Cytogenetic Aspects of Evolution of the Family 1

A. C. TRIANTAPHYLLOU 2

Cytotaxonomy implies the utilization of phylogenetic relationships of the members of cytological information in . Such a group and have indicated the direction of information is mostly limited to the descrip- their evolution. The greatest phylogenetic tion of the karyotype, i.e., chromosome num- interpretation value of the karyotype is often ber, morphology, size, sex chromosomes, at the genus, and usually at or below the presence or absence of supernumerary or species level; this is because the causal rela- other types of chromosomes, and the study tionships of the observed karyotypic varia- of the behavior of the chromosomes during tions are easier to interpret at the genus or the mitotic and the meiotic cycle. With the species level due to a shorter evolutionary adoption of the biological species concept in time scale compared to that of higher taxa. taxonomy, however, it became neces- In keeping with the overall objectives of sary for species delineation to know the this symposium, I should probably discuss mode of reproduction of a particular orga- in general terms the role cytogenetic in- nism and its capabilities for effective inter- formation can play in solving problems in breeding with related organisms. To meet taxonomy. A general discussion, the new requirements, cytotaxonomy ex- however, involving all would be tended its activities to include the study of difficult to comprehend, and would provide the mode of reproduction through karyologi- no solutions to specific taxonomic problems. cal analysis of gametogenesis, and the study For this reason I will limit this discussion of the behavior of the chromosomes in hy- to the family Heteroderidae, an important brids between related organisms. These new family of plant-parasitic nematodes, in which approaches are both cytological and genetic a good deal of cytogenetic work has been in nature. Therefore, in a broader definition, conducted recently. General conclusions will cytotaxonomy utilizes "cytogenetic" informa- undoubtedly be applicable to other families tion for elucidation of taxonomic problems. of nematodes with similar cytogenetic char- Analysis of the karyotype has been valu- acteristics. able in the study of the relationships within The central theme of this discussion is that evolution in the family Heteroderidae has several animal groups such as primates, in- been influenced by extensive modifications sects, rodents, etc. Karyotype comparisons of the basic karyotype, including the estab- have often permitted interpretations of the lishment of polyploidy and aneuploidy, in

Received for publication 23 September 1969. association with the establishment of various 1 Paper number 2983 of the Journal Series of the North types of parthenogenetic reproduction. By Carolina State University Agricultural Experiment Station, studying the karyotypic relationships of the Raleigh, North Carolina. Supported by National Science Foundation Grant GB-7214. present day forms of these organisms, and 2 Department of Genetics, North Carolina State University at Raleigh, North Carolina 27607. considering other available cytogenetic in- 26 SYMPOSIUM: HETERODERIDAE; CYTOGENETIC EVOLUTION ° Triantaphyllou 27

formation, we may be able to make some Miller & Gray, H. mexicana Campos, and inferences as to the actual pathway of evolu- Osborne's cyst nematode. The karyotype tion of the family, and suggest a sound of all these species is quite similar. There taxonomic treatment. Of course, evaluation are some differences in chromosome mor- of the cytogenetic information alone, to the phology, chromosome size, and chromosomal exclusion of other available information, behavior during gametogenesis from species would share the same shortcomings of other to species, but, in the absence of a detailed taxonomic systems relying entirely on in- karyotypic analysis and comparison, such formation of one kind. A better system of differences are of limited value in character- classification may result, if all information izing the species or suggesting relationships available with regard to cytogenetics, mor- among them. phology, physiology, biochemistry, ecology, Another three species of that behavior and distribution of these organisms have been studied cytologically, i.e., H. tri- is considered and evaluated at the same time. folii (Goffart), H. galeopsidis (Goffart), In the present discussion emphasis will be and H. lespedezae Golden & Cobb, as well placed on the following cytogenetic char- as an undescribed species from Rumex cris- acters: karyotype, chromosomal behavior pus L., are morphologically closely related during gametogenesis, and mode of repro- to H. schachtii and H. glycines and constitute duction. Certain morphological and phys- a series of chromosomal forms, with somatic iological characters will be considered in numbers ranging from 24 to 34, and re- some cases. produce by mitotic parthenogenesis (4). According to the latest taxonomic treat- There is tittle doubt that these "species" have ment, the family Heteroderidae comprises been derived from H. schachtii or H. glycines five genera, Heterodera A. Schmidt, Meloi- through various chromosomal changes lead- dogyne Goeldi, Meloidodera Chitwood, Han- ing to polyploidy and aneuploidy in associa- non & Esser, Cryphodera Colbran, and tion with the establishment of parthenogenetic Meloidoderita Poghossian. A sixth genus, reproduction (Table 1). It is very likely Hypsoperine Sledge & Golden, was synony- that all of them have evolved along the same mized with Meloidogyne in 1968 (15), phyletic line, or a number of parallel phyletic but a new species described early in 1969 lines derived from the same basic species was placed in Hypsoperine as H. ottersoni or group of related species, at different Thorne (8). No cytogenetic work has been occasions and probably at different time done with the monotypic Cryphodera and periods. They constitute a parthenogenetic Meloidoderita, and therefore, these genera species complex completely separated re- will not be included in the discussion. productively and, therefore, genetically from Among the species of Heterodera studied the amphimictic species from which they thus far cytologically, 13 are diploid amphi- have been derived. mictic with a haploid number of 9 chromo- From a taxonomic viewpoint, it is an open somes. These include H. schachtii A. question whether members of such a par- Schmidt, H. glycines Ichinohe, H. oryzae thenogenetic species complex should be Luc & Brizuela, H. avenae Wollenweber, H. treated as separate species (as has been done goettingiana Liebscher, H. cruciferae Frank- in the past in nematode taxonomy) without tin, H. carotae Jones, H. weissi Steiner, H. reference to their phylogenetic relationships, rostochiensis Wollenweber, H. tabacum should be regarded as subspecies (14) or as Lownsbery & Lownsbery, H. virginiae properly designated, but unnamed, infra- 28 Journal o[ Nematology, Vol. 2, No. 1, January 1970

specific categories of the same parthenoge- H. betulae indicates that the karyotype of netic species. Nematode taxonomists need to this species has not yet stabilized. Further- find a practical solution to this problem, and more, the occasional appearance of males, at the same time to decide which of the and the meiotic type of maturation of the theoretically numerous morphological and oocytes suggest that reproduction by cross- physiological variants within a parthenoge- fertilization may occur occasionally in this netic group should be recognized as separate species. Consequently, the instability of the taxonomic entities. karyotype, and the facultative type of par- A similar case of parthenogenetic evolu- thenogenesis, would tend to indicate that H. tion in the genus Heterodera involves H. betulae has been derived recently and is still oryzae and H. sacchari Luc & Merny of the in a state of active evolution. This is in H. schachtii species group. These two species contrast to the evolutionary status of the are very closely related morphologically and H. trifolii species complex, in which the have the same geographical distribution. H. complete absence of males, and the obliga- oryzae is a diploid amphimictic species with tory type of mitotic parthenogenesis indicate n = 9 chromosomes, whereas, H. sacchari an advanced state of evolution (although has 2n = 27 and reproduces by mitotic par- degenerative evolution), with no spectacular thenogenesis (6). It is very likely that H. changes being expected in the future. sacchari has evolved from H. oryzae, or These, briefly, are the three separate lines another amphimictic relative as a triploid of parthenogenetic evolution observed thus parthenogenetic form (Table 1 ). H. leuce- far in the genus Heterodera. ilyma Di Edwardo & Perry, is closely related In the genus Meloidodera, the only species morphologically to H. sacchari, has no males studied cytogenetically, M. floridensis Chit- and undoubtedly reproduces by partheno- wood, Hannon & Esser, reproduces by genesis. Although no cytological work has mitotic parthenogenesis and has a somatic been done with H. leuceilyma, it very likely chromosome number of 26 and 27 (A. C. has evolved along the same parthenogenetic Triantaphyllou, unpublished data). Its chro- line with H. sacchari and, consequently, be- mosomes resemble those of mitotic partheno- longs to the same parthenogenetic species genetic species of Heterodera, like H. trifolii, complex. although they are smaller in size. M. [lori- A third but different case of parthenoge- dens& can be regarded as a triploid par- netic evolution in the genus Heterodera in- thenogenetic organism, probably derived cludes H. betulae Hirschmann & Riggs (3) from an amphimictic diploid ancestor with which reproduces by meiotic parthenogenesis n = 9 chromosomes (Table 1). Further and has a haploid number of 12 and 13 cytogenetic work may reveal the existence chromosomes (A. C. Triantaphyllou, un- of some diploid amphimictic species of Me- published data). H. betulae must have loidodera with n = 9 chromosomes, and thus evolved from a diploid (n = 9) amphimictic confirm the previous assumption. Meloido- relative, probably of the H. cacti Filipjev dera charis Hopper, appears to be closely & Schuurmans Stekhoven species group, related morphologically and physiologically through a gradual increase of the basic to M. floridensis, and also is suspected of chromosome number from 9 to 12 and 13, being very similar cytogenetically. Appar- and the establishment of parthenogenetic ently it belongs to the same parthenogenetic reproduction (Table 1 ). The existence of species complex as M. floridensis. individuals with 12 and 13 chromosomes in Turning our attention to the genus Meloi- SYMPOSIUM: HETERODERIDAE; CYTOGENETIC EVOLUTION • Triantaphyllou 29

TABLE 1. Partial phylogenetic scheme, showing M. hapla Chitwood stands at the same three separate lines of parthenogenetic evolution level of evolution as M. graminicola and M. in the genus Heterodera and one in the genus Meloidodera. naasi from the standpoint of mode of repro- duction, but it has gone a few steps further schacht if Diploid [11=tl ) with regard to the evolution of its karyotype. [_~ t~l~', invs Ampl mict it" The chromosomal complement of M. hapla tvifolil ~/] : S Po ] yploid (3- L+n=24- 34) has been reduced from 18 to 17, 16 and 15 l~,spedvza~ 5 totlc lart i~.l]Og~ll..Lic I Sl,. t'~om Aurlex ( 11 ). Moreover, some populations of M. ~t ispus hapla and all populations of M. arenaria ,,rvza, I Dipl,,hl (n=") Amp h it, J, L i c I (Neal) (10) have evolved further with .... + +++ ...... the establishment of various degrees of t-----,I it,t, a::h i,o,,qt tic polyploidy (2n = 45, and 36 or 51-54, re- DipIo;d (n=')) ca~t i .p. ~t ,,,q, A'np 11~,1 ¢ t 1 spectively) in association with mitotic par-

L An upi~,id (n=l" ; 1 ~) thenogenesis. Probably these forms have ht I, lac A/q~hiu3hi*ti~, Pa/'[ht,tu,,tt1~. u:l~ ic been derived secondarily from populations of

}Iu I c i dodL ra M. hapla through occasional fertilization of gametes of different chromosomal comple- L[ iI-[plo~J (Jll~2I,. 27) -t "tld~tll~i~ Z.~ t,q [ 'attlt'u,,~;,'n~tic ment. Thus with 45 chromosomes ~hari,.' M. hapIa may have been derived through fertilization of an unreduced egg of M. hapla with 30 dogyne, we see that almost the entire genus chromosomes by a reduced sperm containing has evolved along a parthenogenetic mode the haploid set of 15 chromosomes. of reproduction. The only species that ap- M. incognita (Kofoid & White) and M. pears to reproduce exclusively by amphimixis javanica (Treub) reproduce by mitotic par- is a new species that will be described as thenogenesis and are polyploids (2n -- 41--44 M. carolinensis, and which probably has 18 and 43-48, respectively) (A. C. Triantaphyl- chromosomes (2) (Table 2). M. carolinen- lou, unpublished data) and (9). Such forms sis' can be considered cytogenetically as an are equivalent to the mitotic parthenogenetic ancestral species, representative of the central populations of M. hapla and M. arenaria but, line of evolution of the genus. Its limited with our present knowledge, we cannot trace host range (it infects only blueberry and their phylogenetic derivation. Further search azalea) however, indicates a highly evolved parasite, at least with respect to host special- ization. TABLE 2. Species relationships in the genus Meloi- dogyne based on their karyotype and mode of The next most advanced species, from the reproduction. standpoint of evolution of reproduction, are M. graminicola Golden & Birchfield, and M. naasi Franklin, which normally repro- i ,arul h,c :,gi ~ n~l,s(?) 2,,,phh*'i :'ti c duce by meiotic parthenogenesis but under ~rar,inic~ la :.L~oti. ] ,h, uogenet~c ,Hc:'s,,ul certain circumstances can also reproduce by i amphimixis (12). They are closely related - ' ' ."l',phimlc r ic morphologically to M. carolinensis and have the same number (n = 18) of chromosomes, 2n=L~'~ 5 N [I ~ ju. ]~al-t[:~,ll ,gtqlot 1¢ which can be regarded as the basic number 3h" I~,J d.,~x ne {?) ~par t [nae n~7 At,,phlrG e t i c for the genus Meloidogyne. 30 Journal of Nematology, Vol. 2, No. 1, January 1970

will probably associate them with an am- Heterodera and Meloidogyne. Although con- phimictic or, at least, meiotic parthenoge- sidered to be taxonomically closely related, netic species of Meloidogyne. these genera are still quite far apart both Three species of the former genus Hypso- morphologically and cytogenetically (13). perine, recently synonymized with Meloi- The genus Heterodera has a basic chromo- dogyne, have been studied cytogenetically. some number of 9 and predominantly am- Six populations of M. graminis (Sledge & phimictic mode of reproduction. It may Golden) of different origin have n = 18 represent the main line of evolution from a chromosomes and reproduce regularly by hypothetical diploid (x = 9) amphimictic meiotic parthenogenesis and occasionally by ancestor, and probably the only line of amphimixis (A. C. Triantaphyllou, unpub- progressive evolution in the family Heter- lished data). Thus, M. graminis is cytogenet- oderidae. The genus Meloidogyne, on the ically similar with M. graminicola and M. other hand, has a basic chromosome number naasi. Its recent transfer to the genus Meloi- of 18, and has followed a predominantly dogyne (15) on the basis of morphological parthenogenetic line of evolution, which by criteria is, therefore, supported by the avail- its nature is a regressive or degenerative type able cytogenetic information. The recently of evolution. Several hypotheses have been described species Hypsoperine ottersoni is expressed with regard to the relationship of indistinguishable cytogenetically from M. the karyotypes of these two genera, but none graminis (A. C. Triantaphyllou, unpublished has been satisfactory. In a recent analysis data) and may also be transferred to the it was proposed that, until more evidence genus Meloidogyne as Meloidogyne otter- becomes available regarding the course of soni, in the same species group with M. karyotypic evolution in each genus, the graminicola and M. naasi (Table 2). MeIoi- karyotypes of these two genera should be dogyne spartinae (Rau & Fassuliotis), on considered separately, without reference to the other hand, has a haploid number of 7 their relationship (12). chromosomes---~e smallest number observed Recent DNA measurements in hypo- in the family Heteroderidae--and reproduces dermal nuclei of second-stage larvae of by amphimixis (A. C. Triantaphyllou, un- various members of these genera confirmed published data) and (1). It is difficult to the hypothesis that polyploidy exists within speculate on its relationship with other each genus, but failed to clarify the relation- Meloidogyne species. It can be assumed ship of the karyotypes of the two genera (5). that, as an amphimictic species, with a The difficulty of establishing a definite chromosome number much different from relationship between these two karyotypes the basic number of the genus Meloidogyne, may actually indicate the lack of a close M. spartinae is a basic species, and has not relationship between them. Therefore, if we evolved from any of the Meloidogyne species postulate a common ancestor for the genera studied cytogenetically thus far. Its cyto- Meloidogyne and Heterodera, branching of genetic characteristics together with existing the evolutionary lines that gave rise to these substantial morphological and physiological genera must have occurred in the very distant differences would probably justify the assign- evolutionary past, so that characterization of ment of M. spartinae to a separate genus. such a common ancestor now, on a cytologi- Now let us discuss the possible relation- cal basis, is almost impossible. ships among the genera of the family Heter- With regard to the genus Meloidodera, the oderidae, and first the two main genera, type species M. floridensis, cannot be re- SYMPOSIUM-" HETEROOERIDAE; CYTOGENETIC EVOLUTION • Triantaphyllou 31

garded as a direct phylogenetic link between and sporadically with other plant-parasitic the genera Meloidogyne and Heterodera, as nematodes. It all supports the general con- has been suggested in the past on the basis clusion that evolution of plant-parasitic of morphological characteristics, because it is nematodes has been influenced by extensive a triploid parthenogenetic organism. As modifications of their karyotype and the such, and because its karyotype is similar establishment of various types of partheno- to that of Heterodera, M. floridensis can be genetic reproduction. Consequently, cyto- regarded as the end product of an evolu- genetic analysis of various groups will tionary line that originated quite early in the undoubtedly yield valuable information for phylogenetic scheme from a diploid (n -- 9) elucidating phylogenetic relationships and amphimictic organism, which was the hypo- clarifying taxonomic problems. thetical common ancestor of the genera Meloidodera and Heterodera. M. floridensb LITERATURE CITED must have evolved slowly compared to the 1. FASSULIOTIS, G., and G. J. RAu. 1966. genus Heterodera due to its parthenogenetic Observations on the embryogeny and his- mode of reproduction, which reduced the topathology of Hypsoperine spartinae on smooth cordgrass roots, Spartina alterni- rate of genetic evolution. For this reason it flora. Nematologica 12:90. (Abstr.) has maintained some of the primitive char- 2. Fox, J.A. 1967. Biological studies of the acteristics of the common ancestor, such as blueberry root-knot nematode (Meloido- gyne carolinensis n. sp.). Diss. Abstr. 28: the subequatorial position of the vulva, the 1311-1312. presence of well-developed transverse cutic- 3. HIRSCHMANN, HEDWIG, and R. D. RaGGS. ular annulations in the female, and the 1969. Heterodera betulae n. sp. (Heter- oderidae), a cyst-forming nematode from absence of a leathery brown, cyst stage. river birch. J. Nematol. 1:169-179. This very briefly is an analysis of the 4. HIRSCHMANN, HEOWIG, and A. C. TRIAN- phylogeny of the family Heteroderidae from TAPHYLLOU. 1965. Comparative cytolog- a cytogenetic viewpoint. The most striking ical and morphometric studies in some parthenogenetic Heterodera species. Phy- characteristic is the active evolution of the topathology 55:1061. (Abstr.) karyotype, and the establishment of poly- 5. LAPP, N. A., and A. C. TRIANTAPHYLLOU. ploidy and aneuploidy in association with 1969. Relative DNA content of some members of Heteroderidae. J. Nematol. parthenogenetic reproduction. Expansion of 1:296. (Abstr.) the cytogenetic work to include other mem- 6. NETSCHER, C. 1969. L'ovogrn~se et la re- bers, particularly the genera Cryphodera and production chez et H. sacchari (Nematoda: Heteroderidae). Ne- Meloidoderita will be very useful. matologica 15:10-14. The contribution of cytogenetics will be 7. ROMAN, J., and A. C. TRIANTAPHYLLOU. significant for many other families of plant- 1969. Gametogenesis and reproduction of seven species of Pratylenchus. J. Nema- parasitic nematodes with similar cytogenetic tol. 1:357-362. characteristics as the family Heteroderidae. 8. THORNE, G. 1969. Hypsoperine ottersoni Recent cytogenetic work with various mem- sp. n. (Nemata, Heteroderidae) infesting canary grass, Phalaris arundinacea (L.) bers of the genus Pratylenchus (7) has shown Reed in Wisconsin. Proc. Helminthol. Soc. that evolution of this genus has been associ- Wash. 36:98-102. ated with changes of the basic chromosome 9. TRIANTAPHYLLOU, A. C. 1962. Oogenesis in the root-knot nematode Meloidogyne number and the establishment of polyploidy ]avanica. Nematologica 7:105-113. and parthenogenesis. Some cytogenetic work 10. TmANTAPHYLLOO, A. C. 1963. Polyploidy has also been done with members of the and parthenogenesis in the root-knot nema- tode . J. Morphol. subfamilies Hoplolaminae and Tylenchinae 113:489-499.

II 32 Journal o[ Nematology, Vol. 2, No. 1, January 1970

11. TRIANTAPHYLLOU, A. C. 1966. Polyploidy matol. Univ. P.R., Rio Piedras, Puerto and reproductive patterns in the root-knot Rico. p. 11-17. nematode Meloidogyne hapla. J. Morphol. 14. TRIANTAPHYLLOU, A. C., and HEDWIG HIRSCH- 118:403-413. MANN. 1964. Reproduction in plant and 12. TRIANTAPHYLLOU, A. C. 1969. Gameto- soil nematodes. Ann. Rev. Phytopathol. genesis and the chromosomes of two root- knot nematodes, Meloidogyne graminicola 2:57-80. and M. naasi. J. Nematol. 1:62-71. 15. WHITEHEAD, A. G. 1968. Taxonomy of 13. TRIANTAPHYLLOU, A.C. 1969. Cytogenetic Meloidogyne (Nematodea: Heteroderidae) evaluation of the phylogeny in the family with descriptions of four new species. Heteroderidae. Proc. Symp. Tropic. Ne- Trans. Zool. Soc. London 31:263-401.