Phycological Research 2010; 58: 69–77
Chamaebotrys prolifera sp. nov. (Rhodymeniaceae,
Rhodophyta) from Puerto Rico, Caribbean Sea, based on
morphology and small subunit rDNA sequences
pre_560
69..77
David L. Ballantine,* Hector Ruiz and Chad Lozada-Troche
Department of Marine Sciences, P.O. Box 9013, University of Puerto Rico. Mayagüez, Puerto Rico 00681
SUMMARY
A new rhodymeniacean species, Chamaebotrys prolifera, is described from a shallow water habitat in Puerto
Rico, representing the first occurrence of the genus in
the Atlantic Ocean. Plants, to 5 cm across, are decumbent and comprised of compressed vesicles that are
originally proliferously branched at the perimeter. Older
vesicles become branched from their dorsal surfaces as
well. Branches are septate at their origin and become
irregularly shaped with age. Anastomoses between adjacent vesicles is common. Individual vesicles measure to
15 mm in broadest dimension. Vesicle walls consist of
two layers of medullary cells and two layers of cortical
cells. Tetrasporangia, which occur in diffuse nemathecia, are spherical, to 30 mm in diameter and are cut
off terminally from an inner cortical cell. Cystocarps are
hemispherical measuring to 800 mm in diameter and
350 mm in height. Spermatangia are apparently cut off
randomly from outer cortical cells across the thallus
surface. Molecular evidence confirms placement of
Chamaebotrys within the Rhodymeniaceae.
Key words: Chamaebotrys prolifera sp. nov., Puerto
Rico, Rhodophyta, Rhodymeniaceae, small subunit
rDNA.
INTRODUCTION
Chamaebotrys was segregated from Coelarthrum
Børgesen by Huisman (1996) with Chamaebotrys
boergesenii (Weber Bosse) Huisman designated as the
generitype. Chamaebotrys boergesenii differs from
Coelarthrum on the basis of habit, that is, decumbent
plants attached at several points, commonly possessing secondary anastomoses, and reproductively in the
formation of tetrasporangia in a terminal rather than
in an intercalary position within nemathecia (Huisman
1996). The transfer of a second species also formerly
treated as Coelarthrum, C. lomentariae (Tanaka
& K.Nozawa) Huisman was also made at that time.
A third species, Chamaebotrys erectus Schils &
© 2010 Japanese Society of Phycology
Huisman, was recently established by Schils et al.
(2003). The erect habit of this later species broadened the generic circumscription somewhat. Currently
all Chamaebotrys species are known from the Pacific
and Indian Oceans: C. boergesenii is widely distributed in the Indo-Pacific (Huisman 1996), C. erectus
is known only from its type locality, Yemen, Indian
Ocean (Schils et al. 2003) and C. lomentariae is
endemic to Japan (Tanaka 1964; Yoshida et al.
1990). Continuing study of the marine algal flora of
Puerto Rico has resulted in recognition of a new
species of Chamaebotrys, which represents the first
occurrence of the genus in the Atlantic Ocean.
MATERIALS AND METHODS
Specimens were collected by snorkeling and then either
preserved in 10% Formalin/seawater or placed in silica
gel in a glass container. Transections (30–40 mm thick)
were made with an American Optical Cryo-Cut freezing
microtome (Leica, Wetzlaar, Germany). Microscope
slide preparations were stained with 1% acidified
aniline blue and mounted in 60% Karo syrup. Photomicrographs were taken with a Spot RE digital camera
through an Olympus BMAX light microscope. The plates
were assembled from digital photographs utilizing
Adobe Photoshop CS2. Voucher specimens have been
deposited in MICH, MSM and US. Herbarium abbreviations follow Holmgren et al. (1990), and authority designations are in accordance with Brummitt and Powell
(1992).
Molecular analyses used the small subunit (SSU
rDNA) and RUBISCO (rbcL) genes. DNA extraction,
polymerase chain reaction (PCR) amplification and
sequencing were carried out using the protocols
described in Ballantine and Lozada-Troche (2008).
Phylogenetic reconstruction was carried out using the
*To whom correspondence should be addressed.
Email: david.ballantine@upr.edu
Communicating editor: O. DE Clerck.
Received 14 November 2008; accepted 13 June 2009.
doi: 10.1111/j.1440-1835.2009.00560.x
70
D. L. Ballantine et al.
maximum parsimony (MP), neighbor joining (NJ) and
maximum likelihood (ML) algorithms as implemented
in PAUP* (Swofford 2002) and Bayesian Inference using
MrBayes (Huelsenbeck & Ronquist 2001). Modeltest
(Posada & Crandall 1998) was used to determine the
correct DNA substitution model to be used as input for
NJ and ML analyses and the Bayesian Information
Criterion for Bayesian Inference. The optimal model
found for the SSU rDNA analysis was the GTR + I + G
evolutionary model (General Time Reversal model +
Invariable Sites + Gamma Distribution) (Lanave et al.
1984; Rodriguez et al. 1990). Assumed nucleotide frequencies were: A = 0.2427, C = 0.2126, G = 0.2850,
T = 0.2597. The assumed substitution rate matrix was:
A-C = 0.9958, A-G = 3.1979, A-T = 1.2154, C-G =
0.5727, C-T = 4.2088, G-T = 1.0000. Proportion of
sites assumed to be invariable = 0.6941; rates for variable sites were assumed to follow gamma distribution
with shape parameter = 0.8019. Bayesian Inference
was done using the K80 + I + G evolutionary model
(Kimura 2-parameter + Invariable sites + Gamma Distribution) (Kimura 1980). Assumed nucleotide frequencies were equal for all bases. The proportion of
sites assumed to be invariable = 0.6956; rates for variable sites were assumed to follow a gamma distribution
with shape parameter = 0.7912.
Table 1.
The optimal model for rbcL data also was:
(GTR + I + G). Assumed nucleotide frequencies were:
A = 0.3043, C = 0.1515, G = 0.2248, T = 0.3195.
The assumed substitution rate matrix was: A-C substitutions = 1.4593, A-G = 6.2946, A-T = 4.0647,
C-G = 1.6844, C-T = 18.0794, G-T = 1.0000; proportion sites assumed to be invariable = 0.5390; rates for
variable sites were assumed to follow a gamma distribution with shape parameter = 2.7617. Bayesian analysis
of rbcL sequences was done following the TIM + G
evolutionary model (Transition model + Gamma Distribution) (Posada & Crandall 2001). The robustness of
18S and rbcL data was determined by bootstrapping the
dataset (Felsenstein 1985) 2000 times for MP and NJ
and 1000 times for ML. Bayesian analysis was conducted running 1 000 000 generations. Trees were
sampled every 100 generations with log-likelihood
scores stabilized at approximately 2500 generations.
For 18S sequence analysis the first 3000 trees of a
possible 10 000 trees were discarded as burn-in. Analysis of rbcL sequences stabilized at 3500 generations
and the first 4000 trees were discarded as burn-in.
Tables 1 and 2 show species used (and source) for the
SSU rDNA and rbcL gene sequences, respectively.
Results from the newly sequenced species are deposited
in GenBank (Table 1, accession numbers in bold).
List of species used in the18S (small subunit [SSU]) gene sequence analysis (numbers in bold are new to GenBank)
Species
Source
Accession Number
Reference
Chamaebotrys prolifera (DLB-7257)
Chamaebotrys prolifera (DLB-7257)
Coelarthrum cliftonii (CLT-197)
Coelarthrum cliftonii (DLB-7260)
Coelarthrum cliftonii (DLB-7260)
Coelarthrum opuntia
Chrysymenia agardhii (DLB-6000)
Chrysymenia nodulosa (CLT-181)
Chrysymenia ornata
Erythrocolon podagricum
Gloiosaccion brownii
Halymenia plana
Sebdenia flabellata
San Juan, Puerto Rico
San Juan, Puerto Rico
Guanica, Puerto Rico
Guanica, Puerto Rico
Guanica, Puerto Rico
GenBank
La Parguera, Puerto Rico
La Parguera, Puerto Rico
GenBank
GenBank
GenBank
GenBank
GenBank
EU715131
EU715132
EU086465
EU670594
EU670595
AF085258
EF690262
EF690261
AF085257
U23953
AF085269
U33133
U33128
This paper
This paper
This paper
This paper
This paper
Saunders et al. 1999
This paper
This paper
Saunders et al. 1999
Millar et al. 1996
Saunders et al. 1999
Saunders & Kraft 1996
Saunders & Kraft 1996
Table 2.
List of species used in the rbcL gene sequence analysis. (numbers in bold are new to GenBank)
Species
Source
Accession Number
Reference
Chamaebotrys prolifera (DLB-7257)
Chamaebotrys prolifera (DLB-7257)
Coelarthrum cliftonii (DLB-7260)
Chrysymenia agardhii (DLB-6000)
Chrysymenia procumbens
Halymenia floresii
Sebdenia integra
San Juan, Puerto Rico
San Juan, Puerto Rico
Guanica, Puerto Rico
La Parguera, Puerto Rico
GenBank
GenBank
GenBank
EU715134
EU715135
EU715136
EU715133
AY294381
AB038603
AY294363
This paper
This paper
This paper
This paper
Gavio et al. 2005
Wang et al. 2000
Gavio et al. 2005
© 2010 Japanese Society of Phycology
A new Chamaebotrys species from Puerto Rico
RESULTS
Chamaebotrys prolifera sp. nov.
Plantae decumbentes, ad sitos numerosos brevibus
hapteronibus paxilliformibus affixi; coloniae integrae
usque ad 5.0 cm latae, e vesiculis compressis
et ovatis usque ad irregularibus constantes, usque
ad 15 mm ut maximum; ramificatio prolificans a
margine, non plerumque e superficiebus dorsalibus
ramose; rami septati; vesiculae contiguae plerumque
anastomosantes; paries vesiculae plerumque crassus
4-cellulas; stratum medullosum, quod crassum
2-cellulas est, e strato interiore magnarum cellularum
rectangularium constans, ut visum in sectione 125–
165 (-200) ¥ 65–100 mm, atque vel e strato partiali
cellularum triangularium medullosarumque conferto
inter cellulas medullosas maiores usque ad 50–75 mm
ut maximum, vel e strato continuo localique cellularum medullosarum minorum; cortex, ex 2 stratis cellularum constans, stratum interiorem cellularum
sphaericarum usque ad ovatas, 15–20 mm lato,
praebens; stratus extimus cellularum 5.0–7.5 mm
diametro; cortex cellulas medullsas omnino tegens;
glandicellulae numerosae (2 ad 12) et pyriformes,
usque ad 17.5 mm longae ¥15 mm diametro, a
magnis cellulis ferentibus et incoloratis abscissae
quae in interioribus cellulis medullosis portata sunt
aut rarius directe a interioribus cellulis medullosis;
tetrasporangia sphaerica, usque ad 30 mm diametro,
a interiore cellula corticali terminaliter abscissa et in
nematheciis diffusis locata; tetrasporangia paraphysibus unicellularibus circumcincta; plantae monoeciae;
cystocarpia hemisphaerica, usque ad 800 mm
diametro et 350 mm alta, intrinsecus et extrinsecus
aeque protrudentia; pauca spermatangia abscissa, ut
videtur fortuito a exterioribus cellulis corticalibus
trans superficiem thalli.
Plants decumbent, attached at numerous sites with
short (to 1.0 mm long) peg-like holdfasts; entire colonies to 5.0 cm across; comprised of compressed ovate
to irregularly-shaped vesicles, measuring to 15 mm
in largest dimension, with proliferous branching from
margins, less commonly branched from the dorsal surfaces; branches septate; adjacent vesicles commonly
anastomosing; vesicle wall generally four cell layers
thick, the two cell-thick medullary layer consisting of an
inner layer of large rectangular cells, when viewed in
section measuring 125–165 (-200) ¥ 65–100 mm,
and either a partial layer of triangular medullary cells
wedged between the larger medullary cells, measuring
50–75 mm in broadest dimension or a locally continuous
layer of smaller medullary cells; the two cell-layer cortex
with an inner layer of spherical to oval cells, 15–20 mm
in broadest dimension, the outermost layer of spherical
cells measuring 5.0–7.5 mm in diameter; the cortex
© 2010 Japanese Society of Phycology
71
completely covering the medullary cells; numerous
(2–12) pyriform gland cells, to 17.5 mm long and 15 mm
in diameter, cut off from large colorless bearing cells that
are borne on inner medullary cells or more rarely, directly
from the inner medullary cells; tetrasporangia spherical,
to 30 mm in diameter cut off terminally from an inner
cortical cell and located in diffuse nemathecia and
surrounded by single-celled paraphyses; plants monoecious, cystocarps hemispherical measuring to 800 mm
in diameter and 350 mm in height; and protruding
equally inwardly and outwardly; spermatangia cut off in
few numbers, apparently randomly from outer cortical
cells across the thallus surface.
Holotype. D.L. Ballantine 7257, San Juan Bay (Pier
8), San Juan, Puerto Rico (18°27.766′N,
66°06.202′W), 1–2 m, coll. H. Ruiz, 4.xi.2007 (Alg.
Coll. # US-209253). Isotypes: MICH: MSM; US.
Paratypes. D.L.B. 7211, ibid, 6.x.2007; D.L.B. 7443,
ibid, 11.ii.2008.
Etymology. The specific epithet refers to the proliferous
generation of new segments from parent vesicles.
Plants originate as single compressed, peltate
vesicles, being attached by one or more peg-like holdfasts. The primary vesicles measure to 15 mm in diameter and are typically ovate. Originally branches are
produced from the margins (Figs 3,4) with young
branchlets being ovate-compressed. The branchlets
enlarge and similarly rebranch (Fig. 5). With age,
branches may also form on the dorsal surface with an
increasing number of vesicles being irregular in shape
(Figs 5,6). Branches are septate at their origin (Figs 3–
5,11). Lateral attachments commonly occur between
adjacent vesicles (Figs 4–6). Mature plants are a chaotic
arrangement of vesicles (Figs 1,2,6) with the entire
plant measuring to 5.0 cm broad. Older vesicles may
tear with the wall remaining viable and supporting continuing branching (Fig. 7). The vesicle walls possess two
layers of colorless medullary cells and two layers of
cortical cells (Figs 8–10). The innermost medullary cells
are rectangular and measure 125–165(-200) ¥ 65–
100 mm. Between the inner medulla and the cortex is
a partial layer of triangular medullary cells wedged
between the larger medullary cells (Fig. 8), measuring
50–75 mm in broadest dimension or a locally continuous
layer of smaller medullary cells. The two-cell layer cortex
consists of an inner layer of spherical to oval cells,
15–20 mm in broadest dimension. The outermost cortical cells are spherical and measure 5.0–7.5 mm in
diameter. The cortex completely covers the medullary
cells. Numerous (to 12) pyriform gland cells, to 17.5 mm
long by to 15 mm in diameter are cut off from large
colorless bearing cells that are borne on inner medullary
cells (Fig. 9) or more rarely directly from the inner
medullary cells (Fig. 10). The septa consist of multilayered medullary cells that increase in layer number
with age (Fig. 11).
72
Figs 1–7.
D. L. Ballantine et al.
Chamaebotrys prolifera sp. nov. (all DLB-7257). 1. Habit of large specimen showing multiple irregularly arranged vesicular
branches. Scale bar, 1.0 cm. 2. Habit of the holotype. Scale bar, 1.0 cm. 3. Primary vesicle with marginal vesicular branches. Scale
bar, 2.0 mm. 4. Primary vesicle with marginal branches, many of which are laterally connected (arrowheads) and a dorsally placed
branch (asterisk). The plant is cystocarpic (arrows). Scale bar, 2.0 mm. 5. Plant in which the primary branches are becoming irregular
in shape and giving rise to a new order of branchlets. Arrowhead denotes lateral fusion. Scale bar, 5.0 mm. 6. Close up of irregularly
shaped branches bearing cystocarps (arrows). Lateral fusions are denoted with arrowheads. Scale bar, 5.0 mm. 7 Portion of vesicle that
has split open, but remains viable. Scale bar, 1.0 cm.
Tetrasporangia are spherical, to 30 mm in diameter,
and are cut off terminally from an inner cortical cell
(Fig. 12) and are located in somewhat diffuse sori.
Adjacent to the tetrasporangia-producing cortical
cells, cortical cells also produce single-celled
sterile paraphyses (Fig. 12). Plants are monoecious.
Cystocarps are hemispherical measuring to 800 mm
in diameter and 350 mm in height; they protrude
equally inwardly and outwardly (Fig. 13). Spermatangia are cut off in few numbers, apparently randomly
from outer cortical cells across the thallus
surface.
© 2010 Japanese Society of Phycology
A new Chamaebotrys species from Puerto Rico
73
Figs 8–13. Chamaebotrys prolifera sp. nov. 8. Cross-section through vesicle wall, showing two cell-layered medulla (DLB-7257). Scale
bar, 100 mm. 9. Cross-section through vesicle wall showing an accessory medullary cell bearing gland cells (DLB-7257). Scale bar,
100 mm. 10. Cross-section through vesicle wall, in which regular medullary cells give rise to small gland cells (DLB-7257). Scale bar,
100 mm. 11. Cross-section through vesicle wall at site of multicellular septum at point of branching (DLB-7257). Scale
bar, 250 mm. 12. Cross-section through vesicle wall showing terminally placed tetrasporangia (arrows) (DLB-7443) with adjacent
paraphyses. Scale bar, 50 mm.
13. Cross-section through cystocarp (DLB-7257). Scale bar, 100 mm.
Lengths of new SSU rDNA sequences obtained
from Chamaebotrys prolifera and other Rhodymeniaceae species varied from 1697 to 1756 bp. A
maximum likelihood tree inferred from the SSU rDNA
sequence data (Fig. 14) indicates that C. prolifera is a
sister taxon of the genera Coelarthrum and Erythroco© 2010 Japanese Society of Phycology
lon. Approximately 1291 bp of the Rubisco (rbcL)
gene were also newly sequenced for specimens of
C. prolifera, Coelarthrum cliftonii (Harv.) Kylin and
Chrysymenia agardhii Harv. A maximum likelihood
tree inferred from rbcL sequence data (not shown)
also indicates a highly supported separation between
74
D. L. Ballantine et al.
Fig. 14. Small subunit (SSU) (18s) sequence phylogram using maximum likelihood. Bootstrap proportions are shown on top of the
branches, left to right: Bayesian inference (1 000 000 generations), maximum parsimony (2000 replicates), neighbor joining (2000
replicates), and maximum likelihood (100 replicates).
Chamaebotrys prolifera and Coelarthrum cliftonii with
an 11% divergence between them and are supportive
of the taxonomic relationship revealed by the SSU
rDNA analysis.
DISCUSSION
Huisman (1996) presented a table of differentiating
characters for hollow rhodymeniacean genera. Four of
these: Chamaebotrys, Coelarthrum, Erythrocolon and
Webevanbossea, as is the new species, are regularly
segmented. Coelarthrum and Erythrocolon differ from
the new species by possessing tetrasporangia that originate in intercalary positions (Huisman 1996). Erythrocolon and Webervanbossea produce gland cells borne
on internal filaments (Huisman 1995) that differ from
gland cell origin in the new entity. Webervanbossea,
however, is now placed in the Faucheaceae (Saunders
et al. 1999; Schneider & Wynne, 2007). On these
bases, the new species would appear to be most closely
related to Chamaebotrys. Unfortunately, there are no
prior molecular data for Chaemobotrys with which to
compare the sequences obtained of the new species.
Nevertheless, the new species does closely group with
Erythrocolon and Coelarthrum, the genus from which
Chamaebotrys was segregated.
Chamaebotrys is currently constituted by species
(Table 3) ‘similar in morphology to Coelarthrum but
differing in tetrasporangial initiation’ (Huisman 1996,
p. 108). Chamaebotrys prolifera differs from all other
members of the genus in lacking a strong resemblance to Coelarthrum. Nevertheless Wynne (2001)
figured two highly irregular thalli of putative Chamaebotrys boergesennii f. minimus (WeberBosse) M.J.
Wynne, neither with gross morphological similarity to
Coelarthrum. Chamaebotrys prolifera further differs
from C. boergesenii on the basis of its chiefly
polychotomous branching as opposed to mostly
© 2010 Japanese Society of Phycology
This paper
Narrow
On elongate
nemathecial
filaments,
20–25 ¥
27–40
To 15 ¥ 17.5 mm To 30 mm
© 2010 Japanese Society of Phycology
W ¥ L, width ¥ length.
Decumbent,
To 15 mm
colony to 5 cm
Puerto Rico
Chamaebotrys
prolifera
Polychotomous,
proliferous
Present
2
6.5–20.5
2
Erect. 20 cm
high
7 ¥ 20 mm
Yemen, Indian
Ocean
Chamaebotrys
erectus
Dichotomous or Absent
verticillate
1
Erect?, 3–5 cm
To 2.5 ¥
To 6 mm
Dichotomous,
irregular
Japan
Chamaebotrys
lomentariae
Unknown
Narrow
Tanaka &
Nozawa (1963)
in Tanaka (1964);
Yoshida et al. 1990
Schils et al. 2003
Broad
15–20 ¥
10–15 mm
Huisman 1996
20–30 ¥ 35–40 Broad
Spherical,
20–25 mm
diameter
Spherical or
ovate
1
Present
Indian, Pacific Decumbent,
2–6 ¥ 2–5 mm Dichotomous,
Oceans
colony to 5 cm
trichotomous
Chamaebotrys
boergesenii
Habit
Segment size
Branching
Distribution
Table 3. Comparative features of Chamaebotrys species
75
Species
Gland cells
Number
Secondary
medullary
lateral
anastomoses layers
Tetrasporangia
size (mm,
W ¥ L)
Constrictions Reference
between
segments
A new Chamaebotrys species from Puerto Rico
di-trichotomous branching and smaller tetrasporangia
(to 30 mm diameter vs. 20–30 ¥ 35–40 mm) in the
latter (Huisman 1996).
Chamaebotrys erectus as the specific epithet indicates, is an erect species. It otherwise differs from C.
prolifera by its substantially larger size (to 20 cm
high), dichotomous branching and lack of secondary
anastomoses (Schils et al. 2003). Furthermore,
Chamaebotrys erectus produces elongate nemathecial
filaments that arise from outer cortical cells and ultimately produce tetrasporangia terminally. These elongate nemathecial filaments are not produced by either
the new species or the generitype, C. boergesenii,
although short single celled paraphysal-like filaments
surround sporangia in C. prolifera (Fig. 12). Chamaebotrys lomentariae is a relatively poorly known entity
that may prove to be conspecific with C. boergesenii
(Huisman 1996), differing by its erect habit, although
that character was questioned by Huisman (1996).
Nevertheless, Masuda et al. (2001) emphatically
regarded the two taxa as separate species. Chamaebotrys lomentariae also differs from the new species
in that the constrictions between segments are barely
noticeable as opposed to being distinct in the new
species.
In our phylogenetic tree (Fig. 14), the clade including Coelarthrum, Chamaebotrys and Erythrocolon is
distinct from that containing Chrysymenia. While the
generic sampling is admittedly low, both clades
contain genera that produce intercalary sporangia
(Chrysymenia and Coelarthrum). The obvious morphological feature that separates them is the presence of
segmentation in the Coelarthrum/Chamaebotrys clade
and its lack in Chrysymenia. Lee (1978) regarded
genera with terminal cruciately divided tetrasporangia
as belonging to Rhodymeniaceae, while Saunders
et al. (1999) placed these genera in the Faucheaceae
and considered that algae with intercalary cruciate
tetrasporangia belong in the Rhodymeniaceae. Nevertheless, Saunders et al. (1999) speculated on the
familial placement of Chamaebotrys, and despite its
seeming affinity with the Facuheaceae, provisionally
assigned the genus (and correctly in our opinion) to
the Rhodymeniaceae. Our molecular evidence clearly
demonstrates that Chamaebotrys is a member of the
Rhodymeniaceae.
While substantial strides have been realized in
understanding Rhodymeniales taxonomy including
the recent recognition of two new families (Le Gall
et al. 2008), systematic difficulties within the
order have been well documented (Saunders & Kraft
1994, Huisman 1995, 1996; Saunders 2004).
One confounding issue in Rhodymeniaceae circumscription remains the fact that many characters
are shared between genetically distinct groups.
This is may be attributable to a highly conserved veg-
76
etative evolution that is illustrated by the many rhodymeniacean genera that possess essentially identical
vesicle walls (Huisman 1996) as well as difficulty in
recognition of evolutionary reversions (Saunders et al.
1999).
ACKNOWLEDGMENTS
The Sequencing and Genotyping facility, University of
Puerto Rico-Río Piedras is supported in part by NCRRAABRE Grant #P20 RR16470, NIH-SCORE Grant
#S06GM08102, University of Puerto Rico Biology
Department, NSF-CREST Grant #0206200. We thank
Sr. Angel García who facilitated permits and collection
in the San Juan Bay port area. Drs Craig Schneider and
John Huisman provided constructive criticism, which
improved the manuscript. Ms Angela Piper provided the
Latin translation.
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