Rediscovery of the Presumed Extinct Philippine Quillwort Isoetes philippinensis Merr. & L.M. Perry (Isoetaceae) and New Insights on its Morphology and Ecology

Abstract

Isoetes philippinensis, a critically endangered site-endemic lycophyte, was rediscovered after the last collection in 1969. The present study elucidates the morphology and ecology of the species-which have not been fully investigated-by describing the detailed morpho-anatomical characters, collecting water samples for physico-chemical analysis, and providing an in situ description of its habitat. The diagnostic characteristics of the species included long microphylls (up to 63 cm long), cobwebby megaspores and microspores, and the presence of velum. The morphology of I. philippinensis exhibits the features of other aquatic, amphibious and terrestrial species of Isoetes-such as the presence of lacuna in the roots, air chambers in the leaves, reduced stele, and abundance of parenchyma cells throughout the organs. Water-quality values are within the minimum acceptable limit. Threats to the population of I. philippinensis are discussed and characterized as Critically Endangered (CR A1c, B2a). The information presented here is vital for both in situ and ex situ conservation of the species.

Full text

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Rediscovery of the Presumed Extinct Philippine Quillwort
Isoetes philippinensis Merr. & L.M. Perry (Isoetaceae)
and New Insights on its Morphology and Ecology
Victor B. Amoroso1,2, Fulgent P. Coritico1,2*, April Joie D. Lagumbay1,
Nathanie Joy S. Chatto3§, and Janece Jean Polizon-Manubag1
1Center for Biodiversity Research and Extension in Mindanao,
Central Mindanao University, Musuan, Bukidnon 8710 Philippines
2Department of Biology, College of Arts and Sciences,
Central Mindanao University, Musuan, Bukidnon 8710 Philippines
3College of Science and Mathematics, Mindanao State University–
Iligan Institute of Technology, Iligan City, Lanao del Norte 9200 Philippines
Isoetes philippinensis, a critically endangered site-endemic lycophyte, was rediscovered after
the last collection in 1969. The present study elucidates the morphology and ecology of the
species – which have not been fully investigated – by describing the detailed morpho-anatomical
characters, collecting water samples for physico-chemical analysis, and providing an in situ
description of its habitat. The diagnostic characteristics of the species included long microphylls
(up to 63 cm long), cobwebby megaspores and microspores, and the presence of velum. The
morphology of I. philippinensis exhibits the features of other aquatic, amphibious and terrestrial
species of Isoetes – such as the presence of lacuna in the roots, air chambers in the leaves, reduced
stele, and abundance of parenchyma cells throughout the organs. Water-quality values are within
the minimum acceptable limit. Threats to the population of I. philippinensis are discussed and
characterized as Critically Endangered (CR A1c, B2a). The information presented here is vital
for both in situ and ex situ conservation of the species.
Keywords: morpho-anatomy, submerged aquatic plant, type locality, water quality
*Corresponding author: cfulgent@cmu.edu.ph
§Ph.D. student
INTRODUCTION
The lycophyte family Isoetaceae consists of a single
extant genus, Isoetes L., which comprises approximately
250 living species worldwide (Troia et al. 2016; Brunton
and Troia 2018). The Isoetes species occur in a variety
of seasonally or permanently inundated habitats – such
as ponds, small ditches, streams, lakes, wetlands, and
terrestrial habitats in the tropical to subarctic biomes
(Merrill and Perry 1940; Hoot et al. 2006; Singh et al.
2021). Some of them are widely distributed, whereas
others are restricted to a few sites or just one specific site
(Alston 1959; Liu et al. 2005; Pereira et al. 2016).
Isoetes philippinensis Merr. and L.M. Perry is the only
species of this genus occurring in the Philippines; it is a
site-endemic species, recently considered to be extinct
(POWO 2022). This species is critically endangered
and is reported only from the municipality of Balo-i
in Lanao del Norte. It is a submerged aquatic plant
growing at the bottom of the Olangu River at about 1–2
m depth (Alston 1959). It is locally called as kabauing-
bauing or lunsay.
Philippine Journal of Science
151 (6A): 2093-2100, December 2022
ISSN 0031 - 7683
Date Received: 01 Apr 2022

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The first collection of I. philippinensis was made on 18
Nov 1938 by A.L. Zwickey; it was later collected by Price
on 24 Aug 1969. After 53 years of the last collection and a
search at the type locality of the species, we rediscovered
this species with Maranao local guides.
Using the combined analysis of plastid and nuclear
ribosomal data, I. philippinensis is phylogenetically
related to the Asia-Tropical species, viz. I. sinensis
(China), I. taiwanensis (Taiwan), I. hopei (Indonesia), and
I. stevensii (New Guinea) (Larsén et al. 2022).
The morphological description of I. philippinensis is
incomplete, being essentially based only on megaspore
morphology, and its anatomy has not been studied.
Moreover, because the species is critically endangered,
there is a need to study its ecology. Thus, the present
communication provides a detailed study of the
morphology, anatomy, and ecology of I. philippinensis,
and an updated description based on materials from the
type locality. This information is vital for the in situ and
ex situ conservation of the species.
METHODOLOGY
Entry Protocol and Permits
The prior informed consent and gratuitous permit to
collect I. philippinensis and explore its habitat were
secured from the head of the barangay, the municipal
mayor, and the Department of Environment and Natural
Resources (DENR) Region X Office.
Collection of Specimens, Herbarium Preparation,
and Morphology
Complete individuals of I. philippinensis were collected
and pressed as herbarium specimens, with some preserved
in 70% ethyl alcohol for anatomical examination. The
detailed quantitative and qualitative morphological
characters of the organs such as roots, corms, microphylls,
and sporangia were described from the collected
specimens. The organs were sectioned with the free-hand
technique for anatomical description and later stained
and mounted on a slide and were examined using a Swift
Optical M38022CB-3 compound binocular microscope.
The megaspores were counted by teasing the sporangium,
and the range and mean of the number of spores per
sporangium were taken. The number of microspores
per sporangium was estimated, and only the range was
taken. Furthermore, a scanning electron microscope
(SEM) (Hitachi SU3800) was used to get images of the
megaspores and microspores. Spores were described,
following the terminology of Diggs and Lipscomb (2014).
Ecology
The coordinates and elevation were recorded with Garmin
GPS. The physico-chemical properties of the water like
temperature, pH, electrical conductivity (EC), turbidity,
dissolved oxygen (DO), and total dissolved solids (TDS)
were measured in situ using a Pro DSS multi-parameter
probe during a single collection day. In the sampling
station, nine sampling points were randomly selected close
to the right and left riverbanks and in the middle of the
river with triplicates, and the mean value was computed.
The associated aquatic plants, including the surrounding
vegetation, were recorded.
RESULTS AND DISCUSSION
Gross Morphology
The plant was herbaceous, perennial, heterosporous,
aquatic, submerged ca. 1–2 m deep in the water, growing
individually, and in clumps. The basal part was buried,
usually with grass-like leaves arising annually in a tuft
from a lobed, corm-like stock. The roots were emerging
from corm, numerous, light brown, and branching at tips.
The stock was divided into corm and rhizophore, as well
as 3–4-lobed. The corm was almost globose, 7.5 cm × 5.5
cm, hard, suberized, with cavities in between, and united
at the base and apex; the mature corm had distinct and
closely packed leaf scars of the previous season – adding
hardness apart from the secondary tissues being formed,
hard at the outer part, and solid and soft toward the center.
The microphylls were 34–84 per clump, flaccid, slender,
and crowded – with the young microphylls innermost and
the older microphylls outermost with overlapping bases,
distichous, flexible, erect in still water, often with the build-
up of microalgae, elongated up to 63 cm long, lamina dark
green to light green, spathulate, tapering to the end, 4 mm
wide at the base, 3 mm at the middle, and 1 mm wide at the
apex. The ligule was flat, membranaceous with a hyaline,
ovate, and filiform margin near the base of the leaf above
the sporangium. The sporophyll bases were covered with
a translucent membrane on the adaxial surface and with
wings, with the abaxial surface depressed containing the
sporangium. All microphylls potentially sporophylls with
sporangia were seated in fovea on the abaxial surface below
the ligule. The abaxial surface of the young megasporophyll
had a distinct solitary and large sporangium containing
spore mother cells. The fovea measured 1–2 mm × 0.5–1
mm; the opening had a velum. The sporangium was solitary,
pinkish when young, light brown becoming dark brown
when mature, 4 mm × 6 mm, elongate-ovate-triangular,
tongue-like or foot-like in appearance when pushed upward,
and covered with a clear membrane; the mature sporangium
had bursting spores and plantlets (Figure 1H), sessile,
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attached distally to the fovea, without chlorophyll, and
opening transversely above middle or near the apex. The
megasporangium was oblate, thin-walled, and with 300–
360 (325) megaspores; the megaspores were trilete with a
bacillate ornamentation (dense cobwebby) having fused
ridges (Figure 5), 410–475 (442) μm, often on germination
forming young sporophytes within the sporangium (Figures
1 and 2); the microsporangium had ca. 400,000–409,600
microspores – which were monolete, elliptic, having a
bacillate ornamentation (densely cobwebby) without ridges,
and 24–26 (25) μm in diameter.
Anatomy
The transection of the mature root was ovate in outline,
with its epidermis single-layered having thickened
outer walls, followed by the cortex with 3–4 layers of
parenchyma cells; the protostele was eccentric in position
(Figure 4B) through the disintegration of inner cortical
cells on one side of stele to form a large central lacuna, as
also observed in other species of Isoetes (Bold 1973). The
corm was thickened by the formation of secondary tissues
from the secondary growth. The lower part of the lamina
was winged with four cavities in the undifferentiated
Figure 1. Habit of Isoetes philippinensis: [A] whole view showing arrangement of the microphylls; [B]
abaxial surface of the fovea with the solitary sporangium and prominent wings; [C] adaxial
surface of fovea; [D] young solitary sporangium (in arrow) with ligule above; [E] mature
solitary sporangium; [F] roots and microphylls arising from the corm; [G] branching tips of
the roots; [H] spores germinating to form plantlets (in arrow) within the sporangium.
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mesophyll. The protostele was centrally placed with
reduced xylem and phloem tissues. The middle part of
the lamina had accessory peripheral hairs arising from
the epidermis (Figure 4E), with the abaxial side flattened
without wings and the adaxial side rounded and with the
innermost laminae having four large cavities, surrounded
mainly by parenchymatous cells and centrally placed
reduced xylem and phloem tissues. The laminar surface
had a median and unbranched single vein (Figure 4).
Notes: our detailed description differed from the one
made by Merrill and Perry (1940) in some morphological
features such as much longer microphylls (63 cm
vs. 50 cm), velum present (vs. absent), ovate and flat
ligule (vs. elongate), larger megaspores up to 475 µm
(vs. 420 µm), cobwebby megaspores with ridges (vs.
smooth or sparingly and minutely rugose), and densely
cobwebby microspores (vs. very minutely scabrous)
(Figure 5). Moreover, we have added more insights on
the morphology of I. philippinensis, such as overlapping
leaf bases and unusual secondary growth of the corm with
Figure 2. [A] Enlarged abaxial surface of sporophylls of I. philippinensis with the lighter colored
spathulate fovea and large solitary sporangium (in arrow). [B] Sporophylls with the associated
sporangia; [C] Enlarged view of the young and large sporangium with the prominent wings
of the basal sporophyll; [D and E] Tongue-like megasporangium attached to the distal end
of the fovea; [F] Young megasporangium with velum (v); [G] Mature megasporangium; [H]
Closely arranged innermost and youngest yellow microphylls; [I] Microphylls arranged from
the outermost to innermost; [J] Mature sporophylls.
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distinct leaf scars and cavities (Figures 3). The leaves
are microphyllous because they are traversed by a single
vein despite being long. Similar to the other species of
Isoetes such as I. iapygia, I. todaroana (Ernandes and
Marchiori 2012), I. nana (Pereira et al. 2017), I. butleri,
I. lithophila, and I. melanopoda (Diggs and Lipscomb
2014), the velum is present. The anatomy of the fully
submerged I. philippinensis showed features typical of
aquatic plants – such as the presence of lacunae in the
root, air cavities in microphylls, a reduced stele, and the
mesophyll mainly composed of parenchyma cells. The
xylem contains many parenchymatous cells with few
tracheids. The sporangium is larger than those of other
lycophytes. The ligule is not directly connected to the
sporangium, unlike the other species of Isoetes. The spores
exhibited unusual germination by forming plantlets while
within the sporangium (Figure 1H).
According to the interviewed locals in the area, the
microphylls are consumed as green vegetables. Another
species of Isoetes (I. sinensis) has a strong antioxidant
activity and flavonoid content, implying that the species is
a potential edible and medicinal plant (Wang et al. 2020).
Figure 3. Mature corm with distinct leaf scars
and cavities.
Figure 4. Morphology and anatomy of I. philippinensis: [A] habit; [B] transections
of the root; [C] thickened corm and soft inner tissues; [D] lower part of the
microphyll; [E] middle part of the microphyll; and [F] peripheral hairs (h)
arising from the epidermis.
Figure 5. SEM micrographs of spore of I. philippinensis: [A]
equatorial view of trilete megaspore with bacillate
ornamentation (fine cobwebby surface) and fused ridges;
[B] close-up of equatorial view of the megaspore; [C]
proximal view of the monolete microspore with bacillate
ornamentation (dense cobwebby surface) and without
ridges; [D] close-up of proximal view of the microspore.
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Figure 6. Type locality of I. philippinensis with associated vegetation at the headwaters of the Olangu River,
Barangay Angayen, Baloi, Lanao del Norte, Philippines.
Table 1. Mean water physico-chemical parameter values of
the Olangu River, Baloi Lanao del Norte, where I.
philippinensis grows.
Parameter Mean value
Temperature, °C 24.00
pH level 6.88
Oxidation-reduction potential, mV 96.17
Specic conductance, μS/cm 0.242
Turbidity (NTU), mg/L 2.00
DO, mg/L 5.59
TDS, mg/L 157.66
Depth, m 1–2
Specimens examined: Philippines, Mindanao, Lanao
Province near Momungan, vicinity of Olangu, 400–500 masl,
18 November 1938, A.L. Zwickey 776, GH (GH00062344-
image! Holotype) MICH (MICH1191336-image! Isotype).
M.G. Price 750 PNH (PNH113530-Topotype).
Additional specimens examined: Philippines, Mindanao,
Lanao del Norte, Municipality of Baloi, Barangay
Angayen, Purok Olangu, Olangu river, 387 masl, 25
May 2022, VB Amoroso et al. Collection No. 31069
(CMUH13447!).
Location, Ecology, and Threats
This species is currently known only from the type locality
in the Olangu River, Purok Olangu, Barangay Angayen,
Municipality of Balo-i, Lanao del Norte, where it grows
fully submerged down to 2 m deep in silty loam to sandy
loam soil. It grows singly, in clumps, or together with other
floating and submerged aquatic macrophytes – such as the
invasive species Ceratophyllum demersum, Eichhornia
crassipes, Hydrilla verticillata, Ottelia alismoides, and
Pistia stratiotes. The emerging vegetation surrounding
I. philippinensis included Pandanus sp., Acalypha
amentacea, Xanthosoma sagittifolium, Sphaerostephanos
unitus, Diplazium esculentum, Christella dentata, Arenga
pinnata, Caryota cumingii, and Cocos nucifera and other
fruit trees (Figure 6).
Quillworts thrive in fragile habitats, are submerged in
slow-moving fresh water throughout their life cycle,
and are known to be highly threatened – and can be a
key indicator species of aquatic ecosystem health (Abeli
et al. 2018). The clean and clear water and its physico-
chemical properties where I. philippinensis was growing
support this statement (Table 1). The temperature, pH,
NTU (nephelometric turbidity units), and DO values are
within the minimum acceptable limits of the standards
for class AA, A, and B rivers (DENR-DAO 1990). With
ca. 100 individuals of I. philippinensis, the population
can be expected to decline because of threats. In the
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Figure 7. Submerged plants of I. philippinensis with associated invasive species (Ceratophyllum demersum) (in
arrows).
same river and contiguous to the natural habitat of I.
philippinensis where water is polluted and disturbed by
mobile and parked pump boats, no individuals of Isoetes
were observed. Moreover, macrophytes were observed
competing with I. philippinensis, appearing to affect its
growth and survival (Figure 7). The presence of Tilapia
spp. further destroys the healthy growing I. philippinensis
by eating the tips of the microphylls.
Environmental disturbances such as eutrophication, water
pollution, habitat loss, and the presence of invasive aquatic
macrophytes appear to have contributed to population
decline. The presence of these threats indicates that the
species is facing serious risks of extinction. Currently, I.
philippinensis is listed as a critically endangered species
(Ebihara et al. 2012; DENR-DAO 2017). Thus, we support
listing the species as Critically Endangered (CR B2a, b)
based on its unique highly restricted population, the area
of occupancy estimated to be < 10 km2, and with ca. 100
mature individuals (IUCN 2019).
CONCLUSION AND
RECOMMENDATIONS
The morphology of I. philippinensis exhibits features of
other species of Isoetes such as the presence of lacuna
and air cavities and a reduced stele composed mainly of
parenchyma cells. The physico-chemical values of the
water are within the minimum range acceptable limit for
not-polluted water. The morphological and ecological
data are useful information for the in situ and ex situ
conservation of the critically endangered I. philippinensis.
It is, therefore, important to protect the natural habitat of
the species by formulating and strictly implementing the
policies and ordinances to save this site-endemic species,
as well as educate the local stakeholders to avoid the
extinction of this species.
ACKNOWLEDGEMENT
We thank the DENR Region X Office for the issuance of the
Gratuitous Permit No. 2022-38; local government officials
of Barangay Angayen, Balo-i, Lanao del Norte for the
prior informed consent and the Maranaos for guiding us to
the site; the National Research Council of the Philippines,
the Philippine Council for Agriculture, Aquatic, and
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Natural Resources Research and Development, and the
United States (US) National Science Foundation (Grant
No. DEB-1754697) for the funding; the Central Mindanao
University for logistical support; Dr. Peter W. Fritsch and
Marsha Stripe of the Botanical Research Institute of Texas
in the US for suggestions on the manuscript and SEM
images of the spores, respectively.
REFERENCES
ABELI T, CAUZZI P, ROSSI G, PISTOJA F, MUCC-
IARELLI M. 2018. A gleam of hope for the critically
endangered Isoetes malinverniana: use of small scale
translocations to guide conservation planning. Aquat
Conserv 28: 501–505.
ALSTON AHG. 1959. Isoetaceae. Flora Malesiana, Vol.
I, Series II.
BOLD H. 1973. Morphology of Plants, Third Edition.
Harper @ Row, Publishers, Incorporated.
BRUNTON DF, TROIA A. 2018. Global review of recent
taxonomic research into Isoetes (Isoetaceae), with
implications for biogeography and conservation. Fern
Gaz 20: 309–333.
[DENR-DAO] Department of Environment and Natural
Resources–Department Administrative Order. 1990.
Revised effluent regulations of 1990. Revising and
amending the effluent. Regulations of 1982. 17p.
[DENR-DAO] Department of Environment and Natural
Resources–Department Administrative Order. 2017.
Updates National List of Threatened Philippine Plants
and their Categories. 30p.
DIGGS GM, LIPSCOMB BL. 2014. The ferns and ly-
cophytes of Texas. first edition. Botanical Research
Institute of Texas. p. 56–62.
EBIHARA A, FRASER-JENKINS CR, PARRIS BS,
ZHANG XC, HONG YY, CHIOU WL, CHANG HM,
LINDSAY S, MIDDLETON D, KATO M, PRAPTO-
SUWIRYO TN, AMOROSO VB, BARCELONA JF,
RAJAPAKSHA RHG, PARK CH, MURAKAMI N,
HOYA A. 2012. Rare and Threatened Pteridophytes
of Asia 1: an Enumeration of Narrowly Distributed
Taxa. Bulletin of the National Museum of Nature and
Science, Series B 38(3): 93–119.
ERNANDES P, MARCHIORI S. 2012. A comparative
study of two endemic Isoetes species from South Italy.
International Scholarly Research Network (ISRN) Bot-
any [Article ID 127250]. 7p. doi: 10.5402/2012/127250
HOOT SB, TAYLOR WC, NAPIER NS. 2006. Phylogeny
and biogeography of Isoetes (Isoetaceae) based on nu-
clear and chloroplast DNA sequence data. Systematic
Botany 31(3): 449–460.
LARSÉN E, WIKSTRÖM N, KHODABANDEH A,
RYDIN C. 2022. Phylogeny of Merlin’s grass (Isoeta-
ceae): revealing an “Amborella syndrome” and the im-
portance of geographic distribution for understanding
current and historical diversity. BMC Ecol Evo 22: 32.
https://doi.org/10.1186/s12862-022-01988-w
LIU X, WANG J-Y, WANG Q-F. 2005. Current status
and conservation strategies for Isoetes in China: a case
study for the conservation of threatened aquatic plants.
Oryx 39: 335–338.
MERRILL ED, PERRY LM. 1940. A New Philippine
Isoetes. American Fern Journal 30(1): 18–20.
PEREIRA JBS, SALINO A, ARRUDA A, STUTZEL T.
2016. Two new species of Isoetes (Isoetaceae) from
northern Brazil. Phytotaxa 272: 141–148.
PEREIRA JBS, STUTZEL T, SCHULZ C. 2017. Isoetes
nana, a new species from the coastal mountains of
southeastern Brazil. PhytoKeys 89: 91–105.
SINGH SK, SHUKLA PK, BRUNTON DF, DUBEY N,
SHUKLA SK. 2021. The taxonomy and conservation
status of Isoetes (Isoetaceae; Lycopodiopsida) in India.
Botany Letters 168(2): 200–226.
[POWO] Plants of the World Online. 2022. Plants of the
World Online. Royal Botanic Gardens, Kew, United
Kingdom. Retrieved on 25 Jul 2022 from http://www.
plantsoftheworldonline.org/
[IUCN] International Union for Conservation of Nature–
Standards and Petitions Committee. 2019. Guidelines
for Using the IUCN Red List Categories and Criteria,
Version 14. Retrieved from http://www.iucnredlist.org/
documents/RedListGuidelines.pdf
TROIA A, PEREIRA JB, KIM C, TAYLOR WC. 2016.
The genus Isoetes (Isoetaceae): a provisional checklist
of the accepted and unresolved taxa. Phytotaxa 277:
101–145.
WANG X, DING G, LIU B, WANG Q. 2020. Flavonoids
and antioxidant activity of rare and endangered fern:
Isoetes sinensis. PLoS One 15(5): e0232185. doi:
10.1371/journal.pone.0232185
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