Rf Genes Research Articles

Marker-assisted breeding for rf1, a nuclear gene controlling A1 CMS in sorghum (Sorghum bicolor L. Moench)

A study on marker-assisted selection (MAS) for the rf1 gene, which controls pollen sterility in the sorghum A1 cytoplasm, was conducted on the offspring population of two crosses between a maintainer line, BTx-622, and two sweet sorghum lines, BJ-299 and Lunen-2, to test the effectiveness of the MAS method and develop maintainer lines with sweet and juicy stalks and corresponding cytoplasmic-nuclear male sterility (CMS) lines. The simple sequence repeat marker Xtxp18 exhibited a high accuracy (95.098 %) for selecting recessive homozygotes for the rf1 gene. The segregation ratio matched the expected ratio calculated according to the reported genetic distance in the F2 population of the two crosses used. Finally, four excellent maintainer lines/CMS line pairs (F5/BC3) with high stalk juice and stalk juice sugar contents were developed. The MAS method based on Xtxp18 for the sorghum rf1 gene could be used for hybrid breeding programs at a low cost in the future.

The mechanism of ORFH79 suppression with the artificial restorer fertility gene Mt‐GRP162

The restoration fertility complex (RFC) was previously identified in Honglian (HL)-cytoplasmic male sterility (CMS) rice (Oryza sativa), and glycine-rich protein 162 (GRP162) is responsible for binding to the CMS-associated transcript atp6-orfH79. Here, we engineered a recombinant GRP162 containing the mitochondrial transit peptide, termed Mt-GRP162, as an artificial restorer of fertility (Rf) gene. Mt-GRP162 was confirmed to bind to CMS-associated RNA and to localize to the mitochondria. The transgenic plants showed restored fertility with partially functional pollen. We found that the expression of ORFH79 decreased in transgenic plants, while the expression of atp6-orfH79 was not changed. These findings indicate that Mt-GRP162 restores fertility by suppressing the expression of the cytotoxic protein ORFH79 at the post-transcriptional level rather than via the cleavage of atp6-orfH79 in the presence of RFC. These findings contribute to our understanding of the mechanisms of restoration through diverse pathways.

A detrimental mitochondrial-nuclear interaction causes cytoplasmic male sterility in rice

Plant cytoplasmic male sterility (CMS) results from incompatibilities between the organellar and nuclear genomes and prevents self pollination, enabling hybrid crop breeding to increase yields. The Wild Abortive CMS (CMS-WA) has been exploited in the majority of 'three-line' hybrid rice production since the 1970s, but the molecular basis of this trait remains unknown. Here we report that a new mitochondrial gene, WA352, which originated recently in wild rice, confers CMS-WA because the protein it encodes interacts with the nuclear-encoded mitochondrial protein COX11. In CMS-WA lines, WA352 accumulates preferentially in the anther tapetum, thereby inhibiting COX11 function in peroxide metabolism and triggering premature tapetal programmed cell death and consequent pollen abortion. WA352-induced sterility can be suppressed by two restorer-of-fertility (Rf) genes, suggesting the existence of different mechanisms to counteract deleterious cytoplasmic factors. Thus, CMS-related cytoplasmic-nuclear incompatibility is driven by a detrimental interaction between a newly evolved mitochondrial gene and a conserved, essential nuclear gene.

Identification of the predominant nonrestoring allele for Owen-type cytoplasmic male sterility in sugar beet (Beta vulgaris L.): development of molecular markers for the maintainer genotype

Hybrid seed production in sugar beet relies on cytoplasmic male sterility (CMS). As time-consuming and laborious test crosses with a CMS tester are necessary to identify maintainer lines, development of a marker-assisted selection method for the rf gene (the nonrestoring allele of restorer-of-fertility locus) is highly desirable for sugar-beet breeding. To develop such a method, we investigated genetic variation at the Rf1 locus, one of two Rf loci known in sugar beet. After HindIII-digestion, genomic DNAs from beet plants known to have a restoring Rf1 allele yielded a range of hybridization patterns on agarose gels, indicating that Rf1 is a multi-allelic locus. However, the hybridization patterns of 22 of 23 maintainer lines were indistinguishable. The nucleotide sequences of the rf1 coding regions of these 22 maintainer lines were found to be identical, confirming that the lines had the same rf1 allele. Two PCR markers were developed that targeted a downstream intergenic sequence and an intron of Rf1. The electrophoretic patterns of both markers indicated multiple Rf1 alleles, one of which, named the dd(L) type, was associated with the maintainer genotype. To test the validity of marker-assisted selection, 147 sugar beet plants were genotyped using these markers. Additionally, the 147 sugar beet plants were crossed with CMS plants to determine whether they possessed the maintainer genotype. Analysis of 5038 F1 offspring showed that 53 % of the dd(L) plants, but none of the plants with other alleles, had the maintainer genotype. Thus, selection for the dd(L) type considerably enriched the proportion of plants with the maintainer genotype.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-013-9854-8) contains supplementary material, which is available to authorized users.

Diversifying Sunflower Germplasm by Integration and Mapping of a Novel Male Fertility Restoration Gene

The combination of a single cytoplasmic male-sterile (CMS) PET-1 and the corresponding fertility restoration (Rf) gene Rf1 is used for commercial hybrid sunflower (Helianthus annuus L., 2n = 34) seed production worldwide. A new CMS line 514A was recently developed with H. tuberosus cytoplasm. However, 33 maintainers and restorers for CMS PET-1 and 20 additional tester lines failed to restore the fertility of CMS 514A. Here, we report the discovery, characterization, and molecular mapping of a novel Rf gene for CMS 514A derived from an amphiploid (Amp H. angustifolius/P 21, 2n = 68). Progeny analysis of the male-fertile (MF) plants (2n = 35) suggested that this gene, designated Rf6, was located on a single alien chromosome. Genomic in situ hybridization (GISH) indicated that Rf6 was on a chromosome with a small segment translocation on the long arm in the MF progenies (2n = 34). Rf6 was mapped to linkage group (LG) 3 of the sunflower SSR map. Eight markers were identified to be linked to this gene, covering a distance of 10.8 cM. Two markers, ORS13 and ORS1114, were only 1.6 cM away from the gene. Severe segregation distortions were observed for both the fertility trait and the linked marker loci, suggesting the possibility of a low frequency of recombination or gamete selection in this region. This study discovered a new CMS/Rf gene system derived from wild species and provided significant insight into the genetic basis of this system. This will diversify the germplasm for sunflower breeding and facilitate understanding of the interaction between the cytoplasm and nuclear genes.

Identification of Fertility Restorer Genes in Cotton through DNA Markers

Molecular markers, particularly, STS markers are highly specific for regions of DNA being used for marker assisted selection (MAS). The present study was carried out with the objective to validate the STS markers, previously reported to be linked with fertility restorer (Rf1) gene for CMS lines of cotton (Gossypium hirsutum). Four sets of primer pairs viz., UBC 1471400, UBC607500, UBC679700 and UBC6591500 were used to identify restorer and non-restorer lines of G. hirsutum with G. harkenssii background. Out of the four marker sets screened, UBC607500 showed confirmatory results by amplifying a single band of size 517 bp in restorer lines and no amplification product was obtained in any of the non-restorer plants by these markers. Other STS markers, however, failed to show confirmatory results.

Efficiency of molecular markers in identifying fertility restoration trait of WA-CMS system in rice

To assess the efficiency of different molecular markers which are linked to fertility restorer genes Rf3 and Rf4 of WA-CMS system, 103 breeding lines with no information on fertility restoration were screened with the help of molecular markers linked to major fertility genes Rf3 and Rf4. The breeding lines were crossed with APMS6A and the F1s were evaluated for pollen and spikelet fertility to identify restorers and maintainers.The SSR primer RM6100 linked to Rf4 gene of chromosome 10 and RM10313 linked to Rf3 gene of chromosome 1 showed eighty five and eight one percentage efficiency respectively in identifying restorer lines. Therefore, these markers are useful tool for evaluating large number of breeding lines to know about their fertility restoration in a short period of time without generating and evaluating large number of test crosses.The potential restorers may be identified with hundred percentage efficiency based on molecular screening itself, if candidate genes based markers are developed and validated for both Rf4 and Rf3 genes.

Contribution of interspecific and intergeneric hybridization to sunflower breeding

SUMMARY This investigation was directed to sunflower improvement using hybrid forms resulted from wide hybridization. The aim was to create new B/A and R lines from interspecific and intergeneric hybrids resistant to diseases, parasite broomrape, herbicides, other stress factors and with high combining ability in highly productive oil-type sunflower hybrids with varied fatty acid contents. The confectionary hybrids should have a high kernel protein content and amino acid content. The investigation was carried out during the period 1983-2010. The programme included 16 cultivars and 18 B lines with their analogues. Interspecific, intraspecific, and intergeneric hybridization produced hybrid materials originating from 38 Helianthus species with different ploidy levels, 9 annuals and 29 perennials, and 28 species from other genera of family Compositae . New sunflower forms and lines created possessed resistance to downy mildew, Phomopsis, Phoma and Alternaria, tolerance to Sclerotinia and total resistance to the different races of parasitic broomrape. The new forms had distinctive plant architecture, different vegetation periods, and seeds of different sizes and coloration. New B/A and R lines, characterized with high combining ability, seed oil, and fatty acid content and varying protein amino acid contents were obtained. Fifteen sources of cytoplasmic male sterility ( cms ) were obtained from interspecific hybrids and 271 sources of fertility restoration ( Rf ) genes from interspecific and intergeneric hybrids. Five new oil type hybrids and one confectionery type were developed and registered. The results from this investigations showed that by wide hybridization new genetic material can be transferred to the cultivated sunflower. These results supplemented the contribution of interspecific and intergeneric hybridization for sunflower breeding.

Maintaining and restoring cytoplasmic male sterility systems in pepper (Capsicum annuum L.)

We studied the efficiency of maintaining and restoring cytoplasmic male sterility (CMS) systems in pepper (Capsicum annuum L.). An Rf-linked molecular marker was employed to analyze the interaction between 6 CMS lines (A), 5 maintainers (B), and 6 restorers (C). Sterility was maintained in the matings of lines 201A x 200B, 203A x 200B, 206A x 200B, 200A x 201B, 206A x 201B, 200A x 202B, 200A x 203B, 200A x 206B, and 201A x 206B. All 6 restorers restored the fertility of lines 200A, 202A, 203A, and 204A, except that 213C could not restore the fertility of lines 200A and 204A. However, the 6 restorers had diverse restoring abilities in individual CMS lines. The Rf-linked molecular marker was amplified by PCR in lines 207C, 208C, and 213C. This DNA marker was only found in the F1 hybrids M39, M14, M19, M25, M13, M20, and M22. We conclude that the restorers 208C and 207C can transmit the Rf gene or the Rf-linked marker to F1 hybrids.

Allelic differentiations and effects of the Rf3 and Rf4 genes on fertility restoration in rice with wild abortive cytoplasmic male sterility

To reveal the allelic differentiations at the two genes for fertility restoration (Rf) on chromosomes 1 (Rf3) and 10 (Rf4), 15 chromosome single segment substitution lines (SSSLs) with the Rf3 locus and 18 SSSLs with the Rf4 locus were crossed with Bobai A (BbA), a cytoplasmic male sterility line with wild abortive type of cytoplasm (WA-CMS), respectively. Based on the pollen and seed fertility of the F1 hybrids, the Rf3 and Rf4 genes were each classified into four alleles, namely Rf3-1, Rf3-2, Rf3-3, and Rf3-4 for Rf3, and Rf4-1, Rf4-2, Rf4-3, and Rf4-4 for Rf4. Out of the 33 SSSLs, an SSSL W23-19-06-06-11 carrying the genotype Rf3-4Rf3-4/Rf4-4Rf4-4 possessed the strongest restoring ability for BbA. To determine the genetic effects of Rf3 and Rf4 for WA-CMS, one BC3F2 population possessing the genetic background of W23-19-06-06-11 was generated from the cross between W23-19-06-06-11 and BbA by backcrossing and marker-assisted selection. In the BC3F2 population, the plants carrying the Rf3Rf3/Rf4Rf4, Rf3Rf3/rf4rf4, and rf3rf3/Rf4Rf4 genotypes were selected and their phenotyping for pollen and spikelet fertility were evaluated. The result showed that under the genetic background of SSSL W23-19-06-06-11, the effect of Rf4 appeared to be slightly larger than that of Rf3 and their effects were additive for WA-CMS system. These studies will lead to the transfer of Rf genes into adapted cultivars through marker-assisted selection in active hybrid rice breeding programs.

Unusual and Typical Features of a NovelRestorer-of-FertilityGene of Sugar Beet (Beta vulgarisL.)

Male gametogenesis in plants can be impaired by an incompatibility between nuclear and mitochondrial genomes, termed cytoplasmic male sterility (CMS). A sterilizing factor resides in mitochondria, whereas a nuclear factor, Restorer-of-fertility (Rf), restores male fertility. Although a majority of plant Rf genes are thought to encode a family of RNA-binding proteins called pentatrico-peptide repeat (PPR) proteins, we isolated a novel type of Rf from sugar beet. Two BACs and one cosmid clone that constituted a 383-kbp contig covering the sugar beet Rf1 locus were sequenced. Of 41 genes borne by the contig, quadruplicated genes were found to be associated with specific transcripts in Rf1 flower buds. The quadruplicated genes encoded a protein resembling OMA1, a protein known from yeast and mammals to be involved in mitochondrial protein quality control. Construction of transgenic plants revealed that one of the four genes (bvORF20) was capable of restoring partial pollen fertility to CMS sugar beet; the level of restoration was comparable to that evaluated by a crossing experiment. However, the other genes lacked such a capability. A GFP-fusion experiment showed that bvORF20 encoded a mitochondrial protein. The corresponding gene was cloned from rf1rf1 sugar beet and sequenced, and a solitary gene that was similar but not identical to bvORF20 was found. Genetic features exhibited by sugar beet Rf1, such as gene clustering and copy-number variation between Rf1 and rf, were reminiscent of PPR-type Rf, suggesting that a common evolutionary mechanism(s) operates on plant Rfs irrespective of the translation product.

Inheritance of the fertility restoration and genotyping of rice lines at the restoring fertility (Rf) loci using molecular markers

The combination of cytoplasmic male sterility (CMS) in one parent and a restorer gene (Rf) to restore fertility in another are indispensable for the development of hybrid varieties. To genotype rice lines at the restoring fertility (Rf) loci, 38 lines were crossed with a sterile tester (rfrf) line. Pollen fertility test was performed to identify sterile and fertile F1 hybrids. Seven lines were identified as restorer lines. Then these F1 hybrids were self-pollinated to obtain F2 seeds, and at flowering stage pollen fertility test was performed to identify sterile and fertile individuals. Bulked segregant analysis (BSA) in F2 showed that fertility in rice WA system is controlled by more than two loci, one on the short arm of chromosome 1, one on the short arm of chromosome 10, one on the long arm of chromosome 10 and an unknown Rf gene in the rice genome. Results also show that lines IR28, Amol1 and Amol2 carry Rf4 gene linked with SSR marker RM171 on the long arm of chromosome 10, lines IR36 and IR60966 carry Rf3 gene linked with SSR marker RM1 on the short arm of chromosome 1, line IR62030 carries Rf5 gene on the short arm of chromosome 10, and finally line IR24 carries Rf4 gene on the long arm of chromosome 10 and an unknown Rf gene, respectively. The results suggested the use of the method deployed here to identify restorer lines and also allelic location of fertility restoration genes in rice lines.

BnC15 and BnATA20, the different putative components, control anther development in Brassica napus L.

In Brassica napus, male fertility depends on proper cell differentiation in the anther. However, relatively little is known about the genes regulating anther cell differentiation and function. Here, we report two floral organ specific genes, BnC15 and BnATA20, derived from a B. napus two-line Rs1046A/B floral subtractive library. Although BnC15 and BnATA20 genes have a different expression pattern in anthers demonstrated by in situ hybridization and real-time PCR analysis, silencing of both genes in B. napus by antisense suppression resulted in pollen abortion after microspore release. Light and electron microscopy observation revealed the lack of plastoglobuli, lipid bodies and sporopollenin secreted from the tapetum leading to aberrations in exine sculpturing and the formation of a pollen coat. In addition, the microspores were squeezed to the irregular shape in the locule in the end. As shown by gene expression analysis in transgenic plants and the comparison of anther development between bnc15 or bnata20 mutants and Rs1046A, BnC15 and BnATA20 were positively regulated downstream of Rf gene controlling the fertility of Rs1046B in the same pathway. The results support the hypothesis that BnC15 and BnATA20 are crucial components of a genetic network that controls tapetum development and exine sculpturing.

Molecular characterization of latent fertility restorer loci for Honglian cytoplasmic male sterility in Oryza species

In Oryza species, several distinct cytoplasmic male sterility (CMS) systems have been identified. Honglian (HL) is one of the mainstream breeding CMS lines in large-scale commercial production in China and southern Asia. In order to increase restorer diversity, we screened 130 Oryza accessions to find new restorer loci for HL-CMS. We analyzed the restoring ability and the diversity of molecular markers cosegregated with Rf5 (Milyang23) and Rf6(t) (9311) for HL-CMS. Molecular and genetic analyses revealed that eight accessions, including one cultivar and seven wild rice, showed polymerase chain reaction (PCR) patterns different from Rf5 and Rf6(t), and each of them harbored a pair of Rf genes unallelic to Rf5 and Rf6(t) for HL-CMS. Furthermore, Northern blot revealed that the Rf loci in the eight accessions showed three types of processing manners of HL-CMS-related transcripts, including endonucleolytic cleavage, partial endonucleolytic cleavage, and degradation of dicistronic atp6-orfH79 messenger RNA (mRNA). The Rf genes for HL-CMS in AA-genome Oryza species elucidated by genetic and molecular analyses will be highly beneficial to sustainable development of hybrid rice.

Inheritance and fine mapping of a restorer-of-fertility (Rf) gene for the cytoplasmic male sterility in soybean

The cytoplasmic male sterility (CMS) line FuCMS5A and its restorer line FuHui9 were crossed to produce a segregating F2 population for pollen fertility assay and the genetic mapping of restorer-of-fertility (Rf) gene. Results showed that the individual F2 plants were fertile or semi-fertile based on their pollen fertility characteristics. The average ratios of viable pollen were 96.90% and 50.00% for each class of individuals. The segregation of F2 plants showed a good fit to a 1:1 ratio, which reflects a typical heredity pattern of gametophytic CMS with fertility restorer being controlled by a single dominant gene. Using bulk segregation analysis (BSA) and genetic mapping, the Rf gene was mapped on molecular linkage group J (chromosome 16), between the simple sequence repeat (SSR) makers BARCSOYSSR-16-1064 and BARCSOYSSR-16-1082 with the distances of 0.59 and 0.83cM, respectively. Four SSR markers (BARCSOYSSR-16-1070, Sctt011, BARCSOYSSR-16-1076 and BARCSOYSSR-16-1077) were cosegregating with this Rf gene in the mapping population. These makers will greatly facilitate the maker assisted selection procedures in CMS breeding programs and it lays a foundation for further map-base cloning of the Rf gene.

The Rice Pentatricopeptide Repeat Protein RF5 Restores Fertility in Hong-Lian Cytoplasmic Male-Sterile Lines via a Complex with the Glycine-Rich Protein GRP162

The cytoplasmic male sterility (CMS) phenotype in plants can be reversed by the action of nuclear-encoded fertility restorer (Rf) genes. The molecular mechanism involved in Rf gene-mediated processing of CMS-associated transcripts is unclear, as are the identities of other proteins that may be involved in the CMS-Rf interaction. In this study, we cloned the restorer gene Rf5 for Hong-Lian CMS in rice and studied its fertility restoration mechanism with respect to the processing of the CMS-associated transcript atp6-orfH79. RF5, a pentatricopeptide repeat (PPR) protein, was unable to bind to this CMS-associated transcript; however, a partner protein of RF5 (GRP162, a Gly-rich protein encoding 162 amino acids) was identified to bind to atp6-orfH79. GRP162 was found to physically interact with RF5 and to bind to atp6-orfH79 via an RNA recognition motif. Furthermore, we found that RF5 and GRP162 are both components of a restoration of fertility complex (RFC) that is 400 to 500 kD in size and can cleave CMS-associated transcripts in vitro. Evidence that a PPR protein interacts directly with a Gly-rich protein to form a subunit of the RFC provides a new perspective on the molecular mechanisms underlying fertility restoration.

Contribution of interspecific hybridization to sunflower breeding

SUMMARY This investigation is directed at improving sunflower using hybrid forms resulted from interspecific hybridization. The aim is to create new B/A and R lines from interspecific hybrid forms that are resistant to diseases, the parasite broomrape, herbicides, and other stress factors and are characterized with high combining ability and to obtain on this basis highly productive oilseed sunflower hybrids with varied fatty acid composition of oil. The investigation was carried out during the period 1983-2010. It involved 16 cultivars and 18 B lines with their analogues as well hybrid material originated from 38 Helianthus species - 9 annuals and 29 perennials. Intraspecific hybridization and purposeful selection were the methods used. Crossing between interspecific hybrids and crossing of interspecific hybrids with sunflower cultivars or lines were applied. Self-pollination, sib-pollination, backcross with pollen from cultivated sunflower and pollination with pollen from different interspecific hybrids were carried out. Phytopathological and biochemical evaluation of seeds and plants and morphological characterization of sunflower forms, lines and hybrids were performed. New sunflower forms and lines were created as a result of this investigation. A greater part of them possess resistance to downy mildew, phomopsis, phoma and alternaria, tolerance to sclerotinia and full resistance to the different races of the parasite broomrape. The new forms were distinguished by new plant architecture, different vegetation period and seeds with different size and coloration. New B/A and R lines having high combining ability and seed oil and fatty acid contents were obtained. Fifteen sources of cytoplasmic male sterility ( cms) were obtained from interspecific hybrid forms, as were 251 sources of genes for restoration of fertility ( Rf genes). Five new hybrid cultivars of the oilseed type that were obtained on the basis of these lines were created and registered. The obtained results from the investigations showed that by interspecific hybridization new genetic material was transferred to the cultivated sunflower. These results have augmented the contribution of interspecific hybridization to sunflower breeding.

Genetic diversity of sources of sunflower pollen fertility restorer genes

The genetic diversity of the sunflower collection with respect to components of the CMS-Rf system is studied with the use of molecular markers specific for mitochondrial genes atp9 and orfH522 and well as SCAR markers closely linked with nuclear gene Rf1 (fertility restorer for CMS PET1).

Molecular mapping of the Rf 3 fertility restoration gene to facilitate its utilization in breeding confection sunflower

The inheritance of a previously identified dominant Rf gene in the confection sunflower line RHA 280 has been determined and designated as Rf 3 . This study reports the mapping of the Rf 3 locus using an F2 population of 227 individuals derived from CMS HA 89-3149 × RHA 280. Bulked segregant analysis with 624 pairs of simple sequence repeat (SSR) primers and sequence tagged site (STS) primers identified two polymorphic SSR markers each of linkage groups (LGs) 7 and 11 from a previous map. Results on 90 F2 individuals with 42 polymorphic markers of LGs 7 and 11 indicated that the Rf 3 gene was linked with eight markers on LG 7, including five SSR markers (ORS328, ORS331, ORS928, ORS966, and ORS1092) and three expressed sequence tag (EST)-SSR markers (HT619-1, HT619-2, and HT1013). Further analysis of the total F2 population of 227 individuals identified a co-dominant marker, ORS328, linked to Rf 3 at a genetic distance of 0.7 cM on one side, and a female-dominant marker HT1013 at 12.6 cM proximal to Rf 3 on the other side; a genetic distance of 47.1 cM for LG 7 was covered. This is the first report of an Rf gene from the confection sunflower. The closely linked marker to Rf 3 will facilitate marker-assisted selection, and provide a basis for cloning of this gene.

Fungos conidiais do bioma Caatinga I. Novos registros para o continente americano, Neotrópico, América do Sul e Brasil

Resumo Durante inventário de fungos conidiais realizado na Serra da Fumaça, Pindobaçu, estado da Bahia, Brasil, foram identificadas 44 espécies. Destas, cinco representam novos registro para o continente americano: Dendryphiosphaera parvula Nawawi & Kuthub., Diplococcium dendrocalami Goh, K.D. Hyde & Umali, Sporidesmiella aspera Kuthub. & Nawawi, S. fusiformis W.P. Wu e Triposporium verruculosum R.F. Castañeda, Gené & Guarro. Stanjehughesia hormiscioides (Corda) Subram. é um novo registro para o Neotrópico; Hemibeltrania decorosa R.F. Castañeda & W.B. Kendr constitui novo registro para a América do Sul e Spadicoides macroobovata Matsush. é um novo registro para o Brasil. Descrições, ilustrações, distribuição geográfica mundial e comentários são apresentados para os novos registros citados. Uma lista com as demais espécies encontradas é adicionada.