CYTOTAXONOMIC OBSERVATIONS IN THE GENUS AESCHYNANTHUS ( GESNERIACEAE )

This study is a contribution to the further understanding of cytological patterns in Aeschynanthus ( Gesneriacaeae ). Chromosome numbers are reported for 12 species from six sections; nine of these are new counts. Two basic numbers, x = 16 and x = 15, are generally encountered. Aeschynanthus gracilis proved to be of exceptional interest, as its rare somatic number, 2n = 28, conﬁrms the occurrence of a third basic number, x = 14, in the genus. Variation in chromosome number in relation to seed morphology is examined.


I 
Aeschynanthus Jack (Gesneriaceae, subfamily Cyrtandroideae, tribe Trichosporeae) is a genus of some 150 species of perennial, usually epiphytic, subshrubs distributed from India and China and throughout SE Asia to the Solomon Islands.The tribe Trichosporeae (consisting of Aeschynanthus, Agalmyla (including Dichrotrichium), Loxostigma, Lysionotus, and the doubtful monotypic genus Micraeschynanthus) is distinguished by the possession of appendages, often hair-like, at each end of the seed.The apical appendage is always single, but in Aeschynanthus the number and form of the hilar appendages are taxonomically important.Bentham (1876) first proposed a sectional classification of Aeschynanthus based almost entirely on seed appendages, and recognized four sections: Polytrichium, Diplotrichium, Haplotrichium, and Holocalyx (now sect.Aeschynanthus).Clarke (1883) added sect.Microtrichium.Schlechter (1923) added sect.Anisocalyx but this was subsumed under Microtrichium by Burtt & Woods (1975), while Wang (1984) created sect.Xanthanthos, based not on seed but on corolla characters, to accommodate a single Chinese species.Recent studies particularly of seed and appendage morphology (Mendum et al., 2001) identified two major groups within the genus, with each group subdividing into sections.Species with Type A seeds encompass sects Microtrichium, Aeschynanthus and Haplotrichium sens.str.; species with Type B seeds comprise sects Polytrichium, Diplotrichium, Xanthanthos, and a section consisting of many of the species previously placed in sect.Haplotrichium.The last group cannot be adequately circumscribed until further studies, particularly on sect.Xanthanthos, are complete so is provisionally referred to as sect.X.This classification has been adopted for this study.1 The first cytological studies on Aeschynanthus were made by Rogers (1954); since then 25 species, two unnamed taxa and two synthetic hybrids have been counted.The basic numbers for the genus appear to be x=16 and x=15 (Ratter, 1975), which as Kiehn & Weber (1997) point out do not neatly correlate with sectional circumscription.The unusual number of 2n=28 occurs in A. longicaulis (Eberle, 1956) as a variant within a species which also shows 2n=30 (Rogers, 1954;Ratter & Prentice, 1964).This has been the only deviation from the pattern of x=16 and 15.Polyploidy has been recorded from all examined sections except Diplotrichium and Haplotrichium sens.str.In the light of increased taxonomic knowledge, the present investigation has been undertaken to seek further elucidation of cytological relationships between sections.

M      M 
The Royal Botanic Garden Edinburgh has an extensive living collection of Aeschynanthus.From this, three species from sect.X, two each from sects Aeschynanthus and Diplotrichium, and one each from sects Haplotrichium sens.str., Polytrichium and Microtrichium were selected.In addition, two species of sect.Microtrichium previously regarded as anomalous, A. magnificus Stapf and A. vinaceus P. Woods, were chosen to provide chromosomal results which might assist in further clarifying their status.The species are listed in the Appendix.Material of species of sect.Xanthanthos was not available.Root tips, cotyledons and ovules were used as sources of meristematic tissue.
Root tips were harvested from cuttings grown in perlite in a propagator unit with bottom heat; usable roots were produced in 3-4 weeks.For ovules, ovaries from very young flower buds were cut in half longitudinally before pretreatment and subsequent processing.Seedlings of most Old World gesneriads develop unequal cotyledons after germination, and the basal meristem of the larger one is a good source of dividing cells.Seed was germinated on filter paper in a growth chamber; 10-14-day-old seedlings were pretreated and fixed whole, and enlarging cotyledons squashed after staining (after Jong & Mo ¨ller, 2000).
Two pretreatment chemicals were normally used concurrently, a-bromonaphthalene for 2-3h at room temperature (20°C ) and 0.002M 8-hydroxyquinoline for 4-6h at 13°C, the latter consistently yielding better chromosome spreads than the former.The material was then fixed in fresh Farmer's Fluid (3:1, ethanol:glacial 1 Aeschynanthus classification according to Mendum et al. (2001).Type A seed: testa cell orientation almost always spiral, papillae formed from single cells, appendages short, not papillose.Subtypes equivalent to sects Haplotrichium sens.str., Microtrichium and Aeschynanthus.Type B seed: testa cell orientation straight, papillae formed from raised ends of two adjacent cells, appendages long, slender, always papillose.Subtypes equivalent to sects.Polytrichium, Diplotrichium, Xanthanthos, and those species with Type B seed that were previously assigned to sect.Haplotrichium, now placed in a provisional sect.X. acetic acid ) and stored until required.Cotyledons and ovules were stained with lactopropionic orcein (after Dyer, 1963) after softening in 5M HCl for 15min.The preferred protocol for root tips was hydrolysis in 5M HCl for 30-50min, washing in several changes of water, and staining with Feulgen Reagent (after Fox, 1969).Additional softening in a 1:1 enzyme mixture of 4% cellulase and 4% pectinase for 20-40min at 35°C greatly improved squashing.Mounting in 0.4% aceto-carmine after Feulgen staining, and viewing under phase-contrast optics, greatly increased visibility of the chromosomes.
All the photomicrographs were taken on 35mm Kodak Technical Pan film.Permanent slides were prepared according to a modified quick-freeze method (after Conger & Fairchild, 1953, in Jong, 1997).
Voucher herbarium specimens and permanent slides are lodged at the Royal Botanic Garden Edinburgh.

R   D 
All counts from this study are listed in Table 1.Most counts were based on root tips, a few on ovules, and some confirmatory ones on cotyledons.Apart from those for A. boschianus (sect.Aeschynanthus) with 2n=64, and A. lineatus (sect.Diplotrichium) and A. longicaulis (sect.Polytrichium), both 2n=30, agreeing with already published data, all are first reports.Previously published chromosome numbers in Aeschynanthus are summarized in Table 2.
The results of the present study, together with previously published counts ( Table 2) confirm the presence of two prevalent basic numbers, x=16 and x=15, in Aeschynanthus, with 2n=32 the dominant sporophytic number (Fig. 1), and the most frequent in sects Diplotrichium and Microtrichium ( Table 3).Gametophytic numbers of n=14, 15, 16 and 32, and sporophytic numbers of 2n=28, 30, 32, 60, 64 and 96, have all been encountered.Of the hybrids examined, one gave 2n=32 and one 2n=48, as expected from the numbers of the parental species.It is worth pointing out that n=11 (for A. parasiticus), attributed to Malla et al. (1978), is most probably a typographical error (in Goldblatt, 1981) and should be ignored.
The chromosome counts listed in Table 3 show that although the sections cannot be separated on basic number, certain cytological patterns of variation occur correlated with the seed group classification proposed by Mendum et al. (2001).TA BL E 3. Summary of all Aeschynanthus chromosome counts to date, relative to sectional classification (all given as 2n for ease of comparison)

A
This study was started as a thesis by the first author for completion of an MSc degree in the Biodiversity and Taxonomy of Plants (run jointly by the University of Edinburgh and Royal Botanic Garden Edinburgh).Completion of the work was supported by financial assistance from DfID and is gratefully acknowledged.The investigation was made possible by RBGE's extensive living collection and thanks are extended to Mr Steve Scott, who maintains it with dedication and interest.We wish also to thank RBGE Trustees Reserve Fund for funding Jo Mendum, who provided counts for all the accessions of A. gracilis cited in this study.We are grateful to Dr J. Ratter for discussion and critical comment.
Type A seed group, basic number x=16, and polyploidy Species with Type A seed (sects Microtrichium, Aeschynanthus, and Haplotrichium sens.str.) are based on x=16, although two species show intraspecific dysploidy (counts of 2n=60 and 64 in A. pulcher, and 2n=30 and 32 in A. radicans).Furthermore, polyploidy appears to be relatively common in sect.Aeschynanthus, occurring in four out of the 10 species counted, and is also recorded in sects Microtrichium, X and Polytrichium.Most of the polyploids are tetraploids (2n=64, 60) but one stock of A. ellipticus (sect.Microtrichium) is hexaploid (2n=96, Ratter & Prentice, 1964).This species, together with A. parvifolius and A. pulcher, are the only examples known so far with intraspecific polyploid series.Aeschynanthus boschianus (sect.Aeschynanthus) is the only polyploid species encountered in the present study.Although cytological data are at present only available for an inadequate representation of species, it does seem that polyploidy has F IG . 1 .Histogram showing the distribution of sporophytic chromosome numbers in all counted named species of Aeschynanthus.(Different cytotypes occurring in the same species have been scored separately.)
series within a single species.In A. longiflorus, polysomatic variations of 2n=21 and 28 have been encountered in addition to the normal 2n=30(Ratter &  Prentice, 1964).Dysploid change through chromosome loss is probably responsible for the relationship between x=16 and x=15(Ratter, 1975), but see below for further discussion.Polyploids are scarce, recorded so far only inA.myrmecophilus  and A. speciosus, both 2n=64.Milne, 1975); the only other report of such a number was as a somatic variation within roots of A. longiflorus, where 2n=30 is the prevalent number(Ratter & Prentice, 1964).Understandably, there has been some hesitancy in accepting the presence of a third basic number, x=14, in the genus( Kiehn & Weber,  1997).However, it has been confirmed in the present study in A. gracilis, a species with a wide geographical range, recorded from China ( Yunnan), Thailand, Myanmar, N Vietnam, N India, and Bhutan.This species has a trailing, flexuous habit and thick leaves, uncommon features in sect.X.Its unusual somatic number of 2n=28 was encountered in five different accessions ( Table1; Appendix).Aeschynanthus chromosomes are small, (0.6-1.5mm; Kiehn & Weber, 1997) and metaphase spreads with clear morphology rather hard to find; it is nevertheless notable that the karyotype of A. gracilis has a large number of acrocentric chromosomes (Fig.2g,h), compared with other species illustrated in Fig.2where metacentrics and submetacentrics predominate.Our study thus clearly demonstrates the existence in Aeschynanthus of a third basic number of x=14, with possibly a distinctive karyotype.Aeschynanthus magnificus and A. vinaceusThe taxonomic assignment of these two species was at one time uncertain, and they were provisionally placed in sect.Microtrichium.Mendum et al. (2001)agree with this placement, and in terms of chromosome number, they both have the same somatic number 2n=32, the predominant number in the Type A seed group.Ratter(1975)deduced that of the two prevalent basic numbers in Aeschynanthus, x=16 is ancestral and x=15 derived through dysploid reduction.Arguments in favour of this view have also been forwarded byKiehn & Weber (1997), namely that x=16 occurs throughout sect.Microtrichium, while Agalmyla and Lysionotus, also in the tribe Trichosporeae, have x=16(Fussell, 1958; Ratter, 1975; Kiehn &  Weber, 1997).On the basis of appendage morphology(Mendum et al., 2001)and molecular data(Denduangboripant & Cronk, 2000), sect.Microtrichium is regarded as basal to the genus.There are dysploid series of 2n=30 and 32 in both Type A seed and Type B seed groups, occurring both inter-and intraspecifically, and this is now extended to 2n=28 in sect.X ( Type B seed).Such variation may have been the result of dysploid change in chromosome number which has become established in sects X, Diplotrichium, and Polytrichium, and perhaps sect.Aeschynanthus, presumably representing many independent dysploid evolutionary lines(Ratter, 1975), and taken even further to x=14 in A. gracilis of sect.X.Chromosome reorganization leading to numerical change appears to have taken two different directions within the genus Aeschynanthus.Polyploidy seems to have played an important role in diversification, especially in the Type A seed group of species, and particularly in sect.Aeschynanthus.Numerical change is achieved mainly through dysploidy, with only limited and sporadic occurrence of polyploidy, in the Type B seed group.On the basis of seed morphology and molecular data, the Type B species appear to be more derived.Counts obtained during this study, together with previous records, suggest the derivation of 2n=28 and 2n=30 through dysploid reduction from an ancestral 2n=32, a number that predominates in species with Type A seed.Available cytological data for the genus are still limited, and further studies, including karyotype analysis, should bring greater cytotaxonomic understanding.
played an important part in species diversification in sect.Aeschynanthus ( Kiehn & Weber, 1997), but not in others where polyploidy is only of sporadic occurrence.dysploid