Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 76(4): 51-57, 2023 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2222 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Irudayaraj, V., Benniamin, A., & Arokia Raj, S. (2023). Apogamous Isoetes coromandelina L.f. (Isoeta- ceae) with asynaptic meiosis. Caryo- logia 76(4): 51-57. doi: 10.36253/caryo- logia-2222 Received: July 02, 2023 Accepted: January 24, 2024 Published: March 14, 2024 Copyright: © 2023 Irudayaraj, V., Ben- niamin, A., & Arokia Raj, S. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Apogamous Isoetes coromandelina L.f. (Isoetaceae) with asynaptic meiosis V. Irudayaraj1,2, A. Benniamin3,*, S. Arokia Raj1 1 Department of Botany, St. Xavier’s College (Autonomous), Palayamkottai, Tamil Nadu – 627002 2 Present Address: 23/116, Darlingnagar, Maharajanagar Post, Tamil Nadu, India-627 011 3 Botanical Survey of India, Western Regional Centre, Pune, India- 411 001 *Corresponding author. E-mail: fernsbenni@gmail.com Abstract. This cytological study on Isoetes coromandelina L.f (Isoetaceae) from Tirunelveli District, Tamil Nadu, South India shows the presence of twenty two indi- vidual chromosomes along with a fragment (n=2n=22+1) during meiosis in megaspore mother cells without the presence of any bivalent. Cytomixis between two megaspores is very common, resulting in the formation of a high percentage of abortive spores. The present material is of an apogamous taxon with asynaptic Meiosis as reported by Abraham and Ninan from Kerala and Karnataka. Keywords: Isoetes coromandelina, asynaptic meiosis, apogamy. INTRODUCTION Lycophytes and Pteridophytes form the dominant vegetation on the earth next to flowering plants. Fern allies and ferns are a monophyletic group and the closest living relatives to seed plants (Pryer et al., 2001). This group of plants, with homosporous clubmosses, heterosporous spikemosses and quillworts, is the ancestor for Lycopodiopsida and Monilophytes Smith et al. (2006). Quillworts are considered by some to be the last remnant of the fos- sil  tree lycopsid with which they share some unusual features including the development of wood and bark, a modified shoot system acting as roots, bipolar growth, and an upright stance (Retallack, 1997). Isoetes, one of the extant members of Lycopodiopsida, is a heterosporous fern. They are small aquatic herbs and they are commonly called quillworts or Merlin grass. Many aquatic plant populations including quillworts are under severe threat in the Western Ghats, particularly in Kerala and Maharashtra. For exam- ple, the endangered Isoetes panchganiensis has been reported from tempo- rary ponds and pools on the high altitude plateaus of Panchgani table lands in Maharashtra and Kemmangundi Hills in Karnataka (Molur et al., 2011). Some aquatic species, often found in mixed populations containing taxa of different ploidy, appear to have evolved abruptly via interspecific hybridiza- http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-2222 https://doi.org/10.36253/caryologia-2222 https://doi.org/10.36253/caryologia-2222 http://www.fupress.com/caryologia http://www.fupress.com/caryologia mailto:fernsbenni@gmail.com 52 V. Irudayaraj, A. Benniamin, S. Arokia Raj tion and chromosome doubling. Evidence from distri- bution patterns, megaspore morphology and viability, chromosome numbers, and electrophoretic profiles of leaf enzymes supports a hypothesis of allopolyploid spe- ciation (Taylor & Hickey, 1992). Isoetes is not only a very complicated genus but also a remarkable genus with lot of things yet to be stud- ied and understood. From India, large number of spe- cies under Isoetes has been reported as new species. After Fraser-Jenkins et al. (2017) it is clear that there are only four different species (I. coromandelina L.f., I. dixitii Shende, I. sahyadrii Magab. ex L. N. Rao, I. udipiensis P. K. Shukla, G. K. Srivast., S. K. Shukla and P. K. Rajago- pal) of Isoetes in India. As the species of Isoetes are mostly aquatic and Tamil Nadu can have low rainfall for several years with rare occurrence of normal rainfall, the occur- rence of Isoetes species is rare and seasonal. But once there is enough rainfall, they flourish immediately and cover large area in several acres as mono-dominant spe- cies which is the characteristic features of several related fossil lycophytes like Pleuromeia rossica, which grew in wet shore side habitats and formed mono-dominant com- munities. Such ecological type of a mono-dominant plant community is quite typical of such hygrophilous vegeta- tion in both the geological past and the present-day world, but it is inhabited by taxonomically different plants. The occurrence of mono-dominat Isoetes coromandelina L. f. has been reported from Kallidaikurichy, Tirunelveli District by Manickam and Irudayaraj (1992) and from a lake near Bharathidasan University, Tiruchirapalli, Tamil Nadu on 1999. They have mentioned that although the species is common throughout India, it is a rare species in Tamil Nadu. With the presence of rainfall above the aver- age amount during the year 2014, this aquatic weed start- ed to grow in and around several ponds and water pits in several localities, including the same locality Kallidaiku- richy of Manickam and Irudayaraj (1992). Thanks to the availability of this seasonal aquatic weed, we aimed to make detailed cytological studies on the quillwort Isoetes coromonandelina L. f. MATERIALS AND METHODS Materials for the present study were collected from Kallidaikurichy, Tirunelveli District, Tamil Nadu, India during 2014 (Figure 1 A,B). Sporangia (Figure 1 C, D) were fixed in the mixture of Absolute alcohol, Chloro- form and Acetic acid (6:3:1). Acetocarmine squash tech- nique was followed to study the chromosomes behaviour in megaspore mother cells. Voucher has been deposited in St. Xavier’s College Herbarium, Palayamkottai. RESULTS AND DISCUSSION Meiosis was observed in megaspore mother cells. Thin, network chromosomes, without the identity of individual chromsosme were observed in the leptotene stage and long, thick, rod like overlapping chromosomes were observed in zygotene stage (Figure 2, A-C). Pachy- tene and diplotene stages were not observed. After zygo- tene stage, each and every chromosome remains as such without pairing of homologous chromosomes. Instead of bivalents formation, they all remain as univalents. Thus all the chromosomes were like that of mitotic ones. There are 22 stout and thick chromosomes of more or less uniform size along with a very short chromosome. The chromosomes are of variable in length and type (Figure 2, D-E). The meiosis is more or less regular from I metaphase to II telophase except the irregular behav- ior of the extra chromosome (22 + 1) which either forms Chromosome Bridge or lies little away from others dur- ing first anaphase and telophase (Figure 2 F-I). There is no indication for the presence of this extra chromosome, in the form of micronucleus, in tetrads or young spores (Figure 2 L-Q). The mature megaspores are in different size and the surface of the megaspores is with prominent tubercles (Figure 1, E-H). The careful observation on number of chromosomes in each group of Anaphase I, clearly shows the unreduc- tion of chromosome number during first meiosis (Figure 2, F, G, H). From the present study on meiosis in mega- spore mother cells of Isoetes coromandelina L.f from Kallidaikurichy, Tirunelveli District, Tamil Nadu, it is concluded that the chromosome number is n=22 +1. This is the first count for I. coromandelina L. f from Tamil Nadu and it is in agreement with Abraham and Ninan (1958), who have observed the presence of complete asynapsis in this species in populations from Kerala and Karnataka. They have concluded that this species from South India is of asynaptic apogamous form. Cytological reports on Indian Quillworts are availa- ble. Plants of I. panchananii, I. indica and Varanasi plants of I. coromandelina are with forty-four chromosomes and a fragment at mitosis in root tip smears but the plants of I. coromandelina growing in Lohgarha and Konark are with only thirty-three chromosomes and a fragment (Pant & Srivastava, 1965). As a rule, the first meiotic divi- sion shows almost complete asynapsis, most of the chro- mosomes being univalents although a few are bivalents and multivalents. The association of bivalent and multi- valent chromosomes takes place in various ways. About 50% of the spores are nucleate and the remaining 50% are enucleate, which again suggests that a second nuclear division is normally absent in the spore mother cells of 53Apogamous Isoetes coromandelina L.f. (Isoetaceae) with asynaptic meiosis Figure 1. A. Large colony of Isoetes coromandelina L.f. in Kallidaidurichi, Tirunelveli District, Tamil Nadu, India, B. Habit, C-D. Sporo- phylls, E-H. Megaspores under light microscope (E, G) and Electron Microscope (F,H). 54 V. Irudayaraj, A. Benniamin, S. Arokia Raj Figure 2. Meiosis in megaspore mother cells of Isoetes coromandelina L. f. A-J Meiosis I. K-N. Diads and tetrads; O,P,Q- Young spores. 55Apogamous Isoetes coromandelina L.f. (Isoetaceae) with asynaptic meiosis Indian species of Isoetes and that their tetrads are formed by cytokinesis of the two dyads. The young larger mega- spores of I. coromandelina, I. indica and I. panchananii usually show one nucleus and one “centrosomic plastid” while the smaller ones in the same species are enucleate with only one “centrosomic plastid”. Karyomorphology on triploid I. coromandelina from South India shows Tri- somy and translocations (Kuriachan and Ninan, 1974a, b). In general, I. coromandelina L.f. in India is a larger species complex with varied rhizome morphology, orna- mentation of megaspore and cytology with the presence of diploid apomictic, triploid apomictic and triploid hybrid, tetraploid apomictic, pentaploid hybrid and hexa- ploid. I. udipiensis P.K.Shukla, G.K.Srivast., S.K.Shukla & P.K.Rajagopal is diploid sexual (Fraser-Jenkins et al. 2017). I. dixitii Shende is with 4x sexual and 3x, 6x sterile hybrids (Bir and Verma 2010). In the present study, considerable percentage of microspores and megaspores are abortive ones with high reduction in size and irregular in shape. Another kind of abnormality in both microspores and megaspores is the presence of cytomixis. Mixing of chromatin mate- rials of two spores through a tubular connection of the spore wall is a common phenomenon in the presently studied quillwort. The tubular connection is of differ- ent size. It is very thin and narrow, or very thick and short. Sometimes two spores are compactly and closely joined together without any tubular connection (Figure 3 A-S). Chromosomal abnormalities including cytomixis occur in meiotic process of pollen mother cells of many angiosperm species at high frequency, but similar phe- nomenon was rarely reported in lower vascular plants. Irregular chromosomal behaviour, especially cytomix- is, is present in Isoetes sinensis Palmer (Heng-Chang et al., 2007). It appeared in each stage of meiotic division from prophase I to telophase II. The cytoplasmic chan- nels between microspore mother cells are narrow and stretched. The genetic material connection is maintained mainly by visible chromatin fiber. Cytomixis occurs between two adjacent microspore mother cells of same stage. A variety of other abnormal chromosomal behav- iors, such as chromosomal stickiness, fragmentation, bridge, laggards, micronuclei are also present but with low frequency. Indeed, the cause of cytomixis formation and the evolutionary significance of it are still source of huge controversy. Bobak and Herich (1978) and Morisset (1978) believed it was just a pathological phenomenon by pharmaceuticals or mechanical pressure. Ghanima and Talaat (2003) stressed the impacts of extreme envi- ronmental factors. Nirmala and Rao (1996) thought cell fusion and chromatin degeneration were probably caused by both environmental and genetic factors. Isoetes species are cytologically much variable with the presence of diploid, triploid, tetraploid and hexa- ploid species/cytotypes based on the base number x=11. Majority of them are sexual with few apogamous taxa. In Isoetes reticulate evolution by complex allopolyploid speciation has well established. So it is necessary to increase the sampling of Isoëtes looking for undescribed, missing species and additional intraspecific variation (Hoot et al., 2004). Apogamy is not common among fern allies. In the present case of the apogamous Isoetes coro- mandelina L.f., it is peculiar in forming diplospores by avoiding normal reduction division during first meiosis in spore mother cells. Usually, the first meiosis is hete- rotypic division by the reduction of chromosome num- ber and the second meiosis is homotypic division with normal mitosis. In the present case, both first and sec- ond meiosis are homotypic division without the reduc- tion of chromosome number resulting in the forma- tion of diplospores. Formation of diplospores with total asynapsis has been reported in several cases and the genetic control of total asynapsis in apogamous taxa has also been explained. The level and mode of diploid megaspore formation has been studied in full-sib diploid potato clones with either normal or desynaptic meiosis (Jongedijk et al., 1991). The present observation of diplo- spory in the apogamous I. coromandelina L.f is similar one of pseudohomotypic division during first meiosis. Apomixis has been repeatedly observed in fern lin- eages that experienced frequent reticulate evolution combined with polyploidization. The apomictic line- ages showed no increase in speciation rate. Instead, all apomictic lineages appeared to be short lived despite some evidence for post origin diversification. In general, apomictic ferns are evolutionary dead ends in the long term but maintain the short-term potential to be highly successful in particular ecological conditions such as cli- mates with strong seasonality (Liu et al., 2012). There are several reports of asynaptic meiosis both in ferns and angiosperms. Manton and Sledge (1954) reported complete absence of chromosome pairing at meiosis in two separate wild collections of filmy fern Hymenophyllum javanicum Sprengel from Ceylon. In another filmy fern Trichomanes insigne v.d.B forma α is with normal meiosis, while forma β is with asynaptic meiosis (Mehra & Singh, 1957). 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