Research Article |
Corresponding author: František Šťáhlavský ( stahlf@natur.cuni.cz ) Academic editor: Marielle Schneider
© 2018 František Šťáhlavský, Vera Opatova, Pavel Just, Leon N. Lotz, Charles R. Haddad.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Šťáhlavský F, Opatova V, Just P, Lotz LN, Haddad CR (2018) Molecular technique reveals high variability of 18S rDNA distribution in harvestmen (Opiliones, Phalangiidae) from South Africa. Comparative Cytogenetics 12(1): 41-59. https://doi.org/10.3897/CompCytogen.v12i1.21744
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The knowledge of cytogenetics in the harvestmen family Phalangiidae has been based on taxa from the Northern Hemisphere. We performed cytogenetic analysis on Guruia africana (Karsch, 1878) (2n=24) and four species of the genus Rhampsinitus Simon, 1879 (2n=24, 26, 34) from South Africa. Fluorescence in situ hybridization with an 18S rDNA probe was used to analyze the number and the distribution of this cluster in the family Phalangiidae for the first time. The results support the cytogenetic characteristics typical for the majority of harvestmen taxa, i.e. the predominance of small biarmed chromosomes and the absence of morphologically well-differentiated sex chromosomes as an ancestral state. We identified the number of 18S rDNA sites ranging from two in R. qachasneki Kauri, 1962 to seven in one population of R. leighi Pocock, 1903. Moreover, we found differences in the number and localization of 18S rDNA sites in R. leighi between populations from two localities and between sexes of R. capensis (Loman, 1898). The heterozygous states of the 18S rDNA sites in these species may indicate the presence of XX/XY and ZZ/ZW sex chromosomes, and the possible existence of these systems in harvestmen is discussed. The variability of the 18S rDNA sites indicates intensive chromosomal changes during the differentiation of the karyotypes, which is in contrast to the usual uniformity in chromosomal morphology known from harvestmen so far.
Karyotype, meiosis, sex chromosomes, FISH, 18S rDNA
The harvestmen (Opiliones) represent one of the oldest terrestrial arachnid lineages (
So far, the chromosomes of 90 species of harvestmen have been examined, which ranks them as the third best cytogenetically explored arachnid order (
The majority of the available chromosome data on harvestmen originates from the suborder Eupnoi (58 species examined) (
The suborder Eupnoi is divided into six families comprising 1822 species (Pinto-da-Rocha et al. 2007,
The material examined in this study is deposited in the National Museum, Bloemfontein, South Africa (
Guruia africana (Karsch, 1878): KwaZulu-Natal: Ndumo Game Reserve (26.8749°S, 32.2109°E), 4 males (NMBAO00900–NMBAO00903).
Rhampsinitus capensis (Loman, 1898): Eastern Cape: Hogsback (32.5888°S, 26.9352°E), 2 males, 2 females (NMBAO01016–NMBAO01019).
Rhampsinitus discolor (Karsch, 1878): Eastern Cape: Port St. Johns (31.5977°S, 29.5346°E), 2 males (NMBAO01023, NMBAO01024).
Rhampsinitus leighi Pocock, 1903: Eastern Cape: Silaka Nature Reserve (31.6529°S, 29.4919°E), 1 male (NMBAO01020); KwaZulu-Natal: Vernon Crookes Nature Reserve (30.27489°S, 30.6092°E), 2 males (NMBAO01021, NMBAO01022).
Rhampsinitus qachasneki Kauri, 1962: KwaZulu-Natal: Royal Natal National Park (28.7101°S, 28.9336°E), 1 male, 1 female (NMBAO01025, NMBAO01026).
The specimens were individually hand collected and kept alive until the gonad dissection. Chromosome preparation follows the “plate spreading” method (
The probe for 18S rDNA was prepared from Euscorpius sicanus (Koch, 1837). The whole genomic DNA was extracted using Genomic DNA Minikit - Tissue (Geneaid), following the manufacturer’s guidelines. The 18S rDNA fragment (GenBank accession number MG761815) were amplified with the following primer combination 18S-Gal forward: 5‘-CGAGCGCTTTTATTAGACCA-3‘ and 18S-Gal reverse: 5‘-GGTTCACCTACGGAAACCTT-3‘ (
The FISH protocol was performed following
The diploid number of chromosomes in the four males analysed was 24 (Fig.
Chromosomes of Guruia africana (2n=24) males after Giemsa staining (A–E) and after FISH with 18S rDNA (A partly, F–I). A karyotype based on mitotic metaphase B pachytene C diplotene D diakinesis E one sister cell of metaphase II F–I are the same cells as B–E after FISH with 18S rDNA. White arrows indicate the position of 18S rDNA and black arrows indicate the same position after Giemsa staining. Bar = 10 μm.
Two pairs of 18S rDNA clusters were detected by FISH in this species. The 18S rDNA probe signals were localized interstitially at approximately one third of the short arm of chromosome pair No. 1 and approximately at one third of the long arm of chromosome pair No. 3 (Fig.
The diploid number of chromosomes in the two males and two females analysed was 34 (Fig.
Chromosomes of Rhampsinitus capensis (2n=34) (A-E) and Rhampsinitus discolor (F–K) (2n=24) after Giemsa staining (A, B, D, F, G, J) and after FISH with 18S rDNA (A and F partly, C, E, H, I, K). A male karyotype based on mitotic metaphase B, C female mitotic metaphase D, E female pachytene, bottom left inset shows a reconstruction of the heteromorphic bivalent; crosses indicate the subterminal position of 18S rDNA; arrowhead indicates bivalent with the expanded positive heteropycnotic area F male karyotype based on two sister cells of metaphase II G, H male mitotic metaphase I diakinesis J, K two sister cells of metaphase II. White arrows indicate the position of 18S rDNA and black arrows indicate the position after Giemsa staining. Asterisks indicate large signal of heteromorphic bivalent. Bar = 10 μm.
The diploid number of chromosomes in the two males analysed was 24 (Fig.
We identified two cytotypes from two different localities in this species. The diploid numbers of chromosomes in both of them were 26 (Fig.
The karyotype of cytotype I from Vernon Crookes comprised six pairs of metacentric, five pairs of submetacentric (pairs No. 2, 4, 8, 9, 13), and one pair of subtelocentric (pair No. 5) chromosomes. Moreover, the karyotype of cytotype I included one heteromorphic bivalent (pair No. 11) composed of one metacentric and one submetacentric chromosome. The chromosomes of the first two pairs were distinctly longer (6.18 % and 5.67 % of the diploid set) than the next chromosomes, which gradually decrease in length from 4.29 % to 2.42 % of the diploid set (Suppl. material
Chromosomes of Rhampsinitus leighi (2n=26) from Vernon Crookes (A, C–F) and from Silaka (B, G–M) after Giemsa staining (A–C, E, G, I, J, L) and after FISH with 18S rDNA (A and B partly, D, F, H, K, M). A, B karyotypes of males based on mitotic metaphase C, D pachytene with heterozygous bivalent (asterisk), bottom right inset shows a reconstruction of the heteromorphic bivalent E, F diplotene with heterozygous bivalent (asterisk) G, H mitotic prophase I diplotene, arrowheads indicate bivalents with two chiasmata. J, K diakinesis L, M one sister cell of metaphase II. White arrows indicate the position of 18S rDNA and black arrows indicate the same position after Giemsa staining. Asterisks indicate heteromorphic bivalent with large signal of rDNA. Bar = 10 μm.
The karyotype of cytotype II from Silaka (Fig.
The diploid number of chromosomes in this species was 24 (Fig.
Chromosomes of Rhampsinitus qachasneki (2n=24) males after Giemsa staining (A, B, E) and after FISH with 18S rDNA (A partly, C, D, F). A male karyotype based on two sister cells of metaphase II B, C mitotic metaphase D diakinesis E, F two sister cells of metaphase II. White arrows indicate the position of 18S rDNA and black arrows indicate the same position after Giemsa staining. Bar = 10 μm.
Phalangiid harvestmen have a centre of diversity in the Northern Hemisphere (
The results of the cytogenetic analyses of Guruia africana (2n=24) and four species of the genus Rhampsinitus (2n=24, 26, 34) show that the African taxa share the basic karyotype characteristics with the Northern Hemisphere phalangiids analysed so far (13 species). However, detailed information about the karyotypes is only available in three species, whereas the records for the remaining species only comprise chromosome numbers (see
Alternatively, the second hypothesis presumes the ancestral number of chromosomes of the whole suborder Eupnoi being between 20 and 24 (the most frequent number in this suborder; see
The data concerning the number and position of the 18S rDNA clusters using the FISH technique in arachnids are still limited (see
Contrastingly, the variability of the number of 18S rDNA sites may be high in some arachnids (e.g.
We also detected intraspecific variability and a heterozygous state in R. capensis and R. leighi that may correspond to population polymorphism. The low dispersal capability, typical for most harvestmen (
Interestingly, we also identified heteromorphic bivalents in both populations of R. leighi caused by distinctly enlarged 18S rDNA clusters on one chromosome. Similarly, the heteromorphic bivalents were also detected in R. capensis, but the enlarged rDNA sites were only identified in females. The heterozygous states in the size of 18S rDNA clusters could indicate the existence of XY sex chromosomes in R. leighi and ZW sex chromosomes in R. capensis. The XY sex chromosome system is known in the harvestmen Sabacon makinoi Suzuki, 1949 (Dyspnoi, Sabaconidae) (
The size variation of rDNA clusters on homologous chromosomes is frequently considered a result of unequal crossing-over or gene duplication that does not represent sex-specific differences (e.g.
We are grateful to J.A. Neethling for technical support during the field work and to J. Štundlová for technical assistance during probe preparation. The project was supported by the Czech Republic Ministry of Education, Youth and Sports grant SVV 260 434 / 2017 and by grants from the National Research Foundation of South Africa in the Knowledge Interchange and Collaboration programme (No.’s 97495 and 105318) and Competitive Funding for Rated Researchers programme (No. 95569) to the last author. The specimens were collected under the collecting permits OP 4072/2016 from Ezemvelo KZN Wildlife (KwaZulu-Natal Province) and RA-0201 from the Eastern Cape Parks and Tourism Agency (Eastern Cape Province). Microscopy was performed in the Laboratory of Confocal and Fluorescence Microscopy, co-financed by the European Regional Development Fund and the state budget of the Czech Republic. Project no. CZ.1.05/4.1.00/16.0347 and CZ.2.16/3.1.00/21515.
Table S1
Data type: Microsoft Excel Worksheet (.xls).
Explanation note: Measurements of the relative chromosome length (% RCL) and arm ratio of South African harvestmen from family Phalangiidae (± standard deviation). Numbers of measured metaphases are given in brackets following the species names.
Table S2
Data type: Microsoft Excel Worksheet (.xls).
Explanation note: Numbers of nuclei with different numbers of chiasmata in diplotene of Rhampsinitus leighi (2n = 26) from Vernon Crookes (cytotype I) and Silaka (cytotype II).