Karyological analysis of order testudines 2016

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Introduction

Here discuss about the biology that lead to studing the number of

chromosome in the order tetudines and inheritance of the

chromosomes in the offsprings and also studying of molecular

structure and function of gene ,gene behavior of a cell or organism is

the genetics

And also studying gene distribution,variation in population

Cytogenetic

that is branch of genetics that considered with studing the structure

and function of cell specially chromosomes

it also give acloser and amore comprehensive studying on changes in

structure and number of chromosomes from one organism to another

and this fact was used in what is called cytotaxonomy.

Cytotaonomy

Is a branch of science that classifies the livinh organisms based on

cytological studies (number of chromosomes meiosis behavior)

Helps to stablish relationships between the different organism one of

the methods of karyotyping

Karyotype

Is Method where total set of chromosomes of an organism is viewed

under microsocope where the number of chromosome along with their

length position of centromeres Banding pattern any differences between

sex chromosomes and any others physical CHARACTERISTIC is

observed (king, stansfield and mulligan 2006)

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kAIM OF WOR

Karyological data are available for 55% of all cryptodiran turtle species

including members of all but one family. Cladistic analysis of these data

as well as consideration of other taxonomic studies, lead us to propose

and phylogeny not greatly different from that a formal classification

suggested by other workers. Werecognize 11 families and three

superfamilies. The platysternid and staurotypidturtles are recognized at

the familial level. Patterns and models of karyotypic

evolution in turtles are reviewed and discussed.

RESONE FOR STUDING TURTLE

The history of where turtles are found is an

important record for conservation and

preservation efforts and an invaluable resource

for anyone interested in turtle research. If you

have ever wondered which turtles are found

where you live, you are interested in turtle

research.

this species of turtle and Because of diversity of

according to a lots of scientists that made research on order testudines

research that will disscuss in the following , so that I want studing this

Within the conversation community, turtles are considered to be in

situation , brought about by human activities {reviewed in van Dijk crisis

et al ., 2000; turte conversation fund 2002}

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Currently , out of 200 species of fresh water turtle and tortoises listed by

iucn} in he conversation of nature {nion for te listed as the world wide u

e their red list {IUCN, 2006} , 24 ar

Listed as critically endangerd

HOWEVER, about 100 species of fresh water turtles are not listed by

either because they are mor common or have not IUCN in the red list ,

yet been evaluated for listing this mean that at least about 42% of

freshwater turtles and tortoises are considered to be facing a high risk

of extinction , and are in need of urgent conversation action

Turtles have been prized as pets or killed for commercial products and

although some of this trade is met by commercial farms illegal harvest

from the wild occurs on a broad scale

et al.,2000} In many {thorbjarnarson

Morphology and taxonomy Introduction of turtles

A turtle is an animal in armor. Much of its body lies within a protective

shell, which has openings for the turtle's four chunky legs, short tail,

and head. When danger threatens, many turtles pull legs, tail, and head

l. But unlike some animals that live in shells, such as hermit into the shel

crabs and snails, a turtle cannot crawl out of its shell. The shell is part of

the turtle's body.

All turtles belong to the class of backboned animals known as reptiles.

s snakes, lizards, and crocodiles. Turtles are the This class also include

oldest group. The first turtles crawled about on earth more than

250,000,000 years ago. Turtles have changed very little since that time.

the Turtles are found in almost all temperature and tropical regions of

world. Many turtles spend all or most of their lives in fresh water. They

may live in swamps, ponds, running streams, or even roadside ditches.

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They come up on dry land to sun themselves or to lay eggs. Other

ers live in warm seas, sometimes turtles live completely on land. Still oth

following warm currents far northward.

The name "turtle" is often used to identify those animals that live in

water. The name "tortoise" frequently refers to a turtle that lives on land.

usually refers to small freshwater The American Indian name "terrapin"

turtles, especially those used for food. But these groupings are not

strictly scientific. In this article, all of these animals will be referred to

generally as turtles, though the proper name for a specific animal, such

Fig1as Galápagos tortoise, will be used.

Fig.1))

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General characteristics (around300species)-

Rigid shell enclosing the soft organs

(Fig 2)

-Carapace= dorsal part

-Plastron= ventral part

-Shell is composed of dermal bony elements covered by keratinous

scutesor leathery skinthe shell incorporates ribs, vertebrae, portions of

pectoral girdle-Plastron can be rigidor hinged -Shell shape –ranges

from domed(in terrestrial species) Flat to hydrodynamic

shaped(aquaticand marine species

(fig3)

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pads (terrestrial Absence of teeth (keratinous beakinstead)

-Freshwater species carnivorous, omnivorous, or herbivorous;

terrestrial usually herbivorous.

-Limb structure –flippers(marine species), webbing between digits

(freshwater species), stout limbs with thickened species)

"classification"

Kingdom Animalia animals

Phylum Chordata chordates

Subphylum Vertebrata vertebrates

Superclass Gnathostomata jawed vertebrates

Class sauropsida

Subclass anapsida

Order testudines

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Suborder Cryptodira

Super family testudinidae

Family Emydidae (Pond Turtles/Box and Water Turtles)

Family Testudinidae (Tortoises)

Family Geoemydidae (Bataguridae)

(Asian River Turtles, Leaf and

RoofedTurtles, Asian Box Turtles)

Family Platysternidae (Big-headed

Turtles)

Family chelydridae ( snapping turtle)

Superfamily Trionychoidea

Family Carettochelyidae (Pignose Turtles)

Family Trionychidae (Softshell Turtles)

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Superfamily Kinosternoidea

Family Dermatemydidae (River Turtles)

Family Kinosternidae (Mud and Musk Turtles)

Superfamily Chelonioidea

Family Cheloniidae (Sea Turtles)

Family Dermochelyidae (Leatherback Turtles)

Suborder Pleurodira

Family Chelidae (Austro-American Sideneck Turtles)

Superfam. Pelomedusoidea

Family Pelomedusidae (Afro-American Sideneck Turtles)

Family Podocnemididae (Madagascan Big-headed and American

Sideneck River Turtles)

Sauropsida ("lizard faces") is a group of amniotes that includes all

existing reptiles and birds as well as their fossil ancestors and relatives.

Sauropsida is distinguished from Synapsida, which

includes mammals and their fossil ancestors.

This clade includes Parareptilia and other extinct clades. All living

sauropsids are members of the sub-group Diapsida.

An anapsid is an amniote whose skull does not have openings near

the temples.[1] Traditionally, the Anapsida are the most primitive

subclass of reptiles, the ancestral stock from

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which Synapsida and Diapsida evolved, making anapsids paraphyletic. It is however doubtful whether all anapsids lack temporal fenestra as a primitive trait, or whether all the groups traditionally seen as anapsids

truly lacked fenestra DeBraga, M. (1996)

Temple indicates the side of the head behind the eyes. The bone beneath the temporal bone as well as part of the sphenoid bone.

(Fig5)

Cryptodira is a suborder of Testudines ;Zug, G. R. 1966) that includes

most living tortoises and turtles. Cryptodira differ from Pleurodira (side-

neck turtles) in that they lower their necks and pull the heads straight

back into the shells, instead of folding their necks sideways along the

body under the shells' margins. They include among their species

freshwater turtles, snapping turtles, tortoises, soft-shell turtles, and sea

turtles.

Two circumscriptions of the Cryptodira are commonly found. One is

used here; it includes a number of primitive extinct lineages known only

from fossils, as well as the Eucryptodira. These are, in turn, made up

from some very basal groups, and the Centrocryptodira contain the

prehistoric relatives of the living cryptodires, as well as the latter, which

are collectively called Polycryptodira. (Gaffney, E. S. 1975)

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The family Testudinidae contains approximately 11 genera and 40-50

species the plastron is usually without a hinge carapace is domed,

Ornat-box turtle (family emydidae) (Fig6)

Adaptations for terrestrial life include thick, elephantine rear legs, short,

web-less feet, and short digits. The forelegs usually have heavy scales

on the anterior surface. Tortoises can be diagnosed by the lack of

glands in the axillary and inguinal regions and the presence of only four

digits on the rear feet.( Gray, J. E. 1870)

The Testudinidae are most closely related to the pond turtles (Emydidae)

and are included along with that family in the Testudinoidea.

the Emydidae is split into two families- Emydidae and Bataguridae

The reduced volume of a fusiform body means sea turtles can not

retract their head, legs, and arms into their shells, like other turtles can.

the number of and shape ofscutes on the carapace, and the type of

inframarginal scutes on the plastron.There are seven extant species of

sea turtles: the green, loggerhead, Kemp's ridley, olive

ridley, hawksbill, flatback, and leatherback. (Nakamura ;1949)sea turtles

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have a more fusiform body plan than their terrestrial or freshwater

counterparts

Chelonia mydas green sea turtle (Fig.7)

Trionychia is a superfamily of turtles which encompasses

the species that are commonly referred to as softshelled turtles as well

as some others. They are found throughout the temperate regions of the

world.( Jordan,1975)

It traditionally consisted of a single family, two subfamilies, and

14 genera. However, more recently it was realized that the supposed

"Kinosternoidea" are actually early offshoots of the Trionychoidea and

not as closely related among (Pablo A. Martinez ; et al 2009)each other

as it was believed. These two families lack the characteristic

trionychoid apomorphies, but possess some highly derived characters

of their own, which they moreover evolved independently from each

other. (Frair, W. 1972)

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apalone spinifera turtle (Fig.8)

Kinosternoidea is a superfamily of aquatic turtles, which included two

families: Dermatemydidae, and Kinosternidae.

These are nowadays usually considered independent families of

the Trionychia, among which they represent ( Laurin,

etal(1996). very plesiomorphicmembers which share a few peculiarly

advanced traits. These apomorphies coupled with the overall

"primitiveness" was what mislead scientists as to their actual

relationships. elongated shells; plastron reduced or hinged;

carnivorous; bottom walkers

-musk glands on underside; barbelson the chin

Sternotherus odoratusstinkpot/ musk turtle( family kinosternon) (Fig 9)

The Pleurodira are identified by the method

with which they withdraw their heads into their shells. In these turtles,

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the neck is bent in the horizontal plane, drawing the head into a space in

front of one of the front legs. A larger overhang of the carapace helps to

protect the neck, which remains partially exposed after retraction. This

differs from the method employed by a cryptodiran, which tucks its

headandneckbetween its forelegs, within the shell. .( Pearse,et al 1947)

.

(Fig 10)

Family chelidea

Like all pleurodirous turtles, the chelids withdraw their necks sideways

into their shells, differing from cryptodires that fold their necks in the

vertical plane. Frank Grützner;2006 They are all highly aquatic species

with webbed feet and the capacity to stay submerged for long periods of

timeThe highly aquatic nature of the group is typified by the presence

of cloacal breathing in some species (Iverson,etal, 2012) are largely

strike-and-gape hunters or foragers feeding on fish,

Matamata turtle (Fig11)

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Pelomedusidae is a family of freshwater turtles native to sub-Saharan

Africa, with a single species, Pelomedusa subrufa, also found inYemen.

They range in size from 12 to 45 cm (4.7 to 17.7 in) in carapace length,

and are generally roundish in shape. They are unable to fully withdraw

their heads into their shells, instead drawing them to the side and

folding them beneath the upper edge of their shells, hence are

called African side-necked turtles. (W. E.Rainey. 1980)

Pleomedusa subrufa turtle (Fig12)

The Podocnemididae are a family of turtles native to Madagascar and

northern South America. They are side-necked turtles(Pleurodira), which

means they do not retract their heads( Clark; 1967) backwards, but hide

them sideways. These turtles are all aquatic, inhabiting streams and

other flowing water. Their shells are streamlined to aid in swimming.

Podocnemis turtle (Fig13)

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(Fig 14)

In the nucleus of each cell, the DNA molecule is packaged into thread-

like structures Called Chromosomes.Each chromosome is made up of

DNA tightly coiled manytimes around proteins called histones that

support its structure. (Gorman, G. C. 1973)Chromosomes are not visible

in the cell’s nucleus—not evenunder a microscope—when the cell is not

dividing. However, the DNA that makes up chromosomes becomes more

tightly packed during cell division and is then visible under a

microscope. Most of what researchers know about chromosomes was

learned by observing chromosomes during cell division.Each

chromosome has a constriction point called the centromere, which

divides the chromosome into two sections, or ―arms.‖ The short arm of

the chromosome is labeled the ―p arm.‖ The long arm of the

chromosome is labeled the ―q arm.‖ The location of the centromere on

each chromosome gives the chromosome its characteristic shape, and

can be used to help describe the location of specific genes

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(Fig 15)

what are type of chromosome?..

Metacentric Chromosomes Metacentric chromosomes have the centromere in the center, such that both sections are of equal length. Human chromosome 1 and 3 are metacentric.

Submetacentric Chromosomes Submetacentric chromosomes have the centromere slightly offset from the center leading to a slight asymmetry in the length of the two sections. Human chromosomes 4 through 12 are submetacentric.

Acrocentric Chromosomes Acrocentric chromosomes have a centromere which is severely offset from the center leading to one very long and one very short section. Human chromosomes 13,15, 21, and 22 are acrocentric.

Telocentric Chromosomes Telocentric chromosomes have the centromere at the very end of the chromosome. Humans do not possess telocentric chromosomes but they are found in other species such as mice.

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(Fig 16)

romosomes from an individual’s A karyotype is a picture of all the ch

abnormalities. chromosomecells. A karyotype is a test used to check for

A picture of a person’s chromosomes is created by staining the

chromosomes with a special dye, photographing them through a

microscope and arranging them in pairs. A karyotype gives information

he structure of their about the number of chromosomes a person has, t

chromosomes and the sex of the individual

(Fig 17)

What bands of chromosome ?.........

Q-Banding

Quinacrine mustard, an alkylating agent, was the first chemical to band chromosomes viewed under a fluorescence microscope. Quinacrine dihydrochloride has subsequently been substituted by quinacrine mustard. The alternating bands of bright and dull fluorescence are

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called Q bands. The bright bands are primary composed of DNA rich in adenine and thymine, while the dull bands are rich in guanine and cytosine.

Q bands are especially useful for distinguishing the human Y chromosome and various chromosome polymorphisms involving satellites and centromeres of specific chromosomes.

G-banding

Giemsa has become the most commonly used stain in human cytogenetic analysis. Unlike Q-banding, G-banding usually requires pre-treating chromosomes with either salt or a proteolytic (protein-digesting) enzyme. When chromosomes are pre-treated with the proteolytic enzyme trypsin the process is called GTG banding. Giemsa stains preferentially regions rich in adenine and thymine. Therefore, G bands correspond closely to Q bands.Standard G band staining techniques allow between 400 and 600 bands to be seen on metaphase chromosomes. With high resolution G-banding techniques, as many as two thousand different bands have been catalogued on the twenty-four human chromosomes.

R-banding Reverse banding (R-banding) involves the incubation of slides containing metaphase chromosomes in hot phosphate buffer and stained with Giemsa. The banding pattern that results is essentially the reverse of G bands. R bands are GC-rich. The AT-rich regions are selectively denatured by heat leaving the GC-rich regions intact. Fluorochromes that are GC specific also produce a reverse chromosome banding pattern. R-banding is helpful for analyzing the structure of chromosome ends, since these areas usually stain light with G-banding. C-Banding stains areas of heterochromatin, which is tightly packed and repetitive DNA. C-banding is specifically useful in humans to stain the centromeric chromosome regions and other regions containing constitutive heterochromatin - secondary constrictions of human chromosomes 1, 9, 16, and the distal segment of the Y chromosome long arm. NOR-banding

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NOR-banding involves silver staining (silver nitrate solution) of the "nucleolar organizing region", which contains rRNA genes.

T-Banding

T-banding involves the staining of telomeric regions of chromosomes using either Giemsa or acridine orange after controlled thermal denaturation. T bands apparently represent a subset of the R bands because they are smaller that the corresponding R bands and are more strictly telomeric.

(Fig 18)

The nucleolus organizer region (NOR) or nucleolar organizer is

a chromosomal region around which the nucleolus forms. This region is

the particular part of a chromosome that is associated with a nucleolus

after the nucleus divides. The region contains several tandem

copies of ribosomal DNA genes. In humans, the NOR contains genes for

5.8S, 18S, and 28S rRNA clustered on the short arms of chromosomes

13, 14, 15, 21 and 22 (the acrocentric chromosomes).Nucleolus organizer

regions (NORs) are head-to-tail arrays of genes encoding the precursor

of the three largest ribosomal RNAs (18S, 5.8S and 25S in plants). NORs

include active rRNA genes, which give rise to secondary constrictions of

metaphase chromosomes, and silent rRNA genes, which are often

highly compacted in dense heterochromatin.

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At metaphase, a proteinaceous remnant of the nucleolus often remains

associated with the secondary constriction. Each rRNA gene at a NOR is

nearly identical in sequence, although variation in size due to

differences in the number of repeated DNA elements in the intergenic

spacer region is common.

In karyotype analysis, a silver stain can be used to identify the

NOR. Silver nitrate inserts into the NOR-associated protein in the stalks

and satellites, staining the proteins dark black. The amount of stain

deposited and the number of NORs differs among the population,

although the cell should normally have a maximum of 10 NORs per cell.

INTRODUCTION of karyological analysis

of testudines

Cytogenetics is the branch of genetics that studies the structure and

behavior of chromosomes and their relation to human disease and

disease processes. During the past three decades, the importance of

clinical cytogenetics to the practice of obstetrics and gynecology has

dramatically increased because clinical cytogenetics has a direct effect

on the diagnosis, management, and prevention of many disorders that

are caused by chromosome aberrations. For many chromosome

disorders, physicians face medicolegal responsibilities in the areas of

counseling, screening, and diagnosis, and obstetricians and

gynecologists therefore must have knowledge about the human

chromosome constitution and be able to apply basic principles of

chromosome behavior to clinical practice. This chapter reviews

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important concepts and developments in cytogenetics and highlights

their applications in the practice of obstetrics and gynecology.

OVER the past 10 years knowledge of turtle karyology has grown to such an

extent that the order Testudines is one of the better known groups of lower

vertebrates (Bickham,1983). Nondifferentially stained karyotypes areknown for

55% of cryptodiran turtle species

and banded karyotypes for approximately 25%(Bickham, 1981). From this body

of knowledge,as well as a consideration of the morphologicalvariation in the

order, we herein present a general review of the cryptodiran karyological

literature and a discussion of the evolutionary relationships of the higher

categories ofcryptodiran turtles. Although this paper focuses on the Cryptodira

(the largest suborder ofturtles), the Pleurodira also has been well studied in

terms of standard karyotypes (Ayres etal., 1969; Gorman, 1973; Bull and etal,

1980)and a few have been studied with banding

Cope recognized the currently widely acceptedsubordersCryptodira and

Pleurodira.Two major differences between these two suborders are in the plane

of retraction of the neckand the relationship between the shell and pelvic girdle.

In the cryptodires ("hidden-necked"turtles), the neck is withdrawn into the body

ina vertical plane and the pelvis is not fused toeither the plastron or

carapace, whereas in the pleurodires ("side-necked" turtles) the

pelvicgirdle is fused to both the plastron and carapaceand the neck is

folded back against the body ina horizontal plane. Cope's suborder

Athecae

includes only the Dermochelyidae and is nolonger recognized. Most

authors include theDermochelyidae among the Cryptodira (Gaffney,

1975a; Mlynarski, 1976; Wermuth and etal, 1977; Pritchard,1979).

The families of the suborder Cryptodira arearranged in various

superfamilies by several authors. The Testudinoidea, Chelonioidea

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andTrionychoidea are superfamilies common tomost of the recent

classifications (Williams, 1950;Romer, 1966;Gaffney, 1975a; Mlynarski,

1976).However, the limits of these taxa are not uniformly agreed

upon.The non-trionychoid freshwater and land cryptodiran turtles

include the Chelydridae,Kinosternidae, Dermatemydidae,

Platysternidae,Emydidae and Testudinidae and are usually placed in the

Testudinoidea (Williams, 1950;

Romer, 1966). Gaffney (1975a) includes the Kinosternidae and

Dermatemydidae in theTrionychoidea. Mlynarski (1976) includes onlythe

Emydidae and Testudinidae in the Testudinoidea.He recognizes the

superfamily Chelydroidea to include the Chelydridae, Dermatemydidae,

Kinosternidae and Platysternidae.The Chelonioidea includes the Cheloniidaeand

the Dermochelyidae (Baur, 1893; Gaffney,1975a). Williams (1950), Romer

(1966), andMlynarski (1976) recognize a separate superfamily,the

Dermochelyoidea, for the familyDermochelyidae, and include only the

Cheloniidae in the Chelonioidea. Most of the currently utilized family orsubfamily

level taxa have been commonly recognized since Boulenger (1889). However,

thereis no complete agreement regarding the levelat which certain taxa should

be recognized. Parsons (1968) reviewed this confusing situationwith regard to

the Chelydridae, Staurotypidae,Kinosternidae, Platysternidae, Emydidae and

Testudinidae, as recognized here. Not mentioned by him are the inclusion of

Platysternonin the Chelydridae (Agassiz, 1857; Gaffney,1975b) and the

recognition of the Staurotypidae (Baur, 1891, 1893; Chkhkvadze, 1970).

The above discussion of the history of cryptodiran taxonomy serves to illustrate

the complexity of the relationships of the inclusive taxa.The taxonomic confusion

seems to result from:

1) extensive convergent evolution in certainmorphological traits,

Karyological analysis of order testudines 2016

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2) the failure of someworkers to distinguish between shared primitive and

shared derived character states

3)the lack of a widely accepted phylogeny of turtles. Chromosomal data are

used in this paperin an attempt to solve some of the evolutionaryand

classificatory problems. Cytogenetic information seems useful at this level

because of thehigh degree of conservatism expressed in cheloniankaryotypes

(Bickham, 1981). Additionally, the application of chromosome

bandingtechniques solves one of the most troublesomeproblems in phylogeny

reconstruction; namely,the determination of homologous characters.When two

chromosomes have identical bandingpatterns it can safely be concluded that

they are homologous. It is sometimes difficult to determine homology among

morphological characters. For example, determination of homologies among the

plastral scales of various turtle families is difficult. The fact that a scale is in the

same position in members of different familiesdoes not necessarily imply

homology (Hutchison et al, 1981

Most karyotypes were prepared by an in vivo technique as follows:

Blood was removed from the animal by means of a capillary tube

inserted into the posterior angleof the eyelids, and pushed posteriorly

into an orbital blood sinus. This was assumed to promote an immune

response involving cell division in the spleen. An intraperitoneal

injection of 0 1% colchicine was administered, at a dosage rate of 0.05

ml per gram body weight. The animal was killed 5 h after

the colchicine injection. The spleen, and in males a testis, was removed,

chopped finely and placedin 0.9% sodium citrate solution for 10 min. Air-

dried smears were prepared by the techniques ofBaker et al. (1971),

Karyological analysis of order testudines 2016

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being fixed in 3 : 1 methanol : acetic acid. The smears were stained in a

4% aqueoussolution of Gurr's improved Giemsa stain for 10- 15 min, and

mounted in canada balsam after beingdried for at least 1 h on a hotplate

at 40-45OC.Some early results were obtained by a method similar to that

of Baker et al. (1971). Bone marrowfrom the femur was flushed out with

0 9% sodium citrate solution, after the animal had been bled

and treated with phytohaemagglutinin for 2 days. Results from this

method were poor, with the exception of those for Physignathus

lesueurii, and the method described above was subsequentlydeveloped.

At least 10 divisions were examined from each preparation. The clearer

of these preparations were photographed under oil immersion. The

photomicrographs were printed and the lengths ofchromosomes

measured with dial calipers used in a stepwise fashion. The total length

of each chromosome arm was recorded, and the percentage of total

macrochromosome length and centromeric

indices were calculated for each division. Where possible somatic

preparations were used for analysis

(Fig 18)

Chromosomes were arranged, according to themethod of Bickham

(1975), into three groups(A:B:C:) where group A

includedmetacentricsubmetacentricmacrochromosomes,

groupBsubtelocentric-telocentric macrochromosomes,and group C

Karyological analysis of order testudines 2016

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microchromosomes. The A:B:C:formula is given after the diploidnumber

inFig. 19 and in the text.This paper represents a synthesis and

reanalysisof (mostly) published data. In reanalyzing the data we

employed cladistic methodology(Hennig, 1966) in which sister groups

were established by the determination of groups thatpossessed shared

derived characters (synapomorphies).Because banded karyotypes were

not available for the most appropriate outgroup

taxon (Suborder Pleurodira: Family Chelidae)we employed an "internal"

method of characterpolarity determination. Specifically, charactersthat

were shared among families considered tobe distantly related, known

from the fossil record to be early derivatives of the cryptodiraradiation,

or thought to be morphologicallyprimitive, were considered as primitive

(plesiomorphic)chromosomal characters. Because ofthe nature of

karyotypic variation in cryptodiresthe analysis was rather

straightforward. For example, dermatemydids are among the

mostprimitive living turtles and their fossil historyextends back to the

Cretaceous, as does the cheloniids

which are thought to be an early offshootof the cryptodiran line. These

two families possess species with apparently identical karyotypes. It is

highly unlikely that these two familiespossess a synapomorphy at this

level of the phylogeny. This would mean that these two familieswere

more closely related to each other than toany other families studied, an

arrangement thatappeared to conflict with every other line ofevidence in

the literature. We therefore considered this karyotype to be primitive, at

leastfor the non-trionychoid families, and the karyotypes of other

families were derived from this

Karyological analysis of order testudines 2016

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(see below)

(Fig 19)

Fig. 19. G-band karyotype of a batagurine emydid (Chinemys reevesi, 2n

= 52). The chromosomes are arranged into group A (metacentric or

submetacentric macrochromosomes), group B (telocentric and

subtelocentric macrochromosomes), and group C (microchromosomes)

Results and Discussion

The following discussion is segmented intothe commonly accepted family groups. In general, we have accepted each of the families asdistinct entities and do not question their validity .

Emydidae

The two subfamilies of emydid turtles are characterized by different

karyotypes.The predominantly New World emydines have2n = 50 and

the predominantly Old World batagurinesmostly have 2n = 52 (Table 1).

A fewbatagurine species also possess 2n = 50 (Table1), including

Siebenrockiella crassicollis, the onlyemydid known to possess sex

chromosomes (Carrand ,etal 1981). Bickham and etal (1976a)concluded

that the primitive karyotype of theEmydidae was 2n = 52 and identical to

that ofSacalia bealei and other Old World batagurines.This has been

supported by recent findings that some testudinids have banded

karyotypes identical to those of Chinemys reevesi and other batagurines

Karyological analysis of order testudines 2016

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(Dowler and etal, 1982). Fig. 19illustrates the karyotype of a batagurine

(Chinemys reevesi) that possesses the proposed primitive emydid

karyotype.The origin of the 2n = 50 emydine

is unclear (Bickham and etal, 1976a). There is no karyotypic evidence to indicate emydinesare at all closely related to Rhinoclemmys, the only New World batagurine genus (Carr, 1981).There may be some hint of the batagurine-emydine transition in the finding of several speciesof Asiatic batagurines with 2n = 50 (Table1). Any relationship of the emydines to the 2n =50 batagurines will require evidence from othercharacter systems in order to establish its existence.

Testudinidae.

The karyology of this family isnot as well studied as that of the Emydidae but

it seems certain that the primitive karyotype is2n = 52. Some species are known

to possess Gbandpatterns identical to those of certain batagurines including

Geochelone pardalis, G. elongate and G. elephantopus (Dowler and etal,1982).

C-band variation exists among species ofGeochelone, and the karyotypes of

Gopherusspecies differ from Geochelone species by themorphology and location

of the nucleolar organizing region (NOR) (Dowler and etal,1982). Although this

family is nearly world-wide

in distribution and morhpologically diverse, theavailable data indicate a high

degree of karyologicalconservatism.

Karyological analysis of order testudines 2016

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Table 1

Karyological analysis of order testudines 2016

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table 2

Karyological analysis of order testudines 2016

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Table 3

Karyological analysis of order testudines 2016

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Table 4

Karyological analysis of order testudines 2016

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(Table 5)

Platysternidae.

The standard karyotype of thesingle species of platysternid

(Platysternon megacephalum) has 2n = 54 (Haiduk and Bickham,1982).

This species appears to have close affinities to the Emydidae but is

karyotypically distinct from all emydids thus far studied. BecauseP.

megacephalum and emydids do apparently havesynapomorphic

chromosomes that are notshared with chelydrids, Haiduk and Bickham

(1982) considered P. megacephalum to comprisea family distinct from

the Chelydridae (sensuGaffney, 1975b) and resurrected the

Platysternidae (Gray, 1870), a move also suggested byWhetstone (1978).

Karyological analysis of order testudines 2016

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Staurotypidae. This group is usually considered to be a subfamily (Staurotypinae) of

theKinosternidae. Standard karyotypes of all three species in this group

are known (Table 1; seeespecially Bull et al., 1974). The two species of

Staurotypus are distinctive in possessing an XX/XY sex chromosome

system (Bull et al., 1974;Sites et al., 1979a). Claudius angustatus,

likenearly all other turtle species studied, does not possess

heteromorphic sex chromosomes but appears to be otherwise

karyotypically identicalto Staurotypus (Bull et al., 1974). Sites et

al.(1979a, b) report banded karyotypes of 5. Salvini and show that this

species possesses abiarmed second group B macrochromosome

that appears to be homologous to an identicalelement in emydids and

testudinids (and platysternidsbased on standard chromosome

morphology). This chromosome is acrocentricinchelydrids,kinosternids,

dermatemydids andcheloniids (Fig. 20). We conclude that the

biarmedcondition is derived. Centric fusion of the ancestral acrocentric

macrochromosome with amicrochromosome accounts for the presence

of a subtelocentric macrochromosome in the common ancester of the

Emydidae, Testudinidae,Platysternidae and Staurotypidae. This is

indicative of the staurotypids belonging to a cladethat does not include

kinosternids (Kinosternonand Sternotherus). This seems irreconcilable

with

(Fig 20)

Fig. 20. G-band patterns of the second group B chromosomes of (left to right) a staurotypid, an emydid,a kinosternid

Karyological analysis of order testudines 2016

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and a cheloniid. The long arms ofall 4 taxa are identical; the short arms of the staurotypid and emydid are euchromatic and identical, however, the short arms of the kinosternid and thecheloniid are small and heterochromatic; see text forfurther discussion

Chelydridae.

The two extant species of thisfamily have been studied for both

standard (Table 1) and banded karyotypes (Haiduk and et al,1982).

Chelydra serpentina and Macroclemystemminckii both have 2n = 52 but

differ in themorphology of certain chromosomes. Haidukand et al (1982)

conclude that these two

species do not share any derived chromosomalcharacteristics with each

other or with any other families of Cryptodira. However, the karyotypeof

M. temminckii could be derived from that

of C. serpentina. The latter is considered theprimitive karyotype for the

family.

Kinosternidae

This family is comprised of twogenera and about 18 species and has been well

studied karyotypically (Table 1). Early, and apparently inaccurate, reports aside

(Table 1), allspecies thus far examined appear to possess2n = 56. Banded

karyotypes (Bickham and etal, 1979; Sites et al., 1979b) indicate all

speciespossess a large, subtelocentric macrochromosome

not found in any other group of turtles.Kinosternids do not share any derived

chromosomal characters with any other turtle family, including the staurotypids

with which they

Karyological analysis of order testudines 2016

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are usually considered confamilial. An interesting variation was found in

this family by Sitesetal. (1979b). Heterochromatin that stains darkin both

G- and C-band preparations was found

in Sternotherus minor, Kinosternon baurii and K.subrubrum, but not

found in K. scorpioides. Thepresence of this type of heterochromatin

wasconsidered to be a derived character (it is not found in closely

related families) shared amongthe three species that possess it,

indicating that the genus Sternotherus has affinities with temperate

species of Kinosternon.

Dermatemydidae.

The single extant speciesofthis family (Dermatemys mawii) possesses 2n = 56

(Table 1). There are no uniquely derived elements and this species shares no

derived chromosomes with any other family.

Cheloniidae.

Members of this family possess2n = 56 (Table 1). Banding data indicate

cheloniidsand dermatemydids are karyotypicallyindistinguishable (Bickham et

al., 1980; Carr etal., 1981). Early reports of other diploid numbers and sex

chromosomes have not been substantiated by recent studies using current

techniques.

Trionychidae. Members of both subfamilies(Cyclanorbinae and Trionychinae) have 2n =66

(Table 1). Reports of other diploid numbershave been unsubstantiated in

subsequent studies. The report of 2n = 52-54 in Trionyx leithii(Singh et al., 1970)

was due to the misidentification

Karyological analysis of order testudines 2016

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of this specimen (Kachuga dhongoka, Emydidae;Singh, 1972). The 2n = 66

karotype wasconsidered by Bickham et al. (1983) to be theprimitive karyotype

for the family. Banding comparisons between Trionyx and Chelonia revealed

little homology between the Trionychidae and Cheloniidae (Bickham et al.,

1983).

Carettochelyidae

The single extant species(Carettochelys insculpta) has 2n = 68 (Bickham et

al., 1983). Although no banding data have beenreported for this species, the

standard karyotype is very similar to the 2n = 66 karyotype of trionychids.

Taxonomy

The acceptability of using karyotypicdata in order to draw phylogenetic

inferences and erect a classification at the level offamily and higher is based

upon the conservatism of the karyotypic character system. Bycharacter system,

we refer to a suite of characters and character states which may be presumed to

be closely enough related to be withinthe realm of influence of the same set

ofevolutionary constraints. According to this line ofresasoning then, karyotypic

data constitute acharacter system separate from the charactersystems

associated with electrophoretic data orcranial osteology, etc. The level at which

characters are relatively constant within a group isthe point at which

thosecharacters are of systematic utility and those characters are said

tobeconservative (Farris, 1966). Our studies anda review of the

pertinentliteratureindicate thatfamily level groups within the Cryptodira

arecharacteristically karyotypically homogeneousand that the significant

variation (in the phylogenetic sense) is observable interfamilially. Itis upon these

premises that we propose the classification in Table 6 based upon our

cladisticanalysis of the karyotypic data.This classification is conservative in that

allfamilies commonly recognized are maintained,even though in two instances

Karyological analysis of order testudines 2016

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there are familypairs which we cannot karyotypically distinguish [i.e.,

Cheloniidae-Dermatemydidae

Table 6

(Fig 21)

Fig 21. Cladogram showing the hypothesized relationships of the higher

categories of cryptodiranturtles. The diploid number and the number of

chromosome pairs in groups A:B:C (Fig. 19) in the proposedprimitive

karyotype of each family (and both subfamilies of Emydidae) are shown.

Because the trionychoidfamilies are so divergent, the A:B:C formulas

are notgiven (Bickham et al., 1983). Characters 1 -5 are listed

and discussed in the text. to recognize the Staurotypinae as a separate

Karyological analysis of order testudines 2016

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family, the Staurotypidae. This conclusion is incongruentwith data from

other character systems. Many morphological studies report similarities

between the Kinosternidae andStaurotypidae (among these Williams,

1950;Parsons, 1968; Zug, 1971). Most such studieshave not attempted

cladistic analyses (two exceptions are Gaffney, 1975; et al, 1981). There

seems no obvious orsimple manner in which to reconcile the conflicting

data from the karyotypic character system and the overwhelming

amount of data fromvarious morphological character systems.

Inrecognizing the Staurotypidae, we have madeexplicit our prediction of

its relationships toother testudinoid families. Independent confirmation

or refutation of these relationships will determine the merit of this

move.The three superfamilies are all considered tobe holophyletic. Fig.

21 presents a cladogram that we believe best reflects the branching

sequence of the evolution of this group. The Testudinoidea and

Chelonioidea may be sistergroups but this is as yet unproved. The

primitive karyotypes of these two taxa are identical,2n = 56 (character 1

in Fig. 21), and very different from that of the Trionychoidea, 2n = 66-68

(character 2 in Fig. 21),but we do not yet know the polarity of these

character states(Bickham et al., 1983). All testudinoid and chelonioid

turtles possessat least seven group A macrochromosomes(character 1

in Fig. 21). Among the testudinoidfamilies, aclade that includes

Staurotypidae,Platysternidae, Testudinidae, and Emydidae canbe

identified by the presence of a biarmed second group B

macrochromosome (character 3in Fig. 21; Fig. 20). Another clade

includes thePlatysternidae, Testudinidae and Emydidae allof which

primitively possess nine group A macrochromosomes (Fig. 1; character

4 in Fig. 21).A clade including the Emydidae and Testudinidae is

characterized by a 2n = 52 9:5:12 primitive karyotype (Fig. 19; character

Karyological analysis of order testudines 2016

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5 in Fig. 21).Species of the emydid subfamily Emydinae allpossess a

karyotype derived from the primitive9:5:12 arrangement (Bickham and

etal,1976a).

The Dermatemydidae, Kinosternidae andChelyridae possess no chromosomal

synapomorphiesand the branching sequence of these families is not obvious

from chromosomal, morphological or serological data. However, the

Chelydridae is usually considered to be mostclosely related to the Emydidae

(McDowell,1964; Zug, 1971;Frair, 1972; Haiduk and etal, 1982) and the

dermatemydids, morphologically one of the most primitive families ofturtles, are

considered closely allied to the Kinosternidae (Zug, 1971; Frair, 1972;

Gaffney,1975b).The Cheloniidae and Dermochelyidae areconsidered to comprise

the suborder Chelonioidea.There are no karyotypic data availablefor

Dermochelys coriacea so the relationship between this species and cheloniids

has yet to betested chromosomally. But, these two families are closely related

morphologically and serologically(Frair, 1979). We follow most other workers in

giving this group full superfamilialstatus, recognizing that they have invaded

anadaptive zone, the marine environment, that is distinctly different from that of

most other turtles. It must be emphasized that Chelonia mydas(Chelonioidea)

and Dermatemys mawii (Testudinoidea) appear karyotypically identical and

weinterpret this to be the primitive karyotype of these two superfamilies.The

superfamily Trionychoidea includes onlythe Trionychidae and Carettochelyidae.

Thesetwo taxa are closely related chromosomally as well as morphologically and

their karyotypesare distinctly different from those of species of the other two

superfamilies. Some workers haveincluded the Kinosternidae

andDermatemydidae in the Trionychoidea (Gaffney, 1975a).The chromosomal

data do not support such anarrangement because of the disparity in diploid

number and chromosome morphology between testudinoids (including

kinosternids and dermatemydids) and trionychoids (Bickhametal.,1983Historical

review of taxonomic relationships.—The primary subdivisions of the order

Karyological analysis of order testudines 2016

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comprisingthe turtles have undergone a great many namechanges and

rearrangements over the last 100years. Cope (1871) presented an

arrangementof the families into suborders which is still widely accepted today.

Until Cope, the subordinal andsuprafamilial classification of turtles was primarily

based on differences in the digits amongthe sea turtles, the aquatic turtles

and/or theterrestrial tortoises. Hoffman (1890) and Kuhn(1967) present reviews

of the early classifications.Cope recognized the currently widely accepted

suborders Cryptodira and Pleurodira.Two major differences between these two

suborders are in the plane of retraction of the neck).and the relationship

between the shell and pelvic girdle. In the cryptodires ("hidden-necked"turtles),

the neck is withdrawn into the body ina vertical plane and the pelvis is not fused

toeither the plastron or carapace, whereas in the pleurodires ("side-necked"

turtles) the pelvicgirdle is fused to both the plastron and carapaceand the neck

is folded back against the body in

a horizontal plane. Cope's suborder Athecaeincludes only the

Dermochelyidaeand is nolonger recognized. Most authors

includetheDermochelyidae among the Cryptodira (Gaffney,1975a; Mlynarski,

1976; Wermuth andMertens, 1977; Pritchard, 1979).A few authors recognize

the Trionychoidea(sensu Siebenrock, 1909) and/or the Chelonioidea(sensu Baur,

1893) at a suprafamilialrank equivalent with the Cryptodira and

Pleurodira (Boulenger, 1889;Lindholm, 1929; Mertens et al., 1934).

The suborder Cryptodira isused here in the sense of Williams (1950) and

subsequent authors and includes all living nonpleurodiranturtles.The families of

the suborder Cryptodira arearranged in various superfamilies by several authors.

The Testudinoidea, Chelonioidea andTrionychoidea are superfamilies common

tomost of the recentclassifications (Williams, 1950;Romer, 1966;Gaffney,

1975a; Mlynarski, 1976).However, the limits of these taxa are not uniformly

agreed upon.The non-trionychoid freshwater and landcryptodiran turtles include

the Chelydridae,Kinosternidae, Dermatemydidae, Platysternidae,Emydidae and

Karyological analysis of order testudines 2016

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Testudinidae and are usually placed in the Testudinoidea (Williams,

1950;Romer, 1966). Gaffney (1975a) includes the Kinosternidae and

Dermatemydidae in the© 1983 by the American Society of Ichthyologists and

HerpetologistsTrionychoidea. Mlynarski (1976) includes onlythe Emydidae and

Testudinidae in the Testudinoidea.

He recognizes the superfamily Chelydroideato include the Chelydridae,

Dermatemydidae,

Kinosternidae and Platysternidae.The Chelonioidea includes the Cheloniidae

and the Dermochelyidae (Baur, 1893; Gaffney,1975a). Williams (1950), Romer

(1966), and Mlynarski (1976) recognize a separate superfamily,the

Dermochelyoidea, for the familyDermochelyidae, and include only the

Cheloniidae in the Chelonioidea.The Trionychoidea usually includes both

theTrionychidae and Carettochelyidae(Mlynarski,1976), but Williams (1950) and

Romer (1966)recognize the Carettochelyidae separately

intheCarettochelyoidea.Most of the currently utilized family orsubfamily level

taxa have been commonly recognized since Boulenger (1889). However, thereis

no completeagreementregarding the level at which certain taxa should be

recognized. Parsons (1968) reviewed this confusing situationwith regard to the

Chelydridae, Staurotypidae ,Kinosternidae, Platysternidae, Emydidae and

Testudinidae, as recognized here. Not mentioned by him are the inclusion of

Platysternonin the Chelydridae (Agassiz, 1857; Gaffney,

1975b) and the recognition of the Staurotypidae (Baur, 1891,

1893;Chkhkvadze, 1970).The above discussion of the history of

cryptodirantaxonomy serves to illustrate the complexity of the relationships of

the inclusive taxa.The taxonomic confusion seems to result from:

1) extensive convergent evolution in certainmorphological traits

, 2) the failure of someworkers to distinguish between shared primitive and

shared derived character states and

Karyological analysis of order testudines 2016

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3)the lack of a widely accepted phylogeny of turtles. Chromosomal data are

used in this paperin an attempt to solve some of the evolutionaryand

classificatory problems. Cytogenetic information seems useful at this level

because of the high degree of conservatism expressed in chelonian karyotypes

(Bickham, 1981). Additionally, the application of chromosome banding

techniques solves one of the most troublesomeproblems in phylogenyre

construction; namely,the determination of homologous characters.When two

chromosomes have identical bandingpatterns it can safely be concluded that

they arehomologous. It is sometimes difficult to determine homology among

morphological characters. For example, determination of homologiesamong the

plastral scales of various turtle families is difficult. The fact that a scale is in

thesame position in members of different familiesdoes not necessarily imply

homology (Hutchison and Bramble, 1981).

(Fig 22)

Karyological analysis of order testudines 2016

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Testudindae 1) Karyotype ofGeochelone denticulate , male 2n =52 , with an 2:6:12

complement of groups A:B:C 2) karyotype of Geochelone carboaria , male 2n

=52, 9:5:12 (bickham and et al 1976b)

(Fig 23)

emydidae 3) karyotype of chryemys terrapin , male 2n =50 8:5:12

4) karyotype of chrysemys decorate, male 2n=50 , 8:5:12

Karyological analysis of order testudines 2016

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5) karyotype of chrysemys stejnegeri vicina ,male , 2n =50, 8:5:12(Bickham and

BAKER 1976b)

(Fig 24)

Batagurinae 6)Karyotype of rhinoclemys pulcherrima ,female 2n= 52 , 6:5:15

7) karyotype of rhinoclemys puctularia female 2n=56 (BICKHAM AND BAKERb)

(Fig25)

Karyological analysis of order testudines 2016

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kinostrenidae 8) karyotype of kinosternon scorpioides , female 2n = 56, 7:6:15 (Bickham an et

al 1976b)

(Fig 26)

dermatemydidae FlG. 26.—Standard karyotype of Dermatemys mawii with chromosomes

arranged into groups: (A) metacentric to submetacentric macrochromosomes, (B) telocentric to subtelocentric macrochromosomes, and (C) microchromosomes. The standard karyotype of D. mawii(2n = 56) is presented in Figure 26. Chromosomes are arranged according to Bickham(1975) into group A metacentric orsubmetacentric macrochromosomes,group B telocentric or subtelocentric macrochromosomes, and group C microchromosomes.There are 7, 5, and 16 pairs of chromosomes in groups A, B, and C, respectively. A heteromorphic pair ofsex chromosomes is not present in themale specimen examined.

Karyological analysis of order testudines 2016

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(Fig 27)

Fig. 27 G- and C-banded metaphase chromosome karyotypes of female (a, c) andmale (b, d) A. spinifera, respectively. e Enlarged images of the highly heterochromatic microchromosomes in A. spinifera, indicating theWin female (Fem) and the heterochromatic microchromosome pair (m) in both females and males. A large block of the female-specific chromosome (W) is Giemsa faint and

Karyological analysis of order testudines 2016

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C-positive. The arrow indicates the female-specific chromosomes with large C-positive block. Note that the Z is morphologically indistinguishable from several other microchromosomes with similar banding pattern. Scale bar=10 μm

Trionychidae 1972; BICKHAM ET AL. 1983). NINE PAIRS OF MACROCHROMOSOMES AND 24

PAIRS OF MICROCHROMOSOMES WEREIDENTIFIED, DIFFERING SLIGHTLY FROM THE

REPORT BYBICKHAM ET AL. (1983) OF EIGHT PAIRS OF MACROCHROMOSOMES.

THE MACROCHROMOSOMES IDENTIFIED HEREINCLUDED TWO PAIRS OF

METACENTRIC, FOUR PAIRS OF SUBMETACENTRIC,AND THREE PAIRS OF

ACROCENTRIC CHROMOSOMES(FIG. 27). THE CENTROMERE POSITION OF THE 24

PAIRS OFMICROCHROMOSOMES COULD NOT BE DETECTED ACCURATELYDUE TO

THEIR SMALL SIZE, WHICH IMPEDES THE UNAMBIGUOUSPAIRING OF SOME

MICROCHROMOSOMES WITH THEIR GBANDED

Fig 28

Chelonoidea

With the method described above, metaphases were identified with a good

distribution and number, allowing the identification of sets of chromosomes. The

Karyological analysis of order testudines 2016

Page 48

non-banded mitotic chromosomes were visualized by Giemsa staining.

Chelonoidis carbonaria revealed a diploid number of 2n = 52

chromosomes, in both sexes, divided into three groups (A, B, C). Group

A was composed of 28 chromosomes (3 metacentric pairs, one

acrocentric and 10 submetacentric pairs), group B consisted of seven

pairs of acrocentric chromosomes, and group C showed five pairs of

microchromosomes (Figure 1A,B). Sex chromosomes were not

observed.

SUBORDER PLEURODIRA INTRODUCTION of pleurodira

Turtles of the suborder Pleurodira are divided into two families, the

Chelidae and the Pelomedusidae, which are clearly separated by both

morphological (Gaffney, 1977) and molecular (Shaffer et al., 1997)

features. The Chelidae consists of nine genera, five of which are found

in Australia and New Guinea and four in South America (Ernst and

Barbour, 1989). Conflicting phylogenies have been proposed for the

Chelidae, but recent phylogenetic analysis based on molecular markers

(Seddon et al., 1997; Fujita et al., 2004) support the monophyly of the

Australian/New Guinea and South American chelid turtles. The chelid

genus Hydromedusa (commonly known as snake-necked turtles)

consists of two species of semi-aquatic turtles that have an extremely

long throat: H. maximiliani, restricted to the southeast region of Brazil;

and et al, distributed throughout southern and southeastern Brazil,

northeastern Argentina, Uruguay and southeastern Paraguay.

The chromosomes of birds, fishes and some reptile groups are highly

variable in terms of size and morphology, and are characterize by

bimodal or asymmetric karyotypes composed of macro and

Karyological analysis of order testudines 2016

Page 49

microchromosomes. Turtle karyotypes show two general tendencies

based on the presence or absence of microchromosomes but there is

much variation between groups. For example, the chromosome number

in the order Chelonia ranges from 2n = 26 in Podocnemis

dumeriliana (Ayres et al., 1969) to 2n = 96 in Platemys platycephala (Bull

and Legler, 1980; Bickham et al., 1985). Also, while karyotypic studies

have frequently been published for turtles from the suborder Cryptodira,

information about Pleurodires is scarce and fragmented and mainly

based on conventional staining techniques.

In this paper describe the almost complete karyotypic characterization

of Hydromedusa tectifera using several staining techniques and in

situ Fluorescence Hybridization (FISH).

SUPERFAMILY PELOMEDUSIDAE

Pelomedusids have low diploid numbers and few microchromosomes

(2n = 26–36); the five largest chromosomes are homologous in the

three genera.

The big-headed side-neck river turtle, Peltocephalusdumerilianus

(Schweigger, 1812), occurs in the Amazonregion and belongs to the

superfamily Pelomedusoides (approximately24 living species), which

comprises the families

Pelomedusidae, with two living genera: Pelomedusaand Pelusios

represented by one, and at least 15 species, respectively;and

Podocnemididae, with three living genera:the monotypic Erymnochelys

and Peltocephalus, andPodocnemis comprising six species (Ayres et al.,

1969;Vitt and et al, 2009). In Podocnemididae cytogeneticdata are scarce

and based mostly on conventional staining.The Podocnemis and

Erymnochelys species (P. erythrocephala,P. expansa, P. lewyana, P.

sextuberculata, P.unifilis, P. vogli and E. madagascariensis) present a

diploidnumber (2n) of 28, with a karyotype composed of

Karyological analysis of order testudines 2016

Page 50

fivemacrochromosomes (M) and nine microchromosomes (m)(Ayres et

al., 1969; Huang and Fred Clark 1969; Rhodin etal., 1978; Bull and Legler,

1980; Fantin andet al, 2011;Gunski et al., 2013). The exception is

Peltocephalus

dumerilianus that presents 2n = 26 with 4 M and9m, the lowest diploid

number in Testudines (ranging from

2n = 26 to 2n = 96) (Ayres et al., 1969; Bull and et al,1980). The available

cytogenetic data for this species report

a karyotype that is similar to those of other Podocnemididae,in which

differentiated sex chromosomes are absentand a conspicuous

secondary constriction is observed

the karyotype of P.dumerillianus was characterized for the first time

using routine differential techniques, such as GTG, CBGbandingand Ag-

NOR staining (Seabright, 1971; Sumner,For all individuals, at least 20

metaphases were analyzedfor determining the 2n = 26 and FN = 52

karyotype,as described by Ayres et al., 1969, with a conspicuous

secondary constriction on pair 1 (Figure 1A). GTG-banding

patterns allowed the identification and the pairing of allchromosomes

(Figure 1B). CBG-bands were tenuous at thepericentromeric region of

most pairs, except for pair1

(Fig 29)

Figure 29- Karyotype (A and B) and metaphases (C-E) of Peltocephalus dumerilianus, 2n = 26 and FN = 52. (A) Conventional staining. Inset, pair

Karyological analysis of order testudines 2016

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1 from other metaphase showing the conspicuous secondary constriction. (B) GTG-banding pattern. (C) CBG-banding pattern. Note the conspicuous C-positive bands on pair 1. Inset, pair 1 bearing positive Ag-NORs.

probes. Positive signals (green) are seen at the termini of all chromosomes. (E) Mapping of 45S rDNA. Positive FISH signals (red) are at the secondary

constrictionregion of pair 1.

FAMILT PODOcnemis

To determine karyotypes we counted 35 cellsin each individuals from each species. Our resultsshowed that the karyotypic number forboth species of Podocnemis is 2n = 28 chromosomes,consisting of 5 pairs of macrochromosomesand 9 pairs of microchromosomes, with the following morphologies: 16m + 2sm + 10aand NF = 46 (Figure 1). Silver-nitrate staining All species of the genusPodocnemis present a chromosomal number of2n=28, which is extremely low compared withkaryotypes described for other species. Few karyotypic studies have been conductedon the genus Podocnemis

(AYRES et al. 1969;RHODIN et al. 1978; BULL and et al1980; ORTIZet al.

2005), the work by Ayres et al. (1969)is the only one that presents

cytogenetic studies

for P. expansa and P. sextuberculata. However,there are still no studies

on chromosomal evolutionwithin the family Podocnemidae, nor arethere

any comparative studies of chromosomal

banding.This work describes the karyotypes and thelocalization of the

nucleolar organizer regions(NORs) in two species of the genus

Podocnemis

(Fig 30)

Karyological analysis of order testudines 2016

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Fig. 30 — Karyotype of Podocnemis sextuberculata (above)microchromosomes and P. expansa with2n = 28.

Family chelidea

Chelids have high diploid numbers and many microchromosomes (2n =

50–64) and are similar in this respect to cryptodires (2n = 50–66).

(Fig 31)

Karyological analysis of order testudines 2016

Page 53

Fig 32

The chromosome complement of all our Hydromedusa

tectifera specimens was 2n = 58, of which 22 were macrochromosomes

and 36 microchromosomes (Figure 1a 33). It was possible to precisely

determine the position of the centromere in the macrochromosomes,

and we observed one submetacentric chromosome pair, one

metacentric pair and nine pairs of acrocentric chromosomes, giving a

total of 62 chromosome arms. No sex chromosome heteromorphism

was observed. This diploid number agrees with the study of Bull and

Legler (1980),

Karyological analysis of order testudines 2016

Page 54

(Fig 33)

(Fig34)

The G-banding permitted the visualization, especially in the

macrochromosomes, of a pattern of bands that enabled better

identification and pairing of the chromosomes as well as the

construction of an ideogram Figure 34). Such a pattern is similar, but not

identical, to that observed in other Pleurodiran turtles, due to the

presence and absence of some bands when compared to the patterns

found by Bull and Legler (1980) in Pelomedusoid turtles (a group related

to the Chelidae). This variation in the G-banding pattern in Pleurodiran

turtles establishes a different karyotypic evolution from that identified

for the suborder Cryptodira. Previous reports have suggested genomic

stability in Cryptodiran turtles, in which both the banded chromosome

Karyological analysis of order testudines 2016

Page 55

morphology (Bickham, 1981) and the DNA sequences inside the

chromosomes (Muhlmann-Díaz et al., 2001) remain unchanged for

millions of years.

WE CONCLUDE, FOR TWOREASONS, THAT THE PRIMITIVE KARYOTYPE OF THE

SUBORDER CRYPTODIRA IS MOST LIKELY THE 2N = 56KARYOTYPE OF CHELONIID AND

DERMATEMYDID TURTLES. FIRST, THESE ARE AMONG THE MOST ANCIENTFAMILIES IN

THE SUBORDER (BOTH DATE FROM THECRETACEOUS), AND SECOND, THIS KARYOTYPE

IS HIGHLY GENERALIZED AND COULD HAVE GIVEN RISE TO THEDIVERSITY OF

KARYOTYPES IN THE SUBORDER BY A MINIMUM NUMBER OF EVENTS. A PRIMITIVE

KARYOTYPEMORE SIMILAR TO THAT OF TRIONYCHOID TURTLES (2N =66-68) CANNOT

ENTIRELY BE RULED OUT (BICKHAMET AL., 1983). COMPARISONS WITH KARYOTYPES

OFTHE SPECIES OF PLEURODIRA DO NOT SOLVE THE PROBLEM BECAUSE SPECIES OF

THE CHELIDAE ARE KNOWNTO POSSESS DIPLOID NUMBERS IN THE 2N = 56 RANGEAS

WELL AS THE 2N = 66 RANGE (BULL AND LEGLER,

1980). HOWEVER, THE PRIMITIVE KARYOTYPE OF THEPLEURODIRA WAS

CONSIDERED BY Bull and Legler

(1980) to be 2n = 50-54 which is consistent withour hypothesis of a 2n =

56 ancestral karyotypefor the Cryptodira.If the above hypothesis is true,

then chromosomal evolution in the Trionychoidea involved an increase

in the diploid number by a

reduction in the number of macrochromosomesand an increase in the

number of microchromosomes.However, chromosomal evolution inthe

Testudinoidea reduced the diploid numberby an increase in the number

of macrochromosomes and reduction of the number of

microchromosomes. Bickham and Baker (1979) note that specieswithin

a family or subfamily possess identical or very similar karyotypes.

However, karyotypic comparisons among families and subfamilies

Karyological analysis of order testudines 2016

Page 56

almost always reveal variation. A more refinedanalysis of the pattern of

karyotypic variationin turtles (Bickham, 1981) suggests that the rateof

karyotypic evolution has decelerated and thatMesozoic turtles evolved

at a rate twice as fastas their descendants. Additionally, the kinds

ofchromosomal rearrangements incorporatedduring the diversification

of cryptodiran families differ from the kinds of rearrangements

incorporated during the evolution of modernspecieThe above described

pattern of karyotypicevolution is consistent with thecanalizationmodel

of chromosomal evolution (Bickham andBaker, 1979). Under this model,

evolution ofthe karyotype is driven by natural selection because the

chromosomal rearrangements altergenetic regulatory systems. Changes

that areadaptive accumulate more rapidly during theearly radiation of a

lineage. As time goes onmore and more adaptive linkage groups

areproduced. Further chromosomal rearrangement tends to break up

adaptive gene sequencesand the rate of chromosomal evolution

slowsdown. Thus, in an ancient group such as turtles,the process of

canalization has had such a longperiod of time to act that karyotypic

evolutionamong modern forms is virtually nonexistent.However, when

karyotypic comparisons aremade of taxa that diverged early during

turtleevolution, such as comparisons of the primitivekaryotypes of

families, variation is found to bemore pronounced.Models that explain

karyotypic evolution bypopulation demography, such as deme size,

donot apply to turtles. The classical model of chromosomal speciation

(White, 1978) requires fixation of chromosomal rearrangements in

smalldemes due to genetic drift orinbreeding. Thereis some question as

to whether chromosomal

speciation is in fact a viable process (Bickhamand Baker, 1979, 1980;

Futuyma and Mayer,1980), but even if it is, it certainly is not operative in

Karyological analysis of order testudines 2016

Page 57

turtles. There are no known chromosomal races in turtles. This could be

explainedby turtles characteristically not having smallpopulation sizes

or other demographic factorsthat promote the fixation of chromosomal

rearrangements by genetic drift or inbreeding.However, turtles display

such a diversity of demographic characteristics (Auffenberg andIverson,

1979; Bury, 1979; Bustard, 1979) thatthis explanation seems untenable.

Turtles exhibit a diverse array of morphological types and occur in

nearly all habitatsavailable to reptiles. Some, such as the migratory sea

turtles, are highly vagile but others,such as tortoises, have relatively low

vagility.Reproductive rates also vary. The green turtlemay lay as many

as 200 eggs in a single clutch,some emydids may lay only a single large

egg.While there are certainly many species thatcharacteristically have

large population sizes, wecan point to many that probably do not. For

example, kinosternids and emydids that occurin the arid western United

States and Mexicos.

often are found in isolated stock tanks, ponds,intermittent streams and

permanent springs.Population sizes are often small and there isprobably

very little migration among populations.Many of the above mentioned

biological characteristics of turtles conceivably could

promotechromosomal speciation. That it does not occurin a major

radiation (Cryptodira) does not meanthat the process is not viable in

other taxa, butits absence is somewhat unexpected. In conclusion,

population parameters are poorly correlated with chromosomal

variability in turtles andin principle we agree with the criticisms of

thechromosomal speciation models espoused by

Bickham and et al (1979, 1980) and Futuyma and et al (1980).

Karyological analysis of order testudines 2016

Page 58

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