Karyotype Description of Two Andean Species of the guarani Group of Drosophila (Díptera: Drosophilidae) and Cytological Notes.

The guarani group of Drosophila genus (Diptera: Drosophilidae) is formed by 24 species however the relationship of these species is not clear. In the present study are described the karyotypes of Drosophila sachapuyu Peñafiel and Rafael, 2018 and Drosophila zamorana Peñafiel and Rafael, 2018, two Andean species members of the guarani group. Mitotic chromosomes from cerebral ganglia of third stand larval were obtained by thermal shock and cell suspension techniques. The karyotype of D. sachapuyu, presents 2n = 10 (4R, 1V; X = R, Y = R) while D. zamorana exhibits karyotype 2n = 12 (5R, 1V; X = V, Y = R).

The guarani group of Drosophila genus (Diptera: Drosophilidae) is a neotropical group proposed by Dobzhansky and Pavan (1943) and nowadays includes 24 species (Bachli 2021): D. limbinervis (Duda, 1925); D. griseolineata (Duda, 1927); D. ornatifrons (Duda, 1927); D. maculifrons (Duda, 1927); D. nigrifemur (Duda, 1927); D. guaru (Dobzhansky & Pavan, 1943); D. subbadia (Patterson, 1943); D. guaraja (King, 1947); D. alexandrei (Cordeiro, 1951); D. araucana (Brncic, 1957); D. huilliche (Brncic, 1957); D. tucumana (Vilela and Pereira, 1985); D. urubamba (Vilela & Pereira 1993); D. ecuatoriana (Vela & Rafael, 2004); D. pichinchana (Vela & Rafael, 2004); D. quitensis (Vela & Rafael, 2004); D. cuscungu (Vela & Rafael, 2005); D. butantan (Ratcov et al., 2017); D. sachapuyu (Peñafiel & Rafael, 2018); D. zamorana (Peñafiel & Rafael, 2018); D. quinarensis (Peñafiel & Rafael, 2018); D. caxarumi (Peñafiel & Rafael, 2018); D. misi (Peñafiel & Rafael, 2018); D. peixoto (Vaz et al., 2018).

Some misidentification have occurred in this group, D. guarani was synonymized to D. ornatifrons, D. guaramunu was synonymized to D. maculifrons (Vilela and Bächli 1990) and D. pulla (Pavan and Cunhas, 1947) was synonymized to D. guaraja (Freire-Maia and Pavan 1950). Also, D. peruensis was classified in the guarani group and posteriorly was moved to the peruensis group which includes spotted thorax species (Ratcov and Vilela 2007), and D. amplipennis (Malloch, 1934) was proposed like an aberrant member (Vilela and Bachli 2004), however it is not included in guarani group (Bachli 2021).

The guarani group was divided into two subgroups based on the shape of X chromosome (V or rod shape) (King 1947) but nowadays, the guarani subgroup has been accepted and the guaramunu subgroup has been dismissed (Vaz et al. 2018). Phylogenetic relationships of guarani group, based on morphology, cytology, and molecular markers suggested that species of guarani and tripunctata group are related and that guarani group is not monophyletic (Throckmorton 1962, Robe et al. 2002).

In this work are described the karyotypes of Drosophila sachapuyu and Drosophila zamorana, two Andean species that recently have been included in the guarani group based on the external morphology and terminalia (Peñafiel-Vinueza and Rafael 2018). Additionally, we compile the information about the karyotype of ten species of guarani group.

Materials and Methods

Drosophila Stocks

Were analyzed natural populations of D. zamorana (QCAZ-I 3266) (location 3°59′16.7ʺS; 79°5′35ʺW) and D. sachapuyu (QCAZ-I 3309) (4°6′53.7ʺS; 79°10′54.6ʺW) collected in the Podocarpus National Park (Zamora Chinchipe and Loja provinces in Ecuador) (Peñafiel and Rafael 2018) and maintained in the Drosophila ceparium of Evolutionary Genetics Laboratory of Pontificia Universidad Católica del Ecuador. The species are maintained in banana culture medium supplemented with fresh fruit, in temperate room at 17°C, 12 h cycle light/dark.

Cytological Preparations

Third instar larvae (10 males, 10 females) of D. sachapuyu and D. zamorana stocks were dissected to obtain the cerebral ganglia and to observe metaphasic nucleus. The male larvae are identified by the presence of the testes (transparent balls) in the last segments of the larva. Chromosomal plates were prepared for cell suspension method (Cardoso and Dutra 1979) and thermic shock (Holmquist 1975), stained with Giemsa and posteriorly the C banding was performed.

Ten metaphasic nucleus for each sex and species were observed and the chromosome number was counted using the Ziess Axioskop 2 plus – HAL 100 microscope,100× objectives lenses, and optovar 2×. Pictures were obtained with a Cannon PowerShot A640 camera. The modal number was considered the chromosome number of the species.

Centromeric index (CI), centromeric ratio (CR), and relative length (RL) of the chromosomes were estimated using the Axio Vision 4.4. Standard deviation of relative length was analyzed using the SPSS statistical package 26.0v.

Results

Karyotype of D. zamorana

The chromosome number of D. zamorana was 2n = 12 (5R, 1V) (X = V, Y = R), five autosomes, and the sexual pair (Fig. 1).

Fig. 1.

D. zamorana (2n = 12): A) karyotype, B) Idiogram, C) Female metaphase plate, D) Male metaphase plate. Scale bar = 5 µm.

The pair 2 rod-shaped, telocentric, completely heteropycnotic. The pairs 3, 4, 5, and 6, all of them are rod-shaped, telocentric, and heterochromatic in the centromeric region. The chromosome X is V-shaped, submetacentric, heterochromatic in the proximal region of p arm and completely the q arm. The chromosome Y is rod-shaped, telocentric, and completely heterochromatic (Table 1).

Table 1.

D. zamorana chromosome measurements and morphology

Chromosome	TL (µm)	σ (n = 10)	RL (%)	CI	Morphology
X	2.77	0,24	24.58	0.29	Submetacentric
2	1.8	0,21	15.93	0.07	Telocentric
3	1.63	0,19	14.43	0.07	Telocentric
4	1.47	0,22	13.03	0.07	Telocentric
5	1.22	0,2	10.83	0.08	Telocentric
6	0.97	0,14	8.57	0.1	Telocentric
Y	1.43	0,03	12.63	0.1	Telocentric

TL: Total Length, RL: Relative Length, CI: Centromeric Index, σ: St. Deviation.

Karyotype of D. sachapuyu

The chromosome number of D. sachapuyu is 2n = 10 (4R, 1V) (X = R, Y = R), four autosomes, and sexual chromosomes (Fig. 2).

Fig. 2.

D. sachapuyu (2n = 10): A) karyotype, B) Idiogram, C) Female metaphase plate, D) Male metaphase plate. Scale bar = 5 µm.

The pair 2, V-shaped, submetacentric, the proximal region of p arm, and almost the total length of q arm are heterochromatic. The pairs 3, 4, and 5, all of them are rod-shaped, telocentric, and heterochromatic in the pericentromeric region of large arms. The chromosome X is rod-shaped, telocentric, heterochromatic in the centromeric region, and p arm. The chromosome Y is rod-shaped, telocentric, and totally heterochromatic (Table 2).

Table 2.

D. sachapuyu chromosome measurements and morphology

Chromosome	TL (µm)	σ (n = 10)	RL	CI	Morphology
2	3.37	0,45	29.18	0.3	Submetacentric
X	2.08	0,22	18.09	0.06	Telocentric
3	1.82	0,21	15.81	0.06	Telocentric
4	1.63	0,16	14.17	0.06	Telocentric
5	1.44	0,21	12.48	0.07	Telocentric
Y	1.1	0,05	10.27	0.08	Telocentric

TL: Total Length, RL: Relative Length, CI: Centromeric Index, σ: Standard deviation.

Karyotype description of D. zamorana and D. sachapuyu is added to the 10 karyotypes previously described for the species of guarani group (Table 3).

Table 3.

Karyotype characteristics of 12 species of guarani group

Species	2n	Karyotype	Chromosomes		References
			X	Y
*D. guaru (Dobzhansky and Pavan 1943)	12	4R, 1V, 1D	V	J	Dobzhansky and Pavan 1943, King 1947
*D. ornatifrons (Duda 1927)	12	5R, 1V	V	V	Dobzhansky and Pavan 1943, King 1947
*D. subbadia(Patterson 1943)	12	5R, 1V	V	J	King 1947
D. maculifrons (Duda 1927)	12	5R, 1D	R	R	Dobzhansky and Pavan 1943, King 1947
D. griseolineta (Duda 1927)	12	5R, 1D	R	R	Dobzhansky and Pavan 1943, King, 1947
D. araucana (Brncic 1957)	12	5R, 1D			Brncic 1957
D. limbinervis (Duda 1925)	12	5R, 1D	R	R	Clayton and Wasserman 1957
D. zamorana (Peñafiel and Rafael 2018)	12	5R, 1V	V	R	Authors
D. guaraja (King 1947)	10	3R, 1V, 1D / 3R, 1V	R	R	Pavan and da Cunha 1947, King 1947
D. butantan (Rarcov et al. 2017)	10	2R, 2V, 1D	V	R	Ratcov et al. 2017
D. sachapuyu (Peñafiel and Rafael 2018)	10	4R, 1V	R	R	Authors
D. alexandrei (Cordeiro 1951)	8	3R, 1V			Cordeiro 1951

*Species members of guarani subgroup; V: metacentric chromosome, J: submetacentric or subtelocentric chromosome, R: telocentric chromosome, D: dot chromosome.

Discussion

Previous studies of guarani group based on morphology, chromosomes, and molecular phylogenies suggested that the relationships among the species included in this group are not clear. In the guarani group are included species with variation in morphology, brownish and spotted thorax (Vilela and Pereira 1985), heterogeneity in terminalia structures (Throckmorton 1962), and different chromosome number (Kastritsis et al. 1970). These characteristics are shared with some species of tripunctata group, therefore these variations open some questions respect the relationships among the species of guarani and tripunctata groups.

The phylogeny proposed by Throckmorton’s (1962) based on the external morphology and the anatomical characteristics of genital structures place the tripunctata and guarani group very close in the tree. This near relationship between guarani and tripunctata species is supported posteriorly by Kastritsis (1969) and Kastritis et al. (1970), they proposed the division of guarani group in two species groups based on the similarity of banding patterns of polytene chromosomes between D. griseolineata (guaramunu subgroup) and D. mediostriata (Duda 1925) (karyotype 2n = 12; 5R, 1D) (tripunctata group) (Dobzhansky and Pavan 1943).

Also, King (1947) proposed the division of guarani group in two subgroups based on the shape of Y chromosome: the guarani subgroup with V shape (D. guaru, D. ornatifrons, and D. subbadia) and guaramunu subgroup with rod shape (D. maculifrons, D. griseolineta, and D. guaraja). Recently Vaz et al. (2018) based on chromosome analysis dismissed the guaramunu subgroup, therefore only the guarani subgroup is accepted in the guarani group.

With the description of the karyotypes of D. sachapuyu and D. zamorana, there is available information about the karyotype of 12 species of guarani group (Dobzhansky and Pavan 1943, King 1947, Pavan and Cunha 1947, Clayton and Wasserman 1957, Vilela and Bächli 1990, Ratcov et al. 2017, Vilela and Pereira 1985, Cordeiro 1951, Brncic 1957) which shown variation in chromosome number from 2n = 8 to 2n = 12 and four types shapes and morphologies: V-shaped (V), J-shaped (J), rods (R), and dots (D) (King 1947, Ratcov et al. 2017, Cordeiro 1951), which correspond V for a metacentric chromosome, J for a submetacentric or subtelocentric chromosome, R for a telocentric chromosome, and D for dot, a small or very small chromosome (Deng et al. 2007). Species of guarani group present the Y chromosome in rod shape, except D. guaru, D. subbadia, and D. ornatifrons. The X chromosome could be V or rod shape (King 1947) (Table 3).

According to Vaz et al. (2018) in the guarani subgroup has been included four species (D. ornatifrons, 5R, 1V; D. subbadia, 5R, 1V; D. guaru 4R, 1V, 1D; and D. peixoto). Three of them present the same chromosome number 2n = 12, however each species showed different shape of sexual chromosomes. D. ornatifrons and D. subbadia present karyotype 5R, 1R, but the morphology of Y chromosome is V-shaped in D. ornatifrons and J-shaped in D. subbadia. Similar morphology (V-shaped) of sexual chromosomes X have the species D. guaru and D. ornatifrons, however D. guaru presents the Y chromosome J-shaped and a dot chromosome that is not present in D. ornatifrons neither in D. subbadia. An important characteristic described only for D. ornatifrons is the presence of satellites in all the rod chromosomes. There is no information available about the karyotype of D. peixoto, the fourth member of guarani subgroup.

The rest of species remain in the guarani group but have not been assigned to a subgroup. Five species present a karyotype 2n = 12, three of them, D. maculifrons (5R, 1D), D. griseolineata (5R, 1D), D. araucana (5R, 1D), present a dot chromosome, while D. zamorana (5R, 1V) not present the dot chromosome similar to D. limbinervis (5R, 1D). On the other hand, D. griseolineata presents X and Y chromosome large rod-shaped, however the Y chromosome presents a satellite. While, D. maculifrons and D. zamorana (5R, 1V) present V-shaped and rod-shaped X chromosome, respectively, and rod-shaped Y chromosome.

D. guaraja (3R, 1V, 1D), D. butantan (2R, 2V, 1D), and D. sachapuyu (4R, 1V) present karyotype 2n = 10, all of them present a large V-shaped autosome but only D. guaraja and D. butantan present a dot chromosome. Respect the sexual chromosomes, D. guaraja and D. sachapuyu show rod-shaped X and Y chromosomes, however the X chromosome of D. sachapuyu is two times longer the Y chromosome, while the X chromosome of D. guaraja is a short rod. In D. butantan, the X chromosome is V-shaped and the Y chromosome is rod-shaped.

Only the species D. alexandrei (3R, 1V) presents a karyotype 2n = 8, which includes a large V-shaped autosome (similar to the species 2n = 10), and dot chromosome is absent. The X and Y chromosomes are rod-shaped, similar size to autosomes.

Molecular phylogeny of guarani group supports the monophyly only for the guarani subgroup (D. subbadia and D. ornatifrons) while the guarani group is not a monophyletic group (Robe et al. 2002, 2005, Izumitani et al. 2016). In two molecular studies has been proposed a tripunctata lineage cluster which includes species of guarani (D. ornatifrons, D. griseolineata, and D. maculifrons), tripunctata, and cardini groups (Robe et al. 2002, Yotoko et al. 2003). Also, a molecular phylogeny of tripunctata radiation, based on mitocondrial genes, suggested the existence of four clades, one of them is the clade tripunctata-guaramunu lineage conformed by the species D. griseolineata, D. maculifrons (guarani group), D. frotapessoai (Vilela and Bachli 1990), and D. paramediostriata (Townsend and Wheeler 1955) (karyotype 2n = 12; 5R, 1D) (tripunctata group) (Hatadani et al. 2009). In other molecular analysis, few species of guarani group has been included however in all of them is observed that species of guarani and tripunctata groups are closely related (Izumitani et al. 2016, Robe et al. 2005).

Comparison of karyotypes of guarani species showed differences in the size of autosomes but especially in the morphology and size of sexual chromosomes which in some species are twice the size of the autosomes (D. maculifrons, D. griseolineata, and D. araucana). This characteristic is described also for the species D. paramediostriata (Townsend and Wheeler 1955) of the tripunctata group. These four species present karyotype 2n = 12 and the molecular phylogenies proposed that are closely related, however are lacking information about more species to confirm the relationship of guarani group.

Morphological and phylogenetic analysis have not provided enough information to clarify the relationship of the guarani group. However, karyotype information and new genomic analysis could explain changes in chromosome number and chromosome structure to clarify the relationships of guarani group and the high radiation of Drosophila species in the Neotropical region.