Cytogenetics of three species of scorpions of the genus Brachistosternus from Argentina (Scorpiones: Bothriuridae).
Abstract: Meiotic studies on three phylogenetically distant species of the genus Brachistosternus Pocock from Argentina were conducted. One species is from the subgenus Ministernus Francke 1985, B. ferrugineus Thorell 1876, and two species are from the subgenus Brachistosternus Pocock 1893, B. montanus Roig-Alsina 1977 (Andean species group), and morphologically different populations of B. pentheri Mello-Leitao 1931 (plains species group). All species showed achiasmatic meiosis, absence of heteromorphic bivalents, and bibrachial and monobrachial chromosomes of different sizes. Males of Brachistosternus ferrugineus, B. montanus, and one population of B. pentheri have 2n = 46. Males of the typical populations of B. pentheri have 2n = 42. These results suggest that B. pentheri may comprise two species.

Keywords: Achiasmatic meiosis, Neotropics
Article Type: Report
Subject: Meiosis (Observations)
Cytogenetics (Research)
Scorpions (Genetic aspects)
Scorpions (Identification and classification)
Authors: Rodriguez-Gil, Sergio G.
Ojanguren-Affilastro, Andres A.
Barral, Leonel M.
Scioscia, Cristina L.
Mola, Liliana M.
Pub Date: 09/01/2009
Publication: Name: Journal of Arachnology Publisher: American Arachnological Society Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2009 American Arachnological Society ISSN: 0161-8202
Issue: Date: Sept, 2009 Source Volume: 37 Source Issue: 3
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: Argentina Geographic Code: 3ARGE Argentina
Accession Number: 253537140
Full Text: The family Bothriuridae contains about 150 described species; it shows a Gondwanan distribution and has diversified mainly in southern South America. The systematics of this family has been resolved satisfactorily and the taxonomic status of most genera and species is well defined (Prendini 2003; Ochoa 2004a; Ojanguren-Affilastro & Ramirez 2009), making it particularly suitable for studying patterns of cytogenetic evolution. Chromosome number in Bothriuridae varies between 28 and 50 (Piza de Toledo 1947; Ferreira 1968; Giacomozzi 1977). Cytogenetic analyses have been performed on seven species, four species from the genus Bothriurus Peters 1861, Timogenes elegans (Mello-Leitao 1931) and two species of Brachistosternus Pocock 1893 (B. pentheri Mello-Leitao 1931 and B. alienus Lonnberg 1898) (Piza de Toledo 1947; Ferreira 1968; Giacomozzi 1977). The present study focuses on exploring cytological diversity among species and populations of Brachistosternus from Argentina.

Brachistosternus is the most diverse genus of the family, with about 40 known species (Ochoa 2002, 2004b; Ochoa & Acosta 2002; Ojanguren-Affilastro 2003a, b, 2005a, b; Ochoa & Ojanguren-Affilastro 2007; Ojanguren-Affilastro et al. 2007a, b; Ojanguren-Affilastro & Scioscia 2007). It inhabits arid areas in the western and southern parts of South America, from Ecuador (Cekalovic 1969) to southern Argentinean Patagonia (Ojanguren-Affilastro 2003b). The genus is divided into two subgenera (Ojanguren-Affilastro & Ramirez 2009), namely Brachistosternus Pocock 1893 and Ministernus Francke 1985. Both subgenera are present in Argentina. The subgenus Brachistosternus includes two large groups, one including lowland or plains species and the other mountain species from the Andes from altitudes between 2500 and 4500 m asl.

Here we report a cytogenetic study of three phylogenetically distant species of Brachistosternus in order to reveal possible chromosome variations in the genus. Brachistosternus ferrugi- neus (Thorell 1876) was selected as a representative of the subgenus Ministernus. This species is widely distributed in central and northern Argentina, eastern Bolivia, Paraguay and possibly in southwestern Brazil (Maury 1974). Two species were selected as representatives of the subgenus Brachistosternus, one belonging to the Andean group and the other to the plains group. Brachistosternus montanus Roig-Alsina 1977 (Andean group) is restricted to high-altitude areas of the Andean region (2700 to 3500 m asl) in central-western Argentina, in the provinces of Mendoza, San Juan and La Rioja (Ojanguren-Affilastro 2003a; Roig-Alsina 1977). Brachistosternus pentheri Mello-Leitao 1931 (plains group) is also found exclusively in Argentina, with a widespread distribution from Salta province to the southern part of Buenos Aires province. The morph of the northernmost B. pentheri found in the provinces of La Rioja, Catamarca and Salta (here designated as the northern morph) differs slightly from the type material by larger size and less pronounced pigmentation (Roig-Alsina & Maury 1984; Ojanguren-Affilastro 2005b). Both morphs of B. pentheri were analyzed cytogenetically to determine whether they also differ in karyotype.

METHODS

Specimens.--Brachistosternus ferrugineus: The specimens belong to two populations from an area near the center of the known distribution of the species: three males from the locality of Chepes, La Rioja province, Argentina (31[degrees]21 '00"S; 66[degrees]35'60"W), and three males from the locality of San Marcos Sierra, Cordoba province, Argentina (30[degrees]46'60"S; 64[degrees]39'00"W). Brachistosternus montanus: Four males were obtained from the locality of Laguna Brava, La Rioja province (28[degrees]25'50"S; 69[degrees]00'31.3'W). This population has been previously mentioned under the name B. affinis montanus (Ojanguren-Affilastro 2003a) because it shows minor morphological differences from the typical morph of the species, which is present in San Juan and Mendoza provinces. However, we consider this population to be the same species after examining many additional specimens.

[FIGURE 1 OMITTED]

Brachistosternus pentheri: Four males were obtained from the locality of Villa Union, northern La Rioja province (29[degrees]18'00"S; 68[degrees]12'00"W) and belong to the northern morph. The typical morph was represented by three males from Chepes (southern La Rioja province) (31[degrees]21'00"S; 66[degrees]35' 60"W), and one male from Oriente (coastal area of southeastern Buenos Aires province) (38[degrees]36 29"S; 60[degrees]37 07.3"W).

Cytogenetic methods.--All specimens were carried alive to laboratory and killed by cooling in a refrigerator. Their gonads were dissected in a physiological saline solution, swollen in hypotonic solution (0.56% KCl) for 10 min, and then fixed in a mixture of ethanol:chloroform:acetic acid (6:3:1). A piece of testis was placed on a slide, dissociated in a drop of 60% acetic acid with tungsten needles, and spread on the slide using a heating histological plate at approximately 45[degrees] C. Finally, the preparations were air-dried and stained with 3% Giemsa solution in water (pH = 7.4) for 10 min. Five postpachytene-prometaphase I of each cytotype of B. pentheri were measured to determine the meiotic karyotype. Bivalents measurements were made using the computer application Micromeasure version 3.3 (Reeves & Tear 2000). The relative length of each bivalent was calculated as a percentage of total haploid complement length (TCL). The idiogram of each cytotype was drawn on the basis of the relative percentage of each bivalent length to the TCL.

RESULTS

Subgenus Ministernus Francke 1985 (Figs. 1a-c; Table 1)

Brachistosternus ferrugineus has a karyotype of 2n = 46. No positively heteropycnotic bodies were observed at early prophase I. After pachytene, bivalents have no chiasma; homologous chromosomes lie parallel to each other, and condense gradually during prophase I and metaphase I. All bivalents are homomorphic (Figs. 1a, b). Chromosome plates of prometaphase and metaphase II consist of bibrachial (meta- or submetacentric) and monobrachial chromosomes of different sizes (Fig. 1c). No differences were observed between the specimens from the two localities.

Subgenus Brachistosternus Pocock 1893 (Figs. 1d-f, 2a-f, 3a-d; Table 1)

The chromosome complement of B. montanus consists of 46 chromosomes. At early prophase I no positively heteropycnotic bodies are present (Fig. 1d). Meiosis is achiasmatic; all bivalents are homomorphic, condensing gradually until metaphase I (Fig. 1e). Bibrachial and monobrachial chromosomes of different sizes were observed at prometaphase and metaphase II (Fig. 1f).

[FIGURE 2 OMITTED]

Morphs of B. pentheri showed different chromosome numbers. The karyotype of the northern morph (Villa Union, northern La Rioja) exhibits 46 chromosomes (cytotype I) (Fig. 2), whereas karyotype of the typical morph from Chepes (southern La Rioja) and Oriente (southeastern Buenos Aires) consisted of 42 chromosomes (cytotype II) (Fig. 3). Chromosomes of both morphs presented the same meiotic behavior. At spermatogonial prometaphases and metaphases chromosomes of different sizes were observed (Fig. 2a). At prophase I no positively heteropycnotic bodies were detected (Figs. 2b, 3a); meiosis is achiasmatic, and bivalents are homomorphic, condensing gradually during prophase I (Figs. 2c-e, 3b-d). Bibrachial (metacentric or submetacentric) and monobrachial chromosomes could be identified at metaphase II of individuals with 2n = 46 (Fig. 2f). No metaphases II were observed in the individuals with 2n = 42. Karyotype of B. pentheri cytotype I (n = 23) was formed by three larger bivalents of different size and the rest of the complement decreasing gradually in size (from medium-sized to small bivalents) (Fig. 4a, Table 2). Karyotype of B. pentheri cytotype II (n = 21) was formed by four larger bivalents of different size, 10 medium-sized bivalents that gradually decreased in size, and seven smaller bivalents that gradually decreased in size (Fig. 4b, Table 2).

[FIGURE 3 OMITTED]

DISCUSSION

This study focuses on cytogenetics of bothriurid scorpions of the genus Brachistosternus, comparing representatives of the subgenera Ministernus (B. ferrugineus) and Brachistosternus (B. montanus and B. pentheri). The karyotype of studied species is formed by a mixture of bibrachial and monobrachial chromosomes of different sizes. Meiotic complements of the three analyzed species of the family Bothriuridae contain no heteromorphic chromosome pair, which indicates the absence of heteromorphic sex chromosomes in males, as is also the case in Buthus occitanus (Amoreux 1789) and Pandinus imperator (C.L. Koch 1841) (Guenin 1957, 1961). Shanahan (1989a, 1989b) showed achiasmatic meiosis in males of Buthidae and Urodacidae. Our study of Brachistosternus, as well as that of Ferreira (1968) on Bothriurus araguayae Vellard 1934, reveals the presence of this derived type of meiosis also in Bothriuridae.

Our study provides the first cytogenetic analysis of B. ferrugineus and B. montanus. The karyotypes of these species consist of 46 chromosomes, a number that has not been found in the family Bothriuridae previously (Table 1). Although B. ferrugineus is widely distributed, there are almost no morphological differences between populations and no variation in chromosome number.

In contrast to B. ferrugineus, B. pentheri shows two different morphs (Roig Alsina & Maury 1984, Ojanguren-Affilastro 2005a), and our analysis revealed karyotypic differences between them. The northern morph of B. pentheri from Villa Union (northern La Rioja) shows the same chromosome number as B. ferrugineus. In contrast, the karyotype of B. pentheri from Chepes (southern La Rioja) and Oriente (southeastern Buenos Aires) consists in 42 chromosomes. These specimens correspond morphologically to the species' holotype from Mendoza province, Argentina, and they belong to the typical morph of the species. Although the range of the relative lengths of the bivalents in both cytotypes was similar, three bivalent groups can be distinguished in cytotype II, but only two in cytotype I (Fig. 4). Since only 1 % of Brachistosternus species were cytogenetically analyzed it was not possible to determine the ancestral karyotype and therefore the rearrangements that lead to the different chromosome numbers (e.g., centric or tandem fusions versus fissions). The intraspecific morphological and karyotypic variations of B. pentheri indicate that its marginal northern populations could be a different subspecies or even species. This proposal could be tested by attempting hybridization between the typical and northern morphs.

[FIGURE 4 OMITTED]

The genus Brachistosternus was studied cytogenetically for the first time by Giacomozzi (1977) (Tab. 1), who mentions that his specimens were collected and determined by Dr. E. Maury as B. psammophilus Maury 1977 and B. alienus Lonnberg 1898. B. psammophilus was considered a possible endemic species confined to coastal dunes in southern Buenos Aires province (Maury 1977). However, some years later Roig Alsina & Maury (1984) synonymized B. psammophilus with B. pentheri, a widespread species from central and northern Argentina. Therefore, the specimens studied by Giacomozzi (1977) as B. psammophilus should be considered B. pentheri. The karyotype of the specimens studied by Giacomozzi as B. psammophilus consisted of 42 chromosomes, like our specimens of B. pentheri from Oriente. Both samples of specimens belong to the same group of populations from coastal dunes of southern Buenos Aires.

On the other hand, the identity of the second species of Brachistosternus studied by Giacomozzi (1977) is uncertain. Maury determined these specimens as B. alienus (Giacomozzi 1977), but it is possible that they belong to B. angustimanus Ojanguren-Affilastro & Roig-Alsina 2001. At the time of Giacomozzi's investigation, most authors based the identification of B. alienus on the redescription by Mello-Leitao (1938, 1945), whose definition of B. alienus encompassed another species now known as B. angustimanus (Ojanguren-Affilastro 2001; Ojanguren-Affilastro & Roig-Alsina 2001). Both species are sympatric over most of their ranges, but B. angustimanus is more commonly found than B. alienus because of its larger size and higher abundance.

Brachistosternus alienus (sensu Giacomozzi 1977) shows the lowest chromosome number known for the genus (n = 14) (Giacomozzi 1977). Recent phylogenetic analyses (Ojanguren-Affilastro 2008; Ojanguren-Affilastro & Ramirez 2009) that include both morphological and molecular data placed B. pentheri as the sister group of the clade (B. angustimanus (B. alienus (Brachistosternus telteca Ojanguren-Affilastro 2000, Brachistosternus multidentatus Maury 1984))). Therefore, the assessment of whether the low chromosome number is a synapomorphy of the clade or an autapomorphy of the specimens determined by Maury as B. alienus should be made using specimens accurately identified as B. alienus and B. angustimanus.

ACKNOWLEDGMENTS

The present work was supported by grants from the National University of Buenos Aires (UBA) (Ex 317, L. Mola) and from the National Council of Scientific and Technological Research (CONICET) (PIP 5927, L. Mola and PIP 5654, C. Scioscia). Fieldwork was supported by CONICET grant PIP 6502 (Mercedes Lizarralde de Grosso), and by a MACN grant for collections improvement to C. Scioscia and A. A. Ojanguren-Affilastro. The authors wish to thank L. Compagnucci, J.J. Martinez, and L. Piacentini for their help in the fieldwork, and H. Dinapoli for maintaining the scorpions in captivity.

Manuscript received 21 November 2008, revised 21 July 2009.

LITERATURE CITED

Cekalovic, K.T. 1969. Brachistosternus pegnai n. sp. de escorpion para el Ecuador (Scorpionida-Bothriuridae). Boletin de la Sociedad Biologica de Conception 41:163-171.

Ferreira, A. 1968. Contribution to the knowledge of cytology of two species of Brazilian scorpions: Opisthacanthus manauarensis Ferreira, 1967 (Scorpiones, Scorpionidae) and Bothriurus asper araguaie (Scorpiones, Bothriuridae). Anais da Academia Brasileira de Ciencias 40(1):97-99.

Giacomozzi, R.O. 1977. Citotaxonomia de Bothriuridae (Scorpionida) de la Argentina. Physis (Buenos Aires), C 37:236.

Guenin, H.A. 1957. Contribution a la connaissance cytologique des Scorpions: les chromosomes de Pandinus imperator Koch. Revue Suisse de Zoologie 64:349-353.

Guenin, H.A. 1961. Contribution a la connaissance cytologique des Scorpions: les chromosomes de Buthus occitanus Amor. (1). Vie et Mileu 12:89-96.

Maury, E.A. 1974. Escorpiofauna Chaquena. 1. La verdadera identidad de Brachistosternus (Microsternus) ferrugineus (Thorell 1876) (Bothriuridae). Physis (Buenos Aires), C 33(86):73-84.

Maury, E.A. 1977. Un nuevo Brachistosternus de los medanos costeros bonaerenses (Scorpiones: Bothriuridae). Physis (Buenos Aires), C 37(93):169-176.

Mello-Leitao, C.F. de. 1938. Notas sobre alacranes argentinos. Notas del Museo de La Plata 3(9):83-95.

Mello-Leitao, C.F. de. 1945. Escorpioes sul-americanos. Arquivos do Museu Nacional 40:7-468.

Ochoa, J.A. 2002. Nueva especie de Brachistosternus Pocock (Scorpiones. Bothriuridae) del sur del Peru. Revista Peruana de Biologia 9:55-63.

Ochoa, J.A. 2004a. Filogenia del genero Orobothriurus y description de un nuevo genero de Bothriuridae (Scorpiones). Revista Iberica de Aracnologia 9:73.

Ochoa, J.A. 2004b. Brachistosternus ninapo una nueva especie (Scorpiones: Bothriuridae) de los Andes occidentales en el sur del Peru. Revista Peruana de Biologia 11:139-148.

Ochoa, J.A. & L.E. Acosta. 2002. Two new Andean species of Brachistosternus Pocock (Scorpiones. Bothriuridae). Euscorpius 2:1-13.

Ochoa, J.A. & A.A. Ojanguren-Affilastro. 2007. Systematics and distribution of Brachistosternus (Brachistosternus) ehrenbergii (Gervais, 1841) with the first record of stridulation in this genus Brachistosternus (Scorpiones: Bothriuridae). Studies on Neotropical Fauna and Environment 41:79-85.

Ojanguren-Affilastro, A.A. 2001. Sistematica y distribution de Brachistosternus alienus Lonnberg (Scorpiones, Bothriuridae). Revista del Museo Argentino de Ciencias Naturales 3:169-174.

Ojanguren-Affilastro, A.A. 2003a. Las especies andinas de Brachistosternus (Leptosternus), con la description de tres nuevas especies (Scorpiones, Bothriuridae). Revista Iberica de Aracnologia 8: 23-36.

Ojanguren-Affilastro, A.A. 2003b. The genus Brachistosternus in Argentina, with the description of a new species from Patagonia (Scorpiones, Bothriuridae). Journal of Arachnology 31:317-331.

Ojanguren-Affilastro, A.A. 2005a. Estudio monografico de los escorpiones de la Republica Argentina. Revista Iberica de Aracnologia 11:75-241.

Ojanguren-Affilastro, A.A. 2005b. Notes on the genus Brachistosternus (Scorpiones, Bothriuridae) in Chile, with the description of two new species. Journal of Arachnology 33:175-192.

Ojanguren-Affilastro, A.A. 2008. Sistematica y filogenia de los escorpiones del genero Brachistosternus Pocock (Arachnida, Scorpiones, Bothriuridae). Thesis. Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires.

Ojanguren-Affilastro, A.A. & M.J. Ramirez. 2009. Phylogenetic analysis of the scorpion genus Brachistosternus (Arachnida, Scorpiones, Bothriuridae). Zoologica Scripta 38:183-198.

Ojanguren-Affilastro, A.A. & A. Roig Alsina. 2001. Brachistosternus angustimanus, una nueva especie del norte de la Patagonia, Argentina (Scorpiones, Bothriuridae). Physis (Buenos Aires), C 58:15-22.

Ojanguren-Affilastro, A.A. & C.L. Scioscia. 2007. New data about genus Brachistosternus in Chile (Scorpiones: Bothriuridae). Journal of Arachnology 35:102-113.

Ojanguren-Affilastro, A.A., C.I. Mattoni & L. Prendini. 2007a. The genus Brachistosternus in Chile (Scorpiones. Bothriuridae), with the description of two new species. American Museum Novitates 3564:1-44.

Ojanguren-Affilastro, A.A., P. Agusto, J. Pizarro Araya & C.I. Mattoni. 2007b. Two new scorpion species of genus Brachistosternus (Scorpiones: Bothriuridae) from northern Chile. Zootaxa 1623:55-68.

Piza de Toledo, S. 1947. Notas sobre crommossomios de alguns escorpioes brasileiros. Anais da Escola Superior de Agricultura "Luiz de Queiroz" 4:169-176.

Prendini, L. 2003. A new genus and species of bothriurid scorpion from the Brandberg Massif, Namibia, with a reanalysis of bothriurid phylogeny and a discussion of the phylogenetic position of Lisposoma Lawrence. Systematic Entomology 28:149-172.

Reeves, A. & J. Tear. 2000. MicroMeasure for Windows, Version 3.3. Free program available online at http://www.colostate.edu/Depts/Biology/MicroMeasure.

Roig-Alsina, A.H. 1977. Una nueva especie de escorpion andino en Mendoza, Republica Argentina. Physis (Buenos Aires), C 37:255-259.

Roig-Alsina, A.H. & E.A. Maury. 1984. Sistematica y distribution geografica de Brachistosternus (L.) pentheri Mello-Leitao, (1931). (Scorpiones, Bothriuridae). Physis (Buenos Aires), C 42:17-21.

Shanahan, C. 1989a. Cytogenetics of Australian scorpions. I. Interchange polymorphism in the family Buthidae. Genome 32:882-889.

Shanahan, C. 1989b. Cytogenetics of Australian scorpions. II. Chromosome polymorphism in species of Urodactus (Family Scorpionidae). Genome 32:890-900.

Sergio G. Rodriguez-Gil (1), Andres A. Ojanguren-Affilastro (2,3-4) , Leonel M. Barral (1), Cristina L. Scioscia (2), and Liliana M. Mola (1,3): (1) Laboratorio de Citogenetica y Evolucion, Departamento de Ecologia, Genetica y Evolucion, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellon 2, Cuarto Piso, (C1428EHA) Buenos Aires, Argentina; (2) Division Aracnologia, Museo Argentino de Ciencias Naturales "Bernardino Rivadavia" (CONICET), Avenida Angel Gallardo 470 (C1405DJR) Buenos Aires, Argentina; (3) Carrera del Investigador (CONICET)

(4) Corresponding author. E-mail: andres.ojanguren@gmail.com; ojanguren@macn.gov.ar)
Table 1.--Karyotype characteristics and collecting localities
of the Bothriuridae species cytogenetically analyzed.

Species                       2n  n   Locality

Bothriurus sp.                36  --  Tres Lagoas, Matto Grosso,
                                         Brazil
Bothriurus araguayae          44  22  Sao Paulo, Brazil
  Vellard 1934
Bothriurus flavidus           48  24  Buenos Aires Province, Argentina
  Kraepelin 1911
Bothriurus prospicuus         50  25  Buenos Aires Province, Argentina
  Mello-Leitao 1934
Brachistosternus alienus      28  14  Chubut Province, Argentina
  Lonnberg 1898
Brachistosternus ferrugineus  46  23  San Marcos Sierra, Cordoba
  (Thorell 1876)                        Province, Argentina
B. ferrugineus                46  23  Chepes, La Rioja Province,
                                        Argentina
Brachistosternus montanus     46  23  Laguna Brava, La Rioja Province,
  Roig-Alsina 1977                      Argentina
Brachistosternus pentheri     46  23  Villa Union, La Rioja Province,
  Mello-Leitao 1931                     Argentina
B. pentheri                   42  21  Chepes, La Rioja Province,
                                        Argentina
B. pentheri                   42  21  Oriente, Buenos Aires Province,
                                        Argentina
B. pentheri                   42  21  Buenos Aires Province, Argentina

Timogenes elegans             48  24  Rio Negro Province, Argentina
  (Mello-Leitao 1931)

Species                       References

Bothriurus sp.                Piza 1947
Bothriurus araguayae          Ferreira 1968 (sub. B.
  Vellard 1934                  asper araguaie)
Bothriurus flavidus           Giacomozzi 1977
  Kraepelin 1911
Bothriurus prospicuus         Giacomozzi 1977
  Mello-Leitao 1934
Brachistosternus alienus      Giacomozzi 1977
  Lonnberg 1898
Brachistosternus ferrugineus  This work
  (Thorell 1876)
B. ferrugineus                This work
Brachistosternus montanus     This work
  Roig-Alsina 1977
Brachistosternus pentheri     This work
  Mello-Leitao 1931
B. pentheri                   This work
B. pentheri                   This work
B. pentheri                   Giacomozzi 1977(sub. B.
                                psammophilus Maury)
Timogenes elegans             Giacomozzi 1977
  (Mello-Leitao 1931)

Table 2.--Relative lengths (RL) of bivalents of Brachistosternus
pentheri cytotypes.

Bivalent number                      RL (%)

                  Cytotype I (n = 23)   Cytotype II (n = 21)

1                        8.17                   8.78
2                        7.22                   7.99
3                        6.57                   7.14
4                        5.70                   6.42
5                        5.34                   5.79
6                        5.13                   5.57
7                        5.00                   5.39
8                        4.85                   5.12
9                        4.65                   4.96
10                       4.41                   4.83
11                       4.29                   4.71
12                       4.08                   4.58
13                       3.91                   4.33
14                       3.68                   4.17
15                       3.63                   3.74
16                       3.48                   3.36
17                       3.28                   3.02
18                       3.11                   2.81
19                       3.00                   2.66
20                       2.89                   2.46
21                       2.78                   2.17
22                       2.53
23                       2.30
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