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First record of the terrestrial nemertean Geonemertes pelaensis Semper, 1863 (Hoplonemertea: Prosorhochmidae) for Cuba

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BioInvasions Records (2020) Volume 9, Issue 2: 399–407
Morffe et al. (2020), BioInvasions Records 9(2): 399–407, https://doi.org/10.3391/bir.2020.9.2.26 399
CORRECTED PROOF
Rapid Communication
First record of the terrestrial nemertean Geonemertes pelaensis Semper, 1863
(Hoplonemertea: Prosorhochmidae) for Cuba
Jans Morffe1,*, Nayla García1 and Karin Breugelmans2
1Instituto de Ecología y Sistemática, Carretera Varona 11835 e/ Oriente y Lindero, La Habana 19, CP 11900, Calabazar, Boyeros, La Habana, Cuba
2Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000, Brussels, Belgium
Author e-mails: jans@ecologia.cu (JM), nayla@ecologia.cu (NG), kbreugelmans@naturalsciences.be (KB)
*Corresponding author
Abstract
The terrestrial nemertean Geonemertes pelaensis Semper, 1863 (Hoplonemertea:
Prosorhochmidae) is recorded for first time from Cuba based on specimens from
Artemisa and La Habana provinces, western Cuba. Both external morphology and
histology are consistent with G. pelaensis features. Maximum likelihood and
Neighbor-joining analysis of mitochondrial cytochrome c oxidase I (COI) obtained
from Cuban individuals form a well-supported clade with other specimens of
G. pelaensis from Bermuda and Japan, as well as with Geonemertes sp. from Panama.
The current record expands the distribution of G. pelaensis in the West Indies.
Key words: ribbon worm, DNA barcoding, cytochrome oxidase I, introduced species,
range expansion, West Indies
Introduction
The terrestrial nemerteans constitute a small group of ribbon worms that
originate from two separate families of marine hoplonemerteans, namely
Plectonemertidae and Prosorhochmidae (Moore et al. 2001). For a long
time all the species remained placed in the single genus Geonemertes, but
further studies showed the existence of an assemblage of species that
evolved independently (Moore and Gibson 1981; Kvist et al. 2014, 2015).
Currently, about 13 species grouped in seven genera are known inhabiting
fully terrestrial or intermediate habitats (i.e. swamp mangroves and upper
littoral) (Moore and Gibson 1981; Moore et al. 2001).
Most of the species of terrestrial nemerteans are recorded from small
oceanic islands, mainly from the Pacific and Indian oceans and show a
restricted distribution. Only a few species, namely Argonemertes dendyi
(Dakin, 1915), Geonemertes pelaensis Semper, 1863 and Leptonemertes
chalicophora (Graff, 1879) present a wider distribution. G. pelaensis is the
most widespread of the aforementioned species and is recorded from
islands in the Pacific and Indian oceans and The Caribbean, as well as Florida,
U.S.A. and Japan (Gibson and Moore 1998). The more probable G. pelaensis
Citation: Morffe J, García N,
Breugelmans K (2020) First record of the
terrestrial nemertean Geonemertes
p
elaensis Semper, 1863 (Hoplonemertea:
Prosorhochmidae) for Cuba. BioInvasions
R
ecords 9(2): 399–407, https://doi.org/10.
3391/bir.2020.9.2.26
Received: 7 December 2019
Accepted: 17 March 2020
Published: 15 April 2020
Handling editor: Tim Adriaens
Thematic editor: Stelios Katsanevakis
Copyright: © Morffe et al.
This is an open access article distributed under terms
of the Creative Commons Attribution License
(Attribution 4.0 International - CC BY 4.0).
OPEN ACCESS.
First record of Geonemertes pelaensis for Cuba
Morffe et al. (2020), BioInvasions Records 9(2): 399–407, https://doi.org/10.3391/bir.2020.9.2.26 400
mode of dispersal (as with other terrestrial nemerteans) appears to be by
human activities such as the transport of exotic plants used in gardening
and nurseries (Moore et al. 2001; Jones and Sterrer 2005). This success of
G. pelaensis dispersal appears to be increased by being a hermaphroditic
species (Moore et al. 2001).
Studies on Cuban nemerteans are quite scarce. Palacios-Lemagne et al.
(2008) found a specimen of a terrestrial nemertean in western Cuba,
identified as Geonemertes sp. Diez (2013) recorded two marine species
from eastern Cuba, namely Baseodiscus delineatus (Delle Chiaje, 1825) and
Paranemertes sp. In both reports the terrestrial and the marine nemerteans
were identified based only on external morphology. The present paper
constitutes the first record of a terrestrial nemertean from Cuba, including
both external morphology and histology, as well as molecular techniques.
Materials and methods
Specimens collecting and fixation
Specimens were collected by hand under stones from Sierra de Anafe,
Artemisa province, Cuba (n = 5) and the Instituto de Ecología y
Sistemática, Havana province, Cuba (n = 5). They were measured alive
with a ruler (± 1 mm) and photographed with a Nikon D5300 camera with
an AF-S DX Micro NIKKOR 85 mm macro lens. The individuals were
narcotized with 10% ethanol and a small fragment of the posterior end was
cut from each specimen and stored in 96% ethanol for DNA studies. Worms
were fixed in phosphate-buffered 4% formaline and stored in 70% ethanol.
Histological studies
Seven specimens were selected for histological studies. Tissue blocks of the
anterior region and the stomach region were embedded in parafin wax.
Sagittal and transversal 5–7 m sections were stained with Cason’s one step
Mallory-Heidenhain trichrome (Winsor and Sluys 2018) and mounted in
Canada balsam or Permount. Micrographs were taken with an AxioCam
digital camera attached to a Carl Zeiss Axioskop 2 Plus compound
microscope. Scale bars of all plates are given in micrometers. The studied
material is deposited in the Colección Helmintológica de las Colecciones
Zoológicas (CZACC), Instituto de Ecología y Sistemática, La Habana, Cuba.
DNA extraction, amplification and sequencing
Genomic DNA was extracted with the NucleoSpin® Tissue kit (Macherey-
Nagel, Düren, Germany) following manufacturer’s protocol. Partial
sequences of the DNA barcode mitochondrial COI gene (cytochrome c
oxidase subunit I) were amplified with the universal primer set LCO1490
(5´-GGT CAA CAA ATC ATA AAG ATA TTG G-3´) and HCO2198
(5´-TAA ACT TCA GGG TGA CCA AAA AAT CA-3´) (Folmer et al. 1994).
First record of Geonemertes pelaensis for Cuba
Morffe et al. (2020), BioInvasions Records 9(2): 399–407, https://doi.org/10.3391/bir.2020.9.2.26 401
PCR reactions were performed in a total volume of 11 µL with the
Multiplex PCR Master Mix (Qiagen, USA). PCR cycling parameters
consisted of an initial denaturation at 95 °C for 15 min followed by 40 cycles
of 95 °C by 30 s, 48 °C by 30 s and 72 °C for 1 min and a final extension
step of 72 °C for 10 min. The results of the PCR reactions were checked by
agarose gel electrophoresis. PCR products were purified with Exonuclease I
and FastAP™ (Thermo Scientific, USA) and the sequences were obtained
from both strands using Big Dye Terminator v3.1 chemistry (Applied
Biosystems, USA) and the same primers as the PCR reactions.
Raw sequences were manually edited with Sequencher 4.1.4
(http://genecodes.com). Sequences were deposited in GenBank NCBI
(http://www.ncbi.nlm.nih.gov/genbank/). The accession numbers are shown
in the phylogram.
Phylogenetic analysis
Several sequences of hoplonemerteans (Prosorhochmidae, Malacobdellidae)
were selected from GenBank for the phylogenetic analyses (accession
numbers in the phylogram). Three species of Amphiporus (Amphiporidae)
were used as the outgroup taxa.
COI sequences were translated to amino acids, examined for stop
codons or gaps, then manually aligned with MEGA6 (Tamura et al. 2013)
and retranslated to nucleotides. MEGA6 was also used to determine the
optimal model of sequence evolution (GTR+G+I) following the Akaike
Information Criterion (AIC), and perform the Maximum likelihood (ML)
analysis. Branch support for the ML tree was inferred by bootstrap using
1,000 iterations. Also, MEGA6 was used to perform a Neighbor-Joining
(NJ) analysis based on Kimura two-parameter distances (K2P) with 1,000
bootstrap replicates.
Results and discussion
Material examined
Voucher CZACC 18.007; Cuba, Artemisa province, Caimito, Sierra de
Anafe (eastern slope); 22°5727.75N; 82°3637.34W; 30/X/2017; L.F. de
Armas coll.; serial sagittal sections (5–7 µm) of the anterior region in 2
slides; a small portion of the posterior end removed for molecular studies;
rest of specimen in 70% ethanol.
Voucher CZACC 18.008; Cuba, Artemisa province, Caimito, Sierra de
Anafe (eastern slope); 22°5727.75N; 82°3637.34W; 30/X/2017; L.F. de
Armas coll.; a small portion of the posterior end removed for molecular
studies; rest of specimen in 70% ethanol.
Voucher CZACC 18.009; Cuba, Artemisa province, Caimito, Sierra de
Anafe (eastern slope); 22°5727.75N; 82°3637.34W; 30/X/2017; L.F. de
Armas coll.; a small portion of the posterior end removed for molecular
studies; rest of specimen in 70% ethanol.
First record of Geonemertes pelaensis for Cuba
Morffe et al. (2020), BioInvasions Records 9(2): 399–407, https://doi.org/10.3391/bir.2020.9.2.26 402
Voucher CZACC 18.010; Cuba, Artemisa province, Caimito, Sierra de
Anafe (eastern slope); 22°5727.75N; 82°3637.34W; 30/X/2017; L.F. de
Armas coll.; serial sagittal sections (5–7 µm) of the anterior region in 4
slides; a small portion of the posterior end removed for molecular studies;
rest of specimen in 70% ethanol.
Voucher CZACC 18.011; Cuba, Artemisa province, Caimito, Sierra de
Anafe (eastern slope); 22°5727.75N; 82°3637.34W; 30/X/2017; L.F. de
Armas coll.; serial transverse sections (5–7 µm) of the stomach region in 6
slides; a small portion of the posterior end removed for molecular studies;
rest of specimen in 70% ethanol.
Voucher CZACC 18.012; Cuba, La Habana, Boyeros, Instituto de
Ecología y Sistemática; 23°0202.41N; 82°2244.60W; J. Morffe, L. Véliz
coll.; serial sagittal sections (5–7 µm) of the anterior region in 1 slide; serial
transverse sections (5–7 µm) of the stomach region in 1 slide; a small
portion of the posterior end removed for molecular studies; rest of
specimen in 70% ethanol.
Voucher CZACC 18.013; Cuba, La Habana, Boyeros, Instituto de
Ecología y Sistemática; 23°0202.41N; 82°2244.60W; J. Morffe, L. Véliz
coll.; serial transverse sections (5–7 µm) of the stomach region in 3 slides; a
small portion of the posterior end removed for molecular studies; rest of
specimen in 70% ethanol.
Voucher CZACC 18.014; Cuba, La Habana, Boyeros, Instituto de
Ecología y Sistemática; 23°0202.41N; 82°2244.60W; J. Morffe, L. Véliz
coll.; serial sagittal sections (5–7 µm) of the anterior region in 2 slides;
serial transverse sections (5–7 µm) of the stomach region in 3 slides; a
small portion of the posterior end removed for molecular studies; rest of
specimen in 70% ethanol.
Voucher CZACC 18.015; Cuba, La Habana, Boyeros, Instituto de
Ecología y Sistemática; 23°0202.41N; 82°2244.60W; J. Morffe, L. Véliz
coll.; a small portion of the posterior end removed for molecular studies;
rest of specimen in 70% ethanol.
Voucher CZACC 18.016; Cuba, La Habana, Boyeros, Instituto de
Ecología y Sistemática; 23°0202.41N; 82°2244.60W; J. Morffe, L. Véliz
coll.; serial sagittal sections (5–7 µm) of the anterior region in 2 slides; a
small portion of the posterior end removed for molecular studies; rest of
specimen in 70% ethanol.
The color pattern of the Cuban specimens corresponds with what has
been described for most of the localities where the species have been
recorded, including the West Indies and Florida, U.S.A. (Gibson and
Moore 1998). The background color of the dorsum is cream coloured, with
a mid-dorsal dark brown stripe (Figure 1). The single lateral stripe is
present on both sides, very faint and incomplete. The ventral side is pale
buff in color (Figure 1D). One specimen from the Instituto de Ecología y
Sistemática (CZACC 18.014) presented the posterior end of the body dark
First record of Geonemertes pelaensis for Cuba
Morffe et al. (2020), BioInvasions Records 9(2): 399–407, https://doi.org/10.3391/bir.2020.9.2.26 403
Figure 1. Geonemertes pelaensis Semper, 1863 (Nemertea: Hoplonemertea: Prosorhochmidae) from Cuba. Individual from Sierra
de Anafe, Caimito, Artemisa province, Cuba deposited in the Helminthological Collection of the Zoological Collections, Instituto
de Ecología y Sistemática, Havana, Cuba under the collection number CZACC 18.011. A. Entire specimen. B. Detail of the head.
Individual from the Instituto de Ecología y Sistemática, La Habana province, Cuba deposited in the Helminthological Collection of the
Zoological Collections, Instituto de Ecología y Sistemática, Havana, Cuba under the collection number CZACC 18.014. C. Entire
specimen. D. Entire specimen showing the ventral side. Scale bars not available. Photographs by Jans Morffe.
brown and the mid-dorsal stripe turned blackish towards that region
(Figure 1C, D). By the aforementioned color pattern G. pelaensis can be
readily differentiated from the other two nominal species of the genus.
G. rodericana (Gulliver, 1879) presents the dorsal background dark green
and the mid-dorsal stripe is white. In the case of G. philippinensis Gibson &
Moore, 1998 the mid-dorsal stripe is cream, on a dark brown background
(Gibson and Moore 1998).
The specimens included in our study present six eyes arranged as an
anterior, larger pair of eyes and two pairs of smaller and less distinct eyes
(Figure 1B). This falls within the most common range of the eye number
recorded for the species: 4–6 (Gibson and Moore 1998). In life, the length
of the specimens from Sierra de Anafe range 24 mm to 48 mm and in the
case of the individuals from the Instituto de Ecología y Sistemática range
40 mm to 55 mm. These measures are within the values of 10–60 mm
observed by Gibson and Moore (1998). Jones and Sterrer (2005) observed
some specimens with a body length of 70 mm.
The flame cells observed in the Cuban specimens are binucleate and
reinforced by longitudinal and transverse support bars (Figure 2A).
This coincides with the morphology of the flame cells typical of the genus
First record of Geonemertes pelaensis for Cuba
Morffe et al. (2020), BioInvasions Records 9(2): 399–407, https://doi.org/10.3391/bir.2020.9.2.26 404
Figure 2. Geonemertes pelaensis Semper, 1863 (Nemertea: Hoplonemertea: Prosorhochmidae) from Cuba. Transversal sections at
level of the stomach region. A. Binucleate flame cell. B. Proboscis, arrowheads point the proboscis nerves. C. Main lateral nerve
cord (ln) and accessory lateral nerve (an). Scale bars: A. 10 µm. B. 100 µm. C. 50 µm. Micrographs by Jans Morffe.
Figure 3. Maximum likelihood (ML) tree inferred from the cytochrome c oxidase subunit I (COI) gene for several species of
hoplonemerteans (Nemertea: Hoplonemertea). Three species of Amphiporus (Amphiporidae) were used as outgroup taxa. Values at
the nodes correspond to ML bootstrap resampling (≥ 70)/NJ bootstrap resampling (≥ 70).
Geonemertes (Moore and Gibson 1985; Gibson and Moore 1998). The
number of proboscis nerves is variable intraspecifically and G. pelaensis
presents a number of 16–23. Our results (19–20) fall within this range
(Figure 2B). Accessory lateral nerves are present, as is characteristic of the
genus Geonemertes (Figure 2C). In our individuals, such structures constitute
ca. 40% of the length of the main lateral nerves. This is consistent with the
observations of Gibson and Moore (1998), which recorded that the length
of the accessory lateral nerves range froma quarter or less to “a third or
slightly more” of the diameter of the main lateral nerve.
The topology of both the ML and NJ phylograms was identical and only
the former is shown (Figure 3). The Cuban specimens belong to the same
haplotype of the available sequences of G. pelaensis from Bermuda and two
islands of the Japanese archipelago, Minami-Daito and Okinawa, forming
First record of Geonemertes pelaensis for Cuba
Morffe et al. (2020), BioInvasions Records 9(2): 399–407, https://doi.org/10.3391/bir.2020.9.2.26 405
a well-supported monophyletic group. Thus, the phylogenetic analyses
support the morphological and histological data and confirm the present
specimens as G. pelaensis.
The native range of G. pelaensis appears to be the Indopacific region
where the other congeneric species, namely G. philippinensis and G. rodericana
occur (Moore and Gibson 1981; Gibson and Moore 1998). These taxa
share characters that contributes to differentiate them from the other
genera of terrestrial nemerteans native from Australia and New Zealand
(Moore and Gibson 1981). The current study extends the distribution of
G. pelaensis in the West Indies. Previously, the species was recorded from
Jamaica (Moore and Gibson 1986) and Dominica (Moore and Moore
1982). In the Americas G. pelaensis is also present in Florida (Gibson and
Moore 1998) and Bermuda (Jones and Sterrer 2005). In addition, Kvist et
al. (2014) included a specimen identified as Geonemertes sp. from Boca del
Toro, Panama in their phylogeny of Nemertea. According to the photograph
shown in the paper, the specimen presented the color pattern typical of
G. pelaensis, with the background of the dorsum cream coloured and the
dorso-median dark brown stripe. In the present study, the COI sequences
of this individual differ in five homologous positions (in an alignment of
561 bp) and the distance with the sequences of G. pelaensis is 0.9%. Still, in
order to clarify if these Geonemertes belong to another haplogroup of
G. pelaensis or constitute a new species, additional studies are required,
including increased sampling of Panamanian individuals from the same
locality and applying histological and molecular techniques.
Geonemertes pelaensis have been found in man-modified areas, such as
nurseries and gardens, under plant pots or soil sacks and beneath moist
cement blocks and bricks (Jones and Sterrer 2005). Coinciding with the
results of these authors, our specimens were also found in areas disturbed
by human activities. The individuals from Sierra de Anafe were collected
under rocks in a highly altered semi-deciduous forest growing on karst
(Figure 4A, B). This is the vegetation unit typical of the hills of western
Cuba. The specimens from the Instituto de Ecología y Sistemática were
collected under a rock in a garden area (Figure 4C, D). This institution is
located in the outskirts of Havana city, in a former country estate that was
also a school of horticulture. Due to that, exotic plants and soil were
routinely brought to the area for gardening purposes.
The previous record of a terrestrial nemertean from Cuba consisted of a
single specimen from the Valley of Viñales, Pinar del Río province, western
Cuba. The specimen, identified as Geonemertes sp., was fragmented during
collection (Palacios-Lemagne et al. 2008). These authors did not provide a
detailed description or photograph of the individual and it was not
properly fixed and deposited in a collection. Thus, information is not
enough to determine whether the record of Palacios-Lemagne et al. (2008)
First record of Geonemertes pelaensis for Cuba
Morffe et al. (2020), BioInvasions Records 9(2): 399–407, https://doi.org/10.3391/bir.2020.9.2.26 406
Figure 4. Collecting localities of Geonemertes pelaensis Semper, 1863 (Nemertea: Hoplonemertea: Prosorhochmidae) from
western Cuba. A, B. Sierra de Anafe, Caimito municipality, Artemisa province. C, D. Instituto de Ecología y Sistemática, Boyeros
municipality, La Habana province. Photographs by Luis F. de Armas (A, B) and Jans Morffe (C, D).
corresponds to a species of Geonemertes (i.e. the herein recorded G. pelaensis)
or belongs to another taxon. Further surveys on the area are needed in
order to collect terrestrial nemerteans and conduct proper morphological,
histological and molecular studies.
Acknowledgements
We thank Dr. Luis F. de Armas for kindly collect and donate the specimens from Sierra de
Anafe and to Laura Véliz (Facultad de Biología, Universidad de La Habana) for her help during
the collection of individuals from the Instituto de Ecología y Sistemática. We are grateful to the
staff of the laboratories of Pathological Anatomy of the Hospital “General Calixto García” and
the Institute of Gastroenterology, Cuba, for their technical help during the histological
processing of the samples. Eduardo Furrazola (Instituto de Ecología y Sistemática) provided
assistance with the micrographs. Byron Adams (Brigham Young University and Monte L. Bean
Museum) provided comments on early drafts of the manuscript. Two anonymous reviewers
provided useful comments that helped improve this manuscript. This research was also
supported by the project “Colecciones Zoológicas, su conservación y manejo III”, Ministerio de
Ciencia, Tecnología y Medio Ambiente, Cuba.
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