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Chromosome diversity in species of the genus Arachis, revealed by FISH and CMA/DAPI banding, and inferences about their karyotype differentiation

Abstract

The species of the genus Arachis (Leguminosae) are ordered into nine sections. The assignment of genome types in this genus has been based on cross-compatibility analysis and molecular cytogenetic studies. The latter has also allowed karyotypically establishing well-defined genomes and reassigning the genome of several species. However, most of these studies have been focused mainly on the sections Arachis and Rhizomatosae. To increase the knowledge about the chromosome diversity of the whole genus, here we performed a detailed karyotype characterization of representative species of most of the sections and genomes of Arachis. This characterization included chromosome morphology, CMA/DAPI chromosome banding, and chromosome marker localization (rDNAloci and one satDNA sequence) by fluorescent in situ hybridization (FISH). Based on the data obtained and other previously published data, we established the karyotype similarities by cluster analysis and defined eleven karyotype groups. The grouping was partly coincident with the traditional genome assignment, except for some groups and some individual species. Karyotype similarities among some genomes were also found. The main characteristics of each karyotype group of Arachis were summarized. Together, our results provide information that may be beneficial for future cytogenetic and evolutionary studies, and also contribute to the identification of interspecific hybrids.

Key words
Heterochromatin patterns; karyotype groups; molecular cytogenetic; rDNAloci; satDNA

INTRODUCTION

Arachis (Leguminosae) is a South American genus that comprises 82 species divided into nine taxonomic sections (Arachis, Caulorrhizae, Erectoides, Extranervosae, Heteranthae, Procumbentes, Rhizomatosae, Trierectoides and Triseminatae), established according to morphological characteristics, geographic distribution, and cross-compatibility (Krapovickas & Gregory 1994KRAPOVICKAS A & GREGORY WC. 1994. Taxonomía del género Arachis (Leguminosae). Bonplandia 8: 1-186., Valls & Simpson 2005VALLS JFM & SIMPSON CE. 2005. New species of Arachis (Leguminosae) from Brazil, Paraguay and Bolivia. Bonplandia 14: 35-64., 2017VALLS JFM & SIMPSON C. 2017. Una nueva especie de Arachis (Fabaceae) de Mato Grosso, Brasil, afín a A. matiensis. Bonplandia 26(2): 143-149., Valls et al. 2013VALLS JFM, COSTA LC & CUSTODIO AR. 2013. A novel trifoliolate species of Arachis (Fabaceae) and further comments on the taxonomic section Trierectoides. Bonplandia 22: 91-97., Santana & Valls 2015SANTANA SH & VALLS JFM. 2015. Arachis veigae (Fabaceae), la especie silvestre del género más dispersa, sin embargo pasada por alto taxonómicamente. Bonplandia 24(2): 139-150.). This genus includes important agronomic species, such as the cultivated peanut (A. hypogaea L., section Arachis), which is cultivated as an oilseed crop and as a direct source of human food, and the forage species A. pintoi Krapov. & WC Gregory (section Caulorrhizae) and A. glabrata Benth (section Rhizomatosae). Thus, carrying out studies to improve the knowledge of the germ plasm of this genus is important both taxonomically and agronomically.

Most Arachis species are diploid with x=10, only four are diploid with x=9, and five are tetraploid with x=10. Initially, classical cytogenetic studies showed high chromosome homology among the species of the genus (Fernández & Krapovickas 1994FERNÁNDEZ A & KRAPOVICKAS A. 1994. Cromosomas y evolución en Arachis (Leguminosae). Bonplandia 8: 187-220., Lavia 1996LAVIA GI. 1996. Estudios cromosómicos en Arachis (Leguminosae). Bonplandia 9: 111-120., 2001, Lavia et al. 2009LAVIA GI, ORTIZ AM AND FERNÁNDEZ A. 2009. Karyotypic studies in wild germplasm of Arachis (Leguminosae). Genet Resour Crop Evol 56: 755-764.). Later, some molecular cytogenetic studies revealed greater karyotype variability (Seijo et al. 2004SEIJO JG, LAVIA GI, FERNÁNDEZ A, KRAPOVICKAS A, DUCASSE D & MOSCONE EA. 2004. Physical mapping of 5 S and 18–25S Rrna genes evidences that Arachis duranensis and A. ipaënsis are the wild diploid species involved in the origin of A. hypogaea (Leguminosae). Am J Bot 91: 1294-1303., Robledo et al. 2009ROBLEDO G, LAVIA GI & SEIJO G. 2009. Species relations among wild Arachis species with the A genome as revealed by FISH mapping of rDNA loci and heterochromatin detection. Theor Appl Genet 118: 1295-1307., Robledo & Seijo 2008ROBLEDO G & SEIJO G. 2008. Characterization of the Arachis (Leguminosae) D genome using fluorescence in situ hybridization (FISH) chromosome markers and total genome DNA hybridization. Genet Mol Biol 31: 717-724., 2010, Silvestri et al. 2015SILVESTRI MC, ORTIZ AM & LAVIA GI. 2015. rDNA loci and heterochromatin positions support a distinct genome type for ‘x = 9 species’ of section Arachis (Arachis, Leguminosae). Plant Syst Evol 301: 555-562., Ortiz et al. 2017ORTIZ AM, ROBLEDO GA, SEIJO JG, VALLS JFM & LAVIA GI. 2017. Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section Rhizomatosae and related diploid species (Arachis, Leguminosae). J Plant Res 130: 791-807.), but most of them were focused only on the species of the sections Arachis and Rhizomatosae. These karyotype characterizations, which were based on fluorescent in situ hybridization (FISH) of ribosomal genes and C-DAPI banding, together with previous crossing data, allowed establishing six different genomes for the species of the section Arachis and to define the R genome for the only diploid species of the section Rhizomatosae. In contrast, due to the lack of detailed karyotype studies on the remaining Arachis species, their genomes are still assigned based on the subgeneric divisions and interspecific crosses, as follows: Am (Heteranthae), C (Caulorrhizae), E (Trierectoides, Erectoides and Procumbentes), Ex (Extranervosae), T (Triseminatae) and R (Rhizomatosae) (Smartt & Stalker 1982SMARTT J & STALKER HT. 1982. Speciation and cytogenetics in Arachis. In: Pattee HE and Young CT (Eds). Peanut science and technology, Yoakun: American Peanut Research Education Society, p. 21-49.). Pucciariello et al. (2013)PUCCIARIELLO O, ORTIZ AM, FERNÁNDEZ A & LAVIA GI. 2013. Análisis cromosómico del híbrido Arachis pintoi x A. repens (Leguminosae) mediante citogenética clásica y molecular. Bonplandia 48(1): 111-119. established the number of ribosomal loci for the species with C genome, but did not determine the CMA/DAPI heterochromatin content and did not perform the physical mapping of the ribosomal loci. More recently, Ortiz et al. (2017)ORTIZ AM, ROBLEDO GA, SEIJO JG, VALLS JFM & LAVIA GI. 2017. Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section Rhizomatosae and related diploid species (Arachis, Leguminosae). J Plant Res 130: 791-807. mapped these markers in the species of the section Rhizomatosae and a few species of the sections Erectoides and Procumbentes. All this indicates that the karyotype characterization and genome assignments of the genus are still incomplete and that, hence, the relationships between them are not yet well established.

Ribosomal loci and CMA/DAPI heterochromatin bands are helpful markers in comparative cytogenetic studies to infer evolutionary relationships between plant species (Acosta et al. 2016ACOSTA MC, MOSCONE EA & COCUCCI AA. 2016. Using chromosomal data in the phylogenetic and molecular dating framework: karyotype evolution and diversification in Nierembergia (Solanaceae) influenced by historical changes in sea level. Plant Biol 18: 514-526., Chalup et al. 2015CHALUP L, SAMOLUK SS, SOLÍS NEFFA V & SEIJO G. 2015. Karyotype characterization and evolution in South American species of Lathyrus (Notolathyrus, Leguminosae) evidenced by heterochromatin and rDNA mapping. J Plant Res 128: 893-908., Do Nascimento et al. 2018DO NASCIMENTO EFMB, DOS SANTOS BV, MARQUES LOC, GUIMARÃES PM, BRASILEIRO ACM, LEAL-BERTIOLI SCM, BERTIOLI D & ARAUJO ACG. 2018. The genome structure of Arachis hypogaea (Linnaeus, 1753) and an induced Arachis allotetraploid revealed by molecular cytogenetics. Comp Cytogenet 12(1): 111-140., Yung et al. 2017YUNG IL, CHUNG MC, KUO H, WANG CN, CHING YL, LIN CY, JIANG H & YEH CH. 2017. The evolution of genome size and distinct distribution patterns of rDNA in Phalaenopsis (Orchidaceae). Bot J Linn Soc 185: 65-80.). However, to perform more detailed karyotypes and so establish more precise chromosome homeologies within a group, it is sometimes necessary to increase the number of chromosomal markers. Satellite DNA (satDNA), which consists of repetitive units of variable length arranged in tandems of up to 100 Mbp, constitutes a significant part of plant nuclear genomes (Charlesworth et al. 1994CHARLESWORTH B, SNIEGOWSKI P & STEPHAN W. 1994. The evolutionary dynamics of repetitive DNA in eukaryotes. Nature 371: 215-220., Schmidt & Heslop-Harrison 1998SCHMIDT T & HESLOP-HARRISON JS. 1998. Genomes, genes and junk: the large-scale organization of plant chromosomes. Trends Plant Sci 3: 195-199.). SatDNA usually show particular chromosomal locations, being an important component of centromeric heterochromatin (Hudakova et al. 2001HUDAKOVA S, MICHALEK W, PRESTING GG, HOOPEN R, DOS SANTOS K, JASENCAKOVA Z & SCHUBERT I. 2001. Sequence organization of barley centromeres. Nucleic Acids Res 29(24): 5029-5035., Urdampilleta et al. 2009URDAMPILLETA JD, DE SOUZA AP, SCHNEIDER DR, VANZELA AL, FERRUCCI MS & MARTINS ER. 2009. Molecular and cytogenetic characterization of an AT-rich satellite DNA family in Urvillea chacoensis Hunz. (Paullinieae, Sapindaceae). Genetica 136: 171-177., Samoluk et al. 2016SAMOLUK SS, ROBLEDO G, BERTIOLI DJ & SEIJO JG. 2016. Evolutionary dynamics of an atrich satellite DNA and its contribution to karyotype differentiation in wild diploid Arachis species. Mol Genet Genomics 292(2): 283-296.) and telomeric heterochromatin (Pich et al. 1996PICH U, FRITSCH R & SCHUBERT I. 1996. Closely related Allium species (Alliaceae) share a very similar satellite sequence. Plant Syst Evol 202: 255-264., Macas et al. 2000MACAS J, POZÁRKOVÁ D, NAVRÁTILOVÁ A, NOUZOVÁ M & NEUMANN P. 2000. Two new families of tándem repeats isolated from genus Vicia using genomic self-priming PCR. Mol Gen Genet 263: 741-751.) and, less frequently, of interstitial heterochromatin (Mukai et al. 1993MUKAI Y, NAKAHARA Y & YAMAMOTA M. 1993. Simultaneous discrimination of the three genomes in hexaploid wheat by multicolor fluorescence in situ hybridization using total genomic and highly repeated DNA probes. Genome 36: 489-494.). Thus, the use of satDNA sequences as chromosome markers has become a useful tool to study major karyotype changes between closely related species and to make inferences about the evolution of genomes (Pich et al. 1996PICH U, FRITSCH R & SCHUBERT I. 1996. Closely related Allium species (Alliaceae) share a very similar satellite sequence. Plant Syst Evol 202: 255-264., Ugarkovic & Plohl 2002UGARKOVIC D & PLOHL M. 2002. Variation in satellite DNA profiles causes and effects. EMBO J 2: 5955-5959., Urdampilleta et al. 2009URDAMPILLETA JD, DE SOUZA AP, SCHNEIDER DR, VANZELA AL, FERRUCCI MS & MARTINS ER. 2009. Molecular and cytogenetic characterization of an AT-rich satellite DNA family in Urvillea chacoensis Hunz. (Paullinieae, Sapindaceae). Genetica 136: 171-177.). Recently, Zhang et al. (2012)ZHANG L, XU C & YU W. 2012. Cloning and Characterization of Chromosomal Markers from a Cot-1 Library of Peanut (Arachis hypogaea L.). Cytogenet Genome Res 137: 31-41. isolated several satDNA sequences from the cot-1 fraction of A. hypogaea, which could be potentially used as probes to chromosome markers in Arachis. The analysis of the distribution of one of them, particularly the one called clone 119 (NCBI: JQ673497), has shown a markedly different distribution between the two genomes of the allotetraploid A. hypogaea (Zhang et al. 2012ZHANG L, XU C & YU W. 2012. Cloning and Characterization of Chromosomal Markers from a Cot-1 Library of Peanut (Arachis hypogaea L.). Cytogenet Genome Res 137: 31-41., 2017ZHANG L, YANG X, TIAN L, CHEN L & YU W. 2017. Identification of peanut (Arachis hypogaea) chromosomes using a fluorescence in situ hybridization system reveals multiple hybridization events during tetraploid peanut formation. New Phytol 211(4): 1424-1439.). Thus, the analysis of the distribution of this satDNA in other species of Arachis would provide additional information about the karyotype variability within the genus, the karyotype characterization of genome types, and the evolutionary relationships among the genomes.

Based on all the above and to increase the genomic knowledge of genus Arachis, in the present study, we aimed to improve the karyotype characterization of the species belonging to the different sections of the genus Arachis, through the mapping of ribosomal genes by FISH in eight species, the CMA/DAPI banding in fourteen species, and the mapping of the satDNA clone 119 by FISH in fifteen species. The data obtained and previous data were jointly analyzed to extend the karyotype characterization to most of the species of the genus, to establish chromosome homeologies among them, to determine the karyotype relationships within the genus and to comprehensively relate these relationships to the current genomic assignments.

MATERIALS AND METHODS

Plant material

Seeds and rhizomes of the Arachis species used in this study were obtained from the peanut germplasm collections of the Instituto Nacional de Tecnología Agropecuaria-Manfredi (Córdoba, Argentina), the Instituto de Botánica del Nordeste (Corrientes, Argentina) and the Centro Nacional de Recursos Genéticos e Biotecnologia (Embrapa Cenargen, Brasília, Brazil). The provenances and voucher specimens of the samples studied are cited in Table I.

Table I
List of the Arachis species karyotypically characterized, collection number and provenance and chromosome marker analyzed.

CMA - DAPI staining

Double staining with the fluorochromes chromomycin A3 (CMA, Sigma Aldrich) and diamino-2-phenyl-indole (DAPI, Sigma Aldrich) was performed to reveal GC-rich and AT-rich heterochromatic regions respectively, according to Schweizer (1976)SCHWEIZER D. 1976. Reverse fluorescent chromosome banding with Chromomycin and DAPI. Chromosoma 58: 307-324. with minor modifications. The aged slides were double-stained at 37 °C with 0.5 mg/mL of CMA for 90 min, and subsequently with 2 μg/μL of DAPI for 30 min. After the staining of each fluorochrome, the slides were washed with distilled water. Finally, the slides were mounted with Vectashield medium (Vector Laboratories).

Probe labeling and fluorescent in situ hybridization (FISH)

The 5S and 45S rDNA loci were localized using the probes pA5S and pA18S-pA26S, respectively, isolated from genomic DNA of A. hypogaea (Robledo & Seijo 2008ROBLEDO G & SEIJO G. 2008. Characterization of the Arachis (Leguminosae) D genome using fluorescence in situ hybridization (FISH) chromosome markers and total genome DNA hybridization. Genet Mol Biol 31: 717-724.), and then labeled by PCR or nick translation technique with digoxigenin-11-dUTP (Roche Diagnostics) or biotin-11-dUTP (Sigma-Aldrich). The satDNA sequence clone 119 was isolated by PCR from genomic DNA of A. duranensis K7988 by using primers designed by Zhang et al. (2012)ZHANG L, XU C & YU W. 2012. Cloning and Characterization of Chromosomal Markers from a Cot-1 Library of Peanut (Arachis hypogaea L.). Cytogenet Genome Res 137: 31-41., and labeled by PCR with biotin-11-dUTP (Sigma-Aldrich). The in situ hybridizations of clone 119 were combined with the probe pA5S with the objective of using this last chromosome marker as reference for mapping the clone 119 loci in relation to previously published positions of the 5S and 45S rDNA loci.

Pretreatment of slides and in situ hybridization were performed according to Seijo et al. (2004)SEIJO JG, LAVIA GI, FERNÁNDEZ A, KRAPOVICKAS A, DUCASSE D & MOSCONE EA. 2004. Physical mapping of 5 S and 18–25S Rrna genes evidences that Arachis duranensis and A. ipaënsis are the wild diploid species involved in the origin of A. hypogaea (Leguminosae). Am J Bot 91: 1294-1303.. The first set of antibodies consisted of anti-biotin produced in goat (Sigma-Aldrich) and monoclonal anti-digoxigenin conjugated to fluorescein isothiocyanate (FITC) produced in mouse (Sigma-Aldrich). The second set consisted of anti-goat conjugated to tetramethyl-rhodamineisothiocyanate (TRITC) produced in rabbit (Sigma-Aldrich) and anti-mouse conjugated to FITC produced in sheep (Sigma-Aldrich). Preparations were counterstained by mounting them with Vectashield medium (Vector Laboratories) containing 2 mg/mL of DAPI. Counterstaining with DAPI reveals a C-banding-like pattern with major heterochromatic bands fluorescing more intensely in Arachis species (Seijo et al. 2004SEIJO JG, LAVIA GI, FERNÁNDEZ A, KRAPOVICKAS A, DUCASSE D & MOSCONE EA. 2004. Physical mapping of 5 S and 18–25S Rrna genes evidences that Arachis duranensis and A. ipaënsis are the wild diploid species involved in the origin of A. hypogaea (Leguminosae). Am J Bot 91: 1294-1303.).

Fluorescence microscopy and image acquisition

Chromosomes were viewed with a Leica DMRX fluorescence microscope (Leica) and digitally photographed with a computer-assisted Leica DC350 digital camera system. Red, green, and blue images were captured in black and white by using the respective filters for TRITC, FITC, CMA and DAPI excitations. Digital images were processed with Photoshop, version 7.0 (Adobe, San Jose, CA, USA).

Karyotype morphometry

For karyotype determination, we used three to six individuals per species and four metaphase plates per individual. Chromosome measurements were made using the MICROMEASURE software version 3.3 (Reeves & Tear 2000REEVES A & TEAR J. 2000. Micro Measure for Windows, version 3.3. Free program distributed by the authors over the Internet from http://www.colostate.edu/Depts/Biology/MicroMeasure.
http://www.colostate.edu/Depts/Biology/M...
). Karyotype description is based on the nomenclature by Levan et al. (1964)LEVAN A, FREDGA K & SANDBERG AA. 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52: 201-220.. Chromosomes were classified in categories according to the centromeric index (CI = short arm x 100/total length of the chromosome): metacentric (m) when CI = 50–37.5, submetacentric (sm) when CI = 37.5–25, and subtelocentric (st) when CI = 25-12.5. SAT chromosomes were classified on the basis of the satellite relative size and position of the centromere (Fernández & Krapovickas 1994FERNÁNDEZ A & KRAPOVICKAS A. 1994. Cromosomas y evolución en Arachis (Leguminosae). Bonplandia 8: 187-220.). Data from homologous chromosomes were combined to mean values, first between chromosomes in the same metaphase and subsequently between chromosomes in different metaphases of the same species, and then the total karyotype length was obtained by summing the average length of each chromosome pair. The mean chromosome length was calculated by dividing the karyotype length by the haploid number of chromosomes of the species. Chromosome bands and rDNA loci were mapped using the index di = dx 100/a, where d = distance of loci center from the centromere and a = length of the corresponding chromosome arm, according to Greilhuber & Speta (1976)GREILHUBER RJ & SPETA F. 1976. C-banded karyotypes in the Scilla hohenackeri group, S. persica and Puschkinia (Liliaceae). Plant Syst Evol 126: 149-188.. The karyotype asymmetry indices were estimated using the intrachromosomal (A1) and interchromosomal (A2) indexes by Romero Zarco (1986)ROMERO ZARCO C. 1986. A new method for estimating karyotype asymmetry. Taxon 35: 526-530.. Mean values for each species were represented as haploid complements in the ideograms. Chromosomes were ordered first by morphology and then by decreasing size. Some chromosomes within each ideogram were re-ordered according to tentative homeologies on the basis of the current nomenclature proposed for other Arachis species (Seijo et al. 2004SEIJO JG, LAVIA GI, FERNÁNDEZ A, KRAPOVICKAS A, DUCASSE D & MOSCONE EA. 2004. Physical mapping of 5 S and 18–25S Rrna genes evidences that Arachis duranensis and A. ipaënsis are the wild diploid species involved in the origin of A. hypogaea (Leguminosae). Am J Bot 91: 1294-1303., Robledo et al. 2009ROBLEDO G, LAVIA GI & SEIJO G. 2009. Species relations among wild Arachis species with the A genome as revealed by FISH mapping of rDNA loci and heterochromatin detection. Theor Appl Genet 118: 1295-1307., Robledo & Seijo 2010ROBLEDO G & SEIJO G. 2010. Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of Rdna loci and heterochromatin detection: a new proposal for genome arrangement. Theor Appl Genet 121: 1033-1046., Ortiz et al. 2017ORTIZ AM, ROBLEDO GA, SEIJO JG, VALLS JFM & LAVIA GI. 2017. Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section Rhizomatosae and related diploid species (Arachis, Leguminosae). J Plant Res 130: 791-807.).

Clustering analysis

To establish species groups sharing greater karyotype similarities, we made a cluster analysis from a matrix of 15 different cytogenetic characters for 46 species of the genus Arachis. The matrix was constructed on the basis of previous cytogenetic data and the data here published, coded either as binary or multistate in the case of qualitative chromosomal characteristics or as continuous characters in the case of quantitative variables (Supplementary Material – Table SI). The chromosome characteristics considered were: basic number (x), total karyotype length by chromosome set, chromosome mean length, intrachromosomal asymmetry index (A1), interchromosomal asymmetry index (A2), total percentage of DAPI heterochromatin, presence/absence of centromeric DAPI bands, type of centromeric DAPI bands (conspicuous, tiny, or absent), percentage of chromosomes with centromeric DAPI bands, presence/absence of distal DAPI bands, presence/absence of centromeric CMA bands, percentage of chromosomes carrying the 5S loci, position of the 5S rDNA loci on pair #3 (long arm vs. short arm), presence/absence of A chromosomes, and co-localization of 5S and 45S rDNA loci on pair #10. The number of 45S rDNA loci was not included in the analysis due to its high variability among species of the same section, and/or inclusive to the same genome. The INFOSTAT software version 2015 (Di Rienzo et al. 2015DI RIENZO JA, CASANOVES F, BALZARINI MG, GONZÁLEZ L, TABLADA M & ROBLEDO CW. 2015. Info Statversion 2015. Grupo Info Stat, FCA, Universidad Nacional de Córdoba. Argentina. http://www.infostat.com.ar
http://www.infostat.com.ar...
) was used to standardize the matrix, to calculate the average distance, and to generate a phenogram. The distances between the species were estimated by applying the coefficient of dissimilarity of Gower, and clustering was performed using the unweighted pair-group method (UPGMA). Phenogram distortion was measured by computing the cophenetic correlation coefficient (r).

RESULTS

Karyotype morphometry

The eight species analyzed had symmetrical karyotypes mainly composed of metacentric chromosomes with lengths ranging from 1.37 to 4.07 µm (Table II). All species had a haploid karyotype formula of 9m + 1sm, except A. triseminata (section Triseminatae) with 10m. The haploid karyotype length varied between 17.51 µm in A. triseminata and 34.82 µm in A. matiensis (section Procumbentes), whereas the mean chromosome length varied between 1.91 µm in A. dardani (section Heteranthae) and 3.63 µm in A. matiensis. The lowest centromeric index was 41.70 and corresponded to A. repens (section Caulorrhizae), whereas the highest was 44.00 and corresponded to A. triseminata. The indexes of intrachromosomal asymmetry (A1) ranged from 0.15 in A. benthamii (section Erectoides) to 0.27 in A. repens, while the interchromosomal asymmetry (A2) ranged from 0.09 in A. vallsii (section Procumbentes) to 0.20 in A. pusilla (section Heteranthae). All species showed only one pair of secondary constrictions located on pair #10. In A. benthamii and A. matiensis, the secondary constrictions were in the short arms, while in the rest they were in the long arms. Generally, the constrictions were extended at early metaphase, and the satellites remained far from the corresponding proximal segments of the chromosome arms (Figures 1 and 2).

Figure 1
Somatic metaphases of Arachis species belonging to different sections after double fluorescent in situ hybridization (FISH) with ribosomal markers. The 5S rDNAloci are illustrated by the green signals and the 18S–26S rDNAloci by the red signals. DAPI counterstaining in gray is highlighting the heterochromatin bands. The arrows illustrate the homologous position of rDNA signals. a- A. triseminata (Triseminatae); b- A. dardani (Heteranthae); c- A. pusillaVFaPzSv 13107 (Heteranthae); d- A. pusilla V13189 (Heteranthae); e- A. pintoi (Caulorrhizae); f- A. repens (Caulorrhizae); g- A. benthamii (Erectoides); h- A. matiensis (Procumbentes); i- A. vallsii (Procumbentes). Scale bar 5 μm.
Figure 2
Idiograms of Arachis species, showing the distribution of 5S (striped) and 18S-26S rDNAloci (black shaded), DAPI heterochromatic bands (white shaded), and CMA heterochromatic bands (point bands). The chromosomes are ordered by morphology according to decreasing size, but some chromosomes were re-arranged according to tentative homologies between the karyotypes. *band observed in only one homologous chromosome. m= metacentric, sm= submetacentric. Scale bar 2 μm.
Table II
Karyotype features of species belonging to different sections of Arachis.

CMA and DAPI heterochromatin distribution

The analysis of CMA+/DAPI- (CMA bands) and DAPI+/CMA- (DAPI bands) heterochromatin distribution in the karyotypes of fourteen

species of Arachis revealed a great heterogeneity, but some peculiarities were common among some of them.

The karyotypes of A. batizocoi, A. duranensis, A. praecox, A. trinitensis (section Arachis), A. benthamii (section Erectoides), A. matiensis and A. vallsii (section Procumbentes) were characterized by the presence of centromeric DAPI bands in 7 to 10 chromosome pairs and only one pair of CMA bands (Figures 2 and 3). In all these species, the CMA bands were associated with the secondary constrictions. In the first three species of the section Arachis (i.e. A. batizocoi, A. duranensis, A. praecox) and in A. vallsii, DAPI heterochromatin was displayed as conspicuous bands, whereas in A. trinitensis, A. benthamii and A. matiensis, it appeared as tiny bands. Additionally, A. matiensis had distal DAPI bands on the short arms of pair #2 and in only one chromosome of pair #7, while A. praecox had interstitial bands on the long arms of pair #3. The total amount of CMA heterochromatin per haploid complement varied from 1.98% of the total karyotype length in A. batizocoi to 3.05% in A. trinitensis, while the total amount of DAPI heterochromatin varied from 7.68% in A. trinitensis to 21.41% in A. praecox (Table III).

Figure 3
Somatic metaphases of Arachis species belonging to different sections after CMA/DAPI banding technique. The yellow signals correspond to CMA heterochromatin bands and the white signals to DAPI heterochromatin bands. a- A. triseminata (Triseminatae); b- A. dardani (Heteranthae); c- A. pusilla V13189 (Heteranthae); d- A. pusillaVFaPzSv 13107 (Heteranthae); e- A. pintoi (Caulorrhizae); f- A. repens (Caulorrhizae); g- A. benthamii (Erectoides); h- A. duranensis (Arachis, A genome); i- A. vallsii (Procumbentes); j- A. matiensis (Procumbentes); k- A. praecox (Arachis, G genome); l- A. batozocoi (Arachis, K genome); m- A. trinitensis (Arachis, F genome); n- A. ipaënsis (Arachis, B genome); o- A. glandulifera (Arachis, D genome). The yellow arrows point some weak CMA bands. Scale bar 5 μm.
Table III
CMA/ DAPI banding features of species belonging different sections of Arachis.

Similar to the preceding species, A. pintoi and A. repens (section Caulorrhizae) had one pair of CMA bands associated with the secondary constrictions (Figures 2 and 3). In contrast, they had tiny centromeric DAPI bands in four chromosome pairs. In A. pintoi, the total amount of CMA and DAPI heterochromatin was 2.25% and 2.78% of the total karyotype length respectively, whereas in A. repens it was 2.27% and 2.93%, respectively (Table III).

Arachis ipaënsis (section Arachis), A. dardani (section Heteranthae) and A. triseminata (section Triseminatae) had karyotypes completely devoid of DAPI heterochromatin and one to three pairs of CMA bands (Figure 3 and Table III). In the three species, one of the pairs of CMA bands was associated with the secondary constrictions, while the others were centromeric and were only detected in one chromosome pair for A. dardani and in two for A. triseminata. The total amount of CMA heterochromatin varied from 2.98% of the total karyotype length in A. ipaënsis to 9.24% in A. triseminata.

The remaining two species (i.e. A. glandulifera and A. pusilla) had particular heterochromatin distribution patterns. Arachis glandulifera (section Arachis) displayed five pairs of CMA bands and seven pairs of DAPI bands (Figure 3). Both the CMA and DAPI bands were heterogeneous in size. All CMA bands were interstitial, and one pair of them was associated with the secondary constrictions, whereas all DAPI bands were located in the centromeres, except two pairs that were interstitial. The total amount of CMA and DAPI heterochromatin was of 4.87% and 8.11% of the total karyotype length, respectively (Table III). Arachis pusilla (section Heteranthae) had centromeric CMA bands in all chromosome pairs and lacked any type of DAPI bands (Figures 2 and 3). The total amount of CMA heterochromatin was 27.62% of the total karyotype length in the accession A. pusilla 13189 and 30.77% in A. pusilla 13107, but the band size ranged from faint bands to conspicuous blocks (Table III).

Chromosomal mapping of rDNA loci

The eight species analyzed had one pair of 5S rDNA loci proximally located on the metacentric pair #3, except A. pusilla, in which it was located on the submetacentric pair #10 (Figures 1 and 2). In A. pintoi, A. repens, A. vallsii and A. triseminata, these loci were on the short arms, while in A. benthamii, A. dardani, A. pusilla and A. matiensis these loci were on the long arms. Regardless of this, all 5S rDNA loci covered from one fourth to one third of the arm length. Additionally, A. pintoi and A. vallsii had one pair of proximal locion the short arms of the metacentric pair #4. In both species, the signals of these additional loci were smaller and fainter than the loci on pair #3 (Figure 1).

Similarly, all the species had one pair of 45S rDNA loci on the long arms of pair #10, except in A. benthamii and A. matiensis, in which it was located on the short arms (Figures 1 and 2). In all species, these loci co-located with the only secondary constrictions detected and covered one third to half of the arm length. Additionally, A. pusilla and A. vallsii had one second pair of interstitial loci on the long arms of the metacentric pair #2, and A. pusilla had a third pair in proximal position on the long arms of the metacentric pair #3. In both species, the signals of these additional loci were smaller and fainter than those on pair #10 (Figure 1). In A. pusilla, one pair of 45S rDNA loci and the only pair of 5S rDNA loci detected were linked on the long arms of pair #10.

Chromosome mapping of satDNA clone 119

The localization of satDNA clone 119 in the karyotype of 15 Arachis species revealed a great variability in the presence, number and position of the hybridization sites (Figure 4) (Supplementary Material – Figure S1). However, some peculiarities were common among some of them. Three species, A. pintoi, A. pusilla and A. triseminata were characterized by having metaphases devoid of hybridization sites. Almost all the remaining species had one pair of proximal sites on the short arms of pair #3, except A. paraguariensis, A. porphyrocalyx (section Erectoides), A. lignosa, A. matiensis (section Procumbentes), A. burkartii and A. nitida (section Rhizomatosae), which had the proximal sites on the long arms (Figure S1). In the case of A. nitida, the number of sites detected was four due to their polyploidy level (4x), but all were located in the same position on chromosomes with identical morphology. In all species, this pair of satDNA clone 119 sites was linked on adjacent position to the 5S rDNA loci, except in A. glandulifera (section Arachis), in which it was linked in different arms. In A. paraguariensis, A. lignosa, A. matiensis, A. burkartiiand A. nitida, these were the only hybridization sites detected. In contrast, A. porphyrocalyx and the five species of section Arachis (A. duranensis, A. ipaënsis, A. glandulifera, A. trinitensis and A. batizocoi) had two to seven pairs of additional sites. Although there was no common pattern in the distribution of additional sites, the chromosome pairs harboring them mainly corresponded to that of pairs #5 and #9, and less frequently to that of pairs #1, #2, #6, #7 and #8 (Figure S1). All additional sites were located on the centromeres, or at least in a position proximal to them. Particularly, pairs #4 and #10 showed no hybridization sites in any of these species.

Figure 4
Somatic metaphases in representative species of different sections of Arachis after double fluorescent in situ hybridization (FISH) with clone 119 and 5S rDNA markers. The clone 119 loci are illustrated by the red signals and the 5S rDNAloci by the green signals. DAPI counterstaining in gray is highlighting the heterochromatin bands. The yellow arrows illustrate the position where both markers are linked signals in chromosome #3, whereas white arrows show when both markers are located on different positions. a- A. batizocoi (Arachis, K genome); b- A. benensis (Arachis, F genome); c- A. duranensis (Arachis, A genome); d- A. glandulifera (Arachis, D genome); e- A. ipaënsis (Arachis, B genome); f- A. praecox (Arachis, G genome); g- A. lignosa (Procumbentes) ; h. A. burkartii(Rhizomatosae); i- A. porphyrocalyx (Erectoides); j- A. nitida (Rhizomatosae); k- A. paraguariensisparaguariensis (Erectoides); l- A. matiensis (Procumbentes). Scale bar 5 μm.

Arachis praecox (2n = 18) was differentiated by having, in addition to the proximal sites in the short arms of pair # 3, a dispersed distribution pattern of satDNA clone 119 signals along six pairs of chromosomes.

Chromosome markers

The chromosome markers detected together with the chromosome morphometry were useful to identify chromosome pairs on some karyotypes. Thus, the mapping of 5S and 45S rDNA loci and DAPI/CMA banding allowed the individualization of four pairs in A. matiensis, A. vallsii and A. triseminata, three in A. dardani and A. pintoi, and two in the remaining species (Figure 2), whereas the hybridization sites of clone 119, the5S rDNA loci and DAPI banding allowed the individualization of three pairs in A. porphyrocalyx, five in A. batizocoi and all pairs of the complement in A. glandulifera (Figure S1).

The combination of chromosome markers was also helpful to evaluate putative chromosome homeologies. Thus, the tentative homeology of pair #3 harboring 5S rDNA loci was confirmed by the additional hybridization of satDNA clone 119, except in A. pintoi, A. pusilla and A. triseminata, in which satDNA clone 119 was not detected. In all species, pair #3 corresponded to metacentric chromosomes (of medium size mainly) harboring one pair of proximal satDNA clone 119 sites and one pair of 5S rDNA loci more distal on the same arms, except in A. glandulifera, where these markers are harbored by different arms of a submetacentric pair. Similarly, pairs #5 and #9 of A. duranensis, A. batizocoi, and A. porphyrocalyx are homeologous by harboring the only two additional sites of satDNA clone 119. In the three species, pair #5 corresponded to metacentric chromosomes of medium-high size, while pair #9 corresponded to the smallest metacentric chromosomes of the karyotype, except in A. batizocoi, in which it corresponded to a submetacentric pair.

Clustering analyses by karyotype similarities

In the dendrogram obtained, the taxa were grouped in two large clusters by the presence or absence of centromeric DAPI bands (Figure 5). Within these clusters, excepting for A. glandulifera and A. porphyrocalyx, all taxa were grouped in seven groups supported by values of Gower dissimilarity coefficient lower than 0.40, as described below.

Figure 5
Dendrogram representing the relationship among the karyotypes of 45 Arachis species from the sections Arachis, Caulorrhizae, Erectoides, Heteranthae, Procumbentes, Rhizomatosae and Triseminatae. Letters are the designations of genome type. Clustering was performed using unweighted pair-group linkage type, with the Gower distance method. Cophenetic correlation coefficient (r) 0.92.

Group 1 is formed by A. pusilla, A. dardani (section Heteranthae) and A. triseminata (section Triseminatae), grouped by the presence of centromeric CMA bands additional to those associated with the secondary constrictions and by having the shortest chromosomes. Arachis triseminata is less related to the rest by having the most symmetrical karyotype and by the location of 5S rDNA loci on the short arms of pair #3.

Group 2 is formed by five species of the section Arachis (A. williamsii, A. valida, A. gregoryi, A. magna and A. ipaënsis) and the only diploid species of the section Rhizomatosae (A. burkartii). These species are grouped by the absence of centromeric CMA bands additional to those associated with the secondary constrictions, and by having longer chromosomes than the previous group. Within this group, A. burkartii appears less related to the rest by the interstitial location of the 5S rDNA loci on the long arms of pair #3 and by having a higher interchromosomal asymmetry.

Group 3 is formed by A. batizocoi, A. cruziana and A. krapovickasii (section Arachis). These species are grouped by having three pairs of 5S rDNA loci, one pair of 5S and 45S rDNA loci linked on the long arms of pair #10, conspicuous centromeric DAPI bands in only nine chromosomes, and high intra-chromosomal asymmetry values and low interchromosomal asymmetry values.

Group 4 is formed by A. decora, A. palustris and A. praecox (section Arachis). These species are grouped by having a basic number x = 9, conspicuous centromeric DAPI bands in all or almost all the chromosomes, the highest DAPI heterochromatin percentages and high interchromosomal asymmetry values.

Group 5 is formed by 12 species of the section Arachis, grouped by having A chromosomes, conspicuous centromeric DAPI bands in all or almost all the chromosomes and high interchromosomal asymmetry values.

Group 6 is formed by A. vallsii (section Procumbentes), A. pintoi, A. repens (section Caulorrhizae), A. benensis and A. trinitensis (section Arachis). These species are grouped by having small to medium karyotype lengths and low interchromosomal asymmetry values. Within this group, A. vallsii appears less related by having conspicuous centromeric DAPI bands in all the chromosomes and by having higher DAPI heterochromatin percentage, whereas A. benensis and A. trinitensis appear more closely related by having tiny centromeric DAPI bands in more than half of the chromosomes, and A. pintoi and A. repens by having tiny centromeric DAPI bands in less than half of the chromosomes and interchromosomal asymmetry values higher than the previous species.

Finally, group 7 is formed by the 13 taxa from sections Erectoides, Procumbentes (except A. vallsii) and Rhizomatosae (except A. burkartii). These species are grouped by having small dot-like centromeric DAPI bands in all or almost all the chromosomes, by the location of 5S rDNA loci on the long arms of the chromosomes of pair #3, and low interchromosomal asymmetry values.

Arachis porphyrocalyx and A. glandulifera were not included in any group because they have particular chromosome characteristics.

DISCUSSION

The present work constitutes the most comprehensive karyotype analysis of the genus Arachis since the initial studies carried out by Fernández & Krapovickas (1994)FERNÁNDEZ A & KRAPOVICKAS A. 1994. Cromosomas y evolución en Arachis (Leguminosae). Bonplandia 8: 187-220.. It comprised a comparative analysis by morphology, fluorescent banding and chromosome markers revealed by FISH of the karyotypes of 45 Arachis species corresponding to almost all sections.

Karyotype morphology

Like the rest of the species of the genus, the species analyzed showed symmetric karyotypes with predominance of metacentric chromosomes. The karyotype formula of 9m + 1sm was common to almost all of them. Similarly, all karyotypes were composed of small chromosomes according to the categories proposed by Lima de Faria (1980). Our results on the morphometric analysis of metaphase chromosomes counterstained with DAPI (after FISH treatment) are the first karyotype data for A. benthamii, A. matiensis and A. triseminata, while those for A. dardani, A. pusilla, A. pintoi, A. repens and A. vallsii are in agreement in most of the details with previous reports by classical and molecular techniques (Fernández & Krapovickas 1994FERNÁNDEZ A & KRAPOVICKAS A. 1994. Cromosomas y evolución en Arachis (Leguminosae). Bonplandia 8: 187-220., Lavia 2001LAVIA GI. 2001. Chromosomal characterization of germplasm of wild species of Arachis L. belonging to sections Trierectoides, Erectoides and Procumbentes. Caryologia 54: 115-119., Silva et al. 2010SILVA SC, MARTINS MIG, SANTOS RC, PEÑALOZA APS, FILHO PAM, BENKO-ISEPPON AM, VALLS JFM & CARVALHO R. 2010. Karyological features and banding patterns in Arachis species belonging to the Heteranthae section. Plant Syst Evol 285: 201-207., Pucciariello et al. 2013PUCCIARIELLO O, ORTIZ AM, FERNÁNDEZ A & LAVIA GI. 2013. Análisis cromosómico del híbrido Arachis pintoi x A. repens (Leguminosae) mediante citogenética clásica y molecular. Bonplandia 48(1): 111-119.).

In terms of the number and size of chromosomes, and karyotype formulae, the data for the eight species analyzed support the high conservation of the karyotype structure in the genus. Similarly, the high symmetry of their karyotypes is also a conserved feature among Arachis species. According to Stebbins (1971)STEBBINS GL. 1971. Chromosomal evolution in higher plants. Addison-Wesley, London., in groups of phylogenetically related species with mainly symmetrical karyotypes, like the genus Arachis, an increase in asymmetry could be interpreted as a derived character. Coinciding with this, A. triseminata (section Triseminatae) and A. benthamii (section Erectoides), which belong to sections considered as primitive according to Krapovickas & Gregory (1994)KRAPOVICKAS A & GREGORY WC. 1994. Taxonomía del género Arachis (Leguminosae). Bonplandia 8: 1-186., showed the least asymmetric karyotypes, whereas A. pintoi and A. repens (section Caulorrhizae), which belong to one of the sections considered most derived, showed the most asymmetric karyotypes. Even in a comparative analysis of the karyotype lengths among most of the Arachis species, the small karyotypes corresponded to species of sections considered primitive and the large karyotypes corresponded to species of sections considered derived. Within this range, and coinciding with the increase in karyotype asymmetry and the evolutionary position of the sections, A. triseminata has the smallest karyotype of the genus, while A. dardani, A. pusilla, A. pintoi and A. repens, in this order, have larger karyotypes. In contrast, A. benthamii, A. matiensis and A. vallsii have karyotype lengths up to 1.5 times larger than the species with more asymmetric karyotypes (particularly A. pintoi and A. repens) and, according to Krapovickas & Gregory (1994)KRAPOVICKAS A & GREGORY WC. 1994. Taxonomía del género Arachis (Leguminosae). Bonplandia 8: 1-186., belong to a derived section. Increases in the karyotype length of this type have also been observed in other species of the sections Procumbentes and Erectoides (Ortiz et al. 2017ORTIZ AM, ROBLEDO GA, SEIJO JG, VALLS JFM & LAVIA GI. 2017. Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section Rhizomatosae and related diploid species (Arachis, Leguminosae). J Plant Res 130: 791-807.). For example, A. appressipila and A. major (sections Procumbentes and Erectoides, respectively) have less asymmetric karyotypes than A. pintoi and A. repens but 1.8 times larger. These discrepancies could be explained by the fact that the three species with large karyotypes (A. benthamii, A. matiensis and A. vallsii), as the rest of species of the sections Procumbentes and Erectoides, have DAPI bands that span up to 14.52% of the length of their karyotypes, whereas the species of the sections Triseminatae, Heteranthae and Caulorrhizae lack or have low amount (less than 2.93% of the length of karyotype) of this type of heterochromatin. Moreover, the DAPI heterochromatin in species of the sections Procumbentes and Erectoides is distributed as centromeric bands with equal size in both arms in all or almost all the chromosomes; therefore, the increases in the chromosome lengths are similar in both chromosome arms and do not affect the karyotype symmetry. So, the conservation of a karyotype structure composed mostly of metacentric chromosomes but with differences in the karyotype lengths suggests that the variations in chromosome lengths rather than the asymmetry were the main changes occurred during their differentiation. On the other hand, the discrepancies between the evolutionary position of these sections (primitive vs. derived) and the values of asymmetry and length of the karyotypes may be due to the fact that the section Erectoides is considered as basal whereas the section Caulorrhizae is considered as derived. In this sense, on the basis of morphology and crossability, Krapovickas & Gregory (1994)KRAPOVICKAS A & GREGORY WC. 1994. Taxonomía del género Arachis (Leguminosae). Bonplandia 8: 1-186. suggested that the sections Triseminatae, Trierectoides, Erectoides, Extranervosae and Heteranthae are the most primitive, while the sections Procumbentes, Caulorrhizae, Rhizomatosae and Arachis have a more recent origin. However, these authors highlighted that the species of the section Erectoides had lower degree of intersectional isolation than those of the remaining primitive sections. From a karyotype approach, the species of the section Erectoides share more chromosome features with the derived sections than with the primitive ones. The length of the chromosomes of the species of the section Erectoides is similar to that of those of the species of the sections Procumbentes, Rhizomatosae and Arachis; therefore, they have a range of karyotype length that overlaps with that observed in them. Moreover, and in relation with the increase in chromosome size, all or almost all their chromosomes have DAPI bands like those harbored by the chromosomes of the derived sections. In contrast, the two species of the section Caulorrhizae have small chromosomes, the lengths of their karyotypes are in the middle of the range and closer to those of the species of primitive sections, and the DAPI heterochromatin forms tiny bands only in four chromosome pairs. A possible derived position of the section Erectoides within the genus is supported by several molecular phylogenies, and their species form a single clade together with the species of the sections Procumbentes, Rhizomatosae and Trierectoides, which are also closely related to the clade of species of the section Arachis (Bechara et al. 2010BECHARA MD, MORETZSOHN MC, PALMIERI DA, MONTEIRO JP, BACCI M, MARTINS J & GIMENES MA. 2010. Phylogenetic relationships in genus Arachis based on ITS and 5.8 S rDNA sequences. BMC Plant Biol 10: 255., Friend et al. 2010FRIEND SA, QUANDT D, TALLURY SP, STALKER HT & HILU KW. 2010. Species, genomes, and section relationships in the genus Arachis (Fabaceae): a molecular phylogeny. Plant Syst Evol 290: 185-199., Wang et al. 2010WANG CT, WANG XZ, TANG YY, CHEN DX, CUI FG, ZHANG JC & WANG SLY. 2010. Phylogeny of Arachis based on internal transcribed spacer sequences. Genet Resour Crop Evo 58: 311-319.). Likewise, in those phylogenies, the two species of the section Caulorrhizae form a clade with an intermediate position between the most basal and the most derived sections.

CMA heterochromatin distribution

The occurrence of GC-rich heterochromatin (CMA bands) adjacent to or interspersed with nucleolus organizing regions (NORs) has been extensively described for many plant species (Guerra 2000GUERRA M. 2000. Patterns of heterochromatin distribution in plant chromosomes. Genet Mol Biol 23: 1029-1041., Hou et al. 2004HOU MH, ROBINSON H, GAO YG & WANG A. 2004. Crystal structure of the [Mg2+-(chromomycin A3)2]± d(TTGGCCAA)2 complex reveals GGCC binding specificity of the drug dimer chelated by a metal ion. Nucleic Acids Res 32(7): 2214-2222.), and the Arachis species are not the exception. Thus, one pair of CMA bands corresponding to the only NORs occurs in all species here analyzed. However, a greater number of CMA bands, which co-located with non-active 45S rDNA loci, have been reported in other Arachis species (Ortiz et al. 2017ORTIZ AM, ROBLEDO GA, SEIJO JG, VALLS JFM & LAVIA GI. 2017. Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section Rhizomatosae and related diploid species (Arachis, Leguminosae). J Plant Res 130: 791-807., Samoluk et al. 2019SAMOLUK SS, CHALUP LMI, CHAVARRO C, ROBLEDO G, BERTIOLI DJ, JACKSON S & SEIJO GJ. 2019. Heterochromatin evolution in Arachis investigated through genome-wide analysis of repetitive DNA. Planta 249(5): 1405-1415.). Considering the diversity in the number of 45S rDNA loci among Arachis species, the patterns of CMA banding observed do not reflect those of the 45S rDNA loci. In most species, the number of 45S rDNA loci exceeds that of the CMA bands detected. This behavior of CMA in relation to 45S rDNA loci could be due to the higher sensitivity of FISH to reveal 45S rDNA loci in comparison with CMA banding. Supporting this, CMA-neutro small 45S rDNA loci have been reported for some Cestrum species (Nunes Fregonezi et al. 2006NUNES FREGONEZI J, FERNANDES T, DOMINGUES TOREZAN JM, VIEIRA AOS & LAFORGA VANZELA AL. 2006. Karyotype differentiation of four Cestrum species (Solanaceae) based on the physical mapping of repetitive DNA. Genet Mol Biol 29(1): 97-104.).

Arachis triseminata (section Triseminatae), A. dardani, A. pusilla (section Heteranthae), and A. glandulifera (section Arachis) were differentiated from the rest by having CMA bands additional to those associated with NORs. In these species, CMA bands have different sizes; but, while the bands in the first three species are all centromeric, in A. glandulifera they are located in different positions. This is the first report of CMA banding for A. triseminata, while the band patterns observed for A. dardani and A. pusilla are in agreement with those published by Silva et al. (2010)SILVA SC, MARTINS MIG, SANTOS RC, PEÑALOZA APS, FILHO PAM, BENKO-ISEPPON AM, VALLS JFM & CARVALHO R. 2010. Karyological features and banding patterns in Arachis species belonging to the Heteranthae section. Plant Syst Evol 285: 201-207., and the pattern of A. glandulifera with that detected by Samoluk et al. (2019)SAMOLUK SS, CHALUP LMI, CHAVARRO C, ROBLEDO G, BERTIOLI DJ, JACKSON S & SEIJO GJ. 2019. Heterochromatin evolution in Arachis investigated through genome-wide analysis of repetitive DNA. Planta 249(5): 1405-1415.. These last authors demonstrated that, in A. glandulifera, each CMA band corresponds to one 45S rDNA locus. In contrast, we observed that all additional CMA bands in A. triseminata, A. dardani and A. pusilla do not co-locate with any 45S rDNA loci. CMA bands not associated with 45S rDNA loci have been reported in some other groups of species, as Solanum (Moyetta et al. 2017MOYETTA NR, URDAMPILLETA JD, CHIARINI FE & BERNARDELO G. 2017. Heterochromatin and rDNA patterns in Solanum species of the Morelloid and Dulcamaroid clades (Solanaceae). Plant Biosyst 151(3): 539-547.), Cactaceae (Castro et al. 2016CASTRO JP, MEDEIROS-NETO E, SOUZA G, ALVES LI, BATISTA FR & FELIX LP. 2016. CMA band variability and physical mapping of 5S and 45S rDNA sites in Brazilian Cactaceae: Pereskioideae and Opuntioideae. Braz J Bot 39(2): 613-620.), Cestrum (Nunes Fregonezi et al. 2006NUNES FREGONEZI J, FERNANDES T, DOMINGUES TOREZAN JM, VIEIRA AOS & LAFORGA VANZELA AL. 2006. Karyotype differentiation of four Cestrum species (Solanaceae) based on the physical mapping of repetitive DNA. Genet Mol Biol 29(1): 97-104.), Citrus (Barros e Silva et al. 2010BARROS E SILVA AE, MARQUES A, DOS SANTOS KGB & GUERRA M. 2010. The evolution of CMA bands in Citrus and related genera. Chromosom Res 18: 503-514.) and Capsicum (Grabiele et al. 2018GRABIELE M, DEBAT H, SCALDAFERRO MA, AGUILERA PM, MOSCONE EA, SEIJO JG & DUCASSE DA. 2018. Highly GC-rich heterochromatin in chili peppers (Capsicum-Solanaceae): A cytogenetic and molecular characterization. Sci Hortic 238: 391-399.); and the nature of the sequences that composed those CMA bands has been analyzed in some cases. In x=12 species of Capsicum, additional CMA bands not associated with 45S rDNA loci are composed mainly of a mega satellite derived from a 45S rDNA sequence; by contrast, in x=13 species of Capsicum, the additional CMA bands do not hybridize with the probes from this mega satellite. Similarly, in some Citrus species, most CMA bands co-locate with 45S rDNA and/or satDNA CsSat sequences (CsSat is a satellite DNA sequence from Citrus, with a high GC content), but other bands do not hybridize with any of the probes from these sequences. So, it is clear that the CMA heterochromatin condition is not related to a specific sequence, but to sequences with GC contents, distribution and chromatin conformation that are particular. Several satellite sequences have been isolated and characterized for Arachis genomes, all from species of the section Arachis (Zhang et al. 2012ZHANG L, XU C & YU W. 2012. Cloning and Characterization of Chromosomal Markers from a Cot-1 Library of Peanut (Arachis hypogaea L.). Cytogenet Genome Res 137: 31-41., 2017, Samoluk et al. 2016SAMOLUK SS, ROBLEDO G, BERTIOLI DJ & SEIJO JG. 2016. Evolutionary dynamics of an atrich satellite DNA and its contribution to karyotype differentiation in wild diploid Arachis species. Mol Genet Genomics 292(2): 283-296.), and, until now, none of them have been associated with CMA bands. Therefore, we cannot discard that the additional CMA bands of A. triseminata, A. dardani and A. pusilla are composed of satellite sequences derived from the 45S rDNA sequence. This is because the probes here used to reveal the 45S rDNA loci cover only the 18S and 26S rDNA genes, the intergenic spacer (IGS) region was not represented and some satellite sequences derived from this region have been reported for several species of Solanaceae (Stupar et al. 2002STUPAR RM, JUNQI S, TEK AL, CHENG Z, DONG F & JIANG J. 2002. Highly Condensed Potato Pericentromeric Heterochromatin Contains rDNA-Related Tandem Repeats. Genetics 162(3): 1435-1444., Lim et al. 2004LIM KY, MATYASEK R, KOVARIK A & LEITCH A. 2004. Genome evolution in allotetraploid Nicotiana. Biol J Linn Soc 82(4) 599-606., Jo et al. 2009JO S-H, KOO D-H, KIM JF, HUR CG, LEE S, YANG T, KWON SY & CHOI D. 2009. Evolution of ribosomal DNA-derived satellite repeat in tomato genome. BMC Plant Biol 9: 42.).

Among the three species with additional centromeric CMA bands not associated with 45S rDNA loci, A. pusilla is the only one that has bands of variable size along all the chromosome complement. This heterochromatin distribution pattern is not strange because it is the same as that shown by DAPI heterochromatin in most Arachis species (Robledo et al. 2009ROBLEDO G, LAVIA GI & SEIJO G. 2009. Species relations among wild Arachis species with the A genome as revealed by FISH mapping of rDNA loci and heterochromatin detection. Theor Appl Genet 118: 1295-1307., Robledo & Seijo 2010ROBLEDO G & SEIJO G. 2010. Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of Rdna loci and heterochromatin detection: a new proposal for genome arrangement. Theor Appl Genet 121: 1033-1046., Silva et al. 2010SILVA SC, MARTINS MIG, SANTOS RC, PEÑALOZA APS, FILHO PAM, BENKO-ISEPPON AM, VALLS JFM & CARVALHO R. 2010. Karyological features and banding patterns in Arachis species belonging to the Heteranthae section. Plant Syst Evol 285: 201-207., Silvestri et al. 2015SILVESTRI MC, ORTIZ AM & LAVIA GI. 2015. rDNA loci and heterochromatin positions support a distinct genome type for ‘x = 9 species’ of section Arachis (Arachis, Leguminosae). Plant Syst Evol 301: 555-562., Ortiz et al. 2017ORTIZ AM, ROBLEDO GA, SEIJO JG, VALLS JFM & LAVIA GI. 2017. Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section Rhizomatosae and related diploid species (Arachis, Leguminosae). J Plant Res 130: 791-807.). In fact, generalized proximal bands are the most common distribution pattern of the heterochromatin in angiosperms with small chromosomes (Guerra 2000GUERRA M. 2000. Patterns of heterochromatin distribution in plant chromosomes. Genet Mol Biol 23: 1029-1041.). However, so far, the GC-rich composition is a feature only of A. pusilla within the genus Arachis. In relation to this particularity, A. pusilla has the longest chromosome length and the highest interchromosomal index among the species with CMA bands not associated with 45S rDNA loci. Therefore, the differential accumulation of some GC-rich sequences, revealed in the form of CMA bands of different size, could have been involved in the karyotype differentiation of A. pusilla.

Patterns of DAPI heterochromatin distribution

Among angiosperms, there is a notable variability in heterochromatin distribution patterns, but drastic or discontinuous changes are not common within groups of related species (Galasso et al. 1996GALASSO MF, MAGGIANI M, CREMONINI R & PIGNONE D. 1996. Chromatin characterization by banding techniques, in situ hybridization, and nuclear content in Cicer L. (Fabaceae). Genome 39: 258-265., Guerra 2000GUERRA M. 2000. Patterns of heterochromatin distribution in plant chromosomes. Genet Mol Biol 23: 1029-1041., Nunes Fregonezi et al. 2006NUNES FREGONEZI J, FERNANDES T, DOMINGUES TOREZAN JM, VIEIRA AOS & LAFORGA VANZELA AL. 2006. Karyotype differentiation of four Cestrum species (Solanaceae) based on the physical mapping of repetitive DNA. Genet Mol Biol 29(1): 97-104.). In the genus Arachis, the heterochromatin distribution on the karyotypes is conserved. In all the species with DAPI heterochromatin, this is predominantly located in the proximal regions of both chromosome arms. However, differences in the number of bands and percentage of heterochromatin among different species groups allow establishing distinct distribution patterns. Thus, taking into account our results and previously published data about DAPI banding (Seijo et al. 2004SEIJO JG, LAVIA GI, FERNÁNDEZ A, KRAPOVICKAS A, DUCASSE D & MOSCONE EA. 2004. Physical mapping of 5 S and 18–25S Rrna genes evidences that Arachis duranensis and A. ipaënsis are the wild diploid species involved in the origin of A. hypogaea (Leguminosae). Am J Bot 91: 1294-1303., Robledo et al. 2009ROBLEDO G, LAVIA GI & SEIJO G. 2009. Species relations among wild Arachis species with the A genome as revealed by FISH mapping of rDNA loci and heterochromatin detection. Theor Appl Genet 118: 1295-1307., Robledo & Seijo 2010ROBLEDO G & SEIJO G. 2010. Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of Rdna loci and heterochromatin detection: a new proposal for genome arrangement. Theor Appl Genet 121: 1033-1046., Silvestri et al. 2015SILVESTRI MC, ORTIZ AM & LAVIA GI. 2015. rDNA loci and heterochromatin positions support a distinct genome type for ‘x = 9 species’ of section Arachis (Arachis, Leguminosae). Plant Syst Evol 301: 555-562., Ortiz et al. 2017ORTIZ AM, ROBLEDO GA, SEIJO JG, VALLS JFM & LAVIA GI. 2017. Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section Rhizomatosae and related diploid species (Arachis, Leguminosae). J Plant Res 130: 791-807.), five patterns of heterochromatin distribution can be established:

Pattern 1 corresponds to a generalized (bands occurring in at least 70% of the chromosomes of the karyotype) and homogeneous distribution of conspicuous bands, which is observed as centromeric blocks of similar size in all or almost all the chromosomes, except pairs #9 and #10, which have longer bands in the A and K genomes. This pattern coincides with the distribution described in the species with A, G, and K genomes of the section Arachis (Robledo et al. 2009ROBLEDO G, LAVIA GI & SEIJO G. 2009. Species relations among wild Arachis species with the A genome as revealed by FISH mapping of rDNA loci and heterochromatin detection. Theor Appl Genet 118: 1295-1307., Robledo & Seijo 2010ROBLEDO G & SEIJO G. 2010. Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of Rdna loci and heterochromatin detection: a new proposal for genome arrangement. Theor Appl Genet 121: 1033-1046., Silvestri et al. 2015SILVESTRI MC, ORTIZ AM & LAVIA GI. 2015. rDNA loci and heterochromatin positions support a distinct genome type for ‘x = 9 species’ of section Arachis (Arachis, Leguminosae). Plant Syst Evol 301: 555-562.), as well as with A. porphyrocalyx of the section Erectoides (Silvestri et al. 2017SILVESTRI MC, ORTIZ AM, ROBLEDO G, VALLS JFM & LAVIA GI. 2017. Genomic characterisation of Arachis porphyrocalyx (Valls & C.E. Simpson, 2005) (Leguminosae): multiple origin of Arachis species with x = 9. Comp Cytogen 11(1): 29-43.) and A. vallsii of the section Procumbentes.

Pattern 2 corresponds to a generalized and homogeneous distribution of tiny bands, which are observed as centromeric blocksin the form of “dots” in species of the sections Erectoides and Procumbentes and in 4x species of the section Rhizomatosae (Ortiz et al. 2017ORTIZ AM, ROBLEDO GA, SEIJO JG, VALLS JFM & LAVIA GI. 2017. Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section Rhizomatosae and related diploid species (Arachis, Leguminosae). J Plant Res 130: 791-807.) or in the form of bands in the species with F genome of the section Arachis (Robledo & Seijo 2010ROBLEDO G & SEIJO G. 2010. Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of Rdna loci and heterochromatin detection: a new proposal for genome arrangement. Theor Appl Genet 121: 1033-1046.).

Pattern 3 corresponds to a homogeneous but restrained (bands occurring in some chromosomes of the karyotype) distribution of tiny bands that is observed in less than half of the chromosome pairs. This pattern is particular of species of the section Caulorrhizae.

Pattern 4 corresponds to a restrained and notably heterogeneous distribution, which is observed as conspicuous bands of very dissimilar size at pericentromeric and interstitial positions in more than half of the chromosome pairs, and without heterochromatin bands in the remaining chromosome pairs. This pattern is particular of A. glandulifera of the section Arachis (Robledo & Seijo 2008ROBLEDO G & SEIJO G. 2008. Characterization of the Arachis (Leguminosae) D genome using fluorescence in situ hybridization (FISH) chromosome markers and total genome DNA hybridization. Genet Mol Biol 31: 717-724.).

Finally, pattern 5 corresponds to karyotypes devoid of centromeric DAPI bands, and is observed in the species of the sections Heteranthae and Triseminatae, the species with B genome of the section Arachis (Robledo & Seijo 2010ROBLEDO G & SEIJO G. 2010. Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of Rdna loci and heterochromatin detection: a new proposal for genome arrangement. Theor Appl Genet 121: 1033-1046.), and the only 2x species of the section Rhizomatosae, i.e. A. burkartii (Ortiz et al. 2017ORTIZ AM, ROBLEDO GA, SEIJO JG, VALLS JFM & LAVIA GI. 2017. Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section Rhizomatosae and related diploid species (Arachis, Leguminosae). J Plant Res 130: 791-807.).

Distribution patterns of ribosomal DNA

In the genus Arachis, the 5S and 45S rDNA loci are commonly located on different chromosome pairs (Raina & Mukai 1999RAINA SN & MUKAI Y. 1999. Detection of a variable number of 18S–5.8S–26S and 5S ribosomal DNA loci by fluorescent in situ Hybridization in diploid and tetraploid Arachis species. Genome 42: 52-59., Seijo et al. 2004SEIJO JG, LAVIA GI, FERNÁNDEZ A, KRAPOVICKAS A, DUCASSE D & MOSCONE EA. 2004. Physical mapping of 5 S and 18–25S Rrna genes evidences that Arachis duranensis and A. ipaënsis are the wild diploid species involved in the origin of A. hypogaea (Leguminosae). Am J Bot 91: 1294-1303., Robledo et al. 2009ROBLEDO G, LAVIA GI & SEIJO G. 2009. Species relations among wild Arachis species with the A genome as revealed by FISH mapping of rDNA loci and heterochromatin detection. Theor Appl Genet 118: 1295-1307., Robledo & Seijo 2010ROBLEDO G & SEIJO G. 2010. Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of Rdna loci and heterochromatin detection: a new proposal for genome arrangement. Theor Appl Genet 121: 1033-1046., Lavia et al. 2011LAVIA GI, ORTIZ AM, ROBLEDO G, FERNÁNDEZ A & SEIJO G. 2011. Origin of triploid Arachis pintoi (Leguminosae) by autopolyploidy evidenced by FISH and meiotic behavior. Ann Bot 108: 103-111., Silvestri et al. 2015SILVESTRI MC, ORTIZ AM & LAVIA GI. 2015. rDNA loci and heterochromatin positions support a distinct genome type for ‘x = 9 species’ of section Arachis (Arachis, Leguminosae). Plant Syst Evol 301: 555-562., Ortiz et al. 2017ORTIZ AM, ROBLEDO GA, SEIJO JG, VALLS JFM & LAVIA GI. 2017. Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section Rhizomatosae and related diploid species (Arachis, Leguminosae). J Plant Res 130: 791-807.). Coinciding with this behavior, the number of loci per karyotype is not correlated. The number of 5S and 45S rDNA loci range from one to three pairs and from one to five, respectively; but almost all species have only one pair of 5S rDNA loci, while one, two and three pairs of 45S rDNA loci are present in a similar frequency. Thus, the number of rDNA loci detected in the eight species here analyzed is in agreement with that reported for other Arachis species. Likewise, in seven of them, the rDNA loci were on chromosomes with morphology similar to that of those harboring them in the rest of the genus, and in the expected positions. Besides, the location of additional 5S rDNA loci in A. pintoi and A. vallsii is in agreement with the distribution pattern observed within the genus, although the presence of more than one pair of these loci is not the rule. In contrast, the location of the 5S rDNA loci of A. pusilla is not common. Until now, 5S rDNA loci on the long arms of the submetacentric chromosomes of pair #10 and linked to one pair of 45S rDNA loci have been observed only in the species with K genome of the section Arachis (Robledo & Seijo 2010ROBLEDO G & SEIJO G. 2010. Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of Rdna loci and heterochromatin detection: a new proposal for genome arrangement. Theor Appl Genet 121: 1033-1046.) and in A. stenophylla of the section Erectoides (Du et al. 2016DU P, LI LI-NA, ZHANG Z, LIU H, QIN L, HUANG B, DONG WZ, TANG F, QI Z & ZHANG X. 2016. Chromosome painting of telomeric repeats reveals new evidence for genome evolution in peanut. J Integ Agr 15(11): 2488-2496.). However, in K genome species, these 5S rDNA loci are additional to those shared by all Arachis species located on pair #3, whereas in A. stenophylla, the 5S rDNA loci are located more distal than 45S rDNA loci, conversely to the proximal position observed in A. pusilla. Thus, the location of the only pair of 5S rDNA loci of A. pusilla is a particularity of this species.

This is the first report on the mapping of 5S and 45S rDNA loci in A. benthamii, A. dardani and A. vallsii, while the number and location of 5S and 45S rDNA loci observed for A. pusilla, A. matiensis and A. repens are coincident with previously reported data (Raina & Mukai 1999RAINA SN & MUKAI Y. 1999. Detection of a variable number of 18S–5.8S–26S and 5S ribosomal DNA loci by fluorescent in situ Hybridization in diploid and tetraploid Arachis species. Genome 42: 52-59., Pucciariello et al. 2013PUCCIARIELLO O, ORTIZ AM, FERNÁNDEZ A & LAVIA GI. 2013. Análisis cromosómico del híbrido Arachis pintoi x A. repens (Leguminosae) mediante citogenética clásica y molecular. Bonplandia 48(1): 111-119.). In contrast, our results differ from those observed by Raina & Mukai (1999)RAINA SN & MUKAI Y. 1999. Detection of a variable number of 18S–5.8S–26S and 5S ribosomal DNA loci by fluorescent in situ Hybridization in diploid and tetraploid Arachis species. Genome 42: 52-59. for A. triseminata, where an additional pair for each rDNA loci has been observed. The difference in the number of 45S rDNA loci may be attributed to the fact that we did not detect a pair described by those authors as “minute-size”, whereas the difference in the number of 5S rDNA loci may be attributed to the fact that they recognized the hybridization signals as constituted by two “tandemly located” loci. For A. pintoi, we detected the same number of rDNA loci as Raina & Mukai (1999)RAINA SN & MUKAI Y. 1999. Detection of a variable number of 18S–5.8S–26S and 5S ribosomal DNA loci by fluorescent in situ Hybridization in diploid and tetraploid Arachis species. Genome 42: 52-59. and as that previously observed (Lavia et al. 2011LAVIA GI, ORTIZ AM, ROBLEDO G, FERNÁNDEZ A & SEIJO G. 2011. Origin of triploid Arachis pintoi (Leguminosae) by autopolyploidy evidenced by FISH and meiotic behavior. Ann Bot 108: 103-111.). However, both in this work and in Lavia et al. (2011)LAVIA GI, ORTIZ AM, ROBLEDO G, FERNÁNDEZ A & SEIJO G. 2011. Origin of triploid Arachis pintoi (Leguminosae) by autopolyploidy evidenced by FISH and meiotic behavior. Ann Bot 108: 103-111., the two pairs of 5S rDNA loci were found to be located in proximal position on the short arms of different chromosome pairs, while in Raina & Mukai (1999)RAINA SN & MUKAI Y. 1999. Detection of a variable number of 18S–5.8S–26S and 5S ribosomal DNA loci by fluorescent in situ Hybridization in diploid and tetraploid Arachis species. Genome 42: 52-59. the two pairs of loci were found to be “tandemly located” on the same chromosome arm. Until now, a tandem arrangement of rDNA loci has been reported only for 45S rDNA loci in A. valida and A. williamsii of the section Arachis (Robledo & Seijo 2010ROBLEDO G & SEIJO G. 2010. Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of Rdna loci and heterochromatin detection: a new proposal for genome arrangement. Theor Appl Genet 121: 1033-1046.) and in A. burkartii of the section Rhizomatosae (Ortiz et al. 2017ORTIZ AM, ROBLEDO GA, SEIJO JG, VALLS JFM & LAVIA GI. 2017. Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section Rhizomatosae and related diploid species (Arachis, Leguminosae). J Plant Res 130: 791-807.), whereby the distribution of 5S rDNA loci in A. triseminata and A. pintoi should be reconsidered.

The variations in the distribution pattern of rDNA loci in groups of closely related species, commonly named “repatterning”, have been explained via structural rearrangement events such as translocations, inversions, duplications and deletions. All these events commonly result in structural changes of karyotype. Contrarily, the transposition of rDNA genes mediated by the activity of transposable elements may be equally important in the “repatterning” of rDNA loci (Raskina et al. 2004aRASKINA O, BELYAYEV A & NEVO E. 2004a. Activity of the En/Spm-like transposons in meiosis as a base for chromosome repatterning in a small, isolated, peripheral population of Aegilops speltoides Tausch. Chromosome Res 12: 153-161., Datson & Murray 2006DATSON PM & MURRAY BG. 2006. Ribosomal DNA locus evolution in Nemesia: transposition rather than structural rearrangement as the key mechanism? Chromosome Res 14: 845-857.) without affecting the karyotype structure. In addition, in situ amplification of pre-existing minor rDNA arrays via unequal crossing over or transposition can also lead to the formation of novel rDNA loci (Datson & Murray 2006DATSON PM & MURRAY BG. 2006. Ribosomal DNA locus evolution in Nemesia: transposition rather than structural rearrangement as the key mechanism? Chromosome Res 14: 845-857., Eickbush & Eickbush 2007EICKBUSH TH & EICKBUSH DG. 2007. Finely orchestrated movements: evolution of the ribosomal RNA genes. Genetics 175: 477-485., Lan & Albert 2011LAN T & ALBERT VA. 2011. Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady’s slipper orchid. BMC Plant Biol 11: 126., Yung et al. 2017YUNG IL, CHUNG MC, KUO H, WANG CN, CHING YL, LIN CY, JIANG H & YEH CH. 2017. The evolution of genome size and distinct distribution patterns of rDNA in Phalaenopsis (Orchidaceae). Bot J Linn Soc 185: 65-80.). Independently of the event type involved in the distribution pattern changes of rDNA loci, “repatterning” has been associated with the speciation of several species and is considered as an integral part of this process (Raskina et al. 2004bRASKINA O, BELYAYEV A & NEVO E. 2004b. Quantum speciation in Aegilops: molecular cytogenetic evidence from rDNA cluster variability in natural populations. P Natl Acad Sci USA 101: 14818-14823., Baum & Feldman 2010BAUM BR & FELDMAN M. 2010. Elimination of 5S DNA unit classes in newly formed allopolyploids of the genera Aegilops and Triticum. Genome 53(6): 430-438., Lan & Albert 2011LAN T & ALBERT VA. 2011. Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady’s slipper orchid. BMC Plant Biol 11: 126.). Previous studies in the section Arachis have related changes in the number of 45S rDNA loci to the differentiation of karyotype groups within the A genome species (Robledo et al. 2009ROBLEDO G, LAVIA GI & SEIJO G. 2009. Species relations among wild Arachis species with the A genome as revealed by FISH mapping of rDNA loci and heterochromatin detection. Theor Appl Genet 118: 1295-1307.), to the diversification of species assigned to the same genome (Robledo & Seijo 2010ROBLEDO G & SEIJO G. 2010. Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of Rdna loci and heterochromatin detection: a new proposal for genome arrangement. Theor Appl Genet 121: 1033-1046.), and even to the differentiation between the two subspecies of A. paraguariensis (Ortiz et al. 2017ORTIZ AM, ROBLEDO GA, SEIJO JG, VALLS JFM & LAVIA GI. 2017. Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section Rhizomatosae and related diploid species (Arachis, Leguminosae). J Plant Res 130: 791-807.). In this study, we showed that “repatterning” could also be associated with the amplification of the number of 5S rDNA loci, as is the differentiation of karyotypes for A. pintoi and A. repens of the section Caulorrhizae. We also showed that “repatterning” events can occur by changes in the location of rDNA loci, as is the case of the 5S rDNA loci in A. pusilla and A. dardani, which can be parsimoniously explained as the result of a single event of chromosomal translocation. Recently, by using telomeric markers and repetitive and single-copy oligonucleotides as probes, Du et al. (2016, 2018) demonstrated structural rearrangements among the karyotypes of Arachis species. Therefore, the “repatterning” events in Arachis species could have occurred not only by amplification of the number of loci but also by chromosomal rearrangements.

Sites of linked 5S and 45S rDNA loci

The location of 5S and 45S rDNA loci on the same chromosome or even in adjacent positions on the same arm has been observed in many plant groups (Garcia et al. 2009GARCIA S, GARNATJE T, PELLICER J, MCARTHUR ED, YAKOVLEV SS & VALLÉS J. 2009. Ribosomal DNA, heterochromatin, and correlation with genome size in diploid and polyploid North American endemic sagebrushes (Artemisia, Asteraceae). Genome 52: 1012-1024., Modin et al. 2007MODIN M, SANTOS-SEREJO J & AGUIAR-PERECIN M. 2007. Karyotype characterization of Crotolaria juncea (L.) by chromosome banding and physical mapping of 18S-5.8S-26S and 5S rRNA gene sites. Genet Mol Biol 30: 65-72., Li et al. 2016LI KP, XIANG YW, ZHAO H, WANG Y, LÜ XM, WANG JM, XU Y, LI ZY & HAN YH. 2016. Cytogenetic relationships among Citrullus species in comparison with some genera of the tribe Benincaseae (Cucurbitaceae) as inferred from rDNA distribution patterns. BMC EvolBiol 16: 85.). Roa & Guerra (2015)ROA F & GUERRA M. 2015. Non-Random Distribution of 5S rDNA sites and Its Association with 45S rDNA in Plant Chromosomes. Cytogenet Genome Res 146(3): 243-249. found that the frequency of linked rDNA loci is directly influenced by the representation of these loci in the karyotypes, so the frequency is much lower in karyotypes with single rDNA loci than in those with multiple loci. Our results and those previously reported about the location of rDNA loci in Arachis species partially support this hypothesis. All species with linked 5S and 45S rDNA loci, either on the same chromosome (A. burkartii, A. ipaensis) or in adjacent positions on the same arm (A. pusilla, A. magna, A. benensis and the three species with K genome), have a high number of ribosomal loci. However, only 29.63% of the diploid species of Arachis with multiple sites presented linked loci. Therefore, although the presence of multiple loci contributes to the occurrence of linked loci, in the genus Arachis, other factors could be involved in their frequency. In this sense, our results showed that most species with multiple sites have large chromosomes and only a few have small chromosomes, but also that only 21% of the species with large chromosomes and almost all the species with small chromosomes present linked loci. These data suggest that another factor that could increase the occurrence of linked loci is the limited space of the chromosome.

Satellite DNA clone 119

The mapping of clone 119 on the karyotypes of representative species of the different sections and genomes showed that this sequence is not equally represented within the genus Arachis. Its absence in the more basal sections suggests that it was originated in an ancestor common to the sections Arachis, Erectoides, Procumbentes and Rhizomatosae. At the same time, the presence of multiple additional sites in species of the section Arachis suggests that the dispersion of this satDNA occurred later or during the differentiation of the genomes of this section, and supports that proposed by Seijo et al. (2017)SEIJO G, SAMOLUK SS, ORTIZ AM, SILVESTRI MC, CHALUP L, ROBLEDO G & LAVIA GI. 2017. Cytological features of peanut genome. In: Varshney R, Pandey M and Puppala N (Eds). The peanut genome, compendium of plant genomes series. Springer, Germany, p. 37-52. that the genomic fraction of satDNA may have been one of the most dynamic in the karyotype evolution of the genus. In this sense, the localizations of clone 119 and 5S rDNA loci on opposite chromosomal arms of pair #3 in A. glandulifera suggest a chromosome rearrangement as a pericentromeric inversion or an intrachromosomal translocation. This hypothesis could also explain the different chromosome morphology between pair #3 in A. glandulifera (sm) with respect to pair #3 of the rest of the species (m). Similarly, the high number of sites observed in A. porphyrocalyx supports that proposed in Silvestri et al. (2017)SILVESTRI MC, ORTIZ AM, ROBLEDO G, VALLS JFM & LAVIA GI. 2017. Genomic characterisation of Arachis porphyrocalyx (Valls & C.E. Simpson, 2005) (Leguminosae): multiple origin of Arachis species with x = 9. Comp Cytogen 11(1): 29-43. that this species does not share the same genome as the remaining species of the section Erectoides, but is more similar to those of the section Arachis.

In many groups of species, the satDNA sequences associated with the heterochromatin are shared only among closely related species, or are species specific or even chromosome specific (Ceccarelli et al. 2010CECCARELLI M, SARRI V, POLIZZI E, ANDREOZZI G & CIONINI PG. 2010. Characterization, Evolution and Chromosomal Distribution of Two Satellite DNA Sequence Families in Lathyrus species. Cytogenet Genome Res 128: 236-244.). The fact that clone 119 hybridized in some DAPI bands, but not in all, revealed that there are differences in the representation and composition of the satellite sequences that form these bands in Arachis species, as suggested by Samoluk et al. (2016)SAMOLUK SS, ROBLEDO G, BERTIOLI DJ & SEIJO JG. 2016. Evolutionary dynamics of an atrich satellite DNA and its contribution to karyotype differentiation in wild diploid Arachis species. Mol Genet Genomics 292(2): 283-296.. Moreover, the hybridization of clone 119 on DAPI+ and DAPI-neutro of centromeric regions suggests that this is a common sequence for this chromosome region, independently of the chromatin conformation and the nucleotide composition of this region.

Congruence among karyotype groups, taxonomic position and genome assignment of Arachis species

The establishment of the genomes in the genus Arachis has been traditionally based on cross-compatibility assays added to morphological and chromosome features (Smartt et al. 1978SMARTT J, GREGORY WC & GREGORY MP. 1978. The genomes of Arachis hypogaea 1. Cytogenetic studies of puntative genome donors. Euphytica 27: 665-675., Gregory & Gregory 1979GREGORY WC & GREGORY MP. 1979. Exotic germplasm of Arachis L. interspecific hybrids. J Heredity 70: 185-193., Singh & Moss 1982SINGH AK & MOSS JP. 1982. Utilization of wild relative in genetic improvement of Arachis hypogaea L. Theor Appl Genet 61: 305-314., 1984SINGH AK & MOSS JP. 1984. Utilization of wild relatives 1 in genetic improvement of Arachis hypogaea L. Part 5. Genome analysis in section Arachis and its implications in gene transfer. Theor Appl Genet 68: 355-364., Singh 1986SINGH AK. 1986. Utilization of wild relatives in the genetic improvement of Arachis hypogaea L. Theor Appl Genet 72: 433-439., Stalker 1991STALKER HT. 1991. A new species in section Arachis of peanuts with a D genome. Am J Bot 78: 630-637., Fernández & Krapovickas 1994FERNÁNDEZ A & KRAPOVICKAS A. 1994. Cromosomas y evolución en Arachis (Leguminosae). Bonplandia 8: 187-220.). Since the first genome designation was based on compatibility crossing, and incompatibilities may occur due to single genes, cytoplasmic effects, or other biological factors, chromosome similarities may be found between species that are assigned to different genomes. Therefore, here we intend to discuss the karyotype characteristics of the genomes.

Our chromosome analyses of basic morphology as the chromosome length and asymmetry indexes, the amount and distribution of DAPI and CMA heterochromatin, and the number and position of 5S rDNA sites enabled us to propose eleven karyotype groups. Some of these groups have been described before whereas others are here described for the first time. Below we summarize the main characteristics that differentiate each karyotype group.

Karyotype group 1: This group has karyotypes with very small chromosomes devoid of DAPI bands (pattern type 5). It includes species of the sections Heteranthae and Triseminatae, with Am and T genome respectively. Since similarities between the Am and T genomes are mainly based on the lack of DAPI heterochromatin and the chromosome size, we assigned them the same karyotype group. Another characteristic was the centromeric CMA bands, but these were not observed in A. interrupta or A. veigae of the section Heteranthae (Silva et al. 2010SILVA SC, MARTINS MIG, SANTOS RC, PEÑALOZA APS, FILHO PAM, BENKO-ISEPPON AM, VALLS JFM & CARVALHO R. 2010. Karyological features and banding patterns in Arachis species belonging to the Heteranthae section. Plant Syst Evol 285: 201-207.); therefore, we cannot include it as a particularity of this karyotype group. On the other hand, these species also share their natural distribution in the northeast of Brazil, which is different from the other sections of the genus. However, A. triseminata is genetically isolated from the rest of the species because no successful crossings with any members of other sections have been obtained, including those with A. dardani (Am genome) (Gregory & Gregory 1979GREGORY WC & GREGORY MP. 1979. Exotic germplasm of Arachis L. interspecific hybrids. J Heredity 70: 185-193., Krapovickas & Gregory 1994KRAPOVICKAS A & GREGORY WC. 1994. Taxonomía del género Arachis (Leguminosae). Bonplandia 8: 1-186.). Besides, in several molecular analyses, A. triseminata was not closely related to Heteranthae species (Hoshino et al. 2006HOSHINO AA, PEREIRA BRAVO J, ANGELICI CMLCD, GOBBI BARBAROSA AV, ROMERO LOPES C & GIMENES MA. 2006. Heterologous microsatellite primer pairs informative for the whole genus Arachis. Gen Mol Biol 2(4): 665-675., Bechara et al. 2010BECHARA MD, MORETZSOHN MC, PALMIERI DA, MONTEIRO JP, BACCI M, MARTINS J & GIMENES MA. 2010. Phylogenetic relationships in genus Arachis based on ITS and 5.8 S rDNA sequences. BMC Plant Biol 10: 255., Friend et al. 2010FRIEND SA, QUANDT D, TALLURY SP, STALKER HT & HILU KW. 2010. Species, genomes, and section relationships in the genus Arachis (Fabaceae): a molecular phylogeny. Plant Syst Evol 290: 185-199.). The diversification between these two genomes could be by gene differentiation and/or cannot be reflected by the chromosome markers used. Therefore, although the cytogenetic data suggest the same karyotype group for these species, the reproductive isolation and the phylogenetic relationships support the conservation of different genomes for each taxonomic section.

Karyotype group 2: This group has karyotypes with medium to long chromosomes devoid of centromeric DAPI bands (pattern type 5). It includes all species with B genome of the section Arachis and A. burkartii with R genome of the section Rhizomatosae. Like the first group, this group includes species with two different genomes, but, in this case, the genetic distance between both genomes would seem to be greater. Arachis burkartii is reproductively isolated from all species of the genus, including the B genome species. In addition, their natural geographical distribution areas are distant from each other, and the taxonomic and phylogenetic relationships have shown that they are not close (Krapovickas & Gregory 1994KRAPOVICKAS A & GREGORY WC. 1994. Taxonomía del género Arachis (Leguminosae). Bonplandia 8: 1-186., Bechara et al. 2010BECHARA MD, MORETZSOHN MC, PALMIERI DA, MONTEIRO JP, BACCI M, MARTINS J & GIMENES MA. 2010. Phylogenetic relationships in genus Arachis based on ITS and 5.8 S rDNA sequences. BMC Plant Biol 10: 255., Friend et al. 2010FRIEND SA, QUANDT D, TALLURY SP, STALKER HT & HILU KW. 2010. Species, genomes, and section relationships in the genus Arachis (Fabaceae): a molecular phylogeny. Plant Syst Evol 290: 185-199.). Therefore, although they have common chromosomal characteristics, their genome assignments are conserved.

Karyotype group 3: This group has karyotypes with long chromosomes, conspicuous centromeric DAPI bands (pattern type 1), three pairs of 5S rDNA loci, and one 5S and one 45S rDNA loci linked on the long arms of pair #10. It includes A. batizocoi, A. cruziana and A. krapovickasii assigned to the K genome (Robledo & Seijo 2010ROBLEDO G & SEIJO G. 2010. Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of Rdna loci and heterochromatin detection: a new proposal for genome arrangement. Theor Appl Genet 121: 1033-1046.).

Karyotype group 4: this group consists of the species x=9 with conspicuous centromeric DAPI bands (pattern type 1). It includes A. decora, A. palustris and A. praecox assigned to the G genome (Silvestri et al. 2015SILVESTRI MC, ORTIZ AM & LAVIA GI. 2015. rDNA loci and heterochromatin positions support a distinct genome type for ‘x = 9 species’ of section Arachis (Arachis, Leguminosae). Plant Syst Evol 301: 555-562.).

Karyotype group 5: This karyotype group consists of the species x=10 that possess the chromosome pair “A” and conspicuous centromeric DAPI bands (pattern type 1). It includes all species assigned to the A genome (Robledo et al. 2009ROBLEDO G, LAVIA GI & SEIJO G. 2009. Species relations among wild Arachis species with the A genome as revealed by FISH mapping of rDNA loci and heterochromatin detection. Theor Appl Genet 118: 1295-1307.).

Karyotype group 6: This karyotype group has small to medium chromosomes with tiny centromeric DAPI bands in more than half of the chromosome pairs (pattern type 2). It includes A. benensis and A. trinitensis assigned to the F genome (Robledo & Seijo 2010ROBLEDO G & SEIJO G. 2010. Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of Rdna loci and heterochromatin detection: a new proposal for genome arrangement. Theor Appl Genet 121: 1033-1046.).

Karyotype group 7: This group has karyotypes with small to medium chromosomes and tiny centromeric DAPI bands in less than half of the chromosome pairs (pattern type 3). It includes A. pintoi and A. repens assigned to the C genome of the section Caulorrhizae.

In the cluster analysis, the species of karyotype groups 6 and 7 were grouped together; however, we propose that they should be considered as different karyotypes because they show distinct banding DAPI pattern. This is supported by the fact that no successful crossings between F genome species of the sections Arachis and Caulorrhizae species have been reported (Krapovickas 1973KRAPOVICKAS A. 1973. Evolution of the genus Arachis. In: Moav R (Ed). Agricultural genetics. Selected Topics, Jerusalem: National Council for Research and Development, p. 135-151., Krapovickas & Gregory 1994KRAPOVICKAS A & GREGORY WC. 1994. Taxonomía del género Arachis (Leguminosae). Bonplandia 8: 1-186.), and by the fact that neither the geographical distribution nor the phylogenies show relations between the F genome and Caulorrhizae species (Krapovickas & Gregory 1994KRAPOVICKAS A & GREGORY WC. 1994. Taxonomía del género Arachis (Leguminosae). Bonplandia 8: 1-186., Friend et al. 2010FRIEND SA, QUANDT D, TALLURY SP, STALKER HT & HILU KW. 2010. Species, genomes, and section relationships in the genus Arachis (Fabaceae): a molecular phylogeny. Plant Syst Evol 290: 185-199.).

The cytogenetic mapping here performed, as well as morphological and molecular data (Krapovickas & Gregory 1994KRAPOVICKAS A & GREGORY WC. 1994. Taxonomía del género Arachis (Leguminosae). Bonplandia 8: 1-186., Gimenes et al. 2000GIMENES MA, ROMERO LOPES C, GALGARO ML, VALLS JFM & KOCHERT G. 2000. Genetic variation and phylogenetic relationships based on RAPD analysis in section Caulorrhizae, genus Arachis (Leguminosae). Euphytica 116: 187-195., Bechara et al. 2010BECHARA MD, MORETZSOHN MC, PALMIERI DA, MONTEIRO JP, BACCI M, MARTINS J & GIMENES MA. 2010. Phylogenetic relationships in genus Arachis based on ITS and 5.8 S rDNA sequences. BMC Plant Biol 10: 255., Friend et al. 2010FRIEND SA, QUANDT D, TALLURY SP, STALKER HT & HILU KW. 2010. Species, genomes, and section relationships in the genus Arachis (Fabaceae): a molecular phylogeny. Plant Syst Evol 290: 185-199.), confirm the high similarities between the two species of the section Caulorrhizae. In relation to the crossing data, the hybrids between these species have 86.8% pollen fertility, which is a high value in interspecific hybrids (Gregory & Gregory 1979GREGORY WC & GREGORY MP. 1979. Exotic germplasm of Arachis L. interspecific hybrids. J Heredity 70: 185-193.). Besides, the pairing and segregation of chromosomes on meiosis and the pollen formation are normal (Pucciariello et al. 2013PUCCIARIELLO O, ORTIZ AM, FERNÁNDEZ A & LAVIA GI. 2013. Análisis cromosómico del híbrido Arachis pintoi x A. repens (Leguminosae) mediante citogenética clásica y molecular. Bonplandia 48(1): 111-119.). Therefore, we consider that these species share the same karyotype group and also the same genome type.

Karyotype group 8: This karyotype group has long chromosomes with small dot-like centromeric DAPI bands (pattern type 2) and the 5S rDNA loci located on the long arms of pair #3. It includes species of the sections Erectoides and Procumbentes assigned to the E genome (except A. vallsii and A. porphyrocalyx), and the 4x species of the section Rhizomatosae.

The E genome has been assigned to the sections Erectoides, Procumbentes and Trierectoides due to reproductive affinity (Smartt & Stalker 1982SMARTT J & STALKER HT. 1982. Speciation and cytogenetics in Arachis. In: Pattee HE and Young CT (Eds). Peanut science and technology, Yoakun: American Peanut Research Education Society, p. 21-49.). Previous chromosome data and molecular phylogenies confirm the close relationships between these species (Bechara et al. 2010BECHARA MD, MORETZSOHN MC, PALMIERI DA, MONTEIRO JP, BACCI M, MARTINS J & GIMENES MA. 2010. Phylogenetic relationships in genus Arachis based on ITS and 5.8 S rDNA sequences. BMC Plant Biol 10: 255., Friend et al. 2010FRIEND SA, QUANDT D, TALLURY SP, STALKER HT & HILU KW. 2010. Species, genomes, and section relationships in the genus Arachis (Fabaceae): a molecular phylogeny. Plant Syst Evol 290: 185-199.). Moreover, the 4x species of the section Rhizomatosae appear close to the E genome species in molecular phylogeny (Bechara et al. 2010BECHARA MD, MORETZSOHN MC, PALMIERI DA, MONTEIRO JP, BACCI M, MARTINS J & GIMENES MA. 2010. Phylogenetic relationships in genus Arachis based on ITS and 5.8 S rDNA sequences. BMC Plant Biol 10: 255.), share chromosome characteristics (Ortiz et al. 2017ORTIZ AM, ROBLEDO GA, SEIJO JG, VALLS JFM & LAVIA GI. 2017. Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section Rhizomatosae and related diploid species (Arachis, Leguminosae). J Plant Res 130: 791-807.) and produce F1 hybrids with diploid species of the sections Procumbentes and Erectoides (Krapovickas & Gregory 1994KRAPOVICKAS A & GREGORY WC. 1994. Taxonomía del género Arachis (Leguminosae). Bonplandia 8: 1-186.). All these data support the fact that these species belong to the same karyotype group.

Finally, three species were considered as monotypic karyotype groups:

Karyotype group 9: Arachis glandulifera (section Arachis) has a karyotype with long chromosomes, heterogeneous centromeric DAPI bands (pattern type 4), the highest intrachromosomal asymmetry index, and interstitial CMA bands associated with 45S rDNA loci. It has been assigned to the D genome (Robledo & Seijo 2008ROBLEDO G & SEIJO G. 2008. Characterization of the Arachis (Leguminosae) D genome using fluorescence in situ hybridization (FISH) chromosome markers and total genome DNA hybridization. Genet Mol Biol 31: 717-724.),

Karyotype group 10: Arachis porphyrocalyx (section Erectoides) has x=9, the chromosome pair “A”, conspicuous centromeric DAPI bands (pattern type 1), a high intrachromosomal asymmetry index and the 5S rDNA loci on the long arms of pair #3 (Silvestri et al. 2017SILVESTRI MC, ORTIZ AM, ROBLEDO G, VALLS JFM & LAVIA GI. 2017. Genomic characterisation of Arachis porphyrocalyx (Valls & C.E. Simpson, 2005) (Leguminosae): multiple origin of Arachis species with x = 9. Comp Cytogen 11(1): 29-43.).

Karyotype group 11: Arachis vallsii (section Procumbentes) has a karyotype with small to medium chromosomes, conspicuous centromeric DAPI bands (pattern type 1) and two pairs of 5S rDNA loci.

The first monotypic karyotype group is in agreement with its genomic assignment, whereas the other two have chromosome features that are different from those of the remaining species of the sections Procumbentes and Erectoides, all assigned to the E genome. It has been reported that A. porphyrocalyx has more similarities to the A genome species than to the E genome ones (Silvestri et al. 2017SILVESTRI MC, ORTIZ AM, ROBLEDO G, VALLS JFM & LAVIA GI. 2017. Genomic characterisation of Arachis porphyrocalyx (Valls & C.E. Simpson, 2005) (Leguminosae): multiple origin of Arachis species with x = 9. Comp Cytogen 11(1): 29-43.). In addition, our results showed that A. porphyrocalyx is the only diploid species not belonging to the section Arachis that has more than one pair of sites of satDNA clone 119. However, there are no interspecific crossing data involving this species and those of section Arachis. In the cluster analysis, A. vallsii grouped with species of the C and F genomes, but shared the pattern of DAPI bands with the A, G and K genomes of section Arachis. In addition, great reproductive affinity between A. vallsii and different species of the section Arachis has been revealed (Wondracek-Lüdke et al. 2015WONDRACEK-LÜDKE DC, CUSTODIO AR, SIMPSON CE & VALLS JFM. 2015. Crossability of Arachis valida and B genome Arachis species. Genet Mol Res 14(4): 17574-17586.). Finally, neither of the last two species were included in the molecular phylogenies of the genus, so new analysis including them could help to review the genome constitution of these species.

In summary, in the present study, we provided detailed karyotypes of eight species belonging to five sections of Arachis through the mapping of ribosomal genes by FISH and the DAPI banding after FISH. In addition, we extended the karyotype characterization to 14 species belonging to seven sections by CMA/DAPI banding and to 15 species belonging to seven sections by the localization of satDNA clone 119 by FISH. Comparative analysis of the data here obtained and previously published data revealed great chromosome variability within the genus Arachis. However, we were able to recognize five patterns of DAPI heterochromatin distribution and several chromosome homeologies, and thus established 11 karyotype groups which may contribute to better understanding the karyotype affinities among Arachis species. Most karyotype groups were in agreement with the genome assignments of their species, while the assignments of the 4x species of the section Rhizomatosae, A. porphyrocalyx and A. vallsii need to be reviewed. The data of chromosome marker distribution and chromosome homologies here provided will be useful to perform evolutionary studies and to identify interspecific hybrids.

ACKNOWLEGMENTS

We thank Dr. J.F.M. Valls (CENARGEN-EMBRAPA) for their courtesy in sending the seeds. This work was supported by grants from the Secretaría General de Ciencia y Técnica de la Universidad Nacional del Nordeste; Consejo Nacional de Investigaciones Científicas y Técnicas and the Agencia Nacional de Promoción Científica y Tecnológica (PI Nº 12F016, PIP No. 859, PICTO No. 2011-0230). M.C. Silvestri has a fellow, and G.I. Lavia, A.M. Ortiz and G. Robledo are research staff members of CONICET, Argentina.

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SUPPLEMENTARY MATERIAL

Table SI.

Publication Dates

  • Publication in this collection
    07 Sept 2020
  • Date of issue
    2020

History

  • Received
    18 Dec 2018
  • Accepted
    11 Mar 2019
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