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Figure 1.1 : Haploid turtle karyotypes. Key: 1=Chelydra serpentina, 2=Macroclemmys temminckii, 3=Platysternon megacephalum, 4=Clemmys guttata, 5=Sacalia bealei, 6=Siebenrockiella crassicolis, 7=Maureyms caspica, 8=Rhinoclemmys punctularia, 9=Rhinoclemmys pulcherrima, 10=Geochelone denticulata, 11=Geochelone carbonaria, 12=Chinemys reevesi, 13=Carettochelys insculpta, 14=Trionyx spiniferus, 15=Kinosternon scorpiodes, 16=Chelonia mydras, 17=Batrachelys dahli, 18=Chelus fimbriatus, 19=Hydromedusa tectifera (first 11 chromosomes), 20=Phyrnops geoffroanus, 21=Platemys platycephala (first 13 chromosomes), 22=Chelodina expansei, 23=Chelodina steindachneri (first 11 chromosomes), 24=Elseya dentate, 25=Rheodytes (first 11 chromosomes only), 26=Emydura australis, 27=Pseudomydura umbrina, 28=Pelusios subniger, 29=Pelomedusa subrufa, 30=Podocnemis unifilis, 31=Erymnochelys madagascariensis (adapted from previously published material Bickham 1975; Killebrew 1975; Bickham 1976; Bickham and Baker 1976; Bickham, Bjorndal et al. 1980; Bull and Legler 1980; Carr and Bickham 1981; Haiduk and Bickham 1982; Bickham, Bull et al. 1983; Bickham and Carr 1983; Bickham, Tucker et al. 1985)...... 21
Figure 1.2a: Haploid crocodilian karyotypes arranged using traditional crocodilian phylogenies. Key 1=Alligator mississippiensis, 2=Tomistoma schlegelii, 3=Gavialis gangeticus, 4=Ostelaemus tetraspis, 5=Paleosuchus palpebrosus, 6=Melanosuchus niger, 7=Caiman latirostris, 8=Crocodylus siamensis, 9=Crocodylus porosus, 10=Crocodylus intermedius, 11=Crocodylus johnsoni, 12=Crocodylus novaeguineae, 13=Crocodylus moreletti, 14=Crocodylus cataphractus, 15=Crocodylus palustris, 16=Crocodylus rhombifer. Adapted from previously published material (Cohen and Gans 1970)...... 23
Figure 1.2b: Haploid Crocodylian karyotypes arranged following an alternate phylogeny (Janke, Gullberg et al. 2005). Key 1=Alligator mississippiensis, 2=Ostelaemus tetraspis, 3=Paleosuchus palpebrosus, 4=Melanosuchus niger, 5=Caiman latirostris, 6=Gavialis gangeticus, 7= Tomistoma schlegelii, 8=Crocodylus siamensis, 9=Crocodylus porosus, 10=Crocodylus intermedius, 11=Crocodylus johnsoni, 12=Crocodylus novaeguineae, 13=Crocodylus moreletti, 14=Crocodylus cataphractus, 15=Crocodylus palustris, 16=Crocodylus rhombifer. Adapted from previously published material (Cohen and Gans 1970)...... 24
Figure 1.3 : Distribution of fossil Sphenodontian material on a modern world map. Created using a variety of sources (Sues and Reisz 1995; Reynoso 1996; Reynoso 1996; Evans and Sigogneau-Russell 1997; Reynoso 1997; Reynoso and Clark 1998; Benton 2000; Reynoso 2000; Apesteguia and Novas 2003)...... 29
Figure 1.4: Present distribution of tuatara (Gaze 2001)...... 30
Figure 1.5: The range of centromeric positions divided into four regions, ‘m’=metacentric, ‘sm’=submetacentric, ‘st’=subtelocentric, ‘t’=telocentric (modified from Levan, Fredga et al. 1964)...... 40
Figure 1.7: Idiogram of Sphenodon indicating total chromosome length (TCL), centromere position and Ag-NOR location (black block)...... 46
Figure 1.6: Karyotypes of Sphenodon. (a) Giemsa stained karyotype of a male S. guntheri; (b) Ag-NOR stained karyotype of a female S. punctatus (north-eastern group, Stanley Island) ...... 49

10
Figure 1.8: Partial karyotypes (chromosomes 1 – 7) of a Ruamahua-iti animal with additional material on chromosome 3. (a) Giemsa staining; (b) C-banding; (c) Ag-NOR staining...... 49
Figure 1.9: Scatter diagram of mutant and normal chromosome 3 using colchicine and colcemid as cell cycle inhibitors, plotting TCL of the long arm against TCL of the short arm. () Normal Ruamahua-iti chromosome 3; () chromosome 3 with additional material inhibited with colchicine; (
) other member of chromosome pair 3 inhibited with colchicine; (
) chromosome 3 with additional material inhibited by colcemid; () other member of chromosome pair 3 inhibited with colcemid...... 50
Figure 1.11: Variation in Ag-NORs in Sphenodon. (a) Stanley Island (heteromorphic); (b) Stephens Island (homomorphic); (c) Stephens Island (heteromorphic); (d) male Stephens Island (homomorphic); (e) North Brothers Island (heteromorphic)...... 51
Figure 1.10: C-banded metaphase in situ spread of Sphenodon...... 52
Figure 1.12: Restriction endonuclease banding in Sphenodon; a) AluI RE digestion; b) HaeIII RE digestion; c) EcoRI RE digestion...... 52
Figure 1.13: G- banded in situ chromosomes of Sphenodon...... 53
Figure 1.14: Partial haploid karyotypes (chromosomes 1-14) of (a) Sphenodon punctatus, 2n=36; (b) Chelydra serpentina (Chelydridae), 2n=52; (c) Clemmys guttata (Emydidae), 2n=50; (d) Geochelone carbonaria (Testudinidae), 2n=52; (e) Rhinoclemmys punctularia (Bataguridae), 2n=56; (f) Carettochelys insculpta (Carettochelyidae), 2n=68; (g) Trionyx spiniferus (Trionychidae), 2n=66; (h) Kinosternon scorpiodes (Kinosternidae), 2n=56; (i) Chelonia mydras (Cheloniidae), 2n=56; (j) Chelodina expansa (Chelidae), 2n=54; (k) Pelusios subniger (Pelomedusidae), 2n=34; (l) Podocnemis unifilis (Podocnemididae), 2n=28; (m) Alligator mississippiensis, 2n=32; (n) Paleosuchus palpebrosus, 2n=42; (o) Crocodylus johnstoni, 2n=32. Chromosomes have been resized from original material to allow comparison (prepared from the current study and Cohen and Gans 1970; Bickham 1975; Bickham and Baker 1976; Bickham, Bjorndal et al. 1980; Bull and Legler 1980; Haiduk and Bickham 1982; Bickham and Carr 1983)...... 54
Figure 2.1: Diagram of anapsid and diapsid skulls demonstrating the major difference between the two (adapted from Dorit, Walker Jr et al. 1991)...... 68
Figure 2.2: Incidence of GSD and TSD using a traditional morphological division of anapsids and diapsids (Smith and Sinclair 2004)...... 76
Figure 2.3: (a) AMH isolation, black bar indicates band of interest in various tuatara, (b) LML to estimate DNA concentration from two samples...... 98
Figure 2.4: (a) WT1 isolation, black bar indicates bar of interest in various tuatara (Coppermine and Tawhiti Rahi samples from two gels aligned), (b) LML gel to estimate isolated DNA concentration, (c) WT1 isolation from Cook Strait samples demonstrating two bands labelled A and B...... 98
Figure 2.5: (a) DMRT1 isolation, the two bands cut out are labelled Fast and Slow based on their migration distances, (b) Estimation of DNA concentration...... 98
Figure 2.6: (a) DMRT1 alternative isolation, bands of interest are marked with a black bar, (b) LML to estimate DNA concentration for BDT sequencing...... 99
Figure 2.7: Estimation of DNA concentration from extracted 28S sequence...... 99
Figure 2.8: Estimation of DNA concentration from extracted FoxG1 sequence...... 99
Figure 2.9: Phylogeny created from sequence extracted by BN DM TUT F/R primers, Neighbour-Joining 1000 bootstrap...... 99

11
Figure 2.10: Maximum Parsimony phylogeny created using FoxG1 ...... 100
sequence, 1000 bootstrap...... 100
Figure 2.11 Maximum Parsimony phylogeny created using 28S sequences, 1000 bootstrap...... 100
Figure 3.1: Partial haploid karyotypes (chromosomes 1-14) of (a) Sphenodon punctatus, 2n=36; (b) Chelydra serpentina (Chelydridae), 2n=52; (c) Clemmys guttata (Emydidae), 2n=50; (d) Geochelone carbonaria (Testudinidae), 2n=52; (e) Rhinoclemmys punctularia (Bataguridae), 2n=56; (f) Carettochelys insculpta (Carettochelyidae), 2n=68; (g) Trionyx spiniferus (Trionychidae), 2n=66; (h) Kinosternon scorpiodes (Kinosternidae), 2n=56; (i) Chelonia mydras (Cheloniidae), 2n=56; (j) Chelodina expansa (Chelidae), 2n=54; (k) Pelusios subniger (Pelomedusidae), 2n=34; (l) Podocnemis unifilis (Podocnemididae), 2n=28; (m) Alligator mississippiensis, 2n=32; (n) Paleosuchus palpebrosus, 2n=42; (o) Crocodylus johnstoni, 2n=32; (p) Gallus domesticus 2n=78. Chromosomes have been resized from original material to allow comparison (prepared from the current study and Cohen and Gans 1970; Takagi and Sasaki 1974; Bickham 1975; Bickham and Baker 1976; Bickham, Bjorndal et al. 1980; Bull and Legler 1980; Haiduk and Bickham 1982; Bickham and Carr 1983)...121
Figure 3.2: DIG labelled ddUTP incorporation using 1:500 dilutions of previously isolated tuatara WT1...... 128
Figure 3.3: Estimation of DIG labelled WT1 probe concentration using a LML...... 128
Figure 3.4: Examples of WT1 probe hybridisation in Sphenodon. (A) DAPI illumination, (B) FITC illumination and (C) composite image from DAPI and FITC images. Possible hybridisation indicated by white bars...... 129
Figure 3.5: Examples of AMH probe hybridisation in Sphenodon. (A) DAPI illumination, (B) FITC illumination and (C) composite image from DAPI and FITC images. Possible hybridisation indicated by white bars...... 130
Figure 3.6: Human telomeric chromosome paint FISH on Sphenodon chromosomes. (a) DAPI excitation demonstrating chromosomes, (b) FITC excitation demonstrating locations of...... 132
hybridisation, and (c) composite image with FITC overlaid on the DAPI image showing location of the telomere probe...... 132
Figure 3.7: Gallus domesticus chromosome 1 hybridisation to Sphenodon chromosomes (a) DAPI, (b) FITC and (c) composite image. Possible hybridisation indicated by white lines...... 133
Figure 3.8: Gallus domesticus chromosome 2 hybridisation to Sphenodon chromosomes (a) DAPI, (b) FITC and (c) composite image. Possible hybridisation indicated by white lines...... 134
Figure 3.9: Gallus domesticus chromosome 3 hybridisation to Sphenodon chromosomes (a) DAPI, (b) FITC and (c) composite image. Possible hybridisation indicated by white lines...... 135
Figure 3.10: Gallus domesticus chromosome 4 hybridisation to Sphenodon chromosomes (a) DAPI, (b) FITC and (c) composite image. Possible hybridisation indicated by white lines...... 135
Figure 3.11: Gallus domesticus chromosome Z hybridisation to Sphenodon chromosomes (a) DAPI, (b) FITC and (c) composite image. Possible hybridisation indicated by white lines...... 135
Figure 3.12: Gallus domesticus chromosome W hybridisation to Sphenodon chromosomes (a) DAPI, (b) FITC and (c) composite image. Possible hybridisation indicated by white lines...... 136

12
Figure 4.1: Reptilian proto-karyotype idiogram based on the Sphenodon karyotype...... 144
Figure 4.2: Traditional morphological phylogeny of Synapsids, Diapsids and Anapsids (modified from Lee 2001)...... 147
Figure 4.3: Phylogeny based on chromosome morphology (A) with a traditional Lepidosauria and Archosauria and (B) based purely on chromosome morphology and presence/absence of microchromosomes...... 149

 

 
Table 1.1: Nomenclature for centromeric position on mitotic chromosomes (Green and Sessions 1991)...... 40
Table 1.2: Quantitative description of Sphenodon chromosomes. The range of total chromosome length (TCL) and centromeric index (CI) is listed...... 46
Table 1.3: Comparison of Wylie et al (1968) and the current study chromosome numbering ...... 56
Table 2.1: Samples used for DNA sequence analysis...... 87
Table 2.2: PCR mastermix for sequence extraction...... 90
Table 2.3: AWC protocol for Big Dye Termination protocol
X...... 92
Table 2.4 Blast results from Sphenodon sequences...... 95
Table 2.5 Summary of sequence data ...... 96
Table 2.6 Genbank accession numbers for DNA sequences used in analysis ...... 97
Table 3.1: PCR labelling mastermix for DIG labelling ...... 123
Table 3.2: WT1 probe hybridisation summary ...... 131
Table 3.3: AMH probe hybridisation summary...... 131

13
 


   






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30
Reptilian chromosome banding and use of banding for evolutionary studies



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2. All chromosomes show similar banding patterns, except in chromosomes with

special functions such as sex chromosomes.

32
3. Pronounced telomeric bands are associated with short chromosomes or

chromosome arms, whereas longer chromosomes or chromosome arms tend to

develop intercalary bands.

4. C-banding in non-homologous members of the diploid set tend to be located at

similar sites with respect to centromere-band distance.

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same distance from the centromere as the proximal border of the band. Thus

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65

 
  

  
 
 

 
 

 



 

 
 

66

  



 


    

   





   

The 
chromosomal  

 


 demonstrated in Chapter 1   arrived at independently of   

methods commonly used for phylogenetic reconstruction, namely DNA sequencing and morphological  . In the current chapter DNA sequences of the genes FoxG1, 28S rRNA, and the sex determining genes AMH, WT1 and DMRT1, were  


Sphenodon. No variation was seen in FoxG1, 28S and AMH DNA sequences. WT1 sequence analysis clearly separated the north-eastern North Island and Cook Strait populations,  a unique  of WT1 within Cook Strait populations.

DMRT1 possessed no obvious population differentiation. Testudines and Sphenodon did not show the same close relationship reported in Chapter 1.

 

Phylogenetic reconstruction using morphological characteristics can obtain data from fossil specimens, unlike molecular analysis where limits exist on obtaining samples from which DNA can be successfully extracted and sequenced " 



!
&$$(
 
 

!
&$$)#. Therefore, one of the advantages of morphological analysis is the ability to collect data from extant and extinct species

"
%***

&$$%

&$$'#.

67 DNA phylogeny and molecular research on reptiles

Regions of the Tree of Life (http://tolweb.org/tree/phylogeny.html) pertaining to


 are reasonably well defined. The four extant and many extinct Orders have been defined and aligned using morphological evidence. Over the last 20 years fine- scale resolution using molecular data has accumulated, clarifying relationships between closely   species and occasionally questioning established morphological relationships. Descriptions of Reptilia relationships have been the subject of much discussion in the literature, and there are still regions within the

Reptilia that are controversial, in particular the placement of turtles ' 


 
*00-$
 


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!

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+)))$

+))*$
!

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+))*$




+)),(. Recent review work on the definition of the Reptilia advanced removal of the formal name Anapsida, due to the limited use outside of referring to morphological structures (Figure 2.1) '


+)),(. The current study follows the definition of Reptilia “…all members of the synapsid sister-group, regardless of the interrelationships of turtles and other extant non-synapsid amniotes”

'


+)),(.

 
+&*%
  

 

 
 
 

 

 

 
'  

#



&
*00*(

One aim of phylogenetic reconstruction is to organise species into monophyletic groupings. Within the Reptilia, the two clades Archosauria (crocodiles, dinosaurs and

68 birds) and Lepidosauria (Sphenodon and Squamata) are generally agreed to be monophyletic ' 

 
*//.$

+)))$

+))*(. Both clades are diapsid, with two temporal openings (Figure 2.1). Although members of the

Reptilia, Testudines (turtles) possess a derived anapsid skull (Figure 2.1) ' 


"
*///(. Four categories for  

the Testudines within the Reptilia are possible: '*(
as an outgroup to the Lepidosauria and Archosauria; '+(
as a sistergroup to the Lepidosauria; ',(
as a sistergroup to the Archosauria; or '-(
within one of the two clades. Depending on what is being analysed, molecular research has supported all four categories.

1. Outgroup to Lepidosauria and Archosauria

  !
 
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+))*(%

2. Sister group to the Lepidosauria

 !


 


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3. Sister group to the Archosauria

Sequencing of iguana and caiman mitochondrial DNA established that the iguana genome evolves more slowly than that of birds and mammals, whereas alligator and caiman mitochondrial genomes are evolving at a higher rate, a result contradicting the correlation between rate of molecular evolution and generation time ' #

 

%
+))*(. Rapidly evolving mtDNA is a problem for phylogenetic analysis as ordering and rates of evolution are not constant between all species.

69 Analysis of mtDNA placed turtles at the base of the archosaurian branch, supporting the hypothesis that the Testudine anapsid condition is secondarily derived from a diapsid state # 


&,,,!

'%%%!
 


"

'%%&$.

Studies using incomplete mtDNA and short tRNA sequences produced ambiguous results # 
 

"
&,,)!



&,,+$. Comparison of full mtDNA sequences from tuatara (Sphenodon punctatus), turtles (Chrysemys picta,

Chelonia mydas, Pelomedusa subrufa), crocodiles (Caiman crocodylus, Alligator mississippiensis), birds (Vidna chalybeata, Buteo buteo, Rhea americana), lizards

(Eumeces egregius, Iguana iguana, Dinodon semicarinatos) and mammals (Didelphis virginiana, Mus musculus) revealed similarities between Sphenodon, birds and crocodiles. Phylogenetic analysis supported a monophyletic Testudines as a sistergroup to the monophyletic Archosauria, with that clade a sistergroup to a monophyletic Lepidosauria # 


"
'%%($.

4. Within one of the Lepidosauria and Archosauria clades

Reptilian relationships using molecular studies often split the traditional Lepidosauria and Archosauria. Haemoglobin (Hb) chains placed tuatara within the aves and turtles

(
A-Hb), with crocodiles as a sister-group. Squamata were well outside this grouping.

i- and
ii- Hb chains showed a similar pattern, although resolution was poor #

&,,($. Pancreatic polypeptide sequence data placed turtles as diverging after snakes.

The authors stated that the results were the same regardless of topology constraints used during analysis #  


&,,*$.

70 Recent work examining the two forms of the giant tortoise Geochelone gigantea
-D globin genes (
-D and
) supported a phylogeny where tortoises and birds were the closest living relatives of each other   
#!!# . Phylogenies created using the
-D globin gene grouped tuatara and birds (rhea and chicken) with turtles as a sister-group. Within the
genes, turtles and aves grouped together, with Homo sapiens as an outgroup. It should be noted that there were no representatives of the

Crocodylia in the phylogeny, which may have changed relationships within the

Reptilia. The Komodo dragon and a snake grouped some distance from the aves, turtles and tuatara, although they possess
-D globins. This was interpreted as indicating the Squamata began diverging around 335 MYA, much earlier than other
type globins   
#!!# . I suggest that the placement is more evidence for a rapidly evolving squamate genome compared to that of other reptiles. Firstly, most evidence suggests the S  
 

"%0 MYA 
  



#!!#
 
#!!% . Secondly, high recombination rates, smaller G-banding regions, smaller chromosomes, and smaller effective deme sizes 
  



#!!# all provide opportunity for rapid evolution within the Squamata, compared to the other reptilian rders.

Spermatozoa studies emphasised strong similarities between tuatara, turtle, crocodilian, and to a lesser extent non-passerine avian spermatozoa  


  
"&&#
  

 
"&&#
 

  
"&&$ . Reptilian eye structure analysis placed Crocodylia as a sister clade to two groupings, Testudines and a Lepidosauria and Aves clade    



#!!$ .

71 Molecular phylogenies appear more likely than traditional morphological analysis to support paraphyletic reptilian clades. DNA phylogenetics have been less successful in determining where in the Reptilia the Testudines fit/ as gene sequences frequently give results at odds with other gene
'%)"'. Larger datasets provide increased resolution, a situation seen with mtDNA where full sequence analysis gave significantly different results to earlier partial analysis by supporting (
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Other methods of phylogenetic analysis including sperm morphology and ophthalmic evolution supported a strong relationship between turtles and Sphenodon.

Mitochondrial DNA variation and rates of change within the archaic Reptilia

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Aside from five Testudine species' Kachuga smithii (Emydidae, ZW/ZZ system),

Siebenrockiella crassicollis (Emydidae, XX/XY system), Staurotypus salvinii

(Kinosternidae, XX/XY system), Staurotypus triporcatus (Kinosternidae, XX/XY system) and Chelodina longicollis (Chelidae, XX/XY system) +  '
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" 
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76 Incidence of TSD and GSD within Reptilian Orders

Order: Squamata


 
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TSD in Reptiles

There are three patterns of TSD: FM (low temperature
, high temperature ); MF

(low temperature , high temperature
); and FMF (low and high temperatures
,

77 midrange temperatures
). FMF patterns occur in all reptilian lineages with the possible exception of Sphenodon, suggesting it is basal $ 

 
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- 169 -

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