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Reproduction and embryonic development in

two species of squaliform sharks, Centrophorus
granulosus and Etmopterus princeps: Evidence
of matrotrophy?

ARTICLE in DEEP SEA RESEARCH PART II TOPICAL STUDIES IN OCEANOGRAPHY · NOVEMBER 2014
Impact Factor: 2.76 · DOI: 10.1016/j.dsr2.2014.10.009

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Reproduction and embryonic development in two species of

squaliform sharks, Centrophorus granulosus and Etmopterus princeps:
Evidence of matrotrophy?
Charles F. Cotton a,b,n, R. Dean Grubbs a, Jan E. Dyb c, Inge Fossen d, John A. Musick e
a
Florida State University Coastal and Marine Laboratory, 3618 Coastal Hwy 98 St. Teresa, FL 32358, USA
b
Department of Natural Sciences, Savannah State University, 3219 College St., Box 20600, Savannah, GA 31404, USA
c
Norwegian Coastal Administration, P.O. Box 1502, 6025 Ålesund, Norway
d
Moere Research, Section of Fisheries, Industriveien 18, N-6517 Kristiansund, Norway
e
Department of Fisheries Science, Virginia Institute of Marine Science, College of William & Mary, P.O. Box 1346, Gloucester Point, VA 23062, USA

art ic l e i nf o a b s t r a c t

Modes of reproduction and embryonic development vary greatly among the elasmobranchs, and prior
Keywords: studies have suggested that the energetic toll of embryogenesis in lecithotrophic species depletes
Lecithotrophy embryonic organic matter by 20% or more. Matrotrophic species experience a lesser reduction or an
Histotrophy increase in organic matter during embryogenesis. To investigate the maternal–embryonic nutritional
Maturity relationship, we measured changes in organic matter from fertilization to near-parturition in embryos of
Fecundity Centrophorus granulosus and Etmopterus princeps. Embryos of C. granulosus experienced a reduction of
Dogfish 19.5% in organic matter, while E. princeps embryos experienced a reduction of 7.7% in organic matter over
Gulper
the course of embryonic development, suggesting some level of matrotrophy occurs, particularly for the
Lanternshark
latter species. Uterine villi were present in both species and developed concurrently with the embryos,
Squaliformes
increasing in length and thickness while becoming progressively vascularized. Embryos of C. granulosus
were dissected to track the partitioning of water, organic matter, and inorganic matter to the liver,
external yolk sac, internal yolk sac, digestive tract, and evicerated body throughout development. Mating
was aseasonal for both species and spatially-mediated segregation by sex and maturity stage was
observed. Ovarian cycles were concurrent for C. granulosus and consecutive for E. princeps. Size at
maturity for C. granulosus was determined to be 111 cm TL for males and 143 cm TL for females, with an
average fecundity of 5.3 embryos (range¼ 4–7). Size at maturity for E. princeps was determined to be
56.5 cm TL for males and 61 cm TL for females north of the Azores and 54 cm TL for males and 69 cm TL
for females near the Charlie Gibbs Fracture Zone. Average fecundity was 11.2 embryos (range¼ 7–18) for
this species. This is the first reporting of reproductive parameters for these two species, and the
information provided will be valuable for informing stock assessment models in areas where these
species are fished.
& 2015 Published by Elsevier Ltd.

1. Introduction supplements the yolk sac. However, several authors note the lack
of available data to properly classify many presumed yolk-sac
Modes of reproduction in elasmobranchs are quite varied and viviparous species as such (Hamlett et al., 2005; Musick and Ellis,
include internal (viviparous) and external (oviparous) forms of 2005). Matrotrophy occurs in the form of four different processes,
embryonic development (Wourms, 1977). Viviparous species exhi- with varying levels of maternal input (Table 1): uterine secretions
bit one of a number of matrotrophic modes of embryonic devel- (histotrophy, either lipid or mucoid), embryonic feeding on nutri-
opment, or the lecithotrophic mode of development known as tional ova (oophagy), intra-uterine cannibalism (adelphophagy),
yolk-sac viviparity (Hamlett et al., 2005; Musick and Ellis, 2005). placental connection (placentotrophy) (Hamlett et al., 2005).
Yolk-sac viviparity is classified as lecithotrophic because even Mossman (1987) defines histotroph as “any nutritive material,
though embryos develop internally, no maternal nourishment including glandular secretions, cells, and cell debris that is available
to the embryo or fetus and is derived directly from maternal tissues
other than blood.” Uterine secretion in the form of “lipid histotro-
n
Corresponding author. phy” is found only in certain batoids and should be differentiated
E-mail address: ccotton@fsu.edu (C.F. Cotton). from “limited histotrophy” (also known as mucoid, incipient, or

http://dx.doi.org/10.1016/j.dsr2.2014.10.009
0967-0645/& 2015 Published by Elsevier Ltd.

Please cite this article as: Cotton, C.F., et al., Reproduction and embryonic development in two species of squaliform sharks,
Centrophorus granulosus and Etmopterus princeps: Evidence of.... Deep-Sea Res. II (2015), http://dx.doi.org/10.1016/j.dsr2.2014.10.009i
2 C.F. Cotton et al. / Deep-Sea Research II ∎ (∎∎∎∎) ∎∎∎–∎∎∎

Table 1 weight gain during embryogenesis occurs in every elasmobranch

Modes of reproduction in elasmobranchs, with relation to maternal nutrition species, regardless of any maternal input, due to the uptake of
supplied during embryogenesis (adapted from Wourms, 1981; Musick and Ellis,
water. However, metabolic losses during embryogenesis result in a
2005).
partial depletion of the organic matter contained in the yolk at the
Reproductive mode Matrotrophy (M) or Relative level of maternal time of fertilization, generally assumed to be a reduction of 20% or
lecithotrophy (L) nutritional supplementationn greater (Hamlett et al., 2005). Measurements of ash-free dry weight
for early- and late-stage embryos can be compared to quantify loss
Oviparous L None
Viviparous
of organic matter over the course of embryogenesis. If final
Yolk-sac viviparity L None (presumed) measurement of organic matter is greater than 80% of that
Lipid histotrophy M Extensive contained in a freshly fertilized egg, it is assumed that some form
Limited, or M Minimal to moderate of maternal nourishment is utilized (Hamlett et al., 2005; Musick
“mucoid”
and Ellis, 2005).
histotrophy
Oophagy M Extensive Centrophorus granulosus (Supplementary Fig. 1) is one of the
Adelphophagy M Extensive largest species of gulper shark (Family Centrophoridae), with a
Placentotrophy M Moderate to extensive maximum size of at least 1760 mm TL (Castro, 2011). This species
n
has a patchy global distribution and is typically found along the
In addition to the yolk-sac.
edges of continental shelves and upper slopes (White et al., 2013).
This species is rarely landed in U.S. waters as bycatch in the squid
minimal histotrophy; Hamlett et al., 2005), which has been docu- fishery (Capt. Jim Ruhle, F/V Darana R, personal communication),
mented in some triakid and squaliform species (Musick and Ellis, in research and monitoring surveys, and other deep-water trawls
2005). Lipid histotrophy is fairly easy to identify in pregnant and longlines (Kiraly et al., 2003).
stingrays (Myliobatoidei) due to the presence of a lipid-producing A great deal of taxonomic uncertainty persists within the genus
trophonemata and copious amounts of white, milky secretions Centrophorus (Compagno et al., 2005). The species examined in the
(histotroph) found in the uterus during embryonic development present study has at times been identified as C. granulosus, C. acus,
(Musick and Ellis, 2005). Limited histotrophy is more difficult to C. uyato, C. lusitanicus, C. machiquensis, and more recently in the
identify through field observations, as it involves the secretion of a Western Atlantic, C. niaukang (White et al., 2013). At the time the
much more dilute substance (e.g. mucus) than is found in the lipid present study was conducted the species was identified as
histotroph and may occur only episodically during gestation C. niaukang (Compagno et al., 2005; Cotton, 2010). However,
(Hamlett et al., 2005). It has been suggested that many shark species a recent study determined this taxon to be a junior synonym
exhibit “limited histotrophy”, yet few have been documented of C. granulosus (White et al., 2013).
(Hamlett et al., 2005). Additionally, it has been suggested that the Etmopterus princeps (Supplementary Fig. 2) is one of the largest
degree of maternal nutritional supplementation follows a conti- species of lanternshark (Family Etmopteridae) and is widely dis-
nuum across species, with no clear quantifiable threshold to tributed in the North Atlantic Ocean (Compagno et al., 2005) at
discriminate one reproductive mode from another (Hamlett et al., depths of 400–4500 m (Dyb and Bergstad, 2004; Kyne and
2005; Musick and Ellis, 2005). When measuring embryonic organic Simpfendorfer, 2007). Much of the biology and behavior of this
matter consumption, differences between “yolk-sac viviparous” and bathydemersal species in unknown (Compagno et al., 2005), yet it is
“limited histotrophic” species might be indistinct, with varying highly abundant in deep waters of the North Atlantic (Fossen et al.,
degrees of yolk dependence found among taxa (Wourms, 1981; 2008). Currently, no conservation measures exist and few landings
Hamlett et al., 2005; Frazer et al., 2012). are reported for this species (Herndon and Burgess, 2006), despite
Other forms of matrotrophy are more readily identifiable during the high bycatch mortality this species likely encounters due to the
embryonic development. Ovatrophic species continue to ovulate trauma associated with capture by deep-water trawl, gillnet, or
nutritional ova during embryonic development and these ova can longline (Cotton, personal observation). This species is routinely
be found free in the oviducts and uterus, as well as in the stomachs caught and discarded at sea, with no record of the bycatch, largely
of developing embryos (Castro, 2009). Adelphophagy has been due to the fact that the meat has little value and the liver is too
documented only in Carcharias taurus and is characterized by small to justify processing (Stale Dyb, captain M/S Loran, personal
intra-uterine cannibalism (Gilmore et al., 1983). Evidence of adel- communication). The liver of this species contains proportionally
phophagy is apparent in the mid to latter stages of embryonic less squalene than other squaliform sharks (Hernández-Pérez et al.,
development since the number of live embryos will decrease from 1997), further reducing the impetus to harvest.
beginning to end of gestation as siblings are cannibalized. Also, the Knowledge of the reproductive biology of sharks is important
surviving embryo in each uterus bears precocious teeth and for clarifying their life history strategies. As higher-trophic level
extremely distended stomachs that contain nutritional ova and predators (Cortés, 1999; Musick and Cotton, this issue), deep-
the digested bodies of siblings (Gilmore et al., 1983). Placentotrophy water sharks require a great deal of energy in an energy-poor
is also easily identified by the presence of a yolk stalk and placental environment to sustain basic life functions. The process of repro-
connection found in mid- and late-stage embryos. duction places further energetic demands on these species, with
Previous studies have suggested that the energetic toll of mature ova largely composed of energy-dense compounds such as
embryogenesis in lecithotrophic species results in a reduction in fatty acids, amino acids, and lipids (Remme et al., 2005). Ova of
organic matter of more than 20% from freshly fertilized eggs to Centrophorus spp. can reach enormous sizes, up to 95 mm in
full-term embryos (Hamlett et al., 2005). However, few studies diameter and 390 g in weight (Guallart and Vicent, 2001; present
have appropriately quantified such changes in organic matter over study), one of the largest cellular sizes described for any verte-
the course of gestation (Storrie et al., 2009; Frazer et al., 2012). brate. The mode of reproduction and embryonic development
Ranzi’s (1932, 1934) seminal studies analyzed changes in water, employed by a given species therefore offers insight into the
organic content, and minerals from egg to term embryos for 15 energetic requirements of the reproductive cycle.
species of oviparous and viviparous elasmobranchs (translated and In this study, we empirically quantify the organic matter
summarized in Needham, 1942). depletion from fertilization to parturition in C. granulosus and
The classical method for determination of matrotrophy is simple E. princeps embryos to test the hypothesis that these species are
and requires relatively few samples (Hamlett et al., 2005). Absolute lecithotrophic. We also characterize for the first time the

Please cite this article as: Cotton, C.F., et al., Reproduction and embryonic development in two species of squaliform sharks,
Centrophorus granulosus and Etmopterus princeps: Evidence of.... Deep-Sea Res. II (2015), http://dx.doi.org/10.1016/j.dsr2.2014.10.009i
 C.F. Cotton et al. / Deep-Sea Research II ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 3

reproductive parameters (i.e. maturity ogives, fecundity, ovarian opportunistically during research cruises using demersal longlines
cycle, and reproductive seasonality) for these two deep-water or demersal trawl nets during one of the following projects: the
shark species. Virginia Institute of Marine Science (VIMS) shark survey, collections of
voucher specimens for the field guide “The Sharks of North America”
(Castro, 2011), the National Marine Fisheries Service (NMFS) Winter
2. Methods Bottom Trawl Survey, and the Deep-C project (www.deep-c.org). One
additional specimen was obtained as bycatch from a commercial
2.1. Centrophorus granulosus squid trawler, taken approximately 50 km south of Norfolk Canyon,
off the coast of Virginia (Capt. Jim Ruhle, F/V Darana R, personal
Specimens of C. granulosus were collected in the Gulf of Mexico communication). Specimens were measured for precaudal length
and in the Atlantic Ocean off the coasts of Virginia, North Carolina, (PCL), fork length (FL), and total length (TL). Determination of
and Florida (Fig. 1A). These specimens were collected maturity (n¼138 specimens) was based on the presence of enlarged

Fig. 1. Locations of (A) C. granulosus collections from the Mid-Atlantic Bight (MAB), eastern Florida shelf (EFS), and Gulf of Mexico (GOM). Locations of (B) E. princeps
collections along the Mid-Atlantic Ridge north of the Azores (AZ), south of the Charlie Gibbs Fracture Zone (SCGFZ) and north of the CGFZ (NCGFZ).

Please cite this article as: Cotton, C.F., et al., Reproduction and embryonic development in two species of squaliform sharks,
Centrophorus granulosus and Etmopterus princeps: Evidence of.... Deep-Sea Res. II (2015), http://dx.doi.org/10.1016/j.dsr2.2014.10.009i
4 C.F. Cotton et al. / Deep-Sea Research II ∎ (∎∎∎∎) ∎∎∎–∎∎∎

oocytes (or uterine eggs/embryos), distended uteri, and enlarged Figs. 6 and 7). Embryos of E. princeps were not dissected, but
oviducal glands (Supplementary Fig. 1) for females and clasper were dried and incinerated intact following the procedure for
calcification, presence of milt, and degree of vas deferens coiling for C. granulosus embryos. Accidental mishandling of ashed samples
males. In assessing female reproductive status, we used the terms from the largest (near-term) litter of embryos led to sample loss
‘oocyte’, ‘ovum, and ‘egg’ to denote the developing gamete, the (post-incineration) and underestimation of inorganic content for
mature (unfertilized) gamete, and the fertilized gamete, respectively. this batch of samples. A single, frozen term-embryo (TL¼ 175 mm)
Eggs and early stage embryos were frozen in utero to prevent rupture was therefore used as a proxy for inorganic content at this final
of the delicate yolk sac (Supplementary Fig. 3). Mid- and late-stage stage of embryonic development. Maturity ogives were con-
embryos were dissected from the uteri and frozen individually structed as for C. granulosus, with data binned in size-class
(Supplementary Fig. 4). In some specimens, the maximum oocyte intervals of 3 cm (female) and 2 cm (male) for the Azores data
diameter was also recorded. Maturity ogives were constructed with and 5 cm (both sexes) intervals for the CGFZ data, reflecting
SigmaPlot software using logistic regression with data for each sex differences in size distributions among specimens collected in
binned in 10-cm size-class intervals. The inflection points of the these two areas. Basic reproductive parameters were plotted and
curves (size at 50% maturity) were determined visually from each analyzed as for C. granulosus.
plot. Plots and regressions of basic reproductive parameters (fecundity
by size, egg weights by size, embryo lengths by month, oocyte
diameter vs embryo lengths) were generated with Microsoft Excel, 3. Results
and regression analysis was used to determine whether the slope of
each regression was significantly different from zero, i.e. no 3.1. Centrophorus granulosus
relationship.
Embryos (if visible) were measured for TL to nearest mm, and A total of 138 individuals were collected (118 female, 20 male),
for embryos larger than 150 mm TL, PCL and FL were also with a size range of 690–1725 mm TL. Sexual dimorphism was
measured. Eggs and embryos smaller than 150 mm TL were noted with females reaching a larger size (1725 mm TL) than
weighed whole and intact to the nearest 0.001 g. Embryos larger males (1240 mm TL). Twenty-two of the females collected were
than 150 mm TL were dissected to remove the external yolk sac pregnant, yielding 88 embryos with sex determined for 35 of these
(EYS) (Supplementary Figs. 4 and 5A), internal yolk sac (IYS), liver (15 females, 20 males), thus embryonic sex ratio was 0.75 females
(L), and digestive tract (DT) from the evicerated body (B) per male. The EYS was nearly resorbed (0.7–39 g) in the litter
(Supplementary Fig. 5B). Each component of those dissected containing the largest embryos (374–392 mm TL), suggesting that
embryos was weighed to the nearest 0.001 g, and the composite this size is near parturition (Supplementary Fig. 5A).
weight of all components was recorded as the embryo’s total
weight. A small loss in weight was unavoidable using this method, 3.1.1. Maturity ogives
but amounted to no more than 1% of the total weight of the The smallest mature female observed was 1460 mm and the
embryo, as verified by weighing some embryos before dissection. largest immature female was 1630 mm TL. The smallest mature
After weighing, samples were dried in either aluminum trays or male observed was 1150 mm TL, and the largest immature male
ceramic crucibles at 60 1C until the weight stabilized, indicating was 1090 mm TL. Maturity ogives indicated that 50% of females
that all water was removed. Dried samples were transferred to a were mature at approximately 1430 mm TL and 50% of males were
muffle furnace for incineration. The ashing protocol outlined by mature at approximately 1110 mm TL (Fig. 2). Overall sex ratio was
Guallart and Vicent (2001) caused excessive bubbling, splattering, skewed with 5.9 females per male collected. Of the total of 138
and loss of sample, so we formulated a more conservative ashing specimens collected, 83 (mostly pregnant) were collected near
protocol. Samples were incinerated for 24 h at each successive Norfolk Canyon off Virginia and no males have been collected in
temperature of 150, 200, 250, 300, and 350 1C, with transitions that area to date (Moore et al., 2003; present study).
implemented slowly (1 1C/min) to prevent boil-over and loss of
sample. Samples were then incinerated at 550 1C for 72 h, with the 3.1.2. Fecundity and reproductive cycle
remnant ash weighed to the nearest 0.001 g. Water content, Fecundity varied between 4 and 7 embryos (mean ¼5.3) and
organic content, and inorganic content were determined by was uncorrelated (R2 ¼0.0746) with maternal size, with a
formulas (1)–(3):
Water content ¼ wet weight–dry weight ð1Þ

Inorganic content ¼ ash weight ð2Þ

Organic content ¼ dry weight–ash weight ð3Þ

2.2. Etmopterus princeps

Specimens of E. princeps were collected during the MAR-ECO

expedition of 2004 (www.mar-eco.no; Bergstad and Godø, 2003;
Fossen et al., 2008). Two general regions along the northern Mid-
Atlantic Ridge (MAR) were sampled: the Charlie Gibbs Fracture
Zone (CGFZ), and an area approximately 400 km north of Faial
Island, Azores (Fig. 1B). Specimens were captured and processed
according to the methods outlined in Fossen et al. (2008). Maturity
stage was assessed following Stehmann (2002) for 459 individuals
(247 females, 212 males). Whole reproductive tracts were Fig. 2. Length-based maturity ogives for male (open circles) and female (closed
removed from 14 pregnant females, wrapped in cheesecloth, and circles) C. granulosus collected in the northwest Atlantic Ocean and Gulf of Mexico.
preserved in 10% neutral-buffered formalin (Supplementary Size-class intervals of 10 cm were used for both sexes.

Please cite this article as: Cotton, C.F., et al., Reproduction and embryonic development in two species of squaliform sharks,
Centrophorus granulosus and Etmopterus princeps: Evidence of.... Deep-Sea Res. II (2015), http://dx.doi.org/10.1016/j.dsr2.2014.10.009i
 C.F. Cotton et al. / Deep-Sea Research II ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 5

8 450

430
7
410
Fecundity (# embryos)

6
390
5

Egg weight (g)

370

4 350

330
3
310
2
290
1 270

0 250
1450 1500 1550 1600 1650 1700 1450 1500 1550 1600 1650 1700
Maternal TL (mm) Maternal TL (mm)

120

450 100

Maximum oocyte diameter (mm)

400
Total length of largest embryo (mm)

350 80

300
60
250

200
40
150

100
20
50

0 0
1 2 3 4 5 6 7 8 9 10 11 12 0 50 100 150 200 250 300 350 400 450
Month of capture Maximum embryo total length (mm)

Fig. 3. Reproductive biology of female C. granulosus: (A) fecundity related to maternal size (TL), (B) freshly fertilized egg weight (n ¼7 females) related to maternal size (TL),
(C) largest embryo size (TL) per litter of pregnant females by month of capture (non-discernable embryos were assigned a size of 0 mm TL), and (D) relationship of maximum
oocyte diameter to maximum embryo length for each pregnant female, showing that ovarian cycle is concurrent with gestation.

regression slope not significantly different from zero (p ¼0.2579) n ¼35 F, 690–1630 mm TL), and 27 embryos (172–392 mm TL,
(Fig. 3A). Mean egg weight was weakly correlated (R2 ¼0.1474) sexes combined) (Supplementary Fig. 8). Equations for these
with maternal size, with a significantly negative regression slope length relationships (measured in mm) are given by formulas
(p ¼0.019), indicating a reduction in mean egg size as female size (4)–(9):
increased (Fig. 3B). TL  PCL relationship ðadultÞ : TL ¼ 1:0715ðPCLÞ þ 192:87 ð4Þ
The distribution of embryo sizes found throughout the year
suggests an aseasonal reproductive cycle, due to the presence of FL  PCLrelationship ðadultÞ : FL ¼ 0:9285ðPCLÞ þ 227:49 ð5Þ
‘size-0’ embryos (indiscernible in size) found year-round, as well
as the wide range of embryo size classes found in the month of TL  PCL relationship ðjuvenileÞ : TL ¼ 1:2084ðPCLÞ þ 36:67 ð6Þ
October (Fig. 3C). Ovarian development was concurrent with
gestation in this species as evidenced by the increase in oocyte FL  PCL relationship ðjuvenileÞ : FL ¼ 1:1109ðPCLÞ þ 1:75 ð7Þ
size with increasing embryo lengths (Fig. 3D). The maximum
TL  PCL relationship ðembryoÞ : TL ¼ 1:4066ðPCLÞ  25:95 ð8Þ
oocyte diameter measured was in a female with near-term
embryos and was equivalent in size with observed freshly ferti-
FL  PCL relationship ðembryoÞ : FL ¼ 1:2267ðPCLÞ  20:69 ð9Þ
lized uterine eggs (  90 mm in diameter).

3.1.4. Embryonic composition

3.1.3. Length relationships Mean wet weight, water content, and inorganic content in
Length relationships (PCL, FL, and TL) were calculated from freshly fertilized eggs were 319.630 7 4.69 g, 162.555 73.91 g, and
measurements of a subset of 25 adults (n ¼ 2 MZ 1150 mm TL; 4.043 70.07 g, respectively, and increased to 363.985 79.55 g
n ¼23 FZ 1500 mm TL), 49 juveniles (n ¼14 M, 840–1090 mm TL; (þ13.9%), 236.7277 9.50 g (þ 45.6%), and 5.412 70.21 g

Please cite this article as: Cotton, C.F., et al., Reproduction and embryonic development in two species of squaliform sharks,
Centrophorus granulosus and Etmopterus princeps: Evidence of.... Deep-Sea Res. II (2015), http://dx.doi.org/10.1016/j.dsr2.2014.10.009i
6 C.F. Cotton et al. / Deep-Sea Research II ∎ (∎∎∎∎) ∎∎∎–∎∎∎

450 300
ȳ = 363.985 g
ȳ = 236.727 g
400
ȳ = 162.555 g
250
350

300 +13.9% 200

Water content (g)

+45.6%
wet weight (g)

250
150
200 ȳ = 319.630 g

150 100

100
50
50

0 0
0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450
Embryo total length (mm) Embryo total length (mm)

7 250
ȳ = 5.412 g
230
6 ȳ = 4.043 g
210

5 190
Inorganic matter (g)

Organic matter (g)

+33.9% ȳ = 121.845 g
170
4
150
3
130

2 110
-19.5%
90 ȳ = 151.390 g
1
70

0 50
0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400
Embryo total length (mm) Embryo total length (mm)

Fig. 4. Relationships between size (TL) of C. granulosus embryos and (A) wet weight, (B) water content, (C) inorganic content, and (D) organic content. The mean TL of
females with freshly fertilized eggs was 1611 mm (1500–1690 mm), and the TL of the female with term embryos was 1600 mm. Note: In panel C, values for embryos in the
30–55 mm TL range are missing due to spillage of samples during incineration.

(þ 33.9%), respectively, in full-term embryos (Fig. 4A–C). The resorbed, leaving a small amount of tissue composing the outer
changes in wet weight, water content, and inorganic matter all membrane of the EYS (Supplementary Fig. 9). The evicerated body
followed similar patterns, decreasing slightly during the first half is composed mostly of water (approximately 80%) at all sizes
of embryonic development, then increasing after embryos reached (Supplementary Fig. 9). Growth of the digestive tract was mostly
approximately 250 mm in length, likely corresponding to the size augmented by the uptake of water, whereas growth of the IYS and
at which gills become functional. Mean organic matter decreased liver was mostly due to the addition of organic matter to these
steadily from 151.390 73.21 g in the egg stage to 121.845 72.51 g structures as the embryo developed (Supplementary Fig. 10). The
in full-term embryos, representing a loss of 19.5% in organic proportion of inorganic matter in the EYS remained fairly constant
matter over the course of gestation (Fig. 4D). as yolk was depleted, indicating no further mineral input into this
structure (Supplementary Fig. 11). The proportion of inorganic
matter in the evicerated body increased with increasing body size,
3.1.5. Embryonic development likely as a result of increased uterine fluid uptake as the embryo
Wet weight of the EYS decreased with growth of the embryo, developed (Supplementary Fig. 11). The proportion of inorganic
while all other components of the embryo (IYS, L, DT, and B) matter in the IYS and liver decreased with increasing size, as yolk
increased exponentially with increased embryonic length, reflect- was relocated from the EYS to the IYS and as lipids were added to
ing the influx of organic matter from the EYS and uptake of uterine the developing liver (Supplementary Fig. 12). No obvious pattern
fluid, particularly after embryos reached 250 mm TL (Fig. 5). of inorganic matter uptake was evident in the growth of the
During the latter stages of development, enlargement of the IYS digestive tract (Supplementary Fig. 12).
was coincident with the depletion of the EYS, as yolk reserves
were relocated from an external to internal position. (Fig. 5A). 3.2. Etmopterus princeps
The composition of embryonic components varied by type. The
EYS is composed of mostly organic material (approximately 60%), A total of 495 individuals were examined for maturity (269
except when nearly depleted and most of the lipid-rich yolk has female, 226 male), with a size range of 295–940 mm TL. Sexual

Please cite this article as: Cotton, C.F., et al., Reproduction and embryonic development in two species of squaliform sharks,
Centrophorus granulosus and Etmopterus princeps: Evidence of.... Deep-Sea Res. II (2015), http://dx.doi.org/10.1016/j.dsr2.2014.10.009i
 C.F. Cotton et al. / Deep-Sea Research II ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 7

70

450
60
External and internal yolk sac wet weight (g)

400

50
350

Liver wet weight (g)

300
40

250
30
200

150 20

100
10
50

0 0
0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450
Embryo total length (mm) Embryo total length (mm)

16 250

14
Evicerated body wet weight (g) 200
Digestive tract wet weight (g)

12

10
150

100
6

4
50

0 0
0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450
Embryo total length (mm) Embryo total length (mm)

Fig. 5. Relationships between size (TL) of C. granulosus embryos and (A) external (closed circles) and internal (open circles) yolk sac wet weights, (B) liver wet weight,
(C) digestive tract wet weight, and (D) evicerated body wet weight. Embryos o75 mm TL were not dissected and their weights were considered to be entirely composed of
external yolk sac (panel A).

Fig. 6. Length-based maturity ogives for male (open circles) and female (closed circles) E. princeps (A) collected north of the Azores (n¼ 88 males and 131 females,
ratio¼1.00:1.49) and (B) collected near the Charlie Gibbs Fracture Zone (CGFZ) (n¼ 124 males and 116 females, ratio¼ 1.00:0.94). Triangles in panel A represent assumed
values, based on data from CGFZ. Size-class intervals of 3 cm (female) and 2 cm (male) were used for Azores data and 5 cm (both sexes) were used for CGFZ data.

Please cite this article as: Cotton, C.F., et al., Reproduction and embryonic development in two species of squaliform sharks,
Centrophorus granulosus and Etmopterus princeps: Evidence of.... Deep-Sea Res. II (2015), http://dx.doi.org/10.1016/j.dsr2.2014.10.009i
8 C.F. Cotton et al. / Deep-Sea Research II ∎ (∎∎∎∎) ∎∎∎–∎∎∎

dimorphism was observed in maximum size, with a maximum (þ49.6%), using data from a single proxy frozen embryo (Fig. 8C).
male TL of 690 mm and maximum female TL of 940 mm. Overall Samples from a litter of near-term embryos was mishandled after
mean size for males examined for maturity was 576 mm TL and for incineration, resulting in underestimation of inorganic content
females was 631 mm TL. A total of 117 E. princeps embryos were (0.15570.01 g;  25.1%) so these samples were not used to calculate
processed from 14 pregnant females and sex was determined for change in inorganic content. Mean organic matter decreased steadily
54 of these (27 males and 27 females), yielding a sex ratio of 1:1. from 11.85270.16 g in the egg stage to 10.93570.32 g in full-term
The EYS was almost completely resorbed (0.01–2.08 g) in the litter embryos, representing a loss of 7.7% in organic matter over the course
containing the largest embryos (157–175 mm TL), indicating this of gestation (Fig. 8D).
was near the size at parturition.

3.2.1. Maturity ogives 4. Discussion

The smallest mature male observed was 550 mm TL, and the
largest immature male was 640 mm TL. The smallest mature female 4.1. Maternal-embryo nutritional relationship
observed was 610 mm and largest immature female was 760 mm TL.
Maturity ogives were initially constructed with data from both regions After fertilization and during the very early stages of develop-
of the MAR combined, indicating that 50% of females were mature at ment C. granulosus embryos are contained within a thin egg
approximately 675 mm TL and 50% of males were mature at approxi- capsule or “candle”, which dissipates later in embryonic develop-
mately 555 mm TL. Further investigation indicated differences in ment. Other authors have noted the presence of an egg capsule in
female size at maturity between the CGFZ and Azores sampling sites. early development of some squaloid sharks (e.g. Squalus acanthias,
Maturity ogives for the Azores region (south) indicated that 50% of Hamlett and Hysell, 1998; C. cf. uyato, McLaughlin and Morrissey,
males were mature at approximately 565 mm TL and 50% of females 2005; Centroscymnus coelolepis, Moura et al., 2011). Conversely, the
were mature at approximately 610 mm TL (Fig. 6A). In the CGFZ region embryos of E. princeps are not retained within an egg capsule at
(north), 50% of males were mature at approximately 540 mm TL and any stage of development. Lack of an egg capsule in the embryonic
50% of females were mature at approximately 690 mm TL (Fig. 6B). stage has also been reported for other squaloid sharks (e.g.
Likewise, sex ratios differed in these two regions, with 1.49 females Centroscymnus owstoni, Yano and Tanaka, 1988; Centroscyllium
per male in the Azores area and 0.9 females per male in the CGFZ area. fabricii, Yano, 1995). Retention of developing embryos within an
egg capsule obviously precludes any maternal nourishment during
3.2.2. Fecundity and reproductive cycle this phase of development. Differences in organic matter depletion
Fecundity varied between 7 and 18 embryos (mean ¼11.2) and observed in the present study might be attributed to the lack of an
was uncorrelated (R2 ¼0.0725) with maternal size, with a regres- egg capsule in one of the two species examined.
sion slope not significantly different from zero (p ¼0.2511) According to Hamlett et al. (2005), a reduction in organic
(Fig. 7A). Mean egg weight was also uncorrelated (R2 ¼0.0668) matter over the course of embryogenesis of greater than 20%
with maternal size, with a regression slope not significantly should be expected in a lecithotrophic species. Some studies of
different from zero (p ¼0.0670) (Fig. 7B). Sampling of this species maternal–embryo nutritional relationships in squaliform sharks
was conducted over the span of one month, and therefore a have found that reductions in organic matter during embryogen-
thorough determination of reproductive seasonality was not esis were 20% or higher (Guallart and Vicent, 2001; Braccini et al.,
possible. However, due to the even distribution in size classes of 2007; Moura et al., 2011) and therefore concluded that these
embryos encountered (Fig. 7C; Supplementary Fig. 2), it is likely species (C. uyato (as C. granulosus), Squalus megalops, C. coelolepis,
that the reproductive cycle is aseasonal in this species. Ovarian respectively) were lecithotrophic. However, E. princeps exhibited
development is not concurrent with gestation in this species, as substantially less than a 20% reduction (  7.7%) in organic matter,
evidenced by the lack of developed oocytes in gravid females presumably due to some form of nutritional augmentation by the
(Supplementary Figs. 2 and 6). maternal organism. The notion that lecithotrophic species deplete
420% of the organic matter in a freshly fertilized egg was based
3.2.3. Length relationships on several studies that examined eggs of the oviparous species
No FL and PCL measurements were obtained during the MAR- Scyliorhinus canicula (Hamlett et al., 2005), but this species likely
ECO cruise, so no length relationships were calculated for adults of has a shorter gestation (Lechenault et al., 1993) than many deep-
E. princeps. Embryonic length relationships (PCL, FL and TL) were water elasmobranchs (Kyne and Simpfendorfer, 2007; 2010) and
calculated from measurements of 16 E. princeps embryos therefore might not be a valid comparison. Other authors have
(Supplementary Fig. 13). Equations for these length relationships expressed concern about using S. canicula as the sole representa-
are given by formulas (10) and (11): tive for all oviparous species (Huveneers et al., 2011; Frazer et al.,
2012). Similar data from a wide range of oviparous species are
TL  PCL relationship ðembryoÞ : TL ¼ 1:0587ðPCLÞ þ 24:122 ð10Þ needed to develop a better understanding of the energetic costs of
embryogenesis in these unequivocally lecithotrophic species. Of
FL  PCL relationship ðembryoÞ : FL ¼ 0:9539ðPCLÞ þ18:855 ð11Þ
particular interest would be data from larger oviparous species,
e.g. Bathyraja spp., with potentially longer gestational periods and
3.2.4. Embryonic composition higher energetic demands. Indeed a recent study of the Port
Mean wet weight and water content in freshly fertilized eggs was Jackson shark Heterodontus portusjacksoni showed that this ovi-
22.92370.29 g and 10.86370.18 g, respectively, and increased to parous species undergoes a 40% depletion in organic matter during
31.80771.01 g (þ38.8%) and 20.71770.74 g (þ90.7%), respectively, embryogenesis, i.e. twice that of S. canicula (Frazer et al., 2012).
in full-term embryos (Fig. 8A and B). The changes in wet weight and Likewise, more data is needed from a variety of yolk-sac viviparous
water content followed a similar pattern over the course of embryonic species to better estimate the full range of organic matter deple-
development, remaining fairly constant during the first half of tion across species.
embryonic development, then increasing some time after the embryos Results from the present study should be cautiously compared
reached approximately 90 mm in length, likely corresponding to the with those of Ranzi (1934) and Guallart and Vicent (2001), as those
size at which gills become functional. Mean inorganic content in studies involved a congeneric species from the Mediterranean Sea.
freshly fertilized eggs was 0.20770.01 g and increased to 0.310 g Though both studies listed the focal species as C. granulosus, recent

Please cite this article as: Cotton, C.F., et al., Reproduction and embryonic development in two species of squaliform sharks,
Centrophorus granulosus and Etmopterus princeps: Evidence of.... Deep-Sea Res. II (2015), http://dx.doi.org/10.1016/j.dsr2.2014.10.009i
 C.F. Cotton et al. / Deep-Sea Research II ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 9

20 32

18
28
16
Fecundity (# embryos)

14
24

Egg weight (g)

12

10 20

8
16
6

4
12
2

0 8
600 620 640 660 680 700 720 740 760 780 640 660 680 700 720 740 760 780
Maternal TL (mm) Maternal TL (mm)

5
# of females

0
< 1 mm

1 - 25 mm

26 - 50 mm

51 - 75 mm

76 - 100 mm

101 - 125 mm

126 - 150 mm

151 - 175 mm

Maximum embryo TL

Fig. 7. Reproductive biology of female E. princeps: (A) fecundity related to maternal size (TL), (B) freshly fertilized egg weight (n ¼6 females) related to maternal size (TL),
(C) distribution of maximum embryo lengths (TL) per litter of E. princeps, collected in July, 2004 along the northern Mid-Atlantic Ridge.

taxonomic revisions have shown that C. granulosus does not occur uteri and embryos from each species. The only other form of
in the Mediterranean Sea (Veríssimo et al., 2014) and that the only matrotrophy known among elasmobranchs is limited histotrophy
species of gulper shark reported from the Mediterranean Sea is so it was assumed that at least one of the species examined in the
currently under taxonomic redescription and will bear the name C. present study utilizes this reproductive mode, as has been reported
uyato upon completion of that study (White et al., 2013; Veríssimo for some squaliform species (Musick and Ellis, 2005). Macroscopic
et al., 2014). detection of limited histotrophy is not possible as the uterine
Limited histotrophy among sharks is likely more widespread than secretions are very dilute and low in organic content (Hamlett et
is currently known and the degree of maternal nourishment may al., 2005). Biochemical analyses of uterine fluids may also prove
occur along a gradient among species (Wourms, 1981). Hamlett et al. difficult in detecting mucoid histotrophy as the secretions may be
(2005) further assert that it is likely that all viviparous species exhibit episodic. Paiva et al. (2012) showed that Deania calcea is a matro-
some degree of histotrophy, which questions the validity of the trophic species, based on the presence of secretory cells in the
classification of “yolk-sac viviparity” as distinct from “limited histo- uterine wall, the changes in organic matter over embryonic devel-
trophy”. The results of the present study support this notion, as the opment, and the tracking of fatty acids and mercury from pregnant
apparent degree of maternal nourishment was quite different in the females to the developing embryos. Tanaka et al. (1990) reported
two squaloid sharks examined. maternal nourishment in Chlamydoselachus anguineus, based on dry
weights, but the authors offered no evidence of the mechanism.
Likewise, Girard and Du Buit (1999) reported maternal nourishment
4.1.1. Potential mode of matrotrophy in C. coelolepis, but this was based solely on wet weights of eggs and
We were unable to definitively identify a source of supplemental term embryos, so the claim is inconclusive as the observed weight
maternal nourishment for either species, but we easily excluded gain could have been attributable to water uptake. Moura et al.
most forms of matrotrophy (oophagy, adelphophagy, placental (2011) examined this same species and concluded it to be lecitho-
viviparity, lipid histotrophy) by macroscopic examination of the trophic. Further investigation (e.g. histological examination of

Please cite this article as: Cotton, C.F., et al., Reproduction and embryonic development in two species of squaliform sharks,
Centrophorus granulosus and Etmopterus princeps: Evidence of.... Deep-Sea Res. II (2015), http://dx.doi.org/10.1016/j.dsr2.2014.10.009i
10 C.F. Cotton et al. / Deep-Sea Research II ∎ (∎∎∎∎) ∎∎∎–∎∎∎

40 25
ȳ = 20.717 g
ȳ = 22.923 g ȳ = 31.807 g
35
20
30 ȳ = 10.863 g

Water content (g)

Wet weight (g)

25
+38.8% +90.7%
15

20

10
15

10
5
5

0 0
0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 160 180 200
Embryo total length (mm) Embryo total length (mm)

18
ȳ = 11.852 g
0.5
16
0.45
ȳ = 0.207 g ȳ = 10.935 g
0.4
14
y = 0.310 g
Organic matter (g)
Inorganic matter (g)

0.35
12
0.3
+49.6%
0.25 ȳ = 0.155 g 10
0.2

8 -7.7%
0.15

0.1 -25.1%

0.05

0 4
0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 160 180
Embryo total length (mm) Embryo total length (mm)

Fig. 8. Relationships between size (TL) of E. princeps embryos and (A) wet weight, (B) water content, (C) inorganic content, and (D) organic content. The mean TL of females
with freshly fertilized eggs was 703 mm (650–760 mm), and the TL of the female with near-term embryos was 700 mm. Note: In panel C, triangles denote a litter of near-
term embryos that was mishandled after incineration, resulting in ash weight underestimation, so these samples were not used to calculate change in inorganic content.
The closed circle denotes the inorganic content of a single term embryo that was used as a proxy for inorganic content at this final stage of development.

uterine lining) is needed to verify the mechanism for limited tracers, or biomarkers, to detect active uptake of uterine fluid by the
histotrophy in the present study. developing embryo (Huveneers et al., 2011).

4.1.2. Uterine fluid 4.1.3. Uterine villi

Needham (1942) noted the presence of “few” mucus-secreting cells It is likely that the uterine villi secrete nutrients as develop-
in uteri of C. granulosus, Dalatias licha, S. acanthias, and Squalus ment of these structures progresses concurrently with embryo-
blainville, and reported the percentage of organic matter contained genesis. This growth was particularly evident in C. granulosus. In
in uterine fluid of these species at 1.8–2.8%, by weight. According to uteri with freshly fertilized eggs, villi were approximately 3–5 mm
Guallart and Vicent (2001), prior studies have also shown secretory in length, pale in color, thin, and arranged in longitudinal rows
activity in the uterine walls of C. uyato (as C. granulosus), and dilute (Supplementary Fig. 3). Uteri with early- and mid-stage embryos
(o2.5%) organic compounds in the uterine fluids, but these were (35–55 mm TL; 170–190 mm TL), had much thicker, denser, and
assumed to serve a “lubricant” function, rather than a nutritive highly vascularized villi of approximately 5 mm in length
function (Bouchet et al., 1982). No study has since confirmed or (Supplementary Fig. 14). Uteri with slightly larger embryos
refuted this assumption. Despite this evidence of organic material in (230 mm TL) had longer villi (  7 mm) with the same general
the uterine fluids and identification of secretory cells in the uterine appearance. Uteri with near-term embryos (375–390 mm TL) had
epithelium of gulper sharks, many authors maintain that C. uyato and longer villi (  10–12 mm) but the color had changed to dark red,
other squaliform sharks are lecithotrophic species (Ranzi, 1934; with brownish or dark purple patches (Supplementary Fig. 15).
Guallart and Vicent, 2001; Braccini et al., 2007; Moura et al., 2011). Uterine villi of E. princeps greatly increased in length over the
Though the present study suggests that maternal nourishment occurs course of gestation (from approximately 3–5 mm to 15–18 mm),
in E. princeps and possibly C. granulosus, the mechanism for nutritional but overall did not become as dense or vascularized as those of
transport was not determined. Future studies may rely on other C. granulosus (Supplementary Figs. 7 and 16). Although the func-
techniques, e.g. histological examination of uterine lining, chemical tion of these uterine villi is not fully understood, they have been

Please cite this article as: Cotton, C.F., et al., Reproduction and embryonic development in two species of squaliform sharks,
Centrophorus granulosus and Etmopterus princeps: Evidence of.... Deep-Sea Res. II (2015), http://dx.doi.org/10.1016/j.dsr2.2014.10.009i
 C.F. Cotton et al. / Deep-Sea Research II ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 11

noted in other studies of squaloid sharks (Needham, 1942; Yano, differed by two orders of magnitude (Fig. 8). The decision to use
1995; Girard and Du Buit, 1999; Veríssimo et al., 2003; Moura et a frozen specimen as a surrogate value for term-embryo inorganic
al., 2011), with most authors noting that length and density of villi content was justifiable because the inorganic content (minerals) in
increases concurrently with embryonic growth. Hamlett and a frozen sample does not degrade or change under adverse
Hysell (1998) suggest uterine villi in S. acanthias are used strictly condition (i.e. desiccation or thawing). This single specimen was
for respiration and the removal of waste products, though direct not used to calculate wet weight, % water or % organic matter since
evidence for this is lacking. A recent study by Ellis and Otway desiccation likely occurred while in frozen storage prior to
(2011) demonstrated that pregnant Orectolobus ornatus periodi- the study.
cally flush the uteri by opening and closing the cloaca, presumably
for the purpose of disposing of metabolic waste from the embryo. 4.2.3. Embryonic growth
If other elasmobranch species are also capable of uterine flushing, By dissecting embryos of C. granulosus, we were able to track
there would be no need for the more energy-intensive processes the growth of individual components of the embryo. The rapid
of respiration, active transport of embryonic waste products, and growth of the IYS as the EYS was depleted has been reported in
development of a dense “carpet” of uterine villi for the sole several other studies (Guallart and Vicent, 2001; Veríssimo et al.,
purpose of respiration and/or removal of waste products, as 2003; Braccini et al., 2007). Repositioning yolk from the external
proposed by Hamlett and Hysell (1998). yolk sac to an internal position before parturition affords the
newborn shark a hydrodynamic advantage by reducing drag.
4.2. Embryonic development These substantial yolk reserves in the IYS sustain the pup until
first feeding (Yano, 1995; Hamlett et al., 2005). The size-related
4.2.1. Size at parturition increase of inorganic material found in the evicerated body
Sizes and weights of C. granulosus near-term embryos (374– (Supplementary Fig. 11) is driven by an increased mineral demand
392 mm TL, 363–405 g) in the present study were in close by the formation of teeth, skeletal elements, finspines, and
agreement with full-term pups collected previously from the Gulf denticles as the embryo grows.
of Mexico (400–415 mm TL, 320–390 g; J. Castro, unpublished Embryonic gills of C. granulosus appear to become functional at
data). Term embryos (350–400 mm TL) with fully resorbed yolk approximately 250 mm TL (Fig. 4A–C) as wet weight, water
sacs have also been reported from pregnant females collected near content, and inorganic content steadily decline until the embryo
Norfolk Canyon (Kiraly et al., 2003). reaches this size and then markedly increase afterward. This
Lengths of E. princeps near-term embryos in the present study increase in water and inorganic material is presumably tied to
(160–175 mm TL) were larger than some free-swimming juveniles the uptake of uterine fluids through the gills. A similar pattern has
from Iceland (120–180 mm TL; Jakobsdóttir, 2001), suggesting a been reported for C. uyato (as C. granulosus; Guallart and Vicent,
wide range in size at parturition. Given that the specimens from 2001) and C. anguineus (Tanaka et al., 1990), with embryos
Iceland were collected by trawl, it is possible that the smaller size exhibiting a sharp uptake of water about midway through
range included aborted embryos. development.
Tracking the changes in wet weight, organic content, and
inorganic content of different components of the developing
4.2.2. Embryonic composition embryo reveals how water, nutrients, and minerals are partitioned
Ranzi (1934) reported a reduction in organic matter of 54% for throughout the body during embryogenesis (Supplementary
C. uyato (as C. granulosus) embryos during embryogenesis. This Figs. 9–12). Water and minerals are largely diverted to the
value was later adjusted (18–25% reduction) in a subsequent study evicerated body and digestive tract while the liver and yolk sacs
with a larger sample size and a full series of developmental stages (external and internal) are largely composed of organic matter. The
of embryos (Guallart and Vicent, 2001). Though taxonomic uncer- tracking of individual somatic components was not possible for
tainty persists within this genus (White et al., 2013), these two E. princeps, since the embryos were not dissected. As observed in
studies likely examined the same species since both studies C. granulosus, embryonic gill function presumably begins about
collected specimens from the Mediterranean Sea, which harbors midway through development, as evidenced by the sharp increase
only one species of Centrophorus (Guallart and Vicent, 2001; in water and mineral uptake at about 90 mm TL (Fig. 8B and C).
Veríssimo et al., 2014). The value reported by Ranzi (1934) was
derived from only one egg and one term embryo, and therefore
did not take into account individual variation. The results of 4.3. Reproductive biology of Centrophorus granulosus
Guallart and Vicent (2001) were more representative since their
data were derived from multiple eggs and term embryos. As such, 4.3.1. Maturity ogives
their reported organic matter reduction for C. uyato (as C. granu- Size at maturity (female ¼1430 mm TL; male¼ 1110 mm TL)
losus) embryos was quite similar to that of the present study. was in close agreement with that proposed by Kiraly et al. (2003)
The reduction in organic matter recorded for E. princeps (female ¼1460 mm TL; male ¼1100 mm TL; as C. niaukang). The
embryos ( 7.7%) was very close to that observed in S. blainville smallest mature female observed in the present study was
(  7.4%; Frazer et al., 2012). This value is much lower than that 1460 mm TL, and largest immature female was 1630 mm TL. This
observed for C. granulosus (  19.5%) in the present study, as well as large immature female had large oocytes (70 mm) and a small
some other studies of squaliform sharks (Guallart and Vicent, uterus (30 mm width), which was not distended or vascularized
2001; Braccini et al., 2007; Moura et al., 2011). Paiva et al. (2012) (indicative of recent gestation). It is not likely that this individual
showed a 3.8% increase in organic matter for embryos of D. calcea, was in a postpartum “resting” state, since there is no resting stage
suggesting a wide range in magnitude of uterine nourishment for for species with a concurrent ovarian cycle as indicated for this
histotrophic squaliform species. Similarly, Frazer et al. (2012) species (Fig. 3D).
suggested a wide range in organic matter depletion (reduction of
20% to  40%) is found among lecithotrophic species. 4.3.2. Segregation by sex and maturity stage
Problems encountered with estimation of inorganic content of Among samples collected from the Gulf of Mexico, mostly
near-term E. princeps embryos were relatively inconsequential to immature specimens were found in the northern area and mostly
the overall estimation of organic content since these values pregnant females were found in the eastern area (off Tampa, FL)

Please cite this article as: Cotton, C.F., et al., Reproduction and embryonic development in two species of squaliform sharks,
Centrophorus granulosus and Etmopterus princeps: Evidence of.... Deep-Sea Res. II (2015), http://dx.doi.org/10.1016/j.dsr2.2014.10.009i
12 C.F. Cotton et al. / Deep-Sea Research II ∎ (∎∎∎∎) ∎∎∎–∎∎∎

(Fig. 1). In 1986, during a “shakedown” cruise (about 225 km east villi, indicating recent activity in each uterus. Aborted embryos
of Melbourne, FL), three mature male C. granulosus were collected, were occasionally observed on the deck of the boat during sample
ranging from 1170 mm to 1210 mm TL (J. Musick, unpublished collection. This presents an unfortunate uncertainty in determin-
data; American Museum of Natural History catalog #’s 78262, ing fecundity, since abortions may also occur prior to hauling
78266, 78265). specimens on deck.
Kiraly et al. (2003) reported aggregated captures (e.g. n ¼ 40) of Ovarian development is concurrent in this species (Fig. 3D)
female C. granulosus in trawls near Norfolk Canyon (off Virginia) such that vitellogenesis occurs during gestation with a sub-
and hypothesized that the animals were moving into shallower sequent batch of oocytes ovulated shortly after parturition. This
waters at night to feed, since daytime trawls did not capture this pattern has also been reported for C. cf. uyato (McLaughlin and
species. The VIMS longline survey found similar diel differences in Morrissey, 2005) and Squalus cf. mitsukurii (Cotton et al., 2011).
catch records with 25 stations fished at Norfolk Canyon, mostly A closer examination of the ovaries of C. granulosus indicated
during daylight hours between 1990 and 1991 yielding only three that there are two distinctly different vitellogenic batches (size
C. granulosus. All three of these specimens were captured during classes) of oocytes found in the ovaries during gestation
one nighttime set (J. Musick, unpublished data). In contrast, 17 (Supplementary Figs. 1 and 4), presumably developing for two
stations fished in the same area overnight between 2006 and 2008 subsequent ovulations.
yielded 26 C. granulosus. On one of these sets 8 pregnant The aseasonal reproductive cycle observed for this species
C. granulosus were captured on 15 hooks. Conversely, the only (Fig. 3C) is common among deep-water sharks (Kyne and
daylight sets (n ¼2) during this 2006–2008 period yielded no Simpfendorfer, 2010), probably owing to the relative stability in
C. granulosus. These results suggest this species undergoes diel environmental conditions of the deep ocean, and has been
migrations into shallower water at night, however a telemetry reported for many deep-water squaloids, e.g. S. cf. mitsukurii
study is needed to confirm this. (Cotton et al., 2011), Etmopterus granulosus (Wetherbee, 1996), C.
Our limited data, along with other catch records for this species fabricii (Yano, 1995), C. coelolepis (Yano and Tanaka, 1988; Girard
indicate that Norfolk Canyon and the adjacent continental shelf and Du Buit, 1999; Veríssimo et al., 2003), and C. owstoni (Yano
break may provide exclusive habitat for gravid females of this and Tanaka, 1988).
species. The VIMS catch in this region was entirely composed of
females (n ¼28), 82% of which were gravid and the remainder
4.4. Reproductive biology of Etmopterus princeps
were near the size at maturity. Moore et al. (2003) reported that
100% of the C. granulosus (as Centrophorus sp.) caught in NOAA
4.4.1. Maturity ogives
trawl surveys off the eastern coast of the U.S. (n 100) were
Size at maturity was approximately the same in both regions
composed of gravid females, some with near-term embryos (Kiraly
(Fig. 1B) for males but females seem to mature at a larger size in
et al., 2003). It seems clear that this outer-shelf habitat is not
the northern region (CGFZ). This discrepancy in female size at
strictly a “pupping ground” (e.g. Carcharhinus plumbeus in Chesa-
maturity might reflect delayed maturity or differences in growth
peake Bay; Grubbs and Musick, 2007) because pregnant females
rates if the sharks occupying these two regions compose distinct
with embryos of all stages of development were found in this area.
populations. These differences in size at maturity might also be
Likewise, this does not appear to be a case of mere sexual
due to data limitations as the samples from the southern area (AZ)
segregation, because large immature females were mostly absent
excluded large, mature individuals such that size at maturity for
from Norfolk Canyon. The segregation observed in the present
the largest sizes classes were assumed rather than measured
study appeared to be limited to gravid females, as has been
(Fig. 6A).
observed in other squaloid sharks. Girard and Du Buit (1999) and
Jakobsdóttir (2001) reported the length at which 50% of E.
Clarke et al. (2001) found only immature and postpartum female
princeps reached maturity around Iceland as 573 mm TL for males
Centrophorus squamosus, along with mostly mature, reproduc-
and 622 mm TL for females. In the northern stations of the MAR-
tively active males on the shelf break west of Scotland and Ireland,
ECO cruise, we found the size at 50% maturity was 540 mm TL for
but no gravid females were found among their samples. Also,
males and at 690 mm TL for females (Fig. 6B). However,
during the MAR-ECO cruise, the C. squamosus catch was composed
Jakobsdóttir (2001) used different criteria to assess maturity,
of 82 mature males and one immature female on the MAR north of
classifying stage “B” (or “maturing”) individuals as mature,
the Azores (Dyb and Bergstad, 2004). McLaughlin and Morrissey
whereas we classified those individuals as immature. Recalcula-
(2005) reported sexual segregation in C. cf. uyato around Jamaica
tion of female maturity ogives using Jakobsdóttir’s method yielded
and hypothesized that males move into the area during the winter
a similar size at 50% maturity for females, 627 mm TL.
months. In addition to geographical segregation, some squaloids
exhibit depth-mediated segregation by maturity stage (Yano and
Tanaka, 1988; Clarke et al., 2001). 4.4.2. Fecundity and reproductive cycle
Observed fecundity in E. princeps ranged from 7 to 18 embryos
and the relationship between female size and fecundity was
4.3.3. Fecundity and reproductive cycle uncorrelated (R2 ¼0.0725), however this may be an artifact of
A paradoxical reduction in mean egg size with increased the small sample size (Fig. 7A), aborted embryos, or the physio-
maternal length was observed in C. granulosus (Fig. 3B). This logical burden of parasitism, observed in some gravid females.
might have been explained as a correlation with increased Though the lowest observed fecundity was 7, this particular female
fecundity (i.e., lipid reserves divided among an increased number (710 mm TL) was covered in 21 ectoparasites (unidentified mono-
of oocytes), however the regression of fecundity and maternal genean flatworms and parasitic goose barnacles, Anelasma squali-
length (Fig. 3A) showed no relationship. However, both of these cola) and thus may have been compromised. Similarly, the largest
regressions, particularly fecundity, may have been limited by the pregnant female (760 mm TL) examined had a relatively low
small samples sizes of each. Two female specimens in the present fecundity (n ¼12), but this female was also covered in 35 ectopar-
study had an observed fecundity of one; however, these were asites. Previous studies reported reduced fecundity in female
omitted from Fig. 3A due to the likelihood of aborted embryos. In etmopterids parasitized by Anelasma squalicola (Hickling, 1963;
both cases, each female had a single mid-term embryo, yet both Yano and Musick, 2000). The ovarian cycle is consecutive in
uteri were highly vascularized and distended, with well-developed this species, as reported for C. fabricii (Yano, 1995), C. owstoni, and

Please cite this article as: Cotton, C.F., et al., Reproduction and embryonic development in two species of squaliform sharks,
Centrophorus granulosus and Etmopterus princeps: Evidence of.... Deep-Sea Res. II (2015), http://dx.doi.org/10.1016/j.dsr2.2014.10.009i
 C.F. Cotton et al. / Deep-Sea Research II ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 13

C. coelolepis (Yano and Tanaka, 1988). The differing ovarian cycles for either species but this mechanism could influence reproductive
between E. princeps and C. granulosus may reflect varying magni- cycles, particularly for E. princeps which has a resting period after
tudes of matrotrophy or perhaps the differences in energetic parturition. Size at maturity for C. granulosus was determined to be
demands of a matrotrophic and a lecithotrophic species, respec- 111 cm TL for males and 143 cm TL for females, with an average
tively. If E. princeps females allocate lipid stores and energy to the fecundity of 5.3 embryos (range¼4–7). Size at maturity for
production of mucoid histotroph throughout gestation, they may E. princeps was determined to be 56.5 cm TL for males and
be incapable of concurrent vitellogenesis. 61 cm TL for females north of the Azores and 54 cm TL for males
and 69 cm TL for females near the CGFZ. Average fecundity was
4.4.3. Segregation by sex and maturity stage 11.2 embryos (range¼7–18) for this species.
Pregnant E. princeps were “rarely found” around Iceland and This is the first reporting of the reproductive biology of
the Reykjanes Ridge (Jakobsdóttir, 2001) and the author suggested C. granulosus and E. princeps and our analyses of embryonic
that segregation by maturity stage occurs in this species. A high development challenge the notion that these are lecithotrophic
abundance of postpartum and “resting stage” females was also species. Future studies like this are needed to clarify the degree to
reported among the catch in that study. In the present study, only which different viviparous species deplete yolk (organic matter)
1 of 247 females examined was postpartum and this individual during gestation (Frazer et al., 2012). Likewise, the hypothesis of
was found in the northernmost extent of the area sampled. matrotrophy (limited histotrophy) needs to be tested for these
Wetherbee (1996) also reported segregation by reproductive stage species, by histological examination of uterine lining and/or the
and found low numbers of pregnant E. granulosus around New use of tracer molecules to track the movement of uterine nutrients
Zealand, suggesting that gravid females segregate from the rest of into the developing embryo. Our characterization of reproductive
the population. parameters such as size at maturity and fecundity will be crucial
Male maturity data collected in the present study also support for informing stock assessment models in areas where these
the hypothesis of segregation by maturity stage. South of the CGFZ, species are fished.
only about 40% of the male E. princeps collected were mature,
whereas 95% of the males north of the CGFZ were mature and
Acknowledgements
mostly milting (reproductively active). As Yano (1995) reported for
C. fabricii around Greenland, male E. princeps likely remain
The authors thank Odd Aksel Bergstad and Mike Vecchione for
reproductively active after reaching maturity since there is appar-
facilitating MAR-ECO cruise logistics. We thank the captains and
ently no mating season for this species (Jakobsdóttir, 2001;
crews of the M/S Loran and M/V Green Reefer for assistance with E.
present study).
princeps sample collection and arranging transport of samples to
Jakobsdóttir (2001) showed a highly MAR-focused distribution
the USA; captains Durand Ward, Wolf Lange, and John Olney, Jr. (R/
of E. princeps around Iceland, including many postpartum females,
V Bay Eagle) and the VIMS longline team for assistance with
very few reproductively active males, and some small neonates
collection of C. granulosus material from Norfolk Canyon; and
(Z 120 mm TL). The Icelandic shelf break and the Reykjanes Ridge
captains and crew of the R/V Apalachee and R/V Weatherbird II for
may therefore serve as a pupping ground for this species.
assistance with collection of C. granulosus material from the Gulf of
Jakobsdóttir's (2001) observations, combined with the results of
Mexico. We thank Jon Moore for providing information regarding
the present study, suggest that females aggregate along the MAR
NMFS captures of C. granulosus, Karsten Hartel for assistance with
near Iceland for pupping. After the subsequent resting period,
examination of C. granulosus voucher specimens at MCZ, Scott
mating possibly occurs in the high concentration of reproductively
Schaefer for assistance with examination of C. granulosus voucher
active males near the CGFZ and these mated females may then
specimens at AMNH; and Eric Hilton for curating voucher speci-
travel further south along the MAR (north of the Azores) to
mens of C. granulosus in the VIMS Ichthyology Collection. We
gestate. This, however, would require females to undergo exten-
especially appreciate John Galbraith who provided invaluable
sive migrations along the MAR, yet this small-bodied species may
advice throughout the project, as well as data from pregnant
be incapable of migrating such great distances.
gulper sharks collected from Norfolk Canyon. The valuable input
and advice of Will Hamlett guided the original concept of the
5. Summary project. The authors appreciate the valuable comments provided
on this manuscript by Jose Castro, Mike Vecchione, and Tracey
In this study, we show that organic matter is depleted by 19.5% Sutton, as well as three anonymous reviewers. Partial funding was
for embryos of C. granulosus and 7.7% for embryos of E. princeps provided by the National Shark Research Consortium, the MAR-
between the time of fertilization and parturition. Though these ECO project (Census of Marine Life), FIO Shiptime Grant Program,
species are presumed lecithotrophic yolk-sac viviparous, our results Department of Education Title VII Award #P382G090003, and the
suggest that E. princeps is matrotrophic, likely utilizing the “limited Deep-C Consortium (www.deep-c.org). This paper is Contribution
histotrophy” mode of reproduction. Alternate interpretation implies no. 3405 of the Virginia Institute of Marine Science, College of
that the range of organic matter depletion from fertilization to William & Mary.
parturition may vary greatly among species, depending on taxon-
specific conversion efficiency of yolk, and that a depletion of 20% in
Appendix A. Supporting information
embryonic organic matter does not necessarily delimit matrotrophic
and lecithotrophic species (Frazer et al., 2012). Likewise, as has been
Supplementary data and figures associated with this article can
suggested by prior authors (e.g. Hamlett et al., 2005), it is possible
be found in the online version at http://dx.doi.org/10.1016/j.dsr2.
that all yolk-sac viviparous species exhibit some level of histotrophy,
2014.10.009.
with the degree of maternal nutritional supplementation following a
continuum across species.
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Centrophorus granulosus and Etmopterus princeps: Evidence of.... Deep-Sea Res. II (2015), http://dx.doi.org/10.1016/j.dsr2.2014.10.009i
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Please cite this article as: Cotton, C.F., et al., Reproduction and embryonic development in two species of squaliform sharks,
Centrophorus granulosus and Etmopterus princeps: Evidence of.... Deep-Sea Res. II (2015), http://dx.doi.org/10.1016/j.dsr2.2014.10.009i