Chromosome behaviour during male meioses has been studied in two bisexual amphimictic populations of two tardigrade species, namely Richtersius coronifer and Macrobiotus richtersi (Eutardigrada, Macrobiotidae). Both bisexual populations exhibit a diploid chromosome number 2n = 12 and no sex chromosomes were identified. DAPI staining and C-banding data indicate that all chromosomes of the bisexual population of R. coronifer are acrocentric. In both species, at male meiotic prophase, all six bivalent homologous chromosomes are aligned side by side along their length and show no evidence of chiasmata. However, in the oocytes of both species a chiasma is generally present in each bivalent at diplotene stage. Lack of recombination is previously unknown in tardigrades, but is a well known phenomenon in many other metazoans where it is always restricted to the heterogametic sex. In tardigrades there is no evidence of heterochromosomes, but it does not mean that in tardigrades, the heterogametic sex does not exist. The adaptive and evolutionary significance of achiasmatic meiosis is discussed

ALTIERO, Tiziana

REBECCHI, Lorena

English

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Hereditas 139: 116–120 (2003)First evidence of achiasmatic male meiosis in the water bearsRichtersius coronifer and Macrobiotus richtersi (Eutardigrada,Macrobiotidae)TIZIANA ALTIERO and LORENA REBECCHIDepartment of Animal Biology, Uniersity of Modena and Reggio Emilia, Modena, ItalyAltiero, T. and Rebecchi, L. 2003. First evidence of achiasmatic male meiosis in the water bears Richtersius coronifer andMacrobiotus richtersi (Eutardigrada, Macrobiotidae). — Hereditas 139: 116–120. Lund, Sweden. ISSN 0018-0661. Re-ceived March 11, 2003. Accepted September 17, 2003Chromosome behaviour during male meioses has been studied in two bisexual amphimictic populations of two tardigradespecies, namely Richtersius coronifer and Macrobiotus richtersi (Eutardigrada, Macrobiotidae). Both bisexual populationsexhibit a diploid chromosome number 2n=12 and no sex chromosomes were identified. DAPI staining and C-bandingdata indicate that all chromosomes of the bisexual population of R. coronifer are acrocentric. In both species, at malemeiotic prophase, all six bivalent homologous chromosomes are aligned side by side along their length and show noevidence of chiasmata. However, in the oocytes of both species a chiasma is generally present in each bivalent at diplotenestage. Lack of recombination is previously unknown in tardigrades, but is a well known phenomenon in many othermetazoans where it is always restricted to the heterogametic sex. In tardigrades there is no evidence of heterochromo-somes, but it does not mean that in tardigrades, the heterogametic sex does not exist. The adaptive and evolutionarysignificance of achiasmatic meiosis is discussed.Tiziana Altiero, Dipartimento di Biologia Animale, Uniersita` di Modena e Reggio Emilia, Via Campi 213/D, 41100Modena, Italy. E-mail: altiero.tiziana@unimore.itMany species of tardigrades exist as a number ofpopulations which are morphologically very similarto each other but which differ in sex ratio (bisexual orunisexual populations), ploidy (di-, tri-, or te-traploidy) and mode of reproduction (amphimixis orparthenogenesis). Parthenogenesis is a frequently oc-curring reproductive mode in tardigrades. It is oftenapomictic, less frequently automictic, the latter withor without genetic recombination (BERTOLANI 2001;REBECCHI et al. 2003). Heterogony has never beenobserved. The bisexual amphimictic populations arealways diploid, whereas the parthenogenetic ones areoften polyploid (tri- and tetraploid) and less fre-quently diploid (BERTOLANI 1994; REBECCHI et al.2003). In diploid strains, the karyotype has very lowvariability both in chromosome number (n=5 andn=6) and in chromosome shape and size(BERTOLANI 1982; REBECCHI et al. 2002). Hete-rochromosomes have never been identified. To date,the karyological analysis of germ cell maturation intardigrades has mainly been focused on the oocytesand very little on the male germ cells, in which onlymitotic and meiotic metaphases were observed(BERTOLANI 1975, 1994; REBECCHI et al. 2002). Malemeiotic metaphases showed very condensed bivalentsattached to one another to form a five-six-pointedstar (BERTOLANI 1975; REBECCHI et al. 2002). Con-sequently, karyotype and chromosome behaviourduring male meioses have in this paper been sub-jected to a deeper analysis, based on observations intwo bisexual populations belonging to two differenteutardigrade species.MATERIAL AND METHODSSexually mature males and some females of a bisex-ual population of Richtersius coronifer (Richters,1903) and sexually mature males of a bisexual popu-lation of Macrobiotus richtersi Murray, 1911 wereexamined. Both species belong to Macrobiotidae (Eu-tardigrada). The bisexual population of R. coroniferwas collected from a moss growing on beech-tree-trunks in a locality near Lago di Pratignano (1480 ma. s. l., northern Apennines, Italy), whilst the bisexualpopulation of M. richtersi was collected from hazelleaf litter at Formigine (80 m a. s. l., Modena, Italy).Chromosome preparations were performed bymeans of air-drying technique according to REBEC-CHI et al. (2002). Slides were stained either by con-ventional Giemsa solution (5 % in 0.01 M phosphatebuffer, pH 7.2 for 20 min), or by DAPI (4,6-di-amidino-2-phenylindole) and/or by CMA3 (Chro-momycin A3) according to SCHWEIZER (1976). Otherslides were C-banded according to SUMNER (1972)and stained with Giemsa, or sequentially stained withmethyl green, DAPI and CMA3, according to REBEC-CHI et al. (2002). Some animals were stained in totowith a drop of lactic acetic orcein. Slides were exam-First eidence of achiasmatic male meiosis 117Hereditas 139 (2003)ined with a Leitz DM RB microscope or with a ZeissAxioplan fluorescence microscope connected to digi-tal cameras Polaroid DMC Ie Low Light Kit andSpot (Diagnostic Instruments) respectively.RESULTSRichtersius coronifer has a karyotype of 2n=12 chro-mosomes which are very similar to each other inshape (roundish or lightly rod-shaped) and size (1.4m in length) in mitoses of both males and females(Fig. 1a). No sex chromosomes were observed.Prophases I and metaphases I of spermatocytes ex-hibit 6 bivalents. A primary constriction was notvisible either with Giemsa or fluorochromes stainingalone, whereas C-banded prophases I, made visiblewith Giemsa, showed a more heterochromatic regionon one telomere of each bivalent (Fig. 1c). Prophaseswere found in different condensation stages (Fig.1b– j). In early condensation stage, highly uncoiledFig. 1a–l. Mitotic and meiotic chromosomes in Richtersius coronifer. a Twelve mitotic chromosomes in a female gonad. bEarly condensation stage of spermatocyte. c-j Mid-late condensation stages, note the bivalents without chiasmata. cC-banded bivalents. e Connected homologous forming a circular feature. d and f-j Bivalents with two (d and f-i) or one only(j) more highly stained telomere, and the same regions DAPI (f and h) and CMA3 (g and i) positive; note that in f and gonly three bivalents are shown. k and l Metaphases I in polar view; in l a metaphase in side view is present (arrow). Lacticacetic orcein (a), Giemsa (c-e and l), DAPI (b, f, h, j and k) and CMA3 (g and i). Bar: 5 m.T. Altiero and L. Rebecchi118 Hereditas 139 (2003)bivalents could be seen (Fig. 1b). In mid-late conden-sation stage the two telomeres of each bivalent, orsometimes only one, often appeared more intenselystained than the other regions (Fig. 1d and f– j). Atthese stages the bivalents consist of parallel-alignedhomologous chromosomes (Fig. 1f and g) and noneof the six bivalents showed chiasmata (Fig. 1c, d andf– j). In only two prophases, of 125 observed, the twohomologous appeared partly overlapped or, in onecase, connected to form a circular feature (Fig. 1e).At metaphase I, in polar view, the six bivalentsshowed a radial arrangement on the equatorial plateto form a six-point star (Fig. 1k and l); they werevery coiled and the homologous chromosomes ofeach bivalent were not distinguishable. In side view,each homologous chromosome was recognisable (butnot the single sister chromatids) and oriented with itslonger axis parallel to the equatorial plate (Fig. 1l).After DAPI staining, the telomere of each bivalenttowards the centre of the plate sometimes fluorescedmore strongly than the other (Fig. 1k).In male meioses of Macrobiotus richtersi, the sixprophasic bivalents looked homogeneous whenGiemsa stained, whereas, sometimes, DAPI stainedspermatocyte prophases revealed an evident fluores-cence at the telomeres of each of the six bivalents(Fig. 2a and b). The two homologous of each biva-lent were very close to each other. In spermatocyteprophases, none of the six bivalents showed chias-mata (Fig. 2a and b). In metaphase I, in side view,the bivalents were disposed as in the former species,with the homologous chromosomes of each bivalentaligned side by side and facing the opposite poles onthe equatorial plate, whereas in polar view, the radialarrangement of the very condensed bivalents lookedlike a six-point star. Single chromatids and cen-tromeres were not visible.DISCUSSIONPrevious studies have shown that populations ofRichtersius coronifer differ in reproductive mode (RE-BECCHI et al. 2003). In fact, these authors found thatone Italian and one Swedish population were au-tomictic, whereas another Italian population, dis-cussed in this paper, was amphimictic. All thesepopulations, irrespective of their reproductive mode,were diploid with the same chromosome number,2n=12 (REBECCHI et al. 2003). Macrobiotusrichtersi, on the other hand, had a diploid (2n=12)amphimictic population and a triploid apomictic onecharacterised by 17-18 univalents in the oocytes (RE-BECCHI et al. 2002). In both cytotypes of M. richtersi(bisexual and unisexual) all chromosomes were acro-centric (REBECCHI et al. 2002). DAPI staining andC-banding data suggest that in bisexual amphimicticpopulation of R. coronifer also, all chromosomes areacrocentric. The greater intensity staining on the twotelomeres of each bivalent at early prophase is due toa different degree of coiling in the various parts of thechromosome, whereas the presence of only one morehighly stained telomere at late prophase, when thecoiling is almost complete, must be interpreted as anAT-rich region of each bivalent.A chiasma was identified in each oocytes bivalentof the two automictic populations of R. coronifer(REBECCHI et al. 2003). Moreover, in the oocytes ofthe amphimictic populations of both M. richtersi andR. coronifer, a chiasma was generally present in eachbivalent at diplotene (REBECCHI et al. 2002, 2003),whereas chiasmata were absent in the spermatocyteprophases of both species. The case of Fig. 1e may bedue to an achiasmatic meiosis of collochore type. It isvery unlikely that it represents a chiasmatic meiosisbecause not all the bivalents show circular or crossshape. Achiasmatic male meiosis of collochore typehas been found in Drosophila melanogaster (COOPER1964) and in the heteropteran Miridae (NOKKALAand NOKKALA 1986).This is the first report of the absence of recombina-tion in tardigrades males although it is a well knownphenomenon in many other invertebrates such asmolluscs, oligochaetes, crustaceans, scorpions, mitesand insects (WHITE 1973; BELL 1982; NOKKALA andGROZEVA 2000). The achiasmatic meiosis is alwaysrestricted to the heterogametic sex (WHITE 1973;BELL 1982; NOKKALA and GROZEVA 2000). In par-ticular, in one species of mites, in one genus ofMecoptera (ULLERICH 1961), in many genera ofmantids (Orthoptera), in many families of dipterans,including the very well known Drosophilamelanogaster (reviewed by WHITE 1973; BELL 1982),in coleopterans (SERRANO 1981, VIRKKI and SAN-Fig. 2. Prophase spermatocytes in Macrobiotus richtersi :bivalents without chiasmata; note that in b only five biva-lents are shown. Giemsa (a), DAPI (b). Bar: 5 m.First eidence of achiasmatic male meiosis 119Hereditas 139 (2003)TIAGO-BLAY 1996), and in several families of Het-eroptera (reviewed by KUZNETSOVA and MARYAN-SKA-NADACHOWSKA 2000; NOKKALA and GROZEVA2000) it occurs only in males. In Copepoda, Lepi-doptera and Trichoptera achiasmatic meiosis occursin females, the heterogametic sex (SUOMALAINEN1966; SUOMALAINEN et al. 1973; WHITE 1973; BELL1982; TRAUT and CLARKE 1996). An exception tothe correlation of achiasmatic meiosis-heterogameticsex occurs in certain hermaphroditic (and thereforewithout heterogametic sex) enchytraeid worms withachiasmatic meioses in both oogenesis and spermato-genesis (CHRISTENSEN 1961). In bisexual gonochorictardigrades there is no evidence of heterochromo-somes (REBECCHI et al. 2002, 2003), but it does notmean that in tardigrades, the heterogametic sex doesnot exist. NOKKALA and NOKKALA (1983) proposeda connection between restriction of achiasmatic meio-sis to the heterogametic sex and the nature of thecontrol of the meiotic mechanism. Probable con-trolling elements of the meiotic mechanism are lo-calised in the X-chromosome, therefore homogametyleads to normal chiasmatic meiosis and heterogametyto achiasmatic meiosis.The adaptive significance of achiasmatic meiosis isnot yet clear. WHITE (1973) proposed two alternativetheoretical explanations: i) a selection for a low levelof recombination ii) a facilitation of paracentric in-version heterozygosity avoiding the homozygosity oflethal alleles. The former explanation was later sus-tained by SERRANO (1981). Nevertheless, these twohypotheses are not supported by cytological data.According to NOKKALA and NOKKALA (1983), onemore likely explanation might be that achiasmaticmeiosis per se does not have adaptive value, but itcan be considered as one of the mechanisms by whichregular segregation of homologous chromosomes isachieved, the reduction of recombination being onlya secondary effect. In tardigrades, the selection for alow level of recombination might represent a morelikely explanation, since in both tardigrade speciesstudied the absence of chiasmata in male germ cellsand a very low frequency of chiasmata in femaleoocytes were observed. The absence of male geneticrecombination represents a further explanation ofheterozygote deficiency and deviation from Hardy-Weinberg equilibrium frequencies observed in thebisexual population of R. coronifer ; in which malesare heterozygous (REBECCHI et al. 2003). Het-erozygote deficiency was previously explained by hy-pothesising a genetic drift or inbreeding (REBECCHIet al. 2003). The presence of automictic parthenogen-esis may, however, explain the almost complete ab-sence of heterozygosity observed in the twoautomictic populations of R. coronifer. In fact, au-tomixis will produce heterozygote deficiencies and, inthe long run, completely homozygous lineages (RE-BECCHI et al. 2003).Acknowledgements – Particular thanks to Sandra K. Clax-ton of Macquarie University (New South Wales, Australia)for English revision of the manuscript. 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First evidence of achiasmatic male meiosis in the water bears Richtersius coronifer and Macrobiotus richtersi (Eutardigrada, Macrobiotidae)

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First evidence of achiasmatic male meiosis in the water bears Richtersius coronifer and Macrobiotus richtersi (Eutardigrada, Macrobiotidae)

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