Research Article |
Corresponding author: Galina V. Roslik ( roslik_g@mail.ru ) Academic editor: Virmantas Stunžėnas
© 2015 Alim P. Anisimov, Galina V. Roslik, Gennady N. Ganin.
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:
Anisimov AP, Roslik GV, Ganin GN (2015) Cytogenetic description of the earthworm Drawida ghilarovi Gates, 1969 (Oligochaeta, Moniligastridae) from the southern Russian Far East. Comparative Cytogenetics 9(4): 565-577. https://doi.org/10.3897/CompCytogen.v9i4.5741
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Sixty-six specimens of the earthworm Drawida ghilarovi Gates, 1969 (Oligochaeta, Moniligastridae) from 15 localities of the southern Russian Far East were studied cytogenetically. We examined chromosome sets during mitosis and diakinesis as well as DNA content in the spermatogenous and somatic cell nuclei. The populations and morphs displayed no differences in karyotype and ploidy levels estimated in terms of both chromosome number and DNA mass index: n = 10, 2n = 20; c = 1.1 pg, 2c = 2.2 pg. We conclude that polyploidy as a species- or race-forming factor is not typical of these earthworms.
Karyotype, chromosomal set, nuclear DNA content, earthworm, Drawida , Oligochaeta , Moniligastridae
The karyology of Oligochaeta is mainly studied in the earthworms of the family Lumbricidae. A distinctive feature of the evolution of their karyotypes is polyploidy, which is widespread in the family. The polyploid races and subspecies occur as frequently as the diploid ones (
There are no similar data on earthworms of the family Moniligastridae, in particular, Drawida ghilarovi Gates, 1969. The members of this family invaded the South-Eastern Asia after the collision of the Indian and Asian lithospheric plates in the Tertiary period, i.e., 66–1.6 m.y.a. (
Polyploidy is known to be accompanied by polymorphism. As the amount of available data increased, new color morphs of D. ghilarovi were described in the Russian literature, in particular, light-bluish (
It was found out that at the northern limit the tropical moniligastridae distribution the Red Book species Drawida ghilarovi Gates, 1969 exists in two distinct life forms: “soil-litter” (=epigeic) inhabitants of the floodplain meadow-wetland biotopes and “aneciques” of the forest biotopes (
We are not aware of any data on the cytogenetics of Drawida or Moniligastridae species in general. Some information was provided in our previous report (
Specimens of D. ghilarovi were collected in fifteen localities of the southern Russian Far East (Fig.
Map showing collection sites (1–15, see Table
Geographical locality, life form and colored morphs of examined D. ghilarovi.
Locality No. | Locality and biotope | Geographical coordinates | Life form and colored morph |
---|---|---|---|
Pra-Amur and Amur Rivers basin from north to south | |||
1 |
Khabarovsk Territory, Nanaysky District, Slavyanka village, marsh | 49°27'N, 136°46'E | epigeic, black |
2 |
Khabarovsk Territory, Nanaysky District, Anyuisky National Park, marsh | 49°20'N, 137°03'E | epigeic, black |
3 |
Jewish Autonomous Province, Bastak Nature Reserve, marsh | 48°59'N, 135°03'E | epigeic, black |
4.1 |
Khabarovsk Territory, Lazo District, Bolshekhekhtsirskii Nature Reserve, floodplain of Chirki River, marsh | 48°09'N, 135°08'E | epigeic, black |
4.2 |
The same place, marsh | 48°09'N, 135°08'E | epigeic, black-reddish |
5 |
Khabarovsk Territory, Lazo District, Bolshekhekhtsirskii Nature Reserve, floodplain of Odyr River, marsh | 48°06'N, 134°52'E | epigeic, black |
6 | Primorsky Territory, Spassky District, Lake Khanka Nature Reserve, meadow | 44°38'N, 132°49'E | epigeic, black |
7 |
Primorsky Territory, Nadezhdinsky District, floodplain of Razdol’naya River, meadow | 43°33'N, 131°54'E | epigeic, black |
West macro-slope of the southern Sikhote-Alin | |||
8.1 | Khabarovsk Territory, Bikinsky District, Shivki Mountain, forest | 47°00'N, 134°22'E | aneciques, grey |
8.2 | The same place, forest | 47°00'N, 134°22'E | aneciques, brownish |
9.1 |
Primorsky Territory, Ussuriysky Nature Reserve, forest | 43°33'N, 132°21'E | aneciques, greenish-grey |
9.2 | The same place, forest | 43°33'N, 132°21'E | aneciques, yellow-brown |
10 | Primorsky Territory, Mountain-taiga Biological Station, forest | 43°41'N, 132°09'E | aneciques, yellow-brown |
Black Mountains, Chanbaishan Plateau | |||
11 |
Primorsky Territory, Khasansky District, Kedrovaya Pad’ Nature Reserve, forest | 42°26'N, 130°38'E | aneciques, bluish-grey |
East macro-slope of the southern Sikhote-Alin | |||
12.1 |
Primorsky Territory, Vostok Biological Station, forest | 42°54'N, 132°44'E | aneciques, brownish, long |
12.2 |
The same place, forest | 42°54'N, 132°44'E | aneciques, brownish, short |
13.1 |
Primorsky Territory, Lazovsky Ridge, forest | 43°30'N, 133°35'E | aneciques, brownish, long |
13.2 |
The same place, forest | 43°30'N, 133°35'E | aneciques, brownish, short |
14.1 | Primorsky Territory, Lasovsky Nature Reserve, forest | 43°00'N, 133°44'E | aneciques, grey |
14.2 | The same place, forest | 43°00'N, 133°44'E | aneciques, brownish |
15.1 | Primorsky Territory, Sikhote-Alin Nature Reserve, forest | 45°14'N, 136°30'E | aneciques, grey |
15.2 | The same place, forest | 45°14'N, 136°30'E | aneciques, yellow-brown |
In accordance with conventional cytogenetic method, 0.04% colchicine solution was introduced into the body cavity for 18–20 h. For chromosome analysis, air-dried preparations of spermatogenous cells were made from suspended content of seminal vesicles incubated in 0.56% KCl solution and fixed with 3:1 mixture of ethanol and glacial acetic acid at 4 °C (
Preparations made from seminal vesicles contained spermatogenous cells of different maturity depending on sampling period (May–October). In the first half of the summer, spermatogonia and lepto-, zygo-, and pachytene spermatocytes-I predominated, while in the second half, diplotene, diakinetic spermatocytes, cells undergoing meiosis, spermatids, and mature sperm cells mostly occurred. Worms collected in autumn (September–October) or spring (May) were sexually inactive. Samples obtained from them contained degenerating spermatogenous cells of different maturity and rare spermatogonia foci. In summer samples, spermatogenous cells at all maturity stages were found.
Karyotypes were determined based on the joint analysis of chromosome sets in mitotic metaphase plates of dividing spermatogonia, in spermatocytes-I at diakinesis and metaphase I of the first meiotic division. The content of nuclear DNA was measured in coelomocytes and spermatogenous cells at all maturity stages, from spermatogonia to spermatids including chromosome plates. It was also measured in spermatozoa. However, due to high optical density of chromatin, values of DNA content in spermatozoa were lower than in spermatids. They were therefore excluded from further analysis.
Photometric estimation of nuclear DNA content and chromosome analysis did not reveal genotypic differences among worms sampled from different regions or biotopes or having differences in coloration.
Nuclear DNA content in spermatogenous cells was trimodally distributed, as expected, with twofold increment between neighboring nuclear classes. In most cases, a sparse fourth class was also observed (Fig.
Distribution of nuclear DNA content in spermatogenous and somatic cells from seminal vesicles of D. ghilarovi sampled in the Kedrovaya Pad’ Nature Reserve. Abscissa, DNA content (conventional units and haploid “c” units). Ordinate, number of nuclei.
In accordance with these results, tetraploid (4n8c) mitotic figures occurred in clusters of normal diploid mitoses (Fig.
Cluster of synchronously dividing spermatogonia from a seminal vesicle of D. ghilarovi sampled in the Lasovsky Nature Reserve. Arrows point at two tetraploid (4n8c) mitoses among ordinary diploid ones (2n4c). Scale bar: 5 µm.
The estimates of DNA content in coelomic fluid cells confirm the above results, in particular, the presence of octoploid spermatocytes. Coelomocytes used as a coarse reference of diploid DNA content normally had the expected DNA amount (2c), coinciding with the second peak of the distribution of DNA content in sexual cells (appr. 450 conventional units). Occasionally, the distribution of DNA content in coelomocytes displayed hypodiploid asymmetry, possibly, due to mass degradation (apoptosis) of these cells.
Polyploidization of a portion of spermatogenous cells may be considered analogous to somatic polyploidy (endopolyploidy, localized polyploidy) widespread in plants and animals. In somatic polyploidization, a portion of a cell population (occasionally, the whole population) switches to incomplete mitotic cycles including abortive mitosis, endomitosis, or DNA endoreplication in polytene chromosomes (
Using cytophotometry of spermatogenous cells and coelomocytes, the averaged diploid DNA content (in conventional units) was estimated for each of D. ghilarovi populations (life forms and colored morphs) (Table
Mean diploid DNA content in D. ghilarovi locality as determined by cytophotometry. SE – standard error.
Locality No. (see table 1) | Mean 2c DNA content ± SE (in conventional units) | Locality No. (see table 1) | Mean 2c DNA content ± SE (in conventional units) |
---|---|---|---|
1 |
442±9 | 9.2 | 436±5 |
2 |
458±9 | 10 | 479±8 |
3 |
463±5 | 11 |
477±5 |
4.1 |
444±8 | 12.1 |
445±8 |
4.2 |
465±9 | 12.2 |
442±6 |
5 |
467±8 | 13.1 |
448±8 |
6 | 474±6 | 13.2 |
460±8 |
7 |
467±9 | 14.1 | 468±7 |
8.1 | 433±6 | 14.2 | 467±7 |
8.2 | 442±6 | 15.1 | 466±8 |
9.1 |
462±7 | 15.2 | 470±5 |
The size of D. ghilarovi genome expressed as absolute DNA mass (pg) was estimated as follows. The photometric amount of 2c DNA averaged for 22 samples was 458 conventional units. Hence, c = 229 conventional units. The photometric amount of rat 2c DNA determined from cultured cell preparations using the same staining protocol was 1284 conventional units, which gives 642 conventional units per haploid amount (c). The absolute haploid DNA mass of the rat genome is 3.1 pg (see reference base in
Chromosome analysis revealed that worms belonging to different populations and color morphs, with dividing spermatogonia having 20 chromosomes in the diploid set (2n = 20) (Fig.
Mitotic metaphase (a) and karyogram (b) of D. ghilarovi from the Sikhote-Alin Nature Reserve. 2n = 20. Scale bars: 5 µm.
Mean length (ML), its standard deviation (SD) and centromere index (CI) of the chromosome pairs in six metaphase plates of D. ghilarovi. SE – standard error; m – metacentric, sm – submetacentric, st – subtelocentric chromosomes.
Chromosome pair | ML ± SE (µm) | SD of ML | CI ± SE | Centromere position |
---|---|---|---|---|
1 | 3.03±0.05 | 0.17 | 28.97 ±1.18 | sm |
2 | 2.71±0.07 | 0.25 | 27.65±1.14 | sm |
3 | 2.46±0.05 | 0.16 | 29.91±1.47 | sm |
4 | 2.33±0.06 | 0.21 | 24.10±0.37 | st |
5 | 2.20±0.05 | 0.17 | 44.93±0.87 | m |
6 | 2.06±0.06 | 0.19 | 30.16±1.62 | sm |
7 | 1.94±0.05 | 0.15 | 22.92±0.38 | st |
8 | 1.75±0.04 | 0.13 | 38.91±0.82 | m |
9 | 1.53±0.04 | 0.13 | 39.80±0.97 | m |
10 | 1.23±0.05 | 0.16 | 23.12±0.62 | st |
At early diakinesis almost all bivalents had a ring-like morphology, except two bivalents which were rod-shaped (Fig.
Early diakinesis (a) and meiotic metaphase-I (b) in D. ghilarovi from the Kedrovaya Pad’ Nature Reserve. n = 10. Scale bars: 5 µm.
The differences between chromosomes in relative DNA content were determined by cytophotometry of separate bivalents in several diakinetic plates. Their ranked series is presented in Table
DNA cytophotometry data of genome mass (%) distribution in chromosomes of individual diakinetic spermatocytes of D. ghilarovi. M – mean, SE – standard error.
Locality No. / specimen No. |
Chromosome No. | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
1/1 | 16.01 | 14.07 | 13.12 | 10.83 | 10.75 | 10.00 | 9.87 | 6.63 | 5.27 | 3.46 |
3/1 | 15.70 | 14.27 | 12.95 | 10.50 | 10.17 | 10.12 | 9.51 | 6.54 | 6.54 | 3.71 |
6/1 | 15.84 | 13.80 | 11.95 | 11.17 | 11.25 | 9.89 | 9.88 | 6.74 | 6.06 | 3.41 |
6/2 | 15.68 | 13.47 | 12.43 | 10.93 | 10.15 | 9.92 | 9.61 | 7.96 | 6.16 | 3.69 |
7/1 | 15.30 | 13.48 | 12.85 | 11.07 | 10.88 | 9.93 | 9.40 | 6.90 | 6.38 | 3.80 |
11/1 | 16.36 | 14.05 | 12.76 | 11.62 | 11.43 | 10.29 | 9.35 | 5.74 | 4.80 | 3.60 |
11/2 | 15.40 | 12.94 | 12.93 | 11.58 | 11.38 | 10.25 | 9.97 | 6.21 | 5.85 | 3.48 |
M ± SE | 15.76 ±0.14 |
13.73 ±0.17 |
12.71 ±0.15 |
11.10 ±0.15 |
10.86 ±0.20 |
10.06 ±0.06 |
9.65 ±0.09 |
6.67 ±0.26 |
5.87 ±0.24 |
3.59 ±0.06 |
As was mentioned above, there are no available data on the cytogenetics of Moniligastridae, in particular, Drawida. However, karyotypes of Lumbricidae worms are relatively well studied. Approximately half of the members of this group are polyploid. In most lumbricids, the basic haploid set includes 18 chromosomes, and diploid, 36 chromosomes (
To summarize the above, all examined D. ghilarovi populations from the southern Russian Far East had the same karyotype and ploidy level in terms of both chromosome number and DNA mass, exactly, n = 10, 2n = 20; c = 1.1 pg, 2c = 2.2 pg. In other words, polyploidization as a species- or race-forming factor is not typical of this group.
The present study was supported in part by the Russian Foundation for Basic Research (grant 12-04-00221-a) and the Science Foundation of the Far Eastern Federal University (project 14-08-01-4_i) as well as by technical support from the Center of Collective Use of Microscopy Objects of the Institute of Biology and Soil Science, Russian Academy of Sciences, Far East Branch, Vladivostok.