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Blackwell Science, LtdOxford, UKBOJBotanical Journal of the Linnean Society0024-4074The Linnean Society of London, 2005?

Original Article


Botanical Journal of the Linnean Society, 2005, 148, 459–464. With 9 figures

A morphometric study of three closely related taxa in the

European Isoetes velata complex
M. I. ROMERO1* and C. REAL2
University of Santiago, Department of Botany, and 2Department of Cellular Biology and Ecology, Escola
Politécnica Superior, 15702 Lugo, Spain

Received March 2004; accepted for publication December 2004

Isoetes velata A. Braun is an amphibious plant with Mediterranean and Ibero-Atlantic distribution, showing marked
morphological variability. Within the same geographical area, closely related restricted-range taxa are also present,
including I. velata ssp. asturicense (restricted to mountainous areas of the north-west Iberian Peninsula) and
I. boryana (restricted to the southern French Atlantic coast). These related taxa have unclear taxonomy and are both
threatened. We performed a morphometric study of specimens of the three taxa. Our results suggest that I. velata
ssp. asturicense is better considered as a distinct species, I. asturicense Laínz (Laínz), distinguishable from the other
two taxa by mean megaspore length (N ≥ 30) < 360 mm. In contrast, our results do not support consideration of
I. boryana as a separate species, and we here propose that this taxon be considered as a variety, I. velata A. Braun
var. boryana M.I. Romero & C. Real stat. nov. © 2005 The Linnean Society of London, Botanical Journal of the
Linnean Society, 2005, 148, 459–464.

ADDITIONAL KEYWORDS: endemic plants – Habitats Directive – Isoetaceae – pteridophytes – taxonomy –

threatened species.

INTRODUCTION 1986; Prelli, 2001), in addition to the complete velum

and the three-lobed corm. Therefore, these taxa are
Isoetes velata A. Braun is an amphibious plant with included in the section Tuberculatae proposed by Pfeif-
Mediterranean and Ibero-Atlantic distribution, grow- fer (1922: 104) and subsequently validated by Fuchs
ing on seasonally waterlogged, siliceous substrates (1962), although certainly this section is phylogeneti-
(Quezel, 1998). Not only does this taxon show great cally artificial (Rydin & Wikström, 2002). In any case,
variability (Pfeiffer, 1922; Reed, 1953; Jermy & Aker- several authors have expressed doubts about the tax-
oyd, 1993), but various other closely related taxa have onomic status of these endemic Isoetes (Rico & Giral-
been described, including I. velata ssp. asturicense dez, 1990; Prelli, 2001), particularly given that the
(Laínz) Rivas-Martínez & Prada ( ∫ I. boryanum very simple architecture of these plants provides few
Durieu ssp. asturicense Laínz, ∫ I. asturicense (Laínz) character states that can be used to distinguish taxa.
Laínz), restricted to the central and north-western This I. velata complex is included in various sections
Iberian Peninsula (Prada, 1983, 1986), and I. boryana in Annex I of the Habitat Directive, while I. boryana is
Durieu, restricted to the French departments of included in Annex II (Anonymous, 1992). In view of
Gironde and Landes on the Bar of Biscay (Mar this conservation importance, we here report a bio-
Cantábrico) (Badré & Deschatres, 1979). metric study of these taxa, with the aim of evaluating
The restricted-range taxa Isoetes boryana and their taxonomic status.
I. velata ssp. asturicense show morphological charac-
teristics of I. velata, such as spinulose microspores
and tuberculate megaspores (Pfeiffer, 1922; Berthet & MATERIAL AND METHODS
Lecocq, 1977; Rivas-Martínez, 1980; Prada, 1983,
Only herbarium material was used. Specimens of
*Corresponding author. E-mail: I. boryana and I. velata were from the Paris Herbar-

© 2005 The Linnean Society of London, Botanical Journal of the Linnean Society, 2005, 148, 459–464 459
460 M. I. ROMERO and C. REAL

ium (Muséum d’Histoire Naturelle de Paris). Speci- Other variables used were the number of leaves on
mens of I. velata ssp. asturicense were from the the plant (entire plants only) and leaf length. Leaf
Herbarium of the Royal Botanical Garden of Madrid length was the length of the longest leaf on each
(Herbarium MA). Wherever possible, we obtained voucher.
megaspores and microspores, and mounted them in
Euparal for microscopic examination. The variables
measured were maximum diameter (D) of
megaspores, and maximum length (L) and width (l) of Possible relationships between megaspore size and
microspores, as per Berthet & Lecocq (1977). In each other plant characteristics were investigated by mul-
preparation, 30 spores were randomly selected using a tiple regression analysis.
stereomicroscope, as per Britton & Brunton (1993).
Spore sizes were then measured with a micrometer RESULTS
under a 2.5¥ objective (megaspores) or a 40 ¥ objective
(microspores); note that the nature of the measure- SPORANGIAL MORPHOLOGY AND ORNAMENTATION
ment procedure means that megaspore length is a dis- Isoetes velata and I. boryana had tetrahedral
crete variable, not a continuous variable (megaspores, megaspores, with marked tuberculate ornamenta-
2.5¥, step 38.46 mm; microspores, 40¥, 2.5 mm steps). tion (Figs 1, 2). In contrast, all I. velata ssp. asturi-
Both mega- and microspores were also examined and cense specimens examined had spherical spores with
photographed with a scanning electron microscope only weak ornamentation (Figs 3, 4). The mono-
(SEM) (see Kott & Britton, 1983). graph on Isoetes in the Flora Ibérica (Prada, 1986)

1 2

3 4

Figures 1–4. Megaspores of Isoetes velata complex. Fig. 1. Isoetes velata, lateral view. Scale bar = 400 mm. Fig. 2.
I. boryana, lateral and distal views. Scale bar = 400 mm. Fig. 3. I velata ssp. asturicense, lateral view. Scale bar = 200 mm.
Fig. 4. I velata ssp. asturicense, distal view. Scale bar = 200 mm.

© 2005 The Linnean Society of London, Botanical Journal of the Linnean Society, 2005, 148, 459–464

likewise describes the megaspores of I. velata ssp. SPORE SIZE, LEAF NUMBER AND LEAF LENGTH
asturicense as ‘smooth, with small tubercles on the
proximal face and occasional tubercles on the distal Microspores of I. velata, I. boryana and I. velata ssp.
face’. asturicense showed similar mean length, minimum
Microspore morphology is similar in all three taxa, length and maximum length (Table 1) and were thus
although the ornamentation appears rather less dense not considered further. However, megaspore size, leaf
in I. velata ssp. asturicense (Figs 5–7). number and leaf length showed some differences

5 6

Figures 5–7. Microspores of Isoetes velata complex. Scale bars = 20 mm. Fig. 5. I. velata. Fig. 6. I. boryana. Fig. 7. I. velata
ssp. asturicense.

Table 1. Megaspore and microspore size data (L = length, l = width) for the three taxa

Isoetes velata I. boryana I. velata asturicense

Megaspores (mm)
Min – Max 463.14–615.36 269.22–538.44 230.76–384.6
Mean ± S.E. (N) 463.62 ± 53.60 (420) 448.846 ± 45.8623 (330) 292.57 ± 36.28 (240)
Microspores (mm)
Min-Max (L ¥ l) 22.5–35 ¥ 12.5–22.5 22.5–32.5 ¥ 12.5–20 22.5–32.5 ¥ 12.5–20
L Mean ± S.E. (N) 28.84 ± 2.15 (360) 28.82 ± 1.87 (420) 28.15 ± 1.8 (300)
l Mean ± S.E. (N) 16.84 ± 1.61 (360) 16.64 ± 1.44 (420) 15.63 ± 1.61 (300)

© 2005 The Linnean Society of London, Botanical Journal of the Linnean Society, 2005, 148, 459–464
462 M. I. ROMERO and C. REAL

among the three taxa. The distributions of these three than maximum leaf length in I. boryana (4–31 cm),
variables in each taxon are represented by box plots in although median values were similar in the two taxa
Figure 8. (10 and 12 cm, respectively). Note that the range of
The plots indicate that the number of leaves per maximum leaf length in I. velata ssp. asturicense falls
individual show some differences between the three completely within that of I. boryana, but shows very
taxa, but that these differences are only minor. Means little overlap with that of I. velata. The ranges for
and medians were between 16 and 20 leaves, and the I. boryana and I. velata show considerable overlap.
overall range was 5–52 leaves. Megaspore diameter likewise showed differences in
Maximum leaf length (i.e. length of the longest leaf distribution between I. velata ssp. asturicense and the
on the plant) was greater in I. velata (median 21 cm) other two taxa; the range of diameter for I. velata ssp.
than in the other two taxa, and had a larger range asturicense overlapped considerably with that for
(13–43 cm). In addition, maximum leaf length in I. boryana, but less with that for I. velata. The mean
I. velata ssp. asturicense had a larger range (6–15 cm) diameter for I. velata ssp. asturicense (290 mm) was
markedly less than those for I. boryana (450 mm) and
I. velata (460 mm). The latter two taxa also showed lit-
tle overlap in overall range.
To investigate in greater detail the differences in
I. boryana
megaspore diameter between I. velata ssp. asturicense
and the other two taxa, we compared data from the
I. velata individual vouchers (see Fig. 9). Figure 9 shows that
the overlap between the ranges for I. velata ssp. astu-
ricense and I. boryana is largely due to a single
I. velata I. boryana voucher. Nevertheless, even if we exclude
this voucher, there is still some overlap between the
maximum values for I. velata ssp. asturicense and the
0 10 20 30 40 50 60
Number of leaves minimum values for the other two species; note,
though, that there is no overlap between mean values
(maximum voucher mean for I. velata ssp. asturicense
I. boryana megaspores is 340 mm in diameter whereas minimum
voucher mean for megaspores of the other two taxa is
380 mm in diameter).
I. velata
As can be seen from Figure 9, mean megaspore size,
0 10 20 30 40 50 and minimum and maximum values, varied consider-
Max. length of leaves (cm) ably among vouchers within each species. This raises
the possibility that spore size may be related to plant
size. To evaluate this possibility, we performed a mul-
tiple regression for each species, with megaspore
I. boryana
diameter as dependent variable and leaf number and
maximum leaf length as candidate predictors. These
I. velata variables can be considered indicators of plant size
asturicense and vigour. Note that in most cases, we knew only
which voucher a megaspore had come from, not which
I. velata individual plant; thus, leaf number and maximum leaf
length were the mean values for each voucher. Some
vouchers contained very different individuals (e.g. one
230 330 430 530 630
voucher of I. boryana contained one individual with 52
Megaspore diameter (mm)
leaves, one individual with 12 leaves and several indi-
Figure 8. Box-plots summarizing the distributions of the viduals of intermediate number of leaves), so that the
three morphometric variables in the three taxa. Boxes use of the voucher mean in this analysis would be
show the 25th percentile, median and 75th percentile, with inappropriate; we therefore excluded from the analy-
the + sign indicating the mean; whiskers show the mini- sis all I. velata and I. boryana vouchers for which the
mum and maximum values. difference in leaf number between the smallest and

© 2005 The Linnean Society of London, Botanical Journal of the Linnean Society, 2005, 148, 459–464


Megaspore length (mm)




I. boryana I. astur. I. velata
Figure 9. Box-plots summarizing the distributions of megaspore diameter in the three taxa, considering each herbarium
voucher separately. Boxes show the 25th percentile, median and 75th percentile, with the + sign indicating the mean;
whiskers show the minimum and maximum values; note that the median in some cases coincides with one of the other

Table 2. Results of multiple regression with megaspore length as dependent variable and leaf number and maximum leaf
length as predictor variables


Species N R2 Intercept Leaf length Leaf number

I. boryana 7 81.0 331.8 3.309 4.017

I. velata asturicense 7 94.6 230.9 -3.552 5.943
I. velata 7 91.9 137.9 4.777 11.762

largest plant was more than ten leaves. Two other may show overlap with the ranges of the other two
vouchers (one of I. velata, one of I. boryana) were species, but the mean for the voucher or the plant
excluded because they were evident outliers from the should be discriminatory. Specifically, our findings
regression plane. None of the I. velata ssp. asturicense suggest that if mean megaspore diameter is £340 mm
vouchers was eliminated, even when the difference in where N ≥ 30, the material should be identified as
leaf number between the smallest and largest plant I. velata ssp. asturicense. In contrast, a mean
was more than ten leaves, as even when these megaspore size of ≥380 mm indicates I. velata or
vouchers were included, a very good fit was obtained I. boryana, which cannot be distinguished on the basis
(Table 2). Table 2 indicates that in all three species, of megaspore size. For identification keys, the cut-off is
megaspore size showed a close, positive relation with thus best placed at 360 mm (see below).
leaf number. In I. velata and I. boryana, megaspore The relationship between leaf number and
size also showed a close, positive relation with maxi- megaspore size indicates that individual megaspores
mum leaf length. In contrast, I. velata ssp. asturicense between 350 and 400 mm in diameter probably come
showed a negative relationship with maximum leaf either from small I. velatum or I. boryana plants with
length. fewer than 15 leaves, or from large I. velata ssp. astu-
ricense plants with 22–25 leaves. However, it should
be noted that these relationships were estimated
using mean leaf number for each voucher, with the
Differences in megaspore ornamentation and diame- exclusion of some vouchers from the analysis.
ter allow I. velata ssp. asturicense to be distinguished These morphometric differences together with other
from the other two taxa. Some individual macrospores distinguishing characteristics of I. velata ssp. asturi-

© 2005 The Linnean Society of London, Botanical Journal of the Linnean Society, 2005, 148, 459–464
464 M. I. ROMERO and C. REAL


Tetrahedral megaspores
mean length (N ≥ 30) > 360 mm, highly ornamented . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. velata
Spherical megaspores
mean length (N ≥ 30) < 360 mm, scarcely ornamented . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. asturicense

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© 2005 The Linnean Society of London, Botanical Journal of the Linnean Society, 2005, 148, 459–464