taxonomic importance of seed morphology in gentiana (gentianaceae

Taxonomic importance of seed morphology inGentiana (Gentianaceae)

NINO DAVITASHVILI and GERHARD KARRER*

Institute of Botany, Department of Integrative Biology and Biodiversity Research, University ofNatural Resources and Applied Life Sciences, Gregor-Mendel Str. 33, Vienna 1180, Austria

Received 18 October 2009; accept for publication 10 November 2009

The seeds of Gentiana show high diversity in size, shape and surface pattern. Until now, seeds of only a limitednumber of taxa have been studied in detail and their utility in the subgeneric classification of Gentiana has notbeen evaluated. In this study we investigated seed micromorphology of the poorly known sections Pneumonanthe,Frigidae and Isomeria. In order to evaluate the relevance of seed sculpturing for taxonomy, we selected qualitativecharacters of seed shape and testa ornamentation of all documented sections and performed cluster analyses basedon presence and absence of coded characters. Several new scanning electron microscopy pictures of seeds provideda number of valuable characters for the subgeneric differentiation of Gentiana. The results of the cluster analysesof seed characters generally support the sectional classification given by former authors. However, the EuropeanGentiana asclepiadea does not group together with other taxa of section Pneumonanthe, but clusters near to thestrictly European section Gentiana. Gentiana froelichii, endemic to the south-east Alps and treated traditionallywithin section Frigidae, clusters together with taxa of the European section Ciminalis. Our results reveal that seedmicromorphology supports the reassignment of some taxa incertae sedis within Gentiana. © 2010 The LinneanSociety of London, Botanical Journal of the Linnean Society, 2010, 162, 101–115.boj_1020 101..116

ADDITIONAL KEYWORDS: cluster analysis – scanning electron microscopy (SEM) – seed micromorphology– subgeneric classification – testa ornamentation.

INTRODUCTION

Data on features of seed micromorphology havebeen reported to be useful for taxonomy in numer-ous plant groups (e.g. Juan, Pastor & Fernández,2000; Metzing & Thiede, 2001; Môro et al., 2001;Segarra & Mateu, 2001; Plaza et al., 2004; Adams,Baskin & Baskin, 2005; Song, Yuan & Küpfer,2005). Seeds can provide relatively stable charactersets which are valuable for comparative studies atevery level of the taxonomic hierarchy. Most taxo-nomic studies of seed micromorphology involve twosteps: (1) the description of seed testa charactersand (2) the definition of seed types. In general, suchdescriptive ‘standardized’ work largely lacks thecomparative aspect, especially in the case of large

data sets. Utilization of the statistical approach forestablishing different types of seeds is an exceptionrather than a rule in taxonomic literature.

The genus Gentiana L. (Gentianaceae) comprisesapproximately 360 species distributed mainly in tem-perate, arctic and alpine habitats of the northernhemisphere. Gentiana was reduced several timessince its first description as a result of the segregationof genera such as Gentianella Moench and Metagen-tiana T.N.Ho and S.W.Liu. Current infrageneric clas-sifications of Gentiana sensu Ho & Liu (2001) start atthe subgeneric (Halda, 1996) or sectional (Pringle,1978, Ho & Liu, 2001) levels, using simple charactersof seed morphology and other features such as growthform, flower morphology, chromosome number, etc.The importance of seed morphology for taxonomicstudies of Gentiana was pointed out by Kusnezow(1894), Müller (1982), Miège & Wüest (1984), Yuan(1993), Omer & Qaiser (1995), Halda (1996), Ho & Liu*Corresponding author. E-mail: [email protected]

Botanical Journal of the Linnean Society, 2010, 162, 101–115. With 6 figures

© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 162, 101–115 101

(1990; 2001) and Bouman et al. (2002). However, theutility of seed micromorphology in the sectional clas-sification of Gentiana has never been tested on acomprehensive set of taxa.

Molecular phylogenetic studies in Gentiana (Yuan,Küpfer & Doyle, 1996; Gilley & Taberlet, 1996)revealed that the sectional positions of G. asclepiadeaL. and G. froelichii Jan ex Rchb. need to be updated.Neither taxon grouped with the clade with which ithad been associated prior to those analyses. Theresults of the above-mentioned molecular analyses aresupported by karyological and growth form characters(Löve & Löve, 1972; Serebryakova, 1979; Karrer, 1997)showing important differences for G. asclepiadea andG. froelichii compared with the other taxa within therespective sections in which they were placed by otherauthors (Ho & Liu, 2001). Nevertheless, seed micro-morphology of these taxa has not been re-evaluated.

Seed testa ornamentation of gentians is relativelywell known from light microscopic studies (e.g. Kus-nezow, 1894; Halda, 1996), but scanning electronicmicroscopy (SEM) offers the possibility to detectdetails of seed coat sculpture, which are not visiblewith light microscopy. Because of the small size of theseeds, SEM has been particularly useful in studyingseed coat ornamentation in Gentiana. In this regard,the best-known sections are Calathianae Froel., Gen-tiana, Ciminalis (Adans.) Dumort., Cruciata Gaudin(see Yuan, 1993; Bouman et al., 2002), whereas formost of the other sections detailed seed data are stilllacking.

As may be seen, several aspects of gentian seedmicrosculpture still need to be resolved. In this paper,we aim to: evaluate the utility of seed micromorpho-logical characters for the infrageneric classification ofGentiana by means of cluster analyses; test whetherdata on seed micromorphology can contribute infor-mation relating to the sectional position of G. ascl-epiadea and G. froelichii; and contribute additionalknowledge about seed shape and testa sculpture intaxa from the poorly studied sections Pneumonanthe(Gleditsch.) Gaudin, Frigidae Kusn. and IsomeriaKusn.

MATERIAL AND METHODSSPECIES SELECTION

Several species from the poorly investigated sectionsPneumonanthe, Frigidae, Isomeria and PhyllocalyxT.N.Ho were selected for seed micromorphologicalanalysis. Additional data on seed micromorphology ofthe taxa from the sections Calathianae, Chondrophyl-lae Bunge, Ciminalis, Cruciata Gaudin, DolichocarpaT.N.Ho, Gentiana, Kudoa (Masam.) Satake & Toykuniex Toykuni, and Monopodiae (Harry Sm.) T.N.Ho we

gained from the papers of Müller (1982), Miège &Wüest (1984), Yuan (1993), Omer & Qaiser (1995) andHo & Liu (2001) and from personal observations.Characters that were used by Yuan (1993) for estab-lishing seed types within section Chondrophyllae(depth of testa cell undulation and the presence ofpapillate structures on the anticlinal and periclinalwalls) were not included in our analysis, because wecould not evaluate them for the whole species set.

In total, the seed microsculpture of 83 taxa at thespecies level has been reanalysed. Only clearly visibleand measurable characters were documented. Finally,we selected 42 species that gave reliable data forcluster analyses. They represent 12 out of 15 sectionsaccepted in the monograph of Ho & Liu (2001), whichwas our starting reference for the taxonomical treat-ment of the taxa used. Taxa from sections OtophoraKusn., Microsperma T.N.Ho and Fimbricorona T.N.Howere not included into analysis because of lack of seedmicromorphological data.

Seeds were collected from mature capsules of livingor herbarium specimens. A list of voucher speci-mens is presented in Appendix 1. Dried seeds werearranged on stubs for investigation. The stubs werespatter-coated with gold and examined using aPhilips XL30 ESEM scanning electron microscope.

This paper follows the terminology of the System-atics Association Committee for Descriptive Terminol-ogy (1962), Barthlott & Ehler (1977), Barthlott (1981),Stearn (1983), Anderberg (1994) and Werker (1997)for the description of seed shape, cell shape and testastructure. Species and sectional names correspond tothe classification of Ho & Liu (2001).

CHARACTER SELECTION AND CODING

Our criteria for character selection were the indepen-dency of characters and their stability within the taxaanalysed (Stuessy, 1990). Seeds provide several quali-tative and few quantitative characters. Their shape isovoid or elongate and lanceolate, or rounded. Therounded seeds in particular tend to be flattened. Allabove noted types can have more or less distinctwings or other testa outgrowths. We selected thecharacters according to their logical independence(Wilkinson, 1995). We focused on qualitative charac-ters of seed micromorphology that are easy to detect.Two quantitative characters (characters 7 and 8) weremeasured for bigger samples and their means weregrouped in magnitudes that could be treated statisti-cally like qualitative characters.

All characters were coded in a binary (presence/absence) matrix. Multistate characters were treatedas series of two-stated characters, as such coding isrecommended for quantitative taxonomic studies(Sneath & Sokal, 1973; Pleijel, 1995).

102 N. DAVITASHVILI and G. KARRER

© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 162, 101–115

Characters of testa outgrowth (characters 1–6)The testa of some gentians form membranous out-growths like wings and membranous lamellae. Inseveral cases, the outgrowth is developed only at thethinned chalazal end of the seeds (Fig. 1A). ‘Wing’ isused for a single thin, flattened bicellular testa layer(Werker, 1997: fig. 5) and ‘membranous lamellae’ isused for more than one, thin flattened bicellularlayers.

Six characters were defined for different types oftesta outgrowth:

1. Chalazal wing: a small wing is developed at thechalazal end of the seed only (Fig. 1A, C; eachwith the winged chalazal end to the right of theelongated seed body).

2. Complete side wing: both the extended chalazaland the micropylar end of the indistinct flattenedseed is part of the wing formed at one long sideonly (Fig. 1E, G, I).

3. Incomplete side wing: the chalazal and micropy-lar end of the seed is not extended therefore noneis part of the wing that develops only at one longside of the seed body (Ho & Liu, 2001: fig. 5).

4. Complete discoid wing: the wing surrounds thewhole seed body including the micropylar end(micropylar end to the right: Fig. 1K, M, but tothe left: Figs 1O, 2A, G, I, K).

5. Incomplete discoid wing: the wing surrounds thewhole seed body except the micopylar end(Figs 2C, 4 G–H).

6. Membranous lamellae (Fig. 3A, C, D, F, G, I).

Characters of testa cells (characters 7–13)7. Isodiametric testa cells – cells with ratio between

width and length > 0.5 (e.g. Fig. 3).8. Elongated testa cells – cells with ratio between

width and length < 0.5 (e.g. Figs 1, 2).9. Testa cells forming honeycomb-like pits (Fig. 3).

10. Prominently and irregularly raised anticlinalwalls (Fig. 4A–F).

11. Straight anticlinal walls (e.g. Fig. 1).12. Curved anticlinal walls (Fig. 2B, H, J, L).13. Anticlinal walls with floccose structures (Yuan,

1993: fig. 14).

Characters of seed surface (characters 14–15)14. Seed surface ribbed (Miège & Wüest, 1984:

fig. 29).15. Seeds compressed.

Character of the micropylar end (character 16)16. The micropylar end is thickened forming a collar-

like structure (Fig. 3D).

Characters of seed size (characters 17–19)We classified the longest diameter of seeds (Fig. 5)into three size classes:

17. Seeds < 1 mm.18. Seeds expanding from 1 to 3 mm.19. Seeds > 3 mm.

The full character matrix based on the presence(1)/absence (0) of all 19 characters is provided inAppendix 2.

ANALYSIS OF MORPHOLOGICAL DATA

The UPGMA analysis was performed using PAUP*4.0b4a (Swofford, 1998) measuring mean characterdifference with ties broken randomly, followed by aUPGMA bootstrap of 1000 replicates. For comparison,the analyses was also performed with the PAUP*4.0b4a program using the neighbor joining (NJ) clus-tering method, with a mean character difference as adistance measure and NJ bootstrap of 1000 repli-cates. Additionally, different similarity coefficientsfor presence/absence coding (Dice coefficient, Jaccardcoefficient and Raup–Crick index) were tested, butproduced identical terminal clusters in all cases. Par-simony analysis was also performed, but it resulted inunresolved trees and is thus not presented here.

RESULTSSPECIES SPECIFIC SEED TESTA CHARACTERS

In the descriptive part of the results, the analysedtaxa are arranged according to the currentlyaccepted classification by Ho & Liu (2001). SectionPneumonanthe is generally characterized by yellow-ish sparkling seeds with elongated testa cells. In G.asclepiadea elongated testa cells occur only on theseed body (Fig. 2E, F), whereas testa cells from thewing are isodiametric (Fig. 2D, F). The inner peri-clinal walls have a clearly visible reticulate second-ary sculpture. The representatives of this sectiondisplay variation in the types of wing and curvatureof anticlinal walls. The following five types can bedistinguished:

1. Narrowly obovate seeds with straight anticlinalwalls and the presence of a chalazal wing – G.calycosa Griseb., G. paradoxa Albov, G. pneumo-nanthe L., G. sceptrum Griseb. and G. septemfidaPall. (Fig. 1A, C; and Davitashvili & Karrer, 2006:fig. 1).

2. Narrowly obovate seeds with straight borders ofanticlinal walls and the presence of a side wing –G. makinoi Kusn., G. scabra Bunge, G. sikokianaMaxim. (Fig. 1E, G, I).

3. Narrowly obovate flattened seeds with straightanticlinal walls and the presence of a completediscoid wing – G. bicuspidata (G.Don.) Biq., G.linearis Froel., G. rubricaulis Schwein (Fig. 1K, M,O).

TAXONOMIC IMPORTANCE OF SEED MORPHOLOGY IN GENTIANA 103


Figure 1. Seeds with a chalazal wing: seed shape and testa cell details of Gentiana calycosa (A–B); G. sceptrum (C–D).Seeds with a complete semi-wing: seed shape and testa details of G. makinoi (E–F); G. scabra (G–H); G. sikokiana (I–J).Seeds with a complete discoid wing and straight anticlinal walls: seed shape and testa details of G. bicuspidata (K–L);G. linearis (M–N); G. rubricaulis (O).



4. Obovate flattened seed with straight and curvedanticlinal walls and the presence of a completediscoid wing – G. andrewsii Griseb., G. boissieriSchott & Kotschy ex Boiss., G. calycina Boiss. &Hauskn. ex Boiss., G. freyniana Bornm. ex Freyn.,G. gelida M.Bieb., G. puberulenta J.S.Pringle, G.saponaria L., G. spathacea Kunth. (Fig. 2A, G, I,K; and Davitashvili & Karrer, 2006: fig. 1).

5. Broadly obovate seeds with straight anticlinalwalls and the presence of an incomplete discoidwing – G. asclepiadea (Fig. 2C–F).

The following species out of section Frigidae werestudied: G. frigida Haenke, G. froelichii, G. nubigenaEdgew. and G. microdonta Franch. (Fig. 3). The seedsof G. frigida, G. nubigena, and G. microdonta areyellowish, elliptic in outline with isodiametric testacells (Fig. 3A–C, G–I). The anticlinal walls are raised.The outer periclinal walls collapse in mature seedsforming honeycomb-like pits. Inner periclinal wallshave a clearly visible secondary sculpture. Addition-ally, the seed body is surrounded by longitudinalmembranous lamellae (Fig. 3C).

Figure 2. Seeds with a complete discoid wing and curved anticlinal walls. Seed shape and testa details of Gentianaandrewsii (A–B); G. puberulenta (G–H); G. saponaria (I–J); G. spathacea (K–L). Seeds with an incomplete discoid wing.G. asclepiadea (C: seed shape; D: isodiametric testa cells from the wing; E: elongated testa cells from seed body; F: testacells from the seed body and the wing).



Seeds of G. froelichii were investigated here for thefirst time (Fig. 3D–F). They differ from seeds of otherrepresentatives of section Frigidae by their darkbrown colour, elliptic shape, the collar-like structureat the micropylar end, the absence of raised anticlinalwalls and honeycomb-like pits. Similar to othermembers of section Frigidae, the testa of G. froelichiiforms membranous lamellae and the testa cells areisodiametric.

Six species were selected for analysis from sectionIsomeria: G. chinensis Kusn. G. glauca Pall., G. sik-kimensis C.B.Clarke, G. wardii W.W.Sm. (Fig. 4), G.depressa Don and G. urnula Harry Sm. (Davitashvili& Karrer, 2007: fig. 2). Previously, only G. tubiflora(Wallich ex. Don) Griseb. has been documented (Ho &Liu, 2001); thus this study gives a better insight tothe seed character variation of section Isomeria.

All species are characterized by uniform ellipticseeds with distinctly and irregularly raised anticlinalwalls and the testa cells of all representatives ofsection Isomeria analysed form honeycomb-like pits.The cells of the epidermal layer are isodiametric and

the inner periclinal walls have a readily visible reticu-late structure.

Section Phyllocalyx consists of only one species –G. phyllocalyx C.B.Clarke (Fig. 4G–I). The seedbody is surrounded by an incomplete discoid wing.The latter is better visible on pressed and flattenedseeds, which stem from herbarium specimens(Fig. 4G). Additionally, the anticlinal walls of thetesta cells are raised forming honeycomb-like pitsand the inner periclinal walls have readily visiblereticulate secondary sculpture.

CLUSTER ANALYSES

The results of the cluster analyses are presented inFigure 6. In the UPGMA dendrogram (Fig. 6A), 13groups show an average distance of 0, indicating highsimilarity between the species included.

The sections do not show intrasectional variabilityexcept for Pneumonanthe and Chondrophyllae. Allsections, with the exceptions of Kudoa and Monopo-diae, cluster separately, underscoring the taxonomic

Figure 3. Seeds with membranous lamellae. Seed shape and testa details of Gentiana frigida (A–C); G. froelichii (D–F);G. nubigena (G–H); G. microdonta (I). C and F: latitudinal cut of seeds.



importance of seed microsculpture characters for thesectional division of Gentiana. Similar grouping canbe seen in the NJ dendrogram (Fig. 6B).

In general, both dendrograms indicate that seedmorphology follows in part the currently applied sec-tional classification of Gentiana by Ho & Liu (2001).Exceptions are G. asclepiadea and G. froelichii,which do not group within sections Pneumonantheand Frigidae, but form separate clusters with taxaof morphologically rather different sections.

DISCUSSION

The seed coat develops from ovular tissues. Like mostother sympetalous taxa, Gentiana has simple andsmall unitegmic ovules (Bouman & Schrier, 1979;Shamrov, 1988; Boesewinkel & Bouman, 1995) andexotestal seed coats (Corner, 1976; Bouman et al.,2002).

All studied specimens of sections Frigidae, Isome-ria, Kudoa, Monopodiae and Phyllocalyx are charac-

terized by more or less strongly raised anticlinal wallsand honeycomb-like undulations of the testa cells.Light microscopic studies of the seed morphologyof taxa from sections Frigidae, Isomeria, Kudoaand Monopodiae also confirm our observation (Halda,1996; Ho & Liu, 2001). To some degree, details of thetesta sculpture display intersectional variation corre-sponding with the classification of Ho & Liu (1990;2001). For instance, section Frigidae is readily distin-guished by its longitudinal, bicellular, membranouslamellae, section Isomeria by irregular, stronglyraised anticlinal walls without distinct membranouslamellae and section Phyllocalyx by winged seeds. Incontrast, our investigation did not reveal significantintersectional differences in seed testa sculpturebetween the sections Kudoa and Monopodiae. Allspecimens studied had uniform seeds with well-developed raised anticlinal walls forming honeycomb-like pits.

Seeds of nearly all the taxa from sections Chondro-phyllae, Dolichocarpa and Cruciata are characterized

Figure 4. Seeds with irregularly raised anticlinal walls. Seed shape and testa details of Gentiana chinensis (A); G. glauca(B–C); G. sikkimensis (D); G. wardii (E–F). Seeds with an incomplete discoid wing and honeycomb-like testa cells. Seedshape and testa details of G. phyllocalyx (G–I). G: seeds pressed.



by a smooth seed surface and the absence of testaoutgrowths (Yuan, 1993; Müller, 1982; Miège &Wüest, 1984). The only exception is G. pudica fromsection Dolichocarpa, which is characterized by thepresence of an incomplete semi-wing. Nevertheless,other characters of seed microsculpture support itsplacement in section Chondrophyllae (Fig. 6). Thisresult is in agreement with the molecular studies ofYuan & Küpfer (1997), suggesting the inclusion ofsection Dolichocarpa in section Chondrophyllae.

Section Cruciata does not show significant varia-tion in seed microsculpture (Müller, 1982; Miège &Wüest, 1984; Omer & Qaiser, 1995). As already docu-mented by Yuan (1993), Miège & Wüest (1984) andOmer & Qaiser (1995), the seed surface is reticulate,with prominent thickenings of the anticlinal wallsand rather narrow elongated testa cells.

Seeds of taxa from sections Ciminalis, Calathianaeand Gentiana are also characterized by a low infra-sectional variability, but they are clearly distinguish-able from other sections (Miège & Wüest, 1984). Forexample, seeds of section Gentiana are relativelylarge and the seed body is always surrounded by anincomplete wing. Section Ciminalis is characterizedby the presence of ribbed seeds with collar-like micro-pylar ends and isodiametric testa cells. These char-acters were already reported by Miège & Wüest(1984) and the distinct cluster of G. acaulis and G.clusii met our expectations. Section Calathianae has

been well studied by Müller (1982) and Miège &Wüest (1984), who documented only a slight variationbetween the species with respect to the number oftesta cells per seed, the outline of testa cells, theoverall shape and the dimension of seeds.

The present study revealed seed morphologicalsimilarity between sections Calathianae and Chon-drophyllae (Fig. 6). Both sections are characterized bysmall and smooth seeds sometimes developing incom-plete side wings. Other morphological characters andmolecular data (Yuan et al., 1996), however, do notsupport, a close relationship between the sections.

The first studies on seed morphology in G. froelichiiwere carried out by Kusnezow (1894), who reportedthat it had unique seeds but left it in section Frigidae.Ho & Liu (2001) accepted a similar sectional treat-ment for G. froelichii, justified by growth architectureand the presence of lamellar seeds. Our cluster analy-ses, on the one hand, do not support the inclusion ofG. froelichii in section Frigidae, with the presence ofmembranous lamellae being the only character of theseed micromorphology that is shared by G. froelichiiand the other taxa of section Frigidae. On the otherhand, G. froelichii does not have raised anticlinalwalls or honeycomb-like pits like all other represen-tatives of section Frigidae.

According to the present analysis, G. froelichiishows some affinity to section Ciminalis (Fig. 6), asthe representatives of this section are also character-ized by seeds with isodiametric cells and a collar-likestructure at the micropylar end. Gross morphologicaldata, however, do not support such a relationshipbetween G. froelichii and taxa of section Ciminalis. Asomewhat isolated position of G. froelichii is sup-ported by karyological (Löve & Löve, 1972), molecular(Gilley & Taberlet, 1996) and morphological studies(Karrer, 1997). Therefore, we propose to exclude G.froelichii from section Frigidae s.s., and to establish anew monotypic section for this species.

All representatives of section Pneumonanthe(excluding G. asclepiadea) are characterized by theseeds with elongated testa cells. At the infrasectionallevel, the seeds vary in shape, the curvature of anti-clinal walls and wing types. Functionally the wing isan adaptation to wind dispersal (Boesewinkel &Bouman, 1984, 1995; Werker, 1997; Fenner & Thomp-son, 2005). Bouman & Devente (1986) concluded thatwind is the prevalent dispersal vector for gentianseeds and this conclusion appears to be true forsection Pneumonanthe. All seeds examined bearspecial adaptations, such as complete discoid wings,complete side wings or chalazal wings favouring winddispersal. Apparently, wing formation of seeds in taxafrom section Pneumonanthe involves an extension ofthe chalazal and micropylar ends, whereas in taxaof other sections characterized by the presence of

Figure 5. Schematic drawing of a gentian seed (Gentianaasclepiadea) showing the relevant measurement of size: ‘l’indicates longest diameter including the wing.



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winged seeds (e.g. section Gentiana and section Doli-chocarpa) no seeds with extensions of the micropylarend have been recorded so far. Kusnezow (1894) pro-posed an infrasectional classification for section Pneu-monanthe based on the geographical distribution ofthe species. He established three groups: NorthAmerican, Euro Siberia–Far Eastern Asian andWestern Asian. The results of our study do notsupport his classification. Unlike the Far Easternspecies, which form a well-defined group character-ized by complete side wings, the seeds of the NorthAmerican and Western Asian groups are character-ized by both complete discoid and chalazal wings.Characters from flower morphology, for example thepresence of symmetrical and fimbriate plicae, alsoconfirm morphological similarities between NorthAmerican and Western Asian groups as well (Kusn-ezow, 1894; Ho & Liu, 2001). There are numerousexamples supporting affinities between the floras ofWestern Asia and North America (see, for example,Gagnidze et al., 2002; Milne, 2004). For better under-standing of the seed character evolution in theWestern Asian and North American groups, the needfor molecular phylogenetic analyses of section Pneu-monanthe is evident.

Gentiana asclepiadea was included in sectionPneumonanthe on the basis of the absence of leafyrosettes, elongated flowering shoots and the pres-ence of winged seeds (Ho & Liu, 2001). The presentstudy, however, showed that, with respect to seedcharacters, G. asclepiadea has more morphologicalaffinities to section Gentiana than to section Pneu-monanthe (Fig. 6). Similar to the taxa included insection Gentiana, seeds of G. asclepiadea are char-acterized by the presence of isodiametric testa cellsand incomplete seed wings. The close affinity of G.asclepiadea and taxa from section Gentiana hasalready been suggested by Yuan et al. (1996) on thebasis of a phylogenetic analysis of internal tran-scribed spacer (ITS) sequences, although gross mor-phological characters apparently did not supportthis opinion.

The position of G. asclepiadea within section Pneu-monanthe has been critically treated by a number ofresearchers. Combining the results from morphologi-cal, chemical, karyological and molecular studies(Löve & Löve, 1972; Serebryakova, 1979; Mészáros,1994; Yuan et al., 1996; Gilley & Taberlet, 1996;Karrer, 1997) with our analyses, it is evident that G.asclepiadea does not fit within section Pneumonanthe.Our results are congruent with those of the above-mentioned authors, who stated that G. asclepiadeashould be excluded from section Pneumonanthe andeven justify the establishment of G. asclepiadea as aseparate section, as already suggested by Tzvelev(1993).

Our cluster analyses of seed micromorphologicalcharacters demonstrated that the great variety ofseed microsculpture in gentians offers extremelyvaluable systematic and evolutionary information.The groups defined by seed testa characteristicsreflect the actual sectional classification of Gentianaby Ho & Liu (2001) to a considerable extent (Fig. 6).As shown for the hitherto unsatisfactory placed taxa(G. asclepiadea and G. froelichii), seed micromorpho-logical data analysed in a multivariate manner giveinteresting results, coinciding well with more elabo-rate morphological and molecular analyses.

Our results from cluster analyses correlate onlypartly with seed types established by previousauthors. For example, the cluster of sections Kudoaand Monopodiae was already recorded as having‘honeycomb-type’ seeds by Yuan (1993) or Boumanet al. (2002), and the cluster of section Ciminalis wasrecorded as having ‘ribbed-type’ seeds by Miège &Wüest (1984) and Bouman et al. (2002). Taxa with‘winged-type’, ‘semi-winged-type’ and ‘compoundlamellar-type’ seeds (Yuan, 1993; Ho & Liu, 1990,2001; Bouman et al., 2002) are, however, scattered indifferent clusters (Fig. 6). Former descriptions ofgentian seed types were based mostly on single char-acters, whereas in the present study we used 19evenly weighted characters of seed microsculpture.This approach, although seldom used in seed morpho-logical studies, confirms that studies of seed testaornamentation in plants can be of high taxonomicvalue, providing information that facilitates not onlythe identification and delimitation of different taxo-nomic levels, but might also give a better understand-ing of the evolution of taxa.

ACKNOWLEDGEMENTS

We are thankful to Christian Baal, Bernhard Dickoré,Irene Karrer, Thomas Denk, Chris Brickell, thepersons responsible for the herbaria of the RoyalBotanic Gardens, Kew, and of the Museum of NaturalHistory at Vienna. Comments by two anonymous ref-erees were very helpful. The study was supported byAustrian Exchange Service (ÖAD).

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APPENDIX 2

Character matrix based on the presence (1)/absence (0) of the 19 selected characters for 42 Gentiana species.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

G. acaulis L. 0 0 0 0 0 0 1 0 0 0 1 0 0 1 0 1 0 1 0G. alsinoides Franch. 0 0 0 0 0 0 0 1 0 0 1 0 1 0 0 0 1 0 0G. andrewsii 0 0 0 1 0 0 0 1 0 0 1 1 0 0 0 0 0 1 0G. aperta Maxim. 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 1 0 0G. asclepiadea 0 0 0 0 1 0 1 1 0 0 1 0 0 0 0 0 0 1 0G. asterocalyx Diels 0 0 0 0 0 0 0 1 0 0 1 0 1 0 0 0 1 0 0G. bicuspidata 0 0 0 1 0 0 0 1 0 0 1 0 0 0 0 0 0 1 0G. brachyphylla Vill. 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 0G. calycosa 1 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 1 0G. clusii C.P.Perrier & Songeon 0 0 0 0 0 0 1 0 0 0 1 0 0 1 0 1 0 1 0G. crassuloides Franch. 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 1 0 0G. cruciata L. 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 1 0G. depressa D.Don 0 0 0 0 0 0 1 0 1 1 1 0 0 0 0 0 0 1 0G. exigua Harry Sm. 0 0 0 0 0 0 0 1 0 0 1 0 1 0 0 0 1 0 0G. frigida 0 0 0 0 0 1 1 0 1 0 1 0 0 0 0 0 0 1 0G. froelichii 0 0 0 0 0 1 1 0 0 0 1 0 0 0 0 1 0 1 0G. gelida M.Bieb. 0 0 0 1 0 0 0 1 0 0 1 1 0 0 0 0 0 1 0G. glauca 0 0 0 0 0 0 1 0 1 1 1 0 0 0 0 0 0 1 0G. leucomelaena Maxim. 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 1 0 0G. lutea L. 0 0 0 0 1 0 1 1 0 0 1 0 0 0 0 0 0 0 1G. makinoi 0 1 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 1 0G. microdonta 0 0 0 0 0 1 1 0 1 0 1 0 0 0 0 0 0 1 0G. nivalis L. 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 0G. nubigena 0 0 0 0 0 1 1 0 1 0 1 0 0 0 0 0 0 1 0G. olivieri Griseb. 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 1 0G. phyllocalyx 0 0 0 0 1 0 1 0 1 0 1 0 0 0 0 0 0 1 0G. pneumonanthe L. 1 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 1 0G. prattii Kusnesow 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 1 0 0G. pseudoaquatica Kusnesow 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 1 0 0G. pudica Maxim. 0 0 1 0 0 0 0 1 0 0 1 0 0 0 0 0 1 0 0G. punctata Vill. 0 0 0 0 1 0 1 1 0 0 1 0 0 0 0 0 0 0 1G. purpurea L. 0 0 0 0 1 0 1 1 0 0 1 0 0 0 0 0 0 0 1G. rigescens Franch. 0 0 0 0 0 0 1 0 1 0 1 0 0 0 0 0 0 1 0G. rubricaulis 0 0 0 1 0 0 0 1 0 0 1 0 0 0 0 0 0 1 0G. scabra 0 1 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 1 0G. septemfida Pall. 1 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 1 0G. squarrosa Ledeb. 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 1 0 0G. straminea Maxim. 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 1 0G. szechenyii Kanitz 0 0 0 0 0 0 1 0 1 0 1 0 0 0 0 0 0 1 0G. urnula 0 0 0 0 0 0 1 0 1 1 1 0 0 0 0 0 0 1 0G. verna L. 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 0G. wardii 0 0 0 0 0 0 1 0 1 1 1 0 0 0 0 0 0 1 0

Selected characters coded in a multistate way.1. Chalazal wing (0); complete side wing (1); incomplete side wing, (2); complete discoid wing, (3); incomplete

discoid wing, (4); without wing (5).2. Membranous lamellae: yes (0) no (1).3. Isodiametric testa cells (0); elongated testa cells (1).4. Testa cells forming honeycomb-like pits: yes (0) no (1).5. Prominently and irregularly raised anticlinal walls: yes (0) no (1).6. Straight anticlinal walls (0); curved anticlinal walls (1).7. Anticlinal walls with floccose structures: yes (0) no (1).



8. Seed surface ribbed: yes (0) no (1).9. Seeds compressed: yes (0) no (1).

10. The micropylar end is thickened forming a collar-like structure: yes (0) no (1).11. Seeds < 1 mm (0); seeds 1–3 mm (1); seeds > 3 mm (2).

Character matrix based on the selected multistate characters for 42 Gentiana species.

1 2 3 4 5 6 7 8 9 10 11

G. acaulis 5 1 0 1 1 0 1 0 1 0 1G. alsinoides 5 1 1 1 1 0 0 1 1 1 0G. andrewsii 3 1 1 1 1 01 1 1 1 1 1G. aperta 5 1 1 1 1 0 1 1 1 1 0G. asclepiadea 4 1 01 1 1 0 1 1 1 1 1G. asterocalyx 5 1 1 1 1 0 0 1 1 1 0G. bicuspidata 3 1 1 1 1 0 1 1 1 1 1G. brachyphylla 5 1 0 1 1 0 1 1 1 1 0G. calycosa 0 1 1 1 1 0 1 1 1 1 1G. clusii 5 1 0 1 1 0 1 0 1 0 1G. crassuloides 5 1 1 1 1 0 1 1 1 1 0G. cruciata 5 1 1 1 1 0 1 1 0 1 1G. depressa 5 1 0 0 0 0 1 1 1 1 1G. exigua 5 1 1 1 1 0 0 1 1 1 0G. frigida 5 0 0 0 1 0 1 1 1 1 1G. froelichii 5 0 0 1 1 0 1 1 1 0 1G. gelida 3 1 1 1 1 01 1 1 1 1 1G. glauca 5 1 0 0 0 0 1 1 1 1 1G. leucomelaena 5 1 1 1 1 0 1 1 1 1 0G. lutea 4 1 0 1 1 0 1 1 1 1 2G. makinoi 1 1 1 1 1 0 1 1 1 1 1G. microdontha 5 0 1 0 1 0 1 1 1 1 1G. nivalis 5 1 0 1 1 0 1 1 1 1 0G. nubigena 5 0 0 0 1 0 1 1 1 1 1G. olivieri 5 1 1 1 1 0 1 1 0 1 1G. phyllocalyx 5 1 0 0 1 0 1 1 1 1 1G. pneumonanthe 0 1 1 1 1 0 1 1 1 1 1G. prattii 5 1 1 1 1 0 1 1 1 1 0G. pseudoaquatica 5 1 1 1 1 0 1 1 1 1 0G. pudica 2 1 1 1 1 0 1 1 1 1 0G. punctata 4 1 0 1 1 0 1 1 1 1 2G. purpurea 4 1 0 1 1 0 1 1 1 1 2G. rigescens 5 1 0 0 1 0 1 1 1 1 1G. rubricaulis 3 1 1 1 1 0 1 1 1 1 1G. scabra 1 1 1 1 1 0 1 1 1 1 1G. septemfida 0 1 1 1 1 0 1 1 1 1 1G. squarrosa 5 1 1 1 1 0 1 1 1 1 0G. straminea 5 1 1 1 1 0 1 1 0 1 1G. szechenyii 5 1 0 0 1 0 1 1 1 1 1G. urnula 5 1 0 0 0 0 1 1 1 1 1G. verna 5 1 0 1 1 0 1 1 1 1 0G. wardii 5 1 0 0 0 0 1 1 1 1 1



taxonomic importance of seed morphology in gentiana (gentianaceae

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