Zoologischer Anzeiger 289 (2020) 77e86

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Research paper

Spermiogenesis and sperm ultrastructure as sources of phylogenetic

characters. The example of characid fishes (Teleostei: Characiformes)
Irani Quagio-Grassiotto a, *, Clarianna Martins Baicere-Silva b,

sar de Oliveira Santana b, Juan Marcos Mirande c
Júlio Ce
a
Department of Morphology, Botucatu Biosciences Institute, State University of Sa~o Paulo (UNESP), Prof. Dr. Antonio Celso Wagner Zanin 250, 18618-689,
Botucatu, SP, Brazil
b
Graduate Program on the Cell and Structural Biology, Institute of Biology, State University of Campinas (UNICAMP), Campinas, SP, Brazil
c
n Miguel Lillo (JTP), CONICET (Inv. Independiente), San Miguel de Tucuma
Fundacio
n, Argentina

a r t i c l e i n f o a b s t r a c t

Article history: Fish sperm morphology is quite diverse. In the last decades our and other studies on Teleostei, mainly on
Received 30 June 2020 Ostariophysi with emphasis in the Characiformes, have showing that the features of these spermatozoa
Received in revised form can be used in phylogenetic analyses. Our purpose in this review is explaining how we can get data from
17 September 2020
sperm to be used for phylogenetic inference. The shape of the nucleus, midpiece and flagellum; the
Accepted 28 September 2020
Available online 7 October 2020
relative position of the centrioles; the number and distribution of a few organelles (mitochondria and
vesicles) are features to be analyzed. To recognize homologies in the ultrastructural features of sper-
Corresponding Editor: J. Ziermann matozoa the types of spermatogenesis and spermiogenesis variations also need to be known. Other
reproductive characters that have phylogenetic signal are also reviewed, among them the presence of
Keywords: insemination, the organization of the germinal epithelium, the internal structure of the testis. Here we
Phylogeny also explain how to prepare testis for extraction of sperm morphological characters and how treat these
Spermatozoa characters. Lastly, we propose (supplementary material) two lists of characters defined for the Char-
Spermatogenesis aciformes, that may serve as examples of how spermatic features may be used for phylogenetic analyses.
Sperm morphology
© 2020 Elsevier GmbH. All rights reserved.
Teleosts

1. Introduction spermatozoa and they are always distributed individually in the

In the first studies based on the structure, cellular organization
and mode of fertilization, male gametes in animals are classified as
basal or derived (Franze
n 1970), also designated by some authors as
primitive and modified, respectively (Ginzburg 1968). In these
basal spermatozoa are generally found in animals with external
fertilization where gametes are released into the aquatic environ-
ment. Such spermatozoa are characterized by having (from anterior
to posterior) the following: acrosome; spherical nucleus; two
centrioles with the distal differentiating into the basal body, which
in turn develops into the axoneme of the flagellum; few rounded
mitochondria and flagellum containing only the basic axoneme of
the 9 þ 2 type (Franze
n 1970).
In fishes which have retained the original method of insemi-
nation by emission of gametes into the water (most Teleostei), male
gametes may be considered as primitive type flagellate
* Corresponding author.
E-mail address: iraniqg@ibb.unesp.br (I. Quagio-Grassiotto).
seminal fluid (Ginzburg 1968). In that way, fish spermatozoa in
general were previously considered to be of the basal or primitive
type.
Most Teleostei spermatozoa have retained the primitive spher-
ical or subspherical head, but have lost the acrosome. The middle
piece is short and generally contains 4 or 5 mitochondria at the base
of the nucleus, which is characteristic for the primitive type of
structure (Franze
n 1956; Ginzburg 1968). However, the early
studies by Jamieson (1991) and Mattei (1991) have shown that the
diversity in the shape of fish sperm is immense. Moreover, sperm
considered to be basal are present in groups of more derived fishes,
whereas some more basal groups produce sperm considered
structurally derived or modified. Both of the authors also showed
that among fishes, spermatozoa of the basal type are found only in
Neopterygii where they constitute the dominant form. In other
words, the primitiveness of this type of basal spermatozoon is
indicated by its wide distribution among systematically distant
groups of animals and its occurrence as a stage in spermatogenesis
in spermatozoa of divergent structure (Ginzburg 1968).

https://doi.org/10.1016/j.jcz.2020.09.006
0044-5231/© 2020 Elsevier GmbH. All rights reserved.
I. Quagio-Grassiotto, C.M. Baicere-Silva, J.C.O. Santana et al.
According to Jamieson (1991), the sperm of Neopterygii may
have developed secondarily from more complex forms. Moreover,
in this group of fishes, sperm do not have acrosomes. This absence
is accompanied by the presence of a micropyle in the eggs of these
animals (Jamieson 1991). The micropyle is an opening in the egg
envelope that allows the passage of the spermatozoon into the egg
just prior to fertilization (Amanze & Yvengar 1990).
Knowledge of spermatozoa ultrastructure provides data about
fish phylogeny and aids in the identification of relationships among
spermatozoa morphology and its reproductive biology (Jamieson
1991, 2009; Lahnsteiner & Patzner 2008).
Jamieson (1991) and Mattei (1991), in summarizing the struc-
ture of the spermatozoa for different groups of fishes, also reported
structural modifications that occur within each lineage. More
recent information regarding the ultrastructure of fish sperm has
been revised and compiled in two volumes by Jamieson (2009).
That publication includes an extensive report on the sperm ultra-
structure of the Ostariophysi (Burns et al., 2009), demonstrating the
high variabilty of sperm morphology among these fishes.
Within a given family or subfamily of fish, sperm structure tends
to be quite conservative (Baccetti et al., 1984; Jamieson 1991; Mattei
1991; Burns et al., 1998; Quagio-Grassiotto et al., 2003; Quagio-
Grassiotto & Oliveira 2008; Burns et al., 2009; Baicere-Silva et al.
2011a, 2011b; Santana et al., 2013a). Morphological characters
derived from sperm structure and ultrastructure can often be a
useful tool in studies of fish evolution. However, in order to
determine the correct use of such characters it is first necessary to
understand sperm ontogeny, that is, the process of spermiogenesis.
This renders them an interesting and potentially useful source of
information in studies on fish evolution, given that this conserva-
tiveness allows the recognition of homologous structures. How-
ever, during this review was evident that some sperms with similar
morphology derived from deeply different processes of spermio-
genesis. Therefore, proposals of characters and states homologies
should be supported by data from the ontogeny of the sperm
(spermiogenesis).
Initially, Mattei (1970) proposed the existence of two distinct
patterns of spermiogenesis for fishes, referred to as type I and type
II. Subsequently, Quagio-Grassiotto & Oliveira (2008) described a
third pattern, type III. The applicability of classifying fish sper-
miogenesis into types (I, II, III) within Teleostei has been presented
by several authors (see Burns et al., 2009 for review). However,
within different groups of the order Characiformes, particularly in
inseminating species within the family Characidae, patterns of
spermiogenesis may show marked variations. This phenomenon
led Baicere-Silva (2013) to classify spermiogenesis in these species
as unique, not using the three types described previously. The
classification of spermiogenesis provided by Baicere-Silva (2013)
has been adopted in all our later studies, including the present
review.
In some species, although the final forms of the spermatozoa are
essentially identical, the changes occurring during spermiogenesis
may be unique (Baicere-Silva et al., 2011a). In these cases, an un-
derstanding of the initial stages of spermiogenesis is crucial for
recognizing homologies among spermatozoa. Therefore, the pro-
cess of spermiogenesis has a direct impact on the hypotheses of
homology, avoiding misinterpretations about the evolution of
spermatozoa in different species of fish (Baicere-Silva et al., 2011a;
Santana et al., 2013b).
The Characiformes, mainly the Characidae (focus of most of our
researches) are the most diverse family of Neotropical fishes, with
more than 1200 described species (Fricke et al., 2020). This family is
interesting for this experiment for two aspects, in addition to their
great diversity. In one hand, they have a great morphological
conservativeness and known cases of parallelisms masking their
78
Zoologischer Anzeiger 289 (2020) 77e86
relationships and making the diagnoses of groups difficult
(Mirande 2009 2010). In this sense, any additional data useful as
synapomorphies of clades would be a great contribution. The sec-
ond reason is that, after the inclusion of molecular data (e.g.

n et al.,
Oliveira et al., 2011; Arcila et al. 2017; Mirande 2019; Tera
2020), whether alone or combined with morphological data,
there is certain convergence in the resultant phylogenetic hy-
potheses. This is useful to evaluate the accuracy of sperm data, at
least relative to the current hypotheses for characid phylogeny.
The main goals of our research over the years have been to
understand the evolution of sperm characters and to define criteria
for the establishment of homologies using these data. This
knowledge has enabled us to finally create a list of such characters
for use in phylogenetic analysis of different groups of fishes. Below
we will provide results from our studies on sperm fine structure of
Characiformes, concentrating on the Characidae. Information on
how to use this information for the construction of character lists
will also be discussed.
2. Characteristics of spermatozoa used in phylogenetic
analysis
In a group as diverse as the Teleostei, the number of sperm
features (Fig. 1) useful in phylogenetics is actually not that great,
being mostly related to:
- Shape of the nucleus, midpiece and flagellum
- Position of the centriolar complex relative to the nucleus and
flagellum
- Relative position of the centrioles
- Number and distribution of a few organelles (mitochondria and
vesicles)
These features can be observed in different combinations among
the members of a given group, usually showing a variegated
distribution.
3. How can we get improved data from sperm?
It is well known that recognition of homologies reduces “noise”
in phylogenetic analyses and is one of the first steps for under-
standing the mechanisms by which new features arise. Assuming
that studies on the ontogenetic development of spermatozoa are
able to uncover homologies in fish sperm ultrastructure, it is
essential to obtain detailed information on the location and
mechanisms of spermatogenesis and spermiogenesis.
4. Spermatogenesis in fishes
4.1. Cystic spermiogenesis
Spermatogenesis in fishes usually occurs within cystic struc-
tures referred to as spermatocysts, formed by the association of the
germ cells and Sertoli cells. The stages of both mitosis and meiosis
take place within these cysts, thus producing the spermatids which
in turn differentiate into the spermatozoa. When spermiogenesis is
complete, cysts open up, releasing spermatozoa into the luminal
compartment of the testis (see Grier 1992; Schulz et al., 2010). This
type of spermatogenesis is found in most Teleostei.
4.2. Semicystic spermiogenesis
In semicystic spermatogenesis (Mattei et al., 1993), spermato-
cysts open at the end of the meiotic divisions of the germ cells, thus
releasing early spermatids into the luminal compartment of the
I. Quagio-Grassiotto, C.M. Baicere-Silva, J.C.O. Santana et al.
Fig. 1. Illustrative scheme of some ultrastructural characteristics that can be obtained from spermatozoa (modified from Lee & Kim 2001).
testis where they then complete their differentiation and the final
form of the spermatozoon is achieved (see also Burns et al., 2009).
This type of spermatogenesis is found in different taxonomic
groups, for example in Opheliidae (Mattei et al., 1993), Scorpaeni-
dae (Mun ~ oz et al. 2002) and Corydoradinae (Spadella et al., 2007).
4.3. Partially cystic spermiogenesis
In partially cystic spermiogenesis, spermatocysts open before
the end of spermatid differentiation, usually during the stage of
nuclear elongation, releasing the spermatids into the luminal
compartment of the testis. Final nuclear elongation and chromatin
condensation occur within the lumen (see Quagio-Grassiotto et al.,
2012). This type of spermatogenesis is found in some Characidae
species, for example Hollandichthys and Rachoviscus (Quagio-
Grassiotto et al., 2012).
5. Types of spermiogenesis in fish
Spermiogenesis is the ontogenetic process of cellular differen-
tiation where spermatids develop into spermatozoa. We propose
that detailed microscopical analysis of spermiogenesis in different
fish species may lead to finding homologous structures in the
spermatozoa. Up to now there are three known types of spermio-
genesis in fishes, classified as Type I, Type II and Type III.
5.1. Spermiogenesis type I
In spermiogenesis Type I (Mattei 1970), in the early spermatid
the nucleus is centrally located and mitochondria are scattered
throughout the cytoplasm. The centriolar complex subsequently
moves along the nucleus, bringing with it the plasma membrane
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Zoologischer Anzeiger 289 (2020) 77e86
and the initial segment of the flagellum contained within an
invagination. This results in the formation of a space between the
flagellar and plasma membranes, referred to as the cytoplasmic
canal. The nucleus then rotates 90 taking up an anterior medial
position relative to the flagellar axis (Fig. 2). This spermatogenesis
type I is found in some Characidae subfamily, for example in
Cheirodontinae (Oliveira 2007).
5.2. Spermiogenesis type II
In spermiogenesis Type II (Mattei 1970), nuclear rotation does
not occur and, as a consequence, the flagellum is parallel to the
anteroeposterior axis of the nucleus (Fig. 3). This spermatogenesis
type II is found for example in Lebiasininae (Santana et al., 2013a).
5.3. Spermiogenesis type III
In spermiogenesis type III (Quagio-Grassiotto & Oliveira 2008),
the development of the flagellum is medial in relation to the nu-
cleus, the nucleus does not rotate and a shallow (Santos et al., 2001)
nuclear fossa and a short cytoplasmic canal may be formed or not
(Fig. 4). This spermatogenesis type III is found in Pimelodidae
(Quagio-Grassiotto & Oliveira 2008).
5.4. Variations in spermiogenesis
Because each type of spermiogenesis may show a large number of
variations, the literature is filled with extensive descriptions of
spermiogenic subtypes, making comparisons among these processes
difficult. For this reason, we have abolished classifications based on
type of spermiogenesis and instead use the physical descriptions of
the events above as phylogenetic characters (for more information
I. Quagio-Grassiotto, C.M. Baicere-Silva, J.C.O. Santana et al. Zoologischer Anzeiger 289 (2020) 77e86

Fig. 2. Illustrative scheme of spermiogenesis of the Type I e drawings of longitudinal sections from early to late spermatids. Note from A to D the nuclear rotation toward the
flagellar axis. Red arrow: position of the flagellar axis in relation to the nucleus. Blue arrow: nuclear rotation - nuclear movement around the axis perpendicular. (For interpretation
of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. Illustrative scheme of spermiogenesis of the Type II e drawings of longitudinal sections from early to late spermatids. Note from A to C the absence of the nuclear rotation
toward the flagellar axis. Red arrow: position of the flagellar axis in relation to the nucleus. (For interpretation of the references to colour in this figure legend, the reader is referred
to the Web version of this article.)

Fig. 4. Illustrative scheme of spermiogenesis of the Type III e drawings of longitudinal sections from early to late spermatids. Note from A to C the absence of the nuclear rotation
and the formation of the nuclear fossa (yellow arrow). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Baicere-Silva 2013). Centriolar migration and nuclear rotation are the - Position of the nucleus in relation to the flagellar axis
first events that occur during spermiogenesis. Together with the - Position of the centrioles relative to one another
relative position of the centrioles to one another, these events are - Nuclear rotation
crucial for the establishment of homologies in sperm data. - Nuclear elongation

6. Our purpose
Therefore, our main purposes are to understand the evolution of
sperm characters and, through the analysis of spermiogenesis, define
criteria for establishing hypotheses of homology and dependence
among characters. In addition, we will review primary sexual char-
acters (testicular structure, insemination) that are phylogenetically
informative and establish methods of analyzing characters to be used
in phylogenetic studies for different groups of fishes.
7.1. Initial position of the nucleus in relation to the flagellar axis
The first variation in spermiogenesis in the spermatids concerns
the initial position of the nucleus relative to the flagellar axis which
may be medial or lateral. This precedes the later events of nuclear
rotation and nuclear elongation.
7.2. Position of the centrioles in the early spermatid

7. Characters of spermiogenesis
These studies will help define characters of spermiogenesis it-
self that are essential for understanding the processes leading to
the final form of the spermatozoon. These characters include:
80
The position of the centrioles varies in different groups of fishes
(Fig. 5). Centrioles occupying a medial position and being perpen-
dicular to one another is considered to be the most basal condition.
This situation is found during mitotic divisions of spermatogonia,
the first cell type in the spermatogenic lineage. Mitosis is
I. Quagio-Grassiotto, C.M. Baicere-Silva, J.C.O. Santana et al.
Fig. 5. Illustrative scheme of the initial position of the centrioles one in relative to the other.
considered to be more basal than the later meiotic divisions. In
early spermatids the position of the centrioles relative to each other
may be: perpendicular and medial, perpendicular and lateral, par-
allel or oblique. According to their proximity to the nucleus the
centrioles are referred to as proximal or distal.
7.3. Initial position of the centrioles may change during the
spermiogenesis
During spermiogenesis, the orientation of the centrioles in early
spermatids may or may not result in new positions within sperma-
tozoa (Fig. 6). Migration of the centriolar complex and nuclear rotation
are the first events occurring during spermiogenesis and, therefore,
together with the relative position of the centrioles in the initial
spermatid, are crucial for the establishment of homologies in sper-
matic data. Spermatozoa in different species may ultimately have
centrioles exhibiting the same relative positions. However, they are
not necessarily homologous because they may have been derived from
different initial orientations in the early spermatids. Thus, although
they may be anatomically “identical” in the spermatozoa, their
developmental history within spermatids could be quite different, and
thus they cannot be considered to be homologous events.
7.4. Nuclear rotation
During spermiogenesis the nucleus can rotate in relation to the
flagellar axis and can assume distinct positions relative to that
(Fig. 7). The amplitude of the rotation influences:
- The angle of the nucleus in relation to the flagellar axis;
- Position of the centriolar complex in relation to the nucleus
(posterior, lateral or anterior);
- Distribution of the midpiece in relation to the flagellar axis.
Lateralization of the nucleus in relation to the flagellar axis can
be due to the lateral position of the flagellum in the initial sper-
matid or to nuclear movements during spermiogenesis. Thus,
lateralization may be either homologous or convergent, depending
on how it arose in each species being studied (see scheme in
Baicere-Silva et al., 2011b: Fig. 1).
7.5. Nuclear elongation
During spermiogenesis the nucleus may elongate (Fig. 7). Nu-
clear elongation can proceed either forward of the flagellar axis
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Zoologischer Anzeiger 289 (2020) 77e86
(anterior final position) or toward the flagellar axis (final position
along the flagellum) (Burns et al., 2009). Many, but not all, insem-
inating species possess spermatozoa with elongate nuclei, a process
that may ultimately facilitate sperm transfer to the female (Burns &
Weitzman 2005).
Inseminating species are those in which the sperm is introduced
into female genital tract. According to Burns & Weitzman (2006),
until we have clear evidence we cannot say that insemination and
internal fertilization are a sine qua no condition. Thus, insemination
is testified to by the presence of sperm in the female ovary.
Here we would like to draw attention to the use of the terms
aquasperm and introsperm. The term aquasperm is used to refer to
spermatozoa that are released directly into the aquatic environ-
ment, whereas the term introsperm refers to spermatozoa that are
introduced into female genital tract. These terms have often been
used to characterize the shape of the sperm nucleus. This situation
arose because most spermatozoa released directly in the aquatic
environment have spherical nuclei, but in most inseminating spe-
cies the spermatozoa have nuclei that are elongated to some extent
(Burns et al., 2009). In reality, these terms should not be based on
the shape of the nuclei given that the spermatozoa of some
inseminating species of Characidae have been shown to possess
spherical nuclei, as reported by Burns & Weitzman (2005).
A detailed analysis of spermiogenesis, the ontogenetic process
of cellular differentiation by which spermatids give rise to sper-
matozoa, has now demonstrated that cells with similar shapes or
organelles with similar arrangements may have actually originated
from different ontogenetic processes and are therefore not ho-
mologous. As shown above, this can be determined by the presence
or absence of centriolar complex migration toward the nucleus and
the presence or absence of nuclear rotation in relation to the
flagellar axis.
8. Characters not directly related to spermiogenesis
Several other characters that can be obtained from sperm
morphology that are not directly related to spermiogenesis include:
- Pattern of chromatin condensation
- Presence of a nuclear fossa
- Midpiece variations
- Mitochondrial morphology
- Presence of vesicles and tubules
- Flagellum
I. Quagio-Grassiotto, C.M. Baicere-Silva, J.C.O. Santana et al.
Fig. 6. Illustrative scheme of the initial position of the centrioles relative to one another.
8.1. Chromatin condensation
Within sperm nuclei of species mainly in the Characidae,
chromatin condensation may be variable with many intermediate
states from floccular to granular, as seen below. Chromatin can also
be homogeneous and highly condensed.
It is therefore difficult to define discrete states for this character.
One way is to consider the character to be continuous, counting the
number of flocci or granules in five 0.5 mm transepts per sperm
nucleus.
8.2. Nuclear fossa
There are two types of nuclear fossa based on the appearance of
the contour of the nuclear membrane in longitudinal section:
regular or with irregular projections which penetrate the nucleus.
The first, in turn, can be subdivided into single or double.
Most characids have a regular fossa with a double concavity.
8.3. Midpiece
In sperm, a distinct midpiece may be present or absent. Several
features of the midpiece can be informative, such as length and
width, asymmetry along the length, asymmetry in the distribution
of organelles, presence or absence of a cytoplasmic sleeve, and
presence or absence of a cytoplasmic canal, among others. A cyto-
plasmic sleeve is a membrane projection at the terminal end of the
midpiece that surrounds the initial segment of the flagellum.
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Zoologischer Anzeiger 289 (2020) 77e86
8.4. Mitochondria
Mitochondria are dynamic organelles. They move around inside
the cells and can fuse with one another (Westermann 2010). This
fusion is common during spermiogenesis and results in mito-
chondria with variable shapes and lengths. Shapes range from
spherical to elongate. Elongate mitochondria can also be branched.
They can spread throughout the midpiece and/or be located at the
nuclear periphery. If we consider fusion as steps that result in
longer and longer mitochondria, it is possible to consider this as an
ordinate character. In this way shorter mitochondria would be a
plesiomorphic condition in relation to longer ones. We consider
that this character should be treated as ordinate since continuous
characters are already sorted by the nature of the character. This
character seems to be quite conserved among closely related spe-
cies of a genus.
8.5. Tubules and vesicles
The midpiece may also contain tubules and/or vesicles. During
spermiogenesis the vesicles can fuse ultimately forming a tubule
vesicular system (Quagio-Grassiotto et al., 2001, 2003; Pecio et al.,
2007; Gusma ~o-Pompiani et al., 2009; Baicere-Silva 2013). Vesicles
are highly variable in number, dimension and distribution within
the midpiece. However, they tend to be conservative within genera
or species complexes and potentially informative. They are coded
according to the shape, distribution and number.
Other types of membranous compartments that can be present
in the cytoplasm of the sperm cells are the lamellae anulata. These
I. Quagio-Grassiotto, C.M. Baicere-Silva, J.C.O. Santana et al.
Fig. 7. Illustrative scheme of the nuclear rotation and elongation (based on Oliveira 2007; Burns et al., 2009).
are linear double membranes, containing pores similar to those of
the nuclear envelope, that can occur isolated or in stacks.
8.6. Flagellum
Flagellar characteristics appear to be highly conserved. Flagella
in most fish sperm possess a classic axoneme with a pair of central
microtubules and nine doublets of peripheral microtubules (9 þ 2).
Variations are also observed in relation to the presence of vesicles
and fins (projections of the flagellar membrane). In addition,
electron-dense structures associated with each peripheral doublet
of the axoneme may be present (Veríssimo-Silveira 2007).
9. Other reproductive characters that have being used in
phylogenetic analysis
The testis, as the organ responsible for sperm production, is
another structure potentially providing characters that have been
used in phylogenetic analyses (Mirande 2010). In the lower Tele-
ostei, the internal organization of the testis is of the anastomosing
tubular type with an unrestricted spermatogonial distribution
(Parenti & Grier 2004). The epithelium of the tubules is a germinal
epithelium formed by the germinal and Sertoli cells organized into
cysts (Grier 1993). When spermatogenesis is completed, sperma-
tozoa are released into the lumen of the tubules. In the insemi-
nating species of Characidae, the testis may also be divided into
distinct regions. As first reported by Javonillo et al. (2007), testified
to by Quagio-Grassiotto et al. (2012) and early suspected by Burns
83
Zoologischer Anzeiger 289 (2020) 77e86
et al. (1995), many inseminating species of Characidae have a
tripartite testicular structure. The tripartite structure is character-
ized by: (1) a spermatogenic cranial region in which the germinal
epithelium forms cysts containing cells of the germinative lineage
in all the stages of spermatogenesis; (2) an intermediate region in
which the epithelium consists solely of Sertoli cells due to the
opening of the cysts and the progressive loss of the germ cell
component; (3) a caudal storage region in which the epithelium of
the tubules is devoid of the germ cell components and the Sertoli
cells become secretory. Packets of unencapsulated spermatozoa
known as spermatozeugmata can be formed in the caudal region of
the tripartite testis.
10. How to prepare the testis for determination of sperm
morphogical characters
In order to obtain testis and/or sperm morphological characters
it is necessary to have sexually mature males, that is, specimens
with testes full of spermatozoa or spermatozeugmata, if formed.
However, for analysis of spermiogenesis it is necessary to have
specimens containing all stages of the reproductive cycle. These can
be freshly collected specimens or those from zoological collections.
Sexually mature females are also necessary in order to determine if
insemination is characteristic of the species.
Determination of insemination may be performed by routine
histological analysis to detect the presence of spermatozoa within
the ovaries. However, for optimal results, ovaries should be fixed in
Karnovsky solution, embedded in Historesin and sectioned at
I. Quagio-Grassiotto, C.M. Baicere-Silva, J.C.O. Santana et al.
approximately 3 mm to allow for better resolution of the material
being studied (for more information see Quagio-Grassiotto et al.,
2012). This same protocol is also preferred for analysis of the testis.
In order to accurately determine phylogentically useful charac-
ters from spermiogenesis and sperm morphology, both trans-
mission and scanning electron microscopy must be performed. In
this case, testes from freshly collected specimens should be fixed in
Karnovsky’s solution for at least 24 h and subsequently prepared
for analysis according to the standard methods of transmission or
scanning electron microscopy (for more information see Quagio-
Grassiotto et al., 2012, for example). More prolonged fixation
times are generally not a problem and under all circumstances the
tissues must be maintained in Karnovsky’s solution until they can
be prepared for analysis.
For optimal results, ovaries and testes of specimens from
zoological collections, most of which will have been previously
fixed in formaldehyde and then stored in ethanol, should be refixed
in Karnovsky before proceeding (for more information see Baicere-
Silva et al., 2011b). Although this material does not offer the best
resolution with electron microscopy, it can still be very informative
and particularly important when dealing with rare specimens.
11. Treatment of the spermatic characters
Phylogenetic characters represent features that are conservative
enough to trace back homologies, in the broadest sense, between
different species but also variable enough to be informative at the
taxonomic level of the analysis. Excepting some divergent species
and clades, the Characidae are morphologically conservative and
the same happens with some of the most frequently used DNA
markers. Therefore, every possible source of inherited variability is
potentially useful to reconstruct the phylogeny and, if morpho-
logical, to provide synapomorphies for taxonomic groups of Char-
acidae. Ideally, phylogenetic characters are defined as alternative
states of features that are comparable between taxa. Thus, ho-
mologies of states nest within hypothesized homologies of char-
acters. State homologies are tested during parsimony searches,
while character homologies are information given by the
researcher and are not further tested in the phylogenetic analyses.
It can be seen that the proposals of character homologies consti-
tute, thus, the most important aspect of character definition. When
tracing back character homologies is simple, transformation series
definitions are relatively straightforward. However, that is not the
case for several of the characters herein analyzed, given that sperm
has an unusual degree of morphological convergence during the
ontogeny, and therefore the study of the spermiogenesis is essen-
tial to assess characters homologies. Thus, characters definitions
should consider not only the final form of the spermatozoa but also
the ontogenetic routes that produced their morphology. Successive
experiences dealing with phylogenetic information derived from
sperm and spermiogenesis produced the two most comprehensive
lists of sperm and spermiogenesis characters for the Characiformes,
provided herein as examples (Appendices 1 and 2) (Baicere-Silva
2013; Santana 2014).
These kinds of phylogenetic characters influenced by their on-
togenies has points in common with other complex phylogenetic
characters, discussed for different taxonomic groups (e.g. Smith
2001; Schulmeister & Wheeler 2004; Harrison & Larsson 2008;
Sharma et al., 2016). Complex transformational series have in
common that the homology uncertainties are not restricted to
states but also involve the whole characters. These complex char-
acters include sequence data arrays, as DNA (e.g. Wheeler 1996
2003) or morphological serial homologous (Ramírez 2007) and
the bi- or tridimensional morphometric configurations (Catalano
et al., 2010; Goloboff & Catalano 2011). The analysis of those
84
Zoologischer Anzeiger 289 (2020) 77e86
kinds of characters involved either simplifications of the problem
or complexification of the methods. The simplification of the
problems was mainly to split the complex characters into many
simple ones aligned by variously explicit methods (as in DNA
characters, serial homologous, or the phylogenetic analysis of
separate morphometric landmarks). It involved the loss of certainty
of character homologies. The complexification of the methods
allowed to analyze complex characters as such, but in most cases
adding a prohibitive computational time. Fortunately, in the case of
sperm morphology is possible to assess characters homologies as
supported hypotheses only by observation of the spermiogenesis.
In this way it is possible to define characters as applicable only to
subsets of species having certain common steps in the spermio-
genesis, discarding the cases in which similar morphology were
obtained through other ontogenetic routes. In other words, it al-
lows to transform moderately complex characters into simple ones.
In this review we demonstrate the importance of the events that
occur during spermiogenesis for the establishment of character
homologies and dependence among them. The lists of characters
herein included may be considered as a starting point, not only for
studying the sperm cells of the Characiformes, mainly the Char-
acidae, but also for other groups of fishes. Some of the characters
have already been described and used by Mirande (2010), Ferreira
et al. (2011) and Santana et al. (2013b) (see mention of this in the
list of characters). Alterations in the description of these characters
are mainly due to morphological variability (due to use of different
taxa) and divergences in the interpretation of the character. These
divergences of interpretation refer to the dependence of characters
that were not addressed in previous analyses and to the character
nature. In the above mentioned hypotheses, all transformational
characters were discretized, that is, treated as classes of measures
coded as a state of character. An option would be to analyze the
continuous characters as such (Goloboff et al., 2006) instead of
discretize their states. Baicere-Silva (2013) found that the use of
continuous characters is especially important when dealing with
phylogenetic analysis of genera, which usually have low variability.
Our approach not only allows to analyze data from sperm and
spermiogensis to produce phylogenetic hypotheses and morpho-
logical synapomorphies, but also to combine this information with
general morphology and DNA data, which was found as valuable by
Baicere-Silva (2013). This also follows the line of most recent and
comprehensive phylogenetic analyses in the Characidae (Mirande
2019; Tera
n et al., 2020).
Funding
This work was supported by Brazilian Agencies: Fundaça ~o de
Amparo a Pesquisa do Estado de S~ao Paulo (FAPESP) [2009/05237-
3; 2010/01626-2; 2010/05104-0]; Conselho Nacional de Desenvol-
vimento Científico e Tecnolo
gico (CNPq); and Coordenaça ~o de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
In addition to discussing spermatic characters in fishes, this
review would also like to pay tribute to Dr. Xavier Mattei and Dr.
Barrie Jamieson, pioneers in the field who have demonstrated the
tremendous variability in fish sperm morphology and their
phylogenetic signals. We also wish to recognize the studies on
I. Quagio-Grassiotto, C.M. Baicere-Silva, J.C.O. Santana et al.
sperm morphology in inseminating fishes by Dr. John Burns and the
priceless contribution of Dr. Luiz Roberto Malabarba that helped
lead us to utilizing these types of characters in a phylogenetic
context. We also want to thank Dr. Nae
rcio A. Menezes for the
encouragement, Dr. John R. Burns for the previous review of the
manuscript, Dr. Talita S. Mazzoni for the technical help in preparing
the manuscript and Jimena Grosso for helping to show us literature
about phylogenetic analyses of ontogenies. Finally, we are
immensely grateful to the research funding agencies in Brazil
CAPES, CNPq and especially to FAPESP whose support over the
years has allowed us to get here.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jcz.2020.09.006.
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