Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(3): 109-122, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1628 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Inês da Fonseca Simão, Hermenegildo Ribeiro da Costa, Hele- na Cristina Correia de Oliveira, Maria Helena Abreu Silva, Paulo Cardoso da Silveira (2022). Nuclear DNA 2C-values for 16 species from Timor-Leste increas- es taxonomical representation in tropi- cal ferns and lycophytes. Caryologia 75(3): 109-122. doi: 10.36253/caryolo- gia-1628 Received: April 14, 2022 Accepted: November 23, 2022 Published: April 5, 2023 Copyright: © 2022 Inês da Fonseca Simão, Hermenegildo Ribeiro da Costa, Helena Cristina Correia de Oliveira, Maria Helena Abreu Silva, Paulo Cardoso da Silveira. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID IS: 0000-0001-6548-8535 HC: 0000-0002-8059-2397 HO: 0000-0002-4673-0696 MS: 0000-0001-8060-2842 PS: 0000-0002-9253-5381 Nuclear DNA 2C-values for 16 species from Timor-Leste increases taxonomical representation in tropical ferns and lycophytes Inês da Fonseca Simão1, Hermenegildo Ribeiro da Costa1,2,3, Helena Cristina Correia de Oliveira1,2, Maria Helena Abreu Silva1,2, Paulo Cardoso da Silveira1,2,* 1 Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal 2 CESAM-Centre for Environmental and Marine Studies, Department of Biology, Univer- sity of Aveiro, 3810-193 Aveiro, Portugal 3Faculty of Education, Arts and Humanities, National University Timor Lorosa’e (UNTL), Avenida Cidade de Lisboa, Dili, East Timor * Ccorresponding author. E-mail: psilveira@ua.pt Abstract. Knowledge regarding genome size allows us to infer relationships between taxa, address questions related to systematics and contribute to biodiversity studies. However, currently, less than 3% of the described Pteridophyta species have genome size estimates reported in databases, and only around one third of these are tropical species, although the tropics are home of 86% of fern diversity. The region of Timor- Leste, included in one of the 25 hotspots of biodiversity, is considered one of the richest areas of the world in terms of pteridophyte species. Nonetheless, biodiversity- driven research focused on this territory’s biodiversity is scarce. This study presents novel 2C-values for 15 species of ferns collected in Timor-Leste, using flow cytometry. Furthermore, one species of the lycophyte Palhinhaea cernua (L.) Vasc. & Franco, was also studied and its estimated genome size compared to a previous report. Estimates ranged from 10.45 pg in Selliguea feei Bory to 29.7 pg in Microsorum punctatum (L.) Copel, and are considered medium-size genomes. The data was compared with previ- ous reports for closely related species. These are the first 2C-values for two families and seven genera of ferns, increasing the number of pteridophytes with reported C-values from 292 to 307. Keywords: genome size, chromosome, cytogenetics, DNA amount, nuclear DNA con- tent, Malesia, geographical distribution. INTRODUCTION Information regarding genome size plays a fundamental role in under- standing a species’ evolutionary history and is a tool that allows us to infer relationships between taxa, address questions related to cellular and devel- opmental biology and systematics, among others, and contributes to biodi- versity studies (Leitch 2005; Kumar et al. 2011). The considerable differences 110 Inês da Fonseca Simão et al. in nuclear DNA content across species can be related to adaptive features, which shows that genome size can be under selective pressure and its variations may be related to the evolutionary history of a given group (Ohri 1998). Currently, flow cytometry is the main technique used to obtain information related to species DNA C-value (Dolezel 2005). However, despite the importance of these studies, and the recent efforts concerning information about genome size in plants, there is still a substantial gap in knowledge, with only a very small portion of spe- cies studied, and more research is required. The majority of values reported in the Plant DNA C-value database (Release 7.1, April 2019:https://cvalues. science.kew.org/) (Leitch et al. 2019) belong to angio- sperms. The 2C-value for 10.770 species of angiosperms is known, corresponding to 3.3% of their global diver- sity (Antonelli et al. 2020). Pteridophytes are even more under-represented, with only 292 species reported in the database. These numbers account for 2.45% of the 11,916 species of pteridophytes described (PPG 2016). In 2001, Bennet & Leitch set the goal of obtaining the C-value for 200 pteridophytes species by 2005, with a special focus on those that maximize systematic and geographic representation (Bennet and Leitch 2001). Although this goal was met, further studies regarding this group are fundamental, since the pteridophytes represent an important evolutionary transition between bryophytes and spermatophytes and, as such, are criti- cal to our understanding of how DNA content has evolved across land plants (Bainard et al. 2011). Fur- thermore, since the laboratories adequately equipped to make 2C-values estimation are mostly located in tem- perate climate areas, with more difficult access to tropi- cal fern species, we suspected such species would be underrepresented in the Plant DNA C-value database. Yet, pteridophyte diversity in the tropics is significantly higher than in any other region of the globe. Estimates point to the existence of 4500 species of ferns and lyco- phytes in Southeast Asia, more than twice the num- ber of species of the entire Holarctic Kingdom (Moran 2008). At the same time, the region of Timor-Leste, located in Southeast Asia, is included in the biogeo- graphic region of Malesia, which is considered one of the richest areas of the world in terms of tropical pteri- dophyte species diversity (Ebihara and Kuo 2012). Addi- tionally, Timor-Leste is included in Wallacea, an area classified as one of the 25 hotspots of biodiversity iden- tified by Myers et al. (2000) as a priority of conservation at a global scale. Despite the rich biological patrimony of Timor-Leste, research focused on the country’s bio- diversity and genetic resources is lacking, mainly due to the military occupation of the territory that took place between 1975 and 1999 (Bouma and Kobryn 2004). In this sense, better coverage of pteridophy tes nuclear DNA values data in this territory is crucial to under- stand the mechanisms behind genome size evolution and their relationship with geographic and ecological factors (Dagher-Kharrat et al. 2013). Therefore, the aims of this paper are: 1. to check what percentage of genome size data from tropical pteridophytes has been estimated, comparing with other biogeographic regions; and 2. to expand knowledge about genome sizes of tropical fern species occurring in Timor-Leste. MATERIALS AND METHODS Plant material Prior to the field work, a search was conducted in the Plant DNA C-value database to establish which pteridophytes species, known to occur in Timor-Leste, had already 2C-values estimations published, and which had not. From the latter list, those species with popula- tions that could more easily be sampled were selected as target species for this study (Table 1). Leaves of 15 ferns and one lycophyte were collected from several field loca- tions in Timor-Leste (Table 1). These samples were kept fresh (at 0-5ºC) for a period no longer than a week and used for flow cytometry analysis. Voucher specimens were prepared and kept in the herbaria of the University of Aveiro (AVE) and Naturalis Biodiversity Center (L). Duplicates were also kept at the National University of East Timor (UNTL, Díli, Timor-Leste). Nuclear DNA content estimation The nuclear DNA content of fresh leaf samples was assessed using flow cytometry, currently the most used technique to estimate C/2C-value in plants for its sim- plicity, accuracy, convenience, and speed (Galbraith et al. 1983, 2009). The methodology used followed Loureiro et al. (2007), which included the preparation of nuclear suspensions by chopping 50 mg of leaf sample tissue and 50 mg of internal standard leaves, Vicia faba “Inovec” (2C= 26.90 pg; Dolezel, Sgorbati and Lucretii 1992) or Pisum sativum “Ctirad” (2C= 9.09 pg; Dolezel et al. 1992), with a razor blade in a glass Petri dish containing 1 mL of WPB isolation buffer (200 mM Tris.HCl, 4mM MgCl2.6H2O, 2 mM EDTA Na2.2H2O, 86 mM NaCl, 10 mM sodium metabisulfite, 1% PVP-10, 1% (v/v) Triton X-100, pH 7.5; Loureiro et al. 2007). The nuclear solution was then filtered through a nylon net of 50 µm, and 50 μg.mL-1 of propidium iodide (PI, Sigma-Aldrich, St. Lou- 1112C-values for pteridophytes from Timor is, MO, USA) and 50 μg.mL-1 of RNAse (Sigma-Aldrich, St. Louis, MO, USA) were added to the sample, to stain nuclear DNA and prevent staining of double stranded RNA, respectively. Samples were analyzed within a 10 min period on an Attune® Acoustic Focusing Cytometer (TermoFisher Scientific) equipped with a 488 nm laser. For each sample, at least 5,000 nuclei were ana- lyzed. As a quality control, nuclear DNA content esti- mates were only considered when the coefficient of vari- ation of G0/G1 peaks (CVpeak) were below 5%. Samples with higher CVpeak values were discarded and a new sample was prepared. For most of the taxa, three to five individuals were analyzed, but for Selliguea feei and Tectaria melanocau- los, only one individual for each of the species survived the time between sampling in Timor and analysis in Aveiro. The number of individuals measured for each population is provided in Table 1. Statistical analysis Descriptive statistics were calculated for each taxa studied namely, mean, standard deviation (SD), coef- ficient of variation (CV), and minimum and maximum values of the holoploid genome size (2C, pg). Table 1. Scientific names and localities of samples collected for this study. Voucher specimens are kept in the Herbarium of the University of Aveiro (AVE) and of the Naturalis Biodiversity Center (L). Family circumscription according with PPG (2016). Taxon Family Localities in Timor-Leste Lycopodiophyta Palhinhaea cernua (L.) Vasc. & Franco Lycopodiaceae Ainaro, roadside between Maubisse and Turiscai, [8°49’33” S, 125°38’10” E], Costa et al. 254 (AVE) Pteridophyta Calochlaena javanica (Blume) M.D.Turner & R.A.White Dicksoniaceae Ainaro, roadside from Maubisse to Turiscai, [8°49’22” S, 125°37’01” E], Costa et al. 245 (AVE, L.3959675) Pityrogramma calomelanos (L.) Link Adiantum philippense L. Pteris ensiformis Burm. Pteridaceae Pteridaceae Pteridaceae Ainaro, roadside between Maubisse and Turiscai, [8°49’33” S, 125°38’10” E], Costa et al. 253 (AVE) Liquiçá, roadside between Tibar and Faiten, [8°36’59” S, 125°29’09” E], Costa et al. 8 (AVE, L.3959700) Manufahi, roadside of Laclo, [8°51’28” S, 125°41’36” E], Costa et al. 320 (AVE) Blechnopsis orientalis (L.) C.Presl Blechnaceae Ainaro, roadside from Maubisse to Turiscai, [8°49’22” S, 125°37’01” E], Costa et al. 244 (AVE) Diplazium esculentum (Retz.) Sw. Athyriaceae Aileu, from Díli to Aileu, after the crossroad to Remexio and Remexio, [8°37’05” S, 125°38’25” E], Costa et al. 195 (AVE, L.3959688) Tectaria melanocaulos (Blume) Copel. Tectariaceae Aileu, Asumau, [8°37’19” S, 125°38’37”], Costa et al. 200 (AVE, L.3959765) Oleandra musifolia (Blume) C.Presl Oleandraceae Ainaro, roadside between Maubisse and Turiscai, [8°48’57” S, 125°38’39” E], Costa et al. 258 (AVE) Goniophlebium subauriculatum (Blume) C.Presl Microsorum punctatum (L.) Copel. Microsorum scolopendria (Burm.f.) Copel. Platycerium bifurcatum subsp. willinckii (T.Moore) Hennipman & M.C.Roos Pyrrosia lanceolata (Wall.) Farw. Pyrrosia longifolia (Burm.f.) C.V.Morton Selliguea feei Bory Polypodiaceae Polypodiaceae Polypodiaceae Polypodiaceae Polypodiaceae Polypodiaceae Polypodiaceae Viqueque, on the Waibua forest at foothills of Mundo Perdido mountain, [8°43’59” S, 126°22’10” E], Costa et al. 303 (AVE) Viqueque, on the Waibua forest at the foothills of Mundo Perdido mountain, [8°43’59” S, 126°22’10” E], Costa et al. 307 (AVE) Viqueque, on the Waibua forest at foothills of Mundo Perdido mountain, [8°43’59” S, 126°22’10” E], Costa et al. 301 (AVE) Díli, Dare, [8°35’38” S, 125°34’07” E], Costa et al. 84 (AVE, L.3959789) Aileu, roadside between Aileu and Maubisse, [8°48’16” S, 125°35’31” E], Costa et al. 238 (AVE) Viqueque, roadside of Urulita, [8°46’21” S, 126°22’11” E], Costa et al. 290 (AVE) Ainaro, Maubisse - Turiscai, at Rita-Uruho, [8°49’22” S, 125°37’01” E], Costa et al. 243 (AVE) 112 Inês da Fonseca Simão et al. Chromosome number The median of the chromosome numbers for 14 taxa was obtained from the online Chromosome Counts Database (CCDB) (Rice et al. 2015). Floristic kingdoms versus 2C values analysis The floristic kingdom’s classification by Takhtajan (1986) was applied to the Pteridophyta taxa whose DNA C-values are available in the Plant DNA C-value data- base. For that, Global Biodiversity Information Facility (GBIF, at https://www.gbif.org/, January 2022) was con- sulted to establish each species’ main occurrence. Finally, the distribution of species listed in the Plant DNA C-val- ue database by each floristic kingdom was compared with the equivalent distribution of the total World number of Pteridophyte species given by Moran (2008). For this comparison, the Paleotropical and the Cape f loristic kingdoms had to be included in the same group, because Moran (2008) gives a single total number for Africa, without segregating the Cape floristic kingdom. The same was not adopted for the Holantarctic kingdom, because Moran (2008), provides separate figures for New Zealand, which allows some separation from other kingdoms. In South America no separation was possible between the Holantarctic and the Neotropical kingdoms, but since the number of Neotropical species should be much greater than the Holantarctic species present in the region, we assumed that the error would not be critical. RESULTS DNA content estimates were obtained for the 16 samples, 15 of them representing taxa with no previ- ous 2C-value reported. These estimates, as well as the chromosome median 2n value that are described in lit- erature, are presented in Table 2. The 2C DNA content ranged from 10.45 pg in Selliguea feei Bory, with the Vicia faba standard, to 29.7 pg in Microsorum punctat- um (L.) Copel. with the Pisum sativum standard. The average 2C-value for Polypodiopsida was 20.62 pg, and for Lycopodiophyta, represented only by one taxon, the 2C-value was 25.65 pg. The coefficients of variation (CVs) for the samples varied between 3.7% and 6.7%. The list of Pteridophyte taxa for which nuclear DNA 2C-values have been published in the Plant DNA C-value database (Leitch 2019) is presented in the Sup- plementary Material 1, alongside with the Tak hta- jan’s floristic kingdoms (Takhtajan 1986) embraced by their geographical distributions ranges. This informa- tion is summarized in Table 3, alongside with the total world estimated number, and percentage, of Pterido- phyte species for each f loristic kingdom, according with Moran (2008). We can see in this table, that the Paleotropical+Cape kingdoms, together with the Neo- tropical floristic kingdoms, with 45% and 42%, respec- tively, include the vast majority of the world’s pterido- phyte diversity (87%). Contrariwise, the most diverse group of pteridophytes whose nuclear DNA 2C-values are known is the Holarctic, with 44%, followed by the Paleotropical+Cape, with only 23% and the Neotropical with 18%. With this study, the percentages of Holarctic species is reduced to 42%, and the percentage of species from Paleotropical+Cape area increases to 25%. DISCUSSION In spite of the long journey between the field in Timor-Leste and the cytometry laboratory in Aveiro, where the analysis was done, we succeed to analyze, at least, three individuals for 14 of the 16 species, and five/ six, for nine of the 16 species. The higher intraspecific variations detected are, most likely, related to difficulties associated to the flow cytometry technique, since the easiness of obtaining data differs between the taxa, as mentioned by Ober- mayer, et al. (2002). Following Leitch, Chase & Bennet (1998) genome size classification, all taxa have “intermediate” genomes (7<2C≤28 pg). The median value established for genome size in ferns is 22.8 pg/2C and it has been related, par- tially, to variation in post-polyploidization processes- such as additional chromosomes and DNA arising from whole genome duplications-, since diploidization is not linked with genome downsizing in ferns in opposition to angiosperms, a group with smaller genomes (median= 3.4 pg/2C) (Liu et al. 2019). Regarding the lycophytes, the median 2C-value for the group is 0.26 pg (Liu et al. 2019), corresponding to a very small genome (≤2.8 pg) (Leitch et al. 1998). Despite the 2C-value previously reported in the literature of 2.75 pg for Palhinhaea cer- nua (L.) Vasc. & Franco (Kuo et al. 2016), the 2C-value estimated for this species is 25.65 pg, corresponding to the “intermediate” category and to the highest genome size in the Lycopodiaceae family reported until present, more than twice that of Huperzia lucidula (Michx.) Tre- vis., which has 11.28 pg (Bainard et al. 2011) and was the previous highest value reported. Considering that the coefficient of variation for this estimate is 5.5%, it doesn’t seem likely that the 2C-value for P. cernua was 1132C-values for pteridophytes from Timor Ta bl e 2. M ea n 2C -v al ue e st im at es ( pg ) fo r 15 f er n sp ec ie s an d 1 ly co ph yt e co lle ct ed in E as t- T im or , w ith s ta nd ar d de vi at io n (S D ), m in im um a nd m ax im um v al ue s, a ve ra ge c oe ffi ci en t of v ar ia ti on ( C V % ) fo r ea ch t ax on . F am ily c ir cu m sc ri pt io n ac co rd in g w ith P PG ( 20 16 ). E st im at es o bt ai ne d us in g th e V ic ia f ab a st an da rd ( 2C = 2 6. 90 ) ar e id en ti fie d w ith “ *” . Th e re m ai ni ng m ea su re m en ts w er e ob ta in ed u si ng t he P is um s at iv um s ta nd ar d (2 C = 9. 09 p g) . R ep or te d ch ro m os om e nu m be r (m ed ia n n va lu e) f or t he t ax a av ai la bl e is a ls o pr es en te d, ac co rd in g to th e C C D B d at ab as e (r el ea se 1 .5 8, h tt p: // cc db .ta u. ac .il /) . Ta xo n Fa m ily M ed ia n 2n v al ue G en om e si ze ( 2C , p g) n. s am pl es M ea n ± SD M in . M ax . A ve ra ge C V ( % ) Ly co po di op hy ta Pa lh in ha ea c er nu a (L .) V as c. & F ra nc o Ly co po di ac ea e 2 08 , 2 20 , 2 72 , 3 12 , 3 30 , 34 0, 4 16 25 .6 5 ± 0. 43 25 .3 2 26 .3 2 5. 52 5 P te ri do ph yt a C al oc hl ae na ja va ni ca ( Bl um e) M .D .T ur ne r & R .A .W hi te D ic ks on ia ce ae ? 11 .4 1 ± 0. 11 11 .3 2 11 .4 3 4. 78 5 Pi ty ro gr am m a ca lo m el an os ( L. ) Li nk Pt er id ac ea e 23 2, 2 40 26 .4 1 ± 0. 36 26 .1 2 26 .8 5 6. 72 4 A di an tu m p hi lip pe ns e L. 60 , 9 0 21 .9 2* ± 2 .3 18 .4 8 23 .2 9 4. 03 4 Pt er is e ns ifo rm is B ur m . 58 , 8 7- 88 , 1 16 , 1 68 , 1 85 19 .1 5* ± 0 .5 5 18 .7 1 19 .8 1 4. 71 5 Bl ec hn op si s or ie nt al is ( L. ) C .P re sl Bl ec hn ac ea e ? 13 .5 7* ± 0 .1 1 13 .4 3 13 .6 1 5. 91 5 D ip la zi um e sc ul en tu m ( R et z. ) Sw . A th yr ia ce ae 82 22 .6 8 ± 0. 70 22 23 .5 7 5. 88 4 Te ct ar ia m el an oc au lo s (B lu m e) C op el . Te ct ar ia ce ae ? 24 .6 8 - - 3. 69 1 O le an dr a m us ifo lia ( Bl um e) C .P re sl O le an dr ac ea e 80 13 .6 5* ± 0 .0 8 13 .5 7 13 .7 5 6. 11 5 G on io ph le bi um s ub au ri cu la tu m ( Bl um e) C .P re sl Po ly po di ac ea e 72 21 .0 6* ± 0 .3 8 20 .5 9 21 .5 2 4. 98 5 M ic ro so ru m p un ct at um ( L. ) C op el . 72 29 .7 2 ± 0. 44 29 .4 3 30 .2 3 4. 56 3 M ic ro so ru m s co lo pe nd ri a (B ur m .f. ) C op el . 36 ** 24 .5 5 ± 1. 92 21 .1 4 25 .7 6 4. 53 5 Pl at yc er iu m b ifu rc at um s ub sp . w ill in ck ii (T .M oo re ) H en ni pm an & M .C .R oo s 74 28 .4 7 ± 2. 88 23 .7 4 30 .8 3. 93 5 Py rr os ia la nc eo la ta ( W al l.) F ar w . 74 23 .7 3 ± 0. 31 23 .4 7 24 .1 6 5. 34 4 Py rr os ia lo ng ifo lia ( Bu rm .f. ) C .V .M or to n 74 28 .7 9 ± 3. 58 26 .0 6 35 .7 4. 96 6 Se lli gu ea fe ei B or y 74 10 .4 5* - - 5. 44 1 ** n v al ue p re se nt ed , n o 2n v al ue r ep or te d. 114 Inês da Fonseca Simão et al. negatively influenced by artefacts such as the presence of interfering secondary metabolites (Hanusová et al. 2014). This novel result shows that genome size within the Lycopodiaceae family may be more variable than what was thought until now In fact, the chromosome numbers reported for this species varies from n=34 to 2n=208-416 (Rice et al. 2015). Comparing the 2C-value of Diplazium esculen- tum (Retz.) Sw. (22.68 pg) with Diplazium pycnocarpon (Sprengel) M. Broun (12.63 pg), the only other species of the same genus that has been screened for its genome size by Bainard et al. (2011), the 2C-value differs by approx. 10 pg. This variation shows that even within the same genus, genome size may vary greatly, regardless of the two species’ chromosome number being very similar, with D. esculentum (2n=82) and D. pycnocarpon (2n=80). The same conclusion can be drawn when comparing our estimate for Adiantum philippense L., (2C= 21.9 pg) with previous work on the genus: 2C-value estimates reported for Adiantum pedantum L. are 10.16 pg (Bainard et al. 2011) and for Adiantum aleuticum (Rupr.) C. A. Paris are 11.42 pg (an approx. difference of 10.5 pg) (Clark et al. 2016). The 2C-value discrepancy between Adiantum spe- cies may be related, most probably, to differences in chro- mosome numbers between taxa, since both 2n=60 and 2n=90 have been reported for A. philippense in literature. Although 2n=60 is similar to chromosome number for A. pedantum and A. aleuticum (2n=58), a 2n=90 could be a reason to explain this variation. The 2C-value discrepancy between Adiantum species may also be related, in part, to the different geographical origin of the material. Some evidence points towards the prevalence of smaller genomes in plant species that exist in harsher, drier, environments, with shorter growing seasons (Knight, Molinari and Petrov 2005). But check- ing this would require investigations out of the scope of this paper. What we could contribute was towards improving the representation of the most diverse phytogeographi- cal kingdoms for this group (Table 3), following Moran’s (2008) suggestion that this group of organisms shows a dominant pattern called “the latitudinal diversity gradi- ent”, which means that species diversity in ferns increas- es from the pole towards the equator (Moran 2008). Despite this pattern, almost half of the studied species found in the Plant DNA C-value database (Leitch et al. 2019) belong to the Holarctic kingdom. Therefore, an already understudied group of plants in terms of genome size lacks, to a great extent, estimates from species of the most representative phytogeographical kingdoms for this group, which we tried to counteract with the new data presented in this study (Table 3). CONCLUSIONS The present work includes novel data that con- tributes to the knowledge regarding genome size of 15 species of ferns and 1 species of lycophytes. Our data increases the taxonomic representation of DNA content in pteridophytes databases by two families- Blechnaceae and Oleandraceae-, as well as seven genera (Blechnopsis, Goniophlebium, Microsorum, Palhinhaea, Pityrogramma, Pyrrosia and Selliguea). Furthermore, the representa- tion of Paleotropical fern species has increased by 2%. However, with almost 12.000 species of pteridophytes described to date, further work focused on the DNA content of more lycophyte and fern species, especially from tropical regions, is crucial to expand taxonomic representation and fill in the phylogenetic gaps within the group. Although we could not perform chromosome counts alongside with the 2C value estimations, this should be a future target, allowing to draw more complete conclu- Table 3. Distribution of the number and percentage of species of Pteridophytes recognized by each of Takhtajan’s floristic kingdoms com- paring with the same distribution in terms of species with published DNA C-values including the contribution of this study. Takhtajan’s floristic kingdoms No. (%) of species estimated* No. (%) of species with known DNA C-values No. of species added in this study** Current No. (%) of species with known DNA C-values Holartic 1470 (9.4) 190 (44) 188 (42) Neotropical 6500 ((41.7) 76 (18) 4 80 (18) Paleotropical + Cape 6980 (44.7) 94 (23) 16 110 (25) Australian 456 (2.9) 37 (8) 5 42 (9) Holantarctic 193 (1.2) 29 (7) 29 (6) Total 15599 (100) 429 (100) 25 454 (100) * Numbers of species estimated to occur taken from Moran (2008: 369); ** the numbers presented exceed the 16 species analyzed, because several of them are distributed among more than one floristic kingdom, as it was also adopted by Moran (2008). 1152C-values for pteridophytes from Timor sions about the genome of the studied species, namely, concerning ploidy levels. Bearing this in mind, in spite of the relatively modest contribution in terms of species numbers (not so modest when we consider the number of new fami- lies and genera), this paper increases the representa- tion of tropical Pteridophy te diversity whose nuclear 2C-va lues are k nown, and highlights t hat f ur t her studies on genome size in ferns are crucial, especial- ly in species from areas that are considered hotspots of tropica l fern biodiversit y, such as Timor-Leste. The lack of studies on the country’s biodiversity cou- pled with the human impact in the region, makes the execution of these studies even more important, since genome size data is basic information for an appropri- ate management and conservation of the plant genetic resources of the area. ACKNOWLEDGEMENTS We are grateful to the Timor-Leste authorities, spe- cially the Minister of Agriculture and Fisheries and the Quarentine, for allowing the collection and the export of samples by H.R.Costa, and to U.N.T.L. 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Genus Species Subspecies/Variety Phytogeographical region(s) Acrostichum aureum Neotropical, Palaeotropical, Australian Adiantum aleuticum Holarctic Adiantum capillus-veneris Holarctic,Neotropical, Palaeotropical, Australian, Holantarctic Adiantum pedatum Holarctic Adiantum venustum Holarctic Alsophila spinulosa Holarctic, Palaeotropical Amauropelta bergiana var. bergiana Palaeotropical Anemia collina Neotropical Anemia phyllitidis Neotropical Anemia rotundifolia Neotropical Anemia tomentosa Neotropical Angiopteris latipinna Holarctic Angiopteris lygodiifolia Holarctic Angiopteris pruinosa Palaeotropical Arthropteris orientalis Palaeotropical Asplenium achilleifolium Neotropical Asplenium adiantum-nigrum var. adiantum-nigrum Holarctic, Palaeotropical Asplenium adulterinum Holarctic Asplenium aethiopicum subsp. tripinnatum Palaeotropical Asplenium Aethiopicum subsp. dodecaploideum Palaeotropical Asplenium billotii Holarctic Asplenium boreale Holarctic Asplenium caucasicum Holarctic Asplenium ceterach Holarctic Asplenium cristatum Neotropical Asplenium cuneifolium Holarctic Asplenium dalhousiae Holarctic Asplenium daucifolium Palaeotropical Asplenium flabellifolium Australian, Holantarctic Asplenium griffithianum Holarctic, Palaeotropical Asplenium hallbergii Neotropical Asplenium hemionitis Holarctic Asplenium javorkeanum Holarctic Asplenium lividum Palaeotropical Asplenium marinum Holarctic Asplenium mauritiensis Palaeotropical Asplenium myriophyllum Neotropical Asplenium neolaserpitifolium Palaeotropical Asplenium nidus Holarctic,Neotropical, Palaeotropical, Australian Asplenium obtusatum Neotropical, Palaeotropical, Australian, Holantarctic Asplenium onopteris Holarctic Asplenium quadrivalens Holarctic Asplenium rhizophyllum Holarctic Asplenium richardii Holantarctic Asplenium ruta-muraria Holarctic Asplenium scolopendrium Holarctic, Holantarctic Asplenium septentrionale Holarctic Asplenium subglandulosum Australian, Holantarctic Asplenium tenerum compl Palaeotropical 118 Inês da Fonseca Simão et al. Genus Species Subspecies/Variety Phytogeographical region(s) Asplenium trichomanes Holarctic, Neotropical, Palaeotropical, Australian, Holantarctic Asplenium trichomanes subsp. quadrivalens Holarctic, Palaeotropical Asplenium varians Holarctic, Palaeotropical Asplenium Víride Holarctic Asplenium viviparum Palaeotropical Asplenium x- lolegnamense Holarctic Asplenium x-lucrosum Holantarctic Asplenium x-poscharskyanum Holarctic Athyrium filix-femina var. angustum Holarctic Azolla microphylla Holarctic, Neotropical Blechnum microphyllum Neotropical Blechnum nudum Australian, Holantarctic Blechnum spicant Holarctic Bolbitis heudelotii Palaeotropical Bolbitis singaporensis Palaeotropical Botrychium neolunaria Holarctic Botrychium alaskense Holarctic Botrychium boreale Holarctic Botrychium echo Holarctic Botrychium hesperium Holarctic Botrychium lanceolatum Holarctic Botrychium lunaria Holarctic, Australian Botrychium matricariifolium Holarctic Botrychium michiganense Holarctic Botrychium minganense Holarctic Botrychium montanum Holarctic Botrychium pallidum Holarctic Botrychium pinnatum Holarctic Botrychium simplex Holarctic Botrychium spathulatum Holarctic Botrychium virginianum Holarctic Botrypus cf. virginianus Holarctic, Neotropical Brainea insignis Palaeotropical Calochlaena dubia Australian Ceratopteris thalictroides Holarctic, Neotropical, Palaeotropical, Australian Ceterach officinarum subsp. officinarum Holarctic Cheilanthes marantae Holarctic Cibotium barometz Palaeotropical Cibotium hawaiense Palaeotropical Cryptogramma crispa Holarctic Ctenitis sinii Holarctic Culcita macrocarpa Holarctic Cyathea crinita Palaeotropical Cyclosorus arbusculus Palaeotropical Cyclosorus asperum Palaeotropical Cyclosorus dentatus Holarctic, Palaeotropical Cystopteris bulbifera Holarctic Cystopteris dickieana Holarctic Cystopteris fragilis agg. Holarctic, Neotropical, Cape, Holantarctic Cystopteris tenuis Holarctic Danaea antillensis Neotropical 1192C-values for pteridophytes from Timor Genus Species Subspecies/Variety Phytogeographical region(s) Danaea kalevala Neotropical Danaea mazeana Neotropical Davallia denticulata var. denticulata Palaeotropical, Australian Davallia tyermanii Holarctic Dendrolycopodium dendroideum Holarctic Dendrolycopodium obscurum Holarctic Dennstaedtia globulifera Neotropical Dennstaedtia wilfordii Holarctic Deparia acrostichoides Holarctic Deparia boryana Holarctic, Palaeotropical Deparia japonica Holarctic, Palaeotropical Dicksonia antarctica Holarctic, Australian Dicranopteris linearis Holarctic, Neotropical, Palaeotropical, Australian, Holantarctic Diphasiastrum alpinum Holarctic Diphasiastrum digitatum Holarctic Diphasiastrum complanatum Holarctic, Neotropical, Palaeotropical Diphasiastrum tristachyum Holarctic Diplazium arborescens Palaeotropical Diplazium australe Palaeotropical, Australian, Holantarctic Diplazium proliferum Palaeotropical, Australian Diplazium pycnocarpon Holarctic Diplopterygium bancroftii Neotropical Dipteris chinensis Holarctic Dracoglossum plantagineum Neotropical Drynaria heraclea Palaeotropical Dryopteris bernieri Palaeotropical Dryopteris carthusiana Holarctic Dryopteris clintoniana Holarctic Dryopteris cristata Holarctic Dryopteris cycadina Holarctic, Holantarctic Dryopteris dilatata Holarctic, Holantarctic Dryopteris filix-mas Holarctic, Neotropical, Holantarctic Dryopteris goldiana Holarctic Dryopteris intermedia Holarctic Dryopteris marginalis Holarctic Elaphoglossum aubertii Palaeotropical Elaphoglossum crinitum Neotropical Elaphoglossum hybridum Neotropical, Palaeotropical Elaphoglossum lepervanchii Palaeotropical Equisetum arvense Holarctic, Holantarctic Equisetum bogotense Neotropical Equisetum moorei Holarctic Equisetum fluviatile Holarctic Equisetum giganteum Neotropical Equisetum hyemale Holarctic, Neotropical, Australian, Holantarctic Equisetum laevigatum Holarctic, Neotropical Equisetum myriochaetum Neotropical Equisetum palustre Holarctic Equisetum pratense Holarctic Equisetum ramosissimum subsp. ramosissimum Holarctic, Palaeotropical Equisetum scirpoides Holarctic 120 Inês da Fonseca Simão et al. Genus Species Subspecies/Variety Phytogeographical region(s) Equisetum sylvaticum Holarctic Equisetum variegatum Holarctic Gymnocarpium dryopteris Holarctic Gymnocarpium fedtschenkoanum Holarctic Gymnocarpium robertianum Holarctic Gymnosphaera podophylla Holarctic, Palaeotropical Huperzia lucidula holarctic Hymenophyllum badium cf Holarctic, Palaeotropical Hymenophyllum sibthorpioides Palaeotropical Isoetes engelmannii Holarctic Isoetes lacustris Holarctic Lepisorus excavatus Palaeotropical Lindsaea ensifolia Palaeotropical, Australian Llavea cordifolia Neotropical Lonchitis occidentalis Palaeotropical Loxsoma cunninghami Holantarctic Lycopodium annotinum Holarctic Lycopodium clavatum Holarctic, Neotropical, Palaeotropical Lycopodium dendroideum Holarctic Lycopodium obscurum Holarctic Lygodium japonicum Holarctic, Neotropical, Palaeotropical, Australian Lygodium microphyllum Holarctic, Palaeotropical, Australian Lygodium volubile Neotropical Marattia purpurascens Holarctic Marsilea quadrifolia Holarctic, Neotropical, Palaeotropical Matteuccia struthiopteris var. pensylvanica Holarctic Megalastrum macrotheca Neotropical Mickelia nicotianifolia Neotropical, Palaeotropical Microgramma percussa Neotropical, Palaeotropical Microlepia speluncae Neotropical, Palaeotropical, Australian Microlepia strigosa Holarctic, Palaeotropical Nephrolepis biserrata Neotropical, Palaeotropical, Australian Nephrolepis cordifolia ‘Duffi’ Holarctic, Neotropical, Palaeotropical, Australian, Holantarctic Nephrolepis exaltata Holarctic, Neotropical, Palaeotropical, Australian Oleandra neriiformis Palaeotropical, Australian Onoclea orientalis Holarctic Onoclea sensibilis Holarctic Onychium lucidum Holarctic, Palaeotropical Ophioglossum gramineum Palaetropical, Australian Ophioglossum pendulum Palaeotropical, Australian Ophioglossum petiolatum Holarctic, Palaetropical, Holantarctic Osmunda cinnamomea Holarctic, Neotropical Osmunda claytoniana Holarctic Osmunda regalis var. spectabilis Holarctic, Neotropical Paragymnopteris marantae Holarctic Paragymnopteris vestita Holarctic Pellaea atropurpurea Holarctic, Neotropical Pellaea glabella subsp. glabella Holarctic Phegopteris connectilis Holarctic Phyllitis scolopendrium subsp. scolopendrium Holarctic Plagiogyria matsumureana Holarctic 1212C-values for pteridophytes from Timor Genus Species Subspecies/Variety Phytogeographical region(s) Platycerium coronarium Palaeotropical Pleopeltis macrocarpa Neotropical, Palaeotropical Polyphlebium capillaceum Neotropical Polypodium australe Holarctic Polypodium cambricum Holarctic Polypodium glycyrrhiza Holarctic Polypodium interjectum Holarctic Polypodium scouleri Holarctic Polypodium virginianum Holarctic Polypodium vulgare Holarctic, Neotropical, Cape, Holantarctic Polypodium Vulgare x interjectum Not defined Polypodium x-font-queri Holarctic Polypodium x-mantoniae Holarctic Polypodium x-shivasiae Holarctic Polystichum acrostichoides Holarctic Psilotum nudum Holarctic, Palaeotropical, Neotropical, Australian, Holantarctic Pteridium aquilinum Holarctic, Neotropical, Palaeotropical, Australian Pteridium revolutum Palaeotropical Pteridium subsp. caudatum var. arachnoideum Neotropical Pteridrys cnemidaria Palaeotropical Pteris croesus Palaeotropical Pteris linearis Palaeotropical, Neotropical Pteris pseudolonchitis Palaeotropical Pteris vittata Holarctic, Neotropical, Palaeotropical, Holantarctic, Australian Ptisana salicina Holantarctic, Palaeotropical Pyrrosia lingua Holarctic, Palaeotropical Saccoloma domingense Neotropical Sadleria cyatheoides Palaeotropical Salvinia molesta Holarctic, Neotropical, Palaeotropical, Holantarctic, Australian Selaginella apoda Holarctic, Neotropical Selaginella arenicola Holarctic Selaginella arizonica Holarctic Selaginella asprella Holarctic Selaginella bigelovii Holarctic Selaginella braunii Holarctic Selaginella cinerascens Holarctic Selaginella densa Holarctic Selaginella eremophila Holarctic Selaginella exaltata Neotropical Selaginella extensa Neotropical Selaginella flabellata Neotropical Selaginella hansenii Holarctic Selaginella helvetica Holarctic Selaginella involvens Holarctic, Palaeotropical Selaginella kraussiana var. poulteri Holarctic Selaginella kraussiana Holarctic, Neotropical, Holantarctic, Palaeotropical, Australian Selaginella landii Neotropical Selaginella lepidophylla Holarctic, Neotropical Selaginella leucobryoides Holarctic Selaginella martensii Neotropical Selaginella moellendorffii Holarctic, Holantarctic, Palaeotropical 122 Inês da Fonseca Simão et al. Genus Species Subspecies/Variety Phytogeographical region(s) Selaginella mutica Holarctic Selaginella oregana Holarctic Selaginella pallescens Holarctic, Neotropical Selaginella peruviana Neotropical Selaginella pilifera Neotropical Selaginella pulcherrima Neotropical Selaginella rupestris Holarctic Selaginella rupincola Holarctic Selaginella selaginoides Holarctic Selaginella sellowii Neotropical, Holarctic Selaginella tortipila Holarctic Selaginella uncinata Holarctic, Palaeotropical Selaginella underwoodii Holarctic Selaginella vogelii Palaeotropical Selaginella wallacei Holarctic Selaginella watsonii Holarctic Selaginella weatherbiana Holarctic Selaginella willdenowii Holarctic, Neotropical, Palaeotropical, Australian Selaginella wrightii Holarctic, Neotropical Serpocaulon triseriale Neotropical Sphaeropteris lepifera Neotropical, Palaeotropical Spinulum annotinum Holarctic Stenochlaena tenuifolia Palaeotropical Tectaria zeilanica Palaeotropical Thelypteris noveboracensis Holarctic Thelypteris palustris var. pubescens Holarctic Thyrsopteris elegans Neotropical Tmesipteris obliqua Australian Tmesipteris tannensis Holantarctic Todea barbara Palaeotropical, Australian, Holantarctic Trichomanes speciosum Holarctic Vandenboschia auriculata Holarctic, Palaeotropical Vandenboschia davallioides Palaeotropical Vittaria lineata Neotropical, Palaeotropical Woodsia alpina Holartic Woodsia ilvensis Holartic Woodsia pulchella Holartic Woodwardia fimbriata Holartic Woodwardia unigemata Holarctic, Palaeotropical Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 75, Issue 3 - 2022 Firenze University Press Chromosome Mapping of Repetitive DNAs in the Picasso Triggerfish (Rhinecanthus aculeatus (Linnaeus, 1758)) in Family Balistidae by Classical and Molecular Cytogenetic Techniques Kamika Sribenja1, Alongklod Tanomtong1, Nuntaporn Getlekha2,* Chromosome number of some Satureja species from Turkey Esra Kavcı1, Esra Martin1, Halil Erhan Eroğlu2,*, Fatih Serdar Yıldırım3 L-Ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations Selma Tabur1,*, Nai̇me Büyükkaya Bayraktar2, Serkan Özmen1 Characterization of the chromosomes of sotol (Dasylirion cedrosanum Trel.) using cytogenetic banding techniques Kristel Ramírez-Matadamas1, Elva Irene Cortés-Gutiérrez2, Sergio Moreno-Limón2, Catalina García-Vielma1,* Contributions of species Rineloricaria pentamaculata (Loricariidae:Loricariinae) in a karyoevolutionary context A Cius¹, CA Lorscheider2, LM Barbosa¹, AC Prizon¹, CH Zawadzki3, LA Borin-Carvalho¹, FE Porto4, ALB Portela-Castro1,4 Cadmium induced genotoxicity and antioxidative defense system in lentil (Lens culinaris Medik.) genotype Durre Shahwar1,2,*, Zeba Khan3, Mohammad Yunus Khalil Ansari1 Biogenic synthesis of noble metal nanoparticles using Melissa officinalis L. and Salvia officinalis L. extracts and evaluation of their biosafety potential Denisa Manolescu1,2, Georgiana Uță1,2,*, Anca Șuțan3, Cătălin Ducu1, Alin Din1, Sorin Moga1, Denis Negrea1, Andrei Biță4, Ludovic Bejenaru4, Cornelia Bejenaru5, Speranța Avram2 Polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel (Foeniculum vulgare Mill.) 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