the ultrastructure of the spermatozoon of the lizard iguana iguana (reptilia, squamata, iguanidae)...

J. Anat.

(2004)

204

, pp451–464

© Anatomical Society of Great Britain and Ireland 2004

Blackwell Publishing, Ltd.

The ultrastructure of the spermatozoon of the lizard

Iguana iguana

(Reptilia, Squamata, Iguanidae) and the variability of sperm morphology among iguanian lizards

Gustavo H. C. Vieira,

1

Guarino R. Colli

2

and Sônia N. Báo

1

1

Departamento de Biologia Celular, and

2

Departamento de Zoologia, Universidade de Brasília, Brazil

Abstract

The spermatozoon of

Iguana iguana

is filiform and resembles that of other iguanian lizards, being most similar to

Tropidurus

. All sperm synapomorphies of Tetrapoda, Amniota and Squamata are present in the sperm of

Iguana

iguana

. By reconstructing the evolution of 30 sperm characters we identified a novel synapomorphy of Iguania:

the presence of a well-developed acrosomal ridge at the level of the epinuclear lucent zone. Because of the

poor topological resolution among iguanian clades we could not discount the possibility of convergence or neutral

selection as determinant of the variability in characteristics of the sperm cell. In agreement with previous studies,

we identified heterogeneous rates of evolution among the three main regions of the sperm cell, namely the head,

midpiece and tail.

Key words

convergence; evolution; phylogeny; polymorphism; reproduction.

Introduction

Iguanians comprise a diverse array of lizards with

poorly understood phylogenetic relationships (Frost

& Etheridge, 1989; Macey et al. 1997; Schulte et al. 1998;

Frost et al. 2001). Despite the use of different data sets

( i.e. morphological, molecular or mixed data sets), the

results suffer from low resolution and disagreement

among phylogenies derived from each data set. More

recently, Schulte et al. (2003) analysed a combination

of molecular and morphological data and obtained

statistical support for the monophyly of many iguanian

clades, but, like all previous studies, arrived at a lack of

topological resolution among the major lineages. These

results probably stem from character incongruence, as

a result of heterogeneity in evolutionary rates among

different data sets or data sets with different phyloge-

netic histories, sampling error, lack of sufficient infor-

mation among the internal branches of the tree, or lack

of phylogenetic structure in the data (Swofford, 1991;

Miyamoto & Fitch, 1995; Wiens & Hollingsworth, 2000;

Schulte et al. 2003).

The ultrastructure of sperm has been used recently in

phylogenetic analysis of squamates (Jamieson, 1995b,

1999; Jamieson et al. 1996; Oliver et al. 1996; Teixeira

et al. 1999b). These studies demonstrated that sperm

ultrastructure characters contain significant phyloge-

netic signal. However, sperm ultrastructure phylogenies

suffer from limited number of characters and a limited rep-

resentation of taxonomic groups (Teixeira et al. 1999c).

The ultrastructure of sperm has been adequately

described in six families of Iguania: Agamidae (

Pogona

barbata

, Oliver et al. 1996), Chamaleonidae (

Bradypodion

karrooicum

, Jamieson, 1995b); Crotaphytidae (

Crota-

phytus bicinctores

and

Gambelia wislizenii

, Scheltinga

et al. 2001), Phrynosomatidae (

Urosaurus ornatus

and

Uta stansburiana

, Scheltinga et al. 2000), Polychrotidae

(

Anolis carolinensis

and

Polychrus acutirostris

, Teixeira

et al. 1999a; Scheltinga et al. 2001) and Tropiduridae

(

Liolaemus austromendocinus

,

Tropidurus semitaeniatus

and

T. torquatus

, Furieri, 1974; Teixeira et al. 1999d).

Saita et al. (1988) provided the first description of the

ultrastructure of reproductive cells of

Iguana iguana

(misidentified as

I. delicatissima

), but their account was

Correspondence

Gustavo H. C. Vieira, Departamento de Biologia Celular, Universidade de Brasília, DF, CEP 70910–900, Brazil. T: 55 61 307 24 24; F: 55 61 347 65 33; E: [email protected]

Accepted for publication

29 March 2004

The ultrastructure of the spermatozoa of

Iguana iguana

, G. H. C. Vieira et al.


452

limited to distinct point events in spermiogenesis such

as acrosome formation, nuclear condensation and elong-

ation, midpiece formation, and association between

spermatids and Sertoli cells, with no reference to the

mature sperm.

Although some authors have discussed the variability

of sperm-derived characters (Oliver et al. 1996; Scheltinga

et al. 2000; Tavares-Bastos et al. 2002), the nature of

the variation and the stability of characters are still

poorly understood in Squamata and particularly among

families of Iguania. Here we describe, for the first time,

the detailed ultrastructure of mature sperm of

Iguana

iguana

, comparing our results with data provided from

the literature for other lizards. In addition, we investigate

the evolution of sperm morphology within iguanian

lizards, using phylogenetic methods.

Methods

We obtained epididymal mature spermatozoa from

three adult specimens of

Iguana iguana

, from Santa

Terezinha, Mato Grosso state (two) and Palmas, Tocantins

state, Brazil (one). Specimens were deposited at the

Coleção Herpetológica da Universidade de Brasília

(CHUNB 10743, 10792 and 21934, respectively).

We killed lizards with Tiopental®, removed epidi-

dymides by dissection and diced them into 2–3-mm

3

pieces. We fixed epididymal tissues overnight at 4

°

C in

a solution containing 2.5% glutaraldehyde, 2% para-

formaldehyde and 3% sucrose in 0.1

M

sodium cacody-

late buffer, pH 7.2. Specimens were then rinsed in 0.1

M

sodium cacodylate buffer, pH 7.2, with 3% sucrose and

post-fixed for 1 h with 1% osmium tetroxide, 0.8%

potassium ferricyanide and 5 m

M

CaCl

2

in 0.1

M

sodium

cacodylate buffer, pH 7.2. We dehydrated the material

in a series of ascending acetone (30–100%) and embed-

ded it in Spurr’s epoxy resin. We stained ultrathin

sections with uranyl acetate and lead citrate, and made

observations in a Jeol 100C transmission electron micro-

scope. We made light microscopic observations of

spermatozoa, from glutaraldehyde–paraformaldehyde-

fixed sperm smears, under Nomarski contrast using a

Zeiss Axiophot microscope.

We took measurements of the sperm cell using

Nomarski light micrographs: total length (TL), head

length (HL) and tail length (TaL), and using transmis-

sion electron micrographs: acrosome complex length

(AL), midpiece length (MPL), epinuclear lucent zone

length (ETL) and width (ETW), nuclear rostrum length

(NRL), nuclear shoulders width (NSW), nuclear base

width (NBW), distal centriole length (DCL), mean

distance between dense bodies (DDB), ratio of anterior

portion of principal piece to midpiece width (PPMR)

and percentage of midpiece occupied by fibrous sheath

(FSOM). The nuclear length (NL) was estimated by

subtracting mean acrosome complex length from head

length. For each variable we report the mean, standard

deviation and number of observations.

We coded sperm characters into discrete states,

optimizing character transformations on the phylogeny

of Iguania provided by Macey et al. (1997), using Fitch

parsimony (Kitching et al. 1998). We identified unam-

biguous character transformations with the ‘Trace

Selected’ option in MacClade v.4.0, treating polytomies

as regions of ambiguous resolution (‘soft polytomies’,

Maddison & Maddison, 2003). We resolved ambiguous

transformations using the ACCTRAN and DELTRAN

character optimization options in

PAUP

v.4.0b10 (Swofford,

1999). It should be noted that

PAUP

treats polytomies as

multiple speciation events (‘hard polytomies’, Maddison

& Maddison, 2003). A list of characters and the list of

character changes are given in Appendices 1 and 2,

respectively. We used

Sphenodon punctatus

(Healy &

Jamieson, 1994),

Cercosaura ocellata

(Teixeira, 2003),

Tupinambis merianae

(Tavares-Bastos et al. 2002) and

Ameiva ameiva

(Giugliano et al. 2002) as outgroups, to

establish the direction of evolutionary changes in the

characters. Outgroup relationships were based on Estes

et al. (1988) and Lee (1998).

Results

The spermatozoon of

Iguana iguana

is filiform, consist-

ing of the head region (nucleus and acrosome cap),

midpiece (containing the mitochondrial gyres) and tail

(the flagellar region). The mean length of the cell is

71.69

µ

m. The whole sperm of

I. iguana

is represented

diagrammatically in Fig. 1, and Fig. 2(K) shows the sperm

observed from Nomarski light microscopy. Morphometric

characters are summarized in Table 1.

Acrosome complex

The acrosome complex is 4.88

µ

m long, curved and flat-

tened apically (Fig. 2A). Its most anterior portion has

a spatulate aspect (Fig. 2I). The acrosome complex

consists of two conical caps, the external acrosome vesicle

and the internal subacrosomal cone (Fig. 2A,D–G). The


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453

acrosome vesicle caps the subacrosomal cone and,

anteriorly, is divided into two portions (Fig. 2C,J): the

internal, moderately electron-dense medulla and the

external, electron-denser and thinner cortex. A light

band joins the two portions (Fig. 2C). Running posteri-

orly through the acrosome complex, the acrosome

vesicle is more homogeneous and presents a unilateral

ridge (Fig. 2D,E). The ridge becomes less evident (Fig. 2F)

and finally disappears at the posterior end of the

acrosome complex (Fig. 2G), conferring on it a circular

shape. Within the medulla, the perforatorium is an

elongate, inclined and narrow rod, with a pointed tip

(Fig. 2I–J). It has a basal modification, immersed into

the subacrosomal cone and that is knob shaped. This

basal modification is clearly distinct, being electron-

denser than the subacrosomal cone (Fig. 2A). The

subacrosomal cone covers the anterior portion of the

nucleus, the nuclear rostrum (Fig. 2A). Anteriorly, it

is clearly separated from the acrosome vesicle by an

electron-lucent region, the subacrosomal space (Fig. 2A,J).

The subacrosomal cone appears paracrystalline and

homogeneous in longitudinal sections (Fig. 2A,D–F),

but in transverse sections it has a radial arrangement

(Fig. 2G). An epinuclear electron-lucent zone is present

from the anterior portion of the nuclear rostrum to the

perforatorium base plate (Fig. 2A,D). A flange of the

subacrosomal cone projects laterally, behind the acrosome

Fig. 1 Schematic drawing of mature spermatozoon of Iguana iguana in longitudinal section and each corresponding transverse section. All structures are proportionally drawn. Drawn from TEM micrographs.

Table 1 Mean, standard deviation (SD) and number of observations (n) of each morphometric character from Iguana iguana sperm. All values are in micrometres, except the percentage of fibrous sheath occupancy into the midpiece (FSOM) and the ratio of the anterior portion of the principal piece and the midpiece width (PPMR)

Character Mean ± SD n

AL 4.88 0.35 15DCL 1.23 0.27 12DDB 0.68 0.09 14ETL 0.47 0.07 16ETW 0.17 0.03 16FSOM 0.57 0.04 10HL 18.22 1.39 12MPL 3.36 0.38 16NBW 0.53 0.04 14NL 13.34 1.39 12NRL 2.62 0.25 10NSW 0.34 0.06 10PPMR 0.49 0.08 10TaL 53.47 2.97 12TL 71.69 2.43 12

Abbreviations: acrosome complex length (AL), distal centriole length (DCL), mean distance between dense bodies (DDB), epinuclear lucent zone length (ETL), epinuclear lucent zone width (ETW), percentage of fibrous sheath occupancy into the midpiece (FSOM), head length (HL), midpiece length (MPL), nuclear base width (NBW), nuclear length (NL), nuclear rostrum length (NRL), nuclear shoulders width (NSW), ratio of the anterior portion of the principal piece and the midpiece width (PPMR), tail length (TaL), total length (TL).


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455

vesicle, and delimits the most posterior portion of the

acrosome complex (Fig. 2A).

Nucleus

The nucleus is cylindrical, elongate (13.34

µ

m) and

slightly curved. It consists of a homogeneous, electron-

dense and highly compacted chromatin (Fig. 2A,H).

The nuclear rostrum is cone shaped and invades a

substantial portion of the subacrosomal cone, from the

subacrosomal flange to the epinuclear electron-lucent

zone (Fig. 2A,E–G). The electron-lucent space above the

nuclear rostrum and delimited by the nuclear mem-

brane is part of the epinuclear electron-lucent zone.

Nuclear shoulders, which hold the acrosome complex,

are round and mark the beginning of the nuclear

rostrum (Fig. 2A). The posterior region of the nucleus is

shaped like a narrow conical hollow, the nuclear fossa,

into which the neck elements rest (Fig. 3A,K,L).

Neck region

The neck region connects the head with the midpiece

and tail. It has two centrioles, the first ring of dense

bodies and the pericentriolar material (Fig. 3A,K,L).

The proximal centriole is closely fitted and centrally

located at the nuclear fossa (Fig. 3A,K,L). It has a

rounded, centrally located, electron-dense structure in

its interior (Fig. 3K). Immediately posterior and with

perpendicular orientation to the proximal centriole,

the distal centriole represents the basal body of the

axoneme and is the first axial component of the

midpiece (Fig. 3A). The distal centriole extends deeply into

the midpiece (approximately one-third of the midpiece

length, Table 1, Fig. 3A). It consists of nine triplets of

microtubules, nine peripheral fibres that partially cover

the triplets and the two central singlets of the axoneme

(Fig. 3B). Associated with the nine triplets of the distal

centriole, there are nine peripheral coarse (dense) fibres

(Fig. 3B). Both centrioles are encircled by a homogeneously

electron-dense material, the pericentriolar material,

that conforms in shape to the nuclear fossa and is

connected with the anterior portion of the distal

centriole as the dense peripheral fibres (Fig. 3A,K). A

discrete laminar structure projects bilaterally from the

pericentriolar material (Fig. 3A).

Midpiece

The midpiece is approximately five times shorter than

the head (Table 1). It begins at the nuclear fossa, incor-

porating the neck elements, and terminates at the most

posterior electron-dense ring, the annulus (Fig. 3A).

The midpiece consists of the neck and flagellar compo-

nents (axoneme and dense fibres), surrounded by mito-

chondrial gyres and dense body rings. The axial element

of the midpiece changes from centriolar to axonemal

at the anteriormost third of the midpiece (Fig. 3A). This

transition coincides with the beginning of the fibrous

sheath, which extends into approximately 57% of the

midpiece (Table 1). The axoneme is characteristically

arranged in a 9 + 2 pattern of double microtubules and

is surrounded by the fibrous sheath (Fig. 3C–E). The

peripheral dense fibres extend from the pericentriolar

material and decrease in size with the exception of

the fibres 3 and 8, which remain conspicuous through

the midpiece. They are apparently double and detached

from their doublets, being closely associated with the

Fig. 2

Head region of mature spermatozoon of

Iguana iguana

. (A–H, I, J) Transmission electron micrographs. (A)

Longitudinal section through the anterior portion of the nucleus and through the acrosome complex, showing the nuclear rostrum, the subacrosomal cone, the acrosome vesicle, the epinuclear electron-lucent zone and the knob-like perforatorium base plate. The arrows indicate the nuclear shoulders and the flange of the subacrosomal cone; the asterisk indicates the subacrosomal space. (B–G) Corresponding transverse sections of the acrosome complex. Note that the acrosome complex becomes highly depressed, from its base (G) to its apex (B). (B) Most anterior portion of the acrosome complex. (C)

Acrosome vesicle at the perforatorium level showing its subdivision into cortex and medulla; the asterisk shows a less electron-dense region joining the cortex and medulla. (D)

Transverse section through the epinuclear electron-lucent zone; note that the acrosome complex is unilaterally ridged. (E, F) Transverse sections through the nuclear rostrum; the acrosome complex is still unilaterally ridged most anteriorly, but shifts to a circular shape most posteriorly. (G)

Transverse section of the most posterior portion of the acrosome complex. The subacrosomal cone is clearly radially paracrystalline and the acrosome vesicle is very discrete. (H)

Transverse section of the nucleus. (I)

Longitudinal section of the acrosome complex showing the whole perforatorium; the arrow indicates the funnel-shaping of the acrosome vesicle. (J)

Longitudinal section through the acrosome complex showing highly electron-dense cortex and moderate electron-dense medulla. (K)

Nomarski light micrograph of the entire sperm cell. Abbreviations: av, acrosome vesicle; bp, perforatorium base plate; c, cortex of the acrosome vesicle; et, epinuclear electron-lucent zone; h, head; me, medulla of the acrosome vesicle; mp, midpiece; n, nucleus; nr, nuclear rostrum; p, perforatorium; pm, plasma membrane; sc, subacrosomal cone; t, tail; ur, unilateral ridge.


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457

fibrous sheath (Fig. 3C–E). The fibrous sheath encircles

the axoneme, forming a complete and electron-dense

ring in transverse sections (Fig. 3C–E). The fibrous

sheath lies just posterior to the distal centriole and

longitudinal sections indicate that it is formed by regularly

spaced, dense, squared blocks (Fig. 3A). Mitochondria

are sinuous tubules (Fig. 3L) that form regular tiers in

sections that have a perfect longitudinal orientation

(Fig. 3A). They surround the distal centriole and axoneme

and have linear cristae (Fig. 3A). In transverse sections,

they appear trapezoidal, usually establishing 5–6

elements around the axoneme (Fig. 3D) and being

eventually separated by dense body remains. Dense

bodies are complete or interrupted rings (ring struc-

tures) interposed among mitochondrial tiers (Fig. 3A–C).

There are four regularly spaced rings (0.68

µ

m apart)

in longitudinal sections, the first being located in the

vicinity of the proximal centriole, in the neck region

(Fig. 3A,K,L). The rings are formed by granular and

dense structures, not delimited by membranes, and

they lie juxtaposed to the fibrous sheath. Associated

with each dense body ring is a posterior ring of mito-

chondria, which gives the midpiece an aspect of four

identical sets of mitochondria/dense bodies, represented

as rs1/m1, rs2/m2, rs3/m3 and rs4/m4 (Fig. 3A). In trans-

verse sections, dense bodies can form regular and

complete rings or incomplete rings interrupted by

mitochondria (Fig. 3C). Finally, the midpiece ends at a

small dense ring, the annulus, with triangular aspect in

longitudinal section and irregular aspect in transverse

section (Fig. 3A,E).

Principal piece

The principal piece starts posteriorly to the annulus and

is composed of the plasma membrane encircling the

fibrous sheath and the axoneme (Fig. 3F–H). Along with

the endpiece, the principal piece forms the sperm tail.

In its anterior region, a large mass of finely granular

cytoplasm is observed between the membrane and the

fibrous sheath (Fig. 3F), which decreases the diameter

of the transition between the midpiece and the princi-

pal piece. Within this transition, fibres 3 and 8 are still

present, but are not as evident (Fig. 3F). Posteriorly, the

principal piece is solely composed of the plasma mem-

brane juxtaposed to the fibrous sheath, with fibres 3

and 8 absent (Fig. 3G). The fibrous sheath becomes

slender within the posterior portion of the principal

piece (Fig. 3H).

Endpiece

The endpiece is characteristically marked by the absence

of the fibrous sheath. This region of the tail has a reduced

diameter, with its anterior portion maintaining the

9 + 2 axonemal microtubule arrangement (Fig. 3I) and

the posterior portion with disordered microtubules,

the doublets being separated (Fig. 3J).

Evolution of characters

Characters and character states used in analyses are

listed in Table 2. Considering that the phylogeny of

Iguania examined contains a polytomy and that dif-

ferent resolutions of the polytomy may yield different

tree lengths, we produced 10 000 random resolutions

of the polytomy using MacClade. Because the polytomy

involves five elements (Figs 4 and 5), it represents 105

possible dichotomous resolutions that should have been

sampled in approximately homogeneous proportions,

considering the large number of random solutions

examined. Mapping of sperm ultrastructure characters

resulted in tree lengths that ranged from 71 to 74

(mean = 72.97

±

1.10). In what follows, we describe the

Fig. 3

Midpiece and tail region of mature spermatozoon of

Iguana iguana

. (A–L) Transmission electron micrographs. (A)

Longitudinal section through the midpiece showing the arrangements of mitochondria and dense bodies. Note the rs1/m1, rs2/m2, rs3/m3, rs4/m4 arrangement of mitochondria and dense bodies. The arrows indicate the stratified laminar structure. (B–J) Series of transverse sections of the tail. (B)

Neck region showing the distal centriole. The arrowheads show the peripheral fibres. (C)

Through a dense body ring (ring structure) surrounding the axoneme, showing fibres 3 and 8 enlarged (arrowheads). (D)

Through a mitochondrial ring. Asterisk indicates the fibrous sheath. (E) Through the annulus. (F)

Anterior portion of the principal piece, with a large portion of cytoplasm. Asterisk indicates the fibrous sheath. (G)

Medial portion of the principal piece. The plasma membrane is closely associated to the fibrous sheath. (H)

Posterior portion of the principal piece, with a discrete fibrous sheath (asterisk). (I)

Anterior region of the endpiece. The axoneme is still organized. (J)

Posterior portion of the endpiece, with no axonemal organization of microtubules. (K, L) Longitudinal sections of the midpiece. (K)

Through the neck region, showing the central electron-dense element of the proximal centriole (arrow). (L) Oblique longitudinal section of the midpiece showing the columnar mitochondria and the dense body rings. Abbreviations: an, annulus; ax, axoneme; cy, cytoplasm; db, dense body; dc, distal centriole; m, mitochondria; pc, proximal centriole; rs, ring structure.


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reconstructions that involve Iguania or its clades. Six

characters changed unambiguously on the phylogeny

(nos. 3, 4, 5, 7, 9 and 11; Figs 4 and 5). Among them, the

presence of a unilateral electron-lucent space in the

acrosome complex (character 3) is a possible synapo-

morphy of Tropiduridae (evolved independently in

Ameiva

); a subacrosomal cone with radial aspect in tran-

sverse section (character 4) is a possible synapomorphy

Table 2 Matrix showing the distribution of states of 30 sperm ultrastructure characters among iguanian families and the outgroups

Species 10 20 30 Source

Sphenodon punctatus 0000000000 0000000001 0022000010 (Healy & Jamieson, 1994)Cercosaura ocellata 0000010010 0111012110 2300011111 (Teixeira, 2003)Ameiva ameiva 1111011201 0122114110 2300022201 (Giugliano et al. 2002)Tupinambis merianae 1101011101 0122114111 2400012201 (Tavares-Bastos et al. 2002)Pogona barbata 1100111200 0121012111 2311011201 (Oliver et al. 1996)Bradypodion karrooicum 1100110000 1121012111 1011011201 (Jamieson, 1995b)Iguana iguana 1101111100 0121112111 2211021211 Present workTropidurus semitaeniatus 1110110001 0121112111 2111021211 (Teixeira et al. 1999b)Tropidurus torquatus 1110110001 0121112111 2111021211 (Teixeira et al. 1999b)Anolis carolinensis 1200111210 0121114111 2211011201 (Scheltinga et al. 2001)Polychrus acutirostris 1100111101 0121014111 2211111201 (Teixeira et al. 1999a)Urosaurus ornatus 1100111200 0121112111 2211011201 (Scheltinga et al. 2000)Uta stansburiana 1100111200 0121113111 2311011201 (Scheltinga et al. 2000)Crotaphytus bicinctores 1100111200 0121115111 2311111201 (Scheltinga et al. 2001)Gambelia wislizenii 1100111200 0121115111 2311111201 (Scheltinga et al. 2001)

Fig. 4 Cladogram depicting the evolution of sperm-derived characters among iguanian lizards (according to Macey et al. 1997), according to the ACCTRAN optimization. Sphenodon punctatus, Cercosaura ocellata, Ameiva ameiva and Tupinambis merianae are used as outgroups. Numbers indicate characters that change along branches. Underlined numbers represent unambiguous character transformations. See Appendix 2 and text for details.

https://www.researchgate.net/publication/14048281_Evolutionary_Shifts_in_Three_Major_Structural_Features_of_the_Mitochondrial_Genome_Among_Iguanian_Lizards?el=1_x_8&enrichId=rgreq-ec24da33-86d3-4f7b-aa84-afd46dec960c&enrichSource=Y292ZXJQYWdlOzg1MDgzNDM7QVM6MTA0MjE1OTE3NjI5NDYwQDE0MDE4NTg0MTk2NTQ=

The ultrastructure of the spermatozoa of Iguana iguana, G. H. C. Vieira et al.


459

of Iguanidae (evolved independently in Teiidae);

a ridge at the epinuclear electron-lucent zone level in

the acrosome complex (character 5) is a synapomorphy

of Iguania; the presence of a perforatorial base plate

(character 7) apparently has multiple origins within

Iguania (evolved independently in Teiidae); a rounded

perforatorium tip (character 9) is a possible synapomor-

phy of Anolis (evolved independently in Cercosaura);

and the presence of a deep nuclear fossa (character 11) is

a possible synapomorphy of Bradypodion (Figs 4 and 5).

Reconstruction of the evolution of the remaining

characters was ambiguous. Such reconstructions are

problematic in trees that have polytomous nodes,

especially if polytomies are interpreted as regions of

ambiguous resolution (‘soft polytomies’, sensu Maddison

& Maddison, 2003). The problem is that, under different

dichotomous resolutions of the polytomy, different

scenarios of character evolution may arise (Maddison &

Maddison, 2003). In 19 characters, however, the ambig-

uous reconstructions are located outside the polytomy

involving Iguania (characters 1, 2, 6, 12–14, 16–21, 23,

24 and 26–30; Figs 4 and 5), therefore enabling the

investigation of character evolution without having

to examine alternative dichotomous resolutions of

the polytomy. Using the ACCTRAN method of character

optimization, the presence of a unilateral ridge projec-

tion in the acrosome complex (character 2) is a possible

synapomorphy of Anolis; the beginning of the fibrous

sheath in the midpiece at ring structure 2 (character

17) is a possible synapomorphy of Polychrotidae;

the beginning of the fibrous sheath in the midpiece

between ring structures 3 and 4 (character 17) is a

possible synapomorphy of Uta; the beginning of the

fibrous sheath in the midpiece at ring structure 3 (char-

acter 17) is a possible synapomorphy of Crotaphytidae;

mitochondria and ring structures not arranged as

regular rings (sets) (character 21) is a possible synapomor-

phy of Bradypodion; the presence of slightly curved

mitochondria in oblique section (character 23) and

mitochondria with rounded ends in longitudinal

section (character 24) are synapomorphies of Iguania; and

dense bodies forming ring structures (character 26) and

Fig. 5 Cladogram depicting the evolution of sperm-derived characters among iguanian lizards (according to Macey et al. 1997), according to the DELTRAN optimization. Sphenodon punctatus, Cercosaura ocellata, Ameiva ameiva and Tupinambis merianae are used as outgroups. Numbers indicate characters that change along branches. Underlined numbers represent unambiguous character transformations. See Appendix 2 and text for details.

https://www.researchgate.net/publication/14048281_Evolutionary_Shifts_in_Three_Major_Structural_Features_of_the_Mitochondrial_Genome_Among_Iguanian_Lizards?el=1_x_8&enrichId=rgreq-ec24da33-86d3-4f7b-aa84-afd46dec960c&enrichSource=Y292ZXJQYWdlOzg1MDgzNDM7QVM6MTA0MjE1OTE3NjI5NDYwQDE0MDE4NTg0MTk2NTQ=



460

the presence of fibres 3 and 8 at the anteriormost

region of the principal piece (character 29) are possible

synapomorphies of Iguanidae and Tropiduridae (Fig. 4).

Using the DELTRAN tracing produced the same evolu-

tionary reconstructions (regarding Iguania and its

clades), with the exception of characters 1 and 2, which

are synapomorphies of Iguania (Fig. 5). Reconstruction

of the evolution of characters 8, 10, 15, 22 and 25

involves the resolution of ambiguities within the

polytomy of Iguania. Considering the lack of resolution

of the relationships within Iguania, we avoided recon-

structing the evolution of those characters and they are

not displayed in Figs 4 and 5.

We examined the number of character-state changes

in different regions of the spermatozoon based on

10 000 random solutions of the polytomy of Iguania.

Changes were distributed approximately in equal

proportions (∼2.5×) among the sperm head (11 characters,

27.64 ± 0.72 changes), midpiece (17, 40.52 ± 0.59) and

tail (two, 4.80 ± 0.40). However, there were significant

differences in the average number of tree steps or the

average consistency index (averaged across 10 000

random solutions) among characters from different

regions of the spermatozoon (Table 3). Our results

suggest higher rates of evolutionary change (and

homoplasy) in the head, followed by the tail and the

midpiece (Table 3), but should be interpreted with

caution given the very (artificially) large number of

degrees of freedom.

Discussion

The general aspect of the sperm of Iguana iguana

resembles those of other iguanian lizards, being more

similar to tropidurids (among iguanians) and Ameiva

ameiva (regarding Squamata as a whole). Nevertheless,

I. iguana also shares some traits with Cercosaura

ocellata, Polychrus acutirostris, Sphenodon punctatus

and Tupinambis merianae, as depicted in Fig. 4 (see

also Appendix 2).

Because lists of derived characters in the sperm of

Iguania (Teixeira et al. 1999a,d; Scheltinga et al. 2000,

2001) and Squamata (Jamieson, 1995b; Jamieson et al.

1996; Oliver et al. 1996) are available in previous works,

we avoid repeating them here. All synapomorphies of

Tetrapoda and Amniota (Jamieson, 1995a, 1999) and

Squamata (Jamieson, 1995b) were also observed in

the sperm of I. iguana. A presumed synapomorphy of

Iguania, the arrangement of intermitochondrial dense

bodies as regular incomplete rings (Scheltinga et al.

2001), was also present in I. iguana. However, this trait is

also present in some members of Autarchoglossa used

in this study and the gymnophtalmid Micrablepharus

maximiliani (Teixeira et al. 1999b). Conversely, species

of the tropidurid genus Liolaemus (Furieri, 1974) show

dense bodies organized as regular complete rings.

The mapping of 30 sperm morphology characters

revealed a novel synapomorphy of Iguania: the deve-

lopment of a well-developed acrosomal ridge at the

level of the epinuclear lucent zone. Nevertheless, as the

relationships among the clades of Iguania become

resolved, it is likely that more unambiguous evolutionary

changes will emerge.

The agreement between mapped characters and the

proposed phylogeny of a group indicates phylogenetic

inertia (Cheverud et al. 1986; Brown et al. 2000) or, in

other words, that the studied traits are in some way

conserved through the evolutionary history of the

group. We show that, at a more exclusive level (e.g. using

lower taxonomical ranks), the greatest agreement

between the mapped characters and the topology

is achieved (as candidate synapomorphies). As more

inclusive ranks are taken into account, this agreement

is reduced. The increasing divergence (variability) seen

with increasing taxonomic rank seems to hold in general

(Foote, 1997). However, variability of the lower rank taxa,

particularly within the ingroup, is of concern. Why does

it consistently not fit the topology? The variability of

characteristics can show a consistent pattern or a

random pattern. Consistency between the variability and

Region of spermatozoon No. of tree steps Consistency index

Head (11) 2.51 ± 0.07 0.59 ± 0.01Midpiece (17) 2.38 ± 0.03 0.84 ± 0.004Tail (2) 2.40 ± 0.20 0.64 ± 0.02ANOVA F 3219.19 1608 786.00Degrees of freedom 2,29997 2,29997P < 0.0001 < 0.0001

Table 3 Statistics indicative of rates of change in characters across three regions of the spermatozoon of Iguana iguana. Values are based on 10 000 random solutions of polytomy within Iguania. See text for details. Number of characters in parentheses

https://www.researchgate.net/publication/234149106_Evolution_of_morphological_diversity?el=1_x_8&enrichId=rgreq-ec24da33-86d3-4f7b-aa84-afd46dec960c&enrichSource=Y292ZXJQYWdlOzg1MDgzNDM7QVM6MTA0MjE1OTE3NjI5NDYwQDE0MDE4NTg0MTk2NTQ=



461

the evolutionary history of the group suggests adapta-

tion as the driving force, and it can be explained by

historical inheritance (Brooks & McLennan, 1991; Harvey

& Pagel, 1991). The random pattern can result from con-

vergence (Wiens, 2000) or neutral selection, without an

adaptive basis. Tavares-Bastos et al. (2002) found a

poor fit between seven polymorphic sperm characters

and the proposed phylogeny of Tupinambis (i.e. no

phylogenetic inertia), discarding the possibility of

convergence as a result of similar reproductive ecologies.

The variability in sperm morphology of Iguania (and

Squamata) can be associated with reproductive charac-

teristics. Among squamates, there is ample variation in

reproductive characteristics, including: enzymes associ-

ated with the transport of sperm, places of sperm

storage in the female reproductive tract (Olsson et al. 1994;

Blackburn, 1998; Server & Hamlett, 2002), fertilization

process, place of fertilization (Blackburn, 1998), oviduct

or sperm secretions for sperm maintenance in the

female reproductive tract (Girling, 2002), and the mech-

anism of sperm release from storage regions (Girling,

2002). However, detailed data regarding female repro-

ductive tract and fertilization among iguanians are still

lacking. There are two plausible mechanisms for female

discrimination of sperm cell: selection by the reproduc-

tive tract or choice of sperm by the ova (Olsson et al.

1997). One could imagine that sperm characteristics

evolved through sperm competition rules but, again,

studies for sperm competition in reptiles are rare. We

propose that the variability in the sperm cell of Iguania

is a product of historical inheritance, even though this

conclusion is superficial owing to the lack of resolution

for relationships among major lineages (Macey et al.

1997; Schulte et al. 2003). Hence, we should not dis-

count the possibility that sperm-derived characters are

evolutionarily labile and selectively neutral in Iguania,

or a product of convergence due to female reproduc-

tive tract characteristics, particularities of fertilization

and/or sperm competition.

The more characters a region contains, the more

likely that it will contain polymorphism (or variability),

the rates of evolutionary change being the same for

all characters or the changes being selectively neutral

(Ridley, 1993; Tavares-Bastos et al. 2002). Our results

suggest that different regions of the spermatozoon

experience heterogeneous rates of evolution (see

Scheltinga et al. 2000). However, the paucity of data

for female reproductive tract characteristics, particular-

ities of fertilization and/or sperm competition precludes

the correlation of sperm variability with any of these

characteristics.

Acknowledgements

We thank Ruscaia D. Teixeira for her valuable theoret-

ical and technical help and for providing the data from

Cercosaura ocellata, Daniel O. Mesquita for field assist-

ance, Ayrton K. Pérez Jr for providing the specimens

used on the present study, and two anonymous reviewers

for their insightful comments and suggestions. Some

characters and character-states used here are derived

from observations by David M. Scheltinga and Ruscaia

D. Teixeira. This study was supported by FINATEC and

a fellowship from PIBIC-UnB-CNPq and a doctorate

fellowship from CNPq to G.H.C.V.

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Appendix 1

Sperm-derived characters and character-states used

(consistency index in parenthesis).

1. Acrosome complex, ridge (0.500): (0) absent; (1)

present.

2. Acrosome complex, ridge projection (0.667): (0) not

applicable; (1) unilateral; (2) bilateral.



463

3. Acrosome complex, unilateral electron-lucent space

(0.500): (0) absent; (1) present.

4. Acrosome complex, radial aspect of subacro-

somal cone in transverse section (0.500): (0) absent;

(1) present.

5. Acrosome complex, ridge at the epinuclear

electron-lucent zone level (1.000): (0) poorly developed;

(1) well developed.

6. Perforatorium, number (1.000): (0) two; (1) one.

7. Perforatorium, base plate (0.250): (0) absent;

(1) present.

8. Perforatorium, shape of the base plate (0.286):

(0) not applicable; (1) knob-like; (2) stopper-like.

9. Perforatorium, tip (0.500): (0) pointed; (1) rounded.

10. Nucleus, lacunae (0.333): (0) absent; (1) present.

11. Nucleus, nuclear fossa (1.000): (0) shallow; (1) deep.

12. Neck region, stratified laminar structure (1.000):

(0) absent; (1) present.

13. Neck region, stratified laminar structure projection

(1.000): (0) not applicable; (1) unilateral; (2) bilateral.

14. Neck region, stratified laminar structure (1.000):

(0) not applicable; (1) poorly developed; (2) well

developed.

15. Neck region, electron-dense structure inside the

proximal centriole (0.333): (0) absent; (1) present.

16. Midpiece, fibrous sheath (1.000): (0) absent;

(1) present.

17. Midpiece, beginning of the fibrous sheath in the

midpiece (0.800): (0) not applicable; (1) level of mito-

chondria tier 1–2; (2) tier 2–3; (3) tier 3–4; (4) at ring

structure 2; (5) at ring structure 3.

18. Midpiece, mitochondrial cristae (1.000): (0) concen-

tric; (1) linear.

19. Midpiece, dense bodies outside mitochondria

(1.000): (0) absent; (1) present.

20. Midpiece, fibres 3 and 8 (0.500): (0) grossly

enlarged; (1) not grossly enlarged.

21. Midpiece, mitochondria and ring structures as

regular rings (sets) (1.000): (0) not applicable; (1) absent;

(2) present.

22. Midpiece, number of sets of mitochondria and ring

structures (0.571): (0) not applicable; (1) three tiers;

(2) four tiers; (3) five tiers; (4) six tiers.

23. Midpiece, mitochondria in oblique section (1.000):

(0) columnar; (1) slightly curved; (2) rounded.

24. Midpiece, mitochondrial shape in longitudinal

section (1.000): (0) trapezoidal; (1) round ends; (2) totally

round.

25. Midpiece, dense bodies (0.500): (0) solid; (1) granular.

26. Midpiece, dense bodies forming ring structures

(0.500): (0) not applicable; (1) absent; (2) present.

27. Midpiece, appearance of dense bodies in the

ring structures in oblique section (1.000): (0) not appli-

cable; (1) fused, forming compact structures; (2) not

fused.

28. Midpiece, intermitochondrial dense bodies in

transverse section (1.000): (0) not applicable; (1) sepa-

rated from fibrous sheath by mitochondria; (2) juxta-

posed to fibrous sheath.

29. Principal piece, fibres 3 and 8 at the anteriormost

region (0.250): (0) absent; (1) present.

30. Principal piece, wide band of cytoplasm in the

anteriormost region (1.000): (0) absent; (1) present.

Appendix 2

List of changes for the 30 characters of the sperm

derived-characters (see Figs 4 and 5 for reference of

labelled stems). Underlined numbers represent unam-

biguous character transformations.

ACCTRAN optimization

Squamata: 1: 0 → 1, 2: 0 → 1, 6: 0 → 1, 12: 0 → 1, 13:

0 → 2, 14: 0 → 1, 16: 0 → 1, 17: 0 → 2, 18: 0 → 1, 19:

0 → 1, 21: 0 → 2, 26: 0 → 1, 27: 0 → 1, 28: 0 → 2, 30:

0 → 1;

Iguania: 5: 0 → 1, 23: 0 → 1, 24: 0 → 1;

Pleurodonta: no changes;

Acrodonta: no changes;

Scleroglossa: 20: 1 → 0;

Teiidae: 4: 0 → 1, 7: 0 → 1, 14: 1 → 2, 17: 2 → 4, 27:

1 → 2;

Crotaphytidae: 7: 0 → 1, 17: 2 → 5;

Phrynosomatidae: 7: 0 → 1;

Polychrotidae: 7: 0 → 1, 17: 2 → 4;

Tropiduridae: 3: 0 → 1, 7: 1 → 0, 26: 1 → 2, 29: 0 → 1;

B. karrooicum (voucher of Chamaeleonidae): 11: 0 → 1,

21: 2 → 1;

P. barbata (voucher of Agamidae): 7: 1 → 0;

I. iguana (voucher of Iguanidae): 4: 0 → 1, 7: 1 → 0, 26:

1 → 2, 29: 0 → 1;

U. stansburiana: 17: 2 → 3;

A. carolinensis: 2: 1 → 2, 9: 0 → 1;

S. punctatus (voucher of Rhynconcephalia): 23: 0 → 2,

24: 0 → 2, 29: 0 → 1;

C. ocellata (voucher of Gymnophthalmidae): 1: 1 → 0,

2: 1 → 0, 9: 0 → 1, 13: 2 → 1, 28: 2 → 1, 29: 0 → 1;



464

T. merianae: 20: 0 → 1;

A. ameiva: 3: 0 → 1, 26: 1 → 2;

DELTRAN optimization

Squamata: 6: 0 → 1, 12: 0 → 1, 13: 0 → 2, 14: 0 → 1, 16:

0 → 1, 17: 0 → 2, 18: 0 → 1, 19: 0 → 1, 21: 0 → 2, 26:

0 → 1, 27: 0 → 1, 28: 0 → 2, 30: 0 → 1;

Iguania: 1: 0 → 1, 2: 0 → 1, 5: 0 → 1, 23: 0 → 1, 24:

0 → 1;

Pleurodonta: no changes;

Acrodonta: no changes;

Scleroglossa: no changes;

Teiidae: 1: 0 → 1, 2: 0 → 1, 4: 0 → 1, 7: 0 → 1, 14: 1 → 2,

17: 2 → 4, 27: 1 → 2;

Crotaphytidae: 7: 0 → 1, 17: 2 → 5;

Phrynosomatidae: 7: 0 → 1;

Polychrotidae: 7: 0 → 1, 17: 2 → 4;

Tropiduridae: 3: 0 → 1, 7: 1 → 0, 26: 1 → 2, 29: 0 → 1;

B. karrooicum (voucher of Chamaeleonidae): 11: 0 → 1,

21: 2 → 1;

P. barbata (voucher of Agamidae): 7: 0 → 1;

I. iguana (voucher of Iguanidae): 4: 0 → 1, 7: 0 → 1, 26:

1 → 2, 29: 0 → 1;

U. stansburiana: 17: 2 → 3;

A. carolinensis: 2: 1 → 2, 9: 0 → 1;

S. punctatus (voucher of Rhynconcephalia): 23: 0 → 2,

24: 0 → 2, 29: 0 → 1;

C. ocellata (voucher of Gymnophthalmidae): 9: 0 → 1,

13: 2 → 1, 20: 1 → 0, 28: 2 → 1, 29: 0 → 1;

A. ameiva: 3: 0 → 1, 20: 1 → 0, 26: 1 → 2;

the ultrastructure of the spermatozoon of the lizard iguana iguana (reptilia, squamata, iguanidae)...

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