hypothesized to produce sexual isolation
(Bush, 1966; Smith & Bush, 2000) there is still potential for gene flow between some
species pairs within the suavis group (Rull et al., 2012; Tadeo et al., 2013) a pattern
suggestive of recent divergence. Here, we observed behavioral differences between R.
completa and R. ramosae males manifested during mating location guarding and malemale contests (figures 5 and 6). Distinctive morphological traits and behavioral mating
patterns can aid in resolving phylogenetic relationships and the direction of evolutionary
history of the suavis group in Mexico as suggested by Kaneshiro (1976) for closely
related species of drosophila in Hawaii. So far, morphological and behavioral evidence
points to a common origin of R. ramosae and R. boycei whose ancestor may have
separated as a result of physical separation of different subspecies of their host J. major
across the Sierra Madre Occidental.
Some noteworthy behavioral patterns observed during our study are the use of
alternative mating locations by R. completa males coupled with greater aggressiveness
during male-male contests. Such patterns were also reported during similar studies
involving R. zoqui (Tadeo et al., 2013) and were hypothesized to increase mating
probability among R. completa individual males. Kaneshiro (1983) proposed that any
behavioral modification conferring a male mating advantage is subject to sexual
selection and may become fixed in a population. In contrast to R. completa, R. ramosae
males were less aggressive and exhibited typical patterns of resource (host fruit)
defense mating behavior for flies in the genus Rhagoletis (Prokopy & Papaj, 2000) and
for other species in the suavis group such as R. zoqui (Tadeo et al., 2013). Apparently,
small sized males of both R. ramosae and R. zoqui avoid male-male contests on fruit, by
81
crouching when confronted by larger males but remain on mating territories and
potentially mate with egg-laying females, although this claim still needs to be
experimentally confirmed.
The hybrid cross of R. completa males x R. ramosae females produced eggs with
lower hatch than other mating combinations. These results are similar to those of Tadeo
et al., (unpublished results) for the hybrid cross of R. zoqui males x R. ramosae females,
whereas no evidence of postzygotic isolation was reported for the crosses of R.
completa x R. zoqui by Rull et al., (2012). These patterns could be explained if R.
ramosae carry Wolbachia infections not shared by R. completa and R. zoqui.
Alternatively, reduced egg hatch could be the product of Bateson- Dobzhansky-Muller
(BDM) epistatic interactions between sets of alleles substituted at different loci during
geographic isolation of ancestral populations (Bateson 1909; Dobzhansky, 1936;1937;
Muller, 1942). The BDM model of postzygotic isolation has received recent attention and
support (Baton & Charlesworth, 1984; Orr & Turreli, 2001; Sweigart et al., 2006; Fierts &
Hansen, 2009; Cattani & Presgraves, 2009). In any case, as for prezygotic isolation, our
results and those of Rull et al., (2012) and Tadeo et al., (unpublished results) appear to
place R. ramosae in a clade with a different evolutionary history than R. completa and R.
zoqui, and again highlight the importance of examining reproductive isolation between
R. boycei and R. ramosae for a comprehensive view of speciation in the suavis group.
This and previous studies examining reproductive isolation between species pairs
in the suavis group have been useful for understanding of the evolutionary history of a
group of recently diverged flies. We would like to stress the value of documenting
behavioral patterns as a valuable trait for phylogenetic reconstruction where previous
conventional molecular studies (Frey et al., 2013; Rull et al., 2013) had failed to
82
separate three closely related species. In general gathering data of diverse nature (e.g.
behavioral, genetic, ecological, and morphological) provides a more comprehensive
picture than merely constructing gene trees. Future studies including further molecular
scrutiny of flies and associated symbionts and other species pairs evaluations of
reproductive isolation involving R. boycei and other walnut infesting species of
Rhagoletis will allow empiric testing of several hypotheses regarding differentiation of
phytophagous insects, a welcome addition to the understanding of speciation largely
based on experiments on a handful of easy to rear species.
AKNOWLEDGEMENTS
We are grateful to Emilio Acosta for assistance in fruit collections. This work was partly
funded by the Mexican Campaña Nacional Contra Moscas de la Fruta (Secretaría de
Agricultura,
Ganadería,
Desarrollo
Rural
y
Pesca-Instituto
Interamericano
de
Cooperación para la Agricultura (to M.A.), Consejo Nacional de Ciencia y Tecnologia
(CONACyT) special grant 1100/596/04 C-837-04 (to J.R.), and CONACyT CB 200525889-50008Q (to J.R.).
83
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FIGURE LEGENDS
Fig.1. Post diapause adult emergence (male and female) per day for pupae recovered
from Juglans hirsuta in Nuevo Léon during September (R. completa) and Juglans
major in Guerrero (R. ramosae) between late September and early October.
Fig. 2. Mean (± SE) number of copulations per replicate per male-female mating
combination in a1.08x1.08x1.78m cage with 20 sexually mature R. completa (c)
and R. ramosae (r) couples.
Bars with different letters represent significant
differences (α=0.05).
Fig. 3. Average individual frequency of copulations per replicate for males and females
of R. completa and R. ramosae during prezygotic isolation tests in large
enclosures.
Fig. 4. Mean (± SE) number of copulations per mating location (fruit- white bars/ meshblack bars) for different male x female, R. completa (c) x R. ramosae (r) mating
combinations Bars with different letters represent significant differences (α=0.05).
Fig. 5. Mean (±SE) number of single and shared fruit guarding events per replicate for
R. completa (c) and R. ramosae (r) males Bars with different letters represent
significant differences (ANOVA: p<0.05). Wing displays during guarding by R.
completa (lifted wings, top picture), R. ramosae (alert, folded wings, middle
picture), shared guarding by two males (no aggression, bottom picture).
Fig. 6. Mean (±SE) number of male/males remaining on fruit after contests per replicate
according to status (resident/invader) and species (R. completa = c / R. ramosae =
r) Bars with different letters represent significant differences (α=0.05). Picture son
the left of figure depict postures during contests. R. ramosae males extended
wings during boxing (top picture) while R. completa males kept their wings folded
(bottom picture).
90
Fig. 7. Mean (±SE) percent egg hatch for different male-female R. completa (c) x R.
ramosae (r) mating combinations. Bars with different letters represent significant
differences (ANOVA: p<0.05).
91
FIGURES
Figure 1
Figure 2
92
Figure 3
Figure 4
93
Figure 5
Figure 6
94
Figure 7
95
VI. DISCUSIÓN GENERAL
Considerando los resultados más relevantes de los tres estudios donde se
comparó el nivel de aislamiento reproductivo entre R. completa, R. zoqui, R. ramosae,
destacan los siguientes aspectos:
Aislamiento precigótico.
Aislamiento temporal (fenología de hospedero)
En primer lugar destaca que R. completa, R. zoqui y R. ramosae infestan
especies y/o variedades distintas de Juglans (Rull et al., 2013). Estos hospederos
presentan variaciones en sus periodos de fructificación como resultado de procesos
adaptativos en ambientes distintos (Via, 2009; Cocroft et al., 2010). Las asincronias en
el periodo de fructificación, causan un desfase en cascada en el periodo de emergencia
de los adultos de las distintas especies de moscas del grupo suavis que las infestan. La
sincronización en la emergencia de adultos durante los periodos de fructificación de sus
hospederos está determinada genéticamente y resulta fundamental para los insectos
altamente especializados como es el caso de los miembros del genero Rhagoletis
(Feder et al., 1997; Prokopy y Papaj, 2000), pues la disponibilidad de frutos es un
aspecto crucial para asegurar la supervivencia de su progenie (Feder et al., 1997).
En este estudio encontramos evidencia de un proceso de aislamiento
reproductivo temporal como resultado de diferencias en los periodos de fructificación de
las distintas especies de Juglans distribuidas en México que causa desfases en la
ocurrencia de adultos (contados a partir del inicio y fin de la emergencia de adultos
post-dipapausa) de entre dos a cuatros semanas en el caso de R. completa y R. zoqui
y entre esta última y los adultos de R. ramosae. Este desfase en la emergencia de
96
adultos evita o reduce la probabilidad de encuentro entre adultos de R. ramosae con los
adultos de las otras dos especies en un mismo periodo de tiempo en zonas de contacto.
Lo cual de acuerdo con Nosil (2003) y Cocroft et al. (2010) las diferencias entre
hospederos pueden causar aislamiento reproductivo y para el caso de los miembros
del genero Rhagoletis se ha demostrado que diferencias en la fenología de fructificación
favorecen la divergencia y formación de nuevas especies y razas (Xie et al., 2008).
Como ejemplo de ello figuran las especies divergentes de R. pomonella asociadas a la
manzana y tejocote en los Estados Unidos, las cuales presentan un desplazamiento de
dos a tres semanas entre los periodos de fructificación de sus plantas de acogida
Crataegus y Malus (Drés y Mallet, 2002; Feder et al., 1997; 2003). En el caso de las
moscas del grupo suavis presentes en México ó por lo menos entre R. zoqui y R.
ramosae este desplazamiento en los periodos de fructificación de las especies de
Juglans resulto en un factor que dificultó los estudios en laboratorio, en la naturaleza
parece ser un factor relevante para evitar el flujo de genes entre estas dos especies en
zonas de contacto.
Cópulas
Otro resultado relevante es la existencia de cierto nivel de aislamiento sexual
entre especies. Este aspecto de acuerdo con el modelo de aislamiento sexual
asimétrico de Kaneshiro (1976; 1983), establece que cuando entre dos especies
estrechamente relacionadas las hembras de una de ellas aceptan los cortejos de los
machos de ambas especies, pero las hembras de la segunda especie exhiben una
fuerte discriminación por los machos de la primera y prefieren aparearse con los
machos de su misma especie se presenta este tipo de aislamiento. Nosotros
97
encontramos distintos grados de aislamiento sexual entre R. completa, R. zoqui y R.
ramosae al comparar las frecuencias de cópula de las distintas combinaciones machohembra posibles. En el caso de la primera comparación que comprendió a R. completa
con R. zoqui el aislamiento se manifestó con una mayor frecuencia de cópula de la
combinación homotípica de R. completa y heterópica de machos de R. completa con
hembras de R. zoqui, las cuales ocurrieron con mayor frecuencia que las combinación
homotípica de R. zoqui y heterópica de machos de R. zoqui con hembras de R.
completa. En la segunda comparación, que comprendió a R. zoqui con R. ramosae, el
aislamiento se manifestó por la discriminación que mostraron las hembras de R. zoqui
en contra de los machos de R. ramosae, prefiriendo aparearse con los machos de su
misma especie y discriminando a los machos de R. ramosae casi por completo, en
tanto que las hembras de R. ramosae se aparearon con los machos de ambas especies
sin mostrar ninguna discriminación por los machos de alguna de ellas. Por último en la
tercera comparación que comprendió a R. completa con R. ramosae el aislamiento se
manifestó por una menor frecuencia de cópula de los machos de R. ramosae con las
hembras de ambas especies, en tanto que los machos de R. completa copularon
indistintamente con las hembras de ambas especies. Los diferentes tipos de
discriminación de pareja encontrados durante la observación de pares de especies
muestran como el aislamiento sexual puede evolucionar en distintas formas, pero
además se traduce en diversos niveles de aislamiento sexual que puede estimarse
mediante el uso de índices de aislamiento reproductivo “Ipsi” (Calvajal-Rodriguez y
Rolan-Alvarez, 2006). De esta forma al comparar R. completa con R. zoqui se obtuvo
un Ipsi=0, es decir que no presentan aislamiento sexual, lo cual concuerda con los
recientes hallazgos que reportan la presencia de una zona hibrida en el noreste de
98
México donde coocurren y se hibridan estas dos especies en forma natural (Rull et al.,
2013). El hecho de que hayamos encontrado diferencias en las frecuencias de cópula
entre especies, puede usarse para inferir la historia de la evolución de barreras
precigóticas como resultado de periodos de contacto secundario que podrían
evolucionar rápidamente como un refuerzo para evitar el flujo de genes (Dobzhansky,
1970). En lo que respecta a la segunda comparación que comprendió a R. zoqui y R.
ramosae se obtuvo un Ipsi=0.34, lo cual muestra ya la evolución de del aislamiento
reproductivo como mecanismo para evitar el flujo de genes entre dos poblaciones
vecinas geográficamente aisladas en el centro de México (R. zoqui se distribuye del
centro hacia el golfo de México y R. ramosae del centro hacia el océano pacífico). Por
ultimo para la comparación entre R. completa y R. ramosae se obtuvo un de Ipsi=0.53,
lo cual concuerda con la hipótesis que postula que durante el evento fundador que
acompaña a la especiación alopátrica las poblaciones más lejanas tienden a desarrollar
mayor diferenciación e incompatibilidad que las poblaciones más cercanas como
resultado de una interrupción en el flujo de genes (Mayr, 1942; Kaneshiro, 1983;
Futuyma, 1989; Badii et al., 2007).
Comportamiento
Este aspecto es quizás el más conspicuo puesto que evidencia claras diferencias
en el comportamiento sexual de los machos. De acuerdo con la teoría de la selección
sexual cualquier cambio morfológico o conductual que proporciona a los machos una
ventaja sobre otros machos para incrementar su probabilidad de apareamiento estará
sujeto a un proceso de selección sexual y será seleccionado para la población que lo
exhibe (Kaneshiro, 1983; Ritchie, 2007; Jennings, et al., 2011; Pérez-Barros et al.,
99
2011). En nuestro estudio encontramos una clara evidencia de un proceso de selección
sexual entre los machos de R. completa, los cuales mostraron el uso de sitios
alternativos de apareamiento y una mayor agresividad en contra de machos de R.
zoqui y R. ramosae durante la defensa del recurso y en los combates.
Esta agresividad, junto con el uso de sitios alternativos, parecen haber sido los
factores clave que permitieron a los machos de R. completa incrementar su frecuencia
de cópula con las hembras de R. zoqui y R. ramosae. Otros comportamientos
importantes a pesar de que no se encontró evidencia de un efecto en la discriminación
de pareja fueron las marcadas diferencias en los despliegues mostrados por los
machos de R. ramosae durante el resguardo de fruto y combates. El hecho de haber
encontrado estas notorias diferencias en el comportamiento de los machos resulta
interesante ya que se ha demostrado que el comportamiento puede analizarse para
establecer la dirección de la evolución (Kaneshiro, 1976).
En nuestro caso, este análisis nos aportó elementos para hipotetizar sobre la
probabilidad de dos líneas evolutivas para moscas del grupo suavis en México. La
primera de estas líneas pudo haberse desarrollado a lo largo de la Sierra Madre
Oriental donde R. completa y R. zoqui presentan comportamientos muy similares, en
tanto que la segunda línea pudo ocurrir a lo lago de la Sierra madre Occidental donde
R. ramosae, R. juglandis y R. boycei, parecen presentar comportamientos muy
similares. Esta hipótesis aun cuando parece aventurada
ha sido planteada con
anterioridad por los trabajos de Rull et al., (2013) al considerar una serie de aspectos
ecológicos y genéticos. Además como lo discute Kaneshiro (1976), nuestros resultados
muestran que el estudio del comportamiento es una pieza importante al abordar
100
estudios de corte evolutivo ya que pueden ser pieza clave para mostrar la dirección de
la evolución.
Aislamiento postcigótico.
Viabilidad de huevos
En el caso del análisis sobre la evolución del aislamiento postcigótico entre R.
completa, R. zoqui y R. ramosae, la evidencia encontrada fortalece los resultados
obtenidos durante el análisis sobre aislamiento precigótico, apoyando el hecho de la
evolución de un mayor grado de aislamiento entre las poblaciones de R. completa y R.
ramosae. Es decir, las especies con mayor aislamiento geográfico cuyas poblaciones
por efecto de la interrupción en el flujo de genes y la presión por selección natural en
ambientes divergentes (Koevoets y Beukeboom, 2008; Jennings et al., 2011) han
evolucionado diferencias genéticas que se traducen en una reducción en la aptitud
hibrida de la progenie resultante de las cruzas heterotípicas (Baton y Charlesworth,
1984; Orr y Turreli, 2001; Sweigart et al., 2006; Fierts y Hansen, 2009; Cattani y
Presgraves, 2009).
101
VII. CONCLUSIONES
En relación a la hipótesis aquí planteada que postula que las especies mexicanas
del grupo suavis, R. completa, R. zoqui y R. ramosae se encuentran en un proceso de
diferenciación incompleto debido a la existencia de barreras reproductivas con distinto
grado evolutivo que no interrumpen por completo el flujo génico entre especies,
podemos concluir lo siguiente:
1. Se acepta la hipótesis de una diferenciación incompleta entre R. completa, R.
zoqui y R. ramosae, las cuales presentan distintos grados de aislamiento
reproductivo parcial entre especies.
2. Los distintos niveles de aislamiento reproductivo encontrados entre R. completa,
R. zoqui y R. ramosae, evidencian la evolución de barreras reproductivas como
consecuencia de la interrupción génica y la presión por selección natural en
ambientes divergentes.
3. Aun cuando no se encontró evidencia de barreras reproductivas absolutas
capaces de evitar el flujo de genes entre especies. Lo cierto es que nuestros
resultados aportan evidencia sobre la evolución de barreras pre y postcigóticas
que podrían reforzarse en zonas de contacto para mantener la integridad
genética de R. completa, R. zoqui y R. ramosae, lo cual representa una
excelente oportunidad para estudiar aspectos evolutivos y de aislamiento
reproductivo en un escenario natural.
4. Los resultados aquí obtenidos confirman la importancia que representa la
inclusión del comportamiento dentro de los estudios de corte evolutivo ya que la
102
información que originan resulta una herramienta importante para discernir entre
líneas de divergencia evolutiva entre especies.
5. Aun
cuando
nuestros
resultados
aportan
valiosa
información
para
el
esclarecimiento del nivel de divergencia y posible camino evolutivo de las
moscas del grupo suavis en México, creemos que es necesario incluir en futuros
estudios a R. juglandis y R. boycei, especies también reportadas para México
pero cuya información actual es desconocida, así como también a las dos
nuevas especies asociadas al nogal recientemente descubiertas por Rull y
colaboradores en el sur del estado de Veracruz y Chiapas. La inclusión de estas
especies en futuras investigaciones serácrucial para esclarecer procesos
evolutivos que dieron origen a las
moscas del grupo
suavis
en México y
Estados Unidos y quizás aportar valiosa información para resolver el origen de
los otros grupos en el género Rhagoletis también refugiados en México durante
el Pleistoceno.
103
MODELO TEÓRICO
Modelo teórico de las posibles líneas evolutivas seguidas por las moscas del grupo suavis en
México. Línea azul de doble cara muestra la posible ruta evolutiva de R. completa y R. zoqui, en
tanto que la línea gris muestra la posible ruta de R. ramosae y R. boycei. Las imágenes circulares
muestran la similitud de los comportamientos mostrados por los machos en cada línea evolutiva.
Los signos de interrogación muestran la ubicación de dos nuevas especies recientemente
descubiertas en Veracruz y Chiapas.
104
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