chromosome evolution in the phylum echinodermata

Chromosome evolution in the phylum Echinodermata 299

sarcoplasmatiques, appliquke A la systbmatique de la famille Cobitidae (Pisces). Trav. Mus. Hist. Nat . “Gr. Antipa”, Bucarest 11, 339-346.

LEONE, C. A,, 1949: Comparative serology in some brachyuran Crustacea and studies in Hemocynin correspondence. Biol. Bull. 97, 273-286.

- (edit.) 1962: Taxonomic Biochemistry and Serology. N e w York: Ronald Press Comp. MAYR, E., 1957: Species Concepts and Definition, in: MAYR, E. (edit.), The Species Problem.

- 1963: Animal Species and Evolution. Cambridge, Massachusetts: Harvard Univers. Press. - 1969: Principles of Systematic Zoology. N e w York: McGraw-Hill Book Company. MESTER, L., 1972: Studiul Familiei Cobitidae (Pisces) din Romania (The Study of the Family

Cobitidae (Pisces) in Romania). Summary of D. S. degree Thesis, University Bucuresti.

Author’s address: Institutul de Stiinte Biologice, Department of Taxonomy a n d Evolution,

Amer. Assoc. Advancement of Science, Washington, 1-22.

Spl. Independentei 296, Bucuresti 17, Romania

Institute of Animal Biology, University of Padua, Italy

Chromosome evolution in the phylum Echinodermata

By DARIO COLOMBERA

With 19 figures

Received on 2 6 . 4 . 1974

There is a varying amount of information concerning the chromosomes of apparently only 29 species (see table) of approximately 6000 living echinoderms. Mitotic chromosomes have been described as acrocentric in Echinocardium mediterraneum (Ubisch, 1923), Asterias amurensis and Aphelasterias nipponica (Makino and Niiyama, 1947), or mainly acrocentric with a few metacentric in Toxopneustes variegatus (Heff ner, 1910), Henricia nipponica (Makino and Niiyama, 1947), or in varying combinations of metacentric, submetacentric and acrocentric in Arbacia punctulata (German, 1966) and Clypeaster rosaceus (Gardiner, 1927). Diff erenciated sex chromosomes, of the type XX or XY, in cleaving eggs of Clypeaster rosaceus (Gardiner, 1927) and in spermatocytes of Henricia nipponica (Makino and Niiyama, 1947) have been tentati- vely reported. The dimensions of echinoderm chromosomes are relatively small and, in Echinaster sepositus, the smallest ones have been considered to be microchromo- somes (DELOBEL 1971). The morphology of spermatpcyte chromosomes, well illustrated for Echinaster sepositus indicates that spermatogenesis is chiasmatic. A haploid number of nine for fifteen species (see table), representing all the five classes of the phylum echinodermata might appear to support the hypothesis that this is the basic haploid number for this phylum as well as indicating an outstanding chromosomal stability. Unfortunately, the scantiness of karyological research, the frequent discrepancies in chromosome counts by different authors studying the same species (table), the low correlation of chromosome numbers even with the lowest taxonomical categories, do not enable us to formulate hypotheses of echinoderm citotaxonomy and chromosomal evolution. A revision and an augmentation of the available reports seems necessary; the present research is addressed to this objective.

2 . zool. Syst. Evo1ut.-ford. 12 (1974) 299-308 @ 1974 Verlag Paul Parey, Hamburg und Berlin ISSN 0044-3808 / ASTM-Coden: ZZSEAA

300 D. Colombera

Table

Chromosome numbers of echinoderms

I Diploid number I Species prior prior Authors

Class Echinoidea Family Centrechinidae

Arbacia lixula Arbacia pustulosa Arbacia pustulosa Arbacia punctulata Arbacia punctulata Arbacia punctulata Arbacia punctulata Arbacia punctulata Ecbinus acutus Ecbinus esculentus Ecbinus esculentus Ecbinus miliaris Ecbinus miliaris Psammecbinus microtuberculatus Psamrnecbinus microtuberculatus Psammecbinus microtuberculatus Psammecbinus microtuberculatus Paracentrotus lividus Paracentrotus lividus Paracentrotus purpuratus Paracentrotus intermedius Sphaerecbinus granularis Sphaerechinus granularis Sphaerecbinus parma Hemicentrotus pulcherrimus Anthocidaris crassispina Toxopneustes variegatus Toxopneustes variegatus Toxopneustes variegatus Hipponoe esculenta

Family Clipeasteridae Clypeaster rosaceus

Family Schizasteridae Moira atropos

Family Brissidae Brissuls unicolor Echinocardium cordatum Ecbinocardium mediterranerm

Class Asteroidea

Asterias amurensis Asterias glacialis Asterias glacialis Asterias vul aris Asterias forfesii Asterias forbesii Aphaelasterias japonica

Ecbinaster sepositus Ecbinaster sepositus Ecbinaster sepositus Henricia nipponica Cribrella sanguinolenta

Family Asteridae

Family Echinasteridae

40 18

38 18 38 20 34 18 38 18

18 18 18

18

38 36 38 32

46

18

18 18 36 18 36

18

36

40 38

34-40

50

44-46

42

30

48

54

44

44 44

42

42 44-46

42 42

42

44 44

BALTZER 1910 WILSON 1895 this report TENNENT 1912 MATSUI 1924 HARVEY 1940 AUCLAIR 1964 GERMAN 1964 DONCASTER and GRAY 1911 DELAGE 1901 DONCASTER and GRAY 1911 MORGAN 1895 DONCASTER and GRAY 191 1 WILSON 1895 ST EVENS^^^^ GODLEWSKI 1905 this report WILSON 1895 DELAGE 1901 HINDLE 1910 MAKINO and NIIYAMA 1947 WILSON 1895 this report AUCLAIR 1965 NISHIKAVA 1961 NISHIKAVA 1961 WILSON 1901 HEFFNER 1910 TENNENT 19 12 PINNEY 1911

GARDINER 1926

PINNEY 1911

this report WILSON 1895 UBISCH 1923

MAKINO and NIIYAMA 1947 WILSON 1895 DELAGE 1901 TENNENT 1907 WILSON 1895 TENNENT and HOGUE 1906 MAKINO and NIIYAMA 1947

WILSON 1895 DELOBEL 1971 this report MAKINO and NIIYAMA 1947 JORDAN 1910


Table (Continuation)

Species

Family Phidiasteridae Ophidiaster ophidianus Hacelia attenuata

Family Astropectinidae Astropectem bispinosus

Family Asterinidae Asterina pectiniformis

Class Ophiuroidea

Ophiomixa pentagona Ophiomixa pentagona

Ophiothrix fragilis Ophiothrix fragilis

Ophioderma longicauda Ophioderma longicauda

Ophiocomina nigra

Family Ophiomixidae

Family Ophiotricidae

Family Ophiodermatidae

Family Ophiocomidae

Class Holothuroidea Family Holothuridae

Stichopus regalis Holothuria polii Holothuria tubulosa

Cucumaria cucumis Class Crinoidea

Family Cucumaridae

Family Antedonidae Antedon rosacea

prior I 1%

18

18

18

18 18

18

18

Diploid number

-

prior

40

44 44

44

42

44

42

44

44

1 Authors

this report this report

this report

MAKINO and NIIYAMA 1947

WILSON 1895 this report



this report

WILSON 1895 WILSON 1895 this report

WILSON 1895

WILSON 1895

Material and methods

The following species of echinoderms have been utilized for karyological analysis:

class Echinoidea

Family Centrechinidae:

Family Brissidae :

Family Echinasteridae: Family Ophidiasteridae: Family Astropectinidae:

A rbacia pustulosa, Psammechinus microtuberculatus, Sphaerechinus granularis; Brissus unicolor;

class Asteroidea

Echinaster sepositus; Ophidiaster ophidianus, Hacelia attenuata; Astropecten bispinosus;

302 D. Colombera

class Ophiuroidea

Family Ophiomixidae: Family Ophiotricidae : Family Ophiodermatidae: Family Ophiocomidae:

Family Holothuridae:

Ophiomixa pentagona; Ophiothrix fragilis; Ophioderma longicauda; Ophicomina nigra;

class Holothuroidea

Holothuria tubulosa.

Of each species successful chromosome preparations were made from at least four specimens, the only exception being Brissus unicolor for which only one specimen gave satis- factory preparations. All the specimens employed were collected in the Gulf of Naples, Italy, with the exception of Ophiocomina nigra which was dragged in Plymouth Sound, England, and Astropecten bispinosus collected in the Lagoon of Venice, Italy. To obtain cleaving eggs of Arbacia pustulosa, Psammechinus microtuberculatus and Sphaerechinus granularis, a few adults of each species were stimulated by a ten volt alternating current applied to the aboral zone with lead electrodes. Eggs were transferred to sea water in a petri dish. Five drops of sperm were diluted in a test tube containing 30 rnl of sea water. Four drops of this suspension of sperm were transferred to the petri dish containing the eggs. A&er dispersion of the sperm among the eggs, practically 100 O/o fecundation was obtained and cleaving eggs were abundant. Chromosome preparations were made as elsewhere described for ascidians (COLOMBERA 1969, 1970). Observation were made employing a Zeiss phase contrast microscope and when a camera was not available, drawings were made with the aid of a camera lucida.

Results

Arbacia pustulosa = lixula (L.)

Several hundred chromosome plates in various stage of division were found in cleaving eggs and when the chromosomes were well separated, (fig. 1) the diploid number 44 was regularly and easily counted. Consequently, it is assumed that both sexes have 44 chromosomes. Evidence of somatic pairing was noted, but confirmation must await statistical elaboration. T w o pairs of chromosomes are distinctive because of their greater thickness and length and were interpreted as metacentric. I n other chromosomes the position of the centromere is denoted by a bend during anaphase. This bend seems to vary in position from central to distal along the chromosomes.

Male bivalents at diakinesis (fig. 2) appear as small, condensed, generally roundish bodies. Counting them is difficult because of their small size though in 15 diakinetic plates a bivalent count of 22 was found ten times.

Psammechinus microtuberculatus (BLV) The array of count of chromosomes in cleaving eggs was 41, 42, 43, 44 with 42 being the most frequent value; therefore 42 is considered, with some reservation, to be the diploid number for this species.

It is of great interest to note that before the chromosomes have reached a metaphase configuration, some of them show a peculiar separation of daughter chromatids (fig. 3).

Sphaerechinus granularis (LMK) As in Psammechinus microtuberculatus, 42 was the most frequent number of chromosomes encountered in cleaving eggs and this number is considered to be the diploid value for this species. The precocious separation of daughter chromatids in prometa-

Fig. 1. Anaphase chromosomes in cleaving egg of Arbacia pustulosa. The chromosomes migrat- ing to a spindle pole are represented. - Fig. 2 . Spermatocyte bivalents of Arbacia pustulosa. - Fig. 3 . Prometaphase chromosomes in dividing egg of Psammechinus rnicrotuberculatus. In some chromosomes daughter kinetochores are distanced (see arrows). - F i g . 4 . Prometaphase chromosomes in dividing egg of Sphaerechinus granularis. In some chromosomes daughter kinetochores are distanced (see arrows). - F i g . 5 . Spermatocyte bivalents of Brissus unicolor. - Fig. 6 . Spermatocyte bivalents of Echinaster sepositus. - Fig. 7 . Spermatocyte bivalents in Hacelia attenuata. - Fig . 8 . Spermatocyte bivalents in Ophidiaster ophidianus. At pachitene

homologous chromosomes are not distinguishable from each other.

304 D. Colombera

phase chromosomes is often evident (fig. 4). Again, two couple of chromosomes are conspicuous for their relatively greater size.

Brissus unicolor Klein

On the basis of counts for 30 diakinetic plates, the haploid number has been estimated to be 21. I have some reservations on the accuracy of this count owing to the tendency of spermatocyte bivalents to stirk together. All the bivalents form a graded series of strongly condensed bodies of dumb-bell or roundish aspect (fig. 5).

Echinaster sepositus Gray

The haploid number 22 was determined with certainty by counting 40 diakinetic plates from the tests of four specimens (fig.6). At diakinesis the largest bivalents, plus a few of the others, are ring-shaped, but most of the bivalents are condensed into two roundish intensively stained bodies. With the exception of the largest bivalents, all the chromosomes form a graded dimensional series. The position of kinetochores was not determinable.

Hacelia attenuata Gray

When the bivalents in the male gonad were well separated (fig. 7), the haploid number 22 was found in 13 of 15 plates. When a haploid number greater than 22 was scored, an examination of bivalent morphology indicated that some of them were broken into two. According to their morphology these bivalents can be distinguished into two different types: ring-shaped and dumb-bell-shaped. Only the largest bivalents stand out from the other by virtue of their size.

Ophidaster ophidianus LMK

Spermatocyte bivalents in the pachytene phase (fig. 8) had a count of 22 in 8 of the 10 plates examined. The chromosomes are rod-shaped, but otherwise undiff erenciated in structure. At diakinesis (fig. 9) rod-shaped and ring-shaped bivalents are recogniz- able and again the number 22 was determined in 12 plates of the 16 examined. Anaphase-I chromosomes are all V-shaped and therefore metacentric (fig. 10).

Astropecten bispinosus Otto

Examining 25 diakinetic plates from testes, the haploid number was determined in 17 plates and in the remaining plates the considerable variation in chromosome numbers was due to the loss or breakage of chromosomes. These bivalents (fig. 11) are exceedingly small and are of the ring- und dumb-bell-shaped type.

Ophiomixa pentagona Muller et Tr. Twentyone bivalents were counted in 15 out of 20 diakinetic plates examined (fig. 12). Because of the frequent displacement of these bivalents this number is given with some reserve. There are ring-shaped, cross-shaped and cross-shaped bivalents. In some bivalents a pre-metaphase stretch is evident.

Ophiothrix fragilis Abildg.

The diploid number 44 was determined by counting four spermatogonial metaphase plates (fig. 13). These chromosomes are homogeneously stained, 2-5 pm in length. At

Fig. 9. Spermatocyte bivalents at kiakinesis in Ophidiaster ophidianus. - F i g . 10. Anaphase-I chromosomes in testicles of Ophidiaster ophidianus. - Fig. 11. Spermatocyte bivalents of Astro- pecten bispinosus. - Fig. 12. Spermatocyte bivalents of Ophiomixa pentagona. - Fig. 23. Sper- matogonial metaphase chromosomes of Ophiothrix fragilis. - F i g . 1 4 . Spermatocyte bivalents of Ophiothrix fragilis. - F i g . 15. Spermatogonial metaphase chromosomes of Ophioderma longicauda. - Fig. 16. Spermatocyte bivalents of Ophioderma longicauda. - F i g . 17. Spermato- cyte bivalents at early diakinesis of Ophiocomina nigra. - F i g . 18. Spermatocyte bivalents at late diakinesis of Ophicomina nigra. - F i g . 29. Spermatocyte bivalents of Holothuria tubulosa

306 D. Colombera

late diakinesis, there were 22 spermatocyte bivalents (fig. 14) in 16 of the 22 plates examined; in another nine plates, probably due to displacement and oversquashing of the chromosomes, there were fewer bivalents noted. The cross-shaped aspect of some bivalents is probably due to kinetochore activity.

Ophioderma longicauda Linck

Spermatogonial metaphase chromosomes (fig. 15) appear as two rod-shaped sister chromatids easily distinguishable from each other. A most probable diploid number of 44 was determined by counting the chromosomes from ten plates. Diakinetic bivalents (fig. 16) are strongly condensed bodies of dumb-bell and cross-shape type.

Ophiocomina nigra (Abildg.)

At early diakinesis, spermatocyte bivalents (fig. 17) tend to clump together. Cross- shaped and rod-shaped bivalents are clearly evident. At late diakinesis (fig. 18) bivalents are all homogeneously stained and condensed. The haploid number of 22 is based on scores for 20 of the 30 diakinetic plates examined. In some elements repulsion between kinetochores occurs.

Holothuria tubulosa Gmelin

From the male gonad of four specimens, 40 diakinetic plates were available for study (fig. 19). The haploid number 22 was determined with certainty in 24 of the 40 plates examined. The counts are often difficult because of the clumping of the bivalents. Only one element is conspicuously larger than the others. Both ring-shaped and dumb- bell-shaped bivalents are present.

Discussion

All the thirteen species investigated by me have a haploid number of 21-22. These values are in perfect accordance with all the other reports after 1947 (table) which based on modern techniques for chromosome preparation. Therefore this research confirms the low reliability of most of the data prior to 1947, either by direct comparison or, by checking the numbers in closely related species. For instance, the chromosomes of Arbacia pustulosa are reported to have a haploid number of nine (WILSON 1895), whereas I found a diploid number of 44 and a haploid number of 22 which agree with those recently found for other species of the genus Arbacia (AUCLAIR 1965; GERMAN 1966). The present evidence indicates that echinoderms possess only haploid numbers of 21 or 22 (table). Although the validity and generality of this uniformity need to be evaluated using a larger number of species, the finding of the same and only the same chromosome numbers in four classes of the phylum Echino- dermata, suggests that echinoderms might represent by far the most extensive example of chromosomal uniformity so far known. In other terms, this uniformity indicate that since Precambrian times (CUBNOT 1948) echinoderms have been capable of evolving into four different classes and several families varying their basic chromosome complement of only one element. This uniformity is not established as far as chromosomal morphologies are concerned (UBISCH 1923 ; MAKINO and NIIYAMA 1947; HEFFNER 1910; GARDINER 1927; GERMAN 1966). The presence of sex-chromosomes in echinoderms reported by earlier authors (GARDINER 1927; MARINO and NIIYAMA 1947) is not supported by the most recent reports (AUCLAIR 1964; GERMAN 1966; DELOBEL


1971) and by this research. In fact, the pre-metaphase repulsion between homologous kinetochores observed in some spermatocyte bivalents is clearly a phaenomenon related to the “pre-metaphase stretch” (HUGHES-SCHRADER 1947) and has nothing to do with the presence of heterotypic chromosomes. The occurrence in cleaving eggs of both somatic pairing of homologous chromosomes and precocious separation of daughter kinetochores, herein described for the first time in the phylum Echino- dermata, together with the absence of heterotypic sex-chromosomes has been pre- viously reported only for ascidians (COLOMBERA 1972, 1973 a, b). Although the signi- ficance and degree of these similarities are still to be evaluated, they indicate a remote relationship between echinoderms and ascidians. Comparing the four classes herein investigated, my results point out that, a t a karyological level, Asteroidea and Echinoidea are closely related whereas a greater variation seems to occur between Ophiuroidea and Holothuroidea. These results are in accordance with data obtained from the studies on gross morphology (CU~NOT 1948). A further distinction can be made between Asteroidea/Echinoidea and Holothuroidea/Ophiuroidea, since in these two latter classes the dimensions of spermatocyte bivalents are distinctly greater than those found in the former two classes. Although spermatogonial metaphase plates are available for only Ophiothrix fragilis (fig. 13) and Ophioderma longicauda (fig. 15), it is interesting to note that considerable morphological and size differencies are evidenciated between these two species belonging to rather closely related families. The study of anaphase-I plates proved to be helpful in the determination of the position of kinetochores when it is not otherwise determinable.

In conclusion it seems that studies on echinoderm chromosomes might be profitably employed in investigating both chromosome evolution and the obscure phylogenetic relationships of the living echinoderms.

Acknowledgements This research was partially supported by grant C N R no. 72.01034/04 115.0542 from the Institute of Marine Biology, CNR, Venice to the Institute of Comparative Anatomy, Uni- versity of Padua, Italy. I would like to thank Dr. W. CANZONIER for correcting the manuscript.

Summary The finding of the haploid complements 21 or 22 in four classes of echinoderms confirms the low reliability of most of the previous reports and indicates that, at present, the haploid number 21 and 22 are the only reliable ones. In consequence the hypothesis is advanced that living echinoderms have been able of evolving with variation in their basic number of 21 or 22 of only one element since Precambrian times. This uniformity in chromosome numbers is not found in chromosome morphology. A close relationship occurs between echinoderms and ascidians in the behaviour and morphology of chromosomes.

Zusammenfassung Chromosomenevolution im Phylum Echinodermata

Fur das Phylum Echinodermata konnen nur die Chromosomenzahlen 21 und 22 als sicher fest- gestellt gelten. Es wird die Hypothese aufgestellt, dafl in der Evolution einiger Echinodermen seit dem Prakambrium keine Veranderung der Chromosomenzahlen stattgefunden hat. Mit die- ser zahlenmafligen Uniformitat geht jedoch eine bemerkenswerte Veranderlichkeit der Form der Chromosomen einher. Grofle Ahnlichkeit im Chromosomenverhalten besteht zwischen Echino- dermen und Ascidien.

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