Delineation and biogeography of semipelagic spotted eagle rays

1. Delineation and biogeography of semipelagic spotted eagle rays
Stephen Bergacker & Jean-François Flot
Université libre de Bruxelles (ULB)

2. Biogeography
Each species of spotted eagle ray occurs in distinct biogeographic regions
separated by soft and hard barriers to dispersal. A. laticeps is restricted
to the Eastern Pacific, separated from A. ocellatus to the west by the
eastern pacific barriers and from A. narinari by the Isthmus of Panama to
the east. The Benguela upwelling system separate Atlantic and Indian
Ocean aetobatids. The Central Indo-Pacific has been proposed as the
centre of origin for the Aetobatidea family.
Pelagic eagle rays (Aetobatidea)
Spotted eagle rays
Uncertainties and goals
The nature and number of barriers to dispersal are not known for certain. The inclusion of samples from key world regions would enable a higher resolution analysis of biogeographic
barriers and population diversity within the spotted eagle ray complex. The level to which Indo-Pacific populations are structured has not yet been evaluated. This study aims to evaluate the
level at which populations are structured/connected through the use of species delimitation methods and to provide an insight into the biogeography of the said populations.
2009
Resurrection of
the Aetobatidea family,
comprising 5 species (White et al, 2016)
A. flagellum (sister group of spotted eagle rays) split
into 2 putative species (White et al, 2013)
Spotted eagle rays (A. narinari) split
into 3 putative species (Richards et al, 2009)
A. ocellatus populations
highly structured (Schluessel et al, 2010 )
2010 2013 2016
The spotted eagle ray species complex
Background and Phylogeny
Spotted eagle rays are large circumtropically distributed chondrichthyans. Trophically
dependent on the benthos, they occur over the continental shelf and are frequently observed
in proximity to coral reefs. Dorsal ocelli distinguish spotted eagle rays from other pelagic
eagle rays. The Aetobatus genus has undergone taxonomical re-evaluation in the past decade
and has led to the description of three new species and the resurrection of the Aetobatidea
family that encompasses the latter.
A. laticeps A. narinari A. ocellatus A. flagellum A. narutobiei

3. Time calibration
The Beauty and Beast programs were used to create calibrated ultrametric phylogenetic trees. The best models for
evolutions were selected based on the AIC criterion implemented in jModelTest (Posada, 2008). Three internal nodes were
used to calibrate the tree based on fossil records (Poortvliet et al, 2014).
GMYC
The generalized mixed Yule-coalescent (GMYC) (Fujisawa et al, 2013) is a coalescent-based method that relies on tree
topology. It is grounded on the premise that intraspecific branches are significantly denser then interspecific ones. Both
single and multiple threshold GMYC approaches were performed, modeling rate heterogeneity among species.
Samples and Data processing
Sequences were obtained from GenBank, compiling data
from 24 independent studies. Unique haplotypes were
retained for four mitochondrial markers.
Sequences were aligned using the E-INS-i algorithm
implemented in the multiple alignment program MAFFT
(Katoh et al, 2013). Two methods for delineating species
were used, one based on tree topology (GMYC) and the
other on genetic distances (ABGD). The lack of diploid
markers did not enable the use allele sharing as a method
for species delimitation.
Pairwise genetic distances
Numberofcomparisons
Barcode gap
Intraspecic variantion
Intrerspecic variantion
COI
Cytb
ND4
ND2
Species 1Species 2Species 3
Materials and Methods
ABGD
Automatic barcode gap discovery (Puillandre et al, 2012) is
a method that relies on genetic distances. It is based on the
premise that genetic distances are smaller between
intraspecific populations than species, the two being
separated by a “barcode gap”. Kimura 2-parameter
distances were selected and intraspecic variation were set
between 0.001 and 0.1.
ABGD’s concept
GMYC’s concept

4. ABGD
When ABGD was applied to COI, A. laticeps and A. narinari, usually
considered as two distinct species, were clustered together. This was
not the case for other genetic markers. Whenever samples from the
Arabian Sea were available for a given marker, they formed separate
hypothetical species units. The marker ND2 further separated South-
African populations from other Indo-Pacific localities.
0.002
A.ocellatus_10
A.ocellatus_2
A.narinari_2
A.ocellatus_8
A.laticeps_2
A.ocellatus_7
A.ocellatus_11
A.ocellatus_5
A.ocellatus_4
A.laticeps_1
A.ocellatus_3
A.ocellatus_6
A.narinari_1
A.ocellatus_1
A.ocellatus_9
GMYC
The GMYC method proved to be more sensitive than ABGD, with
more hypothetical species units being delineated with the use of the
former. Five clusters were delineated. The distinctiveness of A.
narinari and A. laticeps was confirmed whereas A. ocellatus was split
into 3 distinct populations: the Arabian Sea - South-Western Africa -
other Indo-Pacific localities.
Divergence times
Divergence time estimates concurred with the literature (Richards et
al, 2009; Sales et al, 2019):
- A. laticeps / A. narinari: 1.1 mya
- A. laticeps and A. narinari / A. ocellatus: 2.7 mya
- Time of most recent common ancestor of all aetobatids: 16 mya
Results
A. laticeps A. narinari
A. ocellatus

5. Species delineation
The delineation techniques used in the present study
support the existence of at least three species of
spotted eagle rays. Although A. laticeps and A.
narinari consistently form hypothetical species units
across all genetic markers tested, this is not the case
for Indo-Pacific populations. Indeed, A. ocellatus
populations are highly structured, South-African and
Arabian Sea populations being distinct from other
regions.
As maternally inherited markers were used in this
study, the delineation of populations of A. ocellatus
could be evidence of maternal philopatry rather than
genetic isolation. The inclusion of biparentally
inherited genetic markers is thus required to
understand the nature of population structure in the
Indo-Pacific.
Current biogeography
The genetic diversity in the Indo-Pacific, notably the divergence of Arabia Sea and South-African population, suggests that
population connectivity is not as homogenous as previously considered. The divergence of Eastern and Western Indian spotted
eagle rays suggests that there is a barrier to dispersal between the two coasts of India; the permeability or lack thereof is yet to
be evaluated. As Indian Ocean sea-surface temperatures are mainly governed by monsoons that vary cyclically, there does not
seem to be any cold water mass preventing genetic connectivity. As coral reefs are scarce in this world region, however, genetic
structure could be the result of non-linear habitat distribution.
Historical biogeography
The centre of origin for the Aetobatidea familly was considered
to be the Indo-West Pacific based on the shared habitat of A.
ocellatus and A. flagellum. The distinction between A.
flagellum and A. narutobiei (previously considered conspecific)
as well as the inclusion of Arabian Sea samples in this study
point towards an Middle-Eastern centre of origin of pelagic
eagle rays. As the time of most recent common ancestor of the
family is estimated at 16 mya, aetobatids most likely stemmed
from the Tethys sea, with a subsequent radiation into the Indo-
Pacific. Such radiations have been documented for many taxa
(Miller et al, 2018) as the Tethyan marine biodiversity hotspot
shifted toward today’s Indo-Central Pacific.
A. laticeps – East Pacific
A. narinari – Atlantic
A. narutobiei – West Pacific
A. flagellum – Arabian Sea
A. ocellatus – Arabian Sea
A. ocellatus – South-Africa
A. ocellatus – Indo-Pacific
Discussion