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INSTITUTO POLITÉCNICO NACIONAL CENTRO INTERDISCIPLINARIO DE CIENCIAS
MARINAS
ESTRATEGIAS REPRODUCTIVAS DE DOS ESPECIES DE
BATOIDEOS (Narcine entemedor y Rhinoptera
steindachneri) EN LA BAHÍA DE LA PAZ, BCS, MÉXICO
TESIS
QUE PARA OBTENER EL GRADO DE
DOCTORADO EN CIENCIAS MARINAS PRESENTA
MARÍA ITZIGUERI BURGOS VÁZQUEZ
LA PAZ, B.C.S., JUNIO DE 2018
--------
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INSTITUTO POLITÉCNICO NACIONAL SECRETARIA DE INVESTIGACiÓN Y POSGRADO
ACTA DE REVISIÓN DE TESIS
En la Ciudad de La Paz, 8.C.S., siendo las 12:00 horas del día 26 del mes de
Abril del 2018 se reunieron los miembros de la Comisión Revisora de Tesis desjgnada
por el Colegio de Profesores de Estudios de Posgrado e Investigación de CICIMAR
para examinar la tesis titulada:
"ESTRATEGIAS REPRODUCTIVAS DE DOS ESPECIES DE BATOIDEOS
(Narcine entemedor Y Rhinoptera steindachnerl) EN LA BAHÍA DE LA PAZ, BCS, MÉXICO"
Presentada por el alumno: BURGOS VÁZQUEZ MARÍA ITZIGUERI Apellido patemo matemo nombre(sr)----,,---.-_--.-_--.--_--.--_-.--------,
Con registro: I B I 1 I 4 I O 2 I 8 I O
Aspirante de:
DOCTORADO EN CIENCIAS MARINAS
Después de intercambiar opiniones los miembros de la Comisión manifestaron APROBAR LA DEFENSA DE LA TESIS, en virtud de que satisface los requisitos señalados por las disposiciones reglamentarias vigentes.
LA COMISION REVISORA
Directores de Tesis
DA. vI~ .SCALONA Director de Tesis
DRA. CLAUDIA )ANETL HERNÁNDEZ CAMACHO
INSTITUTO POLITÉCNICO NACIONAL SECRETARíA DE INVESTIGACiÓN YPOSGRADO
CARTA CESiÓN DE DERECHOS
En la Ciudad de La Paz, 8.C.5., eldfa 21 del mes de del año 2018
El (la) que suscribe __---=M~C=.~MA=R=lA::..::..:.ITZ=I.=GU;::.E=R:.::::I:.;:8:..:U:.:.R::.::G:.;:O:..:::S__=V..:..:Á=ZQ""'_=_U=EZ=____ Alumno (a) del Programa
DOCTORADO EN CIENCIAS MARINAS
con número de registro 8140280 adscrito al CENTRO INTEROISCIPLlNARIO DE CIENCIAS MARINAS
manifiesta que es autor(a) Intelectual del presente trabajo de tesis, bajo la dirección de:
DR. víCTOR HUGO CRUZ ESCALONA Y DRA. PAOLA ANDREA ME,IA FALLA
y cede los derechos del trabajo titulado:
"ESTRATEGIAS REPRODUCTIVAS DE DOS ESPECIES DE BATOIDEOS
(Nardne en temedor Y Rhlnoptera stelndachnerl) EN LA BAHÍA DE LA PAZ, BCS, MÉXICOH
al Instituto Politécnico Nacional, para su difusión con fines académicos y de Investigación.
Los usuarios de la información no deben reproducir el contenido textual, gráficas o datos del trabajo
sin el permiso expreso del autor y/o director del trabajo. Éste, puede ser obtenido escribiendo a la
siguiente dirección: [email protected] • [email protected] - [email protected]
Si el permiso se otorga, el usuario deberá dar el agradecimiento correspondiente y citar la fuente del
mismo.
MC.
Nombrey firma delalumno
La ciencia no tendría sentido sin el deseo de vivir,
La vida no tendría sentido sin Dios
y Dios, no tiene sentido sin el amor.
2
A Rosario, que siempre hubo esa inexplicable conexión. Te extraño.
A Dios, que siempre está presente.
Y a mis padres, que, con su amor inagotable, llenan mi vida de esperanza.
3
AGRADECIMIENTOS
Muchas gracias al Instituto Politécnico Nacional (IPN) y al Centro Interdisciplinario de
Ciencias Marinas (CICIMAR), por abrirme las puertas a mi educación profesional, y
proporcionarme las herramientas y recursos económicos y académicos necesarios para mi
preparación como investigadora y poder contribuir al desarrollo y bienestar de mi país,
México, mediante la ciencia y la investigación.
A la Comisión Nacional de Ciencia y Tecnología (CONACyT) por la beca otorgada durante
los cuatro años de doctorado. Al proyecto SEP-CONACyT 180894 “Demografía de los
batoideos costeros más abundantes del Pacifico mexicano centro-norte” por el recurso
para la obtención de las muestras y el apoyo económico para las diversas movilidades
académicas durante el programa de estudio.
A mis estimados directores de tesis, el Dr. Víctor H. Cruz Escalona y la Dra. Paola A. Mejía
Falla, gracias por su conocimiento, su tiempo, su dedicación y sobre todo su amistad.
Espero continuar colaborando como hasta ahora. Este trabajo no se hubiera podido
realizar sin su valioso conocimiento y apoyo. Estoy inmensamente agradecida con ambos.
A mis amigos y maestros, el Dr. Andrés F. Navia, la MS. Nancy Brown Peterson y el Dr.
Mark Peterson. Gracias Andrés por tan buenos consejos, por tu disponibilidad para
compartir tu conocimiento. Nancy and Mark, thank you very much for sharing your
knowledge, I felt at home in Mississippi, forever grateful. A los tres, gracias por abrirme
las puertas de su hogar y brindarme su amistad.
Muchas gracias a todos los miembros de mi Comité revisor, por su valioso conocimiento
y aportaciones a mi trabajo de tesis. A todo el personal de CICIMAR, que siempre me han
recibido con una sonrisa, por su valiosa labor. Especialmente a Roberto Aguilera y Susana
Cárdenas, que se convirtieron en mis grandes amigos. Los voy a extrañar.
Don Juan Higuera y esposa, por su tiempo y dedicación en la isla, para poder llevar a cabo
nuestro trabajo de campo. Este trabajo no se hubiera podido realizar sin su conocimiento.
A todos mis compañeros de campo y laboratorio, que durante cuatro años me brindaron
su tiempo (por el procesamiento de muestras), su conocimiento y amistad. Especialmente
a Valeria E. Chávez García y Yutzin A. Jiménez García, por el apoyo en el laboratorio y
permitirme ser su directora de tesis, gracias por la confianza y la paciencia.
A mi hermana Atziri, que ha sido motor en mi vida, que me hace ser mejor y querer
mostrarle que la vida es maravillosa. A mi hermano Miguel, mi hermano pequeño, que lo
amo infinitamente y que siempre estaré para él. Ambos, a través de su mirada me han
demostrado la existencia de Dios.
A Frank, que llego justo en el momento que Dios lo decidió. Gracias mi amor por tu apoyo,
tu ternura y tu comprensión, quiero seguir eternamente a tu lado, juntando triunfos,
juntando hermosos momentos.
4
A mis amigos Anarosa, Surizaray, Jorge, Athziri, Lavinia y Caro, que durante cuatro años
me han apoyado incondicionalmente, muchas gracias por quererme. Este proyecto se
logró porque muchas veces estuvieron aquí, conmigo, apoyándome. A mi cuñada Paty y
su familia, por sus consejos y su amistad, pero sobre todo por amar a mi hermano.
A todos mis amigos del “Camino Feliz”, gracias por tan valiosa enseñanza de vida, siempre
en mi corazón.
A toda mi familia, mis amigos y compañeros de vida, gracias por hacerme feliz y
enseñarme que la vida es maravillosa, en buenas y malas condiciones.
I
CONTENTS
LIST OF FIGURES (III)
LIST OF TABLES (VI)
RESUMEN GENERAL (VIII)
GENERAL ABSTRACT (IX)
GENERAL INTRODUCTION (XI)
BACKGROUND (XV)
CHAPTER I. REPRODUCTIVE STRATEGY OF THE GIANT ELECTRIC RAY
Narcine entemedor IN THE SOUTHERN GULF OF CALIFORNIA
ABSTRACT (2)
1.1 INTRODUCTION (3)
1.2. MATERIAL AND METHODS (4)
1.2.1 Study site and collection of specimens (4)
1.2.2 Sex ratio, length, and mass (5)
1.2.3 Macroscopic observations of reproductive structures and maturity (6)
1.2.4 Histological analysis (10)
1.2.5 Median size of maturity, pregnancy, and maternity (10)
1.2.6 Fecundity and reproductive cycle (11)
1.3. RESULTS (12)
1.3.1 Sex ratio, length, and mass (12)
1.3.2 Male reproductive structures and reproductive phases (12)
1.3.3 Female reproductive structures (14)
1.3.4 Female size at maturity, pregnancy, and maternity (19)
1.3.5 Ovarian and uterine fecundity (20)
1.3.6 Reproductive Cycle (23)
1.4. DISCUSSION (28)
CHAPTER II. REPRODUCTIVE STRATEGY OF THE PACIFIC COWNOSE RAY
Rhinoptera steindachneri IN THE SOUTHERN GULF OF CALIFORNIA
ABSTRACT (38)
2.1. INTRODUCTION (39)
2.2. MATERIAL AND METHODS (40)
2.2.1 Study area, sample collection and laboratory analysis (40)
2.2.2 Sex ratio, disc width, and mass (41)
2.2.3 Reproductive structures and maturity (42)
2.2.4 Median size at maturity and pregnancy (42)
2.2.5 Fecundity and reproductive cycle (43)
2.3. RESULTS (47)
1
37
II
2.3.1 Sex ratio, disc width, and weight (47)
2.3.2 Reproductive structures and maturity (47)
2.3.2.1 Rare and hard structures in females (53)
2.3.3 Size at maturity and pregnancy (54)
2.3.4 Ovarian and uterine fecundity (55)
2.3.5 Reproductive cycle (56)
2.4. DISCUSSION (59)
CHAPTER III. REPRODUCTIVE AND LIFE HISTORY STRATEGIES OF Narcine
entemedor AND Rhinoptera steindachneri: TWO VIVIPAROUS SPECIES WITH
DIFFERENT REPRODUCTIVE MODES
ABSTRACT (67)
3.1. INTRODUCTION (68)
3.2. MATERIAL AND METHODS (71)
3.2.1 Reproductive mode and effort (71)
3.2.2 Life history traits (71)
3.2.3 Population parameter (72)
3.2.4 Definition and comparison of the life history strategies of both species (72)
3.3. RESULTS (73)
3.3.1 Reproductive mode and effort (73)
3.3.2 Life history parameters (81)
3.3.3 Population parameter (86)
3.3.4. Definition and comparison of the life history strategies of both species (86)
3.4. DISCUSSION (87)
GENERAL CONCLUSION (97)
REFERENCES (98)
ANEXOS (118)
66
III
LIST OF FIGURES
.
Page
Figure 1.- Specimens collected in the Bahía de La Paz, from a) mature male of Narcine
entemedor and b) female neonate of Rhinoptera steindachneri. XIV
Chapter I
Figure 1.- Study area (Gulf of California and Bahía de La Paz, Mexico) including primary
sampling locations for Narcine entemedor (star = El Morrito, diamond = El Quelele, square=
Campo Rodríguez).
5
Figure 2.- Relationship between TL (cm) and inner clasper length (ICL; cm) of male Narcine
entemedor.
13
Figure 3.- Micrographs of the reproductive structures of male Narcine entemedor of a)
Longitudinal section of testes in early stages of spermatogenesis in an actively mating male,
b) Longitudinal section of testes in the actively mating phase and c) Longitudinal section of the
seminal vesicle of a male in the actively mating phase. The dotted line indicates packets of
spermatozoa in a spermatozeugmata. Abbreviations are as follows: Sg = spermatogonia, Sc1
= primary spermatocytes, Sc2 = secondary spermatocytes, St = spermatids, Sz =
spermatozoa.
14
Figure 4.- Ovarian follicles (top) and ovary (without the covering tissue) of a mature female
Narcine entemedor in the ovulation capable phase.
15
Figure 5.- Micrographs of reproductive structures of female Narcine entemedor of a)
Transverse section of an ovary in the immature developing phase (ovarian index 2), b)
Transverse section of a vitellogenic oocyte from a female in the ovulation-capable phase
(ovarian index 3), c) Transverse section of the anterior oviduct of a female in the ovulation-
capable phase with oocytes in the uterus, d) Transverse section of the uterus of an immature
developing female (uterine index 2), e) Transverse section of the uterine villi from a female in
the immature developing phase (uterine index 2), f) Transverse section of the uterus from a
female in the mature-not-pregnant phase (uterine index 3), g) Transverse section of the uterus
from a female with late-stage embryos (uterine index 4E), h) Longitudinal section of the uterine
villi from a pregnant female with late-stage embryos (uterine index 4E) and secretory crypts
(C, i.e., within dotted line). Abbreviations are as follows: Ep = peritoneal epithelium, CA =
cortical alveolar oocytes, PG = primary growth oocytes, Os = ovarian stroma, Zp = zona
pelucida, Fe = follicular epithelium, Te = theca externa, V = granules of vitellogenin, Lo =
oviductal lobules, l = lumen, Po = oviductal plates, Vll = uterine villi, BV = blood vessel, Esc =
simple cylindrical epithelium, Sta = stratified cylindrical epithelium, and H = histotroph.
18
IV
Figure 6.- Maturity ogives for female Narcine entemedor to TL (cm). a) Ogive by maturity
phase, b) Ogive based on ovary condition, c) Ogive based on uterus condition, d) Pregnancy
ogive and e) Maternity ogive.
20
Figure 7.- Fecundity relationships in female Narcine entemedor with a) Total length (cm) and
number of ovarian follicles by group and b) TL (cm) and number of embryos by female.
22
Figure 8.- Relationships of lengths of ovarian follicles and embryos in Narcine entemedor with
a) Monthly variation of largest ovarian follicle length in mature females, b) Monthly variation of
intra-uterine embryo TL (filled circles indicate eggs in the uterus). The lower and upper
boundaries of the boxes indicate the 25th and 75th percentiles, respectively, while the square
(□) inside the boxes indicates the medians. Whiskers indicate the nonoutlier range and circles
indicate outliers and c) Relationship between largest ovarian follicle length and embryo
developmental stage. Whiskers indicate the minimum and maximum length while the square
(□) indicates the medians.
24
Chapter II
Figure 1. Relationship between disc width (DW) and a) left testicle weight, b) left seminal
vesicle width for males, and c) left ovary weight, d) left oviducal gland weight and e) left uterus
weight for females of Rhinoptera steindachneri.
50
Figure 2.- Macrostructures of the reproductive system of Rhinoptera steindachneri females.
Longitudinal section of the left ostium, oviduct and uterus in a) stage 1 (immature), b) stage 2
(developing), c) stage 3 (mature-virgin) and d) stage 4 (mature-pregnant). Hard structures
found in e) left oviducal gland, f) right oviducal gland, g) both uteri (inside) and h) extracted of
those organs. Structures were considered in the text in dorsal position but all photos were
taken in ventral position. Abbreviations are as follows: Os = ostium, Ov = oviduct, T =
trophonemata, HS = hard structure, U = uterus, np = narrowest part, wp = widest part.
53
Figure 3.- a) Relationship between disc width (DW) and inner clasper length (cm) in males; b) maturity ogive in relation to maturity condition of males and c) females; d) pregnant ogive in females of Rhinoptera steindachneri.
55
Figure 4.- Relationship between a) total number of ovarian follicles (OF) and disc width (DW);
b) maximum follicular diameter by month (dotted line: ovulation diameter); c) disc width (DW)
of embryos and neonates by month, of Rhinoptera steindachneri.
57
Figure 5.- Percentage of reproductive stages by months in a) males and b) females of
Rhinoptera steindachneri. 59
V
Chapter III
Figure 1.- Uterus and embryo of a) Narcine entemedor (E = in develop stage embryo) and b)
Rhinoptera steindachneri (E = early stage embryo). Abbreviations are as follows: T =
trophonemata; ys = yolk sac.
74
Figure 2.- Proportion between a) maximum follicular mass and maximum embryonic mass
and b) mature and pregnant females in relation to the total females of Narcine entemedor and
Rhinoptera steindachneri captured in Bahía de La Paz.
76
Figure 3.- Proportion of the a) size at first maturity, median size at maturity (TM50), median
size at pregnancy (TP50) and size at birth with the asymptotic size (L∞) [total length (TL in
Narcine entemedor and disc width (DW) for Rhinoptera steindachneri] and b) proportion of
maximum age, age at first maturity, median age at maturity (A50) and median age at pregnancy
(AP50; %: proportion with the maximum age evaluated) of Narcine entemedor and Rhinoptera
steindachneri.
84
Figure 4.- Ogive of a) median age at maturity (A50), and b) median age of pregnancy (AP50) of Narcine entemedor and Rhinoptera steindachneri.
85
VI
LIST OF TABLES
Page
Chapter I
Table 1. Macroscopic and microscopic descriptions of the male reproductive structures defined to distinguish maturity stages of Narcine entemedor.
7
Table 2. Macroscopic and microscopic descriptions of the female reproductive structures defined to distinguish maturity stages of Narcine entemedor.
8
Table 3. Mean monthly values (± SE) of the Gonadosomatic Index in female Narcine entemedor. Superscript numbers indicate homogeneous subsets (Tukey non-parametric post-hoc test with Bonferroni adjustment, P < 0.004). n = sample size.
25
Table 4. Percentage of maturity stages throughout the year in female Narcine entemedor. 27
Chapter II
Table 1. Maturity stages of Rhinoptera steindachneri males, indicating the characteristics and indices of each reproductive organ.
44
Table 2. Maturity stages of Rhinoptera steindachneri females, indicating the characteristics and indices of each reproductive organ.
45
Table 3. Mean and standard deviation values of the right and left reproductive structures in males and females of Rhinoptera steindachneri, and statistical results of Wilcoxon test. * t-studet t was used only for length of oviducal gland.
48
Chapter III
Table 1. Comparison of indicators of reproductive effort based on fecundity and mass among Narcine entemedor, Rhinoptera steindachneri and the optimal qualitative value for each parameter according to the r – K theory.
78
Table 2. Comparison of age, growth, reproductive and population parameters among Narcine entemedor, Rhinoptera steindachneri and the optimal qualitative value for each parameter according to the r – K theory.
82
VII
RESUMEN GENERAL
Uno de los aspectos más importantes dentro de la estrategia de historia de vida de las
especies, se basa en la estrategia reproductiva, la cual tiene como objetivo maximizar
la producción de la descendencia reproductivamente activa para asegurar la
supervivencia de la especie a través del tiempo. El presente estudio describe la
estrategia reproductiva y la estrategia de historia de vida de dos especies de batoideos
simpátricas para la Bahía de La Paz, BCS, México, la raya eléctrica gigante, Narcine
entemedor y la raya gavilán Rhinoptera steindachneri. El estudio se llevó a cabo con
muestreos mensuales mediante la captura de organismos con redes agalleras de
octubre de 2013 a marzo de 2017. Las hembras de N. entemedor presentaron una
talla mediana de madurez de 58.5 cm de longitud total (LT), talla mediana de preñez
de 63.7 cm LT y talla mediana de maternidad de 66.2 cm LT. Las hembras presentaron
entre 1 y 69 folículos ováricos de forma alargada, con una fecundidad ovárica máxima
(folículos vitelogénicos completamente desarrollados) de 17 y fecundidad uterina entre
1 y 24 embriones por hembra. Se estimó la talla de nacimiento entre 12.4 y 14.5 cm
LT. La vitelogénesis ocurrió de manera sincrónica con la gestación. Se definió un ciclo
reproductivo anual continuo, con un período de ovulación de cinco meses, entre mayo
y septiembre, y un periodo de gestación de cinco meses con dos picos de partos, uno
en enero-febrero y otro en agosto-septiembre y con diapausa embrionaria en algunos
individuos. La evidencia histológica de secreciones del tejido glandular de las
vellosidades uterinas, indica que probablemente esta especie presenta histotrofía
limitada. Para R. steindachneri, la talla mediana de madurez se estimó en 68.5 cm de
ancho de disco (AD) para los machos y 71.8 cm AD para las hembras y la talla mediana
de preñez en 84.3 cm AD. La fecundidad ovárica varió entre 1 y 44 folículos ováricos
y 6 folículos pre-ovulatorios vitelogénicos. La fecundidad uterina fue de un embrión por
hembra. El pico de ovulación y parto se presentó de mayo a julio, con tallas de
nacimiento entre 38.1 y 42 cm AD. R. steindachneri presentó un ciclo reproductivo
anual, continuo y sincrónico; la ovulación tiene una duración de tres meses y gestación
entre 10 y 14 meses. En relación con la estrategia de historia de vida, N. entemedor,
es una especie con crecimientor relativamente lento (khembra = 0.17 cm.año-1), tamaño
pequeño a mediano (talla máxima observada = 84 cm LT), edad de madurez temprana
VIII
(5.1 años, 34.5% de la edad máxima, en hembras), fecundidad máxima alta (24
embriones/hembra), talla de nacimiento pequeña (14.5 cm LT, 17.7% de la talla
asintótica) y edad máxima intermedia (hasta 14.8 años); se sugiere la presencia de un
trueque entre el tamaño pequeño de las crías al nacer con la alta fecundidad. R.
steindachneri es una especie con crecimiento intermedio (khembra = 0.21 cm. año-1),
tamaño mediano (talla máxima observada = 94.2 cm AD), edad de madurez temprana
(3.8 años, 38.8% de la edad máxima en hembras), fecundidad extremadamente baja
(un embrión/hembra), talla de nacimiento grande (42 cm AD, 44% de la talla asintótica)
y edad máxima relativamente baja (hasta 9.8 años). En ésta especie probablemente
se presenta un trueque entre la baja fecundidad y la talla de nacimiento grande. Ambas
especies son matrotroficas, N. entemedor es vivípara con saco vitelino e histotrofia
limitada, mientras que R. steindachneri presenta histotrofia lipídica definitiva, lo cual
podría estar relacionado con el tamaño de las crías al nacer. De acuerdo con el
esfuerzo reproductivo, N. entemedor invierte más energía en términos de fecundidad,
mientras que R. steindachneri invierte más energía en la masa corporal del embrión.
N. entemedor mostró valores de supervivencia (Sx = 0.73) y una tasa potencial de
aumento poblacional (r' = 0.48) mayor que R. steindachneri (Sx = 0.62, r' = -0.18). Los
resultados sugieren que la estrategia reproductiva así como de historia de vida de N.
entemedor permiten a esta especie mayor capacidad para responder a las presiones
del ambiente y ser menos susceptible a la explotación por pesca que R. steindachneri.
IX
GENERAL ABSTRACT
One of the most important aspects within the life history strategy of the species, is
based on the reproductive strategy, which aims to maximize the production of
reproductively active offspring to ensure the survival of the species through time. The
present study describes the reproductive strategy and the life history strategy of two
sympatric batoid species in Bahía de La Paz, BCS, Mexico, the Giant electric ray,
Narcine entemedor and the Pacific cownose ray Rhinoptera steindachneri. The study
was carried out with monthly samplings from October 2013 to March 2017, using
gillnets. N. entemedor females presented a median size at maturity of 58.5 cm of total
length (TL), median length at pregnancy of 63.7 cm TL and median size at maternity of
66.2 cm TL. The females presented between 1 and 69 ovarian follicles of elongated
shape, with maximum ovarian fecundity (fully developed vitellogenic follicles) of 17 and
uterine fecundity between 1 and 24 embryos per female. The size at birth was
estimated between 12.4 and 14.5 cm TL. Vitellogenesis occurred synchronously with
gestation. A continuous annual reproductive cycle was defined with an ovulation period
of five months, between May and September and a gestation period of five months,
with two parturition peaks, one in January-February and another in August-September
and with embryonic diapause in some individuals. The histological evidence of
secretions of the glandular tissue of the uterine villi indicates that this species probably
has limited histotrophy. For R. steindachneri, the median size at maturity was estimated
at 68.5 cm of disc width (DW) for males and 71.8 cm DW for females and the median
size at pregnancy in 84.3 cm DW. Ovarian fecundity varied between 1 and 44 ovarian
follicles and 6 pre-ovulatory vitellogenic follicles. The uterine fecundity was one embryo
per female. Ovulation peak and birth occurred from May to July, with birth sizes
between 38.1 and 42 cm DW. R. steindachneri presented an annual reproductive cycle,
continuous and synchronous; ovulation has a duration of three months and gestation
between 10 and 14 months. In relation to the life history strategy, N. entemedor, is a
relatively slow growing species (kfemale = 0.17 cm.year-1), small to medium size
(maximum observed size = 84 cm TL), early age at maturity (5.1 years, 34.5% of
maximum age in females), high maximum fecundity (24 embryos/female), small size at
birth (14.5 cm TL, 17.7% of asymptotic length) and intermediate lifespan (up to 14.8
X
years). There is probably a trade-offs between the small size at birth and high fecundity.
R. steindachneri is a species with intermediate growth (kfemale = 0.21 cm year-1),
medium size (maximum observed size = 94.2 cm DW), early age at maturity (3.8 years,
38.8% of maximum age in females), extremely low fecundityy (one embryo/female),
large birth size (42 cm AD, 44% asymptotic size) and relatively short lifespan (up to 9.8
years); this species probably presents a trade-off between low fecundity and large size
at birth. Both species are matrotrophic, N. entemedor is viviparous with yolk sac and
limited histotrophy, whereas R. steindachneri presents definitive lipid histotrophy, which
could be related to the size of the offspring at birth. According to the reproductive effort,
N. entemedor invests more energy in terms of fecundity, whereas R. steindachneri
invests more energy in the embryo body mass. N. entemedor showed survival values
(Sx = 0.73) and potential population increase rate (r' = 0.48) higher than R.
steindachneri (Sx = 0.62, r' = -0.18). The results suggest that N. entemedor presents a
greater capacity to respond to the pressures of the environment to which it is subjected,
while R. steindachneri is more susceptible to exploitation by fishing.
XI
GENERAL INTRODUCTION
Reproduction is one of the most important biological process that ensure the
survival of the species; it is reflected in the effort of parents to produce fertile offspring
(Awruch, 2016), which inherits the properties of the parents to ensure survival over time
(Muchlisin, 2014). To optimize reproductively active offspring, the species present
diverse reproductive strategies (Roff, 1992). A reproductive strategy is defined as the
general pattern of reproduction common among individuals of a species, with the
purpose of maximizing the reproductively active offspring in relation to the available
energy, through trade-offs to increase the life expectancy of the parents (Wootton,
1984; Roff, 1992; Pianka, 2000).
Elasmobranchs (sharks, skates, and rays) are a group that exhibits a large
number of reproductive modes (Musick & Ellis, 2005), as a result of evolution through
more than 416 million years, and probably its survival success over time is due to that
diversity in their reproduction (Carrier et al., 2004; Holden, 1973; Hoenig & Gruber,
1990). Within this group there are two main parity categories: viviparity and oviparity,
however, it is the viviparity that is represented in more species, approximately 55%
(Wourms, 1981, Gross & Shine, 1981). The viviparity includes two types of embryonic
nutrition: lecitotrophy and matrotrophy (Wourms, 1977, 1981; Compagno, 1990;
Wourms & Lombardi, 1992). The lecitotrophy is characterized by the dependence
exclusively of the yolk throughout all gestation; while in the matrotrophy, the embryonic
development depend initially on the yolk sac, and once the yolk reserve is completely
consumed, the mother will generate and provide the nutrients for the embryonic
feeding. These nutrients can be pass to the embryos from lipid histotrophy, limited
histotrophy, oophagy, intrauterine cannibalism or placentatrophy (Nakaya, 1975,
Gilmore, 1993; Wourms, 1994; Musick & Ellis, 2005).
The viviparity is a highly successful reproductive mode that has evolved over
time, allowing the maximization of the reproductive potential of the species. This is
reflected in the decrease of the production of offspring, females with largers body sizes,
an effective internal fertilization (through the clasper), the process of absorption of
nutrients of the yolk sac and those generated by the mother, and the intrauterine
XII
protection of the embryos (Wourms, 1981). According to Wourms & Lombardi (1992),
all these characteristics demand a high energetic cost of the mothers for the embryonic
development, however, these traits are compensated (through trade-offs) by other
characteristics that will give the species the possibility of surviving and leaving fertile
offspring through maximizing the energy available in the environment (Goodwin et al.,
2002). The characteristics that viviparity implies in elasmobranchs are balanced
through a variety of adaptations in their life history in order to compensate the costs
involved (Goodwin et al., 2002), responding differently to environmental pressures
which results in different life history strategies in the species (Cortés, 2004; Frisk,
2010).
The reproductive aspects of a species contribute greatly to the definition of its
life history. It is recognized that some reproductive aspects determine the productivity
of a species, as well as the resistance of a population to the exploitation or disturbance
of other human activities (Ricker, 1954; Hilborn & Walters, 1992). For example, a single
life history trait as age at maturity, may instead be a good indicator of vulnerability
because this trait is negatively correlated with population growth rate (Smith et al.,
1998; Musick, 1999; Cortés, 2002).
The life history of an organism is represented by its birth until its death,
describing the age, the patterns of maturity, reproduction, survival and finally death
(Braendle et al., 2011). There are several theories that explain such strategies, such
as the r-K selection theory (Dobzhansky, 1950), bed hedging (Stearns, 1976),
triangular life history model (Winemiller & Rose, 1992), age-specific models (Stearns,
1992), among others. According to the r-K theory, the elasmobranchs will tend to
present traits more towards to K type (large body sizes, low fecundity, slow growth,
small offspring size and high lifespan), compared to teleost fish (Holden,1972).
However, due to the great diversity in life history traits within the elasmobranchs,
various theories have been proposed to explain the strategies that make them up
(Compagno, 1990; Cortés, 2000, 2004; Frisk et al., 2001, 2005; Goodwin et al., 2002;
Winemiller, 2005; Frisk, 2010).
XIII
The analysis of life-history parameters is the first step to understanding a
species’ life-history strategy and to provide assessment tools for a sustainable fishery
(Frisk et al., 2001), such as the Giant electric ray, Narcine entemedor and the Pacific
cownose ray, Rhinoptera steindachneri.
The present thesis focuses on two matrotrophic viviparous species with different
embryonic nutrition modes, N. entemedor (Fig. 1a), which presents yolk sac and limited
histotrohy (Burgos-Vázquez et al., 2017), and R. steindachneri (Fig. 1b), which
possesses secretory villi (trophonemata) and definitive lipid histotrophy (Muick & Ellis,
2005).
Considering that both species are viviparous, the main research questions of
this study derive from the way in which the mother invests the energy towards the
development of her offspring. Does the mode of embryonic nutrition have an influence
on the gestation period? How is the large amount of energy invested by the mothers of
R. steindachneri compensated for the embryonic development? Which of these two
species has greater reproductive effort? And based on that reproductive effort, which
could be less effective in the face of recovery from an overfishing event? Could the
mode of embryonic nutrition be related to other life history traits of these two species?
In order to answer these questions, the main goal of this study was to define the
reproductive strategy of N. entemedor and R. steindachneri, to contrast them and
define the life history strategy of each species. Based on this information, I defined a
priori which species could be more susceptible to anthropogenic events as fishing.
This study was divided into three chapters: chapter I defines the reproductive
strategy of N. entemedor; chapter II defines the reproductive strategy of R.
steindachneri; and chapter III defines and compares the life history strategies of both
species using the information reported on chapters I and II and from information
reported on the literature (reproductive, age, growth and population parameters and
reproductive effort).
XIV
Figure 1.- Batoid species of the study collected in Bahía de La Paz, BCS, México. a) Narcine
entemedor (mature male), b) Rhinoptera steindachneri (female neonate).
a
b
XV
BACKGROUND
There are a few comparative studies related to the life history strategies in
elasmonbranchs. Cortés (2000) used life history traits to define life history strategies in
sharks. Specifically for batoids, Frisk (2010) analyzed the life history traits, using
published information about longevity, maturity, growth and reproduction of the most
studied species worldwide, making a global description about life history strategy.
Comparative studies on the reproductive aspects of batoids are also limited.
Yokota & Lessa (2007) described the reproductive biology of three species of the order
Myliobatiformes (Gymnura micrura, Dasyatis guttata and D. marianae), in Northeastern
Brazil, estimating maturity sizes, birth sizes, and characterization of the reproductive
structure. Collonelo (2009) analyzed the reproductive ecology of three species
(Atlantaroja castelnaui, Rioraja agassizi and Zapteryx brevirostris), through macro and
microscopic analysis, sex ratio, maturity size and reproductive cycle. However, this
study focused on estimating and describing the reproductive aspects of each of the
target species, without evaluating interspecific differences through mathematical
models nor comparisons.
Studies about the reproductive biology of batoids in the Gulf of California are
few, and those focused on the life history are even more limited. The only studies are
based on the species Rhinobatus productus (Máquez-Farías, 2007a, 2007b, 2011).
However, most studies on batoids in this region are focused on the description of the
fishery and although there is not official fishery statistical on the captures or population
studies of N. entemedor and R. steindachneri, both species represent resources
frequently captured by artisanal fisheries on both coasts of the Gulf of California
(Márquez-Farías, 2002; Bizzarro et al., 2007; Bizzarro et al., 2009a, 2009b; Smith et
al., 2009; Salomón-Aguilar, 2015; Saldaña-Ruiz, 2017).
Specifically, N. entemedor, for the Gulf of California, has no studies focused on
the reproductive biology or the characterization of the life history strategy, there are
only recent studies on age and growth (Mora-Zamacona, 2017) and feeding habits
(Cabrera-Meléndez, 2017). Other studies about its biological (age and growth,
reproduction and diet) and taxonomic aspects, have been carried out in other areas of
XVI
Mexico (Valadez-González, 2000; Villavicencio-Garayzar, 2000; De La Cruz-Torres,
2017).
Villavicencio-Garayzar (2000) reported for N. entemedor in Bahía Almejas (west
coast of BCS, Mexico) a maximum length of 93 cm in total length (TL), sex ratio of 11.5
females for each male, size at birth between 14 and 16 cm TL, growth rate of 0.372
cm.year-1, size at first maturity between 62 and 63 cm TL, maximum age of 15 years,
maximum fecundity of 20 embryos per female, annual reproductive cycle and
embryonic diapause as a reproductive tactic.
Regarding R. steindachneri, Villavicencio-Garayzar (1996) recorded for both
coasts of the peninsula of Baja California a maximum disc width (DW) of 108 cm in disc
width (DW), size at first maturity of 85 and 70 cm DW for females and males,
respectively, only the ovary and oviduct right were functional, fecundity of one embryo
per female, sizes at birth of 40 to 44 cm DW and gestation period of 10 to 11 months.
Bizzarro et al. (2007), carried out a study for this species in the northern zone of the
Gulf of California, registering a maximum size of 98 cm DW, median size at maturity in
females of 70.2 cm DW and in males of 69.9 cm DW, fecundity of one embryo per
female, size at birth between 38 and 45 cm DW and gestation period of 11 to 12
months. Flores-Pineda et al. (2008) in Bahía Almejas, recorded a maximum size of 105
cm DW, first size at maturity in both sexes of 75 cm DW, one embryo per female, only
the right oviduct was functional and a gestation period of 11 to 12 months. Carrillo-
Colín (2015) for the southeast of the Gulf of California, recorded a maximum size of
96.9 cm DW, sex ratio of one female for each male, a growth rate of 0.118 cm.year-1
for combined sexes and maximum age of 13 years.
The reproductive effort of a fish population has been evaluated in different ways
(Parsons, 1993, Gunderson, 1997, Haag, 2013, Husey et al., 2010, Damon et al.,
2016). However, this is the first study that uses the method proposed by Acuña et al.
(2001), to contrast the effort that N. entemedor and R. steindachneri output in terms of
reproduction, starting on the theoretical basis in which the reproductive effort has a
direct relationship with other traits of life history (Gunderson, 1997), which is necessary
to fulfill one of the objectives of this thesis.
XVII
Both species are referred to in the Red List of Threatened Species of the
International Union for the Conservation of Nature (IUCN) in different categories, N.
entemedor is referred to as a species with Data Deficient, while R. steindachneri is
classified as Near Threatened species. This justifies the need to generate baseline
scientific information for the formulation of adequate management plans for the
sustainable use of both resources. In this sense, knowing the life history strategy,
allows to integrate the necessary biological data to describe the demography of a
species which is necessary to understand the population dynamics and establish
appropriate assessment that allow the population to recover from overfishing (Walker
& Hislop, 1998; Cortés, 1998; Heppell et al., 1999; Brewster-Geisz & Miller, 2000;
Caswell, 2001; Cortés, 2002; Frisk et al., 2002; Mollet & Cailliet, 2002; Frisk et al., 2005;
Serra-Pereira et al., 2015). Other population parameters that need less biological
information, can be useful to evaluate the risk that a population faces to anthropogenic
factors, as it is the case of the potential rate of population increase r', proposed by
Jennings et al. (1998), which has been estimated for elasmobranchs by Frisk et al.
(2001). However, this rate has not been estimated for any batoid species for the Gulf
of California and could be useful due to the lack of biological information for this group
in this area.
1
CHAPTER I
REPRODUCTIVE STRATEGY OF THE GIANT ELECTRIC RAY Narcine
entemedor IN THE SOUTHERN GULF OF CALIFORNIA
María I. Burgos-Vázquez1, Paola A. Mejía-Falla2, Víctor H. Cruz-Escalona1 & Nancy J.
Brown-Peterson3
1Instituto Politécnico Nacional-Centro Interdisciplinario de Ciencias Marinas., Avenida I.P.N. s/n, Colonia Playa Palo de Santa Rita, Apartado Postal 592, C.P. 23096 La Paz, Baja California Sur,
México. 2Fundación Colombiana para la Investigación y Conservación de Tiburones y Rayas, SQUALUS,
Carrera 64A No 11A-53, Cali, Colombia. 3Center for Fisheries Research and Development, School of Ocean Science and Technology, The
University of Southern Mississippi, 703 East Beach Dr., Ocean Springs, MS 39564, USA.
Burgos-Vázquez M. I., P. A. Mejía-Falla, V. H. Cruz- Escalona & Brown Peterson, N. J. 2017.
Reproductive Strategy of the Giant Electric Ray in the Southern Gulf of California. Marine and Coastal
Fisheries, 9(1): 577-596. DOI: 10.1080/19425120.2017.1370042.
2
ABSTRACT
The objective of the present study was to describe and characterize macroscopic and
microscopic aspects of the reproductive biology of the Giant electric Ray Narcine
entemedor, a viviparous elasmobranch targeted by commercial fishers in Mexico. A
total of 305 individual rays were captured (260 females, 45 males); all males were
sexually mature. The median size at maturity for females was estimated to be 58.5 cm
TL, the median size at pregnancy was 63.7 cm TL, and the median size at maternity
was 66.2 cm TL. The range of ovarian follicles recorded per female was 1–69; the
maximum ovarian fecundity of fully grown vitellogenic oocytes was 17, and uterine
fecundity ranged from 1 to 24 embryos per female. The lengths of the oblong ovarian
follicles varied significantly among months, and the largest ovarian follicles were found
in July, August, and September. Median embryo size was largest in August, and the
size at birth was between 12.4 and 14.5 cm TL. Histological evidence of secretions
from the glandular tissue of the uterine villi indicate that this species probably has
limited histotrophy as a reproductive mode. Vitellogenesis in the ovary occurred
synchronously with gestation in the uterus. Narcine entemedor has a continuous annual
reproductive cycle; a period of ovulation occurs between May and September and two
peaks of parturition, one in January and one in August, occur, suggesting that
embryonic diapause occurs in some individuals. These results provide useful
information for the management of this important commercial species in Bahía de La
Paz, Mexico, and will allow possible modification of the current Mexican regulations to
enable better protection of this species.
3
1.1 INTRODUCTION
The elasmobranchs (sharks, skates, and rays) present diverse modes of
reproduction that have contributed to the success of this group for more than 400 million
years (Helfman et al., 1997). Wourms (1981) proposed two reproductive modes, based
on the type of embryonic nutrition: lecithotrophy, where the embryos depend
exclusively on yolk, and matrotrophy, where in addition to yolk the mother secretes
other nutritious substances. Additionally, the group has developed diverse reproductive
tactics, and some species exhibit embryonic diapause (i.e., an interruption of embryonic
development during gestation as defined by Simpfendorfer, 1992), sperm storage in
females (Pratt, 1993; Pratt & Carrier, 2001; Waltrick et al., 2014), and selection of
specific sites for parturition. Such sites are known as nursery areas that provide
protection against predators to ensure better chances of survival of their offspring
(Hueter et al., 2004).
The electric rays (Order Torpediniformes) have been defined as yolk sac
viviparous (lecithotrophs; Ranzi 1932, 1934; Hamlett et al., 2005). However,
Villavicencio-Garayzar (2000) reported that the Giant Electric Ray Narcine entemedor,
in the lagoon complex of Bahía Almejas, Baja California Sur, Mexico, has a viviparous
matrotrophic reproductive mode and exhibits embryonic diapause. Several species in
this order have two functional ovaries and uteri, e.g., Ocellated Torpedo Torpedo
torpedo (Capapé et al., 2000), Pacific Electric Ray T. californica (Neer & Cailliet, 2001),
and Lesser Electric Ray N. brancoftii (Moreno et al., 2010), and all species in the order
lack the oviducal gland (Prasad, 1945).
The Giant Electric Ray is distributed from the Bahía Magdalena, on the west
coast of Baja California Sur, including the Gulf of California, southward to Peru
(Robertson & Allen, 2015). This is one of the species most frequently captured by
artisanal fishers in northwestern Mexican waters and is opportunistically fished
throughout the year (Villavicencio-Garayzar, 2000; Márquez-Farías, 2002). However,
the agency responsible for management of sharks and batoids in Mexico (INAPESCA,
acronym in Spanish) established a closed season between May 1 and July 31
prohibiting the capture of elasmobranchs in the Mexican Pacific Ocean and sharks in
4
the Gulf of Mexico (Official Mexican Standard for Fishing, NOM-029-PESC- 2006; DOF,
2007, 2012).
Considering that biological information of populations of commercial importance
is essential to ensure effective management of these species (Cortés, 2004; Walker,
2005; Lowerre-Barbieri et al., 2011; Dulvy et al., 2014; Simpfendorfer & Wetherbee,
2015), the objective of this study was to evaluate reproductive aspects of the N.
entemedor in Bahía de La Paz, Baja California Sur, Mexico. Specifically, we present
information on the reproductive mode, tactics, and cycle as well as estimates of
characteristics related to maturity, gestation, and ovulation.
1.2 MATERIAL AND METHODS
1.2.1 Study site and collection of specimens
Monthly collections of N. entemedor were made from October 2013 through
December 2015 in the southern zone of Bahía de La Paz, located in the southern
portion of the Gulf of California (24°25′17.55″N, 110°18′31.64″W), in three different
fishing grounds: El Morrito, El Quelele, and Campo Rodríguez (Fig. 1). Bahía de La
Paz is isolated from the majority of the hydrological processes in the Gulf of California
(Salinas-Gonzáles et al., 2003). Mean annual water temperatures vary from 15°C to
22°C, and mean salinity is 35‰ but can increase during summer due to intense
evaporation and little freshwater inflow (Villaseñor, 1979; Salinas-González et al.,
2003).
The rays were captured by artisanal fishers using monofilament gill nets (100 m
long, 1.5 m high, 8–10 in stretch mesh) traditionally called chinchorros, which are set
in the afternoon at depths between 10 and 40 m over sandy bottoms and recovered the
next morning. Each fish was measured for TL (cm), weighed (total mass [TW]) and
eviscerated mass [EW], ±0.01 kg) and the sex determined. For males, the inner clasper
length (CL, cm), the grade of calcification of the clasper (calcified, partially calcified, not
calcified), and the presence or absence of semen was recorded. Gonads were
5
macroscopically staged to define maturity, weighed (gonad mass [GW], 0.001 g), and
fixed in 10% buffered formalin.
1.2.2 Sex ratio, length, and mass
The sex ratio of adults and juveniles (combined) and embryos was evaluated
with a chi-square test to determine whether it differed from 1:1 (Sokal & Rohlf, 1998).
Differences in the length and mass between males and females (excluding the mass
of pregnant females) were evaluated using a Mann–Whitney U-test. Data were tested
for normality and homogeneity of variances prior to analysis with Kolmogorov–Smirnov
and Lilliefors tests, respectively. All differences were considered significant if P < 0.05.
Figure 1.- Study area (Gulf of California and Bahía de La Paz, Mexico) including primary sampling locations for Narcine entemedor (star = El Morrito, diamond = El Quelele, square= Campo Rodriguez).
6
1.2.3 Macroscopic observations of reproductive structures and maturity
Maturity of males was defined following the criteria proposed by Neer & Cailliet
(2001) and Moreno et al. (2010) and adapted for N. entemedor based principally on the
development of the testes, the presence–absence of testicular lobes, and the
presence–absence of semen (Table 1). Each testis was measured (length and width,
±0.001 cm); differences between the length were evaluated using a Student’s t-test,
and width of the left and right testis were evaluated using a Wilcoxon test. Additionally,
the relationship between the inner length of the clasper as a function of TL was plotted.
Maturity of females was evaluated following the criteria of Martin & Cailliet
(1988), Abdel-Aziz (1994), Villavicencio- Garayzar (2000), Moreno et al. (2010), Mejía-
Falla et al. (2012), and Rolim et al. (2015) adapted to specific characteristics of N.
entemedor. We defined four phases considering macroscopic characteristics of both
ovary and uterus as well as the maturity indices for each independently evaluated
structure (Table 2). The width, length (±0.001 cm), and mass (±0.001 g) of each ovary
was recorded, and the ovarian follicles were removed. The anterior oviducts and the
uterus were removed and measured (width, ±0.001 cm) and the presence of ovarian
follicles in the anterior oviduct (completely vitellogenic as evidence of ovulation) and
embryos in the uterus were recorded. The length of each villi in the uterus was
measured (±0.001 cm), and the abundance of villi was evaluated as few (≤ 50 villi) or
abundant (≥ 51 villi). The differences in the length of the right ovary and the width of
the right uterus by maturity phase were evaluated using a Kruskal–Wallis (KW) test for
independent samples. Differences in the length and width between the right and left
reproductive structures (ovary and uterus) of females were assessed using a Wilcoxon
paired test.
7
Table 1. Macroscopic and microscopic descriptions of the male reproductive structures defined to distinguish maturity stages of
Narcine entemedor.
Testes Seminal vesicle
Maturity Phase
(Maturity index)
Testes Index
Macroscopic condition Microscopic condition Seminal vesicle Index
Macroscopic condition Microscopic condition
Immature -Developing
(2) 2
Presence of testicular lobules and up to 30% of
epigonal tissue
Presence of testicular
lobules with spermatogonia and primary and secondary
spermatocytes
2 Slightly differentiated
from vas deferens and not coiled
Without spermatozoa or seminal fluid
Mature - Spawning
Capable (3) 3
Presence of testicular lobules and up to 10% of
epigonal tissue
Presence of testicular lobules with spermatogonia and primary and secondary spermatocytes, spermatids
and spermatozoa
3 Differentiated vas
deferens, thickened Without spermatozoa or
seminal fluid
Mature - Actively
spawning (4)
4 Presence of testicular
lobules and up to 10% of epigonal tissue
Presence of testicular lobules with spermatogonia and primary and secondary spermatocytes, spermatids
and spermatozoa
4 Differentiated vas
deferens, thickened With sperm and seminal
fluid
8
Table 2. Macroscopic and microscopic descriptions of the female reproductive structures defined to distinguish maturity stages of
Narcine entemedor.
Ovarys
Uterus
Maturity Phase (Maturity index)
Ovary index
Macroscopic condition
Microscopic condition
Uterus index
Macroscopic condition
Microscopic condition
Immature – Early Developing (2) 2
With translucent ovarian follicles ≤ 5
cm long and abundant ovarian
stroma
With primary growth and previtellogenic ovarian follicles and abundant epigonal
tissue
2
Little differentiation in anterior oviducts, short
uterine villi (<1 cm), abundant and wide
between 0.2 and 1.2 cm.
Uterine villi of simple cylindrical epithelium, few blood vessels and little blood segregation
from the basal endometrium. Lacks
glandular epithelium, no histotroph secretions.
Mature – Not pregnant (3)
Developing
3
4
With yellow ovarian follicles and lengths between 10-17 cm, little ovarian stroma
With vitellogenic ovarian follicles ≥ 17.01 cm in length
and no ovarian stroma
With primary growth, previtellogenic and
primary and secondary vitellogenic ovarian
follicles.
With primary growth, previtellogenic, and
primary, secondary and tertiary vitellogenic
ovarian follicles
3
Without eggs or embryos, with uterine
villi throughout the endometrium, slightly
narrow and well differentiated from anterior oviducts. Widths > 1.3 cm
Uterine villi of cylindrical stratified epithelium, with
glandular tissue in the periphery of the villi; one blood vessel central in the uterine villi. Small quantity of histotroph
secretion.
Spawning Capable
Mature - Pregnant (4)
Actively Spawning
4
With vitellogenic ovarian follicles ≥ 17.01 cm in length
and no ovarian stroma
With primary growth, previtellogenic, and
primary, secondary and tertiary vitellogenic
ovarian follicles
4A
With eggs, lacking egg capsule, thick and
abundant uterine villi. There can be ovarian
follicles descending into the anterior oviduct.
Width > 1.7 cm
Embryos in formation
3 4B
Lacking egg capsule, uterine walls thick with abundant uterine villi, > 2 cm wide. Embryos < 3
cm.
9
Early embryos
4C
Lacking egg capsule, uterine walls thick with abundant uterine villi, >
2 cm wide. Embryos 3.1-5.8 cm.
Mid embryos
4
4D
Lacking egg capsule, elongated uterine villi (> 1 cm), muscular walls beginning to thin, vascularized, width > 2.4 cm. Embryos 5.9-12.3 cm.
Late embryos
4E
Muscular walls thin and transparent, highly vascularized, width > 2.4 cm Embryos > 12 cm and no yolk sac.
Mature—Regressig (5)
5
With translucent ovarian follicles ≤ 5
cm long and abundant ovarian
stroma and presence of postovulatory
folliciles
With primary growth and previtellogenic and
vitellogenic ovarian follicles and abundant epigonal tissue, atretic
oocytes and corpus leutum can be present
5
Without eggs or embryos, with uterine villi throughout the endometrium, slightly narrow, thick and well differentiated muscular walls of anterior oviducts
Uterine villi composed of stratified cylindrical epithelium and glandular tissue on the periphery of villi; A central blood vessel in each uterine villi. No histotroph present
10
1.2.4 Histological analysis
Histological processing followed Burgos-Vázquez (2013) and consisted of
successive changes of ethanol at increasing concentrations from 70% to 100%,
followed by clearing and infiltration with paraffin in a tissue processor. Tissues were
embedded in paraffin, and transverse and longitudinal sections (3–5 μm) of the ovary,
anterior oviduct, and uterus of females and testis and seminal vesicles of males were
stained using hematoxylin and eosin.
To define male maturity, the process of spermatogenesis in the testes and
seminal vesicles was examined following that defined by Maruska et al. (1996), ICES
(2010), and Brown- Peterson et al. (2011). Males were considered mature when
primary spermatocytes were present in the testis (Brown-Peterson et al., 2011). For
females, the description of oogenesis followed that defined by ICES (2010) and Brown-
Peterson et al. (2011). Slides were examined using a Nikon Eclipse 50i compound
microscope and photographed with a DXM 1200C camera using ACT-1C software.
1.2.5 Median size of maturity, pregnancy, and maternity
The median size at maturity (TL50) for females was calculated using a logistic
regression model with binomial data (0, immature; 1, mature; Table 2) with the equation
P𝑖= (1+𝑒-(a+b*TLi))-1
where pi is the fraction of mature individuals at TL, a and b are model parameters, and
a/b corresponds to the median size of maturity (Mollet et al., 2000). Females were
considered mature if ovaries were classified in the mature–not pregnant (3), mature–
pregnant (4), or regressing (5) phases (Table 2) or if the uterus showed signs of
development (indices 3, 4A, 4B, 4C, 4D, 4E, or 5; Table 2). Similarly, the median size
at pregnancy (TLP50) was calculated using binomial data, where a value of 1
corresponded to females regressing or with eggs or embryos in the uterus (indices 4A,
4B, 4C, 4D, 4E; Table 2), and a value of 0 was assigned to nonpregnant females
(indices 2 and 3, or 5; Table 2). For the median size at maternity (TLM50), females were
considered as maternal (1) if they would have produced a litter the next season, if not
captured, and contained follicles ≥ 16 cm length in the months of May to December,
11
and nonmaternal (0) if they would not have contributed offspring the next season.
These values were defined from analysis of the reproductive cycle following Walker
(2005) and Mejía- Falla et al. (2012).
1.2.6 Fecundity and reproductive cycle
Ovarian follicles were counted and measured for length (±0.001 cm). The
embryos were sexed (male, female, or undetermined), measured (TL, cm), and
classified ontogenetically based on morphological characteristics following Braccini et
al. (2007) and modified for N. entemedor. We defined four stages of embryonic
development: (1) in formation (embryos ≤ 3.0 cm TL with the presence of external
branchial filaments, without pectoral or pelvic fins, with no coloration, and a complete
and large yolk sac); (2) early development (embryos 3.1–5.8 cm TL with defined
pectoral fins, external branchial filaments present, no coloration pattern, and a large
yolk sac; (3) mid-development (embryos 5.9–12.3 cm TL without external branchial
filaments, small yolk sac, defined pelvic and pectoral fins, and the beginning of
coloration patterns on the skin); and (4) Late development (embryos ≥ 12.4 cm TL with
developed fins, no yolk sack, and defined coloration; Table 2).
Three different groups of ovarian follicles based on their size were defined: small
(previtellogenic, sizes ranging from 0 to 5.9 cm in length), medium (early vitellogenesis,
sizes between 6 and 15.9 cm in length), and large (late vitellogenesis, sizes ≥ 16 cm in
length). The mean and maximum number of ovarian follicles by group were estimated,
and differences among them were evaluated using a Kruskal– Wallis test. Ovarian
fecundity was defined considering only large ovarian follicles. Ovarian and uterine
fecundity were estimated using range, mean, and mode of the number of large ovarian
follicles in the ovary and the number of embryos in the uterus, respectively (Pratt, 1979).
We used linear regression to analyze the relationships between ovarian
fecundity or uterine fecundity with TL. For females that presented ovarian follicles and
embryos, the relationship between the length of the ovarian follicles and the size of the
embryos by ontogenic stage was compared in order to evaluate the synchrony (or
asynchrony) in follicular (vitellogenesis) and embryonic growth (gestation).
12
The reproductive cycle was evaluated in three complementary ways. First, we
examined the monthly variation of the largest ovarian follicles (in length) and of the
embryo size to define the months of ovulation and parturition, respectively (Walker
2005; Mejía-Falla et al., 2012). For the embryo development period, embryos ≤ 5 cm
TL, belonging to pregnant females with a fecundity equal to one, were eliminated to
rule out possible sampling errors (aborts or retarded growth). A Kruskall–Wallis test
followed by a nonparametric Tukey post hoc test with Bonferroni adjustment (Siegel &
Castellan, 1988) was used to determine differences among months. Secondly, the
gonadosomatic index (GSI), calculated as GSI = GW/EW × 100 (Gherbi-Barre, 1983),
was used. Monthly differences in GSI were evaluated using a Kruskal–Wallis test, with
differences among months evaluated with a nonparametric Tukey test. Finally, the
percentage of females in each reproductive phase was examined monthly across the
year.
1.3 RESULTS
1.3.1 Sex ratio, length, and mass
We analyzed a total of 305 N. entemedor (260 females and 45 males), resulting
in a female: male sex ratio for juveniles and adults combined of 5.7:1, which was
significantly different than the expected 1:1 ratio (χ2 = 151.55, df = 1, P < 0.001).
Females were present during all collection months and in greater abundance than
males. Males were not collected in January, April, or June.
Females ranged in size from 48.5 to 84.0 cm TL (mean ± SD, 65.9 ± 7.1 cm) and
males from 41.5 to 58.5 cm TL (51.4 ± 4.4 cm). Females were significantly larger (Z =
9.95, P < 0.001) and heavier (Z = 9.96, P < 0.001) than males.
1.3.2 Male reproductive structures and reproductive phases
Both testes in all males examined were functional, of an oval form, completely
covered by the epigonal organ, and suspended in the thoracic cavity by mesenteries.
There was no significant difference in the length of the left and right testis (t = 0.142, df
= 16, P = 0.889), but the left testis was significantly wider (1.87 ± 0.39 cm) than the
13
right testis (1.75 ± 0.40 cm; Z = 2.762, P = 0.005). The claspers of all males examined
were completely calcified, and the relationship between inner clasper length and male
TL appeared to be linear (Fig. 2).
Figure 2.- Relationship between TL (cm) and inner clasper length (cm) of male Narcine entemedor.
All testes analyzed histologically (n = 20) had spermatocysts with different
stages of spermatogenesis, with spermatogonia, primary spermatocytes, and
secondary spermatocytes present in the spermatocysts (Fig. 3a) and were considered
sexually mature. Testes containing late stages of spermatogenesis, including
spermatids and spermatozoa (Fig. 3b), were identified as capable of mating (with
mature spermatozoa in the testis) or actively mating if the seminal vesicles contained
spermatozoa and seminal fluid. The seminal vesicles of these males also contained
aggregations of spermatozoa packets of the spermatozeugmata type, with the sperm
heads orientated toward the center of the packet and the tails along the margins (Fig.
3c). Males in the mating-capable and actively mating reproductive phases were present
during all months that males were captured. However, males in the actively mating
phase dominated during July (28.9%), August (22.2%), and September (20%).
6
7
8
9
10
11
12
40 45 50 55 60
Inn
er
cla
sp
er
len
gth
(c
m)
Total length (cm)
14
Figure 3.- Micrographs of the reproductive structures of male Narcine entemedor of a) Longitudinal section of testes in early stages of spermatogenesis in an actively mating male, b) Longitudinal section of testes in the actively mating phase and c) Longitudinal section of the seminal vesicle of a male in the actively mating phase. The dotted line indicates packets of spermatozoa in a spermatozeugmata. Abbreviations are as follows: Sg = spermatogonia, Sc1 = primary spermatocytes, Sc2 = secondary spermatocytes, St = spermatids, Sz = spermatozoa.
The size at sexual maturity could not be calculated for male, no immature
specimens were captured in this study. The smallest male captured (41.5 cm TL) had
spermatozoa in the testis and semen in the claspers and was actively mating.
1.3.3 Female reproductive structures
All females evaluated (n = 240) had functional ovaries and uterus and the
oviducal glands were absent. There was no significant difference between the length
of the right and left ovary (Z = 0.92, df = 141, P = 0.355), but the left ovary (median =
2.85 cm) was significantly wider than the right ovary (median = 2.60 cm; Z = 2.39, df =
15
136, P = 0.016). There was a significant difference between the length of the right ovary
and maturity reproductive phase (KW (3, 152) = 68.72, P < 0.0001), during which females
in the ovulation capable phase had longer ovaries (median = 5.5 cm).
Both ovaries were in the anterior portion of the thoracic cavity and suspended
by the mesovarium. Ovaries are conical, and in the more advanced phases of
development, follicles filled the entire ovary with little ovarian stroma remaining. All
females had ovarian follicles in both ovaries. Ovarian follicles have an elongated form
in which the germinal zone is located at the semispherical base and the top of the
follicle is slightly pointed (Fig. 4). Ovarian follicles of different lengths (0.02– 31.8 cm)
were present in the same ovary.
Figure 4.- Ovarian follicles (top) and ovary (without the covering tissue) of a mature female Narcine entemedor in the ovulation capable phase.
Histological analysis of the ovaries showed different gametogenic stages
corresponding to the different reproductive phases. A germinal zone is evident in the
periphery of the ovary near the vertebral column, and ovarian follicles are embedded
in ovarian stroma, which is associated with the epigonal organ. The ovary is surrounded
by a peritoneal epithelium composed of simple cylindrical tissues and collagen fibers
16
(Fig. 5a). Histological analysis permitted differentiation of the follicular epithelial layers
surrounding the oocyte, i.e., the zone pelucida, theca interna, and theca externa (Fig.
5b).
There were no significant differences between the width of the left and right
anterior oviduct (Z = 0.95, df = 133, P = 0.338). There were significant differences in
oviduct width during different reproductive phases (KW (4, 161) = 41.112, P < 0.0001);
ovulation capable females had the greatest width (median = 0.45 cm) compared with
other phases. The anterior oviducts have a tubular form and are connected to the
anterior portion of the uterus by a slight widening of the basal portion of the oviduct.
The oviduct is connected to the upper portion of the thoracic cavity by an ostium near
the corner of the mouth of the esophagus. Microscopic analysis of the basal zone of
the oviducts showed several disperse tubules, similar to oviductal tubules in the
oviducal gland. However, the oviduct lacked the plates and secretory ducts
characteristic of oviducal glands, and there was no evidence of spermatozoa in the
oviduct (Fig. 5c).
There were no significant differences between the left and right uteri in length (Z
= 0.313, df = 162, P = 0.754) or width (Z = 1.232, df = 147, P = 0.217). However, there
were significant differences among reproductive phase and uterus width (KW (4, 182) =
91.530, P < 0.0001); females with a uterine index of 4A–E had a greater width (median
= 4.45 cm) than females with a uterine index of 2, 3, or 5.
Uteri with an index of 2 had thin muscle walls with short and abundant uterine
villi, and the anterior oviducts were not completely differentiated. Uteri with an index of
3 had a thick layer of muscle covered by a serosa layer with abundant and short uterine
villi. In uteri of females in the mature–pregnant phase, the muscular tissue expanded
leaving only the serosa layer, which had a venous system originating in the anterior
part of the uterus, and the uterine villi were longer and more dispersed with an
expansion of the endometrium. The amount of uterine villi varied with uterine index;
indices 4A, 4B, and 4C presented abundant uterine villi, while few uterine villi were
present in uteri with indices 4D and 4E. At the microscopic level, the uterus was
composed of muscle fibers under a layer of connective tissue in all reproductive
17
phases, but the uterine villi changed structurally according to the reproductive phase
(Fig. 5d, e, f, g). Uterine villi in uteri having indices of 2 and 3 were composed of simple
cubical tissue with a main blood vessel (Fig. 5e), while villi in those with indices 4A, 4B,
4C, and 4D were composed of stratified cylindrical tissue of approximately six layers of
cells (Fig. 5h). Finally, the lumen of the uterus of the pregnant females showed
acidophilic secretions from the secretory crypts of each uterine villi (Fig. 5h).
18
Figure 5.- Micrographs of reproductive structures of female Narcine entemedor of a) Transverse section of an ovary in the immature developing phase (ovarian index 2), b) Transverse section of a vitellogenic oocyte from a female in the ovulation-capable phase (ovarian index 3), c) Transverse section of the anterior oviduct of a female in the ovulation-capable phase with oocytes in the uterus, d) Transverse section of the uterus of an immature developing female (uterine index 2), e) Transverse section of the uterine villi from a female in the immature developing phase (uterine index 2), f) Transverse section of the uterus from a female in the mature-not-pregnant phase (uterine index 3), g) Transverse section of the uterus from a female with late-stage embryos (uterine index 4E), h) Longitudinal section of the uterine villi from a pregnant female with late-stage embryos (uterine index 4E) and secretory crypts (C, i.e., within dotted line). Abbreviations are as follows: Ep = peritoneal epithelium, CA = cortical alveolar oocytes, PG = primary growth oocytes, Os = ovarian stroma, Zp = zona pelucida, Fe = follicular epithelium, Te = theca externa, V = granules of vitellogenin, Lo = oviductal lobules, l = lumen, Po = oviductal plates, Vll = uterine villi, BV = blood vessel, Esc = simple cylindrical
epithelium, Sta = stratified cylindrical epithelium, and H = histotroph.
19
1.3.4 Female size at maturity, pregnancy, and maternity
Immature, developing females (n = 47, 18.1% of total) ranged in size from 48.5
to 69.0 cm TL, while mature females (n = 213, 81.9% of total) in the mature–not
pregnant, mature– pregnant, and mature–regressing phases ranged in size from 54.5
to 85.0 cm TL. The largest immature female (69.0 cm TL) had undeveloped ovaries,
ovarian follicles < 4.4 cm long, and thin uteri with very short uterine villi (uterine index
2). However, a smaller (54.5 cm TL), sexually mature female had ovarian follicles
completely developed (24.5 cm in length) and narrow but completely differentiated uteri
with abundant uterine villi (uterine index 3).
The TL50 in females based on maturity index (considering all structures) together
was estimated at 58.5 cm TL (95% CI: 51.7–65.4; Fig. 6a), very similar to values
estimated by considering only ovarian development (58.8 cm TL; 95% CI: 52.3–65.3;
Fig. 6b) or uterine development (59.0 cm TL, 95% CI: 53.4–64.7; Fig. 6c). Pregnant
females ranged from 55.0 to 84.0 cm TL and had a TLP50 estimated at 63.7 cm TL
(95% CI: 58.9–68.4; Fig. 6d). Maternal females ranged from 55.0 to 81.0 cm TL, and
the TLM50 was estimated at 66.2 cm TL (95% CI: 61.9–70.5; Fig. 6e).
20
Figure 6.- Maturity ogives for female Narcine entemedor to TL (cm). a) Ogive by maturity phase, b) Ogive based on ovary condition, c) Ogive based on uterus condition, d) Pregnancy ogive and e) Maternity ogive.
1.3.5 Ovarian and uterine fecundity
All females analyzed had ovarian follicles in both ovaries, varying in number from
1 to 69 per female (mean ± SD, 23.6 ± 15.8; mode = 14) and between 1 and 46 follicles
per ovary (right: 12.8 ± 8.3, mode = 10; left: 13.6 ± 8.8, mode = 8). There were no
significant differences in the number of ovarian follicles between the left and right
ovaries (Z = 0.90, df = 151, P = 0.365).
21
There were significant differences among number of ovarian follicles by size-
groups (KW(2, 168) = 64.63, P < 0.0001). The small group presented the highest mean
and maximum number of ovarian follicles (16.8 and 62, respectively), the medium
group presented a mean of 4.7 and maximum of 26, and the large group presented a
mean of 7 and maximum of 17 ovarian follicles (Fig. 7a). The total number of ovarian
follicles showed a clear relationship with female size; however, only females that were
≥57 cm TL had more than 50 follicles of different sizes, and medium and large follicles
were present in size-group ≥ 52.5 cm TL (Fig. 7a).
22
Figure 7.- Fecundity relationships in female Narcine entemedor with a) Total length (cm) and number of ovarian follicles by group and b) TL (cm) and number of embryos by female.
There was a significant difference between the ovarian index and the total
number of ovarian follicles (KW (2, 168) = 12.796, P = 0.0017). The ovarian fecundity
(mean = 7.0, max = 17) had no relationship with the size of the female (r2 = 0.0180, P
= 0.464), but only females ≥ 58 cm TL had 10 or more follicles capable of being ovulated
(Fig. 7a).
A total of 45 females had embryos in the uterus, and sizes ranged from 0.1 to
14.5 cm TL (n = 307; 123 females, 88 males, and 96 undefined). The embryo female:
male sex ratio was 1.3:1, which was not significantly different from the expected 1:1
ratio (χ2 = 5.805, P = 0.984). Embryonic fecundity varied between 1 and 24 embryos
(6.6 ± 5.3, mode = 2), and the number of embryos did not vary significantly between
left and right uteri (Z = 0.95, df = 34, P = 0.431). There was no relationship between
uterine fecundity and female TL (r2 = 0.0009, P = 0.850, n = 41; Fig. 7b), but only
females ≥ 60 cm TL had more than seven embryos and females ≥ 70.5 cm TL had 13
23
or more embryos. The highest fecundity observed, 24 embryos, was in a 75-cm-TL
female.
1.3.6 Reproductive Cycle
Maximum ovarian follicle lengths varied significantly among months (KW (11, 180)
= 87.12, P < 0.0001), and the largest ovarian follicles occurred in July, August, and
September (31.5, 29.3, and 31.8 cm, respectively; Fig. 8a). Follicular growth and
development begins in May and ends (ovulation events) from August to September.
Additionally, September was the only month in which oocytes were found in the anterior
oviduct and large postovulatory follicles were seen in the ovary, evidence of recent
ovulation. However, two females had large ovarian follicles in February (19.8 cm) and
April (20.5 cm).
The median size of embryos varied significantly across months in which they
were present (KW(8, 307) = 249.84, P < 0.0001; Fig. 7b). Additionally, eggs were
observed in the uterus during all months, which can be indicative of embryonic
diapause. There were two periods of embryonic growth: from October to January–
February and from May to August. This later period corresponded to the season in
which the majority of females were captured.
24
Figure 8.- Relationships of lengths of ovarian follicles and embryos in Narcine entemedor with a) Monthly variation of largest ovarian follicle length in mature females, b) Monthly variation of intra-uterine embryo TL (filled circles indicate eggs in the uterus). The lower and upper boundaries of the boxes indicate the 25th and 75th percentiles, respectively, while the square (□) inside the boxes indicates the medians. Whiskers indicate the nonoutlier range and circles indicate outliers and c) Relationship between largest ovarian follicle length and embryo developmental stage. Whiskers indicate the minimum and maximum length while the square
(□) indicates the medians.
25
Embryonic size at birth was between 12.4 and 14.5 cm TL. There was a clear
tendency of synchronous development between vitellogenesis and gestation; females
with embryos in the two earliest stages of development (Formation and Early in Fig. 8c)
also had small ovarian follicles, while females with embryos in the Late developing
stage had the largest ovarian follicle lengths (Fig. 8c).
The male GSI did not vary significantly among months (KW (7, 38) = 4.93, P =
0.667), although GSI was highest in October (2.94). Female GSI did vary significantly
among months (KW(11, 160) = 64.60, P < 0.0001), and the highest mean value occurred
in August. Two homogeneous subsets were observed in female GSI; GSI values in
May, July, and August significantly higher than those in January, February, October,
and December (Bonferroni adjusted P < 0.004; Table 3). The GSI began to decrease
in October and remained low until April, suggesting little ovarian growth during these
months.
Table 3. Mean monthly values (± SE) of the Gonadosomatic Index in female Narcine entemedor. Superscript numbers indicate homogeneous subsets (Tukey non-parametric post-hoc test with Bonferroni adjustment, P < 0.004). n = sample size.
Month n Mean ± SE
January 13 0.79 ± 0.245a
February 33 0.532 ± 0.106a
March 7 0.503 ± 0.124a,b
April 4 0.341 ± 0.102a,b
May 24 0.953 ± 0.378b
June 3 0.625 ± 0.088a,b
July 22 1.53 ± 0.259b
August 19 3.384 ± 0.39b
September 6 2.773 ± 0.882a,b
October 10 0.525 ± 0.076a
November 4 0.432 ± 0.034a,b
December 15 0.455 ± 0.05a
Finally, when considering the percentage of females in each reproductive phase
throughout the year (Table 4), there is one peak of ovulation between July and
September (highest percentage of ovulation-capable females) but two peaks of
26
parturition (presence of Late developing embryos; Table 4). During the first, primary
peak in parturition, the females enter a period of embryonic diapause from October
through April; embryo development reactivates in May and birth occurs between August
and September. During the second, minor peak in parturition, gestation begins in
October, embryonic diapause does not occur, and females give birth in January to
February.
27
Table 4. Percentage of maturity stages throughout the year in female Narcine entemedor.
Immature Mature
Pregnant Not-
Pregnant
Month Total number
of females Developing
Ovulation
Capable/Actively
Ovulating (with
eggs in the
uterus)
Eggs in the
uterus*
Embryos in
Formation
Early
embryos
Mid
embryos
Late
embryos Regressing
January 18 16.7 0.0 66.7 0.0 5.6 0.0 0.0 11.1
February 35 14.3 11.4 54.3 0.0 0.0 2.9 0.0 17.1
March 10 20.0 0.0 80.0 0.0 0.0 0.0 0.0 0.0
April 11 18.2 27.3 54.5 0.0 0.0 0.0 0.0 0.0
May 33 24.2 15.2 15.2 24.2 15.2 3.0 0.0 3.0
June 13 23.1 0.0 15.4 15.4 15.4 23.1 0.0 7.7
July 31 41.9 32.3 3.2 0.0 3.2 6.5 12.9 0.0
August 35 14.3 54.3 2.9 0.0 0.0 8.6 17.1 2.9
September 14 28.6 42.9 28.6 0.0 0.0 0.0 0.0 0.0
October 23 13.0 30.4 34.8 4.3 4.3 4.3 0.0 8.7
November 8 37.5 0.0 25.0 12.5 12.5 0.0 0.0 12.5
December 29 72.4 0.0 20.7 0.0 0.0 3.4 0.0 3.4
28
1.4 DISCUSSION
This study provides evidence that the Narcine entemedor has a continuous
annual reproductive cycle; one peak of ovulation occurs between July and September,
but two peaks of parturition occur (minor peak in January–February and major peak in
August–September). These two peaks of births suggest that a majority of female of N.
entemedor undergo embryonic diapause, as previously suggested by Villavicencio-
Garayzar et al. (2001) for this species, and similar to reports for other species of rays
(Lessa, 1982; Simpfendorfer, 1992; Morris, 1999; Waltrick et al., 2012). Additionally, in
contrast to previous reports of matrotrophy in this species (Villavicencio-Garayzar,
2000), histological evidence of secretory material in endometrial tissue during late
pregnancy suggests the N. entemdor presents limited histotrophy as a reproductive
mode.
The largest sizes of this species examined in this study are smaller to those
reported by Villavicencio-Garayzar (2000) for the Bahía Almejas, Baja California Sur,
Mexico (females = 93 cm TL, males = 67 cm TL) and those reported for the Ecuadorian
Pacific Ocean by Palma-Chávez et al. (2014) (females = 110 cm TL, males = 83 cm
TL), which could mean different populations were sampled. In contrast, it is possible
that the sizes recorded for the present study are smaller than previously reported
because the rays live in more protected areas (which function as mating or nursery
habitats) within a gulf or that the method of capture did not allow collection of larger
individuals.
Although the observed sizes in our study were smaller than those found in
previous studies, they correspond primarily to sexually mature individuals, similar to
that found in previous studies. Thus, the effect of fishing gear selectivity is likely not a
concern when comparing studies. Smaller sized organisms, such as neonates and
juveniles, are likely to inhabit protected areas, such as shallower waters and marshes,
as also suggested by Villavicencio-Garayzar (2000) for Bahía Almejas. However, since
artisanal fishers only target large-sized organisms, we were not able to collect neonates
during this study. Neonates are likely located in areas that are not accessible for fishing
with gill nets, or they may leave the bay immediately after parturition. This is supported
29
by Rudloe (1989) observation that Brazilian Electric Rays N. brasiliensis in the Gulf of
Mexico tend to move to shallower areas during warm seasons and retreat to deeper
areas during the cold seasons for parturition, which would also explain the absence of
neonates in our study.
The greater proportion of female N. entemdor relative to males in the Bahía de
La Paz has also been reported for this species in Bahía Almejas (Villavicencio-
Garayzar, 2000). A possible explanation for this pattern is that males only enter the
shallow, protected waters of the bays for mating, as the months when they were most
abundant (July–September) is the mating season (Villavicencio-Garayzar et al., 2001),
which coincides with the months of greatest follicular length and highest GSI in females.
Other species of Narcinidae also show female-dominated sex ratios; Brazilian Electric
Rays on the coast of São Paulo, southeastern Brazil, had a female: male sex ratio of
2.2:1, a result attributed to fishing gear selectivity (Rolim et al., 2015). Female Lesser
Electric Rays in Santa Marta, Colombia, also have a sex ratio of 2.4 females per male
(Moreno et al., 2010). In general, sexual segregation is a common characteristic among
diverse species of elasmobranchs and has been attributed to differences in sizes
between sexes in order to reduce predation or differences in feeding grounds, although
there are not sufficient studies to support these hypotheses (Wearmouth & Sims, 2010).
This is the first study to microscopically describe testicular development in N.
entemedor. We observed different spermatogenic phases such as those previously
described in mature elasmobranchs (Maruska et al., 1996). Narcine entemedor testes
have multiple germinal zones, similar to previous histological descriptions in batoids
(Pratt, 1988), and the mature spermatocysts were generally concentrated in the
periphery of the testes near the efferent ducts, as described by Hamlett (1999) for
batoid species.
Villavicencio-Garayzar (2000) defined maturity in male N. entemedor in Bahía
Almejas as the presence of semen in the vas deferens; that author did not evaluate the
different stages of spermatogenesis as we did in this study. Here, we defined functional
maturity based on histological examination of the testes and seminal vesicles as well
as the presence of spermatozoa in the claspers. Another criterion to define maturity in
30
male elasmobranchs is the degree of calcification of the clasper (Abdel-Aziz, 1994);
only mature individuals have claspers that are completely calcified, as was found in this
study for all males examined.
In mature male of N. entemedor, the relationship between clasper length and
fish size (TL) seems to have a linear tendency, similar to reports by Villavicencio-
Garayzar (2000) in Bahía Almejas for N. entemedor and for the N. brasiliensis studied
by Rolim et al. (2015). It is likely that this relationship has a inflection point; however,
we cannot define the type of growth in relation to the clasper and TL since we do not
have immature individuals. The absence of juveniles has also been reported in other
zones and in the Mexican Pacific Ocean for the same species (Villavicencio- Garayzar,
2000; Rolim et al., 2015). Females of various species of elasmobranchs have varying
sizes of reproductive structures, including some organs that are dysfunctional (Dodd,
1972; Castro et al., 1988). However, in female N. entemedor, both ovaries are of similar
length and both contain fertile ovarian follicles. The mass of the left and right ovaries
were different, but this may have been an artifact of field sampling since the largest
ovaries tended to break and expel their oocytes and ovarian stroma prior to obtaining
measurements in the laboratory. The presence of two functional ovaries and uteri of
equal size has been previously reported for the same species in Bahía Almejas
(Villavicencio-Garayzar, 2000) as well as for Ocellated Torpedo (Capapé et al., 2000),
N. entemedor (Neer & Cailliet, 2001), and N. brasiliensis (Rolim et al., 2015), and is
thus likely a common feature among electric rays.
The elongated ovarian follicles of N. entemedor is unique among batoid species,
and it was described previously by Villavicencio-Garayzar (2000) for this species. This
form of the ovarian follicles may be related to uterine space as a reproductive tactic
whereby the female can provide a lot of yolk to the embryos in a reduced space. Moreno
et al. (2010) reported for N. brancoftii oocytes as “yellowish threads,” which likely
corresponds to the elongated form present in N. entemedor, although those authors
did not mention the length of the oocytes.
All females evaluated for this study had ovarian follicles in different stages of
follicular development, results that are very different than those reported by
31
Villavicencio-Garayzar (2000) for the same species in Bahía Almejas where females <
61 cm had no gametogenic activity. However, Villavicencio-Garayzar (2000) did report
ovarian follicles of greater length (to 50 cm) than those encountered in the present
study. This may be due to females being larger (up to 93 cm TL) in Bahía Almejas than
in the present study. The presence of ovarian follicles ≤ 15.9 cm in length in completely
developed ovaries (ovary index = 4) suggests a continuous production of ovarian
follicles throughout the year, which further suggests the species has continuous
reproductive activity (Koob & Callard, 1999). In addition, the presence of postovulatory
follicles in the ovary in September suggests recent ovulation or a period of preovulation,
which is common in elasmobranchs (Lutton et al., 2005).
To define the birth size of N. entemedor in Bahía Almejas, Villavicencio-
Garayzar (2000) used the largest size of embryos in the uterus that had small yolk sacs
as well as the size of the smallest neonate captured (15.7 cm TL) and defined a size of
birth from 14 to 16 cm TL, which corresponds to 14.3–16.4% of the asymptotic size in
his study. Unfortunately, we did not capture any neonates in Bahía de La Paz, so the
size at birth was based on the largest embryo in utero without a yolk sac, following
morphological characteristics proposed by Braccini et al. (2007) and evidence from
Moreno et al. (2010) for N. brancoftii. We defined a birth size between 12.4 and 14.5
cm TL, which, although smaller than the defined birth size from rays in Bahía Almejas,
corresponds to 14–16.4% of the asymptotic size of the sampled population, similar to
the asymptotic size reported by Villavicencio-Garayzar (2000). Interestingly, we did not
find a difference in the sex ratio of embryos, similar to reports by Villavicencio-Garayzar
(2000) for Bahía Almejas. These observations support the idea that adults and juveniles
are temporally and spatially segregated by sex, rather than there being a
preponderance of females in the population.
Villavicencio-Garayzar (2000) defined N. entemedor as a matrotrophic species
and considered that one-third of the mass of the embryo depended on the mother
through uterine milk obtained through the uterine villi; however, this investigator did not
carry out studies that defined the percentage of yolk consumed, as proposed by
Guallart &Vicent (2001). In our study, we did not observe uterine milk, and the material
32
secreted by the uterine villi observed in histological sections was only a few droplet
granules located near the glandular crypts. Additionally, the muscular tissue and serosa
layer of the uterus were very thin, while in elasmobranchs with a dependence on uterine
milk these structures are very thick (Colonello et al., 2013). Thus, the characteristics
observed in the present study suggests that N. entemedor is likely a species with limited
histotrophy (matrotrophic), based on the secretions from secretory crypts in the
endometrium and the increased vascularization in that tissue, since according to Moura
et al. (2011) this is evidence of a certain type of nutrition secreted by the mother.
Additionally, these secretory crypts are composed of more than six layers of cells that
make up the glandular tissue, in contrast to the related Ocellated Torpedo that has only
one to two cell layers in the uterine villi and a viviparous reproductive type with vitelline
sac and no histotrophy (Ranzi, 1934; Hamlett et al., 2005).
Histotrophy can be used as supplemental food when the embryo has used up
the yolk sac (Hamlett et al., 2005). However, histochemical analyses are necessary to
define the type of secretion and to determine if it is a nutrient substance secreted by
the mother to provide embryonic nutrition, since the difference between limited
histotrophy and lecitotrophy is very subtle (Huveneers et al., 2011). Alternatively, a
comparison of the dry mass of eggs and embryos at term, as proposed by Guallart &
Vicent (2001), could also help determine the extent of matrotrophy exhibited by N.
entemedor.
The median size at maturity estimated for the species in Bahía de La Paz
represents 66.2% of the estimated maximum asymptotic length (88.4 cm TL), which is
smaller than that proposed by Villavicencio-Garayzar (2000) for this species in Bahía
Almejas (62–63 cm TL; 68–69%). This difference is likely due to the catch sizes for this
study (maximum TL = 84 cm), which were smaller than those from Bahía Almejas
(Villavicencio-Garayzar, 2000). However, in both studies, the mature population is >
60% of the asymptotic size. In contrast, other species of Torpediniformes, such as N.
brancoftii and N. entemedor reach maturity at 53.5% and 53%, respectively, of their
estimated asymptotic length (Neer & Cailliet, 2001; Moreno et al., 2010). It should be
noted that the median size at maturity based on the condition of the ovaries was similar
33
to the median size at maturity estimated considering all reproductive structures
together. Thus, when monitoring the species, the condition of the ovaries (mature or
immature) can be used to define the maturity of the organism. This is the first study to
evaluate the median size at maternity for N. entemedor, which represents 74.9% of
estimated asymptotic length. This suggests that only the largest females in the
population of Bahía de La Paz contribute to recruitment the following year if they are
not captured.
The total number of ovarian follicles encountered for N. entemedor (69) was
similar to that reported for Pacific Electric Rays (Neer & Cailliet, 2001) and Variable
Torpedo Rays T. sinusperisici (Shrikanya & Sujatha, 2014) of 55 oocytes for both
species. However, ovarian fecundity based only on large vitellogenic ovarian follicles is
less than uterine fecundity in N. entemedor. It is likely that the ovarian fecundity of N.
entemedor is underestimated, since the ovarian follicles could have been damaged or
expelled during the manipulation of specimens in field.
The uterine fecundity of N. entemedor in Bahía de La Paz (1–24 embryos) was
slightly greater than that reported for this species in Bahía Almejas (4–20 embryos:
Villavicencio-Garayzar, 2000). This difference could be due to an underestimation in
the Bahía Almejas population as pregnant females could have aborted their embryos
during capture, since specimens were recovered from fishing gear after several hours.
In our study, we identified a female with a contracted cervix and a fecundity of 24
embryos, suggesting she did not abort any embryos, which provides support for our
estimations. Lower fecundities have been observed in other Torpediniformes, such as
N. brancoftii (1–14, Moreno et al., 2010), T. californica (17, Neer & Cailliet, 2001), and
Marbled Electric Ray T. marmorata (3–16, Consalvo et al., 2007), although all these
species are smaller than N. entemedor and thus can be expected to have lower
fecundities. In addition, in some species the number of embryos is related to embryo
size, such as in Ocellated Torpedo, which has a fecundity of 28 small embryos (12.5
cm TL, Capapé et al., 2000).
In contrast to Villavicencio-Garayzar (2000) and Villavicencio-Garayzar et al.
(2001) who only found ovarian follicles from May to August in N. entemedor rays from
34
Bahía Almejas, we found females with ovarian follicles year-round. However, ovarian
follicle growth began in May and follicles achieved their greatest lengths in September,
although previtellogenic follicles were present throughout the year. It is possible that
previous studies did not report the presence of previtellogenic follicles in the ovary.
The presence of oocytes descending into the anterior oviducts during September
in our study was also reported for this species in Bahía Almejas (Villavicencio-
Garayzar, 2000). Females with ovarian follicles ≥ 16 cm were not observed in October
in rays in either study, indicating that ovulation and mating end in September, which
may be closely related to the increase in temperature in the summer months in both
bays. While only two females were recorded with follicles near ovulation size (19.8 and
20.5 cm) in February and April, respectively, it is likely that these follicles are atypical
since both females contained only one follicle of this size and all others were ≤15.9 cm
in length.
Gestation and vitellogenesis occurred synchronously in N. entemedor in both
Bahía de La Paz and in Bahía Almejas (Villavicencio-Garayzar, 2000; Villavicencio-
Garayzar et al., 2001). Furthermore, gametogenic development was observed in
females in all months, and this is mainly because once the mother gives birth, she is
ready to ovulate immediately after parturition suggesting this is a species with a
continuous annual reproductive cycle (Koob & Callard, 1999). However, this was not
the case for a single female in January with not only late developing embryos but also
with ovarian follicles < 16 cm, too small for ovulation. Likely, this small (66 cm TL)
female was reproducing for the first time and did not go through embryonic diapause
like all the other females in the population. This atypical behavior could be a
physiological response to the environment or a respite from the continuous
reproductive periods; this possibility can only be corroborated with hormonal analysis
as discussed by Lopes et al. (2004) and Murphy (2012).
January and August represent the two periods of parturition, although we only
observed one female in January ready to give birth, and another in February with
embryos in the mid-developing stage. Thus, it appears there are two peaks of birth,
following two separate paths of development. The majority of the females fertilized in
35
summer (August– September) pass through a period of embryonic diapause from
October until April and have fertilized eggs in the uterus during this time. Fertilized eggs
can remain in the blastodisc stage from 4 to 10 months (Simpfendorfer, 1992; Morris,
1999). Later, embryo development is reactivated in May and parturition occurs in
August and September. In the second possible pathway without embryonic diapause,
as seen in a minority of females in Bahía de La Paz, embryonic development of
fertilized oocytes begins immediately in October and parturition occurs in January–
February. The period of embryo development is the same in both pathways (5 months),
but one group of the population delays embryo development for 7 months during the
coldest time of the year, followed by activation of the embryo development period in the
summer months. According to Wyffels (2009), it is common to identify an embryonic
diapause period in females with fertilized eggs in the uterus but without visible embryos
for long periods of time, and this is also common in populations with synchronous
reproductive cycles, as we observed for N. entemedor in Bahía de La Paz.
Embryonic diapause was suggested previously for N. entemedor by
Villavicencio-Garayzar (2000) and Villavicencio-Garayzar, Mariano, and Downtonn
(abstract) in Bahía Almejas. Furthermore, other species of rays have also been
reported to have embryonic diapause, such as Bluntnose Stingray Dasyatis say
(Simpfendorfer, 1992; Morris, 1999), Brazilian Guitarfish Rhinobatos horkelii (Lessa,
1982), and Whiptail Stingray D. brevis and Shovelnose Guitarfish R. productus
(Villavicencio-Garayzar et al., 2001).
This study reports previously unknown reproductive data for N. entemedor, an
important commercial species in Bahía La Paz, Mexico. Of particular concern is that
most of the mature individuals caught in the area are pregnant females with eggs or at
different stages of gestation. Furthermore, the principal months of birth are July to
September, yet elasmobranch fishing closures in Mexico only occur from May 1 to July
31 (DOF, 2012). Our data suggest female of N. entemedor are vulnerable to capture
during the primary birthing months, which may negatively impact population recruitment
and jeopardize the population’s recovery from overharvesting. Although the Mexican
law is meant to protect different species of elasmobranchs, the complexity of
36
incorporating biological information with fishing and resource dynamics is challenging,
particularly when biological information is lacking. Thus, information provided here is
important for the evaluation of the population of N. entemedor in Bahía de La Paz and
should be considered when future policies and management plans are drafted to
protect this species.
37
CHAPTER II
REPRODUCTIVE STRATEGY OF THE PACIFIC COWNOSE
RAY Rhinoptera steindachneri IN THE SOUTHERN GULF OF
CALIFORNIA
María I. Burgos-Vázquez1,*, Valeria E. Chávez-García1-2, Víctor H. Cruz-Escalona1, Andrés F. Navia3 and Paola A. Mejía-Falla3,4
1 Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas,. La Paz, Baja California Sur, México. Av. Instituto Politécnico Nacional s/n Col. Playa Palo de Santa Rita
Apdo. Postal 592. Código Postal 23096 La Paz, B.C.S. 2Universidad del Mar, Ciudad Universitaria, Puerto Ángel, Distrito de San Pedro Pochutla,
Oaxaca México C.P. 70902. 3Fundación colombiana para la investigación y conservación de tiburones y rayas,
SQUALUS. Calle 10A No 72-35, Cali, Colombia. 4. Wildlife Conservation Society, WCS-Colombia. Av. 5N No. 22N-11, Cali, Colombia.
Burgos-Vázquez, M. I., Chávez-García, V. E., Víctor H. Cruz-Escalona, V. H., Andrés F. Navia, A. F &
Mejía-Falla, P. A. Reproductive strategy of the Pacific cownose ray Rhinoptera steindachneri in the
southern Gulf of California. Marine & Freshwater Research. In press.
38
Abstract
Rhinoptera steindachneri is one of the most common batoid species in the artisanal
gillnet fishery of the Gulf of California. Its reproductive biology was studied based on
317 specimens caught in Bahía de la Paz, Mexico. Females measured up to 94.2 cm
disc width (DW) and males reached 82.5 cm DW; there were no significant differences
in size or weight between sexes. Median size at maturity was estimated at 68.5 cm DW
for males and 71.8 cm DW for females, and the median size at pregnancy was 84.3 cm
DW. Only the left ovary and uterus were functional, a maximum of six pre-ovulatory
vitellogenic follicles per female were recorded, whereas uterine fecundity was one
embryo per female. The ovulation period and birth occurred in May, June, and July,
with birth sizes ranging from 38.1 to 42 cm DW. Rhinoptera steindachneri presented
low fecundity, large size at maturity and births, and a continuous and synchronic annual
reproductive cycle in Bahía de la Paz.
39
2.1 INTRODUCTION
It has been historically assumed that elasmobranchs as a group present a K life
history strategy, with low fecundity, late maturity, and slow growth (Hoening & Gruber,
1990; King & McFarlane, 2003). Knowledge of the life history of a species, and in
particular, of its reproductive strategy, provides one of the most important contributions
for the evaluation of populations and gives effective tools for decision makers to
establish capture limits and prevent overfishing of species (Walker, 2005). Recent
studies show that anthropogenic pressures on elasmobranch species can affect these
life history strategies (Smith et al., 1998; Cortés 2000; Frisk et al., 2002).
Viviparous elasmobranchs exhibit a wide diversity of reproductive modes, which
is reflected in the number of ways in which the mother contributes to the development
of embryos (Conrath & Musick, 2012). For example, lipid histotrophy occurs only in rays
from the order Myliobatiformes. Mothers secrete a protein- and lipid-rich histotroph from
highly developed secretory structures within the uterine lining called trophonemata
(Hamlett et al., 2005). This mechanism of energy transfer seems to be more efficient,
causing Myliobatiformes to gain more weight during embryonic development than
species that present other reproductive modes (Conrath & Musick, 2012). This
reproductive mode could also be related to low fecundities and large size at birth
(Garayzar et al., 1994; Neer & Thompson, 2005; Jacobsen et al., 2009).
The Pacific cownose ray Rhinoptera steindachneri (Evermann & Jenkins, 1982)
is a batoid from the order Myliobatiformes, and the only representative from this family
distributed in the Eastern Pacific (Robertson & Allen, 2015). It inhabits shallow waters,
especially over soft bottoms, and performs seasonal migrations related to water
temperature (Bizzarro et al., 2007). Few studies on the reproductive biology of this
species have been published. A study carried out in the northern Gulf of California
estimated a median size at maturity of 70 cm disc width, fecundity of a single pup, and
a gestation period of 10 to12 months (Bizzarro et al., 2007).
Rhinoptera steindachneri is one of the most common batoid species in the
artisanal gillnet fishery of the northern (Bizzarro et al., 2007) and southern (González-
González, 2017) Gulf of California. It is also caught as by-catch in the shrimp fishery of
40
the southern Pacific region of Mexico (Navarro et al., 2012). Due to the scarcity of
information and the threats identified in its distribution range, the species is listed as
Near Threatened in the IUCN Red List (Smith & Bizzarro, 2006).
It has been found that reproductive characteristics can vary between populations
of elasmobranchs, even at small spatial scales (Yamaguchi et al., 2000; Lombardi-
Carlson et al., 2003; Walker, 2007; Mejía-Falla, 2012). However, other authors
suggested further invertigation to define whether those spatial differences are real or
apparent (Trinnie et al., 2014) This highlights the importance of obtaining local data to
avoid incorporating bias into demographic models and management strategies based
on reproductive parameters defined for other locations. The objective of the present
study was therefore to quantify reproductive variables of R. steindachneri for the
southern area of the Gulf of California, including sex ratio, size at birth, size at maturity
and pregnancy, fecundity, gestation period, and ovarian cycle.
2.2 MATERIALS AND METHODS
2.2.1 Study area, sample collection and laboratory analysis
Monthly samplings were performed from January 2014 to March 2017 in
southern Bahía de La Paz, located in the southern Gulf of California (24° 25' 17.55'' N,
110° 18' 31.64'' W). Specimens were captured by artisanal fishermen using
monofilament gill nets (100 m long x 1.5 m high, 8-10 inches mesh size) traditionally
called “chinchorros”, which are set in the afternoon, between 10 and 30 m depth over
sandy bottoms and recovered the next morning. The disc width (DW, cm) was
measured and the sex determined by the presence of copulatory organs in males
(claspers). The inner length (CL, cm) of one clasper from each male was measured,
and the degree of calcification (calcified, partially calcified, not calcified) and presence
or absence of semen were recorded. The gonads were weighed (GM, 0.001 g) and
fixed in 10% buffered formalin.
The biometry of testes, seminal vesicles, and claspers of males were evaluated.
The testes length (±0.001 cm), width (±0.001 cm), and mass (±0.001g), as well as
41
seminal vesicle length (±0.001 cm) and width (±0.001 cm) were measured. The length,
width (±0.1 cm), and mass (±0.01g) of ovaries, uterus, and oviducal glands of females
were measured. Visible ovarian follicles were extracted, quantified, measured
(diameter ±0.1 cm), and collected. The length of the longest trophonemata in the uterus
was measured. Embryos were sexed and measured (DW), weighed, and
ontogenetically classified based on morphological characteristics, following criteria
proposed by Hamlett et al. (1985) and Colonello et al. (2013).
Maturity was defined based on the macroscopic observation of the reproductive
organs of both sexes, following proposals by Smith & Merriner (1986) and Poulakis &
Grier (2014), adapted for R. steindachneri males (Table 1) and females (Table 2). The
characteristics used to define maturity in males were: presence/absence and degree
of development of the testicular lobes in testes, degree of development of the epigonal
organ (when present), presence of seminal material, and degree of winding in the
seminal vesicles, as well as absence/presence of the alkaline gland and
absence/presence of fluid in this structure. The characteristics used to define maturity
in females were: presence of ovarian follicles and degree of vitellogenesis,
absence/presence and development of uterine villi and embryos, and thickness and
weight of the muscular wall of the uterus. The total number of ovarian follicles (OF) per
female was counted, but only the pre-ovulatory vitellogenic ovarian follicles (VOF; ≥ 0.8
cm of diameter) were used to evaluate ovarian fecundity.
2.2.2 Sex ratio, disc width, and mass
The sex ratio of adults, juveniles, neonates, embryos, and all individuals together
were evaluated using a Chi-Square test to determine if it differed from 1:1 (Sokal &
Rohlf, 1998). Differences between males and females in disc width and weight
(excluding the weight of pregnant females) were evaluated using a Mann-Whitney U
test. Data were tested for normality and homogeneity of variances with Kolmogorov
Smirnov and Lilliefors tests, respectively, prior to analysis. All differences were
considered significant if P < 0.05.
42
2.2.3 Reproductive structures and maturity
The logistic equation modified by Piner et al. (2005) was used to evaluate the
relationship between DW and CL, using the following equation:
𝐶𝐿𝑖 = 𝑚𝑖𝑛𝐶𝐿 +𝑚𝑎𝑥𝐶𝐿 − 𝑚𝑖𝑛𝐶𝐿
1 + 𝑒𝑏(𝑎−𝐷𝑤𝑖)
where a is the inflection point of the curve, b is another parameter of the model, and
min and max represent the minimum and maximum CL values (Mejía-Falla et al.,
2012).
Differences in length, width, and mass between right and left reproductive
structures (testes, seminal vesicle, ovary, and uterus) were assessed using a Wilcoxon
paired test; and differences in length and width of the oviducal gland were evaluated
using Student´s t test.
Differences in weight of reproductive structures on the left (dorsal position) of
the body (testes, ovaries, oviducal glands, and uterus) were evaluated by maturity
stage using a Kruskal Wallis test for independent samples; for seminal vesicles, the
width was evaluated.
2.2.4 Median size at maturity and pregnancy
The median size at maturity (DW50%) was calculated for males and females using
a logistic regression model with binomial data (0, immature; 1, mature) using the
equation:
P𝑖= (1+𝑒-(a+b*DWi
))-1
where P𝑖 is the fraction of mature individuals at DW i, a and b are model parameters,
and -a/b is the median size at maturity (Mollet et al., 2000). Males were considered
mature if the claspers were partially calcified or calcified, and there was presence of
semen and/or presence of testicular lobes in testes (maturity indices 3 and 4; Table 1).
Females were considered mature if they presented vitellogenic ovarian follicles or
embryos (maturity indices 3, 4 and 5; Table 2). Median size at maturity was also
calculated using binomial data of the left ovary (DWO; the only one with follicular
43
development). Immature ovaries (indices 1 and 2) were assigned a value of “0” and
mature ovaries (indices 3 and 4) a value of “1”. Median size at maturity was calculated
using binomial data of the left uterus (DWU; the only functional one); immature uteri
(indices 1 and 2) were assigned a value of “0” and mature uteri (indices 3, 4b, 4c, 4d
and 5) a value of “1”. The median size at pregnancy (DWP50) was also calculated using
binomial data, where a value of 1 corresponded to females at maturity stages 4 and 5;
the other females were designated as 0 (maturity stages 1, 2, and 3).
2.2.5 Fecundity and reproductive cycle
The range, mean, and mode of the number of vitellogenic ovarian follicles in the
ovary and the embryos in the uterus were estimated to evaluate ovarian and uterine
fecundity (Pratt, 1979). A linear regression was used to analyze the relationship
between ovarian fecundity and uterine fecundity and DW. Differences between OF and
uterine fecundity through maturity stages were evaluated using a Kruskal Wallis test.
The reproductive cycle was defined by ovulation (using only VOF) and gestation
period. Ovulation was evaluated using the diameter of the largest ovarian follicle (pre-
ovulatory vitellogenic) of each female through the months, and differences between
months were evaluated with a Kruskal Wallis test. Gestation period was defined based
on monthly embryo disc width and weight; information of neonates was also analyzed
to infer birth months (disc width and weight). Birth size was evaluated considering the
DW and weight of the largest/heavier embryo and smallest/ lighter neonate. Additional
information from growth bands on neonate vertebrae (Pabón-Aldana, 2016) was used
to define birth months and birth sizes. The percentage of females and males by maturity
stage by month was examined using a histogram.
44
Table 1. Maturity stages of Rhinoptera steindachneri males, indicating the characteristics and indices of each reproductive organ.
Maturity Index
Maturity stage Testes index
Testes condition Seminal vesicle index
Seminal vesicle condition
Clasper condition
1 Immature – Not developed 1
No presence of testicular
lobes, large amount of testicular stroma.
presence of primary spermatogonia.
1 Undifferentiated
Not calcified, no semen
2 Immature - Developing 2
Some testicular lobes, moderate testicular stroma. Secondary spermatogonia and
primary spermatocytes
2 Differentiated and thick, no seminal
fluid
3 Mature – Mating capable
3
Presence of testicular lobes throughout the organ, seminiferous
ampullas throughout the periphery, mature sperm
cells
3 Differentiated and
coiling, no seminal fluid
Partially or completely calcified, without semen
4 Mature - Actively mating 4
Differentiated and coiling, with seminal fluid
Calcified, with semen
45
Table 2. Maturity stages of Rhinoptera steindachneri females, indicating the characteristics and indices of each reproductive organ.
The 4a uterine index was not found in this study.
Maturity Index
Maturity stage
Ovarian index
Ovarian condition
Oviducal
gland index
Oviducal gland
condition
Uterine index
Uterine condition
1 Immature –
Not developed
1 No follicles, large amount of ovarian
stroma.
1
Not visible or slightly differentiated from the
anterior oviducts. 1
Undifferentiated, no uterine villi, weigh ≤ 0.6 g.
2 Immature - Developing
2
Follicles visible, small (≤ 0.79 cm
diameter) and previtelogenic.
2
Slightly differentiated from the anterior oviducts but not
completely developed.
2
Slightly differentiated, tubular form, short uterine villi (≤ 0.02 cm), weight 0.2
– 9 g. No presence of histotroph.
3 Mature- virgen
3
Follicles visible, large (diameter ≥
0.8 cm) and vitellogenic. Small amount of ovarian
stroma
3
Completely developed, widened
and well differentiated from the oviducts.
3
Completely differentiated, long and thick uterine villi
(0.05 – 0.8 cm), weight 2 – 15 g, without histotroph.
4 Mature-
Pregnant
4a
Uterus with eggs.
Uterus with villis (0.7 – 1 cm), differentiated and
widened, weight 56.3 g, with histotroph. and
embryos in early development.
Uterus with villis (0.7 – 1
cm), differentiated and widened, weight 90 – 395 g,
with histotroph and with embryos in mid development.
4b
4c
46
4d
Uterus with long villis (up to 1.8 cm), differentiated and widened, weight ≥ 850 g,
with histotroph. and embryos in late development.
5
Mature - Post-partum
4
Follicles visible, large (diameter ≥
0.8 cm) and vitellogenic, presence of
postovulatory follicles. Large
amount of ovarian stroma.
5
Completely differentiated and flaccid, long and thick uterine villi (0.9 – 1 cm),
weight of 18.8 – 84.7 g, with waste from histotroph. No
embryos.
Mature-Resting
6
Completely differentiated, long and thick uterine villi
(0.9 cm), weight ≥ 34.4 g, no histotroph and no
embryos.
47
2.3 RESULTS
2.3.1 Sex ratio, disc width, and mass
A total of 317 individuals (150 females, 163 males, and 4 undifferentiated) were
recorded, resulting in a sex ratio equal to the expected 1:1 proportion (χ² = 151.55, d.f.
= 1, P < 0.001). The sex ratio evaluated for each developmental stage was also 1:1 (P
> 0.05 for all cases). Females were present during all months of the year, except
November and December, whereas males were not present in December. Females
ranged from 40.1 to 94.2 cm DW (mean ± SD = 65.4 ± 13.8) and 740 to 14900 g mass
(4301 ± 2793.3) and males ranged from 41.8 to 82.5 cm DW (62.7 ± 10.0) and 850 to
8300 g (3754.3 ± 1764.6), with no significant differences between sexes in size (Z =
1.5, P = 0.1) or weight (Z = 0.5, P = 0.6).
2.3.2 Reproductive structures and maturity
Males had paired oval testes fused at the lower end, just above the rectal gland.
Both testes presented epigonal organs next to the vertebral column; the right epigonal
organ was more developed than the left one and was observed only at maturity stages
2, 3, and 4. Highly vascularized filamentous tissue was observed at maturity stages 2
and 3; and there was a tendency to be reduced by stage 4, due to the increase in size
of the testicular lobes. Alkaline glands were identified on the side of the seminal vesicles
above the kidneys; these were only present at stages 3 and 4 of maturity. The left testis
was longer and heavier than the right one; there were no significant differences in width,
however, there were significant differences in mass (Table 3).
At maturity stage 1 testes presented abundant testicular stroma without
testicular lobes. At maturity stage 2 (testes index 2) some testicular lobes were visible
in the ventral part of each testis. At maturity stages 3 and 4 (testes index 3) males had
well-developed testicular lobes with seminiferous ampullas throughout the periphery of
the testes. There were significant differences among maturity stages in the mass of the
left testicle (KW(3, 61)= 44.1, P < 0.0001). The heaviest male was at the actively mating
stage (78.3 cm DW; 82.1 g), whereas the lightest male was at the immature-not
developed stage (52.3 cm DW; 0.8 g; Fig. 1a).
48
Table 3. Mean and standard deviation values of the right and left reproductive structures (dorsal position) in males and females of Rhinoptera steindachneri, and statistical results of Wilcoxon test. * t-student was used only for length of oviducal gland.
No significant differences in length and width were found between the right and
left seminal vesicles (Table 3). Seminal vesicles at maturity stage 1 (index 1) were
elongated, tubular, vascularized, with thin walls, uncoiled, undifferentiated from
extratesticular ducts, and without seminal fluid. At maturity stage 2 seminal vesicles
began to thicken and were also irrigated and without seminal fluid (index 2). At maturity
stage 3 there were irrigated, thickened and coiling seminal vesicles, without seminal
fluid (index 3). At maturity stage 4 seminal vesicles were morphologically equivalent to
maturity stage 3, but with the presence of seminal fluid (index 4). The width of the
seminal vesicle showed significant differences throughout the maturity stages (KW (3, 70)
= 54.4, P < 0.0001). Males with the largest seminal vesicles (≥ 1.5 cm) were at the
actively mating stage (Fig. 1b).
Organ Measurement Right Left Z/t* df p
Males
Testes Length 7.5 ± 2.6 8.3 ± 2.2 2.5 57 0.01
Width 2.5 ± 1.3 2.5 ± 1.1 0.6 72 0.5
Mass 18.1 ± 19.7
21.6 ± 20.8
4.2 59 <
0.0001 Seminal vesicle
Length 5.4 ± 1.3 5.4 ± 1.2 0.6 65 0.57
Width 0.8 ± 0.6 0.9 ± 0.8 1.5 70 0.14
Females
Ovaries Length 6.3 ± 2.7 6.4 ± 2.1 0.4 32 0.7
Width 2.4 ± 1.1 2.6 ± 1.2 0.9 32 0.4
Mass 8.4 ± 6.5 14.3 ± 12.5
3.9 30 <
0.0001 Uterus Length 4.4 ± 0.8 4.9 ± 1.5 2.6 39 0.009
Width 1.3 ± 0.9 1.6 ± 1.4 3.3 54 0.009
Mass
12.5 ± 10.4
20.3 ± 22.4
2.4 36 0.02
Oviducal gland
Length* 1.8 ± 0.3 1.8 ± 0.4 -
0.1 29 0.9
Width 1.1 ± 0.2 1.1 ± 0.3 1.4 23 0.2
Mass 1.5 ± 0.8 1.7 ± 0.8 1.7 25 0.09
49
Males with uncalcified claspers ranged in size between 41.8 and 69.6 cm DW
(59% of all recorded males) and were categorized as maturity stages 1 and 2. Males
with partially calcified claspers ranged in size between 59.5 and 72 cm DW (13%) and
were categorized as maturity stages 2 and 3. Males with calcified claspers ranged in
size between 64.3 and 82.5 cm DW (28%), and were classified as maturity stages 3
and 4. The smallest clasper measured 2.1 cm CL (belonging to a neonate 46 cm DW)
and the largest measured 12 cm CL, (recorded for three reproductively active males
measuring 73, 77.6, and 81.9 cm DW). The inflection point found in the logistic
relationship was 65.6 cm DW, with a clasper length of 7 cm (Fig. 3a). Therefore, males
with claspers ≥ 7 cm CL were considered mature.
Females presented paired ovaries, elongated and fused at the lower end, just
above the rectal gland. The epigonal organ was positioned on the lateral side of each
ovary; it was visible starting at maturity stage 2 and was slightly more protruding in the
left ovary. However, only the left ovary showed evidence of oogenesis (follicular
development), with the right ovary being rudimentary. The right and left ovaries were
similar in size (length and width), but the left ovary was heavier than the left one (Table
3).
Ovaries showed no visible ovarian follicles at maturity stage 1 (ovarian index 1).
The right ovary showed a decrease in the quantity of ovarian stroma and the first
ovarian follicles began to be observed (0.05 to 0.79 cm diameter) at maturity stage 2
(ovarian index 2); however, it was not possible to observe them externally. Three
different follicular diameter cohorts were observed in the mature reproductive stages:
one: ≥ 0.05 – 0.79 and second: 0.8 – 2.2 cm previtellogenic and third: 3.0 – 3.15 cm
pre-ovulatory yellowish, completely vitellogenic. The ovarian follicles could be observed
externally (only ≥ 0.8 cm, protruding from the ovarian covering tissue) at maturity stages
3, 4 and 5 (ovarian index 3, only on the left). Ovarian stroma of the right ovary increased
in size and the epigonal organ widened throughout the maturity stages. There were
significant differences in ovary weight throughout the reproductive stages (KW (4, 36) =
27.6, P= 0.00001), with post-partum stage females ≥ 84.4 cm DW having the greatest
ovary mass (21.8 – 51.8 g; Fig. 1c).
50
Figure 1. Relationship between disc width (DW) and a) left testicle mass, b) left seminal vesicle width for males, and c) left ovary mass, d) left oviducal gland mass and e) left uterus weight for females of Rhinoptera steindachneri.
51
The paired, bell-shaped oviducal glands were positioned in the anterior part of
the uteri and were similar in size (length and width) and weight (Table 3). The oviducal
glands were not visible macroscopically at maturity stage 1 (oviducal gland index 1).
They could be slightly differentiated from the anterior oviducts and uteri at maturity
stage 2 (oviducal gland index 2) but they were not yet completely developed. They were
wider and well differentiated from the oviducts at maturity stages 3, 4, and 5 (oviducal
gland index 3). There were significant differences in oviducal gland mass throughout
the reproductive stages (KW(3, 26) = 11, P = 0.01). The development of the oviducal
glands was notable in mature females at the not pregnant stage (≥ 1.3 g), and the
heaviest oviducal gland (3.2 g) occurred in a mature post-partum female (88.4 cm DW;
Fig. 1d). However, this stage was only significantly different from the developing stage
(P = 0.006). Both uteri had uterine villi, but the left uterus was functional, wider, longer
and heavier than the right one (Table 3), which was rudimentary.
At maturity stage 1 (index 1), both uteri were tubular, undifferentiated from the
oviducal gland, without uterine villi (trophonemata), and the cervix was not
differentiated (Fig. 2a). At maturity stage 2 (index 2), uteri were thin and flaccid, partially
fused at the posterior end, the cervix began to be distinguishable, and villi began to
develop (0 - 0.2 mm) in both uteri (Fig. 2b). At maturity stage 3 (index 3), the left uterus
began to thicken, the muscular layer was thicker, and the uterine villi were longer (0.5
to 8 mm) and homogeneous throughout the endometrium, while the left one became
thicker (Fig. 2c). At maturity stage 4 (uterine index 4b, 4c and 4d) only the left uterus
presented embryos, the uterine villi were well irrigated and longer (7 -18 mm) and
secreted histotroph (uterine milk; Fig. 2d). The right uterus remained the same as in
uterine index 3. At maturity stage 5 (index 5), the left uterus was similar to maturity
stage 4, but lacked embryos, presented a flaccid structure and had histotroph residues.
The uterine weight significantly varied by maturity stage (KW-H(3, 39) = 27, P < 0.0001).
Females in post-partum and pregnant stages had the greatest weights (≥ 56.3 g). An
increase in uterus weight was notorious in non-pregnant females (Fig. 1e).
52
53
Figure 2. Macrostructures of the reproductive system of Rhinoptera steindachneri females. Longitudinal section of the left ostium, oviduct and uterus in a) stage 1 (immature), b) stage 2 (developing), c) stage 3 (mature-virgin) and d) stage 4 (mature-pregnant). Hard structures found in e) left oviducal gland, f) right oviducal gland, g) both uteri (inside) and h) extracted of those organs. Structures were considered in the text in dorsal position but all photos were taken in ventral position. Abbreviations are as follows: Os = ostium, Ov = oviduct, T = trophonemata, HS = hard structure, U = uterus, np = narrowest part, wp = widest part.
2.3.2.1 Rare and hard structures in females
Sixteen females presented hard structures of unknown material in the left and
right oviducal glands and uteri. Only two females (maturity stage 3) presented these
structures in the anterior part of the oviducal glands. The first female (74.1 cm DW)
presented four hard structures in the left oviducal gland shaped like flat capsules with
ringed edges (like roses; Fig. 2e). The second female (85 cm DW) presented a grayish
single structure in the left oviducal gland in the form of a capsule such as seed or
grenade with both extremes ringed (Fig. 2f). The other females that had hard structures
in both uteri (n = 14; 74.1 to 91.6 cm DW) were also mature (maturity index 3, 4, and
5). Each female contained a single brown and translucent structure per uterus (two per
female; Fig. 2g) in the form of an elongated capsule (at its widest part) with wrinkled
54
ends similar to tendrils (at its narrowest part) and empty inside (Fig. 2h). Those
structures were found in February, May and July but it was not possible to determine
how long they last in the ovidual gland or in the uterus.
2.3.3 Size at maturity and pregnancy
Immature males (68.1% of all males sampled) measured between 41.8 and 75.0
cm DW, whereas mature males (31.3%) measured between 63.0 and 82.5 cm DW.
The median size at maturity was estimated at 68.5 cm DW (95% CI = 58.9-78.1, Fig.
3b).
Immature females (63.5%) measured between 40.1 and 75.0 cm DW, whereas
mature females (36.5%) measured between 62.0 and 94.5 cm DW. Female median
size at maturity was estimated at 71.8 cm DW (95% CI = 58-85.7, Fig. 3c). Pregnant
females (10.1%) comprised sizes between 74.4 and 94.5 cm DW and the median size
at pregnancy was estimated at 84.3 cm DW (73.7-95.02, Fig. 3d). The DW50 based on
ovarian development was estimated at 74.4 cm DW (95% CI = 60.3-88.4) and that
based on uteri was estimated at 72.5 cm DW (95% CI = 64.5-73.4).
55
Figure 3. a) Relationship between disc width (DW) and inner clasper length (cm) in males; b) maturity ogive in relation to maturity condition of males and c) females; d) pregnant ogive in females of Rhinoptera steindachneri.
2.3.4 Ovarian and uterine fecundity
In all females, only the left ovary (dorsal position) presented follicular
development. The OF per female varied between 1 and 44 (21.7 ± 11.8, mode = 22).
A significant but weak positive relationship was detected between OF and DW (r2 = 0.4,
P <0.0001); however, a greater number of OF (≥ 30) were present in females ≥ 74.1
cm DW (Fig. 4a). Significant differences were found in the total number of OF per
maturity stage (KW(3, 36) = 14.2, P = 0.0026). The developing stage had the least
number of OF (4- 21), and post-partum (37-41) and pregnant – late females developed
(44) the most quantity.
VOF was estimated at between 1 and 6 (Median ± SD = 3 ± 1.6, mode = 2). No
clear significant relationship was found between VOF and DW (r2 = 0.03, P = 0.6402)
or between VOF and maturity stages (KW(1, 11) = 1.3, P = 0.2539). However, the highest
ovarian fecundity (based on VOF) was found in the largest female (91.6 cm DW) at the
post-partum stage.
A total of 13 embryos (6.8 to 38.1 cm DW) were recorded in 13 females. There
was uterine fecundity of one embryo per female, all in the left uteri (dorsal position). No
56
evidence of abortions and no females with eggs in the uterus was observed. A single
embryo at the early developmental phase was recorded (6.8 cm DW). It presented a
yolk sac with no pigmentation, the cephalic lobes were not yet fused and same body
shape as the adult. Eleven embryos were recorded in mid – developed phase with sizes
ranging between 18.3 and 30.1 cm DW (24.7±4.1), total mass between 85.4 and 386.8
g (234.9 ± 96.0), little pigmentation, and same body shape as the adult. Only one
embryo was found to be in the late - development phase (38.1 cm DW and 841 g),
which was characterized by an absent yolk sac, body completely pigmented, and same
body shape as the adult.
2.3.5 Reproductive cycle
There were significant differences in the maximum follicular diameter throughout
the months (KW(7, 37)= 14.5, P= 0.04). The lowest values were obtained in October (0.7
- 0.8 cm), January (0.7 – 1.0 cm), and February (1 – 1.8 cm); whereas the highest
values were obtained in May (3.2 cm; post-partum stage), June and July (3.0 cm).
These last three months correspond to the period of ovulation, considering only ovarian
follicles ≥ 3.0 cm as those that can be soon ovulated, which corresponds to a follicular
development period of seven to nine months (Fig. 4b).
The smallest embryo (6.8 cm DW) was found in July 21 and the largest (38.1 cm
DW) and heaviest (841 g) in May 21. The smallest neonate (40.1 cm DW) was found
in July 4 (Fig. 4c), and the lightest (740 g; 42 cm DW) in August 2 (Fig. 4d). Based on
this information and the registration of females in post-partum in the May 21 to August
3 period and the high frecuency (n = 24) of neonates before August 4, it was proposed
May to July as the birthing months (Fig. 4c). Considering the ovulation peaks (May to
July) and subsequent start of embryonic growth (June to August) with the defined
birthing months, a gestation period of between 10 and 14 months is suggested for the
species. Although birth sizes could be estimated at between 38 and 42 cm DW, based
on traditional estimates (based on the largest and heaviest embryo and lightest
neonate), the absence of growth bands in neonates (between 40.1 and 52.1 cm DW)
found in July and August (Pabón-Aldana, 2016), suggests a wider range of birth sizes
(38.1 to 52.1 cm DW; Fig. 4c).
57
Figure 4. Relationship between a) total number of ovarian follicles (OF) and disc width (DW); b) maximum follicular diameter by month (dotted line: ovulation diameter); c) disc width (DW) and d) mass of embryos and neonates by month, of Rhinoptera steindachneri.
58
As females present continuous follicular development and they immediately
ovulate once they give birth, the type of reproductive cycle is continuous. The
synchrony of the reproductive cycle was based on two sources of information: 1) the
female with the largest embryo (38.1 cm DW), which contained follicles of 2.2 cm, a
size close to the ovulation diameter (3 cm) registered in May and 2) females at the post-
partum stage during May and June presenting the largest follicular diameters (3.0 – 3.2
cm; Fig. 4b). Both sources indicate that ovulation occurred in the same month or one
month after birth (Fig. 4b, c).
According to the percentages by developmental stage, adult males were more
frequent in the summer months (from May to August; Fig. 5a), whereas adult females
were only frequent in March and May (Fig. 5b). Neonates of both sexes were absent
from April to June and were more frequent in July and August (Fig. 5a, b), which
indicates that births occurred in May, June and July. This information, along with the
synchronic and continuous annual reproductive cycle described for the species,
indicates that the reproductive activity (ovulation, mating, and births) was concentrated
in the summer months. Juveniles of both sexes observed in the months of July to March
represented the recruits of each reproductive event (Fig. 5a, b).
59
Figure 5. Percentage of reproductive stages by months in a) males and b) females of Rhinoptera steindachneri.
2.4 DISCUSSION
This is the first study to report an anatomical description of the gonadal
structures of R. steindachneri males and females and to propose a maturity scale for
the species. Rhinoptera steindachneri is a matrotrophic species, with presence of
trophonemata to nourish the embryo through the secretion of histotroph (uterine milk),
and with continuous and synchronic annual reproduction.
A higher frequency of R. steindachneri individuals in the summer has been
reported for the Gulf of California (Bizzarro et al., 2007). The absence of individuals in
November and December can be explained by migratory activities, as reported by
Schwartz (1990) for the entire Rhinoptera genus.
Although no size differences by sex were identified in the present study, females
reached greater sizes, due to their viviparous condition and the advantages for survival
(Wourms & Lombardi, 1992), as has been previously reported for other viviparous ray
60
species (Smith et al., 2007; Alkusairy et al., 2014; Romero-Caicedo & Carrera-
Fernández, 2015; Burgos-Vázquez et al., 2017). The average sizes found for the Bahía
de La Paz population were similar to those reported in previous studies within the Gulf
of California (Villavicencio-Garayzar, 1996; Bizzarro et al., 2007), but lower than those
recorded for the west coast of BCS (Bizzarro et al., 2007). Since these last two studies
were conducted using similar fishing gear, it is likely that the differences in size were
due to the environmental characteristics of the two areas, as it has already been
reported that batoids within the Gulf of California are smaller (Villavicencio-Garayzar,
1993; Márquez-Farías, 2007; Bizzarro et al., 2007; Burgos-Vázquez et al., 2017).
Although the degree of clasper calcification has been previously used to
evaluate maturity in males, our results suggest that this measure could underestimate
maturity in R. steindachneri. We identified six specimens (63 – 77 cm DW) with mature
testicles but partially calcified claspers, leading to their initial (visual) classification as
immature. This inconsistency was reported formerly by Walker (2005) for Galeorhinus
galeus and by Poulakis (2013) for R. bonasus. We suggest considering the presence
of testicular lobes in the testicles, the thickening of the seminal vesicle, and the
presence of the alkaline gland as the most trustworthy and effective way to assess
maturity in R. steindachneri males. The minimum size at maturity of males in Bahía de
La Paz (63 cm DW) based on the degree of calcification and the inner length of claspers
was similar to that reported by Bizzarro et al. (2007) for R. steindachneri off the Sonora
coast (65 cm DW). The wide range of sizes found in this study in clasper increase (60
to 70 cm DW) was also similar to reports from that study. Martin & Cailliet (1988) found
that the abrupt change in clasper length allowed the identification of maturity in
Myliobatis californica, and the inflection point of the logistic model corresponded to the
middle of the range (65.6 cm DW); however, in the present study the median size at
maturity was different (68.5 cm DW). This could be due to the fact that size at maturity
was evaluated considering characteristics such as condition of the testicles, seminal
vesicles, and presence or absence of seminal fluid. The size at maturity found in this
study was similar to that evaluated by Bizzarro et al. (2007) for R. steindachneri off the
Sonora coast (69.9 cm DW). It is therefore advisable to use qualitative characteristics
61
to evaluate size at maturity, because the exclusive use of the internal clasper length
could lead to underestimating size at maturity.
Differences in weight (but not size) between the right and left ovary may be due
to the fact that as females mature the left ovary increases its follicular development,
while no follicular development was observed in the right ovary. This same condition
has already been reported for other Myliobatiformes such as R. bonasus (Smith &
Merriner, 1986; Pérez-Jiménez, 2011; Poulakis, 2013), M. californica (Martin & Cailliet,
1988), Gymura micrura (Yokota, 2012), and G. altavela (Capapé et al., 1992), and was
attributed to the fact that the non-functional structure is compensatory at a physiological
level, as a hormonal secretion (Møller, 1994). It is probable that due to the low fecundity
of R. steindachneri (one embryo per female), the energy that would have been
dedicated to follicular development of the rudimentary ovary is destined to other
reproductive functions, such as hormonal production.
As was found for R. bonasus (Smith & Merriner 1986, Perez-Jiménez, 2011), M.
goodei (Colonello et al., 2013), and R. steindachneri in the Gulf of California
(Villavicencio-Garayzar, 1996) our results showed that only the left uterus was
functional. It is proble that this is an ancentral condition, derived from a reproductive
mode where both uterus were viable, however, due to the low fecundity, the right uterus
ceased to be functional, Colonello et al. (2013) suggested that asymmetry is not a
condition of Myliobatiformes and that it may be related to the fertility of the species,
which has also been seen in Urolophus paucimaculatus, with only the left uterus
functional and very low fecundity (1 to 2 embryos; White & Potter, 2005).
We describe for the first time the presence of hard structures in the oviducal
glands and uteri of R. steindacheri. It was not possible to identify the origin of the
material making up these structures; however, the ringed patterns, the shape of the
capsule and the rigidity of the material could be explained as a vestige of reproduction,
because one of the functions of the oviducal gland is to produce the tertiary egg
envelope or flexible candle that wraps the fertilized egg in species with a yolk sac
(Hamlett et al., 1998; 1999; 2005; Hamlett & Koob, 1999). In the case of the capsules
found in the uterus of mature females, Smith & Merrinier (1986) reported two R.
62
bonasus females with capsules in their uterus that presented similar morphological
characteristics, but a female had one egg in one capsule and the other female had
three ovules. In the specific case of R. steindachneri the capsules found did not have
any type of material. It is advisable to perform a histochemical analysis to establish the
origin of these hard structures.
The median size at maturity estimated for R. steindachneri males in this study
(68.5 cm DW) represented 83.0% of the maximum size found, whereas for females
(71.8 cm DW) it represented 76.2% of the maximum size found. This represents a high
value for the species, suggesting late size at maturity. Bizzarro et al. (2007) estimated
median size at maturity for males at 69.9 cm DW and at 70.2 cm DW for females off
the Sonora coast, northern Gulf of California, which is similar to our study. Flores-
Pineda et al. (2008) estimated size at maturity of R. steindachneri males in Bahía
Almejas, Mexico at 79.2 cm DW and at 80.4 cm DW for females. Differences observed
in this parameter between the populations of the Gulf of California and Bahía Almejas
are attributed to the temperature differences between the two areas, as Bahía Almejas
has lower temperatures than the Gulf area (Hamlett et al., 1998; 1999; 2005; Hamlett
& Koob, 1999). This could affect metabolic rate and reflect the influence of temperature
on the maximum size that organisms can reach (Brown et al., 2007; Bernal et al., 2012).
Bizzarro et al. (2007) commented that differences between the R. steindachneri
populations of Bahía Almejas and the northern Gulf of California could be due to limited
genetic exchange and is reflected in the life history traits of the two populations.
The evaluation of ovarian fecundity through the total count of ovarian follicles
allowed us to define three different groups or cohorts of follicular production, and
although ovarian fecundity is not equal to uterine fecundity (one), it is likely that the
number of ovarian follicles found was due solely to the result of the meiotic division in
gametogenesis. Additionally, the presence of only one embryo per female and of the
absence of eggs in the uterus, suggest that the other pre-ovulatory vitellogenic follicles
that are not ovulated are reabsorbed in the ovary (atresic follicles; Chávez-García,
Unpub. Data.). It is well documented that in Myliobatiformes uterine fecundity is low
(Musick & Ellis, 2005) and it has been recorded that R. steindachneri has one of the
63
lowest fecundity value within the order (one embryo/female; Villaviencio-Garayzar,
1996; Bizzarro et al., 2007), which also coincides with what was observed in this study.
However, for U. paucimaculatus a similar fecundity has been reported, with one embryo
per female and rarely two (White & Potter, 2005). Although we found no relationship
between the DW of the mother and the DW of the embryo, Bizzarro et al. (2007)
reported a relationship between these two values; however, the authors did not present
statistical evidence to support their findings due to low sample numbers.
The annual reproductive cycle described in this study is similar to what has been
described by other authors for the Mexican northwest (Villavicencio-Garayzar, 1996;
Bizzarro et al., 2007), and even for R. bonasus in the Gulf of Mexico (Poulakis, 2013).
Our results showed that follicular development occurred during almost all months
sampled (nine). This allowed us to corroborate a continuous reproductive cycle. Once
the larger follicles are ovulated, the next cohort begins the subsequent maturation; this
was also demonstrated by the presence of pre ovulatory vitellogenic follicles (VOF = 6)
in a female at the post-partum maturity stage.
The greatest follicular diameters were observed in May, which also coincides
with the greatest presence of mature males in the area. May is probably the month
when mating starts, ending in July, as females with large vitellogenic follicles (3 cm in
diameter) were recorded then. Mating could therefore last three months for R.
steindachneri in Bahía de La Paz. Unlike other previously mentioned reproductive
parameters, there were no differences in ovulation among the populations of R.
steindachneri that have been studied in the Mexican Pacific, for which ovulation always
occurs during the summer months (Bizzarro et al., 2007).
Synchrony of the reproductive cycle has also been reported by other authors for
R. steindachneri (Flores-Pineda et al., 2008; Bizzarro et al., 2007) and for R. bonasus
of the coasts of North Carolina (Smith & Merriner, 1986) and Florida (Poulakis, 2013).
This condition is given by the continuous production of ovarian follicles in the ovary
while gestation occurs. It is very likely then that these species do not present a period
of cessation of the reproductive cycle, with exception of the only report made by Pérez-
Jiménez (2011) for the southeastern Gulf of Mexico, where he found that R. bonasus
64
reproduces biennially without synchrony in its reproductive period, proposing that
females give birth every two years. This last condition could be also presented by R.
steindachneri in this study, since a resting female (87.5 cm DW) was registered in
February. Although the uterus was not flaccid, weigthed 34.4 g, and its uterine villis
measured 0.9 cm in length; t is probable that this female gave birth in July and was not
fertilized, and given that the maximum diameter of its follicles was 1.1 cm, probably it
restarts its reproductive cycle again in May, when it reaches the ovulation diameter.
The range of birth sizes evaluated in this research is similar to that recorded by
other authors for the same species: 38 to 45 cm DW off the Sonora coast (Bizzarro et
al., 2007); 40 to 44 cm DW off both coasts of the BCS peninsula, Mexico (Villavicencio-
Garayzar, 1996); and 40 cm DW in the northern Gulf of California (Villavicencio-
Garayzar, 2000). Although not enough information could be collected to describe the
entire embryonic development, we report the smallest embryonic size for the species
(6.8 cm DW), recorded in July. A 21 cm DW embryo was recorded in October,
suggesting rapid embryo growth. Embryos subsequently reach sizes between 19.9 and
30.1 cm DW in March; the largest 38.1 cm embryo was recorded in May. This suggests
that embryonic growth is rapid during the first months (summer), slow between autumn
and winter, when the presence of the species is reduced in Bahía de La Paz, to finally
increase starting in May. This same temporal behavior was previously reported for R.
bonasus in northern Carolina (Smith & Merriner, 1986). These authors proposed that
the migratory behavior of the species results in mothers needing energy, leading to the
cessation of embryo growth. It is also likely that the decrease in water temperature
during the winter months leads to a decrease in the metabolic rate of embryonic
development.
We propose a gestation period of between 10 to 14 months for R. steindachneri
in Bahía de La Paz (females can be fertilized in May, June or July and give birth in the
same period), this period was defined taking into account all the pregnant females in
March (with embryos in mid-development), the presence of females in post-partum
stage in May and the high frequency of neonates at the beginning of August, which
confirmed recent births (July) in the population. A similar period (11-12 months) has
65
been proposed for this species off the Sonora coast and for Bahía Almejas (Bizzarro et
al., 2007; Flores-Pineda et al., 2008).
Bahía de La Paz is occupied mainly by juvenile animals which enter the bay in
January; these organisms probably represent those born in the previous summer.
Mature animals enter the bay in the summer to copulate and give birth, and once this
activity is over, they begin to migrate in autumn-winter, leaving only new recruits in the
bay, who leave the area in November. Because this bay is occupied mostly by neonates
and juveniles, and because the species reaches maturity at large sizes and has low
fecundity and only reproduces once a year, it is advisable to carry out demographic
studies to assess the degree of vulnerability to overfishing, since according to Bizzarro
et al. (2007) R. steindachneri is a resource that is often caught by artisanal fisheries
throughout the Gulf of California.
66
CHAPTER III
REPRODUCTIVE AND LIFE HISTORY STRATEGIES OF
Narcine entemedor AND Rhinoptera steindachneri: TWO
VIVIPAROUS SPECIES WITH DIFFERENT REPRODUCTIVE
MODES
María I. Burgos-Vázquez1,*, Víctor H. Cruz-Escalona1 and Paola A. Mejía-Falla2
1 Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas.. La Paz, Baja California Sur, México. Av. Instituto Politécnico Nacional s/n Col. Playa Palo de Santa Rita
Apdo. Postal 592. Código Postal 23096 La Paz, B.C.S. 2Fundación colombiana para la investigación y conservación de tiburones y rayas,
SQUALUS. Calle 10A No 72-35, Cali, Colombia.
67
ABSTRACT
Narcine entemedor and Rhinoptera steindachneri are two sympatric batoids species in
Bahía de La Paz, BCS, Mexico, both of commercial importance in the same region.
Differents biological and population parameters related to reproduction, age, growth,
and survival, were considered and estimated in order to describe and contrast the
reproductive and life history strategies of both species, and to define a priori the fishing
susceptibility of each one, contrasted by the characteristics of an "optimum" theoretical
life history strategy. N. entemedor females have relatively slow growth (k = 0.17
cm.year-1), small to medium size (maximum total length observed: 84 cm LT), early age
at maturity (5.1 years), high fecundity (24 embryos/female) and intermediate lifespan
(14.8 years). R. steindachneri females have intermediate growth (k = 0.21 cm. year-1),
medium size (maximum disc width observed = 94.2 cm), early age at maturity (female
= 3.8 years), low fecundity (one embryo/female) and relatively short lifespan (female =
9.8 years). According to the reproductive effort, N. entemedor invests more energy than
R. steindachneri in terms of fecundity, while R. steindachneri invests more energy in
the embryo body mass. According to all the evaluated traits, N. entemedor presents
probably a trade-off between small size at birth and high fecundity, while R
steindachneri a low fecundity by large size at birth. N. entemedor has better capacity
to recover from an overfishing event (r' = 0.48) and a higher survival (Sx = 0.73) than
R. steindachneri (r' = -0.18; Sx = 0.62). From all the variables evaluated, N. entemedor
is closer to the "optimum" theoretical life history than R. steindachneri, therefore the
life history strategy of N. entemedor allows to be less susceptible to the population
decline by fishing.
68
3.1 INTRODUCTION
Reproductive strategy is one of the most important aspects within the life history
strategy of a species, since it depends on the survival of new recruits in the population
(Stearns, 1976; Wootton, 1984; Roff, 1992; Pianka, 2000). The elasmobranchs are
characterized by presenting a great diversity of reproductive strategies and according
to Carrier et al. (2004), this has allowed them to survive through more than 416 million
years. Life history theory tries to explain how the life traits of a species are shaped, so
it can survive to under environmental conditions in which it lives. For explain the life
history strategy of a species, it is necessary to evaluate size at birth, growth rate, age
and size at maturity, number of offspring, reproductive effort, mortality rate and life
expectancy (Stearns, 2000). Since the available energy in the environment is limited,
the organism can not dispose of it freely and not all traits can be maximized (with the
objective of increasing survival), therefore the energy must be distributed among each
trait through compensations (trade-offs) between these traits to optimize the available
energy in the environment and increase the reproductive success and survival of the
species (Stearns, 1989; Frisk et al., 2005; Braendle et al., 2011; Vrtilek, 2014).
There are several theories to describe the life history strategies of the species; r
– K theory (Dobzhansky, 1950), bet-hedging theory (Stearns, 1976), triangular life
history model (Winemiller & Rose, 1992), age-specific models (Stearns, 1992), among
others. As a generality, and compared with teleost fish, elasmobranchs (sharks, skates
and rays) have life history traits characterized by slow growth, late maturity, low fertility,
low mortality rates and long lifespan, what could be classically defined as K-selected
(Holden, 1974); these traits are also related to vulnerability to fishing compared to
teleost fish (Holden, 1974). However, as a result of the great diversity of life history
strategies among elasmobranch (Compagno, 1990; Hoening & Gruber, 1990; Cortés,
2000; Frisk et al., 2005; Cailliet, 2015), not all species respond equally to fishing
pressure (Casey & Myers, 1998; Walker & Hislop, 1998; Stevens, 1999; Mejia-Falla et
al., 2012), Nevertheless, despite to the great diversity of life history strategies that
elasmobranchs present as a group, there are some studies that have tried to explain
these strategies (Compagno, 1990; Cortés, 2000, 2004; Frisk et al., 2005, Frisk, 2010).
69
Frisk (2010), based on the evaluation of life history traits of the four order of batoids,
defined this group with a great diversity of life history strategies, of long lives and slow
growth and with both, maturity and longevity, similar to other elasmobranchs.
Within the reproductive strategies of batoids, two reproductive parity are
presented: oviparity and viviparity (Wourms & Demski, 1993). Viviparity has been
defined as the most evolved and advantageous reproductive mode (Wourms &
Lombardi, 1992), since it increases the probability of survival of the neonates through
the nutrition generated by the mother and allows larger sizes at birth given the decrease
of the fecundity (Shine, 1989; Clutton-Brock, 1991; Roff, 1992). Specifically, in batoids,
the viviparity is represented in the four orders that make up this taxonomic group and
only the Rajidae family is oviparous (Musick & Ellis, 2005).
Among the viviparous batoids there are only two reproductive modes: viviparous
with yolk sac and definitive lipid histrotrophy. Viviparous with yolk sac is present in the
Torpediniformes, Pristiformes and some Rajiformes, whereas the definitive lipid
histotrophy is only found in the Myliobatiformes (Musick & Ellis, 2005). Although both
reproductive modes are viviparous, there are several differences between them. One
of the most conspicuous, is the mode in which the mother feeds the embryos. In the
case of viviparous with yolk sac, the embryo will depend exclusively on the viteline
reserve (with the exception of species that have limited histotrophy, where the mother
produces and secretes low concentrations of nutrients, which serve as additional
supplement to the vitelline reserve), while in definitive lipid histotrophy, the mother
produce and secrete a large portion of the food that the embryo will depend on once
the yolk reserve is finished (in the early stages of gestation; Ranzi, 1932, 1934; Hamlett
& Koob, 1999; Aschliman, 2004; Musick & Ellis, 2005).
Due to the patterns of energy designation in reproduction are related to the life
history strategy of a species (Frisk & Miller, 2009) and considering that the viviparity
could be selectively more advantageous, could be possible to infer which reproductive
mode invests more energy on the offspring to ensure the survival of the descendants.
Acuña et al. (2001) proposed several indicators to evaluate the reproductive effort,
which is defined as the portion of the total energy that an organism possesses for the
70
reproductive processes to ensure the fertile offspring (Thompson, 1984). The method
proposes by Acuña et al. (2001) requires two main parameters to be evaluated: the
fecundity, which is a measure of the fitness of an organism (Charlesworth, 1994), and
the mass off the offspring, since it allows comparing the investment of energy by
cohorts.
The reproductive aspects, as well as the life history traits of a species, are
necessary to assess the vulnerability of a species to an overfishing event (Frisk et al.,
2001), as well as information about the biological aspects of the species of some groups
of batoids are lacking (Frisk, 2010), however, there are studies that allow to assess a
priori the susceptibility of exploited species, through little information, as is the case of
the potential rate of population increase (r'), proposed by Jennings et al. (1998), which
is based only on some reproductive parameters (median age at maturity and fecundity)
of the population: this rate is used to measure the ability to compensate the overfishing,
where low values of r', are associated to populations that are in decline by exploitation.
Narcine entemedor (Torpediniformes) and Rhinoptera steidachneri
(Myliobatiformes) are two sympatric batoid species for in Bahia de la Paz, BCS, Mexico.
Both are viviparous species but differ in the embryonic nutrition. Narcine entemedor
presents yolk sac and limited histotrophy (Musick & Ellis, 2005; Burgos-Vázquez et al.,
2017) while R. steindachneri is a matrotrophic species with definitive lipid histotrophy
(Musick & Ellis, 2005). These species are a resource frequently extracted in the
Mexican northwest: however, the information on their aspects of life history is limited
(Villavicencio-Garayzar, 2000; Márquez-Farías, 2002; Bizzarro et al., 2007). Due to the
fact that at present, there are no demographic studies that allow the definition of an
adequate fisheries assessment for N. entemedor and R. steindachneri, this study
proposes a priori the evaluation of the vulnerability of these species to fishing
exploitation, through the analysis of reproductive effort, survival and the potential rate
of population increase (r'). Finally, the reproductive and life history strategy will be
contrasted to define which of the two populations is less susceptible to decline due to
an overfishing event.
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3.2 MATERIAL AND METHODS
3.2.1 Reproductive mode and effort
Reproductive mode was defined for each species and comparisons between
maximum follicular and embryonic mass were performed considering the net difference
and the proportion between them.
Reproductive effort was evaluated by comparison from percentage of mature
females, pregnant (with eggs and/or embryos) females and total females sampled, as
well as, the active reproductive life through the difference of the age at first maturity
and the maximum age estimated in females. Likewise, each species was evaluated
from indicators based on both uterine fecundity and mass as proposed by Vooren
(1992) and modified by Acuña et al. (2001). Variables related to reproductive effort in
relation to fecundity were estimated based on the number of embryos with which the
females contribute per reproductive cycle and on the other hand, the variables related
to mass were estimated based on the embryonic mass that mothers provide during the
reproductive cycle.
3.2.2 Life history traits
In this work, the previously determined life traits for N. entemedor and R.
steindachneri (in the same study area: Bahía de la Paz, BCS) were resumed from
Pabón-Aldana (2016), Mora-Zamacona (2017), Burgos-Vázquez et al. (2017) and
Burgos-Vázquez et al. (in press). In this way, it was possible to define and contrast the
reproductive strategy and life history of both species by graphs and tables using the
age and growth parameters [maximum size (observed), asymptotic size, growth
coefficient and maximum age], as well as reproductive parameters [sex ratio, size at
birth, maximum ovarian fecundity (all ovarian follicles), ovarian fecundity (only
vitellogenic ovarian follicles), maximum uterine fecundity, size at first maturity, median
size at maturity (TM50) and pregnancy (TP50)]. Parameters related with size (cm) are
given in total length (TL) for N. entemedor and in disc width (DW) for R. steindachneri.
Other parameters were estimated from individual age-maturity data of females
of both species. Age at first maturity was defined by the youngest mature female
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registered; median age at maturity (A50) and median age at pregnancy (AP50) were
calculated using a binomial logistic model (0: immature individuals / females without
eggs or embryos in uterus; 1: mature individuals/females with eggs or embryos in
uterus), as follows:
𝑷𝒊 = (𝟏 + 𝒆 −(𝒂+𝒃∗𝑨𝒊))−𝟏
where P𝑖 is the fraction of mature/pregnant females at age Ai, a and b are model
parameters, and -a/b corresponds to A50 or AP50. (Mollet et al., 2000).
Survival (Sx) in females was evaluated by the method of Campana et al. (2001),
using the value of the maximum age of each species.
3.2.3 Population parameter
The potential population increase rate (r'), proposed by Jennings et al. (1998) as
a measure of the ability of a population to compensate for exploitation was calculated
for each species from the following equation:
𝑟′ = (𝐿𝑛(𝑓𝑒𝑐𝑢𝑛𝑑𝑖𝑡𝑦))
𝐴50
where the fecundity represents the 50% of the estimated average fecundity, and A50
the age at maturity. Comparisons between species were carried out, considering the
assumption that low values of r are associated with the susceptibility to decrease in
abundance by fishing (Frisk et al., 2001).
3.2.4 Definition and comparison of the life history strategies of both species
Based on all the information generated and evaluated, the life history strategy of
the two studied species was defined.
As an attempt to have a point of comparison to be able to determine which of
both species present better biological and population traits and to contrast the life
history strategies between N. entemedor and R. steindachneri were defined as
"indicators" those variables derived from the reproductive mode, reproductive effort, as
well as reproductive, age and growth and population parameters. Due to the great
73
diversity of life history strategies presented by the elasmobranchs as a group, we chose
those indicators that favor survival, and are less vulnerable to decline under fishing
conditions which theoretically tend to maximize the energy available in the
environment, and will allow to reproduce and leave fertile offspring with high probability
of survival were conceived. A total of 45 indicators were analyzed for both species, and
a qualitative value was designated that approaches the "optimum value". These optimal
values were designated based on those life history traits which are less vulnerable to
decline in a population under fishing exploitation conditions (Holden, 1970, Walker &
Hislop, 1998; Musick, 1999; Dulvy et al., 2000; Stevens et al., 2000; Cailliet, 2015),
which are also related to the high r' values (high values are associated with populations
that are more likely to recover from the decline in response to exploitation; Jennigs et
al., 1999; Frisk et al., 2001). Finally, the species that presents more variables close to
the "optimum" was designated as the most advantageous to recover from
environmental or anthrophogenic disturbances.
3.3 RESULTS
3.3.1 Reproductive mode and effort
Narcine entemedor as well as R. steindachneri are viviparous species with
matrotrophy as their mode of embryonic nutrition. Notwithstanding, N. entemedor
presents viviparity with yolk sack and limited histotrophy (Fig. 1a) whereas, R.
steindachneri presents definitive lipid histotrophy (Fig. 1b).
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Figure 1.- Uterus and embryo of a) Narcine entemedor (E = in develop stage embryo) and b) Rhinoptera steindachneri (E = early stage embryo). Abbreviations are as follows: T = trophonemata; ys = yolk sac.
a
b
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The follicular mass (completely vitellogenic ovarian follicles) are similar in both
species; however, the embryonic mass (embryos in late gestation phases) is greater in
R. steindachneri (Fig. 2a). Rhinoptera steindachneri embryos begin their development
("in formation" phase) with approximately 1.3% of contribution of the yolk sac, and the
rest of the mass gained during gestation (11.2 to 841 g; 98.7% increase gained) is
provided by the mother. In contrast, N. entemedor embryos begin their development
with a yolk sac contribution of 28.1% (13.4 to 47.7 g. Additionally, R. steindachneri
embryos completely consumed the yolk reserve in the "mid" phase, while N. entemedor
embryos do it in the "late" phase.
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Figure 2.- Proportion between a) maximum follicular mass and maximum embryonic mass and b) mature and pregnant females in relation to the total females of Narcine entemedor and Rhinoptera steindachneri captured in Bahía de La Paz.
The reproductive cycle of both, N. entemedor and R. steindachneri is annual,
continous and syncronous. Their ovulation period coincides with spring-summer but,
N. entemedor has an ovulation from July to September, whereas in R. steindachneri
from May to July. The gestation period in R. steindachneri is longer (10 to 14 months)
than in N. entemedor (4 to 5 months); only N. entemedor presented embryonic
diapause as a reproductive tactic.
Narcine entemedor presented the largest proportions of mature (71.9%) and
pregnant (51.9%) females in comparison with R. steindachneri in which only 36% of
the females sampled were mature and only 10% had embryos (Fig. 2b).
From the comparisons of indicators of reproductive effort based on fecundity and
mass (Table 1), it was found that the reproductive effort based on fecundity, showed
that average mass (W1) and the maximum eviscerated mass (W2), were higher n R.
steindachneri. Both species have approximately one year for the duration of the
reproductive cylce (R1), but reproductive life (R2) is four years higher in N. entemedor
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(12 years) than in R. steindachneri, which is due to the difference at the maximum age
of both species (A2). In relation to the average number of embryos per litter (An1), N.
entemedor presented a higher value (8 embryo/female) than R. steindachneri (1
embryo/female). As a result of the difference in the number of offspring, the annual
production of offspring (Ap2) and the number of offspring accumulated during the
period of reproductive activity (No3), was higher in N. entemedor for both indicators.
On the other hand, the mass-dependent indicators showed that the mass at first
maturity (W3), the average mass of embryos to term (W4), the average litter mass (W5),
the annual mass of juvenile production (W6), the growth during the life phase of
reproductive activity (W7) and the accumulated mass of the progeny (W8) were higher
in R. steindachneri. The relative production of litter biomass per cycle (Rwlitter) value
was the same for N. entemedor (average fecundity = 8) and R. steindachneri (0.9). The
relative annual biomass production (Rwyear) as well as the relative body mass at birth
(Rwbirth) was higher in R. steindachneri, however, the relative production of biomass
during the reproductive phase of life (RB1), the relative production of body mass during
the reproductive phase of life (RB2) and the relative production of the litter mass during
the reproductive phase of life (RB3) were similar for both species (Table 1)
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Table 1.- Comparison of indicators of reproductive effort based on fecundity and mass among Narcine entemedor, Rhinoptera
steindachneri and the optimal qualitative value for each parameter according to the r – K theory.
Indicator Definition Narcine
entemedor Rhinoptera
steindachneri Optimum value
Specie "closest" to the optimum
Fecundity
A1-Age at first maturity (years) Age of younger mature
organism 2.8 1.7 Low R.s
A2-Maximum age (years) Maximum age recorded 14.8 9.8 High N.e
W1- Average mass (eviscerated; g)
Average mass of all eviscerated females
2709 3737.1 High R.s
W2- Maximum eviscerated mass (g)
Maximum eviscerated mass recorded
5200 12900 High R.s
R1-Duration of the reproductive cycle (years)
Beginning of follicular development until birth
1 1 Short Both
R2-Duration of the reproductive life (years)
A2 - A1 12 8.1 Long N.e
An1- Average number of embryos (embryos per litter/year)
N. entemedor: females with fecundity equal to one were
eliminated to rule out abortions)
8.0 1 High N.e
Ap2- Annual production of offspring (embryos per litter/year)
A1/R1 8.7 1.1 High N.e
No3-Number of offspring accumulated during the period of
reproductive activity (annual production of offspring in the
reproductive life cycle)
R2 ∙ Ap2 104.4 8.8 High N.e
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Mass
W3-Mass at first maturity (g) Mass of the smallest mature
organism registered 1710 3900 Low N.e
W4- Average Mass of embryos to term
Average mass of embryos in the "Late" stage (in R.
steindachneri, the mass of the only embryo registered at the "late" stage was considered)
36.5 841 High R.s
W5-Average litter mass (g) W4 ∙ An1 291 841 High R.s
W6-Annual mass of juvenile production (g)
W4 ∙ An2 317.5 917.5 High R.s
W7-Growth during the life phase of reproductive activity (g)
W2 - W3 3490 9000 High R.s
W8-Accumulated mass of the progeny (g/year)
R2 ∙ W6 3809.6 7422.2 High R.s
Rwlitter-Relative production of litter biomass per cycle (g/year)
W5/W6 0.9 0.9 High Both
Rwyear-Relative annual biomass production (g/year)
W6/W1 0.1 0.2 High R.s
Rwbirth-Relative body mass at birth (g)
W4/W1 0.0 0.2 High R.s
RB1-Relative production of biomass during the reproductive
phase of life (g/year) (W7 + W8)/W3 4.3 4.2 High Both
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RB2-Relative production of body mass during the reproductive
phase of life (g/year) W7/W3 2.0 2.3 High Both
RB3-Relative production of the litter mass during the
reproductive phase of life (g/year)
W8/W3 2.2 1.9 High Both
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3.3.2 Life history parameters
The comparisons of the life history parameters among N. entemedor, R.
steindachneri and the optimal qualitative values are presented in Table 2. The
maximum sizes observed and the asymptotic size (L∞) of females and males in N.
entemedor were lower than R. steindachneri. For both species, females reached larger
sizes, lower growth coefficient (k) and higher maximum ages than males. Maximum
ages were similar between males of both species, while N. entemedor females
presented the highest maximum age.
Only N. entemedor showed significant difference in relation to the sex ratio in the
whole population in comparison with R. steindachneri, however, the embryonic sex
ratio was equal for both species.
The maximum ovarian and uterine fecundity, as well as the ovarian fecundity was
greater in N. entemedor than in R. steindachneri, which presented a fecundity of only
one embryo per female (Table 2). Consequently, the size at birth of R. steindachneri
represented the 44% of its L∞, while that of N. entemedor corresponded only to 17.7%
of the L∞ estimated for the species (Fig. 3a).
Both species reached the size at first maturity and the TM50 at similar sizes in
relation to the proportion of the L∞. However, the TP50 in R. steindachneri is higher
compared to that of N. entemedor in relation to the proportion with the L∞ (Fig. 3a).
The age at first maturity in females, in relation to the maximum age evaluated of
N. entemedor and R. steindachneri was similar (Fig. 3b). The A50 in females of N.
entemedor was estimated at 5.1 years (95% CI: 3.5-6.7; Fig. 4a), whereas in females
of R. steindachneri was of 3.8 years (1.6-5.9; Fig. 4a). The AP50 for N. entemedor and
R. steindachneri was estimated at 6.8 years (5.3-8.3; 5.0-8.6, respectively; Fig. 4b,
Table 2). The proportion of the A50 in relation with the maximum age for both species,
were similar, while the AP50, was highest in R. steindachneri (Fig. 3b).
Finally, survival in females was greater in N. entemedor than in R. steindachneri
(Table 2)
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Table 2.- Comparison of age, growth, reproductive and population parameters among Narcine entemedor, Rhinoptera steindachneri
and the optimal qualitative value for each parameter according to the r – K theory.
Parameters related with size (cm) are given in total length (TL) for N. entemedor and in Disc Width (DW) for R. steindachneri.
Variable Narcine entemedor Rhinoptera
steindachneri Optimum
value
Specie "closest" to the
optimum
Female Male Female Male
Reproduction
Reproductive cylce type Annual-continuous-
synchronous Annual-continuous-
synchronous Short Both
Reproductive mode Yolk-sac Viviparity - Limited histotrophy
Viviparity - Lipid histotrophy
Viviparous Both
Embryonic nutrition Limit histotrophy Matotrophy Matotrophy R.s
Maximum follicular mass (g) 13.4 11.2 High Both
Maximum embryionic mass (g) 47.7 841 High R.s
Reproductive tactic Embryonic diapause None Which
optimizes the reproduction
N.e
Sex ratio (all individuals) 5.7: 1 (p <0.001) 0.9: 1 (p = 0.462) Higher proportion of
females
N.e
(Embryos) 1: 1 (p = 0.984) 3.3: 1 (p = 0.052) R.s
Size at birth (cm) 14.5 42 High R.s
Maximum ovarian fecundity (all ovarian follicles)
69 44 High N.e
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Ovarian fecundity (only vitelogenic ovarian follicles)
17 6 High N.e
Maximum uterine fecundity 24 1 High N.e
Size at first maturity (cm) 54.5 -- 62 63 Low R.s
Median size at maturity (cm) 58.5 -- 71.8 68.5 Low N.e
Median size at pregnancy (cm) 63.7 -- 84.3 -- Low N.e
Age at first maturity (years) 2.8 -- 1.7 1.1 Low N.e
Median age at maturity (years) 5.1 -- 3.8 3.6 Low N.e
Median age at pregnancy (years) 6.8 -- 6.8 -- Low N.e
Age and growth
Maximum size observed (cm) 84 59 94.2 82.5 Small N.e
Asymptotic size (L∞; cm) 82.1 62.3 95.4 79.15 Small N.e
Growth coefficient (k; cm years-1) 0.17 0.32 0.21 0.25 Fast R.s
Maximum age (years) 14.8 6 9.8 7 Low R.s
Population parameters
r' (In) 0.48 -0.18 High N.e
Sxfemales 0.73 0.62 High N.e
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Figure 3.- Proportion of the a) size at first maturity, median size at maturity (TM50), median size at pregnancy (TP50) and size at birth with the asymptotic size (L∞) [total length (TL in Narcine entemedor and disc width (DW) for Rhinoptera steindachneri] and b) proportion of maximum age, age at first maturity, median age at maturity (A50) and median age at pregnancy (AP50; %: proportion with the maximum age evaluated) of Narcine entemedor and Rhinoptera
steindachneri.
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Figure 4.- Ogive of a) median age at maturity (A50), and b) median age of pregnancy (AP50) of Narcine entemedor and Rhinoptera steindachneri.
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3.3.3 Population parameter
The value of the potential rate of population increase (r') was higher in N.
entemedor than in R. steindachneri (Table 2).
3.3.4. Definition and comparison of the life history strategies of both species
Based in all the variables of reproductive effort, reproduction, age and growth
and others population parameters, the life history strategy of N. entemedor, can be
defined as: small to medium size, with slow growth, small size at birth and high
fecundity, large size and early age at maturity, large size at pregnancy and intermediate
age at pregnancy, the lifespan was intermediate with high survival, and with high
potential rate of population increase (r’). Regarding to R. steindachneri, this is a species
of medium size, with intermediate growth, large size at birth and low fecundity, large
size and early age at maturity, large size and late age at pregnancy, but relatively low
lifespan, with low survival and a negative potential rate of population growth (r’) value.
Both species have a high reproductive effort. In the specific case of N. entemedor,
presents a higher output compared to R. steindachneri, when the parametrs are
analized in relation to fecundity however, based on the indicators of mass, R.
steindachneri presented a higher number. The above indicates that both species have
a high reproductive effort, close to the optimum.
Finally, of the total of 45 variables analyzed (related to reproductive effort, age,
growth, reproductive and population parameters), R. steindachneri presented 17
variables close to the "optimum" and N. entemedor 20 and eight variables tie. The
survival (Sx) and the potential rate of population increase (r') were higher in N.
entemedor, therefore it is likely that with the traits that N. entemedor presents, have
better capacity to overcome environmental disturbances and better capacity to
compensate the exploitation than R. steindachneri.
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3.4 DISCUSSION
The results evaluated in this study allowed to compare, based on the
reproductive mode, age, growth, reproductive parameters, and reproductive effort, two
different populations of batoids that are frequently captured in the southern Gulf of
California. It was possible to describe the life history strategy and the reproductive
potential of both species, which allows a priori projection on the susceptibility of these
species to fishing in the study area.
The main difference with respect to the reproductive mode between N.
entemedor and R. steindachneri is the mode in which the mother nourishes the
embryos. Whereas that N. entemedor presents limited histotrophy (Burgos-Vázquez et
al. 2017) namely with a low nutritional intake (mucoproteins) from the mother towards
the embryo (Musick & Ellis, 2005; Hamlett et al., 2005), R. steindachneri presents
matrotrophy with definitive lipidic histotrophy (Musick & Ellis, 2005, Hamlett et al.,
2005), where embryos are nourished in most of the gestation by the nutrients generated
by the mother, at least from the "mid" stage of embryonic development (Burgos-
Vázquez et al., in review). Therefore, R. steindachneri invest more energy for
embryonic nutrition, the production of the viteline reserve and the production and the
secretion of uterine milk (Wallace, 1978; Wourms, 1981; Wallace & Selman,1981;
Wourms & Lombardi, 1992). In this sense, N. entemedor have an advantage by
requiring less energy for embryonic development; however, matrotrophy gives the
advantage to R. steindachneri to produce larger and more developed offspring, with a
greater probability to survive (Wourms, 1981; Qualls & Shine, 1995; Goodwin et al.,
2002).
The reproductive cycle was similar for both species, and although the peaks
of ovulation, mating and parturition were comparable, R. steindachneri presents a
gestation period three times longer than N. entemedor, this could be related to the
reproductive mode (more energy requirements by the mother for embryonic nutrition in
R. steindachneri) and the offspring size at birth (largest sizes in R. steindachneri).
Additionaly, the reproductive cycle, N. entemedor presents embryonic diapause as a
reproductive tactic in response to unfavorable environmental conditions to birth
88
(Burgos-Vázquez et al., 2017), increasing in this way the survival of the offspring once
it is secured the most favorable external conditions for neonates; as well as allowing
the mother to present a recovery time after the previous reproductive event (Marshal
et al., 2007; Waltrick et al., 2012), which to N. entemedor some advantages over R.
steindachneri in reproductive fitness.
Other advantages found for N. entemedor was the high proportion of mature
(able to ovulate) and pregnant females registered in Bahía de la Paz, in this way more
than half of the sampled population could be able to provide litter to the next
reproductive stock. In contrast, only 36% of R. steindachneri females were mature.
However, this difference could be due to the migratory condition of R. steindachneri,
causing mature organisms to migrate to other areas outside of the bay (Bizzarro et al.,
2007; Burgos-Vázquez et al., in review).
The size of the species has a strong impact on the patterns of the traits of the
life history strategy and constrained the "fast-slow" continuum (Stearns, 1983), where
"slow" species will tend to be large-bodied, slow-growing and low-fecundity, while "fast"
species will tend to have opposite traits (Charnov, 1993; Frisk et al., 2005; Frisk et al.,
2001; Dulvy & Reynolds, 2002; Roff, 2002; Frisk, Miller & Dulvy, 2005). For example,
Cortés (2000) found a positive relationship between maternal size with litter number
and size at birth for sharks. Later, in a comparative study of the life history strategies
of batoids, Frisk (2010) mentioned that electric rays (family Narcinidae) have small
bodies and high fecundity in comparison with the eagle rays (family Myliobatidae), that
usually have medium to large bodies and low fecundity which coincides with the present
study. This difference in size between both species could influence two traits of the life
history strategy: reproductive mode and fecundity. In the first case, it has been
established that the viviparity evolution has led to various modifications in the
physiological and morphological patterns of organisms, and one of them is body size,
which also explains why in both species the females had maximum observed sizes
larger than the males, with the purpose of loading the embryos during pregnancy (Hoar,
1969; Amoroso et al., 1979; Wourms, 1981; Dodd, 1983; Nagahama, 1983; Shine,
1985; Callard & Ho, 1987; Wourms et al., 1988; Callard et al., 1988,1989; Cortés,
89
2000). Likewise, the Narcinidae family is characterized by smaller bodies compared to
the Myliobatidae family, and all the rays of the Narcinidae family present viviparity with
yolk sac, which results in smaller size at birth in comparison with the Myliobatidae
(Muick & Ellis, 2005; Frisk, 2010). It is probable that the small size of the electric rays
is compensated with the reproductive mode that leads to high fecundity but small birth
sizes, unlike Myliobatidae, that their reproductive mode allows a single breed, but of
large body. The second case, fecundity, R. steindachneri, who has definitive lipid
histotrophy as a reproductive mode, due to the large amount of nutrients invested in
gestation, will allow to have larger offspring, therefore, the mother's body size will be
related to that aspect. On the other hand, N. entemedor due to having low energy intake
by the mother (limited histotrophy), the offspring born with small body sizes. This
results, its one of the main trade-off in the life history: fecundity due to the size at birth
(Frisk et al., 2005), compensating the small body size at birth with high fecundities for
N. entemedor and high size at birth with low fecundity in R. steindachneri.
According to Cortés (2000) one of the trade-offs in the life history of the
elasmobranchs, occurs between the litter size and the energy invested in each young.
Within the Myliobatiformes, the diamond stingray ray, Hypanus dipterurus presents a
asymptotic size (DW∞) of 76.2 cm (Carmona-Sánchez, 2016), lower than those
estimated for R. steindachneri (95.4 cm DW∞), and although they have the same
reproductive mode, fecundity is greater in H. dipterurus (1-4 embryos/female; Ebert,
2004), probably to compensate the smaller size of the species. Within the
Torpediniformes (all viviparous with yolk sac; Musick & Ellis, 2005), although
Tretonarce californica reach up to 137 cm TL (Ebert, 2003), its fecundity is lower (17
embryos/female; Neer & Cailliet, 2001) than N. entemedor, it is probable that the
greater fecundity in N. entemedor is due to the fact that this species has smaller sizes
of young at birth (14.5 cm TL), compared with T. californica that presents offspring at
23.1 cm TL (Neer & Cailliet, 2001), therefore it is probable that T. californica
compensates the low fecundity (compared with N. entemedor) by larger offspring.
In general, the elasmobranchs have long lifespan (Hoening & Gruber, 1990);
however, there are several studies that have reported differences between the
90
maximum ages through the different taxonomic groups, which is closely related to its
phylogeny (Jennings et al., 1999; Frisk et al., 2001, 2005) and the relationship it has
with other life history traits, such as the k growth rate (Cortés, 2000). In the study carried
out by Cortés (2000), he found a negative correlation between the growth rate and
lifespan, that is, that low values of k will tend to be from long-lived species, while high
values will have lower maximum ages. In the case of the species analyzed in the
present study, it can be observed that although N. entemedor reaches minor maximum
sizes than R. steindachneri, its slower growth rate (0.17 cm.year-1) which coincides with
the proposal by Cortés (2000).
Two different trends were identified in the sex ratio of the sampled population of
N. entemedor and R. steindachneri. In the first case, the greater proportion of females
on males (5.7: 1) of N. entemedor, is due to the fact that males only enter to the bay
when it is the mating season generating an energetic extra cost in males as a result of
the migration. However, for the sampled R. steindachneri population (0.9:1 sex ratio),
no sexual segregation was identified which could be benefical to the population in terms
of energy saving for males that have no need to migrate looking for females.
An evolutionary trend among vertebrates is related in the trade-off within a few
offspring and large sizes at birth, while a large litter will have small sizes at birth
(Sargent et al., 1987; Pavlov et al., 2009). According to the r - K selection theory, those
organisms with r selection, will have a larger litter but of smaller bodies, and those with
selection K, the inverse (Pianka, 1970). However, the elasmobranchs present different
life history strategies, which is characterized by having different sizes at birth through
the taxonomic groups (Cortés, 2000, 2004; Frisk et al., 2001; Frisk & Miller, 2009; Frisk,
2010). In the comparative study, Cortés (2000) found that the size at birth is closely
related to the maximum size, that is, large organisms will have larger offspring and, in
addition, this pattern will also be influenced by the reproductive mode. Although the two
species evaluated in the present study showed similar maximum body sizes and their
sizes at birth were different, which differs from Cortés (2000). However, it is likely that
this difference is mainly due to the reproductive mode, that is, due to the high energy
investment of the mother towards the embryo in R. steindachneri (definitive lipid
91
histotrophy), which favors the development of a larger embryo (at 44% of the
asymptotic size), and thus increasing the survival probability in the first stages of life.
This evidenci the trade-off between fecundity and size at birth, in R. steindachneri;
whereas N. entemedor compensates the small size at birth (to 17.7% of the asymptotic
size) with a large litter what also increases the possibility of survival.
Compared with teleost fish, maturity occurs late in batoids (Frisk, 2010). This
author found an average age at maturity in females at 8.6 years and in males at 6.9
years, and mentions that the ratio between maturity and longevity results in a short
reproductive life (maximum age – A50), similar to what was recorded in this study, 9.7
years and 6 years for N. entemedor and R. steindachneri, respectively, which
represents the active reproductive life. Frisk et al. (2001) described a high relationship
between the age at maturity and maximum age, and according with these authors, the
difference between age at maturity and maximum age represent the portion of time that
the organism will invest in reproduction. This coincides for the present study, where N.
entemedor presents a ratio = 0.34 and R. steindachneri ratio = 0.39 of the maximum
age and A50, therefore both species will invest the relative same portion of time in their
life cycle to the reproductive activity, likewise, considering this ratio, it can be defined
that both species have early maturity ages. In relation to the age at pregnancy, R.
steindachneri presented a higher proportion (0.7) compared to N. entemedor (0.5), this
may be related to the reproductive mode, because R. steindachneri probably need
more time to develop a larger abdominal cavity to develop a single large embryo.
Cortés (2000) mentioned that maturity is reached at large sizes (75% of the
maximum size) which is similar for the present study were both species reach maturity
at large sizes (N. entemedor = 71.3 % and R. steindachneri = 75.3 % of the asymptotic
size). Frisk et al. (2005) explain that both, the large body size and the late ages at
maturity, are an evolutionary mechanism of viviparity to maximize the offspring survival,
through a extensive intrauterine cavity for embryonic development. In this study, the
largest pregnancy sizes in both species were observed, however, R. steindachneri
presented a larger size (88.4%) in relation to the maximum asymptotic size (L∞)
compared to N. entemedor (77.6%) which confirms that at larger maturity and
92
pregnancy sizes, a larger intrauterine cavity (with more capacity for embryonic
development), which is closely related to the reproductive mode of R. steindachneri.
The fecundity in elasmobranchs is related with the optimum maintenance
population abundance (Pavlov et al. 2008) despite the low fecundity compared with
teleost fish (Compagno, 1990; Cortés, 2000; Gruber et al., 2001).Additionaly, the
elasmobranch fecundity varies through phylogenetic groups, and for this reason, some
authors have made comparative studies to define the influence that has a certain life
history trait on others. Cortés (2000) defined that the litter size varies according to the
maximum size of the species and that the reproductive modes will have an influence
on the fecundity. For example, viviparous-lecithotropic species, have a higher fecundity
(N. brasiliensis = 44 embryos/female; Rolim et al., 2015), and lower sizes at birth (e. g.
N. brancoftii = 11.5 cm TL; Moreno et al., 2010) versus matrotrophic species, which
have a lower litter size (D. dipterurus = 1 to 4 embryos/year; Ebert, 2003), but with
larger sizes at birth (D. dipterurus = 21.3 cm DW; Smith et al., 2007). Although the body
sizes between N. entemedor and R. steindachneri are similar (máximum size observed:
N. entemedor = 84 cm TL and R. steindachneri = 94.2 cm DW), we could attribute that
the high difference between fecundity of both species can be related to the reproductive
mode.
This is the first study that evaluates the reproductive effort for N. entemedor and
R. steindachneri, which is an useful indicator to compare the energy that each species
invests in terms of reproduction (Voreen, 1992). The first way to evaluate the
reproductive effort (depending on fecundity), N. entemedor, presented more indicators
closest to the optimal than R. steindachneri (depending on the r - K selection theory)
therefore it is likely that N. entemedor invests more energy to reproduction in terms of
fecundity than R. steindachneri. It can be observed, for example, that the annual
production of offspring indicator (Ap2), N. entemedor showed a value eight times
greater than R. steindachneri, and the number of offspring accumulated during the
period of reproductive activity indicator (No3), 96 times higher, because both indicators
use the duration of the reproductive cycle and the percentage of litters for that time,
which confirms that the high fecundity and the embryonic diapause are advantages to
93
improve the reproductive effort in N. entemedor. Based on the fact that the reproductive
effort has the purpose of distributing the energy between the costs required by the
biological functions of the organism (growth, maintenance, reproduction; Hirshfield &
Tinkle, 1975), and due to the small size of N. entemedor, it is likely that this species
presents a trade-off between fecundity and size at birth, investing a large portion of its
energy budget in increasing litter number and not in body size.
In the second way of evaluating the reproductive effort (depending on mass), of
a total of 12 indicators, R. steindachneri showed 11 indicators close to the optimum
and N. entemedor only five. According to Roff (1992) and Haag (2013), the method of
evaluating the reproductive effort with body mass is an adecuate proxy to estimate the
energy invested towards reproduction. However, it can present some imitations, mainly
because it does not take into account the energy destined for the production of body,
gonadic and embryonic mass. The present study used the body mass of only one
embryo (the only one in the "late" phase), therefore, these results need to be taken
with carefulness. Despite the limitations that the methodology used may have, it is
evident that R. steindachneri compensates the low fecundity with the energy
investment towards embryonic development and it is likely that the reproductive effort
is higher in R. steindachneri, due to the reproductive mode (embryonic nutrition:
definitive lipid histotrophy), therefore, a trade-off between energy investment towards
embryonic development and fecundity for this species is likely to occur.
Jennings et al. (1998), proposed an alternative for the intrinsic rate of natural
increase (r) of teleost fish, based on two population parameters; fecundity, as an index
of reproductive effort, and maturity time per cohort, which is defined as the potential
rate of population increase (r'). This rate has to been used to evaluate some
elasmobranch species (Frisk et al. 2001) and it was contrasted with the values obtained
from Jennings et al. (1999). Both studies related low values of r' with populations that
are declining due to fishing exploitation. According to Frisk et al. (2001), N. entemedor
presented a high r' value (0.48), while R. steindachneri presented a very low (even
negative = - 0.18). Compared to other species (Frisk et al. 2001), N. entemedor is
among the species with the highest r' values, and only below three species: the shark
94
Sphyrna tiburo (r' = 0.60), which has an earlier maturity age, and the skates Leucoraja
erinacea (r' = 0.68) that presents a fecundity of 30 egg/year (six embryos more per year
than N. entemedor), and Amblyraja radiata (r' = 0.43) that presented a similar value,
due to age at maturity and fecundity similar to N. entemedor. In the case of R.
steindachneri, its negative value of r' could be affected by the fecundity, being one of
the lowest among elasmobranchs (one embryo/female). Frisk et al. (2001) not include
species with this fecundity values; the species with the lowest fecundity was Carcharias
taurus (two embryos/female), which had an age at maturity of seven years and r' = 0.0.
Therefore, due to the low fecundity of R. steindachneri, it is probably less productive
than N. entemedor, and in addition, it has likely less capacity to recover from
overexploitation by fishing.
The low value of the potential rate of population increase (r') has also been
associated with organisms of large body sizes, late maturity and low growth rates, are
more susceptible to population decline due to overexploitation by fishing (Frisk et al.,
2001; Frisk & Miller, 2009). Similarly, Musick (1999) related k values below 0.1, have
less chance of recovering from overexploitation, and Frisk et al. (2001) mentions that
the low values of r' are related to the low values of k. However, in the present study,
although N. entemedor presented a growth rate k slower than R. steindachneri, its r'
was higher, which may be due to the high fecundity, additionally some reproductive
effort indicators were related to fecundity, which were higher in N. entemedor (as
annual production of offspring; Ap2 or number of offspring accumulated during the
period of reproductive activity indicator; No3), and could be contributing to the value of
r' being higher in N. entemedor than in R. steindachneri.
Survival of a species is affected by other life history traits, for example, Frisk et
al. (2005), defined an overlap between those species that invest most nutrients to the
embryos (matrotrophic), and those that do not (lecitotrophic), finding that there is a
trade-off between fecundity and survival. It is likely that in the case of N. entemedor
survival is greater due to the high fecundity they present. Likewise, survival is closely
related to the population size, and when a population is artificially reduced, the survival
rate will be affected (Gruber et al., 2001). Possibly the low survival values of R.
95
steindachneri, thus as the negative value of r' results to the decrease of this population
in the bay, given the catches aimed at neonates and juveniles.
To define the life history strategy of a species, there are several theories, that
try to explain how life traits interact to shape that strategy. Among them is the r-K
selection theory (Dobzhansky, 1950), bet-hedging theory (Stearns, 1976), triangular
life history model (Winemiller & Rose, 1992), and age-specific models (Stearns, 1992).
Specifically Winemiller & Rose (1992), proposed a triangular model to explain the
adaptive response to environmental fluctuations, where they define three strategies of
life history. In the first one "Periodic", the organisms present traits as long lifespan,
high fecundity and high variation in recruitment; the second "Opportunistic", are small,
short-lived organisms, high reproductive effort and high demographic resilience; and
the third "Equilibrium" (associated with K strategists), are organisms with parental care,
large offspring and low fecundity. Thus, the traits of N. entemedor and R. steindachneri
are more similar to the "Equilibrium". Winemiller (2005) mentions that the organisms in
"Equilibrium" tend to the K selection, invest more energy in the development of the
offspring or in increasing fecundity. Likewise, Frisk et al. (2001) mention that
elasmobranchs could be defined in "Equilibrium" due to the high investment in ovarian
follicles, embryos of large sizes, long gestation periods and low fecundity, therefore N.
entemedor and R. steindachneri could be classified as species with life history strategy
in "Equilibrium" according to life strategy theory of Winemiller & Rose (1992).
Several studies have been carried out to define and compare life history
strategies among the different elasmobranch taxa (Compagno, 1990; Cortés, 2000,
2004; Goodwin et al., 2002; Winemiller, 2005; Frisk et al., 2001, 2005; Frisk & Miller,
2009; Frisk, 2010). Cortés (2004) indicated that the advantage of the r-K selection
theory is that all organisms will present traits that tend to be of the r or K strategists;
however, given the great diversity of life history strategies and traits exhibited by the
elasmobranchs, is difficult to use only a theory to describe them, even using other
factors such as morphology, physiology, behavior or space, are necessary, this same
author, based on analysis of correlation between different life history traits, clustered
the sharks into three main groups. In the first group are the organisms of large litters
96
(31 – 135 embryo/female), long lifespan (17 - 53), large maximum size (155 - 450 cm
TL), small size at birth (20 - 78 cm TL) and slow growth (k = 0.07 – 0.25 cm.year-1); the
second group is characterized by large organisms (234 - 640 cm TL), large size at birth
(62.5 - 174 cm TL), low fecundity (2 - 4 embryo/female), slow growth (k = 0.04 – 0.12
cm year-1) and long lifespan (14 – 39 years); and finally the third group, characterized
by low fecundity (5 - 15 young/female), small to moderate body (78 - 247 cm TL),
moderate lifespan (4.5 - 22 years), small size at birth (24 - 67 cm TL) and rapid growth
(k = 0.11 – 1.01 cm.year-1). According to these characteristics, N. entemedor and R.
steindachneri, could be considered within the group three; however, not all the traits of
the group three resemble these two species, because the study does not cover the
group of batoids, therefore, not all the traits of the group are being considered.
According to the reproductive characteristics and life history traits that were
evaluated in the present study, N. entemedor can be defined as a species of small to
medium body size, with relatively slow growth, intermediate lifespan, early maturity age,
high reproductive effort depending on fecundity and high capacity to compensate
overexploitation by fishing compared to other elasmobranches. While, R.
steindachneri, is a medium-sized species, with intermediate growing, relatively short
lifespan, early maturity age, high reproductive effort depending on embryonic mass and
low ability to compensate for overexploitation by fishing compared to other
elasmobranchs.
N. entemedor could be more vulnerable according to the low k values that
exhibes and which are condiered as indicator of more vulnerability to overexploitation
(Musick, 1999). When this trait is compared to R. steindachneri, in relation to fecundity,
the low values of this parameter are related to low values of r', which would indicate
that R. steindachneri is more vulnerable to a fishing overexploitation (Jennings et al.,
1998; Frisk et al., 2001). Both species can be considered with high reproductive effort,
N. entemedor through fecundity and R. steindachneri through the embryonic mass.
Finally, according to the survival and the potential rate of population increase
assessment, N. entemedor showed the highest values, therefore it could be defined as
the species more resilient to environmental disturbances and fishing susceptibility.
97
GENERAL CONCLUSION
Narcine entemedor and Rhinoptera steindachneri are species that reproduce
during the summer months in Bahía de La Paz, with annual, continuous and synchronic
reproductive cycles. The reproductive modes that present these species, viviparity with
yolk sac and limited histotrophy in N. entemedor and with definitive lipid histotrophy in
R. steindachneri, both with matrotrophy, have an influence on their life history traits.
For N. entemedor, it allows it to increase fecundity, throughout small size at birth, while
in R. steindachneri the high nutritional contribution of the mother allow it to have a
single breeding but with larger size.
The embryonic diapause presented in N. entemedor allows the species to
increase its reproductive effort depending on fecundity.
Two probable trade-offs were identified, in N. entemedor the high fecundity by the
small body size at birth of the offspring, and in R. steindachneri low fecundity by a large
size of offspring.
The combination of life history traits in N. entemedor have allowed this species to
have more capacity to survive in comparison with R. steindacherni.
According to the potential rate of population increase, R. steindachneri, is more
susceptible to overexploitation by fishing than N. entemedor.
N. entemedor presented more variables close to the "optimal", and the higher
values in the potential rate of population increase, as well as in survival, therefore N.
entemedor is more effective to designate the energy in relation to reproductive effort,
increasing the survival of the species than R. steindachneri.
98
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ANEXOS
PRODUCTOS DERIVADOS DURANTE EL PROGRAMA DE
DOCTORADO 2014 - 2018
Documentos publicados
Burgos-Vázquez, M. I., Chávez-García, V. E., Víctor H. Cruz-Escalona, V. H., Andrés
F. Navia, A. F & Mejía-Falla, P. A. Reproductive strategy of the Pacific cownose ray
Rhinoptera steindachneri in the southern Gulf of California. Marine & Freshwater
Research. In press.
Burgos-Vázquez, M. I., P. A. Mejía-Falla, Víctor H. Cruz- Escalona & Nancy J. Brown-
Peterson (2017) Reproductive Strategy of the Giant Electric Ray in the Southern Gulf
of California, Marine and Coastal Fisheries, 9:1, 577-596, DOI:
10.1080/19425120.2017.1370042
Uriel Rubio Rodríguez, Jessica A. Navarro-Rodríguez and M. Itzigueri Burgos-
Vázquez (2017). The Gregarious Behavior of Marine Fish and Their Relation to
Fishing. In Advances in Marine Biology (Eds. Adam Kovács and Patrik Nagy). NOVA,
Vol. 2.
Ehemann N.R., Pérez-Palafox X. A., Mora-Zamacona P., M. I. Burgos-Vázquez, A. F.
Navia, P. A. Mejía-Falla, V. H. Cruz-Escalona (2017). Size–weight relationships of
batoids captured by artisanal fishery in the southern Gulf of California, Mexico. J.
Applied Ichthyology. 2017;00:1–4. https://doi.org/ 10.1111/jai.13421.
Congresos
Burgos-Vázquez M. I., Mejía-Falla P. A., Cruz-Escalona V. H. & Brown-Peterson N.
Biología Reproductiva de la raya eléctrica Narcine entemedor, en la Bahía de La Paz,
BCS, México. V Encuentro colombiano sobre condrictios. Bogotá, Colombia. 2016.
Burgos – Vázquez, M.I., V. Cruz – Escalona, P.A. Mejía – Falla. Biología reproductiva
de la raya eléctrica, Narcine entemedor, capturada por la pesca artesanal, en la Bahía
119
de La Paz, B.C.S., México. VII Simposium Nacional de Tiburones y Rayas, Puerto
Vallarta, Jalisco. 2016
Ehemann N. R., X. A., Perez Palafox, P. Mora Zamacona, M. I. Burgos Vázquez, A.
F. Navia, P. A. Mejía Falla & V. H. Cruz Escalona. Relaciones de longitud y peso de
nueve especies de batoideos capturados en el sur del Golfo de California, México. XV
Congreso Nacional/ V Simposio Latinoamericano FIGIS. I Simposio Internacional de
Genomica de Peces. Aguascalientes, Aguascalientes, México. 2016.
Burgos – Vázquez, María. I., Mejía – Falla, P.A., Cruz – Escalona & Navia, A.F.
Biología reproductiva de la raya eléctrica gigante de California, Narcine entemedor, de
la pesquería artesanal en La Bahía de La Paz, B.C.S., México. 68TH annual Gulf and
Caribbean Fisheries Institute meeting, Ciudad de Panama. 2015.
Burgos – Vázquez, María. I., Mejía – Falla, P.A., Cruz – Escalona, V. & Navia, A.F.
Aspectos reproductivos de hembras de la raya eléctrica, Narcine entemedor en la
Bahía de La Paz, Baja California Sur, México. XXII Congreso Nacional de Ciencia y
Tecnología Del Mar, Ensenada, BC. 2015.
Carmona-Sánchez, A., Torres-Palacios, K., Restrepo-Gómez, D. C., Burgos-
Vázquez, M. I., Mejía-Falla, P. A., Navia A. F. & V. H. Cruz-Escalona. NOTAS
Biológicas preliminares de la raya látigo Dasyatis dipterura en la Bahía de La Paz, Baja
California Sur, México. XXII Congreso Nacional de Ciencia y Tecnología Del Mar,
Ensenada, BC. 2015.
Cursos
Curso “Ecophysiology of elasmobranchs” impartido por la Dra. Valentina di Santo,
dentro del marco del V Encuentro colombiano sobre Condrictios, Bogotá, Colombia.
Octubre del 2016.
Curso “Análisis Bayesiano” impartido por el Dr. Enrique Morales Bojórquez, en el
Centro Interdisciplinario de Ciencias Marinas (CICIMAR), en La Paz, BCS. Agosto del
2016.
120
Curso-Taller “Herramientas de Microfotografía” impartido por el Ing. German Bazaldua,
en el Centro Interdisciplinario de Ciencias Marinas del Instituto Politécnico Nacional,
en la ciudad de La Paz, BCS, México. Junio del 2015.
Curso “Uso del análisis de productividad y susceptibilidad como método alternativo
para determinar la vulnerabilidad de un stock pesquero” impartido por el Dr. Emmanuel
Furlong, en el Centro Interdisciplinario de Ciencias Marinas del Instituto Politécnico
Nacional, en la Cd. De La Paz, BCS, México. Mayo del 2015.
Curso “Fisiología Animal” en el Centro de Investigaciones Biológicas del Noroeste S.C.
(CIBNOR). Abril – junio del 2015.
Taller “Comprensión de Lectura y Taller de Expresión Escrita”, curso en Línea.
Impartido por el Centro de Lenguas Extranjeras, del Instituto Politécnico Nacional.
Enero -marzo del 2015.
Curso “Anatomía de Rajiformes” impartido por la sociedad mexicana de peces
cartilaginosos. Expositor: Dr. Abraham Kobelkowsky Díaz, en el Instituto de Ciencias
del Mar y Limnología, UNAM, en la Cd. De México. Noviembre del 2014.
Curso “Áreas de crianza” impartido por el Dr. Oscar Sosa Nishizaki (CICESE), dentro
del marco del IV Encuentro Colombiano sobre Condrictios, en Medellín, Colombia.
Octubre del 2014.
Estancias
Estancia de investigación científica en la Fundación colombiana para la investigación
y conservación de tiburones y rayas, SQUALUS, en Cali, Colombia, a cargo de la Dra.
Paola A. Mejía Falla. Octubre – diciembre del 2017.
Estancia de investigación científica en el centro de investigación Gulf Coast Research
Laboratory, en Ocean Springs Mississippi, EUA, a cargo de la Ms. Nancy Brown-
Peterson. Abril – mayo 2016.
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Tesis dirigidas
Yutzin Aimee Jiménez García. Aspectos reproductivos y de crecimiento de la Raya
Látigo Hypanus longus (Garman, 1880) en la Bahía de La Paz, BCS, México. Tesis de
Licenciatura. Universidad Autónoma de Baja California Sur. Noviembre del 2017.
Valeria Edith Chávez García. Descripción macro y microscópica del sistema
reproductor de la Raya Tecolote, Rhinoptera steindachneri (Evermann & Jenkins,
1892), en Bahía de La Paz, BCS, México. Universidad del Mar. En desarrollo.
Premios
“GCFI Student Travel Award” en el 68TH Annual Gulf and Caribbean Fisheries Institute
meeting in Panamá City. 2015.