Gametophyte morphology offour subspecies of
Asplenium trichomanes L.*
ALEERTO HERRERO; CARMEN PRADA; EMILIA PANGIJA; AbRíAN ESCUDERO; AGUSTíN RuBlo ‘~‘ SANTIAGO PAJARON
Dpto. Biología Vegetal 1. Facultad de Biología. 11CM. E-28040 Madrid
Resumen:
HI?RREito, A., PL<AOA, C., PANGUA. E., EScUOERo, A., Rtjn¡o, A. &FAJARON,5. 1993. Mor-foLogía del gametófito de cuatro subespecies de Asplenium trichomanes L.Bol Complaíensrs
18: 67-77
Mediante el cultivo de esporas deAspleniam tr-ic-homanes L. (subsp. trichomanes.subsp.
qucrdr-rr-a/ens DE Meyer. subsp. pachyrac-lris(Christ) Lovis & Reichsl. y subsp.inexpec-tans
Lovis) se ha estudiado el desarrollo de los gamelófitts y sus características morfológicas. Exeeplo en la subsp. irrexpeclansy en dos de las cuatro muestras estudiadas de la subsp.
¡racl;yrahis. se forman pelos pluricelulares marginales en una proporción variable de prolalos según el laxon Se analiza la longitud, número de pelos por prolalo y densidad de los mismos y se disci¡te su valor taxonómico, así como el de otros caracteres morfológicos como el margen y la escotadura.
Abstract:
iIFRRFRO.A.. PRADA,C., PANChA. E., ESCUDERO, A., Run¡o, A. & PAJARON. 5. 1993. Gaíneíopliyíe morphology of four subspeeies of Asplenium trichomanes L. Bat. Conrplatensis
18: 67-77
By cr¡lturing spores of Aspleniuni trichomanes L.<subsp. Ériclioí,,anes, subsp. quadrivaLens DE. Meyer, subsp. pachyrachis (Chrisl) Lovis & Reichst. and subsp. inexpeelans Lovis), we have sludied Ihe developmenl of Ihe gamelhophytes and Iheir morphological fealures. Multice-halar marginal hairs were produced in different numbers, depending on Ihe laxon, excepí [br subsp. inexpecíans and two samples of subsp. pachyrachis. Length, density and number of hairs per gaíneíophyíe weu-e analyzed and Iheir taxonomic value is diseussed as welI as níher morphoiogicaL fealures such as margin and apical nolch.
*This rescarclr was supponed by aDGLCYT graní,numberPE 87-0553
68 Herrero, A.; Pr-ada. C: fiangua. E.; Escudero, A.,- Rubia, A.,- fiajarón.S
INTRODIJCTION
Study of fern gametophytes has become essential to complete morpho-logical, ecological and reproductive knowledge and has contributed to a better understanding of taxonomic relationship(STOKEY, 1951; NAYAR & KAUR, 1969 and 1971; ATKINSON, 1973; WINDHAM &HAUFLER, 1986). In spite of the presence of numerous papers on fern gametophytes, the num-ber of species whose gametophytes are know in detail is still small (TAY-LOR & MICKEL, 1974). The prothallial development of some Asiatic and Furopean Asp/eniaceae was studied by MoMoSE (1959, 1960a, 1960b, 1961a and 1961b), NAYAR & al. (1968), HUREL-PY (1950), ARMENISE (1959) andHENRIET (1970).
Among the naturally occurring Asp/enium species in Spain, we chose
Asp/eniurn trichornanes L. because of the diversification of this taxon of
which four subspecies are recognized: subsp. u-ichornanes and subsp.
znexputans Lovis, both diploid, and subsp. quadriva/ens D.F. Meyer and
subsp. paChvrachis (Christ) Lovis & Reichst., both tetraploid. The morp-hological characteristies and the habitat preferences of the sporophytes are well known (LovíS, 1964; JERMY & PAGE, 1980; Lovís & REICHSTEIN, 1985; PANGUA & al., 1989); gametophytes, however, have been studied only in part. especially a few details of their morphology (NAYAR & al.,
1968; NAYAR & KAUR, 1971). In a previous study (ROA & al., 1987) we examined gametophytes of the two tetraploid subspecies in order to deter-mine if they provide distinctive morphological features; the results poin-ted to some differences that stimulates us to continue and to complete the study of gametophytes of this group, including the diploid subspecies.
There has been described (RASEACH & al., 1990) a fifth, tetraploid, subspecies, subsp. coriaCeífolium from Mallorca. This taxon has not been included in our study.
MATERIAL AND METI-IODS
Spores for gametophyte cultures were obtained from the following plants:
A. trichomanes subsp. triuhornanes
Spain: Madrid, La Pedriza, sustrato silíceo, 22-IV-1991, Pangua &
P¡ada CElSI.
Spain: Madrid, La Pedriza, sustrato silíceo, 22-IV-1991, Pangua &
Gametoplívíe Morphology ofFoar Sabspec/es ofAsplenium ír-ic-homanes L. 69
A. tí-ichomones subsp. inexpeCtans
Austria: Therman-Alpen, bei Gutenstein an der langen Brúcke, limes-tone rocks, 13-VI-1983, leg. Melzer sn., det. II. RasboCh. Fronds pressed
15-1-1991.
Switzerland: Tilfels-Cheller, bei Wettingen, Baden, J. Schnel/er 1536. Fronds pressed 16-V-1991.
A. nichomanes subsp. quadrivalens
Spain: Orense, entre Córgomo y San Vicente, pizarras, 24-IX-1990,
Prado & al.
CE
121.Spain: Orense, Barco de Valdeorras, Coedo, pizarras, 31-III-1991,
Proc/a CEIS?.
A. trichomones subsp. pochyrachis
Spain: Cuenca, Ciudad Encantada, calizas, 29-IV-1991, Prado CE193. Switzerland: St. Wolgang, Balsthat, Hinterflue, 530 m.s.m., 1.
Súhne-//er 1535. Fronds pressed 16-V-1991.
Spain: Cuenca, Friego, “El Martinete”, cerca del río Guadiela, calizas, exposición N., 900 m.s.m., 24-111-1991, Escudero CEI6O.
Spain: Valencia, Benifaró de Valídigna, camino del repetidor, 6-1V-1991, Proc/a & a/. CEI3I.
Spores from each sample were sown in plastic boxes (4,5 x 4,5 x 2,5 cm.) on minera! agar (DYER, 1979) for study of the early stages of deve-lopment; they were also grown on soil autoclaved twice at 1200C during 30 minutes in the same kind of boxes, in order to obtain mature prothalli and sporophytes. Four replications of each sample were made. Cultures were maintained under constant environmental conditíons ín a growth chamber at 230C and continuous illumination with white fluorescent tubes. Cultures on soil were watered once a week.
During the first 45 days after sowing, cultures on agar were checked weekly and samples were studied under light mícroscope; subsequently, about 50 gametophytes growing on soil were removed from each sample every two weeks. AII observations on morphology and development were carried out on gametophytes from these laboratory cultures.
70 flerrero, A.; fir-ada. C; fiangua, E.; Escudero. A.; Rubio, A.; Pajarón,5.
random sampling were nel (Cochran’s C test P-ct0,05). When the assump-tions of homogeneity of variances was violated, a Kruskal-Wallis test was performed.
RES ULTS
AII samples started germinating between 5 and 20 days afler sowing; subsp.quodriva/ens aud subsp. pochyrochis showed a faster development than did subsp. inexpe~§-tons and subsp. trichornanes.
The spore germination pattern follows the Vittoria type, and prothallial development seems to follow the Adianturn type(NAYAR & KAUR, 1971). Spores of aH samples produce a short germ filament of 1 to 3 celís (Fig. 1 a-c) except for suhsp pachvrachis which produces 2 to 6 celís (Fig. 1 e\ The fiLaments bear 1 or 2 rhizoids on the basal cdl. Development of a bidimensional stage occurs in ah samples 8-10 days after spore germina-tion (Fig. 1 d-f), and the apical notch becomes evident three weeks aftcr germination (Fig. 1 g). Mature prothalli are cordate, broader than long as usual in the family (Fig. 1 h-I); in subsp. inexpec-tans, the apical notcb is broader than in 4w other subspecies (Hg. 1 h, 2 a).
The protalli of subsp. tri~i-homanes, subsp. quadrivo/ens and subsp.
pac-hy¡-ochis (excepí forthe samples CEI3I and CEIÓO) bearmulticellular
marginal hairs with (3)4-5(6) green celís (Fig. 2); the terminal ceil is gran-dular, slighíly swollen at Ihe apex and contains a brown secretion. The hairs are straight or curved. The proportion of hairy gametophytes is shown in figure 3. l-lairs appear 30-40 days after germination in ah hair-bearing samples when the prothalli have become distinctly cordate. The
data of parameters studied are shown in table 1.
The number of hairs per ganietophyteis variable-among the taxa; InSO days oid gametophytes, when sex organs are well developed, subsp.
qua-drivo/ens has a mean of 8 hairs, whereas subsp. trichornanes and poch vv-a-<his have only 2 or 3 hairs (Fig. 4 a). In older gametophytes (90 days oid),
when fertilization has begun lo lake place in Ihe cultures, the mean num-ber of bairs increases considerably in subsp. c,ruodriva/ens, but in subsp.
tí-ichornones and poc-hy¡-achis the mean does nol change (Fig. 4 b).
In ihe same way, the density of the hairs vanes among the taxa; there are significaní differences in density in the 50 aud 90 days oíd stages (Table 1) for alí taxa.
Gametophyíe Morphology ofFoar Sabspecies ofAspleniurn trichomanes L. 71
hairs, about 130 ~ím(Fig. 4 c). In 90 days oíd gametophytes hairs are Ion-ger and 5h11 show significant differences among the taxa (Fig. 4 d), but 15 days later, the hairs of subsp. pac-hyraChis have grown, exceeding slightly the mean length of the hairs of subsp. quodrivalens, whereas in subsp.
tri-chomanes the mean does not change (Fig. 4 e).
One month after germination alí samples produced archegonia, but the diploid subspecies had a lower percentage of prothalli bearing archegonia than the tetraploid ones. Antheridia appeared about two weeks later in alí cases.
DISCUSSION
The morphological features of the gametophytes are similar in alí four taxa, but subsp. inexpeCtons has a notably broad apical notch and the mar-ginal celís frequently protrude, giving the prothalli a characteristie appea-rance; the edge is always naked.
NAYAR & al. (1968) found gametophytes of A. trichomanes to be naked, but according to our observations in three of the subspecies the prothalli bear hairs in different proportion. in subsp. quodrivalens hairy gametophytes are produced at a high rate so that practically alí plants of the cultures bear hairs. In Uds case the higher density of hairs imparts a typical appearance to the prothalli as compared with the other taxa.
An unexpected problem was posed by subsp. trichomanes and subsp.
pachyrachis samples CEI6O and CEI3I; in these cases, both hairy and
naked gametophytes were produced. We did not find any reference to this in the consulted literature. Studies on gametophyte niorphology are not usually done over a sufficiently long period of time and with enough gametophytic individuals to establish the characteristic variations in each species. In studies of hairy gametophytes, attention is focused mainly on ihe type of hair. its position and the time of its development, but there are no indications of naked and hairy gametophytes growing together, both produced by spores from the same sporophytic individual. PRAY (1968) mentions morphological variation in a gametophytic population arisen from a single sporophyte of Pci/oea and¡-omedaefo/ia (Kaulf.) Fée var.
puhescens D.C. Faton; the variation consisted in sorne differences in the
shape of the gametophytes.
Wc have observed that size, sex, and age of prothalli are not related to dic presence of hairs, so that at present we do not have any explanation for this striking feature.
72 Herrero, A.; firada, C.—fia,r gua, E.; Lscadero. A.; Rabio, A.; Pa/arón, 5.
10% of the cultures in 135 days oíd individuals; the behaviour coinciden-ce of both samples studied is remarkable. We have observed in these oíd prothalli that the basal celí degenerates and is easily ruptured (Fig. 2 e,
Q.
This might explain why the number of hairy gametophytes decreases. Diploid Phy//ftis sc-olopendrium from Europe has been reported to have unicellular hairs on the gametophytes, whereas American tetraploids have 1, 2, and 3-celled hairs (ATKINSoN & STOKEY, 1964). In our study, the number of celís does not differ significantly both in diploid and tetraploid taxa but the length of the hairs is much less in 4w d~ploid.With regard to subsp. poc/uíyrochis, we must emphasize the different behaviour found in the studied samples. As noticed aboye, two of them bear hairs on some of their gametophytes, the other two being naked.
Lovís & REICHSTEIN (1985) indicated the existence of plants “close but
distinct from true A. tíichomones subsp. pachyrochis”; PANGUA & al.
(1989) also mentioned variability within this taxon, as was illustrated in their fig. 1 G, H. Fronds of typical subsp. pachya<his have pinnae that are serratolobate, tend to be symmetrical, 2-4 times longer than wide with biauriculate base; there are plants in which the main morphological
cha-racters are the same as in the typical forms, but the pinnae are less than twice as long as wide and they are not always biauriculate. In the four samples of subsp. poc/ryrochis studied, two had typical morphology: gametophytes produced by these plants were always naked. The other two sporophytes, whose pinnae were shorter, produced gametophytic cultures of mixed hairy and naked prothalli. These differences in gametophyte morphology agree with the idea that morphologically atypical plants may represent a different taxon.
Gametophyte Mor-pholagy ofFaur Subspecies ofAspleniam trichonranes L. ‘73
Fig. 1: a-g, Early stages of prothaltial development: a, firsí rhizoid; b, germ fila-mení; c, beginning of laminar stage; e-f, development of meristemalie celis; g, begin-ning of apical notch; h-I, silhouette of malure gametophytes: h, subsp.inexpectans;
subsp. trichomanes;
j,
subsp. quadrivalens; kA, subsp. pachyrachis. Scale bars: a-g, 55 I.lm; h-I, lmm.e
h
74 Herrero. A-Pr-ada. C; fiangua, E.; Escudero, A.; Rabio, A.; fiajarón.S
Gametaphyíe Morphalagy ofFaur Subspecies ofAsplenium trichomanes L. ¶3- 100-- 80- 60- 40- 20-cEl2l .sV—CÉ1S7 — — —- —.- —-casi 75 0
40 50 60 70 80 90 100 110 120 130 140 da~
Hg. 3: Perceníage of hairy prothalli: subsp. quad¡-ivafens(CEI2I, CE157); subsp. pachyr-achis (CE 131, CFI 60); subsp. Ir-ichornanes (CE 181, CE 183).
lo 141 la, 121 111 Ial 91 al * 23456 a 230
¡iii
170 150:
1 1
12 34 54
Fig. 4: Meanconfidence intervaJs, LSD(95%)of garnelophyte characíers. a, num-ber of hairs per prothallus (50 days oíd); b, numnum-ber of hairs (90 days oíd); e, hair lengíh (50 days oíd); d, hair lenglh (90 days oíd); e, hair lenth (105 days oId). 1, population CEISI; 2, CE183; both of subsp. trichomanes; 3, CEI2I; 4, CE157; both of sub.sp. quadr-ivalens; 5, CE16O and 6, CEI3I; both subsp. pachyraehis. Length of hairs in prn.
ca131
A A A
~-cE1Bo
ji
1 -l76 herrero, A.; Pr-ada, C; Pangaa, E.; Esc-ader-o, A.; Rubia, A.; Pajarón, 5.
Table 1: Means (X) and standard deviaíions (SD) of gametophyle characters sco-red for Ihe studied populaíions. TesIs of significance refer to ANOVA excepí those rnarked with KW, which ¡udicate results of KW test(*** indicates p<O.OO 1).
Saniple size: len individuals in each population
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trichomanes qnadrivalens pachyrachis
CEISI CE183 CE121 CE157 CEI6O CE13I
Number of hairs X 3,12 3.37 8 8,25 2,28 2.42 ~
(S0days oid) SU ¡.24 .41) 3,02 2,31 1,11 .61
Number of hairs X 2.80 2,80 1§,40 ¡6 2.40 4,10 *5*
(90 days oid) SU 122 076 2,67 2,26 .34 .19
Densily X (>33 0,35 1)72 0.89 0.20 0. LS KW555
(50 (layS oíd) SU 0.12 0.12 017 0.30 1>09 1)09
Densi¡y X 0,17 0, 9 1)8<) 0,89 0,18 1)23 KW555
(91) days oíd) Sn 0.05 1)1)6 0,24 1)16 1), i 3 0.06 llair ienglh 1pm) X 90, Lo 89.41) Li 210 ¡1540 13 i .22 ¡30.20 ~ 150 dar oid) SD 9.1)7 ¡2.48 Li1~ i 1,91 16,99 4.64 Flair lenglO (pm) X ¡44 i46,90 20i.30 87.80 178,411 i77,70 ~ 190 days oíd) 50 22,8(1 29.10 3403 24,53 34,8i 33.24 flair lcngth (peo X 133,90 133.90 190.90 201,80 201.80 199.70 *15
Gameíophyte Morphalagy ofFoar Suhspecies ofAspleniumti-it.-homanes L. 77
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Recibido /2 ¿le erarza de 1992
