CompCytogen |7:273—281 (2023) COMPARATIVE “rrevsestorenssssiourt doi: 10.3897/compcytogen. | 7.108265 Kas Cytogenetics https://compcytogen.pensoft.net International journal of Plant & Animal Cytogenetics, i Karyosystematics, and Molecular Systematics Metaphase chromosomes of five Neotropical species of the genus Drosophila (Diptera, Drosophilidae) Doris Vela', Erika Villavicencio! | Pontificia Universidad Catélica del Ecuador, Facultad de Ciencias Exactas y Naturales, Laboratorio de Gené- tica Evolutiva. Av. 12 de Octubre 1076 y Roca, Quito, Ecuador Corresponding author: Doris Vela (dvela508@puce.edu.ec) Academic editor: Veronika Golygina | Received 20 June 2023 | Accepted 16 October 2023 | Published 5 December 2023 https://zoobank. org/84EFB793-87DD-4A5F-B176-4665224A5DE7 Citation: Vela D, Villavicencio E (2023) Metaphase chromosomes of five Neotropical species of the genus Drosophila (Diptera, Drosophilidae). Comparative Cytogenetics 17: 273-281. https://doi.org/10.3897/compcytogen. 17.108265 Abstract The mitotic metaphases of five Andean species of genus Drosophila are described for the first time. The evo- lutionary and interspecific genetic relationships within three Neotropical Drosophila species groups are ana- lyzed. The diploid chromosome number for each species is as follows: D. cashapamba Céspedes et Rafael, 2012 2n = 6 (2V, 1J) (X= J, Y=R), D. ecuatoriana Vela et Rafael, 2004 2n = 10 (3R, 2V) (X = V, Y=R), D. ninarumi Vela et Rafael, 2005 2n = 10 (3R, 1V, 1D) (X = V, Y=R), D. urcu Vela et Rafael, 2005 2n = 12 (4R, 2V) (X= V, Y=R), D. valenteae Llangari-Arizo et Rafael, 2018 2n = 8 (3R, 1J) X=J, Y=R). Keywords Andean, Drosophila chromosomes, guarani, mesophragmatica, metaphase, tripunctata Introduction The ancestral karyotype for the genus Drosophila Fallén, 1823 (Diptera, Drosophili- dae) consists of five pairs of large chromosomes (V shape or J shape) and one pair of dots (Sturtevant and Novitski 1941). This Drosophila metaphase chromosome con- figuration has been commonly observed, for instance, in some species of Neotropical groups of the type subgenus Drosophila: D. guarani group (King 1947), D. mesophrag- matica group (Brncic and Koref 1957; Hunter and Hunter 1964), D. repleta group (Wasserman 1960) and D. tripunctata group (Pipkin and Heed 1964). The species Copyright Doris Vela & Erika Villavicencio. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 274 Doris Vela & Erika Villavicencio / Comparative Cytogenetics 17: 273-281 (2023) of the type subgenus present a chromosome configuration ranging from three to six pairs of chromosomes. Cytogenetics studies demonstrated that in the genus Drosophila karyotypes of species may differ from the ancestral karyotype by the number of chro- mosomes and the chromosomal configuration, but chromosomal rearrangements do not break the integrity of Muller elements (chromosome arms and associated linkage groups) (Schaeffer 2018). By means of the karyotypes, it is possible to observe the chromosomal rearrange- ments (inversions, translocations, duplications etc.) in species, and how they can limit the genetic exchange and potentially drive speciation (Noor et al. 2001). In addition, it is possible to detect interspecific and intraspecific polymorphism in species of Drosophila (Deng et al. 2007). Therefore, karyotypes are an important tool for understanding the evolutionary history of the Drosophila species, to conduct comparative genomics stud- ies and to allow genome assembly at the chromosome level (Schaeffer 2018). Most of the available cytological data about Neotropical species of Drosophila were reported in the past century (Metz and Moses 1923; Patterson and Wheeler 1942; Wharton 1943; Burla et al. 1949; Clayton and Wasserman 1957; Clayton and Wheeler 1975). In the most recent cytological studies of Neotropical species of Drosophila kar- yotypes of ten species from four sibling species groups have been described: D. chorlavi Céspedes et Rafael, 2012, D. mesophragmatica Duda, 1927 and D. rucux Céspedes et Rafael, 2012 from the D. mesophragmatica group (Mafla 2012), D. butantan Ratcov, Vilela et Goni, 2017, D. sachapuyu Penafiel-Vinueza et Rafael, 2018, and D. zamorana Pefafiel-Vinueza et Rafael, 2018 from the D. guarani group (Ratcov et al. 2017; Vela and Villavicencio 2021), D. huancavilcae Rafael et Arcos, 1989, D. inca Dobzhansky et Pavan, 1943, and D. yangana Rafael et Vela,;2003 from the D. repleta group (Mafla 2005, 2008), and D. montevidensis Goni et Vilela, 2016 from the D. tripunctata group (Goni and Vilela 2016). In this study, the karyotypes of five Andean species of Drosophila from three sibling species groups are described for the first time: D. ecuatoriana Vela et Rafael, 2004 and D. valenteae Liangari-Arizo et Rafael, 2018 from the D. guarani group, D. cashapamba Céspedes et Rafael, 2012 from the D. mesophragmatica group, D. ninarumi Vela et Rafael, 2005 and D. urcu Vela et Rafael, 2005 from the D. tripunctata group. Methods Species stock The species analysed correspond to natural populations of: D. cashapamba (QCAZ-I 2349), Sangolqui Canton (location 0°19'59.3"S, 78°25'51"W DMS); D. ecuatoriana (QCAZ-I 1609), Yanacocha Forest (location 0°7'3.8"S, 78°35'9.4"W DMS); D. ninarumi (QCAZ-I 1765), Cruz Loma Forest (location 0°11'22"S, 78°31'17.2"W DMS); D. urcu (QCAZ-I 1755), Cruz Loma Forest (location 0°11'22"S, 78°31'17.2"W DMS) and D. valenteae (QCAZ-I 3142), Sangolqui Canton (location 0°19'59.3"S, 78°25'51"W DMS). Metaphase karyotypes of Neotropical species of the Drosophila 279 All species were provided by the Evolutionary Genetics Laboratory of Pontificia Uni- versidad Catélica del Ecuador. The flies were maintained in banana culture medium sup- plemented with fresh fruit, in a temperate room at 17 °C, with a 12 h light/dark cycle. Chromosome plates The metaphase nuclei of cerebral ganglia were obtained from third-instar larvae (ten males, ten females) of each species. Chromosomal plates were prepared by the cell sus- pension method (Cardoso and Dutra 1979) and thermic shock (Holmquist 1975) and stained with Giemsa. Ten metaphase nuclei were observed for each sex and species. A Ziess Axioskop 2 plus — HAL 100 microscope and a Cannon PowerShot A640 camera (100x objectives lens and optovar 2x) were used to observe and take the pictures of the mitotic chromosome cells. The modal number was considered the chromosome number of each species. Mitotic chromosome analysis For each species, the total length (TL), relative length (RL) and centromeric index (CI) of the chromosomes were estimated using the Axio Vision 4.4. Standard deviation of relative length was analysed using the SPSS statistical package 26.0v (Table 1). Results The description of new karyotypes of Drosophila species is presented below: The Drosophila guarani group The karyotype of D. ecuatoriana is 2n = 10 (3R, 2V), comprising of four autosomes — a large V-shaped metacentric (pair 2) and three pairs of rod-shaped telocentric chromo- somes (pairs 3, 4 and 5) — and the sexual pair (X = V, Y = R). The X chromosome is V- shaped metacentric and the Y chromosome is rod-shaped telocentric (Fig. 1A, B, Table 1). The karyotype of D. valenteae is 2n = 8 (3R, 1J), comprising of three rod-shaped telocentric autosomes (pairs 2, 3 and 4), and the sexual pair (X = J, Y = R). The X chro- mosome is J-shaped submetacentric, and the Y chromosome is rod-shaped telocentric (Fig. 1C, D, Table 1). The Drosophila mesophragmatica group The karyotype of D. cashapamba is 2n = 6 (2V, 1J) comprising of two V-shaped meta- centric autosomes (pairs 2 and 3) and the sexual pair (X = J, Y = R). The X chromosome is J-shaped submetacentric and the Y chromosome is rod-shaped telocentric (Fig. 1E, F Table 1). 276 Doris Vela & Erika Villavicencio / Comparative Cytogenetics 17: 273-281 (2023) Table |. Measurement of metaphase chromosomes of five Andean Drosophila species. Species Chromosome TL (um) RL (%) CI SD (n= 10) Morphology D. ecuatoriana > 2,49 24,22 0,47 0,27 metacentric 2n = 10 Y 1,85 17,99 0,05 0,03 telocentric 2 1,65 16,05 0,49 0,12 metacentric 3 1,54 14,98 0,06 0,19 telocentric 4 1,42 13,81 0,07 0,21 telocentric 5 1593 12,93 0,08 0,16 telocentric D. valenteae xX 2,09 27,42 0,37 0,23 submetacentric 2n = 8 Y 1,73 22,7 0,06 0,31 telocentric 2 1,4 18,37 0,07 0,21 telocentric 3 1,26 16,53 0,08 0,23 telocentric 4 1,14 14,96 0,09 0,14 telocentric D. cashapamba xX 2,88 26,2 0,38 0,12 submetacentric 2n=6 ¥ 1,94 17,65 0,05 0,04 telocentric 2 3,21 29,2 0,47 0,11 metacentric 3 2,96 26,93 0,49 0,12 metacentric D. ninarumi xX 1,71 27,49 0,46 0,25 metacentric 2n = 10 Y 1,59 25,56 0,06 0,04 telocentric 2 1,12 18 0,09 0,26 telocentric 3 0,95 15,27 0,11 0,18 telocentric 4 0,83 13,34 0,12 0,2 telocentric 5) 0,02 0,32 0,05 0,01 dot D. urcu > 3,09 24,75 0,48 0,23 metacentric 2n= 12 Y 2,65 21,23 0,04 0,07 telocentric 2 1,62 12,98 0,49 O37 metacentric 3 1,58 12,66 0,06 0,27 telocentric 4 1,45 11,61 0,07 0,21 telocentric 5 1,21 9,69 0,08 0,14 telocentric 6 0,88 7,05 0,11 0,29 telocentric TL: Total Length, RL: Relative Length, CI: Centromeric Index, SD: Standard deviation. The Drosophila tripunctata group The karyotype of D. ninarumi is 2n = 10 (3R, 1V, 1D), comprising of four auto- somes — three rod-shaped telocentric (pairs 2, 3 and 4) and one pair of dot-shaped chromosomes (pair 5), and the sexual pair (X = V, Y = R). The X chromosome is V-shaped metacentric and the Y chromosome is rod-shaped telocentric (Fig. 1G, H, Table 1). The karyotype of D. urcu is 2n = 12 (4R, 2V) comprising of five autosomes —a pair of V-shaped metacentric (pair 2) and four pairs of rod-shaped telocentric chromosomes (pairs 3, 4, 5 and 6) — and the sexual pair (X = V, Y = R). The X chro- mosome is V-shaped metacentric and the Y chromosome is rod-shaped telocentric (Fig. 1, JTable). Metaphase karyotypes of Neotropical species of the Drosophila 2d. Figure |. Metaphase karyotype of A D. ecuatoriana female B D. ecuatoriana male C D. valenteae female D D. valenteae male E D. cashapamba female F D. cashapamba male G D. ninarumi female H D. ninaru- mi male I D. urcu female J D. urcu male. Scale bar: 3 um (A-J). 278 Doris Vela & Erika Villavicencio / Comparative Cytogenetics 17: 273-281 (2023) Discussion Considering the high diversity of Drosophila species in the Neotropical region little is known about diploid chromosome numbers of these species. In the Drosophila guarani group, the most common karyotype is 2n = 12. In the present study, the karyotype of D. ecuatoriana is 2n = 10 (Fig. 1A, B). A similar 2n = 10 karyotype was reported in other species of this group: D. guaraja King, 1947 (King 1947), D. butantan (Ratcov et al. 2017) and D. sachapuyu (Vela and Villavice- ncio 2021). The karyotype of D. valenteae is 2n = 8 (Fig. 1C, D) and is similar to D. alexandrei Cordeiro, 1951 (Cordeiro 1951), both species present the lowest diploid chromosome reported for the Drosophila guarani species group. Several reports have shown that the karyotype of Drosophila species of the D. meso- phragmatica group is highly conserved, 2n = 10, including a pair of rod-shaped or a dot-like fifth chromosomes (Brncic 1957). Additionally, paracentric inversions are the principal chromosomal rearrangements attributed to this species group (Brncic and Ko- ref 1957). In our study, the chromosome number of D. cashapamba is 2n = 6, the chro- mosomes are large and present a small pericentromeric heterochromatin (Fig. 1E, F). It has been suggested that D. cashapamba is a junior synonym of D. dreyfusi Dobzhansky et Pavan, 1943 (Dr Carlos Vilela, pers. communication) due to the similarity of the male genitalia and the same chromosome number, 2n = 6 (Dobzhansky and Pavan 1943). However, in this study we maintain the current taxonomical classification until new taxonomic studies confirm the junior synonym status of D. cashapamba. According to the information available in the Drosophila karyotype databases (Mo- relli et al. 2022), the chromosome number 2n = 6 is rarely reported in Drosophila subgenus. Only thirteen species of Drosophila subgenus present three pairs of chromo- somes: D. canalinea Patterson et Mainland, 1944 from D. canalinea group, D. dreyfusi and D. wingei Cordeiro, 1964 from D. dreyfusi group, D. albomicans Duda, 1923, D. annulipes Duda, 1924, D. neohypocausta Lin et Wheeler, 1973 from D. immigrans group, D. atalaia Vilela et Sene, 1982 from D. peruensis group, D. pinicola Sturtevant, 1942 from D. pinicola group, D. quinaria Loew, 1866 from D. quinaria group; D. neoguaramunu Frydenberg, 1956 from D. tripunctata group, D. montana Patterson et Wheeler, 1942 from D. virilis group, D. aracea Heed et Wheeler, 1957 and D. tran- quilla Spencer, 1942 (not grouped). Most species of the D. tripunctata group have a karyotype 2n = 12, the sixth pair is a dot chromosome; some members of D. tripunctata group have a karyotype 2n = 10 (Morelli et al. 2022). In the karyotype of D. ninarumi, 2n = 10, it is present a dot- like fifth pair of chromosome (Fig. 1G, H) which is reported in the most species of Drosophila tripunctata group. This karyotype is similar to D. fairchaldi Pipkin et Heed, 1964 and D. unipunctata Patterson, 1943 (Wharton 1943; Pipkin and Heed 1964; Clayton and Wheeler 1975) but in these species the dot-like chromosome is absent. In the case of D. urcu, the karyotype is 2n = 12, all the chromosomes are large meta- centric or telocentric (Fig. 11, J). Our data show that the karyotype of D. ninarumi and D. urcu have a relevant similitud, the sexual chromosomes are the largest of the chromosome set, with a Y chromosome heteropycnotic (Fig. 1G, J). Metaphase karyotypes of Neotropical species of the Drosophila 279) Traditional studies like genetic crosses, in situ hybridization, polytene chromo- somes maps or karyotype description are not commonly performed. However, for the genus Drosophila, the information provided by cytological studies is the initial tool in understanding the evolutionary history and the high radiation of the Drosophila spe- cies in the Neotropical region and also important in the beginning of genomic studies on these species. Conclusions This study reveals the first karyotype description of five Neotropical species of Dros- ophila. Only the karyotype of D. urcu, 2n = 12, is similar to the ancestral karyotype of Drosophila, but the sixth pair are large chromosomes. The karyotypes of D. ecuatoriana and D. ninarumi are 2n = 10, but only the last one has a dot-like chromosome. The kar- yotype of D. valenteae is 2n = 8; this is the second species of D. guarani group that have this chromosome number. ‘The karyotype of D. cashapamba presents a low chromosome number, 2n = 6, which is only reported in other thirteen species of subgenus Drosophila. Acknowledgements The present research has been supported by the Pontificia Universidad Catdlica del Ecuador through the project QINV0320-HNV529010200. References Brncic D (1957) A comparative study of chromosomal variation in species of the mesophrag- matica group of Drosophila. Genetics 42(6): 798-805. https://doi.org/10.1093/genet- ics/42.6.798 Brncic D, Koref S (1957) The mesophragmatica group of species of Drosophila. Evolution 11(3): 300-310. https://doi.org/10.2307/2405794 Burla H, Da Cunha AB, Cordeiro AR, Dobzhansky T, Malogolowkin C, Pavan C (1949) The willistoni group of sibling species of Drosophila. Evolution 3(4): 300-314. https://doi. org/10.2307/2405716 Cardoso H, Dutra A (1979) The Neo-X Neo-Y sex pair in Acrididae, its structure and associa- tion. Chromosoma 70(3): 323-336. https://doi.org/10.1007/BF00328770 Clayton FE, Wasserman M (1957) Chromosomal studies of several species of Drosophila. Uni- versity of Texas Publications 5721: 125-131. Clayton F, Wheeler M (1975) A catalog of Drosophila metaphase chromosome configurations. In: King R (Ed.) Handbook of Genetics (Vol. 3). Plenum Press. New York, 471-512. https://doi.org/10.1007/978-1-4615-7145-2_18 Cordeiro AR (1951) Drosophila alexandrei, una nova especie brasileira. Universidade do Rio Grande do Sul 3: 1-10. 280 Doris Vela & Erika Villavicencio / Comparative Cytogenetics 17: 273-281 (2023) Deng Q, Zeng Q, Qian Y, Li C, Yang Y (2007) Research on the karyotype and evolution of Drosophila melanogaster species group. Journal of Genetics and Genomics 34(3): 196-213. https://doi.org/10.1016/S1673-8527(07)60021-6 Dobzhansky T, Pavan C (1943) Studies on brazilian species of Drosophila. Boletim da Facul- dade de Filosofia, Ciéncias e Letras da Universidade de S40 Paulo Biol Geral 36(4): 7-72. http://www.drosophila.jp/jdd/class/030703/03070372.pdf Goni B, Vilela CR (2016) Two new neotropical species of drosophilinae (Diptera: Droso- philidae) from Uruguay. Zoologia 33(6): 1-13. https://doi.org/10.1590/s1984-4689z0- ol-20160142 Holmquist B (1975) A revision of the species Archaeomysis grebnitzkii Czernaivsky (sic) and A. maculata (Holmes) (Crustacea, Mysidacea). Okologie und Geographie der Tiere 102: 51-71. Hunter AS, Hunter RA (1964) The mesophragmatica species group of Drosophila in Colombia. Annals of the Entomological Society of America 57(6): 732-736. https://doi.org/10.1093/ aesa/57.6.732 King JC (1947) A comparative analysis of the chromosomes of the guarani group of Dros- ophila. Evolution 1: 48-62. https://doi.org/10.2307/2405403 Mafla AB (2012) Cariologia beta de tres especies pertenecientes al grupo de especies Drosophila mesophragmatica. Revista Ecuatoriana de Medicina y Ciencias Bioldgicas 33(1—2): 38-45. https://doi.org/10.26807/remcb.v33il-2.221 Mafla AB (2005) Cariotipos metafasicos de Drosophila inca y D. yangana, subgrupo inca, grupo repleta. Revista Ecuatoriana de Medicina y Ciencias Biolégicas 27: 21-25. https://doi. org/10.26807/remcb.v27i1-2.190 Mafla AB (2008) Drosophila huancavilcae: ciclo biolégico y cariotipo metafasico. Revista Ec- uatoriana de Medicina y Ciencias Bioldgicas 29(1—2): 7-10. https://doi.org/10.26807/ remcb.v29i1-2.205 Metz CW, Moses MS (1923) Chromosome relationships and genetic behavior in the genus Drosophila: 1. A comparison of the chromosomes of different species of Drosophila. Journal of Heredity 14(5): 195-205. https://doi.org/10.1093/oxfordjournals.jhered.al102315 Morelli MW, Blackmon H, Hjelmen CE (2022) Diptera and Drosophila karyotype databases: A useful dataset to guide evolutionary and genomic studies. Frontiers in Ecology and Evolu- tion 10: 832378. https://doi.org/10.3389/fevo.2022.832378 Noor MAK, Grams KL, Bertucci LA, Reiland J (2001) Chromosomal inversions and the re- productive isolation of species. Proceedings of the National Academy of Sciences 98(21): 12084-12088. https://doi.org/10.1073/pnas.221274498 Patterson J, Wheeler MR (1942) Description of new species of the subgenera Hirtodrosophila and Drosophila. University of Texas Publications 4213: 67-109. Pipkin SB, Heed WB (1964) Nine new members of the Drosophila tripunctata species group (Diptera: Drosophilidae). Pacific Insects 6(2): 256-273. http://www.drosophila.jp/jdd/ class/030703/03070394.pdf Ratcov V, Vilela CR, Gofi B (2017) A new species of neotropical Drosophila (Diptera, Dros- ophilidae) belonging to the guarani group. Revista Brasileira de Entomologia 61(3): 232-238. https://doi.org/10.1016/j.rbe.2017.06.002 Metaphase karyotypes of Neotropical species of the Drosophila 281 Schaeffer SW (2018) Muller “elements” in Drosophila: How the search for the genetic basis for speciation led to the birth of comparative genomics. Genetics 210(1): 3-13. https://doi. org/10.1534/genetics.118.301084 Sturtevant AH, Novitski E (1941) The homologies of the chromosome elements in the genus Drosophila. Genetics 26(5): 517-541. https://doi.org/10.1093/genetics/26.5.517 Vela D, Villavicencio E (2021) Karyotype description of two andean species of the guarani group of Drosophila (Diptera: Drosophilidae) and Cytological Notes. Journal of Insect Sci- ence 21(3). https://doi.org/10.1093/jisesa/ieab032 Wasserman M (1960) Cytological and phylogenetic relationships in the repleta group of the genus Drosophila. Genetics 46: 842-859. https://doi.org/10.1073/pnas.46.6.842 Wharton LT (1943) An analysis of the metaphase and salivary chromosome morphology with- in the genus Drosophila. University of Texas Publications 4313: 282-319. ORCID Doris Vela https://orcid.org/0000-0001-7690-7758