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Hypnea musciformis (Cystocloniaceae) from the Yucatan Peninsula: morphological
variability in relation to life-cycle phase
ERIKA VA
´ZQUEZ-DELF´
IN
1
,GAHUN BOO
2
,DEN´
IRODR´
IGUEZ
3
,SUNG MIN BOO
2
AND DANIEL ROBLEDO
1
*
1
Laboratory of Applied Phycology, Department of Marine Resources, CINVESTAV Unidad Merida, Mexico
2
Laboratory of Algal Biodiversity and Evolution, Department of Biology, Chungnam National University, Korea
3
Laboratory of Phycology, Faculty of Sciences, UNAM, Mexico
ABSTRACT:Hypnea musciformis is one of the most studied species of the genus Hypnea. It has a wide geographic range
along the Atlantic, Pacific and Indian Oceans that would be consistent with an extensive intraspecific variation in
morphology. Hypnea musciformis has been previously reported on coasts of the Yucatan Peninsula; however, neither
morphological characterization nor molecular studies have been done for Gulf of Mexico or Mexican Caribbean
populations. For the first time, we present a detailed morphoanatomical description of the tetrasporophytic,
gametophytic and carposporophytic phases and its reproductive structures for this region, including the description of
the male gametophyte. Mexican Caribbean samples showed morphological variation in branching pattern, branch
abundance and branchlet types, and this was attributed to life-history phase and the reproductive status of the thalli.
Molecular analysis of ribulose-1,5-bisphosphate carboxylase/oxygenase gene and the 50region of the mitochondrial
cytochrome C oxidase subunit I gene markers allows us to confirm the unique genetic identity of Mexican specimens. The
analysis of H. musciformis collected in different tropical areas showed a high degree of intraspecific variation and
suggested the necessity of a combined analysis of morphology and different gene markers for a better understanding of its
taxonomy and phylogeny.
KEY WORDS: Alternating phases, COI-5P, Life cycle, Molecular markers, Morphological diversity, Quintana Roo coast,
rbcL
INTRODUCTION
Hypnea musciformis (Wulfen) J.V.Lamouroux is an eco-
nomically important species because of its content of kappa
carrageenan used in industry (Reis et al. 2006). It was
originally described for the Mediterranean Sea and is
widely distributed along coasts of the Atlantic Ocean
(Taylor 1960; Mshigeni 1978; Mshigeni & Chapman 1994;
Va
´zquez-Delf´
ınet al. 2014). Some reports indicate its
presence in the Indian Ocean (Silva et al. 1996) and in the
Pacific Ocean as a successful introduced species (Smith et
al. 2002). Taxonomic studies with H. musciformis indicate
extensive morphological variation, probably related to its
wide distribution (Table S1). The main morphological
diagnostic character for species identification is the
presence of a hook in the apex, which is reported as
swollen, flattened or thickened (Taylor 1960; Schneider &
Searles 1991; Abbott 1999; Littler & Littler 2000; Mag-
alhaes 2006; Cabioc’h et al. 2007; Littler et al. 2008; Dawes
& Mathieson 2008; Guimar˜
aes 2011; Jesus et al. 2014).
However, it has been reported that identification of the
species only on a morphological basis is not reliable because
of the existence of a H. musciformis worldwide complex of
related species (Nauer et al. 2015).
In general, species delimitation in the genus Hypnea is
complicated because of overlapping characters and the
difficulty of distinguishing between intraspecific and inter-
specific morphological variation (Mshigeni & Chapman
1994). Molecular studies have facilitated the clarification of
some taxonomic aspects. The principal molecular markers
used for this purpose are the mitochondrial gene cox1 that
encodes for cytochrome oxidase 1, the 50region of the gene
cox1 that encodes cytochrome c oxidase subunit 1(COI-5P),
the plastid gene rbcL that encodes for the large subunit of
ribulose-1,5-bisphosphate carboxylase/oxygenase, and the
psaA gene that encodes the reaction center proteins of
photosystem I. Taxonomic studies of Hypnea that include
some of these molecular markers have been carried out for
Asian and Brazilian coasts (Geraldino et al. 2006, 2009,
2010; Guimar˜
aes 2011; Nauer et al. 2014, 2015), reporting
interspecific divergence values of 11–13%for COI-5P and
1.3–6.8%for rbcL for Asian species, and of 10.1–16.3%for
COI-5P and 3.2–6.7%for rbcL for Brazilian species (Nauer
et al. 2014).
On Brazilian coasts, molecular analysis resolved taxonomic
confusions between species identified morphologically as
Hypnea valentiae (Turner) Montagne,H.nigrescensGreville
ex J.Agardh and H. musciformis (Wulfen) J.V.Lamouroux
(Schenkman 1986; Ortega et al. 2001). Guimar˜
aes (2011)
pointed out that H. nigrescens was a morphological variant of
H. musciformis for Brazil on the basis of molecular markers
(rbcL, COI-5Pand universal plastid amplicon ). More recently,
Nauer et al. (2015) showed that specimens previously identified
as H. valentiae,H. nigresens and H. muscifomis from Brazil
corresponded to the same species, a new species named H.
pseudomusciformis Nauer, Cassano & M.C.Oliveira, with
intraspecific divergence values of 0–1.8%for COI-5P and 0–
0.7%for rbcL. Molecular analysis showed that this species is
closely related to H. musciformis from other parts of the world
(Barbados, Italy and USA), where interspecific divergence
* Corresponding author (daniel.robledo@cinvestav.mx).
DOI: 10.2216/15-118.1
Ó2016 International Phycological Society
Phycologia Volume 55 (2), 230–242 Published 8 March 2016
230
values of 3.9–7.1%for COI-5P and 1.7–2.7%for rbcLwere
reported (Nauer et al. 2015). Morphological analysis does not
distinguish between H. pseudomusciformis and H. musciformis,
thus emphasizing the importance of molecular characters in
taxonomic studies (De Clerck et al. 2013; Nauer et al. 2015).
Molecular and morphological evaluation of H. muscifor-
mis is needed from throughout its distribution to discard the
possible presence of cryptic species, and to re-evaluate its
real geographic distribution. It has important implications
for other disciplines, such as the under-/overestimation of
species diversity in ecology (Leliaert et al. 2014), and for the
correct use of a particular species in applied phycology.
Hypnea musciformis was reported previously from Mexico
from the Gulf of Mexico and the Caribbean Sea (Ortega et
al. 2001); however, there are no morphological or molecular
studies for the species in this region. The objective of the
present study was to provide a detailed description of H.
musciformis from the Mexican Caribbean Sea, on the basis
of morphological and molecular data, and to characterize
the intraspecific morphological variation in that region.
Comparisons with the descriptions of specimens from other
localities in the Gulf of Mexico and from the western
Atlantic Ocean and Mediterranean Sea are included.
MATERIAL AND METHODS
Specimens of H. musciformis were obtained from several
localities in Mexico (Fig. 1). Fresh specimens were collected
between February 2012 and December 2013 as part of a
seasonal biomass study from the Yucatan Peninsula, at
Quintana Roo at Playa del Carmen (20839034.2400 N,
87802008.9700 W) and Isla Holbox (21831041.21 00 N,
87822056.87 00 W). These samples were transported live,
cleaned and fixed in seawater formalin (4%) for morphoa-
natomical examination. All representative voucher speci-
mens from the Yucatan Peninsula were deposited in the
National Herbarium (MEXU) at Instituto de Biolog´
ıa (IB),
Universidad Nacional Aut´
onoma de M´
exico (UNAM), as
MEXU 2026, MEXU 2221 and MEXU 2222. In addition,
we examined a total of 43 specimens belonging to the
National Herbarium IB (MEXU) and from the Facultad de
Ciencias, UNAM. Both herbarium and liquid-preserved
specimens were from various regions within the Gulf of
Mexico: Veracruz (29 specimens) and Campeche (11
specimens), and from the Mexican Caribbean Sea: Quintana
Roo (3 specimens).
Approximately 1 g per sample was preserved in silica gel
desiccant for DNA extraction. Additionally, fresh specimens
of H. musciformis were also collected from the Mediterra-
nean Sea (Ma
´laga, Spain), close to the locality from which
the type specimen was reported, to compare amongst
specimens from both Atlantic coasts.
Morphological analysis was done on the basis of the
relevant taxonomic characters defined for the genus Hypnea
and reported by Mshigeni (1978) and Masuda et al. (1997):
(1) plant habit, basal system and plant size; (2) distinctive-
ness of main axes, and their shape and diameter; (3) lateral
Fig. 1. Map of Mexico showing the localities studied in the Caribbean Sea and Gulf of Mexico coasts. Quintana Roo: Playa del Carmen,
Puerto Morelos, Holbox; Campeche: Laguna de T´
erminos; Veracruz: Boca del R´
ıo, Actopan. See Table 2 for details on specimens examined.
Va
´zquez-Delf´
ınet al.: Hypnea musciformis from the Yucatan Peninsula 231
branches, branchlet abundance and morphology; (4) branch-
ing pattern; (5) apex shape; (6) position, number and size of
axial and periaxial cells, and size of medullary, subcortical
and cortical cells; and (7) reproductive structures (position of
tetrasporangial sorus/cystocarps, cystocarps size, tetraspore/
carpospore size). For those specimens collected in the field,
the growth habit was also noted at the time of collection. For
quantitative anatomical characters, comparisons between
life-cycle phases were examined with a Mann–Whitney Utest
using SPSS Statistics (21v).
Observations and measurements were made on fresh
specimens and liquid-preserved samples. Histological sec-
tions were cut by hand with a razor blade. Identification and
description of morphoanatomical characters were made
using a stereomicroscope (Stemi SV 6, Zeiss) and an optical
microscope (PrimoStar, Zeiss). Microphotographs were
obtained with a digital camera coupled to both microscopes
(AxioCam MRc5, Zeiss). Morphological analysis and
measurements of anatomical characters were made using
the Axio Vision software (4.2v).
Information on specimens analyzed in the present study
is in Table 1. DNA extraction, polymerase chain reaction
amplification, and sequencing were performed as described
in Geraldino et al. (2006). The phylogeny of rbcL sequences
was reconstructed using maximum likelihood (ML), and
included previously published data in GenBank (Geraldino
et al. 2009, 2010; Nauer et al. 2014, 2015). Calliblepharis
ciliata (Hudson) K ¨
utzing and Rhodophyllis reptans (Suhr)
Papenfuss were used as outgroups for rbcL, and C. jubata
(Goodenough & Woodward) K¨
utzing was used instead of
Table 1. Specimens used in the study. Dash mark indicates no sequence analyzed.
Species/Sample
GenBank accession number
rbcL COI
Hypnea asiatica P.J.L.Geraldino, E.C.Yang & S.M.Boo EU240837 EU240798
H. boergesenii T.Tanaka AF385634 —
H. caespitosa P.J.L.Geraldino & S.M.Boo FJ694936 FJ694901
H. charoides J.V.Lamouroux EU240845 EU240819
H. chordacea K¨
utzing AB033160 —
H. cornuta (K¨
utz.) J.Agardh AB095911 GQ141878
H. flagelliformis Grev. ex J.Agardh AB033162 —
H. flexicaulis Yamagishi & Masuda AB033163 EF136593
H. japonica T.Tanaka AB033164 EU345986
H. musciformis (Wulfen) J.V.Lamour.
AM1, Mexico
1
KT428780 KT428772
AM2, Mexico
1
KT428781 KT428773
IH0512, Isla Holbox, Quintana Roo, Mexico
1
— KT428774
NM1, Mexico
1
KT428782 KT428775
PC0812, Playa del Carmen, Quintana Roo, Mexico
1
KT428783 KT428776
PC1112, Playa del Carmen, Quintana Roo, Mexico
1
KT428784 KT428777
O51, Playa del Carmen, Quintana Roo, Mexico
1
KT428785 KT428778
O52, Playa del Carmen, Quintana Roo, Mexico
1
KT428786 —
MA0912, Malaga, Spain
1
KT428787 KT428779
North Carolina, USA U04179 —
Villefranche, France EU346011 GQ141881
Cannes, France EU346012 —
Antibes, France EU346013 —
Maui Island, Hawaii — HQ422612
Ohau Island, Hawaii — HQ422630
Kaui Island, Hawaii — HQ422646
Ohau Island, Hawaii — HQ422876
Needham’s Point, Barbados, Nauer et al. 2015 KM509063 KM523203
Ancona, Italy, Nauer et al. 2015 KM509072 KM523207
Ancona, Italy, Nauer et al. 2015 KM509073 —
H. nidifica J.Agardh FJ694932 GQ141879
H. nidulans Setchell FJ694946 FJ694900
H. pannosa J.Agardh FJ694958 FJ694892
H. pseudomusciformis Nauer, Cassano & M.C.Oliveira KM509065 KM523183
RG0612, Rio Grande do Norte, Brazil
1
KT428788 —
H. rosea Papenfuss FJ694935 GQ141883
H. spinella (C.Agardh) K¨
utz. AB033166 EU240818
H. stellulifera (J.Agardh) Yamagishi & Masuda AB095913 EU345984
H. tenuis Kylin FJ694934 GQ141880
H. valentiae (Turner) Mont. FJ694933 GQ141882
H. viridis Papenf. FJ694930 FJ694908
H. yamadae T.Tanaka AB095916 —
Hypnea sp. AB033167 —
Outgroups
Calliblepharis ciliata (Hudson) K¨
utzing KC121120 DQ960360
C. jubata (Goodenough & Woodward) K¨
utzing — DQ442895
Rhodophyllis reptans (Suhr) Papenfuss AF385660 —
1
Sequenced in this study.
232 Phycologia, Vol. 55 (2)
R. reptans in the COI-5P analysis. Modeltest v.3.7 (Posada
& Crandall 1998) determined GTR þGþI model of
evolution as the best fit for rbcL and COI-5P data set. ML
analyses were performed with RAxML v.7.2.8 (Stamatakis
2006) using the GTR þGþI model. We used 300
independent tree inferences. To identify the best tree, we
applied a rearrangement of automatically optimized subtree
pruning regrafting and 25 distinct rate categories in the
program. Statistical support for each branch was obtained
from 1000 bootstrap replications with the same substitution
model.
Bayesian inference was performed for individual data sets
with MrBayes v.3.2.1 (Ronquist et al. 2012) using the
metropolis-coupled Markov chain Monte Carlo with the
GTR þGþI model. For each matrix, 2 million generations
of two independent runs were performed with four chains
and sampling trees every 100 generations. The burn-in period
was identified graphically by tracking the likelihoods at each
generation to determine whether they reached a plateau. The
29,202 trees sampled at the stationary state were used to infer
Bayesian posterior probabilities (BPP).
We generated 12 rbcL sequences. A total of 41 sequences
from Hypnea, including Calliblepharis ciliata and Rodophyllis
reptans as outgroups, was aligned using 1355 base pairs (bp)
of the rbcL gene. A total of 14 COI-5P sequences (465 bp)
generated in the present study was aligned with 26 published
sequences, including two sequences of Calliblepharis as
outgroups.
RESULTS
Morphological analysis and habitat
Morphological observations of Hypnea musciformis from
different regions revealed some general characters that were
always present and were considered stable, whereas others
were not common and were variable between samples (Table
2). For Quintana Roo samples, extensive morphological
variation in several characters occurred, and this was mainly
associated with reproductive status and life-history phase
(Figs 2–9). Three different morphologies occurred and these
corresponded to the different phases of the H. musciformis life
cycle (Figs 10–20). In Table 3 we describe each life-history
phase indicating the main differences, with a detailed
description below.
The different life-history phases of Hypnea musciformis
collected from the Yucatan Peninsula were found in the
intertidal zone. They were attached to different natural (rock
and sand) or artificial substrata (ropes and shore-parallel
geotextile tubes, called geotubes, installed for erosion control).
In general, its distribution pattern was patchy when growing
over sandy or rocky substrates, whereas they formed a
homogeneous turf when growing over the geotubes.
Tetrasporophyte
Thalli grew as erect tufts up to 9 cm high. The main axes were
cylindrical, filiform, very long and thinned toward the apex.
These axes were percurrent, slightly contorted and anasto-
mosed at basal parts. The main axis had rare subdichotomous
branching and exhibited abundant lateral branches (long and
cylindrical) of first and second order, emerging at acute angles;
these were irregularly arranged in different planes around the
axis. The apices were acute and attenuated, occasionally
recurved or with a distal flattened hook with branchlets in the
outer curve. The main axis and branches of all types remained
covered with numerous simple spiny branchlets. The holdfast
was a filiform branched hapteron with basal or lateral discs
like suction pads; these disks were composed of clusters of
elongated cortical cells as rhizoidal filaments. This morph
coincided with the traditional description of Hypnea musci-
formis based on the flattened hooked tips and the numerous
upwardly curved spur-like branchlets (Figs 2–4).
Axial cells were 48–80 lm in diameter and surrounded by
five to seven larger periaxial cells (38–115 lm). Medullary cells
were round or irregular from 60 to 188 lm in diameter. Round
subcortical cells ranged from 24 to 85 lm in diameter and
cortical cells were from 8 to 11 lm long and in one or two
layers.
The nemathecial sori covered totally (ring) or partially
(lateral) the basal or middle portions of the spiniform
branchlets; sometimes they also occurred on lateral second-
order branches. In a transverse section, tetrasporangia were at
different stages of development, organized anticlinally in the
cortex and 36–63 lm long (Figs 10–12).
Female gametophyte including the carposporophyte
Thalli grew as erect tufts up to 7 cm high. The main axes were
cylindrical to complanate and thick, percurrent, slightly
contorted and anastomosed in basal parts. The main axis
had abundant subdichotomous branching, and scarce lateral
branches of first and second order emerged at acute angles;
these were irregularly placed in different planes around the
axis. The apices were acute and recurved or bifurcated,
sometimes highly divided and star shaped. The main axis and
branches of upper orders were covered with numerous
spiniform branchlets at middle and apical portions with
anastomoses between them. These branchlets were slightly
complanate, short, and simply branched with shorter spines.
The holdfasts were similar to those described for the
tetrasporophytes (Figs 5–7).
Axial cells were 21–74 lm in diameter and surrounded by
five to seven larger periaxial cells (18–150 lm). Medullary cells
were round or irregular and 58–177 lminmaximum
dimension. Subcortical cells were round and 13–67 lmin
diameter and cortical cells were 8–11 lm long forming one or
two layers.
The carposporophytes developed within globose and sessile
cystocarps located anticlinally on basal or middle portions of
the female gametophyte branchlets. The cystocarps were
solitary or in groups of two, three or up to four in the
branchlets, ranging from 203 to 903 lm in diameter. An
ostiolar region in the distal end of the cystocarp was observed
(Fig. 16). The pericarp consisted of one or two layers of
photosynthetic cortical cells in anticlinal position and one or
two inner layers of elongated cells in periclinal position. Some
gonimoblastic sterile filaments were joined to the pericarp,
which could have a nutritional function. In longitudinal
section, basal gonimoblast was observed with a placenta
having sterile filaments elongated toward the center of the
Va
´zquez-Delf´
ınet al.: Hypnea musciformis from the Yucatan Peninsula 233
cystocarp. The carposporangia were observed in clusters
developed from the central filaments in the cystocarp that gave
rise to spherical carpospores from 16 to 29 lm in diameter
(Figs 17–20).
Male gametophyte
Thalli grew as erect tufts up to 4 cm high. The main axes were
cylindrical, short and thick, percurrent, slightly contorted and
Table 2. Morphological comparison of Hypnea musciformis from the Mexican Gulf, Mexican Caribbean Sea and Mediterranean Sea and H.
pseudomusciformis from Southwest Atlantic coast.
Region Habit/plant size (cm) Main axes Holdfast Branching
CS (MX1) large tufts (16.5–18) terete, percurrent,
slender in apical
sections
rhizoids at basal parts
and discoid holdfast
dichotomous, acute
angles (73–758)
MG (MX2) large tufts, entangled. terete, percurrent, 1–
1.2 mm diameter
not observed dichotomous, at
angles of 63–87.78
MG (MX3) tufts (3–20) terete, mainly
percurrent,
sometimes not
percurrent; slender
to thick axes
(737.2–1182 lm)
rhizoidal and discoid
holdfast
irregular or
dichotomous at
angles of 1348;
abundant
MG (MX
4
) mainly small tufts
(3.5–6) but
sometimes large
(12–18)
terete, percurrent and
not percurrent;
slender axes
not observed. dichotomous, wide
angles (71.1–109.38)
MS (SP) tufts. terete, percurrent axes. not observed.
AO (BZ) large entangled tufts
(15).
terete, not percurrent. not observed. abundant,
dichotomous
CS (MX1) ¼Caribbean Sea (Playa Carmen, Quintana Roo, Mexico); MG (MX2) ¼Mexican Gulf (Holbox Island, Quintana Roo, Mexico);
MG (MX3) ¼Mexican Gulf (Veracruz, Mexico); MG (MX4) ¼Mexican Gulf (Campeche, Mexico); AO (BZ) ¼Atlantic Ocean (Rio Grande
do Norte, Brazil); MS (SP) ¼Mediterranean Sea (Malaga, Spain); NA ¼not available; it was not possible to include anatomical characters for
herbarium specimens because of the degradation of the tissue. Reference material: Quintana Roo: Playa del Carmen PC0212
1
, PC0812
1
,
PC1112
1
; Puerto Morelos MEXU2037, MEXU2038, MEXU2039; Isla Holbox IH0512
1
; Campeche: Laguna de T´
erminos MEXU1081,
MEXU1082, MEXU1083, MEXU1084, MEXU1085, MEXU1086, MEXU1142, MEXU1145, MEXU1146, MEXU1149, MEXU1337;
Veracruz: Boca del R´
ıo MEXU1465, MEXU1668; Actopan MEXU1094, MEXU1402, MEXU1431, MEXU1468, MEXU1469, MEXU1662,
MEXU1663, MEXU1664, MEXU1665, MEXU1666, MEXU1679, MEXU1670, MEXU1673, MEXU1691, MEXU1963, GM300, GM348,
GM363, GM365, GM366, GM370, GM389, GM492, GM503, GM554, GM560, GM604.
1
Specimens with rbcL and COI-5P molecular confirmation.
234 Phycologia, Vol. 55 (2)
anastomosed in basal parts. The main axis exhibited very
abundant subdichotomous branching and scarce lateral
branches of first and second order emerging at acute angles;
these were irregularly disposed in different planes around the
axis. The apices were acute and recurved, or sometimes
bifurcate. The main axis and lateral branches were covered
with numerous simple, short and thin spiniform branchlets at
middle and apical portions, and occasionally with spines on
them. The holdfasts were similar to those described for the
tetrasporophytes (Figs 8, 9).
Axial cells were 34–86 lm in diameter surrounded by five to
seven larger periaxial cells (18–113 lm). Round or irregular
medullary cells ranged from 56 to 109 lm in diameter. Round
subcortical cells ranged from 13 to 66 lm in diameter. Cortical
cells ranged from 7 to 10 lm in length and developed in one or
two layers.
The spermatangia were like nemathecial elevations that
developed over the cortex in the main axis, branches and
branchlets. A gelatinous matrix covered reproductive portions
of the thallus. In transversal section, the spermatangial stem
cell (4–8 lm in diameter) was observed giving rise to two or
three spermatangia, which produced spermatia dividing
transversely and forming linear chains. The spermatia ranged
from 3 to 4 lm in diameter (Figs 13–15).
Table 2. Extended
Type of branchlets Apices
Transversal section
(diameter in lm)
Reproductive
structures (diameter in
lm)
Species/reference
material
large filiform, very
abundant covering
the entire thalli; in
right angle respect
the main axis
flattened hooks with
spine-like and
filiform branchlets
in outer curve; some
pointed apices
axial cell (55.5)
surrounded by four
to five periaxials
(79.9–112.6); two
layers of pigmented
cortical cells
cystocarp (549) with
carpospores of
22.1–34;
tetrasporangial
sorus girdling basal
part of branchlets
H. musciformis
collected in this
study and from
herbarium (MEXU
and GM)
1
large filiform and
short spine-like
branchlets covering
the entire thalli
flattened hooks with
spine-like and
filiform branchlets
in outer curve
axial cell (76.3)
surrounded by four
to five periaxials
(139–269); two
layers of pigmented
cortical cells
abundant branchlets
with nemathecial
sorus girdling its
middle parts; ovoid
tetrasporangia,
zonately divided,
40.6–47.2 long and
17.3–20.2 wide
H. musciformis
collected in this
study
1
mainly filiform,
curved adaxial,
sometimes spine like
at right angle
respect main axis;
abundant to sparse
flattened or simple
hooks with spine-
like and filiform
branchlets in outer
curve; sometimes
with pointed apices
small axial cell (50.6
lm) surrounded by
five to seven large
periaxials (82.2–
145); one or two
layers of pigmented
cortical cells
tetrasporangial sorus
girdling basal or
middle part of
branchlets; ovoid
tetrasporangia,
zonately divided,
42.7–47 long and
20.6–23.3 wide;
spherical cystocarps
(664–681), solitary
or in groups;
carpospores of
23.9–35.1
H. musciformis from
herbarium (MEXU
and GM)
mainly filiform at
right angles respect
the main axis;
spine-like branchlets
sparse
mainly pointed apices
sometimes divided;
some samples with
flattened or simple
hooks with spine-
like and filiform
branchlets in outer
curve
NA not observed. H. musciformis from
herbarium (MEXU
and GM)
very abundant filiform
branchlets; curved
adaxial respect the
axes; spine-like
branchlets covering
second-order
branchlets
flattened hooks with
spine-like and
filiform branchlets
in outer curve; some
others pointed and
divided
axial cell (49–83)
surrounded by fve
to six larger
periaxials (153–205).
Medullary cells
ranging from 46 to
205. One layer of
pigmented cortical
cells (8–14).
not observed H. musciformis
collected in this
study
1
very abundant filiform
and spinelike
branchlets covering
the entire thallus;
curved adaxial
respect the axes
flattened hooks; some
other pointed apices
NA not observed H. pseudomusciformis
collected in this
study
Va
´zquez-Delf´
ınet al.: Hypnea musciformis from the Yucatan Peninsula 235
Molecular phylogeny
The rbcL tree revealed the monophyly of Hypnea musciformis
(99%for ML, 1.0 for BPP); however, H. musciformis consisted
of two distinct groups (Fig. 21). All the Mexican specimens
had identical sequences and formed a monophyletic group
with a specimen from Barbados (96%for ML, 1.0 for BPP).
The second group included Italy, Spain, France and North
Figs 2–9. Hypnea musciformis external morphology of the life phases. Scale bar ¼1 cm.
Figs 2–4. Tetrasporophyte with nemathecial sorus.
Figs 5–7. Female gametophyte bearing cystocarps.
Figs 8, 9. Male gametophyte with spermatangia covering the surface.
Table 3. Comparisons of morphological characters between reproductive phases of Hypnea musciformis.
Character Tetrasporophyte Female gametophyte Male gametophyte
Thallus size erect tufts up 9 cm erect tufts up 7 cm erect tufts up 4 cm
Axis shape cylindrical, long and narrow cylindrical to complanate, short
and thick
cylindrical, short and thick
Branching subdichotomous, and very
scarce in the main axis, and
lateral branching abundant
subdichotomous, and abundant
in the main axis, and lateral
branching frequent
subdichotomous, and abundant
in the main axis, and lateral
branching frequent
Lateral branches abundant and very long frequent and short frequent and short
Apices acute, thin, sometimes recurved
or with a hook
acute, thin, sometimes recurved
or bifurcate
acute, thin, sometimes recurved
or bifurcate
Branchlets abundant, spiniform, simple,
cylindrical on all thallus
very abundant, spiniform,
branched, cylindrical to
complanate, anastomosed,
short on apical or medium
portions
very abundant, spiniform,
simple or branched,
cylindrical to complanate,
anastomosed, short on apical
or medium portions
Basal diameter (lm)
1
789 6102a 995 6365b 788 6364a
Apical diameter (lm) 492 660a 666 6113b 687 6278a
Periaxial cell diameter (lm) 73 620 89 624a 71 617b
Subcortical cell diameter (lm) 55 616a 41 615b 40 616
1
Mean and standard deviation of quantitative characters are indicated: basal diameter (n11); apical diameter (n14); periaxial cell
diameter (n17); subcortical cell diameter (n20). Different letters indicate statistical differences between reproductive phases.
236 Phycologia, Vol. 55 (2)
Carolina, USA (71%for ML, 0.97 for BPP). The pairwise
divergence between these two groups was 0.8–1.64%(11–22
bp). One Brazilian specimen analyzed in the present study was
identified as H. pseudomusciformis.
The position of H. musciformis in the COI-5P tree was
congruent with that in the rbcL phylogeny (Fig. 22). However,
the Mexican specimens were identical with those from Hawaii
and Barbados, but differed from European specimens by 2.8–
3.2%pairwise divergence (13–15 bp).
DISCUSSION
Morphological variability in Hypnea musciformis has been
reported in the literature, masking the most important
taxonomical characters and making the proper identification
of the species difficult and in some cases uncertain
(Geraldino et al. 2009, 2010). These characters are:
distinctiveness of main axes, branching pattern, branchlet
type, presence–absence of lenticular thickenings, position of
Figs 10–20. Hypnea musciformis reproductive structures.
Figs 10, 11. Tetrasporophyte with nemathecial sorus covering branchlets (arrows). Scale bar ¼500 lm.
Fig. 12. Zonately divided tetrasporangia. Scale bar ¼50 lm.
Fig. 13. Spermatangial thallus with superficial sori covering the branches (arrows). Scale bar ¼500 lm.
Figs 14, 15. Transversal section of the branch shows immature and mature spermatangia on a spermatangial mother cell (smc) and produce
spermatia (s). Scale bar ¼50 lm.
Fig. 16. Female gametophyte bearing cystocarps (arrows). Scale bar ¼500 lm.
Fig. 17. Transversal section of the mature cystocarp shows gonimoblast filaments (g) fusing with multinucleate cells of the nutritive tissue
(nt). Scale bar ¼50 lm.
Figs 18, 19. Carpospores in clusters from carposporangia (c) surrounded by mucilage envelope (arrows). Scale bar ¼50 lm.
Fig. 20. Basal portion of a mature cystocarp showing gonimoblast filaments and placenta (p). Scale bar ¼50 lm.
Va
´zquez-Delf´
ınet al.: Hypnea musciformis from the Yucatan Peninsula 237
Fig. 21. Maximum likelihood tree of the rbcL from the genus Hypnea. The numbers above or below the nodes are RAxML bootstrap values
and Bayesian posterior probabilities. Only bootstrap values 50%and 0.90 Bayesian posterior probabilities are shown on the tree.
238 Phycologia, Vol. 55 (2)
Fig. 22. Maximum likelihood tree of the COI-5P from the genus Hypnea. The numbers above or below the nodes are RAxML bootstrap
values and Bayesian posterior probabilities. Only bootstrap values 50%and 0.90 Bayesian posterior probabilities are shown on the tree.
Va
´zquez-Delf´
ınet al.: Hypnea musciformis from the Yucatan Peninsula 239
nemathecial sori over the branchlets (basal or middle
portions), position of cystocarps and the presence–absence
of a peduncle in cystocarps (Table S1). In Mexican samples,
we found that in addition to the above features, shape and
size of main axes, frequency of branching, lateral branch
type and apex shape were also variable. Furthermore, the
swollen hook apex, reported as diagnostic for identification
of H. muscifomis, is sometimes absent, and specimens
showed an acute or pointed apex, questioning its taxonom-
ical value. Whereas the branching pattern was reported as
irregular or alternate for specimens from different regions
(Magalhaes 2006; Cabioc’h et al. 2007; Littler et al. 2008;
Dawes & Mathieson 2008), in our samples from Mexico,
Brazil and Spain, branching was dichotomous to subdichot-
omous.
Regardless of the different morphologies found in the
Yucatan Peninsula, molecular analysis showed that Mexican
specimens formed a monophyletic group with European
specimens of Hypnea musciformis, including those from the
type locality. Mexican samples differed by 0.8–1.64%(11–22
bp) in rbcL and 2.8–3.2%(13–15 bp) in COI-5P, although no
morphological differences were observed between these two
groups (Table 2). Interspecific divergences ranged from 1.2
to 7.4%in rbcL and 4 to 36.5%in cox1 (Geraldino et al.
2006, 2009, 2010; Nauer et al. 2014). On the basis of a 1.7–
2.7%difference for rbcL and a 3.9–7.1%difference for COI-
5P, Nauer et al. (2015) described the new species, H.
pseudomusciformis, which had been identified as H. musci-
formis in Brazil. However, there are no clear morphological
differences between H. pseudomusciformis and H. muscifor-
mis, and its identification is based only on molecular
analysis. There are no clear limits for species boundaries in
pairwise divergences in red algae (Freshwater et al. 2010).
Divergence values between Mexican and European samples
of H. musciformis are below the interspecific divergence
values mentioned above.
The results of morphological variation found in Mexican
specimens suggest that the reproductive phases in the
Hypnea musciformis life cycle represent an important source
of morphological variability (Table 3). Some characters, like
shape and diameter of axes, branching frequency, lateral
branches, apex shape, branchlet type and diameter of
periaxial and subcortical cells, are different between the
reproductive phases. For example, the main difference
between phases was found in the branching frequency,
which is scarce in the tetrasporophyte, whereas in the
gametophyte (male and female) it is very abundant. On the
other hand, lateral branching in the tetrasporophyte was
more frequent than in the gametophyte, and the size of
laterals was larger on the former. Careful examination of
these results on a seasonal basis indicated that the sterile
branchlets were modified with the onset of reproduction,
changing the aspect of the thalli. Tetrasporophyte branchlets
were spiniform, simple and cylindrical, whereas for the
gametophyte they are cylindrical to complanate and can be
branched or not. Branchlets in the female gametophyte were
mostly branched with rounded apex, whereas in the male
gametophyte they were simple and pointed. In some cases,
these reproductive modifications were the basis for taxo-
nomic confusion; therefore, these morphological features of
the reproductive phases of the species must be considered for
its proper identification.
Morphological differences between phases have been
reported in other species with isomorphic life cycles,
including thallus size (smaller gametophytes relative to the
sporophytes), as well as differences in branching patterns,
basal filament system, pigmentation and in carrageenan
composition (Garbary et al. 1980; Hannach & Santelices
1985; Carrington et al. 2001; Ara´
ujo et al. 2014). Some
degree of heteromorphism has been described between male
and female gametophytes, as well as between gametophytes
and tetrasporophytes in Ceramium rubrum C.Agardh and C.
codicola J.Agardh. Branching pattern and branching angle
were different between phases and in relation to the
development and location of reproductive structures (Gar-
bary et al. 1978, 1980; Lewis & Lanker 2004). In Gelidium
sesquipedale (Clemente) Thuret gametophytes were shorter
and more heavily branched in relation to the tetraspor-
ophytes (Santos & Duarte 1996). The same subtle morpho-
logical differences between gametophytes and
tetrasporophytes are observed in Pterocladiella capillacea
(S.G.Gmelin) Santelices & Hommersand, with the former
reduced in length and more branched than the latter
(Polifrone et al. 2012).
The male gametophyte of Hypnea musciformis has been
reported as rare, having a short lifetime or being absent (Reis
& Yoneshigue-Valentin 2000; Smith et al. 2002; Caires et al.
2013). The male gametophyte is small and sometimes can be
confused with the tetrasporophyte because of the similarity
of the nemathecial sorus. At first glance, the spermatangial
nemathecia can be confused with tetrasporangial nemathecia
by their superficial aspect. However, closer examination of
specimens revealed the presence of a gel matrix in the outer
layer of the spermatangial nemathecia, whereas in the
tetrasporangial nemathecia the protruding larger heads of
the tetrasporangia are distinguishable. Similarity between
male gametophytes and tetrasporophytes was reported in the
red alga Ceramium codicola, where the thallus form appears
to be related to location of the reproductive structures
(spermatia and sporangia, respectively; Lewis & Lanker
2004). In the population at Quintana Roo, male gameto-
phytes were rare, and only observed during the cold season
(December). Phenology of H. musciformis in this area
showed no male gametophyte for most of the year, and
female gametophyte only occurred in low proportions (,
9%), with tetrasporophyte predominating (data not pub-
lished).
The observations reported for the carposporophyte agree
with those previously reported by Hommersand & Fredericq
(1990). They described the Hypnea cystocarpwitha
placenta, defined as a structure conferring nutritional
functions. A cystocarpic basal network of tissue mixed
between the carposporophyte and the gametophyte forms
the placenta.
A second source of morphological variation observed in
this species is plasticity due to the environmental conditions;
however, no studies have evaluated the factors triggering
such possible variation in this species. Hypnea musciformis
tolerates a wide range of environmental conditions. In
Quintana Roo, H. musciformis grew over a broad range of
environmental conditions including different types of sub-
240 Phycologia, Vol. 55 (2)
strata (ropes, sand, rocks and artificial geotubes), at different
wave and light exposure conditions and subjected to high
variable temperature and salinity (data not published). As a
result, thallus colour, branchlet abundance and apex shape
could be related to the environmental factors occurring at
microhabitat levels. However, it was not possible to establish
an association between these environmental factors and
morphological variation. The flattened hook apex, present
only in the tetrasporophytic phase and absent in some
samples, disappears after 1 month in culture (personal
observation), indicating that this is a plastic character that
could serve for attachment, as suggested previously (Smith et
al. 2002). In consequence its utility as a taxonomical
character for H. musciformis must be reconsidered.
ACKNOWLEDGEMENTS
We thank M.L. Zaldivar and J.L. Bortolini for technical
assistance during collection and specimens processing, and
to the National Herbarium and Faculty of Sciences
Herbarium, UNAM for the facilitation of herbarium
specimens, in particular to J.L. God´
ınez and D. Le´
on.
Thanks to Fabio Nauer for sending rbcL and COI-5P
sequences of Hypnea pseudomusciformis. EV-D thanks
Consejo Nacional de Ciencia y Tecnolog´
ıa (CONACyT)
for financial support (No. 225431). Programa Nacional de
Posgrados de Calidad (PNPC) CONACyT is also acknowl-
edged for financial support to attend the CLABA Workshop
in Florianopolis Brazil. Molecular analysis was supported by
the Marine Biotechnology program of the Korean Ministry
of Ocean Sciences and Fishery.
SUPPLEMENTARY DATA
Supplementary data associated with this article can be found
online at http://dx.doi.org/10.2216/15-118.1.s1.
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Received 24 October 2015; accepted 11 January 2016
Associate Editor: Mariana Cabral Oliveira
242 Phycologia, Vol. 55 (2)
