Keywords

1 Introduction

Karyosystematics, or the study of the natural relationships of species using the information provided by chromosomes, can provide valuable information for taxonomic classifications and evolutionary analyses. In primates, during the last three decades, researchers have proposed chromosomal speciation as a probable evolutionary mechanism to interpret diversity of living primates (De Grouchy et al. 1972; Dutrillaux et al. 1975, 1980; Seuánez 1979; Dutrillaux and Couturier 1981; De Grouchy 1987; Clemente et al. 1990). More recently, chromosomal data began to be used as phylogenetic markers, since they are inherited as Mendelian characters and are conserved within species (Sankoff 2003; Dobigny et al. 2004; Stanyon et al. 2008). Following the maximum parsimony criterion, karyological comparisons allow the identification of chromosomal forms shared by common ancestrality (Dobigny et al. 2004).

In order to obtain better taxonomic inferences, a wide battery of variables including genetic, morphological, and ecological should be employed into the theoretical framework referred to as “Total Evidence” (Kluge 1989). The systematics of New World Primates (NWP) is still under discussion (Rylands 2000; Groves 2001, 2005; Schneider et al. 2001; Rylands and Mittermeier 2009; Perelman et al. 2011; Rylands et al. 2012), and the genus Alouatta is not the exception (Cabrera 1957; Hill 1962; Groves 2001; Gregorin 2006; Rylands and Mittermeier 2009; Rylands et al. 2012; Cortés-Ortiz et al. 2014). In this context, cytogenetic studies have become an important tool complementing molecular and morphological data traditionally used in systematic studies.

2 Karyological Features of the Genus Alouatta

2.1 Classical Cytogenetic Analysis

Since the first cytogenetic characterization of the red howler monkey, Alouatta seniculus (Bender and Chu 1963), the karyological features of the genus have attracted the scientists’ attention, due to the multiple interspecific autosomal rearrangements and, particularly, for the multiple sex chromosome systems observed. The first cytogenetic studies employed standard staining techniques (Fig. 4.1), established the diploid numbers, and allowed to arrange the chromosomes by size and morphology (A. seniculus: Bender and Chu 1963; A. palliata: Hsu and Benirschke 1970; A. caraya: Egozcue and de Egozcue 1965, 1966). The advent of the chromosome banding techniques in the 1970s (Giemsa banding or G-banding, reverse banding or R-banding, and restriction enzyme banding or REs banding) made it possible to reveal a characteristic pattern of specific dark and light bands along each chromosome (Fig. 4.2).

Fig. 4.1
figure 1

Female standard karyotype of Alouatta pigra. Chromosome pairs were numbered consecutively following decreasing size within each type of morphology. Inset: sex chromosome system observed in the male Alouatta pigra. Bar = 10 μm

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Fig. 4.2
figure 2

G-banded karyotype of Alouatta caraya female. Inset: sex chromosome system observed Alouatta caraya males. Bar = 10 μm

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Using these differential banding techniques, homologous chromosomes, chromosome segments, and chromosomal rearrangements could be identified with precision in each karyotype, thus allowing the first interspecific comparisons (Koiffmann and Saldanha 1974; Yunis et al. 1976; Mudry et al. 1981, 1984, 1994; Minezawa et al. 1985; Armada et al. 1987; Lima and Seuánez 1991; Stanyon et al. 1995; Rahn et al. 1996; Vassart et al. 1996; Steinberg et al. 2008). Cytogenetic analyses using these staining techniques in somatic cells showed high chromosomal variability with drastic differences in the chromosome number among species (Table 4.1). Howler monkeys exhibit diploid numbers (2N) ranging from 2N = 43 in A. seniculus to 2N = 58 in A. pigra. Several interspecific chromosomal rearrangements such as translocations and inversions have been described (De Boer 1974; Mudry et al. 1994; Consigliere et al. 1996, 1998; de Oliveira et al. 2002). This interspecific chromosomal variation coincides with the one observed in the night monkey, genus Aotus (Ma 1981; Torres et al. 1998; Ruiz Herrera et al. 2005; Defler and Bueno 2007), but contrasts with other platyrrhine genera such as the squirrel monkeys, genus Saimiri (Hershkovitz 1984; Moore et al. 1990), or the capuchin monkeys, genus Cebus (Matayoshi et al. 1987; Mudry et al. 1987; Ponsà et al. 1995), in which interspecific variation in 2N is not observed (species show constant diploid numbers).

Table 4.1 Cytogenetic data published for howler monkeys
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Alouatta chromosomal variability is not restricted to differences among species. A few intraspecific polymorphisms have been described in A. caraya, but other species, such as A. guariba, show multiple intraspecific rearrangements involving differences in diploid number and sexual systems (see Table 4.1). This high intraspecific variability suggests that these may be species complexes rather than single species (Stanyon et al. 1995; Consigliere et al. 1996, 1998; de Oliveira et al. 2000). Within Alouatta, karyological studies have contributed to the taxonomic reassessment of several taxa previously considered as subspecies, elevating them to the species level: A. nigerrima, previously A. seniculus nigerrima; A. macconnelli, previously A. seniculus macconnelli; A. stramineus, previously A. seniculus stramineus (here considered within A. macconnelli); and A. belzebul, previously A. seniculus belzebul (Lima and Seuánez 1991; de Oliveira 1996).

2.1.1 Heterochromatin

Traditionally, the term heterochromatin was used to denote chromosomal regions of the karyotype showing increased condensation (Heitz 1928), revealed as regions of intense staining (denoted as C+ bands) (Fig. 4.3) by C-banding (Sumner 1971). Later, cytomolecular techniques showed that these regions are constituted by distinct medium and highly repetitive DNA sequences (Copenhaver et al. 1999; Fransz et al. 2000; Avramova 2002). In Platyrrhini, heterochromatic regions are highly variable in quantity, quality, and location. C-banding techniques and restriction enzymes digestion have demonstrated that there are different kinds of heterochromatin, and particularly among platyrrhine species, a great variability was described, not only in sequence types but also in their location (centromeric, interstitial, and telomeric blocks) (Matayoshi et al. 1987; Mudry de Pargament and Labal de Vinuesa 1988; Pieczarka et al. 2001; García et al. 2003). Compared with other platyrrhine genera, like Saimiri, Cebus, and Aotus, that show big C+ blocks of extracentromeric heterochromatin, Alouatta is the genus with the lowest heterochromatin proportion (Ma et al. 1976; García et al. 1979; 1983; Matayoshi et al. 1987; Mudry de Pargament and Slavutsky 1987; Mudry de Pargament et al. 1984; Mudry 1990; Mudry et al. 1990; 1991; Ponsà et al. 1995; Rahn et al. 1996; Nieves et al. 2005a; Nieves 2007). Within howler monkeys, A. seniculus group shows the lowest C+ heterochromatin proportion, with centromeric location only (Lima et al. 1990); meanwhile A. guariba clamitans (de Oliveira et al. 1998) and A. palliata (Ma et al. 1975) show the highest heterochromatin content within the genus, with centromeric, interstitial, and telomeric C+ blocks (Table 4.2).

Fig. 4.3
figure 3

C-banded metaphases of male howler monkeys. The arrows indicate the interstitial C+ bands. Single arrowheads indicate telomeric C+ bands. Bar = 10 μm. (a) Alouatta caraya 2N = 52, X1X2Y1Y2, (b) A. guariba 2N = 45, X1X2X3Y1Y2, (c) A. pigra 2N = 58, X1X2Y1Y2, and (d) A. palliata 2N = 53, X1X2Y

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Table 4.2 Heterochromatin location in somatic cells of howler monkeys revealed by C-banding technique
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2.1.2 Microchromosomes

Microchromosomes are supernumerary chromosomes and their presence is unusual among primates. Some authors denominate these chromosomes as “B chromosomes” in contrast to the chromosome complement, generally denominated as “A chromosomes”. Their name is derived from the fact that many of these supernumerary chromosomes are smaller than the smallest A chromosomes (reviewed in Vujoševič and Blagojevič 2004). In Platyrrhini, A. seniculus (Yunis et al. 1976; Torres and Leibovici 2001), A. sara (Minezawa et al. 1985), and A. macconnelli (Lima et al. 1990; Vassart et al. 1996) exhibit this kind of accessory chromosomes. The structure of microchromosomes has mostly been characterized by C-banding technique (Arrighi and Hsu 1971; Sumner 1971). These minute chromosomes have been reported as small segments of heterochromatin (Patton 1977). However, in howler monkeys, there are reports of microchromosomes being either C-band negative (C−) such as in A. seniculus (Yunis et al. 1976) or C-band positive (C+) like in A. macconnelli (previously A. stramineus, Lima and Seuánez 1991; Lima et al. 1990; Vassart et al. 1996). In A. sara, some microchromosomes are C-positive and others are C-negative, and there is no formal hypothesis about this particularity (Minezawa et al. 1985; Torres and Leibovici 2001).

In some howler monkey species, there is variation in microchromosome number between sexes and between individuals (Yunis et al. 1976; Minezawa et al. 1985; Lima et al. 1990; Lima and Seuánez 1991; Vassart et al. 1996). Battaglia (1964) suggested that variation in microchromosome number in mammals could affect the frequency of chiasmata as well as growth, viability, and fertility. Little is known about the meiotic behavior of these microchromosomes, since few analyses in germ cells have been performed. More studies need to be developed in order to elucidate the nature and transmission of these supernumerary chromosomes in howler monkeys.

2.1.3 Sex Chromosomes

In mammals, and particularly in primates, the most frequently described sexual system is the XX/XY (reviewed in Solari 1993). As variants of the ancestral male XY sexual system, Y-autosome translocations that generate multiple sex chromosome systems in males (Fig. 4.4) have been described in Platyrrhini (Hsu and Hampton 1970; Ma et al. 1975; Dutrillaux et al. 1981; Armada et al. 1987; Lima and Seuánez 1991; Rahn et al. 1996; Mudry et al. 1998, 2001; Solari and Rahn 2005; Steinberg et al. 2008). Only the karyological study of germ cells (meiotic analysis) allows the identification and confirmation of these sexual systems; however, meiotic studies are remarkably scarce.

Fig. 4.4
figure 4

Hypothesis on the origin of the sexual system X1X2Y1Y2 in mammalian males. The ancestral X is shown in black, the ancestral Y in white, and the autosomal pair (A) involved in the translocation in gray. From an XY sexual system, two simultaneous breaks on the proximal region of the short arm (Approx) and the terminal region of the long arm (Yqter), followed by a reciprocal translocation (RT), give origin to the chromosomes Y1 and Y2. The homologous chromosome of the autosomal pair not involved in the translocation became known as X 2, and the ancestral X is now denominated X1

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Meiotic karyotypes of platyrrhines have only been described for a small number of species, confirming the sex determination XX/XY in Cebus libidinosus (formerly C. apella paraguayanus; Seuánez et al. 1983; Mudry et al. 2001), Ateles geoffroyi and Ateles paniscus (Mudry et al. 2001; Nieves et al. 2005b), and Saimiri boliviensis boliviensis (Egozcue 1969; Steinberg et al. 2007) and multiple sex chromosome systems in Aotus azarae (Ma et al. 1976), Callimico sp. (Hsu and Hampton 1970), Cacajao sp. (Dutrillaux et al. 1981), and five species of Alouatta (Armada et al. 1987; Lima and Seuánez 1991; Rahn et al. 1996; Mudry et al. 1998, 2001; Solari and Rahn 2005; Steinberg et al. 2008). All meiotic studies of howler monkeys were performed using testes biopsies and confirmed two types of multiple sex chromosome systems (Table 4.3): (1) X1X2Y1Y2 (which forms a chain of four elements or quadrivalent at Metaphase I) in A. macconnelli (Lima and Seuánez 1991), A. caraya (Fig. 4.5a) (Mudry et al. 1998, 2001), and A. pigra (Steinberg et al. 2008) and (2) X1X2Y (which forms a chain of three elements or trivalent at Metaphase I) in A. belzebul (Armada et al. 1987) and A. palliata (Fig. 4.5b) (Solari and Rahn 2005). The multivalent configurations observed in Metaphase I of howler monkey spermatocytes (Fig. 4.5) allow an alternate segregation in Anaphase I, ensuring a balanced gamete production and maintaining the fertility of the individual carriers.

Table 4.3 Confirmation of howler monkey sexual systems through meiotic studies in males
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Fig. 4.5
figure 5

Howler monkey C-banded spermatocytes in Metaphase I showing the location of the C+ heterochromatic regions. This staining technique allows the identification of C+ centromeres, thus revealing the structure of the multivalents. Bar = 5 μm. (a) A. caraya spermatocyte. The arrow indicates the sexual X1X2Y1Y2 (four centromeres C+). Inset: detail of the sexual quadrivalent. The arrows indicate the centromeres of each of the four chromosomal components. (b) A. palliata spermatocyte. The arrow indicates the sexual X1X2Y1 (three centromeres C+). Inset: detail of the sexual trivalent. The arrows indicate the centromeres of each of the three chromosomal components

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Some mitotic studies of Alouatta described the presence of a typical XY male sexual system in A. seniculus (Yunis et al. 1976), A. guariba clamitans (Koiffmann and Saldanha 1974; de Oliveira et al. 1995, 2000), and A. palliata (Torres and Ramírez 2003), and other studies suggested the presence of a X1X2X3Y1Y2 sexual system (which would form a chain of five elements or pentavalent in Metaphase I) in A. guariba guariba and A. g. clamitans (de Oliveira et al. 2002), a X1X2Y system in A. guariba guariba (de Oliveira et al. 1998) and A. sara (Minezawa et al. 1985), and a X1X2Y1Y2 system in A. sara (Consigliere et al. 1998). However, the occurrence of all these sex chromosome systems in Alouatta still awaits confirmation by meiotic analysis.

The meiotic behavior in early meiotic stages of the howler monkey sexual multivalents has been studied in just a few cases (Rahn et al. 1996; Mudry et al. 1998, 2001; Solari and Rahn 2005). The analysis of the sexual quadrivalent of A. caraya at pachytene showed that the maximum extent of synapsis in Y2 is 51 % of the length of X 2, whereas the maximum extent of synapsis of Y1 with X 2 is 42.9 %. The synaptonemal complex (SC) between the X1 axis and Y1 is the smallest pairing segment in the whole quadrivalent (Mudry et al. 1998). In the sexual trivalent of A. palliata, however, the long arms of X 2 and Y1 are paired in almost all their length, and the short arm of Y1 forms the pseudoautosomal region (PAR) with X1p (Solari and Rahn 2005). The end-to-end joining between the X1 and Y1 chromosomes is similar in both multivalents, although the Y1 is a much longer acrocentric in A. palliata. In both A. caraya and A. palliata, the X1 axis has the typical characteristics (branchings, tangling) that are common in spermatocytes at pachytene stages described for other mammalian X axis (Solari 1993), including other Neotropical primates with XX/XY sexual systems (Mudry et al. 2001).

To understand the mechanisms underlying chromosomal evolution and speciation in mammalian species, experimental descriptions of recombination maps are needed. Few studies using “in situ” immunolocalization of recombination proteins have been applied in nonhuman primates (Garcia-Cruz et al. 2009, 2011; Hassold et al. 2009). Only one of such studies has been carried out in howler monkeys (on spermatocytes of A. caraya by Garcia-Cruz et al. 2011), analyzing MLH1 foci, which correspond to recombination spots and are equivalent to the chiasmata observed in Metaphase I. The mean MLH1 foci number per autosomal set was 40.6 ± 4.3 (standard deviation), with a range of 31–50 MHL1 foci per cell. This value is lower than the one observed for human males (49.8 ± 4.3, Sun et al. 2004) but similar to those observed in Cebus libidinosus (41.3 ± 4.8), C. nigritus (39.2 ± 3.3) (Garcia-Cruz et al. 2011), and Macaca mulatta (39.0 ± 3.0) (Hassold et al. 2009). The sexual quadrivalent formed a convoluted sex body, which folded back onto itself, not allowing for a correct visualization of the MLH1 foci. More studies are needed in order to understand the meiotic process in howler monkeys.

Classical cytogenetic analysis showed that the chromosomal pair involved in the sexual systems in Mesoamerican howler monkeys, A. pigra (API) and A. palliata (APA), share no homeology (see Sect. 4.2.2) with the pair involved in the South American species (Steinberg et al. 2008). Chromosomal pair API17 (denominated APIX2 in males) is involved in A. pigra’s multiple sexual system, and APA19 (APA19 in females, APAX2 in males) is involved in the multiple sexual system of A. palliata. These chromosomal pairs share homeology with A. caraya autosome 14 (ACA14) (Fig. 4.6). Therefore, the autosomal pair that is involved in the formation of the sexual systems in A. pigra and A. palliata is not homeologous with the one involved in the sexual system in A. caraya (known as ACA7 in females and ACAX2 in males). ACA7 shares homeology with the autosomal pairs involved in the sexual systems of all South American howler monkey species studied so far (Rahn et al. 1996; Mudry et al. 1998, 2001): A. seniculus (Lima and Seuánez 1991), A. belzebul (Armada et al. 1987), A. sara (Minezawa et al. 1985), A. guariba (de Oliveira et al. 2002), and A. macconnelli (Lima et al. 1990). This suggests that the multiple sexual systems originated independently in South American and Mesoamerican howler monkeys.

Fig. 4.6
figure 6

Chromosome homeologies among A. caraya (ACA), A. pigra (API), A. palliata (APA), and A. guariba (AGU). (a) ACA7 (X 2) shares homeology with API26, API19, APA23, APA18, and AGU7 (X 2). (b) ACA14 shares homeology with API17 (X 2), APA19 (X 2), and AGU6

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2.2 Cytomolecular Analysis

Homeologies at chromosomal level refer to the recognition of chromosome pairs carrying the same information among different organisms. Homologous chromosomes are defined as chromosome pairs of approximately the same length, centromere position, and staining pattern, with genes for the same characteristics at corresponding loci. One homologous chromosome is inherited from the organism’s mother and the other from the organism’s father. When we consider genetic information in different karyotypes of different organisms, we apply the term “homeolog” (Andersson et al. 1996), which becomes useful for phylogenetic analysis. The homeologies identified by G-banding pattern and employed for karyological comparisons are often not informative enough. This is the case when the homeologies involve complex chromosomal rearrangements, small translocated chromosomal fragments or highly rearranged karyotypes, such as Alouatta’s (Dobigny et al. 2004; Stanyon et al. 2008).

In the past 20 years, the Fluorescence in situ hybridization (FISH) technique has proven to be a fast and reliable method to establish chromosomal homeologies between taxa. In this technique, labeled DNA probes specific for entire chromosomes or chromosome regions of a given species are used to hybridize to entire chromosome or chromosome segments in target metaphases (John et al. 1969; Pardue and Gall 1969; Pinkel 1986; Wienberg and Stanyon 1997). Several authors applied this technique to characterize genome conservation in primates (Wienberg et al. 1990; Morescalchi et al. 1997; Consigliere et al. 1998; Stanyon et al. 2004, 2011; Dumas et al. 2007; Amaral et al. 2008). FISH technique provides an unequivocal confirmation of the homeologies previously described by G-banding, giving a higher definition at the cytomolecular level (Wienberg et al. 1990; Wienberg 2005; Müller 2006; Stanyon et al. 2008). Conserved chromosomal syntenies (regions of chromosomes that can be located together side by side on the same chromosome arm) may be used as markers to investigate possible common evolutionary origin. Cytogeneticists refer to a broken chromosomal synteny when a region in a single chromosome of one taxon is found located in different chromosomes of another taxon. Therefore, it is said that the synteny is broken in the later taxon. Chromosomal syntenies can be broken by fissions or translocations. The analysis of these syntenies throughout the phylogeny of a group, such as primates, allows the identification of the chromosomal rearrangements that might be involved in the speciation process. In the last two decades, cross-hybridization using probes of human chromosomes and/or other primate species (Wienberg and Stanyon 1997; Wienberg 2005; Stanyon et al. 2008) has been especially helpful to analyze genomic conservation.

Only five species of Alouatta have been analyzed using FISH: A. caraya (Mudry et al. 2001; Stanyon et al. 2011), A. guariba (both A. g. clamitans and A. g. guariba, de Oliveira et al. 2002; Stanyon et al. 2011), A. sara and A. arctoidea (Consigliere et al. 1996), and A. belzebul (Consigliere et al. 1998). FISH analyses in Alouatta were concordant with the G-banding studies, showing high levels of interspecific chromosomal variability, with interchromosomal rearrangements (Consigliere et al. 1996, 1998; Mudry et al. 2001; de Oliveira et al. 2002; Stanyon et al. 2011). Two syntenic associations (4/15 and 10/16) and the loss of the ancestral association 2/16 were proposed as synapomorphies of Alouatta. All howler monkeys share the syntenies 14/15 from the ancestral mammalian karyotype and 8/18 from the ancestral Platyrrhini (Consigliere et al. 1996, 1998; de Oliveira et al. 2002; Stanyon et al. 2011).

The synteny 3/15 was found in all South American Alouatta species with the exception of A. belzebul (Fig. 4.7), and it is involved in their multiple sexual systems (Consigliere et al. 1996, 1998; Mudry et al. 2001; de Oliveira et al. 2002; Stanyon et al. 2011) (see Sect. 4.2.1.3). The 3/15 synteny was also observed in other atelids, such as Ateles geoffroyi and A. belzebuth hybridus, but is not involved in their XY “human-like” sexual systems (Morescalchi et al. 1997; García Haro 2001; de Oliveira et al. 2005). This synteny was not found in other platyrrhine genera such as Cebus libidinosus or Saimiri boliviensis boliviensis (Mudry et al. 2001). It was then proposed that this synteny could be ancestral for the Atelidae, and an association with multiple sex chromosomes would have only occurred in South American howler monkeys (de Oliveira et al. 2012). However, this 3/15 synteny is not involved in the sexual systems of A. pigra and A. palliata (see Fig. 4.6), and it is not conserved in the autosomes of these species (Steinberg et al. 2014). Considering that in other species of atelids, such as Lagothrix and Brachyteles, the 3/15 synteny has also not been found (Stanyon et al. 2001; de Oliveira et al. 2005), the hypothesis of the ancestrality of this association is not supported. The 3/15 association may had arisen independently in both Ateles and the South American howler monkeys. More cytogenetic studies in both genera are needed in order to confirm this last hypothesis.

Fig. 4.7
figure 7

Fluorescence in situ hybridization with human chromosome probes X, #3, and #15 performed in South American species of Alouatta with male sexual system X1X2Y1Y2. The numbers to the right of the chromosome indicate the hybridization signal of each human chromosome on its corresponding Alouatta homeolog chromosome

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3 Concluding Remarks

Alouatta is a genus with high chromosomal variability, showing multiple interspecific chromosomal rearrangements. C+ heterochromatin is scarce, suggesting that it might not play a prominent role in Alouatta’s chromosomal speciation, which contrasts with observations in other platyrrhines (such as Cebus sp.). Instead, structural rearrangements might be the main factor promoting the karyological evolution of the genus.

The high inter- and intraspecific karyological variability in the genus needs to be considered when assessing the taxonomy of Alouatta. Several species still lack a cytogenetic characterization (either requiring mitotic or meiotic studies, or both). Considering that karyology contributed to the reassessment of several taxa in the past, it seems plausible that the number of species and subspecies could be underestimated (or overestimated) if genetic data are not considered. The characterization of meiotic behavior in A. sara, A. guariba, and A. seniculus, as well as studies in somatic and germ cells in A. p. coibensis and A. nigerrima, would contribute to Alouatta taxonomy and allow testing hypotheses on the chromosomal evolution in the genus.

As stated in Sect. 4.2.1.3, Alouatta is one of the NWP genera that present multiple sex chromosome systems, together with Aotus, Callimico, and Cacajao. In Old World Primates, multiple sexual systems have only been suggested by mitotic studies for one species, the silvered leaf monkey Presbytis cristata (Bigoni et al. 1997). The involvement of the NWP Y-chromosome in multiple sex chromosome systems, together with the absence of homeology with the human Y-chromosome observed by FISH analysis (Consigliere et al. 1996, 1998; Mudry et al. 2001; de Oliveira et al. 2002), highlights the highly different genomic composition and behavior of Y-chromosomes in platyrrhines compared to those of catarrhines.

Finally, the complex multiple sex chromosome systems observed in Alouatta constitute an interesting case study to understand the evolution of sex chromosomes, not only for the diversity of sexual systems but also because it is the only reported case of an independent origin of multiple sex chromosome systems in primates.