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Insights into ontogenetic scaling and morphological variation in sharks
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from near-term brown smooth-hound (Mustelus henlei) embryos
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3
Joel H. Gayford1,2, Phillip C. Sternes2,3, Scott G. Seamone4, Hana Godfrey 5, Darren A. Whitehead5,6
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¹ Department of Life Sciences, Silwood Park Campus, Imperial College London,
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2 Shark Measurements, London, UK,
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3 Department of Evolution, Ecology, and Organismal Biology, University of California,
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Riverside,USA,
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4 Department of Marine Sciences, Bahamas Agriculture and Marine Science Institute, Bahamas,
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5 Investigación Tiburones México A.C, 23010, La Paz, Mexico,
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6 Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas, La Paz, México
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Funding: No funding is declared for this study.
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Conflicts of interest: The authors have no conflicts of interest to disclose.
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Author contributions: JHG conceived the study, JHG, HG and DAW collected the data, JHG analysed
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the data, and all authors contributed to and reviewed the final manuscript.
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Abstract
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Elasmobranchs (sharks and rays) exhibit a wide range of body forms adapted to various
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ecological niches. Body form differs not only between species, but between life stages of
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individual species as a result of ontogenetic allometry. In sharks, it has been proposed that
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these ontogenetic shifts in body form result from shifts in trophic and/or spatial ecology (the
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allometric niche shift hypothesis). Alternatively, it has been suggested that ontogenetic
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allometry may result from intrinsic morphological constraints associated with increasing
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body size – e.g. to counteract shifts in form-function relationships that occur as a function of
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size and could compromise locomotory performance. One major limitation affecting our
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understanding of ontogenetic scaling in sharks is that existing studies focus on postpartum
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ontogeny – ignoring the period of growth that occurs prior to birth/hatching. In this study, we
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report ontogenetic growth trajectories from 39 near-term brown smooth hound (Mustelus
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henlei) embryos taken from manually collected measurements. We found that unlike most
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other species and later ontogenetic stages of M. henlei, these embryos predominantly grow
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isometrically, and appear to display relatively high levels of morphological disparity. These
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results provide rudimentary support for the allometric niche shift hypothesis (as in the
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absence of ontogenetic niche shifts isometry dominates body-form scaling) and provide
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important insight into early shark ontogeny and morphological/developmental evolution.
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Introduction
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Elasmobranchs (sharks and rays) are a large radiation of marine vertebrates that have
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persisted through periods of intense environmental change and represent an ecologically
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important component of marine diversity (Heithaus et al., 2010; Grogan et al., 2012; Flowers
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et al., 2021). Elasmobranchs are extremely diverse in terms of both morphology and ecology
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(Maisey et al., 2004; Navia et al., 2007; Cortés et al., 2008; Sternes and Shimada, 2020;
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Kuraku, 2021; Mull et al., 2022; Andrzejaczek et al., 2022). Whilst many knowledge gaps
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remain regarding our understanding of elasmobranch ecology, it has long been known that
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some species exhibit shifts in spatial and trophic ecology through development (Grubbs,
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2010). It is only relatively recently however that ontogenetic shifts in morphology have been
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documented (e.g. Lingham-Soliar, 2005; Irschick and Hammerschlag, 2015; Fu et al., 2016;
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Irschick et al., 2017; Ahnelt et al., 2020; Sternes and Higham, 2022; Bellodi et al., 2023;
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Gayford et al., 2023a; Gayford et al., 2023b; Seamone et al., 2023; Yun and Watanabe, 2023;
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Gayford et al., 2024). These shifts are important not only from the perspective of functional
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ecology but help us to understand the selective forces underlying the evolution of
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elasmobranch morphology (Gayford et al., 2023b). Various hypotheses exist for observed
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growth trajectories, including selection relating to spatial and trophic ecology and
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fundamental constraints on locomotor performance associated with increasing body size
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(Irschick et al., 2017; Gayford et al., 2023b; Seamone et al., 2023). Typically, a mosaic of
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allometric and isometric growth (where some structure grows disproportionately or
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proportionately relative to body size respectively) is observed in functionally important
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morphological measurements, with the nature of these scaling relationships varying between
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size classes and sexes in some cases (Gayford et al., 2023b). Despite the increasing number
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of studies addressing the topic of ontogenetic allometry in a range of shark species, there
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exist a number of factors that severely limit our understanding of the phenomenon, including
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taxonomic coverage, sample sizes, and an understanding of the genetic/developmental and
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functional basis of the morphological structures in question (Sternes and Higham, 2022;
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Gayford, 2023; Gayford et al., 2023a).
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One potential limitation that has not yet been addressed in the literature is that existing
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ecomorphological studies of scaling in elasmobranchs focus predominantly on postnatal
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ontogeny, despite the evolutionary and ecological significance of morphological changes
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occurring during prenatal ontogeny. Those studies that do address embryogenic scaling focus
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on developing staging tables without much focus on the ecomorphology of scaling itself
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(Tomita et al., 2018; López-Romero et al., 2020; Byrum et al., 2023). They may, as in other
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ontogenetic stages (Gayford et al., 2023b) act to maximise fitness in the context of the trophic
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and spatial ecology of this taxon post-partum. It is also important to consider that embryo
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morphology may also be influenced by prepartum environmental conditions (Kaplan and
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Phillips, 2006; Rodda and Seymour, 2008). Selective pressures associated with prepartum
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conditions could include sibling relatedness (Pfennig and Collins, 1993), constraints relating
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to the anatomy of the mother (Qualls et al., 1995), and even temporal variation in the
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environmental conditions experienced by the mother (Sale et al., 2007; McCoy et al., 2020).
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Each of these factors is yet to be considered from the perspective of elasmobranch taxa,
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representing a significant gap in our understanding of morphological evolution within this
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clade. Studies of scaling in embryos may also provide insight into the selective pressures
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driving the evolution of allometric and isometric growth, as shark embryos typically exhibit
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substantial proportional body-size changes (Tomita et al., 2018; López-Romero et al., 2020;
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Byrum et al., 2023) without undergoing major shifts in the trophic/spatial environment.
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The prepartum environment is important to the process of development across taxa, but is
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arguably of particular interest in elasmobranchs due to the complexity of their reproductive
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biology: not only are multiple mating systems observed amongst extant elasmobranch taxa
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(Bester-van der Merwe et al., 2022), but substantial variation has been reported in parameters
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such as brood size, gestation periods, ovarian cycles and reproductive behaviours (Carrier et
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al., 2004). Elasmobranch reproduction is a complex arena of intense genetic conflict between
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multiple players: sexually antagonistic coevolution (Portnoy and Heist, 2012), male-male
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conflict (Rowley et al., 2019) and sibling conflict (Chapman et al., 2013) have all been shown
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to occur within this clade and have potential consequences for morphological evolution.
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Sexually antagonistic coevolution has been attributed with the evolution of a number of
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sexual dimorphisms (Kajiura and Tricas, 1996; Whitehead et al., 2022), and intrauterine
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cannibalism (one manifestation of sibling conflict) (Gilmore et al., 2011) would logically
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impart strong selection pressures on tooth morphology and the developmental timing of tooth
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acquisition.
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The brown smoothhound shark, Mustelus henlei (Gill, 1863) is a primarily demersal
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Carcharhiniform shark found along the Pacific coast of the Americas, from California to Peru
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(Compagno, 1984; Ebert et al., 2021). This species is heavily fished in Baja California,
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Mexico (Medina-Morales et al., 2020; Smith et al., 2009), which has facilitated several
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studies into its biology and ecology in the region (Pérez-Jiménez and Sosa-Nishizaki, 2008;
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Byrne and Avise, 2012; Pantoja-Echevarría et al., 2020; Gayford et al., 2023a). M. henlei has
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a relatively great distribution compared to other Mustelus species with which it exhibits some
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degree of range overlap (Chabot et al., 2015), however it appears that populations are
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differentiated both genetically (Chabot and Haggin, 2014; Chabot et al., 2015) and
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ecologically, with ontogenetic and sex-based trophic variation, and overall trophic position
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appearing to differ between study sites (Ruso, 1975; Espinoza et al., 2012; Rodríguez-
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Romero et al., 2013; Amariles et al., 2017). There is a paucity of data regarding reproductive
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biology in this species (Pérez-Jiménez and Sosa-Nishizaki, 2008). Multiple paternity is
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known to be present in M. henlei (Chabot and Haggin, 2014; Rendón-Herrera et al., 2022),
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and basic information exists regarding observations of placental viviparity and variation in
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litter size (Yudin, 1987; Pérez-Jiménez and Sosa-Nishizaki, 2008), however no data exist
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regarding the morphogenesis or early ontogeny of M. henlei. A recent study did present
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ontogenetic growth trajectories for this species, using a large dataset of both adult and
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juvenile (but not neonate or embryonic) specimens (Gayford et al., 2023a). For this reason,
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M. henlei presents an ideal case study through which to compare ontogenetic growth
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trajectories in pre and postpartum environments.
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In this study, we use measurements obtained from M. henlei embryos to explore possible
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ontogenetic shifts in body form during this crucial stage of development. We seek to compare
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growth trajectories for various morphological structures with those found from post-natal
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individuals (both juvenile and adult) and propose potential explanations for any differences
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observed. This study provides a vital contribution to existing literature on ontogenetic
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morphological shifts in elasmobranch taxa as existing evolutionary studies have focussed on
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post-natal ontogeny – neglecting the potential selective influence of the pre-natal
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environment on morphology and growth trajectories.
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Methodology
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Ethics statement:
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Data collection and analysis procedures in this study complied with Mexican animal welfare
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laws and guidelines. No permit or ethical approval was necessary as animals were caught as
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part of legal artisanal fisheries and data were only collected after landing with permission of
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the fishers. Participants of this study neither promote nor encourage the harvesting of sharks.
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Data Collection
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M. henlei comprise a major component of total catch at artisanal fishing camps on the Pacific
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coast of Baja California Sur, Mexico. During the month of December 2022, we recovered
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paired uteri from 5 pregnant females at an artisanal fish camp. Full uteri were recovered
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(Figure 1), however due to the rate at which sharks are processed at this camp, it was not
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possible to match uteri to specific adult females. Uteri were stored at 5°C and transported to a
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laboratory for analysis within 24 hours. As we retained full uteri, embryos did not desiccate
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and therefore did not exhibit any significant morphological deformation. At the laboratory,
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uteri were dissected and embryos where all morphological structures of interest were clearly
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present were isolated for data collection. Damaged embryos, or those too small for
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morphological structures to be reliably measured were not included. From each of the
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remaining 39 embryos, 28 morphological measurements capturing variation in body form
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were taken recorded with a tape measure to the nearest millimetre (Table 1).
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Figure 1: Paired uteri from one pregnant M. henlei individual, prior to extraction and
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measurement of embryos.
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Ta bl e 1 : Morphological measurements taken from embryos, their abbreviations and definitions.
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Character
Abbreviation
Definition
Tot al le ngt h
TL
Distance from the tip of the snout to the tip of the upper caudal lobe
Precaudal length
PL
Distance from the tip of the snout to the precaudal pit
Upper caudal
length
UL
Distance from the upper insertion point of the caudal fin to the tip of the caudal fin upper
lobe (posterior tip)
Lower caudal
length
LL
Distance from the lower insertion point of the caudal fin to the tip of the caudal fin lower
lobe (ventral tip)
Caudal height
CH
Distance between the posterior tip and ventral tip of the caudal fin
Caudal keel
CK
Tot al ci rcu mfe re nce at t he cau da l k eel
Lateral span
LS
Distance over the dorsal body surface, measured between the anterior insertion points of the
left and right pectoral fins
Frontal span
FS
Distance over the dorsal body surface at the anterior edge of the insertion point of the first
dorsal fin, measured between points on the flank on the same horizontal plane as the
pectoral fin insertion points
Figure 1: Paired uteri from one pregnant M. henlei individual, prior to extraction of embryos
1cm
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Proximal span
PS
Distance over the dorsal body surface at the posterior edge of the insertion point of the first
dorsal fin, measured between points on the flank on the same horizontal plane as the
pectoral fin insertion points
Frontal span 2
FS2
Distance over the dorsal body surface at the anterior edge of the insertion point of the
second dorsal fin, measured between points on the flank on the same horizontal plane as the
pelvic fin insertion points
Proximal Span 2
PS2
Distance over the dorsal body surface at the posterior edge of the insertion point of the
second dorsal fin, measured between points on the flank on the same horizontal plane as the
pelvic fin insertion points
Dorsal length
DL
Distance from the anterior insertion point of the first dorsal fin to the upper tip of the same
fin
Dorsal width
DW
Horizontal distance from the anterior insertion point of the first dorsal fin to the posterior
tip of the same fin
Dorsal height
DH
Vertical distance from the tip of the first dorsal fin to the base of the first dorsal fin
Second dorsal
length
DL2
Distance from the anterior insertion point of the second dorsal fin to the upper tip of the
same fin
Second dorsal
width
DW2
Horizontal distance from the anterior insertion point of the first dorsal fin to the posterior
tip of the same fin
Second dorsal
height
DH2
Vertical distance from the tip of the first dorsal fin to the base of the second dorsal fin
Pectoral fin
length
PF
Distance from the distal insertion point of the pectoral fin to the fully extended tip of the
pectoral fin
Pectoral fin
width
PFW
Distance from the proximal insertion point of the pectoral fin to the distal insertion point of
the pectoral fin
Pelvic fin length
PLF
Distance from the distal insertion point of the pelvic fin to the fully extended tip of the
pelvic fin
Pelvic fin width
PLFW
Distance from the proximal insertion point of the pelvic fin to the distal insertion point of
the pelvic fin
Anal fin length
AL
Distance from the anterior insertion point of the anal fin to the upper tip of the same fin
Anal fin height
AH
Horizontal distance from the anterior insertion point of the anal fin to the posterior tip of the
same fin
Anal fin width
AW
Vertical distance from the tip of the anal fin to the base of the first dorsal fin
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Eye to eye
EE
Distance over the dorsal body surface from the midpoint of one eye to the midpoint of the
other
Eye height
EH
Ve rt i c al d ia m e te r of t he e ye a t i t s m i dp o i nt
Eye length
EL
Horizontal diameter of the eye at its midpoint
Mouth diameter
MD
Linear distance over the ventral body surface between the posterior most insertion points of
the lower jaw
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Data analysis
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No developmental staging table exists for M. henlei. To assess the general developmental
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stage of embryos, external morphology was compared to that described in embryological
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scaling tables for other shark species (e.g. Ballard et al., 1993; Rodda and Seymour, 2008;
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Onimaru et al., 2018).
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To extract ontogenetic growth trajectories for morphological measurements, simple linear
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regression analysis was performed using the function lm in the R statistical environment (R
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Core Team, 2023). All data were log-transformed prior to statistical analyses, such that
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growth curves are represented by linear (rather than exponential) relationships, the gradient
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of which determines whether growth is allometric or isometric. Each measurement (excluding
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TL and PL) was regressed separately against precaudal length (PL). The observed scaling
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coefficient (gradient) was then compared to a null hypothesis of isometry (equivalent to a
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gradient of 1.00). Where scaling coefficients are found to be significantly (p<0.05) lower or
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higher than 1.00 they represent negative and positive allometric relationships respectively
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(the absence of any significant relationship is hence an extreme case of negative allometry).
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This means that as body size increases, the measurement in question becomes
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disproportionately smaller or larger, respectively.
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Results
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Embryos ranges in size from 168mm to 208mm in total length (Table 2), within the size
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range at which pups are born (Ebert et al., 2021). All embryos were recovered in good
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condition, enabling all measurements to be taken from all individuals. The largest embryos
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were determined to be near term (close to parturition) on the basis of their well-developed
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external morphology, and comparison with existing developmental staging tables for other
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shark species (Ballard et al., 1993; Rodda and Seymour, 2008; Onimaru et al., 2018). Due to
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the lack of any published staging table for any member of the Mustelus genus we were unable
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to ascribe specific stages to each embryo.
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Ta bl e 2 : Su m ma ry st at i st i cs f o r al l m ea su re me nt s in c lu de d i n th e s tu dy , re c or de d t o t he n ea re s t mm.
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Character
Minimum
(mm)
Maximum
(mm)
Mean
(mm)
Standard
Deviation
(mm)
TL
168
208
188
10.1
PL
138
168
151
7.8
UL
30
41
37
3.0
LL
11
18
15
1.6
CH
25
39
31
3.2
CK
7
19
13
2.5
LS
26
36
30
2.5
FS
19
29
23
2.6
PS
11
22
18
2.0
FS2
8
16
13
1.6
PS2
7
12
10
1.2
DL
17
24
21
1.7
DW
20
28
24
2.2
DH
10
18
14
2.1
DL2
12
19
16
1.9
DW2
14
23
19
1.9
DH2
5
14
10
1.7
PF
19
26
22
1.7
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PFW
13
22
18
2.0
PLF
10
14
12
1.3
PLFW
8
13
11
1.2
AL
8
12
10
1.0
AH
3
6
5
0.8
AW
11
16
13
1.2
EE
13
20
17
1.7
EH
3
4
3
0.5
EL
6
9
8
0.8
MD
11
15
13
1.2
197
Linear regression of 26 morphological measurements against precaudal length (PL) failed to identify
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statistically significant departures from isometry in most cases: only three measurements
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demonstrated allometric trajectories (PFW, AL and EL) whereas the remaining 23 measurements scale
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isometrically (Table 3). Each of the three allometric relationships were negative, meaning that they
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become disproportionately smaller through ontogeny (Table 3). Whilst the slope of the scaling
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relationship in these cases is significantly lower than 1, this does not rule out the possibility that there
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is no significant relationship between body size at PFW, AL, or EL. Indeed the low ranges for AL and
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EL (Table 2) relative to the measurement resolution (1mm) mean that scaling relationships for these
205
measurements should be interpreted with caution. The proportion of variance explained by scaling
206
relationships was low in many cases, ranging between 0.03 and 0.35 (Table 3).
207
208
Ta bl e 3 : Li n ea r re g re ss i on r es ul ts fo r t he L o g1 0 t ra ns f or me d t ot a l da ta se t ( n= 3 9) . Si gn i fi ca n t (allometric)
209
results (p
≤
0.05) are indicated in bold.
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Character
Coefficient
Std. error
t value
p value
Residual std. error
𝑹𝟐
Adj. 𝑹𝟐
F va lu e
UL
0.9565
0.2124
0.205
0.839
0.02965
0.354
0.3366
20.28
LL
0.9824
0.3135
0.056
0.956
0.04377
0.2097
0.1884
9.819
CH
1.0991
0.2592
0.382
0.704
0.03618
0.3271
0.3089
17.98
CK
1.3668
0.6503
0.564
0.576
0.09078
0.1067
0.0825
4.418
LS
0. 9182
0.2155
-0.380
0.706
0.03008
0.3292
0.3111
18.16
FS
0.9785
0.3003
0.072
0.943
0.04192
0.223
0.202
10.62
PS
1.0135
0.3426
0.040
0.969
0.04783
0.1913
0.1694
8.75
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FS2
0.7742
0.3945
0.572
0.571
0.05507
0.0943
0.0698
3.851
PS2
0.4952
0.4184
1.206
0.235
0.0584
0.03649
0.0104
1.401
DL
0.6903
0.2330
1.329
0.192
0.03253
0.1917
0.1699
8.777
DW
0.8366
0.2525
0.647
0.522
0.03524
0.2288
0.208
10.98
DH
1.1942
0.4280
0.454
0.653
0.05975
0.1738
0.1515
7.785
DL2
0.6289
0.
3686
1.007
0.321
0.05146
0.07293
0.0479
2.91
DW2
0.9284
0.2888
0.248
0.806
0.04032
0.2183
0.1972
10.33
DH2
0.5780
0.5947
0.710
0.482
0.08302
0.02489
-0.002
0.944
PF
0.59619
0.21537
1.875
0.0687
0.03006
0.1716
0.1492
7.663
PFW
0.1363
0.3663
2.358
0.0238
0.05113
0.00373
-0.023
0.139
PLF
0.4274
0.3416
1.676
0.102
0.04769
0.04058
0.0147
1.565
PLFW
0.5790
0.3545
1.187
0.243
0.04949
0.06724
0.0420
2.667
AL
0.3056
0. 3104
2.237
0.0314
0.04334
0.02553
-0.001
0.969
AH
0.6326
0.5550
0.662
0.512
0.07747
0.03392
0.0078
1.299
AW
0.9035
0.2491
0.388
0.701
0.03477
0.2623
0.2423
13.15
EE
1.1957
0.2439
0.802
0.4276
0.03405
0.3938
0.3774
24.03
EH
0.1336
0.4465
1.941
0.060
0.06233
0.00241
-0.025
0.090
EL
0.1579
0.3257
2.586
0.0138
0.04546
0.00631
-0.021
0.235
MD
0.48595
0.29460
1.745
0.0893
0.04112
0.0685
0.0433
2.721
211
212
Discussion
213
A number of studies have investigated postnatal ontogenetic scaling trends in elasmobranch fishes.
214
Whilst in some cases, specific morphological structures exhibit predominantly isometric or allometric
215
growth respectively (Reiss and Bonnan, 2010; Irschick and Hammerschlag, 2015; Ahnelt et al., 2020),
216
it appears that in most species body form develops through a combination of isometry and allometry
217
(Irschick and Hammerschlag, 2015; Irschick et al., 2017; Sternes and Higham, 2022; Bellodi et al.,
218
2023; Gayford et al., 2023a; Gayford et al., 2023b; Seamone et al., 2023). In this study we instead
219
focus on the latter stages of prenatal ontogeny, finding that isometric growth is present across nearly
220
all functionally important aspects of external morphology (Table 3). Moreover, even where allometry
221
is present,
𝑅!
values are far lower than found in other studies (Table 3) – even across similar size
222
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ranges in the same species (Gayford et al., 2023a). This suggests that across a critical stage of
223
ontogeny, the body form of M. henlei remains broadly unchanged and morphological variation is
224
relatively great compared to postnatal ontogenetic stages. Here, we discuss the implications of these
225
results for our understanding of body form evolution and ontogenetic scaling in sharks and the life
226
history of M. henlei.
227
228
The prevalence of isometry as opposed to allometry in M. henlei embryos lends further credence to
229
the idea that allometric shifts in shark body form result at least in part from ontogenetic niche shifts.
230
Studies investigating ontogenetic shifts in body form in sharks typically relate these changes to
231
differences in trophic or spatial ecology (Lingham-Soliar, 2005; Irschick and Hammerschlag, 2015;
232
Fu et al., 2016; Irschick et al., 2017; Sternes and Higham, 2022; Gayford et al., 2023a; Gayford et al.,
233
2023b; Seamone et al., 2023; Yun and Watanabe, 2023; Gayford et al., 2024). In many species, adults
234
consume larger prey and spend a greater proportion of time in offshore, open-ocean environments –
235
likely resulting in a shift in the selective pressures acting on individuals through ontogeny (Sternes
236
and Higham, 2022; Gayford et al., 2023b). Niche shifts are not the only hypothesised drivers of
237
allometry in sharks – there have also been suggestions that allometric growth may be more prevalent
238
in larger-bodied species as a result of size-related physiological/locomotory constraints (Irschick et
239
al., 2017; Seamone et al., 2023). This hypothesis suggests that allometric growth might act to
240
maintain locomotor performance as body size increases, as opposed to modifying performance to suit
241
different ecological conditions (Seamone et al., 2023). Our results support the allometric niche shift
242
hypothesis given that, in the absence of an ontogenetic niche most measurement scale with isometry
243
(Table 3). Additionally, the few cases of allometry are weak and explain a negligible proportion of
244
variance in the data (Table 3). This is in stark contrast to later ontogenetic stages of M. henlei, (that
245
are thought to undergo some degree of ontogenetic niche shift) where a much greater proportion of
246
measurements scale with allometry, and allometric relationships explain substantial proportions of
247
morphological variance (Gayford et al., 2023a).
248
249
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The observed ~25% increase in body size is unlikely to have similar physiological/locomotory
250
consequences in embryos because they are not subject to the physical constraints of locomotion in
251
water – and therefore we are unable to directly test the hypothesis that constraint associated with
252
locomotor performance drives the evolution of allometry. However, our results (combined with those
253
of Gayford et al., 2023a) suggest that in M. henlei, allometric growth is not an intrinsic consequence
254
of increased body-size alone and support the role of ontogenetic niche shifts in selecting for the
255
evolution of allometry.
256
257
The broad absence of allometric growth in M. henlei embryos does not match findings from other
258
ontogenetic stages of the species, and could be indicative of morphological constraint imposed by the
259
maternal environment. Embryo morphology in other taxa can be influenced by selection pressures that
260
are unique to the prenatal environment (Kaplan and Phillips, 2006; Rodda and Seymour, 2008). There
261
are obvious size and shape constraints relating to the internal anatomy and body size of the mother (or
262
the eggcase in the case of oviparous taxa) that could hypothetically influence morphology. Moreover,
263
selection relating to locomotion, predation, and nutrient acquisition clearly differs substantially
264
between the prenatal and postnatal environments. In several shark species (including both
265
matrotrophic and oviparous taxa), shifts in embryo morphology have been hypothesised to result from
266
differences between these environments: in white sharks (Carcharodon carcharias), embryos undergo
267
a dramatic transition from heterocercal to lunate caudal fins, with the latter is thought to be
268
advantageous to fast-moving active predators such as young white sharks (Tomita et al., 2018).
269
Interestingly, this transition continues well into postnatal ontogeny (Lingham-Soliar, 2005). The early
270
development and subsequent loss of external gill filaments recorded in various elasmobranch taxa is
271
thought to result from potential hypoxia during early stages of development, which is of course
272
unique to the prenatal environment (Rodda and Seymour, 2008). Such shifts are clearly not present in
273
M. henlei, as body form remains broadly constant through the latter stages of prenatal ontogeny (Table
274
3).
275
276
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There are two potential explanations for why M. henlei predominantly exhibits isometric growth
277
during late prenatal ontogeny: either selective constraint on embryo morphology imposed by the
278
maternal environment prevents morphology optimised for the postnatal environment from arising
279
until after parturition, or selective constraint imposed by the maternal environment is largely absent,
280
and isometry results from acquisition of optimal postnatal morphology during early prenatal ontogeny.
281
Measurements included in this study such as DH and PF define the maximum body diameter of
282
embryos, and thus may be under constraint as they could influence the number of embryos able to fit
283
within the uteri, or the probability of injury to the mother during parturition. In line with this, body
284
girth measurements along the trunk grow isometrically in embryos (Table 3), despite showing
285
significant positive allometry in later postnatal life stages (Gayford et al., 2023a). It is not however
286
possible to conclusively distinguish between this hypothetical constraint and an ontogenetic trajectory
287
in which the morphology favoured by selection in the postnatal environment is acquired early in
288
prenatal ontogeny on the basis of our data. Ontogenetic morphological trajectories for neonate M.
289
henlei would enable us to discern between these hypotheses, as a predominance of isometric growth
290
here would rule out postnatal allometry as a mechanism for overcoming prenatal morphological
291
constraint. Contrastingly, predominance of allometric growth would suggest that there are substantial
292
differences between the body forms favoured in prenatal and postnatal environments, providing
293
rudimentary evidence for morphological constraint.
294
295
296
Va r io u s t he o r e ti c a l m o d e ls ha v e be e n p r o du c ed t o d e sc r i b e p at t e r ns of m o r ph o l o gi cal variation
297
through vertebrate embryogenesis and ontogeny (Vo n B a e r , 1 8 2 8 ; Keibel and Abraham, 1900). Whilst
298
not universally accepted, the ‘hourglass model’ is amongst the most supported of these models, and
299
suggests that morphological diversity is greatest during the earliest and latest stages of embryogenesis
300
(Irie and Kurutani, 2014; Irie, 2017). This is thought to be due to more stringent developmental
301
constraints acting on intermediate embryogenic stages (Piasecka et al., 2013). These models have
302
traditionally been used to compare morphological divergence between taxa, however it has previously
303
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been suggested that patterns of intraspecific morphological variation could follow an hourglass model
304
(Pantalacci and Sémon, 2015). We cannot empirically test the validity of an hourglass model with the
305
data in this study, nevertheless our results are consistent with the concept of relatively high
306
morphological variance during the latter stages of embryogenesis, as evidenced by the low
𝑅!
values
307
relative to similar proportional body size ranges in latter ontogenetic stages of the same species (Table
308
3; Gayford et al., 2023a). Comparing our results to these latter ontogenetic stages, this suggests that
309
selection/constraint on morphology may be weakened or relaxed during the latter stages of
310
embryogenesis of M. henlei relative to subsequent life stages (Gayford et al., 2023a). This also
311
provides further indirect support for the niche shift-driven allometry, suggesting that where niche
312
shifts are not present, selection on body form is relatively weak. Importantly, the evolutionary
313
constraint posited by developmental models is not the same as the physical constraints on morphology
314
that could theoretically influence embryo morphology mentioned previously.
315
316
Without additional genetic and morphological information from the earlier stages of embryogenesis it
317
is impossible to prove whether variation in the intensity of developmental constraint is responsible for
318
the morphological variation reported here. There are indeed several plausible alternative explanations
319
for such variation. In the case of some measurements (particularly AL and EL), the measurement
320
resolution (1mm) is fairly low relative to the measurement range (Table 2), and this could lead to
321
spuriously low
𝑅!
values. However this is not the case for all measurements considered here (Table 2)
322
and thus, that measurement resolution alone cannot explain this apparent high morphological
323
variance. Multiple paternity (where a single brood consists of individuals sired by more than one
324
male) is a known phenomenon in M. henlei (Byrne and Avise, 2012; Chabot and Haggin, 2014;
325
Réndon-Herrera et al., 2022) and could hypothetically contribute to morphological variation.
326
However, the potential influence of multiple paternity is difficult to quantify due to uncertainty
327
regarding the genetic basis of morphological traits in sharks (Gayford, 2023), and geographic
328
variation in the extent of multiple paternity (Chabot and Haggin, 2014; Réndon-Herrera et al., 2022).
329
Even if the genotypes of potential sires do not result in substantial morphological variation, multiple
330
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paternity could result in morphological differences between embryos due to latency of fertilisation
331
within a brood. In several shark species where multiple paternity is present, fertilisation is thought to
332
occur over an extended period of time (Schmidt et al., 2010; Marino et al., 2015). In this scenario
333
there may be discrepancies in the maternal provisioning that different embryos receive at a given
334
embryogenic stage, as in matrotrophic species the nutrients provided to embryos by the mother
335
depends to some extent on the trophic characteristics of the environmental conditions to which the
336
mother is exposed (Olin et al., 2011; McCoy et al., 2020). The degree of body size variation in our
337
results (Table 2) combined with the ~10 month gestation period and seasonal migratory behaviour of
338
M. henlei (Pérez-Jiménez and Sosa-Nishizaki, 2008) leads us to suggest that multiple paternity in this
339
taxon likely does result in different embryos within a litter receiving different nutritional profiles from
340
the mother at given embryogenic stages. Whether this contributes to morphological variation remains
341
unknown, but we suggest that future studies should investigate the extent to which models of
342
developmental constraint, multiple paternity, and delayed fertilisation could contribute to intraspecific
343
and interspecific patterns of morphological variation.
344
345
Conclusion
346
347
In this study we have shown that during the latter stages of prenatal ontogeny, M. henlei body form
348
grows predominantly isometrically, in stark contrast with postnatal ontogenetic stages of this species.
349
These results not only improve our understanding of the life history of M. henlei but provide valuable
350
insight into the evolution of ontogenetic scaling and morphological diversity in sharks. In the context
351
of the prenatal environment, isometric growth supports the allometric niche shift hypothesis and
352
relatively high morphological disparity between embryos raises questions about shark reproductive
353
biology, evolutionary genetics and development that warrant further study. Our dataset is undoubtedly
354
limited – both in terms of ontogenetic coverage and measurement resolution. However, similar studies
355
covering similar proportional ranges (body size increases of ~25-50%) of body size (Irschick and
356
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Hammerschlag, 2015; Gayford et al., 2023a) have been key in forming current opinion on ontogenetic
357
scaling in sharks. Future studies considering different ontogenetic stages of M. henlei and similar
358
ontogenetic stages in other shark species will enable us to further discern between some of the
359
hypotheses addressed in this study – particularly in the cases of developmental constraints and
360
morphological disparity between embryos. Ultimately this study acts as a baseline against which
361
ontogenetic body form trajectories from embryos of other shark species can be compared, within the
362
same quantitative and theoretical framework that has been applied to studies of ontogenetic scaling in
363
postnatal life-stages of elasmobranch taxa.
364
365
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