DOI: 10.5061/dryad.612jm640p
title: Data from: Cytogenetics, geographic distribution, conservation and new species of Macrotorus (Mollinedioideae, Monimiaceae) from the Brazilian Atlantic rainforest

description: A new species of Macrotorus (Monimiaceae) from the Brazilian Atlantic
 rainforest is here described and illustrated: M. genuflexus. This species,
 restricted to the Poço das Antas Biological Reserve (situated in the
 central region of Rio de Janeiro state, Brazil), is the second in the
 genus Macrotorus. The new species description is based on morphological
 and cytogenetic (karyotype and genome size - GS) comparative analyses. We
 also report a new record of Macrotorus utriculatus for the state of Bahia,
 confirming a potential distribution modeling prediction and provide
 comments for the conservation of both species.
description: <b><i>Genome size estimation by </i></b><b><i>Flow cytometry </i></b>— For GS estimation, six individuals from each species were analyzed in triplicate. For each sample, approximately 50 mg of young leaf tissue from <i>Macrotorus utriculatus </i>and <i>M. genuflexus</i> was macerated with approximately 25 mg of an appropriate internal reference standard leaf tissue (see Results). Both materials were macerated in 0.5 ml of cold Ebihara buffer (Ebihara et al. 2005) supplied with 0.025 µg mL<sup>-1</sup> RNAse using a scalpel blade to release the nuclei into suspension. Nuclei suspensions were stained by adding 12.5 µL of a 1 mg mL<sup>-1</sup> solution of propidium iodide (PI, Sigma). The analysis was performed using the FACSCanto II cytometer (Becton Dickinson, San Jose, CA, USA) kindly made available by the Microbiology and Immunology Department of IBB-UNESP (Botucatu, Brazil). The histograms were obtained with FACSDiva software based on 5,000 events, and the statistical evaluation was performed using the Flowing Software 2.5.1 (http://www.flowingsoftware.com/). The quality control of samples was based on the coefficient of variation (CV) of each measurement, which should be below 5%, and the standard deviation (SD) among 2C-values, which should be below 3%. Such limits ensure that the variations observed inside and among measurements are due to technical factors and should not represent intraspecific variation among individuals (Pellicer and Leitch 2014). <b><i>Chromosome analysis – Metaphase preparation and idiogram construction</i></b><b><i> </i></b>— The methodological procedures followed Guerra and Souza (2002). The roots collected were pretreated in antimitotic solution [0.002 M 8-hydroxyquinoline (8-HQ)] for 20–24 h at 10°C, fixed in absolute alcohol:glacial acetic acid (3:1, v:v) for 20–24 h, and stored at –20°C. For metaphase preparation, the fixed root tips were washed in distilled water, twice for five minutes each, and digested in a solution of 2% (w/v) cellulase (Onozuka)/20% (v/v) pectinase (Sigma)/1% macerozyme (Sigma) solution at 37°C for 45 min. The meristems were squashed in a drop of 60% acetic acid, and the coverslip was removed in liquid nitrogen. The selected chromosome preparations were stained with DAPI (1 ug ml<sup>-1</sup>) for 30 min and mounted with McIlvaine:glicerol pH 7.0 buffer (Guerra and Souza 2002). The slides were examined using an Olympus BX 53 epifluorescence microscope (Olympus Life Science), photographed with a coupled XM10 camera, and analyzed using Olympus CellSens software. The images were processed uniformly for color balance, contrast, and brightness using Adobe Photoshop CS5 (Adobe Systems, Inc.). The idiograms were constructed based on three metaphases from each species using KaryoType software (Altinordu et al. 2016) and the values of Total Haploid Lenght (THL) and the karyotype type following Stebbing (1971) are presented along with the haploid idiogram.