1. Molecular phylogenetic evidence refuting the hypothesis
Molecular Phylogenetics and Evolution 26 (2003)
Molecular phylogenetic evidence refuting the hypothesis
of Batoidea (rays and skates) as derived sharks
Christophe J. Douady, Mine Dosay, Mahnmood S. Shivji, and Michael J. Stanhope

2. Abstract
Early morphological study:
Elasmobranch (Sharks, Batoids)
Sharks : monophyletic
Batoids (skates and rays): monophyletic
Modern cladistic study: Hypnosqualea hypothesis:
Batoids are derived sharks (sawsharks, angelsharks)
sawshark
angelshark
To address this issue: 2.4kb mtDNA (12S rRNA-tRNA-16S rRNA locus)
-Two order of batoides
-All order of sharks(8)
-Outgroup: holocephali ( sister group to elasmobranchs)
Results:
Sharks: monophyly
Rejection of the Hypnosqualea hypothesis

3. Elasmobranch fishes / Compagno(1973,1977)
Introduction
Elasmobranch fishes / Compagno(1973,1977)
Superorder
Orders
Common name
1.Galeomorphii
1.Orectolobiformes
Carpet sharks
2.Heterodontiformes
Bullhead sharks
3.Carcharhiniformes
Ground sharks
4.Lamniformes
Mackerel sharks
2.Squatinomorphii
5.Squatiniformes
Angel sharks
3.Squalomorphii
6.Hexanchiformes
Frilled and cow sharks
7.Squaliformes
Dogfish shark
8.Pristiophoriformes
Saw sharks
4.Batoidea
9.Myliobatiformes
Stingrays
10.Pristiformes
Sawfishes
11.Torpediniformes
Electric rays
12.Rajiformes
Skates
13.Rhinobatiformes
Guitarfishes
The relative relationships between these orders and superorders remains uncertain

4. History
Early morphological work:
A fundmental split between batoids and sharks
(Bigelow and Schroeder 1948, 1953)
2. Modern morphological cladistic study
- Batoids are derived sharks,
closely related to sawsharks and angelsharks (Compagno, 1977~ Shirai, 1996)
Hypnosqualea hypothesis:
(Squatinomorphii (Pristiophoriformes,Batoidea))
Superorder Hypnosqualea (Carvalho and Maisey, 1996)

5. Study of Interordinal relationships of sharks: very few
3 relevant studies: Dunn and Morrisey(1995), Kitamura et al. (1996), Arnason(2001)
Kitamura : Cytb(732bp), 8 of 13 elasmobranch orders,
No support values, rooted with a very distant outgroup not Holocephali
No chimaeras were not used as an outgroup -> difficult to interpret
- Support a possible distinction of batoids from hypnosqualea
- Not good marker(Cytb)
Dune and Morrissey: 12S rRNA gene(303bp), 5 orders without any taxa related with hypnosqualea hypothesis (no batoids)
Arnason : complete mitogenome, 3 order of sharks, a skate and a ratfish
Rejection of the concept of Squalea(shirai, 1992): including skate.
No squaliform representative on same branch as the skate
=> Poorly represented elasmobranch taxonomic diversity
The superorder Selachimorpha is divided into Galea (or Galeomorphii), and Squalea. The Galeans are the Heterodontiformes, Orectolobiformes, Lamniformes, and Carcharhiniformes. Lamnoids and Carcharhinoids are usually placed in one clade, but recent studies show the Lamnoids and Orectoloboids are a clade. Some scientists now think that Heterodontoids may be Squalean. The Squalea is divided into Hexanchoidei and Squalomorpha. The Hexanchoidei includes the Hexanchiformes and Chlamydoselachiformes. The Squalomorpha contains the Squaliformes and the Hypnosqualea. The Hypnosqualea may be invalid. It includes the Squatiniformes, and the Pristorajea, which may also be invalid, but includes the Pristiophoriformes and the Batoidea.[

6. Purpose:
- To provide a molecular phylogenetic perspective “whether sharks are paraphyletic, due to a derived position for Batoidea,
or whether sharks are monophyletic, to the exclusion of Batoidea.
- An examination of several additional questions pertaining to elasmobranch interordinal affinities

7. 2. Materials and methods
2.1. Taxa and phylogenetic loci
* mitochondrial 12S– 16S and intervening tRNA valine, :
- powerful phylogenetic marker for other groups of vertebrates (Springer et al., 2001)
preliminary sequence data indicated there was little or no transition saturation, even in comparisons involving the chimaera sequence(s) (data not shown).
The sequence alignments: without ambiguous positions /software SOAP
:the effect of removing all the positions that are sensitive to variation
in alignment position.
- Rooting phylogenetic trees: Outgroups increase the noise in the sequence data but are required to polarise the trees.
- single outgroups : more subjective to the affects of long branch
attraction than multiple paraphyletic taxa.
Different outgroup permutations. The more outgroups utilized, the fewer positions were included in the results.
This permutation of outgroups provided an additional perspective on the batoid/shark diphyletic hypothesis

8. Squalo
Squatino
Galeo
Batoidea
(2010)
420
437
440
(1963)

9. 2.2. Phylogenetic reconstruction
* Modeltest software (Posada and Crandall, 1998)
;to objectively determine the best suited model of sequence evolution and the accompanying parameter values for these data
- Maximum likelihood (ML): TN+G+ I : used for the 420, 437, 440 bp data sets
The 1880, 1963, and 2010 data sets all used GTR+G+ I
Maximum parsimony (MP)
Minimum evolution (ME) / using both ML and LogDet distances.
/ using PAUP* 4b8 (Swofford, 1998).
Bayesian phylogenetic analyses in Mr.Bayes 2.01
GTR model allowing a gamma shape of among-site rate variation
- Statistical significance, under a likelihood model, of several phylogenetic hypotheses, was assessed using the KH test
3. Results and discussion

10. much more ancient split between the two groups, with
- all trees support a
much more ancient split between the two groups, with
batoids as the sister group to a clade consisting of all
shark orders
Shark monophyly
Hypnosqualea hypothesis were significantly rejected for all data sets in the KH test
Order: ML MP
ME ml
ME LogDet
Bayesian

11. Despite of lacks representatives of the putative deepest branching batoid orders (e.g.,Torpediniformes), no long branch attraction has generated the basal position of Batoidea.
1, all tree reconstruction methods recover this position, not only maximum parsimony(known to be sensitive to this reconstruction artefact)
2, the batoid-shark branch is far from being the longest, with the branch leading to nurse shark and the Hexanchiformes branch
3.unroot the tree, the branching arrangement remains unchanged
* shark monophyly, to support batoid monophyly: these data represent the first time a skate and ray are included in the same molecular phylogenetic analysisw.
The reduced data sets of 440, 437, and 420 bp also supported distinction between batoids and sharks, for shark monophyly, for the monophyly of Holocephali.
(If additional sequence: Chimaeridae would be paraphyletic),
* All elasmobranch, chondrichthyan,batoid monophyly

12. Several superordinal associations within sharks were strongly supported.
Squaliformes (Piked dogfish and gulper shark) with an angelshark/sawshark clade. =>the earlier morphological hypotheses by Shirai (1992b) and de Carvalho (1996), :Squaliformes as the sister group to Hypnosqualea. Without Batoidea from Hypnosqualea, would agree with this arrangement
Shirai (1992b, 1996) and de Carvalho (1996) suggested the concept of Squalea, which has the six and seven gill sharks as the sister group to Squaliformes/Hypnosqualea.
Mode of reproduction for Squalea (i.e., the association of Hexanchiformes, Squaliformes, Pristiophoriformes, and Squatiniformes) is yolksac viviparous (Compagno, 1990).
Batoid : uterine to oviparous
Compagno(1990)
Extended oviparous -> retained oviparous => yolksac viviparous => cannibal, placental or uterine viviparous.

13. Compagno (1973, 1977) /Shirai (1996)/ de Carvalho (1996) is Galeomorphii:
(((Lamniformes/Carcharhiniformes) Orectolobiformes) Heterodontiformes )))
The only portion of this hypothesis that is clearly evident
from our analyses is the lamniform/carcharhiniform association. (It’s ok)
The rest: Low bootstrap value / high p value
Certainly our data are insufficient to either accept or reject Shirai and de Carvalhos
Galeomorphii arrangement (2110 data set)
Our study provides the first convincing molecular case for rejection
of the Hypnosqualea hypothesis
and suggests that the seven or more putative morphological synapomorphies
used to define this superorder are either symplesiomorphic
or the consequence of convergent evolution in the ancestor
to sawsharks/angelsharks and batoids (depending on the resolution of Galeomorphii).

14. Our data strongly suggest that an evolutionary scenario for the diversification
of sharks and batoids is an early fundamental split into two respectively
monophyletic lineages.
A basal vs. derived position for Batoidea will have significant implications in
the polarization of morphological and life history characteristics for elasmobranchs and means that much of the current thinking
in this regard needs to be re-evaluated.