Henochilus wheatlandii, the only species of this genus, is critically endangered and was considered extinct for over a century. The rediscovery of this fish in 1996 made it possible to study its phylogenetic relationships with other species in the subfamily Bryconinae. The aim of this study was to characterise the karyotype of H. wheatlandii. Standard staining, C-positive heterochromatin and nucleolar organiser region (NOR) banding, chromomycin A3 staining, and fluorescent in situ hybridisation (FISH) using 5S rDNA and 18S rDNA probes were conducted on nineteen specimens collected in the Santo Antonio River, a sub-basin of the Doce River in Ferros municipality, Minas Gerais State, Brazil. Henochilus wheatlandii shared the same diploid number and chromosome morphology as other species of Bryconinae. However, its heterochromatin distribution patterns, NOR localisation, and FISH patterns revealed a cytogenetic profile unique among Neotropical Bryconinae, emphasizing the evolutionary uniqueness of this threatened species.
Citation: Silva PC, Santos U, Travenzoli NM, Zanuncio JC, Cioffi MdB, Dergam JA (2012) The Unique Karyotype of Henochilus wheatlandii, a Critically Endangered Fish Living in a Fast-Developing Region in Minas Gerais State, Brazil. PLoS ONE 7(7): e42278. https://doi.org/10.1371/journal.pone.0042278
Editor: Daniela Cimini, Virginia Tech, United States of America
Received: April 10, 2012; Accepted: July 2, 2012; Published: July 27, 2012
Copyright: © Silva et al. This is an open-access article 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.
Funding: This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (REUNI/CAPES) and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Henochilus wheatlandii is the only species of this genus. It was first collected by the Thayer expedition in 1865 and 1866. The first record was from the Mucuri River Basin , an isolated drainage area in eastern Brazil. The second record did not provide details of its location . Henochilus wheatlandii was described in 1890 , but collection efforts in this type locality during the late 20th century were unsuccessful. Therefore, this species was officially considered to be at risk or extinct , . In 1996, H. wheatlandii was collected in Preto do Itambé River, a tributary of the Santo Antonio River of the Doce River Basin in Minas Gerais State, Brazil. Based on this finding, the absence of new captures in the Mucuri River Basin was considered a record error of the type locality of the holotype , and the current distribution of H. wheatlandii now includes the Santo Antonio River Basin. Alternatively, this species could have become extinct in the type locality .
The systematics of H. wheatlandii have always been a matter of debate, and different authors have placed the species in different subfamilies , , . Today, this genus is considered a member of the subfamily Bryconinae . The close phylogenetic relationship of H. wheatlandii with members of this subfamily was corroborated with molecular ,  and morphological data analyses . The patterns obtained for the 16S ribosomal mitochondrial gene suggested a paraphyletic condition of the genera Brycon within Bryconinae , where Brycon insignis and Brycon ferox are more closely related to H. wheatlandii than other Brycon. Morphologically, the most evident difference between Henochilus and Brycon is the arrangement of premaxillary teeth in a double row in Henochilus and three rows in Brycon , a character variation that has been considered insufficient to justify the existence of the genus Henochilus .
Bryconins are indicators of high quality environmental habitat because they require rapid flowing waters with sandy substrate, highly oxygenated water, riparian vegetation and a moderate amount of nutrients . These habitat conditions also restrict the distribution of H. wheatlandii to the Santo Antonio River Basin , although predatory exotics may also be a limiting factor for the presence of this species in the Doce River. Unfortunately, this species was rediscovered in a drainage area currently threatened by human activities such as mining, logging and hydroelectric projects .
Cytogenetic descriptions of Bryconinae are restricted to eight species that occur in the Amazon, São Francisco and Paraná watersheds  and one species in the Paraíba do Sul drainage. Historically, cytogenetic data on Ostariophysan fish have allowed the identification of cryptic species , the characterization of populations , , and the formulation of phylogenetic and phylogeographic hypotheses –. This study reports the first cytogenetic data for H. wheatlandii and compares the results with other species of Bryconinae from a biogeographic and phylogenetic perspective.
Materials and Methods
Nineteen individuals, six females, eleven males and two juveniles, were collected in the Santo Antonio River, a sub-basin within the Doce River Basin (S19°13′858″ W43°04′905″) in Ferros municipality, Minas Gerais State, Brazil. The specimens were transported to the laboratory and anaesthetised with clove oil . Following dissection, mitotic chromosomes were obtained from cell suspensions of the anterior kidney, using the conventional air-drying method . Chromosomes were analysed with silver nitrate staining  to visualise the nucleolar organiser regions (Ag-NOR) in addition to the standard Giemsa method. C-banding was used to detect C-positive heterochromatin . Chromosome guanine-cytosine (GC) rich regions were identified using chromomycin A3 (CMA3) fluorescence staining . FISH was performed according to Pinkel and colleagues  using 18S rDNA  and 5S rDNA  probes. The probes were labelled by nick translation with biotin-14-dATP. Signal detection and probe amplification were performed using conjugated avidin-fluorescein isothiocyanate (FITC) and anti-avidin-biotin, and the metaphases were analysed with an epifluorescence microscope. The chromosomal images were captured using CoolSNAP-Pro software. The chromosomes were measured using Image Pro Plus® and classified following Levan and colleagues . Voucher specimens were deposited in the João Moojen de Oliveira Museum of Zoology in Viçosa, Minas Gerais State, Brazil (MZUFV3195, MZUFV3744, MZUFV3791, MZUFV3821, MZUFV3827, and MZUFV4011). Collecting permit SISBIO14975-1 was issued to Prof. Jorge A. Dergam.
All specimens had 2n = 50 chromosomes and a karyotypic formula of 26m+12sm+12st (see Figure 1a and S1), with no differences observed between males and females. At least 40 Giemsa-stained, five C-banded, two Ag-NOR, two CMA3 and two FISH metaphases were analysed for each specimen. The mean values of the arm ratios were 1.28–1.61 (s.e. 0.002–0.2) for metacentrics; 1.90–2.67 (s.e. 0.05–0.2) for submetacentrics, and 3.24–4.14 (s.e. 0.04–0.2) for subtelocentrics. One pericentromeric and two telomeric heterochromatic blocks were visible in the largest pair of metacentric chromosomes of H. wheatlandii. Pericentromeric heterochromatin was evident in metacentric chromosome pairs 2 and 9 and submetacentric chromosome pairs 14 and 18, whereas the largest subtelocentric chromosomes (pair 20) presented conspicuous telomeric and pericentromeric heterochromatic blocks. Pericentromeric blocks predominated in most subtelocentrics, except for pairs 22 and 23 (see Figure 1b). At least five C-banded metaphases were analysed for each specimen.
Mean values of arm ratios are in parentheses (sample size = 40).
Sites marked with Ag-NOR, CMA3, and 18S rDNA sites were telomeric and identified in the same first pair of subtelocentric chromosomes (see Figure 2). The 5S rDNA regions were distributed in the short arm of subtelocentric chromosome pair 24 and were congruent with heterochromatic blocks (see Figures 1b and 2c).
The observed 2n = 50 metacentric, submetacentric, and subtelocentric chromosomes of H. wheatlandii are characteristic of other Bryconinae –, . The absence of telocentric chromosomes may represent an apomorphy within the taxon, despite some variation in karyotypic formulae. This karyotypical structure is shared with species of the genus Salminus , suggesting a close phylogenetic relationship with Bryconinae, a pattern supported by molecular data –. In this context, karyological evidence also adds support for the existence of the family Bryconidae, as suggested by Oliveira and colleagues . However, from a morphological standpoint, Salminus species are still considered to be incertae sedis within Characidae . On the other hand, the presence of a metacentric chromosome pair as the largest of the complement in H. wheatlandii and other Bryconinae  is characteristic of the family Characidae , .
The presence of pericentromeric and telomeric heterochromatic blocks on opposite arms of the first pair of metacentric chromosomes of H. wheatlandii differs from the equilocal pattern observed in other Bryconinae, and thus has great relevance for understanding the karyotypical evolution of this species. Equilocal heterochromatic markings are common in Bryconinae species and considered to be plesiomorphic within this taxon . This pattern has been reported in Brycon orthotaenia, Brycon hilarii, Salminus hilarii , and Brycon amazonicus . The different pattern observed in H. wheatlandii suggests the occurrence of paracentromeric inversions in this species. To the best of our knowledge, this is the first record of paracentromeric inversions in Characidae. Chromosomal inversions are crucial for the reproductive isolation of populations  and contribute to the speciation process –. High levels of variability in heterochromatin patterns of Brycon were considered to be relevant factors in chromosome evolution and differentiation of the Bryconinae , , and the presence of large heterochromatic blocks in the first pair of submetacentric chromosomes , , , probably represents a plesiomorphic character state for the subfamily Bryconinae. Based on overall patterns of heterochromatin distribution, Margarido and Galetti Jr.  proposed that Brycon species might be separated into two groups: the first characterised by predominantly pericentromeric heterochromatin in submetacentric chromosomes; and the second diagnosed by telomeric markings in metacentric chromosomes. Henochilus wheatlandii exhibits a third pattern, with markers prevalent in the pericentromeric region of subtelocentric chromosomes. This characteristic seems to be an autapomorphy for this taxon.
The physical congruence of 18S probe sites, CMA3 fluorescence, and Ag-positive NOR locations in H. wheatlandii was similar to patterns observed for Brycon falcatus, Brycon cephalus, B. hilarii, Brycon orbignyanus, B. orthotaenia, Brycon insignis, and B. amazonicus , , indicating the presence of a single 18S rDNA locus in Bryconinae. However, the 18S rDNA site occurred on the subtelocentric chromosome pair in H. wheatlandii, instead of the first pair of submetacentric chromosomes as in all Brycon species. Also, the 5S rDNA site in H. wheatlandii was found in a pair of subtelocentric chromosomes, whereas in Brycon species the 5S rDNA occurs in submetacentric chromosomes , . These differences indicate a long independent karyotypic evolutionary history of H. wheatlandii. Henochilus wheatlandii also showed congruence of the 5S rDNA sites with heterochromatic blocks, which is a common characteristic of Brycon ,  suggesting that this is plesiomorphic trait for Bryconinae.
The occurrence of three heterochromatic karyotype patterns in Bryconinae is partially congruent with some phylogeographic clades based on data from the mitochondrial 16S rDNA gene  and is also consistent with the morphological groups proposed by Howes in 1982  (see Figure 3). Based on molecular data, Hilsdorf and colleagues  obtained some well-supported molecular clades. Within the eastern coastal clade, H. wheatlandii is the sister species of Brycon insignis and Brycon ferox. The C-banding patterns of B. insignis were interpreted by Margarido and Galetti  as an instance of the second heterochromatic pattern . However, this species, like H. Wheatlandii, has a high number of subtelocentrics with heterochromatic blocks and the absence of equilocal heterochromatic blocks in the first chromosome pair, which might be the result of a paracentromeric inversion in the ancestor of both species. Differences in the marking locations of 5S rDNA sites sets H. wheatlandii apart from all other Bryconinae. Whereas in this species 5S rDNA markings were located in a small subtelocentric chromosome, the probe only occurs in submetacentric chromosomes in species endemic to other basins: Brycon orthotaenia (São Francisco River), B. hilarii (Paraguay River), B. orbignyanus (La Plata River), B. cephalus (Amazon drainage), and Brycon sp. (Araguaia River, a specimen collected by Wasko ). 5S rDNA markings on submetacentrics also characterise Brycon amazonicus (from the Amazon and Orinoco basins) and B. falcatus (from the Amazon, Orinoco, and Guyana/Suriname basins) , . The presence of 5S rDNA in two pairs of submetacentric chromosomes in B. insignis and a small subtelocentric chromosome in H. wheatlandii suggests a high level of variation of this ribosomal gene in species that occur in eastern Brazil. This may be the result of a relatively longer geological isolation of the eastern basins compared with the other drainage areas of the Neotropical region, which have a complex history of fragmentation and connections .
Numbers above branches are Bayesian posterior probabilities expressed in percentage, numbers below branches indicate maximum likelihood analyses bootstrap values (modified from Hilsdorf and colleagues ). Morphological groups proposed by Howes : stars for Brycon acuminatus, circles for Brycon falcatus, squares for Brycon orbignyanus, and a triangle for Brycon alburnus. P1 and P2, heterochromatic patterns proposed by Margarido and Galetti . P3 is a new pattern recorded in Henochilus wheatlandii. Bars indicate geographical distribution.
Castro and colleagues  reviewed the historical changes of the systematic position of H. wheatlandii. The species was initially considered by Garman  as closely related to Tetragonopterus and Scissor. Later, Eigenmann and Myers  viewed H. wheatlandii as “an aberrant member of the Tetragonopterinae” and Géry  added the species to the Cheirodontinae. Malabarba  redefined the Cheirodontinae and placed it in incertae sedis condition within Characidae. Currently, due to the presence of premaxillary large teeth and symphysial dentary teeth, the species is considered morphologically within Bryconinae . Molecular-based studies show that H. wheatlandii is closely related to Brycon insignis ,  and Brycon ferox , suggesting the need to include Henochilus within Brycon. Aside from this caveat, the inclusion of H. wheatlandii within Brycon will keep this species as a clear example of adaptive radiation .
The unique cytogenetic features of H. wheatlandii compared to other species of Bryconinae from continental biogeographical units suggest this species (possibly together with other species from the eastern drainage areas) is a separate phylogenetic unit that faces high extinction risks.
Chromosome spread from karyotypes presented in this work. Conventional staining (Giemsa) (a); C-banding protocols (b); Ag-NOR banding protocols (c); chromomycin A3 (d), and fluorescent in situ hybridisation using 18S (pink) and 5S (green) probes (e).
Nicholas J. Walker helped with laboratory work. To Asia Science for English revision and editing this manuscript. Further revisions were done by Journal Editors of America, LLC.
Conceived and designed the experiments: PCS US JAD. Performed the experiments: PCS US NMT MBC JAD. Analyzed the data: PCS US JAD. Contributed reagents/materials/analysis tools: JAD. Wrote the paper: PCS US JAD. Reviewing the manuscript: JCZ JAD.
- 1. Castro RMC, Vari RP, Vieira F, Oliveira C (2004) A phylogenetic analysis and redescription of the genus Henochilus (Characiformes, Characidae). Copeia 3: 496–506.
- 2. Eigenmann CH, Myers GS (1929) The American Characidae. Mem Mus Comp Zool 43: 429–558.
- 3. Garman S (1890) On a genus and species of the characines (Henochilus wheatlandii, gen. N. Et sp. N.). Bull Essex Inst 22: 49–52.
- 4. Rosa RS, Menezes NA (1996) Relação preliminar das espécies de peixes (Pisces, Elasmobranchii, Actinopterygii) ameaçadas no Brasil. Rev Bras Zool 13: 647–667.
- 5. Swerdlow J (1998) Making sense of the millennium. Nat Geog 193: 2–33.
- 6. Vieira F, Alves CBM, Santos GB (2000) Rediscovery and first record of Henochilus wheatlandii (Teleostei: Characiformes) a rare Neotropical fish, in rio Doce basin of southeastern Brazil. Ichthyol Explor Freshw 11: 201–206.
- 7. Géry J (1977) Characoids of the world. Neptune City: TFH Publications. 672.
- 8. Malabarba LR (1998) Monophyly of the Cheirodontinae, characters and major clades (Characiformes: Characidae). In: Phylogeny and classification of Neotropical fishes. Malabarba LR, Reis RE, Vari RP, Lucena ZMS, Lucena CAS, editors. Porto Alegre: Edipucrs. 193–233.
- 9. Lima FCT (2003) Subfamily Bryconinae. In: Malabarba LR, Reis RE, Vari RP, Lucena ZMS, Lucena CAS, editors. Phylogeny and classification of Neotropical fishes. Porto Alegre: Edipucrs. pp. 174–181.
- 10. Hilsdorf AWS, Oliveira C, Lima FCT, Matsumoto CK (2008) A phylogenetic analysis of Brycon and Henochilus (Characiformes, Characidae, Bryconinae) based on the mitochondrial gene 16S rRNA. Genet Mol Biol 31: 366–371.
- 11. Botero-Botero A, Ramírez-Castro H (2011) Ecología trófica de la Sabaleta Brycon henni (Pisces:Characidae) en el río Portugal de Piedras, Alto Cauca. Rev MVZ Cordoba 16: 2349–2355.
- 12. Vieira FC, Alves CBM (2001) Threatened fishes of the world: Henochilus wheatlandii Garman, 1890 (Characidae). Environ Biol Fish 62: 414.
- 13. Mariguela TC, Nirchio M, Ron E, Gaviria JI, Foresti F, et al. (2010) Cytogenetic characterization of Brycon amazonicus (Spix et Agassiz, 1829) (Teleostei: Characidae) from Caicara del Orinoco, Venezuela. Comp Cytogenet 4: 185–193.
- 14. Bertollo LAC, Oliveira C, Molina WF, Margarido VP, Fontes MS, et al. (2004) Chromosome evolution in the erythrinid fish, Erythrinus erythrinus (Teleostei: Characiformes). Heredity 93: 228–233.
- 15. Dergam JA, Bertollo LAC (1990) Karyotypic diversification in Hoplias malabaricus (Osteichthyes, Erythrinidae) of the São Francisco and Alto Paraná basins, Brazil. Genet Mol Biol 13: 755–756.
- 16. Jacobina UP, Paiva E, Dergam JA (2011) Pleistocene karyotypic divergence in Hoplias malabaricus (Bloch, 1974) (Teleostei: Erythrinidae) populations in southeastern Brazil. Neotrop Ichthyol 9: 325–333.
- 17. Kavalco KF, Pazza R, Bertollo LAC, Moreira-Filho O (2005) Karyotypic diversity and evolution of Loricariidae (Pisces, Siluriformes). Heredity 94: 180–186.
- 18. Santos U, Volcker CM, Belei FA, Cioffi MB, Bertollo LAC, et al. (2009) Molecular and karyotypic phylogeography in the Neotropical Hoplias malabaricus (Erythrinidae) fishin eastern Brazil. J Fish Biol 75: 2326–2343.
- 19. Kavalco KF, Pazza R, Brandão KO, Garcia C, Toledo LFA (2011) Comparative cytogenetics and molecular phylogeography in the group Astyanax altiparanae Astyanax aff. Bimaculatus (Teleostei, Characidae). Cytogenet Gen Res 134: 108–119.
- 20. Inoue LAKA, Neto CS, Moraes G (2003) Clove oil as anaesthetic for juveniles of matrinxã Brycon cephalus (Gunther, 1869). Ciênc rural 33: 943–947.
- 21. Bertollo LAC, Takahashi C, Moreira-Filho O (1978) Cytotaxonomic considerations on Hoplias lacerdae (Pisces, Erythrinidae). Braz J Genet 1: 103–120.
- 22. Howell WM, Black DA (1980) Controlled silver staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia 36: 1014–1015.
- 23. Sumner AT (1972) A simple technique for demonstrating centromeric heterocromatin. Exp Cell Res 75: 304–306.
- 24. Sola L, Rossi AR, Laselli V, Rasch EM, Monaco PJ (1992) Cytogenetics of bisexual/unisexual species of Poecilia II. Analysis of heterochromatin and nucleolar organizer regions in Poecilia mexicana mexicana by C-banding and DAPI, quinacrine, chromomycin A3, and silver staining. Cytogenet Cell Genet 60: 229–235.
- 25. Pinkel D, Straume T, Gray J (1986) Cytogenetic analysis using quantitative, high sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83: 2934–2938.
- 26. Cioffi MB, Martins C, Bertollo LAC (2009) Comparative chromosome mapping of repetitive sequences. Implications for genomic evolution in the fish, Hoplias malabaricus. BMC Genet 10: 34.
- 27. Martins C, Ferreira IA, Oliveira C, Foresti F; Galetti Jr PM (2006) A tandemly repetitive centromeric DNA sequence of the fish Hoplias malabaricus (Characiformes: Erythrinidae) is derived from 5S rDNA. Genetica 127: 133–141.
- 28. Levan A, Fredga K, Sandberg AA (1964) Nomenclature for centromeric position on chromosome. Hereditas 1: 201–220.
- 29. Margarido VP, Galetti PM Jr (1996) Chromosome studies in fish of the genus Brycon (Characiformes, Characidae, Bryconinae). Cytobios 85: 219–228.
- 30. Margarido VP, Galetti PM Jr (1999) Heterochromatin patterns and karyotype relationships within and between the genera Brycon and Salminus (Pisces, Characidae). Genet Mol Biol 22: 357–361.
- 31. Wasko AP, Galetti PM Jr (2000) Mapping 18s ribosomal genes in fish of the genus Brycon (Characidae) by fluorescence in situ hibridization (FISH). Genet Mol Biol 23: 135–138.
- 32. Wasko AP, Martins C, Wright JM, Galetti PM Jr (2001) Molecular organization of 5S rDNA in fishes of the genus Brycon. Genome 44: 893–902.
- 33. López DD, Palacio GV, Cortés TR, Angel MO (2008) Caracterización citogenética del pez neotropical Brycon henni (Pisces: Characidae). Rev biol trop 56: 1619–1628.
- 34. Souza IL, Santos-Silva LK, Venere PC, Moreira-Filho O (2008) Molecular cytogenetics of Salminus fish (Characiformes) based on 5S and 18S rRNA genes hybridization, fluorochrome staining and C-banding. Micron 39: 1036–1041.
- 35. Calcagnotto D, Schaefer SA, DeSalle R (2005) Relationships among characiforms fishes inferred from analysis of nuclear and mitochondrial gene sequences. Mol Phylogenet Evol 36: 135–153.
- 36. Javonillo R, Malabarba LR, Weitzman SH, Burns JR (2010) Relationships among major lineages of characid fishes (Teleostei: Ostariophysi: Characiformes), based on molecular sequence data. Mol Phylogenet Evol 54: 498–511.
- 37. Oliveira C, Avelino GS, Abe KT, Mariguela TC, Benine RC, et al. (2011) Phylogenetic relationships within the speciose family Characidae (Teleostei: Ostariophysi: Characiformes) based on multilocus analysis and extensive ingroup sampling. BMC Evol Biol 11: 275.
- 38. Daniel-Silva MFZ, Almeida-Toledo LF (2005) Chromosome evolution in fish: BrdU replication patterns demonstrate chromosome homologies in two species of the genus Astyanax. Cytogenet Genome Res 109: 497–501.
- 39. Kavalco KF, Pazza R, Almeida-Toledo LF (2009) Astyanax bockmanni Vari and Castro, 2007: an ambiguous karyotype in the Astyanax genus. Genetica 136: 135–139.
- 40. Stefansson H, Helgason A, Thorleifsson G, Steinthorsdottir V, Masson G (2005) A common inversion under selection in Europeans. Nature Rev Genet 37: 129–137.
- 41. Hoffmann AA, Rieseberg LH (2008) Revisiting the impact of inversions in evolution: from population genetic markers to drivers of adaptive shifts and speciation? Annu Rev Ecol Evol Syst 39: 21–42.
- 42. Kirkpatrick M, Barton N (2006) Chromosome inversions, local adaptation and speciation. Genetics 173: 419–434.
- 43. White MJD (1978) Modes of speciation. San Francisco: W. H. Freeman and Company.
- 44. King M (1993) Species evolution: the role of chromosome change. Cambridge: Cambridge University Press.
- 45. Howes GJ (1982) Review of the genus Brycon (Teleostei:Characoidei). Bull Br Mus Nat Hist (Zool) 43: 1–17.
- 46. Reis RE, Kullander SO, Ferraris CJ (2003) Check List of the Freshwater Fishes of South and Central America. Porto Alegre: Edipucrs.
- 47. Lundberg GJ, Marshall LG, Horton JGB, Malabarba MCSL, Wesselingh F (1998) The stage for neotropical fish diversification: a history of tropical south American rivers. In: Malabarba LR, Reis RE, Vari RP, Lucena ZMS, Lucena CAS, editors. Phylogeny and classification of Neotropical fishes. Porto Alegre: Edipucrs. pp. 13–48.
- 48. Albert JS, Petry P, Reis RE (2011) Major biogeographic and phylogenetic patterns. In: Albert JS, Reis RE, editors. Historical of neotropical freshwater fishes. Los Angeles: University of California Press. pp. 21–58.