The characterization of the chromosomal morphology of Haplopappus foliosus (Hook. & Arn.) determined a chromosomal number of 2n = 10, with a karyotypic formula of one subtelocentric pair (st) and four submetacentric pairs (sm). The intrachromosomal asymmetry index (A1) and interchromosomal asymmetry index (A2) showed a moderate tendency towards telocentricity and low variability in the sizes of chromosome pairs.
La caracterización de la morfología cromosómica de Haplopappus foliosus (Hook. & Arn.) determinó un número cromosómico de 2n = 10, con una fórmula cariotípica de un par subtelocéntrico (st) y cuatro pares submetacéntricos (sm). Los índices de asimetría intracromosómica (A1) e intercromosómica (A2) mostraron una moderada tendencia a la telocentría y baja variabilidad en los tamaños de los pares cromosómicos.
ABSTRACT
The characterization of the chromosomal morphology of Haplopappus foliosus (Hook. & Arn.) determined a chromosomal number of 2n = 10, with a karyotypic formula of one subtelocentric pair (st) and four submetacentric pairs (sm). The intrachromosomal asymmetry index (A1) and interchromosomal asymmetry index (A2) showed a moderate tendency towards telocentricity and low variability in the sizes of chromosome pairs.
Keywords: endemic shrub, karyotype, karyotypic morphology.
RESUMEN
La caracterización de la morfología cromosómica de Haplopappus foliosus (Hook. & Arn.) determinó un número cromosómico de 2n = 10, con una fórmula cariotípica de un par subtelocéntrico (st) y cuatro pares submetacéntricos (sm). Los índices de asimetría intracromosómica (A1) e intercromosómica (A2) mostraron una moderada tendencia a la telocentría y baja variabilidad en los tamaños de los pares cromosómicos.
Palabras claves: arbusto endémico, cariotipo, morfología cariotípica.
The taxonomic history of the genus Haplopappus Cass (Asteraceae) has been complex, beginning with Hall (1928), who described 149 species for the genus, evenly divided between North and South America. Subsequently, interpretations of generic limits have focused almost exclusively on the North American elements of the genus. Some authors support Hall's treatment (Jackson 1966, Moran 1969, Grau 1976), while others propose that at least the North American sections be treated as separate genera or combined With existing genera (Anderson et al. 1974, Clark et al. 1980). Thus, over the last eight decades, there has been almost complete disintegration of the genus, and all taxa recognized by Hall (1928) have been accommodated within previously named genera, leading to a formal redescription of the genus (Lane & Hartman 1996). Currently, 70 species are recognized, distributed exclusively in South America (Klingenberg 2007).
The main controversies in the taxonomic history of the genus stem from the existence of hybrids, classifications primarily made based on morphological characters, which do not always allow for accurate differentiation between species (Lane & Hartman 1996). Therefore, the incorporation of information from new characters is a significant contribution to the resolution of taxonomic problems (Siljak-Yakovlev & Peruzzi 2012). The inclusion of cytogenetic characters in such cases is of great assistance, as it allows discrimination between parentals and hybrids and is based on traits that are less affected by the environment. One species of the genus Haplopappus is Haplopappus foliosus (Hook. & Arn. 1983), which is an endemic shrub of Chile, distributed along the coastal zone from the Atacama region to the Maule region (Hoffmann 1998, Rodriguez et al. 2018). Despite being a common species in Chile, cytogenetic data is scarce. At the chromosomal level, only a haploid value of n = 5 has been determined (Brown & Clark 1981, Frias 2005) without a detailed description of its karyotype. Therefore, the aim of this study is to characterize the karyotypic morphology of H. foliosus.
Seeds of H. foliosus were collected during the fruiting stage (March/April) from 30 randomly chosen individuals belonging to the population located in the BioParque Puquén (32°14'16"S - 71°31'16"W), in Los Molles, Valparaíso region, Chile. The reference material is deposited in the herbarium of the Facultad de Ciencias Forestales y Conservacion de la Naturaleza, Universidad de Chile.
For obtaining mitotic plates, the seeds were germinated in the laboratory in Petri dishes. The apical root zone of approximately 5-8 mm in length was cut from 60 specimens and pre-fixed with a 0.002 М 8-Hydroxyquinoline solution for five hours at room temperature. Subsequently, the material was washed twice with distilled water. The material was then fixed in an Ethanol-acetic acid solution (3:1 v/v) at room temperature for 20 hours. The material was washed again twice, followed by acid hydrolysis with 5 N HCI for 10 minutes at room temperature. It was then washed twice with distilled water, and finally, the root apex was stained with 2% orcein lactic-acetic to proceed with the squashing of the root meristems. Images of the mitotic plates were captured using an OLYMPUS BX61 video microscope connected to a MS DOS computer (in the Laboratorio de Neurobiología y Biología del Conocer, Universidad de Chile), using the Spot diagnostic instruments program (version 4.5). Fifteen metaphase plates were measured using the MicroMeasure 3.3 program (Reeves 2001). The images of the mitotic plates were later edited to generate the karyotype using Photoshop. Based on the position of the centromere, the length of the long and short arms of each chromosomal pair was determined. The measurements were expressed as a percentage of the total length of the haploid chromosomal set. With these values, the karyotypic morphology was obtained, and karyotypes were prepared according to the chromosomal categories proposed by Levan et al. (1964), visualized in a karyo-ideogram, with the aim of graphically representing the size and morphology of each chromosomal pair. Additionally, intrachromosomal and interchromosomal asymmetry was determined using two numerical parameters, A1 and A2, proposed by Romero-Zarco (1986). The intrachromosomal asymmetry index varies from О to 1 and tends to zero when the chromosomes are metacentric (Cheenaveralah 1973). The interchromosomal asymmetry index measures the variation among chromosomes within the karyotype of a species. When the sizes of the chromosomes within a species are more or less uniform or show little variability, the interchromosomal asymmetry tends to exhibit very low values (Romero-Zarco 1986). Additionally, the total relative chromosome length (LR%) was calculated.
Haplopappus foliosus exhibits a karyotype of 2n = 10 with a haploid karyotypic formula of 4sm + 1st, meaning the first pair is subtelocentric and the remaining four pairs are submetacentric (Figs. 1a-1c, Table 1). The values of the intrachromosomal asymmetry index (A1) are 0.63 + 0.04, indicating a moderate tendency towards telocentricity, and the interchromosomal asymmetry index (A2) is 0.17 + 0.04, revealing uniformity or little variability in the sizes of chromosomal pairs relative to each other (Table 1). The LR% of the species is 20.00 + 3.33 (Table 1).
The diploid chromosome number of 2n = 10 obtained in H. foliosus confirms the chromosomal number previously described by Brown & Clark (1981) and Frías (2005), which is equivalent to that documented for 32 species of the genus in South America (Brown & Clark 1981). Therefore, it is inferred that the ancestral basic number is n = 5, and it is conserved in South America despite the distinctive morphological characteristics of the species. This situation contrasts with what is found in North American species, where the chromosome number is characterized by high heterogeneity (n= 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, and 45), which correlates with a wide spectrum of morphological diversity observed among the species (Brown & Clark 1981).
Although Grau (1976) conducted a karyogram of the species, this is the first study to present the karyotypic morphology of H. foliosus, highlighting the structural characteristics of the chromosomes (Table 1). Furthermore, Grau (1976) reported a chromosomal pair with a satellite in position one. These karyotypic differences in the first chromosomal pair may be attributed to measurement challenges of the satellites or genuine differences between the analyzed populations. Therefore, it is essential to conduct studies on different populations to assess the intraspecific genomic variability that may be occurring at the chromosomal level. On the other hand, the results show similarity in karyotypic morphology for many species within the genus, where taxa exhibit at least four pairs of submetacentric chromosomes. However, this similarity cannot be directly correlated with morphological differences within the genus. Grau (1976) cytologically investigated eight species of the genus in Chile and detected a chromosomal number of 2n = 10. The karyotype of these species was very similar, consisting of a large submetacentric chromosome pair (chromosome pair with satellite) and four smaller submetacentric pairs (Grau 1981). More recent studies, based on species of Haplopappus (H. grindelioides, H. glutinosus, H. macrocephalus, H. stolpii, and H. donianus), have also found a chromosomal number of 2n = 10 (Baeza et al. 2007, Baeza et al. 2016). Other studies have exclusively recorded pairs of submetacentric chromosomes, with satellites in pair 3 for some species and in pair 2 for others (Baeza & Schrader 2005). The continuity in chromosomal similarity among species within the genus is likely related to the group's evolutionary history and the fact that distinguishable chromosomal pairs have originated through rearrangements in shape, size, and specificity within this genus.
The chromosomal asymmetries reported for the species under study (Table 1) align with Romero-Zarco's (1986) assertion regarding Al, as when the indices deviate from zero, chromosomes tend towards telocentricity. The values of A2 revealed little variability in chromosomal sizes within the species. This value is consistent with previous studies on species such as H. glutinosus (A2 = 0.19), H. grindelioides (A2 = 0.15), and H. stolpii (A2 = 0.16), which also exhibit low values (Baeza & Schrader 2005). Therefore, it appears that the karyotypes of this genus are rather symmetrical.
The cytogenetic evidence presented, based on quantitative analysis of karyotypic morphology, suggests that H. foliosus shares the same chromosomal number (2n=10) and considerable similarity in chromosomal morphology with species within the genus Haplopappus that have been studied. Additionally, for an adequate assessment of biodiversity in particular regions and to understand the current structure and dynamics of vegetation, the analysis of chromosomal data is of paramount importance. This is because the number of chromosomes and homology largely determine mating behavior and fertility, and thus reproductive behavior and patterns of variation (Stace 2000), the understanding of which is crucial for achieving a classification that reflects the process of evolution.
stages of the research process. Additionally, we extend our gratitude to Carolina Allendes-Mu oz for the preparation of figures and the support from Dr. Cristian Villagra Gil's FONDECYT Initiation Project No. 11100109. Paola JaraArancio thanks to ANID PIA/BASAL Grant PFB210018 to the International Cape Horn Center (CHIC); ANID PIA/BASAL Grant FB210006 to the Institute of Ecology and Biodiversity (IEB) and FONDECYT project 1230717. Marcelo MirandaCavallieri thanks for the support of the "Beca Doctorado Nacional" ANID 21212225. We thank the anonymous reviewer for their valuable comments, which significantly improved the first version of the manuscript.
ACKNOWLEDGEMENTS
We would like to thank the Laboratorio del Instituto de Entomología - UMCE, the Laboratorio de Sistemática y Ecología Vegetal de la Universidad de Chile, and the Laboratorio de Neurobiología y Biología del Conocer - Universidad de Chile for their support during the different
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